Preparation and characterization of biodegradable

Asian Journal of Pharmaceutical Sciences 2012, 7 (2): 143-148

Preparation and characterization of biodegradable cefpodoxime proxetil nanocapsules Dhiraj Muratkar, Lakshya Untwal*, Jitendra Naik University Department of Chemical Technology, North Maharashtra University, Jalgaon, India Received 14 October 2011; Revised 29 December 2011; Accepted 17 February 2012

_____________________________________________________________________________________________________________

Abstract Owing to poor water solubility, drugs such as cefpodoxime proxetil show poor dissolution characteristics and in turn poor oral bioavailability. The aim of present study was to formulate biodegradable cefpodoxime proxetil nanocapules by interfacial deposition technique using poly-(D,L-lactic acid) (PLA) as polymer and to evaluate the prepared nanocapsules. PLA was selected as biodegradable polymer for the preparation after careful compatibility study between drug and polymer. The drug: polymer ratio and process were modified to get encapsulation efficiency of nearly 94%. Characterization shows that the prepared nanocapsules had round to oval shape with particle size in the range of 40-400 nm and a zeta potential of −50 ± 2 mV. In vitro drug release study shows that the prepared nanocapsules possess sustained release properties. Keywords: PLA; Modified release; Interfacial deposition technique; Bioavailability enhancement; Release kinetics ____________________________________________________________________________________________________________

1. Introduction

biodegradable polymeric nanocarriers, which protect encapsulated drug thus prolong the duration of the desired effects [1,8-10]. Nanocapsules are composed of either an oily or an aqueous core surrounded by a thin polymer membrane. Such nanocapsules can be prepared using techniques such as interfacial polymerization of a monomer or interfacial deposition of a preformed polymer [11,12]. Nanocapsules can act as smart drugs which can impart it property to target cancerous or diseased cells. The various advantages that nanocapsules possess include higher dose loading with smaller dose volumes, longer site-specific dose retention, rapid absorption of active drug substance, increased bioavailability of the drug, higher safety and efficacy [13]. Nanocapsules have been evaluated for their application in ophthalmic [14,15], respiratory [16], and parenteral [17] drug delivery. Various factors that affect the drug loading, drug release characters include properties of drug and polymer used, drug: polymer ratio and method applied [18,19]. Cefpodoxime proxetil (CP) a third generation cephalosporin is BCS class II drug. CP is a prodrug ester that gets hydrolyzed to its active form cefpodoxime in vivo, due to its poor water solubility (400 µg/ml) the drug has problems associated with poor dissolution characteristics and thus results in poor oral bioavailability [20-22]. Researchers have tried to enhance the solubility and bioavailability of cefpodoxime by applying various approaches like preparation of polymeric microcapsules [23], solid dispersion with urea using solvent evaporation method [24], oil in water submicron emulsion [25], self nanoemulsifying drug delivery system [26].

Many drug candidates face problems like poor absorption, rapid metabolism and elimination, toxicity due to drug distribution to other tissues (anticancer drugs), poor drug solubility, unpredictable bioavailability, etc. These problems need to be resolved so as to make the existing drugs successful for therapy [1,2]. One of the promising strategies to overcome these problems is use of nanotechnology. Unique properties like small size, high surface area, ease of suspending in liquids, deep access to cells and organelles, variable optical and magnetic properties are offered by nanoparticles as compared to micro or macro particles [3]. It is a known fact that the ability to target and control the drug release are important characteristics for an ideal drug-delivery [4]. Nanoparticles composed of biodegradable polymer possess several advantages such as improved stability of the therapeutic agents against degradation, controlled drug release, targeted drug delivery or modified biological distribution of drugs both at cellular and organ level. They also fulfill the stringent requirements placed on these delivery systems, such as biocompatibility and degradability within an acceptable period of time [5-7]. Nanocapsulation helps to lengthen the therapeutic window by designing __________ *Corresponding author. Address: University Department of Chemical Technology, North Maharashtra University, Jalgaon 425001, India. Tel: +91-9404458065 E-mail: [email protected]; [email protected]

