Near-Infrared Light Induced Phase Transition of Biodegradable

Dec 27, 2017 - Except for cells, each well also contained Transwell tissue culture plate insert with a small round disc (3 mm in diameter and 2 mm thi...
0 downloads 7 Views 1MB Size
Subscriber access provided by READING UNIV

Article

Near-Infrared Light Induced Phase Transition of Biodegradable Composites for on-Demand Healing and Drug Release Alona Shagan, Tsuf Croitoru-Sadger, Enav Corem-Salkmon, and Boaz Mizrahi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17481 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 30, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Near-Infrared Light Induced Phase Transition of Biodegradable Composites for on-Demand Healing and Drug Release Alona Shagan, Tsuf Croitoru-Sadger, Enav Corem-Salkmon, Boaz Mizrahi* Faculty of Biotechnology and Food Engineering, Technion, Haifa 32000, Israel Corresponding author E-mail: [email protected] KEYWORDS: Stimuli Responsive; Drug Delivery; Gold Nano-shells; Light Triggered; Biodegradable Polymers; Polyethylene glycol; Polycaprolactone

1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 31

ABSTRACT

Light responsive materials play an important role in many biomedical applications. Despite the great potential, commonly available systems are limited by their toxicity and lack of biodegradability. Here, an efficient light triggered system from safe, biodegradable star-polyethylene glycol (star-PEG) and poly ε-caprolactone (PCL) with varying melting points controlled by the length of the CL segments is described. When incorporated with gold nano-shells (GNS) and exposed to near infrared (NIR) irradiation, matrices temporarily disengage, thus allowing efficient on-demand healing, adhesion and drug release. The responsiveness of this system to light, with its tailorable physical and healing properties, biocompatibility, biodegradability and the capability to incorporate drugs and on-demand drug release are all desirable traits for numerous clinical applications.

2 ACS Paragon Plus Environment

Page 3 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

INTRODUCTION Stimuli responsive materials play an important role in many biomedical applications, including drug delivery1, self-healing coatings2-3, medical adhesives4 and antifouling surfaces5-6. These materials are capable of responding to external stimuli, such as pH7, redox8, enzymes9, light10, temperature11, magnetic12 and electrical signal13 by changing their physic-chemical properties. Among these, light is of particular interest, since its parameters (distance, intensity, wavelength etc.) are easy to manipulate14. Recent progress in nanotechnology, and the development of photosensitive materials, such as gold and quantum dots nanoparticles are significant steps that have been taken towards the development of light responsive systems15. Despite the great potential, commonly available light triggered materials are limited by two major concerns. First, toxicity of the light source in use. Early attempts using short UV wavelengths were limited by both low tissue transparency in the UV region and the carcinogenic effects on the tissue16. These shortcomings could be addressed by the use of near infrared (NIR) light that can penetrate deeper into the tissue without causing damage17. Secondly, in order to translate a light triggered molecular behavior into a macroscale system, a polymeric scaffold should be fabricated18. Unfortunately, most commonly used polymers utilized to incorporate the triggered molecules are associated with toxicity and lack of biocompatibility19. For example, acrylamides and acrylic acid polymers such as poly-NIPAAM are not hydrolytically degradable and were associated with neurotoxicity20-21. Consequently, these shortcomings currently prevent the translation of light triggering concepts into an actual medical application22.

3 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 31

Herein, we present a simple NIR-responsive composite from star-polyethylene glycol (star-PEG) and poly ε-caprolactone (PCL), which are both well-known biodegradable, biocompatible and FDA-approved polymers23 incorporated with gold nano-shells (GNS). Gold nanoparticles are widely used for drug delivery purposes, since they have a unique property of absorbing light and in response release heat24-26. Our structure motif is based on the ability of GNS to absorb light in the NIR window, and in response efficiently emit heat that diffuses to the surrounding medium27-29. To this end, we have developed a straightforward synthetic scheme (Scheme 1) to produce a series of multi-armed PEGPCL (star-PEG-PCL) copolymers with tailored melting points, which can be governed by the length of the PCL segments. The effect of molecular weight on melting points of the polymer is pronounced in multi-armed structures in comparison to linear polymers of the same size and composition30-31. Also, it has been suggested that star-shaped copolymers have lower melting points than linear copolymers of similar composition and molecular weight30,

32-33

. Applying these copolymers could potentially decrease the risk of

overheating, and damaging surrounding tissue and could also be exploited for on-demand healing and release of guest molecules (Movie S1).

