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.
Malipeddy Reddy A, Sarkar K, 2019 “Shear-induced collective diffusivity down a concentration gradient in a viscous emulsion,” Journal of Fluid Mechanics, 868, 5-25.
The shear-induced collective diffusivity down a concentration gradient in a viscous
emulsion is computed using direct numerical simulation. A layer of randomly packed
drops subjected to a shear flow, shows the layer width to increase with the 1=3
power of time, consistent with a semi-dilute theory that assumes a diffusivity linear
with concentration. This characteristic scaling and the underlying theory are used
to compute the collective diffusivity coefficient. This is the first ever computation
of this quantity for a system of deformable particles using fully resolved numerical
simulation. The results match very well with previous experimental observations.
The coefficient of collective diffusivity varies non-monotonically with the capillary
number, due to the competing effects of increasing deformation and drop orientation.
A phenomenological correlation for the collective diffusivity coefficient as a function
of capillary number is presented. We also apply an alternative approach to compute
collective diffusivity, developed originally for a statistically homogeneous rigid sphere
suspension – computing the dynamic structure factor from the simulated droplet
positions and examining its time variation at small wavenumber. We show that
the results from this alternative approach qualitatively agree with our computation
of collective diffusivity including the prediction of the non-monotonic variation of
diffusivity with the capillary number.
Pullan J, Confeld M, Osborn J, Kim J, Sarkar K, Mallik S, 2019 “Exosomes as drug carriers for cancer therapy,” Molecular Pharamaceutics, 16, 1789-1798.
Exosomes, biological extracellular vesicles, have recently begun to find use in targeted drug delivery in solid tumor research. Ranging from 30−120 nm in size, exosomes are secreted from cells and
isolated from bodily fluids. Exosomes provide a unique material platform due to their characteristics, including physical properties such as stability, biocompatibility, permeability, low toxicity, and low immunogenicity—all critical to the success of any nanoparticle drug
delivery system. In addition to traditional chemotherapeutics, natural products and RNA have been encapsulated for the treatment of breast, pancreatic, lung, prostate cancers, and glioblastoma. This review discusses current research on exosomes for drug delivery to solid
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.
Singha S, Malipeddy AR, Zurita-Gotor M, Sarkar K, Shen K, Loewenberg M, Migler KB, Blawzdziewicz J 2019 “Mechanisms of spontaneous chain formation and subsequent microstructural evolution in shear-driven strongly confined drop monolayers,” Soft Matter, 15, 4873-4889.
It was experimentally demonstrated by Migler and his collaborators [Phys. Rev. Lett., 2001, 86, 1023; Langmuir, 2003, 19, 8667] that a strongly confined drop monolayer sheared between two parallel plates can spontaneously develop a flow-oriented drop-chain morphology. Here we show that the formation of the chain-like microstructure is driven by far-field Hele-Shaw quadrupolar interactions between
drops, and that drop spacing within chains is controlled by the effective drop repulsion associated with the existence of confinement-induced reversing streamlines, i.e., the swapping trajectory effect. Using
direct numerical simulations and an accurate quasi-2D model that incorporates quadrupolar and swapping-trajectory contributions, we analyze microstructural evolution in a monodisperse drop
monolayer. Consistent with experimental observations, we find that drop spacing within individual chains is usually uniform. Further analysis shows that at low area fractions all chains have the same spacing, but at higher area fractions there is a large spacing variation from chain to chain. These findings are explained in terms of uncompressed and compressed chains. At low area fractions most chains are
uncompressed (spacing equals lst, which is the stable separation of an isolated pair). At higher area fractions compressed chains (with tighter spacing) are formed in a process of chain zipping along
y-shaped structural defects. We also discuss the relevance of our findings to other shear-driven systems, such as suspensions of spheres in non-Newtonian fluids.
Malipeddy AR, Sarkar K 2019 “Collective diffusivity in a sheared viscous emulsion: effects of viscosity ratio,” Physical Review Fluids, 4, 093603.
