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Physicochemical aspects involved in methotrexate release kinetics from biodegradable spray-dried chitosan microparticles
Journal of Physics and Chemistry of Solids 81 (2015) 27–33
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Journal of Physics and Chemistry of Solids journal homepage: www.elsevier.com/locate/jpcs
Physicochemical aspects involved in methotrexate release kinetics from biodegradable spray-dried chitosan microparticles Philippe C. Mesquita a, Alice R. Oliveira b, Matheus F. Fernandes Pedrosa a, Anselmo Gomes de Oliveira b, Arnóbio Antônio da Silva-Júnior a,n a Graduate Program on Pharmaceutical Sciences, Department of Pharmacy, Federal University of Rio Grande do Norte, UFRN, Gal. Gustavo Cordeiro de Farias, Petrópolis, 59072-570 Natal, RN, Brazil b Department of Drug and Medicines, School of Pharmaceutical Sciences, State University of São Paulo, UNESP, Highway Araraquara-Jau, Km 01, 14801-902 Araraquara, SP, Brazil
art ic l e i nf o
a b s t r a c t
Article history: Received 18 July 2014 Received in revised form 12 January 2015 Accepted 30 January 2015 Available online 30 January 2015
Spray dried methotrexate (MTX) loaded chitosan microparticles were prepared using different drug/ copolymer ratios (9%, 18%, 27% and 45% w/w). The physicochemical aspects were assessed in order to select particles that were able to induce a sustained drug release effect. Particles were successfully produced which exhibited desired physicochemical aspects such as spherical shape and high drug loading. XRD and FT-IR analysis demonstrated that drug is not bound to copolymer and is only homogeneously dispersed in an amorphous state into polymeric matrix. Even the particles with higher drug loading levels presented a sustained drug release profile, which were mathematically modeled using adjusted Higuchi model. The drug release occurred predominantly with drug dissolution and diffusion through swollen polymeric matrix, with the slowest release occurring with particles containing 9% of drug, demonstrating an interesting and promising drug delivery system for MTX. & 2015 Elsevier Ltd. All rights reserved.
Keywords: Amorphous materials Polymers Mechanical properties Microstructure Transport properties
1. Introduction Polymeric nanoparticles and microparticles are commonly applied in the chemical, cosmetic, food and pharmaceutical industries as solid dispersed systems. In these particles, substances can be encapsulated, adsorbed or dispersed in order to prevent their degradation, improve specific characteristics or modulate their absorption. In the case of pharmaceuticals, additional advantages can be mucosal penetration, modulation of the time of drug in the organism, improving efficacy and reducing drug toxicity according to physicochemical characteristics of particles [1]. Among different polymers used to produce these particles, chitosan (CH) is a natural copolymer obtained by deacetylation of chitin, a polysaccharide found in the exoskeleton of crustaceans (Fig. 1a). CH is a biodegradable linear copolymer composed of Dglucosamine and N-acetyl-D-glucosamine monomers in different ratios, which gives it interesting properties. In aqueous media this polycationic material can interact with negative charges of mucosal epithelium and enhance bioavailability of drugs limited by a short residence time at the site of absorption [2,3]. Bioadhesion, biocompatibility and biodegradability characteristics allow CH to n
Corresponding author. Fax: þ 55 84 33429833. E-mail address: [email protected] (A.A. da Silva-Júnior).
http://dx.doi.org/10.1016/j.jpcs.2015.01.014 0022-3697/& 2015 Elsevier Ltd. All rights reserved.
be used in microencapsulation to obtain new drug delivery systems. In some cases, the organism eliminates the drug too quickly and this requires the administration of a considerable dose or several doses in a short time interval, causing side effects. This occurs commonly with drugs used for cancer treatment, such as methotrexate (MTX) (Fig. 1b), which is a prototype cytostatic folate antagonist drug used in the treatment of cancer and inflammatory diseases. It is known to be eliminated from organism in a short time and presents side effects and toxicity when high therapeutic doses are administered [4]. Among several microencapsulation methods available to obtain polymeric particles for drug delivery, spray drying is a single step technique that converts a liquid dispersion (solution, emulsion or suspension) into solid particles by exposing the sprayed feed to a heated air flow. Other advantages include total solvent removal, easy scale up and mainly a small and narrow size distribution [5– 7]. Some important encapsulation parameters like rate of spraying, the feed rate of drug/polymer solution, nozzle size, inlet and outlet temperatures have previously studied and standardized in our research group to produce efficient drug loaded-spray dried particles which impacted directly on the presence of residual solvent size, shape and surface of particles, as well as the crystallinity, thermal behavior and drug-polymer distribution in particles [8– 10]. In this work, different MTX/CH ratios were used to produce
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Fig. 1. Schematic representation of chemical structure of chitosan (A) and methotrexate (B).
spray dried microparticles containing a considerable drug amount in particles. Thus, their physicochemical properties were carefully evaluated and correlated with involved drug release kinetics in order to obtain biodegradable microparticles to prolong MTX release and potentially be used in cancer treatment.
