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Fertil Steril. Author manuscript; available in PMC 2017 June 01. Published in final edited form as: Fertil Steril. 2016 June ; 105(6): 1638–1648.e8. doi:10.1016/j.fertnstert.2016.03.001.
Magnetic nanoparticles as a new approach to improve the efficacy of gene therapy against differentiated human uterine fibroid cells and tumor-initiating stem cells
Author Manuscript
Shahinaz Mahmood Shalaby, M.D., M.Sc.1,2, Mostafa K Khater, Pharm D, M.Sc.1, Aymara Mas Perucho, Ph.D.1, Sara A Mohamed, MD, M.Sc.1,3, Inas Helwa, Ph.D.4, Archana Laknaur, M.Sc.1, Iryna Lebedyeva, Ph.D.5, Yutao Liu, Ph.D4, Michael P Diamond, MD1, and Ayman A Al-Hendy, MD,Ph.D.1,* 1Department
of Obstetrics and Gynecology, Georgia Regents University, Augusta, Georgia
30912, US 2Department
of Pharmacology, Tanta Faculty of Medicine, Tanta 31111, Egypt
3Department
of Obstetrics and Gynecology, Mansoura University Hospital, Mansoura Faculty of Medicine, Mansoura 35516, Egypt
4Department
of Cell Biology and Anatomy, Georgia Regents University, Augusta, GA 30912, US
5Department
of Chemistry and Physics, Georgia Regents University, Augusta, GA 30912, US
Author Manuscript
Abstract Uterine fibroid(s) (UF/UFs) are benign tumors commonly found in women of reproductive age. The long-term outcomes of myomectomies are often hampered by high rates of recurrence (up to 60%). Objective—To study whether efficient transduction and subsequent elimination of fibroid tumor initiating stem cells during debulking of tumor cells will aid in completely eradicating the tumor as well as decreasing the likelihood of recurrence.
Author Manuscript
Design—We have developed a localized non-surgical adenovirus-based alternative for the treatment of UFs. Combining viral based gene delivery with nanotechnology provides an opportunity to develop more efficient targeted viral gene therapy. Magnetic nanoparticles (MNPs) complexed to adenovirus, in the presence of an external magnetic field, accelerate adenovirus transduction. Setting—Research laboratory located in Georgia Regents University, an academic research institution.
*
Correspondence address. Tel: +1-706-721-3833; Fax: +1-706-721-6211; [email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
All authors have certified that there are no conflicts of interest(s) to disclose.
Shalaby et al.
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Patients N/A Interventions—MNPs complexed to adenovirus (AD GFP) or (AD LacZ) were used to transfect differentiated human fibroid cells in vitro. Main Outcome Measures rate of transduction and tumor growth inhibition. Results—We observed a significant increase in transduction efficiency among differentiated human fibroid cells at 2 different multiplicities of infection (MOI); 1 and 10 respectively, with MNPs as compared to adenovirus-alone. Human fibroid stem cells transfected with AD-LacZ expressed β-Galactosidaze at (MOI) of 1, 10, and 50 at percentages of 19%, 62%, and 90%, respectively, which were significantly enhanced with MNPs. Conclusion—When applied with adenovirus herpes simplex thymidine kinase, magnetofection significantly suppressed proliferation and induced apoptosis in both cell types. Through the use of magnetofection, we will prove that a lower viral dose will effectively increase the overall safety profile of suicide gene therapy against fibroid tumors.
Author Manuscript
Introduction
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Uterine fibroids (UFs), also known as uterine leiomyomas are benign neoplasms of the myometrium and represent the most common solid tumor in reproductive- aged women (1, 2). These tumors occur in 77% of women overall with clinical manifestation in 25% of those affected by age 45 (2–5). Although benign, they commonly cause severe symptoms such as heavy, irregular and prolonged menstrual bleeding, and anemia. Other common symptoms include pelvic discomfort, bowel and bladder dysfunction caused by pressure due to anatomical placement, and/or positioning of the fibroids. UFs have also been associated with subfertility and recurrent spontaneous abortion (6–10).These clinical complications seriously impact women’s health and quality of life. UFs are the most common indication for the more than 600, 000 hysterectomies performed in the US annually. Hysterectomy is an invasive major surgery. is oftentimes associated with significant morbidity, possible mortality, and imposes a huge economic impact on the US healthcare delivery system.(10, 11)
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For women with symptomatic UFs, that desire future fertility, only limited conservative methods of treatment are available to manage fibroids without compromising subsequent chances of achieving a healthy pregnancy. Due to various factors, more women are delaying childbearing, which has led to an increase in the number of nulligravida patients with symptomatic UFs(5). Despite the burden of suffering, many women affected are averse to surgery and actively seek fertility-preserving alternatives.(12–14) We have previously shown that intratumoral gene therapy, a localized method of UF treatment, has the ability (15–19), to ablate UFs without interfering with ovulation, uterine blood supply or systemic ovarian function. (14, 18) The use of adenovirus complexes for the treatment of UFs serves as a novel and minimally invasive therapeutic option, for this growing group of patients (14). Adenoviruses are among the most robust gene delivery tools and offer immense promise in the field of gene therapy. Our group has a proven track-record in the utilization of these unique vectors for the development of a localized non-surgical alternative for the treatment of UF tumors. (16) (15–18) This technique allows for the successful ablation of UFs without
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interfering with ovulation, uterine blood supply, or systemic ovarian function oftentimes associated with other UF treatment modalities.
