Low temperature activated covalent organic frameworks for fluorescence CRYO-imaging of cancer tissue

Abstract

Provided are covalent organic frameworks (COFs) and methods of making and using same. The COFs may be used in methods to visualize cancer cells and cancerous tissues at low temperatures associated with cryo-imaging and surgery. Visualization may be achieved via fluorescence. A COF of the present disclosure may have the following structure: formula (I)

Description

LOW TEMPERATURE ACTIVATED COVALENT ORGANIC FRAMEWORKS FOR FLUORESCENCE CRYO-IMAGING OF CANCER TISSUE CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 761,551, filed February 21, 2025, the entire disclosure of which is incorporated herein by reference.BACKGROUND OF THE DISCLOSURE

[0002] Cryosurgery is a pivotal advancement in the minimally invasive treatment of cancers resistant to conventional therapies. It uses extreme cold, typically between -20 °C and -40 °C, to effectively eradicate malignant tissue. This technique offers numerous benefits, including reduced pain, minimal bleeding, and lower morbidity rates, and is particularly beneficial for tumors that do not respond to radiotherapy. The less invasive nature of cryosurgery enhances patient comfort and promotes faster recovery and fewer surgical complications. However, the efficacy and safety of cryosurgery depend critically on accurate real-time visualization of the target tissue. Accurate imaging is essential to minimize damage to surrounding healthy tissue and ensure comprehensive removal of cancer cells to optimize the therapeutic outcomes of cryosurgery. Current imaging techniques such as ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI) are essential for modem cryosurgery, but due to their limited resolution at the molecular level, they are often unable to determine the extent of frozen tissue reliably.

[0003] Non-invasive optical fluorescence imaging has emerged as a promising approach to improve tumor visualization during surgical interventions.7This method leverages fluorescent dyes to provide direct, real-time insights at the molecular level, which is very helpful in accurately differentiating cancerous from healthy tissue during diagnosis and therapy. Despite its potential, the application of fluorescence imaging in real-time guided cryosurgery remains largely unexplored. This is mainly due to the challenges of tumorspecific visualization under freezing conditions and the necessary sensitivity to intracellular ice formation, which is crucial for effectively destroying cancer cells. Traditional organic luminescent materials often suffer from reduced stability and sensor sensitivity at low temperatures, which can affect performance and reliability due to issues such as aggregation-induced quenching and low photostability. In response to these challenges, He etal. have developed a novel strategy that utilizes the aggregation-induced emission (AIE) of fluorogens. This innovative approach significantly enhances real-time monitoring capabilitiesin cryosurgery by activating fluorescent probes in cold environments to improve surgical precision and effectively differentiate between malignant and benign tissue. This development underscores the urgent need for advanced imaging technologies that can adapt to the unique requirements of cryogenic conditions, redefining the effectiveness and safety of cryosurgical cancer treatment.

[0004] Recent advances in materials science have highlighted the potential of Covalent Organic Frameworks (COFs) in biomedical fields. These frameworks are characterized by their highly tunable structure and porosity, which can be precisely tailored to meet specific clinical requirements. The inherent crystallinity of COFs provides consistent and predictable behavior, essential for diverse medical applications. Moreover, their adjustable pore sizes enable the encapsulation of various therapeutic agents, ranging from small-molecule drugs to larger biologicals, ensuring controlled release that can be fine-tuned by modifying the framework. These properties, combined with exceptional chemical stability and biocompatibility, make COFs ideal candidates for targeted drug delivery, diagnostic applications, and scaffolds for tissue engineering.

[0005] Compared to conventional fluorescent organic solids, fluorescent COFs possess rigid chemical structures that inherently minimize the intramolecular rotation, vibration, and motion typically observed in standard organic molecules and polymers. This stabilization effectively restricts bond motion and blocks non-radiative pathways, which enhances fluorescence by conserving excited-state energy and reducing energy decay. We have pioneered the field of bioimaging using COFs through the development of fluorescent covalent organic nanosheets capable of selective intracellular localization in the cellular nucleus, providing a unique approach for cancer diagnosis and therapy without the need for external targeting agents. Consequently, through careful design and synthesis, fluorescent COFs can outperform conventional dissolved organic dyes and compete with conventional organic solid-based luminescent materials, providing a robust platform for biomedical imaging.

[0006] The field of image-guided cryosurgery is currently facing major challenges, including the lack of fluorescent probes capable of specifically and sensitively distinguishing tumor tissue from normal tissue in a frozen state — an essential factor in achieving precise surgical margins. Moreover, most existing probes do not work effectively when intracellular ice forms, a crucial process to induce cancer cell death during cryoablation (Table 1).Additionally, there is an urgent need to develop non-toxic, nanoscale materials that can circulate in the bloodstream and target tumors without causing adverse effects.BRIEF SUMMARY OF THE DISCLOSURE

[0007] The present disclosure provides covalent organic frameworks (COFs). Also provided are methods of making the COFs and methods of using the COFs.

[0008] Cryosurgery represents a transformative approach in the treatment of resistant tumors, utilizing extreme cold to selectively ablate malignant tissue. However, the clinical success of this technique is constrained by the limited ability of current imaging techniques to differentiate effectively between cancerous and healthy tissues with high spatial resolution. To overcome this challenge, described are nanoscale Covalent Organic Framework, nTG-DFP-COF, specifically designed to enhance fluorescence-guided cryo-imaging. This framework exhibits a unique temperature-dependent luminescence, that results in enhanced fluorescence emission under cryogenic conditions, enabling precise tissue differentiation during surgical procedures. Engineered for biocompatibility and water dispersibility, nTG-DFP-COF demonstrates minimal cytotoxicity and exceptional specificity toward cancer cells. Comprehensive in vitro, in vivo, and ex vivo evaluations confirm its structural stability and functional efficacy under cryogenic conditions. This innovation not only enhances the precision and safety of cryosurgical procedures but also advances the integration of diagnostic and therapeutic functionalities into a unified platform. By substantially improving tumor targeting accuracy, the use of nTG-DFP-COF will reduce the need for repeat surgeries, facilitate faster recovery, and minimize healthcare costs, thus setting a new standard in oncologic imaging and intervention.

[0009] In an aspect, the present disclosure provides COFs suitable for use in detecting cancerous tissues.

[0010] A suitable COF may have the following structure:wherein each R is independentlyeach R' is independently sA suitable COF may be made from the following monomers:hydrazinecarbohydrazonhydrazideandpyridine-2,6-dicarbaldehydeThe resulting COF may comprise the following structure:where the terminal ends of the pyridyl groups are either formyl groups or are attached to an additional triamino guanidinium group, such as in the following structure:and R' is as provided above. In various examples, at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% of the R groups areFollowing synthesis of the COF, the COF may be exposed to a liquid exfoliation process.

[0011] In an aspect, the present disclosure provides compositions. The composition may comprise one or more COF nanosheets of the present disclosure and a pharmaceutically acceptable carrier.

[0012] In an aspect, the present disclosure provides methods for imaging cancerous cells and / or cancerous tissues in a subject having or suspected of having cancer.

[0013] The method may comprise administering to a subject the COF nanosheets of the present disclosure and visualizing the COF nanosheets. The COF nanosheets may be endocytosed by cancerous cells at rates higher than in non-cancerous cells. The method may further comprise decreasing the temperature of the area to be visualized to a freezing temperature or lower. In various examples, the temperature is a temperature suitable for cryoimaging and surgery (e.g., less than 0 °C, such as, for example, at or around-40 °C). In various examples, the excitation of the COF nanosheet is 430 to 450 nm (including all ranges and values therebetween) and the emission of the COF nanosheet is 500 to 540 nm (including all ranges and values therebetween).

[0014] In an aspect, the disclosure provides kits. A kit may comprise pharmaceutical preparations comprising COF nanosheets and printed material.BRIEF DESCRIPTION OF THE FIGURES

[0015] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.

[0016] Figure 1. Structure and Characterization of nTG-DFP-COF. a) Diagram illustrating the liquid exfoliation processing (LEP) used to convert bulk TG-DFP-COF to nanoscale nTG-DFP-COF. The process involves a two-hour treatment in water, transitioning from a bulky to a finely exfoliated structure. Digital images of TG-DFP-COF solution before (left) and after (right) liquid exfoliation (2 mg / mL). b) High-resolution transmission electron microscopy (HRTEM) images comparing the morphology of bulk TG-DFP-COF (left) with the exfoliated nTG-DFP-COF (right). The right panel includes a size distribution histogram, showing the uniformity and scale of the nanosheets, c) Atomic force microscopy (AFM) images displaying the topographical contrast between the bulk material (left) and the nanostructured nTG-DFP-COF (right), with a distinct reduction in height and smoother surface profile.

[0017] Figure 2. Characterization of nTG-DFP-COF. a) Experimental (yellow) and calculated (black) PXRD patterns of nTG-DFP-COF, showing the primary diffraction peaks at 29 = 5.56° (200) and a wide-angle reflection at 29 = 26.89° (003) indicating the long-range order and TI- TI stacking of the 2D layers. The inset shows a top view of the spacefilling model of the simulated structure observed in the hexagonal space group P3. b) High-resolution transmission electron microscopy (HRTEM) of nTG-DFP-COF with nanosheets of about 240 nm in size, c) Lattice fringes HRTEM image and d) their reconstruction: The images show continuous and consistent lattice fringes with a d-spacing of 0.35 nm and are in close agreement with the wide-angle PXRD data, e) Selected area electron diffraction (SAED) shows the crystalline nature of nTG-DFP-COF with distinct electron diffraction spots corresponding to the doo3 plane with a d-spacing of 0.34 nm. f) Stability analysis of nTG-DFP-COF in various solutions over time. Hydrodynamic diameter distributions of nTG-DFP-COF nanosheets suspended in different solvents: Ethanol, water, phosphate-buffered saline (PBS), fetal bovine serum (FBS), and Dulbecco’s Modified Eagle Medium (DMEM). Measurements were performed at two intervals, 24 hours (left panel) and 1 week (right panel).

[0018] Figure 3. Temperature-Dependent Optical and Fluorescence Properties of nTG-DFP-COF. a) Two-Dimensional heatmap of variable temperature solid-state DR-UV-Visible spectra of nTG-DFP-COF: This heatmap shows the variation in absorption of nTG-DFP-COF over a spectrum of wavelengths (350 nm to 600 nm) and temperatures (from -30°C to 30°C). b) Variable temperature solid-state fluorescence spectra of nTG-DFP-COF over a wide temperature range from -35 °C to 35 °C, showing the thermally modulated fluorescence intensity of the material ( ex / cm = 400 / 540 nm), where order of the lowest temperature displayed the highest intensity and the highest temperature displayed the lowest intensity with fluorescence intensity increasing as temperature decreased. Fluorescent phantom imaging at different concentrations and temperatures: c) IVIS®-based images showing the fluorescence of nTG-DFP-COF at different concentrations, compared at physiological temperature (37°C) and in the frozen state (-40°C), to illustrate the changes in fluorescence intensity (Vx / cm = 465 / 540 nm). d) Quantitative analysis of fluorescence intensity: Plots of fluorescence image intensities versus concentrations of nTG-DFP-COF used to calculate the weight radiant efficiencies, highlighting the relationship between concentration and fluorescence efficiency under different thermal conditions.

[0019] Figure 4. Analysis of nTG-DFP-COF Across Variable Temperatures, a)13C CP / MAS solid-state NMR Spectra: Overlay of the one-dimensional13C CP / MAS solid-state NMR spectra of nTG-DFP-COF at 20 °C (red lines) and at 0 °C (blue lines), showing the stability of the chemical shifts upon temperature reduction, b) ^-^C HETCOR solid-state NMR spectra: Overlay of two-dimensional ^-^C HETCOR solid-state NMR spectra of nTG-DFP-COF at 20 °C (red contours) and 0 °C (blue contours). Notable upfield changes in proton chemical shifts are indicated by a green arrow, suggesting stronger hydrogen bonding at lower temperatures, c) Temperature-Dependent IR Spectra across a temperature range from -40 °C to 35 °C: Evolution of the IR spectra of nTG-DFP-COF with temperature changes. Spectra were recorded for nTG-DFP-COF (lwt.% in KBr) before and after activation under vacuum at 40 °C for 12 hours to remove residual adsorbed water. The spectra subtracted from the baseline recorded at 40 °C highlight changes due to temperature variations. Inset shows the evolution of band areas centered at 3330 cm-1, assigned to N-H hydrogen bonding bands, d) XPS Analysis at room temperature and -40 °C: XPS C is (left) and N Is (right) spectra of nTG-DFP-COF comparing room temperature and -40 °C, illustrating the changes in the electronic environment and bonding properties under extreme temperature conditions.

