Engineering of cell membranes with a bisphosphonate-containing polymer using ATRP synthesis for bone targeting

Biomaterials 35 (2014) 9447e9458

Contents lists available at ScienceDirect

Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Engineering of cell membranes with a bisphosphonate-containing polymer using ATRP synthesis for bone targeting Sonia D'Souza a, 1, Hironobu Murata a, 1, Moncy V. Jose b, c, Sholpan Askarova d, Yuliya Yantsen d, Jill D. Andersen a, Collin D.J. Edington a, William P. Clafshenkel a, Richard R. Koepsel a, Alan J. Russell a, * a

The Institute for Complex Engineered Systems, Carnegie Mellon University, 5000 Forbes Ave, Pittsburgh, PA 15213, USA McGowan Institute for Regenerative Medicine, University of Pittsburgh, 450 Technology Drive, Pittsburgh, PA 15219, USA PolyOne Technology & Innovation Center, 11650 Lake Side Crossing Court, St Louis, MO 63146, USA d Department for Bioengineering, Cell Technologies, and Cell Therapy, Center for Life Sciences, Nazarbayev University, 53 Kabanbay Batyr Ave, Astana 010000, Kazakhstan b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 March 2014 Accepted 23 July 2014 Available online 21 August 2014

The field of polymer-based membrane engineering has expanded since we first demonstrated the reaction of N-hydroxysuccinimide ester-terminated polymers with cells and tissues almost two decades ago. One remaining obstacle, especially for conjugation of polymers to cells, has been that exquisite control over polymer structure and functionality has not been used to influence the behavior of cells. Herein, we describe a multifunctional atom transfer radical polymerization initiator and its use to synthesize water-soluble polymers that are modified with bisphosphonate side chains and then covalently bound to the surface of live cells. The polymers contained between 1.7 and 3.1 bisphosphonates per chain and were shown to bind to hydroxyapatite crystals with kinetics similar to free bisphosphonate binding. We engineered the membranes of both HL-60 cells and mesenchymal stem cells in order to impart polymer-guided bone adhesion properties on the cells. Covalent coupling of the polymer to the non-adherent HL-60 cell line or mesenchymal stem cells was non-toxic by proliferation assays and enhanced the binding of these cells to bone. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Membrane engineering ATRP Bone targeting polymer Cell reactive polymers

1. Introduction Targeting therapeutics to specific tissues can result in higher local drug concentrations and fewer complications at peripheral sites. The factors that are crucial for successful targeting of therapeutics to specific tissues are: unique chemical moieties or biomarkers in the targeted tissue; a binding moiety that recognizes the unique target; and a mechanism to carry the therapeutic cargo. Many attempts to use tissue targeting have centered on cancer and bone, primarily due to the ever increasing array of well-known biomarkers for cancer and the well understood structure and composition of bone. Research in these areas has been the subject of several recent reviews [1e4]. A versatile carrier molecule for cell targeting should be adaptable to nearly any tissue. Due to specific regenerative challenges * Corresponding author. E-mail addresses: alanrussellþ@cmu.edu, [email protected] (A.J. Russell). 1 Authors contributed equally to the work. http://dx.doi.org/10.1016/j.biomaterials.2014.07.041 0142-9612/© 2014 Elsevier Ltd. All rights reserved.

associated with bone injuries and diseases, there are a number of ongoing efforts to target therapeutic agents to bone. In fact, targeting the components of bone repair to injury sites has shown promise in increasing the rate and quality of bone healing. Therapeutic drugs for the treatment of osteoporosis have provided a number of compounds, in particular the bisphosphonates, which bind tightly to the hydroxyapatite (HA) component of bone and are potent inhibitors of osteoclast activity [5e7]. Polymers capable of binding bone through chargeecharge interactions have been synthesized by coupling the cationic prepolymers poly(L-lysine) and poly(ethyleneimine) to thiobisphosphonate side groups [8]. When the water-soluble polymers poly(ethylene glycol) (PEG) and poly[N-(2-hydroxypropyl) methacrylamide] (PHPMA) were modified with bisphosphonate groups, both showed accumulation in bone tissue in mice [9]. The bisphosphonate modified PHPMA was also conjugated to an antiangiogenic drug, TNP-40, and the conjugate targeted calcified bone metastases and significantly inhibited osteosarcoma growth [10].

9448

S. D'Souza et al. / Biomaterials 35 (2014) 9447e9458

Synthetic polymers have been shown to drive small molecule and protein therapeutics to an active repair or disease site. However, the use of polymers as targeting agents for cells has been limited. In the case of bone, regeneration is orchestrated by cells. Mesenchymal stem cells (MSCs) have been an attractive cell source for therapy and tissue engineering because MSCs are recruited to bone injury sites where they proliferate and differentiate into osteoblasts to deposit matrix at the injury sites thus aiding in bone regeneration [11]. MSCs can also differentiate into multiple lineages, are immunosuppressive, and can be easily isolated and expanded ex vivo [11e13]. MSCs have been injected systemically in a number of preclinical and clinical trials, however very few MSCs reach the injury site with most localizing in the liver, spleen, and lungs [14e20]. Considerable effort has been directed towards seeding active repair sites with differentiated cells as well as with stem cells, and a number of reports have shown that polymeric scaffolds pre-seeded with cells improve bone repair [21e25]. However, none of these approaches provided cells to the tissue from circulation and none of the cells had enhanced bone binding properties. A recent study has used a peptide mimetic ligand against a4b1 integrin that contained a terminal alendronate group to selectively bind to MSCs and direct them to bone sites [26]. This elegant approach points to the possibility that a more generalizable targeting material can be developed that can be used to engineer many different cell types to target specific tissues. Herein, we describe the atom transfer radical polymerization (ATRP) synthesis of polymers that can be easily modified with both tissue targeting and cell binding moieties. The objective of this study was to evaluate the interaction of HL-60 cells and bone marrow-harvested MSCs with hydroxyapatite after cell surface modification with a bisphosphonate-containing polymer (Fig. 1). Binding affinities for polymer-modified cells were determined using both hydroxyapatite crystals and rodent femur bone fragments. The effect of polymer modification on proliferation in both cell lines was determined by quantifying the reduction of MTS tetrazolium in viable cells. Additionally, the effect of polymer modification on the adipogenic and osteogenic differentiation of bone marrowharvested MSCs was determined by staining for lipid content and alkaline phosphatase activity, respectively.

Fig. 1. Targeting cells to bone. A multi-functional polymer was designed to partition cells to bone. The polymer contained N-hydroxysuccinimide (pink) and pendant bisphosphonate (purple) groups. The N-hydroxysuccinimide group reacted with cell surface components and the bisphosphonate group bound to the HA component of bone. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

purchased from Corning Incorporated (Corning, NY). Oil red O powder (Cat. No. A12989) was purchased from Alfa Aesar (Ward Hill, MA). Alizarin Red stain was purchased from Lifeline Cell Technology (Frederick, MD). 2.2. Synthesis of bone targeting polymers

2.1. Materials

Three different length bone targeting polymers were generated using ATRP as described below. The polymer with a molecular weight of 10.1 kD was designated as BT-1, the 21.8 kD polymer was designated as BT-2, and the 45.0 kD polymer was designated as BT-3.

