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1.
Semin Cancer Biol ; 67(Pt 1): 12-23, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32380234

RESUMO

Chondrogenesis is a highly coordinated event in embryo development, adult homeostasis, and repair of the vertebrate cartilage. Fate decisions and differentiation of chondrocytes accompany differential expression of genes critical for each step of chondrogenesis. SOX9 is a master transcription factor that participates in sequential events in chondrogenesis by regulating a series of downstream factors in a stage-specific manner. SOX9 either works alone or in combination with downstream SOX transcription factors, SOX5 and SOX6 as chondrogenic SOX Trio. SOX9 is reduced in the articular cartilage of patients with osteoarthritis while highly maintained during tumorigenesis of cartilage and bone. Gene therapy using viral and non-viral vectors accompanied by tissue engineering (scaffolds) is a promising tool to regenerate impaired cartilage. Delivery of SOX9 or chondrogenic SOX Trio into cells produces efficient therapeutic effects on chondrogenesis and this event is facilitated by scaffolds. Non-viral vector-guided delivery systems encapsulated or loaded in mechanically stable solid scaffolds are useful for the regeneration of articular cartilage. Here we review major milestones and most recent studies focusing on regulation and function of chondrogenic SOX Trio, during chondrogenesis and cartilage regeneration, and on the development of advanced technologies in gene delivery with tissue engineering to improve efficiency of cartilage repair process.


Assuntos
Cartilagem/citologia , Diferenciação Celular , Condrogênese , Regeneração , Fatores de Transcrição SOX9/metabolismo , Animais , Cartilagem/metabolismo , Humanos
2.
BMC Infect Dis ; 21(1): 952, 2021 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521365

RESUMO

BACKGROUND: Robust evidenced treatment strategy for Coronavirus disease 2019 (COVID-19) has not been established yet. Early, targeted, comprehensive management approach can be essential. METHODS: A lopinavir/ritonavir (LPV/r)-based antiviral treatment was administered to the patients with computed tomography (CT)-documented pneumonia. Medical records of patients with COVID-19, previously discharged or hospitalized for ≥ 21 days at the Seoul Medical Center from January 29 to April 15, 2020 were reviewed to analyze clinical and virological outcomes. Patients were divided into two groups (PCR-Negative conversion group vs. Non-negative conversion group and requiring oxygen group vs. Non-requiring oxygen group). RESULTS: In total, 136 patients with a mean age of 41.8 ± 18.2 years were included with median 3-day delay of hospitalization after illness. Thirteen (9.56%) were initially asymptomatic, and 5 (3.67%) were persistently asymptomatic. Eighty-five (62.5%) had CT-documented pneumonia, 94% of whom received LPV/r treatments. A total of 53 patients (38.97%) had negative polymerase chain reaction (PCR) results within 28 days. Eight (9.4%) out of 85 pneumonic patients received oxygen supplementation. Patients with initial lower respiratory symptoms showed significant delay in PCR negative conversion (> 28 days) (odds ratio [OR] 0.166; 95% confidence interval [CI] 0.067-0.477; P < 0.001). However, antiviral treatment for pneumonic patients was significantly related with early conversion within 28 days (OR 3.049; 95% CI 1.128-8.243; P = 0.028). Increasing age increased the likelihood of oxygen supplementation requirement in the pneumonic patient group (OR 1.108; 95% CI 1.021-1.202; P = 0.014). CONCLUSIONS: Early, pneumonia targeted LPV/r-based antiviral therapy resulted in a significantly higher probability of negative conversion of PCR within 28 days compared to symptomatic treatment.


