Mechanical properties and immunotherapeutic effects of dissolving microneedles with different drug loadings based on hyaluronic acid

Abstract Improving vaccine immunity and reducing antigen usage are major challenges in the clinical application of vaccines. Microneedles have been proven to be painless, minimally invasive, highly efficient, and have good patient compliance. Compared with traditional transdermal drug delivery, it can effectively deliver a large-molecular-weight drug into the skin, resulting in a corresponding immune response. However, few studies have examined the relationship between microneedle loading dose and immune effects. In this study, the hyaluronic acid (HA) conical and pyramidal dissolving microneedles were prepared by the two-step vacuum drying method, respectively. The model drug ovalbumin (OVA) was added to HA to prepare dissolving microneedles with different loading amounts. The mass ratios of HA to OVA were 5:1, 5:3, and 5:5. The mechanical properties of the dissolving microneedles were characterized using nanoindentation and in vitro puncture studies. The immune effects of the matrix and drug content were studied in Sprague-Dawley (SD) rats. Finally, the diffusion behavior of OVA and the binding mode of HA and OVA in the microneedles were simulated using Materials Studio and Autodocking software. The experimental results showed that the conical microneedles exhibited better mechanical properties. When the mass ratio of HA to OVA was 5:3, the immune effect can be improved by 37.01% compared to subcutaneous injection, and achieved a better immune effect with relatively fewer drugs. This conclusion is consistent with molecular simulations. This study provides theoretical and experimental support for the drug loading and efficacy of microneedles with different drug loadings

Synergistic effect of l-ascorbic acid and hyaluronic acid on the expressions of matrix metalloproteinase-3 and -9 in human chondrocytes.

Proinflammatory cytokines and reactive oxygen species (ROS) are known to be involved in the initiation and progression of osteoarthritis (OA). New evidence clarifying the correlation between ROS and inflammation has indicated that oxidative stress can up-regulate inflammatory cytokines. l-Ascorbic acid (AA), an antioxidant, has been shown to have anti-inflammatory effects and improve matrix deposition in chondrocytes. The purpose of this study was to examine the effects of hyaluronic acid (HA; 100 µg/mL) supplemented with AA (50 µg/mL) on human normal and interleukin-1 beta-stimulated (IL-1ß, 10 ng/mL) chondrocytes. HA, AA, and HA + AA treatment did not change cell morphology, viability, proliferation, and glycosaminoglycan production in normal chondrocytes. HA, AA, and HA + AA, by contrast, partially restored viability and morphology of hypertrophic chondrocytes, and HA and HA + AA further decreased the cytotoxicity of IL-1ß. Real-time PCR revealed that AA and HA + AA had no substantial effects on unstimulated chondrocytes, except for down-regulation of matrix metalloproteinase (MMP)-9 mRNA levels. For IL-1ß-stimulated chondrocytes, significant down-regulation of IL-1ß, tumor necrosis factor-alpha (TNF-α), MMP-3, and MMP-9 mRNA expression was found when cells were cultured in HA-supplemented media. Moreover, HA + AA supplementation further significantly decreased MMP-3 and MMP-9 mRNA expression. The protein production of MMP-3 was decreased, with a significant difference between the HA + AA group and HA group. The antioxidant capacity and superoxide dismutases activity were also partially restored in stimulated chondrocytes. HA supplemented with AA modulates MMPs expression and antioxidant fuction in chondrocytes. AA may enhance the anticatabolic effects of HA on OA chondrocytes. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1809-1817, 2018.

Cyclic phosphatidic acid and lysophosphatidic acid induce hyaluronic acid synthesis via CREB transcription factor regulation in human skin fibroblasts.

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator and an analog of the growth factor-like phospholipid lysophosphatidic acid (LPA). cPA has a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We showed before that a metabolically stabilized cPA derivative, 2-carba-cPA, relieved osteoarthritis pathogenesis in vivo and induced hyaluronic acid synthesis in human osteoarthritis synoviocytes in vitro. This study focused on hyaluronic acid synthesis in human fibroblasts, which retain moisture and maintain health in the dermis. We investigated the effects of cPA and LPA on hyaluronic acid synthesis in human fibroblasts (NB1RGB cells). Using particle exclusion and enzyme-linked immunosorbent assays, we found that both cPA and LPA dose-dependently induced hyaluronic acid synthesis. We revealed that the expression of hyaluronan synthase 2 messenger RNA and protein is up-regulated by cPA and LPA treatment time dependently. We then characterized the signaling pathways up-regulating hyaluronic acid synthesis mediated by cPA and LPA in NB1RGB cells. Pharmacological inhibition and reporter gene assays revealed that the activation of the LPA receptor LPAR1, Gi/o protein, phosphatidylinositol-3 kinase (PI3K), extracellular-signal-regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding protein (CREB) but not nuclear factor κB induced hyaluronic acid synthesis by the treatment with cPA and LPA in NB1RGB cells. These results demonstrate for the first time that cPA and LPA induce hyaluronic acid synthesis in human skin fibroblasts mainly through the activation of LPAR1-Gi/o followed by the PI3K, ERK, and CREB signaling pathway.

Stimulation of hyaluronan synthesis by interleukin-1beta involves activation of protein kinase C betaII in fibroblasts from patients with Graves' ophthalmopathy.

Hyaluronan accumulation in the retroorbital connective tissue is one of the pathological features of Graves' ophthalmopathy. Interleukin-1beta (IL-1beta) is known to stimulate hyaluronan synthesis in orbital fibroblasts. In the present study, the intracellular signal transduction pathways involved in this stimulatory effect were investigated in cultured human retroorbital fibroblasts from patients with Graves' ophthalmopathy. IL-1beta-induced hyaluronan synthesis was significantly inhibited by pretreatment of the cells with two protein kinase C (PKC) inhibitors, chlerythrine chloride and H-7. In addition, treatment with phorbol 12-myristate 13-acetate (PMA), a direct PKC activator, also resulted in increased hyaluronan production. IL-1beta- or PMA-stimulated hyaluronan synthesis was blocked by the protein synthesis inhibitor, cycloheximide. Moreover, the intracellular Ca(2+) concentration of the orbital fibroblasts was also involved in the IL-1beta induced transduction pathway, the effect being completely inhibited by BAPTA, an internal calcium chelator. In addition, A23187, a calcium ionophore, increased hyaluronan synthesis in unstimulated cells. These results suggest that the Ca(2+)-dependent PKC signal transduction pathway plays an important role in the IL-1beta-induced hyaluronan synthesis. Moreover, IL-1beta treatment resulted in increased PKC activity and the rapid translocation of PKC betaII from the cytoplasm to the plasma membrane. These results indicate that cytosolic Ca(2+) and PKC betaII are involved in IL-1beta-induced hyaluronan synthesis in cultured orbital fibroblasts from patients with Graves' ophthalmopathy.