Formulation and Evaluation of Alginate Microcapsules Containing an Uncompetitive Nanomolar Dimeric Indenoindole Inhibitor of the Human Breast Cancer Resistance Pump ABCG2 with Different Excipients

Abstract

Background/Objectives: The ABCG2 transporter actively effluxes anticancer drugs, reducing their efficacy and promoting multidrug resistance (MDR). Developing oral formulations of poorly soluble ABCG2 inhibitors remains challenging due to their low solubility and intestinal permeability. This study aimed to formulate and evaluate an ABCG2 inhibitor using micro- and nanoscale drug delivery systems.

Methods: To address the poor solubility and bioavailability of the corresponding active ingredient, a self-nanoemulsifying drug delivery system (SNEDDS) was developed. The SNEDDS was encapsulated into microcapsules using sodium alginate crosslinked with calcium chloride. Five microcapsule formulations were developed, varying in the inclusion of polyvinylpyrrolidone (PVP), Transcutol^® HP and SNEDDS. The effects of the excipients on encapsulation efficiency, swelling capacity, enzymatic stability, dissolution, cytocompatibility, and permeability were systematically evaluated.

Results: The SNEDDS exhibited monodisperse particle sizes and efficient drug entrapment. Results revealed that formulations incorporating PVP and SNEDDS improved encapsulation efficiency and bioavailability. SNEDDS-containing formulations demonstrated superior enzymatic stability in simulated gastric and intestinal fluids and provided the highest cumulative drug release in vitro. Cytotoxicity studies conducted on Caco-2 and MCF-7 cells demonstrated that our formulations were well tolerated, indicating favorable biocompatibility.

Conclusions: Our findings demonstrate that SNEDDS-loaded alginate microcapsules offer an efficient platform for oral delivery of dimeric ABCG2 inhibitors, combining enhanced solubility, stability, and controlled release. The optimized formulation can be regarded as a promising strategy to enhance the oral bioavailability of efflux pump inhibitors and other poorly soluble drugs.

Introduction

ATP-binding cassette (ABC) transporters are a family of membrane proteins that play a crucial role in cellular homeostasis by facilitating the transport of various substrates across biological membranes. These transporters are widely expressed in different tissues and are involved in the absorption, distribution, and excretion of endogenous and exogenous compounds, including drugs. Among them, ABCG2, also known as the breast cancer resistance protein (BCRP), has garnered significant attention due to its role in multidrug resistance (MDR) in cancer therapy [1]. ABCG2 is highly expressed in several drug-resistant cancer cells, where it actively effluxes chemotherapeutic agents, such as mitoxantrone, topotecan, and anthracyclines, thereby reducing their intracellular accumulation and limiting their cytotoxic effects. This protective function, while beneficial in physiological contexts such as the blood–brain barrier and placenta, poses a major challenge in oncology, as it diminishes the efficacy of anticancer drugs and contributes to tumor progression [1,2,3].

Efforts to overcome MDR have led to the exploration of various ABCG2 inhibitors, which can block or modulate its efflux function, thereby enhancing intracellular drug retention and improving therapeutic outcomes. Indenoindole derivatives have emerged as potent inhibitors of the ABCG2 receptor, demonstrating significant promise as molecular scaffolds for the rational design of more effective and selective modulators. These compounds possess a rigid polycyclic aromatic framework that enables favorable π–π stacking and hydrophobic interactions within the ABCG2 binding pocket. Structural modifications on the indenoindole core have been shown to strongly influence inhibitory potency and selectivity, providing valuable insights into the structure–activity relationship of this compound class. Therefore, indenoindole-based molecules represent an attractive platform for the development of next-generation ABCG2 inhibitors with improved pharmacokinetic and pharmacodynamic profiles [4,5,6,7].

However, many of these inhibitors suffer from poor aqueous solubility, low bioavailability, and potential off-target toxicity, limiting their clinical applicability [8,9]. To address these challenges, advanced drug delivery systems, particularly those at the micro- and nanoscale, have emerged as promising approaches. These systems can improve the solubility, stability, and controlled release of therapeutic agents while enhancing their targeted delivery to cancer cells [10,11].

SNEDDS have gained increasing interest in pharmaceutical research to improve the solubility and bioavailability of poor water-soluble compounds. SNEDDS are isotropic mixtures of oils, surfactants, and cosurfactants that spontaneously form fine oil-in-water nanoemulsions upon contact with gastrointestinal fluids, enhancing drug dissolution and absorption [12,13]. Despite their advantages, liquid SNEDDS formulations may suffer from stability issues, necessitating their conversion into solid dosage forms for better handling and controlled drug release. One effective approach is the encapsulation of SNEDDS into biocompatible polymeric microcapsules, which can further improve drug stability, protect against enzymatic degradation, and provide sustained release profiles [14].

