Article pubs.acs.org/molecularpharmaceutics
Synthesis and Evaluation of Clickable Block Copolymers for Targeted Nanoparticle Drug Delivery Siyan Zhang,† Kiat Hwa Chan,§ Robert K. Prud'homme,† and A. James Link*,†,‡ Departments of †Chemical and Biological Engineering and ‡Molecular Biology, Princeton University, Princeton, New Jersey 08540, United States § Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, 138669, Singapore S Supporting Information *
ABSTRACT: Polymeric nanoparticles with multifunctional capabilities, including surface functionalization, hold great promise to address challenges in targeted drug delivery. Here, we describe a concise, robust synthesis of a heterofunctional polyethylene glycol (PEG), HO-PEGazide. This macromer was used to synthesize polylactide (PLA)-PEGazide, a functional diblock copolymer. Rapid precipitation of this copolymer with a hydrophobic cargo resulted in the generation of monodisperse nanoparticles with azides in the surface corona. To demonstrate conjugation to these nanoparticles, a regioselectively modified alkyne-folate was employed as a model small molecule ligand, and the artificial protein A1 with an alkyne moiety introduced by unnatural amino acid substitution was selected as a model macromolecular ligand. Using the copper-catalyzed azide−alkyne ligation reaction, both ligands exhibited good conjugation efficiency even when low concentrations of ligands were used. KEYWORDS: nanoparticles, click chemistry, bioorthogonal reactions, bioconjugation
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INTRODUCTION In an effort to drive innovation in medical therapy employing nanotechnology, the NCI developed the Cancer Nanotechnology Plan (NIH Publication Number 04-5489, 2004). Among the opportunities identified in the plan are “multifunctional therapeutics”, which combine therapy and diagnostics; enable targeting, control delivery, and release; and monitor effectiveness. A variety of nanocarriers have been introduced including liposomes, microgels, and inorganic and polymeric carriers.1,2 A universal requirement of nanocarriers is a protective, biocompatible coating to prevent premature clearance by the reticulo-endothelial system (RES).3 Most often, these protective layers are polyethylene glycol (PEG) polymer chains.4 These coatings improve the circulation time of nanocarriers and also present a handle for the attachment of targeting ligands to the surface corona of nanoparticles. The addition of surface functionalization opens up the possibility of creating cell-specific or even disease-specific nanocarriers.5−9 Our focus has been on polymeric nanocarriers with multifunctional capabilities that can be efficiently made with rapid precipitation and block copolymer-directed kinetic assembly, a process termed Flash NanoPrecipitation (FNP)10 (Figure 1). The advantage in using block copolymers is that the protective PEG layer is an integral part of the nanocarrier, simplifying the overall nanocarrier design. The surface functionalization of polymer nanoparticles continues to be a challenge in large part due to the inefficiency of conventional bioconjugation strategies such as amine−carboxylic acid coupling and thiol−maleimide reactions.5 When using these © 2012 American Chemical Society
bioconjugation methods, a large excess of the targeting ligand is often used to ensure suitable levels of functionalization.11 This becomes problematic if the targeting ligand is a costly reagent, such as an antibody or other protein. Newer bioconjugation strategies such as the azide−alkyne click reaction12−14 promise to enable more efficient labeling of the surface corona of polymeric nanoparticles. Azide−alkyne click chemistry is bioorthogonal,12 can be carried out in an aqueous environment at physiological temperatures, and also has high efficiency. Ultimately, both small molecules and macromolecules will have utility as targeting ligands, and azide and alkyne functional groups can be readily added to both types of ligands. Here, we have developed a robust, high-yielding synthesis of an asymmetric HO-PEG-azide polymer that is expected to be broadly useful for the synthesis of many different azidefunctionalized nanocarriers. Using this azide-PEG, we generated a polylactide (PLA) copolymer, PLA-PEG-azide, and demonstrated that this copolymer formed monodisperse nanoparticles using FNP (Figure 1). We evaluated the efficiency of azide−alkyne bioconjugation to these particles with both an alkyne-labeled small molecule ligand, alkynefolate, and a model alkyne-labeled protein ligand and found that both ligands were efficiently conjugated to the nanoparticles even with low ligand loadings. Received: Revised: Accepted: Published: 2228
February 8, 2012 June 10, 2012 June 26, 2012 June 26, 2012 dx.doi.org/10.1021/mp3000748 | Mol. Pharmaceutics 2012, 9, 2228−2236
Molecular Pharmaceutics
Article
Figure 1. Schematic of nanoparticle assembly by FNP and surface corona conjugation of ligands using click chemistry. Block copolymer and hydrophobic cargo are dissolved in THF and rapidly mixed with an antisolvent (water) to create supersaturation and form composite nanoparticles. The azide groups provided by the functional copolymer are used for conjugation to either a small molecule ligand or a macromolecule ligand bearing an alkyne moiety.
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Synthesis of HO-PEG-Cl. To allyl-PEG-Cl (4.8 g, 0.93 mmol) in anhydrous THF (50 mL) at 20 °C a solution of 9BBN in THF (3.74 mL, 1.87 mmol; 0.5 M) was injected. This mixture was allowed to stir for 24 h, after which it was reacted with 12 mL of a 1:2 mixture of aqueous 5 M NaOH and aqueous 30% H2O2 and allowed to stir for a further 48 h at 20 °C. The reaction was then quenched with K2CO3 (1 g), diluted with CH2Cl2 (800 mL), and dried with excess Na2SO4/MgSO4. The solids were filtered, and the filtrate was concentrated to 20 mL, which was then added dropwise to ether (600 mL) with vigorous stirring. The white precipitate was filtered, washed with ether (3 × 50 mL), and dried in vacuo to furnish HOPEG-Cl (4.2 g, 0.82 mmol; 88% yield) as a white solid. As expected, the bulky hydroboration reagent 9-BBN led to regioselectivity toward the terminal carbon, although trace amounts (
