Cosilencing Intestinal Transglutaminase-2 and Interleukin-15 Using

Subscriber access provided by UNIV OF NEWCASTLE

Article

Co-Silencing Intestinal Transglutaminase-2 and Interleukin-15 using Gelatin-Based Nanoparticles in an In Vitro Model of Celiac Disease Husain Attarwala, Valerie Clausen, Prasoon Chaturvedi, and Mansoor M. Amiji Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00233 • Publication Date (Web): 24 Jul 2017 Downloaded from http://pubs.acs.org on July 25, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Co-Silencing Intestinal Transglutaminase-2 and Interleukin15 using Gelatin-Based Nanoparticles in an In Vitro Model of Celiac Disease

Husain Attarwala1,2, Valerie Clausen2, Prasoon Chaturvedi2, and Mansoor M. Amiji1*, 1

Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, Boston, MA 02115 USA 2 Alnylam Pharmaceuticals, Inc., Cambridge, MA 02142 USA

Running title: Co-Silencing TG-2 and IL-15 with Gelatin Nanoparticles

*Corresponding author: Tel. (617) 373-3137 Fax (617) 373-8886 Email: [email protected] 1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT In this study, we have developed type B gelatin-based nanoparticle based siRNA delivery system for silencing of intestinal transglutaminase-2 (TG2) and interleukin-15 (IL-15) genes in cultured human intestinal epithelial cells (Caco-2) and murine alveolar macrophage cells (J774A.1). Small interfering RNA (siRNA) targeting the TG2 or IL-15 gene was encapsulated within gelatin nanoparticles using ethanol-water solvent displacement method. Size, charge and morphology of gelatin nanoparticles were evaluated using Zetasizer instrument and transmission electron microscopy. siRNA encapsulation efficiency was determined using an siRNA specific stem-loop quantitative polymerase chain reaction (qPCR) assay. Cellular uptake of siRNA containing gelatin nanoparticles was determined using fluorescent microscopy and stem-loop qPCR assay. siRNA loading in the RISC (RNA-induced silencing complex) was determined by immunoprecipitation of argonaute 2 (AGO2) protein followed by stem-loop qPCR for siRNA quantification. Gene expression analysis of TG2, IL-15 and the proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), was performed using qPCR assays. Efficacy of silencing TG2 and IL-15 knockdown was evaluated in an in vitro model of celiac disease by utilizing immunogenic α-gliadin peptide p31-43 in cultured J774A.1 cells. siRNA containing gelatin nanoparticles were spherical in shape with mean particle size and charge of 217±8.39 nm and -6.2±0.95 mV, respectively. siRNA loading efficiency within gelatin nanoparticles was found to be 89.3±3.05%. Evaluations of cellular uptake using fluorescent microscopy showed rapid internalization of gelatin nanoparticles within 2 h of dosing, with cytosolic localization of delivered siRNA in Caco-2 cells. Gelatin nanoparticles showed greater intracellular siRNA exposure with a longer half-life, when compared to Lipofectamine mediated siRNA delivery. Approximately 0.1% of total intracellular siRNA was associated in the RISC complex. A maximum knockdown of 60% was observed at 72 h post siRNA treatment for both, TG2 and IL-15 genes, which corresponded to ~200 copies of RISC associated siRNA. Further, efficacy of gelatin nanoparticle-mediated knockdown of TG2 and IL15 mRNA was tested in an in vitro model of celiac disease. Significant suppression in the levels of proinflammatory cytokines (TNF-α and IFN-γ) was observed in p31-43 stimulated J774A.1 cells upon either IL-15 or IL-15+TG2 siRNA treatment. The results from this study indicate that gelatin nanoparticle-mediated TG2 and IL-15 siRNA gene silencing is a very promising approach for the treatment of celiac disease.

Keywords: Type B gelatin nanoparticles, transglutaminase 2, interleukin-15, gene silencing, celiac disease. 2 ACS Paragon Plus Environment

