Even though ultrasound remains the safest and the most popular (one in every three imaging in the world) means of imaging, its utility is limited due to poor contrast. 20% of the 17 million echocardiography performed in the United States in 2000 were suboptimal, i.e. did not provide definitive diagnosis for coronary heart disease. Microbubbles intravenously injected into patients’ body can enhance the contrast of ultrasound images. A good contrast agent will enable reliable imaging of abnormal blood flows leading to early diagnosis of heart disease, as well as those of the kidney, liver and brain. They can also deliver drugs to tageted tissues. Current methods of contrast agent design and its use in drug delivery are empirical.

In collaboration with Professor Flemming Forsberg (Thomas Jefferson Medical School) we perform in vitro experiments and develop theoretical models to offer a unique and clear pathway for a rigorous methodology to customize contrast agent design for specific tasks and applications. It will be a useful tool to agent developers (e.g. GE Health Care) and scanner manufacturers (e.g. GE or Phillips). Contrast bubbles are stabilized by a thin encapsulating layer ( ~ 4-10nm) of surface active material, such as protein, lipid or surfactant. The encapsulation plays a critical role in the performance of these agents. In 2003 we first proposed and developed interfacial rheological models for the encapsulation, which has since become popular with the contrast agent reserach community. The encapsulation is treated as an interface having zero thickness and an intrinsic surface rheology (surface viscosity and elasticity). The rationale for our model vis-à-vis ones with a thick layer is the anisotropy and inhomogeneity (in thickness direction) in the molecular structure of the encapsulation. Our hypothesis is that a model, if it retains the essential physics, will offer applicability over a wide range of acoustic excitations. We adopted a two-prongted approach of determination and validation. After we determine the model parameters from one set of experiments, we proceed to validate the model using a different set of experiments. The model is validated by comparing its prediction against the scattered nonlinear (sub- and super-harmonic) response measured at higher amplitudes. We have succesfully applied this approach to model commercialy available agents such as Optison® (Mallinckrodt, St Loius, MO) and Definity® (Bristol Meyer-Squibb Imaging, N. Billerica, MA). Our current focus has been further model development, applicatiosn to newer agents and developing underlying physics of other applications such as subharmonic aided noninvasive pressure estimation (SHAPE).


Related Publication

  • Mukherjee S, Sarkar K 2013Effects of matrix viscoelasticity on the lateral migration deformation of a deformable drop in a wall bounded shear,Journal of Fluid Mechanics, 727, 318-345.

    The dynamics of a drop deforming, orienting and moving in a shear flow of aviscoelastic liquid near a wall is numerically investigated using a front-tracking finite-difference method and a semi-analytic theory. The viscoelasticity is modelled usingthe modified FENE-CR constitutive equation. In a Newtonian system, deformation in adrop breaks the reversal symmetry of the system resulting in a migration away fromthe wall. This study shows that the matrix elasticity reduces the migration velocity, thereduction scaling approximately linearly with viscoelasticity (product of the Deborah number De and the ratio of polymer viscosity to total viscosity β). Similar to a Newtonian system, for small Deborah numbers, the dynamics quickly reaches a quasi-steady state where deformation, inclination, as well as migration and slip velocitiesbecome independent of the initial drop–wall separation. They all approximately scaleinversely with the square of the instantaneous separation except for deformation whichscales inversely with the cube of separation. The deformation shows a non-monotonicvariation with increasing viscoelasticity similar to the case of a drop in an unboundedshear and is found to influence little the change in migration. Two competing effectsdue to matrix viscoelasticity on drop migration are identified. The first stems fromthe reduced inclination angle of the drop with increasing viscoelasticity that tries toenhance migration velocity. However, it is overcome by the second effect inhibitingmigration that results from the normal stress differences from the curved streamlinesaround the drop; they are more curved on the side away from the wall comparedwith those in the gap between the wall and the drop, an effect that is also presentfor a rigid particle. A perturbative theory of migration is developed for small ratioof the drop size to its separation from the wall that clearly shows the migrationto be caused by the image stresslet field due to the drop in presence of the wall. The theory delineates the two competing viscoelastic effects, their relative magnitudes, and predicts migration that matches well with the simulation. Using the simulationresults and the stresslet theory, we develop an algebraic expression for the quasi-steadymigration velocity as a function of Ca, De and β. The transient dynamics of themigrating drop is seen to be governed by the finite time needed for development of theviscoelastic stresses. For larger capillary numbers, in both Newtonian and viscoelasticmatrices, a viscous drop fails to reach a quasi-steady state independent of initialdrop–wall separation. Matrix viscoelasticity tends to prevent drop breakup. Drops that† Email address for correspondence: sarkar@gwu.edu

     

     

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  • Mukherjee S, Sarkar K 2013Effects of matrix viscoelasticity on the lateral migration deformation of a deformable drop in a wall bounded shear,Journal of Fluid Mechanics, 727, 318-345.

    The dynamics of a drop deforming, orienting and moving in a shear flow of aviscoelastic liquid near a wall is numerically investigated using a front-tracking finite-difference method and a semi-analytic theory. The viscoelasticity is modelled usingthe modified FENE-CR constitutive equation. In a Newtonian system, deformation in adrop breaks the reversal symmetry of the system resulting in a migration away fromthe wall. This study shows that the matrix elasticity reduces the migration velocity, thereduction scaling approximately linearly with viscoelasticity (product of the Deborah number De and the ratio of polymer viscosity to total viscosity β). Similar to a Newtonian system, for small Deborah numbers, the dynamics quickly reaches a quasi-steady state where deformation, inclination, as well as migration and slip velocitiesbecome independent of the initial drop–wall separation. They all approximately scaleinversely with the square of the instantaneous separation except for deformation whichscales inversely with the cube of separation. The deformation shows a non-monotonicvariation with increasing viscoelasticity similar to the case of a drop in an unboundedshear and is found to influence little the change in migration. Two competing effectsdue to matrix viscoelasticity on drop migration are identified. The first stems fromthe reduced inclination angle of the drop with increasing viscoelasticity that tries toenhance migration velocity. However, it is overcome by the second effect inhibitingmigration that results from the normal stress differences from the curved streamlinesaround the drop; they are more curved on the side away from the wall comparedwith those in the gap between the wall and the drop, an effect that is also presentfor a rigid particle. A perturbative theory of migration is developed for small ratioof the drop size to its separation from the wall that clearly shows the migrationto be caused by the image stresslet field due to the drop in presence of the wall. The theory delineates the two competing viscoelastic effects, their relative magnitudes, and predicts migration that matches well with the simulation. Using the simulationresults and the stresslet theory, we develop an algebraic expression for the quasi-steadymigration velocity as a function of Ca, De and β. The transient dynamics of themigrating drop is seen to be governed by the finite time needed for development of theviscoelastic stresses. For larger capillary numbers, in both Newtonian and viscoelasticmatrices, a viscous drop fails to reach a quasi-steady state independent of initialdrop–wall separation. Matrix viscoelasticity tends to prevent drop breakup. Drops that† Email address for correspondence: sarkar@gwu.edu

     

     

  • Mukherjee S, Sarkar K 2013Effects of matrix viscoelasticity on the lateral migration deformation of a deformable drop in a wall bounded shear,Journal of Fluid Mechanics, 727, 318-345.

    The dynamics of a drop deforming, orienting and moving in a shear flow of aviscoelastic liquid near a wall is numerically investigated using a front-tracking finite-difference method and a semi-analytic theory. The viscoelasticity is modelled usingthe modified FENE-CR constitutive equation. In a Newtonian system, deformation in adrop breaks the reversal symmetry of the system resulting in a migration away fromthe wall. This study shows that the matrix elasticity reduces the migration velocity, thereduction scaling approximately linearly with viscoelasticity (product of the Deborah number De and the ratio of polymer viscosity to total viscosity β). Similar to a Newtonian system, for small Deborah numbers, the dynamics quickly reaches a quasi-steady state where deformation, inclination, as well as migration and slip velocitiesbecome independent of the initial drop–wall separation. They all approximately scaleinversely with the square of the instantaneous separation except for deformation whichscales inversely with the cube of separation. The deformation shows a non-monotonicvariation with increasing viscoelasticity similar to the case of a drop in an unboundedshear and is found to influence little the change in migration. Two competing effectsdue to matrix viscoelasticity on drop migration are identified. The first stems fromthe reduced inclination angle of the drop with increasing viscoelasticity that tries toenhance migration velocity. However, it is overcome by the second effect inhibitingmigration that results from the normal stress differences from the curved streamlinesaround the drop; they are more curved on the side away from the wall comparedwith those in the gap between the wall and the drop, an effect that is also presentfor a rigid particle. A perturbative theory of migration is developed for small ratioof the drop size to its separation from the wall that clearly shows the migrationto be caused by the image stresslet field due to the drop in presence of the wall. The theory delineates the two competing viscoelastic effects, their relative magnitudes, and predicts migration that matches well with the simulation. Using the simulationresults and the stresslet theory, we develop an algebraic expression for the quasi-steadymigration velocity as a function of Ca, De and β. The transient dynamics of themigrating drop is seen to be governed by the finite time needed for development of theviscoelastic stresses. For larger capillary numbers, in both Newtonian and viscoelasticmatrices, a viscous drop fails to reach a quasi-steady state independent of initialdrop–wall separation. Matrix viscoelasticity tends to prevent drop breakup. Drops that† Email address for correspondence: sarkar@gwu.edu

     

     

  • Paul S, Nahire R, Mallik S, Sarkar K 2014 “Encapsulated microbubbles and echogenic liposomes for contrast ultrasound imaging and targeted drug delivery,” Computational Mechanics, 53,413-435.

    Micron- to nanometer-sized ultrasound agents,like encapsulated microbubbles and echogenic liposomes,are being developed for diagnostic imaging and ultra-sound mediated drug/gene delivery. This review providesan overview of the current state of the art of the mathe-matical models of the acoustic behavior of ultrasound con-trast microbubbles. We also present a review of the in vitroexperimental characterization of the acoustic properties ofmicrobubble based contrast agents undertaken in our lab-oratory. The hierarchical two-pronged approach of model-ing contrast agents we developed is demonstrated for a lipidcoated (SonazoidTM)and a polymer shelled (polyD-L-lactic acid) contrast microbubbles. The acoustic and drugrelease properties of the newly developed echogenic lipo-somes are discussed for their use as simultaneous imagingand drug/gene delivery agents. Although echogenicity is con-clusively demonstrated in experiments, its physical mecha-nisms remain uncertain. Addressing questions raised herewill accelerate further development and eventual clinicalapproval of these novel technologies.

  • Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2014 “pH-Triggered Echogenicity and Contents Release from Liposomes,” Molecular Pharamaceutics, 11, 4059-4068.

    Liposomes are representative lipid nanoparticles widelyused for delivering anticancer drugs, DNA fragments, or siRNA to cancercells. Upon targeting, various internal and external triggers have been usedto increase the rate for contents release from the liposomes. Among theinternal triggers, decreased pH within the cellular lysosomes has beensuccessfully used to enhance the rate for releasing contents. However,imparting pH sensitivity to liposomes requires the synthesis of specializedlipids with structures that are substantially modified at a reduced pH.Herein, we report an alternative strategy to render liposomes pH sensitiveby encapsulating a precursor which generates gas bubblesin situinresponse to acidic pH. The disturbance created by the escaping gasbubbles leads to the rapid release of the encapsulated contents from theliposomes. Atomic force microscopic studies indicate that the liposomalstructure is destroyed at a reduced pH. The gas bubbles also render theliposomes echogenic, allowing ultrasound imaging. To demonstrate the applicability of this strategy, we have successfully targeteddoxorubicin-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 releasedfrom these liposomes are further enhanced by the application of continuous wave ultrasound (1 MHz), resulting in substantiallyreduced viability for the pancreatic cancer cells (14%).

  • Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2013 “Polymer Coated Echogenic Lipid Nanoparticles with dual release triggers,” Biomacromolecules, 14, 841-853.

    Although lipid nanoparticles are promising drugdelivery vehicles, passive release of encapsulated contents atthe target site is often slow. Herein, we report contents releasefrom targeted, polymer-coated, echogenic lipid nanoparticles inthe cell cytoplasm by redox trigger and simultaneouslyenhanced by diagnostic frequency ultrasound. The lipidnanoparticles were polymerized on the external leaflet usinga disulfide cross-linker. In the presence of cytosolicconcentrations of glutathione, the lipid nanoparticles released76% of encapsulated contents. Plasma concentrations ofglutathione failed to releasethe encapsulated contents.Application of 3 MHz ultrasound for 2 min simultaneouslywith the reducing agent enhanced the release to 96%. Folicacid conjugated, doxorubicin-loaded nanoparticles showed enhanced uptake and higher cytotoxicity in cancer cells overexpressingthe folate receptor (compared to the control). With further developments, these lipid nanoparticles have the potential to be usedas multimodal nanocarriers for simultaneous targeted drug delivery and ultrasound imaging.

  • Nahire R, Hossain R, Patel R, Paul S, Ambre AH, Meghnani V, Layek B, Katti KS, Gange KN, Leclarc E, Srivastava D K, Sarkar K, Mallik S 2014 “Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells,” Biomaterials, 35, 6482-6497.

    Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack ofstability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles ofamphiphilic polymers) are considerably more stable compared to liposomes; however, they alsodemonstrate 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 torelease 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 amonolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with theanticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. Withfurther improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offeringa triggered release as well as diagnostic ultrasound imaging.

  • Kumar KN, Sarkar K 2015 “Effects of ambient hydrostatic pressure on the material properties of the encapsulation of an ultrasound contrast microbubble,” Journal of the Acoustical Society of America, 138, 624-634.

