Targeted delivery in breast cancer cells via iodine: nuclear localization sequence conjugate.

The nonviral vector with iodine-nuclear localization sequence (namely, NLS-I) targeting breast cancer cells was fabricated. Ternary complexes were formed via charge interactions among NLS-I peptides, PEI 1800, and DNA, and we investigated their cellular internalization, nuclear accumulation as well as transfection efficiency. All the experiments were assessed by employing MCF-7 cells that express sodium/iodide symporter and HeLa cells that lack the expression of the symporter. In MCF-7 cells, cell internalization and nuclear accumulation of NLS-I was markedly increased compared to that in NLS. In addition, compared to that of the PEI1800/DNA complex, PEI1800/DNA/NLS-I complexes exhibited much enhanced luciferase reporter gene expression by up to 130-fold. By contrast, in HeLa cells, the evident improvements of cellular internalization, nuclear accumulation, and transfection efficiency by NLS-I were not observed. This study demonstrates an alternative method to construct a nonviral delivery system for targeted gene transfer into breast cancer cells.

Inhibition of enhanced green fluorescent protein expression by (dextran-hexamethylenediisocyanate)-g-polyethylenimine/siRNA complexes.

Small interfering RNA (siRNA), as a potential tool for gene therapy, requires suitable vehicles for stable complexation, protection, low cytotoxicity and high efficiency of gene knockdown. Here, a new polycation, (dextran-hexamethylenediisocyanate)-g-polyethylenimine ((Dex-HMDI)-g-PEI), was used as a siRNA vector to knock down the expression of Enhanced Green Fluorescent Protein (EGFP). It was found that the complexation of siRNA with the polycation can significantly knock down the expression of EGFP, but has no remarkable effect on the expression of luciferase.

A potential targeting gene vector based on biotinylated polyethyleneimine/avidin bioconjugates.

PURPOSE: To improve the gene delivery efficiency and safety of non-viral vector in liver cells, avidin, which exhibited good biocompatibility and remarkable accumulation in liver, was bioconjugated with biotinylated polyethylenimine to obtain a novel gene vector. MATERIALS AND METHODS: Biotinylated polyethyleneimine/avidin bioconjugate (ABP) was synthesized through grafting biotin to high molecular weight branched polyethylenimine (PEI, 25 kDa) and then bioconjugating with avidin by the biotin-avidin interaction. Physiochemical characteristics of ABP/pDNA complexes were analyzed, and in vitro cytotoxicity and transfection of ABP were also evaluated in HepG2, Hela and 293 T cells by using 25 kDa PEI as the control. RESULTS: It was found that ABP was able to condense pDNA efficiently at N/P ratio of 4. The particle sizes of ABP/pDNA complexes were less than 220 nm, and the average surface charges were around 27 mV at the N/P ratio ranging from 2 to 60. Among three different cell lines, ABP and its DNA complexes demonstrated much lower cytotoxicity and higher transfection efficacy in HepG2 cells as compared with 25 kDa PEI. CONCLUSION: ABP presented higher transfection efficacy and safety in HepG2 cells due to the biocompatibility of avidin and the specific interactions between avidin and HepG2 cells.

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.

Novel thermoresponsive nonviral gene vector: P(NIPAAm-co-NDAPM)-b-PEI with adjustable gene transfection efficiency.

