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Page 1: 2nd SPLC-CRS Young Scientists Meeting...2nd SPLC-CRS Young Scientists Meeting Novoa Santos Auditorium, School of Medicine (USC) Rua San Francisco s/n Santiago de Compostela January

2nd SPLC-CRS Young Scientists Meeting

Novoa Santos Auditorium, School of Medicine (USC)

Rua San Francisco s/n

Santiago de Compostela

January 23rd, 2019

BOOK of ABSTRACTS

Rectorship of University of Santiago de Compostela Colexio de San Xerome, Praza do Obradoiro

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2nd SPLC-CRS Young Scientists Meeting, January 23rd, 2019

Santiago de Compostela, Spain

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Contents

Contents ....................................................................................................................... 1

Organizing Committees ................................................................................................ 2

Welcome Message and Venue ..................................................................................... 4

Scientific Program ......................................................................................................... 6

Invited Speakers ......................................................................................................... 10

Abstracts: Oral presentations ...................................................................................... 13

Abstracts: Poster presentations .................................................................................. 29

List of authors ............................................................................................................. 63

List of co-authors ........................................................................................................ 67

List of participants ....................................................................................................... 72

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2nd SPLC-CRS Young Scientists Meeting, January 23rd, 2019

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Organizing Committees

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SPLC-CRS Board

Prof. Dr. Dolores Torres, President, University of Santiago de Compostela, Spain

Prof. Dr. María Blanco Prieto, Secretary, University of Navarra, Spain

Prof. Dr. Carmen Maria Évora García, Vice-President, University of La Laguna, Spain

Prof. Dr. Bruno Sarmento, Director, University of Porto, Portugal

Prof. Dr. Helena Florindo, Director, University of Lisbon, Portugal

Prof. Dr. Manuel Santander, Treasurer, University of Castilla La Mancha, Spain

Young Scientist Committee

Alba Calvo Bacaicoa, University of Navarra, Spain

Carlos Rodríguez, University of Navarra, Spain

Flávia Sousa, University of Porto, Portugal

Liane Moura, University of Lisboa, Portugal

María Plaza, University of Castilla La Mancha, Spain

Matilde Durán Lobato, University of Santiago de Compostela, Spain

Patricia Costa Filipe, University of Lisbon, Portugal

Sandra Jesus, University of Coimbra, Portugal

Sonia Vicente-Ruiz Salvador, Centro de Investigación Príncipe Felipe Valencia, Spain

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Welcome Message and Venue

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Dear participants,

On behalf of the Board of the Spanish-Portuguese Local Chapter of the Controlled Release Society (SPLC-CRS), we are delighted to welcome you to the 2nd SPLC-CRS Young Scientists Meeting at the Novoa Santos Auditorium, School of Medicine, University of Santiago de Compostela, on January 23th, 2019.

This initiative comes along with the CRS initiative to award young scientists with travel grants to those local chapters applying for those awards. These travel grants will enable students to participate in the 2019 CRS annual meeting (July 21-24, 2019, Valencia, Spain). Four travel grants will be awarded to the best oral and poster communications. The program includes 2 invited talks by Marta Vives-Pi (UniversitatAutònoma de Barcelona) and João F. Mano (University of Aveiro), well known leaders in their field of research, 15 oral presentations and 33 posters.

In advance, we again thank you for your interest in 2nd SPLC-CRS Young Scientists Meeting.

We look forward to meeting you in Santiago!

On behalf of the Board of SPLC-CRS,

Dolores Torres President of the SPLC-CRS

María J. Blanco-Prieto Secretary of the SPLC-CRS

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2nd SPLC-CRS Young Scientists Meeting, January 23rd, 2019

Santiago de Compostela, Spain

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Scientific Program

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Novoa Santos Auditorium, School of Medicine (USC)

23/01/2019

8:30 Registration

Presentations upload/posters mounting

8.50 Welcome

Dolores Torres (USC; SPLC-CRS President)

Maria José Alonso (USC; CRS President)

9:00 Invited speaker – Marta Vives-Pi “Targeting autoimmune diseases with nanomedicine: The immune re-

education”

Head of the Immunology of Diabetes UnitGermans Trias i Pujol Research Institute (IGTP), Autonomous University of Barcelona

Chairs: María Jesús Vicent (CIPF, Valencia) Bruno Sarmento (i3S; U Porto)

Young Scientists Session I

9:30 Polymeric nanocapsules as a novel tolerogenic treatment for Type 1 Diabetes - Ana Olivera Center for Research in Molecular Medicine and Chronic Diseases (CIMUS, University of Santiago de Compostela

9:39 Development of in vitro dissolution tools predictive of the in vivo behaviour of pharmaceutical formulations: Candesartan in vitro-in vivo correlation (IVIVC) - Bárbara Sánchez Dengra Pharmacokinetics and Pharmaceutical Technology Area. Miguel Hernandez University

9:48 Engineered albumin conjugated nanoparticles are a promising strategy to enhance FcRn-mediated delivery across intestinal barriers – Claudia Azevedo i3S Instituto de Investigação e Inovação em Saúde. Universidade do Porto

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9:57 Nanoencapsulated Bevacizumab Inhibits Glioblastoma Vascularization via

Intratumoral VEGF Trapping – Flávia Sousa i3S Instituto de Investigação e Inovação em Saúde. Universidade do Porto

10.06 A nanotechnological approach to improve the therapeutic efficacy of cytotoxic agents against melanoma – Jacinta Pinho

Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa

10:15 Evaluation of the immunotoxicological properties of PLA nanoparticles– Jessica da Silva Faculty of Pharmacy. University of Coimbra

10:24 Casein nanoparticles as effective vehicles for enhancing the oral bioavailability of resveratrol – Jorge Morales Gracia Department of Chemistry and Pharmaceutical Technology, University of Navarra

10:33 Design of an anti IGFR1 polymer-antibody conjugate in combination with Abiraterone for the treatment of Castration-Resistant Prostate Cancer (CRPC) – Katia Maso Polymer Therapeutics Lab, Centro de investigación Príncipe Felipe (CIPF), Valencia

10:42 Combination of cardiomyocytes derived from human induced pluripotent stem cells with biomaterials restores cardiac function in infarcted mice – Laura Saludas Echauri Department of Chemistry and Pharmaceutical Technology, University of Navarra

11.00 Coffee-break/ Posters Viewing

11:30 Invited speaker – Joao Mano

“Nano to macro- polymeric particles for the release of active molecules”

Full Professor Department of Chemistry, CICECO - Aveiro Institute of Materials University of Aveiro, Portugal

Chairs: Helena Florindo (U Lisboa) Manuel Santander (U Castilla-La Mancha)

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Young scientists session II

12:00 Nanotechnology-based approach for the delivery of glycoconjugate vaccines - Maruthi Prasanna Center for Research in Molecular Medicine and Chronic Diseases (CIMUS, University of Santiago de Compostela

12:09 Rational development and preclinical assessment of bio-inspired Protein-Metal Nanosystems as oral delivery carrier – Matilde Durán Lobato Center for Research in Molecular Medicine and Chronic Diseases (CIMUS, University of Santiago de Compostela

12:18 Control of Loading and Polarized Orientation of Antibodies onto Gold Nanoparticles – Mireya López Borrajo Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela, Spain

12:27 Impact of PEG coating in the distribution and retention of PLGA nanoparticles in colorectal tissues – Rute Nunes I3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto

12:36 Multimodal nanoemulsions for whole-body imaging in metastatic cancer – Sandra Diez VillaresNano-Oncology Unit, Translational Medical Oncology Group (ONCOMET). Health Research Institute of Santiago de Compostela (IDIS) and CIBERONC, Santiago de Compostela

12.45 Combination of polymeric nanoparticles and alginate cryogel towards the development of an in situ cancer vaccine – Tomas Ramos i3S Instituto de Investigação e Inovação em Saúde e Instituto de Engenharia Biomédica, Universidade do Porto

13:00 Award Ceremony for the best oral and poster presentations

13:15 SPLC-CRS General Assembly

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Invited Speakers

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Targeting autoimmune diseases with nanomedicine: The

immune re-education

Marta Vives-Pi

Head of the Immunology of Diabetes Unit

Germans Trias i Pujol Research Institute (IGTP), Autonoma University, Barcelona

Type 1 diabetes (T1D) is a metabolic disease caused by the autoimmune

destruction of insulin-producing β-cells. With its incidence increasing worldwide,

to find a safe approach to permanently cease autoimmunity and allow β-cell

recovery has become vital. Relying on the inherent ability of apoptotic cells to

induce immunological tolerance, we demonstrated that liposomes mimicking

apoptotic β-cells arrested autoimmunity to β-cells and prevented experimental

T1D through tolerogenic dendritic cell (DC) generation. This novel

immunotherapy has the potential to re-establish immunological tolerance,

opening the door to new therapeutic approaches in the field of autoimmunity.

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Nano to macro- polymeric particles for the release of active molecules

João F. Mano

Department of Chemistry. CICECO. University of Aveiro. 3810-193 Aveiro. Portugal.

Email: [email protected]

Biodegradable and biocompatible polymeric particles have been widely used to

encapsulate molecules that could then be released in a controlled or sustained

way. This lecture will explore a range of technologies for the processing and

application of particles for drug release ranging from the nano to the macro-scale

sizes. Although the methodologies proposed are transversal for many

pharmaceutical and bioengineering applications examples will especially focus

the area of tissue engineering and regenerative medicine, in particular for bone

repair. Nanoparticles can be used to deliver differentiation factors to direct the

faith of stem cells. We discovered that naringin, a naturally occurring flavanone,

encapsulated in nanomiceles could be internalised and released in mesenchymal

stem cells, promoting the differentiation and augmenting the pro-osteogenic

effect over that of free drug and standard induction methods, thus indicating the

potential of this system to enhance stem cell–based bone regeneration.

Micro and macro-particles should be prepared using other methodologies. We

proposed the use of superhydrophobic substrates as platforms to produce

hydrogel or polymeric particles able to encapsulate molecules with a high loading

efficiency. Several examples are presented in this context using natural based

polymers and synthetic biodegradable polymers. The mild conditions that the

particles may be formed allows for the encapsulation of living cells, extending the

applicability of the technology.

Liquefied capsules are also an interesting system where drugs or cells may be

encapsulated and released in a controllable fashion. We have been using the

layer-by-layer technique (LbL) for the production of the multilayer shell that is

enveloping the capsules. The mechanical and transport properties of the

multilayers could be tuned, and the use of stimuli-responsive polymers in the

construction of the shells permits to develop smarter delivery systems. The

technology was also adapted to encapsulate living cells, and the resulting

compartmentalized cell microfactories can be produced for distinct biomedical

applications.

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Abstracts: Oral presentations

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O1. Polymeric nanocapsules as a novel tolerogenic treatment for Type 1 Diabetes

Ana Olivera1,2, Cristina Calviño-Sampedro3, Rubén Varela-Calviño3, Dolores Torres2, María José

Alonso1,2

1Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de

Santiago de Compostela, Spain 2Department of Pharmacology, Pharmacy and Pharmaceutical Technology, School of Pharmacy,

Universidade de Santiago de Compostela, Spain 3Department of Biochemistry, School of Pharmacy, Universidade de Santiago de Compostela,

Spain

Type 1 diabetes (T1D) is considered a chronic autoimmune disease caused by the destruction of

β-cells located in the Langerhans islets by the immune system, which leads to the loss of insulin

production in pancreas. Within this context, specific treatments to overcome the immune reaction

against β-cells are desirable. Nanotechnology offers the possibility of modulating the specific

immune response, by developing a tolerogenic profile in the immune competent cells, thus

avoiding the off-target effects1. In this report, we evaluated polymeric nanocapsules (NCs)

containing a tolerogenic molecule as a therapy for Type 1 Diabetes. NCs were prepared using

solvent-displacement technique, showing all the prototypes particle size around 200 nm and a

surface charge around +50 mV. Upon 6 h of incubation of the NCs with human dendritic cells

(hDCs), no signs of toxicity were observed up to a dose of 900 µg/mL. Fluorescence microscopy

images showed the ability of the NCs to interact with hDCs. NCs were able to maintain the activity

of the tolerogenic molecule and were effective at inducing a tolerogenic profile in hDCs. Finally,

NCs were evaluated in NOD mice model, leading to a significant delay in the diabetes onset

compared to controls.

REFERENCES

1. Dacoba T.G. et al., Semin Immunol. 34:78-102, 2017

ACKNOWLEDGEMENTS

Research reported in this publication was supported by the Spanish Ministry of Science,

Innovation and Universities [grant agreement number 646142]. The first author is also supported

by a FPI grant from the Spanish Ministry of Economy, Industry and Competitiveness (grant

number BES-2015-071236).

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O2. Development of in vitro dissolution tools predictive of the in vivo behaviour of

pharmaceutical formulations: Candesartan in vitro-in vivo correlation (IVIVC).

Andrés Figueroa-Campos1,2, Bárbara Sánchez-Dengra1, Virginia Merino2, Isabel González-

Álvarez1, Marta González-Álvarez1, Marival Bermejo1

1Engineering: Pharmacokinetics and Pharmaceutical Technology Area. Miguel Hernandez

University, San Juan de Alicante, Alicante, 03550, Spain. 2Pharmacy and Pharmaceutical Technology. Valencia University, Valencia, Valencia, 46100,

Spain.

Introduction

An in vitro-in vivo correlation (IVIVC) is a mathematical relationship which, after being validated,

is able to relate an in vitro characteristic of a drug (e.g. dissolution), with a characteristic of its

biological behaviour in vivo (e.g. absorption). The objective of this work was to establish a level

A IVIVC for three Candesartan oral immediate release formulations.

Methods

Plasma profiles of Candesartan for all the formulations were deconvoluted by Loo-Riegelman

method to obtain the individual fractions absorbed (fa).

Fractions dissolved (fdiss) were obtained in several conditions in USP II and USP IV apparatus

and the most biopredictive in vitro experiment was chosen by comparison of dissolution

profiles with the f2 similarity factor.

Levy plot was constructed to estimate the time scaling factor and two two-steps IVIVCs (linear

and polynomial) were obtained.

The program Berkeley-Madonna was used for obtaining a one-step IVIVC.

The percentages of prediction error (%PEs) were calculated as a predictability measure for

validating the correlations.

Results and discussion

In vivo biological behaviour of Candesartan can be described by means of a two-compartment

model. Dissolution profiles obtained in USP IV apparatus with media of pH 1.2, 4.5 and 6.8

modified with 0.2% Tween 20 were selected as biopredictive for preparing the IVIVCs.

Three level A IVIVCs were obtained, whose percentages of prediction error are lower than the

pre-established limits (the individual %PE of each formulation for each parameter does not

exceed 15% and the mean %PE of all formulations for each parameter is less than 10%), so they

are valid and biopredictive.

Conclusions

All three IVIVCs could be used as substitutes of human bioequivalence studies, after their

approval by the competent agencies. Nevertheless, the correlation considered more appropriate,

at this moment, is the linear IVIVC obtained by the two-step method.

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O3. Engineered albumin conjugated nanoparticles are a promising strategy to

enhance FcRn-mediated delivery across intestinal barriers

C. Azevedo1,2,3, J. Nilsen4,5, A. Grevys4,6, R. Nunes1,2,3, J. T. Andersen4,6, B. Sarmento1,2,7

1i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal; 2Nanomedicines & Translational Drug Delivery Group, INEB - Instituto Nacional de Engenharia

Biomédica, Portugal; 3Instituto de Ciências Biomédicas Abel Salazar, University of Porto, Portugal; 4Department of Immunology, Centre for Immune Regulation (CIR), Oslo University Hospital

Rikshospitalet and University of Oslo, Oslo, Norway 5Institute of Clinical Medicine, University of Oslo, Oslo, Norway 6Department of Biosciences, University of Oslo, Norway. 7CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde

& Instituto Universitário de Ciências da Saúde, Gandra, Portugal

To maintain patient compliance, oral administration is the preferred drug delivery route [1]. Yet,

many drugs, including biopharmaceuticals do not endure the harsh environment of the

gastrointestinal tract and cross epithelial barriers inefficiently. Functionalization of nanoparticles

(NPs) with ligands that can bind specifically to surface receptors may be one strategy to overcome

these obstacles and to improve the delivery of pharmaceuticals through biological barriers [2]. We

aimed to explore whether polymeric nanoparticles (NPs) conjugated with engineered human

albumin for enhanced FcRn binding [3, 4] could be an attractive strategy for delivery of

encapsulated drugs across mucosal barriers. For this purpose, PLGA NPs were designed using

double emulsion/evaporation technology where engineered human albumin was site-specifically

conjugated to the polymers.

NPs with approximately 150 nm, presented an insulin encapsulation efficiency over 80% and

approximately 100% of the conjugated albumin on the surface. NPs-HSA variants bind with

expected [5] binding hierarchy to hFcRn: NPs-KAHQ < NPs-WT < NPs-KP < NPs-TNNEKP. NPs-

KP and NPs-TNNEKP present improved affinity for FcRn by 6- fold and 12-fold, respectively. This

enhance FcRn-mediated delivery across intestinal barriers and contributes for the NPs extended

half-life. This was confirmed by permeability studies and Human endothelial cell-based recycling

assay (HERA), which suggest that NPs conjugated with high binder albumin variants are

transported and recycled more efficiently, in an FcRn-dependent manner. The proof of concept

was demonstrated in the preliminary pharmacodynamic study, using the state-of-the-art human

FcRn transgenic mouse model. It was verified that NPs-TNNEKP present approximately 18% of

relative hypoglycemic decrease and 6% of pharmacologic availability, after 24h of administration,

when compared with insulin s.c. administered. Next, we will explore the pharmacokinetics as well

as the biodistribution of the formulations.

In short, the FcRn-targeted approach may pave the way for more efficient delivery of NP-

encapsulated drugs.

References

[1] B. Sarmento, et al., L. Jorgensen and H.M. Nielsen, Editors, 2009, 207-227.

[2] P. Fonte, et al, Biotechnology Advances, 2015, 33, 1342-1354.

[3] D.C. Roopenian and S. Akilesh, Nature Reviews Immunology, 2007, 7, 715-725.

[4] J. Martins, et al, Pharmacology & Therapeutics, 2016, 161, 22-39.

[5] J.T. Andersen, et al., Journal of Biological Chemistry, 2014, 289(19): 13492-13502.

