Biodegradable Tri-Block Copolymer Poly(lactic acid)-poly(ethylene glycol)-poly(l-lysine)(PLA-PEG-PLL) as a Non-Viral Vector to Enhance Gene Transfection

Low cytotoxicity and high gene transfection efficiency are critical issues in designing current non-viral gene delivery vectors. The purpose of the present work was to synthesize the novel biodegradable poly (lactic acid)-poly(ethylene glycol)-poly(l-lysine) (PLA-PEG-PLL) copolymer, and explore its applicability and feasibility as a non-viral vector for gene transport. PLA-PEG-PLL was obtained by the ring-opening polymerization of Lys(Z)-NCA onto amine-terminated NH₂-PEG-PLA, then acidolysis to remove benzyloxycarbonyl. The tri-block copolymer PLA-PEG-PLL combined the characters of cationic polymer PLL, PLA and PEG: the self-assembled nanoparticles (NPs) possessed a PEG loop structure to increase the stability, hydrophobic PLA segments as the core, and the primary ɛ-amine groups of lysine in PLL to electrostatically interact with negatively charged phosphate groups of DNA to deposit with the PLA core. The physicochemical properties (morphology, particle size and surface charge) and the biological properties (protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in HeLa and HepG2 cells) of the gene-loaded PLA-PEG-PLL nanoparticles (PLA-PEG-PLL NPs) were evaluated, respectively. Agarose gel electrophoresis assay confirmed that the PLA-PEG-PLL NPs could condense DNA thoroughly and protect DNA from nuclease degradation. Initial experiments showed that PLA-PEG-PLL NPs/DNA complexes exhibited almost no toxicity and higher gene expression (up to 21.64% in HepG2 cells and 31.63% in HeLa cells) than PEI/DNA complexes (14.01% and 24.22%). These results revealed that the biodegradable tri-block copolymer PLA-PEG-PLL might be a very attractive candidate as a non-viral vector and might alleviate the drawbacks of the conventional cationic vectors/DNA complexes for gene delivery in vivo.     

Introducing primary and tertiary amino groups into a neutral polymer: A simple way to fabricating highly efficient nonviral vectors for gene delivery

In this work, a brushed polycationic polymer with primary and tertiary amino groups was designed and synthesized for gene delivery. The backbone polymer was poly(N‐hydroxyethylacrylamide) (PHEAA) by the atom transfer radical polymerization (ATRP), and then 3,3′‐diaminodipropylamine (DPA) was grafted onto the PHEAA by the reaction between hydroxyl and the secondary amine. A brushed PHEAA‐DPA cationic polymer was achieved with primary and tertiary amino groups and the ratio was 2 : 1. The PHEAA₁₀₀‐DPA and PHEAA₂₀₀‐DPA could effectively condense plasmid DNA (pDNA) at the weight ratio of vector/DNA of 0.6 and 0.4, respectively. The cytotoxicity of PHEAA‐DPA/pDNA to COS‐7 cells and HepG‐2 cells within the weight ratio of vector/DNA of 16 : 1 was lower than that of PEI25k, and cell viability decreased with the increment of the weight ratio. Although the cytotoxicity of PHEAA₁₀₀‐DPA/pDNA was lower than PHEAA₂₀₀‐DPA/pDNA, the latter possessed higher transfection efficiency at the same weight ratio both in COS‐7 cells and HepG‐2 cells, compared with PEI25k, the transfection efficiency of PHEAA₂₀₀‐DPA/pDNA was better in COS‐7 cells and HepG‐2 cells with the weight ratio of 12 : 1 and 10 : 1, respectively. These results showed that the PHEAA‐DPA with less cytotoxicity and higher gene transfection efficiency has a broad perspective in gene therapy. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40468.     

