Development and Evaluation of Docetaxel-Loaded Nanostructured Lipid Carriers for Skin Cancer Therapy

This study focuses on the design, characterization, and optimization of nanostructured lipid carriers (NLCs) loaded with docetaxel for the treatment of skin cancer. Employing a systematic formulation development process guided by Design of Experiments (DoE) principles, key parameters such as particle size, polydispersity index (PDI), zeta potential, and entrapment efficiency were optimized to ensure the stability and drug-loading efficacy of the NLCs. Combined XRD and cryo-TEM analysis were employed for NLC nanostructure evaluation, confirming the formation of well-defined nanostructures. In vitro kinetics studies demonstrated controlled and sustained docetaxel release over 48 h, emphasizing the potential for prolonged therapeutic effects. Cytotoxicity assays on human umbilical vein endothelial cells (HUVEC) and SK-MEL-24 melanoma cell line revealed enhanced efficacy against cancer cells, with significant selective cytotoxicity and minimal impact on normal cells. This multidimensional approach, encompassing formulation optimization and comprehensive characterization, positions the docetaxel-loaded NLCs as promising candidates for advanced skin cancer therapy. The findings underscore the potential translational impact of these nanocarriers, paving the way for future preclinical investigations and clinical applications in skin cancer treatment.

1. Introduction

Recent decades have marked a significant shift in pharmaceutical sciences, primarily driven by the need for more effective drug delivery systems, aiming to enhance therapeutic outcomes, extend the duration of drug release, and improve patient adherence. Among these emerging technologies, nanostructured lipid carriers (NLCs) have garnered attention due to their unique ability to optimize drug delivery through a highly controlled and targeted approach [1].

First introduced in the late 1990s, NLCs evolved from solid lipid nanoparticles (SLNs) by integrating a mixture of solid and liquid lipids in a heat-controlled crystallization process [2,3], presenting an innovative solution to several limitations faced by earlier formulations [4]. Compared to SLNs, NLCs offer a disorganized crystalline matrix with more imperfections in crystal to accommodate the drug, which in turn ensures higher drug loading capacity and minimal drug leakage during storage, addressing key issues of drug stability and release rates [5,6,7].

Numerous preparation techniques are currently used for formulating NLCs, enabling the creation of effective and stable formulations. High-pressure homogenization stands as the most prominent technique due to its ability to consistently produce particles with uniform size and distribution [3,8,9,10,11]. Additional methods include emulsification–ultrasonication, which combines high shear mixing with ultrasonic waves to disperse the lipids [5,12,13], microemulsion technique, which employs both water and oil phases to achieve nano-sized carriers [14,15], the film ultrasonication–dispersion technique, that utilizes ultrasonic energy to disperse a preformed thin lipid film [4], and solvent diffusion technique, where the carrier matrix is formed by the diffusion of a solvent, leading to the encapsulation of the drug [16,17,18]. Recent advancements have led to the development of novel techniques such as those utilizing supercritical fluids [19] and microfluidic platforms [20]. These innovative approaches offer advantages in terms of precise control over particle size, morphology, and enhanced drug encapsulation efficiency.

NLCs can encapsulate a wide range of hydrophobic [5,8,9,10,12] or hydrophilic [13,21] drugs, thus having broad applicability across different therapeutic categories. Additionally, the biocompatible and biodegradable nature of the lipid materials used in formulation ensures minimal in vivo toxicity, a critical consideration for clinical applications [4,10,22]. The surface of NLCs can also be specifically modified to target particular tissues or cellular pathways, enhancing therapeutic efficacy and reducing systemic side effects [23,24,25,26].

Currently, NLCs are being explored for their ability to improve the solubility, stability, and bioavailability of poorly water-soluble drugs, as well as to provide controlled and sustained drug release [27,28]. One significant area of focus is the optimization of NLC formulations for improving the systemic bioavailability of poorly soluble drugs, resulting in the formulation of hydrochlorothiazide [2], axitinib [29], carbamazepine [30], atorvastatin [12], or erlotinib [26] NLCs with superior bioavailability to the commercial formulations.

