Formation of Immunoliposomes used for the Targeted Delivery of Mutant KRAS siRNA for the treatment of Pancreatic Cancer   Abstract: Pancreatic cancer is one of the dangerous and deadliest forms of cancer diagnosed in cancer patients worldwide. Pancreatic ductal adenocarcinoma (PDAC) being the most common among different This increase in the mortality rate is mainly because of the lack of early symptoms and early detection of the disease. Gemcitabine along with cetuximab has been the standard form of therapy for the cancer patients but it is still far from the optimal and novel therapy which is required for this disease. Hence, there is a need for the development of a novel drug treatment. Mutations in the Kras gene has been found in about 90% of the pancreatic cancer case and is the major source for the malignancy. Thus, the goal of this study is to investigate the silencing of this gene which will eventually inhibit the pancreatic tumor growth. In order to this, I combined the k-ras small interfering RNA (siRNA) with the lipid molecule to increase the stability of siRNA, which in turn is associated with an anti-EGFR antibody. The pancreatic cells overexpress many protein receptors like the epidermal growth factor receptor (EGFR) so the anti-EGFR antibody will target the siRNA/liposome complex to the target the tumor cells. The liposomes are PEGylated so as to increase the circulation time and effective delivery of the drug target system. This cargo will be tested both in-vitro and in-vivo to evaluate the silencing of the desired gene. Objective: Development of liposomal-based targeted siRNA delivery for the treatment of pancreatic cancer. A. Specific aimsThis project aims to design an effective drug delivery system for pancreatic cancer combining multiple strategies. Pancreatic cancer arises because of the genetic and accumulated alternations (1, 2). It is well known that the most common type of genetic mutation abnormality in pancreatic adenocarcinomas is the kras mutation (3). In 70-90% of the cases point mutation occur in k-ras gene, the majority occurring at codon 12 of oncogene (4,5). Therefore, efforts have been made to define the function of Ras in normal and diseased cells and to target ras for cancer therapy.KRAS being a GTPase is activated by GTP and deactivated by GDP. The activated Kras binds and thus activates the RAF family kinases, BRAF, ARAF and RAF1(6). Activated RAFs is responsible for phosphorylating and activating ERK1 and ERK2 kinases. Later ERK phosphorylates nuclear and cytoplasmic transcription factors like ELK1 and c-JUN which leads to cell proliferation. Therefore, the mutation which causes the activation of K-ras leads to uncontrolled cell growth which eventually leads to cancer development and spreading (7). SiRNA will be used as a therapeutic drug to suppress KRAS expression. This siRNA mediated reduction in the expression of mutated KRAS in the pancreatic cells can reduce cell proliferation thus reducing the tumor growth (8, 9). Thus, to make sure that siRNA will preferentially target the cancer tissues rather than the normal tissues, cancer-specific antibody can be used for targeting purpose. One of the trait seen in cancer is that there is an overexpression of certain protein receptors on the cell surface. Pancreatic cancer cells have high expression of Epidermal Growth Factor Receptor (EGFR), which can be used as a drug delivery target against pancreatic cancer cell lines. Hence, the targeting moiety (anti-EGFR antibody) will be attached on the surface of liposomes to target specific cancer cell delivery.                         Figure 1: KRAS PathwayAim 1: Preparation of liposomes and siRNA complex as a target-specific drug delivery system to treat pancreatic cancera)      Preparation of maleimide-terminated PEG-DSPEb)     Preparation of the liposome and siRNA complex Aim 2: Preparation of antibody-liposome-siRNA- conjugate and evaluation of this complex for the treatment of the cancer cellsThe anti-EGFR antibody will be chemically conjugated to the siRNA-liposome complex and the resulting conjugate will be tested for antibody binding efficiency and its ability to deliver Kras-siRNA to the pancreatic cancer cell.Aim 3: In vitro testing in cell lines for cytotoxic studies.This involves testing the efficacy and toxicity profile of the conjugated cargo. The transfection efficiency, silencing effect and cell viability difference will be measured between the pancreatic cancer cell lines and the normal cell lines. Aim 4: In vivo testing in mice models: Ultimately the therapeutic efficacy will be tested in xenograft mice model. The effects on the tumor in mice will be checked after injection. B. Background and SignificanceCancer is a disease condition in which the cells have the ability to invade other parts of the body. Pancreatic cancer is a condition in which the cells in the glandular organ behind the stomach, the pancreas, starts to grow abnormally and form a mass. Pancreas consists of two main types of cells:Exocrine cells: These cells produce pancreatic enzymes which helps in the intestinal digestion.Endocrine cells: These are present as a small group which forms the Islets of Langerhans. These cells are responsible for the synthesis of insulin and glucagon in the body which helps in maintaining the blood sugar levels in the body.            