壳聚糖复合纳米基因载体的制备

目的制备壳聚糖复合纳米基因载体,并探讨其理化性质、细胞毒性、稳定性及体外转染效率。方法采用复凝聚法制备包封聚乙烯亚胺(Polyethylenimine,PEI)/DNA复合物的壳聚糖复合纳米基因载体,用纳米粒度分析仪测定其粒径和Zeta电位;透射电镜观察其形态;MTT法检测其细胞毒性;在PBS溶液(pH 7.4)及含10%小牛血清的RPMI1640培养基中,于37℃条件下放置0、1、3、5 d,1%琼脂糖凝胶电泳检测其稳定性;体外转染CNE细胞,评价其转染活性。结果当N(PEI的氨基)/P(DNA的磷酸根)≥6时,能够形成稳定的壳聚糖复合纳米粒,平均粒径约为300 nm,表面电荷约为30 mV;壳聚糖复合纳米基因载体呈球形,圆整且分散性好;复合纳米基因载体的细胞毒性较低;1%琼脂糖凝胶电泳分析显示,DNA被完全包裹在复合纳米载体中,且5 d内无游离DNA释放;体外转染活性与壳聚糖/DNA复合物相比,提高了约1 000倍,且转染能力不受血清的干扰。结论制备的壳聚糖复合纳米基因载体是一种高效、低毒的非病毒载体,具有作为体内基因治疗载体的应用潜力。     

Degradable poly(ester amine) based on poly(ethylene glycol) dimethacrylate and polyethylenimine as a gene carrier

Degradable poly(ester amine) (PEA) based on poly(ethylene glycol) dimethacrylate (PMEG) and polyethylenimine (PEI) were synthesized by Michael addition reaction. The ratios of PEI to PMEG in PEAs were 0.99, 1.02, and 1.07 with corresponding number‐average molecular weight of 1.3 × 10⁴, 1.2 × 10⁴, and 0.9 × 10⁴, respectively. Degradation rate of PEA at pH 7.4 was higher than that at pH 5.6. Good plasmid condensation and protection ability was shown when N/P molar ratio of PEA to DNA was above 15 (N: nitrogen element in PEA, P: phosphate in DNA). PEA/DNA complexes had positive zeta potential, narrow size distribution, good dispersity, and spheric shape with size below 250 nm when N/P ratio was above 30, suggestion of their endocytosis potential. Compared with PEI 25 KDa, the PEAs showed essential nontoxic to HeLa, HepG2 and 293T cells. With an increase in the molecular weight of PMEG, the transfection efficiency of PEAs in HeLa, HepG2 and 293T showed a tendency to decrease as well as the percent decrease of gene transfection efficiency with serum. The mechanism of PEA‐mediated gene transfection was attributed to “proton sponge effect” of PEI in the PEA. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Paclitaxel-loaded nanoparticles of star-shaped cholic acid-core PLA-TPGS copolymer for breast cancer treatment

A system of novel nanoparticles of star-shaped cholic acid-core polylactide-d-α-tocopheryl polyethylene glycol 1000 succinate (CA-PLA-TPGS) block copolymer was developed for paclitaxel delivery for breast cancer treatment, which demonstrated superior in vitro and in vivo performance in comparison with paclitaxel-loaded poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles and linear PLA-TPGS nanoparticles. The paclitaxel- or couramin 6-loaded nanoparticles were fabricated by a modified nanoprecipitation method and then characterized in terms of size, surface charge, surface morphology, drug encapsulation efficiency, and in vitro drug release. The CA-PLA-TPGS nanoparticles were found to be spherical in shape with an average size of around 120 nm. The nanoparticles were found to be stable, showing no change in the particle size and surface charge during 90-day storage of the aqueous solution. The release profiles of the paclitaxel-loaded nanoparticles exhibited typically biphasic release patterns. The results also showed that the CA-PLA-TPGS nanoparticles have higher antitumor efficacy than the PLA-TPGS nanoparticles and PLGA nanoparticles in vitro and in vivo. In conclusion, such nanoparticles of star-shaped cholic acid-core PLA-TPGS block copolymer could be considered as a potentially promising and effective strategy for breast cancer treatment.     

