Spray dried chitosan–EDTA superior microparticles as solid substrate for the oral delivery of amphotericin B

International Journal of Biological Macromolecules 58 (2013) 310–319

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Spray dried chitosan–EDTA superior microparticles as solid substrate for the oral delivery of amphotericin B Kuldeep Singh, A.K. Tiwary, Vikas Rana ∗ Pharmaceutics Division, Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala 147002, India

a r t i c l e

i n f o

Article history: Received 15 March 2013 Received in revised form 12 April 2013 Accepted 16 April 2013 Available online 23 April 2013 Keywords: Solid self emulsifying drug delivery Full factorial design Spray drying Microparticles Chitosan–EDTA conjugate Oral amphotericin B

a b s t r a c t The present investigation was aimed at synthesis of chitosan–EDTA superior microparticles (COECH) bearing high oil adsorbing and oil desorbing properties. These superior particles were prepared by thermal amide conjugation of COO− group of EDTA with NH2 group of chitosan employing spray-drying technique. The synthesis was optimized using 42 full factorial design. The particles showed high oil adsorbing capacity as well as oil desorbing capacity with enhanced dispersive components of surface free energy as compared to Aerosil 200. In addition, these COECH microparticles showed higher amphotericin B loading capacity, enhancement in the in vitro dissolution performance (12-fold) and produces nanoemulsion in the size range of 70–90 nm. Further, the results were in consonance with those observed during ex vivo performance. Thus, the findings revealed simple synthesis of COECH microparticles that showed superior properties of solid substrate for the development of amphotericin B nanoemulsion. © 2013 Elsevier B.V. All rights reserved.

1. Introduction In recent years, low-density porous carriers with large surface area like porous silica (Sylysia® ), magnesium aluminometasilicate (Neusilin® ) are used in order to improve dissolution and bioavailability of poorly soluble drugs such as carvedilol, indomethacin, fenofibrate, and ritonavir. Kang et al. [1] used various solid carriers of different properties, i.e. hydrophobic (silicon dioxide, magnesium stearate) and hydrophilic (PVA, Na-CMC and HP-␤-CD) solid carriers. All of these carriers had significant and positive effect on the crystalline property, dissolution rate and oral bioavailability of flurbiprofen in the solid self-nanoemulsifying drug delivery system (S-SNEDDS). Further, Aerosil 200, d-mannitol, gelatin, microcrystalline cellulose and lactose can also used as solid carrier in various formulations of S-SNEDDS [1]. However, these solid carriers bear lower oil carrying capacity, low bulk density and lower compressibility such that their solid dosage form could not be made. In addition, solid carrier for the preparation of S-SNEDDS containing light sensitive and thermolabile drugs is still at the initial stage of research. Hence, an attempt was made to develop a solid carrier that could be challenged to entrap enhanced amount of lipid phase without employing any heat based systems likes spray drying (amphotericin B as a poorly water soluble thermolabile drug) to form solid self nanoemulsifying drug delivery system.

∗ Corresponding author. Tel.: +91 9872023038. E-mail address: vikas [email protected] (V. Rana). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.04.053

Amphotericin B (AMB), a polyene macrolide antibiotic, is a well known fungicidal and a drug of choice for the treatment of visceral leishmaniasis. AMB was characterized by very low solubility of less than 1 mg/l in water or at physiological pH (pH 6–7). Owing to its amphipathic nature, AMB forms aggregates in water at concentrations around 2 × 10−7 M. These aggregates formed well below critical micellar concentration (ca. 3 ␮M) by interaction between neighboring polyene chains (chromophores). This may be the reason for its low water solubility that leads to poor gastrointestinal absorption. Thus, causes minimal oral bioavailability when it was given per os [2]. Hence, AMB belongs to BCS class IV compound (being limited solubility and permeability). Alternatively, AMB is administered via parenteral route in order to achieve concentrations that are sufficient to be effective for instance against systemic fungal infections. Various parenteral AMB products have been developed to overcome its low solubility. The liposomal formulation (AmbisomeTM ), micellar dispersion with deoxycholate (FungizoneTM ) and lipid complex (AbelecetTM ) are parenteral products available in the market. However, for such treatments, a patient needs to bear high cost of AMB injections, pain during and after injection and hospitalization (for parenteral injection). In addition, the acute side effects of parenteral AMB like haemolysis, fever, bone pain, thrombophebitis etc. limits its wide spread application [3,4]. Spray drying of chitosan (CH) salt solution is now a routine technique to prepare microspheres [5–7]. These microspheres were in the size range of 2–5 ␮m, free flowing powder, compressible and therefore most suitable as drug carrier. In addition, spray drying