2. Experimental 2.1. Materials Methotrexate (MTX) was purchased from DEG (Brazil); Chitosan (CH) with a degree of 85% deacetylation from Sigma Co (Saint Louis, USA) and acetic acid from Merck S.A. (Brazil). All other reagents were analytical grade. The purified water (1.3 μS) was prepared from reverse osmosis purification equipment; model OS50 LX, Gehaka (Brazil). 2.2. Sample preparation Suitable amounts of drug and copolymer were dissolved in 0.1 M acetic acid solution in order to obtain MTX-loaded CH microparticles with different MTX/CH ratios (9%, 18%, 27% and 45% w/ w), these were then dried in a mini spray-dryer Buchi-191 with a 0.7 mm nozzle using an inlet temperature of 140 °C and outlet temperature of 90 °C. An air flow of 500 Nl h 1; spray feed rate of 3 ml min 1 and aspirator efficiency of about 90% were selected during all experiments. Dried microparticles were collected and stored under vacuum at room temperature. Physical mixtures were used as control for analysis, which were prepared by mixing MTX and CH in a mortar, using the same mass ratios of MTXloaded CH microparticles (9%, 18%, 27% and 45% w/w). The drug/ polymer ratios were established according to experimental results of drug-loading efficiency. 2.3. Physicochemical aspects The shape and surface aspect of microparticles were accessed using scanning electronic microscopy SEM (SSX 550, Shimadzu). The volume-based mean diameter and the size distribution of the microparticles were determined by using Dynamic Light Scattering, in a Nanotrac NPA252 (Microtrac Inc., City, USA). The particles were suspended in 0.5% of tween 80 aqueous solution. The particle size distribution was characterized by mean diameter (D10, D50, D90), and SPAN was calculated as [(D90 D10)/D50]. For drug loading analysis, samples were dissolved in 0.1 M acetic acid and analyzed by using UV spectrophotometry in a 1 cm path-length cuvette, which had been previously validated. The analyses were carried out in triplicate and drug concentration was calculated using the equation from the standard curve fitted plot. Drugloading efficiency was determined as the ratio between the analytical and theoretical drug content. FT-IR spectra were recorded by KBr disk method (prepared with 2 mg of samples mixed with 300 mg of KBr) using Perkin Elmer FT-IR Spectrum. The X-ray diffraction (Rikugu diffractometer model Dmax 2500PC) was
determined using Cu-Kα radiation (λ ¼ 1.54056 Ǻ). 2.4. In vitro drug release The drug release profile was monitored using an amount of biodegradable microparticles containing 2 mg of drug, which was incubated in 4 mL of phosphate buffer medium (KH2PO4 0.05 mol l 1, pH ¼7.4) at 37 °C 70.2 °C. At specific intervals, the flasks were centrifuged at 3500 rpm and supernatant analyzed by UV spectrophotometry at 303 nm. Cumulative percentage of released MTX was plotted versus time and different mathematical models: zero order, first order and adjusted Higuchi model were used to fit the experimental data.
3. Results and discussion SEM images (Fig. 2) indicating that particles were successfully produced with a predominant spherical smooth shape for different drug-loaded microparticles. The droplet residence time and droplet diameter are important, but mainly the characteristics of products are fundamental to produce regular particles. A smaller amount of particles present some irregularity in shape like toroid cenospheres, mainly for large particles and for samples with lower drug loading. This kind of particle is typically formed after evaporation in two stages. At the first, the instantaneous contact of spray droplets with drying air led to the quick formation of a saturated vapor film around the particle surface, which evaporates at constant rate until reaching a critical point sufficient to form a dried shell. On the other hand, the second stage of evaporation is dependent on the diffusion rate of moisture through this dried shell, which increases causing a continuous reduction in the evaporation rate according to the diameter of the droplet, as was observed in large particles [11,12]. Furthermore, the used inlet temperature during all experiments was superior to the boiling point of the solvent mixture, contributing to vapor formation inside the particles. The rapid crust formation in large particles increases the internal pressure and depending on the nature of the crust, a rupture or collapse can occur, as observed in particles with lower drug loading [13]. Although this effect appeared in a smaller portion of the particles and the majority of particles presented spherical and smooth particles for all studied compositions. In previous studies, Oliveira et al. prepared MTX-loaded poly (D,Llactide-co-glycolide) (PLGA) spray dried microparticles. However, the presence aggregate or agglomerates of particles were observed, mainly for particles with higher amount of drug loading. This was attributed to particles' composition because biodegradable polyesters exhibit low Tg, which make it prone to aggregation during spray drying [10]. Thus, the selected parameters and composition were fundamental to obtain small droplets during drying to produce regular spherical chitosan microparticles, which also showed a dependence on used drug ratio. The biological activity of drug loaded particles is directly connected with particle aspects such as surface and shape, due to precision and accuracy of drug release rate that the system can
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Fig. 2. Scanning electron microscopy images of spray dried microparticles containing different drug loading: (A) 9%; (B) 18%; (C) 27% and (D) 45% for two magnifications, 6000 (left images) and 15,000 (right images).
