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− | <li class="active"><a data-toggle="tab" href="#gsrhome"> | + | <li class="active"><a data-toggle="tab" href="#gsrhome">Introduction</a></li> |
− | <li><a data-toggle="tab" href="#gsrmenu1"> | + | <li><a data-toggle="tab" href="#gsrmenu1">Enzyme Kinetics</a></li> |
− | <li><a data-toggle="tab" href="#gsrmenu2"> | + | <li><a data-toggle="tab" href="#gsrmenu2">Differential Equations & Paramters</a></li> |
− | <li><a data-toggle="tab" href="# | + | <li><a data-toggle="tab" href="#gsrmenu3">Procedure & Assumptions</a></li> |
− | <li><a data-toggle="tab" href="# | + | <li><a data-toggle="tab" href="#gsrmenu4">Results</a></li> |
+ | <li><a data-toggle="tab" href="#gsrmenu5">Discussion</a></li> | ||
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− | < | + | <h3>Introduction</h3> |
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+ | <div id="gsrmenu1" class="tab-pane fade"> | ||
+ | <h3>Enzyme Kinetics</h3> | ||
<p> The following 6 reactions describe the antioxidant system inside the cortex and nucleus.</p> | <p> The following 6 reactions describe the antioxidant system inside the cortex and nucleus.</p> | ||
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<p><b>Reaction 6:</b> By the <b>Law of Passive Diffusion</b>, the rate of diffusion into the cortex and lens is: is: $$r_6=k_6([H_2O_2]_{out}-[H_2O_2]_{in})$$</p> | <p><b>Reaction 6:</b> By the <b>Law of Passive Diffusion</b>, the rate of diffusion into the cortex and lens is: is: $$r_6=k_6([H_2O_2]_{out}-[H_2O_2]_{in})$$</p> | ||
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− | + | <h4>Differential Equations</h4> | |
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<p>We have six reaction rates derived from above. Now, we will form differential equations, where every time a species is used as a reactant, the reaction rate will be subtracted from the species’ derivative, while each time it is formed as a product, the reaction rate will be added to the species’ derivative. We will go through each species in detail:</p> | <p>We have six reaction rates derived from above. Now, we will form differential equations, where every time a species is used as a reactant, the reaction rate will be subtracted from the species’ derivative, while each time it is formed as a product, the reaction rate will be added to the species’ derivative. We will go through each species in detail:</p> | ||
<p>Substituting the rate of each reaction, we get the following system of differential equations. </p> | <p>Substituting the rate of each reaction, we get the following system of differential equations. </p> | ||
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+ | <h3>Procedure</h3> | ||
+ | <p>Listed outside collapsible</p> | ||
+ | <p>1. After building this differential equation model in Mathematica, we change the initial starting concentration of GSR, and numerically solve the equations. </p> | ||
+ | <p>2. The output will be a the concentration of H2O2 as a function of time. Integrating this function over 50 years using the definition in Model 1 returns the total crystallin damage. </p> | ||
+ | <p>3. We repeat this procedure from 0 to 100 uM of initial GSR, and graph the resulting crystallin damage against the initial GSR inputted (Figure 4).</p> | ||
+ | <h3>Assumptions</h3> | ||
+ | <p>In this experiment, we made the following assumptions: | ||
− | + | <ol> | |
− | + | <li>Crystallin damage is linear with respect to both concentration and time, as evidenced by cysteine’s first order reaction with hydrogen peroxide (Domínguez-Vicent). (see Model 1 for full explanation).</li> | |
+ | <li>The amount of total GSR (in both forms) is constant. In reality, for the first days of the treatment the GSR level is being increased, so GSR levels will not be constant until 100 days of the treatment have passed. As we will discuss in Model 3, a one-time treatment will be made as soon as prevention is fully effective.</li> | ||
