Noninvasive assessment of pressure distribution and fractional flow in middle cerebral artery using microbubbles and plane wave in vitro.

Fractional flow has been proposed for quantifying the degree of functional stenosis in cerebral arteries. Herein, subharmonic aided pressure estimation (SHAPE) combined with plane wave (PW) transmission was employed to noninvasively estimate the pressure distribution and fractional flow in the middle cerebral artery (MCA) in vitro. Consequently, the effects of incident sound pressure (peak negative pressures of 86-653 kPa), pulse repetition frequency (PRF), number of pulses, and blood flow rate on the subharmonic pressure relationship were investigated. The radio frequency data were stored and beamformed offline, and the subharmonic amplitude over a 0.4 MHz bandwidth was extracted using a 12-cycle PW at 4 MHz. The optimal incident sound pressure was 217 kPa without skull (sensitivity = 0.09 dB/mmHg; r2 = 0.997) and 410 kPa with skull (median sensitivity = 0.06 dB/mmHg; median r2 = 0.981). The optimal PRF was 500 Hz, as this value affords the highest sensitivity (0.09 dB/mmHg; r2 = 0.976) and temporal resolution. In addition, the blood flow rate exhibited a lesser effect on the subharmonic pressure relationship in our experimental setup. Using the optimized parameters, the blood pressure distribution and fractional flow (FFs) were measured. As such, the FFs value was in high agreement with the value measured using the pressure sensor (FFm). The mean ± standard deviations of the FF difference (FFm - FFs) were 0.03 ± 0.06 without skull and 0.01 ± 0.05 with skull.

Clutter filtering of angular domain data for contrast-free ultrafast microvascular imaging.

Objective. Contrast-free microvascular imaging is clinically valuable for the assessment of physiological status and the early diagnosis of diseases. Effective clutter filtering is essential for microvascular visualization without contrast enhancement. Singular value decomposition (SVD)-based spatiotemporal filter has been widely used to suppress clutter. However, clinical real-time imaging relies on short ensembles (dozens of frames), which limits the implementation of SVD filtering due to the large error of eigen-correlated estimations and high dependence on optimal threshold when used in such ensembles.Approach. To address the above challenges of imaging in short ensembles, two optimized filters of angular domain data are proposed in this paper: grouped angle SVD (GA-SVD) and angular-coherence-based higher-order SVD (AC-HOSVD). GA-SVD applies SVD to the concatenation of all angular data to improve clutter rejection performance in short ensembles, while AC-HOSVD applies HOSVD to the angular data tensor and utilizes angular coherence in addition to spatial and temporal features for filtering. Feasible threshold selection strategies in each feature space are provided. The clutter rejection performance of the proposed filters and SVD was evaluated with Doppler phantom andin vivostudies at different cases. Moreover, the robustness of the filters was explored under wrong singular value threshold estimation, and their computational complexity was studied.Main results. Qualitative and quantitative results indicated that GA-SVD and AC-HOSVD can effectively improve clutter rejection performance in short ensembles, especially AC-HOSVD. Notably, the proposed methods using 20 frames had similar image quality to SVD using 100 frames.In vivostudies showed that compared to SVD, GA-SVD increased the signal-to-noise-ratio (SNR) by 6.03 dB on average, and AC-HOSVD increased the SNR by 8.93 dB on average. Furthermore, AC-HOSVD remained better power Doppler image quality under non-optimal thresholds, followed by GA-SVD.Significance. The proposed filters can greatly enhance contrast-free microvascular visualization in short ensembles and have potential for different clinical translations due to the performance differences.

Effect of tissue viscoelasticity and adjacent phase-changed microbubbles on vaporization process and direct growth threshold of nanodroplet in an ultrasonic field.

