High Intensity Focused Ultrasound Responsive Metallo

Publication Date (Web): July 11, 2014. Copyright ... The development of stimuli-responsive polymeric micelles for effective delivery of chemotherapeut...
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High Intensity Focused Ultrasound Responsive Metallosupramolecular Block Copolymer Micelles Bo Liang,†,‡ Rui Tong,† Zhenhua Wang,† Shengwei Guo,‡ and Hesheng Xia*,† †

State Key Laboratory of Polymer Materials Engineering, Polymer Research Institute, Sichuan University, Chengdu 610065, China School of Material Science and Engineering, Beifang University of Nationalities, Yinchuan 750021, Ningxia Hui Autonomous Region, China

‡

S Supporting Information *

ABSTRACT: The metal−supramolecular diblock copolymer containing mechano-labile bis(terpyridine)−Cu(II) complex linkage in the junction point was synthesized. These metal−ligand containing amphiphilic copolymers are able to self-assemble in aqueous solution to form spherical micelles with poly(propylene glycol) block forming the hydrophobic core. It is found that high intensity focused ultrasound can open the copolymer micelles and trigger the release of the payload in the micelle. The micellar properties and release kinetics of encapsulated guest molecule in response to ultrasound stimuli were investigated. The weak Cu(II)−terpyridine dynamic bond in the copolymer chain can be cleaved under ultrasound and thus leads to the disruption of the copolymer micelle and the release of loaded cargo. This study will open up a new way for the molecular design of ultrasound modulated drug delivery systems.

1. INTRODUCTION The synthesis of multifunctional nanocarriers for drug delivery and the design of new stimuli-responsive means are of great significance.1 A major challenge is the realization of the optimized coupling interaction between the physics stimulus means and the microscopic nanocarrier. Recently, ultrasound triggered release from liposomes,2 polyelectrolyte microcontainers,3 microemulsions,4 multilayered capsules,5 and micelles6−10 has attracted increasing attention. However, the use of ultrasound, especially the high intensity focused ultrasound (HIFU), to control polymer micellar disruption remains largely unexplored as compared to the use of pH or temperature or even light.11−13 Also, the novel interaction mechanism between HIFU and nanocarriers, especially polymer micelles, is needed to develop. Previously, we examined the ultrasound responsive behavior of copolymer micelles under HIFU, but the responsive rate is still not ideal, especially at a lower HIFU power owing to the relatively weak cavitation effect of HIFU.13−16 In order to improve the ultrasound response, it is of fundamental interest to develop block copolymer micelles that can be rapidly and efficiently disrupted by HIFU. For such a purpose, the copolymer should contain weak bonds, ideally mechano-labile ones that are sensitive to the mechanical effects associated with the ultrasonic cavitation. The supramolecular copolymer micelle containing a metal− ligand bond located at the junction between two blocks offers the possibility of constructing this kind of HIFU responsive micelles. In the past few years, self-assembly of metallo© 2014 American Chemical Society

supramolecular block copolymers has made significant progress.17 Schubert et al. developed a series of metallosupramolecular amphiphilic AB block copolymers containing a metal−ligand link, and these copolymers can self-assemble to form the micellar architectures in solution.18−22 The metallosupramolecular copolymer micelle could be used for advanced encapsulation and release of both hydrophilic and hydrophobic molecules in the field of nanoreactors, nanocatalysis, and drug/ gene delivery. Some new block copolymers that could not be prepared by classical polymerization techniques can be obtained by linking metal and ligand groups in two separate polymer blocks, regardless of their chemical structure and the reactivity ratios of the constituting comonomers. The metal− ligand coordination bond is highly directional and can be easily broken by ultrasonic cavitation.23,24 The interaction strength of metal−ligand bond can be tuned by changing the types of metal ion and ligand, which is helpful for the selection of ultrasound intensity. Herein we develop novel ultrasound responsive metallosupramolecular block copolymer micelles by coupling metal− ligand bond with the remote HIFU stimulus and realize HIFUcontrolled release of the encapsulants from micelles. The diblock poly(propylene glycol) (PPG) and poly(ethylene glycol) (PEG) copolymer containing a HIFU responsive mechano-labile Cu(II)−terpyridine (Tpy) bond in the junction Received: March 5, 2014 Revised: July 10, 2014 Published: July 11, 2014 9524

