DEVELOPMENT AND IN VITRO, IN VIVO EVALUATION OF CONTROLLED RELEASE, BIOCOMPATIBLE NANOPARTICLES
AMULYARATNA BEHERA*, SUNIT KUMAR SAHOO
University Department of Pharmaceutical Sciences, Utkal University, Bhubaneswar, Orissa
The intention of the study was to formulate poly (D, L-lactic-co-glycolic acid) PLGA nanoparticles containing glibenclamide and to characterize by both in vitro and in vivo method. Nanoparticles were prepared by emulsion solvent evaporation technique using methanol and dichloromethane in a ratio
2:1 as solvent with (PVA/polysorbate-80) in a fixed concentration as surfactant. The prepared nanoparticles were characterized by transmission electron microscopy (TEM), differential scanning calorimetry (DSC), stability
and for in vitro drug release study. The in vivo antidiabetic study along with biochemical and haematological study was also carried out using streptozotacin induced female albino rats. Stable glibenclamide loaded PLGA nanoparticles were successfully prepared by solvent evaporation technique without any incompatibility as indicated by DSC study. The drug release from prepared nanoparticles continued for 3 days in a controlled zero order fashion. The optimised formulation was able to produce significant antidiabetic activity and the activity continued for 7 days.No significant change in behavioural, biochemical and haematological parameters was observed during the study period.
(Received November 7, 2011; Accepted February 17, 2012)
Keywords: poly(lactic-co-glycolic acid) (PLGA), Glibenclamide, Nanoparticles (NPs), Controlled release
1. Introduction
Nanoparticles (NPs) in the field of nanotechnology have been emerging as key mechanisms by providing a targeted approach for efficient delivery of conventional therapeutic drugs. This targeted approach leads to the delivery of drugs, in low dosages, in a controlled manner for an efficient action within the body [1-4]. NPs are nano-sized stable, colloidal or solid particles, generally made of biodegradable polymer or lipids. They entrap or encapsulate the active principal or adsorb or attach to their surface. NPs can also offer advantages like limiting fluctuation within therapeutic range, reducing side effects, decreasing dosing frequency, and improving patient compliance. In general, rapid gastrointestinal (GI) absorption is required for oral hypoglycemic drugs, in order to prevent a sudden increase in blood glucose level after food intake in patients with diabetes mellitus.
Glibenclamide (GLM) is oral antidiabetic used in the management of type II diabetes mellitus. It causes hypoglycaemia by stimulating release of insulin from pancreatic cells and by increasing the sensitivity of peripheral tissue to insulin. It is rapidly absorbed after oral administration with plasma half life 5.05 h. A slow absorption of a drug usually originates from either poor dissolution of the drug from the formulation or poor permeability of the drug across the
*Corresponding author: [email protected]
GI membrane. It has a history of low bioavailability, which is attributed to poor dissolution.
Glibenclamide is partially soluble in water. The formation of amorphous forms to increase drug solubility and the reduction of particle size to expand surface area for dissolution anddecrease the interfacial tension with the aid of a water‐soluble carrier are among the possible mechanisms for increasing dissolution rates and improving bioavailability of poor water‐soluble drugs [5-14].
Nanoparticulate delivery systems, such as those based on poly(lactic-co-glycolic acid) (PLGA) polymers, have been studied extensively for many years. PLGA polymers have the advantage of being well characterized and already com-mercially used for microparticulate drug delivery systems. PLGA polymers are biocompatible, biodegradable, and bio-resorbable.
Biodegradable materials with PLGA ( poly DL-lactide-co-glyco-lide), have been utilized as a drug delivery system for controlling the release of various drugs. Release kinetics from PLGA and other biodegradable polymers are controlled by diffusion, erosion or a combination there of, and are dependent on the polymer (Mw, copolymer ratio and crystallinity) [15]. The primary goal of this study was to formulate characterize PLGA controlled release nanoparticles containing GLM by emulsification solvent evaporation method and to achieve a better release profile suitable for per oral administration and which could overcome the drawbacks of GLM delivery through conventional dosage forms.
2. Experimental Materials
Poly (lactic-co-glycolic) acid (copolymer ratio 50:50, viscosity 0.41 dL/g) was purchased from Birmingham Polymers Inc. (Birmingham, USA) and glibenclamide (GLM) were procured as gift sample from Alembic Pharma, India. All other chemicals used in this study are of analytical grade from E Merck (India).
