José L. Santos

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.

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.

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.

Osteogenic differentiation of mesenchymal stem cells using PAMAM dendrimers as gene delivery vectors.

This paper reports the use of different generations of polyamidoamine (PAMAM) dendrimers for the in vitro transfection of mesenchymal stem cells (MSCs). A systematic study was carried out on the transfection efficiency achieved by the PAMAM dendrimers using a beta-galactosidase reporter gene system. Transfection results were shown to be dependent upon the generation of dendrimers, the amine to phosphate group ratio and the cell passage number. In all cases, the transfection efficiency was very low. Nevertheless, it was hypothesized that a low transfection level could be sufficient to promote the in vitro differentiation of MSCs towards the osteoblastic lineage. To address this possibility, dendrimers carrying the human bone morphogenetic protein-2 (hBMP-2) gene-containing plasmid were used. All quantitative (alkaline phosphatase activity, osteocalcin secretion and calcium deposition) and qualitative (von Kossa staining) osteogenic markers were significantly stronger in transfected cells when compared to non-transfected ones. This study not only clearly demonstrates that a low transfection level can be sufficient for inducing in vitro differentiation of MSCs to the osteoblast phenotype but also highlights the importance of focusing research on the development of gene delivery vectors in the concrete application.

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.

Bioinspired superhydrophobic poly(L-lactic acid) surfaces control bone marrow derived cells adhesion and proliferation

The aptitude of a cell to adhere, migrate, and differentiate on a compact substrate or scaffold is important in the field of tissue engineering and biomaterials. It is well known that cell behavior can be controlled and guided through the change in micro- and nano-scale topographic features. In this work, we intend to demonstrate that special topographic features that control wettability may also have an important role in the biological performance of biodegradable substrates. Poly(L-lactic acid) surfaces with superhydrophobic characteristics were produced, based on the so-called Lotus effect, exhibiting dual micro- and nano-scale roughness. The water contact angle could be higher than 150 degrees and a value of that order could be kept even upon immersion in a simulated body fluid solution for more than 20 days. Such water repellent surfaces were found to prevent adhesion and proliferation of bone marrow derived cells previously isolated from the femurs of 6-week-old male Wistar rats, when compared with smoother surfaces prepared by simple solvent casting. Such results demonstrate that these superhydrophobic surfaces may be used to control cell behavior onto biodegradable substrates.

Control of polymeric nanoparticle size to improve therapeutic delivery

As nanoparticle (NP)-mediated drug delivery research continues to expand, understanding parameters that govern NP interactions with the biological environment becomes paramount. The principles identified from the study of these parameters can be used to engineer new NPs, impart unique functionalities, identify novel utilities, and improve the clinical translation of NP formulations. One key design parameter is NP size. New methods have been developed to produce NPs with increased control of NP size between 10 and 200nm, a size range most relevant to physical and biochemical targeting through both intravascular and site-specific deliveries. Three notable techniques best suited for generating polymeric NPs with narrow size distributions are highlighted in this review: self-assembly, microfluidics-based preparation, and flash nanoprecipitation. Furthermore, the effect of NP size on the biological fate and transport properties at the molecular scale (protein-NP interactions) and the tissue and systemic scale (convective and diffusive transport of NPs) are analyzed here. These analyses underscore the importance of NP size control in considering clinical translation and assessment of therapeutic outcomes of NP delivery vehicles.

Shape control in engineering of polymeric nanoparticles for therapeutic delivery

Nanoparticle-mediated delivery of therapeutics holds great potential for the diagnosis and treatment of a wide range of diseases. Significant advances have been made in the design of new polymeric nanoparticle carriers through modulation of their physical and chemical structures and biophysical properties. Nanoparticle shape has been increasingly proposed as an important attribute dictating their transport properties in biological milieu. In this review, we highlight three major methods for preparing polymeric nanoparticles that allow for exquisite control of particle shape. Special attention is given to various approaches to controlling nanoparticle shape by tuning copolymer structural parameters and assembly conditions. This review also provides comparisons of these methods in terms of their unique capabilities, materials choices, and specific delivery cargos, and summarizes the biological effects of nanoparticle shape on transport properties at the tissue and cellular levels.

Calcium phosphate-mediated gene delivery using simulated body fluid (SBF).

The present study aimed at developing a new approach in gene delivery of calcium phosphate nanoparticles through simulated body fluid (CaP-SBF). The physicochemical and biological characteristics of the CaP-SBF nanoparticles were compared with those made in pure water (CaP-water) via a similar procedure. The CaP-SBF and CaP-water solutions were then adjusted to two different pH values of 7.4 and 8.0, mixed with plasmid DNA (pDNA), and added in varying amounts to human embryonic kidney (HEK 293T) cells. The transfection efficiency and cell viability were studied in vitro by reporter gene (luciferase and Enhanced Green Fluorescent Protein) expression and the resazurin reduction assay, respectively, 24 and 48 h after the incubation with the nanoparticles. Our results indicated considerably high in vitro transfection efficiency for CaP-SBF/DNA complexes at physiological pH (7.4) with high amounts of CaP. Additionally, the SBF solution exhibited the ability to reduce the rapid growth of CaP particles over time, leading to higher transfection efficiency of CaP-SBF/DNA complexes than those made in water (CaP-water/DNA).

Insight into the role of N,N-dimethylaminoethyl methacrylate (DMAEMA) conjugation onto poly(ethylenimine): cell viability and gene transfection studies

In 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.