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.     

Amino acid-modified PAMAM dendritic nanocarriers as effective chemotherapeutic drug vehicles in cancer treatment: a study using zebrafish as a cancer model

The use of nanomaterials for drug delivery offers many advantages including the targeted delivery of drugs and their controlled release. Nonetheless, entry into the target cells remains a challenge for many nanomaterials used for drug delivery. Moreover, cellular uptake limits the therapeutic efficiency of many anticancer drugs. An important goal is to increase the specific accumulation of these nanoparticles (NPs) at the desired cancerous tissues. Notably, cancer cells show a high demand for some amino acids and we have used this knowledge to develop novel carrier systems. In this study, drug carriers were produced by the conjugation of multiple amino acids such as l-histidine (H) and l-cysteine (C) or single amino acids such as only H with the G4.5 dendrimers (G) to produce GHC aggregates and GH NP carriers, respectively. Doxorubicin was loaded into the G4.5, GH, and GHC dendrimers (G/DOX, GH/DOX and GHC/DOX, respectively) and the release mechanism was demonstrated at pH 7.4 and pH 5.0. GH/DOX and GHC/DOX showed better stability under physiological conditions than the dendrimer alone (G/DOX). GH/DOX and GHC/DOX exhibited higher inhibition of HeLa cell proliferation in in vitro and in vivo studies in zebrafish, confirming the early release of DOX by disrupting the endosomal membrane and triggering the destabilization of carriers at a lower pH of 5.0.

The use of nanomaterials for drug delivery offers many advantages including the controlled release and their targeted delivery.     

Localized ultrasound enhances delivery of rapamycin from microbubbles to prevent smooth muscle proliferation

Microbubble contrast agents have been shown to enhance reagent delivery when activated by ultrasound. We hypothesized that ultrasound would enhance delivery of rapamycin, an antiproliferative agent, from the shell of microbubbles, thus reducing proliferation of vascular smooth muscle cells. Our objective was to determine optimal ultrasound parameters that maximized therapeutic efficacy, maintained cell adherence, and minimized the drug exposure time. In vitro assays determined that ultrasound (1 MHz, 0.5% duty cycle) is required to successfully deliver rapamycin from microbubbles and reduce proliferation. Co-injection of rapamycin with control microbubbles did not result in a reduction in proliferation. Successful reduction in proliferation (> 50%) required pulses at least 10 cycles in length and at least 300 kPa peak negative pressure at which point 90% of cells remained adherent. The anti-proliferative effect was also localized within a 6 mm wide zone by focusing the ultrasound beam.     

Improved Anti-Tumor Effect of Liposomal Doxorubicin After Targeted Blood-Brain Barrier Disruption by MRI-Guided Focused Ultrasound in Rat Glioma

The blood-brain barrier (BBB) inhibits the entry of the majority of chemotherapeutic agents into the brain. Previous studies have illustrated the feasibility of drug delivery across the BBB using focused ultrasound (FUS) and microbubbles. Here, we investigated the effect of FUS-enhanced delivery of doxorubicin on survival in rats with and 9L gliosarcoma cells inoculated in the brain. Each rat received either: (1) no treatment (control; N = 11), (2) FUS only (N = 9), (3) IV liposomal doxorubicin (DOX only; N = 17), or (4) FUS with concurrent IV injections of liposomal doxorubicin (FUS+DOX; N = 20). Post-treatment by magnetic resonance imaging (MRI) showed that FUS+DOX reduced tumor growth compared with DOX only. Further, we observed a modest but significant increase in median survival time after a single treatment FUS+DOX treatment (p = 0.0007), whereas neither DOX nor FUS had any significant impact on survival on its own. These results suggest that combined ultrasound-mediated BBB disruption may significantly increase the antineoplastic efficacy of liposomal doxorubicin in the brain.     