K. Singh et al. / International Journal of Biological Macromolecules 58 (2013) 310–319

of CH salts at various inlet temperature 70–160 ◦ C provides CH microspheres that were varies in their solubility as well as swelling characteristics [5]. However, preparation of solution for spray drying to prepare CH microspheres is a tedious task. CH in acetic acid solution reacts immediately with its polyanionic agents (alginic acid, carboxy methylcellulose, acacia gum, etc.) to form gelatinous precipitates [8–10]. These gelatinous precipitates were unable to spray dry. Therefore, CH carbamate salt in alkaline medium was prepared to obtain clear solution with polyanion [11]. Thus, an attempt was made to identify a polyanionic agent that form clear solution in acetic acid solution of CH. This could avoid the formation of CH carbamate step necessary for the formation of CH microspheres. ethylenediaminetetraacetic acid is well known chelating agent having pH 2.2. It is soluble 1 in 500 parts of water. However, salt form of EDTA, i.e. EDTA disodium (EDTA-diNa) is 1 part soluble in 11 mL water. EDTA-diNa salt has a pH 4.5–4.7 [12]. Therefore, CH in acetic acid forms clear solution with EDTA-diNa [13,14]. Further, various attempts have been made to encapsulate drugs into the CH microspheres prepared by spray drying technology [15]. However, this technology was not suitable for poorly water soluble and thermolabile drugs. A chitosan covalently crosslinked with EDTA was prepared using free radical initiator and was found to show mucoadhesive, antibacterial and antifungal properties [11,16,17]. However, these free radical initiator techniques sometimes cause degradation of the polysaccharide backbone, giving rise to the products with complicated and ambiguous structures. Qu et al. [18] have reported the thermal amide conjugation of CH and d,l-lactic acid first at 80 ◦ C for 4 h to avoid evaporation of the monomer then put under a vacuum at 80 ◦ C for 3 h to promote dehydration of the CH copolymer salts with formation of the corresponding amide linkages. Hence, a possibility was to utilize the surface of microparticles prepared after spray drying should be subjected to drug adsorption in lipid phase. For this purpose, the microparticles should have enhanced hydrophobic surface [19]. Therefore, a need was felt to prepare microparticles via amide linkages to form interpenetrating network that provide microparticles with hydrophobic surface. In the light of the above, the present investigation was aimed to optimize the synthesis of covalently crosslinked EDTA–chitosan (COECH) microparticles employing 42 full factorial experimental design. The synthesized COECH microparticles were characterized and evaluated for the oral delivery of S-SNEDDS containing amphotericin B (a thermolabile, light sensitive and antifungal/antileishmaniasis drug). 2. Materials and methods 2.1. Materials Amphotericin B was purchased from HiMedia Laboratories Pvt. Limited (Mumbai, India). Chitosan (88–89% deacetylated) was purchased from India Sea Foods, (Cochin, India). Labrasol® (caprylocaproylmacrogol glycerides) and Labrafac PG were a gift from Gattefossé Canada (Mississauga, Ontario). Captex 355 was a gift from Abitec Corp. (Wisconsin) USA. Acetic acid, ethylenediaminetetraacetic acid disodium salt and isopropyl alcohol (Loba Chemie, Bombay, India) were used as supplied. All other chemicals used were of analytical grade and used as received. 2.1.1. Synthesis of covalently crosslinked chitosan–EDTA (COECH) microparticles The COECH microparticles were synthesized employing spray drier without the use of proton initiator. For the synthesis, chitosan (CH) was dissolved in 1 N HCl separately, ethylenediaminetetraacetic acid disodium salt (EDTA-diNa) was dissolved in 50 mL distilled water. CH solution was added dropwise to EDTA-diNa

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solution with constant stirring at 2000 rpm. This clear solution was spray dried using a laboratory scale spray drier (Labultima India, Mumbai, India) equipped with a standard 1 mm nozzle. The specifications (inlet temperature = 50–170 ◦ C, cool temperature = 30 ◦ C, inlet high temperature = 185 ◦ C, outlet high temperature = 175 ◦ C, aspirator flow rate = 40 Nm3 /h and feed rate = 1 mL/min) were used to spray dry the CH–EDTA-diNa solution. 2.1.2. Experimental design 42 full factorial design was selected to optimize synthesis of COECH. During preliminary studies, proportions of CH to EDTA (X1 ) and inlet temperature (X2 ) of spray drier were found to be the critical factors (independent variables). Percentage yield (Y1 ), particle size (Y2 ) and zeta potential (Y3 ) of spray dried COECH microparticles were used as dependent variables. 2.2. Identification of COECH 2.2.1. FTIR-ATR analysis A pure sample of CH, EDTA-diNa, COECH microparticles were subjected to FTIR-ATR analysis (RXI-JR, Alpha-E, Bruker, Germany) in the spectral region of 500–4000 cm−1 . 2.2.2. DSC analysis Differential scanning calorimetric thermogram of CH, EDTAdiNa, COECH microparticles were recorded using differential scanning calorimeter (Q10, TA Systems, USA) in the temperature range of 40–400 ◦ C and heating rate of 10 ◦ C per minute in nitrogen atmosphere (50 mL/min). 2.3. Characterization of COECH 2.3.1. Oil adsorbing capacity (OAC) of COECH For the estimation of OAC, ethanolic solution of Captex 355, Labrafac PG, Captex 355:Labrasol (70:30) and Labrafac PG:Labrasol (70:30) were prepared. These ethanolic solutions were mixed with COECH and lyophilized (Allied Frost, New Delhi, India). The OAC was estimated by weighing the pure COECH (Wa ) and weight of dried COECH (Wb ) obtained after the evaporation of ethanol from the blend of COECH and ethanolic solution of Captex 355, Labrafac PG, Captex 355:Labrasol (70:30) and Labrafac PG:Labrasol (70:30). The inclusion criterion for Wb was based on the initial screening of powder blends. Only those COECH/oil:Labrasol powder blends obtained after evaporation of ethanol that showed no significant changes in physical properties as that of pure COECH such as nonoily, non-sticky, free flowing and having similar texture as that of pure COECH were accepted for Wb . The OAC was estimated by the following formula. OAC =

Wb − Wa × 100 Wa

2.3.2. Oil desorbing capacity (ODC) of COECH The oil desorbing capacity was estimated by suspending oil adsorbed COECH (Wb ) in 10 mL of water. After stabilization for 1 h, the suspended particles were recovered by centrifugation (3000 rpm/min), dried and weighed (Wc ). The ODC was estimated by the following formula. ODC =

