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| Year : 2010 | Volume
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| Issue : 3 | Page : 205-211 |
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| Preparation and in vitro characterization of paclitaxel-loaded injectable microspheres |
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Jagadeesh G Hiremath1, V Kusum Devi2
1 Department of Pharmaceutics, East West College of Pharmacy, Hosur Road, Karnataka, Bangalore, India
2 Department of Pharmaceutics, Al-Ameen College of Pharmacy, Hosur Road, Karnataka, Bangalore, India
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| Date of Web Publication | 26-Oct-2010 |
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 Abstract | | |
The main objective of this study was to develop paclitaxel loaded poly (caprolactone) injectable microspheres prepared by solvent evaporation method. Mircoparticles were characterized in terms of particle size and size distribution, surface morphology, drug physical state, and crystalline nature by using master size analyzer, scanning electron microscope, differential scanning calorimetry, and X-ray diffraction. Paclitaxel loading over different concentrations was analyzed by high-performance liquid chromatography. In vitro drug release studies were performed in phosphate buffer saline. Best formulation was selected for in vitro cytotoxic studies by using MCF-7 breast cancer cell lines. Keywords: Cancer, injectable, microspheres, paclitaxel
How to cite this article:
Hiremath JG, Devi V K. Preparation and in vitro characterization of paclitaxel-loaded injectable microspheres. Asian J Pharm 2010;4:205-11 |
| Introduction | |  |
Paclitaxel, the first of new class of microtubule stabilizing agent, has been hailed by National Cancer Institute (NCI) as the most significant advance in chemotherapy of the past 15-20 years. Paclitaxel has shown to be effective for the treatment of different types of tumors, including ovarian, breast, and lung cancer. [1],[2],[3] One of the major challenges when developing paclitaxel formulations is the very low water solubility of the drug 0.6 mM. [4],[5] Moreover, common approaches to improve solubility like addition of charged complexing agents or by producing alternate salts of the drug are not feasible in the case of paclitaxel. [6] However, severe side effects (in particularly hyper sensitivity reactions) can be caused by Cremophor® EL. [7] The objectives of the present study were to prepare drug-loaded and drug-free, PCL-based microspheres and in vitro characterization of prepared injectable microspheres.
| Materials and Methods | | |
Poly(caprolactone) (PCL) (Mn 90,000) was purchased from Sigma Aldrich, Bangalore, India. Paclitaxel was obtained as gift samples from Indena Spa, Milano, Itlay. Sodium chloride, sodium dihydrogen orthophosphate and potassium dihydrogen orthophosphate and poly(vinyl alcohol) (PVA) were purchased from SD Fine Chemicals, Bangalore, India. All HPLC and analytical grade solvents were purchased from Ranbaxy Chemicals, Bangalore, India.
Methods
Preparation of poly (caprolactone) microspheres by solvent evaporation method
Paclitaxel-loaded PCL microspheres were prepared by solvent evaporation method. PCL was dissolved in dichloromethane separately after which paclitaxel was added to the solution. The organic phase was slowly added drop wise (0.8 ml/min) into the external aqueous phase containing 0.1% polyvinyl alcohol (50 ml) and stirred at 1400±5 rpm (mechanical stirrer) using a stainless steel propeller having 16 mm diameter. The resulting emulsion was stirred till dichloromethane evaporated (approximately 3-4 h). The microspheres were collected by filtration using 0.22 μm (Millipore, Bangalore, India.) nylon filters, washed three times with deionized water (Millipore, Bangalore, India) and resuspended into 5 ml of deionized water, frozen in liquid nitrogen and lyophilized. Drug-free microparticles were prepared in the manner without adding paclitaxel. The quantity of polymer and drug used for the preparation of microspheres are illustrated in [Table 1]. | Table 1 :Formulation parameters and characteristics of the investigated paclitaxel PCL microspheres
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Instrument and chromatographic conditions
The HPLC system consists of a Shimadzu SPD-10ATVP, binary pump equipped with a normal sample injector with a 50 μL loop, SPD-10AVP variable wavelength UV detector, and Spincotech station for data analysis. Chromatographic separations were achieved using a Phenomenex C-18 column, (4.6 Χ 250 mm, 5 μm) and Phenomenex C-18 guard column cartridge (KJ0-4282, 4.0 Χ 3.0 mm, 5 μm). The mobile phase, consisting for the estimation of paclitaxel in bulk and formulation, was acetonitrile:water (60:40% v/v) passed through a 0.22 μm membrane filter and degassed by ultrasonication under vacuum before use. The analysis was performed at the flow rate of 1.3 ml min -1 with UV detector at 227 nm and the sensitivity was 0.02, absorbance unit force per second (AUFS).
