Combining hyaluronic acid with chitosan enhances gene delivery

The low gene transfer efficiency of chitosan-DNA polyplexes is a consequence of their high stability and consequent slow DNA release. The incorporation of an anionic polymer is believed to loosen chitosan interactions with DNA and thus promote higher transfection efficiencies. In this work, several formulations of chitosan-DNA polyplexes incorporating hyaluronic acid were prepared and characterized for their gene transfection efficiency on both HEK293 and retinal pigment epithelial cells. The different polyplex formulations showed morphology, size, and charge compatible with a role in gene delivery. The incorporation of hyaluronic acid rendered the formulations less stable, as was the goal, but it did not affect the loading and protection of the DNA. Compared with chitosan alone, the transfection efficiency had a 4-fold improvement, which was attributed to the presence of hyaluronic acid. Overall, our hybrid chitosan-hyaluronic acid polyplexes showed a significant improvement of the efficiency of chitosan-based nonviral vectors in vitro, suggesting that this strategy can further improve the transfection efficiency of nonviral vectors.Fundacao para a Ciencia e Tecnologia [SFRH/BD/52424/2013]; Marie Curie Reintegration Grant [PIRG-GA-2009-249314

Chitosan-Hyaluronic acid hybrid vectors for retinal gene therapy

info:eu-repo/semantics/publishedVersio

Synthesis and evaluation of polymer nanoparticles for delivery in RPE cells

Ocular pathologies are among the most debilitating medical conditions affecting all segments of the population. Traditional treatment options are often ineffective, and gene therapy has the potential to become an alternative approach for the treatment of several pathologies. Methacrylate polymers have been described as highly biocompatible and are successfully used in medical applications. Due to their cationic nature, these polymers can be used to form polyplexes with DNA for its delivery. This work aims to study the potential of PDMAEMA (poly(2-(N,N’-dimethylamino)ethyl methacrylate)) as a non viral gene delivery system to the retina. The first part of this work aimed to study the potential for gene delivery of a previously synthesized PDMAEMA polymer of high molecular weight (354kDa). In the second part, we synthesized by RAFT a PDMAEMA with a lower molecular weight (103.3kDa) and similarly, evaluated its ability to act as a gene delivery vehicle. PDMAEMA/DNA polyplexes were prepared at 5, 7.5, 10, 12.5 and 20 nitrogen/phosphorous (N/P) ratio for the 354kDa PDMAEMA and at 5 and 7.5 for the 103.3kDa PDMAEMA. Dynamic light scattering and zeta potential measurements confirmed the nanosize and positive charge of polyplexes for all ratios and for both polymers. Both high and low Mw PDMAEMA were able to efficiently complex and protect DNA from DNase I degradation. Their cytotoxicity was evaluated using a non-retinal cell line (HEK293) and a retinal pigment epithelium (RPE) cell line (D407). We have found that cytotoxicity of the free polymer is concentration and time dependent, as expected, and negligible for all the concentrations of the PDMAEMA-DNA polyplexes. Furthermore, for the concentrations to be used in vivo, the 354kDa PDMAEMA showed no signs of inflammation upon injection in the intravitreal space of C57BL/6 mice. The transfection efficiency, as evaluated by fluorescence microscopy and flow cytometry, showed that the D407 retinal cells were transfected by polyplexes of both high and low Mw PDMAEMA, but with varied efficiency, which was dependent on the N/P ratio. Althogether, these results suggest that PDMAEMA is a feasible candidate for non-viral gene delivery to the retina, and this work constitutes the basis of further studies to elucidate the bottleneck in transfection and further optimization of the material.Desde o século passado que as doenças relacionadas com o genoma têm vindo a ganhar importância. O projecto de mapeamento do genoma humano permitiu descobrir a origem de muitas doenças. Várias abordagens terapêuticas têm sido objecto de estudo, sendo a terapia génica uma das mais promissoras. A terapia génica tem como objectivo usar material genético, em regra DNA, para manipular as células dos pacientes. Antes do aparecimento da terapia génica existia apenas tratamento dos sintomas ou terapia de substituição para várias patologias, e não uma verdadeira cura. Para as doenças oculares, que estão entre as doenças mais debilitantes que afectam todos os segmentos da