Abstract
Poly(lactic-co-glycolic acid) (PLGA) nanospheres loaded with papain were prepared by the emulsion solvent diffusion in water (ESD) and the w/o/w emulsion solvent evaporation (ESE) methods. The nanosphere loaded with papain from the ESE method gave smaller particle sizes (220–232 nm) and higher encapsulation efficiency of about two-folds than those from the ESD method. The morphology of the nanospheres loaded with papain prepared by the ESE method exhibited spherical shape and smooth surface investigated by SEM and TEM. The release profile of papain from the PLGA nanospheres of the ESD and ESE method indicated two phases with an initial rapid phase of 6 h and followed by the slow release phase of 48 h. The unloaded PLGA nanospheres from the two methods did not show any cytotoxicity in human skin fibroblasts, while the unloaded papain gave toxicity more than the loaded papain of 1.5 times. Papain loaded in PLGA nanospheres prepared by the ESE method was more chemical stable than the unloaded papain of eight and three times when kept at 4°C and 25°C for 6 weeks, respectively. The developed stable and low cytotoxic nanosphere loaded with papain can be further developed as topical products.
1. Introduction
Papain, the enzyme from the latex of the papaya fruit, is one of the proteolytic enzymes. It is widely used as meat tenderization and defibrinating agents. This enzyme may have the collagenolytic activity, resulting in the flatten and the reducible size of the collagenous nodules that can be applied for scar treatment. Papain is a macromolecule that consists of 212 amino acid residues, and has the molecular mass of about 23.5 kDa. For topical application, the reduction of transdermal penetration are not only from the large molecule structure of papain, but also the barrier of the stratum corneum (SC) of the skin. Moreover, papain can be degraded by many epidermal enzymes such as triacrylglycerol hydrolase, acid phosphatase, and phospholipase A2 Citation1. Most peptide drugs including papain also have low chemical stability, which limits their extemporaneous use from its short shelf-life in the formulations. Most marketed formulations containing papain have to be stored at low temperatures. Papain is unstable at high temperature Citation2 and in acidic conditions (at pH values below 2.8). Its activity significantly loses, probably as a result of autolysis and oxidation. Several strategies have been used to enhance the chemical stability of papain. Sankalia et al. demonstrated that the shelf-life of papain can be improved by entrapping in alginate beads. The shelf lives of the entrapped papain, the marketed formulation, and the papain solution were 3.60, 1.01, and 0.48 years, respectively Citation3. Papain entrapped in pectin stored for 6 months showed no loss of activity Citation4. The shelf-life of papain was increased from 1.01 years in the conventional papain formulation to 3.63 years of papain loaded in the κ-carrageenan beads Citation5. Nanoparticles (NPs) are submicron-size colloidal particles that a therapeutic agent can be either entrapped in the polymer matrix or bound on the surface. The small size NPs can be taken up more efficiently by the cells than the large size NPs. NPs can also cross the cell membrane barriers by transcytosis. The cellular uptake and transport of many macromolecules such as peptides across the biological membranes were significantly improved by condensing into nanospheres Citation6. The nanospheres prepared from poly(lactide-co-glycolide) (PLGA) are safe for human because of their biodegradability, increase the shelf-life, protect from degradation, and control the release over a longer period of time of the entrapped substances Citation7,Citation8. PLGA nanospheres have been reported to be effective delivery systems for several peptides such as cyclosporine A Citation9, insulin Citation10,Citation11, and salmon calcitonin Citation12. The commonly utilized techniques for loading hydrophilic molecules such as, papain in PLGA nanospheres are the double emulsion solvent evaporation (ESE) and the emulsion solvent diffusion in water (ESD) techniques. The ESD method has several advantages, such as simplicity, no need for homogenization, high batch-to-batch reproducibility and ease of scaling up Citation13. The ESE method can improve the formulation characteristics including small size, low size distribution, and high encapsulation efficiency Citation14. The aim of this study was to develop a stable and less toxic PLGA nanosphere formulation loaded with papain by these two methods. The characteristics, entrapment efficiency, toxicity to human fibroblasts, chemical stability, and the release profile of papain from the PLGA nanospheres were investigated.
