DESC:TECHNICAL FIELD

The present disclosure relates to nanomaterials and, more particularly, to synthesizing gold nanoparticles coated nanobubbles (AuNBs) for multimodal imaging and theranostic applications.

BACKGROUND
The history of the ultrasound contrast agent can be traced back to the initial observation of the contrast effect of a cloud of echoes after the intracardiac injection of saline in 1968 by Gramiak et al. In the late 80's, the ultrasonic tissue characterization had certain difficulties due to similar ultrasonic characteristics between some tissues, even though these tissues are pathologically different. Thus, the ultrasound contrast agent that enhances the echogenicity of tissues and blood has drawn great attention.

The generation of the contrast agent is classified according to the type of gas filled in micro-bubbles, where the first-generation contrast agent is mainly filled with air that is encapsulated by polymers like albumin or galactose, and the second-generation contrast agent is filled with high-density inert gas that is encapsulated by a soft, thin membrane. Due to difference in elastic prosperities of materials, such microbubbles create a contrast and are used as ultrasound contrast agent in clinical settings. Usually, these particles are gas core stabilized by biocompatible material such as polymers, lipids, proteins, surfactants, carbohydrates of spherical, more than 1 micron size.

Various studies demonstrated that the gold nanoparticles could be loaded into the shell of the microbubbles (ultrasound contrast agents of more than 1 micron size). Such particles were reported to improve the stability of the loaded gas and, thus stability of microbubbles. Similar studies were reported for protein microbubble. Recently gold nanoparticles were reported to be conjugated on the shell using conjugation chemistry (example: EDC and NHS) for ultrasound imaging and photoacoustic applications. Gold nanoparticles also been reported as CT imaging agent. Multimodal contrast agents are important as they can be used for performing imaging of more than one modality. For making CT-Ultrasound or Photoacoustic-Ultrasound, multimodal agent, typically CT contrast agents were loaded into the Ultrasound contrast agent (microbubbles – vesicles of more than 1 micrometre size) or by conjugating CT contrast agent for using conjugation chemistry (example: EDC-NHS, Biotin-Avidin).

OBJECT OF THE INVENTION
The primary object of the present disclosure is to provide a method for synthesizing a contrast agent comprising gold nanoparticles coated nanobubbles (AuNBs) using one pot method.
It is another object of the present disclosure to provide a multimodal imaging agent for US and CT imaging applications.
Another object of the present disclosure is to provide a multimodal imaging agent used as a therapeutic agent for therapeutic delivery application.
It is still another object of the present disclosure to provide a multimodal imaging agent to improved stability of ultrasound contrast agent.

SUMMARY
In an aspect of the present disclosure, a method of synthesizing an agent for multimodal imaging and theranostic applications is disclosed. The agent comprises metal nanoparticles coated nanobubbles. The method comprises the steps of: synthesizing lipid nanobubbles (LNBs) using lipids with a gas purging process, forming a nanobubble suspension by adding at least one metal salt to the lipid nanobubbles (LNBs), followed by mixing the lipid nanobubbles (LNBs), adding at least one reducing agent to the nanobubble suspension and mixing the nanobubble suspension till colour of nanobubble suspension gets changed, and forming a coat on the surface of nanobubbles based on amount of metal nanoparticles grown in the nanobubble suspension, thereby synthesizing metal nanoparticles coated nanobubbles.
The lipid nanobubbles (LNBs) are synthesized using a scalable, one-step, one-pot process. The process comprises: dissolving lipids in an ethanal solution, thereby forming lipid dissolved ethanol, purging the gas into a buffer solution of phosphate buffer saline (PBS) or distilled water obtained from a MiliQ filter system, maintained at a temperature and placing the buffer solution on a hot water bath, simultaneously injecting lipid dissolved ethanol drop by drop into the buffer solution and the purging gas, stirring the result lipid solution continuously using a high-speed homogenizer at a speed thereby forming lipid bubbles, and centrifuging the formed lipid bubbles to remove excess lipid from a shell of nanobubbles and collecting the formulated nanobubbles (NBs).

In another aspect of the present disclosure, a method of synthesizing an agent for multimodal imaging and theranostic applications is disclosed. The agent comprises metal nanoparticles coated nanobubbles. The method comprises the steps of: synthesizing lipid nanobubbles (LNBs) using lipids with a thin-film hydration gas purging process, forming a nanobubble suspension by adding at least one metal salt and at least one reducing agent to the lipid nanobubbles (LNBs) at a ratio and mixing the nanobubble suspension till colour of nanobubble suspension gets changed, and forming a coat on the surface of nanobubbles based on amount of metal nanoparticles grown in the nanobubble suspension, thereby synthesizing metal nanoparticle coated nanobubbles. In the method, the at least one metal salt and at least one reducing agent is added at a ratio selected from 1:1, 1:2, 1:4.

In yet another aspect of the present disclosure, a method for synthesizing a contrast agent comprising gold nanoparticles coated nanobubbles (AuNBs) is disclosed. The method uses lipidic nanobubbles (NB) as a template for synthesizing gold nanoparticles coated nanobubbles (AuNB). Gold nanoparticles (AuNPs) are synthesized using reducing agents on the surface of nanobubbles. The nanobubbles are produced using lipid using at least one technique selected from high-speed homogenizing, thin film hydration liposome-gas purge or any similar method and with Sulphur hexafluoride (SF6), ambient air. Further, gold chloride is added and mixed, followed by adding reducing agent, such as Ascorbic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, thereby producing a nanobubble suspension. Further, the nanobubble suspension is continuously mixed till white coloured nanobubbles turn red and then to green colour. Depending on amount of gold nanoparticles grown, the gold nanoparticles form a coat on the nanobubbles surface, synthesising gold nanoparticle coated nanobubbles (AuNBs).