    Ultrasound contrast microbubbles experience widely varying ambient blood pressure in differentorgans, which can also change due to diseases. Pressure change can alter the material properties ofthe encapsulation of these microbubbles. Here the characteristic rheological parameters of contrastagent Definity are determined by varying the ambient pressure (in a physiologically relevant range0–200 mm Hg). Four different interfacial rheological models are used to characterize the microbub-bles. Effects of gas diffusion under excess ambient pressure are investigated in detail accountingfor size decrease of contrast microbubbles. Definity contrast agent show a change in their interfacialdilatational viscosity (3.6108Ns/m at 0 mm Hg to 4.45108Ns/m at 200 mm Hg) and inter-facial dilatational elasticity (0.86 N/m at 0 mm Hg to 1.06 N/m at 200 mm Hg) with ambient pres-sure increase. The increase results from material consolidation, similar to such enhancement inbulk properties under pressure. The model that accounts for enhancement in material propertieswith increasing ambient pressure matches with experimentally measured subharmonic response asa function of ambient pressure, while assuming constant material parameters does not.

  • Lang X, Porter T, Sarkar K 2015 “Interpreting broadband attenuation measured at different excitation amplitudes to estimate strain-dependent interfacial rheological properties of the encapsulation of lipid-coated mono-disperse microbubbles,” Journal of the Acoustical Society of America, 138, 3994-4003.

    Broadband attenuation of ultrasound measured at different excitation pressures being differentraises a serious theoretical concern, because the underlying assumption of linear and independentpropagation of different frequency components nominally requires attenuation to be independent ofexcitation. Here, this issue is investigated by examining ultrasound attenuation through a monodis-perse lipid-coated microbubble suspension measured at four different acoustic excitation ampli-tudes. The attenuation data are used to determine interfacial rheological properties (surface tension,surface dilatational elasticity, and surface dilatational viscosity) of the encapsulation according tothree different models. Although different models result in similar rheological properties, attenua-tion measured at different excitation levels (4–110 kPa) leads to different values for them; the dila-tation elasticity (0.56 to 0.18 N/m) and viscosity (2.4108to 1.52108Ns/m) both decreasewith increasing pressure. Numerically simulating the scattered response, nonlinear energy transferbetween frequencies are shown to be negligible, thereby demonstrating the linearity in propagationand validating the attenuation analysis. There is a second concern to the characterization arisingfrom shell properties being dependent on excitation amplitude, which is not a proper constitutivevariable. It is resolved by arriving at a strain-dependent rheology for the encapsulation. The limita-tions of the underlying analysis are discussed.

  • Kumar KN, Sarkar K 2016 “Interfacial rheological properties of contrast microbubble Targestar P as a function of ambient pressure,” Ultrasound in Medicine and Biology, 42, 1010-1017.

    In this Technical Note, we determine the interfacial rheological parameters of the encapsulation of thecontrast agent Targestar P using ultrasound attenuation. The characteristic parameters are obtained according totwo interfacial rheological models. The properties—surface dilatational elasticity (0.09 ± 0.01 N/m) and surfacedilatational viscosity (8 ± 0.1E–9 N$s/m)—are found to be of similar magnitude for both models. Contrast micro-bubbles experience different ambient pressure in different organs. We also measure these parameters as functionsof ambient pressure using attenuation measured at different overpressures (0, 100 and 200 mm Hg). For each valueof ambient hydrostatic pressure, we determine the rheological properties, accounting for changes in the size dis-tribution caused by the pressure change. We discuss different models of size distribution change under overpres-sure: pure adiabatic compression or gas exchange with surrounding medium. The dilatational surface elasticityand viscosity are found to increase with increasing ambient pressure.

  • Kumar KN, Mallik S, Sarkar K, 2017 “Role of freeze-drying in the presence of mannitol on the echogenicity of echogenic liposomes,” Journal of the Acoustical Society of America, 142, 3670-3676.

    Echogenic liposomes (ELIPs) are an excellent candidate for ultrasound activated therapeutics andimaging. Although multiple experiments have established their echogenicity, the underlying mech-anism has remained unknown. However, freeze-drying in the presence of mannitol during ELIPpreparation has proved critical to ensuring echogenicity. Here, the role of this key component in thepreparation protocol was investigated by measuring scattering from freshly prepared freeze-driedaqueous solution of mannitol—and a number of other excipients commonly used in lyophiliza-tion—directly dispersed in water without any lipids in the experiment. Mannitol, meso-erythritol,glycine, and glucose that form a highly porous crystalline phase upon freeze-drying generated bub-bles resulting in strong echoes during their dissolution. On the other hand, sucrose, trehalose, andxylitol, which become glassy while freeze-dried, did not. Freeze-dried mannitol and other crystal-line substances, if thawed before being introduced into the scattering volume, did not produce echo-genicity, as they lost their crystallinity in the thawed state. The echogenicity disappeared in adegassed environment. Higher amounts of sugar in the original aqueous solution before freeze-drying resulted in higher echogenicity because of the stronger supersaturation and crystallinity. Thebubbles created by the freeze-dried mannitol in the ELIP formulation play a critical role in makingELIPs echogenic.

  • Xia L, Karandish F, Kumar KN, Froberg J, Kulkarni P, Gange KN, Choi Y, Mallik S, Sarkar K 2017 “Acoustic characterization of echogenic polymersomes prepared from amphiphilic block copolymers,” Ultrasound in Medicine and Biology, 44, 447-457.

    Polymersomes are a class of artificial vesicles prepared from amphiphilic polymers. Like lipid vesicles(liposomes), they too can encapsulate hydrophilic and hydrophobic drug molecules in the aqueous core and thehydrophobic bilayer respectively, but are more stable than liposomes. Although echogenic liposomes have beenwidely investigated for simultaneous ultrasound imaging and controlled drug delivery, the potential of thepolymersomes remains unexplored. We prepared two different echogenic polymersomes from the amphiphilic co-polymers polyethylene glycol–poly-DL-lactic acid (PEG-PLA) and polyethylene glycol–poly-L-lactic acid (PEG-PLLA), incorporating multiple freeze-dry cycles in the synthesis protocol to ensure their echogenicity. We investigatedacoustic behavior with potential applications in biomedical imaging. We characterized the polymeric vesicles acous-tically with three different excitation frequencies of 2.25, 5 and 10 MHz at 500 kPa. The polymersomes exhibitedstrong echogenicity at all three excitation frequencies (about 50- and 25-dB enhancements in fundamental andsubharmonic, respectively, at 5-MHz excitation from 20g/mL polymers in solution). Unlike echogenic lipo-somes, they emitted strong subharmonic responses. The scattering results indicated their potential as contrastagents, which was also confirmed by clinical ultrasound imaging.

  • Aliabouzar M, Kumar KN, Sarkar K, 2018Acoustic vaporization threshold of lipid coated perfluoropentane droplets,Journal of the Acoustical Society of America, 143, 2001-2012.

    Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing micro-bubbles as contrast agentsin situas well as higher stability and the possibility of achieving smallersizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with aperfluoropentane (PFP) core (diameter 400–3000 nm) is acoustically measured as a function of theexcitation frequency in a tubeless setup at room temperature. The changes in scattered responses—fundamental, sub-, and second harmonic—are investigated, a quantitative criterion is used to deter-mine the ADV phenomenon, and findings are discussed. The average threshold obtained using threedifferent scattered components increases with frequency—1.0560.28 MPa at 2.25 MHz,1.8960.57 MPa at 5 MHz, and 2.3460.014 MPa at 10 MHz. The scattered response from vapor-ized droplets was also found to qualitatively match with that from an independently prepared lipid-coated microbubble suspension in magnitude as well as trends above the determined ADV thresh-old value.

  • Aliabouzar M, Kumar KN, Sarkar K, 2018Acoustic vaporization threshold of lipid coated perfluoropentane droplets,Journal of the Acoustical Society of America, 143, 2001-2012.

    Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing micro-bubbles as contrast agentsin situas well as higher stability and the possibility of achieving smallersizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with aperfluoropentane (PFP) core (diameter 400–3000 nm) is acoustically measured as a function of theexcitation frequency in a tubeless setup at room temperature. The changes in scattered responses—fundamental, sub-, and second harmonic—are investigated, a quantitative criterion is used to deter-mine the ADV phenomenon, and findings are discussed. The average threshold obtained using threedifferent scattered components increases with frequency—1.0560.28 MPa at 2.25 MHz,1.8960.57 MPa at 5 MHz, and 2.3460.014 MPa at 10 MHz. The scattered response from vapor-ized droplets was also found to qualitatively match with that from an independently prepared lipid-coated microbubble suspension in magnitude as well as trends above the determined ADV thresh-old value.

  • Kulkarni P, Haldar MK, Karandish F, Confeld M, Hossain R, Borowicz P, Gange KN, Xia L, Sarkar K, Mallik S 2018Tissue-penetrating, hypoxia-responsive echogenic polymersomes for drug delivery to solid tumors,Chemistry A European Journal, 24, 12490-12494.

    Hypoxia in solid tumors facilitates the progres-sion of the disease, develops resistance to chemo and radiotherapy, and contributes to relapse. Due to the lack of tumor penetration, most of the reported drug carriers are unable to reach the hypoxic niches of the solid tumors. We have developed tissue-penetrating, hypoxia-responsive echogenic polymersomes to deliver anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-responsive azobenzene conjugated and a tissue penetrating peptide functionalized polylactic acid-polyethylene glycol polymer. The drug-encapsulated, hypoxia-responsive polymersomes substantially decreased the viability of pancreatic cancer cells in spheroidal cultures. Under normoxic conditions, polymersomes were echogenic at diagnostic ultrasound frequencies but lose the echogenicity under hypoxia. In vivo imaging studies with xenograft mouse model further confirmed the ability of the polymersomes to target, penetrate, and deliver the encapsulated contents in hypoxic pancreatic tumor tissues.

  • Karandish F, Haldar MK, Xia L, Gange KN, Feng L, You S, Choi Y, Sarkar K, Mallik S  2018Nucleus-targeted, echogenic polymersomes for delivering a cancer stemness inhibitor to pancreatic cancer cells,Biomacromolecules, 19,4122-4132.

    Chemotherapeutic agents for treating cancers show considerable sideeffects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport andsubsequently release the encapsulated anticancer drugs within the nuclei of pancreaticcancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3−polyethylene glycol (PEG)−polylactic acid (PLA) copolymer employing the Cu2+catalyzed“Click”reaction. We prepared polymersomes from the dexamethasone−PEG−PLA conjugate along with a synthesized stimuli-responsive polymer PEG−S−S−PLA. Thedexamethasone group dilates the nuclear pore complexes and transports the vesicles to thenuclei. We designed the polymersomes to release the encapsulated drugs in the presence ofa high concentration of reducing agents in the nuclei of pancreatic cancer cells. Weobserved that the nucleus-targeted, stimuli-responsive polymersomes released 70% ofencapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulatedthe cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. Thepolymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medicalultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have thepotential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

  • Aliabouzar M, Kumar KN, Sarkar K, 2019 “Effects of size and boiling point of perfluorocarbon droplets on the frequency dependence of vaporization threshold, Journal of the Acoustical Society of America, 145, 1105-1106.

    Phase shift liquid perfluorocarbon (PFC) droplets vaporizable by ultrasound into echogenicmicrobubble above a threshold pressure, termed acoustic droplet vaporization (ADV), are usedfor therapeutic and diagnostic applications. This study systematically investigated the effect ofexcitation frequency (2.25, 10, and 15 MHz) on the ADV and inertial cavitation (IC) thresholds oflipid-coated PFC droplets of three different liquid cores—perfluoropentane (PFP), perfluorohexane(PFH), and perfluorooctyl bromide (PFOB)—and of two different sizes—average diameters smallerthan 3lm and larger than 10lm—in a tubeless setup. This study found that the ADV thresholdincreases with frequency for the lowest boiling point liquid, PFP, for both large and small sizedroplets. For higher boiling point liquids, PFH and PFOB, this study did not detect vaporization forsmall size droplets at the excitation levels (maximum 4 MPa peak negative) studied here. The largePFOB droplets experienced ADV only at the highest excitation frequency 15 MHz. For large PFHdroplets, ADV threshold decreases with frequency that could possibly be due to the superharmonicfocusing being a significant effect at larger sizes and the higher excitation pressures. ADV thresh-olds at all the frequencies studied here occurred at lower rarefactional pressures than IC thresholdsindicating that phase transition precedes inertial cavitation.

  • Aliabouzar M, Kumar KN, Sarkar K, 2019 “Effects of size and boiling point of perfluorocarbon droplets on the frequency dependence of vaporization threshold, Journal of the Acoustical Society of America, 145, 1105-1106.

    Phase shift liquid perfluorocarbon (PFC) droplets vaporizable by ultrasound into echogenicmicrobubble above a threshold pressure, termed acoustic droplet vaporization (ADV), are usedfor therapeutic and diagnostic applications. This study systematically investigated the effect ofexcitation frequency (2.25, 10, and 15 MHz) on the ADV and inertial cavitation (IC) thresholds oflipid-coated PFC droplets of three different liquid cores—perfluoropentane (PFP), perfluorohexane(PFH), and perfluorooctyl bromide (PFOB)—and of two different sizes—average diameters smallerthan 3lm and larger than 10lm—in a tubeless setup. This study found that the ADV thresholdincreases with frequency for the lowest boiling point liquid, PFP, for both large and small sizedroplets. For higher boiling point liquids, PFH and PFOB, this study did not detect vaporization forsmall size droplets at the excitation levels (maximum 4 MPa peak negative) studied here. The largePFOB droplets experienced ADV only at the highest excitation frequency 15 MHz. For large PFHdroplets, ADV threshold decreases with frequency that could possibly be due to the superharmonicfocusing being a significant effect at larger sizes and the higher excitation pressures. ADV thresh-olds at all the frequencies studied here occurred at lower rarefactional pressures than IC thresholdsindicating that phase transition precedes inertial cavitation.

  • Aliabouzar M, Kumar KN, Sarkar K, 2019 “Effects of size and boiling point of perfluorocarbon droplets on the frequency dependence of vaporization threshold, Journal of the Acoustical Society of America, 145, 1105-1106.

