Microplastics: Can engineered enzymes remove microplastics from the human body?

Methods

Summary

We will test engineered PET-degrading enzymes (PETase and MHETase) in human serum to measure microplastic particle counts, activity, stability, and breakdown products using established analytical techniques (O-PTIR, HPLC, and mass spectrometry). Safety will be assessed in human cell models (HEK293) through cytotoxicity and viability assays. By combining pre- and post-particle counts, enzyme kinetics, stability testing, and cell assays, this study will provide reproducible data on whether enzymes can safely degrade PET microplastics under human-like conditions.

Challenges

Key risks include: (1) reduced enzyme activity or stability in serum due to proteases or matrix effects; (2) assay interference when quantifying particles and monomers; (3) cell-level cytotoxicity from enzymes or PET breakdown products (e.g., terephthalic acid, ethylene glycol); and (4) variability across serum lots and microplastic morphologies.

Mitigations: run time-course kinetics at 37 °C with appropriate controls (enzyme-only, plastic-only, serum blanks), use orthogonal analytics (O-PTIR plus HPLC/MS) with spike-in standards and recovery checks, perform endotoxin testing and dose-response safety assays in HEK293, and include replicate lots/particle types to ensure reproducibility. If activity is low, we’ll optimize buffer conditions and enzyme dosing within the TVA’s predefined, non-proprietary parameters.

Pre Analysis Plan

Hypotheses

• PETase/MHETase hydrolyze PET in human/artificial serum at 37 °C vs controls.

• Enzymes retain activity over the incubation interval.

• Target exposures are non-cytotoxic in HEK293; PET byproducts are non-cytotoxic at assay levels.

Primary endpoints

• PET depolymerization (TPA, MHET, BHET, EG by HPLC/MS), normalized to PET mass/surface & time.

• HEK293 viability across enzyme/byproduct dose–response.

Secondary endpoints

• Enzyme stability over time; PETase vs MHETase vs combo.

• Serum type (human vs artificial) and PET form factor effects.

• Pre/post microplastic particle counts (with size bins) to complement chemistry.

• Orthogonal polymer confirmation via O-PTIR (± FTIR/Raman) as confirmatory only.

Design & data

• Time-course (e.g., 0–72 h) across enzyme doses & PET loadings with enzyme-only, plastic-only, and serum blanks.

• Replication: ≥3 technical/run; ≥2–3 serum lots; repeat on different days.

• Cell assays: predefined ranges with vehicle and positive-control cytotoxins.

• Particle counts: image/flow-imaging with fixed subsampling/SOPs.

Quantitation & QC

• External calibration + internal standards; blank subtraction; LOD/LOQ logged.

• MS/MS ID where applicable; O-PTIR for polymer ID/surface-chemistry checks.

• Particle-count QC via beads/reference images and inter-operator checks.

• Pre-set run acceptance criteria (calibration fit, recoveries, count QC).

Statistics

• Products: initial rates + 24/48/72-hour yields; mixed-effects models (fixed: treatment/time/dose/serum/form factor; random: serum lot/run); report effect sizes with 95% CIs.

• Particle counts: pre vs post and size-bin shifts (GLMM or paired tests; Holm–Bonferroni); supportive to HPLC/MS.

• Cytotoxicity: 4-parameter logistic fits; IC10/IC50 vs target exposure bands.

• Primary outcomes: depolymerization rate at 37 °C and HEK293 viability; adjust secondary comparisons; emphasize estimation over p-values.

Variance/outliers/missing

• Exclude only for pre-specified QC failure or documented assay error; otherwise analyze as-is.

• No imputation for missing time-points; use replicate structure.

Go/no-go (TVA-linked)

• Activity: enzyme > plastic-only by preset margin and ≥ minimum monomer-release threshold.

• Stability: retains preset fraction of activity over the defined interval.

• Safety: no significant HEK293 viability loss at target exposures; byproducts non-cytotoxic.

• Supportive: particle-count reduction consistent with chemical depolymerization.

Reproducibility

• Independent repeats across lots/days; locked analysis scripts and versioned SOPs; anonymized chromatograms, particle-count summaries, and final tables in report.