Multicomponent synergistic immobilization via in-situ and dynamic exchange strategies for constructing hierarchical biopharmaceuticals

Domain: chemistry
Published: 2026-01-31
Editorial outcome: major_revision

Abstract
Hierarchical immobilization of multicomponent biomacromolecules has been demonstrated to be a favorable protocol for constructing advanced biopharmaceuticals, but still with formidable challenges. Herein, we innovate a multicomponent synergistic immobilization approach combining an in-situ method with a structural-transformation-promoted dynamic exchange method in a flexible zeolitic pyrimidine framework (ZPF) platform to construct multicomponent biopharmaceuticals. In-depth mechanism investigation reveals that special functional groups (e.g., carboxyl, sulfhydryl, imidazolyl) on protein surface can significantly promote the dynamic structural transformation of ZPF and allow for the efficient immobilization of proteins on the surface of ZPF crystals via dynamic exchange approach. By combining with the in-situ method, which can immobilize biomacromolecules in the inner part of ZPF crystals, multicomponent biomacromolecules can be regionally localized within ZPF crystals. This controlled spatial preparation provides substantial advantages over conventional core-shell architectures for cascading enzyme immobilization and enables the development of advanced anticancer agents demonstrating enhanced therapeutic efficacy for female Balb/c nu mice. Moreover, this platform can also generate highly efficient hierarchical antibacterial formulations to significantly enhance the wound-healing process of male Sprague-Dawley (SD) rats. This study provides a revolution to immobilize multicomponent biomacromolecules and represents an innovative and effective platform of biopharmaceuticals with immense potential for anticancer and antibacterial formulations.

Introduction
Biological macromolecules, including proteins, nucleic acids, and polysaccharides, are essential and fundamental large molecules that play critical roles in sustaining life, regulating organismal functions, and facilitating the transmission of genetic information. Investigating the synergistic use of biomacromolecules is of great significance for accumulating scientific theories and technological advances in a wider range of fundamental sciences. However, the utilization of such biomacromolecules faces several challenges. For instance, packaging multiple biomacromolecules into one system with a rational design to achieve a synergistic effect remains challenging due to the intrinsically varied characteristics of each molecule. Furthermore, after encapsulation, maintaining stability and biocompatibility of the whole system under various conditions, such as varied temperature, pH, and ionic strength, also requires considerable effort, particularly for in vivo applications.

The applications of these multicomponent biomacromolecules are diverse, including enzyme immobilization and the development of biopharmaceuticals, such as therapeutic agents targeting cancer or bacterial infections. To ensure effective immobilization and delivery, carriers that provide protection, stabilization, and controlled release of the biomacromolecules are often employed. However, conventional immobilization strategies face challenges, including biomacromolecule inactivation, poor carrier stability, and limited versatility. Particularly, the immobilized enzyme carrier often causes the enzyme activity to decrease, and the ordered enzyme distribution inside the carrier is often challenging to achieve. Furthermore, it is difficult to adjust the position of different components in their carriers. To address these issues, it is urgently essential to develop carriers and immobilization methods that can facilitate the positioning, proportion, and quantification of multicomponent biomacromolecules to achieve a synergistic effect.

Over the past two decades, metal-organic frameworks (MOFs) have emerged as one of the most attractive functional supramolecular materials due to their adaptable and defined structures, high porosity, tunable pore size, and customizable functionality. MOFs have demonstrated great potential in diverse research fields, including gas adsorption and separation, chemical sensing, heterogeneous catalysis, and especially the immobilization of biomacromolecules for biopharmaceuticals. The immobilization methods of MOFs for biomacromolecules mainly include pore adsorption, covalent grafting, and in-situ encapsulation. Among them, the in-situ immobilization method, which simulates the phase separation of natural biological macromolecules, offers the significant advantage of being capable of immobilizing biological molecules regardless of their size. However, previously reported MOFs capable of enzyme immobilization under ambient aqueous conditions remain exceedingly rare (<10). Furthermore, conventional methodologies (e.g., core-shell methods) suffer mass transfer barriers and lack precise control over biomolecular spatial distribution within these materials, which presents significant challenges, particularly for multicomponent enzyme systems. Recently, our group has developed a dynamic exchange strategy (also named dynamic defect generation strategy) for enzyme immobilization, utilizing the dissociation equilibrium of MOFs mediated by enzymes. This approach enables the uniform loading of enzymes onto the material surface, circumventing the size limitations of biomacromolecules. Thus, combining the in-situ and dynamic exchange strategies in one system can achieve the regional distribution of multicomponent biomacromolecules and facilitate their immobilization and function, but this has not yet been realized.

In this study, aiming to address the challenges associated with the spatial immobilization of multicomponent biomacromolecules, we have developed a multicomponent synergistic approach integrating in-situ and dynamic exchange methods to construct multicomponent biopharmaceutical formulations within a flexible MOF structure synthesized under ambient aqueous conditions—ZPF-8 (ZPF = zeolitic pyrimidine framework). The resulting bio-composites could realize a hierarchical localization of multicomponent proteins, hence serving as highly efficient anticancer or wound-healing biopharmaceuticals. The discovery and utilization of ZPF-8 advance the design and synthesis of versatile platforms for combining multicomponent biomacromolecules and establish the guiding principles for developing biopharmaceutical platforms.

Results
Immobilization of biomacromolecules by the in-situ method
Pyrimidine plays a crucial role in biological systems, not only as an essential base in nucleic acids but also as a participant in metabolic processes and genetic information transmission. Furthermore, it exhibits significant potential for applications in the pharmaceutical domain. Our group has reported a flexible zinc-based zeolitic pyrimidine framework (ZPF-2), which can be synthesized at room temperature in an aqueous solution, hence benefiting the immobilization of enzymes via an in-situ method. That is, enzymes can be immobilized during the formation process of ZPF-2 crystals. Single-crystal X-ray diffraction (SCXRD) characterization suggested that the actual crystal structure of ZPF-2 fitted with the theoretical simulation. Notably, this is the inaugural instance where the structure of ZPF-2-Zn can be precisely determined by using single-crystal data. The results of Powder X-ray diffraction (PXRD), Thermal gravimetric analysis (TGA), Scanning electron microscope (SEM) images, N2 and CO2 adsorption-desorption of the synthesized ZPF-2 were also found to be consistent with the literature. To explore the ability of ZPF-2 to immobilize enzymes, we then used ZPF-2 to immobilize enzymes (e.g., glucose oxidase (GOx)) via an in-situ assembly approach (GOx@ZPF-2, @=in-situ immobilization). GOx@ZPF-2 retained the initial PXRD pattern and SEM morphology of ZPF-2, while Fourier transform infrared spectroscopy (FT-IR) spectra showed the characteristic peaks of GOx in GOx@ZPF-2. To investigate the distribution of GOx, ZPF-2 was synthesized in FITC-tagged GOx solution, and a Super-Resolution Imaging System (SIM) was conducted. The SIM images demonstrated that FITC-labeled GOx was uniformly distributed inside the GOx@ZPF-2.

Immobilization of biomacromolecules by the dynamic exchange method
Subsequently, we further explored whether ZPF-2 could undergo the traditional dynamic exchange approach to achieve surface immobilization of enzymes, which could further construct a multi-enzyme regional distribution system. However, when ZPF-2 was immersed directly into the solution of GOx, we found that it barely immobilized the enzyme, indicating that ZPF-2 was challenging to be triggered by the enzyme molecules to dissociate and create defects for enzyme immobilization. Interestingly, we discovered that after treating ZPF-2 with acetone, THF, DMSO, 1,4-dioxane, or ethyl acetate, PXRD results revealed that ZPF-2 could transform into a phase (named ZPF-8) exhibiting a distinct PXRD pattern. SCXRD characterization suggested ZPF-8 has nearly the same coordinate environment as ZPF-2 but crystallizes in higher symmetry, i.e., the cubic I-43m space group (a=b=c=15.8963 Å, α=β=γ=90°, V=4016.9 Å3). The topological structure and coordination environment of ZPF-8 are almost identical to those of the well-known ZIF-8. A face-to-face comparison of the structures of ZPF-2 and ZPF-8 found differences between the coordinates of the coordinate bonds and dihedral angles of ligands with 6-membered rings. These results indicate that upon stimulation with specific solvents, the F-pym ligand can rotate, thus leading to structural transformation. It should be noted that it belongs to a single-crystal-to-single-crystal transformation process, not the typical dissolve-regenerate pathway.

The transition process from ZPF-2 to ZPF-8 was then investigated in an in situ fashion. PXRD patterns of the transition states showed the gradual disappearance of the ZPF-2 peaks and the appearance of ZPF-8. Quantitative analysis of each transition state showed the deceleration of these transformations until all ZPF-2 microsized crystals transformed to ZPF-8. Interestingly, by simply immersing ZPF-8 in the water for a certain time (e.g., 24 h), ZPF-8 could transform back to ZPF-2. Both processes could be performed repeatedly upon altering the solvents for five cycles. These interesting structure transformation processes were then investigated in depth via FT-IR spectra. After immersing ZPF-8 in water, the characteristic peaks of Zn–H2O (γ(Zn–O) at 495 cm−1 and ρ (H2O) at 557 cm−1) appeared initially and then disappeared gradually. This phenomena proved that the Zn–N bonds in ZPF-8 could be partially dissociated upon the attack of water molecules. Then, they reformed after extending the immersion time, forming a more stable structure in the water environment, i.e., ZPF-2. TGA traces and SEM images for ZPF-8 showed similar results with ZPF-2, while its CO2 adsorption-desorption isotherm showed a typical type I isotherm and adsorbed ~40 cm3/g of CO2 at ~100 kPa, indicative of a more rigid structure. Besides, the transformed ZPF-2 from ZPF-8 showed a feature very similar to that of pristine ZPF-2, suggesting complete reversibility of the structural transformation.

The dynamic nature and mild structural transformation conditions of ZPF-8 encouraged us to try the immobilization of biomacromolecules. ZPF-8 was immersed in an aqueous solution of GOx as a demonstration. A time-dependent loading curve was displayed, and the loading amount increased gradually with the extension of the loading time and approached a saturation of 0.12 g/g after 20 min. The PXRD pattern of the formed product (named as GOx⊂ZPF-2, ⊂=dynamic exchange immobilization) confirmed the successful transition from ZPF-8 to ZPF-2. FT-IR also showed the characteristic peaks of GOx in GOx⊂ZPF-2. To further investigate the distribution of GOx, ZPF-8 was soaked in FITC-tagged GOx solution for different times. The SIM images demonstrated that FITC-labeled GOx was mainly embedded in the outer layer of ZPF-2 particles, and the thickness of GOx entry increased with increasing loading time.

Accordingly, one can reason that the disintegration and reorganization process of the framework would be the driving force facilitating the encapsulation of GOx during the structural transformation from ZPF-8 to ZPF-2. To further explore the mechanism of enzyme encapsulation during immobilization, we also monitored the changes in soaking solutions via 1H Nuclear Magnetic Resonance (1H NMR). After the same soaking time in GOx solution, the ligand signal of ZPF-8 rose significantly (nearly two times compared with pure D2O), while pristine ZPF-2 released almost no ligands into D2O. This phenomenon indicated the generation of more defects on MOFs and the coordinate bonds mediated by GOx, which should come from the interaction between functional groups on proteins with metal ions. To prove this, 19 natural amino acids (except Tyr, which had a low solubility) were screened to identify which functional groups (-R) could promote this process via different immobilization efficiencies of ZPF-8. The results showed that the significantly high loading capacities of Cys (0.14 g/g), Glu (0.035 g/g), Asp (0.083 g/g), and His (0.10 g/g) suggested that besides predicted carboxyl, sulfhydryl and imidazolyl in proteins could also possibly promote the enzyme immobilization. To further prove this conclusion, seven glycine-based dipeptides and four polypeptides with different sizes were also tested, suggesting that the immobilization efficiencies had a positive correlation with the ratio of Cys+Glu+Asp+His in these peptides total amino acids. These comparisons proved that after “activating” ZPF-2 into ZPF-8 and then immersing it in enzyme solution, proteins could generate more competitive coordination against original ligands during the dynamic structural transformation and enzyme immobilization process. Finally, along with original ligand release and defect generation, the coordination and immobilization of enzymes within the MOF structures were achieved.