+ | <li>The cortex and nucleus are indistinguishable and regarded as a single entity. As cataract damage can occur in both areas, we simplify the model by combining them to form a single system.</li> | ||
+ | <li>The amount of H2O2 in the aqueous humor is constant. As cellular respiration is constantly regenerating ROS and forming H2O2, there will constantly be H2O2 diffusing into the lens.</li> | ||
+ | <li>The amount of H2O2 in the aqueous humor is equal to the initial value of H2O2 in the cortex. The rationale is that initially dynamic equilibrium exists between the lens and the aqueous humor.</li> | ||
+ | </ol> | ||
+ | </p> | ||
+ | </div> | ||
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− | < | + | <h3>Results</h3> |
− | <p> | + | <table class="table table-bordered" style='width: 90%;margin-left:0%;'> |
− | </div> | + | <caption style='caption-side:top;'><b>Table 4: Results of Model 2 - Necessary GSR to prevent cataract from exceeding LOCS for an amount of time.</caption> |
+ | <thead> | ||
+ | <tr> | ||
+ | <th>LOCS</th> | ||
+ | <th>Crystallin Damage (c.d.)</th> | ||
+ | <th>GSR needed to maintain LOCS for 1 year</th> | ||
+ | <th>GSR needed to maintain LOCS for 20 years</th> | ||
+ | <th>GSR needed to maintain LOCS for 50 years</th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tr> | ||
+ | <th>0.0</th> | ||
+ | <th>0</th> | ||
+ | <th>N/A</th> | ||
+ | <th>N/A</th> | ||
+ | <th>N/A</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>0.5</th> | ||
+ | <th>0.1327</th> | ||
+ | <th>25.9</th> | ||
+ | <th>N/A</th> | ||
+ | <th>N/A</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>1</th> | ||
+ | <th>0.2774</th> | ||
+ | <th>23.4</th> | ||
+ | <th>52.49</th> | ||
+ | <th>96.1</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>1.5</th> | ||
+ | <th>0.4610</th> | ||
+ | <th>21.8</th> | ||
+ | <th>40.79</th> | ||
+ | <th>67.0</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>2.0</th> | ||
+ | <th>0.6966</th> | ||
+ | <th>20.4</th> | ||
+ | <th>34.78</th> | ||
+ | <th>52.3</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>2.5</th> | ||
+ | <th>0.9981</th> | ||
+ | <th>18.5</th> | ||
+ | <th>31.19</th> | ||
+ | <th>43.5</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>3.0</th> | ||
+ | <th>1.3840</th> | ||
+ | <th>16.5</th> | ||
+ | <th>28.83</th> | ||
+ | <th>37.8</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>4.0</th> | ||
+ | <th>2.5101</th> | ||
+ | <th>10.0</th> | ||
+ | <th>25.90</th> | ||
+ | <th>31.1</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>5.0</th> | ||
+ | <th>4.3514</th> | ||
+ | <th>0.00</th> | ||
+ | <th>24.05</th> | ||
+ | <th>27.5</th> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div id="gsrmenu5" class="tab-pane fade"> | ||
+ | <h3>Confirmation of Initial Value of GSR</h3> | ||
+ | <p>We expect the lens to have some GSR in the lens naturally as part of the antioxidizing system. There will not be enough GSR to fully prevent cataract damage, however. Based on research, if H2O2 levels remain at 10 uM for 1 year, which is common after traumatic accidents affecting the eye, a moderately severe cataract will develop, expected at around the LOCS 4-4.5 level.</p> | ||
+ | <p>Literature data estimates that the natural concentration is around 10uM. This corresponds to roughly a LOCS 4 cataract after a year, which confirms that our initial value of GSR in the cortex before any treatment. We will consider this as our starting point, and add GSR to reach this point.</p> | ||
+ | </div> | ||
+ | |||
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Revision as of 12:13, 16 October 2016
Model
Cataract prevention occurs over 50 years, so we cannot perform experiments direct on the long-term impact of adding GSR or CH25H. However, computational biology allows us to predict cataract development in the long-term. These models allow our team to: (1) understand the impact of adding GSR-loaded nanoparticles into the lens over a 50 year period and (2) design a full treatment plan on how to prevent and treat cataracts with our project. Therefore, the results of our model are essential in developing a functional prototype.
