Targeted virus-based catalase nanoreactors for potential acatalasemia treatment

Virus-like particles (VLPs) are promising nanoplatforms for the targeted delivery of drugs and other therapeutic agents for a variety of diseases. Among the strategies in which VLPs offer an advantage is Enzyme Replacement Therapy, which aims to exogenously administer an enzyme deficient or absent in the human body that is responsible for a disease. An example of such a disease is acatalasemia, in which catalase activity in erythrocytes is low or absent. Therefore, in this work, we present the synthesis and characterization of nanoreactors with catalase activity (VLP-Cat), which had an average size of 30 nm and remarkable catalytic activity. We also demonstrate the successful functionalization of the viral nanoparticles with the ERY1 peptide, which is complementary to the GYPA1 protein present in the erythrocyte membrane. Using flow cytometry, we quantified targeting, observing a 3-fold increase in the positive population from 3% to 13% relative to the control. We therefore demonstrated that this strategy is effective for specifically targeting catalytic nanoreactors (VLP-Cat) to the erythrocytes.

Acatalasemia, also known as acatalasia, is a rare hereditary metabolic disorder characterized by a marked deficiency or complete absence of the enzyme catalase in red blood cells (RBCs) and other tissues. This inherited condition is autosomal recessive and typically asymptomatic; however, in some populations, including those in Japan and Hungary, it is more common. Catalase plays a critical role in detoxifying hydrogen peroxide (H 2 O 2), a reactive oxygen species, by catalyzing its decomposition into water and oxygen. Without sufficient catalase activity, cells may become vulnerable to oxidative damage due to hydrogen peroxide accumulation (1).

This disease may be associated with ulcerative lesions in the oral cavity or increased susceptibility to oxidative stress-related damage (2). Acatalasemia was first described in Japan by Takahara in 1948, where affected individuals developed gangrenous ulcers following dental extractions, a condition later termed Takahara’s disease (3). Despite the absence of catalase activity, most patients with acatalasemia remain clinically normal, suggesting compensatory mechanisms involving other antioxidant enzymes such as glutathione peroxidase and superoxide dismutase may play a role in mitigating the effects of hydrogen peroxide, providing insight into oxidative stress pathways and the redundancy of antioxidant defense mechanisms in humans (4). Diagnosis typically involves biochemical assays that measure catalase activity in erythrocytes or fibroblasts, and genetic analysis can identify mutations in the CAT gene on chromosome 11p13.

Currently, there is no cure for acatalasemia; treatment is symptomatic and preventative. It focuses on managing complications, primarily through rigorous oral hygiene, periodontal treatments for ulcers, and the use of antibiotics for infections. Most cases are asymptomatic but require medical monitoring. Bone marrow transplantation has been proposed as potential treatment (5), and recently the use of catalase-mimic nanocatalyst cerium oxide was also proposed (6).

Virus‐like particles (VLPs) are self‐assembled protein shell structures derived from viral capsid proteins (but lacking infectious genetic material). Because of their regular architecture, interior cavity, defined pores, and stability, they are attractive as nano‐containers for enzyme encapsulation, creating “nanoreactors” (7).

Encapsulating enzymes within VLPs offers several potential advantages, including protection of the enzyme cargo from proteases, denaturation, and harsh conditions (8); isolation of reaction environments (“compartmentalization”) that could enhance cascade reactions; limitation of side reactions; and control of substrate access (9). In addition, they are uniform and tunable nanostructures, in which the VLP interior environment (size, crowding, porosity) can influence enzyme kinetics (10). Thus, potential biomedical or biotechnological applications of virus-based enzymatic nanoreactors (biocatalysis, enzyme replacement therapy, drug delivery) have been reported by combining enzyme function with the VLP scaffold. Virus-based nanoreactors have been proposed to enhance the activation of prodrugs in cancer treatment (11, 12) and the Enzyme Replacement Therapy (ERT) (13).

To improve the ERT, endogenous enzymes could be encapsulated in VLPs and specifically targeted to the tissues where they are needed, enabling a smart treatment that avoids systemic administration. The encapsulation of β‐glucocerebrosidase (GCase) into brome mosaic virus (BMV)-derived VLPs and mannose functionalization to target macrophages for the treatment of Gaucher disease; encapsulation provided ≈an 11‐fold increase in stability under physiological conditions (14). Encapsulation of Galactose-1-phosphate uridylyltransferase (GALT) into plant‐virus capsids for potential treatment of classic galactosemia (15). Another example is the VLP encapsulation of acid alpha-glucosidase for the treatment of Pompe disease (16).