Page 2 of 33

Page 3 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. INTRODUCTION Celiac disease is a disease of the small intestine and is caused by T-cell mediated immune response against deamidated cereal gluten peptides modified by the enzyme transglutaminase 2 (TG2) (1, 2). Currently an estimated 1% of general population in United States and Europe is affected by this disease. Patients with celiac disease have gastrointestinal abnormalities such as abdominal pain, bloating, diarrhea and constipation along with nutritional malabsorption resulting in deficiencies of iron, vitamin D, vitamin K and weight loss (3), which are reversed in majority of patients by gluten-free diet. A minority of patients suffer from a more severe version of celiac disease, called refractory celiac disease, and are resistant to recovery even after adhering to a gluten-free diet (4, 5). Pathogenesis of celiac disease involves a combination of dietary, genetic and immunological mechanisms (6-9). Gluten peptides produced by the digestion of proteins present in dietary cereal grains (such as wheat, rye and barley) get deamidated by enzyme tissue transglutaminase 2 (TG2), rendering them recognizable by HLADQ2/8 complexes at the antigen presenting cells (6, 10, 11). All patients diagnosed with celiac disease have HLA-DQ2 or –DQ8 alleles at the HLA-DQ locus. The cytokine IL-15 is overexpressed in almost all patients with celiac disease. IL-15 plays a vital role in the activation of intraepithelial lymphocytes (IELs) and in increasing the permeability of the intestinal mucosa. IELs that are activated by IL-15 in patients with celiac disease behave more like natural killer (NK) cells than do typical antigen-specific T cells; upon further activation, these IELs can cause direct mucosal damage (6). Gluten peptides (such as the peptide p31-43) that are in the aminoterminus of α-gliadin cause upregulation of IL-15 (up to 5-fold increase) in epithelial cells (Caco-2 cells) when tested in vitro using a cell culture system (12). α-Gliadin peptides also augment the production of IL-15 by intestinal epithelial cells, leading to an increase in the

3 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

infiltration and activation of IELs, and in turn resulting in apoptosis of intestinal epithelial cells, when evaluated in a human intestinal mucosal organ culture model system (13). In a separate study, an antibody-mediated blockade of IL-15 resulted in the reversal of intestinal damage in a transgenic mouse model of celiac disease (14). Collectively, the underlying mechanism responsible for the development of celiac disease indicates that tissue transglutaminase 2 (TG2) and IL-15 play crucial roles in development and progression of celiac disease. In the current study, we have utilized the RNAi (RNA interference) technology for lowering TG2 and IL-15 genes aimed at treating celiac disease. RNAi is a biological mechanism, where double-stranded small interference RNA (siRNA) cleaves messenger RNA (mRNA) based on complementary base pairing. siRNA based therapy has tremendous potential for the treatment of various conditions caused by overexpression of disease causing proteins, especially those intractable by traditional small molecule approaches. siRNA delivery is not very straightforward due to the presence of a number of extracellular and intracellular barriers. Extracellular barriers include rapid siRNA degradation by physiological enzymes such as nucleases in blood and tissues, hindrance to cellular uptake due to barriers such as electrostatic repulsion between negatively charged cell membrane and siRNA, physical barriers of mucus (in case of mucosal routes of delivery) and lack of stability at various physiological pH values. Once inside the cell, siRNA molecules have to make their way into cellular cytoplasm by overcoming the endo-lysosomal pathway. In order to facilitate siRNA mediated effects on target gene, a delivery system needs to be designed such that these delivery barriers are bypassed and siRNA is released in the cellular cytoplasm for association with RNA induced silencing complex (RISC), needed for target mRNA cleavage. Gelatin based delivery systems have been developed and utilized in our laboratories over the past


Page 4 of 33

Page 5 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


decade for delivery of plasmid DNA, siRNA and microRNA (15-22). In a recent study, Kriegel et al. have developed a multi-compartmental siRNA system for oral delivery of siRNA targeting inflammatory genes. This system, when tested in vivo, showed efficient silencing of TNF-α and cyclin-D1 after oral administration to mice, as well as effective resolution of disease symptoms in an animal model of inflammatory bowel disease (18, 23). In our current study, we have developed gelatin nanoparticle based TG2 and IL-15 siRNA delivery systems for the treatment of celiac disease. This system was evaluated for kinetics of cellular uptake, siRNA loading in RISC and efficacy of TG2 and IL-15 gene silencing in an in vitro model of celiac disease.