[0020] Figure 5. Intracellular uptake and fluorescence response of nTG-DFP-COF in cancerous and non-cancerous cell lines, a) Transmission electron microscopy (TEM) images of HeLa, U251-MG, and HEK-293 cells after 24 hours of incubation with nTG-DFP-COF (arrows) at 10 pg / mL. The images show preserved cellular integrity with minimal debris, highlighting the biocompatibility of nTG-DFP-COF. b) Bright-field andFluorescence microscopy images of HeLa, U251-MG, and HEK-293 cells co-stained with nTG-DFP-COF and LysoTracker red, under 469 nm and 531 nm light, at temperatures of 37 °C and -10 °C. Scale bar: 200 pm c) Graph depicting the fluorescence intensity changes for HeLa, U251-MG, and HEK-293 cells at 37 °C and -10 °C. The increase in green fluorescence intensity of nTG-DFP-COF in HeLa and U251-MG is contrasted with the decrease in red fluorescence of LysoTracker red, illustrating the different stability and response to temperature changes. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant. Green bars are the two bars on the left and the red bars are the two bars on the right in each series, d) Schematic representation of the fluorescence activation mechanism of nTG-DFP-COF at different temperatures and its cellular localization in green and red channels, showing the selective activation of nTG-DFP-COF in cancer cells (HeLa, U251-MG), highlighting its potential for improved specificity of cryo-imaging.

[0021] Figure 6. In vivo Toxicity and Biocompatibility Assessment of nTG-DFP-COF in CD-I Mice, a) Experimental Overview: Schematic representation of administration of nTG-DFP-COF by a single intraperitoneal (i.p.) injection in CD-I mice, followed by detailed analysis of organ tissues after 7 days, b) Body weight of CD-I mice treated with either saline (control) or nTG-DFP-COF at a dose of 20 mg / kg, monitored for 7 days postinjection. c)£x Vivo Bioluminescence Imaging: Images of key organs (liver, heart, kidney, spleen) obtained using the IVIS® Spectrum imaging system one-week after administration of nTG-DFP-COF at a dose of 20 mg / kg. d) Histopathological Analysis: Microscopic examinations of major organs (liver, heart, kidney, spleen) to assess cellular and structural integrity after administration of nTG-DFP-COF administration. These panels confirm the overall biocompatibility and safety of nTG-DFP-COF.

[0022] Figure 7. Evaluation of nTG-DFP-COF as targeted cryo-imaging probe in U251-MG Glioblastoma Models, a) Ex vivo Fluorescence Imaging: U251-MG glioblastoma tumors imaged using the IVIS® system at different temperatures. The sequence shows tumors at physiological temperature (37 °C), in cryogenic conditions (-40 °C), and subsequently returned to 37 °C, with control tumors included for comparison. Dose administered: 20 mg / kg (200 pL). b) Quantitative Analysis of Radiant Efficiency: This graph depicts the total radiant efficiency of nTG-DFP-COF in the U251-MG tumors, illustrating significantly stronger emissions in the frozen state compared to physiological temperature. This enhancement underscores the probe's effectiveness under cryosurgical conditions. The marked decrease in fluorescence upon rewarming the tumors to 37 °C highlights the temperature-responsive properties of nTG-DFP-COF, reinforcing its potential as a dynamiccryo-imaging tool. Significance levels indicated by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. c) In Vivo Targeting and Biodistribution of nTG-DFP-COF.Sequential fluorescence images of mice bearing subcutaneous U251-MG glioblastoma tumors treated with nTG-DFP-COF, captured at 0, 24, and 48 hours post-injection using the IVIS® Spectrum imaging system. These images illustrate the dynamic accumulation and subsequent clearance of nTG-DFP-COF at the tumor site, d) Post-mortem Fluorescence Imaging of U251-MG Glioblastoma Tumors: Sequential fluorescence images of a control mouse treated with PBS and imaged at physiological temperature (37 °C) 24 hours posttreatment; a mouse treated with nTG-DFP-COF (20 mg / kg, 200 pL) under the same conditions showing initial fluorescence distribution in the tumor; and the mouse after cryotreatment at 4 °C and -20 °C. These images highlight the temperature-dependent 'turn-on' property of nTG-DFP-COF. All animals were sacrificed 24 hours post-treatment, and fluorescence imaging was conducted using an IVIS® Spectrum system, demonstrating the probe’s enhanced imaging contrast and specificity under targeted temperature conditions.

[0023] Figure 8. Cartoon displaying the use of the COF of the present disclosure

[0024] Figure 9. Microwave-assisted synthesis of TG-DFP COF in bulk form using a 1 : 1 mixture of 1,4-di oxane and water.

[0025] Figure 10. Stacked FTIR spectra of the nTG-DFP-COF (yellow), triamino guanidium hydrochloride salt (TGH.C1, black), and 2,6-diformyl pyridine (DFP, black).

[0026] Figure 11. a) Transmission Electron Microscopy (TEM) image of nTG-DFP-COF. b) Particle size distribution of nTG-DFP-COF. The overlaid dashed lines represent Gaussian fit profiles, derived using the mean diameter calculated from the analysis of 500 individual particles.

[0027] Figure 12. Atomic force microscopy (AFM) images displaying the topographical contrast between the bulk material (left) and the nTG-DFP-COF (right), with a distinct reduction in height and smoother surface profile.

[0028] Figure 13. Tyndall Effect Observed in a Diluted nTG-DFP-COF Dispersion. The picture shows the Tyndall scattering effect in a translucent solution of nTG-DFP-COF (0.4 mg-mL-1) when exposed to a laser beam, indicating the presence of highly monodisperse ultrathin nanosheets in water.

[0029] Figure 14. Comparative Analysis of nTG-DFP-COF Nanosheets Before and After Freezing, a) TEM images of nTG-DFP-COF nanosheets before (left) and after (right) undergoing a freezing process, demonstrating no significant changes in morphology or structural integrity, b) AFM images before (left) and after (right) freezing, further confirmingthe maintenance of surface morphology with no evident changes in the nanosheets' topography. These images collectively illustrate the robustness of nTG-DFP-COF nanosheets under cryogenic conditions, maintaining their structural characteristics after treatment.

[0030] Figure 15. Variable Temperature Powder X-ray Diffraction (VT-PXRD) patterns of nTG-DFP-COF. The spectra depict the structural consistency of nTG-DFP-COF at various temperatures — room temperature (green line), -40 °C (red line), and after regeneration (blue line) as well as calculated (black line) PXRD patterns. The consistency across the spectra confirms the absence of structural changes in the nTG-DFP-COF, confirming its robustness and stability under varying thermal conditions.

[0031] Figure 16. Stability and dispersibility assessments. Measurements of hydrodynamic diameter before and after liquid exfoliation of nTG-DFP-COF in aqueous solution.

[0032] Figure 17. Temperature-dependent fluorescence heatmap of nTG-DFP-COF. The heatmap depicts the fluorescence intensity across a range of temperatures from -35°C to 35°C and wavelengths from 450 nm to 700 nm. This visualization clearly illustrates the thermally induced changes in luminescence, with a peak in intensity observed at lower temperatures (Xex = 400 nm).

[0033] Figure 18. Excitation-Emission Matrix (EEM) of nTG-DFP-COF. This figure displays the EEM fluorescence landscape for nTG-DFP-COF, showcasing the excitation and emission wavelengths. The main fluorescence peak is observed at an excitation wavelength of 520 nm and an emission wavelength of 570 nm, as indicated by the reddest (most intense) area on the map. The color gradient from blue to red represents increasing fluorescence intensity, quantified on the left y-axis, with corresponding wavelength ranges detailed along the x and right y-axes.

[0034] Figure 19. Visual and Thermal Analysis of nTG-DFP-COF in Water. This figure presents both a photographic and a thermal image of nTG-DFP-COF suspended in water at a concentration of 2 mg / mL. The sample was subjected to freezing by immersing the Eppendorf tube in liquid nitrogen for 10 minutes. The thermal image captures and displays the temperature distribution across the sample immediately following its removal from the liquid nitrogen.

[0035] Figure 20. Visual and Thermal Analysis of Cold Ethanol. This figure includes both a photograph and a thermal image of 10 mL of ethanol after cooling. The sample was frozen by immersing the tube in liquid nitrogen for 10 minutes. The thermalimage shows the temperature distribution of the ethanol immediately after being removed from the liquid nitrogen.

[0036] Figure 21. Molecular Dynamics Simulations Illustrating Temperature-Dependent Binding Affinity of Water Molecules with nTG-DFP-COF at -40 °C and 20 °C. a) The simulation system includes a fragment of nTG-DFP-COF along with 15 explicit water molecules to represent hydrogen bonding interactions between the nTG-DFP-COF and water molecules, which are depicted with dashed red lines, b) the graph represents the radial distribution function between the nTG-DFP-COF hydrogen atoms (labeled as Hdfp in panel a) and oxygen atoms of all water molecules at -40 °C (blue) and 20 °C (red). Inset: Average number of water molecules <NW> in the first solvation shell (3 A from Hdfp) at -40 °C (blue) and 20 °C (red).

[0037] Figure 22. Cell viability of HEK-293 (green and left bar in each series), HeLa (red and middle bar in each series), and U251-MG (blue and right bar in each series) after 48-hour treatment with nTG-DFP-COF at concentrations up to 1 mg / mL. Error bars denote the standard deviations from triplicate measurements.

[0038] Figure 23. Evaluation of Cytotoxicity in HeLa, U251-MG, and HEK-293 Cell Lines via MTS, LDH, and Cell Growth Assays, a) Mitochondrial activity measured by the MTS assay across three concentrations (1, 10, and 100 pg / mL) of nTG-DFP-COF, highlighting cell viability in U251-MG (blue and left bar in each series), HeLa (red and middle bar in each series), and HEK-293 (green and right bar in each series) cell lines, b) Membrane integrity assessed through LDH leakage, indicating minimal perturbation across the same concentrations after 24 hours of exposure, c) Growth curves over 72 hours at 37 °C, showing cell count dynamics in control conditions and following incubation with 10 pg / mL of nTG-DFP-COF for each cell line. Data represent mean ± standard deviation (n=3).

[0039] Figure 24. Hemolysis activity of nTG-DFP-COF. a) Photographs after centrifugation of fresh human blood incubated with different concentrations of nTG-DFP-COF up to 2 mg.mL-1for 1 hour, b) Hemolysis rates (%) induced by different concentrations of nTG-DFP-COF up to 2 mg.mL-1. Physiological saline in the absence or the presence of Triton X-100 (0.3 %) were respectively used as negative (C-) and positive (C+) controls.0.0001, significantly different from negative control. Error bars represent standard deviations of triplicate measurements.

[0040] Figure 25. TEM images of HeLa, U251-MG, and HEK-293 control cells.

[0041] Figure 26. Intracellular Uptake of nTG-DFP-COF in Cancer Cells.Transmission electron microscopy (TEM) images show the interaction of nTG-DFP-COFwith HeLa and U251-MG cells after 24 hours of incubation at a concentration of 10 pg / mL. a) Initial contact and engulfment of the nTG-DFP-COF nanosheets by the plasma membrane in both cell lines, indicated by the blue arrows. This interaction indicates the onset of membrane invagination, b) Progression of nTG-DFP-COF aggregates into deeper plasma membrane invaginations in both cell lines, shown by the red arrows, indicative of early endosomal formation, c) Localization of nTG-DFP-COF within endosomal vesicles inside the cytoplasm of both cell lines, indicated by green arrows. These stages illustrate the active internalization of nTG-DFP-COF in cancer cells.

[0042] Figure 27. Fluorescence images of HeLa cells incubated for 4 hours with nTG-DFP-COF at 4 °C (top panel) and 37 °C (bottom panel) at a concentration of 10 pg / mL. The left column shows the bright field, the middle column shows the fluorescence in the green channel (Xex = 420 nm), and the right column is the overlay of both channels.

[0043] Figure 28. Fluorescence images of U251-MG cells incubated for 4 hours with nTG-DFP-COF at 4 °C (top panel) and 37 °C (bottom panel) at a concentration of 10 pg / mL. The left column shows the bright field, the middle column shows the fluorescence in the green channel (Xex = 420 nm), and the right column is the overlay of both channels.

[0044] Figure 29. Confocal Laser Scanning Microscopy (CLSM) Images of HeLa Cells Following a 4-hour Treatment with nTG-DFP-COF. Cells were treated with nTG-DFP-COF at a concentration of 10 pg / mL and co-stained using red fluorescent markers to label the plasma membrane, lysosomes, and nucleus. Imaging channels include nTG-DFP-COF (Xex = 410 nm) and red markers (kex= 561 nm). This figure illustrates the subcellular localization and the possible intracellular pathways of nTG-DFP-COF. White arrows show co-localization.

[0045] Figure 30. Confocal Laser Scanning Microscopy (CLSM) Images of U251-MG Cells Following a 4-hour Treatment with nTG-DFP-COF. Cells were treated with nTG-DFP-COF at a concentration of 10 pg / mL and co-stained using red fluorescent markers to label the plasma membrane, lysosomes, and nucleus. Imaging channels include nTG-DFP-COF (Xex = 410 nm) and red markers (kex= 561 nm). This figure illustrates the subcellular localization and the possible intracellular pathways of nTG-DFP-COF. White arrows show co-localization.

[0046] Figure 31. Confocal Laser Scanning Microscopy (CLSM) Images of HeLa Cells. These images display HeLa cells after 4 hours of incubation without additives (control) and co-stained with red fluorescent markers to visualize the plasma membrane, lysosomes,and nucleus. Imaging channels include an excitation wavelength of 410 nm and 561 nm for the red markers.