    Phase shift liquid perfluorocarbon (PFC) droplets vaporizable by ultrasound into echogenicmicrobubble above a threshold pressure, termed acoustic droplet vaporization (ADV), are usedfor therapeutic and diagnostic applications. This study systematically investigated the effect ofexcitation frequency (2.25, 10, and 15 MHz) on the ADV and inertial cavitation (IC) thresholds oflipid-coated PFC droplets of three different liquid cores—perfluoropentane (PFP), perfluorohexane(PFH), and perfluorooctyl bromide (PFOB)—and of two different sizes—average diameters smallerthan 3lm and larger than 10lm—in a tubeless setup. This study found that the ADV thresholdincreases with frequency for the lowest boiling point liquid, PFP, for both large and small sizedroplets. For higher boiling point liquids, PFH and PFOB, this study did not detect vaporization forsmall size droplets at the excitation levels (maximum 4 MPa peak negative) studied here. The largePFOB droplets experienced ADV only at the highest excitation frequency 15 MHz. For large PFHdroplets, ADV threshold decreases with frequency that could possibly be due to the superharmonicfocusing being a significant effect at larger sizes and the higher excitation pressures. ADV thresh-olds at all the frequencies studied here occurred at lower rarefactional pressures than IC thresholdsindicating that phase transition precedes inertial cavitation.

  • Mukherjee S, Sarkar K 2013Effects of matrix viscoelasticity on the lateral migration deformation of a deformable drop in a wall bounded shear,Journal of Fluid Mechanics, 727, 318-345.

    The dynamics of a drop deforming, orienting and moving in a shear flow of aviscoelastic liquid near a wall is numerically investigated using a front-tracking finite-difference method and a semi-analytic theory. The viscoelasticity is modelled usingthe modified FENE-CR constitutive equation. In a Newtonian system, deformation in adrop breaks the reversal symmetry of the system resulting in a migration away fromthe wall. This study shows that the matrix elasticity reduces the migration velocity, thereduction scaling approximately linearly with viscoelasticity (product of the Deborah number De and the ratio of polymer viscosity to total viscosity β). Similar to a Newtonian system, for small Deborah numbers, the dynamics quickly reaches a quasi-steady state where deformation, inclination, as well as migration and slip velocitiesbecome independent of the initial drop–wall separation. They all approximately scaleinversely with the square of the instantaneous separation except for deformation whichscales inversely with the cube of separation. The deformation shows a non-monotonicvariation with increasing viscoelasticity similar to the case of a drop in an unboundedshear and is found to influence little the change in migration. Two competing effectsdue to matrix viscoelasticity on drop migration are identified. The first stems fromthe reduced inclination angle of the drop with increasing viscoelasticity that tries toenhance migration velocity. However, it is overcome by the second effect inhibitingmigration that results from the normal stress differences from the curved streamlinesaround the drop; they are more curved on the side away from the wall comparedwith those in the gap between the wall and the drop, an effect that is also presentfor a rigid particle. A perturbative theory of migration is developed for small ratioof the drop size to its separation from the wall that clearly shows the migrationto be caused by the image stresslet field due to the drop in presence of the wall. The theory delineates the two competing viscoelastic effects, their relative magnitudes, and predicts migration that matches well with the simulation. Using the simulationresults and the stresslet theory, we develop an algebraic expression for the quasi-steadymigration velocity as a function of Ca, De and β. The transient dynamics of themigrating drop is seen to be governed by the finite time needed for development of theviscoelastic stresses. For larger capillary numbers, in both Newtonian and viscoelasticmatrices, a viscous drop fails to reach a quasi-steady state independent of initialdrop–wall separation. Matrix viscoelasticity tends to prevent drop breakup. Drops that† Email address for correspondence: sarkar@gwu.edu

     

     

  • Mukherjee S, Sarkar K 2013Effects of matrix viscoelasticity on the lateral migration deformation of a deformable drop in a wall bounded shear,Journal of Fluid Mechanics, 727, 318-345.

    The dynamics of a drop deforming, orienting and moving in a shear flow of aviscoelastic liquid near a wall is numerically investigated using a front-tracking finite-difference method and a semi-analytic theory. The viscoelasticity is modelled usingthe modified FENE-CR constitutive equation. In a Newtonian system, deformation in adrop breaks the reversal symmetry of the system resulting in a migration away fromthe wall. This study shows that the matrix elasticity reduces the migration velocity, thereduction scaling approximately linearly with viscoelasticity (product of the Deborah number De and the ratio of polymer viscosity to total viscosity β). Similar to a Newtonian system, for small Deborah numbers, the dynamics quickly reaches a quasi-steady state where deformation, inclination, as well as migration and slip velocitiesbecome independent of the initial drop–wall separation. They all approximately scaleinversely with the square of the instantaneous separation except for deformation whichscales inversely with the cube of separation. The deformation shows a non-monotonicvariation with increasing viscoelasticity similar to the case of a drop in an unboundedshear and is found to influence little the change in migration. Two competing effectsdue to matrix viscoelasticity on drop migration are identified. The first stems fromthe reduced inclination angle of the drop with increasing viscoelasticity that tries toenhance migration velocity. However, it is overcome by the second effect inhibitingmigration that results from the normal stress differences from the curved streamlinesaround the drop; they are more curved on the side away from the wall comparedwith those in the gap between the wall and the drop, an effect that is also presentfor a rigid particle. A perturbative theory of migration is developed for small ratioof the drop size to its separation from the wall that clearly shows the migrationto be caused by the image stresslet field due to the drop in presence of the wall. The theory delineates the two competing viscoelastic effects, their relative magnitudes, and predicts migration that matches well with the simulation. Using the simulationresults and the stresslet theory, we develop an algebraic expression for the quasi-steadymigration velocity as a function of Ca, De and β. The transient dynamics of themigrating drop is seen to be governed by the finite time needed for development of theviscoelastic stresses. For larger capillary numbers, in both Newtonian and viscoelasticmatrices, a viscous drop fails to reach a quasi-steady state independent of initialdrop–wall separation. Matrix viscoelasticity tends to prevent drop breakup. Drops that† Email address for correspondence: sarkar@gwu.edu

     

     

  • Mukherjee S, Sarkar K 2013Effects of matrix viscoelasticity on the lateral migration deformation of a deformable drop in a wall bounded shear,Journal of Fluid Mechanics, 727, 318-345.

    The dynamics of a drop deforming, orienting and moving in a shear flow of aviscoelastic liquid near a wall is numerically investigated using a front-tracking finite-difference method and a semi-analytic theory. The viscoelasticity is modelled usingthe modified FENE-CR constitutive equation. In a Newtonian system, deformation in adrop breaks the reversal symmetry of the system resulting in a migration away fromthe wall. This study shows that the matrix elasticity reduces the migration velocity, thereduction scaling approximately linearly with viscoelasticity (product of the Deborah number De and the ratio of polymer viscosity to total viscosity β). Similar to a Newtonian system, for small Deborah numbers, the dynamics quickly reaches a quasi-steady state where deformation, inclination, as well as migration and slip velocitiesbecome independent of the initial drop–wall separation. They all approximately scaleinversely with the square of the instantaneous separation except for deformation whichscales inversely with the cube of separation. The deformation shows a non-monotonicvariation with increasing viscoelasticity similar to the case of a drop in an unboundedshear and is found to influence little the change in migration. Two competing effectsdue to matrix viscoelasticity on drop migration are identified. The first stems fromthe reduced inclination angle of the drop with increasing viscoelasticity that tries toenhance migration velocity. However, it is overcome by the second effect inhibitingmigration that results from the normal stress differences from the curved streamlinesaround the drop; they are more curved on the side away from the wall comparedwith those in the gap between the wall and the drop, an effect that is also presentfor a rigid particle. A perturbative theory of migration is developed for small ratioof the drop size to its separation from the wall that clearly shows the migrationto be caused by the image stresslet field due to the drop in presence of the wall. The theory delineates the two competing viscoelastic effects, their relative magnitudes, and predicts migration that matches well with the simulation. Using the simulationresults and the stresslet theory, we develop an algebraic expression for the quasi-steadymigration velocity as a function of Ca, De and β. The transient dynamics of themigrating drop is seen to be governed by the finite time needed for development of theviscoelastic stresses. For larger capillary numbers, in both Newtonian and viscoelasticmatrices, a viscous drop fails to reach a quasi-steady state independent of initialdrop–wall separation. Matrix viscoelasticity tends to prevent drop breakup. Drops that† Email address for correspondence: sarkar@gwu.edu

     

     

  • Mukherjee S, Sarkar K 2013Effects of matrix viscoelasticity on the lateral migration deformation of a deformable drop in a wall bounded shear,Journal of Fluid Mechanics, 727, 318-345.

    The dynamics of a drop deforming, orienting and moving in a shear flow of aviscoelastic liquid near a wall is numerically investigated using a front-tracking finite-difference method and a semi-analytic theory. The viscoelasticity is modelled usingthe modified FENE-CR constitutive equation. In a Newtonian system, deformation in adrop breaks the reversal symmetry of the system resulting in a migration away fromthe wall. This study shows that the matrix elasticity reduces the migration velocity, thereduction scaling approximately linearly with viscoelasticity (product of the Deborah number De and the ratio of polymer viscosity to total viscosity β). Similar to a Newtonian system, for small Deborah numbers, the dynamics quickly reaches a quasi-steady state where deformation, inclination, as well as migration and slip velocitiesbecome independent of the initial drop–wall separation. They all approximately scaleinversely with the square of the instantaneous separation except for deformation whichscales inversely with the cube of separation. The deformation shows a non-monotonicvariation with increasing viscoelasticity similar to the case of a drop in an unboundedshear and is found to influence little the change in migration. Two competing effectsdue to matrix viscoelasticity on drop migration are identified. The first stems fromthe reduced inclination angle of the drop with increasing viscoelasticity that tries toenhance migration velocity. However, it is overcome by the second effect inhibitingmigration that results from the normal stress differences from the curved streamlinesaround the drop; they are more curved on the side away from the wall comparedwith those in the gap between the wall and the drop, an effect that is also presentfor a rigid particle. A perturbative theory of migration is developed for small ratioof the drop size to its separation from the wall that clearly shows the migrationto be caused by the image stresslet field due to the drop in presence of the wall. The theory delineates the two competing viscoelastic effects, their relative magnitudes, and predicts migration that matches well with the simulation. Using the simulationresults and the stresslet theory, we develop an algebraic expression for the quasi-steadymigration velocity as a function of Ca, De and β. The transient dynamics of themigrating drop is seen to be governed by the finite time needed for development of theviscoelastic stresses. For larger capillary numbers, in both Newtonian and viscoelasticmatrices, a viscous drop fails to reach a quasi-steady state independent of initialdrop–wall separation. Matrix viscoelasticity tends to prevent drop breakup. Drops that† Email address for correspondence: sarkar@gwu.edu

     

     

  • Mukherjee S, Sarkar K 2013Effects of matrix viscoelasticity on the lateral migration deformation of a deformable drop in a wall bounded shear,Journal of Fluid Mechanics, 727, 318-345.

    The dynamics of a drop deforming, orienting and moving in a shear flow of aviscoelastic liquid near a wall is numerically investigated using a front-tracking finite-difference method and a semi-analytic theory. The viscoelasticity is modelled usingthe modified FENE-CR constitutive equation. In a Newtonian system, deformation in adrop breaks the reversal symmetry of the system resulting in a migration away fromthe wall. This study shows that the matrix elasticity reduces the migration velocity, thereduction scaling approximately linearly with viscoelasticity (product of the Deborah number De and the ratio of polymer viscosity to total viscosity β). Similar to a Newtonian system, for small Deborah numbers, the dynamics quickly reaches a quasi-steady state where deformation, inclination, as well as migration and slip velocitiesbecome independent of the initial drop–wall separation. They all approximately scaleinversely with the square of the instantaneous separation except for deformation whichscales inversely with the cube of separation. The deformation shows a non-monotonicvariation with increasing viscoelasticity similar to the case of a drop in an unboundedshear and is found to influence little the change in migration. Two competing effectsdue to matrix viscoelasticity on drop migration are identified. The first stems fromthe reduced inclination angle of the drop with increasing viscoelasticity that tries toenhance migration velocity. However, it is overcome by the second effect inhibitingmigration that results from the normal stress differences from the curved streamlinesaround the drop; they are more curved on the side away from the wall comparedwith those in the gap between the wall and the drop, an effect that is also presentfor a rigid particle. A perturbative theory of migration is developed for small ratioof the drop size to its separation from the wall that clearly shows the migrationto be caused by the image stresslet field due to the drop in presence of the wall. The theory delineates the two competing viscoelastic effects, their relative magnitudes, and predicts migration that matches well with the simulation. Using the simulationresults and the stresslet theory, we develop an algebraic expression for the quasi-steadymigration velocity as a function of Ca, De and β. The transient dynamics of themigrating drop is seen to be governed by the finite time needed for development of theviscoelastic stresses. For larger capillary numbers, in both Newtonian and viscoelasticmatrices, a viscous drop fails to reach a quasi-steady state independent of initialdrop–wall separation. Matrix viscoelasticity tends to prevent drop breakup. Drops that† Email address for correspondence: sarkar@gwu.edu

     

     

  • Mukherjee S, Sarkar K 2013Effects of matrix viscoelasticity on the lateral migration deformation of a deformable drop in a wall bounded shear,Journal of Fluid Mechanics, 727, 318-345.

