Design and Evaluation of Chitosan-Based Novel pH-Sensitive Drug Carrier for Sustained Release of Cefixime

Purpose: To formulate and evaluate pH-sensitive controlled release cefixime microspheres based on crosslinked chitosan and acryl amide-grafted-poly ethylene glycol Method: pH sensitive interpenetrating network (IPN) cefixime microspheres based on chitosan, its grafted copolymer, and hydrolyzed grafted copolymer were prepared by precipitation and crosslinking methods. The formulations were characterized by Fourier transform infrared spectroscopy (FTIR),X-ray diffraction(XRD),Differential scanning calorimetry (DSC), Scanning electron microscopy(SEM),Raman spectroscopy for particle size analysis. Swelling ratio, Effect of drug loading on encapsulation efficiency was investigated. FTIR, SEM, DSC, X-ray diffraction XRD, swelling studies were investigated for controlled drug release of drug at pH 2 and pH 7.4. Results: The microspheres were spherical in shape with a narrow particle size distribution (56 – 124 µm) and encapsulation efficiency of up to 93 %. The % swelling ratio for blend microspheres of C-PEG are higher than that of plain chitosan in both pH. In both the pH media release was much faster in plain Chitosan microspheres. For instance, only 83% drug was released at 11.5 h for C-grafted copolymer 50 in pH 7.4 media compared to 41% drug released in pH 2 media for the same time whereas 80% drug was released at 11.5 h for C-grafted copolymer 50 (hydrolyzed) in pH 7.4 media compared to 38% drug released in pH 2. Drug release followed Higuchi release kinetics. FTIR, DSC, XRD data indicate that there was no interaction between the drug and the polymers used. Conclusion: It is evident from this study that chitosan microspheres could be further developed to serve as an effective biodegradable carrier for controlled release of cefixime.


INTRODUCTION
Chitosan is the generic name of a linear random copolymer of β-(1-4) linked -D-glucosamine whose molecular structure comprises a linear backbone linked through glycosidic bonds.The basic amine groups of this polysaccharide are protonated and thus positively charged in most physiological fluids.Chitosan is mostly hydrophilic and the percentage of acetylated monomers and their distribution in the chains has a critical effect on its solubility and conformation in aqueous media.Hence, chitosan exhibits pHdependent behavior in solution and some interesting biopharmaceutical properties such as muco-adhesiveness, ability to open, and epithelial tight junction.These characteristics of chitosan have attracted many scientists working in the biomedical field, particularly drug delivery [1][2][3][4].Due to its unique polymeric cationic character, chitosan-based gels and films have been extensively examined in the pharmaceutical industry for its potential in the development of drug delivery system [5].
In recent years, much attention has been given to the development of interpenetrating polymeric network (IPN) from natural biodegradable and biocompatible polymeric materials [6].Chitosan is biopolymer with immense structural possibilities for chemical and mechanical modification to generate novel properties [7].Among many methods of modifying the original structures of polymers, graft copolymerization is an easier method, which makes the derived polymer as attractive biomaterials in controlled released application [8].Grafting of polar monomers onto chitosan by Graft copolymerization improves solubility which enhances its adsorption capacity [8,9].
Several reagents have been used for the crosslinking of chitosan, including glutardehyde, genipin, ethylene glycol, diglycidylether and diisavanate [9,10].Although the grafting of chitosan modifies its properties, it is possible to retain some interesting characteristics such as mucoadhesivity, biocompatibility and biodegradability [11][12].Besides, chitosan being a linear polysaccharide has been found to be a good chemical entity for preparing microspheres because of its high crosslinking ability due to the presence of amino (-NH 2 ) groups.A good number of approaches to improve the wet strength of hydrogels and IPN polymeric network of chitosan have been reported elsewhere [13].Cefixime is an oral third generation cephalosporin antibiotic.It possess antibactericidal activity and used in the treatment of gonorrhea, tonsillitis and pharyngitis.Its biological half-life is of 3 -4 h.Cefixime has p ka value of 2.5 and being a weak acid is insoluble in water after its oral administration which results into the poor bioavailability.Around 40-50% is excreted through renal and biliary route.In order to improve the therapeutic effect of the drug by increasing its bioavailability the IPNs of chitosan with acrylamide-grafted -PEG and hydrolysized acrylamide-grafted PEG microspheres was synthesized for controlled release of Cefixime in both acidic and basic media.
To the best of our knowledge no work has been done so for by using this drug.The present work deals with the in vitro release of loaded formulation with IPN microspheres.

