Formulation and Pharmacokinetics of a Polymeric MicroRNA Nanoparticle Delivery System for Treating Metastatic Breast Cancer
Walbi, Ismail Ali Y. Ali
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RNA interference (RNAi) is a process in which small non-coding, double-stranded RNA (dsRNA) regulates expression of the protein by altering messenger RNA (mRNA) translation process to protein. Two major types of RNAi are small interfering RNA (siRNA) and microRNA (miRNA). Changes of miRNA level has been correlated with different diseases, including cancer. Replacement therapy with miRNA mimics has a potential to restore the normal biological level of miRNA and treat diseases with deficient miRNA levels. Chitosan (CS) is a naturally occurring amino polysaccharide biopolymer that has been studied in the drug delivery field to deliver different types of medication including RNAi. In chapter 2, we used low molecular weight chitosan to formulate nanoparticles loaded with miRNA. We explored the ionotropic gelation method with different counterions and surface coating. The optimized chitosan nanoparticles were prepared using sodium tripolyphosphate (TPP) as counter ion and mPEG-DSPE as surface coating material. The optimized CS/TPP mPEG-DSPE hybrid nanoparticles have high miRNA incorporation efficiency and they can overcome some limitations associated with cationic nanoparticles and can increase the nanoparticle cellular uptake. Using two different therapeutic miRNAs miR-34a, and miR-195 as model compounds, we studied the cellular uptake of miRNA encapsulated hybrid nanoparticles using flow cytometry and fluorescence imaging in multiple cell lines. Coating the CS/TPP nanoparticle with mPEG-DSPE2K increased the cellular uptake of the nanoparticle about two-fold compared to the uncoated nanoparticle in MDA-MB-231 breast cancer cell line. The coating also protects miR-195 from serum endonuclease as compared to unformulated miR-195 mimic. In chapter 3 and chapter 4, we studied both pharmacokinetics and biodistribution of CS/TPP mPEG-DSPE2K hybrid nanoparticles encapsulated with miR-34a-CY5 and miR-195-CY5. For the pharmacokinetic study, we used low volume blood samples (10 µL) and a simple Heat-in-Triton (HIT) extraction method followed by the stem-loop qRT-PCR method to quantify the concentrations of miRNA in mouse blood and tissues. The pharmacokinetics profile showed a steep decline of miRNA at early time points followed by a slower clearance at later time points. The CS/TPP mPEG-DSPE2K nanoparticles mainly accumulate in kidney with no acute toxicity. In chapter 5, we studied the therapeutic efficacy of miR-195-loaded CS/TPP mPEG-DSPE2K nanoparticles (miR-195-NP) in metastatic breast cancer mouse model. The model was developed by intracardiac injection of MDA-MB-231 bone-seeking luciferase transfected cells (231-BM-luc). Intravenous administration of miR-195-NP via tail vein injection inhibited the in vivo metastasis of breast cancer to the bone and increased the survival of mice when compared to the untreated control mice. No signs of toxicity for miR-195-NP was observed based on changes of body weight and plasma creatinine level when compared to the control group. In summary, we have designed and fabricated a hybrid CS/TPP mPEG-DSPE2K nanoparticles that can efficiently encapsulate miRNA therapeutics such as miR-34a and miR-195. We have demonstrated that nanoformulations improved the miRNA circulation half-life and cellular uptake. We have shown that miR-195 encapsulated CS/TPP mPEG-DSPE2K hybrid nanoparticles displayed antitumor efficacy in metastatic breast cancer mouse models. The chitosan-based miRNA nanoparticle formulations have shown promise and warrant further investigation.