Polylactide-graft-doxorubicin Nanoparticles with Precisely Controlled

Jan 21, 2014 - (2) Incorporation of tumor-targeting ligands and imaging agents into DDSs can further induce active tumor targeting and allow visualiza...
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Polylactide-graf t-doxorubicin Nanoparticles with Precisely Controlled Drug Loading for pH-Triggered Drug Delivery Yun Yu,† Chih-Kuang Chen,†,‡ Wing-Cheung Law,§,∥,⊥ Emily Weinheimer,† Sanghamitra Sengupta,∥ Paras N. Prasad,§,∥ and Chong Cheng*,† †

Department of Chemical and Biological Engineering, §Institute for Lasers, Photonics and Biophotonics, and ∥Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260, United States ‡ Department of Fiber and Composite Materials, Feng Chia University, Taichung, Taiwan 40724, Republic of China S Supporting Information *

ABSTRACT: Nanoparticles (NPs) with high drug loading and pH-responsivity were prepared by nanoprecipitation of a hydrophobic polymer-drug conjugate (PDC). The PDC, polylactide-graf t-doxorubicin (PLA-g-DOX), was synthesized by azide−alkyne click reaction to transform acetylenefunctionalized PLA into PLA-graf t-aldehyde (PLA-g-ALD), followed by DOX conjugation to form acid-sensitive Schiff base linkage between drug moieties and polymer scaffold. The DOX loading amount in PLA-g-DOX PDC was determined to be 32 wt % by 1H NMR and UV−vis spectroscopies. PLA-g-DOX PDC was further used to prepare NPs with precisely controlled drug loading by nanoprecipitation in the presence of a PEGylated surfactant. The effects of organic solvent, PLA-gDOX PDC concentration and PLA-g-DOX/surfactant mass ratio on size and size distribution of NPs were systematically examined based on analysis by dynamic light scattering (DLS) and transmission electron microscopy (TEM). NPs prepared under the optimal conditions exhibited well-defined spherical morphology with volume-average hydrodynamic diameter (Dh) around 100 nm. Due to the Schiff base conjugation linkage in PLA-g-DOX PDC, acid-sensitive drug release behavior of the NPs was observed. In vitro studies against MCF-7 breast cancer cells showed that the NPs can be readily taken up and result in enhanced therapeutic efficiency as compared to DOX·HCl, indicating their promising potential applications as anticancer nanomedicines.

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INTRODUCTION With the development of understanding in drug delivery mechanism and cancer pathology, a set of criteria have gradually been established for guiding the preparation of drug delivery system (DDS) for cancer treatment.1,2 DDSs should not cause severe long-term side effects, and therefore, biocompatible and biodegradable materials have been predominantly used for the preparation of DDSs.3−5 In order to effectively kill cancer cells and suppress tumor proliferation, significant drug loadings are required for achieving remarkable therapeutic effects.6,7 Stimuli-responsive drug release from DDSs in tumor tissue is highly preferred to minimize their systemic toxicity and to improve drug delivery efficiency.8−13 Specifically, because tumor tissue is slightly more acidic than normal tissue, recently DDSs with acid-triggered drug release behavior has attracted broad interest.14 Moreover, the nanoscopic sizes of 10−100 nm can be optimal for DDSs to avoid fast systemic clearance and to enable passive tumor targeting through enhanced permeability and retention (EPR) effect.2 Incorporation of tumor-targeting ligands and imaging agents into DDSs can further induce active tumor targeting and allow visualization of biodistribution of DDSs, respectively.15−17 In addition, other factors, such as stability and size uniformity of scaffolds, should also be considered and optimized in the © XXXX American Chemical Society

structural design of DDSs. Driven by the desire to attain higher therapeutic outcome of cancer therapy, DDSs which can meet as many as these criteria have been actively pursued. As a major class of biodegradable and biocompatible polymer, aliphatic polyesters, such as polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and poly(ε-caprolactone) (PCL), have been widely used to prepare nanoparticulate DDSs.18−20 In most cases, aliphatic polyesters-based DDSs are prepared by assembly approaches and drugs are encapsulated in hydrophobic cores of nanoparticles.15,21,22 For instance, nanoprecipitation can readily allow the formation of drugencapsulated polymer-based NPs via the assembly process of water-insoluble polymers in drug-containing solutions.23 Although the encapsulation process via assembly approaches typically is facile, it may also suffer from significant limitations. Generally only several wt% of drug relative to polymer scaffold can be encapsulated, and accordingly high and unfavorable polymer concentrations are required in the administration of DDSs.21,22 Moreover, drug loading amount cannot be predetermined and may show batch-to-batch variations. Received: October 1, 2013 Revised: January 15, 2014

