Conjugation of polyamidoamine dendrimers on biodegradable microparticles for nonviral gene delivery

We report on the preparation and characterization of poly(D, L-lactide-co-glycolide) (PLGA) microparticles with surface-conjugated polyamidoamine (PAMAM) dendrimers of varying generations. The buffering capacity and zeta-potential of the PLGA PAMAM microparticles increased with increasing generation level of the PAMAM dendrimer conjugated. Conjugation of the PAMAM dendrimer to the surface of the PLGA microparticle removed generation-dependent cytotoxicity in HEK293 and COS7 cell lines. PLGA PAMAM pDNA microparticles displayed similar cytotoxicity profiles to unmodified PLGA pDNA microparticles in COS7 cells. A generation three PAMAM dendrimer conjugated to PLGA microparticles significantly increased transfection efficiencies in comparison to unmodified PLGA microparticles.     

Synthesis and characterization of a head-tail type polycation block copolymer as a nonviral gene vector

A head-tail type polycation block copolymer, which is composed of the polyamidoamine (PAMAM) dendron and poly(L-lysine) (PLL) blocks, was newly designed as a nonviral gene vector in this study. This block copolymer (PAMAM dendron-PLL) was successfully synthesized in two steps: the synthesis of the PAMAM dendron block and the polymerization of the PLL block from the PAMAM dendron block. PAMAM dendron and PLL blocks in block copolymer showed independent deprotonation behavior, and their pK(a) were determined to be 6.8 and 9.0, respectively. The complexation with pDNA was evaluated by gel retardation assay and dye exclusion assay, and both assays indicated that pDNA was selectively complexed with PLL block of block copolymer. Also, the PAMAM dendron-PLL poplyplexes showed 10(2) fold higher transfection efficiency to HeLa cells as that for PLL polyplexes. This might be due to the buffering effect of the PAMAM dendron block. This block copolymer could produce a function share in each block, i.e., tail block complexed with pDNA and head block showed a buffering effect. This molecular design of the head-tail type block copolymer might provide a new approach for realizing in vivo gene therapy.     

Glycopolymer modification on physicochemical and biological properties of poly(L-lysine) for gene delivery

Poly(L-lysine) (PLL) has excellent plasmid DNA (pDNA) condensation capacity. However, the relatively high cytotoxicity and low transfection efficiency limit its application as gene delivery vectors. Here, well-defined glycopolymers are synthesized by reversible addition fragmentation transfer polymerization and grafted onto PLL to improve the gene delivery performance. After glycopolymer modification, PLL shows reduced cytotoxicity. By regulating the glycopolymer length and amino group substitution degree, the glycopolymer modified PLL can condense pDNA with proper strength, protect the condensed pDNA from degradation and release them in time. Transfection with NIH3T3 and HepG2 cells shows that the glycopolymer modified PLL has improved transfection efficiencies. The low cytotoxicity, effective pDNA protection and enhanced transfection efficiencies indicate that glycopolymer modification would be an effective strategy to improve the polycation properties for gene delivery.     

In vitro gene delivery using polyamidoamine dendrimers with a trimesyl core

Polyamidoamine (PAMAM) dendrimer represents one of the most efficient polymeric gene carriers. To investigate the effect of the core structure and generation of dendrimers on the complex formation and transfection efficiency, a series of PAMAM dendrimers with a trimesyl core (DT) at different generations (DT4 to DT8) were developed as gene carriers and compared with the PAMAM dendrimers derived from pentaerythritol (DP) and inositol (DI). The minimal generation number of DTs at which the dendrimer has enough amino group density to effectively condense DNA was higher (generation 6) than those of DPs and DIs (generation 5). DTs of generation 6 or higher condensed DNA into complexes with an average diameter ranging from 100 to 300 nm, but the 4th and 5th generations of DT (DT4 and DT5) formed only a severe aggregate with DNA. Interestingly, the DT6/pDNA complex was determined to be much smaller (100-300 nm) than those prepared with DP5 or DI5 (>600 nm) at N/P ratios higher than 15. The optimal generation numbers at which the dendrimers showed the highest transgene expression in COS-7 cells were 5 for DPs and DIs but 6 for DTs. The DT6/pDNAcomplex with smaller size mediated higher transgene expression in COS-7 cells than those prepared with DP5 or DI5. The in vitro transfection efficiency of the DT dendrimers as evaluated in HeLa cells, COS-7 cells, and primary hepatocytes decreased in the order of DT6 > DT7 > DT8 > DT5 > DT4. The transfection mediated by DT6 was significantly inhibited by bafilomycin A1. The acid-base titration curve for DT6 showed high buffer capacity in the pH range from 5.5 to 6.4 (pK(a) approximately 6). This permits dendrimers to buffer the pH change in the endosomal compartment. However, the transfection efficiency mediated by DT6 decreased significantly in the presence of serum in both HeLa cells and COS-7 cells. The cytotoxicity of DTs evaluated in HeLa cells using the 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide assay showed a trend of increasing toxicity with the polymer generations. The LD50 values of DT4 through DT8 were 628, 236, 79, 82, and 77 microg/mL, respectively, which were higher than that of poly(ethyleneimide) (18 microg/mL) and poly(L-lysine) (28 microg/mL) in the same assay. With a lower cytotoxicity and versatility for chemical conjugation, these PAMAM dendrimers with a DT core warrant further investigation for nonviral gene delivery.     

