(31)P solid-state NMR based monitoring of permeation of cell penetrating peptides into skin.

The main objective of the current study was to investigate penetration of cell penetrating peptides (CPPs: TAT, R8, R11, and YKA) through skin intercellular lipids using (31)P magic angle spinning (MAS) solid-state NMR. In vitro skin permeation studies were performed on rat skin, and sections (0-60, 61-120, and 121-180µm) were collected and analyzed for (31)P NMR signal. The concentration-dependent shift of 0, 25, 50, 100, and 200mg/ml of TAT on skin layers, diffusion of TAT, R8, R11, and YKA in the skin and time dependent permeation of R11 was measured on various skin sections using (31)P solid-state NMR. Further, CPPs and CPP-tagged fluorescent dye encapsulate liposomes (FLip) in skin layers were tagged using confocal microscopy. The change in (31)P NMR chemical shift was found to depend monotonically on the amount of CPP applied on skin, with saturation behavior above 100mg/ml CPP concentration. R11 and TAT caused more shift in solid-state NMR peaks compared to other peptides. Furthermore, NMR spectra showed R11 penetration up to 180µm within 30min. The results of the solid-state NMR study were in agreement with confocal microscopy studies. Thus, (31)P solid-state NMR can be used to track CPP penetration into different skin layers.

Topical delivery of anti-TNFα siRNA and capsaicin via novel lipid-polymer hybrid nanoparticles efficiently inhibits skin inflammation in vivo.

The barrier properties of the skin pose a significant but not insurmountable obstacle for development of new effective anti-inflammatory therapies. The objective of this study was to design and evaluate therapeutic efficacy of anti-nociception agent Capsaicin (Cap) and anti-TNFα siRNA (siTNFα) encapsulated cyclic cationic head lipid-polymer hybrid nanocarriers (CyLiPns) against chronic skin inflammatory diseases. Physico-chemical characterizations including hydrodynamic size, surface potential and entrapment efficacies of CyLiPns were found to be 163±9nm, 35.14±8.23mV and 92% for Cap, respectively. In vitro skin distribution studies revealed that CyLiPns could effectively deliver FITC-siRNA up to 360µm skin depth. Further, enhanced (p<0.001) Cap permeation from CyLiPns was observed compared to Capsaicin-Solution and Capzasin-HP. Therapeutic efficacies of CyLiPns were assessed using imiquamod-induced psoriatic plaque like model. CyLiPns carrying both Cap and siTNFα showed significant reduced expression of TNFα, NF-κB, IL-17, IL-23 and Ki-67 genes compared to either drugs alone (p<0.05) and were in close comparison with Topgraf®. Collectively these findings support our notion that novel cationic lipid-polymer hybrid nanoparticles can efficiently carry siTNFα and Cap into deeper dermal milieu and Cap with a combination of siTNFα shows synergism in treating skin inflammation.

Effect of oleic acid modified polymeric bilayered nanoparticles on percutaneous delivery of spantide II and ketoprofen.

The objective of the present study was to evaluate the effect of oleic acid modified polymeric bilayered nanoparticles (NPS) on combined delivery of two anti-inflammatory drugs, spantide II (SP) and ketoprofen (KP) on the skin permeation. NPS were prepared using poly(lactic-co-glycolic acid) (PLGA) and chitosan. SP and KP were encapsulated in different layers alone or/and in combination (KP-NPS, SP-NPS and SP+KP-NPS). The surface of NPS was modified with oleic acid (OA) ('Nanoease' technology) using an established procedure in the laboratory (KP-NPS-OA, SP-NPS-OA and SP+KP-NPS-OA). Fluorescent dyes (DiO and DID) containing surface modified (DiO-NPS-OA and DID-NPS-OA) and unmodified NPS (DiO-NPS and DID-NPS) were visualized in lateral rat skin sections using confocal microscopy and Raman confocal spectroscopy after skin permeation. In vitro skin permeation was performed in dermatomed human skin and HPLC was used to analyze the drug levels in different skin layers. Further, allergic contact dermatitis (ACD) model was used to evaluate the response of KP-NPS, SP-NPS, SP+KP-NPS, KP-NPS-OA, SP-NPS-OA and SP+KP-NPS-OA treatment in C57BL/6 mice. The fluorescence from OA modified NPS was observed up to a depth of 240µm and was significantly higher as compared to non-modified NPS. The amount of SP and KP retained in skin layers from OA modified NPS increased by several folds compared to unmodified NPS and control solution. In addition, the combination index value calculated from ACD response for solution suggested an additive effect and moderate synergism for NPS-OA. Our results strongly suggest that surface modification of bilayered nanoparticles with oleic acid improved drug delivery to the deeper skin layers.

