Biomacromolecules 2010, 11, 2199–2203
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Rapid Cellular Internalization of Multifunctional Star Polymers Prepared by Atom Transfer Radical Polymerization Hong Y. Cho,† Haifeng Gao,† Abiraman Srinivasan,‡ Joanna Hong,‡ Sidi A. Bencherif,† Daniel J. Siegwart,† Hyun-jong Paik,§ Jeffrey O. Hollinger,‡ and Krzysztof Matyjaszewski*,† Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, Department of Biomedical Engineering, Bone Tissue Engineering Center, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, and Department of Polymer Science and Engineering, Pusan National University, Busan, South Korea Received June 7, 2010; Revised Manuscript Received July 17, 2010
Poly(ethylene glycol) (PEG) star polymers containing GRGDS (Gly-Arg-Gly-Asp-Ser) peptide sequences on the star periphery were synthesized by atom transfer radical polymerization (ATRP) of poly(ethylene glycol) methyl ether methacrylate (PEGMA), GRGDS modified poly(ethylene glycol) acrylate (GRGDS-PEG-Acryl), fluorescein o-methacrylate (FMA), and ethylene glycol dimethacrylate (EGDMA) via an “arm-first” method. Star polymers were approximately 20 nm in diameter, as measured by dynamic light scattering and atomic force microscopy. Conjugation of FMA to the stars was confirmed by fluorescence microscopy, and successful attachment of GRGDS segments to the star periphery was confirmed by 1H NMR spectroscopy. Both fluorescent PEG star polymers with and without peripheral GRGDS peptide segments were cultured with MC3T3-E1.4 cells. These star polymers were biocompatible with g90% cell viability after 24 h of incubation. Cellular uptake of PEG star polymers in MC3T3-E1.4 cells was observed by confocal microscopy. Rapid uptake of PEG star polymers with GRGDS peptides (∼100% of FITC-positive cells in 15 min measured by flow cytometry) was observed, suggesting enhanced delivery potential of these functional star polymers.
Introduction Developing efficient polymer-based delivery systems is becoming increasingly important in the field of drug delivery and therapeutics.1-4 The most commonly used delivery systems may be divided into two groups: biological (i.e., viral) and nonbiological (i.e., nonviral) systems. Each group has its own advantages and limitations. Although biological carriers, such as viruses, possess high transfection efficiency, viral vectors are difficult to produce and may be toxic.5,6 These limitations underscore the need for nonbiological delivery systems such as peptides, lipids (liposomes), dendrimers, and polymers.7,8 Dendrimers are spherical and highly branched polymers that possess several beneficial attributes among nonviral systems, as a consequence of their uniformity and dense structure.9,10 While dendrimers have a well-defined structure, size, stability, and biocompatibility, they have challenges as delivery vectors due to the multistep synthesis, laborious purification, and high preparation cost. Atom transfer radical polymerization (ATRP),11-13 one of the most robust controlled radical polymerization (CRP)14 techniques, has advantages for its simple setting-up and easy incorporation of site specific functionality. ATRP procedures have been used to prepare polymers with diverse architectures (e.g., nanogel and star) and with innovative functionalities for evaluation as potential delivery systems.15-20 Star polymers are one of the possible structures and consisting of a core with multiple polymer arms. Star polymers have distinct physical properties,21,22 including a 3-dimensional globular compact * To whom correspondence should be addressed. E-mail: km3b@ andrew.cmu.edu. † Department of Chemistry, Carnegie Mellon University. ‡ Department of Biomedical Engineering, Carnegie Mellon University. § Pusan National University.