Assuntos
Tratamento Farmacológico da COVID-19 , Pneumonia , Antivirais/uso terapêutico , Combinação de Medicamentos , Humanos , Recém-Nascido , Lopinavir/uso terapêutico , Pneumonia/tratamento farmacológico , Estudos Retrospectivos , Ritonavir/uso terapêutico , SARS-CoV-2
3.
Biol Pharm Bull ; 39(11): 1802-1808, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27803451

RESUMO

Growth and differentiation factor 3 (GDF3), a mammalian-specific transforming growth factor ß ligand, and OCT4, one of key stem cell transcription factors, are expressed in testicular germ cell tumors (TGCTs) as well as pluripotent stem cells. To understand the molecular mechanism by which OCT4 and GDF3 function in tumorigenesis as well as stemness, we investigated the transcriptional regulation of GDF3 mediated by OCT4 in human embryonic carcinoma (EC) NCCIT cells, which are pluripotent stem cells of TGCTs. GDF3 and OCT4 was highly expressed in undifferentiated NCCIT cells and then significantly decreased upon retinoic acid-induced differentiation in a time-dependent manner. Moreover, GDF3 expression was reduced by short hairpin RNA-mediated knockdown of OCT4 and increased by OCT4 overexpression, suggesting that GDF3 and OCT4 have a functional relationship in pluripotent stem cells. A promoter-reporter assay revealed that the GDF3 promoter (-1721-Luc) activity was significantly activated by OCT4 in a dose-dependent manner. Moreover, the minimal promoter (-183-Luc) was sufficient for OCT4-mediated transcriptional activation and provided a potential binding site for the direct interaction with OCT4. Collectively, this study provides the evidence about the regulatory mechanism of GDF3 mediated by OCT4 in pluripotent EC cells.


Assuntos
Carcinoma Embrionário/genética , Fator 3 de Diferenciação de Crescimento/genética , Fator 3 de Transcrição de Octâmero/genética , Neoplasias Testiculares/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Masculino , Fator 3 de Transcrição de Octâmero/metabolismo , Transcrição Gênica
4.
Nano Converg ; 11(1): 1, 2024 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-38170345

RESUMO

Photoporation techniques based on plasmonic nanoparticles such as gold nanoparticles have been extensively studied for the intracellular delivery of substances via cell membrane disruption. However, the clinical application of AuNP is challenging due to its absorption in the 500 nm region of the light spectrum. To overcome this challenge, upconversion nanoparticles were employed to stimulate AuNP at NIR wavelengths. posAuNP@UCNPs nanocomposites were produced by coating 30 nm UCNPs on 80 nm AuNPs using DOPA-PEI, which were then irradiated with 980 nm NIR light to facilitate their intracellular delivery. TEM and DLS confirmed that posAuNP and UCNP combine to form nanocomposites. Additionally, multiphysics simulation was used to analyze the distribution of the posAuNP electric field based on morphological differences that change as the UCNP ratio increases. Next, effective LED irradiation conditions were established by applying upconverting-photon quenching-mediated perforation influx to C28/I2 cells as suspensions or spheroids. posAuNP@UCNP nanocomposites were confirmed to be effective for the delivery of baricitinib as a treatment for osteoarthritis in a three-dimensional osteoarthritis model. Finally, chondrocyte differentiation was induced through intracellular delivery of baricitinib using posAuNP@UCNPs. The findings suggest that posAuNP@UCNPs have great potential as a tool for non-invasive drug delivery via UCPPin.

5.
J Control Release ; 368: 756-767, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38499090

RESUMO

Liposomes are widely used as drug delivery nanoplatforms because of their versatility and biocompatibility; however, their ability to load certain drugs may be suboptimal. In this study, we generated liposomes using a combination of DSPE and DSPE-PEG-2 k lipids and loaded them with doxorubicin (DOX) and paclitaxel (PTX), to investigate the effects of light emitting diode (LED) irradiation on liposome structure and drug loading efficiency. Scanning and transmission electron microscopy revealed that the surface of liposomes irradiated with blue or near-infrared LEDs (LsLipo) was rougher and more irregular than that of non-LED-irradiated liposomes (NsLipo). Nuclear magnetic resonance analysis showed that the hydrogen peak originating from the lipid head groups was lower in LsLipo than in NsLipo preparations, indicating that LED irradiation changed the chemical and physical properties of the liposome. Structural changes, such as reduced rigidity, induced by LED irradiation, increased the loading efficiency of DOX and PTX. In vitro and in vivo experiments showed that LsLipo were more effective at inhibiting the growth of cancer cells than NsLipo. Our findings suggest that LED irradiation enhances the drug delivery efficacy of liposomes and offer new possibilities for improving drug delivery systems.