Skin permeating nanogel for the cutaneous co-delivery of two anti-inflammatory drugs.

The aim of this study was to develop an effective drug delivery system for the simultaneous topical delivery of two anti-inflammatory drugs, spantide II (SP) and ketoprofen (KP). To achieve this primary goal, we have developed a skin permeating nanogel system (SPN) containing surface modified polymeric bilayered nanoparticles along with a gelling agent. Poly-(lactide-co-glycolic acid) and chitosan were used to prepare bilayered nanoparticles (NPS) and the surface was modified with oleic acid (NPSO). Hydroxypropyl methyl cellulose (HPMC) and Carbopol with the desired viscosity were utilized to prepare the nanogels. The nanogel system was further investigated for in vitro skin permeation, drug release and stability studies. Allergic contact dermatitis (ACD) and psoriatic plaque like model were used to assess the effectiveness of SPN. Dispersion of NPSO in HPMC (SPN) produced a stable and uniform dispersion. In vitro permeation studies revealed increase in deposition of SP for the SP-SPN or SP+KP-SPN in the epidermis and dermis by 8.5 and 9.5 folds, respectively than SP-gel. Further, the deposition of KP for KP-SPN or SP+KP-SPN in epidermis and dermis was 9.75 and 11.55 folds higher, respectively than KP-gel. Similarly the amount of KP permeated for KP-SPN or SP+KP-SPN was increased by 9.92 folds than KP-gel. The ear thickness in ACD model and the expression of IL-17 and IL-23; PASI score and TEWL values in psoriatic plaque like model were significantly less (p < 0.001) for SPN compared to control gel. Our results suggest that SP+KP-SPN have significant potential for the percutaneous delivery of SP and KP to the deeper skin layers for treatment of various skin inflammatory disorders.

Translocation of cell penetrating peptide engrafted nanoparticles across skin layers.

The objective of the current study was to evaluate the ability of cell penetrating peptides (CPP) to translocate the lipid payload into the skin layers. Fluorescent dye (DID-oil) encapsulated nano lipid crystal nanoparticles (FNLCN) were prepared using Compritol, Miglyol and DOGS-NTA-Ni lipids by hot melt homogenization technique. The FNLCN surface was coated with TAT peptide (FNLCNT) or control YKA peptide (FNLCNY) and in vitro rat skin permeation studies were performed using Franz diffusion cells. Observation of lateral skin sections obtained using cryotome with a confocal microscope demonstrated that skin permeation of FNLCNT was time dependent and after 24h, fluorescence was observed upto a depth of 120 microm which was localized in the hair follicles and epidermis. In case of FNLCN and FNLCNY formulations fluorescence was mainly observed in the hair follicles. This observation was further supported by confocal Raman spectroscopy where higher fluorescence signal intensity was observed at 80 and 120 microm depth with FNLCNT treated skin and intensity of fluorescence peaks was in the ratio of 2:1:1 and 5:3:1 for FNLCNT, FNLCN, and FNLCNY treated skin sections, respectively. Furthermore, replacement of DID-oil with celecoxib (Cxb), a model lipophilic drug showed similar results and after 24h, the CXBNT formulation increased the Cxb concentration in SC by 3 and 6 fold and in epidermis by 2 and 3 fold as compared to CXBN and CXBNY formulations respectively. Our results strongly suggest that CPP can translocate nanoparticles with their payloads into deeper skin layers.