Galactosylated ternary DNA/polyphosphoramidate nanoparticles mediate high gene transfection efficiency in hepatocytes.

Galactosylated polyphosphoramidates (Gal-PPAs) with different ligand substitution degrees (6.5%, 12.5% and 21.8%, respectively) were synthesized and evaluated as hepatocyte-targeted gene carriers. The in vitro cytotoxicity of Gal-PPA decreased significantly with an increase in galactose substitution degree. The affinity of Gal-PPA/DNA nanoparticles to galactose-recognizing lectin increased with galactose substitution degree. However, decreased transfection efficiency was observed for these galactosylated PPAs in HepG2 cells. Based on the results of gel retardation and polyanion competition assays, we hypothesized that the reduced transfection efficiency of Gal-PPA/DNA nanoparticles was due to their decreased DNA-binding capacity and decreased particle stability. We therefore prepared nanoparticles by precondensing DNA with PPA at a charge ratio of 0.5, yielding nanoparticles with negative surface charge, followed by coating with Gal-PPA, resulting in a Gal-PPA/ DNA/PPA ternary complex. Such a ternary nanoparticle formulation led to significant size reduction in comparison with binary nanoparticles, particularly at low N/P ratios (2 to 5). In HepG2 cells and primary rat hepatocytes, and at low N/P ratios (2 to 5), transfection efficiency mediated by ternary nanoparticles prepared with 6.5% Gal-PPA was 6-7200 times higher than PPA-DPA/DNA nanoparticles. Transgene expression increased slightly at higher N/P ratios in HepG2 cells and reached a plateau at N/P ratios between 5 and 10 for primary rat hepatocytes. Such an enhancement effect was not observed in HeLa cells that lack of asialoglycoprotein receptor (ASGPR). Nevertheless, transfection efficiency of ternary particles decreased dramatically, presumably due to the decreased DNA binding capacity and particle stability, as PPA galactosylation degree increased. This highlights the importance of optimizing ligand conjugation degree for PPA gene carrier.

Ternary complexes comprising polyphosphoramidate gene carriers with different types of charge groups improve transfection efficiency.

To understand the influence of charge groups on transfection mediated by polymer complexes, we have synthesized a series of biodegradable and cationic polyphosphoramidates (PPAs) with an identical backbone but different side chains. Our previous study showed that PPA with a spermidine side chain (PPA-SP) showed high transfection efficiency in culture, whereas PPAs with secondary, tertiary, and quaternary amino groups were significantly less efficient. To investigate whether the coexistence of 1 degrees amino charge groups with 3 degrees and 2 degrees amino charge groups in the DNA/polymer complexes would enhance their transfection efficiency, we evaluated a ternary complex system containing DNA and PPAs with 1 degrees amino groups (PPA-SP) and 3 degrees amino groups (PPA-DMA) and a quaternary complex system containing DNA and PPAs with 1 degrees and 2 degrees and 3 degrees amino groups (PPA-EA/PPA-MEA/PPA-DMA), respectively. Ternary complexes mediated 20 and 160 times higher transfection efficiency in COS-7 cells than complexes of DNA with PPA-SP or PPA-DMA alone, respectively. Similarly, quaternary complexes exhibited 8-fold higher transfection efficiency than PPA-EA/DNA complexes. The mechanism of enhancement in transfection efficiency by the mixture carriers appears to be unrelated to the particle size, zeta potential, or DNA uptake. The titration characterization and the transfection experiments using a proton pump inhibitor suggest that the enhancement effect is unlikely due to the slightly improved buffering capacity of the mixture over PPA-SP. This approach represents a simple strategy of developing polymeric gene carriers and understanding the mechanisms of polymer-mediated gene transfer.

Three-dimensional co-culture of rat hepatocyte spheroids and NIH/3T3 fibroblasts enhances hepatocyte functional maintenance.

Functional maintenance of primary hepatocytes in culture can be improved by several distinct approaches involving optimization of the extracellular matrix microenvironment, media composition and cell-cell interactions, both homotypic and heterotypic. Using a galactose-decorated surface, we have developed a method to combine these two approaches by co-culturing rat primary hepatocyte spheroids with NIH/3T3 mouse fibroblast cells. Spheroids were performed by culturing hepatocytes for 3 days on galactosylated poly(vinylidene difluoride) membrane; NIH/3T3 cells were subsequently seeded and co-cultured with the spheroids. Results showed that although NIH/3T3 cells alone responded poorly to the galactosylated PVDF surface and displayed limited attachment, NIH/3T3 fibroblasts attached to the periphery of the hepatocyte spheroids and proliferated around them. Co-cultured hepatocyte spheroids exhibited significantly higher liver-specific functions as compared to spheroids cultured alone. Albumin secretion level in this co-culture system peaked on day 11, which was 1.8- and 2.9-times higher than the peak expression level in spheroid homo-culture control in serum-free (day 3) and serum-containing media (day 4), respectively. The albumin secretion function was maintained for at least two weeks; it was 5.1 (in serum-free medium) and 17.8 (in serum-containing medium) times higher than spheroid homo-culture on day 13. Similarly, the co-culture system also expressed approximately 5.5- and 3.1-times higher 3-methylcholanthrene-induced cytochrome P450 enzymatic activity on day 14 as compared to the homo-culture control in serum-free and serum-containing medium, respectively. In conclusion, this unique co-culture system demonstrated the synergistic roles of homotypic cell-cell interaction, heterotypic cell-cell interaction, cell-substrate interaction and soluble stimuli in hepatocyte functional maintenance.

