Nanoscale covalent organic frameworks as theranostic platforms for oncotherapy: synthesis, functionalization, and applications

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.


Introduction
Cancer remains a worldwide public health issue with high morbidity and mortality rates. 1 It is estimated that by 2030, the number of cancer cases will increase to 24.6 million, while the number of cancer deaths can reach around 13 million. 2 In recent years, increasing number of researchers in the elds of chemistry, materials science, biology, and medicine have turned their research interest towards rational designing and preparation of nanopharmaceuticals for tumor diagnosis and treatment 3-6 due to drawbacks in conventional therapies, such as chemotherapy, radiotherapy, and surgical resection. 7 Nanoparticle-based drug delivery, which integrates emerging nanotechnologies with traditional chemotherapeutic drugs to get rid of drawbacks in traditional therapies as well as offer new possibilities to optimize cancer treatment, has always been one of the focuses in the eld of nanomedicine. [8][9][10] In general, the key advantages of nanodrug delivery are longer circulating halflives, improved pharmacokinetics, selective intratumoral accumulation, and lower systemic toxicity. Meanwhile, some other emerging minimally invasive therapies, such as photothermal Qun Guan is currently a PhD student. He  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][12][13][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 carbonbased materials, 20,21 semiconducting polymers, 22,23 and liposomes. 24 The design, synthesis, and applications of advanced porous materials with specic structures at the micron-and nanoscales have been a research hotspot in various scientic elds; [25][26][27][28][29][30] further, the development of porous materials ranging from traditional inorganic materials (such as zeolites, silicas, and activated carbons) to organic-inorganic hybrid porous materials (such as metal-organic cages (MOCs), 31 coordination polymers (CPs), 32 and metal-organic frameworks (MOFs)). [33][34][35] Among them, MOFs are crystalline materials formed by the selfassembly 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 eld of oncology and have even entered the stage of clinical trials. [37][38][39] With the development of reticular chemistry, 40 a new generation of crystalline porous materials, namely, covalent organic frameworks (COFs), emerged in 2005 41 and have been booming in recent years. 42 As a natural extension of MOFs, COFs are composed of nonmetallic elements (e.g., C, H, N, O, and B) connected by strong covalent bonds into twodimensional (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 applicationoriented material syntheses. Until now, COFs have been widely used for separation and analysis, [45][46][47][48][49] heterogeneous catalysis, [50][51][52] sensing, 53 optoelectronics, 54 energy and environmental science, [55][56][57][58][59] and biomedicine. 60,61 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, uorescence 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. 62 To sum up, we believe that COFs are becoming a promising and efficient organic material platform for building theranostic systems.
In this review (Fig. 1), we systematically summarized the rational design and preparation strategies of COFs, focusing on their nanocrystallization and functionalization strategies, with emphasis on their specic applications in tumor nanotherapeutics. Finally, the remaining challenges and possible future trends of COFs for tumor nanotherapeutics were discussed, expecting to promote further development of COFs for oncotherapy.

Structures of COFs
COFs are generally dened as crystalline, extended 2D and 3D networks with permanent pores constructed by different organic building blocks connected via 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 p-p interactions. For example, 2D monosheets of COF LZU-1 use the face-to-face eclipsed stacking (Fig. 2), 64 which is also known as AA stacking: this is the most common stacking type for 2D COFs. Besides AA stacking, other stacking types, such as staggered AB, [65][66][67] ABC, 68 and ABCD 69 stacking, can also be formed during the assembly of 2D COFs.
COFs are modular in nature. The reactive functional groups (including species and number) and molecular geometry (e.g., length, directionality, and symmetry) of the monomers enable to predene 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 (Fig. 3). An interesting subject in mathematics, namely, plane tessellation, which refers to completely covering a plane using one or more geometric shapes without overlaps and gaps, may be a useful guide for the topological structure design of COFs, particularly 2D COFs with hierarchical porosity. 71,72 However, in terms of topology, 3D topology 44 is expected to be more colorful and complex than 2D topology. As shown in Fig. 4, using polyhedral instead of polygonal monomers 73 or adding geometric constraints to the 2D monomers 74,75 can possibly afford 3D COFs. In particular, the combination of tetrahedral monomers and triangular linkages results in the formation of ctn or bor topology, whereas the combination of tetrahedral and linear monomers usually leads to the dia topology. 73 In contrast to MOFs based on coordination bonds, a considerable amount of research in the eld of COFs has been devoted toward the development of new chemical bonds that constitute linkages. 76 For each new linkage, nding appropriate crystallization conditions is the rst challenge. In order to fabricate extended crystalline solids, covalent bonds formed between the monomers are usually reversible under the given   Nanoscale Advances Review reaction conditions, and the reaction rate must be sufficiently fast to allow sufficient defect self-correction. 77,78 In recent years, conventionally considered irreversible chemical bonds have also been successfully used to construct COFs, 79

Characterization of COFs
Generally, the rst 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, largesized single crystals that meet the requirements of SC-XRD measurements. 81 In this context, powder diffraction crystallography has become the most powerful technique for the determination of COF structures. By combining experimental and simulation results with structural renements, the structure of a COF can be optimized and perfectly determined. This simulation-experiment-renement trilogy has become almost a standard procedure for structural designation via COF diffraction crystallography. 82 Among them, X-ray diffraction is the most common diffraction technique, such as powder X-ray diffraction (PXRD) 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. 84,85 Certain 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 identication, 13 C cross-polarization magic-angle spinning solid-state nuclear magnetic resonance spectroscopy ( 13 C 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. 87 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 specic 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 specic surface area is the Brunauer-Emmett-Teller (BET)   73 (B) Synthesis of 3D COFs by twisting a monomer from planar to tetrahedral symmetry with steric hindrance. Adapted with permission. 74 Copyright 2020, American Chemical Society. theory based on a multilayer gas adsorption model. 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.
Morphology, including particle shape and size, is signicant 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-eld 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 energydispersive X-ray spectroscopy (EDX), elemental distribution can also be semiquantitatively determined. In addition, dynamic light scattering (DLS) measurements can provide

Synthesis of COFs
Since the group of Yaghi pioneered the preparation of the rst COF material under solvothermal conditions in 2005, 41 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 (Fig. 7).

Solvothermal synthesis
Solvothermal synthesis refers to a method for preparing advanced materials in a sealed pressure container at a certain temperature and solvent autogenous pressure through the process of dissolution and recrystallization of raw materials. 92 So far, most of the reported COFs have been synthesized under solvothermal conditions, including the earliest reported ones, i.e., COF-1 and COF-5. 41 When the solvent is water, it is referred to as hydrothermal synthesis. The hydrothermal synthesis of COFs is exceedingly rare, 93 while the preparation of COFs in a mixed solution of organic solvent and water has been realized. 94,95 For solvothermal synthesis, stainless steel reaction kettles with polytetrauoroethylene (PTFE) 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 (Fig. 8A). The general synthesis steps are shown in Fig. 8B. In brief, the calculated amount of monomers and solvents are added to the Pyrex tube; aer several freeze-pump-thaw cycles, the Pyrex tube is sealed to preserve the produced water molecules to maintain the reversibility of the reaction and placed in the oven under a certain temperature for several days (from 3 to 7 days). Aer cooling down to room temperature, the target COF materials can be nally obtained aer thoroughly washing the crude powders with organic solvents and dried in a vacuum. It should be noted that because of the limitation of the volume of a Pyrex tube, it is relatively difficult to obtain COF materials at a large scale.
The reaction temperature used in the solvothermal reaction has a signicant 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. 73,96 Most COFs based on the Schiff-base reaction that form the C]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 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 (Fig. 10). Through this strategy, the reaction rate can be reduced, and it is relatively easier to obtain highly crystalline COF materials. This strategy has been proven by the successful syntheses of COF-5, 102 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 2,4dihydroxybenzene-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.
Multicomponent reactions have recently proven to be an effective way to optimize solvothermal thermodynamics and kinetics. They combine a reversible covalent bond to allow the crystallinity of COFs and an irreversible covalent bond that imparts stability, reaching a higher level of complexity and precision in covalent assembly. Recently, Wang's and Dong's groups reported the applications of this strategy for COF synthesis. As shown in Fig. 11A, the three-component one-pot Debus-Radziszewski reaction among pyrene-4,5,9,10-tetraone, aromatic trialdehydes, and ammonium acetate afforded a series of imidazole-linked COFs under solvothermal conditions, 106 and ve covalent bonds in each cyclic joint were formed in situ during polymerization. Fig. 11B shows that when a mixture of 1,3,5-tris(4-aminophenyl)benzene, 2,5dimethoxyterephthalaldehyde, and phenylethylene was heated in o-dichlorobenzene/n-butanol (1 : 1, v/v) at 120 C for 72 h in the presence of BF 3 $OEt 2 , 4,5-dichloro-3,6dioxocyclohexa-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 COFs 108 were obtained by adding sulfur to the conventional synthesis system of Schiff-base COFs (Fig. 11C). The reversible imine condensation, irreversible C-H functionalization reaction, and oxidative annulation reaction synergistically afforded a set of TZ-COFs with high crystallinity and excellent robustness. This in situ multicomponent polymerization approach might open a new avenue for constructing COFs that are not possible to be successfully obtained by other conventional methods.