BRIEF DESCRIPTION OF THE DRAWINGS
The detailed description is described with reference to the accompanying figures.
Figure 1a shows a method for synthesizing a contrast agent comprising gold nanoparticles coated nanobubbles (AuNBs) in accordance with an exemplary embodiment of the present disclosure.
Figure 1b shows synthesisation of lipid nanobubbles (LNBs), synthesisation of gold nanoparticles AuNP) and synthesisation of gold nanoparticles coated nanobubbles (AuNBs).
Figure 1c shows characterization of formation of AuNBs using zeta potential at each step of the method.
Figure 2 shows a Tissue-mimicking (1% agarose) phantom for ultrasound imaging of AuNBs.
Figures 3a – 3c show a characterization of synthesized gold nanoparticle coated nanobubbles (AuNBs) in accordance with the present disclosure.
Figures 4a – 4c show another characterization of synthesised gold nanoparticle coated nanobubbles (AuNBs) in accordance with the present disclosure.
Figure 5 shows a Cryo-TEM of AuNBs produced by HEPES as reducing agent.
Figure 6 shows a UV-Vis spectra of AuNBs developed using nanobubble produced using thin-film hydration and gas purging method and by applying varying ratio of gold chloride and ascorbic acid to coat AuNPs on the bubble.
Figure 7 shows an in vitro pharmacokinetics of AuNB showing controlled and the ultrasound trigger responsive release of AuNPs.
Figures 8a – 8 b illustrate in vitro echogenic imaging of AuNB at different dilutions and in vitro CT contrast imaging of AuNB at different dilutions.
Figure 9 shows in vitro echogenicity of freshly prepared AuNB in tissue-mimicking phantom for 10 min.
Figure 10 shows in vitro echogenicity of reconstituted AuNB in tissue-mimicking phantom for 10 min.
Figures 11a – 11b show In vitro echogenic imaging of AuNB at different pH and at different depth in tissue-mimicking phantom.
Figures 12a – 12d show Functionalization of AuNB – for molecular imaging and illustration of drug carrier in accordance with the present disclosure.
Figures 13a – 13b show an illustrated FITC-Streptavidin-conjugated AuNBs for molecular imaging of Osteopontin, and in vitro molecular imaging of Osteopontin using functionalized AuNBs.
Figure 14 shows illustration of AuNB carrying thiol linked drug molecule for image-guided drug delivery application.
Figures 15a – 15b show calibration curve and the loaded efficiency of example urokinase for AuNB as carrier of therapeutic molecule.
Figures 16a – 16d show in vitro biocompatibility of AuNB in accordance with the example of the present disclosure.
Figures 17a – 17b show representative ROS generated in THP-1 macrophage fluorescence microscopic images and quantification of intercellular ROS in AuNB treated THP-1 macrophage cells.
Figures 18a – 18b show quantification of Oil-O-red stained AuNBs treated foam cells and representative images of Oil-O-Red stained foam cells.
Figures 19a – 19d show ultrasound imaging results showing contrast enhancement in accordance with another example of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION
In the present invention, a method comprising a method for synthesizing an agent for multimodal imaging and theranostic applications is disclosed. the agent comprises gold nanoparticles coated nanobubbles (AuNBs). In the present invention, coated gold nanoparticles on the surface of ultrasound contrast agents of less than 1micron size in a one step and in one pot without using conjugation chemistry are developed. The coating is due to the amine and gold affinity chemistry. The reducing agent can be changed from ascorbic acid to HEPES buffer. The change of reducing agent allows an improvement of conjugation of antibodies, therapeutic molecules by 2-5 folds. Hence, the efficacy of the gold nanoparticle coated ultrasound contrast agent is improved as multimodal (ultrasound and CT or X-ray) molecular imaging probe, anti-inflammatory agent, and therapeutic agent.

In one embodiment of the present disclosure, a method for synthesizing an agent for multimodal imaging is disclosed. The agent comprises metal nanoparticle coated nanobubbles. The method for synthesizing the agent comprises the steps of synthesizing of lipid nanobubbles (LNBs) using lipids and gas purge, forming a nanobubble suspension by adding metal salts to the lipid nanobubbles (LNBs) and mixing the nanobubbles, adding at least one reducing agent to the nanobubble suspension and mixing the nanobubble suspension till colour of nanobubbles changes, forming metal nanoparticle coated nanobubbles based on amount of metal nanoparticles grown in the nanobubble suspension. The metal comprises at least one precious metal or metal alloy selected from Gold, Silver, Platinum, and Palladium, alloys of precious metals, or a combination thereof. Lipid comprises at least one Distearoylphosphatidylcholine (DSPC), palmitic acid or lipid having amine group. The gas used in gas purge comprises Sulphur Hexafluoride (SF6) or ambient air. In another embodiment, Perfluorocarbons, oxygen, xenon is used for gas purge. Metal salts comprise chlorides of precious metals and alloys, wherein precious metal is selected from Gold Chloride, Silver, Platinum, and Palladium, alloys of precious metals or a combination thereof. The reducing agent is selected from Ascorbic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer. The selected metal or metal alloy has affinity towards primary, secondary amine, tertiary amine group.