A thermoresponsive cationic copolymer, poly( N-isopropylacrylamide- co- N-(3-(dimethylamino)propyl)methacrylamide)- b-polyethyleneimine (P(NIPAAm- co-NDAPM)- b-PEI), was designed and synthesized as a potential nonviral gene vector. The lower critical solution temperature (LCST) of P(NIPAAm- co-NDAPM)- b-PEI in water measured by UV-vis spectroscopy was 38 degrees C. P(NIPAAm- co-NDAPM)- b-PEI as the gene vector was evaluated in terms of cytotoxicity, buffer capability determined by acid-base titration, DNA binding capability characterized by agarose gel electrophoresis and particle size analysis, and in vitro gene transfection. P(NIPAAm- co-NDAPM)- b-PEI copolymer exhibited lower cytotoxicity in comparison with 25 kDa PEI. Gel retardation assay study indicated that the copolymer was able to bind DNA completely at N/P ratios higher than 30. At 27 degrees C, the mean particle sizes of P(NIPAAm- co-NDAPM)- b-PEI/DNA complexes decreased from 1200 to 570 nm corresponding to the increase in N/P ratios from 10 to 60. When the temperature changed to 37 degrees C, the mean particle sizes of complexes decreased from 850 to 450 nm correspondingly within the same N/P ratio range due to the collapse of thermoresponsive PNIPAAm segments. It was found that the transfection efficiency of P(NIPAAm- co-NDAPM)- b-PEI/DNA complexes was higher than or comparable to that of 25 kDa PEI/DNA complexes at their optimal N/P ratios. Importantly, the transfection efficiency of P(NIPAAm- co-NDAPM)- b-PEI/DNA complexes could be adjusted by altering the transfection and cell culture temperature.

The influence of RGD addition on the gene transfer characteristics of disulfide-containing polyethyleneimine/DNA complexes.

Arginine-glycine-aspartic acid (RGD) ligand is often chemically attached to polycation vector to improve the transfection efficiency. However, the chemical reaction may reduce or even inactivate the biological activities of peptides. In order to retain the targeting ability and biological activities, the RGD peptide was noncovalently introduced into polycations as gene delivery systems. In this paper, the tripeptide sequence RGD was added to disulfide-containing polyethyleneimine (SS-PEI)/DNA binary complexes to evaluate the influence of RGD addition for the particle size, zeta potential, morphology, and transfection efficiency. GelRed was used as a molecular probe to show the effect of RGD addition on the cellular uptake of complexes. In vitro transfection experiments showed that SS-PEI exhibited comparable transfection efficiency, but lower cytotoxicity in comparison with 25kDa PEI. The transfection efficiency of complexes with RGD in HeLa cells was reduced statistically significantly with the increasing content of RGD peptide, but that in 293T cells was not altered significantly with the increasing content of RGD peptide. The reduced transfection efficiency of SS-PEI/DNA complexes with RGD in HeLa cells was attributed to the targeted binding interactions between the surplus RGD and the alpha(nu)beta(3) and alpha(nu)beta(5) integrins in HeLa cells, which would prevent the binding between RGD in complexes and integrin receptor on the surface of cells as well as nonspecific endocytosis of SS-PEI/DNA complexes mediated by proteoglycan in HeLa cells.

Biodegradable and pH-sensitive hydrogels for cell encapsulation and controlled drug release.

Hydrogels with pH-sensitive poly(acrylic acid) (PAAc) chains and biodegradable acryloyl-poly(-caprolactone)-2-hydroxylethyl methacrylate (AC-PCL-HEMA) chains were designed and synthesized. The morphology of hydrogel was observed by scanning electron microscopy. The degradation of the hydrogel in the presence of Pseudomonas lipase was studied. The in vitro release of bovine serum albumin from the hydrogel was investigated. Cytotoxicity study shows that the AC-PCL-HEMA/AAc copolymer exhibits good biocompatibility. Cell adhesion and migration into the hydrogel networks were evaluated by using different cell lines. The hydrogel with a lower cross-linking density and a larger pore size exhibited a better performance for cells migration.

Polyethyleneimine modified biocompatible poly(N-isopropylacrylamide)-based nanogels for drug delivery.

A series of biocompatible and stimuli-sensitive poly(N-isopropylacrylamide-co-propyl acrylic acid) (P(NIPAAm-co-PAAc)) nanogels were synthesized by emulsion polymerization. In addition, polyethyleneimine (PEI) was further grafted to modify the PNIPAAm-based nanogels. The P(NIPAAm-co-PAAc)-g-PEI nanogels exhibited good thermosensitivity as well as pH sensitivity. Transmission electron microscopy (TEM) showed that the P(NIPAAm-co-PAAc)-g-PEI and P(NIPAAm-co-PAAc) nanogels displayed well dispersed spherical morphology. The mean sizes of the nanogels measured by dynamic light scattering (DLS) were from 100 nm to 500 nm at different temperatures. The cytotoxicity study indicated P(NIPAAm-co-PAAc) nanogels exhibited a better biocompatibility than both PNIPAAm nanogel and P(NIPAAm-co-PAAc)-g-PEI nanogel although all the three kinds of nanogels did not exhibit apparent cytotoxicity. The drug-loaded nanogels, especially the PEI-grafted nanogels, showed temperature-trigged controlled release behaviors, indicating the potential applications as an intelligent drug delivery system.