Chitosan-Graft-Polyethylenimine/DNA Nanoparticles as Novel Non-Viral Gene Delivery Vectors Targeting Osteoarthritis

The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes.     

Effects of structure of polyamidoamine dendrimer on gene transfer efficiency of the dendrimer conjugate with alpha-cyclodextrin

To improve gene transfer activity of a new nonviral vector, a polyamidoamine dendrimer (G2) conjugate with alpha-cyclodextrin (alpha-CDE conjugate (G2)), we prepared alpha-CDE conjugates with dendrimer having different generations (G3 and G4), and their gene transfer activities were compared with those of alpha-CDE conjugate (G2) and TransFast, a novel transfection reagent. alpha-CDE conjugates (G2, G3, and G4) formed the complexes with pDNA, changing the zeta-potential and particle size of pDNA complexes and the protection of pDNA from DNase I in a charge ratio-dependent manner, although their differences at higher charge ratios (vector/pDNA) were small. The gene transfer activity of alpha-CDE conjugates (G2, G3, and G4) was higher than that of the corresponding dendrimer alone in NIH3T3 and RAW264.7 cells. Of these CDE conjugates, alpha-CDE conjugate (G3) had a superior gene transfer activity which was comparable to that of TransFast in NIH3T3 cells. The intracellular distribution of pDNA after application of the pDNA complex with alpha-CDE conjugate (G3) to NIH3T3 cells was different from that with dendrimer alone (G3), although the cellular association of pDNA was almost comparable among all vectors. alpha-CDE conjugate (G3) strongly interacted with a fluorescence probe, 2-(p-toluidinyl)-naphthalene-6-sulfonate (TNS), suggesting that the conjugate possesses the inclusion ability with biomembrane constituents such as phospholipids after transfection. These results suggest that alpha-CDE conjugates, particularly the G3 conjugate, could be novel nonviral gene transfer agents.     

Lactosylated chitosan for DNA delivery into hepatocytes: the effect of lactosylation on the physicochemical properties and intracellular trafficking of pDNA/chitosan complexes

Chitosan is a useful nonviral vector for gene delivery. To make a pDNA/chitosan complex specific to hepatocytes, lactose-modified chitosan (lac-chitosan) was synthesized. When the percentage of lactose residues substituted was 8%, lac-chitosan showed excellent DNA-binding ability, good protection of DNA from nuclease, and the suppression of self-aggregation and serum-induced aggregation. Although the cellular uptake efficiency of the pDNA/lac-chitosan complex was almost the same as that of the pDNA/chitosan complex, the cell transfection efficiency of the former was greater for HepG2 cells having asialoglycoprotein receptors. Inhibitor of endocytosis such as bafilomycin A1 and nocodazole significantly reduced the transfection efficiency of the pDNA/lac-chitosan complex. Observations with a confocal laser scanning microscope indicated that the pDNA/lac-chitosan complexes traversed endocytic compartments more rapidly than the pDNA/chitosan complex. Furthermore, the pDNA/lac-chitosan complexes were delivered to the late endosome and have the advantage of delivering DNA to the perinuclear region.     

