Resiquimod-encapsulated MOF525 for Sustained Immunostimulant Release and Synergistic Photodynamic– Immunotherapy in Colorectal Cancer

Resiquimod-encapsulated MOF525 for Sustained Immunostimulant Release and Synergistic Photodynamic– Immunotherapy in Colorectal Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Resiquimod-encapsulated MOF525 for Sustained Immunostimulant Release and Synergistic Photodynamic– Immunotherapy in Colorectal Cancer Na Kyeong Lee, Nea Young Chun, Yu Jin Oh, Ulziituya Batjargal, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8452162/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Metal-organic frameworks (MOFs) represent a novel class of materials with exceptional potential for biomedical applications. In this study, we present MOF525, a porphyrin-based MOF, as a dual-functional platform for photodynamic therapy (PDT) and immunotherapy in the treatment of colorectal cancer. MOF525 encapsulates Resiquimod (R848), a potent Toll-like receptor 7/8 agonist, thereby establishing a synergistic therapeutic strategy. Upon light exposure, MOF525 efficiently generates reactive oxygen species, leading to localized tumor cell death and the release of tumor-associated antigens. Simultaneously, the sustained release of R848 enhances immune activation, effectively overcoming tumor-induced immunosuppression. This dual approach addresses the limitations of conventional PDT and immunotherapy while maximizing their synergistic potential. In vitro and in vivo studies demonstrate that the combination of PDT and R848-loaded MOF525 significantly inhibits tumor growth and elicits a robust antitumor immune response. Moreover, the therapeutic synergy between PDT and R848 is more pronounced under a less frequent treatment regimen, highlighting the potential clinical applicability of this dual-functional nanoplatform. These findings underscore the promise of R848-loaded MOF525 as a versatile and effective therapeutic strategy for colorectal cancer by integrating photodynamic tumor ablation with innate immune stimulation. Colorectal Cancer Photodynamic therapy Immunotherapy Metal Organic Frameworks (MOF) Nanoplatforms Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Metal-Organic Frameworks (MOFs) have emerged as a novel and highly versatile class of materials with extensive applications in biomedical fields [ 1 , 2 ], particularly in cancer therapy [ 3 – 5 ]. MOFs are crystalline structures composed of metal ions coordinated to organic ligands [ 6 – 9 ]. The distinct connectivity and rotation angles of metal ion and organic ligands lead to various topologies of MOFs creating porous materials [ 9 – 11 ]. Additionally, experimental synthesis factors such as temperature, reaction time, concentration, and others contribute to variations in morphology [ 10 , 12 ]. Furthermore, due to the porosity of MOFs, the empty spaces permit interactions with external molecules, resulting in outstanding applications for transport [ 13 – 15 ]. These properties are particularly advantageous in drug delivery systems. Moreover, MOFs can be designed with photoactive organic ligands, which enable their use in photodynamic therapy (PDT) [ 16 , 17 ]. Among these, porphyrin-based ligands have drawn significant attention due to their intrinsic photodynamic activity. Porphyrin photosensitizer (PS) can be activated by specific wavelengths of light to generate reactive oxygen species (ROS), which induce apoptosis by targeting cellular organelles such as mitochondria [ 18 ]. Their ability to preferentially accumulate in tumor tissues and efficiently produce ROS upon irradiation makes them particularly well-suited for PDT [ 19 ]. By incorporating porphyrins as organic linkers within the MOF framework, these materials can inherit the photodynamic properties of porphyrins while also offering structural benefits such as high surface area, tunable porosity, and the capacity to load additional therapeutic agents. This dual functionality allows porphyrinic MOFs to serve both as efficient PS and as drug delivery vehicles, enhancing the potential of combination therapies [ 20 ]. These unique and tunable physical and chemical properties make MOFs exceptional candidates for drug delivery systems and PDT, where the efficient generation of ROS is crucial for inducing localized cell death. MOFs can also be engineered to encapsulate therapeutic agents, enhancing the efficacy of combination therapies [ 13 – 15 , 21 – 23 ]. Given the significant advancements in the application of MOFs for cancer therapy, their potential in treating specific types of cancer, such as colorectal cancer (CRC), is garnering considerable interest [ 24 – 27 ]. CRC stands as the second leading cause of cancer-related deaths globally [ 28 – 30 ]. According to estimates from 2020, there were over 1.9 million new cases of CRC worldwide, resulting in more than 930,000 deaths attributed to the disease [ 31 ]. Traditional treatments such as surgery, chemotherapy, and radiotherapy remain the standard of care for CRC; however, PDT provides distinct therapeutic advantages for CRC treatment. In particular, PDT can be implemented through a minimally invasive approach using endoscopic guidance [ 32 , 33 ], which enables site-specific light delivery to colorectal lesions while minimizing systemic toxicity. This strategy allows selective tumor ablation, preserves adjacent healthy tissues, and permits repeated administration without cumulative dose limitations, underscoring its potential role as a complementary or alternative therapeutic modality in CRC. Consequently, PDT has attracted considerable attention as a promising treatment strategy that employs PS, light, and oxygen to generate ROS, leading to targeted tumor cell destruction [ 34 , 35 ]. Recent advances in cancer therapy highlight the potential of combining PDT with other treatment modalities to enhance overall efficacy[ 36 , 37 ]. While PDT achieves localized tumor ablation through ROS generation, its efficacy is often limited by the immunosuppressive tumor microenvironment and the possibility of tumor recurrence [ 32 , 33 ]. Immunotherapy, on the other hand, requires adequate exposure to tumor-associated antigens (TAAs) to mount a robust immune response, which may be insufficient in poorly immunogenic tumors [ 38 ]. PDT-induced immunogenic cell death provides a critical bridge by releasing TAAs and danger-associated molecular patterns, thereby enhancing antigen presentation [ 39 ]. When integrated with immunotherapy, these signals can be exploited to potentiate adaptive immune responses, sustain long-term tumor control, and overcome immune evasion [ 33 , 38 ]. Consequently, the rationale for combining PDT with immunotherapy lies in their complementary mechanisms: PDT initiates antigen release, while immunotherapy amplifies and prolongs antitumor immunity, ultimately achieving more durable therapeutic outcomes [ 32 , 38 ]. Among various immunotherapeutic strategies, the use of immune adjuvants has emerged as an effective approach to stimulate the body’s immune response and enhance its reaction to tumor-associated antigens [ 40 – 43 ]. Toll-like receptor (TLR) agonists, particularly Resiquimod (R848), have shown promise due to their ability to activate innate immune cells and promote adaptive immune responses [ 44 – 46 ]. TLRs are pattern recognition receptors that detect pathogen-associated molecular patterns and activate antigen-presenting cells (APCs), thereby inducing pro-inflammatory cytokine secretion and recruiting immune cells [ 47 – 49 ]. By engaging TLR7/8 signaling, R848 induces dendritic cell (DC) maturation and bridges innate and adaptive immunity, enhancing the antitumor immune response [ 50 – 52 ]. Nonetheless, systemic administration of R848 poses challenges, including poor solubility and uncontrolled cytokine release, which may lead to severe side effects [ 53 , 54 ]. Combining PDT with R848 immunotherapy therefore offers several advantages compared to using each modality alone. PDT induces immunogenic cell death, releasing TAAs and danger-associated molecular patterns, while R848 can overcome tumor-induced immune suppression and potentiate the immune response initiated by PDT [ 32 , 55 – 58 ]. Exploiting both mechanisms simultaneously provides a more comprehensive approach to tumor eradication, offering a promising strategy for effective cancer treatment. A porphyrin-based MOF, MOF525, offers a dual-functional platform for PDT and immunotherapy [ 3 ]. Among various porphyrin-based MOFs, MOF525 exhibits high photoreactivity, enabling efficient ROS generation upon light exposure to induce tumor cell death and release tumor-associated antigens [ 59 – 61 ]. This coordination supports the successful incorporation of R848 and its potential stabilization within the MOF525 structure through metal–ligand interactions through Lewis-type metal–ligand interactions between the Lewis acidic Zr⁴⁺ centers and the Lewis basic nitrogen atoms of R848, which are weak and reversible under aqueous or physiological conditions, allowing for subsequent drug release., its porous structure allows for the encapsulation and sustained release of R848, enhancing the antitumor immune response and overcoming the limitations of systemic administration. This combination achieves a synergistic effect with PDT, further improving therapeutic efficacy. In this study, we investigate the potential of R848-loaded MOF525 (R848@MOF525) for combined PDT and immunotherapy in CRC treatment ( Scheme 1 ). We hypothesize that the synergistic effects of PDT and immunotherapy mediated by R848@MOF525 will result in enhanced antitumor efficacy, providing a novel therapeutic approach for CRC. Our comprehensive in vitro and in vivo analyses demonstrate the ability of R848@MOF525 to significantly inhibit tumor growth and induce robust immune responses, highlighting its promise as an advanced multifunctional therapeutic platform. Scheme 1. Schematic illustration for fabrication of R848@MOF525 and mechanism of synergistic effect of PDT and immunotherapy. MOF525 is a type of zirconium-based MOF that acts as both the PS and a carrier for the immune adjuvant R848. Upon laser irradiation of the R848@MOF525 injected into the tumor, ROS are generated, leading to tumor cell death and the formation of TAAs. Concurrently, R848 released from MOF525 matures DCs, enhancing the immune response. This synergistic effect between PDT and immunotherapy contributes to effective tumor treatment. Created with BioRender.com. 2. Result and Discussion 2.1 Characterization of MOF525 and R848@MOF525 In this study, we proposed porphyrin based MOFs, specifically MOF525, as PS and carriers for the immune adjuvant R848. MOF525, synthesized through the solvothermal method, is composed of zirconium (Zr) as the inorganic building unit and Tetrakis(4-carboxyphenyl)porphyrin (TCPP) as the organic building unit. Moreover, R848 was loaded into the pores of MOF525 through coordination bonding, resulting in the formation of R848@MOF525 (Fig. 1 A). The Powder X-ray diffraction (PXRD) patterns clearly show the structure and crystallinity of MOF525, which were maintained after R848 loading (Fig. 1 B) (62). The N 2 adsorption (ADS)-desorption (DES) isotherms of MOF525 and R848@MOF525 indicate that both materials displayed type I pattern isotherms. This confirms the preservation of their microporous structure before and after loading the compound (Fig. 1 C). The BET analysis revealed a surface area of 1356.3 m² g⁻¹ and a pore volume of 0.98 cm³ g⁻¹ for pristine MOF525, which decreased to 80.2 m² g⁻¹ and 0.62 cm³ g⁻¹ for R848@MOF525, respectively. This reduction confirms the occupation of internal pores by R848 molecules. Furthermore, the apparent increase in average pore diameter from 2.88 nm to 15.26 nm is not due to physical expansion of the framework but rather to a shift in the pore distribution. The partial occupation of micropores by R848 blocks nitrogen adsorption in smaller pores, resulting in a relative increase in the contribution of mesopores during BET/BJH analysis [ 62 ]. Scanning electron microscopy (SEM) images exhibited that MOF525 has cuboctahedral-shaped, as reported previously (Fig. 1 D) [ 63 ]. This overall shape did not change after R848 loading. Moreover, the size and zeta potential of the MOFs suspended in deionized water were measured by dynamic light scattering. The average sizes of MOF525 and R848@MOF525 were measured as 320.9 nm and 337 nm, respectively, indicating an increase in size following the loading of R848 (Fig. 1 D ) . The zeta potentials of MOF525 and R848@MOF525 were − 12.3 mV and − 9.5 mV, respectively, indicating that they are relatively similar (Fig. 1 E ) . Next, the colloidal stability of MOF525 was evaluated under serum-containing conditions. The hydrodynamic diameter increased only slightly during the incubation period, and no abrupt changes indicative of aggregation were observed ( Figure S1 in supporting data). This gradual size change and stable PDI profile demonstrate that MOF525 remains colloidally stable even under physiologically relevant conditions. In the Fourier transform infrared (FTIR) spectra analysis of MOF525, a notable feature is observed in the peaks at 1600, 1410, and 660 cm − 1 . These peaks signify the asymmetric and symmetric stretching of the carboxylate linker and the stretching mode of the metal-ligand bonding, respectively, within the Zr-porphyrin MOFs [ 64 ]. For R848, the peak at 3112 cm − 1 is related to = C-H bond stretching vibration and the peak at 2975 cm − 1 could be attributed to stretching of -C-H bonds. For R848@MOF525, the characteristic signals observed from MOF525 showed no discernible shift, and the signals from R848 were retained. This indicates that the chemical structures remained unaltered following encapsulation and the MOF525 and R848 coexist (Fig. 1 F). To examine whether R848 interacts with the open metal sites in MOF525, we performed X-ray photoelectron spectroscopy (XPS) analysis of the Zr 3d region before and after drug loading ( Figure S2 in supporting data). In the pristine MOF525, the Zr 3d spectrum displayed characteristic peaks corresponding to Zr–O coordination [ 65 ]. Upon R848 loading, a new signal emerged that is attributable to O–Zr–N bonding, indicating that the nitrogen-containing functional group of R848 likely coordinates to the unsaturated Zr sites within the MOF525 framework [ 66 ]. This coordination supports the successful incorporation of R848 and its potential stabilization within the MOF525 structure through metal–ligand interactions through Lewis-type metal–ligand interactions between the Lewis acidic Zr⁴⁺ centers and the Lewis basic nitrogen atoms of R848, which are weak and reversible under aqueous or physiological conditions, allowing for subsequent drug release [ 67 ]. These changes confirm that R848 molecules occupy part of the internal pores and simultaneously coordinate with surface Zr sites, indicating a coexistence of pore encapsulation and surface binding within the MOF525 structure. 