Percutaneous delivery of α-melanocyte-stimulating hormone for the treatment of imiquimod-induced psoriasis.

PURPOSE: α-Melanocyte-stimulating hormone (α-MSH) is an endogenous peptide hormone with anti-inflammatory responses. We developed topical formulation(s) of α-MSH to reduce psoriasis-related inflammation. METHODS: Transcutol (TC) and n-methyl 2-pyrrolidone (NMP) were used to formulate a gel for α-MSH. Skin permeation and dermal microdialysis of the solution and optimized gel were performed. The inflammatory response of α-MSH gel was investigated in imiquimod-induced psoriasis mouse model. Histology and immunohistochemistry were then performed on treated skin. RESULTS: Solution comprising 50%w/w TC and 10%w/w NMP showed higher (p < 0.05) skin retention (0.27 ± 0.024 µg of α-MSH/mg of skin) than solutions containing either 50% w/w TC or 10% w/w NMP at 24 h. Dispersion of α-MSH in Carbopol Ultrez 10 produced a uniform dispersion. α-MSH gel showed pseudoplastic flow with thixotropic behavior. Dermal microdialysis results suggested that skin permeation of gel after 5 h was 1.9-folds higher than the solution. Further, gel-treated psoriatic-like plaque skin sections showed significant (p < 0.05) decrease in the expression of a melanocortin receptor, in the psoriasis area and severity index score and transepidermal water loss compared to the solution. CONCLUSION: TC, NMP and Carbopol Ultrez 10 form a stable gel with improved skin permeation of α-MSH for a reduction in psoriasis-associated inflammation.

Effect of combination of hydrophilic and lipophilic permeation enhancers on the skin permeation of kahalalide F.

OBJECTIVES: The purpose of this study was to investigate the influence of combination of various lipophilic and hydrophilic chemical enhancers on skin delivery of kahalalide F (KF). METHODS: KF formulations comprising a combination of lipophilic and hydrophilic chemical enhancers with varied per cent were prepared and evaluated for skin permeation studies. In vitro skin permeation of KF formulations was performed using Franz diffusion cell. Stability studies of KF formulations were performed according to the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guideline, and the therapeutic efficacy of KF formulation was evaluated using allergic contact dermatitis animal model. KEY FINDINGS: The efficacy of KF formulations to improve skin delivery of KF was sequenced in the order of: formulation #4 > formulation #2 > formulation #1 > formulation #3, where formulation #4 contains labrasol (40% w/v), ethyl oleate (5% w/v) and span 80 (5% w/v) along with transcutol (40% w/v) and ethanol (10% w/v). Further, all the formulations were stable for 1 month when stored at 30°C/65% relative humidity. CONCLUSIONS: The results of present study suggest that therapeutically effective concentrations of KF can be delivered in the skin using combination of lipophilic and hydrophilic chemical enhancers.

Topical administration of dual siRNAs using fusogenic lipid nanoparticles for treating psoriatic-like plaques.

AIM: Psoriasis is a chronic autoimmune skin disorder with substantial negative impact on the patient's quality of life. The present study was carried out to demonstrate the efficiency of a novel topical delivery system in the transport of two siRNAs for the treatment of psoriatic-like plaques. MATERIALS & METHODS: We designed and developed a novel fusogenic nucleic acid lipid particle (F-NALP) system containing two therapeutic nucleic acids, anti-STAT3 siRNA (siSTAT3) and anti-TNF-α siRNA (siTNF-α). Novel cationic amphiphilic lipid with oleyl chains was synthesized and used in the nanocarrier system. Therapeutic efficacies of F-NALPs were assessed using an imiquimod-induced psoriatic-like plaque model. RESULTS: Hydrodynamic size and surface potential of F-NALPs were 102 ± 6 nm and 32.14 ± 6.21 mV, respectively. F-NALPs delivered fluorescein isothiocyanate-siRNA to a skin depth of 360 µm. F-NALPs carrying siSTAT3 and siTNF-α significantly (p < 0.05) reduced expression of STAT3 and TNF-α mRNAs and IL-23 and Ki-67 proteins compared with solution, and was superior in comparison with Topgraf(®) (GlaxoSmithKline Pharmaceuticals Limited, Maharashtra, India). CONCLUSION: Our observations demonstrate that F-NALPs can efficiently carry siSTAT3 and siTNF-α into the dermis and combination of the two nucleic acids can synergistically treat psoriatic-like plaques.

(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.

Investigation of follicular and non-follicular pathways for polyarginine and oleic acid-modified nanoparticles.