Understanding the behavior of nanodroplets converted into microbubbles with applied ultrasound is an important problem in tumor therapeutical and diagnostic applications. In this study, a comprehensive model is proposed to investigate the vaporization process and the direct growth threshold of the nanodroplet by following the vapor bubble growth, especially attention devoted to the effect of tissue viscoelasticity and adjacent phase-changed microbubbles (PCMBs). It is shown that the ultrasonic energy must be sufficiently strong to counterbalance the natural condensation of the vapor bubble and the tissue stiffness-inhibitory effect. The softer tissue with a lower shear modulus favors the vaporization process, and the nanodroplet has a lower direct growth threshold in the softer tissue. Moreover, the adjacent PCMBs show a suppression effect on the vaporization process due to the negative value of the secondary Bjerknes force, implying an attractive force, preventing the nanodroplet from escaping from the constraint of the adjacent PCMBs. However, according to the linear scattering theory, the attractive force signifies that the constraint is weak, causing the direct growth threshold to increase in the range of 0.09-0.24 MPa. The weak increase in threshold demonstrates that the direct growth threshold is relatively unaffected by the adjacent PCMBs. The prediction results of our model are in good agreement with the experiment results obtained by the echo enhancement method, in which the threshold is relatively independent of the intermediate concentration. The findings presented here provide physical insight that will be further helpful in understanding the complex behavior of the nanodroplet responses to ultrasound in practical medical applications.

Focused acoustic vortex-mediated sonochemotherapy for the amplification of immunogenic cell death combined with checkpoint blockade to potentiate cancer immunotherapy.

Sonodynamic therapy (SDT) as an auxiliary modality of cancer immunotherapy enhances systemic anti-tumor immunity. However, the efficiency of SDT-mediated immunotherapy based on conventional focused ultrasound (FUS) is restricted by the tiny focal region of FUS. Focused acoustic vortex (FAV) possessing a larger focal region, can induce stronger cavitation and thermal effects than FUS with the same parameters, having the potential to overcome this issue. This research investigated the feasibility of FAV-mediated sonochemotherapy combined with the immune checkpoint blockade (ICB) to reshape immunosuppressive tumor microenvironment (TME), inhibit tumor growth and lung metastasis. Sonosensitizer chlorin e6 (Ce6) and chemotherapeutic agent doxorubicin (Dox) were co-loaded into microbubble-liposome complex to compose Ce6/Dox@Lip@MBs (CDLM) for "all-in-one" synergistic sonochemotherapy, whose main components were clinical approved. FAV-activated CDLM significantly enriched immunogenic cell death (ICD) inducers in tumors and amplified ICD of cancer cells compared with FUS-activated CDLM. Furthermore, the amplified-ICD combined with ICB increased the infiltration of cytotoxic T lymphocytes and natural killer cells, polarized M2 macrophages to M1 macrophages, and decreased regulatory T cells. This study provides a multifunctional strategy for enriching ICD inducers in tumors and amplifying ICD to ameliorate immunosuppressive TME and potentiate systemic anti-tumor immunity.

Velocity field estimation in transcranial small vessel using super-resolution ultrasound imaging velocimetry.

Based on the diameter and position information of small vessels obtained by transcranial super-resolution imaging using 3 MHz low-frequency chirp plane waves, a Gaussian-like non-linear compression was adopted to compress the blood flow signals in spatiotemporal filtering (STF) data to a precise region, and then estimate the blood flow velocity field inside the region over the adjacent time intervals using ultrasound imaging velocimetry (UIV). Imaging parameters, such as the mechanical index (MI), frame rate, and microbubble (MB) concentration, are critical during the estimation of velocity fields over a short time at high MB contrast agent concentrations. These were optimized through experiments and algorithms, in which dividing the connected domain was proposed to calculate MB cluster spot centroid spacing (SCS) and the spot-to-flow area ratio (SFAR) to determine the suitable MB concentration. The results of the in vitro experiments showed that the estimation of the small vessel flow velocity field was consistent with the theoretical results; the velocity field resolution for vessels with diameters of 0.5 mm and 0.3 mm was 36 µm and 21 µm, and the error between the mean velocity and the theoretical value was 0.7 % and 0.67 %, respectively.

High-Performance Delivery of a CRISPR Interference System via Lipid-Polymer Hybrid Nanoparticles Combined with Ultrasound-Mediated Microbubble Destruction for Tumor-Specific Gene Repression.