    The dynamics of a drop deforming, orienting and moving in a shear flow of aviscoelastic liquid near a wall is numerically investigated using a front-tracking finite-difference method and a semi-analytic theory. The viscoelasticity is modelled usingthe modified FENE-CR constitutive equation. In a Newtonian system, deformation in adrop breaks the reversal symmetry of the system resulting in a migration away fromthe wall. This study shows that the matrix elasticity reduces the migration velocity, thereduction scaling approximately linearly with viscoelasticity (product of the Deborah number De and the ratio of polymer viscosity to total viscosity β). Similar to a Newtonian system, for small Deborah numbers, the dynamics quickly reaches a quasi-steady state where deformation, inclination, as well as migration and slip velocitiesbecome independent of the initial drop–wall separation. They all approximately scaleinversely with the square of the instantaneous separation except for deformation whichscales inversely with the cube of separation. The deformation shows a non-monotonicvariation with increasing viscoelasticity similar to the case of a drop in an unboundedshear and is found to influence little the change in migration. Two competing effectsdue to matrix viscoelasticity on drop migration are identified. The first stems fromthe reduced inclination angle of the drop with increasing viscoelasticity that tries toenhance migration velocity. However, it is overcome by the second effect inhibitingmigration that results from the normal stress differences from the curved streamlinesaround the drop; they are more curved on the side away from the wall comparedwith those in the gap between the wall and the drop, an effect that is also presentfor a rigid particle. A perturbative theory of migration is developed for small ratioof the drop size to its separation from the wall that clearly shows the migrationto be caused by the image stresslet field due to the drop in presence of the wall. The theory delineates the two competing viscoelastic effects, their relative magnitudes, and predicts migration that matches well with the simulation. Using the simulationresults and the stresslet theory, we develop an algebraic expression for the quasi-steadymigration velocity as a function of Ca, De and β. The transient dynamics of themigrating drop is seen to be governed by the finite time needed for development of theviscoelastic stresses. For larger capillary numbers, in both Newtonian and viscoelasticmatrices, a viscous drop fails to reach a quasi-steady state independent of initialdrop–wall separation. Matrix viscoelasticity tends to prevent drop breakup. Drops that† Email address for correspondence: sarkar@gwu.edu

     

     

  • Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2014 “pH-Triggered Echogenicity and Contents Release from Liposomes,” Molecular Pharamaceutics, 11, 4059-4068.

    Liposomes are representative lipid nanoparticles widelyused for delivering anticancer drugs, DNA fragments, or siRNA to cancercells. Upon targeting, various internal and external triggers have been usedto increase the rate for contents release from the liposomes. Among theinternal triggers, decreased pH within the cellular lysosomes has beensuccessfully used to enhance the rate for releasing contents. However,imparting pH sensitivity to liposomes requires the synthesis of specializedlipids with structures that are substantially modified at a reduced pH.Herein, we report an alternative strategy to render liposomes pH sensitiveby encapsulating a precursor which generates gas bubblesin situinresponse to acidic pH. The disturbance created by the escaping gasbubbles leads to the rapid release of the encapsulated contents from theliposomes. Atomic force microscopic studies indicate that the liposomalstructure is destroyed at a reduced pH. The gas bubbles also render theliposomes echogenic, allowing ultrasound imaging. To demonstrate the applicability of this strategy, we have successfully targeteddoxorubicin-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 releasedfrom these liposomes are further enhanced by the application of continuous wave ultrasound (1 MHz), resulting in substantiallyreduced viability for the pancreatic cancer cells (14%).

  • Paul S, Nahire R, Mallik S, Sarkar K 2014 “Encapsulated microbubbles and echogenic liposomes for contrast ultrasound imaging and targeted drug delivery,” Computational Mechanics, 53,413-435.

    Micron- to nanometer-sized ultrasound agents,like encapsulated microbubbles and echogenic liposomes,are being developed for diagnostic imaging and ultra-sound mediated drug/gene delivery. This review providesan overview of the current state of the art of the mathe-matical models of the acoustic behavior of ultrasound con-trast microbubbles. We also present a review of the in vitroexperimental characterization of the acoustic properties ofmicrobubble based contrast agents undertaken in our lab-oratory. The hierarchical two-pronged approach of model-ing contrast agents we developed is demonstrated for a lipidcoated (SonazoidTM)and a polymer shelled (polyD-L-lactic acid) contrast microbubbles. The acoustic and drugrelease properties of the newly developed echogenic lipo-somes are discussed for their use as simultaneous imagingand drug/gene delivery agents. Although echogenicity is con-clusively demonstrated in experiments, its physical mecha-nisms remain uncertain. Addressing questions raised herewill accelerate further development and eventual clinicalapproval of these novel technologies.

  • Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2013 “Polymer Coated Echogenic Lipid Nanoparticles with dual release triggers,” Biomacromolecules, 14, 841-853.

    Although lipid nanoparticles are promising drugdelivery vehicles, passive release of encapsulated contents atthe target site is often slow. Herein, we report contents releasefrom targeted, polymer-coated, echogenic lipid nanoparticles inthe cell cytoplasm by redox trigger and simultaneouslyenhanced by diagnostic frequency ultrasound. The lipidnanoparticles were polymerized on the external leaflet usinga disulfide cross-linker. In the presence of cytosolicconcentrations of glutathione, the lipid nanoparticles released76% of encapsulated contents. Plasma concentrations ofglutathione failed to releasethe encapsulated contents.Application of 3 MHz ultrasound for 2 min simultaneouslywith the reducing agent enhanced the release to 96%. Folicacid conjugated, doxorubicin-loaded nanoparticles showed enhanced uptake and higher cytotoxicity in cancer cells overexpressingthe folate receptor (compared to the control). With further developments, these lipid nanoparticles have the potential to be usedas multimodal nanocarriers for simultaneous targeted drug delivery and ultrasound imaging.

  • Nahire R, Hossain R, Patel R, Paul S, Ambre AH, Meghnani V, Layek B, Katti KS, Gange KN, Leclarc E, Srivastava D K, Sarkar K, Mallik S 2014 “Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells,” Biomaterials, 35, 6482-6497.

    Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack ofstability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles ofamphiphilic polymers) are considerably more stable compared to liposomes; however, they alsodemonstrate 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 torelease 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 amonolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with theanticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. Withfurther improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offeringa triggered release as well as diagnostic ultrasound imaging.

  • Kumar KN, Sarkar K 2015 “Effects of ambient hydrostatic pressure on the material properties of the encapsulation of an ultrasound contrast microbubble,” Journal of the Acoustical Society of America, 138, 624-634.

    Ultrasound contrast microbubbles experience widely varying ambient blood pressure in differentorgans, which can also change due to diseases. Pressure change can alter the material properties ofthe encapsulation of these microbubbles. Here the characteristic rheological parameters of contrastagent Definity are determined by varying the ambient pressure (in a physiologically relevant range0–200 mm Hg). Four different interfacial rheological models are used to characterize the microbub-bles. Effects of gas diffusion under excess ambient pressure are investigated in detail accountingfor size decrease of contrast microbubbles. Definity contrast agent show a change in their interfacialdilatational viscosity (3.6108Ns/m at 0 mm Hg to 4.45108Ns/m at 200 mm Hg) and inter-facial dilatational elasticity (0.86 N/m at 0 mm Hg to 1.06 N/m at 200 mm Hg) with ambient pres-sure increase. The increase results from material consolidation, similar to such enhancement inbulk properties under pressure. The model that accounts for enhancement in material propertieswith increasing ambient pressure matches with experimentally measured subharmonic response asa function of ambient pressure, while assuming constant material parameters does not.

  • Lang X, Porter T, Sarkar K 2015 “Interpreting broadband attenuation measured at different excitation amplitudes to estimate strain-dependent interfacial rheological properties of the encapsulation of lipid-coated mono-disperse microbubbles,” Journal of the Acoustical Society of America, 138, 3994-4003.

    Broadband attenuation of ultrasound measured at different excitation pressures being differentraises a serious theoretical concern, because the underlying assumption of linear and independentpropagation of different frequency components nominally requires attenuation to be independent ofexcitation. Here, this issue is investigated by examining ultrasound attenuation through a monodis-perse lipid-coated microbubble suspension measured at four different acoustic excitation ampli-tudes. The attenuation data are used to determine interfacial rheological properties (surface tension,surface dilatational elasticity, and surface dilatational viscosity) of the encapsulation according tothree different models. Although different models result in similar rheological properties, attenua-tion measured at different excitation levels (4–110 kPa) leads to different values for them; the dila-tation elasticity (0.56 to 0.18 N/m) and viscosity (2.4108to 1.52108Ns/m) both decreasewith increasing pressure. Numerically simulating the scattered response, nonlinear energy transferbetween frequencies are shown to be negligible, thereby demonstrating the linearity in propagationand validating the attenuation analysis. There is a second concern to the characterization arisingfrom shell properties being dependent on excitation amplitude, which is not a proper constitutivevariable. It is resolved by arriving at a strain-dependent rheology for the encapsulation. The limita-tions of the underlying analysis are discussed.

  • Kumar KN, Sarkar K 2016 “Interfacial rheological properties of contrast microbubble Targestar P as a function of ambient pressure,” Ultrasound in Medicine and Biology, 42, 1010-1017.

    In this Technical Note, we determine the interfacial rheological parameters of the encapsulation of thecontrast agent Targestar P using ultrasound attenuation. The characteristic parameters are obtained according totwo interfacial rheological models. The properties—surface dilatational elasticity (0.09 ± 0.01 N/m) and surfacedilatational viscosity (8 ± 0.1E–9 N$s/m)—are found to be of similar magnitude for both models. Contrast micro-bubbles experience different ambient pressure in different organs. We also measure these parameters as functionsof ambient pressure using attenuation measured at different overpressures (0, 100 and 200 mm Hg). For each valueof ambient hydrostatic pressure, we determine the rheological properties, accounting for changes in the size dis-tribution caused by the pressure change. We discuss different models of size distribution change under overpres-sure: pure adiabatic compression or gas exchange with surrounding medium. The dilatational surface elasticityand viscosity are found to increase with increasing ambient pressure.

  • Kumar KN, Mallik S, Sarkar K, 2017 “Role of freeze-drying in the presence of mannitol on the echogenicity of echogenic liposomes,” Journal of the Acoustical Society of America, 142, 3670-3676.

    Echogenic liposomes (ELIPs) are an excellent candidate for ultrasound activated therapeutics andimaging. Although multiple experiments have established their echogenicity, the underlying mech-anism has remained unknown. However, freeze-drying in the presence of mannitol during ELIPpreparation has proved critical to ensuring echogenicity. Here, the role of this key component in thepreparation protocol was investigated by measuring scattering from freshly prepared freeze-driedaqueous solution of mannitol—and a number of other excipients commonly used in lyophiliza-tion—directly dispersed in water without any lipids in the experiment. Mannitol, meso-erythritol,glycine, and glucose that form a highly porous crystalline phase upon freeze-drying generated bub-bles resulting in strong echoes during their dissolution. On the other hand, sucrose, trehalose, andxylitol, which become glassy while freeze-dried, did not. Freeze-dried mannitol and other crystal-line substances, if thawed before being introduced into the scattering volume, did not produce echo-genicity, as they lost their crystallinity in the thawed state. The echogenicity disappeared in adegassed environment. Higher amounts of sugar in the original aqueous solution before freeze-drying resulted in higher echogenicity because of the stronger supersaturation and crystallinity. Thebubbles created by the freeze-dried mannitol in the ELIP formulation play a critical role in makingELIPs echogenic.

  • Xia L, Karandish F, Kumar KN, Froberg J, Kulkarni P, Gange KN, Choi Y, Mallik S, Sarkar K 2017 “Acoustic characterization of echogenic polymersomes prepared from amphiphilic block copolymers,” Ultrasound in Medicine and Biology, 44, 447-457.

    Polymersomes are a class of artificial vesicles prepared from amphiphilic polymers. Like lipid vesicles(liposomes), they too can encapsulate hydrophilic and hydrophobic drug molecules in the aqueous core and thehydrophobic bilayer respectively, but are more stable than liposomes. Although echogenic liposomes have beenwidely investigated for simultaneous ultrasound imaging and controlled drug delivery, the potential of thepolymersomes remains unexplored. We prepared two different echogenic polymersomes from the amphiphilic co-polymers polyethylene glycol–poly-DL-lactic acid (PEG-PLA) and polyethylene glycol–poly-L-lactic acid (PEG-PLLA), incorporating multiple freeze-dry cycles in the synthesis protocol to ensure their echogenicity. We investigatedacoustic behavior with potential applications in biomedical imaging. We characterized the polymeric vesicles acous-tically with three different excitation frequencies of 2.25, 5 and 10 MHz at 500 kPa. The polymersomes exhibitedstrong echogenicity at all three excitation frequencies (about 50- and 25-dB enhancements in fundamental andsubharmonic, respectively, at 5-MHz excitation from 20g/mL polymers in solution). Unlike echogenic lipo-somes, they emitted strong subharmonic responses. The scattering results indicated their potential as contrastagents, which was also confirmed by clinical ultrasound imaging.

  • Aliabouzar M, Kumar KN, Sarkar K, 2018Acoustic vaporization threshold of lipid coated perfluoropentane droplets,Journal of the Acoustical Society of America, 143, 2001-2012.

    Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing micro-bubbles as contrast agentsin situas well as higher stability and the possibility of achieving smallersizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with aperfluoropentane (PFP) core (diameter 400–3000 nm) is acoustically measured as a function of theexcitation frequency in a tubeless setup at room temperature. The changes in scattered responses—fundamental, sub-, and second harmonic—are investigated, a quantitative criterion is used to deter-mine the ADV phenomenon, and findings are discussed. The average threshold obtained using threedifferent scattered components increases with frequency—1.0560.28 MPa at 2.25 MHz,1.8960.57 MPa at 5 MHz, and 2.3460.014 MPa at 10 MHz. The scattered response from vapor-ized droplets was also found to qualitatively match with that from an independently prepared lipid-coated microbubble suspension in magnitude as well as trends above the determined ADV thresh-old value.

  • Kulkarni P, Haldar MK, Karandish F, Confeld M, Hossain R, Borowicz P, Gange KN, Xia L, Sarkar K, Mallik S 2018Tissue-penetrating, hypoxia-responsive echogenic polymersomes for drug delivery to solid tumors,Chemistry A European Journal, 24, 12490-12494.