população, esta realidade era sobretudo relevante. Em terapia génica existe a necessidade de desenvolver veículos para transporte de material genético, veículo este que tem que proteger o ácido nucleico de degradação, ter especificidade para células/órgão, permitir a expressão do gene de interesse ao longo do tempo e com níveis adequados, não desencadear resposta imunitária (excepto em caso de interesse como no cancro e vacinas) e que seja administrado por injecção sistémica. As duas principais estratégias para entrega de material genético usa vectores virais e não-virais. Os sistemas virais de entrega apresentam diversas vantagens, mas também várias limitações: imunogenicidade, baixa capacidade de empacotamento do material genético, potential inserção aleatória no genoma hospedeiro e toxicidade. Os vectores não virais, de entre os quais se destacam os polímeros, surgiram como uma alternativa para contornar esses problemas, pois estes apresentam maiores benefícios em termos de segurança, versatibilidade química e estrutural para a manipulação das propriedades fisico-químicas, maior capacidade de empacotamento de genes e estabilidade durante o armazenamento. Dentro da classe dos polímeros, os polímeros naturais como o quitosano e alginato têm ganho importância devido à sua biocompatibilidade, mas são os polímeros sintéticos, como a poli(etilenimina) (PEI), a poli(L-lisina) (PLL) e o poli(2-dimetilamino)etil metacrilato (PDMAEMA), que tem maior relevância devido ao controlo preciso da sua síntese e consequentemente das suas propriedades. A natureza catiónica de alguns polímeros, como os indicados acima, permite que estes sejam usados para formar poliplexos com material genético. O objectivo do nosso grupo de investigação é o desenvolvimento de novas estratégias baseadas em terapia génica para tratamento de patologias da retina. Dentro deste enquadramento, o presente trabalho tem como objectivo compreender o potencial do poli(2-dimetilamino)etil metacrilato (PDMAEMA) como sistema de entrega de material genético na retina. Na primeira parte deste trabalho um polímero PDMAEMA com 354kDa, que tinha sido previamente sintetizado, foi avaliado para o seu potencial como veículo para terapia génica. Na segunda parte, sintetizamos um PDMAEMA com um menor peso molecular (103.3kDa) via RAFT (Reversible addition-fragmentation chain-transfer) para efectuar uma comparação entre a eficiência de transfecção de PDMAEMAs com diferentes pesos moleculares. Poliplexos de PDMAEMA/DNA foram preparados nos rácios amina(N)/fosfato(P) de 5, 7.5, 10, 12.5 e 20 com o PDMAEMA 354kDa e nos rácios 5 e 7.5 com o PDMAEMA 103.3kDa. Medições de DLS (Dynamic light scattering) e potencial zeta confirmam que foram preparados poliplexos à escala nanométrica com carga positiva, em todos os rácios. Ambos os polímeros PDMAEMA são capazes de complexar eficientemente e proteger o DNA da degradação por parte da DNAse I. A citoxicidade de ambos os polímeros foi avaliada usando duas linhas celulares: a HEK293 (linha celular de rim embriónico humano) e a D407 (linha celular de epitélio pigmentar da retina). Os resultados revelam que a citoxicidade do polimero livre (não-complexado) é dependente da concentração e do tempo de exposição, como esperado, e os poliplexos PDMAEMA/DNA não apresentam citoxicidade. Foi também observado que nas concentrações usadas in vivo a citoxicidade do PDMAEMA 354kDa é negligível, como demonstrado pela ausência de inflamação após a injecção no espaço intravítreo de ratinhos C57BL6. A eficiência de transfecção foi avaliada qualitativamente por microscopia de fluorescência e quantitativamente por citometria de fluxo e mostrou que os poliplexos PDMAEMA/DNA são capazes de transfectar, e que a eficiência varia de acordo com o rácio N/P utilizado, para os polimeros de alto e baixo peso molecular. Em suma, estes resultados sugerem que o PDMAEMA é um candidato viável para a entrega de genes na retina, e este trabalho constitui a base de estudos futuros que visam elucidar o passo limitante na transfecção e desse modo permitir a optimização deste vector não-viral

Oxidative stress modulates the expression of VEGF isoforms in the diabetic retina

Funding: This work was supported by the Portuguese Foundation for Science and Technology (FCT) with individual grants to S. Simão (SFRH/ BPD/78404/2011), D. Bitoque (SFRH/BD/52424/2013), S. Calado (SFRH/BD/76873/2011), GA Silva (EXPL-BIM-MEC-1433-2013, PIRG05-GA-2009-249314–EyeSee). FCT Research Center Grant UID/ BIM/04773/2013 CBMR 1334.publishersversionpublishe

