High-Intensity Focused Ultrasound-Induced Disulfide Mechanophore Activation in Polymeric Nanostructures for Molecule Release

Open AccessCCS ChemistryRESEARCH ARTICLES30 Apr 2024High-Intensity Focused Ultrasound-Induced Disulfide Mechanophore Activation in Polymeric Nanostructures for Molecule Release Jilin Fan, Kuan Zhang, Mingjun Xuan, Xiang Gao, Rostislav Vinokur, Robert Göstl, Lifei Zheng and Andreas Herrmann Jilin Fan DWI—Leibniz Institute for Interactive Materials, 52056 Aachen Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, 52074 Aachen , Kuan Zhang DWI—Leibniz Institute for Interactive Materials, 52056 Aachen Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, 52074 Aachen Wenzhou Institute, University of Chinese Academy of Sciences, 325001 Wenzhou , Mingjun Xuan *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] DWI—Leibniz Institute for Interactive Materials, 52056 Aachen Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, 52074 Aachen , Xiang Gao DWI—Leibniz Institute for Interactive Materials, 52056 Aachen Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, 52074 Aachen , Rostislav Vinokur DWI—Leibniz Institute for Interactive Materials, 52056 Aachen , Robert Göstl DWI—Leibniz Institute for Interactive Materials, 52056 Aachen Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, 52074 Aachen , Lifei Zheng *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Wenzhou Institute, University of Chinese Academy of Sciences, 325001 Wenzhou and Andreas Herrmann *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] DWI—Leibniz Institute for Interactive Materials, 52056 Aachen Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, 52074 Aachen https://doi.org/10.31635/ccschem.024.202403876 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Ultrasound (US) activation of mechanophores in polymers that initiates cascade chemical reactions is a promising strategy for on-demand molecule release. However, the typical US frequency used for mechanochemistry is around 20 kHz, producing inertial cavitation that exceeds the tolerance threshold of biological systems. Here, high-intensity focused US (HIFU) as a mechanical stimulus is introduced to drive the activation of disulfide mechanophores in hyperbranched star polymers (HBSPs) and microgels (MGLs). The mechanism of molecular release is attributed to the thiol-disulfide exchange reaction and subsequent intramolecular cyclization. We reveal that HBSPs and MGLs effectively transduce HIFU as mechanical input to chemical output, demonstrated by the quantification of the release of fluorescent umbelliferone (UMB). Moreover, an in vitro study of drug release is carried out using camptothecin as the model drug, which is covalently loaded in MGLs, demonstrating the potential of our system for controlled drug delivery to cancer cells. Download figure Download PowerPoint Introduction Polymer mechanochemistry strives to control chemical transformations by rearranging or cleaving specific bonds at precisely defined sites within polymer chains. Mechanical forces, such as tension and compression, or indirect sources, such as US, are being used.1–4 The force-sensitive moieties responsible for this process are known as mechanophores.5–9 The development of diverse mechanophores has expanded the possibilities for designing polymer systems with tailored mechanochemical properties.10–16 While polymer mechanochemistry has found widespread applications in polymer science ranging from damage detection,17–19 stress sensing,20–22 and nanolithography4 to self-regulating materials,23 its utilization in a biomedical context is still in an early stage. Recently, our group24–30 and others31–33 has pioneered the field termed sonopharmacology, which encompasses the application of principles of polymer mechanochemistry for pharmacotherapy.34 Indeed, the deep penetration of US in biological tissues offers the possibility of achieving mechanochemical processes