Wb − Wc × 100 Wc

2.3.3. Surface free energy components of COECH/Aerosil 200 The surface free energy components of COECH/Aerosil 200 were estimated as per the method reported by Chibowski and PereaCarpio [20]. The dispersive and polar components of surface free

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energy of COECH and Aerosil 200 before and after oil adsorption were estimated employing diiodomethane (llw = 50.8, l+ =

0, l− = 0); n-hexane (llw = 18.4, l+ = 0, l− = 0), dimethyl-

sulphoxide (DMSO; llw = 36, l+ = 0.5, l− = 32) and water (llw = 21.8, l+ = 25.5, l− = 25.5) as probe liquids. The advancing weight and reducing weight for each probe liquid obtained were incorporated into the following equation to determine dispersive and total polar components. Wa = 2[(lLW )

1/2

+ (s+ .l− )

1/2

+ (s .l+ )

1/2

]

where Wa is the work of adhesion of the liquid to the solid surface, slw is the dispersive (apolar Lifshitz–van der Waals),  + electron acceptor, and  − is the electron donor interactions. 2.3.4. Dynamic advancing contact angle The dynamic advancing contact angle ( a ) was determined as per the method reported by Chibowski and Perea-Carpio [20] and it is different from contact angle on a flat surface of same solid. During the estimation of surface free energy components, advancing weight of the probe liquid (water) in the capillary (ma ) as well as effective pore radius of solid (Reff.p ) were obtained. The following equation was used to estimate  a . cos a =

ma g 2Reff.p 

where g = 980 cm/s2 and  = 72.6 dyn/cm. 2.4. Fabrication of COECH solid self-nanoemulsifying drug delivery system (COECH-S-SNEDDS) For the preparation of COECH-S-SNEDDS, a homogenous mixture of 10%, w/v AMB in lipid phase (Captex 355: Labrasol::70:30) was prepared and added to ethanol:water (80:20, v/v). This homogenous mixture was adsorbed on COECH microparticles (1 g). This wet mass was subjected to lyophilization to evaporate water and ethanol. The dried product obtained was COECH-S-SNEDDS microparticles. This powder obtained was stored in polyethylene bags until further use. A similar method was used to prepare Aerosil 200 solid self nano emulsifying drug delivery system (Aerosil 200S-SNEDDS, a standard carrier generally used for the preparation of S-SNEDDS) instead of COECH. 2.5. Evaluation of COECH-S-SNEDDS 2.5.1. Morphological evaluation The outer macroscopic structure of COECH and COECH-SSNEDDS, Aerosil 200 and Aerosil 200-S-SNEDDS was investigated by scanning electron microscopy (S-4100, Hitachi, Japan) at 15 keV accelerating voltage. The sample was fixed on a SEM-stub using double-side adhesive tape and then coated with a thin layer of gold. 2.5.2. Flow properties The flow properties of COECH microparticles, COECH-SSNEDDS, Aerosil 200 and Aerosil 200-S-SNEDDS were evaluated by determining angle of repose, bulk density, tapped density, Hausner ratio and Carr’s index (%), as reported earlier [21]. 2.6. Evaluation of reconstituted nanoemulsion obtained from COECH-S-SNEDDS/Aerosil 200-S-SNEDDS 100 mg of COECH COECH-S-SNEDDS/Aerosil 200-S-SNEDDS was suspended in 10 mL of triple distilled water with constant stirring. This suspension was centrifuged at 5000 rpm for 10 min to separate undissolved solid components. The supernatant so obtained was reconstituted nanoemulsion and further characterized.

2.6.1. Determination of droplet size, size distribution and zeta potential The droplet size, size distribution and zeta potential of the reconstituted nanoemulsion was determined by Zetasizer Nano ZS (Malvern Instruments, UK) at a wavelength of 635 nm and scattering angle of 90◦ at 25 ◦ C. Each study was carried out in triplicate to ensure reproducibility. 2.6.2. Transmission electron microscopy Morphological and structural examination of COECH microparticles/Aerosil 200 suspended in triple distilled water and reconstituted nanoemulsion prepared from COECH-S-SNEDDS/Aerosil 200-S-SNEDDS as explained above, was carried out using transmission electron microscopy (TEM; Hitachi, Tokyo, Japan) on a H7500 machine operating at 100 kV capable of point-to-point resolution. A 0.5 mL droplet of these samples were directly positioned on the copper electron microscopy grids, stained with 0.5% (w/v) aqueous solution of phosphotungstic acid for 30 s, and the excess was drawn off. After drying, these grids were observed employing TEM. Combinations of different bright-field imaging at increasing magnification were used to expose the structure as well as the size of the formed oil droplets/solid COECH microparticles/Aerosil 200. 2.6.3. Stability testing of reconstituted nanoemulsion A stability of reconstituted nanoemulsion was examined using emulsification time determination, thermodynamic stability studies and cloud point measurements as per the method reported by Elnaggar et al. [22] and Bandyopadhyay et al. [23], respectively. 2.7. In vitro dissolution studies AMB released from COECH-S-SNEDDS/Aerosil 200-S-SNEDDS was evaluated by using the US pharmacopeia dissolution apparatus II-paddle (Electrolab India, Mumbai, India) at 37 ± 0.5 ◦ C using 500 mL of 0.1 N HCl as a dissolution medium with stirring speed of 50 rpm. Aliquots (5 mL) withdrawn at various time intervals was immediately filtered through whatman filter paper, diluted suitably and analyzed for AMB spectrophotometrically (Beckman DU-640 B UV/vis spectrophotometer) at 405 nm. 2.8. Ex vivo permeation studies The enhancement in the transportation of AMB across biological membrane (porcine small intestine) was examined when AMB was entrapped into nanoemulsion fabricated either from COECH or from Aerosil 200. For this purpose, porcine small intestine was chosen as diffusion barrier [24]. Porcine small intestine was procured from a local slaughter house (Patiala, India) and used within 1 h of slaughter. The tissue was stored in Kreb’s Ringer phosphate buffer (KRPB) at 4 ◦ C continuously aerated with the aid of an electrical aerator. The 10 cm length of porcine small intestine was excised, washed using saline and placed on saline-soaked filter paper. The isolated intestinal tract was cut lengthwise to flatten it using scissors. The serosal membrane was set upward and the muscle layer was removed with the help of a scalpel. This intestinal membrane was mounted on modified franz diffusion cell facing mucosal membrane toward donor compartment. The receptor compartment contains 22.5 mL of phosphate buffer pH 6.8. The receptor medium was stirred at 50 rpm and the temperature was maintained at 37 ± 0.5 ◦ C. Subsequently, a total of 225 mg COECHS-SNEDDS/Aerosil 200-S-SNEDDS formulation was added from the top of the tube (donor compartment) at the mucosal side of isolated intestine. Aliquots of 1 mL sample was withdrawn at different time intervals and it was replaced with fresh 1 mL buffer each time maintained at 37 ± 0.5 ◦ C in the receptor compartment. The amount of AMB diffused across porcine small intestine was determined