The dCas9-based CRISPR interference (CRISPRi) system efficiently silences genes without causing detectable off-target activity, thus showing great potential for the treatment of cancer at the transcriptional level. However, due to the large size of the commonly used CRISPRi system, effective delivery of the system has been a challenge that hinders its application in the clinic. Herein, a combination of pH-responsive lipid-polymer hybrid nanoparticles (PLPNs) and ultrasound-mediated microbubble destruction (UMMD) is used for the delivery of the CRISPRi system. The core-shell structure of PLPNs can effectively be loaded with the CRISPRi plasmid, and increases the time spent in the circulating in vivo, and "actively target" cancer cells. Moreover, the combination of PLPNs with UMMD achieves a higher cellular uptake of the CRISPRi plasmid in vitro and retention in vivo. Furthermore, when PLPNs loaded with a CRISPRi plasmid that targets microRNA-10b (miR-10b) are used in combination with UMMD, it results in the effective repression of miR-10b in breast cancer, simultaneous disturbance of multiple cell migration and invasion-related signaling pathways, and a significant inhibition of lung metastasis. Thus, the established system presents a versatile, highly efficient, and safe strategy for delivery of the CRISPRi system both in vitro and in vivo.

Retraction: Enhanced anti-tumor efficacy of hyaluronic acid modified nanocomposites combined with sonochemotherapy against subcutaneous and metastatic breast tumors.

Retraction of 'Enhanced anti-tumor efficacy of hyaluronic acid modified nanocomposites combined with sonochemotherapy against subcutaneous and metastatic breast tumors' by Pengying Wu et al., Nanoscale, 2019, 11, 11470-11483, https://doi.org/10.1039/C9NR01691K.

Synergistic therapy for orthotopic gliomas via biomimetic nanosonosensitizer-mediated sonodynamic therapy and ferroptosis.

Ferroptosis is an emerging form of programmed cell death, and its combination with sonodynamic therapy (SDT) for anti-tumor activity is gradually attracting attention. However, their application against gliomas has not been studied. Herein, multifunctional cancer homologous targeting biomimetic nanoparticles (PIOC@CM NPs) encapsulating both Fe3O4 and Ce6 were constructed as a nanosonosensitizer. Based on focused ultrasound (US) combined with circulating microbubbles (MBs) to open the blood-brain barrier (BBB) in a safe and transient manner, the development of a therapeutic strategy to integrate the biomimetic nanosonosensitizer-mediated SDT and ferroptosis could achieve synergistic therapeutic effects against gliomas. We demonstrated that the glioma C6 cell membrane (CM) on the surface of the NPs allowed the nanosonosensitizer to accumulate selectively in tumors through homologous targeting in vitro. After efficient internalization in C6 cells, the PIOC@CM NPs could significantly increase the level of reactive oxygen species (ROS) and deplete glutathione (GSH) upon ultrasonic irradiation, resulting in the loss of glutathione peroxidase-4 (GPX4) activity, which facilitated SDT and ferroptosis to kill glioma C6 cells. Furthermore, the PIOC@CM NPs were intravenously injected after noninvasively opening the BBB via US-MBs, which enhanced the accumulation of the nanosonosensitizer in tumor tissues. Crucially, an attractive phenomenon of the significant reduction in orthotopic gliomas after the second US pulse-triggered SDT and ferroptosis was observed. Taken together, this study presents a novel combinatorial glioma therapeutic strategy based on noninvasive BBB opening with a biomimetic sonotheranostic system-mediated SDT and ferroptosis.

Focused Acoustic Vortex-Regulated Composite Nanodroplets Combined with Checkpoint Blockade for High-Performance Tumor Synergistic Therapy.

