Implement Group B

THEME.md

@@ -24,6 +24,14 @@ The notebook text and bubble background colors can be accessed with the `noteboo
 </div>
 ```
 
+The notebook text and bubble background colors can be accessed with the `notebookBg` and `notebookText` keywords:
+
+```html
+<div class="bg-notebookBg">
+    <p class="text-notebookText">Body</p>
+</div>
+```
+
 ## Typography
 
 The Montserrat and Fira Sans fonts have been added to the project. Montserrat has been set as the default font, and Fira Sans can be accessed with `font-title`.

src/components/CustomTable.vue

@@ -8,7 +8,7 @@
   }
 }>()
 
 const border = 'border-notebookText'
 </script>
 
 <template>
@@ -32,7 +32,7 @@
         >
           {{ tableData.rowHeaders[i / tableData.rowsPerRowHeader] }}
         </th>
-        <td v-for="(item, j) in row" :key="j" class="py-1 lg:text-lg">{{ item }}</td>
+        <td v-for="(item, j) in row" :key="j" class="py-1 lg:text-lg text-center">{{ item }}</td>
       </tr>
     </tbody>
   </table>

src/components/lab-notebook/NotebookBody.vue

@@ -7,7 +7,7 @@ import TableOfContents from '@/components/lab-notebook/TableOfContents.vue'
     <div v-if="$windowWidth >= 1024" class="basis-1/5 py-8 ml-8 sticky h-1/3 top-0">
       <TableOfContents />
     </div>
-    <div class="flex flex-col items-center gap-10 w-full lg:basis-4/5 p-8 leading-relaxed">
+    <div class="flex flex-col items-center gap-10 w-full lg:basis-4/5 p-4 md:p-8 leading-relaxed">
       <slot></slot>
     </div>
   </div>

src/components/navbar/NavBar.vue

@@ -48,7 +48,7 @@ const tree: Tree[] = [
           },
           {
             name: 'Liposome Formation',
-            url: '#'
+            url: '/lab-validation/liposome-formation'
           },
           {
             name: 'Octahedron Formation',

src/router/index.ts

@@ -5,6 +5,7 @@ const TeamView = () => import('@/views/TeamView.vue')
 const ELSIView = () => import('@/views/ELSIView.vue')
 const SponsorsView = () => import('@/views/SponsorsView.vue')
 const GroupAView = () => import('@/views/lab-validation/GroupAView.vue')
+const GroupBView = () => import('@/views/lab-validation/GroupBView.vue')
 const GroupCView = () => import('@/views/lab-validation/GroupCView.vue')
 const OctadedronFormation = () => import('@/views/lab-validation/OctahedronFormation.vue')
 
@@ -16,19 +17,16 @@ const router = createRouter({
       name: 'home',
       component: HomeView
     },
-
     {
       path: '/ideas',
       name: 'ideas',
       component: IdeasView
     },
-
     {
       path: '/elsi',
       name: 'elsi',
       component: ELSIView
     },
-
     {
       path: '/team',
       name: 'team',
@@ -44,6 +42,11 @@ const router = createRouter({
       name: 'dnase-i',
       component: GroupAView
     },
+    {
+      path: '/lab-validation/liposome-formation',
+      name: 'liposome-formation',
+      component: GroupBView
+    },
     {
       path: '/lab-validation/thiolation-and-conjugation',
       name: 'thiolation-and-conjugation',

src/views/lab-validation/GroupBView.vue

@@ -0,0 +1,241 @@
+<script setup lang="ts">
+import Notebook from "@/components/lab-notebook/Notebook.vue"
+import SingleColumn from "@/components/lab-notebook/SingleColumn.vue"
+import TripleColumn from "@/components/lab-notebook/TripleColumn.vue"
+import CustomTable from '@/components/CustomTable.vue'
+
+const tableData = {
+  headers: [
+    '',
+    'Before Purification',
+    'Purified',
+    'Purified with Maleimide Groups'
+  ],
+  rowHeaders: ['Z-Average (d.nm)', 'PDI', 'Std. Deviation'],
+  rowsPerRowHeader: 1,
+  data: [
+    [99.03, 100.7, 32.36],
+    [0.250, 0.167, 0.206],
+    [11.73, 18.50, 6.288],
+  ]
+}
+</script>
+
+<template>
+  <Notebook>
+    <template #title>Liposome Formation</template>
+    <template #body>
+      <SingleColumn>
+        <template #title>
+          Intro
+        </template>
+        <template #body>
+          <div class="flex flex-col gap-4">
+            <p>
+              To prevent enzyme degradation by conjugating enzymes to liposomes, both components must be
+              treated to possess compatible functional groups (Nanjwade et al., 2015). Maleimide, or
+              H<sub>2</sub>C<sub>2</sub>(CO)<sub>2</sub>NH, is a chemical compound often used in synthetic
+              products with thiols due to
+              maleimide’s reactivity and ability to be readily conjugated with thiols via the mechanism of a
+              thiol-maleimide Michael addition reaction to form thioether bonds (Darling et al., 2016).
