Article pubs.acs.org/Biomac
A Robust Graft-to Strategy To Form Multifunctional and Stealth Zwitterionic Polymer-Coated Mesoporous Silica Nanoparticles Yongheng Zhu,†,‡,§,⊥ Harihara S. Sundaram,†,⊥ Sijun Liu,∥ Lei Zhang,† Xuewei Xu,† Qiuming Yu,† Jiaqiang Xu,*,§ and Shaoyi Jiang*,†,∥ †
Department of Chemical Engineering, University of Washington, Seattle, Washington 98195, United States School of Urban Development and Environmental Engineering, Shanghai Second Polytechnic University, Shanghai 201209, China § Department of Chemistry, College of Science, Shanghai University, Shanghai 200444, China ∥ Department of Bioengineering, University of Washington, Seattle, Washington 98195, United States ‡
S Supporting Information *
ABSTRACT: Mesoporous silica nanoparticles (MSNs) are a new class of carrier materials promising for drug/gene delivery and many other important applications. Stealth coatings are necessary to maintain their stability in complex media. Herein, a biomimetic polymer conjugate containing one ultralow fouling poly(carboxybetaine) (pCBMA) chain and one surfaceadhesive catechol (DOPA) residue group was efficiently grafted to the outer surface of SBA-15 type MSNs using a convenient and robust method. The cytotoxicity of SBA-15-DOPA-pCBMAs was evaluated by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results showed no significant decrease in cell viability at the tested concentration range. Macrophage cell uptake studies revealed that the uptake ratios of SBA15-DOPA-pCBMAs were much lower than that of parent MSNs. Furthermore, inductively coupled plasma mass spectrometry (ICP-MS) analysis results showed that after SBA-15-DOPA-pCBMAs were conjugated with a targeting cyclo-[Arg-Gly-Asp-D-Tyr-Lys] (cRGD) peptide, uptake by bovine aortic endothelial cells (BAECs) was notably increased. Results indicated that cRGDfunctionalized MSNs were able to selectively interact with cells expressing αvβ3 integrin. Thus, MSNs with DOPA-pCBMAs are promising as stealth multifunctional biocarriers for targeted drug delivery or diagnostics.
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INTRODUCTION There has been a rapid increase in research on ordered mesoporous materials during the past decades.1 Their applications have become highly attractive due to their unique features such as high specific surface area, large pore volume, tunable pore structure, facile multifunctionalization, and excellent physicochemical stability.2−5 In recent years, mesoporous silica nanoparticles (MSNs) have been increasingly exploited for numerous biomedical and technological applications.6 Their utility in these fields ranges from targeted drug and gene delivery7−11 to carrier vehicles for diagnostics.12,13 Despite all the aforementioned examples of the utility of silica, one major obstacle to these applications is nonspecific protein adsorption, which can result in cellular uptake, nanoparticle aggregation, immunological responses, and other serious problems for in vivo applications.14 This lack of a versatile and effective nonfouling material is thus a crucial obstacle for their biomedical applications.15−17 To address this issue, neutral and hydrophilic polymers are often used to reduce nonspecific protein adsorption. Poly(ethylene glycol) (PEG) is the most widely studied polymer for this purpose.18,19 However, PEG is subjected to oxidation in the presence of oxygen and transition metal ions.20 In addition to © 2014 American Chemical Society
fouling resistance, many biomedical applications require a functionalizable surface. This is necessary to immobilize biorecognition elements for targeting specific disease areas or selectively interacting with cells or biomolecules.21 Zwitterionic poly(carboxybetaine) (pCBMA) is attractive for biomedical applications due to its dual capabilities for fouling resistance and functionalization.22−24 The fouling level of pCBMA has been shown to be less than 5 ng/cm2 in the presence of 100% blood plasma and serum.25 Furthermore, unlike other nonfouling materials, pCBMA has abundant functional groups (COOH) that can be employed for the effective and convenient conjugation of biorecognition elements via simple 1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) coupling chemistry under mild aqueous conditions.26 Previously, to coat a zwitterionic polymer onto a surface, the “graft-from” method via atom transfer radical polymerization (ATRP) has achieved surface coatings with excellent ultralow fouling properties.27 However, ATRP requires surface-grafted Received: February 13, 2014 Revised: March 23, 2014 Published: March 26, 2014 1845
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Scheme 1. Scheme of the Synthesis Process of SBA-15-DOPA-pCBMAa
a
Starting with the mesoporous silica nanoparticles, the DOPA-pCBMA polymer is grafted onto their surfaces; SBA-15-DOPA-pCBMA is able to prevent nonspecific protein adsorption and can be activated with NHS groups to react with desired biomolecules for a wide range of biomedical applications.
endothelial cells (BAECs) after a targeting cyclo-[Arg-Gly-Asp(cRGD) peptide was conjugated to SBA-15-DOPApCBMAs. Results indicated that cRGD-functionalized MSNs were able to selectively interact with cells expressing αvβ3 integrin, for which RGD is a ligand.30
