Introduction
Over the years pharmaceuticals have been trying to find multiple ways to improve the drug delivery system for cancer. Microgels and hydrogels were first established, and then the new creations of nanogels were created. Nanogels have grown to have a huge impact for the use of certain anticancer drugs. Although characteristics of nanogels are still being studied by recent researchers they tend to have many advanced features as a drug delivery system. Some features include: simplicity of drug formulation; high drug-loading capacity; exceptional dispersion stability; and prolonged storage of drug formulations in the freeze-dried form. [1] Nanogels are cross linked particles of sub-micrometer size, made up of hydrophilic polymers. There three-dimensional polymer chains are formed by using covalent linkages or the self-assembly processes. [2] A polymer with two different properties such as soluble and insoluble is called an amphiphilic polymer. When the polymer is added to water it creates nanoparticles. The outside of the nanoparticle is called the shell and the center is called the core. When the core is cross linked it then forms a nanogel. Within this experiment cysteamine hydrochloride is added to create crosslink chains in side of the nanogel and TCEP is then added to cleave the disulfide bonds within the gel. Because Nile red is insoluble in water it is added before water during the cross linking reaction. Without the addition of cysteamine hydrochloride the Nile red solvent would not encapsulate inside of the nanogel. To use the nanogel as a drug delivery system, first the drug must be encapsulated inside of the gel. When the gel enters the body, it then enters the blood stream and the drug is release. Depending on the size and the effectiveness of the gel it determines whether the drug is released at a fast or slow paste. If the drug is released at a fast paste it will only affect a small portion of the drug reaching the cancer tissues, causing non-targeted drug release, low drug efficacy, toxicity to healthy tissues, and less medication available to fight the cancer. [3] If the drug is released at a slow paste the drug cannot build up a concentration higher than the cells' ability to purge itself of the drug, leading to ineffective killing of the cancer cells. [3] Nanogels, microgels, and hydrogels, have similar internal structures but varies in size. Because there is no actual define size of a nanogel various research groups, concluded that the average size should range from 100nm-200 nm to have better effect. [3] For an example when drug loaded nanogels with the size of 100nm enter the body, they are able to reach the smallest capillaries and as a result it builds up in the sites with a high density of targeted receptors. Therefore the size of a nanogel should remain small, simply because it has better effects rather than being large in size. My goal is to analyze different size nanogel samples with the addition of different percentages of cystamine hydrochloride and TCEP.
Materials
All chemicals and solvents were purchased from Sigma-Aldrich Company.
Methods
Synthesis of Polymer PDA-co-PEG
Polydopamine and poly (ethylene glycol) was synthesized by adding 250 mg PDA, 478.8 mg PEG, and 14.61 mg AIBN to a 100 ml round flask beaker with 10 ml anisole inside of it. To carry out this synthesis I began by degassing 10 ml of anisole in a 100 ml sealed round flask for ten minutes. While waiting ten minutes I prepared PDA, PEG, and AIBN, by weighing them out into three separate tubes and adding 1 ml of anisole to each tube. After ten minutes of degassing the anisole, PDA, PEG and AIBN was added drop wise into the flask, while waiting 30 minutes in-between each addition. While waiting for all solutions to be added the heater was set to warm at 65 C. Once all of the solutions were added the round bottom flask was placed to heat and stir for 24 hr. After 24 hr the product was precipitated using ice cool ether, to dissolve the polymer. This step was carried out by first adding ether to the product, second vortexting the product, and third centrifuging the product for 10 minutes at a high speed. The product was then removed and the solvent was poured out. Precipitation of the polymer was done 3X. Finally the polymer was placed in a vacuum oven to dry and remove the remaining solvent for 24 hr. All polymers were synthesized using the same technique but with different ratios Polymer | PDA-Aocl | PEG | AIBN | Ratio | I | 250 mg | 478.8 mg | 14.610mg | 1:05 | II | 250 mg | 239.4 mg | 10.89 mg | 1:1 | II | 250 mg | 718.2 mg | 18.69 mg | 1:1.5 |
Table 1. Properties and ratio of Polymers
Table 1. Properties and ratio of Polymers

Nanogel
Nanogel

Figure 1. Schematic representation of PDA-co-PEG synthesis
Figure 1. Schematic representation of PDA-co-PEG synthesis

