Adsorption and release studies of new cephalosporin from chitosan-g-poly(glycidyl methacrylate) microparticles

European Polymer Journal 82 (2016) 132–152

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Adsorption and release studies of new cephalosporin from chitosan-g-poly(glycidyl methacrylate) microparticles Toni Andor Cigu a,b, Silvia Vasiliu c,⇑, Stefania Racovita c, Catalina Lionte d, Valeriu Sunel b, Marcel Popa a, Corina Cheptea e a ‘‘Gheorghe Asachi” Technical University of Iasi, Faculty of Chemical Engineering and Environmental Protection, Department of Natural and Synthetic Polymers, Prof. Dr. Docent Dimitrie Mangeron Street, No. 73, 700050 Iasi, Romania b ‘‘Al. I. Cuza” University, Faculty of Chemistry, Carol I Bvd, No. 11, 700506 Iasi, Romania c ‘‘Petru Poni” Institute of Macromolecular Chemistry, Grigore Ghica Voda Alley No. 41A, 700487 Iasi, Romania d ‘‘Gr. T. Popa” University of Medicine and Pharmacy, Faculty of Medicine, Universitatii Street No. 16, 700115 Iasi, Romania e ‘‘Gr. T. Popa” University of Medicine and Pharmacy, Faculty of Biomedical Bioengineering, Department of Biomedical Sciences, Kogalniceanu Street No. 9-13, 700454 Iasi, Romania

a r t i c l e

i n f o

Article history: Received 12 April 2016 Received in revised form 4 July 2016 Accepted 17 July 2016 Available online 18 July 2016 Keywords: Cephalosporin Chitosan Glycidyl methacrylate Adsorption isotherm Adsorption kinetic Release kinetics

a b s t r a c t Porous microparticles of chitosan-g-poly(glycidyl methacrylate) were obtained by grafting chitosan onto crosslinked networks based on glycidyl methacrylate and ethylene glycol dimethacrylate using suspension polymerization technique. A new cephalosporin from the indazole class prepared by acylation of 7-Aminodesacetoxycephalosporanic acid with mixed anhydride of 5-nitroindazole-1-yl-acetic acid was used as active principle. The cephalosporin-microparticle systems were characterized by FT-IR spectroscopy, SEM and AFM analysis. Batch experiments were carried out to study the influence of initial drug concentration, temperature, contact time, drug:microparticles ratio and pH on the adsorption process of cephalosporin onto porous crosslinked microparticles. Two-parameter and three-parameter isotherm models were used to evaluate the adsorption equilibrium. The values of diffusion coefficients indicate that the cephalosporin adsorption onto microparticles was controlled by both film diffusion and pore diffusion mechanisms. The analysis of the kinetic data of the release process indicate that the release mechanism of cephalosporin from microparticles corresponds to the anomalous transport mechanism. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction The increase of treatment efficiency, as well as the elimination of the side effects of drug caused either by over-doses either sometimes by their aggressiveness to both the disordered cells and the healthy ones are the major problems that medicine needs to solve. Drug administration in free form, no matter if orally, parenterally, by instillations or ointments has the effect of continuous change of their concentration in the body or at the disease site. Short time after administration, the concentration of the active principle exceeds the one related to the therapeutic field, becoming toxic. Then, the concentration of drug quickly drops under the therapeutic field, which requires a new administration. This problem can be solved by the administration

⇑ Corresponding author. E-mail address: [email protected] (S. Vasiliu). http://dx.doi.org/10.1016/j.eurpolymj.2016.07.011 0014-3057/Ó 2016 Elsevier Ltd. All rights reserved.

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of drug immobilised on a polymeric support, when it is observed a variation of its concentration in the body after another kinetics. The drug is released gradually from macromolecular carrier ensuring a constant concentration in the body corresponding to the therapeutic value as well as an increase of the treatment effectiveness. Among the various macromolecular carriers, the grafted chitosan copolymers obtained by several methods such as, grafting initiated by free radicals [1], grafting using radiation [2], enzymatic grafting [3], grafting via polycondensation [4], living cationic polymerization [5], soapless emulsion copolymerization [6], oxidative coupling [7] and ring opening [8] received an increased interest of the scientific community. Chitosan-grafted-poly(acrylic acid) particles and microparticles of poly(acrylamide)-grafted-chitosan crosslinked with glutaraldehyde were prepared to encapsulate indomethacin [9], hydrophilic drugs or sensitive proteins [10] and nifedipine [11]. Also, the graft chitosan copolymers have potential to be used in dialysis [12] and present interesting properties for wound-healing and cardio-vascular applications [13]. Generally, the aim of drug delivery system administration focuses on the prevention of illnesses, improvement of some symptoms and the cure of various diseases such as, localized diseases by applying solutions and ointments or generalized diseases by administrating antibiotics. The b-lactam antibiotics (penicillins, cephalosporins, carbapenems, monobactams) represent a category of drugs with a very wide spectrum in the therapy of bacterial infections [14]. Initially, the cephalosporins (CFS) have been isolated from cultures of bacteria Cephalosporium acremonium to give three types of compounds: the C and N cephalosporins having a similar structure with the penicillins and P cephalosporin, a steroid antibiotic with a similar structure to the one of the fusidic acid [15]. Subsequently, in 1964 the semi-synthetic cephalosporins were synthesized. The semi-synthetic CFS can be obtained by the introduction of different substitutes in position 3 of 7-ADCA structure [16]. Whatever was the method used for the synthesis of CFS, the final goal was to improve the antimicrobial and pharmaceutical properties, such as: broadening of the antibacterial spectrum, increasing of the stability towards acids or absence of side effects [17–19]. The originality of this work consists in the synthesis of the new porous polymer carriers and the new cephalosporin in order to obtain the drug delivery systems for potential applications in the treatment of infectious diseases. Also, the studies of the equilibrium, kinetic and the mechanism of cephalosporin adsorption, as well as the release studies from the porous macromolecular supports in form of microparticles obtained by grafting chitosan (CH) onto crosslinked network based on glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) were realized. Adsorption studies are very important to establish the performance of the adsorbents and hence for the selection of a sustained/controlled release system of active principle with highly efficiency. 2. Experimental 2.1. Materials 5-Nitroindazol, sodium chloroacetate, 7-Aminodesacetoxycephalosporanic acid and hydrochloric acid were purchased from Merck & Co. Trimethyl acetic acid, dimethyl sulfoxide and chitosan (CH, Mw = 600,000 g/mol, degree of acetylation = 13.5%) were purchased from Fluka Chemical Company. Glycidyl methacrylate from Sigma Aldrich was distilled under reduced pressure. Ethylene glycol dimethacrylate, benzoyl peroxide (BOP), ammonium persulfate (APS), toluene, poly(vinyl alcohol) (PVA, Mw = 51,000 g/mol, degree of hydrolysis = 88%), triethylamine, cephalexin, sodium bicarbonate, sodium sulphate, acetic acid and butyl acetate were purchased from Sigma Aldrich. 2.2. Methods 2.2.1. Preparation of the porous microparticles The porous crosslinked microparticles based on GMA (G1 and G2 microparticles) and microparticles based on chitosan-gpoly(glycidyl methacrylate) (C1 and C2 microparticles) were prepared by suspension polymerization technique described elsewhere [20,21]. It is known that, the reaction mixture of suspension polymerization is formed by two phases: aqueous phase and organic phase. In case of G microparticles the aqueous phase was formed by distilled water as dispersion medium and 1.5 wt.% PVA as a suspension stabilizer. In case of C microparticles the aqueous phase consists of 1.5 wt.% solution of PVA and chitosan. Also, in the aqueous phase of C microparticles a free radical initiator (APS) was added in order to create radicals on polysaccharide chain. The organic phase for both types of microparticles was formed by GMA (90 mol% or 80 mol%), EGDMA (10 mol% or 20 mol %), BOP as free radical initiator of polymerization reaction of methacrylic monomers (2.5 wt.% with respect to the total amount of the monomers) and toluene as porogenic agent at a dilution of D = 0.5, where D = ml toluene/(ml toluene + ml monomers). For C microparticles, the CH/methacrylic monomers ratio was 1:23 (w/w) and (BOP + APS) content was 2.5 wt.% with respect to the total amount of the monomers. The mixture of the monomers (GMA and EGDMA), BOP and toluene was added dropwise to the aqueous phase (PVA or PAV + CH) in a 500 cm3 cylindrical reactor fitted with mechanical stirrer,

