Polymeric Nanocarriers for Treatment of Melanoma and Genetically Modified Mesenchymal Stem Cells to Improve Outcome of Islet Transplantation

Melanoma is a lethal malignancy with limited treatment options for advanced metastatic stages. New targeted therapeutic options with discovery of BRAF and MEK inhibitors have shown significant survival benefit. Despite the recent progress, inefficient tumor accumulation and dose limiting systemic toxicity remains pressing challenges for treating metastatic melanoma and there is a need for drug delivery approach to improve therapeutic index of chemotherapeutics. Nanoparticle based drug delivery represents promising approach to enhance efficacy and reduce the dose limiting systemic toxicity. Nanoparticles can be formulated either by physical encapsulation of drugs or by covalent conjugation of drugs to the polymeric backbone. Nanoparticles based strategies for encapsulation and conjugation of drugs to the polymer was reviewed in Chapter 2 where we summarized non-covalent interactions between polymer backbone and drug for physical encapsulation, various polymeric backbones for drug conjugation and application of photodynamic therapy in melanoma. Phototherapy, a light activated treatment modality is a potential therapeutic option for treatment of melanoma. Excitation of photosensitizer by light of specific wavelength can be clinically utilized for fluorescence assisted tumor surgery, photoacoustic imaging, photochemical internalization and phototherapy. Indocyanine green, water soluble FDA approved anionic tricarbocyanine with excellent safety profile and absorption in the near infrared (NIR) range is an excellent photosensitizer. However, short half-life (2-4 minutes) and limited extravascular distribution restricts PT application of ICG. In chapter 3, we have described ICG based phototherapy wherein plasma circulation and tumor accumulation of ICG was improved by designing its micelles formulation. ICG micelles were formulated by covalently conjugating ICG-NH2 to the pendant carboxyl groups of poly (ethylene glycol)-block-poly(2- methyl-2-carboxyl-propylene carbonate) (PEG-PCC) copolymer using carbodiimide coupling. ICG conjugated amphiphillic polymer self-assembled into micelles with particle size of 30-50 nm and high drug loading. These ICG conjugated micelles exhibited significant in vitro photodynamic cytotoxicity. Use of sodium azide and NIR radiation at 4° C revealed photodynamic and photothermal as primary mechanism of cytotoxicity of ICG solution and ICG conjugated micelles respectively. In vivo NIR imaging demonstrated ICG conjugated micelles prolonged ICG circulation and increased its tumor accumulation through enhanced permeability and retention effect Increase in tumor accumulation improved therapeutic efficacy with complete tumor regression in NIR irradiated ICG conjugated micelles compared to free ICG and control in A375 human melanoma tumor model in athymic nude mice. These results suggest that ICG conjugated micelles can be potentially utilized for phototherapy. Clinical translation of tubulin inhibitors for treating melanoma is limited by multidrug efflux transporters, poor aqueous solubility, and dose-limiting peripheral toxicities. Tubulin inhibitors with efficacy in taxane-resistant cancers are promising drug candidates and can be used as single agent or in conjunction with other chemotherapy. In chapter 4, we describe synthesis of tubulin inhibitors with activity in taxane resistant cell lines with IC50 in nanomolar range for the treatment of metastatic melanoma. LY293, a 5 indole derivative analog, binds to colchicine binding site and does not exhibit clinically prevalent drug resistance mechanism such as multidrug resistance (MDR) protein, breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp). Since LY293 is poorly soluble in water, LY293 was formulated as polymeric nanoparticles for systemic therapy of melanoma. Methoxy polyethylene glycol-b-poly (carbonate-co-lactide) (mPEG-b-P (CB-co-LA)) random copolymer was synthesized and characterized by 1H NMR and gel permeation chromatography (GPC). Polymeric nanoparticles were formulated using o/w emulsification method with a mean particle size of 150 nm and loading efficiency of 7.40%. Treatment with LY293 loaded nanoparticles effectively inhibited the proliferation of melanoma cells in vitro and exhibited concentration dependent cell cycle arrest in G2/M phase. In vivo, LY293 loaded nanoparticles significantly inhibited the proliferation of highly aggressive metastasized melanoma in a syngeneic lung metastasis melanoma mouse model without toxicity to vital organs. Islet transplantation has been performed in many patients especially undergoing kidney transplantation to treat Type I diabetes. Proportion of recipients who achieved insulin independence is low and is limited by long-term graft rejection and by primary non-function of islets. Primary non-function is characterized as the loss of islet viability and function caused by non-immune reasons, such as the disruption of islet microvasculature and apoptosis of islets due to production of inflammatory cytokines at the transplantation sites. In chapter 5, we studied the potential of human bone marrow derived mesenchymal stem cells (hBMSCs) as gene carriers for improving the outcome of human islet transplantation. hBMSCs were transduced with Adv-hVEGF-hIL-1Ra to overexpress human vascular endothelial growth factor (hVEGF) and human interleukin-1 receptor antagonist (hIL-1Ra). Viability of human islets co-cultured with hBMSCs was determined by membrane fluorescent method and glucose stimulation test. Transduced hBMSCs and human islets were co-transplanted under the kidney capsule of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ diabetic mice and blood glucose levels were measured over time to evaluate the efficacy of genetically modified hBMSCs. Our in vitro and in vivo results showed hBMSCs can be used as gene delivery vehicles to improve the outcome of islet transplantation without affecting their stemness and differentiation potential

