A Combination of Biocatalysis and Fenton-Like Reaction Induced OH• Burst for Cascade Amplification of Cancer Chemodynamic Therapy.

Chemodynamic therapy (CDT) is a novel antitumor strategy that employs Fenton or Fenton-like reactions to generate highly toxic hydroxyl radical (OH•) from hydrogen peroxide (H2O2) for inducing tumor cell death. However, the antitumor efficacy of the CDT strategy is harshly limited by the redox homeostasis of tumor cells; especially the OH • is easily scavenged by glutathione (GSH) and the intracellular H2O2 level is insufficient in the tumor cells. Herein, we propose the Mn2+-menadione (also known as vitamin K3, MK3) cascade biocatalysis strategy to disrupt the redox homeostasis of tumor cells and induce a OH• storm, resulting in enhanced CDT effect. A nanoliposome encapsulating Mn-MK3 (Mn-MK3@LP) was prepared for the treatment of hepatic tumors in this study. After Mn-MK3@LPs were taken up by tumor cells, menadione could facilitate the production of intracellular H2O2 via redox cycling, and further the cytotoxic OH • burst was induced by Mn2+-mediated Fenton-like reaction. Moreover, high-valent manganese ions were reduced by GSH and the depletion of GSH further disrupted the redox homeostasis of tumor cells, thus achieving synergistically enhanced CDT. Overall, both cellular and animal experiments confirmed that the Mn-MK3@LP cascade biocatalysis nanoliposome exhibited excellent biosafety and tumor suppression efficacy. This study may provide deep insights for developing novel CDT-based strategies for tumor therapy.

Effects of water-soluble additive on the release profile and pharmacodynamics of triptorelin loaded in PLGA microspheres.

A satisfactory drug release profile for gonadotropin-releasing hormone (GnRH) agonist drugs is high initial release followed by small amount of drug release per day. In the present study, three water-soluble additives (NaCl, CaCl2 and glucose) were selected to improve the drug release profile of a model GnRH agonist drug-triptorelin from PLGA microspheres. The pore manufacturing efficiency of the three additives was similar. The effects of three additives on drug release were evaluated. Under the optimal initial porosity, the initial release amount of microspheres containing different additives was comparable, this ensured a good inhibitory effect on testosterone secretion in the early stage. For NaCl or CaCl2 containing microspheres, the drug remaining in the microsphere depleted rapidly after the initial release. The testosterone concentration gradually returned to an uncontrolled level. However, for glucose containing microspheres, it was found that the addition of glucose could not only increase the initial release of the drug but also assist in the subsequent controlled drug release. A good and long-time inhibitory effect on testosterone secretion was observed in this formulation. The underlying cause why the incorporation of glucose delayed the subsequent drug release was investigated. SEM results showed that considerable pores in glucose containing microspheres were healed during the microspheres incubation. After thermal analysis, an obvious glass transition temperature (Tg) depression was observed in this formulation. As Tg decreased, polymer chains are able to rearrange at lower temperatures. This, morphologic change was reflected in the gradual closure of the pores, and is the likely reason that drug release slowed down after the initial release.HIGHLIGHTSThe addition of glucose could not only increase the burst release of the drug but also delay the subsequent drug release.High initial burst and a sustained drug release helped obtain a good inhibitory effect on testosterone secretion.As Tg decreased, polymer chain was prone to rearrange. Morphologic change was reflected in the gradual closure of the pores. This was the reason that drug release slowed down after the initial burst.

Efficacy of Temozolomide-Conjugated Gold Nanoparticle Photothermal Therapy of Drug-Resistant Glioblastoma and Its Mechanism Study.