1 143 1


Polylactic acid (PLA) also known as polylactide has been used as biodegradable polymer for controlled release of drugs. PLA is being presented as one of the promising biodegradable polymers and has been studied and worked upon for a long time. PLA can be subjected to largescale production, it is commercially available, can be manufactured by large number of techniques giving a wide range of grades. As it is relatively cheap with some remarkable properties, it becomes suitable for wide range of applications [27-29]. The present study was devised with an objective of formulating CP nanocapsules that may enhance bioavailability of CP and also provide sustained/controlled release by using a suitable biodegradable polymer. This paper describes formulation of CP nanocapsules using interfacial polymer deposition-solvent displacement method with PLA as the polymer and characterization of the same.

Differential scanning calorimetry (DSC) was carried out using on a Shimadzu DSC instrument. DSC was performed to study the thermal behavior of plain drug, polymer and the mixture of drug and polymer. The samples were heated in hermetically sealed aluminum pans under nitrogen flow (20 ml/min) at a scanning rate of 10˚C/min. 2.3. Preparation of nanocapsules The biodegradable nanocapsules containing CP were prepared as per the procedure described by Fessi et al [33,34]. In a beaker PLA was dissolved in 25 ml acetone, in another beaker CP was dissolved in 0.5 ml benzyl benzoate and then added to the polymer solution. The resulting organic phase was then poured in 50 ml of water containing 100 mg of poloxamer 188 under moderate magnetic stirring. The aqueous phase turned milky after some time and showed bluish opalescence which indicates the formation of nanocapsules. The acetone and water were evaporated under reduced pressure and the final volume adjusted to 15 ml. Several batches were prepared and the effect of change in drug: polymer ratio and oil core concentration on the encapsulation efficiency/drug loading was studied. The details are given in Table 1.

2. Materials and methods 2.1. Materials CP was gift sample from BlueCross Laboratories Pvt. Ltd. Nasik, India. PLA was synthesized in our laboratory, Poloxamer 188 was obtained from BASF global chemical company, benzyl benzoate and solvents of analytical grade used were purchased from various manufacturers/ suppliers.

2.4. Characterization of prepared nanocapsule 2.4.1. Determination of the pH of nanocapsules

2.2. PLA synthesis and characterization

pH of the prepared nanocapsules formulation was measured using a digital pH meter at room temperature. The pH of different batches is given in Table 1.

Synthesis of PLA was carried out using the ring opening polymerization process [30-32]. The synthesized PLA had properties similar to those of commercially available PLA. Its melting point determined by open end capillary method was 175˚C–177˚C, while the molecular weight, determined by the gel permeation chromatography was been found to be ~72000 and the polydispersity 1.3381 g/mol.

2.4.2. Determination of drug content Drug content was determined by dissolving 1 ml of prepared nanocapsules in 20 ml of acetonitrile. Appropriate quantity of sample was then subjected to the UV spectrophotometry at 232 nm. The absorbance for each sample was measured and compared with the standard.

2.3. Drug polymer compatibility studies Compatibility of drug with polymer was studied using DSC and IR spectroscopy. IR spectroscopy of pure CP, PLA and their physical mixture was carried out using FTIR spectrophotometer (Shimadzu). Infrared (IR) spectroscopy was conducted using a Fourier transform infrared spectrophotometer and the spectrum was recorded in the wavelength region of 4,000– 400 cm−1. The procedure consisted of dispersing a sample (drug, polymer and a mixture of drug and polymer) in KBr. The sample was placed in the light path in sample holder, and the spectrum was recorded. All spectra were collected at a resolution of 2 cm-1.

