Photothermally Targeted Thermosensitive Polymer-Masked

Jun 2, 2014 - Mai Nguyen , Xiaonan Sun , Emmanuelle Lacaze , Pamina Martina Winkler , Andreas Hohenau , Joachim R. Krenn , Céline Bourdillon ...
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Photothermally-targeted thermosensitive polymer-masked nanoparticles Aoune Barhoumi, Weiping Wang, David Zurakowski, Robert Langer, and Daniel S. Kohane Nano Lett., Just Accepted Manuscript • DOI: 10.1021/nl403733z • Publication Date (Web): 02 Jun 2014 Downloaded from http://pubs.acs.org on June 4, 2014

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Photothermally-targeted thermosensitive polymer-masked nanoparticles Aoune Barhoumi†‡, Weiping Wang†‡, David Zurakowski§, Robert S. Langer‡ and Daniel S. Kohane*†‡ †

Laboratory for Biomaterials and Drug Delivery, §Department of Anesthesiology, Division of Critical Care Medicine, Children’s Hospital Boston, Harvard Medical School, 300 Longwood Avenue, Boston, Massachusetts 02115, United States ‡ David H. Koch Institutes for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States

Abstract The targeted delivery of therapeutic cargos using noninvasive stimuli has the potential to improve efficacy and reduce off-target effects (toxicity). Here, we demonstrate a targeting mechanism that uses a thermoresponsive copolymer to mask a peptide ligand that binds a widely distributed receptor (integrin β1) on the surface of silica core-gold shell nanoparticles. The nanoparticles could efficiently convert NIR light into heat, which caused the copolymer to collapse, exposing the ligand peptide, allowing cell binding. The use of NIR light could allow targeting of plasmonic nanoparticles deep within tissues. This approach could be extended to a variety of applications including photothermal therapy and drug delivery. Keywords: Au nanoshells, Photo-targeting, pNIPAAm, Shielding, Temperatureresponsive, YIGSR

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Many drug delivery systems are limited in that their therapeutic payloads are distributed throughout the entire body, rather than at the desired site of effect.1,2 This can potentially reduce efficacy, and increase toxicity to off-target cells.3-5 One approach to enhancing site-specific delivery is active targeting wherein, for example, a specific ligand is used to guide a drug or nanoparticle to a particular cell type.6 One difficulty with this approach is that there are frequently conditions or tissues for which no specific ligand is known. Consequently, we have previously developed nanoparticles functionalized with ligands that could bind a wide range of tissues, but in which the ligands are prevented from binding by photocleavable chemical protection groups (“caged”) until irradiated with the appropriate wavelength of light.1,2,6 Upon irradiation, the caging groups come off, exposing the active ligands, which then bind to tissues. By these means, the particles are immobilized at the site of irradiation. A principal shortcoming of photocleavable chemical caging is that ultraviolet (UV) light is required to cleave the covalent bond linking the caging group to the ligand. UV light is readily absorbed by tissues, limiting tissue penetration, and can cause phototoxicity due to its high energy.3-5,7 Near-infrared (NIR) light in the so-called “NIR water window” (690-900 nm) has deeper tissue penetration and causes less photodamage.6,8 However, it does not possess enough energy to break a covalent bond. Here we have developed a NIR-sensitive system that allows phototargeting of nanoparticles using NIR light. Ligands immobilized on Au nanoshells are prevented from binding by a co-immobilized layer of thermosensitive polymer. We have hypothesized that NIR irradiation will heat the nanoshells, causing collapse of the thermosensitive layer, revealing the ligands and allowing binding (Figure 1a). For the nanoparticle, we have selected Au nanoshells, plasmonic nanoparticles where the plasmon-resonance frequencies are readily controlled by the relative inner and outer radii of the metallic shell layer.9 Au nanoshells can be easily engineered to absorb light in the NIR range and efficiently convert it into heat due to their large absorption cross-section and inability to emit light.10 YIGSR was selected as the ligand as it binds to integrin β1 on the cell membrane of a variety cell types including stromal and endothelial cells.6,11 It would be possible to use more specific ligand (binds to specific cell type or line) which may increase the specificity and controllability of the nanoparticle binding however, our approach does not focus on a specific cell type or line but target any cell in a given irradiated location. Poly(NIPAAm-co-AAm), a derivative of the widely used thermosensitive polymer poly(N-isopropylacrylamine) (pNIPAAm)12 was used to create the collapsible thermosensitive layer (Figure 1b). Plasmonic nanoparticles coated with NIPAAm copolymer have been extensively studied for drug delivery applications. The heat generated on the surface of the plasmonic nanoparticle causes the thermoresponsive polymer to collapse and the drug, initially encapsulated within the nanoparticle12 or the polymer, to be released. However, using pNIPAAm to mask a ligand on the surface of plasmonic nanoparticle for nanoparticle targeting has not been reported yet.13,14 Recently, electrical heating of gold microstructures on a macroscopic surface has been used to collapse an overlying layer of NIPAAm copolymer, exposing a biologically active molecule (kinesin-1).15 Moreover, DNA and NIPAAm copolymer were co-assembled on Au nanoparticles where DNA can be reversibly exposed and masked upon exposure to temperature stimulus.16 pNIPAAm has a low critical solution temperature (LCST) of 32 oC,17 meaning that

