Power-Doppler-based NH002 microbubble sonoporation with chemotherapy relieves hypoxia and enhances the efficacy of chemotherapy and immunotherapy for pancreatic tumors.

Pancreatic ductal adenocarcinoma (PDAC) poses challenges due to late-stage diagnosis and limited treatment response, often attributed to the hypoxic tumor microenvironment (TME). Sonoporation, combining ultrasound and microbubbles, holds promise for enhancing therapy. However, additional preclinical research utilizing commercially available ultrasound equipment for PDAC treatment while delving into the TME's intricacies is necessary. This study investigated the potential of using a clinically available ultrasound system and phase 2-proven microbubbles to relieve tumor hypoxia and enhance the efficacy of chemotherapy and immunotherapy in a murine PDAC model. This approach enables early PDAC detection and blood-flow-sensitive Power-Doppler sonoporation in combination with chemotherapy. It significantly extended treated mice's median survival compared to chemotherapy alone. Mechanistically, this combination therapy enhanced tumor perfusion and substantially reduced tumor hypoxia (77% and 67%, 1- and 3-days post-treatment). Additionally, cluster of differentiation 8 (CD8) T-cell infiltration increased four-fold afterward. The combined treatment demonstrated a strengthening of the anti-programmed death-ligand 1(αPDL1) therapy against PDAC. Our study illustrates the feasibility of using a clinically available ultrasound system with NH-002 microbubbles for early tumor detection, alleviating hypoxic TME, and improving chemotherapy and immunotherapy. It suggests the development of an adjuvant theragnostic protocol incorporating Power-Doppler sonoporation for pancreatic tumor treatment.

Concurrent Osteosarcoma Theranostic Strategy Using Contrast-Enhanced Ultrasound and Drug-Loaded Bubbles.

Osteosarcoma (OS) is the most common bone tumor in children and teenagers. The multidrug resistant property of OS produces a major obstacle to chemotherapy, since the effective drug dose cannot be achieved via conventional drug delivery routes without serious systemic cytotoxicity. Microbubbles in conjunction with ultrasound (US) has recently been shown to spatially and temporally permeabilize the cellular membrane, promoting drug penetration into tumors. Here, we investigated whether drug (doxorubicin, DOX)-loaded bubbles (DOX-bubbles) can serve as drug-loaded carriers in combination with US in order to facilitate tumor drug delivery. The proposed bubbles have a high payload capacity (efficiency of 69.4 ± 9.1%, payload of 1.4 mg/mL) for DOX. In vitro data revealed that when used in combination with US (1-MHz), these DOX-bubbles facilitate DOX entering into tumor cells. In tumor-bearing animals, DOX-bubbles + US could provide 3.7-fold suppression of tumor growth compared with the group without insonation (1.8 ± 0.9 cm3 vs. 8.5 ± 2.2 cm3) because of the acceleration of DOX-induced tumor necrosis. In the meantime, the tumor perfusion and volume can be monitored by DOX-bubbles with contrast-enhanced ultrasound imaging. Our data provide useful information in support of translating the use of theranostic US-responsive bubbles for regulated tumor drug delivery into clinical use.

Macrophages as Drug Delivery Carriers for Acoustic Phase-Change Droplets.

The major challenges in treating malignant tumors are transport of therapeutic agents to hypoxic regions and real-time assessment of successful drug release via medical imaging modalities. In this study, we propose the use of macrophages (RAW 264.7 cells) as carriers of drug-loaded phase-change droplets to penetrate ischemic or hypoxic regions within tumors. The droplets consist of perfluoropentane, lipid and the chemotherapeutic drug doxorubicin (DOX, DOX-droplets). The efficiency of DOX-droplet uptake, migration mobility and viability of DOX-droplet-loaded macrophages (DLMs) were measured using a transmembrane cell migration assay, the alamarBlue assay and flow cytometric analysis, respectively. Our results indicate the feasibility of utilizing macrophages as DOX-droplet carriers (DOX payload of DOX-droplets: 459.3 ± 35.8 µg/mL, efficiency of cell uptake DOX-droplets: 88.8 ± 3.5%). The migration mobility (total number of migrated microphages) of DLMs decreased to 32.3% compared with that of healthy macrophages, but the DLMs provided contrast-enhanced ultrasound imaging (1.7-fold enhancement) and anti-tumor effect (70.9% cell viability) after acoustic droplet vaporization, suggesting the potential theranostic applications of DLMs. Future work will assess the tumor penetration ability of DLMs, mechanical effect of droplet vaporization on in vivo anti-tumor therapy and the release of the carried drug by ultrasound-triggered vaporization.

Real-time monitoring of inertial cavitation effects of microbubbles by using MRI: In vitro experiments.

