Team:TAS Taipei/Experimental Summary

Experimental Summary - TAS Taipei iGEM Wiki




C◎UNTERACTS

Experimental Summary

Setting Up a Cataract Model

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.

Figure 1 (left). Priacanthus macracanthus purchased from the market. (right) The two spheres on the left are lens with cortex and nucleus and the two smaller spheres on the right are nucleus.


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.

Figure 3. CT-2400 Spectrophotometer used to measure absorbance value


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.



Figure 4. Percent change in absorbance of lens solutions treated with H2O2

Figure 5. SDS Page. The left lane is lens protein solution seated in room temperature for two days. The middle lane is lens protein solution treated with 10mM of H2O2 for two days. The right lane is the molecular ladder.


Testing Prevention of Cataracts with GSH

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 .

Figure 6. Glutathione and Reduced Glutathione recycling pathway


Figure ???. Caption not provided!!!

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 #)



Testing Treatment of Cataracts with CH25H

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.

Figure 1. The Ocluvet vet eyedrop contains antioxidants that can treat cataracts. We used this eyedrop as our positive control for cataracts treatment.

Figure 2. Lens Treatment Experiment after 48hrs. (left) Control, Vet eyedrop treated cataracts solution, 50uM 25HC treated cataracts solution, 20uM 25HC treated cataracts solution. Each treatment is added after adding H2O2 for 24 hours.


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.



Figure 4. Percent change in absorbance of the treated cataracts lens protein solution after 48 hours

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.

Construct Design


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.



Figure X. Full Construct.


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).

Figure BB. His + term cloning


Figure AA. 1kb ladder, GSR alone, samples after are K880005 + GSR PCR checks. Boxed bands are correct (slightly higher than GSR alone)



Treatment: CH25H-HIS

Figure ???. Caption not provided!!!


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 #.



Figure ???. Caption not provided!!!

Figure ???. Caption not provided!!!


Prototype

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.



Figure 3.1: His-tag allows the proteins to be purified. Both GSR and CH25H (in light blue) constructs include a downstream his-tag (in yellow) for protein purification.


The his-tag has 10 consecutive histidine amino acids that allow the protein to bind to nickel when it passes through a nickel column. In this way, the his-tagged proteins can be eluted to obtain purified proteins.

Since we confirmed the expression of CRYAB-HIS protein (figure 2.15), we used CRYAB and CRYAB-HIS to test a commercial kit (Capturem His-tagged Purification Miniprep Kit from Clontech). We lysed bacteria expressing GFP, CRYAB, and CRYAB-HIS and centrifuged the crude lysates to remove cell debris. Next, we passed the lysates through nickel columns to separate any his-tagged protein. To elute the his-tagged proteins, we added elution buffer with 500mM imidazole into the columns. We expected no protein in the GFP and CRYAB elutions and protein in the CRYAB-HIS elution. However, using the Nanodrop we found no protein in all three elutions.

Through literature research we learned that 6x his-tag are more commonly used and easier to elute off a column compared to a 10x his-tag (Carlsson et al., 2016). Thus, we hypothesized that our tags bound too strongly to the column. If this were the case, we should see a difference in protein concentration before and after passing his-tagged lysates through the column, whereas lysates containing untagged proteins should not change in protein concentration. After testing this hypothesis, we found that the protein concentration of CRYAB-HIS lysate decreased after passing through the column (figure 3.2), which shows that his-tagged proteins were indeed bound to the column. In contrast, the protein concentrations of CRYAB and GFP lysates remained similar.



Figure 3.2: CRYAB-HIS proteins bind to the nickel column. Nanodrop was used to measure protein concentrations. The protein concentration of the CRYAB-HIS lysate decreased after passing through the column, while the protein concentrations of GFP and CRYAB lysates remained almost the same.



Step 2: Packaging in Nanoparticles

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).





Figure 3.3: Nanoparticles containing our proteins embed into the cornea and degrade. The released proteins are then delivered within the eye.
Figure 3.4: Purified proteins can be encapsulated in chitosan nanoparticles.


Below is a video of our nanoparticle synthesis procedure.

Figure C. Video


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).



Figure 3.5: Team members imaging nanoparticles on the scanning electron microscope and atomic force microscope.


Figure 3.6: Scanning electron microscope image of chitosan nanoparticles
Figure 3.7: We imaged chitosan nanoparticles using atomic force microscopy. On the left is the empty silica plate. On the right is an image of the chitosan nanoparticles, which were placed on the silica plate



Protein Encapsulation

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.

Figure 3.8: Proteins were successfully encapsulated into nanoparticles. Figure shows nanoparticle pellets containing no protein, GFP, RFP, and pGRN (left to right) under white light (top) and blue light (bottom). Fluorescence of GFP and RFP-containing pellets shows that proteins are still functional.

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%.



Figure 3.9:The encapsulation efficiency is 72%. Using a Bradford assay, we created a standard curve of known BSA protein concentrations by measuring absorbance at 595 nm. Top: graph shows absorbance values of the supernatant after nanoparticle formation. Bottom: cuvettes containing standard solutions (left) and the sample solution (right).



Protein Release

Figure 3.10:BSA proteins are released from chitosan nanoparticles at 37℃, but almost no change occurred at 4℃.

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.




Step 3: Application (EYE DROP OR CONTACT LENSES)

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.


Eye drops

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).


Contact Lenses

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).

Figure 3.11:A 3D printed mold (left) used to make hydrogel lenses (right).

Final Proof of Concept

Figure 3.12:Experimental flowchart: using GSH or 25HC-packaged nanoparticles to test for prevention and treatment effects in our cataracts model.

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.

Figure 3.13:GSH-containing nanoparticles (blue) prevented an increase in lens solution absorbance after adding H2O2. In the control group (gray), empty nanoparticles were used.
Figure 3.14:25HC-containing nanoparticles (pink) showed a smaller increase in lens solution absorbance after adding H2O2. In the control group (gray), empty nanoparticles were used.


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Prevention

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