initiators/catalysts and oxygen-free conditions. For practical applications, it is desirable to use a simpler and more convenient method to attach zwitterionic polymers onto a surface, for example, by simply treating the surface with a polymer solution using a “graft-to” method,28 while the “graftto” process needs optimization to get higher graft densities that usually depends on the molecular weight of the polymers.28 Polymers used in the “graft-to” method have surface-adhesive moieties, enabling the adhesion of nonfouling polymers onto a surface. Recently, 3,4-dihydroxyphenyl-L-alanine (DOPA) inspired by the adhesive proteins found in mussels has been shown to have high affinity toward many types of surfaces, including noble metals, oxides, polymers, semiconductors, and ceramics.29 In this work, using a polymer conjugate containing one ultralow fouling pCBMA chain and one surface-adhesive DOPA residue group, multifunctional SBA-15 type mesoporous silica nanoparticles with abundant functionalizable groups were prepared via a convenient and robust “graft-to” approach as shown in Scheme 1. The novel organic−inorganic hybrid materials have been characterized by means of X-ray diffraction, nitrogen adsorption/desorption, Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and thermogravimetric measurements, showing the ordered mesoporous structures of SBA-15 and the successful anchoring of DOPA-pCBMA to SBA-15. The cytotoxicity of SBA-15-DOPA-pCBMAs was evaluated by MTT assays. Results showed no significant change in cell viability at the tested concentrations. Macrophage uptake studies revealed that the uptake ratios of SBA-15-DOPA-pCBMAs were much lower than that of parent MSNs. Furthermore, results from inductively coupled plasma mass spectrometry (ICP-MS) analysis showed notable increase of uptake by bovine aortic
D-Tyr-Lys]
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EXPERIMENTAL SECTION
Materials. Chemicals and solvents of analytical grade were purchased and used as received. Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (EO20-PO70-EO20) P123, hydrochloric acid (HCl), tetraethyl orthosilicate (TEOS), sodium hydroxide (99.998%), L-3,4-dihydroxyphenylalanine (DOPA), 2-bromoisobutyric acid, dicyclohexylcarbodiimide (DCC), copper(I) bromide (99.999%), 2,2′-bipyridine (BPY 99%), tetrahydrofuran (THF, HPLC grade), phosphate-buffered saline (PBS, pH 7.4, 0.15 M, 138 mM NaCl, 2.7 mM KCl), NHS, EDC, and hydrofluoric acid (HF, ⩾49%) were purchased from Sigma-Aldrich. Tetrabutylammonium fluoride (TBAF, 1 M solution in THF containing ca. 5% water), 1,3-diamino-2hydropropane, and tert-butyl chlorodimethylsilane (TBDMS, 98%) were purchased from Acros. Tris, crystallized free base (molecular biology grade), was purchased from Fisher Scientific. Cyclo-[Arg-GlyAsp-D-Tyr-Lys] (cRGD) was purchased from Peptides International. The carboxybetaine methacrylate (CBMA) monomer was synthesized using methods reported previously.30 Methods. Synthesis of SBA-15 Nanoparticles. The uniform hexagonal prism SBA-15 nanoparticles were synthesized following procedures reported in literature.31,32 The synthetic procedures began with dissolving P123 (2.0 g) in HCl (75 mL, 2.0 M) at 45 °C with stirring rate (600 rpm). After a homogeneous solution formed, tetraethyl orthosilicate (TEOS) (4.3 g) was added dropwise into the reaction container at 45 °C for 24 h. Subsequently, the resulting mixture was hydrothermally aged for 24 h without stirring at 110 °C. The product was filtered and washed with distilled water, then dried at 100 °C overnight to obtain the mesoporous SBA-15 precursor. Pure 1846
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mesoporous SBA-15 was obtained by heating the precursor at 550 °C for 6 h so as to decompose the template. Synthesis of DOPA-pCBMA. The DOPA-Br initiator and zwitterionic polymers with DOPA chain terminals were prepared using the previously reported methods by our group.33 The synthesis of DOPA-pCBMA was illustrated in Supporting Information Scheme 1S. Initiator (7.52 mg, 0.05 mmol), BPY (44 mg, 0.29 mmol), CuBr (13.6 mg, 0.094 mmol), and CuBr2 (1.03 mg, 0.005 mmol) were placed into a three-necked flask. The system was degassed three times and filled with N2, and then 1 mL of DMF (degassed) was added under N2. The mixture was stirred for 20 min at room temperature. CBMA (1.0 g) dissolved in a mixture of H2O/DMF (2 mL/7 mL) was added into the reaction system. The polymerization continued for 10 h. The resulting polymer precipitate was collected by filtration and dissolved in H2O again. The polymer solution was dialyzed for 2 days with DI water, and white polymer powder (0.85 g, 85%) was obtained after removal of water. The deprotection reaction was performed in methanol using five times excess of TBAF. After deprotection the polymer was recovered by repeated precipitations in THF from methanol and dried. The molecular weight and molecular weight distribution of the polymer were measured with gel permeation chromatography (GPC). The number-average molecular weight (Mw) was 60.8 kDa using PEG standards while the polydispersity index (PDI) was 1.39. Grafting DOPA-pCBMA onto Mesoporous Silica Nanoparticles (SBA-15-DOPA-pCBMA). The deprotected polymer (20 mg) was dissolved in 10 mL of Tris (pH ∼ 8.5) solution in a 20 mL glass tube and stirred for 0.5 h. Then, 10 mg of as-prepared SBA-15 was added into the solution. Subsequently, the resultant mixture was stirred for another 2 h and then washed three times with DI water. Two concentrations of solutions were used for the coating of mesoporous silica nanoparticles SBA-15-DOPA-pCBMA-1 is 1 mg/mL and SBA15-DOPA-pCBMA-2 is 2 mg/mL. Protein Fouling Assays. One milligram pure each of SBA-15, SBA15-DOPA-pCBMA-1, and SBA-15-DOPA-pCBMA-2 MSNs was soaked in a PBS solution for 0.5 h, then all samples were incubated in 1.5 mL of 0.2 mg/mL FITC-labeled BSA protein solution. The mixture was stirred for 0.5 h and then washed three times by 1.5 mL of PBS. After that, all samples were gently washed three times again with DI water. Finally, these three samples were imaged with a confocal fluorescence microscope. Functionalization of SBA-15-DOPA-pCBMA Mesoporous Silica Nanoparticles with cRGD (MSNs-cRGD). Ten milligrams of SBA-15DOPA-pCBMA-1 MSNs was dispersed in 4 mL of DI water. Six milligrams of EDC and 1.0 mg of NHS were then added successively. The mixture was stirred for 0.5 h and then washed two times by DI water. After that, the nanoparticles were redispersed in 4 mL of DI water, and 0.10 mg of cRGD was added. The mixture was stirred for another 3 h at room temperature. The final product was washed three times with DI water. Cytotoxicity Assay. Cell viability of NIH/3T3 fibroblasts, RAW 264.7 macrophages, and BAEC was tested by using a Vybrant MTT Cell Proliferation Assay