K. Singh et al. / International Journal of Biological Macromolecules 58 (2013) 310–319

spectrophotometrically (Beckman DU-640 B UV/vis spectrophotometer) at 405 nm after appropriate dilutions. Each experiment was performed in triplicate. 3. Results and discussion Development of an oral formulation of AMB is important to overcome the drawbacks of safety and high cost of the parenteral formulations, especially in developing countries [25–27]. Various attempts were made to incorporate AMB into oral drug delivery based systems like cochleates [28], nanosuspensions [29], self emulsifying drug delivery system [25–27] polymeric nanoparticles [30] and lipid nanoparticles [31]. Italia et al. [30] developed AMB nanoparticles and achieved 176.0 ng/mL of Cmax in 24 h. In another study, high molecular weight gelatin coated hybrid nanoparticles were prepared showing 94.38 ng/mL of Cmax within 8 h [31]. An AMB cochleate (prepared using l-␣phosphotidyl serine, chloroform and dextran) was also evaluated against mouse model of systemic candidiasis [28]. However, these formulations involve tedious method of preparation with lower entrapment efficiency. Therefore, a need was felt to develop a lipid based oral formulation of AMB. This could not only improve oral bioavailability but also reduced the nephrotoxicity associated with AMB oral lipid based formulations [3,25–27]. In addition, the nephrotoxicity produced by AMB oral lipid based formulation was reported to be significantly lower than those of the commercial injectable products Ambisome® and Fungizone® [3,25,26]. Liquid lipid based formulations prepared using various combinations of oil (Peceol, Captex 355® , etc.), surfactants (distearoyl-phosphatidylamine polyethyleneglycol-2000 (DSPE-PEG-2000, Labrasol® , Gelucire 44/14® , etc.), cosurfactants (Campul® , VitE-TPGS, ethanol, etc.) were attributed to improve absorption profile and efficacy in the treatment of leishmanial and fungal infection [3,25–27]. However, solid formulations are well accepted as compared to liquid dosage forms. Hence, an attempt was made to develop solid self-emulsified drug delivery system of AMB. These superior microparticles/solid substitutes shall adsorb AMB in lipid base over its surface under cold conditions. When these superior microparticles come in contact with stomach fluid, they are converted into AMB nanoemulsion. This nanoemulsion shall be absorbed from gastro intestinal tract and it entered into the body for the treatment of systemic fungal infection. Hence, an attempt was made to synthesize and characterize covalently cross-linked COECH microparticles employing spray drying technique and evaluated as a solid substrate to design solid self nanoemlsifying drug delivery system of poorly water soluble, thermolabile and light sensitive AMB. 3.1. Synthesis of COECH CH form soluble salts with acetic acid. However, CH dissolved in acetic acid immediately reacts with sodium salt of organic acids to produce white gelatinous precipitates. Moreover, EDTA is insoluble in acidic conditions below pH 4.3–4.7. Thus, disodium salt of ethylenediaminetetraacetic acid (EDTA-diNa) was chosen. The addition of EDTA-diNa salt solution into CH acetate** solution leads to gelatinous precipitates after sometime. Therefore, dropwise addition of EDTA-diNa salt solution into CH acetate solution under vigorous stirring at 2000 rpm provides clear ionic solution of CH and EDTA. This clear CH–EDTA solution was spray dried to obtain microparticles. During preliminary investigations, it was evident that spray drying of CH–EDTA-diNa solution with variable concentration and temperature produces mixture of water soluble fraction and water insoluble fraction with different particle size and zeta potential. Hence, process for the synthesis of COECH

313

need to be optimized using experimental design. The most popular and simple experimental design was factorial design at two level (2k ). However, selection of each factor at two levels leads to difficulty in reaching an accurate synthetic method for the synthesis of hydrophobic microparticles. This is probably due to large variation in responses with each 40–45 ◦ C rise in the temperature while moving from lower level to higher level as observed during preliminary studies. Therefore, to achieve optimum process conditions, it was essential to select full factorial design at high level. Therefore, 4k experimental factorial design was chosen. This experimental design works at 4 levels. In 42 factorial design experiment, each factor was assigned four levels (0, 1, 2 and 3) and total two factors namely concentration of CH:EDTA (X1 ) and inlet temperature of spray drier (X2 ) were identified as active independent factors. The percentage yield (Y1 ), particle size (Y2 ) and zeta potential (Y3 ) were taken as dependent variables influenced by X1 and X2 . The results of all the 16 experiments performed are summarized in Table 1. The following quadratic equations were generated after obtaining results of 16 experimental trials. Percentage yield = 12.35 + 5.20X1 + 5.33X2 − 1.81X12 − 0.53X1 X2 + 1.31X22