2. MATERIALS AND METHODS 2.1. Materials Type B gelatin polymer bloom strength 225 was obtained from Sigma-Aldrich (St Louis, MO). siRNA targeting factor VII siRNA was obtained from Alnylam Pharmaceuticals.

siRNA

targeting TG2 and IL-15 were obtained from Life Technologies, NY. α-Gliadin peptide p31-43 was obtained from Peptides, Inc (Rocky Hills, NJ). Antibody against human Argonaut 2 protein was obtained from Wako Chemicals (Richmond, VA). Primers, probes and gene expression assays were purchased from Life Technologies, NY.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 33

2.2 Formulation and Characterization of Gelatin Nanoparticles Gelatin nanoparticles were formulated using an ethanol-water solvent displacement method, as described previously (16). Briefly, 25 mg of type B gelatin of bloom strength 225 (Sigma-Aldrich, St Louis, MO) were dissolved in 20 ml of deionized water at 37°C; the pH of the solution was adjusted to 7.0 (with 0.2 M NaOH solution).

siRNA at 1% w/w final

concentration of gelatin was added to this solution, followed by precipitation of gelatin nanoparticles by addition of absolute ethanol to final concentration of 65% v/v. The resulting gelatin nanoparticles were cross-linked by drop-wise addition of 500 µL of 4% glyoxal solution, with constant stirring. Excess cross-linking reagent was quenched by addition of 100 µl of 1 mM glycine solution. Ethanol was removed by using tangential flow filtration. The aqueous gelatin nanoparticle suspension thus obtained was then concentrated to a final gelatin concentration of 5 mg/mL, and stored at 4°C until used for further studies. Particle size and charge of gelatin nanoparticles were determined using Malvern’s Zeta-sizer® ().

siRNA loading in gelatin

nanoparticles was determined using a custom designed qPCR assay (24) that employed stemloop primers for Factor VII and TG2 siRNA. Gelatin nanoparticles were digested by incubation with protease buffer (0.2 mg/mL) at 37oC for 30 minutes. The resulting solution was heated with 1:1 solution of 0.5% Triton-X 100 in 1x PBS buffer at 95oC for 15 minutes, which was then used for the qPCR analysis. Primers and probes used for the factor VII and TG2 siRNA stem-loop qPCR assay are listed in Supplementary Table 1.

2.3 Qualitative Microscopic Assessment of Cellular Uptake and Distribution To determine whether Caco-2 cells can internalize gelatin nanoparticles, and subsequently release encapsulated siRNA within the cellular cytoplasm, we undertook cellular


Page 7 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


uptake and intracellular trafficking studies. To visualize localization of gelatin nanoparticles, siRNA, and the cell nucleus under a confocal fluorescence microscope, all of the above three were labeled with distinct fluorescent dyes. Prior to formulation into nanoparticles, gelatin polymer was conjugated with Alexa Fluor 488 dye (green) (Life Technologies, CA) as per the manufacturer’s protein-dye conjugation protocol. siRNA labeled with Alexa Fluor 647 dye (red) was obtained from Alnylam Pharmaceuticals (Cambridge, MA), and used for encapsulation in labeled gelatin polymer-based nanoparticles. This formulation was tested for cellular uptake and intracellular trafficking of formulation components. To this end, Caco-2 cells were plated in a 96-well plate at a count of 20,000 cells per well, and allowed to attach to the well surface for 12 h; after this, fluorescently labeled formulations were incubated with Caco-2 cells in a serum-free cell culture medium, for periods of 0.5 h, 1 h, 2 h, 4 h, 6 h or 8 h, at a 0.5 nM siRNA concentration. Cells treated with unlabeled formulation and untreated cells served as controls. At each time point, cell nuclei were stained with the nuclear stain Hoechst 405 dye (blue) (Life Technologies, CA) for ten minutes. following which the cells were fixed with 4% paraformaldehyde and stored at 4⁰C until imaged. An Opera High Content Screening System (manufacturer) was used to capture images for the green, red and blue signals emitted from each well. For each well, a set of 20 random images were captured by the instrument and quantified for the integrated pixel density for green and red signals.

2.4 Quantitative Analysis of Intracellular siRNA Uptake We quantitatively evaluated the intracellular kinetics of siRNA uptake and clearance, after incubation with gelatin nanoparticles or with the commercial transfection reagent Lipofectamine 2000. For this purpose, Caco-2 cells were cultured using Dulbecco's Modified



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Eagle Medium (DMEM), with or without 10% fetal bovine serum at 37°C and 5% CO2. Factor VII siRNA-encapsulated gelatin nanoparticles, or factor VII siRNA complexed with Lipofectamine 2000, were incubated with Caco-2 cells, and seeded at 1x106 cells per T25 flask in serum free media for a period of 24 h, at a final siRNA concentration of 100 nM. Cell culture media containing siRNA formulation was removed, and cells were washed three times with 1xPBS, followed by incubation with formulation-free, serum-containing cell culture media for time points up to 168 h. At each time point, cells were detached using trypsin, centrifuged, and washed 5 times with 1x PBS. After this step, cells were lysed with 0.25% triton in 1xPBS buffer at 95⁰C for 15 minutes, and the lysate was stored at -80⁰C until analyzed. Stem-loop qPCR assay for factor VII siRNA was performed for each sample, in order to quantify cellular siRNA content. Primer and probe sequences used for this assay are listed in Supplementary Table 1.