Figure 3:On and Off switch for KRAS pathway             Figure 2: Anatomy of the pancreas.TYPES OF PANCREATIC CANCERS:                Figure 2: Anatomy of the pancreas.                                          Exocrine Pancreatic cancer: The most common type of cancer due to pancreatic adenocarcinoma as the pancreas are made up of 95% exocrine pancreas. Endocrine Pancreatic Cancer: Cancerous tumors which arise from the other cells of the pancreas that make hormones and are released into the bloodstream, are called pancreatic neuroendocrine tumors. This type of cancer is not very common.Among many types of pancreatic cancer Pancreatic ductal adenocarcinoma (PDAC) is the most common. These adenocarcinomas start within the part of the pancreas which makes the digestive enzymes. Pancreatic ductal adenocarcinoma stands out to be one of the deadliest diseases with worst conditions. Yearly, 37000 patients are diagnosed with cancer and among them 34000 leads to deaths (10). PADAC is among the fourth leading cause of death in the United States (11). From the last two decades, various efforts have been taken to improve the survival rate for this cancer using regimen like chemotherapy, radiation therapy, immunotherapy and gene therapy, but the results were suboptimal. Pancreatic cancer is usually diagnosed at a stage where it cannot be treated because of the lack of early detection methods and the absence of early symptoms (12, 13). Therefore, after the conventional therapies the median survival rate is only 5 to 6 months, resulting in less than 5% overall 5-year survival rate (13). The late diagnosis followed by intrinsic (de novo) and extrinsic (acquired) therapeutic resistance of pancreatic cancer for the conventional therapies becomes the major disappointing survival outcome.KRAS plays a vital role in PDAC and is believed to be a target for treatment. KRAS which is also known as K-Ras2, Kras, c-Kras, or c-Kiras is a small GTPase (21 kDa), responsible for binding guanosine triphosphate and diphosphate nucleotide (7). It is activated by GTP and deactivated by GDP. KRAS is also responsible in regulating another signaling pathway, for instance, PI3K-AKT, PLC-PKC, and RAL, which are also involved in the cancer progression (14). The active KRAS mutates the codons G12, G13 or Q61. Mutations in codon 12 of KRAS occur most frequently. The mutation consists of the substitution of Glycine instead of Aspartate. 95% of pancreatic cancers have active mutations in KRAS and modifications in G12. Clinical studies states that the mutations in KRAS can be significantly used as a biomarker, along with a tool for therapy (7).The  advantage of RNA interference (RNAi)To treat the human cancer various strategies have been developed which target kras. We are proposing to utilize the potential of RNAi as it is emerging as a powerful tool to control the expression of any protein at the post-transcriptional level (15). Small interfering RNA (siRNA) which are typically 21-23 base pair long dsRNA, are designed to degrade a specific mRNA. The siRNA can bind and silence a particular mRNA strand and prevent it from undergoing translation and ultimately blocks the protein expression. Its working can be described as follows. When the ds-siRNA enters the cell, it is cleaved into two strands by an enzyme DICER. After its incorporation into RISC, one of the strands gets degraded and the other strand makes a complementary pair with the mRNA and thus cleaves the mRNA by Argonaute component of the RISC. With the degradation of mRNA, no gene expression occurs. The siRNA can be targeted for any harmful gene, knockdown its expression and stop the tumor growth. Thus siRNA would be a very effective therapeutics and its potential can be tested for the treatment of pancreatic cancer. Since the siRNA is labile in the serum, there are efforts to protect it from the degradation to increase its half-life and overall efficacy. It involves encapsulating it into liposome, cationic nanoparticles or chemically modifying the siRNA itself (16, 17). All of these systems are under investigation and all have their associated benefits and challenges. Many protein targets which are overexpressed in pancreatic cells are identified. Therefore, taking advantage of these overexpressed protein targets we can target our drug-delivery system along with the silencing effect of KRAS which can be identified for the cure of pancreatic cancer. EGFR are highly expressed on the pancreatic tumor cells and hence can be used to target the siRNA for silencing the mRNA. There are two strategies to circumvent the tumor progression, first by using stable anti-KRAS siRNA, siKras sense strand, 5’-AGUUGGAGCUGAUGGCUAdtdt-3’ and a siKras antisense strand, 3’-dtdtTUCAACCUCGACUACCGCAU-5’(18). This is followed by blocking of EGFR with an appropriate antibody which may reduce the proliferation of the tumor and induce apoptosis in cancer cells. This delivery of the siRNA-liposome complex with the targeting moiety, that is the anti-EGFR antibody, which not only provides ligand characteristics for tumor but also downregulates the tumor