Oxaliplatin delivery via chitosan/vitamin E conjugate micelles for improved efficacy and MDR-reversal in breast cancer.

A bioinspired chitosan/vitamin E conjugate (Ch/VES, 1:4) was synthesized, optimized based on chitosan's molecular weight (15, 300 kDa), and was assembled to entrap oxaliplatin (OXPt). 1H NMR, infrared spectroscopy, chromatography, X-ray photoelectron spectroscopy, X-ray diffraction, drug release, hemolysis, and stability studies were performed to characterize OXPt@Ch/VES micelles. The therapeutic efficacy of the micelles was tested in vitro in ER+/PR+/HER2- and triple-negative sensitive/resistant breast cancer cells, MCF-7 and MDA-MB-231 via cellular uptake, cytotoxicity, nuclear staining, DNA fragmentation, mitochondrial membrane potential, ROS generation, apoptosis, and cell cycle assays and in vivo using 4T1(Luc)-tumor-bearing mice. OXPt@Ch/VES Ms exhibited decreased IC50 towards MCF-7, MDA-MB-231 (sensitive/resistant) than OXPt. OXPt@Ch/VES Ms caused extensive DNA damage, mitochondrial depolarization, apoptosis, and cell-growth arrest (G2/M). OXPt@Ch/VES Ms treatment retarded tumor growth significantly, prolonged survival, and decreased nephrotoxicity than OXPt. The OXPt@Ch/VES Ms could serve as a potential nanomedicine to overcome conventional OXPt-mediated drug resistance/nephrotoxicity in breast cancer.

Chitosan derivatives functionalized dual ROS-responsive nanocarriers to enhance synergistic oxidation-chemotherapy.

The efficient triggering of prodrug release has become a challengeable task for stimuli-responsive nanomedicine utilized in cancer therapy due to the subtle differences between normal and tumor tissues and heterogeneity. In this work, a dual ROS-responsive nanocarriers with the ability to self-regulate the ROS level was constructed, which could gradually respond to the endogenous ROS to achieve effective, hierarchical and specific drug release in cancer cells. In brief, DOX was conjugated with MSNs via thioketal bonds and loaded with ß-Lapachone. TPP modified chitosan was then coated to fabricate nanocarriers for mitochondria-specific delivery. The resultant nanocarriers respond to the endogenous ROS and release Lap specifically in cancer cells. Subsequently, the released Lap self-regulated the ROS level, resulting in the specific DOX release and mitochondrial damage in situ, enhancing synergistic oxidation-chemotherapy. The tumor inhibition Ratio was achieved to 78.49%. The multi-functional platform provides a novel remote drug delivery system in vivo.

Zinc ions coordinated carboxymethyl chitosan-hyaluronic acid microgel for pulmonary drug delivery.

Microgel affords a porous and swollen microstructure for the establishment of pulmonary delivery system with sustained released properties. Here, we report a microgel (with the diameter around 4 µm) prepared with a precipitation method, synthesized by coordinating Zn2+ to the Schiff base cross-linked carboxymethyl chitosan and glycol split hyaluronate. The microgel has shown well swollen and pH sensitive behaviors, high safety and biocompatibility in vitro. Besides, the biomaterial could escape from macrophage phagocytosis, a key factor contribute to quick drug clearance in the lung after co-incubated with RAW 264.7 cells. In consist with this, the bovine serum albumin loaded in the microgel showed sustained release behavior in 24 h in vitro; meanwhile, the drug had a retention time up to 36 h in the lung and followed by clearance in ICR mice through pulmonary administration. Thus, our microgel platform provides a promising candidate for pulmonary drug delivery systems with controlled release rate.

Chitosan-based nanoparticle co-delivery of docetaxel and curcumin ameliorates anti-tumor chemoimmunotherapy in lung cancer.

