Arginine-rich polyplexes for gene delivery to neuronal cells

Neuronal gene therapy potentially offers an effective therapeutic intervention to cure or slow the progression of neurological diseases. However, neuronal cells are difficult to transfect with nonviral vectors, and in vivo their transport across the blood–brain barrier (BBB) is inefficient. We synthesized a series of arginine-rich oligopeptides, grafted with polyethyleneimine (PEI) and modified with a short-chain polyethylene glycol (PEG). We hypothesized that the arginine would enhance cellular uptake and transport of these polyplexes across the BBB, with PEG imparting biocompatibility and “stealth” properties and PEI facilitating DNA condensation and gene transfection. The optimized composition of the polyplexes demonstrated hemocompatibility with red blood cells, causing no lysis or aggregation, and showed significantly better cytocompatibility than PEI in vitro. Polyplexes formulated with luciferase-expressing plasmid DNA could transfect rat primary astrocytes and neurons in vitro. Confocal imaging data showed efficient cellular uptake of DNA and its sustained intracellular retention and nuclear localization with polyplexes. Intravenous administration of the optimized polyplexes in mice led to gene expression in the brain, which upon further immunohistochemical analysis demonstrated gene expression in neurons. In conclusion, we have successfully designed a nonviral vector for in vitro and in vivo neuronal gene delivery.     

Characterization of a multifunctional PEG-based gene delivery system containing nuclear localization signals and endosomal escape peptides

Endosomal escape and nuclear localization are two barriers to gene delivery that need to be addressed in the design of new nonviral gene delivery vehicles. We have previously synthesized low-toxicity polyethylene glycol (PEG)-based vehicles with endosomal escape functionalities, but it was determined that the transfection efficiency of PEG-based vehicles that escaped the endosome was still limited by poor nuclear localization. Two different nuclear localization signal (NLS) peptides, SV40 and TAT, were coupled to PEG-based vehicles with DNA-binding peptides (DBPs) to determine the effect of NLS peptides on the transfection efficiency of PEG-based gene delivery vehicles. Coupling one SV40 peptide, a classical NLS, or two TAT peptides, a nonclassical NLS, to PEG-DBP vehicles increased the transfection efficiency of PEG-DBP/DNA particles 15-fold and resulted in similar efficiency to that of a common cationic polymer vehicle, polyethylenimine (PEI). The transfection efficiency of both types of PEG-DBP-NLS particles was further increased 7-fold in the presence of chloroquine, suggesting that the transfection efficiency of PEG-DBP-NLS particles is limited by their ability to escape the endosome. To develop particles that could escape the endosome and target the nucleus, a mixture of PEG-DBP-NLS vehicles and PEG-based vehicles with DBPs and endosomal escape peptides were complexed with plasmid DNA to form multifunctional particles that had a transfection efficiency 2-3 times higher than that of PEI. Additionally, the PEG-based vehicles were less toxic and more resistant to nonspecific protein adsorption than PEI, making them an attractive alternative for nonviral gene delivery.     

PAMAM–Triamcinolone acetonide conjugate as a nucleus-targeting gene carrier for enhanced transfer activity

The excellent transfection efficiency and viability are essential for successful gene therapy. It suggested that when bound to its glucocorticoid receptor, glucocorticoid steroid can dilate the nuclear pore complexes and facilitated the transport of pDNA into the nucleus. In this research, the two different degrees of substitution of PAMAM–triamcinolone acetonide (PAMAM–TA) conjugates were synthesised for efficient translocation of pDNA into the nucleus. The physicochemical properties of the polyplexes were investigated by agarose gel electrophoresis, Zeta-sizer and TEM. They both could form nano-size polyplexes with pDNA. The polyplexes were very stable and showed excellent buffering capacities, facilitating endosomal escape, and no obvious difference was found between them. The TA-conjugated PAMAM-mediated transfection of luciferase and EGFP genes showed better transfer activity than native PAMAM and was comparable to the PEI 25K (polyethylenimine), and lower cytotoxicity in HEK 293 and HepG 2 cells. Even with 10% serum, their transfer activity was still high relatively. In addition, confocal microscopy examination confirmed that the enhancing mechanism for enhanced gene transfer activity of PAMAM–TA conjugate may involve the nuclear translocation of the polyplex. The low substituted degree of TA to 0.22 did not interrupt its nuclear localization potency. These findings demonstrated that the TA-grafted PAMAM dendrimer is a potential candidate as a safe and efficient gene delivery carrier for gene therapy.     