Acryloyl chloride, 6-aminohexanoic acid, N-hydroxysuccinimide (NHS), N-(3dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDCHCl), benzyl alcohol, 4-aminobutanoic acid, p-toluenesulfonic acid monohydrate (TosOH), 2chloropropionyl chloride, N,N0 -diisopropylcarbodiimide (DIC), 4-amino-1-butanol, fluorescein O-methacrylate (fluorescein monomer), copper (I) chloride (Cu(I)Cl), triethylamine (TEA), toluene, dichloromethane (CH2Cl2), ethanol, 2-propanol (IPA), ethyl acetate, 1,4-dioxane, acetonitrile, n-hexane, diethyl ether, hydrochloric acid, sodium hydroxide, Hydroxyapatite (HA; Cat. No. 289396-25G), trypan blue 0.4 (w/v) % solution (Cat. No. T8154), isopropanol, acetone, and alkaline phosphatase kit (Cat. No. 85L3R-1KT) were purchased from Sigma-Aldrich (St Louis, MO). N,N-dimethylacrylamide (DMAA; Sigma-Aldrich) was distilled in the presence of calcium hydride under vacuum. Sodium alendronate was purchased from Spectrum Chemical, Gardena, CA. Tris[2-(dimethylamino)ethyl]amine (Me6TREN) was prepared according to a procedure reported by Ref. [28]. Trypsin/EDTA (Cat. No. SH30042.01), antibiotic/ antimytotic solution e 100 (Cat. No. SV30079.01), and formalin 10% (v/v) solution were purchased from Thermo Fisher Scientific (Logan, UT). Dulbecco's phosphate buffered saline (DPBS) and OsteoAssay™ human bone plate were purchased from Lonza Incorporated (Allendale, NJ). Heat inactivated fetal bovine serum (FBS; Cat. No. 10082147), GlutaMAX™ (Cat. No. 35050), Dulbecco's modified Eagle's Medium High Glucose (DMEM; Cat. No. 31053), Iscove's modified Dulbecco's Medium (IMDM), StemPRO Osteogenesis Differentiation kit (Cat. No. A10072-01) and Stericup filters were purchased from Gibco® Invitrogen Cell Culture (Carlsbad, CA). Rabbit serum (Cat. No. 191357) was purchased from MP Biomedicals (Solon, OH). Cell Titer 96® AQueous One Solution Cell Proliferation assay (Cat. No. G3580) was purchased from Promega (Madison, WI). Dimethyl sulfoxide (DMSO; tissue culture grade, Cat. No. 25-950-CQC) was purchased from Mediatech Incorporated (Manassas, VA). Tissue culture T75 flasks and tissue culture plates (96-well and 24-well plates) were

2.2.1. Synthesis of N-(2-chloropropionyl)-4-butyric acid benzyl ester A mixture of 4-aminobutanoic acid (20.6 g, 200 mmol), benzyl alcohol (100 mL, 965 mmol) and TosOH (45.6 g, 240 mmol) in toluene (200 mL) was refluxed using DeaneStark apparatus at 160  C for 5 h. After the mixture was cooled to room temperature, 100 mL of diethyl ether were added and the precipitated crude compound was filtered off. The benzyl ester intermediate (BEI1; Fig. 2A) was obtained by recrystallization from ethanol and diethyl ether (1/9 volume ratio); yield 62.6 g (88%), mp 106e108  C. 1H NMR (300 MHz, CDCl3) d 1.82 (m, 2H, J ¼ 7.5 Hz,  NHþ 3 CH2CH2CH2COO), 2.30 (s, 3H, CH3C6H4SO3 ), 2.50 (t, 2H, J ¼ 7.5 Hz, þ NHþ 3 CH2CH2CH2COO), 2.83 (t, 2H, J ¼ 7.5 Hz, NH3 CH2CH2CH2COO), 5.12 (s, 2H, COOCH2C6H5), 7.13 and 7.51 (d, 2  2H, J ¼ 7.8 Hz, CH3C6H4SO 3 ), 7.38 (s, 5H, COOCH2C6H5) and 7.71 (broad s, 3H, NHþ 3 CH2CH2CH2COO) ppm. IR (KBr) 3450, 3039, 2941, 2671, 1733, 1644, 1534, 1497, 1453, 1417, 1394, 1344, 1297, 1191, 1146, 1127, 1069, 1036, 1011, 997, 978, 949, 819, 774, 732 and 711 cm1. A solution of 2chloropropionyl chloride (3.2 mL, 33 mmol) in CH2Cl2 (10 mL) was slowly added to a mixture of BEI1 (11 g, 30 mmol) and triethylamine (10 mL, 70 mmol) in CH2Cl2 (150 mL) at 0  C. After stirring at room temperature for 2 h, the mixture was washed with water (100 mL  2), saturated NaHCO3 aq. (100 mL  3), 5% HCl aq. (100 mL  3) and water (100 mL  2). The organic phase was dried with MgSO4 and evaporated to remove any solvent. N-2-chloropropionyl-4-aminobutanoic acid benzyl ester (BEI2) was isolated in vacuo; yield 6.8 g (80%). 1H NMR (300 MHz, CDCl3) d 1.74 (d, 3H, J ¼ 6.9 Hz, eNHC]OCHClCH3), 1.93 (m, 2H, 7.2 Hz, OC] OCH2CH2CH2NHC]O), 2.45 (t, 2H, J ¼ 7.2 Hz, OC]OCH2CH2CH2NHC]O), 3.36 (q, 2H, J ¼ 7.2 Hz, OC]OCH2CH2CH2NHC]O), 4.41 (q, 1H, J ¼ 6.9 Hz, eNHC] OCHClCH3), 5.16 (s, 2H, C6H5CH2OC]O), 6.67 (broad s, 1H, amide proton), and 7.38 (s, 5H, C6H5CH2OC]O) ppm. IR (NaCl) 3306, 3089, 3067, 3034, 2973, 2880, 1734,

2. Materials and methods

S. D'Souza et al. / Biomaterials 35 (2014) 9447e9458

9449

Fig. 2. Schematic representation of the synthesis of a ATRP initiator and tissue targeting polymers. Bone targeting polymers were synthesized by ATRP as described in Materials and methods. The bone targeting polymers contained N-hydroxysuccinimide, bisphosphonate, and fluorescein. Control polymer (C1) had an aminobutanol instead of bisphosphonate and a carboxy-terminal instead of N-hydroxysuccinimide, and thus bound to neither bone nor cells. A second control polymer (C2) had an aminobutanol instead of bisphosphonate and contained N-hydroxysuccinimide, and thus bound cells but not bone.

