Dynamic Contrast Enhanced MRI Assessing the Antiangiogenic Effect

Jun 5, 2016 - The biodegradable macromolecular MRI contrast agent GODP was synthesized as previously described.(18) The hetereobifunctional polyethyle...
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DCE-MRI assessing the anti-angiogenic effect of silencing HIF-1# with targeted multifunctional ECO/siRNA nanoparticles Anthony S. Malamas, Erlei Jin, Maneesh Gujrati, and Zheng-Rong Lu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00227 • Publication Date (Web): 05 Jun 2016 Downloaded from http://pubs.acs.org on June 8, 2016

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DCE-MRI assessing the anti-angiogenic effect of silencing HIF-1α with targeted multifunctional ECO/siRNA nanoparticles Anthony S. Malamas, Erlei Jin, Maneesh Gujrati, and Zheng-Rong Lu*

Case Center for Biomolecular Engineering, Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106, USA

Running Title: Targeted ECO/siHIF-1α Nanoparticles for Cancer Therapy

Key words: multifunctional lipid, DCE-MRI, siRNA, HIF-1, angiogenesis

Corresponding Authors: Zheng-Rong Lu, Case Western Reserve University, Department of Biomedical Engineering, Wickenden 427, Mail stop 7207, 10900 Euclid Avenue, Cleveland, OH 44106. Phone: (216) 368-0187. Email: [email protected]

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ABSTRACT Stabilization of hypoxia inducible factor 1α (HIF-1α), a biomarker of hypoxia, in hypoxic tumors mediates a variety of downstream genes promoting tumor angiogenesis and cancer cell survival as well as invasion, and compromising therapeutic outcome. In this study, dynamic contrast enhanced (DCE)-MRI with a biodegradable macromolecular MRI contrast agent was used to non-invasively assess the antiangiogenic effect of a RGD-targeted multifunctional lipid ECO/siHIF-1α nanoparticles in a mouse HT29 colon cancer model. The RGD-targeted ECO/siHIF-1α nanoparticles resulted in over 50% reduction in tumor size after intravenous injection at a dose of 2.0 mg-siRNA/kg every three days for three weeks as compared to a saline control. DCE-MRI revealed significant decline in vascularity and over a 70% reduction in the tumor blood flow, permeability-surface area product, and plasma volume fraction vascular parameters in the tumor treated with the targeted ECO/siHIF-1α nanoparticles. The treatment with targeted ECO/siRNA nanoparticles resulted in significant silence of HIF-1α expression at the protein level, which also significantly suppressed the expression of VEGF, Glut-1, HKII, PDK-1, LDHA, and CAIX, which are all important players in the tumor angiogenesis, glycolytic metabolism, and pH-regulation. By possessing the ability to illicit a multi-faceted effect on tumor biology, silencing HIF-1α with RGD-targeted ECO/siHIF-1α nanoparticles has great promise as a single therapy or in combination with traditional chemotherapy or radiation strategies to improve cancer treatment.

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INTRODUCTION Tumor hypoxia plays a central role in angiogenesis, proliferation, and resistance to cancer therapies. This state of oxygen deprivation often arises when aggressive tumors outgrow their vascular supply, and tend to develop an abnormal vascular network comprised largely of disorganized, heterogeneous, and structurally immature blood vessels that lack the proper functionality to maintain constant perfusion and oxygen tension throughout the entire lesion

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Although the inefficient supply of oxygen would suggest an eventual decline in tumor proliferation, numerous studies have demonstrated that intratumoral hypoxia in various types of cancer is actually associated with a highly proliferative and aggressive phenotype that is mediated by the transcription factor hypoxia inducible factor-1α (HIF-1α). The up-regulation of HIF-1α expression plays an essential role in tumor progression by inducing the transcription of various downstream genes that guide tumors to adapt and survive under hypoxic conditions. One mechanism by which HIF-1α contributes to cancer progression is by initiating the production and release of pro-angiogenic factors to expand the vascular network and meet the demand for nutrients and oxygen by the growing tumor. It is also responsible for switching cellular metabolism from an oxidative process to one that is highly glycolytic, as wells as imparting protective measures against glycolytic acid buildup, both of which provide selective growth advantages for tumor cells in the stromal tissue. In addition, hypoxic tumors that express high levels of HIF-1α are typically characterized by their increased risk of metastatic invasion and apoptotic resistance.

