Degradable gene delivery systems based on Pluronics-modified low-molecular-weight polyethylenimine: preparation, characterization, intracellular trafficking, and cellular distribution

Wei Fan1,2,*, Xin Wu1,*, Baoyue Ding3,*, Jing Gao4, Zhen Cai1, Wei Zhang1, Dongfeng Yin1, Xiang Wang1, Quangang Zhu1, Jiyong Liu1, Xueying Ding4, Shen Gao1 1Department of Pharmaceutics, Changhai Hospital, Second Military Medical University, Shanghai, 2Department of Pharmaceutics, The 425th Hospital of PLA, Sanya, 3Department of Pharmaceutics, Medical College of Jiaxing University, Jiaxing, 4Department of Pharmaceutics, School of Pharmacy, Second Military Medical University, Shanghai, People's Republic of China*These authors contributed equally to this workBackground: Cationic copolymers consisting of polycations linked to nonionic amphiphilic block polymers have been evaluated as nonviral gene delivery systems, and a large number of different polymers and copolymers of linear, branched, and dendrimeric architectures have been tested in terms of their suitability and efficacy for in vitro and in vivo transfection. However, the discovery of new potent materials still largely relies on empiric approaches rather than a rational design. The authors investigated the relationship between the polymers' structures and their biological performance, including DNA compaction, toxicity, transfection efficiency, and the effect of cellular uptake.Methods: This article reports the synthesis and characterization of a series of cationic copolymers obtained by grafting polyethyleneimine with nonionic amphiphilic surfactant polyether-Pluronic® consisting of hydrophilic ethylene oxide and hydrophobic propylene oxide blocks. Transgene expression, cytotoxicity, localization of plasmids, and cellular uptake of these copolymers were evaluated following in vitro transfection of HeLa cell lines with various individual components of the copolymers.Results: Pluronics can exhibit biological activity including effects on enhancing DNA cellular uptake, nuclear translocation, and gene expression. The Pluronics with a higher hydrophilic-lipophilic balance value lead to homogeneous distribution in the cytoplasm; those with a lower hydrophilic-lipophilic balance value prefer to localize in the nucleus.Conclusion: This Pluronic-polyethyleneimine system may be worth exploring as components in the cationic copolymers as the DNA or small interfering RNA/microRNA delivery system in the near future.Keywords: Pluronics, gene transfer, nonviral vectors, transfection efficiency, cellular uptak

A tumor microenvironment-responsive micelle co-delivered radiosensitizer Dbait and doxorubicin for the collaborative chemo-radiotherapy of glioblastoma

Glioblastoma is rather recalcitrant to existing therapies and effective interventions are needed. Here we report a novel microenvironment-responsive micellar system (ch-K5(s-s)R8-An) for the co-delivery of the radiosensitizer Dbait and the chemotherapeutic doxorubicin (DOX) to glioblastoma. Accordingly, the ch-K5(s-s)R8-An/(Dbait-DOX) micelles plus radiotherapy (RT) treatment resulted in a high degree of apoptosis and DNA damage, which significantly reduced cell viability and proliferation capacity of U251 cells to 64.0% and 16.3%, respectively. The angiopep-2-modified micelles exhibited substantial accumulation in brain-localized U251 glioblastoma xenografts in mice compared to angiopep-2-lacking micelles. The ch-K5(s-s)R8-An/(Dbait-DOX) + RT treatment group exhibited the smallest tumor size and most profound tumor tissue injury in orthotopic U251 tumors, leading to an increase in median survival time of U251 tumor-bearing mice from 26 days to 56 days. The ch-K5(s-s)R8-An/(Dbait-DOX) micelles can be targeted to brain-localized U251 tumor xenografts and sensitize the tumor to chemotherapy and radiotherapy, thereby overcoming the inherent therapeutic challenges associated with malignant glioblastoma

Experimental Conditions That Influence the Utility of 2′7′-Dichlorodihydrofluorescein Diacetate (DCFH2-DA) as a Fluorogenic Biosensor for Mitochondrial Redox Status