2.2 In vitro anticancer effect evaluation We examined in vitro release profile of R848 in phosphate buffered saline (PBS) at pH 7.4 and 37 ℃ with shaking. The encapsulation efficiency was determined after loading 20 mg of R848 into 100 mg of MOF525, resulting in approximately 44 µg of R848 loaded per 1 mg of MOF525. R848 was released in a sustained manner, with the majority of the payload gradually released over 11 days in vitro (Fig. 2 A), suggesting that coordination-mediated interactions do not prevent R848 diffusion under physiological ionic conditions, likely due to competitive coordination from phosphate ions and water molecules. The in vitro cell cytotoxicity of MOF525 and R848@MOF525 was assessed using mouse fibroblast cells, L929 which are recommended by ISO 10993-5 as a standard for biocompatibility evaluation (Fig. 2 B) [ 68 ]. Both types of MOFs show dose-dependent cytotoxicity toward L929. Moreover, concentrations of MOF525 and R848@MOF525 that resulted in cell survival rates of 80% is 50 µg/mL, which is considered non-toxic. Consequently, a dose of 50 µg/mL was chosen for further in vitro and in vivo experiments. We next evaluated whether MOF525-mediated photostimulation could induce immunogenic stress responses in tumor cells. Extracellular ATP, a representative damage-associated molecular patterns (DAMP) marker, was quantified in CT26 cells under four different conditions (non-treated, laser irradiation only, MOF525 only, and MOF525 combined with laser irradiation). Among these groups, MOF525 with laser irradiation induced the highest ATP release, whereas the other groups showed minimal changes ( Figure S3 in supporting data). These results indicate that MOF525 enhances ATP-associated DAMP signaling when combined with laser activation. In vitro ROS generation and the anti-cancer effect were assessed under light exposure. Following the mixing of two types of MOFs with a singlet oxygen sensor green (SOSG) solution and subsequent irradiation with a laser for a total of 10 min, the intensity of SOSG gradually increased, indicating ROS generation (Fig. 2 C). Moreover, treatment of CT26 cells with either MOF525 or R848@MOF525 combined with laser irradiation for the same cumulative duration resulted in higher anticancer efficacy compared to the control, control + Laser, and R848 + Laser groups (Fig. 2 D). These findings suggest that the PS of MOF525 induce cell death by releasing ROS upon laser irradiation. To assess the activation of DCs by MOF525 and R848@MOF525, we co-incubated MOF with bone marrow-derived dendritic cells (BMDCs) differentiated from mouse bone marrow cells and analyzed cell markers using flow cytometry. BMDCs were gated on FSC/SSC parameters, followed by singlet discrimination (FSC-H/FSC-A) and live/dead exclusion, and finally on CD11c⁺ cells ( Figure S4 in supporting information). Additionally, for the group treated with R848 alone, the amount of R848 was based on the highest concentration used for R848@MOF525 treatment. The expression of CD86 in BMDCs treated with the highest concentrations of MOF525 and R848@MOF525 was approximately 1.75 times higher than that in the lipopolysaccharide (LPS)-treated group (Fig. 3 A) At lower concentrations, CD86 expression gradually decreased but still remained consistently higher than that observed in the LPS group ( Figure S5 , Supporting Information). The expression of CD40 increased up to 3.37 times compared to the untreated group (Fig. 3 B), with increased expression even at the lowest treatment concentration (10 µg/mL) ( Figure S6 in supporting information). Lastly, except for the lowest treatment concentration, the expression of major histocompatibility complex class II (MHCII) was over 2.38 times higher in the MOF-treated group than in the untreated group (Fig. 3 C, Figure S7 in supporting data). Moreover, at concentrations above 10 µg/mL, there was little difference in the expression of maturation markers between MOF525 and R848@MOF525, indicating that MOF525 alone was sufficient to induce BMDC maturation at higher doses. This effect may arise from the adjuvant-like property of Zr⁴⁺ within the MOF framework, which can stimulate innate immune responses and promote dendritic cell maturation, consistent with previous reports [ 69 ]. In contrast, at concentrations below 10 µg/mL, R848@MOF525 induced higher CD40 expression compared to MOF525, suggesting that the presence of R848 confers an additional stimulatory effect under submaximal dosing conditions ( Figure S8 in supporting information). DCs play a crucial role in regulating the immune system by mediating both innate and adaptive immunity as APCs [ 70 , 71 ]. Immature DCs differentiated from monocytes engulf pathogens or antigens from the external environment through phagocytosis [ 72 , 73 ]. They digest them and present the resulting fragments on the surface through the MHCII [ 74 ]. Simultaneously, upon recognition of pathogen-associated molecular patterns such as LPS or R848 via TLRs, DCs undergo activation and maturation [ 75 – 78 ]. One of the differences between immature and mature DCs lies in their phenotypical characteristics; mature DCs have up-regulated surface expression of costimulatory molecules such as CD86, CD40, and MHCII compared to immature DCs [ 79 , 80 ]. Mature DCs bind antigens and costimulatory molecules presented on their surface to receptors on adaptive immune cells such as T cells, inducing their activation and enabling them to destroy pathogens or infected cells [ 81 ]. Therefore, the increase in these markers suggests that R848@MOF525 may not only activate DCs through sustained release of R848 but also induce DC activation by MOF525 itself. 2.3 In vivo anticancer effect evaluation We further examined the in vivo anticancer efficiency of R848@MOF525. The therapeutic efficacy of R848@MOF525 was evaluated using a CT26 colon cancer bearing mice model. (Fig. 4 A). Additionally, to assess the therapeutic efficacy of R848@MOFs, groups were included that did not receive laser irradiation, as well as groups that received as the same amount of R848 loaded onto the MOFs and the same volume of PBS. As depicted in Fig. 4 B-D, while the tumor growth inhibition effect without laser irradiation was similar to the control group in the case of MOF525, R848@MOF525 showed a relatively inhibited tumor growth rate, to the sustained release of R848 at the tumor site. In the MOF525 + Laser and R848@MOF525 + Laser groups, tumor regression occurred over a period of 40 days after tumor inoculation. However, in the R848@MOF525 + Laser group, all mice exhibited complete tumor regression by day 25, whereas in the MOF525 + Laser group, complete tumor regression was observed by day 35, indicating a difference in the rate of tumor regression due to the incorporation of R848.Additionally, 60 days after the first tumor inoculation, the same amount of tumor cells was inoculated on the opposite flank to evaluate the long-term immune memory and protective effects induced by the treatment. As a result, no tumors regrew, indicating that the initial treatment not only eliminates the primary tumor but also establishes a systemic immune response capable of recognizing and attacking tumor cells if they reappear ( Figure S9 in supporting information). To further validate the synergistic contribution of R848 in the PDT-based treatment, we conducted an additional in vivo study using an extended treatment interval of 5 days, which was intended to moderate the overall therapeutic intensity and better reveal any potential immunological enhancement (Fig. 5 A). In this experiment, only the MOF525 + Laser and R848@MOF525 + Laser groups were compared. Under this milder treatment schedule, the R848@MOF525 + Laser group exhibited greater tumor growth suppression compared to the MOF525 + Laser group, particularly after the third treatment session (Fig. 5 B, C). This result highlights the immune-stimulatory role of R848 in enhancing the therapeutic efficacy of PDT, which may have been underrepresented under the more intensive 3-day treatment regimen due to rapid tumor clearance. These findings further support the hypothesis that R848-mediated immunomodulation acts synergistically with PDT-induced tumor antigen release, facilitating more robust and durable antitumor responses. While both MOF525 + Laser and R848@MOF525 + Laser groups exhibited complete protection upon tumor rechallenge, indicating that PDT-induced immunogenic cell death alone is sufficient to establish durable antitumor memory, the inclusion of R848 resulted in more rapid tumor regression and enhanced suppression during the active treatment phase, particularly under the extended 5-day interval. This moderated treatment schedule allowed clearer observation of R848’s immunostimulatory contribution, which may have been obscured under the more intensive 3-day regimen due to rapid tumor elimination. These findings suggest that although PDT alone can induce long-term immune protection, co-delivery of R848 can accelerate and amplify early immune priming, potentially offering therapeutic advantages in tumors with higher proliferation rates or stronger immunosuppressive environments. Furthermore, a less frequent treatment schedule may better balance direct tumor cytotoxicity with systemic immune activation, minimizing treatment-associated stress while preserving or enhancing overall therapeutic efficacy. 2.4 Anti-cancer efficacy evaluation using a histopathological examination The experimental groups were necropsied on day 40 after tumor inoculation, the point at which tumors had completely disappeared in the laser-treated groups. Tumors remaining in the control, R848, non-laser-treated MOF525, and R848@MOF525 groups were collected and fixed in 10% neutralized buffered formalin for further histopathological evaluation. Tumor samples were subjected to a general paraffin embedding procedure, followed by hematoxylin and eosin (H&E) staining. To assess the overall size and cellular composition of the tumor, the tumor slide was scanned and a whole mount image was collected (Fig. 6 A). The tumors were found to be larger in the order of control, MOF525, R848, and R848@MOF525 (Fig. 6 A, B). A necrotic lesion was present in the central area of all tumor groups, and the MOF525 had a wider necrotic lesion compared to the R848. The large necrotic lesions observed in MOF525 may have been a factor in increasing tumor size over the actual tumor lesions when measuring tumor size. The R848@MOF525 group also had smaller tumor sizes overall, with smaller foci of necrosis found throughout the tumor in addition to the central necrotic lesion. At higher magnification, necrotic lesions composed of amorphous necrotic material were observed in all groups, with a large number of inflammatory cells infiltrating around these necrotic lesions (Fig. 6 C). Furthermore, while the inflammatory cell infiltration was not prominent in the proliferating part of the tumor in the control group, the treatment groups (R848, MOF525, and R848@MOF525) had varying degrees of inflammatory cell infiltration. In particular, R848@MOF525 showed the formation of small necrotic foci with scattered inflammatory cell infiltration. Compared to MOF525, more mononuclear cell infiltration was observed in middle and periphery lesion of R848. In the quantitative analysis of necrotic lesion (Fig. 6 D), it was observed that the relative necrotic lesion (necrotic lesion: tumor area) was increased in the R848@MOF525 and MOF525 at a statistically significant level compared to the control group. However, no statistical significance was observed between MOF525 and R848. The R848@MOF525 group showed more relative necrotic lesions than R848 and MOF525 as well as the control. This may be due to the smaller tumor size of the R848@MOF525 group, but also the inclusion of central necrotic lesions and sporadic necrotic lesions, resulting in an increase in relative necrotic foci. On TUNEL staining to evaluate the extent of apoptotic cell death in tumor, central necrosis in all groups was observed to be characterized by the presence of multiple TUNEL positive cells (Orange to brown color). In particular, the R848 and R848@MOF525 were observed to have multiple TUNEL positive apoptotic cell death foci in the area between the central necrosis and the periphery. In the quantitative evaluation analysis of TUNEL positive area (Fig. 6 F), TUNEL positive area was observed to be statistically significantly higher in the R848@MOF525 group than in any other group. A statistically significant increase in TUNEL positive area was also observed in the R848 and MOF525 groups compared to the control group, but the difference between the two groups did not reach statistical significance. According to our results, it is suggested that R848@MOF525 exhibits MOF525-based necrotizing lesion induction ability and R848-based inflammatory cell infiltration and apoptotic cell death induction ability, may indicating overall improved tumor treatment efficacy. 