Enhancing Curcumin's therapeutic potential in cancer treatment through ultrasound mediated liposomal delivery.

Improving the efficacy of chemotherapy remains a key challenge in cancer treatment, considering the low bioavailability, high cytotoxicity, and undesirable side effects of some clinical drugs. Targeted delivery and sustained release of therapeutic drugs to cancer cells can reduce the whole-body cytotoxicity of the agent and deliver a safe localized treatment to the patient. There is growing interest in herbal drugs, such as curcumin, which is highly noted as a promising anti-tumor drug, considering its wide range of bioactivities and therapeutic properties against various tumors. Conversely, the clinical efficacy of curcumin is limited because of poor oral bioavailability, low water solubility, instability in gastrointestinal fluids, and unsuitable pH stability. Drug-delivery colloid vehicles like liposomes and nanoparticles combined with microbubbles and ultrasound-mediated sustained release are currently being explored as effective delivery modes in such cases. This study aimed to synthesize and study the properties of curcumin liposomes (CLs) and optimize the high-frequency ultrasound release and uptake by a human breast cancer cell line (HCC 1954) through in vitro studies of culture viability and cytotoxicity. CLs were effectively prepared with particles sized at 81 ± 2 nm, demonstrating stability and controlled release of curcumin under ultrasound exposure. In vitro studies using HCC1954 cells, the combination of CLs, ultrasound, and Definity microbubbles significantly improved curcumin's anti-tumor effects, particularly under specific conditions: 15 s of continuous ultrasound at 0.12 W/cm2 power density with 0.6 × 107 microbubbles/mL. Furthermore, the study delved into curcumin liposomes' cytotoxic effects using an Annexin V/PI-based apoptosis assay. The treatment with CLs, particularly in conjunction with ultrasound and microbubbles, amplified cell apoptosis, mainly in the late apoptosis stage, which was attributed to heightened cellular uptake within cancer cells.

Effect of Poly(ethylene glycol) Configuration on Microbubble Pharmacokinetics.

Microbubbles (MBs) hold substantial promise for medical imaging and therapy; nonetheless, knowledge gaps persist between composition, structure, and in vivo performance, especially with respect to pharmacokinetics. Of particular interest is the role of the poly(ethylene glycol) (PEG) layer, which is thought to shield the MB against opsonization and rapid clearance but is also known to cause an antibody response upon multiple injections. The goal of this study was, therefore, to elucidate the role of the PEG layer in circulation persistence of MBs in the naïve animal (prior to an adaptive immune response). Here, we directly observe the number and size of individual MBs obtained from blood samples, unifying size and concentration into the microbubble volume dose (MVD) parameter. This approach enables direct evaluation of the pharmacokinetics of intact MBs, comprising both the lipid shell and gaseous core, rather than separately assessing the lipid or gas components. We examined the in vivo circulation persistence of 3 µm diameter phospholipid-coated MBs with three different mPEG2000 content: 2 mol % (mushroom), 5 mol % (intermediate), and 10 mol % (brush). MB size and concentration in the blood were evaluated by a hemocytometer analysis over 30 min following intravenous injections of 20 and 40 µL/kg MVD in Sprague-Dawley rats. Interestingly, pharmacokinetic analysis demonstrated that increasing PEG concentration on the MB surface resulted in faster clearance. This was evidenced by a 1.6-fold reduction in half-life and area under the curve (AUC) (p < 0.05) in the central compartment. Conversely, the AUC in the peripheral compartment increased with PEG density, suggesting enhanced MB trapping by the mononuclear phagocyte system. This was supported by an in vitro assay, which showed a significant rise in complement C3a activation with a higher PEG content. In conclusion, a minimal PEG concentration on the MB shell (mushroom configuration) was found to prolong circulation and mitigate immunogenicity.

Ultrasound enhanced siRNA delivery using cationic liposome-microbubble complexes for the treatment of squamous cell carcinoma.

