Integrin-Targeted Zwitterionic Polymeric Nanoparticles with Acid

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Integrin-Targeted Zwitterionic Polymeric Nanoparticles with AcidInduced Disassembly Property for Enhanced Drug Accumulation and Release in Tumor Pingsheng Huang,†,‡ Huijuan Song,†,§ Weiwei Wang,§ Yu Sun,‡ Junhui Zhou,‡ Xue Wang,‡ Jinjian Liu,∥ Jianfeng Liu,∥ Deling Kong,*,§ and Anjie Dong*,‡ ‡

Department of Polymer Science and Technology, Key Laboratory of Systems Bioengineering of the Ministry of Education, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China § Institute of Biomedical Engineering and ∥Tianjin Key Laboratory of Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin 300192, China S Supporting Information *

ABSTRACT: Reasonably structural design of nanoparticles (NPs) to combine functions of prolonged systemic circulation, enhanced tumor targeting and specific intracellular drug release is crucial for antitumor drug delivery. Combining advantages of Arg-Gly-Asp (RGD) for active tumor targeting, zwitterionic polycarboxybetaine methacrylate (PCB) for prolonged systemic circulation, poly(2-(diisopropylamino) ethyl methacrylate) (PDPA) for acid-triggered intracellular release, novel RGD-PCB-b-PDPA (RGD-PCD) block copolymers were prepared via reversible addition−fragmentation chain transfer (RAFT) polymerization and followed by functionalization with RGD. Doxorubicine (DOX) was encapsulated within the RGD-PCD NPs as model medicine (RGD-PCD/DOX NPs). With ultra pH-sensitivity of PDPA, the drug release was restrained at pH 7.4 for only 24% within 36 h, which was increased to 60% at pH 6.0 within 24 h, and released more rapidly at pH 5.0 for 100% within 5 h, indicating that the RGD-PCD/DOX NPs were able to turn drug release “off” at neutral pH (e.g., systemic circulation) whereas “on” under acidic conditions (e.g., inside endo/lysosomes). Furthermore, the results of fluorescence microscopy and flow cytometry analysis demonstrated improved internalization of RGD-PCD/DOX NPs in HepG2 cells via integrin-mediated endocytosis with rapid DOX release intracellularly. Consequently, the RGD-PCD/DOX NPs showed considerable cytotoxicity against HepG2 and HeLa cells in comparison with free DOX. Importantly, the RGD-PCD/DOX NPs exhibited little protein adsorption property with excellent serum stability, which led to prolonged systemic circulation and enhanced tumor accumulation in tumor-bearing nude mice. Therefore, this multifunctional RGD-PCD NPs, which represented the flexible design approach, showed great potential for the development of novel nanocarriers in tumor-targeted drug delivery. therapeutic range for optimal periods of time.8,9 Otherwise, low drug bioavailability could result in decreased drug efficacy and increased possibility of multidrug resistance (MDR).10 Accordingly, in order to achieve better therapeutic efficacy, the development of multifunctional polymeric NPs is a burgeoning research area, which could be expected to reach the tumor site effectively via both passive and active targeting,11−13 then be internalized via specific receptormediated endocytosis,14,15 and finally release the encapsulated drugs quickly triggered by intracellular stimulation.16,17 However, the integration of multiple functions into one DDS is of great challenge, since adding new functionality inevitably elevates complexity (e.g., multistep synthesis, purification, and

1. INTRODUCTION In recent years, the medical application of nanotechnology, especially the nanomedicines, has given crucial impulse to the development of various types of nano drug delivery systems (DDS) such as lipid- or polymer-based NPs for cancer therapy.1−3 Nevertheless, concern on the development of NPs for drug delivery is deepening as its application has encountered two major intractable problems.4−7 First, the accumulation of NPs in tumor tissues is poor, which is mainly due to the clearance by reticuloendothelial system (RES) and lack of tumor targeting ability, inevitably leading to compromised therapeutic effect and causing undesired side effects. Another important issue involved with many NP-based DDS is whether they lead to appropriate drug bioavailability for tumor cells. It is meant that once the NPs are localized to the solid tumor, the encapsulated drugs must be released (become bioavailable) at a rate that maintains free drug levels in the © 2014 American Chemical Society

Received: May 26, 2014 Revised: July 8, 2014 Published: July 23, 2014 3128

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characterization) and cost and lowers controllability (e.g., multicomponent, heterogeneous formulations).6 To date, excellent efforts for multifunctional NPs have been done, while the design of NPs with a flexible and reasonable combination feature of targeted delivery and intracellular responsive drug release is urgently needed to be considered to facilitate clinical applications.18−20 Herein, based on the “from outer to inner” structural design concept, a kind of novel multifunctional NPs was fabricated flexibly for tumor-targeted intracellular drug release by combining advantages of Arg-Gly-Asp (RGD) for active tumor targeting, zwitterionic polycarboxybetaine methacrylate (PCB) for prolonged systemic circulation, poly(2-(diisopropylamino) ethyl methacrylate) (PDPA) for acid-triggered “off-on” drug release (as illustrated in Scheme 1). First, Arg-Gly-Asp

Consequently, it is expected that the reasonable combination of RGD, PCB, and PDPA could synergeticly perform integrate functions of prolonged systemic circulation, tumor targeted accumulation, and specific intracellular release to improve cancer treatment efficiency. In this work, the functional combination of RGD, PCB, and PDPA was realized by facile preparation of RGD-PCB-b-PDPA (termed as RGD-PCD) copolymer via reversible addition− fragmentation chain transfer (RAFT) polymerization and followed by functionalization with RGD via amidation reaction. The physicochemical characteristics of RGD-PCD NPs and drug loading properties were investigated in detail. The pHsensitivity of RGD-PCD NPs and acid-triggered drug release was studied systematically. The cellular uptake and intracellular drug release were confirmed by fluorescence microscope and flow cytometry analysis. The in vitro antitumor effect was evaluated by cytotoxicity experiments. The protein adsorption and serum stability of PCD/DOX NPs and RGD-PCD/DOX NPs were measured using bovine serum albumin (BSA) and fibrinogen (FBG) as the model proteins. The in vivo drug distribution and tumor accumulation of RGD-PCD/DOX NPs was performed on HepG2 tumor bearing nude mice.

