Contribution of hydrophobic/hydrophilic modification on cationic chains of poly(ε-caprolactone)-graft-poly(dimethylamino ethylmethacrylate) amphiphilic co-polymer in gene delivery.

Nanoparticles (NPs) assembled from amphiphilic polycations have been certified as potential carriers for gene delivery. Structural modification of polycation moieties may be an efficient route to further enhance gene delivery efficiency. In this study two electroneutral monomers with different hydrophobicities, 2-hydroxyethyl methacrylate (HEMA) and 2-hydroxyethyl acrylate (HEA), were incorporated into the cationic poly(dimethylamino ethyl methacrylate) (PDMAEMA) side-chains of amphiphilic poly(ε-caprolactone)-graft-poly(dimethylamino ethylmethacrylate) (PCD) by random co-polymerization, to obtain poly(ε-caprolactone)-graft-poly(dimethylamino ethyl methacrylate-co-2-hydroxyethyl methacrylate) (PCD-HEMA) and poly(ε-caprolactone)-graft-poly(dimethylamino ethyl methacrylate-co-2-hydroxyethyl acrylate) (PCD-HEA). Minimal HEA or HEMA moieties in PDMAEMA do not lead to statistically significant changes in particle size, zeta potential, DNA condensation properties and buffering capacity of the naked NPs. However, the incorporation of HEMA and HEA lead to reductions and increases, respectively, in the surface hydrophilicity of the naked NPs and NPs/DNA complexes, which was confirmed by water contact angle assay. These simple modifications of PDMAEMA with HEA and HEMA moieties significantly affect the gene transfection efficiency on HeLa cells in vitro: PCD-HEMA NP/DNA complexes show a much higher transfection efficiency than PCD NPs/DNA complexes, while PCD-HEA NPs/DNA complexes show a lower transfection efficiency than PCD NP/DNA complexes. Fluorescence activated cell sorter and confocal laser scanning microscope results indicate that the incorporation of hydrophobic HEMA moieties facilitates an enhancement in both cellular uptake and endosomal/lysosomal escape, leading to a higher transfection efficiency. Moreover, the process of endosomal/lysosomal escape confirmed in our research that PCD and its derivatives do not just rely on the proton sponge mechanism, but also on membrane damage due to the polycation chains, especially hydrophobic modified ones. Hence, it is proved that hydrophobic modification of cationic side-chains is a crucial route to improve gene transfection mediated by polycation NPs.

Gene transfection efficacy and biocompatibility of polycation/DNA complexes coated with enzyme degradable PEGylated hyaluronic acid.

Coating the polycation/DNA binary complexes with PEGylated polyanions can improve long-circulation and biocompatibility in vivo. However, it has been certificated PEG dilemma can reduces gene transfection efficiency because of inhibition in cellular uptake and endosomal escape. Herein, two PEGylated anionic polymers, PEGylated hyaluronic acid (HgP) and PEGylated polyglutamic acid (PGgP) were synthesized to coat the binary complexes of core-shell cationic polycaprolactone-graft-poly (N, N-dimethylaminoethyl methacrylate) nanoparticles/DNA (NP-D). The effects of polyanion structure were evaluated in terms of particle size, zeta potential, cytotoxicity, cellular uptake and transfect efficiency in vitro and in vivo. In vitro study illustrated that HgP coated complexes showed better efficiencies in both cell uptake and transfection than PGgP coated complexes. The coating of HgP on NP-D improved the biocompatibility without reduction in cell uptake and transfection efficacy, and resulted in higher accumulation and gene expression in tumor after IV injection. The success of HgP coating in overcoming PEG dilemma is attributed to the hyaluronic acid (HA)-receptor-mediated endocytosis and outer shell-detachment through the hyaluronidases catalyzed degradation of HA. These results demonstrated that HgP was a promising anionic polymer for coating the polycation/DNA complexes and ternary complexes (HgP coated NP-D) hold promising potential for cancer therapy.

Polycation-detachable nanoparticles self-assembled from mPEG-PCL-g-SS-PDMAEMA for in vitro and in vivo siRNA delivery.

Long circulation, cell internalization, endosomal escape and small interfering RNA (siRNA) release to the cytoplasm are the prerequisite considerations for siRNA delivery vectors. Herein, a kind of sheddable nanoparticles (NPs) with micelle architecture for siRNA delivery were fabricated by using an intracellular-activated polycation-detachable copolymer (PECssD), which was prepared by introducing highly reducing environment-responsive disulfide linkages between PEGylated polycaprolactone (PCL) and the grafted polycation, poly(2-dimethylaminoethyl methacrylate) (PDMAEMA). The architecture of PECssD self-assembled NPs includes a biodegradable hydrophobic PCL core, a PEG shield and a detachable comb-like polycation surface. The stable nanosized complexes of PECssD NPs with siRNA, termed PECssD/siRNA micelleplexes, were formed, which could prolong circulation, improve accumulation and retention in tumor tissue, and be favorable for internalization. In particular, the cleavage of the disulfide linkages in the intracellular microenvironment and the subsequent dissociation of the PDMAEMA/siRNA polyplexes from the PEGylated PCL cores of PECssD/siRNA micelleplexes were also confirmed, which facilitated the endosomal escape and the efficient release of siRNA. As a result, the distribution of siRNA in cytoplasm was enhanced and subsequently promoted the efficiency of siRNA in gene silencing. Furthermore, systemic administration of the NPs carrying siPlk1 (polo-like kinase 1 specific siRNA) induced a tumor-suppressing effect in the HeLa-Luc xenograft murine model. Therefore, the devised strategy of the polycation-detachable copolymer PECssD NPs could address the requirements of the multistep systemic delivery process of siRNA. The hydrophobic core of the PECssD/siRNA micelleplexes is expected to entrap antitumor drugs or other therapeutic agents for combined therapies.