in biological systems with precise spatiotemporal control. This capability provides a valuable framework for manipulating the activation and release of drug molecules.35,36 However, conventional polymer mechanochemistry initiated by 20 kHz US poses a risk of damaging cells and tissues due to the strong cavitation effect and the formation of liquid microjets.37 In contrast, high-intensity focused US (HIFU) with MHz frequency has been proven to be biocompatible and is commonly associated with biomedical applications.38 Among these, Li and coworkers32 have reported the activation of azo mechanophores embedded in hydrogels by HIFU to generate free radicals for noninvasive cancer therapy. More recently, Robb and coworkers33 developed a seminal synergistic platform that combines the selective activation of masked 2-furylcarbinol mechanophores in linear polymers (LPs) with biocompatible focused US using pressure-sensitive gas vesicles (GVs) as acousto-mechanical transducers. Although this approach allows for precise control over the release of drug molecules, it necessitates systemic coinjection and colocalization of the GVs and polymers in tumor tissues, which might add complexity to practical implementation of this technique. Therefore, further research is needed to explore the full potential of biocompatible US-based polymer mechanochemistry for the development of drug delivery systems. Disulfide mechanophores have recently attracted significant interest due to their low bond dissociation energy and thermal stability. They are commonly incorporated into LPs and can be activated in the presence of US irradiation.24–27,39–44 However, the activation of disulfide mechanophores using HIFU in polymeric nanostructures has been unexplored. In this study, we present the design of hyperbranched star polymers (HBSPs) and microgels (MGLs) containing disulfide mechanophores, as well as inactive fluorescent probes (umbelliferone, UMB) or a small molecule drug (camptothecin, CPT). Within these systems, the disulfide mechanophores are covalently incorporated. Upon HIFU irradiation, the disulfide mechanophores undergo bond scission, generating free thiols. These, in turn, activate a carbonate unit within the polymer scaffolds through thiol-disulfide exchange and intramolecular cyclization, resulting in the release of UMB or CPT (Figure 1a,b). By exposing HeLa cells to the released drug, we can further investigate their cytotoxic effects and potential for cancer treatment. Overall, this work demonstrates the potential of HIFU as a noninvasive method for triggering drug release from polymeric nanomaterials. Figure 1 | (a) Summary of previous work on US-activated LP structures containing disulfide mechanophores. (b) Schematic illustration of the mechanism of disulfide mechanophores activation in branched and cross-linked polymeric structures upon HIFU irradiation involving thiol-disulfide exchange reactions and the following intramolecular cyclization. Download figure Download PowerPoint Experimental Methods Materials All chemical reagents and solvents were used without further purification unless otherwise stated. 2-Hydroxyethyl disulfide (technical grade, Sigma-Aldrich, Steinheim, Baden-Württemberg, Germany), methacryloyl chloride (97%, Sigma-Aldrich), triethylamine (TEA, ≥99%, Sigma-Aldrich), tetrahydrofuran (THF, anhydrous, Sigma-Aldrich), dichloromethane (anhydrous, ≥99.8%, Sigma-Aldrich), N,N′-disuccinimidyl carbonate (≥95%, Sigma-Aldrich), toluene (≥99.5%, Sigma-Aldrich), 4-(dimethylamino)pyridine (>99.0%, TCI Deutschland GmbH, Eschborn, Hesse, Germany), 1,4-dioxane (99.8%, Sigma-Aldrich), triphosgene (98%, Sigma-Aldrich) UMB (99%, ACROS ORGANICS, Bvba, Antwerp, Belgium), CPT (≥95%, Abcr GmbH, Karlsruhe, Baden-Württemberg, Germany), 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (reversible addition fragmentation chain-transfer (RAFT) agent, Sigma-Aldrich), N,N-diisopropylethylamine (≥99%, Sigma-Aldrich), CuBr2 (99%, Sigma-Aldrich), Me6TREN (97%, Sigma-Aldrich), cystamine dihydrochloride (≥97.0%, TCI Deutschland GmbH), 2-mercaptoethanol (MCE, >99.0%, TCI Deutschland GmbH), and bis[2-(2′-bromoisobutyryloxy)ethyl]disulfide (98%, Sigma-Aldrich) were used as received. Pentafluorophenyl methacrylate (PFPMA, 97%, Abcr GmbH) and poly(ethylene glycol) methyl ether methacrylate (PEGMEMA, Mn ∼ 300 Da, Sigma-Aldrich) were purified by a column of activated basic Al2O3 to remove the inhibitor before utilization. 