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thermometer and reflux condenser. The organic/aqueous phase ratio was 1:9 and the copolymerization reactions were allowed to proceed for 8 h at 78 °C and 1 h at 90 °C with stirring rate of 360 r.p.m. Then, the G and C microparticles were separated by decantation, washed with hot water and extracted with ethanol in a Soxhlet apparatus to remove traces of residual monomers, liniar oligomers and porogenic agent. The sample codes and some characteristics of the G and C microparticles are presented in Table 1. 2.2.2. Preparation of 7-(50 -nitroindazole-10 -yl-acetamidil)-cephalosporin sodium salt (7-NAC) 7-Aminodesacetoxycephalosporanic acid (0.048 mol) and triethylamine (0.044 mol) was added to the anhydrous dichloromethane (150 mL) and the resulting solution was stirred for 90 min at 22–24 °C. Separately, the mixed anhydride was prepared by mixing the sodium salt of the 5-nitroindazole-1-yl-acetic acid (0.04 mol) and pivaloyl chloride (0.04 mol) under stirring at 0–5 °C. The solution of triethylammonium salt of 7-ADCA was added dropwise to the mixed anhydride in a period of 120 min at 8–10 °C. Then, the temperature was increased at 20–24 °C and the reaction continued under stirring for 3 h. After completion of the coupling reaction, the dichloromethane was removed by vacuum distillation at 25–30 °C. The distillation residue was dissolved in 600 mL of saturated solution of sodium bicarbonate and then acidified to pH = 1.5–2 with HCl in the presence of butyl acetate (150 mL). The acidified solution was extracted with 50 mL water three times to remove the HCl traces, dried over anhydrous Na2SO4 (30 g) and filtered. The sodium salt of cephalosporin was isolated from organic layer by precipitation with sodium acetate (0.036 mol) or sodium ethylhexanoat at room temperature. The solid is filtered and dried under vacuum. The product is presented as a white crystalline powder, stable at room temperature, highly soluble in water. 2.2.3. Characterization of 7-NAC, porous microparticles and 7-NAC-porous microparticle systems The 1H NMR spectra of 7-NAC were recorded on Brucker ARX 400 Spectrometer (5 mm NP probe 1H/13C/31P/19F). 1 H NMR (DMSO-d6, 400 MHz), d (ppm): 1.82 (s, 3H, CH3); 3.33 (s, 2H, CH2); 3.64 (s, 2H, CH2); 4.94–4.98 (d, 1H, CH); 5.85 (s, 1H, CH); 7.75–7.80 (d, 1H, CHAr); 8.41–8.44 (d, 1H, CHAr); 8.85 (s, 1H, CHAr); 8.96 (s, 1H, CHAr); 9.65 (s, 1H, NH). The elemental analysis of 7-NAC was performed on Exeter Analytical CF-440 Elemental Analyzer. Elemental analysis for C17H14N5O6SNa (M = 439 g/mol): 46.46% C; 3.18% H; 15.94% N; 7.28% S. Found: 46.77% C; 3.47% H, 16.36% N; 7.61% S. The 7-NAC, the porous microparticles and the 7-NAC-porous microparticle systems were characterized by FT-IR spectroscopy (Bruker Vertex FT-IR Spectrometer) in the range of 4000–400 cm1 at a resolution of 2 cm1. FT-IR (7-NAC) mmax: 3256 cm1 (NH amidic); 1739 cm1 (CO lactamic); 1650 cm1 (CO amidic); 1367 cm1 (NO2 sim.); 1537 cm1 (NO2 asim.); 1606 cm1 (COO carboxilat). The surface morphology and topography of the 7-NAC-porous microparticle systems were observed with Environmental Scanning Electron Microscope type Quanta 200-FEI coupled with an energy dispersive X-ray system and with Scanning Probe Microscope Solver Pro-M platform (NT-MDT Co., Zelenograd, Moscow, Russia), respectively. The AFM measurements on 7NAC-porous microparticle systems were made in air, at room temperature (23 °C), in tapping mode, using a rectangular silicon cantilever NSG 10 and 203 Hz oscillation frequency. To analyze and calculate the surface parameters of cephalosporinporous microparticle systems the images were examined using the last version of the NT-MDT Nova software. 2.2.4. Biological activity of 7-NAC 2.2.4.1. Toxicity of 7-NAC. Healthy male and female white mice, weighing 20 ± 1 g each, divided into groups of 10 animals were selected to study the toxicity of synthesized cephalosporin. The animals had free access to feed and water ad libitum. Each experimental group was injected intraperitoneally with a small amount of tested solutions (1 mL), while the control group received injection of sterile distilled water (1 mL). The mortality rate of the experimental and the control lots was recorded at 24, 48 and 72 h. The Spearman-Kärber method was used for determining the LD50 [22]. All experiments were carried out in accordance with Directive 2010/63/EU of European Parliament and Council on the protection of animals used for scientific and experimental purposes. 2.2.4.2. Antimicrobial activity of 7-NAC. The antimicrobial activity of 7-NAC was performed on standard microbial strains, such as Streptococcus pneumoniae, Staphylococcus aureus as gram-positive germs and Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli, as gram-negative germs, respectively.