    Hypoxia in solid tumors facilitates the progres-sion of the disease, develops resistance to chemo and radiotherapy, and contributes to relapse. Due to the lack of tumor penetration, most of the reported drug carriers are unable to reach the hypoxic niches of the solid tumors. We have developed tissue-penetrating, hypoxia-responsive echogenic polymersomes to deliver anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-responsive azobenzene conjugated and a tissue penetrating peptide functionalized polylactic acid-polyethylene glycol polymer. The drug-encapsulated, hypoxia-responsive polymersomes substantially decreased the viability of pancreatic cancer cells in spheroidal cultures. Under normoxic conditions, polymersomes were echogenic at diagnostic ultrasound frequencies but lose the echogenicity under hypoxia. In vivo imaging studies with xenograft mouse model further confirmed the ability of the polymersomes to target, penetrate, and deliver the encapsulated contents in hypoxic pancreatic tumor tissues.

  • Karandish F, Haldar MK, Xia L, Gange KN, Feng L, You S, Choi Y, Sarkar K, Mallik S  2018Nucleus-targeted, echogenic polymersomes for delivering a cancer stemness inhibitor to pancreatic cancer cells,Biomacromolecules, 19,4122-4132.

    Chemotherapeutic agents for treating cancers show considerable sideeffects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport andsubsequently release the encapsulated anticancer drugs within the nuclei of pancreaticcancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3−polyethylene glycol (PEG)−polylactic acid (PLA) copolymer employing the Cu2+catalyzed“Click”reaction. We prepared polymersomes from the dexamethasone−PEG−PLA conjugate along with a synthesized stimuli-responsive polymer PEG−S−S−PLA. Thedexamethasone group dilates the nuclear pore complexes and transports the vesicles to thenuclei. We designed the polymersomes to release the encapsulated drugs in the presence ofa high concentration of reducing agents in the nuclei of pancreatic cancer cells. Weobserved that the nucleus-targeted, stimuli-responsive polymersomes released 70% ofencapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulatedthe cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. Thepolymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medicalultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have thepotential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

  • Aliabouzar M, Kumar KN, Sarkar K, 2019 “Effects of size and boiling point of perfluorocarbon droplets on the frequency dependence of vaporization threshold, Journal of the Acoustical Society of America, 145, 1105-1106.

    Phase shift liquid perfluorocarbon (PFC) droplets vaporizable by ultrasound into echogenicmicrobubble above a threshold pressure, termed acoustic droplet vaporization (ADV), are usedfor therapeutic and diagnostic applications. This study systematically investigated the effect ofexcitation frequency (2.25, 10, and 15 MHz) on the ADV and inertial cavitation (IC) thresholds oflipid-coated PFC droplets of three different liquid cores—perfluoropentane (PFP), perfluorohexane(PFH), and perfluorooctyl bromide (PFOB)—and of two different sizes—average diameters smallerthan 3lm and larger than 10lm—in a tubeless setup. This study found that the ADV thresholdincreases with frequency for the lowest boiling point liquid, PFP, for both large and small sizedroplets. For higher boiling point liquids, PFH and PFOB, this study did not detect vaporization forsmall size droplets at the excitation levels (maximum 4 MPa peak negative) studied here. The largePFOB droplets experienced ADV only at the highest excitation frequency 15 MHz. For large PFHdroplets, ADV threshold decreases with frequency that could possibly be due to the superharmonicfocusing being a significant effect at larger sizes and the higher excitation pressures. ADV thresh-olds at all the frequencies studied here occurred at lower rarefactional pressures than IC thresholdsindicating that phase transition precedes inertial cavitation.

  • Aliabouzar M, Kumar KN, Sarkar K, 2019 “Effects of size and boiling point of perfluorocarbon droplets on the frequency dependence of vaporization threshold, Journal of the Acoustical Society of America, 145, 1105-1106.

    Phase shift liquid perfluorocarbon (PFC) droplets vaporizable by ultrasound into echogenicmicrobubble above a threshold pressure, termed acoustic droplet vaporization (ADV), are usedfor therapeutic and diagnostic applications. This study systematically investigated the effect ofexcitation frequency (2.25, 10, and 15 MHz) on the ADV and inertial cavitation (IC) thresholds oflipid-coated PFC droplets of three different liquid cores—perfluoropentane (PFP), perfluorohexane(PFH), and perfluorooctyl bromide (PFOB)—and of two different sizes—average diameters smallerthan 3lm and larger than 10lm—in a tubeless setup. This study found that the ADV thresholdincreases with frequency for the lowest boiling point liquid, PFP, for both large and small sizedroplets. For higher boiling point liquids, PFH and PFOB, this study did not detect vaporization forsmall size droplets at the excitation levels (maximum 4 MPa peak negative) studied here. The largePFOB droplets experienced ADV only at the highest excitation frequency 15 MHz. For large PFHdroplets, ADV threshold decreases with frequency that could possibly be due to the superharmonicfocusing being a significant effect at larger sizes and the higher excitation pressures. ADV thresh-olds at all the frequencies studied here occurred at lower rarefactional pressures than IC thresholdsindicating that phase transition precedes inertial cavitation.

  • Aliabouzar M, Kumar KN, Sarkar K, 2018Acoustic vaporization threshold of lipid coated perfluoropentane droplets,Journal of the Acoustical Society of America, 143, 2001-2012.

    Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing micro-bubbles as contrast agentsin situas well as higher stability and the possibility of achieving smallersizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with aperfluoropentane (PFP) core (diameter 400–3000 nm) is acoustically measured as a function of theexcitation frequency in a tubeless setup at room temperature. The changes in scattered responses—fundamental, sub-, and second harmonic—are investigated, a quantitative criterion is used to deter-mine the ADV phenomenon, and findings are discussed. The average threshold obtained using threedifferent scattered components increases with frequency—1.0560.28 MPa at 2.25 MHz,1.8960.57 MPa at 5 MHz, and 2.3460.014 MPa at 10 MHz. The scattered response from vapor-ized droplets was also found to qualitatively match with that from an independently prepared lipid-coated microbubble suspension in magnitude as well as trends above the determined ADV thresh-old value.

  • Karandish F, Haldar MK, Xia L, Gange KN, Feng L, You S, Choi Y, Sarkar K, Mallik S  2018Nucleus-targeted, echogenic polymersomes for delivering a cancer stemness inhibitor to pancreatic cancer cells,Biomacromolecules, 19,4122-4132.

    Chemotherapeutic agents for treating cancers show considerable sideeffects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport andsubsequently release the encapsulated anticancer drugs within the nuclei of pancreaticcancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3−polyethylene glycol (PEG)−polylactic acid (PLA) copolymer employing the Cu2+catalyzed“Click”reaction. We prepared polymersomes from the dexamethasone−PEG−PLA conjugate along with a synthesized stimuli-responsive polymer PEG−S−S−PLA. Thedexamethasone group dilates the nuclear pore complexes and transports the vesicles to thenuclei. We designed the polymersomes to release the encapsulated drugs in the presence ofa high concentration of reducing agents in the nuclei of pancreatic cancer cells. Weobserved that the nucleus-targeted, stimuli-responsive polymersomes released 70% ofencapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulatedthe cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. Thepolymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medicalultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have thepotential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

  • Kulkarni P, Haldar MK, Karandish F, Confeld M, Hossain R, Borowicz P, Gange KN, Xia L, Sarkar K, Mallik S 2018Tissue-penetrating, hypoxia-responsive echogenic polymersomes for drug delivery to solid tumors,Chemistry A European Journal, 24, 12490-12494.

    Hypoxia in solid tumors facilitates the progres-sion of the disease, develops resistance to chemo and radiotherapy, and contributes to relapse. Due to the lack of tumor penetration, most of the reported drug carriers are unable to reach the hypoxic niches of the solid tumors. We have developed tissue-penetrating, hypoxia-responsive echogenic polymersomes to deliver anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-responsive azobenzene conjugated and a tissue penetrating peptide functionalized polylactic acid-polyethylene glycol polymer. The drug-encapsulated, hypoxia-responsive polymersomes substantially decreased the viability of pancreatic cancer cells in spheroidal cultures. Under normoxic conditions, polymersomes were echogenic at diagnostic ultrasound frequencies but lose the echogenicity under hypoxia. In vivo imaging studies with xenograft mouse model further confirmed the ability of the polymersomes to target, penetrate, and deliver the encapsulated contents in hypoxic pancreatic tumor tissues.

  • Kulkarni P, Haldar MK, Karandish F, Confeld M, Hossain R, Borowicz P, Gange KN, Xia L, Sarkar K, Mallik S 2018Tissue-penetrating, hypoxia-responsive echogenic polymersomes for drug delivery to solid tumors,Chemistry A European Journal, 24, 12490-12494.

    Hypoxia in solid tumors facilitates the progres-sion of the disease, develops resistance to chemo and radiotherapy, and contributes to relapse. Due to the lack of tumor penetration, most of the reported drug carriers are unable to reach the hypoxic niches of the solid tumors. We have developed tissue-penetrating, hypoxia-responsive echogenic polymersomes to deliver anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-responsive azobenzene conjugated and a tissue penetrating peptide functionalized polylactic acid-polyethylene glycol polymer. The drug-encapsulated, hypoxia-responsive polymersomes substantially decreased the viability of pancreatic cancer cells in spheroidal cultures. Under normoxic conditions, polymersomes were echogenic at diagnostic ultrasound frequencies but lose the echogenicity under hypoxia. In vivo imaging studies with xenograft mouse model further confirmed the ability of the polymersomes to target, penetrate, and deliver the encapsulated contents in hypoxic pancreatic tumor tissues.

  • Kumar KN, Mallik S, Sarkar K, 2017 “Role of freeze-drying in the presence of mannitol on the echogenicity of echogenic liposomes,” Journal of the Acoustical Society of America, 142, 3670-3676.

    Echogenic liposomes (ELIPs) are an excellent candidate for ultrasound activated therapeutics andimaging. Although multiple experiments have established their echogenicity, the underlying mech-anism has remained unknown. However, freeze-drying in the presence of mannitol during ELIPpreparation has proved critical to ensuring echogenicity. Here, the role of this key component in thepreparation protocol was investigated by measuring scattering from freshly prepared freeze-driedaqueous solution of mannitol—and a number of other excipients commonly used in lyophiliza-tion—directly dispersed in water without any lipids in the experiment. Mannitol, meso-erythritol,glycine, and glucose that form a highly porous crystalline phase upon freeze-drying generated bub-bles resulting in strong echoes during their dissolution. On the other hand, sucrose, trehalose, andxylitol, which become glassy while freeze-dried, did not. Freeze-dried mannitol and other crystal-line substances, if thawed before being introduced into the scattering volume, did not produce echo-genicity, as they lost their crystallinity in the thawed state. The echogenicity disappeared in adegassed environment. Higher amounts of sugar in the original aqueous solution before freeze-drying resulted in higher echogenicity because of the stronger supersaturation and crystallinity. Thebubbles created by the freeze-dried mannitol in the ELIP formulation play a critical role in makingELIPs echogenic.

  • Xia L, Karandish F, Kumar KN, Froberg J, Kulkarni P, Gange KN, Choi Y, Mallik S, Sarkar K 2017 “Acoustic characterization of echogenic polymersomes prepared from amphiphilic block copolymers,” Ultrasound in Medicine and Biology, 44, 447-457.

    Polymersomes are a class of artificial vesicles prepared from amphiphilic polymers. Like lipid vesicles(liposomes), they too can encapsulate hydrophilic and hydrophobic drug molecules in the aqueous core and thehydrophobic bilayer respectively, but are more stable than liposomes. Although echogenic liposomes have beenwidely investigated for simultaneous ultrasound imaging and controlled drug delivery, the potential of thepolymersomes remains unexplored. We prepared two different echogenic polymersomes from the amphiphilic co-polymers polyethylene glycol–poly-DL-lactic acid (PEG-PLA) and polyethylene glycol–poly-L-lactic acid (PEG-PLLA), incorporating multiple freeze-dry cycles in the synthesis protocol to ensure their echogenicity. We investigatedacoustic behavior with potential applications in biomedical imaging. We characterized the polymeric vesicles acous-tically with three different excitation frequencies of 2.25, 5 and 10 MHz at 500 kPa. The polymersomes exhibitedstrong echogenicity at all three excitation frequencies (about 50- and 25-dB enhancements in fundamental andsubharmonic, respectively, at 5-MHz excitation from 20g/mL polymers in solution). Unlike echogenic lipo-somes, they emitted strong subharmonic responses. The scattering results indicated their potential as contrastagents, which was also confirmed by clinical ultrasound imaging.

  • Kumar KN, Sarkar K 2016 “Interfacial rheological properties of contrast microbubble Targestar P as a function of ambient pressure,” Ultrasound in Medicine and Biology, 42, 1010-1017.

    In this Technical Note, we determine the interfacial rheological parameters of the encapsulation of thecontrast agent Targestar P using ultrasound attenuation. The characteristic parameters are obtained according totwo interfacial rheological models. The properties—surface dilatational elasticity (0.09 ± 0.01 N/m) and surfacedilatational viscosity (8 ± 0.1E–9 N$s/m)—are found to be of similar magnitude for both models. Contrast micro-bubbles experience different ambient pressure in different organs. We also measure these parameters as functionsof ambient pressure using attenuation measured at different overpressures (0, 100 and 200 mm Hg). For each valueof ambient hydrostatic pressure, we determine the rheological properties, accounting for changes in the size dis-tribution caused by the pressure change. We discuss different models of size distribution change under overpres-sure: pure adiabatic compression or gas exchange with surrounding medium. The dilatational surface elasticityand viscosity are found to increase with increasing ambient pressure.

  • Kumar KN, Sarkar K 2015 “Effects of ambient hydrostatic pressure on the material properties of the encapsulation of an ultrasound contrast microbubble,” Journal of the Acoustical Society of America, 138, 624-634.

