The Formulation And In Vitro/in Vivo Evaluation Of Lornoxicam Loaded Nanostructured Lipid Carriers That Modified With Cell Penetrating Peptides

Nanostructured lipid carriers(NLC)are emerging as the attractive drug carrier in transdermal drug delivery.In recent years,more attention has been paid to NLC because of some advantages such as high encapsulation efficiency and drug loading,good stability,enhancing skin hydration,promoting the drug transdermal absorption and good biocompatibility.Cell penetrating peptides(CPP)modified on the surface of NLC have been investigated widely because of the potential of which to efficiently transport some carriers through the cell membrane in skin comeum further to enhance the percutaneous permeability of drugs.The objective of the current study was to enhance the percutaneous permeability of LN,improve the anti-inflammatory effect of LN and to evaluate the potential of CPP(polyarginine)to translocate the NLC loaded with lornoxicam(LN)into the skin layers and their in vivo anti-inflammatory effect.The NLC were prepared by emulsion-evaporation and low temperature-solidification method.The formulations were optimized by the single factor study and the central composite design-response surface method(CCD-RSM),then R11 peptides were modified on the surface of NLC.The permeation-promoting potential of R11 peptides were investigated by in vitro skin permeation studies using Franz diffusion and cellular uptake assays on HaCaT cells using flow cytometry(FCM)and laser scanning confocal microscopy(LSCM).The mechanism of cell uptake of R11 was investigated on HaCaT cells by determining the concentration of Ca2+ and the change of member potential.The developed NLC formulated with LN and R11(LN-NLC-R11)were incorporated into 2%HPMC gels.The carrageenan-induced rat paw edema and adjuvant-induced arthritis models were adopted to evaluate the anti-inflammatory activity of LN-NLC-R11 gels.The formulations of LN-NLC were optimized by the single factor study and CCD-RSM as follows:ratio of drug to lipid was 1:50,dosage of soy lecithin was 162.5 mg,the percentage of liquid fat was 25%in total lipid and emulsifier dosage was 958.2 mg.The particle size,the zeta potential,the encapsulation efficiency and the drug loading of LN-NLC-R11 prepared by the optimal formulations were 118±3.06 nm,-8.5±0.48 mV,(74.61±1.13)%and(7.922±0.33)%,respectively.In vitro drug release study,the LN in R11-LN-NLC suspension could release continuously for 72 h in phosphate buffers containing 0.5%tween-80.In vitro skin permeation study showed better permeation enhancing potential of R11(0.02%,w/w)by increasing the amount of LN penetrated through skin and retained in skin layers than other content of R11(0.01%or 0.04%).The cell cytotoxicity study showed that the blank NLC and the blank NLC modified with R11 have no cytotoxicity on HaCaT cells and the NLC loaded with LN increased the cell cytotoxicity with the increase of concentration of LN.FCM and LSCM studies suggested that the NLC modified with R11(0.02%,w/w)had enhanced significantly cell internalization of nanoparticles relative to unmodified NLC(P<0.05 or P<0.01),indicting the great penetration-enhancing potential of Rll(0.02%,w/w).The mean fluorescence intensity(MFI)inside HaCaT cells increased with longer time and higher concentrations,demonstrating that HaCaT cells showed time-dependent and concentration-dependent uptake of RhB-NLC with or without R11.Moreover,R11 can enhance the permeability of cell membranes via changing the membrane potential and the degree of intercellular Ca2+.In carrageenan-induced rat paw edema models,LN-NLC-R11 gels inhibited obviously rat paw edema as compared to LN-NLC gels and the diclofenac sodium gels(P<0.01 or P<0.05),indicating that LN-NLC-R11 gels can inhibit effectively the cute inflammation.LN-NLC-R11 gels inhibited obviously the produce of inflammatory cytokines(IL-6,COX-2 and INOS),especially the inhabitation of COX-2 and INOS was greater than IL-6,indicating that the anti-inflammation of LN-NLC-R11 gels was achieved by inhibiting the produce of COX-2,further inhibiting the produce of PGs.In adjuvant-induced arthritis models,LN-NLC-R11 gels inhibited obviously rat paw edema,infiltration of inflammatory cells,and hyperplasia of chondrocytes and reduced the degree of fibrosis of cartilage,indicating the good effect of LN-NLC-R11 gels in treating inflammation.Above all,LN-NLC-R11 gels were developed after being optimized by CCD-RSM and modified with R11.The studies indicated that LN-NLC-R11 gels effectively alleviated the severity of inflammation and were expected to be a new formulation with the advantage of safety,convenience and high effect in transdermal LN delivery.