Microwave synthesis
Microwave synthesis 109 is related to the synthesis approach using microwave heating. When compared with the traditional external heating method, microwave heating is endogenous, that is, the object to be heated is a heat-generating object; further, it does not require heat conduction, and uniform heating can be achieved in a short time.
In 2009, Cooper et al. were the rst to use microwaves to synthesize B-O-linked COF-5 with a high yield of 68%. 110 The reaction time (about 20 min) by microwave heating is more than 200 times faster than that of solvothermal synthesis (3 days). Meanwhile, the BET specic surface area of the obtained COF-5 is higher than that of previously reported COFs under solvothermal conditions (2019 vs. 1590 m 2 g À1 ). Imine-linked TpPa-COF was also synthesized by microwave heating using benzene-1,4-diamine and 2,4,6-trihydroxybenzene-1,3,5tricarbaldehyde as the monomers. 111 Dichtel et al. further synthesized imine-and b-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-tetrauoroisonicotinonitrile with triphenylene-2,3,6,7,10,11-hexaol. 113 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 signicantly difficult to be achieved for solvothermal synthesis.

Ionothermal synthesis
Ionic liquids (ILs) are a class of organic salts that are liquid at room temperature or near room temperature. 114 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. This journal is © The Royal Society of Chemistry 2020 Nanoscale Adv., 2020, 2, 3656-3733 | 3663
The synthesis of COFs that do not contain ILs as the guest in the pores was reported for the rst time by Kang  In addition, ILs [C n mim][BF 4 ] (n ¼ 4, 6, 10) 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.

Atmospheric solution synthesis
The importance of achieving COF synthesis under ambient pressure is self-evident. The rst example of COF synthesis at atmospheric pressure and room temperature was reported by Zamora et al. in 2015. 120 Benzene-1,3,5-tricarbaldehyde and 1,3,5-tris(4-aminophenyl)benzene were used as the monomers and stirred in DMSO for 48 h to obtain a white powder of RT-COF-1 (Fig. 13A). This milestone work opened new avenues for the large-scale production of COFs. Furthermore, COF-300 has also been obtained under atmospheric pressure. As shown in Fig. 13B, with 1,4-dioxane and cyclohexane as the reaction solvents, a gram-scale yellow powder of COF-300 was obtained at 65 C with a yield of up to 90%. 121 The crucial factors for obtaining high-quality crystalline imine-linked COFs mainly include lower temperature (to prevent the oxidation of -NH 2 ), reduction in water within the reaction system (to maintain reversibility of imine condensation), and reduction in solubility (to control nucleation and continuous crystal growth).
Recently, Fang et al. reported the atmospheric pressure synthesis of COFs based on the Michael addition-elimination reaction in aqueous solutions. 122 Typically, b-ketoenamine and arylamine were suspended in an aqueous solution containing acetic acid as the catalyst, followed by maintaining the reaction at ambient temperature and pressure to produce crystalline JUC-520, JUC-521, JUC-522, and JUC-523 solids (Fig. 13C). More importantly, by scaling up, it took only 30 min to afford gram-scale JUC-521 with a yield of up to 93%. This eco-friendly, low-cost, and mild synthesis method provides the possibility of large-scale production of COFs.
To give the readers a better understanding of this promising approach, detailed examples of COF syntheses in an atmospheric solution are summarized in Table 1. 120-134

Mechanochemical synthesis
Mechanical chemistry research originated from the transformation of mechanical energy and chemical energy in biochemistry related to physiological functions. At present, mechanochemistry mainly refers to the process of applying mechanical energy to substances via squeezing, shearing, and friction to induce chemical changes between solids. 135 With the development of the machinery industry, the continuous emergence of various high-energy grinding equipment has enabled the application of mechanical chemistry in many elds such as     14A). Mechanochemical synthesis is also considered as a green synthesis process due to its obvious characteristics of no/low solvents. In 2013, TpPa-1, TpPa-2, and TpBD COFs were synthesized by means of solvent-free mechanochemical grinding using aldehyde-amine condensation reactions. 137 In short, the raw materials were placed in an agate mortar and ground at room temperature. Aer about 5 min, the color of the powder changed to light yellow and gradually turned into orange within 15 min.
Aer 45 min of grinding, the powder became crimson in color, suggesting the successful formation of COFs (Fig. 14B). When compared with the classic solvothermal method, this method is fast, controllable, and environment-friendly; however, the crystallinity of the resulting COFs is normally unsatisfactory. The BET specic surface area is only 61 m 2 g À1 for TpPa-1 COF, 56 m 2 g À1 for TpPa-2 COF, and 35 m 2 g À1 for TpBD COF. In contrast, the COFs synthesized by the solvothermal method afforded BET surface areas of 535, 339, and 537 m 2 g À1 for TpPa-1, TpPa-2, and TpBD COFs, respectively.
In order to further improve the synthesis efficiency, grinding can be performed in a ball mill. According to the report of Banerjee et al., when the frequency of the ball mill with two 7 mm-diameter stainless steel balls is 25 Hz, the yield of TpPa-1 COF can reach 90% at 45 min. 137 Besides, ball milling can also be used to synthesize Tp-MA COF (Fig. 14C) by the reaction of 2,4,6-trihydroxybenzene-1,3,5-tricarbaldehyde and melamine at ambient temperature. 138 The resulting material can be used for degrading various types of organic pollutants. The third method of mechanochemical synthesis of COFs is the extrusion process. A twin-screw extruder has been used for the continuous synthesis of COFs. 139 In a representative synthesis process, p-phenylenediamine and solid catalyst of p-toluenesulfonic acid were mixed in a beaker, manually fed into the extruder, and then 2,4,6-trihydroxybenzene-1,3,5-tricarbaldehyde and a small amount of water were sequentially added; aer mixing for a specic period of time, the mixture was heated at 170 C for 1 min. Highly crystalline and porous COF materials were obtained aer washing and drying (Fig. 14D). This approach provides the possibility of large-scale production of COFs at high throughputs of several kilograms per hour.
More recently, 3D printing technology has also been employed for the preparation and molding of COFs. 140 With the help of a 3D printing template, Pluronic F127 and the raw materials were mixed and subsequently forming hydrogels; then, the COFs could be printed using commercial 3D printers (Fig. 14E). Due to the extremely controllable and operational accuracy of 3D printing, very delicate COF devices could be obtained.
The mechanochemical synthesis of COFs is still at a vigorously developing stage. The advantage of space-time yield overwhelms the lack of relatively high crystallinity, and the resulting COFs have been applied in the elds of separation, 141 detection, 142 and electrochemistry. 143,144

Other synthesis methods
In addition to the aforementioned methods, other synthesis methods have also been developed, such as photochemical synthesis, 145,146 electron-beam irradiation synthesis, 147 and vapor-assisted synthesis. 148 Although these methods are probably not universal, they might be highly effective for the synthesis of task-specic COFs with specic structures. Among these novel methods, it is particularly worth mentioning the efforts to synthesize hcc-COF by Choi et al. 146 Benzene-1,2,4,5tetraamine, cyclohexane-1,2,3,4,5,6-hexaone, water, and acetic acid were mixed in a quartz bottle and exposed to simulated sunlight ($200-2500 nm, 50 mW cm À2 ) irradiation for 3 h to produce hcc-COF. Light energy not only accelerates the imine condensation reaction, but also promotes the conversion reaction from amorphous polyimide precipitation to crystalline COFs via fast and reversible dynamic imine condensation. As a comparison, the reaction in darkness for only 3 h formed an amorphous product.

