Materials Science & Engineering C 81 (2017) 542–550
Contents lists available at ScienceDirect
Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Injectable methotrexate loaded polycaprolactone microspheres: Physicochemical characterization, biocompatibility, and hemocompatibility evaluation
MARK
Mukesh Dhanka, Chaitra Shetty, Rohit Srivastava⁎ Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai 400076, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Methotrexate Polycaprolactone Microspheres Cell viability Hemolysis
In this study, bare polycaprolactone microspheres (PCL MPs) and methotrexate (MTX) loaded PCL microspheres (MTX-PCL MPs) have been developed by oil-in-water emulsion solvent evaporation method using hydroxypropyl methylcellulose (HPMC) as an emulsifier. Encapsulation efficiency and loading capacity of methotrexate were found to be 51.28% ± 0.52 and 2.8% ± 0.06 respectively. Environmental scanning electron microscopy showed the PCL MPs and MTX-PCL MPs to have a spherical shape and smooth surface morphology. The mean size of microspheres (23 μm) was found within injectability criteria. High-Resolution X-ray diffraction of microspheres revealed that PCL retained its semi-crystalline nature after processing in microspheres, but the drug looses its crystallinity. Fourier transmittance infrared spectroscopy and thermogravimetry analysis of the microspheres indicated that no physicochemical modification occurred. In vitro, MTX release study from MTX-PCL MPs in phosphate buffer saline (pH 7.4) showed controlled release profile and only 31% of MTX released in 306 h. The microspheres in lyophilized form are physicochemically stable for 8 months. Furthermore, L929 cells treated with microspheres showed cell viability > 80%. The different concentrations of microspheres found hemocompatible and did not affect the biconcave shape of red blood cells (RBCs). The physiochemical and biological evaluation of microspheres suggests their further use for drug delivery application.
1. Introduction Over the past few decades, the various types of novel biomaterials have been developed for applications in biomedical engineering such as drug delivery, cell encapsulations and tissue engineering. The various types of active molecules (protein, gene, drugs and several others) of delivery systems have been developed including hydrogels, microparticles, nanoparticles, nanofibers, liposomes and micelles, etc. for improving their overall therapeutic efficacy [1–14]. Microspheres prepared from natural as well as synthetic polymers have been widely investigated as a drug delivery carrier [15,16]. Their use has gradually increased in the pharmaceutical field due to its advantages such as controlled release, protection from premature degradations, increased bioavailability, and decreased toxicity, reduced dosage frequency and improved patient compliance [15–17]. Also, they are easily injectable compared to surgical insertion of large implants. Microspheres have been administered through different routes such as intravenous, intramuscular, subcutaneous, and pulmonary, etc. [15–18]. Recently, non-biodegradable polymers have been replaced by biodegradable polymers for the development of polymeric microspheres based ⁎
Corresponding author. E-mail address: [email protected] (R. Srivastava).
http://dx.doi.org/10.1016/j.msec.2017.08.055 Received 25 May 2017; Received in revised form 14 July 2017; Accepted 10 August 2017 Available online 12 August 2017 0928-4931/ © 2017 Published by Elsevier B.V.
delivery systems in pharmaceutical technology. Microspheres made from biocompatible and biodegradable polymer produce a non-toxic product from their biodegradation and avoid the surgical removal from injected site [17–20]. Recently, many reports are available on the injectable microspheres for localized delivery of the drug in solid tumor and joints. Because these injectable depots of microspheres are related with site-specific drug release, the drug toxicity at non-targeted tissues is avoided [21–24]. Among synthetic polymers, polylactic acid (PLA), polyglycolic acid (PGA) and poly (lactide-co-glycolide) acid (PLGA) have been most widely used for the development of microspheres based drug delivery systems owing to their excellent biocompatibility, biodegradability, and non-immunogenicity [17,18,23,25–30]. However, their high cost and generation of the acidic environment of degradation which can produce inflammation are responsible for their limited use in the microsphere development for biomedical applications. Polycaprolactone (PCL) is a synthetic polymer and is suitable for the development of controlled drug delivery systems due to its high permeability for drug molecules and slow degradation kinetics compared to polylactide and polyglycolide. It is a cost-effective polymer and does not create an acidic environment during its degradation. Also, it is
Materials Science & Engineering C 81 (2017) 542–550
M. Dhanka et al.
(Mumbai, India). The Triton X-100 was purchased from Fisher Scientific Ltd. (Mumbai, India), and glutaraldehyde (38%) supplied by Merck Ltd. (Mumbai, India).
biocompatible, biodegradable and non-immunogenic with hydrophobic semi-crystalline nature. It is a Food and Drug Administration-approved polymer for pharmaceutical applications and had been used for a long time for development of drug delivery systems in the pharmaceutical industry. PCL based microspheres have been developed for encapsulation and controlled delivery of hydrophilic and hydrophobic drugs [19,31–34]. Drug-loaded microspheres preparation mainly depends on the solubility of drug and polymer in various solvents. The solvent evaporation is a most widely used simple and reproducible technique for microspheres preparation [21,30,32,35,36]. Methotrexate (MTX) is a potent antimetabolite for folic acid with anti-cancer, anti-inflammatory, anti-rheumatic and disease modifying properties [37–42]. In 1970, MTX was included as disease modifying anti-rheumatic drug (DMARDs) and is currently the most widely used drug for treatment of rheumatoid arthritis. Due to its high effectiveness compared to other drugs for rheumatoid arthritis treatment, it is a popular second line drug among the doctors. The clinical application of MTX has limitations such as poor solubility, large amount excreted by the kidney, short half-life, and rapid diffusion into the body. However, its use is also associated with serious side effects, due to which around 30% patients worldwide leave the treatment of rheumatoid arthritis. The most frequent side effects arising out of MTX dosing includes mouth sores, stomatitis, and a decrease in white blood cells count. A high dose for longer time produces severe hepatotoxicity, nephrotoxicity, and bone marrow suppression. Other side effects caused by MTX include skin rash, itching, dizziness, hair loss, and drowsiness. In the clinical application, regular monitoring of MTX amount in the blood is required to ensure the minimal toxic level [23,39,40,43,44]. To decrease the toxicity the localized intra-articular and intra-tumoral free MTX suspension has been administered, but the efficacy was found low due to rapid clearance from the target site [22,29,38,40,42,45–48]. Therefore, MTX is an ideal drug for microencapsulation into PCL polymer. In this study, bare polycaprolactone microspheres (PCL MPs) and methotrexate loaded PCL microspheres (MTX-PCL MPs) have been developed from single O/W emulsion solvent evaporation method. Here, hydroxypropyl methylcellulose (HPMC) has been used as an emulsifier for the development of PCL MPs and MTX-PCL MPs. The developed microspheres size, shape, and morphology were characterized by Environmental Scanning Electron Microscopy (ESEM), and Fourier Transform Infrared Spectroscopy did physiochemical characterization (FTIR), Thermogravimetric Analysis (TGA) and High-Resolution X-ray Diffractometry (HR-X-RD). The stability study for lyophilized PCL microspheres powder sample was performed up to 8 months. The biocompatibility was performed on the L929 fibroblast cells for 24 h and hemocompatibility was performed on the human blood. This system will pave the way for further advancement in microsphere-based injectable drug delivery system for treatment of localized tumor and joint-rheumatoid arthritis.
2.2. Preparation of microspheres The PCL MPs and MXT-PCL MPs were prepared by using O/W emulsion solvent evaporation method. Briefly, MTX was dissolved into 500uL DMSO and mixed into 2 mL DCM containing 100 mg of dissolved PCL polymer. After polymer and drug containing organic phase was added by using syringe pump with the speed 1 mL/min into 30 mL of water phase containing HPMC (0.2%w/v) as an emulsifier. Simultaneously the resulting emulsion was emulsified by homogenizer with the speed 3000 rpm for 5 min and then the emulsion was kept on a magnetic stirrer at 800 rpm for evaporation of organic phase for 6 h. After complete evaporation of organic phase, the MTX-PCL MPs were collected by centrifuging at 10000 rpm for 15 min and microspheres dried by lyophilisation. PCL MPs were also obtained by the same method but without the addition of MTX. MTX loaded PCL microspheres were prepared in dark condition due to light-sensitive nature of MTX. 2.3. Morphology and size analysis of microspheres The shape and surface morphology of PCL MPs and MTX-PCL MPs were characterized by environmental scanning electron microscope (ESEM) (FEI-ESEM, Quanta 200, Netherlands). The dried samples of microspheres were stacked on double-sided carbon tape, and samples were coated with platinum for 300 s with 10 mA (JFC-1600, Fine Coater, Japan). The microspheres were observed under different magnifications with accelerated voltage from 5 to 20 kV. Average size and size distribution of PCL MPs and MTX-PCL MPs were from ESEM images calculated by using Image J online software. 2.4. Production yield (% PY) of microspheres The total amount of PCL MPs and MTX-PCL MPs were estimated by weighing the obtained lyophilized microspheres powder on a weighing machine. The production yield of PCL MPs and MTX-PCL MPs were calculated by using Eq. (1).
Production yield (%) = (Wa Wb) × 100
(1)
The microparticles production yield was calculated by weight of microspheres produce (Wa) and weight of compounds used in the formulation for microspheres preparation (Wb), PCL or PCL plus MTX. 2.5. Drug encapsulation efficiency and loading capacity
2. Materials and methods
To calculate the encapsulated amount of MTX, the 5 mg of MTX-PCL MPs were dissolved in 2 mL DCM, and then 1 mL DMSO was added to solubilize the drug. After the mixture was kept for bath sonication of 1 min, and then added into 10 mL phosphate buffer saline (PBS, pH 7.4) and stirred until complete evaporation of DCM. The obtained solution was centrifuged at 10000 rpm for 15mins, and collected supernatant was analyzed using UV-spectrometer at 304 nm wavelength. PCL MPs were also analyzed using the same procedure and used as a control. The encapsulation efficiency (%EE) and loading capacity (%LC) was calculated by using the following formulas (2) and (3).
2.1. Materials PCL (molecular weight 14,000 Da) was purchased from SigmaAldrich (Mumbai, India), and hydroxypropyl methylcellulose (HPMC, K15M) procured from Otto Chemie Ltd. (Mumbai, India). Methotrexate was kindly gifted by Sun Pharma Ltd. (Gujarat, India). Organic solvent like dichloromethane (DCM) HPLC grade and dimethyl sulfoxide (DMSO) analytical grade were obtained from Merck Ltd. (Mumbai, India). Chemicals for the preparation of Phosphate Buffer Saline (used for drug release studies) disodium hydrogen phosphate, sodium chloride, and potassium dihydrogen phosphate was purchased from Merck Ltd. (Mumbai, India) and potassium chloride purchased from Sigma-Aldrich (Mumbai, India). Cell culture media (DMEM), antibiotics (Penicillin and streptomycin) and PBS were purchased from HiMedia 543
%EE =
Mass MTX in microspheres × 100 Total mass of MTX used in formulation
(2)
%LC =
Mass of MTX loaded microspheres × 100 Total mass of MTX used in formulation
(3)
Materials Science & Engineering C 81 (2017) 542–550
M. Dhanka et al.
100 μL of fresh media containing 10 μg/mL resazurin dye was added to each well and again the plate was incubated (37 °C, CO₂ incubator) for 4 h. Further, the absorbance was recorded at 570 nm with reference absorbance at 600 nm using plate reader (Tecan infinite M 200 PRO). The PBS (negative control) and 1% Triton X-100 (positive control) treated cell were used for comparison study. The cell viability was calculated by using below Eq. (5).
2.6. FTIR analysis The quality analysis of PCL polymer and drug after formation of microspheres was observed under FTIR spectroscopy (FTIR, Bruker, Japan). The powder samples of pure MTX, PCL MPs, and MTX-PCL MPs were mixed with potassium bromide (KBr) and converted into thin KBr pellet. The FTIR spectra of the samples were recorded using transmission mode of the instrument from 400 to 4000 cm− 1 range of wavenumber and at 4 cm− 1 resolution.
%cell viability =
Absorbance of test sample × 100 Absorbance of control
(5)
2.7. HR-XRD characterization 2.12. Hemolysis assay The X-ray diffraction patterns for pure MTX, PCL MPs, and MTXPCL MPs were recorded by using HR-XRD (Rigaku, Smartlab 9 kW, High resolution-X-ray Diffractometer, Japan) for evaluation of the crystalline structure of drug and PCL polymer after being processed into microspheres. The samples were scanned over the range of 5–40° (2θ) with scanning rate of 6°/s.
The hemocompatibility tests for the microspheres were conducted only after taking institutional ethical clearance (IITB-IEC-2016-028) and informed consent form (ICF). The human blood samples were collected from a healthy volunteer (age, 26 yrs) in trisodium citrate containing vials. The red blood cells (RBCs) were separated from whole blood samples by using centrifugation (1000 rpm, 5mins) followed by three times washing with physiological buffer saline (PBS, pH 7.4). The RBCs collected from 10 mL whole blood sample were then dispersed into 16 mL PBS and 500 μL RBCs containing samples were tested with each test sample. The hemolysis study for PCL MPs and MTX-PCL MPs was studied spectrophotometrically by measuring free hemoglobin (Hb) released from red blood cells (RBCs) lysis upon interaction with test samples. First, for obtaining a pre-saturated solution, different amounts (0.5 mg, 1 mg, 2.5 mg, and 5 mg) of PCL MPs and MTX-PCL MPs microspheres were incubated in 500 μL PBS at 37 °C for 1 h. After presaturation, 500 μL test samples were added into 2 mL Eppendorf containing 500 μL of RBCs solution under aseptic condition. The RBCstreated samples were incubated in a water bath for 3 h under shaking condition (60 rpm) at 37 °C. After incubation, intact RBCs were pelleted down by centrifugation (4000 rpm, 10 min) and 100 μL of the supernatant was used for recording the absorbance at 540 nm using plate reader. RBCs treated with phosphate buffer saline (0% lysis) was used as negative control (NC), and 1% Triton-X (100% lysis) treated cells were used as positive control (PC). According to reported studies, materials that cause < 5% hemolysis can be included into hemocompatible materials [49–52]. The biconcave shape and morphology of erythrocytes after treatment with test samples were observed under E-SEM. Treated RBCs collected after centrifugation were redispersed into 1 mL of PBS. 1% glutaraldehyde solution in PBS (pH 7.4) was used as a fixative for RBCs. The 400 μL of glutaraldehyde solution was added into RBCs solution and kept for fixation at 4 °C for 2 h. The fixed RBC samples were then centrifuged, and the pellets were redispersed in 1 mL of water and drop-casted on aluminum foil and dried at room temperature. The dried RBCs samples on aluminum foil were stacked on double-sided carbon tape and coated with platinum for 300 s. The samples were observed at different magnification with accelerated voltage from 15 kV.