[0047] Figure 32. Confocal Laser Scanning Microscopy (CLSM) Images of U251-MG Cells. These images display U251-MG cells after 4 hours of incubation without additives (control) and co-stained with red fluorescent markers to visualize the plasma membrane, lysosomes, and nucleus. Imaging channels include an excitation wavelength of 410 nm and 561 nm for the red markers.

[0048] Figure 33. Visual and Thermal Analysis of U251-MG Tumors Treated with nTG-DFP-COF. This figure presents both photographs and thermal images of control U251 -MG tumors (untreated) and those treated with nTG-DFP-COF (20 mg / kg, 200 pL) at room temperature and in a frozen state. The tumors were frozen by immersing them in liquid nitrogen for 10 minutes. The thermal images show the temperature distribution of the control tumors and the treated tumors immediately after removal from the liquid nitrogen, highlighting the differences in thermal response. Scale bar: 1 cm.

[0049] Figure 34. Thermal Imaging Analysis of Mice Subjected to Local CryoTreatment with nTG-DFP-COF. This figure shows thermal images of a comparison between a control mouse and a mouse treated with nTG-DFP-COF, both at room temperature and during localized cryo-treatment. The left panel shows the baseline temperature distribution in the body of each mouse at room temperature. The right panel shows the dramatic temperature change in the tumor regions after local cryo-treatment, which selectively cools the tumors to -10.8 °C in the control mouse and -12.0 °C in the nTG-DFP-COF-treated mouse. These images visually demonstrate the precision of targeted cooling.DETAILED DESCRIPTION OF THE DISCLOSURE

[0050] Although claimed subject matter will be described in terms of certain examples, other examples, including examples that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, process step, and electronic changes may be made without departing from the scope of the disclosure.

[0051] As used herein, unless otherwise indicated, “about”, “substantially”, or “the like”, when used in connection with a measurable variable (such as, for example, a parameter, an amount, a temporal duration, or the like) or a list of alternatives, is meant to encompass variations of and from the specified value including, but not limited to, those within experimental error (which can be determined by, e.g., a given data set, an art acceptedstandard, etc. and / or with, e.g., a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as, for example, variations of + / - 10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value), insofar such variations in a variable and / or variations in the alternatives are appropriate to perform in the instant disclosure. As used herein, the term “about” may mean that the amount or value in question is the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, compositions, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error, or the like, or other factors known to those of skill in the art such that equivalent results or effects are obtained. In general, an amount, size, composition, parameter, or other quantity or characteristic, or alternative is “about” or “the like,” whether or not expressly stated to be such. It is understood that where “about,” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0052] Ranges of values are disclosed herein. The ranges set out a lower limit value and an upper limit value. Unless otherwise stated, the ranges include the lower limit value, the upper limit value, and all values between the lower limit value and the upper limit value, including, but not limited to, all values to the magnitude of the smallest value (either the lower limit value or the upper limit value) of a range. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “0.1% to 5%” should be interpreted to include not only the explicitly recited values of 0.1% to 5%, but also, unless otherwise stated, include individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5% to 1.1%; 0.5% to 2.4%; 0.5% to 3.2%, and 0.5% to 4.4%, and other possible sub-ranges) within the indicated range. It is also understood (as presented above) that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent“about, it will be understood that the particular value forms a further disclosure. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0053] The articles “a” and “an” are used in this disclosure to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0054] As used herein, unless otherwise stated or indicated, “s” refers to second(s), “min” refers to minute(s), and “h” refers to hour(s).

[0055] As used herein, unless otherwise stated, the term “group” refers to a chemical entity that is monovalent (i.e., has one terminus that can be covalently bonded to other chemical species), divalent, or polyvalent (i.e., has two or more termini that can be covalently bonded to other chemical species). The term “group” also includes radicals (e.g., monovalent and multivalent, such as, for example, divalent radicals, trivalent radicals, and the like).Illustrative examples of groups include:

[0056] The present disclosure provides covalent organic frameworks (COFs). Also provided are methods of making the COFs and methods of using the COFs.

[0057] Cryosurgery represents a transformative approach in the treatment of resistant tumors, utilizing extreme cold to selectively ablate malignant tissue. However, the clinical success of this technique is constrained by the limited ability of current imaging techniques to differentiate effectively between cancerous and healthy tissues with high spatial resolution. To overcome this challenge, described herein is the nanoscale Covalent Organic Framework, nTG-DFP-COF, specifically designed to enhance fluorescence-guided cryo-imaging. This framework exhibits a unique temperature-dependent luminescence, that results in enhanced fluorescence emission under cryogenic conditions, enabling precise tissue differentiation during surgical procedures. Engineered for biocompatibility and water dispersibility, nTG-DFP-COF demonstrates minimal cytotoxicity and exceptional specificity toward cancer cells. Comprehensive in vitro, in vivo, and ex vivo evaluations confirm its structural stability and functional efficacy under cryogenic conditions. This innovation not only enhances the precision and safety of cryosurgical procedures but also advances the integration of diagnostic and therapeutic functionalities into a unified platform. By substantially improving tumor targeting accuracy, the use of nTG-DFP-COF will reduce the need for repeat surgeries,facilitate faster recovery, and minimize healthcare costs, thus setting a new standard in oncologic imaging and intervention.

[0058] In an aspect, the present disclosure provides COFs suitable for use in detecting cancerous tissues. The COFs may be referred to as “exfoliated COFs.”

[0059] A suitable COF may have the following structure:wherein each R is independentlywherein at least one R ise made from the following monomers:hydrazinecarbohydrazonhydrazideandpyridine-2,6-dicarbaldehydeThe resulting COF may comprise the following structure:where the terminal ends of the pyridyl groups are either formyl groups or are attached to an additional triamino guanidinium group, such as in the following structure:and R' is as provided above. In various examples, at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% of the R groups areFollowing synthesis of the COF, the COF may be exposed to a liquid exfoliation process.

[0060] The COF may be at least partially crystalline. For example, the COF is mostly crystalline, substantially crystalline, or crystalline.

[0061] The COFs of the present disclosure may form nanosheets. Bulk COFs of the present disclosure may be exposed to a liquid exfoliation process. Following liquid exfoliation, the longest linear dimension and thickness of the exfoliated COF nanosheets decreases from its bulk form. The exfoliated COF nanosheets may have a longest lineardimension of about 150 to 300 nm, including all nm values and ranges therebetween. The exfoliated COFs have a decreased thickness. For example, the thickness decreases from the millimeter scale down to less than 75 nm (e.g., 60 nm, 50 nm, 40 nm). Without intending to be bound by any particular theory, at least 90% (e.g., at least at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or all) of the liquid exfoliated COF nanosheets in a composition have a longest linear dimension of 300 nm.

[0062] The exfoliated COF nanosheets (which may be referred to as “COF nanosheets”) may have desirable properties. For example, the COF nanosheets may have temperature-dependent optical and fluorescence properties. The absorbance of the COF nanosheets increases as the temperature decreases. For example, the COF nanosheets may display a peak absorbance at 420 nm at -30 °C. Similarly, the fluorescence properties of the COF nanosheets increases at lower temperatures (e.g., less than -20 °C). For example, the COF nanosheets may display desirable fluorescence at -35 °C. Without intending to be bound by any particular theory, it is considered these optical and spectral properties are due to increased stabilization from hydrogen bonds in the COF networks as temperatures decrease. Also affected at lower temperatures is the binding affinity of water, with a higher affinity for water at lower temperatures (e.g., at less than -20 °C (e.g., -40 °C or lower).

[0063] The exfoliated COF nanosheets may also have desirable properties in the presence of various tissues. COF nanosheets of the present disclosure are internalized in greater numbers by cancer cells and cancerous tissues relative to non-cancerous cells and non-cancerous tissues. Additionally, COF nanosheets of the present disclosure display desirable fluorescence provides in cancer cells at low temperatures (e.g., less than 0 °C). At room temperature, the COFs may emit weak green fluorescence under 469 nm and show strong red fluorescence at 531 nm. Following freezing, the fluorescence of the COF nanosheets may increase by 2-fold, 3 -fold, or more.

[0064] In an aspect, the present disclosure provides compositions. The compositions may comprise a pharmaceutically acceptable carrier.

[0065] The composition can comprise COF nanosheets in a pharmaceutically acceptable carrier (e.g., carrier). The carrier can be an aqueous carrier suitable for administration to individuals including humans. The carrier can be sterile. The carrier can be a physiological buffer. Examples of suitable carriers include sucrose, dextrose, saline, and / or a pH buffering element (such as, a buffering element that buffers to, for example, a pH from pH 5 to 9, from pH 6 to 8, (e.g., 6.5)) such as histidine, citrate, or phosphate. Additionally, pharmaceutically acceptable carriers may be determined in part by the particular compositionbeing administered. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present disclosure. Additional, non-limiting examples of carriers include solutions, suspensions, and emulsions that are dissolved or suspended in a solvent before use, and the like. The composition may comprise one or more diluents.Examples of diluents, include, but are not limited to distilled water, physiological saline, vegetable oil, alcohol, dimethyl sulfoxide, and the like, and combinations thereof.Compositions may contain stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, and the like, and combinations thereof. Compositions may be sterilized or prepared by sterile procedure. A composition of the disclosure may also be formulated into a sterile solid preparation, for example, by freeze-drying, and may be used after sterilization or dissolution in sterile injectable water or other sterile diluent(s) immediately before use. Additional examples of pharmaceutically acceptable carriers include, but are not limited to, sugars, such as, for example, lactose, glucose, and sucrose; starches, such as, for example, com starch and potato starch; cellulose, including sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as, for example, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as, for example, propylene glycol; polyols, such as, for example glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as, for example, ethyl oleate and ethyl laurate; agar; buffering agents, such as, for example, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Additional non-limiting examples of pharmaceutically acceptable carriers can be found in: Remington: The Science and Practice of Pharmacy (2012) 22ndEdition, Philadelphia, PA. Lippincott Williams & Wilkins. For example, a composition comprises a plurality of COF nanosheets and a sterile, suitable carrier for administration to individuals including humans — such as a physiological buffer such as sucrose, dextrose, saline, pH buffering (such as from pH 5 to 9, from pH 7 to 8, from pH 7.2 to 7.6, (e.g., 7.4)) element such as, for example, histidine, citrate, or phosphate. In various examples, the composition may be suitable for injection. Parenteral administration includes infusions and injections, such as, for example, intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous administration, and the like.

[0066] In an aspect, the present disclosure provides methods for imaging cancerous cells and / or cancerous tissues in a subject having or suspected of having cancer.

[0067] The method may comprise administering to a subject the COF nanosheets of the present disclosure and visualizing the COF nanosheets. The COF nanosheets may be endocytosed by cancerous cells at rates higher than in non-cancerous cells. The method may further comprise decreasing the temperature of the area to be visualized to a freezing temperature or lower. In various examples, the temperature is a temperature suitable for cryoimaging and surgery (e.g., less than 0 °C, such as, for example, at or around-40 °C). In various examples, the excitation of the COF nanosheet is 430 to 450 nm (including all ranges and values therebetween) and the emission of the COF nanosheet is 500 to 540 nm (including all ranges and values therebetween).

[0068] In various examples, the method can be used to differentiate cancerous cells / tissues from non-cancerous cells / tissues. For example, the method may be used to determine the borders of a tumor in a subject to differentiate between cancerous and non-cancerous areas in an individual. These borders may be then used by a medical professional to assist during surgical removal of the tumor such that all or nearly all of the cancerous tissue is removed.

[0069] A method of the present disclosure may comprise the following steps: preparing a composition containing Covalent Organic Framework (COF) nanosheets; administering the composition to an area of the subject where cancer cells are suspected, using techniques suitable for the specific type of tissue and cancer, such as injection or topical application; allowing sufficient time for the COF nanosheets to interact with the cancer cells, typically ranging from several minutes to hours depending on the application method and tissue type; cooling the targeted area to a predetermined temperature that enhances the fluorescence of the COF nanosheets, thereby activating their imaging capabilities. This temperature should be low enough to induce a significant increase in fluorescence without causing undue harm to the surrounding healthy tissue.

[0070] Using a fluorescence imaging system calibrated to detect the specific wavelength emitted by the activated COF nanosheets, visualize the area to identify cancer cells distinguished by their enhanced fluorescence (e.g., by measure fluorescence intensity) compared to the surrounding non-cancerous tissues. An increase in fluorescence intensity indicates the presence of cancerous cells. Without intending to be bound by any particular theory, it is considered that cancer cells and cancerous tissues uptake the nanosheets in greater numbers relative to non-cancerous cells and non-cancerous tissues. COF nanosheets of the present disclosure display desirable fluorescence provides in cancer cells at low temperatures.

[0071] In one embodiment, the individual is a human or non-human mammal. Nonhuman animals include ungulates such as bovines. Additional non-limiting examples of non-human mammals include pigs, mice, rats, rabbits, cats, dogs, or other agricultural mammals, pet, or service animals, and the like.

[0072] In various examples, the method comprises administering the composition or COF nanosheets via non-oral routes, such as, for example, injection.