2. Materials and methods
2.1. Materials
PLGA poly(lactide-co-glycolide) with an average molecular weight of 20,000 and the 75:25 copolymer ratio of DL-lactide to glycolide (Wako Pure Chemical Indu. Ltd., Japan) were used as biodegradable polymers. The standard purified papain (95% purity) was purchased from Sigma Chemicals Co. in USA. Polyvinyl alcohol (PVA-403, Kuraray, Japan) was used as a dispersing agent. Dulbecco's modified Eagle's medium (DMEM) and penicillin-streptomycin were purchased from Gibco BRL, USA. Fetal bovine serum (FBS) was from PAA Laboratories GmbH in Austria. Trichloroacetic acid and trifluoroacetic acid from Merck Co. in Germany, and sulforhodamine B (SRB) monosodium salt from Fluka Co. in USA were used. Trypsin was from Gibco Invitrogen Corp., USA. Ammonium molybdate from Fisher Scientific UK Limited, United Kingdom was used. Acetone, chloroform, hydrochloric acid, and glacial acetic acid were analytical grade solvents. Acetonitrile was the HPLC grade.
2.2. Preparation of papain-loaded nanospheres
Four PLGA nanosphere formulations including the unloaded and the papain (0.01 mg papain/1 mg PLGA nanospheres) loaded PLGA nanospheres by the two methods [the emulsion solvent diffusion methods in water (ESD) and the w/o/w emulsion solvent evaporation method (ESE)] were prepared.
2.2.1. Emulsion diffusion method in water (ESD)
PLGA nanospheres were prepared by the modified emulsion diffusion method in water Citation15. Briefly, PLGA (100 mg) and papain (1 mg) were dissolved completely in the mixture of acetone (5 ml) and 0.01 M hydrochloric acid (0.9 ml). The resulting polymer-enzyme solution was poured into 50 ml of an aqueous PVA solution (2.0%w/v) and stirred at 400 rpm for 5 min using a propeller-type agitator with three blades (Heidon 600 G, Shinto Scientific Co., Ltd., Japan). The entire dispersed system was then centrifuged at 43,400 × g for 10 min at 4°C (Kubota 7800, Kubota Co., Ltd., Japan) and resuspended in distilled water to remove the unloaded papain and PVA. This washing process was done in duplicate. The resulting dispersion was dried by a freeze dryer (EYELA FD1000, Tokyo Rikakikai Co., Ltd, Japan).
2.2.2. Water-oil-water (w/o/w) emulsion solvent evaporation method (ESE)
One hundred microliters of papain (1 mg) solution were emulsified in 500 µl of chloroform containing PLGA (100 mg) by sonication (duty cycle 75%, output 2) for 10 s using a Branson Sonifier 250 (Branson Ultrasonic, Danbury, CT, USA). The resulting primary emulsion was added to 2 ml of 10%w/v PVA and sonicated for 60 s to form a double emulsion. The obtained emulsion was added dropwise to 18 ml of 10%w/v PVA and stirred at 400 rpm using a propeller-type agitator with three blades (Heidon 600 G, Shinto Scientific Co., Ltd., Japan) for 3 h at room temperature (25 ± 2°C) under evaporation to completely remove the chloroform. Papain loaded PLGA nanospheres were collected by centrifugation at 43,400 × g for 10 min at 4°C (Kubota 7800, Kubota Co., Ltd., Japan) and resuspended in distilled water to remove the unloaded papain and PVA. This washing process was done in duplicate. The resulting dispersion was dried by a freeze dryer (EYELA FD1000, Tokyo Rikakikai Co., Ltd, Japan).
2.3. Analysis of physicochemical properties of the papain loaded nanospheres
2.3.1. Particle size and zeta potential determination
The average particle size and zeta potential value of the PLGA nanospheres dispersed in distilled water were determined by a laser particle size analyzer (Zetasizer Nano ZS British, Malvern Instruments, UK), based on a dynamic light scattering concept. The surface topography of the freeze dried nanospheres was observed by a scanning electron microscope (JSM-5900LV, JEOL, Tokyo, Japan).
