Elsevier

Biomaterials

Volume 34, Issue 2, January 2013, Pages 598-605
Biomaterials

Tumor suppression via paclitaxel-loaded drug carriers that target inflammation marker upregulated in tumor vasculature and macrophages

https://doi.org/10.1016/j.biomaterials.2012.10.004Get rights and content

Abstract

Clinically approved chemotherapeutic nanoparticles may provide advantages over free drugs by achieving slower clearance and preferential accumulation in tumors. However, the lack of leaky vasculatures can create barriers to the permeation of ∼100 nm-sized nanoparticles in solid tumors. We hypothesized that nanoparticles designed to target both tumor and tumor stroma would penetrate deeper into the tumors. To construct such comprehensive drug carriers, we utilized cross-linked amphiphilic polymer nanoparticles and functionalized them to target ICAM-1, a biomarker prevalent in various tumors and inflamed tumor stroma. The targeting moiety was derived from the modular domain present in αL integrin, which was engineered for high affinity and cross-reactivity with human and murine ICAM-1. ICAM-1-selective delivery of paclitaxel produced potent tumor suppression of not only ICAM-1-positive cervical cancer cells but also ICAM-1-negative tumors, presumably by causing cytotoxicity in tumor-associated endothelium (CD31+) and macrophages (CD68+) over-expressing ICAM-1. Contrary to the strategies of targeting only the tumor or specific tumor stromal constituents, we present a strategy in delivering therapeutics to the major cellular components of solid tumors. Drug carriers against inflammation-biomarkers may be effective against many different types of tumors, while being less susceptible to the highly mutable nature of tumor markers.

Introduction

Achieving site-specific delivery of drug carriers, while minimizing unwanted distribution, has been one of the pursued goals in cancer therapy. Current drug carriers of ∼100 nm in size can be selectively delivered into tumors through the enhanced permeability and retention (EPR) effect [1]. Several different nanoparticles employing the EPR effect have been approved for clinical use, e.g., Doxil and Abraxane. Tumor killing by non-molecularly targeted carriers may be accomplished via slow release of drugs within the tumor interstitial space or from the release of drugs inside cells after non-specific uptake [2]. However, current drug delivery systems fall short on achieving adequate concentrations of drugs in tumor cells in hypoxic regions as well as in malignancies that lack leaky vasculature, promoting the development of drug-resistance [3]. Much of the limitations of non-targeted nanoparticle-based drug carriers come from the fact that (i) leaky pores in tumor vessels are often sparsely distributed and some tumors completely lack well-perfused vasculatures, leading to heterogenous or limited dissemination of nanoparticles [1], [4], (ii) increased interstitial pressure within tumors tends to rapidly clear nanoparticles distributed outside of cells, and (iii) surface modifications of drug carriers to reduce non-specific uptake by phagocytic cells and opsonization also disfavor the uptake of nanoparticles by tumors [2], [5], [6].

To overcome such physiological barriers, local applications of hyperthermia [7], photosensitization [8], and ultrasound [9] have been used to increase the permeability of tumor vessels and thus, the penetration of drug carriers throughout the tumor. Alternatively, new types of nanoparticles to address the aforementioned problems have also been engineered. For example, drug carriers possessing the ability to target tumor stromal constituents, including the vasculature [10], [11] and/or tumor-associated macrophages [12], have shown promising results. Similarly, we have previously found that a molecule called intercellular adhesion molecule (ICAM)-1 was highly induced in endothelial cells within and in the vicinity of tumors [13]. ICAM-1 is a cell adhesion molecule normally expressed at low levels in a variety of cell types but over-expressed in inflammation and neoplastic conditions, likely due to the activation of the transcriptional factor, NF-κB. ICAM-1 is upregulated in many carcinomas including breast, colon, non-small cell lung, and gastric tumors [16], [17], [18], [19], [20], [21], where the over-expression of ICAM-1 is correlated with tumor progression, metastatic capability, and poor prognosis [22], [23], [24]. Upregulation of ICAM-1 within the tumor stroma is, therefore, consistent with the increasing reports on the implication of inflammation in tumor initiation, progression, and metastasis [14], [15]. More importantly, it can be speculated that drug carriers designed to target ICAM-1 would be broadly effective in eradicating not only carcinoma cells expressing ICAM-1 but also those lacking ICAM-1 by killing the tumor stromal cells that are critical to tumor growth and metastasis. The delivery of drugs against multi-cellular components comprising tumors may also be less susceptible to the development of drug-resistance.