Versatile Supermolecular Inclusion Complex Based on Host-Guest Interaction for Targeted Gene Delivery.

A facile and targeted gene delivery system was prepared by conjugating ß-cyclodextrin modified polyethylenimine (PEI-CD) and adamantyl peptide (AdGRGDS) based on host-guest interaction. With the rational design between PEI-CD and AdGRGDS, the PEI-CD/AdGRGDS gene delivery system showed excellent DNA binding capability and exhibited good ability to compact DNA into uniform spherical nanoparticles. In vitro luciferase assay showed that gene expression transfected by PEI-CD/AdGRGDS was stronger than that by PEI-CD in HeLa cells, whereas gene expression transfected by PEI-CD/AdGRGDS and PEI-CD was similar to each other in COS7 cells. Internalization of complexes was qualitatively studied using a confocal laser scanning microscope (CLSM) and quantitatively analyzed by flow cytometry, respectively, and targeting specificity was also evaluated by CLSM. Results of CLSM and flow cytometry indicated that PEI-CD/AdGRGDS had good targeting specificity to tumor cells with integrin αvß3 overexpression. To further evaluate the targeting specificity and transfection efficiency in vivo, a rat model with murine hepatic carcinoma cell line H22 was used. PEI-CD/AdGRGDS showed stronger gene expression efficiency than PEI-CD via in vivo transfection of pORF-LacZ and pGL-3 plasmids after subcutaneous injection. Interestingly, PEI-CD/AdGRGDS also showed high targeting specificity and transfection distribution to tumor xenograft after tail-vein injection. In vitro and in vivo assays highlighted the importance of GRGDS targeting specificity to tumor cells with integrin αvß3 overexpression and demonstrated that the PEI-CD/AdGRGDS gene delivery system would have great potential for targeted tumor therapy.

Fabrication of novel reduction-sensitive gene vectors based on three-armed peptides.

To address the inherent barriers of gene transfection, two reduction-sensitive branched polypeptides (RBPs) are synthesized and explored as novel non-viral gene vectors. The introduced disulfide linkages in RBPs facilitate glutathione-triggered intracellular gene release and reduce polymer degradation-induced cytotoxicity. Furthermore, the highly branched architecture concurrently realizes multivalency for strong DNA binding and elicits conformational flexibility for tight DNA compacting, which are beneficial for cellular entry. To increase the endosomal escape of plasmid DNA, pH-sensitive histidyl residues are incorporated into RBPs to improve buffer capacity in an acidic environment. In vitro study demonstrates that RBPs can efficiently mediate the DNA transfection and avoid apparent cytotoxicity in HeLa and COS7. The present gene delivery system offers a simple and flexible approach to fabricate microenvironment-specific branched gene vectors for gene therapy.

Oligoamines grafted hyperbranched polyether as high efficient and serum-tolerant gene vectors.

To develop low toxic, high efficient, and excellent serum-tolerant polycation gene delivery systems, a series of oligoamines grafted hyperbranched polyether (oligoamines-g-HBP) were synthesized by conjugating different oligoamines, including triethylenetetramine (TETA) and tetraethylenepentamine (TEPA), onto COOH-functionalized hyperbranched poly(3-ethyl-3-oxetanemethanol). It was found that oligoamines-g-HBP exhibited good buffering capacity, strong DNA binding and high resistance against protein adsorption. In vitro cytotoxicity measurement indicated that oligoamines-g-HBP had much lower cytotoxicity as compared with 25 kDa PEI. The transfection efficiency of TEPA-g-HBP/DNA complexes at a certain N/P ratio was significantly higher than that of 25 kDa PEI/DNA complexes. Interestingly, it was found that TEPA-g-HBP had much improved serum-tolerant capability as compared with 25 kDa PEI even when serum concentration was increased to 30%. Confocal laser images further showed that the amount of YOYO-1 labeled DNA in nuclei got increased with increasing the number of secondary amino ethylene groups in oligoamines-g-HBP. The oligoamines-g-HBP presented great potential as gene delivery vectors for further clinical applications.

