RESEARCH ARTICLE

Formulation and development of nateglinide loaded sustainedrelease ethyl cellulose microspheres by O/W solvent emulsificationtechnique

Gokul Khairnar • Vinod Mokale • Jitendra Naik

Received: 31 October 2013 / Accepted: 22 January 2014

� The Korean Society of Pharmaceutical Sciences and Technology 2014

Abstract The aim of this study was to investigate the

combined influence of 3 independent variables in the

preparation of sustained release Nateglinide (NTG)

microspheres by O/W solvent emulsification method. A

3-factor, 3-level Box–Behnken design was used to derive

second order polynomial equation and construct contour

plots to predict responses. The independent variables

selected were polymer concentration (A), surfactant con-

centration (B) and speed of the stirrer (C). Percentage drug

loading (Y1) and Percentage drug release (Y2) were con-

sidered dependent variables. The prepared microspheres

were evaluated for percentage of yield, drug loading, drug

release study in 6.8 phosphate buffer, Fourier transform

infrared (FT-IR), X-ray diffraction (XRD), scanning elec-

tron microscopy analysis. Contour plots were constructed

to show the effects of of A, B, C on Y1 and Y2. The yield

of microspheres was found to be in the range of

42.29–97.22 %. The drug loading was found to be in the

range of 12.18 % (F9) to 24.55 % (F14). FT-IR analysis

revealed no drug excipient interference. The morphology

of evaluated microspheres were found to be spherical and

smooth in nature. In XRD analysis crystalline pattern of

pure NTG was changed to amorphous pattern when con-

verted to microspheres. Out of 17 batches, formulation

batches F1, F4, F12, F14, F17 had percentage drug loading

52.75, 50.78, 43.88, 47.45, 44.78 % at 10 h respectively

which indicated excellent sustained drug release pattern.

From this study it was concluded that NTG loaded ethyl

cellulose sustained release microspheres were developed

using 3-factor, 3-level Box–Behnken design.

Keywords Microsphere � Nateglinide �Box–Behnken design � Sustain drug delivery �Drug loading � Drug release

Introduction

Nateglinide (NTG), a novel oral hypoglycemic agent, is a

nonsulfonylurea anti-diabetic agent that stimulates insulin

secretion via the same mechanism as that by which insulin

secretion is stimulated by sulfonylureas, although NTG

induces a more rapid and briefer decrease in blood glucose

level than do sulfonylureas (Shinkai et al. 1988; Fujitani

and Yada 1994; Akiyoshi et al. 1995; Ikenoue et al. 1997).

After oral administration, NTG is rapidly absorbed with an

average elimination half-life of 1.5 h. The drug demon-

strates linear pharmacokinetics over a dose range of

60–240 mg, and the time to peak concentration is not dose

dependent (Keilson et al. 2000). NTG is metabolized pre-

dominantly by cytochrome P450 2C9 to its hydroxy and

glucuronide metabolites. The major metabolites of NTG are

[3 times less potent antidiabetic agents than nateglinide

(Weaver et al. 2001; Takesada et al. 1996). NTG is a white

powder with a molecular weight of 317.43. It is practically

insoluble in water. NTG immediate release tablets are

available under the brand name of Starlix� containing 60,

or 120 mg, of NTG for oral administration. Starlix� (NTG)

should be taken 1–30 min prior to meals. The recom-

mended starting and maintenance dose of Starlix�, alone or

in combination with Metformin or a Thiazolidinedione, is

120 mg three times daily before meals (Nateglinide tablet,

US FDA label). So to repeat administrations have been

G. Khairnar � V. Mokale � J. Naik (&)

Department of Pharmaceutical Technology, University Institute

of Chemical Technology, North Maharashtra University, Umavi

Nagar, Post Box No. 80, Jalgaon 425 001, Maharashtra, India

e-mail: jitunaik@gmail.com

123

Journal of Pharmaceutical Investigation

DOI 10.1007/s40005-014-0118-3

required to achieve efficient glucose control and patient

relief. Due to the repeated administration of the NTG

complications and patient in compliance may result.

