J Phys Chem B.2016 Jul;120(29):7133-7142
Penetration of Gold Nanoparticles through Human Skin: Unravelling Its Mechanisms at the Molecular Scale
Rakesh Gupta and Beena Rai*
TCS Innovation Labs, TATA Research Development & Design Centre, Pune 411013, India
Recent experimental studies suggest that nanosized gold nanoparticles (AuNPs) are able to penetrate into the deeper layer (epidermis and dermis) of rat and human skin. However, the mechanisms by which these AuNPs penetrate and disrupt the skin’s lipid matrix are not well understood. In this study, we have used computer simulations to explore the translocation and the permeation of AuNPs through the model skin lipid membrane using both unconstrained and constrained coarse-grained molecular dynamics simulations. Each AuNP (1–6 nm) disrupted the bilayer packing and entered the interior of the bilayer rapidly (within 100 ns). It created a hydrophobic vacancy in the bilayer, which was mostly filled by skin constituents. Bigger AuNPs induced changes in the bilayer structure, and undulations were observed in the bilayer. The bilayer exhibited self-healing properties; it retained its original form once the simulation was run further after the removal of the AuNPs. Constrained simulation results showed that there was a trade-off between the kinetics and thermodynamics of AuNP permeation at a molecular scale. The combined effect of both resulted in a high permeation of small-sized AuNPs. The molecular-level information obtained through our simulations offers a very convenient method to design novel drug delivery systems and effective cosmetics.
Researches from both pharmaceutical and cosmetic industries have shown considerable interest in skin models, both in-vitro and in-silico. The accurate prediction of dermal uptake of chemicals is relevant to both transdermal drug delivery as well as topical application of cosmetics. Understanding permeation mechanism of molecules through the skin shall help in designing novel transdermal drug delivery systems. On the similar note, minimization of permeation of nanoparticles is of great interest to the cosmetic industry. The current industry standard, however, both in pharma and cosmetics, is to conduct detailed experimental trials. These obviously incur huge expenses thereby leading to a very few successful candidates that are finally approved by regulatory authority (FDA). The 2-D in-vitro cell culture studies do not accurately reflect the complex interactions that occur between the multiple cells present in the 3-D in-vivo skin environment. In-vivo studies in rodents and other small animals do not translate well to the human situation due to differences in anatomical structures. Though some of the commercially available human skin equivalents EpiSkin® (L’Oreal, Paris) and EpiDermTM (MatTek, Massachusetts) are available in the market, these require highly specialized skills and are very expensive. The European Union (EU) regulation (76/768/EEC, Feb. 2003) prohibits the use of animal or animal-derived substances for the development and testing of cosmetic and pharmaceutical ingredients. Considering the time and costs involved in the development and testing of new drug/cosmetics formulations, it is imperative to supplement/replace some of the elaborate in-vivo/in-vitro tests with in-silico tests.
At TCS innovation labs, we are involved in the development of a realistic in-silico model of human skin based on the multiscale modelling approach. Some of the highlights of our research is presented below:
Skin Barrier Function: The outer layer of skin, also known as Stratum Corneum (SC) is mainly responsible for skin’s barrier properties. The SC mainly consists of corneocytes (brick) and lipid matrix (mortar). The corneocytes are mostly impermeable and molecules penetrate through lipid matrix. These lipid matrix are the key determinant for the barrier functions and is mainly composed of heterogeneous mixture of long chain ceramides (CER), cholesterol (CHOL) and free fatty acid (FFA) in certain ratios.
Model Development and Validation: We first developed a model of skin SC at different molar ratio of individual components and tested it with available experimental data. The paper describing the effect of temperature and molar ratio on skin lipid properties was published in 2015 (1). It was reported that CHOL increases the stability of the mixed bilayer while the pure ceramide bilayer disintegrated around∼390 K. The CHOL molecule provided more rigidity to the mixed bilayer and led to a more ordered phase at elevated temperatures. The CHOL molecule provided fluidity to the bilayer below the phase transition temperature of CER and kept the bilayer rigid above the phase transition temperature. The presence of CHOL increases the compressibility of the bilayer which is responsible for the high barrier function of skin.
Figure 1. Skin model development: Atomistic skin model development at different molar ratio of skin lipids matrix constituents. The ratio (in the inset) stands for CER: CHOL: FFA. Individual molecules were placed randomly on XY plane and then replicated along the Z direction which was followed by solvation by SPC water.