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amine (TEA; >99%), copper sulfate pentahydrate (CuSO4·5H2O; >98%), sodium ascorbate (NaAsc; 99%), dichloromethane (DCM; HPLC), tetrahydrofuran (THF; HPLC), ethyl acetate (HPLC), hexane (HPLC), N,N′-dimethylformamide (DMF; HPLC), and DMSO (HPLC) were purchased from Fisher Scientific. Doxorubicin hydrochloride salt (DOX·HCl; >99%) was purchased from LC laboratories (Woburn, MA). DSPE-PEG2k-MAL was purchased from Creative PEGWorks (Winston-Salem, NC). Instruments. 1H NMR spectra were obtained on a Varian INOVA NMR spectrometer operating at 500 MHz at r.t., and the samples were dissolved in CDCl3 (99.8 atom % D) containing 1.0 vol% tetramethylsilane (TMS) or methyl sulfoxide-d6 (DMSO-d6, >99.5 atom % D). Proton 2D diffusion-ordered spectroscopy (DOSY) data on the PLA-g-DOX and free DOX in DMSO-d6 were acquired on a Varian INOVA NMR spectrometer operating at 500 MHz at r.t., using the vendor supplied bipolar pulse pair stimulated echo (Dbppste) pulse sequence. The gradient was varied nonlinearly from 2.843 to 70.85 gauss/cm (15 steps), using a 120 ms diffusion delay and a diffusion gradient length of 2.0 ms; 64 transients were collected for each of the 15 incremented gradient values, with a one-second relaxation delay. The data were analyzed using the vendor provided software program VNMRj (version 3.2). FT-IR spectra were obtained on a Bruker Tensor 27 system using attenuated total reflectance (ATR) sampling accessories. UV−vis absorbance spectra were obtained on a Shimadzu UV-3600 spectrometer over a wavelength range from 300 to 800 nm. Highresolution mass spectrometry (HRMS) data were recorded on a VG 70-SE mass spectrometer with electron ionization mode. GPC data were obtained from a Viscotek GPC system equipped with a VE-3580 refractive index (RI) detector, a VE-3210 UV−vis detector at wavelength of 490 nm, and a VE 1122 pump and two mixed-bed organic columns (PAS-103M and PAS-105M with polystyrene MW exclusion limit up to 70 × 103 and 4 × 106 Da, respectively). DMF (HPLC) with 0.01 M LiBr was used as the solvent for polymers and the eluent for GPC with a flow rate of 0.5 mL/min at 55 or 65 °C. The GPC instrument was calibrated with ten narrowly dispersed linear polystyrene standards (peak maximum MW (Mp) = 0.58, 1.53, 3.95, 10.21, 29.51, 72.45, 205, 467, 1319, and 2851 kDa) purchased from Varian. The Dh and zeta potential values of NPs were measured by DLS on a nano-ZS90 instrument (Malvern, Inc.) in water at 25 °C. All experiments were conducted using a 4 mW 633 nm HeNe laser as the light source at a fixed measuring angle of 90° to the incident laser beam. The correlation decay functions were analyzed by cumulants method coupled with Mie theory to obtain volume and number distribution. Transmission electron microscopy (TEM) images were obtained using a JEOL 2010 microscope. The TEM sample was prepared by dropwise addition of 10 μL of aqueous solution of NPs (0.05 mg/mL) on a carbon-coated copper grid, followed by negative staining using 10 μL of 0.5% uranyl acetate solution. Cell imaging was obtained using a Leica TCS-SP2/AOBS confocal microscope, equipped with excitation laser lines of 405, 442, 458, 476, 488, 496, 543, and 633 nm and capable of spectral detection in the range of 400−720 nm. All of the confocal images were taken under the same conditions (the parameters of photodetector gain, pinhole size, and exposure time were kept constant). Synthesis of 6-Azido-1-hexanol (1). In a 100 mL flask, 6-bromo-1hexanol (1.39 g, 7.69 mmol) was dissolved in 30 mL of DMF at r.t. and then NaN3 (1.00 g, 15.4 mmol) was added slowly. After stirring for 3 days, DCM (50 mL) was added into the reaction mixture and followed by extraction using water (3 × 30 mL) and then brine (30 mL). The organic layer was collected and dried over Na2SO4 to give 1.0 g of 1 as a slightly yellow oil (yield: 91%). 1H NMR (500 MHz, CDCl3, ppm): δ 3.64 (t, 2H, J = 6.0 Hz, CH2OH), 3.27 (t, 2H, J = 7.0 Hz, N3CH2), 1.67−1.38 (m, 8H, N3CH2(CH2)4CH2OH). FT-IR (cm−1): 3345, 2935, 2861, 2092, 1668, 1456, 1412, 1387, 1350, 1258, 1158, 1056, 894, 805, 729, 663. Synthesis of 6-Azidohexyl 4-formylbenzoate (2). In a 250 mL flask, 1 (0.90 g, 6.3 mmol), 4-carboxybenzaldehyde (1.00 g, 6.61

