Krishna N. Kumar

Effects of ambient hydrostatic pressure on the material properties of the encapsulation of an ultrasound contrast microbubble

Ultrasound contrast microbubbles experience widely varying ambient blood pressure in different organs, which can also change due to diseases. Pressure change can alter the material properties of the encapsulation of these microbubbles. Here the characteristic rheological parameters of contrast agent Definity are determined by varying the ambient pressure (in a physiologically relevant range 0-200 mm Hg). Four different interfacial rheological models are used to characterize the microbubbles. Effects of gas diffusion under excess ambient pressure are investigated in detail accounting for size decrease of contrast microbubbles. Definity contrast agent show a change in their interfacial dilatational viscosity (3.6 × 10(-8) Ns/m at 0 mm Hg to 4.45 × 10(-8) Ns/m at 200 mm Hg) and interfacial dilatational elasticity (0.86 N/m at 0 mm Hg to 1.06 N/m at 200 mm Hg) with ambient pressure increase. The increase results from material consolidation, similar to such enhancement in bulk properties under pressure. The model that accounts for enhancement in material properties with increasing ambient pressure matches with experimentally measured subharmonic response as a function of ambient pressure, while assuming constant material parameters does not.

Acoustic Characterization of Echogenic Polymersomes Prepared From Amphiphilic Block Copolymers

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 the hydrophobic bilayer respectively, but are more stable than liposomes. Although echogenic liposomes have been widely investigated for simultaneous ultrasound imaging and controlled drug delivery, the potential of the polymersomes remains unexplored. We prepared two different echogenic polymersomes from the amphiphilic copolymers 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 investigated acoustic behavior with potential applications in biomedical imaging. We characterized the polymeric vesicles acoustically with three different excitation frequencies of 2.25, 5 and 10 MHz at 500 kPa. The polymersomes exhibited strong echogenicity at all three excitation frequencies (about 50- and 25-dB enhancements in fundamental and subharmonic, respectively, at 5-MHz excitation from 20 µg/mL polymers in solution). Unlike echogenic liposomes, they emitted strong subharmonic responses. The scattering results indicated their potential as contrast agents, which was also confirmed by clinical ultrasound imaging.

Acoustic vaporization threshold of lipid-coated perfluoropentane droplets

Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing microbubbles as contrast agents in situ as well as higher stability and the possibility of achieving smaller sizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with a perfluoropentane (PFP) core (diameter 400-3000 nm) is acoustically measured as a function of the excitation 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 determine the ADV phenomenon, and findings are discussed. The average threshold obtained using three different scattered components increases with frequency-1.05 ± 0.28 MPa at 2.25 MHz, 1.89 ± 0.57 MPa at 5 MHz, and 2.34 ± 0.014 MPa at 10 MHz. The scattered response from vaporized 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 threshold value.

Mechanism of echogenicity of echogenic liposomes

Liposomes prepared by a freeze-drying technique in the presence of mannitol have proved to be echogenic. However, the mechanism of echogenicity is not well understood. Here, we attempt to explain it. It was observed that only freeze-dried mannitol (without lipids) generates a strong scattered response because it generates bubble upon dissolution in water. The bubble generation was confirmed optically under an optical microscope. During the dissolution of the crystalline mannitol, the concentration of mannitol becomes locally very high. As the solute (mannitol) concentration increases, the saturated dissolved gas concentration decreases. Therefore, the dissolved gas in the solution near the dissolving crystal is in a supersaturated state. Upon sufficient supersaturation, bubble nucleation takes place. We found that freeze-dried crystalline excipients such as mannitol facilitate bubble nucleation compared to freeze-dried glassy excipient such as trehalose because of differences in surface morphology.

Study of acoustic droplet vaporization using classical nucleation theory

Lipid coated perfluorocarbon (PFC) nanodroplets can be vaporized by an external ultrasound pulse to generate bubbles in situ for tumor imaging and drug delivery. Here we employ classical nucleation theory (CNT) to investigate the acoustical droplet vaporization (ADV), specifically the threshold value of the peak negative pressure required for ADV. The theoretical analysis predicts that the ADV threshold increases with increasing surface tension of the droplet core and frequency of excitation, while it decreases with increasing temperature and droplet size. The predictions are in qualitative agreement with experimental observations. We also estimate and discuss energy required to form critical cluster to argue that nucleation occurs inside the droplet, as was also observed by high-speed camera.