growth. Liposomal siRNA based delivery system is one of the most studied and most promising delivery system. It is easy and comes with its biocompatible and biodegradable nature. Electrostatic interactions help the lipids to assemble with siRNA easily with charged amines, thereby generating multilamellar lipoplexes with positively charged lipid bilayers which are separated by a sheet of negatively charged siRNA strand. This whole complex is called liposomes. The siRNA being hydrophilic can also be in cooperated inside the core of liposome. However, off-target delivery, poor distribution, efficient and safe delivery of the siRNA is still a challenge. To improve this targeting of siRNA to tumor cells, attachment of PEG (polyethylene glycol) to the liposomes in conjugation of cancer specific ligands to the liposomal surface provides interaction with the cancer cells. (19) Therefore, in order to solve this problem different kinds of ligands were used which includes the antibody fragments, peptides (RGD-sequences) and oligonucleotides aptamers (20,21,22). Among all these delivery systems antibody conjugated with liposomes are used extensively. This conjugation is referred to as immunoliposomes. When we compare the untargeted liposomes with targeted liposomes, the latter is more beneficial with respect to efficient localization to the tumor sites. In addition, it also increases the interaction with the tumor cell population once it reaches the appropriate tumor site (19). Designing the immunoliposomes appropriately with ionizable amino lipid, phosphatidylcholine, cholesterol and lipid coat helps in successful attachment of the target ligand to the surface and hence deliver the siRNA. Therefore, utilizing the basic concepts of molecular biology for cancer metastasis, development of drugs and its formulation may provide a targeted cancer therapy and be therapeutically successful. C. Preliminary StudiesFigure 4: Knockdown in the expression of mutant KRAS using PEG-PLL/siRNA   Figure 5: Knockdown in the expression of Bmi-1 with PEG-PLL/siBA) This preliminary data shows that the siRNA combined with cationic polymer PEG-PLL creates a knockdown in mutant KRAS when transfected into pancreatic cancer cell lines. Zeng et al. in a study showed that the siRNA mediated by PEG-PLL for mutant KRAS was transfected in either Panc-1 or Bxpc-3 cell lines. The experiment was evaluated both the mRNA (Figure A) and the protein expression level (Figure B). The results showed that there was a downregulation of Kras mRNA and protein level in Panc1 cell line as compared to control cells which were transfected with PEG-PLL only. The study also showed that the wild-type Kras expression was not affected by the transfection. In contrast to the Panc-1 cell line, the Bxpc-3 cell lines were not transfected with the PEG-PLL/siRNA or lacked some necessary machinery for RNAi which was proved by using another siRNA that targets Bmi-1 oncogene (used as a control). The expression of Bmi-1 was suppressed using PEG-PLL/siB in both the cell lines    B) Another Preliminary in-vivo data further proved that there was an inhibition of the endogenous k-ras by siRNA. The inhibition of KRAS expression leads to the inhibition of pancreatic tumor growth. Zorde Khvalevsky et al. used LODER-derived siG12D to check the effects on the tumor growth. LODER is a biodegradable polymeric minute matrix which accommodates siRNA. The main aim of designing LODER was to protect siRNA and slow release of drug within the tumor for a period of few months. In the study siG12D LODER was implanted in the subcutaneous implantation (Figure A) followed by the quantification of the necrotic area which showed that it exceeds 60% of the total tumor tissue area (Figure B). Therefore, the study showed silencing of KRAS inhibited subcutaneous tumor growth (23).Thus, these preliminary data show that the siRNA knockdowns the mutant K-ras which in turn inhibits the pancreatic cancer cell proliferation and tumor growth.Figure 6 A Effect on tumor after insertion of LODER derived siG12D. Figure 6 B Necrotic area increase by 60% with LODER-siG12DD. Research Design and Methods:  Aim 1: Preparation of liposomes and siRNA complex as a target-specific drug delivery system to treat pancreatic cancer Aim 1a) Preparation of maleimide-terminated PEG-DSPE (Mal-PEG-DSPE) PEG-DSPE is usually modified with the attachment of a malemide group at the end of PEG chain to modify targeted delivery because it is convenient and reacts quickly with the other ligands like antibody and peptide (24, 25).             