Bioreducible cationic random copolymer for gene delivery

Cationic polymers have been widely investigated for gene delivery, although their low transfection efficiency and high cytotoxicity limit their application. We synthesized a bioreducible cationic random copolymer, poly(cystamine bisacylamide‐aminoethyl piperazine)‐co‐poly(cystamine bisacylamide‐histamine) (denoted as CBA‐AEP‐His) from N,N′‐cystamine bis acrylamide (CBA) with aminoethyl piperazine (AEP) and histamine (His). CBA‐AEP‐His copolymer possesses disulfide linkages that endow it with redox‐responsivity to the intracellular environment. This polymer efficiently condenses pZNF580 into complexes with the size of 160 ± 4 nm to 280 ± 5 nm and positive zeta potential of 20 ± 0.3 mV to 30 ± 0.4 mV. The gel‐retardation assay shows that CBA‐AEP‐His can retard pZNF580 even at a low mass ratio of 1/1. The gene complexes were triggered to release pZNF580 when exposed to the reducing environment of dithiothreitol (DTT). CBA‐AEP‐His random copolymer presented higher buffer capacity owing to its His moieties, which protected pZNF580 from DNase degradation. The gene transfection results reveal that CBA‐AEP‐His can efficiently deliver pZNF580 and transfect EA. Hy926 cells. The MTT assay indicates that CBA‐AEP‐His and its complexes exhibit lower cytotoxicity than PEI25KDa. These results illustrate that CBA‐AEP‐His had promising properties for gene delivery, which may provide a suitable platform for the development of a non‐viral gene carrier.     

A degradable hyperbranched poly(ester amine) based on poloxamer diacrylate and polyethylenimine as a gene carrier

Polyethylenimine (PEI) is a well-known cationic polymer which has high transfection efficiency due to its buffering effect. However, nondegradability, cytotoxicity, aggregation, and short-circulation time in vivo still need to be overcome for a successful gene delivery. Degradable, hyperbranched poly(ester amine)s (PEAs) based on poloxamer diacrylate and low molecular weight branched PEI, were successfully synthesized and evaluated as a nonviral gene carrier. The PEAs were obtained in significant yields through Michael type addition reaction of diacrylate monomers and low molecular weight branched PEI. Analysis of degradation products by the reduction in molecular weight demonstrated that PEAs degrade in a controlled fashion. The PEA showed good DNA binding ability and the sizes of complexes under physiological condition were below 150 nm, implicating its potential for intracellular delivery. It showed lower cytotoxicity in three different cell lines (A549, 293T, and HepG2) compared with PEI 25K. PEAs showed much higher transfection efficiencies in three cell lines compared with PEI 25K and PEI 1.8K, and revealed little serum dependency in A549 cell line when the content of poloxamer in the PEA was increased up to 30%.     

Polyplex Particles Based on Comb‐Like Polyethylenimine/Poly(2‐ethyl‐2‐oxazoline) Copolymers: Relating Biological Performance with Morphology and Structure

The present contribution is focused on feasibility of using comb‐like copolymers of polyethylenimine with poly(2‐ethyl‐2‐oxazoline) (LPEI‐comb‐PEtOx) with varying grafting densities and degrees of polymerization of PEI and PEtOx to deliver DNA molecules into cells. The copolymers form small and well‐defined particles at elevated temperatures, which are used as platforms for binding and condensing DNA. The electrostatic interactions between particles and DNA result in formation of sub‐100 nm polyplex particles of narrow size distribution and different morphology and structure. The investigated gene delivery systems exhibit transfection efficiency dependent on the copolymer chain topology, shape of the polyplex particles, and internalization pathway. Flow cytometry shows enhanced transfection efficiency of the polyplexes with elongated and ellipsoidal morphology. The preliminary biocompatibility study on a panel of human cell lines shows that pure copolymers and polyplexes thereof are practically devoid of cytotoxicity.     

