Transfection Efficiency In HepG2 Cells Research Articles

Diffusion coefficient of cationic liposomes during lipoplex formation determines transfection efficiency in HepG2 cells

Cationic lipid-based lipoplexes are well-known for gene delivery. To determine the relationship between physicochemical characteristics and transfection efficiency, cationic liposomes of different sizes were prepared and incubated with plasmid DNA at different temperatures to form lipoplexes. We found that the liposome diffusion coefficient during lipoplex formation strongly correlated with the physicochemical characteristics of lipoplexes, accessibility of plasmid DNA in lipoplexes, and logarithm of gene expression per metabolic activity. Clathrin-mediated endocytosis was the major route for lipoplexes comprising 100 nm-liposomes, as reported previously. As liposome size increased, the major route shifted to lipid raft-mediated endocytosis. In addition, macropinocytosis was observed for all liposome sizes. The role of reactive oxygen species might depend on liposome size and endocytosis. Information from this study would be useful for understanding cationic lipoplex-mediated transfection.

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing.

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and Pfizer/BioNTech. These vaccines have demonstrated the efficacy of mRNA-LNP therapeutics and opened the door for future clinical applications. In mRNA-LNP systems, the LNPs serve as delivery platforms that protect the mRNA cargo from degradation by nucleases and mediate their intracellular delivery. The LNPs are typically composed of four components: an ionizable lipid, a phospholipid, cholesterol, and a lipid-anchored polyethylene glycol (PEG) conjugate (lipid-PEG). Here, LNPs encapsulating mRNA encoding firefly luciferase are formulated by microfluidic mixing of the organic phase containing LNP lipid components and the aqueous phase containing mRNA. These mRNA-LNPs are then tested in vitro to evaluate their transfection efficiency in HepG2 cells using a bioluminescent plate-based assay. Additionally, mRNA-LNPs are evaluated in vivo in C57BL/6 mice following an intravenous injection via the lateral tail vein. Whole-body bioluminescence imaging is performed by using an in vivo imaging system. Representative results are shown for the mRNA-LNP characteristics, their transfection efficiency in HepG2 cells, and the total luminescent flux in C57BL/6 mice.

Hydrothermal Reduction of Polyethylenimine and Polyethylene Glycol Dual-Functionalized Nanographene Oxide for High-Efficiency Gene Delivery.

In this study, a physiologically stable dual-polymer-functionalized reduced nanographene oxide (nrGO) conjugate (PEG-nrGO-PEI, RGPP) with high efficiency of gene delivery is successfully synthesized through mixing PEGylated nanographene oxide (PEG-nGO, GP) and polyethylenimine (PEI, 25 kDa) solution under 80 °C for 2 h. This hydrothermal reduction of GP during PEIylation promotes the nucleophilic reaction between the amino moieties of PEI and the epoxy groups (or carboxylic groups) in GP and then forms C-NH- groups (or NH-CO groups) to covalently connect PEI and GP, which makes the RGPP nanocomposite more stable in physiological environments and has superior gene transfection efficiency compared with the nonhydrothermally reduced PEG-nGO/PEI conjugate (GPP) obtained by mixing GP and PEI under 20 °C for 2 h. Moreover, 808 nm laser irradiation (2 W/cm2) for 25 min increases ∼1.5-fold of gene transfection efficiency for RGPP but does not increase the gene transfection efficiency of GPP. Finally, RGPP is also able to efficiently deliver functional plasmid GFP-Bax (pGFP-Bax), exhibiting ∼43% of transfection efficiency in HepG2 cells. Collectively, the RGPP developed here is a highly efficient nanocarrier for gene delivery, and this work encourages further explorations of developing functionalized reduced nano-GO for high-efficiency gene therapy.