Galactosylated poly(vinylidene difluoride) hollow fiber bioreactor for hepatocyte culture.

To overcome the limitations of long-term expression of highly differentiated hepatocyte functions, we have developed a novel bioreactor in which hepatocytes are seeded in a ligand-immobilized hollow fiber cartridge. Galactosylated Pluronic polymer is immobilized on poly(vinylidene difluoride) (PVDF) hollow fiber surface through an adsorption scheme yielding a substrate with hepatocyte-specific ligand and a hydrophilic surface layer, which can resist nonspecific protein adsorption and facilitate cell binding to the galactose ligand. Interestingly, the galactosylated PVDF hollow fiber shows enhanced serum albumin diffusion across the membrane. Freshly isolated rat hepatocytes were seeded and cultured in the extralumenal space of the hollow fiber cartridge for 18 days in a continuously circulated system. Albumin secretion function of the seeded hepatocytes was monitored by analyzing circulating medium by enzyme-linked immunosorbent assay. Urea synthesis and P-450 function (7-ethoxycoumarin dealkylase activity) were measured periodically by doping the circulating medium with NH4Cl and 7-ethoxycoumarin, respectively. Hepatocytes cultured on galactosylated PVDF hollow fibers maintained better albumin secretion and P-450 functions than on unmodified and serum-coated PVDF hollow fibers when cultured in serum-containing medium. Morphological examination by scanning electron microscopy showed that hepatocytes cultured on galactosylated PVDF hollow fibers developed significant aggregation, in contrast to those cultured on unmodified PVDF fibers or on serum-coated PVDF fibers. Transmission electron microscopy images revealed that tight junctions and canaliculus-like structures formed in these aggregates. These results suggest the potential application of this galactosylated PVDF hollow fiber cartridge for the design of a bioartificial liver assist device.

Water-soluble and nonionic polyphosphoester: synthesis, degradation, biocompatibility and enhancement of gene expression in mouse muscle.

A nonionic and water-soluble polyphosphoester, poly(2-hydroxyethyl propylene phosphate) (PPE3), was synthesized by chlorination of poly(4-methyl-2-oxo-2-hydro-1,3,2-dioxaphospholane), followed by esterification with 2-benzyloxyethanol and deprotection of the hydroxyl group by catalytic hydrogenation in the presence of Pd-C. PPE3 degraded rapidly in PBS 7.4 at 37 degrees C. The cytotoxicity and tissue compatibility assays suggested good biocompatibility of PPE3 in vitro and in vivo. The expression of pVR1255 Luc plasmid in mouse muscle after intramuscular (i.m.) injection of DNA formulated with PPE3 solution in saline was enhanced up to 4-fold compared with that of naked DNA. These results suggest the potential of this polyphosphoester for naked DNA-based gene therapy. The advantages of this polymer design include the biodegradability of the polyphosphoester and its structural versatility, which allows the fine-tuning of the physicochemical properties to optimize the enhancement of gene expression in muscle.

High density of immobilized galactose ligand enhances hepatocyte attachment and function.

Galactosylated surface is an attractive substrate for hepatocyte culture because of the specific interaction between the galactose ligand and the asialoglycoprotein receptor on hepatocytes. In this study, we described a scheme to achieve high density of immobilized galactose ligands on polyethylene terephthalate (PET) surface by first surface-grafting polyacrylic acid on plasma-pretreated PET film under UV irradiation, followed by conjugation of a galactose derivative (1-O-(6'-aminohexyl)-D-galactopyranoside) to the grafted polyacrylic acid chains. A high galactose density of 513 nmol/cm(2) on the PET surface was used in this study to investigate the behavior of cultured hepatocyte. This engineered substrate showed high affinity to fluorescein isothiocyanate-lectin binding. Primary rat hepatocytes, when seeded at a density of 2 x 10(5) cells/cm(2), attached to the galactosylated PET substrate at a similar efficiency compared with collagen-coated substrate. The hepatocytes spontaneously formed aggregates 1 day after cell seeding and showed better maintenance of albumin secretion and urea synthesis functions than those cultured on collagen-coated surface.

