Affordable Topical Delivery of Biopharmaceuticals to Disrupt/Prevent Oral Biofilm

Objectives: Biopharmaceuticals are prevalent in healthcare to treat various infectious diseases. However, they are produced in prohibitively expensive systems that require high purity and cold storage, resulting in short shelf-life. Biopharmaceuticals produced in plants eliminate expensive production, purification and storage challenges (Molecular Therapy 24:1342-1350, 2016). Biopharmaceuticals used in dentistry face additional challenges of surgical invasive delivery. Caries are the most prevalent biofilm-associated oral diseases worldwide. Antimicrobials are minimally effective against biofilm bacteria that are protected by an exopolysaccharide (EPS) matrix. We reported recently that Plant Made Antimicrobial-Peptides (PMAMPs) combined with EPS-degrading enzymes (dextranase/mutanase) block Streptococcus mutans biofilm, preventing its maturation and disrupt mature biofilms by killing S. mutans (Biomaterials 105:156-166, 2016).
Methods: Codon-optimized mutanase and native dextranase with PG1 was synthesized by overlap-extension PCR. A new codon optimization algorithm was developed based on codon usage frequency of highly expressed chloroplast gene psbA, to enhance heterologous gene expression. Of 1261 amino-acids in mutanase, 586 codons were optimized and both fusion genes were constructed with 6X His-tag, GPGP (Gly-Pro-Gly-Pro) hinge region and furin cleavage site, RRKRSV (Arg-Arg-Lys-Arg-Ser-Val).
Results: Dextranase/mutanase fused with PG1 was cloned into chloroplast vectors. Mutanase is fully functional in E. coli when tested with suitable substrate. The expression cassette is now introduced into lettuce chloroplast genome by particle bombardment. Stable integration of transgene cassettes will be evaluated by PCR and Southern blots. EPS-degrading and antimicrobial activity against S. mutans will be tested by anti-biofilm disruption assays. Results on topical delivery of PMAMP-PG1 fused with dextranase/mutanase to gum tissues in a non-invasive manner will be presented.
Conclusions: As recombinant drug-based treatment with a non-surgical delivery system has not yet been developed in dentistry; this could be the first study on affordable production and non-invasive delivery of biopharmaceuticals for oral applications. This novel approach has potential to increase patient compliance and enhance oral health.