Metformin-Loaded Hyaluronic Acid Nanostructure for Oral Delivery

AAPS PharmSciTech ( # 2018) DOI: 10.1208/s12249-018-1085-1 Research Article Metformin-Loaded Hyaluronic Acid Nanostructure for Oral Delivery Sonal Bhujbal1 and Alekha K. Dash1,2 Received 11 April 2018; accepted 24 May 2018 The objective of this study was to develop a nanodelivery system containing a Abstract. mucoadhesive polymer hyaluronic acid (HA) for oral delivery. Metformin was used as a model drug. Blank and drug-loaded HA nanostructures were prepared by precipitation method and characterized for particle size (PS), zeta potential (ZP), physical stability (over 65 days), surface morphology, moisture content, and physical state of the drug in the nanostructures. The cytotoxicity and hemolysis potential of the delivery system was assessed in Caco-2 cells and whole human blood, respectively. The in vitro release of metformin and its uptake in Caco-2 cells was evaluated using high-performance liquid chromatography. Ex vivo permeability of metformin was measured through goat intestinal membrane. The nanoparticles were physically stable and neutrally charged with an average PS of 114.53 ± 12.01 nm. This nanodelivery system existed as nanofibers containing metformin in a crystalline state. This delivery system released the drug rapidly with > 50% of metformin released within 1 h. Cellular uptake studies on Caco-2 cells indicated higher uptake of metformin from nanoparticle as compared to metformin in solution, up to first 45 min. Ex vivo permeability studies on the other hand showed a higher metformin permeability from solution relative to that from nanoparticles through the goat intestinal membrane. Metformin nanoparticles were non-toxic at therapeutic concentrations in Caco-2 cells and showed no hemolytic effect to RBCs. This study indicates the preparation, characterization, as well as the potential use of HA nanostructures for oral delivery. KEY WORDS: hyaluronic acid; nanostructure; oral delivery; metformin; permeability. INTRODUCTION Globally, an increasing prevalence of people stricken with diabetes occurred with an estimated 422 million adults, living with diabetes in 2014, as compared to 180 million in 1980. The global prevalence of diabetes nearly doubled since 1980, rising from 4.7 to 8.5% in the adult population. In addition, diabetes caused 1.5 million deaths in 2012 (1). In 2014, 29.1 million people in the USA (9.3% of the population) had diabetes and 8.1 million people were undiagnosed (2). Type 2 diabetes is the most common type of diabetes which accounts for 90–95% of those with diabetes around the world (3). It is a chronic condition that results from the body’s ineffective use of insulin and affects the way the body processes blood sugar (glucose) because of beta cell deficiency coupled with peripheral insulin resistance (4). Type 2 diabetes used to occur nearly entirely among adults, but now can be seen in children as well (1). It is usually controlled by 1 Department of Pharmacy Sciences, School of Pharmacy and Health Professions, Creighton University, 2500 California Plaza, Omaha, Nebraska 68178, USA. 2 To whom correspondence should be addressed. (e–mail: [email protected]) oral drug therapy and may require insulin therapy when oral drug therapy alone fails to control the blood glucose level (5). Metformin is generally recommended as a first-line treatment for type 2 diabetes. The glucose-lowering effect of metformin is the result of the drug action on liver, muscle, and adipose tissues. Effect of metformin on hepatic glucose production is considered to be dominant. Explanations offered for its hypoglycemic action are suppressing of hepatic gluconeogenesis and glucose output from liver and enhancement of insulin-mediated glucose disposal in muscle and adipose tissues (6). It seems to alter the location of GLUT4 transport from the intracellular site to plasma membrane (7). In addition, metformin lowers the blood sugar level by increasing glucose uptake in peripheral tissues. Also, it inhibits intestinal glucose absorption by increasing anaerobic glucose metabolism, i.e., by stimulating glycolysis. It