Characterization of Dextran Coated ICG Characterization of Dextran Coated ICG Containing NACsContaining NACs

1:18 ratio, sample 2

Rainya Bridges, Bongsu Jung, and Bahman Anvari

IntroductionIndocyanine 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).

ObjectiveObjective

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

Three-Step Mesocapsule AssemblyThree-Step Mesocapsule Assembly

+PAH Na2HPO4

ICG

Dextran

ICG Containing Mesocapsule

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

0.60

0.55

0.50

0.45

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0.35

0.30E

xtin

ctio

n [

arbi

t.u.]

12001000800600400Wavelength [nm]

ICG_capsule 2 week old

1.8

1.6

1.4

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Ext

inct

ion

[ ar

bit.u

.]

12001000800600400Wavelength [nm]

nonICG_capsule freshly made

Non-ICG Containing NACs ICG Containing NACs

Mesocapsule SizeMesocapsule Size

The first part of our investigation involved The first part of our investigation involved attempting to correlate the PAH: Naattempting to correlate the PAH: Na22HPOHPO44 ratio ratio

with possible trends in mean particle size. with possible trends in mean particle size.

Ratio samples ranging from 1:2 to 1:20 were Ratio samples ranging from 1:2 to 1:20 were prepared and imaged.prepared and imaged.

All images were analyzed using Imagej All images were analyzed using Imagej software.software.

1:2 – 1:10 Ratio Solutions

1:4 Ratio Solution, Sample 1, Image 101:4 Ratio Solution, Sample 1, Image 10 1:6 Ratio Solution, Sample 2, Image 71:6 Ratio Solution, Sample 2, Image 7

1:8 Ratio Solution, Sample 2, Image 21:8 Ratio Solution, Sample 2, Image 2 1:10 Ratio Solution, Sample 1, Image 91:10 Ratio Solution, Sample 1, Image 9

1:2 Ratio Solution, Sample 1, Image 1

1:12 – 1:20 Ratio Solutions1:12 – 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 Trend Graph For Entire Data Set Including Both SamplesIncluding Both Samples

Mean Particle Size in Relation to Ratio Solution

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1:02 1:04 1:06 1:08 1:10 1:12 1:14 1:16 1:18 1:20

Mixture Ratio PAH:Na2HPO4

Par

ticl

e S

ize

(um

)

Particle Size

Morphology of NACsMorphology of NACs

These images demonstrate normal mesocapsule These images demonstrate normal mesocapsule morphology. (a) 1:4 Ratio image taken from sample 1,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

a b

1:4 Ratio Solution (XL30)1:4 Ratio Solution (XL30)

Image of ICG Image of ICG containing NACs containing NACs taken on a XL30 taken on a XL30 SEM. Sample was SEM. Sample was sputter-coated for sputter-coated for 60 seconds before 60 seconds before imagingimaging

(Sample 1)(Sample 1)

1:18 Ratio Solution (XL30)1:18 Ratio Solution (XL30)

Image taken on a Image taken on a XL30 SEM. Note XL30 SEM. Note the smaller the smaller capsules capsules interspersed interspersed among the larger among the larger particles.particles.

Size Difference in ICG Containing Size Difference in ICG Containing NACsNACs

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.

a b

Mesocapsule AggregationMesocapsule Aggregation

The second part of our investigation involved The second part of our investigation involved attempting to correlate the PAH: Naattempting to correlate the PAH: Na22HPOHPO44 ratio to ratio to

mean particle aggregation. Particle count for each mean particle aggregation. Particle count for each ratio sample image was approximated using ImageJ ratio sample image was approximated using ImageJ software.software.

Mesocapsule aggregation throughout the following Mesocapsule aggregation throughout the following samples was random, with only the 1:4 ratio solution samples was random, with only the 1:4 ratio solution showing something approaching consistent showing something approaching consistent aggregation throughout all data points.aggregation throughout all data points.

Examples of Even AggregationExamples 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 AggregationUneven Aggregation

These two images illustrate the uneven aggregation These two images illustrate the uneven aggregation between images from the same ratio set.between images from the same ratio set.

1:8 Ratio Solution, Sample 2 Image 9 1:8 Ratio Solution, Sample 2 Image 2

Heavy Edge AggregationHeavy Edge Aggregation

Another factor that served to hinder analysis was the Another factor that served to hinder analysis was the presence of heavy edge aggregation along the presence of heavy edge aggregation along the perimeter of the sample, as illustrated in these two perimeter of the sample, as illustrated in these two images.images.

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

Aggregation Trend Graph for Entire Aggregation Trend Graph for Entire Data Set Including Both SamplesData Set Including Both Samples

Mean Mesocapsule Aggregation in Relation to Ratio Solution

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1:02 1:04 1:06 1:08 1:10 1:12 1:14 1:16 1:18 1:20

Mixture Ratio PAH:Na2HPO4

Part

icle

Ag

gre

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Particle Aggregation

Conclusions: Mesocapsule SizeConclusions: Mesocapsule Size

Mean particle size increases as the ratio of PAH: Mean particle size increases as the ratio of PAH: NaNa22HPOHPO44 increases, suggesting a direct link increases, suggesting a direct link between particle size and solution ratio.between particle size and solution ratio.Mean particle size increased at an average of 70 Mean particle size increased at an average of 70 nm per ratio change throughout the entire data nm per ratio change throughout the entire data set.set.Increase in particle size is separated into two Increase in particle size is separated into two distinct categories. In ratio solutions 1:4- 1:12 the distinct categories. In ratio solutions 1:4- 1:12 the average increase is 82 nm. This declines average increase is 82 nm. This declines dramatically after the 1:12 mark, with solutions dramatically after the 1:12 mark, with solutions 1:14 – 1:20 having a mean size increase of only 1:14 – 1:20 having a mean size increase of only 20 nm per ratio change.20 nm per ratio change.

Conclusions: AggregationConclusions: Aggregation

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

Viability of ICG Containing NACsViability of ICG Containing NACs

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

Non-ICG Containing MesocapsulesNon-ICG Containing Mesocapsules

a b

Image (a) 34 day old 1:4 ratio solutionImage (a) 34 day old 1:4 ratio solutionImage (b) 34 day old 1:8 ratio solutionImage (b) 34 day old 1:8 ratio solution

ICG Containing MesocapsulesICG Containing Mesocapsules

a b

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

Conclusions: Mesocapsule ViabilityConclusions: Mesocapsule Viability

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

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

Capsule Size in Relation to Differing Precursor Concentration

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Datapoint

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2 mg/ml PAH:.01 M Na2HPO4

1 mg/ml PAH.025 MNa2HPO42 mg/ml PAH ;.025 MNa2HPO41 mg/ml PAH ;.005 MNa2HPO4

a b

c

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

Future ResearchFuture 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.

AcknowledgementsAcknowledgements

I would like to thank the following people for their I would like to thank the following people for their support, time, and understanding.support, time, and understanding.

Bongsu JungBongsu Jung

Prof. Bahman AnvariProf. Bahman Anvari

Jun WangJun Wang

I would also like to thank NSF and UCR for the I would also like to thank NSF and UCR for the research opportunityresearch opportunity

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