2,2′-Azobis(2-methylpropionitrile) (98.0%) was obtained from Sigma-Aldrich and recrystallized twice from MeOH. Dialysis membranes (3.5 kDa MWCO) were obtained from Spectrum Labs (Rancho Dominguez, California, USA). The centrifugal filter (3000 MWCO) was obtained from Sartorius (Göttingen, Lower Saxony, Germany). The HeLa cell line was obtained from ATCC (Manassas, Virginia, USA): The Global Bioresource Center. Ultrapure Milli-Q water (18.2 MΩ·cm) was used. Analytical instrumentation 1H and 13C NMR spectra were measured at room temperature in CDCl3 with a 400 MHz Bruker Avance 400 spectrometer (Bruker, Billerica, Massachusetts, USA; 13C: 101 MHz). The chemical shifts are reported in δ units using residual protonated solvent signals as the internal standard (1H: CDCl3 (δH = 7.26 ppm), 13C: CDCl3 (δC = 77.16 ppm)). Thin-layer chromatography (TLC) was performed on a Merck TLC Silica gel 60 F254 TLC plates (Merck KGaA, Darmstadt, Hesse, Germany) with a fluorescence indicator, employing a 254 nm or 365 nm UV hand lamp for visualization. Silica gel (40–63 μm) for chromatography was used for flash column chromatography. Gel permeation chromatography/size exclusion chromatography (GPC/SEC) with THF [high-performance liquid-chromatography (HPLC) grade] was performed using a HPLC pump (PU-2080plus, Jasco, Hachioji, Tokyo, Japan) equipped with a refractive index (RI) detector (RI-2031plus, Jasco). The sample solvent contained 250 mg/mL 3,5-di-t-4-butylhydroxytoluene (≥99%, Fluka Chemie GmbH, Buchs, St. Gallen, Switzerland) as the internal standard. One precolumn (8 × 50 mm) and four SDplus gel columns (8 × 300 mm, MZ Analysentechnik GmbH, Mainz, Rhineland-Palatinate, Germany) were applied at a flow rate of 1.0 mL min−1 at 20 °C. The diameter of the gel particles was 5 μm, the nominal pore widths were 50, 102, 103, and 104 Å. Calibration was achieved using narrowly distributed poly(methyl methacrylate) (PMA) standards (polymer standards service (PSS)). Molar masses (Mn and Mw) and molar mass distributions (Mw/Mn) were calculated by using the PSS WinGPC UniChrom software (Version 8.1.1; Mainz, Rhineland-Palatinate, Germany). Ultrahigh performance liquid-chromatography-mass spectrometer (UHPLC-MS) system: ACQUITY UPLC I-Class System (Waters, Milford, Massachusetts, USA) with the compatible ACQUITY UPLC PDA eλ Detector and ACQUITY QDa detector (Waters). Solvents: A = water [contained 0.1% trifluoroacetyl (TFA)], B = acetonitrile (contained 0.1% TFA); flow = 0.4 mL min−1; gradient (B): 0–1 min (10%), 1–5 min (10%–90%), 5–7 min (90%), 7–10 min (90%–10%). Electrospray ionization mass spectrometry (ESI MS): micrOTOF-Q II™ ESI-Qq-TOF mass spectrometer system (BRUKER, Billerica, Massachusetts, USA). Transmission electron microscopy (TEM) images were captured on a LIBRA®120 transmission electron microscope (Carl Zeiss, Oberkochen, Baden-Württemberg, Germany) with an accelerating voltage of 120 kV, and images were recorded using a Gatan Ultra Scan 1000 (Gatan Incorporation, Pleasanton, California, USA). TEM sample preparation: one drop (∼10 μL) of sample was deposited onto a carbon-coated copper grid and then air-dried. Dynamic light scattering (DLS) was measured on a Zetasizer instrument (Zetasizer Ultra, Malvern, Worcestershire, United Kingdom). Samples were dispersed in H2O/DMSO (4:1, v/v), and then the mixture was transferred to a disposable plastic cell. Fluorescence spectroscopy Fluorescence spectroscopy was