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Though the efficacy and safety profile of replication-incompetent adenoviruses is outstanding, their clinical use with systemic or even localized delivery is hampered by adverse reactions, including thrombocytopenia (20), acquired immune responses mediated by cytotoxic lymphocytes against viral and/or transgene products,(21, 22) and in some cases, may lead to the potentially life-threatening systemic cytokine syndrome (23–25). The latter acute toxic effects are due to activation of the innate immune system and exhibit a steep dependence on vector dose i.e., decreased viral load, and equates to a lessened likelihood of severe immune reaction. Its occurrence varies substantially among subjects, and the likelihood of eliciting such an immune response cannot be readily predicted. Our research group, has developed a method which targets therapeutic adenoviruses towards fibroid lesions and minimizes any potential delivery beyond the tumor lesion. In our approach, we have genetically modified the adenovirus with a targeting short peptide composed of 3 amino acids (Glycine, Arginine, and Aspartic Acid) collectively referred to as the RGD peptide motif. The RGD peptide motif is expressed on the virus capsid to utilize different internalization pathways other than the well-known Coxacie-adenovirus receptor (CAR), commonly expressed on many normal cells.(17) The advantage of the CAR independent RGD pathway is its utilization of the integrin internalization pathway which is highly expressed on fibroid tumor cells as compared to surrounding normal myometrium(17, 26, 27).
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We have found that the integration of gene therapy and nanotechnology serves as yet another approach that can be used in minimizing the required dosage of tumor-targeted adenovirus while sufficiently increasing the efficiency of transduction. MNPs conjugated to adenoviral vectors, in the presence of an external magnetic field, have been shown to greatly enhance targeted gene transfer into tumor cells (28). These magnetic nanoparticles accelerate transduction kinetics, a technique referred to as magnetofection (29). The magnetofection method was developed to overcome biological barriers against the delivery of efficient gene therapy through the use of nucleic acids or viral vectors associated with MNPs (29, 30).
Author Manuscript
The principle of magnetofection is to associate transfection reagents or viruses with specific magnetic nanoparticles; thereby, forming molecular complexes. Resulting molecular complexes are then concentrated and transported into cells supported by an appropriate magnetic field (29, 30). Through the exertion of a magnetic force upon gene vectors, we were able to rapidly increase the concentration of the applied vector dose on cells, so that 100% of the cells come in contact with a high vector dose, thereby promoting cellular uptake. This approach has not yet been evaluated against human UF tumor cells. Our aim is threefold: 1) to enhance the efficiency of transduction while maintaining or minimizing viral dose, 2) to enable targeting to fibroid tumor tissue and avoid surrounding healthy myometrium, and 3) to validate that our approach can transduce and eliminate fibroid stem cell populations. The latter would be a novel paradigm-shifting improvement in UF therapeutics. Eliminating tumor-forming fibroid stem cells, would likely prevent tumor
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recurrence, a major challenge in the field of UFs, and likely prevent the development of new fibroid lesions. Currently available treatment modalities are not capable of affecting fibroid stem cells. Utilization of magnetofection in fibroid gene therapy is novel and innovative. It represents the natural evolution and progression of therapeutic options available. Our group aims to pursue the design and development of cutting-edge approaches which are localized, effective, safe, and fertility-preserving therapies in the treatment of uterine fibroids. In this work, we demonstrate that magnetofection does indeed enhance adenoviral gene therapy, increase lethality against human fibroid cells, and for the first time, demonstrates that such an approach is efficient in eliminating human fibroid tumor initiating stem cells.
Materials and Methods Cell culture
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All experiments were done in accordance with biosafety guidelines of Georgia Regents University and conducted after protocol approval by Georgia Regents University Institutional Review Board. Human immortalized fibroid cells and uterine smooth muscle cells were in-kind gifts provided by Darlene Dixon, PhD (National Institute of Environmental Health Sciences, Research Triangle Park, NC, US). Cells were cultured and maintained in smooth muscle cell basal media (SmBM, Lonza Walkersville, MD, US) containing 10% fetal bovine serum (FBS, Lonza Walkersville, MD, US), 0.1% insulin, 0.2% human fibroblast growth factor–basic (hFGF-B), 0.1% gentamycin sulfate, amphotericin-B (GA-1000), and 0.1% human epidermal growth factor (h-EGF) (Lonza, Walkersville, MD, US). Human fibroid stem cells were isolated and characterized in our laboratory as previously described (31, 32). For cell proliferation assays, cells were grown in a 24-well culture plate and transduced with adenoviral vectors (0–100 PFU/cell) followed by ganciclovir (GCV) therapy (Sigma Co, St. Louis, MO, US) at a concentration of 10 µg/ml for 3–7 days. Cell growth was measured with the MTT kit (Sigma Co, St. Louis, MO, US) per the manufacturer's instructions. AD-GFP vector was used in our initial proof of concept experiments. We then used adenovirus vector encoding HSV-1TK gene under transcriptional control of the Rous sarcoma virus (AD-HSV1TK), as described by Chen et. al. (33). An adenovirus vector expressing a marker gene coding for bacterial β-galactosidase (AD-Lac Z), was used as a negative control. Both viruses were in-kind gifts from Dr. Savio Woo (Mount Sinai School of Medicine, New York, NY, US). The AD-RGD-TK vector, a modified adenovirus vector contains the herpes simplex virus thymidine kinase gene. This complex was prepared in Dr. David Curiel’s lab (Washington University, St. Louis, MO). A genetic incorporation of the RGD-4C motif into the H1 loop of the fiber was used to enhance the infectivity of the vector by permitting binding to the αvβ3 and αvβ5 integrins as previously described.(34). Magnetic nanoparticles were purchased from OZ Biosciences (Parc Scientifique de Luminy, France). •