The shear-induced collective or gradient diffusivity in an emulsion of viscous drops,
specifically as a function of viscosity ratio, was computed using a fully resolved numerical method. An initially randomly packed layer of viscous drops spreading due to drop-drop interactions in an imposed shear has been simulated. The collective diffusivity coefficient was computed using a self-similar solution of the drop concentration profile. We also obtained the collective diffusivity (the collective diffusivity coefficient multiplied by the average drop volume fraction), computing the dynamic structure factor from the simulated drop positions—an analysis typically applied only to homogeneous systems. The two quantities computed using entirely different methods are in broad agreement, including their predictions of nonmonotonic variations with increasing capillary number and viscosity ratio. The computed values were also found to match with past experimental
measurements. The collective diffusivity coefficient computed here, as expected, is 1 order of magnitude larger than the self-diffusivity coefficient for a dilute emulsion previously computed using pairwise simulation of viscous drops in shear. The collective diffusivity coefficient computed here shows a nonmonotonic variation with viscosity ratio, in contrast to self-diffusivity computed using pairwise computation. The difference might point to an intrinsic difference in physics underlying the two diffusivities. Alternatively, it also might
arise from drops not reaching equilibrium deformation in the period after one interaction and before the next—an effect absent in the pairwise simulation used for the computation of self-diffusivity. We offer a qualitative explanation of the nonmonotonic variation by relating it to average nonmonotonic drop deformation with increasing viscosity ratio. We
also provide empirical correlations of the collective diffusivity as a function of viscosity ratio and capillary number.
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.
Aliabouzar M, Zhang LG, Sarkar K, 2018 “Acoustic characterization of 3D printed PEGDA scaffolds for tissue engineering applications,” Biomedical Materials, 13,055013.
The acoustic and mechanical properties of 3D-printed porous poly-(ethylene glycol)-diacrylate(PEGDA)hydrogel scaffolds were investigated using an ultrasound pulse echo technique on differentscaffold microstructures(solid, hexagonal and square pores). Acoustic parameters such as speed ofsound, acoustic impedance and attenuation coefficient as well as physical parameters such as the porestructure, effective density and elastic moduli were determined. The results show that microstructure(porosity and pore geometry)plays a crucial role in defining properties of 3D-printed scaffolds,achieving the highest attenuation for the scaffold with hexagonal pores and showing a decrease insound speed and elastic moduli with increasing porosity. The properties were also found to be similarto those of soft tissues, making PEGDA scaffolds a suitable candidate for tissue engineeringapplications. To evaluate their cellular performance, adhesion and proliferation of humanmesenchymal stem cells(hMSCs)in these scaffolds were investigated. The porous scaffolds performedbetter than the solid one, recording the highest cell attachment and growth for the scaffold with thesquare pores.
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.
Osborn J, Aliabouzar A, Zhou X, Rao R, Zhang LG, Sarkar K 2018 “Ultrasound and microbubbles enhance osteogenic differentiation of human mesenchymal stem cells on 3D printed scaffolds,” Advanced Biosystems, 2, 1800257.
Lipid-coated microbubbles, clinically approved as contrast enhancing agents for ultrasound imaging, are investigated for the first time for their possible applications in bone tissue engineering. Effects of microbubbles (average diameter 1.1 μm) coated by a mixture of lipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and 1,2-dipalmitoyl-3-trimethylmmonium-propane) in the presence of low intensity pulsed ultrasound (LIPUS) on human mesenchymal stem cells seeded on 3D printed poly(lactic acid) porous scaffolds are investigated. LIPUS stimulation (30 mW cm−2, 1.5 MHz, 20% duty cycle) for 3 min a day with 0.5% v/v microbubbles results in a significant increase in proliferation (up to 19.3%) when compared to control after 1, 3, and 5 d. A 3-week osteogenic differentiation study shows a significant increase in total protein content (up to 27.5%), calcium deposition (up to 4.3%), and alkaline phosphatase activity (up to 43.1%) initiated by LIPUS with and without the presence of microbubbles. The microbubbles are found to remain stable during exposure, and their sustained oscillations demonstrably help focus the LIPUS energy toward enhanced cellular response. Integrating LIPUS and microbubbles promises to be a novel and effective strategy for bone tissue engineering and regeneration therapies.
