The first step of our project was to simulate the formation of cataracts, so that we can test our products in the future. We set up the model by extracting soluble proteins from the lens of Priacanthus macracanthus, a common freshwater fish that we purchased from a market (figure 2.1, left). There are two distinct parts of the lens: an outer soft layer called the cortex and an inner layer called the nucleus (figure 2.1, right). We used the lens nucleus, because it contains older cells and is more prone to cataract formation (Cvekl & Ashery-Padan, 2014). In order to isolate the lens protein, we shook the lens nucleus in Tris buffer overnight and centrifuged the samples (Mello et al., 2012). The supernatant contains dissolved proteins from the lens nucleus. We then added hydrogen peroxide (H2O2), the main factor that oxidizes lens proteins and induces cataract formation.

To quantify the severity of cataracts in our model, we used a spectrophotometer to measure absorbance values of the lens solution (figure 2.2). As lens proteins become oxidized, absorbance should increase because as proteins aggregate, they fall out of solution, which will scatter incoming light. To find a wavelength of light for data collection, we compared the absorbance of lens solution alone (negative control), H2O2-treated lens solution, and heat-denatured lens solution (positive control). We observed an absorbance peak at 397.5 nm for both H2O2-treated and heat-denatured solutions, and chose to collect all future absorbance values at that wavelength.

After adding different concentrations of H2O2, our results show that increasing concentrations of H2O2 lead to more severe cataracts (figure 2.3). We also ran a protein gel to compare the sizes of untreated and H2O2-treated proteins. After treatment with H2O2, there was an increase in higher bands, which is consistent with the idea that proteins are clumping and aggregating (figure 2.4). Together, the protein gel and our absorbance data suggest that our model accurately represents cataract development.

Through literature research, we learned that glutathione (GSH) and 25-hydroxycholesterol (25HC) are potential candidates for the prevention and treatment of cataracts, respectively (Ottonelloa et al., 2000; Makley et al., 2015). Using our cataract model, we wanted to test the effects of these compounds.

GSH is the main antioxidant in the lens and prevents H2O2 from oxidizing crystallin proteins. Glutathione reductase (GSR) is the enzyme responsible for making GSH. Older cells in the lens nucleus carry less GSR (Michael & Bron, 2011), which in turn decreases the amount of GSH. This makes the lens nucleus more prone to cataract formation. In order to restore GSH levels, we want to deliver more GSR into the lens.

We found the concentration of GSH in healthy lenses (Kamei, 1993), and used that to determine how much GSH to use in our cataract model. We prepared two samples: one with GSH (from Sigma Aldrich) and H2O2, and the other with H2O2 alone. GSH-treated solutions showed lower absorbance values compared to the untreated control, which indicates less protein aggregation (figure 2.5). These results suggest that GSH has a preventative effect on cataract formation.

Protein solutions with GSH and H2O2 have an absorbance value lesser than those with just protein solutions with H2O2 . The smaller absorbance value indicates smaller protein aggregation and shows GSH has preventative effect. (figure #)

We selected 25HC to be our treatment because it reverses aggregation and can restore the solubility of lens crystallin proteins (Makley et al., 2015). In order to test this, we bought 25HC from Sigma Aldrich and used this in our cataract model. From veterinarians, we learned that commercial eye drops are available for use in pets (OcluVet; figure 2.6), so we wanted to test the effects of 25HC against these eye drops.

Lens solutions were first incubated with H2O2, then treatments were added. As shown in figure 2.7, absorbance values increased when lens proteins were incubated with H2O2. Adding pet eye drops did not lower the absorbance as much as treatment with 25HC, suggesting that 25HC is more effective at reducing cataracts. In addition, a higher concentration of 25HC lowered the absorbance even further (50 μM compared to 20 μM).

Prevention: GSR-HIS

We want to express glutathione reductase (GSR) to catalyze the reduction of GSSG to GSH, the main antioxidant in the lens. This will prevent crystallin proteins from being oxidized by H2O2 . We also want to extract the proteins produced by E. coli so that no bacteria is used in our final delivery prototype. Thus, we include a histidine tag behind the gene we want to express. Proteins with a histidine tag can be detected and purified.

The purchased GSR cDNA has two internal PstI and three EcoRI cutting sites. After making silent mutations to the sequence, we sent the cDNA to MissionBiotech for mutagenesis to remove these cutting sites. Once we had the correct sequence of GSR (with 5 point mutations), we designed primers to add the BioBrick prefix and suffix in order to clone GSR into a BioBrick backbone. The primer designs were sent to Tri-I Biotech for oligo synthesis, and polymerase chain reaction (PCR) was set up. We first cloned GSR behind BBa_K880005 (strong promoter + strong ribosome binding site; Figure AA), then front-inserted this before a new plasmid (Figure BB) containing 10x Histidine tag (BBa_K844000) and double terminator (BBa_B0015). The sequence of the full construct was confirmed by sequencing (Tri-I Biotech).

We want to extract alpha crystallin B protein, one of the main proteins in the lens, to see if we can create the same aggregation as cataracts lenses.The construct here contains similar components, except the open reading is replaced by CRYAB and 10x Histidine tag. We wanted to express CRYAB, one of the two main crystallin proteins in the lens, to test if h2o2 can aggregate crystallin proteins. CRYAB cDNA was ordered from Origene. We designed primers that were synthesized by Tri-I biotech to move CRYAB into iGEM backbone. The full construct is shown in figure #.

