Construction of Multifunctionalizable, Core-Cross-Linked Polymeric

Mar 11, 2014 - The dye-loaded nanoparticles exhibited pH-triggered release behavior around the acidic tumoral environment, implying that these nanopar...
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Construction of Multifunctionalizable, Core-Cross-Linked Polymeric Nanoparticles via Dynamic Covalent Bond Xiaobei Wang,† Lin Wang,† Shixia Yang,† MingMing Zhang,‡ Qingqing Xiong,‡ Hanying Zhao,† and Li Liu*,† †

Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071, P. R. China, and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, P. R. China ‡ Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300192, P. R. China S Supporting Information *

ABSTRACT: Well-defined hydrazide-containing copolymers poly(poly(ethylene glycol) methacrylate-co-methacryoyl hydrazide) (P(PEG-co-MAH)) via reversible addition−fragmentation chain transfer radical polymerization were used as a reactive scaffold for bioconjugations to prepare polymers for protein recognition. The nucleophilic reaction of hydrazide and glucose generated glycoconjuagted copolymer that can recognize Con A. Biotinylated copolymer was prepared by the conjugation of aldehyde-functionalized biotin to the copolymer via hydrazone bond. Subsequently, dynamic covalent cross-linked nanoparticles were constructed via reversible acylhydrazone linkages by the reaction of copolymer and terephthaldicarboxaldehyde. The crosslinked nanoparticles demonstrated reversible pH-dependent formation/disintegration and adaptive characters. The cross-linked nanoparticles were further adorned through successive reactions of their remaining hydrazide units with aldehyde-functionalized biotin and fluorescein isothiocyanate to generate multifunctional nanoparticles. An in vitro study confirmed that the cross-linked nanoparticles were nontoxic to HeLa cells. These nanoparticles can encapsulate a cargo of small hydrophobic molecules, Nile red. The dye-loaded nanoparticles exhibited pH-triggered release behavior around the acidic tumoral environment, implying that these nanoparticles via hydrazone linkages have promise as therapeutic nanocarriers in a drug delivery system. Therefore, these dynamic covalent nanoparticles generated from hydrazide-containing copolymers can be utilized not only as building blocks for the construction of multifunctional materials with pH-responsive and adaptive characters but also as smart nanocarriers in biomedicine.

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INTRODUCTION The development of smart polymeric nanoparticles possessing environmentally responsive and adaptive properties has attracted great attention in recent decades because of their promising applications in biomedical fields such as diagnostics, bioimaging, and drug delivery.1−3 Conventionally, the thermoand/or pH-responsive nanoparticles are extensively prepared on the basis of polymer micelles formed by the block copolymers which are inherently stimuli-responsive. Wooley and co-workers reported the synthesis of shell cross-linked “knedel” (SCK) nanoparticles with pH-dependent properties.4 By incorporation of functional monomers with different pKa © 2014 American Chemical Society

into block copolymers, Armes and co-workers have prepared zwitterionic shell cross-linked micelles with tunable hydrophilic/hydrophobic cores depending on the environmental temperature, pH, and salt concentration.5 Liu and co-workers developed a series of stimuli-responsive shell or core crosslinked micelles.6 However, there still exists a limited palette of structure, property, and responsiveness of these stimuliresponsive block copolymers. Alternatively, an important and Received: November 21, 2013 Revised: February 28, 2014 Published: March 11, 2014 1999

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aldehyde-modified biotin moiety generated glycoconjugated copolymer and biotinylated copolymer for protein recognition. Subsequently, on the basis of the concept of dynamic covalent chemistry, the core-cross-linked nanoparticles were prepared using intra/intermolecular acylhydrazone formation of hydrazide-containing copolymers and terephthaldicarboxaldehyde (TDA). The pH-reversible and dynamic characters of the nanoparticles were investigated by means of 1H NMR and DLS. Excess hydrazide functions not involved in the cross-linking can be utilized as handles for the postfunctionalization of nanoparticles, which were further adorned with biotin and FITC. The core-cross-linked nanoparticles showed good biocompatibility as estimated by in vitro cytotoxicity studies against HeLa cells. Using hydrophobic dye Nile red as a model drug, the pH-triggered release behavior of dye from the nanoparticles was studied. The incorporation of dynamic acylhydrazone linkages endows the core-cross-linked nanoparticles with pH-responsive and dynamic characters, which may have various potential applications in biotechnology and biomedical fields.