‘Click’ synthesized sterol-based cationic lipids as gene carriers,and the effect of skeletons and headgroups on gene delivery

In this work, we have successfully prepared a series of new sterol-based cationic lipids (1–4) via an efficient ‘Click’ chemistry approach. The pDNA binding affinity of these lipids was examined by EB displacement and agarose-gel retardant assay. The average particle sizes and surface charges of the sterol-based cationic lipids/pDNA lipoplexes were analyzed by dynamic laser light scattering instrument (DLS), and the morphologies of the lipoplexes were observed by atomic force microscopy (AFM). The cytotoxicity of the lipids were examined by MTT and LDH assay, and the gene transfection efficiencies of these lipid carriers were investigated by luciferase gene transfection assay in various cell lines. In addition, the intracellular uptake and trafficking/localization behavior of the Cy3-DNA loaded lipoplexes were preliminarily studied by fluorescence microscopy. The results demonstrated that the pDNA loading capacity, lipoplex particle size, zeta potential and morphology of the sterol lipids/pDNA lipoplexes depended largely on the molecular structure factors including sterol-skeletons and headgroups. Furthermore, the sterol-based lipids showed quite different cytotoxicity and gene transfection efficacy in A549 and HeLa cells. Interestingly, it was found that the cholesterol-bearing lipids 1 and 2 showed 7–10⁴ times higher transfection capability than their lithocholate-bearing counterparts 3 and 4 in A549 and HeLa cell lines, suggested that the gene transfection capacity strongly relied on the structure of sterol skeletons. Moreover, the study on the structure–activity relationships of these sterol-based cationic lipid gene carriers provided a possible approach for developing low cytotoxic and high efficient lipid gene carriers by selecting suitable sterol hydrophobes and cationic headgroups.     

Histidylated polylysine as DNA vector: elevation of the imidazole protonation and reduced cellular uptake without change in the polyfection efficiency of serum stabilized negative polyplexes

We have reported that polylysine substituted with histidyl residues (His) was suited to make complexes with plasmid DNA (pDNA) and to transfect cells in vitro in the presence of serum. The present study was performed to determine whether the acetylation of the alpha-amino group of histidyl residues (AcHis) had an influence on the size and the charge of polyplexes and on their transfection efficiency. We found that the presence of free alpha-amino groups allowed the formation of smaller polyplexes but did not modify the zeta potential of +17 mV. At a physiological salt concentration, the adsorption of many serum proteins on His- and AcHis-polyplexes reduced their size below 100 nm, inhibited their aggregation, and reversed their zeta potential to -25 mV. The acetylation of the alpha-amino groups reduced slightly the adsorption of serum proteins. The presence of the alpha-amino groups increased the pK of the imidazole protonation of histidine bound to polylysine from pH 5.8 to 6.9; in addition, the protonation was further elevated in the presence of pDNA. Serum stabilized negative histidylated polyplexes were less taken up by cells but their transfection efficiency did not decrease; depending on the cell line, His-polyplexes were more efficient than AcHis-polyplexes. The results indicate that (i) the alpha-amino groups of histidyl residues bound to polylysine favorably influence the size and the transfection efficiency of polyplexes, (ii) the alpha-amino groups also elevate the imidazole protonation of His-polyplexes, which is suited to destabilize the membrane of early endocytic vesicles in order to favor pDNA delivery in the cytosol, and (iii) the absorption of selective serum proteins on His-polyplexes could be a way for in vivo gene targeting.     

Synthesis and characterization of folate-PEG-grafted-hyperbranched-PEI for tumor-targeted gene delivery

A great challenge for gene therapy is to develop a high efficient gene delivery system with low toxicity. Nonviral vectors are still attractive although the current agents displayed some disadvantages (i.e., low transfection efficiency, high toxicity). To overcome the high toxicity of poly(ethylene imine) (PEI) and low transfection efficiency of PEGylated PEI (PEG-PEI), we linked a cell specific target molecule folate (FA) on poly(ethylene glycol) (PEG) and then grafted the FA-PEG onto hyperbranched PEI 25 kDa. The FA-PEG- grafted-hyperbranched-PEI (FA-PEG-PEI) effectively condensed plasmid DNA (pDNA) into nanoparticles with positive surface charge under a suitable N/P ratio. Tested in deferent cell lines (i.e., HEK 293T, glioma C6 and hepatoma HepG2 cells), no significant cytotoxicity of FA-PEG-PEI was added to PEG-PEI. More importantly, significant transfection efficiency was exhibited in FA-targeted cells. Reporter assay showed that FA-PEG-PEI/pDNA complexes had significantly higher transgene activity than that of PEI/pDNA in folate-receptor (FR) positive (HEK 293T and C6) cells but not FR-negative (HepG2) cells. These results indicated that FA-PEG-PEI might be a promising candidate for gene delivery with the characteristics of good biocompatibility, potential biodegradability, and relatively high gene transfection efficiency.     