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below 32 oC it is hydrophilic and has an extended conformation in water. Above 32 oC pNIPAAm undergoes a phase transition and becomes hydrophobic, and the polymer collapses. The integration of acrylamide (AAm) into the polymer chain allows tuning of the LCST across a wide range of temperatures. To avoid copolymer collapse at body temperature (37 oC) and even in the context of other medical (e.g. fever), or environmental (e.g. external heat) conditions we synthesized a pNIPAAm-co-pAAm copolymer (NIPAAm:AAm molar ratio of 75:25, MW ≈ 120 000 Da) with a LSCT at 55 o C (Figure 2a).12 The copolymer was synthesized using atom-transfer radical polymerization starting from a disulfide initiator allowing subsequent copolymer covalent binding to Au nanoshells.12 The Au nanoshells’ plasmon-resonance absorption red-shifted by ~ 12 nm (Figure 2b) indicating successful copolymer coating.18 The coating significantly enhanced the particles’ long-term stability in water and phosphate buffered saline (PBS). Suspending non-coated nanoshells in PBS caused them to aggregate within a few minutes, which manifests as changes in the suspension color, precipitation, and broadening of the optical extinction spectrum. The long-term stability was verified by the unchanging optical extinction spectrum of NIPAAm copolymer-coated nanoshells over long periods of time (up to one year). Dynamic light scattering (DLS) measurements showed that the hydrodynamic diameters of copolymer-covered nanoshells in a 0.5% (w/v) suspension decreased from ~ 300 nm at 25 oC (copolymer extended) to ~ 200 nm at 70 oC (copolymer collapsed) when thermally heated. The hydrodynamic diameter of copolymer-coated nanoshell is affected by the shape (dynamic) of the particles as well as the solvent (hydro) thus the hydrodynamic diameter variation due to copolymer collapse does not reveal the real diameter variation. The hydrodynamic diameter variation was reversible (Figure 2c). This reversible size change could also be induced by NIR light (808 nm, 1.2 W/cm2; Figure 2d). The change in hydrodynamic diameter (defined as          ( ) × 100 ± STDEV ) was     

34 ± 8% after thermal heating to 70 oC and 16 ± 2% after laser-induced heating. A lower diameter increase was recorded with photothermal heating is mainly due to the experimental conditions. Note that in the thermal heating experiments, the sample was heated to 70oC by the DLS machine and that temperature was maintained during diameter measurement. However, in laser-induced heating the sample was irradiated outside of the DLS machine then placed inside to perform diameter measurements (which take around 4 minutes), which likely resulted in heat dissipation. Importantly, the temperature of the NIR-irradiated suspension only rose from 26.4 oC (laser off) to 29 oC (laser on), which is lower than the copolymer LCST, suggesting that the collapse of the copolymer coating on the Au nanoshells was due to local heat generation, not bulk heating of the water. This is important in avoiding unwanted tissue hyperthermia. The size of the ligand peptide was critical in this design since it had to be covered when the copolymer was extended and exposed when the copolymer was collapsed (Fig. 1a). The copolymer layer had a uniform thickness of ~ 4 nm in the dry state, as determined by transmission electron microscopy (TEM) (Figure 3). Based on copolymer size, molecular weight, hydrodynamic diameter oscillation, and reported correlation between copolymer MW and size,12 we estimated that the thickness of the copolymer coating in the extended state was ~ 6 nm. Consequently, we designed a 5 nm-long 14amino acids ligand peptide with the YIGSR recognition sequence at one end, 2 cysteines 3