Oxidative stress has been causally linked to various diseases. Electron transport chain (ETC) inhibitors such as rotenone and antimycin A are frequently used in model systems to study oxidative stress. Oxidative stress that is provoked by ETC inhibitors can be visualized using the fluorogenic probe 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH2-DA). Non-fluorescent DCFH2-DA crosses the plasma membrane, is deacetylated to 2′,7′-dichlorodihydrofluorescein (DCFH2) by esterases, and is oxidized to its fluorescent form 2′,7′-dichlorofluorescein (DCF) by intracellular ROS. DCF fluorescence can, therefore, be used as a semi-quantitative measure of general oxidative stress. However, the use of DCFH2-DA is complicated by various protocol-related factors that mediate DCFH2-to-DCF conversion independently of the degree of oxidative stress. This study therefore analyzed the influence of ancillary factors on DCF formation in the context of ETC inhibitors. It was found that ETC inhibitors trigger DCF formation in cell-free experiments when they are co-dissolved with DCFH2-DA. Moreover, the extent of DCF formation depended on the type of culture medium that was used, the pH of the assay system, the presence of fetal calf serum, and the final DCFH2-DA solvent concentration. Conclusively, experiments with DCFH2-DA should not discount the influence of protocol-related factors such as medium and mitochondrial inhibitors (and possibly other compounds) on the DCFH2-DA-DCF reaction and proper controls should always be built into the assay protocol

Mir-382 Promotes Differentiation of Rat Liver Progenitor Cell WB-F344 by Targeting Ezh2

Background/Aims: Liver progenitor cells (LPCs) were considered as a promising hepatocyte source of cell therapy for liver disease due to their self-renewal and differentiation capacities, while little is known about the mechanism of LPC differentiate into hepatocytes. This study aims to explore the effect of miR-382, a member of Dlk1-Dio3 microRNA cluster, during hepatic differentiation from LPCs. Methods: In this study, we used rat liver progenitor cell WB-F344 as LPC cell model and HGF as inducer to simulate the process of LPCs hepatic differentiation, then microRNAs were quantified by qPCR. Next, WB-F344 cell was transfected with miR-382 mimics, then hepatocyte cell trait was characterized by multiple experiments, including that periodic acid schiff staining and cellular uptake and excretion of indocyanine green to evaluate the hepatocellular function, qPCR and Western Blotting analysis to detect the hepatocyte-specific markers (ALB, Ttr, Apo E and AFP) and transmission electron microscopy to observe the hepatocellular morphology. Moreover, Luciferase reporter assay was used to determine whether Ezh2 is the direct target of miR-382. Results: We found that miR-382 increased gradually and was inversely correlated with the potential target, Ezh2, during WB-F344 hepatic differentiation. In addition, functional studies indicated that miR-382 increased the level of hepatocyte-specific genes. Conclusions: This study demonstrates that miR-382 may be a novel regulator of LPCs differentiation by targeting Ezh2

Metallated phthalocyanines and their hydrophilic derivatives for multi-targeted oncological photodynamic therapy

Background and aim: A photosensitizer (PS) delivery and comprehensive tumor targeting platform was developed that is centered on the photosensitization of key pharmacological targets in solid tumors (cancer cells, tumor vascular endothelium, and cellular and non-cellular components of the tumor microenvironment) before photodynamic therapy (PDT). Interstitially targeted liposomes (ITLs) encapsulating zinc phthalocyanine (ZnPC) and aluminum phthalocyanine (AlPC) were formulated for passive targeting of the tumor microenvironment. In previous work it was established that the PEGylated ITLs were taken up by cultured cholangiocarcinoma cells. The aim of this study was to verify previous results in cancer cells and to determine whether the ITLs can also be used to photosensitize cells in the tumor microenvironment and vasculature. Following positive results, rudimentary in vitro and in vivo experiments were performed with ZnPC-ITLs and AlPC-ITLs as well as their water-soluble tetrasulfonated derivatives (ZnPCS4 and AlPCS4) to assemble a research dossier and bring this platform closer to clinical transition. Methods: Flow cytometry and confocal microscopy were employed to determine ITL uptake and PS distribution in cholangiocarcinoma (SK-ChA-1) cells, endothelial cells (HUVECs), fibroblasts (NIH-3T3), and macrophages (RAW 264.7). Uptake of ITLs by endothelial cells was verified under flow conditions in a flow chamber. Dark toxicity and PDT efficacy were determined by cell viability assays, while the mode of cell death and cell cycle arrest were assayed by flow cytometry. In vivo systemic toxicity was assessed in zebrafish and chicken embryos, whereas skin phototoxicity was determined in BALB/c nude mice. A PDT efficacy pilot was conducted in BALB/c nude mice bearing human triple-negative breast cancer (MDA-MB-231) xenografts. Results: The key findings were that (1) photodynamically active PSs (i.e., all except ZnPCS4) were able to effectively photosensitize cancer cells and non-cancerous cells; (2) following PDT, photodynamically active PSs were highly toxic-to-potent as per anti-cancer compound classification; (3) the photodynamically active PSs did not elicit notable systemic toxicity in zebrafish and chicken embryos; (4) ITL-delivered ZnPC and ZnPCS4 were associated with skin phototoxicity, while the aluminum-containing PSs did not exert detectable skin phototoxicity; and (5) ITL-delivered ZnPC and AlPC were equally effective in their tumor-killing capacity in human tumor breast cancer xenografts and superior to other non-phthalocyanine PSs when appraised on a per mole administered dose basis. Conclusions: AlPC(S4) are the safest and most effective PSs to integrate into the comprehensive tumor targeting and PS delivery platform. Pending further in vivo validation, these third-generation PSs may be used for multi-compartmental tumor photosensitization