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O4. Nanoencapsulated Bevacizumab Inhibits Glioblastoma Vascularization via

Intratumoral VEGF Trapping

Flávia Sousa1,2,3,4,5, Andrea Cruz5, Fábio Júnio Ferreira1,6, José Bessa1,6, Bruno Sarmento1,2,4, Inês Mendes Pinto5* 1i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen 208, 4200- 393 Porto, Portugal 2INEB - Instituto Nacional de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen 208, 4200- 393 Porto, Portugal 3ICBAS - Instituto Ciências Biomédicas Abel Salazar, Universidade do Porto, 4150-180 Porto, Portugal 4CESPU - Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Rua Central de Gandra 1317, 4585-116 Gandra, Portugal 5INL, International Iberian Nanotechnology Laboratory, Avenida Mestre José Veiga, 4715-330 Braga, Portugal 6IBMC, Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua Alfredo Allen 208, 4200-393 Porto, Portugal

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumor, being the median survival time of patients at around 15 months after disease diagnosis1. GBM has the constant need of vascularization, making this tumor one of the most vascularized and invasive solid tumors2. Bevacizumab, an anti-VEGF monoclonal antibody, was approved by FDA to be used as a single agent for patients with GBM3. Despite good results in the clinical trials, the low probability of bevacizumab in crossing the BBB limits its CNS accessibility and does not allow an improvement in the overall patient survival4. Therefore, an alternative to improve the efficacy of GBM treatment is highly needed and might be achieved by the combination of nanotechnology through controlled release nanosystems5. In this study, bevacizumab-loaded PLGA NP were successfully developed as an alternative to cross effectively BBB, accomplishing a better therapy6. No significant differences were also found by BrdU and ELISA assay for anti-proliferative and anti-VEGF properties between free and encapsulated bevacizumab, demonstrating the success of encapsulation. In vivo efficacy of bevacizumab-loaded PLGA NP was evaluated using a glioma zebrafish model to study the neoangiogenesis and tumor growth through the injection of GBM cancer cells. In vivo results showed a significant decrease in tumor area just for the bevacizumab-loaded PLGA NP group. Trying to understand the molecular mechanism behind the efficacy of nanoparticles, a cellular uptake in both cell lines was done to study the internalization of bevacizumab and its effect on VEGF secretion. A significant increase in the number of bevacizumab positive cells and a decrease in the number of VEGF producing cells was obtained for the bevacizumab-loaded PLGA NP group. These last results demonstrated that bevacizumab-loaded PLGA NP might cause a disorder in VEGF signaling pathway, being an efficient alternative to deliver intracellularly monoclonal antibodies.

References 1 Ohgaki, H. Epidemiology of brain tumors. Methods Mol. Biol. 472, 323-342, doi:10.1007/978-1-

60327-492-0_14 (2009). 2 Broekman, M. L. et al. Multidimensional communication in the microenvirons of glioblastoma. Nat. Rev. Neurol. 14, 482-495, doi:10.1038/s41582-018-0025-8 (2018).

3 Cohen, M. H., Shen, Y. L., Keegan, P. & Pazdur, R. FDA drug approval summary: bevacizumab (Avastin) as treatment of recurrent glioblastoma multiforme. Oncologist 14, 1131-1138,

doi:10.1634/theoncologist.2009-0121 (2009). 4 Thompson, E. M., Frenkel, E. P. & Neuwelt, E. A. The paradoxical effect of bevacizumab in the therapy of malignant gliomas. Neurology 76, 87-93, doi:10.1212/WNL.0b013e318204a3af (2011).

5 Gomes, M. J., Fernandes, C., Martins, S., Borges, F. & Sarmento, B. Tailoring Lipid and Polymeric Nanoparticles as siRNA Carriers towards the Blood-Brain Barrier - from Targeting to Safe Administration. J. Neuroimmune Pharmacol. 12, 107-119, doi:10.1007/s11481-016-9685-6 (2017). 6 Sousa, F. et al. A new paradigm for antiangiogenic therapy through controlled release of bevacizumab from PLGA nanoparticles. Sci. Rep. 7, 3736, doi:10.1038/s41598-017-03959-4 (2017).

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O5. A nanotechnological approach to improve the therapeutic efficacy of cytotoxic

agents against melanoma

Jacinta O. Pinho1, Eduarda Mendes1, Maria de Jesus Perry1, Joana D. Amaral1, Cecília M.P.

Rodrigues1, Ana P. Francisco1, Angela Casini2, Graça Soveral1, M. Manuela Gaspar1

1Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, 1649-003, Portugal; 2School of Chemistry, Cardiff University, Cardiff, United Kingdom

Melanoma is a major cause of morbidity and mortality, and effective therapeutic options are

needed. Therefore, two distinct compounds have been explored by our research group. First, the

copper complex Cuphen [1,2], recently reported as inhibitor of aquaporins (AQPs). These are a

family of transmembrane proteins overexpressed in several malignancies and promising targets

for cancer treatment [3]. Second, a newly synthesized triazene hybrid molecule (THM) [4]. THM

displays two distinct moieties, acting by two mechanisms: 1) high specificity towards tyrosinase,

an enzyme up-regulated in melanoma cells; 2) toxic alkylating properties.

In vitro studies demonstrated the high cytotoxic effect of Cuphen and THM towards murine and

human cancer cell lines, with IC50 values <10 µM and <30 µM, respectively. To improve in vivo

tumor accumulation, drugs’ stability and therapeutic benefit, these compounds were associated

to long circulating liposomes, with or without pH-sensitive properties [1,2,5]. All developed

nanoformulations were homogeneous and high incorporation efficiencies were achieved for

Cuphen (80-90%) and THM (100%). Their antiproliferative properties were preserved after

association to liposomes, showing IC50 values similar to the corresponding free drug. The

assessment of hemolytic activity in human red blood cells, and hepatic biomarkers in healthy

mice, following i.v. administration, demonstrated their safety for in vivo assays.

Cuphen and THM nanoformulations were evaluated in a syngeneic murine melanoma model

[2,6]. For both drugs, a superior therapeutic benefit was obtained for mice treated with the

compounds associated to liposomes, compared to control group, to mice receiving the free drug,

and to mice receiving 5-fluorouracil (5-FU), a clinically used chemotherapeutic. This was

confirmed by impaired tumor progression, absence of hepatic toxic side effects, and 100%

survival.

Overall, these preclinical data demonstrate the therapeutic advantages of liposomes as a delivery

system to target cytotoxic compounds to solid tumors. Cuphen and THM are considered two

compounds with high potential against melanoma.

[1] Nave M, et al. Nanoformulations of a potent copper-based aquaporin inhibitor with cytotoxic effect against

cancer cells. Nanomedicine. 11(14), 1817–1830 (2016).

[2] Pinho JO, et al. Copper complex nanoformulations featuring highly promising therapeutic potential in

murine melanoma models. Nanomedicine. (2018) (Submitted).

[3] Verkman AS, et al. Aquaporins – new players in cancer biology. J. Mol. Med. 86, 523 (2008).

[4] Granada AM, et al. Evaluation of stability, hepatotoxicity and activation by tyrosinase of new triazene

prodrugs for metastatic malignant melanoma. 1st Symposium on Medicinal Chemistry, University of Minho,

Braga, Portugal (2013).

[5] Simões S, et al. On the formulation of pH-sensitive liposomes with long circulation times. Adv. Drug Deliv.

Rev. 56(7), 947–965 (2004).

[6] Sainz V, et al. α-Galactosylceramide and peptide-based nano-vaccine synergistically induced a strong

tumor suppressive effect in melanoma. Acta Biomater. 76, 193-207 (2018).

Acknowledgements: Fundação para a Ciência e a Tecnologia (FCT) through projects:

UID/DTP/04138/2014, PTDC/BTM-SAL/28977/2017 and PTDC/MED-QUI/31721/2017, as well as by PhD

fellowship SFRH/BD/117586/2016.

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O6. Evaluation of the immunotoxicological properties of PLA nanoparticles

Jessica Da Silva1;2, Sandra Jesus1;2, Natália Bernardi1;2 and Olga Borges1;2

1Faculty of Pharmacy, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra,

Portugal 2Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal

Polylactic acid (PLA) is a biodegradable, biocompatible and a Food and Drug Administration

approved polymer, widely used as a platform for antigen and drug delivery, among others. [1, 2]

Despite generally regarded as safe, the immunotoxicological profile of PLA, when used as a

polymeric nanoparticle (NP), is not well documented. [3] Thus, this study intends to fill this gap, by

evaluating the immunotoxicological profile of PLA NPs, and to compare it with other NPs such as

polycaprolactone (PCL) and PCL/chitosan (PCL/Chi) NPs.

All nanoparticles were produced by a nanoprecipitation method and were characterized regarding

its mean diameter, polydispersity index and zeta potential. Then, several assays were performed

in order to evaluate the immunotoxicological properties of each NP: cell viability, nitric oxide (NO)

production and inhibition, and reactive oxygen species (ROS) production were evaluated in RAW

264.7 cells; cell viability was assessed in human peripheral blood mononuclear cells (PBMCs);

and hemolysis was studied in whole human blood.

Two different size PLA NPs were produced (200 nm and 100 nm), as well as PCL NPs with ~170

nm. PCL/Chi NPs presented ~270 nm and a distinctive positive zeta potential. Cell viability studies

in RAW 264.7 showed higher toxicity for PCL/Chi NPs, with an IC50 of 222 µg/mL. Regarding

ROS production, no effect was observed with the PCL and the PCL/Chi NPs under the

concentration range tested (8.6-689.4 µg/mL). Interestingly, PLA NPs with larger size induced a

dose-dependent ROS production. Also, none of the NPs under test induced NO production or

inhibition (1-100 µg/mL), all induced cell viabilities in PBMCs above 70 % (1.1-1125 µg/mL) and

presented a good hemocompatibility profile.

Therefore, this study emphasizes the importance of establishing the immunotoxicological profile

of different NPs, as we verified that differences in size, composition and zeta potential can

influence the delivery system biological activity.

[1] ESSA, S. et al. Improved antifungal activity of itraconazole-loaded PEG / PLA nanoparticles. v.

30, n. 3, p. 205–217, 2013. [2] LEGAZ, S. et al. ST AC. v. 5390, n. April, 2016. [3] SINGH, R. P.; RAMARAO, P. Accumulated Polymer Degradation Products as Effector

Molecules in Cytotoxicity of Polymeric Nanoparticles. v. 136, n. 1, p. 131–143, 2013.

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O7. Casein nanoparticles as effective vehicles for enhancing the oral

bioavailability of resveratrol

Rebeca Peñalva1, Jorge Morales1, Carlos J. González-Navarro2, Juan M. Irache1

1Department of Chemistry and Pharmaceutical Technology, University of Navarra, 31008

Pamplona, Spain. 2Centre for Nutrition Research, University of Navarra, 31080 Pamplona, Spain.

Resveratrol is a naturally occurring polyphenol that provides several health benefits including

cardioprotection and cancer prevention. However, its biological activity is limited by a poor

bioavailability when taken orally. In addition, after the oral administration of resveratrol, it suffers

a presystemic metabolism, which leads to the formation of a metabolite (resveratrol-3-O-

glucuronide) that also results in a low oral bioavailability. The aim of this work was to evaluate the

capability of casein nanoparticles as oral carriers for resveratrol. Nanoparticles were prepared by

a coacervation process, purified and dried by spray-drying. The mean size of the resveratrol

loaded nanoparticles was around 200 nm, with a payload close to 30 µg/mg nanoparticle.

Resveratrol release from casein nanoparticles was not affected by the pH conditions and followed

a zero-order kinetic. When nanoparticles were administered orally to rats, they remained within

the gut, displaying an important capability to reach the intestinal epithelium. No evidence of

nanoparticle “translocation” were observed. The resveratrol plasma levels were high and

sustained for at least 8 hours, it was also quantified in plasma after 24 hours. Figure 1 shows the

plasma concentration levels of resveratrol as a function of time after a single oral administration

of 15 mg/kg to male Wistar rats of the different formulations tested (solution and casein

nanoparticles). The oral bioavailability of resveratrol when loaded in casein nanoparticles was

calculated to be 26.5%, ten-fold higher than when the polyphenol was administered as oral

solution. The main metabolite of the polyphenol, resveratrol-3-O-glucuronide, was quantified in

plasma after 24 hours. Finally, a good correlation between in vitro and in vivo data was observed.

Figure 1. Resveratrol plasma concentration vs. time after a single oral administration of 15 mg/kg

i) resveratrol PEG400:water solution (●), ii) resveratrol-loaded casein nanoparticles (■). Data

expressed as mean± SD, (n= 6).

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O8. Design of an anti IGFR1 polymer-antibody conjugate in combination with

Abiraterone for the treatment of Castration-Resistant Prostate Cancer (CRPC)

Sonia Vicente-Ruiz1, Katia Maso1, Ana Armiñan1, Elena Gallon1, Julie Movellan1, David

Charbonnier1, Fernanda Rodríguez1, María García-Flores2, José A. López-Guerrero2, María J.

Vicent1

1Polymer Therapeutics Lab, Centro de investigación Príncipe Felipe (CIPF), Valencia, Spain 2Fundación Instituto Valenciano de Oncología, Valencia, Spain

A chromosomal rearrangement between TMPRSS2 (androgen-dependent serine protease) and

ERG (transcription factor belonging to the ETS family) genes, that leds to the overexpression of

TMPRSS2-ERG (T2E) transcript, is present in 50-70% of prostate cancer (PCa) patients.1,2 It has

been showed that the combination of an inhibitor of type 1 insulin-like growth factor receptor

(IGF1R) with an anti-androgen drug (Abiraterone) results in synergistic anti-tumor effects in T2E

positive cells.3 In the present work, we have investigated the anti tumoral activity of a human

monoclonal antibody (mAb) targeting IGF1R and its synergism with the anti-androgen drug

against aggressive PCa. In particular, the anti IGFR1 mAb was covalently conjugated to poly-L-

glutamic acid (PGA) through a reducible disulfide bridge and the polymer conjugate was fully

characterized by GF chromatography, SDS-PAGE, DLS, FUV-CD and amino acid analysis. In

vitro citotoxicity of mAb-PGA was evaluated in a panel of PCa cell lines (VCaP, LNCaP, PC3,

22RV1, DU145 and RWPE1) with the MTS assay. However, only the VCaP cells, which express

the T2E fusion gene, responded to both conjugated and unconjugated mAb. In addition, in vitro

results disclosed that mAb-PGA in combination with Abiraterone displayed enhanced selectivity

for VCaP cells respect to the combination with the unconjugated mAb. To further investigate this

result, the compounds were Cy5.5 labelled and their intracellular fate in VCaP cells was followed

by confocal and STORM microscopy, showing a different cellular trafficking. Moreover, to

determine the cellular pathways related with treatments, we checked the differential proteins

expression by Western Blot assay. We optimized an orthotopic PCa mice model employing

luciferase-expressing VCaP cells and we iv injected unconjugated and conjugated mAb at the

same dose for comparison. The results showed an higher mAb-PGA antitumoral activity respect

to mAb. To corroborate the in vitro synergistic effects, we are currently testing the activity of the

combination therapy with abiraterone in our in vivo model. So far, these results suggest that the

combination anti IGFR1 mAb-PGA + Abiraterone may represent a promising therapeutic strategy

for the T2E PCa patient subtype.

Acknowledgements: This study is funded by PROMETEO/2016/103, SOGUG, Plan Nacional

I+D; SAF2013-44848-R and FPI grant BES-2014-068439.

References:

1. Barbieri, CE et al. (2012) Molecular genetics of prostate cancer: emerging appreciation of genetic

complexity. Histopathology. 60(1):187-98.

2. Tomlins, SA et al. (2009) ETS gene fusions in prostate cancer: from discovery to daily clinical practice.

European urology. 56(2):275-86.

3. Mancarella, C et al. (2015) ERG deregulation induces IGF-1R expression in prostate cancer cells and

effects sensitivity to anti-IGF-1R. Oncotarget. 30;6(18):16611-22.

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O9. Combination of cardiomyocytes derived from human induced pluripotent stem

cells with biomaterials restores cardiac function in infarcted mice

Laura Saludas1,2, Elisa Garbayo1,2, Manuel Mazo2,3, Beatriz Pelacho2,3, Gloria Abizanda2,3, Felipe

Prósper2,3, María Blanco-Prieto1,2

1 Department of Pharmaceutical Technology and Chemistry, Faculty of Pharmacy and Nutrition, University

of Navarra, Pamplona, Spain 2 Instituto de Investigación Sanitaria de Navarra, IdiSNA, Pamplona, Spain 3 Area of Cell Therapy, Center for Applied Medical Research and Clínica Universidad de Navarra, Pamplona,

Spain

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) represent the only cell source with potential to remuscularize the infarcted area of the human heart [1]. Furthermore, they offer several other advantages such as proven effective paracrine effect, integration in the host myocardium and non-immunogenicity [2,3]. However, delivery issues related to the low cell survival and engraftment in the heart have limited the clinical implementation of an effective cell-based therapy for cardiac repair [4,5]. In this study, we proposed that the combination of hiPSC-CMs with microparticles (MPs) could constitute an effective strategy to improve the long-term engraftment of cells and therefore, to enhance their therapeutic effect. A homogenous population of poly(lactic-co-glycolic) acid MPs with a mean size of 10.4 ± 0.8 µm was obtained by the multiple emulsion solvent evaporation method. Next, particles were covered with collagen and poly-D-lysine to provide particles with a favourable biomimetic microenvironment for cell adhesion. CMs were obtained by differentiation of hiPSC by small-molecule manipulation of the Wnt-pathway, purified and adhered to the biomimetic MPs. First, the potential of particles to support cell survival was analyzed in vitro by the Alamar Blue assay, revealing a 1.99-fold increase in cell viability. Then, hiPSC-CM-MPs complexes were administered in a mouse myocardial infarction model. Interestingly, transplantation of hiPSC-CMs adhered to MPs improved the long-term engraftment of these cells up to two months. Furthermore, engrafted cells expressed dystrophin and connexin 43, which suggest that hiPSC-CMs maintained a cardiac phenotype and showed efficient electrical coupling through gap junctions. Finally, the improvement in the long-term retention of cells correlated with an increased heart function recovery. These findings suggest that MPs represent excellent platforms for cell delivery applications as they improve the long-term retention of cells. This strategy could be implemented also in other fields of regenerative medicine.

References

[1] Y.-W. Liu, B. Chen, X. Yang, J.A. et al., Human embryonic stem cell–derived cardiomyocytes

restore function in infarcted hearts of non-human primates, Nat. Biotechnol. 36 (2018) 597–605.

[2] D.A. Pijnappels, S. Gregoire, S.M. Wu, The integrative aspects of cardiac physiology and their

implications for cell-based therapy., Ann. N. Y. Acad. Sci. 1188 (2010) 7–14.

[3] A. Tachibana, M.R. Santoso, M. Mahmoudi, et al., Paracrine Effects of the Pluripotent Stem Cell-

Derived Cardiac Myocytes Salvage the Injured Myocardium., Circ. Res. 121 (2017) e22–e36.

[4] O. Iglesias-García, S. Baumgartner, L. Macrí-Pellizzeri, et al., Neuregulin-1β induces mature

ventricular cardiac differentiation from induced pluripotent stem cells contributing to cardiac tissue

repair., Stem Cells Dev. 24 (2015) 484–96.

[5] J. V Terrovitis, R.R. Smith, E. Marbán, Assessment and optimization of cell engraftment after

transplantation into the heart., Circ. Res. 106 (2010) 479–94.