Drug entrapment in microspheres
The encapsulation efficiency is defined as the ratio of amount of encapsulated drug to that of the drug used for the microsphere preparation.
To determine the paclitaxel content in the microspheres followed by previously reported method [8] 10 mg of paclitaxel-loaded microspheres were dissolved in 5ml of DCM. 10 ml of a mixture containing acetonitrile and water (60:40, v/v) was then added. Nitrogen gas stream was introduced to evaporate DCM until a clear solution was obtained and filtered through 0.22 μ membrane. The solution was suitably diluted and analyzed by HPLC as described for determination of paclitaxel concentration. In order to account for the drug, which could be lost throughout the above procedure, the recovery efficiency of the procedure was determined by dissolving a known quantity of paclitaxel in DCM and subjecting it to the same procedure as described above.
Characterization
Surface morphology
The shape and surface characteristics of microspheres before were observed by scanning electron microscopy. Immediately after manufacturing, blank and drug-loaded microspheres were subjected to surface morphology studies using a scanning electron microscope (SEM). The microspheres were first dried under vacuum. Samples were glued to aluminum sample holders (Indian Institute of Science, Bangalore, India) and gold coated under argon atmosphere. The coated samples were finally analyzed using JSM 840. The surface morphology of microspheres was observed under suitable magnification.
Particle size analysis
In order to analyze particle size, blank and drug-loaded lyophilized (SZY10, Bioasset Technologies, Pvt, Ltd, India) microspheres were dispersed in deionzed water, vortexed for 10 min, and sonicated for 5 min before sampling. Particle size was determined by laser scattering light (Malvern Laser Analyzer Instruments, Strides Arco Lab, Bangalore, India). Polydispersity was determined according to a previously a reported method. [9] In briefly, polydispersity was determined according to the equation below:
where D (0.9) corresponds to particle size immediately above 90% of the sample. D (0.5) corresponds to particle size immediately above 50% of the sample. D (0.1) corresponds to particle size immediately above 10% of the sample. Microsphere size and polydispersity were determined. Each sample was measured in triplicate.
Thermal studies
Differential scanning calorimetry (DSC) was conducted using Mettler Toledo Star system, (Indian Institute of Science, Bangalore, India). Samples were weighed (5.00-8.00±0.5mg) and placed in sealed aluminum pans. The coolant was liquid nitrogen. The samples were scanned at 10ºC/min from 20ºC to 230ºC. The DSC thermograms of pure paclitaxel, pure PCL, physical mixtures of paclitaxel and PCL, and paclitaxel-loaded microspheres were obtained.
X-ray diffraction studies
The X-ray diffraction (XRD) patterns of paclitaxel drug-loaded PCL microspheres were determined using a diffractometer equipped with a rotating target X-ray tube and a wide-angle goniometer (Indian Institute of Science, Bangalore, India). The X-ray source was Ka radiation from a copper target with a graphite monochromater. The X-ray tube was operated at a potential of 50 kV and a current of 150 mA. The range (2q) of scans was from 0 to 70º and the scan speed was 2º per minute at increments of 0.02º. The XRD patterns of pure paclitaxel, pure PCL, physical mixtures of paclitaxel and PCL, and paclitaxel-loaded microspheres were obtained.