2.5 Quantitative Analysis of RISC Associated siRNA in Caco-2 Cells For quantitative evaluation of the intracellular kinetics of RISC associated siRNA after incubation with gelatin nanoparticles or with the commercial transfection reagent Lipofectamine 2000, Caco-2 cells were cultured using Dulbecco's Modified Eagle Medium (DMEM), with or without 10% fetal bovine serum at 37°C and 5% CO2. Factor VII siRNA-encapsulated gelatin nanoparticles, or TG2 siRNA complexed with Lipofectamine 2000, were incubated with Caco-2 cells, and seeded at 3x106 cells per T75 flask in serum free media for a period of 24 h, at a final siRNA concentration of 100 nM. Cell culture media containing siRNA formulation was removed, and cells were washed three times with 1xPBS, followed by incubation with formulation-free, serum-containing cell culture media for time points up to 168 h. At each time point, cells were detached using trypsin, centrifuged, and washed 5 times with 1x PBS. RISC


Page 8 of 33

Page 9 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


loading assay to detect Ago2 (argonaute-2)-associated TG2 siRNA in Caco-2 cells was carried out by sonicating a 1x10^7 cells/mL suspension of Caco-2 cells in Triton X-100 based lysis buffer followed by centrifugation at 14,000 RPM for 15 minutes at 4oC. Supernatants were collected and used for immunoprecipitation. Immunoprecipitation of Ago2 protein was performed by overnight incubation of cell lysates at 4oC with human monoclonal anti-Ago2 antibodies obtained from Wako Chemicals (Richmond, VA). After this step, 60 µL of protein G Dynabeads® (Life Technologies [Fredrick, MD]) were added to cell lysates and incubated at room temperature for 45 minutes for capturing anti-hAgo2 antibodies.

A tube containing

Dynabeads® was placed on a magnetic stand for capturing Dynabead®-antibody complex and removing cell lysate supernatant containing free floating siRNA and proteins. Cell lysate supernatants obtained in this step were stored at -20oC until analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) based western blot. Captured Dynabeads were washed three times with 1xPBS buffer to remove any unbound free floating siRNA. After wash steps, captured Ago2 protein was eluted using a glycine based elution buffer (as per manufacturer’s protocol for Dynabeads), which was then used for SDS PAGE based western blot (for determining Ago2 immunoprecipitation efficiency) and stem-loop qPCR (for quantifying TG2 siRNA levels) based analyses. A schematic representation of RISC loading assay is shown in Supplementary Error! Reference source not found..

2.6 Quantification of In Vitro Gene Silencing To evaluate the efficacy of in vitro gene-silencing mediated by siRNA-containing gelatin nanoparticles, siRNA transfection experiments were performed using Caco-2 cells. TG2 and IL15 siRNA, targeted against the human TG2 and IL-15 mRNA, respectively, were obtained from 9 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 33

Life Technologies, NY, and used to formulate gelatin nanoparticles. Caco-2 cells were seeded in different wells of a 96-well plate (20,000 cells per well). The cells were allowed to adhere to the well surface for 12 h. After cellular attachment, serum containing cell culture medium was replaced with serum-free siRNA containing formulations, and cells were incubated at 37°C, 5% CO2 for 24 h; formulations containing cell culture media were removed, and cells were washed three times with 1xPBS. Following this step, formulation-free, serum-containing cell culture media was added to each well, and the cells were incubated for 24 h, 48 h, 72 h, 96 h or 120 h. At each time point, 100µL of RNAlater solution (Life Technologies, NY) was added to each well, and the cells were stored at -20°C until samples from all time points were ready for RNA isolation and RT-qPCR analysis. A magnetic bead based mRNA isolation kit (Life Technologies, NY) was used to isolate cellular mRNA, which was reverse-transcribed into cDNA, followed by qPCR analysis. A specific human TG2 or IL-15 gene expression assay (Life Technologies, NY) was utilized for this assay as per protocol optimized for ABI 7900HT qPCR instrument (Applied Biosystems, state).. The relative TG2 or IL-15 mRNA expression was computed based on the ∆∆CT method (25) using expression levels of human actin-ß gene as the endogenous control. TG2 or IL-15 expression levels of siRNA-containing formulations were normalized to TG2 or IL-15 mRNA expression in scramble siRNA containing formulation treated cells. 2.7 In Vitro Model of Celiac Disease An in vitro model of celiac disease was developed in two separate cell lines by incubating Caco-2 and J774A.1 cells with the immunogenic α-gliadin p31-43 peptide. The p31-43 peptide, obtained from Peptides, Inc. (KY), was dissolved in sterile water at a concentration of 10 mg/mL. Caco-2 and J774A.1 cells were plated in a 96-well plate at 20,000 cells per well and allowed to attach the plate surface for 12 h. After cellular attachment, cells were treated with a


