Photothermal Effect-Triggered Drug Release from Hydrogen Bonding

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Photothermal Effect-Triggered Drug Release from Hydrogen Bonding-Enhanced Polymeric Micelles Yuce Li, Jianxun Ding, Jintao Zhu, Huayu Tian, and Xuesi Chen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01702 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on January 30, 2018

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Biomacromolecules

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

1 Environment ACS Paragon Plus

Biomacromolecules 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

ABSTRACT: Incorporation of non-covalent 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(Llysine) (mPEG-b-PLL) with similar chemical structures while different hydrogen bonding donors (urethane, urea, and thiourea groups) are synthesized, and their capacities for co-delivery 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 anti-cancer activity. These results demonstrate that the hydrogen bonding-enhanced micelles are promising carriers for controllable chemo-photothermal synergistic therapy. KEYWORDS: Hydrogen bonding, Drug release, Photothermal effect, Synergistic therapy, Selfassembly, Block copolymers


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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, non-covalent interactions (e.g., electrostatic interaction7, host-guest interaction8,

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 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, urea-containing 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-crosslinked 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 uracil-terminated 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 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 coworkers 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 four-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 three-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


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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 bondingenhanced micelles thereby improve their cytotoxicity towards cancer cells, herein, we reported a series of amphiphilic BCPs based on mPEG-b-poly(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 co-loading 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.

Hydrogen bonding

Self-assembly in selective solvent

NIR laser irradiation

Dox ICG Enhanced drug loading and stability

Photothermal triggered drug release

Scheme 1. Schematic illustration showing the drug loading and NIR-triggered drug release from hydrogen bonding-enhanced micelles.



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′,6-diamidino-2phenylindole dihydrochloride (DAPI) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Benzyl isocyanate/isothiocyanate were purchased from Tianjin Heowns Biochemical Technology Co., Ltd (Tianjin, China). Amino-terminated mPEG (mPEG–NH2) and NεBenzyloxycarbonyl-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, USA). Actin-Tracker Green was purchased from Beyotime Biotechnology (Nantong, China). Synthesis and Characterization of BCPs with Different Hydrogen Bonding Donors. mPEG-bpoly(Nε-Benzyloxycarbonyl-L-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 dimethyl formamide (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


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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, mPEG-b-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. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker AV 400 NMR spectrometer (Billerica, MA, USA) 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, USA), an 18 angles laser scattering instrument (Wyatt Technology Co., USA) and an OPTILAB DSP interferometric refractometer (Wyatt Technology Co., Santa Barbara, CA, USA). 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 Mili-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, USA), and stirred for another 12 h. The resultant solutions were dialyzed against Mili-Q water and the ultimate volume was made constant at 10 mL to obtain a 1.0 mg/mL solution.



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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, UK). 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 Bonding-Enhanced Polymeric Micelles. Dox-, ICGor 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 Mili-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 Mili-Q water for 24 h and then lyophilized to obtain the drugloaded 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 fluorospectro photometer (Perkin–Elmer LS50B luminescence spectrometer, Waltham, MA, USA) using a standard curve method (λex = 480 nm). DLC and DLE of the drug-loaded micelles were calculated according to Equations 1 and 2, respectively: DLC wt % = DLE wt % =

× 100%

(1)

× 100%

(2)

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.


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The time-dependent temperature increasing profiles were recorded by a K-type thermocouple (Model SC-GG-K-30-36, Omega Engineering Inc., Stamford, CT, USA). 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 6-well 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, USA) based on the method in previous report.21



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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 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, USA). 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 Equation 3: Cell viability % =

%&'()*+ %,-./0-*

× 100%

(3)

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 re-suspended with PBS, and the cellular uptake efficiency was determined on a Guava EasyCyte® flow cytometer (Merk Millipore, Billerica, MA, USA). 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.


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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

(a)

(b)



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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). 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 (–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)). a

b c d e f

g

PPBTU g

g

PPBU h

8

7

PPZ

6

h g

5

a

b

4

f

3

c, d, e

2

1

0

Chemical shift (ppm)

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


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Table 1. Characteristics of the synthesized BCPs and the corresponding micelles. Polymer

Mn a (kDa)

Mn b (kDa)

PDI c

CMC (mg/L)

Dh d (nm)

PPZ

10.2

11.6

1.10

5.60

81.2

PPBU

10.2

11.7

1.20

3.74

57.4

PPBTU

10.5

12.3

1.13

8.47

92.6

Note: a Molecular weight (Mn) obtained from NMR;

b

Mn obtained from GPC; c Polydispersity

index (PDI) obtained from GPC; d Dh obtained from DLS measurements. 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 (Figures 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 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.