2.3.2. Morphology investigation
A drop of nanosphere dispersion was applied on a 300-mesh formvar copper grid on paraffin and allowed the sample to adhere on the former for 10 min. The remaining dispersion was removed and a drop of 2% aqueous solution of ammonium molybdate was applied for 5 mins. The remaining solution was then removed, air dried and examined by a TEM microscope (TEM 1200S JEOL, JEOL Ltd., Tokyo, Japan) operated at 80 kV. The morphology of the nanospheres was observed.
2.3.3. Encapsulation efficiency determination
The encapsulation efficiency of papain in PLGA nanospheres was defined as the weight ratio of papain loaded in the nanospheres to the initial loading papain amount. The freeze dried nanospheres loaded with papain were dissolved in acetonitrile, into which distilled water was added to preferentially precipitate the polymer. The papain content in the supernatant after centrifugation (43,400 × g at 4°C for 10 min, Kubota 7800, Kubota, Japan) was measured by HPLC using the reversed-phase column (Gemini-NX, 5 µ C18 110 A, 4.6 × 250 mm, Phenomenax, USA). Acetonitrile/distilled water (7:3, v/v) containing 0.05% trifluoroactic acid was used as a mobile phase with the flow rate of 1 ml/min. An amount of 20 µl of the injection volume (autosample, AS300, Thermo Finnigan, USA) was eluted in the column and monitored at 230 nm of the UV-detector. All samples were filtered through a 0.45 -µm membrane filter, prior to injection onto the HPLC column. The encapsulation efficiency was calculated according to the following equation Citation16:
2.4. The release profile of papain from the PLGA nanospheres
Five milligrams of the nanospheres were dispersed in 5 ml of 0.2 M phosphate buffer (pH 7.0) solution and shaken horizontally at 27 ± 2°C and 80 strokes per min. The dispersion (1 ml) was withdrawn from the system at time intervals (0, 2, 4, 6, 8, 24, and 48 h) and centrifuged at 12,000 rpm, 4°C for 10 min. The supernatant was removed and the sediment was dissolved in 0.2 ml acetonitrile to which the distilled water was added to precipitate the polymer and to dissolve the enzyme in the aqueous mixture. The resulting suspension was centrifuged at 12,000 rpm, 4°C for 10 min to remove the precipitated polymer. The papain in the supernatant was determined by HPLC, as described in Section 2.3.3. In order to evaluate the release kinetics, the in vitro release results were fitted to various kinetic equations including zero order (% cumulative drug release vs. time), first order (log % cumulative drug remaining vs. time), and Higuchi matrix (% cumulative drug release vs. square root of time).
2.5. Human skin fibroblast cytotoxicity by SRB assay
2.5.1. Cell cultures
The human skin fibroblast was obtained from the Faculty of Tropical Medicine, Mahidol University, Bangkok in Thailand. The cells were maintained as adherent cells in T75 culture flasks at 37°C in a humidified incubator containing 5% CO2. The Dulbecco's modified Eagle's medium (DMEM) that was supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin was used as the growth medium for the cells. For subculturing, cells were rinsed with phosphate buffer saline (pH 7.4) and finally detached with 0.25% trypsin-EDTA solution. The cells at the 27th to 28th passage were used for the cytotoxicity assay.
