PEGylated PEI-based biodegradable polymers as non-viral gene vectors

Novel functional biodegradable gene vectors, poly(l-succinimide)-g-polyethylenimines-g-poly(ethylene glycol) (PSI-g-PEI-g-PEGs) were synthesized by conjugating methoxy poly(ethylene glycol) (mPEG, M_w = 750 Da) to PEI segments (M_w = 800 Da) of PSI-g-PEI. The physicochemical properties of PSI-g-PEI-g-PEGs, including buffering capability, pDNA binding ability, cytotoxicity, zeta potential and the particle size of polymer/pDNA complexes, were explored. The influence of PEGylation was discussed based on a comparative study of PSI-g-PEI-g-PEGs, PSI-g-PEI and PEI25k (M_w = 25 kDa). SEM images revealed that PSI-g-PEI-g-PEG/pDNA particles have a regular shape with the diameter ranging from 70 to 170 nm. PEGylation could suppress the aggregation occurrence between complexes, resulting in a reduction of the polymer/pDNA complex size. PSI-g-PEI-g-PEGs exhibited remarkably lower cytotoxicity compared to PSI-g-PEI and PEI25k. In 293T and HeLa cells, the obtained PSI-g-PEI-g-PEGs showed very high transfection efficiency compared to PEI25k. Fluorescent confocal microscopy demonstrated that PSI-g-PEI-g-PEGs could effectively transport pGL-3 plasmids into the nuclei of HeLa cells. Taking into account the continued high transfection efficacy and decreased toxicity after PEG modification, PSI-g-PEI-g-PEGs show great potential as the non-viral vectors for gene transfection.     

Structure–transfection activity relationships with glucocorticoid–polyethyl-enimine conjugate nuclear gene delivery systems

Efficient nuclear gene delivery is essential for successful gene therapy. It was previously reported that the transport of DNA into nucleus may be facilitated by glucocorticoid (GC). In this study, five glucocorticoids with different structures and potencies were conjugated with low molecular weight PEI 1800, and the degree of substitution of glucocorticoids was controlled to be close to each other. The glucocorticoid–polyethylenimine (GC–PEI)/pDNA complexes were prepared and their physico-chemical properties and transfection efficiency were investigated. The results showed that the complexes had similar physico-chemical properties, but their transfection activities were different statistically. In order to explore the reason of this difference, the affinity of GC–PEI polymer with GC receptor was analyzed by the application of molecular docking, and the correlation between transfection activity and the potency of five GC was investigated. The result showed that receptor binding of five GC was different and transgene expression enhanced linearly with the increasing GC potency, but log P. In addition, confocal microscopy examination confirmed that GC–PEI/DNA complexes were more effectively translocated in the nucleus than PEI 25 K or PEI 1800 complexes and the cytotoxicities of the GC–PEI polymers were lower than that of PEI 25 K. These results demonstrated that transfection activity of GC–PEI polymer correlated with its GC potency, and this regularity might be useful for the development of more efficient GC substituted polymer as promising nuclear-targeting carrier.     

Arginine-grafted bioreducible poly(disulfide amine) for gene delivery systems

Arginine-grafted bioreducible poly(disulfide amine) (ABP) polymer was synthesized for non-viral gene delivery systems. Its Mw was measured to be 4.45 × 10³ Da/mole by FPLC-SEC and its PDI value was 1.49. ABP was able to retard pDNA from a weight ratio of 2 but ABP could not retard pDNA even at a weight ratio of 10 in the presence of DTT, showing that it can be biodegraded in reducing environment such as cytoplasm. ABP was examined to form positively charged nano-sized particles (<200 nm) with pDNA. ABP showed no significant cytotoxicity and greatly enhanced transfection efficiency in comparison with unmodified poly(cystaminebisacrylamide-diaminohexane) (poly(CBA-DAH)) and PEI25k in mammalian cells. The transfection efficiency of ABP was not much reduced even in the serum condition. Chloroquine treatment was not found to improve the transfection efficiency of ABP. The cellular uptake pattern of ABP polyplexes was almost similar with poly(CBA-DAH), suggesting that greatly enhanced transfection efficiency of ABP is not induced by its high cellular penetrating ability but may be mediated by other factors such as good nuclear localization ability.     

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.     