Microspheres designed for oral treatment target the

gastrointestinal (GI) tract, and encapsulation can enhance

GI treatments. Toxic drugs, which can cause side effects

when administered in large quantities, or insoluble drugs,

which may require large doses to promote absorption, can

be administered with a lower frequency and smaller

quantity (Davis et al. 1984). Sustain release formulations

either microspheres or nanoparticles by single dose would

be clinically important means of extending the duration of

action of NTG. Sustain release formulation with reduced

dose could be an interesting and suitable way to mini-

mizing the complications and improving the patient com-

pliance (Naik et al. 2013; Subhedar et al. 2010).

Traditional experiments require more effort, time, and

materials when a complex formulation needs to be devel-

oped. The various experimental designs are useful in

developing a formulation requiring less experimentation

and providing estimates of the relative significance of

different variables (Gohel and Amin 1998; Li et al. 2001;

Nazzal and Khan 2002). The work reported in this paper, a

Box–Bhenken design (Box and Behnken 1960) was used to

optimize microspheres containing NTG as a drug and EC

20 CP as a sustained release polymer. Independent vari-

ables selected were polymer conc. (A), surfactant conc.

(B) and speed of stirrer (C) to evaluate their separate and

combined effects on drug loading (DL) and drug release

(DR) as dependent variables.

The aim of this work was to prepare NTG loaded ethyl

cellulose (EC) microspheres which would deliver NTG at a

sustained rate for a prolonged period of time. Development

of sustained drug delivery system for oral formulation of

drugs will definitely bring a reduction in daily dose and

cost effective. It can reduce the chance for both under and

overdosing as well as number of repeated administration,

provide more localized to better use of active agents and

increase patient compliance.

Materials and methods

Materials

Nateglinide (NTG) was a kind gift sample from the

Wockhardt Research lab (Aurangabad, India). Ethyl cel-

lulose 20cp viscosity grade (EC), Dichloromethane

(DCM), Tween 80, n-hexane were obtained from Merck

Specialties Private Limited (Mumbai, India). Potassium

dihydrogen phosphate and Disodium hydrogen phosphate

were obtained from RFCL Limited (New Delhi, India). All

other chemicals used were of analytical grade.

Methods

Preparation of NTG loaded microspheres (Guyot

and Fawaz 1998)

The O/W emulsion solvent evaporation method was

applied with slight modification, to the fabrication of NTG

loaded EC microspheres. Briefly, NTG and EC were dis-

solved in 20 ml DCM (Organic Phase) and sonicated for

10 min to get clear, transparent solution. The resulted

organic phase was transferred into a 10 ml syringe and

added drop by drop into 100 ml distilled water containing

varying concentrations of Tween 80 as an emulsifier as an

external aqueous phase maintaining different RPM of

overhead stirrer (Remi electrotechnik Limited, Thane,

India) to produce O/W emulsion. 20 ml of n-hexane was

added to the emulsion for the hardening of the micro-

spheres. Stirring was continued up to complete evaporation

of DCM. Emulsions containing solid microspheres were

separated by vacuum filtration, washed with n-hexane, air

dried and used for further evaluation.

Experimental design

During the development of NTG microspheres multiple

initial trials were conducted to improve the drug loading of

the NTG. The preliminary experiments include the change

in drug: polymer ratio, surfactant concentration and speed

of the stirrer. During these batches we found that these

selected independent variables had a great influence on the

DL and DR (data and shown) and hence decided to select

as an independent variable.

The Box–Behnken design was specifically selected,

since it requires fewer runs than a central composite design

in cases of three or four variables. This cubic design is

characterized by a set of points lying at the midpoint of

each edge of a multidimensional cube and center point

replicates (n = 3), whereas the ‘‘missing corners’’ help the

experimenter to avoid the combined factor extremes (Box

and Behnken 1960). A 3-factor, 3-level Box–Behnken

design was used to derive a second order polynomial

equation and construct contour plots to predict responses.

The independent variables selected were the amount of

polymer (X1, mg), amount of Tween 80 as a surfactant

(X2, % v/v) and speed of stirrer (X3, RPM). Drug loading

(DL) (Y1 %) and Drug release (DR) (Y2 %) were selected

as dependent factors. The transformed values of the inde-

pendent variables and the dependent variable were sub-

jected to multiple regressions to establish a full-model

second-order polynomial equation. Dependent and inde-

pendent variables along with different levels are presented

in table no 3. The polynomial equation generated by this

experiment is as follows:

G. Khairnar et al.

123

Yi = b0þb1 X1þb2 X2þb3 X3þb12 X1 X2þb13 X1 X3

þb23 X2 X3þb11 X1 2þb22 X22þb33 X33:

(where Yi is the dependent variable; b0 is the intercept; b1

to b33 are the regression coefficients; and X1, X2 and X3

are the independent variable that was selected from the

preliminary experiments.)

