Oral Insulin Delivery System Research Articles

In vivo oral insulin delivery via covalent organic frameworks.

With diabetes being the 7th leading cause of death worldwide, overcoming issues limiting the oral administration of insulin is of global significance. The development of imine-linked-covalent organic framework (nCOF) nanoparticles for oral insulin delivery to overcome these delivery barriers is herein reported. A gastro-resistant nCOF was prepared from layered nanosheets with insulin loaded between the nanosheet layers. The insulin-loaded nCOF exhibited insulin protection in digestive fluids in vitro as well as glucose-responsive release, and this hyperglycemia-induced release was confirmed in vivo in diabetic rats without noticeable toxic effects. This is strong evidence that nCOF-based oral insulin delivery systems could replace traditional subcutaneous injections easing insulin therapy.

Pharmacodynamics and pharmacokinetics behaviour of insulin from PEGylated-mucin microparticles coated with pH sensitive polymer: Preparation and characterization

The aim of this study was to develop a mucin-PEG based microparticles for oral delivery system for insulin for diabetes treatment. Mucin-PEG microparticles were prepared using emulsion technique. These microparticles were coated with Hydroxyl propyl methylcellulose acetate succinate (HPMCAS) using solvent evaporation method. The effect of polymer ratios and coating were investigated in terms of size, surface morphology, entrapment efficiency, micrometrics properties, in vitro release and in vivo studies. The size of insulin-loaded MPs is > 80 μm and exhibited rough surface with irregular shape. The maximum and minimum encapsulation efficiency were 89.10 and 75.15 %, respectively. In vitro release study in pH 1.2 revealed insignificantly low (6 %) (p < 0.05) as compared to the significantly high (85 %) released obtained in pH of 7.4 for formulation combined PEG/mucin ratios (batches B1, B2 and B3). Furthermore, in vivo experiments in Wistar Albino rat model demonstrate that insulin-loaded MPs effectively reduced blood glucose levels over a period of 10 h after oral administration. Therefore, these findings clearly suggest that the HPMCP-coated PEG-mucin microparticles offer an interesting mode of insulin delivery orally for the treatment of diabetes.

\u201cOil-soluble\u201d reversed lipid nanoparticles for oral insulin delivery

BackgroundIn this study, we aimed to design a novel oral insulin delivery system, named “oil-soluble” reversed lipid nanoparticles (ORLN), in which a hydrophilic insulin molecule is encapsulated by a phospholipid (PC) shell and dissolved in oil to prevent the enzymatic degradation of insulin. ORLN was characterized by transmission electron microscopy and dynamic light scattering.ResultsIn vitro enzymatic stability studies showed higher concentrations of insulin in cells incubated with ORLN-encapsulated insulin than in those incubated with free insulin solution in artificial intestinal fluid (pH 6.5). The protective effect of ORLN was attributed to its special release behavior and the formulation of the PC shell and oil barrier. Furthermore, an in vivo oral efficacy study confirmed that blood glucose levels were markedly decreased after ORLN administration in both healthy and diabetic mice. In vivo pharmacokinetic results showed that the bioavailability of ORLN-conjugated insulin was approximately 28.7% relative to that of the group subcutaneously administered with an aqueous solution of insulin, indicating enhanced oral absorption.ConclusionsIn summary, the ORLN system developed here shows promise as a nanocarrier for improving the oral absorption of insulin.

Design and Investigation of Penetrating Mechanism of Octaarginine-Modified Alginate Nanoparticles for Improving Intestinal Insulin Delivery

The aim of the study is to design octaarginine (R8)-modified insulin-alginate nanoparticles (INS-SA/R8 NPs) as the oral insulin delivery system, and further investigate its penetrating mechanism. The characterization results indicated that the surface of INS-SA/R8 NPs was smooth and the average diameter was about 300 nm. INS-SA/R8 NPs exhibited a stronger stability in the simulated gastrointestinal fluids and had a better controlled release than unmodified alginate nanoparticles (INS-SA NPs). Moreover, INS-SA/R8 NPs group had the strongest insulin transport capacity and the largest amount of insulin uptake in all experimental groups. Most importantly, the improvement of insulin intestinal uptake was further confirmed in rat intestine in vivo, and its penetrating mechanism might be involved in the production of endogenous nitric oxide (NO) signal molecule. In addition, in vivo hypoglycemic studies showed that orally administrated INS-SA/R8 NPs produced a better hypoglycemic effect as compared with INS-SA NPs in diabetic rats. Meanwhile, from the cytotoxicity analysis, INS-SA/R8 NPs were safe for oral administration. Taken together, INS-SA/R8 NPs was a good oral insulin delivery system, which might also be suitable for other protein drugs.