Table 1 Sample codes and some characteristics of G and C microparticles.

a

Sample codes

Composition

Diameter (D) (lm)

Specific surface area Spa (m2/g)

G1 G2 C1 C2

GMA:EGDMA-90:10 (mol/mol) GMA:EGDMA-80:20 (mol/mol) 90:10 (mol/mol) GMA:EGDMA + CH 80:20 (mol/mol) GMA:EGDMA + CH

316 243 136 124

69 58 160 132

Sp was determined by Dynamic Water Vapor Sorption method (DVS method).

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Microbial strains in suspension form in saline solution were grown on Mueller-Hinton agar, with incubation at 37 °C for 18 h. After that, the synthesized cephalosporin was dissolved in sterile distilled water (100 lg/mL distilled water), this solution being introduced into the culture medium (100 mL/sample). Sterilization was performed at 120 °C for 20 min. A blank sample, including the solvent (sterile distilled water) was performed in order to ensure the control over the transformation of culture medium under the influence of tested compounds. Assessment of antimicrobial activity was performed with the Kirby-Bauer diffusion method [23] and the readings were done after 24 h. Cephalexin was used as reference antibiotic. The Minimum Inhibitory Concentration (MIC) of 7-NAC was determined by the broth microdilution test. Both agar diffusion test and the broth microdilution test were realized according to the National Committee for Clinical Laboratory Standards Institute, NCCLS Approval Standard Document M2-A5, Vilanova, PA, USA (2000). 2.2.5. Batch adsorption studies The batch adsorption method was chosen to study the 7-NAC adsorption on C and G microparticles. Porous microparticles were firstly placed in 250 mL beakers filled with deionised water and allowed to swell for 24 h. Prior to the usage in the adsorption experiments, the microparticles were removed from aqueous solution by decantation and were centrifuged at 1000 rpm for 10 min. The influence of the initial 7-NAC concentration on the adsorption process was investigated by adding a known amount of wet C and G microparticles equivalent to 0,2 g dry microparticles to a set of conical flasks filled with aqueous solution of 100 mg 7-NAC with various concentrations ranging from 50 mg/L to 1000 mg/L. The effect of contact time on the adsorption capacity of C and G microparticles was studied for different period of time ranging from 10 to 1440 min. The influence of temperature on the adsorption rate of 7-NAC onto C and G microparticles was studied at three temperatures, such as 25, 30 and 40 °C. Also, the influence of the pH on cephalosporin loading was performed at various pH values ranging from 3 to 9. Another parameter that influences the cephalosporin loading capacity is cephalosporin:microparticles ratio. In this study the 7-NAC-microparticles systems were realized using the following weight ratios between cephalosporin and microparticles: 1:0.5, 1:1, 1:2. For all experiments the conical flasks were shaken at 180 r.p.m. using a thermostated shaker bath (Memmert, M00/M01, Germany). After the specified period of time, the porous microparticles were removed quantitatively from the 7-NAC solution by centrifugation at 1000 r.p.m. for 10 min. The amount of 7-NAC adsorbed onto the porous microparticles was calculated by the difference between the 7-NAC concentration in the supernatant before and after adsorption process. The retained amount of 7-NAC was determined spectrophotometrically (UV–vis SPEKOL 1300 Spectrophotometer) at the wavelength corresponding to the maximum absorbance (242 nm) using a previously plotted calibration curve. The quantities of the 7-NAC adsorbed at any time (qt) and at equilibrium (qe) were calculated as:

C0  Ct V W C0  Ce V qe ¼ W

qt ¼

ð1Þ ð2Þ

where C0 = initial concentration of 7-NAC solution (mg/L); Ct = concentration of 7-NAC solution at any time (mg/L); Ce = concentration of 7-NAC solution at equilibrium (mg/L), V = volume of 7-NAC solution (L) and W = mass of microparticles (g). All the adsorption data were achieved in triplicate and the average values were taken into consideration. 2.2.6. In vitro release studies The release characteristics of optimized systems were realized by incubation of 500 mg cephalosporin-polymer systems in 50 mL of dissolution media with pH = 1.2 (simulated gastric solution) and pH = 7.4 (phosphate buffer solution) at 37 °C, under gentle shaking (50 rpm) using a thermostated shaker bath. The dissolution medium was collected with microsyringes at predetermined time intervals and the amount of 7-NAC was determined spectrophotometrically. The absorbency was monitored on Nanodrop ND 100 (Wilmington, USA) apparatus at 242 nm. The amount of 7-NAC released at different periods of time was determined using the calibration curves. The release experiments were realized in triplicate for each sample and the average values were used. 3. Results and discussion 3.1. Synthesis and characterization of 7-NAC From structural point of view, the cephalosporins contain a b-lactam ring structure attached to a dihydrotiazynic ring, this structure being called the cephem nucleus. The substitution of R1 and R2 side chain radicals leads to the synthesis of cephalosporins with different antimicrobial activities and pharmacological properties. The literature describes the synthesis of cephalosporins by acylation of the 7-Aminodesacetoxycephalosporanic acid (7-ADCA) with different organic compounds, such as organic acids activated with dicyclohexylcarbodiimide, mixed anhydrides, acid chlorides, polyphenols and thioesters

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[17,18,24–27]. The activation of the organic acids necessary for acylation of 7-ADCA is justified because the carboxyl groups have the low reactivity in the acylation process of the primary amine groups. In addition, this reaction must be achieved without damaging of b-lactam ring. In this work, the semi-synthetic cephalosporin was achieved by acylating the 7-Aminodesacetoxycephalosporanic acid with the mixed anhydride of 5 nitroindazole-1-yl-acetic acid at 5 °C. On this purpose, the sodium salt of the 5 nitroindazole (II) was initially obtained by warm dissolution of the 5 nitroindazole (I) in an alcoholic solution containing sodium ethoxide. The treatment of the compound (II) with sodium monochloroacetate leads to the sodium salt of the 5 nitroindazole-1-yl-acetic acid (III) (Fig. 1) [28].

Fig. 1. Synthesis route of 7-(50 -nitroindazole-10 -yl-acetamidil)-cephalosporin sodium salt.