Gene transfer by chemical vectors, and endocytosis routes of polyplexes, lipoplexes and lipopolyplexes in a myoblast cell line

Chemical vectors are widely developed for providing safe DNA delivery systems. It is well admitted that their endocytosis and intracellular trafficking are critical for the transfection efficiency. Here, we have compared the endocytic pathways of lipoplexes, polyplexes and lipopolyplexes formed with carriers of various chemical compositions. Engineered C2C12 mouse myoblast cells expressing Rab5-EGFP, Rab7-EGFP or Cav1-GFP were used to monitor the location of the plasmid DNA into the endocytic compartments by real time fluorescence confocal microscopy. We observed that (i) DNA complexes made with dioleyl succinyl paromomycin:O,O-dioleyl-N-histamine phosphoramidate (DOSP/MM27) liposomes or histidinylated lPEI (His-lPEI) allowing the highest transfection efficiency displayed a positive ζ potential and were internalized by clathrin-mediated endocytosis, (ii) DOSP/MM27 lipoplexes were 6-times more internalized than His-lPEI polyplexes, (iii) all negatively charged DNA complexes lead to less efficient transfection and entered the cells via caveolae and (iv) lipopolyplexes allowing high transfection efficiency were weakly internalized via caveolae. Our results indicate that the transfection efficiency is better correlated with the nature of the endocytic pathway than with the uptake efficacy. This study shows also that engineered cells expressing specific fluorescent compartments are convenient tools to monitor endocytosis of a fluorescent plasmid DNA by real time fluorescence confocal microscopy.     

Increased expression of DNA cointroduced with nuclear protein in adult rat liver

Conclusion We have developed an efficient in vivo gene transfer system based on liposomes, HVJ, and nuclear proteins. In this system DNA is delivered directly into the cytoplasm using virus-cell fusion, and the DNA is most likely inserted into the nucleus and stabilized there to result in efficient gene expression by the cointroduced nuclear protein. HMG-1 is so far the most successful nuclear proteins in enhancing gene expression. Moreover, our delivery system results in a significant increase in the stability and effectiveness of antisense ODNs. Although there still exist limitations in the delivery system (e.g., transient gene expression, inability to target), the HVJ liposome or vesicle complex will be one of the most practical gene delivery systems not only for the treatment of postnatal disorders but also for the analysis of molecular aspects of diseases.Abbreviations ACE Angiotensin-converting enzyme - AGN Angiotensinogen - BSA Bovine serum albumin - CAT Chloramphenicol acetyltransferase - FITC Fluoroscein isothiocyanate - HMG High-mobility group - HSV-TK Herpes simplex virus thymidine kinase - HVJ Hemagglutinating virus of Japan - ID Inhibiting dose - Ltk- L cells deficient in the TK gene - ODN Oligonucleotide - pActCAT Chicken -actin promoter controlled chloramphenicol acetyltransferase - pActHIN Chicken -actin promoter controlled human insulin vesicle complex - pActSVT Chicken -actin promoter controlled SV40 large T antigen - RBC Red blood cell - TK Thymidine kinase - VSMC Vascular smooth muscle cell [CE2]     

Self-assembled ternary complexes of plasmid DNA, low molecular weight polyethylenimine and targeting peptide for nonviral gene delivery into neurons

