Photoactivation of sulfonated polyplexes enables localized gene silencing by DsiRNA in breast cancer cells.
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ABSTRACT:
SUBMITTER: Puri A
PROVIDER: S-EPMC8117728 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:RNA interference (RNAi) molecules have tremendous potential for cancer therapy but are limited by insufficient potency after intravenous (IV) administration. We previously found that polymer complexes (polyplexes) formed between 3'-cholesterol-modified siRNA (Chol-siRNA) or DsiRNA (Chol-DsiRNA) and the cationic diblock copolymer PLL[30]-PEG[5K] greatly increase RNAi potency against stably expressed LUC mRNA in primary syngeneic murine breast tumors after daily IV dosing. Chol-DsiRNA polyplexes, however, maintain LUC mRNA suppression for ~48 h longer after the final dose than Chol-siRNA polyplexes, which suggests that they are the better candidate formulation. Here, we directly compared the activities of Chol-siRNA polyplexes and Chol-DsiRNA polyplexes in primary murine 4T1 breast tumors against STAT3, a therapeutically relevant target gene that is overexpressed in many solid tumors, including breast cancer. We found that Chol-siSTAT3 polyplexes suppressed STAT3 mRNA in 4T1 tumors with similar potency (half-maximal ED50 0.3 mg/kg) and kinetics (over 96 h) as Chol-DsiSTAT3 polyplexes, but with slightly lower activity against total Stat3 protein (29% vs. 42% suppression) and tumor growth (11.5% vs. 8.6% rate-based T/C ratio) after repeated IV administration of equimolar, tumor-saturating doses every other day. Thus, both Chol-siRNA polyplexes and Chol-DsiRNA polyplexes may be suitable clinical candidates for the RNAi therapy of breast cancer and other solid tumors.
| S-EPMC8788547 | biostudies-literature
Project description:RNA interference molecules have tremendous potential for cancer therapy but are limited by insufficient potency after i.v. administration. We previously found that Chol-DsiRNA polyplexes formed between cholesterol-modified dicer-substrate siRNA (Chol-DsiRNA) and the cationic diblock copolymer PLL[30]-PEG[5K] greatly increase the activity of Chol-DsiRNA against a stably expressed reporter mRNA in primary murine syngeneic breast tumors after daily i.v. dosing. Here, we provide a more thorough preliminary preclinical study of Chol-DsiRNA polyplexes against the therapeutically relevant target protein, STAT3. We found that Chol-DsiSTAT3 polyplexes greatly increase plasma exposure, distribution, potency, and therapeutic activity of Chol-DsiSTAT3 in primary murine syngeneic 4T1 breast tumors after i.v. administration. Furthermore, inactive Chol-DsiCTRL polyplexes are well tolerated by healthy female BALB/c mice after chronic i.v. administration at 50 mg Chol-DsiCTRL/kg over 28 days. Thus, Chol-DsiRNA polyplexes may be a good candidate for Phase I clinical trials to improve the treatment of breast cancer and other solid tumors.
| S-EPMC8184584 | biostudies-literature
Project description:Gene therapy platforms offer a variety of potentially effective solutions for development of targeted agents that can be exploited for cancer treatment. The physicochemical properties of nanocarriers can be tuned to enhance their localization in tumors, and cell specificity can also be increased by appropriate selection of gene targets. A relatively underexploited approach to enhance therapeutic selectivity in cancer tissues is the use of nanocarriers whose nuclear targeting and uptake are triggered by the altered expression of specific endomembrane trafficking proteins in cancer cells. Previously, we showed that histone 3 (H3) peptide-targeted DNA polyplexes traffic to the nucleus efficiently through caveolar endocytosis followed by transfer through the Golgi and endoplasmic reticulum (ER). We hypothesized that these polyplexes would exhibit enhanced activity in inflammatory breast cancer (IBC) cells, which overexpress caveolin-1 as part of their invasive phenotype, and we also posited that this targeting effect could be exploited to facilitate IBC-specific transfection and prodrug conversion in the presence of normal breast epithelial cells. Using cellular transfection experiments, function-blocking assays, and confocal imaging in both IBC SUM149 cell monocultures and IBC SUM149 co-cultures with MCF10A normal breast epithelial cells, we found that our H3-targeted polyplexes selectively transfected IBC SUM149 cells at a 4-fold higher level than normal breast epithelial cells. This selectivity and increased transfection were caused by a 2.2-fold overexpression of caveolin-1 in IBC SUM149 cells, which led to increased polyplex trafficking to the nucleus through the Golgi and ER. We also saw similar enhancements in cell selectivity and transfection when cells were transfected with a suicide gene/prodrug combination, as the increased expression of the suicide gene in IBC SUM149 cells led to a 55% decrease in viability in IBC SUM149 cells as compared to a 25% decrease in MCF10A cells. These findings demonstrate that differences in the expression of the endocytic membrane protein caveolin-1 can be exploited for cell-selective gene delivery, and ultimately, these gene-based targeting approaches may be useful in potential treatments for aggressive cancer types. Biotechnol. Bioeng. 2016;113: 2686-2697. © 2016 Wiley Periodicals, Inc.