9450

S. D'Souza et al. / Biomaterials 35 (2014) 9447e9458

1663, 1609, 1540, 1498, 1454, 1418, 1376, 1356, 1322, 1258, 1236, 1215, 1168, 1093, 1070, 1028, 988 and 751 cm1. 2.2.2. Synthetic procedure for N-acryloyl-6-aminohexanoic acid N0 hydroxysuccinimide ester (NHS monomer) The NHS monomer was synthesized according to the procedure reported by Murata and co-workers [27] from acryloyl chloride, 6-aminohexanoic acid and NHS (Supplemental Fig. 1). A mixture of acryloyl chloride (17.8 mL, 220 mmol) and 1,4dioxane (50 mL) was slowly added to a solution of 6-aminohexanoic acid (26.2 g, 200 mmol) and sodium carbonate (42.4 g, 400 mmol) in water (400 mL) at 0  C, and then stirred at room temperature for 2 h. The mixture was adjusted to pH 2.0 with 1 N HCl in an ice bath and extracted with ethyl acetate, and then the organic phase was dried with MgSO4. After the MgSO4 was filtered out and the organic phase evaporated, N-acryloyl-6-aminohexanoic acid was obtained by recrystallization from ethyl acetate and n-hexane (1/1 volume ratio); yield 33.6 g (91%), mp 79e82  C, 1H NMR (300 MHz, CDCl3) d 1.39e1.47 (m, 2H, NHCH2CH2CH2CH2CH2COO), 1.55e1.74 (m, 4H, NHCH2CH2CH2CH2CH2COO), 2.38 (t, 2H, J ¼ 7.2 Hz, NHCH2CH2CH2CH2CH2COO), 3.37 (q, 2H, J ¼ 7.2 Hz, NHCH2CH2CH2CH2CH2COO), 5.66 (dd, 1H, J ¼ 1.8 and 10.2 Hz, alkene), 5.88 (broad s, 1H, amide proton), 6.12 (dd, 1H, J ¼ 10.2 and 17.1 Hz, alkene) and 6.30 (dd, 1H, J ¼ 1.8 and 17.1 Hz, alkene) ppm. IR (KBr) 3288, 3063, 2947, 2863, 2773, 1703, 1654, 1623, 1547, 1460, 1446, 1434, 1408, 1350, 1295, 1272, 1240, 1173, 1106, 1068, 1001, 960, 937, 810 and 743 cm1. To a solution of N-acryloyl-6aminohexanoic acid (9.3 g, 50 mmol) in CH2Cl2 (250 mL), DIC (7.0 g, 55 mmol) and NHS (6.4 g, 55 mmol) were added at 0  C. The mixture was stirred at room temperature overnight. Precipitated urea was filtered out and the solvent was removed by evaporation. NHS monomer was obtained by recrystallization from IPA; yield 11.5 g (81%), mp 106e110  C, 1H NMR (300 MHz, CDCl3) d 1.46e1.87 (m, 6H, NHCH2(CH2)3CH2COO), 2.66 (t, 2H, J ¼ 7.1 Hz, NHCH2(CH2)3CH2COO), 2.87 (s, 4H, ethylene (succinimide)), 3.39 (q, 2H, J ¼ 6.3 Hz, NHCH2(CH2)3CH2COO), 5.64 (dd, 1H, J ¼ 1.8 and 10.2 Hz, alkene), 5.85 (broad S, 1H, amide proton), 6.13 (dd, 1H, J ¼ 10.2 and 17.1 Hz, alkene) and 6.31 (dd, 1H, J ¼ 1.8 and 17.1 Hz, alkene) ppm. IR (KBr) 3253, 3060, 2930, 2865, 1815, 1793, 1755, 1666, 1650, 1554, 1473, 1450, 1424, 1408, 1380, 1297, 1247, 1220, 1114, 1081, 989, 884, 808 and 727 cm1. 2.2.3. Synthesis of block polymer containing fluorescein (F1) DMAA (5.1 mL, 50 mmol for BT-1, 20.4 mL, 200 mmol for BT-2, or 25.5 mL, 250 mmol for BT-3), fluorescein O-methacrylate (400 mg, 1.0 mmol for BT-1 and BT2, or 200 mg, 0.5 mmol for BT-3), BEI2 (283 mg, 1.0 mmol for BT-1 and BT-2, or 142 mg, 0.5 mmol for BT-3; Fig. 2A), IPA (25 mL) and deionized water (25 mL) were placed in a polymerization tube. The polymerization solution was charged with argon for 30 min and then argon charged solution of Me6TREN (460 mg, 2.0 mmol for BT-1 and BT-2, or 230 mg, 1.0 mmol for BT-3), and Cu(I)Cl (200 mg, 2.0 mmol for BT-1 and BT-2, or 100 mg, 1.0 mmol for BT-3) in water (10 mL) was added under argon flow. The polymerization was carried out at room temperature for 18 h. The resulting mixture was dialyzed by using an Mwco 1000 dialysis tube (Spectra/Por®, Spectrum Laboratories Inc., Rancho Dominguez, CA) in deionized water for 2 days, and then the isolated polymer was lyophilized. IR (KBr) 3462, 2932, 1766, 1631, 1499, 1403, 1357, 1254, 1147 and 1101 cm1. 2.2.4. Synthesis of NHS functionalized block polymers DMAA (103 mL, 1.0 mmol), NHS monomer (142 mg, 0.5 mmol), first block(700 mg (BT-1), 1.7 g (BT-2) or 4.2 g (BT-3) respectively, 0.1 mmol of Cl end group) and IPA (12.5 mL) and acetonitrile (12.5 mL) were placed in a polymerization tube. The polymerization solution was charged with argon for 30 min, and then argon charged solution of Me6TREN (46 mg, 0.2 mmol), Cu(I)Cl (20 mg, 0.2 mmol) in IPA (10 mL) was added under argon flow. After polymerization, the copper catalyst was removed by passing the mixture through silica gel. The second block polymer was obtained by precipitation with diethyl ether. The ether-insoluble fraction was filtered off and dried overnight in vacuo. IR (KBr) 3464, 2933, 1812, 1765, 1470, 1720, 1635, 1499, 1463, 1429, 1402, 1357, 1255, 1144, 1099, 1060 and 995 cm1. 2.2.5. Reaction with amino-bisphosphonate A solution of the second block polymer described in Section 2.2.4 (475 mg, (BT1), 1.07 g (BT-2), or 2.24 g (BT-3), respectively, 0.05 mmol polymers i.e. ca. 0.15 mmol of NHS groups) in DMSO (5 mL) was added into a sodium alendronate solution (120 mg, 0.37 mmol) in 0.1 M sodium phosphate buffer (pH 9.0, 25 mL). The mixture was stirred overnight at room temperature. One milliliter of 1 N NaOH was added to the mixture and stirred at room temperature for 3 h. The resulting aminobisphosphonate block polymer was obtained by lyophilization after dialyzing the solution in deionized water with an Mwco 1000 dialysis tube. IR (KBr) 3474, 2932, 1751, 1630, 1500, 1462, 1432, 1402, 1357, 1256, 1144, 1098 and 1059 cm1. EDCHCl (39 mg, 0.2 mmol) and NHS (24 mg, 0.2 mmol) were then added to solutions of the amino-bisphosphonate block polymer(200 mg (BT-1), 430 mg (BT2), or 900 mg (BT-3), respectively, 0.02 mmol of eCOOH end group) in deionized water (10 mL) and stirred at room temperature for 30 min. The final bone targeting polymers (BT-1, BT-2 and BT-3) were isolated by dialysis using an Mwco 1000 dialysis tube in the refrigerator and then lyophilized. IR (KBr) 3461, 2933, 1766, 1741, 1730, 1630, 1500, 1457, 1432, 1420, 1402, 1357, 1256, 1213, 1145, 1097 and 1059 cm1.

2.2.6. Synthesis of control polymer To obtain the control polymer, the bisphosphonate group was replaced with a butanol group. 4-amino-1-butanol was used in place of sodium alendronate. A solution of the polymers described in Section 2.2.4 (120 mg, (BT-1), 0.013 mmol polymers i.e. ca. 0.04 mmol of NHS groups) in DMSO (5 mL) was added into 4amino-1-butanol solution (10 mL, 0.1 mmol) in deionized water (10 mL). The mixture was stirred at room temperature overnight. One milliliter of 1 N NaOH was added to the mixture and stirred at room temperature for 3 h. The resulting polymer (C1) was dialyzed in deionized water with an Mwco 1000 dialysis tube and then lyophilized. Control polymer (C2) was synthesized from the preceding polymer and prepared by the same procedure used to generate the bone targeting polymers. A ten-fold molar concentration of EDCHCl and NHS were placed into the solution of polymer C1 in deionized water and stirred at room temperature for 30 min. The polymer C2 was isolated by dialysis using an Mwco 1000 dialysis tube in the refrigerator and then lyophilized. 2.3. Measurements 1 H NMR spectra (Supplemental Figs. 2e7) were recorded on a spectrometer (300 MHz) in the NMR facility located in Center for Molecular Analysis, Carnegie Mellon University, (Pittsburgh, PA) with Deuterium oxide (D2O), DMSO-d6 and CDCl3. Routine FT-IR spectra were obtained with a Nicolet Avatar 360 FT-IR spectrometer (Thermo). Melting points (mp) were measured with a Laboratory Devices Mel-Temp. Number average molecular weights (Mn) and the dispersity (Mw/Mn) were estimated by gel permeation chromatography (GPC) on a Water 600E Series with a data processor, equipped with three polystyrene columns (Waters styragel HR1, HR2 and HR4), using DMF with LiBr (50 mM) as an eluent at a flow rate 1.0 mL/ min, with detection by a refractive index (RI) detector. Poly(methyl methacrylate) standards were used for calibration.