Scheme 1. Illustration of the Preparation of RGD-PCBPDPA/DOX NPs, Tumor-Targeted Accumulation, and Receptor-Mediated Endocytosis and pH-Tunable Intracellular Drug Release

2. EXPERIMENTAL SECTION 2.1. Materials. 2-(Dimethylamino) ethyl methacrylate (DMA), 2(diisopropylamino) ethyl methacrylate (DPA), tert-butyl α-bromoisobutyrate, and pyrene were purchased from Sigma-Aldrich. N-(3(Dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC· HCl), trifluoroacetic acid (TFA), 2,2,2-trifluoroethanol (TFE), and Nhydroxysuccinimide (NHS) were obtained from Alfa Aesar (Lancashire, U.K.). The inhibitor in the DPA monomer was stripped off by passing through a short column packed with neutral alumina. Doxorubicin hydrochloride (DOX·HCl) was purchased from Zhejiang Hisun Pharmaceutical Co. Ltd. Acetonitrile and 2,2-azobis(isobutyronitrile) (AIBN) were obtained from Alfa Aesar (Lancashire, U.K.). Anhydrous ethyl ether and dimethylformamide were all analytical grade and used as received from Jiangtian company (Tianjin, China). Arg-Gly-Asp (RGD), 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), bovine serum albumin (BSA), fibrinogen (FBG), and 10% fetal bovine serum (FBS) were purchased from Sigma-Aldrich (St. Louis, U.S.A.). 2.2. Characterization. 1H NMR (Varian Unity-Plus INOVA 500) was employed to characterize the structure and composition of the monomer and polymers. The molecular weight (Mn) and polydispersity index (Mw/Mn) of the copolymers were determined by gel permeation chromatography (GPC), which was equipped with PLgel Oranginc GPC Column (10 um Mixed-B, Org 300 × 7.8 mm) with RI2000 detector. DMF with LiBr (1 g/L) was used as the eluting solvent at a flow rate of 1 mL/min, and polystyrene was used as the standard for calibration. Particle size, zeta potential, and size distribution (PDI) of NPs were determined by Zetasizer 3000HS (Malvern Instrument, Inc., Worcestershire, U.K.) at a wavelength of 633 nm with a constant angle of 173° at room temperature. The micromorphology of blank and DOX-loaded NPs was observed by transmission electron microscopy (TEM, Hitachi H600). The solution of NPs was dropped on the 400-mesh, carbon-coated grids and excess sample was removed by filter paper. Then, the sample-loaded, carboncoated grids were dried at room temperature. The specimens were viewed under the microscope at an accelerating voltage of 100−200.0 kV. 2.3. Synthesis and Characterization of PCD and RGD-PCD Copolymers. Herein, the chain transfer agent S-1-dodecyl-S-(α,α′dimethyl-α″-acetic acid) trithiocarbonate (CTAm) and monomer 2tert-butoxy-N-(2-(methacryloyloxy) ethyl)-N,N-dimethyl-2-oxoethanaminium (CB-tBu) were synthesized as reported.30,38 Then, the RGDPCD copolymers were obtained with a three-step method (shown in Scheme 2). First, the PCB (tBu)-PDPA copolymer was synthesized by

(RGD) was chosen as the homing device for active tumor targeting since RGD could specifically bind to the αvβ3 integrin receptor, which is highly expressed on the surface of proliferating neovascular endothelial cells and malignant cells in tumor, while minimally expressed in normal quiescent endothelial cells.21−24 Second, polycarboxybetaine methacrylate (PCB), a zwitterionic polymer, was chosen as the hydrophilic segments due to its ultralow fouling property and functionalizable convenience for the attachment of various homing devices for targeted drug delivery via EDC/NHS chemistry.25−29 As demonstrated by Jiang and his co-workers, PCB has been successfully used to modify a variety of NPs and nanogels as an alternative of polyethylene glycol (PEG), which could achieve long-term stability in different kinds of protein solutions and even undiluted blood serum.30−33 It was also demonstrated the mix-charged zwitterion can prolong the systemic circulation and enhance the retention and cellular uptake of golden nanoparticles.34 Subsequently, the pH-sensitive function was integrated into polymeric nanoparticle by employing poly(2(diisopropylamino) ethyl methacrylate) (PDPA) as the hydrophobic block. The PDPA segment was highly pH-sensitive which underwent rapid transition from hydrophobic to hydrophilic due to the protonation of the tertiary amine groups at endo/lysosomal environment (pH 4.0−6.0).35−37 3129

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prepared with the same method and the blank PCD NPs were prepared similarly without the addition of DOX. Subsequently, the drug loading content (DLC) and drug loading efficiency (DLE) were determined by UV−visible spectrophotometer (TU-1900, China). A calibration curve was constructed using different concentrations (1−50 μg/mL) of DOX in TFE. DLC and DLE were calculated from the following equations:

Scheme 2. Synthesis Route of RGD-PCD Copolymers

DLC(%) = (weight of loaded DOX/weight of DOX loaded NPs) × 100%

(1)

DLE(%) = (weight of loaded DOX/weight of DOX in feed) × 100%

(2)

In addition, the serum stability of the PCD/DOX NPs and RGDPCD/DOX NPs was studied by mixing the solution of NPs with DMEM culture medium containing 10% fetal bovine serum (FBS) at pH 7.4. The change of particle size was monitored by zetasizer. In order to verify the protein adsorption properties of the NPs, bovine serum albumin (BSA) and fibrinogen (FBG) were used as model proteins.40 The PCD/DOX NPs and RGD-PCD/DOX NPs were incubated with BSA or FBG solution in PBS at 7.4, with the concentration of NPs and proteins at 0.18 and 0.25 mg/mL respectively. After incubation for 4 and 8 h at 37 °C, 5 mL of each sample was withdrawn and centrifuged at 16000 g for 20 min at 20 °C to thoroughly precipitate the protein adsorbed aggregation. The protein concentration of supernatant was determined using UV− visible spectrophotometer (TU-1900, China). Then, the adsorbed proteins on the aggregate were calculated against a standard calibration curve. 2.6. pH-Sensitivity of PCD NPs. First, the pH-sensitivity of PCD NPs was estimated by fluorescent spectrometry using pyrene as a fluorescence probe.41,42 Polymer solutions (1 mg/mL in 0.01 mM PBS pH 7.4) containing pyrene (6 × 10−7 mol/L) were prepared. The pH values were adjusted by adding 1 N HCl solution dropwise, which was monitored with a pH meter, and then equilibrated for 2 h to assess the pH-induced change of fluorescence spectrum. Second, the pHsensitivity of PCD NPs was also carried out by measuring the change of mean diameter of NPs at different pH values, determined by Zetasizer. Furthermore, the shape and morphology of NPs at different pH vales were assessed by TEM. 2.7. In Vitro Drug Release Studies. In vitro drug release of PCD/DOX NPs was studied in PBS, which simulated the natural pH gradient in vivo. The drug release research was carried out in PBS of different pH values of 7.4, 6.8, 6.0, and 5.0. To obtain the drug release profile, 5 mL of PCD/DOX NPs (1 mg/mL) were sealed in a dialysis bag (MWCO: 3500 Da) and incubated in 40 mL of buffer solutions at 37 °C under oscillation. At scheduled time intervals, 5 mL of release medium was withdrawn for testing and replaced with an equal volume of fresh medium. The amount of drug released was detected by UV− visible spectrophotometry. Three groups of replicate measurements were carried out for each time point. The cumulative drug release percentage (Er) was calculated by the following equation:

RAFT polymerization, using AIBN as the initiator and CTAm as the chain transfer agent in DMF under no oxygen conditions. The PCB (tBu)-PDPA copolymer was dialyzed against water and obtained by freeze-drying. Second, RGD peptide was conjugated via EDC/NHS chemistry. Briefly, PCB (tBu)-PDPA (0.01 mM) copolymer was dissolved in 3 mL of DMSO and incubated with 0.1 mM EDC·HCl and 0.2 mM NHS for 30 min, and then the excess amount EDC·HCl was quenched by mercaptoethanol. Then, RGD (0.01 mM) dissolved in 200 μL of DMSO was added dropwise and the reaction was continued for another 24 h at RT. The resulting RGD-PCB (tBu)PDPA solution was dialyzed against pure water for 2 day and then was lyophilized to obtain the powder of RGD-PCB (tBu)-PDPA copolymer. Finally, the obtained RGD-PCB (tBu)-PDPA copolymers were dissolved in TFA to remove the tBu ester groups for 2 h. The resulting RGD-PCD copolymers was precipitated into ethyl ether and redissolved in a small amount of TFE and precipitated into ethyl ether repeatedly. The PCD copolymers were obtained by the same method without the conjugation of RGD. The chemical structure and composition of copolymers was characterized by 1H NMR and GPC. The amount of RGD being conjugated was determined by UV− visible spectrophotometer (TU-1900, China). A calibration curve was constructed using different concentrations (1−50 μg/mL) of RGD in DMSO. 2.4. Determination of Critical Micellar Concentration (CMC). In order to verify the stability of NPs in diluent situation, CMC was measured by the steady-state fluorescent-probe methodology using pyrene as the probe on a Varian fluorescence spectrophotometer at room temperature.39 Samples for fluorescence investigation were prepared in 0.01 M PBS (pH = 7.4). In this experiment, the concentration of copolymers varied from 1.0 × 10−7 to 1 mg/mL. The final concentration of pyrene in copolymer solutions was maintained at 6 × 10−7 mol/L. The excitation wavelength was chosen as 331.5 nm and the fluorescent intensity at 373 and 383 nm was monitored. The CMC was estimated as the cross-point when extrapolating the intensity ratio I373/I383 at low and high concentration regions. 2.5. Preparation and Characterization of PCD and PCD/DOX NPs. DOX·HCl (100 mg) was dissolved in 10 mL of pH 9.0 borax buffer solution and stirred for 12 h at RT. Then the solution was centrifuged at 10000 for 10 min at 20 °C to obtain the precipitate, which was washed by pure water to remove any residual borax salt. Eventually, the precipitate was lyophilized to obtain the base form of DOX. Subsequently, the PCD/DOX NPs was prepared via nanoprecipitation techniques. Briefly, PCD copolymer (20 mg) and DOX (3 mg) were dissolved in 3 mL of TFE, and the mixed solution was added dropwise into 20 mL of 0.01 M PBS (pH 7.4) under magnetic stirring at 300 rpm. Then, the drug-loaded NPs were formed immediately, and the solvent was removed through evaporation at room temperature for 24 h. The resulting dispersions were centrifuged for 10 min at 5000 rpm to eliminate the aggregated particles and nonentrapped drugs. After that, the supernatant fluid was freeze-dried to obtain the drug-loaded NPs. The RGD-PCD/DOX NPs were

