Cancer nanomedicine is one of the most promising domains that has emerged in the continuing search for cancer diagnosis and treatment. The rapid development of nanomaterials and nanotechnology provide a vast array of materials for use in cancer nanomedicine. Among the various nanomaterials, covalent organic frameworks (COFs) are becoming an attractive class of upstarts owing to their high crystallinity, structural regularity, inherent porosity, extensive functionality, design flexibility, and good biocompatibility. In this comprehensive review, recent developments and key achievements of COFs are provided, including their structural design, synthesis methods, nanocrystallization, and functionalization strategies. Subsequently, a systematic overview of the potential oncotherapy applications achieved till date in the fast-growing field of COFs is provided with the aim to inspire further contributions and developments to this nascent but promising field. Finally, development opportunities, critical challenges, and some personal perspectives for COF-based cancer therapeutics are presented. We outline the latest developments in COF-based nanomedicines for use in oncotherapy, including material synthesis, nanocrystallization, and functionalization strategies, as well as their therapeutics applications. Meanwhile, some other emerging minimally invasive therapies, such as photothermal therapy (PTT) and photodynamic therapy (PDT), have also exhibited promising potential in oncotherapy due to their high selectivity, low side-effects, and negligible drug resistance.11–14 Rapid developments in nanomaterials and nanotechnology have provided a vast material reservoir for use in cancer nanomedicine, which mainly include mesoporous silica,15 metal chalcogenides,16 upconversion materials,17 MXenes,18,19 carbon-based materials,20,21 semiconducting polymers,22,23 and liposomes.24Cancer remains a worldwide public health issue with high morbidity and mortality rates.25–30 further, the development of porous materials ranging from traditional inorganic materials  to organic–inorganic hybrid porous materials ,31 coordination polymers (CPs),32 and metal–organic frameworks (MOFs)).33–35 Among them, MOFs are crystalline materials formed by the self-assembly of organic ligands and metal ions (or clusters) through coordination bonds. The highly ordered structures of MOFs allow precise control over their pore shapes and chemical environments, thereby realizing controllable regulation of their properties.36 In the past decade, MOFs have been widely applied in the field of oncology and have even entered the stage of clinical trials.37–39 With the development of reticular chemistry,40 a new generation of crystalline porous materials, namely, covalent organic frameworks (COFs), emerged in 200541 and have been booming in recent years.42 As a natural extension of MOFs, COFs are composed of nonmetallic elements  connected by strong covalent bonds into two-dimensional (2D) or three-dimensional (3D) crystalline frameworks with predictable and periodic structures.43,44 Due to the diversity of organic syntheses, COFs provide promising prospects for materials design, enabling function- and application-oriented material syntheses. Until now, COFs have been widely used for separation and analysis,45–49 heterogeneous catalysis,50–52 sensing,53 optoelectronics,54 energy and environmental science,55–59 and biomedicine.60,61The design, synthesis, and applications of advanced porous materials with specific structures at the micron- and nanoscales have been a research hotspot in various scientific fields;62 To sum up, we believe that COFs are becoming a promising and efficient organic material platform for building theranostic systems.In recent years, COFs, particularly nanoscale COFs (NCOFs), have joined a huge candidate library of biomedical nanomaterials because of their following unique features. (i) On account of their modular structures, COFs can be easily decorated with multiple functional compositions, enabling diverse biomedical applications, such as tumor targeting, fluorescence imaging, and cancer therapy. (ii) Due to their inherent porosity, COF cavities allow the encapsulation of various guest molecules, thereby facilitating controlled drug release. (iii) Owing to their conjugated structures, the energy level structure of a COF monomer is different in the framework. By tuning the topological structures and geometric parameters to optimize the directional energy and charge transport, COFs may have optical properties that cannot be realized within the monomers, which offers additional and unexpected possibilities for imaging and therapeutic applications of COFs. (iv) The metal-free nature of COFs prevents any potential biological toxicity caused by metal elements.In this review , we syst22.1via covalent bonds.63 Until now, most of the reported COFs have been 2D structures. The structure of a 2D COF consists of 2D sheets held together by covalent bonds, which are then stacked together through noncovalent π–π interactions. For example, 2D monosheets of COF LZU-1 use the face-to-face eclipsed stacking  of the monomers enable to predefine the geometry and topology of the resultant frameworks. Therefore, unlike amorphous polymers, COFs provide positional control over their monomers in the spatial dimension,70 thereby realizing the possibility of the oriented design of frameworks and pore structures. For example, in the 2D plane, trigonal planar monomers can co-condense to form sheets with hexagonal pores, while tetragonal monomers can co-condense with linear monomers to form tetragonal, rhombic, or Kagome pores  and molecular geometry  and small-/wide-angle X-ray scattering (SAXS/WAXS).83 For 3D COFs, apart from the aforementioned techniques, electron diffraction also plays an important role for interpenetrated structural analyses.84,85Generally, the first step for the characterization of COFs is determining their crystal structures. Typically, the structure of a crystalline material is determined by the single-crystal X-ray diffraction (SC-XRD) technique. However, almost all the reported COFs are microcrystalline aggregates in their powder form; it is particularly challenging to obtain high-quality, large-sized single crystals that meet the requirements of SC-XRD measurements.13C cross-polarization magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (13C CP-MAS ssNMR)86 is used to designate the chemical environments of carbon atoms, and X-ray photoelectron spectroscopy (XPS) can reveal the chemical structures of COF surfaces.87Certain spectroscopy methods can also be used for auxiliary research on the chemical structures of COFs. For example, Fourier-transform infrared spectroscopy (FT-IR) is widely used for linkage identification, 88 On the other hand, the pore volume and pore size distribution of COFs can be determined by various approaches,89 such as nonlocal density functional theory (NLDFT), quenched solid density functional theory (QSDFT), grand canonical Monte Carlo (GCMC) method, Barrett–Joyner–Halenda (BJH) method, and Horvath–Kawazoe (HK) method. However, the analysis methods should be carefully selected according to the characteristics of different COF materials; otherwise, it can lead to inaccurate or completely incorrect analysis results.90 Since the expected information regarding the pore structure can also be calculated from the crystal structure, it is very meaningful to compare the experimental results with the theoretical predictions.Another important feature of COFs, namely, their permanent pore structures, can be evaluated by gas adsorption and desorption experiments, which can provide valuable information regarding the specific surface area, pore size, and pore volume of COFs. Currently, the most common test gas is nitrogen at 77 K and the optimal analysis approach to obtain the specific surface area is the Brunauer–Emmett–Teller (BET) theory based on a multilayer gas adsorption model.40,91Morphology, including particle shape and size, is significant for COF characterization. It is a common practice to observe the microscopic morphology of particles with electron microscopes, such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), atomic force microscopy (AFM), and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM). Lattice spacing and diffraction pattern obtained by HRTEM can provide additional assistance for the structural analyses of COFs. By combining with energy-dispersive X-ray spectroscopy (EDX), elemental distribution can also be semiquantitatively determined. In addition, dynamic light scattering (DLS) measurements can provide statistical distribution of the hydrodynamic diameters of the particles, and it plays an indispensable role in the study of uniformity and stability of NCOFs.341 various synthesis methods have been employed and reported for the synthesis of COFs to satisfy the needs of extensive applications. By using relevant examples, this section will summarize and discuss the conventional synthesis methods of COFs, including solvothermal synthesis, microwave synthesis, ionothermal synthesis, atmospheric solution synthesis, and mechanochemical synthesis  lining are the most general pressure vessels. Nevertheless, it is difficult to isolate the air, making it unsuitable for the synthesis of COFs. Therefore, typical COF solvothermal synthesis is usually carried out in a Pyrex tube; a thick-walled pressure tube can also be used instead of a disposable Pyrex tube . The gen73,96 Most COFs based on the Schiff-base reaction that form the C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>N bond usually react at 120 °C.78 In some cases, higher temperatures, such as 160 °C (PI-COF-4 and PI-COF-5),97 200 °C (PI-COF-1 and PI-COF-2),98 and even 250 °C (PI-COF-3),98 were adopted for the synthesis of polyimide-based COFs.The reaction temperature used in the solvothermal reaction has a significant impact on the properties of COFs, particularly crystallinity. The more commonly used reaction temperatures range from 85 to 120 °C. For instance, B–O-linked COF-6, COF-8, COF-10, COF-102, COF-103, COF-105, and COF-108 can be obtained at 85 °C.o-dichlorobenzene, and n-butanol. It is necessary to try various solvents with different ratios one by one during synthesis to optimize the synthesis conditions. In 2011, Jiang et al. synthesized porphyrin-based COF of ZnP-COF;99 further, they found that when mesitylene and 1,4-dioxane were used as the solvents, the ratio of these two solvents significantly affected the crystallinity and micromorphology of ZnP-COF. More importantly, apart from the crystallinity of COFs, the solvent may also affect the structures of the COF materials. An interesting example is TPE-COF-I and TPE-COF-II  as the solvent, the conventional [4 + 4] pathway of 4,4′,4′′,4′′′-tetraaniline and 4,4′,4′′,4′′′-tetrabenzaldehyde affords TPE-COF-I with a fully bonded network. However, using 1,4-dioxane/acetic acid  for the synthesis reaction, an unusual [4 + 2] pathway leads to TPE-COF-II with unreacted trans-position aldehyde groups. Recently, Zhao et al. reported solvent-induced COF isomerization tetrakis) in different solvents  formed SP-COF-ED with a single-pore structure and DP-COF-ED with a heteropore structure. Interestingly, these two COFs exhibited significantly different adsorption behaviors toward n-hexane, and in situ structural transformation from DP-COF-ED to SP-COF-ED could be realized by the reaction of DP-COF-ED with 