Difference between revisions of "Team:TecCEM/Improve"

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<p>The red fluorescent protein has an isoelectric point of 4.07 and has a neutral charge when the medium pH is 4. When doing the characterization of RFP encapsulation, this was made under the next conditions: pH of 4 and solution concentrations of 3 mg/mL chitosan and 1 mg/mL TPP. This resulted in an encapsulation with an average size of ~25 nm.</p>
 
<p>The red fluorescent protein has an isoelectric point of 4.07 and has a neutral charge when the medium pH is 4. When doing the characterization of RFP encapsulation, this was made under the next conditions: pH of 4 and solution concentrations of 3 mg/mL chitosan and 1 mg/mL TPP. This resulted in an encapsulation with an average size of ~25 nm.</p>
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                                    <img style="max-height: 70vh;" src="https://static.igem.org/mediawiki/2018/c/c9/T--TecCEM--Figure1Improvement.png "
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                                        class="figure-img img-fluid rounded" alt="IMP-1">
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                                    <figcaption class="figure-caption"><strong>Figure 1. RFP loaded nanoparticles result from TEM imaging at a 150000x magnification in two different quadrants. </strong></figcaption>
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<p>We performed a particle analysis in NanoSight NS300 to obtain the particle size distribution. A dot graph for a triplicate analysis is presented below.<p>
 
<p>We performed a particle analysis in NanoSight NS300 to obtain the particle size distribution. A dot graph for a triplicate analysis is presented below.<p>
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                                    <img style="max-height: 70vh;" src="https://static.igem.org/mediawiki/2018/3/30/T--TecCEM--Figure2Improvement.jpg "
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                                        class="figure-img img-fluid rounded" alt="IMP-1">
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                                    <figcaption class="figure-caption"><strong>Figure 2. Size distribution of RFP-loaded chitosan nanoparticles prepared at pH 4</strong></figcaption>
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<p>The particle sizes obtained from NanoSight NS300 differ from those observed by transmission electron microscopy (TEM), but we hypothesize this discrepancy is caused by particle conglomeration rather than different particle sizes. Water at pH 7.4 was the solvent in which particles were resuspended for the NS300 analysis, while samples were treated beforehand for the observation in TEM. This treatment helps to enable a clear observation of individual nanoparticles. This effect may not be reached in a polar environment like an aqueous solution.</p>
 
<p>The particle sizes obtained from NanoSight NS300 differ from those observed by transmission electron microscopy (TEM), but we hypothesize this discrepancy is caused by particle conglomeration rather than different particle sizes. Water at pH 7.4 was the solvent in which particles were resuspended for the NS300 analysis, while samples were treated beforehand for the observation in TEM. This treatment helps to enable a clear observation of individual nanoparticles. This effect may not be reached in a polar environment like an aqueous solution.</p>
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                                    <img style="max-height: 70vh;" src="https://static.igem.org/mediawiki/2018/d/da/T--TecCEM--Figure3Improvement.png  "
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                                        class="figure-img img-fluid rounded" alt="IMP-1">
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                                    <figcaption class="figure-caption"><strong>Figure 3. Particle conglomeration in NanoSight NS300 analysis. </strong></figcaption>
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<p>To demonstrate the possibility of modifying chitosan solution pH, an encapsulation procedure was carried out using bovine serum albumin (BSA). BSA has a pI ranging from 4.7 to 4.9. As chitosan is soluble in acidic environments while acquiring a positive net charge, by adjusting the pH to 5.5 BSA charges become negative, improving the interaction with chitosan. Encapsulation efficiency may be optimized with this adjustments. </p>
 
<p>To demonstrate the possibility of modifying chitosan solution pH, an encapsulation procedure was carried out using bovine serum albumin (BSA). BSA has a pI ranging from 4.7 to 4.9. As chitosan is soluble in acidic environments while acquiring a positive net charge, by adjusting the pH to 5.5 BSA charges become negative, improving the interaction with chitosan. Encapsulation efficiency may be optimized with this adjustments. </p>
  
 
<p>The standard curve was done with Bradford assay to determine the concentrations of free protein in the supernatant after encapsulation and centrifugation.</p>
 
<p>The standard curve was done with Bradford assay to determine the concentrations of free protein in the supernatant after encapsulation and centrifugation.</p>
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                                    <img style="max-height: 70vh;" src="https://static.igem.org/mediawiki/2018/3/3a/T--TecCEM--Figure4Improvement.jpg"
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                                        class="figure-img img-fluid rounded" alt="IMP-1">
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                                    <figcaption class="figure-caption"><strong>Figure 4. Standard curve for BSA supernatant concentration of nanoparticles.</strong></figcaption>
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Revision as of 20:56, 16 October 2018

Cell Gif

Improvement

Improvement of nanoencapsulation protocol with low molecular weight chitosan and sodium tripolyphosphate (TPP)

The main objective of nanoencapsulation was to create an efficient vehicle for protein delivery. In order to obtain this, we used the same nanoencapsulation protocol from iGEM Team Taipei 2016, and created a smaller nanoparticle with an approximate size of ~25 nm. We assessed the fact that during encapsulation pH may need a modification to optimize the interaction between chitosan and proteins, shifting the encapsulation efficiency. A troubleshoot was applied for this protocol to work with any protein, as pH 5.5 is not a universal ideal condition.

The reason behind this is that chitosan is positively charged, and proteins with low isoelectric value are better encapsulated at a pH greater than the pI value, favoring the interaction between negatively charged proteins and chitosan. Therefore, before encapsulating any protein, the pI has to be consulted to adjust the chitosan solution pH, so that pI < pH < 6.5 (Gan and Wang, 2007).

The red fluorescent protein has an isoelectric point of 4.07 and has a neutral charge when the medium pH is 4. When doing the characterization of RFP encapsulation, this was made under the next conditions: pH of 4 and solution concentrations of 3 mg/mL chitosan and 1 mg/mL TPP. This resulted in an encapsulation with an average size of ~25 nm.

IMP-1
Figure 1. RFP loaded nanoparticles result from TEM imaging at a 150000x magnification in two different quadrants.

We performed a particle analysis in NanoSight NS300 to obtain the particle size distribution. A dot graph for a triplicate analysis is presented below.

IMP-1
Figure 2. Size distribution of RFP-loaded chitosan nanoparticles prepared at pH 4

The particle sizes obtained from NanoSight NS300 differ from those observed by transmission electron microscopy (TEM), but we hypothesize this discrepancy is caused by particle conglomeration rather than different particle sizes. Water at pH 7.4 was the solvent in which particles were resuspended for the NS300 analysis, while samples were treated beforehand for the observation in TEM. This treatment helps to enable a clear observation of individual nanoparticles. This effect may not be reached in a polar environment like an aqueous solution.

IMP-1
Figure 3. Particle conglomeration in NanoSight NS300 analysis.

To demonstrate the possibility of modifying chitosan solution pH, an encapsulation procedure was carried out using bovine serum albumin (BSA). BSA has a pI ranging from 4.7 to 4.9. As chitosan is soluble in acidic environments while acquiring a positive net charge, by adjusting the pH to 5.5 BSA charges become negative, improving the interaction with chitosan. Encapsulation efficiency may be optimized with this adjustments.

The standard curve was done with Bradford assay to determine the concentrations of free protein in the supernatant after encapsulation and centrifugation.

IMP-1
Figure 4. Standard curve for BSA supernatant concentration of nanoparticles.