Novel preparation of PLGA/HP55 nanoparticles for oral insulin delivery
© Wu et al.; licensee Springer. 2012
Received: 29 September 2011
Accepted: 8 June 2012
Published: 8 June 2012
The aim of the present study was to develop the PLGA/HP55 nanoparticles with improved hypoglycemic effect for oral insulin delivery. The insulin-loaded PLGA/HP55 nanoparticles were produced by a modified multiple emulsion solvent evaporation method. The physicochemical characteristics, in vitro release of insulin, and in vivo efficacy in diabetic rats of the nanoparticles were evaluated. The insulin encapsulation efficiency was up to 94%, and insulin was released in a pH-dependent manner under simulated gastrointestinal conditions. When administered orally (50 IU/kg) to diabetic rats, the nanoparticles can decrease rapidly the blood glucose level with a maximal effect between 1 and 8 h. The relative bioavailability compared with subcutaneous injection (5 IU/kg) in diabetic rats was 11.3% ± 1.05%. This effect may be explained by the fast release of insulin in the upper intestine, where it is better absorbed by the high gradient concentration of insulin than other regions. These results show that the PLGA/HP55 nanoparticles developed in the study might be employed as a potential method for oral insulin delivery.
Insulin is the most effective medicine in lowering the glucose level of blood for the treatment of diabetes mellitus . Early introduction of insulin can also protect islets from apoptosis and increase β-cell regeneration in type 2 diabetes . Subcutaneous injections of insulin remain to be the preferred approach for diabetic patients but often result in poor patient compliance [3, 4]. Oral administration of insulin seems to be the most convenient way and can mimic endogenous production of insulin . However, a reliable insulin formulation for the oral delivery is encountered with some barriers in the gastrointestinal (GI) tract that include (a) enzymatic degradation in the GI tract and (b) poor insulin permeability through the GI system . The bioavailability of insulin solution delivered orally is less than 1% .
pH-sensitive polymers have been developed and introduced into the particulate carriers to circumvent the barriers for oral insulin delivery. As early as 1999, insulin was formulated with pH-sensitive microparticles, and a prolonged reduction of hyperglycemia was observed after oral administration to diabetic rats . This reduction was dose dependent and lasted up to 8 h with a dose of 25 IU/kg of encapsulated insulin. These results were confirmed by pH-responsive hydrogel particles and demonstrated a similar manner in the reduction of blood glucose level after oral administration of polymeric dosage form and direct injection of insulin [8, 9]. Generally, nanoparticles have greater intracellular uptake compared with microparticles and are available to a greater range of biologic targets due to their smaller size and mobility . Depending on the pH-sensitive polymer combinations, nanoparticles can be tailored to control release kinetics, facilitate the uptake of insulin, and increase the oral bioavailability [11, 12]. Various pH-sensitive nanoparticles based on pH-sensitive polymers were developed for oral delivery of insulin. These pH-sensitive polymers include polymethacrylic acid [13–18], hydroxypropyl methylcellulose phthalate (HPMCP(HP55)) , dextran sulphate [20–23], alginate [21, 24], poly-γ-glutamic acid [25, 26], and so on. Generally, these formulations prepared with these pH-sensitive polymers, orally administered mostly to diabetic rats, induced a different extent reduction in blood glucose level.
However, absorption of insulin from different regions of the intestine is not uniform due to the cellular morphology of the intestines’ changes from region to region [23, 27–29]. Interestingly, the plasma insulin concentration-time curves after orally delivered nanoparticles showed different insulin peaks. According to the time of insulin peaks, the ways of insulin absorption could be divided into the fast and the slow absorption way (the dividing point is often observed in 8 h of post-administration). The fast absorption way is probably related to insulin directly absorbed through paracellular pathway [23, 24, 28, 30]. The slow absorption way is possibly related to higher insulin residence time at the absorption site and delay of nanoparticle uptake by endocytosis via enterocytes or M cells in the small intestine [23, 24, 29]. Insulin within carrier could be directly absorbed through the intestinal wall exerting a hypoglycemic effect [31, 32]. Insulin level in blood after intraduodenal injection of insulin/carrier solution showed the insulin peak occurred in 20 to 40 min [33, 34]. The upper intestinal area seems to be the most active region for insulin absorption . In addition, nanoparticles orally delivered are an aqueous suspension; they will leave the stomach of fasted animals very rapidly and arrive in the upper intestine where they will be able to release insulin . Hence, the nanoencapsulated insulin rapidly absorbed may occur primarily in the upper intestine.