PURPOSE: To investigate the feasibility of half-Fourier acquisition single-shot turbo spin-echo (HASTE) for real-time monitoring of signal changes because of water flow induced by inertial cavitation (IC) during microbubbles (MBs)-present focused ultrasound (FUS) exposure. THEORY AND METHODS: Strong turbulence produced in MB solution at the onset of IC results in the difficulty to refocus signal echoes and thus the decrease in signal intensity (SI). Fundamental investigations were conducted using an agar phantom containing MB dilutions exposed to 1.85-MHz FUS. The effects of various experimental conditions including MB concentrations, imaging slice thicknesses, chamber diameters, acoustic pressures, duty cycles, and pulse repetition frequencies (PRFs) were discussed. RESULTS: Continuous 2.8 MPa FUS exposure resulted in SI changed from 11% to 55% when MBs concentrations increased from 0.025% to 0.1%. When slice thickness increased from 3 mm to 6 or 8 mm, smaller SI changes were observed (84%, 59%, and 46%). Images acquired with chamber diameter of 6 and 3 mm showed SI changes of 84% and 35%, respectively. In burst modes, higher duty cycles exhibited higher SI changes, and lower PRFs exhibited smaller and longer SI decrease. CONCLUSION: Under various conditions, substantial signal changes were observable, suggesting the feasibility of applying HASTE to real-time monitor IC effect under FUS exposure. Magn Reson Med 77:102-111, 2017. © 2015 Wiley Periodicals, Inc.

Biomimetic Acoustically-Responsive Vesicles for Theranostic Applications.

In recent years, biomimetic cell membrane-derived particles have emerged as a new class of drug delivery system with advantages of biocompatibility, ease of isolation and long circulation profile. Here we report the development and potential theranostic applications of a new biomimetic acoustically-responsive droplet system derived from mammalian red blood cell membrane (RBCM). We hypothesized that drug-loaded RBCM droplets (RBCMDs) would undergo a transition from liquid (droplets) to gas (bubbles) upon high intensity focused ultrasound (HIFU) insonation, resulting in on-demand drug release. The generated microbubbles could also serve as a contrast agent to enhance ultrasound imaging. As-synthesized RBCMDs exhibited uniform size, good dispersity and preservation of RBCM-associated proteins that prevented uptake by macrophages. Camptothecin (CPT), an anti-cancer drug, was successfully loaded in the RBCMDs with a loading efficiency of 2-3% and an encapsulation efficiency of 62-97%. A short (3 min) exposure to HIFU irradiation triggered release of CPT from the RBCMDs and the physical explosion of droplets damaged nearby cancer cells resulting in significant cell death. In addition, the acoustically vaporized RBCMDs significantly increased the ultrasound echo signal to 30 dB. Lastly, we demonstrated that RBCMDs could be acoustically vaporized in vivo in target tissues, and enhancing ultrasound imaging. Taken together, we have developed a new class of naturally derived RBCMDs which show great potential for future application in remotely triggered drug delivery and ultrasound imaging enhancement.

Targeted tumor theranostics using folate-conjugated and camptothecin-loaded acoustic nanodroplets in a mouse xenograft model.

In this study, we aimed to validate the feasibility of receptor-targeted tumor theranostics with folate-conjugated (FA) and camptothecin-loaded (CPT) acoustic nanodroplets (NDs) (collectively termed FA-CPT-NDs). The ND formulation was based on lipid-stabilized low-boiling perfluorocarbon that can undergo acoustic droplet vaporization (ADV) under ultrasound (US) exposure. Conjugation of folate enhanced the selective delivery to tumors expressing high levels of folate receptor (FR) under mediation by the enhanced permeability and retention effect. In vitro and in vivo studies were performed using FR-positive KB and FR-negative HT-1080 cell lines and mouse xenograft tumor models. Simultaneous therapy and imaging were conducted with a clinical US imaging system at mechanical indices of up to 1.4 at a center frequency of 10 MHz. The results demonstrated that FA-CPT-NDs selectively attached to KB cells, but not HT-1080 cells. The targeted ADV caused instant and delayed damage via mechanical disruption and chemical toxicity to decrease the viability of KB cells by up to 45%, a much higher decrease than that achieved by the NDs without folate conjugation. The in vivo experiments showed that FR-mediated targeting successfully enhanced the EPR of FA-CPT-NDs in KB tumors mainly on the tumor periphery as indicated by immunofluorescence microscopy and US B-mode imaging. Treatments with FA-CPT-NDs at a CPT dose of 50 µg/kg inhibited the growth of KB tumors for up to six weeks, whereas treatment with NDs lacking folate produced a 4.6-fold increase in tumor volume. For HT-1080 tumors, neither the treatments with FA-CPT-NDs nor those with the NDs lacking folate presented tumor growth inhibition. In summary, FR-targeted tumor theranostics has been successfully implemented with FA-CPT-NDs and a clinical US unit. The ligand-directed and EPR-mediated accumulation provides active and passive targeting capabilities, permitting the antitumor effects of FA-CPT-NDs to be exerted selectively to FR-positive tumors and simultaneously providing targeted US imaging capabilities.

