Enzymatic Laccase Nanoreactors Induce Apoptosis in MOLT-4-ALL Cells and Activate Prodrugs in a Synergetic Effect

Introduction

Cancer is one of the three diseases with the highest number of deaths worldwide.¹ It can be defined by 14 characteristics, the most common being uncontrolled cell growth, known as neoplasia.² This leads to high cellular heterogeneity within the tissue.

Traditional cancer treatments like surgery, radiotherapy, and chemotherapy have played a central role in combating the disease. However, their usefulness is often limited by their lack of specificity and severe side effects, which can significantly diminish patients’ quality of life. To reduce non-specific toxicity, some chemotherapy drugs are developed as prodrugs, which only become active anticancer agents after being converted inside the patient’s body.^3,4 Cytochrome P450 (CYP) enzymes carry out this conversion.⁵ Unfortunately, in some cases, within the mechanisms of cell death evasion, the expression of these CYP450-type oxidoreductase enzymes is decreased within tumor tissue and adjacent tissue.⁶

For instance, in lymphoblastic leukemia, polymorphism of CYP2B6 has been reported as a main risk factor.⁷ This cytochrome P450 enzyme is known to participate in the oxidative activation of the prodrug procarbazine, leading to the generation of cytotoxic radical intermediates.⁸ Therefore, the low expression levels of CYP2B6 in leukemic cell lines such as MOLT-4 (human Acute Lymphoblastic Leukemia) may limit the intrinsic activation of procarbazine, potentially contributing to reduced drug sensitivity in these cells. This is why strategies have been developed to activate prodrugs using exogenous oxidoreductases, such as fungal laccases.

Laccases (EC 1.10.3.2) are copper-containing enzymes that catalyze the oxidation of organic compounds and simultaneously reduce oxygen to water.⁹ One of the unique aspects of this enzyme is the similarity of its redox reactions to those of cytochrome P450, as mentioned above, that is essential for the metabolism (activation) of chemotherapy prodrugs.¹⁰ Not only has laccase itself been evaluated as a therapeutic protein, but the anti-cancer activity of the reaction products of various organic compounds, such as coumestan and hydroquinone, has also been reported.^11,12

In recent years, the study of laccases from diverse origins has explored them as potential anticancer agents¹⁰ making this enzyme class a potential therapeutic. The first report on the anti-cancer activity of laccase was reported by Ünyayar et al¹³ they evaluated the cytotoxicity of standardized aqueous bioactive extracts prepared from Coriolus versicolor and Funalia trogii on HeLa and fibroblast cell lines. On the other hand, the anti-cancer effect of laccase from Trametes versicolor was reported by Kale Bakir et al,¹⁴ where the cytotoxic effect of laccase on the cell line TT (thyroid cancer), Ishikawa (endometrial cancer), and HUVEC (human umbilical vein endothelial cell line) was analyzed, demonstrating that there is a pro-apoptotic effect and damage to DNA mediated by the dysregulation of the genes Bcl-2, Bax, Rad51, and ATM Even fractions from a culture and purified enzyme from Cerrena unicolor were evaluated against several cell lines (HT-29, CDD 848 CoTr, Caov-3, and NIH: OVCAR-3).^9,15

Laccases exhibit low substrate specificity in their oxidoreductase activity¹⁶ and thus can be used as an agent for activating prodrugs. Recently, we demonstrated that the laccase from Coriolopsis gallica can activate various prodrugs used in chemotherapy.¹⁷

On the other hand, the delivery of therapeutic agents at the cellular level is presented as a promising alternative for performing a more specific and efficient therapy. Enzymatic nanoreactors based on virus-like nanoparticles (VLPs) have been proposed to enhance cancer chemotherapeutic treatments^18,19 and enzyme replacement therapies.^20–22 Specifically, several platforms for the delivery of laccase have already been published, ranging from its immobilization on carbon nanotubes²³ to its confinement within VLPs for the formation of nanoreactors with laccase activity.¹⁷

Here, the antineoplastic effect of laccase from Coriolopsis gallica, in both free (Lac) and nanoconfined (VLP-saLac) forms, was demonstrated explicitly in the context of prodrug activation. The viability and mechanisms of death were evaluated in the MOLT-4 cell line (a Human Acute Leukemia Line), demonstrating the prodrug activation effect of laccase and a pro-apoptotic effect of the enzyme itself.

