pH-activatable polymeric nanodrugs enhanced tumor chemo/antiangiogenic combination therapy through improving targeting drug release

https://doi.org/10.1016/j.jcis.2018.10.039Get rights and content

Abstract

It was widely accepted that polymeric nanodrugs held superiority in enhancing antitumor efficacy, reducing side effect and achieving better long-term prognosis. However, there still existed disputes that whether their therapeutic efficiency was closely related to insure effective release of hydrophobic drug located in their hydrophobic core in tumor site. In order to investigate this controversy, we constructed two polymeric nanodrugs (pH-activatable sLMWH-UOA and non-sensitive LMWH-UOA) with low molecular weight heparin (LMWH) and ursolic acid (UOA) for chemo-and anti-angiogenic combination therapy in hepatocellular carcinoma. The degradation ratio of pH-activatable sLMWH-UOA increased by 33% compared with non-sensitive LMWH-UOA in in vitro degradation study. Besides, confocal microscopy captured that sLMWH-UOA could effectively release drug in acidic microenvironment of lysosome while LMWH-UOA nearly could not. More importantly, in contrast with LMWH-UOA, sLMWH-UOA presented pH-dependent cytotoxicity, indicating that promoting drug release played a key role in enhancing the cytotoxicity of polymeric nanodrugs. Additionally, in vivo pharmacodynamic evaluation showed that although non-sensitive LMWH-UOA had benefited from enhanced tumor targeting drug delivery ability to achieve absolute advantage over free drug combination therapy in antitumor combination therapy, sLMWH-UOA could acquire further optimized combined therapeutic effect with better drug release in tumor. All above, application of tumor-triggered chemical bonds to construct polymeric nanodrugs held vast prospect for improving the therapeutic efficiency for tumor cells.

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 (single bondCHdouble bondNsingle bond) 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

References (51)

  • C. Cárdenas et al.

    Effects of ursolic acid on different steps of the angiogenic process

    Biochem. Biophys. Res. Commun.

    (2004)
  • Y. Li et al.

    Sigma receptor-mediated targeted delivery of anti-angiogenic multifunctional nanodrugs for combination tumor therapy

    J. Controlled Release

    (2016)
  • J. Li et al.

    Effects of ursolic acid and oleanolic acid on human colon carcinoma cell line HCT15

    World J. Gastroenterology

    (2002)
  • J.W. Shao et al.

    In vitro and in vivo anticancer activity evaluation of ursolic acid derivatives

    Eur. J. Med. Chem.

    (2011)
  • Y. Wang et al.

    A nanoparticle-based strategy for the imaging of a broad range of tumours by nonlinear amplification of microenvironment signals

    Nat. Mater

    (2014)
  • J.L. Andres et al.

    Binding of two growth factor families to separate domains of the proteoglycan betaglycan

    J. Biol. Chem

    (1992)
  • J.W. Park et al.

    High antiangiogenic and low anticoagulant efficacy of orally active low molecular weight heparin derivatives

    J. Controlled Release

    (2010)
  • G.L. Logue

    Effect of heparin on complement activation and lysis of paroxysmal nocturnal hemoglobinuria (PNH) red cells

    Blood

    (1977)
  • X. Yan et al.

    Gambogic acid grafted low molecular weight heparin micelles for targeted treatment in a hepatocellular carcinoma model with an enhanced anti-angiogenesis effect

    Int. J. Phamaceut

    (2017)
  • H. Zhang et al.

    pH-sensitive prodrug conjugated polydopamine for NIR-triggered synergistic chemo-photothermal therapy

    Eur. J. Pharm. Biopharm.

    (2018)
  • S. Jafarzadeh-Holagh et al.

    Self-assembled and pH-sensitive mixed micelles as an intracellular doxorubicin delivery system

    J. Colloid. Interf. Sci.

    (2018)
  • W. Zhang et al.

    Polymeric nanoparticles developed by vitamin E-modified aliphatic polycarbonate polymer to promote oral absorption of oleanolic acid

    Asian J. Pharm. Sci.

    (2017)
  • J. Fang et al.

    Enhancement of chemotherapeutic response of tumor cells by a heme oxygenase inhibitor, pegylated zinc protoporphyrin

    Int. J. Cancer

    (2004)
  • C. Li et al.

    Complete regression of well-established tumors using a novel water-soluble poly(L-glutamic acid)-paclitaxel conjugate

    Cancer Res.

    (1998)
  • S. Kaur et al.

    Trigger responsive polymeric nanocarriers for cancer therapy

    Biomater. Sci.

    (2015)
  • Cited by (25)

    • Fluorescence interactions of a novel chalcone derivative with membrane model systems and human serum albumin

      2022, Biophysical Chemistry
      Citation Excerpt :

      On the other hand, the synthesized dyes may cause pollution problems, depletion of natural resource affecting ecological balance. Therefore, it is important to examine the cytotoxic effects of new organic dyes [29–31]. The production of a fluorescent probe, which can be synthesized with an easy and inexpensive technique, is non-cytotoxic, with well-defined photophysical properties will provide an advantage over the molecules in the literature.

    • Preparation and application of pH-responsive drug delivery systems

      2022, Journal of Controlled Release
      Citation Excerpt :

      When the amino group reacts with the aldehyde group, the absorption peaks for the aldehyde group and the amino group disappear [73], or a new absorption peak of C=N stretching vibration of the imine bond appears at about 1600 cm-1 [74,75]. And 8.03 ppm in a 1H NMR spectrum is often assigned to the imine bond [76]. All of them can be used as an indicator for the formation of an imine bond.

    • Carboxymethyl chitosan based redox-responsive micelle for near-infrared fluorescence image-guided photo-chemotherapy of liver cancer

      2021, Carbohydrate Polymers
      Citation Excerpt :

      Compared to monotherapy, the combination of two or more therapeutics with different pharmacological mechanisms could achieve better clinical results (Mokhtari et al., 2017; Phua et al., 2019; Zhang et al., 2015). Combination therapy is the suggested way to increase treatment efficacy, to prevent the development of drug resistance, and to reduce the duration of treatment (Li et al., 2017; Wu et al., 2020; Xiong et al., 2019). The combination use of chemotherapeutics and photosensitizers (PS) has provided an innovative strategy for cancer treatments.

    • Camouflaged, activatable and therapeutic tandem bionanoreactors for breast cancer theranosis

      2020, Journal of Colloid and Interface Science
      Citation Excerpt :

      Nevertheless, NRs with the capability to produce H2O2 in situ will be beneficial for improved therapeutic efficacy in vivo. The smart delivery nanosystems responsive to the tumor microenvironment (TME), including cellular metabolism or the physical attributes (pH, redox species) are desirable in clinical medicine [12–15]. On the other hand, the one-pot cascade reaction of naturally occurring enzymes are highly specific and selective that is exciting from an environmental and economic point of view [4,16–19].

    • Evolution from small molecule to nano-drug delivery systems: An emerging approach for cancer therapy of ursolic acid

      2020, Asian Journal of Pharmaceutical Sciences
      Citation Excerpt :

      The diameter size of sLMWH-UA was 223.1 ± 0.9 nm, and the critical aggregation concentration was 38.30 µg/ml, indicating its excellent stability in the systemic circulation. The addition of Schiff base bond promoted the micelles to hydrolyze and then release drugs under acidic conditions [93]. Overall, these studies demonstrated that conjugating UA with other chemotherapeutic agents (summarized in Fig. 5) may be a promising strategy for cancer therapy.

    View all citing articles on Scopus
    View full text