Consequently, tumor hypoxia, under the guidance of HIF-1α, is

associated with a highly proliferative and aggressive phenotype that is also largely resistant to various radiation and chemotherapy treatments 7. Therefore, silencing HIF-1α constitutes a promising strategy for future cancer therapies. One way to accomplish this is by developing a

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novel gene delivery platform that takes advantage of RNA interference (RNAi). RNAi is an inherent post-transcriptional gene regulatory process in cells, comprised of a complex set of enzymatic machinery that is able to induce sequence-specific degradation of target mRNA transcripts through the use of small interfering RNA (siRNA). The power of RNAi is now being harnessed for the development of new anti-cancer therapies that regulate the expression of cancer-related genes. Due to the ease of siRNA synthesis, RNAi can be utilized to regulate the expression of potentially any protein in the cell, regardless of where it is located. Such versatility is particularly attractive for cancer therapy because it allows for robust silencing of molecular targets that may not be compatible with conventional treatment modalities, including small-molecular inhibitors and monoclonal antibodies 8, 9. However, the therapeutic potential of RNAi is challenged by a number of barriers that impede the delivery of therapeutic siRNA first into the tumor site, and then into individual cancer cells 10. In order to maximize performance, siRNA needs to be formulated in a system that can protect it from serum nucleases in circulation, promote intracellular escape from the endocytic pathway, and then facilitate cytosolic delivery

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multifunctional pH-sensitive lipid carrier ECO that is able to electrostatically complex with siRNA and form stable nanoparticles with the aid of hydrophobic interactions and disulfide crosslinking. Upon cellular uptake in vitro, ECO/siRNA nanoparticles have demonstrated the ability to enhance endosomal escape, cytosolic siRNA release, and robust gene silencing events 13-16

. ECO also allows for facile modification of siRNA nanoparticles with tumor-specific

targeting agents for enhanced delivery of therapeutic siRNA following systemic administration. Intravenous injections of RGD-targeted ECO/siβ3 nanoparticles have recently been shown to effectively silence the expression of β3-integrin expression in tumors, alleviating primary tumor

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burden, and significantly inhibiting metastasis of triple negative breast cancer in mouse tumor models17. In this study, we investigated the effectiveness of targeted ECO nanoparticles of an antiHIF-1α siRNA (siHIF-1α) in antiangiogenic cancer therapy in a HT29 mouse colon cancer model. Dynamic contrast enhanced MRI (DCE-MRI) using the biodegradable macromolecular contrast agent GODP was employed to noninvasively and quantitatively assess the efficacy of the RGD-ECO/determine the anatomical and physiological changes of the tumor vasculature in response to the RNAi treatment

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Parametric values related to blood flow, vascular

permeability, and plasma volume were calculated by applying the adiabiatic approximation to the tissue homogeneity (AATH) model. The overall changes observed in the tumor vasculature with DCE-MRI were verified by analyzing the concurrent effects on CD31 and VEGF protein expression throughout the tumor tissue, the latter of which is known to be a downstream target of the HIF-1α transcription factor. The effect of HIF-1α silencing on the expression of multiple downstream targets involved in tumor metabolism and pH regulation was also investigated in this study 19.

MATERIALS AND METHODS Materials Anti-HIF-1α siRNA (siHIF-1α) (sense sequence: 5′-UCACCAAAGUUGAAUCAGA dTdT-3′, anti-sense sequence 5′-UCUGAUUCAACUUUGGUGAdTdT-3′) and negative control siRNA (siCon) (sense sequence: 5′-UUAGCGUAGAUGUAAUGUGdTdT-3′, antisense sequence: 5′-CACAUUACAUCUACGCUAA-3′) were purchased from Dharmacon (Lafayette, CO). AlexaFluor-647 labeled siHIF-1α siRNA was purchased from Qiagen (Valencia, CA).

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Cyclic RGD (Arg-Gly-Asp-D-Phe-Lys) and cyclic RAD (Arg-Gly-Asp-D-Phe-Lys) peptides were purchased from Peptides International Inc (Louisville, KY). The biodegradable macromolecular MRI contrast agent GODP was synthesized as previously described

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hetereobifunctional polyethylene glycol linker MAL-PEG-SCM (MW=5 kDa) was supplied by Creative PEGWorks (Winston Salem, NC). Primary antibodies for GLUT-1, CAIX, and HIF-1α were purchase from Novus Biologicals (Littleton, CO). The primary antibodies for VEGF and CD31 were purchased from Abcam (Cambridge, MA), while those for LDHA, PDK1, HKII, and β-actin were purchased from Cell Signaling (Danvers, MA). The secondary antibodies Dk-antiRb-HRP and Dk-anti-Rb-Alexa647 were purchased from Jackson ImmunoResearch (West Grove, PA). Pimonidazole hypoxia stain was supplied by Hypoxyprobe Inc (Burlington MA). The HT29 human colon adenocarcinoma cell line was purchased from ATCC (Manassas, VA), and athymic nude mice were purchased from Charles River Laboratories. Targeted ECO/siRNA Nanoparticle formulation The ECO cationic lipid carrier was synthesized using a solid phase reaction scheme that has been previously reported

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. The targeting peptides cyclic RGD (c-RGDfK) and RAD (c-

RADfK) were conjugated to the hetereobifunctional polyethylene glycol (PEG) linker MALPEG-SCM (MW=5 kDa) to yield Mal-PEG(5000)-RGD/RAD (RGD/RAD-PEG) conjugates. Stock solutions of the RGD/RAD-PEG conjugates and siRNA were prepared in nuclease-free water, while the ECO lipid was dissolved in ethanol. To formulate the particles, the maleimide groups on the peptide-PEG conjugates were first reacted with the free thiols on ECO at a 2.5:100 molar ratio (PEG:ECO) for 30 min. siRNA was then added to the mixture and stirred for another 30 min to allow for nanoparticle formation. Each formulation was prepared at an N/P ratio of 10, whereby the final volume for injection was 200 µL and the ratio of ethanol:water was 1:20. The