+              Maleimide derivatives in biological systems often act as lipid anchors binding polypeptides to
+              phospholipid membranes (Elliott & Prestwich, 2000). To synthesize enzymosomes–composed of
+              thiolated enzymes and liposomes containing maleimide–requires maleimide groups that are able to
+              readily react with excess thiols and form thioether bonds (Girão et al., 2021). We needed to
+              synthesize liposomes in a method that preserves the maleimide groups in formulated liposomes and
+              would be able to react with modified enzymes, without compromising the structure of the
+              liposome.
+            </p>
+
+            <p>
+              <b>
+                Aim: To create liposomes for the enzymosome structure that are formulated with maleimide
+                groups
+              </b>
+            </p>
+          </div>
+        </template>
+      </SingleColumn>
+      <TripleColumn>
+        <template #title>Methods</template>
+        <template #left-title>Lipid Stock Mixtures Preparation</template>
+        <template #left-body>
+          <div class="flex flex-col gap-4">
+            <p>
+              DOPC and Maleimide-PEG2000 were purchased through Avanti Polar Lipids.
+              <br />
+              Three initial 10mg/mL stock solutions were prepared:
+            </p>
+            <ul class="list-disc ml-6">
+              <li>10mg of DOPC dissolved in 1.0mL of pure ethanol</li>
+              <li>10mg of cholesterol dissolved in 1.0mL of pure ethanol</li>
+              <li>10mg of Maleimide-PEG2000 dissolved in 1.0mL of pure ethanol</li>
+            </ul>
+            <p>
+              The lipid stock solution was then prepared by reverse pipetting 572.6µL of DOPC solution,
+              134.8µL of cholesterol solution, and 100µL of PEG solution and mixed.
+            </p>
+            <p>
+              Phosphate-buffered saline (PBS) solution prepared by combining 8g of 0.137M sodium chloride,
+              0.2g of 0.0027M potassium chloride, 1.44g of 0.01M sodium phosphate dibasic, 0.245g of 0.0018M
+              potassium phosphate monobasic with water to create a 1L solution.
+            </p>
+          </div>
+        </template>
+        <template #mid-title>Liposome Formation</template>
+        <template #mid-body>
+          <p>
+            Liposome formation methodology was developed based upon the protocol outlined in a paper on lipid
+            nanoparticle synthesis (Wang, et al., 2022). Liposomes were synthesized by mixing 19.44µL of lipid
+            stock mixture and 0.56µL of ethanol in an RNase-free tube at room temperature. The solution was then
+            treated with 60µL of 1x PBS buffer and thoroughly mixed by pipetting up and down rapidly for about
+            30s, to ensure fully formed uniform lipid nanoparticles. Before proceeding, the solution was
+            incubated at room temperature for up to 15 minutes. Initial liposome formation was checked by
+            analyzing the diffraction pattern from a red laser beam.
+          </p>
+        </template>
+        <template #right-title>Ethanol Removal</template>
+        <template #right-body>
+          <div class="flex flex-col gap-4">
+            <p>
+              Amicon Filtration was used to remove ethanol from the liposome solution. After equilibrating
+              100kDa Amicon filter membrane, the liposome sample was added to filter with excess water and
+              centrifuged at 2500g for 30 minutes twice. Then, the filter was reversed in a new tube and
+              centrifuged at 1000g for 2 minutes to produce about a 25µL solution.
+            </p>
+            <p>
+              The solution was then transferred into a cuvette and placed into a Zetasizer Nano ZS Machine
+              with a He-Ne laser (λ = 632 nm) where the particle sizes were measured. The volume of the
+              solution was measured before being stored at 4°C for use, as recommended by the lipid
+              nanoparticle synthesis paper (Wang, et al., 2022).