2.8. TGA analysis Thermal gravimetric analysis (TGA) for the pure drug, drug loaded and blank microspheres were performed by the TGA instrument (TG/ DTA, Perkin Elmer, USA). The heating rate for the sample was 10 °C/ min and nitrogen flow rate 60 mL/min. The percent weight loss study of the sample was done with temperature range from 42 °C to 1000 °C. 2.9. Stability study of microspheres In order to conduct the stability test, dried powder samples of MTX loaded PCL microspheres were stored in the freezer for a different time period. After a specific time interval, the samples were taken under shape, morphology, and FTIR characterization for evaluation of physicochemical properties of microspheres. 2.10. Drugs release study The 10 mg MTX-PCL MPs were kept in the dialysis bag to separate the microspheres from the dissolution medium. The study was carried out in 15 mL PBS (pH 7.4) and incubated at 37 °C in water bath shaker at 60 rpm shaking rate. Determination of MTX release from MTX-PCL MPs the 1 mL release medium was replaced with freshly prepared PBS at a particular time interval. The quantification of release drug was done by UV-spectrophotometer with 1:1 dilution of the original sample. The drug release was calculated in cumulative percentage release (w/w %) by using Eq. (4).
MTX released% =
MTX released at specific time interval × 100 Total MTX into systems
(4)
2.11. Biocompatibility assay
%Hemolysis = The L929 normal mouse fibroblast cells (National Centre for Cell Sciences, Pune, India) were grown in Dulbecco's Modified Eagle's Medium (DMEM) (HiMedia, India) supplemented with 10% FBS (Fetal Bovine Serum) and 1% antibiotics (streptomycin: penicillin). The cells were maintained in standard cell culture condition (37 °C under 5% CO₂, saturated with the humid environment) for further use. Further, to evaluate the cell cytotoxicity of PCL MPs and MTX-PCL MPs a colorimetric alamar assay was used. The cells viability were tested in triplicate using 96 well plates, each well seeded with 4000 cells/100 μL. The plates were then incubated at 37 °C in CO₂ incubator for 24 h. The media was removed, and 100 μL of fresh media containing different concentrations of microspheres (0.5 mg/mL, 1 mg/mL, 2.5 mg/mL, 5 mg/mL) was added to the respective wells and incubated at standard cell culture condition for 24 h. After 24 h, the media was removed and
(Absorbance of test sample−Absorbance of NC) × 100 (Absorbance of PC − Absorbance of NC) (6)
3. Results and discussion 3.1. Preparation of microspheres The PCL based microspheres drug delivery systems have been widely developed for the sustained and controlled release of pharmaceutical drugs. The emulsion solvent evaporation method has been widely used for microencapsulation of hydrophilic and hydrophobic drugs into the PLC microspheres [21,31,34,53,54]. In the reported studies of PCL MPs the PVA have been used as emulsifier, but here we have used HPMC biocompatible polymer as emulsifier. Here, PCL MPs 544
Materials Science & Engineering C 81 (2017) 542–550
M. Dhanka et al.
Fig. 2. FTIR spectra of blank PCL, MTX loaded PCL microspheres and pure MTX.
microspheres were obtained without significant difference (82–85%) as shown in Table 1. In the evaluation, the properties of drug loaded microspheres, the drug loading capacity, and encapsulation efficiency both are very important parameters. MTX loaded PCL microspheres were prepared by O/W solvent evaporation method with varying the concentration of MTX to observe the effect on encapsulation efficiency. The maximum encapsulation efficiency obtained was 51.68% in MTXPCL MPs, and loading capacity was 2.8%. Fig. 1. SEM characterization of PCL microspheres (a) blank microspheres at 1600 × (b) blank microspheres at 6000× (c) MTX loaded PCL microspheres at 1600 × (d) MTX loaded PCL microspheres at 6000 × (e) Pure MTX at 5000 ×.
3.4. FTIR analysis FTIR spectroscopy is a widely used technique for observing the chemical interaction between polymer and drug. Therefore, FTIR characterizations for the samples were done to find out the chemical changes in the structure of PCL polymer and MTX after formation of microspheres. FTIR spectra for pure MTX, PCL MPs, and MTX-PCL MPs were recorded and presented in Fig. 2. The FTIR spectrum of pure MTX showed similarity with the already reported studies [49]. After inspecting the FTIR curve of PCL MPs, all characteristics bands were observed, first a distinct peak at 1725 cm− 1 for carbonyl stretching (C]O), and a broad band at 3440 cm− 1 due to an overtone of the carbonyl stretching, 2950 and 2866 cm− 1 which are due to symmetrical CH₃ and antisymmetrical CH₂ stretching respectively. The vibration for the CH₂ band is observed at 1370, 1420, and 1473 cm− 1 and OeC vibration bands are at 1105, 1043, and 959 cm− 1 respectively. Furthermore, stretching for ester functional group was observed at 1186 and 1242 cm− 1 [34,55,56]. It has already been reported that a band at 1297 cm− 1 occurred due to the backbone of CeC and CeO stretching mode in the crystalline form of PCL, and in amorphous form, this band appears at 1157 cm− 1 [34,57]. However, no peak occurred at 1157 cm− 1 (related to the amorphous form of PCL), thus confirming the high crystalline degree of obtained PCL microspheres [57]. The crystalline nature of PCL polymer is the very important property for controlled release of drugs [34]. The FTIR curve for MTX-PCL MPs, was similar to PCL MPs and no extra peak was observed. This indicated that there was no chemical interaction between polymer and drug after encapsulation, and drug dispersed in its molecular form in the polymer matrix. In the reports, FTIR spectra of PCL polymer did not change after encapsulation of drug into PCL microspheres [34,56]. However, the O/ W emulsion solvent evaporation method is a physical method and do not provide the favorable environment for chemical modification between polymer and drug molecules.
and MTX-PCL MPs has been produced by single solvent evaporation (O/ W) method by using a mixture of DCM and DMSO. 3.2. Morphology and size analysis of microspheres Shape and surface morphology of the microspheres were visualized under ESEM and micrographs are presented in Fig. 1. The PCL MPs (Fig. 1a, b) and MTX-PCL MPs (Fig. 1c, d) were obtained with some important physical properties including, individuality, spherical shape, smooth surface morphology, and no other deformed microspheres occurred. There were no additional effects observed on the surface morphology and shape of the PCL microspheres after the loading of MTX. The crystalline nature of MTX can be seen in XRD studies (Fig. 4) and ESEM images (Fig. 1e), but there was no drug crystal appearance on the surface of MTX-PCL MPs. Size is a very important factor for injectable microspheres because size should be large enough to contain sufficient amount of drug but not too large to cause discomfort after injection [15]. The average size of the PCL MPs was found to be 24.094 ± 2.548 μm, but after loading of drug the mean size was obtained slightly low that is 23.88 ± 1.15 μm. The average size of PCL MPs and MTX-PCL-MPs has shown in Table 1, which is under injectability criteria [15,17]. 3.3. Production yield, encapsulation efficiency and loading capacity Production yield is a very important parameter in the pharmaceutical industry for development of microspheres based drug delivery systems. The production yield for blank and MTX loaded PCL Table 1 Size, EE, LC and PY of PCL microspheres. Formulations
Mean size (μm)
%EE
%LC
%PY
PCL MPs MTX-PCL MPs
24.09 ± 2.54 23.88 ± 1.15
– 51.28 ± 0.52
– 2.8 ± 0.06
82.06 ± 2.81 85.6 ± 1.70
3.5. TGA analysis The thermal stability of PCL MPs and MTX-PCL MPs were determined by thermogravimetry analysis using a TA instrument. TGA 545
Materials Science & Engineering C 81 (2017) 542–550
M. Dhanka et al.
Fig. 3. TGA analysis of Pure MTX, PCL, and MTX loaded PCL microspheres.
thermograms for pure MTX, PCL MPs, and MTX-PCL MPs are shown in the Fig. 3. In thermal degradation study, the percent weight loss as a function of temperature 10 °C/min heating rate under a dynamic nitrogen atmosphere was observed. It was found that PCL MPs and MTXPCL MPs were stable from 42 °C to 250 °C. The % weight loss gradually increased from 250 °C to 500 °C due to thermal decomposition of polymer [58–60]. After 500 °C, remaining weight for PCL MPs and MTX-PCL MPs remained constant till 1000 °C. The remaining weight for drug loaded microspheres and blank microspheres were found to be close to each other. Drug-loaded microspheres did not show significant different thermal degradation pattern compared to blank microspheres which indicated that the MTX-PCL MPs were stable, and no physicochemical change occurred in the polymer structure and the drug directly entrapped into the polymer matrix.
Fig. 5. MTX release from MTX-PCL MPs in PBS (pH 7.4).
peaks of MTX disappeared in MTX-PCL MPs, and only characteristic peaks of PCL appeared. The crystalline structure of MTX might have lost during encapsulation, and only amorphous form was encapsulated inside the MTX-PCL MPs, but PCL maintained its semi-crystalline characteristics throughout the microspheres preparation. The XRD spectra of the samples clearly showed the crystalline nature of the PCL polymer in PCL MPs and MTX-PCL MPs [62,65]. 3.7. Drug release study The direct administration of MTX leads to poor efficacy due to its poor water solubility and short half-life. The entrapment of drug into biocompatible and biodegradable polymers can decrease the fast elimination from the body and enhance its therapeutic effect by releasing the drug in a controlled manner at a localized site [48,66–68]. The implant of PCL and microspheres of PLLA-PEG-PLLA and PLA have been developed and characterized by other groups with the aim to provide the controlled MTX release profile [23,67,69]. Here, spherical injectable MTX-PCL MPs have been prepared and characterized for localized delivery of methotrexate. The in vitro release profile in PBS (pH 7.4) of MTX loaded PCL microspheres are shown in Fig. 5. 10 mg of MTX-PCL microspheres were added into enough volume of PBS to dissolve MTX present in microspheres. Methotrexate was released from the MTX-PCL MPs in the “biphasic” pattern. The first pattern was the burst release due to loosely bound of MTX molecules on the surface of the microspheres. In the burst release effect, around 12% of the drug was released from the microspheres in 10 h. The second pattern was the slow release due to diffusion of the drug from the core of polymer. The MTX-PCL MPs showed the drug release pattern in the desirable slow and sustained release manner, and only 30% of the methotrexate was released from the microspheres in the 306 h. According to reported studies, PCL takes a long time for complete degradation, and the drug release profile majorly depends on diffusion rather than degradation [19,33,34].
3.6. HR-XRD characterization XRD diffractometry is a widely used and most suitable technique to evaluate the crystalline structure of the polymer, drug, and drug-loaded microspheres [61–63]. The XRD spectra for pure MTX, PCL MPs, and MTX-PCL MPs were recorded and presented in Fig. 4. The XRD diffraction pattern for PCL MPs showed two intense peaks in the range of 20–25° which are characteristic of semi-crystalline nature of PCL MPs as shown in the reports [20,34,59,62]. The XRD diffraction pattern of MTX showed multiple intense peaks in the range of 5–40° (2θ) that indicated its high degree of crystallinity [64]. But, these characteristic
3.8. Stability study of MTX-PCL MPs The stability of drug loaded microspheres in powder form is a very important aspect for long term storage of the formulation in the pharmaceutical applications. Here, lyophilized samples of MTX-PCL MPs were stored at 4 °C for a different time interval (4, 6, 8 months) and the physiochemical properties were characterized by using ESEM and FTIR. Fig. 6, shows the ESEM micrograph of the stored MTX loaded PCL
Fig. 4. HR-XRD diffractions patterns of PCL, MTX-PCL MPs and pure MTX.
546
Materials Science & Engineering C 81 (2017) 542–550
M. Dhanka et al.
Fig. 6. ESEM images of stored MTX loaded PCL microspheres for the different time period (a and b) 4 month (c and d) 6 month (e and f) 8 month.
Fig. 8. Cell viability studies of PCL microspheres.
effects of MTX [43]. According to reported study, free MTX did not show high cytotoxicity effect on normal fibroblast cells [70]. Thus, these microspheres are very suitable for targeted injection for providing the localized drug delivery at the disease site. Therefore, the drug will not release at the normal tissue, and cytotoxic effects can be avoided.
Fig. 7. FTIR spectra of MTX-PCL microspheres stored for different time periods.
microspheres for 4 months (a, b), 6 months (c, d) and 8 months (e, f). It was observed that there was no change in the shape, and surface morphology of MTX loaded PCL microspheres, i.e. the microspheres were found to retain their spherical shape with smooth surface morphology even after storing them for different time periods. Also, the size of the microspheres did not show any significant difference compared to original microspheres. Fig. 7, shows the FTIR spectra of the MTX loaded PCL microspheres, and it was observed that no chemical modification occurred after storing for different time intervals. Therefore, it can be inferred that MTX loaded PCL microspheres are stable and can be stored for up to 8 months.
