N-(3-Cyanophenyl)-2-phenylacetamide, an effective inhibitor of morbillivirus-induced membrane fusion with low cytotoxicity

Based on the structural similarity of viral fusion proteins within the family Paramyxoviridae, we tested recently described and newly synthesized acetanilide derivatives for their capacity to inhibit measles virus (MV)-, canine distemper virus (CDV)- and Nipah virus (NiV)-induced membrane fusion. We found that N-(3-cyanophenyl)-2-phenylacetamide (compound 1) has a high capacity to inhibit MV- and CDV-induced (IC(50) muM), but not NiV-induced, membrane fusion. This compound is of outstanding interest because it can be easily synthesized and its cytotoxicity is low [50 % cytotoxic concentration (CC(50)) >/= 300 muM], leading to a CC(50)/IC(50) ratio of approximately 100. In addition, primary human peripheral blood lymphocytes and primary dog brain cell cultures (DBC) also tolerate high concentrations of compound 1. Infection of human PBMC with recombinant wild-type MV is inhibited by an IC(50) of approximately 20 muM. The cell-to-cell spread of recombinant wild-type CDV in persistently infected DBC can be nearly completely inhibited by compound 1 at 50 muM, indicating that the virus spread between brain cells is dependent on the activity of the viral fusion protein. Our findings demonstrate that this compound is a most applicable inhibitor of morbillivirus-induced membrane fusion in tissue culture experiments including highly sensitive primary cells.

Cytotoxicity testing of silver-containing burn treatments using primary and immortal skin cells

A novel burn wound hydrogel dressing has been previously developed which is composed of 2-acrylamido-2-methylpropane sulfonic acid sodium salt with silver nanoparticles (silver AMPS). This study compared the cytotoxicity of this dressing to the commercially available silver products; Acticoat™, PolyMem Silver® and Flamazine™ cream. Human keratinocytes (HaCaT and primary HEK) and normal human fibroblasts (NHF) were exposed to dressings incubated on Nunc™ polycarbonate inserts for 24, 48 and 72h. Four different cytotoxicity assays were performed including; Trypan Blue cell count, MTT, Celltiter-Blue™ and Toluidine Blue surface area assays. The results were expressed as relative cell viability compared to an untreated control. The cytotoxic effects of Acticoat™ and Flamazine™ cream were dependent on exposure time and cell type. After 24h exposure, Acticoat™ and Flamazine™ cream were toxic to all tested cell lines. Surprisingly, HaCaTs treated with Acticoat™ and Flamazine™ had an improved ability to survive at 48 and 72h while HEKs and NHFs had no improvement in survival with any treatment. The novel silver hydrogel and PolyMem Silver® showed low cytotoxicity to all tested cell lines at every time interval and these results support the possibility of using the novel silver hydrogel as a burn wound dressing. Researchers who rely on HaCaT cells as an accurate keratinocyte model should be aware that they can respond differently to primary skin cells.

Effect of biotransformation by liver S9 enzymes on the mutagenicity and cytotoxicity of melanin extracted from Aspergillus nidulans

A mutant that exhibited increased melanin pigment production was isolated from Aspergillus nidulans fungus. This pigment has aroused biotechnological interest due to its photoprotector and antioxidant properties. In a recent study, we showed that melanin from A. nidulans also inhibits NO and TNF-α production. The present study evaluates the mutagenicity and cytotoxicity of melanin extracted from A. nidulans after its exposure to liver S9 enzymes. The cytotoxicity of multiple concentrations of melanin (31.2-500 μg/mL) against the McCoy cell line was evaluated using the Neutral Red assay, after incubation for 24 h. Mutagenicity was assessed using the Ames test with the Salmonella typhimurium strains TA98, TA97a, TA100, and TA102 at concentrations ranging from 125 μg/plate to 1 mg/plate after incubation for 48 h. The cytotoxicity of A. nidulans melanin after incubation with S9 enzymes was less than (CI50 value= 413.4 ± 3.1 μg/mL) that of other toxins, such as cyclophosphamide (CI50 value = 15 ± 1.2 μg/mL), suggesting that even the metabolised pigment does not cause significant damage to cellular components at concentrations up to 100 μg/mL. In addition, melanin did not exhibit mutagenic properties against the TA 97a, TA 98, TA 100, or TA 102 strains of S. typhimurium, as shown by a mutagenic index (MI)  <2 in all assays. The significance of these results supports the use of melanin as a therapeutic reagent because it possesses low cytotoxicity and mutagenic potential, even when processed through an external metabolising system.