Temozolomide (TMZ) is a standard-of-care chemotherapeutic drug for the treatment of glioblastoma (GBM), but TMZ-acquired resistance limits its therapeutic effect. In this study, TMZ-loaded gold nanoparticles (TMZ@GNPs) with anti-EphA3 modification on the surface (anti-EphA3-TMZ@GNPs) were synthesized for chemical and auxiliary plasma photothermal treatment (GNPs-PPTT), aiming to overcome the problem of glioma resistance to TMZ and improve the therapeutic effects of GBM. The prepared anti-EphA3-TMZ@GNPs were spherical with a particle size of 45.88 ± 1.9 nm, and the drug loading was 7.31 ± 0.38%. In vitro, cell-culture-based experiments showed that anti-EphA3 increased the cellular uptake of GNPs in T98G cells. Upon laser irradiation, the cytotoxicity and apoptosis rate in the anti-EphA3-TMZ@GNPs-treated group were significantly higher than those in the GNPs and nonphotothermal groups (p < 0.001). The Western blot analysis showed that the GNPs-PPTT-mediated killing of tumor cells induced apoptosis by regulating the apoptotic signaling molecules and cell cycle inhibitors; the expression of MGMT significantly decreased upon p53 induction, thereby reversing drug resistance. After photothermal treatment, the survival time of the subcutaneous GBM model of nude mice in the anti-EphA3-TMZ@GNPs group was prolonged to 46 days, 1.64-fold longer as compared to that in the TMZ group. Based on H&E and TUNEL staining, GNPs-PPTT could elevate apoptosis in T98G cells. In vivo thermal imaging results showed that GNPs could enter the brain via intranasal administration and be eliminated in 2 days, indicating that GNPs are safe for brain. In conclusion, GNPs-PPTT could effectively induce apoptosis in glioma cells and reverse TMZ resistance, thereby, indicative of a promising treatment strategy for GBM.

Charge reversal nano-systems for tumor therapy.

Surface charge of biological and medical nanocarriers has been demonstrated to play an important role in cellular uptake. Owing to the unique physicochemical properties, charge-reversal delivery strategy has rapidly developed as a promising approach for drug delivery application, especially for cancer treatment. Charge-reversal nanocarriers are neutral/negatively charged at physiological conditions while could be triggered to positively charged by specific stimuli (i.e., pH, redox, ROS, enzyme, light or temperature) to achieve the prolonged blood circulation and enhanced tumor cellular uptake, thus to potentiate the antitumor effects of delivered therapeutic agents. In this review, we comprehensively summarized the recent advances of charge-reversal nanocarriers, including: (i) the effect of surface charge on cellular uptake; (ii) charge-conversion mechanisms responding to several specific stimuli; (iii) relation between the chemical structure and charge reversal activity; and (iv) polymeric materials that are commonly applied in the charge-reversal delivery systems.

Intranasal Delivery of Temozolomide-Conjugated Gold Nanoparticles Functionalized with Anti-EphA3 for Glioblastoma Targeting.

Glioblastoma multiforme (GBM) is a highly lethal and aggressive tumor of the brain that carries a poor prognosis. Temozolomide (TMZ) has been widely used as a first-line treatment for GBM. However, poor brain targeting, side effects, and drug resistance limit its application for the treatment of GBM. We designed a Temozolomide-conjugated gold nanoparticle functionalized with an antibody against the ephrin type-A receptor 3 (anti-EphA3-TMZ@GNPs) for targeted GBM therapy via intranasal administration. The system can bypass the blood-brain barrier and target active glioma cells to improve the glioma targeting of TMZ and enhance the treatment efficacy, while reducing the peripheral toxicity and drug resistance. The prepared anti-EphA3-TMZ@GNPs were 46.12 ± 2.0 nm and suitable for intranasal administration, which demonstrated high safety to the nasal mucosa in a toxicity assay. In vitro studies showed that anti-EphA3-TMZ@GNPs exhibited significantly enhanced cellular uptake and toxicity, and a higher cell apoptosis ratio has been seen compared with that of TMZ (54.9 and 14.1%, respectively) toward glioma cells (C6). The results from experiments on TMZ-resistant glioma cells (T98G) demonstrated that the IC50 of anti-EphA3-TMZ@GNPs (64.06 ± 0.16 µM) was 18.5-fold lower than that of TMZ. In addition, Western blot analysis also revealed that anti-EphA3-TMZ@GNPs effectively down-modulated expression of O6-methylguanine-DNA methyltransferase and increased chemosensitivity of T98G to TMZ. The antiglioma efficacy in vivo was investigated in orthotopic glioma-bearing rats, and the results demonstrated that the anti-EphA3-TMZ@GNPs prolonged the median survival time to 42 days and increased tumor-cell apoptosis dramatically compared with TMZ. In conclusion, anti-EphA3-TMZ@GNPs could serve as an intranasal drug delivery system for efficacious treatment of GBM.

