Bioconjugate Chem. 2008, 19, 1561–1569
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Antibody Targeting of Camptothecin-Loaded PLGA Nanoparticles to Tumor Cells Paul A. McCarron,† Waleed M. Marouf,‡ Derek J. Quinn,† Francois Fay,† Roberta E. Burden,† Shane A. Olwill,§ and Christopher J. Scott*,† School of Pharmacy, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, U.K., Hikma Pharmaceuticals (Jordan), P.O. Box 182400, Amman 11118, Jordan, and Fusion Antibodies Ltd., Springbank Industrial Estate, Pembroke Loop Road, Belfast, BT17 0QL, U.K. Received February 12, 2008; Revised Manuscript Received May 26, 2008
Antibody targeting of drug substances can improve the efficacy of the active molecule, improving distribution and concentration of the drug at the site of injury/disease. Encapsulation of drug substances into polymeric nanoparticles can also improve the therapeutic effects of such compounds by protecting the molecule until its action is required. In this current study, we have brought together these two rationales to develop a novel immunonanoparticle with improved therapeutic effect against colorectal tumor cells. This nanoparticle comprised a layer of peripheral antibodies (Ab) directed toward the Fas receptor (CD95/Apo-1) covalently attached to poly(lactideco-glycolide) nanoparticles (NP) loaded with camptothecin. Variations in surface carboxyl density permitted up to 48.5 µg coupled Ab per mg of NP and analysis of nanoparticulate cores showed efficient camptothecin loading. Fluorescence visualization studies confirmed internalization of nanoconstructs into endocytic compartments of HCT116 cells, an effect not evident in NP without superficial Ab. Cytotoxicity studies were then carried out against HCT116 cells. After 72 h, camptothecin solution resulted in an IC50 of 21.8 ng mL-1. Ab-directed delivery of NP-encapsulated camptothecin was shown to be considerably more effective with an IC50 of 0.37 ng mL-1. Calculation of synergistic ratios for these nanoconstructs demonstrated synergy of pharmacological relevance. Indeed, the results in this paper suggest that the attachment of anti-Fas antibodies to camptothecin-loaded nanoparticles may result in a therapeutic strategy that could have potential in the treatment of tumors expressing death receptors.
INTRODUCTION Targeting of tumor-associated antigens using specific antibodies forms the basis of a drug delivery strategy to circumvent difficulties and side effects associated with nonspecific drug dosing. In the design of any immuno-nanoparticulate system, the choice of peripheral antibody, its conjugation chemistry, and therapeutic payload will depend on the intended target cell and associated cell-surface biomarker. One such cell-surface target, amenable to binding by antibodies, is the Fas (CD95/Apo-1) receptor. Fas is a 45 KDa type 1 membrane protein and a member of the tumor necrosis factor (TNF) super family. It is found on the plasma membrane of a variety of cancer cell lines (1) and has been identified as a target for antibodies that induce apoptosis in various human cell lines (2, 3). Fas is present on the cell surface as a homotrimer (4). Upon Fas ligand (FasL) or agonist antibody binding, activation of the Fas receptor trimer results in recruitment of the death inducing signaling complex (DISC) to its intracellular domain leading to activation of procaspase 8 (5, 6). Pro-caspase 8 is autocatalytically activated (6), and in turn cleaves and activates several downstream substrates including the effector caspases 3 and 7, cumulating in a proteolytic cascade that leads to the death of the cell (7). The cell surface expression of Fas has been shown to be modulated by treatment of cells with drugs such as the quinoline* Corresponding author. Dr. Christopher J. Scott, School of Pharmacy, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, U.K. Tel: +44 (0) 28 90 972 350, Fax:+44 (0) 28 90 247 794, E-mail:
[email protected]. † Queen’s University Belfast. ‡ Hikma Pharmaceuticals (Jordan). § Fusion Antibodies Ltd.
