Reconstruction of TNF-α with specific isoelectric point released from SPIONs basing on variable charge to enhance pH-sensitive controlled-release.

The clinical application of tumor necrosis factor-α (TNF-α) is limited by its short half-life, subeffective concentration in the targeted area and severe systemic toxicity. In this study, the recombinant polypeptide S4-TNF-α was constructed and coupled with chitosan-modified superparamagnetic iron oxide nanoparticles (S4-TNF-α-SPIONs) to achieve pH-sensitive controlled release and active tumor targeting activity. The isoelectric point (pI) of S4-TNF-α was reconstructed to approach the pH of the tumor microenvironment. The negative-charge S4-TNF-α was adsorbed to chitosan-modified superparamagnetic iron oxide nanoparticles (CS-SPIONs) with a positive charge through electrostatic adsorption at physiological pH. The acidic tumor microenvironment endowed S4-TNF-α with a zero charge, which accelerated S4-TNF-α release from CS-SPIONs. Our studies showed that S4-TNF-α-SPIONs displayed an ideal pH-sensitive controlled release capacity and improved antitumor effects. Our study presents a novel approach to enhance the pH-sensitive controlled-release of genetically engineered drugs by adjusting their pI to match the pH of the tumor microenvironment.

Controlled SPION-Exosomes Loaded with Quercetin Preserves Pancreatic Beta Cell Survival and Function in Type 2 Diabetes Mellitus.

Introduction: Quercetin has an ideal therapeutic effect on islet function improvement in type 2 diabetes mellitus (T2DM). However, the therapeutic benefit of quercetin is hindered by its poor bioavailability and limited concentration in pancreatic islets. In this study, superparamagnetic iron oxide nanoparticle (SPION)-modified exosomes were prepared to load quercetin, hoping to endow quercetin with enhanced water solubility and active targeting capacity with the help of magnetic force (MF). Methods: Transferrin-modified SPIONs (Tf-SPIONs) were synthesized by exploiting N-hydroxysuccinimidyl (NHS) conjugation chemistry, and quercetin-loaded exosomes (Qu-exosomes) were acquired by electroporation. Tf-SPION-modified quercetin-loaded exosomes (Qu-exosome-SPIONs) were generated by the self-assembly of transferrin (Tf) and the transferrin receptor (TfR). The solubility of quercetin was determined by high-performance liquid chromatography (HPLC) analysis. The pancreatic islet targeting capacity and insulin secretagogue and antiapoptotic activities of Qu-exosome-SPIONs/MF were evaluated both in vitro and in vivo. Results: The Qu-exosome-SPIONs were well constructed and harvested by magnetic separation with a uniform size and shape in a diameter of approximately 86.2 nm. The water solubility of quercetin increased 1.97-fold when loaded into the SPION-modified exosomes. The application of SPIONs/MF endowed the Qu-exosomes with favorable targeting capacity. In vitro studies showed that Qu-exosome-SPIONs/MF more effectively inhibited or attenuated ß cell apoptosis and promoted insulin secretion in response to elevated glucose (GLC) compared with quercetin or Qu-exosome-SPIONs. In vivo studies demonstrated that Qu-exosome-SPIONs/MF displayed an ideal pancreatic islet targeting capacity, thereby leading to the restoration of islet function. Conclusion: The Qu-exosome-SPIONs/MF nano-delivery system significantly enhanced the quercetin concentration in pancreatic islets and thereby improved pancreatic islet protection.

SPION decorated exosome delivery of TNF-α to cancer cell membranes through magnetism.

Tumor necrosis factor (TNF-α) is capable of inducing apoptosis and is a promising candidate for genetic engineering drugs in cancer therapy; however, the serious side-effects of TNF-α hinder their clinical application. In the present study, a method for preparing fusion proteins of cell-penetrating peptides (CPP) and TNF-α (CTNF-α)-anchored exosomes coupled with superparamagnetic iron oxide nanoparticles (CTNF-α-exosome-SPIONs) with membrane targeting anticancer activity has been demonstrated. To acquire exosomes with TNF-α anchored in its membrane, a CTNF-α expression vector was constructed and a stable mesenchymal stem cell cell line that expressed CTNF-α was established. Conjugating transferrin-modified SPIONs (Tf-SPIONs) onto CTNF-α-exosomes through transferrin-transferrin receptor (Tf-TfR) interaction yields CTNF-α-exosome-SPIONs with good water dispersibility. The incorporation of TNF-α into exosomes and the conjugation of SPIONs significantly enhanced the binding capacity of TNF-α to its membrane-bound receptor TNFR I, thus increasing the therapeutic effects. CTNF-α-exosome-SPIONs significantly enhanced tumor cell growth inhibition via induction of the TNFR I-mediated apoptotic pathway. In vivo studies using murine melanoma subcutaneous cancer models showed that TNF-α-loaded exosome-based vehicle delivery enhanced cancer targeting under an external magnetic field and suppressed tumor growth with mitigating toxicity. Taken together, our results suggest that CTNF-α-exosome-SPIONs showed great potential in membrane targeting therapy.

