Jianfeng Han

Cationic bovine serum albumin based self-assembled nanoparticles as siRNA delivery vector for treating lung metastatic cancer.

It is generally believed that intravenous application of cationic vectors is limited by the binding of abundant negatively charged serum components, which may cause rapid clearance of the therapeutic agent from the blood stream. However, previous studies show that systemic delivery of cationic gene vectors mediates specific and efficient transfection within the lung, mainly as a result of interaction of the vectors with serum proteins. Based on these findings, a novel and charge-density-controllable siRNA delivery system is developed to treat lung metastatic cancer by using cationic bovine serum albumin (CBSA) as the gene vector. By surface modification of BSA, CBSA with different isoelectric points (pI) is synthesized and the optimal cationization degree of CBSA is determined by considering the siRNA binding and delivery ability, as well as toxicity. The CBSA can form stable nanosized particles with siRNA and protect siRNA from degradation. CBSA also shows excellent abilities to intracellularly deliver siRNA and mediate significant accumulation in the lung. When Bcl2-specific siRNA is introduced to this system, CBSA/siRNA nanoparticles exhibit an efficient gene-silencing effect that induces notable cancer cell apoptosis and subsequently inhibits the tumor growth in a B16 lung metastasis model. These results indicate that CBSA-based self-assembled nanoparticles can be a promising strategy for a siRNA delivery system for lung targeting and metastatic cancer therapy.

Enzyme-responsive liposomes modified adenoviral vectors for enhanced tumor cell transduction and reduced immunogenicity

Limitations of adenoviral (Ad) vectors for cancer gene therapy could be overcome by their combination with pharmaceutical technologies. Here we show that an enzyme-responsive liposomal formulation could significantly enhance the tumor cell transduction abilities and reduce the immunogenicity of Ad vectors. In the current research, the enzymatically cleavable PEG-lipids composed of a PEG/matrix metalloproteinase (MMP)-substrate peptide/cholesterol (PPC) were synthesized and characterized by (1)H NMR and TOF MS ES(+). The obtained MMP-cleavable lipids were inserted into the anionic liposomal Ad vectors (AL-Ad) by the post-insertion method. The results of in vitro infection assays indicated that the enzymatically cleavable formulation (PPC-AL-Ad) displayed a much higher gene expression than naked Ad5 and the non-cleavable PEG-lipid modified Ad vectors in tumor cells. More importantly, PPC-AL-Ad induces a lower production of neutralizing antibody and lower innate immune response, as well as significantly reduced liver toxicity in vivo. These findings suggest that PPC-AL-Ad is a promising system for gene delivery in tumor therapy.

Combination of adenovirus and cross-linked low molecular weight PEI improves efficiency of gene transduction

Recombinant adenovirus (Ad)-mediated gene therapy is an exciting novel strategy in cancer treatment. However, poor infection efficiency with coxsackievirus and adenovirus receptor (CAR) down-regulated cancer cell lines is one of the major challenges for its practical and extensive application. As an alternative method of viral gene delivery, a non-viral carrier using cationic materials could compensate for the limitation of adenovirus. In our study, adenovectors were complexed with a new synthetic polymer PEI-DEG-bis-NPC (PDN) based on polyethylenimine (PEI), and then the properties of the vehicle were characterized by measurement of size distribution, zeta potential and transmission electron microscopy (TEM). Enhancement of gene transduction by Ad/PDN complexes was observed in both CAR-overexpressing cell lines (A549) and CAR-lacking cell lines (MDCK, CHO, LLC), as a result of facilitating binding and cell uptake of adenoviral particles by the cationic component. Ad/PDN complexes also promoted the inhibition of tumor growth in vivo and prolonged the survival time of tumor-bearing mice. These data suggest that a combination of viral and non-viral gene delivery methods may offer a new approach to successful cancer gene therapy.

Mannosylated biodegradable polyethyleneimine for targeted DNA delivery to dendritic cells.

Background To establish a potential gene-delivery system with the ability to deliver plasmid DNA to dendritic cells (DCs) more efficiently and specifically, we designed and synthesized a low-molecular-weight polyethyleneimine and triethyleneglycol polymer (PEI–TEG) and a series of its mannosylated derivatives. Methods PEI–TEG was synthesized from PEI2000 and PEI600 with TEG as the cross-linker. PEI–TEG was then linked to mannose via a phenylisothiocyanate bridge to obtain man-PEI–TEG conjugates. The DNA conveyance abilities of PEI–TEG, man-PEI–TEG, as well as control PEI25k were evaluated by measuring their zeta potential, particle size, and DNA-binding abilities. The in vitro cytotoxicity, cell uptake, and transfection efficiency of these PEI/DNA complexes were examined on the DC2.4 cell line. Finally, a maturation experiment evaluated the effect of costimulatory molecules CD40, CD80, and CD86 on murine bone marrow-derived DCs (BMDCs) using flow cytometry. Results PEI–TEG and man-PEI–TEG were successfully synthesized and were shown to retain the excellent properties of PEI25k for condensing DNA. Compared with PEI–TEG as well as PEI25k, the man-PEI–TEG had less cytotoxicity and performed better in both cellular uptake and transfection assays in vitro. The results of the maturation experiment showed that all the PEI/DNA complexes induced an adequate upregulation of surface markers for DC maturation. Conclusion These results demonstrated that man-PEI–TEG can be employed as a DC-targeting gene-delivery system.