To study the effect of soaking time on the catalytic performance of the encapsulated enzyme, we investigated the relationship between enzyme activity and loading time. The activity increased gradually with the extension of the loading time and approached its highest at ~20 min. Notably, GOx⊂ZPF-2 could fully retain the free enzyme activity. Theoretically, immobilized enzymes on the surface of particles would greatly promote catalytic performance because they could expose more active enzymes from the material surface. To highlight the advantages of this dynamic structural transformation approach, we also used GOx@ZPF-2 as a comparison. It was found that the encapsulated enzymes retained 90% of their activity (loading amount: 0.15 g/g). All these findings further demonstrate that the dynamic transformation induces the formation of more defects in the material, resulting in a higher enzyme loading compared to the conventional defect generation strategy (0.12 vs 0.04 g/g), which exhibits meaningless enzymatic activity due to insufficient loading capacity. Compared with free GOx, GOx⊂ZPF-2 retained over half of its initial activity under various harsh conditions (e.g., GOx⊂ZPF-2 exposure to 70 °C, remaining activity = ~100%, trypsin: 56%; while free GOx only retained <15% under the same treating conditions). Furthermore, GOx⊂ZPF-2 could maintain approximately 80% of its catalytic activity after 10 recycles and retain its crystallinity and morphology. However, only 25% of the remaining activity of GOx@ZPF-2 was observed. It is possible because enzymes encapsulated inside the crystal catalyze the substrate, and then the produced H2O2 accumulates and partially damages the carrier from the inside. These results indicated that enzyme⊂ZPF-2 is an excellent enzyme immobilization platform with superior biocatalytic performance and superior cyclic catalytic capacity.

In practical applications, multi-enzyme systems are often required to achieve advanced catalytic functions, presenting significant challenges for rational construction and optimization. For example, GOx can catalyze the oxidation of glucose to gluconic acid while simultaneously generating hydrogen peroxide (H2O2), which subsequently serves as a substrate for catalase (CAT). Co-immobilization of these two enzymes could effectively verify the feasibility of enzyme cascade reactions. To systematically explore the influence of spatial distribution on cascade reaction efficiency, we developed a synergistic immobilization strategy. Initially, GOx was incorporated into the ZPF-2 via an in-situ synthesis method (i.e., GOx@ZPF-2), followed by selective immobilization of CAT onto these composite structures through a dynamic exchange method (during the transformation from GOx@ZPF-2 to GOx@ZPF-8, the enzymatic activity of GOx was fully retained), thereby establishing a GOx@CAT⊂ZPF-2 cascade system. This synergistic strategy exploits the complementary characteristics of both immobilization methods: the in-situ embedded GOx remains stably anchored within the crystal core through multiple coordination interactions established during framework assembly, while the subsequent dynamic exchange process—triggered by the ZPF-8→ZPF-2 transformation—generates surface defects and coordination vacancies that selectively capture CAT at the particle surface. This spatially programmed architecture creates an optimal substrate channeling pathway where glucose diffuses inward to react with core-embedded GOx, and the generated H2O2 is immediately consumed by the surface-localized CAT, minimizing intermediate accumulation and preventing oxidative damage to both enzymes and the framework structure. The hierarchical localization was confirmed by SIM images. The catalytic performance of this hierarchical design was rigorously compared with two approaches: (I) a traditional core-shell configuration (GOx@ZPF-2@CAT@ZPF-2) and (II) simultaneous co-immobilization of both enzymes via dynamic exchange (GOx&CAT⊂ZPF-2). Under standardized conditions, maintaining equivalent enzyme loading capacities and ratios (GOx:CAT = 1:1) across all configurations, the GOx@CAT⊂ZPF-2 system exhibited superior catalytic efficiency, achieving >200% relative activity compared to the alternative materials. These results convincingly demonstrate the critical importance of optimizing both the regional cascade distribution of enzymes and simultaneously maximizing enzyme-substrate interactions for enhanced catalytic performance. The recyclability of the optimized GOx@CAT⊂ZPF-2 system was evaluated through ten consecutive catalytic cycles. The system maintained approximately 80% of its initial activity, demonstrating substantial operational stability attributed to the rapid consumption of the generated H2O2 intermediate via CAT.

The hierarchical immobilization strategy demonstrated above extends beyond enzymatic cascade catalysis to encompass diverse multicomponent biomedical systems requiring precise spatio-temporal coordination. In advanced therapeutic applications, distinct biomolecular components must function in orchestrated sequences—targeting moieties and therapeutic proteins in tumor therapy must operate sequentially to achieve site-specific accumulation followed by localized drug action, while in wound healing applications, antimicrobial agents and tissue repair factors necessitate programmed release profiles to combat infection before promoting regeneration. These complex biomedical scenarios uniformly demand spatially compartmentalized delivery systems that can regulate the sequential presentation of multiple bioactive components, thereby maximizing therapeutic efficacy while minimizing off-target effects.

Construction of multicomponent anti-tumor biopharmaceuticals
The starvation therapy for cancer, as an approach to treating malignant tumors, could consume the glucose reserves of tumor cells to eliminate cancer cells. We then tried to combine in-situ and dynamic exchange methods to construct multicomponent synergistic formulations. For example, transferrin (Tf), known for its strong selective binding to the abundant transferrin receptors on the surface of tumor cells, can be utilized for targeted drug delivery to tumor cells. By employing Tf in a carrier, GOx can be directly transported into cancer cells, inducing apoptosis through intracellular glucose consumption. So we incorporated GOx into ZPF-2, then immobilized Tf onto these composite structures, forming GOx@Tf⊂ZPF-2 multicomponent synergistic system. The hierarchical localization was confirmed by utilizing FITC-labeled GOx and RhB-labeled Tf, which was conducted via SIM images. The pH-responsive property of GOx@Tf⊂ZPF-2 revealed remarkable stability at physiological pH (7.4) — exhibiting merely 15% GOx release after 5 h. Conversely, under acidic conditions (cancer cell’s lysosomal environment), material rapidly released GOx (a notable 90% at pH 4.6, 5 h) — indicating the carrier’s response capability to environmental stimuli and promoting the bioactive interaction between GOx and glucose within the tumor microenvironment.

The cytotoxicity of GOx@Tf⊂ZPF-2 was evaluated in vitro via MTT assay. This study involved two representative cancer cell lines (HeLa and MCF-7) and a normal cell line (3T3) to assess the cytotoxicity. Compared to the control ZPF-2, which exhibited half-maximum inhibitory concentration (IC50) values of 58.05 μg/mL, the viability of HeLa cells notably decreased when treated with GOx@Tf⊂ZPF-2 (IC50 = 39.77 μg/mL). This suggests that the encapsulation of GOx and Tf within the nanoparticles enhanced the toxicity against the cancer cells. Conversely, negligible cytotoxicity was observed in 3T3 cells within the nanoparticle concentration range of 0–40 μg/mL. The study also explored other counterpart multicomponent synergistic systems created through a similar method. Remarkably, adjusting the protein configuration within the nanoparticles could increase the values of IC50. These results confirm that the enhanced cytotoxic effect is primarily due to the specific hierarchical distribution of the anti-tumor multicomponent synergistic system. To further support these findings, FITC-labeled materials were analyzed to assess cellular uptake capabilities through a confocal laser scanning microscope (CLSM). The images revealed a pronounced increase in green fluorescence in cells, indicating that surface-incorporated Tf significantly enhances cellular uptake by effectively targeting transferrin receptors, thereby leading to a marked accumulation of the material in HeLa cells.

The rational design of nanoparticle components and their ratios is crucial for optimizing cancer therapy. By immobilizing both proteins within the ZPF-2 structure, we could also adjust the GOx/Tf ratio to optimize anti-tumor efficiency. The optimized GOx@Tf⊂ZPF-2 nanoparticles (GOx:Tf = 1.1:1) yielded the lowest IC50 values of 39.77 μg/mL and 68.00 μg/mL for HeLa and MCF-7 cells, respectively. Additionally, we tested the universality of this approach by integrating two different anticancer agents—5-fluorouracil (5-FU) and apoptin caspase 3—into similar systems. The assembled composites demonstrated significant anti-tumor activity, surpassing their counterparts without Tf. Furthermore, we combined cancer-starving therapy with small-molecule drugs to enhance the killing effect of the combined formulation on tumor cells, via incorporating 5-FU into GOx@Tf⊂ZPF-2 to assess its anti-tumor properties. Synthesized GOx&5-FU@Tf⊂ZPF-2 nanoparticles exhibited remarkable cytotoxicity at lower concentrations against both HeLa and MCF-7 cells (IC50 = 25.38 or 54.01 μg/mL), confirming a significant enhancement in the combined anti-tumor effects via the design and regulation of this hierarchical multicomponent synergistic system.

After assessing the anti-tumor effect in vitro, the multicomponent synergistic tumor inhibition effect of GOx&5-FU@Tf⊂ZPF-2 was evaluated using a subcutaneous HeLa tumor-bearing female Balb/c nu mouse model. Throughout the therapeutic period (11 days), negligible fluctuations in mice’s body weights were observed following various treatments. Digital photographs and spider plots visually confirmed that the tumors in the GOx&5-FU@Tf⊂ZPF-2 group were nearly eliminated, and the corresponding tumor-bearing ratio had decreased significantly, verifying the prominent multicomponent synergistic therapeutic outcome. The relative tumor volume curves for different groups are presented, and the corresponding tumor growth inhibition (TGI) rates were calculated, while the GOx&5-FU@Tf⊂ZPF-2 group was found to be the highest (73.26%). In contrast, other control groups showed negligible tumor suppression or continued growth, comprehensively demonstrating the synergistic efficacy of our strategy. To investigate the therapeutic mechanism, an H&E staining assay was performed to investigate the histological damage and apoptosis levels in the tumors. As shown, severe morphological changes and apoptosis were observed in the tumors of GOx&5-FU@Tf⊂ZPF-2 group. In contrast, no significant damage were detected in the tumors treated with the counterpart groups, further validating the synergistic effects mediated by the multicomponent system. These results clearly demonstrate the excellent therapeutic outcome exerted by GOx&5-FU@Tf⊂ZPF-2, which can be attributed to several factors. Primarily, Tf is utilized for targeted drug delivery to tumor cells. Subsequently, GOx mediates the competitive consumption of tumorous glucose while, simultaneously, 5-FU directly kills cancer cells. In summary, GOx&5-FU@Tf⊂ZPF-2, as a multicomponent synergistic anti-tumor system, potently inhibits tumor growth through hierarchical localization.