is important to note that it does not stimulate insulin secretion (8). Hence, metformin requires insulin for its action and acts as an anti-hyperglycemic agent and does not cause hypoglycemia. Metformin has a low oral bioavailability (50–60%). Despite its high solubility (50 mg/mL in water), metformin’s oral bioavailability is limited due to its saturable and poor intestinal absorption (9). Active transport is the primary mechanism of transport for metformin (10). Passive diffusion 1530-9932/18/0000-0001/0 # 2018 American Association of Pharmaceutical Scientists Bhujbal and Dash through cells has been reported to be low (11). The high solubility and poor absorption properties have led to classification of metformin as a Biopharmaceutics Classification System (BCS) class III drug (12). Hence, the objective of this study was to develop an oral formulation of metformin, which could enhance its absorption rate across the intestinal membrane. Development of such formulation could reduce the drug dose and the associated side effects of metformin. Previous studies have indicated that it is possible to prepare polymeric nanoparticles in such a manner that they can increase the intestinal absorption of a drug by offering mucoadhesion, permeation enhancing, or sustained release characteristics leading to an increase in the intestinal absorption and thereby increasing the oral bioavailability of the drugs (13). For example, cyclosporine-loaded poly lactic-coglycolic acid (PLGA) nanoparticles showed sustained release of the drug in the plasma and a decrease in the cyclosporineassociated nephrotoxicity. However, these two characteristics were absent in the commercial formulation (Sandimmune Neoral) (14, 15). Chitosan nanoparticles have been shown to exhibit an ability to increase the permeability of drugs (16). Sarmento et al. found that mucoadhesive alginate/chitosan nanoparticles could augment oral absorption and oral bioactivity of insulin (17). Such nanoparticles, with a higher surface-to-volume ratio than that of microparticulate systems, can significantly increase their cellular contact and/or provide greater area for dissolution and hence increase the absorption, thereby increasing the bioavailability. It has also been found that PLGA polymeric nanocarriers have the ability to improve oral bioavailability of drugs with poor, enzymatic or metabolic stability like estradiol (18) and atorvastatin (19). Although there is literature indicating the absorption enhancement capability of nanoparticles for certain drugs, studies indicating the advantage of these nanoparticulate systems for enhancing the absorption of BCS class III drugs like metformin are limited. Therefore, the present research reports the development and characterization of polymeric nanostructures containing metformin with mucoadhesive hyaluronic acid as a polymer that may increase the intestinal absorption of the drug thereby increasing its oral bioavailability. MATERIALS AND METHODS Materials Metformin (commercial grade) was purchased from BioVision, Inc. (San Francisco, CA). Sodium hyaluronate (MW = 1.0 × 106 Da) samples were donated by Bloomage Freda Biopharm Co, Ltd. (Jinan, China). Adipic acid dihydrazide (ADH), 1-[3-(dimethylamino) propyl]-3 ethylcarbodiimide hydrochloride (EDC), Optima grade acetone, and acetonitrile for HPLC analysis were purchased from Fischer Scientific (Fair Lawn, NJ). Both ADH and EDC were reagent grade, acetone was Optima grade, and acetonitrile was of HPLC grade. Monobasic potassium phosphate (reagent grade) was purchased from Spectrum Chemical Mfg Corp (Brunswick, NJ). Phenomenex Luna C18—5-μm (250 × 4.6 mm) column was purchased from Phenomenex (Torrance, CA). Caco-2 cells were purchased from American Type Culture Collection (Manassas, VA). Dulbecco’s modified Eagle’s media (DMEM), Roswell Park Memorial Institute (RPMI) medium, trypsin, penicillin/streptomycin, L-glutamine, sodium pyruvate, and non-essential amino acids (all reagent grade) were purchased from Cellgro Mediatech Inc. (Corning, NY). MTT reagent, dibasic potassium phosphate, sodium dodecyl sulfate (SDS), sodium bicarbonate, magnesium