Evaluation of microspheres

Yield of microspheres:

Nateglinid (NTG) loaded microspheres from each batch

were weighed accurately and yield of microspheres was

calculated by using a formula (Ziyaur et al. 2006).

% Yield ¼Weight of dried microsphere/Weight of drug

+ Weight of polymer� 100

Determination of DL

Microspheres equivalent to 10 mg of Nateglinide were

weighed and stirred in sufficient quantity of DCM to dis-

solve the polymeric coat. To this solution sufficient amount

of 6.8 phosphate buffer was added to the extraction of the

drug. Stirring was continued till complete evaporation of

DCM. This dispersion was filtered through Whatman filter

paper. The filtrate containing drug was analyzed after

dilutions at 210 nm by UV spectrophotometer (HITACHI

U-2900, Tokyo, Japan). From an absorbance DL was cal-

culated by using the following formula (Ziyaur et al. 2006).

% DL = Drug Entrapped / Weight of Microspheres� 100

In vitro dissolution studies

In vitro dissolution studies for 12 h were performed using

XXVIII apparatus, Type I (rotating paddle method). NTG

loaded microspheres equivalent to 60 mg of NTG were

introduced into the dissolution medium (6.8 phosphate

buffer) maintaining a stirring speed of 100 RPM and

temperature of dissolution medium 37 ± 0.5 �C. Aliquots

of the dissolution medium were withdrawn at predeter-

mined time intervals and replenished with fresh dissolution

media to maintain the sink condition. The samples were

filtered through Whatman filter paper no 41. Samples were

analyzed spectrophotometrically by a UV spectrophotom-

eter (HITACHI U-2900, Tokyo, Japan) at 210 nm. The

means of three determinations were given. Corrections

were made for any observance due to EC.

Fourier transform infrared (FTIR) spectroscopy study

The infrared spectra of NTG, EC, and NTG loaded

microspheres were obtained by using a FTIR spectropho-

tometer (FTIR-8400; Shimadzu, Asia Pacific Pvt. Ltd.

Singapore) by the potassium bromide pellet method. For

that, sample (1 mg) was mixed with potassium bromide

(40 mg) and formed into disc in a manual press. Spectra

were recorded in the scan range of 4,400–500 cm-1.

Table 1 % yield of microspheres, % DL and % DR at t = 10 h

Batch No Total solid content Wt of microspheres % Yield % DL % DR at t = 10 h

F1 720 700 97.22 21.00 52.75

F2 1,200 930 77.50 16.42 29.11

F3 960 533 55.52 18.58 25.08

F4 720 548 76.11 22.39 50.78

F5 960 698 72.71 17.09 29.56

F6 1,200 950 79.17 13.97 32.51

F7 960 712 74.17 17.20 30

F8 960 629 65.52 14.48 13.88

F9 1,200 1,056 88.00 12.18 12.9

F10 960 406 42.29 18.80 32.9

F11 960 754 76.54 18.30 35.28

F12 1,200 865 72.08 14.75 43.88

F13 720 390 54.17 20.50 84.27

F14 960 652 67.91 24.55 47.45

F15 960 695 72.39 18.90 28.3

F16 720 332 46.11 23.18 82.75

F17 960 714 74.37 15.52 44.78

Formulation and development of nateglinide loaded sustained release ethyl cellulose

123

X-ray diffraction (XRD) study

The physical state (either crystalline or amorphous) of

NTG, EC and NTG loaded microspheres was explored by

X-ray diffraction. Powder X-ray diffractometer was carried

out with X-ray diffractometer (Miniflex, Rikagu) using Ni

filtered, Cu-ka radiation (k = 1.5406 A), a voltage of

40 kV and a current of 40 mA. The scanning rate was

0.06�/min over a 2h range of 20�–80�.

Scanning electron microscopy (SEM)

Scanning electron microscopy (FESEM-S 4800, Hitachi,

Japan) was used to evaluate the shape, size and surface

characteristics of the microspheres. The SEM was operated

at a distance of 8.6–8.7 mm and accelerating voltage of

1.0 kV.