2.5 In vivo biosafety evaluation To analyze the toxicity of vital organs of MOF525 + Laser and R848@MOF525 + Laser groups, we harvested heart, lung, liver, kidney, and spleen for toxico-pathological analysis 3 weeks after tumor rechallenge test ( Figure S10 in supporting data ). Analyzing the abnormalities of vital organs in three animals from each group, we observed mild proliferation of pulmonary interstitial tissue, mild increase in the number of reactive hepatocytes in the liver, and mild increase in the size of splenic nodules in some individuals. However, these changes were considered to be mild side effects of the body's response to the repetitive tumor cell transplants and subsequent tumor cell therapy, and there was no significant damage to vital organs. These results demonstrated that our MOF525 and R848@MOF525 may be not only effective but also safe for the treatment of recurrent tumor lesion. Furthermore, there was no significant decrease in mouse body weight observed in any of the groups, indicating that MOFs administration was not toxic to the animals ( Figure S11 in supporting data ) . 3. Conclusion This study demonstrates the potential of MOF-525 as an immunotherapeutic cargo–loaded nanoplatform with sustained release capability for the treatment of colorectal cancer (CRC). We developed an R848-encapsulated MOF-525 nanoplatform designed to integrate photodynamic therapy (PDT) with innate immune activation, enabling a synergistic antitumor strategy within a single nanoscale system. The highly porous and photoreactive architecture of MOF-525 facilitated efficient encapsulation and sustained release of the TLR7/8 agonist R848, while simultaneously enabling robust reactive oxygen species (ROS) generation upon light irradiation for direct tumor cell destruction. In vitro and in vivo experiments confirmed that the MOF-525 nanoplatform functions as a dual-functional nanotherapeutic system, capable of sustained immunotherapeutic agent delivery and photodynamically induced tumor ablation. Upon laser irradiation, R848@MOF-525 generated ROS, leading to localized tumor cell necrosis, while the gradual release of R848 promoted antigen-presenting cell activation within the tumor microenvironment. This coordinated nano-enabled delivery of photodynamic and immunostimulatory functions resulted in a pronounced synergistic effect between PDT and immunotherapy, as evidenced by enhanced tumor regression that correlated with R848 loading levels. Notably, this synergistic therapeutic benefit was more clearly revealed under a less frequent treatment regimen, in which PDT was administered at five-day intervals rather than the conventional three-day schedule. Under this milder dosing paradigm, the immunological contribution of sustained R848 release became more distinguishable, highlighting the advantage of nano-controlled immunotherapeutic delivery in achieving durable antitumor responses. Collectively, these findings underscore the promise of R848-loaded MOF-525 as a multifunctional nanoplatform for sustained immunotherapy and photodynamic tumor ablation, offering a clinically relevant and well-tolerated strategy for CRC treatment that leverages the unique capabilities of metal–organic framework–based nanomedicine. 4. Materials and Method 4.1 Materials Benzoic acid (assay = 99.0%), TCPP (assay = 97.0%), R848 (assay = 98.0%) were purchased from TCI (Japan). Dimethylformamide (DMF, assay = 99.5%) was purchased from DAEJUNG (Korea). Zirconyl chloride octahydrate (ZrOCl 2 ㆍ8H 2 O) and LPS ( E. coli O55:B5) were purchased from Simga-Aldrich (USA). Dulbecco’s Modified Eagle Medium (DMEM), RPMI 1640 Medium (RPMI), PBS (pH 7.4) were purchased from Thermo Fisher Scientific (USA). 4.2 Preparation of R848@MOF525 MOF525 was synthesized following a previously reported protocol with slight modifications [ 82 ]. Briefly, TCPP (235 mg, 0.297 mmol) and benzoic acid (6750 mg, 55 mmol) were dissolved in 40 mL DMF and sonicated for 30 min. Then, ZrOCl 2 ㆍ8H 2 O (525 mg, 1.62 mmol) was added to the TCPP solution, and subsequently dissolved for 30 min. Finally, the resulting solution was kept in an oven at 100℃ for 24 hours. The resultant products were collected by centrifugation and washed three times with DMF and ethanol to remove excess reactants, followed by drying at 80℃ for 6 hours. Subsequently, MOF525 was then placed under a vacuum oven at 150℃ to clear the pores as remove residual solvent. To prepare the R848@MOF525, 20 mg R848 was first dissolved and sonicated in 2 mL ethanol, to which 100 mg of the dehydrated MOF525 added. The suspension was kept under RT for 24 h. Then, the resulting suspension was filtered using a 200 nm PVDF membrane filter, washed with ethanol thrice, and dried at 70℃. 4.3 Characterization PXRD patterns of MOF525 and R848@MOF525 were recorded using and X-ray diffractometer (SmartLab, Rigaku, Japan) at 3 kW with CuKα radiation. The surface area, pore size, and pore volume were obtained by a surface area and porosity analyzer (Autosorb IQ, Quanmtachrome, USA) at -196℃. The morphology and size of MOF525 and R848@MOF525 were characterized using a SEM (Dimension ICON, Bruker, USA). The FTIR spectra of MOF525 and R848@MOF525 were determined using a FTIR spectrophotometer (Cary670 (Main bench) + Cary620 (microsocope)), Agilent, USA) over the range of 4000 to 500 cm − 1 at room temperature. The hydrodynamic size and zeta potential were measured by DLS (Zetasizer pro, Malvern Panalytical, UK) and serum stability was evaluated in PBS containing 5% FBS. The XPS spectra of MOF525 and R848@MOF525 were obtained using an X-Ray Photoelectron Spectroscopy System (PHI Quantera-Ⅱ, Ulvac-PHI, Japan). 4.4 In vitro drug release profile In vitro drug release was confirmed by reverse phase high-performance liquid chromatography (1260 Infinity Ⅱ, Agilent, USA). R848@MOF525 was dispersed in PBS at a concentration of 1 mg/mL, and 5 mL of this dispersion was dialyzed using SnakeSkin® dialysis tubing (MWCO: 3500, Thermo Fisher Scienticfic, USA) against 20 mL of same buffer at 37℃ with shaking. At each time point, the PBS outside the tube was recovered and replaced with fresh PBS. To quantify the initial drug loading on the MOF, R848@MOF525 was dispersed in PBS at a concentration of 0.5 mg/mL and sonicated for 3 hours to extract the encapsulated drug. For each time point and loading quantification, the obtained samples were centrifuged at 17,000 rpm for 10 minutes, and the supernatant was injected into the HPLC. The mobile phase consisted of 20 mM PBS containing 60% phosphoric acid (pH 2.5) and 40% acetonitrile, with a flow rate of 1.0 mL/min. R848 was detected at 249 nm, and the injection volume was 10 µL. 4.5 In vitro study cytotoxicity evaluation In vitro cytotoxicity was evaluated using a water-soluble tetrazolium salt-based cell viability assay (EZ-Cytox; Daeil Lab Service, Korea) in L929 mouse fibroblast cells. L929 cells were seeded at a density of 0.9 × 10 4 cells/well in a 96-well plates and incubated at 37℃ with 5% CO 2 for 1 day. The medium was completely removed and replaced with the MOF525 or R848@MOF525 suspension prepared in fresh cell culture medium at varying concentration (50, 25, 10 and 1 µg/mL) and incubated for another 24 h. The cells were washed three times with PBS. After that, EZ-Cytox solution was added to the medium at 10% volume and the cell incubated for 1 h. The absorbance was measured at 450 nm with a reference wavelength of 600 nm, using a microplate reader (Synergy H1, BioTek, USA). The cell viability was calculated by using the following equation: $$\:Cell\:vaibility\:\left(\%\right)=\:\frac{Absobance\:of\:treated\:cells-Absorbance\:of\:media}{Absorbance\:of\:negative\:control\:cells-Absorbance\:of\:media}\:\times\:100$$ 4.6 In vitro study on ROS generation To measure the degree of ROS generation of nanoparticle, Singlet Oxygen Sensor Green (SOSG; Thermo Fisher Scienfitic, USA) was used. Briefly, suspensions of MOF525 in medium at 25 and 50 µg/mL concentrations were prepared. Then, 100 µL of suspension was added in 96-well black plates. Subsequently, 100 µL of 10 µM SOSG solution using medium were mixed to the suspensions of MOF525 with enough pipetting. The laser equipment (808NM-3W, LASERLAB, KOREA) was used with 660nm laser wavelength and 500 mW power output. 50 µg/mL of MOF525 was allowed for 3 min to the laser beam, and 25 µg/mL of MOF525 was irradiated for 10 min. Likewise, the R848@MOF525 was performed at 25 µg/mL concentration for 10 min in the same method as described above. The SOSG intensity was measured at 500 nm with a reference wavelength 530 nm, using the microplate reader. 4.7 In vitro PDT effect evaluation In vitro PDT was assessed using a 10 µM SOSG solution in mouse colon cancer CT26 cells (ATCC, USA), which were used within five passages after thawing. Cells were seeded in a 96-well plate at a density of 1.0 × 10 4 cells/well. After 24 h incubation, the medium was removed and replaced with 50 µg/mL of MOF525 and R848@MOF525. MOF525 was evaluated in two distinct cell types, whereas R848@MOF525 was assessed only in CT26 cells. Each well was irradiated with a 500 mW laser beam for 5 min, allowed to rest at room temperature for 1 min, and then re-exposed for an additional 5 min (total irradiation time: 10 min). After a 24 h incubation, all wells were washed three times with PBS, followed by the addition of 100 µL of 10% Ez-Cytox solution prepared in fresh medium. The cells were then incubated for an additional 1 h, after which cell viability was determined as described above. 4.8 In vitro anticancer effect evaluation To examine the anticancer effect of the R848@MOF525, CT26 cells were seeded in a 96-well plate at a density of 1.0 × 10 4 cells/well and incubated for 24 h. The culture medium was replaced with 100 µL of a R848@MOF525 suspension at various concentration (50, 25, 10, 1 µg/mL). After 24 h of incubation, the medium was removed and the cells were washed with PBS. Subsequently, 100 µL of 10% Ez-Cytox solution prepared in fresh medium was added, and the plate was incubated for an additional 1 h. Cell viability was assessed using the microplate reader and calculated, as above. ATP levels were quantified using a colorimetric assay. CT26 cells were seeded at a density of 1 × 10⁶ cells in 500 µL of complete media per well in a 24-well treated plate and incubated overnight. The cells were then treated with MOF525 (50 µg/mL) followed by laser irradiation, and subsequently incubated overnight. ATP measurement was performed according to the manufacturer’s instructions using the PicoSens™ ATP Assay Kit (BIOMAX, Korea), and absorbance was recorded at 570 nm using a microplate reader. 4.9 In vitro BMDCs activation effect evaluation The mouse bone marrow progenitor cells were flushed from BALB/c femurs and tibias using a syringe and PBS with 2% fetal bovine serum. After removing the red blood cells with ACK lysis buffer, the collected cells were cultured in petri dishes in 10 mL of RPMI 1640 culture medium with 20 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF, Biolegend, USA). On day 6, proliferating immature DCs were harvested as non-adherent and loosely adherent clusters of cells using 2 mM ethylene-diamine-tetraacetic acid (EDTA). The DCs were seeded in non-treated 96-well plates at 1.0 × 10 5 cells/well and treated with MOF525 and R848@MOF525 (50, 25, and 10 µg/mL) or with LPS (100 ng/mL) as a positive control. After 24 hours of incubation, the BMDCs were labeled with fluorescence-conjugated antibodies, including zombie violet, CD11c-PerCP-Cy5.5, CD86-PE, CD40-PE-Cy7, and MHCII-FITC (Biolegend, USA), to analyze their maturation. The maturation of the BMDCs was then assessed using flow cytometry. 4.10 In vivo antitumor efficacy To evaluate the potential antitumor efficacy of R848@MOF525, Female 7 weeks Balb/C mice (Orient Bio, Korea) were subcutaneously inoculated with CT26 (5 × 10 5 cells) suspended in 100 µL of PBS into the right flank. All procedures, including tumor inoculation, were performed under isoflurane inhalation anesthesia. Tumor size was measured using digital calipers, and the volume was calculated as \(\:\:\text{L}\text{e}\text{n}\text{g}\text{t}\text{h}\:\times\:\:{\text{W}\text{i}\text{d}\text{t}\text{h}}^{2}\:\times\:\:0.5\) . When the tumor volume reached approximately 100 mm³, mice were randomly divided into six groups and treated with PBS, R848, PCN525, R848@MOF525, MOF525 + Laser, or R848@MOF525 + Laser, respectively. MOF525 and R848@MOF525 were dispersed in PBS at a concentration of 1 mg/mL, while R848 was diluted to 0.2 mg/mL. Each formulation was then injected intratumorally at a total volume of 100 µL. For the MOF525 + Laser and R848@MOF525 + Laser groups, a laser with 660nm wavelength and 600 mW power output was utilized. The mouse was irradiated for a duration of 5 min, followed by a resting period of 1 min, and then irradiated again for an additional 5 min, resulting in a total irradiation time of 10 min. After each treatment, the tumor size and body weight of the mouse were measured. After 40 days post-tumor inoculation, the control and R848 groups, as well as the non-laser-irradiated MOF525 and R848@MOF525 groups, were CO₂ euthanized, and their tumor tissues were extracted for histopathological analysis to assess anti-cancer efficacy. Additionally, for the MOF525 and R848@MOF525 groups that exhibited complete tumor regression after PDT, a re-challenge test was conducted by inoculating the same number of tumor cells into the opposite flank. Tumor growth was monitored for 3 weeks, and after confirming no tumor growth, the mice were euthanized, and their vital organs were extracted for biosafety evaluation. All animal experiments were conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Sungkyunkwan University College of Medicine (IACUC No. SKKU IACUC 2024-02-40-1). 