PAMAM-pullulan conjugates as targeted gene carriers for liver cell

Targeted nano-carriers are highly needed to promote nucleic acid delivery into the specific cell for therapeutic approaches. Pullulan as a linear carbohydrate has an intrinsic liver targeting property interacting with asialoglycoprotein receptor (ASGPR) found on liver cells. In the present study, we developed polyamidoamine (PAMAM)-pullulan conjugates and investigated their targeting activity in delivering gene into liver cells. The particle size, zeta potential, buffering capacity and ethidium bromide exclusion assays of the conjugates were evaluated. The cytotoxicity and transfection efficiency of new derivatives were assessed following in vitro transfection of HepG2 (receptor positive) and N2A (receptor negative) cell lines. Size of conjugated polymers ranged between 118 and 184 nanometers and their cytotoxicity were similar to PAMAM. Among six produced nanocarriers, G4PU4 and G5PU4 enhanced transfection efficiency in HepG2 cells compared to unmodified PAMAM. Therefore, the PAMAM-pullulan derivatives seem to improve delivery of nucleic acids into the liver cells expressing asialoglycoprotein receptor with minimal transfection in non-targeted cells.

Biocompatible anionic polyelectrolyte for improved liposome based gene transfection

Cationic liposomes have been widely used as efficient gene carriers. However, the serious cytotoxicity caused by exposed positive charges restricts the further application of those kinds of gene vectors. Thus, it is challenging to develop biocompatiable non-positive charge carriers to achieve high gene transfection efficiencies. Herein, we report a novel design by pasting biocompatible anionic polyelectrolyte, namely alginic acid, hyaluronic acid, pectin and polyglutamic acid, to the positive charge surface of liposome/pDNA complex. Through shielding the positive charges, the new gene carriers show decreased cytotoxicity while still maintaining high transfection efficiency. To be noted, the complex formed by coating polyglutamic acid to the surface of liposome/pDNA greatly enhanced the transfection efficiency in HepG2 cells, and the use of pectin shows increased transfection in MCF-7 cells. Hemolysis assay proved a possible mechanism that when the new gene complex was internalized into cells, as acidity increases, more side chains become hydrophobic, and thus destabilizing the endosomal membrane to accelerate DNA escape. The present results suggest that such anionic polyelectrolyte covered liposome based carrier possess promising application for clinical gene delivery.

Glucose-containing diblock polycations exhibit molecular weight, charge, and cell-type dependence for pDNA delivery.

A series of diblock glycopolycations were created by polymerizing 2-deoxy-2-methacrylamido glucopyranose (MAG) with either a tertiary amine-containing monomer, N-[3-(N,N-dimethylamino) propyl] methacrylamide (DMAPMA), or a primary amine-containing unit, N-(2-aminoethyl) methacrylamide (AEMA). Seven structures were synthesized via aqueous reversible addition–fragmentation chain transfer (RAFT) polymerization that varied in the block lengths of MAG, DMAPMA, and AEMA along with two homopolymer controls of DMAPMA and AEMA that lacked a MAG block. The polymers were all able to complex plasmid DNA into polyplex structures and to prevent colloidal aggregation of polyplexes in physiological salt conditions. In vitro transfection experiments were performed in both HeLa (human cervix adenocarcinoma) cells and HepG2 (human liver hepatocellular carcinoma) cells to examine the role of charge type, block length, and cell type on transfection efficiency and toxicity. The glycopolycation vehicles with primary amine blocks and PAEMA homopolymers revealed much higher transfection efficiency and lower toxicity when compared to analogs created with DMAPMA. Block length was also shown to influence cellular delivery and toxicity; as the block length of DMAPMA increased in the glycopolycation-based polyplexes, toxicity increased while transfection decreased. While the charge block played a major role in delivery, the MAG block length did not affect these cellular parameters. Lastly, cell type played a major role in efficiency. These glycopolymers revealed higher cellular uptake and transfection efficiency in HepG2 cells than in HeLa cells, while homopolycations (PAEMA and PDMAPMA) lacking the MAG blocks exhibited the opposite trend, signifying that the MAG block could aid in hepatocyte transfection.