Effect of peptides and their introduction methods on target gene transfer of gene vector based on disulfide-containing polyethyleneimine.

To evaluate the effect of different peptides as well as their introduction methods on target gene transfer of gene vectors based on disulfide-containing polyethyleneimine (SS-PEI), a series of peptides including N(3)-GRGDSF, GRGDSF, and EEEEEEEEGRGDSF (E(8)GRGDSF) were prepared. N(3)-GRGDSF was conjugated to SS-PEI by click chemistry and SS-PEI-GRGDSF was obtained. GRGDSF was non-covalently introduced into SS-PEI/DNA mainly through hydrogen bonding to obtain SS-PEI/DNA/GRGDSF complexes, whereas E(8)GRGDSF was further non-covalently introduced to SS-PEI/DNA through electrostatic force to obtain SS-PEI/DNA/E(8)GRGDSF complexes. Transfection efficiency of all complexes with peptides was lower than that of SS-PEI/DNA in COS-7 cells due to the fact that nonspecific endocytosis was prohibited after peptide introduction. Whereas in HeLa cells, transfection activity of SS-PEI-GRGDSF/DNA and SS-PEI/DNA/E(8)GRGDSF at certain w/w ratios was higher than that of SS-PEI/DNA. But the transfection efficiency of SS-PEI/DNA/E(8)GRGDSF at peptide/DNA w/w ratios higher than 30 dropped due to targeted binding interactions between surplus E(8)GRGDSF and the integrins in HeLa cells, which would prohibit specific endocytosis of E(8)GRGDSF in complexes. Transfection activity of SS-PEI/DNA/GRGDSF was lower than or comparable to that of SS-PEI/DNA because of loose complexes constructed by hydrogen bonding between GRGDSF and SS-PEI/DNA.

Bilayer matrix composed of polycation/DNA complex and sodium alginate gel as a tumor cell catcher.

A bilayer matrix consisting of TABP-SS/DNA complexes and sodium alginate gel is formed via electrostatic interaction. In vitro cell adhesion, proliferation and transfection of the bilayer matrix are investigated in HepG2, HeLa and COS7 cells. Results show that this matrix can only promote tumor cell attachment and growth. Compared with normal cells, the bilayer matrix exhibits significantly higher transfection efficacy in tumor cells. Cell co-culture competitive transfection assay shows that the cell uptake of TABP-SS/DNA complexes is significantly enhanced in tumor cells rather than normal cells under the co-culture competitive condition, which confirms that TABP-SS/DNA complexes have strong tumor cell selectivity and tumor targeting transfection ability.

Poly(ß-amino amine) cross-linked PEIs as highly efficient gene vectors.

To increase the release of DNA into the cytoplasm and further improve transgene expression of nucleic acid novel polymeric gene carriers were prepared which would be biodegradable under the reducing conditions in the cytoplasm. Disulfide-containing poly(ß-amino amine)s were first synthesized and then used to cross-link low molecular weight polyethyleneimine (1800 Da) through Michael addition to obtain SS-PBAA-PEIs as the final gene carriers. The physicochemical characteristics of SS-PBAA-PEI/DNA complexes were characterized. In vitro transfection mediated by the SS-PBAA-PEIs under serum conditions was carried out. Cell uptake of the gene delivery systems was observed by confocal laser scanning microscopy. The results of the physicochemical characterisation demonstrated that the SS-PBAA-PEIs could efficiently condense DNA. In vitro transfection under serum conditions showed that SS-PBAA-PEIs had comparable or even higher transfection efficiencies than 25 kDa PEI. And SS-PBAA-PEIs showed much lower cytotoxicity compared with 25 kDa PEI. In summary, the SS-PBAA-PEIs possess great potential as non-viral gene vectors and exhibit high transfection efficiency under serum conditions.