Nanocrystallization of COFs
In the earlier section, we discussed the general synthesis approaches for fabricating bulk COFs. However, due to the strict restrictions on the size of the materials for biomedical applications, micron-sized bulk COFs cannot be directly applied in the eld of oncology. In this section, we will systematically discuss how to obtain NCOFs to meet the needs of biomedical applications. It should be noted that as long as one spatial dimension of the COF material is in the nanoscale, then it can be classied as NCOF, including, but not limited to, quantum dots, nanorods, nanosheets, and nanoparticles. The process of preparing NCOFs is also called COF nanocrystallization. Similar to other 2D materials, 18,57 according to the differences in raw materials, COF nanocrystallization can be divided into two categories (Fig. 15). One is the top-down method, which uses bulk COFs as the precursor, which destroys the interlayer interaction of the COFs under certain conditions to afford nanosheets. The other is the bottom-up method, which employs the corresponding monomers to directly synthesize NCOFs.

Top-down synthesis
4.1.1 Ultrasonic exfoliation. Ultrasonic exfoliation 149 is a universally applicable and commonly used nanocrystallization strategy, theoretically applicable to all types of COFs. The common practice is that bulk COFs are suspended in a specic solvent with the appropriate surface energy and then simply sonicated. During this process, ultrasound induces bubbles in the solvent; when the bubbles burst due to their huge surface tension, shock waves are induced across the bulk COF surfaces that destroy the interlayer p-p-stacking interactions of the COFs, thereby affording exfoliated nanosheets.
The solvents are signicant for the process of ultrasonic exfoliation. Appropriate solvents can promote exfoliation as well as inhibit aggregation. Generally, polar solvents (e.g., ethanol, water, and 1,4-dioxane) can be used to obtain better exfoliation effectivity. 150 Theoretical studies have indicated that the Hansen's parameter (HSP) of the solvent-a metric that determines the intensity of the molecular forces in the solvent-may be a useful reference for selecting the ultrasonic exfoliation media. 151 Some examples for the preparation of NCOFs by ultrasonic exfoliation are shown in Table 2. [150][151][152][153][154][155][156][157][158][159][160][161][162][163][164][165] Evidently, the weaker the interactions between the COF layers, the easier it becomes to perform ultrasonic exfoliation. TPA-COF constructed with tris(4-aminophenyl)amine and tris(4-formylphenyl)amine exible monomers with the C 3v symmetry conrmed this prediction. 152 Bulk TPA-COF can be synthesized by the solvothermal method in 1,2-dichlorobenzene/ethanol/3 M acetic acid (20 : 5 : 1, v/v/v). Bulk TPA-COF  was sonicated in an ultrasonic bath (110 W, 40 kHz, ethanol) for 3 h, which was naturally sedimented for 24 h to obtain highquality nanosheets in the supernatant liquid (Fig. 16). If the tris(4-formylphenyl)amine in TPA-COF was replaced with 1,3,5tris(4-formylphenyl)benzene, the resulting TPA-COF-2 was relatively difficult to exfoliate into nanosheets under similar conditions. This interesting phenomenon can be attributed to the approximately planar structure and strong p-delocalized system of 1,3,5-tris(4-formylphenyl)benzene, resulting in the increasing p-p interactions between the adjacent layers in TPA-COF-2 as compared to those in TPA-COF. Unfortunately, ultrasonic exfoliation is a very time-and energy-consuming process, and it is challenging to prepare a large number of NCOF sheets within a short time. More importantly, the size of the nanosheets obtained by ultrasonic exfoliation is oen uneven. Therefore, proper postprocessing is necessary, such as removing the large COF particles that have not been completely peeled by standing or low-speed centrifugation. In some cases, ltration is also a feasible option.
4.1.2 Mechanical exfoliation. Mechanical exfoliation mainly refers to the method of exfoliating bulk COFs by grinding, which is widely used for the preparation of 2D nanomaterials. 166 During grinding, the grinding medium exerts impact, friction, and shear force on the COF powders via regular or irregular movements, causing crystallographic slips between the different layers and subsequently resulting in aking. Based on the differences in the media, mechanical exfoliation can be This journal is © The Royal Society of Chemistry 2020 Nanoscale Adv., 2020, 2, 3656-3733 | 3671

Review
Nanoscale Advances divided into wet grinding and dry grinding: the difference between these two methods is the necessity to add a solvent. Generally, wet grinding yields better exfoliating efficiency. NCOF sheets prepared by mechanical exfoliation were reported for the rst time by Banerjee et al. in 2013. 167 They synthesized eight Schiff-base COFs with different pore diameters based on the imine condensation reaction (Fig. 17A), and manually ground them in an agate mortar for about 30 min in the presence of a small amount of methanol. Aer removing the remaining bulk COFs by centrifugation, eight kinds of NCOF sheets (thicknesses ranging from 3 to 10 nm) were obtained with a yield of about 8% ( Fig. 17B and C). The obtained NCOF sheets and bulk COF exhibited almost the same FT-IR spectra, conrming that the intrastratal chemical bonds did not change (Fig. 17D). However, in the PXRD pattern, the diffraction peak attributed to the (001) plane broadened, and the diffraction peak corresponding to the (100) plane decreased in intensity, indicating the reduced number of stacked layers and decreased periodicity along the z-direction because of random slips in these nanosheets (Fig. 17E). Nevertheless, the resulting nanosheets exhibited good stability in strong acid media.
The dry ball-milling method has been used for preparing redox-active DAAQ-TFP-COF nanosheets for lithium-ion electrodes. 168 In a typical synthesis process, bulk DAAQ-TFP-COF was placed in a ball crusher under a vibration frequency of 50 Hz for 0.5 h to prepare DAAQ-TFP-COF nanosheets with a thickness of $3-5 nm (Fig. 18). Electrochemical experiments proved that as compared to bulk DAAQ-TFP-COF, exfoliated DAAQ-TFP-COF nanosheets with shorter lithium diffusion pathways yield signicantly higher utilization efficiency of redox sites and faster lithium storage kinetics. Moreover, 3BD COF and nanosheets prepared by the dry ball-milling method even show the possibility of uorescence sensing of peroxidebased explosives. 169 Since the ball mill has been widely used in industry, NCOF nanosheets prepared by the ball-milling method have been used as modier additives in the elds of polyurethane modi-cation 170 and mixed matrix membranes. 171,172 4.1.3 Chemical exfoliation. The essence of chemical exfoliation is to use chemical reactions to introduce large-sized groups into the COF layer in order to increase the interlayer spacing and weaken the van der Waals force between the layers, thereby inducing exfoliation.
Exfoliation induced by the Diels-Alder cycloaddition reaction between N-hexylmaleimide and anthracene-based DaTp COF is the earliest report on chemical exfoliation. 173 The introduction of N-hexylmaleimide with a length much larger than the interlayer distance in the COF interferes with the p-pstacking interaction and planarity of the COF layer, resulting in exfoliation (Fig. 19). Importantly, although both ultrasonic and mechanical exfoliation methods can afford DaTp COF exfoliation, only DaTp-MA NCOF sheets prepared by the chemical exfoliation method can self-assemble in a layer-by-layer manner between the air-water interfaces to produce centimeter-sized lms. The authors claimed that this was related to the hydrophobic hexyl groups, which reduced their exposure in the water environment. Since surfactant or stability is not necessary, this method exhibits great promise for various applications. For instance, chemically exfoliated anthracene-based COF nanosheets using maleic anhydride as the functionalizing exfoliation reagent 174 have been used to enhance the anodic performance of COF materials in lithium-ion batteries.
Due to the low efficiency of the ultrasonic exfoliation method, it is extremely challenging to prepare TpBD COF nanosheets in a large quantity in water. However, macroscopic suspended solids were invisible aer dissolving FeCl 3 and ultrasonication for 1 h. 175 Then, the TpBD COF nanosheets with a hydrodynamic diameter of $50 nm and thickness of 2.5 nm were successfully obtained with the removal of Fe 3+ by dialysis. Quantitative calculations conrmed that Fe 3+ could coordinate with the b-ketoenamine linkage of TpBD COF, resulting in an increase in the interlayer distance from 3.42 to 9.85Å; further, the interlayer interaction energy changed from À362 to À19 kJ mol À1 (Fig. 20A). This increased interlayer spacing and weakened interlayer interaction energy lead to the facile insertion of solvent molecules between the COF layers and subsequent delamination. Similarly, the axial coordination of 4ethylpyridine with the central metal ion of porphyrin can also coercively increase the spacing between the COF layers ( Fig. 20B), thereby inducing the exfoliation of porphyrin-based COFs. 176 The coordination reaction provides newer possibilities for the chemical exfoliation of COFs.
Photochemical reactions can also induce the chemical exfoliation of COFs. As shown in Fig. 21, when TpAD COF loaded with cis-azobenzene guest molecules was exposed to ultraviolet light at 365 nm for 12 min in isopropanol, cisazobenzene underwent the isomerization reaction. The The hydrolysis reaction of n-butyllithium has also been used for the chemical stripping of TpPa-1 COF. 178 In an n-hexane solution, n-butyllithium was embedded into the TpPa-1 COF   Nanoscale Advances layers. Aer the hydrolysis reaction, TpPa-1 COF nanosheets were obtained with an astonishing productive rate of 80%. Another interesting method is to generate nanoparticles in situ between the COF layers via redox reactions, thereby realizing chemical exfoliation. As shown in Fig. 22, Wang et al. synthesized E-TFPB-COF nanosheets by the reduction reaction of KMnO 4 . 179 Typically, bulk TFPB-COF and perchloric acid were mixed in water; then, KMnO 4 was carefully added into the solution, which was maintained at 30 C for 30 min. Thereaer, the solution was sonicated for 2 h to afford a black powder of E-TFPB-COF/MnO 2 . During the exfoliation process, MnO 2 nanoparticles acted as spacers to effectively prevent the reaggregation of E-TFPB-COF. Finally, light-yellow E-TFPB-COF nanosheets could be obtained aer etching away the MnO 2 nanoparticles with hydrochloric acid. The thickness of the E-TFPB-COF nanosheets, as measured by AFM, was $1.6-2.0 nm.