n−1

Er(%) = (Ve ∑ Ci + V0Cn/mDOX ) × 100% 1

(3)

where Ve is the volume of the replaced medium (Ve = 5 mL), V0 is the whole volume of the release medium (V0 = 40 mL), Cn is the concentration of DOX in the nth sample, and mDOX represents the amount of DOX in the NPs. 2.8. Cell Uptake and Intracellular Drug Release Studies. 2.8.1. Cell Culture. HepG2 cells were maintained in Hyclone Ham’s/ F12 medium. All media were supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 U/mL). All cell lines were maintained at 37 °C and 5% CO2 in humidified atmosphere. 2.8.2. Cellular Uptake Studies. HepG2 cells were seeded in 24 well plates at a density of 2 × 105 cells per well in 0.5 mL of DMEM medium and incubated at 37 °C in a 5% CO2 humidified atmosphere 3130

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Table 1. Structure and Composition of PCD Copolymers composition

a

samples

CBa

DPAa

Mna (Da)

Mnb (Da)

Mwb (Da)

Mw/Mnb

CMCc (mg/mL)

PCD-1 PCD-2 PCD-3

25 25 25

19 28 37

9425 11400 13418

10055 11950 13855

12468 15057 17872

1.24 1.26 1.29

6.31 × 10−4 1.26 × 10−4 3.16 × 10−5

Calculated by 1H NMR spectra. bDetermined by GPC. cMeasured by pyrene-based fluorescent spectrometry. for three times. After centrifugation (12000 g, 15 min, 4 °C), the organic phase was separated and dried by vacuum drying oven. The residue was dissolved in 0.5 mL of mobile phase and followed by filtered with ultrafiltration membrane (220 nm).40 Eventually, the filtrate was collected for HPLC analysis. The amount of DOX was determined by HPLC (Lab Alliance Model 201) with a Hypersil ODS2 (250 mm × 4.6 mm, 5 μm) C18 column and the detection wavelength was set at 480 nm. The mobile phase was composed of acetonitrile and water (50:50, v/v) with pH 2.74 adjusted by HClO4. The injection volume was 20 μL and the flow rate of mobile phase was 1.0 mL/min. To obtain the standard curve for DOX, the samples were prepared by adding free DOX to the tumor tissue from untreated mice. The linear detection range was 0.1−30 μg/mL. All animals were obtained from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed individually in plastic cages in a controlled environment with free access to food and water. All animal experiments were performed in accordance with the People’s Republic of China national standard (GB/T 16886.6−1997).

for 24 h. The original medium was discarded and washed twice with PBS. Then fresh DMEM medium containing free DOX, PCD/DOX NPs, and RGD-PCD/DOX NPs were added. In addition, in order to verify the RGD receptor-mediated endocytosis, competitive cellular uptake study was conducted meanwhile.43 The fresh DMEM medium containing 10 times amount of free RGD was added with the RGDPCD/DOX NPs. Then, the cells were incubated for 4 h at 37 °C, and then washed three times with cold PBS, and harvested by trypsin treatment. The harvested cells were suspended in PBS and centrifuged at 1000 g for 5 min at 4 °C. The supernatants were discarded and the cell pellets were washed with PBS to remove the background fluorescence in the medium. After two cycles of washing and centrifugation, cells were resuspended with 200 μL of PBS for analyses using a FACS Calibur flow cytometer (BD Biosciences, U.S.A.) with an argon laser excitation at 488 nm (Becton Dickinson) and fluorescence (FL3) was detected. Cells treated with PBS were used as control. 2.8.3. Intracellular Drug Release Studies. HepG2 cells were seeded in 24-well plates with 2 × 104 cells per well in 0.5 mL DMEM culture medium and incubated under standard cell culture conditions. Then, cells were washed twice with PBS and fresh medium was added with free DOX, PCD/DOX NPs and RGD-PCD/DOX NPs. After 4 h incubation, cells were washed twice with ice-cold PBS and fixed with fresh 4% paraformaldehyde for 15 min at room temperature. The intracellular release behaviors of RGD-PCD/DOX NPs were further conducted after incubation for 6 h, and 8 h. The cells were counterstained 4,6-diamidino-2-phenylindole (DAPI) for the cell nucleus with an excitation of 405 nm, sequentially following the standard protocol of the manufacturer before imaged on fluorescence microscopy (Leica AF 6500), and the fluorescence of DOX was observed under an excitation wavelength of 543 nm. 2.9. In Vitro Cytotoxicity Assay. The cytotoxicity of PCD NPs, RGD-PCD NPs, PCD/DOX NPs, and RGD-PCD/DOX NPs against HeLa and HepG 2 cells was evaluated by MTT assay. HeLa and HepG 2 cells were seeded in 96-well plates at 5000 cells per well in 100 μL DMEM culture medium and incubated under standard cell culture conditions. The culture medium was replaced with 100 μL of fresh medium containing the NPs at pH 7.4. After treatment for 48 h, the medium was replaced by 100 μL of fresh DMEM medium followed by addition of 25 μL of MTT stock solution (5 mg/mL in PBS). After incubation for an additional 2 h, 100 μL of the extraction buffer (20% SDS in 50% DMF, pH 4.7) was added to the wells and incubated overnight at 37 °C. The absorbance of the solution was measured at 570 nm using a microplate reader. The results were expressed as a percentage of the absorbance of the blank control. 2.10. In Vivo Drug Distribution and Tumor Accumulation Studies. The Balb/c nude mice (female, 6−8 weeks old) bearing HepG2 tumor model was used for drug accumulation studies. When the mean volume of tumor reached 150 mm3, free DOX, PCD/DOX NPs, and RGD-PCD/DOX NPs were administrated intravenously into the tail vein at an equivalent dose of 10 mg/kg. At scheduled time intervals, the mice were sacrificed, and then the major organs and tumor were harvested. These obtained organs were examined by the Kodak IS in vivo FX imaging system. To further quantify the drug accumulation amount in tumor tissues, the excised tumor tissues were washed with cold saline and dried by filter paper and then weighed. The obtained tumors were homogenized in borate buffer solution (10 mM, pH 9.0). Subsequently, 300 μL of tissue homogenate was taken out and extracted with 500 μL of chloroform/isopropanol (6:1, v/v) by vortex