Kit (Molecular Probes). Cells were seeded in 96-well cell culture plates in 100 of μL medium supplemented with 10% serum under 5% CO2 at 37 °C to 90% confluence. On the day of the test, cells were washed with PBS and incubated with 100 μL of fresh medium containing SBA-15-DOPA-pCBMA-1MSNs at various silicon concentrations. After 24 h, cells were washed with PBS and incubated with 100 μL of medium and 10 μL of 12 mM MTT stock solution for another 4 h. Then, 85 μL of medium was removed and 50 μL of DMSO was added and incubated for 10 min. The absorbance at 540 nm was read with a 96-well plate reader (SpectraMax M5, Molecular Devices). Culture of Macrophage Cells and Uptake Quantification. RAW 264.7 cells were cultured in DMEM medium with 10% fetal calf serum (FBS) and 1% antibiotics in a 24-well plate. Prior to the test, cells were washed with PBS three times and then SBA-15 at concentrations of 20 μg/mL, SBA-15-DOPA-pCBMA-1 MSNs at concentrations of 20 μg/ mL (MSNs-1) and 50 μg/mL (MSNs-2) were added in culture. After 4 h of incubation at 37 °C and 5% CO2, cells were washed three times
with PBS and lysed with 1 mL of 50 mM HF solution.34 Intracellular silicon content was determined by ICP-MS. Cell Targeting to BAECs. BAECs were cultured in DMEM supplemented with 10% FBS and 1% antibiotics in a 6-well plate. First, cells were washed with PBS three times. Then SBA-15-DOPApCBMA-1 MSNs with or without RGD peptide in fresh culture media at silicon concentrations of 20 and 50 μg/mL were added. After 4 h of incubation, cells were washed three times with PBS and lysed with 1 mL of 50 mM HF solution. Intracellular silicon content was determined by ICP-MS. Characterization Methodologies. The structural characteristics of the resulting mesoporous materials were determined by powder X-ray diffraction (XRD) analysis using a D/max 2550 V diffractometer with Cu Kα radiation (λ = 1.54056 Å) (Rigaku, Tokyo, Japan). The nitrogen adsorption isotherms were measured at −196 °C by using a Micromeritics ASAP 2000 system. Morphologies and sizes of the samples were characterized by transmission electron microscopy (TEM, JEOL JEM-2010F working at 200 kV) and field emission scanning electron microscopy (FE-SEM, Hitachi S4800 operated at 15 kV). Surface functionalization was monitored by FTIR with a AVATRA370 FTIR instrument using KBr plates within 4500−400 cm−1. All XPS spectra were taken on a Surface Science Instruments Sprobe spectrometer. This instrument has a monochromatized Al Kα X-ray and a low energy electron flood gun for charge neutralization of nonconducting samples. The samples were spread onto double-sided tape and run as insulators. X-ray spot size for these acquisitions was approximately 800 μm. Pressure in the analytical chamber during spectral acquisition was less than 5 × 10−9 Torr. Pass energy for survey spectra (to calculate composition) was 150 eV; pass energy for highresolution spectra was 50 eV. The takeoff angle (the angle between the sample normal and the input axis of the energy analyzer) was ∼55° (55° takeoff angle = 50 Å sampling depth). Thermogravimetry (TG) and differential thermal analysis (DTA) were carried out between 20 and 800 °C in air using a Universal V2.6D TA Instruments. The concentration of all MSNs samples was determined by inductively coupled plasma atomic emission spectroscopy (ICP-MS, Elan DRC-e, PerkinElmer)
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RESULTS AND DISCUSSION The morphology and mesoporous structure of the parent SBA15 material were studied using TEM and FE-SEM. SBA-15 of the hexagonal prism with a uniform size of about 0.4 μm in width and about 0.4 μm in height was observed (Figure 1a−c).
Figure 1. TEM (a,c) and SEM (b,d) of the hexagonal prism parent SBA-15. 1847
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Figure 2. FT-IR spectra of pCBMA, parent SBA-15, SBA-15-DOPApCBMA-1, and SBA-15-DOPA-pCBMA-2.
Figure 4. (A) Picture corresponding to the parent SBA-15 (marked a), SBA-15-DOPA-pCBMA-1 (marked b), and SBA-15-DOPA-pCBMA-2 (marked c) after incubation in 0.2 mg/mL FITC-labeled BSA protein solution. (B−D)Corresponding confocal fluorescence microscopy images of these three samples using the same excitation light intensity and exposure time.
Figure 3. (a) XPS survey spectra and (b) representative XPS high resolution N 1s spectra of the parent SBA-15, SBA-15-DOPApCBMA-1, and SBA-15-DOPA-pCBMA-2.
Figure 1d clearly shows that the sample has uniform and ordered short channels. Supporting Information Figure S1 shows the N2 adsorption/desorption isotherms of SBA-15DOPA-pCBMA-1 and SBA-15-DOPA-pCBMA-2 together with the pure-silica SBA-15 sample. All three samples display a typeIV isotherm with H1 hysteresis and a sharp increase,35 indicating that SBA-15-DOPA-pCBMA samples possess similar uniform mesoporous and narrow pore-size distribution as that of pure SBA-15.36 On the other hand, both modified samples have a lower absorption volume and capillary condensation of N2 occurring over a slightly wider range. The BET surface area and pore volume of SBA-15-DOPA-pCBMA also decreased, as shown in Supporting Information Table S1. The physisorption data in Supporting Information Table S1 indicate that in the presence of a suitable concentration of DOPA-pCBMA the textural properties of SBA-15 were substantially maintained. Supporting Information Figure S2 shows the small-angle XRD patterns of parent SBA-15 and SBA-15-DOPA-pCBMA samples, respectively. It can be seen that the XRD patterns of all the samples exhibited three well-resolved XRD diffraction peaks associated with a 2D p6 mm hexagonal mesostructure in the region of 2θ = 0.5−2.51, confirming the highly ordered mesostructures of the samples.35 The incorporation of DOPA-
Figure 5. (a) Cytotoxicity of SBA-15-DOPA-pCBMA-1 MSNs to NIH/3T3 cells, RAW 264.7 macrophages, and BAECs by MTT assay (n = 3). (b) Macrophage cell uptake of uncoated SBA-15 MSNs at the Si concentration of 20 μg/mL and SBA-15-DOPA-pCBMA-1 MSNs at two Si concentrations: MSNs-1 (50 μg/mL) and MSNs-2 (20 μg/mL) (n = 3).
