Claims:We claim:

1. An one pot method of synthesizing an ultrasound contrast agent, the method comprising: simultaneously purging gas and injecting a lipid dissolved in an organic solvent into a hot aqueous phase placed in a pot sonicated by an ultrasound water bath, thereby forming micro or nano bubbles as the ultrasound contrast agent in a single step.

2. The one pot method as claimed in claim 1, wherein the gas comprises Sulfur hexafluoride (SF6).

3. The one pot method as claimed in claim 1, wherein the solvent comprises Ethanol.

4. The one pot method as claimed in claim 1, wherein the one pot method is performed for a quick period ranging up to a minimum of 15 seconds and to a maximum of 1 minute.

5. The one pot method as claimed in claim 1, wherein the lipid formulation comprises distearoylphosphatidylcholine (DSPC) and Palmitic acid (PA) in a 7:3 molar ratio.

6. The one pot method as claimed in claim 1, wherein the lipid formulation comprises distearoylphosphatidylcholine (DSPC), Palmitic acid (PA) and distearoyl phosphatidyl ethanolamine (DSPE) in a molar ratio of 7:2.5:0.5.

7. The one pot method as claimed in claim 1, wherein the aqueous phase comprises distilled water.

8. The one pot method as claimed in claim 1, wherein the aqueous phase comprises phosphate buffer saline (PBS).

9. The one pot method as claimed in claim 1, wherein the gas is purged at a pressure of 5 psi or 0.2 kg/cm2.

10. The one pot method as claimed in claim 1, wherein bubbles have a size about 400 to 500 nm.

Dated this 11th day of July 2019

MAHUA ROY CHOWDHURY
IN/PA – 496
(Authorized Patent Agent for the Applicant)
, Description:A ONE POT AND ONE STEP METHOD FOR SYNTHESIS OF ULTRASOUND CONTRAST AGENT

TECHNICAL FIELD
[0001] The present disclosure relates to ultrasound contrast agents, and more particularly to an one step process of synthesizing micro or nano bubbles as ultrasound contrast agent.
BACKGROUND
[0002] In recent years, a number of safe and practical ultrasound contrast agents (UCA) have been developed. Most of these are based on gas-filled microbubbles enhancing Doppler signals. The shell is either protein, lipid, surfactant or polymer and agents have a particle size of a few microns, allowing them to pass through the lungs. Furthermore, UCAs based on bubbles are compressible, which causes a non-linear behavior, namely they resonate at specific frequencies of ultrasound typically employed in clinical studies.

[0003] Although microbubble agents have been proven useful, they have significant limitations, such as their limited lifetime and the difficulties of targeting them to specific organs.

[0004] Ideally an ultrasound contrast agent having a particle size of less than 8 microns, has many advantages over other UCAs. Different particles comprising metals or metal oxides with magnetic properties have been developed for use as contrast agents for magnetic resonance imaging (MRI). U.S. Pat. No. 5,985,247 describes a method producing an imaging agent for ultrasound comprises a mixing step and a separation step, and the mixing step further includes two stages, i.e., a low speed mixing stage and a high speed mixing stage.

[0005] U.S. Pat. Application No. 20070189972 describes a method of forming nanobubbles by applying physical irritation to microbubbles contained in a liquid so that the microbubbles are abruptly contracted to form nanobubbles. U.S. Pat. Application No. 20080181853 describes Nanobubbles useful as an ultrasonic contrast agent for the lymphatic system, wherein the nanobubbles are prepared by an emulsification process and the concentrated emulsion is then be diluted in water to form a stable suspension of organic phase droplets without the need for surfactants, viscosity enhancers, or high shear rates.

[0006] Current ultrasound contrast agent formulation methods involves multiple steps: (i) dissolving lipid into a desirable solvent and evaporate the lipid to form a lipid cake or thin film, (ii) hydrating the thin film to develop lipid vesicles and (iii) purging the desirable gas under pressure to get the micro or nanobubble as ultrasound contrast agent. Thus, the current formulation methods involves more than one step and consumes more than few minutes to develop an ultrasound contrast agent.