2-methylbenzene-1,4-diamine for 3 days.Another key factor affecting the synthesis of COFs is the solvent, such as mesitylene, 1,4-dioxane, E-COF-II  based onrization .101 In p102 COF-10,102 NiPc-PBBA-COF,102 LZU-20,103 LZU-21,103 LZU-22,103 and DBC-2P COF.104 In addition, propylamine-protected 2,4-dihydroxybenzene-1,3,5-tricarbaldehyde was used as a precursor for COF synthesis,105 and propylamine inhibited the lateral growth of COF sheets, thereby affording hexagonal COF mesocrystals with rod-like morphology.The formation of COFs is essentially a thermodynamic control reaction. Therefore, in order to avoid the formation of amorphous polymers under the rapid reaction between monomers, the reaction sites in the monomers can be protected in advance with the protecting groups . Through106 and five covalent bonds in each cyclic joint were formed in situ during polymerization. o-dichlorobenzene/n-butanol  at 120 °C for 72 h in the presence of BF3·OEt2, 4,5-dichloro-3,6-dioxocyclohexa-1,4-diene-1,2-dicarbonitrile (DDQ), and acetic acid, the one-pot in situ Povarov reaction afforded P-StTaDm-COF as an orange-red crystalline solid.107 Similarly, by replacing trimethylsilane carbonitrile with phenylethylene in the aforementioned reaction system, S-TmTaDm-COF could be obtained via the three-component in situ Strecker reaction.107 In addition, based on the report by Cooper et al., benzothiazole-linked COFs108 were obtained by adding sulfur to the conventional synthesis system of Schiff-base COFs  by microwave heating is more than 200 times faster than that of solvothermal synthesis (3 days). Meanwhile, the BET specific surface area of the obtained COF-5 is higher than that of previously reported COFs under solvothermal conditions (2019 vs. 1590 m2 g−1). Imine-linked TpPa-COF was also synthesized by microwave heating using benzene-1,4-diamine and 2,4,6-trihydroxybenzene-1,3,5-tricarbaldehyde as the monomers.111 Dichtel et al. further synthesized imine- and β-ketoenamine-linked COFs by microwave synthesis using benzophenone N-aryl imine with higher solubility and stronger oxidation stability to replace the aromatic amine in the imine condensation reaction.112 Very recently, dioxin-linked DH-COF was synthesized under microwave conditions within 30 min using the nucleophilic substitution reaction of 2,3,5,6-tetrafluoroisonicotinonitrile with triphenylene-2,3,6,7,10,11-hexaol.113In 2009, Cooper These examples clearly indicate that microwave heating can immensely increase the reaction rate and shorten the reaction time. Meanwhile, microwave synthesis enables online monitoring, which is significantly difficult to be achieved for solvothermal synthesis.3.3114 As recyclable alternatives to traditional volatile organic solvents, ILs have been widely used as environment-friendly solvents. The synthesis reaction carried out in ILs is referred to as ionothermal synthesis, and it has exhibited great promise for industrial applications due to the avoidance of safety hazards caused by pressure.Ionic liquids (ILs) are a class of organic salts that are liquid at room temperature or near room temperature.2) was chosen as a solvent as well as a catalyst for producing Schiff-base COFs.115 In the ILs, the tetrahedral monomer of 4,4′,4′′,4′′′-methanetetrayltetrabenzaldehyde reacted with the linear monomers of gradually increasing lengths, namely, p-phenylenediamine (6 Å), -4,4′-diamine (11 Å), and -4,4′′-diamine (15 Å), to produce 3D-IL-COF-1, 3D-IL-COF-2, and 3D-IL-COF-3 with interpenetrated dia structures, respectively imide -3-methylimidazolium hydrogen sulfate ([BSMIm]HSO4) as the solvent as well as catalyst, four β-ketoenamine-linked COFs were obtained at ambient pressure  with alkyl chains of different lengths have also been used for the ionothermal synthesis of imine- and hydrazone-linked COFs.119 Apart from the inherent structural pores, alkyl chains with different lengths enable the induction of mesopores with different porosities, thereby exhibiting excellent performance in catalyzing C–C coupling reactions.In addition, ILs uril (CB[7]) lead to the restacking of nanosheets. After adding 1-adamantylamine hydrochloride, the nanosheets get self-exfoliated again. This reversible and controllable exfoliation implies the contribution of quaternary ammonium salt in propidium iodide to charge-mediate the self-exfoliation process.Besides bipyridinium,192 and COFBTC-containing iron phthalocyanine.193Self-exfoliation induced by the charges within the frameworks is also observed in other types of COFs, such as iCOF-A-containing 1-methylpiperazine branched chainet al. confirmed that the protonation of the CN bond caused by acid treatment can induce the self-exfoliation of imine-linked COFs.194 BND-TFB COF , but its size in the other two dimensions is still too large , which cannot meet the needs of biomedical applications. Therefore, it is imperative to develop an effective approach for the bottom-up synthesis of COF nanoparticles.196,197 In short, the synthesis of nanocrystals in a solution involves two important processes: nucleation followed by nanocrystal growth. In the early stages of the reaction, a rapid polymerization reaction occurs in the solution, affording polymer fragments with lower solubility. As the reaction proceeds, the polymer in the solution reaches supersaturation, breaking through the critical value required for nucleation. The nucleation stage is completed with precipitate formation. After that, the solution has a lower degree of supersaturation again, and the monomer continues to polymerize on the surface of the formed crystal nucleus with particles growing and becoming larger followed by a decrease in the monomer concentration in the solution. Finally, due to the reversibility of the COF linkages, the precipitation undergoes a dissolution–reprecipitation process, and the monomers tend to be arranged in a periodic order to form crystalline COF particles  for the preparation of NCOFs.Here, we will illustrate the applications of reaction kinetics regulation trianiline (TTA) with (E)-4-(4-(phenyldiazenyl)phenyl)pyridine-2,6-dicarbaldehyde (AzoDFP) in 1,4-dioxane in the presence of acetic acid (3 M) under solvothermal conditions at 120 °C for 1 h. TTA-AzoDFP NCOF is a spherical nanoparticle with a rough surface and the diameter of the particle is only 117 nm. The shorter reaction time (1 h) inhibits the growth process of nanocrystals, resulting in small-sized particles.The feasibility of the preparation of NCOFs by shortening the reaction time was further verified by TTA-AzoDFP NCOF synthesis.et al. prepared COF-5 with different particle sizes by adding different concentrations of reactants at different rates to a pre-prepared COF-5 seed crystal with a particle size of 400 nm.200 When the monomer was gradually added to the reaction mixture, the monomer concentration became limited. Growth dominated nucleation; therefore, the particles gradually grew without forming newer particles, which resulted in an increased particle size. In contrast, when monomers were rapidly added, their concentration increased above the critical nucleation concentration. The reaction was dominated by the formation of new and small-sized particles  as the modulators, which can affect the reaction rate and thermodynamic equilibrium state by participating in the polymerization reaction. This strategy may be suitable for the synthesis of COF particles with any size. For instance, Bein et al. reported that the addition of (4-mercaptophenyl)boronic acid modulator in COF-5 syntheses increased their crystallinity by slowing down the COF-5 growth and supporting the self-healing of crystal defects.204 Dichtel et al. also demonstrated a similar effect for pyrocatechol in COF-5 synthesis.205 Recently, modulators have been used to maintain a thermodynamically stable state to regulate the morphology of COFs, such as COF spheres,206 hollow fibers,206 single crystals,207,208 and thin films.206,209 Additionally, when the added competitor has a chiral site -1-phenylethan-1-amine), this strategy can possible generate chiral COFs,210 even though the monomers used for the construction of COFs do not exhibit any chirality.Unfortunately, unlike amorphous materials and easily crystallized inorganic materials, the crystallization of COFs is a crucial process that accompanies nucleation and growth processes. For COF growth, particularly boronate-ester-linked COF growth, the decisive step is not condensation, but interlayer polymer stacking by a nucleation–extension process.et al. demonstrated that the addition of high-dosage 4-(tert-butyl)benzene-1,2-diol to the synthesis reaction of COF-5 inhibited nucleation in a concentration-dependent mode and induced subsequent anisotropic growth.211 When compared with the constant addition of monomers, the competitor significantly shortened the synthesis time without reducing the crystallinity. By controlling the reagent concentration and reaction time, the hydrodynamic diameter of COF-5 particles was adjustable within the range of ∼60–450 nm -1,3,5-triazine and benzene-1,3,5-tricarbaldehyde in CTAB/SDS (97 : 3) mixtures, demonstrating the feasibility of this method.In addition to regulating the nucleation and growth processes of COFs, the solubilizing effect of surfactants on organic monomers enables aqueous synthesis. Puigmartí-Luis  mixture . Due to tert-butoxycarbonyl, which is deprotected in situ during the reaction, thereby delaying the growth of the crystal nuclei. As shown in tert-butyl-(4-aminophenyl)carbamate and benzene-1,3,5-tricarbaldehyde reacted in ethanol in the presence of polyvinylpyrrolidone (PVP) and trifluoroacetic acid, affording uniform LZU-1 nanocrystals.215 The protonation of the imine bond rendered the nanocrystals to become polar during the growth process, allowing PVP to bind and passivate its surface, thereby regulating the growth process. By changing the molecular weight and concentration of PVP, the particle size of LZU-1 can be tuned as 500 ± 52, 245 ± 25, and 112 ± 11 nm .64Along this line, in 2019, Dong 4.2.3N-linked NCOFs, showing extraordinary application potential in the bottom-up synthesis of NCOFs.Acetonitrile—a common organic solvent—has been successfully used for the preparation of B–O- and Cet al. synthesized translucent COF-5 dispersions in a ternary mixed solvent of acetonitrile, 1,4-dioxane, and mesitylene at 70 °C.217 When the acetonitrile content increases from 15 to 95 vol%, the hydrodynamic diameter of COF-5 drops from 232 to 50 nm. Acetonitrile is irreplaceable for colloidal stabilization, whereas other alternative solvents for acetonitrile  lead to COF-5 precipitation. The authors speculate that the direct interaction between the cyano group and COF-5 is responsible for colloid formation, although the details for this interaction are still not fairly clear. Moreover, as compared to the poor processability of microcrystalline powders, these stable COF colloidal suspensions provide a new avenue for processing these materials into centimeter-scale thin films from solution. Besides boronate-ester-linked COFs, boroxine-linked Ph-COF, BPh-COF, DBD-COF, Py-COF, and TMPh-COF colloidal dispersions  trifluoromethanesulfonate as a transamination catalyst131 was added to the acetonitrile solutions of 1,3,5-tris(4-aminophenyl)benzene and p-phthalaldehyde  solvent; then, acetic acid (12 M) was slowly added. Finally, TAPA in N,N-dimethylformamide solution was carefully added dropwise to the acetic acid surface. As TAPA moved to the intermediate