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Table 1 Main parameters of particle size distribution of different MTX-CH microparticles. Sample identification
D10 (μm) D50 (μm) D90 (μm) Mean diameter (μm)
Size span
MTX-CH MTX-CH MTX-CH MTX-CH
0.98 1.68 1.46 2.27
2.23 0.81 1.21 0.70
45 27 18 9
2.12 5.67 3.81 5.87
5.71 6.30 6.08 6.37
2.76 4.91 3.86 5.39
supply [8,9,14]. Other important aspect is the particle size (Table 1), which impacts directly the drug dissolution from pharmaceuticals and hence, bioavailability. Small particles have a larger surface area available for dissolution providing faster release kinetics. In addition, particle size also regulates penetration across the physiological drug barriers [15]. Spray dried powder particles are generally obtained in the range of microparticles. Liu et al. chitosan microparticles showed a mean size between 35 and 50 μm using solute concentrations of 0.5%–1.5% [13]. It is well established that spray drying process parameters and the feedstock solution properties, mainly solute concentration, implies on particle properties such as size, e.g. Kašpar et al. obtained particles in the range of 2–9 μm by using solute concentration until 1% [16]. Comparing to reported data, MTX-CH microparticles showed small and narrowed particles size distribution with mean particle size in range of 2.7–5.3 μm (Table 1) that can be attributed to selected parameters of drying process that led to particles with potential parenteral or pulmonary application. SEM images did not reveal the presence of any crystal or considerably irregular particles, which strongly suggests total and homogeneous drug loading into polymeric matrix [6,17]. The first characteristic was confirmed by the high drug loading efficiency analytically assessed for different microparticles, even for samples with greater drug loading (Table 2). This is a characteristic advantage of the spray drying technique, because other microencapsulation methods such as emulsification with solvent evaporation or inotropic gelation, specifically in the case of chitosan, demonstrates low drug loading efficiency when a high drug/chitosan is used. In addition, high chitosan concentrations can complicate the formation of spherical particles, mainly by the high viscosity of the drying solution [18]. Thus, the used parameters in spray drying and used drug/polymer ratio in this work lead to spherical particles with a high drug loading efficiency. Actually, what really contributes to increment viscosity of spray dried solution is the solute concentration and not drug/polymer ratio [5,18]. All samples were prepared with a fixed solute concentration of 0.5% w/v, in order to avoid a considerable viscosity increment of drying solution and to guarantee total dissolution of MTX and CH in the same organic solvent. Fig. 3 shows FT-IR analyses, which were carried out in order to investigate possible drug-polymer binding, because MTX presents two carboxyl groups, while CH presents free primary amine groups (Fig. 1). The spectra of MTX showed that the characteristic Table 2 Encapsulation efficiency, analytical drug content and transport parameters for different polymeric microparticles (n¼ 3). Sample
MTX-CH MTX-CH MTX-CH MTX-CH
Drug loading (%)7 SD
45 27 18 9
39.8 7 0.5 22.5 7 2.2 15.14 70.5 7.7 7 0.3
Encapsulation efficiency (EE) (%)7 SD
88.4 7 1.2 83.5 7 8.2 84.17 2.5 86.0 7 3.2
Mathematical modeling of drug release data kH
r2
11.6 7 5.2 27.0 7 3.3 34.9 7 17.0 16.5 7 1.9
0.92 0.94 0.95 0.97
Fig. 3. FT-IR spectra for MTX, CH, physical mixtures (PM), and microparticles (Mc) with different drug/polymer ratios.
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bands of functional groups corresponded to N‒H stretch of primary amine (3415 cm 1), O‒H stretch of carboxylic acid group (2960 cm 1), C ¼O stretch of secondary amide (1643 cm 1), tertiary amide N‒H (1608 cm 1) and tertiary amine N‒C (1209 cm 1). For CH, alcohol O‒H and amine N‒H peaks are overlapped in 3423 cm 1, C‒H stretch (2898 cm 1), secondary amide C¼ O (1650 cm 1) and N‒H (1598 cm 1) (derived from the N-acetylated monomeric units), and ether C‒O stretch (1074 cm 1). Despite MTX presents two carboxyl groups and CH presents primary amine groups, it was not capable to identify any reaction between the copolymer and drug. Similar behavior was identified for ciprofloxacin-loaded spray dried microparticles, in which the ester carbonyl groups of copolymer did not react with the amino groups of drug [8]. The infrared spectra of the microparticles showed no difference from those of physical mixtures, demonstrating a common characteristic of drug-loaded spray dried microparticles, in which no chemical binding between two compounds or changes in the chemical structure of drug was identified. However, the proximity and overlapping between the characteristic peaks of drug and polymer does not allow a thorough evaluation, except for a particular band in 1209 cm 1, which is attributed to the stretching of tertiary amine bond and, as expected, it is more intense for samples with higher drug ratio. The maintenance of this group is very important for the biological activity of the particles because of differentiates of methotrexate in folic acid (Fig. 1). Fig. 4 shows XRD analysis for different samples. Unprocessed methotrexate occurred in a crystalline state, which was attributed to well-defined diffraction peaks at 2θ of 7.6 and 9.2, characteristic of its trihydrate form [10,19]. In accordance with some reported studies, in the XRD of chitosan diffraction peaks were observed at 10° or 20° corresponding to hydrated and anhydrous crystals respectively [20,21]. Comparing to pure drug and polymer, less crystallinity was observed for prepared physical mixtures, which increase according drug ratio in the sample [22]. The non-similarity among XRD patterns of MTX-loaded CH microparticles and physical mixtures confirmed the absence of crystalline MTX into particles, which indicated total dissolution of drug into polymeric matrix or amorphization during drying [10,23]. Anterior studies with biodegradable spray dried microparticles containing ciprofloxacin or triamcinolone also demonstrated the preparation of amorphous particles even for drug ratio about 50% [8,9]. Solids in amorphous state have molecules randomly arranged, which can be distributed among polymeric chains, requiring less free energy during dissolution and drug release. Generally, drug/polymer ratio strongly influences drug release rate from microparticles, but drug-polymer interactions or drug distribution into particles are parameters that may always be considered in the nano/microparticles development, permitting the evaluation of the real contribution of particle microstructure to drug release rate [24,25]. The established presence of MTX in amorphous phase in CH particles is a promising characteristic for uniformity in the drug release rates of those systems. Fig. 5 shows the in vitro cumulative release profiles of different MTX-loaded CH microparticles. All formulations successfully presented a sustained release for MTX according to the used drug/polymer ratio. Diffusion, swelling and erosion are the most important rate-controlling mechanisms involved with drug release from commercially available controlled release products [26]. A biodegradable hydrogel like CH formed microparticles that were structurally arranged as hydrophilic matrices, in which drug release can involve three different steps according to the environment. Initially, the drug adsorbed on the particles surface is rapidly dissolved causing a “burst effect”, followed by dissolution-diffusion of drug portion distributed into
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Fig. 4. X-ray powder diffraction analysis MTX, CH, microparticles (MC) and their respective physical mixtures (PM) with different drug/polymer ratios.
