{"paper_id":"39393ab0-aa57-4005-a5e3-f8e4473787b7","body_text":"Endometriosis is an estrogen-dependent gynecological\ndisease in which endometrial tissue is found in unusual\nplaces outside the uterus, ranging from minor lesions on\nother healthy pelvic organs to large endometriotic ovarian\ncysts. This condition changes the ovaries and causes the\nviscera to stick to each other, and this adhesion is the\nmain cause of pain and infertility in these people ( 1 ). Ding\net al. ( 2 ) show that the cause of estrogen sensitivity in\nendometriosis tissues is related to the activity of neurons.\nInflammation affects the growth of the oocyte, and the\nunfavorable environment created by the activity of\nmacrophages reduces the quality of the oocyte, resulting\nin infertility ( 3 ). There is also an endocannabinoid system\nin the human ovary that is effective in the neuro protection\nof cells and has an anti-inflammatory function through\nmodulating cell survival and proliferation and inducing\napoptosis in normal cells.\nIn one study a relationship between this system and\novarian pathologies was shown ( 4 ). In endometriosis,\nreceptors of this system are inhibited and increased\ncell inflammation, disease progression, and pain ( 5 ).\nEndometriosis is classified as a tumor disease by the\nWorld Health Organization (WHO) because it has a\ntumor-like structure and behaves like cancer in terms of\ninvading other tissues. In fact, endometriosis, like cancer\ncells, attacks tissues, induces angiogenesis, increases the\nproduction of estrogen, impairs immune function, and\ncauses inflammation ( 6 ). At present, the best method for\nthe diagnosis of endometriosis and isolation of invasive\ntissues is laparoscopic examination with the histological\nconfirmation of glands in removed lesions ( 7 ). Therefore,\npeople’s fear of surgery and assuming dysmenorrhea as\nnormal delay the timely diagnosis of the disease ( 8 ).\nToday, methods such as gene therapy have been used\nin the diagnosis and treatment of diseases. MicroRNAs\n(miRNAs) regulate post-transcriptional gene expression\nplaying a crucial role in proliferation, differentiation, and apoptosis, which are key to the diagnosis of endometriosis\n( 9 ). miRNAs are involved in cell survival, proliferation,\nangiogenesis, and apoptosis, therefore, they are effective in\nthe pathogenesis of endometriosis ( 10 ). In endometriosis,\nprogrammed cell death, which is called apoptosis, is\nreduced. Thus, endometriotic tissues develop in the\nabdominal and pelvic cavities. Accordingly, there is an\ninverse relationship between the severity of endometriosis\nand the rate of apoptosis ( 11 ). Viral and non-viral vectors\nare used for miRNAs transmission. Non-viral gene\ndelivery vectors, such as inorganic nanoparticles (NPs)\nand liposomes, have been used in recent years ( 12 ).\nOne of the best carriers is poly (lactic-co-glycolic acid) (PLGA), which has been widely\nused in drug delivery because it escapes from the endo-lysosome system and keeps its\ncontents longer. Therefore, it is also suitable for gene transfer ( 13 ). Various miRNAs have\nbeen observed in endometrial and atopic lesions ( 14 ). One of the miRNAs inhibited in\nendometriosis is miR-503, which is involved in endometriotic cyst stromal cells (ECSCs) cell\napoptosis. miR-503 inhibits cell cycle in the G0/G1 phase and prevents cell proliferation\n( 15 ). Since to date, there is no study that have been measured the effects of this\nnanoparticle on apoptosis of endometriosis cells, in this study we performed the effect of\nPLGA-micro RNA delivery on the apoptosis of stromal cells of ovarian endometrium cysts\n in vitro .