Cytotoxicity evaluation of Curcuma zedoaria (Christm.) Roscoe fluid extract used in oral hygiene products

Objective. This in vitro study evaluated the cytotoxic effects of the Curcuma zedoaria (Christm.) Roscoe (popular name: zedoary) fluid extract, as used in preparations for oral hygiene, mostly for anti-septic purposes. Materials and methods. The cell viability and cell growth were assessed by Trypan blue dye exclusion assay using the LMF cell line derived from oral mucosa. Cell viability (short-term assay) was measured 0, 6, 12 and 24 h after contact with the fluid extract. Cell growth (long-term assay) was analyzed in 1, 3, 5 and 7 days. The experimental groups were those testing the fluid extract obtained from the zedoary rhizome and the extractor liquid (ethanol 70 degrees GL) in the concentrations of 0.01-0.0001% v/v. Fresh DMEM were used in the control cultures. Results. Short-term assay-all studied cultures maintained stable cell viability; Long-term assay-there was progressive cell growth in all studied cultures. Conclusion. According to the results, the zedoary fluid extract presents low cytotoxicity and probably can be used in the oral hygiene products.

The evaluation of new and isotopically labeled isoindoline nitroxides and an azaphenalene nitroxide for EPR oximetry

Isoindoline nitroxides are potentially useful probes for viable biological systems, exhibiting low cytotoxicity, moderate rates of biological reduction and favorable Electron Paramagnetic Resonance (EPR) characteristics. We have evaluated the anionic (5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl; CTMIO), cationic (5-(N,N,N-trimethylammonio)-1,1,3,3-tetramethylisoindolin-2-yloxyl iodide, QATMIO) and neutral (1,1,3,3-tetramethylisoindolin-2-yloxyl; TMIO) nitroxides and their isotopically labeled analogs ((2)H(12)- and/or (2)H(12)-(15)N-labeled) as potential EPR oximetry probes. An active ester analogue of CTMIO, designed to localize intracellularly, and the azaphenalene nitroxide 1,1,3,3-tetramethyl-2,3-dihydro-2-azaphenalen-2-yloxyl (TMAO) were also studied. While the EPR spectra of the unlabeled nitroxides exhibit high sensitivity to O(2) concentration, deuteration resulted in a loss of superhyperfine features and a subsequent reduction in O(2) sensitivity. Labeling the nitroxides with (15)N increased the signal intensity and this may be useful in decreasing the detection limits for in vivo measurements. The active ester nitroxide showed approximately 6% intracellular localization and low cytotoxicity. The EPR spectra of TMAO nitroxide indicated an increased rigidity in the nitroxide ring, due to dibenzo-annulation.

Two-Photon Fluorescence Microscopy Imaging of Cellular Oxidative Stress Using Profluorescent Nitroxides

