Laser-assisted nanoparticle delivery to promote skin absorption and penetration depth of retinoic acid with the aim for treating photoaging

Abstract

Retinoic acid (RA) is an approved treatment for skin photoaging induced by ultraviolet (UVA). Topically applied RA is mainly located in the stratum corneum (SC) with limited diffusion into the deeper strata. A delivery system capable of facilitating dermal delivery and cellular internalization for RA is critical for a successful photoaging therapy. Two delivery approaches, namely nanoparticles and laser ablation, were combined to improve RA’s absorption efficacy and safety. The nanoparticle absorption enhancement by the lasers was compared between full-ablative (Er:YAG) and fractional (CO₂) modalities. We fabricated poly-L-lactic acid (PLA) and PLA/poly(lactic-co-glycolic acid) (PLGA) nanoparticles by an emulsion-solvent evaporation technique. The mean size of PLA and PLA/PLGA nanocarriers was 237 and 222 nm, respectively. The RA encapsulation percentage in both nanosystems was > 96 %. PLA and PLA/PLGA nanocarriers promoted RA skin deposition by 5- and 3-fold compared to free control. The ablative lasers further enhanced the skin deposition of RA-loaded nanoparticles, with the full-ablative laser showing greater permeation enhancement than the fractional mode. The skin biodistribution assay evaluated by confocal and fluorescence microscopies demonstrated that the laser-assisted nanoparticle delivery achieved a significant dermis and follicular accumulation. The cell-based study indicated a facile uptake of the nanoparticles into the human dermal fibroblasts. The nanoparticulate RA increased type I collagen and elastin production in the UVA-treated fibroblasts. A reduction of matrix metalloproteinase (MMP)-1 was also highlighted in the photoaging cells. The calculation of therapeutic index (TI) by multiplying collagen/elastin elevation percentage and skin deposition predicted better anti-photoaging performance in Er:YAG laser-assisted nanoparticle delivery than CO₂ laser. Nanoencapsulation of RA decreased the cytotoxicity against skin fibroblasts. In vivo skin tolerance test on a nude mouse showed less skin damage after topical application of the nanoparticles than free RA. Our results hypothesized that the laser-mediated nanoparticle delivery provided an efficient and safe use for treating photoaging.

Introduction

The aging sign of skin offers the first mark of the passing time, caused by intrinsic or extrinsic factors. Skin exposure to ultraviolet (UV) irradiation from sunlight is the main source (90 %) of extrinsic factors. The photoaging induced by UV produces DNA and reactive oxygen species (ROS) generation, resulting in increased matrix metalloproteinases (MMPs) and collagen degradation (Lee et al., 2021a, Lee et al., 2014, Lee et al., 2019, Lee et al., 2021b). Photoaged skin shows the appearance of wrinkling, sagging, laxity, discoloration, and roughness. In addition to photodamage on the skin, the aging appearance causes an emotional and psychological impact, reducing life quality (Campione et al., 2021). The topical treatment of retinoic acid (RA), a vitamin A metabolite, is approved for the application of anti-photoaging therapy to mitigate wrinkles, skin damage, and elasticity loss (Spierings, 2021). RA is verified to have the bioactivities of cell differentiation, embryonic development, immune response modulation, and homeostasis of epithelial tissue (de Mendonça Oliveira et al., 2018). Through the RA receptor- and retinoid X receptor-mediated gene activation, RA increases epidermal thickness and inhibits wrinkling via MMP-1 suppression and type I collagen increase in photoaged skin. RA is extensively used to treat skin disorders including psoriasis, acne, ichthyosis, cutaneous cancer, and photoaging (Szymański et al., 2020). Although topical RA is best applied to dermatology, the drawbacks of this drug are the photochemical degradation and cutaneous irritation, such as peeling, erythema, and xerosis (Ferreira et al., 2020). The poor stratum corneum (SC) penetration and insufficient bioavailability in the dermis also restrain the successful therapeutic efficacy of RA (Limcharoen et al., 2020).

The drug delivery into the skin can be enhanced by ablative approaches, such as microneedles, radiofrequency, and laser irradiation (Hsiao et al., 2019). Among these, laser-assisted drug delivery improves dermal bioavailability to accomplish a greater therapeutic effect (Wenande et al., 2020). Ablative lasers partially or completely remove SC and disintegrate skin structure to promote drug permeation. Fractional lasers create an array of microchannels to bypass the SC barrier. The advantages of laser-assisted delivery include the precise control of ablative skin depth with minor cross-contamination risk compared to microneedles and direct SC stripping (Garvíe-Cook et al., 2016). Although the lasers are highly efficient in reducing the cutaneous barriers to enhance drug absorption, the subsequent intracellular drug entrance for exhibiting pharmacological activities is still challenging. Furthermore, conventional vehicles for RA are limited due to poor aqueous solubility, storage instability, and skin damage (Morales et al., 2015). Nanoparticles can be ideal carriers for RA to avoid photochemical degradation and elevate RA internalization into the target cells. The topically applied nanocarriers show the possibility to largely accumulate in the skin, improving the delivery efficiency of the drugs (Carter et al., 2019). The transport of intact nanoparticles into the skin is difficult. Previous studies (Charoenputtakun et al., 2014, Ogunjimi et al., 2021) demonstrate that no intact RA-loaded nanoparticles diffuse into the skin. As evidenced by Bellefroid et al. (2019), the permeation of nanoparticles can only be detected after SC removal. Effective photoaging treatment by RA demands the skin barrier elimination and the facile entrance into the cells. We aimed to investigate the effect of laser-assisted nanoparticle delivery for facile and deeper RA penetration. In particular, a cell-based model estimated the anti-photoaging activity of the nanocarriers.

The most commonly used ablative lasers for drug delivery enhancement are erbium:yttrium-aluminum-garnet (Er:YAG) and CO₂ modalities (Haedersdal et al., 2016). In this study, the full-ablative Er:YAG and fractional-ablative CO₂ lasers were employed to appraise the laser-mediated transport of RA-loaded nanoparticles. We used poly-l-lactic acid (PLA) and poly(lactic-co-glycolic acid) (PLGA) as the main materials to produce polymer-based nanocarriers for entrapping RA. Both polymers are approved by USUDA as biocompatible and biodegradable materials with easy surface modification, targeting capability, good stability, and controlled drug leakage (Qi et al., 2019). The animal-based study for drug tests is a matter of controversy concerning animal sentience. The 3R rule (replacement, reduction, and refinement) about animal welfare is globally accepted as the ethical framework when the animal experiment is conducted. For replacement and reduction, the application of in vitro model as the substitution for in vivo study can be cheaper and faster than the animal model (Hubrecht and Carter, 2019). For these reasons, we reduced the animals used in this work. Instead, the in vitro cell study was performed for the evaluation of anti-photoaging efficacy of the nanocarriers. The in vivo anti-photoaging activity was anticipated by calculating the therapeutic index (TI) based on the multiplication of collagen/elastin inhibition percentage in dermal fibroblasts and in vitro RA absorption amount. We only used 24 nude mice to examine the tolerance of nanocarriers on skin. It was because that the real test of the topical drug formulation on skin is still required to explore the cutaneous irritation. The cell-based study is difficult to understand the authentic skin safety of the drug in the clinical condition until now.