Bioreducible Guanidinylated Polyethylenimine for Efficient Gene Delivery

Cationic polymers are known to afford efficient gene transfection. However, cytotoxicity remains a problem at the molecular weight for optimal DNA delivery. As such, optimized polymeric gene delivery systems are still a sought‐after research goal. A guanidinylated bioreducible branched polyethylenimine (GBPEI‐SS) was synthesized by using a disulfide bond to crosslink the guanidinylated BPEI (GBPEI). GBPEI‐SS showed sufficient plasmid DNA (pDNA) condensation ability. The physicochemical properties of GBPEI‐SS demonstrate that it has the appropriate size (～200 nm) and surface potential (～30 mV) at a nitrogen‐to‐phosphorus ratio of 10. No significant toxicity was observed, possibly due to bioreducibility and to the guanidine group delocalizing the positive charge of the primary amine in BPEI. Compared with the nonguanidinylated analogue, BPEI‐SS, GBPEI‐SS showed enhanced transfection efficiency owing to increased cellular uptake and efficient pDNA release by cleavage of disulfide bonds. This system is very efficient for delivering pDNA into cells, thereby achieving high transfection efficiency and low cytotoxicity.     

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.     

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.     

Guanidinylation: A simple way to fabricate cell penetrating peptide analogue‐modified chitosan vector for enhanced gene delivery

In view of the analogous transmembrane function to cell penetrating peptides, guanidine group was incorporated into chitosan by chemical modification to enhance the transfection performance of chitosan vectors. Guanidinylated chitosan (GCS) was shown to be well soluble in neutral aqueous solution. The interaction between GCS with plasmid DNA was characterized by agarose retardation experiment and ethidium bromide displacement assay. GCS formed more stable complexes with DNA under physiological pH than chitosan. The transfection efficiency of GCS was evaluated employing COS‐7 cell line—GCS polyplexes demonstrated higher transfection efficiency and lower cytotoxicity relative to chitosan. The optimum efficiency of GCS was achieved in the vicinity of the critical complexing ratio. The results of flow cytometry indicated that guanidinylation promoted an eightfold increase in the cell uptake. The study revealed that guanidinylated chitosan is a promising candidate as an effective nonviral vector for in vivo gene delivery. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Characterization and Optimization of Chitosan-Coated Polybutylcyanoacrylate Nanoparticles for the Transfection-Guided Neural Differentiation of Mouse Induced Pluripotent Stem Cells

Gene transfection is a valuable tool for analyzing gene regulation and function, and providing an avenue for the genetic engineering of cells for therapeutic purposes. Though efficient, the potential concerns over viral vectors for gene transfection has led to research in non-viral alternatives. Cationic polyplexes such as those synthesized from chitosan offer distinct advantages such as enhanced polyplex stability, cellular uptake, endo-lysosomal escape, and release, but are limited by the poor solubility and viscosity of chitosan. In this study, the easily synthesized biocompatible and biodegradable polymeric polysorbate 80 polybutylcyanoacrylate nanoparticles (PS80 PBCA NP) are utilized as the backbone for surface modification with chitosan, in order to address the synthetic issues faced when using chitosan alone as a carrier. Plasmid DNA (pDNA) containing the brain-derived neurotrophic factor (BDNF) gene coupled to a hypoxia-responsive element and the cytomegalovirus promotor gene was selected as the genetic cargo for the in vitro transfection-guided neural-lineage specification of mouse induced pluripotent stem cells (iPSCs), which were assessed by immunofluorescence staining. The chitosan-coated PS80 PBCA NP/BDNF pDNA polyplex measured 163.8 ± 1.8 nm and zeta potential measured −34.8 ± 1.8 mV with 0.01% (w/v) high molecular weight chitosan (HMWC); the pDNA loading efficiency reached 90% at a nanoparticle to pDNA weight ratio of 15, which also corresponded to enhanced polyplex stability on the DNA stability assay. The HMWC-PS80 PBCA NP/BDNF pDNA polyplex was non-toxic to mouse iPSCs for up to 80 μg/mL (weight ratio = 40) and enhanced the expression of BDNF when compared with PS80 PBCA NP/BDNF pDNA polyplex. Evidence for neural-lineage specification of mouse iPSCs was observed by an increased expression of nestin, neurofilament heavy polypeptide, and beta III tubulin, and the effects appeared superior when transfection was performed with the chitosan-coated formulation. This study illustrates the versatility of the PS80 PBCA NP and that surface decoration with chitosan enabled this delivery platform to be used for the transfection-guided differentiation of mouse iPSCs.     