A range of varying chromophore nitroxide free radicals and their nonradical methoxyamine analogues were synthesized and their linear photophysical properties examined. The presence of the proximate free radical masks the chromophore’s usual fluorescence emission, and these species are described as profluorescent. Two nitroxides incorporating anthracene and fluorescein chromophores (compounds 7 and 19, respectively) exhibited two-photon absorption (2PA) cross sections of approximately 400 G.M. when excited at wavelengths greater than 800 nm. Both of these profluorescent nitroxides demonstrated low cytotoxicity toward Chinese hamster ovary (CHO) cells. Imaging colocalization experiments with the commercially available CellROX Deep Red oxidative stress monitor demonstrated good cellular uptake of the nitroxide probes. Sensitivity of the nitroxide probes to H2O2-induced damage was also demonstrated by both one- and two-photon fluorescence microscopy. These profluorescent nitroxide probes are potentially powerful tools for imaging oxidative stress in biological systems, and they essentially “light up” in the presence of certain species generated from oxidative stress. The high ratio of the fluorescence quantum yield between the profluorescent nitroxide species and their nonradical adducts provides the sensitivity required for measuring a range of cellular redox environments. Furthermore, their reasonable 2PA cross sections provide for the option of using two-photon fluorescence microscopy, which circumvents commonly encountered disadvantages associated with one-photon imaging such as photobleaching and poor tissue penetration.

Synthesis, molecular docking and anti-mycobacterial evaluation of new imidazo1,2-a]pyridine-2-carboxamide derivatives

New anti-tubercular agents, imidazo1,2-a]pyridine-2-carboxamide derivatives (5a-q) have been designed and synthesized. The structural considerations of the designed molecules were further supported by the docking study with a long-chain enoyl-acyl carrier protein reductase (InhA). The chemical structures of the new compounds were characterized by IR, H-1 NMR, C-13 NMR, HRMS and elemental analysis. In addition, single crystal X-ray diffraction has also been recorded for compound 5f. Compounds were evaluated in vitro against Mycobacterium tuberculosis H37Rv, and cytotoxicity against HEK-293T cell line. Amongst the tested compounds 5j, 5l and 5q were emerged as good anti-tubercular agents with low cytotoxicity. The structure-anti TB activity relationship of these derivatives was explained by molecular docking. (C) 2014 Elsevier Masson SAS. All rights reserved.

Biocompatibilidade do polissacarídeo celulósico sintetizado por zoogloea sp.: um estudo em bexigas de coelho

Atualmente, o material utilizado para o tratamento endoscópico é o Deflux, porém este é um material não-biológico. Sabe-se que a substância ideal deve ser atóxica, biocompatível, não-migratória, não-antigênica e deve causar o mínimo possível de inflamação no local do implante. A bactéria Zoogloea sp. produz um exopolissacarídeo celulósico (CEP) com baixa citotoxicidade e alto biocompatibilidade. O objetivo deste estudo é investigar, na bexiga de coelho, a biocompatibilidade de implantes de exopolissacarídeo de celulose, produzidos pela Zooglea sp. Foram utilizados como modelo experimental, 20 coelhos adultos da raça Califórnia, com média de seis meses de idade. Os animais foram divididos em dois grupos, sendo o grupo G1, composto por animais mortos três dias após a aplicação do implante (n=9), e o grupo G2, composto por animais mortos três meses após a aplicação do implante (n=11). Cada animal recebeu, no total, quatro implantes, sendo dois de gel de biopolímero e dois de gel Deflux. Foram realizadas as técnicas imunohistoquímicas para marcação de colágeno tipos I e III, alfa-actina de músculo liso, PCNA e reação química TUNEL. Nas amostras de três dias, os implantes de CEP e deflux, eram estruturalmente homogêneos e livres de células inflamatórias ou vasos sanguíneos. Por outro lado, nas amostras de três meses, com exceção de algumas áreas, o CEP estava organizado como feixes curtos que eram sugestivos de um tecido fibroso. Apesar disso, o implante de CEP corou negativamente para colágenos tipos I e III, fibras elásticas, enquanto que o tricrômico de masson, não indicou a presença de colágeno. Em contraste as áreas de implante de deflux nas amostras de três meses estavam fragmentadas, mas ainda eram homogêneas, e ainda não havia nenhuma célula nem vaso sanguíneo em seu interior. As células positivas para PCNA podiam ser claramente percebidas dentro dessas ilhotas, dessa forma indicando um processo inflamatórioproliferativo, em curso. No grupo sacrificado aos três meses, os implantes de deflux ainda estavam negativos, mas em torno das áreas de CEP algumas células positivas para a técnica do TUNEL eram perceptíveis. Nos implantes de CEP de três meses, muitos vasos sanguíneos eram visualizados, e a sua densidade era de 23.865.48. A densidade de microvasos na lâmina própria (41.5111.19) foi significativamente diferente (p<0.001) daquela no implante de CEP. Nossos resultados mostraram que o CEP possui pouca imunogenicidade e se integra melhor no tecido hospedeiro quando comparado ao deflux. Portanto o CEP deve ser um material eficiente em casos em que a incorporação ao tecido é desejada como por exemplo em estruturas de suporte na cirurgia de reconstrução