pCBMA groups causes the slight broadness of peaks and a decrease in diffraction peak intensity, indicating the slight 1848
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to that of pristine SBA-15 (Figure 3a). This indicates that the thickness of pCBMA is much less than the sampling depth of XPS, which is about 5−10 nm.38 For high-resolution C 1s spectra, pCBMA has a high binding energy peak at 289.1 eV, corresponding to the O−CO species of polymers (Supporting Information Figure S3). The main C 1s peak for pCBMA is attributed to the overlap of C−O (∼287.3 eV), C−N (∼286.3 eV), and C−C (∼285 eV). The above data confirm the successful attachment of pCBMA on the surface of MSNs. Finally, the thermogravimetric analysis (TGA) of the pure parent SBA-15 and SBA-15-DOPA-pCBMA samples reveals the pCBMA coating (Supporting Information Figure S4). The first weight loss that occurred for all the tested samples around 150 °C is related to the loss of physisorbed water in the mesoporous material surfaces.39 In the case of SBA-15DOPA-pCBMA-1, 12.5% weight loss is seen for the pCBMA groups linked to the surface of MSNs. The corresponding weight percent loss is 24.1% for SBA-15-DOPA-pCBMA-2 indicating more polymer is attached to the MSNs surface as higher concentration of DOPA-pCBMA (2 mg/mL) is used for the coating. To test the ability of SBA-15-DOPA-pCBMAs to resist nonspecific protein adsorption, a protein adsorption assay was carried out to probe the nonfouling property of these materials. Pure SBA-15 MSNs without pCBMA were used as controls. All samples were briefly rinsed with PBS and then incubated in 0.2 mg/mL fluorescein isothiocyanate labeled bovine-serum albumin (FITC-BSA) solution for 30 min. After incubation, all samples were gently washed again with PBS. Finally, the pictures of three samples were taken under a confocal fluorescence microscope. It can been seen in Figure 4A that pure SBA-15 MSNs without pCBMA changes from a white powder to a yellow powder after incubation, showing the obvious adsorption of FITC-BSA, while the other two samples are free from FITC-BSA adsorption. SBA-15-DOPA-pCBMA-1 showed fluorescence intensity similar to that of SBA-15-DOPApCBMA-2, indicating the effective reduction of protein adsorption for these samples. The cytotoxicity of SBA-15-DOPA-pCBMA MSNs was evaluated by an MTT assay.40 Results are shown in Figure 5a. No significant decrease in cell viability can be observed at the tested concentrations. Macrophage cell uptake was also studied. Mouse macrophage cell line, RAW 264.7, was used in this work. As shown in Figure 5b, SBA-15-DOPA-pCBMA MSNs at two different concentrations (MSNs-1 and MSNs-2) showed significantly lower uptake by macrophages compared to that of uncoated MSNs. This test further shows the advantage of the pCBMA coating. It is desirable for multifunctional MSNs to be functionalizable.41 We have shown previously that the abundant carboxyl groups in pCBMA can be efficiently and easily conjugated to biomolecules by conventional EDC/NHS chemistry.42 Furthermore, activated but unreacted NHS groups can be hydrolyzed back to carboxyl groups, ensuring that the excellent nonfouling properties of the coating are maintained in postfunctionalized surfaces (shown in Figure 6a).42 In this study, a cRGD peptide was adopted as the targeting ligand and conjugated to SBA-15-DOPA-pCBMA MSNs (shown in Figure 6a). BAECs were used to test the targeting efficiency of the MSNs by measuring intracellular silicon concentrations. As shown in Figure 6b, at two different MSNs concentrations MSNs-1-cRGD and MSNs-2-cRGD (20 and 50 μg/mL) tested nonfunctionalized SBA-15-DOPA-pCBMA MSNs have a very
Figure 6. (a) Schematic illustration of BAEC cell uptake of SBA-15DOPA-pCBMA MSNs with or without RGD peptide. (b) BAEC uptake of SBA-15-DOPA-pCBMA-1 MSNs with or without cRGD peptide at two different particle concentrations MSNs-1 and MSNs-2 (n = 3).
decrease in crystalline, but not the collapse of the pore structure of mesoporous materials. The FTIR spectra of the synthesized samples are shown in Figure 2. The principal bands observed in all the spectra correspond to the intense silicon−oxygen covalent bond vibrations in the range of 1100−1000 cm−1. The symmetric stretching vibration of Si−O−Si appears at ∼800 cm−1 while the bending mode appears at ∼467 cm−1, confirming the existence of SBA-15 framework.37 The FTIR spectra of the SBA-15-DOPA-pCBMA samples show the appearance of the characteristic absorption peaks associated with pCBMA. Bands resulting from C−H stretching in pCBMA can be observed between ∼2800 cm−1 and ∼3000 cm−1. The new peaks at ∼1730 cm−1 (carbonyl group), ∼ 1680 cm−1 (carboxyl group − O−CO), ∼ 1480 cm−1 (C−N), and ∼1380 cm−1 C−H stretching in pCBMA can be observed between ∼2800 cm−1 (CH3) are clearly observed, indicating the successful grafting of pCBMA chains to the surface of MSNs via this “graft-to” method. The surface composition variations of the pristine SBA-15 and SBA-15-DOPA-pCBMAs were characterized by XPS using the atomic detection of C, O, and N shown in Figure 3. Supporting Information Table S2 lists the detailed data from XPS scans on different surfaces. After the “graft-to” process, the composition of carbon increased, the composition of oxygen decreased, and a small amount of nitrogen (∼3.0%, N 1s,) appeared. High-resolution N 1s spectra exhibited two distinct peaks (N 1s, Pk01 ∼ 399.1 eV and Pk02 ∼ 402.0 eV). The lower binding energy peak was N bound to C as in C−N while the higher binding energy peak was N in an ammonium ion. From a survey scan of XPS, the spectrum of the SBA-15DOPA-pCBMA has sodium, silicon, and oxygen peaks similar 1849
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low uptake level, similar to macrophage studies. In contrast, cRGD-functionalized particles, MSNs-1-cRGD and MSNs-2cRGD, showed much higher uptake levels. These results demonstrate the successful conjugation of cRGD with the nanoparticles and the notable active targeting efficacy of MSNscRGD after conjugation with cRGD targeting ligand.
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CONCLUSIONS In summary, we developed a convenient and robust method to efficiently modify MSNs, using a biomimetic polymer containing one ultralow fouling pCBMA chain and one surface-adhesive catechol residue group. The uniqueness of dual-functional pCBMA (i.e., ultralow fouling and functionalizable) makes this zwitterionic biopolymer attractive as nanoparticle coatings for targeted drug delivery and diagnostics applications. Macrophage uptake studies revealed that the uptake ratios of SBA-15-DOPA-pCBMA were much lower than that of parent MSNs. Furthermore, biorecognition elements can be conjugated to pCBMA via NHS/EDC chemistry such as SBA-15-DOPA-pCBMA MSNs conjugated with a targeting cRGD peptide. Results showed notably increased uptake by BAEC cells, indicating their potential as vectors for targeted therapy.
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ASSOCIATED CONTENT
S Supporting Information *
Characterization of SBA-15 and modified SBA-15: nitrogen adsorption isotherms, small-angle X-ray scattering, C 1s XPS spectra, TGA profile, and elemental surface composition. 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]. Tel.: +1 206 616 6509. *E-mail: [email protected]. Tel.: 86-021-66132701. Author Contributions ⊥
Y.Z. and H.S.S. contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The research is supported by the National Science Foundation (CMMI 1301435), the National Nature Science Foundation of China (No. 61371021), and the Basic Research Foundation of Shanghai Science and Technology Committee (13NM1401300). Y.H.Z was supported by the Chinese Scholar Program.
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REFERENCES
(1) Beck, J. S.; Vartuli, J. C.; Roth, W. J.; Leonowicz, M. E.; Kresge, C. T.; Schmitt, K. D.; Chu, C. T. W.; Olson, D. H.; Sheppard, E. W.; Mccullen, S. B.; Higgins, J. B.; Schlenker, J. L. J. Am. Chem. Soc. 1992, 114, 10834−10843. (2) Cassiers, K.; Linssen, T.; Mathieu, M.; Benjelloun, M.; Schrijnemakers, K.; Van Der Voort, P.; Cool, P.; Vansant, E. F. Chem. Mater. 2002, 14, 2317−2324. (3) Hartmann, M. Chem. Mater. 2005, 17, 4577−4593. (4) Zhang, L.; Qiao, S. Z.; Jin, Y. G.; Yang, H. G.; Budihartono, S.; Stahr, F.; Yan, Z. F.; Wang, X. L.; Hao, Z. P.; Lu, G. Q. Adv. Funct. Mater. 2008, 18, 3203−3212. (5) Chen, C. S.; Chen, C. C.; Chen, C. T.; Kao, H. M. Chem. Commun. 2011, 47, 2288−2290. 1850
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(41) Meng, Q. T.; Zhang, X. L.; He, C.; He, G. J.; Zhou, P.; Duan, C. Y. Adv. Funct. Mater. 2010, 20, 1903−1909. (42) Vaisocherova, H.; Wang, Y.; Zhang, Z.; Cao, Z.; Cheng, G.; Piliarik, M.; Homola, J.; Jiang, S. Anal. Chem. 2008, 80, 7894−7901.