Assuntos
Lipossomos , Neoplasias , Humanos , Lipossomos/química , Sistemas de Liberação de Medicamentos , Paclitaxel/química , Doxorrubicina/química , Neoplasias/tratamento farmacológico , Linhagem Celular Tumoral
6.
J Control Release ; 367: 768-778, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38341178

RESUMO

Immunotherapy based on adoptive transfer of natural killer (NK) cells is a promising strategy for circumventing the limitations of cancer treatments. However, components of the immunosuppressive tumor microenvironment (TME), such as transforming growth factor-beta (TGF-ß), compromise the therapeutic efficacy of NK cells significantly. To address these limitations, we developed a novel method of engineering NK cells for adaptive transfer. The method is based on nanogels that serve two functions: (1) they overcome the TGF-ß-mediated stress environment of the TME, and (2) they enhance the direct anti-tumor activity of NK cells. Previously, we demonstrated that cationic compounds such as 25 K branched polyethylenimine (25 K bPEI) prime NK cells, putting them in a 'ready-to-fight' state. Based on these findings, we designed nanogels that have two primary characteristics: (1) they encapsulate galunisertib (Gal), which is used clinically to inhibit TGF-ß receptor activity, thereby blocking TGF-ß signaling; and (2) they provide cells with a surface coating of 25 K bPEI. When grown in culture medium containing TGF-ß, nanogel-treated NK cells demonstrated greater migration ability, degranulation activity, and cytotoxicity towards cancer cells than untreated NK cells. Additionally, the in vivo efficacy of nanogel-treated NK cells against PC-3 xenografts was significantly greater than that of Chem_NK cells primed by 25 K bPEI alone. These findings suggest that Gal-loaded 25 K bPEI-coated nanogels exert anti-tumor effects via chemical priming, as well suppressing the effects of TGF-ß on NK cells. We also expect 25 K bPEI-based nanogels to have great potential to overcome the suppressive effects of the TME through their NK cell-priming activity and delivery of the desired chemicals.


Assuntos
Citotoxicidade Imunológica , Polietilenoglicóis , Polietilenoimina , Fator de Crescimento Transformador beta , Humanos , Nanogéis , Fator de Crescimento Transformador beta/farmacologia , Linhagem Celular Tumoral , Células Matadoras Naturais , Microambiente Tumoral
7.
Biomater Sci ; 11(8): 2912-2923, 2023 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-36883517

RESUMO

Almost all cells can be exposed to stress, but oocytes, which are female germ cells, are particularly vulnerable to damage. In this study, melatonin, a well-known antioxidant, was loaded into biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and delivered to damaged oocytes in order to improve their quality and restoration. Etoposide (ETP)-induced deteriorated oocytes show poor maturity, mitochondrial aggregation, and DNA damage. Treatment of NPs not only reduced DNA damage but also improved mitochondrial stability, as evidenced by increased ATP levels and mitochondrial homogeneity. When melatonin was added to the culture medium at the same concentration as that present in NPs, DNA and mitochondrial repair was insignificant due to the half-life of melatonin, whereas DNA repair in damaged oocytes upon multiple treatments with melatonin was similar to that observed with melatonin-loaded NPs. Next, we evaluated whether the oocytes treated with NPs could have cryoprotective abilities during vitrification/thawing. Vitrified-oocytes were stored at -196 °C for 0.25 h (T1) or 0.5 h (T2). After thawing, live oocytes were subjected to in vitro maturation. The NP-treated group showed maturity similar to the control group (77.8% in T1, 72.7% in T2) and the degree of DNA damage was reduced compared to the ETP-induced group (p < 0.05).