particles, which occurs when the dissolution medium penetrates into particles with a swelling of polymeric matrix. Finally, the erosion of the swelled matrix occurs with the release of residual drug amount [27–29]. The “burst effect” occurred for all formulations according to the used drug-polymer ratio, which is characteristic of spray dried particles [9,10]. At the moment of drying of molecular dispersions, during self-assembly of compounds, a faster diffusion of small molecules into polymeric matrix can occur by capillary flow, with co-precipitation of drug near the surface of particles [6,30]. It is interesting to observe that the “burst effect” can be a desirable property, when the particles are produced intend pulmonary or
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Fig. 5. MTX release profile (●) for CH microparticles in different drug/polymer ratios: (A) 9%; (B) 18%; (C) 27% and (D) 45% with all data and fitted plots shows (○) mathematical adjusted Higuchi model ft ¼ kh1/√t for release profile after third data (without the burst effect).
parenteral rout. In previous studies, Cardillo et al. identified a similar burst release about 60% in the in vitro release studies of biodegradable spray dried microparticles containing about 30% of ciprofloxacin with mean diameter about of 1 μm. However, a single dose of subconjunctival ciprofloxacin-PLGA microspheres sustained aqueous and vitreous therapeutic levels for up to 10 days. In addition, the particles were able to reduce bacterial recovery and signs of clinical endophthalmitis induced in rabbits [31]. In a following study with humans, a single dose of these same particles was able to prevent ocular infection in 67 patients after cataract surgery. The patients were examined on postoperative days during 28 days [32]. Thus, we believe that results of in vitro release studies is not able to predict in vivo effects, but they are so important to evaluate physicochemical properties and compositions of specific particles in order to select the best system for a desired administration rout. Thus, in order to compare efficiently the effect of this phenomenon in the overall drug release rate, the experimental data were mathematically adjusted by different models and velocity constants extracted [25,33]. Despite subjecting the experimental data to different mathematical models, after the initial burst release, an optimum linear fitting was reached only by using an adjusted Higuchi model, which was described by the equation ft ¼kH1/√t (Fig. 5). The identified kH for different MTX-CH loaded microparticles and respective correlation coefficient (r2) are summarized in Table 2. The Higuchi model describes drug release as a diffusion process based on Fick's law being square root time dependent [33]. However, swellable particles may lead to release that does not only agree with Fickian behavior, due to specific networks formed after polymer relaxation, in this case, the application of “Peppas equation” (Mt/M1 ¼ktn) is recommended, which considers the first 60% of total released drug. In this model, “k” incorporates characteristics of a macromolecular network into the polymeric matrix and “n” is a diffusional indicative of the transport mechanism involved [33]. However, “Peppas equation” was not applied because all MTX-CH loaded microparticles presented a considerable “burst effect” in which more than 60% of drug was released in the first stage. The drug adhered to the surface of the microsphere was rapidly released in the medium, which also contributed forming the water channels in the polymeric matrix. The second stage of MTX release occurred due to swelling is the glassy polymer change to a rubbery state, which expands the mobility of polymer chains with consequent drug diffusion through the swollen layer. These data were fitted by using an adjusted Higuchi model with an inverse of square root time dependence. Thus, during swelling, a layer of gel is formed around the particle, which limits and controls drug release rate [34]. The thickness of the gelling layer is dependent on drug-polymer interactions, environment and drug/ polymer ratio. Moreover, the third and final step of drug release due to copolymer erosion was not observed, in which a residual portion of drug remained entrapped in particles. In previous studies, Qi et al. successfully intercalated MTX into the Mg–Al–LDHs layers by themechanochemical–hydrothermal method. However a burst release about 60% was observed at the first 4 h of experiment, with almost total drug release after 10 h. Different mathematical models were applied, in which a similar model (parabolic diffusion model, Mt/M1/t ¼kt 0.5 þb) was applied and successfully elucidates that the release process was controlled by diffusion such as intra-particle diffusion or surface diffusion [23]. This step of study confirmed the most MTX release by drug dissolution, followed by partial drug diffusion through swollen particles, even for microparticles with highest drug loading. The mathematical modeling made it possible to identify a considerable sustained release effect for particles containing 9% of drug, which
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is fundamental for the selection of a suitable formulation for in vivo studies and establishes a dosage for specific administration or clinical pathology.
4. Conclusion Spray dried MTX-loaded chitosan microparticles were successfully produced using selected parameters. Particles exhibited desired physicochemical aspects such as spherical and smooth shape, high drug loading with non-bounded drug homogeneously dispersed in an amorphous state into polymeric matrix. Even the particles with higher drug loading levels presented a sustained drug release profile. The drug release occurred predominantly through drug dissolution and diffusion in swollen polymeric matrix, with slowest release for particles containing 9% of drug. These results support the idea that spray dried MTX loaded CH microparticles are a very promising drug delivery system, which can be administered by different routes such as oral, subcutaneous, pulmonary or intramuscularly due to the physicochemical aspects of particles.
Acknowledgments The authors wish to thank the Brazilian Agencies CNPq (grant number: 479195/2008; 483073/2010-5, 481767/2012-6) and CAPES (Scholarship of P.C. Mesquita and A.R. Oliveira), for their financial support. The authors also acknowledge the help extended by Andy Cumming in proofreading the English text.
