Enhancing the Photothermal Properties of Indocyanine Green in Melanoma Spheroids via Encapsulation in Span80-Containing Lipid Nanocapsules

Abstract

Indocyanine green (ICG), a well-known photosensitiser, has shown potential in photothermal therapy (PTT) for cancer treatment, but its effectiveness is limited by poor skin penetration and rapid clearance. To address this, lipid nanocapsules (LNCs) were used as nanocarriers to enhance ICG’s cellular uptake and photothermal (PT) performance in melanoma cells. Utilising our recently developed Span 80-modified LNCs (LNC100-S8) with high biocompatibility and enhanced cellular uptake in B16F10 melanoma cells, ICG was loaded into LNC100-S8 using the phase inversion temperature method. The results showed that ICG encapsulation at 4.5 mg/mL maintained LNC sizes (95-105 nm). Moreover, the heating capacity of ICG in LNCs was approximately 1.5 times higher than free ICG, achieving temperature increases over 10°C post-irradiation. In cell cancer monolayers, LNC100-S8 enhanced ICG uptake by 1.5 times compared to free ICG and reduced cell viability to 50% following 808 nm laser irradiation. More promisingly, ICG-LNC100-S8 combined with laser irradiation significantly reduced three-dimensional B16F10 spheroids size up to 11 days post-treatment compared to free ICG. Overall, our findings validate LNC100-S8, as promising nanocarriers for enhancing ICG-based PTT, supporting their potential applications in vivo to treat melanoma and other skin cancers.

Introduction

Photothermal therapy (PTT) is a minimally invasive approach used to ablate tumours by converting light energy into heat, leading to localised hyperthermia and subsequent tumour cell apoptosis and necrosis (Chen et al., 2016; Shiyi Zhou, 2020). Compared to traditional cancer treatment modalities, such as chemotherapy or surgery, PTT is minimally invasive and has fewer side effects (Yaseen et al., 2007). PTT involves introducing a photosensitiser into the body, followed by laser irradiation to achieve the optimal temperature increase for tumour cell ablation (Lopes et al., 2022; Shiyi Zhou, 2020). Near-infrared (NIR) irradiation is advantageous for PTT, as NIR light penetrates deeper into tissues, allowing for more effective and localised tumour ablation (Liang et al., 2021). NIR-induced PTT using the non-invasive delivery of a photosensitiser holds great promise for patients with inoperable topical and near-topical cancers (Totonchy & Leffell, 2017).

Indocyanine green (ICG), is a fluorescent dye approved by the U.S. Food and Drug Administration (FDA) for angiography and lymph node biopsy. Preclinically, it has gained attention for its potential in PTT (Ntziachristos et al., 2000). ICG effectively converts light into thermal energy when exposed to NIR irradiation, making it a key component in laser-mediated PTT (Shirata et al., 2017; Zheng et al., 2011). Once delivered to the tumour site, ICG is exposed to NIR light, generating cytotoxic heat that ablates cancer cells highlighting its potential for treating topical cancers in delicate regions, such as the face or near nerves and blood vessels (Totonchy & Leffell, 2017; Yoon et al., 2017; Yorozu et al., 2022). Apart from cancer, ICG showed potential in phototherapy to treat superficial acne vulgaris with no skin irritation following ICG application to the face and the back (Genina et al., 2004). ICG also has shown a skin rejuvenation effect in vivo with significant improvements in wrinkles, pores, and hyperpigmentation (Jung et al., 2024). All of these reports make ICG not only a potential photosensitiser but also a skin-friendly agent (Topaloğlu et al., 2020). However, to overcome ICG degradation, and thermal instability, and improve its skin penetration (Jung et al., 2018), ICG encapsulation in skin compatible drug delivery system can open the door for ICG repurposing as a photothermal agent to treat skin cancers, such as basal cell carcinoma and melanoma.

Lipid nanocapsules (LNCs) are a promising drug delivery system due to their unique physicochemical properties, which enable efficient drug encapsulation and release (Huynh et al., 2009). LNCs consist of an oily core, a non-ionic surfactant shell and an aqueous continuous phase. They can encapsulate both hydrophobic and amphiphilic drugs (Hoarau et al., 2004). The shell of LNCs is composed of biocompatible and biodegradable excipients, such as the non-ionic surfactant Kolliphor HS15 and the amphiphilic co-surfactant phospholipids. The oil core is composed of caprylic/capric triglyceride (Labrafac), a key component that enhances lipophilic drug delivery by providing a solubilising medium for hydrophobic drugs (Idlas et al., 2021). LNCs are highly suitable for transdermal applications due to their small particle size (20–100 nm), high encapsulation for hydrophobic and amphiphilic drugs and long-term stability (Dabholkar et al., 2021). Moreover, LNCs have been shown to be more suitable for transdermal applications than other nanocarriers, such as nanocrystals (Hatahet et al., 2018). The ability of LNCs to facilitate deeper penetration into the skin is particularly advantageous for photothermal therapy (PTT), ensuring sufficient concentrations of the photosensitiser, such as ICG, at the tumour site for effective tumour ablation.

Recently, our group has recently developed Span 80-modified LNCs (LNC100-S8) with high safety in melanoma cell lines (Wu et al., 2025). We showed that a partial replacement of Kolliphor HS15 with Span 80 (35% Span 80 with 65% Kolliphor HS15 as surfactant) reduced the in vitro cytotoxicity on B16F10 cells compared to conventional LNC100-0 (100% Kolliphor HS15), while improving their cellular uptake (Wu et al., 2025). In the current study, we further investigated LNC100-S8 as a nanocarrier for ICG delivery and assessed their physicochemical properties and heating capacity in solution. Then, ICG-LNC100-S8 cell uptake was assessed in the B16F10 cell line, and their photothermal effect was evaluated in 2D and 3D cell models. Promisingly, our findings confirmed the successful loading of ICG into LNC-100-S8 to enhance ICG thermal properties, resulting in superior cell killing in B16F10 melanoma cells and spheroids. The present study highlights the potential of ICG-LNC100-S8 for PTT in skin cancer, which lays the foundation for future skin permeability testing and PPT in vivo.

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Materials

Kolliphor® HS15 (Macrogol-15-hydroxystearate), Span 80 (Sorbitan monooleate), Acetonitrile (CH3CN), Dimethyl sulfoxide (DMSO), Sodium chloride (NaCl) and Phosphate buffered saline (PBS) tablets were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipoid S 75 (Phospholipids with 70 % phosphatidylcholine) was provided as a free sample from Lipoid GmbH (Ludwigshafen, Germany). Labrafac™ lipophile WL 1349 (Medium chain triglycerides) was obtained as a free sample from Gattefossé (Saint-Priest, France). Indocyanine Green (ICG) was purchased from AdooQ BioScience (Irvine, CA, USA). RPMI 1640 medium, Dulbecco’s Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), penicillin-streptomycin, sodium pyruvate, methylcellulose, resazurin, and trypsin-EDTA were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Sheath fluid was purchased from BD FACSFlow (Franklin Lakes, NJ, USA). PD-10 column was purchased from GE Healthcare (UK).

Siyang Wu, Taher Hatahet, Wafa Al-Jamal, Enhancing the Photothermal Properties of Indocyanine Green in Melanoma Spheroids via Encapsulation in Span80-Containing Lipid Nanocapsules, European Journal of Pharmaceutical Sciences, 2025, 107049, ISSN 0928-0987, https://doi.org/10.1016/j.ejps.2025.107049.

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