The combination of checkpoint blockade with focused ultrasound (FUS) physical therapy can enhance antitumor immune response by improving the precision and efficiency of immunotherapy. However, one of the major disadvantages of conventional FUS treatment is the small lesion size, which prolongs treatment duration. We constructed a focused acoustic vortex (FAV) system with a hollow cylindrical focal region, which exhibited a larger focal region compared to conventional FUS of the same frequency. We developed an all-in-one synergistic therapy against metastatic breast cancer based on integrated FAV double combination sequence-regulated phase-transformation nanodroplets (CPDA@PFH) with checkpoint blockade immunotherapy. A single treatment with FAV + CPDA@PFH resulted in 2.25-fold higher inhibition of tumor growth compared to that with FUS + CPDA@PFH. In addition, FAV-regulated CPDA@PFH combined with ICB induced a systemic immune response that not only inhibited the growth of primary (98.41% inhibition rate) and distal (80.71%) 4T1 tumors but also reduced the progression of lung metastasis. In addition, the synergistic therapy achieved long-term immune memory that effectively prevented tumor growth and improved the survival time of mice. The long-term survival rate of 4T1 tumor-bearing mice treated with FAV + CPDA@PFH + Anti-PD-L1 was 57.14% on day 60 after treatment. Our study is a proof-of-concept of cascade-amplified synergistic tumor therapeutics based on ultrasonic-hyperthermia, cavitation, sonodynamic therapy (SDT), and checkpoint blockade immunotherapy through FAV-regulated CPDA@PFH phase-transformation nanodroplets.

Noninvasive Pressure Estimation Based on the Subharmonic Response of SonoVue: Application to Intracranial Blood Pressure Assessment.

Intracranial blood pressure can directly reflect the status of blood vessels in real time. However, it can only be estimated invasively using a microcatheter during craniotomy. Subharmonic-aided pressure estimation (SHAPE) is a promising technique for estimating cardiac pressures but mainly uses Sonazoid, whereas SHAPE using SonoVue is still in the early stages of development. The aim of this study was to optimize transcranial SHAPE using SonoVue by investigating the relationship between subharmonic signals and middle cerebral artery pressure (MCAP) (20-160 mmHg) in vitro. We examined the effect of acoustic output levels (peak negative pressures (PNPs) of 238, 346, and 454 kPa), time in suspension (time from reconstituting the suspension to extracting it: 0-30 min), and exposure to gas-equilibrated saline (3 min, 1 h, or original gas completely replaced by air) on the subharmonic-pressure relationship. A mean subharmonic amplitude over a 0.4 MHz bandwidth was extracted using a 5 MHz 12-cycle pulse. A PNP of 346 kPa elicited the best subharmonic sensitivity for assessing hydrostatic pressures up to 0.24 dB/mmHg, possibly because compression-only behavior no longer occurs at this pressure. Moreover, the expansion force is not large enough to offset the effects of hydrostatic pressure. A linear monotonic relationship between the subharmonic amplitude and hydrostatic pressure was only observed for just prepared SonoVue. Excessive exposure to gas-equilibrated saline also affected the subharmonic-pressure relationship. Therefore, just prepared SonoVue should be used, and the duration of the pressure estimation process should be strictly controlled.

Study on the Application of Super-Resolution Ultrasound for Cerebral Vessel Imaging in Rhesus Monkeys.

Background: Ultrasound is ideal for displaying intracranial great vessels but not intracranial microvessels and terminal vessels. Even with contrast agents, the imaging effect is still unsatisfactory. In recent years, significant theoretical advances have been achieved in super-resolution imaging. The latest commonly used ultrafast plane-wave ultrasound Doppler imaging of the brain and microbubble-based super-resolution ultrasound imaging have been applied to the imaging of cerebral microvessels and blood flow in small animals such as mice but have not been applied to in vivo imaging of the cerebral microvessels in monkeys and larger animals. In China, preliminary research results have been obtained using super-resolution imaging in certain fields but rarely in fundamental and clinical experiments on large animals. In recent years, we have conducted a joint study with the Xi'an Jiaotong University to explore the application and performance of this new technique in the diagnosis of cerebrovascular diseases in large animals. Objective: To explore the characteristics and advantages of microbubble-based super-resolution ultrasound imaging of intracranial vessels in rhesus monkeys compared with conventional transcranial ultrasound. Methods: First, the effectiveness and feasibility of the super-resolution imaging technique were verified by modular simulation experiments. Then, the imaging parameters were adjusted based on in vitro experiments. Finally, two rhesus monkeys were used for in vivo experiments of intracranial microvessel imaging. Results: Compared with conventional plane-wave imaging, super-resolution imaging could measure the inner diameters of cerebral microvessels at a resolution of 1 mm or even 0.7 mm and extract blood flow information. In addition, it has a better signal-to-noise ratio (5.625 dB higher) and higher resolution (~30-fold higher). The results of the experiments with rhesus monkeys showed that microbubble-based super-resolution ultrasound imaging can achieve an optimal resolution at the micron level and an imaging depth >35 mm. Conclusion: Super-resolution imaging can realize the monitoring imaging of high-resolution and fast calculation of microbubbles in the process of tissue damage, providing an important experimental basis for the clinical application of non-invasive transcranial ultrasound.