EXPERIMENTAL Materials
Cefixime USP was obtained as a gift from Kosher Pharmaceuticals, India.High molecular weight chitosan poly(D-glucosamine) was purchased from Sigma-Aldrich, Belgium; acryl amide was purchased from Qualigens Mumbai, India; while polyethylene glycol 4000, hydrochloric acid, glutaradehyde, acetic acid and sodium hydroxide were purchased from SD Fine Chemicals, India.All other chemicals used in this work were of analytical reagent grade

Synthesis of polyethylene glycol graftedacryl amide
Polyethylene glycol 4000 was dissolved in water at 60 -65 o C and treated with acryl amide under nitrogen gas atmosphere followed by addition of trace quantity of potassium persulfate under continuous stirring at a temperature of 65 o C for 5 h [14].The resulting product was washed with water: methanol (1:1 v/v), filtered and kept in vacuum for drying at 60 o C.About 2 wt % (100ml) solution of PEG-grafted acryl amide was hydrolyzed using 50 ml of equimolar concentration of sodium hydroxide solution at 60 o C for 5 h.The final product was filtered and dried under vacuum at 60 o C.

Drug loading
Approximately 2 g of the polymer blend obtained above was dissolved in 2 % acetic acid containing a required amount of Cefixime (100mg) and was dispersed with equal quantities of light liquid paraffin stirred at high speed and the resulting water-oil emulsion was stabilized by addition of 1 % Tween 80 solution.Thereafter, the aqueous phase of the emulsion was hardened to form a microgel.Glutaraldehyde (5ml) was added drop-wise to the emulsion and stirred for 30 min at room temperature to stabilize the prepared microsphere.The mixture was then left to cool at 10-15 o C for 30 min.The microspheres were collected by filtration using Whatman-41 filter paper (pore size: 25µm).Finally, the microspheres were washed with 30ml of hexane and water to remove paraffin oil, acid, water and excess glutaraldehyde.In order to determine the drug release characteristics of the microgels, the formulation was prepared by varying amount of drug and blend ratio of chitosan with PEG, PEG-grafted copolymer as well as hydrolyzed PEG-grafted copolymer matrices.Drug concentration with different polymeric blend and their formulation codes are given in Table 1.

Fourier transform infrared spectroscopy (FTIR)
The FTIR spectra of Cefixime and drug-loaded microspheres were recorded on an FTIR system (JASCO-4100, Japan) using KBr disc with a scanning range of 4000 -400 cm -1 in order to assess structural changes that could have occurred in the drug due to drug-polymer interaction following microsphere formation.

Evaluation of water content and swelling ratio of microspheres
The microsphere formulations (beads) (~ 50 mg) were placed in 50 mL distilled water and at 30 min intervals, beads were taken out and excess water was removed using filter paper and weighed immediately on an electronic weighing balance (Model AUY220, Shimadzu, Japan).Swelling ratio was expressed as % weight gain.

X-ray diffraction (XRD) studies
X-ray diffraction (XRD) studies (Bruker D8 Advance diffractometer, Germany, CuKά radiation, Nickel filter) were performed to determine crystalline patterns of the drug in the cross-linked polymer network.The XRD patterns of pure cefixime, drug-loaded chitosan microspheres matrix and blank microspheres were recorded and compared.