Miao S, Castro NJ, Nowicki M, Xia L, Cui H, Zhou X, Zhu W, Lee S, Sarkar K, Vozzi G, Tabata Y, Fisher J, Zhang LG 2017 “4D printing of polymeric materials for tissue and organ regeneration,” Materials Today, 20, 577-591.
Four dimensional (4D) printing is an emerging technology with great capacity for fabricating complex,stimuli-responsive 3D structures, providing great potential for tissue and organ engineering applica-tions. Although the 4D concept wasfirst highlighted in 2013, extensive research has rapidly developed,along with more-in-depth understanding and assertions regarding the definition of 4D. In this review,we begin by establishing the criteria of 4D printing, followed by an extensive summary of state-of-the-art technological advances in thefield. Both transformation-preprogrammed 4D printing and 4Dprinting of shape memory polymers are intensively surveyed. Afterwards we will explore and discussthe applications of 4D printing in tissue and organ regeneration, such as developing synthetic tissuesand implantable scaffolds, as well as future perspectives and conclusions.
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, Zhang LG, Sarkar K, 2017 “Effects of scaffold microstructure and low intensity pulsed ultrasound on chondrogenic differentiation of human mesenchymal stem cells,” Biotechnology & Bioengineering, 115, 495-506.
The effects of low intensity pulsed ultrasound (LIPUS) on proliferation andchondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) scaffolds with varying poregeometries (square and hexagonal channels) were investigated. The scaffold withsquare pores resulted in higher hMSC growth and chondrogenic differentiation than asolid or a hexagonally porous scaffold. The optimal LIPUS parameters at 1.5 MHz werefound to be 100 mW/cm2and 20% duty cycle. LIPUS stimulation increasedproliferation by up to 60% after 24 hr. For chondrogenesis, we evaluated key cartilagebiomarkers abundant in cartilage tissue; glycosaminoglycan (GAG), type II collagen andtotal collagen. LIPUS stimulation enhanced GAG synthesis up to 16% and 11% forscaffoldswithsquareandhexagonalpatterns,respectively,after2weeks.Additionally,type II collagen production increased by 60% and 40% for the same patterns,respectively under LIPUS stimulation after 3 weeks. These results suggest that LIPUSstimulation, which has already been approved by FDA for treatment of bone fracture,could be a highly efficient tool for tissue engineering in combination with 3D printingand hMSCs to regenerate damaged cartilage tissues.
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.
Srivastava P, Malipeddi Reddy A, Sarkar K 2016 “Steady shear rheology of a viscous emulsion in the presence of finite inertia at moderate volume fractions: sign reversal of normal stress differences,” Journal of Fluid Mechanics, 85, 494-522.
The shear rheology of an emulsion of viscous drops in the presence of finite inertiais investigated using direct numerical simulation. In the absence of inertia, emulsionsdisplay a non-Newtonian rheology with positive first and negative second normalstress differences. However, recently it was discovered that a small amount ofdrop-level inertia alters their signs – the first normal stress difference becomesnegative and the second one becomes positive, each in a small range of capillarynumbers (Li & Sarkar,J. Rheol., vol. 49, 2005, pp. 1377–1394). Sign reversal wasshown numerically and analytically, but only in the limit of a dilute emulsion wheredrop–drop interactions were neglected. Here, we compute the rheology of a density-and viscosity-matched emulsion, accounting for the interactions in the volume fractionrange of 5 %–27 % and Reynolds number range of 0.1–10. The computed rheologicalproperties (effective shear viscosity and first and second normal stress differences) inthe Stokes limit match well with previous theoretical (Choi–Schowalter in the dilutelimit) and simulated results (for concentrated systems) using the boundary elementmethod. The two distinct components of the rheology arising from the interfacialstresses at the drop surface and the perturbative Reynolds stresses are investigated asfunctions of the drop Reynolds number, capillary number and volume fraction. Thesign change is caused by the increasing drop inclination in the presence of inertia,which in turn directly affects the interfacial stresses. Increase of the volume fractionor capillary number increases the critical Reynolds number for sign reversals due toenhanced alignment of the drops with the flow directions. The effect of increasingthe volume fraction on the rheology is explained by relating it to interactions andspecifically to the contact pair-distribution function computed from the simulation.The excess stresses are seen to show an approximately linear behaviour with theReynolds number in the range of 0.1–5, while with the capillary number and volumefraction, the variation is weakly quadratic.