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Contents lists available at ScienceDirect
Journal of Physics and Chemistry of Solids journal homepage: www.elsevier.com/locate/jpcs
Physicochemical aspects involved in methotrexate release kinetics from biodegradable spray-dried chitosan microparticles Philippe C. Mesquita a, Alice R. Oliveira b, Matheus F. Fernandes Pedrosa a, Anselmo Gomes de Oliveira b, Arnóbio Antônio da Silva-Júnior a,n a Graduate Program on Pharmaceutical Sciences, Department of Pharmacy, Federal University of Rio Grande do Norte, UFRN, Gal. Gustavo Cordeiro de Farias, Petrópolis, 59072-570 Natal, RN, Brazil b Department of Drug and Medicines, School of Pharmaceutical Sciences, State University of São Paulo, UNESP, Highway Araraquara-Jau, Km 01, 14801-902 Araraquara, SP, Brazil
art ic l e i nf o
a b s t r a c t
Article history: Received 18 July 2014 Received in revised form 12 January 2015 Accepted 30 January 2015 Available online 30 January 2015
Spray dried methotrexate (MTX) loaded chitosan microparticles were prepared using different drug/ copolymer ratios (9%, 18%, 27% and 45% w/w). The physicochemical aspects were assessed in order to select particles that were able to induce a sustained drug release effect. Particles were successfully produced which exhibited desired physicochemical aspects such as spherical shape and high drug loading. XRD and FT-IR analysis demonstrated that drug is not bound to copolymer and is only homogeneously dispersed in an amorphous state into polymeric matrix. Even the particles with higher drug loading levels presented a sustained drug release profile, which were mathematically modeled using adjusted Higuchi model. The drug release occurred predominantly with drug dissolution and diffusion through swollen polymeric matrix, with the slowest release occurring with particles containing 9% of drug, demonstrating an interesting and promising drug delivery system for MTX. & 2015 Elsevier Ltd. All rights reserved.
Keywords: Amorphous materials Polymers Mechanical properties Microstructure Transport properties
1. Introduction Polymeric nanoparticles and microparticles are commonly applied in the chemical, cosmetic, food and pharmaceutical industries as solid dispersed systems. In these particles, substances can be encapsulated, adsorbed or dispersed in order to prevent their degradation, improve specific characteristics or modulate their absorption. In the case of pharmaceuticals, additional advantages can be mucosal penetration, modulation of the time of drug in the organism, improving efficacy and reducing drug toxicity according to physicochemical characteristics of particles [1]. Among different polymers used to produce these particles, chitosan (CH) is a natural copolymer obtained by deacetylation of chitin, a polysaccharide found in the exoskeleton of crustaceans (Fig. 1a). CH is a biodegradable linear copolymer composed of Dglucosamine and N-acetyl-D-glucosamine monomers in different ratios, which gives it interesting properties. In aqueous media this polycationic material can interact with negative charges of mucosal epithelium and enhance bioavailability of drugs limited by a short residence time at the site of absorption [2,3]. Bioadhesion, biocompatibility and biodegradability characteristics allow CH to n
Corresponding author. Fax: þ 55 84 33429833. E-mail address: [email protected] (A.A. da Silva-Júnior).
http://dx.doi.org/10.1016/j.jpcs.2015.01.014 0022-3697/& 2015 Elsevier Ltd. All rights reserved.
be used in microencapsulation to obtain new drug delivery systems. In some cases, the organism eliminates the drug too quickly and this requires the administration of a considerable dose or several doses in a short time interval, causing side effects. This occurs commonly with drugs used for cancer treatment, such as methotrexate (MTX) (Fig. 1b), which is a prototype cytostatic folate antagonist drug used in the treatment of cancer and inflammatory diseases. It is known to be eliminated from organism in a short time and presents side effects and toxicity when high therapeutic doses are administered [4]. Among several microencapsulation methods available to obtain polymeric particles for drug delivery, spray drying is a single step technique that converts a liquid dispersion (solution, emulsion or suspension) into solid particles by exposing the sprayed feed to a heated air flow. Other advantages include total solvent removal, easy scale up and mainly a small and narrow size distribution [5– 7]. Some important encapsulation parameters like rate of spraying, the feed rate of drug/polymer solution, nozzle size, inlet and outlet temperatures have previously studied and standardized in our research group to produce efficient drug loaded-spray dried particles which impacted directly on the presence of residual solvent size, shape and surface of particles, as well as the crystallinity, thermal behavior and drug-polymer distribution in particles [8– 10]. In this work, different MTX/CH ratios were used to produce
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Fig. 1. Schematic representation of chemical structure of chitosan (A) and methotrexate (B).
spray dried microparticles containing a considerable drug amount in particles. Thus, their physicochemical properties were carefully evaluated and correlated with involved drug release kinetics in order to obtain biodegradable microparticles to prolong MTX release and potentially be used in cancer treatment.