Avaliação da ação antitumoral in vitro da pterocarpanoquinona LQB 118 e estudo de alguns mecanismos de ação

Tem sido descrito que o acúmulo de mutações em proto-oncogenes e genes supressores de tumor contribui para o direcionamento da célula à carcinogênese. Na maioria dos casos de câncer, as células apresentam proliferação descontrolada devido a alterações na expressão e/ou mutações de ciclinas, quinases dependentes de ciclinas e/ou inibidores do ciclo celular. Os tumores sólidos figuram entre o tipo de câncer mais incidente no mundo, sendo a quimioterapia e/ou hormônio-terapia, radioterapia e cirurgia os tratamentos mais indicados para estes tipos de tumores. Entretanto, o tratamento quimioterápico apresenta diversos efeitos colaterais e muitas vezes é ineficaz. Portanto, a busca por novas moléculas capazes de conter a proliferação destas células e com baixa toxicidade para o organismo se faz necessário. Este trabalho teve por objetivo avaliar a ação antitumoral in vitro de um novo composto sintético, a pterocarpanoquinona LQB118, sobre algumas linhagens tumorais humanas de alta prevalência e estudar alguns dos seus mecanismos de ação. As linhagens tumorais estudadas neste trabalho foram os adenocarcinomas de mama (MCF7) e próstata (PC-3), e carcinoma de pulmão (A549). A citotoxicidade foi avaliada pelo ensaio do MTT e a proliferação celular pela contagem de células vivas (exclusão do corante azul de tripan) e análise do ciclo celular (citometria de fluxo). A expressão gênica foi avaliada por RT-PCR e a apoptose foi avaliada por condensação da cromatina (microscopia de fluorescência-DAPI), fragmentação de DNA (eletroforese) e marcação com anexina V (citometria de fluxo). Das linhagens tumorais testadas, a de próstata (PC3) foi a que se mostrou mais sensível ao LQB 118, e em função deste resultado, os demais experimentos foram realizados com esta linhagem tumoral. O efeito citotóxico do LQB 118 se mostrou tempo e concentração dependente. Esta substância inibiu a proliferação celular e prejudicou a progressão do ciclo celular, acumulando células nas fases S e G2/M. Buscando esclarecer os mecanismos desta ação antitumoral, demonstrou-se que o LQB 118 inibe a expressão do mRNA do fator de transcrição c-Myc e das ciclinas D1 e B1, e induz a apoptose de tais células tumorais. Em suma, o LQB 118 é capaz de inibir a proliferação das células tumorais de próstata, alterando a expressão do mRNA de alguns genes reguladores do ciclo celular, resultando em interrupção do ciclo celular e indução de apoptose, indicando este composto como um potencial candidato a futuro medicamento no tratamento do câncer de próstata.

