Ultrasound-triggered Drug Delivery Research Articles

Paclitaxel-liposome–microbubble complexes as ultrasound-triggered therapeutic drug delivery carriers

Liposome–microbubble complexes (LMC) have become a promising therapeutic carrier for ultrasound-triggered drug delivery to treat malignant tumors. However, the efficacy for ultrasound-assisted chemotherapy in vivo and the underlying mechanisms remain to be elucidated. Here, we investigated the feasibility of using paclitaxel-liposome–microbubble complexes (PLMC) as possible ultrasound (US)-triggered targeted chemotherapy against breast cancer. PTX-liposomes (PL) were conjugated to the microbubble (MB) surface through biotin–avidin linkage, increasing the drug-loading efficiency of MBs. The significant increased release of payloads from liposome–microbubble complexes was achieved upon US exposure. We used fluorescent quantum dots (QDs) as a model drug to show that released QDs were taken up by 4T1 breast cancer cells treated with QD-liposome–microbubble complexes (QLMC) and US, and uptake depended on the exposure time and intensity of insonication. We found that PLMC plus US inhibited tumor growth more effectively than PL plus US or PLMC without US, not only in vitro, but also in vivo. Histologically, the inhibition of tumor growth appeared to result from increased apoptosis and reduced angiogenesis in tumor xenografts. In addition, a significant increase of drug concentration in tumors was observed in comparison to treatment with non-conjugated PL or PLMC without US. The significant increase in an antitumor efficacy of PLMC plus US suggests their potential use as a new targeted US chemotherapeutic approach to inhibit breast cancer growth.

Doxorubicin liposome-loaded microbubbles for contrast imaging and ultrasound-triggered drug delivery

Targeted drug delivery under image guidance is gaining more interest in the drug-delivery field. The use of microbubbles as contrast agents in diagnostic ultrasound provides new opportunities in noninvasive image-guided drug delivery. In the present study, the imaging and therapeutic properties of novel doxorubicin liposome-loaded microbubbles are evaluated. The results showed that at scanning settings (1.7 MHz and mechanical index 0.2), these microbubbles scatter sufficient signal for nonlinear ultrasound imaging and can thus be imaged in real time and be tracked in vivo. In vitro therapeutic evaluation showed that ultrasound at 1 MHz and pressures up to 600 kPa in combination with the doxorubicin liposomeloaded microbubbles induced 4-fold decrease of cell viability compared with treatment with free doxorubicin or doxorubicin liposome-loaded microbubbles alone. The therapeutic effectiveness is correlated to an ultrasound-triggered release of doxorubicin from the liposomes and an enhanced uptake of the free doxorubicin by glioblastoma cells. The results obtained demonstrate that the combination of ultrasound and the doxorubicin liposome-loaded microbubbles can provide a new method of noninvasive image-guided drug delivery.

Doxorubicin loaded superparamagnetic PLGA-iron oxide multifunctional microbubbles for dual-mode US/MR imaging and therapy of metastasis in lymph nodes

Current strategies for tumor-induced sentinel lymph node detection and metastasis therapy have limitations. In this work, we co-encapsulated iron oxide nanoparticles and chemotherapeutic drug into poly(lactic-co-glycolic acid) (PLGA) microbubbles to form multifunctional polymer microbubbles (MPMBs) for both tumor lymph node imaging and therapy. Fe3O4 nanoparticles and doxorubicin (DOX) co-encapsulated PLGA microbubbles were prepared and filled with perfluorocarbon gas. Enhancement of ultrasound (US)/magnetic resonance (MR) imaging and US triggered drug delivery were evaluated both in vitro and in vivo. The MPMBs exhibited characters like narrow size distribution and smooth surface with a mean diameter of 868.0 ± 68.73 nm. In addition, varying the concentration of Fe3O4 nanoparticles in the bubbles did not significantly influence the DOX encapsulation efficiency or drug loading efficiency. Our in vitro results demonstrated that these MPMBs could enhance both US and MR imaging which was further validated in vivo showing that these MPMBs enhanced tumor lymph nodes signals. The anti-tumor effect of MPMBs mediated chemotherapy was assessed in vivo using end markers like tumor proliferation index, micro blood vessel density and micro lymphatic vessel density, which were shown consistently the lowest after the MPMBs plus sonication treatment compared to controls. In line with these findings, the tumor cell apoptotic index was found the largest after the MPMBs plus sonication treatment. In conclusion, we have successfully developed a doxorubicin loaded superparamagnetic PLGA-Iron Oxide multifunctional theranostic agent for dual-mode US/MR Imaging of lymph node, and for low frequency US triggered therapy of metastasis in lymph nodes, which might provide a strategy for the imaging and chemotherapy of primary tumor and their metastases.