Research on the fate of polymeric nanoparticles in the process of the intestinal absorption based on model nanoparticles with various characteristics: size, surface charge and pro-hydrophobics.

BACKGROUND: The use of drug nanocarriers to encapsulate drugs for oral administration may become an important strategy in addressing the challenging oral absorption of some drugs. In this study-with the premise of controlling single variables-we prepared model nanoparticles with different particle sizes, surface charges, and surface hydrophobicity/hydrophilicity. The two key stages of intestinal nanoparticles (NPs) absorption-the intestinal mucus layer penetration stage and the trans-intestinal epithelial cell stage-were decoupled and analyzed. The intestinal absorption of each group of model NPs was then investigated. RESULTS: Differences in the behavioral trends of NPs in each stage of intestinal absorption were found to result from differences in particle properties. Small size, low-magnitude negative charge, and moderate hydrophilicity helped NPs pass through the small intestinal mucus layer more easily. Once through the mucus layer, an appropriate size, positive surface charge, and hydrophobic properties helped NPs complete the process of transintestinal epithelial cell transport. CONCLUSIONS: To achieve high drug bioavailability, the basic properties of the delivery system must be suitable for overcoming the physiological barrier of the gastrointestinal tract.

Intelligent Escape System for the Oral Delivery of Liraglutide: A Perfect Match for Gastrointestinal Barriers.

Epithelial cells are known to impede the oral delivery of polypeptides, and the accumulation of mucus and regular dynamic renewal also significantly impede drug absorption. In this work, we prepared a core-shell (COS) nanosystem using poly-N-(2-hydroxypropyl)methacrylamide (pHPMA)/chitosan (CTS). Liraglutide (NN2211) was isolated from the gastrointestinal environment and smoothly passes through the mucous layer. CSKSSDYQC (CSK) peptide and hemagglutinin-2 (HA2) were introduced into the COS nanosystem to establish a complete path from the oral cavity to the epithelial basal side. The fate of nanocapsules in vivo was studied by fluorescence detection. The results showed that the nanocapsules escaped smoothly from the mucus. Taking into account the characteristics of CSK targeting goblet cells, we conducted cell-level studies, and the results showed that after the modification of CSK and pHPMA, more nanocapsules entered the cells. In vitro and in vivo evaluation results showed that the system successfully established a complete path from mucus to epithelial cells by responding to the gastrointestinal environment multiple times.

Synthesis of CSK-DEX-PLGA Nanoparticles for the Oral Delivery of Exenatide to Improve Its Mucus Penetration and Intestinal Absorption.

The oral absorption of exenatide, a drug for type 2 diabetes treatment, can be improved by using nanoparticles (NPs) for its delivery. To improve the mucus penetration and intestinal absorption of exenatide, we designed a block copolymer, CSKSSDYQC-dextran-poly(lactic-co-glycolic acid) (CSK-DEX-PLGA), and used it for the preparation of exenatide-loaded NPs. The functionalized exenatide-loaded NPs composed of CSK-DEX-PLGA were able to target intestinal epithelial cells and reduce the mucus-blocking effect of the intestine. Moreover, the CSK modification of DEX-PLGA was found to significantly promote the absorption efficiency of NPs in the small intestine based on in vitro ligation of the intestinal rings and an examination of different intestinal absorption sites. Compared to DEX-PLGA-NPs (DPs), the absorption of CSK-DEX-PLGA-NPs (CDPs) was increased in the villi, allowing the drug to act on gobletlike Caco-2 cells through clathrin-, caveolin-, and gap-mediated endocytosis. Furthermore, the enhanced transport ability of CDPs was observed in a study on Caco-2/HT-29-MTX cocultured cells. CDPs exhibited a prolonged hypoglycemic response with a relative bioavailability of 9.2% in diabetic rats after oral administration. In conclusion, CDPs can target small intestinal goblet cells and have a beneficial effect on the oral administration of macromolecular peptides as a nanometer-sized carrier.

Preparation and evaluation of lactic acid acylated exenatide and its long-acting preparation.