2. Experimental 2.1. Materials Methotrexate (MTX) was purchased from DEG (Brazil); Chitosan (CH) with a degree of 85% deacetylation from Sigma Co (Saint Louis, USA) and acetic acid from Merck S.A. (Brazil). All other reagents were analytical grade. The purified water (1.3 μS) was prepared from reverse osmosis purification equipment; model OS50 LX, Gehaka (Brazil). 2.2. Sample preparation Suitable amounts of drug and copolymer were dissolved in 0.1 M acetic acid solution in order to obtain MTX-loaded CH microparticles with different MTX/CH ratios (9%, 18%, 27% and 45% w/ w), these were then dried in a mini spray-dryer Buchi-191 with a 0.7 mm nozzle using an inlet temperature of 140 °C and outlet temperature of 90 °C. An air flow of 500 Nl h 1; spray feed rate of 3 ml min 1 and aspirator efficiency of about 90% were selected during all experiments. Dried microparticles were collected and stored under vacuum at room temperature. Physical mixtures were used as control for analysis, which were prepared by mixing MTX and CH in a mortar, using the same mass ratios of MTXloaded CH microparticles (9%, 18%, 27% and 45% w/w). The drug/ polymer ratios were established according to experimental results of drug-loading efficiency. 2.3. Physicochemical aspects The shape and surface aspect of microparticles were accessed using scanning electronic microscopy SEM (SSX 550, Shimadzu). The volume-based mean diameter and the size distribution of the microparticles were determined by using Dynamic Light Scattering, in a Nanotrac NPA252 (Microtrac Inc., City, USA). The particles were suspended in 0.5% of tween 80 aqueous solution. The particle size distribution was characterized by mean diameter (D10, D50, D90), and SPAN was calculated as [(D90 D10)/D50]. For drug loading analysis, samples were dissolved in 0.1 M acetic acid and analyzed by using UV spectrophotometry in a 1 cm path-length cuvette, which had been previously validated. The analyses were carried out in triplicate and drug concentration was calculated using the equation from the standard curve fitted plot. Drugloading efficiency was determined as the ratio between the analytical and theoretical drug content. FT-IR spectra were recorded by KBr disk method (prepared with 2 mg of samples mixed with 300 mg of KBr) using Perkin Elmer FT-IR Spectrum. The X-ray diffraction (Rikugu diffractometer model Dmax 2500PC) was
determined using Cu-Kα radiation (λ ¼ 1.54056 Ǻ). 2.4. In vitro drug release The drug release profile was monitored using an amount of biodegradable microparticles containing 2 mg of drug, which was incubated in 4 mL of phosphate buffer medium (KH2PO4 0.05 mol l 1, pH ¼7.4) at 37 °C 70.2 °C. At specific intervals, the flasks were centrifuged at 3500 rpm and supernatant analyzed by UV spectrophotometry at 303 nm. Cumulative percentage of released MTX was plotted versus time and different mathematical models: zero order, first order and adjusted Higuchi model were used to fit the experimental data.
3. Results and discussion SEM images (Fig. 2) indicating that particles were successfully produced with a predominant spherical smooth shape for different drug-loaded microparticles. The droplet residence time and droplet diameter are important, but mainly the characteristics of products are fundamental to produce regular particles. A smaller amount of particles present some irregularity in shape like toroid cenospheres, mainly for large particles and for samples with lower drug loading. This kind of particle is typically formed after evaporation in two stages. At the first, the instantaneous contact of spray droplets with drying air led to the quick formation of a saturated vapor film around the particle surface, which evaporates at constant rate until reaching a critical point sufficient to form a dried shell. On the other hand, the second stage of evaporation is dependent on the diffusion rate of moisture through this dried shell, which increases causing a continuous reduction in the evaporation rate according to the diameter of the droplet, as was observed in large particles [11,12]. Furthermore, the used inlet temperature during all experiments was superior to the boiling point of the solvent mixture, contributing to vapor formation inside the particles. The rapid crust formation in large particles increases the internal pressure and depending on the nature of the crust, a rupture or collapse can occur, as observed in particles with lower drug loading [13]. Although this effect appeared in a smaller portion of the particles and the majority of particles presented spherical and smooth particles for all studied compositions. In previous studies, Oliveira et al. prepared MTX-loaded poly (D,Llactide-co-glycolide) (PLGA) spray dried microparticles. However, the presence aggregate or agglomerates of particles were observed, mainly for particles with higher amount of drug loading. This was attributed to particles' composition because biodegradable polyesters exhibit low Tg, which make it prone to aggregation during spray drying [10]. Thus, the selected parameters and composition were fundamental to obtain small droplets during drying to produce regular spherical chitosan microparticles, which also showed a dependence on used drug ratio. The biological activity of drug loaded particles is directly connected with particle aspects such as surface and shape, due to precision and accuracy of drug release rate that the system can
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Fig. 2. Scanning electron microscopy images of spray dried microparticles containing different drug loading: (A) 9%; (B) 18%; (C) 27% and (D) 45% for two magnifications, 6000 (left images) and 15,000 (right images).
