Photothermal Effect-Triggered Drug Release from Hydrogen Bonding

Publication Date (Web): January 30, 2018. Copyright © 2018 American Chemical Society ..... Join ACS Publications in Boston for Fall MRS 2018. The Mat...
0 downloads 0 Views 3MB Size
Download PDF
Article Cite This: Biomacromolecules XXXX, XXX, XXX−XXX

pubs.acs.org/Biomac

Photothermal Effect-Triggered Drug Release from Hydrogen Bonding-Enhanced Polymeric Micelles Yuce Li,†,‡ Jianxun Ding,‡ Jintao Zhu,*,† Huayu Tian,*,‡ and Xuesi Chen‡ †

State Key Laboratory of Materials Processing and Mold Technology, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China ‡ Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China S Supporting Information *

ABSTRACT: Incorporation of noncovalent interactions into hydrophobic cores of polymeric micelles provides the micelles with enhanced physical stability and drug loading efficiency, however, it also creates obstacles for drug release due to the strong interactions between carriers and drugs. Herein, a series of amphiphilic block copolymers based on poly(ethylene glycol)-b-poly(L-lysine) (mPEG-b-PLL) with similar chemical structures, while different hydrogen bonding donors (urethane, urea, and thiourea groups) are synthesized, and their capacities for codelivery of anticancer drug (e.g., doxorubicin) and photothermal agent (e.g., indocyanine green) are investigated. The resulting hybrid micelles display decreased critical micelle concentrations (CMCs) and enhanced micelle stabilities due to the hydrogen bonding between urea groups in the polymers. Moreover, the strong hydrogen bonds between the urea/thiourea groups and drugs provide the carriers with enhanced drug loading efficiencies, decreased micelle sizes, however, slower drug release profiles as well. When exposed to the near-infrared laser irradiation, destabilization of the hydrogen bonding through photothermal effect triggers fast and controlled drug releases from the micelles, which dramatically promotes the aggregation of the drugs in the nuclei, resulting in an enhanced anticancer activity. These results demonstrate that the hydrogen bonding-enhanced micelles are promising carriers for controllable chemophotothermal synergistic therapy.

■

INTRODUCTION Self-assembled polymeric micelles with a size range of 10−200 nm are considered to be ideal drug carriers due to their capacity to prolong blood circulation for passive targeting into solid tumors by the enhanced permeability and retention (EPR) effect.1,2 Although great progress has been made in the past few decades, further in-depth applications of polymeric micelles are limited because of the unsatisfied drug loading efficiency, in vivo stability, drug release behavior, and others.3,4 Hydrophobic interaction is the most common driving force for micellization and drug loading of the amphiphilic block copolymers (BCPs) in aqueous solution; yet, the hydrophobic interaction is often too weak to maintain the self-assembled structures and drug loading under near infinite dilution conditions in the circulatory system, resulting in undesired spontaneous drug releases and side effects.5,6 To enhance the drug loading capacities and stabilities of polymeric micelles, noncovalent interactions (e.g., electrostatic interaction,7 host−guest interaction,8,9 and hydrogen bonding10) have been introduced to the polymeric micelles in recent reports. Among them, hydrogen bonding is directional, selective, and relatively strong interaction that only forms between hydrogen bonding donor and receptor, so that they are promising in these drug delivery systems.5 Yang et al.3,11 © XXXX American Chemical Society

reported a series of diblock copolymers of urea-functionalized polycarbonate (PUC) and poly(ethylene glycol) (PEG), which formed micelles with enhanced stabilities due to the strong hydrogen bonding between urea groups. Besides, ureacontaining micelles displayed promoted drug loading efficiencies along with the increased affinity between the drugs and micellar cores. Huang et al.12,13 conjugated complementary nucleobases adenine (A) and thymine (T) to the hydrophobic cores of amphiphilic copolymers and fabricated hydrogen bonding-cross-linked micelles, which displayed lowered critical micelle concentrations (CMCs). Niu et al.14 prepared hydrogen bonding-based amphiphilic supramolecular prodrug assemblies consisting of theophylline−diosgenin conjugates and uracilterminated PEG, which not only enhanced drug solubility but also prolonged systemic circulation. In addition, Suryanarayanan and co-workers15 also concluded that with the increase of drug−polymer hydrogen bonding strength, the drug loading Special Issue: Biomacromolecules Asian Special Issue Received: December 5, 2017 Revised: January 28, 2018 Published: January 30, 2018 A

DOI: 10.1021/acs.biomac.7b01702 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Scheme 1. Schematic Illustration Showing the Drug Loading and NIR-Triggered Drug Release from Hydrogen BondingEnhanced Micelles

■

efficiency and physical stability of nifedipine solid dispersions increased. Besides above advantages, introduction of strong hydrogen bonding between drugs and carriers intensively hindered drug release from the micelles, resulting in decreased bioavailability of the loaded drugs. For example, Zhang et al.10 demonstrated that only 66.9% of the loaded methotrexate (MTX) could be released from the amphiphilic peptide assemblies at the physiological environments due to the strong complementary hydrogen bonding between cyanuric acids in peptide and melamines in methotrexate. Similarly, Yang and co-workers reported that the maximal release amount of the loaded doxorubicin (Dox, an anticancer drug) reduced from 73% to 41% due to the introduction of urea groups to the hydrophobic cores of the micelles, resulting in a delayed nucleus uptake as well as a 4-fold increase of half maximal inhibitory concentrations (IC50).3 Thus, it is desirable to accelerate the drug releases from the hydrogen bonding-enhanced drug delivery systems. For instance, Pan et al.16 proved that 3-fold complementary hydrogen bonding between thymine and aminotriazine can be disassociated at pH value ranging from 5 to 3. Zhang et al.10 revealed that release amounts of MTX increased from 66.9% to 85% when exposed to strong acidic (pH 5.0) or basic (pH 9.0) environments. Kubo et al.17 indicated that drug release profiles could be regulated by suppressing the formation of hydrogen bonding at high temperature (>60 °C). However, these conditions are so rigorous that they are undesirable and almost unreachable for in vivo applications. To develop facile yet effective strategy to accelerate drug release from hydrogen bonding-enhanced micelles thereby improve their cytotoxicity toward cancer cells, herein, we reported a series of amphiphilic BCPs based on mPEG-bpoly(L-lysine) (mPEG-b-PLL) with similar chemical structures while different hydrogen bonding donors (e.g., urethane, urea, and thiourea groups), and then investigated their capacities for coloading anticancer drug doxorubicin (Dox) and photothermal agents indocyanine green (ICG; see Scheme 1). Also, influences of the different hydrogen bonding donors on the sizes, stabilities, drug loading efficiencies and release profiles of the formed different micelles were studied. Upon near-infrared laser (NIR) irradiation, rapid drug release and aggregation of Dox in nucleus due to the destabilization of the hydrogen bonding by the photothermal effect was observed through confocal laser scanning microscopy (CLSM) investigation.

