Preparation Of Nanobubbles For
Ultrasound Imaging And Intracellular
Drug Delivery
Madan Baral
B. Pharm.
School of Health a...
OVERVIEW
 Introduction
 Materials and Methods
• Materials
• Apparatus
• Preparation of nanobubbles for contrast agent
• ...
Introduction
• Nanomedicine : Fusion of nanotechnology and medicine
• Nanobubble : Nanobubbles are gas cavities in aqueous...
Nanobubbles
Discussion
• Gas-filled bubbles, commonly used as echo-enhances in
ultrasonic diagnosis, were prepared in micrometer size....
Methodology
Materials
• SF6 ( Sulphur Hexafluoride )
• Soyabean Lipid ( SPC )
• Tween 80
• Cholesterol (CHO)
• Coumarin-6
• Other anal...
Apparatus
• Ultrasonic emission instrument (40kHz, 250W)
• B-mode ultrasonic biological microscope (UBM)
• Nano-S90 Zetasi...
Preparation of nanobubbles for contrast agent
Low
Pressur
e
Lipids (SPC)
Additives (Tween
80 & CHO)
Acetonitrile
Mixture
R...
Formula Influence of the Nanobubbles
• An investigation of three important components was carried by
changing one of the a...
B-mode Ultrasonic Biological Microscopy
(UBM)
In vitro ultrasonic imaging of bubbles
and acoustic quality evaluation
• The ultrasound contrast results were observed by ...
In Vivo Ultrasonic Imaging
• A female nude mouse was obtained from Slaccas (Shanghai, CHN) and
used at 6 weeks for in vivo...
Drug Load Nanobubbles Preparation
• Coumarin- 6 dissolved in soybean oil, and the oil phase
was then mixed with lipid solu...
Contd.
• Emulsion was prepared using the method of bubbles preparation without
loading gas.
• Liposome preparation done by...
MCF-7 Cell Culture And Vector Mediated
Drug Uptake
• 2μl of formulation was added in each well of cultured and incubated C...
RESULTS
In Vitro Ultrasonic Contrast Observation of
Nanobubbles
Fig 1: The prepared nanobubbles presents as bright spots viewed by...
Nanobubbles Acoustic Quality Evaluation
• Four parameters used to quantify and investigate
the influence factors:
a) Regio...
Nanobubbles Acoustic Quality Evaluation
Fig 2: Influence of
Tween 80 on the
acoustic property of
nanobubbles. The solid
li...
Influence of Cholesterol on acoustic
property of nanobubbles
Fig 3: The solid line is
the NBS actually
counted, the dashed...
Influence of Lipid Concentration On The
Acoustic Property of Nanobubble
Fig 4: Influence of lipid
concentration on the
aco...
In Vivo Ultrasonic Imaging
Fig 5: B-mode ultrasound image of nanobubbles in a nude mouse after
intravenous injection of bu...
Tumor Cell Uptake of Coumarin -6 Loaded
Bubbles
Fig 6: Treated by
different preparations,
the coumarin-6 cell
uptaken in M...
Influence of Tween Modified Preparations
Fig 7: Treated by
different preparations,
the coumarin-6 cell
uptaken in MCF-7
ce...
Detailed Parameters
Conclusion
• The nanobubbles reported in this paper can work
as an ultrasound contrast agent and a drug delivery
vector. N...
Reference
• Wanga Y., Li X., Zhou Y., Huanga P., Xua Y., Preparation of
nanobubbles for ultrasound imaging and intracellua...
Thank You !!!!!!!!!!!
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Preparation of Nanobubbles for Novel Drug Delivery

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This is a promising technique in novel durg delivery.

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  • Bubbles have explosive property and under ultra sound energy illumination cellular pembrne permeability changes give the effective imaging. Initially OFP filled nanobubbles 450nm-700 nm used in echo enhancement in live spleen tumour etc.
  • This instrument could obtain the real-time ultrasonic signal and photos.
  • After in vitro formula screening of the above section, the in vivo test was done.