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O10. Nanotechnology-based approach for the delivery of glycoconjugate

vaccines

Maruthi Prasanna1,2, Aline Pillot2, Emilie Camberlein2, Cristina Calviño3, Ruben Varela3,

Daphnée Soulard4, François Trottein4, Marcos Garcia-Fuentes1, Cyrille Grandjean2, Noemi

Csaba1

1Center for Research in Molecular Medicine and Chronic Diseases, University of Santiago de Compostela,

Spain; 2Unit Function & Protein Engineering, UMR CNRS 6286, University of Nantes, France; 3Department of Biochemistry and Molecular Biology, University of Santiago de Compostela, Spain; 4Centre d’Infection et d’Immunité de Lille, Université de Lille, CHU Lille- Institut Pasteur de Lille, France

The mortality of pneumonia has been greatly reduced by conjugate vaccines. However, their coverage is still limited due to serotype replacement. To overcome this, a glycoconjugate vaccine based on (pneumococcal surface adhesin-A (mPsaA)) is prepared. mPsaA is a protein antigen present in the surface of all pneumococcal serotypes. A synthetic mimic of pneumococcal serotype 14 tetrasaccharide (Pn14PS) was synthesized chemically and mPsaA was expressed in E. coli BL21 (DE3) strain. The obtained mPsaA and Pn14PS were successfully conjugated by thiol-maleimide coupling chemistry to obtain protein/sugar conjugates at ratio 1/5.4. The secondary structure of mPsaA was evaluated by circular dichroism after conjugation and lyophilization. The mPsaA-Pn14TS conjugate was loaded into chitosan nanoparticles (CNPs) prepared by an ionic gelation method. CNPs had a size of 118 ±3 nm and zeta potential of 31 ±1 mV; the encapsulation efficiency of the conjugate in CNPs was 70 ±3 %. Nanoparticle tracking analysis confirmed that CNPs had a particle count of 1.69 x 1010 per mg. The studies performed by scanning electron microscopy revealed that CNPs were spherical and had smooth surface. The CNPs were stable over a period of 24 h in simulated nasal fluid. The studies performed with human monocyte-derived dendritic cells (MoDCs) confirm the interaction of CNPs with antigen presenting cells. The internalization of fluorescently labelled CNPs by the MoDCs was confirmed by confocal microscopy and flow cytometry. The flow cytometry results showed the maximum uptake of nanoparticles in MoDCs was in less than 2 h. Immunization studies performed in a mouse model revealed that the groups immunized (S.C.) with CNPs produced a significantly higher IgG response against both Pn14TS and mPsaA, when compared to the mice immunized (S.C.) with mPsaA-Pn14TS alone. These results demonstrate the effectiveness of CNPs in generating enhanced immune response against both protein and carbohydrate antigens.

References

[1] Safari D et al., Nanomedicine (Lond) (2012) 7:651–662. [2] Larentis AL, et al., Protein Expr Purif (2011) 78:38–47. [3] Calvo P et al., Pharm Res (1997) 14:1431–1436.

Acknowledgements The project has received funding from NanoFar, Erasmus Mundus Joint Doctorate in Nanomedicine and Pharmaceutical Innovation under European Union’s Horizon 2020 and RETOS-Spanish Ministry of Economy and Competitiveness (SAF2016-79230-R).

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O11. Rational development and preclinical assessment of bio-inspired Protein-

Metal Nanosystems as oral delivery carrier

Matilde Durán-Lobato1, Juan Cunarro1, Sulay Tovar 1, Carlos Diéguez1,María J. Alonso1,2,3

1Center for Research in Molecular Medicine and Chronic Diseases University of Santiago de

Compostela, Spain 2IDIS Research Institute, Santiago de Compostela, Spain 3School of Pharmacy, University of Santiago de Compostela, Spain

In spite of the high relevance of protein/peptide therapeutics in the current healthcare market scenario, the path from bench to beside still remains elusive for an oral peptide product. In order to design an oral protein nanocarrier product, aside from increasing oral bioavailability, other key features frequently sidelined should be considered, such as biocompatibility of materials and methods, reproducibility and scalability of the processes and the possibility of obtaining a final dosage form. Aiming at confronting these challenges, we explored the design of protein-metal nanosystems (Me-NSs) inspired on physiological materials and produced with industry-transferable methodologies. These Me-NSs were produced using an aqueous-based precipitation technique displaying a nanometer size below 100 nm with neutral ZP, association efficiency close to 100% and 10% protein loading. Upon contact with Simulated Intestinal Fluid (SIF), they proved to be colloidally stable while controlling protein release and protecting the protein cargo against enzymatic degradation in the presence of pancreatin. Furthermore, the production was successfully scaled-up by 10- and 100-fold increase in batch size and freeze-dried, leading to a powder product that maintained the physicochemical properties of the original formulation after mid-term RT storage. Moreover, the bioactivity of the associated protein was confirmed in an in vivo study, while the oral administration of the nanocarriers to rats proved their ability to enhance protein absorption. Overall, the results demonstrated the potential of Me-NSs as a bio-inspired and effective oral protein/peptide nanocarrier aiming at industrial translation.

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O12. Control of Loading and Polarized Orientation of Antibodies onto Gold

Nanoparticles

Mireya L. Borrajo1, Víctor Puntes1,2,3

1Vall d’Hebron Institut de Recerca (VHIR), 08035, Barcelona, Spain 2Institut Català de Nanociència i Nanotecnologia (ICN2), CSIC and The Barcelona Institut of

Science and Technology (BIST), Campus UAB, 08193, Bellaterra, Barcelona, Spain 3Institució Catalana de Recerca i Estudis Avançats (ICREA), P. Lluís Companys, 23, 08010,

Barcelona, Spain

After the administration of inorganic nanoparticles (NPs), including gold nanoparticles (Au NPs),

or their dispersion into biological fluids, the exposure to physiological proteins normally withstands

the Protein Corona formation around the NP. The control over which proteins are present on this

corona, their potential conformations and their absorption kinetics have been studied intensively

in the past few years. Among them, antibodies have been conjugated to NP, showing a combined

effect of the properties of both. The possibility of surface functionalization using more than one

antibody can suppose the development of biomaterials with multiple functions (e.g. multiplexed

diagnostics or multicell therapy). To achieve that, different conjugation strategies have been

purposed; however, most of them implied complex and time-consuming chemistry in order to not

only conjugate the antibodies onto the Au NPs, but also to maintain their functionality. Simple

conjugation procedures are based on the hydrophobic and electrostatic interactions between

antibodies and Au NPs, leading often to a poor functionality owing to the uncontrolled binding of

the Fc and Fab regions of the antibodies to the NPs.

Here we show the polarized conjugation of two different antibodies onto Au NPs, based on the

preferential interaction of the Fc with the metallic surface, the spontaneous island growth of

antibodies and their consequent segregation into different antibody domains. To characterize this

Janus polarization, recognition antigen-antibody has been used to attach size different Au NPs.

As it is shown in Fig. 1, Janus Au NPs (60 nm, functionalized with IgG anti-HSA and anti-BSA)

interacts with Au NPs of 25 nm functionalized with HSA and Au NPs of 15 nm conjugated with

BSA, demonstrating the polarized antibody distribution in the Au NP surface.

Figure 1. (A) Schematic representation of the polarization of the antibodies onto the surface of the Au NP.

(B) TEM characterization of the Janus distribution of the antibodies.

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O13. Impact of PEG coating in the distribution and retention of PLGA

nanoparticles in colorectal tissues

Rute Nunes1,2,3, Francisca Araújo1,2, Bruno Sarmento1,2,4, José das Neves1,2,4

1I3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal 2INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal 3ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto,

Portugal 4CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da

Saúde, Gandra, Portugal

Mucus penetrating nanoparticles (NPs) obtained by dense polyethylene glycol (PEG) coating may

be a powerful tool for the delivery of drugs in mucosal surfaces due to their ability to transpose

the mucus barrier more efficiently than mucoadhesive ones. In this work, poly (lactic-co-glycolic

acid) (PLGA NPs) were developed and non-covalently surface modified with poloxamer 407 – a

triblock copolymer comprising polyethylene glycol and polypropylene glycol chains. PLGA NPs

and PEG-PLGA presented sizes around 200 nm and narrow size distribution. The differences in

zeta potential values for PLGA NPs and PEG-PLGA NPs were well-matched with the presence

or not of PEG chains at NPs surface. The success of surface modification was also confirmed by 1H NMR, energy dispersive spectroscopy and contact angle. Developed NPs labeled with a near

infrared probe were rectally administered to mice and the distribution and retention patterns in

colorectal tissues were assessed by in vivo imaging. PEG-PLGA NPs were able to migrate farther

into the colon and provide higher extension of coverage when compared to PLGA NPs after as

little as 15 minutes of administration. PEG-PLGA NPs were able to cover around 40% of the total

length of the colon, while PLGA NPs were restricted to less than 10%. Also, PEG-PLGA NPs

showed higher residence in colorectal tissues between 15 min and 2 hours post-administration,

as assessed by the analysis of the total fluorescence. Histological analysis of colorectal tissues

of mice after rectal daily administration of PLGA NPs and PEG-PLGA NPs over 14 days revealed

no architectural changes or cells infiltrates. Also, no differences in TNF-α, IFN-γ, IL-6 and IL1-β

levels were observed between NPs and PBS treated animals. Overall, our results point out that

PEG-PLGA NPs may be a useful platform for the mucosal delivery of drugs.

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O14. Multimodal nanoemulsions for whole-body imaging in metastatic cancer.

Sandra Díez-Villares1, Juan Pellico2, Ramón Iglesias-Rey3, Santiago Grijalvo4, Miguel A.

Ramos-Docampo5, Verónica Salgueiriño5, Ramón Eritja4, Francisco Campos3, Fernando

Herranz2, Rafael López-López1, María de la Fuente1.

1Nano-Oncology Unit, Translational Medical Oncology Group (ONCOMET). Health Research

Institute of Santiago de Compostela (IDIS) and CIBERONC, Santiago de Compostela, Spain. 2Nanomedicine and Radiochemistry Group, National Centre for Cardiovascular Research Carlos

III (CNIC) and CIBERES, Madrid, Spain. 3Clinical Neurosciences Research Laboratory, Health Research Institute of Santiago de

Compostela (IDIS), Santiago de Compostela, Spain. 4Nucleic Acids Chemistry Group, Institute of Advanced Chemistry of Catalonia (IQAC – CSIC)

and CIBER-BBN, Barcelona, Spain. 5Magnetic Materials Group, University of Vigo, Vigo, Spain.

Early diagnosis of metastatic cancer by whole-body techniques with high resolution and sensibility

such as Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET) could

improve the outcome of patients and increase their survival. On view of this, we aimed to develop

biocompatible nanoemulsions (NEs) that can incorporate MRI and PET probes and specifically

reach disseminated cancer cells.

NEs composed by sphingomyelin and oleic acid, were prepared by ethanol injection and

characterized using a Nanosizer 2000®. NEs had a small colloidal size (130 nm), a monodisperse

population (0.2), and a negative surface charge (-50 mV). Hydrophobic Superparamagnetic Iron

Oxide Nanoparticles (SPIONs) coated with oleic acid were successfully incorporated into the oily

nucleus of the NEs. These magnetic formulations were evaluated by MRI showing an excellent

negative contrast (in vitro and in vivo) due to the Fe content and a high transverse relaxivity. For

PET purposes, we synthesized a stearylamine derivative of the chelating agent NOTA (NOTA-

SA) and it was efficiently associated to the NEs, without modifying their physicochemical

properties. Afterwards, 68Ga was incubated with the NEs and purified by ultrafiltration, obtaining

a high percentage of radioisotope association (86%) and a good stability in mice serum. NEs were

injected intravenously in healthy mice, and PET/CT images were acquired at 2h post-

administration. Biodistribution studies were done by the collection of mice organs and the

quantification of their radioactivity at the end of the experiment. Results showed a good signal of

the PET probe, not cardiotoxicity and a high detoxification by the liver, kidneys and spleen.

In conclusion, we have developed biocompatible nanoemulsions that have potential for the

development of novel tools for MRI and PET diagnosis. Next experiments will be aimed to explore

their potential in a mice model of metastatic disease.

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O15. Combination of polymeric nanoparticles and alginate cryogel towards the development of an in situ cancer vaccine

Tomás Bauleth-Ramos1,2,3,4,5,6, Ting-Yu Shih5,6, Mohammad-Ali Shahbazi4,7, Alexander J. Najibi5,6, Angelo S. Mao5,6, Pedro Granja1,2,3, Hélder A. Santos4,8, Bruno Sarmento1,2,9, David J. Mooney5,6 1 Instituto de Investigação e Inovação em Saúde (I3S), University of Porto, Rua Alfredo Allen, 208, 4200-135, Porto, Portugal 2 Instituto de Engenharia Biomédica (INEB), University of Porto, Rua Alfredo Allen, 208, 4200-135, Porto, Portugal 3 Instituto Ciências Biomédicas Abel Salazar (ICBAS), University of Porto, Rua Jorge Viterbo 228, 4150-180, Porto, Portugal 4 Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, FI-00014, Helsinki, Finland 5 John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA 6 Wyss Institute Biologically Inspired Engineering, Harvard University, Boston, MA, 02115, USA 7Department of Pharmaceutical Nanotechnology, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran 8Helsinki Institute of Life Science 9CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Gandra, Portugal

INTRODUCTION Immunotherapies for cancer had an outstanding development in the past decade. However, the lack of antigen accumulation and proper stimulation of the immune cells led to poor antitumor responses.[1] Biomaterials have shown promising results as tools to surpass these issues.[2] Considering this, we hypothesized that by combining nutlin-3a (Nut3a) loaded nanoparticles (NPs) within an alginate cryogel loaded with granulocyte-macrophage colony-stimulating factor (GM-CSF) and cytosine-phospodiester-guanine-oligonucleotide (CpG-ODN), it would be possible to enhance the delivery of the drug, increase the therapeutic efficacy and simultaneously modulate the immune cells, creating an in situ vaccine. METHODS Nut3a loaded spermine-modified acetalated dextran NPs were prepared by double emulsion[3] and GM-CSF and CpG-ODN loaded alginate cryogels by covalent and ionic crosslinking.[4] NPs were physically adsorbed onto the cryogel and the system characterized by DLS, TEM, confocal microscopy, HPLC and fluorometer. In vitro association studies, biocompatibility, toxicity and immunogenicity were done in EL4 cancer cells. Biodistribution and efficacy studies were performed in vivo in C57BL6 mice bearing EL4 tumors. RESULTS Nut3a encapsulated NPs with positive surface charge and homogenous size distribution (221±4 nm) were formed and successfully adsorbed into the cryogels (Figure 1A). The NPs suffered a burst release in the first 6h and controlled release up to 72h (figure1B). Furthermore, NPs loaded gels enhanced tumor accumulation compared to intravenous injection (Figure 1C). Drug loaded NPs exerted an anticancer effect in vitro (Figure 1D) and Nut3a induced immunogenic cell death and slowed the tumor growth and increased survival in vivo. CONCLUSION Our results show that the developed combinatorial system can promote an anticancer effect, induce immunogenic cell death and increase the accumulation of nanoparticles in the tumor site. We believe that by combining the advantages of the drug loaded NPs with the GM-CSF/CpG loaded cryogel scaffold, it will be possible to exert a strong anticancer effect.

References

1. Wang, H. and D.J. Mooney, Biomaterial-assisted targeted modulation of immune cells in cancer treatment. Nat Mater, 2018. 17(9): p. 761-772.

2. Weber, J.S. and J.J. Mule, Cancer immunotherapy meets biomaterials. Nat Biotechnol, 2015. 33(1): p. 44-5.

3. Bauleth-Ramos, T., et al., Nutlin-3a and Cytokine Co-loaded Spermine-Modified Acetalated Dextran Nanoparticles for Cancer Chemo-Immunotherapy. Advanced Functional Materials, 2017. 27(42).

4. Shih, T.Y., et al., Injectable, Tough Alginate Cryogels as Cancer Vaccines. Advanced Healthcare Materials, 2018. 7(10).

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Abstracts: Poster presentations

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P1. Rational design of Next-Generation Exosomas for targeted drug delivery in

cancer therapeutics.

Abi Judit Vazquez-Rios1,2, Ángela Molina-Crespo3, Sandra Alijas-Pérez2, Belén L Bouzo1,2,

Gema Moreno-Bueno3, María de la Fuente Freire1.

1 University of Santiago de Compostela (USC), Spain. 2 Nano-Oncology Unit, Translational Medical Oncology Group, Health Research Institute of

Santiago de Compostela (IDIS), SERGAS, CIBERONC, 15706 Santiago de Compostela, Spain. 3 Translational Cancer Research Laboratory, Department of Biochemistry, Autonomous

University of Madrid, School of Medicine, "Alberto Sols" Biomedical Research Institute CSIC-

UAM, Madrid, Spain.

In the past decade, exosomes have been proposed as ideal drug delivery systems for a broad

range of pathologies, including cancer, inflammatory and regenerative diseases. Recent reports

have shown impressive results in vivo that have prompted them into early clinical studies.

However, important limitations related to their cumbersome and costly production processes as

well as safety concerns are hampering their translation to clinic. Making use of the well-known

liposome technology we propose the engineering of Next-Generation Exosomes (NGE) with clear

advantages over natural exosomes in terms of yield of production, safety, and cost-effectiveness.

NGE show great similarities to natural exosomes with respect to their physicochemical

properties, ability to interact with the target cells, and to deliver innovative anticancer

therapeutics. Moreover, NGE have been successfully functionalized with membrane proteins with

organotropic properties, and have demonstrated to efficiently transport bioactive macromolecules

(microRNAs) to the target cancer cells, in a similar fashion to tumoral exosomes. We present in

this work a complete characterization of the proposed nanoplatform and evidence of the its

potential for the targeted delivery of therapeutic RNAs to cancer cells in vitro and in vivo in an

orthotopic xenograft model of human lung adenocarcinoma.

We provide the first proof-of-concept of the potential of this technological nanoplatform as a real

alternative to exosomes for the development of safer and more efficient anticancer therapies, a

technology that is versatile and can be adapted as we go deep in the study of exosomes and the

molecular features related to their tumor-homing properties. NGE provide additional opportunities

of being further amendable to surface functionalization as well as loading of therapeutic cargo for

a broader application in other fields.

Taken together, our results suggest that NGE gain competitive advantage for drug delivery of

anticancer therapeutics and provide a real alternative to natural exosomes for a widespread

translation.

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P2. Assessment of immunotoxicological features of chitosan nanoparticles

Alana Duarte1,2#, Ana Patrícia Marques1,2#, Mariana Colaço1,2, Sandra Jesus1,2, Olga Borges1,2

1Center for Neuroscience and Cell Biology, University of Coimbra, Portugal.2Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Portugal.

