Weiwei Wang

Design of Multifunctional Micelle for Tumor‐Targeted Intracellular Drug Release and Fluorescent Imaging

Figure 1. Illustrative preparation of PTX and QD-loaded micelle and pH-tunable drug release. Over the past two decades, nanoscale polymeric micelles have demonstrated great potential in delivering anticancer drugs.[1–3] However, in spite of the successes both in vitro and in vivo,[1a,b,4] application of polymeric micelles for drug delivery has encountered two intractable problems.[1c] At first, premature drug leakage exists in sample storage or during blood circulation. Secondly, accumulation of micelles in tumor tissues and cells is poor due to their easy clearance by reticuloendothelial system and lack of active targeting to tumor cells, which leads to the decreased therapeutic effect and causes undesired side effects of the transported anticancer drugs. So far, only few reports are available on regulating drug release property of polymeric micelles in terms of inhibiting premature drug leakage.[1d] On the other hand, accumulation of drug-loaded micelles in tumor cells can be enhanced by the active targeting strategy utilizing ligands to generate specific interaction between micelles and molecular bio-markers on tumor cell membranes.[1a,b,5] For instance, folic acid (FA) was employed to potentiate the gene and siRNA delivery to folate receptor (FR)-enriched tumors, which resulted in improved therapeutic effect both in vitro and in vivo.[6] Moreover, multifunctional nanocarriers combining imaging function with drug delivery are emerging as the next generation of nanomedicines to improve the outcome of drug therapy.[1b] To date, reports on polymeric micelles with a combined feature of

The investigation of polymer-siRNA nanoparticle for gene therapy of gastric cancer in vitro

Small interfering RNA (siRNA) molecules have significant therapeutic promise for the genetic treatment of cancer. To overcome instability and low transfection efficiency, polyethylene glycol-polyethyleneimine (PEG-PEI) was synthesized and investigated as a non-viral carrier of siRNA targeting CD44v6 in gastric carcinoma cells. The size, surface charge using zeta potential, and morphology via scanning electron microscopy (SEM) of PEG-PEI/siRNA nanoparticles was characterized, and their cytotoxicity, transfection efficiency, and interaction with SGC7901 human gastric carcinoma cells was evaluated. The transfection efficiency of PEG-PEI/siRNA nanocomplexes was dependant on the charge ratio between amino groups of PEG-PEI and phosphate groups of siRNA (N/P) values, which reflected the molar ratio of PEG-PEI to siRNA during complex formation. The transfection efficiency of PEG-PEI/siRNA at N/P 15 was 72.53% ± 2.38%, which was higher than that observed using Lipofectamine 2000 and PEI as delivery carriers. Cytotoxicity of PEG-PEI was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and was obviously lower than that of PEI. Moreover, when N/P was below 15, PEG-PEI/siRNA was less toxic than Lipofectamine 2000/siRNA. RT-PCR (real time polymerase chain reaction) and Western blot analyses of CD44v6 expression demonstrated the gene silencing effect of PEG-PEI/siRNA at N/P 15. These data indicate that PEG-PEI may be a promising non-viral carrier for altering gene expression in the treatment of gastric cancer with many advantages, such as relatively high gene transfection efficiency and low cytotoxicity.

Delivery of cationic polymer-siRNA nanoparticles for gene therapies in neural regeneration.

The therapeutic applications of neural stem cells (NSCs) have potential to promote recovery in many obstinate diseases in central nervous system. Regulation of certain gene expressions using siRNA may have significant influence on the fate of NSC. To achieve the optimum gene silencing effect of siRNA, non-viral vector polyethylene glycol-polyethyleneimine (PEG-PEI) was investigated in the delivery of siRNA to NSCs. The characteristics of PEG-PEI/siRNA polyplexes were detected by scanning electron microscopy (SEM). The effects of nanoparticles on cell viability were measured via CCK-8 assay. In addition, the transfection efficiency was evaluated by fluorescence microscope and flow cytometry, and real-time PCR and Western Blot were employed to detect the gene inhibition effect of siRNA delivered by PEG-PEI. The SEM micrographs showed that PEG-PEI could condense siRNA to form diffuse and spherical nanoparticles. The cytotoxicity of PEG-PEI/siRNA nanocomplexes (N/P=15) was significantly lower when compared with that of Lipofectamine 2000/siRNA (P<0.05). Moreover, the highest transfection efficiency of PEG-PEI/siRNA nanoparticles was obtained at an N/P ratio of 15, which was better than that achieved in the transfection using Lipofectamine 2000 (P<0.05). Finally, the gene knockdown effect of PEG-PEI/siRNA nanoparticles was verified at the levels of mRNA and protein. These results suggest that PEG-PEI may potentially be used as a siRNA delivery vector for neural regeneration therapy.

