# The effect of nicotine delivery system on blood protease levels: a randomized crossover study

## TL;DR

This study compares how different nicotine delivery devices affect blood protease levels, which are linked to lung disease, finding that e-cigarettes may increase harmful protease release more than traditional cigarettes.

## Contribution

The study introduces a novel comparison of protease responses to e-cigarettes, heated tobacco, and traditional cigarettes using graphene-based nanobiosensors.

## Key findings

- E-cigarette use (JUUL) increased serum neutrophil elastase and MMP1 compared to usual brand cigarettes after adjusting for nicotine.
- All devices increased peak serum protease levels, but JUUL showed higher pathogenic protease release than IQOS and traditional cigarettes.

## Abstract

E-cigarettes and heated tobacco products are marketed as safer combustible cigarette alternatives due to their perceived potential for reduced tobacco-related toxicant exposure; however, their relative safety remains controversial. In this study we utilized serum protease levels, established biomarkers of harm contributing to lung disease, to study the effects of alternate tobacco products. Twenty-one adults who smoke cigarettes completed three visits in a randomized crossover design, separated by a 48-h washout period. Participants used their usual brand of cigarette (UBC), e-cigarette (JUUL), and heated tobacco (IQOS). We quantified serum proteases (matrix metalloproteinase (MMP) 1, MMP9, and neutrophil elastase (NE) using graphene-based nanobiosensors. UBC delivered significantly greater peak nicotine concentrations compared to JUUL or IQOS. Every device increased peak serum protease levels. After adjustment for serum nicotine, JUUL use resulted in higher levels of NE and MMP1 compared to UBC. Hierarchical clustering revealed three distinct patterns of systemic protease production that agnostically grouped by device. We demonstrated that e-cigarettes, but not IQOS, exhibited increased risk of potentially pathogenic protease release compared to UBC. These data indicate the need for prospectively designed and fully powered studies of longer duration to better understand the relative risks of e-cigarettes, IQOS and cigarettes on protease activation.

The online version contains supplementary material available at 10.1038/s41598-025-19832-8.