Rationale: Microbubble (MB) contrast agents combined with ultrasound targeted microbubble cavitation (UTMC) are a promising platform for site-specific therapeutic oligonucleotide delivery. We investigated UTMC-mediated delivery of siRNA directed against epidermal growth factor receptor (EGFR), to squamous cell carcinoma (SCC) via a novel MB-liposome complex (LPX). Methods: LPXs were constructed by conjugation of cationic liposomes to the surface of C4F10 gas-filled lipid MBs using biotin/avidin chemistry, then loaded with siRNA via electrostatic interaction. Luciferase-expressing SCC-VII cells (SCC-VII-Luc) were cultured in Petri dishes. The Petri dishes were filled with media in which LPXs loaded with siRNA against firefly luciferase (Luc siRNA) were suspended. Ultrasound (US) (1 MHz, 100-µs pulse, 10% duty cycle) was delivered to the dishes for 10 sec at varying acoustic pressures and luciferase assay was performed 24 hr later. In vivo siRNA delivery was studied in SCC-VII tumor-bearing mice intravenously infused with a 0.5 mL saline suspension of EGFR siRNA LPX (7×108 LPX, ~30 µg siRNA) for 20 min during concurrent US (1 MHz, 0.5 MPa spatial peak temporal peak negative pressure, five 100-µs pulses every 1 ms; each pulse train repeated every 2 sec to allow reperfusion of LPX into the tumor). Mice were sacrificed 2 days post treatment and tumor EGFR expression was measured (Western blot). Other mice (n=23) received either EGFR siRNA-loaded LPX + UTMC or negative control (NC) siRNA-loaded LPX + UTMC on days 0 and 3, or no treatment ("sham"). Tumor volume was serially measured by high-resolution 3D US imaging. Results: Luc siRNA LPX + UTMC caused significant luciferase knockdown vs. no treatment control, p<0.05) in SCC-VII-Luc cells at acoustic pressures 0.25 MPa to 0.9 MPa, while no significant silencing effect was seen at lower pressure (0.125 MPa). In vivo, EGFR siRNA LPX + UTMC reduced tumor EGFR expression by ~30% and significantly inhibited tumor growth by day 9 (~40% decrease in tumor volume vs. NC siRNA LPX + UTMC, p<0.05). Conclusions: Luc siRNA LPXs + UTMC achieved functional delivery of Luc siRNA to SCC-VII-Luc cells in vitro. EGFR siRNA LPX + UTMC inhibited tumor growth and suppressed EGFR expression in vivo, suggesting that this platform holds promise for non-invasive, image-guided targeted delivery of therapeutic siRNA for cancer treatment.

Double-layer hollow mesoporous silica nanoparticles for ultrasound-guided photodynamic treatment.

Cervical carcinoma persists as a major global public health burden. While conventional therapeutic modalities inevitably cause ablation of adjacent non-tumorous tissues, photodynamic therapy (PDT) offers a targeted cytotoxic strategy through a photosensitizing agent (PS). However, the hydrophobicity and lack of selective accumulation of promising PS compounds such as zinc(II) phthalocyanine (ZnPc) impedes their clinical translation as standalone agents. The present study sought to incorporate ZnPc within double-layer hollow mesoporous silica nanoparticles (DHMSN) as nanocarriers to enhance aqueous dispersibility and tumor specificity. Owing to their compartmentalized design, the hollow mesoporous silica nanoparticles (HMSN) demonstrated enhanced ultrasonic imaging contrast. Combined with the vaporization of the perfluorocarbon perfluoropentane (PFP), the HMSN-encapsulated ZnPc enabled real-time ultrasound monitoring of PDT treatment.In vivo, the innate thermal energy induced vaporization of the DHMSN-carried PFP to significantly amplify ultrasound signals from the tumor site. Results demonstrated biocompatibility, efficient PFP microbubble generation, and robust photocatalytic activity. Collectively, this investigation establishes ultrasound-guided PDT utilizing multi-layer HMSN as a targeted therapeutic strategy for cervical malignancies with mitigated toxicity.

Ultrasound­targeted microbubble destruction technology delivering ß­klotho to the heart enhances FGF21 sensitivity and attenuates heart remodeling post­myocardial infarction.

