Topical delivery of low-cost protein drug candidates made in chloroplasts for biofilm disruption and uptake by oral epithelial cells

Abstract

Protein drugs (PD) are minimally utilized in dental medicine due to high cost and invasive surgical delivery. There is limited clinical advancement in disrupting virulent oral biofilms, despite their high prevalence in causing dental caries. Poor efficacy of antimicrobials following topical treatments or to penetrate and disrupt formed biofilms is a major challenge. We report an exciting low-cost approach using plant-made antimicrobial peptides (PMAMPs) retrocyclin or protegrin with complex secondary structures (cyclic/hairpin) for topical use to control biofilms. The PMAMPs rapidly killed the pathogen Streptococcus mutans and impaired biofilm formation following a single topical application of tooth-mimetic surface. Furthermore, we developed a synergistic approach using PMAMPs combined with matrix-degrading enzymes to facilitate their access into biofilms and kill the embedded bacteria. In addition, we identified a novel role for PMAMPs in delivering drugs to periodontal and gingival cells, 13–48 folds more efficiently than any other tested cell penetrating peptides. Therefore, PDs fused with protegrin expressed in plant cells could potentially play a dual role in delivering therapeutic proteins to gum tissues while killing pathogenic bacteria when delivered as topical oral formulations or in chewing gums. Recent FDA approval of plant-produced PDs augurs well for clinical advancement of this novel concept.

Targeting microbial biofilms: current and prospective therapeutic strategies

“…Targeting EPS can also disrupt the viscoelastic properties to further weaken biofilm cohesiveness and enhance antimicrobial efficacy, including host mediated antimicrobial responses. Recent studies showed that glucano-hydrolases, glycoside-hydrolases and DNases enhanced antimicrobial delivery and potentiated killing by antibiotics or antimicrobial peptides when used in combination against pre-formed biofilms in vitro 49,50 . Overall, EPS synthesis inhibitors or EPS-degrading enzymes, which lack intrinsic antibacterial activity, seem to be a promising adjunctive approach for biofilm control that could potentially enhance the killing efficacy of antimicrobial agents and promote biofilm removal when co-administered.…”

“…However, more pre-clinical efficacy studies are required as AMPs can bind to EPS matrix components and to other host molecules, which reduces their effectiveness, and microbial proteases may further diminish AMP potency 106 . Additionally, the high cost of AMPs synthesis is a barrier for clinical development and commercialization, although using chloroplast-based technology for large-scale production in automated greenhouses may mitigate costs 50 . Nevertheless, AMPs can be immobilized onto solid surfaces to enhance efficacy or specificity.…”

“…AMPs can also enhance conventional antimicrobial activity, and the combination with strategies that target the EPS matrix may further increase both the access and permeabilizing properties of AMPs once in the biofilm 50,103 . Although targeting tolerant cells using AMPs is a promising approach, reaching the target cells that are embedded within a biofilm either topically or systemically and for the compound to be active across a spatially and chemically heterogeneous microenvironment remain important challenges in vivo .…”

Expression and functional evaluation of biopharmaceuticals made in plant chloroplasts

“…CTB fusion protein was detected in both human non-immune and immune modulatory cells [7••]. Recent reports also show that antimicrobial peptide (AMP), Protegrin-1 (PG-1) enters various human cell types including periodontal ligament stem cells, head and neck squamous cell carcinoma cells, Gingiva-derived mesenchymal stromal cells and adult gingival keratinocytes more efficiently than any other known cell penetrating peptides [11••]. …”

“…Many human infections are biofilm associated including dental caries caused by S. mutans . The topical application of plant produced AMPs (PMAMP) PG1 and RC101 on tooth mimetic surface effectively inhibited biofilm formation [11••]. In addition, PMAMPs when combined with exopolysaccharide degrading enzymes (dextranase/mutanase) disrupted mature biofilms and killed S. mutans [11••].…”