2.5.2. Cytotoxicity assay
Cells were seeded in 96-well plates at an amount of 1 × 104 cells/well and allowed to incubate overnight. Then, cells were exposed to various concentrations of PLGA nanospheres loaded with papain for 24 h. After incubation, the adherent cells were fixed by adding 50 µl of cold 50%w/v trichloroacetic acid and incubated for further 1 h at 4°C. Then, the cells were rinsed five times with distilled water, air-dried and stained with 50 µl of 0.4% SRB in 1% glacial acetic acid for 30 min at room temperature (27 ± 2°C). The unbound SRB was removed by washing with 1% glacial acetic acid solution for four times. After air-drying, 100 µl per well of 10 mM Tris base were added to disslove the bound dye. After mixing, the absorbance was measured at 540 nm with a microplate reader (Biorad, Milan, Italy). The cells with no treatment were used as a negative control. The assays were done in three independent separate experiments. Cell viability (%) was calculated using the following equation Citation17,Citation18:
2.6. Physico-chemical stability of papain loaded in PLGA nanospheres
The selected PLGA nanosphere loaded with papain which gave the superior characteristics of small size, low polydispersity, high encapsulation efficiency, and low toxicity was dispersed in 0.2 M phosphate buffer (pH 5.0) solution and kept in the tight covered bottles at 4 ± 2, 25 ± 2, and 45 ± 2°C for 6 weeks. The physical characteristics (color and sedimentation) of the dispersions were observed visually. Particle size and zeta potential were determined by a laser particle size analyzer. At 1, 2, 3, 4, and 6 weeks, the samples were withdrawn and the remaining enzyme contents in the samples were analyzed by HPLC as previously described in Section 2.3. The remaining enzyme (%) was calculated by the following equation:
2.7. Statistical analysis
Data were expressed as mean ± SD. Statistical analysis was carried out using the ANOVA using the software SPSS 13.0 for Windows and the p value at less than 0.05 was considered as statistical significance.
3. Results and discussion
3.1. Characteristics of papain-loaded PLGA nanospheres
Papain loaded in PLGA nanospheres was prepared in the solvent system of acetone (5 ml) and 0.01 M hydrochloric acid (0.9 ml). Papain was expected to be stable in the mixed acetone/hydrochloric acid system that had the pH of 4.5 because the low concentration at 0.01 M of hydrochloric acid was used. In fact, papain has been reported to be stable at pH 5.0 and becomes unstable at pH lower than 3.0 and above 11 Citation19. In our first step of the double emulsion method, sonication that has been widely applied to produce stable emulsion Citation20 was used. Sonication can produce the stable emulsion containing small droplets that is critical for nanoencapsulation.
3.1.1. Particle size, zeta potential values, and morphology
All PLGA nanosphere dispersions from the ESD and ESE methods were in colloidal appearance with milky white appearances, and no sedimentation or layer separation. After freeze drying, the white puffy PLGA nanosphere dry powder was obtained. The average particle size and zeta potential values of the unloaded and loaded PLGA nanospheres with papain by the ESD and ESE methods were demonstrated (). The particle sizes of the PLGA nanospheres unloaded and loaded with papain by both methods were in the nanosize range (220–335 nm) and exhibited low polydispersity indicating a relatively narrow particle size distribution. The size of PLGA nanospheres slightly increased when loaded with papain. This may be from the encapsulated or adsorbed papain on the nanosphere surface. PLGA nanospheres that prepared by the ESE method gave smaller particle size and lower polydispersity index than the ESD method showing better physical stability. The process variables such as PVA concentration and sonication may affect the particle size and the polydispersity index Citation21. The ESE method had the sonication process in the emulsification step, while sonication was not used in the ESD method. The lower amount of PVA was incorporated to prepare the nanospheres by the ESD (2% w/v) Citation22 than by the ESE (10% w/v) Citation23. Mukerjee et al. have indicated that the high PVA concentration and the increase sonication time gave small particle size Citation24,Citation25. Several previous reports have also demonstrated that the increase of the PVA concentration in the external aqueous phase decreased the size of the nanoparticles Citation26–28. At high concentration, more PVA can be oriented at the organic solvent/water interface resulting the decrease of the interfacial tension Citation29, the significant increase in the net shear stress at a constant energy density during emulsification and the promotion for the formation of smaller emulsion droplets. The freeze dried nanospheres were readily redispersed in an aqueous medium by shaking manually, reproducing the original particle sizes before drying. It was assumed that PVA introduced into the system as dispersing agent was adsorbed on the surface of the nanospheres during freeze drying Citation30. The PVA on the surface might improve the wettability of the nanospheres on redispersing.
Table 1. The comparison of particle sizes, zeta potential values and encapsulation efficiency (%EE) of PLGA nanospheres loaded with papain prepared by the ESD and ESE methods.