EXPERIMENTAL SECTION

Materials. Nε-Benzyloxycarbonyl-L-lysine (H-Lys(Z)-OH) was supplied by GL Biochem Co. Ltd. (Shanghai, China). Poly(ethylene glycol) monomethyl ether (mPEG, Mn = 5000 Da), Hydrobromic acid solution in acetic acid (33 wt %), trifluoroacetic acid and 4′,6diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma−Aldrich (St. Louis, MO, U.S.A.). Benzyl isocyanate/ isothiocyanate were purchased from Tianjin Heowns Biochemical Technology Co., Ltd. (Tianjin, China). Amino-terminated mPEG (mPEG-NH2) and Nε-benzyloxycarbonyl-L-lysine-N-carboxyanhydride (Lys(Z)-NCA) were synthesized according to the methods in our previous report.7 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were supplied by Amresco (Solon, OH, U.S.A.). Actin-Tracker Green was purchased from Beyotime Biotechnology (Nantong, China). Synthesis and Characterization of BCPs with Different Hydrogen Bonding Donors. mPEG-b-poly(Nε-benzyloxycarbonylL-lysine) polymer (PPZ) was synthesized by the ring opening polymerization of Lys(Z)-NCA monomers with mPEG-NH2 as macroinitiator. In brief, mPEG-NH2 (2.0 g, 0.4 mmol) was azeotropic dehydrated with toluene and dissolved in dry dimethylformamide (DMF, 20.0 mL), and then Lys(Z)-NCA (2.45 g, 8 mmol) in dry DMF (20.0 mL) was added via a syringe under argon. The reaction was maintained for 3 days at 25 °C under argon atmosphere. Then, the PPZ polymer was isolated through repeated precipitation from DMF into excess amounts of diethyl ether, followed by vacuum drying. Subsequently, the PPZ polymer (3.0 g) was dissolved in dichloroacetic acid (30.0 mL). After addition of HBr/acetic acid (33 wt %, 9 mL), the solution was allowed to react at 25 °C under stirring for 1 h. The crude product was obtained by precipitation of the resulting samples into excessive diethyl ether. Finally, the crude product was dialyzed against distilled water (MWCO = 3500 Da) and lyophilized to give the mPEG-b-PLL product. The urea/thiourea-functionalized BCPs (PPBU/PPBTU) were synthesized via the additive reaction between amino groups in mPEG-b-PLL and benzyl isocyanate/isothiocyanate.18 Briefly, mPEGb-PLL (0.30 g, containing 0.81 mmol of amino groups) was dissolved in dry DMF (10.0 mL) along with 50 μL of triethylamine as alkali. Excessive benzyl isocyanate (0.133 g, 1.0 mmol) or benzyl isothiocyanate (0.149 g, 1.0 mmol) was then added dropwise to the above solution. The resultant solution was stirred for another 24 h at room temperature, and the PPBU/PPBTU polymers were isolated by repeated precipitation from DMF into excess amounts of diethyl ether, followed by vacuum drying. Characterization. 1H NMR spectra were recorded on a Bruker AV 400 NMR spectrometer (Billerica, MA, U.S.A.) in trifluoroacetic acid-d (TFA-d). Mw, Mn, and PDI of the BCPs were determined by a Waters 515 GPC system equipped with a linear 7.8 × 300 mm column (Waters Co., Milford, MA, U.S.A.), an 18 angles laser scattering instrument (Wyatt Technology Co., U.S.A.), and an OPTILAB DSP B

DOI: 10.1021/acs.biomac.7b01702 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Scheme 2. Synthesis Route of the Amphiphilic BCPs with Different Hydrogen Bonding Donors (a) and Hydrogen Bonding Formed between Different Hydrogen Bonding Donors and Receptors (b)

Photothermal Effects of the Drug-Loaded Micelles. Solution of Dox/ICG-loaded micelles (200 μL) with various concentrations of ICG (0−100 mg/L) was stored in transparent plastic vials at initial temperature of 25 °C, and then irradiated by a NIR laser (1.0 W/cm2, 808 nm) for 5 min. The time-dependent temperature increasing profiles were recorded by a K-type thermocouple (Model SC-GG-K30−36, Omega Engineering Inc., Stamford, CT, U.S.A.). In Vitro Drug Release. Dox release profiles were performed in PBS buffer (pH = 7.4) by a dialysis method at 37 °C.20 Briefly, weighed drug-loaded NPs were dissolved in 2.0 mL of PBS (10 mmol/ L) and transferred into a dialysis tubing (MWCO = 7000 Da). The release experiment was initiated by placing the dialysis bag into 18.0 mL of PBS at 37 °C with continuous shaking at 70 rpm. At predetermined intervals, 2 mL of incubated solution was taken out and replaced with equal volume of fresh PBS. The amount of released Dox was determined by fluorescence measurement (λex = 480 nm). For the release under NIR laser irradiation, the solution was irradiated with NIR laser (1.0 W/cm2, 808 nm) for 5 min at 4 h. All experiments were performed in triplicate. Intracellular Drug Release by CLSM. Intracellular drug release behaviors of the drug-loaded NPs were observed by CLSM (Olympus FV1000, Tokyo, Japan) toward HeLa cells. HeLa cells were cultured in complete DMEM medium containing 10% (v/v) FBS at 37 °C and 5% (v/v) carbon dioxide. Cells were seeded on sterilized coverslips in 6well plates at a density of 2.0 × 106 cells per well in 2.0 mL of complete DMEM for 12 h, and then incubated with the NPs at a final DOX concentration of 10.0 mg/L for another 4 h. The cells were treated with or without 5 min of NIR irradiation, followed by another incubation for 4 h. Thereafter, the cells were fixed with 4% (w/v) paraformaldehyde for 30 min at 25 °C, and counterstained with DAPI (blue) for cell nuclei and Actin-Tracker Green (FITC, green) for cytoskeleton according to the standard protocols provided by the suppliers. CLSM images of the cells were obtained through a CLSM. The pixel statistical analysis was performed by the Adobe Photoshop CS6 Extended software (Adobe Systems Inc., San José, CA, U.S.A.) based on the method in previous report.21 For measuring the relative fluorescence intensity of Dox in nuclei, the nuclei or whole cell zones were manually selected on the CLSM investigation by the blue or green channel, and the integral quantifications of red channel for Dox-emission in desired areas were calculated by the function “histogram”. In Vitro Cytotoxicity Assay. HeLa cells were seeded in a 96-well plate at a density of 1.0 × 104 cells per well for 12 h and incubated

