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. 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 (Na 2 HPO 4 ).

Objective Determine if a connection exists between the ratio of PAH to Determine if a connection exists between the ratio of PAH to Na 2 HPO 4 and mesocapsule size and aggregation.

Three-Step Mesocapsule Assembly + PAHNa 2 HPO 4 ICG Dextran ICG Containing Mesocapsule

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

Mesocapsule Size The first part of our investigation involved attempting to correlate the PAH: Na 2 HPO 4 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. All images were analyzed using Imagej software.

1:2 – 1:10 Ratio Solutions 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 1:2 Ratio Solution, Sample 1, Image 1

1:12 – 1:20 Ratio Solutions 1:18 Ratio Solution, Sample 2, Image 9 1:16 Ratio Solution, Sample 2, Image 71:14 Ratio Solution, Sample 1, Image 71:12 Ratio Solution, Sample 1, Image 9 1:20 Ratio Solution, Sample 2 Image 8

Trend Graph For Entire Data Set Including Both Samples

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 (b) 1:16 ratio sample image taken from sample 2 ab

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)

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

Size Difference in ICG Containing NACs 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. ab

Mesocapsule Aggregation The second part of our investigation involved attempting to correlate the PAH: Na 2 HPO 4 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.

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

Uneven Aggregation These two images illustrate the uneven aggregation between images from the same ratio set. 1:8 Ratio Solution, Sample 2 Image 91:8 Ratio Solution, Sample 2 Image 2

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 6 1:6 Ratio Solution, Sample 2, Image 10

Aggregation Trend Graph for Entire Data Set Including Both Samples

Conclusions: Mesocapsule Size Mean particle size increases as the ratio of PAH: Na 2 HPO 4 increases, suggesting a direct link between particle size and solution ratio. Mean particle size increases as the ratio of PAH: Na 2 HPO 4 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.

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.

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.

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

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

Conclusions: Mesocapsule Viability Mesocapsules composed solely of PAH, in which ICG has been incorporated. Mesocapsules composed solely of PAH, Na 2 HPO 4 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.

Future Research Using the precursor (PAH, Using the precursor (PAH, Na 2 HPO 4 ) concentration to control capsule size.

ab c Examples of varying capsule size through precursor concentration. (a) 2 mg/ml PAH;.01 M Na 2 HPO 4 (b) 1mg/ml PAH;.005 M Na 2 HPO 4 (c) 1 mg/ml PAH ;.025 M Na 2 HPO 4

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.

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

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