4 ACS Paragon Plus Environment

Page 5 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Scheme 1. Schematic of Near-Infrared (NIR) triggered phase transition. EXPERIMENTAL SECTION Materials and methods. Four-armed polyethylene glycol 2000 Da (star-PEG) was purchased from JenKem Technology Co., Ltd. (Beijing, China). Pentaerythritol, Methoxy-polyethylene glycol (Methoxy-PEG), ε-caprolactone monomer (CL), stannous octoate (Sn(Oct)2), chloroform-d, Dulbecco’s phosphate buffered saline (PBS) pH 7.4, Dulbecco’s modified Eagle’s medium (DMEM), esterase from porcine liver, Gold(III) chloride trihydrate, polyvinylpyrrolidone 40 kDa and Bupivacaine hydrochloride monohydrate (BUPI) were purchased from Sigma Aldrich (MO, USA). (S)-(+)Camptothecin (CPT) was purchased from Chem-Impex International Inc. (IL, USA). Penicillin-streptomycin, fetal bovine serum (FBS) and l-glutamine were purchased from Biological Industries (Israel). Cobalt chloride hexahydrate was purchased from Alfa Aesar (Ward Hill, MA, USA). Sodium borohydride was purchased from Strem Chemicals Inc. (Newburyport, MA, USA). Sodium citrate tribasic dehydrate was

5 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 31

purchased from Riedel-deHaen (Honeywell Inc., Seelze, Germany). A CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS) kit was purchased from Promega (WI, USA). All solvents were of analytical grade and were purchased from Bio-Lab Ltd. (Israel). Synthesis and characterization of star-PEG-PCL. star-PEG or linear Methoxy-PEG (4.4 mmol) was dissolved in 180 mL toluene and the solution was heated to 120oC. Approximately 50 mL of the toluene were collected in the Dean Stark trap to remove water. CL monomers were added (see Table 1 and Table S1) in addition to the Sn(Oct)2 catalyst (0.02:1 molar rate compared to CL). The solution was allowed to reflux for 12 hours, cool to room temperature and the solvent was evaporated by a rotary evaporator. The resulting copolymer was dissolved in 4 mL dichloromethane (DCM) and precipitated in about 500 mL of cold petroleum ether. The final precipitate was dried in a desiccator overnight. Similar procedure was used to synthesize star-PCL (20kDa), using pentaerythritol as an initiator. The chemical structure of the synthesized star-PEG-PCL was evaluated by 1H-Nuclear magnetic resonance (1H-NMR), using Bruker Avance III 400 MHz NMR spectrometer (MA, USA) in CDCl3. CL content was determined by comparing the integral value of the 4 hydrogens of methylene protons of CL (indicated by δ at 1.6 ppm) to the 4 methylene protons of PEG (indicated by β at 3.6 ppm). The molecular weights of the synthesized star-PEG-PCL were evaluated by gel permeation chromatography (GPC) using Viscotek VE 1122 (Malvern instrument, UK) with PSS GRAM 1000 Å + PSS GRAM 30 Å columns (PSS, Germany) in Tetrahydrofuran (THF). The crystallinity of star-PEG, starPCL and all synthesized star-PEG-PCL copolymers was evaluated using X-Ray