Finally, VLP‐based enzymatic nanoreactors represent a promising platform for both biocatalysis and therapeutic enzyme delivery. By encapsulating enzymes inside protein cages derived from viruses, one can benefit from confinement effects, improved stability, and control over reaction microenvironments. In this work, virus-based enzymatic nanoreactors with catalase activity were synthesized and demonstrated erythrocyte targeting via surface functionalization with the erythrocyte-binding peptide ERY1 (Figure 1), which has high affinity through interaction with the extracellular domain of Glycophorin A (GYPA1), which is present in erythrocyte membranes (17, 18).

Reagents

Catalase from bovine liver and all other general reagents were purchased from Sigma-Aldrich (St. Louis, MO). The salts used for buffer solutions were purchased from Fermont (Monterrey, MX). Hydrogen peroxide 30% and the bifunctional polyethylene glycol (NHS-PEG 4 -Mal) were obtained from Sigma-Aldrich (St. Louis, MO). A modified ERY1 peptide containing an additional amino acid with a cysteine at the C-terminus (WMVLPWLPGTLD GGSGCRGC) was synthesized by Peptide 2.0 Inc. (Chantilly, VA). TEM analysis was performed using copper grids (400-mesh) coated with formvar/carbon support film (Ted-Pella, USA). Elution solvents HPLC grade were obtained from Merck KGaA (Darmstadt, Germany). All chemicals were of the highest grade commercially available.

Bioinformatic analysis of proteins

To evaluate the possibility of encapsulating the catalase enzyme into BMV capsids using electrostatic charge complementarity, an in silico analysis of its electrostatic properties was performed. Three-dimensional structures of the BMV capsid (ID: 3J7L) and bovine liver catalase (ID: 1TGU) were obtained from the Protein Data Bank. The surface electrostatic charge distribution of the BMV capsomer and the catalase enzyme was analyzed using Maestro Schrödinger software, which employed the Poisson–Boltzmann method. The analysis focused on the surface distribution of electrostatic charges on catalase, visualized using a color map to facilitate identification of regions with distinct electrostatic characteristics.

Virus production and purification.

Virions of Brome Mosaic Virus (BMV) were obtained by infecting barley plants, according to Cadena-Nava et al. (19). Briefly, the infected leaves were ground in a virus extraction buffer containing 0.5 M sodium acetate and 80 mM magnesium acetate at pH 4.5. The mixture was filtered through cheesecloth, and one volume of chloroform was added to the filtrate. The mixture was then centrifuged at 10,000 rpm for 20 min at 4 °C. The upper aqueous phase was recovered and maintained at 4 °C, shaking for 12 hours to eliminate chloroform residues.

The BMV virions were extracted by centrifugation on a 10% sucrose cushion at 32,000 rpm for 120 min at 4 °C in a Beckman ultracentrifuge (model Optima XPN-100), using the SW-32 Ti rotor. The pellet, containing the virions, was resuspended in the extraction buffer (0.5 M sodium acetate and 0.08 M magnesium acetate, pH 4.5).

The virions were then purified by centrifugation through a 10-40% sucrose gradient at 30,000 rpm for 2 hours. Afterward, a visible blue band, visible under light in the centrifuge tubes, was carefully extracted.

The purified virion suspension was diluted 4-fold with the extraction buffer and centrifuged again at 32,000 rpm using the SW-32 Ti rotor for 3 hours at 4 °C to remove sucrose. The pellet was resuspended, and the concentration and purity were spectrophotometrically evaluated. Samples with an absorbance ratio (A 260 /A 280) higher than 1.5 were considered and stored at -80 °C.

Coat protein purification

The coat protein (CP) was obtained by disassembling the virions through 12 hours of dialysis at 4 °C, using a 3.5 kDa membrane with a disassembling buffer containing 0.5 M CaCl 2 and 50 mM Tris base, adjusted to pH 7.5 with HCl. Then, the resulting disassembled CP was centrifuged at 50,000 rpm, 4 °C for 4 h. After centrifugation, the supernatant containing the CP was collected in 0.5 mL fractions. The CP purity of the fractions was estimated using the A 280 /A 260 quotient. The best fractions (A 280 /A 260 ≥ 1.6) were combined and dialyzed against the protein buffer (1 M NaCl, 20 mM Tris-HCl, pH 7.2) for 2 h. Finally, the dialyzed CP was recovered and then quantified by absorbance using the CP extinction coefficient at 280 nm of 24,200 M −1 cm −1.