Crucial factors and emerging concepts in ultrasound-triggered drug delivery

Time and space controlled drug delivery still remains a huge challenge in medicine. A novel approach that could offer a solution is ultrasound guided drug delivery. “Ultrasonic drug delivery” is often based on the use of small gas bubbles (so-called microbubbles) that oscillate and cavitate upon exposure to ultrasound waves. Some microbubbles are FDA approved contrast agents for ultrasound imaging and are nowadays widely investigated as promising drug carriers. Indeed, it has been observed that upon exposure to ultrasound waves, microbubbles may (a) release the encapsulated drugs and (b) simultaneously change the structure of the cell membranes in contact with the microbubbles which may facilitate drug entrance into cells. This review aims to highlight (a) major factors known so far which affect ultrasonic drug delivery (like the structure of the microbubbles, acoustic settings, etc.) and (b) summarizes the recent preclinical progress in this field together with a number of promising new concepts and applications.

Ultrasound triggered drug release from 10-hydroxycamptothecin-loaded phospholipid microbubbles for targeted tumor therapy in mice

Ultrasound targeted microbubble destruction (UTMD) was one of the most promising strategies to enhance drug delivery in cancer therapy. Microbubbles (MBs) serve as a vehicle to carry anti-tumor drugs and locally release them when exposed to therapeutic ultrasound, resulting in drug accumulation in tumor tissues and enhanced anti-tumor effect. However the ultrasound triggered drug delivery system has been seriously limited due to the poor loading capacity of MBs. Here we present a new strategy to overcome the low drug payload of MBs for ultrasound guided drug delivery. In this study, we developed a novel microbubble carrying 10-HCPT which only needs a particularly low single dose of injection (4–6mg) for tumor therapy in clinical application, therefore, the required high dosing of drug loaded MBs for ultrasound mediated drug delivery is not necessary. We subsequently investigated the combination of ultrasound application with HLMs to achieve therapeutic effect on tumor at a feasible dose of MBs. HLMs were manufactured with a high drug encapsulation and loading content and simultaneously maintained the acoustic properties as an ultrasound contrast agent. After that, tumor-bearing mice were routinely and non-invasively administered with HLMs through the tail vein and were then exposed to ultrasound, resulting in a remarkable drug accumulation in tumor tissues and a significant increase in tumor inhibition rate (70.6%) compared with HLMs alone (47.8%) as well as commercial HCPT injection (49.4). In conclusion, HLMs are expected to improve the therapeutic efficacy of MBs and are worthy of further study for UTMD mediated drug delivery.

Fluorescent microscope system to monitor real-time interactions between focused ultrasound, echogenic drug delivery vehicles, and live cell membranes

Rapid development in the field of ultrasound triggered drug delivery has made it essential to study the real-time interaction between the membranes of live cells and the membranes of echogenic delivery vehicles under exposure to focused ultrasound. The objective of this work was to design an analysis system that combined fluorescent imagining, high speed videography, and definable pulse sequences of focused ultrasound to allow for real time observations of both cell and vehicle membranes. Documenting the behavior of the membranes themselves has not previously been possible due to limitations with existing optical systems used to understand the basic physics of microbubble/ultrasound interaction and the basic interaction between microbubbles and cells. The performance of this new system to monitor membrane behavior was demonstrated by documenting the modes of vehicle fragmentation at different ultrasound intensity levels. At 1.5MPa the membranes were shown to completely fragment while at intensities below 1MPa the membranes pop open and slowly unfold. The interaction between these vehicles and cell membranes was also documented by the removal of fluorescent particles from the surfaces of live cells out to 20μm from the microbubble location. The fluid flow created by microstreaming around ensonated microbubbles was documented at video recording speeds from 60 to 18,000 frames per second. This information about membrane behavior allows the chemical and physical properties of the drug delivery vehicle to be designed along with the ultrasound pulse sequence to cause the most efficient drug delivery.