For sake of clarity, we will discuss each model in detail with respect to prevention (using GSR) only. At the end, we extend these results to treatment. In addition, we include collapsibles for interested readers and judges, in order to fully document our modeling work (eg. assumptions, mathematics, and full analysis) while keeping the main page clear with basic points only.
Introduction
Guiding Questions
How much GSR to maintain in the lens? (GSR Function)
How to maintain that amount of GSR using nanoparticles and eyedrops? (Delivery Prototype)
Focus of Models
Since our construct is not directly placed into the eyes, how our synthesized protein impacts the eye after it is separately transported into the lens is of greater importance. As a result, we create models with the intent on understanding how GSR and CH25H impacts the eye, and how we can control its impact with a well-designed delivery prototype.
Prevention: GSR Function
Model 1: Crystallin Damage
The amount of damage to crystallin by H2O2 determines the severity of a cataract. We relate the amount of crystallin damage to the corresponding rating on the LOCS scale, used by physicians to rate cataract severity. Our goal is to lower LOCS to below 2.5, the threshold for surgery. Through literature research as well as our own experimental data, we find the maximum allowable crystallin damage to prevent a LOCS 2.5 cataract from developing.
Measurement of Cataract Severity
There are four ways of measuring cataract severity, each used for a different purpose.
- Lens Optical Cataract Scale (LOCS): Physicians use this scale, from 0 – 6, to grade the severity of cataracts.
- Absorbance at 397.5 nm: This is the experimental method, used by our team in the lab (c.d.)
- Crystallin Damage: This is a chemical definition. We quantify cataract severity as a function of how much oxidizing agents there are, as well as how long crystallin is exposed to oxidizing agents.
Measurement of Cataract Severity
Numerous studies show how absorbance measurements can be converted to the LOC scale that physicians use. With the results of ________ and ________, we construct the first two columns in Table 2.
Absorbance Equivalence to Crystallin Damage: Experimental Data
We use experimental measurements from our team’s Cataract Lens Model (link). They induced an amount of crystallin damage, and measured the resulting absorbance. With this relation in Figure 2, we calculate the equivalent crystallin damage of each LOCS rating and absorbance.
Conclusion
To guarantee that surgery is not needed for 50 years, we need to limit crystallin damage to 0.9883 units. If crystallin damage goes above this threshold, then surgery is needed. This is the crystallin damage threshold for a LOCS 2.5 cataract.
LOCS | Absorbance (@397.5 nm abs units) |
Crystallin Damage (M-h) |
---|---|---|
0.0 | 0.0000 | 0.0000 |
0.5 | 0.0143 | 0.1243 |
1.0 | 0.0299 | 0.2878 |
1.5 | 0.0497 | 0.4697 |
2.0 | 0.0751 | 0.6949 |
2.5 | 0.1076 | 0.9883 |
3.0 | 0.1492 | 1.3747 |
4.0 | 0.2706 | 2.5259 |
5.0 | 0.4691 | 4.3472 |
Model 2: GSR Pathway
Now that we know how much GSR we need to limit crystallin damage to LOCS 2.5, we model the naturally occurring GSR Pathway in the lens of a human eye. For prevention (2A), we calculate the necessary GSR concentration to be maintained over 50 years so that the resulting cataract is below LOCS 2.5.
Chemical Kinetics Model: Differential Equations
By various enzyme kinetics laws, fully documented in the collapsible, we build a system of 10 differential equations based on 6 chemical reactions. All parameters, constants, and initial conditions are based off literature data. Estimates made are also shown with assumptions and reasoning. The details are shown in the collapsible for interested readers.
Blackbox Approach: Testing GSR Impact
We vary the input, Initial GSR concentration, holding all other variables constant, and numerically solve for the amount of hydrogen peroxide over time. With this graph, we can find the amount of crystallin damage accumulated over 50 years if different levels of GSR is maintained.