+            </p>
+          </div>
+        </template>
+      </TripleColumn>
+
+      <SingleColumn>
+        <template #title>
+          Results
+        </template>
+        <template #body>
+          <div class="flex flex-col gap-y-4">
+            <div class="flex flex-col items-center">
+              <div class="w-full overflow-scroll">
+                <CustomTable :table-data="tableData" />
+              </div>
+              <p class="text-sm text-center">
+                Table 1: Liposome Particle Size Distribution under 3 Conditions - Pre-purification, Post-purification (via
+                Amicon filtration), and Post-incorporation of Maleimide Groups. Samples measured using dynamic light
+                scattering (DLS). Z-Average (mean particle size) and PDI (polydispersity index) characterize particle size
+                and
+                distribution, respectively.
+              </p>
+            </div>
+            <p>
+              Lipid stock solutions were made and used in liposome formulation. Liposome formation was evaluated with a
+              Zetasizer Nano ZS machine to assess consistency in size so that succeeding steps of liposome and enzyme
+              conjugation were not complicated by variable liposome sizes. As seen in Table 1, unpurified liposome samples
+              were on average about 99.03 d.nm in size, as represented by the z-average of the samples based on intensity,
+              and had a poly-dispersive index (PDI) of 0.250. Purified liposome samples show a z-average of 100.7 d.nm and
+              a
+              PDI of 0.167. Liposome samples with attached maleimide groups had an average size of 32.36 d.nm, and a PDI
+              of
+              0.206 (Table 1). It should be noted that the maleimide-treated liposomes were formed with low volume sizing
+              cuvettes, unlike the other samples, which likely led to the smaller liposome size.
+            </p>
+          </div>
+        </template>
+      </SingleColumn>
+
+      <SingleColumn>
+        <template #title>
+          Discussion
+        </template>
+        <template #body>
+          <div class="flex flex-col gap-4">
+            <p>
+              The results of liposome formation found that both purified and unpurified samples of liposomes (without
+              maleimide) yielded similar sized liposome products on average–100.7 d.nm sized liposomes for purified and
+              99.03 d.nm for unpurified samples. However, an important factor to consider is the increased consistency in
+              size seen in trials with purified samples. This can be detected in the low poly-dispersive index (PDI) of
+              0.167, as seen in Table 1, which indicated that the purified liposomes formed in the experimentation were
+              all quite similar in size. Unpurified samples had a slightly higher PDI of 0.250, indicating greater
+              variation and inconsistency in liposome sizes produced within each sample compared to the purified samples.
+              These results align with previous experiments involving formation of comparably sized liposomes by similar
+              manners, such as a 2019 study that prepared liposomes to co-load with antitumor drugs for cancer treatment
+              that characterized the liposomes produced in their trials as being on average about 85-120 d.nm with a PDI
+              of 0.183 to 0.230 (Song et al., 2019). While past literature does support and provide confidence in the
+              consistency and replicability of the results of these trials, our intention is to further reduce this PDI in
+              the future with the use of our DNA n-gonal bipyramid to produce more precise size-controlled liposomes. An
+              interesting result is the increase in the standard deviation of sizes in the purified samples compared to
+              the unpurified samples (Table 1), while concurrently observing a decrease in the PDI. This suggests a more
+              uniform size distribution after purification, with the standard deviation increase likely attributed to a
+              few liposomes with highly deviant sizes. It is our intention, that with the use of our DNA n-gonal bipyramid
+              structures, this inconsistency will be reduced and more homogenous sized liposomes will be produced across
+              samples.
+            </p>
+            <p>
+              Liposomes treated with maleimide, in preparation to being conjugated with DNase I, show uniformity in
+              results with a PDI of 0.206, which is between the PDI of purified and unpurified samples without maleimide
+              (Table 1). While the addition of maleimide does seem to have increased the PDI, it is still within an
+              acceptable range and comparable to past literature results of untreated liposomes between the 0.183 to 0.230
+              PDI range (Song et al., 2019). The average liposome size of the sample was 32.36 d.nm, but the divergence
+              from the previous liposome sample sizes can be cited as a result of the low volume sizing cuvettes that were
+              used for only the liposomes with maleimide. Future experiments could include investigation of whether
+              liposome size remains stable before and after maleimide addition when using the same size cuvettes.
