Design and optimization of inherently cytotoxic nanostructured lipid carriers as drug vehicles with dual anti-cancer activity

Abstract

Cancer remains to be one of the most life-threatening diseases with high rates of mortality and morbidity. The major challenge facing chemotherapy is targeting the drug molecules to malignant tissues and avoiding healthy ones. Optimal design of drug delivery carriers can assist not only in directing the drug moieties to the cancer cells but also in potentiating their action. In this study, we fabricated inherently cytotoxic nanostructured lipid carriers (NLCs) as nanovehicles for chemotherapeutic drugs for dual anticancer activity. Natural oils reported to have anticancer activity were used for the preparation of these cytotoxic nanovehicles, replacing conventionally used oils. Optimized NLCs impregnated with flaxseed or lemon oil had a size of 184 ± 10 and 167 ± 1 nm, respectively. Flaxseed oil impregnated NLCs lowered IC₅₀ against MCF-7 breast cancer cells from 214 ± 65 μg oil/mL for conventional NLCs to 33 ± 12 μg oil/mL (6-fold enhancement), while lemon oil NLCs lowered it to 44 ± 17 μg oil/mL (5-fold enhancement). Loading both NLCs with curcumin, as a natural anticancer model drug, resulted in IC₅₀ of 2.71 ± 1.8 and 2.79 ± 1.5 μg curcumin/mL for flaxseed and lemon oil, respectively. This was lower than IC₅₀ values recorded for free curcumin (12.93 ± 6.9 μg curcumin/mL) and curcumin loaded in conventional NLCs (7.21 ± 1.6 μg curcumin/mL). These results show that these designed cytotoxic nanovehicles can potentiate the chemotherapeutic action of any loaded drug, providing dual anticancer activity. They represent a promising drug delivery carrier in our fight against cancer.

Introduction

Cancer is the leading cause of death worldwide and is responsible for about 10 million deaths in 2020. The most common new cases are due to breast, lung, and colon cancer with breast cancer remaining one of the leading causes of cancer-related morbidity and mortality worldwide [1,2]. Early detection and treatment are critical to reduce mortality rates. Conventional cancer treatment strategies include surgery, radiation and chemotherapy [1].

However, the fundamental problem of chemotherapy is the lack of targeted delivery of the therapeutic agents selectively into the tumor tissue. This is essential to maintain effective drug concentration for a certain period and ensure drug-tumor interaction to perform anticancer activities with minimal systemic side effects. Luckily, tumors have a unique pathophysiological phenomenon called enhanced permeability and retention (EPR). This phenomenon makes the highly vascularized tumors permeable to macromolecular compounds, polymer conjugated drugs as well as nano-sized medications that can progressively accumulate within the tumor. Once inside the tumor, the reduced lymphatic drainage of the tumors keeps these compounds trapped inside the tumor tissue for a prolonged period of time [3]. This phenomenon can be taken advantage of to ensure efficient passive targeted delivery and retention of therapeutic agents to the tumors.

Lipid nanoparticles represent an opportunity as drug delivery carriers against cancer. They are biocompatible, easily manufactured and can be used for passive and active targeting of malignant tissues [4]. An interesting type is solid lipid nanoparticles (SLNs), which are composed of solid lipids stabilized by surfactant molecules on the surface. However, owing to lipid recrystallization and formation of a perfect crystal lattice, drug expulsion may occur upon storage [5]. This was overcome by adding a liquid lipid or oil to the solid lipid to create imperfections in the crystal lattice and reduce the subsequent drug expulsion [6]. These nanoparticles represented the second generation of SLNs and were termed nanostructured lipid carriers (NLCs).

Natural-based compounds can target one or more of the mechanisms involved in cancer initiation and progression such as DNA damage, epigenetic modifications, metabolic alterations and chronic inflammation and thus suppress the initiation, progression, and even metastatic spread and relapse of cancers [1]. Several natural oils have shown antitumor activity against various human cancer cell lines [7,8]. Of particular interest are lemon, black seed, and flaxseed oils as they are edible and commonly consumed in various cuisines worldwide. In addition, their cytotoxicity against different cancer cell lines was previously reported. Lemon oil showed the highest cytotoxicity against human skin (A431), gastric (MKN-45) and brain (U-87 MG) cancer cell lines compared to other oils and was safe till a dose of 2000 mg/kg body weight [8].