Because drug is released from encapsulated DDSs through diffusion, burst effect with rapid drug release within the first few hours is usually observed and can cause severe systemic toxicity. To increase drug loading and suppress burst effect of DDSs, the preparation of polymer−drug conjugates (PDCs),1,24−30 including these using aliphatic polyesters-based scaffolds,31−38 has attracted increasing interest. Typically post-polymerization functionalization strategy was utilized to prepare PDCs with terminal drug moieties. As demonstrated by Cheng and coworkers, drug-initiated polymerization of lactide was also developed to conjugate drug at α-terminals of PLAs, and nanoprecipitation approach was subsequently used to formulate the resulting PDCs into NP delivery vehicles.31,32 To further promote drug loadings, multivalent aliphatic polyesters should be employed as the polymeric scaffolds.33−48 Functional PLAs with multiple hydroxyl groups have been prepared by several groups via copolymerization strategy. Kolishetti et al. further utilized hydroxyl-functionalized PLA to conjugate a cisplatinbased prodrug.37 Jing and co-workers also synthesized a biodegradable amphiphilic diblock copolymer which possesses a hydroxyl-functionalized aliphatic polyester block for further conjugation with doxorubicin.38 However, the preparation of these hydroxyl-functionalized multivalent biodegradable scaffolds generally requires relatively tedious process, because protected monomers are often used for the preparation of these scaffolds and deprotection is needed after polymerization. Our group has prepared alkyne and allyl functionalized PLAs via functional lactide monomers without deprotection step, and further converted them through highly efficient azide−alkyne or thiol−ene click reactions into novel PLA-based conjugates and nanostructures for applications in drug or gene delivery.34−36,49,50 Specifically, as we reported previously, brush polymer-drug conjugates (BPDCs) with PLA-based backbones carrying poly(ethylene glycol) (PEG)-based grafts and paclitaxel (PTXL) drug moieties were synthesized and their properties, especially drug release behavior, were investigated.34,35 Herein, we report our recent study on PLAg-DOX-based NPs with precisely controlled high drug loading for pH-triggered release. This work was incorporated with several important design considerations. First, DOX, one of the most commonly used chemotherapeutic agents, was chosen as the model drug to conjugate with biodegradable PLA-based backbone. Second, because of the reactive amine functionality of DOX, acid-sensitive Schiff base conjugation linkage was designed to link DOX with aldehyde functionalities of PLA-gALD and to enable pH-triggered DOX release.51 Accordingly, PLA-g-ALD needs to be synthesized from alkyne-functionalized PLA through functional group transformation. Third, to achieve NPs with high wt% of conjugated DOX, hydrophobic PLA-gDOX PDC was prepared and then converted into waterdispersible NPs through nanoprecipitation process. Fourth, 1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimidePEG-2000] (DSPE-PEG2k-MAL), a biocompatible surfactant, was selected as the surfactant in nanoprecipitation. As a result, the PEG-based corona of the NPs may remarkably enhance the stability and circulation time of NPs, and the maleimide endgroup of PEG chains can be further utilized to introduce targeting peptides through thiol-maleimide reaction.52

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EXPERIMENTAL SECTION

Materials. 6-Bromo-1-hexanol (97%), 4-dimethylaminopyridine (DMAP; > 99%), and N,N′-dicyclohexylcarbodiimide (DCC; 99%) were purchased from Sigma-Aldrich. Sodium azide (99%), triethylB

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Scheme 1. Synthesis of PLA-g-DOX PDC

mmol), and DMAP (0.154 g, 1.26 mmol) were dissolved in 70 mL of dry THF under N2. Then DCC (1.95 g, 9.45 mmol) was added. After stirring at r.t. for 24 h, the reaction mixture was filtrated to remove the precipitates and then separated by flash chromatography using gradient solvent (hexane/ethyl acetate (v/v) = 9:1 to 7:3) to yield 1.33 g of 2 as a colorless oil (yield: 70%). 1H NMR (500 MHz, CDCl3, ppm): δ 10.11 (s, 1H, C6H4CHO), 8.20 and 7.96 (m, 4H, C6H4CHO), 4.37 (t, 2H, J = 7.0 Hz, CH2CH2OCO), 3.29 (t, 2H, J = 7.0 Hz, N3CH2), 1.84−1.46 (m, 8H, N3CH2(CH2)4). FT-IR (cm−1): 2938, 2861, 2734, 2094, 1705, 1610, 1578, 1504, 1464, 1419, 1385, 1350, 1272, 1201, 1168, 1104, 1016, 963, 855, 816, 759, 732, 688, 631. HRMS: [M + Na]+ calcd for C14H17O3N3, 298.1162; found, 298.1166. Synthesis of PLA-g-ALD (4). As described in our previous publication, alkyne-functionalized PLA (3; MnNMR = 9.3 kDa, Mw/ MnGPC = 1.23; with 54 mol % of alkyne groups relative to backbone repeat units) was obtained by ring-opening copolymerization of alkyne-functionalized lactide monomer with L-lactide.34 To a 10 mL ampule, 3 (100 mg, 0.34 mmol of alkyne groups), 2 (94 mg, 0.34 mmol), and CuSO4·5H2O (4.3 mg, 0.017 mmol) and NaAsc (8.1 mg, 0.041 mmol) were added under N2 followed by addition of N2 bubbled DMF (4 mL). After stirring at r. t. for 24 h, the reaction mixture was diluted by 50 mL of DCM then extracted by 50 mL of deionized water three times and brine twice followed by dialysis (MWCO: 3.5−5.0 kDa) against acetone for 3 days. Finally, 157 mg of the colorless polymer 4 was obtained (yield: 81%). 1H NMR (500 MHz, CDCl3, ppm): δ 10.10 (s, 1H from 2, C6H4CHO), 8.17 and 7.95 (m, 4H from 2, C6H4CHO), 7.50 (s, 1H from triazole, C CHN3), 5.40 and 5.16 (br m, 2H from monomer unit of 3, OCHCOO), 4.33 (s, 4H from 2, NCH2(CH2)4CH2OCO), 3.20−3.40 (br m, 2H from alkyne-functionalized monomer unit of 3, CHCH2), 1.92−1.38 (br m, 8H from 2, NCH2(CH2)4CH2; all CH3 from 3). MnNMR = 18 kDa, Mw/MnGPC = 1.35. Synthesis of PLA-g-DOX PDC (5). In a 10 mL flask, DOX·HCl (12 mg, 0.021 mmol) was dissolved in DMSO (2 mL) and then TEA (6 μL, 0.041 mmol) was added to neutralize HCl. The mixture was stirred in the dark for 2 h and then was dropwise added to a solution of