Poly(ethyleneimine)‐Mediated Large‐Scale Transient Gene Expression: Influence of Molecular Weight,Polydispersity and N‐Propionyl Groups

Three synthesis lots of linear poly(ethyleneimine) (PEI) are compared to a fully hydrolyzed linear PEI (commercially available as PEI “Max”) regarding structure, polyplex formation with plasmid DNA, and transfection of suspension‐adapted HEK‐293E cells. PEI “Max” binds DNA more efficiently than the other PEIs, but it is the least effective in terms of transient recombinant protein yield. One PEI lot is fractionated by means of SEC. The fractions of high‐$\overline {M} _{{\rm n}} $ PEI are the most efficient for complex formation and transfection. Nevertheless, the highest transient recombinant protein yields are achieved with unfractionated PEI. The results demonstrate that the polydispersity and charge density of linear PEI are important parameters for gene delivery to suspension‐adapted HEK‐293E cells.

     

基因载体材料聚乙烯亚胺与磷脂酰胆碱脂质体的相互作用对膜结构的影响

采用动态光散射、荧光光谱、zeta电位测定和等温滴定量热技术分析了分子量分别为25000,10000和1800的聚乙烯亚胺(PEI)与二油酰磷脂酰胆碱(DOPC)脂质体的相互作用及其对脂质体膜内环境极性和膜通透性的影响.结果表明,PEI通过氨基与DOPC的磷脂基团和胆碱基团产生氢键或范德华作用,从而与脂质体结合形成复合物;低浓度PEI(0.075 mg/mL)导致DOPC脂质体的聚集和表面电位的增加,但未引起脂质体膜融合和表面电位反转;进一步增加PEI的浓度对脂质体表面电位的影响很小,而结合在表面的PEI分子链之间的排斥作用阻碍了脂质体聚集.PEI分子与DOPC脂质体的结合降低了脂质分子碳氢链的堆积密度和脂质体膜内环境的疏水性,从而增强了钙黄绿素和槲皮素在脂质体膜中的通透性.PEI与DOPC脂质体的相互作用具有明显的分子尺寸效应,增大PEI的分子量可以增强与脂质体的相互作用及对脂质体膜结构的影响.     

High-molecular-weight polyethyleneimine conjuncted pluronic for gene transfer agents

In order to enhance the gene delivery efficiency and decrease cytotoxicity of polyplexes, copolymers consisting of branched polyethyleneimine (PEI) 25 kDa grafted with Pluronic (F127, F68, P105) were successfully synthesized using a simple two-step procedure. The copolymers were tested for cytotoxicity and DNA condensation and complexation properties. Their polyplexes with plasmid DNA were characterized in terms of DNA size and surface charge and transfection efficiency. The complex sizes were below 300 nm, which implicated their potential for intracellular delivery. The Pluronic-g-PEI exhibited better condensation and complexation properties than PEI 25 kDa. The cytotoxicity of PEI was strongly reduced after copolymerization. The Pluronic-g-PEI showed lower cytotoxicity in three different cell lines (Hela, MCF-7, and HepG2) than PEI 25 kDa. pGL3-lus was used as a reporter gene, and the transfection efficiency was in vitro measured in HeLa cells. Compared with unmodified PEI 25 kDa Pluronic-g-PEI showed much higher transfection efficiency. These results demonstrate that polyplexes prepared using a combined strategy of surface crosslinking and grafted with Pluronic seem to provide promising properties as stable, high transfection efficiency vectors.     

Two novel non-viral gene delivery vectors: low molecular weight polyethylenimine cross-linked by (2-hydroxypropyl)-beta-cyclodextrin or (2-hydroxypropyl)-gamma-cyclodextrin

Two novel polymers of low molecular weight polyethylenimine cross-linked by (2-hydroxypropyl)-beta-cyclodextrin or (2-hydroxypropyl)-gamma-cyclodextrin showed lower cytotoxicity and higher transfection efficiency for the delivery of plasmid DNA compared with those of polyethylenimine (PEI, 25 kDa).     