Gas-driven exfoliation.
The key point of gas-driven exfoliation is to trigger the lattice expansion of a 2D material at high temperatures and then induce exfoliation by the vaporization of liquid nitrogen. Aer several "expansionvaporization" cycles, the 2D material can be exfoliated into several atomic layers. This method has been used for the exfoliation of graphene, 180 hexagonal boron nitride, 181 and MOFs. 182 Recently, three COFs (NUS-30, NUS-31, and NUS-32) with triangular and hexagonal pores were exfoliated by using this method ( Fig. 23A and B). 183 COF bulk powders were heated to 300 C in air and maintained for 10 min, followed by immediately immersing them in liquid nitrogen (Fig. 23C); the above steps were repeated ve times. Thereaer, the as-prepared COF nanosheets were centrifuged in acetonitrile to remove any large particles and to obtain COF nanosheets with thicknesses ranging from 2.4 to 3.1 nm (Fig. 23D). Although a few COF nanosheets have been successfully exfoliated via this method, its general applicability needs to be further explored. Exfoliation induced by gases instead of liquid nitrogen can be a promising alternative in the near future.
4.1.5 Charge-mediated self-exfoliation. Due to electrostatic repulsion, the interactions between polymer chains with an embedded ionic characteristic decrease. Along this line, providing no or less external energy, COFs with positive charges on the framework may get spontaneously exfoliated, which is called charge-mediated self-exfoliation. Generally, the charge is located on the monomers, such as benzimidazolium, 184 guanidinium, 185,186 and viologen. [187][188][189] As reported by Banerjee et al., 185 three COFs, namely, TpTG X (X ¼ Cl, Br, and I), were constructed based on the Schiff-base condensation reaction of triaminoguanidinium halide and 2,4,6-trihydroxybenzene-1,3,5-tricarbaldehyde ( Fig.  24A). Similar to hydrogen bonds, N-H/X interactions exist between the halide ions and guanidinium nitrogen of COFs, thereby   This journal is © The Royal Society of Chemistry 2020 Nanoscale Adv., 2020, 2, 3656-3733 | 3675

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Nanoscale Advances immobilizing halide ions between the COF layers. The presence of interlamellar halide ions and inherent positive charges within the guanidine units causes repulsion and also interferes with the p-p-stacking interactions between the COF layers, leading to self-exfoliation into COF nanosheets with a thickness of only a few nanometers. As expected, TpTG X spontaneously exfoliates in water, thereby affording TpTG X nanosheets with a thickness of $2-5 nm. Due to the interaction of the positively charged nanosheets and negatively charged phospholipid bilayer of bacteria, the resulting TpTG X nanosheets exhibited certain antibacterial activity. Recently, Pal et al. prepared highly uorescent self-exfoliable DATG Cl COF via a similar structural design. 186 The thickness of the exfoliated DATG Cl COF nanosheets was in the range of $5-7 nm, indicating that DATG Cl COF nanosheets had $13-18 layers (Fig. 24B). Pyridinium is another type of structural unit that constitutes self-exfoliated COFs. As shown in Fig. 25, using N,N-dimethylacetamide/mesitylene/6 M acetic acid (1 : 9 : 1, v/v/v) as the solvent, PyVg-COF with a staggered AB-stacking arrangement was prepared by the solvothermal reaction of 4,4 0 ,4 00 ,4 000 -(pyrene-1,3,6,8-tetrayl)tetraaniline and 1,1-bis(4-formylphenyl)-4,4 0bipyridinium dichloride. 188 The bipyridinium structural unit with high-density electrostatic repulsion was encoded into the framework to withstand interlayer p-p stacking, resulting in stronger interlayer interaction of PyVg-COF than that of the skeleton-solvent interaction. Therefore, PyVg-COF could be dispersed in a variety of organic solvents (e.g., N-methyl pyrrolidone, dimethyl sulfoxide, N,N-dimethylformamide, N,Ndiethylformamide, N,N-dimethylacetamide, and 1,3-dimethyl-2-imidazolidinone) by simple shaking. The critical aggregation concentration (CAC) of PyVg-COF in DMSO-d 6 was up to 30 mg mL À1 , below and above which monolayers and multilayers were respectively formed. Because of its highly charged skeleton and desirable dispersibility, ionic COF membranes could be easily prepared by means of the electrophoretic deposition method.
Besides bipyridinium, 188,189 phenanthridinium, such as ethidium and propidium, was also introduced into the frameworks to fabricate self-exfoliated ion-containing COFs. As shown in Fig. 26, the self-exfoliated EB-TFP COF 190 and PI-TFP COF 191 obtained in water showed average layer thickness distributions of 1.6 and 1.5 nm, respectively. Surprisingly, the supramolecular reassembly phenomenon exists in both COF nanosheets. For EB-TFP COF nanosheets, 190 double-stranded DNA (dsDNA) induced their reaggregation, which created a hydrophobic environment over the ethidium unit and prevented the excited-state proton transfer process, thereby enhancing uorescence emission. This phenomenon provides a unique opportunity to distinguish between dsDNA and singlestranded DNA (ssDNA). For PI-TFP COF, 191 the host-guest interactions between PI-TPF COF and cucurbit [7]uril (CB [7]) lead to the restacking of nanosheets. Aer 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.
Self-exfoliation induced by the charges within the frameworks is also observed in other types of COFs, such as iCOF-Acontaining 1-methylpiperazine branched chain 192 and COF BTCcontaining iron phthalocyanine. 193 Self-exfoliation induced by the charges on the COF linkages is relatively scarce. In 2020, Dichtel et al. conrmed that the protonation of the C]N bond caused by acid treatment can induce the self-exfoliation of imine-linked COFs. 194 BND-TFB COF ( Fig. 27) with imine linkages was stirred in a mixture of acetonitrile/tetrahydrofuran/triuoroacetic acid (7 : 3 : 2, v/v/v) overnight, which was delaminated into a suspension. Aer acid treatment, the protonated COF layer was positively charged and the charge repulsion induced their exfoliation. AFM and HRTEM images of BND-TFB COF nanosheets showed that the thickness was $5-50 nm and the diameter was $50-1000 nm. Moreover, two additional imine-linked COFs, namely, TAPB-PDA COF and methyl COF, were also exfoliated by acid treatment. Nanopipette-based electrochemical tests by Wang et al. conrmed that as the pH value decreased, the polarization of C]N bonds, slippage of layers, and exfoliation of COF occurred in sequence, nally leading to the formation of uniform COF nanosheets. 195 Acid concentration and treatment time should be precisely controlled to achieve a balance between the degradation caused by the fracture of the covalent bond and exfoliation induced by the destruction of interlayer interaction.