The nanospheres unloaded and loaded with papain by both methods dispersed in distilled water (pH 7.0) exhibited a negative zeta potential ranging from −29.6 to −36.2 mv. It has been previously reported that the negative charges of the PLGA nanoparticles are due to the ionization of the carboxylic-end group of the polymer on the nanoparticle surface Citation31,Citation32. PLGA nanospheres prepared by the ESE method showed slight lower zeta potential values than those from the ESD method. Sahoo et al. demonstrated that the zeta potential values of the nanoparticles prepared with 0.5% PVA were higher than those with 5% PVA Citation26. Coating of the nanoparticles with some amphiphilic polymers normally decreases the zeta potential value because the coating layers may shield the surface charge and move the shear plane outwards from the particle surface. Since the lower amount of PVA was used to prepare the nanospheres by the ESD (2% w/v) than the ESE (10% w/v) method, less shielding and more carboxyl groups of PLGA were available for ionization. PLGA nanospheres loaded with papain showed less negative zeta potential values than the unloaded nanospheres. This may be due to the positive charge of papain that can interact with the negative polarity of the nanosphere surface (from the carboxylic-end group of polymer). No significant difference in the particle size and zeta potential values of the nanospheres prepared by the ESE method before and after freeze-drying and reconstitution in the form of suspension were observed (). For the ESD method, the nanospheres gave larger diameters, possibly due to the agglomeration of the particles by compression during the ice growth Citation33.
The morphology and surface characterization of the papain loaded PLGA nanospheres were observed by scanning electron (SEM) and transmission electron (TEM) microscopy. showed the SEM and TEM microphotographs of papain-loaded PLGA nanospheres prepared by the ESE method. The nanospheres were in the spherical shape with the mean diameters of 250 nm and a smooth surface morphology.
Figure 1. The scanning electron (A) and transmission electron (B) microphotographs of PLGA nanospheres (composed of 100 mg PLGA and 10%w/v PVA403) loaded with 43 µg papain/mg PLGA nanospheres prepared by the ESE method (12000X).
3.1.2. Encapsulation efficiency of papain in the PLGA nanospheres
Encapsulation efficiency of papain in the PLGA nanospheres was summarized in . The encapsulation efficiencies of papain in the PLGA nanospheres prepared by ESD and ESE method were at 19.42 ± 0.63 and 43.03 ± 0.15%, respectively. The ESE method that gave about two times higher entrapment efficiency than the ESD method appeared to be the suitable method to load papain in the PLGA nanospheres. In the ESE method, papain that is a water-soluble peptide can be introduced into the internal aqueous phase of the multiple emulsions, resulting in an increase of encapsulation efficiency in the nanospheres. The encapsulation efficiency of water-soluble drugs in PLGA nanospheres by the ESD method of less than 20% was reported Citation16,Citation22. This is most probably due to the rapid diffusion of the enzymes into the water phase when their solution containing PLGA was poured into the aqueous medium. However, the high encapsulation efficiency of more than 20% was observed by the ESE method. This method has also been used to prepare water-soluble drug-loaded nanospheres. It is supposed that the oil phase surrounding the internal water phase would prevent the leakage of the drug into the external water phase. Thus, the high encapsulation efficiency of more than 20% by the ESE method was obtained. In addition, the higher PVA concentration in the ESE method can affect the stability of the w/o emulsion providing a higher mass transferring resistance, thereby reducing the amount of the enzyme molecules diffusing from the internal into the external water phase during the emulsification Citation27. Also, when the polymer solution precipitated to form nanospheres, PVA could promote compatibility between the hydrophilic enzyme molecules and the hydrophobic polymer network, which is preferential for enzyme to locate within the nanosphere matrix.
3.2. The release profile of papain from the PLGA nanospheres
The release profile of papain from the PLGA nanospheres prepared by the ESD and ESE method in 0.2 M phosphate buffer (pH 7.0) solution at 27 ± 2°C gave a 2-phase release profile as demonstrated in . Papain released from the nanospheres prepared by the ESD method were 65%, 82%, 84%, and 87% at 2, 6, 8, and 24 h, respectively and almost completed (90%) after 48 h. The slower release rate was observed in the ESE method which gave 48%, 62%, 63%, 64%, and 72% at 2, 6, 8, 24, and 48 h, respectively. The high PVA concentration of the ESE method can not only increase the viscosity of the external phase and the difficulty for the enzyme to diffuse out of the nanospheres, but also gave a more stable emulsion which can hinder the mass transfer of the enzyme to the surroundings Citation34.
