Conditioned medium of induced pluripotent stem cell derived neuromesodermal progenitors enhances cell migration in vitro.

BACKGROUND: Identification of novel cell-based therapy sources has been of great interest in recent years to provide alternative and available therapy options in clinics. Conditioned medium (CM) can be a valuable supply for growth factors, cytokines and chemokines as a source of stem cell secretome. Exploring the role of new CM sources for tissue regeneration might be a promising approach for therapeutic purposes. METHODS AND RESULTS: In the current study, neuromesodermal progenitors (NMPs) derived from induced pluripotent stem cells (iPSCs) were used to collect CM. Fibroblast derived iPSCs were successfully differentiated into NMPs and NMPs were characterized by double positive T/Bra and Sox2 staining. CM was collected from NMPs, and the content was characterized by membrane analysis. In vitro wound healing assay was used as a model system to observe potential activity of CM on cell migration. Fibroblasts, keratinocytes and endothelial cells were used to evaluate the effect of NMP-derived CM (NMP-CM) on cell migration in vitro. Several important proteins related to wound healing such as ANGPT 1, ANGPT 2, MCP-1, PDGF-AA, SDF-1α, TIMP-1 and TIMP-2 were increased in NMP-CM. NMP-CM increased cell proliferation and migration in vitro. CONCLUSIONS: In vitro data obtained from three distinct cell types suggest a promising role of NMP-CM on cell migration. NMP-CM can be used for wound management in the further future after detailed in vitro and in vivo research.

Azoxymethane-induced carcinogenesis-like model of mouse intestine and mouse embryonic stem cell-derived intestinal organoids.

BACKGROUND: Tumor modeling using organoids holds potential in studies of cancer development, enlightening both the intracellular and extracellular molecular mechanisms behind different cancer types, biobanking, and drug screening. Intestinal organoids can be generated in vitro using a unique type of adult stem cells which are found at the base of crypts and are characterized by their high Lgr5 expression levels. METHODS AND RESULTS: In this study, we successfully established intestinal cancer organoid models by using both the BALB/c derived and mouse embryonic stem cells (mESCs)-derived intestinal organoids. In both cases, carcinogenesis-like model was developed by using azoxymethane (AOM) treatment. Carcinogenesis-like model was verified by H&E staining, immunostaining, relative mRNA expression analysis, and LC/MS analysis. The morphologic analysis demonstrated that the number of generated organoids, the number of crypts, and the intensity of the organoids were significantly augmented in AOM-treated intestinal organoids compared to non-AOM-treated ones. Relative mRNA expression data revealed that there was a significant increase in both Wnt signaling pathway-related genes and pluripotency transcription factors in the AOM-induced intestinal organoids. CONCLUSION: We successfully developed simple carcinogenesis-like models using mESC-based and Lgr5 + stem cell-based intestinal organoids. Intestinal organoid based carcinogenesi models might be used for personalized cancer therapy in the future.

Muscle Organoid and Assembloid Systems.

Skeletal muscle is one of the most complex and largest tissues that perform important processes in the body, including performing voluntary movements and maintaining body temperature. Disruption of muscle homeostasis results in the development of several disorders, including diabetes and sarcopenia. To study the developmental and regenerative dynamics of skeletal muscle and the mechanism behind muscle diseases, it is important to model skeletal muscle and diseases in vitro. Since skeletal muscle has a complex structure and interaction with other tissues and cells that are required to perform their function, conventional 2D cultures are not sufficient to model the skeletal muscle with their interactions. Advances in the field of organoids and assembloids will enable the establishment of more complex and realistic tissue or disease models which cannot be fully recapitulated in conventional 2D culture systems for use in several areas, including disease research, regenerative, and tissue biology. To overcome these limitations, 3D organoid systems and assembloid systems are promising because of their success in recapitulating the complex structural organization, function, and cellular interactions of skeletal muscle.

Apelin Receptor Signaling Protects GT1-7 GnRH Neurons Against Oxidative Stress In Vitro.

