Effect of molecular weight and degree of substitution of quaternized cellulose on the efficiency of gene transfection.

Quaternized celluloses (QCs) with different molecular weight (M(w)) and degree of cationic substitution (DS) were homogeneously synthesized in NaOH/urea aqueous solutions and were studied as gene carriers. QCs were evaluated for efficacy of nanoparticle formation, DNA binding efficiency, morphology, and in vitro gene transfection efficiency. The factors affecting the transfection efficiency, e.g., M(w), DS, and N/P ratios, have been evaluated. The cytotoxicity of QCs and QC/DNA complexes were evaluated in 293T cells and were found to be relatively low compared with 25 kDa PEI and PEI/DNA complex, which increased slightly with increasing of M(w) and DS. All QCs obtained could bind DNA efficiently, and QC/DNA complexes exhibited effective transfection in comparison to the naked DNA. More importantly, the QC/DNA complexes, were stable and the transfection efficiency was not inhibited in the presence of serum. The results revealed an important combined effect between M(w) and DS of QCs in determining transgene expression, and QCs with M(w) of about 8 x 10(4) g/mol and DS of about 0.6 displayed relatively higher transfection efficiencies attributed to the intermediate stability.

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.

Targeted gene delivery mediated by folate-polyethylenimine-block-poly(ethylene glycol) with receptor selectivity.

The folate receptor (FR) is a tumor marker overexpressed in large numbers of cancer cells. Folic acid has high affinity to the FR and retains its binding affinity upon derivatization via its gamma-carboxyl. Therefore, in this article, folate-polyethylenimine-block-poly(ethylene glycol) (FOL-PEI-b-PEG) was designed for specific receptor targeted gene delivery. Physicochemical characterizations of resulting FOL-PEI-b-PEG/DNA complexes in terms of agarose gel electrophoresis, particle size, and zeta potential measurements were investigated. The results indicated that FOL-PEI-b-PEG was able to condense plasmid DNA tightly with a suitable particle size. The cytotoxicity study indicated that the copolymer exhibited less toxicity in comparison with that of 25 kDa PEI. Luciferase assay and green fluorescent protein (GFP) detections were also used to confirm that FOL-PEI-b-PEG could be an effective gene vector. Importantly, transfection efficiency of FOL-PEI-b-PEG with free folic acid was much lower than that of the copolymer without free folic acid on FR-positive HeLa cells, suggesting that FOL-PEI-b-PEG has great potential as a targeting gene vector.

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.

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.

π-Extended Benzoporphyrin-Based Metal-Organic Framework for Inhibition of Tumor Metastasis.

We report on the benzoporphyrin-based metal-organic framework (TBP-MOF), with 10-connected Zr6 cluster and much improved photophysical properties over the traditional porphyrin-based MOFs. It was found that TBP-MOF exhibited red-shifted absorption bands and strong near-infrared luminescence for bioimaging, whereas the π-extended benzoporphyrin-based linkers of TBP-MOF facilitated 1O2 generation to enhance O2-dependent photodynamic therapy (PDT). It was demonstrated that poly(ethylene glycol)-modified nanoscale TBP-MOF (TBP-nMOF) can be used as an effective PDT agent under hypoxic tumor microenvironment. We also elucidated that the low O2-dependent PDT of TBP-nMOF in combination with αPD-1 checkpoint blockade therapy can not only suppress the growth of primary tumor, but also stimulate an antitumor immune response for inhibiting metastatic tumor growth. We believe this TBP-nMOF has great potential to serve as an efficient photosensitizer for PDT and cancer immunotherapy.

Photo-Powered Artificial Organelles for ATP Generation and Life-Sustainment.

Adenosine triphosphate (ATP) is the most important immediate energy source for driving intracellular biochemical reactions in nearly all life forms. Controllable generation of ATP in life is still an unrealized goal. Here, thylakoid fragments are recombined with lipid molecules to synthesize a synthetic/biological hybrid proteoliposome, named highly efficient life-support intracellular opto-driven system (HELIOS) for the generation of ATP. With red light irradiation, HELIOS can improve the intracellular ATP concentration to 1.38-2.45 times in various cell lines. Moreover, it is noticed that HELIOS-mediated ATP generation can comprehensively promote cell functions such as protein synthesis and insulin secretion. At organ and individual levels, it is also proved that HELIOS can rescue a mouse heart from myocardial infarction and sustain life of fasting zebrafish Danio rerio models. The photo-powered artificial organelle can deepen our understanding of metabolism and enable the development of optical therapy that targets intracellular energy supply.

Dual stimuli-responsive multi-drug delivery system for the individually controlled release of anti-cancer drugs.

A dual stimuli-responsive multi-drug delivery system was developed for "cancer cocktail therapy". Upon UV irradiation, microcapsules could rapidly release the small-molecule drugs, and thereafter the macromolecular drugs would be released in the presence of MMP in the tumor cells. This system will find great potential as a novel chemotherapeutic combination for cancer treatment.

A boronate-linked linear-hyperbranched polymeric nanovehicle for pH-dependent tumor-targeted drug delivery.

Advanced drug delivery systems, which possess post-functionalization feasibility to achieve targetability and traceability, favorable pharmacokinetics with dynamic but controllable stability, and preferable tumor accumulation with prolonged drug residence in disease sites, represent ideal nanomedicine paradigm for tumor therapy. To address this challenge, here we reported a dynamic module-assembly strategy based on reversible boronic acid/1,3-diol bioorthogonality. As a prototype, metastable hybrid nanoself-assembly between hydrophobic hyperbranched diol-enriched polycarbonate (HP-OH) and hydrophilic linear PEG terminated with phenylboronic acid (mPEG-PBA) is demonstrated in vitro and in vivo. The nanoconstruction maintained excellent stability with little leakage of loaded drugs under the simulated physiological conditions. Such a stable nanostructure enabled the effective in vivo tumor accumulation in tumor site as revealed by NIR imaging technique. More importantly, this nanoconstruction presented a pH-labile destruction profile in response to acidic microenvironment and simultaneously the fast liberation of loaded drugs. Accordingly at the cellular level, the intracellular structural dissociation was also proved in terms of the strong acidity in late endosome/lysosome, thus favoring the prolonged retention of remaining drug-loaded HP-OH aggregates within tumor cells. Hence, our delicate design open up a dynamical module-assembly path to develop site and time dual-controlled nanotherapeutics for tumor chemotherapy, allowing enhanced tumor selectivity through prolonged retention of delivery system in tumor cells followed by a timely drug release pattern.

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.

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.