| S-EPMC5268818 | biostudies-literature
Project description:Therapeutic gene silencing in the brain is usually achieved using highly invasive intracranial administration methods and/or comparatively toxic vectors. In this work, we use a relatively biocompatible vector: poly(ethylene glycol) star-shaped polymer capped with amine groups (4APPA) via the nose to brain route. 4APPA complexes anti- itchy E3 ubiquitin protein ligase (anti-ITCH) siRNA to form positively charged (zeta potential +15 ± 5 mV) 150 nm nanoparticles. The siRNA-4APPA polyplexes demonstrated low cellular toxicity (IC50 = 13.92 ± 6 mg mL-1) in the A431 cell line and were three orders of magnitude less toxic than Lipofectamine 2000 (IC50 = 0.033 ± 0.04 mg mL-1) in this cell line. Cell association and uptake of fluorescently labelled siRNA bound to siRNA-4APPA nanoparticles was demonstrated using fluorescent activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), respectively. Gene silencing of the ITCH gene was observed in vitro in the A431 cell line (65% down regulation when compared to the use of anti-ITCH siRNA alone). On intranasal dosing with fluorescently labelled siRNA-4APPA polyplexes, fluorescence was seen in the cells of the olfactory bulb, cerebral cortex and mid-brain regions. Finally, down regulation of ITCH was seen in the brain cells (54 ± 13% ITCH remaining compared to untreated controls) in a healthy rat model, following intranasal dosing of siRNA-4APPA nanoparticles (0.15 mg kg-1 siRNA twice daily for 3 days). Gene silencing in the brain may be achieved by intranasal administration of siRNA- poly(ethylene glycol) based polyplexes.
| S-EPMC9496157 | biostudies-literature
Project description:ObjectiveTo achieve effective delivery of siRNA into target cells in vivo, we have developed a novel approach of siRNA delivery by using local drug delivery systems.ResultsThe chitosan hydrogel (CH-HG) displayed a liquid-solid phase transition in a temperature-dependent manner and formed an endothermic hydrogel in tumor tissue after intra-tumoral injection. Additionally, we tested the extent of in vivo delivery following a single intra-tumoral injection of Alexa555 siRNA/CH-HG into A375SM-bearing mice. The Alexa555 siRNA demonstrated higher localization into tumor cells compared to control. The Alexa555 siRNA delivery extends to tumor cells outside of CH-HG and some tumor cells also infiltrated into CH-HG. For therapeutic proof-of-concept studies, CH-HG including TG2-targeted siRNA significantly inhibited tumor growth in melanoma (A375SM) and breast (MDA-MB231) tumor models compared to control (A375SM: 72% reduction and MDA-MB231: 92% reduction, p < 0.001).Experimental designwe prepared a CH-HG system loaded with siRNA to enhance localized therapeutic efficacy without risk for systemic side effects. Delivery of siRNA into CH-HG was confirmed by fluorescence microscopy. Antitumor efficacy was examined in mouse models of melanoma (A375SM) and breast (MDA-MD231) cancer.ConclusionsThis study developed a novel local delivery method for siRNA therapy using the CH-HG system. This approach could have broad applications for multiple localized diseases.
| S-EPMC3100632 | biostudies-literature
Project description:Small interfering RNA (siRNA) targeted therapeutics (STT) offers a compelling alternative to tradition medications for treatment of genetic diseases by providing a means to silence the expression of specific aberrant proteins, through interference at the expression level. The perceived advantage of siRNA therapy is its ability to target, through synthetic antisense oligonucleotides, any part of the genome. Although STT provides a high level of specificity, it is also hindered by poor intracellular uptake, limited blood stability, high degradability and non-specific immune stimulation. Since serum proteins has been considered as useful vehicles for targeting tumors, in this study we investigated the effect of incorporation of human serum albumin (HSA) in branched polyethylenimine (bPEI)-siRNA polyplexes in their internalization in epithelial and endothelial cells. We observed that introduction of HSA preserves the capacity of bPEI to complex with siRNA and protect it against extracellular endonucleases, while affording significantly improved internalization and silencing efficiency, compared to bPEI-siRNA polyplexes in endothelial and metastatic breast cancer epithelial cells. Furthermore, the uptake of the HSA-bPEI-siRNA ternary polyplexes occurred primarily through a caveolae-mediated endocytosis, thus providing evidence for a clear role for HSA in polyplex internalization. These results provide further impetus to explore the role of serum proteins in delivery of siRNA.