  • Preparation of Nanobubbles for Novel Drug Delivery

    1. 1. Preparation Of Nanobubbles For Ultrasound Imaging And Intracellular Drug Delivery Madan Baral B. Pharm. School of Health and Allied Sciences Pokhara University, PO Box 427, Lekhnath-12, Kaski, NEPAL
    2. 2. OVERVIEW  Introduction  Materials and Methods • Materials • Apparatus • Preparation of nanobubbles for contrast agent • Formulation influence of nanobubbles • In vitro ultrasonic imaging of bubbles and acoustic quality • In vivo ultrasonic imaging • Drug load nanobubbles preparation • MCF-7 cell culture and vector mediated drug uptake  Results and Discussion  Conclusion
    3. 3. Introduction • Nanomedicine : Fusion of nanotechnology and medicine • Nanobubble : Nanobubbles are gas cavities in aqueous solution with the size ranging 25-1000nm in radius. • Applications: – Ultrasonic imaging contrast agent. – Intracellular drug delivery. – electrolyte stabled ozone nanobubbles to sterilize materials for many months. – Oxygen nanobubbles in the prevention of arteriosclerosis. – Preservation of living tissue and organisms. • Objective : To prepare the nanobubble and assess the ability as contrast agent for ultrasound imaging and drug delivery vector. • In vitro and in vivo tests suggested nanobubbles can work as an ultrasound contrast agent and a drug delivery vector
    4. 4. Nanobubbles
    5. 5. Discussion • Gas-filled bubbles, commonly used as echo-enhances in ultrasonic diagnosis, were prepared in micrometer size. Echo signals are proportional to the bubbles size and acoustic backscatter intensity is negatively correlated. • Since nanoparticles are invisible to the ultrasonic imager an ultrasound microscope device with higher ultrasound frequency is used in order to characterize and optimize the nanobubbles echo intensities. • Nanobubbles showed higher delivery efficiency than both the liposome and the emulsion. • The presence of Tween 80 in the formulation suggests the increase in the drug uptake.
    6. 6. Methodology
    7. 7. Materials • SF6 ( Sulphur Hexafluoride ) • Soyabean Lipid ( SPC ) • Tween 80 • Cholesterol (CHO) • Coumarin-6 • Other analytically purified
    8. 8. Apparatus • Ultrasonic emission instrument (40kHz, 250W) • B-mode ultrasonic biological microscope (UBM) • Nano-S90 Zetasizer • Infinite M200 multi-functional plate reader • MATLAB Software • Origin Software
    9. 9. Preparation of nanobubbles for contrast agent Low Pressur e Lipids (SPC) Additives (Tween 80 & CHO) Acetonitrile Mixture Residue Resuspended in Saline Upper Visible Foam Removed CCl4 Sonication 5min.
    10. 10. Formula Influence of the Nanobubbles • An investigation of three important components was carried by changing one of the agents while the other two unvaried • Generally, soybean lipid (SPC) was chosen as the bubble film. • Tween-80 and cholesterol (CHO) were added as the additives. a) Tween 80 concentration was varied from 0% to 3%, the lipid concentration was 5mg/ml, and SPC: CHO was 8:1 (molar ratio). b) Lipid concentration was varied from 1mg/ml to 8 mg/ml, meanwhile Tween 80 concentration was 1%, and SPC: CHO is 8:1. c) SPC: CHO was varied from 8:0 to 8:8, meanwhile Tween 80 concentration was 1%, and lipid concentration was 5mg/ml.
    11. 11. B-mode Ultrasonic Biological Microscopy (UBM)
    12. 12. In vitro ultrasonic imaging of bubbles and acoustic quality evaluation • The ultrasound contrast results were observed by a B-mode ultrasonic biological microscopy (UBM). • Freshly prepared bubble formulation was transferred into a glass beaker • Pre-covered with a layer of solid agarose to avoid the acoustic reflection of glass. • The probe of the UBM was immersed into the liquid to get the ultrasonic image, the image photos were taken for 10 s to get 100 photos, with sampling interval of 0.1 s. • Matlab software was utilized for counting the bubbles of the UBM.
    13. 13. In Vivo Ultrasonic Imaging • A female nude mouse was obtained from Slaccas (Shanghai, CHN) and used at 6 weeks for in vivo evaluation of the ultrasonic contrast effect on the bubbles. • Optimized formulation was chosen for in vivo testing. 30mg of SPC, CHO and DPPG were mixed and suspended in 10ml of saline (1% Tween 80 contained) with the molar ratio of 8:1:1, sonicatd by probe sonication with final lipid concentration of 3mg/ml. • Formulations were injected into the nude mouse through the tail vein. • The UBM investigation for the contrast enhancement was done at liver region.