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A Robust Graft-to Strategy To Form Multifunctional and Stealth Zwitterionic Polymer-Coated Mesoporous Silica Nanoparticles Yongheng Zhu,†,‡,§,⊥ Harihara S. Sundaram,†,⊥ Sijun Liu,∥ Lei Zhang,† Xuewei Xu,† Qiuming Yu,† Jiaqiang Xu,*,§ and Shaoyi Jiang*,†,∥ †
Department of Chemical Engineering, University of Washington, Seattle, Washington 98195, United States School of Urban Development and Environmental Engineering, Shanghai Second Polytechnic University, Shanghai 201209, China § Department of Chemistry, College of Science, Shanghai University, Shanghai 200444, China ∥ Department of Bioengineering, University of Washington, Seattle, Washington 98195, United States ‡
S Supporting Information *
ABSTRACT: Mesoporous silica nanoparticles (MSNs) are a new class of carrier materials promising for drug/gene delivery and many other important applications. Stealth coatings are necessary to maintain their stability in complex media. Herein, a biomimetic polymer conjugate containing one ultralow fouling poly(carboxybetaine) (pCBMA) chain and one surfaceadhesive catechol (DOPA) residue group was efficiently grafted to the outer surface of SBA-15 type MSNs using a convenient and robust method. The cytotoxicity of SBA-15-DOPA-pCBMAs was evaluated by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results showed no significant decrease in cell viability at the tested concentration range. Macrophage cell uptake studies revealed that the uptake ratios of SBA15-DOPA-pCBMAs were much lower than that of parent MSNs. Furthermore, inductively coupled plasma mass spectrometry (ICP-MS) analysis results showed that after SBA-15-DOPA-pCBMAs were conjugated with a targeting cyclo-[Arg-Gly-Asp-D-Tyr-Lys] (cRGD) peptide, uptake by bovine aortic endothelial cells (BAECs) was notably increased. Results indicated that cRGDfunctionalized MSNs were able to selectively interact with cells expressing αvβ3 integrin. Thus, MSNs with DOPA-pCBMAs are promising as stealth multifunctional biocarriers for targeted drug delivery or diagnostics.
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INTRODUCTION There has been a rapid increase in research on ordered mesoporous materials during the past decades.1 Their applications have become highly attractive due to their unique features such as high specific surface area, large pore volume, tunable pore structure, facile multifunctionalization, and excellent physicochemical stability.2−5 In recent years, mesoporous silica nanoparticles (MSNs) have been increasingly exploited for numerous biomedical and technological applications.6 Their utility in these fields ranges from targeted drug and gene delivery7−11 to carrier vehicles for diagnostics.12,13 Despite all the aforementioned examples of the utility of silica, one major obstacle to these applications is nonspecific protein adsorption, which can result in cellular uptake, nanoparticle aggregation, immunological responses, and other serious problems for in vivo applications.14 This lack of a versatile and effective nonfouling material is thus a crucial obstacle for their biomedical applications.15−17 To address this issue, neutral and hydrophilic polymers are often used to reduce nonspecific protein adsorption. Poly(ethylene glycol) (PEG) is the most widely studied polymer for this purpose.18,19 However, PEG is subjected to oxidation in the presence of oxygen and transition metal ions.20 In addition to © 2014 American Chemical Society
fouling resistance, many biomedical applications require a functionalizable surface. This is necessary to immobilize biorecognition elements for targeting specific disease areas or selectively interacting with cells or biomolecules.21 Zwitterionic poly(carboxybetaine) (pCBMA) is attractive for biomedical applications due to its dual capabilities for fouling resistance and functionalization.22−24 The fouling level of pCBMA has been shown to be less than 5 ng/cm2 in the presence of 100% blood plasma and serum.25 Furthermore, unlike other nonfouling materials, pCBMA has abundant functional groups (COOH) that can be employed for the effective and convenient conjugation of biorecognition elements via simple 1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) coupling chemistry under mild aqueous conditions.26 Previously, to coat a zwitterionic polymer onto a surface, the “graft-from” method via atom transfer radical polymerization (ATRP) has achieved surface coatings with excellent ultralow fouling properties.27 However, ATRP requires surface-grafted Received: February 13, 2014 Revised: March 23, 2014 Published: March 26, 2014 1845
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Scheme 1. Scheme of the Synthesis Process of SBA-15-DOPA-pCBMAa
a
Starting with the mesoporous silica nanoparticles, the DOPA-pCBMA polymer is grafted onto their surfaces; SBA-15-DOPA-pCBMA is able to prevent nonspecific protein adsorption and can be activated with NHS groups to react with desired biomolecules for a wide range of biomedical applications.