Exenatide (EX), a glucagon-like peptide-1 receptor agonist, is used to treat diabetes mellitus. However, its short half-life necessitates frequent administration and fluctuations in its plasma concentration may cause adverse effects. Previously, we developed glycolic acid acylated EX, which showed a good glucose-lowering effect. However, the release of lactic acid (LA) acylated exenatide (LA-EX) as an acylated adduct in EX microspheres has not been studied. Here, we investigated the biological properties of LA-EX. Additionally, LA-EX-loaded microspheres were formulated by an emulsion-solvent evaporation method and their in vitro characteristics, in vivo pharmacokinetic properties, and antidiabetic activities were evaluated. Pharmacokinetic studies revealed that the t1/2 of LA-EX (5.95 h) was 2.3-fold longer than that of EX. The antidiabetic activities of LA-EX in db/db mice were similar to those of EX. LA-EX release from microspheres was fairly well-sustained compared to that of EX microspheres. Additionally, LA-EX-loaded microspheres were more effective in lowering nonfasting blood glucose concentrations than EX microspheres. These findings suggest that LA-EX have the same efficacy as EX and that encapsulating LA-EX into microspheres can achieve better efficacy for the long-term type 2 diabetes mellitus treatment.

IR-780-loaded polymeric micelles enhance the efficacy of photothermal therapy in treating breast cancer lymphatic metastasis in mice.

Cancer metastasis is responsible for over 90% of breast cancer-related deaths, and inhibiting lymph node metastasis is an option to treat metastatic disease. Herein, we report the use of IR-780-loaded polymeric micelles (IPMs) for effective photothermal therapy (PTT) of breast cancer lymphatic metastasis. The IPMs were nanometer-sized micelles with a mean diameter of 25.6 nm and had good stability in simulated physiological solutions. Under 808-nm laser irradiation, IPMs exhibited high heat-generating capability in both in vitro and in vivo experiments. After intravenous injection, IPMs specifically accumulated in the tumor and metastatic lymph nodes and penetrated into these tissues. Moreover, a single IPMs treatment plus laser irradiation significantly inhibited primary tumor growth and suppressed lymphatic metastasis by 88.2%. Therefore, IPMs are an encouraging platform for PTT applications in treatment of metastatic breast cancer.

Development, validation and comparison of surrogate matrix and surrogate analyte approaches with UHPLC-MS/MS to simultaneously quantify dopamine, serotonin and γ-aminobutyric acid in four rat brain regions.

As biomarkers, endogenous neurotransmitters play critical roles in the process of neuropsychiatric diseases, and neurotransmitter levels in different brain regions can contribute to neurological disease diagnosis and treatment. Due to the lack of a blank matrix for endogenous neurotransmitters, surrogate-matrix and surrogate-analyte approaches have been used for the determination of neurotransmitters to solve this problem. In this study, we capitalised on the high accuracy, precision, and throughput of UHPLC-MS/MS and developed new methods based on the two approaches. Both approaches satisfied FDA and EMA validation criterias after an appropriate parallelism assessment, and they were used to further quantify the three endogenous neurotransmitters, including dopamine (DA), serotonin (5-HT) and γ-aminobutyric acid (GABA) in rat brain four regions (cortex, striatum, hypothalamus and hippocampus) which represent the catecholamines, indolamines, and amino acids, respectively. Comparison of the results in the same rats (n = 10) showed there was no significant difference in DA, 5-HT, or GABA levels between the two approaches (P > 0.05). The concentrations of DA and GABA were highest in striatum and hypothalamus, respectively, and the levels of 5-HT were paralleled in striatum and hippocampus almost 2-fold higher than other regions. This is the first study to compare these two approaches in the determination of endogenous neurotransmitter content in the rat brain, and the surrogate-matrix approach proved to be simple, rapid, and reliable, considering cost, matrix similarity, and practicality.

Chitooligosaccharides Modified Reduction-Sensitive Liposomes: Enhanced Cytoplasmic Drug Delivery and Osteosarcomas-Tumor Inhibition in Animal Models.