Construction of multicomponent anti-bacteria biopharmaceuticals for wound healing
Skin, the largest sensory organ, serves as a protective barrier for the human body against external damage. Wounds on the skin frequently develop in various situations, and the blood or blood clot in the early phases of hemostasis provides a nourishing culture media for the growth of invading bacteria. The above promising multicomponent synergistic anti-tumor results have also inspired the further construction of multicomponent synergistic anti-bacteria formulations to form composite agents for application in wound healing. By adjusting the distribution of the components within the system, it is possible to target the various phases of wound healing. Extraordinary, nisin, known for its strong inhibitory effect on many gram-positive bacteria, can be utilized in the initial inflammation phase to eliminate invading bacteria. Zn2+ in MOFs can promote the proliferation of fibroblasts and collagen synthesis, which is conducive to the growth of granulation tissue. Additionally, trypsin (Tps) can provide powerful support for the maturation phase by eliminating inflammatory secretions, breaking down denatured proteins, and promoting tissue regeneration. Considering the above positive factors, Tps@Nisin⊂ZPF-2 multicomponent synergistic system could be formed for wound healing. The hierarchical localization was confirmed by SIM images. The in vitro antibacterial activity of Tps@Nisin⊂ZPF-2 was evaluated using minimum inhibitory concentration (MIC) standard tests against Staphylococcus aureus (ATCC 29213). Under the same nisin concentration (MIC of free nisin = 50 mg/L), the antibacterial effects of Tps@Nisin⊂ZPF-2 were found to be very close to free nisin, while an insignificant antibacterial effect was observed for ZPF-2. The results demonstrated that our reversible deformation strategy did not affect the antimicrobial properties of the encapsulated nisin. These preliminary results showed the feasibility of applying this synergistic immobilization strategy to build antibacterial formulations through hierarchical localization for wound healing.

To further evaluate the wound healing capacity of Tps@Nisin⊂ZPF-2 in vivo, experiments were conducted on the dorsum of male Sprague-Dawley (SD) rats. Results showed the wound contraction for Tps@Nisin⊂ZPF-2 was superior to other groups, exhibiting the best wound healing capacity even on the 3rd day. The percentages of wound area closure at different healing times were calculated, revealing that after 7th days, wounds treated with Tps@Nisin⊂ZPF-2 achieved significant closure of ~95%, compared to the control, which showed ~75% wound closure. Remarkably, after 14th day of treatment with Tps@Nisin⊂ZPF-2, the wounds healed completely, with 100% wound contraction, demonstrating a highly improved wound healing ability. These results indicate that multicomponent synergistic anti-bacteria systems can be beneficial and effective for accelerating wound healing, due to the multicomponent synergies in Tps@Nisin⊂ZPF-2.

Histopathological studies were carried out to further compare the wound healing efficiencies via hematoxylin-eosin (H&E) and Masson’s staining. After 7th day, wounded tissues in the control group still exhibited numerous immune cell infiltrations. Surprisingly, for Tps@Nisin⊂ZPF-2, inflammatory cell infiltration was suppressed, and a significantly higher number of fibroblasts was observed around the impaired region. After 14th day, compared to the irregular structure in control, the wounds treated with Tps@Nisin⊂ZPF-2 showed higher density and volume fraction of collagen, greater regularity of both epithelium and connective tissue with more fibroblasts, a complete epithelium structure, and regenerated hair follicles and blood vessels. Tissue of Tps@Nisin⊂ZPF-2 group exhibited excellent wound healing characteristics, facilitating a faster and more effective re-epithelization process, connective tissue remodeling and arrangement, and the formation of a more regular structure with increased hair follicle formation. This can be attributed to the excellent multicomponent synergistic properties of Tps@Nisin⊂ZPF-2, promoting overall wound healing.

In conclusion, we have demonstrated the construction of a dynamic zeolitic pyrimidine framework to enable precise and efficient regulation of multicomponent combinations of biomacromolecules. This flexible MOF possesses the ability to either immobilize biomacromolecules via an in-situ method or via a structural-transformation-promoted dynamic exchange method. The structural transformation process and enzyme immobilization mechanism were unveiled in-depth via various characterizations, including SCXRD, in situ PXRD, FT-IR, etc. We found that the Zn-N bonds in the ZPF structure could be partially dissociated, then reversibly reformed upon the attack of water molecules, accompanied by ligand movement, rotation, and recombination. This structural transformation allows for the immobilization of enzymes on the outer part of the framework, which is challenging to achieve by the traditional immobilization strategies. Our findings suggest that proteins can produce more competitive coordination from their sulfhydryl, carboxyl, and imidazolyl functional groups, ultimately promoting enzyme immobilization. The resulting biocatalysts exhibited superior biocatalytic performance, protective capabilities, and superior cyclic catalytic capacity. Notably, by combining the in-situ method with the dynamic exchange method in one system, various multicomponent biomacromolecule formulations with hierarchical localization can be constructed, such as cascading enzyme immobilization, anti-tumor and anti-bacteria multicomponent formulations. For instance, cascading immobilized enzyme preparations demonstrated a two-fold enhancement in catalytic activity compared to conventional core-shell structured formulations. Furthermore, a multicomponent biopharmaceutical with co-immobilization of transferrin, GOx and 5-FU led to enhanced therapeutic performance, confirmed by in vitro and in vivo anti-tumor experiments. Further, by incorporating the antibacterial nisin and the wound healing-promoting trypsin, advanced antibacterial formulations for wound healing were successfully constructed through hierarchical localization, while simultaneously utilizing the components of the ZPF to significantly enhance the wound healing process through in vitro and in vivo experiments. This study enriches the toolbox for biomacromolecule immobilization and provides a synergistic approach for fabricating multicomponent biopharmaceuticals.

Methods
General
Except for the special description, chemicals like Zn(NO3)2·6H2O (Innochem, AR), 5-fluoro-2-hydroxypyrimidine (Innochem, 97%), 5-FU (Innochem, 99%), Nisin (Innochem, 98%), organic solvents (Innochem, AR), and biological reagents like GOx (Solarbio, 10 KU), CAT (Solarbio, BR), transferrin (Solarbio, 98%), trypsin (Solarbio, BR) throughout the experiments, were purchased from commercial sources and used as received without further purification. All cell lines certification is provided by the ATCC company from which they originated. Animal experiments on anti-tumor were approved by the Animal Research Committee of Nankai University, China (Approval Number: 2023-SYDWLL-000641), and were carried out strictly according to the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). Animals used in wound healing experiments complied with the National Research Council’s Guide for the Care and Use of Laboratory Animals, and these procedures were reviewed and approved by the Committee for the Care and Use of Animals at the Tianjin University of Science & Technology in China (Approval Number: SYXK2023-0003).

Characterization
SCXRD data were collected at 120 K via an Oxford Cryostream system on a SuperNova (Mo) X-ray Source with a micro-focus sealed X-ray tube. The structure was solved and refined using Olex2 with ‘XS’ and ‘XL’ plug-in. Hitachi UH-5300 UV-vis spectrophotometer was used to record the catalytic process. The absorbance value was measured with the SpectraMax M2e microplate reader. Powder X-ray diffraction (PXRD) patterns of all materials were collected at ambient temperature with a Rigaku dmax 2500 diffractometer operated at 40 kV and 40 mA using Cu Kα (λ=1.5418 Å) radiation, with a scan speed of 1 sec/step, a step size of 0.02° in 2θ, and a 2θ range of 5° to 40°. Crude data were subjected to baseline correction and analyzed by Jade 6. The low-pressure CO2 adsorption and desorption isotherms were measured at 195 K using a Micromeritics ASAP 2460 instrument. Before gas adsorption measurements, the samples were outgassed at 95 °C for 24 h before the measurements. Fourier transform infrared spectrophotometer (FT-IR) spectra of all materials were obtained using a Thermo Fisher spectrometer with ATR modes. The confocal images experiments were performed on a Zeiss Elyra 7 Super-Resolution Imaging System (SIM) with a 63× objective oil lens. The Confocal laser scanning microscopy (CLSM) images were recorded on Leica TCS SP8.

Synthesis of ZPF-8
Zn(NO3)2·6H2O (1 mmol) was dissolved in deionized water (5 mL), and 5-fluoro-2-hydroxypyrimidine (2 mmol dissolved in 5 mL of water) was added under stirring. The clear solutions were kept at room temperature for about 5 min, and then the pH was raised to 4.5 by adding 1M base (NaOH, KOH, or NH3·H2O). The precipitate was harvested by centrifugation at 4000xg for 1 min and then washed twice with deionized water to obtain ZPF-2. As-synthesized ZPF-2 powder was immersed in THF for 24 h to obtain ZPF-8.

Synthesis of GOx⊂ZPFs
GOx solution (3 mg/mL) was prepared by dissolving GOx in 50 mM MES buffer (pH=6.0). 15 mg as-synthesized ZPF-8 was ultrasonically dispersed in 1 mL GOx solution, and incubated in a 37 °C shaker for 1, 2, 4, 7, 10, 15, 20, 30, and 60 min, respectively. The solid was collected by centrifuge and washed with DI water twice. The supernatants were collected to determine the amount of immobilized GOx in MOFs. The uptake amount was determined by the Bradford method. The loading efficiency (LE%) and loading capacity (LC, g/g) were calculated as follows:
LE% = (total protein - protein in supernatant) / total protein × 100% (1)
LC = protein loaded into materials / weight of materials (2)

Catalytic performance of GOx, and GOx⊂ZPFs composites
The activity of GOx was determined through the 3,5-dinitrosalicylic acid (DNS) method, which is used to detect the concentration of glucose. 100 μg GOx (or GOx⊂ZPFs composites containing 100 μg GOx) were added into 0.91 mL aqueous solution. After that, 90 μL of glucose solution (50 mM) was added. After 7 min, 30 μL of the supernatant was taken and mixed with 60 μL DNS solution. The mixture was heated at 100 °C for 5 min. Hitachi UH-5300 UV-vis spectrophotometer was used to record the catalytic process. After cooling to room temperature, the final glucose concentrations were monitored by measuring the absorbance at 540 nm and compared with the standard curve to calculate the final glucose concentration.

Synthesis of GOx@Tf⊂ZPF-2
3 mg GOx was added to the aqueous solutions of 5-fluoro-2-hydroxypyrimidine (0.2 M, 667 μL). Zinc nitrate hexahydrate was dissolved in deionized water (0.2 M, 333 μL). These two aqueous solutions were mixed and placed at room temperature for 20 min. The solid was collected by centrifugation and washed with DI water and DMSO twice, respectively. As-synthesized GOx@ZPF-2 powder was immersed in DMSO and incubated at room temperature on a shaker for 20 min to obtain GOx@ZPF-8, then collected by centrifugation. Transferrin solution (3 mg/mL) was prepared by dissolving transferrin in 50 mM HEPES buffer (pH=7.0). As-synthesized GOx@ZPF-8 was ultrasonically dispersed in 1 mL transferrin solution, and incubated at room temperature on a shaker for 20 min to obtain GOx@Tf⊂ZPF-2. The solid was collected by centrifugation and washed with DI water twice. The supernatants were collected to determine the amount of immobilized GOx or transferrin in MOFs. The uptake amount was determined by the Bradford method.

In vivo anticancer therapeutic evaluation
Tumor (HeLa) bearing Balb/c nu mice (female, 6 weeks of age) were randomly divided into 4 groups (n=5). When the tumor volume reached about 100 mm3, the mice were intratumorally injected with Normal saline (100 µL), 5-FU (156 μg/mL, 100 μL), GOx@Tf⊂ZPF-2, GOx&5-FU@ZPF-2, and GOx&5-FU@Tf⊂ZPF-2 (the corresponding 5-FU was 156 μg/mL, 100 μL), respectively. Prior to the initiation of the experiment, the rats were anesthetized with isoflurane (3% to 4% for induction and 2% to 2.5% throughout the surgical procedure) to ensure the maintenance of a stable state. The tumor volume and body weight of mice were recorded every other day. The tumor volume was calculated as follows: V = W2 × L/2, where W and L represented the tumor width and length, respectively. The mice were sacrificed 11 days post-treatment, and the tumors were collected and photographed. The tumor growth inhibition (TGI) rate was calculated according to the equation.
TGI = (1 - VT / VC) × 100% (3)
Where VC represents the tumor volume of the control group, and VT represents the tumor volume after the treatments.
Simultaneously, the tumors of the mice were collected and used for histological and H&E staining assay, and the tumor size of all experimental animals did not exceed 20 mm as stated in the ethical review documents.

