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Characterization of Dextran Coated ICG Containing NACs

Characterization of Dextran Coated ICG Containing NACs. 1:18 ratio, sample 2. Rainya Bridges, Bongsu Jung, and Bahman Anvari. Introduction Indocyanine green (ICG) is a tricarbocyanine dye used in clinical settings to determine hepatic function and macular degeneration.

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Characterization of Dextran Coated ICG Containing NACs

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  1. Characterization of Dextran Coated ICG Containing NACs 1:18 ratio, sample 2 Rainya Bridges, Bongsu Jung, and Bahman Anvari

  2. Introduction • Indocyanine green (ICG) is a tricarbocyanine dye used in clinical settings to determine hepatic function and macular degeneration. • To increase the therapeutic range of ICG several encapsulation methods have been used • Despite these investigations no serious study has been given to the characterization of ICG containing mesocapsules as affected by the ratio of polyally-lamine hydrochloride (PAH) to disodium hydrogen phosphate heptahydrate (Na2HPO4).

  3. Objective • Determine if a connection exists between the ratio of PAH to Na2HPO4 and mesocapsule size and aggregation.

  4. Three-Step Mesocapsule Assembly + PAH Na2HPO4 ICG Dextran ICG Containing Mesocapsule

  5. Spectrum Analysis of ICG and Non-ICG Containing NACs Non-ICG Containing NACs ICG Containing NACs

  6. Mesocapsule Size • The first part of our investigation involved attempting to correlate the PAH: Na2HPO4 ratio with possible trends in mean particle size. • Ratio samples ranging from 1:2 to 1:20 were prepared and imaged. • All images were analyzed using Imagej software.

  7. 1:2 – 1:10 Ratio Solutions 1:2 Ratio Solution, Sample 1, Image 1 1:4 Ratio Solution, Sample 1, Image 10 1:6 Ratio Solution, Sample 2, Image 7 1:8 Ratio Solution, Sample 2, Image 2 1:10 Ratio Solution, Sample 1, Image 9

  8. 1:12 Ratio Solution, Sample 1, Image 9 1:14 Ratio Solution, Sample 1, Image 7 1:16 Ratio Solution, Sample 2, Image 7 1:18 Ratio Solution, Sample 2, Image 9 1:20 Ratio Solution, Sample 2 Image 8 1:12 – 1:20 Ratio Solutions

  9. Trend Graph For Entire Data Set Including Both Samples

  10. Morphology of NACs • These images demonstrate normal mesocapsule morphology. (a) 1:4 Ratio image taken from sample 1, (b) 1:16 ratio sample image taken from sample 2 a b

  11. 1:4 Ratio Solution (XL30) • Image of ICG containing NACs taken on a XL30 SEM. Sample was sputter-coated for 60 seconds before imaging (Sample 1)

  12. 1:18 Ratio Solution (XL30) • Image taken on a XL30 SEM. Note the smaller capsules interspersed among the larger particles.

  13. Size Difference in ICG Containing NACs a b A side-by-side comparison illustrates the change in mean mesocapsule size. Image (a)1:18 ratio solution. Image (b) 1:4 ratio solution sample.

  14. Mesocapsule Aggregation • The second part of our investigation involved attempting to correlate the PAH: Na2HPO4 ratio to mean particle aggregation. Particle count for each ratio sample image was approximated using ImageJ software. • Mesocapsule aggregation throughout the following samples was random, with only the 1:4 ratio solution showing something approaching consistent aggregation throughout all data points.