\n\nIn this experimental study, ovarian endometrioma cyst walls were removed from women 30 to\n40 years at Hazrat-e Rasool Hospital during laparoscopic surgery. After washing, the\ntissue fragments were enzymatically digested with collagenase and DNAase and incubated at\n37°C with 5% CO 2  for 90 minutes. Then, the tissue pieces were passed through\nfilters (45 µm) ( 16 ). Cells cultured at 37°C in a 5% CO 2  atmosphere in DMEM/F12\nmedium were supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), gentamycin\n(40 µg/mL), and 5% fetal bovine serum for three weeks, and the culture medium was changed\nevery 2-3 days when they reached 80% confluency .The research was approved by the Research\nEthics Committee of Iran University of Medical Sciences (IR.IUMS.REC.1397.1176).\nIn order to confirm the cyst wall endometrial tissue-isolated\ncells, immunocytochemistry was performed for CD10 markers\n( 17 ). After 3 weeks, endometriosis cells were detached from\nthe flask using trypsin/EDTA .The cells were smeared and\nincubated with 4% paraformaldehyde at room temperature\nfor 10 minutes. Triton X-100 was added to the cells to make\nthe cell membrane permeable to antibodies, then antibodies\nwere added to the cells at appropriate concentrations in a\n1:200 ratio overnight at 4°C. Also, the cells were washed\nthoroughly and stained with Fluorescein isothiocyanate\n(FITC)-conjugated anti-mouse Ig antibodies 1:500 for 1 hour\nat room temperature in the dark. After a further wash, they\nwere mounted in glycerol. CD markers were analyzed by\nfluorescence microscopy.\nmiRNAs 503, including primer (sequence [5ˊ → 3ˊ]:\nTAGCAGCGGGAACAGTTCTGCAG) (Fluorescent\nmarker), were purchased from Pishgam Co. (St Kargar,\nTehran, Iran), and PLGA (Resomer RG502H) with\na 50:50 mole ratio of glycolic acid to lactic acid and a\nmolecular weight of 12,000 g/mol, polyvinyl alcohol\n(PVA, 89 mol% hydrolyzed), Span 80, and Tween 80\nwere purchased from Sigma (St Louis, MO, USA).\nPLGA NPs containing miRNAs were synthesized by using\nthe water-in-oil-in-water solvent vaporization technique\nconsisting of an organic phase and two aqueous phases in one\nmedium ( 18 ). To stabilize the dispersed phase, a stabilizer\nis needed, the most commonly used of which is polyvinyl\nalcohol. This substance in the external aqueous phase creates\na thin layer around the particles. In the organic phase, the\nactive substance was used at the Span 80 level, and in the\naqueous external phase, Tween 80 level. All the solutions\nwere prepared in DEPC-treated water, and RNase-free media\nwere used at all stages. To prepare the desired nano capsule,\nthe internal aqueous phase was first obtained by creating a\npolyplex derived from polyethylene imine(PEI 25)KDa\nand miRNA with a 3: 1 mass ratio. Polyethylene imine is\ncapable of compressing high molecular chain genetic content\nthat can produce NPs of appropriate size entering the cell\nas endocytosis. First, 0.1% solution of PEI was prepared in\nDEPC-treated water. A miRNA solution was also prepared\nusing DEPC-treated water at a concentration of 100 pmol/μl.\nIt was then combined in 80 µl of PEI solution containing 90\nµg of PEI and 40 µl of miRNA containing 30 µg of miRNA,\nand the volume was reached to 0.5 ml by phosphate-buffered\nsaline (PBS, Merck, USA) and incubated in a thermocycler\nat 37°C for 30 minutes.\nTo prepare the organic phase in solvent evaporation, 10\nmg of PLGA was dissolved in 2 ml of organic solvent ethyl\nacetate. To form an initial emulsion of 0.5 ml of internal\naqueous phase, 0.5 ml of Span 80 solution at a concentration\nof 5 mg/ml was added to the organic phase using Vertex and\nan ultrasonic bath. The initial water emulsion was created in\nthe oil.