Preparation and Characterization of Cationic PLA-PEG Nanoparticles for Delivery of Plasmid DNA

The purpose of the present work was to formulate and evaluate cationic poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) nanoparticles as novel non-viral gene delivery nano-device. Cationic PLA-PEG nanoparticles were prepared by nanoprecipitation method. The gene loaded nanoparticles were obtained by incubating the report gene pEGFP with cationic PLA-PEG nanoparticles. The physicochemical properties (e.g., morphology, particle size, surface charge, DNA binding efficiency) and biological properties (e.g., integrity of the released DNA, protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in Hela cells) of the gene loaded PLA-PEG nanoparticles were evaluated, respectively. The obtained cationic PLA-PEG nanoparticles and gene loaded nanoparticles were both spherical in shape with average particle size of 89.7 and 128.9 nm, polydispersity index of 0.185 and 0.161, zeta potentials of +28.9 and +16.8 mV, respectively. The obtained cationic PLA-PEG nanoparticles with high binding efficiency (>95%) could protect the loaded DNA from the degradation by nuclease and plasma. The nanoparticles displayed sustained-release properties in vitro and the released DNA maintained its structural and functional integrity. It also showed lower cytotoxicity than Lipofectamine 2000 and could successfully transfect gene into Hela cells even in presence of serum. It could be concluded that the established gene loaded cationic PLA-PEG nanoparticles with excellent properties were promising non-viral nano-device, which had potential to make cancer gene therapy achievable

Poly-lactic acid nanoparticles (PLA-NP) promote physiological modifications in lung epithelial cells and are internalized by clathrin-coated pits and lipid rafts

BackgroundPoly-lactic acid nanoparticles (PLA-NP) are a type of polymeric NP, frequently used as nanomedicines, which have advantages over metallic NP such as the ability to maintain therapeutic drug levels for sustained periods of time. Despite PLA-NP being considered biocompatible, data concerning alterations in cellular physiology are scarce.MethodsWe conducted an extensive evaluation of PLA-NP biocompatibility in human lung epithelial A549 cells using high throughput screening and more complex methodologies. These included measurements of cytotoxicity, cell viability, immunomodulatory potential, and effects upon the cells’ proteome. We used non- and green-fluorescent PLA-NP with 63 and 66 nm diameters, respectively. Cells were exposed with concentrations of 2, 20, 100 and 200 µg/mL, for 24, 48 and 72 h, in most experiments. Moreover, possible endocytic mechanisms of internalization of PLA-NP were investigated, such as those involving caveolae, lipid rafts, macropinocytosis and clathrin-coated pits.ResultsCell viability and proliferation were not altered in response to PLA-NP. Multiplex analysis of secreted mediators revealed a low-level reduction of IL-12p70 and vascular epidermal growth factor (VEGF) in response to PLA-NP, while all other mediators assessed were unaffected. However, changes to the cells’ proteome were observed in response to PLA-NP, and, additionally, the cellular stress marker miR155 was found to reduce. In dual exposures of staurosporine (STS) with PLA-NP, PLA-NP enhanced susceptibility to STS-induced cell death. Finally, PLA-NP were rapidly internalized in association with clathrin-coated pits, and, to a lesser extent, with lipid rafts.ConclusionsThese data demonstrate that PLA-NP are internalized and, in general, tolerated by A549 cells, with no cytotoxicity and no secretion of pro-inflammatory mediators. However, PLA-NP exposure may induce modification of biological functions of A549 cells, which should be considered when designing drug delivery systems. Moreover, the pathways of PLA-NP internalization we detected could contribute to the improvement of selective uptake strategies

Immuno-Isolation of Pancreatic Islet Allografts Using Pegylated Nanotherapy Leads to Long-Term Normoglycemia in Full MHC Mismatch Recipient Mice

Two major hurdles need to be surmounted for cell therapy for diabetes: (i) allo-immune rejection of grafted pancreatic islets, or stem/precursor cell-derived insulin-secreting cells; and (ii) continuing auto-immunity against the diabetogenic endogenous target antigen. Nanotherapeutics offer a novel approach to overcome these problems and here we ask if creation of “stealth” islets encapsulated within a thin cage of pegylated material of 100–200 nanometers thick provides a viable option for islet transplantation. The aims of this study were to test islet viability and functionality following encapsulation within the pegylated cage, and functional efficacy in vivo in terms of graft-derived control of normoglycemia in diabetic mice. We first demonstrated that pegylation of the islet surface, plus or minus nanoparticles, improved long-term islet viability in vitro compared to non-pegylated (naked) control islets. Moreover, pegylation of the islets with nanoparticles was compatible with glucose-stimulated insulin secretion and insulin biogenesis. We next looked for functionality of the created “stealth” DBA/2 (H-2(d)) islets in vivo by comparing glycemic profiles across 4 groups of streptozotozin-induced diabetic C57BL/6 (H-2(b)) recipients of (i) naked islets; (ii) pegylated islets; (iii) pegylated islets with nanoparticles (empty); and (iv) pegylated islets with nanoparticles loaded with a cargo of leukemia inhibitory factor (LIF), a factor both promotes adaptive immune tolerance and regulates pancreatic β cell mass. Without any other treatment, normoglycemia was lost after 17 d (+/−7.5 d) in control group. In striking contrast, recipients in groups (ii), (iii), and (iv) showed long-term (>100 d) normoglycemia involving 30%; 43%, and 57% of the recipients in each respective group. In conclusion, construction of “stealth” islets by pegylation-based nanotherapeutics not only supports islet structure and function, but also effectively isolates the islets from immune-mediated destruction. The added value of nanoparticles to deliver immune modulators plus growth factors such as LIF expands the potential of this novel therapeutic approach to cell therapy for diabetes