MRI guided High Intensity Focused Ultrasound

Local temperature elevation may be used for tumor ablation, gene expression, drug activation, gene and/or drug delivery. High Intensity Focused Ultrasound (HIFU) can be used to achieve a local temperature increase noninvasively. MRI provides continuous temperature mapping during HIFU for spatio-temporal control of the heating procedure and prediction of the final lesion. MRI-HIFU is now clinically accepted for ablation of uterine fibroids. MRI-HIFU is being evaluated in the cancer field. The technology also shows great promise for advanced therapies such as gene therapy and drug delivery. MRI-HIFU, together with the use of a temperature sensitive promoter, provides local, spatio-temporal control of transgene expression. The goal of ultrasound triggered, image guided drug delivery is to increase the therapeutic index of drugs, and decrease adverse effects of drugs. The bio-effects of ultrasound can lead to local tissue heating, cavitation, and radiation force, which can be used for 1) local drug release from nanocarriers circulating in the blood, 2) increased extravasation and cellular uptake of drugs and/or carriers, and 3) enhanced diffusivity of drugs. Thermo-sensitive liposomes have been suggested for local drug release more than 25 years ago. Microbubbles may be designed to enhance cavitation effects. Real-time imaging methods, such as MRI, optical and ultrasound imaging have led to novel insights for ultrasound triggered drug delivery.

Paclitaxel-liposome loaded microbubbles for ultrasound-triggered drug delivery in vitro and in vivo

The aim of this work was to develop paclitaxel-liposome loaded microbubbles (PTX-loaded MBs) and to investigate the efficacy of chemotherapy through ultrasound-triggered drug delivery in vitro and in vivo. PTX-loaded liposomes were prepared by a minor modification of thin film hydration method and further conjugated to the surface of microbubbles through biotin-avidin linkage. The resulting payload-loaded MBs were characterized and applied to ultrasound assisted chemotherapy in breast cancer. Our results showed the MBs were able to achieve satisfactory drug encapsulation efficiency. Under ultrasound exposure, about 9.54-fold higher drug release and a significant improvement of cell uptake than that of loaded liposomes were observed. In addition, PTX-loaded MBs showed significantly greater tumor growth inhibition both in vitro and in vivo xenograft growth of breast tumor cells, compared with PTX-loaded liposomes and unloaded MBs. Drug distributions assay in various organs (heart, liver, spleen, lung, kidney and tumor) indicated about 3-fold higher PTX enriched in tumor. Histological examinations further demonstrated the tumor growth inhibition might contribute to increased apoptosis and reduced angiogenesis in tumor xenografts. In conclusion, the study indicated the PTX-loaded MBs significantly increased the anti-tumor efficacy and can be used as a potential chemotherapy approach for ultrasound assisted breast cancer treatment.

Optical characterization of inidividual liposome-loaded microbubbles

Newly developed liposome-loaded (LPS) microbubbles [1] were characterized by comparing their oscillating response with standard phospholipid-shelled (BARE) microbubbles using the ultra-high speed imaging camera (Brandaris 128). 73 LPS bubbles and 41 BARE bubbles of diameters ranging from 3 μm to 10 μm were insonified by narrow band pulses with a driving frequency ranging from 0.5 MHz to 4 MHz and an acoustic pressure from 5 kPa to 100 kPa. Shell elasticity of LPS bubbles (0.17±0.1 N/m) was nearly the same as that of BARE bubbles (0.19±0.1 N/m) for all investigated bubble sizes. Clear difference of shell viscosity was found for bubbles larger than 6 μm. Averaged viscosity of LPS bubbles (2.5×10-8 kg/s) was almost twice of that of BARE bubbles (1.4×10-8 kg/s). A second finding for LPS bubbles was the dominant “expansion-only” behavior (70% of LPS bubbles), while this was only 13% for BARE bubbles. Results from this study will facilitate future preclinical studies and clinical applications of LPS bubbles for ultrasound triggered drug delivery system. Reference 1. Geers, B., et al., J Control Release, 2011. 152(2): p. 249-56.

The impact of microbubbles on measurement of drug release from echogenic liposomes

Echogenic liposomes (ELIP) are under development to enable ultrasound-triggered drug delivery. The mechanisms of ultrasound-mediated drug release from ELIP are not well understood. The effect of cavitation activity on drug release from ELIP was investigated in flowing solutions using two fluorescent molecules: a lipophilic drug (rosiglitazone) and a hydrophilic drug substitute (calcein). ELIP samples were exposed to pulsed Doppler ultrasound from a clinical diagnostic ultrasound scanner at pressures above and below the inertial and stable cavitation thresholds. Control samples were exposed to Triton X-100, a detergent (positive control), or to flow alone (negative control). Fluorescence techniques were used to detect release. Encapsulated microbubbles reduced the measured fluorescence intensity. This effect should be considered when assessing drug release if microbubbles are present. Release of rosiglitazone or calcein compared to the negative control was only observed with detergent treatment, but not with ultrasound exposure, despite the presence of inertial or stable cavitation activity. Thus, cavitation activity did not correlate with release of rosiglitazone or calcein from ELIP using a clinical diagnostic ultrasound scanner. These findings lay the foundation for future studies of ultrasound-mediated drug delivery with ELIP.