We hypothesized that the insulin, remaining high concentration due to the fast release from the pH-sensitive nanoparticles with high entrapment efficiency, should be better absorbed by the upper intestinal mucosa and, in consequence, should rapidly produce the biological efficacy. In addition, the rapid adsorption of insulin in the upper intestine would decrease the loss of drug across the intestine. Thus, the present study aims to develop an oral dosage of insulin-loaded nanoparticles composed of poly (lactide-co-glycolide (PLGA)/HP55 with strong drug encapsulation ability and good pH-sensitive release property. The nanoparticles were prepared by a novel technique of modified multiple emulsion solvent evaporation (MESE) method. The physicochemical characterization (morphology, size, insulin loading, and in vitro release of insulin) of nanoparticles was examined and evaluated. The biological efficacy after oral administration of insulin-loaded PLGA/HP55 nanoparticles in diabetic rats was studied.
Pure crystalline porcine insulin was purchased from Xuzhou Wanbang Bio-Chemical Co. Ltd., (No. 0312A02, Jiangsu, China), with a nominal activity of 27 IU/mg. PLGA (50/50, Mw is about 20,000) was acquired from Shandong Medical Instrument Institute (Shandong, China). The HPMCP (HP55, Mw is about 45,000) and polyvinyl alcohol (87% hydrolysed, Mw is about 31,000 to 50,000) were purchased from Acros Organics (NJ, USA). All other reagents and solvents used were analytical grade. Streptozotocin (STZ; HPLC) was purchased from Sigma-Aldrich, Corporation, St. Louis, MO, USA. Distilled and deionized water (Mili-Q water systems, Millipore, Bedford, MA, USA) was used for the preparation of all sample solutions.
Male sprague Dawley rats weighing 220 to 250 g, 12 to 13 weeks old, were provided by the pharmacological laboratory of the Hong Kong University of Science and Technology. They received standard laboratory chow diet and tap water, available ad libitum. All experiments were carried out in accordance with the Guideline for the Care and Use of Experiment Animals in Hong Kong University of Science and Technology, Hong Kong.
Preparation of insulin-loaded PLGA/HP55 nanoparticles
The preparation of nanoparticles was carried out by the modified MESE technique. Briefly, the inner aqueous phase of pH 2.0 insulin solution (10 mg/mL, 1 mL) was added into the oil phase of polymer solution (PLGA/HP55, 50 mg/100 mg) using methylene chloride and acetone (3 mL/2 mL) and then the resulting mixture was emulsified to form the primary emulsion by sonification at power of 40 W for 0.5 min (SONOPULS, Bandelin, German). The primary emulsion was thereafter poured into 20 mL of external aqueous phase of polyvinyl alcohol solution (1%, w/v) and sonicated at power of 60 W for 1 min, involving the formation of the multiple emulsions. After evaporation of solvents under reduced pressure, the nanoparticles were collected by centrifugation at 20,000 rpm for 10 min and then washed three times with distilled water. The insulin-loaded PLGA/HP55 nanoparticles were vacuum freeze-dried for 24 h after prefreezing of the resultant dispersion at −20°C overnight.
Characterization of nanoparticles
The size of nanoparticles was determined by photon correlation spectroscopy at 25°C with a detection angle of 90° using a Malvern Zetasizer II (Malvern Instruments Ltd., Worcestershire, UK). Measurements were made on aqueous dilute nanoparticle suspension.
The morphology of nanoparticles was determined by scanning electron microscopy (SEM) (LEO 1530 VP, LEO Elektronenmikroskopie GmbH, Oberkochen, Germany) and transmission electron microscopy (TEM) (Hitachi JEM-100 CXII, Hitachi Ltd., Chiyoda, Tokyo, Japan). For SEM measurement, the powder of nanoparticles was fixed on an aluminum stub as a thin film and coated with gold before observation. SEM was performed at an accelerating voltage of 10 kV and a magnification of × 20,000 in the transmission electron mode. For TEM measurement, an aqueous droplet of nanoparticle solutions was immobilized on copper grids and negatively stained with phosphotungstate solution (2%, w/v). After drying at room temperature, the morphology of the nanoparticles was examined.
where Mtotal is initial amount of insulin (mg); Vsupernatant, volume of supernatant (mL); Csupernatant, concentration of insulin in supernatant (mg/mL); and Wnanoparticle, weight of nanoparticles.
