pH-triggered echogenicity and contents release from liposomes.

Liposomes are representative lipid nanoparticles widely used for delivering anticancer drugs, DNA fragments, or siRNA to cancer cells. Upon targeting, various internal and external triggers have been used to increase the rate for contents release from the liposomes. Among the internal triggers, decreased pH within the cellular lysosomes has been successfully used to enhance the rate for releasing contents. However, imparting pH sensitivity to liposomes requires the synthesis of specialized lipids with structures that are substantially modified at a reduced pH. Herein, we report an alternative strategy to render liposomes pH sensitive by encapsulating a precursor which generates gas bubbles in situ in response to acidic pH. The disturbance created by the escaping gas bubbles leads to the rapid release of the encapsulated contents from the liposomes. Atomic force microscopic studies indicate that the liposomal structure is destroyed at a reduced pH. The gas bubbles also render the liposomes echogenic, allowing ultrasound imaging. To demonstrate the applicability of this strategy, we have successfully targeted doxorubicin-encapsulated liposomes to the pancreatic ductal carcinoma cells that overexpress the folate receptor on the surface. In response to the decreased pH in the lysosomes, the encapsulated anticancer drug is efficiently released. Contents released from these liposomes are further enhanced by the application of continuous wave ultrasound (1 MHz), resulting in substantially reduced viability for the pancreatic cancer cells (14%).

Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells.

Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack of stability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles of amphiphilic polymers) are considerably more stable compared to liposomes; however, they also demonstrate a slow release for the encapsulated contents, limiting their efficacy as a drug-delivery tool. As a solution, we prepared and characterized echogenic polymersomes, which are programmed to release the encapsulated drugs rapidly when incubated with cytosolic concentrations of glutathione. These vesicles encapsulated air bubbles inside and efficiently reflected diagnostic-frequency ultrasound. Folate-targeted polymersomes showed an enhanced uptake by breast and pancreatic-cancer cells in a monolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with the anticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. With further improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offering a triggered release as well as diagnostic ultrasound imaging.

Encapsulated microbubbles and echogenic liposomes for contrast ultrasound imaging and targeted drug delivery.

Micron- to nanometer-sized ultrasound agents, like encapsulated microbubbles and echogenic liposomes, are being developed for diagnostic imaging and ultrasound mediated drug/gene delivery. This review provides an overview of the current state of the art of the mathematical models of the acoustic behavior of ultrasound contrast microbubbles. We also present a review of the in vitro experimental characterization of the acoustic properties of microbubble based contrast agents undertaken in our laboratory. The hierarchical two-pronged approach of modeling contrast agents we developed is demonstrated for a lipid coated (Sonazoid™) and a polymer shelled (poly D-L-lactic acid) contrast microbubbles. The acoustic and drug release properties of the newly developed echogenic liposomes are discussed for their use as simultaneous imaging and drug/gene delivery agents. Although echogenicity is conclusively demonstrated in experiments, its physical mechanisms remain uncertain. Addressing questions raised here will accelerate further development and eventual clinical approval of these novel technologies.

Determination of the interfacial rheological properties of a poly(DL-lactic acid)-encapsulated contrast agent using in vitro attenuation and scattering.

The stabilizing encapsulation of a microbubble-based ultrasound contrast agent (UCA) critically affects its acoustic properties. Polymers, which behave differently from materials commonly used (i.e., lipids or proteins) for monolayer encapsulation, have the potential for better stability and improved control of encapsulation properties. Air-filled microbubbles coated with poly(DL-lactic acid) (PLA) are characterized here using in vitro acoustic experiments and several models of encapsulation. The interfacial rheological properties of the encapsulation are determined according to each model using attenuation of ultrasound through a suspension of microbubbles. Then the model predictions are compared with scattered non-linear (sub- and second harmonic) responses. For this microbubble population (average diameter, 1.9 µm), the peak in attenuation measurement indicates a weighted-average resonance frequency of 2.5-3 MHz, which, in contrast to other encapsulated microbubbles, is lower than the resonance frequency of a free bubble of similar size (diameter, 1.9 µm). This apparently contradictory result stems from the extremely low surface dilational elasticity (around 0.01-0.07 N/m) and the reduced surface tension of the poly(DL-lactic acid) encapsulation, as well as the polydispersity of the bubble population. All models considered here are shown to behave similarly even in the non-linear regime because of the low surface dilational elasticity value. Pressure-dependent scattering measurements at two different excitation frequencies (2.25 and 3 MHz) revealed strongly non-linear behavior with 25-30 dB and 5-20 dB enhancements in fundamental and second-harmonic responses, respectively, for a contrast agent concentration of 1.33 µg/mL in the suspension. Sub-harmonic responses are registered above a relatively low generation threshold of 100-150 kPa, with up to 20 dB enhancement beyond that pressure. Numerical predictions from all models show good agreement with the experimentally measured fundamental response, but not with the experimental second-harmonic response. The characteristic features of sub-harmonic responses and the steady response beyond the threshold are matched well by model predictions. However, prediction of the threshold value depends on estimated properties and size distribution. The variation in size distribution from sample to sample leads to variation in estimates of encapsulation properties: the lowest estimated value for surface dilational viscosity better predicts the sub-harmonic threshold.

Polymer-coated echogenic lipid nanoparticles with dual release triggers.

