João Rodrigues

Functionalization of poly(amidoamine) dendrimers with hydrophobic chains for improved gene delivery in mesenchymal stem cells

A new family of gene delivery vectors is synthesized consisting of a medium-size generation PAMAM dendrimer (generation 5, with amine termini) core randomly linked at the periphery to hydrophobic chains that vary in length (12 to 16 carbon alkyl chains) and number (from 4.2 to 9.7 in average). The idea subjacent to the present work is to join the advantages of the cationic nature of the dendrimer with the capacity of lipids to interact with biological membranes. Unlike other amphiphilic systems designed for the same purpose, where the hydrophobic and hydrophilic moieties coexist in opposite sides, the present vectors have a hydrophilic interior and a hydrophobic corona. The vectors are characterized in respect to their ability to neutralize, bind and compact plasmid DNA (pDNA). The complexes formed between the vectors and pDNA are analyzed concerning their size, zeta-potential, resistance to serum nucleases, capacity of being internalized by cells and transfection efficiency. These new vectors show a remarkable capacity for mediating the internalization of pDNA with minimum cytotoxicity, being this effect positively correlated with the -CH(2)- content present in the hydrophobic corona. Gene expression in MSCs, a cell type with relevancy in the regenerative medicine clinical context, is also enhanced using the new vectors but, in this case, the higher efficiency is shown by the vectors containing the smallest hydrophobic chains.

Biodegradable Polymer Nanogels for Drug/Nucleic Acid Delivery

Yulin Li,*,†,‡ Dina Maciel,† Joaõ Rodrigues,*,† Xiangyang Shi,*,†,§ and Helena Tomaś*,† †CQM-Centro de Química da Madeira, MMRG, Universidade da Madeira, Campus da Penteada 9000-390, Funchal, Portugal ‡The State Key Laboratory of Bioreactor Engineering, Key Laboratory for Ultrafine Materials of Ministry of Education, Engineering Research Centre for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, People’s Republic of China College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, People’s Republic of China

Non-Viral Gene Delivery to Mesenchymal Stem Cells: Methods, Strategies and Application in Bone Tissue Engineering and Regeneration

Mesenchymal stem cells (MSCs) can be isolated from several tissues in the body, have the ability to self-renewal, show immune suppressive properties and are multipotent, being able to generate various cell types. At present, due to their intrinsic characteristics, MSCs are considered very promising in the area of tissue engineering and regenerative medicine. In this context, genetic modification can be a powerful tool to control the behavior and fate of these cells and be used in the design of new cellular therapies. Viral systems are very effective in the introduction of exogenous genes inside MSCs. However, the risks associated with their use are leading to an increasing search for non-viral approaches to attain the same purpose, even if MSCs have been shown to be more difficult to transfect in this way. In the past few years, progress was made in the development of chemical and physical methods for non-viral gene delivery. Herein, an overview of the application of those methods specifically to MSCs is given and their use in tissue engineering and regenerative medicine therapeutic strategies highlighted using the example of bone tissue. Key issues and future directions in non-viral gene delivery to MSCs are also critically addressed.

Redox-responsive alginate nanogels with enhanced anticancer cytotoxicity.

Although doxorubicin (Dox) has been widely used in the treatment of different types of cancer, its insufficient cellular uptake and intracellular release is still a limitation. Herein, we report an easy process for the preparation of redox-sensitive nanogels that were shown to be highly efficient in the intracellular delivery of Dox. The nanogels (AG/Cys) were obtained through in situ cross-linking of alginate (AG) using cystamine (Cys) as a cross-linker via a miniemulsion method. Dox was loaded into the AG/Cys nanogels by simply mixing it in aqueous solution with the nanogels, that is, by the establishment of electrostatic interactions between the anionic AG and the cationic Dox. The results demonstrated that the AG/Cys nanogels are cytocompatible, have a high drug encapsulation efficiency (95.2 ± 4.7%), show an in vitro accelerated release of Dox in conditions that mimic the intracellular reductive conditions, and can quickly be taken up by CAL-72 cells (an osteosarcoma cell line), resulting in higher Dox intracellular accumulation and a remarkable cell death extension when compared with free Dox. The developed nanogels can be used as a tool to overcome the problem of Dox resistance in anticancer treatments and possibly be used for the delivery of other cationic drugs in applications beyond cancer.

