Low Intensity Focused Ultrasound Ignited “Deep-Penetration Nanobomb” (DPNB) for Tetramodal Imaging Guided Hypoxia-Tolerant Sonodynamic Therapy Against Hypoxic Tumors

Introduction

Photodynamic therapy (PDT) has shown great promise in combating tumors, which employs light to activate the photosensitizers and produce highly toxic reactive oxygen species (ROS).1–3 It features the advantages of non-ionization, easy controllability, high efficiency, and low cost. However, the efficacy of PDT seriously depends on the penetration depth of light.4,5 As a mechanical wave, ultrasound (US) has been widely used in the biomedical field, including diagnostic imaging (US imaging) and therapeutic applications (sonodynamic therapy, SDT). Sonosensitizers in SDT generate ROS that promote cell apoptosis and necrosis by low-energy ultrasound activation rather than light energy activation. SDT retains traditional PDT advantages and has a specific killing effect on deep-seated tumors due to the high-penetration US.6–8 Although promising, the dense tumor stroma, increased interstitial fluid pressure, and disordered vasculature in tumors, especially deep-seated tumors, could lead to insufficient nanomedicine accumulation, which is not conducive to initiate SDT for tumor therapy.9–11 Moreover, deep-seated solid tumor cells distant from blood vessels often suffer from insufficient oxygen supply due to tissue oxygen content’s attenuation.12–14 Considering the highly oxygen-dependent manner of SDT, the therapeutic outcome is severely restricted in deep-seated tumors. Therefore, it is necessary, urgent, but challenging to develop a deep-penetration system, which can efficiently induce hypoxic tumor cell death.

Many efforts have been proposed to overcome tumor hypoxia, including delivering oxygen into tumors,15–17 in-situ oxygen generation,18–20 reducing oxygen consumption,21–23 and hypoxia-activated prodrugs chemotherapy.24–26 However, most strategies show limited efficacy in hypoxic tumors and encounter tremendous challenges for clinic applications. Recently, an oxygen-independent free radicals generation strategy is proposed, which employs thermal decomposable initiators (e.g. 2,2-azobis[2-(2-imidazoline-2-yl) propane)] dihydrochloride, benzoyl peroxide, azodiisobutyronitrile, etc.) to generate free radicals for eradiating hypoxic tumors.8,27,28 Although promising, low initiator’s loading capability and high decomposition temperature unavoidably lead to insufficient free radicals generation and diffusion for treating hypoxic tumors. Fortunately, the emergence of ultrasound (US) nanomedicine might provide an ideal choice for constructing next-generation nanoagents to achieve efficient hypoxic tumor therapy, which has been rarely reported.

In contrast to traditional US microbubbles, perfluoropentane (PFP), as a “liquid-to-gas” phase transitional material, was encapsulated in liposomes to prolong their half-life in vivo for effective accumulation in the tumor site.29–31 Acoustic droplet vaporization (ADV) effect could induce a fast phase-transition manner, converting liquid phase PFP into gas phase under ultrasound triggering. We can observe a significantly augmented US and contrast-enhanced ultrasound imaging (CEUS) signal intensity.32,33 Generally, the collapse of bubbles under the treatment of US beams could induce an inert cavitation effect and enhance the therapeutic impact via inherent physical factors, including sheer streets, shock waves, microjets, and ROS production multilevel mechanisms due to their intrinsic mechanical-wave nature. Considering microbubbles could increase the efficacy of high-intensity focused ultrasound (HIFU) ablation with reduced acoustic energy and enhanced pathological lesion in tumor treatment, the phase transitional material with similar therapeutic behavior is anticipated to regulate the acoustic environment in hypoxic tumors.34,35 Notably, the acoustic environment’s change has been continuously developed as advanced oxidation processes for wastewater remediation by oxidizing hazardous organic compounds, which is featured by more efficient cavitation effect and ROS production in anoxic condition with the assistance of N2 than that in the oxic state.36–38 This industrial technology inspires us to develop a theranostic system to combat hypoxic tumors; along this line, we anticipate that PFP based cavitation effect could serve as a newborn strategy for efficiently inducing hypoxic tumor death without the need to deliver oxygen. Moreover, all these approaches still cannot effectively solve deep-seated tumors due to insufficient nanomedicine accumulation. CGNKRTR (tLyP-1), a kind of cell-penetrating peptide, has been regarded as a ligand targeted to the neuropilin-1 receptor (NPR-1) with high affinity and specificity and can penetrate deep-seated tumor cells effectively through endocytic/exocytic transport pathway (CendR pathway).11,31,39 As a result, it is a tendency to integrate deep-penetration peptide into a nanomedicine and explore a fresh therapeutic method toward deep-seated hypoxic tumors by regulating acoustic tumor microenvironment.40–42