Multi-armed poly(aspartate-g-OEI) copolymers as versatile carriers of pDNA/siRNA

To search for potential non-viral nucleic acids carriers, a series of novel cationic polymers, multi-armed poly(aspartate-graft-oligoethylenimine) (MP-g-OEI) copolymers were designed and synthesized by grafting different types of oligoethylenimine (OEI) to a multi-armed poly(l-aspartic acid) backbone. The as-synthesized MP-g-OEI copolymers were characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance and gel permeation chromatography. These MP-g-OEI copolymers (MP423, MP600 and MP1800) exhibited good capacity in condensing nucleic acids (pDNA or siRNA) into nanosized particles (90–150 nm) with positive surface charges. Gene transfection activity of the MP-g-OEI copolymers (especially MP1800) showed improved performance compared with PEI25k in both HeLa and CHO cell lines. The silencing efficiency of MP600/siRNA and MP1800/siRNA complexes showed a superior knockdown effect in CT26 and Huh-7 cell lines. Moreover, the MP-g-OEI copolymers exhibited much lower cytotoxicity than PEI25k. Flow cytometric analysis showed that MP-g-OEI copolymers could efficiently mediate the entry of nucleic acids into cells. These results suggest that MP-g-OEI copolymers may be potential non-viral gene carriers for the delivery of nucleic acids in future gene therapy.     

Gelatin coating to stabilize the transfection ability of nucleic acid polyplexes

Amphiphilic polymers are effective in complexing and delivering therapeutic nucleic acids, such as plasmid DNA (pDNA) and short interfering RNA (siRNA). However, long-term stability of the complexes is not desirable, as it may have an impact on the transfection efficiency in vivo. To develop a method to preserve complex stability we first showed that pDNA complexes formed with the amphiphilic polymer linoleic acid-substituted polyethylenimine (PEI–LA) and incubated at 37 °C lost ～90% of their transfection efficiency after only 24 h of complex formation. Polyethyleneglycol modification of complexes to control the increase in complex size and incubation in scaffolds used for implantation did not preserve the transfection ability of the complexes. Among a variety of approaches explored, gelatin coating of complexes was found to be the best at maintaining the original transfection efficiency. Mechanistic studies suggested that improved complex uptake, not size stability, was responsible for retention of the transfection efficiency. Similarly to the results with pDNA, gelatin coating also prevented the decreases in uptake and silencing efficiency of siRNA complexes observed following incubation at 37 °C. Gelatin-stabilized complexes were, furthermore, effective in vivo and led to subcutaneous transgene expression with a low pDNA dose that was otherwise ineffective. We conclude that a simple gelatin coating approach offers an efficient means to preserve the transfection efficiency of polyplexes.     

A linear-dendritic cationic vector for efficient DNA grasp and delivery

This paper presents an attempt to design an efficient and biocompatible cationic gene vector via structural optimization that favors the efficient utilization of amine groups for DNA condensation. To this end, a linear-dendritic block copolymer of methoxyl-poly(ethylene glycol)-dendritic polyglycerol-graft-tris(2-aminoethyl)amine (mPEG-DPG-g-TAEA) was prepared with specially designed multiple functions including strong DNA affinity, endosomal buffering and expected serum-tolerance. Based on the transfection in serum-free and serum-conditioned media, the influences of the polymer structures including the degree of polymerization of DPG and TAEA substitution degree were explored. As compared to polyethylenimine (M_w = 5 kDa) (PEI5k) with similar molecular weight and higher amine density, mPEG-DPG-g-TAEA displayed comparably high DNA affinity due to the special linear-dendritic architecture. Consequently, at very low N/P ratio, mPEG-DPG-g-TAEA vectors could mediate efficient in vitro luciferase expression at levels that are comparable with or even superior to the commercially available Lipofectamine? 2000, while being apparently higher than PEI5k. The designed vectors exhibit considerably higher cell biocompatibility and better resistance against bovine serum albumin adsorption than PEI5k. The stability of the complexes on coincubation with heparin was found to be largely dependent on the polymer structure. As concluded from the comparative transfection study in the absence/presence of chloroquine, it is likely that the polycation itself could produce endosomal buffering. This linear-dendritic vector shows promising potential for the application of gene delivery.     

