This is a promising technique in novel durg delivery.

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. 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. 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. Nanobubbles

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. Methodology

7. Materials
• SF6 ( Sulphur Hexafluoride )
• Soyabean Lipid ( SPC )
• Tween 80
• Cholesterol (CHO)
• Coumarin-6
• Other analytically purified

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. 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. 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. B-mode Ultrasonic Biological Microscopy
(UBM)

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. 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. 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. 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. 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. RESULTS

18. In Vitro Ultrasonic Contrast Observation of
Nanobubbles
Fig 1: The prepared nanobubbles presents as bright spots viewed by the UBM.

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. 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. 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. 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. 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. 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. Influence of Tween Modified Preparations
Fig 7: Treated by
different preparations,
the coumarin-6 cell
uptaken in MCF-7
cells.

26. Detailed Parameters

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. 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. Thank You !!!!!!!!!!!