Differential scanning calorimetry (DSC)
DSC thermograms of the pure drug and IPN microsphere were recorded with a differential scanning calorimeter (DSC Q1000V9.4Build 287) from 0-400 o C at a heating rate of 10°C/min.

Particle size
The particles size of the microsphere was measured by Raman spectrometer (Lesser 536 Aglitron USA) equipped with camera-microscope system.The test sample was spread flat on a glass microscope slide and the experiment was performed at room temperature at a magnification of 100x200 µm slit width and exposure time of 5 sec.Triplicate tests were carried out for all samples.

Scanning electron microscopy (SEM) studies
The surface and cross sectional morphologies of the microspheres were examined using high resolution scanning electron microscopy (FEI Quanta FEG 200).

Drug release studies
In vitro drug release studies were performed using USP paddle single stage digital apparatus with 900 ml of phosphate buffer (pH 7.4) and HCl/KCl buffer (pH 2) as dissolution medium.At predetermined time intervals, 5 ml samples each were withdrawn from the medium at various time intervals and the medium replenished immediately with the same volume of fresh dissolution medium to maintain sink conditions.The amount of dissolved drug in the sample taken was measured (after filtration through Whatman-41 filter paper (pore size: 25µm) spectrophotometrically (model-V-670PC, Jasco UV-visible-NIR spectrophotometer) at a λ max of 288 nm.Moreover all the four release kinetics model namely, zero order kinetics, first order kinetics, Korsenmeyer Peppas model and Higuchi model were applied to Data obtained from in vitro release studies.It was observed that zero order kinetics model showed a r 2 value of 0.7453 (~0.7182-0.8012);first order kinetics model with r 2 value of 0.8660 (~ 0.8120-0.8710);Higuchi model with r 2 value of 0.990 (~ 0.980-.0.990) and Korsenmeyer peppas model with r 2 value of 0.9215 (~ 0.9210-0.9219).The assessment of the release kinetics revealed that drug release from the chitosan microspheres followed Higuchi model.The mechanism of drug release from microspheres was diffusion controlled.Data obtained from in vitro release studies were fitted to Higuchi model (Q = Kt½) to find out the mechanism of drug release from the chitosan microsphere.The tests were carried out in triplicate .

Determination of drug content
A sample (100 mg) of the formulation was weighed accurately, transferred quantatively into a 100 mL volumetric flask and diluted up to mark using distilled water.The mixture was stirred overnight to allow total release of the drug from the microspheres.After filtration through Whatman-41 filter paper (poresize:25µm) the filtrate was assayed spectrophotometrically at 264 nm.

Statistical analysis
Statistical analysis was performed using Origin 7.0 software.All the tests were run in triplicate and the data were analyzed by one-way ANOVA for drug release.Statistical significance was set at р < 0.05.

FTIR spectra
The FTIR spectra of plain PEG, PEG-grafted copolymer and hydrolyzed PEG-grafted copolymer are shown in Figure 1.The broad band appearing at 3495.09 cm -1 corresponds to the associated -OH stretching vibrations of the hydroxyl group of the grafted copolymer (Fig 1a-A).A new peak appeared at 3071.24 cm -1 and the related peak at 1632.35cm -1 corresponds to -NH bending vibrations of the primary amides of acryl amide.In the spectra of hydrolyzed PEGgrafted copolymer, the shoulder peak (at 856 cm - 1 ) disappeared but two new peaks appeared around 1330 cm -1 and 1091cm -1 , respecrively, which are due to the anti-symmetric vibrations of -COOH groups.
The FTIR spectrum of plain chitosan showed two peaks around 836 and 1020 cm -1 corresponding to saccharin structure in the spectra (Fig 1b-A).The observed sharp peaks at 1385 and 1650 cm -1 are assigned to -CH 3 group (Fig 1b-B).Due to increase in peak intensity at ~1594 cm -1 , there are two distinguishing peaks at ~1398 and ~ 1475cm -1 .The spectrum of chitosan blend with acrylamide grafted with PEG hydrolyzed complex was observed at around 1638 cm -1 and corresponds to -NH 3 + group due to protonation of the primary amino group in the presence of the carboxylic group of the hydrolyzed polymer (Fig 1b-C).The peak at ~1428 cm -1 confirms the presence of carboxylic acid in the polymer.