Enhanced Intracellular Delivery of a Model Drug Using Microbubbles Produced by a Microfluidic Device

Focal drug delivery to a vessel wall facilitated by intravascular ultrasound and microbubbles holds promise as a potential therapy for atherosclerosis. Conventional methods of microbubble administration result in rapid clearance from the bloodstream and significant drug loss. To address these limitations, we evaluated whether drug delivery could be achieved with transiently stable microbubbles produced in real time and in close proximity to the therapeutic site. Rat aortic smooth muscle cells were placed in a flow chamber designed to simulate physiological flow conditions. A flow-focusing microfluidic device produced 8 μm diameter monodisperse microbubbles within the flow chamber, and ultrasound was applied to enhance uptake of a surrogate drug (calcein). Acoustic pressures up to 300 kPa and flow rates up to 18 mL/s were investigated. Microbubbles generated by the flow-focusing microfluidic device were stabilized with a polyethylene glycol-40 stearate shell and had either a perfluorobutane (PFB) or nitrogen gas core. The gas core composition affected stability, with PFB and nitrogen microbubbles exhibiting half-lives of 40.7 and 18.2 s, respectively. Calcein uptake was observed at lower acoustic pressures with nitrogen microbubbles (100 kPa) than with PFB microbubbles (200 kPa) (p < 0.05, n > 3). In addition, delivery was observed at all flow rates, with maximal delivery (>70% of cells) occurring at a flow rate of 9 mL/s. These results demonstrate the potential of transiently stable microbubbles produced in real time and in close proximity to the intended therapeutic site for enhancing localized drug delivery.     

Activation of microbubbles by short-pulsed ultrasound enhances the cytotoxic effect of cis-diamminedichloroplatinum (II) in a canine thyroid adenocarcinoma cell line in vitro

Ultrasound targeted microbubble destruction has succeeded in delivering drugs and genes. This study was designed to explore characteristics of ultrasound targeted microbubble destruction using short-pulsed diagnostic ultrasound. Canine thyroid adenocarcinoma cells were exposed to short-pulsed diagnostic ultrasound in the presence of cis-diamminedichloroplatinum (II) (cisplatin) and ultrasound contrast agent Sonazoid^® microbubbles. The cytotoxic effect of cisplatin was enhanced by short-pulsed diagnostic ultrasound and microbubbles. Incubation time with microbubbles influenced the cytotoxic effect of cisplatin. However, exposure duration did not affect the cytotoxic effect of cisplatin. Therefore, short-pulsed diagnostic ultrasound may activate microbubbles near cells and deliver cisplatin into cells. In addition, activation of microbubbles may be concluded in a short time. Our results suggest that short exposure duration could be potentially sufficient to induce efficient drug delivery by ultrasound targeted microbubble destruction using short-pulsed diagnostic ultrasound.     

Sonodynamic Therapy Using Dacarbazine-Loaded AuSiO2 Nanoparticles for Melanoma Treatment: An In-Vitro Study on the B16F10 Murine Melanoma Cell Line

The use of nanoparticles as a sonosensitizer in cancer sonodynamic therapy has been gaining attention because of their great advantages in drug delivery applications. By conjugating chemotherapy agents with nanoparticles, we can develop a drug delivery platform, control drug release and improve the outcome of treatments. The in-vitro study described here evaluates the combination of AuSiO₂ nanoparticles and dacarbazine (DTIC@AuSiO₂) as a sonosensitizer for sonodynamic therapy of melanoma. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays revealed that the viability of B16F10 melanoma cells was significantly inhibited by the increase in apoptosis induction in treatment with DTIC@AuSiO₂ nanoparticles under ultrasound exposure compared with treatment with the free DTIC or AuSiO₂ nanoparticles. The sonosensitization activity of AuSiO₂ nanoparticles and greater uptake of DTIC by tumor cells after loading in DTIC@AuSiO₂ nanoparticles inhibited the proliferation of melanoma tumor cells effectively. In conclusion, the DTIC@AuSiO₂ nanoparticles established in this study could represent a good drug delivery and sonosensitizer platform for use in melanoma sonodynamic therapy.     

Endothelial Cells,First Target of Drug Delivery Using Microbubble-Assisted Ultrasound

Microbubble-assisted ultrasound has emerged as a promising method for local drug delivery. Microbubbles are intravenously injected and locally activated by ultrasound, thus increasing the permeability of vascular endothelium for facilitating extravasation and drug uptake into the treated tissue. Thereby, endothelial cells are the first target of the effects of ultrasound-driven microbubbles. In this review, the in vitro and in vivo bioeffects of this method on endothelial cells are described and discussed, including aspects on the permeabilization of biologic barriers (endothelial cell plasma membranes and endothelial barriers), the restoration of their integrity, the molecular and cellular mechanisms involved in both these processes, and the resulting intracellular and intercellular consequences. Finally, the influence of the acoustic settings, microbubble parameters, treatment schedules and flow parameters on these bioeffects are also reviewed.     

