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Concept

In our design, antimicrobial peptides complex (AMPC) can be cleaved by V8 protease secreted by S. aureus. The V8 protease would dock on the cleavage site which is the C-terminal of glutamic acid (E). For AMPs, lysostaphin and hBD3 are responsible for inhibiting S. aureuss growth. For the platform, the hyaluronic acid methacrylate (HAMA) is coated on the tip surface, which can be hydrolyzed by hyaluronidase from S. aureus, triggering the filler release. Furthermore, the feature of AMPC can achieve the “slow drug release” property.

Fig.1 Three main concepts (a)V8 protease cleaves the AMPC into AMP (b)AMP kills S. aureus (c)HAMA is degraded by hyaluronidase and release AMPC specifically. (Click ☝ )

How to Prove our Concept?

To prove our concept, we need to perform the five major experiments:

  1. Check whether V8 protease can cleaves AMPC at the designated the cleavage site.
  2. Confirm the specificity of AMPC to S.aureus.
  3. Check whether the AMPC can be filled in gelatin and preserved its antimicrobial ability.
  4. The selectivity of microneedles shell to S. aureus.
  5. Determine the slow release of AMPC from the microneedle patch.

Achievement

Here are just parts of experiment result to be the major experiment for proofing our concept. The complete experimental results are shown in the “result” part.

See Results-AMPC page for detail information.

See Results-Microneedle page for detail information.

Autodock: V8 Protease & Cleavage Site

To confirm that our AMPC can be activated by V8 protease, We performed junction simulations with Autodock, which confirmed that the reaction is kinetically spontaneous.
Note that the name of the cleavage site means the amino acids of the P2-P1-P1' sites.

Fig.2 (a) SEL cleavage site (b) SEA cleavage site

Take protein [LL37] E [LL37]' E [Lysostaphin] (include cleavage sites SEL and SEA) as an example, we referred to the parameter of the research paper and simulated two cleavage sites and V8 junction. Finally, we found their binding energy is negative, which means the reaction is kinetically spontaneous.

See Model page for detail information.

Disc Diffusion Assay: Specificity of AMPC to S. aureus

We have confirmed that lysostaphin and Human-Beta-Defensin-3 (hBD3) have antimicrobial effects on S. aureus. And the hBD3 can kill the E. coli. Therefore, we used No.7 to do the disc diffusion assay on S. aureus and E. coli, respectively. The result shows that this AMPC only affects S. aureus, confirming that AMPC has specificity.

Fig.3 Result of disc diffusion assay

AMPC-Microneedle Test on S.aureus: Successfully Loaded the Drug

The microneedle patch carries the AMPC in its tips with gelatin. The antimicrobial ability of protein No.3 has been proved, so we fabricate four different concentrations of microneedle with protein No.3. After sticking them on the agar and incubating them with S. aureus, we found that the antimicrobial effect was significant with the increase of concentration. The presence of inhibition zones indicated that microneedle loaded as well as released the drug successfully.

Fig.4 The microneedle with various concentration AMPC incubated with S. aureus

The Selectivity of Microneedles Shell

We prepared two HAMA films on the plastic plate to confirm that the HAMA has selectivity. We added the bacterial solution of S. aureus and E. coli respectively and incubate them at 37℃. As expected that HAMA disappears on the S. aureus group as hyaluronidase by S. aureus hydrolyzes the HAMA. HAMA on the E. coli group is still on the plate. Therefore, we can confirm that HAMA coated on tips has a certain barrier property and selectivity.

Fig.5 The time dependent evolution image of HAMA film under various media. Click to enlarge the picture.(Click ☝ )

Drug Release Model: Slow Release of AMPCPC

Take protein [Ranalexin] E [Ranalexin]' E [Lysostaphin] as an example:

Fig.6 Drug release model of [Ranalexin] E [Ranalexin]' E [Lysostaphin]

According to the enzyme kinetics, we construct the model to predict the effective time of the drug releasing. See the graph above, the concentration of Ranalexin is above its minimum inhibitory concentration for about 11 hr, which means it has the ability to kill S. aureus at least 11 hr.

See Model page for detail information.

Reference

  1. Bolatchiev A. Antibacterial activity of human defensins against Staphylococcus aureus and Escherichia coli. PeerJ. 2020 Nov 25;8:e10455. doi: 10.7717/peerj.10455. PMID: 33304659; PMCID: PMC7698690.