From this graph, we can find the GSR concentration needed for the LOCS 2.5 threshold.
Crystallin Damage vs. GSR Level
According to literature data and our model, the naturally occurring GSR concentration is 10 uM. All curves show crystallin damage decreasing as GSR levels are increased, which supports both research and experimental data, and suggests that this prototype is effective in preventing crystallin damage. However, GSR levels need to be raised significantly, up to 40+ uM from the natural 10 uM of GSR in order to show long-term protection.
Table 3 shows the amount of GSR we need to maintain for 50 years in order to prevent a LOCS cataract of a certain severity. The row of interest is LOCS 2.5, the threshold for surgery. Notice that we say “maintain” the level of GSR. This level needs to be constant at all times for 50 years for full prevention. The delivery of GSR to maintain this level is discussed in Model 3.
![](https://static.igem.org/mediawiki/2016/c/cc/T--TAS_Taipei--FinalGSRGraph.png)
Conclusion
We need to maintain (NOT add) 43.5 uM of GSR in the lens so that the crystallin damage recorded over 50 years is below the LOCS 2.5 threshold.
Prevention: Prototype Function
Model 3: Nanoparticle Protein Delivery
To maximize delivery efficiency to the lens, we encapsulate GSR in chitosan nanoparticles. From Models 1-2, we have found the necessary concentration of GSR that needs to be maintained in the lens. Now we design nanoparticles that will maintain those amounts. We build a model find how nanoparticles release GSR at appropriate rates to control the amount of GSR in the lens, and find the best engineered design for nanoparticles.
Single Dose: Change in GSR Concentration
In finding the best engineered design, we take into account variables such as nanoparticle radius and concentration. We build a differential equation model for the impact of a single dose of nanoparticles over time. To generalize the model, instead of using absolute concentrations, we use relative concentration, with respect to the natural amount, or initial amount of GSR in the lens. The full mathematics and details can be found in the collapsible.
We get two curves, concentration of GSR in the nanoparticles, and GSR release from nanoparticles, over time. This allows us to predict nanoparticle delivery rates before we perform the actual experiments.
![](https://static.igem.org/mediawiki/2016/6/6b/T--TAS_Taipei--GSR_SingleDose_Model.png)
Comparison with Experimental Data
Yet in our model, we do not know the thickness of the nanoparticle diffusion layer. After performing experiments, we can use measurements of our prototype device to find this thickness, and refine our model. A direct comparison of our model with our experiment data is shown in Figure ___.
To generalize the model, instead of using absolute concentrations, we use relative concentration, with respect to the natural amount, or initial amount of GSR in the lens.
![](https://static.igem.org/mediawiki/2016/5/5e/T--TAS_Taipei--Nanoparticle_Release_Model_final.png)
Multiple Dose: Change in GSR Concentration
In Figure ____, all curves approach equilibrium, after which the concentration oscillates about equilibrium. We have three goals, in order of importance for best nanoparticle design:
- GSR equilibrium concentration equal to amount we desire (i.e. 43.5 uM from Model 2)
- Stability of concentration at equilibrium (Model 4 goes into deeper depth regarding sensitivity)
- Time to reach equilibrium (time for full prevention to come into effect)
To do so, we can alter different variables: GSR concentration in nanoparticles, nanoparticle radius, and dose frequency. For a full analysis of how each variable impacts the concentration function, see the collapsible. Below is a summary of the results:
Independent Variable | Time to Reach Equilibrium | Equilibrium Concentration | Stability |
---|---|---|---|
Concentration | No impact | Proportional Increase | Slight Increase |
Radius | No impact | Decrease | No impact |
Frequency | No impact | Decrease | Increase |
We find the optimal combination of parameters is: _____________.