Result and discussion

Yield of microspheres

Sustained release NTG loaded microspheres were suc-

cessfully formulated by an O/W solvent evaporation

method using Box-Bhenken experimental design. The yield

of microspheres was found to be in the range of

42.29–97.22 % of total solid content employed during the

formulation of microspheres. Table 1 represents the sum-

mary of the yield of microspheres. The graphical repre-

sentation is illustrated in Fig. 1. Wide variation was found

in yield of microspheres. It was found that during the

collection of microspheres from the external phase some of

the precipitated polymer was stuck on the surface of stirrer

and some of the microspheres were adhered to the wall of

the container this might be the reason for low yield in some

of the batches.

Statistical analysis of DL and DR

DL

The drug loading was found to be in the range of 12.18 %

(F9) to 24.55 % (F14). Table 1 represents the DL of all

batches. Table 2 shows A Box–Behnken experimental

design with 3 independent variables (A, B, C) at 3 different

levels (-1, 0, ?1) was used to study the effects on the

dependent variables (Y1, Y2). Transformed values of all

the batches along with their results are shown in Table 3.

Formulation batches F1, F4, F13, F14, and F16 had highest

DL ([20 %). Table 4 shows the observed and predicted

values with residuals for all the batches. The second order

polynomial equation obtained for DL (Y1) was given by

Fig. 1 Effect of A and B on %

DL at C = 1,000

Table 2 Variable and three levels

Independent variable Low level

(-1)

Medium

level (0)

High

level (?1)

A = Polymer conc. 480 720 960

B = Surfactant conc. 0.2 0.5 0.8

C = Speed 800 1,000 1,200

Dependent variables

Y1 = % DL

Y2 = % DR

G. Khairnar et al.

123

Y1 = 18:11� 3.72A� 0.84Bþ 1.15C� 1.16AB

þ 1.12AC� 2.91BC:

A positive value in the regression equation for a response

represents an effect that favors the optimization (syner-

gistic effect), while a negative value indicates an inverse

relationship (antagonistic effect) between the factor and the

response. The value of correlation coefficient (R2) was

found to be 0.9040, indicating good fit, as shown in

Table 5. The DL values measured for the all batches

showed wide variation from minimum 12.18 % to maxi-

mum 24.55 %. The results showed that DL affected by the

independent variables like A, C and BC. In regression

equation Y1, the main effects of A, B and C represents the

average results of changing 1 variable at a time from its

low level to high level. The interaction terms AB, AC and

BC show how the DL changes when 2 variables are

simultaneously changed. The negative coefficients of the

independent variables indicate an unfavorable effect on the

DL. The positive coefficients of the independent variables

indicate favorable effect on the DL. As shown in Table 6,

the Model F value of 15.69 implies the model is significant.

There is only a 0.01 % chance that a ‘‘Model F Value’’ this

large could occur due to noise. Values of ‘‘Prob [ F’’

\0.0500 indicates model terms are significant. In this case

A, C and BC are significant model terms. Values [0.1000

indicate the model terms are not significant.