Fibroblast growth factor (FGF)21 is a peptide hormone that improves mitochondrial function and energy metabolism, and the deficiency of its co­receptor ß­klotho (KLB) causes decreased FGF21 sensitivity. The present study examined whether the cardiac delivery of plasmids containing the KLB gene via ultrasound­targeted microbubble destruction (UTMD) enhances the efficacy of FGF21 against heart failure post­acute myocardial infarction (AMI). For this purpose, the levels of FGF21 in patients and rats with heart dysfunction post­infarction were determined using ELISA. Sprague­Dawley rats received the 3X UTMD­mediated delivery of KLB@cationic microbubbles (KLB@CMBs) 1 week following the induction of AMI. Echocardiography, histopathology and biochemical analysis were performed at 4 weeks following the induction of AMI. The results revealed that patients with heart failure post­infarction had higher serum FGF21 levels than the healthy controls. However, the downstream signal, KLB, but not α­klotho, was reduced in the heart tissues of rats with AMI. As was expected, treatment with FGF21 did not substantially attenuate heart remodeling post­infarction. It was found that decreased receptors KLB in the heart may result in the insensitivity to FGF21 treatment. In vivo, the UTMD technology­mediated delivery of KLB@CMBs to the heart significantly enhanced the effects of FGF21 administration on cardiac remodeling and mitochondrial dysfunction in the rats following infarction. The delivery of KLB to the heart by UTMD and the administration of FGF21 attenuated mitochondrial impairment and oxidative stress by activating nuclear factor erythroid 2­related factor 2 signals. On the whole, the present study demonstrates that the cardiac delivery of KLB significantly optimizes the cardioprotective effects of FGF21 therapy on adverse heart remodeling. UTMD appears a promising interdisciplinary approach with which to improve heart failure post­myocardial infarction.

Contrast-enhanced Ultrasound Using Intradermal Microbubble Sulfur Hexafluoride in Non-invasive Axillary Staging in Breast Cancer: Are we Missing a Chance?

BACKGROUND/AIM: In the context of surgical de-escalation in early breast cancer (EBC), this study aimed to evaluate the contrast enhancement ultrasound (CEUS) sentinel lymph node (SLN) procedure as a non-invasive axillary staging procedure in EBC in comparison with standard SLN biopsy (SLNB). PATIENTS AND METHODS: A subanalysis of the AX-CES study, a prospective single-arm, monocentric phase 3 study was performed (EudraCT: 2020-000393-20). The study included patients with EBC undergoing upfront surgery and SLN resection, with no prior history of locoregional treatment, and weighing between 40-85 kg. All patients underwent the CEUS SLN procedure as a non-invasive axillary staging procedure, with CEUS SLN accumulation marked using blue dye. After the CEUS SLN procedure, all patients underwent the standard mapping procedure. Data on success rate, systemic reactions, mean procedure time, mean surgical procedure, mean procedure without axillary staging, CEUS SLN appearance (normal/pathological), SLN number, and concordance with standard mapping procedure were collected. RESULTS: After the CEUS SLN procedure, 29 LNs among 16 patients were identified and marked. In all cases, CEUS SLN revealed at least one LN enhancement. Six (37.50%) LNs were defined as pathological after the CEUS SLN procedure. Definitive staining of CEUS SLN pathology revealed metastatic involvement in four (66.67%) of the cases. Two SLNs were identified during the CEUS SLN procedure; however, owing to the low disease burden, no change in the surgical plan was reported. CONCLUSION: The CEUS SLN procedure shows promise as a technique for non-invasive assessment of the axilla, potentially enabling safe axillary de-escalation in EBC by estimating the axillary disease burden.

Hydralazine loaded nanodroplets combined with ultrasound-targeted microbubble destruction to induce pyroptosis for tumor treatment.

Pyroptosis, a novel type of programmed cell death (PCD), which provides a feasible therapeutic option for the treatment of tumors. However, due to the hypermethylation of the promoter, the critical protein Gasdermin E (GSDME) is lacking in the majority of cancer cells, which cannot start the pyroptosis process and leads to dissatisfactory therapeutic effects. Additionally, the quick clearance, systemic side effects, and low concentration at the tumor site of conventional pyroptosis reagents restrict their use in clinical cancer therapy. Here, we described a combination therapy that induces tumor cell pyroptosis via the use of ultrasound-targeted microbubble destruction (UTMD) in combination with DNA demethylation. The combined application of UTMD and hydralazine-loaded nanodroplets (HYD-NDs) can lead to the rapid release of HYD (a demethylation drug), which can cause the up-regulation of GSDME expression, and produce reactive oxygen species (ROS) by UTMD to cleave up-regulated GSDME, thereby inducing pyroptosis. HYD-NDs combined with ultrasound (US) group had the strongest tumor inhibition effect, and the tumor inhibition rate was 87.15% (HYD-NDs group: 51.41 ± 3.61%, NDs + US group: 32.73%±7.72%), indicating that the strategy had a more significant synergistic anti-tumor effect. In addition, as a new drug delivery carrier, HYD-NDs have great biosafety, tumor targeting, and ultrasound imaging performance. According to the results, the combined therapy reasonably regulated the process of tumor cell pyroptosis, which offered a new strategy for optimizing the therapy of GSDME-silenced solid tumors.