+            </p>
+            <p>
+              Additionally, experimentation to quantitatively test for the presence and amount of maleimide groups yielded
+              from the liposome formulation could be done to verify the presence of maleimide on products and ensure the
+              liposome is suitable for reacting with enzymes in successive steps of the enzymosome structure formation.
+              This could be done in further investigations with a maleimide assay. A maleimide assay would involve
+              reacting a known amount of excess thiols to the liposome solution and then treating the solution with a
+              thiol-reactive chemical, such as 4,4'-dithiodipyridine (DTDP), to quantify maleimide presence on the surface
+              of the final liposome structures and show liposomes were successfully formulated. This assay would quantify
+              the presence of maleimide groups, which are highly reactive in the presence of thiols, by measuring the
+              difference between initial excess thiol amounts and the amount of unreacted thiol after the complete
+              reaction to the maleimides by assaying unreacted thiols with another agent (Singh, 1994). Doing so in future
+              experimentations would provide evidence that liposomes formed are capable of being conjugated with enzymes
+              and confirm consistency of results yielded from this methodology.
+            </p>
+          </div>
+        </template>
+      </SingleColumn>
+
+      <SingleColumn :always-dropdown="true">
+        <template #title>
+          References
+        </template>
+        <template #body>
+          <div class="pl-6 -indent-6">
+            <p>Darling, Nicole J., Hung, Yiu-Sun, Sharma, Shruti, & Segura, Tatiana. (2016) “Controlling the
+              Kinetics of Thiol-Maleimide Michael-Type Addition Gelation Kinetics for the Generation of
+              Homogenous Poly(Ethylene Glycol) Hydrogels.” Biomaterials, 101,199–206.
+              https://doi.org/10.1016/j.biomaterials.2016.05.053</p>
+            <p>Elliott, J. T., & Prestwich, G. D. (2000). Maleimide-functionalized lipids that anchor
+              polypeptides to lipid bilayers and membranes. Bioconjugate Chemistry, 11(6), 832–841.
+              https://doi.org/10.1021/bc000022a</p>
+            <p>Girão, L. F. C., Carvalheiro, M. C., Ferreira-Silva, M., da Rocha, S. L. G., Perales, J.,
+              Martins, M. B. F., Ferrara, M. A., Bon, E. P. S., & Corvo, M. L. (2021). ASP-Enzymosomes with
+              Saccharomyces cerevisiae Asparaginase II Expressed in Pichia pastoris: Formulation Design and In
+              Vitro Studies of a Potential Antileukemic Drug. International Journal of Molecular Sciences,
+              22(20), 11120. https://doi.org/10.3390/ijms222011120</p>
+            <p>Nanjwade, B. K., Hundekar, Y., Mohamied, A. S., Idris, N. F., Srichana, T., & Shafioul, A. S. M.
+              (2015). Nanomedicine to Tumor by Enzymosomes. Nanotechnology: Nanomedicine&Nanobiotechnology,
+              2(1), 1–5. https://doi.org/10.24966/NTMB-2044/100004</p>
+            <p>Singh, R. (1994). A Sensitive Assay for Maleimide Groups. Bioconjugate Chemistry, 5(4), 348–351.
+              https://doi.org/10.1021/bc00028a011</p>
+            <p>Song, M., Wang, J., Lei, J., Peng, G., Zhang, W., Zhang, Y., Yin, M., et al. (2019). Preparation
+              and Evaluation of Liposomes Co-Loaded with Doxorubicin, Phospholipase D Inhibitor
+              5-Fluoro-2-Indolyl Deschlorohalopemide (FIPI) and D-Alpha Tocopheryl Acid Succinate (α-TOS) for
+              Anti-Metastasis. Nanoscale Research Letters, 14, 138. https://doi.org/10.1186/s11671-019-2964-4
+          </p>
+          <p>Wang, X., Liu, S., Sun, Y., Yu, X., Lee, S. M., Cheng, Q., Wei, T., Gong, J., Robinson, J.,
+            Zhang, D., Lian, X., Basak, P., & Siegwart, D. J. (2022). Preparation of selective
+            organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for
+            tissue-specific mRNA delivery. Nature Protocols, 18, 265-291.</p>
+        </div>
+      </template>
+    </SingleColumn>
+  </template>
+</Notebook></template>