Preparation of adenovirus−magnetic nanoparticles (MNPs) complexes for magnetofection
Magnetic nanoparticles adenovirus complexes were prepared by mixing 75 µL of Adenomag magnetic nanoparticle suspension with 15 µl of 6.5× 1011 VP of AD-RGD-TK, equal to 6.5 × 109 PFU/ml diluted in 500 µL of serum free culture media, a lower ratio than
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recommended by the manufacturer of Adenomag (OZ Biosciences, Parc Scientifique de Luminy, Marcella, France). After a 20-minute incubation period at room temperature (RT), different dilutions of the complexes were performed to cover the required multiplicities of infection (MOIs), so that 50 µL of the complexes were added to the cells. AD-GFP, ADRGD Luciferase, and AD-RGD-TK were utilized in preparation of the complex for proof of infectivity to wild type adenovirus as well as genetically modified AD-RGD variant, and cytotoxicity of AD-RGD-TK adenovirus complexes. Magnetic complexes were applied to cells in a drop-wise manner. Cells were then incubated for 30 min on a super magnetic plate (OZ Biosciences, France) at 37°C, 5% CO2. The magnetic plate was then removed and cells were cultured under standard cell culture conditions until the end of the experiments. •
MNPs characterization
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The morphological examination of MNP was carried out through the use of a transmission electron microscope (TEM) (JEOL JEM 1230 Transmission Electron Microscope) with 2% phosphotungstic acid negative staining. The mean size and zeta potential of the nanoparticles were determined by nanoparticle tracking analysis with ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) and corresponding software ZetaView 8.02.28. Samples were diluted 1 to 10 in 1× PBS and sonicated prior to ZetaView measurement. Zeta potential was measured using 0.05 × PBS instead of 1× PBS for conductivity at approximately 500 µS/cm. Per OZ Bioscience Company, nanoparticles have a hydrodynamic radius of 200–300 nm, with 10–15 nm, metal cores with positive zeta potential. We also characterized adenovirus size by electron microscope before and after conjugation with the MNP through incubation with the viral particles for 20 minutes at RT. All measurements were taken at pH 7.4, 25° C. •
Comparing Adeno-GFP (AD-GFP) regular transduction versus magnetofection
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Upon completion of both regular transduction and magnetofection protocols, human fibroid tumor cells and human fibroid stem cells with AD-GFP were washed, and fixed with 4% paraformaldehyde (PFA). GFP expression was detected under an inverted fluorescence microscope equipped with a digital camera (Axiovert, Zeiss Carl Zeiss Microscopy Ltd, Cambridge, MA, US). Images were taken with a 20× Plan-Neofluar dry lens. The same exposure for luminosity and contrast were applied to each slide. Images presented, represent three independent experiments. •
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Comparative analysis of AD-RGD luciferase regular transduction versus magnetofection AD-RGD luciferase transfection and magnetofection were performed on human fibroid tumor cells and human fibroid stem cells. Luciferase bioluminescence assays were conducted for group comparisons. –
Boosting suicidal gene therapy of human fibroid tumor cells and human fibroid stem cells by MNPs and applied to a magnetic field Human fibroid tumor cells and human fibroid stem cells were seeded in flat-bottom 24-well plates, at 40,000 cells/ well density for human fibroid tumor cells and 2,000 cells/
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well density for human fibroid stem cells. Cells were then incubated under standard cell culture conditions. After 24h incubation, media in each well was replaced with 600 µL of fresh cell culture media containing 10% FBS; 50 µL of MNPs complexed AD-RGD-TK, AD- Lac Z (negative control) or WT-AD (positive control) virus alone, and prepared as previously described. Cells with MNPs complexed AD-RGD-TK were placed on a magnetic plate in the CO2 incubator for 30 minutes, removed and maintained under regular cell culture conditions. After 24h of infection, media was replenished with fresh cell culture media, and cells were further cultivated until evaluation. All samples were measured in triplicate. Cell survival was assessed by a luciferase assay in stable cells expressing luciferase or by a MTT-based respiration activity assay.
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–
MTT Assay: Cells were Infected with Adenovirus RGD thymidine kinase (AD-RGD-TK), adenovirus Bgalactosidase (AD- Lac Z), considered the wild type adenovirus (WT-AD) (negative control). Untreated cells were washed with PBS and incubated for 3h in 100 µL of 1 mg/mL MTT solution which was prepared in PBS with 5 mg/ mL glucose. Afterwards, 100 µL of solubilization solution with Dimethyl Sulphoxide (DMSO Sigma, St. Louis.MO, US) was added to dissolve formazan. Optical density was measured at 590 nm and cell viability was expressed as respiration activity normalized to the reference data for untreated cells.
–
Luciferase Assay: Cells were washed with PBS and lysed with 100 µL of lysis buffer (0.1% Triton X-100 in 250 mM Tris; pH 7.8) per well. After incubation for 15–20 min at RT, 50 µL of cell lysate was drawn from each well and transferred into a 96-well black flat-bottom plate. Finally, 100 µL of luciferase buffer was added. Cells were then washed with PBS, and 100 µL of 35 µM D-Luciferin in PBS was added.