chloride, and dimethylformamide (DMF) were purchased from Sigma Aldrich (St. Louis, MO) and were reagent grade. Fetal bovine serum (FBS) albumin was purchased from Atlanta Biologicals (Lawrenceville, GA). Whole human blood was obtained commercially from Innovative Research Inc. (Novi, MI). This blood was collected from a 55-year-old Hispanic male. PAMPA plates (donor and acceptor) were obtained from Millipore Corporation (Billerica, MA). Preparation of the Nanoformulation Blank and drug-loaded nanoformulations were prepared by suitable modification and optimization of previously published precipitation method (20). Briefly, 1% (w/v) sodium hyaluronate solution was prepared by overnight hydration of 750 mg sodium hyaluronate in 75 mL filtered and deionized water. EDC (34 mg) was added to the hyaluronic acid solution and stirred on magnetic stirrer at 100 rpm for 30 min. Metformin hydrochloride (200 mg) was added to the above mixture and stirred on magnetic stirrer at 100 rpm. Finally, ADH (30 mg) was added and the mixture was probe-sonicated at 50 W in pulse mode (on time 1 s, off time 0.5 s) for 2 h to ensure complete crosslinking. The cross-linked aqueous phase was added to 225 mL acetone (aqueous phase:organic phase; 1:3 v/v) with continuous magnetic stirring. The colloidal dispersion was centrifuged at 13,000 rpm and at 4°C for 10 min to separate the nanoparticles. The organic solvent was evaporated using a rotatory evaporator and finally lyophilized using a Millrock LD85 freeze dryer (Millrock Technology, NY). The freeze-drying protocol consisted of a slow freeze from room temperature to − 50°C over 240 min under minimal vacuum followed by a final freeze to − 60°C for 20 min at 200 mTorr. Primary drying consisted of a ramp from − 20 to + 20°C at 50 mTorr with 240-min hold for each 10°C and 30min ramps to each hold temperature. Samples were held at 20°C for 480 min at 0–10 mTorr in the secondary drying step. Following the secondary drying, samples were cooled to 5°C and 0–10 mTorr and held until the cycle was completed. Characterization of the Nanoformulation The particle size (PS) and the zeta potential (ZP) were determined for nanosuspensions as well as nanoparticles obtained after freeze-drying using a Brookhaven zetameter (ZetaPlus, Brookhaven Instruments Corporation, Holtsville, NY). For PS measurement, 2 mL of nanosuspension was diluted with 8 mL of acetone. In case of nanoparticles, 2 mg of nanoparticles was suspended in 10 mL of the solvent system containing 2 mL of deionized water and 8 mL of acetone. For ZP measurement, 5 mg of nanoparticles was suspended in 10 mL of deionized water. The physical stability of blank as well as metformin-loaded nanoparticles was Hyaluronic Acid Nanostructure for Oral Delivery determined by measuring PS and ZP of the nanoparticles over a period of 65 days stored at room temperature (25°C) and in a container containing desiccant (Drierite) at 0% relative humidity. Atomic force microscopy (AFM) was performed to study the morphology of blank and metformin-loaded nanoparticles. AFM images in air were acquired using MultiMode AFM NanoScope IV system (Bruker Instruments, Santa Barbara, CA) operating in tapping mode with a 1.5-Hz scanning rate. TESPA probes from Bruker with a resonance frequency between 300 and 320 kHz were used. A previously established HPLC method (21) was suitably modified and validated to analyze metformin content of nanoparticle in the release media (phosphate buffer, pH 7.4). Chromatographic separation of metformin was achieved by isocratic elution on Phenomenex Luna C18, 5-μm (250 × 4.6 mm) column. The mobile phase composition was acetonitrile:potassium phosphate buffer in water (40:60 v/v). The flow rate of 1 mL/min was maintained for a total run time of 6 min. The column effluents were monitored at a detector wavelength of 252 nm. The drug content analysis was performed using HPLC. Briefly, 7.5 mg of metformin-loaded nanoparticles was dispersed in 9 mL of potassium phosphate buffer (pH 7.4) and stirred for 24 h by magnetic stirrer at 100 rpm. The dispersion was centrifuged at 13,000 rpm for 10 min at 4°C to separate the polymer