Article pubs.acs.org/Macromolecules
Polymer-Grafted, Nonfouling, Magnetic Nanoparticles Designed to Selectively Store and Release Molecules via Ionic Interactions Johan Sebastian Basuki,† Hien T. T. Duong,† Alexander Macmillan,‡ Renee Whan,‡ Cyrille Boyer,*,† and Thomas P. Davis*,§ †
Australian Centre for NanoMedicine, School of Chemical Engineering and ‡Biomedical Imaging Facility, Mark Wainwright Analytical Centre, University of New South Wales, 2052 Sydney, Australia § Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia S Supporting Information *
ABSTRACT: Surface functionalization of superparamagnetic iron oxide nanoparticles (IONPs) was achieved by exploiting a grafting “onto” approach simultaneously with an in situ modification of the graft block copolymer. Terminal phosphonic-acidbearing block copolymers composed of pendant-activated ester moieties, that is, poly(pentafluorophenyl acrylate) (P(PFPA)) and poly(oligoethylene glycol acrylate) (P(OEGA)), were synthesized and assembled on IONP surfaces. The assembly was performed in the presence of different primary amines to introduce different functionality to the grafted chains, followed by subsequent thiol−ene Michael additions with acrylates or maleimides to decorate the IONP surface. The aim of this “double”-click chemistry on the polymer-coated nanoparticles was to generate a library of IONPs consisting of an internal layer of functionalized polyacrylamides and an outer shell of antifouling P(OEGA) decorated with fluorescent ligands. The resultant multifunctionalized IONPs were characterized using ATR-FTIR, XPS and TGA, proving the presence of modified polymers on the IONP surfaces. The functionalized nanoparticles proved to be stable in both water and phosphate buffer containing bovine serum albumin. Zeta potentials of the functionalized nanoparticles could be tuned by the judicious choice of functional groups introduced by the primary amines, for example, spermine, 3(dimethylamino)-1-propylamine, L-lysine, L-histidine, L-arginine, β-alanine, and taurine. Depending on the pH of IONP dispersions, the charge induced by functional groups within the polymer shell was used to encapsulate ionic dyes (methyl blue and rhodamine 6G in cationic and anionic layers, respectively), serving as models for drug loading via ionic complexation. The attachment of fluorophore through thiol−ene Michael addition was demonstrated by conjugating fluorescein-O-acrylate, as monitored by fluorescence spectroscopy. Cytotoxicity studies revealed that multifunctionalized IONPs were nontoxic to normal human lung fibroblast cell lines. Fluorescence lifetime imaging microscopy was employed to demonstrate the complexation and release of rhodamine 6G dye from L-lysine-functionalized IONPs.
1. INTRODUCTION Iron oxide nanoparticles (IONPs) have been studied extensively for diverse biomedical applications such as magnetic resonance imaging (MRI) contrast agents, hyperthermia agents, biosensors, protein separation, stem cell tracking, and drug carriers.1−4 In particular, the use of IONPs as a negative contrast agent in T2-weighted MRI has been optimized and investigated in clinical trials.5,6 It is important to understand the interactions that occur when IONPs are placed in a physiological environment and relate this to the physicochemical properties of the nanoparticles.7 Particle size- and morphology-controlled IONPs can be synthesized by thermal decomposition of an organometallic precursor or by using flame-spray pyrolysis.8 An in vivo toxicity study of IONPs indicated that they are relatively safe; however, in a biological environment IONPs tend to agglomerate following salt and protein adsorption.9,10 Nonimmunogenic coating materials like biocompatible polymers can be applied to the IONP surface using physisorption or chemisorption processes to © 2013 American Chemical Society
improve biocompatibility and colloidal stability of IONPs. The presence of hydroxyl groups on IONP surfaces gives a convenient handle for the attachment/adsorption of different functionalities to anchor any desired coating.11 Coating materials such as protein, dextran, and poly(ethylene glycol) can be applied by layer-by-layer assembly (physisorption). Ferridex and Combidex are two examples of clinically used dextran-coated IONPs for liver/spleen imaging and lymph-node metastases imaging, respectively.4,12 Alternatively, high-affinity and biostable IONPs can be engineered using a polymer conjugation approach (chemisorption) using established functionalization chemistry, for example, exploiting silane, dopamine, or phosphonic acid groups.13 Living radical polymerizations such as nitroxide-mediated polymerization (NMP), atom-transfer Received: June 6, 2013 Revised: August 1, 2013 Published: August 22, 2013 7043
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Scheme 1. (A) Synthesis of RAFT Agent 3 and Phosphonic Acid Terminated Block Copolymer P(PFPA)-b-P(OEGA) and (B) Synthesis of Homopolymer P(OEGA).
