Click Conjugation of Peptide to Hydrogel Nanoparticles for Tumor

Aug 15, 2014 - acrylamide (AAm) and 2-carboxyethyl acrylate (CEA). The ... F3 peptide also dramatically enhances the uptake of co(CEA-AAm) NPs by the ...
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Click Conjugation of Peptide to Hydrogel Nanoparticles for TumorTargeted Drug Delivery Ming Qin,† Hong Zong,‡ and Raoul Kopelman*,†,‡ †

Department of Chemistry and ‡Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan, Ann Arbor, Michigan 48109, United States S Supporting Information *

ABSTRACT: Here we introduce a modified peptide-decorated polymeric nanoparticle (NP) for cancer cell targeting, which can deliver drugs, such as doxorubicin (Dox), to several kinds of cancer cells. Specifically, we employ a nucleolintargeting NP, with a matrix based on a copolymer of acrylamide (AAm) and 2-carboxyethyl acrylate (CEA). The negatively charged co(CEA-AAm) NP was conjugated with a nucleolin-targeting F3 peptide using a highly efficient and specific copper(I) catalyzed azide−alkyne click reaction. F3 peptide binds to angiogenic tumor vasculatures and other nucleolin overexpressing tumor cells. Attaching F3 peptide onto the NP increases the NP uptake by the nucleolin-expressing glioma cell line 9L and the breast cancer cell line MCF-7. Notably, the F3-conjugated NPs show much higher uptake by the nucleolin-overexpressing glioma cell line 9L than that by the breast cancer cell line MCF-7, the latter having a lower expression of nucleolin on its plasma membrane surface. Moreover, the F3 peptide also dramatically enhances the uptake of co(CEA-AAm) NPs by the drug-resistant cell line NCI/ADR-RES. Also, with this F3-conjugated co(CEA-AAm) NP, a high loading and slow release of doxorubicin were achieved.

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photosensitizer.15,16 Furthermore, the size of these NPs, on the order of 50 nm, should prevent them from entering the heart tissue, thus avoiding cardiotoxicity.17 Negatively charged NPs are important delivery vehicles for cationic drugs, for example, doxorubicin.18 For example, a new negatively charged hydrogel NP was prepared via the copolymerization of acrylamide (AAm), 2-carboxyethyl acrylate (CEA), with the cross-linker 3-(acryloyloxy)-2-hydroxypropyl methacrylate (AHM), in a microemulsion system.19 This co(CEA-AAm) NP has been used as a delivery vehicle for cationic drugs, for example, doxorubicin (chemotherapy drug) and verapamil (chemosensitizer). However, the uptake of these anionic NPs by tumor cells is challenging, due to repulsion by the anionic cell membrane.4 One possible way to solve this problem is to modify the NP surface with tumor-specific targeting ligands, which exhibit high affinity toward tumor-specific receptors. These targeting ligands can facilitate NP uptake through receptor-mediated endocytosis,20−22 overcome drug resistance by neutralizing the Pglycoprotein-mediated drug efflux pump, and therefore, increase the retention time of NPs in the tumor.12 While the EPR effect serves as the basis for the development of macromolecular anticancer therapy in vivo, the active targeting