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point (PPG-[Cu]-PEG) was synthesized and used as an example to illustrate our idea. Scheme 1 illustrates the HIFU

Scheme 2. Synthesis Route of PPG-[Cu]-PEG Block Copolymer

Scheme 1. Schematic Illustration of HIFU Responsive Process of Block Copolymer Micelles Containing Labile Cu(II)−Terpyridine Bonds

responsive process of the micelles. The process includes the HIFU-induced site-specifically degradation of PPG-[Cu]-PEG chain, micelle disruption, and the controlled payload release as well as the re-formation of metal−ligand bond and reassembly of the copolymer micelle after HIFU was off. PPG-[Cu]-PEG diblock copolymers are assembled into micelles containing a hydrophobic core with labile Cu(II)−Tpy bonds. When the micelles are subjected to HIFU, the Cu(II)−Tpy bonds are cleaved, and the amphiphilic structures are broken, which further leads to the disruption of copolymer micelles and the release of the encapsulated cargo. When HIFU is off, the dynamic Cu(II)−Tpy bonds are re-formed owing to its reversibility and the copolymer micelles are reassemblied again.

were dried over Na2SO4 and removed in vacuo. Then the PPG-Tpy was obtained. After that, PEG-Tpy (0.20 g, 0.1 mmol), PPG-Tpy (0.25 g, 0.1 mmol), and the metal salt copper(II) acetate monohydrate (Cu(OAc)2·H2O) in a molar ratio of 1:1:1 were stirred under reflux in methanol (10 mL) for 4 h, after which an excess of NH4PF6 (0.167 g, 1.0 mmol) was added, which formed the precipitates of PPG-[Cu]PEG and PEG-[Cu]-PEG. The reaction mixture was cooled to room temperature and partitioned between dichloromethane and water. The organic layer containing PPG-[Cu]-PEG was washed three times to remove excess salts, then dried over Na2SO4, and removed in vacuo. Finally, the PPG-[Cu]-PEG was obtained as blue solid (yield: 65%). The comparative sample PPG-[Ru]-PEG complex was synthesized following a similar procedure as PPG-[Cu]-PEG. The obtained PPG[Ru]-PEG is a brown-reddish solid (yield ∼ 60%). Characterizations. Proton nuclear magnetic resonance 1H NMR spectra were recorded at room temperature with a Bruker spectrometer operating at 400 MHz using deuterated chloroform CDCl3 as the solvent and tetramethylsilane as an internal reference. The molecular weight was measured by MALDI-TOF-mass spectra (Bruker Autoflex III) with methanol as solvent. UV/vis measurements were recorded using a UV-Vis 3010 spectrometer with distilled water as solvent. Dynamic light scattering (DLS) was performed on a Brookhaven BI-200 goniometer with vertically polarized incident light of wavelength λ ∼ 532 nm supplied by an argon laser operating at 200 mW and a Brookhaven BI-9000 AT digital autocorrelator. Measurements were made at 25 °C and at the detect angle of 90°. The autocorrelation functions from DLS were analyzed by using the nonnegatively constrained least-squares algorithm (NNLS) method to obtain the diameter distributions. The micellar morphology was observed with scanning electron microscopy (SEM, Inspect F, Fei Company). The specimens for SEM observations were prepared by depositing several drops of micellar solutions onto silicon wafers and were dried by lyophilization. The particle size data were obtained by an Image-Pro Plus analysis software based on the SEM images. Steadystate fluorescence emission spectra of the micelle solutions were