efficient approach of endowing nanoparticles with the virtues of stimuli responsiveness is the utilization of dynamic covalent bonds (DCBs).7 The introduction of DCBs into or between polymer chains can generate polymeric materials with responsive and adaptive characters. DCBs, also referred to as reversible covalent bonds, are in an equilibrium state, meaning that the bond is reversibly formed and broken as exploited in dynamic combinatorial chemistry.8 The reversible nature of DCBs enables products to modify their constitutions by exchanging and reshuffling their building blocks. The implementation of DCBs in polymer chemistry has generated advanced polymeric materials that have the ability to reversibly adapt to their environment and possess a wide range of responses ranging from self-healing to mechanical work.1,9 The most studied DCBs include thermally activated alkoxyamine bonds,10 Diels−Alder adducts,11 acylhydrazones,12 imines,13 boronic esters,14 and disulfide bonds.15 For example, the Lehn group has pioneered the preparation of reversible acylhydrazone-based linear dynamers with tunable mechanical, optical, and thermoresponsive properties.16 They also incorporated biologically related moieties into dynamers to generate biodynamers that combine the functional properties of biomolecules with the adaptive behaviors of constitutional dynamic systems.17 DCBs also possess appealing features in the construction of stimuli-responsive polymeric nanoparticles because of their reversible and adapative characters. Fulton and co-workers untilized reversible acylhydrzaone or imine linkages to synthesize dynamic covalent stars, pH- and redox-responsive nanoparticles, and single-chain polymer nanoparticles.18 They found that the thermoresponsive single-chain nanoparticles could reversibly transform into a chemically cross-linked hydrogel at pH 4.5 upon the temperature above LCST because of the dynamic character of acylhydrazone bonds to undergo component exchange. The synergy of conventional stimuliresponsive polymer with DCBs results in a polymeric material possessing unique adaptive features.19 McCormick and coworkers prepared shell cross-linked (SCL) micelles with cleavable imine linkages. The pH-triggered release of the hydrophobic drug encapsulated in these SCL micelles was clearly observed.20 Thayumanavan and co-workers21 empolyed a facile nonemulsion approach for the preparation of surfacefunctionalizable nanogels via dynamic-covalent disulfide linkages with a wide range of biomedical applications from drug delivery to biosensing. The groups of Haddleton and Davis have prepared stimuli-responsive biodegradable core-crosslinked polymers using dimethacrylate monomer containing a disulfide linkage in the RAFT block copolymerization.22 Sumerlin’s research group developed a series of core-crosslinked dynamic-covalent stars containing Diels−Alder, boronic ester, and disulfide linkages capable of reversibly reconfiguring their composition/structures in response to a stimulus.14b,23,24 Otsuka and Takahara have exploited a TEMPO-based thermally exchangeable radical crossover reaction to construct star-like nanogel assemblies, discrete spherical nanoparticles, and macroscopically cross-linked organo- and hydrogels.10c,25−28 In this paper, the hydrazide-containing copolymers were synthesized by RAFT polymerization. Because of the versatile reactivity of hydrazides, the hydrazide-containing copolymer provides a scaffold where biologically active substances can be attached under mild conditions. In our preliminary studies, the bioconjugation of the copolymer with glucose and the

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EXPERIMENTAL SECTION

Materials. Poly(ethylene glycol) methyl ether methacrylate (PEGMA, Mn = 475 g/mol, Aldrich) was purified by passing through a basic alumina column. Acetone oxime (Alfa Aesar, 98%) was recrystallized from petroleum ether. Terephthaldicarboxaldehyde (TDA) was recrystallized from ethanol/H2O and dried under vacuum. Biotin, fluorescein isothiocyanate (FITC), 2-(4-hydroxyphenylazo)benzoic acid (HABA)/avidin reagent, and picrylsulfonic acid (TNBS) solution were purchased from Sigma and used as received. Aminoacetaldehyde dimethyl acetal (98%), 2-mercaptothiazoline (98%), Nile red (99%), and phenylboronic acid (PBA, 98%) were purchased from J&K Chemical Ltd. and used as received. 4,4′Azobis(4-cyanopentanoic acid) (ACPA, Acros, 97%) was dried under vacuum before use. Alizarin red S sodium salt was purchased from Alfa Aesar and used directly. Cell counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Japan). Dulbecco’s Modified Eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from Thermo Scientific. Penicillin and streptomycin were bought from Solarbio Technology Co., Ltd. All other reagents were commercially available products and used as received. All solvents were redistilled before use. 4-Cyanopentanoic acid dithiobenzoate (CPADB) was synthesized according to the method reported in the literature.29 Methacryloylacetone oxime (MAO) was prepared by the reaction of acetone oxime and methacyloyl chloride.30 1H NMR (CDCl3, 400 MHz) δ (ppm): 6.10 (1H, m), 5.57 (1H, m), 2.02 (3H, s), 1.98 (3H, s), 1.95 (3H, s). Characterizations. 1H NMR spectra were recorded on a Varian UNITY-plus 400 M nuclear magnetic resonance spectrometer using CDCl3, DMSO-d6, or D2O as the solvents. The number-average molecular weights (Mn) and polydispersities (Mw/Mn) of the P(PEGMA-co-MAO) copolymers were determined by GPC at 35 °C with a Waters 1525 chromatograph equipped with a Waters 2414 refractive index detector. Tetrahydrofuran (THF) was used as the mobile phase at a flow rate of 1 mL/min. Polystyrene standards were used for calibration. The molecular weights and polydispersities of P(PEGMA-co-MAH) copolymers were determined by aqueous GPC (VISCOTEK, TDA-302) at 30 °C equipped with a TSK column with a molecular range of 1000−860K, a right angle light scattering, and a refractive index detector. The eluent was PBS (0.10 M, pH = 6.8) at a flow rate of 1 mL/min. Calibration was achieved using a series of poly(ethylene oxide) standards ranging from 2200 to 26 900 g mol−1. UV−vis spectroscopy was performed on a Shimadzu UV-2450 UV−vis spectrophotometer. Fluorescence spectra were recorded on a Shimadzu RF-5301 PC spectrofluorophotometer. Dynamic light scattering (DLS) measurements were conducted on a Zetasizer Nano ZS from Malvern Instruments equipped with a 10 mW HeNe 2000