Zhou X, Castro NJ, Zhu W, Cui H, Aliabouzar M, Sarkar K, Zhang LG 2016 “Improved bone marrow mesenchymal stem cell osteogenesis in 3D bioprinted tissue scaffolds with low intensity pulsed ultrasound stimulation,” Nature Scientific Report, 6, 32876.
3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm2 intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application.
Aliabouzar M, Zhang LG, Sarkar K, 2016 “Lipid-coated microbubbles and low intensity pulsed ultrasound enhance chondrogenesis of human mesenchymal stem cells in 3D printed scaffolds,” Nature Scientific Report, 6, 37728.
Lipid-coated microbubbles are used to enhance ultrasound imaging and drug delivery. Here we apply these microbubbles along with low intensity pulsed ultrasound (LIPUS) for the first time to enhance proliferation and chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in a 3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) hydrogel scaffold. The hMSC proliferation increased up to 40% after 5 days of culture in the presence of 0.5% (v/v) microbubbles and LIPUS in contrast to 18% with LIPUS alone. We systematically varied the acoustic excitation parameters—excitation intensity, frequency and duty cycle—to find 30 mW/cm2, 1.5 MHz and 20% duty cycle to be optimal for hMSC proliferation. A 3-week chondrogenic differentiation results demonstrated that combining LIPUS with microbubbles enhanced glycosaminoglycan (GAG) production by 17% (5% with LIPUS alone), and type II collagen production by 78% (44% by LIPUS alone). Therefore, integrating LIPUS and microbubbles appears to be a promising strategy for enhanced hMSC growth and chondrogenic differentiation, which are critical components for cartilage regeneration. The results offer possibilities of novel applications of microbubbles, already clinically approved for contrast enhanced ultrasound imaging, in tissue engineering.
Singh R, Sarkar K 2015 “Hydrodynamic interactions between pairs of capsules and drops in a simple shear: effects of viscosity ratio and heterogeneous collision,” Physical Review E, 92, 063029.
Hydrodynamic interactions between a pair of capsules in simple shear are numerically investigated using afront-tracking finite difference method. The membrane of the capsule is modeled using different hyperelasticconstitutive relations. We also compare the pair interactions between drops to those between capsules. Anincreased viscosity ratio leads to a reduced net cross-stream separation between capsules as well as drops aftercollision. At low viscosity ratios, for the same capillary number drop-pairs show higher cross-stream separationthan those for capsule-pairs, while substantially large viscosity ratios result in almost the same value for bothcases. We investigate pair-collisions between two heterogeneous capsules C1and C2with two different capillarynumbers. The maximum deformation of C1was seen to increase with increasing stiffness (decreasing capillarynumber) of C2, even though the stiffness of C1was kept fixed. The findings are similar for a drop-pair, however,with a smaller maximum deformation for the same combinations of capillary numbers. The final cross-streamdrift of the trajectory of C1decreases with the increasing stiffness of C2, but the relative trajectory betweenthe capsules remains unchanged. The maximum deformation and the cross-stream drift of the trajectory of C1are shown to approximately vary with power-law functions of the ratio of the capillary numbers of C1andC2. An analytical explanation of the dependence on the two capillary numbers is offered. Different membraneconstitutive laws result in similar deformation and drift in trajectory.
Kumar KN, Sarkar K 2015 “Effects of ambient hydrostatic pressure on the material properties of the encapsulation of an ultrasound contrast microbubble,” Journal of the Acoustical Society of America, 138, 624-634.