interferometric refractometer (Wyatt Technology Co., Santa Barbara, CA, U.S.A.). Preparation of Polymeric Nanoparticles (NPs). Neat NPs of the above BCPs were prepared by a nanoprecipitation method. Briefly, 10.0 mg of each BCP was dissolved in 2.0 mL DMF, and the solution was allowed for stirring overnight. 2.0 mL of Milli-Q water was then added into the above solution at a speed of 50 μL/min by a precise programmable syringe pump (New Era Pump Systems, Inc., Farmingdale, NY, U.S.A.) and stirred for another 12 h. The resultant solutions were dialyzed against Milli-Q water and the ultimate volume was made constant at 10 mL to obtain a 1.0 mg/mL solution. Characterization of Hydrogen Bonding-Enhanced Polymeric Micelles. Size and distribution of the micelles was measured with a dynamic laser scattering (DLS, Malvern Nano-ZS90, Malvern, U.K.). Transmission electron microscopy (TEM) measurements were performed on the TecnaiG2 F20 microscope (FEI, Eindhoven, The Netherlands) at 200 kV. The CMC measurements was performed by fluorescence spectra using pyrene as probe as previously reported.7 Encapsulation of ICG and Dox in Hydrogen BondingEnhanced Polymeric Micelles. Dox-, ICG-, or Dox/ICG-loaded complex NPs were prepared by the nanoprecipitation technique.19 Typically, each BCP (20 mg) were dissolved in DMF (4.0 mL) along with Dox·HCl (6.0 mg)/triethylamine (2.0 mg, 2 equiv to Dox·HCl) with or without ICG (8.0 mg). The mixture was stirred at 25 °C overnight in dark and then 4.0 mL of Milli-Q water was added into the solution at 50 μL/min by the syringe pump, following by stirring for another 12 h. Organic solvents and excess free drugs were removed by dialysis against Milli-Q water for 24 h and then lyophilized to obtain the drug-loaded NPs. Sizes and morphologies of the drug-loaded nanoparticles were determined by DLS and TEM as described above. The drug-loaded NPs were dissolved in DMF to determine the drug loading content (DLC, wt %) and drug loading efficiency (DLE, wt %) with a fluoro-spectro photometer (Perkin−Elmer LS50B luminescence spectrometer, Waltham, MA, U.S.A.) using a standard curve method (λex = 480 nm). DLC and DLE of the drug-loaded micelles were calculated according to eqs 1 and 2, respectively:

DLC(wt%) =

amt of drug in nanoparticle × 100% amt of nanoparticle

(1)

DLE(wt%) =

amt of drug in nanoparticle × 100% amt of feeding drug

(2) C

DOI: 10.1021/acs.biomac.7b01702 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules with the polymers or drug-loaded NPs at different concentrations for 48 h. Thereafter, 10.0 μL of MTT solutions (5.0 g/L) were added into each well and incubated for another 4 h. The medium was carefully discarded, and the resultant formazan crystals were dissolved in 100.0 μL DMSO, and the absorbance at 492 nm was measured by an ELISA microplate reader (Bio-Rad Laboratories, Hercules, CA, U.S.A.). For cytotoxicity measurement under NIR irradiation, the cells were treated by NIR laser (1.0 W/cm2, 808 nm) for 5 min at 4 h. The cell viability was calculated based on eq 3: cell viability(%) =

A sample Acontrol

× 100%

(−CHCH2CH2CH2NH−). The chemical shifts of methylenes in benzyls (−CH2−C6H5) of the three polymers are different: 4.9 ppm for PPZ, 4.2 ppm for PPBU and 4.5 ppm for PPBTU. Average degree of polymerization (DP) of L-lysine block was calculated to be 21 by comparing the integrations of lysine residues and EG segments, agreeing well with the feeding molar ratios. Gel permeation chromatography (GPC) analyses showed that the copolymers possessed narrow molecular weight distributions (PDI = 1.10, 1.20 and 1.13, respectively; Table 1 and Figure S1 in the Supporting Information (SI)).

(3)

Table 1. Characteristics of the Synthesized BCPs and the Corresponding Micelles

Flow Cytometry. HeLa Cells were seeded into 12-well plates (1.0 × 105 cells per well) in 1.0 mL of complete DMEM for 12 h, and then incubated with Dox/ICG-loaded NPs at a final Dox concentration of 10.0 mg/L for 4 h. Thereafter, the cells were carefully washed by PBS thrice, then harvested and resuspended with PBS, and the cellular uptake efficiency was determined on a Guava EasyCyte flow cytometer (Merk Millipore, Billerica, MA, U.S.A.). Statistical Analysis. All experiments were repeated at least three times and data expressed as means ± SD. Statistical significances were determined using Student’s t test. p < 0.05 was considered statistically significant, and p < 0.01 was considered highly significant.

polymer

Mna (kDa)

Mnb (kDa)

PDIc

CMC (mg/L)

Dhd (nm)

PPZ PPBU PPBTU

10.2 10.2 10.5

11.6 11.7 12.3

1.10 1.20 1.13

5.60 3.74 8.47

81.2 57.4 92.6

a

Molecular weight (Mn) obtained from NMR. bMn obtained from GPC. cPolydispersity index (PDI) obtained from GPC. dDh obtained from DLS measurements.