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RGD-targeted ECO/siRNA nanoparticles were 105 ± 6 nm in diameter, as demonstrated by dynamic light scattering, and possessed a zeta-potential of 9.7 ± 1.6 mV at neutral pH. By utilizing the Quant-iT RiboGreen RNA Assay kit (Thermo), we determined that the siRNA loading efficiency of our formulation process was 94%. Chemical structures of ECO, RGD, MalPEG(5000)-NHS, and the RGD-PEG conjugate, as well as a schematic of the final nanoparticle are displayed in Figure 1. Mouse model and siRNA delivery with ECO/siRNA nanoparticles A mouse model bearing subcutaneous HT29 colon adenocarcinoma flank xenografts was developed in accordance a protocol approved by the Institutional Animal Care and Use Committee for Case Western Reserve University. A total of 5x105 cells were inoculated into athymic nude mice in a 250 µL volume of Matrigel (BD Bioscienes). Tumors were allowed to grow to approximately 0.5 – 1.0 cm in diameter for two weeks before the treatment experiments. A total of 2 groups consisting of 3 mice each with HT29 xenografts with a size of approximately 1 cm in diameter were used for assess tumor siRNA delivery efficiency of the RGD- and RADtargeted ECO-siRNA nanoparticles. The nanoparticles were intravenously injected with a siRNA dose of 2 mg/kg. The siRNA was labeled with AlexaFluor-647 and tumor siRNA accumulation was assessed using a Maestro fluorescence imaging system (PerkinElmer) after the mice were sacrificed at 24 hours post-injection. Tumor treatment with RGD-ECO/siHIF-1α delivery system The anti-tumor efficacy of the RGD targeted ECO/siHIF-1α nanoparticles was investigated in a mouse model bearing subcutaneous HT29 colon adenocarcinoma flank xenografts with a size of approximately 0.5 cm in diameter. The mice (4 mice each group) were either treated with RGD-targeted ECO/siHIF-1α, RAD-targeted ECO/siHIF-1α, or RGD-targeted

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ECO/siCon nanoparticles. Another group received a phosphate-buffered saline (PBS) control therapy. Treatments were carried out by tail-vein injections of the therapeutic regimen on days 0, 3, 6, 9, 12, 15, and 18. All mice were sacrificed on day 21 after the start of treatment after DCEMRI. Nanoparticles were administered at a dose of 2 mg/kg of siRNA. Tumor growth was measured at several time points during the therapy with a caliper, and volumes were calculated using the formula V=(1/6)πD12D22, where D1 and D2 were two diameters measured along perpendicular axes of the tumor lesion. Dynamic contrast enhanced magnetic resonance imaging On day 21, DCE-MRI was performed on each mouse from both the saline control and RGD-targeted ECO/siHIF-1α treatment groups. Each mouse was catheterized and injected with a bolus of GODP at a dose of 0.1 mmol-Gd/kg at the start of the imaging sequence. A T1-weighted 3D-FLASH gradient echo (GE) sequence was used for the DCE-MRI data acquisition on a 7T pre-clinical scanner (Bruker). Sequential MR images of the tumor were acquired before, during, and after the injection of the contrast agent using a imaging protocol described in a previous publication 18. The signal intensity of the MR images of the regions of interest was determined and converted into concentration of the contrast agent by using the variable flip angle technique

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Concentration-time curves were formulated for both the tumor tissue and a major artery near the lesion, which served as the arterial input function for pharmacokinetic modeling. These concentration-time curves were parametrically curve-fitted to the AATH model to calculate blood flow (Fp), permeability-surface area product (PS), and plasma volume fraction (Vp) in the tumor. The concentration-time curves from the tumor were also integrated in order to determine the extent of contrast agent accumulation within the lesion. This calculation was performed over

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the entire duration of the scan, and also over only the first 90 seconds, in order to obtain total area-under-the-curve (AUC) and initial AUC (iAUC) measurements, respectively. Immunofluorescence and western blotting After completing DCE-MRI acquisition, each mouse from the control and RGD-targeted ECO/HIF-1α groups was intravenously injected with 60 mg/kg of pimonidazole, an established marker for the detection of tissue hypoxia. The tumors from these mice were resected one hour after pimonidazole administration in order to determine changes in HIF-1α, Glut-1, HKII, PDK1, LDHA, CAIX, VEGF, CD31, and hypoxia using western blotting and immunofluorescence analysis. The western blots from all 4 mice in each group are presented throughout this paper. Image J software was utilized to quantify relative protein expression from the blots. H&E staining was also performed to determine the extent of necrosis in each treatment group. Statistical Analysis Statistical analyses were performed using unpaired, two-tailed Student’s t-tests with a 95% confidence interval, assuming equal variances.

Probability values of p