    14. 14. Drug Load Nanobubbles Preparation • Coumarin- 6 dissolved in soybean oil, and the oil phase was then mixed with lipid solution (1:40, O/W, v/v)followed by similar prior operations with final lipid concentration 3 mg/ml. • Emulsion, liposome and chitosan nanoparticle (CNP) were also prepared for the cell test, they were taken as control agents for nanobubbles. Coumarin-6 and lipid concentration maintained at 20μg/ml and 3ml respectively.
    15. 15. Contd. • Emulsion was prepared using the method of bubbles preparation without loading gas. • Liposome preparation done by dissolving 30 mg of SPC in chloroform together with the drug, then organic solvent was evaporated by a reduced pressure evaporator, then the residual film was washed by 10ml of saline (contained 0.1% of Tween 80) to form the liposome. • CNP was prepared through a crosslinking way. In brief, a certain quantity of drugs were dissolved in 1.5ml of 0.1% sodium poly phosphate (MPP), and then the mixture was added into 8.5ml of 0.5% chitosan acid solution dropwise until the opalescence emerged.
    16. 16. MCF-7 Cell Culture And Vector Mediated Drug Uptake • 2μl of formulation was added in each well of cultured and incubated Castor plate containing human breast carcinoma cells (MCF-7) to quantify the Coumarin-6 uptake my tumor cells. • Wells were then washed by PBS (pH 7.4) twice. • Subsequently, 100 μl of cell lysis buffer was precisely added into the wells to lyse the cells. • The lysate was then centrifuged (10,000 rpm, 5 min), 50μl of supernatant was then added into a black opaque Costar assay plate to detect the concentration of coumarin-6 in an infinite M200 multifunctional plate reader. The excitation and emission wavelength were 456 nm and 504 nm, respectively. • Data analysis was performed by Origin software.
    17. 17. RESULTS
    18. 18. In Vitro Ultrasonic Contrast Observation of Nanobubbles Fig 1: The prepared nanobubbles presents as bright spots viewed by the UBM.
    19. 19. Nanobubbles Acoustic Quality Evaluation • Four parameters used to quantify and investigate the influence factors: a) Region of Interest (ROI) b) Time of interest (TOI) c) Number of bright spots (NBS) d) MNTI (mean NBS and TOI)
    20. 20. Nanobubbles Acoustic Quality Evaluation Fig 2: Influence of Tween 80 on the acoustic property of nanobubbles. The solid line is the NBS actually counted, the dashed line is MNTI calculated based on NBS. MNTI is displayed in the corner figure.
    21. 21. Influence of Cholesterol on acoustic property of nanobubbles Fig 3: The solid line is the NBS actually counted, the dashed line is MNTI calculated based on NBS. MNTI is displayed in the corner figure.
    22. 22. Influence of Lipid Concentration On The Acoustic Property of Nanobubble Fig 4: Influence of lipid concentration on the acoustic property of nanobubbles. The solid line is the NBS actually counted, the dashed line is MNTI calculated based on NBS. MNTI is displayed in the corner figure.
    23. 23. In Vivo Ultrasonic Imaging Fig 5: B-mode ultrasound image of nanobubbles in a nude mouse after intravenous injection of bubble preparation, the red frame demonstrates the echo enhancement in the liver region. (A) Pre-injection; (B) post-injection.
    24. 24. Tumor Cell Uptake of Coumarin -6 Loaded Bubbles Fig 6: Treated by different preparations, the coumarin-6 cell uptaken in MCF-7 cells. Solid line is the real- time depended drug concentration.
    25. 25. Influence of Tween Modified Preparations Fig 7: Treated by different preparations, the coumarin-6 cell uptaken in MCF-7 cells.
    26. 26. Detailed Parameters
    27. 27. Conclusion • The nanobubbles reported in this paper can work as an ultrasound contrast agent and a drug delivery vector. Nanotechnology will allow earlier diagnosis and therapy, the combination of ultrasound imaging and drug delivery could be highly beneficial and may be achievable with further development of nanobubble formulations.
    28. 28. Reference • Wanga Y., Li X., Zhou Y., Huanga P., Xua Y., Preparation of nanobubbles for ultrasound imaging and intracelluar drug delivery, International Journal of Pharmaceutics 384 (2010) 148–153.
    29. 29. Thank You !!!!!!!!!!!

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