2.4.3. Structural characterization Microscopical characterization of nanocapsules was done by using transmission electron microscopy (TEM) and field emission scanning electron microscopy (FE-SEM) to determine various attributes like shape, size, and surface morphology. TEM micrographs of the nanocapsules were obtained using a Phillips CM 200 TEM operated at 20–200 kV while the FE-SEM was carried out using Hitachi S-4800 FE-SEM equipped with energy dispersive spectrometer (EDS). 1 144 1


Table 1 Concentration of components, drug loading and pH for different batches of nanocapsules. Batch No.

Drug (mg)

Polymer (mg)

Oil core (ml)

Surfactant (mg)

Drug loading (%)

pH

B1

10

50

0.5

100

63.38

4.26

B2

10

60

0.5

100

71.64

4.87

B3

10

70

0.5

100

84.58

5.6

B4

10

80

0.5

100

91.24

5.74

B5

10

90

0.5

100

93.46

5.92

B6

10

100

0.5

100

93.64

6.07

B7

10

90

0.4

100

83.26

4.09

B8

10

90

0.6

100

93.42

4.23

B9

10

90

0.8

100

93.45

4.27

B10

10

90

1

100

93.48

4.34

3. Results and discussion

2.4.4. Particle size distribution and particle charge/Zeta potential

3.1. Drug polymer compatibility studies

Particle size distribution (also known as polydispersity index) is an important aspect during the formulations of nanosystems. Nanocapsules were characterized for their particle size distribution and zeta potential using Malvern Zetasizer.

The IR spectra of pure drug and pure polymer were compared with the standard spectra and found to be similar. The spectra of physical mixture showed all characteristic peaks of pure drug and polymer, while no other peaks were observed (Fig. 1). DSC of drug polymer physical mixture was carried out and compared with the DSC of pure drug and pure polymer (Fig. 2). They did not show any considerable change in the melting endotherms. These studies indicated that PLA was compatible with CP and there were no undesired interactions between the physical mixture of drug and polymer.

2.4.5. In vitro drug release In vitro dissolution studies were carried out using USP type II dissolution apparatus (Electrolab TDT-06T). After carefully studying the characteristics of nanocapsules, sample from batch B6 was taken and its drug release was studied. The study was carried out in 100 ml of buffer (pH 3.0). The nanocapsule suspension was placed in dialysis membrane and dipped in dissolution medium which was kept in a thermostatically controlled water bath, maintained at 37 ± 0.5˚C. The stirring rate was maintained at 100 rpm. At predetermined time intervals, 5 ml of samples were withdrawn and assessed for drug release spectrophotometrically (λmax 232 nm). After each withdrawal, 5 ml of fresh dissolution medium was added to dissolution jar.

T (%)

(A)

2.4.6. Study of drug release kinetics Zero order model, first order model, Higuchi model, and Korsemeyer-Peppas model are vital in describing drug release phenomena in best manner. The physicochemical properties of the drug as well as polymer and the drug to polymer ratio modify the release kinetics as they govern the release of drug from the formulation. The data obtained from the in vitro release study was used to determine the best suited model for the release kinetics for the prepared CP nanocapsules.

(B)

(C)

3200

2400

1800 1400 1000 Wavenumber (cm-1)

600

Fig. 1. IR spectra of cefpodoxime proxetil, PLA and their physical mixture.

1 145 1


(A)

(A)

(B) Heat flow

(B)

(C)

50

100

accordance with the particle size obtained by TEM and FE-SEM. Zeta potential study which shows the surface charge on the particles, is an important determinant for the attractive/repulsive forces on the surface of the particles and in turn the stability. Literature reports that zeta potential values, above 30 mV (positive or negative values), leads to more stable nanocapsule suspensions as the repulsion between particles prevents their aggregation [36]. The zeta potential of the prepared nanocapsules was found to be −50 ± 2 mv, which is an indicator for good stability of the preparation. Thus from the zeta potential studies it can be concluded that the nanocapsules have good stability in the suspension form.

(C) 150

200

250

300

Temperature (˚C)

(A)

Fig. 2. DSC of cefpodoxime proxetil, PLA and their physical mixture.