4.11 Histopathological Evaluation Both tumor samples and vital organs were collected after animal sacrifice and stored in 10% neutralized buffered formalin until analysis. Samples were subjected to general paraffin embedding processing (dehydration/clearing/paraffin infiltration) and then prepared for paraffin blocks. 4 um of tissue sections of were deparaffinized, hydrated, and HE stained. Histopathological images were obtained by inverted light microscopy (Amscope, USA), and quantitative analysis of tumor size and necrotic lesions was performed using Image J software. TUNEL staining was performed according to the manufacturer's guideline (Abcam, USA). For TUNEL staining, slides were deparaffinized/hydrated, followed by endogenous peroxidase removal (H 2 O 2 in methanol) and pressurized heat antigen retrieval in citrate buffer before staining. TUNEL staining images were also obtained using inverted light microscopy, and quantitative analysis of TUNEL positive areas was performed using Image J software. 4.12 Statistical analysis Statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software, USA). Data are presented as mean ± standard deviation unless otherwise indicated in the figure legend. The number of samples in each group is specified in the figure legend. For analysis of 3 or more groups, one-way ANOVA followed by Tukey’s post hoc multiple comparison test was used, unless otherwise specified in the figure legend. A 95% confidence level ( p < 0.05) was considered statistically significant. Declarations Ethics approval and consent to participate This research received approval from the Ethics Committee of Sungkyunkwan University, and all procedures with animal models followed established ethical standards (IACUC No.: SKKU IACUC 2024-02-40-1). Consent for publication Consent for publication is not applicable. Availability of data and materials The authors declare that the data supporting the findings of this study are available within the paper and supplementary materials. Competing interests The authors Se-Na Kim , Nae Young Chun and Yu Jin Oh declare that patent applications related to the materials and methods described in this work have been filed in South Korea (Application No. 10-2023-0179991) and under the Patent Cooperation Treaty (PCT/KR2023/020618). Funding This research was supported by the Ministry of Science and ICT (MSIT) of the Republic of Korea, under the Information Technology Research Center support program, supervised by the Institute for Information & Communications Technology Planning & Evaluation (IITP-2023-RS-2023-00258971). This work was also supported and grant funded by Korea University Guro Hospital (KOREA RESEARCH-DRIVEN HOSPITAL) and Korea University (K2325651). Additionally, this work was supported by the Korea Institute of Science and Technology Program (Project No. 2E32351-23-130) and the National Research Foundation of Korea grant funded by the MSIT (RS-2023-00208913, RS-2024-00440714, RS-2025-25424498). Finally, this research was supported by the Bio&Medical Technology Development Program of the National Research Foundation funded by the MSIT (RS-2024-00508402). Authors’ contributions N.K.L., S.N.K., and C.G.P. designed the research; N.K.L., N.Y.C, Y.J.O., U.B., M.S.K, and W.J.J. performed the experiment; N.K.L. S.N.K., and H.J.K. analyzed the data; and N.K.L wrote the paper; T.E.P, W.P, S.N.K, and C.G.P revised the paper. Acknowledgements We extend our gratitude to the other contributors to this work. References Tian R, Ma H, Ye W, Li Y, Wang S, Zhang Z, et al. Se‐Containing MOF Coated Dual‐Fe‐Atom Nanozymes With Multi‐Enzyme Cascade Activities Protect Against Cerebral Ischemic Reperfusion Injury. Adv Funct Mater. 2022;32(36):2204025. Chen Z, Li Z, Tang N, Huang Y, Li S, Xu W, et al. Engineering Ultra‐Small Cerium‐Based Metal–Organic Frameworks Nanozymes for Efficient Antioxidative Treatment of Dry Eye Disease. Adv Funct Mater. 2023;34(6):2307569. Wang A, Walden M, Ettlinger R, Kiessling F, Gassensmith JJ, Lammers T, et al. Biomedical Metal-Organic Framework Materials: Perspectives and Challenges. Adv Funct Mater. 2024;34(43):2308589. 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Additional Declarations Competing interest reported. The authors Se-Na Kim, Nae Young Chun and Yu Jin Oh declare that patent applications related to the materials and methods described in this work have been filed in South Korea (Application No. 10-2023-0179991) and under the Patent Cooperation Treaty (PCT/KR2023/020618). Supplementary Files JournalofNanobiotechnologySupportingdataF.docx Supporting Information Supporting Information is available from the Wiley Online Library or from the author. GraphicalAbstract.jpg Scheme1.jpg Scheme 1. Schematic illustration for fabrication of R848@MOF525 and mechanism of synergistic effect of PDT and immunotherapy. MOF525 is a type of zirconium-based MOF that acts as both the PS and a carrier for the immune adjuvant R848. Upon laser irradiation of the R848@MOF525 injected into the tumor, ROS are generated, leading to tumor cell death and the formation of TAAs. Concurrently, R848 released from MOF525 matures DCs, enhancing the immune response. This synergistic effect between PDT and immunotherapy contributes to effective tumor treatment. Created with BioRender.com. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8452162","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":574225244,"identity":"3fca32e7-979b-40dc-b046-7f7e78890f7f","order_by":0,"name":"Na Kyeong Lee","email":"","orcid":"","institution":"Sungkyunkwan University","correspondingAuthor":false,"prefix":"","firstName":"Na","middleName":"Kyeong","lastName":"Lee","suffix":""},{"id":574225245,"identity":"4cf88be6-e4ed-4c00-9b93-7015e1306f74","order_by":1,"name":"Nea Young Chun","email":"","orcid":"","institution":"MediArk 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1","display":"","copyAsset":false,"role":"figure","size":106418,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of MOF525 and R848@MOF525. (A) Schematic illustration of MOF525 and R848@MOF525 fabrication. (B) PXRD patterns. (C) N\u003csub\u003e2\u003c/sub\u003e adsorption-desorption isotherms. (D) SEM images with corresponding size distribution histograms. (E) Zeta potential measurements. (F) FTIR spectra. The data collectively demonstrate the morphology, size, surface charge, and structural properties of MOF525 before and after R848 loading.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452162/v1/1f50b154530150c652e0d1fa.jpg"},{"id":100265231,"identity":"4590bb80-b7cb-491e-9dc2-7cc41281f9f5","added_by":"auto","created_at":"2026-01-14 18:08:57","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":78221,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e anticancer efficacy of MOF525 and R848@MOF525. (A) Drug release profiles (n=3). Inset: Detailed release kinetics within the first 24 hours; Main graph: Extended-release pattern over 11 days. (B) Viability of L929 cells exposed to varying concentrations of MOFs (n=6). (C) ROS generation induced by MOF525 and R848@MOF525 (n=3). (D) PDT effect of MOF525 on CT26 cells (n=5, \u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452162/v1/eebf45d532938b87ac11354e.jpg"},{"id":100372937,"identity":"f0603b5f-e67a-49f0-8edf-03e25fa5a9d2","added_by":"auto","created_at":"2026-01-16 08:13:25","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":141769,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e BMDCs activation by MOF525 and R848@MOF525. Expression levels of (A) CD86, (B) CD40, and (C) MHC II on DCs (n=5). \u003csup\u003e####\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001 compared to the non-treated group; \u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 compared to the LPS-treated group.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452162/v1/0e82b2ac8c123c3a5cf206f8.jpg"},{"id":100372896,"identity":"569dd23f-e4c3-4e0e-b8bc-fff95fac0ede","added_by":"auto","created_at":"2026-01-16 08:13:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":148658,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e antitumor efficacy of MOF525 and R848@MOF525 under laser irradiation. (A) Schematic of CT26 tumor model establishment and treatment timeline. (B) Tumor growth curves of mice from different treatment groups. Data are presented as mean ± standard error of the mean, analyzed by two-way ANOVA with Tukey’s multiple comparisons (n=5, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001). (C) \u003cem\u003eEx vivo\u003c/em\u003e images of excised tumors collected from each group at day 40 after treatment initiation. (D) Representative photographs of tumor sites from each treatment group at the first and final treatment time points\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452162/v1/3ad09640fc1fa2b9b50cbc9d.jpg"},{"id":100265227,"identity":"c1d7bb9b-88ea-4f9d-946c-305ac7963f3e","added_by":"auto","created_at":"2026-01-14 18:08:57","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":76178,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of PDT-induced therapeutic response under a 5-day treatment interval. (A) Schematic representation of the treatment schedule administered to CT26 tumor-bearing mice. (B) Average tumor volume profiles of MOF525+Laser and R848@MOF525+Laser groups. Data are presented as mean ± standard error of the mean, analyzed by two-way ANOVA with Tukey’s multiple comparisons (n=4, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001). (C) Individual tumor growth curves of mice in each treatment group.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452162/v1/907a3751a4fab9f3873fee0d.jpg"},{"id":100265239,"identity":"6bea150d-a86d-4129-9084-86d5b157c7a2","added_by":"auto","created_at":"2026-01-14 18:08:57","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":178254,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological analysis of CT26 tumors treated with R848@MOF525. (A) Representative whole-mount tumor images. Scale bar: 2 mm. (B) Quantification of microscopic tumor areas. (C) H\u0026amp;E stained tumor sections (200× magnification) showing central necrosis, inflammatory infiltration (middle), and active tumor proliferation (periphery). Scale bar: 100 μm. (D) Quantitative analysis of necrotic lesions in tumor areas across experimental groups. (n=5, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; \u003csup\u003e*\u003c/sup\u003ecompared to control, \u003csup\u003e#\u003c/sup\u003ecompared to R848, \u003csup\u003e§\u003c/sup\u003ecompared to MOF525) (E) Representative TUNEL staining images showing 1) central necrosis, 2) middle, and 3) peripheral regions. Scale bar: 50 μm. (F) Quantification of TUNEL-positive cells. (n=3, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; \u003csup\u003e*\u003c/sup\u003ecompared to control, \u003csup\u003e#\u003c/sup\u003ecompared to R848, \u003csup\u003e§\u003c/sup\u003ecompared to MOF525).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452162/v1/d1e605e4639f7a43b4e9aded.jpg"},{"id":100910112,"identity":"6ea1f098-ba70-45d1-9e3b-01d31326f41a","added_by":"auto","created_at":"2026-01-22 16:41:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1720955,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8452162/v1/b7767c54-c2a0-42c8-94fa-f6ed55bd5264.pdf"},{"id":100265256,"identity":"2084bad0-bdf4-4ae0-a0d1-3c43e9cdbfc5","added_by":"auto","created_at":"2026-01-14 18:08:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12829485,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupporting Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupporting Information is available from the Wiley Online Library or from the author.\u003c/p\u003e","description":"","filename":"JournalofNanobiotechnologySupportingdataF.docx","url":"https://assets-eu.researchsquare.com/files/rs-8452162/v1/5a40c83d0a1a2899a869cafb.docx"},{"id":100371100,"identity":"0cb3d58f-79c9-4b07-b38c-562498fab04f","added_by":"auto","created_at":"2026-01-16 08:09:25","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":72728,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452162/v1/ab7fa951a4493c0a85e314d0.jpg"},{"id":100265228,"identity":"3d31d93e-4dcc-49c7-b6e4-5a29693d6c07","added_by":"auto","created_at":"2026-01-14 18:08:57","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":113347,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1. Schematic illustration for fabrication of R848@MOF525 and mechanism of synergistic effect of PDT and immunotherapy. MOF525 is a type of zirconium-based MOF that acts as both the PS and a carrier for the immune adjuvant R848. Upon laser irradiation of the R848@MOF525 injected into the tumor, ROS are generated, leading to tumor cell death and the formation of TAAs. Concurrently, R848 released from MOF525 matures DCs, enhancing the immune response. This synergistic effect between PDT and immunotherapy contributes to effective tumor treatment. Created with BioRender.com.