Wound healing assay
A full-layer skin defect infection model was used to evaluate the wound healing of Sprague-Dawley (SD) rats (male, 180–200 g, 6–8 weeks of age), and 25 rats were randomly divided into 5 groups. Prior to the initiation of the experiment, the rats were anesthetized with isoflurane (3% to 4% for induction and 2% to 2.5% throughout the surgical procedure) to ensure the maintenance of a stable state. After anesthesia, shave the back and disinfect it with medical alcohol. A circular full-layer excision wound (8 mm diameter) was produced with a biopsy perforator (Japan, BP-80F). Each wound was inoculated with 50 μL Staphylococcus aureus suspension (OD600=0.3). Leave the bacterial suspension on the wound for 30 min. They were treated with PBS (Control), nisin, ZPF-2, Nisin⊂ZPF-2, and Tps@Nisin⊂ZPF-2, respectively. After that, the wound area was immobilized with sterile gauze. Wound photos were taken on days 0, 3, 7, 10 and 14, and the wound size was quantified by Image J software. The healing process takes about 14 days. Freshly regenerated skin tissue was collected on days 7 and 14. These tissues were immobilized in 4% PFA solution for 48 h. Wound healing was evaluated by H&E and Masson staining. Sex was considered in the study design, and detailed information was described in the manuscript.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this work, Chen’s group elucidate a cascade immobilization methodology that synergistically integrates in-situ\nimmobilization processes with dynamic exchange immobilization techniques for the fabrication of hierarchically-structured\nmulticomponent biopharmaceutical systems. The investigation employs zeolitic pyrimidine frameworks as versatile and\nreversible matrices, facilitating the immobilization of biomacromolecules with precise spatial orientation and distribution.\nThis strategic approach demonstrates significant implications for the advancement of complex biopharmaceutical\nformulations where spatial control of bioactive components is important for therapeutic efficacy and functional performance.\nThus, I highly recommend it be accepted after a minor revision.\nComments 1: Obviously, this cascade immobilization approach shows higher advantages over the core-shell method. The\ncomparison should be emphasized in the introduction part.\nComments 2: A comparative analysis of the enzymatic activities between GOx⊂ZPF-2 and materials from conventional\ndefect generation strategy should be incorporated into the manuscript. In the event that materials from conventional defect\ngeneration strategy exhibit meaningless enzymatic activity due to insufficient loading capacity, authors should add the\ncorresponding description in the corresponding part.\nComments 3: For recyclability of GOx@CAT⊂ZPF-2, the authors should clarify what the initial activity is.\nComments 4: During the enzyme immobilization, the pH of the enzyme solution should also be considered as to whether it\naffects the structural transformation and defect generation of the MOFs\nComments 5: Some figures are too small, e.g., Supplementary Figure 47. The authors should make some adjustments.", "reviewer_2": "The current manuscript presents a strategy for achieving reversible encapsulation of enzymes and proteins by exploiting the\ndynamic conformational response of ZPF-2 to various solvents, including acetone, THF, DMSO, 1,4-dioxane, and ethyl\nacetate. By combining an in-situ encapsulation approach for inner-layer enzymes with a dynamic exchange method for\nouter-layer enzymes, the authors developed several systems: a hierarchical cascade enzyme system (GOx@CAT⊂ZPF-2),\nmulti-component anti-tumor agents (GOx@Tf⊂ZPF-2), and antibacterial formulations (Tps@Nisin⊂ZPF-2). Among these,\nonly the first qualifies as a cascade system, whereas the latter two consist of independently functioning proteins that do not\nengage in interdependent interactions. The manuscript has several critical shortcomings. Most notably, it fails to clearly\ndemonstrate the innovation, necessity, and uniqueness of the proposed reversible encapsulation strategy when compared\nwith existing methods (Nature Catalysis, 2018, 1(9): 689–695; Angew. Chem.\nInt. Ed., 2023, 62, e202302436). The preparation of GOx@ZPF-2 via an in-situ method followed by organic solvent-induced outer-layer enzyme replacement\ndoes not appear to offer substantial advantages over established techniques. The current data do not provide convincing\nevidence to demonstrate the spatial distribution of the different components within the hierarchical cascade enzyme system.\nThe manuscript also suffers from a lack of narrative coherence. The first half focuses on a cascade enzyme system\n(GOx@CAT⊂ZPF-2), while the second half abruptly transition to multi-component systems (GOx@Tf⊂ZPF-2 and\nTps@Nisin⊂ZPF-2) that are not cascade-related, in which the relevance of these systems to the initial design rationale\nremains unclear. Moreover, the necessity of employing the complex reversible encapsulation strategy in the latter systems is\nnot sufficiently justified, further weakening the overall conceptual continuity. In conclusion, I do not recommend this\nmanuscript for publication in its current form. While it explores the use of ZPF-2 for\nencapsulating cascade enzymes and multicomponent protein systems, it does\nnot convincingly demonstrate the benefits or uniqueness of the proposed\ndynamic encapsulation strategy. Instead, it adds unnecessary complexity and\nleaves key design rationales insufficiently justified. A transfer to a more\nspecialized journal may be more appropriate.\nOther important issues should also be considered and addressed:\n(1) While the authors point out the limited spatial control in traditional enzyme loading strategies using MOFs—such as in\nsitu encapsulation and covalent attachment—the nanoparticles prepared in this study exhibit significant heterogeneity in\nboth size and morphology. This raises concerns about whether the proposed approach truly offers improved precision in\nenzyme distribution and loading as claimed.\n(2) During the construction of the GOx@CAT⊂ZPF-2 system, GOx@ZPF-2 undergoes a conformational transformation into\nGOx@ZPF-8 upon exposure to organic solvents such as acetone, THF, DMSO, 1,4-dioxane, or ethyl acetate. It is unclear\nwhether this solvent treatment affects the catalytic activity of GOx. Given the known sensitivity of enzymes to external\nenvironment, this warrants further clarification and possibly experimental validation.\n(3) For the cascade enzyme system (GOx@CAT⊂ZPF-2), as well as the multi-component anti-tumor agent (GOx@Tf⊂ZPF-\n2) and antimicrobial agent (Tps@Nisin⊂ZPF-2), what is the loading capacity of each enzyme per nanoparticle? How was\nthis determined, and was the quantification accurately performed using fluorescence quantification methods? Considering\nthat the fluorescent molecules are not covalently linked to the enzyme molecules, questions arise regarding the precision\nand reliability of this approach. Was this fluorescence quantification cross-validated against thermogravimetric analysis\n(TGA) or other independent methods? The quantification process should be thoroughly detailed, including the specific\nprotocols and controls used.\n(4) Some data analysis lacks depth and is insufficient to fully support the conclusions; for instance, FTIR characterization did\nnot reveal significant differences, and the relationship between enzyme fluorescence quantification data and\nthermogravimetric data was not thoroughly investigated. In Figure S7, both samples (GOx@ZPF-2 and ZPF-2) show\nessentially identical peaks with only minor shifts. This similarity undermines the claim that GOx is exclusively present in one\nsample. Likewise, the interpretations based on FTIR spectra (Figures S21, S30b, and S31b) are not sufficiently supported by\nthe data and may not be accurate.\n(5) For the cascade enzyme system (GOx@CAT⊂ZPF-2), as well as the multi-component anti-tumor agent (GOx@Tf⊂ZPF-\n2) and the antimicrobial agent (Tps@Nisin⊂ZPF-2), apart from SIM images of GOx@Tf⊂ZPF-2 in Fig 3c, there is no direct\nevidence provided regarding the spatial distribution of other enzymes. The authors are advised to provide more visually\nintuitive characterization results to substantiate their claims of controlled enzyme localization.\n(6) The manuscript highlights limitations of conventional immobilization strategies, including difficulties in achieving ordered\nenzyme distribution and precise spatial control. However, prior studies, particularly those involving MOFs and DNA origami\nhave demonstrated the feasibility of spatially organized, multi-enzyme assemblies. In this context, the manuscript does not\nconvincingly demonstrate how the proposed strategy offers a clear advantage over these existing approaches, especially in\nterms of spatial accuracy, individual enzyme activity, or cascade reaction efficiency.\n(7) In the stability comparison shown in Figure S30, the enzyme activities of free GOx (5%), GOx⊂ZPF-2 (55%), and\nGOx@ZPF (79%) were reported. While the GOx⊂ZPF-2 system immobilizes GOx on the outer surface of ZPF crystals using\na dynamic exchange method, a notably higher reduction in GOx activity was observed under trypsin treatment. This\nobservation contradicts the authors’ claim regarding the superior stability of this system.\n(8) I understand that GOx@ZPF-2 could exhibit improved environmental stability compared to free GOx. However, the\nGOx⊂ZPF-2 system, prepared via dynamic exchange and thus not tightly encapsulated within the MOF framework, still\nretains most of its initial activity under various harsh conditions, which is somewhat unexpected and deserves further\nclarification.\n(9) The reported enhancement of the MOF-confined biocascade system is intriguing. However, considering the relatively\nsmall aperture size of ZPF-2, it is expected that substrate and intermediate diffusion would be restricted, especially given the\nspatially separated enzyme arrangement. This apparent improvement in catalytic performance requires further explanation.\n(10) In comparing bioactivity across different materials, how to ensure the loadings of each enzyme or protein were\nconsistent among different groups, particularly in co-encapsulation system. the manuscript does not clarify how this\nconsistency was achieved, and the reported enzyme/protein loadings appear to vary across different materials, which may\nconfound the interpretation of bioactivity differences.\n(11) For the multi-component anti-tumor agent (GOx@Tf⊂ZPF-2) and the antimicrobial agent (Tps@Nisin⊂ZPF-2), the term\n\"cascading\" is not accurate; it would be more appropriate to describe their mechanism as a \"multi-component synergistic\neffect\".\n(12) This strategy does not provide clear advantages over cancer treatment methods involving cascade enzymes,\nphotothermal therapy, and drug targeting. Since the manuscript discusses the preparation of GOx@CAT⊂ZPF-2, why not\nutilize GOx@CAT⊂Tf⊂ZPF-2 for cancer therapy?\n(13) For GOx@Tf⊂ZPF-2 with an average particle size of 150 nm based on DLS measurements (Figure S37),\nparticularly TEM images reveal significant particle size heterogeneity. Considering this, can the Super-Resolution Imaging\nSystem (SIM) reliably distinguish between GOx and Tf?\n(14) Many cascade catalytic systems have been reported previously. The authors should cite relevant studies and provide a\nquantitative comparison of their results with the existing literature. Such a comparison is essential to justify the necessity and\nadvantages of the hierarchical spatial arrangement strategy proposed in this work.\n(15) In evaluating the anti-tumor effects, digital images (Figure 4b) demonstrate that the tumors in the GOx&5-FU@Tf⊂ZPF-\n2 group were almost entirely eradicated, with a significant reduction in tumor burden, which validates the therapeutic effect.\nTo provide a more comprehensive illustration of the treatment process, the authors are advised to include digital images\nshowing the tumor growth suppression over time (e.g., on days 3, 5, and 7). Moreover, the mice in v (11 d) appear\nexcessively large with swollen abdomens, raising concerns about whether this is a normal observation. How does the\nsynergistic tumor suppression efficacy of the GOx&5-FU@Tf⊂ZPF-2 system compare with that of other anti-tumor\nstrategies?