INTRODUCTION Doxorubicin (Dox) is one of the most effective chemotherapy drugs.1 It has been widely used for the treatment of several forms of cancer: leukemia, bladder, breast, and other cancers. The clinical use of Dox is limited by its side effects, for example, cardiotoxicity, as well as by multidrug resistance.2,3 These side effects are related with the poor biodistribution and unfavorable pharmacokinetics of Dox. Targeted nanoparticle (NP) delivery has been an approach aimed at overcoming the above problems.4−6 As a drug delivery vehicle, hydrogel NPs offer a strategy to (1) prolong drug circulation time, (2) control drug release kinetics, and (3) enhance solubility of hydrophobic drugs.4,6 Due to the enhanced permeability and retention (EPR) effect of macromolecules, hydrogel NPs can preferentially accumulate in tumor tissue, particularly the tumor vasculatures.7−9 These NPs can also alleviate cancer drug resistance via various mechanisms.10−12 For example, Pluronic P85 micelle NPs can interact with P-glycoprotein, which could change the cell membrane structure and thus induce cell membrane permeability.13 HER2 antibody-conjugated poly(D,L-lactide coglycolide) (PLGA) NPs have been shown to improve the uptake of Dox by the drug-resistant ovarian cancer cells, SKOV-3, via receptormediated endocytosis, compared to unmodified PLGA NPs.14 AOT-alginate NPs have been shown to deliver both Dox and methylene blue to drug-resistant NCI/ADR-RES cells, for combining chemotherapy and photodynamic therapy, where methylene blue serves as both P-glycoprotein inhibitor and © XXXX American Chemical Society

Received: July 15, 2014 Revised: August 14, 2014

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Gibco 100× penicillin-streptomycin-glutamine (PSG), and Gibco heat-inactivated fetal bovine serum (HI-FBS) were purchased from Invitrogen. Acetylene-Fluor 488 (AF488) was purchased from click chemistry tools. 1-(Prop-2-yn-1-yl)pyrrole-2,5-dione was purchased from Enamine Ltd. (Ukraine). 1-Azido-3-aminopropane was synthesized as reported.32 F3 peptide (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKKC) was purchased from SynBioSci. The 96-well microplates were purchased from BD Biosciences. All chemicals were used as received, without further purification. All the water used was purified with a Milli-Q system from Millipore. Synthesis of co(CEA-AAm) NPs 4. Deoxygenated hexane (45 mL), AOT (1.6 g), and Brij 30 (4.3 mL) were mixed and stirred vigorously to produce a microemulsion. The mixture of AAm (639 mg), CEA (144 mg), and AHM (428 mg) was dissolved in DI water (1.3 mL), which was sonicated until completely dissolved. The monomer solution was added into the hexane solution under an argon atmosphere. After 20 min, fresh prepared APS solution in DI water (100 μL, 10 wt %) and TEMED (100 μL) were added to initiate polymerization. After 2 h, hexane was removed by rotary evaporation. The residue was suspended in ethanol and transferred into an Amicon ultrafiltration cell (300 kDa, Millipore Corp.). In order to remove the small molecule impurities, the NPs were washed with ethanol (7×) and DI water (7×). The NP solution was lyophilized and stored in the freezer at −20 °C. The polyacrylamide (PAAm) NPs (without CEA) were synthesized in the same manner. Synthesis of Alkyne-F3 Peptide 3. F3 peptide 1 (2 mg, 10 mg/ mL in PBS buffer), 1-(prop-2-yn-1-yl)pyrrole-2,5-dione 2 (3.8 mg, 7.6 mg/mL in DMF), and TCEP (2.8 mg, 50 mg/mL in PBS) were mixed together and stirred overnight (Scheme 1a). TCEP was serving as a reducing agent to prevent the free thiol of F3 peptides 1 from forming disulfide bonds. The mixture was diluted 10× in PBS buffer. Then the NPs were purified using an Amicon ultrafiltration cell (1000 Da; 5× in DI water), freeze-dried, and stored in a freezer. The molecular weights