\nThis water emulsion was then added dropwise to an oil\ncontaining 5 ml of 8% PVA and 10 mg of Tween 80 as an\nouter phase for 3 minutes using a probed ultrasonic device.\nThe power of 50 watts was subjected to sonication to\nform a secondary emulsion. In the final step, the final dual\nemulsion solvent diffusion was added to 4 ml of 0.5% PVA\nand subjected to magnetic stirring for 4 hours until the ethyl\nacetate solvent was used to diffuse the external aqueous\nphase containing solid polymer particles. After forming\nthe nanoparticles, they were separated twice and purified\nby centrifugation at 12,000 rpm for 30 minutes and also suspended in distilled water to remove unloaded miRNA\nand additional surfactants in the external aqueous phase\nfrom the surface of NPs (the supernatant was investigated\nby dynamic light scattering to determine the separation\nefficiency). The nano capsules were finally dried for 24\nhours and stored in a refrigerator at a temperature of 4°C\nfor 24 hours. All of the above steps were performed with\n0.5 ml distilled water without drug as internal aqueous\nphase ( 19 ). Poly lactic-co-glycolic (Resomer RG502H)\nwith a 50:50 mole ratio of glycolic acid to lactic acid\nand a molecular weight of 12,000 g/mol, PVA (89 mol%\nhydrolyzed), Span 80 and Tween 80 were purchased from\nSigma (St Louis, MO, USA).\nSome of the dried powder of PLGA/PEI/miRNA was\ndispersed in 1 ml of saline phosphate buffer at pH=7.4 using\nan ultrasonic bath, and the zeta potential was measured using\na zeta meter device. The size and morphology of the PLGA\nNPs and PLGA modified with PEI /miRNA complexes\nonto a copper sheath, carbon coated, were characterized via\ntransmission electron microscopy (TEM). Finally, the cellular\nuptake of NPs was examined by TEM. For the TEM technique,\nECSCs were washed with PBS, then 2.5% glutaraldehyde was\nused as a primary fixation for 2 hours. The cells were rinsed\n2-3 times with PBS, and free glutaraldehyde was removed.\nThen, 1% osmium tetroxide was used as a secondary fixation\nfor 1.5 hours. The cells were dehydrated in acetone (50%,\n70%, 90%, 100%), infiltrated by resin, and finally, embedded\nin pure resin (Epon 812, TAAB, UK). Then, 50 nm sections\nwere stained with uranyl acetate and lead citrate on copper\ngrade and then imaged with TEM (LEO 906, Zeiss).\nIn this study, ECSCs were divided into the five groups of control and experimental\ngroups, with cells distributed in a 96 well plate at a cell density of 20×10 3 \ncells per well in the different concentrations of PLGA/miRNA (25, 50, 75, and 100 μM) and\ndifferent incubation periods (12, 24, 48, and 72 hours). We performed the MTT assay to\ndetermine the toxicity of PLGA/miRNA. To evaluate the survival rate, the cells were\ncentrifuged and washed with PBS and incubated with 100 µl of MTT solution (MTT tetrazolium\nsalt 5 mg/ ml) for 3-4 hours. Finally, the cells were centrifuged, and the supernatant was\nremoved. Next, 100 µl of dimethyl sulfoxide (DMSO, Merck, USA) was added to the wells, and\nthe plates were shaken for 10 minutes in a microplate shaker before observation with the\nELISA reader at 570 nm. The cells were then treated with the optimum dose obtained, and\nsurvival rates compared with and without nanoparticles.\nAfter determining the effective dose of microRNA and\nPLGA in cell viability ,the cells were cultured with a dose\nof NPs and microRNA/NPs with the lowest viability for\n48 hours. Then, the apoptosis rate of cells was assessed\nusing Annexin V-FITC Apoptosis Detection Kit. That way,\n500μl of the binding buffer was added to the cell plate.