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- Gene delivery using dendrimer/pDNA complexes immobilized in electrospun fibers using the Layer-by-Layer techniquePublication . Ramalingam, Kirthiga; Castro, Rita; Pires, Pedro; Shi, Xiangyang; Rodrigues, João; Xiao, Shili; Tomás, HelenaA gene delivery platform for potential use in tissue engineering applications was developed by surface functionalization of biodegradable electrospun poly(lactic-co-glycolic acid) (PLGA) fibers with nanolayers of chitosan (cationic polymer) and alginate (anionic polymer) using the Layer-by-Layer (LbL) technique. The developed system not only supported the attachment and growth of human Mesenchymal Stem Cells (hMSCs), but also was capable of delivering pDNA/dendrimer complexes and inducing cell differentiation towards the osteogenic lineage when a pDNA codifying for human Bone Morphogenetic Protein-2 (BMP-2) was used. Beyond providing a means for pDNA/dendrimer complex immobilization, the polyelectrolyte coating conferred sustained release properties to the scaffold that resulted in pDNA protection from degradation. The polyelectrolyte coating, by itself, also contributed to enhance cell differentiation.
- Poly(alkylidenimine) Dendrimers Functionalized with the Organometallic Moiety [Ru(η5-C5H5)(PPh3)2]+ as Promising Drugs Against Cisplatin-Resistant Cancer Cells and Human Mesenchymal Stem CellsPublication . Gouveia, Marisol; Figueira, João; Jardim, Manuel G.; Castro, Rita; Tomás, Helena; Rissanen, Kari; Rodrigues, JoãoHere and for the first time, we show that the organometallic compound [Ru(η5-C5H5)(PPh3)2Cl] (RuCp) has potential to be used as a metallodrug in anticancer therapy, and further present a new approach for the cellular delivery of the [Ru(η5-C5H5)(PPh3)2]+ fragment via coordination on the periphery of low-generation poly(alkylidenimine) dendrimers through nitrile terminal groups. Importantly, both the RuCp and the dendrimers functionalized with [Ru(η5-C5H5)(PPh3)2]+ fragments present remarkable toxicity towards a wide set of cancer cells (Caco-2, MCF-7, CAL-72, and A2780 cells), including cisplatin-resistant human ovarian carcinoma celllines(A2780cisRcells). Also,RuCpandthepreparedmetallodendrimersareactiveagainsthuman mesenchymal stem cells (hMSCs), which are often found in the tumor microenvironment where they seem to play a role in tumor progression and drug resistance.
- Fine tuning of the pH-sensitivity of laponite–doxorubicin nanohybrids by polyelectrolyte multilayer coatingPublication . Xiao, Shili; Castro, Rita; Maciel, Dina; Gonçalves, Mara; Shi, Xiangyang; Rodrigues, João; Tomás, HelenaDespite the wide research done in the field, the development of advanced drug delivery systems with improved drug delivery properties and effective anticancer capability still remains a great challenge. Based on previous work that showed the potentialities of the nanoclay Laponite as a pH-sensitive doxorubicin (Dox) delivery vehicle, herein we report a simple method to modulate its extent of drug release at different pH values. This was achieved by alternate deposition of cationic poly(allylamine) hydrochloride and anionic poly(sodium styrene sulfonate) (PAH/PSS) polyelectrolytes over the surface of Dox-loaded Laponite nanoparticles using the electrostatic layer-by-layer (LbL) self-assembly approach. The successful formation of polyelectrolyte multilayer-coated Dox/Laponite systems was confirmed by Dynamic Light Scattering and zeta potential measurements. Systematic studies were performed to evaluate their drug release profiles and anticancer efficiency. Our results showed that the presence of the polyelectrolyte multilayers improved the sustained release properties of Laponite and allowed a fine tuning of the extension of drug release at neutral and acidic pH values. The cytotoxicity presented by polyelectrolyte multilayer-coated Dox/Laponite systems towards MCF-7 cells was in accordance with the drug delivery profiles. Furthermore, cellular uptake studies revealed that polyelectrolyte multilayer-coated Dox/Laponite nanoparticles can be effectively internalized by cells conducting to Dox accumulation in cell nucleus.