Ultrasound contrast microbubbles experience widely varying ambient blood pressure in differentorgans, which can also change due to diseases. Pressure change can alter the material properties ofthe encapsulation of these microbubbles. Here the characteristic rheological parameters of contrastagent Definity are determined by varying the ambient pressure (in a physiologically relevant range0–200 mm Hg). Four different interfacial rheological models are used to characterize the microbub-bles. Effects of gas diffusion under excess ambient pressure are investigated in detail accountingfor size decrease of contrast microbubbles. Definity contrast agent show a change in their interfacialdilatational viscosity (3.6108Ns/m at 0 mm Hg to 4.45108Ns/m at 200 mm Hg) and inter-facial dilatational elasticity (0.86 N/m at 0 mm Hg to 1.06 N/m at 200 mm Hg) with ambient pres-sure increase. The increase results from material consolidation, similar to such enhancement inbulk properties under pressure. The model that accounts for enhancement in material propertieswith increasing ambient pressure matches with experimentally measured subharmonic response asa function of ambient pressure, while assuming constant material parameters does not.
Lang X, Porter T, Sarkar K 2015 “Interpreting broadband attenuation measured at different excitation amplitudes to estimate strain-dependent interfacial rheological properties of the encapsulation of lipid-coated mono-disperse microbubbles,” Journal of the Acoustical Society of America, 138, 3994-4003.
Broadband attenuation of ultrasound measured at different excitation pressures being differentraises a serious theoretical concern, because the underlying assumption of linear and independentpropagation of different frequency components nominally requires attenuation to be independent ofexcitation. Here, this issue is investigated by examining ultrasound attenuation through a monodis-perse lipid-coated microbubble suspension measured at four different acoustic excitation ampli-tudes. The attenuation data are used to determine interfacial rheological properties (surface tension,surface dilatational elasticity, and surface dilatational viscosity) of the encapsulation according tothree different models. Although different models result in similar rheological properties, attenua-tion measured at different excitation levels (4–110 kPa) leads to different values for them; the dila-tation elasticity (0.56 to 0.18 N/m) and viscosity (2.4108to 1.52108Ns/m) both decreasewith increasing pressure. Numerically simulating the scattered response, nonlinear energy transferbetween frequencies are shown to be negligible, thereby demonstrating the linearity in propagationand validating the attenuation analysis. There is a second concern to the characterization arisingfrom shell properties being dependent on excitation amplitude, which is not a proper constitutivevariable. It is resolved by arriving at a strain-dependent rheology for the encapsulation. The limita-tions of the underlying analysis are discussed.
Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2014 “pH-Triggered Echogenicity and Contents Release from Liposomes,” Molecular Pharamaceutics, 11, 4059-4068.
Liposomes are representative lipid nanoparticles widelyused for delivering anticancer drugs, DNA fragments, or siRNA to cancercells. Upon targeting, various internal and external triggers have been usedto increase the rate for contents release from the liposomes. Among theinternal triggers, decreased pH within the cellular lysosomes has beensuccessfully used to enhance the rate for releasing contents. However,imparting pH sensitivity to liposomes requires the synthesis of specializedlipids with structures that are substantially modified at a reduced pH.Herein, we report an alternative strategy to render liposomes pH sensitiveby encapsulating a precursor which generates gas bubblesin situinresponse to acidic pH. The disturbance created by the escaping gasbubbles leads to the rapid release of the encapsulated contents from theliposomes. Atomic force microscopic studies indicate that the liposomalstructure is destroyed at a reduced pH. The gas bubbles also render theliposomes echogenic, allowing ultrasound imaging. To demonstrate the applicability of this strategy, we have successfully targeteddoxorubicin-encapsulated liposomes to the pancreatic ductal carcinoma cells that overexpress the folate receptor on the surface.In response to the decreased pH in the lysosomes, the encapsulated anticancer drug is efficiently released. Contents releasedfrom these liposomes are further enhanced by the application of continuous wave ultrasound (1 MHz), resulting in substantiallyreduced viability for the pancreatic cancer cells (14%).
Singh R, Li X, Sarkar K 2014 “Lateral migration of an elastic capsule in a wall-bounded shear,” Journal of Fluid Mechanics, 739, 421-443.