    Ultrasound contrast microbubbles experience widely varying ambient blood pressure in differentorgans, which can also change due to diseases. Pressure change can alter the material properties ofthe encapsulation of these microbubbles. Here the characteristic rheological parameters of contrastagent Definity are determined by varying the ambient pressure (in a physiologically relevant range0–200 mm Hg). Four different interfacial rheological models are used to characterize the microbub-bles. Effects of gas diffusion under excess ambient pressure are investigated in detail accountingfor size decrease of contrast microbubbles. Definity contrast agent show a change in their interfacialdilatational viscosity (3.6108Ns/m at 0 mm Hg to 4.45108Ns/m at 200 mm Hg) and inter-facial dilatational elasticity (0.86 N/m at 0 mm Hg to 1.06 N/m at 200 mm Hg) with ambient pres-sure increase. The increase results from material consolidation, similar to such enhancement inbulk properties under pressure. The model that accounts for enhancement in material propertieswith increasing ambient pressure matches with experimentally measured subharmonic response asa function of ambient pressure, while assuming constant material parameters does not.

  • Kumar KN, Sarkar K 2016 “Interfacial rheological properties of contrast microbubble Targestar P as a function of ambient pressure,” Ultrasound in Medicine and Biology, 42, 1010-1017.

    In this Technical Note, we determine the interfacial rheological parameters of the encapsulation of thecontrast agent Targestar P using ultrasound attenuation. The characteristic parameters are obtained according totwo interfacial rheological models. The properties—surface dilatational elasticity (0.09 ± 0.01 N/m) and surfacedilatational viscosity (8 ± 0.1E–9 N$s/m)—are found to be of similar magnitude for both models. Contrast micro-bubbles experience different ambient pressure in different organs. We also measure these parameters as functionsof ambient pressure using attenuation measured at different overpressures (0, 100 and 200 mm Hg). For each valueof ambient hydrostatic pressure, we determine the rheological properties, accounting for changes in the size dis-tribution caused by the pressure change. We discuss different models of size distribution change under overpres-sure: pure adiabatic compression or gas exchange with surrounding medium. The dilatational surface elasticityand viscosity are found to increase with increasing ambient pressure.

  • Kumar KN, Sarkar K 2015 “Effects of ambient hydrostatic pressure on the material properties of the encapsulation of an ultrasound contrast microbubble,” Journal of the Acoustical Society of America, 138, 624-634.

    Ultrasound contrast microbubbles experience widely varying ambient blood pressure in differentorgans, which can also change due to diseases. Pressure change can alter the material properties ofthe encapsulation of these microbubbles. Here the characteristic rheological parameters of contrastagent Definity are determined by varying the ambient pressure (in a physiologically relevant range0–200 mm Hg). Four different interfacial rheological models are used to characterize the microbub-bles. Effects of gas diffusion under excess ambient pressure are investigated in detail accountingfor size decrease of contrast microbubbles. Definity contrast agent show a change in their interfacialdilatational viscosity (3.6108Ns/m at 0 mm Hg to 4.45108Ns/m at 200 mm Hg) and inter-facial dilatational elasticity (0.86 N/m at 0 mm Hg to 1.06 N/m at 200 mm Hg) with ambient pres-sure increase. The increase results from material consolidation, similar to such enhancement inbulk properties under pressure. The model that accounts for enhancement in material propertieswith increasing ambient pressure matches with experimentally measured subharmonic response asa function of ambient pressure, while assuming constant material parameters does not.

  • Kumar KN, Sarkar K 2016 “Interfacial rheological properties of contrast microbubble Targestar P as a function of ambient pressure,” Ultrasound in Medicine and Biology, 42, 1010-1017.

    In this Technical Note, we determine the interfacial rheological parameters of the encapsulation of thecontrast agent Targestar P using ultrasound attenuation. The characteristic parameters are obtained according totwo interfacial rheological models. The properties—surface dilatational elasticity (0.09 ± 0.01 N/m) and surfacedilatational viscosity (8 ± 0.1E–9 N$s/m)—are found to be of similar magnitude for both models. Contrast micro-bubbles experience different ambient pressure in different organs. We also measure these parameters as functionsof ambient pressure using attenuation measured at different overpressures (0, 100 and 200 mm Hg). For each valueof ambient hydrostatic pressure, we determine the rheological properties, accounting for changes in the size dis-tribution caused by the pressure change. We discuss different models of size distribution change under overpres-sure: pure adiabatic compression or gas exchange with surrounding medium. The dilatational surface elasticityand viscosity are found to increase with increasing ambient pressure.

  • Kumar KN, Sarkar K 2015 “Effects of ambient hydrostatic pressure on the material properties of the encapsulation of an ultrasound contrast microbubble,” Journal of the Acoustical Society of America, 138, 624-634.

    Ultrasound contrast microbubbles experience widely varying ambient blood pressure in differentorgans, which can also change due to diseases. Pressure change can alter the material properties ofthe encapsulation of these microbubbles. Here the characteristic rheological parameters of contrastagent Definity are determined by varying the ambient pressure (in a physiologically relevant range0–200 mm Hg). Four different interfacial rheological models are used to characterize the microbub-bles. Effects of gas diffusion under excess ambient pressure are investigated in detail accountingfor size decrease of contrast microbubbles. Definity contrast agent show a change in their interfacialdilatational viscosity (3.6108Ns/m at 0 mm Hg to 4.45108Ns/m at 200 mm Hg) and inter-facial dilatational elasticity (0.86 N/m at 0 mm Hg to 1.06 N/m at 200 mm Hg) with ambient pres-sure increase. The increase results from material consolidation, similar to such enhancement inbulk properties under pressure. The model that accounts for enhancement in material propertieswith increasing ambient pressure matches with experimentally measured subharmonic response asa function of ambient pressure, while assuming constant material parameters does not.

  • Kumar KN, Sarkar K 2015 “Effects of ambient hydrostatic pressure on the material properties of the encapsulation of an ultrasound contrast microbubble,” Journal of the Acoustical Society of America, 138, 624-634.

    Ultrasound contrast microbubbles experience widely varying ambient blood pressure in differentorgans, which can also change due to diseases. Pressure change can alter the material properties ofthe encapsulation of these microbubbles. Here the characteristic rheological parameters of contrastagent Definity are determined by varying the ambient pressure (in a physiologically relevant range0–200 mm Hg). Four different interfacial rheological models are used to characterize the microbub-bles. Effects of gas diffusion under excess ambient pressure are investigated in detail accountingfor size decrease of contrast microbubbles. Definity contrast agent show a change in their interfacialdilatational viscosity (3.6108Ns/m at 0 mm Hg to 4.45108Ns/m at 200 mm Hg) and inter-facial dilatational elasticity (0.86 N/m at 0 mm Hg to 1.06 N/m at 200 mm Hg) with ambient pres-sure increase. The increase results from material consolidation, similar to such enhancement inbulk properties under pressure. The model that accounts for enhancement in material propertieswith increasing ambient pressure matches with experimentally measured subharmonic response asa function of ambient pressure, while assuming constant material parameters does not.

  • Lang X, Porter T, Sarkar K 2015 “Interpreting broadband attenuation measured at different excitation amplitudes to estimate strain-dependent interfacial rheological properties of the encapsulation of lipid-coated mono-disperse microbubbles,” Journal of the Acoustical Society of America, 138, 3994-4003.

    Broadband attenuation of ultrasound measured at different excitation pressures being differentraises a serious theoretical concern, because the underlying assumption of linear and independentpropagation of different frequency components nominally requires attenuation to be independent ofexcitation. Here, this issue is investigated by examining ultrasound attenuation through a monodis-perse lipid-coated microbubble suspension measured at four different acoustic excitation ampli-tudes. The attenuation data are used to determine interfacial rheological properties (surface tension,surface dilatational elasticity, and surface dilatational viscosity) of the encapsulation according tothree different models. Although different models result in similar rheological properties, attenua-tion measured at different excitation levels (4–110 kPa) leads to different values for them; the dila-tation elasticity (0.56 to 0.18 N/m) and viscosity (2.4108to 1.52108Ns/m) both decreasewith increasing pressure. Numerically simulating the scattered response, nonlinear energy transferbetween frequencies are shown to be negligible, thereby demonstrating the linearity in propagationand validating the attenuation analysis. There is a second concern to the characterization arisingfrom shell properties being dependent on excitation amplitude, which is not a proper constitutivevariable. It is resolved by arriving at a strain-dependent rheology for the encapsulation. The limita-tions of the underlying analysis are discussed.

  • Kulkarni P, Haldar MK, Karandish F, Confeld M, Hossain R, Borowicz P, Gange KN, Xia L, Sarkar K, Mallik S 2018Tissue-penetrating, hypoxia-responsive echogenic polymersomes for drug delivery to solid tumors,Chemistry A European Journal, 24, 12490-12494.

    Hypoxia in solid tumors facilitates the progres-sion of the disease, develops resistance to chemo and radiotherapy, and contributes to relapse. Due to the lack of tumor penetration, most of the reported drug carriers are unable to reach the hypoxic niches of the solid tumors. We have developed tissue-penetrating, hypoxia-responsive echogenic polymersomes to deliver anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-responsive azobenzene conjugated and a tissue penetrating peptide functionalized polylactic acid-polyethylene glycol polymer. The drug-encapsulated, hypoxia-responsive polymersomes substantially decreased the viability of pancreatic cancer cells in spheroidal cultures. Under normoxic conditions, polymersomes were echogenic at diagnostic ultrasound frequencies but lose the echogenicity under hypoxia. In vivo imaging studies with xenograft mouse model further confirmed the ability of the polymersomes to target, penetrate, and deliver the encapsulated contents in hypoxic pancreatic tumor tissues.

  • Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2014 “pH-Triggered Echogenicity and Contents Release from Liposomes,” Molecular Pharamaceutics, 11, 4059-4068.

    Liposomes are representative lipid nanoparticles widelyused for delivering anticancer drugs, DNA fragments, or siRNA to cancercells. Upon targeting, various internal and external triggers have been usedto increase the rate for contents release from the liposomes. Among theinternal triggers, decreased pH within the cellular lysosomes has beensuccessfully used to enhance the rate for releasing contents. However,imparting pH sensitivity to liposomes requires the synthesis of specializedlipids with structures that are substantially modified at a reduced pH.Herein, we report an alternative strategy to render liposomes pH sensitiveby encapsulating a precursor which generates gas bubblesin situinresponse to acidic pH. The disturbance created by the escaping gasbubbles leads to the rapid release of the encapsulated contents from theliposomes. Atomic force microscopic studies indicate that the liposomalstructure is destroyed at a reduced pH. The gas bubbles also render theliposomes echogenic, allowing ultrasound imaging. To demonstrate the applicability of this strategy, we have successfully targeteddoxorubicin-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 releasedfrom these liposomes are further enhanced by the application of continuous wave ultrasound (1 MHz), resulting in substantiallyreduced viability for the pancreatic cancer cells (14%).

  • Nahire R, Hossain R, Patel R, Paul S, Ambre AH, Meghnani V, Layek B, Katti KS, Gange KN, Leclarc E, Srivastava D K, Sarkar K, Mallik S 2014 “Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells,” Biomaterials, 35, 6482-6497.

    Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack ofstability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles ofamphiphilic polymers) are considerably more stable compared to liposomes; however, they alsodemonstrate 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 torelease 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 amonolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with theanticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. Withfurther improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offeringa triggered release as well as diagnostic ultrasound imaging.

  • Paul S, Nahire R, Mallik S, Sarkar K 2014 “Encapsulated microbubbles and echogenic liposomes for contrast ultrasound imaging and targeted drug delivery,” Computational Mechanics, 53,413-435.

    Micron- to nanometer-sized ultrasound agents,like encapsulated microbubbles and echogenic liposomes,are being developed for diagnostic imaging and ultra-sound mediated drug/gene delivery. This review providesan overview of the current state of the art of the mathe-matical models of the acoustic behavior of ultrasound con-trast microbubbles. We also present a review of the in vitroexperimental characterization of the acoustic properties ofmicrobubble based contrast agents undertaken in our lab-oratory. The hierarchical two-pronged approach of model-ing contrast agents we developed is demonstrated for a lipidcoated (SonazoidTM)and a polymer shelled (polyD-L-lactic acid) contrast microbubbles. The acoustic and drugrelease properties of the newly developed echogenic lipo-somes are discussed for their use as simultaneous imagingand drug/gene delivery agents. Although echogenicity is con-clusively demonstrated in experiments, its physical mecha-nisms remain uncertain. Addressing questions raised herewill accelerate further development and eventual clinicalapproval of these novel technologies.

  • Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2013 “Polymer Coated Echogenic Lipid Nanoparticles with dual release triggers,” Biomacromolecules, 14, 841-853.

    Although lipid nanoparticles are promising drugdelivery vehicles, passive release of encapsulated contents atthe target site is often slow. Herein, we report contents releasefrom targeted, polymer-coated, echogenic lipid nanoparticles inthe cell cytoplasm by redox trigger and simultaneouslyenhanced by diagnostic frequency ultrasound. The lipidnanoparticles were polymerized on the external leaflet usinga disulfide cross-linker. In the presence of cytosolicconcentrations of glutathione, the lipid nanoparticles released76% of encapsulated contents. Plasma concentrations ofglutathione failed to releasethe encapsulated contents.Application of 3 MHz ultrasound for 2 min simultaneouslywith the reducing agent enhanced the release to 96%. Folicacid conjugated, doxorubicin-loaded nanoparticles showed enhanced uptake and higher cytotoxicity in cancer cells overexpressingthe folate receptor (compared to the control). With further developments, these lipid nanoparticles have the potential to be usedas multimodal nanocarriers for simultaneous targeted drug delivery and ultrasound imaging.

  • Aliabouzar M, Kumar KN, Sarkar K, 2019 “Effects of size and boiling point of perfluorocarbon droplets on the frequency dependence of vaporization threshold, Journal of the Acoustical Society of America, 145, 1105-1106.