Polyethylene glycol‐polyethylenimine‐tetrachloroplatinum (IV): A novel conjugate with good abilities of antitumor and gene delivery

It is much importance to develop novel multifunctional delivery systems for the combination therapy of drug and gene. In this work, a novel conjugate, polyethylene glycol‐polyethylenimine‐tetrachloroplatinum (IV) (PEG‐PEI‐Pt), with good abilities of antitumor and gene delivery was proposed by combining PEG (Mw 3400 Da), low molecular weight PEI (Mw 800 Da), and tetrachloroplatinum (IV). The antitumoral and gene transfection activities of PEG‐PEI‐Pt were analyzed in many tumor (A549, A375, HepG‐2, HuH‐7, and B16 cells) and normal (COS‐7 cells) cell lines. Similar to cisplatin (one platinum anticancer drug), PEG‐PEI‐Pt showed much higher sensitivity in tumor cells than in normal cells. More importantly, PEG‐PEI‐Pt had a potential to treat drug‐resistant tumors. Almost no transfection efficiency was observed for PEI (Mw 800 Da) and PEG‐PEI. Very interestingly, PEG‐PEI‐Pt could condense plasmid DNA efficiently, and exhibited good transfection efficiency in B16, HepG‐2, A375 and COS‐7 cells, comparable to even higher than PEI 25 kDa. In addition, PEG‐PEI‐Pt could also effectively deliver siRNA into the cytoplasm of tumor cells. With the good antitumoral and gene delivery abilities, PEG‐PEI‐Pt may have a great potential for combination therapy of drug and gene. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Folic‐Acid‐Targeted Self‐Assembling Supramolecular Carrier for Gene Delivery

A targeting gene carrier for cancer‐specific delivery was successfully developed through a “multilayer bricks‐mortar” strategy. The gene carrier was composed of adamantane‐functionalized folic acid (FA‐AD), an adamantane‐functionalized poly(ethylene glycol) derivative (PEG‐AD), and β‐cyclodextrin‐grafted low‐molecular‐weight branched polyethylenimine (PEI‐CD). Carriers produced by two different self‐assembly schemes, involving either precomplexation of the PEI‐CD with the FA‐AD and PEG‐AD before pDNA condensation (Method A) or pDNA condensation with the PEI‐CD prior to addition of the FA‐AD and PEG‐AD to engage host–guest complexation (Method B) were investigated for their ability to compact pDNA into nanoparticles. Cell viability studies show that the material produced by the Method A assembly scheme has lower cytotoxicity than branched PEI 25 kDa (PEI‐25KD) and that the transfection efficiency is maintained. These findings suggest that the gene carrier, based on multivalent host–guest interactions, could be an effective, targeted, and low‐toxicity carrier for delivering nucleic acid to target cells.     

Hyperbranched PEI grafted by hydrophilic amino acid segment poly[N‐(2‐hydroxyethyl)‐L‐glutamine] as an efficient nonviral gene carrier

A polyethylenimine‐poly(hydroxyethyl glutamine) copolymer (PEI‐PHEG) was designed and synthesized as a gene delivery system. The molecular structure of PEI‐PHEG was characterized using nuclear magnetic resonance. Moreover, PEI‐PHEG/pDNA complexes were fabricated and characterized by gel retardation assay, particle size analysis, and zeta potential analysis. The transfection efficiency and cytotoxicity of PEI‐PHEG were evaluated using human cervical carcinoma (HeLa), human embryonic kidney (HEK293), and murine colorectal adenocarcinoma (CT26) cells in vitro. The results show that PEI‐PHEG could effectively form positively charged nano‐sized particles with pDNA; the particle size was in a range of 130.2 to 173.0 nm and the zeta potential was in a range of 27.6 to 41.0 mV. PEI‐PHEG exhibited much lower cytotoxicity and higher gene transfection efficiency than PEI‐25K with different cell lines in vitro. An animal test was also conducted on a Lewis Lung Carcinoma tumor model in C57/BL6 mice by using subcutaneous intratumoral administration. The results show that in vivo transfection efficiency of PEI‐PHEG was improved greatly compared with that of commercial PEI‐25K. These results demonstrate that PEI‐PHEG can be a potential nonviral vector for gene delivery systems both in vitro and in vivo. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Poly-L-Lysine–Lactobionic Acid-Capped Selenium Nanoparticles for Liver-Targeted Gene Delivery