Optimization of chitosan-coated W/O/W multiple emulsion stabilized with Span 80 and Tween 80 using Box–Behnken design

The present study aimed to optimize insulin-loaded chitosan-coated W/O/W multiple emulsions (MEs) by investigating the effect of process variables on the response using Box–Behnken design. Effect of three independent factors, which are, the concentration of the lipophilic surfactant (Span 80) in the primary emulsion, the concentration of the hydrophilic surfactant (Tween 80) in the secondary emulsion, and phase volume ratio (W/O) in the primary emulsion, was studied on three dependent responses, which are particle size, zeta potential, and entrapment efficiency. The optimized ME showed particle size of 193.7 nm and −64.58 mV zeta potential with maximum entrapment efficiency of 91.84%. The in vitro drug release profile from W/O/W ME was studied under two simulated physiological conditions. In vitro drug release behavior followed the Peppas–Sahlin model and showed an initial and rapid release followed by a slower release. The results of the present investigation suggest the potential of the chitosan-coated W/O/W MEs as a promising oral drug delivery system for insulin.

Fabrication and characterization of a novel semi-interpenetrating network hydrogel based on sodium carboxymethyl cellulose and poly(methacrylic acid) for oral insulin delivery.

In this research, pH-sensitive semi-interpenetrating polymer network hydrogels based on sodium carboxymethyl cellulose and poly(methacrylic acid) were synthesized using free radical polymerization and semi-interpenetrating polymer network approach for oral administration of insulin. The chemical structure and thermal stability of the hydrogels were characterized using Fourier transform infrared spectroscopy, X-ray diffraction, and thermogravimetric analysis measurements. The interior morphology was observed by scanning electron microscopy and the inner structure exhibited a porous honeycomb-like shape. The investigations on the swelling properties of hydrogels revealed their ability to response to pH value change. The in vitro release behavior of insulin was pH dependent and the release of insulin was much lower at pH 1.2 compared to pH 6.8. In vitro cytotoxicity assay indicated that the hydrogels were noncytotoxic to HeLa cells. A sustained reduction in blood glucose level was observed after oral administration of insulin-loaded hydrogel to diabetic rats at 75 IU/kg. These results indicated that the hydrogel would be a promising vehicle for oral insulin delivery systems.

Biodistribution and pharmacokinetics of thiolated chitosan nanoparticles for oral delivery of insulin in vivo

To improve the quality of life of diabetic patients, oral delivery of insulin would be better than subcutaneous injection, and the encapsulation of insulin for its oral delivery is a promising alternative one. In this study, we prepared an oral insulin delivery system using thiolated chitosan nanoparticles (TCNPs) loaded with insulin (Ins) and tested under in vitro and in vivo systems. TCNPs prepared from CS and pentaerythritol tetrakis (3-mercaptopropionate) (PETMP) at 4:1 ratio showed 220 ± 4 nm, 2.3 ± 1 mV, and 119 ± 4 μmol g−1 in their size, charge and sulfhydryl content, respectively. There was a sustained release of insulin from the TCNPs at pH 5.3. TCNPs treatment did not alter cell viability in vitro and oral administration of TCNPs reached over the tip of the microvilli near the intestinal mucosa in vivo. There were increased and decreased the levels of insulin and glucose in the blood, respectively when Ins-TCNPs were orally administered in the diabetes induced rats. Thus, our results suggested that the insulin stays significantly for a prolonged period to make bio-distribution and bioavailability due to its interaction with the mucus of the intestine, thus offering a better oral insulin delivery system for diabetic patients.