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of PCD and RGDPCD Copolymers. The PCD copolymers were synthesized via

Figure 1. (A) 1H NMR spectra of PCB (tBu)-PDPA and (B) PCD copolymer in DMSO-d6.

RAFT polymerization followed by selective hydrolysis of the tert-butyl groups of CB-tBu. As shown in Scheme 2, the chain transfer agent CTAm and CB-tBu monomer were synthesized according to a previous report and characterized with 1H NMR (shown in Figure S1).30,38 Then, the PCB (tBu)-PDPA copolymer was synthesized by RAFT polymerization, using AIBN as the initiator and CTAm as the chain transfer agent in DMF under no oxygen conditions. In order to endow the NPs with tumor targeting ability, RGD peptide was conjugated via EDC/NHS chemistry. Finally, the hydrolysis of the tBu ester groups was rapidly accomplished in TFA within 2 h to obtain the final RGD-PCD copolymers. The chemical structures and compositions of PCD copolymers were determined by 1H NMR and GPC. The 3131

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pyrene in PCD-2 copolymer solutions is shown in Figure 3A. The fluorescence intensity increased as the concentration of PCD-2 copolymer increased. Figure 3B showed the intensity ratio (I373/I383) of the pyrene excitation spectra versus the logarithm of the concentration of PCD copolymer, and the CMC value was determined by extrapolation method. Accordingly, the CMC value of PCD-1, PCD-2, and PCD-3 copolymers were found to be 6.31 × 10−4, 1.26 × 10−4, and 3.16 × 10−5 mg/mL. Due to the extremely low CMC values, the PCD NPs could be expected to maintain original morphology in despite of highly diluted by a large volume of body fluids in systemic circulation before reach the targeting site. 3.2. Preparation and Characterization of PCD and PCD/DOX NPs. The PCD and PCD/DOX NPs were prepared via nanoprecipitation techniques. The particle size, zeta potential, PDI, and micromorphology of the NPs were examined by Zetasizer and TEM. The results are listed in Table 2. As illustrated in Figure 4A,B, the size distributions were narrow and the particles were all spherical in shape with obvious core−shell structure. The sizes of DOX-loaded NPs were larger than those of blank NPs, which was mainly due to the loading of DOX in hydrophobic core of NPs.46 In addition, both the DLC and DLE increased with the increase of the PDPA segment. It is known that physical entrapment of hydrophobic drugs in polymeric NPs is mainly driven by the hydrophobic interactions between hydrophobic segments of polymers and the drug molecules.47,48 Generally, the hydrophobicity of drugs plays a decisive role in the drug-loading process. However, DOX is inherently not so hydrophobic owing to its hydroxyl and amino groups, and thus is less likely to be encapsulated in the hydrophobic core. Apart from the hydrophobic interactions between PDPA and DOX, the deprotonated DOX contains hydroxyl and amino groups, which may form hydrogen bonds with the carbonyl and tertiary amine groups. For longer PDPA segments, H-bonding interactions may enhance the DOX loading. Furthermore, the zeta potential of both PCD and PCD/DOX NPs was approximately 0 mV (shown in Figure S3C). It is reported that neutrally charged NPs with a diameter between 30 and 200 nm could be expected to obtain prolonged systemic circulation and accumulate into the tumor tissues more effectively mediated by EPR effect.1,3,5 Thus, the PCD-2/DOX NPs with commendable drug loading content and particle size were employed in the following studies. As illustrated in Scheme 2, RGD was conjugated to the terminal carboxyl group via conventional EDC/NHS chemistry

Figure 2. Ultraviolent adsorption of RGD and RGD-PCD copolymers.