■

RESULTS AND DISCUSSION BCPs with Different Hydrogen Bonding Donors. Synthesis route of the amphiphilic BCPs with different hydrogen bonding donors is shown in Scheme 2a. PPZ was synthesized by the ring opening polymerization of Lys(Z)-NCA with a macroinitiator mPEG-NH2, while PPBU/PPBTU were synthesized through the addition reactions between benzyl isocyanate/isothiocyanate and amino groups in the deprotected mPEG-b-PLL as reported in our previous work.18 Interestingly, although only a few atoms are different in the resultant polymers, their capacities to form hydrogen bonding are significantly different (Scheme 2b). The urethane groups can only form relatively weak hydrogen bonding (dissociation energy: 18.4 kJ/mol)22 with carbonyl groups in either another urethane group or other structures. Both urea and thiourea groups can form relatively strong bifurcated hydrogen bonding (dissociation energy: about 58.5 kJ/mol)23−26 with carbonyl groups. However, a urea group can also bind to another urea group, while there is no hydrogen bonding between two thiourea groups due to the significantly lower electronegativity of sulfur than oxygen.27 Structures of the resultant BCPs were confirmed by 1H NMR (Figure 1) and all peaks are well assigned. The resonant peaks at about 3.7 ppm refer to the methylenes of PEG (−CH2CH2O−). The peaks at 1.0−1.8, 3.0, and 4.4 ppm are the characteristic resonances of L -lysine residues

Micellization of the BCPs with Different Hydrogen Bonding Donors. To investigate the influences of hydrogen bonding on self-assembling behaviors of the amphiphilic BCPs, we prepared micelles of the copolymers containing different hydrogen bonding donors through the nanoprecipitation method and investigated their formations and stabilities. Sizes and morphologies of the micelles were determined by DLS and TEM investigation. The BCPs formed spherical micelles in aqueous phase with average hydrodynamic diameters (Dh) of 81.2, 57.4, and 92.6 nm for PPZ, PPBU, and PPBTU, respectively (Figure 2a,b and Table 1). Diameters of the micelles obtained from TEM images are slightly smaller than those obtained by DLS because of the collapse of the PEG corona after drying; however, the relative sizes of the micelles are in accordance to the Dh. Differences of the micellar sizes could be attributed to their different capacities to form hydrogen bonding in the hydrophobic cores. In other words, compared with the PPZ micelles, PPBU micelles formed more condensed hydrophobic cores due to the stronger hydrogen bonding between the urea groups, while the PPBTU micelles formed relatively looser cores since no hydrogen bonding was formed between the thiourea groups. Amphiphilic BCPs with lower CMCs can keep selfassembled structures at lower concentrations and thus make the micelles more stable in the near infinite dilution conditions in vivo.28 Here, CMCs of the BCPs were determined by fluorescence spectra using pyrene as the probe (Figure 2c and Table 1). With the increase of hydrogen bonding strength in the hydrophobic cores, the CMC values of the BCPs gradually decreased from 8.47 mg/L of PPBTU, 5.60 mg/L of PPZ to 3.74 mg/L of PPBU. This result indicated that incorporation of hydrogen bonding among the hydrophobic segments endowed the micelles with extra interactions to confront dilution thereby stabilized the micellar structures in aqueous solutions. Drug Loading in the Hydrogen Bonding-Enhanced Micelles. Dox is one of the most effective anthracycline antitumor drugs that inhibits the biosynthesis of nucleic acids by inserting into the double strands of DNAs.29 The multiple carbonyl and hydroxyl groups of Dox provide multiple sites to form hydrogen bonding with the donors in the micelle cores. To evaluate the drug loading capacities of the micelles with

Figure 1. Characterization of the synthesized polymers. 1H NMR spectra of PPZ, PPBU, and PPBTU were performed in trifluoroacetic acid-d (TFA-d). D

DOI: 10.1021/acs.biomac.7b01702 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 2. Micellization of the BCPs with different hydrogen bonding donors. Representative TEM images (a), hydrodynamic diameter (Dh) (b), and CMCs (c) of the micelles were displayed. The scale bar in the right TEM image can be applied to the others.

different hydrogen bonding donors, Dox was first encapsulated in the micellar cores (Table 2). Although the strong hydrogen

results agree well with that of the micelles loaded with neat Dox (Table 2). After coloading of Dox and ICG, all the micelles displayed increased sizes compared with the control micelles (Figure 3,

Table 2. Drug Loading Capacities of the BCPs with Different Hydrogen Bonding Donors DLC (%, w/w) drugs Dox

ICG

Dox/ ICG

DLE (%, w/w)

polymers

Dha (nm)

DOX

ICG

DOX

ICG

PPZ PPBU PPBTU PPZ PPBU PPBTU PPZ PPBU PPBTU

143.9 117.1 126.4 101.7 91.6 150.0 166.4 126.1 140.7

5.8 ± 0.6 7.5 ± 0.3 9.9 ± 1.5 − − − 8.9 ± 1.4 10.3 ± 1.6 13.6 ± 2.1

− − − 7.9 ± 0.8 8.2 ± 1.1 8.5 ± 1.3 10.5 ± 1.3 13.5 ± 2.0 16.1 ± 3.2

20.5 27.0 36.6 − − − 36.8 45.6 64.0

− − − 21.4 22.3 23.2 32.6 44.9 57.2

Average hydrodynamic diameters were determined by DLS. “−” represents not available.

Figure 3. Characterization of the drug-loaded hydrogen bondingenhanced micelles. Representative TEM images (a) and hydrodynamic diameter (Dh) (b) of the drug-loaded micelles were revealed. The scale bar in the right TEM image can be applied to the others.

bonding between Dox and the urea/thiourea groups endowed the PPBU and PPBTU micelles with remarkably higher drug loading contents (DLCs) and drug loading efficiencies (DLEs) than that of the PPZ micelles, they strongly hindered the drug release from the micelles as well, resulting in about 1.9-fold higher IC50 toward cancer cells (Figures S2 and S3), which is dramatically accordant with the previous reports.3 It was reported that coloading of photothermal agents into the drug-loaded micelles could promote the drug release under NIR laser irradiation by increasing local temperature of the micelles via photothermal effects.30−33 Here, ICG, the only near-infrared (NIR) fluorescence dye that is approved by the United States Food and Drug Administration (FDA) for photothermal therapy, was coloaded into the hydrogen bonding-enhanced micelles along with Dox. As shown in Table 2, the DLCs and DLEs of both ICG and Dox significantly increased when they were simultaneously encapsulated into three kinds of micelles due to the electrostatic interaction between the sulfonate groups in ICG and amino groups in Dox. Specifically, compared with the relatively low Dox/ICG DLCs of the PPZ micelles (8.9%/10.5%), the PPBU and PPBTU micelles gave much higher DLCs of 10.3%/13.5% and 13.6%/ 16.1%, respectively, implying that additional hydrogen bonding between the carbonyl groups and urea/thiourea groups could effectively improve the interactions between the drugs and polymers, thus increasing their drug loading efficiencies. The relatively lower DLCs of PPBU than PPBTU can be ascribed to the competitive hydrogen bonding between urea groups. These

