Semi-permeable polymer vesicle-based prooxidative and lactate-depleting nanoreactors with sustained activity against pancreatic cancer

Abstract

Polymer vesicles, also known as polymersomes, consist of polymer membranes enclosing an aqueous core and have attracted significant interest for biomedical applications. The aqueous core is particularly advantageous for encapsulating and stabilizing fragile cargo, such as proteins, to maintain long-term activity. Among these, enzyme-encapsulated polymersomes function as therapeutic nanoreactors and have gained increasing attention in recent years, especially for cancer treatment. A critical factor in their catalytic performance is ensuring semipermeability of the membrane, allowing selective exchange of small-molecule substrates while maintaining stable enzyme encapsulation. However, achieving a balance between prolonged structural integrity and optimal permeability to sustain catalytic activity remains a challenge. Here, we present oxidation-sensitive polyion complex vesicles (PICsomes) encapsulating lactate oxidase as prooxidative and lactate-depleting nanoreactors. The membrane's built-in semipermeability and crosslinked network contribute to the prolonged enzymatic activity of lactate oxidase. Notably, in response to reactive oxygen species (ROS), the nanoreactors undergo swelling, further enhancing membrane permeability to amplify enzymatic catalysis-specifically, ROS production and lactate depletion. This self-amplifying function enhances cytotoxic effects against pancreatic cancer cells. Interestingly, the prooxidative activity also induces immunogenic cell death, as evidenced by elevated levels of calreticulin and HMGB1, suggesting the potential to stimulate antitumor immunity. It is important to note that lactate not only serves as a key respiratory fuel but also facilitates immune evasion. Given these findings, the reported nanoreactors offer a promising strategy for disrupting tumor energy and redox metabolism through lactate depletion and prooxidation, while also priming antitumor immunity for combination immunotherapy.

Keywords: enzyme delivery; immunogenic cell death; lactate depletion; nanoreactors; polymer vesicles; prooxidation; semipermeable polymersomes; sustained activity.

FIGURE 1

Schematic illustration of the construction of lactate oxidase (LOD)-loaded PICsomes as prooxidative and lactate-depleting nanoreactors with potent cytocidal effects. The self-assembly process occurs via electrostatic complexation between oppositely charged P(Asp-ATK) and PEG-b-PAsp in the presence of LOD.

FIGURE 2

Characterization of LOD@PICsomes. (a) The process of electrostatic complexation of P(Asp-ATK) and PEG-b-PAsp in the presence of LOD forming LOD@PICsomes nanoreactors; (b) Intensity-weighted size distribution of empty PICsomes measured by DLS; (c) Intensity-weighted size distribution of LOD@PICsomes measured by DLS; (d) Comparison of Z-average size of empty PICsomes and LOD@PICsomes; (e) Cryo-TEM measurement of empty PICsomes and LOD@PICsomes. Scale bar: 100 nm; (f) Conventional TEM measurement of empty PICsomes and LOD@PICsomes. Scale bar: 200 nm. In conventional TEM sample preparation, the drying process disrupts the aqueous environment essential for vesicle stabilization. This leads to aggregation due to capillary forces and the loss of hydration-mediated repulsion. In contrast, cryo-TEM employs vitrification to flash-freeze samples in their native hydrated state, preserving their original morphology within a glass-like ice matrix. (g) Zetapotential of empty PICsomes and LOD@PICsomes measured by DLS. Data represent mean ± standard deviation (n = 3 for both (d,g)).

FIGURE 3

Evaluation of enzyme activity of LOD@PICsomes and swelling behavior of LOD@PICsomes. (a) LOD activity of LOD@PICsomes with or without H₂O₂ pre-treatment, with free LOD used as a control for comparison; (b) Intensity-weighted size distribution of LOD@PICsomes and LOD@PICsomes with H₂O₂ treatment measured by DLS; (c) Comparison of Z-average size of LOD@PICsomes and LOD@PICsomes with H₂O₂ treatment; (d) Cryo-TEM measurement of LOD@PICsomes with H₂O₂ treatment. Scale bar: 100 nm; (e) Relative enzyme activity of free LOD and LOD@PICsomes after treatment with proteinase K for 6 or 24 h. Samples (free LOD or LOD@PICsomes) without proteinase K treatment were used as controls. Data represent mean ± standard deviation (n = 3 for (a,c,e)).

FIGURE 4

Cytotoxicity of LOD@PICsomes. (a) LOD concentration-dependent cytotoxicity of free LOD, LOD@PICsomes, and LOD@PICsomes with H₂O₂ pre-treatment against Panc02 cells; Data represent mean ± standard deviation (n = 4); (b) IC50 values of free LOD, LOD@PICsomes, and LOD@PICsomes with H₂O₂ pre-treatment, calculated using GraphPad Prism.

FIGURE 5

ROS generation, DNA damage, and live/dead cell assays. (a) ROS generation, DNA damage, and Live/dead cell staining in Panc02 cells treated with LOD@PICsomes or LOD@PICsomes plus catalase, assessed using the ROS probe DCFH-DA, comet assay, and Calcein-AM (live cell)/PI (dead cell) probes, respectively; (b) Quantification of ROS generation based on fluorescence intensity; (c) Quantification of DNA damage expressed as the percentage of tail DNA; (d) Quantification of the percentage of PI-positive cells (red color). The complete inactivation of LOD@PICsomes—evidenced by similar levels of intracellular ROS, DNA damage, and PI-positive cells in the ‘LOD@PICsomes plus catalase’ and PBS groups—confirms the absence of catalase toxicity. Data represent mean ± standard deviation (n = 4 randomly selected images for (b,c,d). Statistical significance was assessed using one-way ANOVA followed by Tukey’s post hoc test.

FIGURE 6

ROS generation ability and cytotoxicity and of recycled samples. (a) The process of recycling free LOD or LOD@PICsomes from old culture medium for repeated use in cytotoxicity and ROS generation evaluation; (b) ROS generation assessed using the ROS probe DCFH-DA; (c) Quantification of ROS generation based on fluorescence intensity in (b); (d) Cytotoxicity of recycled samples. The concentrations used were 1.33 μg/mL for free LOD and 2.14 μg/mL LOD-equivalent for LOD@PICsomes. The samples were collected and concentrated using an ultrafiltration tube with a molecular weight cutoff (MWCO) of 50 kDa for repeated use for cytotoxicity evaluation in Panc02 cells. Data represent mean ± standard deviation (n = 4 for both (c,d)). Statistical significance was assessed using one-way ANOVA followed by Tukey’s post hoc test.

FIGURE 7

Characterization of immunogenic cell death. (a) Calreticulin expression in Panc02 cells treated with LOD@PICsomes or LOD@PICsomes plus catalase, analyzed using flow cytometry with an Alexa Fluor^® 647 anti-calreticulin antibody (mean fluorescence intensity); (b) HMGB1 concentration in culture supernatants of Panc02 cells treated with LOD@PICsomes or LOD@PICsomes plus catalase, measured using an HMGB1 ELISA Kit. Data represent mean ± standard deviation (n = 3 for both (a,b)). Statistical significance was assessed using one-way ANOVA followed by Tukey’s post hoc test.