Construction of Polycation‐Based Non‐Viral DNA Nanoparticles and Polyanion Multilayers via Layer‐by‐Layer Self‐Assembly

Summary: The multilayers of polycation‐based non‐viral DNA nanoparticles and biodegradable poly(L ‐glutamic acid) (PGA) were constructed by a layer‐by‐layer (LbL) technique. Poly(ethyleneimine) (PEI) was used to condense DNA to develop non‐viral DNA nanoparticles. AFM, UV‐visible spectrometry, and TEM measurements revealed that the PEI‐DNA nanoparticles were successfully incorporated into the multilayers. The well‐structured, easily processed multilayers with the non‐viral DNA nanoparticles may provide a novel approach to precisely control the delivery of DNA, which may have great potential for gene therapy applications in tissue engineering, medical implants, etc.

A TEM image of the cross section of a (PGA/PEI‐DNA nanoparticle)₂₀ multilayer.     

Comparison of random and gradient amino functionalized poly(2‐oxazoline)s: Can the transfection efficiency be tuned by the macromolecular structure?

Poly(ethylene imine) can be considered as the gold standard for DNA delivery into cells in vitro, but severe cytotoxic side‐effects and inapplicability for targeted approaches in vivo urgently call for the design of new gene carriers. Since poly(2‐oxazoline)s (P(Ox)s) can be easily synthesized and modified, this polymer class might be ideal for the optimization of polymeric transfection processes. The utilization of 2‐methyl‐2‐oxazoline (MeOx) and 2‐ethyl‐2‐oxazoline (EtOx) is also known to be beneficial because these monomers were suggested to overcome solubility issues, mediate stealth behavior and, consequently, facilitate a reduction of cytotoxicity. A series of amino (AmOx) functionalized P(Ox) copolymers with either MeOx (gradient copolymers) or EtOx (random copolymers) was synthesized, deprotected and biochemically characterized regarding cytotoxicity, polyplex formation ability, cellular uptake, and transfection efficiency. Polymers with percentages of AmOx higher than 35 mol % showed stable polyplex formation and also an increase in cytotoxicity. All elucidated P(Ox)s revealed a poor transfection efficiency in both L929 and Hepa1‐6 cell lines. However, the investigations contribute to the understanding of the influence of stealth units (MeOx and EtOx) and their distribution within the polymer chain on selected properties of polyplexes and describe characteristics of amino functionalized P(Ox)s in different cell lines. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018 , 56, 1210–1224     

Branched and linear poly(ethylene imine)-based conjugates: synthetic modification, characterization, and application

Poly(ethylene imine)s (PEIs) are widely used in different applications, but most extensively investigated as non-viral vector systems. The high ability of cationic PEIs to complex and condense negatively charged DNA and RNA combined with their inherent proton sponge behavior accounts for the excellent efficiency in gene delivery. Further chemical modifications of the polymer expand the application potential, primarily aiming at increased transfection efficiency, cell selectivity and reduced cytotoxicity. Improvements in the synthesis of tailor-made PEIs in combination with new in-depth analytical techniques offer the possibility to produce highly purified polymers with defined structures. The contemporary strategies towards linear and branched poly(ethylene imine)s with modified surface characteristics, PEI-based copolymers as well as conjugates with bioactive molecules will be discussed. In this regard, the versatile branched PEIs have been successfully modified in a statistical manner, whereas the linear counterparts open avenues to design and synthesize well-defined architectures, in order to exploit their high potential in gene delivery.     

(Coixan polysaccharide)-graft-polyethylenimine folate for tumor-targeted gene delivery

CP-PEI-FA was prepared as an effective vector for in vitro and in vivo tumor-targeted gene delivery. The structures of the polymers were characterized, and their DNA condensation capability, particle sizes, zeta potentials, cytotoxicity and in vitro/in vivo transfection were examined. The cytotoxicity of CP-PEI-FA was significantly lower than that of PEI 25 kDa and close to that of PEI 1200. The in vitro transfection of CP-PEI-FA was tested in C6 and HeLa cells (FR-positive cells) and A549 cells (FR-negative cells). CP-PEI-FA showed a high targeting specificity and good gene transfection efficiency in FR-positive cells. These results indicate that CP-PEI-FA is a safe and effective polyplex-forming agent for both in vitro and in vivo transfection of plasmid DNA.     