matrix and the supernatant was analyzed for metformin content. Differential scanning calorimetry (DSC) studies were performed on sodium hyaluronate, metformin, and blank and drug-loaded nanoparticles using DSC (Shimadzu, DSC 60, Kyoto, Japan). About 3 to 5 mg of each sample was crimped in an aluminum pan, which was treated as the sample pan and was analyzed from room temperature to 300°C at a rate of 10°C/min with nitrogen purge (flow rate of 20 mL/min). X-ray diffraction (XRD) analysis was performed on nanoparticles using a powder X-ray diffractometer (D8 ADVANCE; Bruker AXS, Madison, WI) equipped with Si strip onedimensional detector (LynxEye). Samples were exposed to Cu Kα radiation (40 kV × 40 mA) over an angular range of 5– 40° 2θ with a step size of 0.02° and a dwell time of 0.5 s. The moisture content of the nanoformulation was determined by Karl Fisher titrimetry. Percent moisture content was determined for the known weight (25–30 mg) of the sample. Thermogravimetric analysis (TGA) was used to determine weight loss as a function of temperature for nanoparticles (Shimadzu thermogravimetric analyzer, TGA 50, Kyoto, Japan). About 3–5 mg of sample was filled into aluminum pans and heated from room temperature to 300°C at a rate of 10°C/min with nitrogen purge (flow rate of 20 mL/ min). The percent weight loss was analyzed from 25 to 120°C. In vitro Release Studies Dialysis tubing (Biotech CE DialyBiotech CE Dialysis Tubing, 1.0 kD MWCO, 16-mm flat-width) was used as sample holder. Twenty milligrams of nanoparticles dispersed in 2 mL of phosphate buffer (pH 7.4) solution was introduced into a dialysis bag. The dialysis bag was sealed from both ends and introduced in 17 mL phosphate buffer solution in a glass scintillation vial. The release assemblies were placed into an incubated shaker at 37°C. Samples (100 μL) were collected at definite time intervals and replaced with equal volume of phosphate-buffered solution. The drug content in the release medium was determined using the HPLC method described earlier. MTT Toxicity Assay Cytotoxicity of metformin solution and nanoparticles was determined in Caco-2 cells (epithelial cells from colon tissue) using MTT assay. Cells were seeded with 100-μL media/well into 96 well culture plates and incubated overnight in a humidified chamber with 5% CO2 atmosphere at 37°C. After 24 h, cells were treated with different concentrations of the samples being tested. Treatments were applied on top of existing media in equal volume to the media in wells (100:100 μL) and allowed to incubate for 8 h. The treatments and media were removed, 100 μL fresh media was added to each well, and plates were returned to the incubation chamber for 24 and 72 h. Cells were treated with 30 μL solution of MTT reagent (5 mg/mL) in phosphate-buffered solution (pH 7.4) and incubated for additional 4 h. The treatment was removed and a solution of 20% (w/v) SDS:DMF in 1:1(v/v) ratio was used to lyse the cells and solubilize the formazan crystals formed. The plates were mixed on an incubated shaker for 1 h at 37°C and absorbance was analyzed at 540 nm using a plate reader. Hemolysis Assay RBCs were isolated from whole human blood and washed with 0.9% (w/v) normal saline until a clear supernatant was obtained. The RBCs were diluted with 1:10 (v/v) with normal saline and tested for hemolysis with drug solution (500 μM), drug-loaded hyaluronic acid nanoparticles (500 μM), and blank nanoparticles (amount equivalent to drug-loaded nanoparticles). The treatment volume was 200 μL of the RBC solution and 800 μL of the test solution. The treated RBCs were incubated for 1 h and centrifuged at 13,000 rpm for 5 min on the microcentrifuge. The supernatant was collected and was analyzed on a microplate reader. The percent hemolysis was calculated as a mean of two wavelengths (404 and 540 nm) using the following formula: %Hemolysis ¼ Sample absorbance‐Blank absorbance  100 absorbance of positive control‐Blank absorbance Determination of Cellular Uptake Caco-2 cells were cultured in a standard Falcon™ 24-well tissue culture plates at seeding density of approximately 3 × 105 cells per