(1)

Zeta potential = 7.74 + 6.91X1 + 5.01X2 − 1.87X12 − 1.56X1 X2 + 0.62X22

(2)

Particle size = 24.96 − 2.066.91X1 − 4.41X2 + 0.62X12 + 0.06X1 X2 − 0.87X22

(3)

In the above equations, negative sign of coefficients signifies an inverse correlation of responses with independent variables. However, positive sign of coefficients signifies an additive effect (Fig. 1A). Therefore, percentage yield of COECH was found to be increased with increase in proportion of CH:EDTA-diNa from lower level (40:60) to highest level (70:30) suggesting highest amount of CH was essential as compared to EDTA. In addition, 170 ◦ C inlet temperature was essential to obtain highest yield of COECH (Fig. 1B). In addition, particle size of COECH was found to be higher at 50 ◦ C inlet temperature. However, the particle size was significantly (P < 0.05) decreased to 1–5 ␮m when microparticles prepared at 170 ◦ C. Further, the coefficients of X1 as compared to X2 of Eq. (2) suggested 2-fold effect of inlet temperature as compared to concentration of CH:EDTA-diNa (Fig. 1C). However, equal effect of X1 and X2 was observed, when zeta potential of COECH microparticles was compared (Fig. 1D). Thus, to obtain COECH with highest yield and particle size 1–5 ␮m, it was necessary to set the inlet temperature of spray drier to 170 ◦ C, cool temperature = 30 ◦ C, aspirator flow rate = 40 Nm3 /h, flow rate of 1 mL/min and equal proportion of CH and EDTA-diNa. 3.2. Identification of COECH FTIR spectra of COECH and the reactants (CH and EDTA-diNa) are shown in Fig. 2. The FTIR spectra of CH (Fig. 2A) showed a broad OH stretching absorption band between 3450 cm−1 and 3100 cm−1 and the C H stretching between 2990 cm−1 and 2850 cm−1 . Another major absorption band observed between 1220 cm−1 and 1020 cm−1 represents the free amino group ( NH3 + ) at C2 of glucosamine, a major group present in CH. Peak at 1610 cm−1 representing acetylated amino group of CH indicated that the sample was not fully deacetylated. The FTIR spectra of EDTA-diNa

3 0 13 ± 0.3 24 ± 1.3 14 ± 0.1 3 2 24 ± 0.2 11 ± 0.7 14 ± 0.1 3 3 36 ± 0.3 05 ± 0.07 18 ± 1.1 1 3 48 ± 0.4 01 ± 0.01 36 ± 1.5 1 1 26 ± 0.3 18 ± 0.9 18 ± 1.3 2 3 37 ± 1.2 03 ± 0.8 23 ± 1.2 2 2 25 ± 0.1 13 ± 0.4 18 ± 1.1 0 2 28 ± 1.2 14 ± 0.9 19 ± 1.4 2 1 18 ± 1.1 19 ± 0.7 13 ± 0.7 0 1 19 ± 0.9 21 ± 0.8 14 ± 0.9 2 0 12 ± 0.4 26 ± 0.1 11 ± 0.1 1 2 32 ± 0.8 09 ± 0.1 26 ± 1.2 0 3 11 ± 0.1 4 ± 0.02 24 ± 1.3

E6 E1

42 Full factorial design

E2

E3

E4

E5 Proportion of CH to EDTA disodium salt Inlet temperature (◦ C) of spray drier.

X1 X2 Y1 Y2 Y3

E8 E7

2 60:40 130 1 50:50 90 X1 X2

0 0 11 ± 0.1 24 ± 1.2 9 ± 0.1

E14 E13 E12 E11 E10 E9

3 70:30 170 High Low

0 40:60 50

1 0 18 ± 0.1 22 ± 0.9 12 ± 0.1

E16 E15

37 26 −36 11 1 −9 Yield of COECH microparticles (%) Particle size (␮m) Zeta potential (mV) Y1 Y2 Y3

Objective Levels Dependent variables Codes Level Independent variables Codes

Detail of variables and levels

Table 1 42 Full factorial design for the synthesis of spray dried COECH microparticles.

3 1 19 ± 0.1 18 ± 1.1 15 ± 0.1

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Maximum Minimum Maximum (–)