A Two Stage Eyedrop Approach
As shown in Table ____, we cannot alter the time to reach equilibrium, or reach full prevention. As supported by ______, the time to reach equilibrium is a property of the lens that we cannot change. However, we propose a two-step eyedrop approach, of two differing nanoparticle concentrations, to decrease the time needed for full prevention. A full explanation is found in the collapsible.
Generalized Nanoparticles: Customizer
We built a full nanoparticle customizer, which generalizes the model to beyond delivery into the eye, found at the end of the page (Software). We hope that other iGEM teams who are interested in nanoparticle drug delivery can utilize this customizer to help them develop their own prototype.
Conclusion
???
Model 4: Eyedrop Prototype
We have found a nanoparticle design to deliver GSR. We also need to model the function of eyedrops, to determine the concentration of GSR-loaded nanoparticles to put in eyedrops, and analyze how sensitive the resulting system is.
Bioavailability of GSR Delivery
The eye is well protected from foreign material attempting to enter the eye. The corneal epithelium is the most essential barrier against topical drugs in eyedrops, and as a result, much of drugs in eyedrops are lost in tear drainage.
Bioavailability describes the proportion of the drug that reaches the site of action, regardless of the route of administration. For example, it is estimated that only 1-5% of an active drug with small solutes in an eyedrop penetrates the cornea (Schoenwald 1997). In the case of nanoparticles, which are much larger than chemical molecules, more is lost. (Clinical Ocular Toxicology)
The results show that the bioavailability of nanoparticles is about 8.35 x 10-5%, which means that for every gram of GSR (or any drug) we place into nanoparticles, approximately 0.835 ug of the drug reach the aqueous humor. The variance is 0.125 ug/g.
Necessary Adjustments in Eyedrops
To ensure that sufficient concentrations of GSR are delivered, we must place an excess of GSR. To determine how much, we simply divide the concentration of GSR in nanoparticles we found in Model 3 by the fraction of GSR that reaches the aqueous humor.
[Calculations]
We conclude that we need ________ mM of GSR in nanoparticles to maintain 43.5 uM GSR and thus 2.5 LOCS.
Sensitivity Analysis: Revisiting Nanoparticles Model
The mechanism for eyedrop delivery is complex, and there are variances in the bioavailability depending on the conditions of the eye. The thickness of the cornea, lens, other eye diseases, age, and even time of day may impact the bioavailability of the drug. We use a stochastic model to simulate Model 3 again, but this time, add a degree of variance.
The variance is impacted by the frequency of eyedrops. By giving eyedrops more frequently with less amounts given each time, the variance is decreased.
Ideally, we wish to deliver 100% of the GSR concentration of the amount found in Model 2 (43.5 uM). Because of variance, the actual amount maintained in the lens is different, shown in Figure 5. The full details and mathematics of the stochastic model can be found in the collapsible.
CALCULATOR
Prevention
LOCS Score Threshold:We guarentee that by applying this prevention eyedrop daily, your LOCS score will remain below your threshold for 50 years.
Prevention Results
Variable | Value | Source |
---|---|---|
Allowable LOCS | ||
Crystallin Damage | c.d. | Model 1 |
GSR Maintained | uM | Model 2 |
Nanoparticle Conc. | uM | Model 3 |
Eyedrop Conc. | mM | Model 4 |
Eyedrop Result | mg/mL |
Treatment
LOCS Score Threshold:By applying the following treatment, leaving an hour before each dose of eyedrops, we guarentee that it will lower your LOCS score to essentially 0.
Treatment Results
Variable | Value | Source |
---|---|---|
Allowable LOCS | ||
Crystallin Damage | c.d. | Model 2 |
Absorbance | a.u. | Model 1 |
CH25H | uM | Model 5 |
Eyedrop Conc. | uM | Model 4 |
Eyedrop Result | mg/mL | Model 4 |
# of Eyedrops | drops | (of 0.8 mg/mL eyedrop) |
Conclusion
![](https://static.igem.org/mediawiki/2016/e/e5/T--TAS_Taipei--Taipei_Background_1.0.jpg)
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