6 ACS Paragon Plus Environment

Page 7 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Diffraction (XRD), using Rigaku SmartLab 3kW x-ray diffractometer instrument (Rigaku Corporation, Japan) equipped with sealed tube. Data was recorded in the range of 0-40 degrees, scanning speed of 1 deg/min, and step size of 0.01 degree. Melting temperatures of the synthesized star-PEG-PCL or their linear equivalent were measured using differential scanning calorimetry (DSC) equipped with a high-sensitivity sensor HSS7 (Mettler Toledo, Philippines Inc.). Synthesis and Characterization of GNS. Synthesis of GNS was performed according to Timko et. al.29 In brief, cobalt chloride hexahydrate (38 mg) was dissolved in 400 ml of double distilled water (DDW) followed by the addition of sodium citrate tribasic dehydrate (47 mg). The solution was purged with nitrogen for 40 min to prevent premature oxidation. Next, 2 ml of 1 wt% polyvinylpyrrolidone (PVP) solution and 0.4 ml ice-cold 1M-sodium borohydride were added to the degassed solution. Suspension was then purged for an additional 15 min to ensure complete oxidation of the borohydride. The resulting Co nanoparticle solution was added dropwise to an open flask containing 180 µL 0.1M HAuCl4 in 120 mL DDW. The resulting nanoparticles were washed three times with 0.1 wt% PVP solution, and lyophilized to obtain the final GNS particles. The absorbance spectrum of GNS in the range of 400-900 nm was studied by Ultraspec 2100 pro UV/Vis Spectrophotometer (Amersham Pharmacia Biotech, UK) in DDW or DCM. The surface composition of GNS and coated GNS were analyzed by Energy-dispersive X-ray spectroscopy (EDX) software, using tabletop Phenom ProX (Phenom-World, Netherlands).

7 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 31

Mechanical strength. Mechanical strength measurements were carried out by a Lloyd universal testing machine (AMETEK Inc. Berwyn, PA) with a 100 N load cell. Sample area was about 39 mm2 and extension rate was 5 mm/sec. Tensile test was conducted with a 10 mm/min speed. Each star-PEG-PCL sample was held together with clips and the probe was withdrawn from the upper moving grip. Young modulus was calculated from the slope of the stress plotted against the applied strain (n=4). Next, similar samples were cut into two equal specimens, which were held together to allow healing with a NIR laser (WSLS-808-007-H fiber coupled laser system, Wave Spectrum, China) for 1 min. The new Young modulus was then calculated using the same protocol described above. Temperature measurements. Star-PEG-PCL-14 samples containing various GNS concentrations (w/w) were irradiated under various laser intensities from 5 cm distance. Temperature increase was monitored under 2.1 W cm-2 using a thermocouple (VWR, Radnor, Pennsylvania). Cytotoxicity of PEG-PCL. For cytotoxicity measurement of star-PEG-PCL composites, NIH 3T3 cells were seeded in 24-wells plate at a density of 6 × 104 cells/well, incubated for 24 h. Except for cells, each well also contained Transwell™ tissue culture plate insert with a small round disc (3 mm in diameter and 2 mm thickness) of one of the various star-PEG-PCL and GNS composites. Cell viability was evaluated relatively to unexposed cells by MTS assay using CellTiter 96® solution, according to manufacturer’s protocol. Briefly, the inserts were pulled out and the cells media was aspirated. Next, 500 ml working media containing fresh media and reagent (5:1) was added to each well, and the plate was incubated for an additional 1 h. Last, the

8 ACS Paragon Plus Environment

Page 9 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

reaction products were tested for their absorbance at 490 nm, compared to unreacted working media. Drug release. Release experiment from composites (0.1 % GNS) incorporated with CPT was performed in dialysis membrane tubes (Midi GeBaFlex-tube, MWCO = 3.5 kD, Gene Bio-Application Ltd., Israel). The tubes were kept in PBS pH 7.4 containing 1% DMSO at 37oC with constant shaking of 60 RPM. A laser treatment was applied every 1 h for 5 min. The media was sampled and replaced by fresh media at pre-determined times and the released CPT was evaluated using fluorimeter at excitation of 369 nm and emission of 437 nm (n=3). Release of bupivacaine-HCl (BUPI-HCl) was performed in 12-wells plate. A laser treatment was applied for 3 cycles of 5 min each, after 30, 60 and 120 min. The released bupivacaine-HCl was evaluated using high performance liquid chromatography (HPLC) with C18 XBridge column (4.6*150 mm2, 3.5 um) and 2489 UV/Vis detector (Waters, MA, USA), at 204 nm. The mobile phase contained K2HPO4 Buffer and Acetonitrile (70:30), flow rate was 1 ml/min (n=4).

