Functionalized Folate-Modified Graphene Oxide/PEI siRNA Nanocomplexes for Targeted Ovarian Cancer Gene Therapy.
ABSTRACT: Gene therapy is emerging as a valid method for the treatment of ovarian cancer, including small interfering RNA (siRNA). Although it is so powerful, few targeting efficient gene delivery systems seriously hindered the development of gene therapy. In this study, we synthesized a novel gene vector PEG-GO-PEI-FA by functionalized graphene oxide (GO), in which folic acid (FA) can specifically bind to the folate receptor (FR), which is overexpressed in ovarian cancer. Characterizations of the nanocomplexes were evaluated by dynamic light scattering (DLS), atomic force microscopy (AFM), and Fourier transform infrared spectroscopy (FTIR). The siRNA condensation ability and stability were assessed by agarose gel electrophoresis. Cellular uptake efficiency and lysosomal escape ability in ovarian cancer cells were investigated by confocal laser scanning microscopy. Furthermore, cellular biosafety of the system and inhibitory of the siRNA tolerability were evaluated by CCK-8 assay. The size of the PEG-GO-PEI-FA nanocomplexes was 216.1 ± 2.457?nm, exhibiting mild cytotoxicity in ovarian cancer cells. With high uptake efficiency, PEG-GO-PEI-FA can escape from the lysosome rapidly and release the gene. Moreover, PEG-GO-PEI-FA/siRNA can effectively inhibit the growth of ovarian cancer cells. By and large, the PEG-GO-PEI-FA/siRNA may offer a promising strategy for siRNA delivery in the treatment of FR-positive ovarian carcinoma or similar tumors.
Project description:This paper focuses on the ability of a folate-decorated triblock copolymer to deliver a targeted dose of siRNA in order to overcome chemotherapy resistance which can commonly cause complications in ovarian cancer patients. The micelleplexes that are formed upon electrostatic interaction with siRNA are used to deliver siRNA in a targeted manner to SKOV-3 ovarian cancer cells that overexpress folate receptor-? (FR?). The triblock copolymer consists of polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol) (PEI-g-PCL-b-PEG-Fol). In this work, polymers of different molecular weights of PEG, as well as different grafting degrees of the (g-PCL-b-PEG-Fol) chains to PEI, were analyzed to optimize targeted siRNA delivery. The polymers, their micelleplexes, and the in vitro performance of the latter were characterized by nuclear magnetic resonance, dynamic light scattering, transmission electron microscopy, flow cytrometry, western blot, confocal microscopy, and in luciferase assays. The different PEI-g-PCL-b-PEG-Fol conjugates showed suitable sizes below 260 nm, especially at N/P 5, which also allowed for full siRNA condensation. Furthermore, flow cytometry and Western blot analysis demonstrated that our best polymer was able to effectively deliver siRNA and that siRNA delivery resulted in efficient protein knockdown of toll-like receptor 4 (TLR4). Consequently, TLR4 knock down within SKOV-3 cells resensitized them toward paclitaxel (PTX) treatment, and apoptotic events increased. This study demonstrates that PEI-g-PCL-b-PEG-Fol conjugates are a reliable delivery system for siRNA and are able to mediate therapeutic protein knockdown within ovarian cancer cells. Additionally, this study provides further evidence to link TLR4 levels to chemotherapy resistance.
Project description:Systemic administration of chemotherapy for cancer often faces drug resistance, limiting its applications in cancer therapy. In this study, we developed a simple multifunctional nanocarrier based on polyethylenimine (PEI) to codeliver doxorubicin (DOX) and BCL2 small interfering RNA (siRNA) for overcoming multidrug resistance (MDR) and enhancing apoptosis in MCF-7/Adr cancer cells by combining chemotherapy and RNA interference (RNAi) therapy. The low-molecular-weight branch PEI was used to conjugate hydroxypropyl-?-cyclodextrin (HP-?-CD) and folic acid (FA), forming the codelivery nanocarrier (FA-HP-?-CD-PEI) to encapsulate DOX with the cavity HP-?-CD and bind siRNA with the positive charge of PEI for tumor-targeting codelivering drugs. The drug-loaded nanocomplexes (FA-HP-?-CD-PEI/DOX/siRNA) showed uniform size distribution, high cellular uptake, and significant gene suppression of BCL2, displaying the potential of overcoming MDR for enhancing the effect of anticancer drugs. Furthermore, the nanocomplexes achieved significant cell apoptosis through a mechanism of downregulating the antiapoptotic protein BCL2, resulted in improving therapeutic efficacy of the coadministered DOX by tumor targeting and RNA interference. Our study indicated that combined RNAi therapy and chemotherapy using our functional codelivery nanocarrier could overcome MDR and enhance apoptosis in MDR cancer cells for a potential application in treating MDR cancers.
