Efficient Delivery of Antimicrobial Peptides in an Innovative, Slow-Release Pharmacological Formulation.

Both nanostructure and multivalency enhance the biological activities of antimicrobial peptides (AMPs), whose mechanism of action is cooperative. In addition, the efficacy of a particular AMP should benefit from a steady concentration at the local place of action and, therefore, from a slow release after a dynamic repository. In the context of emerging multi-resistant bacterial infections and the urgent need for novel and effective antimicrobial drugs, we tested these concepts through the engineering of four AMPs into supramolecular complexes as pharmacological entities. For that purpose, GWH1, T22, Pt5, and PaD, produced as GFP or human nidogen-based His-tagged fusion proteins, were engineered as self-assembling oligomeric nanoparticles ranging from 10 to 70 nm and further packaged into nanoparticle-leaking submicron granules. Since these materials slowly release functional nanoparticles during their time-sustained unpacking, they are suitable for use as drug depots in vivo. In this context, a particular AMP version (GWH1-NIDO-H6) was selected for in vivo validation in a zebrafish model of a complex bacterial infection. The GWH1-NIDO-H6-secreting protein granules are protective in zebrafish against infection by the multi-resistant bacterium Stenotrophomonas maltophilia, proving the potential of innovative formulations based on nanostructured and slowly released recombinant AMPs in the fight against bacterial infections.

How modular protein nanoparticles may expand the ability of subunit anti-viral vaccines: The spring viremia carp virus (SVCV) case.

Spring viremia of carp (SVC) remains as a vaccine orphan disease mostly affecting juvenile specimens. Young fish are especially difficult to vaccinate and oral administration of vaccine combined with food would be the election system to minimise stress and the vaccination costs associated to injection. However, administration of prophylactics with food pellets faces off several drawbacks mainly related with vaccine degradation and weak protection correlates of oral vaccines. Here we present a platform based on recombinant proteins (subunit vaccines) manufactured as highly resistant nanostructured materials, and providing excellent levels of protection against SVC virus in a preliminary i.p injection challenge. The G3 domain of SVCV glycoprotein G was overexpressed in E. coli together with IFNγ and the modular protein was purified from bacterial aggregates (inclusion bodies) as highly organised nanostructured biomaterial (nanopellets, NP). These SVCV-IFNNP were taken up by zebrafish cells leading to the enhanced expression of different antiviral and IFN markers (e.g vig1, mx, lmp2 or ifngr1 among others) in zebrafish liver cells (ZFL). To monitor if SVCVNP and SVCV-IFNNP can be taken up by intestinal epithelia and can induce antiviral response we performed experiments with SVCVNP and SVCV-IFNNP in 3 days post fertilization (dpf) zebrafish larvae. Both, SVCVNP and SVCV-IFNNP were taken up and accumulated in the intestine without signs of toxicity. The antiviral response in larvae showed a different induction pattern: SVCV-IFNNP did not induce an antiviral response while SVCVNP showed a good antiviral induction. Interestingly ZF4, an embryonic derived cell line, showed an antiviral response like ZFL cells, although the lmp2 and ifngr1 (markers of the IFNγ response) were not overexpressed. Experiments with adult zebrafish indicated an excellent level of protection against a SVCV model infection where SVCV-IFNNP vaccinated fish reached 20% cumulative mortality while control fish reached over 80% cumulative mortality.

Biofabrication of functional protein nanoparticles through simple His-tag engineering.

We have developed a simple, robust, and fully transversal approach for the a-la-carte fabrication of functional multimeric nanoparticles with potential biomedical applications, validated here by a set of diverse and unrelated polypeptides. The proposed concept is based on the controlled coordination between Zn2+ ions and His residues in His-tagged proteins. This approach results in a spontaneous and reproducible protein assembly as nanoscale oligomers that keep the original functionalities of the protein building blocks. The assembly of these materials is not linked to particular polypeptide features, and it is based on an environmentally friendly and sustainable approach. The resulting nanoparticles, with dimensions ranging between 10 and 15 nm, are regular in size, are architecturally stable, are fully functional, and serve as intermediates in a more complex assembly process, resulting in the formation of microscale protein materials. Since most of the recombinant proteins produced by biochemical and biotechnological industries and intended for biomedical research are His-tagged, the green biofabrication procedure proposed here can be straightforwardly applied to a huge spectrum of protein species for their conversion into their respective nanostructured formats.