RESULTS AND DISCUSSION We synthesized a series of star-PEG-PCLs, consisting of a low molecular weight (Mw = 1,613 Da, analyzed by gel permeation chromatography (GPC)) PEG core and PCL segments of various lengths in the presence of stannous octoate (Sn(Oct)2) as a catalyst (Figure 1A)34. Obtained copolymers were precipitated in petroleum ether and identified by 1H-NMR (Table 1, Figure S1). All copolymers were white solids in room temperature. Molecular weight values, analyzed by GPC, varied between 6,628 Da for

9 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 31

the star-PEG-PCL with the shortest PCL segment to 13,932 Da for the star-PEG-PCL with the longest PCL segment (Table 1, Figure S2). Melting temperatures of the synthesized copolymers were analyzed by differential scanning calorimetry (DSC). Melting point (Mp) increased with increasing molecular weights, from 20oC for the pure star-PEG to 56oC for the star copolymer with the longest PCL segment (Table 1, Figure 1B). By way of comparison linear PEG-PCL copolymers of similar composition and molecular weighs resulted in a narrower range of melting points (Table S1), and demonstrated that these star shaped matrices are promising as tunable systems. Two distinct endotherm peaks evident in all copolymers, but not in the homopolymer star-PEG. At 20°C, star-PEG is liquid, probably due to the low average number of the repeating units per arm (~500 Da each arm)35. The addition of the linear PCL possesses a Tg of -60oC and a melting temperature (Tm) of 60oC36-37 showed melting point values that increase with increasing PCL chain length, attributable to the crystallization of PCL38. A similar trend between PCL block length and melting points has been found for linear PEG-PCL block copolymers, where by increasing the length of the PCL block, the melting temperature increased from 30 to 60°C39. Since PEG segment may play a destructive role in the crystalinity of PCL, XRD curves of all copolymers were compared with data of pure star-PCL (i.e. similar structure without PEG segment) (Figure S3). XRD analysis of all tested polymers showed a very similar pattern, typical of PCL40-41 suggesting that PEG segments play a negligible role in the crystalinity of PCL. The lower melting points of star-shaped polymers as well as their enhanced stimuli-responsiveness have been attribute to the lower molecular weights, to

10 ACS Paragon Plus Environment

Page 11 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

the fewer arm entanglements and to the higher density of their functional groups compared with their linear analogs32.

Figure 1. A) Synthesis scheme of star-polyethylene glycol and poly ε-caprolactone (starPEG-PCL). B) Differential scanning calorimetry (DSC) analysis of various star-PEGPCL and star-PEG.

Hollow GNS were fabricated by reacting cobalt with sodium citrate in reduction conditions using poly (vinylpyrrolidone) (PVP) as stabilizing agent29, 42. The solution was added dropwise into HAuCl4, and the formed GNS were concentrated. Based on transmission electron microscopy (TEM) measurements, GNS were uniform in size, with an average outer diameter in the range of 10 to 35 nm (Figure 2A). The surface composition of PVP or DDW washed GNS confirmed the presence of gold (Au) on the 11 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 31

surface of the DDW washed GNS (>97%), while the PVP washed GNS presented a high carbon peak, attributed to the PVP layers analyzed by Energy-dispersive X-ray spectroscopy (EDX) software (Phenom ProX, Figure S4). The resultant GNS absorbed light within the NIR window (UV/Vis Spectrophotometer, Figure 2B), confirming the successful synthesis43.

Figure 2. A) Transmission electron microscopy (TEM) imaging of gold nano-shells (GNS). B) Absorptions of GNS prepared with polyvinyl pirrolidone (PVP), suspended in dichloromethane (DCM) or double distilled water (DDW). Gold nanoparticles have a unique property termed surface plasmon resonance (SPR) which enables them to absorb light, of specific wavelengths, and release heat in response44-45. NIR wavelengths are favorable for biomedical applications, since they have deeper tissue penetration46, low skin absorption47 and lower toxicity48. Hence, GNS with NIR SPR peak are suitable for drug delivery purposes44.