\nAfterward, 5 μl of Annexin V-FITC and 5 μl of PI at room\ntemperature were added to the cells and incubated in foil\nfor 10 minutes. Finally, flow cytometry was performed,\nand the rate of apoptosis in cells was evaluated ( 20 ).\nIn this experiment, 12 mice NMRI (n=6 in each groups) ( 6 - 8 -weeks-old) with 25 ± 1 g\nweight were divided into two groups. In the first group, cells that had only PLGA added to\ntheir culture medium were injected, and in the second group, cells treated with PLGA/miRNA\nwere injected. The animals were kept in university laboratory’s animal house. To weaken\nthe immune system of the mice, they were treated with a single dose of 7.5 Gy\nγ-irradiation for 6 min ( 21 ). After 72 hours, cells were transplanted to the back of the\nthigh of the mice. The mice were anesthetized with the intra peritoneal injection of a\nmixture of 100 mg/kg ketamine hydrochloride 10% (Rotexmedica, Germany) and 10 mg/kg\nxylazine 2% (Alfasan, Holland). Then, 20 µL of suspension, including 2×10 6 \ncells at the fourth passage, was injected subcutaneously into the back of the right front\nlimb in each group. All the mouses were observed for 3 weeks.\nData were analyzed using One-way analysis of variance\n(ANOVA) to compare different groups. The analysis was\nperformed by using of SPSS, version 16 (Chicago, IL,\nUSA). Results were expressed as mean ± SEM, and a\nP<0.05 was considered significant.\n\nThe stromal cells were harvested from endometriosis cell. One week after digestion and\nculture, size and morphology of them were similar to fibroblast cells. At the end of third\nweek cell confluency was 2×10 5  cells/ml. An aspect on the phase contrast\nmicroscopy of the third passage of the culture derived from an ovarian endometrioma is\npresented in Figure 1A, B. The cultured cells were confirmed to be positive for CD10\nantigen ( Fig .1C ).\nThe particle size and surface morphology of the NPs were\nexamined by TEM, DLS, and Zeta potential ( Fig .2A-C ).\nPLGA NPs with a size below 100 nm are effective in gene\ntransfer ( 22 ). In this study, the mean diameter of the sole\nPLGA NPs was 60 ± 4 nm, whereas the size of PLGA/\nmiRNA complex with DLS was increased to 70 ± 5.1 nm.\nThe surface charge index of NPs is determined by Zeta\npotential. For the endocytosis of particles into the cell,\na more positive particle load leads to a stronger bond to\nthe cell membrane surface and easier penetration to the\ncell. In this study, zeta potential values of the PLGA/PEI/\nmiRNA complexes were 27.9 mV. The cellular uptake of\nNPs is shown by TEM ( Fig .2D ).\nThe structure of endometriosis cells in the culture medium. Size and morphology of them were\nsimilar to fibroblast cells.  A.  Endometriosis cells one week after\nplanting,  B.  The end of the third week of culture (scale bar: 50 µm).\n C.  Immunocytochemistry of endometriosis cells with CD10 marker (scale\nbar: 30 µm).\nNanoparticle evaluation tests.  A.  Zeta potential,  B.  The particle size\nbased on the DLS test,  C.  Electron microscope image of PLGA showed\nspherical surface in all nanoparticles (scale bar: 500 nm), and  D.  As\nshown, the nanoparticles have accumulated in the nucleus and cytoplasm (scale bar: 1\nµm). DLS; Dynamic light scattering and PLGA; Poly lactic-coglycolic acid.\nThe viability indices of cells for the control and 25,\n50, 75, and 100 µM PLGA/miRNA concentrations were\n97.3%, 91.3%, 85.6%, 81.4%, and 70.2% in 12 hours,\n98%, 88.4%, 80.4%, 78.8%, and 68.1% in 24 hours, and\n97.3%, 70.3%, 64.2%, 52.6%, and 47.9% in 48 hours,\nand 98%, 66.8%, 60.3%, 49.9%, and 44.6% in 72 hours,\nrespectively. The survival rate of stromal cells at the\nconcentrations of 25, 50, 75, and 100 µM PLGA/miRNA\ndecreased compared to the control group at 12, 24, 48, and\n72 hours in a time- and dose- dependent manner.