# These authors contributed equally to this work

Nanoparticles (NPs) conquered an important role in drug delivery. Nonetheless, their effects on the immune system is poorly understood. So, it is necessary to develop trials and guidelines for the immunotoxicological assessment of nanomaterials to develop safe-by-design (SbD) NPs, minimizing health risks. Chitosan (Chi) is a natural polymer, that has been explored as a drug delivery vehicle. However, its immunotoxicological evaluation show contradictory results. In several reports, different physicochemical characteristics of Chi (molecular weight (MW) or deacetylation degree (DD)), the NPs size or endotoxin contamination are not reported, and can influence the immunotoxicity of the delivery system. The aim of this work is to study the immunotoxicity of NPs produced with chitosans with different DD (80 % and 93 %) and MW, to establish methods for immune function evaluation for SbD NP development. Chi NPs were prepared using a coacervation method and its size and zeta potential were characterized. Studies of toxicity were performed in human peripheral blood mononuclear cells (PBMCs) for MTT assay, in RAW 264.7 cells for MTT assay, for reactive oxygen species (ROS) and nitric oxide (NO) production, and in whole blood for hemolysis and coagulation time assays. The Chi NPs 80 % showed to be more toxic than Chi NPs 93 % in PBMCs. Both NPs and polymers decreased LPS-induced NO production but showed no stimulation in NO production and caused no hemolysis. Only Chi NPs 80 % increased ROS production and had a significant effect on plasma coagulation times, affecting the intrinsic pathway.

We concluded that the DD and MW of Chi in the NPs can influence the results of cytotoxicity, coagulation times and ROS. These results together with further studies will contribute to develop guidelines to implement the SbD approach for nanobiomaterials, with focus on polymeric drug delivery systems.

This work is funded by FEDER funds through the Operational Programme Competitiveness

Factors - COMPETE 2020 and national funds by FCT - Foundation for Science and Technology

under the project PROSAFE/0001/2016 and strategic project POCI-01-0145-FEDER-007440.

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P3. Antibiotic and anti-inflammatory controlled delivery from intraocular lenses for

prophylaxis of endophthalmitis

Ana Topete1,2, Benilde Saramago1, Ana Paula Serro2

1Centro de Química Estrutural, Instituto Superior Técnico, University of Lisbon, Lisbon, Portugal; 2Centro de Investigação Interdisciplinar Egas Moniz, Instituto Universitário Egas Moniz, Monte da

Caparica, Portugal

Abstract:

Cataract is the leading cause of blindness, affecting more than 20 million people worldwide each

year. Cataracts surgery involves the removal of the opacified natural lens and implantation of an

intraocular lens (IOL). In the post-operatory period, complications such as endophthalmitis can

appear. To prevent this, antibiotics and anti-inflammatories eye drops are administered with a

frequent posology. This drug delivery method is not very effective and leads to low patient

compliance. In this work, it is explored the possibility of using drug-loaded IOLs to ensure a

simultaneous release of both antibiotic and anti-inflammatory drugs.

An antibiotic, moxifloxacin (MXF), and an anti-inflammatory, ketorolac (KTL) were loaded in

acrylic IOLs by soaking in drugs solutions at 60°C for 2 weeks. Loading was carried out in a

double drug solution of MXF+KTL and sequentially, in solutions containing just one of the drugs

for comparison purposes. The loaded lenses were sterilized by autoclaving (121ºC, 1bar, 1h). In

vitro drug release experiments were carried out in sink conditions. The effect of the drug loading

and of the sterilization on lenses properties such as the swelling capacity, optical properties

(transmittance) and mechanical properties (Young’s modulus) were evaluated.

It was observed that, the double loaded lenses released a significant higher amount of each drug

than the single loaded lenses. The devices were able to release effective amounts of the drugs

for 2-4 weeks. Sterilization did not affect the release. It was found that the presence of drugs

increases the swelling capacity of the lenses. Concerning the optical and mechanical properties,

the changes observed do not compromise the use of the lenses.

The sterilized double loaded lenses seem promising devices for the post-cataract surgery

prophylaxis, complying with both antibiotic and anti-inflammatory therapeutic needs.

Acknowledgements:

The authors acknowledge funding from Fundação para a Ciência e Tecnologia (FCT) [projects

UID/QUI/00100/2013 and PTDC/CTM-BIO/3640/2014] and support from PhysIOL for providing

the materials for IOLs.

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P4. Chitosan modifications into new amphiphilic derivatives for polymeric

micelles production as drug delivery systems

Andreia Almeida1,2,3, Marco Araújo1,2, Ramon Novoa-Carballal4,5, Cristina C. Barrias1,2,3, Marlene

Lúcio6, Bruno Sarmento1,2,7

1I3S – Institute for Research and Innovation in Health, University of Porto, Portugal2INEB – Institute of Biomedical Engineering, University of Porto, Rua Alfredo Allen, 208, 4200-

135 Porto, Portugal 3ICBAS – Institute of Biomedical Sciences Abel Salazar, University of Porto, Rua de Jorge Viterbo

Ferreira 228, 4050-313 Porto, Portugal 43B’s Research Group − Biomaterials, Biodegradables and Biomimetics, University of Minho,

Headquarters of the European Institute of Excellence in Tissue Engineering and Regenerative

Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 5ICVS/3B’s PT Government Associate Laboratory, Braga/Guimarães, Portugal 6CFUM – Centre of Physics of University of Minho and Porto, Campus de Gualtar, 4710-057

Braga, Portugal 7CESPU, IINFACTS – Institute for Research and Advanced Training in Health Sciences and

Technologies, Rua Central de Gandra 1317, 4585-116 Gandra, Portugal

Chitosan is a biocompatible and biodegradable polymer extensively explored in the development

of drug delivery systems. The production of chitosan derivatives with amphiphilic properties have

been widely investigated for delivery of anticancer agents. Polymeric micelles can be produced

using block copolymers composed of hydrophilic and hydrophobic polymers or using lipids-graft-

hydrophilic polymers. These systems allow to encapsulate drugs with different polarities, but with

higher stability and have been explored as solubility enhancers of many anticancer drugs. Despite

the promising results, the bioavailability and pharmacokinetic profile of oral administered

formulations can be compromised by a release and degradation of the drug at gastric environment

or reduced retention at the intestine and colon. These drawbacks can be overcome by the

development of mucoadhesive formulations that also protect and retain the drug at gastric pH.

Our work proposes to address these inadequacies using nanotechnology to develop oral

mucoadhesive polymeric micelles based on chitosan for delivery of anticancer drugs. Chitosan

derivatives were synthesized by a carbodiimide reaction by amidation and the success of the

reactions was confirmed by Fourier transform infrared spectroscopy (FTIR) and hydrogen nuclear

magnetic resonance analysis (1H NMR). Micelles were produced by solvent evaporation method,

and the critical micelle concentration was also investigated. The obtained micelles presented a

mean particle size in the range of 200-300 nm and positive surface charge. The morphology of

micelles assessed by transmission electron microscopy (TEM) revealed round and smooth

surface, in agreement with dynamic light scattering measurements. The association efficiency

was determined by high-performance liquid chromatography (HPLC) and the cytotoxicity studies

against Caco-2 and HT29-MTX intestinal epithelial cells demonstrated absence of cell toxicity for

all formulations. Moreover, the intestinal permeability in Caco-2 monolayer and Caco-2/HT29-

MTX co-culture model was also determined where chitosan systems presented higher intestinal

permeability compared with the free drug.

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P5. Monodisperse glycosaminoglycan-based microgels for in situ protein

delivery from tissue-mimicking implants

Anna Abbadessa1,2, Carl C. L. Schuurmans1,3, Mikkel A. Bengtson1, Galja Pletikapic4, Huseyin

Burak Eral5,6, Gijsje Koenderink4, Rosalinde Masereeuw3, Wim E. Hennink1 and Tina Vermonden1

1Department of Pharmaceutics, Utrecht University, Utrecht, The Netherlands 2Later post-doc at Department of Fibre and Polymer Technology, Royal Institute of Technology (KTH), Stockholm, Sweden 3Department of Pharmacology, Utrecht University, Utrecht, The Netherlands 4Biological Soft Matter Group, AMOLF, Amsterdam, The Netherlands 5Process and Energy Department, Delft University of Technology, Delft, The Netherlands 6Van’t Hoff Laboratory for Physical and Colloid Chemistry, Utrecht University, Utrecht, The Netherlands

Biological augmentation, via e.g. growth factors, of tissue-mimicking implants is a fundamental

aspect in regenerative medicine. In this study1, we have investigated the potential of

glycosaminoglycans (GAGs) microgels as protein carriers, as well as their protein release kinetics

upon incorporation into hydrogel scaffolds designed for articular cartilage regeneration.

Monodisperse microgels (500-700 μm) based on methacrylated hyaluronic acid (HAMA) and

chondroitin sulfate (CSMA) were fabricated using a microfluidic device. They were subsequently

loaded with a cationic model protein, i.e. lysozyme (Lys), via complex coacervation under two

different buffer conditions (20 and 170 mM ionic strength, pH 7.4). Protein up-take, distribution

and release kinetics from the microgel matrices were studied. Composite hydrogels made of Lys-

loaded CSMA microgels embedded into hydrogel scaffolds were investigated in terms of Lys

release, and were compared to Lys-loaded, CSMA-free hydrogel scaffolds and CSMA/hydrogel

blends. In 20 mM buffer a higher Lys loading (up to 82%) was found for CSMA microgels

compared to HAMA (76%), reasonably due to the higher CSMA charge density. Unlike CSMA

microgels, HAMA microgels showed a significant decrease in Lys loading (51%) in 170 mM

buffer, likely caused by the charge shielding originating from the higher ion concentration. Lys

was homogeneously distributed throughout HAMA and CSMA microgels. In 170 mM PBS buffer

(pH 7.4), Lys was released slower from CSMA than from HAMA microgels (total release around

day 28 and 14, respectively), likely due to the stronger Lys-CSMA electrostatic interaction.

Composite hydrogels released fully active Lys within 58 days, which is a much longer time frame

than that showed by CSMA-free hydrogels (3 days) and CSMA/hydrogel blends (41 days). This

study demonstrates that GAG microsphere/hydrogel composites loaded with cationic proteins via

complex coacervation, are promising for the in situ protein delivery from tissue-engineered

constructs.

References [1] Soft Matter, 2018, 14 (30) 6327-6341.

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P6. Caffeic Acid skin absorption: delivery of microparticles to hair follicles

Lia Carolina Oliveira dos Santos1, Antonio José Guillot 2, Caroline Magnani Spagnol1, Marcos Antonio Corrêa1, Ana Melero Zaera 2.

1Department of Drugs and Medicines, São Paulo State University (UNESP), School pf Pharmaceutical Sciences, Araraquara, SP, Brasil. Rod. Araraquara Jau Km 1- Campus Ville- Araraquara, São Paulo, Brazil CEP:14800-903 - Araraquara, SP. Tel.: (16) 3301-6880 Fax: (16) 3322-0073.

2Department of Pharmacy and Pharmaceutical Technology and Parasitology. University of Valencia. Avda. Vicente A. Estelles SN. 46100, Burjassot. Valencia, Spain

Caffeic acid (CA) is a polyphenol that can be found in many vegetal dietary sources. CA has a

remarkable antioxidant potential and its antimicrobial action has been demonstrated through in

vitro studies. Emulsions, especially O / W, are commonly used topical vehicles for active

substances, since they present several advantages over other dosage forms. The challenge of

dermal application for a drug is to overcome the anatomical and physiological barrier and to

provide the effective concentration of the compound at active site. Therefore, it is necessary to

investigate alternative routes, such as the transfollicular one. The objective of this work was to

investigate the potential of transfollicular delivery of a topical emulsion containing CA formulated

in microparticles and also non-encapsulated for comparative reasons. The tape stripping

technique is widely used and accepted to study the kinetics and penetration of active substances

via the skin, allowing to determine the location and distribution of substances within the Stratum

corneum (SC). The Differential Stripping method is the most straightforward technique to

determine follicular uptake quantitatively. Emulsions containing free CA and microencapsulated

CA were applied over a predetermined area in the outer side of porcine ear skin. After the

predetermined incubation time under constant temperature conditions, the SC was removed by

the tape-stripping technique. To analyze the extent of follicular drug penetration, cyanoacrylate

glue was applied over the pretreated skin area followed by its subsequent removal from the skin

surface. The results of the retention studies demonstrated that, compared to the free drug, the

microparticles allow a more even distribution of CA through the SC. Furthermore, as they are

retained in the hair follicle, the drug can be released over a prolonged time in the hair follicle,

where it can diffuse through the SC, which is much thinner in this appendage. Furthermore, this

route can be explored to locally target the hair follicles with CA for the treatment of acne and hair

loss, due to its pharmacological activity.

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P7. Polyphosphazene nanocarriers for gene delivery in cancer

Carla Garcia-Mazas, Noemi Csaba, Marcos Garcia-Fuentes

Center for Research in Molecular Medicine and Chronic Disease (CiMUS) and Dept.

Pharmacology, Pharmacy and Pharmaceutical Technology, University of Santiago de

Compostela

Gene therapy has enormous potential for the treatment of a large number of diseases, but is still

limited by the need of an efficient vector to allow entry in the cells. While viral vectors typically

offer higher efficacy, there are still concern on their safety. Non-viral viral vectors are quickly

improving their efficacy and they provide a safer alternative. Polymeric nanocarries are widely

used to control the release of different therapeutic agents, both in time and space, which, in turn

enhances drug efficacy and safety. Particularly, synthetic polymers offer the possibility of tailoring

their properties through rational design, and possibility to obtain well-defined structures.

In this work, we have developed and characterized new polymeric nanoparticles for gene therapy

based on polyphosphazenes (PPZ). Polyphosphazenes are characterized by an inorganic

phosphorous/nitrogen backbone, and by two side chains that can be modulated. Some

polyphosphazenes have been investigated previously for gene therapy showing remarkable

activity/toxicity ratio. Herein, we used this platform material to synthesize a cationic homopolymer

(HoCat), a anionic homopolymer (HoAn), and a random copolymer based on HoCat were part of

the amine groups were substituted by neutral moieties. These polymers were characterized by

NMR for their chemical structure and then were used to form polyplexes with pDNA alone or in

combination. The resulting nanoparticles were characterized for their size, pDNA

association/dissociation capacity, toxicity and transfection capacity.

The results showed that the nanosystems showed small particle size (90-100nm) and the capacity

to condense the genetic material and to release it in presence of a polyanionic competitor. Toxicity

tests indicated that the association of cationic with anionic PPZs resulted in improved cell viability

as compared to cationic polyplexes alone. The same tendency was observed for cell transfection,

where polyplexes containing cationic and anionic polymers improved the transfection in

comparison with the cationic polyplexes alone.

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P8. Alkyl-lysophospholipid edelfosine and squalenoyl-gemcitabine form a

novel nanomedicine applicable to cancer therapy

Carlos Rodríguez-Nogales1,2, Víctor Sebastián3,4, Silvia Irusta3,4, Didier Desmaële5, Patrick Couvreur5, María J. Blanco-Prieto1,2

1Pharmacy and Pharmaceutical Technology Department, University of Navarra, Pamplona

31008, Spain2Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona 31008, Spain 3Department of Chemical and Environmental Engineering & Institute of Nanoscience of Aragon

(INA), University of Zaragoza, Zaragoza 50018, Spain 4Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN,

Madrid 28029, Spain. 5Institut Galien Paris-Sud, UMR CNRS 8612, Université Paris-Sud, Université Paris-Saclay,

Châtenay-Malabry Cedex 92290, France

The bioconjugation of drugs to squalenic acid, a nontoxic and biocompatible lipid squalene-

derived molecule, leads to the formation of squalenoyl prodrugs able to self-assemble in

supramolecular structures to form stable nanocomposites [1,2]. Among them, the prodrug

squalenoyl-gemcitabine has been chosen to formulate a novel multidrug nanocomposite in

combination with edelfosine, an alkyl-lysophopholipid with proven anticancer activity [3,4]. The

main objective is to study if the physical mixture of both compounds could lead to the formation

of a new anticancer nanomedicine. Chemical synthesis of squalenoyl-gemcitabine was performed

according to previous studies [5,6] and nanocomposites were formulated by nanoprecipitation

method. The physico-chemical properties of the nanoassemblies and its efficacy in pediatric

osteosarcoma cells were evaluated. Designed and optimized squalenoyl-gemcitabine/edelfosine

nanoassemblies showed a final mean particle size of 51±1 nm, PDI of 0.12 ±0.03 and zeta

potential of -12.56 ±1.15 mv. High angle annular dark field scanning transmission electron

microscopy showed the multilamellar assembly of squalenoyl-gemcitabine/edelfosine

nanoassemblies, displaying a concentric or spiral disposition of the layers. Hemolysis

experiments demonstrated that the nanoassemblies protected erythrocytes from the inherent

hemolysis caused by non-encapsulated edelfosine. Cell culture viability tests in U2-OS confirmed

that the co-assembly of edelfosine and squalenoyl-gemcitabine was not detrimental to the

inherent antitumor activity of squalenoyl-gemcitabine nanoassemblies. Moreover, the novel

nanoformulation killed the remaining plateau cells that were non-responsive to higher doses of

squalenoyl gemcitabine nanoassemblies. Compared to the previous formulation, the combination

of squalenoyl gemcitabine with edelfosine has resulted in a smaller particle size with higher

stability and drug content whereas its antitumoral potential has remained intact. All in all, this

concept may represent a step ahead toward a feasible multidrug nanoassembly that embodies

the properties of a new generation of only drug-based nanomedicines.

References

[1] L.H. Reddy, J.-M. Renoir et al., Mol. Pharm. 6 (2009) 1526–1535.

[2] F. Dosio, L.H. Reddy et al,, Bioconjug. Chem. 21 (2010) 1349–1361.

[3] Y. González-Fernández, E. Imbuluzqueta et al., Cancer Lett. 388 (2017) 262–268.

[4] A. Estella-Hermoso de Mendoza, V. Préat et al., J. Control. Release. 156 (2011) 421–426.

[5] L.H. Reddy, C. Dubernet et al., J. Control. Release. 124 (2007) 20–27.