Although lipid nanoparticles are promising drug delivery vehicles, passive release of encapsulated contents at the target site is often slow. Herein, we report contents release from targeted, polymer-coated, echogenic lipid nanoparticles in the cell cytoplasm by redox trigger and simultaneously enhanced by diagnostic frequency ultrasound. The lipid nanoparticles were polymerized on the external leaflet using a disulfide cross-linker. In the presence of cytosolic concentrations of glutathione, the lipid nanoparticles released 76% of encapsulated contents. Plasma concentrations of glutathione failed to release the encapsulated contents. Application of 3 MHz ultrasound for 2 min simultaneously with the reducing agent enhanced the release to 96%. Folic acid conjugated, doxorubicin-loaded nanoparticles showed enhanced uptake and higher cytotoxicity in cancer cells overexpressing the folate receptor (compared to the control). With further developments, these lipid nanoparticles have the potential to be used as multimodal nanocarriers for simultaneous targeted drug delivery and ultrasound imaging.

Ultrasound enhanced matrix metalloproteinase-9 triggered release of contents from echogenic liposomes.

The extracellular enzyme matrix metalloproteinase-9 (MMP-9) is overexpressed in atherosclerotic plaques and in metastatic cancers. The enzyme is responsible for rupture of the plaques and for the invasion and metastasis of a large number of cancers. The ability of ultrasonic excitation to induce thermal and mechanical effects has been used to release drugs from different carriers. However, the majority of these studies were performed with low frequency ultrasound (LFUS) at kilohertz frequencies. Clinical usage of LFUS excitations will be limited due to harmful biological effects. Herein, we report our results on the release of encapsulated contents from substrate lipopeptide incorporated echogenic liposomes triggered by recombinant human MMP-9. The contents release was further enhanced by the application of diagnostic frequency (3 MHz) ultrasound. The echogenic liposomes were successfully imaged employing a medical ultrasound transducer (4-15 MHz). The conditioned cell culture media from cancer cells (secreting MMP-9) released the encapsulated dye from the liposomes (30-50%), and this release is also increased (50-80%) by applying diagnostic frequency ultrasound (3 MHz) for 3 min. With further developments, these liposomes have the potential to serve as multimodal carriers for triggered release and simultaneous ultrasound imaging.

In vitro measurement of attenuation and nonlinear scattering from echogenic liposomes.

Echogenic liposomes (ELIP) are an excellent candidate for concurrent imaging and drug delivery applications. They combine the advantages of liposomes-biocompatibility and ability to encapsulate both hydrophobic and hydrophilic drugs-with strong reflections of ultrasound. The objective of this study is to perform a detailed in vitro acoustic characterization - including nonlinear scattering that has not been studied before - along with an investigation of the primary mechanism of echogenicity. Both components are critical for developing viable clinical applications of ELIP. Mannitol, a cryoprotectant, added during the preparation of ELIP is commonly believed to be critical in making them echogenic. Accordingly, here ELIP prepared with varying amount of mannitol concentration are investigated for their pressure dependent linear and non-linear scattered responses. The average diameter of these liposomes is measured to be 125-185nm. But they have a broad size distribution including liposomes with diameters over a micro-meter as observed by TEM and AFM. These larger liposomes are critical for the overall echogenicity. Attenuation through liposomal solution is measured with four different transducers (central frequencies 2.25, 3.5, 5, 10MHz). Measured attenuation increases linearly with liposome concentration indicating absence of acoustic interactions between liposomes. Due to the broad size distribution, the attenuation shows a flat response without a distinct peak in the range of frequencies (1-12MHz) investigated. A 15-20dB enhancement with 1.67 µg/ml of lipids is observed both for the scattered fundamental and the second harmonic responses at 3.5MHz excitation frequency and 50-800kPa amplitude. It demonstrates the efficacy of ELIP for fundamental as well as harmonic ultrasound imaging. The scattered response however does not show any distinct subharmonic peak for the acoustic excitation parameters studied. Small amount of mannitol proves critical for echogenicity. However, mannitol concentration above 100mM shows no effect.

Material characterization of the encapsulation of an ultrasound contrast microbubble and its subharmonic response: strain-softening interfacial elasticity model.

Two nonlinear interfacial elasticity models--interfacial elasticity decreasing linearly and exponentially with area fraction--are developed for the encapsulation of contrast microbubbles. The strain softening (decreasing elasticity) results from the decreasing association between the constitutive molecules of the encapsulation. The models are used to find the characteristic properties (surface tension, interfacial elasticity, interfacial viscosity and nonlinear elasticity parameters) for a commercial contrast agent. Properties are found using the ultrasound attenuation measured through a suspension of contrast agent. Dynamics of the resulting models are simulated, compared with other existing models and discussed. Imposing non-negativity on the effective surface tension (the encapsulation experiences no net compressive stress) shows "compression-only" behavior. The exponential and the quadratic (linearly varying elasticity) models result in similar behaviors. The validity of the models is investigated by comparing their predictions of the scattered nonlinear response for the contrast agent at higher excitations against experimental measurement. All models predict well the scattered fundamental response. The nonlinear strain softening included in the proposed elastic models of the encapsulation improves their ability to predict subharmonic response. They predict the threshold excitation for the initiation of subharmonic response and its subsequent saturation.