Polymeric Nanoparticles Induce NLRP3 Inflammasome Activation and Promote Breast Cancer Metastasis

Polymeric nanoparticles gain enormous interests in cancer therapy. Polyethylenimine (PEI) 25 kD is well known for its high transfection efficiency and cytotoxicity. PEI‐CyD (PC) was previously synthesized by conjugating low molecular PEI (M _w 600) with β‐cyclodextrin (β‐CyD), which is shown to induce lower cytotoxicity than PEI 25 kD. In the current study, the in vivo immune response of branched PEI 25 kD and PC is investigated. Compared to PC/pDNA, exposure of PEI 25kD/pDNA induces higher level of immune‐stimulation evidenced by the increased spleen weight, phagocytic capacity of peritoneal macrophage, and proinflammatory cytokines in serum and liver. Importantly, administration of PEI 25 kD can greatly promote breast cancer metastasis in liver and lung tissues, which correlates with its ability to induce high oxidative stress and NLRP3‐inflammasome activation. These results suggest that polymeric nanocarriers have the potential to induce immune‐stimulation and cancer metastasis, which may affect their efficiency for cancer therapy.     

High Molecular Weight Poly(ethylenimine)‐Based Water‐Soluble Lipopolymer for Transfection of Cancer Cells

Over the past decade, search for novel materials for nucleic acid delivery has prompted a special interest in polymeric nanoparticles (NPs). In this study, the biological applicability of a water‐soluble cationic lipopolymer (WSLP) obtained by the modification of high molecular weight branched poly(ethylenimine) (PEI) with cholesteryl chloroformate is characterized and assessed for better cellular membrane permeability. To test the delivery efficiency of the produced lipopolymer, plasmid DNA (pDNA) encoding the enhanced green fluorescent protein and WSLP are mixed at different charge ratios. WSLP and WSLP/pDNA complexes are characterized by dynamic and static light scattering, particle charge detection, scanning electron microscopy, and transmission electron microscopy. The pDNA loading of WSLP is also verified by agarose gel electrophoresis. Cytotoxicity of PEI, WSLP, and of WSLP/pDNA is evaluated on human A549 and HeLa cells. A remarkable dependence of the toxicity on the dose, cholesterylation, and charge ratio is detected. Transfection is monitored by flow cytometry and by fluorescence microscopy. Importantly, cholesterylation decreases the toxicity of the polymer, while promoting high transfection efficiency in both cell lines. This work indicates a possible optimization mode of the high molecular weight PEI‐based WSLP rendering it a promising candidate for gene delivery.     

PEG-detachable polyplex micelles based on disulfide-linked block catiomers as bioresponsive nonviral gene vectors

PEG-based polyplex micelles, which can detach the surrounding PEG chains responsive to the intracellular reducing environment, were developed as nonviral gene vectors. A novel block catiomer, PEG-SS-P[Asp(DET)], was designed as follows: (i) insertion of biocleavable disulfide linkage between PEG and polycation segment to trigger PEG detachment and (ii) a cationic segment based on poly(aspartamide) with a flanking N-(2-aminoethyl)-2-aminoethyl group, P[Asp(DET)], in which the Asp(DET) unit acts as a buffering moiety inducing endosomal escape with minimal cytotoxicity. The polyplex micelles from PEG-SS-P[Asp(DET)] and plasmid DNA (pDNA) stably dispersed in an aqueous medium with a narrowly distributed size range of approximately 80 nm due to the formation of hydrophilic PEG palisades while undergoing aggregation by the addition of 10 mM dithiothreitol (DTT) at the stoichiometric charge ratio, indicating the PEG detachment from the micelles through the disulfide cleavage. The PEG-SS-P[Asp(DET)] micelles showed both a 1-3 orders of magnitude higher gene transfection efficiency and a more rapid onset of gene expression than PEG-P[Asp(DET)] micelles without disulfide linkages, due to much more effective endosomal escape based on the PEG detachment in endosome. These findings suggest that the PEG-SS-P[Asp(DET)] micelle may have promising potential as a nonviral gene vector exerting high transfection with regulated timing and minimal cytotoxicity.     