based plant alkaloids found in the wood, bark, and fruit of the Asian tree Camptotheca acuminate. The most utilized of these is camptothecin (CPT) which selectively inhibits mammalian topoisomerase I, a DNA replication enzyme overexpressed in a wide range of tumors, including advanced human colon, ovarian, and esophageal carcinomas (8). CPT exhibits antitumorigenic effects by trapping topoisomerase-I with DNA in topoisomerase I-cleavage complexes (9). This inhibition results in the stalling of DNA replication, subsequent S-phase arrest, and initiation of apoptotic cell death in tumor cells (10). Previously, it has been observed that agonistic Fas antibodies potentiated the cytotoxicity caused by CPT treatment (11). Specifically, results demonstrated that sublethal concentrations of CPT up-regulated Fas and other pro-apoptotic proteins, such as Fas ligand, Bax, and p21. Similarly, Xia and co-workers demonstrated that CPT significantly increased the glioblastoma cell death response to Fas receptor activation using agonist antibodies (12). Furthermore, a study using CPT-11 (irinotecan), a water-soluble, semisynthetic prodrug of CPT, found upregulation of cell-surface Fas expression in colorectal cancer cells, independent of p53 status. Interestingly, by combination treatments with CPT-11 and agonistic Fas antibody, it was shown that this surface up-regulation could be exploited to produce synergistic tumor cell cytotoxicity (13). Despite the demonstration that CPT has efficacious activity toward tumor cells, drawbacks remain with its clinical application. CPT is particularly active when in its lactone form, but is easily converted at physiological conditions to a less active carboxylate form, which gives rise to increased side effects (14–16). Moreover, human serum albumin binds to the carboxylate form preferentially, driving the equilibrium in favor of carboxylate formation in the systemic circulation (17, 18).
10.1021/bc800057g CCC: $40.75 2008 American Chemical Society Published on Web 07/16/2008
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Additionally, CPT is poorly water-soluble (19), and formulations frequently require addition of cosolvents, such as poly(ethylene glycol) (PEG) 400, prior to administration (20). It is believed that these drawbacks contribute to the considerable variability in patient response after oral or intravenous administration, observed for the entire CPT drug class (21). Furthermore, as an S phase-specific drug, CPT requires prolonged exposure to the tumor to ensure efficacy (11, 22, 23). In ViVo studies examining the pharmacokinetic profile of CPT have shown that large doses administered over extended intervals are poorly effective when compared to continual low doses or frequently fractioned dosing (24). Consequently, irinotecan (CPT-11) was developed in an effort to circumvent these problems. Although intentionally more water-soluble than CPT, it exhibits much lower antitumor activity, with an IC50 of 1200 µM compared to CPT with an IC50 of 23 µM in L1210 murine leukemia cells. Furthermore, CPT-11 has a lower activity on topoisomerase-I inhibition, with an IC50 > 100 µM, compared to an IC50 of 0.6-1.4 µM for CPT (25). Although clinically effective, the reduced potency of CPT11 means that the ability to use CPT still remains an attractive goal for cancer therapy. As an alternative to cosolvent approaches, the in Vitro antitumor activity of CPT has been enhanced by loading into colloidal drug delivery systems, such as mixed micellar systems (PEG)-phosphatidylethanolamine conjugate with D-R-tocopheryl poly(ethylene glycol) 1000 succinate (26) and sterically stabilized phospholipid micelles (23). Similarly, the improved efficacy of CPT-loaded methoxypoly(ethylene glycol)-poly(DL-lactic acid) nanoparticles (NP) lead to a combination of longer plasma retention times with more efficient tumor localization (27). In this present study, the application of antibody-targeted nanoparticle conjugates containing CPT has been investigated. It was hypothesized that a nanoparticulate system containing CPT and bearing antibodies toward Fas would be simultaneously beneficial. The study aimed to investigate if the combination of Fas targeting and intracellular CPT delivery would improve the efficacy of the drug over solution-based delivery. The results show that entrapment of CPT within the NP improves their efficacy toward tumor cells which is further improved by direct targeting and triggering of receptor-mediated endocytosis via antibody directed interaction with Fas receptor to deliver CPT intracellularly to its site of action.
EXPERIMENTAL PROCEDURES Materials. (S)-(+)-CPT was purchased from Sigma-Aldrich, Germany. PLGA (Resomer RG 502 H) with an acid value of 9 mg KOH per g PLGA, molecular weight 12 kDa and inherent viscosity of 0.22 dL g-1 at 0.1% (w/v) in chloroform at 25 °C, was a generous gift from Mr. Markus Weinga¨rtner, Boehringer Ingelheim, Germany. PLGA (Resomer RG 505 S) with an acid value of
