Contrast Ultrasound Imaging

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

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

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

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.

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.

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, 2018 “Acoustic 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, 2018 “Acoustic 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 2018 “Tissue-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  2018 “Nucleus-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 2013 “Effects of matrix viscoelasticity on the lateral migration deformation of a deformable drop in a wall bounded shear,” Journal of Fluid Mechanics, 727, 318-345.

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

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

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

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

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

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.

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.

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, 2018 “Acoustic 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 2018 “Tissue-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  2018 “Nucleus-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, 2018 “Acoustic 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  2018 “Nucleus-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 2018 “Tissue-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 2018 “Tissue-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 2018 “Tissue-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.

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.

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 2013 “Effects of matrix viscoelasticity on the lateral migration deformation of a deformable drop in a wall bounded shear,” Journal of Fluid Mechanics, 727, 318-345.

Aliabouzar M, Kumar KN, Sarkar K, 2018 “Acoustic 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 2018 “Tissue-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  2018 “Nucleus-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, 2018 “Acoustic 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 2018 “Tissue-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  2018 “Nucleus-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.

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.

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 2013 “Effects of matrix viscoelasticity on the lateral migration deformation of a deformable drop in a wall bounded shear,” Journal of Fluid Mechanics, 727, 318-345.

Karandish F, Haldar MK, Xia L, Gange KN, Feng L, You S, Choi Y, Sarkar K, Mallik S  2018 “Nucleus-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.

Mobadersany N, Sarkar K 2019 “Acoustic microstreaming near a plane wall due to a pulsating free or coated bubble: velocity, vorticity and closed streamlines,” Journal of Fluid Mechanics, 875 781-806.

Acoustic microstreaming due to an oscillating microbubble, either coated or free, is analytically investigated. The detailed flow field is obtained and the closed streamlines of the ring vortex generated by microstreaming are plotted in both Eulerian and Lagrangian descriptions. Analytical expressions are found for the ring vortex showing that its length depends only on the separation of the microbubble from the wall
and the dependence is linear. The circulation as a scalar measure of the vortex is computed quantitatively identifying its spatial location. The functional dependence of circulation on bubble separation and coating parameters is shown to be similar to that of the shear stress.