performed on a SpectraMax iD3 spectrometer (Molecular Devices, San Jose, California, USA) at room temperature. For fluorescence spectra measurements of HBSPs and MGLs-UMB, samples were excited at 325 nm. The spectral bandwidths were set to 1 nm (370 ∼ 600 nm) for emission. To obtain the standard curve of UMB, the fluorescence value was collected at the emission wavelength of 465 nm. For fluorescence spectra measurements of MGLs-CPT, samples were excited at 335 nm. The spectral bandwidths were set to 2 nm (375 ∼ 610 nm) for emission. To obtain the standard curve of CPT, the fluorescence value was collected at the emission wavelength of 449 nm. The integration time was 0.1 s, and all spectroscopic measurements were carried out with a pureGrade™ 96-well plate purchased from BRAND GmbH (Frankfurt, Hesse, Germany). For the sonicated solution of HBSPs or MGLs, filtration steps were needed before the fluorescence measurements. Filtration was carried out as follows: Centrifugation three times at 10,000 rpm (10 min); then the suspensions were filtered through a centrifugal filter (3000 MWCO) by centrifugation at 5000 rpm. Sonication experiments Sonication experiments were performed with a home-built HIFU setup. The core devices include waveform generator (33511B, Keysight Technologies, Santa Rosa, California, USA), RF amplifier (AG1021, T&C Power Conversion, Inc., Rochester, New York, USA), and transducers (0.66 MHz, 1.5 MHz and 2.5 MHz, Precision Acoustics Ltd., Dorchester, Dorset, United Kingdom). A 0.5 mm needle hydrophone (Precision Acoustics Ltd., UK) was used for locating the transducer focal point. A custom-made motorized 3D-manipulator/positioning system for controlling the well plate submerged in water was employed. Pulsed sonication (2 s on, 1 s off) was used. 10 mg of polymer were dissolved in 1 mL mixture of H2O/DMSO (4:1, v/v). 1 mL of this solution was added into the 24-well plate with a transparent base made from ultrathin film (lumox® multiwall 24, SARSTEDT, Nümbrecht, North Rhine-Westphalia, Germany). Then, the well plate was placed in the well plate holder. Samples were exposed to constant sonication for a certain time. The samples were then kept for 72 h to complete the downstream release reactions at room temperature before the fluorescence measurements. Cell imaging HeLa cells used for imaging were cultured in Dulbecco's modified Eagle's Medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 U mL−1 of penicillin and 100 μg mL−1 streptomycin at 37 °C under a humidified atmosphere containing 5% CO2. The HeLa cells were seeded in an ibidi μ-Slide 8 Well (with glass bottom) at a density of 2.5 × 105 cells per well in 500 μL culture medium. After 24 h, HeLa cells were incubated with different concentrated sonicated samples or nonsonicated samples in phosphate-buffered saline (PBS) at 37 °C, then washed with PBS three times. Then, PBS was added into the wells (500 μL per well), and 2 μL (1 mg mL−1) calcein acetomethoxy (AM) and 2 μL (1 mg mL−1) propidium iodide (PI) were transferred to the wells and mixed for 5 min. PBS was used to wash out the free cell imaging agents. Then the fluorescence imaging of cells was performed on a confocal laser scanning microscope (STP8, Leica, Wetzlar, Hesse, Germany) (confocal excitation: Calcein AM: 496 nm, PI: 561 nm) and analyzed by ImageJ. MTS proliferation assays HeLa cells were used to evaluate the cytotoxicity of different samples. HeLa cells were cultured in a basal medium containing DMEM (supplemented with 10% FBS and 1% antibiotics/antimycotics) at 37 °C. Actual cell viability was monitored by using a tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (inner salt, MTS reagent) and a chemical electron acceptor dye (phenazine ethosulfate) (Promega, Germany), using an assay according to the manufacturer's instructions. Briefly, approximately 5000 cells in 100 μL of medium were seeded into 96-well plates. After overnight incubation, the culture