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showed a broad OH stretching absorption band 3450–3100 cm−1 and absorption peaks at 2586 cm−1 and 2451 cm−1 representing OH stretching of carboxylic acid (Fig. 2B). Absorption peaks at 1673 cm−1 representing C O stretching, 1476 cm−1 representing CH2 bending and 1395 cm−1 representing OH bending were also present in EDTA-diNa sample. Fig. 2C shows FTIR spectra of COECH microparticles that exhibited peaks at 1645 cm−1 characteristic of amide linkage. However, the occurrence of peak at 2367 cm−1 suggested presence of free acetate moieties that supported all the acetate moieties had not participated in the amide linkages. This indicated that the COECH microparticles were covalently crosslinked CH–EDTA-diNa (COECH). Further, the DSC analysis was performed to understand the purity of synthesized products. In all the thermograms, the occurrence of an endotherm near 100 ◦ C could be attributed to the moisture present in the samples or the presence of acetic acid moieties (Fig. 3). DSC thermogram of CH powder indicated one exothermic transition at 310.5 ◦ C probably due to degradation of CH (Fig. 3A). The thermogram of pure EDTA-diNa showed sharp endotherm at 224.45 ◦ C indicating the melting of EDTA-diNa (Fig. 3B). However, COECH microparticles showed endothermic transition at 321.03 ◦ C (Fig. 3C) followed by degradation exothermic transition at 375.28 ◦ C. This supported the purity of COECH. Therefore, it could be envisaged from DSC and FTIR analysis that COECH was successfully synthesized employing spray drying technique with an inlet temperature of 170 ◦ C. Synthesis of COECH is shown as Scheme 1. 3.3. Characterization of COECH Ideally, a solid carrier should bear the property of high lipid adsorbing capacity (at room temperature) on its surface and same quantity of lipid has been desorbed from its surface (at room temperature) with reduced particle size of oil droplets transported into the solvent. Therefore, to explore this property, oil adsorbing capacity and oil desorbing capacity of COECH were estimated and it was compared with Aerosil 200 (a standard solid carrier used for preparation of S-SNEDDS). Fig. 4A depicts the result of oil adsorbing capacity (OAC) and oil desorption capacity (ODC) of COECH and Aerosil 200. The results suggested that there was no significant difference (P > 0.05) between OAC of COECH and Aerosil 200 irrespective of lipid phase (Captex 355, Labrafac PG, Captex 355:Labrasol (70:30) and Labrafac PG:Labrasol; 70:30) used to adsorb at the surface of solid carrier. However, the ODC of COECH was found to be enhanced as compare to ODC of Aerosil 200. This suggested that COECH had a capacity to adsorb hydrophobic materials and almost same amount was desorbed from its surface. To find out this paradoxical surface behavior of COECH, surface free energy of COECH either before or after oil/oil:surfactant adsorption was estimated. Dispersive component of surface free energy is an indicator of hydrophobic surface. The polar component of surface free energy is a reflection of hydrophilic surface. This concept of surface free energy was applied by Matsumaru [32] and Yoshihashi et al. [33] to explain the correlation of disintegration time of tablets with wetting time of tablets. Therefore, surface free energy components can be used to understand the adsorption/desorption mechanism that had occurred at the surface of COECH microparticles/Aerosil 200. The results suggested surface free energy components of COECH were 40.43 ± 1.20 mJ/m2 and 11.57 ± 0.94 mJ/m2 respectively, for dispersive and polar components. However, the dispersive component of Aerosil 200 was 46.06 ± 1.21 mJ/m2 and polar component was 7.26 ± 0.27 mJ/m2 . Interestingly, the adsorption of Captex 355 and labrafac PG over COECH enhanced the dispersive component and decreased the polar component of COECH (Fig. 4B). However,

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315

Fig. 1. (A) Correlation of factors with responses obtained after multiple linear regression analysis. The response surface plots between concentration of CH:EDTA and inlet temperature of spray drier verses (B) percentage yield, (C) particle size and (D) Zeta potential.

adsorption of Captex 355:Labrasol (70:30) or Labrafac PG:Labrasol (70:30) mixture on COECH enhanced polar components and decreased dispersive component (Fig. 4B). In addition, the polar component of lipid phase adsorbed COECH was higher as compared to Captex 355:Labrasol (70:30) and Labrafac PG:Labrasol (70:30) adsorbed Aerosil 200. The enhancement in the polar component of lipid phase adsorbed COECH showed increase in the ODC of COECH as compared to Aerosil 200. This indicated potential of

Fig. 2. FTIR spectra of CH powder (A), EDTA-diNa salt (B), and COECH (C).

COECH that had provided sufficient surface free energy necessary for the stability of nanoemulsion. In addition, enhanced dispersive component of COECH after the adsorption of Captex 355:Labrasol (70:30) or Labrafac PG:Labrasol (70:30) could reduce the size of oil particles formed after reconstitution of nanoemulsion. Further, the dynamic advancing contact angle ( a ) was estimated to understand the surface behavior (hydrophilic/ hydrophobic) of solids (COECH/Aerosil 200) when come in contact with water. The results ensured hydrophobic surface of

Fig. 3. DSC thermogram of CH powder (A), EDTA-diNa salt (B), and COECH (C).

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Scheme 1. Synthesis of COECH by spray drying at different inlet temperatures.

COECH. This hydrophobicity was reduced on Captex 355:Labrasol (70:30) and Labrafac PG:Labrasol (70:30) adsorbed COECH microparticles. However, Aerosil 200 provides hydrophobic surface and this hydrophobicity was enhanced upon adsorption of Captex 355:Labrasol (70:30) and Labrafac PG:Labrasol (70:30) necessary for the development of nanoemulsion. This is an indicative of suitability of COECH in the adsorption and desorption of lipid phases necessary for the development of nanoemulsion. Thus, this

property of COECH could be useful in the formulation development of self nano emulsifying micro/nanoemulsion (Fig. 4B). 3.4. Surface morphology Scanning electron microscopy was undertaken in order to understand the surface characteristics of COECH microparticle. Fig. 5A and B shows spherical shaped microparticles in the range

Fig. 4. (A) Oil adsorbing and desorbing behavior of COECH-S-SNEDDS and Aerosil 200-S-SNEDDS and (B) surface free energy components and dynamic advancing contact angle of COECH-S-SNEDDS and Aerosil 200-S-SNEDDS. Where CP = Captex 355, LP = Labrafac PG, CP:LS = Captex 355:Labrasol (70:30) and LP:LS = Labrafac PG:Labrasol; 70:30.