\n(16) How should one interpret the sentence \"However, previously reported MOFs which were capable of being synthesized\nunder ambient aqueous conditions while simultaneously facilitating enzyme encapsulation remains exceedingly limited\n(<10)32.\" in the manuscript (on page 4 line 79)?\n(17) Can the dynamic behavior be leveraged to control the amount of enzyme or protein encapsulated within and outside the\nnanoparticles, particularly by modulating the inner-to-outer diameter ratio? How is the loading capacity related to kinetic\nbehavior? Does the influence of different solvents on the dynamic properties differ?\n(18) The references to the SI figures in lines 123–131 are incorrectly numbered. The authors should carefully verify and\ncorrect all SI figure citations to ensure accuracy and clarity."}, "author_response": "Rebuttal to Reviewer #2\nReviewer 2: The current manuscript presents a strategy for achieving\nreversible encapsulation of enzymes and proteins by exploiting the dynamic\nconformational response of ZPF-2 to various solvents, including acetone, THF,\nDMSO, 1,4-dioxane, and ethyl acetate. By combining an in-situ encapsulation\napproach for inner-layer enzymes with a dynamic exchange method for outer-\nlayer enzymes, the authors developed several systems: a hierarchical cascade\nenzyme system (GOx@CAT⊂ZPF-2), multi-component anti-tumor agents\n(GOx@Tf⊂ZPF-2), and antibacterial formulations (Tps@Nisin⊂ZPF-2). Among\nthese, only the first qualifies as a cascade system, whereas the latter two\nconsist of independently functioning proteins that do not engage in\ninterdependent interactions. The manuscript has several critical shortcomings.\nMost notably, it fails to clearly demonstrate the innovation, necessity, and\nuniqueness of the proposed reversible encapsulation strategy when compared\nwith existing methods (Nature Catalysis, 2018, 1(9): 689–695; Angew. Chem.\nInt. Ed., 2023, 62, e202302436). The preparation of GOx@ZPF-2 via an in-situ\nmethod followed by organic solvent-induced outer-layer enzyme replacement\ndoes not appear to offer substantial advantages over established techniques.\nThe current data do not provide convincing evidence to demonstrate the spatial\ndistribution of the different components within the hierarchical cascade enzyme\nsystem. The manuscript also suffers from a lack of narrative coherence. The\nfirst half focuses on a cascade enzyme system (GOx@CAT⊂ZPF-2), while the\nsecond half abruptly transition to multi-component systems (GOx@Tf⊂ZPF-2\nand Tps@Nisin⊂ZPF-2) that are not cascade-related, in which the relevance of\nthese systems to the initial design rationale remains unclear. Moreover, the\nnecessity of employing the complex reversible encapsulation strategy in the\nlatter systems is not sufficiently justified, further weakening the overall\nconceptual continuity. In conclusion, I do not recommend this manuscript for\npublication in its current form. While it explores the use of ZPF-2 for\nencapsulating cascade enzymes and multicomponent protein systems, it does\nnot convincingly demonstrate the benefits or uniqueness of the proposed\ndynamic encapsulation strategy. Instead, it adds unnecessary complexity and\nleaves key design rationales insufficiently justified. A transfer to a more\nspecialized journal may be more appropriate.\nRebuttal: We sincerely appreciate your valuable time and effort in evaluating\nour manuscript and providing constructive comments. However, we respectfully\ndisagree with the comment, especially “it fails to clearly demonstrate the\ninnovation, necessity, and uniqueness of the proposed reversible\nencapsulation strategy when compared with existing methods”\n(1) Our strategy represents a fundamental advancement beyond current\napproaches:\nDistinction from ZIF-8 systems (Nature Catalysis, 2018): While conventional\none-pot co-immobilization strategies successfully achieve multi-enzyme\nencapsulation, they fundamentally cannot address the critical challenge of\nspatial distribution of multi-enzymes. CLSM analysis in the literature\nconfirms uniform enzyme distribution throughout the framework. Subsequent\nstudies have required multi-step synthetic approaches to generate core-\nshell architectures for achieving spatial distribution (Nat. Commun. 2022, 13,\n305; Small 2025, 21, 2503059), thereby demonstrating the necessity of\nspatial distribution for enhanced cascade efficiency. In contrast, our\napproach exploits ZPFs' unique solvent-responsive properties to achieve, for\nthe first time, hierarchical immobilization through in-situ encapsulation (inner-\nlayer enzymes) and dynamic exchange (outer-layer enzymes) under ambient\nconditions while preserving MOF structural integrity.\nDistinction from UiO-66 systems (Angew. Chem. Int. Ed., 2023): The dynamic\ndefect generation strategy is limited to zirconium-based MOFs such as UiO-\n66. Moreover, the ball-milling method for in-situ enzyme immobilization is\nseverely restricted to mechanically robust enzymes (e.g., lipase, GOx) and\nlimited MOFs such as UiO-66 and ZIF-8. Thus, developing new platforms to\nconstruct hierarchical multicomponent biocatalysts or\nbiopharmaceuticals is of great importance. Our ZPF-2-based dynamic\nconformational strategy integrates the advantages of both approaches while\nextending applicability to virtually any protein cargo, significantly\nbroadening the scope of immobilization strategies and enabling applications in\nadvanced pharmaceutical formulations.\n(2) We have employed SIM as the primary characterization technique to\ndemonstrate spatial distribution. By differentially labeling inner-layer enzymes\n(FITC-GOx, green fluorescence) and outer-layer enzymes (RhB-Tf, red\nfluorescence), we observed distinct layered fluorescence signals in the\nGOx@Tf⊂ZPF-2 system (Figure 3c), providing direct evidence for the spatial\ndistribution capability of our strategy.\nFigure 3c. SIM images of GOx@Tf⊂ZPF-2.\nTo further strengthen this evidence, we conducted additional SIM images on\nthe GOx@CAT⊂ZPF-2 and Tps@Nisin⊂ZPF-2 (Figure S33, S52), which\ndemonstrated clear spatial distribution between CAT or Nisin (outer layer), and\nGOx or Tps (inner layer).\nFigure S1. SIM images of GOx@CAT⊂ZPF-2.\nFigure S2. SIM images of Tps@Nisin⊂ZPF-2.\n(3) We acknowledge your critique regarding narrative coherence. The core\ninnovation of our manuscript is the development of a versatile MOF-based\nimmobilization platforms that enable the spatial distribution of diverse\nbiomolecules. To demonstrate the broad applicability of this strategy, we\nsystematically explored its implementation in various contexts, including\nenzymatic cascade catalysis and synergistic therapeutic formulations for\ncancer treatment and wound healing.\nWe appreciate your suggestion and enhanced the manuscript's coherence by\nadding a transitional section between the cascade enzyme system\n(GOx@CAT⊂ZPF-2) and multi-component systems (GOx@Tf⊂ZPF-2,\nTps@Nisin⊂ZPF-2) as \"The hierarchical immobilization strategy demonstrated\nabove extends beyond enzymatic cascade catalysis to encompass diverse\nmulti-component biomedical systems requiring precise spatiotemporal\ncoordination. In advanced therapeutic applications, distinct biomolecular\ncomponents must function in orchestrated sequences—targeting moieties and\ntherapeutic proteins in tumor therapy must operate sequentially to achieve site-\nspecific accumulation followed by localized drug action (Adv. Funct. Mater.\n2025, 2419395.), while in wound healing applications, antimicrobial agents and\ntissue repair factors necessitate programmed release profiles to combat\ninfection before promoting regeneration (ACS Appl. Mater. Interfaces 2019, 11,\n37, 33725.). These complex biomedical scenarios uniformly demand spatially\ncompartmentalized delivery systems that can regulate the sequential\npresentation of multiple bioactive components, thereby maximizing therapeutic\nefficacy while minimizing off-target effects\" in page 12, paragraph 2. We\nsupported this rationale with recent literature, demonstrating the necessity of\nspatiotemporal strategies in tumor microenvironment modulation and wound\nhealing.\n(4) Regarding your observation that \" Among these, only the first qualifies as a\ncascade system, whereas the latter two consist of independently functioning\nproteins that do not engage in interdependent interactions.\" we respectfully\nclarify that the anticancer and wound healing formulations are designed\nfor spatiotemporal synergy targeting different therapeutic phases. In the\nanti-tumor system, Tf (outer layer) provides tumor cell targeting while GOx\n(inner layer) generates reactive oxygen species through glucose oxidation to\nkill tumor cells, creating a sequential \"targeting-then-treatment\" mechanism.\nSimilarly, in the antibacterial system, Nisin (outer layer) rapidly eliminates\nbacteria while Tps (inner layer) sustains deep tissue repair, establishing an\n\"antimicrobial-then-regenerative\" sequential effect. These multi-stage\ntherapeutic approaches achieve comprehensive treatment efficacy through\ncomponent synergy. Following your suggestion, we consistently described all\nsystems as exhibiting \"multicomponent synergistic effect,\" emphasizing how\nspatiotemporal coordination enhances catalytic or therapeutic outcomes.\nOther important issues should also be considered and addressed:\nResponse: Thanks for your suggestions. We have revised the manuscript point\nby point as follows.\n(1) While the authors point out the limited spatial control in traditional enzyme\nloading strategies using MOFs—such as in situ encapsulation and covalent\nattachment — the nanoparticles prepared in this study exhibit significant\nheterogeneity in both size and morphology. This raises concerns about whether\nthe proposed approach truly offers improved precision in enzyme distribution\nand loading as claimed.\nResponse: We thank the reviewer for the comments. The primary objective of\nour study is not to achieve absolute uniformity in nanoparticle size or\nmorphology, but rather to establish precise enzyme positioning through rational\nMOF structural design. Specifically, our work innovatively integrates in-situ\nimmobilization with dynamic ligand exchange strategies. This dual approach\nenables both the immobilization of enzymes within the MOF interior through in-\nsitu immobilization and the selective fixation of enzymes on MOF surfaces via\ndynamic ligand exchange. This methodology effectively constructs functional\n\"enzyme1@enzyme2⊂ZPF-2\" units, shifting the paradigm of spatial control to\nthe precise positioning of enzymes. The observed particle heterogeneity,\nincluding aggregation and morphological variations, originates from inherent\ncrystal growth behaviors during MOF self-assembly processes (Chem. Soc.\nRev. 2020, 49, 7406-7427). Critically, however, the enzyme immobilization\nmechanisms-whether through in-situ immobilization or dynamic ligand\nexchange-operate nearly independently of particle size or morphology. SIM\nimages provide direct evidence of regionalized enzyme distribution within the\nMOF architecture (Figure S33), presenting a marked contrast to the random\ndistribution patterns characteristic of conventional approaches (e.g., as\ndemonstrated in the cited Nature Catalysis, 2018, 1(9): 689–695).\nFigure S3. SIM images of GOx@CAT⊂ZPF-2.\nFunctional validation further substantiates our claims: the GOx@CAT⊂ZPF-2\nsystems prepared through our method demonstrate a 100% enhancement in\ncatalytic activity compared to both core-shell (GOx@ZPF-2@CAT@ZPF-2) and\nco-immobilized via dynamic exchange (GOx&CAT⊂ZPF-2) controls (Nat.\nCommun. 2022, 13, 305; Small 2025, 21, 2503059, Figure S35). This significant\nperformance improvement unequivocally demonstrates that particle\nheterogeneity does not compromise the precision of enzyme spatial distribution.\nFigure S4. Comparison of GOx@CAT⊂ZPF-2 with GOx@ZPF-\n2@CAT@ZPF-2 and GOx&CAT⊂ZPF-2 for the conversion of glucose\noxidation.\nIn summary, while acknowledging the presence of particle heterogeneity,\nour approach successfully elevates enzyme spatial control through strategic MOF\nstructural design. The heterogeneity observed at the particle level does not\ndiminish the local precision of enzyme distribution within individual MOF\nframeworks, as evidenced by both structural characterization and functional\nperformance data.