4 (24 mg, 0.042 mmol of aldehyde groups) in 2 mL of DMSO. After stirring for 24 h, the reaction mixture was diluted by 20 mL of DCM and then extracted by deionized water three times and brine twice to remove DMSO, TEA, and TEA/HCl salt. The resulting organic solution was dried under Na2SO4 overnight and then concentrated to give 30 mg of red polymer (yield: 86%). 1H NMR (500 MHz, DMSOd6, ppm): δ 10.10 (s, 1H from benzaldehyde of 3, C6H4CHO), 8.40 (s, 1H from benzyl imine, NCH), 7.30−8.30 (br m, all Ar−H from 3 and DOX, and 1H from triazole of 3, CCHN3), 5.00−5.60 (br m, 2H from monomer unit of 3, OCHCOO; 2H from DOX, CH and OH), 4.80−5.00 (br, 2H from DOX, 2 × OH), 4.60 (s, 2H from DOX, C O C H 2 OH ), 4.0 5 −4 .3 5 ( b r , 4 H f r o m 2 u n i t o f 3, NCH2(CH2)4CH2OCO), 3.75−4.00 (br m, 4H from DOX, NCH and OCH3), 3.53 (s, 1H from DOX, CHOH), 2.70−3.40 (br m, 2H from alkyne-functionalized monomer unit of 3, CHCH2; 2H from DOX, ArCH2C), 2.00−2.20 (br, 2H from DOX, CHCH2), 0.95−1.95 (br m, all CH3 from 3 and 8H from 2 unit of 3, NCH2(CH2)4CH2; 5H from DOX, CHCH2CH and CH3). MnNMR = 26 kDa. Preparation of PLA-g-DOX-Based NPs. PLA-g-DOX-based NPs were prepared using a modified nanoprecipitation technique with the presence of DSPE-PEG2k-MAL. The typical procedure is as follows: PLA-g-DOX PDC 5 was first dissolved in THF with concentration of 3 mg/mL, and DSPE-PEG2k-MAL was dissolved in 4% ethanol/water as the half mass concentration of 5. Then 0.4 mL of organic solution of 5 was dropwise added into 0.4 mL of 4% ethanol/water solution of DSPE-PEG-MAL under stirring. The solution was vortexing for 3 min followed by addition of 2.0 mL of deionized water dropwise. The dilute solution was stirred for 1 day to allow the evaporation of organ solvents. Then the solution was dialyzed against water using molecularporous dialysis tube with MW cutoff (MWCO) of 12−14 kDa for 1 day to remove trace amounts of THF and ethanol, as well as excess DSPE-PEG-MAL. DOX Release Study of PLA-g-DOX-Based NPs. The NPs were dispersed in 2 mL of in phosphate buffer pH 7.4 and pH 5.5 (10 mM), respectively, with a concentration of 0.5 mg/mL and was put into a dialysis bag (MWCO: 3.5−5 kDa). The solutions of NPs in the C

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Figure 1. 1H NMR spectra of (a) alkyne-functionalized PLA 3, (b) PLA-g-ALD 4, (c) DOX·HCl, (d) mixture of PLA-g-ALD 4 and DOX·HCl, and (e) PLA-g-DOX PDC 5 in DMSO-d6. dialysis bag were incubated in 40 mL of the same buffer medium at 37 °C. At each time interval, 5 mL of medium was withdrawn for UV−vis measurement. The concentration of DOX was determined based on a calibration curve which was acquired from a series of DOX solutions with predetermined concentrations. To study drug release rate versus degradation rate, the NPs were incubated in 4 mL of in phosphate buffer pH 7.4 and pH 5.5 (10 mM) with a concentration of 0.5 mg/ mL at 37 °C. Aliquots were withdrawn and concentrated after 24 and 48 h of incubation and then analyzed by GPC. Cytotoxicity Assay. The cytotoxicity of PLA-g-DOX-based NPs against MCF-7 human breast cancer cells was determined by 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium (MTS) assay (Promega, Madison, WI). The cells were seeded onto a 96-well plate at a density of 5−10 × 103 cells per well in 180 μL of medium, and then 20 μL of samples with different concentration of NPs in water were added. The cells were incubated for 48 h at 37 °C in 5% CO2 atmosphere followed by adding 100 μL of MTS solution. After incubating the cells for another 2 h, the resulting solution was measured for absorbance at 490 nm by a multiwell plate reader (Opsys MR, Dynex). The cell viability was calculated as a percentage of absorbance of sample well compared to that of the control well with untreated cells. The cytotoxicity of DOX·HCl and DOX-free NPs were evaluated with a similar procedure.