Anionic liposomes increase the efficiency of adenovirus-mediated gene transfer to coxsackie-adenovirus receptor deficient cells.

Despite remarkable progress in the research of both viral and nonviral gene delivery vectors, the drawbacks in each delivery system have limited their clinical applications. Therefore, one of the concepts for developing novel vectors is to overcome the limitations of individual vectors by combining them. In the current study, adenoviral vectors were formulated with anionic liposomes to protect them from neutralizing antibodies and to improve their transduction efficiency in Coxsackievirus-adenovirus receptor (CAR) deficient cells. A calcium-induced phase change method was applied to encapsulate adenovirus 5 (Ad5) into anionic liposomes to formulate the complexes of Ad5 and anionic liposomes (Ad5-AL). Meanwhile, the complexes of Ad5 and cationic liposomes (Ad5-CL) were also prepared as controls. LacZ gene expression in CAR overexpressing cells (A549) and CAR deficient cells (CHO and MDCK) was measured by either qualitative or quantitative detection. Confocal laser scanning microscopy was performed to determine intracellular location of Ad5 after their infection. Human sera with a high titer of antiadenovirus antibody were used to assess the neutralizing antibody protection ability of the complexed vectors. Accompanying the enhanced gene expression, a high ability to introduce Ad5 into cytoplasm and nucleus mediated by Ad5-AL was also observed in CAR deficient cells. Additionally, antibody neutralizing assay indicated that neutralizing serum inhibited naked Ad5 and Ad5-CL at rather higher dilution than Ad5-AL, which demonstrated Ad5-AL was more capable of protecting Ad5 from neutralizing than Ad5-CL. In conclusion, anionic liposomes prepared by the calcium-induced phase change method could significantly enhance the transduction ability of Ad5 in CAR deficient cells.

Protection of adenovirus from neutralizing antibody by cationic PEG derivative ionically linked to adenovirus.

Background The generation of anti-adenovirus neutralizing antibody (NAb) in humans severely restricts the utilization of recombinant adenovirus serotype 5 (Ad5) vectors in gene therapy for a wide range of clinical trials. To overcome this limitation, we ionically complexed Ad5 with a newly synthesized copolymer, which we called APC, making an adenovirus shielded from NAb. Methods APC, a cationic polyethylene glycol derivative, was synthesized via two steps of ring-opening copolymerization of ethylene oxide and allyl glycidyl ether, followed by the addition of 2-mercaptoethylamine. The copolymer or the control PEI-2k was ionically complexed to anionic Ad5 in 5% glucose, and in vitro transduction assays were carried out in coxsackievirus and adenovirus receptor-positive cells (A549) and coxsackievirus and adenovirus receptor-negative cells (B16 and SKOV3). The physical properties and morphology of adenovirus alone or the complexes were investigated respectively by zeta potential, size distribution, and transmission electron microscopy image. Then cytotoxicity of APC was examined using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assays. Finally, the ability of APC to protect adenovirus from NAb was evaluated by transfection assays after a neutralizing effect. Results APC was successfully synthesized and showed a low cytotoxicity. Positively charged Ad5/APC exhibited slightly increased diameter (130.2 ± 0.60 nm) than naked Ad5 (115.6 ± 5.46 nm) while Ad5/PEI-2k showed severe aggregation (1382 ± 79.9 nm). Ad5/APC achieved a gene transfection level as high as Ad5/PEI-2k in A549 or B16 cells, and significantly higher than Ad5/PEI-2k in SKOV3 cells. Most importantly, after the exposure to the neutralizing antibody, naked Ad5 and Ad5/PEI-2k exhibited poor gene expression while Ad5/APC still showed significantly efficient gene expression. Conclusion Our results demonstrated that Ad5/APC complex offered good protection for Ad5 against NAb in vitro and suggested a potential strategy of resistance to NAb in vivo.