PURPOSE: To investigate the potential of a reduction-sensitive and fusogenic liposomes, enabled by surface-coating with chotooligosaccharides (COS) via a disulfide linker, for tumor-targeted cytoplasmic drug delivery. METHODS: COS (MW2000-5000) were chemically tethered onto the liposomes through a disulfide linker (-SS-) to cholesterol (Chol). Doxorubicin (DOX) was actively loaded in the liposomes. Their reduction-sensitivities, cellular uptake, cytotoxicity, pharmacokinetics and antitumor efficacy were investigated. RESULTS: The Chol-SS-COS/DOX liposomes (100 nm) had zeta potential of 33.9 mV and high drug loading (13% w/w). The liposomes were stable with minimal drug leakage under physiological conditions but destabilized in the presence of reducing agents, dithiothreitol (DTT) or glutathione (GSH) at 10 mM, the cytosolic level. MTT assay revealed that the cationic Chol-SS-COS/DOX liposomes had higher cytotoxicity to MG63-osteosarcoma cells than non-reduction sensitive liposome (Chol-COS/DOX). Flow cytometry and confocal microscopy revealed that Chol-SS-COS/DOX internalized more efficiently than Chol-COS/DOX with more content to cytoplasm whereas Chol-COS/DOX located around the cell membrane. Chol-SS-COS/DOX preferentially internalized into MG63 cancer cell over LO2 normal liver cells. In rats both liposomes produced a prolonged half-life of DOX by 4 - 5.5 fold (p < 0.001) compared with the DOX solution. Chol-SS-COS/DOX exhibited strong inhibitory effect on tumor growth in MG63 cell-bearing nude mice (n = 6), and extended animal survival rate. CONCLUSIONS: Reduction-responsive Chol-SS-COS liposomes may be an excellent platform for cytoplasmic delivery of anticancer drugs. Conjugation of liposomes with COS enhanced tumor cell uptake, antitumor effect and survival rate in animal models.

Increased Active Tumor Targeting by An αvß3-Targeting and Cell-Penetrating Bifunctional Peptide-Mediated Dendrimer-Based Conjugate.

PURPOSE: A bifunctional RGDTAT peptide-modified PEG-PAMAM dendrimer conjugate RGDTAT-PEG-PAMAM (RTPP) was established for the targeted treatment of αvß3-overexpressing tumor cells. METHODS: The RGDTAT peptide was synthesized and attached to PAMAM using PEG to construct the RTPP conjugate. The methotrexate (MTX) encapsulated RTPPM complex was prepared and characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM) and in vitro release. The targeting ability was then studied in cells and tumor-bearing nude mice using fluorescence microscopy, confocal fluorescence microscopy, flow cytometry, and in vivo imaging. The cytotoxicity and pharmacokinetics of the RTPPM complex was also evaluated in cells and rats. RESULTS: The successful synthesis of the RTPP conjugate was confirmed by 1H-NMR. DLS and TEM measurements revealed that the size was 37 nm and the complex had a spherical shape. RTPP and RTPPM were taken up by αvß3-overexpressing cells more efficiently than by αvß3-lowexpressing cells. The RTPP conjugate localized to the cell nucleus and accumulated in the tumor more efficiently than did the conjugates without RGDTAT. The pharmacokinetic study of the RTPPM complex showed sustained drug release. CONCLUSIONS: The bifunctional peptide-mediated dendrimer-based RTPP conjugate can serve as a promising nanocarrier for targeted drug delivery to improve anti-tumor activity.

Colon-Targeted Delivery of IgY Against Clostridium difficile Toxin A and B by Encapsulation in Chitosan-Ca Pectinate Microbeads.

This study investigated the use of a newly developed chitosan-Ca pectinate microbead formulation for the colon-targeted delivery of anti-A/B toxin immunoglobulin of egg yolk (IgY) to inhibit toxin binding to colon mucosa cells. The effect of the three components (pectinate, calcium chloride, and chitosan) used for the microbead production was examined with the aim of identifying the optimal levels to improve drug encapsulation efficiency, swelling ratio, and cumulative IgY release rate. The optimized IgY-loaded bead component was pectin 5% (w/v), CaCl2 3% (w/v), and chitosan 0.5% (w/v). Formulated beads were spherical with 1.2-mm diameter, and the drug loading was 45%. An in vitro release study revealed that chitosan-Ca pectinate microbeads inhibited IgY release in the upper gastrointestinal tract and significantly improved the site-specific release of IgY in the colon. An in vivo rat study demonstrated that 72.6% of biologically active IgY was released specifically in the colon. These results demonstrated that anti-A/B toxin IgY-loaded chitosan-Ca pectinate oral microbeads improved IgY release behavior in vivo, which could be used as an effective oral delivery platform for the biological treatment of Clostridium difficile infection (CDI).