layer and got protonated, its color gradually changed from red to green. At the same time, 2-hydroxybenzene-1,3,5-tricarbaldehyde in the lower layer slowly diffused to the intermediate layer, thereby generating a large amount of COF quantum dots. When the reaction time was 3 days, the yield was about 29%, and the average particle size was 4.2 nm.Diffusion-based COF synthesis at the solid–liquid,tum dots .230 The 4.2.5et al. prepared hollow silica nanospheres using the template method in 1998,231 it has become one of the most efficient methods for nanomaterial synthesis. The material and structure of the template can be customized according to the property and morphological requirements of the target materials. For the materials that are difficult to reduce to the nanoscale, this method is facile, straightforward, and highly effective.The template method uses a readily synthesized nanostructure as the core; then, a shell material is deposited on its surface. After removing the inner core, hollow nanoparticles can be obtained. Since Möhwald 3O4 was the first template used for the synthesis of NCOFs. Since Fe3O4 is usually prepared by the hydrothermal reaction of sodium citrate with iron salts, the surface of Fe3O4 nanoparticles normally adsorbs citrates, which further facilitates the adsorption of arylamine molecules via hydrogen bonds, resulting in polymerization reactions on the Fe3O4 surface. As shown in et al. employed this method to grow an amorphous polyimide network on the Fe3O4 surface, which was further converted into crystalline COFs via solvothermal synthesis.232 By varying the concentration of COF monomers, the thickness of the COF shell can be controlled. If further etching of Fe3O4 is undertaken with hydrochloric acid, hollow COF nanoshells can be obtained.233 In addition, considering the magnetic property of Fe3O4, core–shell Fe3O4@COFs have shown great promise for magnetic separation and enrichment.234,235Fevia covalent bonds as compared to that via hydrogen bonds. Therefore, templates with amino groups on the surface may facilitate the controlled template synthesis of CN-linked COFs. Zhang et al. were the first to pre-modify the amino group on the surface of indium-based MOF NH2-MIL-68 template with an aldehyde monomer via imine condensation and then extended the polymerization of TPA-COF using formyl groups on the surface as the reactive sites; finally, the NH2-MIL-68@TPA-COF core–shell structure was successfully prepared ,239,240 and NH2-MIL-101(Fe),241 can also be used as templates to anchor aldehyde monomers via covalent bonds.The pre-modification of monomers on the templates is more robust to provide COF growth sites prepared .236 Otheet al. prepared hollow COF capsules using MOFs as the template to encapsulate biomacromolecules 3 as the transamination catalyst. Finally, the MOF core was etched away to afford hollow COF capsules loaded with biomacromolecules. The COF shells protect the biomacromolecules and prevent interference from the external environment. More importantly, its spacious interior maintains the conformational freedom of the biomacromolecules while allowing the efficient diffusion of substrates and products. The generalizability of this approach was confirmed by selecting different MOFs , COFs , and biomacromolecules . In addition, in CO2-dissolved water, hollow LZU-1 and COF-42 were synthesized using ZIF-8 and ZIF-67 as the templates.243Very recently, Chen olecules , which ce.g., Fe3O4 and MOFs), soft templates, such as oil-in-water (O/W) microemulsions,244 can also be used to fabricate hollow NCOFs. Unlike the demanding etching conditions of hard templates, soft templates can be readily removed from the core–shell structures by evaporating or adding demulsifiers.In addition to the aforementioned hard templates -O,O′-diacetyl-l-tartaric anhydride by an esterification reaction to form the chiral-functionalized monomer of CTp. Then, p-phenylenediamine, 2,5-dimethylbenzene-1,4-diamine, and -4,4′-diamine were condensed with CTp to afford the CCOFs of CTpPa-1, CTpPa-2, and CTpBD, respectively. These CCOFs exhibited highly regular chiral pore environments, while their bound capillary columns showed higher resolution for the separation of enantiomers. In addition, for asymmetric catalysis, a CCOF library263 was formulated based on 4,7-dibromo-2-chloro-1H-benzo[d]imidazole (DBCBI) as the platform molecule through nucleophilic substitution, Suzuki coupling reaction, and imine formation  for chiral separation and catalysis applications. For example, the CCOFs reported in 2016 were used for chiral separation.ormation . This fu68,264–266 thioether,267 free radical,268 chiral pyrrolidine,269,270 imidazolium,271 8-hydroxyquinoline,272 and crown ether,273 are also introduced into COFs via pre-SM, which considerably enriches the types and applications of COFs.In addition to the above representative examples, other functional groups, such as alkyl chain,5.1.2274–286 However, most coordination compound molecules cannot be stabilized within the COFs under harsh solvothermal conditions, significantly limiting their widespread applications.When using monomers containing coordination atoms to construct COFs, metal ions can be incorporated into the monomers in advance, which is called pre-synthesis coordination modification. Some porphyrin- and phthalocyanine-based COFs have been metallized by using this method .274–286 5.2287,288 the post-SM of COFs generally involves the embedding of reactive functional groups into the frameworks of pre-synthesized COF materials to achieve task-specific applications via different post-SM strategies, such as post-synthesis covalent modification, post-synthesis functional group conversion, bonding defect functionalization (BDF), and post-synthesis linkage transformation. Here, we will provide detailed summary and discussion of each strategy, expecting to give the readers a better understanding of this promising post-SM approach.Similar to the post-SM of MOFs,5.2.1via covalent bonds. Till now, the reaction sites within the COF structures mainly include ethynyl, vinyl, hydroxyl, amino, and carboxyl groups  scale" fill="currentColor" stroke="none"><path d="M80 600 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 440 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 280 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z"/></g></svg>C]x-H2P-COFs containing content-tunable and reactive ethynyl groups in the pores by the imine condensation reaction of 2,5-dihydroxyterephthalaldehyde, 2,5-bis(ethynyloxy)terephthalaldehyde, and 4,4′,4′′,4′′′-tetraaniline  were successfully incorporated into the COFs with controllable loading contents. The effects of functional groups on their gas adsorption properties were confirmed by CO2 adsorption measurements. For example, the CO2 adsorption capacity could reach up to 157 mg g−1 at 273 K and 1 bar for functionalized COFs with 50% amino groups, which was three times higher than that of pristine COFs. Covalent post-SM based on a click reaction to form a triazole ring is an extremely versatile and efficient method that enables the development of custom-built COFs with tunable porosities and functionalities while maintaining their high crystallinity.164,290–298To achieve the covalent modification of COFs, a high-yield modification reaction under mild conditions is highly desired, aaniline .289 Quan299 Since the enhanced hydrophobicity of the materials can be realized at the expense of porosity and crystallinity of COF-V, by optimizing the reaction conditions, the modification rate of vinyl can be controlled at 4% to balance the hydrophobicity and inherent properties of COF-V. The water contact angle increased from 113° for COF-V to 167° for COF-VF, demonstrating the superhydrophobicity of COF-VF. In addition, other fascinating examples, such as ethane-1,2-dithiol,300,301 (4-mercaptophenyl)boronic acid,302 4-mercaptobenzoic acid,303 and glutathione,253 have been modified into COFs by the thiol–ene reaction.The click reaction of vinyl with thiol is another typical click-chemistry-based post-SM method. For instance, vinyl-functionalized COF-V and 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecane-1-thiol with low surface free energy reacted in the presence of azobisisobutyronitrile (AIBN) as the catalyst to yield perfluoroalkane-modified COF-VF .299 Sincet al. prepared sulfonic-group-modified COFs with the maximum adsorption capacity for diclofenac of up to 770 mg g−1.304Noteworthily, electrophilic addition reactions of vinyl groups, such as the reaction of vinyl groups with sulfonic acids to generate sulfonate esters, can also be used for the post-SM of COFs. For example, Lv et al. synthesized hydroxyl-rich [HO]X%-Py-COFs using X-shaped tetraamine and two linear dialdehydes as the monomers.305 Subsequently, [HO]X%-Py-COFs were reacted with a quaternary ammonium salt for fixing the IL on the pore walls of the COFs. The obtained [Et4NBr]X%-Py-COFs could catalyze the formylation of amine with CO2 and phenylsilane under metal-free conditions. Besides, the post-SM reaction of the hydroxyl group with iodine-substituted perfluoroalkane could convert hydrophilic COF-DhaTab into hydrophobic COF-DTF.306The hydroxyl group has become a promising reaction center for post-SM because of its versatile reaction chemistry . By the et al. studied the reaction of T-COF-OH with fluorescein isothiocyanate (FITC) in acetone to modify nonfluorescent T-COF-OH into T-COF-OFITC with green-light emission via a thiocarbamate bond -camphoric acid dichloride (d-cam-ClO) has been loaded into the pores of TzDa COF by this reaction; the resulting CTzDa COFs were used as an adsorbent for the chiral isolation of amino acid enantiomers bis can integrate ethylenediaminetetraacetic acid (EDTA) into the pores, thereby improving the heavy-metal-ion adsorption capacity of the COF material.311 TpBD(NH2)2-COF-containing amino groups enable the reaction with acetic anhydride to achieve the acetylation of the pore walls.312 In addition, TpBD(NH2)2 COFs can be functionalized by the imine condensation reaction of the amino group with (2-formylphenyl) boronic acid.252There are two different ways for performing the covalent post-SM of amino groups, 313 With the help of certain condensating agents, such as 1-(bis(dimethylamino)methylene)-1H-benzo[d]triazole-1-ium 3-oxide hexafluorophosphate (HBTU) and 3-(((ethylimino)methylene)amino)-N,N-dimethylpropan-1-amine (EDC), the post-SM of carboxyl-functionalized COF-616 can be realized via amidation, esterification, and thioesterification at room temperature, and several chelating groups can be introduced into COF-616 to afford functional materials for the adsorption of pollutants in water 2 COF by the direct reaction of -3,3′,4,4′-tetraamine and 2,4,6-trihydroxybenzene-1,3,5-tricarbaldehyde, because the four amino groups of the former can randomly react with the trialdehyde monomer and interfere with the formation of a periodic ordered network structure. However, using 3,3′-dinitro--4,4′-diamine as the precursor, TpBD(NO2)2 COF was initially prepared (2 was used to reduce the nitro groups to obtain TpBD(NH2)2 COF with high crystallinity.312 Dichtel et al. reported imide-linked COFs containing different amounts of amino groups by reducing the azide groups on the monomer side chain  to obtain other types of functional COF materials.In particular, the reduction of azide and nitro groups is a feasible method to insert amino groups into COFs prepared ; subsequde