The application of traditional chemotherapy drugs for lung cancer has obvious limitations, such as toxic side effects, uncontrolled drug-release, poor bioavailability, and drug-resistance. Thus, to address the limitations of free drugs and improve treatment effects, we developed novel T7 peptide-modified nanoparticles (T7-CMCS-BAPE, CBT) based on carboxymethyl chitosan (CMCS), which is capable of targeted binding to the transferrin receptor (TfR) expressed on lung cancer cells and precisely regulating drug-release according to the pH value and reactive oxygen species (ROS) level. The results showed that the drug-loading content of docetaxel (DTX) and curcumin (CUR) was approximately 7.82% and 6.48%, respectively. Good biosafety was obtained even when the concentration was as high as 500 µg/mL. More importantly, the T7-CMCS-BAPE-DTX/CUR (CBT-DC) complexes exhibited better in vitro and in vivo anti-tumor effects than DTX monotherapy and other nanocarriers loaded with DTX and CUR alone. Furthermore, we determined that CBT-DC can ameliorate the immunosuppressive micro-environment to promote the inhibition of tumor growth. Collectively, the current findings help lay the foundation for combinatorial lung cancer treatment.

Polyvinyl Alcohol/Chitosan Single-Layered and Polyvinyl Alcohol/Chitosan/Eudragit RL100 Multi-layered Electrospun Nanofibers as an Ocular Matrix for the Controlled Release of Ofloxacin: an In Vitro and In Vivo Evaluation.

A novel nanofiber insert was prepared with a modified electrospinning method to enhance the ocular residence time of ofloxacin (OFX) and to provide a sustained release pattern by covering hydrophilic polymers, chitosan/polyvinyl alcohol (CS/PVA) nanofibers, with a hydrophobic polymer, Eudragit RL100 in layers, and by glutaraldehyde (GA) cross-linking of CS-PVA nanofibers for the treatment of infectious conjunctivitis. The morphology of the prepared nanofibers was studied using scanning electron microscopy (SEM). The average fiber diameter was found to be 123 ± 23 nm for the single electrospun nanofiber with no cross-linking (OFX-O). The single nanofibers, cross-linked for 10 h with GA (OFX-OG), had an average fiber diameter of 159 ± 30 nm. The amount of OFX released from the nanofibers was measured in vitro and in vivo using UV spectroscopy and microbial assay methods against Staphylococcus aureus, respectively. The antimicrobial efficiency of OFX formulated in cross-linked and non-cross-linked nanofibers was affirmed by observing the inhibition zones of Staphylococcus aureus and Escherichia coli. In vivo studies using the OFX nanofibrous inserts on a rabbit eye confirmed a sustained release pattern for up to 96 h. It was found that the cross-linking of the nanofibers by GA vapor could reduce the burst release of OFX from OFX-loaded CS/PVA in one layer and multi-layered nanofibers. In vivo results showed that the AUC0-96 for the nanofibers was 9-20-folds higher compared to the OFX solution. This study thus demonstrates the potential of the nanofiber technology is being utilized to sustained drug release in ocular drug delivery systems.

Chitosan coated nanoparticles for efficient delivery of bevacizumab in the posterior ocular tissues via subconjunctival administration.

In several ocular diseases, vascular endothelial growth factor (VEGF) level has been found to be unregulated. Bevacizumab, an anti-VEGF drug, is the most commonly used off level drug for diabetic retinopathy (DR). The present study was to evaluate the chitosan-coated poly (lactide-co-glycolic acid) nanoparticles (CS-PLGA NPs) for sustained and effective delivery of bevacizumab to posterior ocular tissues. The penetration of NP through sclera was studied by confocal laser scanning microscopy (CLSM). For pharmacokinetic study, bevacizumab loaded NPs were administered into the rat eye through subconjunctival injection (SCJ) and pharmacokinetic parameters were compared to drug solution. CLSM and pharmacokinetic study showed better penetration of formulation and higher concentration of bevacizumab in posterior ocular tissues. In retinopathy model, CS-PLGA NPs by SCJ route showed more reduction of VEGF level in retina than the topical and intravitreal administration of formulation. Thus, CS-coated PLGA NPs can be potentially useful as carriers to target retina.

Experimental study on preparation and anti-tumor efficiency of nanoparticles targeting M2 macrophages.