Tumor cell imaging using the intrinsic emission from PAMAM dendrimer: a case study with HeLa cells

HeLa 229 cells were treated with methotrexate (MTX) and doxorubicin (DOX), utilizing fourth generation (G4), amine terminated poly(amidoamine) {PAMAM} dendrimer as the drug carrier. In vitro kinetic studies of the release of both MTX and DOX in presence and absence of G4, amine terminated PAMAM dendrimers suggest that controlled drug release can be achieved in presence of the dendrimers. The cytotoxicity studies indicated improved cell death by dendrimer-drug combination, compared to the control experiments with dendrimer or drug alone at identical experimental conditions. Furthermore, HeLa 229 cells were imaged for the first time utilizing the intrinsic emission from the PAMAM dendrimers and drugs, without incorporating any conventional fluorophores. Experimental results collectively suggest that the decreased rate of drug efflux in presence of relatively large sized PAMAM dendrimers generates high local concentration of the dendrimer-drug combination inside the cell, which renders an easy way to image cell lines utilizing the intrinsic emission properties of PAMAM dendrimer and encapsulated drug molecule.     

Gene Transfer by DNA/mannosylated Chitosan Complexes into Mouse Peritoneal Macrophages

Chitosan is a biodegradable and biocompatible polymer and is useful as a non-viral vector for gene delivery. In order to deliver pDNA/chitosan complex into macrophages expressing a mannose receptor, mannose-modified chitosan (man-chitosan) was employed. The cellular uptake of pDNA/man-chitosan complexes through mannose recognition was then observed. The pDNA/man-chitosan complexes showed no significant cytotoxicity in mouse peritoneal macrophages, while pDNA/man-PEI complexes showed strong cytotoxicity. The pDNA/man-chitosan complexes showed much higher transfection efficiency than pDNA/chitosan complexes in mouse peritoneal macrophages. Observation with a confocal laser microscope suggested differences in the cellular uptake mechanism between pDNA/chitosan complexes and pDNA/man-chitosan complexes. Mannose receptor-mediated gene transfer thus enhances the transfection efficiency of pDNA/chitosan complexes.     

新型阳离子基因传递载体PEI-PBLG的细胞转染研究

对新型阳离子聚合物PEI(10kD)-PBLG进行研究,重点考察其基因转染效率与细胞毒性,探讨其作为基因载体的可能性。通过粒径分析及扫描电镜(SEM)观察PEI(10kD)-PBLG与质粒pEGFP自组装形成的颗粒形态及粒径,预测其进入细胞的可能性。使用MTT比色法分析PEI(10kD)-PBLG、PEI(25kD)-PBLG、PEI(10kD)和PEI(25kD)的细胞毒性差异。选用表达增强型绿色荧光蛋白的质粒pEGFP作为报告基因模型,将其与PEI(10kD)-PBLG自组装后,分别转染真核细胞株Hela、COS-7、Vero-E6和ECV304,应用流式细胞术检测细胞转染效率,并比较了血清、缓冲液、细胞谱等多种因素对基因转染效率的影响。PEI(10kD)-PBLG可包裹质粒形成粒径100~120nm的纳米复合物,适合介导质粒进入细胞。该纳米粒复合物对转染缓冲液的敏感度较低,并能够在10%血清存在的条件下,转染全部实验用细胞株,尤其对Hela的转染效率最高,其次是COS-7、Vero-E6和ECV304;其中PEI-PBLG(10kD)/pEGFP复合物转染Hela细胞的比率为45.02%,高于PEI(10kD)/pEGFP的29.16%;PEI(10kD)-PBLG的细胞毒性作用显著低于PEI(25kD)、PEI(10kD)和PEI(25kD)-PBLG。新型阳离子多聚物PEI(10kD)-PBLG在提高PEI介导的基因转染效率的同时降低了其细胞毒性,提高了生物相容性,有望成为基因转移的有效载体。     

Self-assembled terplexes for targeted gene delivery with improved transfection

To improve transfection efficiency and to incorporate target ligands to the gene delivery systems, heparin and heparin-biotin were introduced to complexes of polyamidoamine dendrimer and DNA (PAMAM/DNA) via electrostatic interactions to form self-assembled PAMAM/DNA/heparin and PAMAM/DNA/heparin-biotin terplexes, respectively. The self-assembled terplexes were characterized by agarose gel electrophoresis and particle size analysis. The MTT assay indicated that, after incorporation of heparin and heparin-biotin, the terplexes exhibited decreased cytotoxicity. In addition, as compared with PAMAM/DNA and PAMAM/DNA/heparin complexes, the PAMAM/DNA/heparin-biotin complexes exhibited much higher cellular uptake into HeLa cells due to the specific interactions between biotin and biotin receptors on HeLa cells, which led to the enhanced transfection activity. The PAMAM/DNA/heparin-biotin complexes would be a promising targeting gene delivery system.     