Nanoparticle Preparation To determine the quantification of pyridine in each polymer, nanoparticles were dissolved in DMSO, and measured using UV spectroscopy at 375nm. This was done by preparing a 1mg/1ml solution of the polymer and DTT. Three tubes were labeled one to three and the other three tubes were labeled one DTT to three DTT. 3 mg of the polymer was weighed out and 3ml of DMSO was added to form the solution. 4.5 mg of DTT was weighed and 300 ul of DMSO was added to form the solution. After waiting ten minutes, 500 ul of the polymer was added to all six tubes, and 100 ul of DTT was added only to the three tubes that were labeled with DTT. To make all solutions equal 100 ul of DMSO was then added to the other three tubes that were labeled one to three. After all measurements were final the solutions sat for 30 minutes and then were taken to be measured. The three tubes that were labeled with one to three were used as a blank while the tubes with DTT were used as the samples. Nanogel Preparation
Based on the quantification calculations of all three polymers, I was able to determine what percentages of TCEP and cysteamine were needed to create each nanogel sample. Each of the polymers was divided upon the different percentages to evaluate which sample s would form a better or smaller nanogel in size. To form the nanogel samples, each polymer one was divided into groups of 5%, 10%, and 20% cysteamine and then groups of 20%,40%, and 80% TCEP, which gave me a variety of dissimilar samples. In each sample 2 mg / 300 ul DMSO of the polymer was added to each tube, and then addition of cysteamine was added in the tubes. After adding both the polymer and cysteamine together in each tube, they were left overnight to react.
Nanogel Preparation Finishing Point
To complete the samples, TCEP and Neil red was added to the tubes. Finally after adding all mixtures I added 50 ul of Nile red to stain the samples. After vortexting the solutions to make sure that they are all mixed I waited 15 minutes then removed the solutions into a small glass bottle. 2ml of H2O was added to make the polymer soluble, and then placed on a stir for 3 hrs. Once the 3hrs was up the nanoparticle solutions was dialyzed through spectator dialysis tube with a sodium phosphate buffer for 12 hrs. Subsequent to the 12hrs the nanoparticle solution was removed. 1ml was centrifuge for 5 min with a speed of 40000 rcf, and then was measured.
Results and Discussion
Characterization of Nanogel
Zeta Sizer machine was used to measure the size and zeta potential of each nanogel samples.
Figure 1. Visual characteristics of different nanogel samples.
Sample 1 resulted in larger size and darker color due to less cysteamine and TCEP. Sample 2 resulted in smaller size due to less cysteamine and TCEP
Sample 3 resulted in-between size due to increase of cysteamine and TCEP
Figure 1. Visual characteristics of different nanogel samples.
Sample 1 resulted in larger size and darker color due to less cysteamine and TCEP. Sample 2 resulted in smaller size due to less cysteamine and TCEP
Sample 3 resulted in-between size due to increase of cysteamine and TCEP

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Nanogel Characterization Size
Figure 2. (A) Polymer I : Size increase as TCEP increase (B) Polymer II : Size decrease as TCEP increase (C) Polymer II : Size increase as TCEP increase
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(B)
(C)
Nanogel Characterization Size
Figure 2. (A) Polymer I : Size increase as TCEP increase (B) Polymer II : Size decrease as TCEP increase (C) Polymer II : Size increase as TCEP increase

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(B)
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Figure 3. In each case all nanogel samples were negative. Increase of TCEP resulted in increase of negative surface charge.
Surface Charge
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Figure 3. In each case all nanogel samples were negative. Increase of TCEP resulted in increase of negative surface charge.
Surface Charge

Conclusion We designed three compositions of PDA-co-PEG to make a variety of nanogel samples. Due to the different amount of cysteamine and TCEP added to each sample, we were able to visualize and analyze the different sizes of each nanogel. The polymer with the 1:1 ratio and 5% cysteamine and 40% TCEP had the smallest size nanogel. As TCEP is increased, the zeta potential (surface charge) decreases. All of these results concluded that the 1:1 ratio polymer would be used to load the drug into the nanogel, optimize its loading efficiency, and then test it’s in virto and in vivo anticancer effect.

Acknowledgment
I would like to thank Dr. Peisheng Xu for giving me the opportunity to work in his laboratory. I have gained many experiences while working in the environment of attentive graduate students. I would also like to appreciate Remant Badhur K.C. for taking the time out to help guide me as I worked on my experiment. Finally I would like to express and give thanks to Dr. Ali and the Department of Environmental Sciences for allowing me to experience a nice summer of interning at University of South Carolina.

References
[1] Serguei V Vinogradov. Polymeric nanogel formulations of nucleoside analogs. Expert Opin Drug Deliv. 2007 January; 4(1): 5–17.
[2] Murali Mohan Yallapu, et al. Design and engineering of nanogels for cancer treatment. Drug Discov Today. 2011 May; 16(9-10): 457–463.
[3] Douglass Davona. Cancer-Cell-Activated Instant-Intracellular-Drug-Releasing Nanoparticles for Chemotherapy and a Degradable Nanogel for Drug Delivery