\n(2) During the construction of the GOx@CAT⊂ZPF-2 system, GOx@ZPF-2\nundergoes a conformational transformation into GOx@ZPF-8 upon exposure\nto organic solvents such as acetone, THF, DMSO, 1,4-dioxane, or ethyl acetate.\nIt is unclear whether this solvent treatment affects the catalytic activity of GOx.\nGiven the known sensitivity of enzymes to external environment, this warrants\nfurther clarification and possibly experimental validation.\nResponse: We thank the reviewer for the comments. Extensive literature on\nMOF-immobilized enzymes demonstrates that brief exposure to organic\nsolvents does not significantly impair encapsulated enzyme activity (Chem. Rev.\n2021, 121, 1077.; ACS Appl. Mater. Interfaces 2021, 13, 56752.). Also, we\nspecifically selected DMSO as the structural transformation agent due to its\nestablished biocompatibility and widespread use in biological applications,\nincluding as a cryoprotectant for cell preservation (e.g., stem cells, primary\ncells), as a penetration enhancer in transdermal drug delivery systems (e.g.,\nPennsaid®), and as additive in PCR amplification. Furthermore, DMSO is\nroutinely employed for long-term enzyme storage at low temperatures (e.g.,\nDNA Polymerase, Restrictive endonuclease). Per the suggestion of the\nreviewer, we conducted comparative activity assays between GOx@ZPF-2 and\nGOx@ZPF-8, and added these contents as “during the transformation from\nGOx@ZPF-2 to GOx@ZPF-8, the enzymatic activity of GOx was fully retained”\nin page 12, paragraph 1.\n(3) For the cascade enzyme system (GOx@CAT⊂ZPF-2), as well as the multi-\ncomponent anti-tumor agent (GOx@Tf⊂ZPF-2) and antimicrobial agent\n(Tps@Nisin⊂ZPF-2), what is the loading capacity of each enzyme per\nnanoparticle? How was this determined, and was the quantification accurately\nperformed using fluorescence quantification methods? Considering that the\nfluorescent molecules are not covalently linked to the enzyme molecules,\nquestions arise regarding the precision and reliability of this approach. Was this\nfluorescence quantification cross-validated against thermogravimetric analysis\n(TGA) or other independent methods? The quantification process should be\nthoroughly detailed, including the specific protocols and controls used.\nResponse: We thank the reviewer for the comments. Contrary to what may\nhave been implied, we did not employ fluorescence quantification for\ndetermining enzyme loading due to the inherent limitations the reviewer\ncorrectly identifies—namely, the potential for non-covalent fluorophore-enzyme\ninteractions that could compromise quantification accuracy. Instead, we utilized\nBradford assay for proteins (with BCA assay specifically for nisin) that have\nbeen extensively validated in the literature for MOF-protein systems (Chem.\nRev. 2021, 121, 1077.; ACS Appl. Mater. Interfaces 2021, 13, 56752.), as\ndetailed in the Supporting Information. The methodology is as follows: The\nsupernatants were collected to determine the amount of immobilized proteins\nin MOFs. The uptake amount was determined by the Bradford method. The\nloading efficiency (LE %) and loading capacity (LC, g/g) were calculated as\nfollows:\ntotal protein−protein in supernatant\nLE %= ×100%\ntotal protein\nprotein loaded into materials\nLC=\nweight of materials\nOur quantitative analysis reveals loading capacities of approximately 0.1 g/g for\nGOx, Tf, and Nisin, and 0.2 g/g for CAT and Tps in their respective systems.\nThese data was added in Supporting Information as “Loading capacities for\nGOx, Tf, and Nisin: ~0.1 g/g; for CAT and Tps: ~ 0.2 g/g.”\n(4) Some data analysis lacks depth and is insufficient to fully support the\nconclusions; for instance, FTIR characterization did not reveal significant\ndifferences, and the relationship between enzyme fluorescence quantification\ndata and thermogravimetric data was not thoroughly investigated. In Figure S7,\nboth samples (GOx@ZPF-2 and ZPF-2) show essentially identical peaks with\nonly minor shifts. This similarity undermines the claim that GOx is exclusively\npresent in one sample. Likewise, the interpretations based on FTIR spectra\n(Figures S21, S30b, and S31b) are not sufficiently supported by the data and\nmay not be accurate.\nResponse: We thank the reviewer for the comments. We acknowledge that the\nFTIR spectral differences between GOx@ZPF-2 and ZPF-2 appear subtle in\nthe current presentation. Upon careful examination, GOx@ZPF-2 exhibits\ncharacteristic protein signatures at ~3300 cm⁻¹ (O-H and N-H stretching) and\n1500-1700 cm⁻¹ (amide I and II bands). Per the suggestion of the reviewer, we\nacquired spectra at higher resolution to improve peak definition, and enhanced\nthe spectral presentation to clearly highlight these diagnostic features in Figure\nS7, S21, S30, and S31.\nFigure S5. FT-IR spectra of ZPF-2, GOx, and GOx@ZPF-2.\nFigure S6. FT-IR spectra of ZPF-2, GOx, and GOx⊂ZPF-2.\nFigure S7. b-d FT-IR curves of GOx⊂ZPF-2 before and after cycling test.\nFigure S8. b-d FT-IR curves of GOx@ZPF-2 before and after cycling test.\nFurthermore, we conducted a comprehensive re-evaluation of the\ncharacterizations in the main text and Supporting Information, and conducted\na thorough analysis of the results to fully support our conclusions.\n(5) For the cascade enzyme system (GOx@CAT⊂ZPF-2), as well as the multi-\ncomponent anti-tumor agent (GOx@Tf⊂ZPF-2) and the antimicrobial agent\n(Tps@Nisin⊂ZPF-2), apart from SIM images of GOx@Tf⊂ZPF-2 in Fig 3c,\nthere is no direct evidence provided regarding the spatial distribution of other\nenzymes. The authors are advised to provide more visually intuitive\ncharacterization results to substantiate their claims of controlled enzyme\nlocalization.\nResponse: We thank the reviewer for the comments. Per the suggestion of the\nreviewer, we conducted additional SIM images on the GOx@CAT⊂ZPF-2 and\nTps@Nisin⊂ZPF-2 (Figure S33, S52).\nFigure S9. SIM images of GOx@CAT⊂ZPF-2.\nFigure S10. SIM images of Tps@Nisin⊂ZPF-2.\n(6) The manuscript highlights limitations of conventional immobilization\nstrategies, including difficulties in achieving ordered enzyme distribution and\nprecise spatial control. However, prior studies, particularly those involving\nMOFs and DNA origami have demonstrated the feasibility of spatially organized,\nmulti-enzyme assemblies. In this context, the manuscript does not convincingly\ndemonstrate how the proposed strategy offers a clear advantage over these\nexisting approaches, especially in terms of spatial accuracy, individual enzyme\nactivity, or cascade reaction efficiency.\nResponse: We thank the reviewer for the comments. As detailed in our\nprevious responses, our approach offers distinct advantages over established\nMOF systems. Compared to ZIF-8 systems, conventional one-pot strategies\nachieve multi-enzyme encapsulation but fail to provide spatial distribution.\nRegarding UiO-66 systems, dynamic defect generation strategy are limited to\nzirconium-based MOFs and applicable only to mechanically robust enzymes,\nseverely restricting their utility. Our strategy combines advantages of both\napproaches while offering spatial accuracy and universal applicability to diverse\nprotein cargoes.\nWhile DNA origami offers partially spatial control, several intrinsic limitations\nconstrain their practical application. First, the synthesis of DNA scaffolds and\nsubsequent enzyme conjugation requires sophisticated molecular biology\ntechniques, resulting in substantial economic and technical barriers. More\ncritically, enzymes tethered to DNA scaffolds through conventional\nbioconjugation strategies retain significant conformational flexibility due to the\ninherent flexibility of enzymes. This molecular mobility, fundamentally\ncompromises the precision of spatial control and long-term stability of the\nenzyme assembly. Furthermore, DNA-based systems exhibit limited chemical\nstability under physiological conditions and are susceptible to nuclease\ndegradation, restricting their applicability in complex biological environments.\n(Nanomaterials 2016, 6, 139; Appl. Microbiol. Biotechnol. 2023, 107, 9.).\nRegarding individual enzyme activity, our data demonstrate that GOx⊂ZPF-2\nretains 100% activity while GOx@ZPF-2 maintains 90% activity. Critically, the\nGOx@CAT⊂ZPF-2 cascade system exhibits over 100% activity enhancement\ncompared to both core-shell (GOx@ZPF-2@CAT@ZPF-2) and co-immobilized\nvia dynamic exchange (GOx&CAT⊂ZPF-2) controls, substantiating superior\ncascade reaction efficiency through spatial distribution (Figure S35).\nFigure S11. Comparison of GOx@CAT⊂ZPF-2 with GOx@ZPF-\n2@CAT@ZPF-2 and GOx&CAT⊂ZPF-2 for the conversion of glucose\noxidation.\n(7) In the stability comparison shown in Figure S30, the enzyme activities of\nfree GOx (5%), GOx⊂ZPF-2 (55%), and GOx@ZPF (79%) were reported.\nWhile the GOx⊂ZPF-2 system immobilizes GOx on the outer surface of ZPF\ncrystals using a dynamic exchange method, a notably higher reduction in GOx\nactivity was observed under trypsin treatment. This observation contradicts the\nauthors’ claim regarding the superior stability of this system.\nResponse: We thank the reviewer for the comments. While GOx⊂ZPF-2\nretains 55% activity under trypsin treatment compared to 79% for GOx@ZPF-\n2, it represents an 10-fold improvement over free GOx (5% retention),\ndemonstrating substantial protective effects. Per the suggestion of the reviewer,\nwe revised our description to more accurately state as \"Compared with free\nGOx, GOx⊂ZPF-2 retained over half of its initial activity under various harsh\nconditions\" in page 11, paragraph 1.\n(8) I understand that GOx@ZPF-2 could exhibit improved environmental\nstability compared to free GOx. However, the GOx⊂ZPF-2 system, prepared\nvia dynamic exchange and thus not tightly encapsulated within the MOF\nframework, still retains most of its initial activity under various harsh conditions,\nwhich is somewhat unexpected and deserves further clarification.\nResponse: We thank the reviewer for the comments. As documented in the\nliterature (Angew. Chem. Int. Ed., 2023, 62, e202302436) and our manuscript,\n“Enzymes can act as “macro ligands” to generate competitive coordination\nagainst original ligands, along with the release of metal of MOFs to generate\ndefects, hence promoting the immobilization of enzymes”, and “proteins can\nproduce more competitive coordination from their sulfhydryl, carboxyl, and\nimidazolyl functional groups, ultimately promoting enzyme immobilization.”\nI.e., proteins function as \"macro-ligands\" that generate competitive coordination\nwith MOF metal centers, inducing defect formation. Subsequently, proteins are\nnot merely embedded within defects but establish multiple coordination\ninteractions through sulfhydryl, carboxyl, and imidazolyl functional groups with\nexposed metal sites. This multivalent binding creates a robust enzyme-MOF\ninterface, significantly enhancing stability beyond simple physical entrapment.\n(9) The reported enhancement of the MOF-confined biocascade system is\nintriguing. However, considering the relatively small aperture size of ZPF-2,\nit is expected that substrate and intermediate diffusion would be restricted,\nespecially given the spatially separated enzyme arrangement. This apparent\nimprovement in catalytic performance requires further explanation.\nResponse: We thank the reviewer for the comments. ZPF shares identical\ntopology (sod) with ZIF-8, exhibiting similar pore dimensions (~0.35 nm), which\nexceed glucose molecular size (~0.30 nm). As demonstrated in the cited Nature\nCatalysis, 2018, 1(9): 689–695, ZIF-8 successfully facilitates glucose cascade\noxidation with encapsulated GOx and HRP, confirming substrate accessibility\nthrough framework pores. Moreover, the ZIF-8 multi-enzyme cascade system\nwith a core-shell structure also confirmed that spatial distribution of enzyme can\nalso improve the catalytic effect (Nat. Commun. 2022, 13, 305). Our observed\nperformance enhancement in the ZPF-2 system, utilizing CAT instead of HRP,\nis therefore consistent with established precedent while benefiting from our\nspatial distribution strategy.