Combinatorial Delivery of Docetaxel- and Erlotinib-Loaded Functionalized Nanostructured Lipid Carriers for the Treatment of Triple-Negative Breast Cancer Using Quality-by-Design Approach

Abstract

This study explored the combined administration of docetaxel (DOC) and erlotinib (ERL) using nanostructured lipid carriers (NLCs), with folic acid (FA) conjugation to enhance their synergistic anticancer efficacy against triple-negative breast cancer. NLCs were developed through hot melt homogenization–ultrasound dispersion, and optimized by a quality-by-design (QbD) approach using Plackett–Burman design and Box–Behnken design. Plots were generated based on maximum desirability. Spherical, nanosized dispersions (<200 nm) with zeta potential ranging from −16.4 to −14.15 mV were observed. These nanoformulations demonstrated ~95% entrapment efficiency with around 5% drug loading. Stability tests revealed that the NLCs remained stable for 6 months under storage conditions at 4 °C. In vitro release studies indicated sustained release over 24 h, following Higuchi and Korsmeyer–Peppas models for NLCs and FA NLCs, respectively. Additionally, an in vitro pH-stat lipolysis model exhibited a nearly fivefold increase in bioaccessibility compared to drug-loaded suspensions. The DOC–ERL-loaded formulations exhibited dose- and time-dependent cytotoxicity, revealing synergism at a 1:3 molar ratio in MDA-MB-231 and 4T1 cells, with combination indices of 0.35 and 0.37, respectively. Co-treatment with DOC–ERL-loaded FA NLCs demonstrated synergistic anticancer effects in various in vitro assays.

Introduction

Breast cancer stands as a leading cause of mortality in women, with approximately 297,790 newly diagnosed cases and an estimated 43,170 deaths in 2023, ranking it second among global cancer-related fatalities [1]. Among breast cancer types, triple-negative breast cancer (TNBC) represents the rarest and most aggressive subtype, comprising 10–15% of total cases. TNBC lacks standard hormonal receptors, rendering endocrine treatments ineffective and leaving chemotherapy as the primary therapeutic option [2]. In this context, taxanes, specifically paclitaxel and docetaxel, serve as widely employed chemotherapeutic agents against TNBC. Docetaxel (DOC), a second-generation taxane, exhibits heightened potency compared to paclitaxel, preventing microtubule depolymerization and disrupting the cell cycle at the G2 and M phases, ultimately inducing cell death [3]. Multi-omics approaches have unveiled various molecular pathways implicated in TNBC progression, presenting potential targets for therapy [4]. Epidermal growth factor receptor (EGFR) overexpression is observed in 60% of TNBC cases [5]. EGFR’s autophosphorylation activates downstream pathways, such as PI3K/AKT and RAS/MAPK, associated with cell survival, proliferation, and drug resistance in TNBC. EGFR tyrosine kinase inhibitors (EGFR TKIs) and monoclonal antibodies (mAbs) are used in the treatment of cancers with activated EGFR mutations [6]. Combination therapy gains prominence over monotherapy in cancer treatment due to its potential to enhance therapeutic efficiency by targeting multiple pathways simultaneously, providing a synergistic mechanism while limiting dose-related toxicity [7].

Numerous studies have explored combination therapies for TNBC, demonstrating synergistic effects. For instance, the combination of docetaxel and cisplatin, as well as docetaxel and carboplatin, showed promising anticancer activity [8,9]. Additionally, co-delivering erlotinib with doxorubicin enhanced doxorubicin’s apoptotic activity [7]. The current study proposes a combination of docetaxel (chemotherapeutics) and erlotinib (EGFR TKIs) to potentially induce synergistic anticancer activity against TNBC. However, differences in physicochemical and pharmacokinetic attributes between the drugs have limited their application in combination therapy. To address this, various nanoparticles were fabricated to homogenize the delivery of multiple therapeutic agents at appropriate doses, aiming for an enhanced synergistic effect with reduced toxicity in healthy organs and tissues [10]. Nanostructured lipid carriers (NLCs), second-generation lipid-based nanoparticles, have evolved to overcome drawbacks associated with first-generation lipid nanoparticles like solid lipid nanoparticles (SLNs). NLCs comprise a lipid matrix, a blend of solid and liquid lipids, stabilized by surfactants. The inclusion of a liquid lipid within the matrix results in an imperfect, less organized structure, facilitating increased loading capacity and preventing drug leakage during storage [3,11].

Moreover, it has been noted that triple-negative breast cancer (TNBC) exhibits elevated folate receptor expression in comparison to normal cells. Therefore, the conjugation of nanostructured lipid carriers (NLCs) with folic acid (FA) enhances tumor site specificity [12]. In this study, our objective is to formulate a combination of docetaxel (DOC) and erlotinib (ERL) encapsulated in NLCs and functionalized with FA to achieve a synergistic anticancer effect with enhanced targeting precision. The material characteristics and process variables essential for fabricating NLCs for oral delivery were systematically screened using the Plackett–Burman design. Subsequently, optimization was conducted using the Box–Behnken design (BBD), and the formulations were assessed for physicochemical parameters, long-term stability, in vitro drug release under simulated gastrointestinal conditions, and relevant attributes in in vitro cell culture settings.

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Excipients used in the study: Compritol 888 ATO, Precirol ATO 5, Gelucire 44/14, Gelucire 50/15, Labrafac PG, Labrafac WL 1349, Labrafil M 2125CS, Labrasol, Tween 80, Tween 20

Chaudhuri, A.; Kumar, D.N.; Srivastava, S.K.; Kumar, D.; Patil, U.K.; Parmar, A.S.; Singh, S.; Agrawal, A.K. Combinatorial Delivery of Docetaxel- and Erlotinib-Loaded Functionalized Nanostructured Lipid Carriers for the Treatment of Triple-Negative Breast Cancer Using Quality-by-Design Approach. Pharmaceutics 2024, 16, 926. https://doi.org/10.3390/pharmaceutics16070926