- Homocysteine metabolism in children and adolescents: influence of age on plasma biomarkers and correspondent genotype interactionsPublication . Araújo, Helena Caldeira; Ramos, Ruben; Florindo, Cristina; Rivera, Isabel; Castro, Rita; Almeida, Isabel Tavares deBackground: Imbalance of homocysteine (Hcy) metabolism links with several pathologies; nevertheless, it is poorly characterized in pediatric populations. This study investigated the impact of age on plasma concentrations of Hcy and relevant biomarkers along with correspondent genotype interactions. Methods: A healthy pediatric cohort aged 9 (n = 195) and 17 (n = 128) years old (yo) was studied. Immunoassays and GC-MS-SIM-mode quantified plasma levels of Hcy and biomarkers. PCR-RFLP or quantitative-PCR assays assessed common variations in related genes. Results: Age impacted on levels of Hcy and metabolic markers: older children presented with the lowest folates and total-cobalamin (tCbl), while with the highest Hcy concentrations, whereas methylmalonic acid (MMA) and holotranscobalamin (Holo-TC) levels remained similar in 9-yo and 17-yo children. The relationships between B-vitamins and metabolic markers were also dependent on age. Only in the older children, MMA correlated with tCbl and Holo-TC, and MMA levels were markedly higher in the 17-yo subjects presenting with the lowest quartiles of Holo-TC concentrations. Lastly, age also impacted on the correlations between genotype and biomarkers. In the 17-yo group, however not in the 9-yo children, tHcy differed between MTHFR 677 genotypes, with subjects who had the MTHFR 677TT genotype displaying the highest tHcy concentrations. Conclusions: Age impacts on the Hcy metabolism dynamics in a pediatric population.
- The effect of PAMAM dendrimers on mesenchymal stem cell viability and differentiationPublication . Gonçalves, M.; Castro, R.; Rodrigues, J.; Tomás, H.Stem cells and nanomaterials are two new and exciting fields of science that are evolving very fast and that are starting to establish ties. Nanomaterials should, however, be designed to interact with stem cells without compromising their biological characteristics, in other words, without affecting their viability and differentiation potential. In the present report and for the first time, the effects of poly(amidoamine) (PAMAM) dendrimers on the viability and differentiation ability towards the osteogenic and adipogenic lineages of human mesenchymal stem cells (hMSCs) are systematically evaluated. Studies were done as a function of the cell culture media composition and PAMAM dendrimer surface functionalization, generation, and concentration. hMSCs were exposed to amino and hydroxyl (generations 2, 4 and 6), and carboxylate (generations 1.5, 3.5 and 5.5) functionalized dendrimers, at two different concentrations (10 μg/mL and 0.5 μg/mL), for a period of 21 days. Overall, the results revealed that amino functionalized dendrimers can be severely cytotoxic, the extension of cell death being dependent on the concentration of amino groups in solution. However, in all cases, the differentiation of hMSCs towards the osteogenic and adipogenic phenotypes seems not to be affected as demonstrated by staining in in vitro cultures.
