pH-activatable polymeric nanodrugs enhanced tumor chemo/antiangiogenic combination therapy through improving targeting drug release
Graphical abstract
Introduction
The use of polymer-drug conjugates-based nanosystems (or called polymeric nanodrugs) was an established approach for improvement of cancer combination therapy. There were a lot of polymers which not only showed adjuvant antitumor activities but also could form tumor specific nanosystems through being conjugated to hydrophobic antitumor drugs [1], [2], [3]. In this way, the pharmacokinetics of the drug attaching to the polymeric carrier could also be modulated [2], [4]. For example, the long-circulating effect could be realized by covalently bonding chemotherapeutic agents to polyethylene glycol (PEG) [5]. Besides, active tumor targeting accumulation could also be achieved by conjugating hyaluronic acid to antitumor drugs [6]. Early clinical trials showed several advantages of polymer-drug conjugates over the corresponding antitumor drugs, including enhanced antitumor efficacy, reduced side effects, ease of drug administration, improved patient compliance and better long-term prognosis [3]. However, there was a controversy whether the therapeutic efficiency of polymeric nanodrugs was closely related to insure effective release of hydrophobic drug located in the hydrophobic core of polymeric nanodrugs in tumor site. Some previous studies showed that polymeric nanodrugs without sensitive response linker for smart drug release also could achieve combination therapy effect as intact nanodrugs [7], [8]. Xiong et al. have conjugated doxorubicin (DOX) to polyethylene oxide-b- polycaprolactone (PEO-b-PCL) core using the stable amide bonds to construct nanoparticles decorated with αvβ3 intergrin-targeting ligand(RGD) called RGD4C-PEO-b-P(CL-Ami-DOX) [9]. The results of cytotoxicity test showed that RGD4C-PEO-b-P(CL-Ami-DOX) could significantly enhance the cytotoxic response of DOX, suggesting that non-sensitive polymeric nanodrugs which might keep intact in tumor cells could still remain and even improve the antitumor activities of free drug [9]. Contrastively, other studies showed that it was necessary to introduce microenviroment-sensitive linkers (e.g. pH-sensitive or enzyme-sensitive bonds) to connect polymer and hydrophobic drug for accelerating the drug release from polymeric nanodrugs in tumor site and thereby achieving prominent antitumor efficiency [10], [11], [12], [13], [14], [15]. To explore the above controversy, we constructed a range of polymer-drug conjugate based nanoparticles with or without pH-sensitive linkers.
Anti-angiogenesis therapy was considered to be a promising approach for antitumor therapy since that tumor vessels were necessary for transporting nutrient required for tumors growth [16], [17]. Inhibition of the pro-angiogenic growth factors such as vascular endothelial growth factor (VEGF) offered remarkable advantages to achieve effective antiangiogenic therapy for most tumor types [17]. Interestingly, drugs that specifically target VEGF pathway such as bevacizumab, sunitinib and aflibercept have shown remarkable therapeutic activity in cancer treatment [18]. However, it was also reported that monotherapy using anti-angiogenic drug alone could not achieve satisfactory antineoplastic efficacy to benefit the patients’ life in clinic [19]. Accordingly, it was necessary to develop therapeutic regimen by combining anti-angiogenic drug and chemotherapeutic agent for enhancing their anticancer efficiency through synergism [20]. Low molecular weight heparin (LMWH) is a water-soluble natural mucopolysaccharide with non-cytotoxic and good biocompatibility [21], [22]. It was known that LMWH could inhibit tumor angiogenesis by interacting with growth factors such as the VEGF and basic fibroblast growth factor (bFGF) that could promote tumor angiogenesis [22]. In addition, modifying LMWH with hydrophobic moiety was found to not only improve its anti-angiogenesis activities but also reduce its hemorrhagic risk [23].