well. Confluent cell monolayers were treated with a single dose of 500 μL of metformin solution (1000 μM) or nanoparticles loaded with equivalent metformin in RPMI. The cells were washed thrice with ice cold RPMI and lysed with 200 μL of 1% (v/v) Triton X-100. Cell lysates were collected in microcentrifuge tubes, and 25 μL of lysate was assayed for total cellular protein content using bicinchoninic acid protein assay (Pierce, Rockford, IL). Remaining portion of lysate was centrifuged at 13,000 rpm for 10 min at 4°C and Bhujbal and Dash the supernatant was analyzed for metformin content by HPLC. Drug Permeability Study Sections of goat gastrointestinal tissues were slowly washed with a large amount of 0.9% sterile phosphate buffer (pH 7.4) and were mounted between the donor and receiver compartments of the diffusion chambers (PermeGear Inc., Hellertown, PA). Three milliliters of phosphate buffer (pH 7.4) was placed in the receiver compartment. A stock solution of 1000 μM metformin solution or nanoparticle dispersion with equivalent amount of metformin was prepared in phosphate buffer, (pH 7.4) and placed in the donor compartment. Samples (200 μL) were collected from the receiver compartment and replaced with equal volume of fresh media at specific time points (up to 120 min). These samples were subsequently analyzed using HPLC method. The apparent apical to basolateral permeability coefficients (Papp), expressed in centimeter per second, were calculated by using the following Eq. 1 (22): Papp ¼ dQ 1  dt AC0 ð1Þ where dQ/dt is the rate of appearance of drugs on the basolateral side (mmole s −1 ), C 0 is the initial drug concentration on the apical side (mM), and A is the area of the goat intestinal membrane (cm2). Statistical Data Analysis The experimental data was statistically analyzed for the purpose of comparison using Student’s t test using Microsoft Excel 2016. The differences were termed statistically significant at P < 0.05. RESULTS AND DISCUSSION Preparation of Nanoformulation Hyaluronic acid was the polymer of choice because firstly, along with exhibiting no cytotoxicity and being biocompatible, hyaluronic acid also exhibits mucoadhesive properties in an intestinal environment [21–23]. These mucoadhesive properties of hyaluronic acid were found to increase residence time of the formulation by producing an intimate contact with intestinal mucosa, possibly resulting in an increased bioavailability (24). Hyaluronic acid has also been associated with an increase in penetration across the Caco-2 cell monolayer (23), pointing to an occurrence of a deeper interaction between hyaluronic acid and the mucosal surfaces. Secondly, its solubility characteristics (soluble in water, insoluble in acetone) made it as an ideal choice for the nanoprecipitation method of nanoparticle synthesis. Lastly, hyaluronic acid nanoparticles could increase cellular uptake of metformin in intestinal epithelial cells via hyaluronic acidCD44 interaction (25). Thus, hyaluronic acid could be used as both matrix and ligand. Therefore, these nanoparticles would be of low cost in comparison with either IgA- or vitamin B12conjugated nanoparticles. EDC and ADH were used as coupling agent and crosslinking agent, respectively. Once metformin, hyaluronic acid, EDC, and ADH were dispersed in the water, carbodiimidemediated cross-linking leads to the formation of a crosslinked hyaluronic acid nanostructure. During the precipitation step, water diffused in acetone causing insoluble hyaluronic acid nanoparticles to precipitate. Metformin, which does not participate in the cross-linking reaction, is also insoluble in acetone, and hence is physically entrapped in the cross-linked hyaluronic acid molecules during this step. Characterization of Nanoformulations The average of PS and ZP measurements is represented in Table I. There was no significant (p < 0.05) increase in the PS of metformin-loaded nanoparticles after freeze-drying, which could be attributed to non-occurrence of aggregation during the process of freeze-drying. Metformin nanoparticles were well dispersed in water and in the size range of 114.53 ± 12.01 nm, which is