  15. Examples of Even Aggregation 1:14 Ratio Solution, Sample 2 Image 7 1:4 Ratio Solution, Sample 1, Image 1 1:14 Ratio Solution, Sample 1 Image 10

  16. Uneven Aggregation • These two images illustrate the uneven aggregation between images from the same ratio set. 1:8 Ratio Solution, Sample 2 Image 9 1:8 Ratio Solution, Sample 2 Image 2

  17. Heavy Edge Aggregation • Another factor that served to hinder analysis was the presence of heavy edge aggregation along the perimeter of the sample, as illustrated in these two images. 1:6 Ratio Solution, Sample 2, Image 10 1:6 Ratio Solution, Sample 2, Image 6

  18. Aggregation Trend Graph for Entire Data Set Including Both Samples

  19. Conclusions: Mesocapsule Size • Mean particle size increases as the ratio of PAH: Na2HPO4 increases, suggesting a direct link between particle size and solution ratio. • Mean particle size increased at an average of 70 nm per ratio change throughout the entire data set. • Increase in particle size is separated into two distinct categories. In ratio solutions 1:4- 1:12 the average increase is 82 nm. This declines dramatically after the 1:12 mark, with solutions 1:14 – 1:20 having a mean size increase of only 20 nm per ratio change.

  20. Conclusions: Aggregation • Both SEM and bright-field imaging proved ineffective means of determining particle aggregation • Another method must be found to more accurately quantify particles, preferably in solution • A coulter counter (range > 400 nm) or dynamic light scattering may prove more effective in determining aggregation.

  21. Viability of ICG Containing NACs • A brief imaging study was done to compare the viability of ICG containing NACs with those of non-ICG containing NACs. Two 34 day old samples of non-ICG containing mesocapsules were vortexed and imaged to assess viability. As a comparison, 34 day old and 29 day old samples of ICG containing NACs were also vortex and imaged.

  22. Non-ICG Containing Mesocapsules a b • Image (a) 34 day old 1:4 ratio solution • Image (b) 34 day old 1:8 ratio solution

  23. ICG Containing Mesocapsules a b • Image (a) 34 day old 1:6 ratio solution sample containing ICG. • Image (b) 29 day old ratio solution sample containing ICG

  24. Conclusions: Mesocapsule Viability • Mesocapsules composed solely of PAH, Na2HPO4 and dextran are less stable than those in which ICG has been incorporated. • The positively charged salt-aggregate core of the mesocapsules is unable to sustain itself over extended periods of time. • By adding negatively charged ICG the overall charge of the particle becomes neutral, creating a stable capsule.

  25. Future Research • Using the precursor (PAH, Na2HPO4) concentration to control capsule size.

  26. a b Examples of varying capsule size through precursor concentration. (a) 2 mg/ml PAH; .01 M Na2HPO4 (b) 1mg/ml PAH; .005 M Na2HPO4 (c) 1 mg/ml PAH ; .025 M Na2HPO4 c

  27. Future Research • Investigating new polymers for the mesocapsule shell, such as iron oxide • Cell studies to determine the behavior of mesocapsules in living tissue. • ‘Programming’ mesocapsules to target cancer cells.

  28. Acknowledgements I would like to thank the following people for their support, time, and understanding. Bongsu Jung Prof. Bahman Anvari Jun Wang I would also like to thank NSF and UCR for the research opportunity

  29. References • “Indocyanine green solution”, National Cancer Institute Drug Dictionary [online]; accessed 29 July 2008; available from http://www.cancer.gov/Templates/drugdictionary.aspx?CdrI D=540122. • J.Yu, M.A. Yaseen, B. Anvari, and M.S. Wong, “Synthesis of near-infrared-absorbing nanoparticle-assembled capsules”, Chem. Matter, 2007, 19, 1277-1284 • M.A. Yaseen, J. Yu, and B. Anvari, “Stability assessment of I ndocyanine green within dextran-coated mesocapsules by absorbance spectroscopy”, J. of Biomed. Optics, 12(6), (2007). • M.A. Yaseen, J. Yu, M.S. Wong, and B. Anvari, “Laster-induced heating of dextran-coated mesocapsules containing indocyanine green”, Biotechnol. Prog. 2007, 23, 1431-1440. • N. Kudo and K. Yamamoto, “Impact of bubbles on ultrasound safety”, International Congress Series, [online]; accessed 29 July 2008; available from http://dx.doi.org/10.1016/j.ics.2004.07.036.

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