Age-Related Changes of the Synucleins Profile in the Mouse Retina

OK Funding Information: This research was funded by Fundação para a Ciência e a Tecnologia, grant number PTDC/MED-PAT/29656/2017. L.S. was funded by FCT PhD studentship (SFRH/BD/144743). Additionally, it was supported by iNOVA4Health—UIDB/04462/2020 and UIDP/04462/2020 and by the Associated Laboratory LS4FUTURE (LA/P/0087/2020), two programs financially supported by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior. This work was developed with the support from the research infrastructure Congento, co-financed by Lisboa Regional Operational Programme (Lisboa2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and Fundação para a Ciência e Tecnologia (Portugal) under the project LISBOA-01-0145-FEDER-022170. Publisher Copyright: © 2023 by the authors.Alpha-synuclein (aSyn) plays a central role in Parkinson’s disease (PD) and has been extensively studied in the brain. This protein is part of the synuclein family, which is also composed of beta-synuclein (bSyn) and gamma-synuclein (gSyn). In addition to its neurotoxic role, synucleins have important functions in the nervous system, modulating synaptic transmission. Synucleins are expressed in the retina, but they have been poorly characterized. However, there is evidence that they are important for visual function and that they can play a role in retinal degeneration. This study aimed to profile synucleins in the retina of naturally aged mice and to correlate their patterns with specific retinal cells. With aging, we observed a decrease in the thickness of specific retinal layers, accompanied by an increase in glial reactivity. Moreover, the aSyn levels decreased, whereas bSyn increased with aging. The colocalization of both proteins was decreased in the inner plexiform layer (IPL) of the aged retina. gSyn presented an age-related decrease at the inner nuclear layer but was not significantly changed in the ganglion cell layer. The synaptic marker synaptophysin was shown to be preferentially colocalized with aSyn in the IPL with aging. At the same time, aSyn was found to exist at the presynaptic endings of bipolar cells and was affected by aging. Overall, this study suggests that physiological aging can be responsible for changes in the retinal tissue, implicating functional alterations that could affect synuclein family function.publishersversionpublishe

Graphene Oxide Thin Films with Drug Delivery Function

Funding Information: Funding: Funding was obtained through the Fundação para a Ciência e Tecnologia (FCT) through the projects iNOVA4Health, Translational Medicine program—UIDB/Multi/04462/2020; UIDB/50008/2020 and PTDC/CTM-REF/2679/2020.Graphene oxide has been used in different fields of nanomedicine as a manager of drug delivery due to its inherent physical and chemical properties that allow its use in thin films with biomedical applications. Several studies demonstrated its efficacy in the control of the amount and the timely delivery of drugs when it is incorporated in multilayer films. It has been demonstrated that oxide graphene layers are able to work as drug delivery or just to delay consecutive drug dosage, allowing the operation of time-controlled systems. This review presents the latest research developments of biomedical applications using graphene oxide as the main component of a drug delivery system, with focus on the production and characterization of films, in vitro and in vivo assays, main applications of graphene oxide biomedical devices, and its biocompatibility properties.publishersversionpublishe

Human-derived NLS enhance the gene transfer efficiency of chitosan

Nuclear import is considered as one of the major limitations for non-viral gene delivery systems and the incorporation of nuclear localization signals (NLS) that mediate nuclear intake can be used as a strategy to enhance internalization of exogenous DNA. In this work, human-derived endogenous NLS peptides based on insulin growth factor binding proteins (IGFBP), namely IGFBP-3 and IGFBP-5, were tested for their ability to improve nuclear translocation of genetic material by non-viral vectors. Several strategies were tested to determine their effect on chitosan mediated transfection efficiency: co-administration with polyplexes, co-complexation at the time of polyplex formation, and covalent ligation to chitosan. Our results show that co-complexation and covalent ligation of the NLS peptide derived from IGFBP-3 to chitosan polyplexes yields a 2-fold increase in transfection efficiency, which was not observed for NLS peptide derived from IGFBP-5. These results indicate that the integration of IGFBP-NLS-3 peptides into polyplexes has potential as a strategy to enhance the efficiency of non-viral vectors.FCT: PTDC/BTM/ORG/28121/2017; PD/BD/52424/2013; SFRH/BD/76873/2011;PIRG-GA-2009-249314info:eu-repo/semantics/publishedVersio