2. EXPERIMENTAL SECTION Materials. Poly(ethylene glycol) methyl ether (PEG, Mn ∼ 2000), poly(propylene glycol) monobutyl ether (PPG, Mn ∼ 2500), 4′chloro-2,2′:6′,2″-terpyridine, Nile Red, and pyrene were obtained from Sigma-Aldrich Chemical Co. Copper(II) acetate monohydrate (Cu(OAc)2·H2O) was purchased from Aladdin Industrial Corporation. PEG was dried for 6 h in vacuum at 60 °C before use. Pluronic F-108, i.e. poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEO−PPO−PEO) with a number-average molecular weight of ∼14 600 and a PEO weight percentage of 82.5 wt %, was purchased from Aldrich. Tetrahydrofuran (THF) was dried by refluxing over sodium wire and distilled to remove the moisture and oxidative impurity prior to use. All chemicals and solvents were used as received unless stated. Synthesis of Metallo-supramolecular Block Copolymer PPG[Cu]-PEG. As shown in Scheme 2, PPG-[Cu]-PEG was synthesized as follows. The terpyridine-terminated poly(ethylene glycol) monomethyl ether (PEG-Tpy) was synthesized according to previous literatures.25,26 Powdered KOH (0.9 g, 16.07 mmol) and poly(ethylene glycol) monomethyl ether (1 g, 0.5 mmol) were stirred in the DMSO solvent at 70 °C. After 30 min, 4′-chloro-2′:6′,2″terpyridine (0.134 g, 0.5 mmol) was added. The mixture was stirred for 24 h, then poured into cold water, and extracted with CH2Cl2. The combined organic layers were dried over Na2SO4 and removed in vacuo. The compounds were followed by a double precipitation from tetrahydrofuran (THF) into diethyl ether. For the terpyridineterminated poly(propylene glycol) monobutyl ether (PPG-Tpy), the powdered KOH (0.5 g, 8.93 mmol) and poly(propylene glycol) monobutyl ether (1 g, 0.4 mmol) were stirred in the DMSO solvent at 65 °C. After 30 min, 4′-chloro-2,2′:6′,2″-terpyridine (0.107 g, 0.4 mmol) was added. The mixture was stirred for 48 h, then poured into cold water, and extracted with CH2Cl2. The combined organic layers 9525

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Figure 1. SEM images of PPG-[Cu]-PEG micelles in solution (a) before and (b) after HIFU for 30 min. DLS curves of PPG-[Cu]-PEG micelles (c) before and (d) after HIFU for 30 min.

(Cu(OAc)2·H2O) to obtain metallo-supramolecular block copolymer PPG-[Cu]-PEG. The selection of copper has some advantages: (1) Copper is a required nutrient, and it is found naturally in foods such as shellfish, beef liver, whole grains, beans, peas, nuts, potatoes, and green vegetables. Copper can help regulate blood pressure and heart rate. (2) Copper has antioxidant properties and may have some anticancer effects, which may help stop cancer growth.28 (3) Copper and its complex have been widely used in medicine.29 For example, recently, the copper−pyridine poly(amidoamine) complex was used as a chemical nuclease in nanomedicine.30 The 1H NMR spectra are shown in Figure S1. The chemical shifts at 8.79, 8.68, 8.12, 7.99, and 7.35 could be attributed to H6, H6″; H3, H3″; H3′, H5′; H4, H4″; and H5, H5″ of the aromatic region from the PPG-Tpy and PEG-Tpy. Of particular interest is the singlet attributed to the 3′,5′ protons on the middle ring, which shifts from 8.49 to 8.12 ppm due to the substitution from a halogen to an oxygen atom at the 4′position. The chemical shifts at ∼4.5 (a′) and ∼4.4 (d′) could be attributed to the methine groups connected with oxygen atoms (−O−CH−) of PPG and the methylene groups connected with oxygen atoms (−O−CH2−) of PEG segments, respectively. Peaks d, a, and b (3.83−3.37) could be attributed to the protons of methylene groups (−CH2−O−) of PEG and methine groups (−O−CH−) and methylene groups (−O− CH(CH3)−CH2−) of PPG. Peaks c (1.17−1.07) could be attributed to the protons of methyl groups (−CH(CH3)−) of PPG. It is noted that no peaks appear in the aromatic region of PPG-[Cu]-PEG spectra, possibly due to the paramagnetic

recorded on a 970CRT spectrophotometer (Shanghai Precision & Scientific Instrument Co., Ltd.). HIFU Experiments. As we reported previously,27 the high intensity focused ultrasound apparatus comprises three main components: arbitrary waveform generator (Agilent 33220A Function Generator), RF power amplifier (A150, Electronics & Innovation), and acoustic lens transducer (H-101, Sonic Concept). The acoustic lens transducer with a high acoustic focal pressure within a long focal volume of Φ 1.26 mm × 11 mm and a geometric focal length of 62.6 mm was mounted at the bottom of a tank filled with water, and the beams of ultrasound were pointed upward and focused on a special spot. The ultrasound output can be adjusted in the range of 0−150 W, and the frequency of ultrasound is 1.1 MHz. The focused beams of ultrasound can penetrate through latex membrane and act on the micelle solution in the cuvette reactor.