strategy further enhances the drug delivery’s selectivity, efficacy, and therapeutic index.20,23 Nucleolin, a shuttle protein between the nucleus and the cell surface, is highly expressed in angiogenic tumor vasculatures and certain types of tumor cells.24,25 The nucleolin-targeting F3 peptide has previously been attached onto several kinds of NPs, which significantly increased their uptake by such tumor and tumor endothelial cells, both in vitro and in vivo.11,23,26,27 For example, F3-conjugated polyacrylamide NPs bind to human tumor endothelial cells in vitro and to human tumor vessels in vivo.11 In addition, the attachment of the F3 peptide reduces the trafficking of the polyacrylamide NPs to lysosomes.28 With this property, F3-conjugated NPs prevent the encapsulated drugs from entering the acidic lysosomes and thus avoid their potential degradation.28 The present studies show that this F3 peptide may enhance the uptake of negatively charged NPs into tumor cells. Chemically, the conjugation of F3 peptide on negatively charged co(CEA-AAm) NPs has been a challenge. Previously, the F3 peptide was attached onto amine-functionalized hydrogel NPs with a bifunctional PEG as a cross-linker.29 This bifunctional PEG can react with amine groups from the NPs via an NHS ester reaction, while their maleimide group can react with the thiol groups of the peptide. This peptide conjugation method, however, is not suitable for the attachment of F3 peptide onto the negatively charged co(CEA-AAm) NPs. For the conjugation of F3 peptide onto co(CEA-AAm) NPs, we found the click reaction to be a very good option because it is modular, high yielding, stereospecific, and can be performed under ambient conditions.30,31 In this study, we planned to attach F3 peptides onto the surface of the negatively charged co(CEA-AAm) NPs via a straightforward click reaction. The F3 peptide was expected to enhance the uptake of these NPs in cancer cells. The co(CEAAAm) NPs were synthesized via a free radical polymerization, performed in a reverse microemulsion system.19 The NPs were then modified with an azide linker for downstream modification.31 The alkyne-F3 peptide was conjugated to the azide-NPs via a copper(I) catalyzed azide−alkyne click reaction.30 The uptake efficiency of F3-conjugated co(CEAAAm) NPs (F3-NPs) by the glioma cell line 9L (high expression of nucleolin), human breast cancer cell line MCF7 (low expression of nucleolin), and human ovarian cell line NCI/ADR-RES (drug-resistant) was evaluated by confocal microscopy. The loading and release behaviors of doxorubicin from F3-conjugated NPs were also evaluated.

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Scheme 1. (a) Synthesis of Alkyne-F3 Peptide; (b) Synthesis of F3-Conjugated co(CEA-AAm) NPs via Carbodiimide Chemistry and Click Chemistry

MATERIALS AND METHODS

Materials. Acrylamide (AAm), 2-carboxyethyl acrylate (CEA), 3(acryloyloxy)-2-hydroxypropyl methacrylate (AHM), ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED), sodium dioctyl sulfosuccinate (AOT), Brij 30, dimethyl sulfoxide (DMSO), acetonitrile, L-ascorpic acid, copper(II) sulfate, phosphatebuffered saline tablet (PBS), and Tris(2-carboxyethyl)phosphine (TCEP) were purchased from Sigma-Aldrich. Ethanol (95%) and hexane were purchased from Fisher Scientific. 1-Ethyl-3-(3(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) were purchased from Thermo Scientific. Human ovarian adenocarcinoma cell line NCI/ADR-RES was purchased from the U.S.A. National Cancer Institute. The 9L rat gliosarcoma and human breast adenocarcinoma cell line MCF-7 were obtained from American Type Culture Collection (Manassas, VA, U.S.A.). Roswell Park Memorial Institute medium (RPMI-1640), 4′,6diamidino-2-phenylindole (DAPI), Gibco 0.05% Trypsin-EDTA, B

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and washed with fresh DPBS buffer three times. The fluorescence signal of the NPs was analyzed with an Olympus DSU (disk scanning unit) confocal microscope. Pixel intensity of the fluorescence picture was analyzed with the ImageJ program. A total of 40 cells per measurement were used to obtain the quantitative pixel intensity analysis data. Competitive Uptake Tests. 9L cells were preincubated with various concentrations of F3 peptide 1 (0, 2, 10, and 50 μM). After 30 min, AF488-labeled F3-NPs 9 (1.0 mg/mL) were added to the cells and the cells were incubated for an additional 2 h. After that, the cells were washed with DPBS buffer three times and incubated in colorless RPMI medium. The fluorescence intensity of F3-NPs 9 was studied with fluorescence microscopy and analyzed with the ImageJ program. Statistical Analysis. Results are presented as mean ± standard deviation from at least three separate experiments.

