Methods for producing microbubbles
View Patent ↗The present invention provides methods for the preparation of gas-filled microbubbles, and methods of using for therapeutic and/or diagnostic applications. In particular, the methods of the invention allow for the preparation of gas-filled microbubbles having narrow size distributions and defined ultrasonic properties.
1. A method for preparing a plurality of microbubbles filled with a perfluorocarbon gas, the plurality of microbubbles having an average, single microbubble acoustic collapse threshold of about 0.3-30 MPa, at least 80% of the microbubbles having diameters within about 10% of a selected mean diameter, the method comprising:
(a) saturating a solution comprising serum albumin and dextrose with the perfluorocarbon gas; and
(b1) delivering a first round of ultrasound energy at a power level of about 150 W within the solution and delivering a second round of ultrasound energy at a power level of about 400-450 W outside the solution, wherein the plurality of microbubbles has a mean diameter of about 1.0 micron;
(b2) delivering a first round of ultrasound energy at a power level of about 125-250 W within the solution and delivering a second round of ultrasound energy at a power level of about 300-375 W outside the solution, wherein the plurality of microbubbles has a mean diameter of about 2.0 microns;
(b3) delivering a first round of ultrasound energy at a power level of about 250 W within the solution and delivering a second round of ultrasound energy at a power level of about 450 W outside the solution, wherein the plurality of microbubbles has a mean diameter of about 3.0 microns;
(b4) delivering a first round of ultrasound energy at a power level of about 250-300 W within the solution and delivering a second round of ultrasound energy at a power level of about 250-350 W outside the solution, wherein the plurality of microbubbles has a mean diameter of about 4.0 microns;
(b5) delivering a first round of ultrasound energy at a power level of about 250-300 W within the solution and delivering a second round of ultrasound energy at a power level of about 200-250 W outside the solution, wherein the plurality of microbubbles has a mean diameter of about 5-6 microns; or
(b6) delivering only one round of ultrasound energy at a power level of about 400-500 W outside the solution, wherein the plurality of microbubbles has a mean diameter of about 0.5-1.0 micron.
2. The method of claim 1 , wherein the first round of ultrasound energy delivered in steps (b1), (b2), (b3), (b4), or (b5) is delivered by a sonic probe immersed and positioned at the midpoint of the solution volume; and the second round of ultrasound energy delivered in steps (b1), (b2), (b3), (b4), or (b5) or the one round of ultrasound energy delivered in step (b6) is delivered by a sonic probe that is positioned no more than about 3 mm above the surface of the solution.
3. The method of claim 1 , wherein the solution in step (b1) comprises about 1-2% of serum albumin and about 3-4% of dextrose; the first round of energy is delivered for about 20-30 seconds; the second round of energy is delivered for about 70 seconds.
4. The method of claim 1 , wherein the solution in step (b2) comprises about 1-2% of serum albumin and about 3-4% of dextrose; the first round of energy is about 125 W and is delivered for about 30 seconds; the second round of energy is about 375 W and is delivered for about 45 seconds.
5. The method of claim 1 , wherein the solution in step (b2) comprises about 3-6% of serum albumin and about 9-12% of dextrose; the first round of energy is about 250 W and is delivered for about 25 seconds; the second round of energy is about 300 W and is delivered for about 20 seconds.
6. The method of claim 1 , wherein the solution in step (b3) comprises about 1-6% of serum albumin and about 9-12% of dextrose; the first round of energy is delivered for about 30 seconds; the second round of energy is delivered for about 20 seconds.
7. The method of claim 1 , wherein the solution in step (b3) comprises about 1-6% of serum albumin and about 9-12% of dextrose; the first round of energy is delivered for about 40 seconds; the second round of energy is delivered for about 30 seconds.
8. The method of claim 1 , wherein the solution in step (b4) comprises about 1-2% of serum albumin and about 3-4% of dextrose; the first round of energy is about 250 W and is delivered for about 30 seconds; the second round of energy is about 350 W and is delivered for about 40 seconds.
9. The method of claim 1 , wherein the solution in step (b4) comprises about 3-6% of serum albumin and about 9-12% of dextrose; the first round of energy is about 300 W and is delivered for about 25 seconds; the second round of energy is about 250 W and is delivered for about 40 seconds.
10. The method of claim 1 , wherein the solution in step (b5) comprises about 1-2% of serum albumin and about 3-4% of dextrose; the first round of energy is about 250 W and is delivered for about 35 seconds; the second round of energy is about 250 W and is delivered for about 45 seconds.
11. The method of claim 1 , wherein the solution in step (b5) comprises about 3-6% of serum albumin and about 9-12% of dextrose; the first round of energy is about 300 W and is delivered for about 25 seconds; the second round of energy is about 200 W and is delivered for about 30 seconds.
12. The method of claim 1 , wherein the solution in step (b6) comprises about 1-2% of serum albumin and about 3-4% of dextrose, the power level is about 450 W, the energy is applied continuously for about 70-90 seconds, and the mean diameter of the plurality of microbubbles is about 1 micron.
13. The method of claim 1 , wherein the solution in step (b6) comprises about 1-2% of serum albumin and about 3-4% of dextrose, the power level is about 500 W, the energy is applied discontinuously for about 70-90 seconds, and the mean diameter of the plurality of microbubbles is about 0.5 microns.
14. The method of claim 1 , wherein the plurality of microbubbles has a size stability in which the mean diameter does not change more than about 10% for up to about two years at 5° C.
15. The method of claim 14 , further comprising treating the plurality of microbubbles with a buffer having a pH of about 3 to about 6, wherein the size stability and the average, single microbubble acoustic collapse threshold of the plurality of microbubbles are increased.
16. The method of claim 1 , wherein the plurality of microbubbles further comprises a biological agent selected from the group consisting of a contrast agent, a pharmaceutical agent, a prodrug, a protein, a peptide, an antibody, and a nucleic acid.