Liver cancer is currently regarded as the second leading cause of cancer-related mortality globally and is the sixth most diagnosed malignancy. Selenium nanoparticles (SeNPs) have attracted favorable attention as nanocarriers for gene therapy, as they possess beneficial antioxidant and anticancer properties. This study aimed to design, functionalize and characterize SeNPs to efficiently bind, protect and deliver pCMV–Luc DNA to hepatocellular carcinoma (HepG2) cells. The SeNPs were synthesized by ascorbic acid reduction and functionalized with poly-L-lysine (PLL) to stabilize and confer positive charges to the nanoparticles. The SeNPs were further decorated with lactobionic acid (LA) to target the asialoglycoprotein receptors abundantly expressed on the surface of the hepatocytes. All SeNPs were spherical, in the nanoscale range (<130 nm) and were capable of successfully binding, compacting and protecting the pDNA against nuclease degradation. The functionalized SeNP nanocomplexes exhibited minimal cytotoxicity (<30%) with enhanced transfection efficiency in the cell lines tested. Furthermore, the targeted SeNP (LA–PLL–SeNP) nanocomplex showed significant (* p < 0.05, ** p < 0.01, **** p < 0.0001) transgene expression in the HepG2 cells compared to the receptor-negative embryonic kidney (HEK293) cells, confirming receptor-mediated endocytosis. Overall, these functionalized SeNPs exhibit favorable features of suitable gene nanocarriers for the treatment of liver cancer.     

A novel nonviral gene delivery vector: Low‐molecular‐weight polyethylenimine‐graft‐ovalbumin

A novel vector for gene delivery was synthesized. Here the ovalbumin (OVA) acts as a core and low‐molecular‐weight PEI600 was grafted to its surface. The finally product was characterized (¹H‐NMR, UV, and TGA) and its biophysical properties such as DNA condensing, particle size, and zeta potential were determined. The agarose gel assay indicated that OVA‐PEI600 could efficiently condense plasmid DNA. Its particle size was about 150 nm and zeta potential was around +20 mV. The MTT assay showed that the cytotoxicity of OVA‐PEI600 was less than PEI25 kDa. Its transfection efficiency in SKOV‐3 and HepG2 cell lines was higher than that of PEI600 and comparable to PEI25 kDa. In vivo, luciferase activity could be tested in liver, spleen, kidney, lung, and blood serum, respectively, in mice. The core‐shell structure of OVA‐PEI600 provided a novel strategy for nonviral gene delivery. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Guanidinylated poly(allyl amine) as a gene carrier

Guanidinylated poly(allyl amine) (GA) was synthesized and used as a gene carrier. The degree of guanidinylation in GA increased linearly when the feed ratio of guanidino groups in 1H‐pyrazole‐1‐carboxamidine to amino groups in poly(allyl amine) (PA) was below 0.7, and the amino groups of poly(allyl amine) with a weight‐average molecular weight of 15,000 (PA15) were almost replaced with guanidino groups when the ratio reached 2. GA showed good plasmid condensation and protection ability. Nanoparticles with a narrow size distribution, good dispersity, and spherical shape could be assembled between GA and DNA. With an increase in the N/P ratio [where N is the amount of nitrogen in the polycation (for GA, three nitrogens per guanidino group) and P is the amount of plasmid phosphate in the DNA as moles] or the degree of guanidinylation, the ζ‐potential of GA/DNA nanoparticles increased, whereas the sizes of GA/DNA nanoparticles decreased sharply with increasing N/P ratios. Compared with polyethylenimine with a weight‐average molecular weight of 25,000 and PA15, GA essentially showed decreased cytotoxicity to HeLa, 293T, and HepG2 cell lines, and guanidinylated PA15 exhibited the lowest cytotoxicity. Guanidinylation of PA enhanced its gene transfection. This enhancement was dependent on the degree of guanidinylation and the cell lines. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