Water soluble poly(histamine acrylamide) with superior buffer capacity mediates efficient and nontoxic in vitro gene transfection

Water‐soluble cationic polymers, poly(histamine acrylamide)s (PHAs), with superior buffer capacity at the endosomal pH range were designed, prepared, and investigated for non‐viral gene transfection. PHAs were obtained with molecular weights ranging from 9.2 to 28.7 kDa through controlled radical polymerization of histamine acrylamide (HA). Acid–base titration results displayed that all PHA polymers had a remarkably high buffer capacity of about 70% at pH 5.1–7.2. 12.7–28.7 kDa PHAs were able to effectively condense DNA into nano‐sized (<220 nm) polyplexes with moderate positive surface charges (+13–+19 mV) at N/P ratios ≥10/1. CCK assays indicated that polyplexes of 12.7 and 17.5 kDa PHAs were non‐toxic to COS‐7 cells up to a tested N/P ratio of 20/1. Interestingly, the in vitro transfection using pCMV‐Luc and pEGFP‐C1 plasmid DNA as reporter genes showed that polyplexes of 12.7 kDa PHA formed at an N/P ratio of 20/1 mediated efficient transfection in COS‐7 cells under 10% serum conditions, with transfection efficiencies comparable to that of 25 kDa polyethylenimine control. Their versatile design of structures, controlled synthesis, low cytotoxicity, and high transfection activity render PHA‐based cationic polymers particularly interesting for the development of safe and efficient non‐viral gene delivery systems. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011.     

A Serum‐Tolerant Hydroxyl‐Modified Polyethylenimine as Versatile Carriers of pDNA/siRNA

The polyethylenimine (PEI) derivatives (PTn) are prepared by treating PEI25k with Tris(hydroxymethyl) acrylamidomethane via the Michael addition. These PTns can effectively condense nucleic acids into nanosized particles with positive surface charges. The PTns show lower cytotoxicity and better serum‐resistant capacity than PEI25k. Specially, the transfection efficiency of PT26/DNA is 29‐fold higher than that of PEI25k in HeLa cells in serum‐containing medium. The PTn/siRNA complexes show superior knockdown effect in CT26 cells in serum‐containing medium. In addition, flow cytometry analysis shows that the PTns can efficiently mediate the entry of nucleic acids into the cell. Thus, PTns are potentially applicable as non‐viral carriers of nucleic acids and warrant further development for use in gene therapy.

     

Characterization of polyplexes involving small RNA

The purpose of the present study is to provide a tool for an efficient design and synthesis of non-viral vectors for small RNA delivery. The effects of properties of the polycation, such as molecular weight, charge density and backbone structure, to polyplex structure and physicochemical behavior were systematically evaluated. The condensing agents, polyethylenimine (PEI), chitosan (CS) and poly(allylamine) (PAA) were added to sRNA molecules at different N/P ratio. The efficiency of encapsulation and protection of sRNA, as well as polyplex size, zeta potential and morphology were followed and compared. The results show that PEI/sRNA polyplexes display a small size and positive zeta potential. However, for low molecular weights, this polycation is unable to protect sRNA in the presence of a decompacting agent. With chitosan, sRNA is efficiently compacted at high N/P ratios. The CS/sRNA complexes display small sizes, ca. 200 nm, positive surface charge and also good stability. Finally, the PAA/sRNA polyplexes were found to be the smallest at low N/P ratios, displaying a good encapsulation efficiency and high stability. A rationale for the experimental observations is provided using Monte Carlo simulation for systems with polycations of different length and charge density. The simulations showed that there is an interplay between the size of polycation chains and its charge density that define the degree of condensation for sRNA.     