architecture with functionalities either on the periphery or in the core.23,24 Star polymers may be compared to dendrimers due to some structural similarities, but are easier to prepare with a lower synthetic cost.25 Star polymers can be prepared by ATRP using three synthetic strategies.26 The first approach is a “core-first” method that uses multifunctional core-based initiators.27 The molecular weight of star polymers increases along with the monomer consumption as arms grow from the functional core. The second is a “coupling-onto” method that uses living polymer chains with terminal reactive site that binds to a multifunctional coupling agent; the coupling efficiency should be quantitative. The third method for star polymer synthesis is an “arm-first” method.28-32 With this method, presynthesized macroinitiators (MI) or macromonomers (MM) are cross-linked with divinyl compound to form the core. Star polymers have a wide variety of potential applications for membranes, coatings, lithography, and drug delivery.25,33-44 In this study, we report the synthesis of poly(ethylene glycol) (PEG)-based multifunctional star polymers by an ATRP “armfirst” method in one-pot approach. Poly(ethylene glycol) methyl ether methacrylate (PEGMA), glycine(G)-arginine(R)-glycine(G)aspartic acid(D)-serine(S) modified poly(ethylene glycol) acrylate (GRGDS-PEG-Acryl), and fluorescein o-methacrylate (FMA) were cross-linked with ethylene glycol dimethacrylate (EGDMA). The GRGDS peptide sequence is widely used in biomedical engineering45-51 and was utilized here to increase cellular uptake. Similar specific cell target delivery was reported by using folate-conjugated block copolymers.52 GRGDS modified PEG star polymers were utilized as a peptide model to study targeted internalization. The biocompatibility of these star polymers was tested and the rate of cellular uptake was compared to that of the star polymers without GRGDS peptides.
10.1021/bm1006272 2010 American Chemical Society Published on Web 08/12/2010
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Biomacromolecules, Vol. 11, No. 9, 2010
Experimental Section Materials. PEGMA (Mn ) 2080, 50 wt % in water) was extracted with methylene chloride. The organic solution was stirred with anhydrous Na2SO4 to remove residual water before passage through a basic alumina column. GRGDS-PEG-Acryl was synthesized via a previously reported method.53 CuCl (98%, Acros) was purified by stirring with acetic acid and washed with ethyl alcohol before being dried for 12 h at 60 °C. All other chemicals, ethyl 2-bromoisobutyrate (EBiB, 99%, Aldrich), 2,2′-bipyridine (bpy, g99%, Aldrich), ethylene glycol dimethacrylate (EGDMA, 98%, Aldrich), and fluorescein o-methacrylate (FMA, 97%, Aldrich), and solvents were used as received without further purification. Measurements. The polymer samples were separated by GPC (Polymer Standards Services (PSS) columns (guard, 105, 103, and 102 Å) with DMF eluent at 50 °C, flow rate ) 1.00 mL/min and differential refractive index (RI) detector (Waters 2410)). The apparent molecular weights (Mn) and polydispersities (Mw/Mn) were determined with a calibration based on linear poly(methyl methacrylate) (polyMMA) standards using WinGPC 6.0 software from PSS. 1H NMR spectra of the polymer solutions in D2O were collected on Bruker Avance 500 MHz spectrometer at 25 °C. Tapping mode atomic force microscopy (AFM) experiments were carried out using a Multimode Nanoscope V system (Veeco instruments). The measurements were performed in air using commercial Si cantilevers with a nominal spring constant and resonance frequency of 40 N/m and 330 kHz respectively. The star polymers were dissolved in methanol (0.002 wt %) before drop casting onto freshly cleaved mica surface. The sample on mica substrate was dried overnight under ambient conditions before AFM measurement. Polymer fluorescence was measured by fluorometer (λex ) 490 nm, λem ) 500-600 nm Fluoromax-2 (Horiba, Jobin-Yvon Tokyo, Japan). Particle size and size distribution of star polymers were determined by dynamic light scattering (DLS) on a high performance particle sizer, Model HP5001 from Malvern Instruments, Ltd. Fluorescence microscopy was performed using a Zeiss Axiovert 200 microscope. Confocal imaging was done on an Olympus FV1000 microscope