This study aimed to develop an effective therapy against M2 macrophages and to investigate the effects of imidazole and mannose modified carboxymethyl chitosan-nanoparticles (MIC-NPs) on tumor growth and antitumor immune responses. MIC-NPs were constructed and analyzed through 1H NMR, nano-laser particle size analyzer, and transmission electron microscopy. The nanoparticles were mainly distributed in 75-85 nm, and zeta potential was 1.5 mV. Cytotoxicity studies in vitro and in vivo indicated that MIC-NPs were safe. The targeting effect of MIC-NPs on M2 macrophages was observed through fluorescence microscope and microplate system. The results demonstrated the uptake of a large amount of FITC-loaded MIC-NPs by M2. Cell growth inhibition experiments showed that MIC-NPs significantly inhibited M2 through cell apoptosis. The evaluation of anti-tumor activity in vivo showed that MIC-NPs could accumulate in the tumor site to exert an anti-tumor effect. Flow cytometry showed that the proportion of M2 macrophages at the tumor site in the experimental group was significantly lower than that in the control group, while the Treg cells and cytotoxic T cells (CTL) were found to be increased. PCR detection showed that the cDNA of FIZZ, MR, TGF-ß, and arginase, closely related to M2 macrophages, in the experimental group, was significantly lower than that in the control group, but there was no significant difference in the cDNA of Treg cell characteristic Foxp3 between the two groups. These results suggest that MIC-NPs are expected to provide a new and effective treatment for tumor.

Hemostatic performance of chitosan-based hydrogel and its study on biodistribution and biodegradability in rats.

Hemostasis is of great significance regardless of the smooth operation or postoperative recovery. Therefore, it is urgent to develop a hemostatic material with excellent biodegradability and biocompatibility. It is well known that both carboxymethyl chitosan and hyaluronic acid with biodegradability and biocompatibility have wound healing promoting property. Here, a degradable chitosan-based hydrogel was prepared based on carboxymethyl chitosan and cross-linked by oxidized hyaluronic acid. The hemostatic performance of the hydrogel in rat liver resection injury was evaluated which results showed that the hydrogel exhibited comparable hemostatic properties compared with Fibrin Sealant. In addition, the hydrogel proved to be rapidly absorbed by the body without significant accumulation in vivo, demonstrating good biodegradability and biocompatibility. The overall results suggested the hydrogel will be a promising hemostatic hydrogel for controlling bleeding.

Chitosan-based systems aimed at local application for vaginal infections.

Vaginal administration is a promising route for the local treatment of infectious vaginal diseases since it can bypass the first-pass metabolism, drug interactions, and adverse effects. However, the commercial products currently available for topical vulvovaginal treatment have low acceptability and do not adequately explore this route. Mucoadhesive systems can optimize the efficacy of drugs administered by this route to increase the retention time of the drug in the vaginal environment. Several polymers are used to develop mucoadhesive systems, among them chitosan, a natural polymer that is highly biocompatible and technologically versatile. Thus, the present review aimed to analyze the studies that used chitosan to develop mucoadhesive systems for the treatment of local vaginal infections. These studies demonstrated that chitosan as a component of mucoadhesive drug delivery systems (DDS) is a promising device for the treatment of vaginal infectious diseases, due to the intrinsic antimicrobial activity of this biopolymer and because it does not interfere with the effectiveness of the drugs used for the treatment.

Microfluidic-based synthesized carboxymethyl chitosan nanoparticles containing metformin for diabetes therapy: In vitro and in vivo assessments.

This work was aimed to synthesize novel crosslinked carboxymethyl chitosan nanoparticles (CMCS NPs) containing metformin hydrochloride (MET) using microfluidics (MF) and evaluate their performance for diabetes therapy. The field emission-scanning electron microscopy (FE-SEM) images and dynamic light scattering (DLS) results showed that the NPs average size was 77 ± 19 nm with a narrow size distribution. They exhibited a high encapsulation efficiency (∼90 %) and the controlled drug release while crosslinking using CaCl2. Eventually, the in vivo assessments dedicated an increased body weight up to 7.94 % and a decreased blood glucose level amount of 43.58 % for MF MET-loaded CMCS NPs with respect to the free drug in diabetic rats. Also, the results of histopathological studies revealed the size of the pancreatic islets to be 2.32 µm2 and ß cells intensity to be 64 cells per islet for the diabetic rats after treating with the MF-based sample. These data were close to those obtained for the healthy rats.

Insight into chitosan/zeolite-A nanocomposite as an advanced carrier for levofloxacin and its anti-inflammatory properties; loading, release, and anti-inflammatory studies.