Since NLCs are particularly advantageous for delivering hydrophobic drugs, which are often used in cancer treatment, they are currently placed in the forefront of innovative cancer treatment strategies, with extensive research and development efforts aimed at enhancing drug delivery, bioavailability, and therapeutic efficacy. This focus is especially relevant for skin cancers (including melanoma and non-melanoma types), which represent a growing global health concern with rising incidence rates [31,32]. Despite advancements in treatment modalities, there remains a critical need for innovative therapeutic approaches with enhanced efficacy and reduced side effects. Nanotechnology-based drug delivery systems, with NLCs as a prime example, offer a promising platform to address this need. By improving drug solubility, bioavailability, ensuring high stratum corneum permeability, and enabling controlled and targeted delivery to the tumor site, while minimizing system toxicity, NLCs have the potential to transform skin cancer treatment and alleviate its global burden [4,10,22,23]. For instance, riluzole-loaded NLCs for topical application have shown potential in treating hyperproliferative skin conditions by inhibiting keratinocyte cell proliferation and providing sustained drug release [33], doxorubicin-loaded NLCs have also shown increased cytotoxicity and drug uptake compared to the plain drug [34], whereas topotecan-loaded NLCs have been found to maintain drug stability and enhance skin permeation, which is crucial for achieving therapeutic drug levels in the skin [35].

One such drug that could benefit significantly from NLC-based delivery is docetaxel, a semi-synthetic taxane second-generation anti-neoplastic agent derived from Taxus baccata [36,37,38]. Docetaxel disrupts cancer cell growth by interfering with microtubule polymerization, ultimately triggering apoptosis [37,38,39]. However, its low water solubility (4.93 µg/mL) [36,38,39,40] and high lipophilicity (logp value of 4.1) [36,40] pose significant challenges for formulation and administration. Additionally, commercial products containing docetaxel have been associated with several adverse reactions, including severe allergic reactions, systemic toxicity, hypotension, bronchospasm, urticaria, fluid retention, extravasation, and thrombosis [36,37,40,41,42,43].

While docetaxel has shown efficacy in treating a range of malignancies, including breast, lung, and prostate cancers [44,45,46], its clinical utility in skin cancer treatment remains largely unexplored. This is primarily due to two key challenges: the drug’s limited ability to penetrate the skin’s outermost layer, the stratum corneum [47], hindering topical application, and the systemic toxicity associated with conventional docetaxel products, which severely restricts its use for systemic therapy. Advanced lipid nanoparticle systems, such as NLCs and SLNs, offer novel approaches to overcome these limitations by encapsulating docetaxel within a lipid matrix, enhancing its delivery and therapeutic efficacy.

For docetaxel parenteral use, studies have demonstrated that the lipid nanoparticles can improve a drug’s pharmacokinetics and biodistribution, leading to enhanced bioavailability, better tumor targeting, and reduced systemic toxicity [4,8,10,36,48]. Similarly, studies have shown an increased intestinal absorption and lymphatic uptake after oral administration of cysteine-functionalized docetaxel NLCs [40], as well as for surface-modified docetaxel SLNs [37].

While the potential of NLCs and SLNs for systemic docetaxel delivery is well-documented, their application in topical delivery, particularly for skin cancer, remains largely unexplored. This approach holds significant promise as it allows for localized treatment directly at the tumor site, potentially minimizing systemic exposure and side effects, while ensuring sustained drug release. A few recent studies have demonstrated the feasibility of topical docetaxel delivery using lipid nano carriers. One such approach involved development of docetaxel-loaded nano liquid crystals using an emulsification solvent diffusion method, which showed enhanced drug uptake, enhanced drug penetration through the skin, prolonged drug release, and increased cytotoxicity against skin cancer cells compared to the plain drug [49]. Furthermore, de Moura et al. described a hybrid NLC-in-hydrogel system designed for the topical treatment of melanoma, which demonstrated improved drug delivery and therapeutic outcomes [5].

Building upon this foundation, our research aims to advance the development of novel NLC formulations for sustained delivery of docetaxel, specifically for topical administration in skin cancer treatment. By optimizing formulation parameters such as lipid composition and drug loading, we aim to tailor optimized NLCs able to achieve sustained drug release, prolonged retention at the tumor site, and enhanced intracellular uptake within cancer cells, ultimately aiming to bridge the gap between promising preclinical results and clinical translation for topical nanomedicine in skin cancer treatment.