Chemical conjugation of targeting ligands to polycation/plasmid DNA complexes has been widely used to improve the transfection efficiency of nonviral gene delivery vectors. However, conjugation reactions may reduce or even inactivate the biological activities of chemically sensitive moieties, such as proteins and peptides. Here we describe a new method for introducing targeting ligands into nonviral vectors, in which ternary complexes are formed via charge interactions among polyethylenimine (PEI) of 600Da, plasmid DNA and targeting peptides with positively charged DNA-binding sequence. Owing to the nerve growth factor (NGF) loop 4 hairpin motif in the targeting peptide, these ternary complexes are capable of mediating gene delivery efficiently and specifically into cells expressing the NGF receptor TrkA. In in vitro experiments, the complexes improved luciferase reporter gene expression by up to 1000-fold while comparing with that produced by complexes with nontargeting control peptide. In an in vivo experiment, the ternary complexes with the targeting peptide was 59-fold more efficient than the control ternary complexes in transfecting dorsal root ganglia (DRG), the peripheral nervous sites with TrkA-expressing neurons. In a cell viability study, the ternary complexes were remarkably different from DNA complexes by PEI of 25 kDa, the gold standard for nonviral gene carriers, displaying no toxicity in tested neuronal cells. Thus, this study demonstrates an alternative method to construct nonviral delivery system for targeted gene transfer into neurons.     

Sustained release of PEG-g-chitosan complexed DNA from poly(lactide-co-glycolide)

Chitosan and its derivatives have emerged as promising gene-delivery vehicles because of their capability to form polyplexes with plasmid DNA and enhance its transport across cellular membranes through endocytosis. Evidently, polyplexes of chitosan and DNA significantly improve transfection efficiency; however, these polyplexes are not capable of sustained DNA release and, thus, prolong gene transfer. In order to achieve prolonged delivery of DNA/chitosan polyplexes, we have formulated microspheres by physically combining poly(ethylene glycol)-grafted chitosan (PEG-g-CHN) with poly(lactide-co-glycolide) (PLGA) using a modified conventional in-emulsion solvent evaporation method. Electrophoretic analysis of materials released from these microspheres suggests the presence of PEG-g-CHN complexed DNA and these microspheres are capable of sustained release of DNA/PEG-g-CHN for at least 9 weeks. The rate of DNA release can be modulated by varying the amount of PEG-g-CHN. The release products from these microspheres are bioactive and show enhanced transfection in vitro over DNA released from conventional PLGA microspheres containing no PEG-g-CHN. In vivo experiments also show that these microspheres are capable of achieving gene transfer in a rat hind limb muscle model.     

Functional Poly(Dimethyl Aminoethyl Methacrylate) by Combination of Radical Ring‐Opening Polymerization and Click Chemistry for Biomedical Applications

In this work, the macromolecular design and modular synthesis of degradable and biocompatible copolymers via radical polymerization and click chemistry is highlighted and the resulting systems are evaluated as gene delivery carriers. Poly(ethylene glycol) (PEG) grafted poly[2‐methylene‐1,3‐dioxepane (MDO)‐co‐propargyl acrylate (PA)‐co‐2‐(dimethyl aminoethyl methacrylate (DMAEMA)] (MPD) is synthesized using radical polymerization and azide‐alkyne click chemistry. The polymers are less cytotoxic and are able to condense plasmid DNA into nanosized particles. The low transfection efficiency of polyplexes in HepG2 cells is significantly improved by mixing Tat peptide with polyplexes.     

Branched polyethylenimine-based PKCα-responsive gene carriers

We examined in vitro performance of the branched polyethylenimine (bPEI)-based gene carriers which respond to cancer-specific activation of protein kinase Cα (PKCα) to express plasmid DNA. The carriers were synthesized straightforward by using amide bond formation between a peptide terminal carboxyl and a primary amine group of bPEI. To examine the effect of the peptide contents in the carrier, we prepared several carriers with various peptide contents. The obtained polymers form polyplexes with tighter condensation of plasmid DNA than our previous gene carriers. After internalization of the polyplexes via endocytosis, the polyplexes effectively escaped from the endosome into cytosol. Then, the polyplexes showed a clear-cut response to PKCα to release plasmid DNA for gene expression. We determined the optimum contents of the peptides in carriers as 5?mol% to achieve the clear-cut response to PKCα.     

