Nano-vectors for efficient liver specific gene transfer

Figures & data

Figure 1 Schematic representation of hepatocytes.

Figure 2 Schematic representation of internalization of ligand anchored system with cell surface lectins.

Figure 3 In vitro transfection activity of DNA/liposome complexes at a charge ratio of 2.3 in HepG2 cells. Cells were transfected with DNA/liposome complexes in the absence (†) and presence (†) of 20 mM galactose. DOPE containing liposome/DNA complexes (A) and DOPE noncontaining liposome/DNA complexes (B) were applied to the cells. DNA concentration was fixed at 0.5 μg/ml in all experiments. Statistical analysis was performed by analysis of variance (**indicated p < 0.01; NS, not significant). Each value represents the mean 6 SD values (n = 3) (Adapted from Kawakami et al 2000).

Figure 4 Biodistribution of gene expression 24 h after intravenous injection with plasmid DNA (50 μg) complexed with liposomes or Tfx into mice (Adapted from Hwang et al 2001).

Figure 5 N/P ratio dependent transfection efficiency of gal-LMWC/plasmid DNA complexes (a) and gal-LMWC/ASO complexes (b) in HepG2 cell line (Adapted from Gao et al 2005).

Figure 6 MTT assay for cytotoxicity of CaP, Lipofectin, HMWC, LMWC, and gal-LMWC in HepG2 cell line (Adapted from Gao et al 2005).

Figure 7 Effect of bafilomycin A1 on the gene transfection in 293 T cells. WSC/DNA complex was prepared at charge ratio 10 and the amount of PEI used was 0.5 Ag. Different amounts of bafilomycin A1 diluted in DMSO were put into the wells. After 10 min incubation period, transfection solutions were added into the wells. The cells were incubated in the transfection solution for 4 h and then in the growth medium for 48 h (Adapted from Kim et al 2005).

Table 1 Studies performed with various nanocarriers for hepatocytes specific gene delivery