Table 2). In contrast to larger Dh of the neat PPBTU micelles than that of neat PPZ micelles (92.6 vs 81.2 nm), the Dox/ICG coloaded PPBTU micelles gave a smaller Dh than that of Dox/ ICG coloaded PPZ micelles (140.7 vs 166.4 nm). Similar results were also demonstrated by TEM investigation (Figure 3a). Variation of the relative sizes could be attributed to the cross-linking hydrophobic cores of PPBTU by multiple carbonyl groups in Dox through hydrogen bonding, resulting in more compact hydrophobic cores. Interestingly, after loading with Dox/ICG, the PPZ micelles displayed similar spherical morphology with the control micelles, while the PPBU and PPBTU micelles gave significant different morphologies from the control micelles. Similar morphologies were also found in the Dox-loaded PPBU/PPBTU micelles (Figure S4). Notably, the reason for the variation of PPBU/PPBTU micelles after loading Dox or Dox/ICG is still not well understood. Yet, hint can be obtained from the report from Hedrick et al.,34 where they revealed that introduction of hydrogen bonding to the BCPs could induce a significant transformation of morphology on the amphiphilic BCP micelles. Further detailed studies on the self-assembly behaviors of the hydrogen bonding-enhanced copolymers will be performed in the next step. Drug Release from the Hydrogen Bonding-Enhanced Micelles. In vitro Dox release from the Dox/ICG-loaded micelles was performed in PBS (pH 7.4) at 37 °C (Figure 4a). Compared to the micelles loaded with neat Dox, all of the micelles coloaded with Dox and ICG displayed slower drug release rate and less accumulative Dox release amounts (Figure S2). Presumably, the reason can be owed to the electrostatic

a

E

DOI: 10.1021/acs.biomac.7b01702 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 4. In vitro drug release from the hydrogen bond-enhanced micelles. Dox release profiles of the Dox/ICG-loaded micelles in absence (a) and in presence (b) of NIR laser irradiation were performed at 37 °C. Arrow represents the time that NIR irradiation performed. Data was presented as mean ± standard deviation (n = 3).

Figure 5. Intracellular drug release from the hydrogen bonding-enhanced micelles. (a) Experimental settings; (b) CLSM images of HeLa cells at 8 h treated with or without NIR laser irradiation. The nuclei and cytoskeleton were labeled by DAPI (blue) and phalloidin-FITC (green), respectively. The scale bar in the upper left CLSM image applies to the others. (c) Intranuclei accumulative Dox fluorescence calculated by pixel statistical analyses. **p < 0.01, ***p < 0.001.

interactions between Dox and ICG, which was also demonstrated by Wan et al.31 Specifically, the drug release rates from the Dox/ICG-loaded PPBU and PPBTU micelles were much slower than that of the Dox/ICG-loaded PPZ micelles, and the cumulative Dox release after 48 h were 64.6%, 50.5%, and 43.1% for PPZ, PPBU, and PPBTU micelles, respectively. The reduced drug release rates and amounts from Dox/ICG-loaded PPBU and PPBTU micelles could be attributed to the robust hydrogen bonding between Dox and hydrophobic cores. Hydrogen bonding was reported to be destroyed under high temperatures, for example, the double helix structure of DNA can be uncoiled when heated to 70−80 °C because of the dissociation of hydrogen bonding between the base pairs, which is the basic principle of polymerase chain reaction (PCR).35,36 However, such high temperature is almost undesirable in vivo by direct heating. The photothermal effect of ICG provides an indirect method to increase the local temperature around ICG

molecules, making it possible to destroy the hydrogen bonding between the drugs and carriers. As shown in Figure S5, the solution temperature of Dox/ICG-loaded micelles increased rapidly when exposed to the NIR laser irradiation, and no significant differences were found between the free ICG and the Dox/ICG-loaded micelles at the same ICG concentration of 100 mg/L, indicating that encapsulation of ICG to these micelles negligibly influences in the photothermal conversion efficiency of ICG, based on previously reported algorithms.37 The maximum temperature of the solutions rises from 45 to 61 °C as the ICG doses increase from 10 to 100 mg/L (Figure S5b). At concentrations lower than 25 mg/L, the solution temperature started to decrease at 250 s due to the exhausting of active ICG. To confirm whether the increase of temperature could trigger the drug release from these drug carriers, the Dox/ICG-loaded micelles were exposed to NIR laser irradiation for 5 min at defined time points, and the drug release profiles were determined (Figure 4b). A burst drug F

DOI: 10.1021/acs.biomac.7b01702 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 6. Cell viability of the micelles toward HeLa cells. (a) Cytotoxicity of the control micelles; (b−d) Cytotoxicity of the Dox/ICG-loaded PPZ, PPBU and PPBTU micelles with or without NIR laser irradiation. Data were presented as mean ± standard deviation (n = 3).

laser power density of 1.0 W/cm2), the solution temperature just raised from 25 °C to maximal 43 °C (Figure S5), which was much lower than the reported dissociation temperature of carbonyl−urea hydrogen bonding in aqueous solution (approximately 60 °C).39 As reported by Yang et al., when exposed to NIR laser irradiation, the intramicellar temperature of the photothermal agent-loaded micelles rapidly raised within just a few seconds, and the temperature differences between the micelle cores and total solutions increased with the increase of laser power density.40 Therefore, it is reasonable to believe that the carbonyl−urea hydrogen bonding was destroyed at these conditions. These results indicated that indirect heating through the photothermal agents (e.g., ICG) is much superior to the direct heating for the controlled drug release from the hydrogen bonding-enhanced polymeric micelles and avoid the unnecessary side effects to the normal tissues as well. Cellular Internalization and Cytotoxicity of the Hydrogen Bonding-Enhanced Micelles. Cellular uptake of the drug-loaded micelles was evaluated by flow cytometry (Figure S6). All of the Dox/ICG-loaded micelles could be efficiently internalized by the HeLa cells. A slightly higher average intracellular Dox fluorescence intensity of the PPBTU and PPBU micelles than that of the PPZ micelles could be attributed to the higher drug content in each micelle. To investigate the synergistic effect of chemo-photothermal therapy by the drug-loaded hydrogen bonding-enhanced micelles, the in vitro cytotoxicity of the micelles toward HeLa cells was evaluated by the MTT assay (Figure 6). No cytotoxicity of the synthesized polymers was observed even at the concentration of 400 g/L by the cell viability test due to the biocompatibilities of both PEG and lysine derivates (Figure 6a). All the ICG-loaded micelles displayed undetectable dark cytotoxicity and similar dose-dependent cell-killing abilities in present of NIR laser irradiation (1.0 W/cm2) due to the hyperthermia at relatively high ICG concentrations (Figure S7). In the absence of NIR laser irradiation, the Dox/ICG-loaded PPBU/PPBTU micelles displayed less dark cytotoxicity and higher IC50 (4.53 and 4.21 mg/L) of the micelle-enveloped Dox toward the HeLa cells than the PPZ micelles (3.36 mg/L),

