The Entrapment of natural peptides in nanoparticles for scar treatment

Abstract

This study aimed to develop optimum transdermal delivery systems for peptide enzymes from natural sources loaded in nanocarrier for scar treatment. Low cytotoxic elastic niosomal formulation loaded with salmon calcitonin (a model peptide) which composed of Tween 61, cholesterol and edge activators was developed. The 5% mole sodium cholate (NaC) elastic niosome appeared to be the most appropriate niosomal system to load the peptide because of its high elasticity and low cytotoxicity on human skin fibroblasts. Both calcitonin loaded in elastic and non-elastic niosomes did not only show higher chemical stability at high storage temperatures, but also superior transdermal absorption in comparing to calcitonin in solution. Papain and bromelain, the protease enzymes from Carica papaya latex and pineapple (Ananas comosus) fruits were extracted and purified by the simple precipitation with 95% ethanol and the saturated ammonium sulfate, respectively. The molecular weight of the extracted enzymes was compared with their standards and the protein marker by SDS-PAGE. The purity of papain and bromelain of the extracted enzymes determined by HPLC were 82.31 and 38.03%, respectively. The physicochemical properties including solubility and stability in various reagents of the extracted enzymes were similar to their standards. The standard and extracted papain exhibited higher antioxidant activity than bromelain both in free radical scavenging (SC50) and lipid peroxidation inhibition (IPC50) activity. However, the extracted papain showed lower antioxidant activity than the standard papain. All protease enzymes at 25 µg/ml did not only show no cytotoxicity, but also exhibited an interesting relative MMP-2 stimulation on human skin fibroblasts. The extracted papain gave the highest MMP-2 stimulatory activity at 2.10 folds of the control which were higher than the standard papain, the standard and extracted bromelain of 1.04, 1.31 and 1.24 times, respectively. The papain and bromelain loaded in 5% mole sodium cholate (NaC) elastic niosome were developed. They gave the vesicular sizes in the range of 109.5 to 143.9 nm with the negative zeta potential of -14.7 to -30.1 mv. The morphology of the niosomes was in spherical shape with unilamellar structure observed under TEM microscope. The elastic niosomes did not only improve the entrapment efficiency of the enzymes over the non-elastic niosomes, but also reduced the toxicity on skin human fibroblasts. The MMP-2 stimulatory activity of papain and bromelain was still existing similar to their free enzymes even they were loaded in elastic niosomes. The relative MMP-2 stimulation of the standard papain, extracted papain, standard bromelain and extracted bromelain loaded in elastic niosomes were 1.26, 1.34 1.09 and 1.20 for the pro MMP-2 and 1.26, 1.41, 1.01 and 1.03 for the active MMP-2, respectively in comparing to the control. The extracted papain loaded in elastic niosomes gave superior characteristics (low cytotoxicity and high MMP-2 stimulation) to other enzymes. The elastic niosomes can enhance the chemical stability of the extracted papain, by exhibiting higher remaining contents than the free enzyme and that loaded in non-elastic niosomes when kept at various temperatures (4±2, 27±2 and 45±2 C) for 8 weeks. The standard papain loaded in PLGA nanospheres prepared by the water-oil-water emulsion solvent evaporation method (ESE) gave superior characteristics (small particle size and low polydispersity index) to the emulsion solvent diffusion in water method (ESD). 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 encapsulation efficiency of papain in the nanospheres by the ESE method was about 2 times higher than those by the ESD method. The release profile of papain from the PLGA nanospheres of the ESD and ESE methods indicated the 2 phases with an initial rapid phase of 6 h and followed by the slow release phase of 48 h. Papain loaded in the nanospheres prepared by the ESD method showed more rapid initial release than those by the ESE method. The blank PLGA nanospheres from the two methods did not show any cytotoxicity in human skin fibroblasts, while the free 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 free papain of 8 and 3 times when kept at 4±2 and 25±2 C for 6 weeks, respectively. The gels incorporated with papain loaded in non-elastic niosomes (GNN), elastic niosomes (GEN) and PLGA nanospheres (GPN) were developed. These formulations were investigated for physicochemical characteristics and transdermal penetration through rat skin by Franz diffusion cells. The vesicular sizes of all niosomes and nanospheres in the gel formulations were in the range of 220.7 to 520.2 nm. Papain loaded in elastic niosomes and incorporated in gel exhibited the accumulate amounts and fluxes in the whole rat skin and in the receiving solution more than those from gel containing papain loaded in non-elastic niosomes, PLGA nanospheres and in solution. The calculated PII values of all gel formulations containing papain in rabbit skin irritation closed patch test were in the range of 0.00-0.44 indicating of no irritation, except gel containing free papain (PII = 0.78, slight irritation). GEN gave superior chemical stability to gel containing free papain (GS) of 1.13, 1.29 and 1.35 times when stored at 4±2, 27±2 and 45±2 C after 3 months, respectively. After 28 day application by the hypertrophic scar model in rabbit ears, the scar applied with GEN exhibited the percentage reduction of hypertrophic scars at 29.17% which was higher than gel base, GS, and GNN of 10.20, 2.73 and 2.31 times, respectively, but slightly lower than commercial product of 1.10 times. This study has demonstrated the potential of niosomes, especially the elastic niosomes, for the enhancement of rat skin transdermal absorption and the improvement of scar reduction in rabbit ear model of papain which will be beneficial for topical application in scar treatment.