medium in the 96-well plates was removed and exchanged with fresh medium (100 μL) containing different concentrated testing samples. Control cultures were treated with dimethyl sulfoxide (DMSO) alone. The final concentration of DMSO in the medium did not exceed 0.5%. After 48 h incubation, the cell culture media were removed, the cells were washed with 100 μL PBS buffer, and then 20 μL MTS reagent with 100 μL fresh medium was added to the cells. The mixture of MTS reagent with cell culture medium served as negative control. The resulting solution was mixed thoroughly, and the absorbance was monitored using a microplate spectrophotometer at 490 nm (Synergy™ HT microplate reader, BioTek Instruments, Winooski, Vermont, USA). MTS signals were used for survival and proliferation determination. All the sample cultures were performed at least in triplicate. Results and Discussion HBSPs synthesis and HIFU-induced disulfide mechanophore activation Initially, we assessed whether cargo molecules could be released from the disulfide-carbonate structure. Therefore, the monomer containing a disulfide, carbonate, and an UMB unit ( A2-1) as well as a model compound containing only a hexanol, carbonate, and UMB unit ( A3) were synthesized ( Supporting Information Scheme S1). UMB was the designated fluorescent reporter, allowing facile quantification of molecule release. In compound A2-1, a carbonate linker was positioned in the β-position to the disulfide moiety. Upon thiol-initiated thiol-disulfide exchange and the following intramolecular cyclization, UMB was released, and its fluorescence was turned on due to breakage of the carbonate linker. As shown in Supporting Information Figures S1, S5, and S6, strong fluorescence of A2-1 solution was observed under 365 nm UV light irradiation after the addition of MCE, indicating the occurrence of the thiol-disulfide exchange reaction, followed by the intramolecular cyclization ( Supporting Information Scheme S2). The formation of the released fluorescent UMB was further confirmed by 1H NMR spectroscopy and ultrahigh-performance liquid chromatography (UPLC) ( Supporting Information Figures S3 and S4). As a control sample, A3 without the disulfide group was also mixed with MCE. As expected, the reaction mixture containing A3 and MCE showed no enhancement of fluorescence ( Supporting Information Figures S1 and S2). Next, HBSPs containing disulfide mechanophores were synthesized by combining RAFT copolymerization and chain extension from the endpoints of the HBSP-core with the biocompatible PEG-analogue PEGMEMA. We hypothesized that chain extension would increase the molar mass of the constructs and facilitate the force transmission from the solvated chain ends to the mechanophore-loaded core. The synthesis involved two steps: the fabrication of the polymeric core architecture and the growth of PEGMEMA chains on the surface of the polymeric core (Figure 2a and Supporting Information Scheme S3). A2-1 containing UMB fluorophores were copolymerized into the HBSPs structures for monitoring the release process. The molar mass of the HBSPs was roughly estimated to 113 kDa by GPC using a RI detector ( Supporting Information Figure S7). The molar ratio of PEGMEMA to A2-1 was approximately 59:1 ( Supporting Information Figure S8), meaning that each HBSP polymer contained between 6 and 7 A2-1 molecules. The morphology and size distribution of the HBSPs were studied using TEM (Figure 2b and Supporting Information Figure S9) and DLS, respectively. The diameter of HBSPs was found to be in the range of 20–50 nm ( Supporting Information Figures S10 and S11). Figure 2 | Synthesis and characterization of HBSPs, and HIFU-induced disulfide mechanophore activation. (a) RAFT polymerization of PEGMEMA, A2-1, and B1 for the synthesis of HBSPs-core, followed by chain extension on HBSPs-core surface with PEGMEMA. (b) TEM micrograph of HBSPs. (c) Photograph of HBSPs solutions under 365 nm UV light