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laser at a wavelength of 633 nm. Transmission electron microscopy (TEM) observations were carried out on a Tecnai G2 20 S-TWIN electron microscope equipped with a Model 794 CCD camera. The nanoparticle samples were deposited on a carbon-coated copper grid. Water was evaporated in air. To increase the contrast, the samples were stained by OsO4 vapor. Synthesis of P(PEGMA-co-MAO) Copolymer by RAFT Polymerization. Typically, PEGMA (0.51 g, 1.08 mmol), MAO (0.15 g, 1.08 mmol), CPADB (12.1 mg, 0.0432 mmol), and ACPA (2 mg, 0.0072 mmol) were dissolved in DMF (1 mL) in a flask. After degassed by three freeze−vacuum−thaw cycles, the polymerization was performed at 70 °C for 24 h. The reaction was stopped by cooling the solution in ice water and exposing to air. P(PEGMA-co-MAO) copolymer was precipitated in excess of cold ethyl ether, centrifuged, and dried overnight under vacuum at room temperature. Synthesis of P(PEGMA-co-methylacryloyl hydrazide) (P(PEGMA-co-MAH)) Copolymer. Briefly, hydrazine hydrate solution (1.1 mL, 17.6 mmol) was added to the solution of P(PEGMA-coMAO) (0.19 g) in DMF (3.7 mL). The reaction was carried out at room temperature for 48 h. The solution was dialyzed extensively against DI water (MWCO 1000) for 2 days. P(PEGMA-co-MAH) copolymer was recovered by lyophilization. The content of hydrazide was determined by a modified TNBS assay.31 Synthesis of Glycoconjugated Copolymer. P(PEGMA-coMAH) copolymer (80.9 mg) dissolved in acetate buffer (0.87 mL, 100 mM, pH = 5.5) containing aniline (1 mM) and glucose (11.8 mg, 0.066 mmol) was added. The solution was stirred at 50 °C for 24 h. After dialysis against DI water (MWCO 1000) for 2 days, the glucoseconjugated copolymer was recovered by lyophilization. The attachment of glucose moiety to copolymer was analyzed by an Alizarin Red S (AR) assay.32 Fluorescence spectra were measured at λex = 460 nm. The excitation and emission band widths were 3 and 1.5 nm, respectively. Synthesis of Biotinylated Copolymer. Aldehyde-functionalized biotin was prepared in situ from the deprotection of acetal-modified biotin. Briefly, acetal-modified biotin (14 mg, 0.050 mmol) was dissolved in DMSO (3 mL). After addition of a small amount of TFA, the solution was stirred at 50 °C for 1 h to get aldehyde-functionalized biotin. After the addition of triethylamine (TEA), the solution of aldehyde-functionalized biotin was added to the solution of P(PEGMA-co-MAH) copolymer (20 mg, CHO/hydrazide = 1:1) in DMSO (15 mL). Aniline (137 μL) as catalyst was added to the solution. The reaction was carried out at room temperature for 24 h and then dialyzed against DMF/H2O (2:3 v/v, MWCO 1000) for 1 day and DI water subsequently for 2 days. The biotinylated copolymer was recovered by lyophilization. The amount of available biotin was evaluated by an HABA/avidin binding assay. Synthesis of Core-Cross-Linked Nanoparticles via Reversible Acylhydrazone Linkages (NP1−NP4). The preparations of corecross-linked nanoparticles were carried out at the P(PEGMA-coMAH) concentration of 1 mg/mL and at −CHO/hydrazide = 1:4 (NP1), 1:2 (NP2), 3:4 (NP3), and 1:1 (NP4) equiv. A typical procedure was as follows. An aliquot solution of terephthaldicarboxaldehyde (TDA) in acetone was added to a flask and heated to evaporate acetone. The aqueous solution of P(PEGAM-co-MAH) copolymer was added to the flask. The cross-linking reaction proceeded at 50 °C for 24 h and stopped by cooling to the room temperature. The solution was dialyzed against DI water for 2 days (MWCO 1000). An aliquot solution was lyophilized to obtain dried nanoparticles for characterization. Preparation of Multifunctional Core-Cross-Linked Nanoparticles. To the aqueous solution of core-cross-linked nanoparticles NP2 (7 mg, 10 mL) was added DMSO (3 mL), aniline (1.3 mmol, 118 μL), and aldehyde-functionalized biotin (1.1 mg, 0.0039 mmol, in 0.2 mL of DMSO). The solution was allowed to stir at room temperature for 24 h and then dialyzed against DMSO/DI water (2:3, v/v, MWCO 1000) for 2 days, and DI water subsequently for 2 days to obtain biotinylated NP5. The amount of biotin on nanoparticles was evaluated by an HABA/Avidin assay.