Spectroscopic characterization of protein-wrapped single-wall carbon nanotubes and quantification of their cellular uptake in multiple cell generations

We study the spectral characteristics of bovine serum albumin (BSA) protein conjugated single-wall carbon nanotubes (SWNTs), and quantify their uptake by macrophages. The binding of BSA onto the SWNT surface is found to change the protein structure and to increase the doping of the nanotubes. The G-band Raman intensity follows a well-defined power law for SWNT concentrations of up to 33 μg ml-1 in aqueous solutions. Subsequently, in vitro experiments demonstrate that incubation of BSA-SWNT complexes with macrophages affects neither the cellular growth nor the cellular viability over multiple cell generations. Using wide spot Raman spectroscopy as a fast, non-destructive method for statistical quantification, we observe that macrophages effectively uptake BSA-SWNT complexes, with the average number of nanotubes internalized per cell remaining relatively constant over consecutive cell generations. The number of internalized SWNTs is found to be ∼30 × 106 SWNTs/cell for a 60 mm-2 seeding density and ∼100 × 10 6 SWNTs/cell for a 200 mm-2 seeding density. Our results show that BSA-functionalized SWNTs are an efficient molecular transport system with low cytotoxicity maintained over multiple cell generations. © 2013 IOP Publishing Ltd.

Microwave synthesis of fluorescent carbon nanoparticles with electrochemiluminescence properties

We present a facile, economical microwave pyrolysis approach to synthesize fluorescent carbon nanoparticles with electrochemiluminescence properties.

Sugars-grafted aliphatic biodegradable poly(L-lactide-co-carbonate)s by click reaction and their specific interaction with lectin molecules

A novel biodegradable aliphatic poly(L-lactide-co-carbonate) bearing pendant acetylene groups was successfully prepared by ring-opening copolymerization of L-lactide (LA) with 5-methyl-5-propargyloxycarbonyl-1,3-dioxan-2-one (PC) in the presence of benzyl alcohol as initiator with ZnEt2 as catalyst in bulk at 100 degrees C and subsequently used for grafting 2-azidoethyl beta-D-glucopyranoside and 2-azidoethyl beta-lactoside by the typical "click reaction," that is Cu(I)-catalyzed cycloaddition of azide and alkyne. The density of acetylene groups in the copolymer can be tailored by the molar ratio of PC to LA during the copolymerization. The aliphatic copolymers grafted with sugars showed low cytotoxicity to L929 cells, improved hydrophilic properties and specific recognition and binding ability with lectins, that is Concanavalin A (Con A) and Ricinus communis agglutinin (RCA). Therefore, this kind of sugar-grafted copolymer could be a good candidate in variety of biomedical applications.

Novel targeting of PEGylated liposomes for codelivery of TGF-β1 siRNA and four antitubercular drugs to human macrophages for the treatment of mycobacterial infection: a quantitative proteomic study

Tuberculosis (TB) is still a major public health issue in developing countries, and its chemotherapy is compromised by poor drug compliance and severe side effects. This study aimed to synthesize and characterize new multimodal PEGylated liposomes encapsulated with clinically commonly used anti-TB drugs with linkage to small interfering RNA (siRNA) against transforming growth factor-β1 (TGF-β1). The novel NP-siRNA liposomes could target THP-1-derived human macrophages that were the host cells of mycobacterium infection. The biological effects of the NP-siRNA liposomes were evaluated on cell cycle distribution, apoptosis, autophagy, and the gene silencing efficiency of TGF-β1 siRNA in human macrophages. We also explored the proteomic responses to the newly synthesized NP-siRNA liposomes using the stable isotope labeling with amino acids in cell culture approach. The results showed that the multifunctional PEGylated liposomes were successfully synthesized and chemically characterized with a mean size of 265.1 nm. The novel NP-siRNA liposomes functionalized with the anti-TB drugs and TGF-β1 siRNA were endocytosed efficiently by human macrophages as visualized by transmission electron microscopy and scanning electron microscopy. Furthermore, the liposomes showed a low cytotoxicity toward human macrophages. There was no significant effect on cell cycle distribution and apoptosis in THP-1-derived macrophages after drug exposure at concentrations ranging from 2.5 to 62.5 μg/mL. Notably, there was a 6.4-fold increase in the autophagy of human macrophages when treated with the NP-siRNA liposomes at 62.5 μg/mL. In addition, the TGF-β1 and nuclear factor-κB expression levels were downregulated by the NP-siRNA liposomes in THP-1-derived macrophages. The Ingenuity Pathway Analysis data showed that there were over 40 signaling pathways involved in the proteomic responses to NP-siRNA liposome exposure in human macrophages, with 160 proteins mapped. The top five canonical signaling pathways were eukaryotic initiation factor 2 signaling, actin cytoskeleton signaling, remodeling of epithelial adherens junctions, epithelial adherens junction signaling, and Rho GDP-dissociation inhibitor signaling pathways. Collectively, the novel synthetic targeting liposomes represent a promising delivery system for anti-TB drugs to human macrophages with good selectivity and minimal cytotoxicity.