Subsequently, 4 was prepared by the copper-catalyzed azide− alkyne click reaction of alkyne-functionalized PLA 3 with 2. The precursor polymer 3 (Mn = 9.3 kDa, Mw/MnGPC = 1.23; with 54 mol % of alkyne groups relative to backbone repeat units) was synthesized by ring-opening copolymerization of alkyne-functionalized lactide monomer with L-lactide.34 Number-average degree of polymerization (DPn) of 59 for 3 was estimated by 1H NMR analysis in CDCl3 based on comparing the resonance intensities of the CH protons from comonomer units at 5.1−5.4 ppm with these of the terminal CH2 protons from ethanol (initiator) at 4.2 ppm.34 With the feed ratio of [alkyne of 3] 0 /[2] 0 /[CuSO 4 ·5H 2 O] 0 /[NaAsc] 0 = 1:1:0.05:0.12, the reaction mixture was stirred in DMF at r.t. for 24 h. The mixture was then extracted, followed by dialysis against acetone for 3 days to remove catalysts and other impurities. Although 81% isolated yield of 4 indicated a minor product loss during workup procedure, high reaction conversion was verified by 1H NMR analysis of 4 (Figure 1b). 1H NMR spectrum of 4 exhibits all of the characteristic proton resonances corresponding to its molecular structure. Along with the essential disappearance of the resonances of alkyne protons at 2.7−3.0 ppm, the presence of resonances of the triazole proton at 7.8 ppm and the aldehyde proton at 10.1 ppm confirmed the successful click functionalization. Moreover, by comparing the integrals of resonance intensities of protons from side chain (such as protons d, e, f, g, h, or i) to these of protons from backbone (such as protons a or b), nearly quantitative click efficiency (>96%) and aldehyde grafting density of ∼54% relative to the backbone repeating unit can be determined. Based on DPn of precursor polymer 3 and the aldehyde grafting density in 4 measured by 1H NMR analysis, Mn of 4 of 18 kDa was further obtained. According to GPC analysis using DMF (with 0.01 M LiBr) as eluent at 55 °C, 4 had a Mw/Mn of 1.35 relative to linear polystyrenes (Figure 2). PLA-g-DOX PDC, 5, was prepared by the reaction of aldehyde group of PLA-g-ALD, 4, with the primary amine group of DOX to form acid-sensitive Schiff base linkage

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RESULTS AND DISCUSSION Synthesis of PLA-g-DOX PDC (5). PLA-g-DOX 5 was synthesized through multistep reaction using PLA-g-ALD 4 as precursor (Scheme 1). To obtain 4 by transformation of the pendant alkyne groups of 3 into aldehyde functionlities via azide−alkyne click reaction, a small molecule functionalization agent 2 as the unimolecular combination of azide and benzaldehyde was prepared at first via two steps of reactions. 6-Azido-1-hexanol (1) was synthesized in 91% yield by the nucleophilic substitution reaction of 6-bromo-1-hexanol with sodium azide (2.0 equiv) at r.t. for 3 days. Then 2 was obtained in 70% yield by the esterification reaction of 1 with a slight excess of 4-carboxybenzaldehyde (1.05 equiv) in THF at r.t. for 24 h, using DCC (1.5 equiv) and DMAP (0.2 equiv) as catalysts. D

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NMR analysis, PLA-g-DOX PDC 5 has a Mn of 26 kDa and a DOX loading of 32 wt %. The conjugation of DOX onto PLA-g-ALD can also be confirmed by GPC measurement using both RI detector and UV−vis detector, with DMF (with 0.01 M LiBr) as eluent (Figure 2). GPC analysis was conducted initially at 55 °C. PLAg-ALD exhibited a monomodal GPC peak by RI detector and no signal by UV−vis detector at 490 nm (the GPC peaks in RI channel at 22−27 mL were system peaks not belonging to the polymer sample). Surprisingly, DOX·HCl did not show distinct signal by using RI detector, presumably because its elution peak overlapped with system peaks. On the other hand, a broad and retarded GPC curve with peak at 25.9 mL attributed to DOX was observed using UV−vis detector at 490 nm, indicating DOX·HCl was somewhat absorbed by GPC columns under the elution conditions. Using RI detector, PLA-g-DOX PDC showed a broad GPC peak at ∼18 mL which apparently overlapped with system peaks; using UV−vis detector, PLA-gDOX also showed a broad monomodal GPC peak at 18.0 mL. Comparison of GPC curves of PLA-g-ALD and PLA-g-DOX indicated that the conjugation of DOX onto PLA-based scaffolds greatly changed the GPC elution behavior of polymers. Similar to DOX·HCl, PLA-g-DOX PDC was also somewhat adsorbed by GPC columns, and therefore, the GPC results could not indicate its hydrodynamic sizes, molecular weights and molar mass dispersity any more. Subsequently, the PDC was further studied by GPC analysis using DMF (with 0.01 M LiBr) as eluent at 65 °C. Relative to its GPC results at 55 °C (Figure 2), the monomodal GPC peaks of PLA-g-DOX observed at 65 °C became narrower and shifted to smaller elution volumes (17.4 mL in RI channel and 17.8 mL in UV− vis channel; Figure S2). Evidently, the interaction between the PDC and GPC columns was significantly reduced at higher temperature. Although the GPC results indicated that the interaction would not completely disappear even at 65 °C, the absence of GPC shoulders or side peaks on the side of larger elution volume suggested no considerable occurrence of backbone degradation event during the synthesis of the PDC. Preparation of PLA-g-DOX-Based NPs. After the successful synthesis of PLA-g-DOX PDC, it was then utilized to prepare NPs by a modified nanoprecipitation approach.31 The NPs were obtained by slow addition of a solution of PLAg-DOX PDC into a solution of surfactant, then slow addition of water into the resulting mixed solution, followed by dialysis against water. Several types of water-miscible organic solvents, including THF, DMSO, acetone, and acetonitrile, were tested for the preparation of the solution of PLA-g-DOX PDC. Among them, THF was determined to be the best solvent, because DMSO eventually led to broad size distributions of NPs (data not shown), while acetone and acetonitrile could not dissolve PLA-g-DOX PDC very well due to its high content of DOX. With amphiphilic structure and MAL functionality, DSPE-PEG-MAL was selected as surfactant, and it was dissolved in 4% ethanol in water. Through the nanoprecipitation process, DSPE-PEG-MAL adsorbed by the hydrophobic core of PLA-g-DOX-based NPs can form a hydrophilic PEG shielding corona to stabilize the NPs in aqueous solution and to increase their blood circulation time. Although different mass ratios (0.25−1.0) of surfactant (i.e., DSPE-PEG-MAL) to PDC (i.e., PLA-g-DOX) were used, eventually about 10 wt % of surfactant relative to PDC was present after the final dialysis step. The surfactant amount in NPs was estimated through 1H NMR analysis of the NPs by