Methods
2.1 Methods of Preparation of Glibenclamide loaded nanoparticles NPs
Glibenclamide loaded PLGA NPs were prepared by emulsification solvent evaporation method. An attempt was made to optimize the nanoparticle formulation using various formulation parameters including varying drug/polymer ratio organic solvent (methanol/dichloromethane) ratio, stirring speed (300 – 3000 rpm) and surfactant ratio (PVA/polysorbate-80) in a fixed concentration (0.5 %w/v), using a modified emulsification solvent evaporation technique. PLGA and GB were dissolved in 25 ml of solvent mixture containing methanol to dichloromethane in a ratio 2:1, using a vortex shaker to form a homogeneous solution of drug and polymer. This homogeneous solution was added slowly to 120 ml of aqueous surfactant solution containing 0.5%(w/v) PVA, Polysorbate 80 and homogenised using a high pressure homogenizer (Ika, Japan) to prepare the emulsion [16-18]. The emulsion formed was stirred with a laboratory magnetic stirrer (Remi, India) for 5 h at 25ºC followed by centrifugation (Remi, India) for 22 min. The supernatant was discharged and the pellets obtained were washed using the same volume of distilled water as the supernatant and then centrifuged for 6 min. Washing was repeated three times and the resulting NPs were freeze-dried (Lyphlock, Labconco).
2.2 Determination of drug incorporation efficiency
Freeze-dried NPs (10gm) were dissolved in dichloromethane (50 ml), the common solvent for both drug and polymer. The amount of drug in the solution was determined using Perkin-Elmer
ultraviolet spectrophotometer at 243.5 nm. Drug content (%w/w) and drug entrapment (%) were calculated using Eqs 1 and 2, respectively.
% Drug content = (M/Mr)100 ...(1) where M is the mass of drug in the NPs and Mr the mass of NPs recovered.
% Drug entrapment = (D/Df)100 ... (2) where D = amount of drug in the nanoparticles
Df = Total amount of drug used for the preparation of the nanoparticles.
2.3 Particle size analysis and zeta potential measurement
To evaluate particle size, zeta potential and polydispersity index (PI), freeze dried NPs were reconstituted in distilled water. The size of the NPs was determined by Zetasizer (Nano ZS, Malvern Instruments, Malvern, UK) based on dynamic light scattering technique. The Polydispersity Index(PI) which is a dimensionless no indicating the width of the size distribution was also measured. The mean particle size of the formulation was determined by photo correlation spectroscopy with a zeta master (Malvern Instruments, UK) equipped with the Malvern PCS software. Every sample was diluted with phosphate buffered saline pH 7.4. The surface charge (Zeta potential) was determined by measuring the electrophoretic mobility of the NPs using a Malvern zeta sizer (Malvern Instruments, UK). Samples were prepared by dilution with phosphate buffer saline pH 7.4.
2.4 Differential scanning calorimetric (DSC) studies
The physical state of glibenclamide encapsulated in NPs was characterized by differential scanning calorimetry (DSC, Shimadzu, DSC-50, Japan). Each sample containing drug equivalent to 10 mg was sealed separately in a standard aluminium pan the samples were purged with nitrogen gas at a flow rate of 10ml/min, and heated at 100c/min from 0 to 350 °C.
2.5 Transmission electron microscopic (TEM) studies
NPs were also evaluated for size with a transmission electron microscope (Philips/FEI Inc, Barcliff, Manor, NY, USA). For this purpose, a sample of NPs was suspended in water (0.5 mg/ml) and sonicated for 30 s. One drop of this suspension was placed over a carbon-coated copper TEM grid (150 mesh, Ted Pella Inc., Rodding, CA) and negatively stained with 1 % uranyl acetate for 10 min and then allowed to dry. Images were visualized at 120 kV under the microscope.