Assuntos
Melatonina , Nanopartículas , Feminino , Masculino , Animais , Vitrificação , Melatonina/farmacologia , Oócitos , Antioxidantes , Etoposídeo
8.
Biomaterials ; 302: 122350, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37864947

RESUMO

Many attempts have been made to use mitochondria (MT) to treat human diseases; however, MT are large, making them difficult to deliver effectively. Therefore, a transfer strategy based on membrane fusion was established. Fusogenic mitochondrial capsules (FMCs) comprising a neutral lipid (PE), a cationic lipid (DOTAP), an aromatic lipid (Liss Rhod PE), and three types of liposome (FMC0, FMC1, and FMC2), were designed and synthesized. The amount of DOTAP, which affects membrane fusion efficiency, differed between FMC preparations. The characteristics of these FMCs were analyzed by DLS, TEM, and AFM, and the encapsulation and fusion efficiency between FMC-MT and FMC-chondrocytes were confirmed by FRET, mtDNA copy number, and CLSM, respectively. Compared with naked MT, delivery of FMCs to chondrocytes was faster and more efficient. Moreover, fusion was a more stable delivery method than endocytosis, as evidenced by reduced induction of mitophagy. In vitro and in vivo experiments revealed that FMCs reduced expression of inflammatory cytokines and MMP13, increased expression of extracellular matrix components, and promoted cartilage regeneration. These findings suggest that FMCs are a highly effective and promising strategy for delivery of MT to promote cartilage regeneration, and highlight their potential as a novel platform for MT transfer therapy.


Assuntos
Ácidos Graxos Monoinsaturados , Lipossomos , Humanos , Lipossomos/metabolismo , Compostos de Amônio Quaternário , Mitocôndrias/metabolismo
9.
Biomater Sci ; 10(2): 514-523, 2022 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-34904600

RESUMO

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system recognizes and deletes specific nucleotide sequences in cells for gene editing. This study aimed to edit and knockdown the RUNX2 gene, a key transcription factor that is directly involved in all stages of stem cell differentiation into osteoblasts. The RUNX2 gene was depleted using the CRISPR-Cas9 system to inhibit osteoblast differentiation of stem cells. shRNA vectors targeting RUNX2 were used as a control. The surface of nanoparticles (NPs) was coated with the cationic polymer linear polyethyleneimine. Thereafter, negatively charged CRISPR-Cas9 and shRNA vectors were complexed with positively charged NPs via ionic interactions. Several analytical methods were used to determine the size, surface charge, and morphology of NPs and to characterize the complexed genes. NPs complexed with CRISPR-Cas9 and shRNA vectors were delivered into human mesenchymal stem cells (hMSCs) via endocytosis. The mRNA and protein expression patterns of various genes in hMSCs were measured over time following internalization of NPs complexed with CRISPR-Cas9 and shRNA vectors in two- and three-dimensional culture systems. Knockdown of the RUNX2 gene decreased osteogenic differentiation and increased chondrogenic differentiation of hMSCs. As a result of investigating the efficiency of NPs complexed with CRISPR-Cas9 (CASP-NPs), Runx2 effectively knocked down in mesenchymal stem cells to enhance differentiation into chondrocytes, therefore CASP-NPs proved to be an effective gene carrier in hMSCs.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core , Osteogênese , Sistemas CRISPR-Cas/genética , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Humanos , Osteogênese/genética , Células-Tronco
10.
Theranostics ; 12(14): 6409-6421, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36168629

RESUMO

The efficiency of gene therapy is often dictated by the gene delivery system. Cationic polymers are essential elements of gene delivery systems. The relatively cheap cationic polymer, polyethyleneimine, has high gene delivery efficiency and is often used for gene delivery. However, the efficiency of gene therapy with polyethyleneimine-pDNA polyplex (PEI) is low. Human mesenchymal stem cells transfected with polyethyleneimine and a plasmid carrying the important osteogenic differentiation gene runt-related transcription factor 2 (RUNX2) accumulated DNA double-strand breaks and mitochondrial damage proportional to the amount of polyethyleneimine, reducing viability. Genomic/cellular stabilizer mediating RUNX2 delivery (GuaRD), a new reagent incorporating RS-1 NPs developed in this study, promoted DNA repair and prevented the accumulation of cell damage, allowing the delivery of pRUNX2 into hMSCs. while maintaining genome and mitochondrial stability. DNA damage was significantly lower and the expression of DNA repair-related genes significantly higher with GuaRD than with PEI. In addition, GuaRD improved mitochondrial stability, decreased the level of reactive oxygen species, and increased mitochondrial membrane potential. Osteogenic extracellular matrix (ECM) expression and calcification were higher with GuaRD than with PEI, suggesting improved osteogenic differentiation. These results indicate that lowering the cytotoxicity of PEI and improving cell stability are key to overcoming the limitations of conventional gene therapy, and that GuaRD can help resolve these limitations.