- PAMAM dendrimer/pDNA functionalized-magnetic iron oxide nanoparticles for gene deliveryPublication . Xiao, Shili; Castro, Rita; Rodrigues, João; Shi, Xiangyang; Tomás, HelenaHerein, we report an easy and ingenious method to functionalize magnetic iron oxide nanoparticles (MNPs) with plasmid DNA (pDNA) to obtain nanohybrid systems suitable for nucleic acid therapy. The nanohybrids were prepared by combining complexes of dendrimers and pDNA (dendriplexes) and poly(styrene) sulfonate-coated MNPs through electrostatic interactions. The effects of the dendrimer generation (generations 2, 4 and 6) and the amine to phosphate group (N/P) ratio on the hydrodynamic diameter, zeta potential, cell viability, cellular internalization and transfection efficiency of the nanohybridswere systematically investigated at different transfection conditions (including incubation time, pDNA concentration, presence or absence of an external magnetic field, and presence or absence of fetal bovine serum). The results confirmed that the nanohybrids were able to transfect NIH 3T3 cells, and that the level of gene expression (the luciferase protein reporter gene was used) was strongly dependent on the dendrimer generation, the N/P ratio, and the pDNA concentration. The best system was based on dendriplex-coated MNPs formed by generation 6 dendrimers at an N/P ratio of 10 that, at optimized conditions, led to a gene expression level which was not significantly different from that obtained only using dendriplexes. In summary, a coherent set of results was reached indicating the potential of the developed nanohybrids as effective gene delivery nanomaterials.
- Novos vectores baseados em dendrímeros com aplicações em terapia antisensePublication . Castro, Rita Maria de; Tomás, Helena Maria Pires Gaspar; Rodrigues, João Manuel CunhaA terapia genética tem se revelado uma ferramenta potente na Medicina, na tentativa de revolucionar o tratamento de várias doenças hereditárias e adquiridas. A introdução de genes em células pretende a expressão estável e prolongada de proteínas com efeitos terapêuticos. O silenciamento de genes, através da terapia genética que faz uso de oligonucleótidos antisense, pequenos RNA de interferência (siRNA) ou ribozimas, visa o decréscimo ou anulação do funcionamento de um gene cuja expressão amplificada, por algum motivo, leva ao desenvolvimento de umapatologia. A internalização de material genético nas células, usualmente, carece de métodos e/ou sistemas de entrega (vectores). Estes podem pertencer a duas categorias, designadamente, métodos virais e métodos não-virais. O primeiro é considerado o mais eficiente, apresentando porém, sérias desvantagens como o risco de carcinogénese. A solução é a utilização de métodos não virais,que podem ser físicos ou químicos. O objectivo principal desta dissertação foi a utilização de dendrímeros para o silenciamento do gene da proteína fluorescente optimizada (EGFP) em células HeLa, previamente modificadas para expressarem esta proteína. Dendrímeros poli(amidoamina) geração 5 (PAMAM G5) modificados com 4 ou 8 moléculas de ácidos gordos de diferentes comprimentos foram complexados com oligonucleótidos antisense. A vantagem que estes apresentam em relação aos dendrímeros nativos é que são capazes de interagir com os lípidos da membrana celular, esperando-se, por isso, uma melhor eficiência de transfecção e efeitos antisense. Isto foi efectivamente verificado, sendo que o nível de silenciamento do gene da EGFP obtido, está directamente relacionado com o aumento da razão NP, o número e o comprimento das cadeias hidrofóbicas. O silencimento de genes tem sofrido grandes avanços, havendo actualmente uma série de ensaios clínicos para a sua utilização no tratamento de doenças como cancros de origem hereditária ou viral, prevendo-se que venha para ficar, juntamente com o silenciamento mediado por siRNA.
- Insight into the role of N,N-dimethylaminoethyl methacrylate (DMAEMA) conjugation onto poly(ethylenimine): cell viability and gene transfection studiesPublication . Nouri, Alireza; Castro, Rita; Kairys, Visvaldas; Santos, José L.; Rodrigues, João; Li, Yulin; Tomás, HelenaIn the present study, the effect of N,N-dimethylaminoethyl methacrylate (DMAEMA) conjugation onto branched poly(ethylenimine) (PEI) with different grafting degree was examined for gene delivery applications. The DMAEMA-grafted-PEI conjugates were characterized and complexed with plasmid DNA (pDNA) at various concentrations, and the physicochemical properties, cell viability, and in vitro transfection efficiency of the complexes were evaluated in HEK 293T cells. Computational techniques were used to analyze the interaction energies and possible binding modes between DNA and conjugates at different grafting degrees. The cytotoxicity analysis and in vitro transfection efficiency of the conjugate/pDNA complexes exhibited a beneficial effect of DMAEMA conjugation when compared to PEI alone. The computational results revealed that the DNA/vector interaction energy decreases with increasing grafting degree, which can be associated to an enhanced release of the pDNA from the carrier once inside cells. The results indicate the significance of DMAEMA conjugation onto PEI as a promising approach for gene delivery applications.