The concentration of insulin was determined by reverse phase HPLC method (Agilent 1200, ZORBAX 300 SB-C18 column 150 mm × 4.6 mm, 5 μm, Agilent Technologies Inc., Santa Clara, CA, USA). The mobile phase consisted of a premixed isocratic mixture of 0.2 M sodium sulfate anhydrous solution adjusted to pH 2.3 with phosphoric acid and acetonitrile (73:27, v/v). The injection volume for samples and standards were 20 μL and eluted at a flow rate of 0.8 mL min−1 at 30°C. The absorbance of insulin was determined using the UV trace at 214 nm.
In vitro release study
Five milligrams of the insulin-loaded nanoparticles were dispersed in 5-mL simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 7.4), respectively, and shaken at 50 rpm at 37.8°C using a constant-temperature shaker (SHA-B, Guohua Co. Ltd., Jiangsu, China). At specified time intervals (0, 30, 60, 120, 180, 240, and 480 min), supernatants were collected by centrifugation. The concentrations of insulin in the supernatant were determined by reverse phase HPLC method, and total amount of insulin released from the nanoparticles was calculated. Each experiment was carried out in triplicate.
In vivo study in diabetic rats
Male sprague Dawley rats were made diabetic prior to the study by intravenous injection of 60-mg/kg STZ dissolved in citrate buffer at pH 4.5. They were considered to be diabetic when the fasted glucose levels exceeded 250 mg/dL 2 weeks after STZ treatment. The rats were then used to evaluate the hypoglycemic effects of the insulin-loaded PLGA/HP55 nanoparticles.
Diabetic rats were fasted for 12 h prior to and remained fasted during the experiment but were allowed free access to water throughout the whole experiment. Blood samples were collected from the tail veins of rats prior to drug administration and at different time intervals after dosing. The blood glucose was then determined by a glucose meter using ACCU-Chek Active (Roche Diagnostics, Mannheim, Germany).
Hypoglycemic effect was evaluated by the decrease of plasma glucose levels relative to the basal values in rats divided in three groups (n = 3/group) corresponding to (a) subcutaneous (SC)-injected free-form insulin at dose of 5 IU/kg and (b) orally delivered insulin-loaded nanoparticles and (c) orally delivered blank nanoparticles.
where AUCoral is the total area under the serum insulin concentration vs. time curve of oral administration of insulin nanoparticles; AUCSC, total area under the serum insulin concentration vs. time curve of pure insulin injection; DoseSC, dosage of pure insulin injection (IU/kg); and Doseoral, dosage of oral administration of insulin nanoparticles (IU/kg).
Data are presented as mean ± standard deviation (S.D.). Comparison between two groups was analyzed by the one-tailed Student’s t-test. A statistical difference was considered when p value was less than 0.05.
Results and discussion
Preparation and characterization of insulin-loaded PLGA/HP55 nanoparticles
Properties of insulin-loaded PLGA/HP55 nanoparticles prepared via MESE and SESD methods (mean ± S.D., n = 3)
Particle size (nm)
169 ± 16
65.41 ± 2.3
3.17 ± 0.24
181.9 ± 19.0
94.25 ± 1.24
5.89 ± 0.17
Drug entrapment ability of PLGA/HP55 nanoparticles
The drug entrapment ability of PLGA/HP55 nanoparticles prepared by SESD and MESE techniques is compared in Table 1. Entrapment efficiency (EE) was significantly dependent on the preparation method . The reason for this lower EE (65.41% ± 2.3%) by SESD is due to the low affinity between the polymer and hydrophilic drug, leading to the drug substance tendency to move from the organic phase to the outer aqueous phase during the single emulsion diffusion process. In order to avoid this, the hydrophilic insulin partitioned between the two immiscible phases (the inner aqueous phase and the oil phase), allowing the drug to be initially dissolved in an inner aqueous phase. Hence, the high EE (94.25% ± 1.24%) was obtained by MESE technique in the study.
The novel MESE preparation technique described here for the encapsulation of hydrophilic macromolecule in PLGA/HP55 nanoparticles resulted in improved encapsulation efficiency in comparison to the SESD technique. Obviously, the success of this technique hinges upon the ability to construct biocompatible nanoparticles that allow high loading of insulin molecules without premature release before reaching the destination, i.e., intestine.