Catalase nanoreactors synthesis and purification

For the synthesis of VLP-catalase nanoreactors, 1 mL of purified CP at 2.9 mg/mL was mixed with different molar proportions of catalase (1:6, 1:12, and 1:30 Cat: CP). Then, the mixture was dialyzed against the autoassembly buffer (50 mM sodium acetate, 750 mM NaCl, and 8 mM magnesium acetate, at pH 5.1) for 12 h at 4 °C. After assembly, the VLP-Cat nanoreactors were purified by HPLC (Series 1100, Agilent Technologies, USA) using a size exclusion column (SEC-3000, 300 x 7.8 mm, Phenomenex, USA), eluted with autoassembly buffer, and monitored at 280 nm, thereby eliminating non-encapsulated catalase. The nanoreactor concentration was evaluated by using Bio-Rad Bradford Protein Assay (Hercules, CA).

VLP-Cat nanoreactors characterization

To examine the size, distribution, and shape of the VLP-Cat nanoreactors, they were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The hydrodynamic diameter of the nanoreactors was estimated by DLS using a ZetaSizer NanoZS instrument (Malvern Instruments, UK). All measurements were performed at least in triplicate.

Subsequently, a sample was prepared for TEM analysis to examine its surface structure and estimate nanoparticle size. A drop (6 µL) of the nanoparticle suspension was placed onto a 400-mesh copper TEM grid coated with Formvar and a carbon sheet (Ted-Pella, USA). The sample was incubated for two minutes at room temperature, allowing the particles to settle on the grid. The liquid was then carefully removed with absorbent paper. A negative stain was then performed by placing a 6 µL drop of 1% uranyl acetate onto the grid and incubating it under the same conditions. The uranyl acetate was removed after two minutes. Finally, the preparations were observed using a JEM-2010 TEM instrument (JEOL, Japan) operated at 100 kV.

Catalase activity determination

The catalytic activity of the nanoreactors was measured by the change in hydrogen peroxide concentration using a Lambda 25 UV-Vis spectrophotometer (PerkinElmer, USA) at 240 nm using an extinction coefficient of 43.6 M -1 cm -1. The kinetic constants were determined by varying the H 2 O 2 concentration from 2 mM to 14 mM, and the data were fitted to the Lineweaver-Burk double-reciprocal equation. All measurements were performed at least in triplicate.

Functionalization of VNPs

In order to test internalization in erythrocytes, catalase-free viral particles (VNPs) were functionalized with Cy5.5 as a fluorescence label and with the ligand ERY1 peptide. The peptide was modified and synthesized to contain a cysteine sequence at its C-terminal end, allowing direct interaction with the maleimide group of the NHS-PEG 4 -MAL compound, which was used as a crosslinker between the ERY1 peptide and the viral particle. The final sequence of the peptide is WMVLPWLPGTLDGGSGCRGC, in which the underlined part is the recognition fragment of the glycoprotein A of the mouse erythrocyte membrane, and the bold part is the sequence targeted for functionalization and correct orientation on the BMV (20). First, the VNPs were labeled with the fluorophore Cy5.5 in MES buffer pH 6.5. The molar proportion of VPN-Cy5.5 used was 1:1, and the reaction was performed at 4 °C for 16 hrs; afterward, the reaction mixture was purified by dialysis. Then, the ERY1 was conjugated with the crosslinker NHS-PEG 4 -MAL to obtain NHS-PEG 4 -MAL-ERY1 in the same buffer and temperature at pH 7 for 4 hours using a molar ratio of 1:10. Once both components of the system (VNPs@Cy5.5 and NHS-PEG4-MAL-ERY1) were ready, the mixture was prepared at a 1:100 molar ratio of VNPs@Cy5.5 to NHS-PEG4-MAL-ERY1 in saline solution (NaCl 0.9%) and incubated at room temperature for 4 hours. At the end of this period, the sample was concentrated and purified by centrifugation through a 30 kDa Amicon filter. Finally, the sample was quantified and characterized using UV-vis spectroscopy, DLS for hydrodynamic diameter and zeta potential, and TEM measurements.