In recent years, photosensitizers have shown great promise in tumor theranostics due to their advantages in photoacoustic (PA) imaging and near-infrared fluorescence (NIRF) imaging.43–46 NIRF holds the benefits of monitoring the distribution of NPs in the whole body with high sensitivity and short acquisition time. Comparatively, PA can provide images with higher contrast and spatial resolution due to the optical absorption. Although promising, the integration of PA and NIRF still lacks deep-tissue penetration and detailed anatomic information. Both US and magnetic resonance (MR) imaging could achieve deep-seated tumor imaging with high resolution nonradiatively. Simultaneously, the US could rapidly diagnose diseases at a low cost, and MR could provide high spatial information without penetration limited. Therefore, the integration of all these imaging modalities into a single nanoplatform is highly expected to obtain complementary information and achieve “mistake-free diagnosis” in a tailor-made “all-in-one” contrast agent.47,48

Herein, we report, for the first time, a LIFU triggered hypoxic-tolerant sonotherapeutic strategy to combat deep-seated hypoxic and normoxic tumors indiscriminately under multimodal imaging guidance. tLyP-1 functionalized phase-transition liposomes was successfully prepared in this strategy, which can actively target MDA-MB-231 tumor cells and subsequently penetrate deeply into the tumors. Moreover, hematoporphyrin monomethyl ether gadolinium (H(Gd)), as the sonosensitizer, was loaded in phospholipid bilayer (designated as [email protected](Gd)) to initiate the SDT process. Notably, PFP, acting as the main functional phase transitional materials to achieve efficient “liquid-to-gas” transformation under an ADV mechanism, can serve as ideal materials to exert a “bomb-like” therapeutic effect on tumor cells under LIFU irradiation without oxygen-dependence. Because of the excellent targeting abilities and deep penetrating capabilities, tLyP-1 functionalized phase transitional nanoparticles performed the functions of a “deep-penetration nanobomb” for combating tumors. When irradiated with LIFU, which is targeted and focused, [email protected](Gd) could transform from liquid to gas and subsequently achieve contrast-enhanced ultrasound imaging and induce cell death via cavitation effect and SDT effect. Therefore, the LIFU activated [email protected](Gd) could realize hypoxic-tolerant sonodynamic therapy, which provided a low-cost and efficient approach against hypoxic tumors. Unlike other versatile nanoagents that integrate theranostic iron via either encapsulated in the shell or modified on the surface of NPs, Gd3+ was previously incorporated into the matallohematoporphyrin monomethyl ether. H(Gd) was used as sonosensitizers because of its high potent in PA/MRI dual-modal imaging and NIRF imaging was achieved after doping hydrophobic fluoroprobe DiR without additional modifications. Therefore, [email protected](Gd), which acts as a “Deep-Penetration Nanobomb” (DPNB), will enable a  US/NIRF/PA/MRI  tetramodal imaging-guided hypoxic-tolerant sonodynamic therapy against hypoxic tumors (Figure 1).

Figure 1 The generation of ROS and ADV mechanisms for synergistic hypoxia-tolerant sonodynamic therapy against solid hypoxic tumors under multimodal imaging guidance. The [email protected](Gd) could actively target the tumor site and penetrate deeply into the tumors, which exerts a “bomb-like” ADV effect and SDT mechanisms to destroy tumor cells under the irradiation of low intensity focused ultrasound (LIFU).

Materials and Methods Materials

Dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylglycerol (DPPG), distearoyl phosphatidyl ethanolamine-polyethylene glycol–maleimide (DSPE-PEG3400-mal), and ethanolamine-polyethylene glycol (DSPE-PEG3400) were purchased from Avanti Polar Lipids Inc. tLyP-1 was obtained from Chinapeptide. Perfluoropentane (PFP) was purchased from Elf Atochem. Dulbecco’s modified eagle medium (DMEM) cell culture medium and fetal bovine serum (FBS) were purchased from Sigma-Aldrich Co. (USA). 2-(4-Amidinophenyl)-6-indolecarbamidinedihydrochloride (DAPI) was bought from Beyotime Technology. Agarose was purchased from Invitrogen (Thermo Fisher Scientific). Calcein AM and propidium iodide (PI) and cell counting kit-8 (CCK-8) assay were obtained from Dojindo (Japan). All the chemical reagents in this study were analytical grade and used without any purification.