estimated to be optimum for permeation across the intestinal epithelium (26). The PS data of physical stability analysis as represented in Fig. 1 indicated a significant increase (p < 0.05) in PS at certain time intervals, for both blank and drug-loaded nanoparticles. Throughout the tested period, PS remained < 200 nm and ZP of both blank and drug-loaded nanoparticles was found to be in neutral range (± 1 mV), suggesting that nanoparticles are physically stable over a period of 65 days. No change in PS is generally expected in case of solid nanoparticles. The reason behind this is that freeze-drying converts the liquid nanosuspension into solid nanoparticles, thereby ensuring that there is no Ostwald ripening, which generally is the major cause for physical instability in nanosystems (27). Ostwald ripening is a thermodynamically Table I. Particle Size, Polydispersity, and Zeta Potential of Metformin Nanoformulations Measurement Particle size (nm) Polydispersity index Zeta potential (mV) *Mean ± SD; n = 3 Blank nanoparticles Metformin-loaded nanoparticles Nanosuspensions (before freeze-drying)* Nanoparticles (after freeze-drying)* Nanosuspensions (before freeze-drying)* Nanoparticles (after freeze-drying)* 76.99 ± 10.24 0.24 ± 0.02 – 155.52 ± 18.70 0.30 ± 0.05 0.06 ± 0.34 178.89 ± 20.45 0.11 ± 0.03 – 114.53 ± 12.01 0.34 ± 0.01 − 0.14 ± 0.39 Hyaluronic Acid Nanostructure for Oral Delivery Fig. 1. Particle size as determined by Malvern ZetaPlus that determines particle size by dynamic light scattering for blank hyaluronic acid nanostructure (a) at room temperature (n = 3) and metformin nanoformulations (b) at room temperature (n = 3). Plus sign indicates significant increase driven spontaneous process that can be observed in solid solutions or dispersions. Smaller particles in solution dissolve and deposit on larger particles to reach a more thermodynamically stable state wherein the surface-to-area ratio is minimized. In general, the larger particles grow at the expenses of smaller particles. This shrinking of smaller particles and growing of larger particles results in a larger mean diameter of a particle size distribution. The results from the AFM studies are shown in Fig. 2, which indicated that particles were present in the form of nanofibers and were in good agreement with the results obtained from PS analysis using a Brookhaven zetasizer. The drug load and drug entrapment efficiency of metformin nanoparticles as determined by HPLC was 22.84 ± 0.74% (w/w) and 91.4 ± 1.29% (w/w), respectively. During nanoparticle preparation, hyaluronic acid nanoparticles were dispersed in an organic phase in which both hyaluronic acid and metformin were insoluble. The excess organic solvent was removed from the system by using a rotatory evaporator. Thus, there was minimal loss of the drug during the process resulting in a formulation with more than 90% of metformin content. This high metformin content could be due to drug entrapment as well as due to the presence of drug in free or adsorbed form in the system. The sharp endothermic peak in the DSC thermogram as shown in Fig. 3 at 231.5°C corresponds to the melting point of metformin. Blank and drug-loaded nanoparticulate formulations displayed diffused (broad) endothermic peaks around 100°C. To characterize these broad endothermic peaks obtained in DSC thermograms, weight loss of these formulations was analyzed up to a temperature of 120°C by TGA which showed a weight loss of 8.45 ± 0.72% and 7.20 ± 2.08% for blank and drugloaded nanoparticles, respectively. However, this weight loss could have been due to dehydration or desolvation. Therefore, Karl Fischer titration was performed which indicated moisture content of the blank and drug-loaded nanoparticles to be 13.85 ± 1.21% (w/w) and 6.81 ± 0.54% (w/w), respectively. As there was a good degree of agreement in the percent weight loss detected by TGA and percent moisture content determined by Karl Fischer titration, the broad endothermic peak in DSC thermograms of the formulations was confirmed to be from the moisture in the formulation. The metformin-loaded nanoparticles showed an endothermic peak around 