Referring to figure 1a, illustrated is a method for synthesizing a contrast agent comprising gold nanoparticles coated nanobubbles (AuNBs) in accordance with an exemplary embodiment of the present disclosure. The method uses lipidic nanobubbles (LNB) as a template for synthesising gold nanoparticles coated nanobubbles (AuNB). Gold nanoparticles (AuNPs) are synthesized using reducing agents on the surface of nanobubbles. The nanobubbles are produced using lipid using at least one technique selected from high-speed homogenizing, thin film hydration liposome-gas purge or any similar method and with Sulphur hexafluoride (SF6), ambient air.

Further, gold chloride is added and mixed, followed by adding reducing agent, such as Ascorbic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, thereby producing a nanobubble suspension. After the gold chloride and reducing agent are added, the nanobubble suspension is continuously mixed till white coloured nanobubbles turn red and then to green colour. Depending on amount of gold nanoparticles grown, the gold nanoparticles form a coat on the nanobubbles surface synthesising gold nanoparticle coated nanobubbles (AuNBs). In an embodiment, the nanobubble suspension is continuously mixed till white coloured nanobubbles turn red or blue or green colour.

In an example of the present disclosure, gold nanoparticle coated nanobubbles (AuNBs) were synthesized using lipidic nanobubbles as a template. The lipid nanobubbles (LNBs) were synthesized using a scalable, one-step, one-pot method, 8.77 mg of DSPC and 2.45 mg of Palmitic acid were dissolved in 1 ml ethanol, and SF6 gas was purged into 10ml of 1 PBS (pH 7.4) solution (60°C) and placed on a 60 °C hot water bath, and simultaneously solvent phase (lipid dissolved ethanol) was injected drop by drop into the PBS and SF6 gas. At the same time, the solution stirred continuously for 120 seconds using a high-speed homogenizer at 10000 RPM, forming bubbles. In alternative embodiment, SF6 gas was purged into distilled water obtained from a MiliQ filter system. The resulted bubbles were centrifuged at 1000 G for 3 minutes to collect the formulated nanobubbles (NBs) using centrifugal flotation to remove excess free lipid not associated with the nanobubbles (NBs) shell. The bubbles were collected into fresh type II distilled water (DW) or phosphate buffer saline (PBS) store at 4 oC until further use.

In the example, gold nanoparticle coated nanobubbles (AuNBs) were synthesized using ascorbic acid buffer. 31.25µl of 100mM HAuCl3.4H2O was added to the 5ml lipid nano bubbles (LNBs) suspension with a final lipid concentration of 500µg and mixed gently. Then, 500µl of 100mM ascorbic acid was added to the above nanobubble suspension and mixed gently till colour of the nanobubble suspension changes. Alternatively, gold chlorides to ascorbic acid are used at 1:1, 1:2, 1:4 ratio for coating gold nanoparticles on the nanobubbles, and nanobubbles are produced using thin-film hydration gas purging process.

In another example, gold nanoparticle coated nanobubbles (AuNBs) were synthesized using HEPS buffer. 31.25µl of 100mM HAuCl3.4H2O was added to the 5ml suspension of lipid nano bubbles (LNBs) with a final lipid concentration of 500µg and mixed gently. Then, 500µl of 20mM HEPES buffer was added and mixed gently till of the suspension colour changes.

Referring to figure 1b, illustrated are different dilutions showing synthesisation of lipid nanobubbles (LNBs), synthesisation of gold nanoparticles AuNP) and synthesisation of gold nanoparticles coated nanobubbles (AuNBs).

Referring to figure 1c, illustrated, is a characterization of formation of AuNBs using zeta potential at each step of the method.

In vitro pharmacokinetics of AuNB:
Release of Gold (Au) from the surface of gold nanoparticle coated nanobubbles (AuNB) were studied by injecting 2.5 × 1010 AuNBs into release media having 10% FBS and 2% antibiotics. In a rotary incubatory, the AuNBs were incubated at 300 RPM, 37oC. After a predefined interval, the know concentration of aliquots was collected and stored at 4ºC till further processing and refilled with an equal volume of fresh sink media. Post experiment, the collected aliquots were digested in aqua regia (1:5 diluted). Digested samples were analysed using ICP-AES (ARCOS, Simultaneous ICP Spectrometer, SPECTRO Analytical Instruments GmbH, Germany). The release Au quantified as follows:
% Au released=[(Au released from AuNB at given time)/(Total Au on the AuNB)]×100

AuNB for molecular imaging and therapeutic delivery application:
Gold nanoparticle coated nanobubbles (AuNBs) produced using reducing agent - ascorbic acid or HEPES were incubated with 5ug/ml Osteopontin monoclonal antibody or 10000 Units of urokinase for 30 minutes to 24 hours. AuNBs incubated with Osteopontin monoclonal antibody (OPN mAb-AuNBs) or any similar imaging probe (other type of antibody or aptamer or peptide, single chain antibody, etc.) further treated with ox-LDL derived THP-1 foam cells (or any targeted cells) for 10-15 minutes and then centrifuged for 3000 RPM for 5 minutes and then analysed using FACS. FACS analysis indicates OPN mAb-AuNBs shows 75% conjugation efficacy to detect the foam cells; thus, OPN mAb-AuNBs can be used to detect vulnerable atherosclerosis.

For developing urokinase conjugated AuNBs (producing using HEPES buffer) (urokinase-AuNB): After incubating urokinase with AuNBs for 2 hrs, AuNBs were centrifuged, collected, and analysed for urokinase using BCA assay. The AuNB producing using HEPES buffer shows 5-fold conjugation of urokinase compared to AuNBs produced using ascorbic acid.