Combining magnetic field/temperature dual stimuli to significantly enhance gene transfection of nonviral vectors.

Monodisperse magnetic nanoparticles (MNPs) were prepared through an organic phase process, and the obtained MNPs were capped with poly[2-(2-methoxyethoxy)ethyl methacrylate]-b-poly[2-(dimethylamino)ethyl methacrylate] synthesized by surface-initiated atom transfer radical polymerization (ATRP). The MNPs-polymer brushes exhibited both superparamagnetic and thermoresponsive behaviors, and could condense plasmid DNA into nanocomplexes with a size of 100-120 nm at appropriate complexing ratios. Enhanced gene expression in COS-7 cells and HepG-2 cells was achieved under a magnetic field and variable temperature conditions due to magnetic force-facilitated internalization of nanocomplexes, and temporary cooling-triggered intracellular gene unpacking. Amazingly, combining magnetic field and temperature dual stimuli contributed to a 50-100- and 25-45-fold increase of the transfection efficiency in HepG-2 cells compared to conventional protocol and PEI25k, respectively.

Delivery of miRNA Using Fe<sub>2</sub>O<sub>3</sub> Nanoparticles Capped Polyethyleneimine as a Nonviral Carrier

An efficient and safe delivery system of RNA interfering is required for clinical application of gene therapy. The study aimed to develop Fe2O3-based nanoparticles for gene delivery to overcome the disadvantages of polyethyleneimine (PEI) or cationic liposome as gene carrier including the cytotoxicity caused by positive charge and aggregation in the cells surface. PEI-capped Fe2O3 nanoparticles are successfully manufactured utilizing Fe2O3 as core, PEI as carapace, which bind miRNA at an appropriate weight ratio by electrostatic interaction and result in well-dispersed nanoparticles. The synthesized GFP tag with miR-26a expression plasmid was used for monitoring transfection efficiency in HepG2 cells. The nanocomplex exhibited higher transfection efficiency and lower cytotoxicity in HepG2 cells than the PEI/DNA complex and commercially available liposome. The delivery resulted in a significantly upregulation of miR-26a in HepG2 cells. Our results offer an alternate delivery system for RNA interfering that can be used on any gene of interest.

Ternary Complexes of Poly(Vinyl Pyrrolidone)-Graft-Poly(2-Dimethylaminoethyl Methacrylate), DNA and Bovine Serum Albumin for Gene Delivery

Novel well-defined hydrophilic cationic polymers with different length of polycation chain for gene delivery, poly(vinyl pyrrolidone)-graft-poly(2-dimethylaminoethyl methacrylate)s (PPDs), were synthesized by atom transfer radical polymerization (ATRP). The chemical structures and compositions of these polymers were characterized by FT-IR, 1H-NMR and GPC. The experimental results of dynamic light scattering (DLS), ζ-potential and transmission electron microscopy (TEM) indicated that PPD could condense plasmid DNA (pDNA) to form nanocomplexes. Agarose gel retardation assays demonstrated that PPD could encapsulate plasmid DNA completely when the N/P ratio is equal to or above 3. MTT assay and in vitro gene transfection results indicated that PPD/pDNA complexes exhibited high transfection efficiency concomitant with obvious cytotoxicity. Furthermore, bovine serum albumin (BSA) was utilized to assembly with the binary complexes of PPD/pDNA to screen the residual surface positive charges of complexes in order to decrease cytotoxicity of the binary complexes. Physicochemical properties were characterized and the results indicated that the coating of BSA was able to decrease the zeta potential of the nano-sized PPD/pDNA complexes nearly to electroneutrality without interfering with DNA condensation ability. The ternary complexes of BSA/PPD/pDNA demonstrated no cytotoxicity and also maintained high gene transfection efficiency in HepG2 cells in 10% serum compared with that in serum-free condition.