The biotinylated NP5 were further labeled by FITC. Aliquot (25 μL) of the FITC solution in DMSO (2 mg/mL) was added to the aqueous solution of biotinylated NP5 (0.7 mg/mL, 4 mL). The solution was stirred in dark at room temperature for 24 h and then dialyzed against DI water (MWCO 1000) for 2 days. Fluorescence spectra were measured at λex = 490 nm. The excitation and emission band widths were 3 and 1.5 nm, respectively. Reversible Formation of Core-Cross-Linked Nanoparticles. Briefly, a drop of TFA was added to the aqueous solution of NP2 (1.4 mg, 2 mL), and the solution was stirred at room temperature. After 24 h, triethylamine was added to set pH to about 7, and then the solution was heated at 50 °C for 24 h. The hydrodynamic diameter of nanoparticles at each step was determined by DLS. Disassembly of Core-Cross-Linked Nanoparticles via Chain Exchange with Pyridoxal 5′-Phosphate (PLP). PLP (2.2 mg, 0.008 56 mmol) was added to the aqueous solution of NP2 (1.4 mg, 2 mL), and pH was set to 4.5 by adding TFA. The solution was stirred at room temperature for 24 h. The hydrodynamic diameter of nanoparticles was determined by DLS. Preparation of Nile Red-Loaded Core-Cross-Linked Nanoparticles. P(PEGAM-co-MAH) copolymer (5 mg) and TDA (0.45 mg, [hydrazide]/[−CHO] = 0.75:1 equiv) were dissolved in DI water (5 mL); the solution of Nile red (NR) (0.1 mg) in THF (750 μL) was added dropwise. The reaction was left to stir for 24 h at 50 °C, open to the atmosphere to evaporate THF. The reaction solution was filtered through a syringe filter (0.45 μm) to remove excess insoluble NR. The incorporated efficiency and the loading capability of NR in the crosslinked nanoparticles were about 5 and 0.1 wt % (Supportiong Information). The NR-loaded nanoparticles were characterized by DLS, fluorescence spectroscopy, and TEM. Release of Nile Red from Core-Cross-Linked Nanoparticles. The release of Nile red from core-cross-linked nanoparticles was studied under three different pH values: (a) pH 4.5 acetate buffer solution, (b) pH 5.5 acetate buffer solution, and (c) pH 7.4 phosphate buffer solution. In a typical experiment, the aqueous solution of NRloaded cross-linked nanoparticles was mixed with equal volume of buffer solution with various pH values. The solution was stirred at 37 °C. A fluorescence spectrophotometer was used to monitor the release of Nile red with time. Fluorescence emission spectra were recorded at λex = 550 nm. Lectin Recognition. A stock solution of glycoconjugated copolymer (1 mg/mL) was prepared in Tris-HCl solution (0.1 M, pH 7.4). A stock solution of Con A (1 mg/mL) was prepared in TrisHCl solution (0.1 M, pH 7.4, containing 1 mM Ca2+, Mg2+). After the addition of Con A solution (0.5 mL) to the solution of glycoconjugated copolymer (0.5 mL) in a vial, the thoroughly mixed solution was immediately transferred to a cuvette. The lectin recognition activity of the glycoconjugated copolymer was evaluated by changes in the turbidity of solution with time at 420 nm under room temperature. Avidin−HABA Assay. The amount of available biotin on polymer or nanoparticles was determined by an HABA/avidin binding assay. The HABA/avidin reagent was reconstituted with 10 mL of deionized water. In a 1 mL cuvette, pipet 900 μL of HABA/avidin reagent and ) by a UV−vis measure the absorbance at λ = 500 nm (AHABA/avidin 500 spectrophotometer. To this solution, 100 μL of sample was added, the solution was mixed by inversion and the absorbance at λ = 500 ) was read. The amount of the available biotin was (AHABA/avidin+sample 500 calculated by the following formula:33 μmol biotin/mL = (ΔA500/34) × 10, which corresponds to the μmoles of biotin per milliliter of the blank ) + Asample − sample solution, where ΔA500 = 0.9 × (AHABA/avidin 500 500 HABA/avidin+sample . A500 Cytotoxicity Assay. Cytotoxicity of core-cross-linked nanoparticles and the corresponding copolymer precursors were evaluated using a standard cell-counting kit-8 (CCK-8) assay. The assay was carried out in triplicate in the following manner. The cells were cultured in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL) at standardized conditions (37 °C, 5% CO2) in a 96-well plate with a density of 8000 cells/well. After 24 h incubation, fresh medium 2001

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Scheme 1. Outline for the Synthesis of Hydrazide-Containing Copolymer P(PEGMA-co-MAH) via RAFT Polymerization

containing the cross-linked nanoparticles and corresponding polymer precursors (concentration: 10−300 μg/mL) was added into each well, and the cells were incubated for another 24 h. CCK-8 solutions were added to each tested well and incubated for 2 h; subsequently, the absorbance of each sample was measured at 450 nm using a multifunctional ELISA plate reader (Thermo Varioskan Flash). All experiments were carried out in triplicate. Cell viability was calculated by the equation

cell viability (%) =

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A sample − A blank Acontrol − A blank

× 100%

RESULTS AND DISCUSSION Synthesis of Hydrazide-Containing Copolymer by RAFT Polymerization. The hydrophilic hydrazide-containing copolymer P(PEGMA-co-MAH) was prepared in two steps (Scheme 1). First, copolymer of PEGMA and methacryloylacetone oxime (MAO) was synthesized by CPADB-mediated RAFT polymerization with ACPA as initiator at 70 °C in DMF. P(PEGMA-co-MAO) copolymers with different compositions were prepared by changing the PEGMA/MA ratio (Supporting Information, Figure S1), indicating that the copolymerization proceeded in a controlled fashion. The copolymer compositions were calculated from 1H NMR data by comparing the integration of signals at 4.2 ppm (CH2OC(O) protons of PEGMA) with that at 1.5−2.4 ppm ((CH3)2C protons of MAO and CH protons of backbone) (Figure 1A). The composition and molecular weight data of the copolymers are summarized in Table 1. By treatment with excess hydrazine hydrate, P(PEGMA-co-MAO) copolymer was converted to the corresponding hydrazide-containing copolymer P(PEGMA-coMAH), as exhibited by the dramatically decreasing intensity of signals at δ 2.0 ppm associated with methyl protons of oxime (Figure 1B). The results from GPC analyses demonstrated that there was no cross-linking or coupling reaction occurring during this step. The contents of hydrazide groups in copolymers were also determined by a modified TNBS assay and are listed in Table 1, and the data were in agreement with the contents of MAO in P(PEGMA-co-MAO) copolymers calculated on the basis of 1H NMR analysis. Hydrazide-Containing Copolymer P(PEGMA-co-MAH) as a Versatile Scaffold for Bioconjugation. Glycoconjugate is very important for high-throughput diagnostics or screening of carbohydrate-binding proteins associated with various biological functions or disease states. 34 Weakly basic nucleophiles like aminooxy ethers (H2NO-R, MeHNOR) and hydrazides (H2NNHCO-R) have been shown to condense with hemiacetals in aqueous solutions for efficient glycoconjugation.35 For example, a series of biotinylated glycopolymers were prepared on the basis of the bioconjugation of an acryloyl hydrazide polymer scaffold to free reducing sugars including glucose, galactose, mannose, etc.36 These glycopolymers were microarrayed on the streptavidin-coated glass slides and recognized specifically by lectins according to their pendant