The characteristics and transfection efficiency of cationic poly (ester–co–urethane) – short chain PEI conjugates self-assembled with DNA

To improve the transfection efficiency of polycations with DNA, we synthesized poly(ester–co–urethane)(PEU-g-PEI800) with short chain PEI800 in the side chain, and poly(ester–co–urethane)(PEU) without short chain PEI800. Both PEU-g-PEI800 and PEU, readily self-assembled with plasmid DNA (pCMV-βgal) in a HEPES buffer, were characterized by dynamic light scattering and zeta-potential. The results reveal that PEU-g-PEI800 and PEU were able to self-assemble particles with DNA and yield nano-sized complexes (<200 nm) with positive charge at N/P ratios of 20/1 and 120/1, respectively. The degradation studies indicate that the half-life of PEU-g-PEI800 and PEU in the HEPES buffer were 14 and 35 h at pH 7.4, respectively. Titration studies were performed to determine the buffering capacities of the polymers. The COS-7 cell viabilities in the presence of PEU-g-PEI800/DNA, PEU/DNA, and PEI25k/DNA were studied. In addition, The PEU-g-PEI800/DNA complexes were able to transfect COS-7 cells in vitro with a high efficiency comparable to a well-known gene carrier PEI25k. The results indicate that PEU-g-PEI800 is an attractive cationic poly (ester–co–urethane) for gene delivery and an interesting candidate for further study.     

Transfection of macrophages by collagen hollow spheres loaded with polyplexes: A step towards modulating inflammation

Macrophages are key orchestrators of inflammation as they secrete proteases and inflammatory cytokines. To date, therapies aimed at modulating macrophage phenotype have failed due to the short half-life of biomolecules in the body. Therefore, inhibition of inflammation by gene therapy constitutes a new hope.In the present study, we have assessed collagen hollow spheres as a reservoir system for polyplexes in order to transfect human macrophages while preserving cell viability. Polyplexes were formed by complexing G-Luc plasmid with a poly(2-dimethylaminoethyl methacrylate) poly(ethylene glycol) based hyperbranched polymer. Several ratios of polymer/pDNA (5:1, 8:1, 10:1 w/w) complexes in two different sphere sizes (1.24 and 4.5 μm) were tested. Collagen hollow spheres were loaded with polyplexes up to 80 μg of pDNA per mg of microspheres. The release of polyplexes from the spheres was delayed and prolonged i.e. 20% of the initial amount released in 5 days. Following incubation with polyplex-loaded microspheres, macrophages were transfected (polyplex pDNA:polymer ratio 1:10 w/w). In addition, collagen hollow spheres maintained cell viability as more than 80% of cells were viable after 4 days in culture. In contrast, when used alone, polyplexes were seen to be toxic, while there was no transfection detected. Taken together, these results show that collagen hollow spheres may be used as a reservoir for controlled gene delivery to macrophages. Unlike existing gene delivery systems, this system allows for macrophage transfection with minimal toxicity. Hence, this system has a potential for the delivery of a therapeutic gene in order to modulate inflammation.     

Poly(ethylene glycol) analogs grafted with low molecular weight poly(ethylene imine) as non-viral gene vectors

A novel class of non-viral gene vectors consisting of low molecular weight poly(ethylene imine) (PEI) (molecular weight 800 Da) grafted onto degradable linear poly(ethylene glycol) (PEG) analogs was synthesized. First, a Michael addition reaction between poly(ethylene glycol) diacrylates (PEGDA) (molecular weight 258 Da) and d,l-dithiothreitol (DTT) was carried out to generate a linear polymer (PEG–DTT) having a terminal thiol, methacrylate and pendant hydroxyl functional groups. Five PEG–DTT analogs were synthesized by varying the molar ratio of diacrylates to thiols from 1.2:1 to 1:1.2. Then PEI (800 Da) was grafted onto the main chain of the PEG–DTTs using 1,1′-carbonyldiimidazole as the linker. The above reaction gave rise to a new class of non-viral gene vectors, (PEG–DTT)–g-PEI copolymers, which can effectively complex DNA to form nanoparticles. The molecular weights and structures of the copolymers were characterized by gel permeation chromatography, ¹H nuclear magnetic resonance and Fourier transform infrared spectroscopy. The size of the nanoparticles was <200 nm and the surface charge of the nanoparticles, expressed as the zeta potential, was between +20 and +40 mV. Cytotoxicity assays showed that the copolymers exhibited much lower cytotoxicities than high molecular weight PEI (25 kDa). Transfection was performed in cultured HeLa, HepG2, MCF-7 and COS-7 cells. The copolymers showed higher transfection efficiencies than PEI (25 kDa) tested in four cell lines. The presence of serum (up to 30%) had no inhibitory effect on the transfection efficiency. These results indicate that this new class of non-viral gene vectors may be a promising gene carrier that is worth further investigation.     