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In order to increase the reactivity of carboxyl group, the sodium salt of the acid (III) was transformed by treatment with pivaloyl chloride into the mixed anhydride (IV), able to react fast and under mild conditions with 7-ADCA. The pivaloyl chloride is an ideal partner for this transformation due to the steric and +I effects of the three methyl groups located on the same carbon atom. As the mixed anhydride (IV) is unstable and difficult to dissolve, its use ‘‘in situ” was chosen, in a dichloromethane solution, where 7-ADCA is dissolved as triethylammonium salt (V). After completing the coupling reaction, the dichloromethane was removed under vacuum distillation and the distillation residue was dissolved in a saturated sodium bicarbonate solution, at this point the cephalosporin passed as sodium salt in the alkaline solution (VI). The structure of the compound (VI) was established by elemental and spectral analyses (FT-IR and 1H NMR). Based on elemental analysis it was established that the molecular formula and the molecular weight of 7-NAC are C17H14N5O6SNa and 439 g/mol, respectively. In the 1H-RMN spectrum (Fig. 2), the protons belonging to the CH2 group from the side chain present signals at 3.33 ppm, while for the protons from the cephalosporin ring the signals are present at d = 3.64 ppm and d = 4.94–5.85 ppm, respectively. The ACH3 group from position 3 of cefem ring generates a singlet at 1.82 ppm, and the proton from the ANH amide group appears at 9.65 ppm. The signals attributed to the aromatic protons from the indazolic ring appear within the range of 7.75–8.44 ppm and 8.85–8.96 ppm, respectively. FT-IR spectrum (Fig. 3) has revealed the presence of two carbonyl absorption bands at 1739 and 1650 cm1 assigned to the C@O stretching vibrations from b-lactam ring and amide group. The absorption band at 3256 cm1 is attributed to the NH stretching vibration from amide group, while the absorption bands at 1367 and 1537 cm1 correspond to the symmetric and asymmetric stretching vibrations of NO2 group.

3.2. Toxicity and antimicrobial activity of 7-NAC The LD50 value of synthesized cephalosporin was determined by using the Spearman-Karber method [22] and is shown in Table 2. According to the experimental data, the LD50 value of 7-NAC is smaller than that of cephalexin, indicating that the new cephalosporin can be included in the category of substances with moderate toxicity. The antimicrobial activity of cephalosporins is characterized by their selective toxicity against the microorganism generating the infection [29–32]. In this study, the antimicrobial activity of 7-NAC was realized using both gram positive and gram-negative germs. Antimicrobial test results for 7-NAC and cephalexin were expressed by inhibition zone diameter (Table 2). 7-NAC shows appreciable activity against S. pneumoniae comparable with cephalexin and exhibited no activity against P. aeruginosa. It was evident that the S. aureus, E. coli and K. pneumoniae were less sensitive to the action of 7-NAC compared to the cephalexin. The antimicrobial effectiveness, described in terms of Minimum Inhibitory Concentration is also presented in Table 3.

Fig. 2. 1H NMR spectrum of 7-NAC.

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Fig. 3. FT-IR spectra of: (a) 7-NAC; (b) 7-NAC-G1 system and (c) G1 microparticles. Table 2 The LD50 values and antimicrobial activity of 7-NAC and cephalexin (reference drug). Sample codes

7-NAC Cephalexin

LD50 (mg/kg body)

393 420

Inhibition zone diameter (mm) K. pneumoniae

P. aeruginosa

S. pneumoniae

S. aureus

E. coli

9–10 20–21

– –

20–21 22–23

18–19 32–33

8–9 23–24

Table 3 MIC values for 7-NAC. Sample code

7-NAC

MIC (lg/mL) K. pneumoniae

P. aeruginosa

S. pneumoniae

S. aureus

E. coli

52

–

14

12

54

Determining the MIC values of cephalosporin indicate that the introduction of 5-nitroindazole nucleus in the side chain of b-lactam antibiotic led to an effective activity against certain Gram positive germs. 3.3. Characterization of the porous microparticles and 7-NAC-porous microparticle systems The presence of 7-NAC in G and C microparticle structures was highlighted by FT-IR, SEM and AFM analyses.

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In the spectrum of 7-NAC-G1 microparticles system (Fig. 3) the appearance of new absorption band at 1557 cm1 (C@O symmetric stretching vibrations) as well as a shift of the absorption band position from 1730 cm1 to the higher frequency (1734 cm1) confirm the adsorption of 7-NAC within the structure of the porous microparticles. One of the main methods of characterization of microparticles from morphological point of view is the scanning electron microscopy which helps the investigation of the shape, dimensions and internal and surface structure of the microparticles. Fig. 4 illustrates the comparison between SEM images for the G1, G1-7-NAC, C1 and C1-7-NAC microparticles, both from the point of view of the surface differences as well as the internal structures. The SEM microscopy emphasizes the fact that the absorption of 7-NAC does not change the porous structure and spherical shape of the microparticles. The surface topography has been studied by atomic force microscopy. The adsorption of 7-NAC in the porous structures generates a decrease of the microparticles roughness as well as a decrease of pore diameters (Fig. 5). 3.4. Adsorption studies 3.4.1. Selection of optimized systems The influence of different parameters on the adsorption process of 7-NAC was studied in order to find the optimal conditions for 7-NAC loading onto porous microparticles as well as to select the suitable cephalosporin delivery system. The effect of the initial concentration of 7-NAC solution on the adsorption capacity of porous microparticles is presented in Fig. 6. From Fig. 6 it is clear that the adsorption capacity of porous microparticles increases with the increase of the initial 7-NAC concentration. At high concentration of the 7-NAC solution the number of the interactions between sorbent and sorbat ions increases, leading to the increase of the amount of 7-NAC adsorbed. The highest amount of 7-NAC adsorbed was observed for C microparticles due to their high specific surface area (Table 1) and also to the presence of chitosan in the structure of porous microparticles that is a hydrophilic polymer and increase the hydrophilicity of the C microparticles compared to that of G microparticles. The influence of contact time on the adsorption process is plotted in Fig. 6. The adsorption capacity of microparticles increases with the increase of contact time, reaching adsorption equilibrium within 600 min (C microparticles) and 720 min (G microparticles), respectively. The uptake of 7-NAC is fast at the first hour because a large number of binding sites

Fig. 4. SEM images of: (a) G1 microparticles; (b) C1 microparticles; (c) G1-7-NAC system and (d) C1-7-NAC system.

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Fig. 5. AFM images (3-D) of: (a) G1 microparticles; (b) G1-7-NAC system; (c) C1 microparticles and (d) C1-7-NAC system.

Fig. 6. Influence of the initial concentration of 7-NAC on the adsorption process.

from the surface is available for adsorption and thereafter it becomes slower until the equilibrium is established. At longer contact times, the change in the amount of the drug adsorbed is negligible. Another parameter that has an influence on the adsorption process is temperature (Fig. 7). As it can be seen from Fig. 7 an increase of adsorption capacities of G and C microparticles takes place as follows: from 64.1 mg/g to 81.3 mg/g (G1 microparticles); 61.3 mg/g to 78.6 mg/g (G2 micropaticles); 74.67 mg/g to 104.3 mg/g

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Fig. 7. Influence of temperature on the adsorption process.