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Detection of the bystander effect in human fibroid stem cells Tumor cells transduced with an adenovirus vector expressing the herpes simplex virus thymidine kinase (HSV-TK) gene are rendered sensitive to the anti-herpetic drug, ganciclovir. The bystander effect refers to the observation that not all cells need to be transduced, in order to be killed. This is attributed to the capacity to transfer small cytotoxic molecules from transfected to non-transfected cells due to direct contact and the presence of gap junctions, thus conferring the cytotoxicity to them. We wanted to check for this bystander effect in human fibroid stem cells so that we could account for its action in
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deducing the viral load required for their complete eradication. Human fibroid stem cells were grown in 100 mm tissue culture plates until 70–80% confluence was achieved and then transfected with the AD-RGD TK as previously described. The next day, cells (as well as cells from uninfected plates) were trypsinized and counted. Different ratios of uninfected/ infected cells were cocultured, for a total of 4,000 cells and plated in 24-well plates. 24h later media was removed and replaced with media containing GCV 10ug/ml. Five days later, viable cell counts were determined using MTT Assay. Isolation of protein and immunoblots
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Proliferating cell nuclear antigen (PCNA), and BCL2-associated X protein (BAX) expression was examined using western blot analysis. Ice-cold radio-immune-precipitation assay (RIPA) buffer supplemented with phosphatase and protease inhibitors (50 mM sodium vanadate, 0.5 mM phenylmethylsulphonyl fluoride, 2 mg/ml aprotinin, and 0.5 mg/ml leupeptin) (Sigma, St. Louis, MO, US) was used for protein extraction and for the elimination of homogenates and cell lysate. Protein concentrations were measured by Bradford protein assay (Bio-Rad, Hercules, CA, US). Total protein samples (40 µg) were filtered via SDS-PAGE (Polyacrylamide Gel Electrophoresis) (10% acrylamide gel) using the Bio-Rad Trans-Blot system (Bio-Rad, Hercules, CA, US) and transferred to membranes. Membranes were blocked with 5% non-fat milk in phosphate-buffered saline (Gibco, Grand Island, NY, US) containing 0.1% Tween 20 (Sigma, St. Louis, MO, US)(PBS-T), incubated for 1h, washed in PBS-T, and hybridized with primary antibodies, specific antibodies for PCNA (BAX Santa Cruz Biotechnology, Inc. Dallas, Texas US). In addition, membranes were incubated with a secondary antibody to bind β-actin at a 1:15,000 dilution (Sigma, US), which served as an internal control. Incubation with secondary antibodies and the detection of the antigen-antibody complex were performed using a Super Signal™ West Dura Extended Duration Substrate (Life Technologies, Grand Island, NY, US). Immunoblots of PCNA (36 kDa), BAX (27 kDa), β-actin (43 kDa) were quantified with a Bio-Rad Image Lab. densitometer (Bio-Rad, Hercules, CA US). Caspase-3 Assay
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Caspase-3 enzyme activity was measured in human fibroid stem cells using the Caspase assay system (Abcam, Cambridge, MA, US) based on Caspase 3 enzyme’s ability to release yellow chromophore p-nitro aniline (pNA) from the colorimetric substrate (Ac-DEVD-pNA) provided in the Caspase assay system. Tissue lysates were centrifuged at 13,000 rpm. Reading was at 405 (excitation)/510(emission) using the 96-plate reader Synergy HT with Gen 5 software (Biotek Instruments, Inc., Winooski, VT, US) according to the manufacturer’s instructions. Caspase-3 activity values were normalized against the total tissue protein content measured Data analysis Statistical analysis of the samples were done using a Student’s t-test, where P-values ≤0.05 were considered statistically significant with 95% confidence intervals. All statistical analyses were performed using GraphPad Prism version 6.00 for Mac (GraphPad Software, San Diego, US).
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Results MNPs size and surface charge identification MNPs size ranged from 115 to 214 nm in size, with a mean of 129±71nm and zeta potential of 18.12 mV by ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) and corresponding software ZetaView 8.02.28.Transmision electron microscopy (JEOL JEM 1230, Peabody, MA) showed the shape of adenovirus and MNPs. Covalent conjugation with adenovirus and MNPs was confirmed by the close intimate approximation of nanoparticles around the surface of adenovirus (Fig. 1). MNPs enhance AD-GFP transduction of human fibroid tumor cells
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The purpose of conducting this experiment was to evaluate whether conjugating adenoviral vectors with MNPs, would enhance the ability of adenoviral vectors to transfect human fibroid tumor cells. We compared the transduction efficiency of non-conjugated AD-GFP to that of MNPs conjugated AD-GFP at two different multiplicities of infection (MOI), 5 and 10 PFU/cell (Fig.2A). As shown in Fig.2B, there was a significant increase in the number of transfected cells from 8.06% in unconjugated AD GFP group to 28.56 % ± 8.9% in the MNPs conjugated group (p < 0.005), for MOI 5 and an increase from 25.38% in the unconjugated AD GFP group to 85.65 ± 7.57% in the MNPs conjugated group for MOI 10 (p= 0.005). MNPs enhance AD-RGD-luciferase transduction into human fibroid tumor cells
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A previous report from our group (16) demonstrated that by genetically altering adenovirus to express RGD peptide in the viral capsid, transduction efficiency of the adenovirus was markedly enhanced against human fibroid tumor cells. In this study, we aimed to investigate whether further enhancement of transduction efficiency could be accomplished by means of MNPs-adenoviral conjugations. As shown in Supplementary Fig. 1, MNPs significantly enhanced transduction efficiency of the modified virus AD-RGD-Luc (as reflected in bioluminescence intensity measured by luciferase assay) from 23.43 % in the unconjugated AD RGD LUC to 46.20 ± 5.7% in the MNPs group at MOI 5 and from 44.67±7.8% to 85.77%± 8.6% at MOI 10 10, respectively when compared to the unconjugated AD-RGD-Luc against human fibroid tumor cells (P = 0.005). MNPs enhance the ability of AD-RGD-TK to suppress proliferation of human fibroid tumor cells
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We recently used AD-RGD-TK followed by ganciclovir treatment to efficiently induce apoptosis and inhibit proliferation of human fibroid cells (16). In order to assess the ability of MNPs to further enhance the anti-fibroid ability of AD-RGD-TK, ascending MOIs of MNPs-conjugated versus unconjugated virus were tested against human fibroid cells in vitro. As shown in Supplementary Fig. 2, there was a significant decrease in the percentage of viable cells from 45% ± 1.34 % to 33% ± 1.38% at MOI 25, from 39± 2.6 % to 25 ± 1.43% at MOI 50 and from 32 ± 2.15 % to 21 ± 0.98% at MOI 75 (p
Fertil Steril. Author manuscript; available in PMC 2017 June 01. Published in final edited form as: Fertil Steril. 2016 June ; 105(6): 1638–1648.e8. doi:10.1016/j.fertnstert.2016.03.001.