chain .314 More79,80 JUC-505 was synthesized by the irreversible nucleophilic aromatic substitution reaction between 2,3,5,6-tetrafluoroterephthalonitrile and triphenylene-2,3,6,7,10,11-hexaol, which forms 1,4-dioxin linkages. The cyano groups within the JUC-505 framework can be converted to carboxylic acid, amide, amidoxime, and methanamine in NaOH/ethanol/water, NaOH/water, NaOH/ethanol/water, hydroxylamine/tetrahydrofuran/water, and LiAlH4/tetrahydrofuran solution  as the model.solution . Functio318 viologen reduction,319p-phenol oxidation,168 photoinduced cis–trans isomerization,320 and photoinduced pericyclic reaction.321In addition to the aforementioned generic examples of functional group conversion, there are some other post-SM examples that are only compatible with specific COF structures to some extent. For example, methyl bromination,Above all, a considerable number of organic chemical reactions can be carried out on COF platforms to achieve functional group conversion, which can considerably enrich the types of functional groups in COFs and expand the applications of advanced COF-based functional materials.5.2.3e.g., dust). Among them, the bonding defect located at the crystal grain edge and caused by unreacted functional groups on the monomer remains reactive, providing an opportunity for surface post-SM. This covalent modification method using the bonding defects of COFs is called BDF.Despite the high crystallinity of COFs, defects are still inevitable, such as vacancy defects due to monomer deficiency in periodic ordered structures, edge dislocations because of squeezing or stretching of lattices, extrinsic defects as a result of the doping of extraneous materials trianiline, has carboxyl groups on its surface and can be used for BDF. Generally, the formation of NKCOF-1 occurs in two steps. First, the anhydride reacts with the primary amine via ring opening to form amic acid. When the synthesis temperature rises above 150 °C, amic acid gets further dehydrated to form imide linkages. During the two-step condensation reaction, a portion of the carboxyl groups is not condensed with the amino groups, thereby resulting in bonding defects. Ma et al. found that when the synthesis temperature was 200 °C, the content of carboxyl groups was approximately 5.7% in NKCOF-1, as determined by the acid–base titration analysis.323 Furthermore, these carboxyl groups continued to react with the amino groups, making NKCOF-1 as the ideal platform for the covalent modification of biomolecules, such as lysozyme, tripeptide Lys–Val–Phe, and lysine )tetraboronic acid that self-condenses to form COF-102 was modified via the substitution of one of its four arylboronic acid moieties by the appointed functional group. Thereafter, the resulting triangular triboronic acid molecule was co-condensed with the original tetraboronic acid monomer to afford truncation-unit-containing COF-102. When the incorporated truncation unit is )triboronic acid, the vinyl group is introduced into COF-102, and the covalent attachment of other molecules can be obtained by the thiol–ene click reaction of the vinyl group with the sulfhydryl group boronic acid. Due to the high affinity of phenylboronic acid and diol, the resulting B-COF can be used to selectively enrich riboflavin, luteolin, and pyrocatechol for mass spectrometry analysis; further, the limits of detection are as low as femtogram per milliliter.326 In addition, covalently doping photoactive hydroxyquinoline–Pt(ii)327 and protocatechualdehyde–Fe(iii)328 complexes into imine-linked COFs as the truncation units have also been reported.The feasibility of this highly inspiring method has been proven by subsequent reports and studies. For example, partly replacing 1,4-phenylenediboronic acid with 4-boronobenzoic acid to synthesize COF-5 can introduce carboxyl groups into COF-5. At room temperature, EDC-activated carboxyl groups can easily react with amino-functionalized ATTO 633 fluorescent dye to endow COF with near-infrared fluorescence properties.et al. synthesized dCOF-NH2-X with exposed amino groups  The truncation unit itself may contain anchoring sites for post-SM as long as these sites do not participate in the condensation reaction to form COFs. (ii) The doped truncation unit results in the failure of linkage formation at the truncation location, thereby inducing well-controlled defects and active anchoring sites from the COF monomers. (iii) The content of the truncation unit was determined by the feed ratio of the two monomers, which can prevent higher contents of truncation units while maintaining higher crystallinity and porosity of the pristine COFs. Therefore, the TUF strategy can effectively overcome the limitation of the BDF strategy.5.2.5via post-synthesis linkage transformation  or NaOH (1 M) solutions for 24 h, the crystallinity of the amide-linked COFs was retained, while the corresponding imine-linked COFs were dissolved or turned into amorphous polymers. The NaClO2 oxidation of 3D COFs based on tetrahedral tetraamine and chiral tetraaldehyde monomers331 also achieved the conversion of imines to amides, suggesting the generalization of this method.The oxidation of C4 can reduce imine- to amine-linked COFs, as demonstrated by COF-300 and COF-366-M .332 For instance, after the reduction of COF-300 with NaBH4 in methanol, the 13C and 15N CP-MAS ssNMR of the obtained COF-300-AR verified the quantitative conversion of imine to amine .337 The reaction of TPB-DMTP-COF, BF3·OEt2, and chloranil in the presence of toluene with phenylacetylene at 110 °C for 72 h resulted in the formation of deep-yellow quinoline-linked MF-1a COF (4 in water/acetonitrile for 24 h), and reductant (NaBH4 in methanol at 65 °C for 24 h). More importantly, this method also provides a good platform for introducing other functional groups by simply using substituted phenylacetylene for post-SM.Imine linkage has also been converted to quinoline F-1a COF . Due to et al. further extended the application scope of this strategy by using phenylethylene instead of phenylacetylene to realize the quinolinization of TPB-DMTP-COF.107 Moreover, since olefinic bonds can appear in the ring, when 3,4-dihydro-2H-pyran was used for the reaction, the 2,3-dihydropyranoquinoline structure can be introduced into the framework. More importantly, the synthesis can be performed not only gradually through post-SM, but also in situ under solvothermal conditions. Therefore, this approach provides an unprecedented new way to build condensed ring-linked COFs. Dong et al. used the Strecker reaction for COF post-SM.107 With BF3·OEt2 as the catalyst and DDQ as the dehydrogenated agent, the reaction of trimethylsilane carbonitrile or diethyl phosphorocyanidate with TPB-DMTP-COF converted the imine bond into α-aminonitrile, thereby introducing cyano groups into the framework.More recently, Dong 338 Further, with regard to B-COF-1, there is a thienyl group in the ortho position of the imine bond. With trifluoroacetic acid as the catalyst, B-COF-1 was heated in a sealed tube containing oxygen at 100 °C for 2 days , thienopyridothienoquinoline, and 3,4-dihydro-2H-benzo[e]oxazin-2-(thi)one heterocyclic structures into the COFs. On the contrary, it is almost impossible to introduce such a complicated heterocyclic system by de novo COF synthesis. Notably, for chemistry and materials science concepts, this multistep post-SM process including covalent modification and linkage transformation actually transferred the classic solution of organic chemistry from the flask to the COFs, representing a significant step toward bringing the accuracy of organic solution-phase synthesis to extended solid-state materials.Finally, we emphasize upon the role of neighboring-group participation in the establishment of heterocyclic linkages in COFs. I-COF, a 2D COF formed by the condensation of 2,4,6-triaminobenzene-1,3,5-triol and terephthalaldehyde through Schiff-base condensation, was oxidatively cyclized by DDQ, which was converted into a benzoxazole-linked BO-COF with significantly improved thermal and chemical stability .338 Furtr 2 days . The coumidazole .340 With5.2.6via monomer exchange.341,342The high crystallinity of most COFs is due to the reversibility of linkages. The reversible condensation reaction allows the monomer to connect and disconnect from the framework under thermodynamically controlled conditions to endow COFs with the self-correcting ability during the formation process, thereby minimizing system energy, maximizing crystallinity, and preventing the formation of amorphous polymers. Similarly, this dynamic reaction also allows COF-to-COF transformation 343 In the presence of acetic acid as the catalyst, TP-COF-BZ was immersed in benzene-1,4-diamine at 120 °C and was almost completely converted to TP-COF-DAB within 4 h tribenzaldehyde, respectively, due to the lack of differential reactivity, but it can be prepared by co-heating PTPA and PTBD COFs with the corresponding monomers via monomer exchange  was generated.Combining monomer exchange with linkage transformation results in the generation of newer linkages . For exa346 Imine-linked COF TzBA and terephthaloyl dichloride were immersed in dioxane/mesitylene  for 2 days at 4 °C, and the -4,4′-dicarbaldehyde on the framework was gradually replaced by terephthaloyl dichloride, leading to the formation of amide-linked JNU-1 COF with irreversible linkage. Due to the formation of the N–H⋯Cl hydrogen bond and the coordinated interaction between O and Au, JNU-1 COF afforded highly selective adsorption capacity for gold recovery, which was not possible in TzBA COF.Monomer exchange can also occur between monomers with different types of functional groups.5.2.7Via rational design, electrocyclization reactions between two adjacent layers of monomers become possible.The classic electrocyclic reaction has strict requirements on the spatial distance and orientation of the reactant molecules, while regular ordered structures of COFs provide tight constraints on the spatial distribution of monomers. 