In vitro ultrasound and CT imaging:
Referring to Figure 2, illustrated is a Tissue-mimicking (1% agarose) phantom for ultrasound imaging of AuNBs. For in vitro ultrasound (US) imaging, 1% agarose-based tissue mimicking phantom were produced. Briefly, molten agarose was poured into the container, and an array of pipettes was inserted to form arrays of pockets/wells. The agarose was left to solidify at RT. After solidification, the array of pipettes removed. Using ultrasound imaging, the agarose gel now has pockets/wells for loading particles. Degassed water was added to fill the void and keep phantom hydrated until further use. AuNBs were echogenically characterized for time, depth, dilutions, reconstituted AuNBs' echogenicity in acidic pH conditions. The ultrasound contrast enhances images were captured using Philips’s ultrasound machine and 12-3 MHz linear probe placed at 90 o to the well of interest in tissue mimicking phantom.

The CT imaging was performed using a Siemens SOMATOM Definition AS Open CT machine. The 1×1010 AuNBs, AuNPs, and blank NB in a plastic vail were kept under the scanner, and images were obtained using a predefined imaging sequence used for an adult abdominal scan.

Cellular internalization of AuNB:
Biocompatibility of AuNB were evaluated on L929 cells and THP-1 macrophage cells. For the biocompatibility experiment, 1×104 cells/well were seeded in 96 wells plate and incubated for 24 h at 37? in a 5% CO2 incubator. After 24 hours, the 1×105 to 1×1010 AuNBs/ml were added in media supplemented with 10% FBS and 1% antibiotic. The seeded cells incubated for 24 h at 37?, in a 5% CO2 incubator. After 48 hours, the cells were subjected to MTT-assay. The developed colour intensity was measured using a multi-plate reader at 570 nm, and cell viability was measured.

Further, hemocompatibility of AuNBs were evaluated. The whole blood was incubated with 1×1010 NPs for 1 h and 24 h at 37 °C at 150 RPM. After incubation, the samples were centrifuged for 5 minutes at 3000 RPM using a benchtop centrifuge (Eppendorf 5427R). Hemolysis was quantified by measuring the absorbance of the blood plasma at 550 nm using a UV-Vis spectrometer (EnSpire® Multimode Plate Reader, Massachusetts, USA). The hemolysis percentage was calculated using the following formula: PBS (pH 7.4) was used as the negative control and Triton X-100 was used as the positive control:
% Hemolysis=((Abs.of test sample – Abs.of negative control))/((Abs.of positive control – Abs.of Negative control) )×100

In vitro therapeutic effect of AuNB:
The therapeutic effect of AuNB was studied on THP-1 cells-derived macrophage cells. THP-1 macrophage cells, 1×104 cells per wells were seeded 96 wells plate and incubated for 72 hours, at 37oC, in a 5% CO2 incubator. After 24 hours, the 1×1010,1×109,1×108 AuNB per ml was added in RPMI media supplemented with 10% FBS and 1% antibiotic and subjected to ultrasound trigger. Non-triggered AuNBs treated cells were used as controls. The cells are incubated at 37oC, in a 5% CO2 incubator. After 30 minutes, the cells were subjected to MTT assay as described elsewhere. The intensity of the developed purple colour was measured using a multi-plate reader (Varioskan Flash, thermo-fisher, USA) at 570 nm, and cell viability was measured.

In vitro ROS induction by AuNB:
PMA(5ng/ml) treated THP-1 macrophage cells, 1×104 cells per wells were seeded 96 wells plate and incubated for 72 hours, at 37?, in a 5% CO2 incubator. Post 72 hours, THP-1 cells were treated with AuNB with and without US trigger. Post-treatment cells were incubated for 1 hour, and post-incubation, the spent media was removed and replaced by serum free media having 3mM DCFDA. The cells were incubated for 40 minutes and imaged under epifluorescence microscope for analysis of induction of ROS using Image J.

In vitro immunomodulatory action of AuNB:
PMA treated THP-1 macrophage cells, 1×104 cells per wells were seeded 96 wells plate and incubated for 72 hours, at 37?, in a 5% CO2 incubator. Post 72 hours, THP-1 cells were treated with LPS and 1×109 AuNB per ml + US, US, AuNB, NB groups. Post 24 hours of incubation, supernatant were quantified using expression of IL-6 and TGF-ß1 cytokines using a DuoSet® ELISA kit.

AuNB induced reverse cholesterol effluxion:
PMA(5ng/ml) treated THP-1 macrophage cells, 1×104 cells per wells were seeded 96 wells plate and incubated for 72 hours, at 37?, in a 5% CO2 incubator. Post 72 hours, THP-1 cells were treated 50 µg/ml Ox-LDL and incubated for 48 h. Foam cells were treated with AuNB with and without US trigger. post-treatment cells were incubated for 6 hours, and the spent media was removed post-incubation. The cells were fixed with 4% formaldehyde, and then cells were stained with Oil Red O dye, and then the cells were observed under the microscope, and the Oil Red O was quantified using UV-vis spectrometer.

In vivo Ultrasound imaging:
For the study, all mice are purchased and maintained as per the animal ethical guidelines, at the Baker Heart and Diabetes Institute, Melbourne, Australia, under Alfred Plus Alliance Animal Ethics Committee no. P8453. AuNB injected into mouse vein and ultrasound images were collected before and after from heart of mouse using MS-250 probe attached to visualsonics 2100 ultrasound machine.