Intracellular cleavable poly(2-dimethylaminoethyl methacrylate) functionalized mesoporous silica nanoparticles for efficient siRNA delivery in vitro and in vivo.

A low cytotoxicity and high efficiency delivery system with the advantages of low cost and facile fabrication is needed for the application of small interfering RNA (siRNA) delivery both in vitro and in vivo. For these prerequisites, cationic polymer-mesoporous silica nanoparticles (ssCP-MSNs) were prepared by surface functionalized mesoporous silica nanoparticles with disulfide bond cross-linked poly(2-dimethylaminoethyl methacrylate) (PDMAEMA). In vitro and in vivo evaluations were performed. The synthesized ssCP-MSNs are 100-150 nm in diameter with a pore size of 10 nm and a positively charged surface with a high zeta potential of 27 mV. Consequently, the ssCP-MSNs showed an excellent binding capacity for siRNA, and an enhancement in the cell uptake and cytosolic availability of siRNA. Furthermore, the intracellular reducing cleavage of the disulfide bonds cross-linking the PDMAEMA segments led to intracellular cleavage of PDMAEMA from ssCP-MSNs, which facilitated the intracellular triggered release of siRNA. Therefore, promoted RNA interference was observed in HeLa-Luc cells, which was equal to that of Lipofectamine 2000. Significantly, compared to Lipofectamine 2000, the ssCP-MSNs were more biocompatible, with low cytotoxicity (even non-cytotoxicity) and promotion of cell proliferation to HeLa-Luc cells. The in vivo systemic distribution studies certified that ssCP-MSNs/siRNA could prolong the duration of siRNA in vivo, and that they accumulated in the adrenal gland, liver, lung, spleen, kidney, heart and thymus after intravenous injection. Encouragingly, with the ability to deliver siRNA to a tumor, ssCP-MSNs/siRNA showed a tumor suppression effect in the HeLa-Luc xenograft murine model after intravenous injection. Therefore, the ssCP-MSNs cationic polymer-mesoporous silica nanoparticles with low cytotoxicity are promising for siRNA delivery.

Binary and ternary complexes based on polycaprolactone-graft-poly (N, N-dimethylaminoethyl methacrylate) for targeted siRNA delivery.

Small interfering RNA (siRNA) is a powerful gene silencing tool and has promising prospects in basic research and the development of therapeutic reagents. However, the lack of an effective and safe tool for siRNA delivery hampers its application. Here, we introduced binary and ternary complexes that effectively mediated siRNA-targeted gene silencing. Both complexes showed excellent siRNA loading even at the low N/P/C ratio of 3:1:0. FACS and confocal microscopy demonstrated that nearly all cells robustly internalized siRNAs into the cytoplasm, where RNA interference (RNAi) occurred. Luciferase assay and Western blot verified that silencing efficacy reached >80%, and introducing folate onto the ternary complexes further enhanced silencing efficacy by about 10% over those without folate at the same N/P/C ratio. In addition, the coating of PGA-g-mPEG decreased the zeta potential almost to electroneutrality, and the MTT assay showed decreased cytotoxicity. In vivo distribution measurement and histochemical analysis executed in C57BL/6 and Hela tumor-bearing BALB/c nude mice showed that complexes accumulated in the liver, lungs, pancreas and tumors and were released slowly for a long time after intravenous injection. Furthermore, ternary complexes showed higher siRNA fluorescence intensity than binary complexes at the same N/P ratio in tumor tissues, those with folate delivered more siRNAs to tumors than those without folate, and more folate induced more siRNA transport to tumors. In addition, in vivo functional study showed that both binary and ternary complexes mediated down-regulation of ApoB in liver efficiently and consequently blocked the secretion of fatty acids into the blood, resulted in lipid accumulation in liver, liver steatosis and hepatic dysfunction. In conclusion, these complexes provided a powerful means of administration for siRNA-mediated treatment of liver-related diseases and various cancers, especial for pancreatic and cervical cancer.

Structural contributions of blocked or grafted poly(2-dimethylaminoethyl methacrylate) on PEGylated polycaprolactone nanoparticles in siRNA delivery.