of the F3 peptide 1 and alkyne-F3 peptide 3 were analyzed with an Agilent Q-TOF HPLC/MS system. The purity of F3 peptide 1 and alkyne-F3 peptide 3 was assessed using ultraperformance liquid chromatography (UPLC), which was carried out on a Waters Acquity Peptide Mapping System equipped with a photodiode array detector. The peptide solution (1 mg/mL) was run on an Acquity BEH C4 column (100 × 2.1 mm, 1.7 μm). Gradient elution was a mixture of water/acetonitrile, in which the ratio of water/acetonitrile ranged from 99:1 (v/v) to 20:80. Flow rate was maintained at 0.208 mL/min. Trifluoroacetic acid (TFA) at 0.14 wt % in water as well as in acetonitrile was used as a counterion. The column temperature was maintained at 35 °C. Synthesis of Azide-NPs 6. co(CEA-AAm) NPs 4 (10 mg) were dissolved in PBS buffer at 10 mg/mL, followed by EDC (6.3 mg) and Sulfo-NHS (7.3 mg; Scheme 1b). 1-Azido-3-aminopropane 5 (3.4 mg, 50 mg/mL in acetonitrile) was added into the NP solution dropwise. The mixed solution was kept stirring for 4 h. The azide-NPs (6 in Scheme 1) were purified by a centrifuge filter (100 k Da, 4000 g), using PBS buffer (4×) and collected at 10 mg/mL. The purified sample was stored at 4 °C prior to use. Synthesis of F3-Conjugated NPs 8. Azide-NPs 6 (10 mg, 10 mg/mL in PBS), alkyne-F3 3 (1 mg, 10 mg/mL in H2O), CuSO4 (20 μL, 10 mM in water), and L-ascorbic acid (10 μL, 50 mM in water) were mixed together and stirred at room temperature overnight (Scheme 1b). The mixture was purified using a centrifuge filter (100 kDa, 4000 g, PBS 5×, DI water 4×). The F3-NP 8 solution was adjusted to 10 mg/mL and stored at 4 °C. The amount of F3 peptide on the F3-conjugated NP 8 was analyzed by quantitative amino acid analysis (QAAA, Protein Chemistry Lab, Texas A&M University). Synthesis of AF488-Labeled NPs 7. In order to track NPs, the NPs were labeled with alkyne-functionalized fluorescence dye AF488 via click chemistry. AF488 (20 μg, 10 mg/mL in DMSO), CuSO4 (20 μL, 10 mM in water), and L-ascorbic acid (10 μL, 50 mM in water) were added into an azide-NP solution 6 (10 mg, 10 mg/mL in PBS) and stirred for 4 h. The mixture was purified by a centrifuge filter (100 kDa, 4000 g, PBS 4×). AF488-labeled NPs 7 (10 mg/mL) were stored at 4 °C prior to use. Characterization of NPs. The size and zeta potential of the co(CEA-AAm) NPs (4, Scheme 1) before and after modification were measured using a Delsa Nano (Beckman Coulter). The FTIR spectrum of the NPs was analyzed with a Spectrum BX FTIR (PerkinElmer). 1H NMR spectra of the NPs were analyzed with a Varian Inova 500 MHz spectrometer. Loading of Dox into co(CEA-AAm) NPs. Dox was loaded into the co(CEA-AAm) NPs 4 or F3-NPs 8 via postloading. The particle solution in DI water (10 mg, 10 mg/mL) and Dox solution in DI water (1 mg, 10 mg/mL) were mixed together and kept stirring overnight. The Dox-loaded NP solution was purified by a centrifuge filter (100 kDa, 4000 g, DI water 3×) to remove the unloaded drug. Release of Dox from co(CEA-AAm) NPs. Dox-loaded NP stock solution (0.2 mL, 10 mg/mL) was diluted 50× with PBS buffer. After that, this drug-loaded NP solution (10 mL, 0.2 mg/mL) was incubated in a water bath at 37 °C. After incubation for 0, 1, 3, 5, 8, and 24 h, 1 mL of Dox-loaded NP solution was taken out and transferred into a centrifuge filter (100 k Da). The NP solution was centrifuged at 4000 g for 15 min at room temperature and the filtrate was collected for UV-vis analysis. In view of the easy degradation of free Dox in PBS buffer, a calibration showing the degradation of free Dox was performed. Cell Culture and Confocal Microscopy Imaging. The human breast adenocarcinoma cell line MCF-7, rat gliosarcoma cell line 9L, and human ovarian adenocarcinoma cell line NCI/ADR-RES were cultivated in RPMI 1640 medium with 10% HI-FBS and 1% PSG. The cells were cultivated on an eight-well chambered cover glass system (Nunc, Lab-Tek) for 2 days. After that, the cells were incubated with AF488-labeled NPs 7 and 9 (1.0 mg/mL in PBS buffer) for 2 h. After incubation, unbound NPs were removed by rinsing with fresh DPBS buffer three times. The cells were fixed with paraformaldehyde solution (4% in DI water) for 15 min and washed with fresh DPBS buffer three times. Then the cells were stained with DAPI for 5 min