3.10. Hemolysis assay The injectable polymeric microspheres for biomedical application come in contact with blood after injection at the targeted site so, this application attracts our attention to conduct RBC compatibility studies. Hemocompatibility of the PCL MPs was evaluated by studying two important factors, hemolysis and RBCs morphology study. The chemical composition, size, shape, and charge of polymeric microspheres can be a reason of hemolysis. Therefore, to evaluate the hemolysis by developed microspheres, the extracted RBCs were treated with different concentration of PCL MPs and MTX-PCL MPs (0.5 mg/mL, 1.0 mg/mL, 2.5 mg/mL, 0.5 mg/mL). It was observed from Fig. 9 that hemolysis for PCL MPs and MTX-PCL MPs were below 5% and under the hemocompatibility criteria [49–52]. But in the case of Triton X treated RBCs the 100% hemolysis occurred. Previously studies have also reported that PCL polymer is completely hemocompatible [51,52,54,71,72]. According to the reported studies, MTX is a non-hemolytic drug but, after loading into the chitosan nanoparticles it induces hemolysis, and the same property of MTX has also been reported with liposomes [70,73].But here MTX–PCL MPs was found to be hemocompatible and this may be because the encapsulated MTX did not alter the physiochemical properties PCL polymer. It was also clearly observed from
3.9. Biocompatibility assay To achieve the biomedical applications of the developed PCL microspheres, they must be non-toxic in nature. In order to evaluate the biocompatibility of developed PCL MPs and MTX-PCL MPs, the L929 (Mouse fibroblast) cells were treated with different concentrations (0.5 mg/mL, 1 mg/mL, 2.5 mg/mL, 5 mg/mL) of both the microparticles for 24 h. The % cells viability analysis is presented in Fig. 8. The cell viability of cells treated with PCL MPs and MTX-PCL MPs were found to be > 90% and > 80% respectively. The decrease in the viability of cells treated with MTX-PCL MPs may be due to the cytotoxic 547
Materials Science & Engineering C 81 (2017) 542–550
M. Dhanka et al.
Fig. 9. Hemolysis study for blank and MTX loaded PCL microspheres for 3 h, Negative control (NC), positive control (PC), PCL MPs (B), MTX-PCL MPs (M).
Fig. 10. ESEM images of RBCs after 3 h treatment with different concentrations of blank and MTX loaded PCL microspheres, (a and b) negative control (NC) (c and d) 0.5 mg/mL PCL MPs (e and f) 1.0 mg/mL PCL MPs (g and h) 2.5 mg/mL PCL MPs (i and j) 5 mg/mL PCL MPs (k and l) 0.5 mg/mL MTX-PCL MPs (m and n) 1 mg/mL MTX-PCL MPs (o and p) 2.5 mg/mL MTXPCL MPs (q and r) 5 mg/mL MTX-PCL MPs.
microspheres showed the good encapsulation efficiency and loading capacity. The physicochemical characterizations of microspheres showed the absence of physicochemical modifications, and the drug molecules were found to be in dispersed form inside the polymeric matrix. PCL did not lose its crystallinity like MTX after being processed into microspheres. The controlled and prolonged MTX release from PCL microspheres makes them more suitable for in vivo application. The in vitro biocompatibility studies on cells L929 depicts that the material is cytocompatible and is appropriate for in vivo injection. The hemocompatibility of microspheres studies also showed no hemolytic property as well as no change in biconcave shape of RBCs. Therefore, the biocompatible and hemocompatible nature of these microspheres makes them suitable for in vivo applications in the future for treatment of rheumatoid arthritis.
photographic images (Fig. 8 B) that the microsphere-treated RBCs were undamaged, and sedimented in the bottom like negative control (RBCs in PBS), but positive control showed the leakage of hemoglobin into PBS. The further confirmation of hemolysis was done by E-SEM imaging of treated and non-treated RBCs shown in Fig. 10. The ESEM images of blank (Fig. 10, a, d, c, d) and MTX-PCL MPs (Fig. 10, e, f, g, h) showed the intact and biconcave morphology of RBCs like negative control (Fig. 10, i). Therefore, it was concluded that treatment of RBCs with developed PCL MPs and MTX-PCL MPs did not affect the morphology of RBCs after 3 h treatment. These microspheres were found to be completely hemocompatible and thus can be used for in vivo drug delivery as a localized injectable system. 4. Conclusion
Acknowledgments
PCL polymer-based microspheres have been developed and characterized for the delivery of different active pharmaceutical ingredients. Here, we have developed PCL MPs, and MTX-PCL MPs by a single solvent evaporation method. The prepared microspheres were found in a spherical shape with smooth surface morphology. The PCL
Authors acknowledge the Sun Pharmaceutical Industries Ltd. (Gujarat, India) for providing the Methotrexate drug as gift sample for our research work. Mr. Dhanka acknowledges Ministry of Human 548
Materials Science & Engineering C 81 (2017) 542–550
M. Dhanka et al.
Resources and Development (MHRD), India for providing financial support. Authors acknowledge Sophisticated Analytical Instrumentation Facility (SAIF), Industrial Research Consultancy Centre (IRCC), Indian Institute of Technology Bombay (IITB), for providing central instrumentation facility.
ejpb.2009.06.009. [23] L.S. Liang, J. Jackson, W. Min, V. Risovic, K.M. Wasan, H.M. Burt, Methotrexate loaded poly(L-lactic acid) microspheres for intra-articular delivery of methotrexate to the joint, J. Pharm. Sci. 93 (2004) 943–956, http://dx.doi.org/10.1002/jps. 20031. [24] V. Natarajan, P. Saravanakumar, B. Madhan, Collagen adsorption on quercetin loaded polycaprolactone microspheres: approach for “stealth” implant, Int. J. Biol. Macromol. 50 (2012) 1091–1094, http://dx.doi.org/10.1016/j.ijbiomac.2012.03. 003. [25] C. Zhang, G. Li, Y. Wang, F. Cui, J. Zhang, Q. Huang, Preparation and characterization of 5-fluorouracil-loaded PLLA-PEG/PEG nanoparticles by a novel supercritical CO2 technique, Int. J. Pharm. 436 (2012) 272–281, http://dx.doi.org/10. 1016/j.ijpharm.2012.06.022. [26] B. Sayin, S. Çalifi, Influece of accelerated storage conditions on the stability of vancomycin-loaded poly (D,L-lactide-co-glycolide) microspheres, J. Pharma.Sci. 29 (2005) 111–116. [27] G.C. Bazzo, A.T. De Macedo, J.P. Crenca, V.E. Silva, E.M. Pereira, M. Zétola, B.R. Pezzini, Microspheres prepared with biodegradable PHBV and PLA polymers as prolonged-release system for ibuprofen: in vitro drug release and in vivo evaluation, Brazilian J. Pharm. Sci. 48 (2012) 773–780. [28] N. Butoescu, O. Jordan, P. Burdet, P. Stadelmann, A. Petri-fink, H. Hofmann, E. Doelker, Dexamethasone-containing biodegradable superparamagnetic microparticles for intra-articular administration: physicochemical and magnetic properties, in vitro and in vivo drug release, Eur. J. Pharm. Biopharm. 72 (2009) 529–538, http://dx.doi.org/10.1016/j.ejpb.2009.03.003. [29] R.K. Averineni, G.V. Shavi, A.K. Gurram, P.B. Deshpande, K. Arumugam, N. Maliyakkal, S.R. Meka, U. Nayanabhirama, PLGA 50: 50 nanoparticles of paclitaxel: development, in vitro anti-tumor activity in BT-549 cells and in vivo evaluation, Bull. Mater. Sci. 35 (2012) 319–326, http://dx.doi.org/10.1007/ s12034-012-0313-7. [30] S. Freitas, H.P. Merkle, B. Gander, Microencapsulation by solvent extraction/evaporation: reviewing the state of the art of microsphere preparation process technology, J. Control. Release 102 (2005) 313–332, http://dx.doi.org/10.1016/j. jconrel.2004.10.015. [31] V.R. Sinha, K. Bansal, R. Kaushik, R. Kumria, A. Trehan, Poly-ɛ-caprolactone microspheres and nanospheres: an overview, Int. J. Pharm. 278 (2004) 1–23, http:// dx.doi.org/10.1016/j.ijpharm.2004.01.044. [32] T.K. Dash, V.B. Konkimalla, Poly-??-caprolactone based formulations for drug delivery and tissue engineering: a review, J. Control. Release 158 (2012) 15–33, http://dx.doi.org/10.1016/j.jconrel.2011.09.064. [33] M.A. Woodruff, D.W. Hutmacher, The return of a forgotten polymer—polycaprolactone in the 21st century, Prog. Polym. Sci. 35 (2010) 1217-125, http://dx.doi.org/10.1016/j.progpolymsci.2010.04.002. [34] P. Arunkumar, S. Indulekha, S. Vijayalakshmi, R. Srivastava, Synthesis, characterizations, in vitro and in vivo evaluation of etoricoxib-loaded poly (caprolactone) microparticles — a potential intra-articular drug delivery system for the treatment of osteoarthritis — a potential intra-articular drug deliverys, J. Biomater. Sci. Polym. Ed. 27 (2016) 303–316, http://dx.doi.org/10.1080/09205063.2015. 1125564. [35] M. Dasaratha, N. Sathyamoorthy, Preparation of poly (epsilon-caprolactone) microspheres containing etoposide by solvent evaporation method, Asian J. Pharm. Sci. 5 (2010) 114–122. [36] Y. Zhang, Y. Zhang, S. Guo, W. Huang, Tyrosine kinase inhibitor loaded PCL microspheres prepared by S/O/W technique using ethanol as pretreatment agent, Int. J. Pharm. 369 (2009) 19–23, http://dx.doi.org/10.1016/j.ijpharm.2008.10.032. [37] A. Chen, T. Dang, S. Wang, N. Tang, Y. Liu, W. Wu, The in vitro and in vivo antitumor effects of MTX-Fe3O4-PLLA-PEG-PLLA microspheres prepared by suspensionenhanced dispersion by supercritical CO2, Sci. China Life Sci. 57 (2014) 698–709, http://dx.doi.org/10.1007/s11427-014-4680-8. [38] J.M. Kremer, Methotrexate pharmacogenomics, Ann. Rheum. Dis. 65 (2006) 1121–1123, http://dx.doi.org/10.1136/ard.2006.051789. [39] J.S. Jerzy Swierkot, Methotrexate in rheumatoid arthritis, Inst. Pharmacol. Polish Acad. Sci. 58 (2006) 473–492. [40] Z.A. Khan, R. Tripathi, B. Mishra, Methotrexate: a detailed review on drug delivery and clinical aspects, Expert Opin. Drug Deliv. 9 (2012) 151–169, http://dx.doi.org/ 10.1517/17425247.2012.642362. [41] S.M. Lee, H.J. Kim, Y.J. Ha, Y.N. Park, S.K. Lee, Y.B. Park, K.H. Yoo, Targeted chemo-photothermal treatments of rheumatoid arthritis using gold half-shell multifunctional nanoparticles, ACS Nano 7 (2013) 50–57, http://dx.doi.org/10.1021/ nn301215q. [42] J.R. O'Dell, Methotrexate use in rheumatoid arthritis, Rheum. Dis. Clin. N. Am. 23 (1997) 779–796, http://dx.doi.org/10.1016/S0889-857X(05)70360-4. [43] J. Albuquerque, C.C. Moura, B. Sarmento, S. Reis, Solid lipid nanoparticles: a potential multifunctional approach towards rheumatoid arthritis theranostics, Molecules 20 (2015) 11103–11118, http://dx.doi.org/10.3390/ molecules200611103. [44] P. Prabhu, R. Shetty, M. Koland, K. Vijayanarayana, K.K. Vijayalakshmi, M.H. Nairy, G.S. Nisha, Investigation of nano lipid vesicles of methotrexate for antirheumatoid activity, Int. J. Nanomedicine 7 (2012) 177–186, http://dx.doi.org/10. 2147/IJN.S25310. [45] S.M. Wigginton, B.C. Chu, M.H. Weisman, S.B. Howell, Methotrexate pharmacokinetics after intraarticular injection in patients with rheumatoid arthritis, Arthritis Rheum. 23 (1980) 119–122, http://dx.doi.org/10.1002/art.1780230121. [46] S.H.R. Edwards, Intra-articular drug delivery: the challenge to extend drug residence time within the joint, Vet. J. 190 (2011) 15–21, http://dx.doi.org/10.1016/ j.tvjl.2010.09.019.