Tumor-Targeted Gene Transfer with DNA Polyplexes

Systemic gene delivery systems are needed for therapeutic applications; in some situations, target cells might be spread throughout the organism, as in the case of cancer metastases, which can be reached only via the systemic route. Within the class of nonviral vectors, polymer-based transfection particles named DNA polyplexes and lipid-based systems named DNA lipoplexes are being developed for this purpose. For systemic circulation, masking the surface charge of DNA complexes has to be accomplished to avoid interactions with plasma components, erythrocytes, and the reticuloendothelial system. Among other vector formulations, polyplexes based on polyethylenimine (PEI), shielded with polyethylene glycol (PEG), and linked to the receptor binding ligands transferrin (Tf) or epidermal growth factor (EGF) have been developed. Complexes were found to mediate efficient gene transfer into tumor cell lines in a receptor-dependent and cell-cycle-dependent manner. Systemic administration of surface-shielded Tf-PEI polyplexes into the tail vein of mice resulted in preferential gene delivery into distantly growing subcutaneous tumors. In contrast, application of positively charged PEI polyplexes directed gene transfer primarily to the lung.     

The mechanism of selective transfection mediated by pentablock copolymers; part II: nuclear entry and endosomal escape

Transfection efficiencies of non-viral gene delivery vectors commonly vary with cell type, owing to differences in proliferation rates and intracellular characteristics. Previous work demonstrated that the poly(diethylaminoethylmethacrylate) (PDEAEM)/Pluronic F127 pentablock copolymers exhibit transfection in vitro selectively in cancer cell lines as opposed to non-cancerous cell lines. This study continues the investigation of intracellular barriers to transfection using this vector in "normal" and cancer cell lines to understand the underlying mechanisms of the selectivity. Results from Part I of this investigation showed, using conjugated epidermal growth factor, that cellular uptake of these polyplexes is not a major barrier in these systems. Part II of this work continues the investigation into the other potential intracellular barriers, endosomal escape and nuclear entry, using a lysosomotropic agent chloroquine (CLQ), and a nuclear localization signal (NLS) SV40, respectively. Lack of effectiveness of NLS peptide in improving the transfection efficiency suggests that nuclear uptake might not be the major intracellular barrier using the pentablock copolymer vectors, or that the nuclear transport might not be primarily achieved through nuclear pores. However, inclusion of CLQ led to a dramatic enhancement in the level of gene expression, with an almost two orders of magnitude increase in expression seen in normal cell lines, compared with that the increase observed in cancer cell lines. The different lysosomal pH values in normal vs cancer cells was believed to cause the pentablock copolymer vectors to behave distinctly during transport through endocytic pathways, with greater loss of functional DNA occurring in normal cells containing more acidic endocytic vesicles in contrast to cancer cells with less acidic vesicles. Interestingly, CLQ introduced almost no enhancement in the transfection with the control vector ExGen which lacked selectivity of transfection. Exploiting intracellular differences between normal and cancer cells for gene delivery vector design offers a new paradigm to achieve transfection selectivity based on intracellular differences rather than conventional approaches involving vector modification using specific ligands for targeted delivery.     

All-trans-retinoic acid (ATRA)-grafted polymeric gene carriers for nuclear translocation and cell growth control

Polyethyleneimine (PEI)-g-All-trans-retinoic acid (ATRA) (designated as PRA) was synthesized as a gene carrier. ATRA at its low concentration is known to be linked to nuclear translocation and cell cycle control (either proliferation or growth arrest) depending on its binding protein in cells. The cytotoxicity of PRA conjugates was lower than that of PEI and was gradually reduced as increasing ATRA graft ratios. The resulting nanosized and positively charged PRA/pDNA complexes showed lower transfection efficiency than the PEI/pDNA complexes (N/P = 10) against NIH3T3 which is less sensitive to ATRA in cell growth and more sensitive HeLa cells. However, when a mixed gene complex of PEI and PRA was applied in an effort to reduce the ATRA contents, their NIH3T3 transfection evidenced effective nuclear translocation and induced 2- to 4-fold better transfection efficiency as compared with the PEI/pDNA complexes. When the PEI/pDNA complexes were utilized to transfect HeLa cells, free ATRA treatment reduced their cellular uptake and transfection efficiency. These findings show that the NIH3T3 cells against ATRA-mediated growth arrest would not damage the PRA-mediated transfection enhancement resulting from the facilitated nuclear translocation of polyplexes or pDNA. The more ATRA-sensitivity in growth arrest of HeLa cells would reduce the transfection efficiency of ATRA-incorporated polyplexes. The transfection capability of gene by newly synthesized PRA conjugates to cells is differentiated by their ATRA-sensitivity to nuclear translocation and cell growth control.     