Erythrocytes purification

Animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health, and were approved by the CNyN-UNAM bioethical committee (approval resolution 0087-2025). Female BALB/c mice aged 6–8 weeks were used for blood samples. Animals were housed in cages under pathogen-free conditions with food and water available ad libitum and maintained on a 12-h dark–light cycle. To allow adequate acclimation, mice were housed within the animal facility for 1 week before experimentation. Blood from healthy mice was obtained by cardiac puncture. The collected blood was diluted 1:1 with sterile saline solution (0.9% NaCl). Erythrocytes were isolated using a density gradient (Ficoll-Paque PLUS). Briefly, 3 mL of Ficoll-Paque PLUS were placed at the bottom of a centrifuge tube, and 4 mL of the diluted blood were carefully layered on top. Samples were centrifuged at 400 × g for 30 min at room temperature without a break. After centrifugation, the erythrocyte fraction was collected, washed twice with saline solution, and resuspended in 0.9% NaCl. The isolated red blood cells were stored at 4 °C and used within 1 week, as cell viability declines thereafter.

Flow cytometry analysis

Flow cytometry was used to analyze 2% erythrocyte suspensions exposed to various treatments, with saline solution and Cy5.5 solution as controls, and with untargeted VNP@Cy5 and targeted VNP@ERY1/Cy5. After 1 hour of exposure at 4 °C under static conditions, 20 µL of the sample was collected and suspended in 1 mL of saline solution to reduce the total cell count and maintain an optimal working threshold for the equipment. The cytometer (LongCyte C3140 flow cytometer) was calibrated with the erythrocyte target (saline solution only), and the BL1 (530/30 nm, FITC) and RL2 (710/20 nm, Cy5.5) detection channels were used. Each reading was performed in triplicate, with a total volume of 100 µL and a speed of 10 µL/min. 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 identify individual cells and eliminate doublets (cells that are fused). Using this filter, cells were plotted on a double-indicator graph (showing different dye populations) to determine quadrants and positive populations, and a compensation matrix was generated for each system. All measurements were performed at least in triplicate.

Statistics

Data was analyzed using GraphPad Prism 8.0 software (Dotmatics, UK). Catalytic constants were analyzed using a two-way analysis of variance (ANOVA) with Sídák multiple comparisons. Variations in the results were considered statistically significant at p < 0.05.

In silico study

The hydrodynamic diameter of catalase was estimated using Maestro (Schrödinger) software, yielding an average value of 104.4 Å, consistent with dynamic light scattering measurements (11.13 ± 2.48 nm) and previously reported values (~90 Å) (21). In addition, in silico analysis of the electrostatic charge distribution revealed a predominance of negative charges on the catalase surface (Figure 2B), confirming its electrostatic complementarity with the positively charged inner surface of BMV VLPs (16).

Based on the hydrodynamic diameter of catalase (11.13 nm) and the internal diameter of BMV capsids (19 nm) (22), and assuming spherical geometry, the theoretical encapsulation capacity was estimated to be approximately three catalase molecules per BMV nanoreactor.

Synthesis of nanoreactors (VLP-Cat)

The encapsidation procedure was performed using three different catalase concentrations. VLP: Cat molar ratios used were 1:6, 1:12, and 1:30. All preparations showed a similar hydrodynamic diameter (Table 1). After nanoreactor purification to eliminate free and non-encapsulated catalase, the specific activity was determined at 10 mM hydrogen peroxide; unexpectedly, the nanoreactors from a 1:6 molar ratio showed the highest specific activity (Table 1). Thus, this ratio was used for further experiments. It is important to note that the specific activity was estimated from the total protein content of the nanoreactors, in which catalase is a minor component.

To estimate the amount of catalase molecules encapsulated in each VLP-based nanoreactor, SDS-PAGE electrophoresis was performed, and the proportions of CP and catalase proteins were estimated by densitometry. The results showed that CP accounts for 88% of total protein and catalase for 12%. Considering the molecular mass of both proteins and 180 CP monomers forming the VLP capsid, an average of 2.09 molecules of catalase were encapsulated inside each nanoreactor (Figure 3).

Considering only the amount of catalase enzyme in each preparation, the catalytic constants were calculated using the Lineweaver-Burk method, and the results are shown in Table 2. The k cat of free catalase was 30,800 s - ¹, which was 10 times greater than the k cat of the nanoreactors (2,800 s - ¹). On the other hand, as expected for mass transfer limitations, the affinity constant (K M) of the VLP-Cat nanoreactor was (27.3 mM) significantly higher than that of the free enzyme (7.9 mM).