Influential factors associated with biotinylated disulfide containing PEI/avidin bioconjugate mediated gene delivery in HepG2 cells.

The influences of environmental factors on how PEI derivatives mediate gene delivery are absolutely essential to obtaining high transfection efficacy, but have been rarely investigated until now. In this study, biotinylated PEI-SS/avidin bioconjugate (ABP-SS) mediated gene transfection in HepG2 cells was exposed to varying environmental factors, such as pH, NaCl, serum, temperature and time. Physicochemical characteristics of ABP-SS/DNA complexes were evaluated in terms of agarose gel electrophoresis, particle size measurements, and in vitro transfection assays. The DNA binding ability of ABP-SS was weakened when pH value was decreasing. It was inferred that ABP-SS/DNA complexes could form compact structures at pH 7.4 and looser structures at lower pH values. Transfection efficiencies were largely dependent on the pH of the culture medium, and the optimal pH of culture medium for ABP-SS mediated gene delivery in HepG2 was 7.4. The particle sizes of ABP-SS/pDNA complexes formed in 150 mM NaCl solution were less than 280 nm, and were larger than those of complexes formed in deionized water. The transfection ability of ABP-SS/pGL-3 complexes formed in deionized water was much weaker than that formed in 150 mM NaCl solution. The presence of lower serum contents had no obvious effect on the transfection efficiency of ABP-SS/pGL-3 complexes. The optimum complex-forming temperature and time for ABP-SS/pGL-3 complexes were 37 degrees C and 30 min. Cell morphology was observed by live cell confocal microscopy. It can be concluded that the cytotoxicity of ABP-SS in HepG2 cells was the result of apoptosis, and naked DNA had no negative impact on cells.

Biotinylated transferrin/avidin/biotinylated disulfide containing PEI bioconjugates mediated p53 gene delivery system for tumor targeted transfection.

As mutation and dysfunction of p53 gene could induce most of human cancers, the p53 tumor suppressor gene was used to replace them and recover their normal functions in cancer cells. In this paper, biotinylated transferrin/avidin/biotinylated disulfide containing PEI bioconjugates (TABP-SS) mediated p53 gene delivery system was formed attributed to the 'avidin-biotin bridge'. Characteristics of the obtained TABP-SS and its p53 complexes were evaluated in terms of acid-base titration, agarose gel electrophoresis, SEM, particle size and zeta-potential measurements. The acid-base titration results showed that TABP-SS had good buffer capability. The results of gel electrophoresis indicated that TABP-SS could fully condensed DNA and would be degraded by reducing agent inside cells. In vitro cell viability and transfection of TABP-SS were investigated in COS7, HepG2, and HeLa cells. Among the three different cell lines, TABP-SS exhibited much lower cytotoxicity and higher transfection efficacy in HepG2 and HeLa cells due to the specific interactions between transferrin ligands and their receptors on tumor cells. Apoptotic morphology was observed using confocal microscopy, and the expression of p53 protein in transfected cells was evaluated by western blotting. All the results indicated that TABP-SS/p53 complex could be considered as a low toxic and high efficient tumor targeted gene delivery system, which has great potential for further clinical application.

Poly(methyl methacrylate)-graft-oligoamines as low cytotoxic and efficient nonviral gene vectors.