Improved biocompatibility of poly(vinylpyrrolidone)-graft-poly(2-dimethylaminoethyl methacrylate)/DNA complexes by coating with bovine serum albumin

Abstract Bovine serum albumin (BSA) was utilized to assemble with the binary complexes of poly(vinylpyrrolidone)-graft-poly(2-dimethylaminoethyl methacrylate) (PVP-g-PDMAEMA)/DNA formed by layer-by-layer electrostatic interactions to screen the residual surface positive charges of complexes. The coating of BSA was able to decrease the zeta potential of binary complexes nearly to electroneutrality without interfering with DNA condensation ability. The ternary complexes of BSA/PVP-g-PDMAEMA/DNA demonstrated lower cytotoxicity compared with the binary complexes and also maintained high gene transfection efficiency in HepG2 cells.

Combined pluronic P85- and ultrasound contrast agents-mediated gene transfection to HepG2 cells

This study examined the effect of P85 (a pluronic block copolymer) and microbubble (MB) ultrasound contrast agents under ultrasound irradiation on gene transfection and expression. The pEGFP plasmids that can encode enhanced green fluorescent protein (pEGFP) served as a report gene and were mixed with different concentrations of MB/0.05% (w/v) P85. Then the plasmids were transfected into human hepatoma G2 (HepG2) cells. The HepG2 cells treated with MB/P85 or without treatment were exposed to ultrasound (US parameters: 1 MHz, 1.0 W/cm(2), 20 s, 20% duty cycle). Twenty-four hours later, the transfection efficiency was assessed by fluorescence microscopy and fluorescence activated cell sorting (FACS) analysis. The cell viability was evaluated by Trypan blue exclusion test. The results showed that the gene transfection efficiency in HepG2 cells under ultrasound irradiation was significantly higher than that without ultrasound irradiation. HepG2 cells in the MB or P85 group in the absence of ultrasound expressed less amount of green fluorescent protein. The expression efficiency reached (22.14 ± 3.06)% and the survival rate was as high as (55.73 ± 3.32)% in the 30% MB plus P85 group. It was concluded that MB and P85 in the presence of ultrasound can enhance gene transfection and expression.

Supramolecular assembly of cyclodextrin-based nanospheres for gene delivery

Polymeric hollow nanospheres were prepared by self-assembly of rod-coil complexes, in which the rod-like segments were formed by inclusion complexes of β -cyclodextrins ( β -CDs) and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) diamines. Nanospheres encapsulating DNA or polyethylenimine/DNA (PEI/DNA) were synthesized and characterized in terms of their supramolecular structures, DNA binding and condensation ability, cytotoxicity, and in vitro gene transfection efficiency in HepG2 cells.

Amphoteric hyaluronic acid derivative for targeting gene delivery

The study aimed to develop an amphoteric hyaluronic acid (HA) derivative with polyethyleneimine (PEI) chains (HAP) for gene delivery to overcome the disadvantages of PEI as gene carrier including the cytotoxicity caused by excess of positive charge, non-special interaction and aggregation in the blood, and non-target gene delivery. The HAP was synthesized by an imine reaction between periodate-oxidized HA and PEI. The HAP/DNA complex was prepared, and its characterization was investigated. The size of complex with higher molecular weight HA in PBS was about 200 nm at optimal charge ratio. No apparent aggregation among the particles was observed. The HAPs also showed high protection of DNA from nuclease, better dissociation of DNA from the complex and lower cytotoxicity. It also exhibited higher transfection efficiency in HepG2 cells than the PEI/DNA complex. Among all complexes, the HAP50/DNA complex was especially found to be most efficient, yielding comparable transfection efficiency with that of Lipofectamine/DNA lipoplexes. Moreover, the HAP-IR820 obviously accumulated in tumor after i.v. administration as compared to the PEI-IR820, which indicated that the HAP could assist the DNA targeting to the tumor. Therefore, HAP should be a promising non-viral gene vector.