Figure 2. GPC curves of PLA-g-ALD, DOX·HCl and PLA-g-DOX PDC obtained at 55 °C using RI detector and UV−vis detector at 490 nm (with system peaks at 22−27 mL in RI channel).

between polymer scaffold and drug.51 In this reaction, DOX· HCl was neutralized by mixing with 2 equiv of TEA for 2 h and then added dropwise into the solution of 4. After stirring for 24 h in the dark, the Schiff base linkage was formed. Due to the feed ratio of [DOX]0/[aldehyde of 4]0 = 1:2, no free DOX needs to be further removed after the completion of reaction, and therefore, no other purification is necessary except extraction to remove TEA and TEA/HCl salt. Chemical structure of the resulting PLA-g-DOX PDC was verified by 1H NMR spectroscopy. 1H NMR spectra of PLA-g-ALD and DOX· HCl were shown in Figure 1b and 1c, while the 1H NMR spectrum of the mixture of PLA-g-aldehyde and DOX·HCl was given in Figure 1d. It is interesting that without addition of TEA into the mixture of PLA-g-ALD and DOX·HCl, no reaction between these two chemicals was observed, because Figure 1d essentially can be interpreted as the overlay of Figure 1b and 1c. However, after the addition of TEA, the reaction occurred; as demonstrated in 1H NMR spectrum of the resulting purified polymer (Figure 1e), distinct peaks from DOX proton and broad peaks from PLA-g-ALD proton are merged together to form broad peaks, suggesting the absence of free DOX in the sample after the simple workup process. Moreover, the obvious decrease of resonance intensity of aldehyde proton at 10.1 ppm and the appearance of resonance from imine proton at 8.4 ppm indicated that a portion of aldehyde group was converted to imine group. Different diffusion coefficients of the PDC and free DOX in DMOS-d6 were revealed by DOSY NMR analysis, further verifying the successful DOX grafting reaction and the absence of free DOX in the PDC sample (Figure S1).53,54 Based upon the ratio of integrals of 1H NMR resonance intensities of aldehyde and imine protons, DOX grafting density (x) of ∼27% was further determined, corresponding to essentially complete conversion of DOX in the conjugation reaction. Thus, based on DPn of precursor polymer 3 and composition of 5 measured by 1H E

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further deduced that the surface density of DSPE-PEG-MAL was essentially constant for NPs, regardless of the nanoprecipitation conditions. With a Dh of 98 nm and a PDI of 0.18 (Figure 3a), the NPs prepared at PDC concentration of 3 mg/mL and surfactant to PDC mass ratio of 0.5, i.e. NP-5 in Table 1, were chosen for further investigations. TEM imaging was utilized to visualize the morphology and uniformity of these NPs (Figure 3b). They showed uniform spherical morphology with average diameter of around 100 nm, which was in good agreement with their DLS results. In addition to size and homogeneity, colloidal stability of NPs should also be considered for their applications in drug delivery. Due to the presence of charged surfactant molecules on surface, the NPs can be readily suspended in water and Dulbecco’s Modified Eagle Medium (DMEM; with 10% Fetal bovine serum (FBS)), as well as phosphate buffer (pH 5.5 and 7.4), without noticeable occurrence of aggregation. Moreover, their colloidal stability can be maintained for extended time periods in these media. A suspension of the NPs in deionized water (pH = 7.0) was monitored by DLS. As shown by Figure 4, Dh and scattering intensity of the NPs were essentially

comparing resonance intensities of maleimide protons from the surfactant at 6.7 ppm with these of PDC protons at 5.00−5.60 ppm (data not shown). According to UV−vis analysis of the systems before and after dialysis, around 10% of loss of conjugated DOX was estimated during the entire workup process, presumably due to the slow release of DOX through the cleavage of imine linker under the workup conditions. Thus, with surfactant-coating corona and PDC-based core, the freshly prepared NPs possessed about 26 wt % of DOX. Based upon DLS analysis, the effects of PDC and surfactant concentrations on the size and size distribution of PLA-g-DOXbased NPs were investigated. PDC concentrations in THF of 1, 3, and 5 mg/mL and the mass concentration ratio of surfactant to PDC as 1, 0.5, and 0.25 were chosen in the study. From Table 1, it can be easily observed that the volume-average Table 1. Preparation of PLA-g-DOX-Based NPs via a Modified Nanoprecipitation Approacha NPs NP-1 NP-2 NP-3 NP-4 NP-5 NP-6 NP-7 NP-8 NP-9 a

concentration of PDC in THF (mg/mL)

surfactant/ PDC mass ratio

Dhb (nm)