(C1 microparticles) and from 68.3 mg/g to 89.7 mg/g (C2 microparticles). These results indicate that a high temperature favors 7-NAC adsorption onto porous microparticles because the increasing of temperature leads to the swelling of the microparticles and to the increase of the diffusion rate which facilitates transport of the 7-NAC molecules across the surface and in the internal pores of the adsorbent [33]. A similar behavior has been observed for the adsorption of cefotaxime sodium salt on polymer coated ion exchange resin microparticles [34]. The effect of GMA:EGDMA ratio on the adsorption of 7-NAC onto porous microparticles was also investigated. In order to study the influence of the EGDMA amount on 7-NAC adsorption process, the porous microparticles were prepared by varying the ratio between GMA and EGDMA. It was observed that the adsorption capacity of microparticles decreased with increase of the EGDMA content. This can be explained by the fact that increasing of crosslinking density leads to a compact structure of microparticles and therefore the adsorption of 7-NAC by microparticles is decreased. The optimal conditions for the preparation of 7-NAC – porous microparticle systems (G1 and C1) are: GMA: EGDMA = 90:10 (mol/mol), 7-NAC:microparticles ratio = 1:2 (g/g), temperature = 40 °C, pH = 4.5, initial concentration of drug solution = 1000 mg/L. 3.4.2. Kinetic studies It is known from literature that the mechanism of sorbate adsorption onto porous adsorbent can be described by three consecutive steps [35]: 1. transport of the adsorbate molecules from the bulk solution across the boundary layer surrounding the porous microparticles to the surface of the adsorbent. In this case the adsorption rate is governed by film diffusion; 2. adsorption of the adsorbate molecules at binding sites on the adsorbent surface. This step is very fast in the case of physical adsorption and much slower when a chemical reaction is established between adsorbate molecules and adsorbent [36]; 3. diffusion of the adsorbate molecules within the internal structure of the adsorbent by a pore diffusion or solid surface diffusion or branched pore diffusion mechanisms [37]. In this case the rate of adsorption is governed by particle diffusion. In order to study the mechanisms of 7-NAC adsorption onto G and C microparticles the following mathematical models were used: Lagergren model (pseudo-first order kinetic model), Ho model (pseudo-second order kinetic model) and intraparticle diffusion model (Weber-Morris). The kinetic data interpretation was done for C7-NAC = 1000 mg/L, which is the concentration for which the highest amount of 7-NAC was adsorbed. For a most accurate interpretation of the experimental data, in case of Lagergren and Ho models, a nonlinear regression technique was used while for the Weber-Morris model the linear fitting technique was applied. The nonlinear regression method using Origin PRO 7.5 software was chosen for the determination of the parameters derived from the isotherm and kinetic models because this method introduces less errors compared with linear fitting. To evaluate the best fitting kinetic models and sorption isotherms to the experimental data two statistical error functions namely, correlation coefficient (R2) and chi-square test (v2) have been used. The chi-square value can be calculated using the following equation:

v2 ¼

X ðq  q Þ2 e em qem

ð3Þ

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where qem = equilibrium capacity derived from theoretical data of the studied model (mg/g) and qe = equilibrium capacity obtained from the experimental data [38]. Pseudo first order kinetic model was proposed in 1898 by Lagergren and is frequently used to describe the adsorption process in solid/liquid systems [39]. The nonlinear form of this model can be expressed as:

qt ¼ qe ð1  ekt t Þ

ð4Þ

where qe and qt are the adsorbat amounts adsorbed at equilibrium and at time t (mg/g), k1 is the rate constant of the pseudofirst order model (min1). The nonlinear fitted results of the Lagergren model in case of 7-NAC adsorption onto G and C microparticles at 40 °C are shown in Fig. 8. The parameter values corresponding to Lagergren model (k1, qe) as well as the values of the error functions (R2 and v2) are presented in Table 4.

Fig. 8. Adsorption kinetics of 7-NAC onto G1 and C1 microparticles.

Table 4 Kinetic parameters and diffusion coefficients for the adsorption of 7-NAC onto G1 and C1 microparticles. Sample codes

G1

C1

qe,exp (mg/g)

81.3

104.3

75.98 2.14 0.976 18.18

99.47 3.37 0.979 26.04

v2

81.54 3.6 0.998 0.71

104.28 5.0 0.999 0.78

Weber-Morris intraparticle diffusion model Ki2 (mg/(g min1/2)) Ci2 R2

0.92 55.71 0.994

0.81 85.09 0.993

Diffusion coefficients Df (109 cm2/s) Dp (1012 cm2/s)

1.49 1.82

0.49 0.45

Lagergren pseudo-first order model qe.calc (mg/g) k1  102 (min1) R2

v2 Ho pseudo-second order model qe.calc (mg/g) k2  104 (g/mg min) R2

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The values of the 7-NAC amount adsorbed onto microparticles obtained by applying the Lagergren model are much lower compared with the experimental ones. Also, from Table 4 one can notice that the R2 and v2 values are in the range from 0.976 to 0.979 and from 18.18 to 26.04, respectively suggesting that this model does not describe the process of 7-NAC absorption onto G and C microparticles. Pseudo-second order kinetic model is described by the nonlinear equation proposed by Ho and McKay [40].

qt ¼

k2  q2e  t 1 þ k2  qe  t

ð5Þ

where k2 is the rate constant of the pseudo-second order model (g/mg min). In Fig. 8, the nonlinear plot of the Ho model is presented and the values of k2, qe, R2and v2 are listed in Table 4. Only a small difference between qe values corresponding to Ho model and experimental values of qe for all types of microparticles was observed. Also, it can be seen that the values of the rate constant (k2) increases with increasing temperature, indicating a higher adsorption rate of 7-NAC at higher temperatures. The increased values achieved for R2 (0.998–0.999) correlated with low values for v2 (0.71–0.78) indicates that the Ho model describes better the experimental data. To identify the exact diffusion mechanism the experimental data were analyzed by Weber-Morris model using the linear form of the following equation [41]:

qt ¼ kid  t 1=2 þ C i

ð6Þ

where kid is the intraparticle diffusion rate constant (mg/g min1/2) and Ci is the constant that gives information about the thickness of the boundary layer. The plot qt versus t1/2 enables the determination of constants kid and Ci as slope and intercept, respectively (Fig. 9). If the plot qe versus t0.5 represents a straight line passing through the origin, then the absorption is exclusively controlled by the intra-particle diffusion. If the plot shows multiple linear segments the absorption process may have two or more steps. From Fig. 9 it can be observed that the studied adsorption process has three stages: (1) rapid adsorption of 7-NAC on the adsorbent surface or instantaneous adsorption stage; (2) gradual adsorption where the rate-limiting step is probably the intra-particle diffusion; (3) final equilibrium stage due to the low concentration of 7-NAC in the liquid phase, as well as to the smaller number of available adsorption sites. The values of the diffusion rate constants kid and the correlation coefficients are given in Table 4.

Fig. 9. Intraparticle diffusion plots of 7-NAC adsorption onto G1 and C1 microparticles.