Receptor-mediated gene delivery using PAMAM dendrimers conjugated with peptides recognized by mesenchymal stem cells.

As mesenchymal stem cells (MSCs) can differentiate into multiple cell types, the delivery of exogenous genes to this type of cell can be an important tool in tissue regeneration and engineering. However transfection of MSCs using nonviral gene delivery vectors is difficult, the development of more efficient and safe DNA vehicles being necessary. Moreover, specific transfection of MSCs may be required to avoid unwanted side effects in other tissues. In this study, a novel family of gene delivery vectors based on poly(amidoamine) (PAMAM) dendrimers functionalized with peptides displaying high affinity toward MSCs was prepared. The vectors were characterized with respect to their ability to neutralize, bind and compact plasmid DNA (pDNA). The complexes formed between the vectors and pDNA were analyzed concerning their size, zeta-potential, capacity of being internalized by cells and transfection efficiency. These new vectors exhibited low cytotoxicity, receptor-mediated gene delivery into MSCs and transfection efficiencies superior to those presented by native dendrimers and by partially degraded dendrimers.

Gene Delivery into Mesenchymal Stem Cells: A Biomimetic Approach Using RGD Nanoclusters Based on Poly(amidoamine) Dendrimers

Poly(amidoamine) dendrimers (generations 5 and 6) with amine termini were conjugated with peptides containing the arginine-glycine-aspartic acid (RGD) sequence having in view their application as gene delivery vectors. The idea behind the work was to take advantage of the cationic nature of dendrimers and of the integrin targeting capabilities of the RGD motif to improve gene delivery. Dendrimers were used as scaffolds for RGD clustering and, by controlling the number of peptides (4, 8, and 16) linked to each dendrimer, it was possible to evaluate the effect of RGD density on the gene delivery process. The new vectors were characterized in respect to their ability to neutralize and compact plasmid DNA (pDNA). The complexes formed by the vectors and pDNA were studied concerning their size, zeta potential, capacity of being internalized by cells and ability of transferring genes. Transfection efficiency was analyzed, first, by using a pDNA encoding for Enhanced Green Fluorescent Protein and Firefly Luciferase and, second, by using a pDNA encoding for Bone Morphogenetic Protein-2. Gene expression in mesenchymal stem cells was enhanced using the new vectors in comparison to native dendrimers and was shown to be dependent on the electrostatic interaction established between the dendrimer moiety and the cell surface, as well as on the RGD density of nanoclusters. The use of dendrimer scaffolds for RGD cluster formation is a new approach that can be extended beyond gene delivery applications, whenever RGD clustering is important for modulating cellular responses.

RGD peptide-modified multifunctional dendrimer platform for drug encapsulation and targeted inhibition of cancer cells

Development of multifunctional nanoscale drug-delivery systems for targeted cancer therapy still remains a great challenge. Here, we report the synthesis of cyclic arginine-glycine-aspartic acid (RGD) peptide-conjugated generation 5 (G5) poly(amidoamine) dendrimers for anticancer drug encapsulation and targeted therapy of cancer cells overexpressing αvβ3 integrins. In this study, amine-terminated G5 dendrimers were used as a platform to be sequentially modified with fluorescein isothiocyanate (FI) via a thiourea linkage and RGD peptide via a polyethylene glycol (PEG) spacer, followed by acetylation of the remaining dendrimer terminal amines. The developed multifunctional dendrimer platform (G5.NHAc-FI-PEG-RGD) was then used to encapsulate an anticancer drug doxorubicin (DOX). We show that approximately six DOX molecules are able to be encapsulated within each dendrimer platform. The formed complexes are water-soluble, stable, and able to release DOX in a sustained manner. One- and two-dimensional NMR techniques were applied to investigate the interaction between dendrimers and DOX, and the impact of the environmental pH on the release rate of DOX from the dendrimer/DOX complexes was also explored. Furthermore, cell biological studies demonstrate that the encapsulation of DOX within the G5.NHAc-FI-PEG-RGD dendrimers does not compromise the anticancer activity of DOX and that the therapeutic efficacy of the dendrimer/DOX complexes is solely related to the encapsulated DOX drug. Importantly, thanks to the role played by RGD-mediated targeting, the developed dendrimer/drug complexes are able to specifically target αvβ3 integrin-overexpressing cancer cells and display specific therapeutic efficacy to the target cells. The developed RGD peptide-targeted multifunctional dendrimers may thus be used as a versatile platform for targeted therapy of different types of αvβ3 integrin-overexpressing cancer cells.