Figure 2. Comparision of profile release of papain loaded in PLGA nanospheres prepared by ESD (emulsion solvent diffusion method in water) and ESE (w/o/w emulsion solvent evaporation method) in 0.2 M phosphate buffer (pH 7.0) solution at 27 ± 2°C.
The initial burst release of papain was observed within the first 6 h with the steep slope of 12.385 and 9.4899% h−1 and followed by a plateau with the slope of 0.1678 and 0.2128% h−1 of the ESD and ESE method, respectively. The first phase was faster than the second phase of about 73 and 45 times, respectively. The burst release in the first phase may be caused by a rather high mobility of the enzyme in the particles especially the small particle size, which has the short diffusion distance. The slow release of papain from the nanospheres may be not only from the electrostatic interaction between the enzyme and the polymer Citation35, but also the charge interaction between papain that has the positive charge (pI = 8.75) in phosphate buffer (pH 7.4) and the negative charge of the carboxylic-end group of the polymer on the nanoparticle surface. Other possibilities including the total content of drug in the nanospheres that became less and less over the release time, the drug molecules located in the center of the nanospheres that have longer distance to diffuse and the crystallinity of the polymer that may have increased during incubation time in phosphate buffer may be affected to slow release rate of papain in the second phase Citation36–39. showed the release kinetics of papain from the PLGA nanospheres. According to various kinetic models, the first phase of the enzyme release from the nanospheres prepared by the ESD and ESE method can be estimated for linearity fitting as the Higuchi's equation plot that gave the r 2 values of 0.9456 and 0.9554, respectively. These kinetics demonstrated the release of the enzyme from the matrix as a square root of the time-dependent process base on the Fickian diffusion that described the release by the pure diffusion of the enzyme from the nondegradable matrix Citation40. However, the second phase of the enzyme released from the nanospheres prepared by the ESE method was best fitted in the zero-order kinetic with the r 2 value of 0.9409, while that by the ESD method was the Higuchi kinetics that gave the r2 values of 0.9566. The release rate of papain from the PLGA nanospheres of the ESD and ESE method gave 13.73% h−1/2 and 10.47% h−1/2 of the first phase and at 1.88% h−1/2 and 1.56% h−1 of the second phase, respectively. This different release behavior may be resulted from the differences of the nanosphere structures depending on the method of preparation. In the ESD method, the enzyme dispersed evenly in the matrix of the polymer due to the coprecipitation of the polymer and enzyme Citation22. The nanospheres prepared by the ESE method may construct a reservoir-like structure Citation41, that papain entrapped in the core of the nanospheres can be released by diffusing through the polymer shell indicating the zero-order kinetics Citation42.
Table 2. Release kinetics of papain from the PLGA nanospheres prepared by the ESD and ESE method in 0.2 M phosphate buffer (pH 7.0) solution at 27 ± 2°C to 48 h.
3.3. In vitro cytotoxicity of papain loaded in PLGA nanospheres
The human skin fibroblasts incubated with the unloaded papain at 10 µg/ml showed cytotoxicity on human skin fibroblasts of 16.17 ± 0.28% cell viability with the IC50 value at 6.22 µg/ml. It has been shown that when human skin contacted with papain at 0.2%w/v for 24 h, the large amount of the intercellular material in the stratum corneum was lost and most of the extracellular components were digested Citation43. The unloaded PLGA nanospheres at more than 1 µg/ml gave of cell viability more than 80%. PLGA that is the main composition of the nanospheres is a biodegradable and biocompatible polymer, thereby being well tolerated by the cells. The percentages of the human skin fibroblast viability treated with the unloaded nanospheres and nanospheres loaded with papain prepared by both ESD and ESE methods were shown in . The percentages of cell viability were compared between the two methods at different nanosphere concentrations. When the concentrations of the nanospheres loaded with papain increased from 0.001 to 10 µg/ml, cell viability was decreased. The loaded nanospheres at the concentration range of 0.001–0.1 g/ml gave no significant difference at p > 0.05 of cell viability of more than 80% in comparing to the untreated cells. At 0.001, 0.01, and 0.1 µg/ml of the PLGA nanospheres loaded with papain prepared by the ESD method gave cell viability at 89.62 ± 2.31, 85.73 ± 6.95, and 80.64 ± 2.38%, respectively, while those by the ESE method were 90.15 ± 5.82, 86.01 ± 5.11, and 81.11 ± 7.73%, respectively. At higher concentrations (1–10 µg/ml), PLGA nanospheres loaded with papain prepared by the ESD method gave slight higher cell viability (but no significant difference p > 0.05) than those prepared by the ESE method. This may be due to the no difference in zeta potential values of the PLGA nanospheres loaded with papain prepared by both methods after freeze drying and rehydration Citation44. The zeta potential values of papain loaded PLGA nanospheres may relate to the cell viability. The larger the zeta potential values of the PLGA nanospheres, the stronger the interaction between the nanosphere membrane and the cell membrane leading to higher cytotoxicity Citation45.