Metallated phthalocyanines and their hydrophilic derivatives for multi-targeted oncological photodynamic therapy

Background and aim: A photosensitizer (PS) delivery and comprehensive tumor targeting platform was developed that is centered on the photosensitization of key pharmacological targets in solid tumors (cancer cells, tumor vascular endothelium, and cellular and non-cellular components of the tumor microenvironment) before photodynamic therapy (PDT). Interstitially targeted liposomes (ITLs) encapsulating zinc phthalocyanine (ZnPC) and aluminum phthalocyanine (AlPC) were formulated for passive targeting of the tumor microenvironment. In previous work it was established that the PEGylated ITLs were taken up by cultured cholangiocarcinoma cells. The aim of this study was to verify previous results in cancer cells and to determine whether the ITLs can also be used to photosensitize cells in the tumor microenvironment and vasculature. Following positive results, rudimentary in vitro and in vivo experiments were performed with ZnPC-ITLs and AlPC-ITLs as well as their water-soluble tetrasulfonated derivatives (ZnPCS4 and AlPCS4) to assemble a research dossier and bring this platform closer to clinical transition. Methods: Flow cytometry and confocal microscopy were employed to determine ITL uptake and PS distribution in cholangiocarcinoma (SK-ChA-1) cells, endothelial cells (HUVECs), fibroblasts (NIH-3T3), and macrophages (RAW 264.7). Uptake of ITLs by endothelial cells was verified under flow conditions in a flow chamber. Dark toxicity and PDT efficacy were determined by cell viability assays, while the mode of cell death and cell cycle arrest were assayed by flow cytometry. In vivo systemic toxicity was assessed in zebrafish and chicken embryos, whereas skin phototoxicity was determined in BALB/c nude mice. A PDT efficacy pilot was conducted in BALB/c nude mice bearing human triple-negative breast cancer (MDA-MB-231) xenografts. Results: The key findings were that (1) photodynamically active PSs (i.e., all except ZnPCS4) were able to effectively photosensitize cancer cells and non-cancerous cells; (2) following PDT, photodynamically active PSs were highly toxic-to-potent as per anti-cancer compound classification; (3) the photodynamically active PSs did not elicit notable systemic toxicity in zebrafish and chicken embryos; (4) ITL-delivered ZnPC and ZnPCS4 were associated with skin phototoxicity, while the aluminum-containing PSs did not exert detectable skin phototoxicity; and (5) ITL-delivered ZnPC and AlPC were equally effective in their tumor-killing capacity in human tumor breast cancer xenografts and superior to other non-phthalocyanine PSs when appraised on a per mole administered dose basis. Conclusions: AlPC(S4) are the safest and most effective PSs to integrate into the comprehensive tumor targeting and PS delivery platform. Pending further in vivo validation, these third-generation PSs may be used for multi-compartmental tumor photosensitization

MiR-376a and Histone Deacetylation 9 Form A Regulatory Circuitry in Hepatocellular Carcinoma

Background/Aims: Our previous study has demonstrated that down-regulation of miR-376a might contribute to the development of hepatocellular carcinoma (HCC), but the mechanism underlying this down-regulation remains obscure. Methods/Results: histone deacetylase (HDAC) inhibitor increased the level of miR-376a in L02 and Huh7 cells by up-regulating the acetylation level of histone 3 at the Maternally expressed 3 (Meg3) differentially methylated region (DMR). Interestingly, HDAC9, a histone deacetylase responsible for deacetylating lysine 18 of histone 3 (H3K18), was identified as the target of miR-376a. In addition, HDAC9 siRNA increased the expression of miR-376a by up-regulating the global histone H3K18 acetylation level, with Meg3 DMR included. Finally, miR-376a and HDAC9 were inversely correlated in HCC. Conclusion: HDAC9 plays an important role both as effects and targets of miR-376a