Chitosan complexed carboxymethylated iota-carrageenan oral insulin particles: Stability, permeability and in vivo evaluation

We previously reported that insulin-entrapped chitosan complexed carboxymethylated iota-carrageenan (CS/CMCi) particles exhibit pH-responsive swelling behavior. However, the particles’ stability in the enzymatic gastrointestinal environment, their drug permeability mechanism, and related in vivo studies have not been discussed to date. In this study, we investigated the stability, muco-adhesiveness, transport mechanism and in vivo assessment of the particles. The particles retained their bioactivity and displayed a generally stable behavior in the simulated enzymatic environment of the gastrointestinal tract with high muco-adhesiveness (79.1 ± 4.3%). The results of cellular membrane permeability experiments further suggested that insulin from the insulin-entrapped particles was transported across the Caco-2 cell monolayers mainly via the paracellular pathway. This activity was inferred by the trans-epithelial electrical resistance (TEER) and the apparent permeability coefficient (Papp) of the insulin-entrapped particles (22-fold greater than control insulin solution), suggesting that the opening of tight junctions (TJs) of Caco-2 cells was involved in the process. The particles did not exhibit significant cytotoxicity at 0.5–10.0 mg/mL based on 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salts (MTS) and lactate dehydrogenase (LDH) assays. Additionally, an in vivo study with diabetic Sprague Dawley (SD) rats revealed an extended blood glucose-lowering effect for up to 36 h (Cmax: 175.1 ± 23.7 mIU/L, Tmax: 5 h, AUC: 1789.4 ± 158.6). The estimated bioavailability of insulin from CS/CMCi particles in humans was 44.7–46.9%, which may be increased three fold compared with rats. Thus, the above results support the effectiveness of chitosan-complexed carboxymethylated iota-carrageenan particles as an oral insulin delivery system for extended glycemic control in basal insulin therapy.

Cp1-11 peptide/insulin complex loaded pH-responsive nanoparticles with enhanced oral bioactivity

To improve the oral efficiency of insulin, a novel oral insulin delivery system was developed, to protect the insulin from destruction, and to deliver monomeric insulin with higher bioactivity. The oral insulin delivery system was developed by using chitosan/alginate nanoparticles as carriers for the oral delivery of Cp1-11 peptide/insulin complex (Cp1-11 peptide/Insulin Loaded Nanoparticle, CILN). There is a supramolecular interaction between the insulin and the Cp1-11 peptide, which could inhibit insulin aggregation and improve its bioactivity. In vitro release studies showed that the pH-responsive CILN system could retain insulin in a simulated gastric buffer solution, and exhibited sustained release of the insulin in simulated intestinal buffer solution. The stability studies indicated that CILN could also protect insulin against degradation by proteases in the gastrointestinal tract. Moreover, the insulin oral delivery system appeared to show excellent hypoglycemic effect, and led to higher pharmacological availability of insulin, compared with free insulin loaded nanoparticles, in diabetic rats. Thus, this study has suggested that insulin complexed with Cp1-11 peptide, followed by encapsulating in chitosan/alginate nanoparticles, providing an effective strategy for an oral insulin delivery system to treat diabetes in the future.

Oral delivery of insulin via mesoporous carbon nanoparticles for colonic release allows glycemic control in diabetic rats

In this article, a new type of mesoporous carbon nanoparticles (MCN) was fabricated as a potential oral delivery system of insulin to reduce the adverse reactions by hypodermic injection. The mesoporous carbon nanoparticles-carried insulin (MCNI) was studied using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FT-IR) compared with the blank MCNs. The Brunauer–Emmett–Teller (BET) method was utilized to calculate the specific surface area. The pore volume and pore size distribution (PSD) curves were calculated by Barrett–Joyner–Halenda (BJH) model. The entrapment efficiency (EE%) and loading content (LC%) of insulin onto the MCNs were determined by RP-HPLC. In vitro insulin release from MCNI was determined in simulated intestinal fluid. To evaluate the pharmacodynamics of MCNIs orally, the variation of glycemia of diabetic rats after oral administration of MCNIs was compared with the rats receiving hypodermic injection of insulin. Besides, the absorption of FITC-labeled MCNs in HCT-116 cells was tested. The results showed that there is significant difference between MCNs and MCNIs through SEM, TEM, and FT-IR. The entrapment efficiency, loading content and in vitro insulin release met the requirements of the pharmacodynamic study. The specific surface area, pore volume and pore size of MCNIs were significantly decreased compared to that of MCNs. The pharmacodynamics study showed that the blood sugar level was significantly decreased after the oral administration of MCNIs. The FITC-labeled MCNs showed significant absorption in HCT-116 cells. The MCNIs were successfully synthesized with commendable entrapment efficiency and loading content which preferably decreased the blood sugar in diabetes rats via oral administration.