[0073] In an aspect, the disclosure provides kits. A kit may comprise pharmaceutical preparations comprising COF nanosheets and printed material.

[0074] In various examples, a kit comprises a closed or sealed package that contains the pharmaceutical preparation. In various examples, the package comprises one or more closed or sealed vials, bottles, blister (bubble) packs, or any other suitable packaging for the sale, or distribution, or use of the compounds and compositions comprising compounds of the present disclosure. The printed material may include printed information. The printed information may be provided on a label, or on a paper insert, or printed on the packaging material itself. The printed information may include information that identifies the compound in the package, the amounts and types of other active and / or inactive ingredients, and instructions for taking the composition, such as the number of doses to take over a given period of time, and / or information directed to a pharmacist and / or another health care provider, such as a physician, or a patient. The printed material may include an indication that the pharmaceutical composition and / or any other agent provided with it is for treatment of a subject. In various examples, the product includes a label describing the contents of the container and providing indications and / or instructions regarding use of the contents of the container to treat a subject. A kit may comprise a single dose or multiple doses.

[0075] The steps of the method described in the various embodiments and examples disclosed herein are sufficient to carry out the methods of the present disclosure. Thus, in an embodiment, the method consists essentially of a combination of the steps of the methods disclosed herein. In another embodiment, the method consists of such steps.

[0076] The following Statements provide various examples of the present disclosure. They are not intended to be limiting in any way.Statement 1. A composition comprising a covalent organic framework (COF) nanosheet (e.g., an exfoliated COF nanosheet) comprising a co-condensate of triaminoguanidinium chloride (TGH.C1) and 2,6-diformylpyridine (DFP), wherein the COF nanosheet has a longest linear dimension of 150-300 nm, including all nanometer values and ranges therebetween.Statement 2. A composition according to Statement 1, wherein at least 90% of the COF nanosheets have a longest linear dimension of 300 nm or less.Statement 3. A composition according to claim 2, wherein at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or all of the COF nanosheets have a longest linear dimension of 300 nm or less.Statement 4. A composition according to any one of the preceding Statements, wherein the COF nanosheet comprises the following structure:wherein each R is independentlyStatement 5. A composition according to any one of the preceding Statements, wherein the COF nanosheet comprises the following structure:Statement 6. A composition according to any one of the preceding Statements, wherein the COF nanosheet is porous and at least partially crystalline.Statement 7. A composition according to Statement 6, wherein the COF is substantially crystalline or crystalline.Statement 8. A composition according to Statement 7, wherein the COF is substantially crystalline or crystalline at -40 °C.Statement 9. A composition according to any one of the preceding Statements, wherein the longest linear dimension remains the same at -40°C.Statement 10. A composition according to any one of the preceding Statements, wherein the COF nanosheets have a binding affinity for water molecules that increases as temperature decreases.Statement 11. A method for imaging one or more cancer cells comprising contacting a composition according to any one of the preceding Statements to i) an area of a subject suspected of having or having cancer cells or to ii) a sample suspected of having or having cancer cells and visualizing the COF nanosheets.Statement 12. A method according to Statement 11, wherein the COF is used during a cryoimaging (e.g., visualization is performed via cryo-TEM).Statement 13. A method according to Statement 11, wherein the nanoscale materials can circulate in the bloodstream and target tumors.Statement 14. A method according to Statement 11, wherein fluorescence intensity is selective for cancerous cells at low temperatures.Statement 15. A method according to Statement 14, wherein the low temperatures correspond to a range of -40 to -10 °C.Statement 16. A method according to Statement 14, wherein the composition is used to label and track tumors in a subject in need.Statement 17. A method according to Statement 11, wherein cancerous tissues and healthy / normal tissues are differentiated via fluorescence.Statement 18. A method according to Statement 17 wherein the fluorescence distinguishes boundaries between cancerous and healthy tissues.Statement 19. A method according to Statement 11, wherein the method is used for cancer diagnosis.Statement 20. A method according to Statement 11, wherein the subject in need has a cancer associated with solid tumors.Statement 21. A method according to Statement 11, further comprising measuring fluorescence or otherwise visualizing the COF nanosheets (e.g., via microscopy or imaging).

[0077] The following example is presented to illustrate the present disclosure. It is not intended to be limiting in any matter.EXAMPLE

[0078] This example provides a description of COFs of the present disclosure.

[0079] Described is a Covalent Organic Framework (COF), as a robust and biocompatible fluorescent probe designed specifically for real-time cryo-imaging. Its ability to maintain enhanced fluorescence at low temperatures, combined with superior biocompatibility and high specificity for cancer tissue, makes it an ideal tool for the precise detection of tumors while sparing adjacent healthy tissue. Additionally, nTG-DFP-COF retains its fluorescent properties in the presence of intracellular ice, enabling effective realtime monitoring during cryo-imaging. Our comprehensive in vitro, in vivo, and ex vivo studies confirm the structural integrity and functional efficacy of the COF, underpinning its potential for clinical use. This development underscores the versatility and transformativepotential of COFs in medical imaging, particularly in addressing the unique challenges of oncologic surgery.

[0080] Results and Discussion.

[0081] Exfoliation of TG-DFP-COF for Real-time Cryo-imaging Applications. Bulk TG-DFP-COF was synthesized according to previously established methods, primarily by the condensation of triaminoguanidinium chloride (TGH.C1, 8.46 mg, 0.06 mmol) with 2,6-diformylpyridine (DFP, 12.15 mg, 0.09 mmol) in 1,4-dioxane (2 mL, Figure 9). In the FT-IR spectra of TG-DFP COF, the lack of a C=O stretching vibration band at 1723 cm-1and a new band at 1629 cm-1indicate the formation of a C=N bond. The absence of the N-H stretching vibration at 3185 cm-1, usually linked to the amino group in TGH, further confirms an imine bond formation (Figure 10). For biomedical applications, converting the TG-DFP-COF in bulk form into a nanoscale form while ensuring water solubility and stability was crucial. To this end, a liquid exfoliation process (LEP) by ultrasonication for two hours was used to convert TG-DFP-COF in bulk form into nanosheets (nTG-DFP-COF, Figure la). This process resulted in 2D nanosheets with an average size of approximately 239 nm ± 47, as verified by TEM (Figures lb and 11). AFM analysis showed that after liquid exfoliation, the thickness of the sheets was reduced from micrometers to 40 nm, creating uniformly thin and shaped nanosheets (Figures 1c and 12). The resulting nTG-DFP-COF was dispersible in water, making it suitable for biological applications. The translucent solution shows a pronounced Tyndall effect (Figure 13), confirming the presence of highly monodispersed ultrathin nanosheets suspended in water.

[0082] Powder X-ray diffraction (PXRD) measurements were recorded to confirm the crystallinity of nTG-DFP-COF (Figure 2a). The PXRD pattern shows the most intense diffraction peaks at 20 = 5.56° (200), indicating the long-range order in the framework. In the wide-angle region, the reflection at 20 = 26.89° associated with the (003) plane suggests the adjacent TI- TI stacking of the 2D layers. Following the principles of reticular chemistry, crystal structure models were constructed based on the geometry of the building blocks; the COF structure was observed in the hexagonal space group P3, following a triangular sequence arrangement. The completed Pawley refinement supports the obtained unit cell with lattice parameters of a = 37.273 A, b = 35.525 A, and c = 12.30 A.

[0083] The crystalline nature of nTG-DFP-COF was further evidenced by latticeresolution TEM images, which showed consistent and continuous lattice fringes throughout the COF structure (Figures 2b and 2c). With lattice spacings of 0.35 nm, these fringescorresponded well to the dbo3 plane interlayer distances and the 7t- it stacking, closely aligning with the experimental wide-angle PXRD data at 0.34 nm (Figure 2d). Furthermore, the selected area electron diffraction (SAED, Figure 2e) demonstrates the well-crystallized feature of nTG-DFP-COF and has distinct electron diffraction spots that fit well with the bo3 plane with a tZ-spacing of 0.34 nm.

[0084] Given the material is designed for use at extremely low temperatures, possible structural changes in nTG-DFP-COF were evaluated under freezing conditions using TEM, AFM, and Variable Temperature (VT)-PXRD. TEM and AFM analyses showed no changes in the morphology or dimensions of nTG-DFP-COF nanosheets after freezing to-40 °C, confirming the robust structural stability (Figure 14). Additionally, VT-PXRD experiments conducted before, during, and after freezing showed consistent peak positions and intensities at different temperatures, indicating that the crystal lattice remains intact without any changes in lattice parameters (Figure 15). Overall, these results confirm the structural integrity and stability of nTG-DFP-COF and emphasize its suitability for cryogenic applications.

[0085] The aqueous solutions of the exfoliated nTG-DFP-COF nanosheets show stability and dispersibility, with no signs of precipitation over time. At a neutral pH of 7.4 in water, the hydrodynamic diameter of these nanosheets was measured to be 295 nm with a low poly dispersity index (PDI) of 0.1 compared to the bulk material, indicating a reduced and uniform particle size distribution (Figure 16). Larger particle sizes are generally observed in DLS than in TEM (240 nm) due to hydration layers and the potential for aggregation in solution, reflecting the interaction of the nanosheets with the solvent environment. The improved stability compared to the bulk material can be attributed to the significant decrease in the (^-potential of nTG-DFP-COF, which shifted from -4.8 ± 1.8 mV in the bulk material to -15.6 ± 0.3 mV. The stronger negative charge on the nanosheets promotes better interparticle repulsion and thus contributes to their stability. The hydrodynamic diameter of the nanosheets was measured in different solvents, including ethanol, PBS, FBS, and DMEM, after 24 hours and 1 week, as shown in Figure 2f. Initially, the stability in the different solvents varied, with DMEM showing the highest stability and ethanol having the lowest stability. The size generally increases slightly over one week, except in DMEM, where it decreases slightly, indicating strong colloidal stability. These results show that the nanosheets aggregate only minimally and are very stable, especially in physiological environments, emphasizing their suitability for biomedical applications.

[0086] Temperature-Dependent Optical and Fluorescence Properties of nTG-DFP-COF for Enhanced Cryo-imaging Applications. To utilize nTG-DFP-COF for cryo-imaging, the electronic properties of the exfoliated nanosheets were studied by UV-visible and steadystate fluorescence spectroscopy as a function of temperature.

[0087] The absorbance wavelength change on nTG-DFP-COF suspended in acetonitrile in a temperature range from -30 °C to 30 °C was measured. The data presented in Figure 3a indicate that absorbance increases as the temperature decreases, reaching a peak intensity at 420 nm when the temperature is at -30 °C. These data provide important insights into the thermal stability and responsive behavior of nTG-DFP-COF under various thermal conditions, underscoring its potential for temperature-sensitive applications. Also measured was the solid-state luminescence of nTG-DFP-COF over a broad temperature range from -35 °C to 35 °C. At -35 °C, nTG-DFP-COF exhibits strong green fluorescence (Aem= 545 nm, Figures 3b, 17, and 18) when excited at 400 nm, highlighting its robust photophysical properties. The inherent fluorescence of nTG-DFP-COF is enhanced at lower temperatures, likely due to the stabilization of hydrogen bonds within the COF networks, which reduces the non-radiative relaxation pathways and increases the emission intensity. As the temperature increases, the disruption of the weak hydrogen bonds leads to a significant reduction in emission intensity, decreasing by a factor of 5. The results demonstrate that the photoluminescence behavior is strongly temperature-dependent, suggesting that higher temperatures may increase structural flexibility, thereby promoting non-radiative relaxation processes.

[0088] To evaluate the optical properties of nTG-DFP-COF as fluorescent bioimaging probes in water, we used the IVIS® spectrum imaging system. Phantom imaging was performed with nTG-DFP-COF samples diluted in water at different concentrations, kept at 37 °C and in a frozen state (-40 °C, verified with a thermal imaging camera, Figure 19). A 465 nm bandpass excitation filter and a 540 nm emission filter were used to selectively capture fluorescence images (Figure 3c). The analysis revealed that the fluorescence intensities of the nTG-DFP-COF samples increase with concentration. All fluorescence intensities were normalized to photons / second / centimeter2 / steradian (p / s / cm2 / sr), with background intensities subtracted for accuracy. At 37 °C, the increase in fluorescence intensity was moderate and linear with concentration. In contrast, the increase was more pronounced in the frozen state and followed a linear trend. Using the data shown in Figure 3d, the average radiant efficiencies were quantified at 37 °C and -40 °C. The slopes of the radiant efficiency curves were 1.9 x 109and 6.0 x 109p / s / cm2 / sr per mg for the samples at37 °C and in the frozen state, respectively. This significant increase in efficiency in the presence of ice crystals suggests that ice formation does not affect the fluorescence intensity of the probe at low temperatures but can even enhance it. These results are consistent with our steady-state fluorescence studies (Figure 3a-b), which confirm that the structural integrity and fluorescence response of nTG-DFP-COF are maintained or even improved under frozen conditions.