Chitosan/zeolite-A nanocomposite (CH/ZA) was synthesized as a potential carrier for levofloxacin (LVOX) of enhanced technical properties. The CH/ZA composite displayed enhanced loading capacity (425 mg/g) as compared to chitosan (188.8 mg/g) and zeolite-A (234.6 mg/g). The loading behavior follows Pseudo-Second-order and Langmuir as kinetic and isotherm models. The equilibrium studies, Gaussian energy (8.15 KJ/mol), and thermodynamic parameters demonstrate homogenous and monolayer loading by complex chemical and physical reactions that are of spontaneous and exothermic nature. The CH/ZA composite is of slow and continuous release profile (200h) with 94.3% as the maximum release percentage. The release reactions are of non-Fickian behavior involving both diffusion and erosion mechanisms. The loading of LVOX into CH/ZA induced its anti-inflammatory effect against the cytokine production (IL-6 and IL-8) within the human bronchial epithelia cells (NL20). The cytotoxicity studies on the normal cells demonstrated a high safety value for the composite.

Chitosan-based nanocarriers for encapsulation and delivery of curcumin: A review.

To overcome the poor aqueous solubility and bioavailability of curcumin, emphasize its functional features, and broaden its applications in the food and pharmaceutical industries, many nanoscale systems have been widely applied for its encapsulation and delivery. Over many decades, chitosan as a natural biopolymer has been extensively studied due to its polycationic nature, biodegradability, biocompatibility, non-toxicity, and non-allergenic. Various chitosan-based nanocarriers with unique properties for curcumin delivery, including but not limited to, self-assembled nanoparticles, nanocomposites, nanoemulsions, nanotubes, and nanofibers, have been designed. This review focuses on the most-recently reported fabrication techniques of different types of chitosan-based nanocarriers. The functionalities of chitosan in each formulation which determine the physicochemical properties such as surface charge, morphology, encapsulation driving force, and release profile, were discussed in detail. Moreover, the current pharmaceutical applications of curcumin-loaded chitosan nanoparticles were elaborated. The role of chitosan in facilitating the delivery of curcumin and improving the therapeutic effects on many chronic diseases, including cancer, bacterial infection, wound healing, Alzheimer's diseases, inflammatory bowel disease, and hepatitis C virus, were illustrated. Particularly, the recently discovered mechanisms of action of curcumin-loaded chitosan nanoparticles against the abovementioned diseases were highlighted.

Use of combined nanocarrier system based on chitosan nanoparticles and phospholipids complex for improved delivery of ferulic acid.

A novel nanocarrier system of phospholipids complex loaded chitosan nanoparticles (FAPLC CNPs) was developed to improve the oral bioavailability and antioxidant potential of FA. FAPLC CNPs were optimized using a Box-Behnken Design (BBD). FAPLC CNPs were characterized using differential scanning calorimetry, Fourier transforms infrared spectroscopy, powder x-ray diffractometry, proton nuclear magnetic resonance, solubility, in vitro dissolution, ex vivo permeation, and in vivo antioxidant activity in carbon tetrachloride (CCl4)-induced albino rat model. The characterization studies indicated a formation of the complex as well as FAPLC CNPs. The FAPLC CNPs exhibited a lower particle size ~123.27 nm, PDI value ~0.31, and positive zeta potential ~32 mV respectively. Functional characterization studies revealed a significant improvement in the aqueous solubility, dissolution, and permeation rate of FAPLC and FAPLC CNPs compared to FA and FA CNPs. The FAPLC CNPs showed significant enhancement of in vivo antioxidant activity of FA by restoring the elevated marker enzymes in the CCl4-intoxicated rat model compared to FA CNPs. Moreover, the pharmacokinetic analysis demonstrated a significant enhancement of oral bioavailability of FA from FAPLC CNPs compared to FA CNPs. These findings show that FAPLC CNPs could be used as an effective nanocarrier for improving the oral delivery of FA.

An overview of gastrointestinal mucus rheology under different pH conditions and introduction to pH-dependent rheological interactions with PLGA and chitosan nanoparticles.

This review discusses the physicochemical and mechanical properties of porcine gastrointestinal mucus from a rheological point of view. Considering mucus as a viscoelastic gel that functions as a biological barrier by limiting particles passage, lubricating the gastrointestinal tract, and protecting the stomach from gastric acids. The viscoelastic and protective properties of mucus are mainly produced by its mucin network, which is stabilized through electrostatic, hydrophobic and hydrogen bonding interactions. Otherwise, mucus rheology is determined by its polyanionic nature at physiological pH. At neutral pH, mucus presents a viscous behavior produced by chains crosslinking. While, at acidic pH, mucus exhibits an elastic behavior related with the extended conformation that produces mucus gelation at the stomach. Additionally, rheology studies the degree of adhesion between a polymer-mucus mixture through rheological synergism, and how it varies at different pH conditions. Finally, mucoadhesion phenomenon is exemplified with chitosan (cationic) and poly (lactic-co-glycolic) acid (anionic) polymers.