347 The boronate-ester-linked 2D COF of Ph-An-COF was obtained by the condensation of anthracene-2,3,6,7-tetraol and benzene-1,3,5-triyltriboronic acid under solvothermal conditions. The AA-stacking structure caused the reactive anthracene to overlap with each other with an appropriate distance of 3.4 Å, which led to 9,10-photodimerization under light irradiation at 360 nm, disrupting the original conjugated system. As this conversion is reversible, the re-aromatization of the anthracene ring upon heating at 100 °C promotes the formation of the original COF. In addition, for anthracene-based IISERP-COF7 with polychromatic light emission, Vaidhyanathan et al. observed blue-light quenching due to interlayer [4 + 4] cycloaddition.348The first example of a reversible COF interlayer reaction is the [4 + 4] cycloaddition reaction of anthracene-based COFs .347 The et al. reported changes in the optical properties caused by [2 + 2] cycloaddition based on vinylene-linked V-COF-1.349 However, the resulting cycloaddition product was amorphous, and this conversion was not reversible. Partially reversible interlayer [2 + 2] reaction of COFs was reported by Perepichka et al. in 2020.350 By the alkali-catalyzed aldol condensation of 2,4,6-trimethyl-1,3,5-triazine and terephthalaldehyde, vinylene-linked P2PV COF with a layer spacing of 3.4 Å was synthesized ethylenediamine  is an attractive structural unit for coordination chemistry due to its stable planar square structure after chelating metal ions.370–372On the other hand, even though there are no chelating coordination sites on the monomers, COF metallization can be performed  ligands , particun+@COF) can be further reduced or hydrolyzed to produce M@COF or MxOy@COF composite materials, which have been widely studied for a variety of heterogeneous catalytic applications.373–377The post-synthesis coordination modification of COFs provides valuable opportunities to deposit metal or metal oxide nanoparticles in the COF pores. COFs coordinated with metal ions imide (TFSI−) ion.380For ionic COFs with charges on the monomer, their counter ions can be exchanged by other isoelectric ions, thereby generating new COF materials. For example, EB-COF:Br contains ethidium cations; therefore, the counteranion Bret al. reported anionic COFs as single-ion-conducting COF solid electrolyte materials.381 The treatment of benzimidazole-containing R-ImCOF  with n-butyllithium in n-hexane deprotonated the imidazole ring to form lithium-imidazolate-containing COFs. The imidazolate anions formed loose ion pairs with lithium cations, leading to high lithium-ion conductivity.In 2019, Zhang 5.2.10382–386 organic molecules,387–389 and even biological macromolecules.390–393Host–guest encapsulation is a versatile and effective modification strategy to endow COFs with more functions, which utilizes accessible and permanent pores of COFs to capture guest molecules, including a variety of functional inorganic molecules,394,395 First, the pores of COFs have a precise polygonal reticular structure with well-defined angles. The pore size can be predesigned from micropores to mesopores, which makes it possible to predesign the COF structures according to the size of the guest molecules to meet different needs. Next, morphologically, the pores of 2D COFs are one-dimensional (1D) pore channels instead of a closed cavity, which allows guest molecules to readily enter the pore from the top or sides. Finally, these 1D pore channels are spatially independent and isolated from each other. Moreover, the pore walls can also be pre-decorated via pore surface engineering (including pre-SM and post-SM) to establish a tailored interface to precisely regulate the interactions between the framework and guest molecules, facilitating the regulation of the release of guest molecules under different conditions. In conclusion, the pore shape, size, and chemical environment of COFs can be precisely predicted, which are significantly important structural parameters to control the interactions between COFs and guest molecules.The structural basis of host–guest encapsulation is the pores of COFs, which have unique characteristics.392,393 Meanwhile, the chemical modification of the pore walls of COFs can easily regulate the pore environment, thereby improving the interaction and compatibility of COFs with specific enzymes, as well as optimizing the orientation of the active sites of the enzyme, thereby further improving the catalytic activity of the enzymes.391Host–guest encapsulation is the prerequisite of a COF-based drug delivery system, which will be systematically discussed in the next section. Here, we mainly emphasize upon the encapsulation of biological macromolecules by COFs. Due to the large and regular pores of COFs, the controlled loading and release of enzymes on the COF platform allows the enzyme to remain viable under harsh environmental conditions, which is imperative for enzyme catalysis and undoubtedly expands the application ranges of the enzymes.6221,396–403 phototherapy reagents,163,216,223,261,404–410 and combination therapy platforms161,162,220,411–415 for tumor therapeutics  and tools  for making bread. Here, we will introduce the different kinds of methods to make the bread: COF-based oncotherapy examples. Till now, COFs have been used as drug delivery carriers,apeutics .6.16.1.1416,417 However, the efficacy of many chemotherapeutic drugs is limited by many factors. (i) Low solubility: to some extent, the low solubility of a large number of chemotherapeutic drugs  in water has led to insufficient bioavailability, forcing larger injectable doses to be given to keep the drug concentration in the lesion within the therapeutic range, which undoubtedly increases off-target toxicity. (ii) Low chemical stability: the photostability of platinum-based anticancer drugs is poor. For example, cisplatin should be strictly protected from light during injection, but photohydration and photoredox reactions cannot be completely avoided.418 The gradual deepening of the yellow color of a cisplatin solution during intravenous infusion is still an unavoidable phenomenon. Again, this reduces the concentration of active ingredients, and the photoreactive product has even higher side-effects than cisplatin itself. (iii) Non-selectivity: the in vivo distribution of conventional chemotherapeutic drugs is not tumor-selective. The toxicity of chemotherapeutic drugs to healthy cells has been criticized, e.g., the cardiotoxicity of anthracyclines such as doxorubicin (DOX)419,420 and the toxicity of platinum-based anticancer drugs  toward the renal and digestive systems.421,422 (iv) Drug resistance: the proportion of drug-resistant cells that overexpress efflux transporter proteins increases during treatment, and cancer chemotherapy often struggles to achieve the desired results.423Chemotherapy is one of the dominant means for clinical treatment of cancer.in vivo circulation, which can be divided into three parts: phagocytosis escape, passive targeting, and active targeting.At the level of tissues and organs, nanopharmaceuticals have overcome the challenges faced by conventional chemotherapy drugs to some extent. Among them, the most important one is the biodistribution characteristics exhibited by nanoparticles during 424–426First, nanoparticles with surface functionalization can be prevented from being recognized and eliminated by the mononuclear phagocytic system (MPS) and reticuloendothelial system (RES), which increases the circulation time of the active ingredient and therefore extends the drug half-life.427 that increases the amount of accumulation at the tumor site, which is called passive targeting (−), prostaglandin, and tumor necrosis factor, which stimulate tumor vasodilation.Second, nanoparticles have an enhanced permeability and retention (EPR) effectargeting . Althoug428 aptamers,429,430 and antibodies431 to promote their interaction with the tumor cells and overexpress receptors in the extracellular matrix, which is called active targeting. However, latest research has shown that the endothelial pathway and trans-cell transportation pathway are far more important than expected for the enrichment of nanoparticles in tumor tissues.432–434Finally, in order to further enhance the enrichment of nanoparticles at the tumor site, they can also be functionalized with targeting groups,via endocytosis, thereby bypassing the limitations of the selective permeability of the cell membrane toward small-molecule drugs.435 However, after uptake by cells, nanoparticles get restricted to acidic organelles such as endosomes and lysosomes and are gradually degraded, which limits their subsequent functionalities.436 Fortunately, for NCOFs, particularly imide-linked COF, the N atoms in the linkage are alkaline,437 causing an increase in the endo-/lysosomal pH, which induces the formation of Cl− ions and water molecules into the endo-/lysosome. When exceeding the endo-/lysosomal self-adjusting ability, endo-/lysosomes swell osmotically and eventually lead to endo-/lysosomal rupture, which release NCOFs into the cytoplasm. This process is known as endo-/lysosomal escape facilitated by the proton sponge effect.438 In addition, some nanoparticles with subcellular targeting properties can precisely act on specific organelles to achieve subcellular-targeting therapy.439,440 This is beneficial for minimizing drug doses, improving therapeutic benefits, and reducing side-effects.At the cellular and molecular levels, nanoparticles are taken up by the cells almost indiscriminately 6.1.2As mentioned in the earlier section, the structural basis of COF-related drug delivery is the availability of tunable permanent pores. The loading and release kinetics of small-molecule drugs based on host–guest systems have been extensively studied.et al. prepared 3D PI-COF-4 and PI-COF-5 .97 PI-COF-4 and PI-COF-5 have non- and fourfold-interpenetrated diamond nets, respectively, and their nitrogen adsorption isotherm analyses reveal that their BET specific surface areas are 2403 and 1876 m2 g−1 with pore sizes of 13 and 10 Å, respectively. Due to their huge specific surface areas, PI-COF-4 and PI-COF-5 can effectively load captopril, ibuprofen, and caffeine model drugs. For example, PI-COF-4 and PI-COF-5 were stirred in ibuprofen n-hexane solution for 2 h, and the loading amounts were up to 24 and 20 wt%, respectively. Further, for the drug release process, the release rates of ibuprofen in PI-COF-4 and PI-COF-5 were 60% and 49% at 12 h, respectively, indicating that the drug release is directly related to the pore size and geometry of COFs. For both PI-COF-4 and PI-COF-5, after 6 days, over 95% of the drug molecules were released 2 COF for ibuprofen in water was 119 mg g−1, while the equilibrium adsorption capacity for the more hydrophilic acetaminophen and ampicillin was less than 20 mg g−1.441 Besides, MICOF@SiO2 core–shell material not only exhibited superior adsorption capacity toward ibuprofen, but also showed excellent adsorption capacity for other nonsteroidal antiinflammatory drugs such as ketoprofen, diclofenac, indomethacin, flurbiprofen, and naproxen.254 The adsorption of other model molecules in COFs has also been studied, such as sulfamerazine389 and Congo red.442 These results provide valuable information for understanding the interactions between small molecules and COF pores and for formulating relationships among the molecular structure, hydrophilicity, molecular configuration, and adsorption and desorption behaviors of the molecules in the COF pores.The adsorption and release behaviors of ibuprofen in other COFs have been extensively studied. Salonen 6.1.3et al. investigated the potential of imine-linked PI-3-COF and PI-2-COF (in vitro.396 The antitumor drug fluorouracil (5-FU) was stirred with PI-3-COF and PI-2-COF in n-hexane for drug loading. As shown in In 2016, Zhao PI-2-COF  as drug 397 was prepared by the imine condensation of 4,4′,4′′-tribenzaldehyde and 4,4′,4′′-trianiline, where the electron pairs on imine nitrogen reversibly anchored the quercetin guest molecules through noncovalent interactions benzene was added to form TAPB-DMTP-COF. In this way, the drug loading of DOX was as high as 32.1 wt%. For in vitro experiments, DOX@TAPB-DMTP-COF was effectively taken in by HeLa cells, and it inhibited cell proliferation. For in vivo experiments in a xenograft model of H22 cells, an intratumoral injection of DOX@TAPB-DMTP-COF also showed significant tumor-suppressive effects. Notably, the authors believed that the reaction of the amino in DOX with 2,5-dimethoxyterephthalaldehyde resulted in a shift in the main diffraction peak of TAPB-DMTP-COF, possibly suggesting an alteration in the structure of COFs that requires further study.In addition to using the principle of host–object envelopment to load drugs into COFs in vitro and in vivo. However, due to the low dispersibility and inadequate bioavailability of bulk COFs, it