Assuntos
Nanopartículas , Osteogênese , Cátions , Subunidade alfa 1 de Fator de Ligação ao Core/genética , DNA/metabolismo , Reparo do DNA , Técnicas de Transferência de Genes , Humanos , Plasmídeos , Polietilenoimina , Espécies Reativas de Oxigênio , Transfecção
11.
Stem Cell Res ; 59: 102643, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34971932

RESUMO

Beta2-microglobulin (B2M) is a subunit of human leukocyte antigen class-I (HLA-I) heterodimer that mediates immune rejection through activation of cytotoxic T cells. B2M binding to HLA-I proteins is essential for functional HLA-I on the cell surface. Here, we generated a B2M homozygous knockout somatic cell nuclear transfer-induced embryonic stem cell (SCNT-ESC) line using CRISPR/Cas9-mediated gene targeting. B2M KO cell line, which does not express HLA-I molecules on cell surface, has pluripotency and differentiation ability to three germ layers. This cell line provides a useful cell source for investigating immunogenicity of allogeneic ESCs and their derivatives for tissue regeneration.

12.
J Immunother Cancer ; 10(8)2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-36028281

RESUMO

BACKGROUND: Due to their powerful immune surveillance activity and ability to kill and clear cancer cells, natural killer (NK) cells are an emerging anticancer immunotherapeutic agent. Therefore, there is much interest in developing efficient technologies that further enhance the therapeutic antitumor efficacy of NK cells. METHODS: To produce chemically primed NK cells, we screened polymers with various electric charges and examined their ability to enhance the cytotoxicity of NK cells. The effect of primary amine and electric charges of 25 kDa branched polyethylenimine (25KbPEI) was investigated by fluorination of the chemical. The role of 25KbPEI in determining the major priming mechanism was investigated in terms of calcium influx into NK cells. In vivo therapeutic efficacy of chemically primed NK cells was evaluated against solid tumor mouse model of triple negative breast and ovarian cancers. RESULTS: Chem_NK that was produced by the priming activity of 25KbPEI showed potent antitumor activity to various cancer cells. Chem_NK showed an activated phenotype, which manifests as increased expression of activating/adhesion/chemokine receptors and perforin accumulation, leading to enhanced migration ability and antitumor activity. Chem_NK display potent therapeutic efficacy against in vivo mouse model of triple negative breast and ovarian cancers. Fluorination of the primary amine group reduces the activity of 25KbPEI to prime NK cells, indicating that the cationic charge on the chemical plays a critical role in NK cell activation. A major priming mechanism was 25KbPEI-mediated calcium influx into NK cells, which occurred mainly via the Ca2+-permeable non-selective cation channel transient receptor potential melastatin 2. CONCLUSIONS: NK cells can be chemically primed with 25KbPEI to express potent antitumor activity as well as enhanced migration ability. Because PEI is a biocompatible and Food and Drug Administration-approved chemical for biomedical use, these results suggest a cost-effective and simple method of producing therapeutic NK cells.


Assuntos
Antineoplásicos , Neoplasias Ovarianas , Neoplasias de Mama Triplo Negativas , Aminas , Animais , Cálcio , Linhagem Celular Tumoral , Feminino , Humanos , Imunoterapia , Células Matadoras Naturais , Camundongos , Polietilenoimina , Estados Unidos
13.
Biomaterials ; 277: 121104, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34478934