irradiation before and after HIFU treatment (f = 1.5 MHz, 2 s on, 1 s off, 30 min, 3 d equilibration). (d) Mass spectrometry of released UMB after HIFU treatment. The molar mass of UMB at 163.04 Da was retrieved. (e) UPLC elugrams of released UMB with a retention time of 2.82 min. (f) Excerpt from 1H NMR spectrum of released UMB (400 MHz, CDCl3). Color-coded signals represent the characteristic peaks of UMB. (g) Schematic of the degradation of HBSPs under HIFU irradiation. (h) The mechanism of HIFU-induced disulfide scission and subsequent intramolecular reaction pathway for activating disulfide mechanophores and fluorescent UMB release. Download figure Download PowerPoint Once the HBSPs were obtained, we investigated whether the incorporated disulfide mechanophores could be activated by HIFU. Upon ultrasonication (1.5 MHz, pulse sequence 2 s on, 1 s off) for 30 min, a blue fluorescence from the HBSPs solution was observed (Figure 2c), indicating that the mechanochemical transduction occurred and generated free thiols, which subsequently induced the release of the fluorescent UMB molecules. This was further confirmed by UPLC-MS measurements (Figure 2d,e) and the appearance of characteristic proton peaks of UMB in the 1H NMR spectrum (Figure 2f). The degradation of HBSPs under HIFU irradiation is schematically shown in Figure 2g. In addition, the mechanism of disulfide mechanophore activation and UMB release from HBSPs under HIFU irradiation is illustrated in Figure 2h. To quantify the amount released upon US activation, the fluorescence of sonicated HBSPs solution was measured. The release efficiency was calculated by using the fluorescence of a HBSPs solution that was treated with a large excess of reducing agent MCE as a reference ( Supporting Information Figures S15–S17). It was found that the fluorescence intensity increased rapidly in the first 10 min, and the release efficiency of UMB reached the maximum of 49% after 20 min. For comparison, LPs with a molar mass of 48 kDa and a central disulfide mechanophore were synthesized ( Supporting Information Scheme S4 and Figures S12 and S13). No fluorescence was detected after 30 min of HIFU treatment ( Supporting Information Figure S14), suggesting no activation of the disulfide mechanophore in LPs. This difference can be attributed to the fact that HBSPs have more disulfide scission sites compared to LPs, allowing for a larger number of mechanically active disulfide bonds to facilitate the destruction of HBSPs into fragments for UMB release. Another reason could be the multimechanophore architecture and the higher molar mass of the HBSPs that allows for more efficient transfer of the force to the mechanophores in comparison to linear chain polymers.45 Subsequently, the experimental parameters affecting the efficiency of molecule release were systematically investigated. The acoustic power and mechanical index (MI) of the HIFU are detailed in Supporting Information Table S1. At a certain US frequency, the fluorescence intensity of the sonicated HBSPs solutions increased upon enhancing the acoustic power from 4 to 32 W (Figure 3a–c). Moreover, we found that the response sensitivity of HBSPs to 1.5 MHz HIFU was higher compared to 0.66 or 2.5 MHz HIFU. For example, at a power of 32 W, around 50% of UMB, was released under 1.5 MHz HIFU irradiation in 20 min, while increasing the US frequency to 2.5 MHz or decreasing the US frequency to 0.66 MHz decreased the release efficiency to 28% and 16%, respectively (Figure 3d). Additionally, no UMB release was observed from HBSPs when the HIFU acoustic power was lower than 4 W using the output frequencies of 0.66 or 2.5 MHz, whereas 1.5 MHz HIFU still facilitated more than 9% UMB release. Figure 3 | Quantification of disulfide mechanophore activation and UMB release using different HIFU parameters. Fluorescence spectra of HBSPs solution after 20 min HIFU irradiation with the frequency of (a) 0.66 MHz, (b) 