Hypothalamic-pituitary-adrenal (HPA) axis regulates stress response in the body and abnormal increase in oxidative stress contributes to the various disease pathogenesis. Although hypothalamic distribution of Apelin receptor (APLNR) has been studied, the potential regulatory role in hormone releasing function of hypothalamus in response to stress is not well elucidated yet. To determine whether APLNR is involved in the protection of the hypothalamus against oxidative stress, gonadotropin-releasing hormone (GnRH) cells were used as an in vitro model system. GT1-7 mouse hypothalamic neuronal cell line was subjected to H2O2 and hypoxia induced oxidative stress under various circumstances including APLNR overexpression, knockdown and knockout. Overexpression and activation of APLNR in GnRH producing neurons caused an increase in cell proliferation under oxidative stress. In addition, blockage of APLNR function by siRNA reduced GnRH release. Activation of APLNR initiated AKT kinase pathway as a proliferative response against hypoxic culture conditions and blocked apoptosis. Although expression and activation of APLNR have not been related to GnRH neuron differentiation during development, positive contribution of activated APLNR signaling to GnRH release in mouse embryonic stem cell derived GnRH neurons was observed in the present study. Sustained overexpression and complete deletion of APLNR in mouse embryonic stem cell derived GnRH neurons reduced GnRH release in vitro. The present findings suggest that expression and activation of APLNR in GnRH releasing GT1-7 neurons might induce a protective mechanism against oxidative stress induced cell death and APLNR signaling may play a role in GnRH neurons.

Parathyroid Cell Differentiation from Progenitor Cells and Stem Cells: Development, Molecular Mechanism, Function, and Tissue Engineering.

Parathyroid glands are endocrine organs which are located posterior to thyroid glands and control secretion of parathyroid hormone (PTH) in order to regulate blood calcium level. PTH maintains calcium homeostasis by acting on the bone, kidney, and small intestine. PTH deficiency leads to chronic hypocalcemia, organ calcinosis, kidney and heart failure, painful muscle spasms, neuromuscular problems, and memory problems. Since parathyroid cells have inadequate proliferation potential in culture conditions, their utilization as a cellular therapy option is very limited. Although studies conducted so far include parathyroid cell differentiation from various cell types, problems related to successful cellular differentiation and transplantation still remain. Recently, parathyroid tissue engineering has attracted attention as a potential treatment for the parathyroid-related diseases caused by hypoparathyroidism. Although major progression is made in the construction of tissue engineering protocols using parathyroid cells and biomaterials, PTH secretion to mimic its spontaneous harmony in the body is a challenge. This chapter comprehensively defines the derivation of parathyroid cells from various cell sources including pluripotent stem cells, molecular mechanisms, and tissue engineering applications.

Organoids in Tissue Transplantation.

Improvements in stem cell-based research and genetic modification tools enable stem cell-based tissue regeneration applications in clinical therapies. Although inadequate cell numbers in culture, invasive isolation procedures, and poor survival rates after transplantation remain as major challenges, cell-based therapies are useful tools for tissue regeneration.Organoids hold a great promise for tissue regeneration, organ and disease modeling, drug testing, development, and genetic profiling studies. Establishment of 3D cell culture systems eliminates the disadvantages of 2D models in terms of cell adaptation and tissue structure and function. Organoids possess the capacity to mimic the specific features of tissue architecture, cell-type composition, and the functionality of real organs while preserving the advantages of simplified and easily accessible cell culture models. Thus, organoid technology might emerge as an alternative to cell and tissue transplantation. Although transplantation of various organoids in animal models has been demonstrated, liöitations related to vascularized structure formation, cell viability and functionality remain as obstacles in organoid-based transplantation therapies. Clinical applications of organoid-based transplantations might be possible in the near future, when limitations related to cell viability and tissue integration are solved. In this review, the literature was analyzed and discussed to explore the current status of organoid-based transplantation studies.

Apelin Receptor Signaling During Mesoderm Development.