2.4. Polymer binding to hydroxyapatite crystals HA crystals (10 mg/ml) (Sigma-Aldrich) were incubated with varying concentrations of polymer BT-1 and C2 solutions (0.05e1.0 mM) prepared in 0.1 M DPBS (Sigma-Aldrich) for a period of 2 h at 37  C on an orbital shaker. After incubation, the solutions were centrifuged at 14,000 RPM for 2 min and the supernatant was removed. The HA crystals were then washed once with DPBS and twice with deionized water. After each wash, the solution was centrifuged at 14,000 RPM to remove the supernatant. The washed HA crystals were then freeze-dried for further use. To determine the amount of polymer adsorbed to the HA crystals, the crystals were dissolved in 0.1 M HCl solution to obtain a 5 mg/ml of HA-HCl solution. Aliquots of 300 mL of the HA-HCl solution were used to measure the adsorbed polymer with a fluorescence plate reader (Molecular Devices, Sunnyvale, CA) with excitation at 480 nm and emission at 520 nm. The observed relative fluorescence was compared to an appropriate calibration curve developed by preparing different concentrations of polymer BT-1 in 0.1 M HCl solution containing dissolved HA (5 mg/ml) for evaluating the bound polymer concentration. The saturation binding levels and the dissociation constant were determined using non-linear regression. To obtain images of polymer interactions with HA, HA crystals (10 mg/ml) were incubated in a 0.3 mM polymer BT-1 solution prepared in 0.1 M DPBS for a period of 1 h at 37  C on an orbital shaker. After incubation, the solution was centrifuged at 14,000 RPM for 2 min and the supernatant was removed. This was followed by washing once in DPBS (0.1 M) and twice in deionized water. After each wash, the solution was centrifuged and the supernatant removed. After washing, 10 mL of the HA crystal solution were transferred onto a glass slide and imaged using a Leica DM IRB inverted microscope (Leica Microsystems, GmbH, Wetzlar, Germany) under white light and fluorescence. 2.5. Cell culture HL-60 cells were purchased from ATCC (Manassas, VA). HL-60 cells were cultured to confluence at 37  C in a 5% (v/v) CO2 atmosphere in complete medium containing IMDM (Invitrogen) supplemented with 20% (v/v) FBS (Invitrogen), and 1% (v/v) penicillin/streptomycin (Invitrogen). HL-60 cell cultures were maintained by the addition or replacement of fresh medium every 2e3 days. C57BL6 MSCs (passage 8) were purchased from Invitrogen, Carlsbad, CA. The MSCs were cultured at 37  C in a 5% (v/v) CO2 atmosphere in complete medium containing DMEM (Invitrogen) supplemented with 10% (v/v) FBS (Invitrogen), GlutaMAX™ (Invitrogen), and 1% (v/v) Antibiotic/antimytotic (Thermo Fisher Scientific). Cultures were maintained by the addition or replacement of fresh medium every 2e3 days. Murine MSCs were not used past passage 12. The MSCs were lifted from the plate by incubating the cell layer with trypsin/EDTA for approximately 5 min at 37  C. 2.6. Bone marrow mesenchymal stem cell extraction Primary bone marrow MSCs were isolated from the tibias of Sprague Dawley rats aged 11 weeks. Bone marrow was harvested from the tibias of mice with DMEM containing 1% (v/v) antibiotic/antimytotic solution. The bone marrow aspirates were centrifuged at 1000 RPM for 10 min and resuspended in DMEM containing 10% (v/v) FBS, GlutaMAX™, and 1% (v/v) antibiotic/antimytotic solution. The cell suspension was added to T75 tissue culture treated flasks. Cultures were maintained by the

S. D'Souza et al. / Biomaterials 35 (2014) 9447e9458

9451

replacement of fresh medium every 2e3 days. The adherent bone marrow MSCs were cultured at 37  C and were passaged either three or four times before assessing their differentiation potential. 2.7. Polymer modification of cells In order to characterize HL-60 cell-polymer interactions, the HL-60 cells (1  106) were incubated with polymer BT-1 (1 mg) in 1 ml of phosphate buffer solution pH 8.0 for 10 min in a 37  C water bath. The cells were then washed three times in DPBS to remove any residual polymer. Cells were centrifuged between each wash at 100 g for 5 min. To visualize the polymer modified HL-60 cells a few microliters of the cell suspension were transferred to a glass slide and the cells were imaged with a Leica DM IRB inverted microscope (Leica Microsystems, GmbH, Wetzlar, Germany). In order to characterize MSC-polymer modification, MSCs (1  106) were washed with DPBS (5e10 ml) twice. After washing the cells with DPBS, the cell pellet was resuspended in a polymer solution (0.00001 M) prepared in DPBS pH 8.0 in a total volume of 1 mL. The cell polymer solution was incubated for 10 min in a 37  C water bath. After 10 min, the solution was washed three times with DPBS (10 mL). The cells were centrifuged at 300 g for 5 min between each wash. After the final wash, the cells were resuspended in approximately 1 mL DPBS. To visualize the cells, 25 mL of the cell suspension were transferred to a glass slide and a cover slip was placed on the solution. The cells were imaged with a Leica DM IRB inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany) under white light and fluorescence. Control (unmodified) MSCs were treated in a similar manner with the exception of polymer addition. 2.8. Quantitative analysis of images obtained from polymer modified/unmodified MSCs To determine the relative fluorescence per square micron, we modified MSCs as described above. Trypan blue was used to exclude dead cells. A program was developed to streamline the quantification of cell fluorescence using the Image J software tool. The pseudo-code test file used a combination of simple programming conditions and loops, as well as macro-calls to the Image J interface to rapidly analyze images and minimize human bias. The threshold was manually set for the images to isolate individual cells from neighboring cells and background noise and then the built-in “Analyze Particles” function was used to automatically isolate cells. The outlines of the cells were saved in a separate file. The area and integrated density of the fluorescent color channel were measured and exported to a CSV file which was further analyzed using Microsoft Excel. All output units were automatically converted from pixels to microns using a global scale which was calibrated with a scale bar from one of the images. 2.9. Cell proliferation assay HL-60 cells (1  106) were suspended in 1.0 mL of DPBS pH 8.0 and added to a tube containing polymer BT-1 (1 mg) that was dissolved in 10 mL of tissue culture grade DMSO (Mediatech Inc., Manassas, VA). The cell-polymer solution was incubated for 10 min in a 37  C water bath. The HL-60 cells were centrifuged at 100 g for 5 min and the cell pellet was washed three times with DPBS (Invitrogen). The cells were maintained in a T75 tissue culture flask (Corning Inc., Corning, NY) at a concentration of 1  105 cells per mL in a 37  C incubator containing 5% (v/v) CO2. Each T75 flask contained a total of 10 mL of either modified or unmodified HL-60 cells. Control (unmodified) cells were treated in a similar manner with the exception of polymer BT-1 addition. At each of the indicated time points, an aliquot of the cell suspension was used to count the number of cells. We also performed proliferation assays to determine if the polymer engineering of MSCs affected its proliferative ability. Briefly, after polymer modification, MSCs (5  103) were plated in a 96-well plate. MSC proliferation was determined using the Cell Titer 96® AQueous

Fig. 3. Binding kinetics and binding of polymer BT-1 to HA. A) Binding kinetics for polymer BT-1 was measured by dissolving the HA e polymer complexes in 0.1 M HCl and comparing the fluorescence of the samples with those from a series of known polymer concentrations. B) Micrographs of HA with or without polymer BT-1 in white light (a and c) and fluorescence (b and d).