3.2. Drug content and pH of nanocapsules Cefpodoxime is known to exhibit a pH dependent solubility behavior, with highest solubility in acidic pH [35], PLA too shows different degradation behavior at different pH. Hence the pH of the nanocapsule preparation is important and must be maintained to acidic or neutral. To achieve a better drug content and optimum pH we tried to modify the drug: polymer: oil core ratio and also adjusted the pH. Table 1 shows the drug content and pH of different batches. From the results it can be observed that the drug: polymer ratio in the range 1:8 showed the best results for drug loading, almost 94%. While the oil core (benzyl benzoate) gave the best results at 0.5 ml and any further increase in its concentration did not show any benefit. Batch B6 was selected as an ideal batch for carrying out further characterization as it had the most promising results for drug loading along with optimum drug: polymer: oil core ratio.

(B)

3.3. Structural characterization The morphological characterization was carried out using TEM and FE-SEM. TEM and SEM not only give a clear picture about the size and shape but also help in analyzing any defects or irregularities on the surface of the nanocapsules. The TEM (Fig. 3) and FE-SEM (Fig. 4) images showed round to oval shape nanocapsules with size ranging from 40 to 400 nm.

Fig. 3. TEM image of prepared cefpodoxime proxetil nanocapsules.

3.5. In vitro drug release study and study of drug release kinetics The percent drug release (concentration) versus time plot (Fig. 5), shows that the release from nanocapsules in the first hour was 10.66% (0.710 µg/ml). The study was carried out for 8 h and nearly 34% (2.26 µg/ml) drug was released from the nanocapsules during this time span. Assuming that the release follows this pattern, it can be

3.4. Particle size distribution and Zeta potential By using the zeta sizer, the average particle size of the nanocapsules was found to be ~333 nm, which is in 1 146 1


4. Conclusion

predicted that the drug will be released completely from the nanocapsules in about 29–30 h. The R2 values for the zero order, first order, Higuchi model and Korsemeyer-Peppas models were 0.920, 0.924, 0.935 and 0.940, respectively. As these values were very close and had marginal difference, it was difficult to predict exact fit model for the nanocapsules but as the R2 value for Korsemeyer-Peppas was highest (0.940) it can be proposed that the prepared CP nanocapsules followed the release kinetics for diffusion model. Another approach can be proposed for the release kinetics that the release first follows first order followed by zero order release. From these observations it can be concluded that the nanocapsules possessed characteristics of a sustained release formulation.

Nanoparticles prepared using biodegradable polymers have several advantages; they not only help to improve stability and therapeutic activity but also prolong the duration of action without any additional undesired effects. Nanocapsules have wide range of applications in drug delivery and present an interesting avenue for drugs like CP which have problems associated with poor dissolution characteristics and poor oral bioavailability. With this approach in mind and proper literature review we selected PLA as the biodegradable polymer and interfacial deposition technique for the preparation of CP nanocapsules. We can conclude that we were successful in preparing biodegradable CP nanocapsules. The prepared nanocapsules had round to oval shape with particle size in the range of 40–400 nm and a zeta potential of −50 ± 2 mv. The in vitro drug release and release kinetics studies show that the nanocapsules possess sustained release properties. With suitable modifications and addition of appropriate additives the prepared nanocapsules can be formulated to various dosage forms for oral, pulmonary or parenteral administration. Large surface area of the nanocapsules results in increased mass transfer rate and this in turn increases the dissolution rate as well as bioavailability of CP.

Acknowledgements We would like to acknowledge BlueCross Laboratories Pvt. Ltd. Nasik for providing the gift sample of cefpodoxime proxetil. The authors wish to thank Central Instrumentation Laboratory, UDCT NMU for IR, DSC studies, SAIF, IIT, Bombay for the TEM studies, NIPER, Mohali for Zeta Potential characterization, School of Life Sciences, NMU, Jalgaon for UV-Visible and Nanodrop spectrophotometric studies.