Effect of palmitic acid on the characteristics and release profiles of rotigotine-loaded microspheres.

BACKGROUND: The initial burst release is a major obstacle to the development of microsphere-formulated drug products. PURPOSE: To investigate the influence of palmitic acid on the characteristics and release profiles of rotigotine-loaded poly(d,l-lactide-co-glycolide) microspheres. MATERIALS AND METHODS: Rotigotine-loaded microspheres (RMS) were prepared using the oil-in-water emulsion solvent evaporation technique. The in vitro characteristics of the RMS were evaluated with scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and a particle size analyzer. The in vitro drug release and in vivo pharmacokinetics of the RMS were investigated. RESULTS AND DISCUSSION: The SEM results showed that the addition of palmitic acid changed the surface morphology of the microspheres from smooth to dimpled and then to non-smooth as the palmitic acid content increased. DSC revealed the existence of molecularly dispersed forms of palmitic acid in the microspheres. The in vitro and in vivo release profiles indicated that the addition of 5% and 8% palmitic acid significantly decreased the burst release of rotigotine from the microspheres, and the late-stage release was delayed as the palmitic acid content increased across the investigated range (5-15%). CONCLUSION: The addition of palmitic acid to the microspheres significantly affects the release profile of rotigotine from RMS.

Application of hot-melt extrusion technology for designing an elementary osmotic pump system combined with solid dispersion for a novel poorly water-soluble antidepressant.

TP1 is a novel antidepressant with poor solubility. To reduce fluctuations in blood concentration and increase oral bioavailability, a controlled-release system was developed by combining a solid dispersion (SD) and an elementary osmotic pump (EOP). The study compared different methods of preparing SDs. Hot-melt extrusion (HME) exhibited clear advantages over the traditional melting technique. An in vitro release study demonstrated that HME-EOP tablets released TP1 in a zero-order manner over 12 h and the drug release was in dependent of the release medium and agitation speed, whereas release from molten-EOP tablets lasted only 8 h. In contrast to immediate-release tablets, the HME-EOP tablets exhibited less fluctuation in blood concentration and higher bioavailability in vivo. In summary, the osmotic pump system combined with an HME-based SD of TP1 presented controlled release in vitro, high bioavailability in vivo and a good in vivo-in vitro correlation.

Preparation and evaluation of rotigotine-loaded implant for the treatment of Parkinson's disease and its evolution study.

PURPOSE: To develop rotigotine-loaded implants (RI) to achieve continuous release of rotigotine for long duration for the treatment of Parkinson's disease (PD). METHODS: RI was prepared by hot-melt extrusion method using poly (lactide-co-glycolide) (PLGA) as the matrix. In vitro drug release was optimized by drug loading, melting temperature during preparing process and additives. The surface and internal morphology of RI was imaged by SEM. The in vivo release profile of RI was investigated on rat. RESULTS: RI prepared with PLGA 7525 5A showed sustained release of 40 days while suffering a lag phase, which was significantly shortened by blending 5050 2A and mannitol in the matrix. RI prepared by 7525 5A/5050 2A/mannitol = 55:10:5 (rotigotine 30%) showed a 40-day sustained release in vivo with no lag phase. The drug release from RI was also affected by drug loading and melting temperature probably due to the drug state existed in the implant. The evolution of implants during release process was correlated well with the drug release kinetics. CONCLUSION: RI could achieve sustained drug release for 40 days which could supply an alternative of continuous dopaminergic stimulation (CDS) for the treatment of PD.

The glucose-lowering potential of exenatide delivered orally via goblet cell-targeting nanoparticles.