Bottom-up synthesis
Although many strategies have been developed to prepare NCOFs by the top-down synthesis, these exfoliation techniques usually yield 2D nanosheets. The 2D nanosheet is very thin (<10 nm), but its size in the other two dimensions is still too large (up to several microns), 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.
The nucleation and growth theories of nanocrystals are extremely complicated. 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. Aer 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  This journal is © The Royal Society of Chemistry 2020 Nanoscale Adv., 2020, 2, 3656-3733 | 3677

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Nanoscale Advances a dissolution-reprecipitation process, and the monomers tend to be arranged in a periodic order to form crystalline COF particles (Fig. 28A). Therefore, according to this model, to prepare uniform-sized nanoparticles, a large amount of nucleation should be explosively formed in the shortest possible time such that the above process is separated as much as possible.
The simultaneous nucleation and growth processes may result in the formation of particles with nonuniform sizes. In the nucleation stage, according to the theory of crystal nucleation, for spherical crystal nuclei, where DG represents the total free energy change when a new phase is formed, v corresponds to the molar volume of the crystal, r is the radius of the nucleus, k B represents the Boltzmann constant, S is the solution supersaturation, and g represents the surface free energy per surface area. The functional relationship between DG and r is shown in Fig. 28B. When S > 1, the maximum value of DG is obtained at This critical radius r crit corresponds to the minimum size at which a particle can survive in solution without being redissolved (Fig. 28B). Therefore, it is possible to reduce r crit by increasing S or decreasing g to promote nucleation. Besides, rate control is also signicant during growth, and ripening in addition to avoiding secondary nucleation can improve the uniformity of nanoparticles and increase in crystallinity.
According to the aforementioned nanocrystal nucleation theory and growth model, we will comprehensively introduce and discuss the bottom-up synthesis strategies of NCOFs and summarize the related examples in the following section.

Reaction kinetics regulation.
Here, we will illustrate the applications of reaction kinetics regulation (e.g., changing the reaction time, controlling the rate of monomer addition, and adding nucleation modulators) for the preparation of NCOFs.
Deng et al. prepared COF-606 with different particle sizes ( Fig. 29) by varying the temperature and reaction time of the solvothermal reaction. 198 In order to prepare COF-606 with particle sizes of 500 nm and 1 mm, a Pyrex tube containing the building blocks and solvents was placed in an oven and heated at a programmed temperature at a constant rate of 0.1 to 90 C and maintained for 7 days. When the precipitate was immediately separated, the particle size of the obtained COF-606 was 500 nm. If the Pyrex tube was cooled down to room temperature under programmed temperature at a constant rate of 0.1 C, the particle size of the obtained COF-606 was 1 mm. More importantly, if the Pyrex tube was directly heated at 65 C for 12 h, the particle size of COF-606 was reduced to 100 nm. Indeed, this synthesis strategy is fully consistent with the theory of nanocrystal nucleation and growth. A slow increase in temperature inhibits the nucleation process, and a slow decrease in temperature promotes the growth process such that large-sized particles can be obtained. The rapid and short-term reaction favors explosive nucleation, retards growth, and tends to form small-sized COF nanoparticles.
In 2018, Dichtel 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 (Fig. 30). Furthermore, the quantitative analysis of COF-5 nucleation and growth by kinetic Monte Carlo (KMC) simulations 201,202 showed that there was a threshold of the monomer concentration below which growth dominated nucleation and nucleation and growth rates had second-and rst-order dependence on the monomer concentration, respectively. Besides COF-5, studies on COF-10 and TP-COF also conrmed the above trends. 200 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. 203 Therefore, indiscriminately promoting nucleation and inhibiting growth may reduce the crystallinity of COFs and even lead to the formation of amorphous polymers. In this context, a reasonable balance between nucleation and growth is imperative. One effective strategy is to add monofunctional species (e.g., phenylboronic acid, catechol, benzaldehyde, and phenylamine) 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 bers, 206 single crystals, 207,208 and thin lms. 206,209 Additionally, when the added competitor has a chiral site (e.g., (S)-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.
Among them, Dichtel 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 concentrationdependent mode and induced subsequent anisotropic growth. 211 When compared with the constant addition of monomers, the competitor signicantly 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 (Fig. 31). By adding the competitor in a similar way, three other boronate-ester-linked COF materials, namely, TP-COF, DPB-COF, and COF-10, were also obtained within a hydrodynamic diameter range of $110-1400, $90-260, and $110-800 nm, respectively. 4.2.2 Surfactant-assisted synthesis. According to the earlier discussion, in the eqn (1), reducing g can reduce r crit , thereby affording small-sized nuclei. To our delight, surfactants can reduce g by reducing the tension at the solid-liquid interface. 212,213 Therefore, surfactant-assisted synthesis is one of the most effective methods for synthesizing NCOFs.
In addition to regulating the nucleation and growth processes of COFs, the solubilizing effect of surfactants on organic monomers enables aqueous synthesis. Puigmartí-Luis et al. synthesized imine-linked TAPB-BTCA COF nanoparticles in water using a surfactant mixture (Fig. 32). Due to the formation of micelles, the water-insoluble benzene-1,3,5tricarbaldehyde and 1,3,5-tris(4-aminophenyl)benzene monomers were effectively dissolved in aqueous solutions containing cationic hexadecyltrimethylammonium bromide (CTAB) and anionic sodium dodecyl sulfate (SDS) surfactants, thereby forming two homogeneous solutions. When acetic acid was added to the mixture, the mixture turned orange, indicating the formation of the C]N bond. An aqueous colloidal solution of TAPB-BTCA COF was obtained aer reaction for 72 h at 30 C. This journal is © The Royal Society of Chemistry 2020 Nanoscale Adv., 2020, 2, 3656-3733 | 3679 Review Nanoscale Advances The particle size of TAPB-BTCA COF strongly depended on the ratio of CTAB to SDS; by increasing the amount of SDS, the hydrodynamic diameter of TAPB-BTCA COF could range from 15 to 73 nm. Additionally, another imide-based COF, i.e., Tz-COF, with a particle size of about 20 nm, could also be prepared via the reaction of 2,4,6-tris(4-aminophenyl)-1,3,5-  Rapid heating and slow growth are benecial to COF nanocrystallization. For this purpose, two strategies have been developed for surfactant-assisted COF nanocrystal synthesis. First, microwaves provide speedy and uniform heating, which facilitates a nucleation burst. Second, the amine monomer is protected with tert-butoxycarbonyl, which is deprotected in situ  This journal is © The Royal Society of Chemistry 2020 Nanoscale Adv., 2020, 2, 3656-3733 | 3681