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P9. Essential oil-loaded nanocapsules to fight against multidrug resistant bacteria

Carmen Fernández-Varela1, Juan Carlos Vázquez-Ucha2, José Crecente-Campo1, Alejandro

Beceiro2 and María J. Alonso1,3,4

1Center for Research in Molecular Medicine and Chronic Diseases University of Santiago de

Compostela, Spain 2Institute for Biomedical Research of A Coruña – CHUAC 3IDIS Research Institute,Santiago de Compostela, Spain 4School of Pharmacy, University of Santiago de Compostela, Spain

Currently, multidrug-resistant bacteria are gaining ever-increasing importance mainly because of

the abuse of antibiotics (1). Classic antibiotics will be soon unable to kill several highly pathogenic

bacterial. In this study, we have developed polymer-based nanosystems containing different

essential oils (EOs), aiming to increase their microbicide activity. A total of 4 different EOs were

evaluated. Nanocapsules have been characterized and tested against two bacterial strains of

Klebsiella pneumoniae and Acinetobacter baumannii, which are two of the most multiresistant

nosocomial pathogens, in M9 culture media. The maximum loading of EO offering adequate

physicochemical properties and stability was calculated. The nanosystems were isolated and

concentrated by tangential flow filtration, and characterized in terms of particle size,

polydispersity, zeta potential, and morphology. The nanosystems showed a particle size below

100 nm (2) that is considered adequate for interaction with bacteria, positive zeta potential and

acceptable loading of EOs. The nanosystems were stable in M9 culture media for at least 24

hours. Finally, the nanosystems were tested against several multidrug-resistant bacteria

observing a reduction of the mean inhibitory concentration between 32- and 16-fold depending

on the EO against A. baumannii.

1. Rios AC, Moutinho CG, Pinto FC, Del Fiol FS, Jozala A, Chaud M V., et al. Alternatives toovercoming bacterial resistances: State-of-the-art. Microbiol Res. 2016;191:51–80.

2. Hadinoto K, Cheow WS. Nano-antibiotics in chronic lung infection therapy againstPseudomonas aeruginosa. Colloids Surfaces B Biointerfaces. 2014;116:772–85.

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P10. Co-formulating and co-delivering protein therapeutics using PLGA

nanocarriers: a model study

Cláudia Martins1,2,3,4, Veeren M. Chauhan4, Amjad A. Selo4, Mohammad Al-Natour4, Hongyu

Zhang5, Paul Dalby5, Jonathan W. Aylott4, Bruno Sarmento1,2,6

1I3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto,

Portugal 2INEB - Instituto de Engenharia Biomédica, Universidade do Porto, 4200-135 Porto, Portugal 3ICBAS - Instituto Ciências Biomédicas Abel Salazar, Universidade do Porto, 4050-313 Porto,

Portugal 4School of Pharmacy, Boots Science Building, University of Nottingham, NG7 2RD Nottingham,

United Kingdom 5Department of Biochemical Engineering, University College London, WC1H 0AH London, United

Kingdom 6CESPU - Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde,

4585-116 Gandra, Portugal

Co-formulation of multiple therapeutics into single nanocarriers has been investigated to simplify

delivery to patients, realize synergetic bioresponses and personalized dosages, as well as co-

target therapeutics through all-in-one nanocarriers’ functionalization [1]. A yet poorly explored

field is the co-formulation of proteins, which consist on a multibillion-dollar class of therapeutics

known to provide high specificity and potency [2].

Herein, poly(lactic-co-glycolic acid) (PLGA) nanocarriers were used to mono- and co-formulate

two albumin-based model proteins. Nanocarriers’ manufacturing was performed through a novel

microfluidic modified-nanoprecipitation technique. PLGA concentration and flow rates were

optimized in order to obtain protein-loaded nanocarriers of around 100 nm diameter, low

polydispersity index (<0.1), and negatively charged (-15 mV). These physicochemical properties

were independent on the batch volume (up to 100 mL), proving the scalability of the manufacturing

process. PLGA nanocarriers demonstrated an association efficiency up to 80%/70% and

maximum protein loading close to 1.5%/0.7% for the mono-/co-formulation, respectively. Both

formulations presented a controlled protein release over time under simulated intravenous

conditions. The secondary structure of proteins was not compromised by the mono- or co-

formulation process, as investigated through circular dichroism (minimum mean residue ellipticity

of around -20,000 deg·cm2·dmol-1). Studies in macrophage cells proved that protein mono-/co-

loaded PLGA nanocarriers did not impair metabolic activity (>70%). The intracellular transport of

the proteins was around 4- and 2-times higher when mono- and co-formulated into nanocarriers,

respectively, compared to the free protein controls, according to flow cytometry analysis.

Moreover, the intracellular transport of the co-formulated proteins was 4-times higher than the

physical mixture of nanocarriers individually loaded with each protein.

This work proved the effectiveness of PLGA nanocarriers for co-formulating and enhancing the

intracellular transport of co-delivered model proteins, hopefully serving as a proof-of-concept for

future protein combination therapies.

[1] Huang et al. Adv.Drug.Deliv.Rev. 2017. 115:82-97

[2] Pakulska et al. Science 2016. 351:aac4750-1-aac4750-6

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P11. Evaluation of mucus-permeating nanocarriers for the oral delivery of

proteins.

Cristian Reboredo1, Ana Luisa Martínez-López1, Carlos Javier González-Navarro2, Juan Manuel

Irache1.

1Department of Chemistry and Pharmaceutical Technology, University of Navarra, 31008

Pamplona, Spain 2Centre for Nutrition Research, University of Navarra, 31080, Pamplona,

Oral delivery of proteins and peptides remains an important challenge with many developmental

issues to solve. The physico-chemical properties and enzymatic sensitivity significantly hamper

the absorption of therapeutic proteins and peptides. As a consequence, their oral bioavailability

is, in general, very low (< 1 %). The general aim of this work is to develop and evaluate

nanocarriers with mucus-permeating properties for the oral delivery of therapeutic proteins, using

insulin as model. In this context, the use of the animal organism C. elegans may be an interesting

tool to facilitate the optimization and selection of nanocarriers for the oral delivery of bioactive

proteins. Zein nanoparticles were prepared by a desolvation method, purified by tangential

filtration and dried in a spray-drier apparatus. PEG-coated nanoparticles were obtained by simple

incubation of the just formed nanoparticles with poly(ethylene glycol) 35,000 (PEG) before

purification and drying. The mucus-permeating properties of nanoparticles were evaluated in vivo

after fluorescently labelling with Lumogen red. PEG-coated nanoparticles displayed the capability

of reaching the intestinal epithelium whereas bare nanoparticles remained trapped in the

protective mucus layer. Nanoparticles were also evaluated in C. elegans, which is sensitive to

human insulin through the Daf-2 receptor involved in lipid accumulation (1,2). In this model, the

nanoencapsulation of insulin in PEG-coated nanoparticles significantly decreased the

accumulation of lipids by worms, compared with insulin free or loaded in bare nanoparticles. In

summary, PEG-coated nanoparticles appear to be a suitable nanocarrier for oral delivery of

therapeutic proteins and will be evaluated in rodents soon.

Bibliography:

1. Pierce SB et al, Genes Dev. 15(6):672–86 (2001)

2. Kimura KD et al, Science, 277(5328):942-6 (1997).

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P12. Pollen grain as a drug delivery system

Diego Pan Delgado1, Jose Manuel Ageitos1, Noemi Csaba1

1Center for Research in Molecular Medicine and Chronic Diseases University of Santiago de

Compostela, Spain

The oral route is one of the most popular routes of medication administration by patients. The

presence of physical and biological barriers prevents the retention and absorption of

biomacromolecules administered by this route. Different physical methods are currently under

evaluation to improve the adhesion and penetration of drugs in the mucosa. In this sense, the

use of pollen grains (PG) due to its characteristic morphology, can serve as a method of

anchoring and release of drugs.

Our objective is to develop a platform based on the use of PG to improve mucointeraction and

improve the absorption of therapeutic agents nanoencapsulated across the intestinal barriers.

PGs were processed by different purification methods (water, cyclohexane, acids and

enzymatic treatments) in order to eliminate possible contaminants and allergens. Moreover, this

process did not change in the morphology of the GPs were studied using infrared (IR)

spectroscopy, elemental analysis, and electronic and confocal microscopy. In the final of the

process, PGs were obtained free of contaminants, without compromising their structure, with

adequate stability in the simulated intestinal fluid.

In the second step, we have associated a different kind of nanosystems (nanocapsules and

nanoparticles) with the PGs. We had obtained around of 60% of association efficiency when we

used protamine nanocapsules using a passive method of loading with different incubation times.

Finally, in order to improve this association, we have used a freeze-drying process.

As a conclusion, PGs can be treated to eliminate contaminants without modifying their main

structure and thus be used for the administration of nanoencapsulated drugs, which are

associated in their surface.

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P13. Adequate safety and biocompatibility of PLGA-based nanoparticles for drug delivery

Fátima Fernández-Álvarez1, Gracia García-García1, Silvia Fuerte-Rodríguez1, Consolación

Melguizo2-4, José C. Prados2-4, José Luis Arias1,3,4.

1Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of

Granada, Spain 2Department of Human Anatomy and Embryology, Faculty of Medicine, University of Granada,

Spain. 3School Institute of Biopathology and Regenerative Medicine (IBIMER), Biomedical Research

Centre (CIBM), University of Granada, Spain 4Biosanitary Institute of Granada (ibs.GRANADA), Andalusian Health Service (SAS) – University

of Granada, Spain

Development of multifunctional nanoplatforms for the combined imaging and treatment of cancer

has been postulated to be an efficient antitumor approach. Polymers are commonly employed to

that aim, given their capability and versatility for surface and internal functionalizations. In this

biomedical scene, poly(D,L-lactide-co-glycolyde) (PLGA) is the frequently used polymer.

First steps in the development of PLGA multifunctional nanostructures are reported in the present

contribution. Concretely, 300nm-sized PLGA nanoparticles (NPs) were formulated by a

reproducible methodology based on the well-known emulsion/evaporation technique. The method

led to a homogeneous NP population suitable for a parenteral administration. Fourier-Transform

infrared spectrometry analysis demonstrated that the characteristics chemical groups and

structure of the copolymer was maintained after formation of the NPs.

The interaction of the PLGA particles with blood components was investigated to hypothesize

their potential use for therapeutic purposes, i.e. anticancer drug delivery. Interestingly, they were

found to be haemocompatible, given that a negligible effect on platelet activation, complement

system activation, haemolysis (even after 24 hours), and plasma clotting time of blood samples

was observed (n = 4). Complementarily, it was evaluated the in vitro cytotoxicity of the NPs on

the CCD-18 and T-84 cell lines, by the 3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyl tetrazolium

bromide (MTT) proliferation assay. The NP concentrations investigated varied from 50 ng/mL to

100 μg/mL. It was found that the PLGA particles exhibited null cytotoxicity in both cell lines.

Based on these findings, it could be hypothesized that the polymeric nanoparticulate system

presents an adequate biocompatibility and safety for drug delivery purposes. Work on

functionalization of the particles on the basis of a passive and active targeting design is currently

on progress.

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P14. Poly(ε-caprolactone)-based colloids to be used in drug delivery

Gracia García-García1, Silvia Fuerte-Rodríguez1, Fátima Fernández-Álvarez1, Laura Cabeza2-4,

Consolación Melguizo2-4, José L. Arias1,3,4

1Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of

Granada, Spain. 2Department of Human Anatomy and Embryology, Faculty of Medicine, University of Granada,

Spain. 3Institute of Biopathology and Regenerative Medicine (IBIMER), Biomedical Research Centre

(CIBM), University of Granada, Spain. 4Biosanitary Institute of Granada (ibs.GRANADA), Andalusian Health Service (SAS), University

of Granada, Spain.

Nanotechnology approaches in Biomedicine are principally based on nanoparticle engineering

with a great variety of materials. Currently, polymers have attracted great attention mainly in the

field of drug delivery due to their capability for a controlled release and targeted delivery. In this

area, one of the very promising polymers is the aliphatic polyester poly(ɛ-caprolactone) (PCL)

because of its safety profile in addition to other advantages, e.g. a very low degradation rate, and

high compatibility and permeability to drug molecules. These advantages along with its capability

for being blended to other polymers (leading copolymeric particles) and/or being surface

functionalized with targeting moieties, demonstrates that PCL nanoparticles are a versatile

material to obtain drug nanocarriers.

In this work, it was to developed a reproducible interfacial polymer disposition procedure to

formulate PCL-based colloids exhibiting adequate properties for a targeted and controlled drug

delivery. Photon correlation spectroscopy and transmission electron microscopy analysis proved

that 150nm-sized particles were obtained, while Fourier-transform infrared spectroscopy data

demonstrated that the typical chemical groups of the polymer are maintained upon nanoparticle

preparation. Cell viability experiments carried out in CCD-18 colon fibroblast cells and T-84

colonic adenocarcinoma cells demostrated the almost null toxicity of the PCL particles (at the

concentrations investigated). Finally, the polymeric particles proved to be haemocompatible,

since a negligible effect was detected (n = 3) on haemolysis (even after 24 hours), platelet

activation, complement system activation, and plasma clotting time of blood samples. Thus, the

PCL-based nanoparticulate system has demonstrated a great potential for biomedical

applications.

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P15. Polysaccharide nanocapsules as potential antibody delivery carriers for

cancer treatment.

I. Fernández Mariño1, Geir Klinkenberg2, Ane Marit Wågbø2, J. Crecente-Campo1, R. Schmid2,

M.J. Alonso1

1 Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Campus Vida,

Universidade de Santiago de Compostela, Spain.

2 Department of Biotechnology and Nanomedicine, SINTEF Industry, Trondheim, Norway.

Polysialic acid (PSA) has been reported as a polysaccharide able to improve the stability and the

average circulation time of proteins [1]. In this sense, PSA could be an interesting polymer in the

development of nanostructures for cancer treatment, promoting a passive targeting. Previously,

we have shown the interest of hyaluronic acid (HA) nanocapsules (NCs) for the association and

delivery of monoclonal antibodies for cancer treatment [2]. In this work, we have investigated a

panel of new PSA and HA based nanosystems in common cytotoxicity assays.

Nanocapsules were prepared by a self-emulsifying method. Different molecular weights and

hydrophobized versions of HA and PSA have been employed: 1) PSA of 8, 30, 94 kDa and C12-

PSA (30 kDa); 2) HA of 50, 330, 1500 kDa and C16-HA (55 kDa). The particle size of the NCs

were comprised in the range of 105 nm and 170 nm, the PDI <0.30 and zeta potential of -2 to -

17. Systems were stable in culture cell medium at 37 ºC for at least 24 hours.

The cytotoxicity of the NCs was tested using the MTT, WST-8 and LDH leakage technique in

different neoplastic (HCT116, HT29, HEP-G2, LLC-PK1, SW620) and macrophage (Raw264.7,

THP1) cell lines. Regardless the molecular weight, and the polymer of the shell the NCs showed

a good safety profile being non-toxic in all cell lines at 1.25 mg/mL except for the SW620 cell line.

A tendency was observed for modified polymer-based NCs to be less toxic.

Next steps will evaluate the interaction and intracellular trafficking of the different NCs looking for

the best prototype to delivery monoclonal antibodies to the intracellular level.

References:

[1] G. Gregoriadis et al. Cellular and Molecular Life Sciences. 2000, 57 (13-14), 1964-1969.

[2] Cadete A. “Hyaluronic acid nanocapsules for the intracelular delivery of anticancer drugs”.

2016

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P16. Antibiotic-Lipid Based Nanosystems as a Tool to Specifically Target

Staphylococcus aureus Biofilms

Magda Ferreira1, Isabel A. Ribeiro1, Sandra Pinto2,3, Frederico Aires-da-Silva4, António J.

Almeida1, Sandra I. Aguiar4,5, Ana Bettencourt1, Maria M. Gaspar1

1 Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisboa,

Portugal 2 Centro de Química-Física Molecular e IN, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco

Pais 1049-001 Lisboa, Portugal 3 iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico,

Universidade de Lisboa, Av. Rovisco Pais 1049-001 Lisboa, Portugal 4 Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária,

Universidade de Lisboa, Lisboa, Portugal 5 Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal

Infections caused by Staphylococcus aureus biofilms are a major public health concern.

Treatment is hampered by reduced penetration of antibiotics into biofilms, leading to resistant

strains emergence [1]. Liposomes can constitute an innovative and alternative therapeutic

strategy for these infections, enabling preferential interaction with biofilms and release of

incorporated antibiotics [2].

As a first step, the microbiological activity of antibiotics in free form against planktonic and biofilm

S. aureus strain (ATCC®25923) was tested. Selected antibiotics were: Rifabutin (RFB),

Levofloxacin (LEV) and Vancomycin (VCM). The minimal inhibitory concentrations (MICs) for

planktonic cells, and minimal biofilm inhibitory concentrations (MBIC50) were determined by MTT

assay. MICs and MBIC50 were defined as the lowest concentration that reduced bacterial growth

>95% and >50%, respectively. MICs obtained for RFB, LEV and VCM were 0.02, 0.1 and 1.6

µg/mL, and MBIC50 were <0.07, 0.4 and >6.25 µg/mL, respectively.

Secondly, the antibiotics were nanoformulated in lipid-based systems aiming to evaluate the

advantages of liposomes on biofilm interaction. Higher Incorporation Efficiencies (I.E.) were

obtained for VCM and RFB, ranging from 32 to 88% and antibiotic loading between 23 and 47

µg/µmol of lipid, respectively.

Preliminary in vitro studies of S. aureus antibiofilm effect were performed for the most promising

antibiotic, RFB, in liposomal form by MTT assay. The anti-biofilm activity of RFB liposomes was

lipid composition dependent: positively charged liposomes presented the highest MBIC (>20

µg/mL) while the lowest values were achieved for fusogenic liposomes (<0.07 µg/mL). Confocal

microscopy imaging techniques were performed to validate the in vitro assays. This included the

selectively staining of live and dead bacteria in biofilms and the use of rhodamine-labeled

liposomes [3].

Overall, the use of lipid-based systems, enabling a preferential targeting to infected sites and

allowing a deep interaction with biofilm, is expected to improve S. aureus infections control.

Acknowledgments: The authors acknowledge Fundação para a Ciência e Tecnologia (FCT), Portugal, for the financial support

under iMED.ULisboa project Pest-UID/DTP/04138/2014 and PTDC/MED-QUI/31721/2017.

References:

[1] Ferreira, M.; Rzhepishevska, O.; Grenho, L.; Malheiros, D.; Gonçalves, L.; Almeida, A. J.; Jordão, L.; Ribeiro, I. A.; Ramstedt, M.; Gomes, P.; Bettencourt, A., Int J Pharm., 2017, 532(1), 241–8. [2] Gaspar, M. M.; Cruz, A.; Penha, A. F.; Reymão, J.; Sousa, A. C.; Eleuterio, C. V.; Domingues, S. A.; Fraga, A. G.; Filho, A. L.; Cruz, M. E. M.; Pedrosa, J., Int J Antimicrob Agents., 2008, 31(1), 37–45. [3] Jefferson K. K.; Goldmann, A. D.; Pier B. G., Antimicrob Agents Chemother, 2005, 49(6), 2467-73.

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P17. Ascorbyl dipalmitate nanoemulsions as novel delivery systems for curcumin.