The multiformity in polymer structure and conformation design provides a great potential in improving the gene silencing efficiency of siRNA by polymer vectors. In order to provide information on the polymer design for siRNA delivery, the structural contributions of blocked or grafted poly(2-dimethylaminoethyl methacrylate) on PEGylated polycaprolactone nanoparticles (NPs) in siRNA delivery were studied. Herein, two kinds of self-assembly nanoparticles (NPs) formed by amphiphilic cationic polymers, methoxy poly(ethylene glycol)-block-polycaprolactone-block-poly(2-dimethylaminoethyl methacrylate) (mPEG-PCL-b-PDMAEMA, PECbD) and methoxy poly(ethylene glycol)-block-(polycaprolactone-graft-poly(2-dimethylaminoethyl methacrylate)) (mPEG-PCL-g-PDMAEMA, PECgD), were used to deliver siRNA for in vitro and in vivo studies. The physiochemical properties including size and zeta potential of PECbD NPs/siRNA and PECgD NPs/siRNA complexes were characterized. In vitro cytotoxicity, cellular uptake and siRNA knockdown efficiency were evaluated in HeLa-Luc cells. The endosome escape and intracellular distribution of PECbD NPs/siRNA and PECgD NPs/siRNA in HeLa-Luc cells were also observed. In vivo polymer mediated siRNA delivery and the complexes distribution in isolated organs were studied using mice and tumor-bearing mice. At the same total degree of polymerization (DP) of DMAEMA, PECgD NPs/siRNA complexes possessed higher zeta potentials than PECbD NPs/siRNA complexes (at the same N/P ratio), which may be the reason that PECgD NPs/siRNA complexes can deliver more siRNA into the cytoplasm and lead to higher in vitro luciferase and lamin A/C silencing efficiency than PECbD NPs/siRNA complexes. The in vivo imaging measurement and histochemical analysis also confirmed that siRNA could be delivered to lungs, livers, pancreas and HeLa-Luc tumors more efficiently by PECgD NPs than PECbD NPs. Meanwhile, the PDMAEMA chains of PECgD could be shortened which provides benefits for clearing. Therefore, PECgD NPs have great potential to be used as efficient non-viral carriers for in vivo siRNA delivery.

Ternary complexes of amphiphilic polycaprolactone-graft-poly (N,N-dimethylaminoethyl methacrylate), DNA and polyglutamic acid-graft-poly(ethylene glycol) for gene delivery.

Binary complexes of cationic polymers and DNA were used commonly for DNA delivery, whereas, the excess cationic charge of the binary complexes mainly leads to high toxicity and unstability in vivo. In this paper, ternary complexes by coating polyglutamic acid-graft-poly(ethylene glycol)(PGA-g-mPEG) onto binary complexes of polycaprolactone-graft-poly(N,N-dimethylaminoethyl methacrylate) (PCL-g-PDMAEMA) nanoparticles (NPs)/DNA were firstly developed for effective and targeted gene delivery. The coating of PGA-g-mPEG was able to decrease the zeta potential of the nano-sized DNA complexes nearly to electroneutrality without interferring with DNA condensation ability. As a result, the stability, the escape ability from endosomes and the transfection efficiency of the complexes were enhanced. The ternary complexes of PCL-g-PDMAEMA NPs/DNA/PGA-g-mPEG demonstrated lower cytotoxicity in CCK-8 measurements and higher gene transfection efficiency than the binary complexes in vitro. In addition, Lactate dehydrogenase (LDH) assay was performed to quantify the membrane-damaging effects of the complexes, which is consistent with the conclusion of CCK-8 measurement for cytotoxicity assay. The in vivo imaging measurement and histochemical analysis of tumor sessions confirmed that the intravenous administration of the ternary complexes with red fluorescent protein (RFP) as payload led to protein expression in tumor, which was further enhanced by the targeted coating of PGA-g-PEG-folate.

Amphiphilic and biodegradable methoxy polyethylene glycol-block-(polycaprolactone-graft-poly(2-(dimethylamino)ethyl methacrylate)) as an effective gene carrier.

A group of amphiphilic cationic polymers, methoxy polyethylene glycol-block-(polycaprolactone-graft-poly(2-(dimethylamino)ethyl methacrylate)) (PECD), were synthesized by combining ring-opening polymerization (ROP) and atom transfer radical polymerization (ATRP) methods to form nanoparticles (NPs). The structures of these amphiphilic cationic polymers were characterized by (1)H NMR measurement. The PECD NPs have hydrophobic cores covered with hydrophilic PEG and cationic PDMAEMA chains. These self-assembly nanoparticles were characterized by dynamic light scattering (DLS) technique. PECD NPs can effectively condense DNA to form compact complexes of the size 65-160 nm suitable for gene delivery. The in vitro gene transfection studies of HeLa and HepG2 cells show that PECD NPs have better transfection efficiency compared to polyethylenimine (PEI) and Lipofectamine 2000 at low dose (N/P = 5). The cytotoxicity result shows that PECD NPs/DNA complexes at the optimal N/P ratio for transfection have comparable toxicity with PEI and Lipofectamine. These results indicate that PECD NPs have a great potential to be used as efficient polymeric carriers for gene transfection.