The Apelin receptor (Aplnr) is a G-protein coupled receptor which has a wide body distribution and various physiological roles including homeostasis, angiogenesis, cardiovascular and neuroendocrine function. Apelin and Elabela are two peptide components of the Aplnr signaling and are cleaved to give different isoforms which are active in different tissues and organisms.Aplnr signaling is related to several pathologies including obesity, heart disases and cancer in the adult body. However, the developmental role in mammalian embryogenesis is crucial for migration of early cardiac progenitors and cardiac function. Aplnr and peptide components have a role in proliferation, differentiation and movement of endodermal precursors. Although expression of Aplnr signaling is observed in endodermal lineages, the main function is the control of mesoderm cell movement and cardiac development. Mutant of the Aplnr signaling components results in the malformations, defects and lethality mainly due to the deformed heart function. This developmental role share similarity with the cardiovascular functions in the adult body.Determination of Aplnr signaling and underlying mechanisms during mammalian development might enable understanding of regulatory molecular mechanisms which not only control embryonic development process but also control tissue function and disease pathology in the adult body.

Gene Editing in Human Pluripotent Stem Cells: Recent Advances for Clinical Therapies.

The identification of human embryonic stem cells and reprogramming technology to obtain induced pluripotent stem cells from adult somatic cells have provided unique opportunity to create human disease models, gene editing strategies and cell therapy options.Development of pluripotent stem cells from somatic cells and genomic manipulation tools enabled to use site specific nucleases in the cell therapy research. Identification of efficient gene manipulation, safe differentiation and use will provide a novel strategy to treat many diseases in the near future. Current available registered clinical trials clearly indicate the need for pluripotent stem cell and gene therapy treatment options. Although gene editing based pluripotent stem cell research is a popular field for research worldwide, improvement of clinical approaches for treatment still remains to be investigated. In this review, we summarized the current situation of gene editing based pluripotent cell therapy developments and applications in clinics.

Boron containing compounds promote the survival and the maintenance of pancreatic ß-cells.

Diabetes mellitus is worldwide disease. The life of diabetic patients are dependent on exogenous insulin. Pancreas or particularly islet transplantations are performed for reducing external insulin dependency. External substances are also used to protect the ß-cells from the death or increase insulin secretion. In the current study, two different boron containing compounds (sodium pentaborate pentahydrate-NaB and boric acid-BA) were investigated for their effect on pancreatic cells in terms of pro-apoptotic and anti-apoptotic markers, genes related to insulin production mechanism, pancreatic development and glucose metabolism, some antioxidant enzymes, and genes for the initiation of diabetes, insulin secretion and antioxidant enzyme activities in vitro. The results revealed that boron containing compounds did not lead to apoptosis. On the contrary, they increased cell viability, antioxidant enzyme activities and the level of genes related to insulin production. Overall evaluation, data in the current study showed that boron containing compounds might be promising therapeutic agents for type 1 diabetes. However, additional investigations are strictly needed to elucidate molecular mechanisms of boron containing compounds.

The effects of bisphosphonates on osteonecrosis of jaw bone: a stem cell perspective.

Bisphosphonate-induced osteonecrosis of the jaw (BIONJ) is a commonly encountered side effect of Bisphosphonates (BPs). Although certain aspects of BIONJ have been studied, the effects of BPs on the proliferation, differentiation, and maintenance of dental stem cells (DSC) in way that might account for development of BIONJ have not been evaluated. In the current study, Dental Pulp Stem Cells (DPSCs), Periodontal Stem Cells (PDLSCs), and human Tooth Germ Stem Cells (hTGSCs) were characterized and then each stem cell type were treated with selected BPs: Zoledronate (ZOL), Alendronate (ALE), and Risedronate (RIS). Negative effect on osteogenesis capacity of DSCs has not been observed after differentiation experiments in vitro. BPs exerted inhibitory effect on the migratory capacities of stem cells confirmed by in vitro scratch assay analysis. Angiogenesis of endothelial cells was blocked by BPs treatment in tube formation analysis. In conclusion, inhibitory effects of BPs on migration capacity of DSCs localized in close proximity to the jaw bone might be the primary reason for the side effects of BPs in the development of BIONJ process. Therefore, further in vivo evidence is required to investigate DSC properties in BP treated animals which might elucidate the importance of DSCs in BIONJ formation.