3. RESULTS AND DISCUSSION The metallo-supramolecular block copolymer PPG-[Cu]-PEG was synthesized as shown in Scheme 2. The linker consists of two 2,2′:6′,2″-terpyridine ligands that can chelate around a copper(II) ion in an octahedral fashion. Terpyridine is known to form stable bis-complexes with a wide range of transition metal ions. Polymers can be introduced at the 4′-position of the ligand by a nucleophilic substitution reaction. The PEG-Tpy was obtained through the reaction between the poly(ethylene glycol) monomethyl ether and 2,2′:6′,2″-terpyridine in a DMSO solvent. For the preparation of PPG-Tpy, the procedure is similar to that for PEG-Tpy, but the poly(propylene glycol) monobutyl ether was used instead. PEG-Tpy and PPG-Tpy were mixed and reacted with copper acetate monohydrate 9526

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Figure 2. (a) Variation of fluorescence emission spectra of pyrene/PPG-[Cu]-PEG micelle solutions (λex = 337 nm) with different time at a HIFU power output of 2 W. (b) Decrement percentage in fluorescence emission intensity for pyrene/PPG-[Cu]-PEG micelle with the time at different HIFU powers output.

Figure 3. (a) Decrement percentage in fluorescence emission intensity for pyrene/PPG-[Cu]-PEG micelle with time at different temperatures. (b) Decrement percentage in fluorescence emission intensity with time at different HIFU powers output for pyrene/PPG-[Cu]-PEG (A), pyrene/PPG[Ru]-PEG (B), and pyrene/PEO−PPO−PEO (C) micelles.

The SEM image (Figure 1a) shows that the blank PPG-[Cu]PEG micelle is spherical with an average diameter of ∼100 nm and the size distribution is relatively uniform. After HIFU treatment for 30 min, the spherical micelle diameter decreases to ∼70 nm (Figure 1b). The DLS result shows that the mean diameter of blank PPG-[Cu]-PEG micelle is ∼120 nm (Figure 1c), and the mean diameter of PPG-[Cu]-PEG micelle after HIFU for 30 min is ∼80 nm by DLS (Figure 1d). HIFU leads to a slight reduction of the blank PPG-[Cu]-PEG micelle. To verify the HIFU responsive process shown in Scheme 1, HIFU triggered release behavior of the PPG-[Cu]-PEG micelle was first examined. The hydrophobic pyrene and Nile Red (NR) were chosen as the payload because the release of both substances can be easily characterized by the change in the fluorescence emission intensity. 13 This is because the quenching of fluorescence can occur if the fluorescent dye entrapped in the core is released or exposed to water, in which it is insoluble. So the release percentage could be evaluated according to the variation of fluorescence intensity before and after HIFU treatment. Figure 2a shows that the fluorescence emission intensity for the pyrene/PPG-[Cu]-PEG micelle decreases with HIFU time, which indicates that the pyrene was released. It has been

nature of the metal complexes. This also confirms no existence of uncomplexed terpyridine.31 UV/vis spectra (Figure S2) show that after the end-group functionalization of PEG with Tpy the terpyridine absorption bands at 234 and 276 nm appear. The specific Cu(II) terpyridine metal−ligand charge-transfer (MLCT) band at 315 and 327 nm can be found in the spectra of the PPG-[Cu]PEG. In addition, the PEG-Tpy aqueous solution was colorless, while the PPG-[Cu]-PEG solution is blue due to the formation of the Cu(II)−terpyridine bond. The copolymer micelles were prepared by adding water dropwise to the copolymer/THF solution. The addition of water makes the hydrophobic PPG block aggregate together, and the hydrophilic PEG block will prevent the further aggregation of the hydrophobic block. After the dropwise addition of water, a large volume of water is added immediately to stabilize the micelle morphology. The THF in the solution is removed by evaporation, and then the micelle solution with light blue color is obtained. The copolymer micelles containing model payload (pyrene or NR) in the inner hydrophobic core were also prepared for the investigation on the release behavior. The size and morphology of the blank micelles before and after the stimuli of HIFU were characterized by DLS and SEM. 9527