PURPOSE: To investigate the percutaneous permeation pathways of cell penetrating peptide modified lipid nanoparticles and oleic acid modified polymeric nanoparticles. METHODS: Confocal microscopy was performed on skin cultures (EpiDermFT™) for modified and un-modified nanoparticles. Differential stripping was performed following in vitro skin permeation of Ibuprofen (Ibu) encapsulated nanoparticles to estimate Ibu levels in different skin layers and receiver compartment. The hair follicles (HF) were blocked and in vitro skin permeation of nanoparticles was then compared with unblocked HF. The surface modified nanoparticles were investigated for response on allergic contact dermatitis (ACD). RESULTS: Surface modified nanoparticles showed a significant higher (p < 0.05) in fluorescence in EpiDermFT™ cultures compared to controls. The HF play less than 5% role in total nanoparticle permeation into the skin. The Ibu levels were significantly high (p < 0.05) for surface modified nanoparticles compared to controls. The Ibu levels in skin and receiver compartment were not significantly different when HF were open or closed. Modified nanoparticles showed significant improvement in treatment of ACD compared to solution. CONCLUSIONS: Our studies demonstrate that increased skin permeation of surface modified nanoparticles is not only dependent on a follicular pathway but also occur through non-follicular pathway(s).

Dermal microdialysis technique to evaluate the trafficking of surface-modified lipid nanoparticles upon topical application.

PURPOSE: To evaluate the skin pharmacokinetics and tissue distribution of cell penetrating peptides (CPP) modified nano-structured lipid carrier (NLC) using an in vivo dermal microdialysis (MD) technique. METHODS: Celecoxib (Cxb) encapsulated NLCs (CXBN), CPP modified CXBN (CXBN-CPP) and Cxb-Solution (CXBS) formulations were prepared and tested for in vitro skin distribution. MD was used to assess pharmacokinetic parameters of Cxb after topical application of Cxb formulations. The effect of pre-treatment with Cxb formulations was evaluated for expression of prostaglandin-E2 (PGE(2)) and Interleukin-6 (IL-6) after exposure of xylene using MD. Allergic contact dermatitis (ACD) model was used to confirm in vivo therapeutic response of Cxb formulations. RESULTS: The cumulative permeation of Cxb in MD dialysate after 24 h for CXBN-CPP was significantly higher (p < 0.001) than CXBN and CXBS. Further, pre-treatment with CXBN-CPP significantly inhibited PGE(2) and IL-6 expression compared to CXBS and CXBN (p < 0.001). In ACD model, CXBN-CPP showed significant reduction (p < 0.001) in ear thickness compared to controls. CONCLUSIONS: Surface modification of NLC with CPPs can enhance the skin permeation of Cxb and MD can be used to investigate pharmacokinetics of Cxb nanoparticles in the skin.

Enhanced skin permeation using polyarginine modified nanostructured lipid carriers.

The objective of the present study was to investigate the effect of polyarginine chain length on topical delivery of surface modified NLCs. Design of experiments (DOE) was used to optimize number of arginines required to deliver active drug into deeper skin layers. The NLCs were prepared by hot-melt technique and the surface of NLCs was modified with six-histidine tagged cell penetrating peptides (CPPs) or YKA. In vivo confocal microscopy and Raman confocal spectroscopy studies were performed using fluorescent dye encapsulated NLCs and NLC-CPPs. Spantide II (SP) and ketoprofen (KP) were used as model drugs for combined delivery. In vitro skin permeation and drug release studies were performed using Franz diffusion cells. Inflammatory response corresponding to higher skin permeation was investigated in allergic contact dermatitis (ACD) mouse model. NLCs had a particle size of 140±20nm with higher encapsulation efficiencies. The negative charge of NLC was reduced from -17.54 to -8.47 mV after surface modification with CPPs. In vivo confocal microscopy and Raman confocal spectroscopy studies suggested that a peptide containing 11 arginines (R11) had significant permeation enhancing ability than other polyarginines and TAT peptides. The amount of SP and KP retained in dermis after topical application of NLC-R11 was significantly higher than solution and NLC after 24 h of skin permeation. SP was not found in receiver compartment. However, KP was found in receiver compartment and the amount of KP present in receiver compartment was increased approximately 7.9 and 2.6 times compared to the control solution and NLCs, respectively. In an ACD mouse model, SP+KP-NLC-R11 showed significant reduction (p<0.05) in ear thickness compared to SP+KP solution and SP+KP-NLC. Our results strongly suggest that the surface modification of NLC with R11 improved transport of SP and KP across the deeper skin layers and thus results in reduction of inflammation associated with ACD.

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.

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.

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.