CHO-DG44细胞瞬时转染条件的优化

目的优化CHO-DG44细胞瞬时转染的条件。方法采用TubeSpin一次性生物反应器,以绿色荧光蛋白(Green fluorescence protein,GFP)为报告基因,氯醚酰亚胺(Polyether imide,PEI)为转染试剂,将质粒pIRESneo3-eGFP瞬时转染CHO-DG44细胞,优化瞬时转染的基本条件[细胞密度、DNA浓度和DNA:PEI比例(w/w)]以及其他条件(换液、渗透压和温度),采用优化的条件转染质粒pIRESneo3-eGFP,流式细胞术检测细胞转染效率,生物发光仪检测相对荧光强度。结果 CHO-DG44细胞瞬时转染的最佳条件为:采用XLG-P8培养基进行转染,细胞密度为2×106个/ml,DNA浓度为6.25μg/5 ml,DNA∶PEI(w/w)比例为1∶5;转染后4 h更换新鲜培养基,添加30 mmol/L NaCl,并于31℃继续培养。在此条件下,CHO-DG44细胞的瞬时转染效率可达81.45%,相对荧光强度可达9×105RFU/106cells。结论优化了CHO-DG44细胞瞬时转染的条件,为下一步药物蛋白的研发奠定了基础。     

Novel Cholesterol-Based Cationic Lipids as Transfecting Agents of DNA for Efficient Gene Delivery

The design, synthesis and biological evaluation of the cationic lipid gene delivery vectors based on cholesterol and natural amino acids lysine or histidine are described. Cationic liposomes composed of the newly synthesized cationic lipids 1a or 1b and neutral lipid DOPE (1,2-dioleoyl-l-α-glycero-3-phosphatidyl-ethanolamine) exhibited good transfection efficiency. pEGFP-N₁ plasmid DNA was transferred into 293T cells by cationic liposomes formed from cationic lipids 1a and 1b, and the transfection activity of the cationic lipids was superior (1a) or parallel (1b) to that of the commercially available 3β-[N-(N'',N''-dimethylaminoethyl)-carbamoyl] cholesterol (DC-Chol) derived from the same cholesterol backbone with different head groups. Combined with the results of agarose gel electrophoresis, transfection experiments with various molar ratios of the cationic lipids and DOPE and N/P (+/−) molar charge ratios, a more effective formulation was formed, which could lead to relatively high transfection efficiency. Cationic lipid 1a represents a potential agent for the liposome used in gene delivery due to low cytotoxicity and impressive gene transfection activity.     

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.     

A systemic gene vector constructed by zwitterionic polymer modified low molecular weight PEI