Enhanced HIFU Theranostics with Dual-Frequency-Ring Focused Ultrasound and Activatable Perfluoropentane-Loaded Polymer Nanoparticles.

High-intensity focused ultrasound (HIFU) has been widely used in tumor ablation in clinical settings. Meanwhile, there is great potential to increase the therapeutic efficiency of temporary cavitation due to enhanced thermal effects and combined mechanical effects from nonlinear vibration and collapse of the microbubbles. In this study, dual-frequency (1.1 and 5 MHz) HIFU was used to produce acoustic droplet vaporization (ADV) microbubbles from activatable perfluoropentane-loaded polymer nanoparticles (PFP@Polymer NPs), which increased the therapeutic outcome of the HIFU and helped realize tumor theranostics with ultrasound contrast imaging. Combined with PFP@Polymer NPs, dual-frequency HIFU changed the shape of the damage lesion and reduced the acoustic intensity threshold of thermal damage significantly, from 216.86 to 62.38 W/cm2. It produced a nearly 20 °C temperature increase in half the irradiation time and exhibited a higher tumor inhibition rate (84.5% ± 3.4%) at a low acoustic intensity (1.1 MHz: 23.77 W/cm2; 5 MHz: 0.35 W/cm2) in vitro than the single-frequency HIFU (60.2% ± 11.9%). Moreover, compared with the traditional PFP@BSA NDs, PFP@Polymer NPs showed higher anti-tumor efficacy (81.13% vs. 69.34%; * p < 0.05) and better contrast-enhanced ultrasound (CEUS) imaging ability (gray value of 57.53 vs. 30.67; **** p < 0.0001), probably benefitting from its uniform and stable structure. It showed potential as a highly efficient tumor theranostics approach based on dual-frequency HIFU and activatable PFP@Polymer NPs.

Plasmid-loadable magnetic/ultrasound-responsive nanodroplets with a SPIO-NP dispersed perfluoropentane core and lipid shell for tumor-targeted intracellular plasmid delivery.

Using ultrasound activating contrast agents to induce sonoporation is a potential strategy for effective lesion-targeted gene delivery. Previous reports have proven that submicron nanodroplets have a better advantage than microbubbles in that they can pass through tumor vasculature endothelial gaps by passive targeting; however, they cannot achieve an adequate dose in tumors to facilitate ultrasound-enhanced gene delivery. Additionally, a few studies focused on delivering macromolecular genetic materials (i.e. overexpression plasmid and CRISPR plasmid) have presented more unique advantages than small-molecular genetic materials (i.e. miRNA mimics, siRNA and shRNA etc.), such as enhancing the expression of target genes with long-term effectiveness. Thereby, we constructed novel plasmid-loadable magnetic/ultrasound-responsive nanodroplets, where superparamagnetic iron oxide nanoparticle dispersed perfluoropentane was encapsulated with lipids to which plasmids could be adhered, and branched polyethylenimine was used to protect the plasmids from enzymolysis. Furthermore, in vitro and in vivo studies were performed to verify the magnetic tumor-targeting ability of the plasmid-loadable magnetic/ultrasound-responsive nanodroplets and focused ultrasound enhanced intracellular plasmid delivery. The plasmid-loadable magnetic/ultrasound-responsive nanodroplets, carrying 16-19 plasmids per droplet, had desirable diameters less than 300 nm, and integrated the merits of excellent magnetic targeting capabilities and phase transition sensitivity to focused ultrasound. Under programmable focused ultrasound exposure, the plasmid-loadable magnetic/ultrasound-responsive nanodroplets underwent a phase-transition into echogenic microbubbles and the subsequent inertial cavitation of the microbubbles achieved an ∼40% in vitro plasmid delivery efficiency. Following intravenous administration, T2-weighted magnet resonance imaging, scanning electron microscopy and inductively coupled plasma optical emission spectrometry of the tumors showed significantly enhanced intratumoral accumulation of the plasmid-loadable magnetic/ultrasound-responsive nanodroplets under an external magnetic field. And a GFP ELISA assay and immunofluorescence staining indicated that focused ultrasound-induced inertial cavitation of the plasmid-loadable magnetic/ultrasound-responsive nanodroplets significantly enhanced the intracellular delivery of plasmids within the tumor after magnet-assisted accumulation, while only lower GFP levels were observed in the tumors on applying focused ultrasound or an external magnet alone. Taken together, utilizing the excellent plasmid-loadable magnetic/ultrasound-responsive nanodroplets combined with magnetism and ultrasound could efficiently deliver plasmids to cancer cells, which could be a potential strategy for macromolecular genetic material delivery in the clinic to treat cancer.