[0089] To investigate the effect of temperature on the binding affinity of water molecules to the nTG-DFP-COF, molecular dynamics (MD) simulations were performed on a segment of the framework at two distinct temperatures, -40 °C and 20 °C. These simulations used the xTB-GFN2 method, which combines semi-empirical and tight binding quantum mechanical approaches, along with an implicit water solvent model. To further investigate the hydrogen bonding interactions, 15 explicit water molecules were introduced into the model, enabling a detailed analysis of the interactions between the water molecules and the nTG-DFP-COF (Figure 21a). The simulation system is confined to a sphere with a radius of 10.5 A by a repulsive potential to prevent the escape of water molecules. The MD simulations are carried out for 200 ps with a time step of 2 fs using an extended tight binding (XTB) program, and the trajectories are collected every 10 fs. The first 10 ps of the trajectory was discarded for equilibration, and the final 190 ps trajectory was used for analysis. The radial distribution function (RDF) between the oxygen atoms of water and the hydrogen atoms of nTG-DFP-COF was illustrated to illustrate different binding affinity at different temperatures (Figure 21b). In the radial distribution function (RDF), it was found that the peak intensity is significantly higher at -40 °C than at 20 °C, indicating that the binding affinity between water molecules and nTG-DFP-COF increases as the temperature decreases (Figure 21b).Moreover, the peak position shifts by 0.07 A at 20 °C compared to -40 °C, indicating stronger interactions between water molecules and nTG-DFP-COF at the lower temperature (Figure 21b). Integration of the RDFs reveals that at -40 °C, there are, on average, 2.26 water molecules in the first solvation shell (within a 3 A distance), whereas at 20 °C, there are only 1.45 water molecules. These results show that temperature significantly affects the binding affinity of water molecules to nTG-DFP-COF.

[0090] Mechanisms of Temperature-Dependent Fluorescence Enhancement in nTG-DFP-COF: Insights from Advanced Spectroscopic The fluorescence enhancement mechanism of nTG-DFP-COF at low temperatures was investigated using variabletemperature (VT) techniques, including NMR, FTIR, and XPS.

[0091] Variable temperature solid-state NMR experiments were first performed.Figure 4a presents the one-dimensional13C CP / MAS solid-state NMR spectra of nTG-DFP-COF at 20 °C (red lines) and 0 °C (blue lines). The spectra predominantly show peaks of imine (145 to 155 ppm) and aromatic carbons (115 to 145 ppm), with no significant shifts in carbon chemical shifts observed upon cooling. This stability in carbon chemical shifts can be attributed to the low sensitivity of13C nuclei to non-covalent interactions and the inherently broad13C signals in solid-state NMR, which may obscure subtle changes. However, the increased13C CP / MAS signal at 0 °C indicates reduced molecular motion and increased rigidity of the framework compared to room temperature. The enhancement of13C CP / MAS signal intensity at 0 °C reflects the increased efficiency of cross-polarization (CP), as molecular rigidity strengthens dipolar couplings between abundant nuclei (1H) and13C nuclei. The reduction in dynamic disorder at lower temperatures allows for more effective magnetization transfer, leading to improved polarization transfer efficiency and enhanced signal intensity. These observations confirm that the nTG-DFP-COF framework becomes more rigid and exhibits reduced molecular motion at lower temperatures.

[0092] Because1H chemical shifts are more sensitive to hydrogen bonding, changes in proton chemical shifts were monitored by two-dimensional3H-13C HETCOR (heteronuclear correlation) solid-state NMR experiments at 20 °C and 0 °C (Figure 4b). The spectra at both temperatures show correlation peaks between aromatic, imine, and amine protons with imine and aromatic carbons. Additionally, peaks corresponding to water molecules (~4.6 ppm) interacting with imine carbon atoms (-150 ppm) are observed, indicating the close proximity of water molecules to the guanidine moiety of the imine carbon atoms. At 0 °C, significant upfield chemical shifts (-2 ppm) for protons were observed, suggesting that non-covalent interactions become stronger and more ordered as the temperature decreases.

[0093] The nTG-DFP-COF framework, consisting of planar, electron-rich guanidine units with multiple nitrogen atoms, exhibits exceptional hydrogen bonding capabilities. As the temperature decreases, these non-covalent interactions become stronger, leading to increased proton shielding and corresponding upfield shifts in solid-state NMR. Research indicates that at low temperatures, the cationic guanidinium groups in arginine can selfassemble through hydrophobic interactions, forming stacked pair configurations. This assembly helps to offset the anticipated Coulomb repulsion. The resulting reduction in molecular vibrations and rotations at lower temperatures leads to a more rigid structure that enhances the shielding effects for protons involved in non-covalent interactions such as 7t-7tstacking or hydrogen bonding. As a result, nTG-DFP-COF shows enhanced fluorescence at lower temperatures due to the reduced non-radiative decay pathways. At higher temperatures, molecular vibrations and rotations dissipate energy non-radiatively, reducing fluorescence intensity. At lower temperatures, suppression of these processes allows more energy to be emitted as fluorescence. Additionally, enhanced non-covalent interactions improve electronic conjugation within the COF framework, further boosting fluorescence.

[0094] Low-temperature transmission FT-IR analysis was also performed on nTG-DFP-COF in a temperature range of -40 °C to 35 °C, with the sample diluted in KBr to prevent the saturation of the structural bands (Figure 4c). The self-supported pellet was first activated under vacuum at 40 °C for 12 hours to remove residual adsorbed water. The results show that at low temperatures, the mobility within the nTG-DFP-COF structure decreases, leading to stronger hydrogen bonding between the N-H groups and other electronegative atoms, such as nitrogen, within the COF structure. This increased hydrogen bonding leads to a contraction of the COF framework, effectively densifying the vibrational modes and decreasing the flexibility of the structure. This densification is reflected by an increase in the intensity of the structural vibrational bands in the range of 3100-2700 cm-1and 1700-1100 cm-1. Furthermore, stronger hydrogen bonding elongates the N-H bond, which reduces its bond strength and consequently lowers the vibrational energy. This change is manifested by the presence of an isobestic wavenumber at 3445 cm-1, demonstrating a shift of the N-H bond vibration from 3590 cm-1to 3300 cm-1with a symmetrical evolution of both vibrational bands with the temperature (Figure 4c). The densification of the structure explains the enhancement of both absorption / excitation and photoluminescence (PL) of nTG-DFP COF with decreasing temperature. The enhanced PL efficiency of nTG-DFP-COF at lower temperatures may be due to the COF’s reduced structural flexibility, which limits non-radiative relaxation pathways. This results from improved 7t-7t interactions, reduced non-radiative decay, stabilized excited states, and confined excitons, leading to increased PL intensity as the temperature drops.

[0095] The X-ray photoelectron spectroscopy (XPS) analysis of C Is and N Is levels in nTG-DFP-COF provides critical insights into its chemical structure and bonding environment, highlighting its significant temperature-dependent properties (Figure 4d and Table 2). At room temperature, the C Is spectrum features distinct peaks at 283.95 eV (C=C), 284.90 eV (C-N), and 286.13 eV (N~C=N), constituting 19.0%, 24.0%, and 51.25% of the spectrum respectively. These peaks underscore a highly ordered aromatic backbone essential for the COF’s structural integrity, ensuring robustness under ambient conditions.Additionally, the carbon-nitrogen bonds not only enhance the electronic properties but also contribute to the COF's fluorescence capabilities. The low residual standard deviation of 0.916029 across the material suggests a consistent and uniform chemical environment. At -40 °C, the primary peak at 284.55 eV increases to 77.19%, indicating the aromatic backbone's stability in colder conditions. Minor shifts and slight broadening of the secondary peak at 286.05 eV, accounting for 16.87% of the spectrum, suggest enhanced hydrogen bonding and reduced thermal vibrations, thus reinforcing the structure. The increased residual standard deviation to 1.25917 at this temperature points to a more complex electronic environment, likely a result of cryogenic stabilization. In the N Is spectrum, the room temperature profile decomposes into three primary peaks at 397.99 eV (C=N Pyridine), 399.64 eV (C-N*), and 401.4 eV (C-NH-N*), contributing 13.6%, 68.11%, and 10.8%, respectively (Figure 4d and table 3). These peaks play a vital role in the electronic stability and fluorescence enhancement of the COF. An additional weaker peak around 401.2 eV, likely representing protonated or oxidized nitrogen species, indicates environmental interactions. However, at -40 °C, the spectrum simplifies significantly; the C=N Pyridine and C-N* peaks merge into a broad peak at 398.45 eV, representing 96.94% of the spectrum, signifying enhanced nitrogen stabilization under cryogenic conditions. The diminished secondary peak at about 402.1 eV, contributing only 3.06%, underscores stronger hydrogen bonding and reduced thermal vibrations. The absence of the peak for protonated or oxidized nitrogen at this temperature suggests stabilization of these species within the cryogenic framework.

[0096] This comprehensive analysis using solid-state NMR, VT-FTIR, and XPS demonstrates the exceptional stability and functional adaptability of nTG-DFP-COF across various temperature ranges. The COF retains its structural integrity even under cryogenic conditions, exhibiting significant enhancements in hydrogen bonding and electronic stabilization. The fluorescence enhancement observed in nTG-DFP-COF at cryogenic temperatures arises from several interrelated factors that influence its photophysical properties. At low temperatures, the significant reduction in thermal energy decreases molecular vibrations and motions within the COF matrix. This reduction in molecular motion facilitates the formation and stabilization of hydrogen bonds, leading to a more rigid molecular structure. The increased rigidity effectively minimizes the non-radiative relaxation pathways, which are typically driven by molecular motions such as rotations and vibrations. With these non-radiative pathways becoming less active, the material predominantly favors radiative decay processes, resulting in increased photon emission rather than energydissipation as heat. This shift significantly enhances fluorescence intensity at lower temperatures. This direct link between increased molecular rigidity and a shift towards predominantly radiative relaxation processes underlines the unique photophysical characteristics of nTG-DFP-COF in cryogenic conditions.

[0097] In vitro Evaluation of nTG-DFP-COF as a Fluorescent Probe in Cancer and Normal Cell Lines. In vitro studies were performed on three different cell lines — HEK-293 (non-cancerous), HeLa (cervical cancer), and U251-MG (glioblastoma) — to evaluate nTG-DFP-COF as a biocompatible fluorescent probe for cryo-imaging. HEK-293 cells were used as a control to assess the probe’s response in normal cells, while HeLa and U251-MG cell lines were used to evaluate the efficacy and specificity of the probe in different cancer types. These experiments validate the suitability of nTG-DFP-COF for real-time cryo-imaging in both normal and cancerous cells.

[0098] The material showed minimal cytotoxicity in all cell lines at concentrations up to 1 mg / mL after 48 hours of incubation, confirming its excellent biocompatibility and potential for cellular labeling (Figure 22). Additionally, cytotoxicity assessments were further confirmed through MTS and lactate dehydrogenase (LDH) assays conducted after 24 hours of exposure to different concentrations of nTG-DFP-COF (Figure 23). The MTS assay indicated normal mitochondrial function, suggesting healthy cellular metabolism, while the LDH assay showed minimal release of LDH, indicating that cell membranes were largely intact. Longterm toxicity and cell proliferation studies tracked the growth of HeLa, U251-MG, and HEK-293 cells over a three-day period and analyzed daily cell counts and population doubling times (PDTs), which revealed no significant differences between treated and control groups, confirming that nTG-DFP-COF does not negatively affect cell proliferation (Figure 22 and Table 4). To evaluate the biocompatibility and immunotoxicity of our material designed for bloodstream penetration, we conducted a hemolysis assay on human erythrocytes. The hemolysis rates (HR) were below 3%, indicating non-hemolytic properties as per ASTM F 756-08 standards, which set a threshold of <5%. These results confirm that nTG-DFP-COF is biocompatible and does not induce immunotoxicity in human blood erythrocytes, likely due to the negatively charged surface of our nanoparticles that reduces hemolytic effects.Altogether, these results underscore the probe’s suitability for long-term cellular studies and its potential for safe biomedical applications.

[0099] We investigated the intracellular uptake of nTG-DFP-COF by HeLa, U251 -MG, and HEK-293 cells using TEM after 24 hours of incubation at a concentration of 10 pg / mL (Figure 5a). TEM analyses showed that the structural integrity of all cell lines waslargely preserved with minimal evidence of cell debris, indicating that nTG-DFP-COF is nontoxic and does not affect cell function (see Figure 25 for comparison with control cells). In HeLa and U251-MG cells, significant amounts of nTG-DFP-COF were detected both inside the cells and on their surface, indicating active uptake (Figures 5a and 26). Initially, the nTG-DFP-COF nanosheets come into contact with the cell membrane, followed by their engulfment. This progresses into deeper invaginations of the cell membrane, indicating early endosomal formation. Subsequently, the nanosheets are transported into large vacuoles, primarily located near the perinuclear region and in the cytoplasm. This sequence from plasma membrane engagement to endosomal localization highlights the active internalization process of nTG-DFP-COF in these cancer cell lines (Figure 5a and 26). Conversely, HEK-293 cells showed lower uptake of nTG-DFP-COF, suggesting it selectively interacts with cancer cells rather than non-cancerous cells. nTG-DFP-COF nanosheets target cancer cells effectively by exploiting their altered physiological pathways. These nanosheets use their size and surface charge to enhance entry into cells through endocytosis, a process more pronounced in cancer cells because of their higher metabolic activity. This leads to more internalization in cancer cells than in normal cells, potentially improving therapeutic outcomes. nTG-DFP-COF is preferentially taken up by cancer cells over HEK-293 cells, attributed to variations in cell surface charge, membrane thickness, and turnover rates, underscoring their selective targeting properties.