Preparation of [email protected](Gd), [email protected](Gd) and [email protected]

[email protected](Gd), [email protected](Gd), and [email protected] were prepared by a facile one-step filming-rehydration method. To prepare [email protected](Gd), DSPE-PEG3400-tLyP-1 was firstly synthesized by Chongqing Protein Way Biotechnology Co. LTD. via a maleimide-thiol coupling reaction. Briefly, 10 mg DPPC, 4 mg DSPE-PEG3400-tLyP-1, 3 mg DPPG, 3 mg cholesterol were dissolved in 10 mL trichloromethane and 2 mg H(Gd) was dissolved in 10 mL methyl alcohol, and the mixture was transferred into a round-bottomed flask. After rotary vacuum evaporation at 50°C for 1 h, a uniform layer film was fabricated. After that, 4 mL PBS was used to rehydrate the film, and 400 μL PFP was added under pulsed sonication (100 W, 5 s on, and 5 s off) for 6 min in an ice bath. Finally, the [email protected](Gd) was fabricated after repeated centrifugation (8000 rpm, 10 min) and stored at 4 °C for further use. To prepare [email protected](Gd), DSPE-PEG3400 was used to replace DSPE-PEG3400-tLyP-1 with the same additive dose. [email protected] was ready with the same method except for the addition of H(Gd).

Loading Efficiency Calculation of H(Gd)

UV-Vis spectra measured the H(Gd) content and organic aqueous phases in the supernatant after repeated centrifugation. The standard curve of H(Gd) was established by detecting the absorbance curves at various concentrations. Loading efficiency of H(Gd) was calculated as follows: Loading efficiency (%) = weight of encapsulated H(Gd)/total weight of H(Gd) × 100 %.

In vitro US/PA/MRI/NIRF Tetramodal Imaging

To evaluate the US imaging capability of [email protected](Gd) in vitro, a 3 % agar (w/v) gel mold was carried out as a test model. First, [email protected](Gd) with the concentration of 0.25 mg mL−1 dispersed in 1 mL of solutions were placed and set in the gel. Both B-mode and CEUS-mode were performed to evaluate the imaging properties (Vevo LAZR, Canada). After that, the NPs were exposed to the various intensity of LIFU (0, 0.8, 1.6, and 3.2 W cm−2) for different times (1, 2, 3, and 4 min). The images of the samples were collected. The signal intensity of each sample was determined by the software provided by the imaging system.

To evaluate the PA imaging capability of [email protected](Gd) in vitro, a 3% agar (w/v) gel mold was carried out as a test model. Various concentrations (0.15625, 0.3125, 0.625, 1.25, 2.5, and 5 mg mL−1) of [email protected](Gd) dispersed in 200 μL of solutions were placed in the gel for PA imaging using the same system at the excitation wavelength of 700 nm. The samples’ images were collected, and the imaging system’s software determined the corresponding signal intensity.

An MR imaging system (Siemens Medical System, Chongqing People’s Hospital) at 3.0 T was used to examine the T1-weighted MRI ability in vitro. 2 mL of [email protected](Gd) (with the Gd3+ concentration of 0.015– 0.1 mM-1) samples with various concentrations were placed in 4 mL of Eppendorf tubes. The corresponding T1 relaxation time was calculated according to the signal intensity. MR images were obtained using a T1-weighted sequence, and the parameters were set as follows: slice thickness = 3 mm, 320×320 matrices, repetition time (TR)/echo time (TE)=790/15 ms.

A Xenogen IVIS Spectrum Imaging System (PerkinElmer, USA) was employed to evaluate the NIRF imaging ability in vitro. 200 μL of DiR labeled [email protected](Gd)(0.01– 5 mg mL−1) samples with various concentrations were placed in a 96-well plate, and NIRF images were collected at the excitation wavelength of 790 using an 845 nm filter.

Cell Culture and Tumor Xenograft Models

MDA-MB-231 cells and HUVEC cells were obtained from Chongqing Key Laboratory of Ultrasound Molecular Imaging, which were approved by committee of Chongqing Medical University. Cells were cultured in a DMEM medium supplemented with 10 % fetal bovine serum and 1 % penicillin-streptomycin. Healthy female balb/c nude mice (6 weeks old) were purchased from Beijing Huafukang Biotechnology. This study’s protocols were conducted following the protocol approved by the department of animal care and use committee of Chongqing Medical University according to the guidelines (GB_T 35892–2018). The tumor xenograft models were established by subcutaneous injection of MDA-MB-231 cells (2×106 dispersed in 100 μL of PBS solution) into each mice’s right hind limb.