Swelling of microgels
In plain chitosan microspheres in pH 2 media, % equilibrium swelling of microspheres is higher than the value of it in pH 7.4.Swelling increased from 185-190% in pH 7.4 media compared to swelling in pH 2 media which increased about 196-201%.The % equilibrium swelling for blend microsphere of chitosan and PEG are higher than plain chitosan microspheres, formulations of C-PEG-50 to C-PEG-100.The % equilibrium swelling of C-grafted copolymer 50 to C-grafted copolymer 100 increased to 245-258% in pH 7.4 and 226-239% in pH 2 respectively.The % equilibrium swelling of formulations containing Cgrafted copolymer 50 (hydrolyzed) to C-grafted copolymer 100 (hydrolyzed) increased to 273-283% in pH 7.4 and to 261-271% in pH 2.

X-ray diffraction (XRD)
XRD aids in assessing changes in the crystallinity of a drug after formulation or processing.As indicated in Fig 2 b, cefixime showed characteristic intense peaks at 10 and 30 o due to its crystalline nature.However, these peaks were not seen in the drug-loaded matrix complex, placebo microgels and drug-loaded microgels which indicates that the encapsulated drug became amorphous.

Differential scanning calorimetry (DSC) thermograms
DSC is a useful tool that could assist in determining if any changes have taken place in the drug itself and the drug matrix.Figure 2b-A indicates that the thermogram of the pure drug exhibited an endothermic peak at 202 o C, corresponding to its melting point, but this peak was absent from the thermogram of the drugloaded microspheres which suggests that most of the drug was well dispersed within the matrix.

Particle size
The size of the microsphere was dependent on the amount of drug loaded, extents of drug loading and degree of crosslinking.All the microspheres were spherical with diameter ranging from 56 to 124 µm.The size of the microspheres increased from 103 to 124 µm for 100 % drug-loaded chitosan-grafted copolymer 100 (hydrolyzed).

Drug content and entrapment efficiency
In developing an effective formulation, it is important to achieve high encapsulation efficiency (EE).In the present case, % EE data show a dependence on % drug loading, For instance, formulation containing high amount of drug exhibited higher value of EE and also % EE is higher for chitosan microspheres of blends of hydrolyzed grafted copolymer as compared to other formulations.The effect of cross linking was studied by adding different amount of glutaraldehyde ranging from 2.5 mL to 7.5 mL.It was observed that increasing amount of glutardehyde in the microspheres decreased %EE (Table 1).

Scanning electron micrographs (SEM)
SEM microgram indicates that the microspheres were fairly spherical in shape and that polymeric material surrounded the microspheres (Fig 3A).The surface was, however, rough and rugged (Fig 3B).The type and blend of polymeric used in microsphere formulation did alter surface properties.

Drug release
The release data for the various formulations are shown in Figure 4.In both the pH media release was much faster in plain chitosan microspheres.For instance, only 83% drug was released at 11.5 h for C-grafted copolymer 50 in pH 7.4 media compared to 41% drug released in pH 2 media for the same time whereas 80% drug was released at 11.5 h for C-grafted copolymer 50 (hydrolyzed) in pH 7.4 media compared to 38% drug released in pH 2. On subjecting the drug release data to Higuchi model (Q = Kt½) in order to ascertain the drug release mechanism, a linear relationship was observed with a regression coefficient close to 1 (r 2 = 0.990).