Multipotent miRNA Sponge-Loaded Magnetic Nanodroplets with Ultrasound/Magnet-Assisted Delivery for Hepatocellular Carcinoma Therapy.

Gene therapy is likely to be the most promising way to tackle cancer, while defects in molecular strategies and delivery systems have led to an impasse in clinical application. Here, it is found that onco-miRNAs of the miR-515 and -449 families were upregulated in hepatocellular carcinoma (HCC), and the sponge targeting miR-515 family had a significant probability to suppress cancer cell proliferation. Then, we constructed non-toxic sponge-loaded magnetic nanodroplets containing 20% C6F14 (SLMNDs-20%) that are incorporated with fluorinated superparamagnetic iron oxide nanoparticles enhancing external magnetism-assisted targeting and enabling a direct visualization of SLMNDs-20% distribution in vivo via magnetic resonance imaging monitoring. SLMNDs-20% could be vaporized by programmable focused ultrasound (FUS) activation, achieving ∼45% in vitro sponge delivery efficiency and significantly enhancing in vivo sponge delivery without a clear apoptosis. Moreover, the sponge-1-carrying SLMNDs-20% could effectively suppress proliferation of xenograft HCC after FUS exposure because sponge-1-suppressing onco-miR-515 enhanced the expression of anti-oncogenes (P21, CD22, TIMP1, NFKB, and E-cadherin) in cancer cells. The current results indicated that ultrasonic cavitation-inducing sonoporation enhanced the intracellular delivery of sponge-1 using SLMNDs-20% after magnetic-assisted accumulation, which was a therapeutic approach to inhibit HCC progression.

Cavitation characteristics of flowing low and high boiling-point perfluorocarbon phase-shift nanodroplets during focused ultrasound exposures.

This work investigated and compared the dynamic cavitation characteristics between low and high boiling-point phase-shift nanodroplets (NDs) under physiologically relevant flow conditions during focused ultrasound (FUS) exposures at different peak rarefactional pressures. A passive cavitation detection (PCD) system was used to monitor cavitation activity during FUS exposure at various acoustic pressure levels. Root mean square (RMS) amplitudes of broadband noise, spectrograms of the passive cavitation detection signals, and normalized inertial cavitation dose (ICD) values were calculated. Cavitation activity of low-boiling-point perfluoropentane (PFP) NDs and high boiling-point perfluorohexane (PFH) NDs flowing at in vitro mean velocities of 0-15 cm/s were compared in a 4-mm diameter wall-less vessel in a transparent tissue-mimicking phantom. In the static state, both types of phase-shift NDs exhibit a sharp rise in cavitation intensity during initial FUS exposure. Under flow conditions, cavitation activity of the PFH NDs reached the steady state less rapidly compared to PFP NDs under the lower acoustic pressure (1.35 MPa); at the higher acoustic pressure (1.65 MPa), the RMS amplitude increased more sharply during the initial FUS exposure period. In particular, the RMS-time curves of the PFP NDs shifted upward as the mean flow velocity increased from 0 to 15 cm/s; the RMS amplitude of the PFH ND solution increased from 0 to 10 cm/s and decreased at 15 cm/s. Moreover, amplitudes of the echo signal for the low boiling-point PFP NDs were higher compared to the high boiling-point PFH NDs in the lower frequency range, whereas the inverse occurred in the higher frequency range. Both PFP and PFH NDs showed increased cavitation activity in the higher frequency under the flow condition compared to the static state, especially PFH NDs. At 1.65 MPa, normalized ICD values for PFH increased from 0.93 ± 0.03 to 0.96 ± 0.04 and from 0 to 10 cm/s, then decreased to 0.86 ± 0.05 at 15 cm/s. This work contributes to our further understanding of cavitation characteristics of phase-shift NDs under physiologically relevant flow conditions during FUS exposure. In addition, the results provide a reference for selecting suitable phase-shift NDs to enhance the efficiency of cavitation-mediated ultrasonic applications.