The Formation of Chitosan-Coated Rhamnolipid Liposomes Containing Curcumin: Stability and In Vitro Digestion.

There is growing interest in developing biomaterial-coated liposome delivery systems to improve the stability and bioavailability of curcumin, which is a hydrophobic nutraceutical claimed to have several health benefits. The curcumin-loaded rhamnolipid liposomes (Cur-RL-Lips) were fabricated from rhamnolipid and phospholipids, and then chitosan (CS) covered the surface of Cur-RL-Lips by electrostatic interaction to form CS-coated Cur-RL-Lips. The influence of CS concentration on the physical stability and digestion of the liposomes was investigated. The CS-coated Cur-RL-Lips with RL:CS = 1:1 have a relatively small size (412.9 nm) and positive charge (19.7 mV). The CS-coated Cur-RL-Lips remained stable from pH 2 to 5 at room temperature and can effectively slow the degradation of curcumin at 80 °C; however, they were highly unstable to salt addition. In addition, compared with Cur-RL-Lips, the bioavailability of curcumin in CS-coated Cur-RL-Lips was relatively high due to its high transformation in gastrointestinal tract. These results may facilitate the design of a more efficacious liposomal delivery system that enhances the stability and bioavailability of curcumin in nutraceutical-loaded functional foods and beverages.

The role of Chito-oligosaccharide in regulating ovarian germ stem cells function and restoring ovarian function in chemotherapy mice.

In recent years, the discovery of ovarian germ stem cells (OGSCs) has provided a new research direction for the treatment of female infertility. The ovarian microenvironment affects the proliferation and differentiation of OGSCs, and immune cells and related cytokines are important components of the microenvironment. However, whether improving the ovarian microenvironment can regulate the proliferation of OGSCs and remodel ovarian function has not been reported. In this study, we chelated chito-oligosaccharide (COS) with fluorescein isothiocyanate (FITC) to track the distribution of COS in the body. COS was given to mice through the best route of administration, and the changes in ovarian and immune function were detected using assays of organ index, follicle counting, serum estrogen (E2) and anti-Mullerian hormone (AMH) levels, and the expression of IL-2 and TNF-α in the ovaries. We found that COS significantly increased the organ index of the ovary and immune organs, reduced the rate of follicular atresia, increased the levels of E2 and AMH hormones, and increased the protein expression of IL-2 and TNF-α in the ovary. Then, COS and OGSCs were co-cultured to observe the combination of COS and OGSCs, and measure the survival rate of OGSCs. With increasing time, the fluorescence intensity of cells gradually increased, and the cytokines IL-2 and TNF-α significantly promoted the proliferation of OGSCs. In conclusion, COS could significantly improve the ovarian and immune function of chemotherapy model mice, and improve the survival rate of OGSCs, which provided a preliminary blueprint for further exploring the mechanism of COS in protecting ovarian function.

An overview of chitosan and its application in infectious diseases.

Infectious diseases, such as the coronavirus disease-19, SARS virus, Ebola virus, and AIDS, threaten the health of human beings globally. New viruses, drug-resistant bacteria, and fungi continue to challenge the human efficacious drug bank. Researchers have developed a variety of new antiviral and antibacterial drugs in response to the infectious disease crisis. Meanwhile, the development of functional materials has also improved therapeutic outcomes. As a natural material, chitosan possesses good biocompatibility, bioactivity, and biosafety. It has been proven that the cooperation between chitosan and traditional medicine greatly improves the ability of anti-infection. This review summarized the application and design considerations of chitosan-composed systems for the treatment of infectious diseases, looking forward to providing the idea of infectious disease therapy.

Formulation of Quaternized Aminated Chitosan Nanoparticles for Efficient Encapsulation and Slow Release of Curcumin.