Project description:Approaches for the synthesis of biomaterials to facilitate the delivery of "biologics" is a major area of research in cancer therapy. Here we designed and characterized a hyaluronic acid (HA) based self-assembling nanoparticles that can target CD44 receptors overexpressed on multidrug resistance (MDR) ovarian cancer. The nanoparticle system is composed of HA-poly(ethyleneimine)/HA-poly(ethylene glycol) (HA-PEI/HA-PEG) designed to deliver MDR1 siRNA for the treatment of MDR in an ovarian cancer model.HA-PEI/HA-PEG nanoparticles were synthesized and characterized, then the cellular uptake and knockdown efficiency of HA-PEI/HA-PEG/MDR1 siRNA nanoparticles was further determined. A human xenograft MDR ovarian cancer model was established to evaluate the effects of the combination of HA-PEI/HA-PEG/MDR1 siRNA nanoparticles and paclitaxel on MDR tumor growth.Our results demonstrated that HA-PEI/HA-PEG nanoparticles successfully targeted CD44 and delivered MDR1 siRNA into OVCAR8TR (established paclitaxel resistant) tumors. Additionally, HA-PEI/HA-PEG nanoparticles loaded with MDR1 siRNA efficiently down-regulated the expression of MDR1 and P-glycoprotein (Pgp), inhibited the functional activity of Pgp, and subsequently increased cell sensitivity to paclitaxel. HA-PEI/HA-PEG/MDR1 siRNA nanoparticle therapy followed by paclitaxel treatment inhibited tumor growth in MDR ovarian cancer mouse models.These findings suggest that this CD44 targeted HA-PEI/HA-PEG nanoparticle platform may be a clinicaly relevant gene delivery system for systemic siRNA-based anticancer therapeutics for the treatment of MDR cancers.
Project description:A Mn3O4 nanoparticle (NP)-based dual-modality probe has been developed for tumor positron emission tomography (PET)/magnetic resonance (MR) imaging. The dual-modality imaging probe was constructed by modifying multifunctional polyethyleneimine (PEI)-coated Mn3O4 NPs with folic acid (FA), followed with the radiolabeling with 64Cu. The formed imaging probe was utilized for PET/MR imaging of human cervical cancer mouse xenografts, which overexpress folate receptor (FR). The PEI-coated Mn3O4 NPs were synthesized using a solvothermal approach via decomposition of acetylacetone manganese. Multifunctional groups, including fluorescein isothiocyanate (FI), PEGylated FA, and NOTA chelator, were then sequentially loaded onto the surface of the amine groups of the Mn3O4 NPs. The remaining PEI amines were neutralized by the acetylation reaction. The resulting NOTA-FA-FI-PEG-PEI-Ac-Mn3O4 NPs were fully characterized and evaluated in vitro and successfully radiolabeled with 64Cu for tumor PET/MR imaging in small animals. In vivo blocking experiments were performed to determine the FR binding specificity of NPs. PET imaging results demonstrated that 64Cu-labeled Mn3O4 NPs display good tracer uptake in the FR-expressing HeLa tumors (tumor-to-muscle (T/M) ratio: 5.35 ± 0.31 at 18 h postinjection (pi)) and substantially reduced tracer uptake in the FR-blocked HeLa tumors (T/M ratio: 2.78 ± 0.68 at 18 h pi). The ex vivo data, including PET imaging and biodistribution, further confirmed the tumor binding specificity of the 64Cu-labeled Mn3O4 NPs. Moreover, the FR-targeted Mn3O4 NPs exhibited efficient T1-weighted MR imaging (MRI), leading to the precise tumor MRI at 18 h pi. PET/MR imaging with the 64Cu-NOTA-FA-FI-PEG-PEI-Ac-Mn3O4 NPs may offer a new quantitative approach to precisely measure the FR in tumors. The strategy of incorporating PEI nanotechnology into the construction of new biomaterials may be applied for the construction of novel nanoplatforms for cancer diagnosis and therapy.