Figure 1. 1H NMR spectra of (A) P(PEGMA-co-MAO) in CDCl3 and (B) P(PEGMA-co-MAH) in D2O.

glycans. Because most of the free reducing glycans are available from natural sources, the ligation of reducing glycans to hydrazide-containing polymers eliminates the complicated synthesis of functionalized glycosides required for glycopolymer synthesis and provides a facile access to various glycopolymers suitable for high-throughput microarray applications. Here, we investigated the conjugation of underivatized glycan to P(PEGMA-co-MAH) copolymer C2, containing 35% of PEGMA and 65% of MAH. The conjugation of glucose proceeded smoothly in acetate buffer (pH 5.5) with aniline as a catalyst to generate glycoconjugated copolymer (Scheme 2). Because of the overlapped signals of PEGMA protons and glucose protons, the attachment of glucose moiety can not be characterized by 1H NMR (Supporting Information, Figure S2). By means of alizarin red S (AR) assay, the incorporation of glucose units into the copolymer was qualitatively analyzed. The reversible covalent binding of phenylboronic acid (PBA) with the catechol diol groups of AR induces fluorescent emission in alkaline conditions.32 After the addition of glycoconjugated copolymer solution to the solution of the AR−PBA complex, the fluorescence intensity was remarkably decreased (Figure 2A), suggesting that glucose moieties on copolymer displaced AR from the AR−PBA complex because of the high affinity of glucose to PBA. This result confirmed the 2002

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Table 1. Characteristics of P(PEGMA-co-MAO) and P(PEGMA-co-MAH) Copolymers A P(PEGMA-co-MAO)

C P(PEGMA-co-MAH)

sample

f MAOa (mol %)

FMAOb (mol %)

Mnc

PDIc

Mnd

PDId

hydrazidee (mol %)

1 2 3

70 50 30

70.5 63.5 35.7

12 700 16 400 16 300

1.13 1.22 1.16

14 800 19 900 24 000

1.16 1.26 1.23

68.8 64.8 42.5

a

Molar fraction of MAO in feed. bMolar fraction of MAO units in P(PEGMA-co-MAO) copolymer calculated by 1H NMR. cDetermined by GPC in THF. dAbsolute molecular weight determined by aqueous GPC-MALLS in PBS (pH 6.8). eDetermined by a modified TNBS assay.

Figure 2. (A) Fluorescence spectra of (a) ARS−PBA complex solution, (b) mixed solution of ARS−PBA and glycoconjugated copolymer, and (c) mixed solution of ARS−PBA and P(PEGMA-co-MAH) copolymer. (B) Interaction of glycoconjugated copolymer with Con A in Tris-HCl solution (pH 7.4, 0.5 mM Ca2+, Mg2+).

successful conjugation of glucose to P(PEGMA-co-MAH) copolymer. After conjugation, the content of hydrazide units in copolymer decreased to 19 mol % on the basis of TNBS assay; therefore, the glycoconjugated copolymer contained 46 mol % saccharide units. The biological recognition of glucose moieties on copolymer was evaluated by the interaction with Con A. After the addition of Con A to the solution of glycoconjugated copolymer, the transmittance of the mixed solution was monitored by UV−vis spectroscopy (Figure 2B). It was observed that the transmittance gradually decreased with time indicating the specific binding of glucose moieties to Con A.

Hydrazides have been widely used for conjugation to aldehyde (or ketone)-containing biomolecules through reversible hydrazone. Biotin is a vitamin that can bind to the proteins avidin and streptavidin, yielding stable complexes with a Kd of 10−15 M, one of the strongest noncovalent interactions known.37 Since avidin has four binding sites for biotin, it can serve as a universal linkage between biotinylated materials and other proteins. Biotin/avidin (or streptavidin) linking system has been widely used in biotechnology for purification, localization, and disgnostics.38 In our previous studies, biotin was attached to the polymer by “click” chemistry to generate bitinylated polymers that can interact with avidin effectively.33b,39 In this study, biotinylated copolymer was prepared by the coupling of P(PEGMA-co-MAH) copolymer C2 with aldehyde-functionalized biotin via reversible hydrazone bond at CHO/hydrazide = 1:1 equal. The aldehyde- functionalized biotin was prepared in situ from the hydrolysis of acetal derivative of biotin by addition of 0.5% TFA. After being neutralized with TEA, the DMSO solution of aldehydefunctionalized biotin was added to the solution of C2. The conjugation of biotin was confirmed by an HABA/avidin competitive binding assay. The amount of available biotin to avidin was calculated to be ca. 1.1 μmol biotin/mg polymer (Figure 3). Synthesis of Core-Cross-Linked Polymeric Nanoparticles via Reversible Acylhydrazone Linkages. Chemically cross-linked, stimuli-responsive polymer nanoparticles constitute a promising scaffold in therapeutic delivery applications, offering potential to circumvent stability issues and improve release behavior.40 Dynamic covalent chemistry offers a promising alternative for the construction of polymeric nanoparticles with the stimuli-responsive and adaptive characters.14b,18b,20−24 The reversible formation and chain exchange

