Team:NWU-CHINA-A/Implementation
Experimental characterization
We proved that the transformed E. coli can express PhaP-AMP successfully(figure 1), PhaP-HD5 and PhaP-HD5d5 are the most expressed complexs among them.
Figure 1 the expression of PhaP-AMP
In order to verify the adhesion between PHA and PhaP-AMPs ,we did the following experiments:
First, we used electrospinning technology to make PHA films. Secondly the films was immersed in the supernatant after bacterial sonication for 2 hours. Thirdly, we washed the films gently with lysis buffer, then put them into a centrifuge tube and added SDS-PAGE buffer, heat at 90℃ until the thin membrane softens.
In order to make the adhesion between PhaP-AMPs and PHA better, we used nano-level PHA for verification. Added PHA and supernatant in a ratio of 1:20, stirred two hours by the magnetic stirrer, then centrifuged at 8000rpm for 2min, rinsed twice with lysate, and added SDS-PAGE buffer.
We took the liquid from the above product for SDS-PAGE, as we can see, PhaP-HD5d5 combines the most products with PHA，whether it is electrospinning or nanoparticles.
Figure 2 Combination of PHA and PhaP-AMP
Electrospinning
Electrospinning, as the most promising process method for preparing polymer nanofibers, has received widespread attention from academia and industry. The nanofiber cloth prepared by electrospinning has a porous structure of fibers. Its smaller pore size can block contact between bacteria and wound surface. More importantly, the nanofibers prepared by electrospinning technology can structurally mimic the extracellar matrix (ECM) secreted by cells and accelerate wound healing. Electrospinning is a very good technique for making adhesive bandages.
Figure 3 the micrograph of
electrospinning
Elasticity
Electrospinning
Waterproofness
At this stage, we have made shaped adhesive bandages using electrospinning technology. In the experiment, we proved that PHA electrospun films can be combined with PhaP-HD5d5 tightly (figure 2).
Nanoparticles
Controlled drug release is the most convenient way of drug delivery currently, and the use of nano-particles has received special attention in the past two decades. Controlled drug delivery (CDD) technology is one of the most prominent fields in the field of human health science.
Polyesters have been widely used in drug delivery because of their biodegradability and adjustable mechanical properties. Polyesters have been used in various delivery systems for low molecular weight drugs as well as peptide and protein drugs. Most of the nano-particles based on polyester are focused on tumor-targeted drug delivery. Nano-particles made from polyester materials include microspheres, capsules, cubes and other shapes, and the active drug component (API) is usually uniformly dispersed in the polyester matrix[1].
Nano-particles are partly internalized by liquid phase endocytosis and grid protein-mediated endocytosis. Within 10 minutes after incubation, they quickly escape lysosomes and enter the cytoplasm, and nano-particles can effectively incorporate drugs into their structures[2]. Compared with large particles with small surface area, microns and nano-particles with large surface volume ratio can provide more reaction sites, so PHA nanoparticles can better combine with PhaP, thus carrying more AMP. Because the concentration of AMP is positively related to the antibacterial effect, the use of PHA nano-particles can have a better antibacterial effect.
We may express and use PHA nano-particles by cell administration in the future. Through the genetic engineering of the cells, the engineered cells can be targeted to specific infection sites in the body for targeted drug administration. Because the intestinal tract has a special mechanism for absorbing particles of a specific size, nano-particles are considered to be one of the best ways to absorb particles from the gastrointestinal tract after oral administration. And because of the sustained release effect of cell administration, we can keep the concentration of PhaP-AMP at a certain level without worrying about the inhibitory effect of excessive high concentration on surrounding normal tissue and low concentration on invasive bacteria.
Patent
While conducting the project feasibility test in the laboratory, we are also working hard to gain market recognition. At present, the patent of our project is under application and has been professionally recognized(figure 4).
Figure 4 the payment notice for patent
In order to implement our project from the laboratory to the real world, we have the following plans
1. As far as the product itself is concerned, the choice and the safety of raw materials are very important, such as whether GMO strains are legal and whether there are corresponding national regulations; It is necessary to consider the realizability of the design scheme, the application data of the product and the development plan of the follow-up product.
2. As far as production is concerned, special raw materials should be bound with exclusive authorization as much as possible; In the choice of the foundry factory, we should pay attention to the confidentiality of the formula and the constraints on the foundry factory, and the final cost should be calculated carefully.
3. As far as documents are concerned, the project involves patents and trademarks, production licenses and approvals, etc., which should be planned from the beginning.
4. In terms of marketing, the concept of this product is environmentally friendly and novel, so marketing is very important. After the completion of the market positioning, if the production cost is positioned at the high-end products, you can use your own advantages to publicize them.
After listening to our plan, Li Yang, the manager of Coway International TechTrans Co.,Ltd., expressed her agreement, and added that it is not difficult to turn this project into a product. The most basic thing is the funds and the team. We already have the team. A clear division of labor and the expansion of their respective fields are more important and the capital actually does not require large-scale capital at the beginning, and small capital can also start this project.
References
[1]Lassalle V, Ferreira ML. PLA nano- and microparticles for drug delivery: an overview of the methods of preparation. Macromol Biosci. 2007 Jun 7;7(6):767-83. doi: 10.1002/mabi.200700022. PMID: 17541922.
[2] Lee BK, Yun Y, Park K. PLA micro- and nano-particles. Adv Drug Deliv Rev. 2016 Dec 15;107:176-191. doi: 10.1016/j.addr.2016.05.020. Epub 2016 Jun 1. PMID: 27262925; PMCID: PMC5133193.