Combined Delivery of Temozolomide and siPLK1 Using Targeted Nanoparticles to Enhance Temozolomide Sensitivity in Glioma.
ABSTRACT: Introduction:Temozolomide (TMZ) is the first-line chemotherapeutic option to treat glioma; however, its efficacy and clinical application are limited by its drug resistance properties. Polo-like kinase 1 (PLK1)-targeted therapy causes G2/M arrest and increases the sensitivity of glioma to TMZ. Therefore, to limit TMZ resistance in glioma, an angiopep-2 (A2)-modified polymeric micelle (A2PEC) embedded with TMZ and a small interfering RNA (siRNA) targeting PLK1 (siPLK1) was developed (TMZ-A2PEC/siPLK). Materials and Methods:TMZ was encapsulated by A2-PEG-PEI-PCL (A2PEC) through the hydrophobic interaction, and siPLK1 was complexed with the TMZ-A2PEC through electrostatic interaction. Th?en, an angiopep-2 (A2) modified polymeric micelle (A2PEC) embedding TMZ and siRNA targeting polo-like kinase 1 (siPLK1) was developed (TMZ-A2PEC/siPLK). Results:In vitro experiments indicated that TMZ-A2PEC/siPLK effectively enhanced the cellular uptake of TMZ and siPLK1 and resulted in significant cell apoptosis and cytotoxicity of glioma cells. In vivo experiments showed that glioma growth was inhibited, and the survival time of the animals was prolonged remarkably after TMZ-A2PEC/siPLK1 was injected via their tail vein. Discussion:The results demonstrate that the combination of TMZ and siPLK1 in A2PEC could enhance the efficacy of TMZ in treating glioma.
Project description:The addition of temozolomide (TMZ) to radiotherapy (RT) improves survival of patients with glioblastoma (GBM). However, TMZ?+?RT causes excess toxicity in patients. In this study, we prepared angiopep-2 (A2) modified lipid-poly (hypoxic radiosensitized polyprodrug) nanoparticles for TMZ delivery (A2-P(MIs)25/TMZ) to achieve synergistic effects against glioma. This A2-P(MIs)25/TMZ display highly promising advantages: (1) a hydrophobic P-(MIs)25 core where poorly water-soluble TMZ can be encapsulated; (2) nitro groups of the hydrophobic P-(MIs)25 core that are converted into hydrophilic amino groups (P(NH<sub>2</sub>s)25) under low oxygen conditions to mimic the oxygen-increased sensitization to RT; (3) a lipid monolayer at the interface of the core and the shell to modify the A2 (a specific ligand for low-density lipoprotein receptor-related protein-1 (LRP-1), which are expressed in the blood-brain barrier (BBB) and human glioma cells), thereby enhancing the drug encapsulation efficiency in glioma. These nanoparticles appear as a promising and robust nanoplatforms for TMZ and hypoxic cell radiosensitization delivery.
Project description:Mismatch repair (MMR) deficiency through MSH6 inactivation has been identified in up to 30% of recurrent high-grade gliomas, and represents a key molecular mechanism underlying the acquired resistance to the alkylating agent temozolomide (TMZ). To develop a therapeutic strategy that could be effective in these TMZ-refractory gliomas, we first screened 13 DNA damage response modulators for their ability to suppress viability of MSH6-inactivated, TMZ-resistant glioma cells. We identified a PLK1 selective inhibitor, Volasertib, as the most potent in inhibiting proliferation of glioblastoma cells. PLK1 inhibition induced mitotic catastrophe, G2-M cell-cycle arrest, and DNA damage, leading to caspase-mediated apoptosis in glioblastoma cells. Importantly, therapeutic effects of PLK1 inhibitors were not influenced by MSH6 knockdown, indicating that their action is independent of MMR status of the cells. Systemic treatment with Volasertib potently inhibited tumor growth in an MMR-deficient, TMZ-resistant glioblastoma xenograft model. Further in vitro testing in established and patient-derived cell line panels revealed an association of PLK1 inhibitor efficacy with cellular Myc expression status. We found that cells with deregulated Myc are vulnerable to PLK1 inhibition, as Myc overexpression sensitizes, whereas its silencing desensitizes, glioblastoma cells to PLK1 inhibitors. This discovery is clinically relevant as glioma progression post-TMZ treatment is frequently accompanied by MYC genomic amplification and/or pathway activation. In conclusion, PLK inhibitor represents a novel therapeutic option for recurrent gliomas, including those TMZ-resistant from MMR deficiency. Genomic MYC alteration may serve as a biomarker for PLK inhibitor sensitivity, as Myc-driven tumors demonstrated pronounced responses.