PDIb

zeta potentialb (mV)

1

1 0.5 0.25 1 0.5 0.25 1 0.5 0.25

60 ± 6 63 ± 7 67 ± 8 91 ± 6 98 ± 4 102 ± 8 132 ± 6 149 ± 5 163 ± 6

0.28 0.37 0.40 0.17 0.18 0.21 0.11 0.11 0.12

−33 −32 −30 −34 −35 −35 −37 −36 −33

3

5

PDC: PLA-g-DOX; surfactant: DSPE-PEG-MAL. bBy DLS.

hydrodynamic diameter (Dh) of the NPs, ranging from 60 to 163 nm, significantly increased with polymer concentration. At the same PDC concentration, the Dh of NPs also decreased slightly with the increase of surfactant amount. Besides size of NPs, size distribution of NPs is also an important criterion to assess the quality of formulation. According to Table 1, with the increase of PDC concentration and the surfactant to PDC ratio, the polydispersity indexes (PDI) of NPs decreased, indicating the increase of uniformity of NPs. Additionally, as illustrated by zeta potential measurement, all of the NPs showed moderately high negative surface charge (−30 to −36 mV) which was not significantly affected by PDC concentration or the surfactant to PDC mass ratio. The results can be ascribed to the surface presence of DSPE-PEG-MAL on all these NPs, because of the negatively charged DSPE unit of the surfactant. It can be

Figure 4. Colloidal stability of PLA-g-DOX-based NP-5 in deionized water at r.t.

consistent during 60 days under r.t., indicating relatively slow degradation of the PLA-based polymer backbones and significant colloidal stability of the NPs under the experimental conditions.55 Noteworthily, the NPs can also be readily suspended in plasma without noticeable occurrence of aggregation, although their colloidal stability in plasma for extended time periods could not be probed through DLS monitoring due to the intrinsic instability of plasma.

Figure 3. (a) DLS size distribution of PLA-g-DOX-based NP-5 in water and (b) TEM images of PLA-g-DOX-based NP-5. F

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Drug Release Study. The extracellular fluid in tumor tissue (pH = 6.5−7.2) is more acidic than that in nomal tissue (pH ∼ 7.4), while intracellular endosome and lysosome (pH = 4.5− 6.5) also possess acidic environments.56 Therefore, after the expected accumulation of nanoscale DDSs in tumor due to EPR effect, their acid-sensitive drug release behavior can be highly preferred. In our study, we designed Schiff base linkage (benzyl imine) between DOX and PLA-based backbone because of its high acid-lability.11,57 Drug release behavior of PLA-g-DOX from the NPs was studied at 37 °C in phosphate buffer pH 5.5 and pH 7.4. Using dialysis approach and based upon the UV−vis measurement of DOX concentrations in the buffer media outside dialysis bags, the pH-triggered DOX release property of PLA-g-DOX was verified. As shown in Figure 5, with half-time release of 14 h, DOX was quickly

Figure 6. GPC curves of PLA-g-DOX-based NP-5 after incubation in phosphate buffer at 37 °C at pH 5.5 for 24 h (a), at pH 7.4 for 24 h (b), at pH 5.5 for 48 h (c), and at pH 7.4 for 48 h (d). The curves were obtained at 55 °C using UV−vis detector at 490 nm.

could only be attributed to the cleavage of acid-sensitive imine linkage. Comparison of GPC curves of PLA-g-DOX after incubating the NPs under pH 5.5 and pH 7.4 further reveals that degradation of PLA-based backbone underwent faster in pH 7.4 than in pH 5.5 as the major peak of the conjugate drifted to 18.3 mL after 48 h of incubation in pH 7.4. Similar trend of PLA-based backbone degradation in pH 5.5 and pH 7.4 was also observed in our previous study.34 Although in vivo drug release from the NPs was not probed, their noticeable backbone degradation and slow drug release at 37 °C at pH 7.4 suggest the possibility that a portion of DOX moieties may not release from the NPs under in vivo conditions before DOXcontaining degradation residues are eliminated through systemic clearance. Cytotoxicity and Cell Internalization Studies. With well-defined chemical structure, uniform morphology and pHresponsive drug release profile, PLA-g-DOX-based NPs were further assessed through in vitro cytotoxicity and cell uptake studies. MCF-7 breast cancer cell line was selected as the experimental model for cancer cells. DOX·HCl and PLA NPs prepared by nanoprecipitation were used as positive and negative controls, respectively. As shown in Figure 7, relative to DOX·HCl, PLA-g-DOX-based NPs exhibited even higher therapeutic efficacy in killing MCF-7 cells at the entire concentration range of DOX moieties ([DOX]0 = 0.1−10

Figure 5. DOX release profile from PLA-g-DOX-based NP-5 at 37 °C under different pH (5.5 and 7.4). Error bars represent the standard deviation (n = 3).