Engineered constructs	Purpose/result	Reference
Gal-PEG-grafted PLL polymeric gene carrier	Form stable and soluble complexes with nucleic acids (which in turn are able to efficiently transform cells) PEG attached to the PLL gives better solubility to the carrier complex and improved transfection efficiency without considerable cytotoxicity.	United States Patent 6177274
Lactosylated PLL	The transfection efficiency of HepG2 cells with pSV2Luc/lactosylated PLL complexes was greatly enhanced in the presence of chloroquine/fusogenic peptide. In the presence of the fusogenic peptide, the luciferase activity in HepG2 cells was 10 fold larger than that of cells transfected with pSV2Luc/lactosylated PLL complexes in the presence of chloroquine.	Midoux et al (1993)
Asialofetuin-PLL conjugate [(Gal-6)3Lys2-bA] for hepatocytes targeting Nonenzymatic glycosylation of poly-l-lysine	Asialofetuin-PLL conjugate efficiently internalize ODN in hepatoma cell line PLC/PRF/5. Trigalactosylated bisacridine [(Gal-6)3Lys2-bA] compound could mediate the binding of DNA to both the ricin lectin and to primary hepatocytes. PLL was crosslinked to an asialoglycoprotein, and the resulting conjugate was complexed with the DNA. The electrostatic binding between DNA and PLL asialoglycoprotein ensures efficient i.v. delivery complex to the liver by receptor mediated endocytosis.	Reinis et al (1993) Haensler and szoka (1993) Martinez-Fong et al (1994)
DNA/lactosylated polylysine complexes	At high concentrations of chloroquine induced the dissociation of plasmid DNA/lactosylated PLL complexes and enhanced the transfection. The luciferase activity was enhanced in the presence of primaquine, a chloroquine analogue, but was not increased when transfection was performed in the presence of ammonium chloride, methylamine, spermine, or monensin, compounds known to neutralize the pH of the endocytotic vesicle lumen as chloroquine does.	Erbacher et al (1996)
Lactose-PEG-grafted PLL	Transfection experiments showed that Lac-PEG-PLL efficiently delivers DNA to a hepatoma cell line in vitro (at a 1:3 weight ratio of DNA to carrier). As the lactose-PEG substitution content increased up to 30%, the transfection efficiency increased, which demonstrated that the lactose serves as a targeting moiety. No considerable cytotoxicity was observed due to Lac-PEG-PLL.The use of chloroquine increased transfection efficiency that indicated the involvement of hydrolytic degradation of the system in lysosome.	Choi et al (1998)
PLL/DNA polyplexes	Attachment of asialoorosomucoid to PLL increased the PLL chain length required for efficient DNA binding in saline and for efficient DNA condensation. Attachment of asialoorosomucoid\lessened, but did not eliminate, the aggregation of PLL polyplexes, and did not result in efficient delivery of polyplexes to hepatocytes. Conjugation of PEG to PLL sterically stabilized resulting polyplexes at neutral charge ratios by shielding the surfaces.	Kwoh et al (1999)
Lactose-PEG grafted PLL	Lac-PEG-PLL was shown to protect DNA against nuclease action in a DNase I protection assay. Lac-PEG-PLI, formed complexes with plasmid DNA gave little cytotoxicity, and showed increased efficiency of gene transfer into hepatoma cells in vitro. Lac-PEG-PLL was more efficient than Lipofectin or galactose-PEG-PLL in transfection efficiency.	Choi et al (1999)
Galactosylated PEI/DNA complexes	5% of the PEI nitrogens were grafted with a linear tetragalactose structure (lGal4), small and stable particles were formed upon complexation with plasmid DNA. Slightly charged PEI-lGal4/DNA complexes were very selectively galactosylated-PEI vector to transfect hepatocytes.	Bettinger el al (1999)
Glycosylated-PEI	Gal-PEI was prepared using titanium (IV) isopropoxide and sodium borohydride, as a substitute for the highly toxic sodium cyanoborohydride method. Systems were found to be nontoxic and efficient transfection agent in HepG2 cells.	Leclercq et al (2000)
Poly(2-(dimethylamino)ethylmethacrylate - N-vinyl-2-pyrrolidone (DMAEMA-NVP)-b-PEG-galactose as gene delivery vector for hepatocytes	Poly(DMAEMA-NVP)-b-PEG-galactose in combination with an endosomolytic peptide, KALA demonstrated sufficient transfection efficiency as high as that of commercial agent.	Lim et al (2000)
Pullulan derivatives with chelate residues based on metal coordination	i.v. injection of the pullulan derivatives-plasmid DNA conjugates with Zn^2+ coordination significantly enhanced the level of gene expression in the liver. A fluorescent-microscopic study revealed that the plasmid DNA was localized at the liver after injection of the pullulan-DTPA-plasmid DNA conjugate with Zn^2+ coordination.	Hosseinkhni et al (2002)
Galactose-PEI-DNA	MTT and LDH assay confirmed that cytotoxicity of gal-PEI was found to decrease with increasing galactosylation. Require necessity of careful optimization of polyplex composition for active gene targeting.	Kunath et al (2003)
Lactose-appended schizophyllan	Schizophyllan (beta-1,3-glucan) derivative carrying lactose-appendages prepared by reductive amination, effectively mediates gene transfection into hepatocytes.	Hasegawa et al (2004)
Surface modified albumin nanoparticles	In this study, Calcein loaded bovine serum albumin nanoparticle (Cal-BSA-NP) and bovine serum albumin nanoparticles with their surface modified by glycyrrhizin (Cal-BSA-NP-GL) were developed as a novel hepatocyte targeting based on active targeting technology mediated by specific binding site of GL on rat cellular membrane. The uptake amount of Cal-BSA-NP-GL by rat hepatocytes was 4.43-fold higher than that of Cal-BSA-NP and results shows significant difference in the uptake of Cal-BSA-NP-GL and Cal-BSA-NP by hepatocytes.	Mao et al (2005)
β-(1–3)-D-glucan schizophyllan for AS-ODN delivery	Galactose moieties were conjugated to the side chain of SPG to enhance cellular ingestion through endocytosis mediated by ASGP-Rs.	Karinaga et al (2006)
Chitosan-DNA nanoparticles	Chitosan-DNA nanoparticles showed several times higher luciferase expression in the liver after retrograde intrabiliary infusion (RII). Luciferase expression by RII of PEI-DNA nanoparticles was 17-fold lower than that of chitosan-DNA nanoparticles. RII of chitosan-DNA nanoparticles did not yield significant toxicity.	Dai et al (2006)
Surface modified magnetite nanoparticles	In this report, superparamagnetic magnetite nanoparticles were surface-modified with lactobionic acid (LA) to improve their intracellular uptake and ability to target hepatocytes. Cell culture experiment showed that LA-modified nanoparticles were internalized into hepatocytes and atomic absorption spectrometer (AAS) measurement indicated that the uptake amount of LA-modified magnetite into hepatocytes was higher than that of unmodified and control MA-modified nanoparticles. LA-modified nanoparticle solution was injected in rabbit and the magnetic resonance (MR) images obtained showed that LA-coated nanoparticles were selectively accumulated onto the hepatocytes	Kamruzzaman et al (2007)
Synthetic PEGylated glycoproteins for hepatocyte targeting	PEGylated glycoproteins (PGPs) were synthesized by copolymerizing a Cys-terminated PEG-peptide, glycopeptide, and melittin peptide by varying the ratio of PEG-peptide (20%–90%) and melittin (0%–70%) with a constant amount of glycopeptide (10%). Results shows that the level of gene expression mediated by PGP-DNA was 5000-fold less than direct hydrodynamic dosing of an equivalent amount of DNA and was independent of the mol percent of melittin incorporated into the polymer, but dependent on the presence of galactose on PGP.	Chen et al (2007)

Scheme 1 Synthetic scheme for PPA-DPA and galactosylated PPA-DPA (adapted from Zhang and coworker 2005).

Scheme 2 Preparation of binary and ternary nanoparticles containing galactosylated PPA-DPA (adapted from Zhang and coworker2005).

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