234.2°C corresponding to the melting point of metformin and indicating that drug might be present in crystalline state. The physical state of the drug in the formulation was Bhujbal and Dash Fig. 2. Atomic force microscopic images obtained using a MultiMode AFM NanoScope IV system of blank hyaluronic acid nanostructure (dilution 5 times) (a), blank hyaluronic acid nanostructure (dilution 50 times) (b), metformin-loaded hyaluronic acid nanostructure (dilution 5 times) (c), and metformin-loaded hyaluronic acid nanostructure (dilution 10 times) (d) further confirmed by powder X-ray diffraction studies. The powder XRD patterns of blank as well as drugloaded nanoformulations are shown in Fig. 4. The drugloaded nanoformulations showed distinct metformin peaks which were absent in blank formulations. This confirms the crystalline state of metformin in drug-loaded nanoformulations. In vitro Drug Release The in vitro release profiles of metformin from the nanoparticles and metformin solution are shown in Fig. 5. The metformin release from nanoparticles was rapid as exhibited by high percent cumulative release (over 90% w/ w) in the first 2 h. The release of metformin from nanoparticles was observed over a period of 8 h after which the release seemed to be complete and had reached a plateau. The release of the drug from the particles was seen within 5 min of the beginning of the study. This quick release could be due to the presence of free as well as adsorbed drug on the surface of nanoformulation. MTT Toxicity Assay The MTT toxicity assay on Caco-2 cells as represented in Fig. 6 indicated that the metformin-loaded nanoparticulate system exhibited toxicity only at its highest tested concentration (1000 μM). The increased toxicity of metformin nanoparticles as compared to the toxicity of metformin solution or blank nanoparticles may be attributed to the higher accumulation of metformin within the Caco-2 cells. For metformin nanoparticles, percent cell death was significant (p < 0.05) only after 72 h of incubation, and not at 24 h, suggesting possible apoptosis caused by metformin in Caco-2 cells. Metformin solution and blank nanoparticles indicated no toxicity even at highest Hyaluronic Acid Nanostructure for Oral Delivery Fig. 3. Differential scanning calorimetric curves of sodium hyaluronate, metformin hydrochloride, blank nanostructure, and metformin-loaded nanostructure analyzed with blank nanoparticles as reference. Empty aluminum pans were used as reference for rest of the DSC studies concentrations up to 72 h. This suggests that the hyaluronic acid nanoparticulate formulation by itself is safe and metformin nanoparticles were safe at therapeutic concentrations (< 50 μM). Hemolysis Assay The results of hemolysis assay at two wavelengths of detection (404 and 540 nm) are shown in Fig. 7. Hemolysis by Fig. 4. An overlay plot of the powder XRD patterns of blank nanostructures, drug-loaded nanostructures, and the pure drug (metformin) Bhujbal and Dash Fig. 5. Comparison of the in vitro release characteristics of metformin from solution and hyaluronic nanostructure Triton X was used a positive control and normal saline as a negative control. The hemolysis caused by the drug solution and nanoparticles was lower than that of the vehicle control indicating no hemolysis from nanoparticles and drug solution. This study ensured that the formulation was safe in the event of its entry into the circulatory system. Additionally, it confirms that this nanodelivery system could be administered intravenously, if necessary. Fig. 6. Determination of cytotoxicity profile of metformin solution, blank nanostructure, and metformin-loaded nanostructure after 24-h incubation (a) and metformin solution, blank nanostructures, and metformin-loaded nanostructures after 72-h incubation (b). Plus sign indicates significant cell death Hyaluronic Acid Nanostructure for Oral Delivery Fig. 7. Hemolysis assay as determined by the percent hemolysis at two wavelengths of detection (404 and 540 nm) for drug solution and blank and drug-loaded nanostructures Cellular Uptake of Metformin The in vitro cellular uptake results as