Modified linear polyethylenimine-cholesterol conjugates for DNA complexation

Linear polyethylenimine (LPEI) is an effective nonviral gene carrier with transfection levels equal or above branched polyethylenimine (BPEI) and exhibits a lower cytotoxicity profile than BPEI. High molecular weight LPEI M(w) 25 k was modified with cholesterol in three different geometries: linear shaped (L), T-shaped (T), and a combined linear/T-shaped (LT) forming the LPEI-cholesterol (LPC) conjugates LPC-L, LPC-T, and LPC-LT, respectively. Physical characterization of LPC/pDNA complexes included particle size, zeta potential, DNase protection, mIL-12 p70 expression, and cytotoxicity. The particle size was further confirmed by atomic force microscopy (AFM). The LPC-T/pDNA complexes were optimal at N/P 10/1 that resulted in a particle size of approximately 250 nm, which was confirmed by AFM, and a surface charge of +10 mV. These complexes also effectively protected the pDNA for up to 180 min in the presence of DNase I. B16-F0 cells transfected with LPC-L and LPC-T showed protein expression levels higher than LPEI alone and twice that of BPEI but without any significant loss in cell viability. These results were confirmed with EGFP flow cytometry and transfection of Renca cells. The differences in rates of transfection of the LPC/pDNA complexes is due in part to conformational changes from the point of complex formation to interaction with the plasma membrane. These conformation changes provide protection for unprotonated secondary amines in the LPEI backbone by hydrophobic protection of the cholesterol moiety that we termed "unprotonated reserves". Finally, we show that LPC conjugates exploit receptor-mediated endocytosis via the LDL-R pathway with transgene expression levels decreasing nearly 20% after saturating the LDL-R sites on MCF-7 cells with hLDL-R-Ab.     

Physicochemical characteristics associated with transfection of cationic cholesterol-based gene delivery vectors in the presence of DOPE

The physicochemical properties of a novel series of cholesterol-based cationic lipids in the presence of DOPE were studied by various techniques in an effort to correlate cationic lipid structure with transfection efficacy. It was found that while DOPE improves the β-gal activity of the active AC and MC derivatives, the overall zeta potential of the particles, pDNA complexation and condensation is not improved. This is in stark contrast with the tertiary amine derivative DC whose dispersion properties were improved and its monolayer surface potential is restored at high molecular surface density in the presence of DOPE. Overall the transfection activity mediated by DC and the quaternary ammonium TC derivative was greatly improved in the presence of DOPE and is attributed to decreased cytotoxicity, improved fusogenicity and cellular association.     

Modified branched peptides with a histidine-rich tail enhance in vitro gene transfection

Successful gene therapy depends on the development of efficient, non-toxic gene delivery systems. To accomplish this objective, our laboratory has focused on solid-phase synthesized peptide carriers, in which the amino acid sequence can be varied precisely to augment intracellular DNA transport. We previously determined that linear and branched co-polymers of histidine and lysine in combination with liposomes enhanced the efficiency of gene transfection. In this study, we have modified two branched histidine-lysine (HK) peptides by adding a histidine-rich tail. In a variety of cell lines, this histidine-rich tail markedly improved transfection efficiency, presumably by increasing the buffering capacity of the polymer. One polymer with a histidine-rich tail, H²K4bT, compared favorably with the commonly used transfection agents. Together with modification of our transfection protocol, these improved HK peptides alone, without liposomes, are the effective carriers of plasmids into a variety of cells. We anticipate that branched HK peptides will continue to be developed as carriers of nucleic acids for in vitro and in vivo applications.     

Plasmid DNA delivery by arginine-rich cell-penetrating peptides containing unnatural amino acids

Cell-penetrating peptides (CPPs) have been developed as drug, protein, and gene delivery tools. In the present study, arginine (Arg)-rich CPPs containing unnatural amino acids were designed to deliver plasmid DNA (pDNA). The transfection ability of one of the Arg-rich CPPs examined here was more effective than that of the Arg nonapeptide, which is the most frequently used CPP. The transfection efficiencies of Arg-rich CPPs increased with longer post-incubation times and were significantly higher at 48-h and 72-h post-incubation than that of the commercially available transfection reagent TurboFect. These Arg-rich CPPs were complexed with pDNA for a long time in cells and effectively escaped from the late endosomes/lysosomes into the cytoplasm. These results will be helpful for designing novel CPPs for pDNA delivery.