Sonoporation as a Cellular Stress: Induction of Morphological Repression and Developmental Delays

For sonoporation to be established as a drug/gene delivery paradigm, it is essential to account for the biological impact of this membrane permeation strategy on living cells. Here we provide new insight into the cellular impact of sonoporation by demonstrating in vitro that this way of permeating the plasma membrane may inadvertently induce repressive cellular features even while enhancing exogenous molecule uptake. Both suspension-type (HL-60) and monolayer (ZR-75-30) cells were considered in this investigation, and they were routinely exposed to 1-MHz pulsed ultrasound (pulse length, 100 cycles; pulse repetition frequency, 1 kHz; exposure period, 60 s) with calibrated field profile (spatial-averaged peak negative pressure, 0.45 MPa) and in the presence of microbubbles (cell:bubble ratio, 10:1). The post-exposure morphology of sonoporated cells (identified as those with calcein internalization) was examined using confocal microscopy, and their cell cycle progression kinetics were analyzed using flow cytometry. Results show that for both cell types investigated, sonoporated cells exhibited membrane shrinkage and intra-cellular lipid accumulation over a 2-h period. Also, as compared with normal cells, the deoxyribonucleic acid synthesis duration of sonoporated cells was significantly lengthened, indicative of a delay in cell cycle progression. These features are known to be characteristics of a cellular stress response, suggesting that sonoporation indeed constitutes as a stress to living cells. This issue may need to be addressed in optimizing sonoporation for drug/gene delivery purposes. On the other hand, it raises opportunities for developing other therapeutic applications via sonoporation.     

Drug delivery in pluronic micelles: effect of high-frequency ultrasound on drug release from micelles and intracellular uptake.

The effect of high-frequency ultrasound on doxorubicin (DOX) release from Pluronic micelles and intracellular DOX uptake was studied for promyelocytic leukemia HL-60 cells, ovarian carcinoma drug-sensitive and multidrug-resistant (MDR) cells (A2780 and A2780/ADR, respectively), and breast cancer MCF-7 cells. Cavitation events initiated by high-frequency ultrasound were recorded by radical trapping. The onset of transient cavitation and DOX release from micelles were observed at much higher power densities than at low-frequency ultrasound (20-100 kHz). Even a short (15-30 s) exposure to high-frequency ultrasound significantly enhanced the intracellular DOX uptake from PBS, RPMI 1640, and Pluronic micelles. The mechanisms of the observed effects are discussed.     

PEGylated ethyl cellulose micelles as a nanocarrier for drug delivery

Natural polymers provide a better alternative to synthetic polymers in the domain of drug delivery systems (DDSs) because of their renewability, biocompatibility, and low immunogenicity; therefore, they are being studied for the development of bulk/nanoformulations. Likewise, current methods for engineering natural polymers into micelles are in their infancy, and in-depth studies are required using natural polymers as controlled DDSs. Accordingly, in our present study, a new micellar DDS was synthesized using ethyl cellulose (EC) grafted with polyethylene glycol (PEG); it was characterized, its properties, cell toxicity, and hemocompatibility were evaluated, and its drug release kinetics were demonstrated using doxorubicin (DOX) as a model drug. Briefly, EC was grafted with PEG to form the amphiphilic copolymers EC-PEG1 and EC-PEG2 with varying PEG concentrations, and nano-micelles were prepared with and without the drug (DOX) via a dialysis method; the critical micelle concentrations (CMCs) were recorded to be 0.03 mg mL⁻¹ and 0.00193 mg mL⁻¹ for EC-PEG1 and EC-PEG2, respectively. The physicochemical properties of the respective nano-micelles were evaluated via various characterization techniques. The morphologies of the nano-micelles were analyzed via transmission electron microscopy (TEM), and the average size of the nano-micelles was recorded to be ∼80 nm. In vitro, drug release studies were done for 48 h, where 100% DOX release was recorded at pH 5.5 and 52% DOX release was recorded at pH 7.4 from the micelles. In addition, cytotoxicity studies suggested that DOX-loaded micelles were potent in killing MDA-MB-231 and MCF-7 cancer cells, and the blank micelles were non-toxic toward cancerous and normal cells. A cellular uptake study via fluorescence microscopy indicated the internalization of DOX-loaded micelles by cancer cells, delivering the DOX into the cellular compartments. Based on these studies, we concluded that the developed material should be studied further via in vivo studies to understand its potential as a controlled DDS to treat cancer.

