No abstract

Background: Supramolecular micelles as drug-delivery vehicles are generally unable to enter the nucleus of nondividing cells. In the work reported here, nuclear localization signal (NLS)-modified polymeric micelles were studied with the aim of improving nuclear drug delivery. Methods: In this research, cholesterol-modified glycol chitosan (CHGC) was synthesized. NLSconjugated CHGC (NCHGC) was synthesized and characterized using proton nuclear magnetic resonance spectroscopy, dynamic light scattering, and fluorescence spectroscopy. Doxorubicin (DOX), an anticancer drug with an intracellular site of action in the nucleus, was chosen as a model drug. DOX-loaded micelles were prepared by an emulsion/solvent evaporation method. The cellular uptake of different DOX formulations was analyzed by flow cytometry and confocal laser scanning microscopy. The cytotoxicity of blank micelles, free DOX, and DOX-loaded micelles in vitro was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HeLa and HepG2 cells. Results: The degree of substitution was 5.9 cholesterol and 3.8 NLS groups per 100 sugar residues of the NCHGC conjugate. The critical aggregation concentration of the NCHGC micelles in aqueous solution was 0.0209 mg/mL. The DOX-loaded NCHGC (DNCHGC) micelles were observed as being almost spherical in shape under transmission electron microscopy, and the size was determined as 248 nm by dynamic light scattering. The DOX-loading content of the DNCHGC micelles was 10.1%. The DOX-loaded micelles showed slow drug-release behavior within 72 hours in vitro. The DNCHGC micelles exhibited greater cellular uptake and higher amounts of DOX in the nuclei of HeLa cells than free DOX and DOX-loaded CHGC (DCHGC) micelles. The half maximal inhibitory concentration (IC 50 ) values of free DOX, DCHGC, and DNCHGC micelles against HepG2 cells were 4.063, 0.591, and 0.171 µg/mL, respectively. Moreover, the IC 50 values of free DOX (3.210 µg/mL) and the DCHGC micelles (1.413 µg/mL) against HeLa cells were nearly 6.96-and 3.07-fold (P , 0.01), respectively, higher than the IC 50 value of the DNCHGC micelles (0.461 µg/mL). Conclusion:The results of this study suggest that novel NCHGC micelles could be a potential carrier for nucleus-targeting delivery.

Skin wound healing is a complex and dynamic process that involves angiogenesis and growth factor secretion. Newly formed vessels can provide nutrition and oxygen for skin wound healing. Growth factors in skin wounds are important for keratinocytes and fibroblasts proliferation, epithelialization, extracellular matrix remodeling, and angiogenesis, which accelerate skin wound healing. Therefore, treatment strategies that enhance angiogenesis and growth factors secretion in skin wounds can accelerate skin wound healing. This study investigated the effects of a SIKVAV (Ser-Ile-Lys-Val-Ala-Val) peptide-modified chitosan hydrogel on skin wound healing. Hematoxylin and eosin (H&E) staining demonstrated that the SIKVAV-modified chitosan hydrogel accelerated the re-epithelialization of wounds compared with that seen in the negative and positive controls. Masson’s trichrome staining showed that more collagen fibers were deposited in the skin wounds treated with the SIKVAV-modified chitosan hydrogel than in the negative and positive controls. Immunohistochemistry assays demonstrated that more myofibroblasts were deposited and more angiogenesis occurred in skin wounds treated with the SIKVAV-modified chitosan hydrogel than in the negative and positive controls. In addition, ELISA assays showed that the SIKVAV-modified chitosan hydrogels promoted the secretion of growth factors in skin wounds. Taken together, these results suggest that the SIKVAV-modified chitosan hydrogel has the potential to be developed as synthesized biomaterials for the treatment of skin wounds.

Background: The process of bone repair is heavily dependent on the ability of human bone marrow mesenchymal stem cells (hMSCs) to undergo osteogenic differentiation. MicroRNAs have been shown to regulate this osteogenic process. This study aimed to investigate the role of miR-765 in the osteogenic differentiation of hMSCs. Methods: We transfected hMSCs with lentiviral constructs to knock down or overexpress this miRNA, allowing us to assess its role in osteogenesis via assessing the expression of the relevant markers alkaline phosphatase (ALP), runt-related gene-2 (RUNX-2), and osteocalcin (OCN), with further functional measurements made via quantifying ALP activity and conducting Alizarin Red S staining. The targeting of the 3′-untranslated region (UTR) of BMP6 by miR-765 was examined via luciferase assay. We used hMSCs with altered miR-765 expression to assess p-Smad1/5/9 levels via Western blotting over the course of osteogenic differentiation. We also assessed the osteogenic differentiation of hMSCs in which miR-765 was knocked down and at the same time as a BMP/Smad signaling inhibitor was added to disrupt Smad1/5/9 phosphorylation. Results: We found miR-765 overexpression to inhibit osteogenesis-associated gene upregulation during osteogenic differentiation of hMSCs, whereas knockdown of this miRNA was associated with increased expression of these genes. Using luciferase reporter assays, we confirmed direct miR-765 binding to the 3′-untranslated region (UTR) of BMP6. We also found that miR-765 overexpression reduced Smad1/5/9 phosphorylation, and knockdown of this miRNA enhanced this phosphorylation on BMP6/Smad1/5/9 signaling. The osteogenic differentiation of hMSCs in which miR-765 had been knocked down was further weakened upon the addition of a BMP/Smad signaling inhibitor relative to miR-765 knockdown alone. Conclusions: Together, these results thus suggest that miR-765 is able to inhibit hMSC osteogenic differentiation by targeting BMP6 via regulation of the BMP6/Smad1/5/9 signaling pathway. Our findings may offer molecular insights of value for the development of novel therapeutic treatments for bone diseases including osteoporosis.

Ubiquitin-specific processing enzyme 22 (USP22) plays a direct role in regulating cell cycle, and its overexpression has been reported to be involved in tumor progression. However, little is known about the regulation of USP22 transcription. In this study, we cloned and characterized the human USP22 promoter. Using 5′ RACE (rapid amplification of cDNA ends) analysis, the transcriptional initiation site was identified. Promoter deletion analysis showed that the sequence between −210 and −7 contains the basal promoter for USP22 in human fibroblast and tumor cells. Surprisingly, mutations in a putative Sp1 binding site immediately upstream of the USP22 transcriptional start site (−13 to −7) resulted in a significant induction of promoter activity. Further study revealed that Sp1 binds to this site in human normal fibroblast cells, and treatment with the Sp1 inhibitor mithramycin A led to a marked increase in USP22 transcript levels. Forced expression of exogenous Sp1 repressed the USP22 promoter activity in HeLa cells. In contrast, knockdown of Sp1 enhanced USP22 promoter activity and mRNA levels. These data suggest that Sp1 is a crucial regulator of USP22 transcription.