The effects of A and B with their interaction on the DL

at C = 1,000 are shown in Fig. 1. It was determined from

the contour plot that the maximum DL ([20 %) could be

obtained with an A level below 603 and B level 0.40 at

C = 1,000. From this it was clear that as polymer and

surfactant concentration decreases DL increases when

speed was maintained at 1,000. The effects of A and C with

their interaction on DL at B = 0.50 are shown in Fig. 2. It

was determined from the contour plot that maximum DL

([20 %) could be obtained with an A level below 591 and

C level below 978 when B = 0.50 %. The effects of B and

C with their interaction on the DL at A = 720 are shown in

Fig. 3. Maximum DL could be obtained with B level below

0.38 and speed above 1,143. Collectively from contour

plots it was concluded that when polymer concentration

was maintained at ?1 level % DL was found to be

12.18–16.42 % which was least as compared to the other

levels. Also when polymer concentration was maintained at

0 level DL was found to be in the range of 14.48–18.90 %

except 24.55 % in batch no F14. The highest level of DL

was achieved when polymer concentration was maintained

at -1 level. In this case DL was found to be in the range of

20.50–23.18 %. Therefore DL was found in this order

-1 [ 0 [ ? 1. In case of surfactant concentration there

was no linear relationship between surfactant concentration

and DL. When the surfactant concentration was maintained

at -1 level the DL was found to be in the range of

14.48–24.55 %. 12.18–22.39 % DL was achieved when

surfactant concentration was maintained at 0 levels. The

DL 13.97–23.18 % was achieved with surfactant concen-

tration maintained at ?1 level. A linear relation was found

Table 3 Formulation of NTG microspheres using Box–Behnken design

Batches Factor Resopnse

A B C Y1 Y2

F1 -1 -1 0 21.00 52.75

F2 ?1 -1 0 16.42 29.11

F3 0 0 0 18.58 25.08

F4 -1 0 -1 22.39 50.78

F5 0 ?1 -1 17.09 29.56

F6 ?1 ?1 0 13.97 32.51

F7 0 0 0 17.20 30

F8 0 -1 -1 14.48 13.88

F9 ?1 0 -1 12.18 12.9

F10 0 0 0 18.80 32.9

F11 0 0 0 18.30 35.28

F12 ?1 0 ?1 14.75 43.88

F13 -1 0 ?1 20.50 84.27

F14 0 -1 ?1 24.55 47.45

F15 0 0 0 18.90 28.3

F16 -1 ?1 0 23.18 82.75

F17 0 ?1 ?1 15.52 44.78

Formulation and development of nateglinide loaded sustained release ethyl cellulose

123

in case of speed of the stirrer. As the speed of the stirrer

increases DL also increases. The effect of speed on the DL

was found to be significant (P = 0.0368). 13.97–23.18 %

DL was achieved when speed was maintained at -1 level.

DL was found to be in the range of 13.97–23.18 % when

speed was maintained at 0 levels. Highest DL was found

when speed was maintained at ?1 level.

In vitro dissolution studies

In vitro dissolution study was performed in 6.8 phosphate

buffer because the media shows three times solubility of

maximum dose of NTG (180 mg) in liter of the buffer.

Formulation batches F1, F4, F12, F14, F17 had DR 52.75,

50.78, 43.88, 47.45, 44.78 % at 10 h respectively (Table 1).

The graphical representation was illustrated in Fig. 4. All

batches showed the initial burst release due to surface bound

drug on microspheres which was unremoved during wash-

ing of the microspheres with n-hexane. These four batches

showed excellent sustain drug release pattern in 6.8 phos-

phate buffer. In batch F1, F4 and F17, initial sudden increase

in drug release were observed; it might be due to the

immediate release of surface associated drug. Further, a very

slow release phase of encapsulated nateglinide was

observed in 10 h from EC microspheres. The reason behind

the decrease in drug release content after 2 h of study might

Table 4 Diagnostics case

statistics for various response

variables

Batch no Response variables Actual value Predicted Value Residual

F1 Y1 21.00 21.50 -0.50

Y2 52.75 55.85 -3.10

F2 Y1 16.42 16.38 0.039

Y2 29.11 31.11 -2.00

F3 Y1 18.58 18.11 0.47

Y2 25.08 30.11 -5.23

F4 Y1 22.39 21.79 0.60

Y2 50.78 52.19 -1.41

F5 Y1 17.09 19.03 -1.94

Y2 29.56 30.15 -0.59

F6 Y1 13.97 12.39 1.58

Y2 32.51 29.41 3.10

F7 Y1 17.20 18.11 -0.91

Y2 30.00 30.31 -0.31

F8 Y1 14.48 14.89 -0.41

Y2 13.88 9.37 4.51

F9 Y1 12.18 12.13 0.055

Y2 12.90 15.41 -2.51

F10 Y1 18.80 18.11 0.69

Y2 32.90 30.31 2.59

F11 Y1 18.30 18.11 0.19

Y2 35.28 30.31 4.97

F12 Y1 14.75 16.65 -1.90

Y2 43.88 42.47 1.41

F13 Y1 20.50 21.86 -1.36

Y2 84.27 81.76 2.51

F14 Y1 24.55 23.00 1.55

Y2 47.45 46.86 0.59

F15 Y1 18.90 18.11 0.79

Y2 28.30 30.31 -2.01

F16 Y1 23.18 22.15 1.03

Y2 82.75 80.75 2.00

F17 Y1 15.52 15.51 0.012

Y2 44.78 49.29 -4.51

G. Khairnar et al.

123

be due to the dilution of the dissolution media in order to

maintain the sink condition and very sustain release of drug

from the microspheres during this period. The polynomial

equation was also developed for second dependent factor Y2

i.e. DR. The second order polynomial equation given by

Y2 ¼ 30.31� 19.02Aþ 5.80Bþ 14.16Cþ 16.50A2

þ 2.46B2 þ 1.14C2 � 6.65AB� 0.63AC� 59BC:

The value of correlation coefficient (R2) was found to be

0.9768, indicating good fit, as shown in Table 5. The DR

values measured for all the batches showed wide variation

from minimum 12.9–84.27 %. As shown in Table 6 The

Model F value of 32.81 implies the model is significant.