In vitro Active-Targeting Effect of [email protected](Gd)

MDA-MB-231 cells and HUVEC cells were seeded in CLSM-specific cell culture dishes (1×105 cells per well) and cultured for 24 h to adhere to the dish’s wall to investigate the active targeting effect of [email protected](Gd). Then [email protected](Gd) or [email protected](Gd) dispersed in DMEM (1 mg mL−1) was added into each well and incubated various times (0.5, 1, 2, and 4 h). After that, the cells were washed with fresh DMEM three times and incubated with DAPI for another 15 min. Finally, the cells were fixed with 4% paraformaldehyde and observed under CLSM (Nikon A1, Japan). To build the three-dimensional spheroids, MDA-MB-231 cells (105 mL-1) were seeded into agarose-coated plates. 10 days later, the spheroids were created for further use. After that, 250 μg mL-1 of DiI-labeled nanoparticles were introduced to the plates containing spheroids. After 24 hours of incubation, spheroids were rinsed three times with PBS and fixed for 30 minutes with 4 % paraformaldehyde. CLSM was used to observe the fluorescence intensity at different depths of spheroids.

To further evaluate the uptake behavior of MDA-MB-231 cells and HUVEC cells, the cells were seeded (1×105 cells per well). After 24 h incubation, [email protected](Gd) or [email protected](Gd) were added and incubated for different times (0.5, 1, 2, and 4 h) respectively. Then the medium was discarded, and the cells were washed with fresh DMEM. The intracellular uptake behavior was analyzed with flow cytometry.

In vitro ROS Production Under the Irradiation of LIFU

3 mL of [email protected] at a concentration of 1 mg mL−1 containing methylene blue (10 μg mL−1) was added into an eppendorf tube and irradiated with LIFU (1.6 W cm−2) for different time intervals (5, 10, 15, 20, and 25 min) followed by observation with a UV-Vis spectrometer ranging from 500–750 nm. Moreover, 3 mL of different NPs at various concentrations containing SOSG (50 × 10−6 M) was added into a cuvette and irradiated with LIFU (1.6 W cm−2) for a different time interval followed by observation with a multimode reader. MDA-MB-231 cells were incubated with different NPs (2 mg mL−1) for 4 h. Then the cells were incubated with fresh DMEM medium (containing 10 μM of DCFH-DA) at 37°C for 20 min. Then the cells were exposed to LIFU (1.6 W cm−2, 2 min) and observed by CLSM. The cells were put in a GENbox Jar to a hypoxic group until the color of the anaerobic indicator changed from pink to colorless.

In vitro Synergistic Therapeutic Effect Induced by LIFU

MDA-MB-231 cells were seeded in 96-well plates (1×104 per well) and cultured overnight to evaluate the therapeutic effect of NPs in both normoxic and hypoxic conditions. Then the medium was replaced by fresh DMEM with various concentrations with [email protected](Gd), [email protected], and tLyP-1-LIP, respectively. The NPs concentrations were ranged from 0.375–3.0 mg mL−1. After 4 h incubation, the cells were treated with LIFU (1.6 W cm−2, 3 min) and cultured for another 8 h. Cells without irradiation with LIFU were set as controls. For the hypoxic group, the cells were treated with identical conditions except for the GENbox Jar treatment. The environment would become hypoxia since the indicator’s color changed from pink to colorless. After that, the samples were washed with fresh DMEM three times and incubated with 10 μL CCK-8 solution for 2 h. The cell viability was determined according to the results obtained from the microplate reader at 450 nm.

MDA-MB-231 cells (1×104 per well) were seeded in CLSM-specific plates and cultured for 12 h to adhere to the plate. [email protected](Gd), tLyP-1-LIP-H(Gd), and [email protected](Gd) were added respectively and coincubated for 4 h and exposed to LIFU (1.6 W cm−2, 3 min). After another 6 h, the cells were stained with Calcein-AM and PI for 15 min and imaged by CLSM.

In vivo NIRF/US/PA/MRI Tetramodal Imaging

The MDA-MB-231 tumor-bearing mice were intravenously injected with DiR [email protected](Gd) or [email protected](Gd). The images were collected at different times (0, 2, 6, 12, and 24 h) postinjection. Moreover, major organs (including heart, liver, spleen, lungs, kidneys, and brain) were collected at 24 h postinjection. The images were observed using a Xenogen IVIS Spectrum Imaging System. The biodistribution of NPs was quantified by the software provided by the Xenogen IVIS Spectrum Imaging System.