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on the other end to enable binding to the Au nanoshell, and 7 glycines acting as a spacer in the middle. A fluorescent dye (5-FAM, C21H12O7) was incorporated toward the outward-facing end of the glycine spacer (H-Cys-Cys-Gly-Gly-Gly-Gly-Gly-Lys(5FAM)-Gly-Gly-Tyr-IIe-Gly-Ser-Arg-OH) to minimize fluorescence quenching by the nanoshell Au surface.19 The ratio of copolymer to peptide ligand was set at 100:1 to encourage covering of the binding peptide when the thermosensitive copolymer is in the extended conformation inhibiting their binding functionality. Au nanoshells were then incubated with an excess of the copolymer/ligand peptide mixture for 24 hours. To verify the presence of peptide, washed Au nanoshells coated in peptide and copolymer were incubated with freshly prepared displacing solution (12 mM mercaptoethanol in PBS, pH = 7) overnight.10 After centrifugation, the fluorescence intensity of the collected supernatant was measured and compared to the fluorescence intensity of the baseline (supernatant of the nanoshells coated in peptide and NIPAAm copolymer before incubation with displacing solution). The mercaptoethanol molecules readily displace the copolymer and peptide molecules bound to Au nanoshells via a thiol exchange reaction. This process is rapid and efficient because mercaptoethanol can form a tightly packed self-assembling monolayer due to its greater packing energy via Van der Waals forces, removing all peptide and copolymer molecules.20 Therefore the fluorescent intensity of the supernatant is correlated to the number of peptide molecules that were bound to Au nanoshells. The baseline showed no fluorescence, indicating the absence of free peptide in the nanoparticle suspension after thorough washing. The supernatant of the nanoshells coated with peptide and copolymer after incubation with displacing solution showed a strong fluorescence intensity demonstrating the presence of peptide on nanoshells surface. Au nanoshells coated with peptide and copolymer were added to human umbilical vein endothelial cells (HUVECs) were incubated. The cells were exposed to NIR light (808 cw laser, 1.2 W/cm2) for 4 min or maintained in the dark, then incubated for 30 min in the dark. Unbound nanoparticles were discarded by decantation. To assess nanoshellcell binding, inductively coupled plasma mass spectroscopy (ICP MS) was used to measure the concentration of Au in cell samples incubated with copolymer-coated nanoshells (with and without peptide). Both groups were irradiated or not. Higher Au concentration indicated increased nanoshells binding. Two-way ANOVA revealed very strong effects of light (F = 148.46, P < 0.001) and peptide (F = 13.07, P < 0.001) on Au concentration. However, the two-way interaction between light and peptide (F = 5.03, P = 0.028) indicated that the effect of peptide was conditionally dependent on whether or not light was used (Figure 4). When light was not used, the mean Au concentrations were 24.3 ± 9.3 µg/kg for particles without peptide and 30.8 ± 5.3 µg/kg for particles with peptide (P = 0.62; i.e. the presence of peptide had no statistically significant effect). In contrast, when light was used the Au concentrations were 71.4 ± 20.9 µg/mg for particles without peptide and 99.2 ± 34.0 µg/mg for particles with peptide (P < 0.001). The magnitude of the increase in Au concentration between the two groups (with and without peptide) was more significant when light was used, indicating that nanoshell binding was enhanced by the presence of the peptide only when cells are exposed to light. Au nanoparticles have been extensively studied as vehicles for drug delivery. The system described here provides a platform for remotely controlling the delivery of such nanoparticle carriers using NIR light. For drug delivery applications, active molecules

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can be anchored to the nanoshell surface through thermally-10,13 or pH-sensitive21 bonds, by disulfide bonds that will be reduced in the presence of glutathione (GSH),22 or by photocleavable moieties.23,24 Drug molecules can also be anchored to NIPAAm copolymer via pH-sensitive linkers.25 The active payloads can be released (actively or passively) once the copolymer-covered nanoshells are delivered to the desired location. These nanoparticles could also provide targeted photothermal therapy26 with or without concurrent drug delivery. The approach demonstrated here could be generalized to other plasmonic nanoparticles including Au nanorods, nanocages, and hollow nanoshells since it is based on the plasmonic property of the nanoparticle rather than the shape. We have designed and synthesized copolymer-covered Au nanoshells that expose targeting ligands upon NIR irradiation, enabling cell binding. This process exploits the photothermal properties of plasmonic nanoparticles to control nanoparticle binding to cells. This approach has the potential to provide spatiotemporal control of nanoparticle binding, enhancing therapeutic efficacy and minimizing toxicity.