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. Amphiphilic BCPs with lower CMCs can keep self-assembled 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 anti-tumor drugs which 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 different hydrogen bonding donors, Dox was firstly encapsulated in the micellar cores (Table 2). Although the strong hydrogen bonding between


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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 folds higher IC50 towards cancer cells (Figures S2 and S3), which is dramatically accordant with the previous reports.3 Table 2. Drug loading capacities of the BCPs with different hydrogen bonding donors.

Drugs

Dox

ICG

Dox/ICG

Polymers

Dh (nm) a

DLC (%, w/w)

DLE (%, w/w)

DOX

ICG

DOX

ICG

PPZ

143.9

5.8 ± 0.6

–

20.5

–

PPBU

117.1

7.5 ± 0.3

–

27.0

–

PPBTU

126.4

9.9 ± 1.5

–

36.6

–

PPZ

101.7

–

7.9 ± 0.8

–

21.4

PPBU

91.6

–

8.2 ± 1.1

–

22.3

PPBTU

150.0

–

8.5 ± 1.3

–

23.2

PPZ

166.4

8.9 ± 1.4

10.5 ± 1.3

36.8

32.6

PPBU

126.1

10.3 ± 1.6

13.5 ± 2.0

45.6

44.9

PPBTU

140.7

13.6 ± 2.1

16.1 ± 3.2

64.0

57.2

Note: a Average hydrodynamic diameters were determined by DLS. “–” represents not available. It was reported that co-loading 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 approved by the United State Food and Drug Administration (FDA) for photothermal therapy, was co-loaded 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 kind 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 results agree well with that of the micelles loaded with neat Dox (Table 2). After co-loading of Dox and ICG, all the micelles displayed increased sizes compared with the control micelles (Figure 3, Table 2). In contrast to larger Dh of the neat PPBTU micelles than that of neat PPZ micelles (92.6 nm vs 81.2 nm), the Dox/ICG co-loaded PPBTU micelles gave a smaller Dh than that of Dox/ICG co-loaded PPZ micelles (140.7 nm vs 166.4 nm). Similar results were also demonstrated by TEM investigation (Figure 3a). Variation of the relative sizes could be attributed to the crosslinking 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, 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.


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Further detailed studies on the self-assembly behaviors of the hydrogen bonding-enhanced copolymers will be performed in the next step.

Figure 3. Characterization of the drug-loaded hydrogen bonding-enhanced 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. 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 co-loaded 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 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.


Biomacromolecules

(a) 100

(b) 100

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

80

Accumulative DOX Release (%)

Accumulative DOX Release (%)

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

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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). Hydrogen bonding was reported to be destroyed under high temperatures, e.g., 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 influence 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 concentration lower than 25 mg/L, the solution


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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/ICGloaded 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 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 diffusion-dependent.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, 5b). 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 co-localized 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.

Figure 5. The 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 was labelled by DAPI (blue) and phalloidin-FITC (green), respectively. The scale bar in upper left CLSM image applies to the others. (c) Intra


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nuclei accumulative Dox fluorescence calculated by pixel statistical analyses. **p < 0.01, ***p < 0.001. Interestingly, at the same conditions of the intracellular drug release experiments (ICG concentration of 10 mg/L and the 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 were 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 towards 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 towards the HeLa cells than the PPZ micelles (3.36 mg/L), 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 Dox/ICGloaded 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.


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(a)

PPZ

PPBU

(b)

PPBTU

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Cell viability (%)

Cell Viability (%)

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PPBU@Dox/ICG(–) PPBU@Dox/ICG(+)

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PPBTU@Dox/ICG(–) PPBTU@Dox/ICG(+)

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1

1

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10



Figure 6. Cell viability of the micelles towards 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). 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. IC50 (mg/L) Group NIR laser (−)

NIR laser (+)

Free Dox

1.51

–

PPZ@Dox/ICG

3.36

2.07

PPBU@Dox/ICG

4.53

1.50

PPBTU@Dox/ICG

4.21

1.12



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 Supporting Information The Supporting Information provides additional 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/ICGloaded micelles; cell viabilities of ICG-loaded micelles towards HeLa cells. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected] (J. Z.). Tel: +86-27-8779 3240


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* E-mail: [email protected] (H. T.). Tel: +86-431-8526 2539. Notes The authors declare no competing financial interest.

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).

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For the Table of Contents Use Only: Title: Photothermal Effect-Triggered Drug Release from Hydrogen Bonding-Enhanced Polymeric Micelles Authors: Yuce Li, Jianxun Ding, Jintao Zhu, Huayu Tian, and Xuesi Chen

Graph:

NIR laser irradiation

Dox ICG Hydrogen bonding enhanced drug loading and stability

Photothermal effect triggered destabilization of hydrogen bonding and drug release


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Photothermal Effect-Triggered Drug Release from Hydrogen Bonding

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