Materials and Methods

Laccase Purification

Laccase from Coriolopsis gallica UAMH 8260 was produced as previously reported.²⁴ Furthermore, it was stored in 10% glycerol at −80°C. Before use, the enzyme was dialyzed against 100 mM phosphate buffer at pH 6 to remove glycerol and further purified by Size Exclusion Chromatography (Hiprep 16/60 Sephacryl S-100 HR, GE Healthcare), by selectively collecting the central fractions to obtain maximum purity, as evaluated by SDS-PAGE electrophoresis, visualized with Coomassie blue staining.

Enzymatic Nanoreactor Production (VLP-saLac)

The enzymatic nanoreactors (VLP-saLac) were produced by encapsulating functionalized laccase with succinic anhydride (saLac) into VLPs from Brome Mosaic Virus, as reported by Medrano-Villagomez et al¹⁷ The ratio of laccase to virus coat protein was 1:9 (w/w). Initially, the mixture of two proteins was dissolved in a protein storage buffer (1 M NaCl, 20 mM Tris, pH 7.2) and dialyzed against an assembly buffer (50 mM NaCl, 10 mM KCl, 5 mM MgCl₂, 10 mM Tris, pH 7.2) for 24 h at 4°C. Subsequently, the mixture was dialyzed against an acidification buffer (50 mM sodium acetate and 8 mM magnesium acetate, pH 4.5) for 24 h at 4°C to induce capsid formation in the presence of laccase. A 14-kDa membrane (Spectrum Laboratories) was employed for this two-step dialysis process. Then, the nanoreactors were purified, and the non-encapsulated laccase and free CP proteins were removed by dialysis with a 100 kDa membrane. Finally, to eliminate aggregates, the nanoreactor suspension was centrifuged at 13,000 rpm for 10 min at 4°C in a Thermo Scientific™ Micro CL17R centrifuge, and the supernatant was then passed through a 0.22 μm filter (Verex Vial; Phenomenex) to obtain purified nanoreactors.

MOLT-4 Cell Line Maintenance

The MOLT-4 cell line, derived from human T cells originating from acute lymphoblastic leukemia, was obtained from a certified cell bank (ATCC, MOLT-4-CRL-1582). Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (100 U/mL and 100 μg/mL), and 2 mM L-glutamine. Cells were maintained in an incubator at 37°C in a humid atmosphere with 5% CO₂ in T-25 culture flasks. The medium was renewed every 2–3 days, and the cells were subcultured when the cell density reached 1.5–2.0×10⁴ cells/mL. Work began with pass 11 of the cells, which were tested for mycoplasma and found negative using the PlasmoTest™ - Mycoplasma Detection Kit in October 2024.

Calibration Curve for Neutral Red

Cell viability was evaluated using neutral red as an indicator according to the protocol described by Repetto et al²⁵ Neutral red accumulates in living cells, reflecting cell viability. Initially, a quantitative relationship between the number of viable cells and the absorbance at 540 nm (the characteristic wavelength absorbed by neutral red dye) was established by generating a calibration curve. Different numbers of cells were seeded in 96-well plates. Then, they were incubated for 4 hours in medium supplemented with neutral red (final concentration: 40 µg/mL). After incubation, the plate was centrifuged at 120 g for 7 minutes, and the medium was removed. The cells were then washed at least twice with saline phosphate buffer (PBS), which maintains cell osmotic balance. The internalized dye was solubilized by cell lysis using a solution of ethanol, acetic acid, and water (5:1:4), and the absorbance was measured at 540 nm. Absorbance was plotted against cell number, yielding a linear regression curve (r² > 0.98) for converting assay absorbance into relative cell numbers.

Evaluation of Cytotoxicity and IC₅₀ Estimation with Neutral Red

To determine the IC₅₀ values (the concentration causing 50% inhibition of cell viability) of each of the elements involved, dose-response curves of the drugs and laccase were performed; this was done by seeding 1×10⁴ cells (MOLT-4) per well in 96-well plates (100 µL/well) and exposing cells to different treatments; the evaluation was performed after at 24 hours incubation. Treatments were performed in triplicate, and the controls included phosphate-buffered saline (PBS) and dimethyl sulfoxide (DMSO) at a final concentration of 20%, as well as a blank control (medium without cells). At the end of incubation with the compounds, 20 µL of neutral red solution (final concentration, 40 µg/mL) was added to each well, and the mixture was incubated for 4 hours. Subsequently, the medium was removed, and the wells were washed with PBS. The adsorbed dye was released by adding 100 µL of lysis solution, and the absorbance was measured at 540 nm using a microplate reader. Viability was then calculated using the following formula:

Viability data (%) were analyzed and plotted against the logarithm of drug and laccase concentrations to generate dose-response curves. Curve fitting utilized a four-parameter logistic model (4PL) in GraphPad Prism software, represented by the following equation:

Where Y is the relative viability (% of viable cells), X is the drug or laccase concentration, t and d are the maximum and minimum viability values, respectively. Hillslope is the slope of the curve, and IC₅₀ is the concentration at which 50% inhibition occurs. The fit was considered adequate if the coefficient of determination (R², a measure of how well the model fits the data) was at least 0.95. The IC₅₀ value was reported in nM or µM, with a 95% confidence interval (the statistical range within which the actual IC₅₀ value falls with 95% certainty).

Flow Cytometry Analysis

A total of 5×10⁴ cells were precisely seeded in 24-well plates in a total volume of 500 μL of medium, allowed to stand for 16 hours, and then the corresponding treatments were added to the wells for a 24-hour incubation period. The measurements were performed using the LongCyte C3140 flow cytometer (Beijing Challen Biotechnology Co., Ltd). The first analysis involved direct double staining: live-cell staining with fluorescein diacetate (FDA, which becomes fluorescent in living cells) and dead-cell staining with propidium iodide (PI, a dye that penetrates only dead cells). The conditions evaluated here were the IC₂₀ (the concentration causing 20% inhibition of cell viability) of the free drug in the presence of free laccase and in the presence of nanoreactors, both with the same final volumetric activity. The controls considered at this stage were PBS, DMSO, and Brome Mosaic Virus (BMV).

After incubation, propidium iodide (PI), a red-fluorescent dye that stains only dead cells, was added at a concentration of 10 μg/mL, and the samples were immediately read in the cytometer. A fluorescence compensation matrix was applied to correct dye spectrum overlap. Data were analyzed using the cytometer’s software, and percentage viability was calculated as follows:

For the second analysis, we employed the same experimental design and culture conditions as previously mentioned, differing only in the staining procedure. Caspase activation was assessed using the commercial CellEvent™ Caspase-3/7 kit (Thermo Fisher Scientific) (caspases are enzymes active during apoptosis). Cells treated with 100 µM irinotecan served as the positive control for apoptosis; together with the DMSO control, these were used to generate the compensation matrix. The percentage of the population in each cell death phase (Qx) was calculated as follows:

All cytometry data were processed using the same workflow as for the scatter plots. Initially, events associated with cellular debris were excluded in the FSC vs SSC graph (FSC: forward scatter, relates to cell size; SSC: side scatter, refers to cell granularity). The remaining events were plotted on SSC-A vs. SSC-H to define individual cells and eliminate doublets (cells that are stuck together). With this filter, cells were plotted on the double indicator graph (showing different dye populations) to determine quadrants and positive populations, considering the compensation matrix generated for each system.

All events detected by the equipment were collected, and a threshold of 20,000 events per reading was used for the final analysis. Biological (n=3) and analytical (n=3) replicates were performed, resulting in n=9.

All results from in vitro cell experiments were plotted using GraphPad Prism software (version 10.6.1), and statistical analyses were performed accordingly. A one-way ANOVA (analysis of variance) and a Sidak-type post hoc analysis (a statistical test for multiple comparisons) were performed for bar comparisons. The comparisons made are indicated by the staples in each graph, considering controls for each comparison. The error bars represent the standard deviation (±SD), based on analytical and experimental triplicates (n=9). The results were considered statistically significant when p < 0.05.

Results

Since laccase activity interfered with the conventional MTT viability assay, the cytotoxicity of each element in the system was evaluated using neutral red as a viability indicator, following the protocol described above. The results are shown in Figure 1, and the IC₅₀ values are presented in Table 1. The dose-response curve data were fitted to a 4PL model.

Table 1 Data of the Dose-Response Curve in Cell Line MOLT-4 (Adjusted to 4PL) with the Different Prodrugs and Laccase

Figure 1 Curve dose-response on MOLT-4 leukemia model cells. (A) Pro-drugs and (B) laccase from C. gallica.