Good blood compatibility and long-term circulation are very important to polycationic systemic gene vectors. In this work, polysulfobetaine-modified low molecular weight polyethyleneimine (LMW PEI, 1.8k) was synthesized and investigated as a vector for gene delivery in vitro and in vivo. PHEAA-b-PMPDSAH was synthesized via atomic transfer radical polymerization method, and then LMW PEI was grafted to PHEAA-b-PMPDSAH by an amido–hydroxy reaction. Incorporation of PMPDSAH into PEI was shown to retain the uncompromised ability to condense DNA into nanocomplexes. MTT assays revealed that the cytotoxicity of LMW PEI-PHEAA-b-PMPDSAH/DNA complexes was lower than that of PEI (25k)/DNA and LMW PEI-PHEAA/DNA complexes. LMW PEI-PHEAA-b-PMPDSAH₅₀ was much superior to PEI (25k) in mediating gene transfection in the presence of 10% serum. At higher serum contents, the transfection of LMW PEI-PHEAA and PEI (25k) was deteriorated, whereas LMW PEI-PHEAA-b-PMPDSAH₅₀ still retained better transfection efficiency, 8-fold more effective than PEI (25k). The expression of red fluorescence protein (RFP) was evaluated by small animal in vivo fluorescence imaging system and the results showed that the expression of RFP was much higher in the mice injected with LMW PEI-PHEAA-b-PMPDSAH₅₀/pDNA-RFP than with LMW PEI-HEAA/pDNA-RFP. Both in vitro and in vivo results suggested that LMW PEI-PHEAA-b-PMPDSAH_X copolymer holds a great potential as a vector for systemic gene therapy.     

EGFP-PDGFRβ融合基因表达载体的构建及其转染COS-7细胞条件的优化

目的构建EGFP-PDGFRβ融合基因表达载体,并优化PEI介导基因转染COS-7细胞的条件。方法PCR扩增EGFP和PDGFRβ基因,依次连接至pcDNA3.1(+)质粒中,构建融合基因表达载体H-E-P-pcDNA3.1(+),经PEI介导转染COS-7细胞,并对转染条件进行优化。结果融合基因表达载体经酶切及测序证明构建正确。经PEI介导转染COS-7细胞的最佳条件为:DNA浓度2μg/ml,N/P比值15.5,在无血清条件下转染。此条件适用于滚瓶培养细胞的转染。结论已成功构建了EGFP-PDGFRβ融合基因表达载体,并优化了经PEI介导转染COS-7细胞的条件。     

Cytotoxicity mechanism of non-viral carriers polyethylenimine and poly-l-lysine using real time high-content cellular assay

Cationic polymers polyethylenimine (PEI) and poly-l-lysine (PLL) used as non-viral gene/drug delivery vehicles, showed high cytotoxicity but their molecular mechanisms of toxicity have been inadequately understood. Therefore, we tried to investigate the toxicity pathway triggered by these polymers through a high-content cellular imaging technique. The results revealed that PEI induced apoptosis via an intrinsic pathway, whereas PLL showed cytotoxicity through both intrinsic and extrinsic caspase cascade. Both PEI and PLL provide different apoptotic activities on HepG2 cells depending on their molecular weight. The degree of apoptosis of PEI also depends on its structure. The branched PEI showed higher cytotoxicity than linear PEI. This observation was verified through Annexin V-FITC/PI assay and real-time high-content monitoring of cytosolic calcium, mitochondrial membrane disruption, and caspase-3 activation methods. The study therefore provides important implications on the molecular mechanisms of PEI and PLL induced cytotoxicity.     

Transgene therapy for glomerulonephritis with rat anti-Thy1.1 introduced by mesangial cell vector with a polyethylenimine/decorin nanocomplex

ABSTRACT: Polyethylenimine (PEI), a cationic polymer, is one of the most efficient non-viral vectors for transgene therapy. Decorin (DCN), a leucine-rich proteoglycan secreted by glomerular mesangial cells (MC), is a promising anti-fibrotic agent for the treatment of glomerulonephritis. In this study, we used PEI-DCN nanocomplexes with different N/P ratios to transfect MC in vitro and deliver the MC vector with PEI-DCN expressing into rat anti-Thy1.1 nephritis kidney tissue via injection into the left renal artery in vivo. The PEI-plasmid DNA complex at N/P 20 had the highest level of transfection efficiency and the lowest level of cytotoxicity in cultured MC. Following injection, the ex vivo gene was transferred successfully into the glomeruli of the rat anti-Thy1.1 nephritis model by the MC vector with the PEI-DCN complex. The exogenous MC with DCN expression was located mainly in the mesangium and the glomerular capillary. Over-expression of DCN in diseased glomeruli could result in the inhibition of collagen IV deposition and MC proliferation. The pathological changes of rat nephritis were alleviated following injection of the vector. These findings demonstrate that the DCN gene delivered by the PEI-DNA nanocomplex with the MC vector is a promising therapeutic method for the treatment of glomerulonephritis.     