and flow cytometry was performed using a Coulter Epix Elite flow cytometer. Synthesis and Characterization of GRGDS-(PEG)n-polyEGDMA(F). GRGDS-(PEG)n-polyEGDMA(F) star polymers containing GRGDS at the periphery of the star shell were synthesized by ATRP via an “arm-first” method. PEGMA MM (Mn ) 2080, 0.4 g, 0.2 mmol), GRGDS-PEG-Acryl (Mn ∼ 3800, 38 mg, 0.01 mmol), FMA (32.0 mg, 0.08 mmol), EGDMA (37.7 µL, 0.2 mmol), bpy (12.5 mg, 0.08 mmol), and DMF were charged to a Schlenk flask. The flask was degassed by five freeze-pump-thaw cycles and filled with nitrogen. CuCl (3.69 mg, 0.04 mmol) was quickly added to the frozen mixture under nitrogen. No special care was taken to avoid moisture condensation. The flask was sealed with a glass stopper then evacuated and backfilled with nitrogen five times before being immersed in a 60 °C oil bath. The deoxygenated initiator EBiB (4.4 µL, 0.03 mmol) was injected into the reaction system via a nitrogen purged syringe through the side arm of the Schlenk flask. At timed intervals, samples were withdrawn via a syringe fitted with stainless steel needle and immediately diluted with DMF. The samples were used to measure polymer molecular weights by GPC. The reaction was stopped after 9 h by exposure to air. The final star polymers were purified by dialysis against methanol and distilled water for 2 days, respectively, by using a dialysis bag with MWCO ) 50000. Cell Culture. Mouse calvarial preosteoblast-like cells (embryonic day 1, subclone 4 (MC3T3-E1.4)) were obtained from American Type Culture Collection (ATCC) and cultured in R-minimum essential medium (R-MEM), supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen). Cell culture flasks, culture plates with flat bottoms (Corning), and 96-well suspension culture plates with U-shaped bottoms (Greiner) were used. Trypsin-ethylenediaminetetraacetic acid (EDTA) for cell passaging and a live/dead viability/ cytotoxicity kit was purchased from Invitrogen. N-TER, a commercially available peptide-based delivery system was obtained from Sigma.
Communications Cytotoxicity. GRGDS-(PEG)n-polyEGDMA(F) and (PEG)m-polyEGDMA(F) star polymers were tested for cell viability and cytotoxicity using the live/dead cytotoxicity assay kit. A total of 5 × 104 cells per well were seeded in a 24-well tissue culture polystyrene plate containing 900 µL of R-MEM media and 100 µL of respective polymer (200 µg/ mL) sample was added. After 24 h culturing, the cell culture media was aspirated and washed with PBS-Tween. Thereafter, 0.5 mL of live/ dead stain was added (calcein 1:2000 and ethidium homodimer 1:500, diluted in PBS) to the cells and incubated at 37 °C for 30 min in the dark and images were captured using a Zeiss Axiovert 200 fluorescence microscope. Cell Internalization. A total of 200 µL/well of 1 × 105 of MC3T3E1.4 cells were seeded directly onto a 96-well U-shaped bottom, nonadherent tissue culture plate. The cells were incubated with 200 µg/mL of GRGDS-(PEG)n-polyEGDMA(F) and (PEG)m-polyEGDMA(F) star polymers at 37 °C for 24 h. The media was aspirated and placed in centrifuge tubes, and the remaining attached cells were collected by trypsinization and seeded onto sterile 12-well tissue culture polystyrene culture plates. After overnight attachment, the cells were washed with PBS and were fixed with 4% paraformaldehyde for 10 min, and examined by confocal microscopy (Olympus FV1000 microscope) after staining with DAPI. All imaging conditions for the confocal microscopy, including laser power, photomultiplier tube, and offset settings, remained constant for each comparison set. Flow Cytometry. A total of 5 ×105 MC3T3-E1.4 cells were suspended in 990 µL of R-MEM media, and were incubated with 10 µL of a 5 mg/mL of GRGDS-(PEG)n-polyEGDMA(F) or (PEG)mpolyEGDMA(F) star polymers in a 96-well U-shaped bottom nonadherent tissue culture plate. The cells were incubated with the star polymers for 15, 30, and 45 min at 37 °C. The control group did not receive any polymers. The cells were then collected into 1.5 mL centrifuge tubes and centrifuged at 2000 rpm for 3 min to pellet the cells. The supernatant was removed and the cells were resuspended in 1 mL of sterile PBS and transferred to a borosilicate tubes. Each sample was analyzed using a Coulter Epix Elite Flow cytometer.