2.6 In Vitro Drug Release Studies
The in vitro release of GB-loaded NPs were performed using USP type II dissolution test apparatus (Electrolab, India) in 900 ml of medium(0.1N Hydrochloric acid) for the first 2 hour and then in Phosphate buffer( PH 7.5) from 3-72h at 37± 0.50c and stirring rate of 100 rpm. 5ml samples were collectedperiodically and replaced with equal volume of fresh dissolution medium on each occasion. After filtration through Whatman Grade No. 41 Quantitative Filter Paper (pore size 25 µm), the concentration of GB was determined spectrophotometrically at 300 nm on UV- Visible spectrophotometer (Jasco V530, Japan). In vitro release profile was analyzed by various kinetic models (zero order, first order and Higuchi) in order to determine the mechanism of drug release [20-22].
2.7 Stablility Study
The selected formulations were subjected to accelerated stability studies to evaluate effect of stress conditions according to ICH guidelines. Formulations were packed on 0.44mm dilaminated aluminium foil and subjected to elevated temperature and relative humidity (RH) conditions of 40 ± 2 ºC / 75 ± 5% RH, 30 ± 2 ºC / 65 ± 5 % RH, 30 ± 2 ºC / 65 ± 5 % RH. Samples were withdrawn at the end of 1, 3 and 6 months and evaluated for physical properties, drug content and in vitro drug release.
2.8 In-Vivo studies
Meticulous analysis of the results and discussion brings us to draw the conclusion that the formulation F2 which has the desirable particle size, drug entrapment efficiency and control release rate profile is the best among all others and is chosen for carrying out In Vivo studies.
Study protocol
The protocol for the In vivo study was approved by Institutional animal ethical committee of Shree Santkrupa College of Pharmacy, Ghogaon, Shivajinagar, Karad, Maharastra) (IAEC/1110/ac/07). All ethical manners regarding the use of animals in scientific research were
carefully considered. Adult female wistar albino rats selected with average body weight of 200- 250g were used throughout the study. Rats were kept in polypropylene cages. They were maintained under standard environment conditions (25±300C and 70±5% relative humidity under natural 12-h light and 12 h dark conditions). They were maintained with free access to water and standard laboratory rat pellet diet (Hindustan Lever Ltd., Bangalore). The animals were divided into five groups, each group containing 6 animals (n=6 / group).
Induction of Diabetes
Streptozotocin (Himedia) was dissolved in 100 mmol⁄L citrate buffer (pH 4.5) and 60 mg⁄
kg fresh solutions was injected intraperitoneally to overnight-fasted rats. Blood glucose levels were measured after 48 h later using Glucometer (One Touch, Johnson & Johnson, India). Each animal with a blood glucose concentration level above 250mg/dL (13.8 mM) was considered to be diabetic and used in the experiments.
Experimental Procedure
Animals were randomly divided into five groups of six rats each group. Pure Glibenclamide and optimized formulation of nanoparticles was administered orally through intragastric tube. Animals divided with five different groups were kept as follows: group I:
Normal controlled rats treated with drinking water; group II: Diabetic controlled rats treated with drinking water; group III: Diabetic controlled rat administered with pure Glibenclamide(2 mg/kg body weight); group IV; Diabetic rats administered with drug loaded PLGA Nanoparticles containing glibenclamide equivalent to 1 mg/kg body weight, incorporated in a single dose; and group V; Diabetic rats administered with optimised formulation of Glibenclamide loaded PLGA Nano Particles equivalent to 4 mg/kg body weight, incorporated in a single dose. Blood glucose levels were measured in all rats using One touch®HorizonTM, Blood Glucose monitoring system, Life Scan, Inc, Milpitas, USA marketed by Johnson & Johnson, India at 2 hours interval for 12hours, next 12hours upto day one and then at 24 hours interval upto 7 days of the treatment period. In all experiments, approximately 0.1 mL blood was drawn each time from the tail using aseptic procedure. Blood samples were immediately placed on the Glucostrips and blood glucose levels were determined using a glucometer. Behavioural parameters (body weight, skin colour, salivation, lacrimation, respiration, motor activity and Muscle Spasm) were studied during treatment of animals with comaprision with normal control animal. At the end of the experimental period blood samples were collected by cardiac puncture techniques and mixed well with anticoagulant. Collected samples were analysed for different Biochemical and Haematological parameters.
2.9 Statistical analysis
Results are expressed as mean ± S.D for triplicate samples. The results were statistically analyzed and significant differences among formulation parameters were determined by one-way analysis of variance using ‘Graph Pad Instate®’Version 3.05 (USA), statistical analysis program.
