Elsevier

Journal of Controlled Release

Volume 212, 28 August 2015, Pages 94-102
Journal of Controlled Release

Enhancing cancer targeting and anticancer activity by a stimulus-sensitive multifunctional polymer-drug conjugate

https://doi.org/10.1016/j.jconrel.2015.06.024Get rights and content

Abstract

Undesirable physicochemical properties, low tumor targeting, insufficient cell internalization, acquired drug resistance, and severe side effects significantly limit the applications of anticancer drugs. In this study, to improve the tumor targeting and drug efficacy of the poorly water-soluble drug, doxorubicin (DOX), a novel drug delivery platform (PEG-ppTAT-DOX) was developed, which contained a polyethylene glycol (PEG), a matrix metalloproteinase 2 (MMP2)-sensitive peptide linker (pp), a cell penetrating peptide (TAT), and a model drug (doxorubicin). The prepared drug platform possessed several key features, including: (i) the nanoparticle formation via the self-assembly; (ii) prevention of the non-specific interaction via the PEGylation; (iii) tumor targeting via the MMP2-mediated PEG deshielding and exposure of the TAT; (iv) the TAT-mediated cell internalization; (v) the TAT-induced endosomal escape; (vi) the inhibition of P-glycoprotein mediated drug efflux; and (vii) the TAT-medicated nuclear translocation. These cooperative functions ensured the improved tumor targetability, enhanced tumor cell internalization, improved intracellular distribution, and potentiated anticancer activity. Compared to the multi-component nanocarriers, the proposed simple but multifunctional polymer-drug conjugate might have greater potential for tumor-targeted drug delivery and enhanced chemotherapy.

Introduction

Chemotherapy is considered as one of the most effective approaches for treating human cancers. However, the severe drug-related side effects have limited the application of anticancer drugs. To decrease side effects, the cancer-specific therapies are preferred. Usually, the anticancer therapeutics are designed to recognize and bind to cancer cells via the cell surface receptors (known as the receptor-mediated tumor targeting) [1]. Although these strategies have improved the drug efficacy, the overall clinical outcomes remain unsatisfactory. Obviously, these strategies will not be effective when treating the cancer without well-defined cell surface receptors, like triple-negative breast cancer (TNBC) [2].

Matrix metalloproteinase (MMP) especially MMP2, is involved in the cancer growth, invasion, angiogenesis and metastasis and found up-regulated in many human cancers [3]. It has been used as a cancer biomarker and therapeutic target, and also provides a strategy for the on-demand delivery of drugs and imaging agents via an enzyme-triggered mechanism [4], [5], [6]. In our previous studies, the octapeptide (pp, GPLGIAGQ) sensitive to the human MMP2 was successfully used in drug nanocarriers including liposomes [4], polymeric micelles [5], and siRNA delivery nanocarriers [7] to improve tumor targetability. Compared to the receptor-mediated tumor targeting, the MMP2-sensitive strategy is cancer pan-specific and suitable for most types of cancers including the cancer without well-defined cell surface receptors.

Other undesirable properties, such as poor water solubility, insufficient cell internalization, and acquired drug resistance, also compromise the outcomes of anticancer drugs. Cell-penetrating peptides (CPPs) such as the trans-activating transcriptional activator (TAT) peptide have shown excellent membrane translocation capability and been widely used in drug nanocarriers and conjugates [4], [5], [8], [9]. Among several possible mechanisms, the interaction between the positive charge of the TAT and the negative charge of the cell membrane plays an important role in the membrane translocation. The strategies, like PEGylation [4], [5] or charge neutralization [6] have been used to prevent TAT-modified nanocarriers from the interaction with non-target tissues/cells and prolong their circulation time in bloodstream. However, various studies, including ours, showed that the protective PEG shell was not always beneficial for drug delivery. Ideally, the PEG should be deshielded before cell internalization [4], [5], [7].

To achieve an optimal clinical outcome of chemotherapy, all the issues associated with anticancer drugs have to be addressed. In this study, we proposed a simple but multifunctional polymer-drug conjugate system which contains a PEG, an MMP2-sensitive peptide linker (pp), a cell penetrating peptide (TAT), and a drug, for tumor targeted drug delivery. Using doxorubicin (DOX), a poorly water-soluble anthracycline, as a model drug, we developed a self-assembly polymer-drug conjugate, PEG-ppTAT-DOX, possessing multiple functions from its components to deal with the aforementioned challenges. Herein, the synthesis, purification, and characterization of PEG-ppTAT-DOX were described. The physicochemical properties and MMP2 sensitivity of the DOX conjugate were studied. Then, the cellular uptake, intracellular localization, drug efflux inhibition, anticancer activity, and apoptosis-inducing capability of the PEG-ppTAT-DOX were determined in various cancer cells including drug resistant cell lines.

Section snippets

Materials

Polyethylene glycol 2000-succinimidyl valerate ester (PEG2000-SVA) was from Laysan Bio (Arab, AL). Succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), chloroform, and acetonitrile were from Thermo Scientific (Rockford, IL). Dimethylformamide (DMF), triethylamine (TEA), and methanol were from Sigma-Aldrich Chemicals (St. Louis, MO). Human active MMP2 protein and analytical TLC plates were from EMD Biosciences (La Jolla, CA). Dialysis tubing (MWCO 2000 and 3500 Da) were from

Design and synthesis of PEG-ppTAT-DOX

In our recent work, the combined use of the PEG, TAT peptide and the pp in the polymeric micelles has shown excellent tumor targeting and improved anticancer activity of the loaded drug [5]. In that study, these functional moieties were on different nanocarrier building blocks, which made the drug carrier complex. In the current study, to simplify the drug delivery system, a novel MMP2-sensitive cell penetrating peptide (ppTAT) (GPLGIAGQYGRKKRRQRRRC) containing both TAT and pp was used as a

Conclusions

In the study, we prepared a simple but multifunctional drug platform, PEG-ppTAT-DOX, containing a PEG, an MMP2-sensitive cell penetrating peptide (ppTAT), and a model drug (DOX), for tumor-targeted drug delivery. The PEG-ppTAT-DOX showed the MMP2-mediated tumor targetability, the improved cell internalization and intracellular distribution, the P-gp inhibiting capability, and the potentiated anticancer efficacy in both drug sensitive and resistant cancer cells. Besides, due to its excellent

Acknowledgments

Dr. Lin Zhu, the recipient of the Controlled Release Society (CRS) T. Nagai Postdoctoral Research Achievement Award, was partially supported by The Nagai Foundation Tokyo.

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