Page 11 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


peptide p31-43 at the final concentration of 100 µg/mL for 24 h. At the end of 24 h, cells were harvested and processed for qPCR based IL-15 and TG2 mRNA expression analysis.

2.8 TG2 and IL-15 Co-silencing in an In Vitro Model of Celiac Disease We next wanted to test whether gelatin nanoparticle mediated silencing of TG2 and IL-15 gene expression, either alone or in combination, was effective in down-regulating the elevated levels of TG2 and IL-15 mRNA expression, along with reduction in levels of mRNA expression for TNF-α and IFN-γ, which are the major proinflammatory cytokines responsible for cellular damage in the pathogenesis of celiac disease. To this end, we first tested the effect the solo TG2 and IL-15 gene silencing, followed by a combined silencing effect of TG2 and IL-15 genes in both Caco-2 and J774A.1 cell lines. For these experiments, Caco-2 and J774A.1 cells were treated with siRNA containing formulations, with use of a protocol similar to the one mentioned in section 2.6, followed by incubation for 72 h. P31-43 peptide was added to the cell culture media 24 h prior to the end of the 72 h time point. Cells were then processed for qPCR-based gene expression analysis, using assays from Life Technologies, NY.

2.9 Data Analysis The quantitative results are reported as mean ± standard deviation. Comparisons between the two groups were made using student’s t-test, and with more than two groups, ANOVA was used to compare the results. The values were considered statistically significant at 95% confidence interval (i.e., p80% TG2 mRNA silencing at the 24 h post treatment time point), but the duration of effect was not sustained at later time points; instead, at the later time points, gelatin nanoparticles showed a greater silencing effect relative to siRNA delivered via Lipofectamine. For example, even at the 96 h and 120 h time points, gelatin nanoparticles produced a greater than 40% TG2 mRNA knockdown, whereas with Lipofectamine, TG2 expression was back to baseline at the 96 h time point. A similar experiment was performed using siRNA targeted against human IL-15 mRNA in Caco-2 cells, which showed similar kinetics of mRNA knockdown (Figure 5b). Together, these results indicate that gelatin nanoparticles protect siRNA from intracellular degradation, allow slow and sustained cytosolic release of encapsulated siRNA, and thereby facilitate efficient and prolonged suppression of targeted mRNA. These results are very encouraging for the further development of gelatin nanoparticle-based siRNA delivery systems. Of note, a good pharmacokinetic-pharmacodynamic (PK-PD) correlation was observed between copies of siRNA associated with RISC complex (PK) and target mRNA knockdown (PD). This PK-PD correlation was described by a sigmoid inhibitory response model (Figure 6), which gave an estimated IC50 value of 207 siRNA copies/cell suggesting that approximately 207 15 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 33

copies of RISC associated siRNA per cell are needed for inducing a 50% reduction in target mRNA expression.

3.6 Co-Silencing TG2 and IL-15 in the Celiac Disease Model We next evaluated the efficacy of TG2 and IL-15 mRNA silencing in an in vitro model of celiac disease. Since maximal silencing of target mRNA was observed at 72 h post treatment for both TG2 and IL-15 siRNAs (Figure 5), and based on our previous studies with gelatin nanoparticles (23, 26), we selected this time point for evaluation of possible anti-inflammatory effects upon singular or co-treatment of siRNA containing gelatin nanoparticles in J77A1.4, assuming similar delay in gene silencing effect.