基于低分子量聚乙烯亚胺和油酸的负电性基因载体

构建负电性的基因载体、发展基于低分子量聚乙烯亚胺(PEI)的基因载体对基因传递研究具有重要意义. 本文基于低分子量聚乙烯亚胺(2 kDa)和油酸构建了负电性的基因载体. 它通过混合聚乙烯亚胺(2 kDa)、dsDNA和油酸胶束而自发形成. 该基因载体在血清中很稳定,细胞毒性非常低,可包封80%以上DNA. 通过1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇2000)]铵盐(DSPE-PEG)对其表面进行修饰,发现多达90%的基因可被细胞摄取.     

Combined Delivery of a Lipopolysaccharide‐Binding Peptide and the Heme Oxygenase‐1 Gene Using Deoxycholic Acid‐Conjugated Polyethylenimine for the Treatment of Acute Lung Injury

A ternary complex comprising plasmid DNA, lipopolysaccharide‐binding peptide (LBP), and deoxycholic acid‐conjugated polyethylenimine (PEI‐DA) is prepared for combinational therapy of acute lung injury (ALI). The LBP is designed as an anti‐inflammatory peptide based on the lipopolysaccharide (LPS)‐binding domain of HMGB‐1. In vitro cytokine assays show that LBP reduces levels of proinflammatory cytokines by inhibiting LPS. PEI‐DA is synthesized as the gene carrier by conjugation of deoxycholic acid to low‐molecular weight polyethylenimine (2 kDa, PEI2k). PEI‐DA has higher transfection efficiency than high‐molecular weight polyethylenimine (25 kDa, PEI25k). The ternary complex of an HO‐1 plasmid (pHO‐1), PEI‐DA, and LBP is prepared as a combinational system to deliver the therapeutic gene and peptide. The transfection efficiency of the ternary complex is higher than that of the pHO‐1/PEI‐DA binary complex. The ternary complex also reduces TNF‐α secretion in LPS‐activated Raw264.7 macrophage cells. Administration of the ternary complex into the lungs of an animal ALI model by intratracheal injection induces HO‐1 expression and reduces levels of proinflammatory cytokines more efficiently than the pHO‐1/PEI‐DA binary complex or LBP alone. In addition, the ternary complex reduces inflammation in the lungs. Therefore, the pHO‐1/PEI‐DA/LBP ternary complex may be an effective treatment for ALI.

     

普鲁兰多糖为骨架的新型阳离子非病毒基因载体

通过琥珀酸酐将低分子量支化聚乙烯亚胺(PEI, 分子量1000)偶联到普鲁兰多糖(Pullulan)上, 合成了新型基因载体P-PEI. 利用 ¹H NMR、 FTIR、 粒度仪、 Zeta电位仪、 透射电镜和凝胶电泳对聚阳离子载体及其与质粒pDNA 的复合物进行了表征. 凝胶阻滞实验结果证明, 载体P-PEI在体外可以通过静电相互作用稳定结合pDNA, 并能有效抑制DNA水解酶及血清成分对pDNA的降解. 噻唑蓝(MTT)细胞毒性测试、 绿色荧光蛋白表达质粒(pGFP)及荧光素酶表达质粒(pGL3)转染实验结果表明, 载体P-PEI在N/P高达12.5时对细胞MCF-7, HeLa和COS-7的毒性低于PEI; 当N/P 为6.25时能有效将pGFP和pGL3带入Hela 细胞并表达, 最佳转染效率及荧光素酶活分别为, 比Lipo 2000[(49.13±0.61)%, (58.47±7.62)×10⁸ RLU/mg蛋白) 略低. 因此以Pullulan为骨架材料的P-PEI是一种新的有潜在应用价值的非病毒基因载体.