Enhanced non-viral gene delivery via calcium phosphate/DNA co-precipitates with low-voltage pulse electroporation in NK-92 cells for immunocellular therapy.

Achieving high cell transfection efficiency is essential for various cell types in numerous disease applications. However, the efficient introduction of genes into natural killer (NK) cells remains a challenge. In this study, we proposed a design strategy for delivering exogenous genes into the NK cell line, NK-92, using a modified non-viral gene transfection method. Calcium phosphate/DNA nanoparticles (pDNA-CaP NPs) were prepared using co-precipitation methods and combined with low-voltage pulse electroporation to facilitate NK-92 transfection. The results demonstrated that the developed pDNA-CaP NPs exhibited a uniform diameter of approximately 393.9 nm, a DNA entrapment efficiency of 65.8%, and a loading capacity of 15.9%. Furthermore, at three days post-transfection, both the transfection efficiency and cell viability of NK-92 were significantly improved compared to standalone plasmid DNA (pDNA) electroporation or solely relying on the endocytosis pathway of pDNA-CaP NPs. This study provides valuable insights into a novel approach that combines calcium phosphate nanoparticles with low-voltage electroporation for gene delivery into NK-92 cells, offering potential advancements in cell therapy.

The synthesis and evaluation of thiolated alginate as the barrier to block nutrient absorption on small intestine for body-weight control.

Obesity is the most common health concern all over the world. However, till now, there is no promising way to manage obesity or body-weight control. The aim of the study is to develop an edible gel as a health supplement that temporarily attaches to the mucus of the intestines, forming an absorption barrier to block the nutrients. We modify the alginate with the thiol group as thiolated alginate (TA) that may stay on the mucosa layer for a much longer time to reduce nutrient absorption. In this study, the TA is synthesized successfully and proved a good mucosal adhesion to serve as a barrier for nutrient absorption both in vitro and in vivo. The results of in vivo imaging system (IVIS) show that the synthesized TA can be exiled from the gastrointestinal tract within 24 h. The animal study shows that the TA by daily oral administration can effectively reduce body weight and fat deposition. The biosafety is evaluated in vitro at the cellular level, based on ISO-10993, and further checked by animal study. We do believe that the TA could have a greater potential to be developed into a safe health supplement to manage obesity and for body-weight control.

The combination of resveratrol and Bletilla striata polysaccharide decreases inflammatory markers of early osteoarthritis knee and the preliminary results on LPS-induced OA rats.

Osteoarthritis (OA) of the knee is characterized by progressive deterioration and loss of articular cartilage with associatedstructural and performance changes in the entire joint, and current treatments for OA only aim to relieve symptoms, rather than to prevent or reverse disease progression. Recently, treatments targeting "early osteoarthritis" (EOA) have attracted attention. However, during EOA stage, chondrocytes may change behaviors to express pro-inflammatory cytokines and free radicals, which would cause detrimental effects to the synovial cavity and further cartilage wear. In this study, we combined resveratrol (Res) and Bletilla striata polysaccharide (BSP) as anti-inflammatories and antioxidants to diffuse free radicals and to alleviate inflammation from the synovial cavity both short term and long term. The current study introduced a new method for harvesting BSP from as-received Bletilla striata to achieve high yields, shortened extraction times, and maintained structure/functions. In addition, it combined Res and home-extracted BSP (Res-BSP) to alleviate oxidative stress and inflammation in a Lipopolysaccharide (LPS)-induced OA model. The gene expressions of inflammatory genes iNOs, IL-1ß, IL-6, and MMP-13 were upregulated 5.7-fold, 6.5-fold, 8.6-fold, and 4.5-fold, respectively on OA-like chondrocytes and the gene expressions were significantly downregulated to 3.3-fold, 2.1-fold, 4.9-fold, and 0.1-fold, respectively, once OA-like chondrocytes were treated with Res-BSP (p < 0.05, compared with OA-like chondrocytes). The gene expressions of chondrogenic genes TGFß1, SOX9, and type II collagen were downregulated by 0.8-fold, 2.2-fold, and 0.8-fold, respectively, based on the control group as a baseline. While it was significantly upregulated by 3.4-fold, 0.32-fold, and 0.4-fold, respectively, once OA-like chondrocytes were treated with Res-BSP. (p < 0.05, compared with OA-like chondrocytes). Finally, we elucidated the role of Res-BSP in EOA in suppressing COX-2 and activating p-Smad 2/3 and p-Erk1/2. We believe that the combination of Res and BSP has great potential as an alternative therapeutic strategy for EOA treatment in future.