Disclosure statement Authors declare no conflict of interest. References [1] D. Kai, S.S. Liow, X.J. Loh, Biodegradable polymers for electrospinning: towards biomedical applications, Mater. Sci. Eng. C 45 (2015) 659–670, http://dx.doi.org/ 10.1016/j.msec.2014.04.051. [2] Z. Li, X.J. Loh, Water soluble polyhydroxyalkanoates: future materials for therapeutic applications, Chem. Soc. Rev. 44 (2015) 2865–2879, http://dx.doi.org/10. 1039/c5cs00089k. [3] H. Ye, A.A. Karim, X.J. Loh, Current treatment options and drug delivery systems as potential therapeutic agents for ovarian cancer: a review, Mater Sci Eng C Mater Biol Appl 45 (2014) 609–619, http://dx.doi.org/10.1016/j.msec.2014.06.002. [4] H. Ye, C. Owh, S. Jiang, C.Z.Q. Ng, D. Wirawan, X.J. Loh, A thixotropic polyglycerol sebacate-based supramolecular hydrogel as an injectable drug delivery matrix, Polymers (Basel) 8 (2016), http://dx.doi.org/10.3390/polym8040130. [5] Q.Q. Dou, S.S. Liow, E. Ye, R. Lakshminarayanan, X.J. Loh, Biodegradable thermogelling polymers: working towards clinical applications, Adv. Healthc. Mater. 3 (2014) 977–988, http://dx.doi.org/10.1002/adhm.201300627. [6] J. Li, X.J. Loh, Cyclodextrin-based supramolecular architectures: syntheses, structures, and applications for drug and gene delivery, Adv. Drug Deliv. Rev. 60 (2008) 1000–1017, http://dx.doi.org/10.1016/j.addr.2008.02.011. [7] X.J. Loh, J. Li, Biodegradable thermosensitive copolymer hydrogels for drug delivery, Expert Opin. Ther. Pat. 17 (2007) 965–978, http://dx.doi.org/10.1517/ 13543776.17.8.965. [8] C.O. Ghislaine Barouti, Sing Shy Liow, Qingqing Dou, Hongye Ye, X.J.L. Sophie, M. Guillaume, New linear and star-shaped thermogelling poly([R]-3-hydroxybutyrate) copolymers ghislaine, Chem. Eur. J. 22 (2016) 10501–10512, http:// dx.doi.org/10.1002/chem.201601404. [9] J. Pushpamalar, A.K. Veeramachineni, C. Owh, X.J. Loh, Biodegradable polysaccharides for controlled drug delivery, Chem. Aust. 81 (2016) 504–514, http:// dx.doi.org/10.1002/cplu.201600112. [10] L. Jiang, C.R.R. Gan, J. Gao, X.J. Loh, A perspective on the trends and challenges facing porphyrin-based anti-microbial materials, Small 12 (2016) 3609–3644, http://dx.doi.org/10.1002/smll.201600327. [11] Y. Xiang, N.N.L. Oo, J.P. Lee, Z. Li, X.J. Loh, Recent development of synthetic nonviral systems for sustained gene delivery, Drug Discov. Today 0 (2017), http:// dx.doi.org/10.1016/j.drudis.2017.04.001. [12] X.J. Loh, S.J. Ong, Y.T. Tung, H.T. Choo, Co-delivery of drug and DNA from cationic dual-responsive micelles derived from poly(DMAEMA-co-PPGMA), Mater. Sci. Eng. C 33 (2013) 4545–4550, http://dx.doi.org/10.1016/j.msec.2013.07.011. [13] X.J. Loh, T.C. Lee, Q. Dou, G.R. Deen, Utilising inorganic nanocarriers for gene delivery, Biomater. Sci. 4 (2016) 70–86, http://dx.doi.org/10.1039/c5bm00277j. [14] Z. Li, E. Ye, David, R. Lakshminarayanan, X.J. Loh, Recent advances of using hybrid nanocarriers in remotely controlled therapeutic delivery, Small 12 (2016) 4782–4806, http://dx.doi.org/10.1002/smll.201601129. [15] V. Tran, J. Benoît, M. Venier-julienne, Why and how to prepare biodegradable, monodispersed, polymeric microparticles in the field of pharmacy? Int. J. Pharm. 407 (2011) 1–11, http://dx.doi.org/10.1016/j.ijpharm.2011.01.027. [16] E. Campos, J. Branquinho, A.S. Carreira, A. Carvalho, P. Coimbra, P. Ferreira, M.H. Gil, Designing polymeric microparticles for biomedical and industrial applications, Eur. Polym. J. 49 (2016) 2005–2021, http://dx.doi.org/10.1016/j. eurpolymj.2013.04.033. [17] R.A. Jain, The manufacturing techniques of various drug loaded biodegradable poly (lactide-co-glycolide) (PLGA) devices, Biomaterials 21 (2000) 2475–2490, http:// dx.doi.org/10.1016/S0142-9612(00)00115-0. [18] F. Chen, G. Yin, X. Liao, Y. Yang, Z. Huang, J. Gu, Y. Yao, X. Chen, H. Gao, Preparation, characterization and in vitro release properties of morphine-loaded PLLA-PEG-PLLA microparticles via solution enhanced dispersion by supercritical fluids, J. Mater. Sci. Mater. Med. 24 (2013) 1693–1705, http://dx.doi.org/10. 1007/s10856-013-4926-1. [19] D.R. Chen, J.Z. Bei, S.G. Wang, Polycaprolactone microparticles and their biodegradation, Polym. Degrad. Stab. 67 (2000) 455–459, http://dx.doi.org/10.1016/ S0141-3910(99)00145-7. [20] D. Pepic, M.S. Nikolic, S. Grujic, M. Lausevic, J. Djonlagic, Release behaviour of carbamazepine-loaded poly (″-caprolactone)/poly(ethylene oxide) microspheres, 30 (2013) 151–160. http://dx.doi.org/10.3109/02652048.2012.704954. [21] V. Natarajan, N. Krithica, B. Madhan, P.K. Sehgal, Formulation and evaluation of quercetin polycaprolactone microspheres for the treatment of rheumatoid arthritis, J. Pharm. Sci. 100 (2011) 195–205, http://dx.doi.org/10.1002/jps.22266 ABSTRACT. [22] N. Butoescu, O. Jordan, E. Doelker, Intra-articular drug delivery systems for the treatment of rheumatic diseases: a review of the factors influencing their performance, Eur. J. Pharm. Biopharm. 73 (2009) 205–218, http://dx.doi.org/10.1016/j.
549
Materials Science & Engineering C 81 (2017) 542–550
M. Dhanka et al.
10.1016/j.jdent.2006.03.002. [61] S. Jana, M.K. Trivedi, R.M. Tallapragada, A. Branton, D. Trivedi, G. Nayak, R. Kumar, Pharmaceutica characterization of physicochemical and thermal properties of chitosan and sodium alginate after biofield treatment, Pharm. Anal. Acta. 6 (2015) 1–9, http://dx.doi.org/10.4172/21532435.1000430. [62] S. Wang, S. Guo, L. Cheng, Disodium norcantharidate loaded poly(??-caprolactone) microspheres. I. Preparation and evaluation, Int. J. Pharm. 350 (2008) 130–137, http://dx.doi.org/10.1016/j.ijpharm.2007.08.030. [63] J. Jingou, H. Shilei, L. Weiqi, W. Danjun, W. Tengfei, X. Yi, Preparation, characterization of hydrophilic and hydrophobic drug in combine loaded chitosan/cyclodextrin nanoparticles and in vitro release study, Colloids Surf. B: Biointerfaces 83 (2011) 103–107, http://dx.doi.org/10.1016/j.colsurfb.2010.11.005. [64] Y. Huang, J. Liu, Y. Cui, H. Li, Y. Sun, Y. Fan, X. Zhang, Reduction-triggered breakable micelles of amphiphilic polyamide amine-G-polyethylene glycol for methotrexate delivery, Biomed. Res. Int. 2014 (2014), http://dx.doi.org/10.1155/ 2014/904634. [65] J.C. Jeong, J. Lee, K. Cho, Effects of crystalline microstructure on drug release behavior of poly(ε-caprolactone) microspheres, J. Control. Release 92 (2003) 249–258, http://dx.doi.org/10.1016/S0168-3659(03)00367-5. [66] S. Ray, M. Joy, B. Sa, pH dependent chemical stability and release of methotrexate from a novel nanoceramic carrier, RSC Adv. 5 (2015) 39482–39494, http://dx.doi. org/10.1039/C5RA03546E. [67] A.Z. Chen, G.Y. Wang, S. Bin Wang, L. Li, Y.G. Liu, C. Zhao, Formation of methotrexate-PLLA-PEG-PLLA composite microspheres by microencapsulation through a process of suspension-enhanced dispersion by supercritical CO2, Int. J. Nanomedicine 7 (2012) 3013–3022, http://dx.doi.org/10.2147/IJN.S32662. [68] S.A. Dhanaraj, S. Muralidharan, V. Venugopal, P. Kanniappan, Formulation and evaluation of chitosan nanospheres containing methotrexate targeted drug delivery system, J. Young Pharm. 8 (2016) 330–334, http://dx.doi.org/10.5530/jyp.2016. 4.7. [69] A. de Fatima Pereira, V. Mara da Costa, M. Cristina Magalhaes Santos, F. Carmo Horta Pinto, G. Rodrigues Da Silva, Evaluation of the effects of methotrexate released from polymeric implants in solid Ehrlich tumor, Biomed Pharmacother 68 (2014) 365–368, http://dx.doi.org/10.1016/j.biopha.2013.12.012. [70] D.R. Nogueira, L. Tavano, M. Mitjans, L. Pérez, M.R. Infante, M.P. Vinardell, In vitro antitumor activity of methotrexate via pH-sensitive chitosan nanoparticles, Biomaterials 34 (2013) 2758–2772, http://dx.doi.org/10.1016/j.biomaterials. 2013.01.005. [71] H. Jiang, X.B. Wang, C.Y. Li, J.S. Li, F.J. Xu, C. Mao, W.T. Yang, J. Shen, Improvement of hemocompatibility of polycaprolactone film surfaces with zwitterionic polymer brushes, Langmuir 27 (2011) 11575–11581, http://dx.doi.org/10. 1021/la202101q. [72] C. Besteiro, A.J. Guiomar, C.A. Gonc, M.N. De Pinho, M.H. Gil, Characterization and in vitro hemocompatibility of bi-soft segment, polycaprolactone-based poly(ester urethane urea) membranes, J. Biomed. Mater. Res. Part A. (2009), http://dx.doi. org/10.1002/jbm.a.32594. [73] N.R. Kuznetsova, C. Sevrin, D. Lespineux, N.V. Bovin, E.L. Vodovozova, T. Meszaros, J. Szebeni, C. Grandfils, Hemocompatibility of liposomes loaded with lipophilic prodrugs of methotrexate and melphalan in the lipid bilayer, J. Control. Release 160 (2012) 394–400, http://dx.doi.org/10.1016/j.jconrel.2011.12.010.
[47] A. Trapani, N. Denora, G. Iacobellis, J. Sitterberg, U. Bakowsky, T. Kissel, Methotrexate-loaded chitosan- and glycolchitosan-based nanoparticles: a promising strategy for the administration of the anticancer drug to brain tumors, AAPS Pharm. Sci. Tech. 12 (2011) 1302–1311, http://dx.doi.org/10.1208/s12249-011-9695-x. [48] D. Seo, Y. Jeong, D. Kim, M. Jang, M. Jang, J. Nah, Colloids and surfaces B: biointerfaces methotrexate-incorporated polymeric nanoparticles of methoxy poly (ethylene glycol)-grafted chitosan, Colloids Surf. B: Biointerfaces 69 (2009) 157–163, http://dx.doi.org/10.1016/j.colsurfb.2008.10.020. [49] T. Muthukumar, S. Prabhavathi, M. Chamundeeswari, T.P. Sastry, Bio-modified carbon nanoparticles loaded with methotrexate possible carrier for anticancer drug delivery, Mater. Sci. Eng. C 36 (2014) 14–19, http://dx.doi.org/10.1016/j.msec. 2013.11.046. [50] M. Notara, C.A. Scotchford, D.M. Grant, N. Weston, G.A.F. Roberts, Cytocompatibility and hemocompatibility of a novel chitosan-alginate gel system, J. Biomed. Mater. Res. A 89 (2008) 854–864, http://dx.doi.org/10.1002/jbm.a. 32027. [51] A.R. Padalhin, N. Thuy, B. Linh, Y.K. Min, B. Lee, A.R. Padalhin, N. Thuy, B. Linh, Y.K. Min, Evaluation of the cytocompatibility hemocompatibility in vivo bone tissue regenerating capability of different PCL blends, J. Biomater. Sci. Polym. Ed. 25 (2014) 487–503, http://dx.doi.org/10.1080/09205063.2013.878870. [52] A.K. Jaiswal, H. Chhabra, S.S. Kadam, K. Londhe, V.P. Soni, J.R. Bellare, Hardystonite improves biocompatibility and strength of electrospun polycaprolactone nano fi bers over hydroxyapatite: a comparative study, Mater. Sci. Eng. C 33 (2013) 2926–2936, http://dx.doi.org/10.1016/j.msec.2013.03.020. [53] R. Yoganathan, R. Mammucari, N.R. Foster, Impregnation of ibuprofen into Polycaprolactone using supercritical carbon dioxide, J. Phys. Conf. Ser. 12087 (2010) 1–6, http://dx.doi.org/10.1088/1742-6596/215/1/012087. [54] L. Chen, T. Shi, X. Xia, J. Miao, C. Mao, B. Huang, J. Shen, Mild anticoagulant effect of heparin-loaded Polycaprolactone microspheres, J. Nanosci. Nanotechnol. 15 (2015) 144–150, http://dx.doi.org/10.1166/jnn.2015.8772. [55] C. Guo, L. Zhou, J. Lv, Effects of expandable graphite and modified ammonium polyphosphate on the flame-retardant and mechanical properties of wood flourpolypropylene composites, Polym. Polym. Compos. 21 (2013) 449–456, http://dx. doi.org/10.1002/app. [56] T. Elzein, M. Nasser-Eddine, C. Delaite, S. Bistac, P. Dumas, FTIR study of polycaprolactone chain organization at interfaces, J. Colloid Interface Sci. 273 (2004) 381–387, http://dx.doi.org/10.1016/j.jcis.2004.02.001. [57] M. Stevanovic, A. Radulovic, V. Pavlovic, Facile synthesis of poly(e-caprolactone) micro and nanospheres using different types of polyelectrolytes as stabilizers under ambient and elevated temperature, Compos. Part B (2012), http://dx.doi.org/10. 1016/j.compositesb.2012.07.008. [58] U.O.H. Yi Zhong, Victor Leung, Lynn Yuqin Wan, Silvio Dutz, Frank K. Ko, Electrospun magnetic nanofibre mats — a new bondable biomaterial using remotely activated magnetic heating, J. Magn. Magn. Mater. 380 (2015) 330–334, http://dx. doi.org/10.1016/j.jmmm.2014.09.069. [59] M.F. Canbolat, A. Celebioglu, T. Uyar, Drug delivery system based on cyclodextrinnaproxen inclusion complex incorporated in electrospun polycaprolactone nanofibers, Colloids Surf. B: Biointerfaces 115 (2014) 15–21, http://dx.doi.org/10.1016/j. colsurfb.2013.11.021. [60] A. Elzubair, C. Nelson, M. Suarez, The physical characterization of a thermoplastic polymer for endodontic obturation, J. Dent. 34 (2006) 784–789, http://dx.doi.org/
550
Contents lists available at ScienceDirect
Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Injectable methotrexate loaded polycaprolactone microspheres: Physicochemical characterization, biocompatibility, and hemocompatibility evaluation
MARK
Mukesh Dhanka, Chaitra Shetty, Rohit Srivastava⁎ Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai 400076, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Methotrexate Polycaprolactone Microspheres Cell viability Hemolysis
In this study, bare polycaprolactone microspheres (PCL MPs) and methotrexate (MTX) loaded PCL microspheres (MTX-PCL MPs) have been developed by oil-in-water emulsion solvent evaporation method using hydroxypropyl methylcellulose (HPMC) as an emulsifier. Encapsulation efficiency and loading capacity of methotrexate were found to be 51.28% ± 0.52 and 2.8% ± 0.06 respectively. Environmental scanning electron microscopy showed the PCL MPs and MTX-PCL MPs to have a spherical shape and smooth surface morphology. The mean size of microspheres (23 μm) was found within injectability criteria. High-Resolution X-ray diffraction of microspheres revealed that PCL retained its semi-crystalline nature after processing in microspheres, but the drug looses its crystallinity. Fourier transmittance infrared spectroscopy and thermogravimetry analysis of the microspheres indicated that no physicochemical modification occurred. In vitro, MTX release study from MTX-PCL MPs in phosphate buffer saline (pH 7.4) showed controlled release profile and only 31% of MTX released in 306 h. The microspheres in lyophilized form are physicochemically stable for 8 months. Furthermore, L929 cells treated with microspheres showed cell viability > 80%. The different concentrations of microspheres found hemocompatible and did not affect the biconcave shape of red blood cells (RBCs). The physiochemical and biological evaluation of microspheres suggests their further use for drug delivery application.