By employing the principles of Quality by Design, we aim to meticulously tailor and improve the properties of these carriers. To achieve this, we will utilize experimental designs, notably the Design of Experiments (DoE) methodology, facilitating a systematic approach towards formulation development and optimization.

2.1. Materials

Docetaxel (ScinoPharm, Tainan, Taiwan), Lipoid S-PC-3 (Lipoid GmbH, Ludwigshafen, Germany), Gelucire^® 43/01 (Gattefosse, Saint-Priest, France), glyceryl caprylate (Ellemental, Oradea, Romania), Miglyol 812 (MCT) (IOI Oleo GmbH, Witten, Germany), and Tween 80 (Scharlau, Barcelona, Spain) were used for the preparation of NLCs. Additional lipids and surfactants included stearic acid, palmitic acid, cetyl palmitate, alpha tocopheryl acetate, retinyl palmitate, oleic acid, DL-limonene, 1,2 propanediol, and isopropyl myristate (all purchased from Merck, Darmstadt, Germany); decanoic acid, sorbitan monostearate, tetradecanoic acid, sodium taurocholate, sodium deoxycholate, sodium cholate, Brij^® 35, Triton^® X 100, Tween 60, Tween 20, and Span 80 (obtained from Sigma Aldrich, Saint Louis, MO, USA), Gelucire^® 50/13, Gelucire^® 48/16, Gelucire^® 44/14, Suppocire^® NAS 50, and Transcutol^® (Gattefosse, Saint-Priest, France); Lipoid E80, Phospholipon^® 90G, Lipoid PG 18:0/18:0, and Lipoid E PC S (Lipoid GmbH, Ludwigshafen, Germany). Kolliphor^® P188, Kolliphor^® P407, and Kolliphor^® EL were obtained from BASF, Ludwigshafen am Rhein, Germany. Glyceryl monostearate (GMS) was obtained from Cognis GmbH, Monheim am Rhein, Germany, whereas self-emulsifying glyceryl monostearate (GMS-SE) was purchased from Ellemental, Oradea, Romania. Oat oil, Neem oil, Pomegranate oil, Sesame oil, Grape seed oil, Jojoba oil, and Wheat germ oil were obtained from various commercial sources.

HPLC gradient grade acetonitrile and methanol were procured from Honeywell Research Chemicals, Seelze, Germany. LC-MS grade formic acid was obtained from Fisher Chemical, Pardubice, Czech Republic. Potassium phosphate monobasic, disodium hydrogen phosphate, sodium chloride, and ethanol for analysis were acquired from Merck, Darmstadt, Germany, while potassium chloride was sourced from Lach:ner, Germany. Ultrapure water (resistivity of 18.2 MΩ·cm at 25 °C and a total organic carbon (TOC) content below 5 ppb), was produced using a Milli-Q EQ 7008 water purification system (Merck Millipore, Burlington, MA, USA).

Finally, 5-Fluorouracyl (5-FU), PBS/1 mM EDTA, L-Glutamine (Glu), Penicillin (100 units/mL), Streptomycin (100 μg/mL), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Propidium Iodide (PI) (stock solution 4 mg/mL PI in PBS), and RNase A (stock solution 10 mg/mL RNase A) were purchased from Sigma Aldrich (St. Louis, MO, USA). Annexin V-FITC kit and CycleTEST PLUS DNA Reagent kit were purchased from Becton Dickinson Biosciences (San Jose, CA, USA).

Download the full study as PDF here: Development and Evaluation of Docetaxel-Loaded Nanostructured Lipid Carriers for Skin Cancer Therapy

or read it here

Cocoș, F.-I.; Anuța, V.; Popa, L.; Ghica, M.V.; Nica, M.-A.; Mihăilă, M.; Fierăscu, R.C.; Trică, B.; Nicolae, C.A.; Dinu-Pîrvu, C.-E. Development and Evaluation of Docetaxel-Loaded Nanostructured Lipid Carriers for Skin Cancer Therapy. Pharmaceutics 2024, 16, 960.
https://doi.org/10.3390/pharmaceutics16070960

excipients formulation