ORAL DRUG DELIVERY OF INSULIN IN DIABETES MELLITUS: AN ATTRACTIVE ALTERNATE TO OVERCOME INVASIVE ROUTE

The subcutaneous injection of insulin for the treatment of diabetes mellitus can lead to patient non-compliance, discomfort, pain and local infection is a chronic metabolic health disease affecting the homeostasis of blood sugar levels in human beings. Oral route of drug delivery system has been the most widely accepted means of drug administration other than invasive drug delivery systems. For the development of an oral insulin delivery system, we have to focus on overcoming the various gastro-intestinal barriers for insulin uptake from the gastrointestinal tract. To overcome these barriers various types of formulations such as insulin conjugates, micro/nanoparticles, liposomes, hydrogel, capsule, and tablets are designed to deliver insulin orally. Various potential ways to administer insulin orally has been explored over years but a fluctuating level of insulin release have been recorded. A number of advancement has taken place in the recent years for understanding the needs of improved oral delivery systems of insulin. This review article concentrates on the challenges for oral drug delivery of insulin as well as various carriers used for the oral drug delivery of insulin and also provides the relevant information about the clinical tested formulations of oral insulin and its patents.

Self-assembly pH-sensitive chitosan/alginate coated polyelectrolyte complexes for oral delivery of insulin

Polyelectrolyte complexes (PEC) provide new opportunities for controlled release system of drugs, and have potentials to address challenges on the way to effective oral insulin delivery. Here, an innovative pH-sensitive PEC for insulin oral administration was developed, which was formed by self-assembly of two oppositely charged nanoparticles (chitosan-coated nanoparticles and alginate-coated nanoparticles) through electrostatic interaction via optimised double emulsion method. The encapsulation efficiency of insulin-loaded alginate-coated and chitosan-coated nanoparticles were 81.5 ± 7.4% and 55.2 ± 7.0%, respectively, and the particle size of these nanoparticles were in 200–300 nm range. The pH-dependent morphology of PEC was observed by transmission electron microscopy. The PEC exhibited insulin release profile triggered by pH in vitro and was non-cytotoxicity against Caco-2 cell. The insulin-loaded PEC could decrease blood glucose levels effectively and prolong insulin release after oral administration to diabetic rats. The results illustrated that the as-prepared PEC may be employed as a potential oral insulin delivery system.

Enzymatically cross-linked arabinoxylan microspheres as oral insulin delivery system

Arabinoxylans (AX) microspheres with different insulin/AX mass ratio were prepared by formation of phenoxy radical issued from the ferulic acid by enzymatic oxidation (entrapped in situ of insulin). Phenolic acid content and FT-IR spectrum of unloaded and insulin-loaded AX microspheres revealed that the phenoxy radical issued from the ferulic acid by enzymatic oxidation did not interact covalently with insulin. The microspheres showed a spherical shape, smooth surface and an average diameter of particles of 320 μm. In vitro control release found that AX microspheres minimized the insulin loss in the upper GI tract, retaining high percentage (~75%) of insulin in its matrix. The stability of the secondary structure of insulin was studied by dichroism circular (CD). The CD spectra of insulin released from AX microspheres did not change according to the insulin/AX mass ratio of the microsphere. Significant hypoglycemic effects with improved insulin-relative bioavailability tested on an in vivo murine model revealed the efficacy of these enzymatically cross-linked arabinoxylans microspheres as a new oral insulin carrier.

Glucose-responsive oral insulin delivery for postprandial glycemic regulation

Controlling postprandial glucose levels for diabetic patients is critical to achieve the tight glycemic control that decreases the risk for developing long-term micro- and macrovascular complications. Herein, we report a glucose-responsive oral insulin delivery system based on Fc receptor (FcRn)-targeted liposomes with glucose-sensitive hyaluronic acid (HA) shell for postprandial glycemic regulation. After oral administration, the HA shell can quickly detach in the presence of increasing intestinal glucose concentration due to the competitive binding of glucose with the phenylboronic acid groups conjugated with HA. The exposed Fc groups on the surface of liposomes then facilitate enhanced intestinal absorption in an FcRn-mediated transport pathway. In vivo studies on chemically-induced type 1 diabetic mice show this oral glucose-responsive delivery approach can effectively reduce postprandial blood glucose excursions. This work is the first demonstration of an oral insulin delivery system directly triggered by increasing postprandial glucose concentrations in the intestine to provide an on-demand insulin release with ease of administration.

