Shirshendu Paul

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.

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.

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.

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.

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%).

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.

Acoustic characterization of polymer-encapsulated microbubbles with different shell-thickness-to-radius ratios using in vitro attenuation and scattering: Comparison between different rheological models

Acoustic behaviors of five different polymer (polylactide) encapsulated microbubbles—PB127 (Point Biomedical), PH37, PH43, PH44, PH45 (Phillips Healthcare) with different shell-thickness-to-radius ratios (STRR) of 3.5, 30, 40, 65, and 100 nm/μm have been characterized. In vitro attenuation data were used to determine the interfacial rheological properties of their shells. Use of different models—Newtonian, viscoelastic, strain-softening, Marmottant, and Church—resulted in similar rheological properties. The shell elasticity and shell viscosity were found to increase with increasing shell thickness as expected. The nonlinear scattered response from these microbubbles were measured. Experimentally measured scattered subharmonic response were compared with the model predictions.

Ambient pressure estimation using subharmonic emissions from contrast microbubbles

In cancerous tumors, the interstitial fluid pressure is higher than that in normal tissues, and therefore can be used as a diagnostic marker. Here we are presenting the results of an in vitro study aimed at developing an ultrasound-aided noninvasive pressure estimation technique using contrast agents—Definity®, a lipid coated microbubble, and an experimental poly lactic acid (PLA) microbubbles. Scattered responses from these bubbles have been measured in vitro as a function of ambient pressure using a 3.5 MHz acoustic excitation of varying amplitude. Definity bubbles produced stronger subharmonic than the PLA coated ones, and therefore, are better suited for this application. At an acoustic pressure of 500 kPa, Definity® microbubbles showed a linear decrease in subharmonic signal with increasing ambient pressure, registering a 12 dB reduction at an overpressure of 120 mm Hg. However, at other frequencies, the variation of subharmonic emission with ambient pressure is nonmonotomic as was also predicted by ...

In vitro acoustic characterization of echogenic liposomes with a polymerized lipid bilayer (Pol-ELIPs)

Liposomes are typically lipid bilayer vesicles with an aqueous interior. A modified preparation protocol can make them echogenic, i.e., capable of scattering incident acoustic pulses, by incorporating gas pockets in their structure. Here we study echogenic liposomes with a polymerized lipid bilayer that can potentially improve their stability and nonlinear behavior. The Pol-ELIPs prepared were found to have a very polydisperse size distribution with an average size of 3μm. The frequency dependent attenuation experiment did not show any distinct peak due to the high polydispersity. They showed echogenicity in our in vitro scattering experiments, even without the presence of bovine serum albumin in the reconstituting media. Scattered response measured from Pol-ELIP suspension showed non-linear behaviors with distinct second-harmonic and subharmonic peaks, in contrast to non-polymerize ELIPs that did not show any subharmonic response. Scattered fundamental, second-harmonic and subharmonic responses at a lipid concentration of 1µg/ml showed up to 35, 30, and 35 dB enhancement, respectively. The subharmonic response showed all its characteristic features—its appearance only above a threshold excitation level (150 kPa) and then a sharp rise with a subsequent saturation. The study proves the echogenicity of the novel Pol-ELIPs with interesting nonlinear properties. [Work partially supported by NSF.]Liposomes are typically lipid bilayer vesicles with an aqueous interior. A modified preparation protocol can make them echogenic, i.e., capable of scattering incident acoustic pulses, by incorporating gas pockets in their structure. Here we study echogenic liposomes with a polymerized lipid bilayer that can potentially improve their stability and nonlinear behavior. The Pol-ELIPs prepared were found to have a very polydisperse size distribution with an average size of 3μm. The frequency dependent attenuation experiment did not show any distinct peak due to the high polydispersity. They showed echogenicity in our in vitro scattering experiments, even without the presence of bovine serum albumin in the reconstituting media. Scattered response measured from Pol-ELIP suspension showed non-linear behaviors with distinct second-harmonic and subharmonic peaks, in contrast to non-polymerize ELIPs that did not show any subharmonic response. Scattered fundamental, second-harmonic and subharmonic responses at a lipi...

Acoustic characterization and modeling of poly-lactic acid-encapsulated contrast microbubbles

Biodegradable polymers like polylactic acid hold potential for better stability and control over encapsulation properties of ultrasound contrast microbubbles. We report here several interesting acoustic properties of air-filled PLA shelled microbubbles through both in vitro experiments and mathematical modeling. Attenuation measurements with PLA microbubbles (average diameter 1.9 micrometer), indicated a resonance frequency of 2.5–3 MHz, which, in contrary to other encapsulated microbubbles, is lower than the resonance frequency of a free bubble of similar size. Pressure dependent scattering measurements at two different excitation frequencies (2.25 and 3 MHz) show strongly non-linear behavior with distinct second and subharmonic responses. Subharmonic responses are registered above a relatively low generation threshold of 100–150 kPa. To investigate the underlying mechanisms, we utilized several preexisting interfacial models describing encapsulated bubble dynamics. The attenuation data were utilized to ...