One Solution Cell Proliferation assay kit as per the manufacturer's instructions. The absorbance was read at 490 nm using a Synergy H1 Hybrid Multi-Mode Microplate reader (BioTek, Winooski, VT). 2.10. Preparation of bone fragments and binding of polymer modified HL-60 cells to bone fragments Femur bones were obtained from mice. The bones were cut into small pieces with bone cutters. Most of the surrounding tissue was removed. The bone pieces were placed in a jar containing ethanol and then placed in 6 M NaOH for a week. The bone pieces were then placed in deionized water for a week to remove any residual NaOH. The bone pieces were then ground with a mortar and pestle and used in bone binding assays. To determine binding of polymer modified HL-60 cells to bone fragments, HL-60 cells were modified with polymers BT-1 or C2 as described above. After the final wash, the cells were resuspended to a final concentration of 1  106

Table 1 Physical characterization of bone targeting polymers. Sample

BT-1 BT-2 BT-3 a b c d e f g h i j

First blocka [I]0/[DMAA]0a 1/50 1/200 1/500

Second blocke b

Yield (%)

Mn

b

PDI

70 79 77

6,500 25,000 56,000

1.47 1.43 1.43

Mn

c

6,900 17,300 42,000

Fluorescein units 0.63 0.80 0.72

d

Third blockh f

Yield (%)

Mn

58 75 86

9,500 21,300 44,800

NHS units 3.1 3.3 3.0

[I]0/[Cu(I)Cl]0/[Me6TREN]0/[Fluorescein monomer]0 ¼ 1/2/2/1, IPA/water ¼ 1/1, r.t., 18 h. determined by GPC. determined by 1H NMR. estimated by 1H NMR, signal area of Fluorescein unit (8.1 ppm) vs. area of C6H5CH2Oe of initiator end (5.1 ppm). [First block polymer]0/[DMAA]0/[NHS monomer]0/[Cu(I)Cl]0/[Me6TREN]0 ¼ 1/10/5/2/2, IPA/AN ¼ 1/1, 40  C, 18 h. determined by 1H NMR. estimated by 1H NMR, signal area of succinimide (2.8e2.9 ppm) vs. area of C6H5CH2Oe of initiator end (5.1 ppm). [NHS]0/[alendronate]0 ¼ 1/2.5, 0.1 M phosphate buffer (pH 9.0) at r.t. overnight. determined by 1H NMR. estimated by 1H NMR, signal area of methylene protons from alendronate (1.85 ppm) vs. area of proton signals (0.9e1.8 ppm).

g

Yield (%)

M ni

BP unitsj

95 82 88

10,100 21,800 45,000

3.1 2.8 1.7

9452

S. D'Souza et al. / Biomaterials 35 (2014) 9447e9458

cells per mL in DPBS. Polymer modified HL-60 cells were incubated with 3 mg of bone fragments in a test tube for 20 min on an orbital shaker at room temperature. The cell and bone fragments were gently washed three times in DPBS. The bone cell clusters were imaged with a multispectral microscope and analyzed with a software program developed by our laboratory for use with Image J. The average areas of bone fragment clusters and the area of green fluorescence over the total area of bone fragments were calculated and graphed.

Osteogenesis was assessed using an alkaline phosphatase kit (Sigma-Aldrich) as per the manufacturer's protocol. Images of the osteogenic cells were obtained with a Leica DM IRB (Leica Microsystems GmbH, Wetzlar, Germany) inverted microscope under white light.

2.11. Adipogenic differentiation assay

After bone marrow extraction, the tibias from the Sprague Dawley rats were cut into small pieces using bone cutters. The bones were placed in a 50 mL tube containing 6 M NaOH for 3 days. The NaOH solution was replaced each day. The bones were then placed in a 50 mL tube containing deionized water for another 3 days to remove the residual NaOH. The deionized water was replaced each day. The bone chips were then placed at 37  C for 4 h to remove of excess water. The dried bone fragments were placed in a Ziploc bag and crushed. To determine binding of polymer modified MSCs to bone, we incubated 5  105 polymer modified MSCs with 0.5 mg of bone chips in an 1.5 mL eppendorf tube in a total volume of 0.5 mL. The modified MSCs and bone were incubated for 20 min on a shaker. An aliquot (25 mL) of the solution was transferred onto a glass slide. The aggregates were visualized using a Nikon E600 fluorescent microscope with a Nuance FX multispectral imaging system (Perkin Elmer, Waltham, MA). For the bone binding experiments with human bone plates, 1  105 polymer modified or unmodified MSCs were added to a bone well. The wells were placed on a shaker for the indicated time. The unbound cells were washed with DPBS three times after which 100 mL of DPBS were added to the wells to prevent the wells from drying. The bone wells were imaged under white light. The number of cells per field and the total area per field after excluding the large bone fragments were

Polymer-modified or unmodified primary bone marrow MSCs were plated in 96-well plates and cultured in adipogenic medium containing DMEM, 10% (v/v) rabbit serum, and 1% (v/v) antibiotic/antimytotic solution. Cells were cultured in a 37  C incubator containing 5% (v/v) CO2 for 10 days. Adipogenesis was assessed using Oil red O. Briefly, after removing the media from the wells, the cells were washed twice with DPBS. The cells were then fixed in 10% (v/v) formalin for 30 min at room temperature after which the cells were washed twice with distilled water. Oil red O solution was added to the cells for 30 min after which the excess stain was removed by washing the cells with water. Mayer's Hematoxylin (Sigma-Aldrich) was added to the cells for 10 min to visualize the nuclei. Excess stain was washed with water. Images of the adipogenic cells were obtained with a Leica DM IRB inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany) under white light. 2.12. Osteogenic differentiation assay Polymer-modified or unmodified primary bone marrow mesenchymal stem cells were plated in 96-well plates and cultured in osteogenic medium (Invitrogen). Cells were cultured in a 37  C incubator containing 5% (v/v) CO2 for 10 days.

2.13. Preparation of bone fragments and binding of polymer modified MSCs to bone fragments/human bone plates

Fig. 4. Engineering of MSCs with bisphosphonate-containing polymers. Polymers of different lengths (BT-1, BT-2, and BT-3) were incubated with MSCs for 10 min in a 37  C water bath. The cell polymer solution was washed 3 times and the cell pellet was resuspended in PBS. Cells were imaged under either fluorescence (A, C, E, G) or under brightfield (B, D, F, H). A and B e MSCs modified with polymer BT-1; C and D e MSCs modified with polymer BT-2; E and F e MSCs modified with polymer BT-3; G and H e unmodified MSC controls.

S. D'Souza et al. / Biomaterials 35 (2014) 9447e9458 counted using Image J. The number of cells per square cm was calculated and graphed. 2.14. Binding of polymer modified MSCs to HA Polymer modified MSCs (3  105) were incubated with 0.3 mg of HA in a 1.5 mL eppendorf tube in a total volume of 0.6 mL. The modified or unmodified MSCs and HA were incubated for 20 min on a shaker. An aliquot (25 mL) of the solution was transferred onto a glass slide. A small volume of DAPI was mixed with the solution to visualize the cells. The aggregates were visualized using a Leica DM IRB inverted microscope. 2.15. Statistical analysis

9453

Table 2 Degree of modification of MSCs with polymers. MSCs were modified with polymers (BT-1, BT-2, and BT-3) as described in Materials and methods. To determine the number of polymer molecules on each MSC, we measured the relative fluorescence of the MSCs after modification and compared the relative fluorescence of each sample to the respective calibration curves. Polymers

BT-1 BT-2 BT-3

Polymer molecules per mouse MSC Avg

SD

6.923Eþ07 1.935Eþ07 3.842Eþ07

2.252Eþ06 2.508Eþ06 7.313Eþ06

The quantitative data were represented as mean ± SEM. Statistical differences were determined by the unpaired student t-test.