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Figure 4. (a) Variation of fluorescence emission spectra of Nile Red/PPG-[Cu]-PEG micelle solutions with different time at a HIFU power output of 25 W. (b) Decrement percentage in fluorescence emission intensity for Nile Red/PPG-[Cu]-PEG micelle with the time at different HIFU powers output.

confirmed that the decrease in the fluorescence emission intensity is not owing to the degradation of pyrene or Nile Red.14,15 The percentage of released pyrene or Nile Red was estimated based on the decrement percentage in the fluorescence emission intensity using the following equation: % release = (I0 − It)/I0, where I0 and It are the fluorescence emission peak intensity of the micelle solution at ∼393 nm for pyrene or ∼530 nm for NR recorded before and after HIFU treatment for t min, respectively. The decrement percentages in the fluorescence emission intensity at different HIFU powers output with HIFU time are shown in Figure 2b. It is clear that increasing HIFU power output leads to a more rapid release of pyrene due to a relatively stronger cavitation effect at a higher acoustic intensity. At a higher HIFU power output of 25 W, the estimated released percentage of pyrene reaches ∼90% in 30 min. It should be noted that the estimated released percentage of pyrene reaches ∼25% in 5 min and ∼75% in 30 min at a low HIFU power output of 2 W. For our previously investigated HIFU responsive poly(ethylene glycol) (PEG) and poly(Llactic acid) (PLA) block copolymer micelle containing central labile disulfide linkage (PEG-S-S-PLA), nearly no pyrene release occurs after HIFU treatment for 10 min at a power output of 20 W.14 This suggests that PPG-[Cu]-PEG has a more sensitive and rapid ultrasound responsiveness than PEGS-S-PLA, which is important for controlled drug release. Also, the required HIFU power (∼2 W) to realize the payload release for PPG-[Cu]-PEG micelle is the lowest among all reported HIFU responsive block copolymer micelle up to now. In order to exclude the thermal effect on the release behavior, we conducted the pyrene release experiment at different temperatures: 40, 60, and 90 °C. The results are shown in Figure 3a. The released percentage of pyrene reaches only ∼2% at 40 °C, ∼10% at 60 °C, and ∼28% at 90 °C in 30 min, which is much lower than that by HIFU stimulus. The temperature of the micelle solution during HIFU treatment at a power output of 25 W is ∼40 °C in this study. The experiment suggests ultrasound plays an important role in the pyrene release possibly due to the weak cavitation effect in the focal point. To confirm the effect of the dynamic metal−ligand bond, the release behaviors of pyrene from three kinds of micelles(A)

PPG-[Cu]-PEG with a weak metal−ligand bond, (B) PPG[Ru]-PEG with a strong metal−ligand bond, and (C) PEO− PPO−PEO with covalent bond were compared. The decrement percentages in fluorescence emission intensity at different HIFU powers output with time are shown in Figure 3b. Clearly, for both PPG-[Ru]-PEG and PEO−PPO−PEO micelles, the fluorescence emission intensity does not decrease at HIFU powers output of 2 and 7 W, which indicates no release occurs in 30 min for the two systems. While for PPG-[Cu]-PEG micelle, the released percentage of pyrene reaches ∼75% in 30 min at a HIFU power output of 2 W. The control experiment suggests the PPG-[Cu]-PEG has a quick response to HIFU owing to the more labile metal−ligand bond. The reason should be that the weaker Cu(II)−Tpy bonds of PPG-[Cu]PEG can be cleaved by HIFU and leads to the micelle disruption while the strong Ru(II)−Tpy bonds of PPG-[Ru]PEG and covalent bonds of PEO−PPO−PEO could not be broken and micelle disruption does not occur during HIFU treatment. Meier et al. determine the relative binding strength of terpyridine ligand with a series of transition metal ions including Cd, Co, Cu, Fe, Mn, Ni, and Ru by using matrixassisted laser desorption/ionization time-of-flight mass spectrometry, which follows this sequence: Co > Ru > Fe > Ni > Cu > Mn > Cd. This suggests that the Ru(II)−Tpy bond is stronger than the Cu(II)−Tpy bond.32 The observed slight increase in the fluorescence emission intensity under HIFU for PPG-[Ru]-PEG and PEO−PPO−PEO micelle may be due to the ultrasound induced micelle adsolubilization. In order to investigate the generality of HIFU triggered payload release from PPG-[Cu]-PEG micelle, the release experiment of hydrophobic substance Nile Red was conducted. Figure 4a shows that the fluorescence emission intensity decreases with HIFU time, indicating that Nile Red was released. Figure 4b shows that the release rate increases with increasing HIFU power output. Comparing Figure 4b with Figure 2b, an interesting phenomenon can be observed that pyrene is much more rapidly released from the PPG-[Cu]-PEG micelle than Nile Red. For example, the estimated release percentage for Nile Red from PPG-[Cu]-PEG micelle is only ∼50% in 30 min at a HIFU power output of 25 W, while for pyrene it is ∼88.5% at the same condition. This should be 9528