| S-EPMC4391875 | biostudies-literature
Project description:BackgroundThe application of non-viral systems for delivering genes to cells is becoming a very interesting issue, especially in the treatment of neoplasms such as Breast Cancer (BC). Polymer-based non-viral systems are safe and feasible gene carriers to be used in targeted cancer therapy. SALL4 gene encodes a transcription factor and is overexpressed in some cancers.MethodsIn this study, carboxyalkylated-PEI25 (25 kDa) was used to deliver plasmids expressing SALL4-siRNA into MCF-7 cells. DLS and AFM were applied to determine the size of nanoparticles. The MTT method was used to assess cytotoxicity, and the efficiency of transfection was confirmed both qualitatively and quantitatively. Finally, the effect of silencing SALL4 was investigated on the migration of MCF7 cells using the scratch test.ResultsThe results showed that transferring the SALL4-siRNA using PEI25G10C50 reduced the expression of the corresponding transcription factor by 14 folds which attenuated the migration of MCF-7 cells by 58%.ConclusionIn conclusion, PEI25G10C50 can serve as an effective gene delivery system for treating BC by targeting SALL-4.
| S-EPMC7903432 | biostudies-literature
Project description:In previous studies, we identified a distantly related rhomboid homologue gene known as RHBDD2 (Rhomboid domain containing 2) to be markedly overexpressed in the advanced stages of the breast and colorectal cancer diseases. In order to identify RHBDD2 modulated pathways, we analyzed two breast cancer cell lines (MCF7 and T47D) from control and RHBDD2-siRNA transient gene silencing followed by gene expression profiling analysis using the whole genome Toray 3D-GeneTM Human Oligo Chip. Statistical analysis of the Toray's 3D gene expression profiling data identified 566 commonly differentially expressed genes in association to the RHBDD2 knockdown in both breast cancer cell lines. Among the statistically significant over-represented biological process, we found the apoptosis, cell cycle and response to DNA damage process related genes. In addition, categories of genes found in the ubiquitin-proteasome and oxidative phosphorylation were also highly enriched related genes in the commonly deregulated gene list. We further used a lentivirus-based system (shRNA-pLKO.1) for stable silencing of RHBDD2 mRNA in the T47D breast cancer cell line. Using a staurosporine-induced apoptosis model, we demonstrate that RHBDD2 abrogation resulted in an apoptosis-resistant phenotype of T47D breast cancer cell line. These data are in line with a recent study, suggesting that RHBDD2 expression could be up-modulated in response to 5FU-induced apoptosis in colorectal cancer cells. Taken together, these data suggest that RHBDD2 could be involved in the modulation of the programmed cell death in cancer cells. In order to analyze differential gene expression profiling of RHBDD2 silencing and control cells, total RNA was isolated from replicate experiments from two breast cancer cell lines (MCF7 and T47D) derived from the negative control-siRNA and the RHBDD2-siRNA treatments in duplicate experiments.
| S-ECPF-GEOD-43015 | biostudies-other
Project description:Branched peptides containing histidines and lysines (HK) have been shown to be effective carriers for DNA and siRNA. We anticipate that elucidation of the binding mechanism of HK with siRNA will provide greater insight into the self-assembly and delivery of the HK:siRNA polyplex. Non-covalent bonds between histidine residues and nucleic acids may enhance the stability of siRNA polyplexes. We first compared the polyplex biophysical properties of a branched HK with those of branched asparagine-lysine peptide (NK). Consistent with siRNA silencing experiments, gel electrophoresis demonstrated that the HK siRNA polyplex maintained its integrity with prolonged incubation in serum, whereas siRNA in complex with NK was degraded in a time-dependent manner. Isothermal titration calorimetry of various peptides binding to siRNA at pH 7.3 showed that branched polylysine, interacted with siRNA was initially endothermic, whereas branched HK exhibited an exothermic reaction at initial binding. The exothermic interaction indicates formation of non-ionic bonds between histidines and siRNA; purely electrostatic interaction is entropy-driven and endothermic. To investigate the type of non-ionic bond, we studied the protonation state of imidazole rings of a selectively (15)N labeled branched HK by heteronuclear single quantum coherence NMR. The peak of N?1-H tautomers of imidazole shifted downfield (in the direction of deprotonation) by 0.5-1.0 ppm with addition of siRNA, providing direct evidence that histidines formed hydrogen bonds with siRNA at physiological pH. These results establish that histidine-rich peptides form hydrogen bonds with siRNA, thereby enhancing the stability and biological activity of the polyplex in vitro and in vivo.
| S-EPMC3920840 | biostudies-literature
Project description:Small interfering RNA (siRNA) has found many applications in tissue regeneration and disease therapeutics. Effective and localized siRNA delivery remains challenging, reducing its therapeutic potential. Here, we report a strategy to control and prolong siRNA release by directly tethering transfection-capable siRNA to photocrosslinked dextran hydrogels. siRNA release is governed via the hydrolytic degradation of ester and/or disulfide linkages between the siRNA and hydrogels, which is independent of hydrogel degradation rate. The released siRNA is shown to be bioactive by inhibiting protein expression in green fluorescent protein-expressing HeLa cells without the need of a transfection agent. This strategy provides an excellent platform for controlling nucleic acid delivery through covalent bonds with a biomaterial and regulating cellular gene expression, which has promising potential in many biomedical applications.
| S-EPMC6713499 | biostudies-literature