All4Nutra
Abstract
Rapamycin holds significant therapeutic potential for various diseases; however, its clinical application is limited by several formulation challenges, primarily its extremely low aqueous solubility (2.6 μg/mL). To address this, nanoparticle-based delivery systems have emerged as a promising strategy to enhance solubility and enable sustained drug release. Currently, Fyarro® (Aadi Bioscience, Inc.), an albumin-bound nanoparticle formulation, is the only FDA-approved injectable rapamycin product. In this study, we aimed to develop and evaluate novel poly(ethylene oxide)-block-poly(ε-caprolactone)-α-tocopheryl succinate (PEO-b-PCL-α-TS) micelles and assess their potential as a delivery system for rapamycin. PEO-b-PCL copolymers with varying PCL/PEO ratios were prepared via ring-opening polymerization and modified by α-TS conjugation, as confirmed by 1H NMR, GPC, XRD, DSC analyses. The optimum rapamycin-loaded micelles (PEO2000–b-PCL4000-α-TS) exhibited nano-sized particles (< 22 nm) with a narrow polydispersity index (<0.29), high drug encapsulation efficiency (≥92 %), and enhanced solubility (>1.3 mg/mL). Stability studies demonstrated that encapsulation protected rapamycin from degradation, maintaining over 90 % drug retention for three months at 4 °C, while in vitro release studies showed sustained release, with 50 % of rapamycin released from PEO2000–b-PCL4000-α-TS micelles over 72 h. In vitro cytotoxicity assays revealed anticancer activity against lung carcinoma epithelial cells (A549), and the human colon adenocarcinoma cell line (HCT116). Minimal toxicity (≥70 % viability) was observed in normal human fibroblast cells (HFF1). These results point to the potential of PEO-b-PCL-α-TS micelles as a promising nanocarrier system, offering improved rapamycin solubility, enhanced stability, sustained release, and effective anticancer activity.
Introduction
Many newly discovered chemical compounds have a problem of low solubility in water, with more than 40 % of active ingredients being hydrophobic. This poses difficulties in formulating them for parenteral administration and leads to delays in bringing these drugs to market (Lipinski, 2002). In the case of anticancer drugs, the majority suffer from issues like low bioavailability, instability in biological environments, challenges in achieving effective concentrations at the site of action, and the emergence of multidrug resistance (Chidambaram et al., 2011).
Rapamycin, also known as sirolimus, is a compound derived from Streptomyces hygroscopicus, belonging to the polyketide macrolide family (Sehgal et al., 1975). It interacts with FK505 binding protein 12 (FK505–12) to form a complex that inhibits the activity of mTOR kinase. As a result, signaling pathways dependent on interleukin-2 (IL-2) receptor and CD28 are blocked (Dowling et al., 2010). Preclinical and clinical studies have shown that rapamycin is effective in treating various diseases related to the immune system (e.g., autoimmune diseases and graft rejection) (Hartford and Ratain, 2007), neurodegenerative disorders (e.g., Alzheimer’s disease) (Li et al., 2014), metabolic disorders (e.g., obesity and diabetes) (Chang et al., 2009; Krebs et al., 2007; Ong et al., 2016; Um et al., 2004), and several types of cancer including breast cancer, renal cell carcinoma, colon cancer, lung cancer, and lymphoma (Law, 2005; Li et al., 2014).
Rapamycin holds significant potential for treating various health conditions; however, its clinical application faces several challenges. These include its limited water solubility (2.6 μg/mL) (Simamora et al., 2001), susceptibility to gastric acid degradation (Kim et al., 2013), and being a substrate for intestinal and hepatic cytochrome P450 enzymes as well as the P-glycoprotein efflux pump (Lampen et al., 1998). These factors contribute to its low and variable oral bioavailability, estimated to be approximately 14 % with the oral solution (MacDonald et al., 2000). Commercially, rapamycin is available under the trade name Rapamune® as an oral solution (1 mg/mL; 60 mL) and tablets (0.5, 1, and 2 mg).
These drawbacks highlight the need for the development of pharmaceutical nanotechnology in order to improve the delivery of rapamycin, making it more accurate and effective. One promising approach involves incorporating rapamycin into nanoparticle systems, which can enhance its solubility and enable sustained drug release (Haeri et al., 2018). Among the efforts in this field, ABI-009 (Fyarro®, Aadi Bioscience, Inc.) is the first and only FDA-approved rapamycin injectable product. It is an albumin-bound nanoparticle formulation (∼100 nm) developed with nab®-technology, which was approved to treat adult patients with locally advanced unresectable or metastatic malignant perivascular epithelioid cell tumors (PEComa). Preclinical studies demonstrated that Fyarro® achieves significantly improved tumor growth inhibition, higher drug accumulation within tumors, and more effective suppression of the mTOR target phospho-S6 compared to oral inhibitors (Wagner et al., 2021). It is currently undergoing several clinical trials for the treatment of other types of malignancies and conditions.