Magnetic nanoparticles as a new approach to improve the efficacy of gene therapy against differentiated human uterine fibroid cells and tumor-initiating stem cells
Author Manuscript
Shahinaz Mahmood Shalaby, M.D., M.Sc.1,2, Mostafa K Khater, Pharm D, M.Sc.1, Aymara Mas Perucho, Ph.D.1, Sara A Mohamed, MD, M.Sc.1,3, Inas Helwa, Ph.D.4, Archana Laknaur, M.Sc.1, Iryna Lebedyeva, Ph.D.5, Yutao Liu, Ph.D4, Michael P Diamond, MD1, and Ayman A Al-Hendy, MD,Ph.D.1,* 1Department
of Obstetrics and Gynecology, Georgia Regents University, Augusta, Georgia
30912, US 2Department
of Pharmacology, Tanta Faculty of Medicine, Tanta 31111, Egypt
3Department
of Obstetrics and Gynecology, Mansoura University Hospital, Mansoura Faculty of Medicine, Mansoura 35516, Egypt
4Department
of Cell Biology and Anatomy, Georgia Regents University, Augusta, GA 30912, US
5Department
of Chemistry and Physics, Georgia Regents University, Augusta, GA 30912, US
Author Manuscript
Abstract Uterine fibroid(s) (UF/UFs) are benign tumors commonly found in women of reproductive age. The long-term outcomes of myomectomies are often hampered by high rates of recurrence (up to 60%). Objective—To study whether efficient transduction and subsequent elimination of fibroid tumor initiating stem cells during debulking of tumor cells will aid in completely eradicating the tumor as well as decreasing the likelihood of recurrence.
Author Manuscript
Design—We have developed a localized non-surgical adenovirus-based alternative for the treatment of UFs. Combining viral based gene delivery with nanotechnology provides an opportunity to develop more efficient targeted viral gene therapy. Magnetic nanoparticles (MNPs) complexed to adenovirus, in the presence of an external magnetic field, accelerate adenovirus transduction. Setting—Research laboratory located in Georgia Regents University, an academic research institution.
*
Correspondence address. Tel: +1-706-721-3833; Fax: +1-706-721-6211; [email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
All authors have certified that there are no conflicts of interest(s) to disclose.
Shalaby et al.
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Author Manuscript
Patients N/A Interventions—MNPs complexed to adenovirus (AD GFP) or (AD LacZ) were used to transfect differentiated human fibroid cells in vitro. Main Outcome Measures rate of transduction and tumor growth inhibition. Results—We observed a significant increase in transduction efficiency among differentiated human fibroid cells at 2 different multiplicities of infection (MOI); 1 and 10 respectively, with MNPs as compared to adenovirus-alone. Human fibroid stem cells transfected with AD-LacZ expressed β-Galactosidaze at (MOI) of 1, 10, and 50 at percentages of 19%, 62%, and 90%, respectively, which were significantly enhanced with MNPs. Conclusion—When applied with adenovirus herpes simplex thymidine kinase, magnetofection significantly suppressed proliferation and induced apoptosis in both cell types. Through the use of magnetofection, we will prove that a lower viral dose will effectively increase the overall safety profile of suicide gene therapy against fibroid tumors.
Author Manuscript
Introduction
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Uterine fibroids (UFs), also known as uterine leiomyomas are benign neoplasms of the myometrium and represent the most common solid tumor in reproductive- aged women (1, 2). These tumors occur in 77% of women overall with clinical manifestation in 25% of those affected by age 45 (2–5). Although benign, they commonly cause severe symptoms such as heavy, irregular and prolonged menstrual bleeding, and anemia. Other common symptoms include pelvic discomfort, bowel and bladder dysfunction caused by pressure due to anatomical placement, and/or positioning of the fibroids. UFs have also been associated with subfertility and recurrent spontaneous abortion (6–10).These clinical complications seriously impact women’s health and quality of life. UFs are the most common indication for the more than 600, 000 hysterectomies performed in the US annually. Hysterectomy is an invasive major surgery. is oftentimes associated with significant morbidity, possible mortality, and imposes a huge economic impact on the US healthcare delivery system.(10, 11)
Author Manuscript
For women with symptomatic UFs, that desire future fertility, only limited conservative methods of treatment are available to manage fibroids without compromising subsequent chances of achieving a healthy pregnancy. Due to various factors, more women are delaying childbearing, which has led to an increase in the number of nulligravida patients with symptomatic UFs(5). Despite the burden of suffering, many women affected are averse to surgery and actively seek fertility-preserving alternatives.(12–14) We have previously shown that intratumoral gene therapy, a localized method of UF treatment, has the ability (15–19), to ablate UFs without interfering with ovulation, uterine blood supply or systemic ovarian function. (14, 18) The use of adenovirus complexes for the treatment of UFs serves as a novel and minimally invasive therapeutic option, for this growing group of patients (14). Adenoviruses are among the most robust gene delivery tools and offer immense promise in the field of gene therapy. Our group has a proven track-record in the utilization of these unique vectors for the development of a localized non-surgical alternative for the treatment of UF tumors. (16) (15–18) This technique allows for the successful ablation of UFs without
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interfering with ovulation, uterine blood supply, or systemic ovarian function oftentimes associated with other UF treatment modalities.