A literature review reveals that only doxorubicin studies have been conducted in MOLT-4; Svensson et al report a value of 60 ±20 nM, which is consistent with our finding.²⁶ No IC₅₀ values are available for Irinotecan and Procarbazine in MOLT-4 cells, neither for laccase.

The IC₂₀ values were also estimated, and these concentrations were used to evaluate the synergy between prodrugs and free or nanoconfined laccase from C. gallica. The production procedure and full characterization of enzymatic nanoreactors containing laccase activity (VLP-saLac) was previously reported.¹⁷ The synergistic effect was analyzed using flow cytometry, where FDA-marked live cells and PI-marked dead cells were distinguished. The cytograms from this stage are shown in Figure 2, which show an enhanced drug effect on the enzyme. Two main patterns are identified: an increase in a subpopulation within the FDA-positive region compared to the control (Figure S1), which may indicate metabolic lethargy potentially corresponding to early apoptosis; however, further confirmation with an assay explicitly detecting this cell death mechanism was performed.

Figure 2 Flow cytometry analysis to determine cell viability. The green zone indicates the death cells with a stain-positive PI, and the red zone indicates the alive cell with a stain-positive FDA. Every panel corresponds to a different treatment of MOLT-4-ALL cell: (A) Doxorubicin, (B) Doxorubicin + Lac, (C) Doxorubicin + VLP-saLac, (D) Irinotecan, (E) Irinotecan + Lac, (F) Irinotecan + VLP-saLac; (G) Procarbazine, (H) Procarbazine + Lac, (I) Procarbazine + VLP-saLac. The percentage in every square indicates the percentage of total events.

This assay also showed an increase in PI-positive cells, indicating cell death. Figure 3 shows increased cell death in all systems, whether with free or nanoconfined enzymes, relative to the prodrug alone. These findings confirm that the combination of laccase and drugs produces an effect greater than the sum of their individual effects, suggesting a possible synergistic effect related to activation of the prodrug.¹⁷

Figure 3 Cell death by flow cytometry. Evaluation of cell death population (PI+) between treatments with different drugs: (A) Doxorubicin, (B) Irinotecan, and (C) Procarbazine. Statistical analysis was one-way ANOVA with a Sidak post hoc test. Significance values *<10−1, **<10−2, ***<10−3. ****<10−4. The error bars ± SD, p < 0.05. Abbreviation: ns, no significant.

These results, along with the FDA-positive populations, suggest that diverse death mechanisms are involved. These death mechanisms in MOLT-4 cells were studied using the same treatments as in the FDA/PI system. Cytograms are presented in Figure S2 (controls) and Figure S3 (treatments); Figure 4 summarizes the percentage of each death mechanism by treatment. Unlike previous analyses, this study distinguishes between different types of death, confirming the FDA+ subpopulation as characterized by early apoptosis. The assay enables more precise analysis of cell death.

Figure 4 Evaluation of the mechanism of cell death. Accumulate the bar chart of the percentage of each population in different mechanisms of cell death; Q1, represented by light gray, is for viable cells, Q2, represented by red, is for apoptotic cells, Q3, represented by blue, is for necroptotic cells, and Q4, represented by dark gray, is for necrotic cells. The comparison is between apoptotic cells (Q2). Statistical analysis was performed using one-way ANOVA with a Sidak post hoc test. Significance values ****<10−4. The error bars ± SD, p < 0.05. Abbreviation: ns, no significant.

Initially, an apparent decrease in the percentage of viable cells was observed, which was equivalent to the population in Q1 (as indicated by the gray bar in Figure 4). This phenomenon is seen in most treatments, except the PBZ + VLP-saLac treatment; on the other hand, in this assay, the data for necrotic cells (Q4, dark gray bars) do not show a significant difference in any of the cases, which indicates that a large part of the cell death represented in Figure 3 is by a route other than necrosis.