Polymeric micelles are self-assembled nanoparticles consisting of a core and shell structure, formed from amphiphilic block copolymers. Polymeric micelles have attracted considerable attention, primarily due to their low critical micelle concentration (CMC), and relatively rigid core structure. These characteristics impart higher thermodynamic stability to the formulation in physiological solutions, a slower rate of dissociation. As a result, the drug is retained for a longer time, allowing for greater accumulation at the tumor site by evading the reticuloendothelial system that has been made possible due to small size and stealth properties of the most commonly used polymeric micellar structures (Aliabadi and Lavasanifar, 2006).
One of the widely investigated type of block copolymer micelles are those composed of methoxy poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) (Grossen et al., 2017). These micelles have gained prominence due to the biocompatibility of PEO, which imparts “stealth” behavior to the formulation (Lin et al., 2005; Otsuka et al., 2003). On the other hand, PCL, with its poly(ester) core, is considered safe for human application due to its susceptibility to hydrolysis and conversion to biocompatible building blocks (Grossen et al., 2017). PEO-b-PCL micelles have been extensively studied as long-circulating drug carriers that passively accumulate in tumor tissues, taking advantage of the enhanced permeability and retention effect (Lin et al., 2005; Otsuka et al., 2003). Additionally, these micelles enhance the solubilization of hydrophobic drugs (Grossen et al., 2017).
D–α–tocopheryl succinate (α-TS) is a succinic acid derivative of α–tocopherol (vitamin E). Studies in the last four decades have shown that α-TS possess anticancer activity both in vitro and in vivo (Prasad et al., 2003). The most intriguing aspect of this α-TS effect is its selectivity toward cancer cells (i.e., does not affect the proliferation of most normal cells). Indeed, numerous studies have shown that α-TS enhances the growth-inhibitory effects of ionizing radiation, chemotherapeutic agents, hyperthermia, and some biological response modifiers selectively on cancer cells, but not on normal cells (Neuzil et al., 2001; Prasad and Edwards-Prasad, 1982; Prasad et al., 2003; Sylvester, 2007; Turley et al., 1997; Turley et al., 1995; Weber et al., 2002; Yu et al., 1997).
In this study, novel block copolymers consisting of PEO-b-PCL-α-TS were synthesized and comprehensively characterized using various analytical techniques. These copolymers were utilized to prepare micelles, which were systematically evaluated for their ability to encapsulate rapamycin, enhance its aqueous solubility and stability, provide sustained drug release, and selectively affect the viability of cancer cells with minimal toxicity to normal cells.
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Materials
Methoxy poly(ethylene oxide) (2 kDa and 5 kDa), ε-caprolactone, α-TS, oxalyl chloride (reagent grade, 98 %), triethylamine (TEA) (≥99 %), diethyl ether (reagent grade), dichloromethane (DCM; reagent grade), and dimethyl sulfoxide (DMSO; HPLC grade) were obtained from Sigma Aldrich Co., (St. Louis, MO, USA). Rapamycin was obtained from PKC Pharmaceuticals Inc. (Woburn, MA, USA). Deuterated chloroform (CDCl3, 99.8 %) was purchased from Cambridge Isotope Laboratories Inc. (Tewksbury, MA, USA). Human breast cancer cells (MCF-7) were purchased from the American Type Culture Collection (Manassas, VA, USA). Acetone and acetonitrile (HPLC grade) were acquired from BDH Laboratory Supplies (BDH Chemicals, Poole, UK). Chloroform (HPLC grade) was purchased from Acros Organics (Morris Plains, NJ, USA).
Ziyad Binkhathlan, Abdullah K. Alshememry, Ahmad M. Balkhair, Raisuddin Ali, Sulaiman S. Alhudaithi, Saad Alobid, Wajhul Qamar, Alhassan H. Aodah, Mohammad Reza Vakili, Afsaneh Lavasanifar, Development, characterization, and in vitro evaluation of poly(ethylene oxide)-block-poly(ε-caprolactone)-α-tocopheryl succinate micelles as a novel nanocarrier for rapamycin delivery, International Journal of Pharmaceutics: X, Volume 9, 2025, 100341, ISSN 2590-1567, https://doi.org/10.1016/j.ijpx.2025.100341.
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