Team:TAS Taipei/Experimental Summary

Experimental Summary - TAS Taipei iGEM Wiki




C◎UNTERACTS

Experimental Summary

We set up a cataract model using fish lenses, and tested the prevention and treatment effects of two compounds, glutathione (GSH) and 25-hydroxycholesterol (25HC), respectively. Once we established their effects, we designed DNA constructs to express glutathione reductase (GSR) and cholesterol-25-hydroxylase (CH25H), enzymes that produce GSH and 25HC. We want to directly deliver the proteins, but not bacteria, into the eye. To this end, we made nanoparticles that can package our protein products and deliver them through the cornea and into the eye. In a final proof of concept experiment, we encapsulated GSH and 25HC inside nanoparticles, added these to fish lens solutions, and showed that cataract development is reduced.

Setting Up a Cataract Model

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.

Figure 2.1: Left Our cataract model was set up using fish lens. Left: Priacanthus macracanthus purchased from the market. Right: the two spheres on the left are entire lenses and the two smaller spheres on the right are lens nuclei.




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.

Figure 2.2: CT-2400 Spectrophotometer was used to measure absorbance and determine severity of cataracts.


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.



Figure 2.3: Increasing the amount of H2O2 leads to a greater increase in lens solution absorbance.

Figure 2.4 H2O2 causes protein aggregation. We ran a protein gel with our fish lens solutions, with or without H2O2. Compared to the untreated solution, treatment with H2O2 resulted in higher bands (red asterisk). The right lane is the molecular ladder (showing protein sizes in kDa).

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.



Prevention of Cataracts with GSH

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 2.5).

Figure 2.5: Addition of GSH results in a smaller increase in lens solution absorbance. Lens solutions were treated with H2O2 only, or with H2O2 and GSH together. Graph shows percent change in absorbance after 48 hours.




Treatment of Cataracts with 25HC

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).

Figure 2.6: OcluVet eye drops are prescribed for pets with cataracts.


Figure 2.7: 25HC is more effective at treating cataracts compared to pet eye drops. Increasing the concentration of 25HC increases effectiveness. Two trials were conducted and the error bars showed a significant difference between untreated and 25HC-treated samples.

Construct Design


Prevention: GSR-HIS

GSR catalyzes the formation of GSH, the main antioxidant in the lens. This will prevent crystallin proteins from being oxidized by H2O2. Since people oppose putting bacteria in their bodies, we included a polyhistidine-tag (his-tag) to purify the desired proteins.

The final construct (figure 2.10) includes a strong promoter, strong ribosome binding site, GSR (mutated to remove internal restriction sites), 10x his-tag, and a double terminator. We acquired a strong promoter + strong ribosome binding site part (BBa_K880005) from the iGEM distribution kit to maximize protein production. We ordered the cDNA of GSR from Origene and used the 10x histidine-tag part from the distribution kit (BBa_K844000). A double terminator (BBa_B0015) was added at the end to stop transcription.



Figure 2.8: Our construct includes a strong promoter, strong ribosome binding site, GSR, his-tag, and double terminator.


The purchased GSR cDNA had two internal PstI and three EcoRI cutting sites. After making silent mutations to the sequence, we sent the cDNA to Mission Biotech 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 2.9), then front-inserted this before a new plasmid (Figure 2.10) containing 10x his-tag (BBa_K844000) and double terminator (BBa_B0015). Sequencing results from Tri-I Biotech show that our final construct was correct.



Figure 2.9: PCR check for BBa_K880005 + GSR. After inserting GSR (pink box, 1.7kb) behind a strong promoter and strong RBS (BBa_K880005), the expected ligation size is around 2kb (blue box).
Figure 2.10: PCR check for his-tag (BBa_K844000) + double terminator (BBa_B0015). The double terminator (~130 bps; or ~400 bps after PCR check, pink box) was inserted behind 10x his-tag (~30 bps). After PCR, the expected ligation size is ~450 bps (blue box).



Treatment: CH25H-HIS

Figure 2.11 Our construct includes a strong promoter, strong ribosome binding site, CH25H, his-tag, and double terminator.


CH25H is the enzyme that converts lens cholesterol into 25HC, which reverses protein aggregation (Makley et al., 2015). The final construct contains similar components as the prevention construct, except the first part of the open reading frame is replaced by CH25H. CH25H was also mutated (by Mission Biotech) to remove internal cutting sites and put into a Biobrick backbone to make a new basic part. The sequence of the final construct was sent to Integrated DNA Technologies (IDT) for synthesis and cloned into a Biobrick backbone.




Crystallin Protein: Alpha-crystallin B (CRYAB)-HIS

Figure 2.12 Our construct includes a strong promoter, strong ribosome binding site, CRYAB, his-tag, and double terminator.


CRYAB is one of the main proteins in the human lens that aggregates to form cataracts. In our fish lens model, we observed protein aggregation after adding H2O2. However, we also wanted to make sure that the fish model results represent what happens with human crystallin proteins, so we designed a CRYAB construct (figure 2.12).

