Team:TAS Taipei/Proof
Proof of Concept
We made a functional prototype to show how chitosan nanoparticles can both encapsulate and release desired proteins for delivery to the eye. We encapsulated green fluorescent protein (GFP, BBa_K741002), red fluorescent protein, and green pigment (Bba_K274003) into nanoparticles. Because the nanoparticles are translucent, the pelleted nanoparticles displayed the color of the protein. When we viewed the nanoparticles under blue light, the GFP and RFP containing pellets glowed, showing that protein encapsulation was successful and that the proteins remained functional inside nanoparticles. We also determined the percent of proteins that were encapsulated into the nanoparticles by measuring the amount of protein left in the solution outside the nanoparticles. We found that 72% of proteins was loaded into nanoparticles. We tested if the nanoparticles would degrade and release proteins by incubating resuspended nanoparticles in phosphate buffered saline at 4℃ and 37℃. Using Coomassie Brilliant Blue to stain proteins, we found that proteins were released at 37℃ but not at 4℃. Finally, we made eye drops containing nanoparticles and GFP-containing nanoparticles.
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).
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).
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 add 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.
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%.
Protein Release
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.
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).
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