For US and PA imaging, the tumor-bearing mice were intravenously injected with [email protected](Gd) or [email protected](Gd). The images were collected at different times (0, 2, 6, 12, and 24 h) postinjection. The images were observed using a PA imaging system (Vevo LAZR, Canada). The tumor-site accumulation of NPs was quantified by the software provided by the PA Imaging System. At 2 h postinjection, LIFU was performed to the tumor-site and imaged using the B-mode and CEUS-mode in the PA Imaging System.

For T1-weighted imaging, the tumor-bearing mice were intravenously injected with [email protected](Gd). The mice injected with identical concentrations of [email protected] were set as a control. The images were observed using a 3.0 T MRI imaging system (Siemens Medical System, Chongqing People’s Hospital) at different times (4, 6, 12, and 24 h) postinjection. The tumor-site accumulation of NPs was quantified by the software provided by the exact MR Imaging System.

In vivo Synergistic Tumor Therapy and Biocompatibility Evaluation

When the tumors grew to about 80 mm3 in volume, the tumor-bearing mice were randomly divided into 6 groups (n=6 in each group). The groups were set as follows: 1) Control group, 2) LIFU only group, 3) tLyP-1-LIP+LIFU group, 4) tLyP-1-LIP-H(Gd) + LIFU group, 5) [email protected](Gd) group, 6) [email protected](Gd) + LIFU group. The power intensity of LIFU was set as 1.6 W cm−2, 3 min. The tumor size and body weight were recorded every other day. The representative mice were sacrificed at 1 d post-treatment. The H&E, PCNA and TUNEL staining of tumors were performed to evaluate the therapeutic effect. The biocompatibility was evaluated by H&E staining of the major organs (heart, liver, spleen, lungs, kidneys, and brain). To further assess the in vivo tumor-site accumulation of [email protected](Gd) and [email protected](Gd), the tumor-bearing mice were intravenously injected with DiI [email protected](Gd) or [email protected](Gd). The mice were sacrificed at different times (1, 2, 6, 12, and 24 h) postinjection. The tumor-site accumulation of NPs was imaged using CLSM.

Results and Discussion Preparation and Characterization of [email protected](Gd) NPs (DPNB)

The synthesis scheme of [email protected](Gd) NPs as “DPNB” was shown in (Figure 2A). Our study prepared [email protected](Gd) NPs in a simple one-step emulsion method with a typical core-shell structure with lipid(LIP)/H(Gd) shell and PFP as the core; tLyP-1 was modified on the surface via a maleimide-thiol coupling reaction as the targeting component for active targeting and deep penetration. After sonication in an ice bath and subsequent centrifugation to remove the excess gradients, transmission electron microscopy (TEM) images were obtained. A uniformly distributed core-shell structure of [email protected](Gd) NPs was observed (Figure 2B). The absorbance spectra of H(Gd) with different concentrations were obtained. According to the UV-Vis absorbance values at the wavelength of 411 nm (Figure 2C and D), H(Gd) ‘s loading efficiency was determined to be 93.3 ± 3.1 % by comparing unloaded H(Gd) to the pre-established standard curve (Figure S1). Moreover, the UV-Vis spectra demonstrated that both [email protected](Gd) and [email protected](Gd) retain the absorption peak of H(Gd) at around 400 nm and several specific wavelengths between 450 and 650 nm (Figure 2E). In comparison, [email protected] showed a smooth UV-Vis spectrum between 300 and 700 nm without a prominent absorption peak, further indicating the success loading of H(Gd) (Figure 2E). The average diameter of the [email protected](Gd) was 260.9 ± 4.3 nm (PDI=0.016), and the zeta potential was −15.7 ± 2.2 mV according to the dynamical light scattering (DLS) results (Figure 2F and G and Figure S2), which is following the TEM results. Moreover, the zeta potential changed from −29.2 mV to −15.7 mV after decoration with positive potential tLyP-1. These results suggest the successful construction of [email protected](Gd) and meet the criterion for subsequent applications.

Figure 2 Characterization of [email protected](Gd). (A) Schematic illustration of [email protected](Gd) preparation process. (B) TEM images of [email protected](Gd). The scale bar is 500 nm, and the insertion scale bar is 200 nm. (C) Calibration curve of H(Gd) based on UV-Vis spectra. (D) UV-Vis spectra of H(Gd) at different concentrations of H(Gd). (E) UV-Vis spectra of H(Gd), [email protected], [email protected](Gd) and [email protected](Gd). (F) Particle sizes of [email protected], [email protected](Gd) and [email protected](Gd). (G) Zeta potential of [email protected], [email protected](Gd), and [email protected](Gd).

