Team:TecCEM/Improve

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 attain this, we applied the nanoencapsulation protocol from iGEM TAS Taipei 2016 and produced a smaller nanoparticle with an approximate size of ~27 nm. The creation of smaller particles yields a higher internalization rate of several drugs, which acts inside the cell, rather than on the surface receptors, small nanoparticles ( < 39 nm) are able to pass through the nuclear pore complexes via passive diffusion and have a higher endocytosis rate. Nuclear target nanoparticles are an example of such a design. (Tamman, 2015)

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 better with any protein, as pH 5.5 is not a universal ideal condition. The reason behind this is that chitosan is positively charged when dissolved in an acidic environment, 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 as an ideal condition, though not all proteins will meet such constraint (Gan and Wang, 2007). Our improvement proposal is demonstrated by the encapsulation of a standard protein that meets this suggested condition.

The employed red fluorescent protein (BBa_J04450) has an isoelectric point of around 5.6. The characterization of RFP encapsulation was made under the following conditions as stated by TAS Taipei 2016: pH of 4 to avoid chitosan aggregation and solution concentrations of 3 mg/mL chitosan and 1 mg/mL TPP. This resulted in an encapsulation with an average size of ~27 nm. Like proposed by team TAS Taipei 2016, RFP encapsulation was verified by centrifugation of particle suspension and UV irradiation of the formed pellet.

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Figure 1. Fluorescence detected after centrifugation of RFP loaded chitosan nanoparticles at pH=4.
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Figure 2. 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.

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Figure 3. 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 an environment like an aqueous solution.

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Figure 4. Particle conglomeration in NanoSight NS300 analysis.

To proof 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 derived with the Bradford assay to determine the concentrations of free protein in the supernatant after encapsulation and centrifugation.

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Figure 5. Standard curve for BSA supernatant concentration of nanoparticles.

Encapsulation efficiency was then determined from:

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The resulting efficiency for BSA loaded nanoparticles was 42% from a four encapsulation sample assay. BSA has a molecular weight (MW) of 66.5 kDa and a pI of 4.6. As it has been proved, encapsulation efficiency increases with decreasing MW. Though BSA has a high MW, the pI < pH effect generates a high-efficiency percentage according to literature. (Jarudilokkul, 2011)

Chromoproteins

In many systems for the control of cloning and transformation reporter protein are used, but what happens when two different reporter proteins are cloned in the same vector and expressed at the same time? That is what here wanted to answer by fusioning two reporter cassettes. In this case a constitutive blue chromoprotein and a repressible Red Fluorescent Protein were cloned in a pSB1C3 plasmid to observe how both reporters interact and if our hypothesis of obtaining a purple color would be true. To prove this idea, after the ligation of the both parts with the plasmid, previously mentioned, this complex needs to be transformed into an appropriate Escherichia coli strain and then, the coloration of the colonies has to be measured.

Another experiment was the repression of Red Fluorescent Protein to prove the expression of Blue Chromoprotein one. This kind of system allows the design of complex multiple reporter proteins to simplify the selection process and developed stronger controls during the creation of synthetic biology projects.

The register number of the part involved in this improvement are:

  • RFP cassette: BBa_I13521
  • Blue chromoprotein: BBa_K592015
  • Fusion of the reporter proteins: BBa_K2719013

References:

  1. 2016 iGEM Taipei Team. Nanoencapsulation protocol.
  2. Tamman, S., Azzazy, H., Breitinger, H., Lamprecht, A. (2015) Chitosan Nanoparticles for Nuclear Targeting: The Effect of Nanoparticle Size and Nuclear Localization Sequence Density. doi: 10.1021/acs.molpharmaceut.5b00478
  3. Jarudilokkul, S. (2011) Preparation of chitosan nanoparticles for encapsulation and release of protein. doi.org/10.1007/s11814-010-0485-z