Ursolic acid (UOA), a pentacyclic triterpenoid with pleiotropic biological effects, has demonstrated the capability to inhibit key steps of angiogenesis, including endothelial cell proliferation, migration and differentiation [24], [25]. Previous study illustrated that modifying LMWH with UOA could effectively improve the anti-angiogenesis activities and antitumor efficiency of nanodrugs in vivo and in vitro through combining UOA induced inhibition of the downregulation of matrix metalloproteinase (MMP) activity and LMWH caused VEGF signal pathway blockade [26]. Moreover, UOA also had strong cytotoxicity against tumor cells through blocking the G0/G1 cycle [27]. Especially, after modifying the 17-COOH of UOA, UOA derivatives further gained remarkably enhanced cytotoxicity against HepG2 cells through improving caspase-3 enzyme activity and strengthening tumor cell cycle blockage [28]. It was also known that the treatment efficiency of liver cancer was always hampered by microvascular invasion [29]. Accordingly, modifying LMWH with UOA and thereby forming polymeric nanodrugs will provide new opportunities for liver cancer treatment through simultaneously inhibiting tumor blood vessels and tumor cells proliferation.
Furthermore, in order to investigate whether it could significantly strengthen the combination antitumor efficiency of LMWH and UOA through accelerating the drug release in tumor, a pH-activatable polymeric nanodrugs(sLMWH-UOA) and a non-sensitive polymeric nanodrug (LMWH-UOA) were prepared. Specifically, schiff base (CHN) with pH-triggered hydrolysis properties and non-sensitive amido bond were employed to connect the LMWH and UOA to form sLMWH-UOA and LMWH-UOA respectively [30]. As displayed in Scheme 1, compared with non-sensitive LMWH-UOA, sLMWH-UOA remained stable before reaching tumor sites and was disassembled after entering into lysosomes with acid microenvironment to spontaneously release UOA and LMWH for exerting dual inhibition of angiogenesis and cytotoxicity. In this study, the structures, particle sizes and drug release behaviors of both LMWH-UOA and sLMWH-UOA were characterized. Besides, we mainly compared the capacity of LMWH-UOA and sLMWH-UOA for inhibition of angiogenesis and tumor proliferation in vitro and in vivo to deduce the correlation between the drug release behaviors and the therapeutic efficiency of polymeric nanodrugs.
Section snippets
Materials and methods
UOA was purchased from Wuhan Yuancheng Co-created Technology Development Co. Ltd. (Wuhan, China) and LMWH (100 IU/mg, average molecular weight 5795 Da) was obtained from Nanjing University. p-Hydroxybenzaldehyde (PHBA) was acquired from Aladdin Industrial Corporation (Shanghai, China). 1-ethyl-3- (3-dimethylaminopropyl)-carbodiimide (EDC), N-Hydroxysuccinimide (NHS), N, N-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) were provided by Sinopharm Chemical Reagent Co. Ltd.
Synthesis and characterization of sLMWH-UOA
LMWH-UOA was synthesized by chemically conjugating hydrophobic UOA to hydrophilic LMWH using non-pH sensitive ethylenediamine as the linker (Fig. 2A) [26]. Besides, the amphiphilic pH-sensitive sLMWH-UOA conjugates were synthesized by chemically conjugating hydrophobic UOA to hydrophilic LMWH using ethylenediamine and p-hydroxybenzaldehyde as the linker (Fig. 2B). The structures of LMWH-UOA and sLMWH-UOA were characterized using 1H NMR as shown in Fig. S1. Compared with LMWH, the spectrum of
Conclusions
A lot of polymeric nanodrugs prepared through connecting hydrophobic chemotherapeutics to multifunctional polymers incorporating either stimuli-responsive or non-sensitive covalent linkers had shown encouraging combination therapy effect in vivo [47], [48]. However, previous studies did not enunciate whether the therapeutic efficiency of polymeric nanodrugs was closely related to their drug release behavior in tumor site [49], [50], [51]. In this study, in order to explore this issue, we
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 81773655), the 12th of Six Talent Peak Foundation of Jiangsu Province (YY-001), the “333” Project Talent Training Fund of Jiangsu Province (BRA2017432), the Open Project of Jiangsu Key Laboratory of Druggability of Biopharmaceuticals (JKLDBKF201702), and the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (JKGQ201107, SKLNMZZJQ201605). We appreciate Dickson Pius Wande
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