Mesenchymal Stem Cells as Regulators of Carcinogenesis.

Mesenchymal Stem Cells (MSCs) are adult stem cells; isolated from various body parts including bone marrow, adipose tissue and dental tissue, have been characterized well and used in regenerative medicine applications. The promising potential of MSCs makes them great candidates in many disorders. It has been well known in the literature that MSCs interact with cancer cells and regulate the carcinogenesis process at different stages. The dual role of MSCs in cancer progression should be clearly identified at the physiological and molecular level to identify clinical potential in cancer treatment. The promoting or suppressive role of MSCs in cancer is controlled by various growth factors, cytokines and chemokines which affect the cell proliferation, angiogenesis and metastasis. Although many studies have been conducted to explore MSC-cancer cell interactions, it is still unclear how MSCs communicate with cancer cells and tumor microenvironment. Further studies are required to investigate secreted factors and paracrine effects, tumor stroma environment, molecular regulators and downstream pathways that are involved in MSC-cancer interaction loop. MSC type, cancer type and stage specific phenotypic and transcriptomic profile changes should be identified in detail to improve clinical use of MSCs in cancer either as a target or as a tool.In the current book chapter, we review the literature to summarize current information about the MSC-cancer cell interactions in terms of soluble factors, angiogenesis, metastasis and drug resistance. The role of MSCs in tumor progression or suppression was discussed based on the current literature.

Cytoglobin inhibits migration through PI3K/AKT/mTOR pathway in fibroblast cells.

Cell proliferation and migration are crucial in many physiological processes including development, cancer, tissue repair, and wound healing. Cell migration is regulated by several signaling molecules. Identification of genes related to cell migration is required to understand molecular mechanism of non-healing chronic wounds which is a major concern in clinics. In the current study, the role of cytoglobin (CYGB) gene in fibroblast cell migration and proliferation was described. L929 mouse fibroblast cells were transduced with lentiviral particles for CYGB and GFP, and analyzed for cell proliferation and migration ability. Fibroblast cells overexpressing CYGB displayed decreased cell proliferation, colony formation capacity, and cell migration. Phosphorylation levels of mTOR and two downstream effectors S6 and 4E-BP1 which take part in PI3K/AKT/mTOR signaling declined in CYGB-overexpressing cells. Microarray analysis indicated that CYGB overexpression leads to downregulation of cell proliferation, migration, and tumor growth associated genes in L929 cell line. This study demonstrated the role of CYGB in fibroblast cell motility and proliferation. CYGB could be a promising candidate for further studies as a potential target for diseases related to cell migration such as cancer and chronic wound treatment.

Embryonic Stem Cells in Development and Regenerative Medicine.

After progressive improvement in embryonic stem (ES) cell field, several studies have been conducted to explore the usage of ES cells in regenerative medicine. Unlimited self renewal and pluripoteny properties, combined with encouraging preclinical trials, remark that ES cell technology might be promising for clinical practice. ES cells, which can form three germ layers in vitro, are potential candidates to study development at the cellular and molecular level. Understanding the cell fate decision and differentiation processes during development might enable generating functional progenitor cells for tissue restoration. Progression in gene modifications and tissue engineering technology has facilitated the derivation of desired cells for therapy. Success in differentiation protocols and identification the regulatory pathways simplify the research for clinical applications. Although there are established protocols for cell differentiation in vitro and promising preclinical studies in vivo, many challenges need to be adressed before clinical translation. In this review, ES cells are discussed as a model of development in vitro and as a potential candidate for regenerative medicine. This review also dissusses current challenges for ES cell based therapy.

Poloxamer P85 increases anticancer activity of Schiff base against prostate cancer in vitro and in vivo.