Project description:Radiation sensitizers that can selectively act on cancer cells hold great promise to patients who receive radiation therapy. We developed a novel targeted therapy and radiation sensitizer for non-small cell lung cancer (NSCLC) based on cetuximab conjugated nanoparticle that targets epidermal growth factor receptor (EGFR) and delivers small interfering RNA (siRNA) against polo-like kinase 1 (PLK1). EGFR is overexpressed in 50% of lung cancer patients and a mediator of DNA repair, while PLK1 is a key mitotic regulator whose inhibition enhances radiation sensitivity. The nanoparticle construct (C-siPLK1-NP) effectively targets EGFR + NSCLC cells and reduces PLK1 expression, leading to G2/M arrest and cell death. Furthermore, we show a synergistic combination between C-siPLK1-NP and radiation, which was confirmed in vivo in A549 flank tumors. We also demonstrate the translational potential of C-siPLK1-NP as a systemic therapeutic in an orthotopic lung tumor model, where administration of C-siPLK1-NP reduced tumor growth and led to prolonged survival. Our findings demonstrate that C-siPLK1-NP is effective as a targeted therapy and as a potent radiation sensitizer for NSCLC. Potential application to other EGFR + cancer types such as colorectal and breast cancer is also demonstrated.
Project description:Glioblastomas (GBM) are associated with high rates of relapse. These brain tumors are often resistant to chemotherapies like temozolomide (TMZ) and there are very few treatment options available to patients. We recently reported that polo-like kinase-1 (PLK1) is associated with the proliferative subtype of GBM; which has the worst prognosis. In this study, we addressed the potential of repurposing disulfiram (DSF), a drug widely used to control alcoholism for the past six decades. DSF has good safety profiles and penetrates the blood-brain barrier. Here we report that DSF inhibited the growth of TMZ resistant GBM cells, (IC90=100 nM), but did not affect normal human astrocytes. At similar DSF concentrations, self-renewal was blocked by ~100% using neurosphere growth assays. Likewise the drug completely inhibited the self-renewal of the BT74 and GBM4 primary cell lines. Additionally, DSF suppressed growth and self-renewal of primary cells from two GBM tumors.These cells were resistant to TMZ, had unmethylated MGMT, and expressed high levels of PLK1. Consistent with its role in suppressing GBM growth, DSF inhibited the expression of PLK1 in GBM cells. Likewise, PLK1 inhibition with siRNA, or small molecules (BI-2536 or BI-6727) blocked growth of TMZ resistant cells. Our studies suggest that DSF has the potential to be repurposed for treatment of refractory GBM.
Project description:Here, we report the hypoxia-responsive ionizable liposomes to deliver small interference RNA (siRNA) anticancer drugs, which can selectively enhance cellular uptake of the siRNA under hypoxic and low-pH conditions to cure glioma. For this purpose, malate dehydrogenase lipid molecules were synthesized, which contain nitroimidazole groups that impart hypoxia sensitivity and specificity as hydrophobic tails, and tertiary amines as hydrophilic head groups. These malate dehydrogenase molecules, together with DSPE-PEG2000 and cholesterol, were self-assembled into O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 16-bis(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl) di(hexadecanedioate) liposomes (MLP) to encapsulate siRNA through electrostatic interaction. Our study showed that the MLP could deliver polo-like kinase 1 siRNA (siPLK1) into glioma cells and effectively enhance the cellular uptake of MLP/siPLK1 because of increased positive charges induced by hypoxia and low pH. Moreover, MLP/siPLK1 was shown to be very effective in inhibiting the growth of glioma cells both in vitro and in vivo. Therefore, the MLP is a promising siRNA delivery system for tumor therapy.
Project description:Despite multimodal therapy with radiation and the DNA alkylating agent temozolomide (TMZ), malignant gliomas remain incurable. Up to 90% of grades II-III gliomas contain a single mutant isocitrate dehydrogenase 1 (IDH1) allele. IDH1 mutant-mediated transformation is associated with TMZ resistance; however, there is no clinically available means of sensitizing IDH1 mutant tumors to TMZ. In this study we sought to identify a targetable mechanism of TMZ resistance in IDH1 mutant tumors to enhance TMZ efficacy. IDH1 mutant astrocytes rapidly bypassed the G2 checkpoint with unrepaired DNA damage following TMZ treatment. Checkpoint adaptation was accompanied by PLK1 activation and IDH1 mutant astrocytes were more sensitive to treatment with BI2536 and TMZ in combination (<20% clonogenic survival) than either TMZ (~60%) or BI2536 (~75%) as single agents. In vivo, TMZ or BI2536 alone had little effect on tumor size. Combination treatment caused marked tumor shrinkage in all mice and complete tumor regression in 5 of 8 mice. Mutant IDH1 promotes checkpoint adaptation which can be exploited therapeutically with the combination of TMZ and a PLK1 inhibitor, indicating PLK1 inhibitors may be clinically valuable in the treatment of IDH1 mutant gliomas.