Additionally, it was formulated in nanoemulsion and induced apoptosis in human lung cancer cells (A549) [9]. Black or Nigella sativa seed extracts were reported to have antitumor activity [10]. Moreover, its oil demonstrated cytotoxicity against human liver (HepG2) and breast (MCF-7) cancer cells with reported IC50 of 44.6 and 46.2 μg/mL, respectively and induced mitochondrial-mediated apoptosis in HepG2 cells [11]. Furthermore, chitosan [12] and gold [13] nanoparticles loaded with black seed oil recorded enhanced cytotoxicity and lower IC50 compared to free oil. Flaxseed oil inhibited proliferation of several cell lines of breast cancer, cervical cancer, and melanoma. Flow cytometry studies showed that flaxseed oil successfully induced apoptosis in murine melanoma (B16-BL6) and breast cancer cells (MCF-7) [14]. Moreover, it was safe on non-malignant cell lines and did not inhibit the growth of embryonic kidney cells (HEK293), human epithelial cells (HSG), and breast epithelial cells (HBL-100) [14]. Incorporating these oils into a suitable drug delivery system should facilitate their administration, protect them from enzymatic breakdown in physiological environment and target them to malignant tissues. It was previously reported that incorporating essential oil of Pistacia atlantica into NLCs significantly potentiated its anticancer activity. This was evident by declining the viability of SKBR3 breast cancer cells via cell cycle arrest and apoptosis, compared to cells treated with placebo and the free essential oil [15].

Our aim was to replace the commonly used oils in NLCs with natural oils having native anticancer activity namely lemon, black seed, and flaxseed oils to formulate NLCs with inherent cytotoxic activity, in other words cytotoxic nanovehicles. We hypothesize that this should augment the cytotoxic activity of NLCs and provide dual acting nanovehicles once loaded with an anticancer molecule. To the best of our knowledge, no one has attempted to prepare inherently cytotoxic NLCs and study the effect of combining these pharmacologically active nanovehicles with curcumin-a model natural cytotoxic drug- and study their combined cytotoxic effect. The effect of varying lipid concentration, type of oil and surfactant on the particle size and surface charge of NLCs was studied and analysed using design of experiments. Moreover, cytotoxicity of optimized formulae against human colon (Caco-2) and breast (MCF-7) cancer cell lines was evaluated. Furthermore, selected cytotoxic NLCs formulations were loaded with curcumin as a model natural drug with reported anticancer activity [16], to get a measure of the potential enhancement in cytotoxicity. This designed drug delivery system would offer dual anticancer activity against malignant tissues, where both the drug and the nanovehicles are cytotoxic to the cancer cells.

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Materials

Glyceryl behenate (Compritol® 888 ATO) and medium chain triglycerides (MCT) (Labrafac Lipophilie® WL 1349) were kindly gifted by Gattefosse (Saint-Priest Cedex, France). Curcumin was a kind gift from Eva Pharma (Cairo Egypt). Lemon oil, black seed oil and flaxseed oil were purchased from Imtenan (Cairo, Egypt). Poloxamer 188 (P188), sulforhodamine B and dimethylsulfoxide (DMSO) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Penicillin/streptomycin were purchased from Capricorn scientific (Ebsdorfergrund, Germany). Fetal bovine serum (FBS) was purchased from Thermo Fisher scientific (Massachusetts, USA). Trypsin was purchased from Lonza (Verviers, Belgium). Roswell Park Memorial Institute (RPMI) and Dulbecco’s Modified Eagle Medium (DMEM) were purchased from Serana (Pessin, Germany). Polysorbate 80 (P80), disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and absolute ethanol were purchased from Adwic, El-Nasr Pharmaceutical Co. (Cairo, Egypt).

John Youshia, Mera Medhat, Mariam George, Haidy Maged, Maureen Sherif, Salma Ashraf, Sara Ahmed, Marina George, Felopateer Karam, Wafaa Ibrahim, Mariam Samir, Dina O. Helal, Design and optimization of inherently cytotoxic nanostructured lipid carriers as drug vehicles with dual anti-cancer activity, Journal of Drug Delivery Science and Technology, Volume 115, Part 2, 2026, 107769, ISSN 1773-2247, https://doi.org/10.1016/j.jddst.2025.107769.

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