Dermal And Cellular Delivery Of An Endogenous Anti-ageing Peptide Using Niosomes

Abstract

Aim: The overall aim of this Thesis was to investigate the physical modification by encapsulation of GHK-Cu, a hydrophilic anti-ageing peptide, with lipid based nano-vesicles to address the barriers to cutaneous and cellular delivery. The influence of the surface charge of the vesicles was particularly studied as it was hypothesized that a cationic charge within the niosomes would increase both cellular and trans-epidermal delivery. The specific objectives were to; (1) evaluate the key physicochemical properties of GHK-Cu for the eventual development of formulations for dermal delivery of this peptide, (2) develop niosomes as a nano-carrier system for GHK-Cu for the potential delivery into the skin, (3) evaluate whether niosomes could act as a potential delivery system to increase cellular absorption as well as trans-epidermal delivery, and (4) evaluate whether the biological effect of GHK-Cu can be improved through niosomal delivery, particularly with cationic niosomes. The mechanisms by which were explored. Methods: The physicochemical properties of GHK-Cu were characterized using conventional methods. High performance liquid chromatography and mass spectrometry was utilised for quantification of the parent compound and identification of degradation products. GHK-Cu was incorporated into niosomal carriers characterised by laser diffraction, transmission electron microscopy (TEM) and zeta potential analysis. Cationic niosomes were chosen and used for the experiments in this Thesis following optimization. A non-ionic niosome of the same composition was used as a reference. Human dermal fibroblasts were used for cellular uptake and intracellular trafficking investigations, imaged with confocal scanning laser microscopy (CLSM) and TEM. All trans-epidermal investigations were undertaken using ex vivo viable human skin using calcein as a fluorescent marker enabling observation with CLSM. The biological response of both niosomal delivery systems was firstly assessed using metalloproteinases (MMP-1, collagenase and MMP-2, gelatinase) and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) as markers in cultured primary human dermal fibroblasts. These markers were measured through their gene expression by real time quantitative polymerase chain reaction (RT-qPCR), normalised to the control. Additionally, the collagen and elastin contents was measured in fibroblast cells following GHK-Cu incubation. The Thesis culminated in a double blind placebo split face clinical trial of 40 participants to determine the effect of the encapsulated niosomal GHK-Cu on wrinkle depth and volume reduction. Results: Preformulation studies indicated GHK-Cu was freely water soluble (325.09 ± 4.38 mg mL-1) and hydrophilic with a logD of -2.49 through -2.38 over pH 7.4 to 4.5. Decreasing the pH caused the reversible dissociation of Cu ions from GHK. This effect proceeded rapidly from pH 4.26 downwards and was primarily caused by the protonation of the imidazole and glycyl residues. Therefore at the HPLC mobile phase with a pH of 2, it was only the GHK peptide that was measured. GHK eluted at 8.02 ± 0.10 min under isocratic conditions making it ideal for routine analysis. The LOD and LOQ were determined to be 0.55 and 1.8 μg mL-1, respectively. The validation data, including that from mass spectrometry, proved the assay to be stability-indicating. Forced degradation studies identified hydrolysis as the main pathway of degradation, leading to the identification of three new products. The final product was verified with injections of pure histidine, a component amino acid. GHK was stable in water and in phosphate bufferered saline (0.1 M) at pH 7.4. The logD and solubility results incentivized the consideration of a lipid-based carrier system, in particular niosomes, to enhance the dermal delivery of GHK-Cu. A niosomal lipid based carrier system was subsequently developed for encapsulating GHKCu. The size, surface charge and loading efficiency of the niosomes were optimized to enhance dermal delivery. A positive charge was obtained following post-insertion of didecyldimethylammonium bromide (DDAB) into the surface of Span 60/cholesterol based niosomes. The niosomes had a final size of 225.8 ± 7.7 nm for cationic and 193.3 ± 5.2 nm for non-ionic niosomes. A positive surface charge of 49.4 ± 1.2 mV was observed in cationic niosomes with a maximum entrapment efficiency of 9.7 ± 0.2% (w/w) of GHK-Cu was achieved for both systems. Calcein, having similar physicochemical properties