3. Results and discussion 3.1. Synthesis of bone binding polymer One could reasonably predict that the covalent attachment of a polymer to a cell membrane could have a wide range of biological effects on that cell. In order to minimize unpredictable effects of membrane engineering with polymers, we sought to control the molecular weight dispersity of a cell-reactive polymer as precisely as possible. ATRP is among the most precisely controlled and versatile polymerizations available [29]. To ensure flexibility for membrane engineering reactions, we used an ATRP initiator, N-(2-chloropropionyl)-4-butyric acid benzyl ester (BEI2), with a blocked carboxyl group that simplified postpolymerization end-group modifications with a variety of celltargeting moieties. The initiator BEI2 (Fig. 2A) was used to synthesize a family of multi-functional polymers for targeting a variety of cargos to specific sites. The multi-functional base polymer was synthesized as a di-block polymer using a two-stage ATRP reaction scheme (Fig. 2B). The first block, a spacing segment between bone and cell surface binding segments, was initiated from BEI2 with the monomers DMAA and fluorescein O-methacrylate, which provided the final polymer with a fluorescence tag as a tracer. As shown in Table 1, the molecular weight of the first block polymer was dependent on the ratio of the initial molar concentration of the ATRP initiator to DMAA. This suggested that the chain length of the spacing segment could be tuned by varying the initial molar ratio of the ATRP initiator and monomer. The first block was terminated with chlorine allowing it to act as a macro-initiator for further ATRP. The second block of the polymer was synthesized with DMAA and an acryl NHS monomer at approximately a 2:1 ratio. The molecular weight and number of NHS groups per polymer chain were determined by 1H NMR (Table 1 and Supplemental Figs. 2e7). The second block polymer contained NHS groups so that it could be used to conjugate a number of different targeting or therapeutic entities (Fig. 2B-2). The benzyl ester of the initiator terminus of the molecule was converted to an active carboxyl group that could be then further modified with binding or targeting moieties. Herein, we describe the first use of the base polymer to generate a bone targeting cell-reactive construct. To synthesize the bone targeting polymer, the pendant NHS groups on the second block polymer were reacted with the aminobisphosphonate, alendronate, followed by cleavage of the benzyl group on the other end of the polymer, thus producing the third block polymer (Figs. 2B-3). Bisphosphonates are avid HA binding molecules that have been shown to provide HA binding capabilities to polymers. The molecular weight and the number of bisphosphonate units per polymer chain for the second block polymer were estimated by 1H NMR (Table 1 and Supplemental Figs. 2e7). To provide cell binding capability to the third block polymer, the

Fig. 5. Polymer loss from MSC surface. MSCs were modified with polymers of different lengths (BT-1, BT-2, and BT-3) as described in Materials and methods. Cells were imaged at the indicated times and the fluorescence of live cells was measured using a cell analyzer program with Image J. The fluorescence per square microns was calculated at the indicated time points.

9454

S. D'Souza et al. / Biomaterials 35 (2014) 9447e9458

carboxyl group was converted to an NHS group by using EDC and NHS (Fig. 2B-4). The terminal NHS allowed the polymer to readily react with amine groups such as those exposed on membrane proteins. Thus, we generated three bone targeting polymers with molecular weights of 10.1 kD (BT-1), 21.8 kD (BT-2) and 45.0 kD (BT-3) using ATRP, each containing 3.1, 2.8 and 1.7 bisphosphonate unit per polymer chain, respectively (Table 1). In addition to the bone targeting polymers, we also synthesized control polymers based on BT-1. The first control, polymer C1, substituted aminobutanol for alendronate and had a carboxylterminal group instead of the NHS and thus would bind to neither bone nor cells (Figs. 2B-5). The second control, polymer C2, with a molecular weight of 10.1 kD, had the same aminobutanol substitution but also contained a terminal NHS group that would bind to cells but not to HA or bone (Fig. 2B-5). 3.2. Binding affinity of polymers to HA The affinity of the bisphosphonate modified polymer for HA was determined in an equilibrium binding assay. We incubated increasing amounts of polymer BT-1 with HA crystals for 2 h at 37  C. The HA crystals were then washed to remove unbound polymer and the polymer-HA complex was dissolved in 0.1 M HCl. The relative fluorescence was measured and the values of the test samples were calibrated with known polymer concentrations (Fig. 3A). The dissociation constant (Kd) of the polymer was determined to be 0.45 mM. The observed Kd of alendronate to rat bone fragments has been reported to be 1 mM [5] and alendronate is known to bind to human bone much more tightly (Kd of 0.072 mM) [30]. Fluorescent and brightfield images of the binding of polymers BT-1 to HA showed that all the HA crystals in the experiment were coated with polymer BT-1 (Fig. 3B). When the control polymer C2 was mixed with HA, only a small amount of background fluorescence was detected. The background signal was likely due, in part, to non-specific adsorption of the polymer on the HA. Thus, our data demonstrated that the ATRP-synthesized bone targeting polymer had an affinity for HA crystals in vitro. 3.3. Modification of cells with bone targeting polymer Mixing NHS-containing polymers with cells resulted in the reaction of the polymer NHS groups with membrane proteins, as we

have shown previously for cells and tissues [31]. The HL-60 cell line used in these experiments had very low levels of intrinsic adherence to any surface [32]. Mixing polymer BT-1 with HL-60 cells resulted in labeling of all or nearly all of the cells with polymer (Supplemental Fig. 8A). HL-60 cells were incubated with varying concentrations of the polymer BT-1 (1 mg/mL, 2 mg/mL, 3 mg/mL and 4 mg/mL) and we determined that the optimal concentration of the polymer for adequate binding to the cell surface of HL-60 cells was 1 mg/mL at an average cell count of 1  106 mL1 for 10 min at 37  C in a phosphate buffered pH 8.0 solution. The observed relative fluorescence was compared to a calibration curve and we found that under these conditions, approximately 1.76  107 ± 7  105 molecules of polymer BT-1 reacted with each HL-60 cell. The BT-1, BT-2, and BT-3 polymers were individually tested for surface modification with MSCs. Comparison of the fluorescent and brightfield images showed that all the MSCs present were modified by the polymers (Fig. 4). Prior studies have shown that the length of polymer affected the fluorescence intensity of polymers [33,34]. We speculate that the longer polymers BT-2 and BT-3 may have shielded the fluorescein molecule and thus the fluorescence emitted by these polymers was not as intense as the shorter polymer (BT-1). To determine the number of polymer molecules on each MSC, we measured the relative fluorescence of the MSCs after modification and compared the relative fluorescence of each sample to the respective calibration curves. We found that once again approximately 107 polymer molecules reacted per MSC (Table 2). Our data demonstrated that irrespective of polymer length, similar numbers of polymer molecules bound to the surface of MSCs. Based on the diameter of the MSCs (~15 microns) and the number of polymer molecules per MSC (~107), we calculated that each polymer molecule occupied approximately 80 nm2 of the cell surface. In order to analyze the residence time of polymer modified proteins in MSC cell membranes, we imaged the polymer engineered MSCs over the course of 24 h. We found that for all the polymers tested approximately 50% of the fluorescence was detected after 4e8 h post-modification (Fig. 5). Shedding of cell surface components is an essential aspect of membrane turnover. A number of studies have utilized radiolabeling of proteins to study membrane protein turnover [35e37]. These studies have demonstrated that the majority of cells exhibit a biphasic pattern of protein shedding with many proteins being turned over within 2e24 h, and thereafter proteins are turned over gradually with a half-life of up to 7 days. Further studies have shown that the initial loss of cell surface proteins could be due to the preparatory steps including changing of media, washing, and centrifugation [38,39]. In our experiments, we performed a number of pre-modification steps, including washing the cells, centrifugation, and trypsinization. These pre-modification steps could have contributed to the turnover of cell surface proteins on the MSCs which we observed over the first 24 h post-modification (Fig. 5).

Table 3 MSC survival postemodification. MSCs were modified with polymers (BT-1, BT-2, and BT-3) as described in Materials and methods. To determine viability of cells, we focused on MSCs and incorporated trypan blue in the cell suspension after modification and imaged the cells. We counted the number of clear cells and blue cells and determined the percentage of dead cells. % Dead MSC Fig. 6. Proliferation of polymer modified MSCs. MSCs were modified with polymers of varying lengths (BT-1, BT-2, and BT-3) as described in Materials and methods. Cells were plated in a 96 well plate and incubated at 37  C for different lengths of time as indicated in the graph above and proliferation was assayed by a MTS assay. The Y-axis represents absorbance at 490 nm.