In vitro drug release studies
In vitro release studies of paclitaxel-loaded microspheres were carried out at 37±2ºC at pH 7.4 phosphate buffer saline for a period of 30 days using an apparatus was indigenously designed and fabricated to conduct in vitro release studies [Figure 1]. [10] The screw capped bottles containing paclitaxel-loaded microspheres in 25 ml of phosphate buffer saline of pH. 7.4 as release medium were fixed to stainless steel holders attached to a mechanical stirrer and the platform was immersed in water maintained at 37±2ºC. The platform was rotated at an average speed of 100 rpm to induce mixing in the release medium. At periodic intervals, initially at 24 h and then followed by every 2 days, 5 ml of the release medium was sampled and 5 ml of fresh release medium was replaced to provide the necessary sink condition. Paclitaxel extracted with DCM drug content was determined by the HPLC method. In order to determine the order of drug release, drug release profile of all the formulations evaluated were fitted to zero-order, first-order, Higuchi, and Krosmeyer Peppas models. | Figure 1 :Scanning electron micrograph: (a) blank PCL microspheres, (b) paclitaxel-loaded PCL microspheres (Formulation F3). Original magnification of the electron micrograph was 1000× and scale bar represents a distance of 10 μm
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In vitro cytotoxicity studies by MCF-07 breast cancer cell-lines
Human breast carcinoma MCF-7 (procured from National Cell Science Centre, Pune, India) cells were grown in monolayer in Dulbecco's modified Eagle's medium (DMEM). The cell lines were cultured in media supplemented with 10% heat-inactivated fetal bovine serum and antibiotics at 37±1ºC in a humidified atmosphere containing 5% carbon dioxide (CO 2 ). MCF-7 cells (100μL) at a density of 4 Χ 10 4 cells/ml growing cells were seeded in 96-well plates in the complete growth culture medium. After culturing for 12h they were treated for 48 h with various concentrations of paclitaxel 10 to 200 ng/ml. The prepared formulation F3 microspheres were subjected for cell lines studies. The microspheres containing 100, 250, 500, and 1000 μg/ml of paclitaxel-loaded and blank microspheres (control) 1.0 mg/ml were transferred with 100 μL of medium. After predetermined incubation periods, a 20 μL MTT solution was added to each well. After 48 h of incubation at 37±1ºC, the medium was removed and any formazan crystals formed were solubilized with DMSO. After slow shaking for 5 min, the absorbance of each well was determined at 540 nm using a microplate reader (EL310, Bio-Tek Instruments Inc., Winooski, VT, Bangalore, India). All the experiments were performed in triplicate. Cell viability was assessed by the MTT assay. This assay depends on the cellular reductive capacity to metabolize the yellow tetra zolium salt, (3- [4,5-dimethylthiazol-2-yl]-3, 5-diphenyltetrazolium bromide dye (MTT), a highly colored formazan product. Cytotoxicity was expressed as percentage of control. The IC 50 value was defined as the drug concentration required to inhibit the growth by 50% relative to controls. Cell Viability % = N p /N c x100. Where N p and N c are the number of surviving cells in the group treated with paclitaxel-loaded microspheres and in the untreated cell group (pure microspheres) (control), respectively. All experiments were performed in triplicate.
| Results and Discussion | | |
Characterization of microspheres
Three different drug to polymer ratios were used to prepare microspheres. The formulation F3 was found to be best percentage yield; high encapsulation with very less percentage unentraped (free drug) of paclitaxel was obtained. Scanning electron micrograph (A) blank PCL microspheres, (B) paclitaxel loaded PCL microspheres (Formulation F3) shown in [Figure 2]. However, in formulations F1 and F2, the obtained results found that high unentraped (free drug), low percentage yield, and 49-70% encapsulation efficiency, which might be due to the reason that the quantity of polymer used was insufficient to completely cover the drug particles and also the viscosity of PCL solutions depends directly on polymer concentration, fixed concentration of surfactant (PVA acting as surfactant), organic solvent, stirring rate, and temperature. [11] This can be explained in a way that an increase in the polymer concentration produced a significant increase in viscosity, thus leading to an increase in emulsion droplet size and finally to a higher microparticle size. Moreover, higher concentration of polymer in the emulsion droplets leads to an enhancement of the microencapsulation efficiency, because high viscosity of the organic phase tends to restrict partitioning of drug into the external aqueous phase. The results are summarized in [Table 1]. Formulation F3 was selected further for all the physicochemical characterization (SEM, DSC, XRD, and in vitro cytotoxicity) studies due to its promising results. | Figure 2 :Results of particle size analysis by the Malvern size analyzer
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Particle size and polydispersity
The particle size was analysed for PCL-based paclitaxel-loaded microspheres prepared using dichloromethane solvent in different drug to polymer ratios. Average particle size distribution (volume mean diameter) in formulations F1, F2, and F3, was found to be 36.9±15.6, 46.3±28.7 and 29.1±09.9, respectively [Figure 3]. The polydispersity of microsphere was found within the range between 0.57 and 0.64 [Table 1]. It is important because sustained/controlled drug release rate depends upon the polydispersity of the microparticulates. The particle size distribution (mean diameter) and polydispersity variation observed in the drug-loaded PCL microspheres due to four factors are deemed essential in the ultimate determination of microspheres particle size, namely the concentration of polymer in the organic phase, the polarity of the solvents, the internal/external phase ratio, and concentration of surfactant. [12] | Figure 3 :Generated DSC thermogram represents: (a) pure PCL, (b) pure paclitaxel, (c) physical mixture of PCL and paclitaxel, (d) paclitaxelloaded microspheres. The experiment was carried with crimped aluminum pans and a heating rate of 10 °C/min; the samples were scanned at 10 C/ min from 10 °C to 230 °C