Improvement of adenoviral vector-mediated gene transfer to airway epithelia by folate-modified anionic liposomes

Despite remarkable progress in the development of both viral and nonviral gene delivery vectors for airway disease treatment, poor gene transfer efficiency to the airway epithelium is a major obstacle in clinical application. To take advantage of the unique features of viral and nonviral vectors, we have developed complexes of adenovirus vector and anionic liposomes (AL-Ad5) by the calcium-induced phase change method. In the current study, based on the fact that there are overexpressed folate receptors on the surface of airway epithelia, we further modified the AL-Ad5 complexes with folate (F-AL-Ad5) to improve the transduction ability of Ad5 in airway epithelia. The transduction efficiencies of the obtained F-AL-Ad5 and AL-Ad5 complexes were assessed in primary-cultured airway epithelia in vitro. Our results indicated that compared with naked adenovirus vector, both AL-Ad5 and F-AL-Ad5 could significantly enhance the gene transduction efficiency of adenovirus vector in primary-cultured airway epithelial cells. Moreover, the enhancement mediated by F-AL-Ad5 was more dramatic than that by AL-Ad5. These results suggested that F-AL-Ad5 may be a useful strategy to deliver therapeutic genes to the airway epithelia and is promising in clinical application.

Co-delivery of Adenovirus and Carmustine by Anionic Liposomes with Synergistic Anti-tumor Effects

ABSTRACTPurposeTo improve gene transducibility mediated by adenovirus (Ad) in cancer cells and further enhance anti-tumor effects by co-delivery.MethodsCalcium-induced phase change method was used to prepare the complex of anionic liposomes and adenovirus (AL/Ad5). Gene expression was qualitatively detected by X-gal staining and quantitatively detected by ELISA. Taking adenovirus-mediated stromal cell-derived factor-1α (Ad5-SDF1α) as therapeutic gene and carmustine (BCNU) as chemotherapeutic agent, a co-delivering system of AL/Ad5-SDF1α/BCNU was prepared and administered to tumor-bearing mice by intratumor injection.ResultsEnhanced LacZ gene transduction was obtained in B16 and Lewis lung carcinoma cells in vitro and in vivo. Complexes of AL/Ad5-SDF1α improved SDF1α gene expression and led to accumulation of dendritic cells among the murine B16 melanoma cells in vivo. This co-delivery system of AL/Ad5-SDF1α/BCNU could significantly suppress tumor growth and prolong survival of tumor-bearing mice.ConclusionsThrough the co-delivering system, AL/Ad5-SDF1α could synergize with BCNU to improve the antitumor effect. It may be a promising strategy for solid tumor therapy.

Anionic Liposomes Enhance and Prolong Adenovirus-Mediated Gene Expression in Airway Epithelia in Vitro and in Vivo

Adenoviral vector mediated gene therapy has received extensive attention in airway disease treatment. However, the lack of the requisite coxsackie-adenovirus receptor (CAR) on the apical surface of airway epithelium and the host immune response to adenoviruses limit their in vivo application. In our study, we developed for the first time a novel formulation composed of anionic liposomes and adenoviruses (AL-Ad5) using a calcium-induced phase change method. The obtained formulation was employed to enhance the transduction efficiency of airway gene delivery. Our results indicated that primary cultured airway epithelial cells infected by AL-Ad5 displayed higher LacZ gene expression compared to naked adenovirus. Importantly, AL-Ad5 significantly improved and prolonged LacZ gene expression in murine airway tissues when delivered in vivo by intratracheal instillation. Additionally, it was found that anionic liposomes provided immunoprotection to the adenovirus from neutralizing antibody, thus slowing down the elimination of Ad5 particles meanwhile reducing the inflammatory reaction caused by the Ad5 vector. These results suggested that the combination of anionic liposomes with adenovirus may be a useful strategy to deliver therapeutic genes into the airway epithelia and is promising in clinical application.

Controlled Production of Poly (3-Hydroxybutyrate-co-3-Hydroxyhexanoate) (PHBHHx) Nanoparticles for Targeted and Sustained Drug Delivery

The ability to control the size and quality of nanoparticles (NPs) during production is critical for their success as a commercial product for clinical applications. Here, we employed a statistical design of experiment approach to identify the key process variables affecting the size of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) NPs during production via the solvent evaporation method. The number of sonication cycles had a standardzed effect on NP size of 55, with sonication power at 25, and PHBHHx concentration at 27 with a combination of these variables having a lower yet significant effect on NP size (p < 0.05). The PHBHHx NPs were stable for at least 7 days with an average polydispersity index of 0.18, a zeta potential of -10 to -40 mV, and an encapsulation efficiency of 63.5 ± 2%. These data were utilized to produce a prediction graph whereby particles could be produced with sizes ranging from 90 to 205 nm with a low mean curve prediction error of 1.96% for Haperzine-A-loaded NPs. Furthermore, a range of drug encapsulates NPs were produced and showed a sustained release of the encapsulated drug. This study demonstrates the ability to control the size of drug-loaded particles by manipulation of the production variables, which will allow targeted and controlled drug release to fit a variety of applications.