6‐O‐dodecyl‐chitosan carbamate–based pH‐responsive polymeric micelles for gene delivery

pH‐responsiveness is highly desirable in the stimuli‐responsive controlled release because of the distinct advantages of the fast response of pH‐triggered release and the available pH‐difference between intra‐ and extra‐cells. The present work reported a kind of novel pH‐responsive polymeric micelles, which was derived from biopolymer of 6‐O‐dodecyl‐chitosan carbamate (DCC) and evaluated as gene‐controlled release vector. The amphiphilic and amino‐rich DDC was synthesized through a protection‐graft‐deprotection method. ¹³C CP/MAS NMR, FTIR, and elemental analysis identified that dodecyls were chemoselectively grafting at 6‐hydroxyls of chitosan via the pH‐responsive bonds of carbamate, and the substitute degree (SD) was 14%. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) showed that DCC self‐assembled into polymeric micelles in aqueous solutions. The DCC polymeric micelles formed complexes with pDNA, which was elucidated by Gel retardation, TEM, and DLS. Transfection and cytotoxicity assays in A549 cells showed that DCC polymeric micelles were suitable for gene delivery. The improved transfection was attributed to the pH‐responsiveness and the moderate pDNA‐binding affinity, which led to easier release of pDNA intra‐cells. The synthesized DCC polymeric micelles might be a promising and safe candidate as nonviral vectors for gene delivery. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 42469.     

Development of Covalent Chitosan-Polyethylenimine Derivatives as Gene Delivery Vehicle: Synthesis,Characterization, and Evaluation

There is an increasing interest in cationic polymers as important constituents of non-viral gene delivery vectors. In the present study, we developed a versatile synthetic route for the production of covalent polymeric conjugates consisting of water-soluble depolymerized chitosan (dCS; M_W 6–9 kDa) and low molecular weight polyethylenimine (PEI; 2.5 kDa linear, 1.8 kDa branched). dCS-PEI derivatives were evaluated based on their physicochemical properties, including purity, covalent bonding, solubility in aqueous media, ability for DNA condensation, and colloidal stability of the resulting polyplexes. They were complexed with non-integrating DNA vectors coding for reporter genes by simple admixing and assessed in vitro using liver-derived HuH-7 cells for their transfection efficiency and cytotoxicity. Using a rational screening cascade, a lead compound was selected (dCS-Suc-LPEI-14) displaying the best balance of biocompatibility, cytotoxicity, and transfection efficiency. Scale-up and in vivo evaluation in wild-type mice allowed for a direct comparison with a commercially available non-viral delivery vector (in vivo-jetPEI). Hepatic expression of the reporter gene luciferase resulted in liver-specific bioluminescence, upon intrabiliary infusion of the chitosan-based polyplexes, which exceeded the signal of the in vivo jetPEI reference formulation by a factor of 10. We conclude that the novel chitosan-derivative dCS-Suc-LPEI-14 shows promise and potential as an efficient polymeric conjugate for non-viral in vivo gene therapy.     

Monitoring the functionalization of single-walled carbon nanotubes with chitosan and folic acid by two-dimensional diffusion-ordered NMR spectroscopy

A conjugate between single-walled carbon nanotubes, chitosan and folic acid has been prepared. It was characterized by diffusion ordered two-dimensional hydrogen-1 nuclear magnetic resonance and hydrogen-1 nuclear magnetic resonance spectroscopy which revealed the presence of a conjugate that was generated by the linkage between the carboxyl moiety of the folic acid and the amino group of the chitosan, which in turn was non-covalently bound to the single-walled carbon nanotubes. The obtained diffusion coefficient values demonstrated that free folic acid diffused more rapidly than the folic acid conjugated to single-walled carbon nanotubes–chitosan. The values of the proton signal of hydrogen-1 nuclear magnetic resonance spectroscopy and two-dimensional hydrogen-1 nuclear magnetic resonance spectroscopy further confirmed that the folic acid was conjugated to the chitosan, wrapping the single-walled carbon nanotubes.     