In vitro acoustic characterization of echogenic polymersomes with PLA-PEG and PLLA-PEG shells

Echogenic liposomes (ELIPs), lipid bilayer-coated vesicles, have been widely studied as an acoustically triggerable drug delivery agent or an ultrasound contrast agent. Instead of liposomes, polymersomes, amphiphilic vesicles, offer additional stability and chemical flexibility. Here, we report the acoustic behaviors of echogenic polymersomes made of block copolymers PLA-PEG and PLLA-PEG, which are stereo-isomers. Polymersomes were excited with three different frequencies, 2.25 MHz, 5 MHz and 10 MH, and their scattered responses were measured. Both PLA-PEG and PLLA-PEG shell polymersomes produce strong acoustic responses as high as 50 dB in the fundamental component, thus demonstrating their potential as contrast agents. Significant subharmonic as well as second harmonic responses were observed at excitation frequencies of 2.25 MHz and 5 MHz. The gas dissolved in the suspension was found to be essential for the echogenicity of polymersomes.

Acoustic and atomic force microscopy characterization of microbubbles with varying shell chemistry

Applications of microbubbles (MBs) in diagnostic and therapeutic interventions critically depend on their stability and scattering properties. The shell chemistry of MBs defines these properties. We investigated the effects of shell chemistry on the size, abundance, acoustic response, and mechanical properties of MBs by varying the poly(ethylene glycol) (PEG) molar ratio (0 to 100%) in a two-lipid (DPPC and DPPE-PEG2000) component shell formulation. Increasing PEG concentration from 0% to 10% resulted in an increase in the number of MBs by at least 10-fold, with adverse effects upon further increases. Microbubbles made with 5–10% PEG generated the strongest fundamental as well as nonlinear (subharmonic and second harmonic) components at the excitation frequency of 2.25 MHz. We used interfacial rheological models to determine the mechanical properties of MB shells as functions of PEG concentration using experimentally measured attenuation values. We also employed atomic force microscopy (AFM) to perform th...

Role of mannitol on the echogenicity of echogenic liposomes (ELIPs)

Although echogenic liposomes (ELIP), specially prepared lipid-bilayer coated vesicles, have proved quite effective as scatterers, the exact mechanism of their echogenicity is not well understood. However, freeze-drying in presence of mannitol has proved to be a critical component of the elaborate preparation protocol of these ELIPs. Here, we investigate the role played by mannitol in ensuring echogenicity. We investigated mannitol along with other similar sugars, such as sucrose, trehalose, and xylitol. Mannitol when freeze-dried assumes a crystalline state, while other sugars adopt glassy states. Aqueous solutions of each sugar were prepared and freeze-dried. The freeze-dried samples were re-dissolved in water and the scattered response from the solution was measured. While the solution of mannitol was found echogenic, indicating production of bubbles, others were not. If the sample was freeze-thawed before dissolution, it was not echogenic. The crystalline state of the excipient, mannitol, was necessary...

Effects of acoustic parameters on nanodroplet vaporization

Phase shift nanodroplets offer a number of advantages over ordinary microbubbles due to their enhanced stability and smaller size distribution. These nanodroplets undergo a phase transition from liquid to highly echogenic gaseous state when activated by sufficient acoustic energy through a process termed acoustic droplet vaporization (ADV). In this study, we synthesized lipid-coated perfluoropentane (PFP) filled nanodroplets via sonication method. We investigated the ADV threshold of these nanodroplets as a function of acoustic parameters such as excitation pressure, frequency, pulse length, and pulse repetition period (PRP). Our results indicate that ADV threshold varies significantly with PRP; while at PRP of 10 ms, the ADV threshold was found to be 3.6 MPa (pk-pk), for PRP of 1 ms, 100 μs, and 500 μs, ADV was not observed even at 10 MPa. At ADV, fundamental and odd harmonics were found to be significantly higher than the background noise. The acoustic response of ordinary perfluorobutane (PFB) filled m...

Experimental and theoretical studies on acoustic droplet vaporization

Phase shift nanodroplets are better alternatives to microbubbles due to their enhanced stability and smaller size distribution. These nanodroplets undergo phase transition from liquid to highly echogenic gaseous state under acoustic excitation through a process termed acoustic droplet vaporization (ADV). In this study, we synthesized lipid-coated perfluoropentane (PFP)-filled nanodroplets via sonication and mechanical agitation methods. We investigated the ADV threshold of these nanodroplets as a function of acoustic parameters such as excitation frequency and pulse repetition period (PRP). Experiments were performed at frequencies 2.25, 5, and 10 MHz. The acoustic signature of droplet vaporization was observed to be a broadband signal at all the studied frequencies. The ADV threshold was studied by increasing the excitation pressure in steps of 200 kPa. The ADV threshold at 2.25 and 5 MHz was found to be 1.8 and 2.2 MPa, respectively. The scattered response from droplets were studied at different PRPs of...