Functionalization of dendrimers for improved gene delivery to mesenchymal stem cell

Disease, injury, and age problems compromise human quality of life and continuously motivate the search for new and more efficacious therapeutic approaches. The field of Tissue Regeneration and Engineering has greatly evolved over the last years, mainly due to the combination of the important advances verified in Biomaterials Science and Engineering with those of Cell and Molecular Biology. In particular, a new and promising area arose – Nanomedicine – that takes advantage of the extremely small size and especial chemical and physical properties of Nanomaterials, offering powerful tools for health improvement. Research on Stem Cells, the self-renewing progenitors of body tissues, is also challenging to the medical and scientific communities, being expectable the appearance of new and exciting stem cell-based therapies in the next years. The control of cell behavior (namely, of cell proliferation and differentiation) is of key importance in devising strategies for Tissue Regeneration and Engineering. Cytokines, growth factors, transcription factors and other signaling molecules, most of them proteins, have been identified and found to regulate and support tissue development and regeneration. However, the application of these molecules in long-term regenerative processes requires their continuous presence at high concentrations as they usually present short half-lives at physiological conditions and may be rapidly cleared from the body. Alternatively, genes encoding such proteins can be introduced inside cells and be expressed using cell’s machinery, allowing an extended and more sustained production of the protein of interest (gene therapy). Genetic engineering of stem cells is particularly attractive because of their self-renewal capability and differentiation potential. For Tissue Regeneration and Engineering purposes, the patient’s own stem cells can be genetically engineered in vitro and, after, introduced in the body (with or without a scaffold) where they will not only modulate the behavior of native cells (stem cell-mediated gene therapy), but also directly participate in tissue repair. Cells can be genetically engineered using viral and non-viral systems. Viruses, as a result of millions of years of evolution, are very effective for the delivery of genes in several types of cells, including cells from primary sources. However, the risks associated with their use (like infection and immunogenic reactions) are driving the search for non-viral systems that will efficiently deliver genetic material into cells. Among them, chemical methods that are promising and being investigated use cationic molecules as carriers for DNA. In this case, gene delivery and gene expression level remain relatively low when primary cells are used. The main goal of this thesis was to develop and assess the in vitro potential of polyamidoamine (PAMAM) dendrimers based carriers to deliver genes to mesenchymal stem cells (MSCs). PAMAM dendrimers are monodispersive, hyperbranched and nanospherical molecules presenting unique characteristics that make them very attractive vehicles for both drug and gene delivery. Although they have been explored for gene delivery in a wide range of cell lines, the interaction and the usefulness of these molecules in the delivery of genes to MSCs remains a field to be explored. Adult MSCs were chosen for the studies due to their potential biomedical applications (they are considered multipotent cells) and because they present several advantages over embryonic stem cells, such as easy accessibility and the inexistence of ethical restrictions to their use. This thesis is divided in 5 interconnected chapters. Chapter I provides an overview of the current literature concerning the various non-viral systems investigated for gene delivery in MSCs. Attention is devoted to physical methods, as well as to chemical methods that make use of polymers (natural and synthetic), liposomes, and inorganic nanoparticles as gene delivery vectors. Also, it summarizes the current applications of genetically engineered mesenchymal stem cells using non-viral systems in regenerative medicine, with special focus on bone tissue regeneration. In Chapter II, the potential of native PAMAM dendrimers with amine termini to transfect MSCs is evaluated. The level of transfection achieved with the dendrimers is, in a first step, studied