Quality assurance for focused ultrasound-induced blood-brain barrier opening procedure using passive acoustic detection.

BACKGROUND: Focused ultrasound (FUS) combined with microbubbles is a promising technique for noninvasive, reversible, and spatially targeted blood-brain barrier opening, with clinical trials currently ongoing. Despite the fast development of this technology, there is a lack of established quality assurance (QA) strategies to ensure procedure consistency and safety. To address this challenge, this study presents the development and clinical evaluation of a passive acoustic detection-based QA protocol for FUS-induced blood-brain barrier opening (FUS-BBBO) procedure. METHODS: Ten glioma patients were recruited to a clinical trial for evaluating a neuronavigation-guided FUS device. An acoustic sensor was incorporated at the center of the FUS device to passively capture acoustic signals for accomplishing three QA functions: FUS device QA to ensure the device functions consistently, acoustic coupling QA to detect air bubbles trapped in the acoustic coupling gel and water bladder of the transducer, and FUS procedure QA to evaluate the consistency of the treatment procedure. FINDINGS: The FUS device passed the device QA in 9/10 patient studies. 4/9 cases failed acoustic coupling QA on the first try. The acoustic coupling procedure was repeatedly performed until it passed QA in 3/4 cases. One case failed acoustic coupling QA due to time constraints. Realtime passive cavitation monitoring was performed for FUS procedure QA, which captured variations in FUS-induced microbubble cavitation dynamics among patients. INTERPRETATION: This study demonstrated that the proposed passive acoustic detection could be integrated with a clinical FUS system for the QA of the FUS-BBBO procedure. FUNDING: National Institutes of Health R01CA276174, R01MH116981, UG3MH126861, R01EB027223, R01EB030102, and R01NS128461.

Ultrasound imaging guided targeted sonodynamic therapy enhanced by magnetophoretically controlled magnetic microbubbles.

Sonodynamic therapy (SDT) utilizes ultrasonic excitation of a sensitizer to generate reactive oxygen species (ROS) to destroy tumor. Two dimensional (2D) black phosphorus (BP) is an emerging sonosensitizer that can promote ROS production to be used in SDT but it alone lacks active targeting effect and showed low therapy efficiency. In this study, a stable dispersion of integrated micro-nanoplatform consisting of BP nanosheets loaded and Fe3O4 nanoparticles (NPs) connected microbubbles was introduced for ultrasound imaging guided and magnetic field directed precision SDT of breast cancer. The targeted ultrasound imaging at 18 MHz and efficient SDT effects at 1 MHz were demonstrated both in-vitro and in-vivo on the breast cancer. The magnetic microbubbles targeted deliver BP nanosheets to the tumor site under magnetic navigation and increased the uptake of BP nanosheets by inducing cavitation effect for increased cell membrane permeability via ultrasound targeted microbubble destruction (UTMD). The mechanism of SDT by magnetic black phosphorus microbubbles was proposed to be originated from the ROS triggered mitochondria mediated apoptosis by up-regulating the pro-apoptotic proteins while down-regulating the anti-apoptotic proteins. In conclusion, the ultrasound theranostic was realized via the magnetic black phosphorus microbubbles, which could realize targeting and catalytic sonodynamic therapy.

Focused ultrasound-mediated blood-brain barrier opening is safe and feasible with moderately hypofractionated radiotherapy for brainstem diffuse midline glioma.