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The Ci2 values increase on increasing temperature, indicating the increase of the thickness of the boundary layer and decrease of external mass transfer. Also, the Ci2 values increase in the following order: C1 > G1 which confirms that the best adsorbent is the C1 microparticles. Based on these results, the conclusion that can be drawn is that the intra-particle diffusion is not the only rate limiting step. Knowing that the mechanism of adsorption of various adsorbents can be explained either by pore or film diffusion a model based on Fick’s law can be applied to evaluate the diffusion mechanism. The film diffusion and pore diffusion mechanisms can be expressed by the following equation [42]:



lnð1  FÞ ¼ ln

F¼

6



p2

 1=2 6 Dp  t  r0 p



  Df  p2  t r 20

ð7Þ

ð8Þ

where Df and Dp are the film diffusion coefficient and the pore diffusion coefficient (cm2 s1), F is the ratio between amount adsorbed at time t and the amount adsorbed at equilibrium, r0 is the radius of the adsorbent microparticles assumed to be spherical (lm) (Table 1). The values of the diffusion coefficients (Df and Dp) were determined from the slope (Pi and Pii) of linear plots of ln(1  F) versus t and F versus t0.5, respectively and can be expressed as:

Df ¼ 

Dp ¼

Pi  r20

p2

P2ii  p  r 20 36

ð9Þ

ð10Þ

The calculated values of Df and Dp are presented in Table 4. When the rate-determining step is the film diffusion, Df has values in the range of 106–108 cm2/s and if the pores diffusion is the one limiting the adsorption rate, then the Dp values are in the range of 1011–1013 cm2/s [43]. In this study, the analysis of experimental data shows that the values of the film and pore diffusion coefficients are 109 and 1012 cm2/s, respectively which indicates that the adsorption of 7-NAC onto microparticles was controlled both by the film and pore diffusion mechanisms. The values of the Df and Dp coefficients are also noticed to increase as the temperature and radius of spherical microparticles increase. Similar behaviours have been observed for the adsorption of Reactive Red-4 onto Sterculia Quadrifida seed shell waste [44] or in case of chromium ions sorption onto chitin [45]. 3.4.3. Adsorption isotherms An understating of the interaction mode between an adsorbate (liquid phase containing the dissolved substances to be adsorbed) and an adsorbent (solid phase) is crucial in the design of pharmaceutical forms and for the evaluation of the potential applications. Description of the adsorption equilibrium characteristics of 7-NAC on G and C microparticles was performed by means of three two-parameter isotherm models and four three parameters isotherm models using the nonlinear regression technique. 3.4.3.1. Two parameter isotherm models. Langmuir isotherm: Langmuir isotherm is widely used to describe the adsorption of adsorbate molecules onto various adsorbents [46]. This model starts from assumption that monolayer adsorption occurs at a finite number of the specific site of adsorption. Langmuir isotherm is described by the equation:

qe ¼

qm  K L  C e 1 þ K L  Ce

ð11Þ

where qe is the 7-NAC amount adsorbed at equilibrium (mg/g); qm is the maximum adsorption capacity (mg/g), KL is the Langmuir constant that reflects the affinity between adsorbate and adsorbent. In 1974, Weber and Chakravorti [47] introduced an equilibrium constant, known as dimensionless separation factor (RL) in order to indicate if the adsorption process is unfavorable (RL > 1), favorable (0 < RL < 1), linear (RL = 1) or irreversible (RL = 0). The separation factor can be calculated according to the relationship:

RL ¼

1 1 þ K L  C0

ð12Þ

where C0 is the initial concentration. The nonlinear fitted results for Langmuir model are shown in Fig. 10 while the Langmuir equation parameters, R2 and v2 values are given in Table 5.

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145

Fig. 10. Two-parameter (a) and three-parameter (b) isotherm plots of 7-NAC adsorption onto G1 and C1 microparticles.

From the analysis of Langmuir isotherm parameters, the following conclusions can be drawn: – the values of the maximum amount adsorbed (qm) determined by using the Langmuir model are close to the experimental values shown in Table 4; – the capacity of saturation increases with increasing temperature, which reflects a better accessibility of the adsorption centres; – the highest value of Langmuir constant (KL) was achieved in case of C1 microparticles, which demonstrates a higher capacity of antibiotic adsorption compared to the G1 microparticles; – the values of KL increase with increasing temperature which indicates a higher efficiency of 7-NAC adsorption at higher temperatures; – the RL values calculated at 25, 30 and 40 °C and for all cephalosporin solution concentrations were in the range of 0 and 1, which indicates the fact that the adsorption process is favorable in the studied working conditions; – for each type of microparticle, the RL value decreases once the temperature gets higher, suggesting an increase of affinity between 7-NAC and microparticles; – the high values achieved for R2 (0.996–0.999) and smaller values for v2 (0.03–0.16) show that the Langmuir model describes well the adsorption of 7-NAC onto the G1 and C1 microparticles. Freundlich isotherm: Another model commonly used in adsorption studies is the Freundlich model. This model can be applied to multilayer sorption of adsorbate on heterogeneous surface [48]. Freundlich isotherm is described by nonlinear equation as: F qe ¼ K F  C 1=n e

ð13Þ

where KF represents the adsorption capacity for a unit equilibrium concentration; 1/nF is a constant that suggests the favorability and capacity of the adsorbent-adsorbate system. Similarly to RL values from Langmuir isotherm, the 1/nF value gives the information about the type of adsorption as follows: (1/nF) > 1 adsorption is unfavorable; 0 < (1/nF) < 1 adsorption is favorable and (1/nF) = 0 adsorption is irreversible. The nonlinear plot of qe versus Ce for 7-NAC adsorption onto all types of microparticles is shown in Fig. 10 and the values of Freundlich constants (KF and 1/nF) are presented in Table 5. Although the values of 1/nF are in the range of 0–1, which indicates that Freundlich model is favorable, the values of statistical error functions R2 (0.884 to 0.885) and v2 (19.55 to 37.34) obtained by applying this model show that the Freundlich model does not fit well the experimental adsorption data. Temkin isotherm: This isotherm was proposed by Temkin and Pyzhev [49] for describing the hydrogen adsorption on platinum electrodes in acid solutions and is based on the following assumptions: (a) the adsorption heat of all molecules in the