Specific effects of PEGylation on gene delivery efficacy of polyethylenimine: Interplay between PEG substitution and N/P ratio

While an effective non-viral gene carrier, 25 kDa branched polyethylenimine (PEI) is cytotoxic, and decreasing its toxicity while maintaining its functionality is vital. Conjugation of carriers with polyethylene glycol (PEG) is a common approach to decreasing toxicity and improving biodistribution; however, the effect of PEGylation on PEI transfection efficacy is contradictory at present. The aim of this work was to reveal the details of this dependence. Polymers were synthesized by grafting 2 kDa PEG to 25 kDa PEI at multiple ratios. Unlike typical investigations, parallel studies based on either total polymer weight or PEI-backbone weight were employed at the same time for accurate investigation into the specific effects of PEGylation. Polymers were assessed for toxicity and plasmid DNA (pDNA) binding, while polyplexes were formed at various polymer/pDNA weight ratios and monitored by dynamic light scattering (DLS) in the presence of serum. The efficacy of the polyplexes for pDNA delivery and transgene expression in HEK293 cells was assessed by flow cytometry. This approach unexpectedly revealed that increased PEG substitution caused lower toxicity and pDNA-binding on a per total polymer weight basis, but not on a per PEI-backbone weight basis. DLS indicated that high PEGylation prevents an increase in polyplex size in the presence of serum. Plasmid uptake and transgene expression were found to have a complex relationship with PEG substitution, dependent on the polymer/plasmid-DNA weight ratio. PEGylation generally decreased the transfection efficacy of PEI, but under ideal conditions of PEG substitution and polymer/pDNA ratio, PEGylation provided more effective carrier formulations than the native PEI itself.     

Ternary complexes of pDNA,polyethylenimine, and γ-polyglutamic acid for gene delivery systems

We discovered a vector coated by γ-polyglutamic acid (γ-PGA) for effective and safe gene delivery. In order to develop a useful non-viral vector, we prepared several ternary complexes constructed with pDNA, polyethylenimine (PEI), and various polyanions, such as polyadenylic acid, polyinosinic–polycytidylic acid, α-polyaspartic acid, α-polyglutamic acid, and γ-PGA. The pDNA/PEI complex had a strong cationic surface charge and showed extremely high transgene efficiency although it agglutinated with erythrocytes and had extremely high cytotoxicity. Those polyanions changed the positive ζ-potential of pDNA/PEI complex to negative although they did not affect the size. They had no agglutination activities and lower cytotoxicities but most of the ternary complexes did not show any uptake and gene expression; however, the pDNA/PEI/γ-PGA complex showed high uptake and gene expression. Most of the pDNA/PEI/γ-PGA complexes were located in the cytoplasm without dissociation and a few complexes were observed in the nuclei. Hypothermia and the addition of γ-PGA significantly inhibited the uptake of pDNA/PEI/γ-PGA by the cells, although l-glutamic acid had no effect. These results strongly indicate that the pDNA/PEI/γ-PGA complex was taken up by γ-PGA-specific receptor-mediated energy-dependent process. Thus, the pDNA/PEI/γ-PGA complex is useful as a gene delivery system with high transfection efficiency and low toxicity.     

Polyethyleneimine-lipid conjugate-based pH-sensitive micellar carrier for gene delivery

A low molecular weight polyethyleneimine (PEI 1.8 kDa) was modified with dioleoylphosphatidylethanolamine (PE) to form the PEI-PE conjugate investigated as a transfection vector. The optimized PEI-PE/pDNA complexes at an N/P ratio of 16 had a particle size of 225 nm, a surface charge of +31 mV, and protected the pDNA from the action of DNase I. The PEI-PE conjugate had a critical micelle concentration (CMC) of about 34 μg/ml and exhibited no toxicity compared to a high molecular weight PEI (PEI 25 kDa) as tested with B16-F10 melanoma cells. The B16-F10 cells transfected with PEI-PE/pEGFP complexes showed protein expression levels higher than with PEI-1.8 or PEI-25 vectors. Complexes prepared with YOYO 1-labeled pEGFP confirmed the enhanced delivery of the plasmid with PEI-PE compared to PEI-1.8 and PEI-25. The PEI-PE/pDNA complexes were also mixed with various amounts of micelle-forming material, polyethylene glycol (PEG)-PE to improve biocompatibility. The resulting particles exhibited a neutral surface charge, resistance to salt-induced aggregation, and good transfection activity in the presence of serum in complete media. The use of the low-pH-degradable PEG-hydrazone-PE produced particles with transfection activity sensitive to changes in pH consistent with the relatively acidic tumor environment.     