Project description:The success of clinically relevant immunotherapies requires reversing tumor-induced immunosuppression. Here we demonstrated that linear polyethylenimine-based (PEI-based) nanoparticles encapsulating siRNA were preferentially and avidly engulfed by regulatory DCs expressing CD11c and programmed cell death 1-ligand 1 (PD-L1) at ovarian cancer locations in mice. PEI-siRNA uptake transformed these DCs from immunosuppressive cells to efficient antigen-presenting cells that activated tumor-reactive lymphocytes and exerted direct tumoricidal activity, both in vivo and in situ. PEI triggered robust and selective TLR5 activation in vitro and elicited the production of hallmark TLR5-inducible cytokines in WT mice, but not in Tlr5-/- littermates. Thus, PEI is a TLR5 agonist that, to our knowledge, was not previously recognized. In addition, PEI-complexed nontargeting siRNA oligonucleotides stimulated TLR3 and TLR7. The nonspecific activation of multiple TLRs (specifically, TLR5 and TLR7) reversed the tolerogenic phenotype of human and mouse ovarian tumor-associated DCs. In ovarian carcinoma-bearing mice, this induced T cell-mediated tumor regression and prolonged survival in a manner dependent upon myeloid differentiation primary response gene 88 (MyD88; i.e., independent of TLR3). Furthermore, gene-specific siRNA-PEI nanocomplexes that silenced immunosuppressive molecules on mouse tumor-associated DCs elicited discernibly superior antitumor immunity and enhanced therapeutic effects compared with nontargeting siRNA-PEI nanocomplexes. Our results demonstrate that the intrinsic TLR5 and TLR7 stimulation of siRNA-PEI nanoparticles synergizes with the gene-specific silencing activity of siRNA to transform tumor-infiltrating regulatory DCs into DCs capable of promoting therapeutic antitumor immunity.
Project description:Development of multidrug resistance (MDR) is an almost universal phenomenon in patients with ovarian cancer, and this severely limits the ultimate success of chemotherapy in the clinic. Overexpression of the MDR1 gene and corresponding P-glycoprotein (Pgp) is one of the best known MDR mechanisms. MDR1 siRNA based strategies were proposed to circumvent MDR, however, systemic, safe, and effective targeted delivery is still a major challenge. Cluster of differentiation 44 (CD44) targeted hyaluronic acid (HA) based nanoparticle has been shown to successfully deliver chemotherapy agents or siRNAs into tumor cells. The goal of this study is to evaluate the ability of HA-PEI/HA-PEG to deliver MDR1 siRNA and the efficacy of the combination of HA-PEI/HA-PEG/MDR1 siRNA with paclitaxel to suppress growth of ovarian cancer. We observed that HA-PEI/HA-PEG nanoparticles can efficiently deliver MDR1 siRNA into MDR ovarian cancer cells, resulting in down-regulation of MDR1 and Pgp expression. Administration of HA-PEI/HA-PEG/MDR1 siRNA nanoparticles followed by paclitaxel treatment induced a significant inhibitory effect on the tumor growth, decreased Pgp expression and increased apoptosis in MDR ovarian cancer mice model. Our findings suggest that CD44 targeted HA-PEI/HA-PEG/MDR1 siRNA nanoparticles can serve as a therapeutic tool with great potentials to circumvent MDR in ovarian cancer.
Project description:Using an orthotopic model of ovarian cancer, we studied the delivery of siRNA in nanoparticles of tri-block copolymers consisting of hyperbranched polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol) (hyPEI-g-PCL-b-PEG) with and without a folic acid targeting ligand. A SKOV-3/LUC FR? overexpressing cell line was employed to mimic the clinical manifestations of ovarian cancer. Both targeted and non-targeted micelleplexes were able to effectively deliver siRNA to the primary tumor and its metastases, as measured by gamma scintillation counting and confocal microscopy. Stability of the micelleplexes was demonstrated with a serum albumin binding study. Regarding biodistribution, intravenous (I.V.) administration showed a slight advantage of FR? targeted over non-targeted micelleplex accumulation within the tumor. However, both formulations displayed significant liver uptake. On the other hand, intraperitoneally (I.P.) injected mice showed a modest 6% of the injected dose per gram (ID/g) uptake within the primary and most interestingly also in the metastatic lesions which subsequently resulted in a 62% knockdown of firefly luciferase expression in the tumor after a single injection. While this is, to the best of our knowledge, the first paper that correlates quantitative tumor accumulation in an orthotopic tumor model with in vivo gene silencing, these data demonstrate that PEI-g-PCL-b-PEG-Fol conjugates are a promising option for gene knockdown in ovarian cancer.