[0007] Thus, there exists a need for a faster and single step method for synthesizing ultrasound contrast agents.

OBJECT OF THE INVENTION
[0008] It is the primary object of the present disclosure to provide an one step and one pot method of synthesizing nanobubbles or microbubbles as ultrasound contrast agent.
[0009] It is another object of the present disclosure to provide a rapid method of synthesizing of nanobubbles or microbubbles in 15 seconds to 1 minute.
SUMMARY
[0010] In an aspect of the present disclosure, an one pot method of synthesizing an ultrasound contrast agent is disclosed. The method comprises the step of simultaneously purging gas and injecting a lipid dissolved in an organic solvent into a hot aqueous phase placed in a pot sonicated by an ultrasound water bath, thereby forming micro or nano bubbles as the ultrasound contrast agent in a single step.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The detailed description is described with reference to the accompanying figures.
[0012] Figure 1, illustrates a setup for one pot and one step method of synthesizing an ultrasound contrast agent in accordance with an exemplary embodiment of the present disclosure.
[0013] Figures 2a – 2f , illustrate Cryo-SEM images of formulation of an example 1 with a 7:3 molar ratio of DSPC and Palmitic acid, micro/nano bubbles formulated and liposome of formulation in the example 1 of the present disclosure.
[0014] Figure 3, illustrates a dynamic light scattering (DLS) characterization of bubble formulation in the example 1 of the present disclosure.
[0015] Figure 4, illustrates acquired sonographs of agar phantom injected with bubble formulation as per the example 1 of the present disclosure.
[0016] Figure 5, illustrates a plot of biocompatibility of formulation as per the example 1 against L929 cell line using MTT assay.
[0017] Figure 6a- 6b, illustrates illustrate Cryo-SEM images of formulation of bubbles with a 7:2.5:0.5 molar ratio of DSPC: PA: DSPE as per the example 2 and micro/nano bubbles formulated in the example 2 of the present disclosure.
[0018] Figure 7, illustrates a dynamic light scattering (DLS) characterization of bubble formulation in the example 2 of the present disclosure.
[0019] Figure 8, illustrates acquired sonographs of agar phantom injected with bubble formulation as per example 2 of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION
[0020] In the present invention, a method of synthesizing an ultrasound contrast agent in one step is disclosed. Further, the method is one pot method using a single pot setup for synthesizing the ultrasound contrast agent. The single step method forms micro-sized or nano-sized bubbles, and the formulated micro or nanobubbles are used as ultrasound contrast agent. Further, the present disclosure provides a fastest method for synthesizing the ultrasound contrast agent.

[0021] Referring to figure 1, illustrates a setup for one pot and one step method of synthesizing an ultrasound contrast agent in accordance with an exemplary embodiment of the present disclosure. The setup comprises an ultrasound water bath filled with hot water, a beaker or pot. The method of synthesizing the ultrasound contrast agent comprises simultaneously purging gas and injecting a lipid dissolved in ethanol, in a hot aqueous phase under sonication mode of the ultrasound water bath for a quick period, ranging up to a minimum period of 15 seconds and to a maximum period of 1 minute..

[0022] In a preferred embodiment of the present disclosure, the hot aqueous phase being a solution comprises distilled water or phosphate buffer saline (PBS) above glass transition temperature of lipids, lipids being the biocompatible shell material dissolve in absolute ethanol in an organic phase and the gas comprises Sulfur hexafluoride (SF6). In the preferred embodiment of the present disclosure, Ethanol is used as a solvent and a size controlling agent, aqueous phase is used as reorient hydrophobic and hydrophilic lipid layer and the gas provide echogenicity to the bubbles formed.

[0023] In the present formulation method, the hydrophobic gas trapped in self-assembling lipid vesicles and stabilized with lipid layer and forms the bubbles instead of liposomes in a single step. The present disclosure involves with simply one-pot-gas injection method and rapid method requiring only 15 seconds to 1 minute. Further, the present methodology uses Ethanol for dissolving lipids and Ethanol is an acceptable solvent for human use.