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Table 1 Main parameters of particle size distribution of different MTX-CH microparticles. Sample identification
D10 (μm) D50 (μm) D90 (μm) Mean diameter (μm)
Size span
MTX-CH MTX-CH MTX-CH MTX-CH
0.98 1.68 1.46 2.27
2.23 0.81 1.21 0.70
45 27 18 9
2.12 5.67 3.81 5.87
5.71 6.30 6.08 6.37
2.76 4.91 3.86 5.39
supply [8,9,14]. Other important aspect is the particle size (Table 1), which impacts directly the drug dissolution from pharmaceuticals and hence, bioavailability. Small particles have a larger surface area available for dissolution providing faster release kinetics. In addition, particle size also regulates penetration across the physiological drug barriers [15]. Spray dried powder particles are generally obtained in the range of microparticles. Liu et al. chitosan microparticles showed a mean size between 35 and 50 μm using solute concentrations of 0.5%–1.5% [13]. It is well established that spray drying process parameters and the feedstock solution properties, mainly solute concentration, implies on particle properties such as size, e.g. Kašpar et al. obtained particles in the range of 2–9 μm by using solute concentration until 1% [16]. Comparing to reported data, MTX-CH microparticles showed small and narrowed particles size distribution with mean particle size in range of 2.7–5.3 μm (Table 1) that can be attributed to selected parameters of drying process that led to particles with potential parenteral or pulmonary application. SEM images did not reveal the presence of any crystal or considerably irregular particles, which strongly suggests total and homogeneous drug loading into polymeric matrix [6,17]. The first characteristic was confirmed by the high drug loading efficiency analytically assessed for different microparticles, even for samples with greater drug loading (Table 2). This is a characteristic advantage of the spray drying technique, because other microencapsulation methods such as emulsification with solvent evaporation or inotropic gelation, specifically in the case of chitosan, demonstrates low drug loading efficiency when a high drug/chitosan is used. In addition, high chitosan concentrations can complicate the formation of spherical particles, mainly by the high viscosity of the drying solution [18]. Thus, the used parameters in spray drying and used drug/polymer ratio in this work lead to spherical particles with a high drug loading efficiency. Actually, what really contributes to increment viscosity of spray dried solution is the solute concentration and not drug/polymer ratio [5,18]. All samples were prepared with a fixed solute concentration of 0.5% w/v, in order to avoid a considerable viscosity increment of drying solution and to guarantee total dissolution of MTX and CH in the same organic solvent. Fig. 3 shows FT-IR analyses, which were carried out in order to investigate possible drug-polymer binding, because MTX presents two carboxyl groups, while CH presents free primary amine groups (Fig. 1). The spectra of MTX showed that the characteristic Table 2 Encapsulation efficiency, analytical drug content and transport parameters for different polymeric microparticles (n¼ 3). Sample
MTX-CH MTX-CH MTX-CH MTX-CH
Drug loading (%)7 SD
45 27 18 9
39.8 7 0.5 22.5 7 2.2 15.14 70.5 7.7 7 0.3
Encapsulation efficiency (EE) (%)7 SD
88.4 7 1.2 83.5 7 8.2 84.17 2.5 86.0 7 3.2
Mathematical modeling of drug release data kH
r2
11.6 7 5.2 27.0 7 3.3 34.9 7 17.0 16.5 7 1.9
0.92 0.94 0.95 0.97
Fig. 3. FT-IR spectra for MTX, CH, physical mixtures (PM), and microparticles (Mc) with different drug/polymer ratios.
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bands of functional groups corresponded to N‒H stretch of primary amine (3415 cm 1), O‒H stretch of carboxylic acid group (2960 cm 1), C ¼O stretch of secondary amide (1643 cm 1), tertiary amide N‒H (1608 cm 1) and tertiary amine N‒C (1209 cm 1). For CH, alcohol O‒H and amine N‒H peaks are overlapped in 3423 cm 1, C‒H stretch (2898 cm 1), secondary amide C¼ O (1650 cm 1) and N‒H (1598 cm 1) (derived from the N-acetylated monomeric units), and ether C‒O stretch (1074 cm 1). Despite MTX presents two carboxyl groups and CH presents primary amine groups, it was not capable to identify any reaction between the copolymer and drug. Similar behavior was identified for ciprofloxacin-loaded spray dried microparticles, in which the ester carbonyl groups of copolymer did not react with the amino groups of drug [8]. The infrared spectra of the microparticles showed no difference from those of physical mixtures, demonstrating a common characteristic of drug-loaded spray dried microparticles, in which no chemical binding between two compounds or changes in the chemical structure of drug was identified. However, the proximity and overlapping between the characteristic peaks of drug and polymer does not allow a thorough evaluation, except for a particular band in 1209 cm 1, which is attributed to the stretching of tertiary amine bond and, as expected, it is more intense for samples with higher drug ratio. The maintenance of this group is very important for the biological activity of the particles because of differentiates of methotrexate in folic acid (Fig. 1). Fig. 4 shows XRD analysis for different samples. Unprocessed methotrexate occurred in a crystalline state, which was attributed to well-defined diffraction peaks at 2θ of 7.6 and 9.2, characteristic of its trihydrate form [10,19]. In accordance with some reported studies, in the XRD of chitosan diffraction peaks were observed at 10° or 20° corresponding to hydrated and anhydrous crystals respectively [20,21]. Comparing to pure drug and polymer, less crystallinity was observed for prepared physical mixtures, which increase according drug ratio in the sample [22]. The non-similarity among XRD patterns of MTX-loaded CH microparticles and physical mixtures confirmed the absence of crystalline MTX into particles, which indicated total dissolution of drug into polymeric matrix or amorphization during drying [10,23]. Anterior studies with biodegradable spray dried microparticles containing ciprofloxacin or triamcinolone also demonstrated the preparation of amorphous particles even for drug ratio about 50% [8,9]. Solids in amorphous state have molecules randomly arranged, which can be distributed among polymeric chains, requiring less free energy during dissolution and drug release. Generally, drug/polymer ratio strongly influences drug release rate from microparticles, but drug-polymer interactions or drug distribution into particles are parameters that may always be considered in the nano/microparticles development, permitting the evaluation of the real contribution of particle microstructure to drug release rate [24,25]. The established presence of MTX in amorphous phase in CH particles is a promising characteristic for uniformity in the drug release rates of those systems. Fig. 5 shows the in vitro cumulative release profiles of different MTX-loaded CH microparticles. All formulations successfully presented a sustained release for MTX according to the used drug/polymer ratio. Diffusion, swelling and erosion are the most important rate-controlling mechanisms involved with drug release from commercially available controlled release products [26]. A biodegradable hydrogel like CH formed microparticles that were structurally arranged as hydrophilic matrices, in which drug release can involve three different steps according to the environment. Initially, the drug adsorbed on the particles surface is rapidly dissolved causing a “burst effect”, followed by dissolution-diffusion of drug portion distributed into
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Fig. 4. X-ray powder diffraction analysis MTX, CH, microparticles (MC) and their respective physical mixtures (PM) with different drug/polymer ratios.