In vitro release profiles from insulin-loaded PLGA/HP55 nanoparticles
Oral administration of insulin-loaded PLGA/HP55 nanoparticles in diabetic rats
Oral administration of the insulin-loaded PLGA/HP55 nanoparticles and SC injection of the free-form insulin solution produced a significant hypoglycemic effect, suggesting that the preparation and freeze-drying processes did not lead to the fragmentation of insulin molecules. SC injection of the insulin solution produced a sharp decrease in blood glucose levels (90% in 2 h), which gradually returned to the basal levels after 8 h. The fast and prolonged reduction in blood glucose levels was investigated between 1 and 8 h after oral administration of the nanoparticles. This phenomenon of fast reduction of blood glucose levels was also conformed within 2 h after oral delivered aspart-insulin or insulin-loaded nanoparticles, which could be attributed to the initial release of insulin from the nanoparticles [24, 28]. The quick pass of nanoparticle suspension through the stomach of fasted rats also has the promoted effect on the rapid reduction of blood glucose. The advantages of the present pH-sensitive nanoparticles are the use of a multiple emulsion method increasing the drug entrapment ability and addition of HP55 that greatly increase pH-sensitivity of nanoparticles. Compared to the nanoparticles developed by Cui et al. , showing similar PLGA/HP55-based nanoparticles, the present nanoparticles showed an improved entrapment ability by increasing the encapsulation efficacy by 1.4-fold and improved release property by increasing the release of insulin from nanoparticles at pH = 7.4 within 1 h by 1.3-fold. These improvements would promote the insulin absorbed as a free peptide in the upper intestine either by a paracellular pathway or a receptor-mediated pathway.
Insulin could be directly absorbed through the intestinal cell exerting a hypoglycemic effect [31, 32]. The direct uptake of insulin has been attributed to specific insulin receptors in intestinal enterocytes and rapid internalization of the nanoparticles by the epithelial cells [23, 40, 41]. The upper intestinal area seems to be the most active region for insulin absorption, improved under fasting conditions . Hence, the fast reduction of blood glucose level after oral insulin-loaded PLGA/HP55 nanoparticles could be attributed to the fast release of insulin in the upper intestine, wherein the pH is approximately 6.0 to 7.0 .
Pharmacokinetic parameters of insulin in diabetic rats
SC-administered insulin solution
Orally administered insulin-loaded nanoparticles
129.21 ± 8.49
69.93 ± 7.64
AUC(0 to 8 h) (μIU h/mL)
143.76 ± 21.37
161.69 ± 28.58
11.3 ± 1.05
The bioavailability for insulin-loaded PLGA/HP55 nanoparticles obtained by the SESD technique is 6.27% ± 0.42%. By introducing the MESE method into the preparation of PLGA/HP55 nanoparticles, the physiological effect was improved by increasing the bioavailability by 1.8-fold. Compared with PLGA/HP55 nanoparticles prepared by SESD technique, the encapsulated insulin by MESE technique can be released very rapidly allowing a high drug gradient concentration in the upper intestine followed by a good absorption.
In this study, novel PLGA/HP55 nanoparticles with a pH-sensitive characteristic were prepared by MESE technique for oral delivery of insulin. The PLGA/HP55 nanoparticles exhibited an excellent insulin entrapment ability and a good pH-sensitive release behavior. Additionally, the pharmacodynamic and pharmacokinetic evaluations of orally administered PLGA/HP55 nanoparticles in diabetic rats indicated that absorption of insulin in the upper intestine was fast, and the hypoglycemic effect was significant. These results suggested that the PLGA/HP55 nanoparticles developed in the study might be employed as a potential approach for the multiple daily oral delivery of insulin.
ZMW is a doctoral candidate, LL and WJ are master students, XDG is an assistant professor, and YQ and LJZ are professors in the School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou, People’s Republic of China. LYZ is a research assistant in the Department of Chemical and Bio-molecular Engineering, School of Engineering, The Hong Kong University of Science and Technology, Hong Kong. KQL is an associate professor in the Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore.
This work was financially supported by National Natural Science Foundation of China (no. 21176090 and no. 20906028), Hong Kong ITF (no. ITP/006/08NP), Team Project of Natural Science Foundation of Guangdong Province, China (no. S2011030001366), Guangzhou Municipal Bureau of Science and Technology (no. 2009 J1-C511-2), and the Fundamental Research Funds for the Central Universities (no. 2011ZM0041).
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