Extremely long tumor retention, multi-responsive boronate crosslinked micelles with superior therapeutic efficacy for ovarian cancer

Abstract

Mortality rates for ovarian cancer have declined only slightly in the past forty years since the “War on Cancer” was declared. The current standard care of ovarian cancer is still cytoredutive surgery followed by several cycles of chemotherapy. The severe adverse effect from chemotherapy drug is a leading cause for the patients to fail in long term therapy post-surgery. New nanocarriers able to minimize the premature drug release in blood circulation while releasing drug on-demand at tumor site have profound impact on the improvement of the efficacy and toxicity profile of the chemotherapeutic drugs. Here we reported a unique type of extremely long tumor retention, multi-responsive boronate crosslinked micelles (BCM) for ovarian cancer therapy. We systemically investigated the stability of BCM in serum and plasma, and their responsiveness to acidic pH and cis-diols (such as mannitol, a safe FDA approved drug for diuresis) through particle size measurement and förster resonance energy transfer (FRET) approach. Paclitaxel (PTX) loaded BCM (BCM-PTX) exhibited higher stability than non-crosslinked micelles (NCM) in the presence of plasma or serum. BCMs possessed a longer in vivo blood circulation time when compared to NCM. Furthermore, BCM could be disassembled in an acidic pH environment or by administrating mannitol, facilitating drug release in an acidic tumor environment and triggered by exogenous stimuli after drug enrichment in tumor mass. Near infra-red fluorescence (NIRF) imaging on SKOV-3 ovarian cancer mouse model demonstrated that the NIR dye DiD encapsulated BCM could preferentially accumulate in tumor site and their tumor retention was very long with still 66% remained on 12th day post injection. DiD-NCM had similar high-level uptake in tumor with DiD-BCM within the first 3 days, its accumulation, however, decreased obviously on 4th day and only 15% dye was left 12 days later. In both formulations, the dye uptake in normal organs was mostly washed away within the first 24–48 h. In in vivo tumor treatment study, PTX loaded BCM showed superior therapeutic efficacy than that of NCM and Taxol. The mice could tolerate 20 mg/kg PTX formulated in nano-formulations, which doubled the maximum tolerated dose (MTD) of Taxol. The administration of mannitol 24 h after BCM-PTX injection further improved the tumor therapeutic effect and elongated the survival time of the mice. The novel boronate-catechol crosslinked nanocarrier platform demonstrated its superior capability in targeted drug delivery, which is not only useful for ovarian cancer treatment but will also be beneficial for the therapy of many other solid tumors.

Introduction

Ovarian cancer exits as the 5th malignancy resulting in death in female patients. The American Cancer Society estimated that in 2017, about 22,440 new cases of ovarian cancer would be diagnosed and 14,080 women would die of ovarian cancer in the United States [1]. Most epithelial ovarian cancers are diagnosed at an advanced stage where the tumor has seeded the abdominal cavity (stage 3) [2], [3]. The current therapeutic treatments for epithelial ovarian cancers include the use of combination chemotherapy with a platinum based drug and paclitaxel (PTX) [4], [5], [6], [7], [8], [9]. If the patient has had an optimal cytoreductive surgery (< 1 cm of residual tumor burden) then the current gold standard of treatment involves intravenous (IV) PTX on Day 1, intraperitoneal (IP) delivery of cisplatin on Day 2, and IP PTX on Day 8, 3 weeks a cycle, repeated a total of 6 cycles. Many patients are unable to complete six cycles due to the debilitating side effects of the chemotherapy. For PTX, these include hypersensitivity reactions, neurotoxicity, and myelosuppression [4], [5], [6], [7], [8], [9], [10]. Optimizing drug delivery while decreasing side effects is critical to improve the therapeutic nature of these drugs while also improving a patient's quality of life [11], [12], [13], [14], [15].

In the past 10–15 years, nanotechnology has been intensively developed to apply in the field of cancer therapy [12], [13], [14], [15]. Polymeric micelles demonstrated their superior potential in drug delivery for cancer therapy in several aspects, such as superior capability to encapsulate water insoluble chemotherapeutic drugs, prolonged in vivo circulation time and preferential accumulation at tumor site via the enhanced permeability and retention (EPR) effect due to their relatively smaller particle size (< 100 nm) [11], [16], [17], [18], [19], [20], [21]. However, there are some challenges that have hampered the clinical translation of this type of nanoparticles. Polymeric micelles typically are a thermo-dynamic system because they are formed through self-assembled procedure in certain buffer system. It is well-known that a delicate equilibrium exhibits between micelles and unimers in different buffer conditions [22], [23]. Blood is the first biological barrier for micelle-based drug delivery systems via IV administration. It has been demonstrated that the interaction with blood proteins and lipoproteins (e.g. HDL, LDL, VLDL and chylomicron) may cause the dissociation of these thermo-dynamic nanoparticles and lead to premature drug release [24]. Furthermore, conventional polymeric micelles may be dissociated into unimers after IV administration owing to their susceptibility to dilution below the critical micelle concentration (CMC) [22], [23]. This is another factor that may result in early dissociation of the micelles and premature drug release before micelles reaching and accumulating in the tumor location.

Herein, researchers have put more efforts to search for feasible ways to improve the stability of polymeric micelles for in vivo drug delivery. Cross-linking approaches have exhibited as one of an ideal choice [25], [26]. A programmable cross-linking strategy to control the release rate of the entrapped drugs in different environments (e.g. normal organs versus tumor) is ideal to minimize the systemic toxicity and enhance the therapeutic efficacy of the chemotherapeutic agents. This led to the development of stimuli-responsive cross-linked micelles (SCMs), a smart nanocarrier system for tumor-targeting drug delivery and on demand drug release [19], [23], [27], [28], [29]. SCMs possessed minimal premature drug release due to their superior structural stability in blood stream while they could be triggered to release drug payloads in response to the local environment of the tumor (e.g. tumor extra-cellular pH 6.5–7.2 and endosomal/lysosomal pH 4.5–6 [30], [31], tumor reductive intra-cellular conditions [19], [23], [26], [32], [33], [34], [35], adenosine triphosphate (ATP) [36], [37], [38] and enzymes [23]) or exogenous reagents (e.g. N-Acetylcysteine and cis-diols [22], [23], [39]. SCMs have shown great potential to decrease drug accumulation at normal organs to minimize the systemic toxicity and increase the therapeutic index due to their on-demand drug releasing nature at tumor sites.

Boronic acids and cis-diols can form reversible boronate esters, which is responsive dually to external pH value and competing diols [31], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48]. Boronate esters have been developed as building blocks in designing stimuli-responsive drug delivery systems. Due to their syn-peri-planar arrangement of the aromatic hydroxy groups and electron-donating character, catechols are an ideal type of diols to form stable boronate ester bond with boronic acids in physiological condition [31]. In the present manuscript, we describe the preclinical development of a unique type of extremely long tumor retention, multi-responsive boronate crosslinked micelles (BCM) for ovarian cancer therapy (Fig. 1). We utilized förster resonance energy transfer (FRET) approach and particle size measurement to systemically investigate the stability and release profiles of BCM in blood related media such as serum and plasma, and their responsiveness to acidic pH and mannitol. The long-term in vivo biodistribution and blood elimination kinetics of BCM were evaluated in SKOV-3 ovarian cancer mouse models. The therapeutic efficacy and toxicity profiles of BCM entrapped with PTX were further evaluated. To the best of our knowledge, this is the first demonstration of a multi-responsive micelle-based drug delivery system with such long tumor retention (up to 12 days).