In vitro Cell Uptake Behavior of [email protected](Gd)

It has been demonstrated that the cell-penetrating peptide tLyP-1 was responsible for the specific adhesion nature to specific cells that overexpressed NPR-1. The tLyP-1 functionalized nanoplatform could target specific tumors through the endocytic/exocytic transport pathway (CendR pathway). As a result, [email protected](Gd) 's dynamic targeting effect against MDA-MB-231 cells was evaluated using human umbilical vein endothelial cells (HUEVC) as the control. As shown in (Figure 3A), MDA-MB-231 cells showed a much higher fluorescence than that in HUEVC cells in 4 h observation after co-incubation with [email protected](Gd), indicating the specific targeting effect of [email protected](Gd) to MDA-MB-231 cells. Additionally, compared to [email protected](Gd) group, [email protected](Gd) group displayed more vigorous fluorescence intensity due to the active targeting effect in the presence of tLyP-1 (Figure 3A). Similarly, it was found that MDA-MB-231 cells exhibited stronger fluorescence in the [email protected](Gd) group than that in [email protected](Gd) group; the fluorescence in MDA-MB-231 cells is higher than that in HUEVC cells when treated by [email protected](Gd) with identical treatment as determined by flow cytometry (Figure S3). All these results indicated the active targeting effect of [email protected](Gd) against MDA-MB-231 cells.

Figure 3 Internalization of [email protected](Gd) in different cell lines. (A) Uptake of [email protected](Gd) and [email protected](Gd) by MDA-MB-231 cells or HUVEC cells at different incubation times observed by CLSM. (B) Three-dimensional reconstruction of the MDA-MB-231 spheroids model incubated with [email protected](Gd) or [email protected](Gd). The scale bar is 50μm.

In vitro, three-dimensional tumor spheroid is a classical model to mimic the microenvironment of solid tumors. In this study, we established three-dimensional spheroids of MDA-MB-231 cells. After co-incubation with [email protected](Gd) or [email protected](Gd) for 24 h, we found that the red fluorescence of [email protected](Gd) distributed more evidently than that of [email protected](Gd) in the spheroids. The [email protected](Gd) could reach more than 90 μm away from the bottom of the tumor spheroids, whereas [email protected](Gd) only in the surface layer of the tumor spheroids. The above results indicated that tLyP-1 peptide could facilitate the transport of NPs into the deep-seated region of tumors (Figure 3B).

In vitro ROS Generation of [email protected](Gd)

[email protected](Gd) includes a core of the PFP molecule, which could undergo a fast liquid-to-gas transition under LIFU irradiation. Notably, the acoustic environment’s change has been continuously developed as advanced oxidation processes for wastewater remediation by oxidizing hazardous organic compounds, which is featured by more efficient cavitation effect and ROS production in anoxic condition with the assistance of N2 than that in the oxic state.[14] Therefore, we anticipate the gaseous PFP could combat hypoxic tumors by assisting ROS production in anoxic condition. Subsequently, the ROS generation of [email protected] and tLyP-1-LIP in the hypoxia conditions was evaluated by methylene blue, whose fluorescence decreased sharply in the presence of ROS. The ROS generation of [email protected](Gd) was performed in distilled water, which is an environment lack of different kinds of gas and can act as hypoxia conditions. As shown in (Figures 4A and B), [email protected] showed potent ROS generation, in sharp contrast to single tLyP-1-LIP, indicating that PFP can induce ROS generation by cavitation effect in a hypoxia environment. Therefore, we anticipate that PFP based cavitation effect could serve as a newborn strategy for efficiently inducing hypoxic tumor death without the need to deliver oxygen.

Figure 4 Evaluation of ROS generation. (A) [email protected] and (B) tLyP-1-LIP. (C) Fluorescence spectrum of SOSG under different [email protected](Gd) concentrations at a fixed LIFU intensity (1.0 MHz, 1.6 W cm−2, 120 s). (D) Quantitative results of the fluorescence of DCF after different treatment in normoxia and hypoxia and (E) corresponding CLSM images. (F) ROS production and hypoxia status of MDA-MB-231 cancer cells incubated with [email protected](Gd) with/without LIFU. The scale bar is 50 μm. (group 1: Control, group 2: tLyP-1-LIP(hypoxia), group 3: [email protected](hypoxia), group 4: [email protected](Gd)(normoxia), group 5: [email protected](Gd)(hypoxia)).(***p < 0.001 compared by one-way ANOVA analysis, n=5).