Figure 3. The percentages of human skin fibroblast viability by the SRB assay of (A) the unloaded nanospheres and (B) the PLGA nanospheres loaded with papain prepared by the ESD (emulsion solvent diffusion in water) method (19 µg papain/mg PLGA nanosphere) and the ESE (w/o/w emulsion solvent evaporation) method (43 µg papain/mg PLGA nanosphere).
Cytotoxicity of papain loaded in nanospheres prepared by these two methods was also compared. The papain contents in the nanospheres prepared by ESD were different from those by the ESE method (19 and 43 µg papain/mg PLGA nanosphere, respectively). When the IC50 values of the papain cytotoxicity were determined, the cytotoxicity of papain loaded in the nanospheres prepared by the ESD method gave higher than those prepared by the ESE. Papain loaded in PLGA nanospheres prepared by the ESD method gave the IC50 values at 6.19 µg/ml that appeared to be more toxic than those by the ESE method with the IC50 values at 9.13 µg/ml. This may be due to the higher release rate of papain from the PLGA nanospheres prepared by the ESD method than that by the ESE method as mentioned above (). The higher released papain of the PLGA nanospheres prepared by the ESD method in the cell culture medium gave higher cytotoxicity to the cells than that of the nanospheres by the ESE method. The cell cytotoxicity of the unloaded papain was more than papain loaded in the nanospheres prepared by the ESE method of 1.5 times, while no significant difference between the unloaded papain and papain loaded in the nanospheres prepared by the ESD method. The results from this study have suggested that the cytotoxicity of papain was reduced when loaded in PLGA nanospheres prepared by the ESE method.
3.4. Physicochemical stability at various storage temperatures of papain loaded in PLGA nanospheres
The physico-chemical stability of papain loaded in PLGA nanospheres was performed in phosphate buffer of pH 5.0, while the released study of papain from PLGA nanospheres was done by using phosphate buffer of pH 7.0. Papain is expected to release from PLGA nanospheres after penetration into the skin. The pH environments of the inner skin layer is similar to the physiological pH. So, the release study of papain from PLGA nanospheres was done by using phosphate buffer at pH 7.0 (the physiological pH is in the range of 6.4 to 7.5) Citation46. However, the stability of papain has been studied at pH 5.0 since this pH value gave the highest activity of papain and this pH will be proposed to formulate the topical preparation of papain loaded in nanospheres Citation47. Papain loaded in PLGA nanospheres prepared by the ESE method determined by visual observation showed good physical stability with no sedimentation, layer separation and color change at 4 ± 2 and 25 ± 2°C for 6 weeks. At 45 ± 2°C, some aggregation of the particles was observed after 1 week. After stored for 3 weeks at 45 ± 2°C, the milky colloidal nanosphere dispersion became clear solution by visual observation and the particle size can not be detected by a laser particle size analyzer (data not shown). The nanospheres were completely degraded possible due to the increase hydrolytic degradation of the PLGA polymer at high temperature.