There is only a 0.01 % chance that a ‘‘Model F Value’’ this

large could occur due to noise. Values of ‘‘Prob [ F’’

\0.0500 indicates model terms are significant. In this case

A, B, C, A2 AB are significant model terms. Values

[0.1000 indicate the model terms are not significant.

The relationship between the dependent and indepen-

dent variables was further elucidated by constructing the

contour plots. The effects of A and C with their interaction

on DR at surfactant conc. 0.50 are shown in Fig. 5. The

plots were found to be linear up to 61.93 % DR, above this

Table 5 Summary of results of regression analysis for responses Y1 and Y2

Models R2 Adjusted R2 Predicted R2 SD Press Remarks

Response Y1

Linear 0.6703 0.5942 0.3210 2.19 128.42 –

2FI 0.9040 0.8464 0.5533 1.35 84.49 Suggested

Quadratic 0.9215 0.8206 -0.1120 1.46 210.31 –

Cubic 0.9900 0.9602 0.69 –

Response Y2

Linear 0.7458 0.68 0.5449 11.18 2908.72 –

2FI 0.7869 0.6590 0.2253 11.67 4951.40 –

Quadratic 0.9768 0.9471 0.7716 4.60 1460.05 Suggested

Cubic 0.9902 0.9606 – 3.97 – –

Regression equations of the fitted models

Y1 = 18.11 - 3.72A - 0.84B ? 1.15C - 1.16AB ? 1.12AC - 2.91BC

Y2 = 30.31 - 19.02A ? 5.80B ? 14.16C ? 16.50A2 ? 2.46B2 ? 1.14C2 - 6.65AB - 0.63AC - 4.59BC

Table 6 ANOVA of models for Y1 and Y2

Source DF Sum of squares Mean square F value P value

Model for Y1 6 170.97 28.49 15.69 0.0001

A 1 110.63 110.63 60.93 <0.0001

B 1 5.59 5.59 3.08 0.1097

C 1 10.53 10.53 5.80 0.0368

AB 1 5.36 5.36 2.95 0.1165

AC 1 4.97 4.97 2.74 0.1289

BC 1 33.87 33.87 18.66 0.0015

Model for Y2 9 6243.24 693.69 32.81 <0.0001

A 1 2893.70 2893.70 136.86 <0.0001

B 1 269.24 269.24 12.73 0.0091

C 1 1603.48 1603.48 75.84 <0.0001

A2 1 1146.87 1146.87 54.24 0.0002

B2 1 25.56 25.56 1.21 0.3079

C2 1 5.49 5.49 0.26 0.6261

AB 1 176.89 176.89 8.37 0.0232

AC 1 1.58 1.58 0.074 0.7928

BC 1 84.18 84.18 3.98 0.0862

The model P value should be less than 0.05 indicate the significance (Bold) of the model and terms

Formulation and development of nateglinide loaded sustained release ethyl cellulose

123

value, the plots were found to be nonlinear indicating a

nonlinear relationship. It was determined from the contour

plot that sustain drug release pattern (43–53 % at 10 h)

could be obtained with an A level range 563–628, and the

B level in the range of 1,040 to 1,044. Figure 6 shows the

contour plots drawn at -1 level of C. The contours of all

the %DR were found to be curvilinear and indicated that

sustain drug release pattern (43–53 % at 10 h) could be

obtained with an A level range 560–618, and the B level in

the range of 0.57–0.55. Figure 7 shows the contour plots

drawn at 0 level of A. The contours of the entire DR were

found to be curvilinear and 43 % DR at 10 h could be

obtained when B is 0.56 and C is 1,165. The prediction of

53 % DR is out of scope of this contour plot.