\nThe results showed that with increasing incubation time\nfrom 24 hoursto 48 hours, the survival rate decreased, and\nwith increasing time to 72 hours, cell survival decreased,\nbut no significant difference was observed in this regard\nbetween 24 hours and 72 hours. Therefore, the incubation\ntime was 48 hours. Also, in comparison with the survival\nrate in different doses, the survival rate decreased with\nincreasing concentration, but as the figures shows, no\nsignificant difference was observed between 75 µm and\n100 µm concentrations; therefore, 75 µm doses were\nselected (P≤0.001,  Fig .3 ). The cells were then incubated\nat a dose of 75 µm of PLGA for 48 hours, and the survival\nrate was assessed. The cell viability rates in the control\nand treated groups were 98.4% and 89.9%, respectively, which showed a significant difference in the survival of\ncells treated with PLGA/miRNA ( Fig .4 ).\nBased on MTT test, treatment with 75 µm miR-PLGA for 48 hours\nwas selected; significant differences between groups were observed\n(P≤0.001). MTT; (3-( 4 , 5 -dimethylthiazol-2-yl)-2,5-diphenyltetrazolium\nbromide and PLGA; Poly lactic-co-glycolic acid.\nBased on MTT test, significant differences in survival rate with and\nwithout miRNA were observed (P≤0.001). MTT; (3-( 4 , 5 -dimethylthiazol-2-\nyl)-2,5-diphenyltetrazolium bromide and PLGA; Poly lactic-co-glycolic acid.\nApoptosis was measured using the annexin V-FITC\napoptosis detection kit. The rate of apoptosis in control\ngroup was 0.98 ± 0.1 ( Fig .5A ). The result showed that total\napoptosis in ECSCs treated with PLGA̸ miRNA503 (35.66\n± 4.6%) were significantly higher than those of cells treated\nwith PLGA (3.76 ± 1.19%, P≤0.01,  Fig .5B, C ).\nThe macroscopic observation of endometriosis lesions\nin the two models are presented in Figure 6. In the first\ngroup, cells were treated with PLGA alone, and in the\nsecond group, they were treated with PLGA/miRNA. As\nshown in the figure, in the second group, the rate of cell\napoptosis was higher, and tumor size was smaller. These\nlesions had a cystic morphology and were distinguished\nfrom the surrounding tissues.\nBased on the annexin assay.  A.  Flow cytometry of cells in the control group,\n B.  PLGA-treated group, and  C.  PLGA/miRNA 503-treated\ngroup, result show that the apoptotic rates of the ECSCs treated with PLGA/miRNA 503\nwere significantly higher than those of cells treated with PLGA (P≤0.01).\nIn both groups, cells was injected subcutaneously into the back of the right front limb. After 3\nweeks, the endometriosis lesions in two models were shown.  A.\n PLGA-treated cell injection and  B.  PLGA/miRNA503- treated cell\ninjection.\n\nEndometriosis is a benign disease of the female reproductive system, which is associated\nwith increased angiogenesis and defects in cellular apoptosis ( 23 ) that behaves like a\ncancer in terms of aggressiveness ( 24 ). In one study, plant compounds with anti-inflammatory\nproperties have been used to upturn the apoptotic effect of drugs in the treatment of\ncancer, and it has been observed that cell proliferation and angiogenesis were inhibited\n( 25 ). Due to increase in the percentage of women with endometriosis and its common\ncomplications, including chronic pelvic pain and infertility, a non-surgical diagnosis is\nabsolutely desirable ( 26 ). As the standard diagnostic modality for endometriosis is still\nlaparoscopy, which carries many risks for the patient ( 27 ), a number of studies have been\nperformed in this regard. For example, Samartzis et al. ( 28 ) used Doxycycline to inhibit the\nprogression of endometriotic stromal cells  in vitro .