Materials and Methods: Chemicals and Materials: N-isopropylacrylamide NIPAAm and Acrylamide AAm were obtained from Sigma-Aldrich (MO, USA) and recrystallized in hexane and methanol respectively prior to use. N,N,N’,N’,N’-pentamethyldiethylenetriamine (PMDETA) and copper(I) bromide and bis[2-(2′-bromoisobutyryloxy)ethyl]disulfide were obtained from Sigma-Aldrich and used as received. HUVECs were obtained from Lonza (NJ, USA). The ligand peptide was ordered from Ananspec (CA, USA) Instruments: Delsa Nano C particle analyzer (Beckman Coulter, CA, USA) was used for nanoparticle sizing. 8453 UV-Vis spectrophotometer (Agilent, CA, USA) was used to acquire UV-Vis spectra. FSX 100 (Olympus, PA, USA) was used for fluorescence imaging. CW Laser diode LDX 3415- 808 from RPMC laser Inc. (MO, USA) fibercoupled to a 12.7 nm collimator from Ceramoptec (MA, USA) was used for laser irradiation. Copolymer synthesis: 1.53 g of NIPAAm, 0.32 g of AAm, 35µl of PMDETA, 0.015 g of bis[2-(2′-bromoisobutyryloxy)ethyl]disulfide, 18 ml of deionized water and 12 ml of methanol were mixed in a three-neck round-bottom flask. The reaction solution was degassed through 3 cycles of freeze–pump–thaw. Next, 0.01 g of CuBr was added. The flask was filled with nitrogen and the mixture was left to melt at room temperature. The reaction solution was left overnight at room temperature under magnetic stirring. After evaporation of the solvent, the crude product was dissolved in water and purified by dialysis to yield the pNIPAAm-co-pAAm copolymer. Au Nanoshells Fabrication: Au nanoshells were synthesized as previously described.10 Copolymer and ligand peptide binding to Au nanoshells: copolymer and peptide ligand were mixed at 100:1 molar ratio and incubated with Au nanoshells. After 24 hours incubation, the solution mixture was centrifuged and the supernatant was discarded. Photo-controlled cell binding experiments: HUVECs were seeded into 48-well plate (20,000 cells per well) in EGM-2 media and grown for 24 h. The media was then

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removed and copolymer-coated Au nanoshells (with and without peptide) suspended in media (200 µl, 5mg/ml) was added to each well. Each well was irradiated (CW laser, 808 nm at 1.2 W/cm2) for 4 min. Negative control cells were kept in dark without irradiation. After incubation at 37 oC for 30 min in dark, the media was removed and the cells were gently washed with PBS buffer (10 mM, pH 7.4) two times to remove unbound nanoshells. For ICP-MS, cells were trypsinized and collected. For each sample 4 wells were treated and Five readings where taken from each well. Statistical Analysis: Two-way analysis of variance (ANOVA) with repeated measures was used to assess the effects of light and peptide on Au concentration. The light × peptide interaction F-test was calculated to ascertain whether the change in Au concentration for the two groups (with and without peptide) depended significantly on whether light was used or not. Statistical analysis was performed using IBM SPSS Statistics (version 21.0, IBM, Armonk, NY). Two-tailed P < 0.05 was considered statistically significant with post-hoc multiple comparisons using the Tukey procedure.27

References: (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12)