Genetically Modified Human Bone Marrow Derived Mesenchymal Stem Cells for Improving the Outcome of Human Islet Transplantation

<div><p>The objective of this study was to determine the potential of human bone marrow derived mesenchymal stem cells (hBMSCs) as gene carriers for improving the outcome of human islet transplantation. hBMSCs were characterized for the expression of phenotypic markers and transduced with Adv-hVEGF-hIL-1Ra to overexpress human vascular endothelial growth factor (hVEGF) and human interleukin-1 receptor antagonist (hIL-1Ra). Human islets were co-cultured with hBMSCs overexpressing hVEGF and hIL-1Ra. Islet viability was determined by membrane fluorescent method and glucose stimulation test. Transduced hBMSCs and human islets were co-transplanted under the kidney capsule of NOD.Cg-<i>Prkdc^scid Il2rg^tm1Wjl</i>/SzJ (NSG) diabetic mice and blood glucose levels were measured over time to demonstrate the efficacy of genetically modified hBMSCs. At the end of study, immunofluorescent staining of kidney section bearing islets was performed for insulin and von Willebrand Factor (vWF). hBMSCs were positive for the expression of CD73, CD90, CD105, CD146 and Stro-1 surface markers as determined by flow cytometry. Transduction of hBMSCs with adenovirus did not affect their stemness and differentiation potential as confirmed by mRNA levels of stem cell markers and adipogenic differentiation of transduced hBMSCs. hBMSCs were efficiently transduced with Adv-hVEGF-hIL-1Ra to overexpress hVEGF and hIL-1Ra. Live dead cell staining and glucose stimulation test have shown that transduced hBMSCs improved the viability of islets against cytokine cocktail. Co-transplantation of human islets with genetically modified hBMSCs improved the glycemic control of diabetic NSG mice as determined by mean blood glucose levels and intraperitoneal glucose tolerance test. Immunofluorescent staining of kidney sections was positive for human insulin and vWF. In conclusion, our results have demonstrated that hBMSCs may be used as gene carriers and nursing cells to improve the outcome of islet transplantation.</p></div

Intraperitoneal glucose tolerance test after 15 days of islet transplantation.

<p>Mice were fasted overnight and then injected intraperitoneally with glucose (2 g/kg of body weight). Blood glucose levels were measured by tail pricking at indicated time points with a glucometer. Data are presented as the mean ± SD (n = 5).</p

Effect of Adv-hVEGF-hIL-1Ra transduced hBMSCs on human serum insulin and C-peptide levels of diabetic NSG.

<p>Mice transplanted with either islet alone or with hBMSCs before or after transduction with Adv-hVEGF-hIL-1Ra at 35 days post transplantation (A) Human serum insulin (B) Human serum C-peptide. Data are presented as the mean ± SD, n = 5. p<0.05 under t-test.</p

Immunofluorescence staining of the kidney sections bearing human islets 35 days after islet transplantation.

<p>Insulin was stained in red to indicate the functional human islets after transplantation. vWF was stained in green to indicate the revascularization of transplanted human islets. Kidney section of mice bearing human islets; human islets co-transplanted with hBMSCs; human islets co-transplanted with Adv-hVEGF-hIL-1Ra transduced hBMSCs.</p

Expression of stem cell markers and differentiation of hBMSCs after transduction with Adv-hVEGF-hIL-1Ra at 200 MOI.

<p>(A) Real Time RT-PCR analysis to quantify change in the expression of Nanog and Oct4 stemness markers. Data are represented as the mean ± SD, n = 3. (B) Oil Red and alizarin red S staining of hBMSCs after culture in adipogenic and osteogenic differentiation media, respectively.</p

Stimulation index (SI) of human islets after incubation with inflammatory cytokine cocktail of 5 ng/ml IL-1β, 10 ng/ml TNF-α and 50 ng/ml IFN-γ for 10 days.

<p>Non-transduced islets were used as controls. SI was determined as the ratio of insulin released from islets when they were incubated in Kreb’s buffer containing 22 mM and 2.2 mM glucose. Data are presented as the mean ± SE (n = 3).</p

Protection of islets against cytokine cocktail as shown by Calcein AM and PI staining.

<p>(A) Islets, (B) Islets with hBMSCs, (C) Islets with hBMSCs transduced with Adv-hVEGF-hIL-1Ra at the ratio of 1 islet equivalent to 100 hBMSCs.</p

Effect of Adv-hVEGF-hIL-1Ra transduced hBMSCs on the outcome of human islet transplantation.

<p>(A, B, C) The blood glucose level of every mouse after receiving 2000 human islet equivalent transplanted either alone or hBMSCs before or after transduction with Adv-hVEGF-hIL-1Ra (A–C). (D) Diabetes reversal ratio of the diabetic mice after human islet transplantation. Blood glucose ≤250 mg/dl (dashed line) was identified as reversed-diabetes (dashed line in A, B, C). Black triangles indicate mice receiving islets with Adv-hVEGF-hIL-1Ra transduced hBMSCs; gray squares indicate mice receiving islets with hBMSCs; black diamond’s indicate mice receiving islets only.</p

Phenotypic characterization of cultured hBMSCs.

<p>hBMSCs are positive for CD73, CD146, CD105, CD90 and Stro1. Open peaks indicate the isotype of each cell and solid peaks represent expression of each marker. Numbers in the box specify the percentage of positive cells as compared to isotype control.</p