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Figure 5. SEM images of pyrene/PPG-[Cu]-PEG micelles (a) before and (b) after HIFU for 30 min. DLS curves of pyrene/PPG-[Cu]-PEG micelles in solution (c) before and (d) after HIFU for 30 min.

Figure 6. SEM images of NR/PPG-[Cu]-PEG micelle before (a) and after HIFU treatment (b).

attributed to different interaction between the payload and copolymer. Nile Red containing phenoxazine structure possibly has a stronger interaction with metal−ligand copolymer than pyrene containing the polycyclic aromatic hydrocarbons structure. The release mechanism can be attributed to the cavitation effect of HIFU in the focal point and the weak strength of Cu(II)−Tpy metal−ligand bond. It is well accepted that the solvodynamic shear produced by ultrasonic cavitation can lead to the polymer chain scission.33 The velocity gradient resulting from the collapse of cavitation bubbles causes the polymer molecule to elongate, setting up stresses along the backbone. The chain scission occurs preferentially at the prescribed

dynamic reversible bonds in the chain, which was also named “mechanophore”.34−36 It is supposed that the PPG-[Cu]-PEG chain with a weak Cu(II)−terpyridine dynamic bond can be cleaved under HIFU; as a result, the hydrophilic−hydrophobic balance of the micelle shifts to an increased hydrophilicity and thus leads to the disruption of PPG-[Cu]-PEG micelle and the release of loaded cargo. When HIFU is removed, it is possible for the copolymer micelles to reassemble again due to the reformation of dynamic metal−ligand bond. In the past few years, there were some reports on the ultrasound-induced breaking of the metal−ligand bond. Liu et al. use high-intensity ultrasound to initiate the metal−ligand cleavage of metalloporphyrins.37 Sijbesma et al. found that 9529

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Figure 7. (a) Variation of UV/vis spectroscopy of PPG-[Cu]-PEG micelle solution at different HIFU power output for 30 min and (b) the color change of the micelle solution before and after HIFU for 30 min at a power output of 25 W.

Figure 8. MALDI-TOF-MS spectra of PPG-[Cu]-PEG before (a) and (b) after HIFU for 30 min at a power output of 25 W.

silver(I) N-heterocyclic carbene complexes and ruthenium bis(carbene) complex can be cleaved when exposed to ultrasound in solution to initiate activation of the latent catalyst.38 Groote et al. investigated mechanochemical scission of supramolecular polymer complexes by ultrasound using viscosity measurements and molecular dynamics simulations combined with constrained geometry optimization calculations. They confirmed that the limiting molecular weight for ultrasound-induced chain scission is lower for metal−ligand polymers than for their covalent analogues. The calculations indicated that the force required to break the chain in the metal−ligand polymers is between 400 and 500 pN, which is much lower than the force of several nN that is typically required to break the covalent C−C bonds in polymer backbones.39 In order to further understand the release mechanism, the HIFU-induced change in the micelle morphology was investigated. The SEM image (Figure 5a) shows that pyrene/ PPG-[Cu]-PEG micelle is spherical and has a mean diameter of ∼110 nm. The mean diameter of the micelle determined by DLS is ∼200 nm (Figure 5c), which is bigger than that by SEM due to the water swelling of micelle. Upon HIFU treatment, the size and morphology for pyrene/PPG-[Cu]-PEG micelle change. Figure 5b shows that the mean diameter of pyrene/ PPG-[Cu]-PEG micelle determined by SEM decreases to ∼60 nm. The DLS result (∼150 nm, Figure 5d) also shows the decrease trend. The similar HIFU-induced size reduction can also be observed for the Nile Red/PPG-[Cu]-PEG micelle (Figure 6).