Prostate cancer is the second most common cancer among men and the leading cause of death after lung cancer. Development of hormone-refractory disease is a crucial step for prostate cancer progression for which an effective treatment option is currently unavailable. Therefore, there is a need for new agents that can efficiently target cancer cells, decrease tumor growth, and thereby extend the survival of patients in late-stage castration-resistant prostate cancer. In the current study, a novel heterodinuclear copper(II)Mn(II) Schiff base complex combined with P85 was used to evaluate anticancer activity against prostate cancer in vitro and in vivo. Cell proliferation and cytotoxicity were evaluated by cell viability, gene, and protein expression assays in vitro. Results showed that the heterodinuclear copper(II)Mn(II) complex-P85 combination decreased cell proliferation by upregulating the apoptotic gene expressions and blocking the cell proliferation-related pathways. Tramp-C1-injected C57/B16 mice were used to mimic a prostate cancer model. Treatment combination of Schiff base complex and P85 significantly enhanced the cellular uptake of chemicals (by blocking the drug transporters and increased life time), suppressed tumor growth, and decreased tumor volume steadily over the course of the experiments. Overall, heterodinuclear copper(II)Mn(II) complex-P85 showed remarkable anticancer activity against prostate cancer in in vitro and in vivo.

Anticandidal activity of hetero-dinuclear copper(II) Mn(II) Schiff base and its potential action of the mechanism.

Invasive fungal infections are one of the major challenges especially for immunosuppressed patients since they are drug resistant and pathogen to patients. Therefore, developing new, efficient and nonresistant antifungal agents have been a primary focus of international research. In the current study, a novel Schiff base [hetero-dinuclear copper(II) Mn(II) complex] (SB) derivative was investigated for its anticandidal activity against Candida albicans and possible mechanisms inducing cell death. The results revealed that SB treatment induces apoptotic and necrotic pathways in C. albicans ATCC10231 strain. Intracellular reactive oxygen species production determined by 2',7'-dichlorofluorescein diacetate staining was triggered by SB and amphotericin B administrations in a dose-dependent manner. Gene expression analysis demonstrated that SB exposure resulted in regulation of critical development and stress related gene expressions. SB treatment directly upregulated expression of stress related genes, DDR48 and RIM101, while suppressed important cell signaling and antibiotic resistance acquiring related genes such as HSP90, ERG11 and EFG1. Furthermore, CaMCA1 mRNA levels were found to be significantly high in SB-treated yeast cells, indicating possible caspase-like mechanism activation. Scanning electron microscopy analysis confirmed that SB treatment led to severe cell wall integrity disruption and wrinkling. The study will encourage development of SB-based anticandidal regimens but further studies are highly warranted to understand limitations and the extended use in the routine.

Schiff Base-Poloxamer P85 Combination Prevents Prostate Cancer Progression in C57/Bl6 Mice.

BACKGROUND: Prostate cancer which is the second most common cause of death among men has a high incidence in recent years. Current therapeutic regimens should be improved to overcome drug resistance. At the metastatic stage, tumors become refractory to established chemotherapeutic treatments and cause serious problems at the clinics. Development of new drug molecules that are able to transport through the membrane easily and kill tumor cells rapidly is of great interest. METHOD: In the current study, a novel Heterodinuclear copper(II)Mn(II) Schiff base complex combined with P85 was used for prostate cancer treatment in vivo. Tramp-C1 cells injected animals were subjected to chemotherapeutic formulation treatment and results were analyzed by toxicology analysis, tumor volume measurements, and histopathological analysis. 0.5 mg/kg Schiff base was selected and combined with 0.05% P85 according to the toxicology analysis showing the enzyme levels, blood parameters, and multiple organ toxicity. RESULTS: Results demonstrated that Heterodinuclear copper(II)Mn(II) complex-P85 combination decreased tumor formation and tumor volume steadily over the course of experiments. CONCLUSIONS: Overall, Heterodinuclear copper(II)Mn(II) complex-P85 exerted remarkable anti-cancer activity in vivo in C57/B16 mice. Prostate 76:1454-1463, 2016. © 2016 Wiley Periodicals, Inc.