EXPERIMENTATION RESULTS
Characterization of Gold nanoparticle coated nanobubbles (AuNB): The formed gold nanoparticle coated nanobubbles (AuNBs) were characterized using zeta potential at each step using a Zeta sizer-ultra (Malvern Panalytical, Malvern, UK). The AuNBs were further characterized for dispersity using a Zetasizer-ultra (Malvern Panalytical, Malvern, UK), and the shape of nanobubbles was visualized using a Cryo-TEM (JEM 2100, Tokyo, Japan). The absorption and X-ray photon spectra (XPS) were recorded on a UV-VIS-NIR spectrophotometer (Varioskan flash, thermo-fisher, USA) and an X-ray photon spectrophotometer (ULVAC – Pilipils 5000, Versa Probe II, Kanagawa, Japan), respectively.

Referring to Figures 3a – 3c, illustrated are characterization of synthesized gold nanoparticle coated nanobubbles (AuNBs) in accordance with the present disclosure. Figure 3a shows a Cryo-TEM micrograph of AuNBs highlighting coated AuNPs around Nanobubbles NB. The Cryo-TEM micrograph of AuNB shows that formulated bubbles were 300-500 nm in size. The darker small dots indicated AuNP coated on the surface of the NB formulation. Figure 3b shows HR-TEM image of 25 nm gold nanoparticles synthesized using a similar step used for AuNBs. HR-TEM micrograph shows that similar methods of present invention developed in-situ coated AuNPs with a range of 30-40 nm in size /diameter and mostly spherical. Further, to confirm the presence of Au on the surface of AuNB, the XPS spectra were obtained. Figure 3c shows XPS of AuNB highlighting the presence of AuNPs on the surface. The XPS spectra of lipid nanobubbles (LNB) in narrow scan show peaks at 88 eV and 90 eV attributed to no evidence of prominent peaks but the AuNB XPS spectra exhibit the peaks at 83 eV and 92 eV, which belong to Au4f7/2 and Au4f4/2 respectively, confirming that the reduced gold (Au°) on the surface of nanobubbles.

Referring to Figures 4a – 4c, illustrated are another characterization of synthesized gold nanoparticle coated nanobubbles (AuNBs) in accordance with the present disclosure. Figure 4a shows a hydrodynamic size of LNB, AuNB, and ultrasound triggered AuNB in DW, Figure 4b shows UV-Vis’s spectra showing ?-max at 630 nm, and Figure c shows HR-TEM micrograph of ultrasound triggered AuNB showing shattered lipid clumps and clumps of gold nanoparticles. Further, Figure 4a shows that the hydrodynamic size of AuNBs is in the range of representative Cryo-TEM images of AuNB. The UV-Vis spectra show AuNP synthesized using ascorbic acid as a reducing and stabilizing agent showing plasmonic absorbance at 520 nm, and the AuNBs show plasmonic absorbance at 630 nm, whereas blank nanobubbles do not exhibit any absorbance maxima. The shift plasmonic absorbance in the case of AuNBs could be due to the couple plasmonic absorbance phenomenon exhibited by AuNPs when assembled in proximity. Interestingly, the ultrasound trigged AuNB also shows a similar profile, indicating that the assembled AuNP does not detach from the lipid shell component of the AuNBs. HR-TEM micrograph, as shown in Figure 4c, shows expected the clumps or aggregated peace of AuNB, showing clusters of AuNPs and separate AuNPs.

In the synthesis of AuNB, instead of ascorbic acid, HEPES is used as reducing agent and produces AuNB. Referring to Figure 5, illustrated is a Cryo-TEM of AuNBs produced by HEPES as reducing agent. Figure 5 highlights that AuNBs are spherical in shape and having diameter of 380 nm.

Further in the exemplary embodiments, synthesized AuNBs of shows coupled plasmonic absorbance at 630 nm when 3:1 (gold chloride: ascorbic acid) used for in suite coating of gold nanoparticles on the nanobubble surface. The coupled plasmonic absorbance is tuneable, depending on the ratio of the gold chloride and ascorbic acid. For example, AuNB developed using nanobubble produced using thin-film hydration and followed by gas purging method produce AuNBs with couple plasmon absorbance at 530 nm, 580 nm, and 830 nm by applying varying ratio of 1:1, 1:2, 1:4, respectively of gold chloride and ascorbic acid to coat AuNPs on the bubble. Figure 6 illustrates a UV-Vis spectra of AuNBs developed using nanobubble produced using thin-film hydration and gas purging method and by applying varying ratio of gold chloride and ascorbic acid to coat AuNPs on the bubble.

In vitro release kinetics of AuNB
To evaluate in vitro release kinetics of AuNB, the bubbles were injected into sink media [10% FBS in PBS (pH 7.4)], and a sample was collected after predefined time points. After each time point, sample centrifuges collected supernatant and released gold quantified using ICP-AES. Referring to Figure 7, illustrated is an in vitro pharmacokinetics of AuNB showing controlled and the ultrasound trigger responsive release of AuNPs. In the Figure 7, data are presented as mean ± SD [n = 3]. From the Figure 7, 3.79 ± 0.677 % of Au was released from AuNBs post 72 hours. Whereas after ultrasound-trigger, the bubble releases 86.6±4.46 % of Au from the AuNB post 120 seconds of post-trigger in the sink media. Thus, the AuNBs can be controlled and trigger delivery of AuNP or therapeutic at a desired site.