RESUMO

This study developed a three-layer consecutive gene delivery system (T-CGDS) for timely gene delivery into human mesenchymal stem cells (hMSCs). The timing of transcription factor expression is important to effectively induce bone differentiation. Therefore, a three-layered nanocomposite was fabricated using differently sized gold nanoparticles to promote bone regeneration and osteogenic differentiation. The core layer comprised 80 nm gold nanoparticles coupled with ATF4 pDNA. Following coating with heparin-conjugated Pluronic F-127 (HP-F127), 50 nm gold nanoparticles coupled with SP7 pDNA were added to fabricate a bi-layer system. After further coating with HP-F127, 20 nm gold nanoparticles combined with RUNX2 pDNA were added. Consequently, a T-CGDS measuring 350-450 nm was fabricated. Genes were released for more than 8 days, while the size of the T-CGDS decreased over time. When the T-CGDS was applied to hMSCs, the gene in the outer layer (RUNX2) was expressed first, followed by those in the middle (SP7) and core (ATF4) layers. The T-CGDS effectively induced bone differentiation and regeneration in vitro and in vivo. Timely delivery of the ATF4 gene to stem cells via the T-CGDS can greatly assist osteogenic differentiation involved in bone regeneration.


Assuntos
Células-Tronco Mesenquimais , Nanopartículas Metálicas , Regeneração Óssea , Diferenciação Celular , Células Cultivadas , Técnicas de Transferência de Genes , Ouro , Humanos , Osteogênese
14.
Int J Pharm ; 610: 121282, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34774691

RESUMO

The characteristics of oocytes, which are female germ cells, have not been studied using optical materials. The structural layers (zona pellucida, ZP) around oocytes make it difficult to deliver drugs aimed at treating infertility. Here, we investigated whether the fluorescent probes sulforhodamine, fluorescein 5(6)-isothiocyanate, tetramethylrhodamine isothiocyanate, cyanine 3 carboxylic acid, and cyanine 5 carboxylic acid penetrate oocytes. By targeting the ZP layer of the oocyte, the characteristics of the model drug, a fluorescent probe, were analyzed, and the position of the probe in the oocyte was confirmed for differences in the characteristics. Penetration of the ZP and delivery into the cytoplasm differed between the fluorescent probes. This was due to their different physiochemical properties, including hydrophobicity (contact angle and surface tension), surfactant activity, and electrical charge. Among the fluorescent probes delivered to cytoplasm, unlike TRITC, Cy3 and Cy5 perturbed oocyte development. These results suggest that in oocytes with high physical barriers (cell membrane, zona pellucida), the delivery efficiency can be estimated by considering the properties (molecular weight and structure, solubility and functional structure, etc.) of the drug. In addition, it suggests that an encapsulated or bound carrier of a drug with properties similar to that of a fluorescent probe can be efficiently delivered into oocytes.


Assuntos
Corantes Fluorescentes , Zona Pelúcida , Feminino , Fluoresceína , Humanos , Oócitos , Organelas
15.
Biochimie ; 186: 33-42, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33865902

RESUMO

The epithelium-specific ETS transcription factor-1 (ESE-1) plays multiple roles in pathogenesis and normal development of epithelial tissues. NANOG, a key mediator of stem cell self-renewal and pluripotency, is also expressed in various cancers and pluripotent cells. In this study, we investigated how ESE-1 influences NANOG expression and NANOG-induced proliferation in human germ cell-derived embryonic carcinoma NCCIT cells. Endogenous ESE-1 expression in NCCIT cells significantly increased during differentiation, whereas NANOG expression decreased. In addition, NANOG expression was downregulated by exogenous overexpression of ESE-1, and increased by shRNA-mediated knockdown of ESE-1. NANOG transcriptional activity was reduced by dose-dependent ESE-1 overexpression and a putative ESE-1 binding site (EBS) was mapped within conserved region 2. Site-directed mutagenesis of the putative EBS abrogated the repressive effect of ESE-1 on NANOG promoter activity. ESE-1 directly interacted with the putative EBS to regulate transcriptional activity of NANOG. Furthermore, NANOG-induced proliferation and colony formation of NCCIT cells were inhibited by ESE-1 overexpression and stimulated by ESE-1 shRNA-mediated knockdown. Altogether, our results suggest that ESE-1 exerts an anti-proliferative effect on NCCIT cells by acting as a novel transcriptional repressor of NANOG.