using a plasmid DNA (pDNA) encoding for the β-galactosidase reporter gene. The effect of dendrimer’s generation, cell passage number, and N:P ratio (where N= number of primary amines in the dendrimer; P= number of phosphate groups in the pDNA backbone) on the level of transfection is evaluated, being the values always very low. In a second step, a pDNA encoding for bone morphogenetic protein-2, a protein that is known for its role in MSCs proliferation and differentiation, is used. The BMP-2 content produced by transfected cells is evaluated by an ELISA assay and its effect on the osteogenic markers is analyzed through several classical assays including alkaline phosphatase activity (an early marker of osteogenesis), osteocalcin production, calcium deposition and mineralized nodules formation (late osteogenesis markers). Results show that a low transfection level is enough to induce in vitro osteogenic differentiation in MSCs. Next, from Chapter III to Chapter V, studies are shown where several strategies are adopted to change the interaction of PAMAM dendrimers with MSCs cell membrane and, as a consequence, to enhance the levels of gene delivery. In Chapter III, generations 5 and 6 of PAMAM dendrimers are surface functionalized with arginine-glycine-aspartic acid (RGD) containing peptides – experiments with dendrimers conjugated to 4, 8 and 16 RGD units were performed. The underlying concept is that by including the RGD integrin-binding motif in the design of the vectors and by forming RGD clusters, the level of transfection will increase as MSCs highly express integrins at their surface. Results show that cellular uptake of functionalized dendrimers and gene expression is enhanced in comparison with the native dendrimers. Furthermore, gene expression is dependent on both the electrostatic interaction established between the dendrimer moiety and the cell surface and the nanocluster RGD density. In Chapter IV, a new family of gene delivery vectors is synthesized consisting of a PAMAM dendrimer (generation 5) core randomly linked at the periphery to alkyl hydrophobic chains that vary in length and number. Herein, the idea is to take advantage of both the cationic nature of the dendrimer and the capacity of lipids to interact with biological membranes. These new vectors show a remarkable capacity for internalizing pDNA, being this effect positively correlated with the –CH2– content present in the hydrophobic corona. Gene expression is also greatly enhanced using the new vectors but, in this case, the higher efficiency is shown by the vectors containing the smallest hydrophobic chains. Finally, chapter V reports the synthesis, characterization and evaluation of novel gene delivery vectors based on PAMAM dendrimers (generation 5) conjugated to peptides with high affinity for MSCs membrane binding - for comparison, experiments are also done with a peptide with low affinity binding properties. These systems present low cytotoxicity and transfection efficiencies superior to those of native dendrimers and partially degraded dendrimers (Superfect®, a commercial product). Furthermore, with this biomimetic approach, the process of gene delivery is shown to be cell surface receptor-mediated. Overall, results show the potential of PAMAM dendrimers to be used, as such or modified, in Tissue Regeneration and Engineering. To our knowledge, this is the first time that PAMAM dendrimers are studied as gene delivery vehicles in this context and using, as target, a cell type with clinical relevancy. It is shown that the cationic nature of PAMAM dendrimers with amine termini can be synergistically combined with surface engineering approaches, which will ultimately result in suitable interactions with the cytoplasmic membrane and enhanced pDNA cellular entry and gene expression. Nevertheless, the quantity of pDNA detected inside cell nucleus is always very small when compared with the bigger amount reaching cytoplasm (accumulation of pDNA is evident in the perinuclear region), suggesting that the main barrier to transfection is the nuclear membrane. Future work can then be envisaged based on the versatility of these systems as biomedical molecular materials, such as the conjugation of PAMAM dendrimers to molecules able to bind nuclear membrane receptors and to promote nuclear translocation.

Polymeric alginate nanoparticles containing the local anesthetic bupivacaine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)