María Plaza-Oliver1,2, *, Ana Beloqui 3, Manuel J. Santander-Ortega1,2, Virginia Rodríguez-

Robledo1,2, Lucía Castro-Vázquez1,2, Joaquín González-Fuentes1,2, Pilar Marcos1,2, María del

Mar Arroyo-Jiménez1,2, M. Victoria Lozano1,2 and Veronique Préat 3

1Cellular Neurobiology and Molecular Chemistry of the Central Nervous System group. Faculty of Pharmacy, University of Castilla-La Mancha, Albacete (Spain). 2Regional Centre of Biomedical Research (CRIB), University of Castilla-La Mancha, Albacete (Spain). 3Université Catholique de Louvain, Louvain Drug Research Institute, Advanced Drug Delivery and Biomaterials, 1200 Brussels (Belgium).

* Correspondence: [email protected]

Curcumin is an antioxidant molecule with anti-inflammatory, antimicrobial and anticarcinogenic

activity [1]. Despite its promising potential, curcumin poor solubility and instability in physiological

media lead to low bioavailability of the drug limiting its application in clinics. Several efforts using

nanotechnology have improved curcumin behaviour and led to promising carriers for this drug [2,

3]. Nanoemulsions are exceptional colloidal carriers for hydrophobic drugs which core-shell

versatility facilitate their behaviour in biological media [4][5]. Therefore, we have designed a new

nanoemulsion based on ascorbyl dipalmitate as delivery system of curcumin and evaluated its

behavior in the well-stablished Caco-2 intestinal model. The physicochemical characterization of

the nanoemulsions showed size values of 170 nm, barely polydispersed and negatively charged.

The incubation of the formulations with the cells produced no toxicity nor decrease of the trans-

epithelial electrical resistance (TEER) values after two hours. The results obtained in the transport

studies pointed out to a local delivery of curcumin to the cell monolayer, where the drug was

accumulated, as curcumin was not detectable at the basolateral chamber. This was confirmed by

confocal microscopy showing high fluorescence at the cell monolayer due to curcumin. These

results were consistent with the determination of the ROS scavenging capacity of the

nanoemulsions, which showed that they were able to significantly decrease the intracellular ROS

levels. These results position ascorbyl dipalmitate nanoemulsions as novel and promising delivery

systems for the local intestinal delivery of curcumin in disorders such as inflammatory bowel

disease.

Acknowledgments: M. Plaza thanks the financial support given by the UCLM (OE154), Spain. A. Beloqui is a Research Associate of the Fonds de la Recherche Scientifique-FNRS (Belgium).

1. Anand, P., et al., Bioavailability of curcumin: Problems and promises. MolecularPharmaceutics, 2007. 4(6): p. 807-818.

2. Bisht, S., et al., Polymeric nanoparticle-encapsulated curcumin ("nanocurcumin"): Anovel strategy for human cancer therapy. Journal of Nanobiotechnology, 2007. 5.

3. Yallapu, M.M., et al., Therapeutic Applications of Curcumin Nanoformulations. AAPSJournal, 2015. 17(6): p. 1341-1356.

4. Plaza-Oliver, M., et al., Design of the interface of edible nanoemulsions to modulate thebioaccessibility of neuroprotective antioxidants. International Journal of Pharmaceutics,2015. 490(1-2): p. 209-218.

5. Santander-Ortega, M.J., et al., PEGylated Nanoemulsions for Oral Delivery: Role of theInner Core on the Final Fate of the Formulation. Langmuir, 2017. 33(17): p. 4269-4279.

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P18. Studying the adhesion of epithelial cells to collagen hydrogels to develop a new 3D intestinal model

Maria Helena Macedo1,2,3, Elena Martinez4,5,6, Cristina Barrias1,2,3, Bruno Sarmento1,2,7

1i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal 2INEB – Instituto Nacional de Engenharia Biomédica, Universidade do Porto, Porto, Portugal 3ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal 4IBEC – Institute for Bioengineering of Catalonia, Barcelona, Espanha 5CIBER-BBN – Consorcio Centro de Investigación Biomédica en Red de Bioingeniería,

Biomateriales y Nanomedicina, Madrid, Espanha 6Electronics and Biomedical Engineering Department, Universitat de Barcelona, Barcelona,

Espanha 7CESPU - Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde

& Instituto Universitário de Ciências da Saúde, Gandra, Portugal

The drug development field is increasingly requesting for reliable tools for high-throughput

analysis of drug absorption profiles. This work aims to establish and characterize a new

tridimensional intestinal model developed using Transwell inserts, encompassing epithelial cells

laid over fibroblasts embedded in extracellular matrix, closely resembling the intestinal mucosa

assembly. Immortalized epithelial cells, namely Caco-2 clone and HT29-MTX will be used in the

preliminary model set up, on top of human intestinal fibroblasts embedded in Collagen type I

providing 3D support. Raji B cells will be used to induce the differentiation of Caco-2 cells into M-

cells. After a full characterization of the model, the immortalized epithelial cells will be replaced

by IPSC-derived enterocyte-like cells and a comparison of performance will be performed.

Preliminary results show that when collagen is placed on the Transwell membrane, a meniscus

is formed due to capillarity effect. As a result, cells do not adhere to the periphery. Different

approaches have been tested to solve the problem of capillarity effect and the use of spacers

seem to be the most promising one. Collagen disks with 1 mm thickness have been tested and

epithelial cells seem to adhere better; nevertheless, a coating with laminin, a protein present in

the basement membrane that showed to increase the TEER of epithelial monolayers when

compared to collagen type IV and fibronectin, will be added. It was also observed that epithelial

cells do not leak through the borders of the gel, what is extremely important for the permeability

studies because shows that although the gels are not polymerized inside the insert, they are able

to cover the all surface without leakages.

The establishment of this model is expected to return new insights regarding the crosstalk

between stromal and epithelial cells and the pharmacokinetic profile of any tested compound.

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P19. Nanosized Glucan Particles as a Delivery System for Curcumin: the effect of size on immunotoxicity

Mariana Colaço1,2, Ana Patrícia Marques1,2, Sandra Jesus1,2, Olga Borges1,2

1Faculty of Pharmacy, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra,

Portugal 2Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal

Curcumin, known for its multiple health benefits, primarily its antioxidant and anti-inflammatory

properties [1], does not have a good clinical efficacy due to its low aqueous solubility, poor

bioavailability and rapid systemic elimination [2]. A promising approach to solve this problem and

enhance curcumin’s clinical relevance is to encapsulate it, using a drug delivery system. Glucan

(from Alcaligenes faecalis) is a polymer consisting of β-1,3-linked glucose residues [3] and has

been addressed in different medical fields, as a vaccine or gene delivery system. This work aims

to describe the production of different glucan nanoparticles (NPs) as a delivery system for

curcumin, and to evaluate in vitro the influence of the NPs size on immunotoxicity.

Different glucan NPs were produced using a nanoprecipitation technique, followed by curcumin

encapsulation. The NPs suspensions were characterized regarding its size and zeta potential and

their morphology was visualized by Scanning Electron Microscopy. Immunotoxicity of glucan NPs

was studied by evaluating cell viability, nitric oxide (NO) production and reactive oxygen species

(ROS) production in RAW 264.7 cells, hemolysis in whole human blood and lymphocyte

proliferation in human peripheral blood mononuclear cells (PBMCs).

Glucan NPs with 120 nm presented greater toxicity than glucan NPs with 250 nm. Smaller sizes

were also related to higher ROS production and to the inhibition of NO production after cell

stimulation. Both NP sizes caused hemolysis and induced lymphocyte proliferation, although the

concentration required to observe such effect was lower for the 120 nm glucan NPs. Curcumin-

encapsulated NPs presented an immunotoxicity profile similar to the corresponding curcumin free

NPs.

In conclusion, NPs size is an important factor for immunotoxicity, as smaller glucan NPs

presented higher toxicity. Moreover, curcumin encapsulation did not change the glucan NPs

immunotoxicity profile, and can be considered as a promisor strategy to improve curcumin low

bioavailability.

This work was funded by FEDER funds through the Operational Programme Competitiveness

Factors - COMPETE 2020 and national funds by FCT - Foundation for Science and Technology

under the project PROSAFE/0001/2016 and strategic project POCI-01-0145-FEDER-007440.

[1] M. Rai, R. Pandit, S. Gaikwad, A. Yadav, A. Gade, Nanotechnology Reviews 2015, 161.

[2] L.M. Huong, H.P. Thu, N.T.B. Thuy, T.T.H. Ha, H.T.M. Thi, M.T. Trang, T.T.N. Hang, D.H.

Nghi, N.X. Phuc, D.T. Quang, Chemistry Letters 2011, 40, 846-848.

[3] K. Chandra Hembram, S. Prabha, R. Chandra, B. Ahmed, S. Nimesh, Artificial Cells,

Nanomedicine, and Biotechnology 2014, 44 305-314.

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P20. Evaluation of immunotoxicological properties of PCL and PCL/Glucan

nanoparticles

N. Bernardi1;2, S. Jesus 1;2 , J. Da Silva, and O. Borges1;2

1Faculty of Pharmacy, University of Coimbra, Azinhaga de Santa Comba, 3000-548

Coimbra,Portugal 2Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal

Poly-ε-caprolactone (PCL) is a synthetic semi-crystalline and highly hydrophobic polymer,

attractive for its biodegradability, biocompatibility, low toxicity and its high versatility, such as the

ability to blend with other polymers [3]. In fact, it has been used in large set of applications from

food packaging to tissue engineering [4]. Glucan is a polymer composed of β-1,3-linked glucose

residues [1] produced by Alcaligenes faecalis, known for increasing host immune defense and

used in different medical fields as a delivery system for drugs and vaccines [2]. The aim of this

study is to evaluate in vitro the immunotoxicity of two nanosized polymeric delivery systems: PCL

and PCL/glucan nanoparticles (NPs).

Both NPs were produced using a nanoprecipitation technique: PCL precipitation was induced in

an aqueous media; Glucan precipitation was induced by acetic acid around the already formed

PCL NPs. The NPs were characterized regarding its size and zeta potential and their

immunotoxicity profile was assessed by evaluating cell viability, reactive oxygen species (ROS)

and nitric oxide (NO) production in RAW 264.7 cells, cell viability in human peripheral blood

mononuclear cells (PBMC) and hemolysis in whole blood.

PCL and PCL/glucan NPs presented a similar toxicity profile in RAW 264.7 cell line, with no

induction of ROS or NO production the concentration range tested (3,5 µg/mL - 70 µg/mL). On

the other hand, PCL NPs showed to be slightly more toxic in PBMCs than PCL/Glucan NPs.

Regarding hemocompatibility, concentrations higher than 25 µg/mL of PCL/Glucan NPs induce

hemolysis while PCL nanoparticles were hemocompatible up to 75 µg/mL.

In this study, we reported the production of PCL and PCL/Glucan NPs and the evaluation of its

immunotoxicity in vitro. Although PCL/glucan NPs presented a better viability profile in PBMCs,

they were found to be more hemolytic than PCL NPs.

[1] K. Chandra Hembram, S. Prabha, R. Chandra, B. Ahmed, S. Nimesh, Artificial Cells, Nanomedicine, and Biotechnology 2014, 44 305-314. [2] Akramiene, D., Kondrotas, A., Didziapetriene, J., & Kevelaitis, E. (2007). Effects of beta-glucans on the immune system. Medicina. [3] Dash TK, Konkimalla VB. Polymeric modifcation and its implication in drug delivery: poly-epsilon-caprolactone (PCL) as a model polymer. Mol. Pharm. 9(9), 2365–2379 (2012) [4] Woodruff, M.A. and D.W. Hutmacher, The return of a forgotten polymer—Polycaprolactone in the 21st century. Progress in Polymer Science, 2010. 35(10): p. 1217-12.

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P21. Determination of α-tocopherol by LC-ESI-MS/MS as an alternative to the use

of fluorescent molecules

Noemí Villaseca-González1,2, Virginia Rodríguez Robledo1,3, Lucía I. Castro-Vázquez1,3, Mª

Victoria Lozano1,2, María Plaza-Oliver1,2, Manuel J. Santander-Ortega1,2, Joaquín González-

Fuentes1,2, Pilar Marcos1,2 & Mª del Mar Arroyo-Jiménez1,2

1 Cell Neurobiology and Molecular Chemistry in Central Nervous System, Faculty of Pharmacy and Regional Center for Biomedical Research. 2 Department of Medical Sciences, University of Castilla-La Mancha. 3 Department of Analytical Chemistry and Food Technology, University of Castilla-La Mancha.

Vitamin E is one of the most important biomolecules antioxidants studied in recent years due to

the complexity of its composition chemistry and its metabolism. It consists of four tocopherols and

tocotrienols (α-, β-, γ- and δ-), that contains a 6-chromanol ring structure methylated to varying

degrees with a C16 saturated side chain in position 2. Alpha-Tocopherol (α-T) is one of the isomers

with greatest biological activity because it is selectively recognized by the α-T transfer protein (α-

TTP)1. Although there are many studies for quantification of α-T by liquid chromatography

coupling mass spectrometry in biological samples currently, Villaseca-Gonzalez et al.2 have

published an ultrafast analytical method for its determination using double detection, diode-array

on line electrospray ionisation (ESI) by triple quadrupole as analyser. This analytical methodology

can be used both, in biopharmaceutical and biological samples such as serum, plasma or tissue.

For that, a whole validation procedure including precision (RSD < 5%) (repeatability and

reproducibility), selectivity, limits of detection and quantitation (0,12 and 0.4 µg mL-1,respectively),

linearity range (0.6125 to 20 µg mL-1), accuracy (98%) and matrix effect (< 10%) was rigorously

carried-out, in this work.

Finally, the validated chromatographic method was used as valuable analytical tool for the direct

determination of α-tocopherol based drug delivery systems. However, the use of tissue biological

samples such as blood, liver, kidney, brain or muscle (among others), requires complete

evaluation of every step including in the samples pre-treatment. In this sense, the quantification

of α-T in different tissues allows us to evaluate its biodistribution and bioaccumulation in vivo. In

addition, this approach enables the identification of one component of the system itself, and then

helps to understand the fate of the systems after their administration without their modification

with fluorescent tracers.

References:

Eitenmiller, R.R.; Lee, J. Vitamin E, Food Chemistry, Composition and Analysis. Marcel Dekker Inc., New York, 2004. Villaseca-Gonzalez, N.; Robledo, V. R.; Castro-Vazquez, L.; Lozano, M. V.; Santander-Ortega, M. J.; Gonzalez-Fuentes, J.; Marcos, P.; Mar Arroyo-Jimenez, M. D. Ultrafast determination of vitamin E using LC-ESI-MS/MS for preclinical development of new nutraceutical formulations; Bioanalysis, 2018, 10 (4), 215-227.

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P22. Putrescine nanoemulsions for cancer gene therapy

Sainza Lores1, Sandra Alijas1, Rafael López1, Ana Dávila2, María de la Fuente1

1Nano-Oncology Unit, Translational Medical Oncology Group (ONCOMET), Health Research

Institute of Santiago de Compostela (IDIS), 15706, Santiago de Compostela, Spain. CIBERONC 2Roche-CHUS Mixed Unit. Translational Medical Oncology Group (ONCOMET), Health Research

Institute of Santiago de Compostela (IDIS), 15706, Santiago de Compostela, Spain.

Introduction: Gene therapy has been proposed as a promising therapeutic approach for cancer.

Non-viral vectors are easy to manufacture with good yield, and are considered to be safe. We

propose the use of the cationic polyamine putrescine for the development of nanoemulsions for

cancer gene therapy.

Objectives: To develop and characterize putrescine nanoemulsions for gene therapy applications.

Materials and Methods: Putrescine nanoemulsions were prepared by ethanol injection. The

reporter plasmid mCherry was subsequently associated onto preformed nanoemulsions.

Characterization was performed with a Nanosizer2000®, with a NanoSight NS3000 System and

by Transmission Electron Microscopy. The association efficiency was determined by agarose gel

electrophoresis. After transfection, the expression of mCherry was observed under the

fluorescence microscope. Cell viability was determined with the MTT assay.

Results and Discussion: Putrescine nanoemulsions have a mean small size below (80 ± 7 nm)

and a positive zeta potential (+45 ± 3 mV). According to the electrophoresis gel, pDNA was

efficiently associated to nanoemulsions and stable for at least one week. It was also observed an

increase in the mean size (150 ± 5 nm) and a decrease in the zeta potential (+30 ± 2 mV). With

respect to the morphology, a change towards a more rigid structure was observed upon

association of pDNA. Transfection studies show that nanoemulsions can successfully mediate

the internalization of the associated pDNA, and expression of mCherry was observed for at least

72h. Formulations were non-toxic at these experimental conditions.

Conclusions: Putrescine nanoemulsions can efficiently associate nucleic acids and transport

them to the target cancer cells. Next experiments will be aimed to replace the reported gene with

a therapeutic gene.

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P23. Multi-layered polymeric nanocapsules for brain delivery of ribo-nucleic acid

therapeutics

Sakthikumar Ragupathy1, Vanessa Castro-López1, Eleni Samaridou1, Maria J. Alonso1,2,3

1Center for Research in Molecular Medicine and Chronic Diseases University of Santiago de

Compostela, Spain 2IDIS Research Institute,Santiago de Compostela, Spain 3School of Pharmacy, University of Santiago de Compostela, Spain

Alzheimer’s disease has no cure or treatment and the discovery of new conventional drugs have

so far failed owing to the complex pathology. RNA therapeutics are ideal candidates to treat and

control such neurodegenerative diseases. However, the clinical success of this strategy is limited

by the lack of safe and efficient brain-homing RNA delivery vehicles. The objective of this work

was therefore to develop a nanoscale RNA -carrier with biocompatible materials that is suitable

for systemic and intra-nasal brain delivery. Such nano-systems should be stable in serum/plasma,

achieve sufficient drug loading with low or no toxicity. A transfer RNA was used as a model drug

and was successfully associated on to the outer-polymeric-shell surrounding a stable hydrophobic

core. The RNA was covered by layers of protamine and hyaluronic acid which might provide

protection from degradation. These multi-layered polymeric nanocapsules had desired

physicochemical properties and achieved a size below 200 nm, a narrow size distribution

(polydispersity index less than 0.2) and a negative zeta-potential (-23mV). The particles were able

to maintain their size without considerable changes in cell culture media for 24 hours at 37°C. In

conclusion, the physico-chemical properties of multilayer polymeric nanocapsules shows the

potential to be efficient delivery vehicles of RNA.

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P24. Protamine nanoparticles for gene therapy to treat glioblastoma multiforme.

*Barrios S.; Reimondez-Trotitiño S., Garcia-Fuentes M., Csaba N.

Center for Research in Molecular and Chronic Disease (CiMUS), Department of Pharmacology,

Pharmacy and Pharmaceutical Technology, University of Santiago de Compostela, Av. Barcelona

s/n, Santiago de Compostela, 15706, Spain.

*[email protected]

Glioblastoma multiforme is the most aggressive brain tumor. The conventional treatment of

this disease consists of three steps: surgical resection, radiotherapy and chemotherapy.

However, due to the potentially severe side effects and low therapeutic benefits of this treatment,

alternative therapies such as gene therapy1 are currently under investigation.