represented in Fig. 8 indicate that cellular uptake of metformin from the nanoparticulate system was significantly higher compared to that from drug solutions at p < 0.05, up to 45 min. The maximum metformin uptake from the nanoparticles was found to be at 30 min, after which it somewhat decreased with time. The trend observed during uptake studies showed a gradual increase, followed by decrease in the amount of drug uptake by the cells, with respect to time for the drugloaded nanoparticles, which could possibly be due to the rapid drug release from the nanoparticles (> 50% in 1 h). Permeability Studies The Papp values as shown in Table II indicate a higher permeability for the drug from metformin solution as compared to metformin nanostructures at all the three time points. A possible reason for this could be the rapid drug release from the nanostructure and their cellular uptake. Mucoadhesive polymers like hyaluronic acid are known to adhere and interact with the intestinal mucous layer and reversibly open the intercellular tight junctions (28). Although the pore of size of this intercellular space is relatively small for large particles like nanostructures to cross, however, small molecules like metformin can easily pass through this space (29). There is a possibility that metformin is being released from the nanostructure prior to the mucoadhesion and interaction of nanoparticles with the intestinal mucous layer. A layer of empty polymeric nanoparticles adhering to intestinal cell may act as additional barrier to the released drug and could contribute for low permeability of metformin from nanoparticles with respect to that from a solution. Studies with metformin have indicated a positive correlation between a sustained release formulation and metformin permeability from mucoadhesive particles (30). Further optimization of the formulation is required to obtain a sustained release of metformin which could possibly lead to an increased Fig. 8. Comparison of the cellular uptake of metformin from the solution and nanoparticulate system by Caco-2 cells at different time points Bhujbal and Dash Table II. Permeability of Metformin Through Goat Gastrointestinal Tissues 5. 6. Time (min) *Apparent permeability coefficient (Papp ×10–4 (cm/s)) 60 90 120 Metformin solution Metformin nanoparticles 17.18 42.60 68.08 5.05 10.17 33.87 7. 8. 9. *Papp as calculated using Eq. 1 10. permeability and subsequent bioavailability of metformin from the nanoparticulate system. CONCLUSION In conclusion, the current study dealt with the development and characterization of mucoadhesive hyaluronic acid nanostructures containing metformin for oral delivery. The nanostructures showed a uniform size distribution and high drug content. They were stable over a period of 2 months and were found to be non-toxic at therapeutic concentrations (≤ 100 μM) to Caco-2 cells. They were also compatible with RBCs and did not cause cell lysis. Metformin was released rapidly from the system, over 50% (w/w) in 1 h which could probably be a reason for low ex vivo permeability of metformin from hyaluronic acid polymeric system relative to metformin solution. Future studies need to be directed to sustain the release of metformin from such systems. ACKNOWLEDGEMENTS The authors would like to thank Dr. Alexey Krasnoslobodtsev (UNMC) for the AFM analysis and Dr. Raj Suryanarayanan (University of Minnesota) for the XRD analysis. FUNDING INFORMATION Financial support from the School of Pharmacy and Health Professions is greatly appreciated. REFERENCES 1. 2. 3. 4. Roglic G. WHO Global report on diabetes: A summary. International Journal of Noncommunicable Diseases. 2016;1(1):3. Centers for Disease Control and Prevention. National diabetes statistics report, 2014: estimates of diabetes and its burden in the United States. Atlanta, GA: US Department of Health and Human Services. 2014. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes care. 2004;27(suppl 1):s5–10. Kahn SE. The relative contributions of insulin resistance and beta-cell dysfunction to the pathophysiology of type 2 diabetes. 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