\n(10) In comparing bioactivity across different materials, how to ensure the\nloadings of each enzyme or protein were consistent among different groups,\nparticularly in co-encapsulation system. the manuscript does not clarify how this\nconsistency was achieved, and the reported enzyme/protein loadings appear to vary across different materials, which may\nconfound the interpretation of bioactivity differences.\nResponse: We thank the reviewer for the comments. Our protocol ensures\nuniformity through batch preparation: Initial in-situ protein loading is performed\non a large batch of ZPF-2 materials, which is subsequently divided into equal\naliquots for structural transformation and secondary protein loading. Protein\nsolutions for secondary loading are likewise prepared from a single stock and\ndispensed in equal volumes. This approach minimizes inter-sample variability\nwithin experimental series. Furthermore, all characterization and evaluation\nexperiments will be conducted simultaneously using materials from the same\nsynthesis batch containing the same quality of drugs (e.g., GOx@ZPFs\ncomposites containing 100 μg GOx), ensuring rigorous comparability across\nexperimental groups. Per the suggestion of the reviewer, we included this\npreparation protocols in the Supporting Information to ensure reproducibility.\n(11) For the multi-component anti-tumor agent (GOx@Tf⊂ZPF-2) and the\nantimicrobial agent (Tps@Nisin⊂ZPF-2), the term \"cascading\" is not accurate;\nit would be more appropriate to describe their mechanism as a \"multi-\ncomponent synergistic effect\".\nResponse: We thank the reviewer for the comments. Per the suggestion of the\nreviewer, we consistently described all systems as exhibiting \"multi-component\nsynergistic effect,\" emphasizing how spatiotemporal coordination enhances\ncatalytic or therapeutic outcomes.\n(12) This strategy does not provide clear advantages over cancer treatment\nmethods involving cascade enzymes, photothermal therapy, and drug targeting.\nSince the manuscript discusses the preparation of GOx@CAT⊂ZPF-2, why not\nutilize GOx@CAT⊂Tf⊂ZPF-2 for cancer therapy?\nResponse: We thank the reviewer for the comments. It is important to clarify\nthat our spatial distribution strategy represents a platform technology that\ncomplements, rather than competes with, existing therapeutic modalities such\nas cascade enzymes, photothermal therapy, and drug targeting. These\nestablished anticancer strategies typically employ multi-component\nformulations that could theoretically benefit from spatial distribution (INT J BIOL\nMACROMOL, 2025, 306, 141581.; INT J BIOL MACROMOL, 2024, 278,\n134746.; Neoplasia, 2024, 56, 101024.). Our methodology provides a versatile\nframework for reconstructing these formulations with enhanced spatial control,\npotentially improving their therapeutic efficacy through optimized component\ndistribution and sequential release profiles. We envision future applications\nwhere our platform technology could be integrated with these various\ntherapeutic approaches to create next-generation combination therapies.\nRegarding GOx@CAT@Tf⊂ZPF-2, our preliminary investigations have indeed\ndemonstrated its feasibility and enhanced anticancer efficacy. However, for this\nmanuscript, we strategically selected the combination of small-molecule drugs\nwith macromolecular therapeutics to comprehensively demonstrate the\nversatility of our loading strategy across diverse molecular architectures. This\ndeliberate choice establishes the versatility of our platform, providing a\nfoundation for future investigations that may incorporate cascade enzyme\nsystems, photothermal agents, drug targeting, or other advanced therapeutic\nmodalities.\n(13) For GOx@Tf⊂ZPF-2 with an average particle size of 150 nm based on\nDLS measurements (Figure S37), particularly TEM images reveal significant\nparticle size heterogeneity. Considering this, can the Super-Resolution Imaging\nSystem (SIM) reliably distinguish between GOx and Tf?\nResponse: We thank the reviewer for the comments. The SIM typically\nachieves resolution of approximately 50 nm, while our SEM characterization\nindicates particle sizes ranging from 50-500 nm, providing adequate resolution\nfor accurate discrimination. Furthermore, to enhance observational reliability,\nwe employed differential fluorescent labeling with FITC-GOx (green\nfluorescence) and RhB-Tf (red fluorescence), and selected particles with a size\nclose to 200 nm for observation. The distinct spectral separation between these\nfluorophores ensures unambiguous differentiation and accurate spatial\nlocalization data.\n(14) Many cascade catalytic systems have been reported previously. The\nauthors should cite relevant studies and provide a quantitative comparison of\ntheir results with the existing literature. Such a comparison is essential to justify\nthe necessity and advantages of the hierarchical spatial arrangement strategy\nproposed in this work.\nResponse: We thank the reviewer for the comments. In our study, we\nspecifically employed core-shell (GOx@ZPF-2@CAT@ZPF-2) and co-\nimmobilization via dynamic exchange (GOx&CAT⊂ZPF-2) systems as internal\ncontrols, demonstrating 100% activity enhancement in our hierarchical system,\nsubstantiating superior cascade reaction efficiency (Figure S35).\nFigure S12. Comparison of GOx@CAT⊂ZPF-2 with GOx@ZPF-\n2@CAT@ZPF-2 and GOx&CAT⊂ZPF-2 for the conversion of glucose\noxidation.\nPer the suggestion of the reviewer, we expanded our literature comparison to\ninclude additional relevant studies (e.g., Nat. Commun. 2022, 13, 305; Small\n2025, 21, 2503059) to strengthen the comparative analysis and highlight the\nadvantages of our spatial organization strategy.\n(15) In evaluating the anti-tumor effects, digital images (Figure 4b) demonstrate\nthat the tumors in the GOx&5-FU@Tf⊂ZPF-2 group were almost entirely\neradicated, with a significant reduction in tumor burden, which validates the\ntherapeutic effect. To provide a more comprehensive illustration of the\ntreatment process, the authors are advised to include digital images showing\nthe tumor growth suppression over time (e.g., on days 3, 5, and 7). Moreover,\nthe mice in v (11 d) appear excessively large with swollen abdomens, raising\nconcerns about whether this is a normal observation. How does the\nsynergistic tumor suppression efficacy of the GOx&5-FU@Tf⊂ZPF-2 system compare with\nthat of other anti-tumor strategies?\nResponse: We thank the reviewer for the comments. We appreciate the\nreviewer's suggestion regarding the inclusion of time-course digital images to\ncomprehensively illustrate the tumor growth suppression dynamics. We fully\nrecognize the scientific value of visual documentation in demonstrating\ntemporal changes during therapeutic intervention. In accordance with our\nexperimental protocol, we conducted systematic tumor volume measurements\nat predefined intervals (days 0, 3, 6, 9, and 11) throughout the treatment period.\nPhotographic documentation was acquired at the initial time point (day 0) and\nterminal endpoint (day 11) to capture the overall therapeutic outcome.\nRegarding the supplementation of intermediate time-point imaging (days 3, 6,\nand 9), we would like to respectfully highlight the current discourse in the\noncological literature concerning optimal data presentation methodologies.\nWhile certain studies have employed sequential photographic documentation\nto capture morphological changes, substantial evidences indicate that\nquantitative tumor growth kinetics, presented as continuous curves, provide\nmore robust and statistically rigorous assessment of therapeutic efficacy (e.g.,\nAdv. Healthc. Mater., 2022, 11, 2102298.; Nat. Commun., 2024, 15, 2954.; Nat.\nCommun., 2024, 15, 8695.; Adv. Healthc. Mater., 2024, 13, 2303911.; ACS\nnano, 2022, 16, 20975-20984.; Adv. Mater., 2021, 33, 2100333.; ACS Appl.\nMater. Interfaces, 2024, 16, 12289-12301.). This approach minimizes potential\nobservational bias associated with single-time-point imaging and ensures\nreproducibility of results. To comprehensively address the reviewer's\nsuggestion while maintaining methodological rigor, we have incorporated a new\nFigure S47 in the revised manuscript, presenting the data as a spider plot—a\nvisualization method widely adopted in oncological literature. This\nmultidimensional representation integrates temporal dynamics with individual\ntumor growth trajectories, enabling simultaneous assessment of both individual\nresponse heterogeneity and population-level therapeutic trends. This analytical\napproach preserves the granularity of individual data points while facilitating\nstatistical interpretation of treatment efficacy across the entire experimental\ncohort.\nFigure S13. Spider plots of individual tumor growth curves.\nRegarding the appearance of mice in group v (day 11), the apparent abdominal\ndistension may be attributed to photographic angle and handling position. Per\nthe suggestion of the reviewer, we selected another representative image to\nensure accurate visual documentation.\nFigure 4c. Photos of tumor dissection.\nThe GOx&5-FU@Tf⊂ZPF-2 system, integrating small-molecule drugs,\nmacromolecular therapeutics, and targeting proteins in spatially segregated\ndomains, achieved a tumor growth inhibition (TGI) rate of 73.26%. In contrast,\ncontrol groups (I: control; II: 5-FU; III: GOx@Tf⊂ZPF-2 without small-molecule\nloading; IV: GOx&5-FU@ZPF-2 without targeting protein loading) showed\nnegligible tumor suppression or continued growth, comprehensively\ndemonstrating the synergistic efficacy of our strategy. Per the suggestion of the\nreviewer, we incorporated these contents into the main text as “In contrast,\nother control groups showed negligible tumor suppression or continued growth,\ncomprehensively demonstrating the synergistic efficacy of our strategy” in page\n16, paragraph 2.\n(16) How should one interpret the sentence \"However, previously reported\nMOFs which were capable of being synthesized under ambient aqueous\nconditions while simultaneously facilitating enzyme encapsulation remains\nexceedingly limited (<10)32.\" in the manuscript (on page 4 line 79)?\nResponse: We thank the reviewer for the comments. We apologize for the\nambiguous phrasing. The intended meaning is among reported literature,\nMOFs capable of enzyme immobilization under ambient aqueous conditions\nremain exceedingly rare (<10). Per the suggestion of the reviewer, we revised\nthis sentence as “However, previously reported MOFs capable of enzyme\nimmobilization under ambient aqueous conditions remain exceedingly rare\n(<10)” in page 3, paragraph 1, to enhance clarity and readability.\n(17) Can the dynamic behavior be leveraged to control the amount of enzyme\nor protein encapsulated within and outside the nanoparticles, particularly by\nmodulating the inner-to-outer diameter ratio? How is the loading capacity\nrelated to kinetic behavior? Does the influence of different solvents on the\ndynamic properties differ?\nResponse: We thank the reviewer for the comments. The progressive\nenhancement of fluorescence intensity from outer-layer enzymes, as\ndocumented in Figures S22 and 2a, provides compelling evidence for time-\ndependent loading augmentation and spatial distribution expansion.\nFigure S22, 2a. SIM images of A FITC-GOx⊂ZPF-2 (1 min), B FITC-\nGOx⊂ZPF-2 (10 min), and C FITC-GOx⊂ZPF-2 (20 min).\nThis observation is also corroborated by Angew. Chem. Int. Ed., 2023, 62,\ne202302436, which employed SIM to visualize similar loading dynamics (Figure\nR1).\nFigure R1. SIM images of FITC-lipase@UiO-66 for different immobilization\ntimes.\nThese observations confirm that the ZPF system exhibits dynamic protein-\nframework interactions that can be leveraged to modulate both the quantity and\nspatial distribution of proteins within the outer layer. The time-dependent nature\nof this process provides a controllable parameter for tuning protein loading\ndensity and distribution volume. Complementarily, conventional in-situ\nencapsulation methods offer precise control over enzyme loading through\nadjustment of initial enzyme concentrations during synthesis (Chem. Rev. 2021,\n121, 1077.; ACS Appl. Mater. Interfaces 2021, 13, 56752.). This established\napproach provides direct regulation of the enzyme quantity within the inner core\nregion. By strategically combining these two approaches, we can effectively\nmodulate both the enzyme loading and the spatial distribution between inner\nand outer regions, i.e., enable systematic control over the inner-to-outer\ndiameter ratio.