Ultrasound-Mediated Gene Therapy of Hepatocellular Carcinoma Using Pre-microRNA Plasmid-Loaded Nanodroplets.

The PIK3 CA gene encodes the p110α protein subunit and is one of the most efficient cancer genes in solid and hematological tumors including hepatocellular carcinoma (HCC). There are currently ongoing therapies against tumors based on PIK3 CA inhibition. Because microRNAs (miRNAs) play an important role in post-transcriptional regulation and are also involved in the inhibition of PIK3 CA expression to suppress cancer cell proliferation, overexpression of tumor-suppressive miRNA is a promising therapeutic approach for HCC therapy. The successful and localized delivery of miRNA overexpression vectors (pre-miRNA plasmids) is very important in improving the therapeutic efficacy of this miRNA therapy strategy. In the study described here, submicron acoustic phase-shifted nanodroplets were used to efficiently deliver pre-miRNA plasmid in vitro and in vivo for HCC therapy under focused ultrasound (US) activation. Briefly, six miRNAs, inhibiting PIK3 CA and downregulated in HCC, were selected through summary and analysis of the currently existing literature data. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blot and cell apoptosis assay revealed that pre-miR-139, -203a, -378a and -422a plasmids among the six miRNA overexpression vectors could suppress growth of the hepatoma cell line SMMC-7721. These four pre-miRNA plasmids were then electrostatically adhered to positively charged lipid-shelled nanodroplets to obtain plasmid-loaded nanodroplets (PLNDs). The PLND-generated microbubbles oscillated and even collapsed under US exposure to release the loaded pre-miRNA plasmids and enhance their cellular uptake through consequent sonoporation, that is, formation of small pores on the cell membrane induced by the mechanical effects of PLND cavitation. Fluorescence microscopy results revealed that PLNDs could effectively deliver the aforementioned four pre-miRNA plasmids into SMMC-7721 cells in vitro under 1.2-MHz 60-cycle sinusoid US exposure with a peak negative pressure >5.5 MPa at a 40-Hz pulse repetition frequency. Plasmid delivery efficiency and cell viability positively correlated with the inertial cavitation dose that was determined mainly by peak negative pressure. Furthermore, PLNDs combined with US were evaluated in vivo to deliver these four pre-miRNAs plasmids and verify their therapeutic efficacy in subcutaneous tumor of the mouse xenograft HCC model. The results revealed that the PLNDs loaded with pre-miR-139 and -378a plasmids could effectively suppress tumor growth after US treatment. Thus, combination of pre-miRNA PLNDs with US activation seems to constitute a potential strategy for HCC therapy.

ROS-Responsive Blended Nanoparticles: Cascade-Amplifying Synergistic Effects of Sonochemotherapy with On-demand Boosted Drug Release During SDT Process.