particles, which occurs when the dissolution medium penetrates into particles with a swelling of polymeric matrix. Finally, the erosion of the swelled matrix occurs with the release of residual drug amount [27–29]. The “burst effect” occurred for all formulations according to the used drug-polymer ratio, which is characteristic of spray dried particles [9,10]. At the moment of drying of molecular dispersions, during self-assembly of compounds, a faster diffusion of small molecules into polymeric matrix can occur by capillary flow, with co-precipitation of drug near the surface of particles [6,30]. It is interesting to observe that the “burst effect” can be a desirable property, when the particles are produced intend pulmonary or
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Fig. 5. MTX release profile (●) for CH microparticles in different drug/polymer ratios: (A) 9%; (B) 18%; (C) 27% and (D) 45% with all data and fitted plots shows (○) mathematical adjusted Higuchi model ft ¼ kh1/√t for release profile after third data (without the burst effect).
parenteral rout. In previous studies, Cardillo et al. identified a similar burst release about 60% in the in vitro release studies of biodegradable spray dried microparticles containing about 30% of ciprofloxacin with mean diameter about of 1 μm. However, a single dose of subconjunctival ciprofloxacin-PLGA microspheres sustained aqueous and vitreous therapeutic levels for up to 10 days. In addition, the particles were able to reduce bacterial recovery and signs of clinical endophthalmitis induced in rabbits [31]. In a following study with humans, a single dose of these same particles was able to prevent ocular infection in 67 patients after cataract surgery. The patients were examined on postoperative days during 28 days [32]. Thus, we believe that results of in vitro release studies is not able to predict in vivo effects, but they are so important to evaluate physicochemical properties and compositions of specific particles in order to select the best system for a desired administration rout. Thus, in order to compare efficiently the effect of this phenomenon in the overall drug release rate, the experimental data were mathematically adjusted by different models and velocity constants extracted [25,33]. Despite subjecting the experimental data to different mathematical models, after the initial burst release, an optimum linear fitting was reached only by using an adjusted Higuchi model, which was described by the equation ft ¼kH1/√t (Fig. 5). The identified kH for different MTX-CH loaded microparticles and respective correlation coefficient (r2) are summarized in Table 2. The Higuchi model describes drug release as a diffusion process based on Fick's law being square root time dependent [33]. However, swellable particles may lead to release that does not only agree with Fickian behavior, due to specific networks formed after polymer relaxation, in this case, the application of “Peppas equation” (Mt/M1 ¼ktn) is recommended, which considers the first 60% of total released drug. In this model, “k” incorporates characteristics of a macromolecular network into the polymeric matrix and “n” is a diffusional indicative of the transport mechanism involved [33]. However, “Peppas equation” was not applied because all MTX-CH loaded microparticles presented a considerable “burst effect” in which more than 60% of drug was released in the first stage. The drug adhered to the surface of the microsphere was rapidly released in the medium, which also contributed forming the water channels in the polymeric matrix. The second stage of MTX release occurred due to swelling is the glassy polymer change to a rubbery state, which expands the mobility of polymer chains with consequent drug diffusion through the swollen layer. These data were fitted by using an adjusted Higuchi model with an inverse of square root time dependence. Thus, during swelling, a layer of gel is formed around the particle, which limits and controls drug release rate [34]. The thickness of the gelling layer is dependent on drug-polymer interactions, environment and drug/ polymer ratio. Moreover, the third and final step of drug release due to copolymer erosion was not observed, in which a residual portion of drug remained entrapped in particles. In previous studies, Qi et al. successfully intercalated MTX into the Mg–Al–LDHs layers by themechanochemical–hydrothermal method. However a burst release about 60% was observed at the first 4 h of experiment, with almost total drug release after 10 h. Different mathematical models were applied, in which a similar model (parabolic diffusion model, Mt/M1/t ¼kt 0.5 þb) was applied and successfully elucidates that the release process was controlled by diffusion such as intra-particle diffusion or surface diffusion [23]. This step of study confirmed the most MTX release by drug dissolution, followed by partial drug diffusion through swollen particles, even for microparticles with highest drug loading. The mathematical modeling made it possible to identify a considerable sustained release effect for particles containing 9% of drug, which
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is fundamental for the selection of a suitable formulation for in vivo studies and establishes a dosage for specific administration or clinical pathology.
4. Conclusion Spray dried MTX-loaded chitosan microparticles were successfully produced using selected parameters. Particles exhibited desired physicochemical aspects such as spherical and smooth shape, high drug loading with non-bounded drug homogeneously dispersed in an amorphous state into polymeric matrix. Even the particles with higher drug loading levels presented a sustained drug release profile. The drug release occurred predominantly through drug dissolution and diffusion in swollen polymeric matrix, with slowest release for particles containing 9% of drug. These results support the idea that spray dried MTX loaded CH microparticles are a very promising drug delivery system, which can be administered by different routes such as oral, subcutaneous, pulmonary or intramuscularly due to the physicochemical aspects of particles.
Acknowledgments The authors wish to thank the Brazilian Agencies CNPq (grant number: 479195/2008; 483073/2010-5, 481767/2012-6) and CAPES (Scholarship of P.C. Mesquita and A.R. Oliveira), for their financial support. The authors also acknowledge the help extended by Andy Cumming in proofreading the English text.
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