\u003c/p\u003e","description":"","filename":"Scheme1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8452162/v1/738a8138e55d66ab5edc8ab9.jpg"}],"financialInterests":"Competing interest reported. The authors Se-Na Kim, Nae Young Chun and Yu Jin Oh declare that patent applications related to the materials and methods described in this work have been filed in South Korea (Application No. 10-2023-0179991) and under the Patent Cooperation Treaty (PCT/KR2023/020618).","formattedTitle":"Resiquimod-encapsulated MOF525 for Sustained Immunostimulant Release and Synergistic Photodynamic– Immunotherapy in Colorectal Cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMetal-Organic Frameworks (MOFs) have emerged as a novel and highly versatile class of materials with extensive applications in biomedical fields [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], particularly in cancer therapy [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. MOFs are crystalline structures composed of metal ions coordinated to organic ligands [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The distinct connectivity and rotation angles of metal ion and organic ligands lead to various topologies of MOFs creating porous materials [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, experimental synthesis factors such as temperature, reaction time, concentration, and others contribute to variations in morphology [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Furthermore, due to the porosity of MOFs, the empty spaces permit interactions with external molecules, resulting in outstanding applications for transport [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These properties are particularly advantageous in drug delivery systems.\u003c/p\u003e \u003cp\u003eMoreover, MOFs can be designed with photoactive organic ligands, which enable their use in photodynamic therapy (PDT) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Among these, porphyrin-based ligands have drawn significant attention due to their intrinsic photodynamic activity. Porphyrin photosensitizer (PS) can be activated by specific wavelengths of light to generate reactive oxygen species (ROS), which induce apoptosis by targeting cellular organelles such as mitochondria [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Their ability to preferentially accumulate in tumor tissues and efficiently produce ROS upon irradiation makes them particularly well-suited for PDT [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. By incorporating porphyrins as organic linkers within the MOF framework, these materials can inherit the photodynamic properties of porphyrins while also offering structural benefits such as high surface area, tunable porosity, and the capacity to load additional therapeutic agents. This dual functionality allows porphyrinic MOFs to serve both as efficient PS and as drug delivery vehicles, enhancing the potential of combination therapies [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These unique and tunable physical and chemical properties make MOFs exceptional candidates for drug delivery systems and PDT, where the efficient generation of ROS is crucial for inducing localized cell death. MOFs can also be engineered to encapsulate therapeutic agents, enhancing the efficacy of combination therapies [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the significant advancements in the application of MOFs for cancer therapy, their potential in treating specific types of cancer, such as colorectal cancer (CRC), is garnering considerable interest [\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. CRC stands as the second leading cause of cancer-related deaths globally [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. According to estimates from 2020, there were over 1.9\u0026nbsp;million new cases of CRC worldwide, resulting in more than 930,000 deaths attributed to the disease [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Traditional treatments such as surgery, chemotherapy, and radiotherapy remain the standard of care for CRC; however, PDT provides distinct therapeutic advantages for CRC treatment. In particular, PDT can be implemented through a minimally invasive approach using endoscopic guidance [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], which enables site-specific light delivery to colorectal lesions while minimizing systemic toxicity. This strategy allows selective tumor ablation, preserves adjacent healthy tissues, and permits repeated administration without cumulative dose limitations, underscoring its potential role as a complementary or alternative therapeutic modality in CRC. Consequently, PDT has attracted considerable attention as a promising treatment strategy that employs PS, light, and oxygen to generate ROS, leading to targeted tumor cell destruction [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent advances in cancer therapy highlight the potential of combining PDT with other treatment modalities to enhance overall efficacy[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. While PDT achieves localized tumor ablation through ROS generation, its efficacy is often limited by the immunosuppressive tumor microenvironment and the possibility of tumor recurrence [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Immunotherapy, on the other hand, requires adequate exposure to tumor-associated antigens (TAAs) to mount a robust immune response, which may be insufficient in poorly immunogenic tumors [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. PDT-induced immunogenic cell death provides a critical bridge by releasing TAAs and danger-associated molecular patterns, thereby enhancing antigen presentation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. When integrated with immunotherapy, these signals can be exploited to potentiate adaptive immune responses, sustain long-term tumor control, and overcome immune evasion [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Consequently, the rationale for combining PDT with immunotherapy lies in their complementary mechanisms: PDT initiates antigen release, while immunotherapy amplifies and prolongs antitumor immunity, ultimately achieving more durable therapeutic outcomes [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong various immunotherapeutic strategies, the use of immune adjuvants has emerged as an effective approach to stimulate the body\u0026rsquo;s immune response and enhance its reaction to tumor-associated antigens [\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Toll-like receptor (TLR) agonists, particularly Resiquimod (R848), have shown promise due to their ability to activate innate immune cells and promote adaptive immune responses [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. TLRs are pattern recognition receptors that detect pathogen-associated molecular patterns and activate antigen-presenting cells (APCs), thereby inducing pro-inflammatory cytokine secretion and recruiting immune cells [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. By engaging TLR7/8 signaling, R848 induces dendritic cell (DC) maturation and bridges innate and adaptive immunity, enhancing the antitumor immune response [\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Nonetheless, systemic administration of R848 poses challenges, including poor solubility and uncontrolled cytokine release, which may lead to severe side effects [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Combining PDT with R848 immunotherapy therefore offers several advantages compared to using each modality alone. PDT induces immunogenic cell death, releasing TAAs and danger-associated molecular patterns, while R848 can overcome tumor-induced immune suppression and potentiate the immune response initiated by PDT [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan additionalcitationids=\"CR56 CR57\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Exploiting both mechanisms simultaneously provides a more comprehensive approach to tumor eradication, offering a promising strategy for effective cancer treatment.\u003c/p\u003e \u003cp\u003eA porphyrin-based MOF, MOF525, offers a dual-functional platform for PDT and immunotherapy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among various porphyrin-based MOFs, MOF525 exhibits high photoreactivity, enabling efficient ROS generation upon light exposure to induce tumor cell death and release tumor-associated antigens [\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. This coordination supports the successful incorporation of R848 and its potential stabilization within the MOF525 structure through metal\u0026ndash;ligand interactions through Lewis-type metal\u0026ndash;ligand interactions between the Lewis acidic Zr⁴⁺ centers and the Lewis basic nitrogen atoms of R848, which are weak and reversible under aqueous or physiological conditions, allowing for subsequent drug release., its porous structure allows for the encapsulation and sustained release of R848, enhancing the antitumor immune response and overcoming the limitations of systemic administration. This combination achieves a synergistic effect with PDT, further improving therapeutic efficacy.\u003c/p\u003e \u003cp\u003eIn this study, we investigate the potential of R848-loaded MOF525 (R848@MOF525) for combined PDT and immunotherapy in CRC treatment (\u003cb\u003eScheme 1\u003c/b\u003e). We hypothesize that the synergistic effects of PDT and immunotherapy mediated by R848@MOF525 will result in enhanced antitumor efficacy, providing a novel therapeutic approach for CRC. Our comprehensive \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e analyses demonstrate the ability of R848@MOF525 to significantly inhibit tumor growth and induce robust immune responses, highlighting its promise as an advanced multifunctional therapeutic platform.\u003c/p\u003e \u003cp\u003e \u003cb\u003eScheme 1.\u003c/b\u003e Schematic illustration for fabrication of R848@MOF525 and mechanism of synergistic effect of PDT and immunotherapy. MOF525 is a type of zirconium-based MOF that acts as both the PS and a carrier for the immune adjuvant R848. Upon laser irradiation of the R848@MOF525 injected into the tumor, ROS are generated, leading to tumor cell death and the formation of TAAs. Concurrently, R848 released from MOF525 matures DCs, enhancing the immune response. This synergistic effect between PDT and immunotherapy contributes to effective tumor treatment. Created with BioRender.com.\u003c/p\u003e"},{"header":"2. Result and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Characterization of MOF525 and R848@MOF525\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this study, we proposed porphyrin based MOFs, specifically MOF525, as PS and carriers for the immune adjuvant R848. MOF525, synthesized through the solvothermal method, is composed of zirconium (Zr) as the inorganic building unit and Tetrakis(4-carboxyphenyl)porphyrin (TCPP) as the organic building unit. Moreover, R848 was loaded into the pores of MOF525 through coordination bonding, resulting in the formation of R848@MOF525 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The Powder X-ray diffraction (PXRD) patterns clearly show the structure and crystallinity of MOF525, which were maintained after R848 loading (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) (62). The N\u003csub\u003e2\u003c/sub\u003e adsorption (ADS)-desorption (DES) isotherms of MOF525 and R848@MOF525 indicate that both materials displayed type I pattern isotherms. This confirms the preservation of their microporous structure before and after loading the compound (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The BET analysis revealed a surface area of 1356.3 m\u0026sup2; g⁻\u0026sup1; and a pore volume of 0.98 cm\u0026sup3; g⁻\u0026sup1; for pristine MOF525, which decreased to 80.2 m\u0026sup2; g⁻\u0026sup1; and 0.62 cm\u0026sup3; g⁻\u0026sup1; for R848@MOF525, respectively. This reduction confirms the occupation of internal pores by R848 molecules. Furthermore, the apparent increase in average pore diameter from 2.88 nm to 15.26 nm is not due to physical expansion of the framework but rather to a shift in the pore distribution. The partial occupation of micropores by R848 blocks nitrogen adsorption in smaller pores, resulting in a relative increase in the contribution of mesopores during BET/BJH analysis [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eScanning electron microscopy (SEM) images exhibited that MOF525 has cuboctahedral-shaped, as reported previously (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. This overall shape did not change after R848 loading. Moreover, the size and zeta potential of the MOFs suspended in deionized water were measured by dynamic light scattering. The average sizes of MOF525 and R848@MOF525 were measured as 320.9 nm and 337 nm, respectively, indicating an increase in size following the loading of R848 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. The zeta potentials of MOF525 and R848@MOF525 were \u0026minus;\u0026thinsp;12.3 mV and \u0026minus;\u0026thinsp;9.5 mV, respectively, indicating that they are relatively similar (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. Next, the colloidal stability of MOF525 was evaluated under serum-containing conditions. The hydrodynamic diameter increased only slightly during the incubation period, and no abrupt changes indicative of aggregation were observed (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e in supporting data). This gradual size change and stable PDI profile demonstrate that MOF525 remains colloidally stable even under physiologically relevant conditions.