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Nanoscale Advances during the reaction, thereby delaying the growth of the crystal nuclei. As shown in Fig. 33A, under microwave treatment, tertbutyl-(4-aminophenyl)carbamate and benzene-1,3,5tricarbaldehyde reacted in ethanol in the presence of polyvinylpyrrolidone (PVP) and triuoroacetic 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 AE 52, 245 AE 25, and 112 AE 11 nm (Fig. 33B-D). When toluene was added to the reaction system to reduce the polarity of the solution, LZU-1 assumed a hexagonal shape (Fig. 33E). This method is also used for the synthesis of Por-COF and TFPB-PDA COF nanocrystals using 4,4 0 ,4 00 ,4 000 -(porphyrin-5,10,15,20-tetrayl)tetrabenzaldehyde and 1,3,5-tris(4formylphenyl)benzene as the monomers (Fig. 33F-H).
Along this line, in 2019, Dong et al. successfully synthesized NCOF LZU-1 under solvothermal conditions instead of the aforementioned microwave method. 216 More importantly, the obtained LZU-1 NCOF exhibited a high surface area of 822 m 2 g À1 , which is twofold higher than that of the original report (410 m 2 g À1 ). 64 4.2.3 Acetonitrile method. Acetonitrile-a common organic solvent-has been successfully used for the preparation of B-O-and C]N-linked NCOFs, showing extraordinary application potential in the bottom-up synthesis of NCOFs.
As shown in Fig. 34, by the cocondensation of triphenylene-2,3,6,7,10,11-hexaol and 1,4-phenylenediboronic acid, Dichtel et 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 (e.g., toluene, tetrahydrofuran, N,N-dimethylformamide, chloroform, and methylene chloride) 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 lms from solution. Besides boronate-ester-linked COFs, boroxine-linked Ph-COF, BPh-COF, DBD-COF, Py-COF, and TMPh-COF colloidal dispersions (Fig. 35) were also synthesized in mixed solvents containing acetonitrile by the trimerization of diboronic acids. 218 According to the study reported by Dichtel et al., acetonitrile was also used as a solvent for the synthesis of imine-linked COFs. 219 Briey, scandium(III) triuoromethanesulfonate as a transamination catalyst 131 was added to the acetonitrile solutions of 1,3,5-tris(4-aminophenyl)benzene and p-phthalaldehyde (Fig. 36). Then, the mixture was stirred for 20 h at room temperature. The particle size of the obtained TAPB-PDA COF varied from the minimum of 200 nm to the maximum of 500 nm with changes in the initial monomer concentration. This approach has been widely applied to the synthesis of various Schiff-base COFs, 220-224 implying its widespread feasibility.

Interfacial synthesis.
Diffusion-based COF synthesis at the solid-liquid, 225 liquid-liquid, [226][227][228] or liquid-gas 229 interfaces usually yields nanosheets or thin lms. Although the thickness can be controlled to a few nanometers or a dozen nanometers, the length and width are usually in the range of several microns. An interesting exception is the interfacial synthesis of TAPA-Sa COF quantum dots (Fig. 37). 230 The monomer of 2-hydroxybenzene-1,3,5-tricarbaldehyde was dissolved in dichloromethane/N,N-dimethylformamide (40 : 1, v/v) solvent; then, acetic acid (12 M) was slowly added. Finally, TAPA in N,Ndimethylformamide 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. 4.2.5 Template method. The template method uses a readily synthesized nanostructure as the core; then, a shell material is deposited on its surface. Aer removing the inner core, hollow nanoparticles can be obtained. Since Möhwald et 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.  232 By varying the concentration of COF monomers, the thickness of the COF shell can be controlled. If further etching of Fe 3 O 4 is undertaken with hydrochloric acid, hollow COF nanoshells can be obtained. 233 In addition, considering the magnetic property of Fe 3 O 4 , core-shell Fe 3 O 4 @COFs have shown great promise for magnetic separation and enrichment. 234,235 The pre-modication of monomers on the templates is more robust to provide COF growth sites via covalent bonds as compared to that via hydrogen bonds. Therefore, templates with amino groups on the surface may facilitate the controlled template synthesis of C]N-linked COFs. Zhang et al. were the rst to pre-modify the amino group on the surface of indiumbased MOF NH 2 -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; nally, the NH 2 -MIL-68@TPA-COF core-shell structure was successfully prepared (Fig. 39). 236 Other MOFs with amino groups, such as UiO-66-NH 2 , 237,238 NH 2 -MIL-125(Ti), 239,240 and NH 2 -MIL-101(Fe), 241 can also be used as templates to anchor aldehyde monomers via covalent bonds.
Very recently, Chen et al. prepared hollow COF capsules using MOFs as the template to encapsulate biomacromolecules (Fig. 40), which could not be directly loaded onto solid COFs by simple adsorption. 242 First, biomacromolecules were encapsulated into acid-labile MOFs by in situ biomimetic mineralization to yield a template for COF growth. Then, considering the fact that acetic acid readily leads to the premature dissolution of MOFs, the COF shells were grown at room temperature with Sc(OTf) 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 This journal is © The Royal Society of Chemistry 2020 Nanoscale Adv., 2020, 2, 3656-3733 | 3683

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Nanoscale Advances 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 conrmed by selecting different MOFs (e.g., ZIF-90, ZIF-8, and ZPF-2), COFs (e.g., COF-42-B and COF-43-B), and biomacromolecules (e.g., bull serum albumin, catalase, and glucose oxidase). In addition, in CO 2 -dissolved water, hollow LZU-1 and COF-42 were synthesized using ZIF-8 and ZIF-67 as the templates. 243 In addition to the aforementioned hard templates (e.g., Fe 3 O 4 and MOFs), so 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, so templates can be readily removed from the coreshell structures by evaporating or adding demulsiers.
Finally, some of the representative examples of COF coreshell structures synthesized using different templates are summarized in Table 3. 94,233-261

Functionalization of COFs
In the earlier section, we discussed methods for preparing NCOFs. However, to some extent, NCOFs are only platforms; additional functional active sites have to be integrated with them to realize some task-specic biomedical applications. The process of incorporating functional components is better known as COF functionalization. Therefore, in this section, we will systematically discuss the functionalization approaches involving COFs.
Generally, approaches for COF functionalization (Fig. 41) can be divided into pre-synthesis modication (pre-SM) and post-synthesis modication (post-SM). For pre-SM, the functionality is rstly introduced into the monomer, which can be further used to synthesize functionalized COFs. Although the obtained functionalized COFs possess abundant functionalities within the frameworks, this approach is not appropriate for all kinds of COFs and it is even incompatible with the synthesis of some COFs, signicantly limiting widespread applications. For post-SM, pre-prepared COFs can be used as the scaffold and then assembled with a variety of functionalities. In contrast, post-SM can introduce some task-specic functionalities that are impossible to be incorporated into the frameworks of COFs via pre-SM. However, due to the inherent deciencies of solidliquid reactions, it is sometimes difficult to achieve the complete functionalization of the frameworks from the inside out. In addition, when the sizes of the functionalized molecules exceed the pore size of the COFs, only surface functionalization can be realized.

Pre-SM
Active sites or functional groups can be introduced into the organic monomers in advance before they can be used for the construction of COFs. In this way, the active sites can be uniformly distributed throughout the COFs, which is benecial for diverse applications such as heterogeneous catalysis and separation. Theoretically, any active group can be designed and incorporated into the monomers for the construction of functionalized COFs, although synthesis conditions need to be optimized on a case-by-case basis.

Post-SM
Similar to the post-SM of MOFs, 287,288 the post-SM of COFs generally involves the embedding of reactive functional groups into the frameworks of pre-synthesized COF materials to achieve task-specic applications via different post-SM strategies, such as post-synthesis covalent modication, post-synthesis functional group conversion, bonding defect functionalization (BDF), and post-synthesis linkage transformation. Here, we will Nanoscale Advances Review provide detailed summary and discussion of each strategy, expecting to give the readers a better understanding of this promising post-SM approach. 5.2.1 Post-synthesis covalent modication. With regard to covalent post-SM, the desired functional groups or active sites can be integrated within the frameworks of pre-synthesized COFs via covalent bonds. Till now, the reaction sites within the COF structures mainly include ethynyl, vinyl, hydroxyl, amino, and carboxyl groups (Fig. 43).
To achieve the covalent modication of COFs, a high-yield modication reaction under mild conditions is highly desired, e.g., click chemistry. Jiang et al. synthesized [HC^C] x -H 2 P-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 0 ,4 00 ,4 000 -(porphyrin-5,10,15,20-tetrayl)tetraaniline (Fig. 44). 289 Quantitative click reactions between the ethynyl units and azide compounds were performed with CuI as the catalyst to anchor the desired groups into the pores. The groups with different hydrophilicities and acid-base properties (including ethyl, ester, hydroxyl, carboxyl, and amino groups) were successfully incorporated into the COFs with controllable loading contents. The effects of functional groups on their gas adsorption properties were conrmed by CO 2 adsorption measurements. For example, the CO 2 adsorption capacity could reach up to 157 mg g À1 at 273 K and 1 bar for functionalized COFs with 50% amino This journal is © The Royal Society of Chemistry 2020 Nanoscale Adv., 2020, 2, 3656-3733 | 3687

