Biomaterials xxx (2014) 1e11
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Efficacy of gemcitabine conjugated and miRNA-205 complexed micelles for treatment of advanced pancreatic cancer Anupama Mittal a, Deepak Chitkara a, Stephan W. Behrman b, Ram I. Mahato a, *,1 a b
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA Department of Surgery, University of Tennessee Health Science Center, Memphis, TN 38163, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 February 2014 Accepted 12 April 2014 Available online xxx
Clinical effectiveness of gemcitabine in pancreatic cancer is hindered due to its rapid plasma metabolism and development of chemo-resistance. We have previously delineated the significant role of miRNAs in mediating the growth and proliferation of cancer stem cells (CSCs) which in turn result in chemoresistance, invasion and metastasis. Here, we designed self-assembling, gemcitabine conjugated cationic copolymers for co-delivery of a tumor suppressor miRNA-205 (miR-205) and evaluated their in vivo efficacy in a pancreatic cancer ectopic tumor model developed using gemcitabine resistant MIA PaCa-2R cells. Combination formulations showed mean a particle size of 10% w/w, exhibited miRNA complexation at N/P ratio of 4/1, sustained release of gemcitabine for >10 days, transfection efficiency of >90%, extended miRNA and drug stability in serum. Functional assays in gemcitabine resistant MIA PaCa-2R and CAPAN-1R pancreatic cancer cells revealed that the combination formulations effectively reversed chemo-resistance, invasion and migration. In pancreatic tumor model, the combination formulation treated group showed significant inhibition of tumor growth. Immuno-hiostochemical analysis revealed decreased tumor cell proliferation with increased apoptosis in the animals treated with miR-205 and gemcitabine combination. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: miR-205 Gemcitabine Pancreas Cancer Micelles miRNA
1. Introduction Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies being the fourth leading cause of cancerrelated deaths in the United States, with a 5 year survival rate of 70% transfection efficiency while in case of SP and TP, complete complexation was achieved at 8:1 and 16:1 N/P ratios, respectively. To assess the efficacy of the combination formulation two gemcitabine resistant cell lines, MIA PaCa-2R and CAPAN-1R were used. Cell viability assessment indicated a significant improvement in the cytotoxicity of gemcitabine when incubated simultaneously with miR-205 using our gemcitabine conjugated cationic polymer. miR-205 can target several genes which are involved in cell proliferation, invasion and EMT and thus could potentially enhance the cytotoxicity of gemcitabine [44e47]. MIA PaCa-2R cells showed an upregulation of ZEB-1, SIP-1, HRAS and LRP-1 and downregulation of CAV-1 and E-CAD which could be potential targets for miR-205. Pancreas is the source of MIA PaCa-2 cells and exhibit epithelial morphology however its resistant version i.e. MIA PaCa-2R has fibroblastic morphology. The downregulation of E-CAD and CAV-1 and up-regulation LRP-1 in MiaPaCa-2R cells could play a significant role in EMT and make the cells more invasive as observed in the invasion and migration assays [44,47e49]. Further, CAV-1 could function in an oncogenic transformation of cells and metastasis and its loss may result in EMT and metastasis [54,55]. On transfection with miR-205 mimic using our combination formulations, we observed a reversal in the expression of these genes indicating their targeting by miR-205. Thus, the combination of miR-205 mimic with gemcitabine holds potential to provide a viable treatment strategy for advanced pancreatic tumor. Gemcitabine resistant MIA PaCa-2R cells were more invasive compared to the sensitive MIA PaCa-2 cells. Transfection with miR205 polyplexes had significantly reduced the invasive as well as migratory potential of these cells which is in line with our gene expression data. Tellez et al. [42] have previously reported that EMT and stem cell like properties in carcinogen induced transformation of human lung epithelial cells could be associated with the sustained epigenetic silencing of miR-205. The transformed cells were more migratory and invasive compared to non-transformed cells. Transient re-expression of miR-205 in these transformed cells prevented their growth in soft agar [42]. Further, Song and Bu have demonstrated the inhibition of tumor cell migration through ectopic expression of miR-205 [48]. Recently, in breast cancer cells also, re-expression of miR-205 has resulted in their decreased migration and invasion potential [44]. miR-205 has a broader interrelated role in inhibiting cell proliferation, EMT and reversing chemo-resistance. This in part is also associated with the small subpopulation of tumor initiating cells having stem cell like properties. The role of miR-205 in cell proliferation and resistance has been explored for other cancers such as prostate cancer [40]. In our