Scheme 2. Outline for the Conjugation of Biomolecules to P(PEGMA-co-MAH) Copolymer Scaffolda

a

Reagents and conditions: (i) glucose, acetate buffer (0.1 M, pH 5.5, 1 mM aniline), 50 °C, 24 h; (ii) (1) biotin, 2-mercaptothiazoline, EDC· HCl, DMAP, DMF, room temperature, 24 h; (2) aminoacetaldehyde dimethyl acetal, TEA, DMF, room temperature, 24 h; (3) 0.5% TFA (v/v), DMSO, 50 °C, 1 h; (4) TEA, 100 mM aniline, DMSO, room temperature, 24 h. 2003

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functionalized further by the reaction of its remaining hydrazide units with molecules containing hydrazide-reactive functionalities, and for this model study the aldehyde-functionalized biotin and FITC were chosen. Cross-linking was investigated with the P(PEGMA-co-MAH) copolymer scaffold C2 at a concentration of 1 mg/mL. The aqueous solutions of C2 were added to the flasks containing various quantities of TDA at −CHO/hydrazide = 0.25:1 (NP1), 0.5:1 (NP2), 0.75:1 (NP3), and 1:1 (NP4) equiv, to generate a range of nanoparticles possessing various densities of cross-linker. The reactions were performed at 50 °C for 24 h. DLS and TEM were utilized to investigate the nanoparticle formation (Figure 4). For example, under the condition of −CHO/hydrazide = 0.5:1 equiv, DLS analysis indicated an increase in Dh from (5 nm) for the unimers to 119 nm after the formation of nanoparticles. Spherical aggregates with a size range from 50 to 100 nm were observed by TEM. At a constant concentration of P(PEGMA-co-MAH) copolymer, an remarkable increase in Dh of nanoparticles was observed by DLS as the ratio of aldehyde to hydrazide changed from 0.25:1 to 0.5:1 equiv. However, increasing the concentration of cross-linker to 0.75:1 and 1:1 equiv did not result in a further increase in the size of nanoparticles. These results suggested that efficient nanoparticle formation under the conditions considered required a minimum −CHO/hydrazide ratio. The obtained nanoparticles kept intact in the dilute solution. After the aqueous solution of nanoparticles (0.7 mg/mL) was diluted to 0.3 and 0.1 mg/mL, the resulting nanoparticles showed a similar size (Supporting Information, Figure S7), indicating that the cross-linked structures are stable and have no concentration dependency. The influence of temperature on the stability of the cross-linked nanoparticles was also investigated. The aqueous solution of nanoparticles (∼pH 6.5) was kept at 60 °C for 24 h. DLS data showed that the size of the nanoparticles at specified time intervals was similar to that before being heated (Supporting Information, Figure S8), indicating that the

Figure 3. UV−vis spectra of the HABA/avidin complex before (a) and after the addition of biotinylated copolymer (b).

of dynamic covalent bond can endow nanoparticles with the ability to reconfigure and optimize their structures. The above studies demonstrate that P(PEGMA-co-MAH) copolymer can be used as a scaffold onto which the aldehydecontaining molecule is efficiently attached through hydrazone bond formation. Hydrazone bond is a kind of well-studied pHsensitive covalent bond in the field of dynamic covalent chemistry, which can be formed under acid catalyst or elevated temperature.2,3,12,41 We envisioned that intermolecular crosslinking of the P(PEGMA-co-MAH) copolymer through hydrazone bond could be accomplished using the bis-aldehyde molecule TDA as a chain cross-linker. In this study, we report a facile approach that allows for the synthesis of polymeric nanoparticles in aqueous media. The water-soluble hydrazidecontaining copolymer P(PEGMA-co-MAH) chains were crosslinked through acylhydrazone bond formation with TDA at 50 °C (Scheme 3). The copolymer nanoparticles could be

Scheme 3. Schematic Representation of Core-Cross-Linked Polymeric Nanoparticles via Reversible Acylhydrazone Linkages and Subsequent Functionalizations with Biotin and FITC

2004

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Figure 4. (A) Size distributions of the cross-linked nanoparticles with different cross-linking densitites. (B) TEM image of NP2.

apparent decrease in the hydrodynamic diameter of the nanoparticles (Figure 5A), which was indicative of dissociation of NP2 resulting from the acylhydrazone exchange reaction. Therefore, the addition of excess monoaldehyde molecule can trigger the dissociation of the present cross-linked polymeric nanoparticles because of the dynamic character of reversible acylhydrazone linkages. Dynamic Covalent Nanoparticle as a Reactive Scaffold for Multifunctionalization. The resulting cross-linked nanoparticles NP2 were further bioconjugated with biotin and subsequently adorned by FITC to generate multifunctional nanoparticles (Scheme 3). The DMSO solution of in situ prepared aldehyde-functionalized biotin was added to the aqueous solution of NP2. The bioconjugation reaction was carried out at room temperature for 24 h to prepare biotinylated nanoparticles NP5. After the solution was purified by dialysis, the bioavailability of biotin present on NP5 to avidin was evaluated by an HABA/avidin assay. The available amount of biotin was estimated about 0.022 μmol biotin/mg polymer (Figure 6A). The interaction of biotin and free avidin was further investigated by mixing NP5 with free avidin. After avidin solution was added to the aqueous solution of biotinylated nanoparticles, the size of nanoparticles became much larger as shown by DLS data (Figure 6B). The formation of large aggregates was also clearly observed on the TEM image (Figure 6C,D). Because there are four biotin binding sites on avidin, it acts as a cross-linking agent and induces the formation of internanoparticle aggregates. These results indicate that biotin moieties on the nanoparticles can effectively recognize avidin. Owing to the diverse reactivity of hydrazide group, the biotinylated nanoparticles were subsequently labeled by fluorescein isothiocyanate (FITC). The conjugation of biotinylated nanoparticles NP5 and FITC performed in the aqueous media. After purified by dialysis against H2O, the biotinylated nanoparticles emitted strong fluorescence at λ 520 nm (Figure 7), suggesting the successful attachment of FITC to nanoparticles. In Vitro Cytotoxicity. In order to apply the cross-linked nanoparticles in biologically relevant fields, their toxicity was evaluated. The relative cytoxicity of P(PEGMA-co-MAH) copolymers (C1 and C2) and the corresponding cross-linked nanoparticles (C1-NP and C2-NP) was investigated in HeLa cells by CCK-8 assays. The cells were incubated with nanoparticles or copolymers for 24 h. The results revealed that copolymer C2 with 64 mol % content of hydrazide groups