Ethyl cellulose was developed as an amphiphilic polymer by PEGylation and fabricated as nanomicelles for delivery of active molecules. This polymeric system can be used as next generation nano drug delivery system (nanoDDS) for cancer therapy.     

Evaluation of Loading Strategies to Improve Tumor Uptake of Gemcitabine in a Murine Orthotopic Bladder Cancer Model Using Ultrasound and Microbubbles

In this study we compared three different microbubble-based approaches to the delivery of a widely used chemotherapy drug, gemcitabine: (i) co-administration of gemcitabine and microbubbles (Gem+MB); (ii) conjugates of microbubbles and gemcitabine-loaded liposomes (GemlipoMB); and (iii) microbubbles with gemcitabine directly bound to their surfaces (GembioMB). Both in vitro and in vivo investigations were carried out, respectively, in the RT112 bladder cancer cell line and in a murine orthotopic muscle-invasive bladder cancer model. The in vitro (in vivo) ultrasound exposure conditions were a 1 (1.1) MHz centre frequency, 0.07 (1.0) MPa peak negative pressure, 3000 (20,000) cycles and 100 (0.5) Hz pulse repetition frequency. Ultrasound exposure produced no significant increase in drug uptake either in vitro or in vivo compared with the drug-only control for co-administered gemcitabine and microbubbles. In vivo, GemlipoMB prolonged the plasma circulation time of gemcitabine, but only GembioMB produced a statistically significant increase in cleaved caspase 3 expression in the tumor, indicative of gemcitabine-induced apoptosis.     

Encapsulated ultrasound microbubbles: therapeutic application in drug/gene delivery.

Encapsulated gas microbubbles are well known as ultrasound contrast agents for medical ultrasound imaging. Nonetheless, not only do these microbubbles help to image, but they can also be used as drug/gene carriers. The microbubbles as drug/gene carriers have an average size less than that of red blood cells, i.e. they are capable of penetrating even into the small blood capillaries and releasing drug and genes under the action of ultrasound field. The application of ultrasound and microbubbles to targeted drug and gene delivery has been the subject of intense experimental research. Under exposure of sufficiently high-amplitude ultrasound, these targeted microbubbles would rupture, spewing drugs or genes, which are contained in its encapsulating layer, to targeted cells or tissues. Recently, targeting ligands are attached to the surface of the microbubbles (i.e. targeted-microbubbles), which have been widely used in cardiovascular system and tumor diagnosis and therapy. In this paper, the characterization of novel targeted ultrasonic contrast agents or microbubbles and their potential applications in drug delivery or gene therapy are reviewed.     

Ultrasound Imaging of Microbubble Activity during Sonoporation Pulse Sequences

Ultrasound-mediated drug delivery using the mechanical action of oscillating and/or collapsing microbubbles has been studied on many different experimental platforms, both in vitro and in vivo; however, the mechanisms remain to be elucidated. Many groups use sterile, enclosed chambers, such as Opticells and Clinicells, to optimize acoustic parameters in vitro needed for effective drug delivery in vivo, as well as for mechanistic investigation of sonoporation or the use of sound to permeate cell membranes. In these containers, cell monolayers are seeded on one side, and the remainder of the volume is filled with a solution containing microbubbles and a model drug. Ultrasound is then applied to study the effect of different parameters on model drug uptake in cell monolayers. Despite the simplicity of this system, the field has been unable to appropriately address what parameters and microbubble concentrations are most effective at enhancing drug uptake and minimizing cellular toxicity. In this work, a common in vitro sonoporation experimental setup was characterized through quantitative analysis of microbubble-dependent acoustic attenuation in combination with high-frame-rate and high-resolution imaging of bubble activity during sonoporation pulse sequences. The goal was to visualize the effect that ultrasound parameters have on microbubble activity. It was observed that under literature-derived sonoporation conditions (0.1–1 MPa, 20–1000 cycles and 10,000 to 10,000,000 microbubbles/mL), there is strong and non-linear acoustic attenuation, as well as bubble destruction, gas diffusion and bubble motion resulting in spatiotemporal pressure and concentration gradients. Ultimately, it was found that the acoustic conditions in common in vitro sonoporation setups are much more complex and confounding than often assumed.     