SPION-Decorated Exosome Delivered BAY55-9837 Targeting the Pancreas through Magnetism to Improve the Blood GLC Response.

BAY55-9837, a potential therapeutic peptide in the treatment of type 2 diabetes mellitus (T2DM), is capable of inducing glucose (GLC)-dependent insulin secretion. However, the therapeutic benefit of BAY55-9837 is limited by its short half-life, lack of targeting ability, and poor blood GLC response. How to improve the blood GLC response of BAY55-9837 is an existing problem that needs to be solved. In this study, a method for preparing BAY55-9837-loaded exosomes coupled with superparamagnetic iron oxide nanoparticle (SPIONs) with pancreas islet targeting activity and an enhanced blood GLC response with the help of an external magnetic force (MF) is demonstrated. The plasma half-life of BAY55-9837 loaded in exosome-SPION is 27-fold longer than that of BAY55-9837. The active targeting property of SIPONs enables BAY-exosomes to gain a favorable targeting property, which improves the BAY55-9837 blood GLC response capacity with the help of an external MF. In vivo studies show that BAY-loaded exosome-based vehicle delivery enhances pancreas islet targeting under an external MF and markedly increases insulin secretion, thereby leading to the alleviation of hyperglycemia. The chronic administration of BAY-exosome-SPION/MF significantly improves glycosylated hemoglobin and lipid profiles. BAY-exosome-SPION/MF maybe a promising candidate for a peptide drug carrier for T2DM with a better blood GLC response.

Dihydrodiosgenin inhibits endothelial cell-derived factor VIII and platelet-mediated hepatocellular carcinoma metastasis.

Background: Our previous studies have demonstrated that diosgenin and diosgenin derivatives exhibit excellent antithrombotic activity via regulating platelet function and coagulation factor level. Platelets and blood coagulation system are highly associated with tumor hematogenous metastasis. Therefore, the purpose of this study was to evaluate whether dihydrodiosgenin (dydio) mediated-platelet inhibition or coagulation factor level modulation is involved in hepatocellular carcinoma cell (HCC) metastasis. Methods: Cell viability was examined by MTT and colony formation assays. Platelet aggregation text and morphology were used to assess dydio's role on tumor cell-induced platelet activation (TCIPA). Scratch assay, adhesion assay and Western blot were used to evaluate dydio's role on platelet-mediated metastasis. Western blot and fluorescence detection were performed to clarify dydio's role on endothelial cell (EC) function. The mice lung metastasis model was constructed to investigated dydio's function on coagulation factor and platelet-mediated metastasis. Results: This study found that pretreatment with dydio caused a significant inhibition of TCIPA. Platelets exposed to dydio significantly inhibited their adhesion to tumor cells, meanwhile, releasates of platelets that pretreated with dydio led to diminished cancer cell proliferation and migration along with the increase of epithelial markers E-cadherin and loss of mesenchymal phenotype. Additionally, ECs pretreated with dydio suppressed factor VIII (FVIII) level which in turn restrained the activation of platelets and the adhesion of cancer cells or platelets to ECs. Interestingly, our study demonstrated that FVIII could promote HCC proliferation. In vivo study revealed that mice intragastrical (i.g.) administration with dydio significantly inhibited the lung metastasis of hepal-6 cells which is highly correlated with the altered platelet function and coagulation level. Conclusion: Taken together, these results demonstrated that dydio altered platelet function and coagulation FVIII level, resulting in decreased metastatic potential of HCC. Thus, our study reveals that dydio exerts novel mechanisms of antitumor action beside its direct antitumor activity.

Oxidative stress mediated by lipid metabolism contributes to high glucose-induced senescence in retinal pigment epithelium.

Retinal pigment epithelium (RPE) dysfunction is thought to increase the risk of the development and progression of diabetic retinopathy (DR), the leading cause of blindness. However, the molecular mechanism behind high glucose-induced RPE cell damage is still blurred. We reported that ARPE-19 exposed to 25 mM glucose for 48 h did not induce apoptosis, but senescence validated by SA-ß-Gal staining, p21 expression and cell cycle distribution. High glucose also increased oxidant species that exerted a pivotal role in senescence, which could be relieved by the treatment with antioxidant N-acetylcysteine (NAC). The accumulation of lipid droplets and the increase of lipid oxidation were also observed in ARPE-19 treated with high glucose. And the supplementation of free fatty acids (FFAs) indicated that lipid metabolism was associated with the generation of hydrogen peroxide (H2O2) and subsequent senescence in ARPE-19. PI3K/Akt/mTOR signaling pathway was shown to be responsible for the accumulation of intracellular lipids by regulating fatty acid synthesis, which in turn controlled senescence. Furthermore, high glucose induced autophagy in ARPE-19 with the treatment of glucose for 48 h, and autophagy inhibitor hydroxychloroquine (HCQ) or bafilomycin further aggravated the senescence, accompanying by an increase in oxidant species. Whereas, prolonged high glucose exposure inhibited autophagy and increased apoptotic cells. Experiments above provide evidence that lipid metabolism plays an important role in oxidative stressed senescence of RPE.