Preparation of pH-sensitive CaP nanoparticles coated with a phosphate-based block copolymer for efficient gene delivery

We have synthesized a phosphate-based block copolymer, PEG-b-PMOEP (poly(ethylene glycol)-b-poly(2-methacryloyloxyethyl phosphate)), with a narrow molecular weight distribution (PD = 1.06) by atomic transfer radical polymerization (ATRP), and have constructed calcium phosphate nanoparticles (CaPNs) coated with the block copolymer as an efficient and safe intracellular gene delivery carrier. The phosphate-mimic PMOEP block could be incorporated into the calcium phosphate (CaP) core to entrap pDNA, with the PEG block forming a shell to prevent uncontrolled growth of CaP precipitates and aggregates in physiological fluids. The CaPNs showed high colloidal stability at pH 7.4, but released entrapped pDNA at an endosomal pH of 5.0 through a pH-dependent protonation of phosphate moieties for efficient endosomal escape. The PEG-b-PMOEP/CaP/pDNA nanoparticles, which were formed simply by mixing, exhibited great potential as gene delivery carriers for future gene therapy applications due to their high transfection efficiency, low toxicity, and good stability under physiological conditions.     

Depolymerized chitosan nanoparticles for protein delivery: Preparation and characterization

In this article we describe our preliminary work involving the use of depolymerized, low molecular weight chitosan nanoparticles as carriers for proteins and peptides. We hypothesized that the molecular weight of chitosan could favorably modulate the particle and protein release characteristics for the delivery of certain bioactive macromolecules. Our primary objectives were to develop nanoparticle formulations that were stable and reproducible across a range of chitosan molecular weights and then characterize the physicochemical and in vitro release properties as functions of the polymer size. Using depolymerized fragments generated by NaNO₂ degradation of different chitosan salts, we prepared nanoparticle formulations based on ionotropic gelation with sodium tripolyphosphate (TPP). Regardless of the formulation, the nanoparticle size decreased with decreasing molecular weight and the ζ‐potential values remained unchanged. Similar comparisons were made with the encapsulation of insulin and tetanus toxoid as model proteins. The results indicated that the quantity of TPP in a given formulation has a greater effect on the protein encapsulation than the chitosan molecular weight. In fast release environments (i.e., buffered media), there was no significant molecular weight effect that could be discerned. These data lead to the conclusion that, under these experimental conditions, the chitosan molecular weight has a measurable effect on the particle properties, although this effect is modest relative to other formulation parameters (e.g., TPP content, type of protein loaded). Because these subtle differences could have dramatic effects physiologically, work is currently underway to elucidate the possible applications of depolymerized chitosans for peptide delivery in vivo. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 12: 2769–2776, 2003     

DNA Targeting Sequence Improves Magnetic Nanoparticle-Based Plasmid DNA Transfection Efficiency in Model Neurons

Efficient non-viral plasmid DNA transfection of most stem cells, progenitor cells and primary cell lines currently presents an obstacle for many applications within gene therapy research. From a standpoint of efficiency and cell viability, magnetic nanoparticle-based DNA transfection is a promising gene vectoring technique because it has demonstrated rapid and improved transfection outcomes when compared to alternative non-viral methods. Recently, our research group introduced oscillating magnet arrays that resulted in further improvements to this novel plasmid DNA (pDNA) vectoring technology. Continued improvements to nanomagnetic transfection techniques have focused primarily on magnetic nanoparticle (MNP) functionalization and transfection parameter optimization: cell confluence, growth media, serum starvation, magnet oscillation parameters, etc. Noting that none of these parameters can assist in the nuclear translocation of delivered pDNA following MNP-pDNA complex dissociation in the cell’s cytoplasm, inclusion of a cassette feature for pDNA nuclear translocation is theoretically justified. In this study incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid improved transfection efficiency in model neurons, presumably from increased nuclear translocation. This observation became most apparent when comparing the response of the dividing SH-SY5Y precursor cell to the non-dividing and differentiated SH-SY5Y neuroblastoma 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     

Improved cell viability of linear polyethylenimine through gamma-cyclodextrin inclusion for effective gene delivery