Author Manuscript
Though the efficacy and safety profile of replication-incompetent adenoviruses is outstanding, their clinical use with systemic or even localized delivery is hampered by adverse reactions, including thrombocytopenia (20), acquired immune responses mediated by cytotoxic lymphocytes against viral and/or transgene products,(21, 22) and in some cases, may lead to the potentially life-threatening systemic cytokine syndrome (23–25). The latter acute toxic effects are due to activation of the innate immune system and exhibit a steep dependence on vector dose i.e., decreased viral load, and equates to a lessened likelihood of severe immune reaction. Its occurrence varies substantially among subjects, and the likelihood of eliciting such an immune response cannot be readily predicted. Our research group, has developed a method which targets therapeutic adenoviruses towards fibroid lesions and minimizes any potential delivery beyond the tumor lesion. In our approach, we have genetically modified the adenovirus with a targeting short peptide composed of 3 amino acids (Glycine, Arginine, and Aspartic Acid) collectively referred to as the RGD peptide motif. The RGD peptide motif is expressed on the virus capsid to utilize different internalization pathways other than the well-known Coxacie-adenovirus receptor (CAR), commonly expressed on many normal cells.(17) The advantage of the CAR independent RGD pathway is its utilization of the integrin internalization pathway which is highly expressed on fibroid tumor cells as compared to surrounding normal myometrium(17, 26, 27).
Author Manuscript
We have found that the integration of gene therapy and nanotechnology serves as yet another approach that can be used in minimizing the required dosage of tumor-targeted adenovirus while sufficiently increasing the efficiency of transduction. MNPs conjugated to adenoviral vectors, in the presence of an external magnetic field, have been shown to greatly enhance targeted gene transfer into tumor cells (28). These magnetic nanoparticles accelerate transduction kinetics, a technique referred to as magnetofection (29). The magnetofection method was developed to overcome biological barriers against the delivery of efficient gene therapy through the use of nucleic acids or viral vectors associated with MNPs (29, 30).
Author Manuscript
The principle of magnetofection is to associate transfection reagents or viruses with specific magnetic nanoparticles; thereby, forming molecular complexes. Resulting molecular complexes are then concentrated and transported into cells supported by an appropriate magnetic field (29, 30). Through the exertion of a magnetic force upon gene vectors, we were able to rapidly increase the concentration of the applied vector dose on cells, so that 100% of the cells come in contact with a high vector dose, thereby promoting cellular uptake. This approach has not yet been evaluated against human UF tumor cells. Our aim is threefold: 1) to enhance the efficiency of transduction while maintaining or minimizing viral dose, 2) to enable targeting to fibroid tumor tissue and avoid surrounding healthy myometrium, and 3) to validate that our approach can transduce and eliminate fibroid stem cell populations. The latter would be a novel paradigm-shifting improvement in UF therapeutics. Eliminating tumor-forming fibroid stem cells, would likely prevent tumor
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recurrence, a major challenge in the field of UFs, and likely prevent the development of new fibroid lesions. Currently available treatment modalities are not capable of affecting fibroid stem cells. Utilization of magnetofection in fibroid gene therapy is novel and innovative. It represents the natural evolution and progression of therapeutic options available. Our group aims to pursue the design and development of cutting-edge approaches which are localized, effective, safe, and fertility-preserving therapies in the treatment of uterine fibroids. In this work, we demonstrate that magnetofection does indeed enhance adenoviral gene therapy, increase lethality against human fibroid cells, and for the first time, demonstrates that such an approach is efficient in eliminating human fibroid tumor initiating stem cells.
Materials and Methods Cell culture
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All experiments were done in accordance with biosafety guidelines of Georgia Regents University and conducted after protocol approval by Georgia Regents University Institutional Review Board. Human immortalized fibroid cells and uterine smooth muscle cells were in-kind gifts provided by Darlene Dixon, PhD (National Institute of Environmental Health Sciences, Research Triangle Park, NC, US). Cells were cultured and maintained in smooth muscle cell basal media (SmBM, Lonza Walkersville, MD, US) containing 10% fetal bovine serum (FBS, Lonza Walkersville, MD, US), 0.1% insulin, 0.2% human fibroblast growth factor–basic (hFGF-B), 0.1% gentamycin sulfate, amphotericin-B (GA-1000), and 0.1% human epidermal growth factor (h-EGF) (Lonza, Walkersville, MD, US). Human fibroid stem cells were isolated and characterized in our laboratory as previously described (31, 32). For cell proliferation assays, cells were grown in a 24-well culture plate and transduced with adenoviral vectors (0–100 PFU/cell) followed by ganciclovir (GCV) therapy (Sigma Co, St. Louis, MO, US) at a concentration of 10 µg/ml for 3–7 days. Cell growth was measured with the MTT kit (Sigma Co, St. Louis, MO, US) per the manufacturer's instructions. AD-GFP vector was used in our initial proof of concept experiments. We then used adenovirus vector encoding HSV-1TK gene under transcriptional control of the Rous sarcoma virus (AD-HSV1TK), as described by Chen et. al. (33). An adenovirus vector expressing a marker gene coding for bacterial β-galactosidase (AD-Lac Z), was used as a negative control. Both viruses were in-kind gifts from Dr. Savio Woo (Mount Sinai School of Medicine, New York, NY, US). The AD-RGD-TK vector, a modified adenovirus vector contains the herpes simplex virus thymidine kinase gene. This complex was prepared in Dr. David Curiel’s lab (Washington University, St. Louis, MO). A genetic incorporation of the RGD-4C motif into the H1 loop of the fiber was used to enhance the infectivity of the vector by permitting binding to the αvβ3 and αvβ5 integrins as previously described.(34). Magnetic nanoparticles were purchased from OZ Biosciences (Parc Scientifique de Luminy, France). •