The migration of a capsule enclosed by an elastic membrane in a wall-bounded linearshear is investigated using a front-tracking method. A detailed comparison with themigration of a viscous drop is presented varying the capillary number (in the caseof a capsule, the elastic capillary number) and the viscosity ratio. In both cases,the deformation breaks the flow reversal symmetry and makes them migrate awayfrom the wall. They quickly go through a transient evolution to eventually reach aquasi-steady state where the dynamics becomes independent of the initial positionand only depends on the wall distance. Previous analytical theories predicted thatfor a viscous drop, in the quasi-steady state, the migration and slip velocities scaleapproximately with the square of the inverse of the drop–wall separation, whereasthe drop deformation scales as the inverse cube of the separation. These power lawrelations are shown to hold for a capsule as well. The deformation and inclinationangle of the capsule and the drop at the same wall separation show a crossoverin their variation with the capillary number: the capsule shows a steeper variationthan that of the drop for smaller capillary numbers and slower variation than thedrop for larger capillary numbers. Using the Green’s function of Stokes flow, asemi-analytic theory is presented to show that the far-field stresslet that causes themigration has two distinct contributions from the interfacial stresses and the viscosityratio, with competing effects between the two defining the dynamics. It predicts thescaling of the migration velocity with the capsule–wall separation, however, matchingwith the simulated result very well only away from the wall. A phenomenologicalcorrelation for the migration velocity as a function of elastic capillary number, walldistance and viscosity ratio is developed using the simulation results. The effects ofdifferent membrane hyperelastic constitutive equations – neo-Hookean, Evans–Skalak,and Skalak – are briefly investigated to show that the behaviour remains similar fordifferent equations.
Paul S, Nahire R, Mallik S, Sarkar K 2014 “Encapsulated microbubbles and echogenic liposomes for contrast ultrasound imaging and targeted drug delivery,” Computational Mechanics, 53,413-435.
Micron- to nanometer-sized ultrasound agents,like encapsulated microbubbles and echogenic liposomes,are being developed for diagnostic imaging and ultra-sound mediated drug/gene delivery. This review providesan overview of the current state of the art of the mathe-matical models of the acoustic behavior of ultrasound con-trast microbubbles. We also present a review of the in vitroexperimental characterization of the acoustic properties ofmicrobubble based contrast agents undertaken in our lab-oratory. The hierarchical two-pronged approach of model-ing contrast agents we developed is demonstrated for a lipidcoated (SonazoidTM)and a polymer shelled (polyD-L-lactic acid) contrast microbubbles. The acoustic and drugrelease properties of the newly developed echogenic lipo-somes are discussed for their use as simultaneous imagingand drug/gene delivery agents. Although echogenicity is con-clusively demonstrated in experiments, its physical mecha-nisms remain uncertain. Addressing questions raised herewill accelerate further development and eventual clinicalapproval of these novel technologies.
Katiyar A, Duncan R L, Sarkar K 2014 “Ultrasound stimulation increases proliferation of MC3T3-E1 preosteoblast-like cells,” Journal of Therapeutic Ultrasound, 2, 1, 1-10.
Background: Mechanical stimulation of bone increases bone mass and fracture healing, at least in part, throughincreases in proliferation of osteoblasts and osteoprogenitor cells. Researchers have previously performedin vitrostudies of ultrasound-induced osteoblast proliferation but mostly used fixed ultrasound settings and have reportedwidely varying and inconclusive results. Here we critically investigated the effects of the excitation parameters oflow-intensity pulsed ultrasound (LIPUS) stimulation on proliferation of MC3T3-E1 preosteoblastic cells in monolayercultures.
Methods:We used a custom-designed ultrasound exposure system to vary the key ultrasound parameters—intensity,frequency and excitation duration. MC3T3-E1 cells were seeded in 12-well cell culture plates. Unless otherwise specified,treated cells, in groups of three, were excited twice for 10 min with an interval of 24 h in between after cell seeding.Proliferation rates of these cells were determined using BrdU and MTS assays 24 h after the last LIPUS excitation.All data are presented as the mean ± standard error. The statistical significance was determined using Student'stwo-sample two-tailedttests.