details of obtained polymers are listed in Table 1. As shown in Figure 1A, all characteristic signals of CTAm, PCB (tBu), and PDPA could be seen clearly. After hydrolysis reaction, the characteristic resonance of -C (CH3)3 in the CB-tBu unit at 1.48 ppm disappeared, indicating that PCB (tBu)-PDPA copolymers were successfully converted to PCD copolymers. There were 10 repeated methylene (-CH2-) on the CTAm molecule. Therefore, the repeating units of PCB and PDPA and molecular weights could be calculated by comparing the integral areas of characteristic peaks of the PDPA unit at 1.02 ppm (-N-CH-(CH3)2) and the PCB unit at 3.45 ppm (-N(CH3)2) with the known CTAm unit (-CH2-) at 1.22 ppm. The composition and molecular weights calculated by 1HNMR agreed well with the feeding ratio. GPC was employed to determine the molecular weight (Mn) and molecular weight distribution (Mw/Mn) of all the obtained PCD polymers, and the results were summarized in Table 1. As shown in Figure S2, the results showed narrow distribution of all the obtained PCD polymers, indicating the well-controlled polymerization process and effective purification methods. Furthermore, the characteristic ultraviolet absorption at 260 nm of RGD was observed on RGD-PCD copolymers, indicating the successful conjugation of RGD (shown in Figure 2). In addition, CMC was an important parameter of amphiphilic copolymers for drug delivery, which was related to the dilution stability of NPs in the bloodstream and the drug release behavior.44,45 The CMC of PCD copolymers was measured by fluorescence spectroscopy using pyrene as hydrophobic fluorescence probe.39 Once the NPs were formed, pyrene molecules were preferentially incorporated into the hydrophobic core of the NPs and its fluorescent characteristic was changed dramatically. The typical fluorescence spectrum of

Figure 3. (A) Fluorescence emission spectra of pyrene in the different concentration of PCD-2 copolymer solutions; (B) Intensity ratios of I373/I383 as a function of copolymer concentrations. 3132

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Table 2. Characteristics of PCD NPs and PCD/DOX NPs PCD NPs

a

PCD/DOX NPs

sample

sizea (nm)

PDIa

sizea (nm)

PDIa

DLCb (%)

DLEb (%)

PCD-1 PCD-2 PCD-3

145 ± 3.5 157 ± 4.8 186 ± 3.2

0.11 ± 0.05 0.12 ± 0.02 0.13 ± 0.04

153 ± 4.4 170 ± 5.7 212 ± 9.2

0.15 ± 0.02 0.12 ± 0.01 0.13 ± 0.02

5.5 ± 0.6 6.7 ± 0.4 7.4 ± 0.8

36.7 ± 0.6 44.6 ± 0.4 49.3 ± 0.8

Determined by Zetasizer. bDetermined by UV−vis spectrum. The feed ratio of DOX was 15%.

Figure 5B, both PCD-2/DOX NPs and RGD-PCD-2/DOX NPs showed little protein adsorption, even after 24 h incubation, which was mainly due to the fact that the hydrophilic PCB block with anionic and cationic groups at the microscopic range could form a hydration layer via electrostatic interactions.31−33,49 Consequently, these results confirmed that the RGD-functionalization had negligible influence on the nonfouling property of PCB segment. The NPs with commendable serum stability and low protein adsorption property would be expected to be stealth in blood plasma and exhibit prolonged systemic circulation time in vivo.12 3.3. pH-Sensitivity of PCD NPs. First, in order to evaluate the pH-sensitivity of PCD NPs, pyrene was taking as the probe due to its dramatic change of fluorescence spectrum according to the change of polarity.39 Obviously, sharp decrease of fluorescence intensity at 373 nm and dramatic change of I373/ I383 was observed at pH 6.5, corresponding to the significant change of the surrounding polarity encountered with pyrene (shown in Figure 6A). Since the critical pH (termed as pKa) was reached, the hydrophobic DPA chains were partially protonated and its hydrophilicity was increased. Upon further lowering the pH, the PDPA blocks were totally positively charged and hence became hydrophilic and swelling, resulting in the further lowering of the fluorescence intensity. Subsequently, the pH induced variation of particle size was measured as a function of pH in PBS. As can be seen clearly in Figure 6B, there was an increasing tendency of the particle size with the decrease of pH from 7.4 to 6.5. This might be due to the partial protonation of the DPA blocks, which afford the electrostatic repulsion among the charged amine groups and consequently hindered the dense packing of the hydrophobic core of NPs.41,42 Upon further decreasing the pH to 5.0, the PCD-2 NPs were hardly detected because the DPA blocks

Figure 4. Size distribution and morphology of (A) PCD-2 NPs and (B) PCD-2/DOX NPs.

to endow the PCD NPs with tumor targeting ability. Hence, it was inferred that the PCB segment could still maintain the excellent nonfouling properties in postfunctionalized surfaces, which was confirmed by serum stability and protein adsorption studies. As shown in Figure 5A, no obvious change of particle size for both the PCD-2/DOX NPs and RGD-PCD-2/DOX NPs was observed after incubation in the presence of 10% FBS for 72 h, which indicated the excellent serum stability of these NPs. Subsequently, evaluation of the interaction of NPs with proteins was conducted using bovine serum albumin (BSA) and fibrinogen (FBG) as model plasma proteins.33 As shown in

Figure 5. (A) Change in particle size of PCD-2/DOX and RGD-PCD-2/DOX NPs incubated with DMEM culture medium with 10% FBS at pH 7.4 at 37 °C; (B) adsorption of BSA and FBG on the PCD-2/DOX and RGD-PCD-2/DOX NPs after incubation for 4, 8, and 24 h at pH 7.4 at 37 °C. The data were expressed as mean ± SD, n = 3. 3133

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Figure 6. (A) Fluorescence intensity of pyrene at 373 nm in PCD-2 NPs solutions and I373/I383 as a function of pH; (B) Variation of PCD-2 NPs size as a function of pH; (C) TEM images of PCD-2 NPs at pH 7.4 and (D) 5.0.