[0100] To distinguish passive from active internalization mechanisms of nTG-DFP-COF in HeLa and U251-MG cells, we analyzed the fluorescence signals under varying cell incubation temperatures (4 °C versus 37 °C). This approach takes advantage of the fact that nanomaterial entry into cells can occur via energy -independent passive mechanisms, such as direct translocation, or via energy -dep endent active mechanisms, such as endocytosis (Figures 27 and 28). In cells incubated at 4 °C, fluorescence was significantly weaker, indicating minimal passive uptake. In contrast, fluorescence significantly increased at 37 °C (HeLa: 90.1%, U251-MG: 88.5%), indicating that active mechanisms, predominantly endocytosis, are responsible for the uptake of nTG-DFP-COF. Further, subcellular localization was assessed in HeLa and U251-MG cells treated with nTG-DFP-COF for 4 hours. Confocal microscopy and co-staining with far-red organelle-specific markers revealed significant co-localization of nTG-DFP-COF with cell membranes and lysosomes, suggesting active internalization and intracellular transport (Figures 29 and 30). This localization supports the hypothesis that nTG-DFP-COF enters cells primarily by endocytosis. These results, supported by TEM data, confirm the effective intracellular transport and localizationof nTG-DFP-COF by endocytosis and highlight its potential for targeted bioimaging applications.

[0101] Next, it was demonstrated that nTG-DFP-COF has a specific freezing-induced turn-on function in cancer cells. Fluorescence micrographs (Figure 5b) show HeLa, U251-MG, and HEK-293 cells incubated with nTG-DFP-COF (10 pg / mL, Ux / cm = 469 / 525 nm) and LysoTracker Red (250 nM, Ac / cm= 531 / 593 nm), both at room temperature and in frozen condition (-10 °C, using cold ethanol, Figure 20). LysoTracker Red was chosen for comparison because, as a traditional organic dye, it faces challenges under cryo-imaging conditions, such as dye aggregation and fluorescence quenching due to ice formation. In addition, although LysoTracker Red passively diffuses into cells and accumulates in the acidic organelles of all cell types, it does not selectively target cancer cells. By comparing the luminescence responses of LysoTracker and nTG-DFP-COF at different temperatures and in different cell types, we aim to evaluate the selective cancer-targeting and turn-on properties of nTG-DFP-COF.

[0102] At room temperature, HeLa and U251-MG cells co-stained with nTG-DFP-COF and LysoTracker red emit weak green fluorescence under 469 nm light and show strong red fluorescence under 531 nm light (Figure 5b). After freezing, the fluorescence intensity of nTG-DFP-COF in HeLa and U251-MG cells increases significantly. In contrast, the fluorescence intensity of LysoTracker red decreases considerably due to ice formation in the cells (Figure 5b). The fluorescence intensity of nTG-DFP-COF increases by 3.8- and 3.7-fold in HeLa and U251-MG cells, respectively, while that of LysoTracker red decreases by almost 3.0- and 3.7-fold in HeLa and U251-MG cells, respectively (Figure 5c). This decrease in fluorescence of the conventional dye is mainly attributed to aggregation-induced quenching and low photostability. These results clearly show that nTG-DFP-COF has a turn-on feature that contrasts with the turn-off property typical of conventional dyes (Figure 5d). This turn-on property of nTG-DFP-COF provides lower background interference and a higher signal-to-noise ratio, which improves real-time imaging capabilities.

[0103] Another essential prerequisite for real-time imaging during cryosurgery is distinguishing tumor tissue from the surrounding normal tissue. Using the non-cancerous HEK-293 cells, we investigated the imaging cancer specificity of nTG-DFP-COF. HEK-293 cells show weak green fluorescence at room temperature when stained with nTG-DFP-COF and strong red fluorescence when stained with LysoTracker Red. LysoTracker Red passively diffuses into cells, both malignant and non-malignant. This dye does not selectively target cancer cells. In contrast, due to their higher endocytosis rate, HeLa and U251-MG cells showgreen fluorescence under the same conditions (Figure 5b). This demonstrates that nTG-DFP-COF selectively targets cancer cells. To further evaluate the utility of nTG-DFP-COF for selective imaging of cancer cells after freezing, it was observed that green and red fluorescences are particularly weak in frozen HEK-293 cells. In contrast, frozen HeLa and U251-MG cells exhibit strong green emission (Figure 5b-c). The selective behavior of nTG-DFP-COF was confirmed with cancer cells at room temperature and sub -freezing temperatures (Figures 5b-c), highlighting the excellent specificity of the probe for cancer cells. This clear contrast between normal and cancer cells enables precise cryo-imaging planning (Figure 5d).

[0104] In Vivo Evaluation of nTG-DFP-COF Biosafety. To assess the biosafety of nTG-DFP-COF in vivo, comprehensive toxicity studies in CD-I mouse model were performed. Mice received a single intraperitoneal injection of nTG-DFP-COF at a dose of 20 mg / kg, while a saline-injected group served as a control (Figure 6a). Over an observation period of 7 days, there were no significant changes in body weight, behavior, or survival rates in the treated mice, indicating no acute toxicity (Figure 6b). The injection sites showed no signs of irritation, and behavioral monitoring confirmed no distress in the treated animals. Seven days after treatment, the animals were sacrificed and major organs were harvested for analysis. Ex vivo bioluminescence imaging and subsequent pathohistological evaluations, including H&E staining of organs, showed no significant differences or morphological changes compared to controls (Figures 6c and 6d). These results indicate effective clearance of nTG-DFP-COF and confirm the absence of systemic toxicity, confirming the compound's biocompatibility and safety in an acute toxicity model.

[0105] Ex Vivo Evaluation of nTG-DFP-COF for Tumor Cryo-imaging. To assess the utility of nTG-DFP-COF for cryo-imaging, we conducted an ex vivo study using an orthotopic U251-MG glioblastoma model. Tumors in mice reached approximately 75-100 mm3before the mice were humanely euthanized and the tumors excised for analysis. Each tumor was injected with either nTG-DFP-COF ([nTG-DFP-COF] = 20 mg / kg, 200 pL) or PBS as a control. Following imaging at 37 °C, the tumors were flash-frozen in liquid nitrogen for 10 minutes to mimic cryosurgery and assess the response of nTG-DFP-COF in a frozen state (-40 °C, verified with a thermal camera, Figure 33), the necessary temperature to destroy malignant cells in vivo.

[0106] Fluorescence imaging used the IVIS® Spectrum to quantitatively analyze the emitted fluorescence of nTG-DFP-COF before and after the freezing process. At 37 °C, both the control tumors and the tumors treated with nTG-DFP-COF showed weak emissions.However, tumors injected with nTG-DFP-COF and subsequently frozen exhibited significantly increased fluorescence (Figure 7a). The fluorescence intensity decreased significantly when the tumor temperature was brought back to the physiological level of 37 °C. We quantified the total radiant efficiency of nTG-DFP-COF in tumors. We observed a substantial increase in emission in the frozen state compared to physiological temperature, underscoring the enhanced radiance efficiency of nTG-DFP-COF under cryosurgical conditions (Figure 7b). After rewarming to physiological value, the fluorescence intensity decreased significantly. This distinct variance in fluorescence between physiological and frozen states suggests that nTG-DFP-COF are promising probes for real-time cryo-imaging. This ex vivo approach has preliminarily validated the efficacy of nTG-DFP-COF for cryo-imaging, demonstrating its capability to activate fluorescence at cold temperatures — a key property for visualizing cancer tissues under cryo-surgical conditions.

[0107] Post-mortem Tumor Targeting and Cryo-imaging in Tumor-Bearing Mice. Athymic NU / J nude mice bearing subcutaneous U251-MG glioblastoma tumors (75-100 mm3) were used to evaluate the tumor-targeting efficacy of nTG-DFP-COF. Mice received a single intraperitoneal injection of nTG-DFP-COF at a dose of 20 mg / kg (200 pL volume). Tumor-specific fluorescence was monitored at 0, 24, and 48 hours using the IVIS® Spectrum imaging system. For comparison, a control group received intraperitoneal injections of PBS solution. Animals were euthanized at three time points — immediately after injection and 24 and 48 hours post-injection — to assess the biodistribution of a probe within the tumors using post-mortem whole-mouse IVIS® Spectrum imaging (Figure 7c). The results quantified the radiance efficiency of nTG-DFP-COF at physiological temperature (37 °C). No fluorescence emission was observed in the control tumors, while the tumors treated with nTG-DFP-COF showed a progressive increase in fluorescence signal that reached its maximum after 24 hours of injection. This peak indicates efficient accumulation and retention of nTG-DFP-COF in the tumor, demonstrating its potential for targeted tumor therapy. After 48 hours, a decrease in fluorescence intensity was observed, indicating that the probe was being cleared from the tumor site.

[0108] Additionally, targeted cryo-imaging was conducted on living mice with implanted subcutaneous U251-MG glioblastoma tumors (Figure 7d). Mice received intraperitoneal injections of nTG-DFP-COF at a dose of 20 mg / kg, 24 hours before imaging to ensure localization of nanoparticles in the tumor cells. Post-mortem IVIS® imaging at 37 °C showed significantly stronger fluorescence in tumors treated with nTG-DFP-COF compared to controls, demonstrating effective passive accumulation and targeted delivery ofthe probe. When the temperature was lowered to 4 °C and -20 °C, the fluorescence intensity in the treated tumors increased by 10% and 20%, respectively. This temperature-sensitive fluorescence enhancement significantly improves the visibility of tumors, potentially facilitating more precise and effective cryogenic treatment.

[0109] Conclusion. As described herein, a nanoscale Covalent Organic Framework (nTG-DFP-COF) was developed and optimized for real-time cryo-imaging. By liquid exfoliation, water-dispersible nanosheets of nTG-DFP-COF were prepared. These nanosheets that are biocompatible and can selectively accumulate in cancer cells without inducing cytotoxic effects. These nanosheets exhibit turn-on fluorescence at lower temperatures, which significantly improves their ability to delineate cancer tissue precisely in cryogenic conditions. The lower thermal energy at these temperatures reduces molecular vibrations and promotes the formation and stabilization of hydrogen bonds. This leads to a more rigid molecular structure that reduces non-radiative relaxation pathways like rotations and vibrations, thereby enhancing the likelihood of radiative decay processes. The increased molecular rigidity and the dominance of radiative relaxation contribute to a significant increase in fluorescence intensity, which greatly improves the visibility of tumors during cryogenic procedures.

[0110] The structural integrity and functional efficacy of nTG-DFP-COF, as well as its proven biosafety, provide a solid foundation for its potential clinical applications. This innovative material has been extensively tested for biocompatibility and has shown minimal toxicity and adverse reactions in biological settings, which supports its use in clinical settings. This characteristic is critical to the further development of nTG-DFP-COF as a safe tool that significantly improves the accuracy and safety of surgical procedures by enabling targeted visualization of cancerous tissue in real-time, thereby minimizing the risk to adjacent healthy tissue.[OHl] The selection of excitation and emission wavelengths (400-450 nm for excitation and 520 nm for emission) is tailored to surface tumor imaging, where lower penetration depths are beneficial and sufficient for precise imaging. This method improves resolution and contrast in superficial tissues and provides clearer images of surface-level structures.

[0112] The breakthrough properties of nTG-DFP-COF represent a significant advance in surgical oncology, particularly in the cryogenic treatment of resistant cancers. This advance not only holds the potential to improve surgical outcomes but also paves the way for integrating diagnostic and therapeutic functions, including drug delivery, in a single platform.This makes nTG-DFP-COF a transformative cryo-imaging tool that could revolutionize oncologic surgery and improve the effectiveness of cancer treatments.