9
previous study [18], miR-205 transfection using Lipofectamine 2000Ò in resistant pancreatic cancer cells resulted in their sensitization however, this may not be a viable approach for in vivo application as the stability of miR-205 lipoplexes is severely compromised in the presence of serum (Fig. 3). The polyplexes prepared in the present study were able to reverse chemoresistance along with improving miR-205 stability. In vivo efficacy of the polyplexes was studied in ectopic tumor model developed using gemcitabine resistant MIA PaCa-2R cells in athymic nude mice. Promising results were obtained wherein miR205 polyplexes containing gemcitabine resulted in a significant reduction in tumor growth rate and tumor weight compared to the control groups (Fig. 8). Immuno-histochemical analysis also revealed reduction in cell proliferation and induction of apoptosis in the test group of miR-205 polyplexes. In addition to the significant improvement conferred by miR-205 replacement therapy, the experimental framework may be employed for assessing other tumor suppressive miRNAs as well as their combinations. In the past few years, several delivery strategies have been employed for delivering miRNAs including miR-34a [56], miR-29b [57] and let-7b [56,58]. However, the present system offers additional advantage of co-delivering highly hydrophilic drug gemcitabine in the same carrier system. This also ensures their similar in vivo biodistribution which may not be feasible by delivering these two agents in different carrier systems. In the present study, we used intra-tumoral route to first establish the proof-of-concept that these polyplexes were efficacious in the resistant cells growing in 3D tumor environment. To completely understand the applicability of the developed formulations, systemic delivery is warranted both in terms of pharmacokinetic and bio-distribution as well as efficacy. Although these formulations contain PEG shell which may provide stealth effect on systemic delivery and could increase the mean residence time, to widen the benefit of the approach, active targeting may be achieved by suitably tailoring the PEG end groups making them amenable to conjugation with small molecules, peptide or antibodies for active targeting to the pancreatic tumor. 5. Conclusions Our self-assembling, gemcitabine conjugated cationic copolymers efficiently form complexes with miR-205 of particle size
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Efficacy of gemcitabine conjugated and miRNA-205 complexed micelles for treatment of advanced pancreatic cancer Anupama Mittal a, Deepak Chitkara a, Stephan W. Behrman b, Ram I. Mahato a, *,1 a b
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA Department of Surgery, University of Tennessee Health Science Center, Memphis, TN 38163, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 February 2014 Accepted 12 April 2014 Available online xxx
Clinical effectiveness of gemcitabine in pancreatic cancer is hindered due to its rapid plasma metabolism and development of chemo-resistance. We have previously delineated the significant role of miRNAs in mediating the growth and proliferation of cancer stem cells (CSCs) which in turn result in chemoresistance, invasion and metastasis. Here, we designed self-assembling, gemcitabine conjugated cationic copolymers for co-delivery of a tumor suppressor miRNA-205 (miR-205) and evaluated their in vivo efficacy in a pancreatic cancer ectopic tumor model developed using gemcitabine resistant MIA PaCa-2R cells. Combination formulations showed mean a particle size of 10% w/w, exhibited miRNA complexation at N/P ratio of 4/1, sustained release of gemcitabine for >10 days, transfection efficiency of >90%, extended miRNA and drug stability in serum. Functional assays in gemcitabine resistant MIA PaCa-2R and CAPAN-1R pancreatic cancer cells revealed that the combination formulations effectively reversed chemo-resistance, invasion and migration. In pancreatic tumor model, the combination formulation treated group showed significant inhibition of tumor growth. Immuno-hiostochemical analysis revealed decreased tumor cell proliferation with increased apoptosis in the animals treated with miR-205 and gemcitabine combination. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: miR-205 Gemcitabine Pancreas Cancer Micelles miRNA