An in vitro model of celiac disease was

developed in two separate cell lines by incubating Caco-2 and J774A.1 cells with the immunogenic α-gliadin p31-43 peptide. Caco-2 cells, when incubated with the p31-43 peptide resulted in a significant increase in the expression of both IL-15 and TG2 mRNA expression (Figure 7a). A 3.5- and 1.75-fold induction from baseline was observed for IL-15 and TG2 mRNAs, respectively. Similar results were obtained in the case of the J774A.1 cell line (Figure 7b), where a more pronounced upregulation of IL-15 (3.32-fold increase from baseline) was observed compared to TG2 upregulation (1.75-fold increase in baseline) upon peptide p31-43 treatment. In J774A.1 cells, expression of TNF-α or INF-γ was not altered after treatment with peptide p31-43, whereas in Caco-2 cells, TNF-α or INF-γ message was not detected before or after p31-43 treatment. Gelatin nanoparticles encapsulating either TG2 or IL-15 siRNA were effective in suppressing the elevation of TG2 and IL-15 mRNA expressions after peptide p31-43 treatment, in both Caco-2 and J774A.1 cells (Figure 8 and 9). In both Caco-2 and J774A.1 cells, siRNA mediated inhibition of IL-15 had no effect on expression profile of TG2. Similarly, no change in 16 ACS Paragon Plus Environment

Page 17 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the expression profile of TG2 mRNA was observed when IL-15 levels were lowered via siRNA in both Caco-2 and J774A.1 cells.

qPCR analysis for TNF-α and IFN-γ expressions was

performed for the J774A.1 cell line treated with different siRNA-containing formulations, the siRNA-mediated IL-15 suppression had a greater impact on down-regulating TNF-α and INF-γ levels, when compared with TG2 siRNA treatment in J774A.1 cells. Additionally, a combination treatment of IL-15+TG2 containing siRNA gelatin nanoparticles produced a greater suppression of TNF-α and IFN-γ when compared to individual treatments in the J774A.1 cell line (Figure 9). It should be noted that these co-silencing effects are at ~40% silencing of TG2 and IL-15 genes. Co-silencing effects may vary at differing gene silencing levels for individual genes. These results suggest that suppression of TG2 and IL-15 gene expressions, either alone or in combination, can be efficacious in suppressing the proinflammatory response toward gluten peptides, and thus might prove to be helpful in the treatment of celiac disease. These results are very encouraging for further developing of TG2 and IL-15 siRNA delivery systems for the in vivo treatment of celiac disease.

4. CONCLUSIONS The results of this study showed that gelatin nanoparticles are capable of efficiently encapsulating siRNA, deliver siRNA to the cellular cytoplasm, enable RISC loading of delivered siRNA in intestinal epithelial cells (Caco-2), and cause efficient target gene knockdown in the Caco-2 and murine alveolar macrophage (J774A.1) cell lines. Further, when tested in an in vitro model of celiac disease, knockdown of IL-15 either specific or adjunct with TG2, showed significant down-regulation in expression of proinflammatory cytokines - TNF-α and IFN-γ. Further evaluations are aimed at developing a gelatin nanoparticle based oral multi-



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 33

compartmental system for silencing TG2 and IL-15 expression in the small intestine for the treatment of celiac disease.

5. ACKNOWLEDGEMENTS We deeply appreciate the assistance of Dr. Amit Singh with the transmission electron microscopy analysis that was performed at the Electron Microscopy Center of Northeastern University. We would also like to thank Dr. Gavrav Sahay for his assistance with qualitative cellular uptake study.


Page 19 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ABBREVIATIONS Abbreviation AGO2 DMEM IEL IFN-γ IL-15 mRNA NK qPCR RISC RNAi SDS PAGE siRNA TG2 TNF-α

Definition Argonaut 2 Dulbecco's Modified Eagle Medium Intra-Epithelial Lymphocytes Interferon gamma Interleukin-15 Messenger RNA Natural Killer cells Quantitative Polymerase Chain Reaction RNA Induced Silencing Complex RNA interference Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Small Interference Ribonucleic Acid Transglutaminase 2 Tumor Necrosis Factor alpha



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 33

TABLES Table 1: Particle size and siRNA encapsulation efficiency in type B gelatin nanoparticles

Formulation

Hydrodynamic Diameter (nm)*

Blank Type B Gelatin 206.0 ± 8.3 Nanoparticles siRNA Containing Type B 217.3 ± 8.4 Gelatin Nanoparticles *Mean ± SD (n=3); PDI=Poly dispersity index