1. Introduction Over the past few decades, the various types of novel biomaterials have been developed for applications in biomedical engineering such as drug delivery, cell encapsulations and tissue engineering. The various types of active molecules (protein, gene, drugs and several others) of delivery systems have been developed including hydrogels, microparticles, nanoparticles, nanofibers, liposomes and micelles, etc. for improving their overall therapeutic efficacy [1–14]. Microspheres prepared from natural as well as synthetic polymers have been widely investigated as a drug delivery carrier [15,16]. Their use has gradually increased in the pharmaceutical field due to its advantages such as controlled release, protection from premature degradations, increased bioavailability, and decreased toxicity, reduced dosage frequency and improved patient compliance [15–17]. Also, they are easily injectable compared to surgical insertion of large implants. Microspheres have been administered through different routes such as intravenous, intramuscular, subcutaneous, and pulmonary, etc. [15–18]. Recently, non-biodegradable polymers have been replaced by biodegradable polymers for the development of polymeric microspheres based ⁎
Corresponding author. E-mail address: [email protected] (R. Srivastava).
http://dx.doi.org/10.1016/j.msec.2017.08.055 Received 25 May 2017; Received in revised form 14 July 2017; Accepted 10 August 2017 Available online 12 August 2017 0928-4931/ © 2017 Published by Elsevier B.V.
delivery systems in pharmaceutical technology. Microspheres made from biocompatible and biodegradable polymer produce a non-toxic product from their biodegradation and avoid the surgical removal from injected site [17–20]. Recently, many reports are available on the injectable microspheres for localized delivery of the drug in solid tumor and joints. Because these injectable depots of microspheres are related with site-specific drug release, the drug toxicity at non-targeted tissues is avoided [21–24]. Among synthetic polymers, polylactic acid (PLA), polyglycolic acid (PGA) and poly (lactide-co-glycolide) acid (PLGA) have been most widely used for the development of microspheres based drug delivery systems owing to their excellent biocompatibility, biodegradability, and non-immunogenicity [17,18,23,25–30]. However, their high cost and generation of the acidic environment of degradation which can produce inflammation are responsible for their limited use in the microsphere development for biomedical applications. Polycaprolactone (PCL) is a synthetic polymer and is suitable for the development of controlled drug delivery systems due to its high permeability for drug molecules and slow degradation kinetics compared to polylactide and polyglycolide. It is a cost-effective polymer and does not create an acidic environment during its degradation. Also, it is
Materials Science & Engineering C 81 (2017) 542–550
M. Dhanka et al.
(Mumbai, India). The Triton X-100 was purchased from Fisher Scientific Ltd. (Mumbai, India), and glutaraldehyde (38%) supplied by Merck Ltd. (Mumbai, India).
biocompatible, biodegradable and non-immunogenic with hydrophobic semi-crystalline nature. It is a Food and Drug Administration-approved polymer for pharmaceutical applications and had been used for a long time for development of drug delivery systems in the pharmaceutical industry. PCL based microspheres have been developed for encapsulation and controlled delivery of hydrophilic and hydrophobic drugs [19,31–34]. Drug-loaded microspheres preparation mainly depends on the solubility of drug and polymer in various solvents. The solvent evaporation is a most widely used simple and reproducible technique for microspheres preparation [21,30,32,35,36]. Methotrexate (MTX) is a potent antimetabolite for folic acid with anti-cancer, anti-inflammatory, anti-rheumatic and disease modifying properties [37–42]. In 1970, MTX was included as disease modifying anti-rheumatic drug (DMARDs) and is currently the most widely used drug for treatment of rheumatoid arthritis. Due to its high effectiveness compared to other drugs for rheumatoid arthritis treatment, it is a popular second line drug among the doctors. The clinical application of MTX has limitations such as poor solubility, large amount excreted by the kidney, short half-life, and rapid diffusion into the body. However, its use is also associated with serious side effects, due to which around 30% patients worldwide leave the treatment of rheumatoid arthritis. The most frequent side effects arising out of MTX dosing includes mouth sores, stomatitis, and a decrease in white blood cells count. A high dose for longer time produces severe hepatotoxicity, nephrotoxicity, and bone marrow suppression. Other side effects caused by MTX include skin rash, itching, dizziness, hair loss, and drowsiness. In the clinical application, regular monitoring of MTX amount in the blood is required to ensure the minimal toxic level [23,39,40,43,44]. To decrease the toxicity the localized intra-articular and intra-tumoral free MTX suspension has been administered, but the efficacy was found low due to rapid clearance from the target site [22,29,38,40,42,45–48]. Therefore, MTX is an ideal drug for microencapsulation into PCL polymer. In this study, bare polycaprolactone microspheres (PCL MPs) and methotrexate loaded PCL microspheres (MTX-PCL MPs) have been developed from single O/W emulsion solvent evaporation method. Here, hydroxypropyl methylcellulose (HPMC) has been used as an emulsifier for the development of PCL MPs and MTX-PCL MPs. The developed microspheres size, shape, and morphology were characterized by Environmental Scanning Electron Microscopy (ESEM), and Fourier Transform Infrared Spectroscopy did physiochemical characterization (FTIR), Thermogravimetric Analysis (TGA) and High-Resolution X-ray Diffractometry (HR-X-RD). The stability study for lyophilized PCL microspheres powder sample was performed up to 8 months. The biocompatibility was performed on the L929 fibroblast cells for 24 h and hemocompatibility was performed on the human blood. This system will pave the way for further advancement in microsphere-based injectable drug delivery system for treatment of localized tumor and joint-rheumatoid arthritis.
2.2. Preparation of microspheres The PCL MPs and MXT-PCL MPs were prepared by using O/W emulsion solvent evaporation method. Briefly, MTX was dissolved into 500uL DMSO and mixed into 2 mL DCM containing 100 mg of dissolved PCL polymer. After polymer and drug containing organic phase was added by using syringe pump with the speed 1 mL/min into 30 mL of water phase containing HPMC (0.2%w/v) as an emulsifier. Simultaneously the resulting emulsion was emulsified by homogenizer with the speed 3000 rpm for 5 min and then the emulsion was kept on a magnetic stirrer at 800 rpm for evaporation of organic phase for 6 h. After complete evaporation of organic phase, the MTX-PCL MPs were collected by centrifuging at 10000 rpm for 15 min and microspheres dried by lyophilisation. PCL MPs were also obtained by the same method but without the addition of MTX. MTX loaded PCL microspheres were prepared in dark condition due to light-sensitive nature of MTX. 2.3. Morphology and size analysis of microspheres The shape and surface morphology of PCL MPs and MTX-PCL MPs were characterized by environmental scanning electron microscope (ESEM) (FEI-ESEM, Quanta 200, Netherlands). The dried samples of microspheres were stacked on double-sided carbon tape, and samples were coated with platinum for 300 s with 10 mA (JFC-1600, Fine Coater, Japan). The microspheres were observed under different magnifications with accelerated voltage from 5 to 20 kV. Average size and size distribution of PCL MPs and MTX-PCL MPs were from ESEM images calculated by using Image J online software. 2.4. Production yield (% PY) of microspheres The total amount of PCL MPs and MTX-PCL MPs were estimated by weighing the obtained lyophilized microspheres powder on a weighing machine. The production yield of PCL MPs and MTX-PCL MPs were calculated by using Eq. (1).
Production yield (%) = (Wa Wb) × 100
(1)
The microparticles production yield was calculated by weight of microspheres produce (Wa) and weight of compounds used in the formulation for microspheres preparation (Wb), PCL or PCL plus MTX. 2.5. Drug encapsulation efficiency and loading capacity
2. Materials and methods
To calculate the encapsulated amount of MTX, the 5 mg of MTX-PCL MPs were dissolved in 2 mL DCM, and then 1 mL DMSO was added to solubilize the drug. After the mixture was kept for bath sonication of 1 min, and then added into 10 mL phosphate buffer saline (PBS, pH 7.4) and stirred until complete evaporation of DCM. The obtained solution was centrifuged at 10000 rpm for 15mins, and collected supernatant was analyzed using UV-spectrometer at 304 nm wavelength. PCL MPs were also analyzed using the same procedure and used as a control. The encapsulation efficiency (%EE) and loading capacity (%LC) was calculated by using the following formulas (2) and (3).
2.1. Materials PCL (molecular weight 14,000 Da) was purchased from SigmaAldrich (Mumbai, India), and hydroxypropyl methylcellulose (HPMC, K15M) procured from Otto Chemie Ltd. (Mumbai, India). Methotrexate was kindly gifted by Sun Pharma Ltd. (Gujarat, India). Organic solvent like dichloromethane (DCM) HPLC grade and dimethyl sulfoxide (DMSO) analytical grade were obtained from Merck Ltd. (Mumbai, India). Chemicals for the preparation of Phosphate Buffer Saline (used for drug release studies) disodium hydrogen phosphate, sodium chloride, and potassium dihydrogen phosphate was purchased from Merck Ltd. (Mumbai, India) and potassium chloride purchased from Sigma-Aldrich (Mumbai, India). Cell culture media (DMEM), antibiotics (Penicillin and streptomycin) and PBS were purchased from HiMedia 543
%EE =
Mass MTX in microspheres × 100 Total mass of MTX used in formulation
(2)
%LC =
Mass of MTX loaded microspheres × 100 Total mass of MTX used in formulation
(3)
Materials Science & Engineering C 81 (2017) 542–550
M. Dhanka et al.
100 μL of fresh media containing 10 μg/mL resazurin dye was added to each well and again the plate was incubated (37 °C, CO₂ incubator) for 4 h. Further, the absorbance was recorded at 570 nm with reference absorbance at 600 nm using plate reader (Tecan infinite M 200 PRO). The PBS (negative control) and 1% Triton X-100 (positive control) treated cell were used for comparison study. The cell viability was calculated by using below Eq. (5).
2.6. FTIR analysis The quality analysis of PCL polymer and drug after formation of microspheres was observed under FTIR spectroscopy (FTIR, Bruker, Japan). The powder samples of pure MTX, PCL MPs, and MTX-PCL MPs were mixed with potassium bromide (KBr) and converted into thin KBr pellet. The FTIR spectra of the samples were recorded using transmission mode of the instrument from 400 to 4000 cm− 1 range of wavenumber and at 4 cm− 1 resolution.
%cell viability =
Absorbance of test sample × 100 Absorbance of control
(5)
2.7. HR-XRD characterization 2.12. Hemolysis assay The X-ray diffraction patterns for pure MTX, PCL MPs, and MTXPCL MPs were recorded by using HR-XRD (Rigaku, Smartlab 9 kW, High resolution-X-ray Diffractometer, Japan) for evaluation of the crystalline structure of drug and PCL polymer after being processed into microspheres. The samples were scanned over the range of 5–40° (2θ) with scanning rate of 6°/s.
The hemocompatibility tests for the microspheres were conducted only after taking institutional ethical clearance (IITB-IEC-2016-028) and informed consent form (ICF). The human blood samples were collected from a healthy volunteer (age, 26 yrs) in trisodium citrate containing vials. The red blood cells (RBCs) were separated from whole blood samples by using centrifugation (1000 rpm, 5mins) followed by three times washing with physiological buffer saline (PBS, pH 7.4). The RBCs collected from 10 mL whole blood sample were then dispersed into 16 mL PBS and 500 μL RBCs containing samples were tested with each test sample. The hemolysis study for PCL MPs and MTX-PCL MPs was studied spectrophotometrically by measuring free hemoglobin (Hb) released from red blood cells (RBCs) lysis upon interaction with test samples. First, for obtaining a pre-saturated solution, different amounts (0.5 mg, 1 mg, 2.5 mg, and 5 mg) of PCL MPs and MTX-PCL MPs microspheres were incubated in 500 μL PBS at 37 °C for 1 h. After presaturation, 500 μL test samples were added into 2 mL Eppendorf containing 500 μL of RBCs solution under aseptic condition. The RBCstreated samples were incubated in a water bath for 3 h under shaking condition (60 rpm) at 37 °C. After incubation, intact RBCs were pelleted down by centrifugation (4000 rpm, 10 min) and 100 μL of the supernatant was used for recording the absorbance at 540 nm using plate reader. RBCs treated with phosphate buffer saline (0% lysis) was used as negative control (NC), and 1% Triton-X (100% lysis) treated cells were used as positive control (PC). According to reported studies, materials that cause < 5% hemolysis can be included into hemocompatible materials [49–52]. The biconcave shape and morphology of erythrocytes after treatment with test samples were observed under E-SEM. Treated RBCs collected after centrifugation were redispersed into 1 mL of PBS. 1% glutaraldehyde solution in PBS (pH 7.4) was used as a fixative for RBCs. The 400 μL of glutaraldehyde solution was added into RBCs solution and kept for fixation at 4 °C for 2 h. The fixed RBC samples were then centrifuged, and the pellets were redispersed in 1 mL of water and drop-casted on aluminum foil and dried at room temperature. The dried RBCs samples on aluminum foil were stacked on double-sided carbon tape and coated with platinum for 300 s. The samples were observed at different magnification with accelerated voltage from 15 kV.