Results:Using discrete LIPUS intensities ranging from 1 to 500 mW/cm2(SATA, spatial average-temporal average), wefound that approximately 75 mW/cm2produced the greatest increase in osteoblast proliferation. Ultrasound exposuresat higher intensity (approximately 465 mW/cm2) significantly reduced proliferation in MC3T3-E1 cells, suggesting thathigh-intensity pulsed ultrasound may increase apoptosis or loss of adhesion in these cells.Variation in LIPUS frequency from 0.5 MHz to 5 MHz indicated that osteoblast proliferation rate was not frequencydependent. We found no difference in the increase in proliferation rate if LIPUS was applied for 30 min/day or 10 min/day, indicating a habituation response.
Conclusion:This study concludes that a short-term stimulation with optimum intensity can enhance proliferation ofpreosteoblast-like bone cells that plays an important role in bone formation and accelerated fracture healing, alsosuggesting a possible therapeutic treatment for reduced bone mass.
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 2014 “Lateral migration of a viscoelastic drop in a Newtonian fluid in a shear flow near a wall,” Physics of Fluids, 26, 103102.
Wall induced lateral migration of a viscoelastic (FENE-MCR) drop in a Newtonianfluid is investigated. Just like a Newtonian drop, a viscoelastic drop reaches a quasi-steady state where the lateral velocity only depends on the instantaneous distancefrom the wall. The drop migration velocity and the deformation scale inversely withthe square and the cube of the distance from the wall, respectively. The migration ve-locity varies non-monotonically with increasing viscoelasticity (increasing Deborahnumber); initially increasing and then decreasing. An analytical explanation has beengiven of the effects by computing the migration velocity as arising from an imagestresslet field due to the drop. The semi-analytical expression matches well with thesimulated migration velocity away from the wall. It contains a viscoelastic stressletcomponent apart from those arising from interfacial tension and viscosity ratio. Themigration dynamics is a result of the competition between the viscous (interfacialtension and viscosity ratio) and the viscoelastic effects. The viscoelastic stressletcontribution towards the migration velocity steadily increases. But the interfacialstresslet—arising purely from the drop shape—first increases and then decreases withrising Deborah number causing the migration velocity to be non-monotonic. The ge-ometric effect of the interfacial stresslet is caused by a corresponding nonmonotonicvariation of the drop inclination. High viscosity ratio is briefly considered to showthat the drop viscoelasticity could stabilize a drop against breakup, and the increase inmigration velocity due to viscoelasticity is larger compared to the viscosity-matchedcase.
Sarkar K, Singh R 2013 “Spatial ordering due to hydrodynamic interactions between a pair of colliding drops in a confined shear,” Physics of Fluids, 25, 051702.
Pair-collision between viscous drops in a confined shear is simulated to show that the confinement alters the trajectories of the drops spatially ordering them at a finite separation in the center of the domain. In contrast to free shear where drops eventually adopt free streamlines with a finite cross-stream separation, here they move towards the centerline achieving zero cross-stream separation but a net stream-wise separation. The latter varies as inverse of capillary number and cube of the confinement (distance between the walls). The final stream-wise separation does not depend on the initial positions of the drops when the drops are in the same shear plane. The separation decreases approximately linearly with the initial separation in the vorticity direction. An analytical theory explaining the phenomenon is presented. Effects of the ratio of drop to matrix viscosity are briefly investigated
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.