In vitro evaluation of AuNB as a multimodal imaging agent:
Figures 8a – 8b illustrate in vitro echogenic imaging of AuNB at different dilutions and in vitro CT contrast imaging of AuNB at different dilutions. To evaluate echogenicity of AuNB, a 3-12 MHz transducer probe and 1% tissue mimicking-agarose phantom are used. Figure 8a shows a linear reduction in the echogenicity of the bubbles with dilutions from 1×1010 to 1×106 / ml. Figure 8a shows the representative ultrasound images showing ultrasound contrast enhancement in brightness mode (B-mode) (right side panel) and contrast enhancement mode (CE-mode) (left side panel). Further, the Figure 8b, in vitro CT contrast imaging of AuNB at different dilutions, showed that AuNB enhances the CT contrast in vitro. As expected, AuNP (positive control) showed contrast enhancement in CT, and blank NP (negative control) did not show any enhancement in CT. Results echogenic imaging and CT contrast imaging indicate that the AuNB can be used as a Ultrasound (US) and a Computerized Tomography (CT) contrast agent. It was also reported that the AuNP conjugated microbubble shell and nanoparticles had been investigated for photoacoustic imaging. Thus, AuNBs can also be used as potential photoacoustic imaging agents and, hence, AuNBs can be multimodal agents.

Figure 9 illustrates in vitro echogenicity of freshly prepared AuNB in tissue-mimicking phantom for 10 minutes. Figure 9 shows representative ultrasound (US) contrast-enhanced images of the freshly prepared AuNB bubble formulation (undiluted). The representative ultrasound images showing ultrasound contrast enhancement in brightness mode (B-mode) and contrast enhancement mode (CE-mode). The echogenicity till 10 minutes was compared with commercial formulation SonoVue®, and the figure 9 shows comparable echogenic intensity. Further, to test the long-term storage possibility, the freeze-dried vail of AuNB was stored at RT for three months. The echogenicity of reconstituted AuNBs (recon AuNB) were studied using the echogenic parameters used for freshly prepared AuNBs. Figure 10 illustrates in vitro echogenicity of reconstituted AuNB in tissue-mimicking phantom for 10 min. The ultrasound images were captured for 10 min, and results of the reconstituted AuNB show that AuNBs were stored longer and can be used as ultrasound contrast agent.

Figures 11a- 11b illustrates in vitro echogenic imaging of AuNB at different pH and at different depth in tissue-mimicking phantom. Figure 11a shows that the echogenic response of AuNB was observed at acidic pH of 6.5 and 5.5. It was observed that the AuNB remains stable and shows a comparable echogenic response at acidic pH of 6.5 and 5.5 as compared to the echogenicity of AuNB at pH 7.5 from the Figure 11a. Further, the effect of depth on echogenicity of freshly prepared AuNBs were studied. AuNBs injected in wells developed at different depths. Figure 11b shows that the echogenicity of the AuNB in both B and contrast enhance mode till 8 cm depth.

Molecular imaging ready AuNB
Figures 12a – 12d illustrates functionalization of AuNB – for molecular imaging and illustration of drug carrier in accordance with the present disclosure. To functionalize the AuNB, AuNB conjugated with thiol linked or Streptavidin linked AuNB were used. Figure 12a shows FTIR of AuNB, indicating the presence of Amine and carboxyl groups on the surface of AuNP coated on the nanobubble surface. The FTIR of AuNB shows the presence of the amine group on the AuNB aid in conjugated Streptavidin using EDC-NHS conjugation chemistry. Similar groups were found to be present on AuNPs. Thus, AuNB can be used for covalent conjugation of Streptavidin. Figure 12b shows UV-Vis absorbance spectra at 488 nm showing AuNB conjugation of FITC labelled Streptavidin. Further optimisation of the AuNB conjugation with FITC-Streptavidin were studied using FACS. Figure 12c shows FACS analysis of AuNB conjugated with various concentrations of FITC- Streptavidin (red dots, green dots in dot plot represents negative and positive events, respectively). Dot plots in Figure 12c show that as the concentration of streptavidin increases, conjugation of streptavidin with AuNB increases. Figure 12d shows quantification of FACS analysis of FITC- Streptavidin-AuNB. Data are presented as mean ± SD [n = 3 for b and d]). With 5µg/ml FITC-Streptavidin incubated with AuNBs, the streptavidin conjugation efficiency increases up to average 32.5% as shown in Figure 12d. Thus, the AuNBs can be used for molecular imaging.

Figures 13a - 13b show AuNB as a molecular imaging agent for vulnerable atherosclerotic plaque. Figure 13a illustrates the potential of AuNB as a molecular imaging agent for the detection of foam cells. Foam cells play an instrumental role in atherosclerotic plaque progression in every stage. A high number of foam cells is also an indicator of vulnerable atherosclerotic plaque. FITC-Streptavidin-conjugated AuNBs can be used for molecular imaging of Osteopontin, an extracellular protein secreted by foam cells, as shown in Figure 13a. Recently Osteopontin-targeted nanoparticles were developed to detect vulnerable atherosclerotic plaque using MRI/Photoacoustic imaging. As Osteopontin, a membrane protein, overexpresses on the foamy macrophages or smooth muscle cells. As illustrated application of AuNBs for molecular imaging of Osteopontin from Figure 13a. Biotinylated monoclonal antibodies against Osteopontin conjugated with Streptavidin linked AuNB to detect foam cells and, thus, vulnerable atherosclerotic plaque. Functionalized AuNB treated with foam cells for 15 minutes. Figure 13b shows in vitro molecular imaging of Osteopontin using functionalized AuNBs using FACS analysis. The FACS analysis data shows 75% conjugation efficacy of AuNB Osteopontin expressed by the foam cells. Thus, AuNBs can be used for molecular imaging of vulnerable atherosclerotic plaque.

Apart from molecular imaging, AuNB can carry other therapeutic molecules, including thiol-linked drug molecules or other biopharmaceutical molecules, for image-guided drug delivery applications. Figure 14 shows illustration of AuNB carrying thiol linked drug molecule for image-guided drug delivery application.