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Timko, B.; Whitehead, K.; Gao, W.; Kohane, D.; Farokhzad, O.; Anderson, D.; Langer, R. Annu. Rev. Mater. Res. 2011, 41, 1–20. Gelperina, S.; Kisich, K.; Iseman, M. D.; Heifets, L. Am J Respir Crit Care Med 2005, 172, 1487–1490. Kadam, R. S.; Bourne, D. W. A.; Kompella, U. B. Drug Metabolism and Disposition 2012, 40, 1380–1388. Sinha, R. Molecular Cancer Therapeutics 2006, 5, 1909–1917. Zhang, L.; Gu, F. X.; Chan, J. M.; Wang, A. Z.; Langer, R. S.; Farokhzad, O. C. Clin Pharmacol Ther 2007, 83, 761–769. Dvir, T.; Banghart, M. R.; Timko, B.; Langer, R.; Kohane, D. Nano Lett. 2010, 10, 250–254. Youn, H.-Y.; McCanna, D. J.; Sivak, J. G.; Jones, L. W. Molecular vision 2011, 17, 237. Weissleder, R. Nature biotechnology 2001, 19, 316–317. Oldenburg, S. J.; Averitt, R. D.; Westcott, S. L.; Halas, N. J. Chemical Physics Letters 1998, 288, 243–247. Barhoumi, A.; Huschka, R.; Bardhan, R.; Knight, M.; Halas, N. J. Chemical Physics Letters 2009, 482, 171–179. Wang, Y. G.; Samarel, A. M.; Lipsius, S. L. The Journal of physiology 2004, 527, 3–9. Yavuz, M. S.; Cheng, Y.; Chen, J.; Cobley, C. M.; Zhang, Q.; Rycenga, M.; Xie, J.; Kim, C.; Song, K. H.; Schwartz, A. G.; Wang, L. V.; Xia, Y. Nature Materials 2009, 8, 935–939. Sershen, S. R.; Westcott, S. L.; Halas, N. J.; West, J. L. Journal of biomedical materials research 2000, 51, 293–298.

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Figure Legends: Figure 1: The photothermally targeted nanoparticle. (a) Schematic illustration. (b) pNIPAAm-co-pAAm copolymer synthesized through atom-transfer radical polymerization starting with a disulphide initiator. pNIPAAm is poly(Nisopropylacrylamine), pAAm is polyacrylamide and disulphide initiator is bis[2-(2′bromoisobutyryloxy)ethyl]disulfide. Figure 2: Representative plots of polymer characterization. (a) Light transmittance of a 0.5% (w/v) solution of pNIPAAm-co-pAAm (NIPAAm:AAm = 75:25) as a function of temperature, demonstrating the LCST of the synthesized copolymer (55 ºC). (b) Red shift of the Au nanoshell absorption peak caused by coating with pNIPAAm-co-pAAm. (c) Cycling of the diameter of NS coated with pNIPAAm-co-pAAm in response to repeated

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heating and cooling. (d) Cycling of the diameter of NS coated with pNIPAAm-co-pAAm in response to NIR irradiation. Figure 3: TEM of Au nanoshells/NIPAAm. Figure 4: Binding of peptide/pNIPAAm-co-pAAm-coated nanoshells to human umbilical vein endothelial cells. ICP MS of HUVECs incubated with Au nanoshells coated with a mixture of pNIPAAm-co-pAAm and peptide (labeled peptide) and pure pNIPAAm-copAAm (labeled no peptide). To evaluate the role of light, light non-treated samples were also assessed for nanoshells binding. Data are means ± standard deviations.

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Figure 1: The photothermally targeted nanoparticle. (a) Schematic illustration. (b) pNIPAAm-co-pAAm copolymer synthesized through atom-transfer radical polymerization starting with a disulphide initiator. pNIPAAm: poly(N-isopropylacrylamine), pAAm: polyacrylamide and disulphide initiator: bis[2-(2′bromoisobutyryloxy)ethyl]disulfide. 254x190mm (96 x 96 DPI)

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Figure 2: Representative plots of polymer characterization. (a) Light transmittance of a 0.5% (w/v) solution of pNIPAAm-co-pAAm (NIPAAm:AAm = 75:25) as a function of temperature, demonstrating the LCST of the synthesized copolymer (55 ºC). (b) Red shift of the Au nanoshell absorption peak caused by coating with pNIPAAm-co-pAAm. (c) Cycling of the diameter of NS coated with pNIPAAm-co-pAAm in response to repeated heating and cooling. (d) Cycling of the diameter of NS coated with pNIPAAm-co-pAAm in response to NIR irradiation. NS: nanoshells, pNIPAAm- co-pAAM: poly(N-isopropylacrylamine)-co- polyacrylamide. 254x190mm (96 x 96 DPI)

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Figure 3: TEM of Au nanoshells with pNIPAAm-co-pAAm coating (arrow). 254x190mm (96 x 96 DPI)

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Figure 4: Binding of peptide/pNIPAAm-co-pAAm-coated nanoshells to human umbilical vein endothelial cells. ICP MS of HUVECs incubated with Au nanoshells coated with a mixture of pNIPAAm-co-pAAm and peptide (labeled peptide) and pure pNIPAAm-co-pAAm (labeled no peptide). To evaluate the role of light, light nontreated samples were also assessed for nanoshells binding. Data are means ± standard deviations. 254x190mm (96 x 96 DPI)

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