BACKGROUND: Diffuse midline glioma (DMG) is a pediatric tumor with dismal prognosis. Systemic strategies have been unsuccessful and radiotherapy (RT) remains the standard-of-care. A central impediment to treatment is the blood-brain barrier (BBB), which precludes drug delivery to the central nervous system (CNS). Focused ultrasound (FUS) with microbubbles can transiently and non-invasively disrupt the BBB to enhance drug delivery. This study aimed to determine the feasibility of brainstem FUS in combination with clinical doses of RT. We hypothesized that FUS-mediated BBB-opening (BBBO) is safe and feasible with 39 Gy RT. METHODS: To establish a safety timeline, we administered FUS to the brainstem of non-tumor bearing mice concurrent with or adjuvant to RT; our findings were validated in a syngeneic brainstem murine model of DMG receiving repeated sonication concurrent with RT. The brainstems of male B6 (Cg)-Tyrc-2J/J albino mice were intracranially injected with mouse DMG cells (PDGFB+, H3.3K27M, p53-/-). A clinical RT dose of 39 Gy in 13 fractions (39 Gy/13fx) was delivered using the Small Animal Radiation Research Platform (SARRP) or XRAD-320 irradiator. FUS was administered via a 0.5 MHz transducer, with BBBO and tumor volume monitored by magnetic resonance imaging (MRI). RESULTS: FUS-mediated BBBO did not affect cardiorespiratory rate, motor function, or tissue integrity in non-tumor bearing mice receiving RT. Tumor-bearing mice tolerated repeated brainstem BBBO concurrent with RT. 39 Gy/13fx offered local control, though disease progression occurred 3-4 weeks post-RT. CONCLUSION: Repeated FUS-mediated BBBO is safe and feasible concurrent with RT. In our syngeneic DMG murine model, progression occurs, serving as an ideal model for future combination testing with RT and FUS-mediated drug delivery.

How Microbubble-Enhanced Shock Waves Promote the Delivery of Lipid-siRNA across Neuronal Plasma Membrane: A Computational Study.

In this computational study, we examine the potential of microbubble-enhanced shock waves to improve the delivery of lipid-siRNA nanoparticles across neuronal plasma membranes with the ultimate aim of enhancing brain tumor treatment. We critically evaluate several variables related to experiments, including the bubble size, the shock speed and action time, and the amount of siRNA encapsulated in the liposome. Our findings reveal that microbubble-enhanced shock waves are essential for the high delivery of small lipid vesicles (under 30 nm diameter); its corresponding variables significantly impact drug penetration and absorption rates and influence the overall efficacy of the drug delivery system. Long-time recovery simulations further provide valuable insights into the self-healing ability of the plasma membrane following shock wave exposure and the subsequent absorption dynamics of siRNA. This work provides the dynamic process of siRNA released from lipid vesicles with shock wave and nanobubbles, thereby serving as a molecular mechanism support for developing tunable delivery systems for RNA-based therapy in brain tumors.

Ultrasound-targeted microbubble destruction rapidly improves left ventricular function in rats with ischemic cardiac dysfunction.

BACKGROUND: Previous studies have demonstrated the efficacy of ultrasound-targeted microbubble destruction (UTMD) in the treatment of ischemic heart failure (HF). The purpose of this study was to explore the mechanism by which UTMD improves ischemic HF. METHODS: An ischemic heart failure model was established using Sprague-Dawley rats. Rats were randomly divided into 7 groups: sham group, HF group, HF + MB group, HF + ultrasound (US) group, HF + UTMD group, HF + UTMD+LY294002 group, and HF + LY294002 group. Serum BNP level and echocardiographic parameters were measured to evaluate cardiac function. PI3K/Akt/eNOS signaling pathway protein levels were detected by immunohistochemistry (IHC) and western blotting. The concentrations of nitrous oxide (NO) and ATP were detected by ELISA, and hematoxylin and eosin (HE) staining was used to evaluate myocardial tissue. RESULTS: UTMD rapidly improved ejection fraction (EF) (HF: 37.16 ± 1.21% vs. HF + UTMD: 46.31 ± 3.00%, P < 0.01) and fractional shortening (FS) (HF: 18.53 ± 0.58% vs. HF + UTMD: 24.05 ± 1.84%, P < 0.01) in rats with ischemic HF. UTMD activated the PI3K/AKT/eNOS signaling pathway (HF vs. HF + UTMD, P < 0.01) and promoted the release of NO and ATP (HF vs. HF + UTMD, both, P < 0.05). Inhibition of the PI3K/AKT/eNOS signaling pathway by LY294002 worsened EF (HF: 37.16 ± 1.21% vs. HF + LY294002: 32.73 ± 3.05%, P < 0.05), and the release of NO and ATP by UTMD (HF + UTMD vs. HF + UTMD+LY294002, P < 0.05). CONCLUSIONS: UTMD can rapidly improve cardiac function in ischemic HF by activating the PI3K/Akt/eNOS signaling pathway and promoting the release of NO and ATP.