endothelial cells (BAECs) after a targeting cyclo-[Arg-Gly-Asp(cRGD) peptide was conjugated to SBA-15-DOPApCBMAs. Results indicated that cRGD-functionalized MSNs were able to selectively interact with cells expressing αvβ3 integrin, for which RGD is a ligand.30
initiators/catalysts and oxygen-free conditions. For practical applications, it is desirable to use a simpler and more convenient method to attach zwitterionic polymers onto a surface, for example, by simply treating the surface with a polymer solution using a “graft-to” method,28 while the “graftto” process needs optimization to get higher graft densities that usually depends on the molecular weight of the polymers.28 Polymers used in the “graft-to” method have surface-adhesive moieties, enabling the adhesion of nonfouling polymers onto a surface. Recently, 3,4-dihydroxyphenyl-L-alanine (DOPA) inspired by the adhesive proteins found in mussels has been shown to have high affinity toward many types of surfaces, including noble metals, oxides, polymers, semiconductors, and ceramics.29 In this work, using a polymer conjugate containing one ultralow fouling pCBMA chain and one surface-adhesive DOPA residue group, multifunctional SBA-15 type mesoporous silica nanoparticles with abundant functionalizable groups were prepared via a convenient and robust “graft-to” approach as shown in Scheme 1. The novel organic−inorganic hybrid materials have been characterized by means of X-ray diffraction, nitrogen adsorption/desorption, Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and thermogravimetric measurements, showing the ordered mesoporous structures of SBA-15 and the successful anchoring of DOPA-pCBMA to SBA-15. The cytotoxicity of SBA-15-DOPA-pCBMAs was evaluated by MTT assays. Results showed no significant change in cell viability at the tested concentrations. Macrophage uptake studies revealed that the uptake ratios of SBA-15-DOPA-pCBMAs were much lower than that of parent MSNs. Furthermore, results from inductively coupled plasma mass spectrometry (ICP-MS) analysis showed notable increase of uptake by bovine aortic
D-Tyr-Lys]
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EXPERIMENTAL SECTION
Materials. Chemicals and solvents of analytical grade were purchased and used as received. Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (EO20-PO70-EO20) P123, hydrochloric acid (HCl), tetraethyl orthosilicate (TEOS), sodium hydroxide (99.998%), L-3,4-dihydroxyphenylalanine (DOPA), 2-bromoisobutyric acid, dicyclohexylcarbodiimide (DCC), copper(I) bromide (99.999%), 2,2′-bipyridine (BPY 99%), tetrahydrofuran (THF, HPLC grade), phosphate-buffered saline (PBS, pH 7.4, 0.15 M, 138 mM NaCl, 2.7 mM KCl), NHS, EDC, and hydrofluoric acid (HF, ⩾49%) were purchased from Sigma-Aldrich. Tetrabutylammonium fluoride (TBAF, 1 M solution in THF containing ca. 5% water), 1,3-diamino-2hydropropane, and tert-butyl chlorodimethylsilane (TBDMS, 98%) were purchased from Acros. Tris, crystallized free base (molecular biology grade), was purchased from Fisher Scientific. Cyclo-[Arg-GlyAsp-D-Tyr-Lys] (cRGD) was purchased from Peptides International. The carboxybetaine methacrylate (CBMA) monomer was synthesized using methods reported previously.30 Methods. Synthesis of SBA-15 Nanoparticles. The uniform hexagonal prism SBA-15 nanoparticles were synthesized following procedures reported in literature.31,32 The synthetic procedures began with dissolving P123 (2.0 g) in HCl (75 mL, 2.0 M) at 45 °C with stirring rate (600 rpm). After a homogeneous solution formed, tetraethyl orthosilicate (TEOS) (4.3 g) was added dropwise into the reaction container at 45 °C for 24 h. Subsequently, the resulting mixture was hydrothermally aged for 24 h without stirring at 110 °C. The product was filtered and washed with distilled water, then dried at 100 °C overnight to obtain the mesoporous SBA-15 precursor. Pure 1846
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mesoporous SBA-15 was obtained by heating the precursor at 550 °C for 6 h so as to decompose the template. Synthesis of DOPA-pCBMA. The DOPA-Br initiator and zwitterionic polymers with DOPA chain terminals were prepared using the previously reported methods by our group.33 The synthesis of DOPA-pCBMA was illustrated in Supporting Information Scheme 1S. Initiator (7.52 mg, 0.05 mmol), BPY (44 mg, 0.29 mmol), CuBr (13.6 mg, 0.094 mmol), and CuBr2 (1.03 mg, 0.005 mmol) were placed into a three-necked flask. The system was degassed three times and filled with N2, and then 1 mL of DMF (degassed) was added under N2. The mixture was stirred for 20 min at room temperature. CBMA (1.0 g) dissolved in a mixture of H2O/DMF (2 mL/7 mL) was added into the reaction system. The polymerization continued for 10 h. The resulting polymer precipitate was collected by filtration and dissolved in H2O again. The polymer solution was dialyzed for 2 days with DI water, and white polymer powder (0.85 g, 85%) was obtained after removal of water. The deprotection reaction was performed in methanol using five times excess of TBAF. After deprotection the polymer was recovered by repeated precipitations in THF from methanol and dried. The molecular weight and molecular weight distribution of the polymer were measured with gel permeation chromatography (GPC). The number-average molecular weight (Mw) was 60.8 kDa using PEG standards while the polydispersity index (PDI) was 1.39. Grafting DOPA-pCBMA onto Mesoporous Silica Nanoparticles (SBA-15-DOPA-pCBMA). The deprotected polymer (20 mg) was dissolved in 10 mL of Tris (pH ∼ 8.5) solution in a 20 mL glass tube and stirred for 0.5 h. Then, 10 mg of as-prepared SBA-15 was added into the solution. Subsequently, the resultant mixture was stirred for another 2 h and then washed three times with DI water. Two concentrations of solutions were used for the coating of mesoporous silica nanoparticles SBA-15-DOPA-pCBMA-1 is 1 mg/mL and SBA15-DOPA-pCBMA-2 is 2 mg/mL. Protein Fouling Assays. One milligram pure each of SBA-15, SBA15-DOPA-pCBMA-1, and SBA-15-DOPA-pCBMA-2 MSNs was soaked in a PBS solution for 0.5 h, then all samples were incubated in 1.5 mL of 0.2 mg/mL FITC-labeled BSA protein solution. The mixture was stirred for 0.5 h and then washed three times by 1.5 mL of PBS. After that, all samples were gently washed three times again with DI water. Finally, these three samples were imaged with a confocal fluorescence microscope. Functionalization of SBA-15-DOPA-pCBMA Mesoporous Silica Nanoparticles with cRGD (MSNs-cRGD). Ten milligrams of SBA-15DOPA-pCBMA-1 MSNs was dispersed in 4 mL of DI