Statistical significant was considered at p < 0.05.
3. Results and discussion
3.1 Entrapment efficiency, drug content, particle size and TEM study
Formulation codes with entrapment efficiency, drug content and particle size were as given in table 1. It has been observed that as concentration of polymer increases entrapment efficiency, drug content and particle size also increases. Also for formulation F4 increase in drug concentration has also increased the entrapment efficiency, and particle size. Lower particle size was observed when stirring speed was increased. TEM photographs (Fig 1) indicate that NPs were spherical in shape and in the nanosize range with discrete spherical outline. DSC thermograms of native PLGA, GLM and GLM-loaded NPs showed the characteristic peaks of the respective compounds, as shown in Figure 2. An endothermic peak of PLGA occurred at approximately at 480c due to glass transition temperature. The peak of PLGA was slightly shifted in drug loaded nanoparticles compared to the native PLGA. The endothermic peak of GLM was occurred to
approximately 172.80c. In the formulation F2 such peaks were observed. . In vitro release profile and kinetics of GB from the NPs are shown in Figure 3 and Table 2, respectively. As polymer concentration increased, drug release decreased. Also, when the drug concentration of the NPs was increased as in case of formulation F2 (drug: polymer ratio 1:2) as compared to formulation F1(drug: polymer ratio 1:1), drug release increased. In order to determine the release pattern of GLM from prepared NPs, various invitro release kinetic models such as Zero order, Higuchi and First order were analysed. In all formulations highest regression co-efficient was observed in zero order equations (table 2).
Table 1: Batch codes with D (drug) P (polymer) ratio, SP (stirring speed), EE (entrapment efficiency), DC (drug content) and PS (particle size) of Glibenclamide loaded NPs.
Sr.No. Batch code D:P ratio SP(rpm) EE(%) DC(%) PS(nm)
1 F1 1:1 2500 46.32 ± 2.14* 11.21 ± 2.43* 532 ± 11.29*
2 F2 1:2 2500 54.24 ± 3.15 26.52 ± 3.16 562 ± 5.28 3 F3 1:3 2500 62.63 ± 1.87* 28.51 ± 2.29* 812 ± 14.72**
4 F4 2:1 2500 90.52 ± 4.52** 36.73 ± 3.16* 1453 ± 13.26**
5 F5 1:2 250 33.21 ±3.12** 10.28 ± 1.13* 1081 ± 23.26**
6 F6 1:2 1000 42.24 ± 2.57* 13.14 ± 2.12* 993 ± 14.28*
7 F7 1:2 1500 48.25 ± 2.21* 21.23 ± 3.31* 803 ± 11.16**
Significantly different from the value of batch F2 at p < 0.01 (**) and p < 0.05 (*)
Fig. 1. Transmission electron microscopy (TEM) for batch with 1:2 drug polymer ratio
3.2 DSC Studies
DSC study shows the nature of the drug encapsulated in the NPs. This analysis was performed on native PLGA, native Glibenclamide and Glibenclamide loaded NPs. Different compounds show their characteristic peaks in DSC (figure 2). The endothermic peaks of PLGA and was found approximately at 48 ºC due to glass transition temperature (Tg) of PLGA. The peak of PLGA was slightly shifted in drug-loaded NPs as compared to that of native PLGA. The endothermic peak of native Glibenclamide was found approximately at 172.75 ºC.
Fig. 2: DSC of Pure GLM (A), PLGA (B), Formulation(C)
3.3 In Vitro Dissolution Study
In vitro release profile and release kinetics of Glibenclamide through Glibenclamide- loaded PLGA NPs were as shown in figure 3 and table 2 respectively. It has been observed that as concentration of polymers increase drug release decreases. But for formulation with increased drug concentration drug release has increased. The in vitro release behaviour of formulations follows zero-order kinetics.
Fig. 3. In vitro release of GB from formulation F1 ( ), F2 ( ), F3 ( ), and F4 ( ) Table2: Release kinetics of GLM loaded PLGA NPs.