The dynamics of a drop deforming, orienting and moving in a shear flow of aviscoelastic liquid near a wall is numerically investigated using a front-tracking finite-difference method and a semi-analytic theory. The viscoelasticity is modelled usingthe modified FENE-CR constitutive equation. In a Newtonian system, deformation in adrop breaks the reversal symmetry of the system resulting in a migration away fromthe wall. This study shows that the matrix elasticity reduces the migration velocity, thereduction scaling approximately linearly with viscoelasticity (product of the Deborah number De and the ratio of polymer viscosity to total viscosity β). Similar to a Newtonian system, for small Deborah numbers, the dynamics quickly reaches a quasi-steady state where deformation, inclination, as well as migration and slip velocitiesbecome independent of the initial drop–wall separation. They all approximately scaleinversely with the square of the instantaneous separation except for deformation whichscales inversely with the cube of separation. The deformation shows a non-monotonicvariation with increasing viscoelasticity similar to the case of a drop in an unboundedshear and is found to influence little the change in migration. Two competing effectsdue to matrix viscoelasticity on drop migration are identified. The first stems fromthe reduced inclination angle of the drop with increasing viscoelasticity that tries toenhance migration velocity. However, it is overcome by the second effect inhibitingmigration that results from the normal stress differences from the curved streamlinesaround the drop; they are more curved on the side away from the wall comparedwith those in the gap between the wall and the drop, an effect that is also presentfor a rigid particle. A perturbative theory of migration is developed for small ratioof the drop size to its separation from the wall that clearly shows the migrationto be caused by the image stresslet field due to the drop in presence of the wall. The theory delineates the two competing viscoelastic effects, their relative magnitudes, and predicts migration that matches well with the simulation. Using the simulationresults and the stresslet theory, we develop an algebraic expression for the quasi-steadymigration velocity as a function of Ca, De and β. The transient dynamics of themigrating drop is seen to be governed by the finite time needed for development of theviscoelastic stresses. For larger capillary numbers, in both Newtonian and viscoelasticmatrices, a viscous drop fails to reach a quasi-steady state independent of initialdrop–wall separation. Matrix viscoelasticity tends to prevent drop breakup. Drops that† Email address for correspondence: firstname.lastname@example.org
Paul S, Russakow D, Rodger T, Sarkar K, Cochran M, Wheatley M 2013 “Determination of the interfacial rheological properties of a Poly(DL-Lactic Acid)-encapsulated contrast agent using in vitro attenuation and scattering,” Ultrasound in Medicine and Biolog
The stabilizing encapsulation of a microbubble-based ultrasound contrast agent (UCA) critically affectsits acoustic properties. Polymers, which behave differently from materials commonly used (i.e.,lipids or proteins)for monolayer encapsulation, have the potential for better stability and improved control of encapsulation prop-erties. Air-filled microbubbles coated with poly(DL-lactic acid) (PLA) are characterized here usingin vitroacousticexperiments and several models of encapsulation. The interfacial rheological properties of the encapsulation aredetermined according to each model using attenuation of ultrasound through a suspension of microbubbles.Then the model predictions are compared with scattered non-linear (sub- and second harmonic) responses. Forthis microbubble population (average diameter, 1.9mm), the peak in attenuation measurement indicatesa weighted-average resonance frequency of 2.5–3 MHz, which, in contrast to other encapsulated microbubbles,is lower than the resonance frequency of a free bubble of similar size (diameter, 1.9mm). This apparently contra-dictory result stems from the extremely low surface dilational elasticity (around 0.01–0.07 N/m) and the reducedsurface tension of the poly(DL-lactic acid) encapsulation, as well as the polydispersity of the bubble population. Allmodels considered here are shown to behave similarly even in the non-linear regime because of the low surface dila-tional elasticity value. Pressure-dependent scattering measurements at two different excitation frequencies (2.25and 3 MHz) revealed strongly non-linear behavior with 25–30 dB and 5–20 dB enhancements in fundamentaland second-harmonic responses, respectively, for a contrast agent concentration of 1.33mg/mL in the suspension.Sub-harmonic responses are registered above a relatively low generation threshold of 100–150 kPa, with up to 20dB enhancement beyond that pressure. Numerical predictions from all models show good agreement with theexperimentally measured fundamental response, but not with the experimental second-harmonic response. Thecharacteristic features of sub-harmonic responses and the steady response beyond the threshold are matchedwell by model predictions. However, prediction of the threshold value depends on estimated properties and sizedistribution. The variation in size distribution from sample to sample leads to variation in estimates of encapsula-tion properties: the lowest estimated value for surface dilational viscosity better predicts the sub-harmonicthreshold.
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.