Figure 7. (A) In vitro drug release of PCD-2/DOX NPs at 37 °C in PBS at different pH; (B) Quantitive measurement of the mean fluorescence intensity after incubated with free DOX, PCD-2/DOX NPs, RGD-PCD-2/DOX NPs, and RGD-PCD-2/DOX NPs with free RGD for 4 h via flow cytometry (blank cells as the control, *p < 0.05 in comparation with PCD-2/DOX NPs). The data were expressed as mean ± SD, n = 3. (C) Fluorescence microscopy images of HepG2 cells incubated with free DOX, PCD-2/DOX NPs and RGD-PCD-2/DOX NPs for 4 h. (D) Fluorescence microscopy images of HepG2 cells incubated with RGD-PCD-2/DOX NPs for 4, 6, and 8 h, respectively. For each panel, images from left to right show DOX fluorescence in cells (red), cell nuclei stained by DAPI (blue), and overlays of two images.

became completely protonated and the PCD-2 copolymers were dissolved completely. Furthermore, the morphology

change of NPs was detected by TEM (shown in Figure 6C,D). At pH 7.4, the NPs were all spherical with obvious 3134

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Figure 8. (A) Viability of HepG2 and (B) HeLa cells after incubation with DOX free PCD-2 NPs, RGD-PCD-2 NPs for 48 h; (C) viability of HepG2 and (D) HeLa cells after incubation with free DOX, PCD-2/DOX NPs, RGD-PCD-2/DOX NPs for 48 h. (*p < 0.05 in comparation with PCD-2/DOX NPs). The data were expressed as mean ± SD, n = 6.

would be restrained in systemic circulation. On the contrary, significantly accelerated drug release was triggered by lowering the pH. Approximately 60% of encapsulated drugs were released at pH 6.0 within 24 h and nearly 100% at pH 5.0 within 5 h, respectively. It was meant that the PCD-2/DOX NPs could regulate the drug release effectively, which was turned release “off” in systemic circulation, whereas “on” in the endo/lysosomal compartments. In order to achieve ideal therapeutic effect, it was crucial for NPs to be trapped inside endo/lysosomes via endocytosis and rapidly intracellular drug release. First, to qualify the amount of DOX delivered in the cells, the intracellular mean fluorescence intensity was evaluated by flow cytometry. As shown in Figure 8B, the fluorescence intensity of RGD-PCD-2/DOX NPs treated group was significantly higher than that of PCD-2/ DOX NPs group (p < 0.01), which was comparable with free DOX, although it is well-known that DOX has strong ability to enter into cells via passive diffusion.52,53 The lower level of DOX delivered by PCD-2/DOX NPs was mainly attributed to the nonfouling property of PCB segment which hindered the endocytosis of PCD-2/DOX NPs. Significantly, the conjugation of RGD ligand enhanced the endocytosis of the RGD-PCD-2/ DOX NPs since the RGD targeting ligand could specifically bind to the αvβ3 integrin receptor highly expressed at the surface of malignant cells, which was further verified by the competitive cellular uptake study.21,23,24 Therefore, without the cost of the nonfouling property of PCB segment, the conjugation of RGD ligand could efficiently overcome the dilemma of PCB in endocytosis to provide enhanced specific tumor cell internalization of the NPs.

Table 3. IC50 Value of Free DOX and NPs Formulations IC50 (μg/mL) sample

HepG2 cell

HeLa cell

free DOX PCD-2/DOX NPs RGD-PCD-2/DOX NPs

0.72 7.15 1.05

0.70 5.72 0.98

core−shell structure. However, random polymeric aggregates were observed at pH 5.0, which indicated that the NPs were disintegrated and the polymer chains were completely soluble. The result of fluorescence measurement was consistent with that of pH induced size and morphology changes. This excellent pH-sensitive property of PCD-2 NPs was significant for the acid-triggered intracellular drug release. 3.4. In Vitro and Intracellular Drug Release Studies. The most obvious and spectacular microenvironments of cancer cells were the slightly acid pH in the endosomal (5.0−6.0) and lysosomal (4.0−5.0) compartments. One of the major strategies was to endow polymers with ionizable groups that underwent conformational and solubility changes in response to environmental pH variation.50,51 However, highly effective DDS must give a sharp response to the subtle pH change between systemic circulation and tumor intracellular microenvironment. Consequently, the drug release behavior of PCD-2/DOX NPs was investigated systematically under conditions that simulated the pH gradient in vivo. As shown in Figure 7A, the drug release profile was clearly pH-dependent. At pH 7.4, about 24% of the loaded drug was released within 36 h, which indicated that the drug release 3135

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Figure 9. (A) Fluorescence images of major organs and tumor after administration of different formulations at scheduled time intervals. (B) Mean fluorescence intensities of DOX in tumor tissues. (C) Quantitative analyses the concentration of DOX in tumor tissues (a = in comparison with free DOX, b = in comparison with PCD/DOX NPs, *p < 0.05, **p < 0.01). The data were expressed as mean ± SD, n = 3.