Islet protection and amelioration of type 2 diabetes mellitus by treatment with quercetin from the flowers of Edgeworthia gardneri.

BACKGROUND AND PURPOSE: The traditional Chinese medicine - the flower of Edgeworthia gardneri - is reported as an effective therapeutic for type 2 diabetes mellitus (T2DM). Nevertheless, most constituents of the flowers of E. gardneri have not yet been studied. This study was conducted to investigate the effect of quercetin extracted from the flowers of E. gardneri on islet protection and amelioration in T2DM and explore its mechanism. METHOD: Quercetin was extracted from the flowers of E. gardneri and verified by high-performance liquid chromatography. Quercetin or crude extract's effect on insulin secretion was investigated. ERK1/2 and phospho-ERK1/2 were detected by Western blot analysis, and fluo-3 AM was used to detect intracellular Ca2+. The anti-apoptosis effect of quercetin or crude extract on MIN-6 cells was investigated by thiazolyl blue tetrazolium bromide (MTT) assay and flow cytometry analysis. Activation of caspases and expression of Bcl-2 and BAX were tested by Western blot analysis. In addition, the mitochondrial membrane potential was determined by JC-1 probe. Moreover, in vivo activity was also tested in db/db mice. RESULTS: A quercetin level of >10 µmol/L could induce insulin secretion. Intracellular Ca2+ and ERK1/2 were involved in the signaling pathway of quercetin-induced insulin secretion. We also observed that quercetin could inhibit palmitic acid-induced cell apoptosis via suppressing the activation of caspase-3, -9, -12; increasing the ratio of Bcl-2/BAX and reversing the impaired mitochondrial membrane potential. Crude extract's effect on insulin secretion was similar to that of pure extracted quercetin, while it possessed higher anti-apoptosis activity. Additionally, intraperitoneal glucose tolerance, plasma insulin level, hepatic triglyceride, hepatic glycogen and the pathological histology of both pancreatic islet and liver in db/db mice were significantly improved by the administration of the extracted quercetin. CONCLUSION: Our study indicated that quercetin extracted from the flowers of E. gardneri exerted excellent properties in islet protection and amelioration.

Chitosan-decorated selenium nanoparticles as protein carriers to improve the in vivo half-life of the peptide therapeutic BAY 55-9837 for type 2 diabetes mellitus.

PURPOSE: As a potential protein therapeutic for type 2 diabetes mellitus (T2DM), BAY 55-9837 is limited by poor stability and a very short half-life in vivo. The purpose of this study was to construct a novel nanostructured biomaterial by conjugating BAY 55-9837 to chitosan-decorated selenium nanoparticles (CS-SeNPs) to prolong the in vivo half-life of BAY 55-9837 by reducing its renal clearance rate. MATERIALS AND METHODS: BAY 55-9837-loaded CS-SeNPs (BAY-CS-SeNPs) were prepared, and their surface morphology, particle size, zeta potential, and structure were characterized. The stability, protein-loading rate, and in vitro release of BAY 55-9837 from CS-SeNPs were also quantified. Additionally, a sensitive high-performance liquid chromatography (HPLC) assay was developed for the quantification of BAY 55-9837 in mouse plasma. Thereafter, mice were injected via the tail vein with either BAY 55-9837 or BAY-CS-SeNPs, and the plasma concentration of BAY 55-9837 was determined via our validated HPLC method at different time intervals postinjection. Relevant in vivo pharmacokinetic parameters (half-life, area under the curve from time 0 to last sampling point, observed clearance) were then calculated and analyzed. RESULTS: BAY-CS-SeNPs were successfully synthesized, with diameters of approximately 200 nm. BAY-CS-SeNPs displayed good stability with a high protein-loading rate, and the release process of BAY 55-9837 from the CS-SeNPs lasted for over 70 hours, with the cumulative release reaching 78.9%. Moreover, the conjugation of CS-SeNPs to BAY 55-9837 significantly reduced its renal clearance to a rate of 1.56 mL/h and extended its half-life to 20.81 hours. CONCLUSION: In summary, our work provides a simple method for reducing the renal clearance rate and extending the half-life of BAY 55-9837 in vivo by utilizing CS-SeNPs as nanocarriers.