may encounter serious defects such as premature clearance and ambiguous targeting when used for intravenous injections. As mentioned earlier, the surface modification of functional ingredients to ameliorate the deficiency of bulk COFs becomes a possible solution.The above examples demonstrate the feasibility of using COFs as drug delivery vehicles in vitro propan-1-amine (APTES) to afford the amine-functionalized TpASH-APTES. Finally, the amino group underwent a condensation reaction with FA to produce TpASH-FA as the target product. Besides, the amino groups on the surface of TpASH-APTES can also conjugate with the fluorescent dye of rhodamine-B-isothiocyanate (RITC) to perform fluorescent labeling. It should be noted that continuous post-SM led to the weakening of the π–π stacking between the COF layers, resulting in exfoliation and enhanced water dispersion of the COFs. Drug loading was achieved by simply stirring COF and 5-FU in water. When the concentration of TpASH-FA-5-FU was 50 μg mL−1, the cell viability of MDA-MB-231 was reduced to 14%, while the cell viability of nontargeted TpASH-APTES-5-FU was approximately 30%. A mechanistic study confirmed that the specific targeting effect of FA on MDA-MB-231 tumor cells enhanced the cell uptake, thereby increasing cell death. In addition, TpASH-FA-5-FU exhibited an inhibitory effect on cell migration.Due to the strong chemical stability and abundant functionalization potential of COFs, it is feasible to use multistep post-SM for functionalization. The first report in this regard discussed the preparation of folic acid (FA)-coupled TpASH-FA COF nanosheets for 5-FU-targeted drug delivery in vitro  by the tet al. prepared a series of water-dispersible PEGylated COF nanodrugs of PEGX-CCM@APTES-COF-1@DOX . A PEGX-CCM coating not only imparted fluorescence imaging capabilities to the nanodrug, but also significantly improved the drug loading and release kinetics, cell uptake, blood circulation time, and tumor accumulation capacity. The DOX content in PEG2000-CCM@APTES-COF-1@DOX was 9.71 ± 0.13 wt% and the encapsulation efficiency was as high as 90.5 ± 4.1%. The in vitro experiments indicated that PEGX-CCM@APTES-COF-1@DOX was broken down in lysosomes, resulting in slow DOX release. Even at very low DOX concentrations (0.25 μg mL−1), PEGX-CCM@APTES-COF-1@DOX significantly inhibited cell proliferation  derivatives have been modified onto the COF surface to enhance hydrophilicity and tumor accumulation. As shown in feration . The in  ability . These reriments .350-CCM@APTES-COF-1@pazopanib could penetrate the blood–brain barrier of mice and achieve intracranial tumor accumulation in the orthotopic models of brain metastasis from renal cancer.400 Due to the brain-targeting characteristic, as compared to the direct drug administration of pazopanib, PEG350-CCM@APTES-COF-1@pazopanib nanomedicine more significantly inhibited angiogenesis and tumor growth, protected mice from systemic drug toxicity, and prolonged survival time.Furthermore, the latest research confirms that PEGet al. prepared Pluronic F68-modified F68@SS-COF for DOX drug delivery.401 SS-COF was prepared by the imine condensation reaction between benzene-1,3,5-tricarbaldehyde and 4,4′-disulfanediyldianiline. The disulfide bond enabled F68@SS-COF to decompose in the presence of glutathione, thereby releasing the encapsulated DOX. DOX-loaded F68@SS-COF was confirmed to have a significant inhibitory effect on HepG2 cells.In addition to PEG, Pluronic F68 can also achieve a similar result. For example, Zhang 402 obtained by the reflux of 2,4,6-trihydroxybenzene-1,3,5-tricarbaldehyde and 4,4′-dianiline at 150 °C in N,N-dimethylformamide for 12 h, exhibited fibrous morphology with a length of about 200 nm and diameter of ∼22–30 nm, as confirmed by its SEM images. The half-maximal inhibitory concentrations (IC50) of EDTFP-1 on HCT-116, A549, HepG2, and MIA PaCa-2 cells were 9.89 ± 1.16, 11.88 ± 1.82, 14.38 ± 2.01, and 14.30 ± 1.32 μg mL−1, respectively. These data indicate that the toxicity of EDTFP-1 is comparable to that of 5-FU, a small-molecule broad-spectrum antitumor drug. In 2018, Bhaumik et al. further found that TrzCOF, with 5′′---4,4′′′′-dicarbaldehyde and 4,4′,4′′-trianiline as the monomers, exhibited similar cytotoxicity.403 The IC50 values of TrzCOF on B16F10, HepG2, HCT-116, and HEK293 cells were 10.47 ± 2.63, 9.55 ± 2.13, 8.31 ± 1.67, and 14.39 ± 2.25 μg mL−1, respectively. At the same concentration, the monomers constituting EDTFP-1 and TrzCOF did not show any activity to induce cell death. Although some mechanistic studies have shown that EDTFP-1 and TrzCOF can lead to cell apoptosis via a mitochondrial-dependent pathway, the relationship between toxicity and COF structure is not fully understood. In addition, these studies implied that COFs with certain specific structures may have inherent cytotoxicity. Therefore, it is necessary to evaluate their systemic toxicity and biocompatibility of COFs prior to their applications as drug carriers. In addition, it is regrettable that the authors did not mention the toxicity of COFs to normal cells, which is worthy of more attention.In most COF-based drug delivery systems, COFs themselves have been reported to be nontoxic or less toxic. However, thus far, two exceptional cases have been reported by Bhaumik and co-workers. EDTFP-1,6.26.2.1443,444 As shown in e.g., porphyrin,445 phthalocyanine,446 BODIPY,447 and cyanine,448 to induce the formation of reactive oxygen species (ROS) under specific wavelengths of light, leading to cytotoxicity.449 According to the photochemical mechanism of ROS generation , hydroxyl radical (˙OH), and superoxide anion (˙O2−). For the type II mechanism, the triplet excited PS directly transfers energy to oxygen to form singlet oxygen (1O2), which is considered to be the more common ROS in most cases.452PDT is a novel alternative method for cancer treatment that has shown superior potential for the minimally invasive treatments of various types of cancers, particularly superficial cancers.neration , PDT can453 are shown in via fiber optics and other means.454 (iv) PDT can be easily combined with other treatments, such as chemotherapy and radiotherapy.The general procedures of PDT455 Traditional small-molecule PSs are usually organic molecules with wide-range conjugated systems. They are poorly water-soluble and can aggregate easily. After systemic administration, the PS has insufficient accumulation in tumor tissues, making it difficult to meet in vivo applications.456 Nano-PSs make up for the abovementioned shortcomings via the EPR effect and active targeting abilities. On the other hand, improvements in the photochemical properties of PSs by nanotechnology cannot be ignored. The uniform modification of PSs on the nanoparticles prevents PS aggregation at the molecular level, thereby preventing fluorescence quenching and improving the 1O2 quantum yield.457Similar to drug delivery, the improvement effect of nanotechnology on PDT can be mainly reflected in the optimization of tumor accumulation.6.2.2216 Dong et al. used the BDF method to modify two amino-substituted BODIPY PSs on the surface of LZU-1 via imine condensation, and they successfully prepared LZU-1-BODIPY-2I containing an iodine atom and LZU-1-BODIPY-2H without an iodine atom , LZU-1-BODIPY-2I afforded higher 1O2 generation efficiency than that by LZU-1-BODIPY-2H. For in vitro PDT, under green LED irradiation, when the BODIPY concentration was 0.5 μM, LZU-1-BODIPY-2I almost completely killed HeLa and MCF-7 cancer cells, while LZU-1-BODIPY-2H-induced cell viability was still higher than 50%. The same trend was observed in the MCF-7 xenograft model, suggesting the significant value of the heavy-atom effect at the animal level. Mechanistic studies confirmed that LZU-1-BODIPY-2I entered the cancer cells primarily via the energy-dependent endocytosis pathway; then, it was mainly localized at the lysosomes and mitochondria and induced cell death by enhancing the lysosomal membrane permeabilization (LMP) and inducing loss of mitochondrial membrane potential (MMP).In 2019, COFs were used for cancer PDT for the first time.ine atom , where tet al. used APTES-COF-1 nanosheets to adsorb phthalocyanine PS to prepare PcS@COF-1.404 Because phthalocyanine can be highly dispersed on the surface of APTES-COF-1, PcS@COF-1 exhibits good photodynamic property under laser irradiation at 660 nm, and it can efficaciously reduce the cell viability of CT26 cells to 35% when the phthalocyanine concentration is only 3 μg mL−1. The PDT therapeutic effect of PcS@COF-1 has also been confirmed in CT26-tumor-bearing mice.It is also feasible to use the π–π interactions between COFs and PS to adsorb PSs onto the COF surface to prepare COF-based nano-PS. Yuan 283,458–460 however, their PDT applications in vitro and in vivo are still rare. Until recently, Qu et al. synthesized ultrasmall porphyrin-based TphDha COF nanodots tetraaniline and 2,5-dihydroxyterephthalaldehyde were used to synthesize bulk TphDha COF in Pyrex tubes; subsequently, ultrasonic exfoliation, surface modification using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG), and filtering separation were performed to obtain DSPE-PEG-coated TphDha COF nanodots (size: ∼3 nm). When the nanodots were irradiated by a laser at 638 nm, the nanodots induced ˙OH and 1O2 production at the same time via the type I and type II mechanisms  enables PDT excited by near-infrared (NIR) light at 980 nm  with a diameter of 30 nm were carboxylated by polyacrylic acid (PAA) to obtain PAA-UCNPs. Subsequently, TphDha COF was grown in situ under the catalysis of the surface carboxyl group to obtain UCCOFs, and the thickness of the shell was adjusted by changing the reaction conditions. When the UCNP core was excited with a 980 nm laser, the emissions at 541 and 654 nm were absorbed by the TphDha COF shell to produce 1O2, while the emission at 800 nm was not affected. Further study showed that when the thickness of the shell was 15 nm, the TphDha COF shell gave the best 1O2 generation capability, leading to the best cytostatic effect of HeLa cells. By loading 1O2-unstable indocyanine green (ICG) fluorescent dye into the pore of the TphDha COF shell, the in situ monitoring of 1O2 location and therapeutic response could be achieved in vivo. This is the first report of COF-based PDT excited by upconversion fluorescence, providing the possibility of deep PDT.t 980 nm .261 Olei406,461 have been successfully synthesized and have shown potential in PDT. Recently, Zhang et al. synthesized TPAPC-COF with a distinctive approximately T-shaped 5,10,15-tris(4-aminophenyl)corrole (H3TPAPC) molecule and a linear terephthalaldehyde molecule.406 TPAPC-COF adopts the staggered AB-stacking form with elliptical pores. The B-band absorption of TPAPC-COF is located at 399 nm, and the extended Q-band absorption is up to 2000 nm, which can be attributed to the huge π-electron delocalization system in the TPAPC-COF layer. When excited by a 635 nm laser, TPAPC-COF exhibited stronger photosensitivity properties than the H3TPAPC monomer and induced more efficient 1O2 production, which may be related to the reduced fluorescence emission and enhanced ISC of TPAPC-COF. For in vitro experiments, DSPE-PEG-coated TPAPC-COF effectively inhibited MCF-7 proliferation through PDT.Corrole-based COFs462 In 2019, Deng et al. synthesized COF-808 and COF-909 with photodynamic properties using inactive 5′,5′′′-bis(4-formylphenyl)--4,4′′′′-dicarbaldehyde (L-3C) and 4′,4′′′′-bis) (L-3N) monomers  to convert light energy into heat energy, leading to elevated temperatures at the tumor sites to kill the tumor cells. Because NIR light has a better tissue penetration ability,464 the ideal PTA should have high absorption in the NIR region. The remarkable feature of inorganic PTAs,11,465e.g., gold nanorods, platinum quantum dots, and graphene nanosheets, is their ability to absorb and manipulate light at the subwavelength scale by supporting coherent electronic oscillation, which is called localized surface plasmon resonance (LSPR).466 As energy transfers from light to electron and then from electron to lattice, the lattice transfers the energy to the environment in the form of heat, resulting in the photothermal effect. Furthermore, organic PTAs,467,468 such as cyanine and phthalocyanine, have a larger π-conjugated system, which can efficiently absorb NIR light and get excited. When the energy is released through a nonradiative transition, a thermal effect is induced.PTT is another potential phototherapy method.6.3.2232 The second is to design and synthesize COFs with light–heat conversion capabilities, e.g., copper(ii) tetraphenylporphyrin-based COFs.69,469Till now, COF-based PTT mainly includes two implementation methods. The first one is to combine PTA with COFs and the COF material, in our case, is used as a PTA carrier.et al. synthesized LZU-1 nanoparticles loaded with CuSe223 and Ag2Se.408 Under an 808 nm laser (1.5 W cm−2), the photothermal conversion efficiency of CuS-loaded LZU-1 (200 μg mL−1) was 26.3%, while that of Ag2Se-loaded LZU-1 (500 μg mL−1) was 37.9%. The authors also reported that the photodynamic properties could be attributed to LZU-1, but further cautious confirmation is necessary. Pang et al. also prepared micron-sized flower-like HCOF linked by β-ketoenamine.409 After metalation with Fe3+, the photothermal conversion efficiency of Fe-HCOF (800 μg mL−1) was 13.9% under an 808 nm laser (1.9 W cm−2).In 2019, Pang in vivo photoacoustic imaging and PTT.410 By performing two sequential post-SM processes of quaternization and one-electron reduction, 2,2′-bipyridine-based Py-BPy-COF was converted into cationic-radical-containing Py-Bpy+˙-COF  and the complex heterogeneity of tumors, it is difficult to achieve consummate therapeutic effects using monotherapy. In order to improve the treatment effects, clinically, combination therapy—also referred to as cocktail therapy—has been used as a standard method for the treatment of various cancers.470,471 In many cases, combining two or more therapeutic approaches not only increases the chances of a cure or long-term remission, but also reduces damage to vital organs and tissues more than that in monotherapy. In the context of nanomedicine, the realization of combination therapy heavily relies on integrating multiple treatments into a single nanoparticle. At this point, because of easy functionalization, COFs have become a fascinating nanoplatform to integrate various treatment strategies.Nanomaterial-based drug delivery and phototherapy have achieved certain success in preclinical tumor treatments. However, due to the inherent shortcomings of monotherapy -10,15,20-triphenylporphyrin (Por) and the PTA of vanadyl 2,11,20,29-tetra(tert-butyl)-2,3-naphthalocyanine (VONc) into TPB-DMTP-COF NCOF to obtain PDT/PTT dual-functional VONc@COF-Por nanomedicine  or PTT (93 μg mL−1) monotherapy. This intensive inhibitory effect of combination therapy toward MCF-7 could be attributed to the fact that an increase in temperature enhanced PDT-induced lysosomal and mitochondrial damage, but there was no significant change in the intracellular 1O2 level when the temperature increased. In vivo experiments conducted in MCF-7 xenograft models revealed that despite the fact that the antitumor effect could be enhanced by increasing the light intensity and drug dose of monotherapy, it caused irreversible skin damage. However, the combination therapy effectively inhibited tumor growth and delayed tumor recurrence, while minimizing side-effects.Modifying the desired PS and PTA into COFs through post-SM is one of the most feasible methods to achieve COF-based combination therapy. For example, Dong medicine .220 VONcoduction . When exoduction . In vitreriments  showed tet al. synthesized NCOF-366 (size: 100 nm) by the ultrasonic exfoliation of bulk COF-366.411 The porphyrin monomer was regularly arranged in the framework to reduce PS quenching. Upon laser irradiation at 635 nm (1.5 W cm−2), NCOF-366 displayed efficient 1O2 generation. On the other hand, the conjugated structure in the layer broadened the absorption band of NCOF-366, making NCOF-366 (200 μg mL−1) yield photothermal conversion efficiency of 15.1% under laser irradiation at 635 nm (1.5 W cm−2). Besides, flow cytometry experiments revealed that NCOF-366 (25 μg mL−1) caused 70.4% of 4T1 cells undergo apoptosis under laser irradiation at 635 nm . Photoacoustic imaging in vivo showed that NCOF-366 spread to the entire tumor within 1.5 h after an intratumoral injection. At this time, irradiating the tumor with a laser at 635 nm (1.5 W cm−2) for 5 min resulted in almost completely inhibiting tumor growth within 14 days.Generally, the combination of PDT and PTT required two functional components and excitation with two light sources of different wavelengths, which can lead to complicated and cumbersome applications. Recently, a multifunctional phototherapy system using single-wavelength excitation was constructed based on a porphyrin-based COF . Chen etet al. also reported the donor–acceptor TP-Por COF nanosheets  were prepared by adding IR783 during the ultrasonic exfoliation of TP-Por (cis-aconityldoxorubicin (CAD) in COF@IR783 achieved chemotherapy. As expected, COF@IR783@CAD inhibited the progress of murine breast cancer both in vitro and in vivo, as well as had photoacoustic imaging capability in vivo.The TP-Por COF mentioned above was also used as a carrier for PTA and chemotherapeutic drug.f TP-Por . Herein,6.4.3412 COFTTA-DHTA with a poly(lactic-co-glycolic-acid)–poly(ethylene glycol) (PLGA–PEG) amphiphilic polymer coating was used to deliver the anti-fibrotic drug of pirfenidone (PFD) to the tumor site . This is a landmark study that combines tumor physiology with nanomedicine for the first time, providing a promising and readily scalable anticancer strategy for targeting the extracellular matrix.The COF-based combination therapy of drug delivery and PDT was also achieved by a more exquisite strategy.mor site . PFD@COF6.4.4472 It aims to use T-cell-activating cytokines, immune checkpoint inhibitors, regulatory T cell depletion, and chimeric antigen receptor to selectively inhibit tumor growth.473,474 Cancer immunotherapy cannot directly kill tumor cells, but it can indirectly kill tumor cells by stimulating immune cell activation, proliferation, and differentiation. A large number of clinical trials and clinical practices have confirmed the effectiveness of this method for specific populations, although the side-effects are not less than those of traditional chemotherapy to a certain extent.475–477 In fact, the effectiveness of immunotherapy is largely restricted by the limited activation of the immune system and nonspecific off-target activation.478 In theory, a therapy that selectively kills cancer cells while activating the immune response of the local host is perfect. The immunogenicity of phototherapy provides a feasible direction for achieving this goal.479–481 Phototherapy can efficiently activate antitumor host immunity, realizing the combination of phototherapy and immunotherapy. This combination therapy is also known as photoimmunotherapy.482,483Cancer immunotherapy is one of the most advanced clinical cancer treatment methods.484 However, research on COF-based photoimmunotherapy is still in its infancy. ICG-loaded COF-1 nanosheets were surface-modified with polydopamine (PDA) and PEG to afford ICG@COF-1@PDA nanosheets with a size of 170 nm  was coated on the surface of COF@ICG via electrostatic interactions. The resulting COF@ICG@OVA had a photothermal conversion efficiency of 35.8% and the ability to generate ROS under laser irradiation at 650 and 808 nm. The combination of PDT and PTT induced tumor-associated antigen production. When further combined with α-PD-L1 therapy, the three-in-one combination therapy not only inhibited the growth of the primary tumor, but also delayed distant tumor growth and cancer lung metastasis.Combining phototherapy with immune checkpoint inhibitors can further enhance the activation of the immune system, enhancing antitumor therapy. In 2020, Pang 6.4.5e.g., Na+, K+, Ca2+, Zn2+, Mg2+, Fe2+, Cl−, H2PO4−, and HCO3−) participate in many important processes in cell biology, such as maintaining the osmotic pressure and acid–base balance, activating signal pathways, getting involved in cellular communication, and constituting enzymes.485 Their abnormal distribution and accumulation can interfere with these processes and induce irreversible cell damage.486,487 In this context, inorganic ions can be effectively exploited against cancers, which is called ion-interference therapy.488 However, due to their limitation of short circulation time and regulation of exogenous ions by cells, inorganic ions are relatively difficult to be directly used in antitumor therapy. Therefore, a combination therapy has come into legitimacy.489–493Some metal and nonmetal ions  CD44-target coating  Preparing NCOFs with high crystallinity and appropriate size is a huge challenge, which even becomes a major obstacle hindering the fundamental laboratory research of COFs. Despite the great efforts that have been made to develop various nanocrystallization methods, the difficulty of large-scale production and poor batch-to-batch consistency are still the bottlenecks restricting NCOF preparation. Unsatisfactory NCOF materials have questioned the uniformity and homogeneity of drug carriers, as well as the reliability of drug release kinetics studies. This is regarded as one of the most challenging issues facing the entire field of nanomedicine.in vivo. The enrichment of these exogenous COF particles in the body may cause serious health risks. Therefore, it is necessary to thoroughly evaluate the decomposition of COFs in vitro and in vivo to balance their stability and shelf life: completing the intended function and degradation at the right time in the right way is the most ideal state.(ii) The stability of COFs is a double-edged sword. Since most linkages are reversible chemical bonds, COFs can be broken down into organic small molecules or polymer fragments in the body, which leads to a limited shelf life but reduces the physiological toxicity of COFs. On the other hand, COFs involving irreversible chemical bonds may be difficult to decompose in vitro results mentioned in the literature, mainly focusing on the cytotoxicity of COFs. Due to their relatively short development history, a thorough assessment of their hemocompatibility, histocompatibility, cytotoxicity, neurotoxicity, and genotoxicity at the cellular and tissue levels are necessary. Furthermore, evaluating their acute toxicity, carcinogenicity, reproductive toxicity, and immunogenicity at the animal level is also a problem that needs to be resolved in the future.495 Considering that most of the degradation products of COFs are aromatic compounds, it is still unknown whether they are toxic or not.