The percentages remaining of papain in the PLGA nanospheres prepared by the ESE method stored at 4°C, 25°C, and 45°C for 6 weeks were shown in . At 4°C and 25°C, papain in the nanospheres exhibited higher remaining amount than in the solution. However, papain loaded in the nanospheres was unstable at 45°C because of the degradation of the PLGA polymer resulting in the leakage of the enzyme from the nanospheres and the destruction of the enzyme by heat. The percentages of papain remaining in PLGA nanospheres stored at 4°C for 3 weeks significantly lower than those stored at 25°C for 3 weeks and the percentages of papain remaining in phosphate buffer at 4°C became lower at 2.5 to 6 weeks in comparing to that kept at 25°C, because papain might form large insoluble materials and aggregation at low temperature Citation48. Another possible reason was also from the special features of low temperature denaturation Citation49. After 3 weeks at 45°C, all PLGA nanospheres were completely degraded and the remaining papain was only the released enzyme in the supernatant. After 6 weeks at 4°C, the enzyme contents in the nanospheres (71.50%) were about 8 times higher than those in the solution (9.32%). The nanospheres can protect papain against aggregation at low temperature by increasing electrostatic repulsion between the particular membranes. At 25°C, the enzyme remained in the nanospheres were 61.88% which were 3 times higher than those in the solution (22.66%). PLGA nanospheres can shield papain from thermal degradation at high temperature. Thus, the PLGA nanospheres appeared to protect the enzyme from peptide aggregation and degradation at low (4°C) and high (25°C) storage temperature. Thus, chemical stability and the shelf life of papain can be improved when loaded in PLGA nanospheres. This result agreed with the previous study that demonstrated that nafarelin stability was improved when loaded in nanospheres in comparing to the drug in the acidic medium [50].
Figure 4. The percentages remaining of the unloaded papain and loaded in PLGA nanospheres prepared by the ESE (w/o/w emulsion solvent evaporation) method stored at different temperatures (25 ± 2, 4 ± 2 and 45 ± 2°C) for 6 weeks; Pn-4: papain loaded in PLGA nanospheres kept at 4°C; Pn-25: papain loaded in PLGA nanospheres kept at 25°C; Pn-45: papain loaded in PLGA nanospheres kept at 45°C; Ps-4: papain solution in 0.2 M phosphate buffer (pH 5.0) kept at 4°C; Ps-25: papain solution in 0.2 M phosphate buffer (pH 5.0) kept at 25°C and Ps-45: papain solution in 0.2 M phosphate buffer (pH 5.0) kept at 45°C.
4. Conclusion
This study has demonstrated that papain loaded in PLGA nanospheres prepared by the ESE method gave superior characteristics (small particle size and low polydispersity index) to the ESD method. The encapsulation efficiency of papain in the nanospheres by the ESE method was about two times higher than those by the ESD method. The release profile of papain from the PLGA nanospheres prepared by both methods demonstrated an initial burst release for 6 h and followed by the sustain release for 48 h. Papain loaded in the nanospheres prepared by the ESD method showed more rapid initial release than those by the ESE method. However, these was no significant difference (p > 0.05) of cell viability on human skin fibroblasts of the unloaded nanospheres prepared by the two methods. The unloaded papain showed cytotoxicity on human skin fibroblasts with the IC50 value of 6.22 µg/ml, while papain loaded in PLGA nanospheres prepared by the ESE method, showed an increase IC50 value of 9.13 µg/ml which was 1.5 times less toxic than the unloaded papain. Papain loaded in the PLGA nanospheres showed higher chemical stability than papain in solution of eight and three times when kept for 6 weeks at 4°C and 25°C, respectively. In summary, this study has indicated that papain (43 µg) loaded in PLGA nanosphere (1 mg) prepared by the ESE method showed lower cytotoxicity to human skin fibroblast and higher chemical stability than the unloaded papain, which can be further developed as topical products.
Acknowledgements
This work was supported by Natural Products Research and Development Center (NPRDC), Science Technology Research Institute (STRI), Faculty of Pharmacy, Chiang Mai University in Thailand, School of Pharmaceutical Sciences, Aichi Gakuin University, Chikusa-ku, Nagoya, Japan and the Thailand Research Fund (TRF) under the Royal Golden Jubilee (RGJ)-Ph.D. program.
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