\nOur kinetic analysis (Figure 2a) reveals that enzyme loading follows a\ncharacteristic saturation profile, with loading capacity increasing gradually over\ntime and approaching an equilibrium value of 0.12 g/g after 20 minutes. This\nfurther proves from another perspective that the timing of controlling dynamic\nbehaviors can regulate the amount of enzyme or protein encapsulated within\nand outside the nanoparticles.\n0.40 120\n0.35\n100\n)\n%\n0.30\n(\ng/ g ) 0.25 80 vi t y\ng\n(\n0.20 60 c\nti\nna\ndi e\na0.15 v\no 40 ti\nL a\n0.10 el\nR\n20\n0.05\n0.00 0\n0 10 20 30 40 50 60\nTime (min)\nFigure R2. Loading capability and relative activity of ZPF-2 with different\nloading times toward GOx.\nRegarding solvent-dependent transformation kinetics, we acknowledge that\ndifferent solvents possess distinct physicochemical properties (e.g., molecular\nsize, lipophilicity, dielectric constant) that theoretically influence structural\ntransformation rates. To ensure complete and reproducible transformations, we\nsystematically extended treatment durations for each solvent system. As\ndemonstrated in Figure S9, treatment with acetone, THF, DMSO, 1,4-dioxane,\nor ethyl acetate results in quantitative conversion to the ZPF-8 phase, with no\ndetectable ZPF-2 diffraction peaks remaining. This complete structural\ntransformation ensures uniform material properties upon re-equilibration in\naqueous media, thereby maintaining consistent enzyme loading behavior\nacross different solvent treatments.\nFigure S9. PXRD patterns of soaked in different solvents.\n(18) The references to the SI figures in lines 123–131 are incorrectly numbered.\nThe authors should carefully verify and correct all SI figure citations to ensure\naccuracy and clarity.\nResponse: We thank the reviewer for the comments. Per the suggestion of the\nreviewer, we carefully verified and corrected all SI figure citations to ensure\naccuracy and clarity.\nWe hope that this response addresses your concerns and highlights the\nsignificance and rigor of our study. We remain committed to improving our\nmanuscript and welcome any further suggestions you may have.\nResponse to reviewer 1\nReviewer 1: In this work, Chen’s group elucidate a cascade immobilization\nmethodology that synergistically integrates in-situ immobilization processes\nwith dynamic exchange immobilization techniques for the fabrication of\nhierarchically-structured multicomponent biopharmaceutical systems. The\ninvestigation employs zeolitic pyrimidine frameworks as versatile and reversible\nmatrices, facilitating the immobilization of biomacromolecules with precise\nspatial orientation and distribution. This strategic approach demonstrates\nsignificant implications for the advancement of complex biopharmaceutical\nformulations where spatial control of bioactive components is important for\ntherapeutic efficacy and functional performance. Thus, I highly recommend it\nbe accepted after a minor revision.\nResponse: We thank the reviewer for the high comment, and we will revise the\nmanuscript point by point as follows.\nQ1. Obviously, this cascade immobilization approach shows higher advantages\nover the core-shell method. The comparison should be emphasized in the\nintroduction part.\nResponse: We thank the reviewer for the comments. Per the suggestion of the\nreviewer, we emphasized the comparison in the introduction part as\n“Furthermore, conventional methodologies (e.g., core-shell methods) suffer\nmass transfer barriers and lack precise control over biomolecular spatial\ndistribution within these materials, which presents significant challenges\nparticularly for multicomponent enzyme systems” in page 3, paragraph 1.\nQ2. A comparative analysis of the enzymatic activities between GOx⊂ZPF-2\nand materials from conventional defect generation strategy should be\nincorporated into the manuscript. In the event that materials from conventional\ndefect generation strategy exhibit meaningless enzymatic activity due to\ninsufficient loading capacity, authors should add the corresponding description\nin the corresponding part.\nResponse: We thank the reviewer for the comments. Per the suggestion of the\nreviewer, we added related content as “All these findings further demonstrate\nthat the dynamic transformation induces the formation of more defects in the\nmaterial, resulting in a higher enzyme loading compared to the conventional\ndefect generation strategy (0.12 vs 0.04 g/g), which exhibit meaningless\nenzymatic activity due to insufficient loading capacity” in page 11, paragraph 1.\nQ3. For recyclability of GOx@CAT⊂ZPF-2, the authors should clarify what the\ninitial activity is.\nResponse: We thank the reviewer for the comments. Per the suggestion of the\nreviewer, we added related content as “the initial activity (defined as 100%)\ncorresponds to the conversion rate measured during the first catalytic cycle”\nin page 11, paragraph 1.\nQ4. During the enzyme immobilization, the pH of the enzyme solution should\nalso be considered as to whether it affects the structural transformation and\ndefect generation of the MOFs\nResponse: We thank the reviewer for the comments. The enzyme solutions\nwere maintained at pH = 6.0 (for MES buffer) or 7.0 (for HEPES buffer), thereby\nminimizing potential pH-induced structural perturbations during immobilization.\nPer the suggestion of the reviewer, we added this experimental detail in the\nSupporting Information to ensure complete reproducibility of our protocols.\nQ5. Some figures are too small, e.g., Supplementary Figure 47. The authors\nshould make some adjustments.\nResponse: We thank the reviewer for the comments. Per the suggestion of the\nreviewer, we systematically reviewed and resized all figures throughout the\nmanuscript and Supporting Information to ensure optimal clarity and\naccessibility (e.g., Figure S49, S54, S55).\nFigure S14. Representative H&E-stained images of tumor slices after\ndifferent treatments.\nFigure S15. Representative H&E-stained images of rat epithelial wound\ntissue. 1: control; 2: nisin; 3. ZPF-2; 4. Nisin⊂ZPF-2; 5. Tps@Nisin⊂ZPF-2.\nthe black arrow: inflammatory cell; white arrows: granulation tissue; yellow\ncircle: microvessels; red dotted line: new epithelium.\nFigure S16. Representative Masson's stained images of rat epithelial wound\ntissue. 1: control; 2: nisin; 3. ZPF-2; 4. Nisin⊂ZPF-2; 5. Tps@Nisin⊂ZPF-2.\nOverall, I want to emphasize our innovation points again. In this work, we\ninnovate a multi-component synergistic immobilization approach via in-\nsitu and dynamic exchange strategies for constructing hierarchical\nbiopharmaceuticals in a Zeolitic metal-organic framework (MOF). We find that\nmulticomponent proteins and drugs can be effectively gathered and\nimmobilized in distinct regions of the MOF crystals, enabling the creation\nof cascading enzyme immobilization or hierarchical advanced anticancer\nagents and antibacterial formulations. This study should provide a revolution\nto construct a versatile platform for facilitating the precise regulation and\nimmobilization of multicomponent biomacromolecules, and establish the\nguiding principles for developing biopharmaceutical platforms.\nConsidering the high novelty and importance, we believe this manuscript\ndeserves reconsideration.\nWith my best regards,\nYao Chen, PhD\nProfessor of Key Laboratory of Biopharmaceutical Preparation and Delivery\nInstitute of Process Engineering, Chinese Academy of Sciences"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I recommend this revised manuscript for publication as is.", "reviewer_2": "The resubmitted manuscript from Yang et al provides further experimental data and explanations that address most of the\ncomments raised by the referees. The manuscript is overall ready for publication, provided remaining comment is\naddressed.\n1.Yang et al innovates a multi-component synergistic immobilization approach via in-situ and dynamic exchange strategies\nfor constructing hierarchical biopharmaceuticals in a Zeolitic MOF. The authors claim that 1) treatment with acetone,\ntetrahydrofuran, dimethyl sulfoxide, 1,4-dioxane, or ethyl acetate can all induce the transformation into the ZPF-8 phase; 2)\nthe specific functional groups on the protein surface (e.g., carboxyl, thiol, imidazole) significantly promote the dynamic\nstructural transition of ZPF. However, the mechanisms underlying the “in-situ and dynamic exchange strategy” are not\nclearly described, and the authors are advised to elaborate further.\n2.Scheme 1 does not sufficiently demonstrate the uniqueness of the dynamic encapsulation strategy and its innovative\napplications."}, "author_response": "Response to reviewer 1\nReviewer 1: I recommend this revised manuscript for publication as is.\nResponse: Once again, we would like to express our gratitude for your\nvaluable suggestions.\nReviewer 2: The resubmitted manuscript from Yang et al provides further\nexperimental data and explanations that address most of the comments raised\nby the referees. The manuscript is overall ready for publication, provided\nremaining comment is addressed.\nResponse: We thank the reviewer for the high comment, and we will revise the\nmanuscript point by point as follows.\nQ1. Yang et al innovates a multi-component synergistic immobilization\napproach via in-situ and dynamic exchange strategies for constructing\nhierarchical biopharmaceuticals in a Zeolitic MOF. The authors claim that 1)\ntreatment with acetone, tetrahydrofuran, dimethyl sulfoxide, 1,4-dioxane, or\nethyl acetate can all induce the transformation into the ZPF-8 phase; 2) the\nspecific functional groups on the protein surface (e.g., carboxyl, thiol, imidazole)\nsignificantly promote the dynamic structural transition of ZPF. However, the\nmechanisms underlying the “in-situ and dynamic exchange strategy” are not\nclearly described, and the authors are advised to elaborate further.\nResponse: We thank the reviewer for the comments. Per the suggestion of the\nreviewer, we described the mechanisms underlying the “in-situ and dynamic\nexchange strategy” as “This synergistic strategy exploits the complementary\ncharacteristics of both immobilization methods: the in-situ embedded GOx\nremains stably anchored within the crystal core through multiple coordination\ninteractions established during framework assembly, while the subsequent\ndynamic exchange process—triggered by the ZPF-8→ZPF-2 transformation—\ngenerates surface defects and coordination vacancies that selectively capture\nCAT at the particle surface. This spatially programmed architecture creates an\noptimal substrate channeling pathway where glucose diffuses inward to react\nwith core-embedded GOx, and the generated H₂O₂ is immediately consumed\nby the surface-localized CAT, minimizing intermediate accumulation and\npreventing oxidative damage to both enzymes and the framework structure.” in\npage 12, paragraph 1.\nQ2. Scheme 1 does not sufficiently demonstrate the uniqueness of the dynamic\nencapsulation strategy and its innovative applications.\nResponse: We thank the reviewer for the comments. Per the suggestion of the\nreviewer, we redrew the Scheme 1 to demonstrate the uniqueness of the\ndynamic encapsulation strategy and its innovative applications, including the\ndetailed and specific process and principles of the dynamic structural\ntransformation process, as well as the step-by-step and staged therapeutic\neffects of the resulting hierarchical biopharmaceutical formulations for anti-\ntumor and wound healing applications.\nAgain we thank the referees, editor, and editorial office for the constructive\ncomments and suggestions, which have made our manuscript much improved.\nThank you very much for your favorable consideration of our manuscript.\nWith my best regards,\nYao Chen, PhD"}, {"round": 3, "reviewer_comments": {"reviewer_2": "I recommend this revised manuscript for publication as is."}, "author_response": ""}]

Decision letter