Neuronal gene delivery by negatively charged pullulan–spermine/DNA anioplexes

Nonviral gene transfer to neurons remains unreliable due to a lack of effective and nontoxic vectors. Here, we achieved effective neuronal gene delivery through salt-free complexation of plasmid DNA and pullulan–spermine, a conjugate prepared from a naturally derived polysaccharide and polyamine. Specifically, at low spermine nitrogen:DNA phosphate (N:P) ratios, complexes formed with ζ-potential and diameter of approximately?40 mV and 350 nm, respectively. Higher N:P ratios increased the ζ-potential to approximately +10 mV. All complexes were stable for at least 1 week and protected DNA from degradation. In vitro transfection of rat sensory neurons occurred at all N:P ratios, but uniquely, efficiency was highest for anionic complexes (anioplexes). Subsequent analyses revealed the inhibition of reporter gene expression by asialofetuin (1 mg/ml) and methyl-beta-cyclodextrin (5 mm), indicating utilization of glycoprotein-specific interactions and lipid rafts for uptake and intracellular trafficking. In marked contrast to a commercial cationic lipid reagent, anioplexes did not exhibit measurable cytotoxicity at up to 20 μg/ml DNA. Additionally, transfection efficiency was maintained in the presence of serum and antibiotics. Based on these favorable properties, we successfully established two transfection methods for cultured adult sensory neurons and tissue explants. Collectively, these data suggest that negatively charged pullulan–spermine/DNA anioplexes could represent an effective gene delivery technology, particularly for neurons.     

Enhanced initial bone regeneration with inorganic polyphosphate-adsorbed hydroxyapatite

Inorganic polyphosphate (poly(P)) can promote binding between fibroblast growth factors and their receptors and enhance osteoblastic cell differentiation and calcification. This study evaluated the possibilities for poly(P) adsorbed onto interconnected porous calcium hydroxyapatite (IP-CHA) as a new bone regeneration material. Prepared 1%, 5%, 25% and 50% poly(P)/IP-CHA composites showed the elution peak of poly(P) between 15 and 20 min, respectively, with the highest value from 50% poly(P)/IP-CHA in vitro. Histologically, at 1 week of placement into the femur of rabbits, granulation tissue had penetrated into the pores in all composites and IP-CHA as a control. In contrast, at 2 weeks of placement, newly formed lamellar bone was found in all groups, although a higher amount of bone regeneration was obviously formed in the 25% and 50% poly(P)/IP-CHA with a significantly higher value of bone regeneration ratio of 50% poly(P)/IP-CHA. These results indicate that 25% and 50% poly(P)/IP-CHA composites may enhance initial bone regeneration.     

Effective delivery of p65 shRNA by optimized Tween 85-polyethyleneimine conjugate for inhibition of tumor growth and lymphatic metastasis

To maximize the interference efficacy of pGPU6/Neo-p65 shRNA-expressing pDNA (p65 shRNA) and subsequently more effectively inhibit tumor growth and lymphatic metastasis through blocking the nuclear factor-kappa B (NF-κB) signaling pathway, seven Tween 85-polyethyleneimine (PEI) conjugates (TnPs, n = 2, 3, 4, 5, 6, 7 and 8), which differed in the length of the polymethylene [–(CH₂)_n–] spacer between Tween 85 and PEI, were synthesized and investigated. The results showed that the transfection efficiency and cytotoxicity both increased with the spacer chain length. Then, TnPs with a [–(CH₂)₆–] spacer (T₆P) were chosen to deliver p65 shRNA to a tumor and subsequently inhibit tumor growth and lymphatic metastasis. The T₆P/p65 shRNA complex nanoparticles (T₆Ns) could significantly down-regulate p65 expression in breast cancer cells, and consequently inhibit cell invasion and disrupt the tube formation. Most importantly, T₆Ns accumulated greatly in tumor tissue, and as a result, significantly inhibited the growth and lymphatic metastasis of breast cancer xenograft. All these results indicated that the transfection efficacies of cationic amphiphiles could be significantly modulated by minor structural variations, and that T₆P was promising for the effective delivery of p65 shRNA to knock down the expression of the key metastasis-driving genes and inhibit tumor growth and metastasis.     