Pluronic-based cationic block copolymer for forming pDNA polyplexes with enhanced cellular uptake and improved transfection efficiency

PEGylated cationic polymers have been extensively studied for substituting virus as gene delivery vehicles. These polymers can produce water-soluble polyionic complexes (polyplexes) with plasmid DNA (pDNA) and show enhanced stability compared to non-PEGylated polyplexes. However, PEGylation always diminishes the transfection efficiency of polyplexes probably due to poor cellular internalization of the particles and difficulty in releasing the pDNA cargo from the complexes intracellularly for gene expression. As non-ionic surfactants, Pluronic block copolymers have been shown to interact with plasma membrane and promote cellular uptake of various small molecules and biomacromolecules. To evaluate whether Pluronic could improve the transfection efficiency of polyplexes, Pluronic P85- and PEG-based cationomers comprising poly{N-[N-(2-aminoethyl)-2-aminoethyl] aspartamide (P[Asp(DET)]) cationic blocks were synthesized and tested for their transfection ability. In this study, it was demonstrated that although the stability of the PEG-based polyplexes was better than that of the P85-based polyplexes based cationic polymers, the P85-based polyplex could achieve significantly higher transfection than the PEG counterparts. The improvement of gene delivering ability was shown to be correlated with the enhanced cellular internalization of the P85-based polyplexes.     

The gene transfection efficiency of a folate–PEI600–cyclodextrin nanopolymer

The success of gene therapy relies on a safe and effective gene delivery system. In this communication, we describe the use of folate grafted PEI₆₀₀–CyD (H₁) as an effective polyplex-forming plasmid delivery agent with low toxicity. The structures of the polymer and polyplex were characterized, and the in vitro transfection efficiency, cytotoxicity, and in vivo transfection of H₁ were examined. We found that folate molecules were successfully grafted to PEI₆₀₀–CyD. At N/P ratios between 5 and 30, the resulting H₁/DNA polyplexes had diameters less than 120 nm and zeta potentials less than 10 mV. In various tumor cell lines examined (U138, U87, B16, and Lovo), the in vitro transfection efficiency of H₁ was more than 50%, which could be improved by the presence of fetal bovine serum or albumin. The cytotoxicity of H₁ was significantly less than high molecular weight PEI-25 kDa. Importantly, in vivo optical imaging showed that the efficiency of H₁-mediated transfection (50 μg luciferase plasmid (pLuc), N/P ratio = 20/1) was comparable to that of adenovirus-mediated luciferase transduction (1 × 10⁹ pfu) in melanoma-bearing mice, and it did not induce any toxicity in the tumor tissue. These results clearly show that H₁ is a safe and effective polyplex-forming agent for both in vitro and in vivo transfection of plasmid DNA and its application warrants further investigation.     

Transfection and intracellular trafficking characteristics for poly(amidoamine)s with pendant primary amine in the delivery of plasmid DNA to bone marrow stromal cells