Efficient Gene Delivery with Osmotically Active and Hyperbranched Poly(ester amine)s

Degradable and hyperbranched poly (ester amine)s (PEAs) were successfully synthesized by Michael addition reaction between hydrophilic glycerol triacrylate (GTA) and low-molecular-weight polyethylenimine (LMW-PEI) and evaluated as nonviral gene carriers. PEAs effectively condensed DNA with particle sizes below 200 nm and suitable surface charges (15-45 mV), suitable for intracellular delivery. PEAs degraded in a controlled fashion showing half-lives of more than 12 days and were essentially nontoxic in three different cell lines. Elevated transfection levels by luciferase assay revealed the superiority of PEAs over PEI 25K and Lipofectamine. PEAs synthesized using 1:4 mol ratio of GTA to PEI [GTA/PEI-1.2(1:4)] showed highest transfection efficiency in HepG2 cells. PEAs showed significant gene expression in vitro as well as in vivo through aerosol administration. Reduction in packed cell volume (PCV) of cells when treated with polyplexes supported the hyperosmotic effect of PEAs. Effect of bafilomycin A1 on transfection efficiency of PEAs on 293T cells indicated its endosomal buffering capacity. High transfection efficiency was attributed to the synergism from hyperosmotic glycerol backbone in the PEAs and endosomal buffering capacity of PEI amine groups. Therefore, this convergence of osmotically active biodegradable PEAs suggests their potential as a safe and efficient gene delivery vector.

Biotinylated disulfide containing PEI/avidin bioconjugate shows specific enhanced transfection efficiency in HepG2 cells

Targeting of non-viral gene vectors to liver cells could offer the opportunity to cure liver diseases. In this paper, disulfide-containing polyethylenimine (PEI-SS) was synthesized from low molecular weight branched PEI and cystamine bisacrylamide (CBA), and then grafted with biotin. The obtained biotinylated PEI-SS was bioconjugated with avidin via the biotin-avidin interaction to form a novel gene vector, biotinylated PEI-SS/avidin bioconjugate (ABP-SS). Characteristics of ABP-SS and its pDNA complexes were evaluated in terms of acid-base titration, agarose gel electrophoresis, SEM morphology observation, particle size and zeta-potential measurements, and PEI-SS was used as the control. The acid-base titration results showed that ABP-SS exhibited comparable buffer capability to 25 kDa PEI. The results of gel electrophoresis indicated that ABP-SS was able to condense pDNA efficiently at an N/P ratio of 6 and could be degraded by reducing agent DTT. The ABP-SS/pDNA complexes had a mean particle size of 226 +/- 40 nm and surface charges of 25 mV. The SEM images showed that the complexes had compact structures with spherical or quadrate shapes. In vitro cell viability and transfection of ABP-SS and PEI-SS were compared in HepG2, 293T and H446 cells. Among the three different cell lines, compared with PEI-SS, ABP-SS exhibited much lower cytotoxicity and higher transfection efficacy in HepG2 cells due to the biocompatibility of avidin and the specific interactions between avidin and HepG2 cells. Molecular probes were used to reveal the cellular uptake of complexes, and the results demonstrated that ABP-SS contributes to more cellular uptake of complexes in HepG2 cells, which was consistent with the transfection results.

Biodegradable poly(ester amine) based on glycerol dimethacrylate and polyethylenimine as a gene carrier