Boron promotes streptozotocin-induced diabetic wound healing: roles in cell proliferation and migration, growth factor expression, and inflammation.

Acute wounds do not generally require professional treatment modalities and heal in a predictable fashion, but chronic wounds are mainly accompanied with infection and prolonged inflammation, leading to healing impairments and continuous tissue degradation. Although a vast amount of products have been introduced in the market, claiming to provide a better optimization of local and systemic conditions of patients, they do not meet the expectations due to being expensive and not easily accessible, requiring wound care facilities, having patient-specific response, low efficiency, and severe side-effects. In this sense, developing new, safe, self-applicable, effective, and cheap wound care products with broad-range antimicrobial activity is still an attractive area of international research. In the present work, boron derivatives [boric acid and sodium pentaborate pentahydrate (NaB)] were evaluated for their antimicrobial activity, proliferation, migratory, angiogenesis, gene, and growth factor expression promoting effects on dermal cells in vitro. In addition, boron-containing hydrogel formulation was examined for its wound healing promoting potential using full-thickness wound model in streptozotocin-induced diabetic rats. The results revealed that while both boron compounds significantly increased proliferation, migration, vital growth factor, and gene expression levels of dermal cells along with displaying remarkable antimicrobial effects against bacteria, yeast, and fungi, NaB displayed greater antimicrobial properties as well as gene and growth factor expression inductive effects. Animal studies proved that NaB-containing gel formulation enhanced wound healing rate of diabetic animals and histopathological scores. Overall data suggest a potential promising therapeutic option for the management of chronic wounds but further studies are highly warranted to determine signaling pathways and target metabolisms in which boron is involved to elucidate the limitations and extend its use in clinics.

A Schiff base derivative for effective treatment of diethylnitrosamine-induced liver cancer in vivo.

Hepatocellular carcinoma is one of the most prevalent cancers, with a high morbidity rate, even in developed countries. In the present study, the curative effect of the Schiff base (SB) heterodinuclear copper(II)Mn(II) complex on diethylnitrosamine (DEN)-induced liver carcinoma was investigated. Hepatocarcinoma was initiated by an injection of DEN and promoted by phenobarbital (0.05%) in the diet. In addition, the potential nephrotoxicity of SB was evaluated in a cisplatin-induced nephrotoxicity model. Rats were administered the SB complex (1 and 2 mg/kg body weight/day) for 24 weeks, and cancer progression was investigated by macroscopic, histopathological, and western blot examinations. The administration of SB decreased the incidence and the number of hepatic nodules in a dose-dependent manner by regulating inflammation response and the apoptotic pathway. Western blot analyses from the livers of rats treated with SB after DEN induction showed significantly enhanced Bax and caspase-3 levels, with a marked decrease in the levels of Bcl-2, NF-κB p65 and cyclooxygenase (COX)-2. Results from the nephrotoxicity study showed that, whereas cisplatin increased serum urea nitrogen and creatinine levels, no increase in serum biochemical parameters was detected in SB-treated animals. Moreover, protein levels of NF-E2-related factor-2 (Nrf2) and heme oxygenase-1 were lower, whereas nuclear factor-κB (NF-κB p65) and activator protein-1 levels were higher in the kidneys of cisplatin-treated animals compared with that of the SB groups. Therefore, the SB complex could be an alternative chemotherapeutic option for liver cancer treatment once its safety in clinical applications has been examined.

In vitro differentiation of human tooth germ stem cells into endothelial- and epithelial-like cells.