Following the analysis, a significant increase is observed in the two mechanisms associated with programmed cell death. For Q3, this is primarily associated with necroptosis or late apoptosis. In contrast, for Q2, it is associated with early apoptosis; in both cases, the population percentage increases with the presence of the laccase-drug combination. The double-positive population in these assays (Caspase 3/7+/7-ADD+) indicates caspase 3 activation and membrane permeability for 7-ADD internalization. These conditions are met when necroptosis and pyroptosis are activated. In both cases, membrane pores are generated (through which 7ADD can be internalized and give a positive signal); in the case of necroptosis, caspase 8 is activated, which is also an effect of caspase 3, which would give a positive signal, while pyroptosis directly activates caspase 3. This is why we classify the double-positive population as necrosis. The cellular pathway was described by Zhu et al.²⁷

A deeper analysis of the Q2 population reveals several phenomena, primarily that the presence of laccase leads to a significant increase in all cases, even in the controls. This phenomenon is observed when comparing the VLP-saLac control (third bar from left to right) and the free laccase (fourth bar from left to right) in Figure 4. The presence of this protein alone induces an apoptotic effect, but in all cases, it is less pronounced than that observed when the analysis is performed in combination with a prodrug. Therefore, this confirms that the effect observed in the prodrug-enzyme combinations is due to a synergistic interaction that coincides with the activation of the prodrug by the laccase enzyme.

However, to demonstrate a synergistic effect among the evaluated elements, the combination index (CI) was calculated for different models, depending on the nature of the results.^28–30

For the first stage, in which an increase in cell death was observed due to the presence of iodide propidium cells, the independent Bliss model equation was used (CI_Bliss), which is used to evaluate the synergy between two therapeutic elements that act independently, this being the most common model for determining synergy between drugs, and the results show a strong synergic effect in the presence of free laccase, in contrast to VLP-saLac where in combination with doxorubicin and irinotecan show a slightly synergism but not with procarbazine (Figure 5).

Figure 5 The Combination Index to determine synergism. The matrix in the upper left shows the values obtained by the Bliss Independence model, indicating synergy when the CI < 1; the remaining three matrices were formed using a CI calculated by the HSA model, which indicates synergy when the value is less than 1.

In addition, the CI for each death mechanism was calculated. Due to the nature of the effect, the most appropriate method to determine de CI is the model HAS (Highest Single Agent) because this model only compares the drug combination effect to the most effective individual drug, not taking into account the expected additive effect of bot drugs involved in the combination. This statement fits with the hypothetical effect of activation of prodrugs. The results for each mechanism are shown in Figure 5, where it is demonstrated that the combination of free laccase and the drugs doxorubicin and procarbazine produces a synergic effect to induce apoptosis. Additionally, late apoptosis (necroptosis) has been enhanced by irinotecan and procarbazine in the presence of both forms of laccase: free and nanoreactors. The results of necrosis did not show any effect.

On the other hand, it is essential to highlight the results obtained with the laccase nanoreactors, since when analyzing the results presented in Figures 3–5, in the comparison against the free version, it is hypothesized a less extent effect due to the catalytic nature of the nanoreactor, in which laccase is confined within a VLP, presenting slower catalytic transformation rate associated with diffusion limitations that have already been widely described for nanoreactors.³¹ This raises the possibility of using these nano-systems to induce programmed cell death with greater control, allowing for targeted cellular intervention.

Discussion

Given their biomedical applications, laccases possess two important properties: the capacity to induce cytotoxicity in tumor tissues^13,14 and the ability to activate prodrug compounds used in cancer chemotherapy treatments.¹⁷

As expected, the three selected prodrugs proved to be cytotoxic to the MOLT-4 cell line (Table 1). Doxorubicin (DOX) is a chemotherapeutic drug used for the treatment of different tumor types, mainly breast cancer. The mechanism of cytotoxicity is not fully understood and remains controversial. It appears that rather than a single main cytotoxic mechanism, multiple mechanisms may be involved, including DNA intercalation and adduct formation, topoisomerase II poisoning, the generation of free radicals and oxidative stress, and altered sphingolipid metabolism, all of which can lead to membrane damage.³² The cytotoxicity of doxorubicin in MOLT-4 cells was reported to have an IC₅₀ value of 60 nM,²⁶ which agrees with our results (Table 1). For the other prodrugs and laccase, no data are available for this cell line.

Irinotecan is a topoisomerase inhibitor that has been extensively studied, and its clinical use was approved for the treatment of cervical, lung, and ovarian cancer. Topoisomerases are nuclear enzymes that play a crucial role in maintaining the proper DNA topology during replication and transcription. There are diverse proposed mechanisms of inhibition, including substrate competitiveness, in which the compound directly binds to the enzyme’s active site, preventing the native substrate from binding. Another is based on interactions between the protein, DNA, and the topoisomerase inhibitor, which result in the blockage of DNA replication and the formation of cleavage complexes. Finally, a possible mechanism involves preventing ATP hydrolysis.³³ The cytotoxicity of irinotecan in two human colorectal tumor cell lines, LoVo and HT-29, showed IC₅₀ values of 15.8 µM and 5.17 µM, respectively,¹¹ which are significantly higher than those found (0.18 µM) on the MOLT-4 cell line (Table 1).