release from the PPBU and PPBTU micelles was observed after the laser exposure. As mentioned above, the increased drug loading efficiency of PPBU and PPBTU was attributed to the hydrogen bonding between drug and carrier, thus, the breakage of this interaction resulted in fast drug release. In contrast, only a slight increase of drug release rate was observed in the PPZ micelles. This can be attributed to the enhanced drug molecular diffusion at high temperature, since most drug release behavior from amphiphilic spherical nanoparticles were diffusiondependent.38 As the intracellular free Dox could spontaneously and rapidly enter the nuclei and insert into the DNA double strands, while the micelle-enveloped Dox would stay in cytoplasm due to the selective permeability of nuclear membrane, we investigated the intracellular drug release from the drug-loaded micelles by CLSM (Figure 5). The HeLa cells were incubated with Dox or the drug-loaded micelles for 4 h, followed by treating with or without 5 min of NIR laser irradiation, and then the CLSM images were obtained after 4 h of further incubation (Figure 5a,b). The percentage of drugs intra the nuclei was calculated by a pixel statistical analysis (Figure 5c). In the free Dox-treated group, more than 95% of drugs were colocalized within the nuclei, revealing the strong affinity of Dox to the nuclei. More than 70% of the drugs in the PPZ micelles were localized in the cytoplasm, and no remarkable differences were observed with or without the NIR laser irradiation, indicating that the photothermal effect of ICG rarely affected the drug release from the PPZ micelles (Figure 5c). In comparison, distributions of Dox in the PPBU/PPBTU micelles treated cells with NIR laser irradiation were significantly different from the one without NIR laser irradiation. After NIR laser irradiation, an additional 22.3/28.6% of the loaded Dox in PPBU/PPBTU micelles moved from the cytoplasm to the nuclei, indicating that large amount of the drugs were released from the micelles and intercalated into the nuclear DNA within 4 h after the NIR laser irradiation. These results were consistent with the above in vitro drug release profiles. Interestingly, at the same conditions of the intracellular drug release experiments (ICG concentration of 10 mg/L and the G

DOI: 10.1021/acs.biomac.7b01702 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules ORCID

which could be ascribed to the much slower drug release profiles of Dox/ICG-loaded PPBU/PPBTU micelles (Figure 6b−d, Table 3). When exposed to the NIR laser irradiation, the

Jintao Zhu: 0000-0002-8230-3923 Huayu Tian: 0000-0002-2482-3744 Xuesi Chen: 0000-0003-3542-9256

Table 3. IC50 Values of the Dox/ICG-Loaded PPZ, PPBU, and PPBTU Micelles after Incubation with HeLa Cells With or Without NIR Laser Irradiation

Notes

The authors declare no competing financial interest.

■

IC50 (mg/L) group

NIR laser (−)

NIR laser (+)

free Dox PPZ@Dox/ICG PPBU@Dox/ICG PPBTU@Dox/ICG

1.51 3.36 4.53 4.21

− 2.07 1.50 1.12

ACKNOWLEDGMENTS We thank the fund support from the National Natural Science Foundation of China (Grant Nos. 51703074, 51473059, 51673190, 21474104, and 51520105004), the China Postdoctoral Science Foundation (2017M612454), and Open Research Fund of State Key Lab of Polymer Physics & Chemistry, CIAC, CAS (2017-27).

■

Dox/ICG-loaded PPZ micelles only displayed slightly decreased IC50 (2.07 mg/L) due to the unremarkable influence of the drug release of Dox by the photothermal effect and inferior cell-killing abilities of ICG under such low concentration. In contrast, the cytotoxicity of Dox/ICG-loaded PPBU/PPBTU micelles significantly increased under NIR laser irradiation, and their IC50 decreased to 1.50 and 1.12 mg/L, respectively. The reason can be attributed to the photothermal effect-triggered fast drug release from the hydrogen bonding-enhanced micelles, which coincides with the drug release results.