Internal polymer scaffolding in lipid-coated microbubbles for control of inertial cavitation in ultrasound theranostics.

A lipid-polymer composite structure was developed for tuning of inertial cavitation activity of microbubbles under ultrasound exposure. The incorporation of a thin layer of polymer networks inside the lipid monolayer resulted in marked reduction in the inertial cavitation dose. This strategy has the potential to increase the safety of ultrasound theranostic applications assisted by microbubble cavitation.

Using microbubbles as an MRI contrast agent for the measurement of cerebral blood volume.

The susceptibility differences at the gas-liquid interface of microbubbles (MBs) allow their use as an intravascular susceptibility contrast agent for in vivo MRI. However, the characteristics of MBs are very different from those of the standard gadolinium-diethylenetriaminepentaacetic acid (Gd-DPTA) contrast agent, including the size distribution and hemodynamic properties, which could influence MRI outcomes. Here, we investigate quantitatively the correlation between the relative cerebral blood volume (rCBV) derived from Gd-DTPA (rCBV(Gd)) and the MB-induced susceptibility effect (ΔR(2*MB)) by conventional dynamic susceptibility contrast MRI (DSC-MRI). Custom-made MBs had a mean diameter of 0.92 µm and were capable of inducing 4.68 ± 3.02% of the maximum signal change (MSC). The MB-associated ΔR(2*MB) was compared with rCBV(Gd) in 16 rats on 4.7-T MRI. We observed a significant effect of the time to peak (TTP) on the correlation between ΔR(2*MB) and rCBV(Gd), and also found a noticeable dependence between TTP and MSC. Our findings suggest that MBs with longer TTPs can be used for the estimation of rCBV by DSC-MRI, and emphasize the critical effect of TTP on MB-based contrast MRI.

Controlling the size distribution of lipid-coated bubbles via fluidity regulation.

Lipid-coated bubbles exhibit oscillation responses capable of enhancing the sensitivity of ultrasound imaging by improving contrast. Further improvements in performance enhancement require control of the size distribution of bubbles to promote correspondence between their resonance frequency and the frequency of the ultrasound. Here we describe a size-controlling technique that can shift the size distribution using a currently available agitation method. This technique is based on regulating the membrane dynamic fluidity of lipid mixtures and provides a general size-controlling variable that could also be applied in other fabrication methods. Three materials (1,2-dihexadecanoyl-sn-glycero-3-phosphocholine, 1,2-dioctadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) and polyethylene glycol 40 stearate) with distinct initial fluidities and phase behaviors were used to demonstrate the use of fluidity regulation to control bubble sizes. Bubble size distributions of different formulations were determined by electrical impedance sensing, and bubble volumes and surface areas were calculated. To confirm the relationship between regulated fluidity and mean bubble size, the membrane fluidity of each composition was determined by fluorescence anisotropy, with the results indicating linear relations in the compositions with similar main transition temperatures. Compositions with a higher molar proportion of polyethylene glycol 40 stearate showed higher fluidities and larger bubbles. B-mode ultrasound imaging was performed to investigate the echogenicity and lifetime of the fabricated bubbles, with the results indicating that co-mixing a high-transition-temperature charged lipid (i.e., 1,2-dioctadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)) extends the tailoring range of this fluidity regulation technique, allowing refined and continuous changes in mean bubble size (from 0.93 to 2.86 µm in steps of ∼0.5 µm), and also prolongs bubble lifetime. The polydispersity of each composition was also determined to evaluate practicality in particular applications. Our study demonstrates a feasible approach to naturally controling bubble size distribution and provides a practical reference for other fabrication systems and ultrasound imaging applications.

SPIO-conjugated, doxorubicin-loaded microbubbles for concurrent MRI and focused-ultrasound enhanced brain-tumor drug delivery.

The blood-brain barrier (BBB) can be temporarily and locally opened by focused ultrasound (FUS) in the presence of circulating microbubbles (MBs). Currently, contrast-enhanced magnetic resonance imaging (CE-MRI) is used to monitor contrast agent leakage to verify BBB-opening and infer drug deposition. However, despite being administered concurrently, MBs, therapeutic agent, and contrast agent have distinct pharmacodynamic behaviors, thus complicating the quantification and optimization of BBB-opening and drug delivery. Here we propose multifunctional MBs loaded with therapeutic agent (doxorubicin; DOX) and conjugated with superparamagnetic iron oxide (SPIO) nanoparticles. These DOX-SPIO-MBs were designed to concurrently open the BBB and perform drug delivery upon FUS exposure, act as dual MRI and ultrasound contrast agent, and allow magnetic targeting (MT) to achieve enhanced drug delivery. We performed burst-tone FUS after injection of DOX-SPIO-MBs, followed by MT with an external magnet attached to the scalp in a rat glioma model. Animals were monitored by T2-weighted MRI and susceptibility weighted imaging and the concentration of SPIO particles was determined by spin-spin relaxivity. We found that DOX-SPIO-MBs were stable and provided significant superparamagnetic/acoustic properties for imaging. BBB-opening and drug delivery were achieved concurrently during the FUS exposure. In addition, MT increased local SPIO deposition in tumor regions by 22.4%. Our findings suggest that DOX-SPIO-MBs with FUS could be an excellent theranostic tool for future image-guided drug delivery to brain tumors.