2.8. TGA analysis Thermal gravimetric analysis (TGA) for the pure drug, drug loaded and blank microspheres were performed by the TGA instrument (TG/ DTA, Perkin Elmer, USA). The heating rate for the sample was 10 °C/ min and nitrogen flow rate 60 mL/min. The percent weight loss study of the sample was done with temperature range from 42 °C to 1000 °C. 2.9. Stability study of microspheres In order to conduct the stability test, dried powder samples of MTX loaded PCL microspheres were stored in the freezer for a different time period. After a specific time interval, the samples were taken under shape, morphology, and FTIR characterization for evaluation of physicochemical properties of microspheres. 2.10. Drugs release study The 10 mg MTX-PCL MPs were kept in the dialysis bag to separate the microspheres from the dissolution medium. The study was carried out in 15 mL PBS (pH 7.4) and incubated at 37 °C in water bath shaker at 60 rpm shaking rate. Determination of MTX release from MTX-PCL MPs the 1 mL release medium was replaced with freshly prepared PBS at a particular time interval. The quantification of release drug was done by UV-spectrophotometer with 1:1 dilution of the original sample. The drug release was calculated in cumulative percentage release (w/w %) by using Eq. (4).
MTX released% =
MTX released at specific time interval × 100 Total MTX into systems
(4)
2.11. Biocompatibility assay
%Hemolysis = The L929 normal mouse fibroblast cells (National Centre for Cell Sciences, Pune, India) were grown in Dulbecco's Modified Eagle's Medium (DMEM) (HiMedia, India) supplemented with 10% FBS (Fetal Bovine Serum) and 1% antibiotics (streptomycin: penicillin). The cells were maintained in standard cell culture condition (37 °C under 5% CO₂, saturated with the humid environment) for further use. Further, to evaluate the cell cytotoxicity of PCL MPs and MTX-PCL MPs a colorimetric alamar assay was used. The cells viability were tested in triplicate using 96 well plates, each well seeded with 4000 cells/100 μL. The plates were then incubated at 37 °C in CO₂ incubator for 24 h. The media was removed, and 100 μL of fresh media containing different concentrations of microspheres (0.5 mg/mL, 1 mg/mL, 2.5 mg/mL, 5 mg/mL) was added to the respective wells and incubated at standard cell culture condition for 24 h. After 24 h, the media was removed and
(Absorbance of test sample−Absorbance of NC) × 100 (Absorbance of PC − Absorbance of NC) (6)
3. Results and discussion 3.1. Preparation of microspheres The PCL based microspheres drug delivery systems have been widely developed for the sustained and controlled release of pharmaceutical drugs. The emulsion solvent evaporation method has been widely used for microencapsulation of hydrophilic and hydrophobic drugs into the PLC microspheres [21,31,34,53,54]. In the reported studies of PCL MPs the PVA have been used as emulsifier, but here we have used HPMC biocompatible polymer as emulsifier. Here, PCL MPs 544
Materials Science & Engineering C 81 (2017) 542–550
M. Dhanka et al.
Fig. 2. FTIR spectra of blank PCL, MTX loaded PCL microspheres and pure MTX.
microspheres were obtained without significant difference (82–85%) as shown in Table 1. In the evaluation, the properties of drug loaded microspheres, the drug loading capacity, and encapsulation efficiency both are very important parameters. MTX loaded PCL microspheres were prepared by O/W solvent evaporation method with varying the concentration of MTX to observe the effect on encapsulation efficiency. The maximum encapsulation efficiency obtained was 51.68% in MTXPCL MPs, and loading capacity was 2.8%. Fig. 1. SEM characterization of PCL microspheres (a) blank microspheres at 1600 × (b) blank microspheres at 6000× (c) MTX loaded PCL microspheres at 1600 × (d) MTX loaded PCL microspheres at 6000 × (e) Pure MTX at 5000 ×.
3.4. FTIR analysis FTIR spectroscopy is a widely used technique for observing the chemical interaction between polymer and drug. Therefore, FTIR characterizations for the samples were done to find out the chemical changes in the structure of PCL polymer and MTX after formation of microspheres. FTIR spectra for pure MTX, PCL MPs, and MTX-PCL MPs were recorded and presented in Fig. 2. The FTIR spectrum of pure MTX showed similarity with the already reported studies [49]. After inspecting the FTIR curve of PCL MPs, all characteristics bands were observed, first a distinct peak at 1725 cm− 1 for carbonyl stretching (C]O), and a broad band at 3440 cm− 1 due to an overtone of the carbonyl stretching, 2950 and 2866 cm− 1 which are due to symmetrical CH₃ and antisymmetrical CH₂ stretching respectively. The vibration for the CH₂ band is observed at 1370, 1420, and 1473 cm− 1 and OeC vibration bands are at 1105, 1043, and 959 cm− 1 respectively. Furthermore, stretching for ester functional group was observed at 1186 and 1242 cm− 1 [34,55,56]. It has already been reported that a band at 1297 cm− 1 occurred due to the backbone of CeC and CeO stretching mode in the crystalline form of PCL, and in amorphous form, this band appears at 1157 cm− 1 [34,57]. However, no peak occurred at 1157 cm− 1 (related to the amorphous form of PCL), thus confirming the high crystalline degree of obtained PCL microspheres [57]. The crystalline nature of PCL polymer is the very important property for controlled release of drugs [34]. The FTIR curve for MTX-PCL MPs, was similar to PCL MPs and no extra peak was observed. This indicated that there was no chemical interaction between polymer and drug after encapsulation, and drug dispersed in its molecular form in the polymer matrix. In the reports, FTIR spectra of PCL polymer did not change after encapsulation of drug into PCL microspheres [34,56]. However, the O/ W emulsion solvent evaporation method is a physical method and do not provide the favorable environment for chemical modification between polymer and drug molecules.
and MTX-PCL MPs has been produced by single solvent evaporation (O/ W) method by using a mixture of DCM and DMSO. 3.2. Morphology and size analysis of microspheres Shape and surface morphology of the microspheres were visualized under ESEM and micrographs are presented in Fig. 1. The PCL MPs (Fig. 1a, b) and MTX-PCL MPs (Fig. 1c, d) were obtained with some important physical properties including, individuality, spherical shape, smooth surface morphology, and no other deformed microspheres occurred. There were no additional effects observed on the surface morphology and shape of the PCL microspheres after the loading of MTX. The crystalline nature of MTX can be seen in XRD studies (Fig. 4) and ESEM images (Fig. 1e), but there was no drug crystal appearance on the surface of MTX-PCL MPs. Size is a very important factor for injectable microspheres because size should be large enough to contain sufficient amount of drug but not too large to cause discomfort after injection [15]. The average size of the PCL MPs was found to be 24.094 ± 2.548 μm, but after loading of drug the mean size was obtained slightly low that is 23.88 ± 1.15 μm. The average size of PCL MPs and MTX-PCL-MPs has shown in Table 1, which is under injectability criteria [15,17]. 3.3. Production yield, encapsulation efficiency and loading capacity Production yield is a very important parameter in the pharmaceutical industry for development of microspheres based drug delivery systems. The production yield for blank and MTX loaded PCL Table 1 Size, EE, LC and PY of PCL microspheres. Formulations
Mean size (μm)
%EE
%LC
%PY
PCL MPs MTX-PCL MPs
24.09 ± 2.54 23.88 ± 1.15
– 51.28 ± 0.52
– 2.8 ± 0.06
82.06 ± 2.81 85.6 ± 1.70
3.5. TGA analysis The thermal stability of PCL MPs and MTX-PCL MPs were determined by thermogravimetry analysis using a TA instrument. TGA 545
Materials Science & Engineering C 81 (2017) 542–550
M. Dhanka et al.
Fig. 3. TGA analysis of Pure MTX, PCL, and MTX loaded PCL microspheres.
thermograms for pure MTX, PCL MPs, and MTX-PCL MPs are shown in the Fig. 3. In thermal degradation study, the percent weight loss as a function of temperature 10 °C/min heating rate under a dynamic nitrogen atmosphere was observed. It was found that PCL MPs and MTXPCL MPs were stable from 42 °C to 250 °C. The % weight loss gradually increased from 250 °C to 500 °C due to thermal decomposition of polymer [58–60]. After 500 °C, remaining weight for PCL MPs and MTX-PCL MPs remained constant till 1000 °C. The remaining weight for drug loaded microspheres and blank microspheres were found to be close to each other. Drug-loaded microspheres did not show significant different thermal degradation pattern compared to blank microspheres which indicated that the MTX-PCL MPs were stable, and no physicochemical change occurred in the polymer structure and the drug directly entrapped into the polymer matrix.
Fig. 5. MTX release from MTX-PCL MPs in PBS (pH 7.4).
peaks of MTX disappeared in MTX-PCL MPs, and only characteristic peaks of PCL appeared. The crystalline structure of MTX might have lost during encapsulation, and only amorphous form was encapsulated inside the MTX-PCL MPs, but PCL maintained its semi-crystalline characteristics throughout the microspheres preparation. The XRD spectra of the samples clearly showed the crystalline nature of the PCL polymer in PCL MPs and MTX-PCL MPs [62,65]. 3.7. Drug release study The direct administration of MTX leads to poor efficacy due to its poor water solubility and short half-life. The entrapment of drug into biocompatible and biodegradable polymers can decrease the fast elimination from the body and enhance its therapeutic effect by releasing the drug in a controlled manner at a localized site [48,66–68]. The implant of PCL and microspheres of PLLA-PEG-PLLA and PLA have been developed and characterized by other groups with the aim to provide the controlled MTX release profile [23,67,69]. Here, spherical injectable MTX-PCL MPs have been prepared and characterized for localized delivery of methotrexate. The in vitro release profile in PBS (pH 7.4) of MTX loaded PCL microspheres are shown in Fig. 5. 10 mg of MTX-PCL microspheres were added into enough volume of PBS to dissolve MTX present in microspheres. Methotrexate was released from the MTX-PCL MPs in the “biphasic” pattern. The first pattern was the burst release due to loosely bound of MTX molecules on the surface of the microspheres. In the burst release effect, around 12% of the drug was released from the microspheres in 10 h. The second pattern was the slow release due to diffusion of the drug from the core of polymer. The MTX-PCL MPs showed the drug release pattern in the desirable slow and sustained release manner, and only 30% of the methotrexate was released from the microspheres in the 306 h. According to reported studies, PCL takes a long time for complete degradation, and the drug release profile majorly depends on diffusion rather than degradation [19,33,34].
3.6. HR-XRD characterization XRD diffractometry is a widely used and most suitable technique to evaluate the crystalline structure of the polymer, drug, and drug-loaded microspheres [61–63]. The XRD spectra for pure MTX, PCL MPs, and MTX-PCL MPs were recorded and presented in Fig. 4. The XRD diffraction pattern for PCL MPs showed two intense peaks in the range of 20–25° which are characteristic of semi-crystalline nature of PCL MPs as shown in the reports [20,34,59,62]. The XRD diffraction pattern of MTX showed multiple intense peaks in the range of 5–40° (2θ) that indicated its high degree of crystallinity [64]. But, these characteristic
3.8. Stability study of MTX-PCL MPs The stability of drug loaded microspheres in powder form is a very important aspect for long term storage of the formulation in the pharmaceutical applications. Here, lyophilized samples of MTX-PCL MPs were stored at 4 °C for a different time interval (4, 6, 8 months) and the physiochemical properties were characterized by using ESEM and FTIR. Fig. 6, shows the ESEM micrograph of the stored MTX loaded PCL
Fig. 4. HR-XRD diffractions patterns of PCL, MTX-PCL MPs and pure MTX.
546
Materials Science & Engineering C 81 (2017) 542–550
M. Dhanka et al.
Fig. 6. ESEM images of stored MTX loaded PCL microspheres for the different time period (a and b) 4 month (c and d) 6 month (e and f) 8 month.
Fig. 8. Cell viability studies of PCL microspheres.
effects of MTX [43]. According to reported study, free MTX did not show high cytotoxicity effect on normal fibroblast cells [70]. Thus, these microspheres are very suitable for targeted injection for providing the localized drug delivery at the disease site. Therefore, the drug will not release at the normal tissue, and cytotoxic effects can be avoided.
Fig. 7. FTIR spectra of MTX-PCL microspheres stored for different time periods.
microspheres for 4 months (a, b), 6 months (c, d) and 8 months (e, f). It was observed that there was no change in the shape, and surface morphology of MTX loaded PCL microspheres, i.e. the microspheres were found to retain their spherical shape with smooth surface morphology even after storing them for different time periods. Also, the size of the microspheres did not show any significant difference compared to original microspheres. Fig. 7, shows the FTIR spectra of the MTX loaded PCL microspheres, and it was observed that no chemical modification occurred after storing for different time intervals. Therefore, it can be inferred that MTX loaded PCL microspheres are stable and can be stored for up to 8 months.