pH sensitive Laponite/alginate hybrid hydrogels: swelling behaviour and release mechanism

Alginate hydrogels containing biocompatible Laponite (LP) were prepared and dried under vacuum or in an oven, and their drug release properties at different pH values were studied in vitro using methylene blue (MB) as a model of a cationic drug. These hybrid hydrogels showed a greater encapsulation efficiency of MB, a better sustained release and higher pH sensitivity in MB release, compared to the pure AG gels. The oven-dried hybrid gels were more pH sensitive in MB release than the vacuum-dried ones. Furthermore, the presence of LP, the drying mode and the pH value were shown to influence the mechanism of MB release. LP/AG hybrid gels are, thus, good candidates for the controlled delivery of cationic drugs under acidic conditions.

Hyper-Rayleigh scattering study of η5-monocyclopentadienyl–metal complexes for second order non-linear optical materials

A series of ionic η5-monocyclopentadienyl–metal compounds possessing p-substituted benzonitrile ligands has been studied by hyper-Rayleigh scattering at the fundamental wavelength of 1.064 µm. Upon systematic variation of the metal ion, the first hyperpolarizability β was found to increase along the sequence Co, Ni, Ru, Fe, with about a three-fold increase from Ru to Fe. This yields very high values for the iron complexes, e.g., β=410×10–30 esu for [Fe(η5-C5H5)(dppe)( p-NCC6H4NO2)]+ [PF6 ]– dissolved in methanol. The high β values are attributed to π back-donation resulting in an extension of the conjugated π-system from the FeII organometallic fragment, acting as a good donor group, via the nitrile to the acceptor group NO2 . Complexes with single phenyl rings as conjugated chains perform better than their biphenyl analogues, which is explained in terms of non-planarity of the coordinated biphenyl ligands in solution. By comparing complexes with electron donor and acceptor substituted ligands it is demonstrated that the organometallic moiety can be used as an extremely effective donor group but not as a good acceptor group.

Dendrimer-assisted formation of fluorescent nanogels for drug delivery and intracellular imaging.

Although, in general, nanogels present a good biocompatibility and are able to mimic biological tissues, their unstability and uncontrollable release properties still limit their biomedical applications. In this study, a simple approach was used to develop dual-cross-linked dendrimer/alginate nanogels (AG/G5), using CaCl2 as cross-linker and amine-terminated generation 5 dendrimer (G5) as a cocrosslinker, through an emulsion method. Via their strong electrostatic interactions with anionic AG, together with cross-linker Ca(2+), G5 dendrimers can be used to mediate the formation of more compact structural nanogels with smaller size (433 ± 17 nm) than that (873 ± 116 nm) of the Ca(2+)-cross-linked AG nanogels in the absence of G5. Under physiological (pH 7.4) and acidic (pH 5.5) conditions, the sizes of Ca(2+)-cross-linked AG nanogels gradually decrease probably because of their degradation, while dual-cross-linked AG/G5 nanogels maintain a relatively more stable structure. Furthermore, the AG/G5 nanogels effectively encapsulate the anticancer drug doxorubicin (Dox) with a loading capacity 3 times higher than that of AG nanogels. The AG/G5 nanogels were able to release Dox in a sustained way, avoiding the burst release observed for AG nanogels. In vitro studies show that the AG/G5-Dox NGs were effectively taken up by CAL-72 cells (a human osteosarcoma cell line) and maintain the anticancer cytotoxicity levels of free Dox. Interestingly, G5 labeled with a fluorescent marker can be integrated into the nanogels and be used to track the nanogels inside cells by fluorescence microscopy. These findings demonstrate that AG/G5 nanogels may serve as a general platform for therapeutic delivery and/or cell imaging.