Preparation of adenovirus−magnetic nanoparticles (MNPs) complexes for magnetofection
Magnetic nanoparticles adenovirus complexes were prepared by mixing 75 µL of Adenomag magnetic nanoparticle suspension with 15 µl of 6.5× 1011 VP of AD-RGD-TK, equal to 6.5 × 109 PFU/ml diluted in 500 µL of serum free culture media, a lower ratio than
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recommended by the manufacturer of Adenomag (OZ Biosciences, Parc Scientifique de Luminy, Marcella, France). After a 20-minute incubation period at room temperature (RT), different dilutions of the complexes were performed to cover the required multiplicities of infection (MOIs), so that 50 µL of the complexes were added to the cells. AD-GFP, ADRGD Luciferase, and AD-RGD-TK were utilized in preparation of the complex for proof of infectivity to wild type adenovirus as well as genetically modified AD-RGD variant, and cytotoxicity of AD-RGD-TK adenovirus complexes. Magnetic complexes were applied to cells in a drop-wise manner. Cells were then incubated for 30 min on a super magnetic plate (OZ Biosciences, France) at 37°C, 5% CO2. The magnetic plate was then removed and cells were cultured under standard cell culture conditions until the end of the experiments. •
MNPs characterization
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The morphological examination of MNP was carried out through the use of a transmission electron microscope (TEM) (JEOL JEM 1230 Transmission Electron Microscope) with 2% phosphotungstic acid negative staining. The mean size and zeta potential of the nanoparticles were determined by nanoparticle tracking analysis with ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) and corresponding software ZetaView 8.02.28. Samples were diluted 1 to 10 in 1× PBS and sonicated prior to ZetaView measurement. Zeta potential was measured using 0.05 × PBS instead of 1× PBS for conductivity at approximately 500 µS/cm. Per OZ Bioscience Company, nanoparticles have a hydrodynamic radius of 200–300 nm, with 10–15 nm, metal cores with positive zeta potential. We also characterized adenovirus size by electron microscope before and after conjugation with the MNP through incubation with the viral particles for 20 minutes at RT. All measurements were taken at pH 7.4, 25° C. •
Comparing Adeno-GFP (AD-GFP) regular transduction versus magnetofection
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Upon completion of both regular transduction and magnetofection protocols, human fibroid tumor cells and human fibroid stem cells with AD-GFP were washed, and fixed with 4% paraformaldehyde (PFA). GFP expression was detected under an inverted fluorescence microscope equipped with a digital camera (Axiovert, Zeiss Carl Zeiss Microscopy Ltd, Cambridge, MA, US). Images were taken with a 20× Plan-Neofluar dry lens. The same exposure for luminosity and contrast were applied to each slide. Images presented, represent three independent experiments. •
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Comparative analysis of AD-RGD luciferase regular transduction versus magnetofection AD-RGD luciferase transfection and magnetofection were performed on human fibroid tumor cells and human fibroid stem cells. Luciferase bioluminescence assays were conducted for group comparisons. –
Boosting suicidal gene therapy of human fibroid tumor cells and human fibroid stem cells by MNPs and applied to a magnetic field Human fibroid tumor cells and human fibroid stem cells were seeded in flat-bottom 24-well plates, at 40,000 cells/ well density for human fibroid tumor cells and 2,000 cells/
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well density for human fibroid stem cells. Cells were then incubated under standard cell culture conditions. After 24h incubation, media in each well was replaced with 600 µL of fresh cell culture media containing 10% FBS; 50 µL of MNPs complexed AD-RGD-TK, AD- Lac Z (negative control) or WT-AD (positive control) virus alone, and prepared as previously described. Cells with MNPs complexed AD-RGD-TK were placed on a magnetic plate in the CO2 incubator for 30 minutes, removed and maintained under regular cell culture conditions. After 24h of infection, media was replenished with fresh cell culture media, and cells were further cultivated until evaluation. All samples were measured in triplicate. Cell survival was assessed by a luciferase assay in stable cells expressing luciferase or by a MTT-based respiration activity assay.
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MTT Assay: Cells were Infected with Adenovirus RGD thymidine kinase (AD-RGD-TK), adenovirus Bgalactosidase (AD- Lac Z), considered the wild type adenovirus (WT-AD) (negative control). Untreated cells were washed with PBS and incubated for 3h in 100 µL of 1 mg/mL MTT solution which was prepared in PBS with 5 mg/ mL glucose. Afterwards, 100 µL of solubilization solution with Dimethyl Sulphoxide (DMSO Sigma, St. Louis.MO, US) was added to dissolve formazan. Optical density was measured at 590 nm and cell viability was expressed as respiration activity normalized to the reference data for untreated cells.
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Luciferase Assay: Cells were washed with PBS and lysed with 100 µL of lysis buffer (0.1% Triton X-100 in 250 mM Tris; pH 7.8) per well. After incubation for 15–20 min at RT, 50 µL of cell lysate was drawn from each well and transferred into a 96-well black flat-bottom plate. Finally, 100 µL of luciferase buffer was added. Cells were then washed with PBS, and 100 µL of 35 µM D-Luciferin in PBS was added.