Interaction Between Microbubbles and Microwave Ablation: A Phantom and Rabbit Model.

OBJECTIVES: This study aimed to explore the interactions between microbubbles and microwave ablation (MWA). METHODS: The study employed custom-made phantoms (in vitro) and white New Zealand rabbits (in vivo). MWA was performed with or without microbubbles in the phantoms (2 × 105 particles mL-1) and rabbit livers (intravenous injection of 0.05 mL/kg SonoVue). During the MWA, K-type thermocouple probes were used to monitor the MWA-induced temperature increase. Contrast-enhanced ultrasound imaging (CEUS) was used to monitor and analyze the microbubbles signal intensity. After MWA, the ablation-zone volumes were evaluated and compared between the groups with and without microbubbles. RESULTS: In both the phantom models and rabbits, microbubbles showed no significant influence on MWA, including the ablation range and MWA-induced temperature increase. In phantoms and rabbit livers filled with microbubbles, MWA caused the formation of a gradually expanding microbubble-defect region over the ablation time. An increase in the temperature caused microbubble destruction. CONCLUSIONS: Microbubbles had no significant influence on MWA. However, MWA induced the destruction of microbubbles in a temperature-dependent manner. Thus, the poor thermotolerance of microbubbles is a non-negligible barrier when using CEUS to monitor the ablation range during MWA in real-time.

mRNA Sonotransfection of Tumors with Polymeric Microbubbles: Co-Formulation versus Co-Administration.

Despite its high potential, non-viral gene therapy of cancer remains challenging due to inefficient nucleic acid delivery. Ultrasound (US) with microbubbles (MB) can open biological barriers and thus improve DNA and mRNA passage. Polymeric MB are an interesting alternative to clinically used lipid-coated MB because of their high stability, narrow size distribution, and easy functionalization. However, besides choosing the ideal MB, it remains unclear whether nanocarrier-encapsulated mRNA should be administered separately (co-administration) or conjugated to MB (co-formulation). Therefore, the impact of poly(n-butyl cyanoacrylate) MB co-administration with mRNA-DOTAP/DOPE lipoplexes or their co-formulation on the transfection of cancer cells in vitro and in vivo is analyzed. Sonotransfection improved mRNA delivery into 4T1 breast cancer cells in vitro with co-administration being more efficient than co-formulation. In vivo, the co-administration sonotransfection approach also resulted in higher transfection efficiency and reached deeper into the tumor tissue. On the contrary, co-formulation mainly promoted transfection of endothelial and perivascular cells. Furthermore, the co-formulation approach is much more dependent on the US trigger, resulting in significantly lower off-site transfection. Thus, the findings indicate that the choice of co-administration or co-formulation in sonotransfection should depend on the targeted cell population, tolerable off-site transfection, and the therapeutic purpose.

Luminal Delivery of Pectin-Modified Oxygen Microbubbles Mitigates Rodent Experimental Intestinal Ischemia.

INTRODUCTION: Ischemic gut injury is common in the intensive care unit, impairs gut barrier function, and contributes to multiorgan dysfunction. One novel intervention to mitigate ischemic gut injury is the direct luminal delivery of oxygen microbubbles (OMB). Formulations of OMB can be modified to control the rate of oxygen delivery. This project examined whether luminal delivery of pectin-modified OMB (OMBp5) can reduce ischemic gut injury in a rodent model. METHODS: The OMBp5 formulation was adapted to improve delivery of oxygen along the length of small intestine. Adult Sprague-Dawley rats (n = 24) were randomly allocated to three groups: sham-surgery (SS), intestinal ischemia (II), and intestinal ischemia plus luminal delivery of OMBp5 (II + O). Ischemia-reperfusion injury was induced by superior mesenteric artery occlusion for 45 min followed by reperfusion for 30 min. Outcome data included macroscopic score of mucosal injury, the histological score of gut injury, and plasma biomarkers of intestinal injury. RESULTS: Macroscopic, microscopic data, and intestinal injury biomarker results demonstrated minimal intestinal damage in the SS group and constant damage in the II group. II + O group had a significantly improved macroscopic score throughout the gut mucosa (P = 0.04) than the II. The mean histological score of gut injury for the II + O group was significantly improved on the II group (P ≤ 0.01) in the proximal intestine only, within 30 cm of delivery. No differences were observed in plasma biomarkers of intestinal injury following OMBp5 treatment. CONCLUSIONS: This proof-of-concept study has demonstrated that luminal OMBp5 decreases ischemic injury to the proximal small intestine. There is a need to improve oxygen delivery over the full length of the intestine. These findings support further studies with clinically relevant end points, such as systemic inflammation and vital organ dysfunction.