Assuntos
Carcinoma Embrionário/metabolismo , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína Homeobox Nanog/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Fatores de Transcrição/metabolismo , Carcinoma Embrionário/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Humanos , Proteína Homeobox Nanog/genética , Proteínas Proto-Oncogênicas c-ets/genética , Fatores de Transcrição/genética
16.
ACS Appl Mater Interfaces ; 13(5): 5975-5988, 2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33502166

RESUMO

The structural layers around oocytes make it difficult to deliver drugs aimed at treating infertility. In this study, we sought to identify nanoparticles (NPs) that could easily pass through zona pellucida (ZP), a special layer around oocytes, for use as a drug delivery carrier. Three types of NPs were tested: quantum dot NPs, PE-polyethylene glycol (PEG)-loaded poly(lactic-co-glycolic acid) (PLGA) NPs (PEG/PL), and tetramethylrhodamine-loaded PLGA NPs (TRNPs). When mouse oocytes were treated with NPs, only TRNPs could fully pass through the ZP and cell membrane. To assess the effects of TRNPs on fertility and potential nanotoxicity, we performed mRNA sequencing analysis to confirm their genetic safety. We established a system to successfully internalize TRNPs into oocytes. The genetic stability and normal development of TRNP-treated oocytes and embryos were confirmed. These results imply that TRNPs can be used as a drug delivery carrier applicable to germ cells.


Assuntos
Sistemas de Liberação de Medicamentos , Nanopartículas/química , Oócitos/efeitos dos fármacos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Rodaminas/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Estrutura Molecular , Tamanho da Partícula , Polietilenoglicóis/química , Rodaminas/farmacologia , Propriedades de Superfície
17.
J Control Release ; 332: 337-345, 2021 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32905800

RESUMO

We prepared pocket-type micro-carriers (PMc) with pores larger than 30 µm for use in cell delivery by adding 40 mg pluronic F-127 copolymers (F-127) to biodegradable PLGA dissolved in dichloromethane solution. The controlling the size of the pockets in this way facilitates the adhesion of cells by regulating the size of the pockets according to the cells having various sizes. The size of PMc pores could be controlled within a range of 2 to 30 µm by varying the F-127 content. The ratio of F-127 to DOPA-bPEI was most appropriate at 1: 1, and the pocket size at 10 mg/ml of F-127 was appropriate for adhering 20-30 µm stem cells. F-127 containing SOX9 pDNA, in combination with DOPA-polyethylene-coated gold nanoparticles and dexamethasone loaded in PMcs, promoted cartilage differentiation. Gold nanoparticles complex and dexamethasone (DEX) loaded in PMcs were identified by micro-CT imaging and fluorescence imaging, respectively. By captured in pore generated on/in microspheres, the stem cells were safe and stable for use in delivery, both in vitro and in an animal model. Thus, microsphere pores can safely capture stem cells, and at the same time provide a microenvironment in which the captured stem cells can differentiate into chondrocytes.


Assuntos
Nanopartículas Metálicas , Preparações Farmacêuticas , Animais , Cartilagem , Ouro , Microesferas , Regeneração , Células-Tronco
18.
Adv Sci (Weinh) ; 7(7): 1901818, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32274291

RESUMO

Using direct conversion technology, normal adult somatic cells can be routinely switched from their original cell type into specific differentiated cell types by inducing the expression of differentiation-related transcription factors. In this study, normal human dermal fibroblasts (NHDFs) are directly converted into cardiomyocyte-like cells by drug and gene delivery using carboxymethylcellulose (CMC) nanoparticles (CiCMC-NPs). CMC-based multifunctional nanogels containing specific cardiomyocyte-related genes are designed and fabricated, including GATA4, MEF2C, and TBX5 (GMT). However, GMT alone is insufficient, at least in vitro, in human fibroblasts. Hence, to inhibit proliferation and to induce differentiation, 5-azacytidine (5-AZA) is conjugated to the hydroxyl group of CMC in CiCMC-NPs containing GMT; in addition, the CMC is coated with polyethylenimine. It is confirmed that the CiCMC-NPs have nanogel properties, and that they exhibit the characteristic effects of 5-AZA and GMT. When CiCMC-NPs-containing 5-AZA and GMT are introduced into NHDFs, cardiomyocyte differentiation is initiated. In the reprogrammed cells, the mature cardiac-specific markers cardiac troponin I and α-actinin are expressed at twofold to threefold higher levels than in NHDFs. Engineered cells transplanted into live hearts exhibit active pumping ability within 1 day. Histology and immunohistology of heart tissue confirm the presence of transplanted engineered NHDF cells at injection sites.