(1) Li, H. J.; Du, J. Z.; Liu, J.; Du, X. J.; Shen, S.; Zhu, Y. H.; Wang, X.; Ye, X.; Nie, S.; Wang, J. Smart Superstructures with Ultrahigh pHSensitivity for Targeting Acidic Tumor Microenvironment: Instantaneous Size Switching and Improved Tumor Penetration. ACS Nano 2016, 10, 6753−6761. (2) Li, Y.; Thambi, T.; Lee, D. S. Co-Delivery of Drugs and Genes Using Polymeric Nanoparticles for Synergistic Cancer Therapeutic Effects. Adv. Healthcare Mater. 2018, 7, 1700886. (3) Tan, J. P. K.; Kim, S. H.; Nederberg, F.; Fukushima, K.; Coady, D. J.; Nelson, A.; Yang, Y. Y.; Hedrick, J. L. Delivery of Anticancer Drugs Using Polymeric Micelles Stabilized by Hydrogen-Bonding Urea Groups. Macromol. Rapid Commun. 2010, 31, 1187−1192. (4) Yao, C.; Wang, X.; Liu, G.; Hu, J.; Liu, S. Distinct Morphological Transitions of Photoreactive and Thermoresponsive Vesicles for Controlled Release and Nanoreactors. Macromolecules 2016, 49, 8282−8295. (5) Ding, J.; Chen, L.; Xiao, C.; Chen, L.; Zhuang, X.; Chen, X. Noncovalent Interaction-Assisted Polymeric Micelles for Controlled Drug Delivery. Chem. Commun. 2014, 50, 11274−11290. (6) Rösler, A.; Vandermeulen, G. W. M.; Klok, H. A. Advanced Drug Delivery Devices via Self-Assembly of Amphiphilic Block Copolymers. Adv. Drug Delivery Rev. 2012, 64, 270−279. (7) Lv, S.; Song, W.; Tang, Z.; Li, M.; Yu, H.; Hong, H.; Chen, X. Charge-Conversional PEG-Polypeptide Polyionic Complex Nanoparticles from Simple Blending of A Pair of Oppositely Charged Block Copolymers as An Intelligent Vehicle for Efficient Antitumor Drug Delivery. Mol. Pharmaceutics 2014, 11, 1562−1574. (8) Zhao, D.; Yi, X. Q.; Yuan, G. D.; Zhuo, R. X.; Li, F. Design and Construction of A Smart Targeting Drug Delivery System Based On Phototriggered Competition of Host-Guest Interaction. Macromol. Biosci. 2017, 17, 1700150. (9) Zhu, J. L.; Liu, K. L.; Wen, Y. T.; Song, X.; Li, J. Host-Guest Interaction Induced Supramolecular Amphiphilic Star Architecture and Uniform Nanovesicle Formation for Anticancer Drug Delivery. Nanoscale 2016, 8, 1332−1337. (10) Cheng, H.; Cheng, Y. J.; Bhasin, S.; Zhu, J. Y.; Xu, X. D.; Zhuo, R. X.; Zhang, X. Z. Complementary Hydrogen Bonding Interaction Triggered Co-Assembly of An Amphiphilic Peptide and An AntiTumor Drug. Chem. Commun. 2015, 51, 6936−6939. (11) Kim, S. H.; Tan, J. P. K.; Nederberg, F.; Fukushima, K.; Colson, J.; Yang, C.; Nelson, A.; Yang, Y. Y.; Hedrick, J. L. Hydrogen BondingEnhanced Micelle Assemblies for Drug Delivery. Biomaterials 2010, 31, 8063−8071. (12) Kuang, H.; Wu, S.; Xie, Z.; Meng, F.; Jing, X.; Huang, Y. Biodegradable Amphiphilic Copolymer Containing Nucleobase: Synthesis, Self-Assembly in Aqueous Solutions, and Potential Use in Controlled Drug Delivery. Biomacromolecules 2012, 13, 3004−3012. (13) Kuang, H.; Wu, S.; Meng, F.; Xie, Z.; Jing, X.; Huang, Y. CoreCrosslinked Amphiphilic Biodegradable Copolymer Based on the Complementary Multiple Hydrogen Bonds of Nucleobases: Synthesis,

■

CONCLUSIONS In summary, a series of mPEG-b-PLL-based amphiphilic BCPs with similar chemical structures while different hydrogen bonding donors (e.g., urethane, urea, and thiourea groups) were synthesized. The additional strong hydrogen bonding among the hydrophobic cores provided the carriers with decreased CMCs and enhanced micellar stabilities. Moreover, the strong hydrogen bonding between the hydrophobic segments and drugs enhanced the drug loading capacities and decreased micelle sizes of the BCPs, however, slowed drug release profiles as well. Furthermore, the incorporation of ICG not only provided the system with potentials of photothermal therapy, but also accelerated the Dox release from the hydrogen bonding-enhanced micelles under NIR laser irradiation, resulting in significant increase of cytotoxicity. These results demonstrated that the hydrogen bonding-enhanced micelles are promising carriers for chemo-photothermal synergistic therapy.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b01702. Figures S1−S7 of following data: GPC curves of the polymers; drug release profiles, cell viabilities, and typical micrographs of Dox-loaded hydrogen bond-enhanced micelles; photothermal effects, cellular uptake analyses of Dox/ICG-loaded micelles; cell viabilities of ICG-loaded micelles toward HeLa cells (PDF).

■

REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +86-27-8779 3240. *E-mail: [email protected]. Tel.: +86-431-8526 2539. H