The morphology of the VLP-Cat nanoreactors was characterized by transmission electron microscopy (TEM) (Figure 4). The VLP-Cat showed icosahedral, quasi-spherical particles of approximately 31 ± 3 nm, similar to those of the native virus (T = 3, 27.7 nm).

Targeting in vitro analysis

To demonstrate erythrocyte targeting, viral nanoparticles (VNPs) were surface functionalized with the ERY1 peptide and labeled with the fluorophore Cy5.5. ERY1 is a peptide with 12 amino acids conforming the recognition sequence (WMVLPWLPGTLD) identified by phage display that specifically binds erythrocytes through interaction with the extracellular domain of Glycophorin A (GYPA1). Phage displaying ERY1 showed ~95-fold higher binding to mouse and rat erythrocytes than wild-type phage, with negligible binding to leukocytes. When conjugated to polymeric nanoparticles, ERY1 enabled efficient erythrocyte binding (~70% after 24 h in a flow model) without causing detectable toxicity or significant alterations in erythrocyte rheology (17, 18).

Figure 1 shows a schematic of the viral nanoparticles synthesis designed to demonstrate targeting red blood cells (RBCs). A modified ERY1 peptide was used, containing an additional amino acid with a cysteine at the C-terminus (WMVLPWLPGTLDGGSGCRGC). This cysteine enables functionalization via a maleimide group while preserving exposure of the C-terminal region, which is necessary for interaction with GYPA.

The presence of ERY1 peptide in VNP@ERY1/Cy5.5 was confirmed by UV-vis spectrophotometry (Figure 5A). In addition, a significant increase in hydrodynamic diameter from an average of 28 nm (VNP@Cy5.5) to an average of 35 nm (VNP@ERY1/Cy5.5) was found (Figure 5B), and a change in the zeta potential both determined by dynamic light scattering (DLS) (Figure 5D), confirm the effective peptide functionalization of viral particles. The Polydispersity Index (PDI) in the DLS technique is a dimensionless value that describes the degree of heterogeneity (or lack of uniformity) in the particle size of a sample (Figure 5C). A PDI < 0.1 indicates a highly monodisperse sample with a very uniform particle size, while a PDI 0.1–0.4 indicates a narrow size distribution, common in many nanoparticle formulations.

Flow cytometry analysis was performed with different erythrocyte treatments (Figure 6). The control treatment, which consisted of erythrocytes in saline buffer, showed Cy5-negative cells. Erythrocyte suspension treated with free Cy5.5 and viral particles functionalized with only Cy5.5, previously washed, showed only 3.77% and 2.96% of Cy5-positive cells, respectively. However, the erythrocytes treated with Cy5.5-labeled and functionalized with ERY1 peptide showed 13.63% Cy5-positive cells. This confirms that the presence of ERY1 on the viral particle surface facilitates the cell internalization of the VLP-Cat nanoreactor.

Acatalasemia is a rare disease with an estimated prevalence of 1–9 per 100,000 individuals. It is more common in specific populations, such as Japan (where the frequency of heterozygotes varies, approximately 0.09% in Hiroshima/Nagasaki and approximately 1.4% in other areas), and has also been reported in Switzerland, Israel, and Hungary, although at lower rates (23, 24). The administration of exogenous enzymes, known as Enzyme Replacement Therapy, has been successfully used to treat several diseases, particularly lysosomal storage diseases such as Pompe and Gaucher (13, 14, 16). VLP-based enzymatic nanoreactors have been proposed to enhance treatment efficiency, enable targeted delivery to cells or tissues where it is needed, and avoid systemic administration (11, 25, 26).

Encapsulating enzymes within nanocarriers is a promising strategy for improving enzyme stability and activity under adverse conditions (27). Furthermore, nanocarriers can be designed to allow the targeted release of their bioactive cargo at specific sites (25). One strategy for enzyme encapsulation is electrostatic charge complementarity, which leverages enzymes’ tendency to ionize amino acids as a function of pH, thereby imparting an electrical charge. These proteins are then encapsulated within nanocarriers with opposite charge via electrostatic interactions (28).