Current clinical techniques in dental practice include stem cell and tissue engineering applications. Dental stem cells are promising primary cell source for mainly tooth tissue engineering. Interaction of mesenchymal stem cell with epithelial and endothelial cells is strictly required for an intact tooth morphogenesis. Therefore, it is important to investigate whether human tooth germ stem cells (hTGSCs) derived from wisdom tooth are suitable for endothelial and epithelial cell transformation in dental tissue regeneration approaches. Differentiation into endothelial and epithelial cell lineages were mimicked under defined conditions, confirmed by real time PCR, western blotting and immunocytochemical analysis by qualitative and quantitative methods. HUVECs and HaCaT cells were used as positive controls for the endothelial and epithelial differentiation assays, respectively. Immunocytochemical and western blotting analysis revealed that terminally differentiated cells expressed cell-lineage markers including CD31, VEGFR2, VE-Cadherin, vWF (endothelial cell markers), and cytokeratin (CK)-17, CK-19, EpCaM, vimentin (epithelial cell markers) in significant levels with respect to undifferentiated control cells. Moreover, high expression levels of VEGFR1, VEGFR2, VEGF, CK-18, and CK-19 genes were detected in differentiated endothelial and epithelial-like cells. Endothelial-like cells derived from hTGSCs were cultured on Matrigel, tube-like structure formations were followed as an indication for functional endothelial differentiation. hTGSCs successfully differentiate into various cell types with a broad range of functional abilities using an in vitro approach. These findings suggest that hTGSCs may serve a potential stem cell source for tissue engineering and cell therapy of epithelial and endothelial tissue.

Process and formulation variables of pregabalin microspheres prepared by w/o/o double emulsion solvent diffusion method and their clinical application by animal modeling studies.

Pregabalin is an anticonvulsant drug used for neuropathic pain and as an adjunct therapy for partial seizures with or without secondary generalization in adults. In conventional therapy recommended dose for pregabalin is 75 mg twice daily or 50 mg three times a day, with maximum dosage of 600 mg/d. To achieve maximum therapeutic effect with a low risk of adverse effects and to reduce often drug dosing, modified release preparations; such as microspheres might be helpful. However, most of the microencapsulation techniques have been used for lipophilic drugs, since hydrophilic drugs like pregabalin, showed low-loading efficiency and rapid dissolution of compounds into the aqueous continous phase. The purpose of this study was to improve loading efficiency of a water-soluble drug and modulate release profiles, and to test the efficiency of the prepared microspheres with the help of animal modeling studies. Pregabalin is a water soluble drug, and it was encapsulated within anionic acrylic resin (Eudragit S 100) microspheres by water in oil in oil (w/o/o) double emulsion solvent diffusion method. Dichloromethane and corn oil were chosen primary and secondary oil phases, respectively. The presence of internal water phase was necessary to form stable emulsion droplets and it accelerated the hardening of microspheres. Tween 80 and Span 80 were used as surfactants to stabilize the water and corn oil phases, respectively. The optimum concentration of Tween 80 was 0.25% (v/v) and Span 80 was 0.02% (v/v). The volume of the continous phase was affected the size of the microspheres. As the volume of the continous phase increased, the size of microspheres decreased. All microsphere formulations were evaluated with the help of in vitro characterization parameters. Microsphere formulations (P1-P5) exhibited entrapment efficiency ranged between 57.00 ± 0.72 and 69.70 ± 0.49%; yield ranged between 80.95 ± 1.21 and 93.05 ± 1.42%; and mean particle size were between 136.09 ± 2.57 and 279.09 ± 1.97 µm. Pregabalin microspheres having better results among all formulations (Table 3) were chosen for further studies such as differential scanning calorimetry, Fourier transform infrared analysis and dissolution studies. In the last step, the best pregabalin microsphere formulation (P3) was chosen for in vivo animal studies. The pregabalin-loaded microspheres (P3) and conventional pregabalin capsules were applied orally in rats for three days, resulted in clinical improvement of cold allodynia, an indicator of peripheral neuropathy. This result when evaluated together with the serum pregabalin levels and in vitro release studies suggests that the pregabalin microspheres prepared with w/o/o double emulsion solvent diffusion method can be an alternative form for neuropathic pain therapy. Conclusively, a drug delivery system successfully developed that showed modified release up to 10 h and could be potentially useful to overcome the frequent dosing problems associated with pregabalin conventional dosage form.