- PAMAM dendrimers: blood-brain barrier transport and neuronal uptake after focal brain ischemiaPublication . Santos, Sofia D.; Xavier, Miguel; Leite, Diana M.; Moreira, Débora A.; Custódio, Beatriz; Torrado, Marília; Castro, Rita; Leiro, Victoria; Rodrigues, João; Tomás, Helena; Pêgo, Ana P.Drug delivery to the central nervous system is restricted by the blood-brain barrier (BBB). However, with the onset of stroke, the BBB becomes leaky, providing a window of opportunity to passively target the brain. Here, cationic poly(amido amine) (PAMAM) dendrimers of different generations were functionalized with poly(ethylene glycol) (PEG) to reduce cytotoxicity and prolong blood circulation half-life, aiming for a safe in vivo drug delivery system in a stroke scenario. Rhodamine B isothiocyanate (RITC) was covalently tethered to the dendrimer backbone and used as a small surrogate drug as well as for tracking purposes. The biocompatibility of PAMAM was markedly increased by PEGylation as a function of dendrimer generation and degree of functionalization. The PEGylated RITC-modified dendrimers did not affect the integrity of an in vitro BBB model. Additionally, the functionalized dendrimers remained safe when in contact with the bEnd.3 cells and rat primary astrocytes composing the in vitro BBB model after hypoxia induced by oxygen-glucose deprivation. Modification with PEG also decreased the interaction and uptake by endothelial cells of PAMAM, indicating that the transport across a leaky BBB due to focal brain ischemia would be facilitated. Next, the functionalized dendrimers were tested in contact with red blood cells showing no haemolysis for the PEGylated PAMAM, in contrast to the unmodified dendrimer. Interestingly, the PEG-modified dendrimers reduced blood clotting, which may be an added beneficial function in the context of stroke. The optimized PAMAM formulation was intravenously administered in mice after inducing permanent focal brain ischemia. Twenty-four hours after administration, dendrimers could be detected in the brain, including in neurons of the ischemic cortex. Our results suggest that the proposed formulation has the potential for becoming a successful delivery vector for therapeutic application to the injured brain after stroke reaching the ischemic neurons.
- DNA/Dendrimer-based films: a novel material with potential biomedical applicationsPublication . Castro, Rita Maria de; Tomás, Helena Maria Pires Gaspar; Pêgo, Ana Paula Gomes MoreiraNesta tese de doutoramento, foram desenvolvidos novos materiais híbridos, sob a forma de filmes finos, essencialmente baseados em interações eletrostáticas estabelecidas entre moléculas de ácido desoxirribonucleico (ADN) e dendrímeros de poli(amidoamina) (PAMAM). Os dendrímeros PAMAM são moléculas à escala nanométrica que apresentam multi-valência e um baixo índice de polidispersão. Esta classe de moléculas tem sido muito estudada para a entrega de genes e fármacos em células, para além de muitas outras aplicações na área biomédica. Isto apenas é possível devido às suas propriedades físicas, químicas e estruturais, que permitem a interação eletrostática com ácidos nucleicos e conjugação à superfície e/ou encapsulamento de fármacos no seu interior. Neste trabalho, foi tirado proveito da capacidade destas moléculas interagirem com o ADN para obter novos materiais com aplicações biomédicas promissoras.