\u003c/p\u003e \u003cp\u003eIn the Fourier transform infrared (FTIR) spectra analysis of MOF525, a notable feature is observed in the peaks at 1600, 1410, and 660 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. These peaks signify the asymmetric and symmetric stretching of the carboxylate linker and the stretching mode of the metal-ligand bonding, respectively, within the Zr-porphyrin MOFs [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. For R848, the peak at 3112 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is related to =\u0026thinsp;C-H bond stretching vibration and the peak at 2975 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e could be attributed to stretching of -C-H bonds. For R848@MOF525, the characteristic signals observed from MOF525 showed no discernible shift, and the signals from R848 were retained. This indicates that the chemical structures remained unaltered following encapsulation and the MOF525 and R848 coexist (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). To examine whether R848 interacts with the open metal sites in MOF525, we performed X-ray photoelectron spectroscopy (XPS) analysis of the Zr 3d region before and after drug loading (\u003cb\u003eFigure S2\u003c/b\u003e in supporting data). In the pristine MOF525, the Zr 3d spectrum displayed characteristic peaks corresponding to Zr\u0026ndash;O coordination [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Upon R848 loading, a new signal emerged that is attributable to O\u0026ndash;Zr\u0026ndash;N bonding, indicating that the nitrogen-containing functional group of R848 likely coordinates to the unsaturated Zr sites within the MOF525 framework [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. This coordination supports the successful incorporation of R848 and its potential stabilization within the MOF525 structure through metal\u0026ndash;ligand interactions through Lewis-type metal\u0026ndash;ligand interactions between the Lewis acidic Zr⁴⁺ centers and the Lewis basic nitrogen atoms of R848, which are weak and reversible under aqueous or physiological conditions, allowing for subsequent drug release [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. These changes confirm that R848 molecules occupy part of the internal pores and simultaneously coordinate with surface Zr sites, indicating a coexistence of pore encapsulation and surface binding within the MOF525 structure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 \u003cem\u003eIn vitro\u003c/em\u003e anticancer effect evaluation\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe examined \u003cem\u003ein vitro\u003c/em\u003e release profile of R848 in phosphate buffered saline (PBS) at pH 7.4 and 37 ℃ with shaking. The encapsulation efficiency was determined after loading 20 mg of R848 into 100 mg of MOF525, resulting in approximately 44 \u0026micro;g of R848 loaded per 1 mg of MOF525. R848 was released in a sustained manner, with the majority of the payload gradually released over 11 days \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), suggesting that coordination-mediated interactions do not prevent R848 diffusion under physiological ionic conditions, likely due to competitive coordination from phosphate ions and water molecules. The \u003cem\u003ein vitro\u003c/em\u003e cell cytotoxicity of MOF525 and R848@MOF525 was assessed using mouse fibroblast cells, L929 which are recommended by ISO 10993-5 as a standard for biocompatibility evaluation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Both types of MOFs show dose-dependent cytotoxicity toward L929. Moreover, concentrations of MOF525 and R848@MOF525 that resulted in cell survival rates of 80% is 50 \u0026micro;g/mL, which is considered non-toxic. Consequently, a dose of 50 \u0026micro;g/mL was chosen for further \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments.\u003c/p\u003e \u003cp\u003eWe next evaluated whether MOF525-mediated photostimulation could induce immunogenic stress responses in tumor cells. Extracellular ATP, a representative damage-associated molecular patterns (DAMP) marker, was quantified in CT26 cells under four different conditions (non-treated, laser irradiation only, MOF525 only, and MOF525 combined with laser irradiation). Among these groups, MOF525 with laser irradiation induced the highest ATP release, whereas the other groups showed minimal changes (\u003cb\u003eFigure S3\u003c/b\u003e in supporting data). These results indicate that MOF525 enhances ATP-associated DAMP signaling when combined with laser activation.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e ROS generation and the anti-cancer effect were assessed under light exposure. Following the mixing of two types of MOFs with a singlet oxygen sensor green (SOSG) solution and subsequent irradiation with a laser for a total of 10 min, the intensity of SOSG gradually increased, indicating ROS generation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Moreover, treatment of CT26 cells with either MOF525 or R848@MOF525 combined with laser irradiation for the same cumulative duration resulted in higher anticancer efficacy compared to the control, control\u0026thinsp;+\u0026thinsp;Laser, and R848\u0026thinsp;+\u0026thinsp;Laser groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These findings suggest that the PS of MOF525 induce cell death by releasing ROS upon laser irradiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the activation of DCs by MOF525 and R848@MOF525, we co-incubated MOF with bone marrow-derived dendritic cells (BMDCs) differentiated from mouse bone marrow cells and analyzed cell markers using flow cytometry. BMDCs were gated on FSC/SSC parameters, followed by singlet discrimination (FSC-H/FSC-A) and live/dead exclusion, and finally on CD11c⁺ cells (\u003cb\u003eFigure S4\u003c/b\u003e in supporting information). Additionally, for the group treated with R848 alone, the amount of R848 was based on the highest concentration used for R848@MOF525 treatment. The expression of CD86 in BMDCs treated with the highest concentrations of MOF525 and R848@MOF525 was approximately 1.75 times higher than that in the lipopolysaccharide (LPS)-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) At lower concentrations, CD86 expression gradually decreased but still remained consistently higher than that observed in the LPS group (\u003cb\u003eFigure S5\u003c/b\u003e, Supporting Information). The expression of CD40 increased up to 3.37 times compared to the untreated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), with increased expression even at the lowest treatment concentration (10 \u0026micro;g/mL) (\u003cb\u003eFigure S6\u003c/b\u003e in supporting information). Lastly, except for the lowest treatment concentration, the expression of major histocompatibility complex class II (MHCII) was over 2.38 times higher in the MOF-treated group than in the untreated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cb\u003eFigure S7\u003c/b\u003e in supporting data). Moreover, at concentrations above 10 \u0026micro;g/mL, there was little difference in the expression of maturation markers between MOF525 and R848@MOF525, indicating that MOF525 alone was sufficient to induce BMDC maturation at higher doses. This effect may arise from the adjuvant-like property of Zr⁴⁺ within the MOF framework, which can stimulate innate immune responses and promote dendritic cell maturation, consistent with previous reports [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. In contrast, at concentrations below 10 \u0026micro;g/mL, R848@MOF525 induced higher CD40 expression compared to MOF525, suggesting that the presence of R848 confers an additional stimulatory effect under submaximal dosing conditions (\u003cb\u003eFigure S8\u003c/b\u003e in supporting information). DCs play a crucial role in regulating the immune system by mediating both innate and adaptive immunity as APCs [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Immature DCs differentiated from monocytes engulf pathogens or antigens from the external environment through phagocytosis [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. They digest them and present the resulting fragments on the surface through the MHCII [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Simultaneously, upon recognition of pathogen-associated molecular patterns such as LPS or R848 via TLRs, DCs undergo activation and maturation [\u003cspan additionalcitationids=\"CR76 CR77\" citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. One of the differences between immature and mature DCs lies in their phenotypical characteristics; mature DCs have up-regulated surface expression of costimulatory molecules such as CD86, CD40, and MHCII compared to immature DCs [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Mature DCs bind antigens and costimulatory molecules presented on their surface to receptors on adaptive immune cells such as T cells, inducing their activation and enabling them to destroy pathogens or infected cells [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. Therefore, the increase in these markers suggests that R848@MOF525 may not only activate DCs through sustained release of R848 but also induce DC activation by MOF525 itself.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 \u003cem\u003eIn vivo\u003c/em\u003e anticancer effect evaluation\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further examined the \u003cem\u003ein vivo\u003c/em\u003e anticancer efficiency of R848@MOF525. The therapeutic efficacy of R848@MOF525 was evaluated using a CT26 colon cancer bearing mice model. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Additionally, to assess the therapeutic efficacy of R848@MOFs, groups were included that did not receive laser irradiation, as well as groups that received as the same amount of R848 loaded onto the MOFs and the same volume of PBS. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D, while the tumor growth inhibition effect without laser irradiation was similar to the control group in the case of MOF525, R848@MOF525 showed a relatively inhibited tumor growth rate, to the sustained release of R848 at the tumor site. In the MOF525\u0026thinsp;+\u0026thinsp;Laser and R848@MOF525\u0026thinsp;+\u0026thinsp;Laser groups, tumor regression occurred over a period of 40 days after tumor inoculation. However, in the R848@MOF525\u0026thinsp;+\u0026thinsp;Laser group, all mice exhibited complete tumor regression by day 25, whereas in the MOF525\u0026thinsp;+\u0026thinsp;Laser group, complete tumor regression was observed by day 35, indicating a difference in the rate of tumor regression due to the incorporation of R848.Additionally, 60 days after the first tumor inoculation, the same amount of tumor cells was inoculated on the opposite flank to evaluate the long-term immune memory and protective effects induced by the treatment. As a result, no tumors regrew, indicating that the initial treatment not only eliminates the primary tumor but also establishes a systemic immune response capable of recognizing and attacking tumor cells if they reappear (\u003cb\u003eFigure S9\u003c/b\u003e in supporting information).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further validate the synergistic contribution of R848 in the PDT-based treatment, we conducted an additional \u003cem\u003ein vivo\u003c/em\u003e study using an extended treatment interval of 5 days, which was intended to moderate the overall therapeutic intensity and better reveal any potential immunological enhancement (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In this experiment, only the MOF525\u0026thinsp;+\u0026thinsp;Laser and R848@MOF525\u0026thinsp;+\u0026thinsp;Laser groups were compared. Under this milder treatment schedule, the R848@MOF525\u0026thinsp;+\u0026thinsp;Laser group exhibited greater tumor growth suppression compared to the MOF525\u0026thinsp;+\u0026thinsp;Laser group, particularly after the third treatment session (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). This result highlights the immune-stimulatory role of R848 in enhancing the therapeutic efficacy of PDT, which may have been underrepresented under the more intensive 3-day treatment regimen due to rapid tumor clearance. These findings further support the hypothesis that R848-mediated immunomodulation acts synergistically with PDT-induced tumor antigen release, facilitating more robust and durable antitumor responses.