Formulation Zero order(r2) First Order(r2) Higuchi (r2)
F1 0.9818 0.9422 0.9413
F2 0.9812 0.9462 0.9326
F3 0.9726 0.9532 0.9524
F4 0.9831 0.9321 0.9623
3.4 STABILITY STUDY
GLM loaded PLGA NPs containing 0.5% PVA (formulation A) as surfactant was subjected to stability study. Stability studies according to ICH guidelines reveal that NPs were stable at the end of 6 months in all the test conditions. No significant changes in particle size, GLM entrapment, GLM content and release profile were observed after the end of 1, 3 and 6 months. The release kinetics was also found identical in stablility studies.
Table 3: Stability study of PVA nanoparticles Test
Duration
Test Condition
0c/RH
Entrapment Efficiency
GLM Content
Mean Particle Size
Kinetics Zero-
order(r2) First- order(r2)
Higuchi(r2) 1 40±20/75±5% 54.21 ± 3.15 26.52 ± 3.14 562 ± 5.28
0.9823 0.9583 0.9824 30±20/65±5% 56.02±2.10 25.08±1.28 560±6.24
25±20/60±5% 56.11±2.62 25.06±3.25 561±5.23 3 40±20/75±5% 56.12±1.35 24.38±1.02 558±4.54
0.9827 0.9552 0.9802 30±20/65±5% 56.54±2.30 25.04±2.11 557±6.13
25±20/60±5% 56.02±2.08 23.20±2.48 557±3.22 6 40±20/75±5% 57.07±1.28 24.91±2.33 559±4.20
0.9829 0.9575 0.9813 30±20/65±5% 57.16±2.09 25.81±2.02 558±4.22
25±20/60±5% 56.51±2.26 23.81±1.52 558±6.10
3.5 In Vivo Evaluation
In vivo evaluation of the Glibenclamide loaded PLGA nanoparticles were carried out in healthy female albino rat. Measurement of blood glucose level were done after oral administration of drug loaded nanoparticles equivalent to the dose of the drug, 2 mg/kg body weight, 2 mg/kg body weight, and 4mg/kg body weight in comparison with administration of pure Glibenclamide at same dose and with double dose. The antihyperglycemic effect of formulation and pure drug in diabetic rat were assessed at different time intervals. When pure Glibenclamide solution was given orally, it was observed that the blood glucose level started to decrease from the second hour, after the sixth hour the blood glucose level reached almost normal level but after the sixth hour, blood glucose level started to increase again. On the other side, the optimized formulation of Glibenclamide loaded PLGA nanoparticles, the blood glucose level started to decrease from the second hour and this decrease continued up to 7 days [23].
Fig. 4: Blood glucose levels in the five study groups over 24 hrs.
(Group1: Control Group2: Diabetic Control Group3: 2mg/kg of PLGA loaded glibenclamide nanoparticle Group 4: 1mg/kg of PLGA loaded glibenclamide
nanoparticle Group5: 4mg/kg of PLGA loaded glibenclamide nanoparticle.)
Table4: Blood glucose concentration (mg/dl) of PLGA Loaded Glibenclamide Nanoparticle compared with control rat
Time GROUP I GROUP II GROUP III GROUP IV GROUP V
0h 88.5±2.59 307.67±4.32 307.33±6.15 323.17±2.64 327.67±4.72 2h 87.83±3.06 317±4.29 125.33±2.73 308.67±3.27 311.5±7.84 4h 91.67±3.08 331.17±2.4 135.17±2.32 295.67±4.63 296.5±5.79 6h 89.5±3.78 341.83±4.45 144.33±2.94 279.5±3.27 284.67±5.53 8h 88±1.79 352.5±5.13 167.67±6.62 263±5.83 268.83±4.26 10h 89.5±3.02 357.83±4.79 268.33±3.5 241.83±6.34 252.5±4.13 12h 91.17±3.06 368.67±5.32 277.67±3.98 228±3.63 237.17±5.63 1day 89.5±3.73 381±5.97 285.5±4.23 212.17±3.76 216.83±6.5 2days 89±4.15 387.5±5.47 295.33±4.03 199.5±6.28 201.33±4.09 3days 92.33±3.2 394.5±4.42 308.33±4.63 190.5±8.41 191.67±5.28 4days 90.67±3.27 394.33±4.37 317.5±5.39 176.83±5.34 176±4.34 5days 90.5±1.22 375.83±4.54 319.5±9.14 166.67±5.85 166.5±5.82 6days 88.5±2.07 365.33±5.72 314.17±10.35 151.83±5.78 149.67±4.93 7days 89.83±3.06 347.33±4.76 307.17±7.99 136.17±6.15 133.67±3.14
(Group I: Control Group II: Diabetic Control Group III: 2mg/kg of PLGA loaded glibenclamide nanoparticle Group IV: 1mg/kg of PLGA loaded glibenclamide nanoparticle Group V: 4mg/kg of
PLGA loaded glibenclamide nanoparticle)
3.6 Behavioural Parameters
There was no significant changes were observed in physical parameters in all groups. No mortality was observed in all treatment groups throughout the experimental period. There was no significant change in the body weight of all the groups as compared with control group.