Superparamagnetic iron oxide and drug complex-embedded acoustic droplets for ultrasound targeted theranosis.

Ultrasound-triggered acoustic droplet vaporization (ADV) has been reported as a mechanical and chemical theranostic strategy for tumor treatment. However, targeting of sufficient amounts of droplets to solid tumors to direct effective mechanical force toward tumor cells remains a major challenge. In this study, we incorporated superparamagnetic iron oxide (SPIO) nanoparticles into acoustic droplets to allow both magnetism-assisted targeting and magnetic resonance (MR)-guided ultrasound-triggered ADV. The multi-functionality of these droplets was further increased by co-encapsulation of the chemotherapeutic drug doxorubicin (DOX) and surface conjugation of anti-vascular endothelial growth factor receptor 2 antibody, to serve as an additional targeting moiety. Maximum loading capacities of 7.69 mg SPIO and 1.53 mg DOX per mL were achieved, and magnetic properties were characterized by determination of magnetic hysteresis curves and transverse relaxation rates. In vitro and in vivo MR imaging demonstrated the feasibility of dual modal imaging of SPIO-embedded droplets. Finally, a vessel-mimicking phantom model with live C6 glioma cells was used to demonstrate a 5.4-fold improvement in targeting efficacy by magnetism-assisted targeting of the SPIO-embedded droplets, and effective disruption of cells by insonation-induced ADV, suggesting the potential of developing this system for future clinical applications.

Aptamer-conjugated and drug-loaded acoustic droplets for ultrasound theranosis.

Tumor therapy requires multi-functional treatment strategies with specific targeting of therapeutics to reduce general toxicity and increase efficacy. In this study we fabricated and functionally tested aptamer-conjugated and doxorubicin (DOX)-loaded acoustic droplets comprising cores of liquid perfluoropentane compound and lipid-based shell materials. Conjugation of sgc8c aptamers provided the ability to specifically target CCRF-CEM cells for both imaging and therapy. High-intensity focused ultrasound (HIFU) was introduced to trigger targeted acoustic droplet vaporization (ADV) which resulted in both mechanical cancer cell destruction by inertial cavitation and chemical treatment through localized drug release. HIFU insonation showed a 56.8% decrease in cell viability with aptamer-conjugated droplets, representing a 4.5-fold increase in comparison to non-conjugated droplets. In addition, the fully-vaporized droplets resulted in the highest DOX uptake by cancer cells, compared to non-vaporized or partially vaporized droplets. Optical studies clearly illustrated the transient changes that occurred upon ADV of droplet-targeted CEM cells, and B-mode ultrasound imaging revealed contrast enhancement by ADV in ultrasound images. In conclusion, our fabricated droplets functioned as a hybrid chemical and mechanical strategy for the specific destruction of cancer cells upon ultrasound-mediated ADV, while simultaneously providing ultrasound imaging capability.

Aptamer-conjugated nanobubbles for targeted ultrasound molecular imaging.

Targeted ultrasound contrast agents can be prepared by some specific bioconjugation techniques. The biotin-avidin complex is an extremely useful noncovalent binding system, but the system might induce immunogenic side effects in human bodies. Previous proposed covalently conjugated systems suffered from low conjugation efficiency and complex procedures. In this study, we propose a covalently conjugated nanobubble coupling with nucleic acid ligands, aptamers, for providing a higher specific affinity for ultrasound targeting studies. The sgc8c aptamer was linked with nanobubbles through thiol-maleimide coupling chemistry for specific targeting to CCRF-CEM cells. Further improvements to reduce the required time and avoid the degradation of nanobubbles during conjugation procedures were also made. Several investigations were used to discuss the performance and consistency of the prepared nanobubbles, such as size distribution, conjugation efficiency analysis, and flow cytometry assay. Further, we applied our conjugated nanobubbles to ex vivo ultrasound targeted imaging and compared the resulting images with optical images. The results indicated the availability of aptamer-conjugated nanobubbles in targeted ultrasound imaging and the practicability of using a highly sensitive ultrasound system in noninvasive biological research.