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Fig. 5. Scanning electron micrographs: (A) COECH microparticles, (B) Aerosil 200, (C) COECH-S-SNEDDS, and (D) Aerosil 200-S-SNEDDS.

of 1–2 ␮m size and irregular shaped particles in the size range of 4–5 ␮m for COECH microparticle and Aerosil 200, respectively. The adsorption behavior of Captex 355:Labrasol (70:30) mixture over the surface of COECH microparticles (COECH-S-SNEDDS) was evident from SEM (Fig. 5C). However, this behavior was absent in S-SNEDSS when prepared from Aerosil 200 (Fig. 5D). This suggested ability of spherical shaped micro size COECH to completely spread lipid phase over its surface. Thus, it could be accepted as an ideal solid carrier to deliver nanoemulsion. 3.5. Flow properties The angle of repose of the synthesized COECH microparticles remained between 29◦ and 32◦ , indicating satisfactory flow properties. The moisture content of COECH microparticles was less than 2.15%, The bulk density, tapped density, Hausners ratio, Carrs index of Captex 355, Labrafac PG, Captex 355:Labrasol (70:30) or Labrafac PG:Labrasol (70:30) adsorbed COECH microparticles were found to be within acceptable limits and therefore the powders were free flowing. It was quite noticeable that there was no significant difference observed in the flow behavior of pure COECH microparticles and COECH-S-SNEDDS. In contrast, Aerosil 200 was fluffy powder with poor flowability. No significant change in the flowing behavior of Aerosil 200 was observed after the entrapment of Captex 355, Labrafac PG, Captex 355:Labrasol (70:30) or Labrafac PG:Labrasol (70:30). 3.6. Preparation and evaluation of solid self-nanoemulsifying drug delivery system of AMB The surface free energy components, oil adsorbing capacity and oil desorbing capacity suggested potential of COECH for the fabrication of solid self-nanoemulsifying drug delivery system. The previous studies had revealed that Labrasol® has been successfully incorporated as surfactant into micro/nanoemulsion systems from 30 to 60% with or without cosurfactant [1,3,34]. Therefore, lower concentration of Labrasol® was taken to challenge the potential of COECH/Aerosil 200 to form nanoemulsion. The solubility studies showed 13 ± 1.2 mg/mL and 8.24 ± 1.3 mg/mL of AMB was soluble in Captex 355:Labrasol (70:30) and Labrafac PG:Labrasol (70:30), respectively. Therefore, Captex 355:Labrasol (70:30) was selected to evaluate COECH as a solid carrier for nanoemulsion drug delivery system.

In traditional method, drug with solid carrier was mixed properly and then suspended in ethanol:water (80:20, v/v). This ethanolic suspension was spray dried to form solid selfnanoemulsifying drug delivery system. Thus, this heat based method is limited for thermolabile drugs. However, in the present investigation COECH was blended with mixture of Captex 355:Labrasol (70:30) and AMB in ethanol. This mixture was subjected to lyophilization. This method avoids the exposure of AMB to high temperature and hence enhanced stability of drug. Moreover, the developed COECH as solid carrier has an ability to adsorb lipid phase at lower temperature conditions. Thus, ensures the versatility of this solid carrier in the development of S-SNEDDS containing thermolabile AMB. 3.7. Evaluation of reconstituted nanoemulsion 3.7.1. Determination of droplet size, size distribution and zeta potential The droplet size of the emulsion is a critical factor in selfnanoemulsification performance because it determines the rate and extent of drug release as well as absorption. The smaller is the droplet size, the larger the interfacial surface area provided for drug absorption. It was observed from Table 2 that the lowest PDI as well as smallest particle size of 90 nm was obtained from reconstituted nanoemulsion generated from COECH-S-SNEDDS surface Table 2 Physical characterization nanoemulsion.

Physical characterization Particle size (nm) PDI Zeta potential (mV) Emulsification time (s) Thermodynamic stability Cloud temperature (◦ C) Centrifugationa Phase separation Precipitation Freeze–thaw cycle 4 ◦C 40 ◦ C a

and

thermodynamic

stability

of

reconstituted

COECH-SSNEDDS

Aerosil200-SSNEDDS

90 0.316 1.1 32–34

225 0.462 −12.3 68–72

65

46

X X

Y Y

Clear Clear

Clear Clear

X, no phase separation/precipitation; Y, phase separation/precipitation.

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Fig. 6. Transmission electron micrographs: (A) COECH microparticles, (B) Aerosil 200, (C) COECH-S-SNEDDS, and (D) Aerosil 200-S-SNEDDS.

as compare to Aerosil 200-S-SNEDDS. The zeta potential is an electrostatic value measured by surface electrostatic double layer of droplets. The neutral zeta potential of reconstituted nanoemulsion generated from COECH-S-SNEDDS suggested its stability. Thus, the nanoemulsion prepared from COECH-S-SNEDDS bears nanosize particles. This indicated potential of COECH to transport AMB in nanosize droplets prepared under cold conditions. 3.7.2. Transmission electron microscopy Fig. 6 shows TEM images of COECH/Aerosil 200 and reconstituted nanoemlusion prepared from COECH-S-SNEDDS/Aerosil 200-S-SNEDDS. The image of COECH microparticles showed a homogeneous internal morphology suggesting a matrix type structure (Fig. 6A). Aerosil 200 showed aggregates (5–8 ␮m) formed by smaller primary particles (16.5 nm) and showed similar size as reported earlier [12] (Fig. 6B). Fig. 6C and D portrays the electron microscopic images depicting the morphology of desorbed nano size Captex 355:Labrasol (70:30) droplets from COECH-SSNEDDS and Aerosil 200-S-SNEDDS. As it is evident from Fig. 6C, all the globules formed from COECH microparticle were of spherical shape with globule size of most of them as nearly 90 nm (Table 2). Further, it was clearly illustrated that there is no signs of coalescence, indicating thereby the enhanced physical stability of the fine droplets formation. Yet the globules formed by Aerosil 200 were spherical in shape, 215 nm size but coalescence was observed which may further proceeds to unstable droplet formation. 3.7.3. Stability testing of reconstituted nanoemulsion The important criterion for selection of the solid carrier for S-SNEDDS is not only its high oil adsorbing capacity but also the ability of solid carrier to desorb maximum, nanosize and stable oil phase spreaded over its surface. Further, the nature of oil and surfactant phase also affects the stability of nanoemulsion. Therefore, stability testing of reconstituted nanoemulsion was evaluated. The results of stability testing are summarized in Table 2. The COECH containing Captex 355:Labrasol (70:30) was found to disperse quickly (within 31–35 s) and completely when subjected to aqueous environment under mild agitation. This suggested COECH has an inherent capacity to generate nanoemulsion successfully when come in contact with aqueous environment. Further, the cloud temperature, centrifugation and freeze thaw cycle suggested that the reconstituted nanoemulsion was found to be thermodynamically