PDI

siRNA Loading (%)*

0.05

NA

0.14

89.3 ± 3.05

Page 21 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FIGURE LEGENDS Figure 1: Transmission Electron Microscopy (TEM) of siRNA containing gelatin nanoparticles. Figure 2: (a) Intracellular localization of fluorescently labeled gelatin and siRNA relative to cell nucleus. Gelatin – Alexa fluor 488 (green signal), siRNA – Alexa-Fluor 647 (red signal), nucleus – Hoechst 405 (blue signal); (b) Quantification of integrated spot signal intensity per object area for green and red fluorescent signals at different time points. Figure 3: Quantitative determination of siRNA cellular uptake. Cellular concentrations of siRNA (y-axis) after treatment with siRNA formulated with either gelatin nanoparticles or Lipofectamie Figure 4: Concentrations (pg/10^8 cells) of RISC loaded TG2 siRNA in Caco-2 cells after treatment with formulations containing TG2 siRNA. Figure 5: (A) Relative expression levels of TG2 mRNA after treatment with control and TG2 siRNA containing formulations in Caco-2 cells. (B) Relative expression levels of IL-15 mRNA after treatment with control and IL-15 siRNA containing formulations in Caco-2 cells. Figure 6: Relationship between siRNA copies per cell loaded in the RISC complex and TG2 mRNA reduction from baseline described through a sigmoid inhibitory response model. Figure 7: α-Gliadin peptide p31-43 mediated upregulation of TG2 and IL-15 mRNA expression in (A) Caco-2 cells and (B) J774A.1 cells. Figure 8: (A) Effect of TG2 knockdown on IL-15 expression, and (B) effect of IL-15 knockdown on TG2 expression in p31-43 stimulated Caco-2 cells at 72 h post treatment with gelatin nanoparticles containing TG2 siRNA. Figure 9: Effect of (A) TG2 knockdown, (B) IL-15 knockdown and (C) TG2+IL-15 knockdown on TNF-α and IFN-γ expressions in p31-43 stimulated J774A.1 cells at 72 h post treatment with siRNA containing gelatin nanoparticles.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 33

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10.

11.

12.

13.

14.

15.

16.

L.M. Sollidand B. Jabri. Celiac disease and transglutaminase 2: a model for posttranslational modification of antigens and HLA association in the pathogenesis of autoimmune disorders. Current opinion in immunology. 23:732-738 (2011). L.M. Sollidand C. Khosla. Future therapeutic options for celiac disease. Nature clinical practice Gastroenterology & hepatology. 2:140-147 (2005). J.M. Barkerand E. Liu. Celiac disease: pathophysiology, clinical manifestations, and associated autoimmune conditions. Advances in pediatrics. 55:349-365 (2008). B.M. Ryanand D. Kelleher. Refractory celiac disease. Gastroenterology. 119:243-251 (2000). S. Daum, C. Cellier, and C.J. Mulder. Refractory coeliac disease. Best Practice & Research Clinical Gastroenterology. 19:413-424 (2005). M.F. Kagnoff. Overview and pathogenesis of celiac disease. Gastroenterology. 128:S1018 (2005). S. Martin. Against the grain: an overview of celiac disease. Journal of the American Academy of Nurse Practitioners. 20:243-250 (2008). A. Mubarak, R.H. Houwen, and V.M. Wolters. Celiac disease: an overview from pathophysiology to treatment. Minerva pediatrica. 64:271-287 (2012). D. Williamsonand M.N. Marsh. Celiac disease : a brief overview. Methods in molecular medicine. 41:1-9 (2000). Y. van de Wal, Y.M. Kooy, P. van Veelen, W. Vader, S.A. August, J.W. Drijfhout, S.A. Pena, and F. Koning. Glutenin is involved in the gluten-driven mucosal T cell response. European journal of immunology. 29:3133-3139 (1999). O. Molberg, N. Solheim Flaete, T. Jensen, K.E. Lundin, H. Arentz-Hansen, O.D. Anderson, A. Kjersti Uhlen, and L.M. Sollid. Intestinal T-cell responses to highmolecular-weight glutenins in celiac disease. Gastroenterology. 125:337-344 (2003). M.V. Barone, D. Zanzi, M. Maglio, M. Nanayakkara, S. Santagata, G. Lania, E. Miele, M.T. Ribecco, F. Maurano, R. Auricchio, C. Gianfrani, S. Ferrini, R. Troncone, and S. Auricchio. Gliadin-mediated proliferation and innate immune activation in celiac disease are due to alterations in vesicular trafficking. PloS one. 6:e17039 (2011). L. Maiuri, C. Ciacci, I. Ricciardelli, L. Vacca, V. Raia, S. Auricchio, J. Picard, M. Osman, S. Quaratino, and M. Londei. Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease. Lancet. 362:30-37 (2003). S. Yokoyama, N. Watanabe, N. Sato, P.Y. Perera, L. Filkoski, T. Tanaka, M. Miyasaka, T.A. Waldmann, T. Hiroi, and L.P. Perera. Antibody-mediated blockade of IL-15 reverses the autoimmune intestinal damage in transgenic mice that overexpress IL-15 in enterocytes. Proceedings of the National Academy of Sciences of the United States of America. 106:15849-15854 (2009). C. Kriegel, H. Attarwala, and M. Amiji. Multi-compartmental oral delivery systems for nucleic acid therapy in the gastrointestinal tract. Adv Drug Deliv Rev. 65:891-901 (2013). H. Attarwalaand M. Amiji. Multi-compartmental nanoparticles-in-emulsion formulation for macrophage-specific anti-inflammatory gene delivery. Pharm Res. 29:1637-1649 (2012).