    Phase shift liquid perfluorocarbon (PFC) droplets vaporizable by ultrasound into echogenicmicrobubble above a threshold pressure, termed acoustic droplet vaporization (ADV), are usedfor therapeutic and diagnostic applications. This study systematically investigated the effect ofexcitation frequency (2.25, 10, and 15 MHz) on the ADV and inertial cavitation (IC) thresholds oflipid-coated PFC droplets of three different liquid cores—perfluoropentane (PFP), perfluorohexane(PFH), and perfluorooctyl bromide (PFOB)—and of two different sizes—average diameters smallerthan 3lm and larger than 10lm—in a tubeless setup. This study found that the ADV thresholdincreases with frequency for the lowest boiling point liquid, PFP, for both large and small sizedroplets. For higher boiling point liquids, PFH and PFOB, this study did not detect vaporization forsmall size droplets at the excitation levels (maximum 4 MPa peak negative) studied here. The largePFOB droplets experienced ADV only at the highest excitation frequency 15 MHz. For large PFHdroplets, ADV threshold decreases with frequency that could possibly be due to the superharmonicfocusing being a significant effect at larger sizes and the higher excitation pressures. ADV thresh-olds at all the frequencies studied here occurred at lower rarefactional pressures than IC thresholdsindicating that phase transition precedes inertial cavitation.

  • Aliabouzar M, Kumar KN, Sarkar K, 2019 “Effects of size and boiling point of perfluorocarbon droplets on the frequency dependence of vaporization threshold, Journal of the Acoustical Society of America, 145, 1105-1106.

    Phase shift liquid perfluorocarbon (PFC) droplets vaporizable by ultrasound into echogenicmicrobubble above a threshold pressure, termed acoustic droplet vaporization (ADV), are usedfor therapeutic and diagnostic applications. This study systematically investigated the effect ofexcitation frequency (2.25, 10, and 15 MHz) on the ADV and inertial cavitation (IC) thresholds oflipid-coated PFC droplets of three different liquid cores—perfluoropentane (PFP), perfluorohexane(PFH), and perfluorooctyl bromide (PFOB)—and of two different sizes—average diameters smallerthan 3lm and larger than 10lm—in a tubeless setup. This study found that the ADV thresholdincreases with frequency for the lowest boiling point liquid, PFP, for both large and small sizedroplets. For higher boiling point liquids, PFH and PFOB, this study did not detect vaporization forsmall size droplets at the excitation levels (maximum 4 MPa peak negative) studied here. The largePFOB droplets experienced ADV only at the highest excitation frequency 15 MHz. For large PFHdroplets, ADV threshold decreases with frequency that could possibly be due to the superharmonicfocusing being a significant effect at larger sizes and the higher excitation pressures. ADV thresh-olds at all the frequencies studied here occurred at lower rarefactional pressures than IC thresholdsindicating that phase transition precedes inertial cavitation.

  • Mukherjee S, Sarkar K 2013Effects of matrix viscoelasticity on the lateral migration deformation of a deformable drop in a wall bounded shear,Journal of Fluid Mechanics, 727, 318-345.

    The dynamics of a drop deforming, orienting and moving in a shear flow of aviscoelastic liquid near a wall is numerically investigated using a front-tracking finite-difference method and a semi-analytic theory. The viscoelasticity is modelled usingthe modified FENE-CR constitutive equation. In a Newtonian system, deformation in adrop breaks the reversal symmetry of the system resulting in a migration away fromthe wall. This study shows that the matrix elasticity reduces the migration velocity, thereduction scaling approximately linearly with viscoelasticity (product of the Deborah number De and the ratio of polymer viscosity to total viscosity β). Similar to a Newtonian system, for small Deborah numbers, the dynamics quickly reaches a quasi-steady state where deformation, inclination, as well as migration and slip velocitiesbecome independent of the initial drop–wall separation. They all approximately scaleinversely with the square of the instantaneous separation except for deformation whichscales inversely with the cube of separation. The deformation shows a non-monotonicvariation with increasing viscoelasticity similar to the case of a drop in an unboundedshear and is found to influence little the change in migration. Two competing effectsdue to matrix viscoelasticity on drop migration are identified. The first stems fromthe reduced inclination angle of the drop with increasing viscoelasticity that tries toenhance migration velocity. However, it is overcome by the second effect inhibitingmigration that results from the normal stress differences from the curved streamlinesaround the drop; they are more curved on the side away from the wall comparedwith those in the gap between the wall and the drop, an effect that is also presentfor a rigid particle. A perturbative theory of migration is developed for small ratioof the drop size to its separation from the wall that clearly shows the migrationto be caused by the image stresslet field due to the drop in presence of the wall. The theory delineates the two competing viscoelastic effects, their relative magnitudes, and predicts migration that matches well with the simulation. Using the simulationresults and the stresslet theory, we develop an algebraic expression for the quasi-steadymigration velocity as a function of Ca, De and β. The transient dynamics of themigrating drop is seen to be governed by the finite time needed for development of theviscoelastic stresses. For larger capillary numbers, in both Newtonian and viscoelasticmatrices, a viscous drop fails to reach a quasi-steady state independent of initialdrop–wall separation. Matrix viscoelasticity tends to prevent drop breakup. Drops that† Email address for correspondence: sarkar@gwu.edu

     

     

  • Aliabouzar M, Kumar KN, Sarkar K, 2018Acoustic vaporization threshold of lipid coated perfluoropentane droplets,Journal of the Acoustical Society of America, 143, 2001-2012.

    Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing micro-bubbles as contrast agentsin situas well as higher stability and the possibility of achieving smallersizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with aperfluoropentane (PFP) core (diameter 400–3000 nm) is acoustically measured as a function of theexcitation frequency in a tubeless setup at room temperature. The changes in scattered responses—fundamental, sub-, and second harmonic—are investigated, a quantitative criterion is used to deter-mine the ADV phenomenon, and findings are discussed. The average threshold obtained using threedifferent scattered components increases with frequency—1.0560.28 MPa at 2.25 MHz,1.8960.57 MPa at 5 MHz, and 2.3460.014 MPa at 10 MHz. The scattered response from vapor-ized droplets was also found to qualitatively match with that from an independently prepared lipid-coated microbubble suspension in magnitude as well as trends above the determined ADV thresh-old value.

  • Kulkarni P, Haldar MK, Karandish F, Confeld M, Hossain R, Borowicz P, Gange KN, Xia L, Sarkar K, Mallik S 2018Tissue-penetrating, hypoxia-responsive echogenic polymersomes for drug delivery to solid tumors,Chemistry A European Journal, 24, 12490-12494.

    Hypoxia in solid tumors facilitates the progres-sion of the disease, develops resistance to chemo and radiotherapy, and contributes to relapse. Due to the lack of tumor penetration, most of the reported drug carriers are unable to reach the hypoxic niches of the solid tumors. We have developed tissue-penetrating, hypoxia-responsive echogenic polymersomes to deliver anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-responsive azobenzene conjugated and a tissue penetrating peptide functionalized polylactic acid-polyethylene glycol polymer. The drug-encapsulated, hypoxia-responsive polymersomes substantially decreased the viability of pancreatic cancer cells in spheroidal cultures. Under normoxic conditions, polymersomes were echogenic at diagnostic ultrasound frequencies but lose the echogenicity under hypoxia. In vivo imaging studies with xenograft mouse model further confirmed the ability of the polymersomes to target, penetrate, and deliver the encapsulated contents in hypoxic pancreatic tumor tissues.

  • Karandish F, Haldar MK, Xia L, Gange KN, Feng L, You S, Choi Y, Sarkar K, Mallik S  2018Nucleus-targeted, echogenic polymersomes for delivering a cancer stemness inhibitor to pancreatic cancer cells,Biomacromolecules, 19,4122-4132.

    Chemotherapeutic agents for treating cancers show considerable sideeffects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport andsubsequently release the encapsulated anticancer drugs within the nuclei of pancreaticcancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3−polyethylene glycol (PEG)−polylactic acid (PLA) copolymer employing the Cu2+catalyzed“Click”reaction. We prepared polymersomes from the dexamethasone−PEG−PLA conjugate along with a synthesized stimuli-responsive polymer PEG−S−S−PLA. Thedexamethasone group dilates the nuclear pore complexes and transports the vesicles to thenuclei. We designed the polymersomes to release the encapsulated drugs in the presence ofa high concentration of reducing agents in the nuclei of pancreatic cancer cells. Weobserved that the nucleus-targeted, stimuli-responsive polymersomes released 70% ofencapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulatedthe cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. Thepolymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medicalultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have thepotential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

  • Aliabouzar M, Kumar KN, Sarkar K, 2019 “Effects of size and boiling point of perfluorocarbon droplets on the frequency dependence of vaporization threshold, Journal of the Acoustical Society of America, 145, 1105-1106.

    Phase shift liquid perfluorocarbon (PFC) droplets vaporizable by ultrasound into echogenicmicrobubble above a threshold pressure, termed acoustic droplet vaporization (ADV), are usedfor therapeutic and diagnostic applications. This study systematically investigated the effect ofexcitation frequency (2.25, 10, and 15 MHz) on the ADV and inertial cavitation (IC) thresholds oflipid-coated PFC droplets of three different liquid cores—perfluoropentane (PFP), perfluorohexane(PFH), and perfluorooctyl bromide (PFOB)—and of two different sizes—average diameters smallerthan 3lm and larger than 10lm—in a tubeless setup. This study found that the ADV thresholdincreases with frequency for the lowest boiling point liquid, PFP, for both large and small sizedroplets. For higher boiling point liquids, PFH and PFOB, this study did not detect vaporization forsmall size droplets at the excitation levels (maximum 4 MPa peak negative) studied here. The largePFOB droplets experienced ADV only at the highest excitation frequency 15 MHz. For large PFHdroplets, ADV threshold decreases with frequency that could possibly be due to the superharmonicfocusing being a significant effect at larger sizes and the higher excitation pressures. ADV thresh-olds at all the frequencies studied here occurred at lower rarefactional pressures than IC thresholdsindicating that phase transition precedes inertial cavitation.

  • Aliabouzar M, Kumar KN, Sarkar K, 2018Acoustic vaporization threshold of lipid coated perfluoropentane droplets,Journal of the Acoustical Society of America, 143, 2001-2012.

    Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing micro-bubbles as contrast agentsin situas well as higher stability and the possibility of achieving smallersizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with aperfluoropentane (PFP) core (diameter 400–3000 nm) is acoustically measured as a function of theexcitation frequency in a tubeless setup at room temperature. The changes in scattered responses—fundamental, sub-, and second harmonic—are investigated, a quantitative criterion is used to deter-mine the ADV phenomenon, and findings are discussed. The average threshold obtained using threedifferent scattered components increases with frequency—1.0560.28 MPa at 2.25 MHz,1.8960.57 MPa at 5 MHz, and 2.3460.014 MPa at 10 MHz. The scattered response from vapor-ized droplets was also found to qualitatively match with that from an independently prepared lipid-coated microbubble suspension in magnitude as well as trends above the determined ADV thresh-old value.

  • Kulkarni P, Haldar MK, Karandish F, Confeld M, Hossain R, Borowicz P, Gange KN, Xia L, Sarkar K, Mallik S 2018Tissue-penetrating, hypoxia-responsive echogenic polymersomes for drug delivery to solid tumors,Chemistry A European Journal, 24, 12490-12494.

    Hypoxia in solid tumors facilitates the progres-sion of the disease, develops resistance to chemo and radiotherapy, and contributes to relapse. Due to the lack of tumor penetration, most of the reported drug carriers are unable to reach the hypoxic niches of the solid tumors. We have developed tissue-penetrating, hypoxia-responsive echogenic polymersomes to deliver anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-responsive azobenzene conjugated and a tissue penetrating peptide functionalized polylactic acid-polyethylene glycol polymer. The drug-encapsulated, hypoxia-responsive polymersomes substantially decreased the viability of pancreatic cancer cells in spheroidal cultures. Under normoxic conditions, polymersomes were echogenic at diagnostic ultrasound frequencies but lose the echogenicity under hypoxia. In vivo imaging studies with xenograft mouse model further confirmed the ability of the polymersomes to target, penetrate, and deliver the encapsulated contents in hypoxic pancreatic tumor tissues.

  • Karandish F, Haldar MK, Xia L, Gange KN, Feng L, You S, Choi Y, Sarkar K, Mallik S  2018Nucleus-targeted, echogenic polymersomes for delivering a cancer stemness inhibitor to pancreatic cancer cells,Biomacromolecules, 19,4122-4132.

    Chemotherapeutic agents for treating cancers show considerable sideeffects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport andsubsequently release the encapsulated anticancer drugs within the nuclei of pancreaticcancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3−polyethylene glycol (PEG)−polylactic acid (PLA) copolymer employing the Cu2+catalyzed“Click”reaction. We prepared polymersomes from the dexamethasone−PEG−PLA conjugate along with a synthesized stimuli-responsive polymer PEG−S−S−PLA. Thedexamethasone group dilates the nuclear pore complexes and transports the vesicles to thenuclei. We designed the polymersomes to release the encapsulated drugs in the presence ofa high concentration of reducing agents in the nuclei of pancreatic cancer cells. Weobserved that the nucleus-targeted, stimuli-responsive polymersomes released 70% ofencapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulatedthe cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. Thepolymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medicalultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have thepotential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

  • Kumar KN, Mallik S, Sarkar K, 2017 “Role of freeze-drying in the presence of mannitol on the echogenicity of echogenic liposomes,” Journal of the Acoustical Society of America, 142, 3670-3676.