As shown in Figure 7C, the result of fluorescence microscopy observation was inconsistent with the result of cellular uptake studies. Notably, intense DOX fluorescence covered all nuclear regions after incubation with free DOX for 4 h. Generally, DOX, as a widely used antineoplastic agent in the treatment of cancers, such as leukemia, lymphomas, breast carcinoma, and many other solid tumors, is known to exert drug effects via intercalation with DNA and inhibition of macromolecular biosynthesis.54,55 In addition, the DOX fluorescence in cells treated with PCD-2/DOX NPs was discernible, but less intense, while DOX fluorescence treated with RGD-PCD-2/ DOX NPs was evident and intense, indicating higher amount of DOX was delivered and released into these cells. To further evaluate the intracellular drug release, RGD-PCD2/DOX NPs were incubated with HepG2 cells for 4, 6, and 8 h, followed by observation using fluorescence microscopy. As shown in Figure 7D, DOX was mainly located around the cell nucleus in cytoplasm after incubated with RGD-PCD-2/DOX NPs for 4 h. However, certain increase of fluorescence intensity of DOX was observed in cell nucleus with the increase of incubation time to 6 and 8 h, indicating that DOX can be released effectively within tumor cells triggered by acid microenvironment in endo/lysosomal compartment and be delivered into cell nucleus. This result was in consistent with the in vitro drug release studies. 3.5. In Vitro Cytotoxicity Assay. The cytotoxicity of DOX-free and loaded NPs was evaluated on HepG2 and HeLa cell lines. As shown in Figure 8A,B, blank PCD-2 and RGDPCD-2 NPs showed no obvious cytotoxicity even the concentration of NPs reached 5 mg/mL, indicating the well cell compatibility of the NPs. Figure 8C,D represented the viability of HepG2 and HeLa cells after incubation with free DOX, PCD-2/DOX NPs, and RGD-PCD-2/DOX NPs for 48 h, respectively. The cell viability was clearly dose-dependent. The cytotoxicity of RGD-PCD-2/DOX NPs was significantly higher than PCD-2/DOX NPs, which could be explained by the result of cell uptake studies (p < 0.05). As shown in Table 3, the IC50 values were 0.72 and 0.70 μg/mL for free DOX on

HepG2 and HeLa cells, respectively. In terms of RGD-PCD-2/ DOX NPs, the IC50 values were 1.05 and 0.98 μg/mL correspondingly, which was just slightly lower than free DOX. Notably, this commendable cytotoxicity of RGD-PCD-2/DOX NPs in comparison with free DOX might be attributed to the enhanced cell uptake and acid-triggered rapid release, which was inconsistent with the results of in vitro and intracellular drug release studies. 3.6. In Vivo Drug Distribution and Tumor Accumulation Studies. In the above protein absorption studies, the NPs showed especially low protein adsorption property, which would be expected to exhibit prolonged blood circulation time in vivo.56 However, protein adsorption of the NPs in the blood is much more sophisticated than that from model protein, as plasma is full of multiple proteins, whose adsorption is regarded as a selective and competitive process.57 In order to evaluate the accumulation of DOX in tumor tissue, free DOX, PCD-2/ DOX NPs, and RGD-PCD-2/DOX NPs were injected intravenously into nude mice bearing HepG2 tumor xenografts. Ex vivo fluorescence imaging showed the distribution of DOX in major organs and tumor tissues. As shown in Figure 9A, it could be seen clearly that both NP formulations enhanced the accumulation of DOX in the tumor tissue in comparison with free DOX, which was inconsistent with the previous report that NPs with similar small sizes (30−200 nm) accumulated preferentially in tumors through the enhanced permeability and retention (EPR) effect and at the reticuloendothelial sites such as liver.57 In addition, as shown in Figure 9B, significantly stronger fluorescence of DOX was detected in the tumor tissue with the administration of the both NPs formulations after injected for a longer time. Subsequently, the quantitative analyzation of DOX concentration in tumor tissues is shown in Figure 9C. The concentration of DOX in tumor tissues with administration of RGD-PCD-2/DOX NPs showed a 1.29 and 1.78 times higher level than with PCD-2/DOX NPs and free DOX administrations for 5 h, respectively. Particularly, the concentration of DOX in tumor with the administration of RGD-PCD-2/DOX NPs were 1.52- and 2.25-fold higher than 3136

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that of PCD-2/DOX NPs and free DOX after injection for 24 h. Obviously, PCB and RGD cooperated to facilitate the improvement of tumor accumulation of the RGD-PCD-2/ DOX NPs by prolonging systemic circulation and tumor site retention, respectively. Normally, NPs with zwitterionic polymer surfaces are expected to accumulate equally into tumor interstitials through the EPR effect; however, it is very likely that RGD-PCD-2/DOX NPs specifically bind to the αvβ3 integrin receptor highly expressed on the surface of proliferating neovascular endothelial cells and malignant cells in tumor, which subsequently promoted the internalization into tumor cells. As reported, NPs that could not be internalized by tumor cells were easily eliminated from the tumor site due to the high interstitial fluid pressure (IFP).4,58

4. CONCLUSIONS In this work, a reasonable combination of RGD, zwitterionic polycarboxybetaine methacrylate (PCB), and poly(2-(diisopropylamino) ethyl methacrylate) (PDPA) into nanoparticles (NPs) for antitumor drug delivery was implemented by facile preparation of RGD-PCB-b-PDPA (termed as RGD-PCD) copolymers via RAFT polymerization and followed by functionalization with RGD via amidation reaction. The in vitro and in vivo evaluation results fully verified the multiple functions of RGD-PCD/DOX NPs, which was realized as RGD for active tumor targeting with enhanced endocytosis, PCB for prolonged systemic circulation, and PDPA for rapid acidtriggered intracellular release. Therefore, this multifunctional RGD-PCD/DOX NPs showed pronounced cytotoxicity against HeLa and HepG2 cells compared with free DOX and predominantly accumulated in tumor tissues after intravenous injection. Consequently, the facilely prepared multifunctional RGD-PCD NPs, which represented flexible design approach, showed great potential for the development of nanocarrier in tumor targeted drug delivery.

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectra of chain transfer agent CTAm and CB-tBu monomer, GPC of PCD copolymers, size distribution and zeta potential of PCD NPs and PCD/DOX NPs, correlation data of DLS measurements, and fluorescence emission spectrum of pyrene in PCD-2 NPs solutions. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions

† These authors contributed equally to this work (P.H. and H.S.).

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by the National Natural Science Foundation of China (31271073, 81301309, and 81171371). REFERENCES

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