Unmodified-MSC BT-1 BT-2 BT-3

Avg

SD

3.43 3.62 15.76 7.05

0.69 2.17 1.08 1.81

S. D'Souza et al. / Biomaterials 35 (2014) 9447e9458

9455

Fig. 7. Osteogenic and adipogenic differentiation of polymer modified MSCs. MSCs were modified with polymers of varying lengths (BT-1, BT-2, and BT-3) as described in Materials and methods and then cultured under osteogenic or adipogenic conditions. Osteogenic differentiation of the MSCs was determined by staining cells for alkaline phosphatase and adipogenic differentiation was determined by staining the cells with Oil red O. Cells were cultured in osteogenic media (A, C, E, G) or control media (B, D, F, H). Cells were cultured in adipogenic media (I, K, M, O) or control media (J, L, N, P). [A, B, I and J e MSCs modified with polymer BT-1; C, D, K and L e MSCs modified with polymer BT-2; E, F, M and N e MSCs modified with polymer BT-3; G, H, O and P e unmodified MSCs]. Scale bar for A-H located in bottom right of H; scale bar for I-P located in bottom right of P.

3.4. Characterization of polymer modified cells In order to determine if polymers (BT-1, BT-2, and BT-3) were toxic to cells, HL-60 cells were labeled with polymer BT-1, and

MSCs were labeled with polymers BT-1, BT-2, and BT-3, and the impact of the polymers on cell proliferation was determined. Comparison of the growth of unlabeled cells with the polymer labeled cells showed that the polymers were not cytotoxic to either

Fig. 8. Binding of polymer modified MSCs to rat bone fragments. MSCs were modified with polymer BT-1 or control polymer C2, and polymer modified MSCs were incubated with bone for 20 min as described in Materials and methods. The cell-polymer-bone aggregates were imaged using a multispectral microscope. A and B e MSCs modified with polymer BT-1; C and D e MSCs modified with control polymer C2.

9456

S. D'Souza et al. / Biomaterials 35 (2014) 9447e9458

HL-60 cells or MSCs (Fig. 6 and Supplemental Fig. 8B). The cell membrane is, of course, selective in the transport of materials across the cell. Viable cells exclude trypan blue, a dye that is normally used to determine cell viability, however when the cell membrane is disrupted, trypan blue can pass through the membrane and the cells stain blue. To determine viability of cells, we focused on MSCs and incorporated trypan blue in the cell suspension after modification and imaged the cells under fluorescence and white light. We counted the number of clear cells and blue cells and determined the percentage of dead cells. Our data demonstrated that polymer (BT-1, BT-2, and BT-3) modification did not substantially affect the survival of MSCs (Table 3). Cell survival of BT-1 and BT-3 modified MSCs was comparable to unmodified MSCs. The survival of BT-2 modified MSCs was slightly lower compared to BT1 and BT-3 modified MSCs and unmodified MSCs, however the proliferative potential of the 21.8 kD modified MSCs appeared to be on par with the BT-1 and BT-3 modified MSCs and unmodified MSCs (Fig. 6). MSCs have the ability to differentiate into a number of different cell types [11]. To determine if the bone targeting polymer modified MSCs were able to differentiate; we isolated primary MSCs from rat tibias and passaged the MSCs four times before using the MSCs in differentiation assays. We characterized the polymers (BT-1, BT-2 and BT-3) modified rat MSCs and determined that the modification of rat MSCs was comparable to the modification of MSCs obtained from mice. We determined that approximately 107 polymer molecules reacted per rat MSC and that polymer modification of rat MSCs did not affect proliferation (Supplemental Fig. 9). We cultured the polymer modified rat MSCs under osteogenic or adipogenic conditions. Osteogenesis and adipogenesis was determined by alkaline phosphatase and Oil red O staining, respectively. Polymer modified MSCs successfully differentiated into osteoblasts and adipocytes (Fig. 7). Thus, collectively, these data suggest that selective modification of the cell membrane proteins by the bone targeting polymers did not impede the ligand-receptor signaling cascades that are crucial for proliferation and differentiation of MSCs.

Fig. 10. Binding of polymer modified MSCs to human bone plates. MSCs were modified with the polymers (BT-1, BT-2, and BT-3) and then incubated with human bone plates for different times as indicated. The adherent cells were imaged under brightfield and the number of cells/cm2 was calculated as described. The surface coverages of the cells were plotted as a function of time.

3.5. Binding affinity of polymer modified cells to bone and HA We explored the selectivity of binding modified HL-60 cells to bone by comparing cells labeled with polymer BT-1 to cells labeled with polymer C2 (Supplemental Fig. 8CeE). We showed that the amount of fluorescence of polymer BT-1 labeled HL-60 cells to bone was significantly higher than C2 labeled HL-60 cells to bone (Supplemental Fig. 8C and D). Additionally, 13 random images from each condition were scanned and the amount of fluorescence was quantified (Supplemental Fig. 8E). We also determined the selective binding of polymer BT-1 modified MSCs to bone fragments (Fig. 8). Comparison of images from polymer BT-1 modified MSCs to polymer C2 modified MSCs demonstrated that polymer BT-1 aided in

Fig. 9. Binding of polymer modified MSCs to HA. MSCs were modified with polymer BT-3 and incubated with HA particles as described in Materials and methods. DAPI was used to detect the bound MSCs. Cells were imaged under fluorescence (A, C, E, G, I, K) and brightfield (B, D, F, H, J, K). A, B, E, F, I, and J e HA incubated with polymer BT-3 modified MSCs; C, D, G, H, K, and L e HA incubated with control MSCs.

S. D'Souza et al. / Biomaterials 35 (2014) 9447e9458

binding of cells to bone fragments (Fig. 8). The polymer BT-1 modified MSCs caused the bone fragments to aggregate thus acting as a bridge between bone fragments (Fig. 8A and B). Incubating HA with BT-3 engineered MSCs resulted in an increased number of MSCs that bound to HA compared to unmodified MSCs (Fig. 9). We also found that MSCs modified with the longer polymer chains (polymer BT-2 and BT-3) bound more effectively to human bone plates than unmodified or BT-1 modified MSCs (Fig. 10 and Supplemental Fig. 10). Previous reports have shown that bisphosphonate can carry proteins to the bone [40,41]. In our study, we report a dual functioning polymer that covalently binds the surface of cells as well as binds hydroxyapaptite/bone. Thus, our studies collectively demonstrated that membrane engineering of HL-60 cells and MSCs with the ATRP-synthesized bone binding polymer resulted in a bone targetable cell. 4. Conclusions We have described the synthesis of a versatile polymer capable of increasing the binding of cells to a target tissue. The binding of the bone targeting polymers to HA crystals was consistent with the binding behavior of the targeting moiety (alendronate) for bone fragments. When the bisphosphonate-containing polymer was covalently coupled to cells and then exposed to bone fragments, we found that cells associated with the bone fragments in significantly higher numbers than cells that had been bound conjugated with a control polymer that lacked bisphosphonate. Fluorescence microscopy showed that cells were uniformly coated with the ATRP designed polymers. The cells modified with the bone binding polymers caused the bone fragments to aggregate and the longer bone binding polymers bound bone considerably better versus the shorter polymers. We have initiated a study of, to what degree ATRP-synthesized bone binding polymers that are attached to MSCs will partition to a bone injury site and enhance healing in vivo. Acknowledgments The original artwork in Fig. 1 was designed by Randall Mckenzie. The manuscript was written through contributions of all authors. The authors would like to thank Dr. Neill Turner for assisting with multispectral imaging (University of Pittsburgh, Pittsburgh, PA) and Helena Zimmermann for assisting with bone binding analysis. Funding Sources: This work was supported by the Advanced Regenerative Medicine grant from Pittsburgh Tissue Engineering Initiative (Grant no. W81XWH-10-1-0618), The Institute for Transfusion Medicine, and by an award from the Government of the Republic of Kazakhstan via Nazarbayev University. NMR instrumentation at CMU was partially supported by NSF (CHE-0130903 and CHE-1039870). Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2014.07.041. References [1] Ruoslahti E, Bhatia SN, Sailor MJ. Targeting of drugs and nanoparticles to tumors. J Cell Biol 2010;188:759e68. [2] Kopecek J, Kopeckova P. HPMA copolymers: origins, early developments, present, and future. Adv Drug Deliv Rev 2010;62:122e49. [3] Zhang S, Gangal G, Uludag H. ‘Magic bullets’ for bone diseases: progress in rational design of bone-seeking medicinal agents. Chem Soc Rev 2007;36: 507e31. [4] Wang D, Miller SC, Kopeckova P, Kopecek J. Bone-targeting macromolecular therapeutics. Adv Drug Deliv Rev 2005;57:1049e76.