The final CRYAB construct contains similar components, except the first part of the open reading frame is replaced by CRYAB. CRYAB cDNA was ordered from Origene. We designed primers that were synthesized by Tri-I biotech to clone CRYAB into a Biobrick backbone, similar to how the GSR construct was made. Sequencing results (Tri-I Biotech) show that the construct was correct, and we confirmed protein expression of both CRYAB (without a poly-his tag; yellow asterisk in (figure 2.13) and CRYAB-HIS (blue asterisk in figure 2.13).

To test that CRYAB aggregates in response to H2O2, we cultured bacteria expressing CRYAB and CRYAB-HIS. Liquid cultures were grown overnight and detergent was added to lyse the bacteria cultures. Different concentrations of H2O2 were added to the lysates and a protein gel was prepared (figure 2.13). With increasing concentration of H2O2, the original band for CRYAB-HIS (blue asterisk) becomes lighter as higher bands (indicated by the blue bracket) increase in concentration. This shows that H2O2 has the same effect on human crystallin proteins, causing them to aggregate and clump.

Figure 2.13 H2O2 causes human CRYAB proteins to aggregate. The expected size for CRYAB (yellow asterisk) is 20 kDa and 21 kDa for CRYAB-HIS (blue asterisk). Adding H2O2 results in a decrease of the original bands at 20 and 21 kDa, and an increase in larger proteins (blue bracket). In addition, the bands in the blue bracket also appear as a smear, which suggests that proteins may be clumping together to form various sizes.




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: 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.



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 added 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. We dispersed GFP-nanoparticles in the polymer solution to test if protein containing nanoparticles can be embedded in the contact lenses. We 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).
Figure 3.12:  Contact lenses embedded with GFP-containing nanoparticles (left) and without GFP nanoparticles (right) in mold.

Proof of Concept in Fish Lenses

Figure 3.13:  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 3.13, 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 3.14). Similarly, when 25HC-containing nanoparticles were added, the increase in absorbance was significantly lower than with only H2O2 (figure 3.15). These result show that, in our cataracts model, the compounds carried inside nanoparticles were released and effective at treating and preventing cataracts.

Figure 3.14:  GSH-containing nanoparticles (blue) prevented an increase in lens solution absorbance after adding H2O2. In the control group (gray), empty nanoparticles were used.
Figure 3.15:  25HC-containing nanoparticles (pink) showed a smaller increase in lens solution absorbance after adding H2O2. In the control group (gray), empty nanoparticles were used.


Citations

Behl, G., Iqbal, J., O’Reilly, N. J., McLoughlin, P., & Fitzhenry, L. (2016). Synthesis and Characterization of Poly (2-hydroxyethylmethacrylate) Contact Lenses Containing Chitosan Nanoparticles as an Ocular Delivery System for Dexamethasone Sodium Phosphate. Pharmaceutical research, 1-11.


Bio-Rad. (n.d.). Quick Start™ Bradford Protein Assay [PDF]. Hercules: Bio-Rad Laboratories, Inc.


Campos, A. M., Diebold, Y., Carbalho, E. L., Sánchez, A., & Alonso, M. J. (2005). Chitosan Nanoparticles as New Ocular Drug Delivery Systems: In Vitro Stability, in Vivo Fate, and Cellular Toxicity. Pharm Res Pharmaceutical Research, 22(6), 1007-1007. doi:10.1007/s11095-005-4596-x


Carlsson, M. (2016, May 18). WEBINAR: Tips for successful purification of his-tagged proteins [PDF]. Retrieved from https://www.google.com.tw/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact=8&ved=0ahUKEwiDuqymvtzPAhXEGJQKHaY4DG0QFggiMAE&url
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Cholkar, K., Patel, S. P., Vadlapudi, A. D., & Mitra, A. K. (2013). Novel Strategies for Anterior Segment Ocular Drug Delivery. Journal of Ocular Pharmacology and Therapeutics, 29(2), 106–123.


Cvekl, A., & Ashery-Padan, R. (2014). The cellular and molecular mechanisms of vertebrate lens development. Development, 141(23), 4432-4447.


Enriquez De Salamanca, A., Diebold, Y., Calonge, M., García-Vazquez, C., Callejo, S., Vila, A., & Alonso, M. J. (2006). Chitosan nanoparticles as a potential drug delivery system for the ocular surface: toxicity, uptake mechanism and in vivo tolerance. Investigative ophthalmology & visual science, 47(4), 1416-1425.


Falsini, B., Iarossi, G., Chiaretti, A., Ruggiero, A., Manni, L., Galli-Resta, L., ... & Abed, E. (2016). NGF eye-drops topical administration in patients with retinitis pigmentosa, a pilot study. Journal of translational medicine, 14(1), 1.


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Patel, A., Cholkar, K., Agrahari, V., & Mitra, A. K. (2013). Ocular drug delivery systems: an overview. World journal of pharmacology, 2(2), 47.


Wilson, T., Aeschlimann, R., Tosatti, S., & Lorenz, K. E. (2014). Defining ‘Fresh’ Corneal Tissue for Utilization in Determining Human Cornea Coefficient of Friction Values. Investigative Ophthalmology & Visual Science, 55(13), 1506-1506.





Prevention

GSR Eyedrop

Treatment

25HC Eyedrop

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Eyedrops




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