Review
Nanoscale Advances 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][291][292][293][294][295][296][297][298] The click reaction of vinyl with thiol is another typical clickchemistry-based post-SM method. For instance, vinylfunctionalized COF-V and 3, 3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecauorodecane-1-thiol with low surface free energy reacted in the presence of azobisisobutyronitrile (AIBN) as the catalyst to yield peruoroalkane-modied COF-VF (Fig. 45). 299 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 modication 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 modied into COFs by the thiol-ene reaction.
Noteworthily, 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. prepared sulfonic-group-modied  COFs with the maximum adsorption capacity for diclofenac of up to 770 mg g À1 . 304 The hydroxyl group has become a promising reaction center for post-SM because of its versatile reaction chemistry (Fig. 46). By the Williamson ether synthesis reaction of hydroxyl groups with halohydrocarbon, the etherication of COFs can be achieved. For example, Gao 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 xing the IL on the pore walls of the COFs. The obtained [Et 4 NBr] X% -Py-COFs could catalyze the formylation of amine with CO 2 and phenylsilane under metal-free conditions. Besides, the post-SM reaction of the hydroxyl group with iodine-substituted peruoroalkane could convert hydrophilic COF-DhaTab into hydrophobic COF-DTF. 306 Esterication is another effective strategy for hydroxyl modication. For example, Bein et al. studied the reaction of T-COF-OH with uorescein isothiocyanate (FITC) in acetone to modify nonuorescent T-COF-OH into T-COF-OFITC with green-light emission via a thiocarbamate bond (Fig. 47A). 307 Another attempt involved the esterication of hydroxyl groups with acyl halides. Chiral D-(+)-camphoric acid dichloride (Dcam-ClO) has been loaded into the pores of TzDa COF by this

Review
Nanoscale Advances reaction; the resulting CTzDa COFs were used as an adsorbent for the chiral isolation of amino acid enantiomers (Fig. 47B). 130 The hydroxyl group can also be esteried by an anhydride and the versatility of this method has been demonstrated by the esterication of hydroxyl groups in 2D COFs 308,309 and 3D COFs. 310 As shown in Fig. 47C, the ringopening esterication reaction of succinic anhydride not only esteries the hydroxyl group, but also introduces a carboxyl group into the COFs. 310 There are two different ways for performing the covalent post-SM of amino groups, i.e., amidation and imidization. For example, the amidation reaction between TpPa-NH 2 and 4,4 0 -(ethane-1,2-diyl)bis(morpholine-2,6-dione) can integrate ethylenediaminetetraacetic acid (EDTA) into the pores, thereby improving the heavy-metal-ion adsorption capacity of the COF material. 311 TpBD(NH 2 ) 2 -COF-containing amino groups enable the reaction with acetic anhydride to achieve the acetylation of the pore walls. 312 In addition, TpBD(NH 2 ) 2 COFs can be functionalized by the imine condensation reaction of the amino group with (2-formylphenyl) boronic acid. 252 Very recently, the modication of carboxyl groups was successfully reported by Yaghi and co-workers. 313 With the help of certain condensating agents, such as 1-(bis(dimethylamino) methylene)-1H-benzo[d] [1,2,3]triazole-1-ium 3-oxide hexa-uorophosphate (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, esterication, and thioesterication at room temperature, and several chelating groups can be introduced into COF-616 to afford functional materials for the adsorption of pollutants in water (Fig. 48).

Post-synthesis
functional group conversion. Although the amino group is a good candidate for post-SM, the de novo preparation of imine-bonded COFs containing free amino groups is a formidable challenge. Post-synthesis functional group conversion (Fig. 49) is regarded as a feasible choice to introduce amino groups into imine-linked COFs.
In particular, the reduction of azide and nitro groups is a feasible method to insert amino groups into COFs via post-SM. For example, it is difficult to prepare TpBD(NH 2 ) 2 COF by the direct reaction of [1,1 0 -biphenyl]-3,3 0 ,4,4 0 -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 0dinitro-[1,1 0 -biphenyl]-4,4 0 -diamine as the precursor, TpBD(NO 2 ) 2 COF was initially prepared (Fig. 50A); subsequently, SnCl 2 was used to reduce the nitro groups to obtain TpBD(NH 2 ) 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 (Fig. 50B). 314 More importantly, the obtained amino groups could be further modied by covalent modication 252,311,312 or functional group conversion (e.g., conversion to isothiocyanate 315 ) to obtain other types of functional COF materials.
Post-SM based on cyano-chemistry has been fully proven by using COF JUC-505 (also known as COF-316) as the model. 79,80 JUC-505 was synthesized by the irreversible nucleophilic aromatic substitution reaction between 2,3,5,6-tetra-uoroterephthalonitrile 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 LiAlH 4 /tetrahydrofuran solution (Fig. 51). Functional group conversion based on the reaction of cyano groups with hydroxylamine has also been demonstrated in   Nanoscale Advances Review structures to some extent. For example, methyl bromination, 318 viologen reduction, 319 p-phenol oxidation, 168 photoinduced cistrans isomerization, 320 and photoinduced pericyclic reaction. 321 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.

BDF.
Despite the high crystallinity of COFs, defects are still inevitable, such as vacancy defects due to monomer deciency in periodic ordered structures, edge dislocations because of squeezing or stretching of lattices, extrinsic defects as a result of the doping of extraneous materials (e.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 modication method using the bonding defects of COFs is called BDF.
The presence of free amino and aldehyde groups in imine-COFs is a widely accepted consensus, as revealed by ssNMR and FTIR measurements. For instance, the ssNMR peak of the aldehyde group in LZU-1 COF is at 191 ppm and the FTIR characteristic peak is at 1695 cm À1 ; in contrast, these peaks are located at 191 ppm and 1682 cm À1 , respectively, for TPB-DMTP-COF. Consequently, Dong et al. further demonstrated that the aldehyde groups on the surface of imine-linked LZU-1 COF 216 and TPB-DMTP-COF 220 can gra with amino-containing functional molecules via Schiff-base condensation reaction (Fig. 52A). Similarly, by the immobilization of polyvinylamine onto the frameworks of LZU-1 COF, the obtained polyCOF hybrid materials with the side chain of amino group exhibited superior CO 2 /N 2 , CO 2 /CH 4 , and CO 2 /H 2 gas separation selectivity. 322 In addition, imide-linked NKCOF-1, which was generated from 1H,3H-benzo[1,2-c:4,5-c 0 ]difuran-1,3,5,7-tetraone and 4,4 0 ,4 00 -(1,3,5-triazine-2,4,6-triyl)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 modication of biomolecules, such as lysozyme, tripeptide Lys-Val-Phe, and lysine (Fig. 52B).

Truncation unit functionalization (TUF).
The concepts of truncation unit and TUF dates back to 2012. 324 Dichtel et al. proposed the truncation unit strategy for the rst time to introduce task-specic functional groups into COFs. The tetrahedral monomer (methanetetrayltetrakis(benzene-4,1diyl))tetraboronic acid that self-condenses to form COF-102 was modied via the substitution of one of its four arylboronic acid moieties by the appointed functional group. Thereaer, the resulting triangular triboronic acid molecule was co-condensed with the original tetraboronic acid monomer to afford  This journal is © The Royal Society of Chemistry 2020 Nanoscale Adv., 2020, 2, 3656-3733 | 3693

Review
Nanoscale Advances truncation-unit-containing COF-102. When the incorporated truncation unit is (but-3-ene-1,1,1-triyltris(benzene-4,1-diyl)) 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 sulydryl group (Fig. 53). 325 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 uorescent dye to endow COF with near-infrared uorescence properties. 204 Further, imine-linked B-COF containing the truncation unit of phenylboronic acid can be obtained by the co-condensation of benzidine, 2,4,6-trihydroxybenzene-1,3,5-tricarbaldehyde, and (4-aminophenyl)boronic acid. Due to the high affinity of phenylboronic acid and diol, the resulting B-COF can be used to selectively enrich riboavin, 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.
Very recently, Han et al. synthesized dCOF-NH 2 -X with exposed amino groups (Fig. 54) using a three-component condensation system with 1,3,5-tris(4-aminophenyl)benzene and 2,5-dihydroxyterephthalaldehyde as the COF monomers and 2,5-dihydroxybenzaldehyde as the truncation unit. 329 More importantly, the amount of free amino groups could be precisely regulated by changing the amount of truncation unit. As expected, free amino groups can be decorated by ILs with aldehyde groups via imine condensation. The resulting materials possess not only well-dened pore channels that provide pathways for ion transport, but also an optimized pore environment containing ILs for higher ion conduction, which can be used as solid-state electrolytes with a wide temperature range from 303 to 423 K. Besides, the authors synthesized dCOF-CHO-X with free aldehyde groups using 5 0 -phenyl-[1,1 0 :3 0 ,1 00terphenyl]-4-amine as the truncation unit, which can be used as post-SM platforms for amino-containing functional molecules. It should be noted that unlike several earlier examples, in this work, the truncation unit itself does not contain reaction sites and the free reaction groups are entirely derived from COF monomers, implying the exibility and feasibility of this strategy.
In conclusion, by simply adding a truncation unit to the reaction system without changing the crystallization conditions of COF synthesis, the truncation unit can be uniformly doped into the entire lattice. The reasons that the truncation unit can provide possibilities for post-SM are as follows. (i) 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.