to GHK-Cu, was chosen for CLSM studies. The mechanism of intracellular uptake, confirmed by TEM imaging, was clathrinmediated endocytosis. This evidence was primarily supported by the size, lysosome formation and clathrin pit formation in fibroblasts. ImageJ analysis of the confocal images showed a slight increase in intensity (83 vs 79) in the cationic treated samples over the nonionic treated ones. A larger number of lysosomes were imaged in cationic niosome treated cells. Endosomal escape was determined to be by destabilisation resulting from both a cationic lipid and a surfactant interaction with the endosomal membrane without affecting the viability of the cells. This was determined using a MTT test, which resulted in all concentrations tested, except that of cationic niosomes at 10-7 M, having no detrimental effect on cell viability. CLSM images revealed that free calcein could not cross the stratum corneum. Niosomes however, both cationic and non-ionic, caused adsorption onto the surface forming a critical thermodynamic activity gradient, driving permeation into the dermis. Upon incubation with niosomes for 24 hours, fibroblasts showed greater expression of MMPs (> 2-fold, p<0.05) and TIMPs (>2-fold, p<0.05). The GHK-Cu solution showed a dosage dependent response in all MMPs and TIMPs whereby higher concentrations caused a decreased expression. The collagen and elastin production with cells incubated with the same concentrations of GHK-Cu, also showed a dosage dependent response for the solution. The higher the concentration of GHK-Cu the less collagen and more elastin were measured. Correlation between production and expression results showed that decreased MMP and TIMP gene expression results in more elastin and less collagen synthesis. The ratios of TIMP-1/MMP-1 were all >1 suggesting a trend towards decreases in collagen degradation. The ratios of TIMP-2/MMP-2 were <1, suggesting a trend towards increases in elastin degradation. Expression results from viable human skin upon the direct topical application of GHK-Cu, in solution and niosomes, could not be correlated to the in vitro results. In most cases, there was an overall decreased expression of MMPs and TIMPs in the skin after 24 hours application. At a concentration of 10-9 M, GHK-Cu, the cationic and non-ionic niosomes produced the lowest response in MMP-1 expression. Lower MMP-1 expression is indicative of slower collagen degradation. MMP-2 and TIMP-2 results were strikingly similar, providing further evidence to existing proof that these two proteins are linked. Cationic niosomes were chosen for in vivo clinical studies based on the TIMP-1/MMP-1 ratio, which was the highest of all the formulations examined. Considering that the dermis is between 70 – 80% collagen, an excess of collagenase inhibitors would be most beneficial for cosmetic anti-wrinkle effects. The double blind placebo split face clinical trial compared the nano-carrier (cationic niosome) encapsulated GHK-Cu, a lipophilic derivative of GHK (Matrixyl® 3000, Strivectin SD Advanced Concentrate) and a placebo (the serum without the GHK-Cu or niosomes). As a measure of activity, there was significant improvements (p<0.005) in wrinkle volume reduction compared to both the placebo and lipophilic derivative. Wrinkle depth also improved the most, however this was only moderately significant (p=0.057). The difference in wrinkle depth and volume versus the placebo indicate the effectiveness of the cationic niosomal GHK-Cu in topical delivery. The cationic encapsulated GHK-Cu was tolerated as well as the marketed Matrixyl® 3000 product with only one participant reacting to both. Unfortunately the non-ionic niosome formulation was not tested due to financial limitations of the company. Conclusion: GHK-Cu was effectively encapsulated into cationic and non-ionic niosomes and effectively delivered into cells and passed the epidermis with significant biological effect. The in vitro and ex vivo studies essentially proved that cationic encapsulation of GHK-Cu was marginally better than the non-ionic counterpart for intracellular and trans-epidermal delivery. This research, for the first time, found that GHK-Cu could promote the cellular expression of MMP-1, MMP-2 and their inhibitors TIMP-1, TIMP-2 in human fibroblasts, and that it is their ratios that decided the changes in their substrates, collagen and elastin content on/in the cells. The in vivo clinical evidence suggests that dermal delivery of GHKCu in cationic niosomes results in significant wrinkle volume and depth reduction with accumulative improvements.