Results and Discussion Synthesis of GRGDS-(PEG)n-polyEGDMA(F). The reaction route is shown in Scheme 1. The procedure starts with the ratio of reagents [PEGMA MM]0/[GRGDS-PEG-Acryl]0/[FMA]0/ [EGDMA]0/ [EBiB]0/[CuCl]0/[bpy]0 ) 1.0/0.05/0.4/1.0/0.15/0.2/ 0.4 in DMF at 60 °C. Molecular weight and molecular weight distribution were measured by DMF GPC with RI detector at 50 °C. The evolution of the GPC traces for the synthesized GRGDS-(PEG)n-polyEGDMA(F) star polymers is shown in Figure 1. Before the polymerization, the mixture of two MMs, PEGMA and GRGDS-PEG-Acryl, showed one peak due to their similar Mn. During the cross-linking reaction, the increase in star yield can be observed in the higher molecular weight region, accompanied by a decrease in the initial MMs peak. After removal of the unreacted MMs via extensive dialysis, the purified star polymers were analyzed by GPC. As shown in Figure 1, unreacted MMs were successfully removed and the apparent molecular weight for GRGDS-(PEG)n-polyEGDMA(F) star polymers was Mn ) 58000 and Mw/Mn ) 1.55. The size of the star polymers containing GRGDS is shown in Figure 2. The hydrodynamic diameter of the GRGDS-(PEG)npolyEGDMA(F) stars was Dh ) 17.2 nm (Figure 2A), measured by DLS in water. When drop casting the star polymers from dilute methanol solution onto mica surface, the AFM result in Figure 2B shows a flattened structure of the star polymers due to the strong adsorption of PEG segments to the mica surface.54 The width of the flattened star polymers was approximately 20 nm, while its height was only about 1 nm. Introduction of functional groups on the periphery and in the core of star
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Scheme 1. Synthesis of Multifunctional PEG-Based Star Polymers by ATRP via an “Arm-First” Methoda
a Glycine(G)-arginine(R)-glycine(G)-aspartic acid(D)-serine(S) peptide sequence and fluorescein were introduced on the periphery and in the core of star polymers, respectively.
Figure 1. GPC traces during the synthesis of GRGDS-(PEG)npolyEGDMA(F) star polymers by an “arm-first” method, experimental conditions: [PEGMA MM]0/[GRGDS-PEG-Acryl]0/[FMA]0/[EGDMA]0/ [EBiB]0/[CuCl]0/[bpy]0 ) 1.0/0.05/0.4/1/0.15/0.2/0.4 in DMF at 60 °C, [PEGMA MM]0 ) 0.125 M, linear polyMMA standards were used for DMF GPC calibration.
polymers was confirmed by 1H NMR and fluorescence spectroscopy analysis, respectively. The presence of the GRGDS peptide on the periphery of star polymers was validated by 500 MHz 1H NMR spectroscopy in D2O. The peaks for the GRGDS peptide sequence are indentified in Figure S1 in the Supporting Information. Peaks at 2.58, 3.17, 3.85, 4.12, and 4.26 ppm from the peptide were confirmed in agreement with the previous reports.47,53 It was anticipated that GRGDS-(PEG)n-polyEGDMA(F) would enhance cellular internalization because of the successful peptide functionality and the size of star polymers.55-57 In Figure S2, the incorporated fluorescein functionality in the core of GRGDS-(PEG)n-polyEGDMA(F), used to visualize the cell uptake of stars, showed an emission curve similar to that
recorded for the fluorescent monomer FMA (maximum absorption at λ ) 490 nm). This similarity in emission behavior indicated that the incorporation of FMA to star core was successful. The concentration of star polymers with GRGDS and monomer were 1.5 mg/mL and 1.0 × 10-3 mg/mL, respectively. As a control sample, non-GRGDS star polymers, (PEG)mpolyEGDMA(F), were synthesized in a similar method by copolymerization of PEGMA, EGDMA, and FMA with EBiB as initiator. The obtained star polymers had Mn ) 59500 and Mw/Mn ) 1.87. Cytotoxicity of Star Polymers. The cell viability of both GRGDS-(PEG)n-polyEGDMA(F) and (PEG)m-polyEGDMA(F) was determined using MC3T3-E1.4 cells in Vitro. The star polymers were dispersed in sterile culture media and incubated with MC3T3-E1.4 cells. The cell viability was determined after 24 h incubation using live/dead cell staining (Figure S3). More than 90% viable cells (green stain) and