released from PLA-g-DOX-based NPs under pH 5.5 in the first ∼30 h, although the release became slow at the late stage due to the significantly reduced difference of drug concentrations between the media inside and outside the dialysis bag. On the other hand, only 20% of DOX released after 48 h under pH 7.4. Thus, the pH-triggered DOX release property of PLA-g-DOX was verified. To further distinguish DOX release mechanism, after incubation of PLA-g-DOX-based NPs at 37 °C in phosphate buffer pH 5.5 and pH 7.4 for 24 and 48 h, the aliquots were withdrawn, concentrated, and then analyzed by GPC using DMF (with 0.01 M LiBr) as eluent at 55 °C. Without system peaks, the GPC curves obtained by using UV−vis detector provided critical information regarding DOX release and PLAbased backbone degradation under the incubation conditions. As shown in Figure 6, significant DOX peak at around 26.0 mL was found after 24 h of incubation of NPs in buffer 5.5 and DOX peak become larger after 48 h, while much smaller DOX peaks appeared after incubation of NPs in buffer 7.4 for 24 and 48 h. Such results agreed qualitatively with the drug release profiles in Figure 5. Moreover, the peaks attributed to the remaining PLA-g-DOX species were also observed in the GPC curves at around 18.1 mL. As compared with the GPC curve of the original PLA-g-DOX species with peak position at 18.0 mL (Figure 2), the GPC results demonstrated that PLA-based backbone of the conjugate did not degrade considerably while DOX release occurred substantially in buffer 5.5 within 48 h. Thus, it can be deduced that the pH-responsive DOX release was not induced by PLA-based backbone degradation and

Figure 7. Cytotoxicity of DOX free NPs, DOX·HCl and PLA-g-DOXbased NP-5 against MCF-7 cells after 48 h incubation. Error bars represent the standard deviation (n = 4). G

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Figure 8. Confocal images of PLA-g-DOX-based NP-5 against MCF-7 cells after 1, 4, and 8 h of incubation (top frame: fluorescence; bottom frame: fluorescence + bright field).

μg/mL) after 48 h of incubation. On the other hand, without conjugated drug moieties, PLA NPs did not cause considerable cytotoxicity against MCF-7 cell line at all experimental concentrations, due to the biocompatible properties of the PLA scaffold. The results suggested that PLA-g-DOX-based NPs potentially can be used for the effective treatment of cancer, and their PLA-based delivery scaffold may not cause significant cytotoxic effects. To visualize the cell uptake and internalization of PLA-gDOX-based NPs, using free DOX as control, the NPs were incubated with MCF-7 cells for 1, 4, and 8 h, and then examined by confocal laser scanning microscopy (CLSM) based on the fluorescence of DOX moieties. As revealed in Figure 8, NPs can be effectively taken up by MCF-7 cells. Although NPs were predominately located at cell membrane after 1 h of incubation, confocal images obtained after 4 h of incubation clearly showed high fluorescence intensities of DOX moieties within cells, indicating that a major portion of NPs were taken up by cells. After 8 h of incubation, the fluorescence intensity within cells further increased. As shown in control experiment (Figure S3), free DOX could be quickly taken up by MCF-7 cells, and accumulation of DOX within cells was observed just after 1 h of incubation. Such difference between cell uptake of NPs and that of free DOX further suggested that NPs can internalize MCF-7 cells to release DOX intracellularly, and this feature may help to reduce side effects of these NPs when they are used for in vivo application. Thus, the cell internalization results further supported the significant potential of PLA-g-DOX-based NPs as a nanomedicine for anticancer treatment.

PLA-g-DOX PDC was further formulated into PLA-g-DOXbased NPs via a modified nanoprecipitation approach in the presence of DSPE-PEG-MAL. Under optimal nanoprecipitation conditions, the resulting NPs possessed relatively narrow size distributions. With PEG-based negatively charged shielding corona, the NPs exhibited remarkable colloidal stability in various aqueous solutions. Due to acid-labile Shiff base linkage between DOX and polymer scaffold, DOX was much more effectively released in pH 5.5 buffer than pH 7.4 buffer. In vitro study against MCF-7 breast cancer cells illustrated that the NPs can be readily taken up and result in higher therapeutic efficiency than DOX·HCl. Our data encourage further in vivo investigation of these NPs for cancer treatment. Potentially, the nanoscopic sizes of the PLA-g-DOX-based NPs may facilitate passive tumor targeting via EPR effect, and the biomedical functions of these NPs can be further enhanced by multifunctionalization through the MAL functionality of surfaceadsorbed DSPE-PEG-MAL to introduce targeting ligands and diagnostic moieties.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

* Supporting Information S

Additional figures as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*Tel.: 716-645-1193. E-mail: ccheng8@buffalo.edu. Present Address ⊥

Department of Industrial and Systems Engineering, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, P. R. China (W.-C.L.).

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CONCLUSION Well-defined PLA-g-DOX PDC was successfully synthesized through the conjugation reaction between DOX and PLA-gALD in the presence of TEA. PLA-g-ALD was prepared by copper-catalyzed azide−alkyne click reaction with high efficiency. With multiple DOX moieties grafted onto each PLA-g-ALD macromolecule, a high DOX loading of 32 wt % was achieved for the resulting PLA-g-DOX PDC. Moreover, the

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Science Foundation (DMR-1206715; CBET-1133737) and the Mark Diamond Research Fund of the Graduate Student Association at H

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University at Buffalo. The authors thank Dr. Dinesh K. Sukumaran for kind support on NMR analysis.

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