Drug release
The release data for the various formulations are shown in Figure 4.At both pH (2.0 and 7.4), drug release was considerably faster non-crosslinked chitosan microspheres than from crosslinked microspheres, with 89.7 and 53.4 % release in 11.5 h from C-50 at pH 2.0 and 7.4, respectively.On the other hand, 35 % drug was released in 11.5 h for C-grafted copolymer 50 (hydrolyzed) at pH 7.4.On subjecting the release data to the Higuchi model in order to ascertain the release mechanism, a linear relationship was observed with a regression coefficient of 0.990.

DISCUSSION
Chitosan exhibits a pH-sensitive behavior as a weak polybase due to the large number of amino groups on its chain.It dissolves easily at low pH but is insoluble at high pH.[16].The mechanism of pH-sensitive swelling involves protonation of the amine groups in chitosan at low pH conditions.This protonation leads to chain repulation, diffusion of proton and counter ions together with water inside gel and dissociation of secondary interaction.This property helps in the delivery of drugs to the stomach and chitosan has been widely investigated as a delivery matrix.[17,18].
The carboxylic groups of acryl amide moieties are hardly ionized and PEG chains are unable to interact with non-ionized carboxylic groups by hydrogen bonding.As a result, copolymers present a collapsed conformation; on the other hand, as the pH value approaches to the pKa value and above, carboxylic acid ionization leads to the breakage of the hydrogen bonds and the number of ionized units increases dramatically.The ionization of the carboxylic groups results in a repulsive interaction between the PEG chains and acrylic acid molecules at high pH.
In case of the swelling-controlled mechanism when diffusion of a drug is significantly faster than hydrogel distention, swelling is considered to be controlling the release behavior [19].Micro gels are hydrophilic in nature and swell considerably in phosphate buffer solution.pHdependent study suggests that swelling ratio at pH 7.4 was higher than that of micro gels at pH 6. Almost all chitosan was in its ionized form i.e., -COOH forms COO -groups which could form intermolecular H-Bonding with OH -groups within the network during swelling.The polymeric complex has been expanded more in size at pH 7.4 compared to pH 2.0.
Increase in the amount of glutaraldehyde incorporated in the matrix lowered encapsulation efficience due to reduced free volume space available in the polymeric matrix as the microsphere became more rigid due to higher crosslink density and complexation.
XRD and DSC results indicate that there were no interactions between drug, chitosan and other ingredients used in preparing the microspheres.Thus, it suggests that the prepared microsphere would be stable.
Sustained drug release over a period of 12 h was achieved by crosslinking chitosan with the release data fitting well to the Higuchi model.It suggests that the drug released was controlled by diffusion mechanism.Uptake of water significantly hindered and slowed down drug diffusion due to strong electrostatic interactions between the polymeric matrix and the drug.Chitosan is deprotonated at pH 7.4, leading to disruption of the interaction between drug and polymeric matrix.The carboxylic groups of chitosan become gradually ionized at pH 7.4 which favors microsphere drug diffusion from the microsphere into the surrounding medium [15].Thus, the collapsed polymeric matrix played an important role in hindering drug diffusion, effectively contributing to sustained release at pH 7.4.This can be attributed to complexation of the amino group of chitosan at this pH, while at pH 2; the complex might have been deformed.

CONCLUSION
The hydrophilic nature of polyacrylamidemodified PEG has been successfully utilized to develop crosslinked chitosan microspheres.The resulting pH-sensitive interpenetrating network of the microsphere matrix facilitated sustained release of cefixime.Further work, including in vivo evaluation, is however, required to ascertain the suitability of the procedure used on a large scale.

Fig 3 :
Fig 3: SEM images of microspheres showing (A) polymeric materials around microspheres and (B) roughness of microsphere surface

Table 1 :
Encapsulation efficiency and equilibrium swelling of Cefixime formulation SD = standard deviation; *mean ± SD (n = 3); GA = glutaraldehyde crosslinked; C = chitosan.Drug concentrations used were 50 mg and 100 mg in each formulation.