Gene transfection of hyperbranched PEI grafted by hydrophobic amino acid segment PBLG

The complex copolymer of hyperbranched polyethylenimine (PEI) with hydrophobic poly(gamma-benzyl L-glutamate) segment (PBLG) at their chain ends was synthesized. This water-soluble copolymer PEI-PBLG (PP) was characterized for DNA complexation (gel retardation assay, particle size, DNA release and DNase I protection), cell viability and in vitro transfection efficiency. The experiments showed that PP can effectively condense pDNA into particles. Size measurement of the complexes particles indicated that PP/DNA tended to form smaller nanoparticles than those of PEI/DNA, which was caused by the hydrophobic PBLG segments compressing the PP/DNA complex particles in aqueous solution. The representative average size of PP/DNA complex prepared using plasmid DNA (pEGFP-N1, pDNA) was about 96 nm. The condensed pDNA in the PP/pDNA complexes was significantly protected from enzymatic degradation by DNase I. Cytotoxicity studies by MTT colorimetric assays suggested that the PP had much lower toxicity than PEI. The in vitro transfection efficiency of PP/pDNA complexes improved a lot in HeLa cells, Vero cells and 293T cells as compared to that of PEI-25K by the expression of Green Fluorescent Protein (GFP) as determined by flow cytometry. Thus, the water-soluble PP copolymer showed considerable potential as carriers for gene delivery.     

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.     

Polypeptide-based combination of paclitaxel and cisplatin for enhanced chemotherapy efficacy and reduced side-effects

A novel methoxy poly(ethylene glycol)-b-poly(l-glutamic acid)-b-poly(l-phenylalanine) (mPEG-b-P(Glu)-b-P(Phe)) triblock copolymer was prepared and explored as a micelle carrier for the co-delivery of paclitaxel (PTX) and cisplatin (cis-diamminedichlo-platinum, CDDP). PTX and CDDP were loaded inside the hydrophobic P(Phe) inner core and chelated to the middle P(Glu) shell, respectively, while mPEG provided the outer corona for prolonged circulation. An in vitro release profile of the PTX + CDDP-loaded micelles showed that the CDDP chelation cross-link prevented an initial burst release of PTX. The PTX + CDDP-loaded micelles exhibited a high synergism effect in the inhibition of A549 human lung cancer cell line proliferation over 72 h incubation. For the in vivo treatment of xenograft human lung tumor, the PTX + CDDP-loaded micelles displayed an obvious tumor inhibiting effect with a 83.1% tumor suppression rate (TSR%), which was significantly higher than that of a free drug combination or micelles with a single drug. In addition, more importantly, the enhanced anti-tumor efficacy of the PTX + CDDP-loaded micelles came with reduced side-effects. No obvious body weight loss occurred during the treatment of A549 tumor-bearing mice with the PTX + CDDP-loaded micelles. Thus, the polypeptide-based combination of PTX and CDDP may provide useful guidance for effective and safe cancer chemotherapy.     

Gene delivery in vitro and in vivo from bioreducible multilayered polyelectrolyte films of plasmid DNA

Layer-by-layer (LbL) films were assembled on flexible stainless steel substrate using plasmid DNA and reducible hyperbranched poly(amido amine) (RHB) polycation. The films were characterized by XPS and their disassembly in reducing conditions confirmed by ellipsometry. Fibroblast and smooth muscle cell attachment and proliferation on DNA/RHB films were indistinguishable from those on control DNA/poly(ethylenimine) (PEI) films. In vitro transfection activity was evaluated using reporter plasmids encoding for secreted alkaline phosphatase (SEAP) and green fluorescent protein (GFP). DNA/RHB films showed higher and longer lasting transfection activity than control DNA/PEI films using SEAP plasmid. It was revealed through the use of GFP plasmid that DNA/RHB films transfected almost the entire cell population growing on the films. In vivo transfection activity was evaluated by subcutaneously implanting a stainless steel substrate coated with the DNA/RHB films containing SEAP plasmid DNA and measuring the levels of SEAP secreted into the blood circulation of rats. It was found that the plasma levels of SEAP peaked at ～160 ng SEAP/mL five days post-implantation.