19.
ACS Appl Mater Interfaces ; 12(27): 30750-30760, 2020 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-32539331

RESUMO

Electrically conductive materials can stimulate stem cells through electric shock and thereby contribute to the regulation of cell proliferation and differentiation. Recently, polymer-metal complexes composed of polyaniline and gold nanoparticles have emerged as novel candidates for use in regenerative medicine. By mixing two different materials, such composites maximize the benefits while alleviating the disadvantages of using either material alone. Based on their excellent conductivity, these complexes can be applied to nerve regeneration using stem cells. In this study, we investigated a method for producing hybrid nanocomposites by complexing gold nanoparticles to polyaniline and tested the resultant composites in a model of nerve regeneration. We manipulated the shape, size, and electrical conductivity of the hybrid composites by compounding the component materials at various ratios. The most efficient nanocomposite was named conductive reinforced nanocomposites (CRNc's). When the CRNc was delivered directly to cells, no cytotoxicity was observed. After the intracellular delivery of the CRNc, the stem cells were electrically stimulated using an electroporator. As a result of performing mRNA-sequencing (Seq) analysis after electrical stimulation (ES) of the CRNc-internalized cells, it was confirmed that the CRNc-internalized cells have a pattern similar to that of the positive group-induced neuron cells. In particular, microtubule-associated protein 2 is more than twice that of the control group (negative control), and the nerve fiber protein is strongly expressed as in the positive control group. In addition, we verified that neural differentiation progressed by monitoring the growth of neurites from stem cells. Together, these findings show that the CRNc can be used to induce the formation of neuron-like cells by applying ES to stem cells.


Assuntos
Compostos de Anilina/química , Ouro/química , Nanopartículas Metálicas/química , Nanocompostos/química , Estimulação Elétrica , Humanos
20.
Biomater Sci ; 8(12): 3392-3403, 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32377654

RESUMO

The use of nanoscale materials (NMs) could cause problems such as cytotoxicity, genomic aberration, and effects on human health, but the impacts of NM exposure during pregnancy remain uncharacterized in the context of clinical applications. It was sought to determine whether nanomaterials pass through the maternal-fetal junction at any stage of pregnancy. Quantum dots (QDs) coated with heparinized Pluronic 127 nanogels and polyethyleneimine (PEI) were administered to pregnant mice. The biodistribution of QDs, as well as their biological impacts on maternal and fetal health, was evaluated. Encapsulation of QDs with a nanogel coating produces a petal-like nanotracer (PNt), which could serve as a nano-carrier of genes or drugs. PNts were injected through the tail vein and accumulated in the liver, kidneys, and lungs. QD accumulation in reproductive organs (uterus, placenta, and fetus) differed among phases of pregnancy. In phase I (7 days of pregnancy), the QDs did not accumulate in the placenta or fetus, but by phase III (19 days) they had accumulated at high levels in both tissues. Karyotype analysis revealed that the PNt-treated pups did not have genetic abnormalities when dams were treated at any phase of pregnancy. PNts have the potential to serve as carriers of therapeutic agents for the treatment of the mother or fetus and these results have a significant impact on the development and application of QD-based NPs in pregnancy.


Assuntos
Portadores de Fármacos/administração & dosagem , Heparina/administração & dosagem , Poloxâmero/administração & dosagem , Polietilenoimina/administração & dosagem , Pontos Quânticos/administração & dosagem , Animais , Portadores de Fármacos/farmacocinética , Feminino , Heparina/farmacocinética , Humanos , Cariótipo , Troca Materno-Fetal , Células-Tronco Mesenquimais , Camundongos Endogâmicos ICR , Poloxâmero/farmacocinética , Polietilenoimina/farmacocinética , Gravidez , Distribuição Tecidual
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