Fig. 4. FE-SEM image of prepared cefpodoxime proxetil nanocapsules. Query: Please include the release of reference formulation and test B1-B10 40

Drug release (%)

30

References

20

[1] Jain N.K. Pharmaceutical technology: pharmaceutical nanotechnology. Available at: http://nsdl.niscair.res.in/bitstr eam/123456789/748/1/revised+Pharmaceuticall+Nanotech. pdf. Accessed on 23 November 2010. [2] Mohanraj V J, Chen Y. Nanoparticles-a review. Trop J Pharm Res. 2006, 5: 561-573. [3] Gupta R B. Fundamentals of drug nanoparticles. In: Gupta R B, Kompella U B. ed. Nanoparticle technology for drug delivery. Taylor & Francis Group, New York. 2006: 17. [4] Thassu D, Pathak Y, Deleers M. Nanoparticulate drugdelivery dystems: an overview. In: Thassu D, Deleers M, Pathak Y. eds. Nanoparticulate drug-delivery Systems.

10 0 0

3

6

9

Time (h)

Fig. 5. Drug release for cefpodoxime proxetil nanocapsules (Batch B6).

1 147 1


[20] Borin M T. A review of the pharmacokinetics of cefpodoxime proxetil. Drugs. 1991, 42: 13-21. [21] Kakumanu V K, Arora V, Bansal A K. Investigation of physicochemical and biological differences of cefpodoxime proxetil enantiomers. Eur J Pharm Biopharm. 2006, 64: 255-259. [22] Kakumanua V K, Arora V, Bansal A K. Investigation of factors responsible for low oral bioavailability of cefpodoxime proxetil, Int J Pharm. 2006, 317: 155-160. [23] Khan F, Katara R., Ramteke S. Enhancement of bioavailability of cefpodoxime proxetil using different polymeric microparticles. AAPS PharmSciTech, 2010 doi: 10.1208/s12249-010-9505-x. [24] Arora S C, Sharma P K, Irchhaiya R. et al. Development, characterization and solubility study of solid dispersion of cefpodoxime proxetil by solvent evaporation method. Int J Chemtech Res., 2010. 2: 1275-1280. [25] Decroix M O, Manciet S C, Brossard D, et al. Cefpodoxime proxetil protection from intestinal lumen hydrolysis by oilin-water submicron emulsions. Int J Pharm. 1998, 165: 97-106. [26] Date A A, Nagarsenker M S, Design and evaluation of selfnanoemulsifying drug delivery systems (SNEDDS) for cefpodoxime proxetil. Int J Pharm. 2007, 329: 166-172. [27] Sinha V R, Trehan A. Biodegradable microspheres for protein delivery. J. Contr. Rel. 2003. 90: 261-280. [28] Kotwal V, Maria S, Inamdar N, et al. Biodegradable polymers: which, when and why? Ind. J. Pharm. Sci. 2009, 69: 615-622. [29] Krause, H.J., Schwartz, A., Rohdewald, P., Polylactic acid nanoparticles, a colloidal drug delivery system for lipophilic drugs. Int. J. Pharm. 1985, 27: 145-155. [30] Cheng Y, Deng S, Chen P, et al.,Polylactic acid (PLA) synthesis and modifications: a review. Front. Chem. China 2009, 4; 259-264 [31] Ajioka M, Enomoto K, Suzuki K. Basic properties of polylactic acid produced by the direct condensation polymerization of lactic acid. Bull.Chem.Soc. Jpn, 1995, 68: 2125 [32] Garlotta D. A literature review of poly(lactic acid) J Polym Environ, 2001, 9: 63-84. [33] Fessi H, Puisieux F, Devissaguet J P. Procédé de preparation de systems colloid aux dispersible d’une substance sous forme de nanocapsules. European Patent 274961 A1, 1988. [34] Fessi H, Puisieux F, Devissaguet J P, et al. Nanocapsule formation by interfacial polymer deposition following solvent displacement. Int J Pharm. 1989, 55: R1-R4. [35] Hamamura T, Ohtani T, Kusai A, et al. Unusual dissolution behavior of cefpodoxime proxetil: effect of pH and ionic factors. S.T.P. Pharm. Sci. 1995, 5: 332–338. [36] Aboubakar M, Puisieux F, Couvreur P, et al. Study of the mechanism of insulin encapsulation in poly(isobut ylcyanoacrylate) nanocapsules obtained by interfacial polymerization. J. Biomed. Mater. Res. 1999, 47: 568-576.