\nNPs through structural mitochondrial damage are\neffective in causing apoptosis and cell necrosis ( 29 ).\nNanomaterials in the treatment of endometriosis are\naccumulation in endometriotic tissues. Chaudhury et al.\n( 30 ) used cerium oxide NPs in an endometriosis-induced\nmouse model and observed the inhibition of angiogenesis.\nIn another study, plant nanocomposites were used to\ninduce apoptosis and necrosis of endometriotic stromal\ncells ( 31 ). NPs can be synthesized from various natural\nor synthetic lipids, proteins, metals, and polymers, one\nof the synthetic polymers used in numerous biomedical\napplications is PLGA ( 32 ). Poly lactic-co-glycolic\nNPs as drug delivery systems in antibiotic therapy,\nchemotherapy, and anti-inflammatory drugs have proven\ntheir potential ( 33 ). Shabani et al. ( 34 ) investigated\nthe anticancer activity of cisplatin conjugated with\nPLGA NPs for elimination of mouse malignant cells\nfrom normal cell and observed that apoptosis rate of\ntumor cell was higher than free drug. Singh et al. ( 35 )\nused the combination of doxycycline and PLGA NPs\nfor the treatment of endometriosis and observed that\nangiogenesis was inhibited. Also used from letrozole\nand curcumin loaded-PLGA NPs for endometriosis in a\nmouse model. Guo et al. ( 36 ) injected endometriosis cells\nsubcutaneously, and then the animal model was treated\nwith two different types of NPs of different sizes (10\nnm vs. 40 nm) by intravenous injection; they observed a\nregression of endometriosis. Li et al. ( 37 ) evaluated iron\noxide NPs (15 nm) modified with hyaluronic acid in a rat\nmodel of endometriosis and reported that these NPs could\naccumulate in CD44 expressing tumors.\nOne biomarker that can be used in research is miRNAs,\nwhich raise as potent regulators of gene expression in\nproliferation, cell survival, and angiogenesis in some\ndisease such as endometriosis ( 38 ). Shams et al. ( 19 ,  39 )\nhave shown that miRNAs are tumor suppressors, they used\ntwo effective miRNAs (143 and 206) to induce apoptosis\nin cancer cells. Hirakawa et al. ( 15 ) found that one miRNA\nin stromal cell of ovarian endometriosis, which inhibits\ncell proliferation and induction of apoptosis, is miRNA\n503 that was epigenetically inhibited. Thus, we developed\na PLGA-based nanoparticle polyplex with miRNA\nexpression to induce apoptosis in endometriosis cells. Our\nresults demonstrated that the cytotoxicity PLGA/miRNA\n503 increased in ECSCs in comparison to PLGA. After the\nincubation of ECSCS with PLGA/miRNA 503, apoptosis\nevaluation was performed by using an annexin V–FITC\napoptosis detection kit. The results showed that the\napoptotic rates of PLGA̸ miRNA 503 were significantly\nhigher than those of PLGA, which is consistent with other\nstudies ( 40 ). After the incubation of cells with PLGA̸\nmiRNA and imaging by TEM, the presence of NPs in the\nnucleus and cytoplasm of cells was confirmed, which is\nin line with studies that showed that NPs with a size of\n10-150 nm and surface charge of +30 – −20 mV could\naccumulate in endometriotic tissues ( 36 ).\n\nWe reported the synthesis and characterization of PLGA/miRNA and its  in\nvitro  effects on the viability of ECSCs. In addition, our results demonstrated\nthat miRNA 503 reduced cell proliferation and progressed apoptotic rate in endometriotic\ncells. The obtained results support the use of the optimal dose of PLGA /miRNA as an\neffective approach for preventing the progression of endometriosis.","source_license":"CC0","license_restricted":false}