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T.A. Cigu et al. / European Polymer Journal 82 (2016) 132–152 Table 5 Two and three-parameter isotherm values for the adsorption of 7-NAC onto G1 and C1 microparticles. Sample codes Two parameter isotherms Langmuir qm (mg/g) KL  102 (L/mg) RL R2

v2 Freundlich KF (L/g) 1/nF R2

v2 Temkin bT (J/mol) aT (L/g) R2

v2 Three-parameter isotherms Sips qm (mg/g) aS  102 (L/mg) nS R2

v2 Redlich-Peterson KRP (L/g) aRP  102 (L/mg) bRP R2

v2 Khan qm (mg/g) bK  102 nK R2

v2 Toth qm (mg/g) bT  102 nT R2

v2

G1

C1

82.65 5.12 0.02–0.28 0.996 0.03

106.325 6.026 0.02–0.25 0.999 0.16

41.23 0.10 0.885 19.55

51.30 0.11 0.884 37.34

75.28 49.96 0.915 14.51

56.13 58.87 0.916 16.98

82.53 5.27 0.96 0.999 0.05

106.64 0.58 0.99 0.999 0.28

4.27 5.21 0.99 0.999 0.14

5.39 5.42 0.99 0.999 0.01

82.21 5.19 0.99 0.999 0.11

105.79 5.49 0.99 0.999 0.01

82.73 5.70 1.003 0.999 0.05

106.64 5.87 1.001 0.999 0.12

layer may linearly decrease together with the covering, due to the interactions between the adsorbent and adsorbate; (b) the adsorption features a uniform distribution of the binding energies. The nonlinear form of Temkin equation is:

qe ¼

RT lnðaT  C e Þ bT

ð14Þ

where bT is the Temkin constant related to the heat of adsorption (J/mol), aT is the equilibrium binding constant corresponding to the maximum binding energy (L/g); R is the gas constant (8.314 J/mol K) and T is absolute temperature (K). The Temkin plot of 7-NAC adsorption onto G1 and C1 microparticles is illustrated in Fig. 10. The values of aT, bT, R2 and v2 are listened in Table 5. The values of equilibrium constant related to the maximum binding energy (aT) increase as temperature increases while the values of Temkin constant describing the adsorption heat (bT) decrease as temperature increases for each of the G1 and C1 microparticles. However, the low values achieved for R2 (0.915–0.916), respectively the high values for v2 (14.51–16.98) show that Temkin model does not describe well the experimental data. 3.4.3.2. Three-parameter isotherm models. The adsorption equilibrium data were analyzed using four three-parameter isotherm models (Sips, Redlich-Peterson, Khan and Toth). All model parameters were evaluated by nonlinear regression and the equation forms can be written as:

T.A. Cigu et al. / European Polymer Journal 82 (2016) 132–152

Sips :

qe ¼

qm  aS  C ne S 1 þ aS  C ne S

Redlich-Peterson :

Khan :

qe ¼ qm

Toth :

qe ¼ qm

qe ¼

147

ð15Þ K RP  C e 1 þ aRP  C be RP

bK  C e n ð1 þ bK  C e Þ K

ð17Þ

bT  C e 1=nT nT

½1 þ ðbT  C e Þ

ð16Þ



ð18Þ

where aS is the Sips constant (L/mg); nS is the Sips model exponent; KRP and aRP are the Redlich-Peterson constants; bRP is the Redlich-Peterson model exponent; bK is the Khan model constant and nK is the Khan model exponent; bT is the Toth model constant and nT is the Toth model exponent. All these models represent the combined feature of Langmuir and Freundlich isotherm equations and under certain conditions can be simplified to one of the two-parameter isotherm models. Therefore: – at low adsorbate concentration the Sips isotherm predicts a multilayer sorption capacity being similarly to the Freundlich isotherm equation, while at high adsorbate concentrations the Sips isotherm is specific to a monolayer sorption capacity corresponding to the Langmuir isotherm model [50]; – for bRP = 1, Eq. (16) reduces to the Langmuir isotherm equation and for aRP  C be RP > 1 the Redlich-Peterson equation can be expressed as Freundlich type isotherm equation [51]; – if nK = 1, Eq. (17) can be simplified to the Langmuir isotherm equation, whereas Eq. (17) can be approximated by Freundlich type isotherm equation if the value of bK  Ce is much bigger than unity [52]; – if nT = 1, Eq. (18) can be simplified to the Langmuir type isotherm equation, but is not any connection between Toth equation an Freundlich model [53]. The three parameter isotherm model plots of 7-NAC adsorption onto G1 and C1 microparticles are illustrated in Fig. 10 while the parameters and the statistical error functions values are presented in Table 5. Analysing the data from the Fig. 10 and Table 5, the following conclusions can be drawn: – the values of the maximum adsorbed amount (qm), determined by using the Sips model, are close to the experimental ones (Table 4); – the values of exponents nS, bRP, nK and nT are very close to the unit, which suggests that the data achieved during the adsorption process fit the Langmuir model better than Freundlich model, as the data from Table 5 confirmed. – higher values obtained for the correlation factor (0.999) and lower values for v2 (0.01–0.28) show that all four models describe well the adsorption of 7-NAC onto G1 and C1 microparticles. – constant (KRP, aPR, bK, bT) and exponent (bRP) values increase with increasing of the temperature demonstrating that the adsorption of 7-NAC onto G1 and C1 microparticles is favored at high temperature and the most efficient adsorbent for 7NAC is represented by C1 microparticles. 3.4.4. Estimation of thermodynamic parameters To describe the thermodynamic behavior of the G1 and C1 microparticles the thermodynamic parameters [Gibbs free energy change (DG), enthalpy (DH) and entropy change (DS)] were calculated. The thermodynamic parameters were estimated using Van’t Hoff equation:

ln K ¼

DS DH  R RT

DG ¼ DH  T  DS

ð19Þ ð20Þ

where K is the Langmuir adsorption equlibrium constant related to the energy of adsorption [54]. The enthalpy and entropy values were calculated from the slope and intercept of plot ln K against 1/T (Fig. 11a) and the results were listed in Table 6. It is known from the literature that the DH and DG values can give information about the type of adsorption process. Thus, for the physical adsorption the DH < 40 kJ/mol and DG values are in the range of 0 to 20 kJ/mol, while for the chemical adsorption process the DH and DG values are in the range of 40–120 kJ/mol and 80 to 400 kJ/mol, respectively [55]. From Table 6 it can be observed that the DH and DG values are in the range of 14.62–16.48 kJ/mol and 10.68 to 9.06 kJ/mol, indicating that the interactions between G1 and C1 microparticles and 7-NAC are physical.

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Fig. 11. Plots of ln K versus 1/T (a) and Arrhenius plots (b) for adsorption of 7-NAC onto G1 and C1 microparticles.