Effect of side-chain structures on gene transfer efficiency of biodegradable cationic polyphosphoesters.

Cationic polyphosphoesters (PPEs) with different side-chain charge groups were designed and synthesized as biodegradable gene carriers. Poly(N-methyl-2-aminoethyl propylene phosphate) (PPE-MEA), with a secondary amino group (-CH(2)CH(2)NHCH3) side chain released DNA in several hours at N/P (amino group of polymer to phosphate group of DNA) ratios from 0.5 to 5; whereas PPE-HA, bearing -CH(2)(CH2)(4)CH(2)NH(2) groups in the side chain, did not release DNA at the same ratio range for 30 days. Hydrolytic degradation and DNA binding results suggested that side chain cleavage, besides the polymer degradation, was the predominant factor affected the DNA release and transfection efficiencies. The side chain of PPE-MEA was cleaved faster than that of PPE-HA, resulting poor cellular uptake and no transgene expression for PPE-MEA/DNA complexes in COS-7 cells at charge ratios from 4 to 12. In contrast, PPE-HA/DNA complexes were stable enough to be internalized by cells and effected gene transfection (3400 folds higher than background at a charge ratio of 12). Interestingly, gene expression levels mediated by PPE-MEA and PPE-HA in mouse muscle following intramuscular injection of complexes showed a reversed order: PPE-MEA/DNA complexes mediated a 1.5-2-fold higher luciferase expression in mouse muscle as compared with naked DNA injection, while PPE-HA/DNA complexes induced delayed and lowered luciferase expression than naked DNA. These results suggested that the side chain structure is a crucial factor determining the mechanism and kinetics of hydrolytic degradation of PPE carriers, which in turn influenced the kinetics of DNA release from PPE/DNA complexes and their transfection abilities in vitro and in vivo.

Galactosylated PVDF membrane promotes hepatocyte attachment and functional maintenance.

One of the major challenges in BLAD design is to develop functional substrates suitable for hepatocyte attachment and functional maintenance. In the present study, we designed a poly(vinylidene difluoride) (PVDF) surface coated with galactose-tethered Pluronic polymer. The galactose-derived Pluronic F68 (F68-Gal) was adsorbed on PVDF membrane through hydrophobic-hydrophobic interaction between PVDF and the polypropylene oxide segment in Pluronic. The galactose density on the modified PVDF surface increased with the concentration of the F68-Gal solution, reaching 15.4 nmol galactosyl groups per cm2 when a 1 mg/ml of F68-Gal solution was used. The adsorbed F68-Gal remained relatively stable in culture medium. Rat hepatocytes attachment efficiency on F68-Gal modified PVDF membrane was similar to that on collagen-coated surface. The attached hepatocytes on PVDF/F68-Gal membrane self-assembled into multi-cellular spheroids after 1 day of culture. These attached hepatocytes in spheroids exhibited higher cell functions such as albumin synthesis and P450 1A1 detoxification function compared to unmodified PVDF membrane and collagen-coated surface. These results suggest the potential of this galactose-immobilized PVDF membrane as a suitable substrate for hepatocyte culture.

New polyphosphoramidate with a spermidine side chain as a gene carrier.

A new cationic polymer (PPA-SP), polyphosphoramidate bearing spermidine side chain, was prepared as a non-viral vector for gene delivery. PPA-SP was synthesized from poly(1,2-propylene H-phosphonate) by the Atherton-Todd reaction. The weight average molecular weight of PPA-SP was 3.44x10(4) with a number average degree of polymerization of 90, as determined by GPC/LS/RI method. The average net positive charge per polymer chain was 102. PPA-SP was able to condense plasmid DNA efficiently and formed complexes at an N/P ratio (free amino groups in polymer to phosphate groups in DNA) of 2 and above, as determined by agarose gel electrophoresis. This new gene carrier offered significant protection to DNA against nuclease degradation at N/P ratios above 2, and showed lower cytotoxicity than PLL and PEI in cell culture. The LD(50) of PPA-SP was 85 microg/ml in COS-7 cells, in contrast to 20 and 42 microg/ml for PLL and PEI, respectively. The complexes prepared in saline at N/P ratios of 5 approximately 10 had an average size of 250 nm and zeta-potential of 26 mV. PPA-SP mediated efficient gene transfection in a number of cell lines, and the transfection protocol was optimized in HEK293 cells using a luciferase plasmid as a marker gene. Gene expression mediated by PPA-SP was greatly enhanced when chloroquine was used in conjunction at a concentration of 100 microM. Under the optimized condition, PPA-SP/DNA complexes yield a luciferase expression level closed to PEI/DNA complexes or Transfast mediated transfection. In a non-invasive CNS gene delivery model, PPA-SP/DNA complexes yielded comparable bcl-2 expression as PEI/DNA complexes in mouse brain stem following injection of the complexes in the tongue.