Gene therapy includes the regulation of specific genes involved in the development of cancer.

This requires two fundamental aspects: therapeutic genes and effective, safe and cost-effective

delivery systems2. Our research group has been exploring protamine/ dextran nanoparticles as

gene delivery systems, using different nucleic acid cargos (siRNA/miRNA) for the inhibition of

tumor cell growth. The nanoparticles were prepared by an ionic gelation method, which yielded

spherical protamine:dextran nanoparticles with homogeneous size distribution at the optimal

polymer ratio of 4:1. These nanoparticles were stable under storage (4ºC) and physiological

conditions (37 ºC pH 7.4), with a size below 200 nm (112 ± 60 nm) and a positive potential (+36

± 7 mV). These optimized nanoparticles have high affinity for the different nucleic acids, with

encapsulation efficiencies over 90%. Moreover, our results in 2D cultures and 3D spheroids of

U87MG glioblastoma cells showed that these nano-vehicles have good cellular penetration

capacity and low toxicity. The results indicate the promise of these nano-vehicles for the delivery

of gene therapy against cancer. Future studies in animal models will be carried out to evaluate

the biopharmaceutical properties of this system and its efficacy in vivo.

(1). Reguera-Nuñez E. et al., Biomaterials. 35:2859-2867 (2014)

(2). Reimondez-Troitiño S. Doctoral Thesis. Chapter IV. 151-17

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P25. Biocompatible poly(butylcyanocrylate) nanoparticles for drug and gene

delivery

Silvia Fuerte-Rodríguez1, Fátima Fernández-Álvarez1, Gracia García-García1, Raúl Ortiz2,3, José

C. Prados2-4, José L. Arias1,3,4

1Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of

Granada, Spain. 2Department of Human Anatomy and Embryology, Faculty of Medicine, University of Granada,

Spain. 3Institute of Biopathology and Regenerative Medicine (IBIMER), Biomedical Research Centre

(CIBM), University of Granada, Spain. 4Biosanitary Institute of Granada (ibs.GRANADA), Andalusian Health Service (SAS), University

of Granada, Spain.

Basic aspects in the formulation of biodegradable colloids for the controlled delivery of drugs and

genes are related to the definition of the best formulation conditions to reproducibly obtain nano-

sized and biocompatible particles. In this work, it has been developed a reproducible methodology

to formulate 100nm-sized poly(butylcyanoacrylate) (PBCA) particles. The procedure is based on

the well-known anionic polymerization method. Transmission electron microscopy and photon

correlation spectroscopy characterizations demonstrated the very small size of the particles, while

infrared analysis proved that the structure of butylcyanoacrylate monomer was maintained upon

formation of the nanoparticles. The PBCA particles proved to be haemocompatible. In fact, a

negligible effect on haemolysis (even after 24 hours), platelet activation, complement system

activation, and plasma clotting time of blood samples was observed (n = 3). Finally, the polymeric

nanoparticles exhibited negligible cytotoxicity in the CCD-18 human fibroblast cell line isolated

from normal colon tissue, and in the T-84 human colon cancer cells, for the particle concentrations

assayed. Based on these findings, it may be postulated that the PBCA-based nanosystem is

characterized by an adequate biocompatibility and safety for drug and gene delivery purposes.

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P26. Comparative study of the antioxidant activity of ferulic and vanillic acids by

the ABTS radical inhibition method.

Amanda Manzini Carneiro1, Gabriel Urbino Phelipe1, Danieli Camilo Marcato1, Caroline Magnani

Spagnol1, Marcos Antonio Corrêa1, Ana Melero2, Antonio J. Guillot2, Silvia Mir-Palomo2.

1Department of Drugs and Medicines, Faculty of Pharmaceutical Sciences, Araraquara,

Universidade Estadual Paulista (UNESP).

2Department of Pharmacy and Pharmaceutical Technology and Parasitology. Faculty of

Pharmacy, University of Valencia, Spain.

Phenolic compounds are widely distributed in plants and ferulic and vanillic acids are two

representatives of this group. Among the different attributed pharmacological activities, their

antioxidant potential can be highlighted, because, according to the literature, they are able to

prevent the harmful effects of solar radiation both as a UV absorber and as a scavenger of free

radicals. The objective of the present study was to determine the antioxidant activity of these

active substances through the ABTS (2,2'azinobis- (3-ethylbensothiazoline) -6-sulfonic acid)

inhibition, so that they can be used as cosmetic actives with anti-ageing purposes. The capture

of the ABTS radical by antioxidants is the basis of its inhibition method. The results were

expressed as percentage of ABTS radical inhibition based on the absorbance values obtained at

734nm. From the percent inhibition values it was possible to obtain the analytical curves and

calculate the inhibition percentages expressed as IC50 values (amount of substance required to

inhibit 50% of the ABTS radical). Ferulic acid showed IC50 2.84 μg.mL-1, while vanillic acid showed

IC50 1.32 μg.mL-1 and ascorbic acid and gallic acid presented IC50 2.55 μg.mL-1 and IC50 0.47

μg.mL-1, respectively. According to the results obtained, it can be observed that vanillic acid has

better antioxidant activity compared to ferulic acid. Although they present the same amount of

hydroxyl groups, responsible for the antioxidant activity, ferulic acid presents a greater branching

and is unsaturated, thus being able to generate greater steric hindrance and stiffness and

therefore reducing the accessibility of the radical. Further studies will be conducted to design the

appropriate dosage forms for cosmetic purposes.

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P27. Poly(I:C)-loaded nanocomplexes for the re-education of tumor-associated

macrophages

Tamara G Dacoba1,2, Fernando T Andón1,4, Clément Anfray4, José Crecente-Campo1,2, Paola

Allavena4, Maria J Alonso1,2,3

1Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidade de

Santiago de Compostela, Santiago de Compostela, Spain

2Department of Pharmacology, Pharmacy and Pharmaceutical Technology (School of

Pharmacy), Universidade de Santiago de Compostela, Santiago de Compostela, Spain

3Health Research Institute of Santiago de Compostela (IDIS), Santiago de Compostela, Spain

4Istituto Clinico Humanitas, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Milan, Italy

The modulation of the tumor microenvironment to direct the immune system towards fighting

against tumors is under investigation as a new cancer treatment. In this context, tumor-associated

macrophages (TAMs), which have lost their innate ability to fight against the tumor, are an

interesting population to target1. Poly(I:C), a dsRNA that mimics viral genetic material and that

activates the toll-like receptor 3, can drive macrophage polarization towards a pro-inflammatory

and antitumoral phenotype. Unfortunately, the systemic administration of poly(I:C) causes

unwanted side effects2, making essential its inclusion in a carrier to only target the disease sites.

Bearing this in mind, our goal has been to engineer a series of nanosystems considering the

affinity of poly(I:C) for arginine-rich polymers. The resulting nanocomplexes were further coated

with different negatively-charged polymers to provide them with stealth properties and targeting

capacity.

Developed nanocomplexes presented low and narrow particle sizes (<200 nm and PDI <0.2),

with surface charges ranging from highly positive to slightly negative values. All nanosystems

were able to efficiently encapsulate poly(I:C), that remained attached into de nanocomplex after

24h of incubation in cell culture media. In vitro studies in primary human macrophages showed

a satisfactory toxicological profile and the ability of some nanocomplexes to enhance the secretion

of the T-cell attracting chemokine CXCL10. Furthermore, pretreatment of macrophages with the

poly(I:C)-loaded nanocomplexes was able to increase their cytotoxicity towards human cancer

cells.

In summary, complexation of the immunomodulator poly(I:C) to arginine-based polymers with

different coatings is a promising tool for the re-education of TAMs towards antitumoral

macrophages.

1. Andón, F. T. et al. Targeting tumor associated macrophages: The new challenge fornanomedicine. Semin. Immunol. 34, 103–113 (2017).

2. Hafner, A. M., Corthésy, B. & Merkle, H. P. Particulate formulations for the delivery ofpoly(I:C) as vaccine adjuvant. Adv. Drug Deliv. Rev. 65, 1386–1399 (2013).

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P28. Development of budesonide polymeric mucoadhesive particles for the local

management of oral mucositis

João Campos1, Domingos Ferreira1, Sofia Lima2, Salette Reis2, Paulo Costa1

1UCIBIO, REQUIMTE, Laboratory of Pharmaceutical Technology, Department of Drug Sciences,

Faculty of Pharmacy, University of Porto, Portugal 2LAQV, REQUIMTE, Laboratory of Applied Chemistry, Department of Chemical Sciences, Faculty

of Pharmacy, University of Porto, Portugal

Oral mucositis, a common side effect of oncological treatments, is an inflammatory condition that

can gravelly hinder the patient quality of life and the ability to endure radiotherapy and/or

chemotherapy. Although several strategies have been studied, management options are few and

still unsatisfactory. Budesonide oral mouthwash has been suggested for its management,

showing some potential in alleviating the symptoms. Yet, the simple delivery design may hamper

beneficial clinical results. Among the several issues regarding the treatment, topical drug delivery

is one of the main challenges. Since the oral cavity represents a well-known difficult location, due

to its mechanical and environmental features, local drug action can be limited. In an appetent to

resolve this issue, budesonide mucoadhesive polymeric particles have been developed. These

particles were obtained by the atomization of a 1:1 ratio budesonide-polymer (chitosan and

eudragit® E PO) solution in a Nano Spray Dryer B-90. The efficiency of the drug entrapment was

50.89±0.58%. A positive zeta potential for the stand-alone particles (C:EPO), +35.1±1.1 mV, and

for the budesonide:polymeric particles (C:EPO:BUD), +36.02±0.27 mV, was observed. A

decrease of these values was obtained after 2-hour incubation with mucin type II, which is

indicative of the mucoadhesive capability. SEM images of the dry powder displayed spherical

nanometric structures. While laser diffraction analysis of the hydrated state showed sizes of

8.17±0.30 µm for the C:EPO and 9.93±0.65 µm for the C:EPO:BUD, showing the ability to

increase their size by polymeric swelling. Finally, a drug release assay was conducted in the

dissolution apparatus II, using dialysis bags to confine the C:EPO:BUD. The particles showed the

ability for a modified 12-hour slow release, fitting the Korsmeyer-Peppas mathematical model

(k=7.64, b=-10.96, n=0.46, R2=0.998). The exponent of the equation is near to 0.43, suggesting

a release mainly depended on Fickian diffusion with some evidence of anomalous transport.

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P29. Double-layered protamine nanocapsules as iRNA delivery systems

Sofia M. Saraiva1, Maria José Alonso1, 2

1 Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Av. Barcelona s/n,

Campus Vida, Universidade de Santiago de Compostela, 15706 Santiago de Compostela, Spain. 2 Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, Universidade

de Santiago de Compostela, 15782 Santiago de Compostela, Spain

Polynucleotide-based therapies, and notably those based on interfering RNA (iRNA), hold great

promise due to their capacity to knockdown specific genes whose misfunction is responsible of

severe diseases. The RNA’s fast degradation in biological fluids and low capacity to enter cells

stresses the need for the development of carriers able to protect the cargo and deliver it at the

target site. Our research group as a background on the development of nanocapsules, intended

for the delivery of both, drugs and polynucleotides. The main goal of this study was the

development of nanocapsule formulations, with an iRNA high loading capacity (achieving a final

iRNA concentration of mg/mL instead of µg/mL), able to hamper its degradation and deliver it to

the site of action (cell cytoplasm). Bearing this in mind, double layered (model iRNA and

protamine) nanocapsules, were developed. iRNA molecules were associated to the positively

charged oily cores and protected by protamine. The effect of different parameters (type of cationic

surfactant, iRNA and polymer concentrations) on the carrier properties (average size, zeta

potential, loading capacity, colloidal stability in biorelevant media) were evaluated. Depending on

the type of cationic surfactant, different iRNA concentrations were achieved (1-2 mg/mL). The

nanocapsules presented a mean size of 100-200 nm and positive zeta potential. Moreover, they

were stable upon incubation with biorelevant media, maintaining their size and avoiding

premature iRNA release. Overall, the results show the capacity of these carriers to associate high

concentrations of iRNA, which would increase the chances of a higher bioavailability of the drug

at the target site.

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P30. Development of a nanoparticle-based hydrogel for topical delivery of insulin for wound healing

Patrícia Filipe1, 2, Ana Macedo1, Catarina Rosado1, Pedro Fonte1,3,*

1CBIOS-Research Center for Biosciences and Health Technologies, Lusófona University, Lisbon,

Portugal 2Department of Biomedical Sciences, University of Alcalá, Madrid, Spain 3LAQV, REQUIMTE, Department of Chemical Sciences – Applied Chemistry Lab, Faculty of

Pharmacy, University of Porto, Porto, Portugal

*Corresponding author: [email protected]

Insulin is a peptide hormone that may be used as a growth factor in wound healing. In Europe,

the cost for wound healing ranges from 6.000€ to 10.000€ per patient per year [1]. The main aim

of this work was to develop an insulin-loaded chitosan-coated PLGA nanoparticle hydrogel

system for wound healing. Chitosan-coated PLGA nanoparticles were developed by a modified

solvent evaporation method based on a w/o/w double emulsion technique [2]. The coating of

chitosan was performed at 0.25%, 0.5% and 1% and the physical-chemical properties were

evaluated to obtain nanoparticles in the nanosize range, low polydispersity and good colloidal

stability (Table 1). Further, the nanocarriers were loaded into a hydrogel for topical administration.

It was obtained a hydrogel, composed of sodium alginate and sodium carboxymethylcellulose

with good uniformity and rheological properties. The developed protocol allowed to produce an

insulin-loaded chitosan-coated PLGA nanoparticles hydrogel with good stability and without

heating. It is expected that these formulations may be a promising delivery system to treat a wide

array of wounds.

Table 1. Physical-chemical properties of PLGA nanoparticles at different concentrations of

chitosan.

Diameter (nm) PdI ZP (mV)

PLGA NP uncoated 270 ± 15 0.21 ± 0,01 -14.12 ± 1.17

PLGA NP coated chitosan 0.25% 749 ± 30 0.29 ± 0.01 30.43 ± 2.43

PLGA NP coated chitosan 0.5% 1072 ± 47 0.34 ± 0.02 31.72 ± 2.58

PLGA NP coated chitosan 1% 1289 ± 24 0.25 ± 0.04 32.54 ± 2.63

Acknowledments:

The authors would like to thank to Fundação para a Ciência e a Tecnologia, Portugal and

COMPETE 2020 (PTDC/MEC-DER/32610/2017) for financial support.

References:

[1] Posnett, J. et al., Journal of Wound Care, 18(4), 154–161, 2009.

[2] Fonte, P. et al., Pharm Res, 33(11), 2777-2793, 2016.

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P31. Evaluation of the stability of therapeutic proteins loaded into nanostructured lipid carriers

Ana Macedo1, Saúl Pereira1, Patrícia Filipe1, Pedro Fonte1,2,*

1CBIOS-Research Center for Biosciences and Health Technologies, Lusófona University, Lisbon, Portugal. 2LAQV, REQUIMTE, Department of Chemical Sciences – Applied Chemistry Lab, Faculty of Pharmacy, University of Porto, Porto, Portugal. *Corresponding author: [email protected]

Nanostructured lipid carriers (NLC) present several advantages over solid lipid nanoparticles

(SLN), mainly regarding their higher drug loading capacity and better controlled drug release

profile. In this work we propose a novel production method of NLC produced without heating,

allowing the incorporation of thermolabile therapeutic proteins. Bovine serum albumin was used

as a model of a therapeutic protein, and its structural stability was assessed upon encapsulation

into NLC and SLN. BSA-loaded NLCs and SLN were prepared using an adapted solvent-

evaporation double emulsion technique [1]. Different solid and liquid lipids were used at a 70/30

w/w ratio with the surfactant Tween 80, at 1%, 2% and 3% w/v. The delivery systems were

physico-chemically characterized. The structural stability of BSA was evaluated by FTIR following

a previously described methodology [2].

After formulation optimization the NLC with both lowest size and higher association efficiency

(AE) were made from the lipids, glyceryl distearate and oleic acid. SLN were produced using only

the solid lipid. The NLC had a size of 311±13,3 nm (PdI of 0,30±0,02) and zeta potential of -

31.2±2.4 mV and the SLN had a size of 360±58 nm (PdI of 0,29±0,01) and zeta potential of -

25.7±3.2 mV. The encapsulation efficiency was about 64% for SLN and 81% for NLC. Additionally,

the NLC showed to preserve the stability of the loading protein in about 90%. The developed

delivery system may be a good carrier for the delivery of therapeutic proteins.

References

1. Fonte, P., et al., Stability study perspective of the effect of freeze-drying usingcryoprotectants on the structure of insulin loaded into PLGA nanoparticles.Biomacromolecules, 2014. 15(10): p. 3753-3765.

2. Fonte, P., et al., Effect of the Freezing Step in the Stability and Bioactivity of Protein-Loaded PLGA Nanoparticles Upon Lyophilization. Pharmaceutical Research, 2016.33(11): p. 2777-93.

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P32. Arginine-rich carriers as mRNA vaccine delivery systems for HIV

Ana Oliveira1,2, Alberto Guardo2, Beatriz Perdiguero3, José Crecente-Campo1,4, Mariano

Esteban3, Montserrat Plana2, Felipe García6 and María J. Alonso1,4,5

1Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidade de

Santiago de Compostela, Spain 2Institut d’Investigacions Biome`diques August Pi i Sunyer (IDIBAPS)-HIVACAT, Barcelona,

Spain 3Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, Consejo

Superior de Investigaciones Científicas (CNB-CSIC), Campus de Cantoblanco, Madrid, Spain4IDIS Research Institute,Santiago de Compostela, Spain 5School of Pharmacy, Universidade de Santiago de Compostela, Spain 6Infectious Diseases Department, Hospital Clinic de Barcelona, Barcelona, Spain

Despite the important human and economical efforts that have been made, vaccination against

HIV has not been successful so far. Recently there have been some promising developments

using mRNA-based therapeutic vaccine. However, further developments are needed to improve

mRNA delivery and the vaccine applicability. Here we evaluated different novel arginine-rich

carriers: coated octaarginine-based nanocomplexes, polyarginine and protamine nanocapsules;

and cationic nanoemulsions, the later as a control.

These carriers have a nanometric size below 200 nm and highly positive surface charge allowing

efficient association of different mRNA molecules, with loadings between 2 and 18% (wt%).

Moreover, the developed carriers were able to protect mRNA from RNases degradation for

extended time periods. In vitro experiments showed dose and pH-dependent toxicity, where

nanocapsules where the most cytocompatible.

The three best performing formulations coated octaarginine-based nanocomplexes, polyarginine

and protamine nanocapsules, will be further tested in vivo for their immunization potential as HIV

therapeutic mRNA vaccines.

