Elsevier

Journal of Controlled Release

Volume 171, Issue 3, 10 November 2013, Pages 280-287
Journal of Controlled Release

CD133-targeted paclitaxel delivery inhibits local tumor recurrence in a mouse model of breast cancer

https://doi.org/10.1016/j.jconrel.2013.07.014Get rights and content

Abstract

Expression of the membrane protein CD133 marks a subset of cancer cells with drug resistant phenotype and enhanced tumor initiating ability in xenotransplantation assays. Because drug resistance and tumor relapse are significant problems, approaches to eliminate these cells are urgently needed. As a step towards achieving this goal, we developed polymeric nanoparticles targeting CD133 by conjugating an anti-CD133 monoclonal antibody to nanoparticles formulated using poly(D,L lactide-co-glycolide) polymer. Nanoparticles were loaded with paclitaxel, a microtubule-stabilizing anticancer agent, as well as with 6-coumarin, a fluorescent probe. CD133-targeted nanoparticles (CD133NPs) were efficiently internalized by Caco-2 cells, which abundantly express CD133 (> 9-fold higher uptake than non-targeted control nanoparticles). The effectiveness of CD133NPs in reducing tumor initiating cell (TIC) fraction was investigated using mammosphere formation and soft-agar colony formation assays. Free paclitaxel treatment was not effective in decreasing the TIC population relative to untreated control, whereas CD133NPs effectively decreased the number of mammospheres and colonies formed. In vivo studies in the MDA-MB-231 xenograft model showed that free paclitaxel was initially effective in inhibiting tumor growth but the tumors rebounded rapidly once the treatment was stopped. Tumor regrowth was significantly lower when paclitaxel was delivered through CD133NPs (tumor volume was 518.6 ± 228 vs. 1370.9 ± 295 mm3 for free paclitaxel at 63 days; P < 0.05). Our studies thus show that encapsulation of paclitaxel in CD133NPs results in a significant decrease in the TIC population and improved therapeutic efficacy compared to that with free paclitaxel treatment. These results indicate the potential of targeting anticancer therapeutics to CD133 + cells for reducing tumor recurrence.

Graphical abstract

A small subset tumor cells, known as tumor initiating cells (TICs) are responsible for tumor recurrence. Targeting drug loaded nanoparticles to CD133 protein expressed on TICs leads to inhibition of tumor recurrence.

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Introduction

Disease recurrence after an initial response to treatment is a significant problem in breast and other solid tumors [1], [2]. Recent studies suggest that a small subset of tumor cells, known as tumor initiating cells (TICs) or cancer stem cells (CSCs), are responsible for tumor recurrence [3], [4]. TICs have the ability to initiate new tumors and drive their subsequent growth [5], [6]. TICs are highly resistant to conventional treatments including chemotherapy [7], [8] and radiation [9]. Many factors including overexpression of drug efflux proteins [10] as well as DNA-repair proteins [11], elevated levels of antioxidants [12], and slower cell division [13] contribute to resistance. New therapies that can efficiently eradicate TICs are needed to prevent tumor recurrence.

Expression of CD133 (Prominin-1) is widely used as a marker to identify neural, endothelial and hematopoietic stem cells [14], [15], [16] as well as TICs in brain [17], colon [18], prostate [19], pancreatic [20] and breast tumors [21]. CD133 is a 120 kDa glycoprotein that localizes to plasma membrane [22]. Its molecular function is yet to be completely characterized. However, its expression is used to enrich for TICs in breast cancer [23]. CD133 + tumor cells are able to form complete tumors under limiting dilution. Importantly, CD133 expression has been correlated with tumor recurrence in breast cancer patients [24].

The objective of this study was to evaluate the therapeutic effectiveness of delivering paclitaxel, a microtubule-stabilizing anticancer drug, targeted to CD133 + tumor cells. Nanoparticles formulated using the biodegradable poly(D,L lactide-co-glycolide) (PLGA) polymer and surface functionalized with an anti-CD133 antibody (CD133NPs) were developed for targeting TICs. The formulation was characterized for its effectiveness against TICs in cell culture studies and for its ability to inhibit tumor recurrence in a mouse model of breast cancer. Our studies show that encapsulation of paclitaxel in CD133-targeted nanoparticles results in significantly improved therapeutic efficacy and reduced local tumor recurrence.

Section snippets

Materials

PLGA (lactide to glycolide ratio 50:50; average Mw 40 kDa) was purchased from Absorbable polymers, Pelham, AL; hydroxyl PEG maleimide was purchased from Laysan Bio Inc., Arab, AL; polyvinyl alcohol (PVA; molecular weight 30–70 kDa) was obtained from Sigma, St. Louis, MO; HPLC grade organic solvents and plasticware were obtained from Fisher Scientific, Pittsburgh, PA. All general reagents were purchased from Sigma, St. Louis, MO, unless indicated otherwise. Caco-2 (colorectal adenocarcinoma) and

Characterization of antibody conjugated nanoparticles

The mean hydrodynamic diameter of non-functionalized nanoparticles was about 215 nm. The particle size increased to ~ 320 nm following antibody conjugation (Table 1). All the nanoparticle formulations had a net negative surface charge. Paclitaxel was efficiently encapsulated in nanoparticles (> 90% encapsulation efficiency at 12 ± 0.6% w/w loading). Conjugation of anti-CD133 antibody or IgG isotype control to nanoparticles was confirmed by Western blotting (Fig. 1B). Bands corresponding to the heavy

Discussion

The concept of a TIC implies the existence of a hierarchy within tumor cells, in which only a small subset of cells are endowed with stem cell-like features of self-renewal, asymmetric cell division and partial differentiation [6], [34]. TICs are considered to be responsible for tumor origin and its subsequent uncontrolled growth. The cellular origin of TICs is not completely understood. However, the existence of TICs is firmly established in several tumor types including myelogenous leukemia

Conclusion

Polymeric nanoparticles loaded with paclitaxel and surface functionalized with anti-CD133 antibody demonstrated efficient elimination of TICs in vitro and significant inhibition of tumor-regrowth in vivo. The results of our study thus suggest that CD133 is a potential target for directed delivery of anticancer therapeutics and that by eliminating CD133 + cells, it is possible to reduce tumor recurrence in breast cancer. Additional studies investigating a more suitable targeting ligand and

Acknowledgments

We thank the following people at the University of Minnesota: Brenda Koniar and Steve Kalscheuer for help with animal studies; Dr. Deepali Sachdev for help with soft agar colony assay protocol; Colleen Forster for assistance with immunohistochemistry; Dr. David Largaespada for allowing us to use the hematology analyzer. We thank the flow cytometry core facility and the Biological Image Processing Laboratory at the University of Minnesota for their assistance. Funding from Randy Shaver foundation

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