Model Testing and Comparison: Later we used this model to compute the permeability, of molecules by employing constrained MD simulation on skin lipids bilayer. The developed model provides molecular mechanism of permeation of hydrophilic and hydrophobic molecules and also gives qualitative trend of permeability with experiments (2). In this study, the lipid matrix was modelled as an equimolar mixture of ceramide (CER), cholesterol (CHOL), and free fatty acid (FFA). The permeation of water, oxygen, ethanol, acetic acid, urea, butanol, benzene, dimethyl sulfoxide (DMSO), toluene, phenol, styrene, and ethylbenzene across this layer is studied using constrained MD simulations technique. The main resistance for the permeation of hydrophilic and hydrophobic permeants has been found to be in the interior of the lipid bilayer and near the lipid−water interface, respectively. The obtained permeability is found to be a few orders of magnitude higher than experimental values for hydrophilic molecules while for hydrophobic molecules more discrepancy was observed. Overall, the qualitative ranking is consistent with the experiments.
Figure 2. Skin lipid bilayer model testing. a) Resistance of permeation of hydrophobic and hydrophilic molecules along the bilayer normal Z. b) Snapshot of system of multiple molecules constrained in single window and c) comparison of computed permeability of hydrophobic and hydrophilic molecules with experiments.
Skin Disease Mechanism and Healing: The skin barrier function is mainly attributed to the presence of ceramides. In fact, experiments have showed that the reduced or altered levels of ceramides were found in the skins that were diseased with atomic dermatitis, ichthyosis, and psoriasis. Moreover, these lipid barrier abnormalities were corrected to a promising extent by topical application of ceramides or their synthetic analogues. To understand the mechanism of this whole process, we have modelled the skin bilayer comprising of ceramide of different chain length and reported on the water permeability through this skin layer (3). The barrier properties were examined by means of permeation studies of water through the model membrane using steered MD simulations. It was shown that shorter chains of one leaflet of the bilayer do not interdigitate with the chains of the other leaflet and lead to more free space in the middle of the bilayer, thereby leading to higher permeability. In CERs with dissimilar chain lengths, the lipids on one chain interdigitate with the other leaflet lipids and increasing the electron density in the middle of the bilayer. The bilayer thickness increases with increase in the CER chain length. The permeability of smaller-chain CERs was found to be an order of magnitude higher than that of the longer-chain CERs.
Figure 3. Skin barrier function deficiency: a) Snapshot of each bilayer system at the end of 500 ns unconstrained simulation. The CER chains, head group oxygen, nitrogen and hydrogen and water are shown in red, blue, white and pink colour, respectively. The mid plane of tail and mid plane of bilayer correspond to z ~ 1.2-1.4 and z~0, respectively. b) Electron density profile along the bilayer normal Z in each bilayer and c) change of permeability of skin lipid bilayer with ceramide chain length. The sn1 chain length was fixed while sn2 chain length was varied from 8 to 24.
Permeation of Gold Nanoparticle and Co-Delivery of Drugs: The paper describing permeation mechanism of gold nanoparticle of different sizes was published recently in 2016 (4). Each gold nanoparticle (2-6 nm) disrupted the bilayer packing and entered in the interior of the bilayer rapidly. There was a trade-off between the kinetics and thermodynamics of AuNP permeation at a molecular scale. The combined effect of both resulted in a high permeation of small-sized AuNPs. The molecular-level information obtained through our simulations offers a very convenient method to design novel drug delivery systems and effective cosmetics. Currently we are designing nanoparticles for co-delivery of proteins and drugs through skin.
Figure 4. Gold nanoparticle permeation and self- healing: a) Self-healing of the skin after the permeation of the 6nm gold nanoparticle b) final snapshot of the skin bilayer in presence of the 6nm gold nanoparticle c) skin permeability of different sized gold nanoparticles.
The importance of these studies is two-fold. First, one can obtain the molecular mechanism of drug/molecules permeation through skin which could be used for designing or developing new drugs/vehicle/carrier. Second, the developed models and permeability protocols can be used as supplement to experiments to cut down the screening and testing efforts in terms of cost and time.
- Gupta, R. and Rai, B., 2015. Molecular Dynamics Simulation Study of Skin Lipids: Effects of the Molar Ratio of Individual Components over a Wide Temperature Range.The Journal of Physical Chemistry B, 119(35), pp.11643-11655.
- Gupta, R., Sridhar, D.B. and Rai, B., 2016. Molecular Dynamics Simulation Study of Permeation of Molecules through Skin Lipid Bilayer.The Journal of Physical Chemistry B, 120(34), pp.8987-8996.
- Gupta, R., Dwadasi, B.S. and Rai, B., 2016. Molecular Dynamics Simulation of Skin Lipids: Effect of Ceramide Chain Lengths on Bilayer Properties.The Journal of Physical Chemistry B.
- Gupta, R. and Rai, B., 2016. Penetration of Gold Nanoparticles through Human Skin: Unraveling Its Mechanisms at the Molecular Scale.The Journal of Physical Chemistry B, 120(29), pp.7133-7142.
Beena Rai, Ph.D.
Principal Scientist & Head
Tata Research, Development & Design Centre
Tata Consultancy Services
54B, Hadapsar Industrial Estate, Pune – 411013
Pune – 411013
Tel: 91-20- 6608 6203 (direct)