Project description:Uncontrollable cell proliferation and irreversible neurological damage make glioma one of the most deadly diseases in clinic. Besides the multiple biological barriers, glioma stem cells (GSCs) that are responsible for the maintenance and recurrence of tumor tissues also hinder the therapeutic efficacy of chemotherapy. Therefore, all-stage precisional glioma targeted therapy regimens that could efficiently deliver drugs to glioma cells and GSCs after overcoming multiple barriers have received increasing scrutiny. Methods: A polymeric micelle-based drug delivery system was developed by modifying a "Y-shaped" well-designed ligand of both GRP78 protein and quorum sensing receptor to achieve all-stage precisional glioma targeting, then we evaluated the targeting ability and barrier penetration ability both in vitro and in vivo. In order to achieve all-stage precisional therapy, we need kill both GSCs and glioma related cells. Parthenolide (PTL) has been investigated for its selective toxicity to glioma stem cells while Paclitaxel (PTX) and Temozolomide (TMZ) are widely used in experimental and clinical therapy of glioma respectively. So the in vivo anti-glioma effect of combination therapy was evaluated by Kaplan-Meier survival analysis and immunohistochemical (IHC) examination of tumor tissues. Results: The "Y-shaped" well-designed peptide, termed DWVAP, exhibited excellent glioma (and GSCs) homing and barrier penetration ability. When modified on micelle surface, DWVAP peptide significantly enhanced accumulation of micelles in brain and glioma. In addition, DWVAP micelles showed no immunogenicity and cytotoxicity, which could guarantee their safety when used in vivo. Treatment of glioma-bearing mice with PTL loaded DWVAP modified PEG-PLA micelles plus PTX loaded DWVAP modified PEG-PLA micelles or PTL loaded DWVAP modified PEG-PLA micelles plus TMZ showed improved anti-tumor efficacy in comparison to PTL and PTX loaded unmodified micelles or PTL loaded unmodified micelles plus TMZ. Conclusion: Combination of all-stage targeting strategy and concomitant use of chemotherapeutics and stem cell inhibitors could achieve precise targeted therapy for glioma.
Project description:<h4>Introduction</h4>Specific polo-like kinase (PLK1) silencing with small interface RNA (siRNA) may be an effective approach for PLK1-overexpressed lung cancer. However, low siRNA concentration into cytoplasm of tumor tissue severely limits its application.<h4>Materials and methods</h4>In this study, a novel triblock copolymer methoxy poly(ethylene glycol)-poly(histidine)-poly(sulfadimethoxine) (mPEG-PHis-PSD, shorten as PHD) was synthesized and used to construct novel nonviral gene vector with cationic liposomes.<h4>Results</h4>The resulting hybrid nanoparticles (PHD/LR) loaded with siPLK1 possessed excellent physiochemical properties. In vitro study indicated that PHD/LR could be efficiently internalized into human lung adenocarcinoma A549 cells and downregulated PLK1 protein expression to induce cell apoptosis, which was attributed to pH-induced instantaneous dissociation, efficient endo/lysosomal escape arose from PHD copolymer. Furthermore, in vivo antitumor activity demonstrated that PHD/LR could efficiently accumulated into tumor tissue and silenced PLK1 expression to possess antitumor activity.<h4>Conclusion</h4>Taken all these together, PHD/LR was expected to be a suitable carrier for specific delivering siRNA for lung cancer therapy.
Project description:Temozolomide (TMZ), used to treat glioblastoma and malignant glioma, induces autophagy, apoptosis and senescence in cancer cells. We investigated fibrin glue (FG) as a drug delivery system for the local administration of high-concentration TMZ aimed at preventing glioma recurrence. Our high-power liquid chromatography studies indicated that FG containing TMZ (TMZ-FG) manifested a sustained drug release potential. We prepared a subcutaneous tumor model by injecting groups of mice with three malignant glioma cell lines and examined the antitumor effect of TMZ-FG. We estimated the tumor volume and performed immunostaining and immunoblotting using antibodies to Ki-67, cleaved caspase 3, LC3 and p16. When FG sheets containing TMZ (TMZ-FGS) were inserted beneath the tumors, their growth was significantly suppressed. In mice treated with peroral TMZ plus TMZ-FGS the tumors tended to be smaller than in mice whose tumors were treated with TMZ-FGS or peroral TMZ alone. The TMZ-FGS induced autophagy, apoptosis and senescence in subcutaneous glioma tumor cells. To assess the safety of TMZ-FG for normal brain, we placed it directly on the brain of living mice and stained tissue sections obtained in the acute and chronic phase immunohistochemically. In both phases, TMZ-FG failed to severely damage normal brain tissue. TMZ-FG may represent a safe new drug delivery system with sustained drug release potential to treat malignant glioma.
Project description:The use of nanoparticle-stabilized nanocapsules (NPSCs) for the direct cytosolic delivery of siRNA is reported. In this approach, siRNA is complexed with cationic arginine-functionalized gold nanoparticles by electrostatic interactions, with the resulting ensemble self-assembled onto the surface of fatty acid nanodroplets to form a NPSC/siRNA nanocomplex. The complex rapidly delivers siRNA into the cytosol through membrane fusion, a mechanism supported by cellular uptake studies. Using destabilized green fluorescent protein (deGFP) as a target, 90% knockdown was observed in HEK293 cells. Moreover, the delivery of siRNA targeting polo-like kinase?1 (siPLK1) efficiently silenced PLK1 expression in cancer cells with concomitant cytotoxicity.