Fig. 2 Effect of A and C on %

DL at B = 0.50 %

Fig. 3 Effect of B and C on %

DL at A = 720

Fig. 4 % DR profile of NTG loaded Microspheres in 6.8 Phosphate

buffer

G. Khairnar et al.

123

Briefly when polymer concentration was maintained at

-1 level DR was found to be 50.75–84.27. At 0 level the %

DR was 13.88–47.45. At ?1 level DR was found to be

12.9–43.88. There was no linear relationship between

surfactant concentration and DR. When the surfactant

concentration was maintained at -1 level the DR was

found to be in the range of 13.88–52.75 %. 12.9–84.27 %

of DR was achieved when surfactant concentration was

maintained at 0 levels. 29.56–82.75 % of DR was achieved

when surfactant concentration maintained at ?1 level. A

linear relationship was found in case of speed of the stirrer.

As speed increases the DR also increases from

12.9–84.27 %. 12.9–50.78 % of DR was achieved when

speed maintained at 0 level. 29.11–84.27 and

44.78–84.27 % at -1 level.

FTIR study

As shown in Fig. 8, the characteristic bands of NTG were

observed at 1,647 cm-1 (C=O), 1,713 cm-1 (–COOH),

2,862–3,096 cm-1 (–CH2–cycloalkane) and 3,296 cm-1

Fig. 5 Effect of A and C on %

DR at B = 0.50 %

Fig. 6 Effect of A and B on %

DR at C = 1,000

Formulation and development of nateglinide loaded sustained release ethyl cellulose

123

(–NH stretching). However, the IR spectrum of the NTG-

EC 20 CP microspheres showed the respective character-

istic bands of NTG at 1,647, 1,713, 2,862–3,096 and

3,296 cm-1. The results confirmed that there was no

chemical interaction between NTG and EC 20 CP polymer.

Surface morphology study

The surface morphology of NTG loaded microspheres were

analyzed by scanning electron microscopy. The FE-SEM

photographs of the surface of microspheres are shown in

the Fig. 9 The microspheres were discrete and free flowing

in nature. The sphericity of the microspheres was good.

The particle size of the microspheres was in the range of

58.5–95.8 l. The surface of the microsphere was smooth

and regular without any erosion and cracking. No pores

were spotted on the surface of the microsphere and this

could be the reason for the sustain release of the drug from

the microspheres. Due to the lack of pores and cracks there

would have been less chances of the drug to leach out from

the polymeric coat of the ethyl cellulose.

X-ray diffraction study

As can be seen from Fig. 10 crystalline peaks of the pure

NTG drug clearly disappears in NTG loaded microspheres.

Pure NTG was found to be 82.8 % crystalline and 17.2 %

amorphous in nature. But when NTG was loaded in EC

microspheres, the crystalline peaks of NTG were disap-

peared. NTG was found to be 42.9 % crystalline and

57.1 % amorphous in nature when converted into micro-

spheres of EC. These evidences indicated that NTG had

been highly disordered and distributed homogeneously in

amorphous form in microspheres.

Fig. 7 Effect of B and C on %

DR at A = 720

Fig. 8 FTIR study: IR of NTG loaded EC microspheres (a), IR of EC

20 CP (b), IR of pure NTG (c)

G. Khairnar et al.

123

Conclusion

The present study reports the development of sustained

release Nateglinide loaded ethyl cellulose microspheres by

an O/W solvent evaporation method using Box-Bhenken

statistical design with the percentage DL of 12.18–24.55.

The 4 batches showed 43.88–52.75 drug release within

10 h. The release of NTG drug was slow and extended over

a longer period of time. The prepared microspheres were

found to be smooth, spherical and without any cracks and

pores on the surface. Pure NTG had been highly disordered

and distributed homogeneously in amorphous form in

microspheres. From the present study it was concluded that

sustained release microspheres of NTG could be developed

and it would bring a reduction in dose and possible side

effects of the NTG.

Fig. 9 SEM analysis: Images showing sphericity, particle size and smooth surface of microspheres

Fig. 10 XRD pattern: XRD of pure NTG (a), XRD of EC 20 CP (b),

XRD of NTG loaded microspheres

Formulation and development of nateglinide loaded sustained release ethyl cellulose

123

Acknowledgement The authors are grateful to Defense Research

and Development Organization, New Delhi, Govt. Of India, for

providing financial support, in terms of Extramural Research Project

[ERIP/ER/0903820/M/01/1379]. The authors also like to thank Uni-

versity Institute of Chemical Technology, North Maharashtra Uni-

versity, Jalgaon 425 001, Maharashtra, India for providing best

facility to carry out this research work.

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