The SDT efficiency of [email protected](Gd) was then confirmed with singlet oxygen sensor green (SOSG) as the ROS indicator in vitro, whose fluorescence increased sharply in the presence of ROS. First, the ROS production was determined by testing fluorescence intensity at various concentrations and prolonged LIFU irradiation duration. We found that the SOSG fluorescence in [email protected](Gd) NPs exhibits a concentration-dependent manner at selected time points after LIFU irradiation (Figure 4C). Interestingly, [email protected](Gd) also showed an irradiation duration-dependent ROS generation manner at a fixed concentration of [email protected](Gd) (Figure S4), which could benefit the precise control of SDT efficiency for personal requirements. The highly efficient ROS production of [email protected](Gd) in solution inspired us to investigate the cellular level’s SDT efficiency further under hypoxic conditions. 2’-7’-dichlorodihydrofluorescein diacetate (DCFH-DA) was used as the indicator, whose fluorescence would be turned on and produce fluorescence DCF by ROS, to evaluate SDT efficacy. As shown in (Figure 4D and E), compared to [email protected], [email protected](Gd) showed a more remarkable ROS production ability with higher DCF fluorescence, which can ascribe to the SDT effect after loading H(Gd). Moreover, [email protected] showed an enhanced ROS production compared to tLyP-1-LIP, confirming ROS generation of [email protected] originated from the cavitation effect during the phase-transition process in the presence of PFP. To estimate the ROS production in a hypoxia environment, we evaluated the SDT efficiency under the same conditions. After LIFU irradiation, a comparable green fluorescence was observed in hypoxia compared to that in normoxia, confirming the ROS generation by [email protected](Gd) is hypoxia-tolerant (Figure 4D and E). In the groups without LIFU irradiation, no noticeable fluorescence could be observed under the same condition. These results revealed that NPs alone without LIFU irradiation could not induce ROS generation, indicating the controlled ROS release ability of [email protected](Gd). These results showed [email protected](Gd) potential as sonosensitizers and cavitation nuclei to achieve hypoxia-tolerant sonodynamic therapy against cancer cells. A hypoxia/oxidative stress detection kit confirmed the hypoxic environment in vitro and intracellular ROS generation (Figure 4F).

LIFU Activated SDT and “Bomb-Like” Cavitation Effect of [email protected](Gd)

Having proved the hypoxia-tolerant ROS generation based on the cavitation effect in the presence of PFP, the therapeutic efficacy of [email protected](Gd) was evaluated by a standard CCK-8 assay. MDA-MB-231 cells were chosen as the representative cell line to assess the cytotoxicity of [email protected](Gd) under normoxia and hypoxia environment. First, the biocompatibility of [email protected](Gd), [email protected](Gd), and [email protected] NPs were tested, and negligible cytotoxicity was observed (Figure S5), indicating the high biocompatibility of the NPs. Subsequently, the antitumor efficacy of [email protected](Gd), tLyP-1-LIP-H(Gd), and tLyP-1-LIP were assessed after treatment with LIFU under normoxic and hypoxic condition. As expected, [email protected] showed enhanced cytotoxicity compared to tLyP-1-LIP, indicating the cavitation effect of [email protected] post “liquid-to-gas” phase-transitional process could induce cell death more efficiently under our testing conditions (Figure 5A and B). Moreover, the [email protected] + LIFU group’s cell viability was significantly lower than that in the [email protected](Gd) + LIFU group at the same concentration, indicating the SDT effect in the presence of H(Gd). Furthermore, the SDT effect of [email protected](Gd) was higher than that of [email protected](Gd) due to the active targeting efficacy originated from tLyP-1 as mentioned above (Figure S6). Notably, for the [email protected] groups, both hypoxia and normoxia environments showed comparable therapeutic efficacy in MDA-MB-231 cells, indicating the hypoxia-tolerant therapeutic process induced by the cavitation effect (Figure 5A and B). To examine the therapeutic effect of LIFU more intuitively, CLSM was carried out, and minimal cell death was observed for all groups without LIFU irradiation (Figure 5C). Moreover, the tLyP-1-LIP-H(Gd)+LIFU group showed minimal therapeutic effect under hypoxia. In contrast, [email protected](Gd) showed the most cell death in both hypoxia and normoxia conditions after LIFU irradiation, further proving the hypoxia-tolerant therapeutic efficacy of SDT in inducing cell death against hypoxic tumors, which was following the CCK-8 results.

Figure 5 In vitro cell experiments. LIFU induced therapeutic efficacy of [email protected](Gd), [email protected], and tLyP-1-LIP in MDA-MB-231 cells under (A) normoxia and (B) hypoxia. (C) CLSM images of MDA-MB-231 cells were costained with Calcein AM (green fluorescence) and PI (red fluorescence) after different treatments in normoxia and hypoxia. The scale bar is 50 μm. (**p < 0.01, ***p < 0.001 compared by one-way ANOVA analysis, n=5).