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RESULTS AND DISCUSSION Synthesis and Characterization of co(CEA-AAm) NPs 4. The co(CEA-AAm) NPs 4 were prepared by reverse microemulsion polymerization.19 The dynamic light scattering (DLS) analysis showed that the hydrodynamic size of NPs was 57.5 ± 0.1 nm in PBS buffer (Table 1), indicating the successful Table 1. Size and Zeta Potential of co(CEA-AAm) NPs 4, Azide-NPs 6, and F3-NPs 8 from DLS

size in PBS (nm) zeta potential (mV)

co(CEA-AAm) NPs 4

azide-NPs 6

F3-NPs 8

57.5 ± 0.1 −61.6 ± 3.2

45.5 ± 0.7 −53 ± 5.1

48.5 ± 1.6 −50.5 ± 5.4

synthesis of the NPs. The zeta potential of these hydrogel NPs was also analyzed, which was −61.6 ± 3.2 mV in DI water (Table 1). The negative charge on the NP surface is related with the carboxyl group of CEA (8 mol % in NPs). We also compared the 1H NMR spectra of co(CEA-AAm) NPs 4 (8 mol %) and PAAm NPs (no CEA). Compared to that of PAAm NPs, a strong peak at 4.25 ppm appeared on the 1H NMR spectrum of co(CEA-AAm) NPs 4 (Figure S1), which also demonstrates the incorporation of CEA into the NPs. Synthesis of Alkyne-F3 Peptide 3. To attach F3 peptide to azide-NPs 6 via click chemistry, F3 peptide has been modified with an alkyne linker. We used a modified F3 peptide, which has an extra Cys at the terminal. The alkyne linker 2 containing a maleimide group was attached to the Cys of F3 peptide 1 through maleimide−thiol coupling (Scheme 1a). Since only one Cys exists in the F3 peptide 1, no isomer would be generated in this synthesis, making the following purification very easy. The mass spectrometry of the alkyne-F3 peptide 3 showed a major peak at 3669.0 m/z (Figure 1b). It is 135.0 larger than that of the F3 peptide 1 (3534.0), which matches the attachment of an alkyne linker. In addition, UPLC results (Figure 2) demonstrated a single peak of alkyne-F3 3, which is separated from that of the F3 peptide, indicating the high purity and successful synthesis of the alkyne-F3 3. Click Conjugation of Alkyne-F3 Peptide onto co(CEAAAm) NPs. The typical method for the attachment of peptides onto NPs is using amine-acid coupling reaction. Due to multiple functional groups in the peptides, this method suffers from side reactions and inefficient coupling yields, making the purification a challenging task. The azide−alkyne click chemistry is highly specific and efficient, which is ideal for NPs surface modifications. The co(CEA-AAm) NPs 4 was first modified with an azide linker, 1-azido-3-aminopropane, through coupling reaction. The successful attachment of the azide linker C