In order to create ICAM-1-specific drug carriers, we used a modular domain called the inserted (I) domain derived from the integrin, leukocyte function associated antigen-1 (LFA-1; also known as αLβ2 or CD11a/CD18), where the I domain is the only region that provides molecular contact with ICAM-1. The I domain in its native sequence, separated from LFA-1, exhibits a low level of binding to ICAM-1, and was therefore engineered for high affinity and cross-reactivity with human and murine ICAM-1 [25], [26], [27]. LFA-1 I domain’s reaction with murine ICAM-1 is an important property that permits selectivity studies of drug carriers against both the ICAM-1-expressing human tumors and the inflamed murine tumor microenvironment in mouse models of human cancer. At the same time, this feature enables us to analyze the unintended delivery to the low, yet widespread, expression of ICAM-1 in many different cellular types. As a drug carrier, we employed nanoparticles synthesized with cross-linked amphiphilic copolymer, providing a hydrophobic core for encapsulation of hydrophobic molecules and hydrophilic corona for stability in vivo as well as facile conjugation with targeting moieties [28]. Drug carriers designed to target markers prevalent in carcinomas and inflamed tumor microenvironment may prove effective against many different types of cancers.

Section snippets

Production of high-affinity LFA-1 I domain

Recombinant LFA-1 I domain with a His-tag at the N-terminal was produced in BL21(DE3) Escherichia coli cells (Invitrogen) as described previously [26]. Briefly, after protein induction with 1 mm IPTG (isopropyl-β-d-thiogalactoside), E. coli cells were resuspended in 10 ml of the washing buffer (50 mm Tris (pH 8.0), 23% w/v sucrose, 0.5% w/v Triton X-100, 1 mm ethylenediaminetetraacetic acid (EDTA)), sonicated, and centrifuged again to purify the inclusion body. The inclusion bodies were solubilized

Biodistribution, selectivity, and toxicity of drug carriers

Prior to testing the efficacy of ICAM-1-specific nanoparticles in eradicating tumors, we first evaluated their biodistribution, selectivity to ICAM-1, and toxicity with or without surface conjugation with I domain. The engineered I domain we utilized in this study contains F265S/F292G mutations and binds human and murine ICAM-1 with high affinity (2–6 nm KD) [29]. Cross-linked UAN nanoparticles contain a core formed by polypropylene oxide networks, which can retain hydrophobic molecules at up to

Discussion

Utilizing polymeric nanoparticles as drug carriers, here we report that molecular markers induced in pro-inflammatory tumor microenvironment may provide a therapeutic strategy that is applicable to the suppression of different types of tumors independent of their tumor surface antigens. Among the surface markers that closely follow the degree of inflammation, we have chosen ICAM-1, a molecule that is rapidly upregulated in response to inflammation. With a targeting moiety against ICAM-1

Conclusions

In this work, we have successfully engineered polymeric drug carriers to target ICAM-1-expressing tumors as well as inflamed tumor microenvironment. UAN has exhibited its advantages as a targeted drug carrier for its ability to evade the mononuclear phagocytic system and cause low levels of systemic toxicity. By targeting an inflammatory marker in tumor vessels, Id-UAN particles may act as a double-edged sword: Not only will they be able to localize drug delivery to the tumor site, they may

Acknowledgments

This work was funded by a National Science Foundation (NSF) GK-12 Fellowship Award and Business for International Cooperative R&D between Industry, Academy, and Research Institute (Korea Small and Medium Business Administration, Grant No. 00042115).

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