Simultaneous diagnosis and gene therapy of immuno-rejection in rat allogeneic heart transplantation model using a T-cell-targeted theranostic nanosystem.

As the final life-saving treatment option for patients with terminal organ failure, organ transplantation is far from an ideal solution. The concomitant allograft rejection, which is hardly detectable especially in the early acute rejection (AR) period characterized by an intense cellular and humoral attack on donor tissue, greatly affects the graft survival and results in rapid graft loss. Based on a magnetic resonance imaging (MRI)-visible and T-cell-targeted multifunctional polymeric nanocarrier developed in our lab, effective co-delivery of pDNA and superparamagnetic iron oxide nanoparticles into primary T cells expressing CD3 molecular biomarker was confirmed in vitro. In the heart transplanted rat model, this multifunctional nanocarrier showed not only a high efficiency in detecting post-transplantation acute rejection but also a great ability to mediate gene transfection in T cells. Upon intravenous injection of this MRI-visible polyplex of nanocarrier and pDNA, T-cell gathering was detected at the endocardium of the transplanted heart as linear strongly hypointense areas on the MRI T(2)*-weighted images on the third day after cardiac transplantation. Systematic histological and molecular biology studies demonstrated that the immune response in heart transplanted rats was significantly suppressed upon gene therapy using the polyplex bearing the DGKα gene. More excitingly, the therapeutic efficacy was readily monitored by noninvasive MRI during the treatment process. Our results revealed the great potential of the multifunctional nanocarrier as a highly effective imaging tool for real-time and noninvasive monitoring and a powerful nanomedicine platform for gene therapy of AR with high efficiency.

Plasmid-encapsulated polyethylene glycol-grafted polyethylenimine nanoparticles for gene delivery into rat mesenchymal stem cells

Background: Mesenchymal stem cell transplantation is a promising method in regenerative medicine. Gene-modified mesenchymal stem cells possess superior characteristics of specific tissue differentiation, resistance to apoptosis, and directional migration. Viral vectors have the disadvantages of potential immunogenicity, carcinogenicity, and complicated synthetic procedures. Polyethylene glycol-grafted polyethylenimine (PEG-PEI) holds promise in gene delivery because of easy preparation and potentially targeting modification. Methods: A PEG8k-PEI25k graft copolymer was synthesized. Agarose gel retardation assay and dynamic light scattering were used to determine the properties of the nanoparticles. MTT reduction, wound and healing, and differentiation assays were used to test the cytobiological characteristics of rat mesenchymal stem cells, fluorescence microscopy and flow cytometry were used to determine transfection efficiency, and atomic force microscopy was used to evaluate the interaction between PEG-PEI/plasmid nanoparticles and mesenchymal stem cells. Results: After incubation with the copolymer, the bionomics of mesenchymal stem cells showed no significant change. The mesenchymal stem cells still maintained high viability, resettled the wound area, and differentiated into adipocytes and osteoblasts. The PEG-PEI completely packed plasmid and condensed plasmid into stable nanoparticles of 100–150 nm diameter. After optimizing the N/P ratio, the PEG-PEI/plasmid microcapsules delivered plasmid into mesenchymal stem cells and obtained an optimum transfection efficiency of 15%–21%, which was higher than for cationic liposomes. Conclusion: These data indicate that PEG-PEI is a valid gene delivery agent and has better transfection efficiency than cationic liposomes in mesenchymal stem cells.

Development of an MRI-visible nonviral vector for siRNA delivery targeting gastric cancer.