Solvent-free synthesis of acetylated cashew gum for oral delivery system of insulin

Cashew gum (CG) is a biopolymer that presents a favorable chemical environment for structural modifications, which leads to more stable and resistant colloidal systems. The gum was subjected to an acetylation reaction using a fast, simple, solvent-free and low cost methodology. The derivative was characterized by infrared and NMR spectroscopy, elemental analysis, coefficient of solubility and zeta potential. The modified biopolymer was used as a platform for drug delivery systems using insulin as a model drug. Nanoparticles were developed through the technique of polyelectrolytic complexation and were characterized by size, surface charge, entrapment efficiency and gastrointestinal release profile. The nanoparticles presented size of 460 nm with a 52.5% efficiency of entrapment of insulin and the electrostatic stabilization was suggested by the zeta potential of + 30.6 mV. Sustained release of insulin was observed for up to 24 h. The results showed that acetylated cashew gum (ACG) presented potential as a vehicle for sustained oral insulin release.

In Silico Study of Phospholipids as An Oral Insulin Delivery System

Phospholipids have been applied as a material for an oral insulin delivery system that either in the form of solid lipid nanoparticles, liposomes, or microemulsions. However, the administration of this delivery system for diabetic treatment requires twenty times higher of dosage than the injection one. In the present study, we performed molecular dynamics simulation to evaluate how phospholipids can protect insulin at the molecular level and why its efficiency for the diabetic treatment is low. Phosphatydilcholine group, such as DPPC, OPPC, LPC and combine of DPPC-LPC, was used as the phospholipid material to model the delivery system in our simulations because in vitro and in vivo studies of these phospholipids as the delivery system have been well-conducted. In these simulations, the waters-phospholipid-insulin system was prepared by randomly mixing phospholipids molecules inside the solvent box containing an insulin molecule. NPT ensemble was used in all simulations with the setting temperature at 310 K and 1 atm for the pressure. The simulation results showed that DPPC and LPC delivery systems were able to maintain both secondary and tertiary structures of the encapsulated insulin within the time range of simulation. However, all phospholipid delivery system models failed to fully encapsulate insulin surface within 150 ns simulation. This study may explain the reason for the low success rate of using phospholipid as the delivery system of oral insulin in the experiment.

Preparation and characterization of hydroxyapatite nanoparticles carrying insulin and gallic acid for insulin oral delivery

Although nanoparticles carriers for oral delivery of insulin have been researched for many years, this method still fails to solve issues with toxicity, biocompatibility, and degradability in the organism. We therefore developed an innovative conjugation system to solve this problem. Nano hydroxyapatite (HAP) particles were used as the core, then polyethylene glycol (PEG) was wrapped onto the surface of hydroxyapatite, and, finally, insulin (INS) and gallic acid (GA) were conjugated with PEG. PEG functionalized HAP was increased the hydrophilicity of the nanoparticles, also protected them from degradation in the gastrointestinal (GI) tract. Most importantly, the in vivo absorption of nanoparticles in rat small intestines revealed that HAP-PEG-GA-INS was absorbed by the small intestine epithelium. The blood glucose of the type 1 diabetes (T1D) rats that were given intragastrically HAP-PEG-GA-INS showed an obvious downward trend. Overall, we synthesized a safe, non-toxic, and effective oral insulin delivery system.