Ultrasound-Responsive Cavitation Nuclei for Therapy and Drug Delivery

Therapeutic ultrasound strategies that harness the mechanical activity of cavitation nuclei for beneficial tissue bio-effects are actively under development. The mechanical oscillations of circulating microbubbles, the most widely investigated cavitation nuclei, which may also encapsulate or shield a therapeutic agent in the bloodstream, trigger and promote localized uptake. Oscillating microbubbles can create stresses either on nearby tissue or in surrounding fluid to enhance drug penetration and efficacy in the brain, spinal cord, vasculature, immune system, biofilm or tumors. This review summarizes recent investigations that have elucidated interactions of ultrasound and cavitation nuclei with cells, the treatment of tumors, immunotherapy, the blood–brain and blood–spinal cord barriers, sonothrombolysis, cardiovascular drug delivery and sonobactericide. In particular, an overview of salient ultrasound features, drug delivery vehicles, therapeutic transport routes and pre-clinical and clinical studies is provided. Successful implementation of ultrasound and cavitation nuclei-mediated drug delivery has the potential to change the way drugs are administered systemically, resulting in more effective therapeutics and less-invasive treatments.     

Effects of pore size on in vitro and in vivo anticancer efficacies of mesoporous silica nanoparticles

Mesoporous silica nanoparticles (MSN) have been widely applied for drug delivery systems. To investigate the effects of pore size on anticancer efficacies, MSN with different pore sizes but similar particle sizes and surface charges were synthesized via a microemulsion method. The pore structures of MSN were characterized by transmission electron microscopy (TEM), small-angle X-ray scattering (SAXS), and N₂ adsorption–desorption isotherms. Doxorubicin loaded MSN (DOX/MSN) were prepared and the minimum drug loading capacity was detected in DOX/MSN with a pore size of 2.3 nm (DOX/MSN2). DOX/MSN with a pore size of 8.2 nm (DOX/MSN8) showed a comparable drug loading amount in comparison with ones with a pore size of 5.4 nm (DOX/MSN5). In vitro drug release profiles showed that DOX/MSN5 could release DOX quickly and completely. Compared with DOX/MSN2 and DOX/MSN8, DOX/MSN5 showed the higher cellular uptake and nucleic concentration of DOX in QGY-7703 cells, which led to efficient cell-apoptosis induction and anti-proliferation effect, and thus the optimal in vivo anticancer activities. Taken together, these results highlighted the importance of pore size in anticancer efficacies, which would serve as a guideline in the rational design of MSN for cancer therapy.

MSN with suitable pore sizes achieved an outstanding performance for in vitro and in vivo antitumor efficacies.     

Enhanced intracellular delivery and improved antitumor efficacy of doxorubicin by sterically stabilized liposomes modified with a synthetic RGD mimetic.

While sterically stabilized liposomes (SSL) can passively accumulate into tumor tissue due to the effect of enhanced permeability and retention (EPR), the intracellular uptake of the entrapped anticancer drugs by the tumor cells should be a determinant step for their antitumor activities. Therefore, strategies that can enhance the intracellular uptake of SSL into tumor cells could lead to an improved therapeutic efficacy for the drugs. To check this possibility, RGD-mimetic-modified SSL (RGDm-SSL) were constructed aimed to achieve tumor accumulation as well as enhanced intracellular delivery, and were loaded with doxorubicin (DOX), an anticancer drug. Flow cytometry and confocal microscopy reveal that RGDm-SSL facilitated the DOX uptake into the melanoma cells via integrin-mediated endocytosis. DOX-loaded RGDm-SSL (RGDm-SSL-DOX) displayed higher cytotoxicity on melanoma cells than DOX-loaded SSL (SSL-DOX). Tissue distribution and therapeutic experiments were examined in C57BL/6 mice carrying melanoma B16 tumors. RGDm-SSL-DOX displayed similar DOX accumulation in tumor tissue to that of SSL-DOX but showed significantly lower DOX level in blood and remarkably higher DOX level in spleen than SSL-DOX. Administration of RGDm-SSL-DOX at a dose of 5 mg DOX/kg resulted in effective retardation of tumor growth and prolonged survival times compared with SSL-DOX. These results suggest that RGDm-modified SSL may be a promising intracellular targeting carrier for efficient delivery of chemotherapeutic agents into tumor cells.     