water. Six milligrams of EDC and 1.0 mg of NHS were then added successively. The mixture was stirred for 0.5 h and then washed two times by DI water. After that, the nanoparticles were redispersed in 4 mL of DI water, and 0.10 mg of cRGD was added. The mixture was stirred for another 3 h at room temperature. The final product was washed three times with DI water. Cytotoxicity Assay. Cell viability of NIH/3T3 fibroblasts, RAW 264.7 macrophages, and BAEC was tested by using a Vybrant MTT Cell Proliferation Assay Kit (Molecular Probes). Cells were seeded in 96-well cell culture plates in 100 of μL medium supplemented with 10% serum under 5% CO2 at 37 °C to 90% confluence. On the day of the test, cells were washed with PBS and incubated with 100 μL of fresh medium containing SBA-15-DOPA-pCBMA-1MSNs at various silicon concentrations. After 24 h, cells were washed with PBS and incubated with 100 μL of medium and 10 μL of 12 mM MTT stock solution for another 4 h. Then, 85 μL of medium was removed and 50 μL of DMSO was added and incubated for 10 min. The absorbance at 540 nm was read with a 96-well plate reader (SpectraMax M5, Molecular Devices). Culture of Macrophage Cells and Uptake Quantification. RAW 264.7 cells were cultured in DMEM medium with 10% fetal calf serum (FBS) and 1% antibiotics in a 24-well plate. Prior to the test, cells were washed with PBS three times and then SBA-15 at concentrations of 20 μg/mL, SBA-15-DOPA-pCBMA-1 MSNs at concentrations of 20 μg/ mL (MSNs-1) and 50 μg/mL (MSNs-2) were added in culture. After 4 h of incubation at 37 °C and 5% CO2, cells were washed three times
with PBS and lysed with 1 mL of 50 mM HF solution.34 Intracellular silicon content was determined by ICP-MS. Cell Targeting to BAECs. BAECs were cultured in DMEM supplemented with 10% FBS and 1% antibiotics in a 6-well plate. First, cells were washed with PBS three times. Then SBA-15-DOPApCBMA-1 MSNs with or without RGD peptide in fresh culture media at silicon concentrations of 20 and 50 μg/mL were added. After 4 h of incubation, cells were washed three times with PBS and lysed with 1 mL of 50 mM HF solution. Intracellular silicon content was determined by ICP-MS. Characterization Methodologies. The structural characteristics of the resulting mesoporous materials were determined by powder X-ray diffraction (XRD) analysis using a D/max 2550 V diffractometer with Cu Kα radiation (λ = 1.54056 Å) (Rigaku, Tokyo, Japan). The nitrogen adsorption isotherms were measured at −196 °C by using a Micromeritics ASAP 2000 system. Morphologies and sizes of the samples were characterized by transmission electron microscopy (TEM, JEOL JEM-2010F working at 200 kV) and field emission scanning electron microscopy (FE-SEM, Hitachi S4800 operated at 15 kV). Surface functionalization was monitored by FTIR with a AVATRA370 FTIR instrument using KBr plates within 4500−400 cm−1. All XPS spectra were taken on a Surface Science Instruments Sprobe spectrometer. This instrument has a monochromatized Al Kα X-ray and a low energy electron flood gun for charge neutralization of nonconducting samples. The samples were spread onto double-sided tape and run as insulators. X-ray spot size for these acquisitions was approximately 800 μm. Pressure in the analytical chamber during spectral acquisition was less than 5 × 10−9 Torr. Pass energy for survey spectra (to calculate composition) was 150 eV; pass energy for highresolution spectra was 50 eV. The takeoff angle (the angle between the sample normal and the input axis of the energy analyzer) was ∼55° (55° takeoff angle = 50 Å sampling depth). Thermogravimetry (TG) and differential thermal analysis (DTA) were carried out between 20 and 800 °C in air using a Universal V2.6D TA Instruments. The concentration of all MSNs samples was determined by inductively coupled plasma atomic emission spectroscopy (ICP-MS, Elan DRC-e, PerkinElmer)
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RESULTS AND DISCUSSION The morphology and mesoporous structure of the parent SBA15 material were studied using TEM and FE-SEM. SBA-15 of the hexagonal prism with a uniform size of about 0.4 μm in width and about 0.4 μm in height was observed (Figure 1a−c).
Figure 1. TEM (a,c) and SEM (b,d) of the hexagonal prism parent SBA-15. 1847
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Figure 2. FT-IR spectra of pCBMA, parent SBA-15, SBA-15-DOPApCBMA-1, and SBA-15-DOPA-pCBMA-2.
Figure 4. (A) Picture corresponding to the parent SBA-15 (marked a), SBA-15-DOPA-pCBMA-1 (marked b), and SBA-15-DOPA-pCBMA-2 (marked c) after incubation in 0.2 mg/mL FITC-labeled BSA protein solution. (B−D)Corresponding confocal fluorescence microscopy images of these three samples using the same excitation light intensity and exposure time.
Figure 3. (a) XPS survey spectra and (b) representative XPS high resolution N 1s spectra of the parent SBA-15, SBA-15-DOPApCBMA-1, and SBA-15-DOPA-pCBMA-2.
Figure 1d clearly shows that the sample has uniform and ordered short channels. Supporting Information Figure S1 shows the N2 adsorption/desorption isotherms of SBA-15DOPA-pCBMA-1 and SBA-15-DOPA-pCBMA-2 together with the pure-silica SBA-15 sample. All three samples display a typeIV isotherm with H1 hysteresis and a sharp increase,35 indicating that SBA-15-DOPA-pCBMA samples possess similar uniform mesoporous and narrow pore-size distribution as that of pure SBA-15.36 On the other hand, both modified samples have a lower absorption volume and capillary condensation of N2 occurring over a slightly wider range. The BET surface area and pore volume of SBA-15-DOPA-pCBMA also decreased, as shown in Supporting Information Table S1. The physisorption data in Supporting Information Table S1 indicate that in the presence of a suitable concentration of DOPA-pCBMA the textural properties of SBA-15 were substantially maintained. Supporting Information Figure S2 shows the small-angle XRD patterns of parent SBA-15 and SBA-15-DOPA-pCBMA samples, respectively. It can be seen that the XRD patterns of all the samples exhibited three well-resolved XRD diffraction peaks associated with a 2D p6 mm hexagonal mesostructure in the region of 2θ = 0.5−2.51, confirming the highly ordered mesostructures of the samples.35 The incorporation of DOPA-
Figure 5. (a) Cytotoxicity of SBA-15-DOPA-pCBMA-1 MSNs to NIH/3T3 cells, RAW 264.7 macrophages, and BAECs by MTT assay (n = 3). (b) Macrophage cell uptake of uncoated SBA-15 MSNs at the Si concentration of 20 μg/mL and SBA-15-DOPA-pCBMA-1 MSNs at two Si concentrations: MSNs-1 (50 μg/mL) and MSNs-2 (20 μg/mL) (n = 3).