It must be pointed out that the size reduction and morphology change of PPG-[Cu]-PEG micelle after HIFU treatment are different from our previous investigated systems. Our previous work on PEG-S-S-PLA and PEG-PTHPMA micelle15,16 shows that after HIFU treatment micelle will enlarge or disappear and finally the aggregates will form. This phenomenon, i.e. HIFU-induced size reduction and morphology change of pyrene/PPG-[Cu]-PEG micelle, supports our hypothesis illustrated in Scheme 1. During HIFU treatment, the pyrene/PPG-[Cu]-PEG micelle was broken due to the cleavage of Cu(II)−Tpy bonds and thus leads to the release of the encapsulated payload; once HIFU treatment is finished, the dynamic Cu(II)−Tpy bonds can be recombined again and the formed copolymer will self-assemble into a new micelle. The UV/vis spectroscopy analysis of PPG-[Cu]-PEG micelles before and after HIFU treatment was conducted. The characteristic band of Cu(II)−terpyridine charge-transfer complex at 234, 275, 315, and 327 nm can be observed in Figure 7a and Figure S2. The absorptions in those regions are attributed to the rotation of the outer pyridine rings along the center C−C bonds that connect them. After HIFU treatment for 30 min at different powers output, the copolymer micelle does not show remarkable changes in the Cu(II)−terpyridine absorption band. The color of the resulted solution slightly fades after HIFU treatment (Figure 7b), which indicates that the copolymer micelle structure changes due to a possible reassembly process of micelle after HIFU treatment. In order to confirm the recombination of dynamic Cu(II)− Tpy bonds in the PEG-[Cu]-PPG. MALDI-TOF mass 9530

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spectrometry is used for the investigation of the change in the molecular weight of the copolymers before and after HIFU. Figure 8 shows MALDI-TOF mass spectra of the PPG-[Cu]PEG before and after HIFU treatment. The number-average molecular weight (Mn) and the weight-average molecular weight (Mw) of the obtained PPG-[Cu]-PEG are 4449 and 4451, respectively, which shows that the Mn of PPG-[Cu]-PEG is nearly equal to the sum of the Mn of block PEG (∼2000) and PPG (∼2500). After HIFU, the MALDI-TOF mass spectrometry of PPG-[Cu]-PEG does not change. The Mn and Mw of the resulted products after HIFU for 30 min at a power output of 25 W are ∼4518 and ∼4523, respectively, which are similar to those of the original PPG-[Cu]-PEG. The results suggest that the reversibility of the bis(terpyridine) Cu(II) complexes after HIFU irradiation.

4. CONCLUSIONS In conclusion, HIFU was used to open the copolymer micelles assembled by the metal−supramolecular diblock copolymer containing mechano-labile Cu(II)−Tpy linkage and trigger the release of the payload in the micelle. The HIFU responsive characteristics for metal−supramolecular diblock copolymer were disclosed. The results showed the payloads can be rapidly released from PPG-[Cu]-PEG micelles in several minutes by HIFU in a remote way at room temperature, while the release does not occur for PPG-[Ru]-PEG with a stronger Ru(II)−Tpy bond and PEO−PPO−PEO with covalent bonds. The release behavior was attributed to a dynamic micelle disruption process resulted from the breakage of the weak Cu(II)−Tpy bonds in the PPG-[Cu]-PEG chain due to the cavitation in the HIFU focal spot. The incorporation of labile metal−ligand bond into the copolymer micelle system would provide a unique opportunity for HIFU triggered drug release from the polymer micelle due to the adjustability of metal−ligand bond strength. This study will open up a new way for the molecular design of ultrasound modulated drug delivery system.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected] (H.X.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS H. Xia acknowledges financial support from National Natural Science Foundation of China (51073100), National High-Tech Research and Development Program of China (863 Program, 2012AA020504), and Program for Changjiang Scholars and Innovative Research Team in University (IRT1163).

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