Biopolymeric Coatings for Local Release of Therapeutics from Biomedical Implants

Funding Information: S.T., B.M., and J.C. contributed equally to this work. The authors are grateful for funding received from the Australian Research Council Centre of Excellence program (Project Number CE 140100012). J.C. acknowledges the European Research Council Starting Grant (ERC‐StG‐2019‐848325). S.N. and F.D. acknowledge the financial support of Australian Research Council through DP200102164. Publisher Copyright: © 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.The deployment of structures that enable localized release of bioactive molecules can result in more efficacious treatment of disease and better integration of implantable bionic devices. The strategic design of a biopolymeric coating can be used to engineer the optimal release profile depending on the task at hand. As illustrative examples, here advances in delivery of drugs from bone, brain, ocular, and cardiovascular implants are reviewed. These areas are focused to highlight that both hard and soft tissue implants can benefit from controlled localized delivery. The composition of biopolymers used to achieve appropriate delivery to the selected tissue types, and their corresponding outcomes are brought to the fore. To conclude, key factors in designing drug-loaded biopolymeric coatings for biomedical implants are highlighted.publishersversionepub_ahead_of_prin

Cationic polyene phospholipids as DNA carriers for ocular gene therapy

Recent success in the treatment of congenital blindness demonstrates the potential of ocular gene therapy as a therapeutic approach. The eye is a good target due to its small size, minimal diffusion of therapeutic agent to the systemic circulation, and low immune and inflammatory responses. Currently, most approaches are based on viral vectors, but efforts continue towards the synthesis and evaluation of new nonviral carriers to improve nucleic acid delivery. Our objective is to evaluate the efficiency of novel cationic retinoic and carotenoic glycol phospholipids, designated C20-18, C20-20, and C30-20, to deliver DNA to human retinal pigmented epithelium (RPE) cells. Liposomes were produced by solvent evaporation of ethanolic mixtures of the polyene compounds and coformulated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol (Chol). Addition of DNA to the liposomes formed lipoplexes, which were characterized for binding, size, biocompatibility, and transgene efficiency. Lipoplex formulations of suitable size and biocompatibility were assayed for DNA delivery, both qualitatively and quantitatively, using RPE cells and a GFP-encoding plasmid. The retinoic lipoplex formulation with DOPE revealed a transfection efficiency comparable to the known lipid references 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC) and GeneJuice. The results demonstrate that cationic polyene phospholipids have potential as DNA carriers for ocular gene therapy.info:eu-repo/semantics/publishedVersio

Understanding the potential of pdmaema-based vectors for gene therapy of the retina