Polyethylenimine (PEI) vectors are widely used in gene delivery because of their high transfection efficiency owing to a unique proton sponge effect. An increase in molecular weight increases transfection efficiency, but simultaneously results in increased toxicity. Therefore, the design and synthesis of new degradable gene delivery carriers having high transfection efficiencies and reduced cytotoxicity are necessary. In the present study degradable poly(ester amines) (PEAs) based on glycerol dimethacrylate (GDM) and low molecular weight branched polyethylenimine (LMW-PEI) were synthesized in anhydrous methanol at 60 degrees C following Michael addition reaction. The transfection efficiencies of the synthesized PEA/DNA complexes were evaluated using three different cell lines (HeLa, HepG2 and 293T cells) in vitro. PEAs with zeta potential in the range of 30-55 mV (at physiological pH) condensed plasmid DNA into nanosized particles (<150 nm) suitable for intracellular delivery. The PEAs degraded in a controlled fashion (t(1/2) of approximately 9-10 days). Compared with PEI 25K, the PEAs showed significantly lower cytotoxicity in three different cells. The PEAs demonstrated much higher transfection efficiency compared to conventional PEI 25K and Lipofectamine. The PEA synthesized using a 1 : 4 mole ratio of GDM to PEI [GDM/PEI-1.2 (1:4)] showed the highest transfection efficiency in HepG2 cells. Significantly higher pEGFP-N(2) reporter gene expression in 293T cells was achieved using these PEAs. The hyperosmotic effect of PEAs was demonstrated by the reduction in packed cell volume (PCV). The GDM/PEI-1.2 (1:4) showed comparable reduction in PCV with respect to glycerol in 293T cells. The effect of bafilomycin A(1) on transfection efficiency of PEAs on 293T cells indicated its endosomal buffering capacity. We hypothesized that the higher transfection efficiency of PEAs was the synergistic effect arising from hyperosmotic glycerol and endosomal buffering capacity of PEAs resulting from the presence of a glycerol backbone and PEI amine groups, respectively.

Characterization of Transferrin-Modified Procationic-Liposome Protamine-DNA Complexes

We developed a novel transferrin modified non-viral gene delivery system, transferrin-modified procationic-liposome-protamine-DNA complexes (Tf-PLPD) and investigated its characteristics. Blank procationic liposomes were prepared using the film dispersion filter method. Protamine was used to condense plasmid DNA to form protamine-DNA complexes and the complexes were further incubated with blank procationic liposomes to form PLPD. Transferrin was adsorbed onto the surface of PLPD via an electrostatic interaction, and thus Tf-PLPD was produced. Characteristics such as stability in rat serum, morphology, average particle size, zeta potential, and transfection efficiency in HepG2 cells were further investigated. The results indicated that the procationic liposomes remained stable in rat serum for 24 h. Tf-PLPD protected plasmid DNA from enzymatic degradation even after lyophilization. The size distribution of Tf-PLPD was in the range of 240+/-12 nm and the zeta potential was -24.10+/-2.5 mV (n=3), respectively. The transfection efficiencies of Tf-PLPD were 24.26+/-2.6 mU beta-galactosidase/mg protein. Lyophilization and the presence of serum did not affect the transfectivity of Tf-PLPD and the procationic liposomes also had low cytotoxicity to cells.

Chitosan‐g‐PEG/DNA complexes deliver gene to the rat liver via intrabiliary and intraportal infusions

Chitosan has been shown to be a non-toxic and efficient vector for in vitro gene transfection and in vivo gene delivery through pulmonary and oral administrations. Recently, we have shown that chitosan/DNA nanoparticles could mediate high levels of gene expression following intrabiliary infusion 1. In this study, we have examined the possibility of using polyethylene glycol (PEG)-grafted chitosan/DNA complexes to deliver genes to the liver through bile duct and portal vein infusions. PEG (Mw: 5 kDa) was grafted onto chitosan (Mw: 47 kDa, deacetylation degree: 94%) with grafting degrees of 3.6% and 9.6% (molar percentage of chitosan monosaccharide units grafted with PEG). The stability of chitosan-g-PEG/DNA complexes was studied by measuring the change in particle size and by agarose gel electrophoresis against bile or serum challenge. The influence of PEG grafting on gene transfection efficiency was evaluated in HepG2 cells using luciferase reporter gene. Chitosan and chitosan-g-PEG/DNA complexes were delivered to the liver through bile duct and portal vein infusions with a syringe pump. Gene expression in the liver and the distribution of gene expression in other organs were evaluated. The acute liver toxicity of chitosan and chitosan-g-PEG/DNA complexes was examined by measuring serum alanine aminotranferase (ALT) and aspartate aminotransferase (AST) activities as a function of time. Both chitosan and chitosan-g-PEG displayed comparable gene transfection efficiency in HepG2 cells. After challenge with serum and bile, chitosan-g-PEG/DNA complexes, especially those prepared with chitosan-g-PEG (GD = 9.6%), did not form large aggregates like chitosan/DNA complexes but remained stable for up to 30 min. In addition, chitosan-g-PEG prevented the degradation of DNA in the presence of serum and bile. On day 3 after bile duct infusion, chitosan-g-PEG (GD = 9.6%)/DNA complexes mediated three times higher gene expression in the liver than chitosan/DNA complexes and yielded background levels of gene expression in other organs. On day 1 following portal vein infusion, gene expression level induced by chitosan/DNA complexes was hardly detectable but chitosan-g-PEG (GD = 9.6%) mediated significant transgene expression. Interestingly, transgene expression by chitosan-g-PEG/DNA complexes in other organs after portal vein infusion increased with increasing grafting degree of PEG. The ALT and AST assays indicated that grafting of PEG to chitosan reduced the acute liver toxicity towards the complexes. This study demonstrated the potential of chitosan-g-PEG as a safe and more stable gene carrier to the liver.