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Detection of the bystander effect in human fibroid stem cells Tumor cells transduced with an adenovirus vector expressing the herpes simplex virus thymidine kinase (HSV-TK) gene are rendered sensitive to the anti-herpetic drug, ganciclovir. The bystander effect refers to the observation that not all cells need to be transduced, in order to be killed. This is attributed to the capacity to transfer small cytotoxic molecules from transfected to non-transfected cells due to direct contact and the presence of gap junctions, thus conferring the cytotoxicity to them. We wanted to check for this bystander effect in human fibroid stem cells so that we could account for its action in
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deducing the viral load required for their complete eradication. Human fibroid stem cells were grown in 100 mm tissue culture plates until 70–80% confluence was achieved and then transfected with the AD-RGD TK as previously described. The next day, cells (as well as cells from uninfected plates) were trypsinized and counted. Different ratios of uninfected/ infected cells were cocultured, for a total of 4,000 cells and plated in 24-well plates. 24h later media was removed and replaced with media containing GCV 10ug/ml. Five days later, viable cell counts were determined using MTT Assay. Isolation of protein and immunoblots
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Proliferating cell nuclear antigen (PCNA), and BCL2-associated X protein (BAX) expression was examined using western blot analysis. Ice-cold radio-immune-precipitation assay (RIPA) buffer supplemented with phosphatase and protease inhibitors (50 mM sodium vanadate, 0.5 mM phenylmethylsulphonyl fluoride, 2 mg/ml aprotinin, and 0.5 mg/ml leupeptin) (Sigma, St. Louis, MO, US) was used for protein extraction and for the elimination of homogenates and cell lysate. Protein concentrations were measured by Bradford protein assay (Bio-Rad, Hercules, CA, US). Total protein samples (40 µg) were filtered via SDS-PAGE (Polyacrylamide Gel Electrophoresis) (10% acrylamide gel) using the Bio-Rad Trans-Blot system (Bio-Rad, Hercules, CA, US) and transferred to membranes. Membranes were blocked with 5% non-fat milk in phosphate-buffered saline (Gibco, Grand Island, NY, US) containing 0.1% Tween 20 (Sigma, St. Louis, MO, US)(PBS-T), incubated for 1h, washed in PBS-T, and hybridized with primary antibodies, specific antibodies for PCNA (BAX Santa Cruz Biotechnology, Inc. Dallas, Texas US). In addition, membranes were incubated with a secondary antibody to bind β-actin at a 1:15,000 dilution (Sigma, US), which served as an internal control. Incubation with secondary antibodies and the detection of the antigen-antibody complex were performed using a Super Signal™ West Dura Extended Duration Substrate (Life Technologies, Grand Island, NY, US). Immunoblots of PCNA (36 kDa), BAX (27 kDa), β-actin (43 kDa) were quantified with a Bio-Rad Image Lab. densitometer (Bio-Rad, Hercules, CA US). Caspase-3 Assay
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Caspase-3 enzyme activity was measured in human fibroid stem cells using the Caspase assay system (Abcam, Cambridge, MA, US) based on Caspase 3 enzyme’s ability to release yellow chromophore p-nitro aniline (pNA) from the colorimetric substrate (Ac-DEVD-pNA) provided in the Caspase assay system. Tissue lysates were centrifuged at 13,000 rpm. Reading was at 405 (excitation)/510(emission) using the 96-plate reader Synergy HT with Gen 5 software (Biotek Instruments, Inc., Winooski, VT, US) according to the manufacturer’s instructions. Caspase-3 activity values were normalized against the total tissue protein content measured Data analysis Statistical analysis of the samples were done using a Student’s t-test, where P-values ≤0.05 were considered statistically significant with 95% confidence intervals. All statistical analyses were performed using GraphPad Prism version 6.00 for Mac (GraphPad Software, San Diego, US).
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Results MNPs size and surface charge identification MNPs size ranged from 115 to 214 nm in size, with a mean of 129±71nm and zeta potential of 18.12 mV by ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) and corresponding software ZetaView 8.02.28.Transmision electron microscopy (JEOL JEM 1230, Peabody, MA) showed the shape of adenovirus and MNPs. Covalent conjugation with adenovirus and MNPs was confirmed by the close intimate approximation of nanoparticles around the surface of adenovirus (Fig. 1). MNPs enhance AD-GFP transduction of human fibroid tumor cells
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The purpose of conducting this experiment was to evaluate whether conjugating adenoviral vectors with MNPs, would enhance the ability of adenoviral vectors to transfect human fibroid tumor cells. We compared the transduction efficiency of non-conjugated AD-GFP to that of MNPs conjugated AD-GFP at two different multiplicities of infection (MOI), 5 and 10 PFU/cell (Fig.2A). As shown in Fig.2B, there was a significant increase in the number of transfected cells from 8.06% in unconjugated AD GFP group to 28.56 % ± 8.9% in the MNPs conjugated group (p < 0.005), for MOI 5 and an increase from 25.38% in the unconjugated AD GFP group to 85.65 ± 7.57% in the MNPs conjugated group for MOI 10 (p= 0.005). MNPs enhance AD-RGD-luciferase transduction into human fibroid tumor cells
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A previous report from our group (16) demonstrated that by genetically altering adenovirus to express RGD peptide in the viral capsid, transduction efficiency of the adenovirus was markedly enhanced against human fibroid tumor cells. In this study, we aimed to investigate whether further enhancement of transduction efficiency could be accomplished by means of MNPs-adenoviral conjugations. As shown in Supplementary Fig. 1, MNPs significantly enhanced transduction efficiency of the modified virus AD-RGD-Luc (as reflected in bioluminescence intensity measured by luciferase assay) from 23.43 % in the unconjugated AD RGD LUC to 46.20 ± 5.7% in the MNPs group at MOI 5 and from 44.67±7.8% to 85.77%± 8.6% at MOI 10 10, respectively when compared to the unconjugated AD-RGD-Luc against human fibroid tumor cells (P = 0.005). MNPs enhance the ability of AD-RGD-TK to suppress proliferation of human fibroid tumor cells
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We recently used AD-RGD-TK followed by ganciclovir treatment to efficiently induce apoptosis and inhibit proliferation of human fibroid cells (16). In order to assess the ability of MNPs to further enhance the anti-fibroid ability of AD-RGD-TK, ascending MOIs of MNPs-conjugated versus unconjugated virus were tested against human fibroid cells in vitro. As shown in Supplementary Fig. 2, there was a significant decrease in the percentage of viable cells from 45% ± 1.34 % to 33% ± 1.38% at MOI 25, from 39± 2.6 % to 25 ± 1.43% at MOI 50 and from 32 ± 2.15 % to 21 ± 0.98% at MOI 75 (p