Sonodynamic therapy (SDT) not only has greater tissue-penetrating depth compared to photo-stimulated therapies, but also can also trigger rapid drug release to achieve synergistic sonochemotherapy. Here, reactive oxygen species (ROS)-responsive IR780/PTL- nanoparticles (NPs) are designed by self-assembly, which contain ROS-cleavable thioketal linkers (TL) to promote paclitaxel (PTX) release during SDT. Under ultrasound (US) stimulation, IR780/PTL-NPs produce high amounts of ROS, which not only induces apoptosis in human glioma (U87) cells but also boosts PTX released by decomposing the ROS-sensitive TL. In the U87 tumor-bearing mouse model, the IR780/PTL-NPs releases the drug at the target sites in a controlled manner upon US irradiation, which significantly inhibits tumor growth and induces apoptosis in the tumor tissues with no obvious toxicity. Taken together, the IR780/PTL-NPs are a novel platform for sonochemotherapy, and can control the spatio-temporal release of chemotherapeutic drugs during SDT.

Time and Frequency Characteristics of Cavitation Activity Enhanced by Flowing Phase-Shift Nanodroplets and Lipid-Shelled Microbubbles During Focused Ultrasound Exposures.

This study investigated and compared the time and frequency characteristics of cavitation activity between phase-shift nanodroplets (NDs) and lipid-shelled microbubbles (MBs) exposed to focused ultrasound (FUS) under physiologically relevant flow conditions. Root-mean-square (RMS) of broadband noise, spectrograms of the passive cavitation detection signals and inertial cavitation doses (ICDs) were calculated during FUS at varying mean flow velocities and two different peak-rarefactional pressures. At a lower pressure of 0.94 MPa, the mean values of the RMS amplitudes versus time for the NDs showed an upward trend but slowed down as the mean flow velocity increased. For flowing NDs, the rate of growth in RMS amplitudes within 2-5 MHz decreased more obviously than those within 5-8 MHz. At a higher pressure of 1.07 MPa, the increase in RMS amplitudes was accelerated as the mean flow velocity increased from 0 to 10 cm/s and slowed down as the mean flow velocity reached 15 cm/s. The general downward trends of RMS amplitudes for the MBs were retarded as the mean flow velocity increased at both acoustic pressures of 0.94 MPa and 1.07 MPa. At 0.94 MPa, the mean ICD value for the NDs decreased from 57 to 36 as the mean flow velocity increased from 0 to 20 cm/s. At 1.07 MPa, the mean ICD value initially increased from 45 to 57 as the mean flow velocity increased from 0 to 10 cm/s and subsequently decreased to 43 as the mean flow velocity reached 20 cm/s. For the MBs, the mean ICD value increased with increasing mean flow velocity at both acoustic pressures. These results could aid in future investigations of cavitation-enhanced FUS with the flowing phase-shift NDs and encapsulated, gas-filled MBs for various applications.

Enhanced anti-tumor efficacy of hyaluronic acid modified nanocomposites combined with sonochemotherapy against subcutaneous and metastatic breast tumors.

Sonochemotherapy is a promising strategy for inhibiting tumor growth. However, achieving highly targeted and effective sonochemotherapy is still an enormous challenge. In this study, a novel chemotherapeutic-carrying nanocomposite (HPCID) was developed, which can effectively target metastatic cancer cells and provide an enhanced therapeutic effect. In detail, HPCID was composed of hyaluronic acid (HA), carboxyl-terminated PAMAM dendrimer, fluorochrome indocyanine green (ICG), and doxorubicin hydrochloride (Dox). The efficacy of this drug delivery system (DDS) in sonochemotherapy was assessed on the CD44-overexpressing metastatic breast cancer cell line 4T1 both in vitro and in vivo. The HA modification significantly improved the cellular internalization of HPCID, and the degradation of the HA shell by hyaluronidase that is abundant in the 4T1 cells resulted in enzyme-responsive drug release. Under ultrasound (US) stimulation, HPCID produced a high amount of reactive oxidant species (ROS), which induced significant cell apoptosis when combined with chemotherapy. In addition, the administration of HPCID in 4T1 xenograft-bearing mice combined with ultrasonic exposure significantly inhibited tumor growth and pulmonary metastasis, with no systemic toxicity. Taken together, the proposed HPCID-mediated sonodynamic therapy (SDT) is a novel strategy against breast cancer progression and metastasis.