Stable polyplexes based on arginine-containing oligopeptides for in vivo gene delivery.

In this study, we investigated to what extent the stability and transduction capacity of polyplexed DNA can be improved by optimizing the condensing peptide sequence. We have synthesized a small library of cationic peptides, at which the lysine/arginine ratio and the cation charge were varied. All peptides were able to compact DNA, at which polyplexes of short lysine-rich sequences were considerably larger than those of elongated or arginine-rich peptides (GM102 and GM202). In addition, the arginine-rich peptides GM102 and GM202 rendered the polyplexes resistant to plasma incubation or DNase I-mediated digestion. While all peptides were found to improve the transfection efficiency in HepG2 cells, only the GM102- and GM202-derived polyplexes could be specifically targeted to HepG2 cells by incorporation of a ligand-derivatized YKAK(8)WK peptide. We propose that GM102 and GM202 combine the advantage of small condensing peptides to give small-sized polyplexes with the superior stability of condensing polymers, which makes GM102 and GM202 excellent candidates for future in vivo gene therapy studies.

Molecular weight-dependent gene transfection activity of unmodified and galactosylated polyethyleneimine on hepatoma cells and mouse liver

To optimize a receptor-mediated and cell-selective gene transfer with polyethyleneimine (PEI)-based vector, we synthesized three galactosylated PEIs (Gal-PEI) with different molecular weights (PEI(1800), PEI(10,000), and PEI(70,000)) and investigated their potential as a targetable vector to asialoglycoprotein receptor-positive cells. All PEI derivatives formed complexes with plasmid DNA (pDNA), whereas the particle size of the complex became smaller on increasing the molecular weight of PEI. Transfection efficiency in HepG2 cells with PEI was highest with PEI(1800); efficiency was next highest with PEI(10,000), although the cellular association was similar. After galactosylation, Gal(19)-PEI(10,000)/pDNA and Gal(120)-PEI(70,000)/pDNA showed considerable agglutination with a galactose-recognizing lectin, but Gal(9)-PEI(1800) did not, suggesting that galactose units on the Gal(9)-PEI(1800)-pDNA complex are not sufficiently available for recognition. Gal(19)-PEI(10,000)-pDNA and Gal(120)-PEI(70,000)-pDNA complexes showed galactose-inhibitable transgene expression in HepG2 cells. Transfection efficiency was greatest with Gal(19)-PEI(10,000)/pDNA, a result that highlights the importance of obtaining a balance between the cytotoxicity and the transfection activity, both of which are found to be a function of the molecular weight of PEI. After intraportal injection, however, Gal(153)-PEI(70,000)/pDNA having a low N/P ratio was most effective, suggesting that additional variables, such as the size of the complex, are important for in vivo gene transfer to hepatocytes.