pCBMA groups causes the slight broadness of peaks and a decrease in diffraction peak intensity, indicating the slight 1848
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to that of pristine SBA-15 (Figure 3a). This indicates that the thickness of pCBMA is much less than the sampling depth of XPS, which is about 5−10 nm.38 For high-resolution C 1s spectra, pCBMA has a high binding energy peak at 289.1 eV, corresponding to the O−CO species of polymers (Supporting Information Figure S3). The main C 1s peak for pCBMA is attributed to the overlap of C−O (∼287.3 eV), C−N (∼286.3 eV), and C−C (∼285 eV). The above data confirm the successful attachment of pCBMA on the surface of MSNs. Finally, the thermogravimetric analysis (TGA) of the pure parent SBA-15 and SBA-15-DOPA-pCBMA samples reveals the pCBMA coating (Supporting Information Figure S4). The first weight loss that occurred for all the tested samples around 150 °C is related to the loss of physisorbed water in the mesoporous material surfaces.39 In the case of SBA-15DOPA-pCBMA-1, 12.5% weight loss is seen for the pCBMA groups linked to the surface of MSNs. The corresponding weight percent loss is 24.1% for SBA-15-DOPA-pCBMA-2 indicating more polymer is attached to the MSNs surface as higher concentration of DOPA-pCBMA (2 mg/mL) is used for the coating. To test the ability of SBA-15-DOPA-pCBMAs to resist nonspecific protein adsorption, a protein adsorption assay was carried out to probe the nonfouling property of these materials. Pure SBA-15 MSNs without pCBMA were used as controls. All samples were briefly rinsed with PBS and then incubated in 0.2 mg/mL fluorescein isothiocyanate labeled bovine-serum albumin (FITC-BSA) solution for 30 min. After incubation, all samples were gently washed again with PBS. Finally, the pictures of three samples were taken under a confocal fluorescence microscope. It can been seen in Figure 4A that pure SBA-15 MSNs without pCBMA changes from a white powder to a yellow powder after incubation, showing the obvious adsorption of FITC-BSA, while the other two samples are free from FITC-BSA adsorption. SBA-15-DOPA-pCBMA-1 showed fluorescence intensity similar to that of SBA-15-DOPApCBMA-2, indicating the effective reduction of protein adsorption for these samples. The cytotoxicity of SBA-15-DOPA-pCBMA MSNs was evaluated by an MTT assay.40 Results are shown in Figure 5a. No significant decrease in cell viability can be observed at the tested concentrations. Macrophage cell uptake was also studied. Mouse macrophage cell line, RAW 264.7, was used in this work. As shown in Figure 5b, SBA-15-DOPA-pCBMA MSNs at two different concentrations (MSNs-1 and MSNs-2) showed significantly lower uptake by macrophages compared to that of uncoated MSNs. This test further shows the advantage of the pCBMA coating. It is desirable for multifunctional MSNs to be functionalizable.41 We have shown previously that the abundant carboxyl groups in pCBMA can be efficiently and easily conjugated to biomolecules by conventional EDC/NHS chemistry.42 Furthermore, activated but unreacted NHS groups can be hydrolyzed back to carboxyl groups, ensuring that the excellent nonfouling properties of the coating are maintained in postfunctionalized surfaces (shown in Figure 6a).42 In this study, a cRGD peptide was adopted as the targeting ligand and conjugated to SBA-15-DOPA-pCBMA MSNs (shown in Figure 6a). BAECs were used to test the targeting efficiency of the MSNs by measuring intracellular silicon concentrations. As shown in Figure 6b, at two different MSNs concentrations MSNs-1-cRGD and MSNs-2-cRGD (20 and 50 μg/mL) tested nonfunctionalized SBA-15-DOPA-pCBMA MSNs have a very
Figure 6. (a) Schematic illustration of BAEC cell uptake of SBA-15DOPA-pCBMA MSNs with or without RGD peptide. (b) BAEC uptake of SBA-15-DOPA-pCBMA-1 MSNs with or without cRGD peptide at two different particle concentrations MSNs-1 and MSNs-2 (n = 3).
decrease in crystalline, but not the collapse of the pore structure of mesoporous materials. The FTIR spectra of the synthesized samples are shown in Figure 2. The principal bands observed in all the spectra correspond to the intense silicon−oxygen covalent bond vibrations in the range of 1100−1000 cm−1. The symmetric stretching vibration of Si−O−Si appears at ∼800 cm−1 while the bending mode appears at ∼467 cm−1, confirming the existence of SBA-15 framework.37 The FTIR spectra of the SBA-15-DOPA-pCBMA samples show the appearance of the characteristic absorption peaks associated with pCBMA. Bands resulting from C−H stretching in pCBMA can be observed between ∼2800 cm−1 and ∼3000 cm−1. The new peaks at ∼1730 cm−1 (carbonyl group), ∼ 1680 cm−1 (carboxyl group − O−CO), ∼ 1480 cm−1 (C−N), and ∼1380 cm−1 C−H stretching in pCBMA can be observed between ∼2800 cm−1 (CH3) are clearly observed, indicating the successful grafting of pCBMA chains to the surface of MSNs via this “graft-to” method. The surface composition variations of the pristine SBA-15 and SBA-15-DOPA-pCBMAs were characterized by XPS using the atomic detection of C, O, and N shown in Figure 3. Supporting Information Table S2 lists the detailed data from XPS scans on different surfaces. After the “graft-to” process, the composition of carbon increased, the composition of oxygen decreased, and a small amount of nitrogen (∼3.0%, N 1s,) appeared. High-resolution N 1s spectra exhibited two distinct peaks (N 1s, Pk01 ∼ 399.1 eV and Pk02 ∼ 402.0 eV). The lower binding energy peak was N bound to C as in C−N while the higher binding energy peak was N in an ammonium ion. From a survey scan of XPS, the spectrum of the SBA-15DOPA-pCBMA has sodium, silicon, and oxygen peaks similar 1849
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low uptake level, similar to macrophage studies. In contrast, cRGD-functionalized particles, MSNs-1-cRGD and MSNs-2cRGD, showed much higher uptake levels. These results demonstrate the successful conjugation of cRGD with the nanoparticles and the notable active targeting efficacy of MSNscRGD after conjugation with cRGD targeting ligand.
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CONCLUSIONS In summary, we developed a convenient and robust method to efficiently modify MSNs, using a biomimetic polymer containing one ultralow fouling pCBMA chain and one surface-adhesive catechol residue group. The uniqueness of dual-functional pCBMA (i.e., ultralow fouling and functionalizable) makes this zwitterionic biopolymer attractive as nanoparticle coatings for targeted drug delivery and diagnostics applications. Macrophage uptake studies revealed that the uptake ratios of SBA-15-DOPA-pCBMA were much lower than that of parent MSNs. Furthermore, biorecognition elements can be conjugated to pCBMA via NHS/EDC chemistry such as SBA-15-DOPA-pCBMA MSNs conjugated with a targeting cRGD peptide. Results showed notably increased uptake by BAEC cells, indicating their potential as vectors for targeted therapy.
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ASSOCIATED CONTENT
S Supporting Information *
Characterization of SBA-15 and modified SBA-15: nitrogen adsorption isotherms, small-angle X-ray scattering, C 1s XPS spectra, TGA profile, and elemental surface composition. 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]. Tel.: +1 206 616 6509. *E-mail: [email protected]. Tel.: 86-021-66132701. Author Contributions ⊥
Y.Z. and H.S.S. contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The research is supported by the National Science Foundation (CMMI 1301435), the National Nature Science Foundation of China (No. 61371021), and the Basic Research Foundation of Shanghai Science and Technology Committee (13NM1401300). Y.H.Z was supported by the Chinese Scholar Program.
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dx.doi.org/10.1021/bm500209a | Biomacromolecules 2014, 15, 1845−1851
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