Table 6 Thermodynamic parameters of adsorption process. Sample codes

Ea (kJ/mol)

DH (kJ/mol)

DS (J/(mol K))

DG (kJ/mol) Temperature (K) 298

303

313

G1 C1

16.85 19.63

14.62 16.48

79.47 86.78

9.06 9.38

9.46 9.81

10.25 10.68

Negative values of DG show that the 7-NAC adsorption process onto G1 and C1 microparticles at all temperatures is spontaneous and favorable. Also, the DG values decrease with increasing of the temperature which demonstrates that the adsorption is favorable at higher temperatures. Positive values of DH indicate that the adsorption process is endothermic. The affinity between G1 or C1 microparticles and 7-NAC is reflected by the positive value of DS. The positive value of DS also, reflects a energy redistribution between the adsorbent and cephalosporin. Because DH > 0 and DS > 0 adsorption occurs spontaneously at all temperatures. The values of activation energy (Ea) can provide information regarding the nature of the adsorption process which may be physical or chemical. The physisorption process is characterized by low activation energy values (5–40 kJ/mol) while in case of chemisorption the activation energy has higher values (40–800 kJ/mol) [56]. The activation energy can be calculated from the linear form of Arrhenius equation [57]:

ln k2 ¼ ln A 

Ea RT

ð21Þ

where k2 is the rate constant of pseudo-second order kinetic model (g/mg min); A- Arrhenius pre-exponential factor that is a temperature independent factor (g/mol s); Ea is the activation energy (kJ/mol); R is gas constant (J/K mol); T = temperature (K). The Ea and A values are obtained from the slope and the intercept of plot ln k2 against 1/T (Fig. 11b, Table 6). When the rate adsorption process is controlled by intraparticle diffusion mechanism, the activation energy is characterized by lower values [58]. In this study the values of activation energy were found to be in the range of 16.85–19.63 kJ/mol, indicating that the 7-NAC adsorption onto G1 and C1 microparticles is governed by the interactions of physical nature. Also, the positive value suggests that the Ea is an endothermic process and increasing temperature favours cephalosporin adsorption onto porous microparticles.

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3.5. In vitro release studies In vitro release studies were realized in phosphate buffer solution (pH = 7.4) and in the simulated gastric fluid (pH = 1.2). The release profiles were shown in Fig. 12. From the graphical representation it can be observed that the release profiles of 7-NAC from G1 microparticles are characterized by a biphasic behavior. The first phase can be attributed to the fast release of 7-NAC probably due to the release of cephalosporin from the surface of G1 microparticles. The second phase characterized by a slower release rate is attributed to the swelling of polymer network and the simultaneous diffusion of 7-NAC through the pores of microparticles. Also, from Fig. 12 it can be observed that the release process of 7-NAC from G1 microparticles was very little affected by the pH of the release medium. For C1 microparticles, the release rate is higher in simulated gastric fluid than in phosphate buffer solution, this phenomenon being explained by higher swelling degree of polymer networks at pH = 1.2 compared to that at pH = 7.4, due to the presence of the chitosan in the microparticle structures. At pH = 1.2, the NH2 groups of chitosan becomes NH+3 groups leading to the repulsions between polymer chains as well as an increase of the amount released. At pH = 7.4 deprotonation of amino groups diminishes the repulsions between polymer chains inducing a decrease in swelling and consequently a decrease of the amount of 7-NAC released. Chitosan grafting onto crosslinked networks based on GMA-co-EGDMA decreases the release rate of 7-NAC in both fluids. The lowest release rate for C1 microparticles can be attributed to the enhanced cephalosporin – polymer carrier interactions through ionic interactions between carboxylate groups of 7-NAC and amino groups of chitosan, as well as through H-bonding. To analyze the diffusion mechanism the release data were fitted according to the Higuchi and Korsmeyer-Peppas models. The Higuchi model describes the release of water soluble and poorly soluble drugs from polymeric matrix according to the equation [59].

Q t ¼ kH  t 1=2

ð22Þ

where kH = Higuchi dissolution constant. Korsmeyer-Peppas model [60] describes the drug release from the polymeric matrix as follows:

Mt ¼ kr  t n M1

ð23Þ

Mt/M1 = fraction of drug released at time t; kr = release rate constant that is characteristic to polymer-drug interactions; n = the diffusion exponent is characteristic to the different release mechanisms. The values of the release parameters and the release profiles of 7-NAC from G1 and C1 microparticles in pH = 1.2 and pH = 7.4 were shown in Table 7 and Fig. 13.

Fig. 12. Release profiles of 7-NAC in phosphate buffer solution (pH = 7.4) and in the simulated gastric fluid (pH = 1.2).

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Table 7 Kinetic parameters of 7-NAC release from G1 and C1 microparticles. Sample codes

G1, pH = 1.2 G1, pH = 7.4 C1, pH = 1.2 C1, pH = 7.4

Higuchi model

Korsmeyer-Peppas model

kH (h1/2)

R2

kr (minn)

n

R2

4.32 4.28 3.51 3.21

0.979 0.986 0.977 0.985

0.041 0.028 0.016 0.011

0.601 0.657 0.745 0.787

0.974 0.975 0.988 0.994

Fig. 13. Release kinetics of 7-NAC from G1 and C1 microparticles.

The release exponent ‘‘n” from Korsmeyer-Peppas equation is situated between 0.601 and 0.787, indicating that the release mechanism of 7-NAC from microparticles corresponds to anomalous transport and was controlled by both swelling and diffusion processes. 4. Conclusions In the present study, the adsorption and release studies of new cephalosporin from porous microparticles obtained by grafting a cationic polysaccharide (chitosan) onto crosslinked networks based on glycidyl methacrylate and ethylene glycol dimethacrylate were studied. The new semi-synthetic cephalosporin by acylation of 7-Aminodesacetoxycephalosporanic acid with mixed anhydride of 5-nitroindazole-1-yl-acetic acid was reported and the structure was confirmed by elemental and spectral analyses (FT-IR and 1H NMR). The optimal conditions for cephalosporin loading onto porous microparticles were established by studying the influence of different parameters, such as initial concentration of 7-NAC solution, temperature, pH and cephalosporin: microparticles ratio. From the adsorption studies it can be observed that the presence of chitosan in the structure of micropaticles leads to a higher adsorption capacity. The adsorption process is endothermic and spontaneous which means that high temperature facilitates the adsorption process of cephalosporin onto porous microparticles. The Langmuir isotherm (two parameter isotherm model) and all four three parameter isotherm models (Sips, RedlichPeterson, Khan and Toth) represent a better fit of experimental data. Adsorption kinetic was found to follow pseudosecond order kinetic model. Based on the analysis of the kinetic data of the release process it can be concluded that the release mechanism of 7-NAC from porous microparticles was controlled by swelling and diffusion processes. The cephalosporin-C microparticle system can be used as sustained drug delivery system for oral administration in infectious diseases. Acknowledgments Paper dedicated to the 150th anniversary of the Romanian Academy. This research was financially supported by the Internal research grant of University of Medicine and Pharmacy ‘‘Grigore T. Popa” Iasi Nr. 30878/30.12.2014. References [1] T. Sun, P. Xu, Q. Liu, J. Xue, W. Xie, Graft copolymerization of methacrylic acid onto carboxymetyl chitosan, Eur. Polym. J. 39 (2003) 189–192. [2] V. Singh, D.N. Tripathi, A. Tiwari, R. Sanghi, Microwave promoted synthesis of chitosan-graft-poly(acrylonitrile), J. Appl. Polym. Sci. 95 (2005) 820–825.

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