PURPOSE: Exenatide, a potent insulinotropic agent, can be used for the treatment of non-insulin-dependent diabetes mellitus. However, the need for frequent injections seriously limits its therapeutic utility. The aim of present report was to develop an orally available exenatide formulation using goblet cell-targeting nanoparticles (NPs). METHOD: The exenatide-loaded nanoparticles were prepared with modified chitosan which was conjugated with a goblet cell-target peptide, CSKSSDYQC (CSK) peptide. RESULTS: The CSK-chitosan nanoparticles shown reduced chitosan toxicity and enhanced the permeation of drugs across the Caco-2/HT-29 co-cultured cell monolayer, which simulated the intestinal epithelium. Following the oral administration of near-infrared fluorescent probe Cy-7-loaded NPs to mice, the distribution of the drugs was investigated with a near-infrared in vivo image system (FX Pro, Bruker, USA). The results showed that Cy-7 fluorescence disseminated from the oesophagus, then to stomach and small intestine and then was absorbed into hepatic, finally into the bladder; over time, Cy-7 was metabolized and excreted. The bioavailability of the modified nanoparticles was found to be 1.7-fold higher compared with the unmodified ones, and the hypoglycemic effect was also better. CONCLUSION: CSK peptide-modified chitosan nanoparticles could be a potential therapeutics for Type II diabetes patients.

Acylation of exenatide by glycolic acid and its anti-diabetic activities in db/db mice.

PURPOSE: To prepare acylated exenatide analogues and investigate their biological properties for guiding the development of PLGA formulations of exenatide. METHODS: The acylated exenatide analogues were prepared by reaction with glycolic acid (GA), one constitutional unit of PLGA, and characterized by HPLC-MS/MS and Circular Dichroism (CD). The pharmacokinetic properties and anti-diabetic activities were studied in SD rats and db/db mice, respectively. RESULTS: Structural characterizations of the acylated products showed that one to four glycolic acids (GAs) were connected to the primary amine groups of exenatide, and there was a conversion of α-helix to ß-sheet to some extent. Pharmacokinetic studies in SD rats revealed that acylated exenatides had a similar Tmax with that of the prototype drug, whereas the Cmax and the AUC values of the adducts were significantly decreased. Biological activity tests demonstrated that exenatide and acylated exenatide analogues had similar in vivo antidiabetic activities in terms of controlling blood glucose concentration, HbA1c level, body weight and food intake. CONCLUSIONS: These findings suggest that GA conjugated exenatide had no influence on the peptide efficacy, therefore it's not necessary to inhibit exenatide acylation in PLGA formulations during the peptide release process.

An analytical strategy to characterize the pharmacokinetics and pharmacodynamics of triptorelin in rats based on simultaneous LC-MS/MS analysis of triptorelin and endogenous testosterone in rat plasma.

Triptorelin, a gonadotropin-releasing hormone agonist, has been used in the treatment of hormone-responsive prostate cancer by inducing testosterone suppression. Research on the relationship between the time courses of triptorelin and testosterone is very important, but accurate quantification of triptorelin and testosterone simultaneously in biological specimens is a challenging analytical problem. In the present study, a rapid, sensitive, and selective method for simultaneous determination of triptorelin and testosterone in rat plasma by solid-phase extraction and liquid chromatography-tandem mass spectrometry was developed using a ZORBAX RRHD Eclipse Plus C8 column (2.1 × 50 mm, 1.8 µm) with a 0.05% propionic acid/methanol gradient. In view of the polarity difference between the two analytes, two internal standards, i.e., leuprolide and testosterone-(13)C3, were used for individual quantitation of triptorelin and testosterone. Endogenous testosterone was determined by reference to a calibration curve prepared using testosterone-D3 as a surrogate analyte. The method exhibits excellent linearity over three orders of magnitude for each analyte. The lower limit of quantification was 0.01 ng/mL for triptorelin and 0.05 ng/mL for testosterone, with consumption of 100 µL of plasma. The method was successfully applied to characterize the pharmacokinetics and pharmacodynamics of slow-release 28-day form triptorelin acetate biodegradable microspheres in rats after intramuscular injections of three consecutive doses of 0.6 mg/kg per 28 days. The results revealed that the pharmacokinetic profile of triptorelin produced an initial flare-up in testosterone levels, rapid castration within 5 days after injection, and long-term castration until the next dose.