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Figure 1. Deconvoluted mass spectra of F3 peptide 1 (top) and alkyne-F3 peptide 3 (bottom).

confirmed the attachment of the azide linker onto the co(CEAAAm) NPs 4. The successful preparation of azide-NPs 6 was also supported by the 1H NMR results. After conjugation with the azide linker, two new peaks appeared at 3.2 and 3.3 ppm (Figure S1). We conjugated the azide-NPs 6 with the alkyne-F3 peptide 3 via click chemistry. The presence of the F3 peptide on the NPs was confirmed by QAAA, which showed that we have 17 μg of the F3 peptide per mg of NP. We further calculated the number of peptides per nanoparticle, assuming that the density of the hydrogel NPs is approximately 1 g/cm3. The volume of the hydrogel NPs is calculated from its diameter (17 nm) from the SEM image.19 Based on this information, we determined the average molecular weight of our hydrogen NPs to be about 1.5 × 106, resulting in about 7 peptides/NP. Additionally, the azide-NPs were labeled with AF488 (7 and 9 in Scheme 1) via click chemistry for intracellular tracking by confocal microscopy. DLS studies on the co(CEA-AAm) NPs 4, azide-NPs 6, and F3-NPs 8 (Table 1 and Figure S2) demonstrated that the attachment of both the azide linker and the F3 peptide did not significantly change the sizes of NPs (57.5 ± 0.1 nm for co(CEA-AAm) NPs 4, 45.5 ± 0.7 nm for azide-NPs 6, and 48.5 ± 1.6 nm for F3-NPs 8). The zeta potentials of the azide-NPs 6 and F3-NPs 8 were −53 ± 5.1 mV and −50.5 ± 5.4 mV, respectively. Compared to the unmodified NPs 4 (−61.6 ± 3.2 mV), the less negative charge of the modified NPs can be attributed to (i) partial carboxyl groups on the NP surface were used for the conjugation of azide linkers and (ii) the attachment of the positively charged F3 peptide.26 Uptake of F3-NPs 8 and Azide-NPs 6 by Tumor Cells: 9L, MCF-7, and NCI/ADR-RES. One important property of a targeted drug delivery vehicle is its ability to selectively

Figure 2. UPLC chromatograms of F3 1 and alkyne-F3 3. Chromatograms were adjusted using solvent control to reduce background.

was confirmed by FTIR (Figure 3) and 1H NMR (Figure S1). Compared to that of co(CEA-AAm) NPs 4, a new peak at 2100 cm−1 was found in the FTIR spectrum of azide-NPs 6, which is the absorption of the azide group (Figure 3).33,34 This result

Figure 3. FTIR spectra of co(CEA-AAm) NPs 4 and azide-NPs 6. D

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between the nucleus and the cell surface in proliferating cells, while in resting cells nucleolin is primarily expressed at the nucleus.25 Such overexpression of nucleolin was found on the surface of numerous kinds of tumor cells, for example, human breast carcinoma cells (MDA-MB-231, MDA-MB-435), human carcinoma (LNCaP, HeLa, G401), and leukemia (Jurkat, HuT 78, CEM) cell lines.37 These results suggest that the F3-NPs 9 may bypass the P-glycoprotein pathway (drug efflux pump), demonstrating their potential as a targeted drug delivery vehicle for drug-resistant cells. The intracellular signals of azide-NPs 7 and F3-NPs 9 were analyzed quantitatively with ImageJ (Table 2). The results

accumulate inside tumor cells. Confocal microscopy was used to evaluate the uptake of AF488-modified NPs by tumor cells. The glioma cell line 9L has high surface expression of nucleolin, while the human breast cancer cell line MCF has a lower expression of nucleolin.35 In order to study the potential of F3NPs to deliver drugs to drug-resistant cells, the human ovarian adenocarcinoma cell line, NCI/ADR-RES, which is well-known to be Dox-resistant, was also included in this study.35,36 Figure 4 shows the confocal microscopy images of 9L, MCF7, and NCI/ADR-RES cells after incubation with azide-NPs 7