Project description:Therapeutic antiangiogenesis strategies have demonstrated significant antitumor efficacy in ovarian cancer. Recently, RNA interference (RNAi) has come to be regarded as a promising technology for treatment of disease, especially cancer. In this study, vascular endothelial growth factor (VEGF)-small interfering RNA (siRNA) was encapsulated into a magnetic mesoporous silica nanoparticle (M-MSN)-based, polyethylenimine (PEI)-capped, polyethylene glycol (PEG)-grafted, fusogenic peptide (KALA)-functionalized siRNA delivery system, termed M-MSN_VEGF siRNA@PEI-PEG-KALA, which showed significant effectiveness with regard to VEGF gene silencing in vitro and in vivo. The prepared siRNA delivery system readily exhibited cellular internalization and ease of endosomal escape, resulting in excellent RNAi efficacy without associated cytotoxicity in SKOV3 cells. In in vivo experiments, notable retardation of tumor growth was observed in orthotopic ovarian tumor-bearing mice, which was attributed to significant inhibition of angiogenesis by systemic administration of this nanocarrier. No obvious toxic drug responses were detected in major organs. Further, the magnetic core of M-MSN_VEGF siRNA@PEI-PEG-KALA proved capable of probing the site and size of the ovarian cancer in mice on magnetic resonance imaging. Collectively, the results demonstrate that an M-MSN-based delivery system has potential to serve as a carrier of siRNA therapeutics in ovarian cancer.
Project description:Polymeric nanoparticles have been studied for gene and drug delivery. These nanoparticles can be modified to utilize a targeted delivery approach to selectively deliver their payload to specific cells, while avoiding unwanted delivery to healthy cells. One commonly over-expressed receptor which can be targeted by ligand-conjugated nanoparticles is the folate receptor alpha (FR?). The ability to target FR? remains a promising concept, and therefore, understanding the binding dynamics of the receptor with the ligand of the nanoparticle therapeutic can provide valuable insight. This manuscript focuses on the interaction between self-assembled nanoparticles decorated with a folic acid (FA) ligand and FR?. The nanoparticles consist of micelles formed with a FA conjugated triblock copolymer (PEI-g-PCL-b-PEG-FA) which condensed siRNA to form micelleplexes. By combining biological and biophysical approaches, this manuscript explores the binding kinetics and force of the targeted siRNA containing nanoparticles to FR? in comparison with free FA. We demonstrate via flow cytometry and atomic force microscopy that multivalent micelleplexes bind to FR? with a higher binding probability and binding force than monovalent FA. Furthermore, we revisited why competitive inhibition studies of binding of multivalent nanoparticles to their respective receptor are often reported in literature to be inconclusive evidence of effective receptor targeting. In conclusion, the results presented in this paper suggest that multivalent targeted nanoparticles display strong receptor binding that a monovalent ligand may not be able to compete with under in vitro conditions and that high concentrations of competing monovalent ligands can lead to measurement artifacts.
Project description:Photodynamic therapy (PDT) is considered an innovative and attractive modality to treat ovarian cancer. In the present study, a biodegradable polymer poly (ethylene glycol) (PEG)-poly (lactic acid)(PLA)-folate (FA-PEG-PLA) was prepared in order to synthesize an active-targeting, water-soluble and pharmacomodulated photosensitizer nanocarrier. Drug-loading content, encapsulation efficiency, in vitro and in vivo release were characterized, in which hypocrellin B (HB)/FA-PEG-PLA micelles had a high encapsulation efficiency and much slower control release for drugs compared to free drugs (P < .05). To evaluate the targeting ability of the HB/FA-PEG-PLA micelles, a cellular uptake study in vitro was carried out, which showed significantly enhanced uptake of HB/FA-PEG-PLA micelles in SKOV3 (FR+) compared to A2780 cancer cells (FR-). The enhanced uptake of HB/FA-PEG-PLA micelles to cancer cells resulted in a more effective post-PDT killing of SKOV3 cells compared to plain micelles and free drugs. Binding and uptake of HB/FA-PEG-PLA micelles by SKOV3 cells were also observed in vivo after ip injection of folate-targeted micelles in tumor-bearing ascitic ovarian cancer animals. Drug levels in ascitic tumor tissues were increased 20-fold (P < .001), which underscored the effect of a regional therapy approach with folate targeting. Furthermore, the HB-loaded micelles were mainly distributed in kidney and liver (the main clearance organs) in biodistribution. These results showed that our newly developed PDT photosensitizer HB/FA-PEG-PLA micelles have a high drug-loading capacity, good biocompatibility, controlled drug release, and enhanced targeting and antitumor effect, which is a potential approach to future targeting ovarian cancer therapy.