Gene Transfection Mediated by Catiomers Requires Free Highly Charged Polymer Chains To Overcome Intracellular Barriers

The prospective use of the block copolymers poly­(ethylene oxide)₁₁₃-<i>b</i>-poly­[2-(diethylamino)­ethyl methacrylate]₅₀ (PEO₁₁₃-<i>b</i>-PDEA₅₀) and poly­[oligo­(ethylene glycol)­methyl ether methacrylate]₇₀-<i>b</i>-poly­[oligo­(ethylene glycol)­methyl ether methacrylate₁₀-<i>co</i>-2-(diethylamino)­ethyl methacrylate₄₇-<i>co</i>-2-(diisopropylamino)­ethyl methacrylate₄₇] (POEGMA₇₀-<i>b</i>-P­(OEGMA₁₀-<i>co</i>-DEA₄₇-<i>co</i>-DPA₄₇)) as nonviral gene vectors was evaluated. The polymers are able to properly condense DNA into nanosized particles (<i>R</i>_H ≈ 75 nm), which are marginally cytotoxic and can be uptaken by cells. However, the green fluorescent protein (GFP) expression assays evidenced that DNA delivery is essentially negligible meaning that intracellular trafficking hampers efficient gene release. Subsequently, we demonstrate that cellular uptake and particularly the quantity of GFP-positive cells are substantially enhanced when the block copolymer polyplexes are produced and further supplemented by BPEI chains (branched polyethylenimine). The dynamic light scattering/electrophoretic light scattering/isothermal titration calorimetry data suggest that such a strategy allows the adsorption of BPEI onto the surface of the polyplexes, and this phenomenon is responsible for increasing the size and surface charge of the assemblies. Nevertheless, most of the BPEI chains remain freely diffusing in the systems. The biological assays confirmed that cellular uptake is enhanced in the presence of BPEI and principally, the free highly charged polymer chains play the central role in intracellular trafficking and gene transfection. These investigations pointed out that the transfection efficiency versus cytotoxicity issue can be balanced by a mixture of BPEI and less cytotoxic agents such as for instance the proposed block copolymers

Nanoparticle–Cell Interactions: Surface Chemistry Effects on the Cellular Uptake of Biocompatible Block Copolymer Assemblies

The development of nanovehicles for intracellular drug delivery is strongly bound to the understating and control of nanoparticles cellular uptake process, which in turn is governed by surface chemistry. In this study, we explored the synthesis, characterization, and cellular uptake of block copolymer assemblies consisting of a pH-responsive poly­[2-(diisopropyl­amino)­ethyl methacrylate] (PDPA) core stabilized by three different biocompatible hydrophilic shells (a zwitterionic type poly­(2-methacryl­oyloxyethyl phosphorylcholine) (PMPC) layer, a highly hydrated poly­(ethylene oxide) (PEO) layer with stealth effect, and an also proven nontoxic and nonimmunogenic poly­(<i>N</i>-(2-hydroxypropyl)­methacrylamide) (PHPMA) layer). All particles had a spherical core–shell structure. The largest particles with the thickest hydrophilic stabilizing shell obtained from PMPC₄₀-<i>b</i>-PDPA₇₀ were internalized to a higher level than those smaller in size and stabilized by PEO or PHPMA and produced from PEO₁₂₂-<i>b</i>-PDPA₄₃ or PHPMA₆₄-<i>b</i>-PDPA₇₂, respectively. Such a behavior was confirmed among different cell lines, with assemblies being internalized to a higher degree in cancer (HeLa) as compared to healthy (Telo-RF) cells. This fact was mainly attributed to the stronger binding of PMPC to cell membranes. Therefore, cellular uptake of nanoparticles at the sub-100 nm size range may be chiefly governed by the chemical nature of the stabilizing layer rather than particles size and/or shell thickness

Efficient Condensation of DNA into Environmentally Responsive Polyplexes Produced from Block Catiomers Carrying Amine or Diamine Groups

The intracellular delivery of nucleic acids requires a vector system as they cannot diffuse across lipid membranes. Although polymeric transfecting agents have been extensively investigated, none of the proposed gene delivery vehicles fulfill all of the requirements needed for an effective therapy, namely, the ability to bind and compact DNA into polyplexes, stability in the serum environment, endosome-disrupting capacity, efficient intracellular DNA release, and low toxicity. The challenges are mainly attributed to conflicting properties such as stability vs efficient DNA release and toxicity vs efficient endosome-disrupting capacity. Accordingly, investigations aimed at safe and efficient therapies are still essential to achieving gene therapy clinical success. Taking into account the mentioned issues, herein we have evaluated the DNA condensation ability of poly­(ethylene oxide)₁₁₃-<i>b</i>-poly­[2-(di­iso­propyl­amino)­ethyl meth­acrylate]₅₀ (PEO₁₁₃-<i>b</i>-PDPA₅₀), poly­(ethylene oxide)₁₁₃-<i>b</i>-poly­[2-(di­ethyl­amino)­ethyl meth­acrylate]₅₀ (PEO₁₁₃-<i>b</i>-PDEA₅₀), poly­[oligo­(ethylene glycol)­methyl ether meth­acrylate]₇₀-<i>b</i>-poly­[oligo­(ethylene glycol)­methyl ether meth­acrylate₁₀-<i>co</i>-2-­(diethylamino)­ethyl meth­acrylate₄₇-<i>co</i>-2-(diisopropylamino)­ethyl meth­acrylate₄₇] (POEGMA₇₀-<i>b</i>-P­(OEGMA₁₀-<i>co</i>-DEA₄₇-<i>co</i>-DPA₄₇), and poly­[oligo­(ethylene glycol)­methyl ether meth­acrylate]₇₀-<i>b</i>-poly­{oligo­(ethylene glycol)­methyl ether meth­acrylate₁₀-<i>co</i>-2-methyl­acrylic acid 2-[(2-(di­methyl­amino)­ethyl)­methyl­amino]­ethyl ester₄₄} (POEGMA₇₀-<i>b</i>-P­(OEGMA₁₀-<i>co</i>-DAMA₄₄). Block copolymers PEO₁₁₃-<i>b</i>-PDEA₅₀ and POEGMA₇₀-<i>b</i>-P­(OEGMA₁₀-<i>co</i>-DEA₄₇-<i>co</i>-DPA₄₇) were evidenced to properly condense DNA into particles with a desirable size for cellular uptake via endocytic pathways (<i>R</i>_H ≈ 65–85 nm). The structure of the polyplexes was characterized in detail by scattering techniques and atomic force microscopy. The isothermal titration calorimetric data revealed that the polymer/DNA binding is endothermic; therefore, the process in entropically driven. The combination of results supports that POEGMA₇₀-<i>b</i>-P­(OEGMA₁₀-<i>co</i>-DEA₄₇-<i>co</i>-DPA₄₇) condenses DNA more efficiently and with higher thermodynamic outputs than does PEO₁₁₃-<i>b</i>-PDEA₅₀. Finally, circular dichroism spectroscopy indicated that the conformation of DNA remained the same after complexation and that the polyplexes are very stable in the serum environment