Table 2. Pixel Intensity Analyses of Confocal Microscopy Images of 9L, MCF-7, and NCI/ADR-RES Cells after Incubation with Azide-NPs 7 or F3-NPs 9 for 2 ha

a

sample

9L

MCF-7

NCI/ADR-RES

azide-NPs 7 F3-NPs 9

0.9 ± 0.4 17.7 ± 5.7

0.5 ± 0.2 5.6 ± 2.3

0.4 ± 0.2 11.9 ± 3.4

NP concentration: 1 mg/mL. NPs were labeled with Fluor 488.

showed that the signals of F3-NPs 9 were 20× higher in 9L cells, 10× higher in MCF-7 cells, and 15× higher in NCI/ADRRES cells compared to the corresponding signals of the nontargeted azide-NPs 7. This means an enhanced cell uptake by an order of magnitude or more, including the multi-drugresistant cells. Generally, the uptake of anionic NPs by tumor cells is challenging. Due to electrostatic repulsion, presumably, anionic NPs cannot easily penetrate the negatively charged cell membrane. However, targeting ligands can improve the intracellular uptake of anionic NPs. It is reported that the attachment of folate improved the uptake of negatively charged poly(acrylic acid)-iron oxide NPs by the folate receptor overexpressing A549 lung cancer cells.31 Our findings here provide another avenue to improve on the uptake of anionic NPs by tumor cells. Uptake of F3-NPs via Receptor-Mediated Endocytosis and Competition Test. To confirm that the increased uptake of F3-NPs 9 is related with nucleolin-mediated endocytosis, we verified its cellular binding by a competition assay in 9L cells with F3 peptide 1. After incubation with varying concentrations of F3 peptide 1 for 30 min, the cells were treated with AF488labeled F3-NPs 9 (1.0 mg/mL) for another 2 h. The results show that the fluorescence signal from AF488-labeled F3-NPs 9 decreased with increasing concentration of F3 peptide (Figure 5), and 50 μM F3 peptide 1 solution can completely block the uptake of the NPs. These results apparently confirm our long held notion that the uptake of F3-NPs 9 by 9L cells is mainly via nucleolin-mediated endocytosis.6,28,38 Loading and Release of Dox from F3-NPs 8. The hydrophobic chemotherapy drug Dox was loaded onto both unmodified co(CEA-AAm) NPs 4 and F3-NPs 8 effectively through postloading, with loading ratios of 8.7 ± 0.2 wt % and 8.5 ± 0.2 wt %, respectively. We also studied the releasing kinetics of Dox from both co(CEA-AAm) NPs 4 and F3-NPs 8 in PBS buffer. The results show that around 42% of Dox was released from the NPs in 24 h, which is similar as that from unmodified NPs (39%; Figure 6), suggesting that the attachment of F3 peptide does not affect the release behavior of Dox from the co(CEA-AAm) NPs. These results demonstrate the effective loading and satisfactory release of

Figure 4. Confocal microscopy images of 9L, MCF-7, and NCI/ADRRES cells after incubation with F3-NPs 9 and azide-NPs 7 for 2 h, respectively. NPs were labeled with Fluor 488 (green), while the cell nucleus was stained with DAPI (blue). NP concentration: 1.0 mg/mL; scale bar: 25 μm.