Preparation and in vitro evaluation of an ultrasound-triggered drug delivery system: 10-Hydroxycamptothecin loaded PLA microbubbles

10-Hydroxycamptothecin (HCPT) loaded PLA microbubbles, used as an ultrasound-triggered drug delivery system, were fabricated by a double emulsion–solvent evaporation method. The obtained microbubbles were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and confocal laser scanning microscope (CLSM). In addition, the effect of diagnostic ultrasound exposure on BEL-7402 cells combined with HCPT-loaded PLA microbubbles was evaluated using cytotoxicity assay, CLSM and flow cytometry (FCM). It was found that the HCPT-loaded PLA microbubbles showed smooth surface and spherical shape, and the drug was amorphously dispersed within the shell and the drug loading content reached up to 1.69%. Nearly 20% of HCPT was released upon exposure to diagnostic ultrasound at frequency of 3.5MHz for 10min. Moreover, HCPT fluorescence in the cells treated only with the HCPT-loaded PLA microbubbles was discernible, but less intense, while those treated with the microbubbles in conjunction with ultrasound exposure was evident and intense, indicating an increased cellular uptake of HCPT by ultrasound exposure. Cytotoxicity test on BEL-7402 cells indicated that the HCPT-loaded PLA microbubbles combined with ultrasound exposure were more cytotoxic than the microbubbles alone. The results suggest that the combination of drug loaded PLA microbubbles and diagnostic ultrasound exposure exhibit an effective intracellular drug uptake by tumor cells, indicating their great potential for antitumor therapy.

Localized activation and cellular effects of ultrasound triggered drug delivery vehicles with encapsulated microbubbles

The harmful side effects of chemotherapy originate from indiscriminate exposure of healthy tissue to the drugs. The goal of targeted drug delivery is to reduce these side effects by encapsulating concentrated drug in a vehicle which releases it only in the tumor region. Low intensity focused ultrasound can be used as a trigger to specifically activate these vehicles by highlighting only tumor tissue, creating a stark differentiation with healthy tissue. A new injectable drug delivery vehicle has been developed with a stabilized nested lipid shell geometry that encapsulates a high capacity chemotherapy payload, and a stabilized microbubble into one structure. Ultrasound affects the microbubble only in the small focal volume, creating a localized shockwave which ruptures the vehicle's outer membrane triggering pinpoint release in tissue phantoms. These shockwaves, and their interactions with the delivery vehicle membranes and live cells, have been documented for the first time using a custom system which co...

Self-assembled liposome-loaded microbubbles: The missing link for safe and efficient ultrasound triggered drug-delivery

Liposome-loaded microbubbles have been recently introduced as a promising drug delivery platform for ultrasound guided drug delivery. In this paper we design liposome-loaded (lipid-shelled) microbubbles through the simple self-assembly of the involved compounds in a single step process. We thoroughly characterized the liposome-loading of the microbubbles and evaluated the cell killing efficiency of this material using doxorubicin (DOX) as a model drug. Importantly, we observed that the DOX liposome-loaded microbubbles allowed killing of melanoma cells even at very low doses of DOX. These findings clearly prove the potential of liposome-loaded microbubbles for ultrasound targeted drug delivery to cancer tissues.

Ultrasound triggered, image guided, local drug delivery

Ultrasound allows the deposition of thermal and mechanical energies deep inside the human body in a non-invasive way. Ultrasound can be focused within a region with a diameter of about 1mm. The bio-effects of ultrasound can lead to local tissue heating, cavitation, and radiation force, which can be used for 1) local drug release from nanocarriers circulating in the blood, 2) increased extravasation of drugs and/or carriers, and 3) enhanced diffusivity of drugs. When using nanocarriers sensitive to mechanical forces (the oscillating ultrasound pressure waves) and/or sensitive to temperature, the content of the nanocarriers can be released locally. Thermo-sensitive liposomes have been suggested for local drug release in combination with local hyperthermia more than 25years ago. Microbubbles may be designed specifically to enhance cavitation effects. Real-time imaging methods, such as magnetic resonance, optical and ultrasound imaging have led to novel insights and methods for ultrasound triggered drug delivery. Image guidance of ultrasound can be used for: 1) target identification and characterization; 2) spatio-temporal guidance of actions to release or activate the drugs and/or permeabilize membranes; 3) evaluation of bio-distribution, pharmacokinetics and pharmacodynamics; and 4) physiological read-outs to evaluate the therapeutic efficacy.