Page 23 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


17.

18.

19.

20.

21. 22. 23.

24.

25. 26.

J. Xuand M. Amiji. Therapeutic gene delivery and transfection in human pancreatic cancer cells using epidermal growth factor receptor-targeted gelatin nanoparticles. J Vis Exp:e3612 (2012). C. Kriegeland M. Amiji. Oral TNF-alpha gene silencing using a polymeric microspherebased delivery system for the treatment of inflammatory bowel disease. J Control Release. 150:77-86 (2011). P. Magadalaand M. Amiji. Epidermal growth factor receptor-targeted gelatin-based engineered nanocarriers for DNA delivery and transfection in human pancreatic cancer cells. AAPS J. 10:565-576 (2008). M.D. Bhavsarand M.M. Amiji. Oral IL-10 gene delivery in a microsphere-based formulation for local transfection and therapeutic efficacy in inflammatory bowel disease. Gene Ther. 15:1200-1209 (2008). S. Kommareddyand M.M. Amiji. Protein nanospheres for gene delivery. CSH Protoc. 2008:pdb top30 (2008). G. Kauland M. Amiji. Long-circulating poly(ethylene glycol)-modified gelatin nanoparticles for intracellular delivery. Pharm Res. 19:1061-1067 (2002). C. Kriegeland M.M. Amiji. Dual TNF-alpha/Cyclin D1 Gene Silencing With an Oral Polymeric Microparticle System as a Novel Strategy for the Treatment of Inflammatory Bowel Disease. Clin Transl Gastroenterol. 2:e2 (2011). Y. Landesman, N. Svrzikapa, A. Cognetta, X. Zhang, B.R. Bettencourt, S. Kuchimanchi, K. Dufault, S. Shaikh, M. Gioia, and A. Akinc. In vivo quantification of formulated and chemically modified small interfering RNA by heating-in-Triton quantitative reverse transcription polymerase chain reaction (HIT qRT-PCR). Silence. 1:16 (2010). T.D. Schmittgenand K.J. Livak. Analyzing real-time PCR data by the comparative CT method. Nature protocols. 3:1101-1108 (2008). S. Shah. Hypoxia in tumor angiogenesis and metastasis: evaluation of VEGF and MMP over-expression and down-regulation of HIF-1a with RNAi in hypoxic tumor cells. (2010).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Figure 1

ACS Paragon Plus Environment

Page 24 of 33

Page 25 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


Figure 2



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Figure 3


Page 26 of 33

Page 27 of 33

Figure 4

T G 2 s iR N A p g /1 0 ^ 8 C e lls

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


L ip o fe c ta m in e 1000

G e la tin N a n o p a ritc le s

100 0

50

100

T im e (h )


150

Figure 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42



Page 28 of 33

Page 29 of 33


Figure 6

120

R e la t iv e T G 2 m R N A ( % )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

C u rv e F it 100

O b s e rv a tio n s 80 60 40 20 0 0

100

200

300

400

500

C o p ie s o f s iR N A in R IS C (C o p ie s /C e ll)


Figure 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42



Page 30 of 33

Figure 8

Page 31 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42



Figure 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42



Page 32 of 33

Page 33 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


82x44mm (300 x 300 DPI)


Cosilencing Intestinal Transglutaminase-2 and Interleukin-15 Using

Recommend Documents