\u003c/p\u003e \u003cp\u003eWhile both MOF525\u0026thinsp;+\u0026thinsp;Laser and R848@MOF525\u0026thinsp;+\u0026thinsp;Laser groups exhibited complete protection upon tumor rechallenge, indicating that PDT-induced immunogenic cell death alone is sufficient to establish durable antitumor memory, the inclusion of R848 resulted in more rapid tumor regression and enhanced suppression during the active treatment phase, particularly under the extended 5-day interval. This moderated treatment schedule allowed clearer observation of R848\u0026rsquo;s immunostimulatory contribution, which may have been obscured under the more intensive 3-day regimen due to rapid tumor elimination. These findings suggest that although PDT alone can induce long-term immune protection, co-delivery of R848 can accelerate and amplify early immune priming, potentially offering therapeutic advantages in tumors with higher proliferation rates or stronger immunosuppressive environments. Furthermore, a less frequent treatment schedule may better balance direct tumor cytotoxicity with systemic immune activation, minimizing treatment-associated stress while preserving or enhancing overall therapeutic efficacy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Anti-cancer efficacy evaluation using a histopathological examination\u003c/h2\u003e \u003cp\u003eThe experimental groups were necropsied on day 40 after tumor inoculation, the point at which tumors had completely disappeared in the laser-treated groups. Tumors remaining in the control, R848, non-laser-treated MOF525, and R848@MOF525 groups were collected and fixed in 10% neutralized buffered formalin for further histopathological evaluation. Tumor samples were subjected to a general paraffin embedding procedure, followed by hematoxylin and eosin (H\u0026amp;E) staining. To assess the overall size and cellular composition of the tumor, the tumor slide was scanned and a whole mount image was collected (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The tumors were found to be larger in the order of control, MOF525, R848, and R848@MOF525 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). A necrotic lesion was present in the central area of all tumor groups, and the MOF525 had a wider necrotic lesion compared to the R848. The large necrotic lesions observed in MOF525 may have been a factor in increasing tumor size over the actual tumor lesions when measuring tumor size. The R848@MOF525 group also had smaller tumor sizes overall, with smaller foci of necrosis found throughout the tumor in addition to the central necrotic lesion.\u003c/p\u003e \u003cp\u003eAt higher magnification, necrotic lesions composed of amorphous necrotic material were observed in all groups, with a large number of inflammatory cells infiltrating around these necrotic lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Furthermore, while the inflammatory cell infiltration was not prominent in the proliferating part of the tumor in the control group, the treatment groups (R848, MOF525, and R848@MOF525) had varying degrees of inflammatory cell infiltration. In particular, R848@MOF525 showed the formation of small necrotic foci with scattered inflammatory cell infiltration. Compared to MOF525, more mononuclear cell infiltration was observed in middle and periphery lesion of R848. In the quantitative analysis of necrotic lesion (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), it was observed that the relative necrotic lesion (necrotic lesion: tumor area) was increased in the R848@MOF525 and MOF525 at a statistically significant level compared to the control group. However, no statistical significance was observed between MOF525 and R848. The R848@MOF525 group showed more relative necrotic lesions than R848 and MOF525 as well as the control. This may be due to the smaller tumor size of the R848@MOF525 group, but also the inclusion of central necrotic lesions and sporadic necrotic lesions, resulting in an increase in relative necrotic foci.\u003c/p\u003e \u003cp\u003eOn TUNEL staining to evaluate the extent of apoptotic cell death in tumor, central necrosis in all groups was observed to be characterized by the presence of multiple TUNEL positive cells (Orange to brown color). In particular, the R848 and R848@MOF525 were observed to have multiple TUNEL positive apoptotic cell death foci in the area between the central necrosis and the periphery. In the quantitative evaluation analysis of TUNEL positive area (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), TUNEL positive area was observed to be statistically significantly higher in the R848@MOF525 group than in any other group. A statistically significant increase in TUNEL positive area was also observed in the R848 and MOF525 groups compared to the control group, but the difference between the two groups did not reach statistical significance. According to our results, it is suggested that R848@MOF525 exhibits MOF525-based necrotizing lesion induction ability and R848-based inflammatory cell infiltration and apoptotic cell death induction ability, may indicating overall improved tumor treatment efficacy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 \u003cem\u003eIn vivo\u003c/em\u003e biosafety evaluation\u003c/h2\u003e \u003cp\u003eTo analyze the toxicity of vital organs of MOF525\u0026thinsp;+\u0026thinsp;Laser and R848@MOF525\u0026thinsp;+\u0026thinsp;Laser groups, we harvested heart, lung, liver, kidney, and spleen for toxico-pathological analysis 3 weeks after tumor rechallenge test (\u003cb\u003eFigure S10 in supporting data\u003c/b\u003e). Analyzing the abnormalities of vital organs in three animals from each group, we observed mild proliferation of pulmonary interstitial tissue, mild increase in the number of reactive hepatocytes in the liver, and mild increase in the size of splenic nodules in some individuals. However, these changes were considered to be mild side effects of the body's response to the repetitive tumor cell transplants and subsequent tumor cell therapy, and there was no significant damage to vital organs. These results demonstrated that our MOF525 and R848@MOF525 may be not only effective but also safe for the treatment of recurrent tumor lesion. Furthermore, there was no significant decrease in mouse body weight observed in any of the groups, indicating that MOFs administration was not toxic to the animals (\u003cb\u003eFigure S11\u003c/b\u003e in supporting data\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003eThis study demonstrates the potential of MOF-525 as an immunotherapeutic cargo\u0026ndash;loaded nanoplatform with sustained release capability for the treatment of colorectal cancer (CRC). We developed an R848-encapsulated MOF-525 nanoplatform designed to integrate photodynamic therapy (PDT) with innate immune activation, enabling a synergistic antitumor strategy within a single nanoscale system. The highly porous and photoreactive architecture of MOF-525 facilitated efficient encapsulation and sustained release of the TLR7/8 agonist R848, while simultaneously enabling robust reactive oxygen species (ROS) generation upon light irradiation for direct tumor cell destruction.\u003c/p\u003e \u003cp\u003eIn vitro and in vivo experiments confirmed that the MOF-525 nanoplatform functions as a dual-functional nanotherapeutic system, capable of sustained immunotherapeutic agent delivery and photodynamically induced tumor ablation. Upon laser irradiation, R848@MOF-525 generated ROS, leading to localized tumor cell necrosis, while the gradual release of R848 promoted antigen-presenting cell activation within the tumor microenvironment. This coordinated nano-enabled delivery of photodynamic and immunostimulatory functions resulted in a pronounced synergistic effect between PDT and immunotherapy, as evidenced by enhanced tumor regression that correlated with R848 loading levels.\u003c/p\u003e \u003cp\u003eNotably, this synergistic therapeutic benefit was more clearly revealed under a less frequent treatment regimen, in which PDT was administered at five-day intervals rather than the conventional three-day schedule. Under this milder dosing paradigm, the immunological contribution of sustained R848 release became more distinguishable, highlighting the advantage of nano-controlled immunotherapeutic delivery in achieving durable antitumor responses. Collectively, these findings underscore the promise of R848-loaded MOF-525 as a multifunctional nanoplatform for sustained immunotherapy and photodynamic tumor ablation, offering a clinically relevant and well-tolerated strategy for CRC treatment that leverages the unique capabilities of metal\u0026ndash;organic framework\u0026ndash;based nanomedicine.\u003c/p\u003e"},{"header":"4. Materials and Method","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Materials\u003c/h2\u003e \u003cp\u003eBenzoic acid (assay\u0026thinsp;=\u0026thinsp;99.0%), TCPP (assay\u0026thinsp;=\u0026thinsp;97.0%), R848 (assay\u0026thinsp;=\u0026thinsp;98.0%) were purchased from TCI (Japan). Dimethylformamide (DMF, assay\u0026thinsp;=\u0026thinsp;99.5%) was purchased from DAEJUNG (Korea). Zirconyl chloride octahydrate (ZrOCl\u003csub\u003e2\u003c/sub\u003eㆍ8H\u003csub\u003e2\u003c/sub\u003eO) and LPS (\u003cem\u003eE. coli\u003c/em\u003e O55:B5) were purchased from Simga-Aldrich (USA). Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM), RPMI 1640 Medium (RPMI), PBS (pH 7.4) were purchased from Thermo Fisher Scientific (USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Preparation of R848@MOF525\u003c/h2\u003e \u003cp\u003eMOF525 was synthesized following a previously reported protocol with slight modifications [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. Briefly, TCPP (235 mg, 0.297 mmol) and benzoic acid (6750 mg, 55 mmol) were dissolved in 40 mL DMF and sonicated for 30 min. Then, ZrOCl\u003csub\u003e2\u003c/sub\u003eㆍ8H\u003csub\u003e2\u003c/sub\u003eO (525 mg, 1.62 mmol) was added to the TCPP solution, and subsequently dissolved for 30 min. Finally, the resulting solution was kept in an oven at 100℃ for 24 hours. The resultant products were collected by centrifugation and washed three times with DMF and ethanol to remove excess reactants, followed by drying at 80℃ for 6 hours. Subsequently, MOF525 was then placed under a vacuum oven at 150℃ to clear the pores as remove residual solvent.\u003c/p\u003e \u003cp\u003eTo prepare the R848@MOF525, 20 mg R848 was first dissolved and sonicated in 2 mL ethanol, to which 100 mg of the dehydrated MOF525 added. The suspension was kept under RT for 24 h. Then, the resulting suspension was filtered using a 200 nm PVDF membrane filter, washed with ethanol thrice, and dried at 70℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Characterization\u003c/h2\u003e \u003cp\u003ePXRD patterns of MOF525 and R848@MOF525 were recorded using and X-ray diffractometer (SmartLab, Rigaku, Japan) at 3 kW with CuKα radiation. The surface area, pore size, and pore volume were obtained by a surface area and porosity analyzer (Autosorb IQ, Quanmtachrome, USA) at -196℃. The morphology and size of MOF525 and R848@MOF525 were characterized using a SEM (Dimension ICON, Bruker, USA). The FTIR spectra of MOF525 and R848@MOF525 were determined using a FTIR spectrophotometer (Cary670 (Main bench)\u0026thinsp;+\u0026thinsp;Cary620 (microsocope)), Agilent, USA) over the range of 4000 to 500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at room temperature. The hydrodynamic size and zeta potential were measured by DLS (Zetasizer pro, Malvern Panalytical, UK) and serum stability was evaluated in PBS containing 5% FBS. The XPS spectra of MOF525 and R848@MOF525 were obtained using an X-Ray Photoelectron Spectroscopy System (PHI Quantera-Ⅱ, Ulvac-PHI, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.4 \u003cem\u003eIn vitro\u003c/em\u003e drug release profile\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e drug release was confirmed by reverse phase high-performance liquid chromatography (1260 Infinity Ⅱ, Agilent, USA). R848@MOF525 was dispersed in PBS at a concentration of 1 mg/mL, and 5 mL of this dispersion was dialyzed using SnakeSkin\u0026reg; dialysis tubing (MWCO: 3500, Thermo Fisher Scienticfic, USA) against 20 mL of same buffer at 37℃ with shaking. At each time point, the PBS outside the tube was recovered and replaced with fresh PBS. To quantify the initial drug loading on the MOF, R848@MOF525 was dispersed in PBS at a concentration of 0.5 mg/mL and sonicated for 3 hours to extract the encapsulated drug. For each time point and loading quantification, the obtained samples were centrifuged at 17,000 rpm for 10 minutes, and the supernatant was injected into the HPLC. The mobile phase consisted of 20 mM PBS containing 60% phosphoric acid (pH 2.5) and 40% acetonitrile, with a flow rate of 1.0 mL/min. R848 was detected at 249 nm, and the injection volume was 10 \u0026micro;L.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.5 \u003cem\u003eIn vitro\u003c/em\u003e study cytotoxicity evaluation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e cytotoxicity was evaluated using a water-soluble tetrazolium salt-based cell viability assay (EZ-Cytox; Daeil Lab Service, Korea) in L929 mouse fibroblast cells. L929 cells were seeded at a density of 0.9 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well in a 96-well plates and incubated at 37℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e for 1 day. The medium was completely removed and replaced with the MOF525 or R848@MOF525 suspension prepared in fresh cell culture medium at varying concentration (50, 25, 10 and 1 \u0026micro;g/mL) and incubated for another 24 h. The cells were washed three times with PBS. After that, EZ-Cytox solution was added to the medium at 10% volume and the cell incubated for 1 h. The absorbance was measured at 450 nm with a reference wavelength of 600 nm, using a microplate reader (Synergy H1, BioTek, USA). The cell viability was calculated by using the following equation:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Cell\\:vaibility\\:\\left(\\%\\right)=\\:\\frac{Absobance\\:of\\:treated\\:cells-Absorbance\\:of\\:media}{Absorbance\\:of\\:negative\\:control\\:cells-Absorbance\\:of\\:media}\\:\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.6 \u003cem\u003eIn vitro\u003c/em\u003e study on ROS generation\u003c/h2\u003e \u003cp\u003eTo measure the degree of ROS generation of nanoparticle, Singlet Oxygen Sensor Green (SOSG; Thermo Fisher Scienfitic, USA) was used. Briefly, suspensions of MOF525 in medium at 25 and 50 \u0026micro;g/mL concentrations were prepared. Then, 100 \u0026micro;L of suspension was added in 96-well black plates. Subsequently, 100 \u0026micro;L of 10 \u0026micro;M SOSG solution using medium were mixed to the suspensions of MOF525 with enough pipetting. The laser equipment (808NM-3W, LASERLAB, KOREA) was used with 660nm laser wavelength and 500 mW power output. 