Localized in Vivo Model Drug Delivery with Intravascular Ultrasound and Microbubbles

An intravascular ultrasound (IVUS) and microbubble drug delivery system was evaluated in both ex vivo and in vivo swine vessel models. Microbubbles with the fluorophore DiI embedded in the shell as a model drug were infused into ex vivo swine arteries at a physiologic flow rate (105 mL/min) while a 5-MHz IVUS transducer applied ultrasound. Ultrasound pulse sequences consisted of acoustic radiation force pulses to displace DiI-loaded microbubbles from the vessel lumen to the wall, followed by higher-intensity delivery pulses to release DiI into the vessel wall. Insonation with both the acoustic radiation force pulse and the delivery pulse increased DiI deposition 10-fold compared with deposition with the delivery pulse alone. Localized delivery of DiI was then demonstrated in an in vivo swine model. The theoretical transducer beam width predicted the measured angular extent of delivery to within 11%. These results indicate that low-frequency IVUS catheters are a viable method for achieving localized drug delivery with microbubbles.     

Coupling of drug containing liposomes to microbubbles improves ultrasound triggered drug delivery in mice

Local extravasation and triggered drug delivery by use of ultrasound and microbubbles is a promising strategy to target drugs to their sites of action. In the past we have developed drug loaded microbubbles by coupling drug containing liposomes to the surface of microbubbles. Until now the advantages of this drug loading strategy have only been demonstrated in vitro. Therefore, in this paper, microbubbles with indocyanine green (ICG) containing liposomes at their surface or a mixture of ICG-liposomes and microbubbles was injected intravenously in mice. Immediately after injection the left hind leg was exposed to 1 MHz ultrasound and the ICG deposition was monitored 1, 4 and 7 days post-treatment by in vivo fluorescence imaging. In mice that received the ICG-liposome loaded microbubbles the local ICG deposition was, at each time point, about 2-fold higher than in mice that received ICG-liposomes mixed with microbubbles. We also showed that the perforations in the blood vessels allow the passage of ICG-liposomes up to 5 h after microbubble and ultrasound treatment. An increase in tissue temperature to 41 °C was observed in all ultrasound treated mice. However, ultrasound tissue heating was excluded to cause the local ICG deposition. We concluded that coupling of drug containing liposomes to microbubbles may increase ultrasound mediated drug delivery in vivo.     

Acoustic activation of drug delivery from polymeric micelles: effect of pulsed ultrasound.

The effect of a continuous wave (CW) and pulsed 20-kHz ultrasound on the Doxorubicin (DOX) uptake by HL-60 cells from the phosphate buffered saline solution (PBS) and Pluronic micellar solutions was studied. Both CW and pulsed ultrasound enhanced DOX uptake from PBS and Pluronic micelles. The main factor that effected drug uptake was ultrasound power density; however, with increasing power, the enhanced drug uptake was accompanied by the extensive cell sonolysis. For PBS, no significant effect of duration of the ultrasound pulse or inter-pulse interval on the drug uptake was observed. For Pluronic micelles, the uptake increased with increasing pulse duration in the range 0.1-2 s, overall sonication time being the same. For 2-s pulses, the uptake was close to that under CW ultrasound. There was no significant effect of the duration of the inter-pulse interval on the drug uptake from Pluronic micelles. The data on the effect of pulse duration on drug uptake suggest that the characteristic times of drug release from micelles and drug uptake by the cells are comparable. The results point to two independent mechanisms controlling acoustic activation of drug uptake from Pluronic micelles. Both mechanisms work in concert. The first one is related to the acoustically-triggered drug release from micelles that results in higher concentration of the free drug in the incubation medium. The second mechanism is based on the perturbation of cell membranes that results in the increased uptake of the micellar-encapsulated drug. The intracellular uptake of Pluronic micelles was confirmed by fluorescence microscopy.