9457

[5] Sato M, Grasser W, Endo N, Akins R, Simmons H, Thompson DD, et al. Bisphosphonate action. alendronate localization in rat bone and effects on osteoclast ultrastructure. J Clin Invest 1991;88:2095e105. [6] Russell RG, Rogers MJ. Bisphosphonates: from the laboratory to the clinic and back again. Bone 1999;25:97e106. [7] Woo SB, Hellstein JW, Kalmar JR. Narrative [corrected] review: bisphosphonates and osteonecrosis of the jaws. Ann Intern Med 2006;144:753e61. [8] Zhang S, Wright JE, Ozber N, Uludag H. The interaction of cationic polymers and their bisphosphonate derivatives with hydroxyapatite. Macromol Biosci 2007;7:656e70. [9] Wang D, Miller S, Sima M, Kopeckova P, Kopecek J. Synthesis and evaluation of water-soluble polymeric bone-targeted drug delivery systems. Bioconjug Chem 2003;14:853e9. [10] Segal E, Pan H, Ofek P, Udagawa T, Kopeckova P, Kopecek J, et al. Targeting angiogenesis-dependent calcified neoplasms using combined polymer therapeutics. PLoS One 2009;4. e5233. [11] Caplan AI. Mesenchymal stem cells. J Orthop Res 1991;9:641e50. [12] Colter DC, Class R, DiGirolamo CM, Prockop DJ. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci U S A 2000;97:3213e8. [13] Sekiya I, Larson BL, Smith JR, Pochampally R, Cui JG, Prockop DJ. Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells 2002;20: 530e41. [14] Fouillard L, Chapel A, Bories D, Bouchet S, Costa JM, Rouard H, et al. Infusion of allogeneic-related HLA mismatched mesenchymal stem cells for the treatment of incomplete engraftment following autologous haematopoietic stem cell transplantation. Leukemia 2007;21:568e70. [15] Chen SL, Fang WW, Ye F, Liu YH, Qian J, Shan SJ, et al. Effect on left ventricular function of intracoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. Am J Cardiol 2004;94:92e5. [16] Lee PH, Kim JW, Bang OY, Ahn YH, Joo IS, Huh K. Autologous mesenchymal stem cell therapy delays the progression of neurological deficits in patients with multiple system atrophy. Clin Pharmacol Ther 2008;83:723e30. [17] Lee RH, Pulin AA, Seo MJ, Kota DJ, Ylostalo J, Larson BL, et al. Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell 2009;5:54e63. [18] Bang OY, Lee JS, Lee PH, Lee G. Autologous mesenchymal stem cell transplantation in stroke patients. Ann Neurol 2005;57:874e82. [19] Kraitchman DL, Tatsumi M, Gilson WD, Ishimori T, Kedziorek D, Walczak P, et al. Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction. Circulation 2005;112:1451e61. [20] Phinney DG, Prockop DJ. Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repairecurrent views. Stem Cells 2007;25:2896e902. [21] Hasegawa T, Miwa M, Sakai Y, Niikura T, Lee SY, Oe K, et al. Efficient cellseeding into scaffolds improves bone formation. J Dent Res 2010;89: 854e9. [22] Panza JL, Wagner WR, Rilo HL, Rao RH, Beckman EJ, Russell AJ. Treatment of rat pancreatic islets with reactive PEG. Biomaterials 2000;21:1155e64. [23] Deible CR, Petrosko P, Johnson PC, Beckman EJ, Russell AJ, Wagner WR. Molecular barriers to biomaterial thrombosis by modification of surface proteins with polyethylene glycol. Biomaterials 1998;19:1885e93. [24] Burchenal JE, Deible CR, Deglau TE, Russell AJ, Beckman EJ, Wagner WR. Polyethylene glycol diisocyanate decreases platelet deposition after balloon injury of rabbit femoral arteries. J Thromb Thrombolysis 2002;13: 27e33. [25] Helfer BM, Balducci A, Nelson AD, Janjic JM, Gil RR, Kalinski P, et al. Functional assessment of human dendritic cells labeled for in vivo (19)F magnetic resonance imaging cell tracking. Cytotherapy 2010;12:238e50. [26] Guan M, Yao W, Liu R, Lam KS, Nolta J, Jia J, et al. Directing mesenchymal stem cells to bone to augment bone formation and increase bone mass. Nat Med 2012;18:456e62. [27] Murata H, Pruker O, Ruhe J. Synthesis of functionalized polymer monolayers from active ester brushes. Macromolecules 2007;40:5497e503. [28] Ciampolini M, Nardi N. Five-coordinated high-spin complexes of bivalent cobalt, nickel, and copper with tris(2-dimethylaminoethyl)amine. Inorg Chem 1966;5:41e4. [29] Matyjaszewski K, Xia J. Atom transfer radical polymerization. Chem Rev 2001;101:2921e90. [30] Leu CT, Luegmayr E, Freedman LP, Rodan GA, Reszka AA. Relative binding affinities of bisphosphonates for human bone and relationship to antiresorptive efficacy. Bone 2006;38:628e36. [31] Xu H, Kaar JL, Russell AJ, Wagner WR. Characterizing the modification of surface proteins with poly(ethylene glycol) to interrupt platelet adhesion. Biomaterials 2006;27:3125e35. [32] Erzurum SC, Kus ML, Bohse C, Elson EL, Worthen GS. Mechanical properties of HL60 cells: role of stimulation and differentiation in retention in capillarysized pores. Am J Respir Cell Mol Biol 1991;5:230e41. [33] Miki K, Oride K, Kimura A, Kuramochi Y, Matsuoka H, Harada H, et al. Influence of side chain length on fluorescence intensity of ROMP-based polymeric nanoparticles and their tumor specificity in in-vivo tumor imaging. Small 2011;7:3536e47.

9458

S. D'Souza et al. / Biomaterials 35 (2014) 9447e9458

[34] Gaylord BS, Wang S, Heeger AJ, Bazan GC. Water-soluble conjugated oligomers: effect of chain length and aggregation on photoluminescencequenching efficiencies. J Am Chem Soc 2001;123:6417e8. [35] Baumann H, Doyle D. Turnover of plasma membrane glycoproteins and glycolipids of hepatoma tissue culture cells. J Biol Chem 1978;253:4408e18. [36] Doetschman TC. Cell surface turnover and shedding of fucosyl-glycoprotein in L6 cells. Cell Biol Int Rep 1980;4:379e90. [37] Chu FF, Doyle D. Turnover of plasma membrane proteins in rat hepatoma cells and primary cultures of rat hepatocytes. J Biol Chem 1985;260: 3097e107.

[38] Hubbard AL, Cohn ZA. Externally disposed plasma membrane proteins. II. Metabolic fate of iodinated polypeptides of mouse L cells. J Cell Biol 1975;64: 461e79. [39] Tweto J, Doyle D. Turnover of the plasma membrane proteins of hepatoma tissue culture cells. J Biol Chem 1976;251:872e82. [40] Gil L, Han Y, Opas EE, Rodan GA, Ruel R, Seedor JG, et al. Prostaglandin E2bisphosphonate conjugates: potential agents for treatment of osteoporosis. Bioorg Med Chem 1999;7:901e19. [41] Uludag H, Yang J. Targeting systemically administered proteins to bone by bisphosphonate conjugation. Biotechnol Prog 2002;18:604e11.