1. Introduction Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies being the fourth leading cause of cancerrelated deaths in the United States, with a 5 year survival rate of 70% transfection efficiency while in case of SP and TP, complete complexation was achieved at 8:1 and 16:1 N/P ratios, respectively. To assess the efficacy of the combination formulation two gemcitabine resistant cell lines, MIA PaCa-2R and CAPAN-1R were used. Cell viability assessment indicated a significant improvement in the cytotoxicity of gemcitabine when incubated simultaneously with miR-205 using our gemcitabine conjugated cationic polymer. miR-205 can target several genes which are involved in cell proliferation, invasion and EMT and thus could potentially enhance the cytotoxicity of gemcitabine [44e47]. MIA PaCa-2R cells showed an upregulation of ZEB-1, SIP-1, HRAS and LRP-1 and downregulation of CAV-1 and E-CAD which could be potential targets for miR-205. Pancreas is the source of MIA PaCa-2 cells and exhibit epithelial morphology however its resistant version i.e. MIA PaCa-2R has fibroblastic morphology. The downregulation of E-CAD and CAV-1 and up-regulation LRP-1 in MiaPaCa-2R cells could play a significant role in EMT and make the cells more invasive as observed in the invasion and migration assays [44,47e49]. Further, CAV-1 could function in an oncogenic transformation of cells and metastasis and its loss may result in EMT and metastasis [54,55]. On transfection with miR-205 mimic using our combination formulations, we observed a reversal in the expression of these genes indicating their targeting by miR-205. Thus, the combination of miR-205 mimic with gemcitabine holds potential to provide a viable treatment strategy for advanced pancreatic tumor. Gemcitabine resistant MIA PaCa-2R cells were more invasive compared to the sensitive MIA PaCa-2 cells. Transfection with miR205 polyplexes had significantly reduced the invasive as well as migratory potential of these cells which is in line with our gene expression data. Tellez et al. [42] have previously reported that EMT and stem cell like properties in carcinogen induced transformation of human lung epithelial cells could be associated with the sustained epigenetic silencing of miR-205. The transformed cells were more migratory and invasive compared to non-transformed cells. Transient re-expression of miR-205 in these transformed cells prevented their growth in soft agar [42]. Further, Song and Bu have demonstrated the inhibition of tumor cell migration through ectopic expression of miR-205 [48]. Recently, in breast cancer cells also, re-expression of miR-205 has resulted in their decreased migration and invasion potential [44]. miR-205 has a broader interrelated role in inhibiting cell proliferation, EMT and reversing chemo-resistance. This in part is also associated with the small subpopulation of tumor initiating cells having stem cell like properties. The role of miR-205 in cell proliferation and resistance has been explored for other cancers such as prostate cancer [40]. In our
9
previous study [18], miR-205 transfection using Lipofectamine 2000Ò in resistant pancreatic cancer cells resulted in their sensitization however, this may not be a viable approach for in vivo application as the stability of miR-205 lipoplexes is severely compromised in the presence of serum (Fig. 3). The polyplexes prepared in the present study were able to reverse chemoresistance along with improving miR-205 stability. In vivo efficacy of the polyplexes was studied in ectopic tumor model developed using gemcitabine resistant MIA PaCa-2R cells in athymic nude mice. Promising results were obtained wherein miR205 polyplexes containing gemcitabine resulted in a significant reduction in tumor growth rate and tumor weight compared to the control groups (Fig. 8). Immuno-histochemical analysis also revealed reduction in cell proliferation and induction of apoptosis in the test group of miR-205 polyplexes. In addition to the significant improvement conferred by miR-205 replacement therapy, the experimental framework may be employed for assessing other tumor suppressive miRNAs as well as their combinations. In the past few years, several delivery strategies have been employed for delivering miRNAs including miR-34a [56], miR-29b [57] and let-7b [56,58]. However, the present system offers additional advantage of co-delivering highly hydrophilic drug gemcitabine in the same carrier system. This also ensures their similar in vivo biodistribution which may not be feasible by delivering these two agents in different carrier systems. In the present study, we used intra-tumoral route to first establish the proof-of-concept that these polyplexes were efficacious in the resistant cells growing in 3D tumor environment. To completely understand the applicability of the developed formulations, systemic delivery is warranted both in terms of pharmacokinetic and bio-distribution as well as efficacy. Although these formulations contain PEG shell which may provide stealth effect on systemic delivery and could increase the mean residence time, to widen the benefit of the approach, active targeting may be achieved by suitably tailoring the PEG end groups making them amenable to conjugation with small molecules, peptide or antibodies for active targeting to the pancreatic tumor. 5. Conclusions Our self-assembling, gemcitabine conjugated cationic copolymers efficiently form complexes with miR-205 of particle size
