Preferential Accumulation of Phospholipid-PEG and Cholesterol-PEG Decorated Gold Nanorods into Human Skin Layers and Their Photothermal-Based Antibacterial Activity

www.nature.com/scientificreports OPEN Received: 14 November 2018 Accepted: 21 March 2019 Published: xx xx xxxx Preferential Accumulation of Phospholipid-PEG and CholesterolPEG Decorated Gold Nanorods into Human Skin Layers and Their Photothermal-Based Antibacterial Activity Nouf N. Mahmoud1, Ala A. Alhusban1, Jamila Isabilla Ali1, Amal G. Al-Bakri2, Rania Hamed1 & Enam A. Khalil2 Herein, a library of gold nanorods (GNR) decorated with polyethylene glycol-thiol (PEG-SH) containing different functionalities were synthesized and characterized by optical absorption spectroscopy, zeta potential, dynamic light scattering (DLS), transmission electron microscope (TEM) and proton nuclear magnetic resonance (1H-NMR). The colloidal stability of GNR when exposed to skin, and their preferential accumulation into excised human skin layers were investigated. Confocal laser scanning microscopy, transmission electron microscope (TEM) and inductively coupled plasmaoptical emission spectroscopy (ICP-OES) were utilized to track the penetration of GNR into different skin layers. The results demonstrated that cholesterol-PEG coated GNR were preferentially loaded up in the upper layers of skin (stratum corneum), while phospholipid-PEG coated counterparts were drastically deposited in skin dermis. Neutral methoxy-PEG-coated GNR were distributed in both SC and dermis skin layers, while charged GNR (anionic-carboxylic acid-PEG-GNR and cationic-amine-PEGGNR) revealed a minimal accumulation into skin. DSPE-PEG-GNR and Chol-PEG-GNR demonstrated antibacterial activities against Staphylococcus aureus (S aureus) at MIC values of 0.011 nM and 0.75 nM, respectively. Photothermal treatment for S. aureus at sub-MIC concentrations resulted in a significant bactericidal effect when using Chol-PEG-GNR but not DSPE-PEG-GNR. Gold-based nanoscale systems have great value as a promising platform for skin diseases therapy. Skin is considered as the ideal site for non-invasive skin therapeutic platforms1,2. Stratum corneum (SC) consists of proteins and lipid matrix and limits the penetration of the topically applied drugs3–5. Chemical and physical penetration enhancers are commonly used to modulate the skin morphology at the molecular level6,7. Furthermore, dermal and transdermal drug delivery nanocarriers, such as polymers, liposomes, lipids and inorganic nanoparticles, can retard drugs’ degradation, prolong their release, and enhance their solubility8–11. Among nano-systems, gold nanoparticles (GNP) and in particular, gold nanorods (GNR) have important applications in nanomedicine such as imaging, sensing, bio-tracking and photothermal therapy owing to their optical properties12–15. GNP of different shapes, sizes and surface functionalities were employed to probe the skin-nano interface16. For example, Sonavane et al. have demonstrated that the small-sized GNP penetrated into skin dermis17, whereas Fernandes et al. revealed that GNP with rod-like shape have better penetration through skin compared to spherical counterparts18. Besides, negatively-charged skin components have repelled anionic nanoparticles, while higher affinity to SC has been reported using cationic nanoparticles19. Moreover, the surface 1 Department of Pharmacy, Faculty of Pharmacy, Al-Zaytoonah University of Jordan, Amman, 11733, Jordan. Department of Pharmaceutics & Pharmaceutical Technology, School of Pharmacy, The University of Jordan, Amman, 11942, Jordan. Correspondence and requests for materials should be addressed to N.N.M. (email: nouf. ) 2 Scientific Reports | (2019) 9:5796 | https://doi.org/10.1038/s41598-019-42047-7 1 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 1. Illustration of different ligands containing PEG-SH used to functionalize the surface of GNR. (A) Methyl; (B) Amine; (C) Carboxylic acid; (D) Phospholipid (DSPE); and (E) Cholesterol. modification of nanoparticles, such as proteins, lipids and oleic acids has increased the permeability and accumulation of nanoparticles into different skin compartments18,20–23. Phospholipids, the main component of the cell membrane, have been used as a biocompatible surface coating agent for many nanoparticles such as graphene, silica, inorganic nanoparticles and quantum dots24– 27 . Lipid-coated nanomaterials have great potentials for biomedical applications. For example, clusters of phospholipid-coated GNP and lipid-coated GNP have been used to enhance the theranostic effects, cellular uptake, drug delivery and cancer therapy of GNP28–32. Accumulation of nanomaterials at specific skin sites has a great impact on dermal drug delivery and tracking, in addition to their applications in diagnosis and skin diseases therapy33. In our earlier work, hydrophobic GNR coated with polystyrene-thiol were designed to be preferentially accumulated into human skin hair follicles, with minimal dermal distribution, as a promising platform for photothermal treatment of acne34. However, the interaction of phospholipid and cholesterol-modified-GNR with human skin and their accumulation potentials into skin layers have not been investigated fully in the literature. The unique photothermal properties of GNR make them attractive anticancer and antibacterial candidates. GNR can convert the absorbed light into intense localized heat that could be effective for cancer and bacterial photothermal ablation15. Recently, the photo-thermolysis activity of GNR of hydrophilic or hydrophobic surface properties was evaluated against common skin bacteria such as Propionibacterium acnes and Staphylococcus aureus (S. aureus)35. In addition, photothermal ablation was reported for GNR conjugated to antibodies against Pseudomonas aeruginosa36. Taking into consideration the unique advantages of GNR as a “nano-model”, their superiority in synthesis, colloidal stability, drug delivery and therapy, GNR decorated with PEG-thiol containing different functionalities were synthesized in this study, and their colloidal stability upon human skin contact was investigated. Employing confocal laser scanning microscopy, transmission electron microscopy (TEM) and inductively coupled plasma-optical emission spectroscopy (ICP-OES), the skin penetration and preferential accumulation of GNR decorated with phospholipid or cholesterol conjugated to PEG-thiol (SH) were probed and compared to that of GNR decorated with methoxy (m)-PEG or charged PEG moieties (carboxylic acid or amine groups). The effect of particle size and vehicle on the accumulation of GNR into skin layers was evaluated. Moreover, the antibacterial and photothermal-based antibacterial activities of DSPE-PEG-GNR and Chol-PEG-GNR were evaluated against S. aureus, a common skin pathogen. Results and Discussion Synthesis and characterization of GNR of different particle sizes and coated with PEG-SHcontaining different functionalities. CTAB-assisted synthesis protocol was used to prepare GNR having aspect ratio (length/width; AR) of ~4 and ~2. (...truncated)