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Thermal characterization of PCL microspheres
The DSC technique can provide qualitative and quantitative information about the physicochemical status of drug in microspheres, which is reported to be involved in the endothermic or exothermic process. Using the DSC analysis of drug, polymer materials, and produced microspheres, the nature of the drug inside the polymer matrix can be assessed, which may emerge in solid solution, metastable molecular dispersion, or crystallization. [13],[14] In order to identify the mechanism of sustained drug release, we first characterized the physical state of the drug within the microparticles. Samples were subjected for DSC studies. A sharp and large melting onset/peak/endset peak of pure paclitaxel and physical mixture of drug and polymer at 216.86/224.94/230.14 and 54.92/65.26/70.05/214.96/225.13/231.30ºC are studied. However, the melting peak was absent on the DSC thermograms of microparticles containing paclitaxel, indicating that the drug was dispersed in the microparticles in an amorphous form. This amorphous nature of the drug may have pronounced pharmaceutical significance as it could lead to increased solubility and finally to an improved biological activity. [13],[14] The generated thermograms are shown in [Figure 4]. | Figure 4 :XRD pattern represents: (a) pure PCL, (b) pure paclitaxel, (c) physical mixture of PCL+ paclitaxel, (d) paclitaxel-loaded PCL microspheres. The X-ray tube was operated at a potential of 50 kV and a current of 150 Ma and the range (2è) of scans was from 0 to 70° and the scan speed was 2° per minute at increments of 0.02°
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X-ray diffraction patterns of drug-loaded PCL microspheres
The DSC studies revealed that the drug-loaded microparticles are amorphous in nature. This phenomenon was further confirmed by XRD patterns. The crystal peak of paclitaxel is clearly observed by X-ray data shown in [Figure 5]B and C. However, the diffraction patterns of paclitaxel-loaded microparticles [Figure 5]D did not contain any peaks associated with crystals of the drug, suggesting that the drug was in the amorphous/dissolution state in the polymer matrix. | Figure 5 :Equipment designed and fabricated for drug release studies. Complete unit of dissolution unit (left) and platform holding screw capped bottles rotating at an average speed of 100±4 rpm at 37±2 ºC (right)
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In vitro drug release studies
The release behavior of paclitaxel from PCL microparticles is illustrated in [Figure 6], which indicates sustained pattern for upto 30 days. At the initial stage, PCL-based microspheres' small burst effect reason is the release of some drug loosely bound on the surface of the microspheres. This loosely bound drug would be released by a mechanism of diffusion through the aqueous pores on the surface of the microspheres created by the water uptake by PCL microspheres immediately after being exposed. At the later stage, the drug release was more slow, whose rate is determined by the diffusion of the polymer matrix. This fact can be explained as follows. As the PCL concentration increased, the drug release behavior showed a more sustained pattern. However, the release of drug from PCL microspheres that depends upon the diffusion path must be filled up by water, in other words, PCL causes the delay of water penetration; hence, the diffusion of the drug through the amorphous region into the release medium is retarded, which results in a small burst effect, then the drug is released more slowly once the water is filled by the diffusion pathlength, then the release rate is determined not by the erosion of polymer but by the diffusion of the drug by the amorphous polymeric matrix. This phenomenon can be explained as follows, although PCL is a biodegradable polymer, biodegradation of the PCL is considerably slower than that of other degradable polymers. [11] Therefore, since PCL is hardly degraded during the diffusion process, diffusion is through the polymer is the only possible mechanism of drug release, and other factors can also be considered such as crystalline microstructure and particle size. [11] This initial small burst drug release was later followed by more controlled/sustained release for the 30 days study period and increasing release rate when drug loading was increased was also observed. [15] The percentage cumulative drug release at the end of 30 day period from F1, F2, and F3 formulations was 62.54±1.6, 48.59±2.14, and 30.58 ±1.5, respectively. Microspheres are spherical particles composed of suitable biodegradable polymer; their release profile in vitro helps us to understand the behavior of these systems in terms of drug release, and therefore its efficacy. | Figure 6 :In vitro release of paclitaxel from microspheres. Dissolution studies were carried in PBS buffer (pH 7.4) (n = 6). F1 (), F2 () and F3 ()
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Zero-order and Korsmeyer Peppas model gave a good fit for all drug release profiles of microspheres with greater regression coefficients in comparison to other models. The fitting of these data to the Korsmeyer-Peppas model demonstrated that drug release occurred mainly through diffusion and partially erosion process, since the value of n was between 0.98 and 1.04. But among all the formulations, F3 showed a very good regression coefficient in both zero-order and Korsmeyer Peppas models [Table 2]. Hence, F3 formulation was selected for further in vitro characterization of cytotoxicity by MCF-7 cancer cell line studies.