DOI: 10.1021/acs.biomac.7b01702 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules Self-Assembly and in vitro Drug Delivery. J. Mater. Chem. 2012, 22, 24832−24840. (14) Li, W. E.; Wang, H.; Wei, Z.; Ning, J.; Ma, L.; Zheng, H.; Niu, H.; Huang, W. Supramolecular Prodrug Micelles Based on the Complementary Multiple Hydrogen Bonds as Drug Delivery Platform for Thrombosis Therapy. RSC Adv. 2016, 6, 62478−62484. (15) Kothari, K.; Ragoonanan, V.; Suryanarayanan, R. The Role of Drug−Polymer Hydrogen Bonding Interactions on the Molecular Mobility and Physical Stability of Nifedipine Solid Dispersions. Mol. Pharmaceutics 2015, 12, 162−170. (16) Chang, X.; Ma, C.; Shan, G.; Bao, Y.; Pan, P. Poly(Lactic Acid)/ Poly(Ethylene Glycol) Supramolecular Diblock Copolymers Based on Three-Fold Complementary Hydrogen Bonds: Synthesis, Micellization, and Stimuli Responsivity. Polymer 2016, 90, 122−131. (17) Kubo, T.; Koterasawa, K.; Naito, T.; Otsuka, K. Molecularly Imprinted Polymer with A Pseudo-Template for Thermo-Responsive Adsorption/Desorption Based on Hydrogen Bonding. Microporous Mesoporous Mater. 2015, 218, 112−117. (18) Li, Y. C.; Tian, H. Y.; Ding, J. X.; Dong, X.; Chen, J.; Chen, X. S. Thiourea Modified Polyethylenimine for Efficient Gene Delivery Mediated by the Combination of Electrostatic Interactions and Hydrogen Bonds. Polym. Chem. 2014, 5, 3598−3607. (19) Deng, Z.; Qian, Y.; Yu, Y.; Liu, G.; Hu, J.; Zhang, G.; Liu, S. Engineering Intracellular Delivery Nanocarriers and Nanoreactors from Oxidation-Responsive Polymersomes via Synchronized Bilayer Cross-Linking and Permeabilizing Inside Live Cells. J. Am. Chem. Soc. 2016, 138, 10452−10466. (20) Wang, C.; Zhang, G.; Liu, G.; Hu, J.; Liu, S. Photo- and Thermo-Responsive Multicompartment Hydrogels for Synergistic Delivery of Gemcitabine and Doxorubicin. J. Controlled Release 2017, 259, 149−159. (21) Eggert, D.; Naumann, M.; Reimer, R.; Voigt, C. A. Nanoscale Glucan Polymer Network Causes Pathogen Resistance. Sci. Rep. 2014, 4, 4159. (22) Król, P. Synthesis Methods, Chemical Structures and Phase Structures of Linear Polyurethanes. Properties and Applications of Linear Polyurethanes in Polyurethane Elastomers, Copolymers and Ionomers. Prog. Mater. Sci. 2007, 52, 915−1015. (23) Ashworth, C. R.; Matthews, R. P.; Welton, T.; Hunt, P. A. Doubly Ionic Hydrogen Bond Interactions within the Choline Chloride-Urea Deep Eutectic Solvent. Phys. Chem. Chem. Phys. 2016, 18, 18145−18160. (24) Buckwalter, D. J.; Hudson, A. G.; Moore, R. B.; Long, T. E. Synthesis and Characterization of Poly(Propylene Glycol) Polytrioxamide and Poly(Urea Oxamide) Segmented Copolymers. Polym. Int. 2014, 63, 1184−1191. (25) Jin, R.; Sun, W. Theoretical Study of Thiourea Derivatives as Chemosensors for Fluoride and Acetate Anions. Sci. China: Chem. 2012, 55, 1428−1434. (26) Zheng, W. R.; Fu, Y.; Shen, K.; Liu, L.; Guo, Q. X. Theoretical Study on Hydrogen Bonding Interaction of Ureas and Thioureas with Imines. J. Mol. Struct.: THEOCHEM 2007, 822, 103−110. (27) Sun, H.; Mei, H.; Xie, C.; Lu, Z.; Han, J.; Pan, Y. Modified Thiourea Used as the Ligand for the Synthesis of Novel Coordination Polymers: Based on Its Significant Conformational Behavior and HBonding Preference. Inorg. Chem. Commun. 2010, 13, 1361−1365. (28) Li, H.; Li, J.; Ke, W.; Ge, Z. A Near-Infrared Photothermal Effect-Responsive Drug Delivery System Based on Indocyanine Green and Doxorubicin-Loaded Polymeric Micelles Mediated by Reversible Diels−Alder Reaction. Macromol. Rapid Commun. 2015, 36, 1841− 1849. (29) Ding, J.; Li, C.; Zhang, Y.; Xu, W.; Wang, J.; Chen, X. ChiralityMediated Polypeptide Micelles for Regulated Drug Delivery. Acta Biomater. 2015, 11, 346−355. (30) Huang, M.; Li, H.; Ke, W.; Li, J.; Zhao, C.; Ge, Z. Finely Tuned Thermo-Responsive Block Copolymer Micelles for Photothermal Effect-Triggered Efficient Cellular Internalization. Macromol. Biosci. 2016, 16, 1265−1272.

(31) Wan, Z.; Mao, H.; Guo, M.; Li, Y.; Zhu, A.; Yang, H.; He, H.; Shen, J.; Zhou, L.; Jiang, Z.; Ge, C.; Chen, X.; Yang, X.; Liu, G.; Chen, H. Highly Efficient Hierarchical Micelles Integrating Photothermal Therapy and Singlet Oxygen-Synergized Chemotherapy for Cancer Eradication. Theranostics 2014, 4, 399−411. (32) Zheng, M.; Yue, C.; Ma, Y.; Gong, P.; Zhao, P.; Zheng, C.; Sheng, Z.; Zhang, P.; Wang, Z.; Cai, L. Single-Step Assembly of Dox/ ICG Loaded Lipid−Polymer Nanoparticles for Highly Effective Chemo-Photothermal Combination Therapy. ACS Nano 2013, 7, 2056−2067. (33) Zhu, C.; Huo, D.; Chen, Q.; Xue, J.; Shen, S.; Xia, Y. A Eutectic Mixture of Natural Fatty Acids Can Serve as the Gating Material for Near-Infrared-Triggered Drug Release. Adv. Mater. 2017, 29, 1703702. (34) Kim, S. H.; Nederberg, F.; Jakobs, R.; Tan, J. P. K.; Fukushima, K.; Nelson, A.; Meijer, E. W.; Yang, Y. Y.; Hedrick, J. L. A Supramolecularly Assisted Transformation of Block-Copolymer Micelles into Nanotubes. Angew. Chem., Int. Ed. 2009, 48, 4508−4512. (35) Zhang, D. D.; Ruan, Y. B.; Zhang, B. Q.; Qiao, X.; Deng, G.; Chen, Y.; Liu, C. Y. A Self-Healing PDMS Elastomer Based on Acylhydrazone Groups and the Role of Hydrogen Bonds. Polymer 2017, 120, 189−196. (36) Izanloo, C.; Parsafar, G. A.; Abroshan, H.; Akbarzade, H. Calculation of Melting Temperature and Transition Curve for Dickerson DNA Dodecamer on the Basis of Configurational Entropy, Hydrogen Bonding Energy, and Heat Capacity: A Molecular Dynamics Simulation Study. J. Iran. Chem. Soc. 2011, 8, 708−716. (37) Yoon, H. J.; Lee, H. S.; Lim, J. Y.; Park, J. H. Liposomal Indocyanine Green for Enhanced Photothermal Therapy. ACS Appl. Mater. Interfaces 2017, 9, 5683−5691. (38) Hu, Y.; Wang, Q.; Wang, J.; Zhu, J.; Wang, H.; Yang, Y. Shape Controllable Microgel Particles Prepared by Microfluidic Combining External Ionic Crosslinking. Biomicrofluidics 2012, 6, 026502. (39) Feula, A.; Tang, X.; Giannakopoulos, I.; Chippindale, A. M.; Hamley, I. W.; Greco, F.; Paul Buckley, C.; Siviour, C. R.; Hayes, W. An Adhesive Elastomeric Supramolecular Polyurethane Healable at Body Temperature. Chem. Sci. 2016, 7, 4291−4300. (40) Wang, J.; Liu, Y.; Ma, Y.; Sun, C.; Tao, W.; Wang, Y.; Yang, X.; Wang, J. NIR-Activated Supersensitive Drug Release Using Nanoparticles with A Flow Core. Adv. Funct. Mater. 2016, 26, 7516−7525.

I

DOI: 10.1021/acs.biomac.7b01702 Biomacromolecules XXXX, XXX, XXX−XXX