During the total treatment period the parameter like body weight, skin colour, salivation, lacrimation, respiration, motor activity and Muscle Spasm were observed and it is reported that only muscle spasm shows negative and all other parameter shows no significant changes.
Table 5: Behavioural Parameters
Parameters Results Body Weight No significant change
Skin Colour Normal
Salivation Normal Lacrimation Normal
Respiration Normal
Motor activity Normal
Muscle spasm Normal
Piloerection Negative
Righting reflex Positive
3.7 Biochemical and Haematological parameters
Levels of serum glutamate transaminase (SGPT), Serum alkaline Phosphate (SAP), serum glutamic pyruvic transaminage (SGPT), lactate, glucose, urea, cholesterol, triglycerides, were estimated in Merck semi auto analyzer by using Merck analytical kits. All the parameters level are with-in the normal range and no significant changes were observed.
Routine hematological parameters were performed on ACT diff-2 Hematology Analyzer (Beckman Coulter India, Ltd., Mumbai. Parameters like BT(sec), CT(sec), Hemoglobin (g/dL), RBC (mill/mcl), WBC (Thous/mcl), Hemoglobin Concentraion(g/dl) were studied. All the parameters are with-in the normal range and no significant changes were marked.
Table 6: Biochemical and haematological report
Parameters Group I Group II Group III Group IV Group V
BT(sec) 71±2.1602 72.5±2.6457 75.75±3.86221 73±3.5590 71.25±3.4034 CT(sec) 49.25±2.21735 59.5±3.1091 63.75±3.0956 49.25±3.8622 50.5±1.9148 Hb (g %) 14.2±1.29 13.22±0.56 13.13±1.09 13.65±0.49 14.5±3.1091 Total RBC
(mm3)
7.29±0.22 6.49±1.67 6.95±0.62 7.59±0.40 7.25±1.2583 Total WBC
(mm3)
7.15±0.49 6.89±0.3 6.88±0.80 7.51±0.99 10±1.8257 ESR (mm/h) 6.50±1.05 6.83±0.75 7.00±0.89 7.00±0.89 6.89±0.3 SGPT (IU/L) 63.33±2.70 61.07±4.35 62.62±2.82 62.39±3.70 62.25±3.7749 SGOT (IU/L) 57.68±1.69 57.5±1.89 56.68±1.02 57.45±1.89 64.25±3.7749 ALP (IU/L) 351.17±8.35 338.5±15.93 343±6.78 340.83±12.45 157.5±3.7859
Glucose (%)
100.00±9.42 103.50±7.01 91.67±2.8 93.17±10.6 91.75±3.0956
4. Conclusion
Stable Glibenclamide loaded PLGA NPs were productively prepared by emulsification solvent evaporation method with varying the Glibenclamide PLGA ratio. Encapsulation efficiency of the prepared NPs was substantially improved with adequate drug content. Also the release of drug was sustained with the polymer concentration indicating its prospective application in drug delivery. Thus Glibenclamide loaded PLGA NPs could reduce dosing frequency leading to improve patient compliance. These NPs can be promising agents for rational drug delivery in diabetes. The in vivo results obtained after oral administration of nanoparticles to healthy female rats were found to be satisfactory. The developed nanoparticles are safer, and are the need of the hour for pharmaceutical industry as an alternative drug delivery system for the treatment of highly prevalent and chronic disease like type II diabetes mellitus.
Acknowledgments
The authors are thankful to Alembic research Centre, Gujarat, India, for providing free gift sample of glibenclamide. The authors are also thankful to Head of the Department, University Department of Pharmaceutical Sciences, Utkal University, Bhubaneswar, Orissa, India for providing research facility.
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