50 \u0026micro;g/mL of MOF525 was allowed for 3 min to the laser beam, and 25 \u0026micro;g/mL of MOF525 was irradiated for 10 min.\u003c/p\u003e \u003cp\u003eLikewise, the R848@MOF525 was performed at 25 \u0026micro;g/mL concentration for 10 min in the same method as described above. The SOSG intensity was measured at 500 nm with a reference wavelength 530 nm, using the microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.7 \u003cem\u003eIn vitro\u003c/em\u003e PDT effect evaluation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e PDT was assessed using a 10 \u0026micro;M SOSG solution in mouse colon cancer CT26 cells (ATCC, USA), which were used within five passages after thawing. Cells were seeded in a 96-well plate at a density of 1.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well. After 24 h incubation, the medium was removed and replaced with 50 \u0026micro;g/mL of MOF525 and R848@MOF525. MOF525 was evaluated in two distinct cell types, whereas R848@MOF525 was assessed only in CT26 cells. Each well was irradiated with a 500 mW laser beam for 5 min, allowed to rest at room temperature for 1 min, and then re-exposed for an additional 5 min (total irradiation time: 10 min). After a 24 h incubation, all wells were washed three times with PBS, followed by the addition of 100 \u0026micro;L of 10% Ez-Cytox solution prepared in fresh medium. The cells were then incubated for an additional 1 h, after which cell viability was determined as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.8 \u003cem\u003eIn vitro\u003c/em\u003e anticancer effect evaluation\u003c/h2\u003e \u003cp\u003eTo examine the anticancer effect of the R848@MOF525, CT26 cells were seeded in a 96-well plate at a density of 1.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well and incubated for 24 h. The culture medium was replaced with 100 \u0026micro;L of a R848@MOF525 suspension at various concentration (50, 25, 10, 1 \u0026micro;g/mL). After 24 h of incubation, the medium was removed and the cells were washed with PBS. Subsequently, 100 \u0026micro;L of 10% Ez-Cytox solution prepared in fresh medium was added, and the plate was incubated for an additional 1 h. Cell viability was assessed using the microplate reader and calculated, as above. ATP levels were quantified using a colorimetric assay. CT26 cells were seeded at a density of 1 \u0026times; 10⁶ cells in 500 \u0026micro;L of complete media per well in a 24-well treated plate and incubated overnight. The cells were then treated with MOF525 (50 \u0026micro;g/mL) followed by laser irradiation, and subsequently incubated overnight. ATP measurement was performed according to the manufacturer\u0026rsquo;s instructions using the PicoSens\u0026trade; ATP Assay Kit (BIOMAX, Korea), and absorbance was recorded at 570 nm using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.9 \u003cem\u003eIn vitro\u003c/em\u003e BMDCs activation effect evaluation\u003c/h2\u003e \u003cp\u003eThe mouse bone marrow progenitor cells were flushed from BALB/c femurs and tibias using a syringe and PBS with 2% fetal bovine serum. After removing the red blood cells with ACK lysis buffer, the collected cells were cultured in petri dishes in 10 mL of RPMI 1640 culture medium with 20 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF, Biolegend, USA). On day 6, proliferating immature DCs were harvested as non-adherent and loosely adherent clusters of cells using 2 mM ethylene-diamine-tetraacetic acid (EDTA). The DCs were seeded in non-treated 96-well plates at 1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well and treated with MOF525 and R848@MOF525 (50, 25, and 10 \u0026micro;g/mL) or with LPS (100 ng/mL) as a positive control. After 24 hours of incubation, the BMDCs were labeled with fluorescence-conjugated antibodies, including zombie violet, CD11c-PerCP-Cy5.5, CD86-PE, CD40-PE-Cy7, and MHCII-FITC (Biolegend, USA), to analyze their maturation. The maturation of the BMDCs was then assessed using flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.10 \u003cem\u003eIn vivo\u003c/em\u003e antitumor efficacy\u003c/h2\u003e \u003cp\u003eTo evaluate the potential antitumor efficacy of R848@MOF525, Female 7 weeks Balb/C mice (Orient Bio, Korea) were subcutaneously inoculated with CT26 (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells) suspended in 100 \u0026micro;L of PBS into the right flank. All procedures, including tumor inoculation, were performed under isoflurane inhalation anesthesia. Tumor size was measured using digital calipers, and the volume was calculated as\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\text{L}\\text{e}\\text{n}\\text{g}\\text{t}\\text{h}\\:\\times\\:\\:{\\text{W}\\text{i}\\text{d}\\text{t}\\text{h}}^{2}\\:\\times\\:\\:0.5\\)\u003c/span\u003e\u003c/span\u003e. When the tumor volume reached approximately 100 mm\u0026sup3;, mice were randomly divided into six groups and treated with PBS, R848, PCN525, R848@MOF525, MOF525\u0026thinsp;+\u0026thinsp;Laser, or R848@MOF525\u0026thinsp;+\u0026thinsp;Laser, respectively. MOF525 and R848@MOF525 were dispersed in PBS at a concentration of 1 mg/mL, while R848 was diluted to 0.2 mg/mL. Each formulation was then injected intratumorally at a total volume of 100 \u0026micro;L. For the MOF525\u0026thinsp;+\u0026thinsp;Laser and R848@MOF525\u0026thinsp;+\u0026thinsp;Laser groups, a laser with 660nm wavelength and 600 mW power output was utilized. The mouse was irradiated for a duration of 5 min, followed by a resting period of 1 min, and then irradiated again for an additional 5 min, resulting in a total irradiation time of 10 min. After each treatment, the tumor size and body weight of the mouse were measured. After 40 days post-tumor inoculation, the control and R848 groups, as well as the non-laser-irradiated MOF525 and R848@MOF525 groups, were CO₂ euthanized, and their tumor tissues were extracted for histopathological analysis to assess anti-cancer efficacy. Additionally, for the MOF525 and R848@MOF525 groups that exhibited complete tumor regression after PDT, a re-challenge test was conducted by inoculating the same number of tumor cells into the opposite flank. Tumor growth was monitored for 3 weeks, and after confirming no tumor growth, the mice were euthanized, and their vital organs were extracted for biosafety evaluation. All animal experiments were conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Sungkyunkwan University College of Medicine (IACUC No. SKKU IACUC 2024-02-40-1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.11 Histopathological Evaluation\u003c/h2\u003e \u003cp\u003eBoth tumor samples and vital organs were collected after animal sacrifice and stored in 10% neutralized buffered formalin until analysis. Samples were subjected to general paraffin embedding processing (dehydration/clearing/paraffin infiltration) and then prepared for paraffin blocks. 4 um of tissue sections of were deparaffinized, hydrated, and HE stained. Histopathological images were obtained by inverted light microscopy (Amscope, USA), and quantitative analysis of tumor size and necrotic lesions was performed using Image J software.\u003c/p\u003e \u003cp\u003eTUNEL staining was performed according to the manufacturer's guideline (Abcam, USA). For TUNEL staining, slides were deparaffinized/hydrated, followed by endogenous peroxidase removal (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in methanol) and pressurized heat antigen retrieval in citrate buffer before staining. TUNEL staining images were also obtained using inverted light microscopy, and quantitative analysis of TUNEL positive areas was performed using Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.12 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism 8 software (GraphPad Software, USA). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation unless otherwise indicated in the figure legend. The number of samples in each group is specified in the figure legend. For analysis of 3 or more groups, one-way ANOVA followed by Tukey\u0026rsquo;s post hoc multiple comparison test was used, unless otherwise specified in the figure legend. A 95% confidence level (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received approval from the Ethics Committee of Sungkyunkwan University, and all procedures with animal models followed established ethical standards (IACUC No.: SKKU IACUC\u0026nbsp;2024-02-40-1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsent for publication is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the data supporting the findings of this study are available within the paper and supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors Se-Na Kim\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eNae Young Chun and Yu Jin Oh declare that patent applications related to the materials and methods described in this work have been filed in South Korea (Application No. 10-2023-0179991) and under the Patent Cooperation Treaty (PCT/KR2023/020618).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Ministry of Science and ICT (MSIT) of the Republic of Korea, under the Information Technology Research Center support program, supervised by the Institute for Information \u0026amp; Communications Technology Planning \u0026amp; Evaluation (IITP-2023-RS-2023-00258971). This work was also supported and grant funded by Korea University Guro Hospital (KOREA RESEARCH-DRIVEN HOSPITAL) and Korea University (K2325651). Additionally, this work was supported by the Korea Institute of Science and Technology Program (Project No. 2E32351-23-130) and the National Research Foundation of Korea grant funded by the MSIT (RS-2023-00208913, RS-2024-00440714, RS-2025-25424498). Finally, this research was supported by the Bio\u0026amp;Medical Technology Development Program of the National Research Foundation funded by the MSIT (RS-2024-00508402).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN.K.L., S.N.K., and C.G.P. designed the research; N.K.L., N.Y.C, Y.J.O., U.B., M.S.K, and W.J.J. performed the experiment; N.K.L. S.N.K., and H.J.K. analyzed the data; and N.K.L wrote the paper; T.E.P, W.P, S.N.K, and C.G.P revised the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our gratitude to the other contributors to this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTian R, Ma H, Ye W, Li Y, Wang S, Zhang Z, et al. Se‐Containing MOF Coated Dual‐Fe‐Atom Nanozymes With Multi‐Enzyme Cascade Activities Protect Against Cerebral Ischemic Reperfusion Injury. Adv Funct Mater. 2022;32(36):2204025.\u003c/li\u003e\n\u003cli\u003eChen Z, Li Z, Tang N, Huang Y, Li S, Xu W, et al. Engineering Ultra‐Small Cerium‐Based Metal\u0026ndash;Organic Frameworks Nanozymes for Efficient Antioxidative Treatment of Dry Eye Disease. Adv Funct Mater. 2023;34(6):2307569.\u003c/li\u003e\n\u003cli\u003eWang A, Walden M, Ettlinger R, Kiessling F, Gassensmith JJ, Lammers T, et al. 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Adv Mater. 2024;36(12):e2210613.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Colorectal Cancer, Photodynamic therapy, Immunotherapy, Metal Organic Frameworks (MOF), Nanoplatforms","lastPublishedDoi":"10.21203/rs.3.rs-8452162/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8452162/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMetal-organic frameworks (MOFs) represent a novel class of materials with exceptional potential for biomedical applications. In this study, we present MOF525, a porphyrin-based MOF, as a dual-functional platform for photodynamic therapy (PDT) and immunotherapy in the treatment of colorectal cancer. MOF525 encapsulates Resiquimod (R848), a potent Toll-like receptor 7/8 agonist, thereby establishing a synergistic therapeutic strategy. Upon light exposure, MOF525 efficiently generates reactive oxygen species, leading to localized tumor cell death and the release of tumor-associated antigens. Simultaneously, the sustained release of R848 enhances immune activation, effectively overcoming tumor-induced immunosuppression. This dual approach addresses the limitations of conventional PDT and immunotherapy while maximizing their synergistic potential. \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies demonstrate that the combination of PDT and R848-loaded MOF525 significantly inhibits tumor growth and elicits a robust antitumor immune response. Moreover, the therapeutic synergy between PDT and R848 is more pronounced under a less frequent treatment regimen, highlighting the potential clinical applicability of this dual-functional nanoplatform. These findings underscore the promise of R848-loaded MOF525 as a versatile and effective therapeutic strategy for colorectal cancer by integrating photodynamic tumor ablation with innate immune stimulation.\u003c/p\u003e","manuscriptTitle":"Resiquimod-encapsulated MOF525 for Sustained Immunostimulant Release and Synergistic Photodynamic– Immunotherapy in Colorectal Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-14 18:08:44","doi":"10.21203/rs.3.rs-8452162/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"841d379d-de16-460d-ad3c-f705df553cb6","owner":[],"postedDate":"January 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-22T16:40:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-14 18:08:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8452162","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8452162","identity":"rs-8452162","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}