and F3-NPs 9. There was a minimal nonspecific internalization of the nontargeted azide-NPs 7 by 9L, MCF-7, and NCI/ADRRES cells. The attachment of the targeting F3 peptide 1 greatly enhanced the uptake of NPs by the nucleolin overexpressing 9L cells. Most of the fluorescence-labeled F3-NPs 9 were located in the perinuclear region of the 9L cells, demonstrating their successful internalization into the cytoplasm. Notably, the co(CEA-AAm) NPs cannot accumulate inside the nucleus because of their large size.35 Notably, the attachment of F3 peptide can prevent the hydrogel NPs from being trapped in the acidic lysosome.28 The F3-NPs 9 also show improved uptake by the MCF-7 cells, compared to the azide-NPs 7. Most of the F3-NP signal was found in the membrane of MCF-7 cells, suggesting incomplete internalization into the cytoplasm. These results are consistent with the hypothesis that the uptake of F3-NPs 9 is nucleolin-mediated. More importantly, the surface decoration with the F3 peptide 1 also significantly enhanced the uptake of F3-NPs 9 by the drug-resistant NCI/ ADR-RES cells, where the F3-NPs 9 were found to be located in both the cell membrane and the cytoplasm. To the best of our knowledge, this is the first observation that the F3 peptide 1 improves the uptake of NPs by a drug-resistant cell line. This improved uptake may again be related to the overexpression of nucleolin on the NCI/ADR-RES cell surface. The expression of nucleolin on the surface of NCI/ADR-RES cells is highly probable. This is because nucleolin behaves as a shuttle protein E

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AUTHOR INFORMATION

Corresponding Author

*Fax: 1-731 936 2778. Tel.: 1-734 764 7541. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grant NIH/NCI 1R21NS084275. We thank Dr. Martin Philbert and Kristen Russ for allowing us to use the confocal microscope in their lab. We thank Leshern Karamchand for his help with the confocal microscopy and also thank Leshern Karamchand and Aniruddha Ray for discussions on the intracellular studies of hydrogel NPs. We also thank James Windak and Paul Lennon for their help with mass spectrometry and FTIR.

Figure 5. Blocking effect of free F3 peptide on nucleolin sites, preventing cell-incorporation of F3-NPs 9. The cells were preincubated with varying concentration of F3 peptide for 30 min; after that, the cells were incubated with F3-NPs for 2 h. NPs were labeled with Fluor 488. The results showed that, with increasing concentration of F3 peptide, the binding of F3-NPs to 9L cells was inhibited significantly. NP concentration: 1.0 mg/mL.

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Figure 6. Release profile of Dox from co(CEA-AAm) NPs 4 and F3NPs 8 in PBS buffer. NP concentration: 0.2 mg/mL; temperature: 37 °C; loading of Dox/NPs: 8.7 ± 0.2 wt %; loading of Dox/F3-NPs: 8.5 ± 0.2 wt %.

Dox from F3-NPs, suggesting its potential as a drug delivery vehicle.

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CONCLUSIONS The development of a click chemistry synthesis of F3 peptideconjugated co(CEA-AAm) NPs, for the targeted delivery of Dox into tumor cells, was successful. The nucleolin-targeting F3 peptide was straightforwardly conjugated onto co(CEA-AAm) NPs, through the copper(I) catalyzed click reaction. This modular method is efficient and applicable to other peptide− nanoparticle conjugations. The attached F3 peptide dramatically enhances the intracellular uptake of co(CEA-AAm) NPs by nucleolin-overexpressing cancer cell lines. This enhanced uptake of F3-conjugated NPs is believed to be related to the nucleolin-mediated endocytosis. Compared to the nontargeted NPs, F3-conjugated NPs show a substantially higher in vitro uptake by three examples of tumor cells, including a drugresistant cell line. They also have the potential of improved therapeutic index in vivo.

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REFERENCES

ASSOCIATED CONTENT

S Supporting Information *

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H NMR spectra of the NPs, size distribution of the NPs, confocal microscopy images of 9L cells after incubation with Dox-F3-NPs, and viability of 9L cells after incubation with DoxF3-NPs. This material is available free of charge via the Internet at http://pubs.acs.org. F

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