Encapsulation of enzymes by charge complementarity presents several advantages over other enzyme immobilization methods: (i) It avoids covalent modification of the enzymes, preserving their active conformation; (ii) The interactions are reversible, allowing the controlled release of the enzyme in response to physicochemical changes; (iii) It prevents non-specific intermolecular aggregation of the enzymes by restricting their mobility and orientation on the surface of the nanomaterial (29, 30).

According to the literature, the N-terminal region of the BMV capsid is located inside the capsid and has an abundance of basic amino acids, such as arginine and lysine. This results in a predominantly positive charge on the inner surface of BMV VLPs, which facilitates the encapsulation of negatively charged molecules (22, 31, 32). Based on this, an in silico analysis of the electrostatic properties of the catalase enzyme was performed. The results of this analysis indicated that catalase has a predominantly negative surface charge, highlighting the importance of electrostatic complementarity for achieving successful encapsulation of catalase in BMV VLPs, which is essential for generating nanoreactors via self-assembly.

The hydrodynamic diameter measured by DLS of the nanoreactor (VLP-Cat) was 33.3 ± 12.95 nm (Table 1), very similar to the 31 ± 3 nm obtained from the TEM micrographs from 100 measurements (Figure 4). It is worth noting that DLS measurements show the hydrodynamic diameter, which is generally 2 nm larger than the TEM measurement (33). Similarly, the TEM micrographs reveal the quasi-spherical structure characteristic of VLPs. This morphology and size suggest that VLPs with T = 3 symmetry were formed, meaning they are composed of 180 identical CP molecules.

DLS analyses and computational modeling indicated that the hydrodynamic diameter of the active catalase tetramers is approximately 11 nm (Figure 2A). On the other hand, previous studies estimate that the internal diameter of icosahedral VLPs (T = 3) derived from BMV is between 17–21 nm (17, 34, 35). Considering these dimensions, since for the nanoreactors to show activity it was necessary for the encapsulated catalase to be in its tetrameric state, since all the dimers and monomers of catalases from mammals are enzymatically inactive (36, 37), sterically it would only be feasible to encapsulate a maximum of 3 catalase tetramers inside each VLP. This reasoning aligns with the experimental results, which show an average of two catalase molecules (tetramers) encapsulated per VLP (2.09 catalase molecules per VLP).

Catalytic considerations should be taken into account for highly confined enzymes, as in the case of enzymatic nanoreactors (38). Kinetic analyses revealed an increase in the K M value of catalase after encapsulation in VLPs compared to the free enzyme (Table 2), as anticipated. A low K M value indicates that the enzyme requires small amounts of substrate to reach saturation. In contrast, a high value indicates that the enzyme requires high substrate concentrations to achieve its maximum velocity. This value is inversely related to the enzyme’s affinity for the substrate; therefore, the increase in K M after catalase’s encapsulation in VLPs indicates a decrease in catalase’s affinity for its substrate (H 2 O 2). This increase in K M is primarily attributed to limited substrate diffusion, which reduces substrate availability at the enzyme’s active site (8, 14).

In support of this observation, the k cat value of encapsulated catalase decreased compared to that of free catalase (Table 2). This decrease in enzyme activity may be due to encapsulation within a rigid or compact structure that limits the molecule’s flexibility, making it difficult for the substrate to transform. This steric effect reduces catalytic activity due to mass-transfer resistance and hindered substrate diffusion, a phenomenon previously reported in the literature (39). It is important to note that, despite being a semi-closed system due to its porous structure, the VLP-Cat nanoreactor permits limited diffusion of substrates and products through pores with a diameter of 2 nm (34, 40). These results are consistent with findings in the literature, which have reported reductions in k cat and increases in K M after enzyme encapsulation in various systems, including VLPs (14, 39). It is important to note that, despite the increase in K M of the encapsulated enzyme, this value was only 3 times greater than that of the free enzyme, compared with increases of up to 15 times reported in the literature (41). Although catalytic activity initially decreases due to encapsulation, enzymes confined in VLPs may exhibit better long-term retention of their biocatalytic capacity (8, 14). Finally, the covalent coating of the nanoreactor with the ERY1 peptide induces targeting to erythrocytes in vitro (Figure 6). Further studies are needed to fully evaluate the in vivo efficacy of VLP-Cat@ERY1 nanoreactors in acatalasemia.

In conclusion, these results provide valuable insights into the kinetic effects of catalase encapsulation within viral capsids and its functionalization with the ERY1 peptide, which targets erythrocytes. VLP-based enzymatic nanoreactors appear to be a potential alternative for treating acatalasemia.