Figure 7:  Synthesis of Mal-PEG-DSPEThere were two methods utilized for the synthesis of PEG-DSPE-Mal. The first method consisted of amino-PEG-DSPE and N-succinimidyl-3-(N-maleimido)-propionate which was dissolved in methylene chloride and DMF followed by trimethylamine. In the second method, MP-PEG-SC was reacted with DSPE and trimethylamine in the presence of chloroform for 4 hours at 45?C (26)Aim 1 b) Preparation of the liposome and siRNA complexFor the preparation of liposomes: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)- 2000] (DSPE-PEG2000-Mal) (2:1:0.1) will be dissolved in a mixture of chloroform and methanol (4:1 v/v). The organic part of solvent will be evaporated under pressure at 40?C for 30 min and the lipid film will be flushed with N2 to remove residual solvent. The dried lipid film will be hydrated using a solution of siRNA in 5% dextrose (w/v) prepared using RNase-free water. The amount of siRNA used will be calculated according to the charge. Size reduction will be performed with the conventional liposomes: sonication in a water bath sonicator for 1 min, which will be followed by extrusion through a 0.2?m Anotop 10 filter. The PEGylated liposome/ siRNA solution will be incubated at room temperature for around 30 min to allow stabilization. This complex will be maintained in a sterile environment for subsequent gene silencing experiments. (27)Aim 1 c) Liposome size and Zeta potential measurement.Using laser Doppler velocimetry and photon correlation spectroscopy (PCS)/ dynamic light scattering, in a Zetasizer the particle size and zeta potential of the liposome/siRNA complex can be confirmed.  (27)Aim 1 d) Evaluation of the siRNA complex with liposomes: The complex will be evaluated by gel retardation assay. The liposomes will be loaded on 1% agarose gel, electrophoresed and visualized under UV. We are assuming that the encapsulated siRNA in the well will be completely retarded in the wells than the ones which are not encapsulated.Aim 2: Preparation of antibody-liposome-siRNA- conjugate and evaluation of this complex for the treatment of the cancer cells. Aim 2 a) Preparation of the antibody-liposome conjugate through sulfide bond linkage.                                                       Figure 8: Synthesis of an antibody-liposome conjugate DSPE-PEG-Mal will be used to attach the thiol terminal of the modified fragment of the antibody, for the conjugation on the surface of liposomes. Anti-EGFR antibody will be reacted with Traut’s reagent thiolated in the presence of phosphate buffer for 1 hr at room temperature followed by immediate conjugation of the thiolated antibodies to the maleimide groups at the distal termini of PEG chains (0.2:1 molar ratio of Ab and maleimide), and overnight incubation at 4?C with continuous shaking.Aim 2 b) Evaluation of Ab-liposome conjugationTo evaluate the presence and integrity of antibody on the liposome, SDS-PAGE will be run. Compared to the control sample the antibody-liposome complex is expected to have slightly larger molecular weight. The experiment will confirm that the antibody conjugated to liposome has slower swimming motility in the gel. No conjugated antibody should be detected by a higher motility rate. (Mention the reference)Aim 3: In vitro testing in cell lines for cytotoxic studies.Aim 3 a) Cellular uptake and silencing effect: Human pancreatic cancer cell lines PANC-1 will be cultured in Dulbecco’s modified Eagle’s medium and was supplemented with 10% Fetal Bovine serum with 4mM L-glutamine and penicillin and streptomycin at 37?C in 5% CO2 and 95% humidity (18).In-vitro gene silencing- 5×104 PANC-1 cells lines will be seeded into 24 well plates and the culture will be maintained for 24 hours. After 24 hours, the immunoliposomes and the control will be added in the medium with varying concentration ranging from 25-100 nM. The transfected cells will be washed with PBS and RNA isolation will be done using Trizol reagent. cDNA will be synthesized using the reverse transcription kit. The gene silencing will be measured using quantitative PCT (RT-PCR), using primers for Kras (forward primer: 5’-CAGGGTACTTCATTGATGCCACAAC-3’) and (reverse primer: 5’-GCTGGTCTCCAGGTAACGAACAATA-3’) with ?-actin used a control with primer pairs (forward primer: 5’ TGGCACCCAGCACAATGAA-3’) and (reverse primer: 5’-CTAAGTCATAGTCCGCCTAGAAGCA-3’). A negative control will be used (without cDNA) to detect any contamination. The relative transcription level of the Kras gene will be calculated by the  2–??Ct. normalized to the level of the housekeeping gene B-actin (18).To check the protein expression level western blot will be done. The cells will be exposed to the siRNA for 24 hours and will be washed with the cold PBS twice and then lysis of the cells will be done. The lysate will be loaded on SDS-PAGE and later transferred to the polyvinylidene difluoride membrane. The membrane will be blocked with 5% non-fat milk powder and will be incubated overnight at 4?C with primary antibody. After 24 hours, the membrane will be incubated with secondary antibody which is peroxidase conjugated for 2 hours at room temperature. Later the bands for proteins will be visualised. The control used will be ?-actin (18).Aim 3 b) In-vitro cytotoxicity assay:The MTT cytotoxicity assayTo evaluate the anti-tumor activity of the siRNA conjugated immunoliposomes, the viability of the transfected PANC-1 cells will be tested by MTT assay. Briefly, 1×104 Panc1 cells will be added per well in 96 well plate and cultured for 24 hours at 37?C. The immunoliposome and siRNA complex will be added with a concentration range from 25-100 nM. MTT solution will be added in the cells and will be incubated for 4 hrs. The media will be later removed from each well and the formazan crystals formed during the process will be dissolved using Dimethyl sulfoxide. 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