[0024] Example 1:
In an example of implementation, the temperature of hot water ultrasound bath and the aqueous phase were adjusted to a temperature above the glass transition temperature of lipids. The aqueous phase may comprise PBS or Distilled water. 10mg of lipids formulation (distearoylphosphatidylcholine (DSPC) and Palmitic acid (PA)) was made to dissolve in 1 mL ethanol in a 7:3 molar ratio. The SF6 gas was purged at a pressure of 5 psi or 0.2 kg/cm2 purged into the aqueous phase and ethanol into the 10 ml of aqueous phase 1X PBS, pH 7.4; under constant sonication irradiation (0.021 W/cm2) in the ultrasonic water bath for 15 seconds. Further, the formulated bubbles were characterized using Cryo-SEM, DLS (dynamic light scattering), and sonography. As also biocompatibility of nanobubbles were performed using MTT assay against L929 cell line.

[0025] Referring to figures 2a, 2b and 2d, illustrated are Cryo-SEM images of formulation of bubbles with a 7:3 molar ratio of DSPC and Palmitic acid as per the example 1 and figure 2e shows a Cryo-SEM image of micro/nano bubbles formulated in the example 1 of the present disclosure. Referring to figures 2c and 2f, illustrated are Cryo-SEM images of liposome of formulation in the example 1 of the present disclosure.

[0026] Referring to figures 3, illustrated is a dynamic light scattering (DLS) characterization of bubble formulation in the example 1 of the present disclosure. Referring to figure 4, illustrated are acquired sonographs of agar phantom injected with bubble formulation as per example 1 of the present disclosure. Referring to figure 5, illustrated is a biocompatibility of DSPC:PA (7:3) formulation as per example 1 against L929 cell line using MTT assay.

[0027] Example 2:
In another example of implementation 10ml of degassed, 60C Warm 1X PBS (pH 7.4) was placed in to a sonicator water bath and simultaneously SF6 gas was purged and a 1ml ethanol solution injected in a 10mg of lipids formulation of DSPC: Palmitic acid: DSPE (distearoyl phosphatidyl ethanolamine) in a molar ratio of 7:2.5:0.5 in the beaker and the aqueous phase was sonicated in the sonication water bath (0.021 W/cm2) for 15 seconds. The final concentration of lipid was 1mg/ml. The SF6 gas at a pressure of 5 psi or 0.2 kg/cm2 was purged into the aqueous phase for 15 seconds. Further, the micro/nano bubbles were characterized using Cryo-SEM, DLS, and sonography.

[0028] Referring to figures 6a, illustrated is Cryo-SEM images of formulation of bubbles with a 7:2.5:0.5 molar ratio of DSPC: PA: DSPE as per the example 2. Figure 6b shows a Cryo-SEM image of micro/nano bubbles formulated in the example 2 of the present disclosure. From the figure 6b, it is observed that the bubbles have a size about 400 to 500 nm.

[0029] Referring to figures 7, illustrated is a dynamic light scattering (DLS) characterization of bubble formulation in the example 2 of the present disclosure. From the figure 7, it is observed that the hydrodynamic size of bubbles is about 577.3 nm with a polydispersity index of 0.005.

[0030] Referring to figure 8, illustrated are acquired sonographs of agar phantom injected with bubble formulation as per example 2 of the present disclosure. In the sonographs of example 2, the formulation was injected in 1% agar phantom.

[0031] The above description along with the accompanying drawings is intended to disclose and describe the preferred embodiments of the invention in sufficient detail to enable those skilled in the art to practice the invention. It should not be interpreted as limiting the scope of the invention. Those skilled in the art to which the invention relates will appreciate that many variations of the exemplary implementations and other implementations exist within the scope of the claimed invention. Various changes in the form and detail may be made therein without departing from its spirit and scope. Similarly, various aspects of the present invention may be advantageously practiced by incorporating all features or certain sub-combinations of the features.