Poly(amidoamine)s with pendant primary amine (polymer 1a–1c) were evaluated as in vitro non-viral gene delivery vectors for bone marrow stromal cells (BMSCs). The cytotoxicity of these poly(amidoamine)s, measured by MTT assay, increased with increasing length of side chain, however, they were less toxic than branched polyethylenimine (PEI) 25 kDa. Using pGL-3 and pEGFP-C1 as luciferase gene and green fluorescent protein (GFP) gene, among all polycations including polymer 1a–1c and PEI, polymer 1b at optimal N/P ratio showed highest luciferase expression (1.92 × 10⁸ RLU/mg protein) as well as percentage of cells expressing GFP (29.01 ± 2.33%). For all polycations, intracellular trafficking of Cy3-labelled plasmid DNA (pDNA) was similar. Fluorescent particles attached to cell membrane at 0.5 h after adding the polycation/DNA complexes, aggregated in cytoplasm after 2 h, and then stayed around the perinuclear region after 4 h. pDNA nuclear localization appeared at 4 h post-transfection, but much more pDNA entered into nucleus at 24 h. At high N/P ratio, polymer 1a–1c could deliver pDNA into 70–80% of BMSCs after 24 h transfection, however, labelled pDNA was observed in only 4–25% of cells at the same time. Compared to PEI, polymer 1b showed comparable or even higher percentage of pDNA uptake and nuclear localization. We concluded that poly(amidoamine)s with pendant primary amine, especially polymer 1b, are new kind of promising candidates of less toxic and highly efficient non-viral gene delivery vectors for BMSCs.     

Peptides containing antigenic and cationic domains have enhanced,multivalent immunogenicity when bound to DNA vaccines

We explored strategies to codeliver DNA- and peptide-based vaccines in a way that enhances the immunogenicity of both components of the combination vaccine for T cells. Specific CD8⁺ T cell responses to an antigenic peptide are primed when the peptide is fused to a cationic peptide domain that is bound to plasmid DNA or oligonucleotides (ODN; with or without CpG motifs). Plasmid DNA mixed with antigenic/cationic peptides or histones forms large complexes with different biological properties depending on the molar ratios of peptide/protein and polynucleotide. Complexes containing high (but not low) molar ratios of cationic peptide to DNA facilitate transfection (DNA uptake and expression of the plasmid-encoded product) of cells. In contrast, complexes containing low (but not high) molar ratios of cationic peptide to DNA prime potent multispecific T cell responses after a single intramuscular injection of the complexes. The general validity of this observation was confirmed mixing different antigenic/cationic peptides with different DNA vaccines. In these vaccine formulations, multispecific CD8⁺ T cell responses specific for epitopes of the peptide- as well as the DNA-based vaccine were efficiently coprimed, together with humoral antibody responses to conformational determinants of large viral antigens encoded by the DNA vaccine. The data indicate that mixtures of DNA vaccines with antigenic, cationic peptides are immunogenic vaccine formulations particularly suited for the induction of multispecific T cell responses.Abbreviations EGFP Enhanced green fluorescent protein - HBcAg Hepatitis B core antigen - HBsAg Hepatitis B surface antigen - HEK Human embryonal kidney - ISS Immune-stimulating sequence - NSS Nonstimulating sequence - ODN Oligodeoxynucleotide     

Nonviral Transfection of Adipose Tissue Stromal Cells: An Experimental Study

Delivery of plasmid DNA and interfering RNA into adipose tissue stromal cells was carried out by the methods of lipofection, calcium phosphate method, and by electroporation. The percent of transfected cells after delivery of plasmid DNA by the calcium phosphate method and lipofection was 0 and 15%, respectively, vs. more than 50% after electroporation. Similar results were obtained for delivery of short-strand RNA into cells. These data indicate that electroporation is the most effective method of nonviral transfection of adipose tissue stromal cells.     

Easy stable transfection of a human cancer cell line by electrogene transfer with an Epstein–Barr virus-based plasmid vector

We report an easy and stable transfection technique using electrogene transfer with a nonviral Epstein–Barr (EB) virus-based vector. To achieve stable transfection of human breast cancer cells, we conducted electrogene transfer of an EB virus-based plasmid vector (reduced size of oriP) containing the enhanced green fluorescence protein (eGFP) gene. Because the EB virus-based vector exhibits high transfer efficiency and strong persistent transgene expression as a result of autonomous replication in human cells, and as Nucleofector electrogene transfer can achieve highly efficient gene transfection, this method is particularly suitable for generation of stably transfected cell lines.