The current method of treatment for cataracts is surgery, which is not only invasive and costly, but also unavailable in areas with limited medical facilities. Taking biosafety, cost, and delivery effectiveness into account, we designed and built a prototype with three distinct steps: Step 1) proteins must be purified and separated from bacteria, Step 2) proteins are packaged in nanoparticles that aid delivery through the cornea and into the lens, and Step 3) delivery of nanoparticles through eye drops or contact lenses.

STEP 1: POLYHISTIDINE-TAG PROTEIN PURIFICATION

Our survey results show that people are reluctant to put anything bacteria-related into their bodies, so we aimed to separate our protein products from the bacteria that produced them. As shown in figure 3.1, our constructs include a downstream 10x polyhistidine-tag (his-tag), which allows the protein to be purified.

The cornea is the outermost layer of the eye and protects the eye from foreign materials, but also prevents drugs from reaching the lens (Gaudana et al., 2010). Scientists have developed several methods to penetrate the cornea and deliver drugs to the lens, but many are invasive, such as implants (Patel et al., 2013). The most promising method is using nanoparticles as drug carriers (Cholkar et al., 2013). so we chose to use nanoparticles to deliver our proteins to the lens.

Nanoparticles can be made from a variety of materials, but we selected chitosan for several reasons. Researchers have used chitosan nanoparticles in the eye; its low toxicity to somatic cells makes it safe and it does not affect the anatomy of the eye (Enriquez de Salamanca et al., 2006). We also learned that chitosan nanoparticles can embed in the cornea, and its biodegradability allows the drug to be released continuously into the eye (figure 3.3) (Enriquez de Salamanca et al., 2006; Campos et al., 2005). Therefore, we want to load our purified proteins into chitosan nanoparticles (figure 3.4).

Below is a video of our nanoparticle synthesis procedure.

Following the procedure of Quan and Wang (2007), we made nanoparticles and imaged them using scanning electron microscopy and atomic force microscopy (figure 3.5). This revealed our nanoparticles to be spherical and at the optimal size of 200-600 nm (figure 3.6 and 3.7).

Next, we wanted to load our purified proteins into the nanoparticles. We first used colored proteins to qualitatively test whether proteins could be successfully encapsulated. To do so, we lysed bacteria expressing green fluorescent protein (GFP), red fluorescent protein (RFP), and green pigment (from pGRN, Bba_K274003). We then add the proteins to the chitosan solution. After nanoparticles were made, our results showed that we successfully encapsulated the colored proteins. When we further viewed the nanoparticles under blue light, the GFP- and RFP-containing pellets glowed (figure 3.8), suggesting that the proteins remain functional. Thus, our nanoparticles can serve as protein carriers to enhance drug delivery.

In order to quantitatively determine encapsulation efficiency, we measured protein concentration in the supernatant before and after nanoparticle formation. We started with 1 mg/mL of bovine serum albumin (BSA). After nanoparticle formation, we performed a Bradford assay and found that the concentration decreased to 0.28 mg/mL. As shown in figure 3.9, the encapsulation efficiency was 72%.

After proteins are encapsulated, nanoparticles should embed in the cornea and release proteins as they degrade over time. To test whether nanoparticles degrade, we measured the release of proteins. After BSA-containing nanoparticles were made, they were spun down and the solution was replaced with phosphate buffered saline (PBS) (Wilson, 2014). Using a Bradford assay, we could then measure protein concentration in the PBS over a 72-hour period.

Trials were performed at two different temperatures: 4°C and 37°C. Our results show that proteins are released from nanoparticles at 37°C , but almost no change could be detected at 4°C (figure 3.10). This finding suggests that we can store a final functional product (e.g., eye drop) at 4°C without nanoparticle degradation, while the proteins can be released from nanoparticles when the eye drop is applied at body temperature.

Our goal is to package GSR and CH25H in nanoparticles to deliver these proteins to the lens using a safe and non-invasive method. We have considered two drug delivery mechanisms to administer the nanoparticles: eye drops and contact lenses.

After packaging our proteins in nanoparticles, the nanoparticles can be spun down and resuspended in saline, since it is commonly used in eye drops (Falsini, 2016).

We found a method to make chitosan nanoparticle-embedded hydrogel contact lenses (Behl, 2016). Following their protocol, we created a polymer solution containing all the necessary components, and then transferred this solution into a 3D-printed mold (figure 3.11, left). After exposure to UV for 40 minutes, we successfully made hydrogel contact lenses (figure 3.11, right).

In a final experiment, we wanted to test if our delivery prototype can actually be used to prevent and treat cataracts in our model. As shown in figure 3.12, we first solubilized fish lens proteins and treated these samples with H2O2 to simulate cataracts. Nanoparticles encapsulating the compounds GSH or 25HC were made. Next, we added the nanoparticles into our cataracts model to test for prevention and treatment effects. We compared the absorbance values after 48 hours.

After adding H2O2 to the lens solution, absorbance values increased. However, when GSH-containing nanoparticles were also added, the absorbance values remained unchanged (figure 3.13). Similarly, when 25HC-containing nanoparticles were added, the increase in absorbance was significantly lower than with only H2O2 (figure 3.14). These result show that, in our cataracts model, the compounds carried inside nanoparticles were released and effective at treating and preventing cataracts.