incorporated in the polymer layer to enable positron emission tomography (PET) imaging.26 The targeting of nanoparticles to specific tissues of interest is often accomplished via conjugation using targeting ligands attached to the nanoparticles surface, for example, RGD peptide or folic acid.27−29 Multiple receptors expressed on the surface membranes of cancer cells can be utilized to bind selectively to IONP for receptor-mediated cell uptake.30,31 In addition, targeted therapeutic delivery can be facilitated by the application of an external magnetic field.32,33 Alternating magnetic fields can be used to kill cancer cells through local heating (hyperthermia) or to induce on-demand drug release.34,35 Superparamagnetic IONPs have many material properties advantageous for theranostic applications.36 Theranostic magnetic nanoparticles can be used to track the distribution of nanoparticles in vivo, allowing the drug delivery to be verified at specific sites where therapy is required.37,38 This concept of multifunctionalized magnetic nanoparticles for theranostic applications has been described by a number of research groups.39−41 In our current work, we have developed a versatile multifunctionalization approach to stabilized magnetic nanoparticle carriers using a RAFT-synthesized block copolymer with activated ester pendent groups; poly(pentafluorophenyl acrylate) (P(PFPA)). We introduced different functional groups to the polymeric layer of IONPs to facilitate the incorporation of therapeutic molecules and fluorophores.42,43 During the postmodification of P(PFPA) blocks, RAFT end groups were also converted into thiols via aminolysis, which we subsequently capped using either acrylates or maleimides via thiol−ene Michael addition reactions.44,45 The objective of our work was to generate a library of multifunctionalized IONPs with high colloidal stability via a versatile and simple one-pot grafting “to” approach combined with an in situ polymer modification.36,46 Our library of IONPs was then subjected to a stability-testing and characterization regime.47,48 Cross-linking of the internal polymer layer of the IONPs was achieved using diamine molecules (e.g., spermine). Multifunctionalized IONPs with
radical polymerization (ATRP), or reversible addition−fragmentation transfer (RAFT) have previously been employed to synthesize polymer architectures bearing anchoring moieties for convenient attachment to the surface of IONPs using grafting “onto” approach.14−16 Polymer coated/functionalized magnetic nanoparticles designed for use as advanced MRI contrast agents need to be optimized to yield high relaxivities and long blood circulation times (to facilitate accumulation in target tissues via the enhanced permeation retention (EPR) effect) and to effect high colloidal stability.5,17 The physicochemical properties of functionalized IONPs, such as size, charge, polymer density, and morphology, are essential factors in determining the IONP serum half life.14,15 Functionalized magnetic nanoparticles have also been widely investigated as therapeutic nanocarriers. Chemotherapeutic drugs or genes have been attached to IONPs via covalent conjugation or ionic complexation. Gene delivery via polyplexes is an established research field exploiting cationic polymers, for example, poly(ethylene imine) or poly(dimethylamino ethyl acrylate) (P(DMAEA)). Cationic polymers grafted onto IONPs have been previously studied for gene delivery and transfection.18,19 Ionic polymer layers need to be shielded to ensure nanoparticle stability in biological fluids, so antifouling polymers such as PEG have been used to shield the charge, thereby preventing IONP agglomeration.20 Similarly, complexation of cationic cargo, for example, adriamycin, using anionic polymers has also been described by others.21−23 In addition to therapeutics, fluorophores can be incorporated into the IONP shells to complement the MRI “tracking” capabilities of the nanocarriers. Thus optical imaging (near-infrared or NIR), for example, Cy5.5 or Cy7 capability, can be built in to the IONP design.24 The potential combined facility for MRI and optical tracking extends the diagnostic capability of IONPs by combining the high-resolution (spatial and temporal) and deep-tissue penetration of MRI with the rapid response and sensitivity of NIR optical imaging.16,25 Radioactive compounds such as 64Cu or 18F can also be 7044
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Figure 1. 1H NMR of (A) RAFT agent 3, (B) homopolymer P(PFPA), and (C) block copolymer P(PFPA)-b-P(OEGA). (D) 19F-NMR and (E) 31P NMR of P(PFPA)-b-P(OEGA).
