Enhancing In Vitro Stability of Albumin Microbubbles Produced Using Microfluidic T-Junction Device.

Microfluidics is an efficient technique for continuous synthesis of monodispersed microbubbles. However, microbubbles produced using microfluidic devices possess lower stability due to quick dissolution of core gas when exposed to an aqueous environment. This work aims at generating highly stable monodispersed albumin microbubbles using microfluidic T-junction devices. Microbubble generation was facilitated by an aqueous phase consisting of bovine serum albumin (BSA) as a model protein and nitrogen (N2) gas. Microbubbles were chemically cross-linked using dilute glutaraldehyde (0.75% v/v) solution and thermally cross-linked by collecting microbubbles in hot water maintained at 368 (±2) K. These microbubbles were then subjected to in vitro dissolution in an air-saturated water. Microbubbles cross-linked with a combined treatment of thermal and chemical cross-linking (TC & CC) had longer dissolution time compared to microbubbles chemically cross-linked (CC) alone, thermally cross-linked (TC) alone, and non-cross-linked microbubbles. Circular dichroism (CD) spectroscopy analysis revealed that percent reduction in alpha-helices of BSA was higher for the combined treatment of TC & CC when compared to other treatments. In contrast to non-cross-linked microbubbles where microbubble shell dissolved completely, a significant shell detachment was observed during the final phase of the dissolution for cross-linked microbubbles captured using high speed camera, depending upon the extent of cross-linking of the microbubble shell. SEM micrographs of the microbubble shell revealed the shell thickness of microbubbles treated with TC & CC to be highest compared to only thermally or only chemically cross-linked microbubbles. Comparison of microbubble dissolution data to a mass transfer model showed that shell resistance to gas permeation was highest for microbubbles subjected to a combined treatment of TC & CC.

Effectiveness of Oil-Layered Albumin Microbubbles Produced Using Microfluidic T-Junctions in Series for In Vitro Inhibition of Tumor Cells.

This work focuses on the synthesis of oil-layered microbubbles using two microfluidic T-junctions in series and evaluation of the effectiveness of these microbubbles loaded with doxorubicin and curcumin for cell invasion arrest from 3D spheroid models of triple negative breast cancer (TNBC), MDA-MB-231 cell line. Albumin microbubbles coated in the drug-laden oil layer were synthesized using a new method of connecting two microfluidic T-mixers in series. Double-layered microbubbles thus produced consist of an innermost core of nitrogen gas encapsulated in an aqueous layer of bovine serum albumin (BSA) which in turn, is coated with an outer layer of silicone oil. In order to identify the process conditions leading to the formation of double-layered microbubbles, a regime map was constructed based on capillary numbers for aqueous and oil phases. The microbubble formation regime transitions from double-layered to single layer microbubbles and then to formation of single oil droplets upon gradual change in flow rates of aqueous and oil phases. In vitro dissolution studies of double-layered microbubbles in an air-saturated environment indicated that a complete dissolution of such bubbles produces an oil droplet devoid of a gas bubble. Incorporation of doxorubicin and curcumin was found to produce a synergistic effect, which resulted in higher cell deaths in 2D monolayers of TNBC cells and inhibition of cell proliferation from 3D spheroid models of TNBC cells compared to the control.