Procarbazine, as a prodrug, requires activation for its antitumoral activity. Procarbazine is used in the treatment of Hodgkin’s lymphoma, malignant melanoma, and brain tumors. Cytochrome P450 and monoamine oxidase should activate it to form its azo derivative, and subsequently, to the azoxy derivative, which in turn forms a methyl carbonium ion (CH₃⁺) that reacts with DNA and proteins, inhibiting DNA and protein synthesis and causing tumor cell death.¹² As in the case of cytochrome P450, laccase could produce the free radical intermediates.¹⁷ The highly reactive CH₃• can bind to DNA and proteins, inhibiting both DNA and protein synthesis, and leading to cell death. Procarbazine itself exhibits low direct cytotoxicity, whereas its metabolite, methylazoxyprocarbazine, is responsible for its significant anticancer effects, with an IC₅₀ of 0.15–0.2 mM in L1210 cells. In contrast, procarbazine was effective only at higher concentrations (approximately 1.5 mM).³⁴ Nevertheless, these doses are higher than the value obtained on MOLT-4 cells (Table 1). However, MOLT-4 cells highly express cytochromes P450 to transform procarbazine.³⁵

Quantitative cytotoxicity of laccase is reported in this work for the first time. An IC₅₀ of 1.18 µM was found on MOLT-4 cells. Laccase cytotoxicity is considerable, and its mechanism remains to be studied. This is a challenging task due to laccase’s ability to oxidize a wide variety of compounds.

To protect and target the enzyme, laccase was encapsulated inside a VLPs. The use of functionalized nanovehicles to specifically target cells and tissues that require treatment and to avoid systemic administration of therapeutics is of particular interest for “smart medicine”. Enzymatic nanoreactors have been proposed to enhance cancer chemotherapy^18,19 and for enzyme replacement therapies,^20–22 in which it is possible to protect the enzymatic activity under physiological conditions and, importantly, direct the treatment by the nanocarrier functionalization with specific ligands to target cell receptors.

In this work, laccase alone showed an apoptotic effect at a concentration of 0.35 μM, and this effect was enhanced by the presence of prodrugs, indicating a synergistic effect on cell death, as corroborated by the combination index (CI) using two models: Bliss independence and Highest Single Agent. There are several mechanisms by which enzymes and drug treatments can act synergistically: i) Enzyme-mediated activation of prodrugs, in which the therapy uses prodrugs that are inactive until an enzyme activates them.^18,19 ii) Degradation or modification of barriers or protective structures. Many pathogens or tumors have protective envelopes, extracellular matrices, or cell walls that enzymes can degrade, making the drug more accessible. Example in tuberculosis.³⁶ iii) Modulation of the microenvironment, as in the case of tumor hypoxia, that reduces the effectiveness of some treatments. Enzymes such as catalase can alleviate hypoxia, thereby improving the effectiveness of drugs or radiation.³⁷ iv) Improved drug delivery or penetration by using enzymes that degrade the extracellular matrix or other physical barriers to enhance drug penetration.³⁷

Conclusions

Research into new cancer treatment strategies is a field of science with significant socioeconomic and public health impacts. The enhancement of prodrug-based treatments is crucial. A new alternative, utilizing laccase enzymes as anticancer agents, has generated significant interest as an alternative to cytochrome P450 prodrug activation.

This work demonstrates that Coriolopsis gallica laccase alone induces apoptosis in the human leukemia cell line MOLT-4. In addition, the activation effect of prodrugs within the cell, showing a synergistic effect on cell death, was demonstrated with the CI. The nanoconfined version of this enzyme (VLP-saLac) exhibits a similar effect to that obtained with the free enzyme preparation.

The use of nanoplatforms or enzyme nanocarriers offers several important advantages, including the ability to functionalize with specific ligands to direct cargo to target cells, protect the enzyme cargo from proteolytic systems, and modulate cytotoxicity. Finally, the groundwork is laid for future work utilizing these nanosystems and this type of enzyme to study and promote cell death, which could lead to an efficient treatment against cancer.