Through literature research, we found that glutathione (GSH) and 25-hydroxycholesterol (25HC) should prevent and treat cataracts, respectively. We wanted to first see if we could simulate cataract formation and reproduce the effects of GSH and 25HC seen by other researchers. If successful, then we can later test our own constructs using the same cataract model.

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 01 left). There are two distinct parts to the lens: an outer soft layer called the cortex and an inner layer called the nucleus (Figure 01 right). We focused on the lens nucleus, because that part contains older cells and is more prone to cataract formation. The lens nucleus was placed into Tris Buffer and gently shaken overnight. After centrifuging, the supernatant contains dissolved protein from the lens nucleus. We then incubated the lens solution with hydrogen peroxide (H2O2), since H2O2 is the main reactive oxygen species that oxidizes lens proteins and induces cataracts.

To quantify the severity of cataracts in our model, we used spectrophotometer to measure absorbance of the protein solution (Figure 03). As lens proteins get oxidized, absorbance should increase because protein clumps that form will scatter the emitted light. To find a wavelength of light for data collection, we compared the absorbance of an untreated proteins, H2O2-treated proteins, and heat-denatured proteins as a positive control. We observed an absorbance peak at 397.5 nm for insoluble lens proteins, and chose to collect all future absorbance values at that wavelength.

We tested different concentrations of H2O2 on this protein solution to see if cataracts form. Our results show that increasing concentrations of H2O2 lead to more severe cataracts (Figure 04). 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 05). Together, the protein gel and our lens cataract model suggest that our model accurately represents cataract development.

GSH is the main antioxidant in the lens and prevents H2O2 from oxidizing crystallin proteins. After GSH converts H2O2 into water, it becomes GSSG, which will be recycled back to GSH with the help of glutathione reductase (GSR) (figure 06). Older cells in the nucleus are unable to produce GSR efficiently, so over time GSSG builds up. Our project is to deliver the GSR into the lens in order to facilitate the conversion of GSSG to GSH. We purchased GSH from Sigma Aldrich. 8 mg of GSH was added to the protein solution prior to the addition of H2O2 .

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 select 25 HC (25 hydroxycholesterol) to be our treatment because it reverses aggregation and restores solubility of the lens crystallin protein by stabilizing the natural state of the proteins. In order to verify our research, we use commercially bought 25 HC from Sigma Aldrich to treat the cataracts model. Commercial 25HC came in powder form, and was dissolved in 95% ethanol; thus, for our negative control, we added both H2O2 and ethanol into the protein solution. We also bought vet eyedrops for cataracts from OcluVet to be the positive control (Figure 1). Figure 2 shows the setup of the experiment.

Two sets of experiments were conducted to prove that 25 HC treats cataracts. For the first set, we added H2O2 into fresh fish lens protein solution, and waited for 24 hours before adding the treatment. After adding 25HC into the cataracts model, we measured the absorbance of the treated protein solutions with a UV spectrophotometer in 24 hours increments (F3). We used Tris buffer to blank the UV spectrophotometer before measuring the absorbances for the 25HC treated tubes, and since the vet eyedrop is yellow, we blanked the spectrophotometer with Tris buffer containing vet eyedrop before measuring vet eyedrop treated cataracts solution. Absorbance increases due to increase of opaqueness of the protein solution. Thus, we assume that, the higher the absorbance, the more cataracts is formed due to protein aggregation.

As shown in Figure 4, absorbance values increased when lens proteins were incubated with H2O2 for 48 hours. Adding vet eyedrops 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). Two trials were conducted and the error bars show that the difference between untreated and 25HC-treated proteins is significant.

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 possibly unavailable in areas with limited medical facilities. Taking biosafety, cost, and delivery effectiveness into account, we designed and built a prototype with two distinct steps. First, proteins must be purified and separated from bacteria, and then packaged in nanoparticles that aid delivery through the cornea and into the lens.

Step 1: His-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. Once isolated, pure proteins can be directly used to avoid introducing foreign bacteria into our bodies. As shown in Figure A, both constructs for prevention and treatment include a downstream 10x histidine tag. The encoded GSR-HIS or CH25H-HIS proteins can then be isolated using a commercial kit (Capturem His-tagged Purification Miniprep Kit from Clontech).

The cornea is the outermost layer of the eye which protects the eye by preventing entry of foreign materials, but this function also largely prevents drugs from reaching the lens (Gaudana et al., 2010). This problem has challenged researchers and driven them to search for a way to deliver drugs through the cornea and into the lens. Currently, the most promising ocular drug delivery method is using chitosan nanoparticles as drug carriers (Cholkar et al., 2013).

We selected chitosan as the optimal material to make nanoparticles for several reasons. First, chitosan nanoparticles can embedded in and adhere to the cornea, this minimizes drainage loss. Also, these nanoparticles can penetrate the cornea and deliver the drug directly to the target area via (Campos et al., 2005). Its low toxicity to somatic cells makes it safe, and its biodegradability allows the drug to be released continuously in the eye (Enriquez de Salamanca et al., 2006).

We dissolved chitosan into 1% by volume glacial acetic acid aqueous solution. We adjusted the pH of the chitosan solution to 5.5 by adding 1M NaOH in order to account for the stability of the desired proteins when they are added. We dissolved sodium tripolyphosphate (TPP) in distilled water. Chitosan and TPP were dissolved in equal volumes and their mass ratio was 3:1. We stirred the chitosan solution at 600 rpm while adding 3 ml of TPP solution dropwise. To collect the nanoparticles, we centrifuged the suspension at 17,000 xg for 40 minutes at 4C, and nanoparticles were collected as pellets. The specific protocol can be found in our lab notebook.

To ensure our nanoparticles were correct in size and configuration, we imaged the nanoparticles using scanning electron microscopy and atomic force microscopy.

Next, we wanted to load our protein into the nanoparticles. In order to determine how successfully our nanoparticles encapsulated proteins, we measured the change in protein concentration in the supernatant before and after nanoparticle formation. By performing a Bradford Assay with the nanoparticle supernatant (Bio-Rad), we found the encapsulation efficiency to be 50%.

More BSA remained in the supernatant after nanoparticle formation when Chitosan was added to TPP. It is inferred that more proteins are encapsulated in the nanoparticles for those made from TPP added to Chitosan.

We synthesized nanoparticles containing fluorescent proteins to prove that our nanoparticles can be used to encapsulate our protein drugs. To do so, we lysed bacteria expressing green fluorescent protein (GFP), red fluorescent protein (RFP), and green pigment (from pGRN, Bba_K274003). We then extracted and encapsulated the proteins in nanoparticles using the procedure described above.

As nanoparticles degrade, they release the proteins inside. We measured and mathematically modeled the release rate to determine the optimal frequency of drug administration.

To measure release rate, we suspended nanoparticles containing bovine serum albumin (BSA), our standard protein, in phosphate buffered saline (PBS). PBS models the conditions of our cornea on which the nanoparticles that we deliver will be embedded to degrade and release the proteins. We measured protein concentration of the solution outside the nanoparticles over time. We performed trials at three different temperatures, -20C, 4C, and 37C to reflect long term storage, short term storage, and drug application.

Procedure for this experiment is illustrated in our lab notebook.

We engineered nanoparticles to deliver glutathione reductase (GSR) and cholesterol 25-hydroxylase (CH25H) to the lens through a safe, cost effective, and non-invasive method. We purified the proteins from bacteria using histidine tag purification method to minimize toxicity and loaded them into nanoparticles to maximize drug delivery. We designed two drug delivery prototypes to apply our nanoparticles: eye drops and contact lenses.