AuNB as a carrier of therapeutic molecule:
Figures 15a- 15b illustrate calibration curve and the loaded efficiency of example urokinase for AuNB as carrier of therapeutic molecule. Figure 15a shows calibration curve for urokinase obtained from BCA assay. Figure 15b shows the loaded efficiency of urokinase after reducing AuNP on nanobubble surface using HEPES instead of ascorbic acid. The loaded efficiency of urokinase were increased by 5-folds. The rise of loading efficiency noticed due to potential thiol group of HEPES reduced AuNPs, which helps in conjugating enzyme or protein showing therapeutic effect. Hence, AuNBs can be used as carrier of therapeutic molecules for image-guided drug delivery applications.

In vitro cellular interaction of AuNB:
To study cellular interaction of AuNB, AuNBs were incubated with L929 and THP-1 macrophages cells. Further, for studying biocompatibility, AuNBs were incubated for 48 h. MTT assay shows more than 80% of the cell were viable, indicating that up to 1×1010. Figure 16a – 16b show cellular internalization of AuNB with cells. Figure 16a shows in vitro biocompatibility of AuNB on L929 and THP-1 macrophage, Figure 16b shows in vitro biocompatibility of AuNB + US on THP-1 macrophage. As observed from Figure 16, AuNBs/ml were biocompatible. Figure 16b shows that post 30 min of treatment of AuNB at US trigger (1 MHz frequency, at Intensity 1 for 30 seconds, 50% duty cycle) and THP-1 macrophages cell treated with 1X109/ml, 1X108/ml cells show more than 80% cell viability whereas at 1X1010 AuNB/ml treated THP-1 macrophages cell shows 60% cell viability. These results show that the 1×1010 and above concentrations of AuNBs with the US triggered can be used for reducing the macrophage population in the early stage of atherosclerotic plaque had shown positive results in stabilization of atherosclerotic plaque. Further, the hemolytic study with human RBCs (Figure 16c-d) indicates that RBCs incubated 1X1010 AuNB/ml for 24 h at 37 oC, shaking at 100 RPM shows 4.93±1.25 % homolysis. Thus, AuNB at a concentration up to 1X1010 /ml were biocompatible and can be safe to use for intravenous (i.v.) applications.

Further, as observed in Figure 16b, when 1X104 per well THP-1 macrophages treated with AuNB irradiated with US, cells shows reduced cell viability to 60%, and the ultrasound (US) trigger induces ROS-mediated apoptosis into the macrophages, thus studied the ROS activation effect on THP-1 cells treated with AuNBs (Figure 17a). Semiquantitative measurement of green fluorescence (Figure 17b) shows that after incubating 1X1010 AuNB/ml with US trigger, the intercellular ROS level in THP-1 macrophages increases. The US-triggered AuNB (Figure 17a-b) shows the highest generation of ROS compared to other treatment groups. The N-Acetylcysteine (NAC) was used as a ROS inhibitor. The US-triggered AuNB + NAC treated cells showed reduced ROS. In the case of US triggered, AuNB/ml treated cells show relatively lesser, but slightly higher ROS production when compared to AuNB/ml, and ultrasound only treated groups.

Furthermore, as observed in Figure 17, AuNB induces reactive oxygen species (ROS) in THP-1 macrophages, and with the US-triggered AuNB treated cells further increases ROS. Figure 17a shows representative ROS generated in THP-1 macrophage fluorescence microscopic images. Images at scale bar 75 µm is shown; blue florescence indicates nucleolus, green florescence indicates ROS. Figure 17b shows quantification of intercellular ROS in AuNB treated THP-1 macrophage cells. ROS plays a crucial role in mediating the biological effects induced by Sonodynamic therapy (SDT). STD is a promising technique that utilises US waves to deliver and activate a sonosensitizer locally. Because STD is non-invasive, strong tissue-penetrating, repeatable, and region-focusing properties, various research groups have recently investigated SDT application as an alternative non-chemotherapeutic treatment for atherosclerosis. Typically, in SDT, activating the sonosensitizer by the US generates ROS. Hence, the efficacy of AuNBs as reverse cholesterol efflux in Ox-LDL induced THP-1 foam cells were investigated. After 6 hours of treatment, the cells were fixed and stained with Oil-Red-O.

Figures 18a – 18b show quantification of Oil-O-red stained AuNBs treated foam cells and representative images of Oil-O-Red stained foam cells. The Oil-Red-O stinting quantification indicated that Ultrasound-triggered AuNBs promote foam cell regression, and US-triggered AuNBs show significant reduction as compared to other treatment groups (Figure 18a). Figure 18b represents Oil-O-red-stained Ox-LDL induced THP-1 foam cells treated with AuNBs. Similar results were reported for AuNP loaded with aminolevulinic acid. Further, no significant difference was observed in the case of cells treated with AuNB and US groups. Thus, these results infer that US-treated AuNB could be used as SDT agents and reduced in foamy cells.

Figures 19a – 19d show ultrasound imaging results demonstrating contrast enhancement after the introduction of AuNBs in accordance with another example of the present disclosure. For example and study, AuNBs (50ug/ml lipid concentration) were injected into mouse artery. Sonographs of mouse’s heart before and after injection of AuNBs were obtained. For the study, all mice are purchased and maintained as per the animal ethical guidelines at the Baker Heart and Diabetes Institute, Melbourne, Australia, under Alfred Plus Alliance Animal Ethics Committee no. P8453. As shown in Figures 19a – 19d, expected ultrasound contrast enhancement in mouse’s heart after injecting AuNBs into mouse artery was observed. Figure 19a shows side view Ultrasound image of mouse’s heart before injecting ultrasound contrast agent, AuNBs, and Figure 19b shows side view Ultrasound image of mouse’s heart after injecting AuNBs showing contrast enhancement. Figure 19c and 19d show sonographs obtained from mouse’s heart by placing ultrasound probe placed at 90 ? to the imaging site. Figure 19c shows Ultrasound image of mouse’s heart before injecting AuNBs, and Figure 19d shows Ultrasound image of mouse’s heart after injecting AuNBs showing contrast enhancement in mouse’s heart in left and right ventricles (highlighted by arrows), indicating AuNBs can be used as ultrasound contrast agent.