12 ACS Paragon Plus Environment

Page 13 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Based on the low amount of PVP (0.01-0.3%) presented in the matrix and its glass transition temperature around 163°C, we speculate that PVP do not play any significant role in the phase transition process of the final product. As a way of comparison, we synthesized a batch of GNS without the PVP. The SPR peak was in the visible region (~600 nm), owing to its thicker shell, compared to GNS formed in the presence of PVP42. Moreover, size and size distribution was larger in the absence of PVP (Figure S5). The final GNS incorporated star-PEG-PCL composites were formed by physical mixing. Star-PEG-PCL composites (~1 gr) were melted at 60oC and pre-weighted GNS (~1 mg) were added. The mixture was stirred until all GNS was covered with star-PEGPCL followed by cooling to room temperature, which caused re-solidification of the copolymers. The procedure of melting and mixing was repeated at least 3 times until a homogenous distribution was achieved, as evident by the SEM imaging and local elemental analysis by energy-dispersive X-ray (EDX) (Figure 3A). An ideal biomaterial should be non-toxic and biodegradable49. Therefore, the cytotoxic effect of the GNS incorporated copolymers was evaluated on NIH 3T3 fibroblast cell line by MTS assay (Figure 3B). We added small round discs (3 mm in diameter, 2 mm in thickness) into each well, containing cells. Cells showed remarkable viability (as a percentage, relative to unexposed cells) above 80%, suggesting biocompatibility with fibroblast cells. These results are in agreement with our previous results with star-PEGPCL of similar and shorter PCL segments30. While PEG is non-toxic polymer, PCL is biocompatible, biodegradable and non-toxic polyester50. Both PEG-PCL copolymers51 and gold nanoparticles52 were extensively tested for various biomedical applications and 13 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 31

all studies demonstrated low cytotoxicity and complete clearance from the body. Thus, the excellent cytotoxicity results combined with the excellent record of the composite components illustrate the high potential of GNS incorporated star-PEG-PCL as a safe carrier in biomedical systems.

Figure 3. A) Energy dispersive X-ray spectroscopy (EDX) mapping of dispersed GNS (light blue), incorporated in a star-PEG-PCL-14 matrix (grey). B) NIH 3T3 cell viability (relatively to unexposed cells) evaluated by MTS assay, 24h after exposure to star-PEGPCL incorporated with GNS (1%). Data are means and SD of n = 4.

Further investigations were carried out on star-PEG-PCL-14 (melting points 44/51oC) based on a trade-off between the clinical needs, i.e. to be well above body temperature, and safety concerns, i.e. a melting point unlikely to damage tissues and biologic molecules29. We started by demonstrating the responsiveness of star-PEG-PCL-14 with GNS (0.1% w/w) to NIR irradiation (5 cm distance). Temperature increase was monitored by thermal

14 ACS Paragon Plus Environment

Page 15 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

camera (Figure 4A) together with phase transition, limited to the irradiation site (lower region, Figure 4B). For comparison, a similar composition without GNS showed no significant temperature change (Figure 4A) or structural changes. Thus, supporting our hypothesis that GNS incorporated in star-PEG-PCL matrix can absorb NIR light and induce phase transition in the appropriate matrix. Furthermore, our materials demonstrated a more pronounced response compared to other heat responsive biomaterials such as poly-NIPAAM-based hydrogels.53 This can be attributable to the simple melting mechanism of star-PEG-PCL and to the absence of a slow-kinetics volume phase transition between a swollen and a shrunken form.54 To better assess the effect of irradiation on the phase transition pathway, star-PEGPCL-14 composites with various GNS concentrations were irradiated at increased intensities and the time until phase transition was recorded (Figure 4C). Irrespective of GNS concentrations, increasing irradiation intensities decreased the time required for melting. However, while composites containing 0.5 or 1 %w/w GNS showed similar patterns, 0.1% GNS generally required longer irradiation time to achieve the same effect (p