A polypseudorotaxane consisting of a linear polyethylenimine with Mn of 22,000 (LPEI22k) and gamma-cyclodextrins (gamma-CDs; LPEI22k/gamma-CD) has been examined as a gene carrier. The polyplex formation with luciferase-encoding plasmid DNA (pDNA), intracellular trafficking of polyplex, cytotoxicity, and transfection efficiency were evaluated by various characteristic methods. LPEI22k/gamma-CD formed a pDNA polyplex at higher N/P ratios than LPEI22k; this suggests that the gamma-CD threading sterically interfered with the polyplex formation. In addition, the zeta potentials of the polyplex significantly decreased due to the reduction in charge density of LPEI22k caused by gamma-CD threading. The cellular uptake of pDNA in the LPEI22k/gamma-CD polyplex was enhanced by free gamma-CDs released from the polyplex that might accelerate the cellular uptake through enhanced membrane affinity. LPEI22k/gamma-CD significantly increased cell viability even at high N/P ratios, and the polyplex showed high transfection efficacy. The low cytotoxicity and high gene expression of LPEI22k/gamma-CD are advantageous to polyplex administration in vivo.     

Effect of degree of deacetylation on solubility of low‐molecular‐weight chitosan produced via enzymatic breakdown of chitosan

Chitosan has emerged as a unique biomaterial, possessing scope in diverse applications in the biomedical, food and chemical industries. However, its high molecular weight is a concern when handling the polymer. Various techniques have been explored for depolymerization of this polymer, wherein enzymes have emerged as the most economic method having minimum degrading effect on the polymer and resulting in formation of side products. Chitosan can be depolymerized using a broad range of enzymes. In this study, various enzymes like α‐amylase, papain, pepsin and bromelain were employed to depolymerize chitosan and convert it into its lower molecular weight counterpart. Further, attempts were made to elucidate the process of depolymerization of chitosan, primarily by determining the change in its viscosity and hence its molecular weight. The process of depolymerization was optimized using a one‐factor‐at‐a‐time approach. The molecular weight of the resultant chitosan was estimated using gel permeation chromatography and infrared spectroscopy. These studies revealed a considerable decrease in molecular weights of chitosan depolymerized by pepsin, papain, bromelain and α‐amylase, resulting in recovery of the low‐molecular‐weight chitosan of 76.09 ± 5, 74.18 ± 5, 55.75 ± 5 and 49.18 ± 5%, respectively. Maximum yield and depolymerization were obtained using pepsin and papain due to their enzymatic recognition pattern, which was also validated using studies involving molecular dynamics. © 2019 Society of Chemical Industry     

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     

Improved transfection efficiency of chitosan‐DNA complexes employing reverse transfection

The present study was designed to systematically compare the conventional and reverse transfection methodologies for chitosan/DNA complexes using a low molecular weight (MW) chitosan. The hydrodynamic diameter of the complexes, measured by Dynamic Light Scattering (DLS) was found to be ～ 216 nm and TEM investigations showed spherical and compact complexes with an average size of 200 nm. The transfection efficiency of chitosan using the two methodologies was assessed by employing reporter gene coding for green fluorescent protein (GFP) and luciferase. More than 50% of HEK 293 cells were transfected when transfection done using reverse transfection strategy at pH 6.5 with 10% serum for 24 h followed by media replenishment with pH 7.4 with 10% serum for an additional 24 h period. Also, the cytotoxicity of chitosan/DNA complexes was also considerably lower than the commercially available transfection reagent lipofectamine. Our investigation concludes that maximal transgene expression levels could be achieved using reverse transfection where the chitosan/DNA complexes are pre‐incubated on the plate surface followed by plating of cells at pH 6.5 with 10% serum for 24h and media resupplemented with pH 7.4 with 10% serum for an additional 24 h period. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Graphene based gene transfection

Graphene as a star in materials research has been attracting tremendous attentions in the past few years in various fields including biomedicine. In this work, for the first time we successfully use graphene as a non-toxic nano-vehicle for efficient gene transfection. Graphene oxide (GO) is bound with cationic polymers, polyethyleneimine (PEI) with two different molecular weights at 1.2 kDa and 10 kDa, forming GO-PEI-1.2k and GO-PEG-10k complexes, respectively, both of which are stable in physiological solutions. Cellular toxicity tests reveal that our GO-PEI-10k complex exhibits significantly reduced toxicity to the treated cells compared to the bare PEI-10k polymer. The positively charged GO-PEI complexes are able to further bind with plasmid DNA (pDNA) for intracellular transfection of the enhanced green fluorescence protein (EGFP) gene in HeLa cells. While EGFP transfection with PEI-1.2k appears to be ineffective, high EGFP expression is observed using the corresponding GO-PEI-1.2k as the transfection agent. On the other hand, GO-PEI-10k shows similar EGFP transfection efficiency but lower toxicity compared with PEI-10k. Our results suggest graphene to be a novel gene delivery nano-vector with low cytotoxicity and high transfection efficiency, promising for future applications in non-viral based gene therapy.     