Informa Healthcare USA, Inc. New York, 2007: 6. [5] Rytting E, Nguyen J, Wang X, et al. Biodegradable polymeric nanocarriers for pulmonary drug delivery. Expert Opin Drug Deliv. 2008, 5: 629-639. [6] Beck-Broichsitter M, Gauss J, Packhaeuser C B, et al. Pulmonary drug delivery with aerosolizable nanoparticles in an ex vivo lung model, Int J Pharm. 2009, 367: 169-178. [7] Mehnert W, Mader K. Solid lipid nanoparticles production characterization and applications. Adv Drug Deliv Rev. 2001, 47: 165-196. [8] Chakravarthi S S, Robinson D H, De S, Nanoparticles prepared using natural and synthetic polymers. In: Nanoparticulate drug-delivery systems, Thassu D., Deleers, M., Pathak, Y. eds. Informa Healthcare USA, Inc. New York, 2007: 51. [9] Quintanar-Guerrero D, Allemann E, Fessi H, et al. Preparation techniques and mechanisms of formation of biodegradable nanoparticles from preformed polymers. Drug Dev Ind Pharm. 1998, 24: 1113-1128. [10] Soppimath K S, Aminabhavi T M, Kulkarni A R, et al. Biodegradable polymeric nanoparticles as drug delivery devices. J Control Release. 2001, 70: 1-20. [11] Couvreur P, Barratt G, Fattal E, et al. Nanocapsule technology: a review, Crit Rev Ther Drug Carrier Syst. 2002, 19: 99-134. [12] Pathak Y, Thassu D, Deleers M. Pharmaceutical applications of nanoparticulate drug-delivery systems. In: Thassu D, Deleers M, Pathak Y. eds. Nanoparticulate drugdelivery systems. Informa Healthcare USA, Inc. New York, 2007: 187-188. [13] Institute of Nanotechnology: Nanocapsules and dendrimers -properties and future applications. Available at: http:// www.azonano.com/article.aspx?ArticleID=1649 Accessed on 20 August 2011. [14] Zimmer A, Kreuter J. Microspheres and nanoparticles used in ocular delivery systems. Adv Drug Deliv Rev. 1995, 16: 61-75. [15] Bourlais C L, Acar L, Zia H, Sado P A, et al. Ophthalmic drug delivery systems-recent advances. Prog Retin Eye Res. 1998, 17: 33-58. [16] Hureaux J, Lagarce F, Gagnadoux F, et al. Lipid nanocapsules: ready-to-use nanovectors for the aerosol delivery of paclitaxel. Eur J Pharm Biopharm. 2009, 73: 239-246. [17] Wissing S A, Kayser O, Muller R H. Solid–lipid nanoparticles for parenteral drug delivery. Adv Drug Deliv Rev. 2004, 56: 1257-1272. [18] Cauchetier E, Deniau M, Fessi H, et al. Atovaquone loaded nanocapsules: influence of the nature of the polymer on their in vitro characteristics. Int J Pharm. 2003, 250: 273-281. [19] Guterres S S, Fessi H, Barratt G, et al. Poly-(D,L-lactide) nanocapsules containing diclofenac: I. Formulation and stability study. Int J Pharm. 1995, 113: 57-63.

1 148 1