An effective drug nanocarrier was developed on the basis of a quaternized aminated chitosan (Q-AmCs) derivative for the efficient encapsulation and slow release of the curcumin (Cur)-drug. A simple ionic gelation method was conducted to formulate Q-AmCs nanoparticles (NPs), using different ratios of sodium tripolyphosphate (TPP) as an ionic crosslinker. Various characterization tools were employed to investigate the structure, surface morphology, and thermal properties of the formulated nanoparticles. The formulated Q-AmCs NPs displayed a smaller particle size of 162 ± 9.10 nm, and higher surface positive charges, with a maximum potential of +48.3 mV, compared to native aminated chitosan (AmCs) NPs (231 ± 7.14 nm, +32.8 mV). The Cur-drug encapsulation efficiency was greatly improved and reached a maximum value of 94.4 ± 0.91%, compared to 75.0 ± 1.13% for AmCs NPs. Moreover, the in vitro Cur-release profile was investigated under the conditions of simulated gastric fluid [SGF; pH 1.2] and simulated colon fluid [SCF; pH 7.4]. For Q-AmCs NPs, the Cur-release rate was meaningfully decreased, and recorded a cumulative release value of 54.0% at pH 7.4, compared to 73.0% for AmCs NPs. The formulated nanoparticles exhibited acceptable biocompatibility and biodegradability. These findings emphasize that Q-AmCs NPs have an outstanding potential for the delivery and slow release of anticancer drugs.

Design and in vitro antibiofilm activity of propolis diffusion-controlled biopolymers.

In this study, a novel pH-sensitive hydrogel beads that is based on gelatin/sodium alginate/chitosan (GEL/SA/CS) loaded with propolis ethanolic extracts (PE) were synthesized. The swelling behavior of GEL/SA/CS hydrogel beads was studied in different pH solutions and compared with unloaded CS (GEL/SA) hydrogel beads. The in vitro release studies have been revealed using four different pH (1.3, 5.0, 6.0, and 6.8), a saliva environment (pH 6.8), a simulated gastric fluid (SGF) (pH 1.3), and a simulated intestinal fluid (SIF) (pH 6.8) to simulate the physiological conditions in gastrointestinal (GI) tract. Propolis-loaded hydrogel beads were found to be stable at pH 1.3, 5.0, 6.0, simulated saliva, SGF, and SIF mediums, whereas the beads lose their stability at pH 6.8 buffer solution. Tested microorganisms displayed greater sensitivity to PE-loaded hydrogel beads compared with pure propolis. Contrary to antimicrobial activity results, antibiofilm activity results of PE-loaded GEL/SA and GEL/SA/CS hydrogel beads were found at low levels. According to the obtained results, the propolis-loaded GEL/SA/CS hydrogel beads synthesized within this study can be used in the treatment of GI tract diseases such as oral mucositis, gastric ulcer, ulcerative colitis, and GI cancer, as controlled releasing carriers of propolis.

Synthesis and characterization of thermosensitive hydrogel based on quaternized chitosan for intranasal delivery of insulin.

Nasal administration is a form of systemic administration in which drugs are insufflated through the nasal cavity. Steroids, nicotine replacement, antimigraine drugs, and peptide drugs are examples of the available systematically active drugs as nasal sprays. For diabetic patients who need to use insulin daily, the nasal pathway can be used as an alternative to subcutaneous injection. In this regard, intranasal insulin delivery as a user-friendly and systemic administration has recently attracted more attention. In this study, a novel formulation consists of chitosan, chitosan quaternary ammonium salt (HTCC), and gelatin (Gel) was proposed and examined as a feasible carrier for intranasal insulin administration. First, the optimization of the chitosan-HTCC hydrogel combination has done. Afterward, Gel with various amounts blended with the chitosan-HTCC optimized samples. In the next step, swelling rate, gelation time, degradation, adhesion, and other mechanical, chemical, and biological properties of the hydrogels were studied. Finally, insulin in clinical formulation and dosage was blended with optimized thermosensitive hydrogel and the release procedure of insulin was studied with electrochemiluminescence technique. The optimal formulation (consisted of 2 wt% chitosan, 1 wt% HTCC, and 0.5 wt% Gel) showed low gelation time, uniform pore structure, and the desirable swelling rate, which were resulted in the adequate encapsulation and prolonged release of insulin in 24 H. The optimal samples released 65% of the total amount of insulin in the first 24 H, which is favorable for this study.