In vitro US/NIRF/PA/MRI Tetramodal Imaging Capability

PFP is a “liquid-to-gas” phase transitional material and acoustic droplet vaporization effect could induce a fast phase-transition manner, converting liquid phase PFP into gas phase under ultrasound triggering. Herein, the cavitation effect induced by the acoustic droplet vaporization effect was intuitively observed by a microscope (Figure 6A). [email protected](Gd) includes a core of the PFP molecule, which could undergo a fast liquid-to-gas transition under LIFU irradiation. After LIFU irradiation, [email protected](Gd) realized a gradual increase in size compared to the control NPs for a prolonged duration of 1–4 min at the power intensity 1.6 W cm−2. In vitro therapeutic efficacy of [email protected](Gd) has demonstrated the inert cavitation effect enhanced sonodynamic therapy at the cellular level in both normoxia and hypoxia. The produced microbubbles contribute to an enhancement in US imaging, which could be examined using both B-mode and contrast-enhanced ultrasound (CEUS)-mode ultrasound imaging. As expected, we observed an intensified signal increase in both B-mode and CEUS-mode with the increasing irradiation duration in 4 min observations (Figure 6B–D), indicating the presence of phase-transition effect and the formation of gaseous PFP. In contrast, the microbubbles could be further exploded at a higher intensity of LIFU, and a decreased imaging signal was observed due to the sputtering of microbubbles. As a control, tLyP-1-LIP-H(Gd) without the encapsulation of PFP showed no noticeable change in ultrasound signal under ultrasound treatment, suggesting PFP could induce a phase-transition behavior. These results supported the powerful ADV effect and the liquid-gas transition of [email protected](Gd) for US imaging under LIFU irradiation.

Figure 6 Multimodal imaging capability in vitro. (A) Optical microscopy images of LIFU-responsive phase-transition process of [email protected](Gd) at prolonged irradiated durations. (B) B-mode and CEUS-mode ultrasound images of [email protected](Gd) under LIFU irradiation with different intensities at different times. Corresponding quantitative echo intensity of (C) B-mode and (D) CEUS-mode. (E) In vitro FL images, (F) PA images, and (G) MR images at different concentrations.

To further evaluate the NIRF imaging properties of [email protected](Gd), the various concentration of DiR-labeled [email protected](Gd) were placed in a 96-well plate. The fluorescence of [email protected](Gd) increased in the range of 0.01 mg mL−1–0.3125 mg mL−1; however, the fluorescence remains unchanged in the field of 0.3125 mg mL−1– 5 mg mL−1 due to the self-quenching effect of DiR, indicating the effectiveness of [email protected](Gd) as a NIRF contrast for imaging applications (Figure 6E). Moreover, considering the PA signal of H(Gd), we evaluated the PA signal of [email protected](Gd), and it increased linearly with the increasing concentration of [email protected](Gd) (Figure 6F and Figure S7). As classical MRI imaging agents, Gd3+ has been widely used as a contrast agent for T1-weighted MRI of tumors in the clinic. Thus, the MRI imaging of [email protected](Gd) was measured quantitatively. As shown in Figure 6G, a linearly brightening effect was observed, and the relaxation time was calculated to be 21.29 mM−1 s−1. Therefore, the above results demonstrated [email protected](Gd) potential as contrast agents for US/NIRF/PA/MRI tetramodal imaging.

In vivo Active-Targeting Capability and US/NIRF/PA/MRI Tetramodal Imaging

[email protected](Gd)’s targeting effect in vitro inspired us to further examine tumor-specific accumulation in tumor-bearing mice. Fluorescence imaging was first performed to evaluate the bio-distribution in the whole body using DiR-labeled [email protected](Gd) as a contrast agent. After intravenous injection of [email protected](Gd) or [email protected](Gd), prominent fluorescence was observed in the [email protected](Gd) group at 2 h postinjection and remain steady at the tumor-site in 24 h observation (Figure 7A). However, no obvious fluorescence signal was found in the [email protected](Gd) group, which could be ascribed to the enhanced tumor-specific targeting of [email protected](Gd) endowed by tLyP-1. Moreover, the fluorescence signal of tumor ex vivo was also improved in [email protected](Gd) group. Simultaneously, a decreased liver accumulation was observed (Figure 7B and C), which further supported the feasibility of decorating tLyP-1 for tumor-specific accumulation.

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