Ultrasound-triggered release of materials entrapped in microbubble–liposome constructs: A tool for targeted drug delivery

We investigated the preparation of ultrasound-triggered drug delivery system, based on a pendant complex of microbubble coated with liposomes. Biotinylated decafluorobutane microbubbles were coated with biotinylated liposomes via a streptavidin linker. Liposomes were prepared incorporating calcein and thrombin. Based on initial concentration of calcein, over 1um3 payload volume per each microbubble–liposome particle was achieved, when 100nm liposomes were used.Insonation of microbubble–liposome pendants in vitro resulted in the complete destruction of microbubbles and triggered release of a significant fraction of the entrapped material. Treatment with 1MHz ultrasound (5 pulses, 100ms, 7MPa peak negative acoustic pressure) resulted in the release of ~30% of entrapped calcein, as estimated by the fluorescence quenching assay.Thrombin release from liposomes complexed with microbubbles (11% of entrapped material) due to ultrasound treatment was estimated by a chromogenic substrate study. Prior to insonation, substrate hydrolysis was at background level. Ultrasound-triggered release of thrombin from the pendant complexes caused an acceleration of blood clotting.

Contrast-enhanced ultrasound for molecular imaging of angiogenesis

Molecular imaging of angiogenesis using contrast-enhanced ultrasound allows for functional, real-time, inexpensive imaging of angiogenesis. The addition of stabilized microbubbles as contrast agents greatly improves ultrasound signal to noise ratio/signal strength/image quality (up to 25 dB) and allows for imaging of angiogenic vasculature. In this article recent advances in the usage of contrast-enhanced ultrasound for molecular imaging of angiogenesis are reviewed. The usage of commercially available agents and correlations between their imaging parameters and molecular markers of angiogenesis are reviewed. Recent developments in ultrasound contrast agents targeted to angiogenic markers for both diagnosis and monitoring are discussed. Finally, a brief overview of the emerging field of chemotherapeutic-loaded agents, which can be used with ultrasound-triggered drug delivery, is provided.

A microcomposite hydrogel for repeated on-demand ultrasound-triggered drug delivery

Here we develop an injectable composite system based for repeated ultrasound-triggered on-demand drug delivery. An in situ-cross-linking hydrogel maintains model drug (dye)-containing liposomes in close proximity to gas-filled microbubbles that serve to enhance release events induced by ultrasound application. Dye release is tunable by varying the proportions of the liposomal and microbubble components, as well as the duration and intensity of the ultrasound pulses in vitro. Dye is minimal at baseline. The composite shows minimal cytotoxicity in vitro, and benign tissue reaction after subcutaneous injection in rats. Ultrasound application also triggers drug release for two weeks after injection in vivo.

Reelin MRNA is decreased in neurons of the hippocampus and temporal cortex in schizophrenia: A cellular in situ hybridisation study

Current strategies for tumor-induced sentinel lymph node detection and metastasis therapy have limitations. In this work, we co-encapsulated iron oxide nanoparticles and chemotherapeutic drug into poly(lactic-co-glycolic acid) (PLGA) microbubbles to form multifunctional polymer microbubbles (MPMBs) for both tumor lymph node imaging and therapy. Fe3O4 nanoparticles and doxorubicin (DOX) co-encapsulated PLGA microbubbles were prepared and filled with perfluorocarbon gas. Enhancement of ultrasound (US)/magnetic resonance (MR) imaging and US triggered drug delivery were evaluated both in vitro and in vivo. The MPMBs exhibited characters like narrow size distribution and smooth surface with a mean diameter of 868.0 ± 68.73 nm. In addition, varying the concentration of Fe3O4 nanoparticles in the bubbles did not significantly influence the DOX encapsulation efficiency or drug loading efficiency. Our in vitro results demonstrated that these MPMBs could enhance both US and MR imaging which was further validated in vivo showing that these MPMBs enhanced tumor lymph nodes signals. The anti-tumor effect of MPMBs mediated chemotherapy was assessed in vivo using end markers like tumor proliferation index, micro blood vessel density and micro lymphatic vessel density, which were shown consistently the lowest after the MPMBs plus sonication treatment compared to controls. In line with these findings, the tumor cell apoptotic index was found the largest after the MPMBs plus sonication treatment. In conclusion, we have successfully developed a doxorubicin loaded superparamagnetic PLGA-Iron Oxide multifunctional theranostic agent for dual-mode US/MR Imaging of lymph node, and for low frequency US triggered therapy of metastasis in lymph nodes, which might provide a strategy for the imaging and chemotherapy of primary tumor and their metastases.