cross-linked nanoparticles are stable at high temperature in the aqueous solution (∼pH 6.5). pH-Reversible and Dynamic Characters of CrossLinked Nanoparticles via Hydrazone Linkage. It was well-known that the acylhydrazone bond was an acid-sensitive linkage with reversible and dynamic characters. DLS was employed to investigate the pH-dependent cleavage and reformation of hydrazone linkage. A drop of TFA was added to NP2 aqueous solution at room temperature (pH ∼3). It was observed that the size of nanoparticles became much smaller after 24 h, indicating the dissociation of nanoparticles because of the cleavage of acylhydrazone bonds (Figure 5A). After being neutralized with TEA (pH ∼7), the solution was heated at 50 °C for 24 h. It was found that the size shifted back to the original diameter, implying the reformation of cross-linked nanoparticles. To understand pH-triggered cleavage of hydrazone bond more clearly, the structures of nanoparticles under various pH conditions were studied by 1H NMR (Figure 5B). The NP2 solid nanoparticles were recovered by lyophilization after the aqueous solution of NP2 was purified by dialysis against H2O and characterized by 1H NMR. The proton signals at 8.83 ppm (peak (v) and 10.10 ppm (peak w) corresponded to acylhydrazone bonds, indicating the successful formation of polymeric nanoparticles. After the addition of DCl, the solution was incubated at room temperature for 24 h. The signal at 10.15 ppm (peak p) corresponding to aldehyde protons of free TDA and the signal at 10.04 (peak q) corresponding to aldehyde proton of TDA attached to nanoparticles were present in the 1H NMR spectrum, along with the decreased intensity of peak w and peak v attributed to acylhydrazone bonds. This result demonstrated that the acylhydrazone linkages were cleaved under the acidic condition. The cleavage of acylhydrazone bonds caused the dissociation of cross-linked nanoparticles. Therefore, NP2 became much smaller after the addition of TFA. As the pH changed to neutrality, which was favored for the formation of acylhydrazone bonds at high temperature, the nanoparticles were reconstructed and the size shifted back. The results from DLS and 1H NMR confirmed the pH-reversible behavior of the cross-linked nanoparticles via acylhydrazone linkages. Demonstration of the dynamic character of the cross-linked nanoparticles was achieved by addition of an excess of monoaldehyde-containing molecule pyridoxal 5′-phosphate (PLP) to the NP2 aqueous solution. After NP2 was incubated with PLP at pH 4.5 for 24 h, DLS measurement showed an 2005

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Figure 6. (A) UV−vis spectra of the HABA/avidin complex before (a) and after addition of NP5 (b). (B) Hydrodynamic size distributions of biotinylated nanoparticles NP5 before (a) and after addition of avidin (b). TEM images of (C) biotinylated nanoparticles NP5 and (D) aggregates formed by NP5 and avidin.

Figure 7. Fluorescence emission spectra of biotinylated nanoparticles NP5 (A) and FITC-labeled nanoparticles (B). Figure 5. (A) DLS curves of copolymer NP2 (a), after addition of TFA (pH ∼3) (b), re-cross-linked nanoparticle (pH ∼7) (c), and chain exchange with PLP (d). (B) 1H NMR spectra of TDA (a), crosslinked nanoparticles NP2 before (b) and after addition of DCl (c).

hydrazone-based cross-linking reaction in the presence of Nile red (NR), a hydrophobic dye. “pH-Triggered” Release of NR. As described above, the cross-linked nanoparticles disintegrated under acidic condition because of the hydrolysis of acid-labile hydrazone linkages, and therefore, it is expected that pH can trigger the release of a model hydrophobic dye, Nile red entrapped in the nanoparticles. The dye-loaded nanoparticles were prepared with copolymers C1−C3 at CHO/hydrazide = 0.75:1 (for C1N) and CHO/hydrazide = 1:1 (for C2N and C3N). DLS studies revealed that the dye-loaded nanoparticles C1N obtained are ∼120 nm in size (Figure 9). The TEM image revealed welldefined spherical structures with slightly smaller diameter than those observed by DLS, which was attributed to the possible shrinkage of nanoparticles in dried state. The dye-loaded nanoparticles were dispersed in three buffer solutions at pH 7.4 (0.1 M PBS), pH 5.5 (0.1 M sodium acetate buffer solution), and pH 4.5 (0.1 M sodium acetate buffer solution) to evaluate the pH-triggered release profiles of NR at

and nanoparticles C2-NP were nontoxic to HeLa cells at the tested concentrations up to 300 μg/mL (Figure 8), but the copolymer C1 with higher hydrazide contents showed toxic to HeLa cells. After being cross-linked by TDA, it is anticipated that the nanoparticles would be relatively nontoxic because biocompatible poly(ethylene glycol) components are present on the surface of nanoparticles. Actually, the cross-linked nanoparticles C1-NP exhibited high cell viability and no concentration-dependent toxicity up to the nanoparticle concentration of 300 μg/mL. This result indicates that the nanoparticle material shows low toxicity and thus a potential candidate for biological applications. Next, to investigate the possibility of encapsulating a hydrophobic guest molecule within the interiors of these nanoparticles, we carried out the 2006

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Figure 8. Cell viability data of cross-linked nanoparticles and the corresponding copolymer precursors. The cell viability was determined by CKK-8 assay (n = 3).