[0113] Table 1. Comparative Analysis of Fluorescent Probes for Cryo-imaging.This table presents a detailed comparison of various fluorescent probes highlighting their test conditions, models used, temperature sensitivity, temperature range, biocompatibility, specificity, cytotoxicity, and additional features.<<

[0114] General Materials and Methods. All reagents and starting materials were purchased from Sigma-Aldrich and used without further purification. Deionized water was used from Millipore Gradient Milli-Q water purification system. Thin-layer chromatography (TLC) was performed on silica gel 60 F254 (E. Merck). The plates were inspected under UV light. Column chromatography was performed on silica gel 60F (Merck 9385, 0.040-0.063mm). Infrared spectra were recorded on an Agilent Technologies Cary 600 Series FTIR Spectrometer using the ATR mode. The samples’ PXRD patterns were recorded using an X-ray Panalytical Empyrean diffractometer. High-resolution transmission electron microscopy (HRTEM) images were obtained using a Talos F200X Scanning / Transmission Electron Microscope (STEM) with a lattice-fringe resolution of 0.14 nm at an accelerating voltage of 200 kV equipped with CETA 16M camera. The high-resolution images of periodic structures were analyzed using Velox software. The topography of the samples was analyzed by atomic force microscopy (JPK Bio AFM; Bruker Nano GmbH, Berlin, Germany). AFM scans were collected using JPK NanoWizard® software in QI™ advance imaging mode. In this mode, optimal image setting was achieved by autocalibration of cantilever and sample properties. Silicon cantilevers (NanosensorsTM, Neuchatel, Switzerland) with resonant frequencies of 250-300 kHz and force constants of 100-130 Nm-1were used. The set point value of 80 nN was used for imaging. AFM scans were collected at 512 points / lines with a scan speed of 50 pm / s at a fixed scan angle of 0°. Scan artifacts were minimized by acquiring a typical scan at an angle of 90° under identical image acquisition parameters. After imaging, the sample data was loaded to JPK Data processing software and further post-processed using Gwyddion™ free software (version 2.47), an SPM data visualization and analysis tool. Dynamic light scattering (DLS) measurements were performed on a Malvern Zetasizer NanoSeries to determine the Zeta(Q-potential as well as the hydrodynamic size of the particles. All samples were analyzed at room temperature in 10 mM phosphate-buffered saline (PBS). Each experiment was performed in triplicate. In vitro, ex vivo, and post-mortem fluorescence imaging was performed using the IVIS Spectrum (Revvity / Perkin Elmer, USA), and images were analyzed with Living Image software. The IVIS Spectrum is equipped with a cooled CCD camera (- 90°C) featuring a 2048 x 2048 sensor and a pixel size of 13.5 pm. Cells images were acquired using a Lionheart FX automated microscope.

[0115] Synthesis.

[0116] Synthesis of the linkers. 2,6-diformylpyridine (DFP) was synthesized according to published procedures with no modification.

[0117] Triaminoguanidinium chloride (TGH.C1) was synthesized according to published procedures with no modification.

[0118] Synthesis of TG-DFP-COF. TG-DFP-COF was synthesized according to a published procedure by mixing an aqueous solution (0.5 mL) of triaminoguanidinium chloride (TGH.C1, 8.46 mg, 0.06 mmol) with a solution of 2,6-diformylpyridine (DFP, 12.15 mg, 0.09 mmol) in 1,4-di oxane (2 mL), which was gradually added. The resulting mixturewas then stirred and subjected to microwave irradiation at 100 °C for 30 minutes (Figure SI). After heating, the mixture was allowed to cool to room temperature, the yellow-colored product, TG-DFP-COF, was collected via centrifugation and washed sequentially with 1,4-dioxane, ethanol, and water.

[0119] Characterizations.

[0120] Fourier Transform Infrared (FTIR) Spectroscopy. In the FT-IR spectra of nTG-DFP-COF, the absence of the C=O stretching vibration band at 1723 cm-1and the emergence of a new band at 1629 cm-1confirm the formation of a C=N bond. Additionally, the disappearance of the N-H stretching vibration band at 3185 cm-1, typically associated with the amino group in TGH, further substantiates the formation of imine bonds.

[0121] High-Resolution Transmission Electron Microscopy (HRTEM). The samples were prepared on holey carbon film mounted on a copper grid. A drop of diluted particle solution was spotted on the grid and dried overnight at room temperature (298 K).

[0122] Atomic Force Microscopy (AFM) was performed. See Figures 12-13.

[0123] Stability of nTG-DFP-COF After Freezing Process. See Figures 14-16.

[0124] Solid State Temperature-Dependent Luminescent Study. Solid state emission spectra were recorded using an FLS1000 spectrometer (Edinburgh, UK) equipped with an SC- 10 as the sample holder with a front-face geometry, which also used a Xenon lamp to excite the sample at 320 and 375 nm with long pass filters (LPFs) at 375 (or 395 nm) and 420 nm, respectively, which were placed between the sample holder and the emission monochromator. A thermoelectrically cooled four-window cuvette holder was used together with an instrument software-powered controller to enable stable control of the sample temperatures, from 263 to 378 K.

[0125] Thermal Imaging. The thermal images were recorded using a FLIR E60bx thermal camera.

[0126] In vitro Fluorescence Imaging of nTG-DFP-COF. Fluorescence imaging of nTG-DFP-COF at varying concentrations (0.2 mg / mL to 1 mg / mL) was performed using the IVIS Spectrum system (Revvity, USA) with a 465 nm excitation filter and a 540 nm emission filter. The nTG-DFP-COF solutions were first warmed to 37 °C to simulate physiological conditions, then rapidly cooled to -40 °C using liquid nitrogen. Fluorescence images were analyzed by drawing regions of interest (ROIs) of consistent size within the Eppendorf tubes.

[0127] Solid-State NMR Spectroscopy. Magic Angle Spinning (MAS) solid-state NMR experiments were carried out on a Bruker Avance-HD 600 MHz spectrometer operating at a static field of 14.1 T using a 4.0 mm MAS probe. Powdered dry samples werepacked into 4.0 mm zirconia rotors and were spun at a MAS frequency of 14 kHz. Cross-Polarization Magic Angle Spinning (CP / MAS) experiments were performed using a standard linearly ramped cross-polarization pulse sequence.13C chemical shifts were externally referenced to the adamantane CH2 signal at 38.48 ppm on the TMS scale. NMR data were processed using TopSpin software.

[0128] In-Situ -FTIR Spectroscopy. The in-situ FTIR measurement was performant using a homemade in-situ FTIR reactor equipped with CaF2 windows (PELICAEN cell). 1 wt.% of nTG-DFP-COF was diluted in KBr and prepared as a self-supported pellet (107 Pa / cm2, S = 2 cm2, m~ 100 mg). The pellet was activated at 40°C under vacuum overnight in order to eliminate the physisorbed water. Then, the measurement was conducted under He to enhance thermal conductivity, and the temperature of the pellet was controlled using an external cryostat connected to the cell body. IR spectra were recorded with a Nicolet IS50 spectrophotometer (Thermo Fisher Scientific), equipped with a DTGS detector and an extended-KBr beam splitter in the region between 400 and 5500 cm-1.

[0129] X-Ray Photoelectron (XPS) Spectroscopy. X-ray photoelectron spectroscopy (XPS) experiments were carried out on a Kratos Axis Ultra DLD spectrometer under a base pressure of ~ 2xlO-10mbar. A monochromated Al Ka X-ray source (1486.69 eV) irradiated samples at room temperature. XPS spectra were recorded from an analysis area of 700 pm x 300 pm. High-resolution XPS data of core levels were obtained with an energy resolution of 0.05 eV. For consistency, XPS measurements were calibrated to Cis (~ 285 eV). Data were analyzed using CasaXPS package with Shirley background subtraction.

[0130] Table 2. Binding energy (eV) and peak % for C Is spectrum deconvolution curves of nTG-DFP-COF at room temperature and -40 °C (instrumental error is ±0.2eV).

[0131] Table 3. Binding energy (eV) and peak % for N Is spectrum deconvolution curves of nTG-DFP-COF at room temperature and -40 °C (instrumental error is ±0.2eV).

[0132] In vitro Biological Studies

[0133] Cell Culture. Human malignant cervical carcinoma (HeLa, ATCC No. CCL-2), glioblastoma (U251-MG, ATCC No. 09063001) and non-cancer Human Embryonic Kidney 293 (HEK293; ATCC No. CRL-1573) cell lines were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 1 % penicillin / streptomycin and 20 mL L-glutamine at 5 % CO2 and 37 °C.

[0134] In vitro Biocompatibility Assessment

[0135] Cell Viability. Cell viability was assessed using CellTiter-Blue® Cell Viability assay (CTB, Promega). The assay measures the metabolic reduction of a non-fluorescent compound, resazurin, into a fluorescent product, resofurin, in living cells. As non-viable cells rapidly lose their metabolic activity, the amount of the resofurin product can be used to estimate the number of viable cells following treatment. Once produced, resofurin is released from living cells into the surrounding medium. Thus, the fluorescence intensity of the medium is proportional to the number of viable cells present.

[0136] 96-well plates were seeded with HEK-293, HeLa, and U251-MG (-5,000 cells per well in 100 pL of DMEM) and incubated at 37 °C for 24 hours. The medium was removed and replaced with fresh medium (control) or various concentrations of nTG-DFP-COF and incubated at 37 °C for 48 hours. After that, cells were incubated with 80 pL DMEM and 20 pL of CTB per well for 6 hours at 37 °C. The fluorescence of the resofurin product (^ex / em 560 / 620) was measured. Untreated wells were used as control.

[0137] The percentage of cell viability was calculated using the following formula:Viability (%)=[(Ftreated - Fblank) / (F control-Fblan k)] x 100All assays were conducted in triplicate.

[0138] MTS assay. Cells were seeded at a density of 500 cells per well in 96-well tissue culture plates and incubated at 37°C in a humidified 5% CO2 atmosphere to facilitatecell attachment. After 24 hours, cells were treated with either nTG-DFP-COF (10 pg / mL) or no additive (control). Following 24 hours of treatment, 20 pl of CellTiter 96® AQueous One Solution Reagent was added to each well, which already contained 100 pl of culture medium, and further incubated at 37 °C for 1-4 hours in a humidified 5% CO2 atmosphere. Cell viability was then assessed by measuring the absorbance at 490 nm using a Cytation 5 Microplate Reader (BioTek Instruments, Inc.). Statistical analyses were performed using GraphPad Prism software, with one-way ANOVA utilized to evaluate differences between treatment groups; significance was set at p < 0.05. All experiments were conducted in triplicate, and results were presented as mean ± standard deviation.

[0139] LDH Release Experiment. To assess the ability of nTG-DFP-COF to disrupt the plasma membrane of the cancer cells, the detection of lactate dehydrogenase (LDH) release from treated cells was performed using LDH-Glo™ Cytotoxicity Assay (Promega #J2380). Cells were incubated for 24 hours with no additives (control) or nTG-DFP-COF (10 pg / mL). After 24 hours of incubation, 5 pL of the medium was diluted in 50 pL of LDH storage buffer and transferred into a new 96 well plates, and then, 50 pL of LDH detection reagent was added. Luminescence was recorded After 30 min of incubation using a Cytation 5 multimode reader (Biotek)

[0140] Cell Growth Analysis. Cells were seeded at a density of 500 cells per well in 96-well tissue culture plates and incubated at 37 °C in a humidified 5% CO2 atmosphere to facilitate cell attachment. After 24 hours, the cells were treated with either nTG-DFP-COF (10 pg / mL) or received no additive as a control. Subsequently, the plates were transferred to a Lionheart FX Automated Microscope (BioTek Instruments, Inc.) for live-cell imaging over a period of 72 hours. Images were captured every 4 hours using 4 / brightfield settings to dynamically monitor cell growth.

[0141] Cell numbers in each well were quantified using Gen5 software (BioTek Instruments, Inc.) from the acquired images. Growth curves illustrating cell count over time were generated, and the cell growth dynamics were analyzed using the exponential Malthusian growth equation via GraphPad Prism software. All experiments were conducted in triplicate, and the results were reported as the mean ± standard error of the mean (SEM).

[0142] Table 4. Population Doubling Tinies (PDTs) for Control and nTG-DFP-COF Treated Cells. This table presents the population doubling times (PDTs) for HeLa, U251-MG, and HEK-293 cell lines over a three-day period. PDTs, calculated from the slope of the natural log of cell numbers plotted against time, indicate the time required for the cell population to double during the early log growth phase. The data compares PDTs betweencontrol groups and cells treated with 10 pg / mL of nTG-DFP-COF. As shown, the PDTs in treated groups are similar to those in control conditions, demonstrating that nTG-DFP-COF does not significantly affect cell proliferation.

[0143] Hemolysis Assay. When the external membrane of the erythrocytes is destroyed, hemoglobin is released. It is possible to estimate the amount of destroyed erythrocytes in a given test by measuring the quantity of hemoglobin in a sample by spectrophotometry.

[0144] Human blood was obtained from 3 healthy donors. 2.0 mL of an ethylenediaminetetraacetate-stabilized blood sample was added into 4 mL of physiological saline buffer (PBS), and then red blood cells were isolated by centrifugation (3000 rpm, 8 min). The red blood cells were washed five times with physiological saline and diluted into 2 % red blood cell suspensions.

[0145] Subsequently, nTG-DFP-COF (0.5, 1.0 and 2.0 mg.mL-1) was added into the red blood cell suspensions at the predetermined concentration and mixed using a gentle vortex. Meanwhile, physiological saline with or without Triton X-100 (0.3 %) was added into the red blood cell suspensions as negative and positive controls, respectively. Samples were placed in a static condition at 37 °C for 1 h. Finally, all samples were centrifuged at 5000 rpm, and 100 pL of the supernatant was placed into a 96-well plate for detection at the wavelength of 540 nm. The hemolysis ratio (HR) represents the degree of red blood cell membranes destroyed in the samples." "100^ Apos .it .ive contro .l — ^ Anegative contro .lAsampie, Apositive control, and Anegative control represented the absorbance of the sample, the positive control, and the negative control, respectively. These tests were performed in triplicate.