Development of resveratrol with thiolated alginate as a supplement to prevent nonalcoholic fatty liver disease (NAFLD).

Nowadays, nonalcoholic fatty liver disease is a common metabolic liver disease of all ages worldwide. However, current pharmacological and surgical treatments are accompanied with side effects and complications. EndoBarrier, a less invasive bariatric surgery, blocks the upper portion of the intestine to reduce nutrition absorption. To mimic the nutrient restriction effect of EndoBarrier, thiol-containing materials may bind to the thiol groups of the mucus with an enhanced mucoadhesive property. Here, we develop thiolated alginate with cysteine conjugation via an N-(3-dimethylaminopropyl)-N-ethylcarbodiimide/N-hydroxysuccinimide reaction. The alginate-cysteine (AC) exhibits excellent mucoadhesive properties and forms a physical barrier in the intestine to reduce absorption significantly, which was tested with both in vitro and in vivo mucoadhesive test and barrier function test. The nontoxicity property of AC was also proven with WST-1 and live and dead stain. In addition, AC demonstrates potent carrier properties of extending the release of resveratrol to improve the efficacy with the test of the transwell system in the release profile. In the long-term therapeutic evaluation, alginate cysteine with resveratrol (ACR) is orally administrated daily to mice with an methionine choline-deficient diet. The results of this in vivo study show that developed ACR could effectively alleviate fat degeneration in the liver and improve fat-related metabolic parameters in serum without hepatocellular damage and kidney dysfunction. In sum, AC was found to be mucoadhesive, reduce glucose absorption, alleviate inflammation, and decrease fatty degradation. This promising material exhibits the potential to be a supplement for nonalcoholic fatty liver disease.

Development of di(2-ethylhexyl) phthalate-containing thioglycolic acid immobilized chitosan mucoadhesive gel as an alternative hormone therapy for menopausal syndrome.

Menopausal syndrome includes the symptoms that most women experience owing to hormone changes after menopause. Although hormone replacement therapy is a common treatment for menopausal syndrome, there are still many side effects and challenges hindering research. In this study, thioglycolic acid (TGA)-immobilized chitosan mucoadhesive gel was synthesized by a new method of low concentration of 1,4-butanediol diglycidyl ether (BDDE) would encapsulate di(2-ethylhexyl) phthalate (DEHP) as an alternative hormone replacement therapy for menopausal syndrome. The efficacies of the DEHP-containing TGA-chitosan gel (CT-D) were confirmed and evaluated by materials characterization and in vitro study. Results showed that CT-D was not cytotoxic and had better mucoadhesive ability than chitosan. The animal model was constructed 1 month after bilateral ovariectomy in SD rats. CT-D was administered intravaginally every 3 days. Bodyweight, wet weight of the uterus and vagina, vaginal smears, histology, blood element analysis, and serological analysis was used to assess the ability of the material to relieve menopausal syndrome. The results indicated that the combination of the sustained release of DEHP and mucoadhesive TGA-immobilized chitosan allows the developed CT-D to relieve the menopausal syndrome through low concentrations of DEHP, which falls in the safety level of the tolerable daily intake of DEHP.

A One-Step, Monolayer Culture and Chemical-Based Approach to Generate Insulin-Producing Cells From Human Adipose-Derived Stem Cells to Mitigate Hyperglycemia in STZ-Induced Diabetic Rats.

The global population of individuals afflicted with diabetes mellitus has been increasing year by year, and this disease poses a serious threat to human health as well as the economies worldwide. Pancreatic or islet transplantations provide one of the most effective and long-term therapies available to treat diabetes, but the scarcity and quality of pancreatic islets limit their use in treatments. Here, we report the development of a one-step, monolayer culture, and chemical-based protocol that efficiently mediates the differentiation of human adipose-derived stem cells (hADSCs) into insulin-producing cells (IPCs). Our data indicate that hADSCs in monolayer culture that are allowed to differentiate into IPCs are superior to those in suspension cultures with respect to insulin secretion capacity (213-fold increase), cell viability (93.5 ± 3.27% vs. 41.67 ± 13.17%), and response to glucose stimulation. Moreover, the expression of genes associated with pancreatic lineage specification, such as PDX1, ISL1, and INS (encoding insulin), were expressed at significantly higher levels during our differentiation protocol (6-fold for PDX1 and ISL1, 11.5-fold for INS). Importantly, in vivo studies demonstrated that transplantation with IPCs significantly mitigated hyperglycemia in streptozotocin-induced diabetic rats. Our results indicate that this one-step, rapid protocol increases the efficiency of IPC generation and that the chemical-based approach for IPC induction may reduce safety concerns associated with the use of IPCs for clinical applications, thereby providing a safe and effective cell-based treatment for diabetes.