An antibody-directed nonviral vector, polyethylene glycol-grafted polyethylenimine functionalized with superparamagnetic iron oxide nanoparticles and a gastric cancer-associated CD44v6 single-chain variable fragment (scFvCD44v6,-PEG-g-PEI-SPION), was constructed as a gastric cancer-targeting and magnetic resonance imaging (MRI)-visible nanocarrier for small interfering RNA (siRNA) delivery. Biophysical characterization of PEG-g-PEI-SPION and scFvCD44v6-PEG-g-PEI-SPION was carried out, including siRNA condensation capacity, cell viability, and transfection efficiency. Both the targeting and nontargeting nanocarriers were effective for transferring siRNA in vitro. The cellular uptake and distribution of nanoparticles complexed with siRNA was analyzed by fluorescence imaging and immunofluorescent staining. Moreover, the gastric cancer-targeting effect was verified in vivo by MRI and histology analysis. These results indicate that scFvCD44v6-PEG-g-PEI-SPION is a promising nonviral vector for gastric cancer gene therapy and diagnosis.

Characterization of polyethylene glycol-grafted polyethylenimine and superparamagnetic iron oxide nanoparticles (PEG-g-PEI-SPION) as an MRI-visible vector for siRNA delivery in gastric cancer in vitro and in vivo.

BackgroundGene therapy is a promising therapeutic method but is severely hampered due to its lack of an ideal delivery system. Therefore, in this study, a nonviral and magnetic resonance imaging (MRI) visible vector, polyethylene glycol-grafted polyethylenimine and superparamagnetic iron oxide nanoparticles (PEG-g-PEI-SPION) was used as a nanocarrier for small interfering RNA (siRNA) delivery in gastric cancer.MethodsBiophysical characterization of PEG-g-PEI-SPION was systematically analyzed, including size, zeta potential, siRNA condensation capacity, cell viability, transfection efficiency, cellular uptake, and MRI-visible function in vivo. Besides, CD44 variant isoform 6 (CD44v6), a protein marker for metastatic behavior in gastric cancer, and was chose as the target gene to further analyze the siRNA delivery function of PEG-g-PEI-SPION.ResultsUnder comprehensive analysis, the appropriate N/P ratio of PEG-g-PEI-SPION/siRNA was 10,. and siRNA targeting at human CD44v6 (siCD44v6) transferred by PEG-g-PEI-SPION was effective at downregulating the CD44v6 expression of gastric carcinoma cell line SGC-7901 in vitro. Moreover, knockdown of CD44v6 impaired migrating and invasive abilities of SGC-7901 cells. Furthermore, PEG-g-PEI-SPION was a highly efficient contrast agent for MRI scan in vivo.ConclusionPEG-g-PEI-SPION was a promising nonviral vector with molecular image tracing capacity for cancer gene therapy. And CD44v6 was a potential target gene for the prevention and detection of metastatic behavior in gastric cancer.

PinX1-siRNA/mPEG-PEI-SPION combined with doxorubicin enhances the inhibition of glioma growth

Resistance to chemotherapy and the side effects of anticancer drugs are the major obstacles for glioma treatment. The aim of the present study was to develop a novel approach for the treatment of gliomas that improved the therapeutic effect; the anticancer drug, doxorubicin (DOX), was combined with short interfering (si)RNA and monomethoxy polyethylene glycol polyethylenimine superparamagnetic iron oxide nanoparticle (mPEG-PEI-SPION), a magnetic resonance imaging (MRI)-visible nanoparticle. Specific siRNA molecules, delivered by mPEG-PEI-SPION, were employed to knockdown the PIN2-interacting protein 1 (PinX1) gene in C6 glioma cells. PinX1 is a nucleolar protein associated with telomere and telomerase. C6 cells were treated with DOX and/or PinX1-siRNA. The results of the transfection experiments revealed that siRNA/mPEG-PEI-SPION was transfected into C6 cells with high efficiency. PinX1-siRNA was unable to inhibit C6 cells, while in the PinX1-siRNA + DOX group, the same dose of DOX caused an increased loss of cell viability. Therefore, mPEG-PEI-SPION was shown to be viable for siRNA delivery into C6 cells and coadministration of DOX with PinX1-siRNA may be a potential therapeutic method for inhibiting gliomas.