[0146] Intracellular Distribution Study Using TEM. For TEM analysis, HEK-293, HeLa, and U251-MG cells were seeded in T75 flasks in complete DMEM and incubated for 24 hours with cell-medium alone (control) or nTG-DFP-COF (10 pg / mL) in DMEM. After harvesting, cell pellets were washed twice with PBS. The cells were cryo-fixed within a fewmilliseconds at a pressure of 2000 bar under liquid nitrogen using a high-pressure freezer (Leica Microsystems, Germany). After freezing, the sample pod was released automatically into a liquid nitrogen bath. While still in liquid nitrogen, the sample carrier was separated from the specimen pod using precooled fine-tipped tweezers and transferred to the cryotransfer storage box for the flat specimen carrier, where the samples were stored in preparation for freeze substitution. Freeze substitution was performed using an automatic freeze substitution (AFS) unit (Leica EM AFS2, Heerbrugg, Switzerland) in a 10 mL solution of cold, dry absolute acetone (v / v) containing 1 % osmium tetroxide (w / v), 0.5 % uranyl acetate (w / v) and 5 % distilled water (v / v). The AFS unit was slowly warmed from -90 °C to 0 °C (2 °C / hour), with the temperature being held at both -60 °C and -30 °C for 8 hours. Samples were transferred to room temperature in a closed container to prevent condensation, rinsed with absolute acetone (3 x 5 minutes), and infiltrated with 30, 60, and 100 % Epon resin for 3 hours each. Epon was exchanged, and individual samples were embedded in 1 mL Eppendorf® lids for 24 hours at 60 °C. Finally, the samples were sectioned with an ultramicrotome at room temperature using a diamond knife, and the ultrathin sections were examined under TEM (Talos F200X STEM). The experiment was performed in triplicate.

[0147] In vitro Internalization Study by Fluorescence Microscopy. HeLa and U251-MG cells were seeded on sterile coverslips in complete DMEM and incubated for 24 hours to allow cell attachment. Cells were then treated either with no additives (control) or with nTG-DFP-COF (10 pg / mL) and incubated at temperatures of 4 °C and 37 °C for 4 hours. After treatment, the cells were washed with PBS, fixed with a 3.7% paraformaldehyde solution for 10 minutes, and then washed three times with PBS. The coverslips were mounted onto microscope slides using mounting medium. The samples were then analyzed using fluorescence microscopy. Each sample was analyzed in duplicate, and the entire experiment was repeated three times.

[0148] In vitro Organelle Co-Localization Study by Confocal Microscopy. HeLa and U251-MG cells were seeded on sterile coverslips in complete DMEM and incubated for 24 hours. Cells were incubated for 4 hours without additives (control) or with nTG-DFP-COF (10 pg / mL). Cells were stained with organelle markers to understand the internalization of nTG-DFP-COF. Cells were incubated for 30 min with either LysoTracker™ 647 Deep Red (labeling lysosomes and endosomes), NucleusTracker™ Deep Red FM (labeling the nucleus), or CellMask™ 647 Deep Red (labeling the membrane), followed by three cycles of PBS washing. Then, for each experiment, the cells were fixed with formaldehyde solution(3.7 %) for 10 min, followed by washing thrice with PBS. The cells were kept for 5 min in PBS during washing cycles. The coverslips were then fixed onto a microscope slide.The intracellular internalization of nTG-DFP-COF was observed by confocal microscopy (Olympus FV1000MPE), measuring the fluorescence signal of nTG-DFP-COF (kex= 410 nm) in the cells and the fluorescence emission of the 3 organelle markers labeling the plasmic membrane, lysosomes, and mitochondria (kex= 561 nm).

[0149] Bioimaging Experiments. HeLa, U251-MG, and HEK-293 cells were seeded in 6-well plates at a density of 2* 105cells per well in complete DMEM and incubated for 24 hours to allow attachment and growth. Following the initial incubation, cells were subjected to two different treatments. For the control group, cells were incubated for an additional 24 hours without any additives. For the treatment group, cells were incubated with nTG-DFP-COF (10 pg / mL) and simultaneously stained with LysoTracker Red (250 nM) for 15 minutes during the last part of the 24-hour period. After treatment, cells in all groups were washed with PBS and fixed with a 3.7% paraformaldehyde solution for 10 minutes, then washed three times with PBS.

[0150] Both treated and control samples were analyzed using a Lionheart FX automated microscope at room temperature and in a frozen state (-10°C, using cold ethanol, with the temperature verified by a thermal camera). We acquired green fluorescence images utilizing a GFP filter cube (Agilent, Part Number: 1225101, excitation wavelength 469 / 35 nm, emission wavelength 525 / 39 nm). For imaging with LysoTracker Red, we employed an RPF filter cube (Agilent, Part Number: 1225103, excitation wavelength 531 / 40 nm, emission wavelength 593 / 40 nm). Each sample was assayed in duplicate, and the entire experiment was repeated three times to ensure the reproducibility and reliability of the results.

[0151] In vivo Biological Studies. All animal experiments were conducted in compliance with the policies of the New York University Institutional Animal Care and Use Committee (IACUC). Athymic NU / J nude mice and CD-I mice (4-6 weeks old, approximately 20 g) were housed under standard conditions with 12-hour light / dark cycles and provided ad libitum access to food and water. The study protocols were approved by the Institutional Animal Care and Use Committee of NYU AD, and protocols were conducted in accordance with the guidelines outlined in the National Institute of Health Guide for Care and Use of Laboratory Animals (IACUC protocol number: 21-0003).

[0152] In Vivo Toxicity and Biocompatibility Assessment of nTG-DFP-COF in CD-I Mice. CD-I mice were randomly divided into two groups (n=3) and treated with either 0.1 mL saline (control) or nTG-DFP-COF (20 mg / kg in 200 pL) by intraperitoneal injection.Body weight was recorded every 2 days for 7 days. No behavioral changes were observed in the treated mice compared to the control group, indicating that no acute toxicity occurred at the administered dose. The survival rate was 100% throughout the study, with no visible signs of irritation at the injection site, such as swelling, redness, pain, or heat. Behavioral observation also confirmed that the treated animals experienced no pain or discomfort.

[0153] Seven days after treatment, the mice were sacrificed, and the major organs, such as the liver, spleen, kidneys, and heart, were removed. Each organ was rinsed with PBS. Ex vivo bioluminescence imaging was performed using the IVIS® Spectrum imaging system to investigate possible organ-specific accumulation and toxicity. Images were analyzed with Living Image software, which used a spectral unmixing approach to distinguish the specific fluorescence signal of nTG-DFP-COF from the inherent fluorescence background. The autofluorescence background was quantified for accurate comparison with untreated control mice.

[0154] Histopathology: The main organs (liver, spleen, kidneys, and heart) were harvested and processed for histologic examination. The tissue sections were stained with hematoxylin and eosin (H&E) and viewed under an optical microscope (Leica DMI 6000) to assess any histopathological changes.

[0155] Tumor Model Establishment. The U251-MG cancer model was employed as an example of cancer. 5* 106U251-MG cells suspended in 200 pL of DMEM medium were injected subcutaneously into the right axillary region of nude mice. Tumor volumes were consistently monitored using calipers every two days post-injection. The volume was calculated using the formula V=0.5*length* (width)2. Once the tumors reached a size of approximately 75-100 mm3— typically about 10 days post-inoculation.

[0156] Ex vivo Cryo-imaging. To evaluate the potential of nTG-DFP-COF for image-guided cryosurgery, we conducted an ex vivo study utilizing an orthotopic U251-MG glioblastoma model. Upon the tumors reaching a volume of approximately 75-100 mm3, the tumor-bearing mice were humanely euthanized using CO2, and the tumors were excised for further analysis.

[0157] Each excised tumor was then treated with one of two treatments: nTG-DFP-COF at a concentration of 20 mg / kg in 200 pL (n=4) or phosphate-buffered saline (PBS) as a control (n=4). Initial imaging of the tumors was performed at a physiological temperature of 37 °C using the IVIS® Spectrum system (Revvity, USA).

[0158] Following the initial imaging, the tumors from each treatment group were rapidly frozen in liquid nitrogen for 10 minutes and imaged again. This step was intended to simulate the effects of cryosurgery.

[0159] Ex vivo fluorescence imaging of the excised tumors was carried out both at 37 °C and after the freezing process. Imaging settings on the IVIS® Spectrum were configured as follows: a 465 nm excitation filter, 540 nm emission filter, binning factor of 8, f / stop of 2, field of view set to ‘C’, and automatic exposure time determined by the system. For quantitative analysis, regions of interest (ROIs) of uniform dimensions were drawn around each tumor, and the total radiance efficiency (normalized data) was calculated.

[0160] Local Cryotherapy in Tumor-Bearing Mice. To investigate the feasibility of local cryotherapy in tumor-bearing mice, we employed a nitrogen freeze spray in postmortem experiments to cool tumors to -10 °C and carefully monitored them with a thermal imaging camera to ensure precision and safety (Figure 34). This method allowed the targeted application of cold temperatures specifically to tumor sites without affecting the adjacent tissue.

[0161] Post-mortem Biodistribution and Cryo-Imaging Study on Tumor-Bearing Mice. To evaluate the tumor-targeting properties of nTG-DFP-COF, tumor-bearing mice were randomly divided into two groups and injected intraperitoneally with 0.2 mL saline (control) or nTG-DFP-COF (20 mg / kg, 200 pL). 0, 24, and 48 hours after injection of nTG-DFP-COF, the animals were humanely sacrificed and subjected to post-mortem fluorescence imaging using the IVIS Spectrum.

[0162] To evaluate the tumor cryo-imaging properties of nTG-DFP-COF, tumorbearing mice were randomly divided into two groups and injected intraperitoneally with 0.2 mL saline (control) or nTG-DFP-COF (20 mg / kg, 200 pL). 24 hours post-injection of nTG-DFP-COF, animals were humanely sacrificed and subjected to post-mortem fluorescence imaging using the IVIS Spectrum at 37 °C, 4 °C, and -20 °C.

[0163] Imaging parameters included 430 nm and 465 nm excitation, 500-540 nm emission, a binning factor of 8, f-stop of 2, a field of view set to 'C and automatic exposure time. Spectral unmixing was performed with Living Image software to isolate the fluorescence signal of the nTG-DFP-COF from tissue autofluorescence. Tumors from untreated control mice were used to establish background autofluorescence. Images were analyzed by drawing regions of interest (ROIs) around the tumors in the unmixed images, and total radiance efficiency (normalized data) was used for quantification.

[0164] Statistical Analysis. All statistical analysis was performed with GraphPad PRISM 8. All data are expressed as mean ±SD. Data were analyzed using one-way ANOVA with post hoc Tukey tests SPSS (IBM, SPSS Statistics, version 23, USA). *p <0.05; **p <0.01; ***p <0.001.

[0165] Although the present disclosure has been described with respect to one or more particular embodiments and / or examples, it will be understood that other embodiments and / or examples of the present disclosure may be made without departing from the scope of the present disclosure.

Claims

Claims:

1. A composition comprising an exfoliated covalent organic framework (COF) nanosheet comprising a co-condensate of triaminoguanidinium chloride (TGH.C1) and 2,6- diformylpyridine (DFP), wherein the COF nanosheet has a longest linear dimension of 1 SO- SOO nm.

2. The composition according to claim 1, wherein at least 90% of the COF nanosheets have a longest linear dimension of 300 nm or less.

3. The composition according to claim 2, wherein at least 95% of the COF nanosheets have a longest linear dimension of 300 nm or less.

4. The composition according to claim 1, wherein the COF nanosheet comprises the following structure:wherein each R is independently5. The composition according to claim 4, wherein the COF nanosheet comprises the following structure:

6. The composition according to claim 1, wherein the COF nanosheet is porous and at least partially crystalline.

7. The composition according to claim 6, wherein the COF is substantially crystalline or crystalline.

8. The composition according to claim 7, wherein the COF is substantially crystalline or crystalline at -40 °C.

9. The composition according to claim 1, wherein the longest linear dimension remains the same at -40°C.

10. The composition according to claim 1, wherein the COF nanosheets have a binding affinity for water molecules that increases as temperature decreases.

11. A method for imaging one or more cancer cells comprising contacting a composition according to claim 1 to i) an area of a subject suspected of having or having cancer cells or to ii) a sample suspected of having or having cancer cells and visualizing the COF nanosheets.

12. The method according to claim 11, wherein the method comprises utilizing cryoimaging.

13. The method according to claim 11, wherein the nanoscale materials can circulate in the bloodstream and target tumors.

14. The method according to claim 11, wherein fluorescence intensity is selective for cancerous cells at low temperatures.

15. The method according to claim 14, wherein the low temperatures correspond to a range of -40 to -10 °C.

16. The method according to claim 14, wherein the composition is used to label and track tumors in a subject in need.

17. The method according to claim 11, wherein cancerous tissues and healthy / normal tissues are differentiated via fluorescence.

18. The method according to claim 17, wherein the fluorescence distinguishes boundaries between cancerous and healthy tissues.

19. The method according to claim 11, wherein the method is used for cancer diagnosis.

20. The method according to claim 11, wherein the subject in need has a cancer associated with a solid tumor.

21. The method according to claim 11, further comprising measuring fluorescence or otherwise visualizing the COF nanosheets.

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