Thiolated Chitosan as an Intestinal Absorption Carrier with Hesperidin Encapsulation for Obesity Treatment.

Obesity is characterized as abnormal or excessive fat accumulation harmful to one's health, linked to hormonal imbalances, cardiovascular illness, and coronary artery disease. Since the disease stems mainly from overconsumption, studies have aimed to control intestinal absorption as a route for treatment. In this study, chitosan-thioglycolic acid (CT) was developed as a physical barrier in the gastrointestinal tracts to inhibit nutrient uptake. CT exhibits a superior mucoadhesive property compared to chitosan both in vitro and in vivo for the ability to form disulfide bonds with the intestinal mucosa. For CT as a potential drug delivery platform, hesperidin, a herb for bodyweight control in traditional Chinese medication, is encapsulated in CT and can be released consistently from this absorption barrier. In animal studies, CT encapsulated with hesperidin (CTH) not only results in a weight-controlling effect but limits adipose accumulation by hindering absorption, suggesting a potential role in obesity treatment. Neither CT nor CTH exhibit cytotoxicity or produce adverse immunological reactions in vivo.

Synthesis, characterization, and evaluation of BDDE crosslinked chitosan-TGA hydrogel encapsulated with genistein for vaginal atrophy.

Vagina atrophy is a common symptom in women after menopause owing to decreasing estrogen levels. The most conventional treatment for this condition is estrogen cream. The shortcoming is its weak adhesion to the vagina mucus, thus requiring frequent daily application. In this study, BDDE was selected to crosslink and graft chitosan with thioglycolic acid, to form thiolated chitosan (CT) and improve the mucoadhesive properties of chitosan. Genistein was selected as the bioactive molecule that could exhibit estrogen-like properties for long-term treatment of vaginal atrophy. The efficacies of the materials were characterized and evaluated both in vitro and in vivo. Results showed that the mucoadhesive property of CT was approximately two-fold stronger against the constant flow than unmodified chitosan. CT with genistein (CT-G) was administered intravaginally every three days in vivo. It showed that the developed CT-G recover 54 % of the epithelium thickness of an atrophic vagina and ease vaginal atrophy.

Curcumin-Loaded Hydrophobic Surface-Modified Hydroxyapatite as an Antioxidant for Sarcopenia Prevention.

Oxidative stress and later-induced chronic inflammation have been reported to play an important role on the progression of sarcopenia. Current treatments for sarcopenia are mainly administered to patients whom sarcopenia already developed. However, there has been no promising results shown in therapy. Therefore, the development of therapeutic and preventive strategies against sarcopenia would be necessary. Curcumin is a traditional medicine that possesses anti-inflammatory and antioxidative properties. In the present study, hydroxyapatite was subjected to hydrophobic surface modifications for curcumin loading (Cur-SHAP). It was, subsequently, utilized for delivery to the patient's body via intramuscular injection in order to achieve constant release for more than 2 weeks, preventing the progression of the sarcopenia or even leading to recovery from the early stage of the illness. According to the results of WST-1, LIVE/DEAD, DCFDA, and gene expression assays, Cur-SHAP exhibited good biocompatibility and showed great antioxidant/anti-inflammatory effects through the endocytic pathway. The results of the animal studies showed that the muscle endurance, grip strength, and fat/lean mass ratio were all improved in Cur-SHAP-treated rats from LPS-induced sarcopenia. In summary, we successfully synthesized hydrophobic surface modification hydroxyapatite for curcumin loading (Cur-SHAP) and drug delivery via the IM route. The LPS-induced sarcopenia rats were able to recover from disease after the Cur-SHAP treatment.