stable when generated from COECH-S-SNEDDS in comparison to Aerosil 200-S-SNEDDS. Thus, pointed toward overwhelming influence of COECH in the fabrication of nanoemulsion containing AMB. 3.8. In vitro drug dissolution study An in vitro release performance of AMB incorporated into nanoemulsion was studied. A control nanoemulsion containing AMB dissolved in Captex 355:Labrasol (70:30) in 80% (v/v) ethanol was prepared (SNEDDS). The in vitro release of AMB from reconstituted nanoemulsion generated from COECH-S-SNEDDS was found to be 70% within 15 min (Fig. 7A). This was similar to AMB release from SNEDDS. However, just 5% was released from pure AMB sample. Thus, nearly 12-fold enhancement of dissolution behavior of AMB was observed when incorporated into nanoemulsion prepared from COECH-S-SNEDDS. Further, the release of AMB reconstituted nanoemulsion generated from Aerosil 200-SSNEDDS was 8-folds enhanced in comparison to AMB released from its pure form. This suggested COECH could be used as a solid carrier to enhance dissolution behavior and deliver lipid soluble drugs. 3.9. Ex vivo permeation studies Ex vivo permeation studies were performed employing porcine small intestine method. The permeability of AMB from COECH-SSNEDDS was examined and compared with permeation of AMB from SNEDDS or Aerosil 200-S-SNEDDS. The results are summarized in Fig. 7B. The flux is an indicator of amount of drug permeated per unit area per unit time across a biological membrane. The higher flux suggested enhancement of drug permeation across membrane. The relative permeability suggested rate with which AMB transferred from serosal side of the intestine. The flux of AMB when released from COECH-S-SNEDDS was not significantly different (P > 0.05) from flux of AMB released from SNEDDS. However, AMB obtained in the donor compartment when released from Aerosil 200-S-SNEDDS was significantly different (P < 0.05) than AMB entrapped into COECH-S-SNEDDS. Similar results were obtained with relative permeability and absorption enhancement ratio. Hence, the results suggested overwhelming influence of COECH as solid carrier in transporting AMB in lipid dispersion across biological membrane via nano-sized droplet transportation.

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Acknowledgements The authors would like to acknowledge the financial assistance provided by UGC, New Delhi, under major research (file no. 39168/2010 (SR). The scholarship provided to Kuldeep Singh (SRF) by Council of Scientific and Industrial Research, New Delhi, India, vide sanction no. 09/140(0154)/2010/EMR-I is acknowledged. We are also thankful to SAIF, AIIMS, New Delhi, for extending the facility of TEM. References

Fig. 7. (A) In vitro AMB release from pure AMB, L-SNEDDS, COECH-S-SNEDDS and Aerosil 200-S-SNEDDS and (B) Ex vivo permeability evaluation of AMB across porcine small intestine incorporated in COECH-S-SNEDDS/Aerosil 200-S-SNEDDS, pure AMB and L-SNEDDS.

4. Conclusion The current investigation pointed toward successful synthesis of covalently crosslinked CH–EDTA without the use of proton initiator employing 42 full factorial design. The 42 full factorial design technique suggested 170 ◦ C inlet temperature of spray drier as well as mixing of CH:EDTA (1%, w/v) as spraying solution produces highest yield of COECH. The FTIR-ATR and DSC analysis identified formation of amide linkages between NH2 of CH and COO− of EDTA along with presence of free acetate moieties. The synthesized COECH microparticles showed high oil desorbing capacity as compared to Aerosil 200. This was due to enhancement in the polar component of surface free energy of Captex 355:Labrasol (70:30) adsorbed microparticles. The high Captex 355:Labrasol (70:30) carrying capacity of COECH could be associated with high dispersive component of surface free energy. This was also evident from SEM analysis. The high AMB loading capacity of COECH-S-SNEDDS as compared to Aerosil 200-S-SNEDDS supported the potential of COECH in the fabrication of nanoemulsion. Further, the reconstituted nanoemulsion generated from COECH-S-SNEDDS produce stable and smaller size nanoemulsion as revealed from TEM analysis. Furthermore, the in vitro dissolution profile of AMB was 12-folds enhanced as compared to pure AMB when incorporated into COECH-S-SNEDDS. This behavior was also observed during ex vivo performance studies. Overall, the synthesis of this macromolecule (COECH) employing spray drying technology was simple and showed high level of industrial potential. The COECH was found to be a useful solid carrier for the development of nanoemulsion containing heat sensitive drug AMB.

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