37 °C. Fluorescence spectroscopy was employed to monitor the release of NR by tracing the decrease of the dye’s spectral emission intensity at λ = 615 nm (Figure 10A). The fluorescence experiments also gave an indication of the kinetics of the release (Figure 10B−D). The rate of dye release was slower at pH 7.4 and became faster with the decreasing of pH. This is attributed to the hydrolysis of hydrazone linkages under acidic media and the resulting disintegration of nanoparticles. For example, approximately 54% of dye was released from C3N nanoparticles in 24 h at pH 7.4. At pH 5.5 and 4.5, 60% and 63% of dye were released after 24 h, respectively. The variation of medium pH value had little effect on the release of dye from C3N. Release from C2N nanoparticles showed slightly difference between pH 7.4 (41%) and 4.5 (52%). The desired difference in the release of dye at various pH values was most pronounced for C1N nanoparticles. C1N released about 46% of dye at pH 7.4 after 24 h, while the released dye increased to 54% at pH 5.5 and further increased to 63% at pH 4.5. C1N nanoparticles, prepared from copolymer C1 containing higher contents of hydrazide functionalities, showed better pH-

Figure 9. (A) Hydrodynamic size distribution of cross-linked nanoparticles loaded with Nile red (C1N nanoparticles) and (B) TEM image of C1N loaded with Nile red.

Figure 10. (A) Fluorescence emission spectra at λex = 550 nm displaying NR release behavior of C1N in acetate buffer (pH 4.5). (B−D) Time dependence of the relative emission intensity of C1N (B), C2N (C), and C3N (D). 2007

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of dye-loaded nanoparticles C2N and C3N. This material is available free of charge via the Internet at http://pubs.acs.org.

responsive dye release behavior. The nanoparticles constructed by copolymer with higher hydrazide contents have a denser cross-linked core, so the release of encapsulated dye is slower at pH 7.4 because of the diffusion-controlled release. Under acidic medium, the hydrolysis of hydrazone linkages results in the disintegration of nanoparticles and facilitates the release of dye. The change of pH values has a remarkable effect on the nanoparticles with dense cross-linked cores via hydrazone linkages. By designing the content of hydrazide units in the copolymers, it is possible to construct cross-linked nanoparticles with favorable pH-triggered release behavior around physicological conditions for cell interactive applications.

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Corresponding Author

*E-mail: [email protected] (L.L.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was financially supported by the National Natural Science Foundation of China under Contract 21174066 and PCSIRT (IRT1257).

CONCLUSIONS

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In this study, the well-defined hydrazide-containing copolymer scaffolds P(PEGMA-co-MAH) for bioconjuagtion were synthesized via RAFT polymerization. The pendant hydrazide groups were reactive to saccharide and the aldehyde-functionalized biotin derivative to generate glycoconjugated and biotinylated copolymers for protein recognition. The attachment of biologically active substances to the P(PEGMA-co-MAH) copolymer via reversible hydrazone bond offers a facile approach for the preparation of biodynamers with structural and functional diversity, which may be considered as biohybrid dynamic materials. The cross-linking of copolymer P(PEGMAco-MAH) through reversible acylhydrazone bonds generated dynamic nanoparticles possessing environmentally responsive and adaptive features. The cross-linked nanoparticles demonstrated pH-reversible formation and disintegration. The dynamic character of nanoparticles was exhibited by a simple chain exchange reaction with PLP molecule. Fluorescencelabeled biotinylated nanoparticles were obtained by further functionalization via the reaction of the residual hydrazide groups with an aldehyde-modified biotin derivative and subsequently with FITC. Because of the versatile reactions of hydrazide group, the nanoparticles can be utilized as a platform for the attachment of hydrazide-reactive biomolecules such as targeting molecules, peptides, and proteins. The cross-linked nanoparticles are nontoxic and thus a potential candidate for biological applications. With Nile red as a model hydrophobic drug, these nanoparticles can encapsulate a cargo of dye molecules, which demonstrate pH-triggered release behavior. The dye-loaded nanoparticles prepared by the copolymer with higher hydrazide contents exhibited better pH-responsive release behavior. Therefore, on the basis of hydrazidecontaining copolymer, it is attractive to construct dynamic covalent nanoparticles with functional properties of biomolecules. These “smart” nanoparticles can be used as promising building blocks for advanced materials. The synergetic combination of the adaptive behavior of constitutional dynamic systems and the biological functionalities of biomolecules allows the nanoparticles numerous applications in the fields of nanotechnology, biotechnology, and therapeutics.

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AUTHOR INFORMATION

REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Syntheses of methacryloylacetone oxime (MAO) and acetalmodified biotin; preparations of poly(methacrylyol hydrazide) (PMAH) homopolymer and glucose-conjugated PMAH polymer; modified TNBS assay and Alizarin Red S (AR) assay; GPC curves of copolymers in Table 1; determination of the amount of Nile red incorporated in the nanoparticles; DLS 2008

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