Using 19F-NMR, the PFPA conversion was calculated using the following equation: αPFPA = [∫ −158.0ppm/(∫ −158.0ppm + ∫ −152.5ppm)] × 100, with ∫ −158.0ppm and ∫ −152.5ppm corresponding to integral of the signal at −158.0 (polymer) and −152.5 (monomer) ppm, respectively. The resultant P(PFPA) macroRAFT was then chain-extended with oligoethyleneglycol acrylate (OEGA) using a ratio of the P(PFPA) macroRAFT concentration to the OEGA concentration of 1:60. Under optimized condition (i.e., anhydrous acetonitrile, 60 °C, 18 h) block copolymer P(PFPA)-b-P(OEG-A) was obtained after an overall monomer conversion of 71%. The polydispersity of the molecular weight distribution remained low (PDI = 1.2), consistent with a wellcontrolled RAFT polymerization. OEGA conversions were calculated from 1H NMR data using the following equation: αOEGA = [∫ 4.1ppm/(∫ 4.1ppm + ∫ 4.3ppm)] × 100, with ∫ 4.1ppm and ∫ 4.3ppm corresponding to integrals of the signals at 4.1 (polymer) and 4.3 (monomer) ppm. Using the signal at 0.9 ppm attributed to the RAFT agent (Figure 1), the integrals of signals at 3.0 and 4.1 ppm were used to determine the average number of PFPA and OEGA units, respectively. On the basis of the following equation MnP(PFPA)‑b‑P(OEGA) = [(∫ 4.1ppm/2)/(∫ 0.9ppm/3)] × MWOEGA + [∫ 3.0ppm/(∫ 0.9ppm/3)] × MWPFPA + MWRAFT 3, the molecular weights of P(PFPA)-b-P(OEGA) block copolymers were calculated, and the ratio between PFPA and OEGA units was determined to be 1:1.5.
cationic or anionic charges were utilized to complex both anionic methyl blue and cationic rhodamine 6G.49,50 This study demonstrates an approach to potential theranostic IONPs, where the dyes can be seen as probes to model the future extension of this work to therapeutics delivery. (siRNA or doxorubicin hydrochloride can also be incorporated via electrostatic interactions.)23,51,52
2. EXPERIMENTAL SECTION The synthesis and characterization of phosphonic-acid-bearing RAFT agent, block copolymers, and multifunctionalized IONPs are reported in the Supporting Information.
3. RESULTS AND DISCUSSION 3.1. Synthesis of Phosphonic-Acid-Terminated Diblock Copolymer. Phosphonic-acid-bearing trithiocarbonate RAFT agent (3) was synthesized (see Scheme 1). Bromotrimethylsilane was employed to selectively deprotect the phosphonate ester.53 The purified RAFT agent was utilized in the polymerization of monomers with an activated ester group, pentafluorophenyl ester acrylate (PFPA).54 PFPA was polymerized in anhydrous dioxane at 80 °C with a ratio of [RAFT 3]:[AIBN]:[PFPA] 1:0.2:40. The molecular weight distribution was monitored by size-exclusion chromatography (SEC), and the monomer conversion was monitored by nuclear magnetic resonance (1H and 19F). 7045
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19
F and 31P NMR data were used to confirm the presence of activated ester (PFPA) pendent groups and terminal phosphonic acid groups in the P(PFPA)-b-P(OEGA) block copolymers (Figure 1D,E). On the basis of measured molecular weight distributions obtained using DMAc SEC (Figure 2), we observed
L -histidine
were also used as amino acids to introduce zwitterionic characteristics. It is noteworthy to comment on our reason for not prefunctionalizing the copolymers prior to polymer brush assembly on the IONP surfaces. In previous work, we found low grafting densities for the assembly of charged polymers on IONPs caused by electrostatic repulsion between polymer chains.19 In addition, cross-linking cannot be performed prior to brush assembly. We also found that the chemical stability of the P(PFPA) block on IONPs could be problematic; the grafting “onto” process involves ultrasonication of the nanoparticles in DMSO, followed by purification of copolymer-functionalized nanoparticles using multiple washing−centrifugation cycles in water. The grafting and purification steps could lead to premature hydrolysis of PFPA block or cleavage of pentafluorophenyl ester groups. We studied the instability of P(PFPA)-b-P(OEGA) copolymers in both DMSO and water using 19F-NMR. After 1 h at 50 °C, ∼17% of activated ester groups were hydrolyzed, and free pentafluorophenol was observed (−162, −165, and −172 ppm signals in 19FNMR). After the grafting “onto” process, IONPs functionalized with P(PFPA)-b-P(OEGA) were isolated and characterized by ATR-FTIR (Figure 3). Before the grafting “onto” process, block copolymer P(PFPA)-b-P(OEGA) exhibited absorptions at 1780 and 1720 cm−1, consistent with ester groups from both PFPA and OEGA, respectively. After brush assembly, the PFPA absorption at 1780 cm−1 disappeared, indicating that the PFPA block hydrolyzed during the grafting “onto” reaction and subsequent purification. We therefore investigated a simultaneous grafting/ functionalization procedure avoiding the prehydrolysis of PFPA. The degradation kinetic of P(PFPA) block was monitored in parallel with the grafting “onto” process (Figure S1, Supporting Information). The kinetic of grafting “onto” process was monitored by thermal gravimetric analysis (TGA) after purification of IONPs@P(OEGA). After 5 min of grafting “onto”, the weight loss of the nanoparticles (or grafting density) appears constant versus grafting “onto” time, demonstrating that the reaction of grafting “onto” is very fast. Because the grafting “onto” IONPs process could be achieved rapidly under ultrasonication, amine addition was performed subsequently (