The present invention discloses a CT contrast agent developed (gold nanoparticles) directly on nanobubbles (gas stabilized vehicles of less than 1 micron size) due to self-assembly principle and amine-gold chemistry. The gold nanoparticles were directly developed (grown) and organized on the shell of nanobubble. This also improves the stability of ultrasound contrast agent. The developed gold nanoparticle coated nanobubbles (AuNBs) exhibits contrast enhancement in Ultrasound and CT. If required also can be used as photoacoustic imaging agent and similar imaging. With gold nanoparticle coating, the nanobubbles becomes inherent particle for functionalization, ready for molecular imaging or image guided delivery application. AuNBs can be used as ultrasound and CT contrast agents and also can be used as sonodynamic agent for treatment of diseases such as atherosclerosis and other inflammatory diseases such as arthritis and cancer. In the present invention, AuNBs enhance contrast in ultrasound and CT imaging as well as exhibit excellent therapeutic effect; hence AuNBs can also be used as theranostic agents.
,CLAIMS:1. A method of synthesizing an agent for multimodal imaging and theranostic applications, the agent comprising metal nanoparticles coated nanobubbles, the method comprising the steps of:
synthesizing lipid nanobubbles (LNBs) using lipids with a gas purging process;
forming a nanobubble suspension by adding at least one metal salt to the lipid nanobubbles (LNBs), followed by mixing the lipid nanobubbles (LNBs);
adding at least one reducing agent to the nanobubble suspension and mixing the nanobubble suspension till colour of nanobubble suspension gets changed; and
forming a coat on the surface of nanobubbles based on amount of metal nanoparticles grown in the nanobubble suspension, thereby synthesizing metal nanoparticles coated nanobubbles.
2. The method as claimed in claim 1, wherein lipid nanobubbles (LNBs) are synthesized using a scalable, one-step, one-pot process, wherein the process comprises:
dissolving lipids in an ethanal solution, thereby forming lipid dissolved ethanol;
purging the gas into a buffer solution of phosphate buffer saline (PBS) or distilled water obtained from a MiliQ filter system, maintained at a temperature and placing the buffer solution on a hot water bath;
simultaneously injecting lipid dissolved ethanol drop by drop into the buffer solution and the purging gas;
stirring the result lipid solution continuously using a high-speed homogenizer at a speed thereby forming lipid bubbles; and
centrifuging the formed lipid bubbles to remove excess lipid from a shell of nanobubbles and collecting the formulated nanobubbles (NBs).
3. The method as claimed in claim 1, wherein lipids comprise at least one of Distearoylphosphatidylcholine (DSPC), palmitic acid or any lipid having an amine group.
4. The method as claimed in claim 1, wherein the gas used in gas purge comprises one of Sulphur Hexafluoride (SF6) and ambient air.
5. The method as claimed in claim 1, wherein the gas used in gas purge comprises one of Perfluorocarbons, oxygen, and xenon.
6. The method as claimed in claim 1, wherein at least one metal salt comprises chlorides of at least one precious metal or metal alloys.
7. The method as claimed in claim 1, wherein the at least one precious metal or metal alloy is selected from Gold, Silver, Platinum, and Palladium, alloys of precious metals or a combination thereof, wherein the at least one precious metal or metal alloy has an affinity towards primary, secondary amine, tertiary amine group.
8. The method as claimed in claim 1, wherein the agent comprises gold nanoparticles coated nanobubbles (AuNBs).
9. The method as claimed in claim 1, wherein the reducing agent is selected from Ascorbic acid, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer.
10. The method as claimed in claim 1, wherein the nanobubble suspension is continuously mixed till white coloured nanobubbles turn red or blue or green colour.
11. The method as claimed in claim 1, wherein the metal nanoparticles coated nanobubbles are spherical in shape.
12. The method as claimed in claim 1, wherein a diameter of the metal nanoparticles coated nanobubbles is in a range of 300 nm – 500 nm.
13. The method as claimed in claim 1, wherein the size of metal nanoparticles is in a range of 30-40 nm.
14. The method as claimed in claim 1, wherein the agent is one of an Ultrasound contrast agent and a Computerized Tomography (CT) contrast agent.
15. A method of synthesizing an agent for multimodal imaging and theranostic applications, the agent comprising metal nanoparticles coated nanobubbles, the method comprising the steps of:
synthesizing lipid nanobubbles (LNBs) using lipids with a thin-film hydration gas purging process;
forming a nanobubble suspension by adding at least one metal salt and at least one reducing agent to the lipid nanobubbles (LNBs) at a ratio and mixing the nanobubble suspension till colour of nanobubble suspension gets changed; and
forming a coat on the surface of nanobubbles based on amount of metal nanoparticles grown in the nanobubble suspension, thereby synthesizing metal nanoparticle coated nanobubbles.
16. The method as claimed in claim 1, wherein at least one metal salt and at least one reducing agent is added at a ratio selected from 1:1, 1:2, 1:4.