Redox‐responsive polyethyleneimine‐coated magnetic iron oxide nanoparticles for controllable gene delivery and magnetic resonance imaging

Recently, theranostic candidates that provide a combination of gene delivery and image diagnosis have attracted much interest in medical research. However, there are still many challenges for their clinical applications, such as uncontrollable gene delivery, high cytotoxicity, low transfection efficiency and reduced image contrast. Herein, redox‐responsive polyethyleneimine‐coated magnetic iron oxide nanoparticles (IONs@rPEI) were prepared for both efficient gene delivery and magnetic resonance (MR) imaging. Firstly, crosslinked rPEI was synthesized by Michael addition reaction with N,N‐bis(acryloyl)cystamine, dopamine and low‐molecular‐weight branched PEI. The rPEI was then coated onto IONs by ligand exchange reaction forming IONs@rPEI. The physicochemical properties of the IONs@rPEI, such as chemical structure, size, zeta potential and DNA condensation ability, were investigated. In addition, a rapid degradation of the as‐prepared nanoparticles was observed, which was triggered by reducing glutathione via destruction of disulfide linkages suggesting a potential controllable DNA release in tumor cells. In MR imaging detection, the IONs@rPEI had a high T₂ relaxivity of 81 L mmol^?1 s^?1 indicating a potential usage as MR imaging contrast reagent. In cell assay, the IONs@rPEI exhibited low cytotoxicity and good transfection efficiency. In conclusion, the as‐prepared crosslinked IONs@rPEI can be used as a promising technology platform for gene therapy and MR imaging in theranostics. © 2019 Society of Chemical Industry     

Antheraea pernyi Silk Fibroin-Coated PEI/DNA Complexes for Targeted Gene Delivery in HEK 293 and HCT 116 Cells

Polyethylenimine (PEI) has attracted much attention as a DNA condenser, but its toxicity and non-specific targeting limit its potential. To overcome these limitations, Antheraea pernyi silk fibroin (ASF), a natural protein rich in arginyl-glycyl-aspartic acid (RGD) peptides that contains negative surface charges in a neutral aqueous solution, was used to coat PEI/DNA complexes to form ASF/PEI/DNA ternary complexes. Coating these complexes with ASF caused fewer surface charges and greater size compared with the PEI/DNA complexes alone. In vitro transfection studies revealed that incorporation of ASF led to greater transfection efficiencies in both HEK (human embryonic kidney) 293 and HCT (human colorectal carcinoma) 116 cells, albeit with less electrostatic binding affinity for the cells. Moreover, the transfection efficiency in the HCT 116 cells was higher than that in the HEK 293 cells under the same conditions, which may be due to the target bonding affinity of the RGD peptides in ASF for integrins on the HCT 116 cell surface. This result indicated that the RGD binding affinity in ASF for integrins can enhance the specific targeting affinity to compensate for the reduction in electrostatic binding between ASF-coated PEI carriers and cells. Cell viability measurements showed higher cell viability after transfection of ASF/PEI/DNA ternary complexes than after transfection of PEI/DNA binary complexes alone. Lactate dehydrogenase (LDH) release studies further confirmed the improvement in the targeting effect of ASF/PEI/DNA ternary complexes to cells. These results suggest that ASF-coated PEI is a preferred transfection reagent and useful for improving both the transfection efficiency and cell viability of PEI-based nonviral vectors.