MnO2/Ce6 microbubble-mediated hypoxia modulation for enhancing sono-photodynamic therapy against triple negative breast cancer.

Sono-photodynamic therapy (SPDT) has emerged as a promising treatment modality for triple negative breast cancer (TNBC). However, the hypoxic tumor microenvironment hinders the application of SPDT. Herein, in this study, a multifunctional platform (MnO2/Ce6@MBs) was designed to address this issue. A sono-photosensitizer (Ce6) and a hypoxia modulator (MnO2) were loaded into microbubbles and precisely released within tumor tissues under ultrasound irradiation. MnO2in situ reacted with the excess H2O2 and H+ and produced O2 within the TNBC tumor, which alleviated hypoxia and augmented SPDT by increasing ROS generation. Meanwhile, the reaction product Mn2+ was able to achieve T1-weighted MRI for enhanced tumor imaging. Additionally, Ce6 and microbubbles served as a fluorescence imaging contrast agent and a contrast-enhanced ultrasound imaging agent, respectively. In in vivo anti-tumor studies, under the FL/US/MR imaging guidance, MnO2/Ce6@MBs combined with SPDT significantly reversed tumor hypoxia and inhibited tumor growth in 4T1-tumor bearing mice. This work presents a theragnostic system for reversing tumor hypoxia and enhancing TNBC treatment.

Ultrasound-mediated HGF Gene Microbubbles Mitigate Hyperkinetic Pulmonary Arterial Hypertension in Rabbits.

AIM: Hyperkinetic pulmonary arterial hypertension (PAH) is a complication of congenital heart disease. Gene therapy is a new experimental treatment for PAH, and ultrasound-mediated gene-carrying microbubble targeted delivery is a promising development for gene transfer. METHODS: This study successfully established a hyperkinetic PAH rabbit model by a common carotid artery and jugular vein shunt using the cuff style method. Liposome microbubbles carrying the hepatocyte growth factor (HGF) gene were successfully constructed. An in vitro experiment evaluated the appropriate intensity of ultrasonic radiation by Western blots and 3H-TdR incorporation assays. In an in vivo experiment, after transfection of ultrasound-mediated HGF gene microbubbles, catheterisation was applied to collect haemodynamic data. Hypertrophy of the right ventricle was evaluated by measuring the right ventricle hypertrophy index. Western blot and immunohistochemistry analyses were used to detect the expression of human (h)HGF and angiogenic effects, respectively. RESULTS: The most appropriate ultrasonic radiation intensity was 1.0 W/cm2 for 5 minutes. Two weeks after transfection, both systolic pulmonary arterial pressure and mean pulmonary arterial pressure were attenuated. Hypertrophy of the right ventricle was reversed. hHGF was transplanted into the rabbits, resulting in a high expression of hHGF protein and an increase in the number of small pulmonary arteries. Ultrasound-mediated HGF gene microbubble therapy was more effective at attenuating PAH and increasing the density of small pulmonary arteries than single HGF plasmid transfection. CONCLUSIONS: Ultrasound-mediated HGF gene microbubbles significantly improved the target of gene therapy in a rabbit PAH model and enhanced the tropism and transfection rates. Thus, the technique can effectively promote small pulmonary angiogenesis and play a role in the treatment of PAH without adverse reactions.

A study combining microbubble-mediated focused ultrasound and radiation therapy in the healthy rat brain and a F98 glioma model.