ROS-sensitive selenium-containing cationic brush polymer with potent gene transfection efficiency and biocompatibility

Smart gene delivery vectors are gaining increasing attention in gene therapy, owing to their low cytotoxicity and intrinsic responsiveness. Our previously fabricated novel cationic brush polymer, comprising C Se bonds and tertiary amine EGIn-g-PDMAEMA, shows potential for gene transfection. In this study, its high efficiency for siRNA/pDNA transfection and low cytotoxicity in reactive oxygen species (ROS)-rich microenvironments is substantiated in vitro. Its superior binding capacity with siRNA/pDNA is confirmed by agarose gel electrophoresis assay. The threshold weight ratios for siRNA/pDNA migration delay are 15 and 3 (polymer-to-nucleic acid, w/w), respectively. Fluorescence microscopy and ribonucleotide reductase regulatory subunit M2 gene silencing essay verify the biodegradability and responsive control release of nucleic acids under hydrogen peroxide stimulation in Huh-7 cells. Compared with the gold standard, polyethylenimine 25 kDa, the target polymer displays superior transfection efficiency in ROS-rich tumor cells under serum-free conditions. Furthermore, the vector–nucleic acid complexes exhibit over 90% cell viability at a high concentration of 12 μg mL⁻¹ and good colloidal stability in phosphate-buffered saline (PBS) and 10% fetal bovine serum-PBS for 24 h. The efficient control release and expression of nucleic acids in ROS environments and reduced cytotoxicity highlight the superiority of EGIn-g-PDMAEMA as a gene delivery platform for tumor gene therapy.     

Calcium Phosphate—DNA Nanocomposites: Morphological Studies and Their Bile Duct Infusion for Liver-Directed Gene Therapy

In this study, liver-directed gene transfer in rats with calcium phosphate (CaP) nanoparticles and the effect of the route of administration and surgical manipulations on transfection efficiency is reported. Formulations of CaP nanoparticles entrapping plasmid DNA (pDNA) were prepared by the reverse micellar method using two different surfactants. Transmission electron microscopy, scanning electron microscopy and dynamic light scattering were used to characterize the CaP–DNA nanocomposites. The morphological characteristics of the formulations showed a strong dependency on temperature. Gel electrophoresis experiments indicated that there was no degradation of the encapsulated pDNA, and in vitro cell transfection in HEK-293 and primary hepatocytes from rats as well as in vivo intraductal delivery experiments suggested that CaP nanoparticles led to significant and prolonged transgene expression. Therefore, our methodology gives a stable and viable formulation for hepatic gene therapy. Low-DNA dosage entrapped in CaP nanoparticles makes it an effective gene delivery system for clinical applications.     

Improvement of hepatocyte-specific gene expression by a targeted colchicine prodrug

Colchicine, an established tubulin inhibitor, interferes with the trafficking of endocytotic vesicles and thereby promotes the escape of lysosome-entrapped compounds. To improve its potency and cell specificity, a targeted prodrug of colchicine was synthesized by conjugation to a high-affinity ligand (di-N(alpha),N(epsilon)-(5-(2-acetamido-2-deoxy-beta-D-galactopyranosyloxy)pentanomido)lysine, K(GalNAc)(2)) for the asialoglycoprotein receptor on parenchymal liver cells. The resulting colchicine-K(GalNAc)(2) conjugate bound to this receptor with an affinity of 4.5 nM. Confocal microscopy studies confirmed rapid uptake and receptor dependency of a prodrug conjugated with fluorescein isothiocyanate. Colchicine-K(GalNAc)(2) substantially increased the transfection efficiency of polyplexed DNA in parenchymal liver cells in a concentration- and receptor-dependent fashion. Colchicine-K(GalNAc)(2) was found to enhance the transfection efficiency by 50-fold at 1 nM, whereas the parental colchicine was ineffective. In conclusion, this nontoxic colchicine-K(GalNAc)(2) conjugate can be a useful tool to improve the transfection efficiency of hepatic nonviral gene transfer vehicles.