RESUMO: As patologias oculares estão entre as condições médicas mais debilitantes que afetam todos os segmentos da população. Como as opções tradicionais de tratamento são frequentemente ineficazes e por vezes inexistentes, a terapia génica tem potencial para se tornar uma abordagem alternativa para o tratamento de diversas patologias. Os primeiros veículos desenvolvidos para entregar material genético eram de origem viral. Devido a algumas complicações clínicas iniciais, começaram a ser desenvolvidos os veículos não-virais, baseados em lipídios e polímeros. Os polímeros de metacrilato foram descritos como altamente biocompatíveis e utilizados com sucesso em aplicações médicas, como cimento ósseo e lente intraocular. Alguns destes polímeros possuem propriedades catiónicas que podem ser usadas para formar poliplexos com DNA de forma a proceder à sua entrega. O presente trabalho tem como objetivo estudar o potencial do PDMAEMA (poli(2-dimetilamino)etil metacrilato) como um sistema de administração não viral de genes à retina. Para atingir esse propósito, primeiro tentámos entender o potencial de entrega de genes do PDMAEMA que anteriormente sintetizamos. Para melhorar o PDMAEMA, é fundamental primeiro esclarecer a entrada e o mecanismo intracelular pelo qual o polímero entrega o material genético. Elucidamos o mecanismo de entrada de polyplexes PDMAEMA em células RPE ao determinar que a endocitose mediada por clatrina é a via principal para a absorção de poliplexos PDMAEMA. Em relação ao tráfico intracelular, os poliplexos escapam do sistema degradativo lisossomal 24 horas após incubação com células RPE e acumulam-se ao redor do núcleo. O estudo de dissociação sugere, desta forma, que o polímero conseguiu liberar o DNA nas primeiras 24 horas após a entrada na célula. A segunda parte deste trabalho teve como finalidade avaliar a eficiência de transfeção in vivo do PDMAEMA, usando para tal os ratinhos rd10, um modelo de Retinite Pigmentosa. Os poliplexos PDMAEMA/DNA foram preparados em duas proporções de nitrogênio/fósforo (N/P).Para validar melhor o PDMAEMA como um potencial vetor não viral na retina, injetámos poliplexos PDMAEMA subretinalmente, os quais foram capazes de entrar e promover a expressão de GFP na camada RPE da retina. O passo seguinte foi testar os poliplexos PDMAEMA num modelo de doença. Procedemos a uma caracterização do modelo da doença de Retinite Pigmentosa, dos ratinhos rd10 com marcadores para fotorreceptores: bastonetes (rodopsina e PDE6β) e cones (arrestina do cone) e observámos alterações nos padrões de expressão começando a P18. Também medimos a espessura das camadas nucleares externas (ONL) e internas (INL) e observámos uma diminuição significativa, que começava a P18 e ia até P35. Uma vez que uma mutação “missense” no gene PDE6β leva à morte de fotorreceptores, dando o rd10 o seu fenótipo, clonámos o gene PDE6β no plasmídeo pEPito-hCMV e realizámos injeções subretinais. Observámos que os polyplexos PDMAEMA foram capazes de expressar o gene PDE6β na camada nuclear dos fotorreceptores. Ao caracterizar os murganhos rd10, observamos que a proteína PDE6β não estava a ser detetada por imunofluorescência, o que nos levou a questionar se a proteína estava realmente a ser produzida. Aproveitando a PDE6β clonada no plasmídeo pEPito, realizámos mutagénese direcionada no local, de forma a explorar melhor a observação anterior. Mutámos o gene com a mutação rd10 (c> t, posição 1678) e também o codão STOP. Construímos quatro plasmídeos: pEPito-hCMV-PDE6β (PDE6β normal); pEPito-hCMV-PDE6βR560C (PDE6β mutado); pEPito-hCMV-PDE6βeGFP (PDE6β normal fundido com GFP) e pEPito-hCMV-PDE6βR560CeGFP (PDE6β mutado fundido com GFP). Os nossos resultados sugerem assim que a formação do complexo de PDE6 in vivo com PDE6βR560C mascara o site que o anticorpo reconhece, prevenindo assim a deteção de PDE6β mutada. Uma vez que o mecanismo da doença no ratinho rd10 ainda é pouco compreendido, consideramos que essas ferramentas genéticas poderão ser utilizadas para estudá-lo melhor. No seu conjunto, estes resultados sugerem, portanto, que o PDMAEMA é um candidato viável para a administração de genes não-virais à retina. No entanto, torna-se ainda necessário realizar mais injeções subretinais do nosso vetor não viral, do modo a aumentar o número de repetições e a avaliar a função da retina.ABSTRACT: It is known that an efficient gene therapy vector must overcome several steps to be able to express the gene of interest: (I) enter the cell by crossing the cell membrane; (II) escape the endo-lysosomal degradation pathway; (III) release the genetic material; (IV) traffic through the cytoplasm and enter the nucleus; and last (V), enable gene expression to synthetize the protein of interest. In recent years, we and others have demonstrated the potential of poly(2-(N,N’- dimethylamino)ethylmethacrylate) (PDMAEMA) as a gene therapy vehicle. Further optimization of gene transfer efficiency requires the understanding of the intracellular pathway of PDMAEMA. Therefore the goal of this study was to determine the cellular entry and intracellular trafficking mechanisms of our PDMAEMA vectors and determine the gene transfer bottleneck. For this, we have produced rhodamine-labeled PDMAEMA polyplexes that were used to transfect retinal cells and the cellular localization determined by co-localization with cellular markers. Our vectors quickly and efficiently cross the cell membrane, and escape the endo-lysosomal system by 24h. We have observed the PDMAEMA vectors to concentrate around the nucleus, and the DNA load to be released in the first 24h after transfection. These results allow us to conclude that although the endolysosomal system is an important obstacle, PDMAEMA gene vectors can overcome it. The nuclear membrane, however, constitutes the bottleneck to PDMAEMA gene transfer ability