Focused Ultrasound (FUS) has been shown to sensitize tumors outside the brain to Radiotherapy (RT) through increased ceramide-mediated apoptosis. This study investigated the effects of FUS + RT in healthy rodent brains and F98 gliomas. Tumors, or striata in healthy rats, were targeted with microbubble-mediated, pulsed FUS (220 kHz, 102-444 kPa), followed by RT (4, 8, 15 Gy). FUS + RT (8, 15 Gy) resulted in ablative lesions, not observed with FUS or RT only, in healthy tissue. Lesions were visible using Magnetic Resonance Imaging (MRI) within 72 h and persisted until 21 days post-treatment, indicating potential applications in ablative neurosurgery. In F98 tumors, at 8 and 15 Gy, where RT only had significant effects, FUS + RT offered limited improvements. At 4 Gy, where RT had limited effects compared with untreated controls, FUS + RT reduced tumor volumes observed on MRI by 45-57%. However, survival benefits were minimal (controls: 27 days, RT: 27 days, FUS + RT: 28 days). Histological analyses of tumors 72 h after FUS + RT (4 Gy) showed 93% and 396% increases in apoptosis, and 320% and 336% increases in vessel-associated ceramide, compared to FUS and RT only. Preliminary evidence shows that FUS + RT may improve treatment of glioma, but additional studies are required to optimize effect size.

Enhancement of Tumor Perfusion and Antiangiogenic Therapy in Murine Models of Clear Cell Renal Cell Carcinoma Using Ultrasound-Stimulated Microbubbles.

OBJECTIVE: To explore the effect of ultrasound-stimulated microbubble cavitation (USMC) on enhancing antiangiogenic therapy in clear cell renal cell carcinoma. MATERIALS AND METHODS: We explored the effects of USMC with different mechanical indices (MIs) on tumor perfusion, 36 786-O tumor-bearing nude mice were randomly assigned into four groups: (i) control group, (ii) USMC0.25 group (MI = 0.25), (iii) USMC1.4 group (MI = 1.4) (iv) US1.4 group (MI = 1.4). Tumor perfusion was assessed by contrast-enhanced ultrasound (CEUS) before the USMC treatment and 30 min, 4h and 6h after the USMC treatment, respectively. Then we evaluated vascular normalization(VN) induced by low-MI (0.25) USMC treatment, 12 tumor-bearing nude mice were randomly divided into two groups: (i) control group (ii) USMC0.25 group. USMC treatment was performed, and tumor microvascular imaging and blood perfusion were analyzed by MicroFlow imaging (MFI) and CEUS 30 min after each treatment. In combination therapy, a total of 144 tumor-bearing nude mice were randomly assigned to six groups (n = 24): (i) control group, (ii) USMC1.4 group, (iii) USMC0.25 group, (iv) bevacizumab(BEV) group, (v) USMC1.4 +BEV group, (vi) USMC0.25 +BEV group. BEV was injected on the 6th, 10th, 14th, and 18th d after the tumors were inoculated, while USMC treatment was performed 24 h before and after every BEV administration. We examined the effects of the combination therapy through a series of experiments. RESULTS: Tumor blood perfusion enhanced by USMC with low MI (0.25)could last for more than 6h, inducing tumor VN and promoting drug delivery. Compared with other groups, USMC0.25+BEV combination therapy had the strongest inhibition on tumor growth, led to the longest survival time of the mice. CONCLUSION: The optimized USMC is a promising therapeutic approach that can be combined with antiangiogenic therapy to combat tumor progression.

Pattern recognition of microcirculation with super-resolution ultrasound imaging provides markers for early tumor response to anti-angiogenic therapy.

Rationale: Cancer treatment outcome is traditionally evaluated by tumor volume change in clinics, while tumor microvascular heterogeneity reflecting tumor response has not been fully explored due to technical limitations. Methods: We introduce a new paradigm in super-resolution ultrasound imaging, termed pattern recognition of microcirculation (PARM), which identifies both hemodynamic and morphological patterns of tumor microcirculation hidden in spatio-temporal space trajectories of microbubbles. Results: PARM demonstrates the ability to distinguish different local blood flow velocities separated by a distance of 24 µm. Compared with traditional vascular parameters, PARM-derived heterogeneity parameters prove to be more sensitive to microvascular changes following anti-angiogenic therapy. Particularly, PARM-identified "sentinel" microvasculature, exhibiting evident structural changes as early as 24 hours after treatment initiation, correlates significantly with subsequent tumor volume changes (|r| > 0.9, P < 0.05). This provides prognostic insight into tumor response much earlier than clinical criteria. Conclusions: The ability of PARM to noninvasively quantify tumor vascular heterogeneity at the microvascular level may shed new light on early-stage assessment of cancer therapy.