    Echogenic liposomes (ELIPs) are an excellent candidate for ultrasound activated therapeutics andimaging. Although multiple experiments have established their echogenicity, the underlying mech-anism has remained unknown. However, freeze-drying in the presence of mannitol during ELIPpreparation has proved critical to ensuring echogenicity. Here, the role of this key component in thepreparation protocol was investigated by measuring scattering from freshly prepared freeze-driedaqueous solution of mannitol—and a number of other excipients commonly used in lyophiliza-tion—directly dispersed in water without any lipids in the experiment. Mannitol, meso-erythritol,glycine, and glucose that form a highly porous crystalline phase upon freeze-drying generated bub-bles resulting in strong echoes during their dissolution. On the other hand, sucrose, trehalose, andxylitol, which become glassy while freeze-dried, did not. Freeze-dried mannitol and other crystal-line substances, if thawed before being introduced into the scattering volume, did not produce echo-genicity, as they lost their crystallinity in the thawed state. The echogenicity disappeared in adegassed environment. Higher amounts of sugar in the original aqueous solution before freeze-drying resulted in higher echogenicity because of the stronger supersaturation and crystallinity. Thebubbles created by the freeze-dried mannitol in the ELIP formulation play a critical role in makingELIPs echogenic.

  • Xia L, Karandish F, Kumar KN, Froberg J, Kulkarni P, Gange KN, Choi Y, Mallik S, Sarkar K 2017 “Acoustic characterization of echogenic polymersomes prepared from amphiphilic block copolymers,” Ultrasound in Medicine and Biology, 44, 447-457.

    Polymersomes are a class of artificial vesicles prepared from amphiphilic polymers. Like lipid vesicles(liposomes), they too can encapsulate hydrophilic and hydrophobic drug molecules in the aqueous core and thehydrophobic bilayer respectively, but are more stable than liposomes. Although echogenic liposomes have beenwidely investigated for simultaneous ultrasound imaging and controlled drug delivery, the potential of thepolymersomes remains unexplored. We prepared two different echogenic polymersomes from the amphiphilic co-polymers polyethylene glycol–poly-DL-lactic acid (PEG-PLA) and polyethylene glycol–poly-L-lactic acid (PEG-PLLA), incorporating multiple freeze-dry cycles in the synthesis protocol to ensure their echogenicity. We investigatedacoustic behavior with potential applications in biomedical imaging. We characterized the polymeric vesicles acous-tically with three different excitation frequencies of 2.25, 5 and 10 MHz at 500 kPa. The polymersomes exhibitedstrong echogenicity at all three excitation frequencies (about 50- and 25-dB enhancements in fundamental andsubharmonic, respectively, at 5-MHz excitation from 20g/mL polymers in solution). Unlike echogenic lipo-somes, they emitted strong subharmonic responses. The scattering results indicated their potential as contrastagents, which was also confirmed by clinical ultrasound imaging.

  • Kumar KN, Sarkar K 2016 “Interfacial rheological properties of contrast microbubble Targestar P as a function of ambient pressure,” Ultrasound in Medicine and Biology, 42, 1010-1017.

    In this Technical Note, we determine the interfacial rheological parameters of the encapsulation of thecontrast agent Targestar P using ultrasound attenuation. The characteristic parameters are obtained according totwo interfacial rheological models. The properties—surface dilatational elasticity (0.09 ± 0.01 N/m) and surfacedilatational viscosity (8 ± 0.1E–9 N$s/m)—are found to be of similar magnitude for both models. Contrast micro-bubbles experience different ambient pressure in different organs. We also measure these parameters as functionsof ambient pressure using attenuation measured at different overpressures (0, 100 and 200 mm Hg). For each valueof ambient hydrostatic pressure, we determine the rheological properties, accounting for changes in the size dis-tribution caused by the pressure change. We discuss different models of size distribution change under overpres-sure: pure adiabatic compression or gas exchange with surrounding medium. The dilatational surface elasticityand viscosity are found to increase with increasing ambient pressure.

  • Kumar KN, Sarkar K 2015 “Effects of ambient hydrostatic pressure on the material properties of the encapsulation of an ultrasound contrast microbubble,” Journal of the Acoustical Society of America, 138, 624-634.

    Ultrasound contrast microbubbles experience widely varying ambient blood pressure in differentorgans, which can also change due to diseases. Pressure change can alter the material properties ofthe encapsulation of these microbubbles. Here the characteristic rheological parameters of contrastagent Definity are determined by varying the ambient pressure (in a physiologically relevant range0–200 mm Hg). Four different interfacial rheological models are used to characterize the microbub-bles. Effects of gas diffusion under excess ambient pressure are investigated in detail accountingfor size decrease of contrast microbubbles. Definity contrast agent show a change in their interfacialdilatational viscosity (3.6108Ns/m at 0 mm Hg to 4.45108Ns/m at 200 mm Hg) and inter-facial dilatational elasticity (0.86 N/m at 0 mm Hg to 1.06 N/m at 200 mm Hg) with ambient pres-sure increase. The increase results from material consolidation, similar to such enhancement inbulk properties under pressure. The model that accounts for enhancement in material propertieswith increasing ambient pressure matches with experimentally measured subharmonic response asa function of ambient pressure, while assuming constant material parameters does not.

  • Lang X, Porter T, Sarkar K 2015 “Interpreting broadband attenuation measured at different excitation amplitudes to estimate strain-dependent interfacial rheological properties of the encapsulation of lipid-coated mono-disperse microbubbles,” Journal of the Acoustical Society of America, 138, 3994-4003.

    Broadband attenuation of ultrasound measured at different excitation pressures being differentraises a serious theoretical concern, because the underlying assumption of linear and independentpropagation of different frequency components nominally requires attenuation to be independent ofexcitation. Here, this issue is investigated by examining ultrasound attenuation through a monodis-perse lipid-coated microbubble suspension measured at four different acoustic excitation ampli-tudes. The attenuation data are used to determine interfacial rheological properties (surface tension,surface dilatational elasticity, and surface dilatational viscosity) of the encapsulation according tothree different models. Although different models result in similar rheological properties, attenua-tion measured at different excitation levels (4–110 kPa) leads to different values for them; the dila-tation elasticity (0.56 to 0.18 N/m) and viscosity (2.4108to 1.52108Ns/m) both decreasewith increasing pressure. Numerically simulating the scattered response, nonlinear energy transferbetween frequencies are shown to be negligible, thereby demonstrating the linearity in propagationand validating the attenuation analysis. There is a second concern to the characterization arisingfrom shell properties being dependent on excitation amplitude, which is not a proper constitutivevariable. It is resolved by arriving at a strain-dependent rheology for the encapsulation. The limita-tions of the underlying analysis are discussed.

  • Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2014 “pH-Triggered Echogenicity and Contents Release from Liposomes,” Molecular Pharamaceutics, 11, 4059-4068.

    Liposomes are representative lipid nanoparticles widelyused for delivering anticancer drugs, DNA fragments, or siRNA to cancercells. Upon targeting, various internal and external triggers have been usedto increase the rate for contents release from the liposomes. Among theinternal triggers, decreased pH within the cellular lysosomes has beensuccessfully used to enhance the rate for releasing contents. However,imparting pH sensitivity to liposomes requires the synthesis of specializedlipids with structures that are substantially modified at a reduced pH.Herein, we report an alternative strategy to render liposomes pH sensitiveby encapsulating a precursor which generates gas bubblesin situinresponse to acidic pH. The disturbance created by the escaping gasbubbles leads to the rapid release of the encapsulated contents from theliposomes. Atomic force microscopic studies indicate that the liposomalstructure is destroyed at a reduced pH. The gas bubbles also render theliposomes echogenic, allowing ultrasound imaging. To demonstrate the applicability of this strategy, we have successfully targeteddoxorubicin-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 releasedfrom these liposomes are further enhanced by the application of continuous wave ultrasound (1 MHz), resulting in substantiallyreduced viability for the pancreatic cancer cells (14%).

  • Paul S, Nahire R, Mallik S, Sarkar K 2014 “Encapsulated microbubbles and echogenic liposomes for contrast ultrasound imaging and targeted drug delivery,” Computational Mechanics, 53,413-435.

    Micron- to nanometer-sized ultrasound agents,like encapsulated microbubbles and echogenic liposomes,are being developed for diagnostic imaging and ultra-sound mediated drug/gene delivery. This review providesan overview of the current state of the art of the mathe-matical models of the acoustic behavior of ultrasound con-trast microbubbles. We also present a review of the in vitroexperimental characterization of the acoustic properties ofmicrobubble based contrast agents undertaken in our lab-oratory. The hierarchical two-pronged approach of model-ing contrast agents we developed is demonstrated for a lipidcoated (SonazoidTM)and a polymer shelled (polyD-L-lactic acid) contrast microbubbles. The acoustic and drugrelease properties of the newly developed echogenic lipo-somes are discussed for their use as simultaneous imagingand drug/gene delivery agents. Although echogenicity is con-clusively demonstrated in experiments, its physical mecha-nisms remain uncertain. Addressing questions raised herewill accelerate further development and eventual clinicalapproval of these novel technologies.

  • Nahire R, Hossain R, Patel R, Paul S, Ambre AH, Meghnani V, Layek B, Katti KS, Gange KN, Leclarc E, Srivastava D K, Sarkar K, Mallik S 2014 “Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells,” Biomaterials, 35, 6482-6497.

    Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack ofstability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles ofamphiphilic polymers) are considerably more stable compared to liposomes; however, they alsodemonstrate 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 torelease 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 amonolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with theanticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. Withfurther improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offeringa triggered release as well as diagnostic ultrasound imaging.

  • Mukherjee S, Sarkar K 2013Effects of matrix viscoelasticity on the lateral migration deformation of a deformable drop in a wall bounded shear,Journal of Fluid Mechanics, 727, 318-345.

    The dynamics of a drop deforming, orienting and moving in a shear flow of aviscoelastic liquid near a wall is numerically investigated using a front-tracking finite-difference method and a semi-analytic theory. The viscoelasticity is modelled usingthe modified FENE-CR constitutive equation. In a Newtonian system, deformation in adrop breaks the reversal symmetry of the system resulting in a migration away fromthe wall. This study shows that the matrix elasticity reduces the migration velocity, thereduction scaling approximately linearly with viscoelasticity (product of the Deborah number De and the ratio of polymer viscosity to total viscosity β). Similar to a Newtonian system, for small Deborah numbers, the dynamics quickly reaches a quasi-steady state where deformation, inclination, as well as migration and slip velocitiesbecome independent of the initial drop–wall separation. They all approximately scaleinversely with the square of the instantaneous separation except for deformation whichscales inversely with the cube of separation. The deformation shows a non-monotonicvariation with increasing viscoelasticity similar to the case of a drop in an unboundedshear and is found to influence little the change in migration. Two competing effectsdue to matrix viscoelasticity on drop migration are identified. The first stems fromthe reduced inclination angle of the drop with increasing viscoelasticity that tries toenhance migration velocity. However, it is overcome by the second effect inhibitingmigration that results from the normal stress differences from the curved streamlinesaround the drop; they are more curved on the side away from the wall comparedwith those in the gap between the wall and the drop, an effect that is also presentfor a rigid particle. A perturbative theory of migration is developed for small ratioof the drop size to its separation from the wall that clearly shows the migrationto be caused by the image stresslet field due to the drop in presence of the wall. The theory delineates the two competing viscoelastic effects, their relative magnitudes, and predicts migration that matches well with the simulation. Using the simulationresults and the stresslet theory, we develop an algebraic expression for the quasi-steadymigration velocity as a function of Ca, De and β. The transient dynamics of themigrating drop is seen to be governed by the finite time needed for development of theviscoelastic stresses. For larger capillary numbers, in both Newtonian and viscoelasticmatrices, a viscous drop fails to reach a quasi-steady state independent of initialdrop–wall separation. Matrix viscoelasticity tends to prevent drop breakup. Drops that† Email address for correspondence: sarkar@gwu.edu

     

     

  • Karandish F, Haldar MK, Xia L, Gange KN, Feng L, You S, Choi Y, Sarkar K, Mallik S  2018Nucleus-targeted, echogenic polymersomes for delivering a cancer stemness inhibitor to pancreatic cancer cells,Biomacromolecules, 19,4122-4132.

    Chemotherapeutic agents for treating cancers show considerable sideeffects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport andsubsequently release the encapsulated anticancer drugs within the nuclei of pancreaticcancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3−polyethylene glycol (PEG)−polylactic acid (PLA) copolymer employing the Cu2+catalyzed“Click”reaction. We prepared polymersomes from the dexamethasone−PEG−PLA conjugate along with a synthesized stimuli-responsive polymer PEG−S−S−PLA. Thedexamethasone group dilates the nuclear pore complexes and transports the vesicles to thenuclei. We designed the polymersomes to release the encapsulated drugs in the presence ofa high concentration of reducing agents in the nuclei of pancreatic cancer cells. Weobserved that the nucleus-targeted, stimuli-responsive polymersomes released 70% ofencapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulatedthe cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. Thepolymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medicalultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have thepotential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

  • Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2013 “Polymer Coated Echogenic Lipid Nanoparticles with dual release triggers,” Biomacromolecules, 14, 841-853.

    Although lipid nanoparticles are promising drugdelivery vehicles, passive release of encapsulated contents atthe target site is often slow. Herein, we report contents releasefrom targeted, polymer-coated, echogenic lipid nanoparticles inthe cell cytoplasm by redox trigger and simultaneouslyenhanced by diagnostic frequency ultrasound. The lipidnanoparticles were polymerized on the external leaflet usinga disulfide cross-linker. In the presence of cytosolicconcentrations of glutathione, the lipid nanoparticles released76% of encapsulated contents. Plasma concentrations ofglutathione failed to releasethe encapsulated contents.Application of 3 MHz ultrasound for 2 min simultaneouslywith the reducing agent enhanced the release to 96%. Folicacid conjugated, doxorubicin-loaded nanoparticles showed enhanced uptake and higher cytotoxicity in cancer cells overexpressingthe folate receptor (compared to the control). With further developments, these lipid nanoparticles have the potential to be usedas multimodal nanocarriers for simultaneous targeted drug delivery and ultrasound imaging.

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