Cytotoxicity studies by MCF-07 breast cancer cell lines
Inhibitory concentration (IC 50 ) of paclitaxel producing 50% of cell inhibition or dead with various concentrations of paclitaxel compared with control (air). Inhibitory concentration (IC 50 ) of paclitaxel on MCF-7 cells line was 100±2 ng/ml after 48 h, (P< 0.05). [16],[17] The F3 microspheres containing 100, 250, 500, and 1000 μg/ml of paclitaxel showed 79.82±8.6, 61.17±5.77 and 39.95±7.5 and 10.58±2.97% cell viability, when compared to the blank microspheres (without paclitaxel) was 97.52±2.4% after 48 h MTT assay (P< 0.05) one-way analysis of variance ANOVA). The cell line was sensitive to the released drug when it was exposed continuously to paclitaxel for 48h. The reduction rate of cell viability from paclitaxel-loaded F3 microspheres (100, 250, 500, and 1000 μg/ml) could be explained with respect to the dependence on release of paclitaxel from the microspheres (F3). Increasing the paclitaxel concentration in biodegradable F3 microspheres, which results in a proportionate increase in dose, resulted in increased reduction in cell proliferation. As a result of this, we supposed that paclitaxel was released from F3 microspheres sustained and continuously. The maximum efficacy, with reduction in cell proliferation of > 90%, [Figure 8]G and H, was seen with 1000 μg/ml of paclitaxel-loaded microspheres. The cytotoxicity against the MCF-7 cell lines were affected significantly by the released amount of paclitaxel. These studies revealed drug-loaded biodegradable F3 microspheres on MCF-7 cell lines; paclitaxel release from microspheres was sustained efficacy and activity. The results obtained are shown in [Figure 7] and [Figure 8]. | Figure 7 :Different concentrations of paclitaxel treated with MCF-7 cell lines. A: air, B: blank microspheres. Pure paclitaxel treatment of C: 10 ng/ml, D: 50 ng/ml, E: 100 ng/ml. Paclitaxel-loaded microspheres treatment containing different concentrations of F: 150 ng/ml, G: 200 ng/ml, H: 100 ìg/ml I: 250 ìg/ml, J: 500 ìg/ml, K: 1000 ìg/ml
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 | Figure 8 :Cell morphology of MCF-7 breast cancer cell lines treated with control and paclitaxel-loaded PCL microspheres during 48 h: (a) Control (non treated), control after 48 h (b), (c) paclitaxel 100 ng/ml and after 48h (d), (e) Blank microspheres (1000 μg/ml), after 48 h observation (f), (g) F3 microspheres (1000 μg/ml), same sample after 48h observation (h)
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| Conclusion | | |
We have successfully prepared and characterized the PCL-based paclitaxel-loaded injectable microspheres. Among all the three formulations, based on the in vitro characterization formulation F3 was found to be the most promising formulation. It can be concluded that drug-loaded microsphere appears to be a promising treatment modality for cancer (either locally or systemic). However, the continuous release of an anticancer agent from a controlled delivery device for 1 week to 1 month is usually required for effective treatment of the cancer. The obtained results indicated the potential use of microsphere using PCL for sustained release of lipophilic drug such as paclitaxel. Optimization of formulations with different polymers, concentrations, and surfactants and in vivo trial evaluations are may be future subjects.
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Correspondence Address:
Jagadeesh G Hiremath
Department of Pharmaceutics, East West College of Pharmacy, B.E.L. Layout, 63, Magadi Road, Viswaneedam Post, Bangalore - 560 091
India
DOI: 10.4103/0973-8398.72119
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2] |
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| This article has been cited by | | 1 |
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| Hiremath, J.G., Devi, V.K. | | Asian Journal of Pharmaceutics. 2011; 5(1): 9-14 | | [Pubmed] | |
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