3.10. Hemolysis assay The injectable polymeric microspheres for biomedical application come in contact with blood after injection at the targeted site so, this application attracts our attention to conduct RBC compatibility studies. Hemocompatibility of the PCL MPs was evaluated by studying two important factors, hemolysis and RBCs morphology study. The chemical composition, size, shape, and charge of polymeric microspheres can be a reason of hemolysis. Therefore, to evaluate the hemolysis by developed microspheres, the extracted RBCs were treated with different concentration of PCL MPs and MTX-PCL MPs (0.5 mg/mL, 1.0 mg/mL, 2.5 mg/mL, 0.5 mg/mL). It was observed from Fig. 9 that hemolysis for PCL MPs and MTX-PCL MPs were below 5% and under the hemocompatibility criteria [49–52]. But in the case of Triton X treated RBCs the 100% hemolysis occurred. Previously studies have also reported that PCL polymer is completely hemocompatible [51,52,54,71,72]. According to the reported studies, MTX is a non-hemolytic drug but, after loading into the chitosan nanoparticles it induces hemolysis, and the same property of MTX has also been reported with liposomes [70,73].But here MTX–PCL MPs was found to be hemocompatible and this may be because the encapsulated MTX did not alter the physiochemical properties PCL polymer. It was also clearly observed from
3.9. Biocompatibility assay To achieve the biomedical applications of the developed PCL microspheres, they must be non-toxic in nature. In order to evaluate the biocompatibility of developed PCL MPs and MTX-PCL MPs, the L929 (Mouse fibroblast) cells were treated with different concentrations (0.5 mg/mL, 1 mg/mL, 2.5 mg/mL, 5 mg/mL) of both the microparticles for 24 h. The % cells viability analysis is presented in Fig. 8. The cell viability of cells treated with PCL MPs and MTX-PCL MPs were found to be > 90% and > 80% respectively. The decrease in the viability of cells treated with MTX-PCL MPs may be due to the cytotoxic 547
Materials Science & Engineering C 81 (2017) 542–550
M. Dhanka et al.
Fig. 9. Hemolysis study for blank and MTX loaded PCL microspheres for 3 h, Negative control (NC), positive control (PC), PCL MPs (B), MTX-PCL MPs (M).
Fig. 10. ESEM images of RBCs after 3 h treatment with different concentrations of blank and MTX loaded PCL microspheres, (a and b) negative control (NC) (c and d) 0.5 mg/mL PCL MPs (e and f) 1.0 mg/mL PCL MPs (g and h) 2.5 mg/mL PCL MPs (i and j) 5 mg/mL PCL MPs (k and l) 0.5 mg/mL MTX-PCL MPs (m and n) 1 mg/mL MTX-PCL MPs (o and p) 2.5 mg/mL MTXPCL MPs (q and r) 5 mg/mL MTX-PCL MPs.
microspheres showed the good encapsulation efficiency and loading capacity. The physicochemical characterizations of microspheres showed the absence of physicochemical modifications, and the drug molecules were found to be in dispersed form inside the polymeric matrix. PCL did not lose its crystallinity like MTX after being processed into microspheres. The controlled and prolonged MTX release from PCL microspheres makes them more suitable for in vivo application. The in vitro biocompatibility studies on cells L929 depicts that the material is cytocompatible and is appropriate for in vivo injection. The hemocompatibility of microspheres studies also showed no hemolytic property as well as no change in biconcave shape of RBCs. Therefore, the biocompatible and hemocompatible nature of these microspheres makes them suitable for in vivo applications in the future for treatment of rheumatoid arthritis.
photographic images (Fig. 8 B) that the microsphere-treated RBCs were undamaged, and sedimented in the bottom like negative control (RBCs in PBS), but positive control showed the leakage of hemoglobin into PBS. The further confirmation of hemolysis was done by E-SEM imaging of treated and non-treated RBCs shown in Fig. 10. The ESEM images of blank (Fig. 10, a, d, c, d) and MTX-PCL MPs (Fig. 10, e, f, g, h) showed the intact and biconcave morphology of RBCs like negative control (Fig. 10, i). Therefore, it was concluded that treatment of RBCs with developed PCL MPs and MTX-PCL MPs did not affect the morphology of RBCs after 3 h treatment. These microspheres were found to be completely hemocompatible and thus can be used for in vivo drug delivery as a localized injectable system. 4. Conclusion
Acknowledgments
PCL polymer-based microspheres have been developed and characterized for the delivery of different active pharmaceutical ingredients. Here, we have developed PCL MPs, and MTX-PCL MPs by a single solvent evaporation method. The prepared microspheres were found in a spherical shape with smooth surface morphology. The PCL
Authors acknowledge the Sun Pharmaceutical Industries Ltd. (Gujarat, India) for providing the Methotrexate drug as gift sample for our research work. Mr. Dhanka acknowledges Ministry of Human 548
Materials Science & Engineering C 81 (2017) 542–550
M. Dhanka et al.
Resources and Development (MHRD), India for providing financial support. Authors acknowledge Sophisticated Analytical Instrumentation Facility (SAIF), Industrial Research Consultancy Centre (IRCC), Indian Institute of Technology Bombay (IITB), for providing central instrumentation facility.
ejpb.2009.06.009. [23] L.S. Liang, J. Jackson, W. Min, V. Risovic, K.M. Wasan, H.M. Burt, Methotrexate loaded poly(L-lactic acid) microspheres for intra-articular delivery of methotrexate to the joint, J. Pharm. Sci. 93 (2004) 943–956, http://dx.doi.org/10.1002/jps. 20031. [24] V. Natarajan, P. Saravanakumar, B. Madhan, Collagen adsorption on quercetin loaded polycaprolactone microspheres: approach for “stealth” implant, Int. J. Biol. Macromol. 50 (2012) 1091–1094, http://dx.doi.org/10.1016/j.ijbiomac.2012.03. 003. [25] C. Zhang, G. Li, Y. Wang, F. Cui, J. Zhang, Q. Huang, Preparation and characterization of 5-fluorouracil-loaded PLLA-PEG/PEG nanoparticles by a novel supercritical CO2 technique, Int. J. Pharm. 436 (2012) 272–281, http://dx.doi.org/10. 1016/j.ijpharm.2012.06.022. [26] B. Sayin, S. Çalifi, Influece of accelerated storage conditions on the stability of vancomycin-loaded poly (D,L-lactide-co-glycolide) microspheres, J. Pharma.Sci. 29 (2005) 111–116. [27] G.C. Bazzo, A.T. De Macedo, J.P. Crenca, V.E. Silva, E.M. Pereira, M. Zétola, B.R. Pezzini, Microspheres prepared with biodegradable PHBV and PLA polymers as prolonged-release system for ibuprofen: in vitro drug release and in vivo evaluation, Brazilian J. Pharm. Sci. 48 (2012) 773–780. [28] N. Butoescu, O. Jordan, P. Burdet, P. Stadelmann, A. Petri-fink, H. Hofmann, E. Doelker, Dexamethasone-containing biodegradable superparamagnetic microparticles for intra-articular administration: physicochemical and magnetic properties, in vitro and in vivo drug release, Eur. J. Pharm. Biopharm. 72 (2009) 529–538, http://dx.doi.org/10.1016/j.ejpb.2009.03.003. [29] R.K. Averineni, G.V. Shavi, A.K. Gurram, P.B. Deshpande, K. Arumugam, N. Maliyakkal, S.R. Meka, U. Nayanabhirama, PLGA 50: 50 nanoparticles of paclitaxel: development, in vitro anti-tumor activity in BT-549 cells and in vivo evaluation, Bull. Mater. Sci. 35 (2012) 319–326, http://dx.doi.org/10.1007/ s12034-012-0313-7. [30] S. Freitas, H.P. Merkle, B. Gander, Microencapsulation by solvent extraction/evaporation: reviewing the state of the art of microsphere preparation process technology, J. Control. Release 102 (2005) 313–332, http://dx.doi.org/10.1016/j. jconrel.2004.10.015. [31] V.R. Sinha, K. Bansal, R. Kaushik, R. Kumria, A. Trehan, Poly-ɛ-caprolactone microspheres and nanospheres: an overview, Int. J. Pharm. 278 (2004) 1–23, http:// dx.doi.org/10.1016/j.ijpharm.2004.01.044. [32] T.K. Dash, V.B. Konkimalla, Poly-??-caprolactone based formulations for drug delivery and tissue engineering: a review, J. Control. Release 158 (2012) 15–33, http://dx.doi.org/10.1016/j.jconrel.2011.09.064. [33] M.A. Woodruff, D.W. Hutmacher, The return of a forgotten polymer—polycaprolactone in the 21st century, Prog. Polym. Sci. 35 (2010) 1217-125, http://dx.doi.org/10.1016/j.progpolymsci.2010.04.002. [34] P. Arunkumar, S. Indulekha, S. Vijayalakshmi, R. Srivastava, Synthesis, characterizations, in vitro and in vivo evaluation of etoricoxib-loaded poly (caprolactone) microparticles — a potential intra-articular drug delivery system for the treatment of osteoarthritis — a potential intra-articular drug deliverys, J. Biomater. Sci. Polym. Ed. 27 (2016) 303–316, http://dx.doi.org/10.1080/09205063.2015. 1125564. [35] M. Dasaratha, N. Sathyamoorthy, Preparation of poly (epsilon-caprolactone) microspheres containing etoposide by solvent evaporation method, Asian J. Pharm. Sci. 5 (2010) 114–122. [36] Y. Zhang, Y. Zhang, S. Guo, W. Huang, Tyrosine kinase inhibitor loaded PCL microspheres prepared by S/O/W technique using ethanol as pretreatment agent, Int. J. Pharm. 369 (2009) 19–23, http://dx.doi.org/10.1016/j.ijpharm.2008.10.032. [37] A. Chen, T. Dang, S. Wang, N. Tang, Y. Liu, W. Wu, The in vitro and in vivo antitumor effects of MTX-Fe3O4-PLLA-PEG-PLLA microspheres prepared by suspensionenhanced dispersion by supercritical CO2, Sci. China Life Sci. 57 (2014) 698–709, http://dx.doi.org/10.1007/s11427-014-4680-8. [38] J.M. Kremer, Methotrexate pharmacogenomics, Ann. Rheum. Dis. 65 (2006) 1121–1123, http://dx.doi.org/10.1136/ard.2006.051789. [39] J.S. Jerzy Swierkot, Methotrexate in rheumatoid arthritis, Inst. Pharmacol. Polish Acad. Sci. 58 (2006) 473–492. [40] Z.A. Khan, R. Tripathi, B. Mishra, Methotrexate: a detailed review on drug delivery and clinical aspects, Expert Opin. Drug Deliv. 9 (2012) 151–169, http://dx.doi.org/ 10.1517/17425247.2012.642362. [41] S.M. Lee, H.J. Kim, Y.J. Ha, Y.N. Park, S.K. Lee, Y.B. Park, K.H. Yoo, Targeted chemo-photothermal treatments of rheumatoid arthritis using gold half-shell multifunctional nanoparticles, ACS Nano 7 (2013) 50–57, http://dx.doi.org/10.1021/ nn301215q. [42] J.R. O'Dell, Methotrexate use in rheumatoid arthritis, Rheum. Dis. Clin. N. Am. 23 (1997) 779–796, http://dx.doi.org/10.1016/S0889-857X(05)70360-4. [43] J. Albuquerque, C.C. Moura, B. Sarmento, S. Reis, Solid lipid nanoparticles: a potential multifunctional approach towards rheumatoid arthritis theranostics, Molecules 20 (2015) 11103–11118, http://dx.doi.org/10.3390/ molecules200611103. [44] P. Prabhu, R. Shetty, M. Koland, K. Vijayanarayana, K.K. Vijayalakshmi, M.H. Nairy, G.S. Nisha, Investigation of nano lipid vesicles of methotrexate for antirheumatoid activity, Int. J. Nanomedicine 7 (2012) 177–186, http://dx.doi.org/10. 2147/IJN.S25310. [45] S.M. Wigginton, B.C. Chu, M.H. Weisman, S.B. Howell, Methotrexate pharmacokinetics after intraarticular injection in patients with rheumatoid arthritis, Arthritis Rheum. 23 (1980) 119–122, http://dx.doi.org/10.1002/art.1780230121. [46] S.H.R. Edwards, Intra-articular drug delivery: the challenge to extend drug residence time within the joint, Vet. J. 190 (2011) 15–21, http://dx.doi.org/10.1016/ j.tvjl.2010.09.019.