(iii) The biological safety of COFs has not been studied in detail. Until now, there are only some preliminary 496 Unfortunately, as compared to other nanomaterials such as metal oxides and MOFs,497 COFs themselves do not contain metals, resulting in their inapplicability in such imaging techniques. To our delight, COFs can be metalized through pre-SM or post-SM, which opens an avenue for imaging applications. In addition, studies on COFs for optical imaging may be a growing research area of COF-based tumor imaging, such as two-photon fluorescence imaging.498(iv) The application of COFs in tumor imaging is limited by the composition of light elements. Magnetic resonance imaging (MRI), ultrasound imaging (USI), computed tomography (CT), and positron emission tomography (PET), which are widely used clinically, rely on high-atomic-number metal or nonmetal contrast media .(v) Due to the complexity and diversity of cancers, it is necessary to develop an intelligent and versatile integrated system for diagnosis and targeting treatment (theranostics platform) to achieve accurate cancer treatment based on COFs. Although currently reported active targeting groups can partially improve the uptake of COFs by tumor cells, it is urgently needed to study exclusive targeting materials that only identify tumor cells to achieve effective tumor inhibition without any side-effects on healthy cells. Fortunately, the diversity of the functional groups in COFs ensures that multiple functions of COFs can be readily achieved through post-SM.75,499 This is a serious challenge for the resolution of COFs with complex structures. In addition, the stacking of COF layers may not be simple overlapping or staggered stacking. Some COFs with interlayer slips have been reported,500–507 which further increases the difficulty of structural analysis.(vi) Difficulties in the preparation of COF single crystals may lead to unreasonable structural analyses. Structural analysis based on PXRD follows the hypothesis–validation pattern. However, due to the broadening of the diffraction peaks, COFs with different structures may have similar PXRD patterns.Overall, COFs are a new member of the crystalline porous material family. In recent years, studies on COFs have mainly focused on the development of new structures and new synthesis strategies, and the applications of COFs in a large part has concentrated on heterogeneous catalysis and separation, while the study of COFs in the cancer biomedical field is still in its infancy. We anticipate that COFs can become a new growth point for cancer treatment and promote the development of nanomedicine, particularly clinical medicine.There are no conflicts to declare.


Poxviruses are large DNA viruses with a linear double-stranded DNA genome circularized at the extremities. The helicase-primase D5, composed of six identical 90 kDa subunits, is required for DNA replication. D5 consists of a primase fragment flexibly attached to the hexameric C-terminal polypeptide (res. 323–785) with confirmed nucleotide hydrolase and DNA-binding activity but an elusive helicase activity. We determined its structure by single-particle cryo-electron microscopy. It displays an AAA+ helicase core flanked by N- and C-terminal domains. Model building was greatly helped by the predicted structure of D5 using AlphaFold2. The 3.9 Å structure of the N-terminal domain forms a well-defined tight ring while the resolution decreases towards the C-terminus, still allowing the fit of the predicted structure. The N-terminal domain is partially present in papillomavirus E1 and polyomavirus LTA helicases, as well as in a bacteriophage NrS-1 helicase domain, which is also closely related to the AAA+ helicase domain of D5. Using the Pfam domain database, a D5_N domain followed by DUF5906 and Pox_D5 domains could be assigned to the cryo-EM structure, providing the first 3D structures for D5_N and Pox_D5 domains. The same domain organization has been identified in a family of putative helicases from large DNA viruses, bacteriophages, and selfish DNA elements. Orthopoxviruses very recently regained attention with the 2022 outbreak of monkeypox virus (MPXV) infections all over the world with a new mode of human to human transmission . This ouThe size of the orthopoxvirus genome varies from 220 kb coding for 223 proteins in cowpox virus to 186 kb for variola virus with 180 open reading frames  and teco323–785 construct [The DNA replication machinery consists of five similarly conserved essential proteins: the three proteins that form the DNA polymerase holoenzyme, E9, A20, and D4; the single-stranded DNA-binding protein I3; and the helicase-primase D5. The domain organization of the 95 kDa D5 protein has been analyzed by Hutin and co-workers . We attrThe C-terminal part of D5 is characterized by the presence of a D5_N motif described in the Pfam database of protein folds   comprising the D5_N domain and the AAA+ helicase domain has already been characterized extensively by small-angle scattering (SAXS) and negative stain electron microscopy [As the hexameric construct of D5 D5323–85 compriFull-length D5 (D5fl) has been expressed and purified as described in . Two pur323–785 fragment was cloned as a His-tagged tobacco etch virus (TEV) protease-cleavable construct using the pProEx HTb vector with the primers 5′-gcgccatggg taataaactg tttaatattg cac-3′ and 5′-atgcaagctt ttacggagat gaaatatcct ctatga-3′, resulting in two additional N-terminal residues Ala-Met from the TEV cleavage site. It was produced and expressed as described in [2, and 1 mM DTT, and the protein was eluted with a gradient ending in 20 mM Tris-HCl pH 7.0, 166 mM NaCl, 334 mM KCl, 2 mM MgCl2, and 1 mM DTT. Fractions of the second peak were pooled, and the buffer was exchanged to buffer K containing 5% glycerol, 50 mM Tris-HCl pH 7.0, 50 mM NaCl, 100 mM KCl, 2 mM MgCl2, and 10 mM β-mercaptoethanol by 2 concentrations to 100 µL in a 2 mL centrifugal concentrator .The 53.4 kDa D5−1 D5fl) were added an additional 10 mM MgCl2, 1 mM ADP, and 2 mM BeF3. About 4 µL of a D5fl sample were applied to a mica sheet covered with a film of evaporated carbon. The carbon film was floated off the mica in glycerol-free buffer, retrieved, and placed onto a 400-mesh copper electron microscopy grid. The sample was subsequently stained with 4 µL of 2% sodium silicotungstate (SST), blotted, and dried. Imaging was performed on an FEI F20 transmission electron microscope operating at 200 kV. Images were recorded on a 4k × 4k Eagle digital camera with a pixel size of 2.17 Å.To the sample  and blotted for 2.5 s at blot force 1 with 0.5 s drain time using a Vitrobot Mark 4  prior to flash-freezing in liquid ethane.A complex with an end-labelled dsDNA 30mer was used. An oligonucleotide with the sequence biotin-5′-ccgaatcaggaagataacagcggtttagcc-3′-digoxigenin (DIG) was annealed to an unlabeled oligomer 5′-ggctaaaccg ctgttatctt cctgattcgg-3′  after heating the mix to 95 °C for 5 min and overnight slow cooling to room temperature in a beaker. Then, 7.5 µM D5The parameters are described in Cryo-EM Data treatment used Relion 3.1 ,23 in a https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb accessed on 27 July 2022).The amino acid sequence of vaccinia virus (Copenhagen strain) D5 was submitted to a Colab notebook running a simplified version of Alphafold v2.1.0 (323–785 construct was split into three domains according to the observed domain boundaries: collar domain (res. 323–403), AAA+ helicase domain (res. 404–705), and C-terminal domain (res. 706–785). The domains were fitted as rigid bodies into the cryo-EM electron density using Chimera [The Alphafold2 model of D5 corresponding to the D5 Chimera . Manual  Chimera . Real-sp Chimera  were car Chimera . The Phe Chimera . We opte Chimera .2+, ATP, ADP, or ADP, and BeF3 showed the flexible attachment  in negative stain under different conditions regarding the presence of Mgtachment  of the Nre of D5 a with a re of D5 b and all323–785 yielded a variety of projections , the β-hairpin loop (16 Å), and the DNA binding loop in the AAA+ helicase domain (18 Å). The channel would just be sufficiently wide for the passage of dsDNA. The lysine residues of the basic cluster and the loops inside the AAA+ helicase domain are flexible and correspond to the least well described parts of the structure, so that the passage of dsDNA cannot be excluded. The sample used for the cryo-EM data collection contained a roughly equimolar complex of D5observed .323–785 reconstructed without symmetry  and two phage-related proteins from K. pneumoniae (A0A0H3GIU0 and A0A0H3GLV7).The best hit of the Dali search with the AAA+ helicase domain was the helicase domain of bacteriophage NrS-1. This similarity extends over all its structure, which could be matched with D5 FATCAT . HowD5 shares with Nrs-1 helicase, E1, the hexameric structure of Rep68, and LTA a planar arrangement of the collar and AAA+ domain subunits, which are in turn in close contact in contrast to the more remote PCV2 Rep protein, where the subunits show a staircase arrangement and the collar and AAA+ domain are connected by a loose linker. For E1 helicase, a helical staircase mechanism of DNA translocation has been proposed, where the β-hairpin loops play a central role as they descend (if the collar domain is placed at the top) progressively from the initial ATP-bound state over an ADP-bound state to a final nucleotide-free state. When the β-hairpin has reached its bottom position, it disengages from the DNA. Conformational changes induced by the bound nucleotide are transmitted to the β-hairpin and the DNA binding loops, whereas positional changes of the arginine finger trigger nucleotide binding and ATP hydrolysis of the neighboring subunit. So far, it is assumed, but not proven, that D5 uses a similar mechanism shared within the above mentioned SF3 helicases but different from the one of PCV2 Rep . RegardiWhereas, globally, the model from Alphafold2 agreed very well with the experimental electron density, the refined model only deviates for the more flexible parts of the helicase domain a from th323–785 would be just wide enough to accommodate dsDNA (323–785 is able to bind dsDNA oligomers at the entrance to the central channel, even though it has not been possible to demonstrate an helicase activity using different forked substrates ([323–785 hexamer with one strand running on the top surface of the collar domain and one strand running inside the central channel, as described recently for E1 [323–785 affects the interaction with DNA.The central channel of D5te dsDNA d as the y for E1  (Figure 323–785 explaining the observed electron density. The flexibility of the C-terminal domains already suggested from negative stain EM (With the limited quality of the electron density, we prefer not to hypothesize about the presence of ssDNA or dsDNA in the central channel of D5stain EM g may refstain EM . The higMost aspects of the helicase action of D5 still remain enigmatic but the available structure will help greatly to orient future experiments required to establish the mechanisms of helicase loading and translocation. The combination of cryo-EM and Alphafold2 structural calculations has been proven to be very powerful for structure determination even in the context of oligomeric proteins, which are not fully implemented into the Alphafold2 algorithm yet. Our structure indirectly validates Alphafold2 calculations of thousands of viral helicase structures related to D5, which are waiting to be released.