Team:TAS Taipei/Design
Applied Design
A main obstacle for protein delivery into the eye is that the cornea acts as a barrier and blocks materials from entering the eye. To increase the amount of proteins that reach the lens, we made biodegradable chitosan nanoparticles that can package and deliver proteins. According to literature research, chitosan nanoparticles can embed in the cornea, where the encapsulated proteins can be released as the particles degrade. This is a better solution than commercially available eye drops (since more proteins can be delivered through the cornea) and surgery (because it is non-invasive). In addition, the nanoparticles do not affect vision or the normal protective functions of the cornea. We show that our nanoparticles successfully encapsulated proteins. Proteins remain inside nanoparticles at 4℃, which allows for storage, but can be released at body temperature. Finally, we envision using these nanoparticles in eye drops or contact lenses.
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, and 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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