Disclosure statement Authors declare no conflict of interest. References [1] D. Kai, S.S. Liow, X.J. Loh, Biodegradable polymers for electrospinning: towards biomedical applications, Mater. Sci. Eng. C 45 (2015) 659–670, http://dx.doi.org/ 10.1016/j.msec.2014.04.051. [2] Z. Li, X.J. Loh, Water soluble polyhydroxyalkanoates: future materials for therapeutic applications, Chem. Soc. Rev. 44 (2015) 2865–2879, http://dx.doi.org/10. 1039/c5cs00089k. [3] H. Ye, A.A. Karim, X.J. Loh, Current treatment options and drug delivery systems as potential therapeutic agents for ovarian cancer: a review, Mater Sci Eng C Mater Biol Appl 45 (2014) 609–619, http://dx.doi.org/10.1016/j.msec.2014.06.002. [4] H. Ye, C. Owh, S. Jiang, C.Z.Q. Ng, D. Wirawan, X.J. Loh, A thixotropic polyglycerol sebacate-based supramolecular hydrogel as an injectable drug delivery matrix, Polymers (Basel) 8 (2016), http://dx.doi.org/10.3390/polym8040130. [5] Q.Q. Dou, S.S. Liow, E. Ye, R. Lakshminarayanan, X.J. Loh, Biodegradable thermogelling polymers: working towards clinical applications, Adv. Healthc. Mater. 3 (2014) 977–988, http://dx.doi.org/10.1002/adhm.201300627. [6] J. Li, X.J. Loh, Cyclodextrin-based supramolecular architectures: syntheses, structures, and applications for drug and gene delivery, Adv. Drug Deliv. Rev. 60 (2008) 1000–1017, http://dx.doi.org/10.1016/j.addr.2008.02.011. [7] X.J. Loh, J. Li, Biodegradable thermosensitive copolymer hydrogels for drug delivery, Expert Opin. Ther. Pat. 17 (2007) 965–978, http://dx.doi.org/10.1517/ 13543776.17.8.965. [8] C.O. Ghislaine Barouti, Sing Shy Liow, Qingqing Dou, Hongye Ye, X.J.L. Sophie, M. Guillaume, New linear and star-shaped thermogelling poly([R]-3-hydroxybutyrate) copolymers ghislaine, Chem. Eur. J. 22 (2016) 10501–10512, http:// dx.doi.org/10.1002/chem.201601404. [9] J. Pushpamalar, A.K. Veeramachineni, C. Owh, X.J. Loh, Biodegradable polysaccharides for controlled drug delivery, Chem. Aust. 81 (2016) 504–514, http:// dx.doi.org/10.1002/cplu.201600112. [10] L. Jiang, C.R.R. Gan, J. Gao, X.J. Loh, A perspective on the trends and challenges facing porphyrin-based anti-microbial materials, Small 12 (2016) 3609–3644, http://dx.doi.org/10.1002/smll.201600327. [11] Y. Xiang, N.N.L. Oo, J.P. Lee, Z. Li, X.J. Loh, Recent development of synthetic nonviral systems for sustained gene delivery, Drug Discov. Today 0 (2017), http:// dx.doi.org/10.1016/j.drudis.2017.04.001. [12] X.J. Loh, S.J. Ong, Y.T. Tung, H.T. Choo, Co-delivery of drug and DNA from cationic dual-responsive micelles derived from poly(DMAEMA-co-PPGMA), Mater. Sci. Eng. C 33 (2013) 4545–4550, http://dx.doi.org/10.1016/j.msec.2013.07.011. [13] X.J. Loh, T.C. Lee, Q. Dou, G.R. Deen, Utilising inorganic nanocarriers for gene delivery, Biomater. Sci. 4 (2016) 70–86, http://dx.doi.org/10.1039/c5bm00277j. [14] Z. Li, E. Ye, David, R. Lakshminarayanan, X.J. Loh, Recent advances of using hybrid nanocarriers in remotely controlled therapeutic delivery, Small 12 (2016) 4782–4806, http://dx.doi.org/10.1002/smll.201601129. [15] V. Tran, J. Benoît, M. Venier-julienne, Why and how to prepare biodegradable, monodispersed, polymeric microparticles in the field of pharmacy? Int. J. Pharm. 407 (2011) 1–11, http://dx.doi.org/10.1016/j.ijpharm.2011.01.027. [16] E. Campos, J. Branquinho, A.S. Carreira, A. Carvalho, P. Coimbra, P. Ferreira, M.H. Gil, Designing polymeric microparticles for biomedical and industrial applications, Eur. Polym. J. 49 (2016) 2005–2021, http://dx.doi.org/10.1016/j. eurpolymj.2013.04.033. [17] R.A. Jain, The manufacturing techniques of various drug loaded biodegradable poly (lactide-co-glycolide) (PLGA) devices, Biomaterials 21 (2000) 2475–2490, http:// dx.doi.org/10.1016/S0142-9612(00)00115-0. [18] F. Chen, G. Yin, X. Liao, Y. Yang, Z. Huang, J. Gu, Y. Yao, X. Chen, H. Gao, Preparation, characterization and in vitro release properties of morphine-loaded PLLA-PEG-PLLA microparticles via solution enhanced dispersion by supercritical fluids, J. Mater. Sci. Mater. Med. 24 (2013) 1693–1705, http://dx.doi.org/10. 1007/s10856-013-4926-1. [19] D.R. Chen, J.Z. Bei, S.G. Wang, Polycaprolactone microparticles and their biodegradation, Polym. Degrad. Stab. 67 (2000) 455–459, http://dx.doi.org/10.1016/ S0141-3910(99)00145-7. [20] D. Pepic, M.S. Nikolic, S. Grujic, M. Lausevic, J. Djonlagic, Release behaviour of carbamazepine-loaded poly (″-caprolactone)/poly(ethylene oxide) microspheres, 30 (2013) 151–160. http://dx.doi.org/10.3109/02652048.2012.704954. [21] V. Natarajan, N. Krithica, B. Madhan, P.K. Sehgal, Formulation and evaluation of quercetin polycaprolactone microspheres for the treatment of rheumatoid arthritis, J. Pharm. Sci. 100 (2011) 195–205, http://dx.doi.org/10.1002/jps.22266 ABSTRACT. [22] N. Butoescu, O. Jordan, E. Doelker, Intra-articular drug delivery systems for the treatment of rheumatic diseases: a review of the factors influencing their performance, Eur. J. Pharm. Biopharm. 73 (2009) 205–218, http://dx.doi.org/10.1016/j.
549
Materials Science & Engineering C 81 (2017) 542–550
M. Dhanka et al.
10.1016/j.jdent.2006.03.002. [61] S. Jana, M.K. Trivedi, R.M. Tallapragada, A. Branton, D. Trivedi, G. Nayak, R. Kumar, Pharmaceutica characterization of physicochemical and thermal properties of chitosan and sodium alginate after biofield treatment, Pharm. Anal. Acta. 6 (2015) 1–9, http://dx.doi.org/10.4172/21532435.1000430. [62] S. Wang, S. Guo, L. Cheng, Disodium norcantharidate loaded poly(??-caprolactone) microspheres. I. Preparation and evaluation, Int. J. Pharm. 350 (2008) 130–137, http://dx.doi.org/10.1016/j.ijpharm.2007.08.030. [63] J. Jingou, H. Shilei, L. Weiqi, W. Danjun, W. Tengfei, X. Yi, Preparation, characterization of hydrophilic and hydrophobic drug in combine loaded chitosan/cyclodextrin nanoparticles and in vitro release study, Colloids Surf. B: Biointerfaces 83 (2011) 103–107, http://dx.doi.org/10.1016/j.colsurfb.2010.11.005. [64] Y. Huang, J. Liu, Y. Cui, H. Li, Y. Sun, Y. Fan, X. Zhang, Reduction-triggered breakable micelles of amphiphilic polyamide amine-G-polyethylene glycol for methotrexate delivery, Biomed. Res. Int. 2014 (2014), http://dx.doi.org/10.1155/ 2014/904634. [65] J.C. Jeong, J. Lee, K. Cho, Effects of crystalline microstructure on drug release behavior of poly(ε-caprolactone) microspheres, J. Control. Release 92 (2003) 249–258, http://dx.doi.org/10.1016/S0168-3659(03)00367-5. [66] S. Ray, M. Joy, B. Sa, pH dependent chemical stability and release of methotrexate from a novel nanoceramic carrier, RSC Adv. 5 (2015) 39482–39494, http://dx.doi. org/10.1039/C5RA03546E. [67] A.Z. Chen, G.Y. Wang, S. Bin Wang, L. Li, Y.G. Liu, C. Zhao, Formation of methotrexate-PLLA-PEG-PLLA composite microspheres by microencapsulation through a process of suspension-enhanced dispersion by supercritical CO2, Int. J. Nanomedicine 7 (2012) 3013–3022, http://dx.doi.org/10.2147/IJN.S32662. [68] S.A. Dhanaraj, S. Muralidharan, V. Venugopal, P. Kanniappan, Formulation and evaluation of chitosan nanospheres containing methotrexate targeted drug delivery system, J. Young Pharm. 8 (2016) 330–334, http://dx.doi.org/10.5530/jyp.2016. 4.7. [69] A. de Fatima Pereira, V. Mara da Costa, M. Cristina Magalhaes Santos, F. Carmo Horta Pinto, G. Rodrigues Da Silva, Evaluation of the effects of methotrexate released from polymeric implants in solid Ehrlich tumor, Biomed Pharmacother 68 (2014) 365–368, http://dx.doi.org/10.1016/j.biopha.2013.12.012. [70] D.R. Nogueira, L. Tavano, M. Mitjans, L. Pérez, M.R. Infante, M.P. Vinardell, In vitro antitumor activity of methotrexate via pH-sensitive chitosan nanoparticles, Biomaterials 34 (2013) 2758–2772, http://dx.doi.org/10.1016/j.biomaterials. 2013.01.005. [71] H. Jiang, X.B. Wang, C.Y. Li, J.S. Li, F.J. Xu, C. Mao, W.T. Yang, J. Shen, Improvement of hemocompatibility of polycaprolactone film surfaces with zwitterionic polymer brushes, Langmuir 27 (2011) 11575–11581, http://dx.doi.org/10. 1021/la202101q. [72] C. Besteiro, A.J. Guiomar, C.A. Gonc, M.N. De Pinho, M.H. Gil, Characterization and in vitro hemocompatibility of bi-soft segment, polycaprolactone-based poly(ester urethane urea) membranes, J. Biomed. Mater. Res. Part A. (2009), http://dx.doi. org/10.1002/jbm.a.32594. [73] N.R. Kuznetsova, C. Sevrin, D. Lespineux, N.V. Bovin, E.L. Vodovozova, T. Meszaros, J. Szebeni, C. Grandfils, Hemocompatibility of liposomes loaded with lipophilic prodrugs of methotrexate and melphalan in the lipid bilayer, J. Control. Release 160 (2012) 394–400, http://dx.doi.org/10.1016/j.jconrel.2011.12.010.
[47] A. Trapani, N. Denora, G. Iacobellis, J. Sitterberg, U. Bakowsky, T. Kissel, Methotrexate-loaded chitosan- and glycolchitosan-based nanoparticles: a promising strategy for the administration of the anticancer drug to brain tumors, AAPS Pharm. Sci. Tech. 12 (2011) 1302–1311, http://dx.doi.org/10.1208/s12249-011-9695-x. [48] D. Seo, Y. Jeong, D. Kim, M. Jang, M. Jang, J. Nah, Colloids and surfaces B: biointerfaces methotrexate-incorporated polymeric nanoparticles of methoxy poly (ethylene glycol)-grafted chitosan, Colloids Surf. B: Biointerfaces 69 (2009) 157–163, http://dx.doi.org/10.1016/j.colsurfb.2008.10.020. [49] T. Muthukumar, S. Prabhavathi, M. Chamundeeswari, T.P. Sastry, Bio-modified carbon nanoparticles loaded with methotrexate possible carrier for anticancer drug delivery, Mater. Sci. Eng. C 36 (2014) 14–19, http://dx.doi.org/10.1016/j.msec. 2013.11.046. [50] M. Notara, C.A. Scotchford, D.M. Grant, N. Weston, G.A.F. Roberts, Cytocompatibility and hemocompatibility of a novel chitosan-alginate gel system, J. Biomed. Mater. Res. A 89 (2008) 854–864, http://dx.doi.org/10.1002/jbm.a. 32027. [51] A.R. Padalhin, N. Thuy, B. Linh, Y.K. Min, B. Lee, A.R. Padalhin, N. Thuy, B. Linh, Y.K. Min, Evaluation of the cytocompatibility hemocompatibility in vivo bone tissue regenerating capability of different PCL blends, J. Biomater. Sci. Polym. Ed. 25 (2014) 487–503, http://dx.doi.org/10.1080/09205063.2013.878870. [52] A.K. Jaiswal, H. Chhabra, S.S. Kadam, K. Londhe, V.P. Soni, J.R. Bellare, Hardystonite improves biocompatibility and strength of electrospun polycaprolactone nano fi bers over hydroxyapatite: a comparative study, Mater. Sci. Eng. C 33 (2013) 2926–2936, http://dx.doi.org/10.1016/j.msec.2013.03.020. [53] R. Yoganathan, R. Mammucari, N.R. Foster, Impregnation of ibuprofen into Polycaprolactone using supercritical carbon dioxide, J. Phys. Conf. Ser. 12087 (2010) 1–6, http://dx.doi.org/10.1088/1742-6596/215/1/012087. [54] L. Chen, T. Shi, X. Xia, J. Miao, C. Mao, B. Huang, J. Shen, Mild anticoagulant effect of heparin-loaded Polycaprolactone microspheres, J. Nanosci. Nanotechnol. 15 (2015) 144–150, http://dx.doi.org/10.1166/jnn.2015.8772. [55] C. Guo, L. Zhou, J. Lv, Effects of expandable graphite and modified ammonium polyphosphate on the flame-retardant and mechanical properties of wood flourpolypropylene composites, Polym. Polym. Compos. 21 (2013) 449–456, http://dx. doi.org/10.1002/app. [56] T. Elzein, M. Nasser-Eddine, C. Delaite, S. Bistac, P. Dumas, FTIR study of polycaprolactone chain organization at interfaces, J. Colloid Interface Sci. 273 (2004) 381–387, http://dx.doi.org/10.1016/j.jcis.2004.02.001. [57] M. Stevanovic, A. Radulovic, V. Pavlovic, Facile synthesis of poly(e-caprolactone) micro and nanospheres using different types of polyelectrolytes as stabilizers under ambient and elevated temperature, Compos. Part B (2012), http://dx.doi.org/10. 1016/j.compositesb.2012.07.008. [58] U.O.H. Yi Zhong, Victor Leung, Lynn Yuqin Wan, Silvio Dutz, Frank K. Ko, Electrospun magnetic nanofibre mats — a new bondable biomaterial using remotely activated magnetic heating, J. Magn. Magn. Mater. 380 (2015) 330–334, http://dx. doi.org/10.1016/j.jmmm.2014.09.069. [59] M.F. Canbolat, A. Celebioglu, T. Uyar, Drug delivery system based on cyclodextrinnaproxen inclusion complex incorporated in electrospun polycaprolactone nanofibers, Colloids Surf. B: Biointerfaces 115 (2014) 15–21, http://dx.doi.org/10.1016/j. colsurfb.2013.11.021. [60] A. Elzubair, C. Nelson, M. Suarez, The physical characterization of a thermoplastic polymer for endodontic obturation, J. Dent. 34 (2006) 784–789, http://dx.doi.org/
550