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P33. Establishment of bioengineered glucose-responsive nanoparticles for type 2

diabetes mellitus therapy

Filipa Fonseca1, Ana Oliveira1, Bruno Sarmento1

1Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal

Diabetes mellitus (DM) is one of the biggest health problems in the world with a high socio-

economic impact. Type-1 (T1DM) is characterized by insuficient insulin secretion due to the

destruction of pancreatic beta-cells and it accounts for 10% of the total cases of DM. Current

treatment options do not restore pancreatic functions and new clinical approaches are urgently

needed. Glucagon-like peptide-1 (GLP-1), an endogenous gastrointestinal incretin hormone that

stimulates insulin secretion and β-cell proliferation, is considered an attractive therapeutic agent

for T1DM treatment. Nevertheless, its very short half-life has led to the development of

nanocarriers-based strategies using degradation resistant GLP-1 analogous to ensure the

intended biodistribution profile of the drug. Here, we propose the production of polymeric pH-

sensitive nanoparticles (NP) loaded with exenatide, an FDA-approved GLP-1 analogous, surface-

functionalized with Glucose Oxidase (GOx) that will release its payload only at acidic pH. The pH-

sensitive release will be achieved by the use of both PLGA and Eudragit E100 as polymeric matrix

which will be soluble in the acidic environment created by the increased amounts of gluconic acid

produced from glucose degradation by GOx. Monodisperse exenatide-loaded PLGA/Eudragit

E100 NP with different polymer proportions were successfully produced and characterized,

revealing sizes between 110 and 180 nm and zeta potentials between 40 and 50 mV. Also, a pH-

dependent degradation study showed that NP disruption started at pH 6 and it increased with

decreasing pH. Currently, GOx is being chemically conjugated to NP surface through two different

methods, EDC/NHS chemistry and using BS3 as a crosslinker. Finally, the release profile of

exenatide at different pH and the long-term stability will be evaluated and the best formulation will

be part of a pancreas surrogate that could be part of a suitable and inovative cell therapy treatment

for T1DM.

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List of authors

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Name Affiliation

Abi Judit Vasquez

Nano-Oncology Unit, Translational Medical Oncology Group (ONCOMET). Health Research Institute of Santiago de Compostela (IDIS) and CIBERONC, Santiago de Compostela, Spain

Alana Duarte Center for Neuroscience and Cell Biology, University of Coimbra, Portugal

Ana Macedo CBIOS-Research Center for Biosciences and Health Technologies, Lusófona University, Lisbon, Portugal

Ana Oliveira Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Ana Olivera Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Ana Topete Centro de Química Estrutural, Instituto Superior Técnico, University of Lisbon, Lisbon, Portugal

Andreia Almeida

I3S – Institute for Research and Innovation in Health, University of Porto, Portugal

Anna Abadessa

Department of Pharmaceutics, Utrecht University, Utrecht, The Netherlands

Antonio José Guillot

Department of Drugs and Medicines, São Paulo State University (UNESP), School pf Pharmaceutical Sciences, Araraquara, Brasil

Bárbara Sánchez

Pharmacokinetics and Pharmaceutical Technology Area. Miguel Hernandez University

Carla Garcia Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Carlos Rodriguez

Department of Chemistry and Pharmaceutical Technology, University of Navarra

Carmen Fernández

Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Claudia Azevedo

i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal;

Claudia Martins

i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal;

Cristian Reboredo

Department of Chemistry and Pharmaceutical Technology, University of Navarra

Diego Pan Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Fátima Fernández

Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada

Filipa Fonseca i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal;

Flavia Sousa i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto

Gracia Garcia Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada

Iago Fernández Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

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Jacinta Pinho Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon

Jessica Da Silva

Faculty of Pharmacy, University of Coimbra

Joao Campos UCIBIO, REQUIMTE, Laboratory of Pharmaceutical Technology, Department of Drug Sciences, Faculty of Pharmacy, University of Porto, Portugal

João F. Mano Department of Chemistry, CICECO, University of Aveiro, Aveiro, Portugal

Jorge Morales Department of Chemistry and Pharmaceutical Technology, University of Navarra

Katia Maso Polymer Therapeutics Lab, Centro de investigación Príncipe Felipe (CIPF), Valencia, Spain

Laura Saludas Department of Chemistry and Pharmaceutical Technology, University of Navarra

Magda Ferreira Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon

Maria Helena Macedo

i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal;

Maria Plaza Cellular Neurobiology and Molecular Chemistry of the Central Nervous System group. Faculty of Pharmacy, University of Castilla-La Mancha

Mariana Colaço Faculty of Pharmacy, University of Coimbra

Marta Vives-Pi Germans Trias i Pujol Research Institute (IGTP), Autonoma University, Barcelon

Maruthi Prasanna

Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela, Spain

Matilde Duran Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela, Spain

Mireya Lopez Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela, Spain

Natalia Bernardi

Faculty of Pharmacy, University of Coimbra

Noemi Villaseca

Cellular Neurobiology and Molecular Chemistry of the Central Nervous System group. Faculty of Pharmacy, University of Castilla-La Mancha

Patricia Filipe CBIOS-Research Center for Biosciences and Health Technologies, Lusófona University, Lisbon, Portugal

Rute Nunes I3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal

Sainza Lores Nano-Oncology Unit, Translational Medical Oncology Group (ONCOMET). Health Research Institute of Santiago de Compostela (IDIS) and CIBERONC, Santiago de Compostela, Spain

Sakthikumar Ragupathy

Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Sandra Diez Nano-Oncology Unit, Translational Medical Oncology Group (ONCOMET). Health Research Institute of Santiago de

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Compostela (IDIS) and CIBERONC, Santiago de Compostela, Spain

Sheila Barrios Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Silvia Fuerte Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada

Silvia Mir Department of Drugs and Medicines, Faculty of Pharmaceutical Sciences, Universidade Estadual Paulista (UNESP).

Sofia Mendes Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Tamara Gomez Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Tomas Ramos Instituto de Investigação e Inovação em Saúde (I3S), University of Porto, Rua Alfredo Allen, 208, 4200-135, Porto, Portugal

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List of co-authors

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Name Affiliation

A. Grevys Department of Immunology, Centre for Immune Regulation (CIR), Oslo University Hospital Rikshospitalet and University of Oslo, Oslo, Norway

Alexander J. Najibi

John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA

Aline Pillot Unit Function & Protein Engineering, UMR CNRS 6286, University of Nantes, France;

Ana Armiñan Polymer Therapeutics Lab, Centro de investigación Príncipe Felipe (CIPF), Valencia, Spain

Ana P. Francisco Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon

Andrea Cruz INL, International Iberian Nanotechnology Laboratory, Avenida Mestre José Veiga, 4715-330 Braga, Portugal

Andrés Figueroa-Campos

Pharmacokinetics and Pharmaceutical Technology Area. Miguel Hernandez University

Angela Casini School of Chemistry, Cardiff University, Cardiff, United Kingdom

Angelo S. Mao John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA

Bruno Sarmento i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal

Beatriz Pelacho Instituto de Investigación Sanitaria de Navarra, IdiSNA, Pamplona, Spain

Carlos Diéguez Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela, Spain

Carlos J. González-Navarro

Centre for Nutrition Research, University of Navarra, 31080 Pamplona, Spain.

Cecília M.P. Rodrigues

Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon

Cristina Calviño Department of Biochemistry and Molecular Biology, University of Santiago de Compostela, Spain

Cyrille Grandjean Unit Function & Protein Engineering, UMR CNRS 6286, University of Nantes, France;

Daphnée Soulard Centre d’Infection et d’Immunité de Lille, Université de Lille, CHU Lille- Institut Pasteur de Lille, France

David Charbonnier

Polymer Therapeutics Lab, Centro de investigación Príncipe Felipe (CIPF), Valencia, Spain

David J. Mooney John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA

Dolores Torres Department of of Pharmacology, Pharmacy and Pharmaceutical Technology, School of Pharmacy, Universidade de Santiago de Compostela, Spain

Eduarda Mendes Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon

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Elena Gallon Polymer Therapeutics Lab, Centro de investigación Príncipe Felipe (CIPF), Valencia, Spain

Elisa Garbayo Department of Chemistry and Pharmaceutical Technology, University of Navarra

Emilie Camberlein

Unit Function & Protein Engineering, UMR CNRS 6286, University of Nantes, France;

Fábio Júnio Ferreira

i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal

Felipe Prósper Instituto de Investigación Sanitaria de Navarra, IdiSNA, Pamplona, Spain

Fernanda Rodríguez

Polymer Therapeutics Lab, Centro de investigación Príncipe Felipe (CIPF), Valencia, Spain

Fernando Herranz Nanomedicine and Radiochemistry Group, National Centre for Cardiovascular Research Carlos III (CNIC) and CIBERES, Madrid, Spain.

Francisca Araújo i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal

Francisco Campos

Clinical Neurosciences Research Laboratory, Health Research Institute of Santiago de Compostela (IDIS), Santiago de Compostela, Spain.

François Trottein Centre d’Infection et d’Immunité de Lille, Université de Lille, CHU Lille- Institut Pasteur de Lille, France

Gloria Abizanda Instituto de Investigación Sanitaria de Navarra, IdiSNA, Pamplona, Spain

Graça Soveral Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon

Hélder A. Santos Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, FI-00014, Helsinki, Finland

Inês Mendes Pinto

INL, International Iberian Nanotechnology Laboratory, Avenida Mestre José Veiga, 4715-330 Braga, Portugal

Isabel González-Álvarez

Pharmacokinetics and Pharmaceutical Technology Area. Miguel Hernandez University

J. Nilsen Department of Immunology, Centre for Immune Regulation (CIR), Oslo University Hospital Rikshospitalet and University of Oslo, Oslo, Norway

J.T. Andersen Department of Immunology, Centre for Immune Regulation (CIR), Oslo University Hospital Rikshospitalet and University of Oslo, Oslo, Norway

Joana D. Amaral Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon

José A.Lopez-Guerrero

Fundación Instituto Valenciano de Oncología, Valencia, Spain

José Bessa i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal

José das Neves i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal

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Juan Cunarro Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela, Spain

Juan M. Irache Department of Chemistry and Pharmaceutical Technology, University of Navarra

Juan Pellico Nanomedicine and Radiochemistry Group, National Centre for Cardiovascular Research Carlos III (CNIC) and CIBERES, Madrid, Spain.

Julie Movellan Polymer Therapeutics Lab, Centro de investigación Príncipe Felipe (CIPF), Valencia, Spain

M. Manuela Gaspar

Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon

Manuel Mazo Department of Chemistry and Pharmaceutical Technology, University of Navarra

Marcos Garcia-Fuentes

Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela, Spain

María Blanco-Prieto

Department of Chemistry and Pharmaceutical Technology, University of Navarra

Maria de Jesus Perry

Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon

María de la Fuente

Nano-Oncology Unit, Translational Medical Oncology Group (ONCOMET). Health Research Institute of Santiago de Compostela (IDIS) and CIBERONC, Santiago de Compostela, Spain

María García-Flores

Fundación Instituto Valenciano de Oncología, Valencia, Spain

María J. Vicent Polymer Therapeutics Lab, Centro de investigación Príncipe Felipe (CIPF), Valencia, Spain

María José Alonso

Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Marival Bermejo Pharmacokinetics and Pharmaceutical Technology Area. Miguel Hernandez University

Marta González-Álvarez

Pharmacokinetics and Pharmaceutical Technology Area. Miguel Hernandez University

Miguel A. Ramos-Docampo

Magnetic Materials Group, University of Vigo, Vigo, Spain.

Mohammad-Ali Shahbazi

Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, FI-00014, Helsinki, Finland

Natália Bernardi Faculty of Pharmacy, University of Coimbra

Noemi Csaba Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela, Spain

Olga Borges Faculty of Pharmacy, University of Coimbra

Pedro Granja i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal

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R. Nunes i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal

Rafael López-López

Nano-Oncology Unit, Translational Medical Oncology Group (ONCOMET). Health Research Institute of Santiago de Compostela (IDIS) and CIBERONC, Santiago de Compostela, Spain

Ramón Eritja Nucleic Acids Chemistry Group, Institute of Advanced Chemistry of Catalonia (IQAC – CSIC) and CIBER-BBN, Barcelona, Spain.

Ramón Iglesias-Rey

Clinical Neurosciences Research Laboratory, Health Research Institute of Santiago de Compostela (IDIS), Santiago de Compostela, Spain.

Rebeca Peñalva Department of Chemistry and Pharmaceutical Technology, University of Navarra

Ruben Varela Department of Biochemistry and Molecular Biology, University of Santiago de Compostela, Spain

Rubén Varela-Calviño

Department of Biochemistry, School of Pharmacy, Universidade de Santiago de Compostela, Spain

Sandra Jesus Faculty of Pharmacy and Center for Neurosciences and Cell Biology, University of Coimbra

Santiago Grijalvo Nucleic Acids Chemistry Group, Institute of Advanced Chemistry of Catalonia (IQAC – CSIC) and CIBER-BBN, Barcelona, Spain.

Sonia Vicente-Ruiz

Polymer Therapeutics Lab, Centro de investigación Príncipe Felipe (CIPF), Valencia, Spain

Sulay Tovar Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela, Spain

Ting-Yu Shih John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA

Verónica Salgueiriño

Magnetic Materials Group, University of Vigo, Vigo, Spain.

Victor Puntes Institut Català de Nanociència i Nanotecnologia (ICN2), CSIC (Barelona)

Virginia Merino Pharmacy and Pharmaceutical Technology. Valencia University, Valencia, Valencia, 46100, Spain.

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List of participants

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Name Affiliation

Abi Judit Vasquez Nano-Oncology Unit, Translational Medical Oncology Group (ONCOMET). Health Research Institute of Santiago de Compostela (IDIS) and CIBERONC, Santiago de Compostela, Spain

Alana Duarte Center for Neuroscience and Cell Biology, University of Coimbra, Portugal

Ana Baião University of Algarve

Ana Igea University of Vigo

Ana Macedo CBIOS-Research Center for Biosciences and Health Technologies, Lusófona University, Lisbon, Portugal

Ana Maria Lopez University of Santiago de Compostela

Ana Melero University of Valencia

Ana Oliveira Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Ana Topete Centro de Química Estrutural, Instituto Superior Técnico, University of Lisbon, Lisbon, Portugal

Andreia Almeida I3S – Institute for Research and Innovation in Health, University of Porto, Portugal

Anna Abadessa Department of Pharmaceutics, Utrecht University, Utrecht, The Netherlands

Antonio José Guillot Department of Drugs and Medicines, São Paulo State University (UNESP), School pf Pharmaceutical Sciences, Araraquara, Brasil

Bárbara Sánchez Pharmacokinetics and Pharmaceutical Technology Area. Miguel Hernandez University

Bruno Sarmento University of Porto

Carla Garcia Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Carlos Rodriguez Department of Chemistry and Pharmaceutical Technology, University of Navarra

Carmen Fernández Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Carmen

Remuñan-LópezUniversity of Santiago de Compostela

Claudia Azevedo i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal;

Claudia Martins i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal;

Ana Olivera Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Carlos Garcia-Gonzalez

University of Santiago de Compostela

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David Panao University of Coimbra

Diana Patricia Gaspar

University of Lisbon

Diego Pan Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Dolores Torres University of Santiago de Compostela

Edna Soares University of Coimbra

Elisa Garbayo University of Navarra

Fátima Fernández Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada

Filipa Fonseca i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal;

Flavia Sousa i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto

Gracia Garcia Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada

Helena Florindo University of Lisbon

Iago Fernández Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Jacinta Pinho Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon

Jessica Da Silva Faculty of Pharmacy, University of Coimbra

Joao Campos UCIBIO, REQUIMTE, Laboratory of Pharmaceutical Technology, Department of Drug Sciences, Faculty of Pharmacy, University of Porto, Portugal

João F. Mano Department of Chemistry, CICECO, University of Aveiro, Aveiro, Portugal

Jorge Morales Department of Chemistry and Pharmaceutical Technology, University of Navarra

José das Neves INEB-Instituto Nacional de Engenharia Biomédica

Katia Maso Polymer Therapeutics Lab, Centro de investigación Príncipe Felipe (CIPF), Valencia, Spain

Laura Saludas Department of Chemistry and Pharmaceutical Technology, University of Navarra

Magda Ferreira Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon

Manoli Igartua University of the Basque Country

Manuel Santander University of Castilla-La Mancha

Maria Blanco University of Navarra

Maria Helena Macedo

i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal;

Cristian Reboredo Department of Chemistry and Pharmaceutical Technology, University of Navarra

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María Jesús Vicent CIPF-Centro de Investigación Príncipe Felipe

Maria Jose Alonso University of Santiago de Compostela

Maria Plaza Cellular Neurobiology and Molecular Chemistry of the Central Nervous System group. Faculty of Pharmacy, University of Castilla-La Mancha

Mariana Colaço Faculty of Pharmacy, University of Coimbra

Marta Vives-Pi Germans Trias i Pujol Research Institute (IGTP), Autonoma University, Barcelona

Maruthi Prasanna Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela, Spain

Matilde Duran Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela, Spain

Mireya Lopez Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela, Spain

Natalia Bernardi Faculty of Pharmacy, University of Coimbra

Noemi Villaseca Cellular Neurobiology and Molecular Chemistry of the Central Nervous System group. Faculty of Pharmacy, University of Castilla-La Mancha

Patricia Filipe CBIOS-Research Center for Biosciences and Health Technologies, Lusófona University, Lisbon, Portugal

Raneem Jatal University of Angers

Rosa Hernández University of the Basque Country

Rute Nunes I3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal

Sainza Lores Nano-Oncology Unit, Translational Medical Oncology Group (ONCOMET). Health Research Institute of Santiago de Compostela (IDIS) and CIBERONC, Santiago de Compostela, Spain

Sakthikumar Ragupathy

Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Sandra Alijas Fundación Jimenez Diaz

Sandra Diez Nano-Oncology Unit, Translational Medical Oncology Group (ONCOMET). Health Research Institute of Santiago de Compostela (IDIS) and CIBERONC, Santiago de Compostela, Spain

Mariana Landin University of Santiago de Compostela

Patricia Díaz University of Santiago de Compostela

María Luisa Juanes University of Santiago de Compostela

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2nd SPLC-CRS Young Scientists Meeting, January 23rd, 2019

Santiago de Compostela, Spain

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Sheila Barrios Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Silvia Fuerte Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada

Silvia Mir Department of Drugs and Medicines, Faculty of Pharmaceutical Sciences, Universidade Estadual Paulista (UNESP).

Sofia Mendes Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Surasa Nagachinta University of Santiago de Compostela

Tamara Gomez Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, Spain

Tomas Ramos Instituto de Investigação e Inovação em Saúde (I3S), University of Porto, Rua Alfredo Allen, 208, 4200-135, Porto, Portugal

Sandra Jesus Faculty of Pharmacy & Center for Neurosciences and Cell Biology University of Coimbra

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2nd SPLC-CRS Young Scientists Meeting, January 23rd, 2019

Santiago de Compostela, Spain

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Thank you for your participation.

School of Medicine of University of Santiago de Compostela Rúa de San Francisco s/n