In-vitro and in-vivo cytotoxicity and efficacy evaluation of novel glycyl-glycine and alanyl-alanine conjugates of chitosan and trimethyl chitosan nano-particles as carriers for oral insulin delivery

PurposeThe aim of this research work was to explore the possibility of providing multifunctional oral insulin delivery system by conjugating several types of dipeptides on chitosan and trimethyl chitosan to be used as drug carriers. MethodConjugates of Glycyl-glycine and alanyl-alanine of chitosan and trimethyl chitosan (on primary alcohol group of polymer located on carbon 6) were synthesized and nanoparticles containing insulin were prepared for oral delivery. Preparation conditions of nanoparticles were optimized and their performance to enhance the permeability of insulin as well as cytotoxicity of nanoparticles in Caco-2 cell line was evaluated. To evaluate the efficacy of orally administered nanoparticles, nanoparticles with the most permeability enhancing ability were studied in male Wistar rats as animal model by measuring insulin and glucose Serum levels. ResultStructural study of all the conjugates by infrared spectroscopy and nuclear magnetic resonance confirmed the successful formation of the conjugates with the desirable substitution degree. By optimizing preparation conditions, nanoparticles with expected size (157.3–197.7 nm), Zeta potential (24.35–34.37 mV), polydispersity index (0.365–0.512), entrapment efficiency (70.60–86.52%) and loading capacity (30.92–56.81%), proper morphology and desirable release pattern were obtained. Glycyl-glycine and alanyl-alanine conjugate nanoparticles of trimethyl chitosan showed 2.5–3.3 folds more effective insulin permeability in Caco-2 cell line than their chitosan counterparts. In animal model, oral administration of glycyl-glycine and alanyl-alanine conjugate nanoparticles of trimethyl chitosan demonstrated reasonable increase in Serum insulin level with relative bioavailability of 17.19% and 15.46% for glycyl-glycine and alanyl-alanine conjugate nanoparticles, respectively, and reduction in Serum glucose level compared with trimethyl chitosan nanoparticles (p < 0.05). ConclusionIt seems that glycyl-glycine and alanyl-alanine conjugate nanoparticles of trimethyl chitosan have met the aim of this research work and have been able to orally deliver insulin with more than one mechanism in animal model. Hence, they are promising candidates for further research studies.

Preparation and Characterization of Hypoglycemic Nanoparticles for Oral Insulin Delivery.

Polymeric nanoparticles have been widely investigated as insulin delivery systems for oral administration. However, the toxic nature of many artificial polymers hampers their effective application, creating a demand for the further exploration of alternative natural polymers. In addition, ethnobotanical research has reported that over 800 plant species have a hypoglycemic function, some of which are polymers. For the advantages of both areas to be combined, the aim of this work was to choose an organic hypoglycemic polymer and prepare it into an insulin carrier to build a dual-functional oral insulin delivery system. We found that the insulin loading rate, release mode, thermostability, and both in vitro and in vivo absorption and efficacy varied with the different modifications of polygalacturonic acid (PGLA) nanoparticulate backbones. By in vivo pharmaceutical testing and constantly monitoring the symptoms of type 1 diabetic (T1D) rats, we ascertained the hypoglycemic function of the nanoparticles and showed that overall diabetic symptoms were ameliorated after the long-term daily administration of nanoparticles with no significant damage to organ structure or cell viability.

Controlled swelling and in vitro release of insulin from konkoli grafted polymethylacrylaminde hydrogel

The extended study of konkoli grafted polymethylacrylaminde (KG-g-poly (MAAm) hydrogel synthesized in our earlier work from methacrylamide (MAAm) grafted and N,N-methylenebisacrylamide (N,N-MBAAm) crosslinked Konkoli ( Maesopsis eminni ) galactomannan (KG), is necessary to establish its potential application as an oral insulin delivery system. In this study, the kg-g-poly (MAAm) Hydrogel swelling in typical pH media show an initial rapid swelling before stabilizing. The hydrogel attains its parabolic peak swelling in distill water (pH = 7). Swelling rises from a lower pH medium (pH = 2.2), and in a medium with a slight pH above this (pH = 7.4), swelling starts dropping. Comparing the media for the insulin release studies, the cumulative release drops to its parabolic trough in the distill water medium (pH = 7). A higher cumulative release was recorded at a lower pH (pH = 2.2), and the highest cumulative release was recorded at a higher pH (pH = 7.4). The kinetics and mechanisms of these processes were presented while using the descriptive statistic of regression to fit the results from the processes to a power law. The release of insulin from kg-g-poly (MAAm) Hydrogel was also shown to increase with increase in glucose concentration in the media with an initial rapid release in all solutions, while in a typical intestinal pH media range, higher release at all concentrations was recorded at pH = 6.8.