A series of poly(methyl methacrylate)-graft-oligoamines (PMMA-g-oligoamines), including PMMA-g-DETA, PMMA-g-TETA and PMMA-g-TEPA, were synthesized through aminolysis of the PMMA with diethylenetriamine, triethylenetetramine and tetraethylenepentamine. Agarose gel retardation assay indicated that PMMA-g-oligoamines had good binding capability with plasmid DNA, and the binding capability increased with increasing length of oligoamines and content of nitrogen (N%). The results of particle size, zeta potential and morphology observation further showed that the PMMA-g-oligoamines could condense DNA efficiently and the PMMA-g-oligoamine/DNA complexes were uniform nanospheres. The in vitro cell viability indicated that PMMA-g-oligoamines were less toxic than 25 kDa PEI, though the cytotoxicity of PMMA-g-oligoamines increased slightly with increasing length of oligoamines as well as the N% of PMMA-g-oligoamines. The transfection efficiency of PMMA-g-oligoamines/DNA complexes in 293 T and HeLa cells demonstrated that PMMA-g-oligoamines could transfect cells efficiently with increasing the length of oligoamines, especially PMMA-g-TEPA with highest N%, and showed similar transfection capability as 25 kDa PEI. The cellular uptake study showed that the distribution of YOYO-1 labeled DNA in the cytoplasm and nuclei increased gradually with increasing length of oligoamines.

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.

N-Succinyl-chitosan grafted with low molecular weight polyethylenimine as a serum-resistant gene vector.

Low transfection efficiency and inactivation by serum are the major drawbacks for cationic polymers when used as non-viral gene vectors. Here, a series of N-succinyl-chitosan-graft-polyethylenimine (NSC-g-PEI) copolymers with different compositions were synthesized through grafting low molecular weight PEI (800 Da) to N-succinyl-chitosan. An agarose gel electrophoresis assay showed NSC-g-PEIs had good binding capability with DNA and the particle size of the NSC-g-PEI-DNA complexes was between 150 to 300 nm as determined by a Zeta sizer. In vitro transfection of NSC-g-PEI-DNA complexes for 293T, HeLa and CHO cells was investigated. It was found that the transfection efficiency of NSC-g-PEI-DNA complexes was higher than that of DNA combined PEI (25 kDa) and the transfection efficiency increased with the increasing GD of PEI. More importantly, the NSC-g-PEI-DNA complexes were stable and the transfection efficiency was not affected obviously in the presence of serum with different concentrations. In addition, NSC-g-PEIs had a lower cytotoxicity than PEI (25 kDa) and the toxicity increased with increasing GD of PEI. The NSC-g-PEI copolymers will have a good potential as efficient non-viral gene vectors in the presence of serum.

Synthesis of (Dex-HMDI)-g-PEIs as effective and low cytotoxic nonviral gene vectors.

Gene vectors, (dextran-hexamethylenediisocyanate)-g-polyethylenimines ((Dex-HMDI)-g-PEIs), were synthesized through grafting low molecular weight (800 Da) branched polyethylenimine (PEI) to HMDI functionalized dextrans with two different molecular weights. The buffer capabilities of (Dex-HMDI)-g-PEIs were examined by acid-base titration. The titration profiles show that both (Dex-HMDI)-g-PEIs have the similar buffer capability regardless of the different molecular weight of dextran. Physiochemical characteristics of (Dex-HMDI)-g-PEI/DNA complexes were analyzed by agarose gel electrophoresis, and particle size and zeta-potential measurements. The result of gel electrophoresis suggests that both (Dex-HMDI)-g-PEIs are able to condense DNA efficiently at N/P ratios higher than 4. The particle sizes of (Dex-HMDI)-g-PEI/DNA complexes are around 160-250 nm, and the surface charges are around 19-23 mV at the N/P ratios ranging from 10 to 60. The morphology of complexes was observed by scanning electron microscopy (SEM) and the images show that nano-sized complexes display a regular spherical shape. In vitro cell viability and transfection were evaluated in 293T and HeLa cells using 25 kDa PEI as a control. The cytotoxicity of (Dex-HMDI)-g-PEIs is lower than that of 25 kDa PEI. The gene transfection efficiency of (Dex-HMDI)-g-PEI/DNA complexes at certain N/P ratios in 293T cells is higher than or comparable to 25 kDa PEI/DNA complex at its optimal N/P ratio of 10. In addition, comparing with (Dex-HMDI)-g-PEI with a high molecular weight dextran, (Dex-HMDI)-g-PEI with a low molecular weight dextran demonstrates lower cytotoxicity and higher transfection efficiency.