1.5 MHz, and (c) 2.5 MHz. (d) UMB release from HBSPs with different HIFU frequencies and acoustic power. (e) The change in Mn of HBSPs using different HIFU frequencies and acoustic power. (f) The rate constant of HBSPs degradation. (g) Fluorescence spectra of HBSPs solutions after HIFU treatment (1.5 MHz, 32 W) for different durations. (h) The release profile of UMB after HIFU treatment (1.5 MHz) for different durations. (i) Size distribution of HBSPs after HIFU (1.5 MHz, 32 W) treatment for different durations. Download figure Download PowerPoint The molar mass and size of HBSPs after HIFU irradiation was also studied using GPC and DLS, respectively. The change in Mn of HBSPs with 0.66, 1.5, and 2.5 MHz HIFU transducers was recorded ( Supporting Information Table S2) and plotted against power (Figure 3e). We observed that there was an inversely proportional relationship between Mn and HIFU power.46,47 The molar mass of HBSPs decreased more rapidly when using the 1.5 MHz HIFU transducer. To determine the degradation rate constants of polymers, kinetic analysis was conducted using a previously reported method ( Supporting Information Table S3).48,49 The rate constant of HBSPs decomposition was plotted as a function of Mn against sonication time. The trend of the rate constant with acoustic power is shown in Figure 3f. Under 1.5 MHz HIFU irradiation, the maximum rate constant was around 60·10−5 min−1, which was higher than those using the other two frequencies. Then, fluorescence intensity measurements were conducted to quantify molecular release at varied HIFU durations using an acoustic power of 32 W (Figure 3g), revealing that more than 50% of fluorescent molecules were released within 30 min sonication (Figure 3h). According to DLS measurements, the size of HBSPs dramatically decreased within 5 min and dropped to less than 10 nm after 20 min, which is consistent with the release kinetics of UMB molecules (Figure 3i and Supporting Information Figure S18). Synthesis of MGLs-UMB and HIFU-induced disulfide mechanophore activation Encouraged by the results obtained with HBSPs, we attempted to broaden the carrier scope to MGLs. MGLs-UMB were synthesized in two steps, as illustrated in Figure 4a and Supporting Information Scheme S5. The RAFT polymerization process involved the use of PEGMEMA, PFPMA, and acrylate-disulfide-UMB ( A2-1) to produce copolymer P1 ( Supporting Information Figure S19) with a molecular weight of 12.9 kDa. The molar ratio of PEGMEMA to PFPMA to A2-1 was approximately 14:5:1 ( Supporting Information Figure S20). Each P1 polymer contained approximately two acrylate-UMB molecules. Subsequently, P1 was cross-linked using cystamine to form MGLs-UMB. Notably, the leaving group pentafluorophenolate facilitated the cross-linking reaction, which was characterized by 19F NMR spectroscopy ( Supporting Information Figure S21). The morphology and size distribution of MGLs-UMB were examined using TEM imaging (Figure 4b and Supporting Information Figure S22). The MGLs-UMB exhibited a size range of 150–450 nm ( Supporting Information Figure S23). Figure 4 | Synthesis and characterization of MGLs-UMB and HIFU activation of disulfide mechanophore in MGLs-UMB. (a) The synthesis of copolymer (P1) using RAFT polymerization of PEGMEMA, PFPMA and A2-1 and the following cross-linking by cystamine to form MGLs-UMB. (b) TEM micrograph of MGLs-UMB. (c) The mechanism of disulfide mechanophore activation in MGLs-UMB. Fluorescence spectra of MGLs-UMB samples after (d) 0.66 MHz, (e) 1.5 MHz, and (f) 2.5 MHz HIFU treatment with an acoustic power of 32 W. (g) The release profile of UMB under HIFU treatment with different frequencies. (h) Fluorescence spectra of MGLs after 30 min HIFU sonication at different acoustic power. (i) UMB release from MGLs-UMB with different HIFU frequencies and acoustic power. Download figure Download PowerPoint The mechanism of disulfide mechanophore activation and UMB release