Excipients such as Transcutol® HP, Labrasol®, Capryol® 90, and polyvinylpyrrolidone (PVP) have gained considerable attention for their ability to enhance the solubility, stability, and bioavailability of poorly water-soluble drugs. Transcutol® HP (diethylene glycol monoethyl ether) is a well-known solubilizer and penetration enhancer that improves drug diffusion across biological membranes [15]. Labrasol® (caprylocaproyl macrogol-8 glycerides) and Capryol® 90 (propylene glycol monocaprylate (type II)) are lipid-based excipients commonly employed in self-emulsifying and nano/microemulsion systems, where they facilitate lipid solubilization and promote efficient drug absorption through the gastrointestinal tract [16,17]. PVP is a widely used hydrophilic polymer in microencapsulation due to its excellent film-forming ability, biocompatibility, and capacity to enhance the solubility of poorly water-soluble drugs. In microcapsule formulations, PVP can act as a stabilizing and matrix-forming agent, contributing to the formation of uniform and mechanically stable shells around the encapsulated core [18]. Its strong hydrogen-bonding potential allows favorable interactions with both hydrophilic and lipophilic drug molecules, thereby improving encapsulation efficiency and controlling drug release profiles. Moreover, PVP can prevent drug crystallization during microcapsule drying and storage, ensuring a more stable amorphous state that enhances dissolution and bioavailability. The polymer’s molecular weight and concentration can be optimized to tailor the release kinetics and degradation behavior of the microcapsules, making PVP a versatile excipient in the design of sustained and controlled drug delivery systems [19,20]. Together, these excipients offer complementary functionalities that can be strategically combined to develop advanced drug delivery systems with optimized solubility and controlled-release characteristics.

Alginate, a natural polysaccharide widely used in drug delivery, is particularly suitable for microencapsulation due to its non-toxic, biodegradable, and mucoadhesive properties [21,22,23]. In addition, they offer efficient targeting, sustained delivery, and controllable release profiles. During the preparation of alginate-based microbeads, the active substance is entrapped within an alginate gel matrix, which is formed through ionic crosslinking between the uronic acid residues of alginate and divalent cations such as Ca2+, Ba2+, or Sr2+ [24,25,26].

In this study, we aimed to develop and characterize an innovative drug delivery system for an ABCG2 inhibitor by incorporating it into SNEDDS and further encapsulating this formulation into alginate-based microcapsules. The model compound used in this study was a dimeric indenoindole-based ABCG2 inhibitor synthesized and previously published by the Cancer Research Center of Lyon (CRCL) [6]. We systematically investigated the physicochemical properties of the developed formulations, including particle size, surface morphology, encapsulation efficiency, and swelling behavior. Additionally, we evaluated the cytocompatibility of microcapsules using Caco-2 and MCF-7 cell lines to assess their safety for potential biomedical applications. By enhancing the bioavailability and stability of ABCG2 inhibitors, this research contributes to the ongoing efforts to develop more effective strategies for combating MDR in breast cancer and other chemoresistant malignancies.

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Materials

The active pharmaceutical ingredient (API) selected for this study, ABCG2 inhibitor 7b, was synthesized by the CRCL research laboratory [6]. The SNEDDS ingredients—Labrasol® (caprylocaproyl macrogol-8 glycerides), Transcutol® HP (diethylene glycol monoethyl ether) and Capryol® 90 (propylene glycol monocaprylate (type II))—were purchased from Gattefossé (Lyon, France). Low viscosity grade sodium alginate was sourced from BÜCHI Labortechnik AG (Flawil, Switzerland). The human adenocarcinoma cancer cell line (Caco-2) and the human breast cancer cell line (MCF-7) were acquired from the European Collection of Authenticated Cell Cultures (ECACC, Public Health England, Salisbury, UK). Culturing flasks and 96-well cell culture plates were supplied by VWR International (Debrecen, Hungary). Transwell® 24-well cell culture inserts were obtained from Greiner Bio-One Hungary Kft. (Mosonmagyarovar, Hungary). All other reagents and chemicals were procured from Sigma-Aldrich (Budapest, Hungary).

Bodnár, K.; Marminon, C.; Perret, F.; Haimhoffer, Á.; Papp, B.; Fehér, P.; Ujhelyi, Z.; Jose, J.; Le Borgne, M.; Bácskay, I.; et al. Formulation and Evaluation of Alginate Microcapsules Containing an Uncompetitive Nanomolar Dimeric Indenoindole Inhibitor of the Human Breast Cancer Resistance Pump ABCG2 with Different Excipients. Pharmaceutics 2025, 17, 1587. https://doi.org/10.3390/pharmaceutics17121587