Post-synthesis linkage transformation.
Essentially, the linkage of COFs is a chemical bond, and it is also widespread in reaction chemistry, providing a unique channel for ne-tuning the pore environment via post-synthesis linkage transformation (Fig. 55). More importantly, this approach makes it possible to prepare COFs with new linkages, particularly for some linkages that are extremely difficult or even impossible to be synthesized de novo. So far, the diversity of linkage modication has been realized in various Schiff-base COFs. The oxidation of C]N in COFs was reported for the rst time in 2016. 330 Two imide-linked 2D COFs, namely, TPB-TP-COF and 4PE-1P-COF, were quantitatively oxidized to amides by NaClO 2 in an acetic acid solution. The additionally added 2-methyl-2butene was used to remove the hypochlorous acid generated from the reaction system. Both PXRD and N 2 adsorption measurements of the COF materials indicated that the amidation process did not lead to signicant changes in the COF structures. Amide-linked COFs exhibited enhanced chemical stability in acid and base solutions. For example, aer treatment in hydrochloric acid (12 M) 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 NaClO 2 oxidation of 3D COFs based on tetrahedral tetraamine and chiral tetraaldehyde monomers 331 also achieved the conversion of imines to amides, suggesting the generalization of this method. NaBH 4 can reduce imine-to amine-linked COFs, as demonstrated by COF-300 and COF-366-M (M ¼ Cu, Zn). 332 For instance, aer the reduction of COF-300 with NaBH 4 in methanol, the 13 C and 15 N CP-MAS ssNMR of the obtained COF-300-AR veried the quantitative conversion of imine to amine (Fig. 56). The peak of imine carbon at 159 ppm completely disappeared, and a secondary amine carbon emerged at 50 ppm. The imine nitrogen peak at 328 ppm disappeared, while the secondary amine nitrogen appeared at 67 ppm. As expected, the reduction of the C]N bond signicantly enhanced the chemical stability of COF-300-AR. When compared with pristine COF-300, on the silver electrode coated with COF-300-AR, the secondary amine bond provided chemisorption sites for the selective adsorption of CO 2 by forming carbamates, thereby improving the faradaic efficiency of CO 2 reduction. Noteworthily, the generated secondary amine linkage can be further modied. For example, the amine linkages underwent an aminolysis reaction with 1,3-propanesultone, affording a sulfonic-acid-functionalized COF. 333 Moreover, with regard to the applications of COF-300-AR, a recent study conrmed that COF-300-AR can also act as a light-responsive oxidase mimic, which can detect glutathione in HL60 cells when excited by purple light at 400 nm. 334 Imine-linked TTI-COF reacts with sulfur at higher temperatures to convert the imine bond into a thiazole ring. 335 During the reaction, rst, the imine bond gets oxidized into thioamide and then oxidative cyclization occurs to form thiazole (Fig. 57A). The resulting TTT-COF was stable in hydrazine, NaOH, and NaBH 4 solutions, while TTI-COF became amorphous under identical conditions, suggesting the contribution of the thiazole ring to the ordered structure of TTT-COF. In addition, when TTI-COF was exposed to the high-energy electron beam of HRTEM, the lattice fringes of TTI-COF gradually decreased with a half- This enhanced stability enabled the study of the formation mechanism and crystal defects of COFs by HRTEM. The signicance of sulfuration is also reected in the preparation of energy-storage COFs (Fig. 57B). By exercising reasonable control over the sulfuration temperature, the imine linkage of COFs can be converted into polysulde-modied thioamide for energy storage. 336 Polysulde chains, rather than sulfur guest molecules, can accelerate the redox kinetics and activity, facilitate ion transport, and facilitate charge conduction. Imine linkage has also been converted to quinoline via the Povarov reaction (also known as aza-Diels-Alder reaction). 337 The reaction of TPB-DMTP-COF, BF 3 $OEt 2 , 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 (Fig. 58). Due to the conversion of dynamic imines to strong quinolines, MF-1a was stable under a variety of extremely harsh chemical conditions, such as strong protonic acid (12 M HCl at 50 C for 8 h), superacid (98 wt% tri-uoromethanesulfonic acid for 72 h), strong base (14 M NaOH in water/methanol at 60 C for 24 h), strong oxidant (KMnO 4 in water/acetonitrile for 24 h), and reductant (NaBH 4 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.
More recently, Dong 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 olenic bonds can appear in the ring, when 3,4dihydro-2H-pyran was used for the reaction, the 2,3-dihydropyrano[2,3-c]quinoline 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 BF 3 $OEt 2 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 a-aminonitrile, thereby introducing cyano groups into the framework.
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 2,4,6triaminobenzene-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 signicantly improved thermal and chemical stability (Fig. 59A). 338 Further, with regard to B-COF-1, there is a thienyl group in the ortho position of the imine bond. With triuoroacetic acid as the catalyst, B-COF-1 was heated in a sealed tube containing oxygen at 100 C for 2 days (Fig. 59B). The coupled process of the imine carbon and thiophene b-position was carried out to form a conjugated heterocyclic system. 339 Furthermore, COF-170 was transformed into cyclic carbamate and thiocarbamate-linked COFs aer three steps, namely, demethylation by BBr 3 , imine reduction by NaCNBH 3 , and cyclization by 1,1 0 -carbonyldiimidazole or 1,1 0 -thiocarbonyldiimidazole (Fig. 59C). 340 With regard to these inspiring examples, the hydroxyl and thienyl groups adjacent to the imine linkage were involved in the construction of heterocyclic linkage in post-SM, which nally incorporated benzo [1,2- [1,3]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 modication and linkage transformation actually transferred the classic solution of organic chemistry from the ask to the COFs, representing a signicant step toward bringing the accuracy of organic solution-phase synthesis to extended solid-state materials.
5.2.6 Post-synthesis monomer exchange. The 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 selfcorrecting 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 via monomer exchange. 341,342 The construction of an imine-linked COF from [1,1 0 :3 0 ,1 00terphenyl]-3,3 00 ,5,5 00 -tetracarbaldehyde and benzidine monomers yields TP-COF-BZ with three distinct kinds of pores. 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 (Fig. 60A). Evidently, this COF-to-COF conversion is a heterogeneous process: in the presence of a large amount of benzene-1,4diamine in solution, benzene-1,4-diamine readily diffuses into the pore and then nucleophilically attacks the imine bond protonated by acetic acid, causing benzidine in this framework to be replaced by benzene-1,4-diamine while retaining its crystallinity. The nucleophilicity of benzene-1,4-diamine is stronger than that of benzidine due to the electron-donating effect of the amino group at the para position, and therefore, monomer exchange is difficult to reverse on a macro level. Besides, other impressive examples have also been reported, including 2D-to-2D, 341 3D-to-2D, 342 and 3D-to-3D 342 COF transformation via monomer exchange. A signicant advantage of post-synthesis monomer exchange is that it allows access to de novo unreachable COFs. For example, imine-based PTBD-NH 2 and PTPA-NH 2 COFs are difficult to fabricate by the direct polymerization of [1,1 0biphenyl]-3,3 0 ,4,4 0 -tetraamine and benzene-1,2,4-triamine with 4,4 0 ,4 00 -(1,3,5-triazine-2,4,6-triyl)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 (Fig. 60B). 344 Combining monomer exchange with linkage transformation results in the generation of newer linkages (Fig. 61). For example, when ILCOF-1 was treated with 4 equivalents of 2,5dimercaptobenzene-1,4-diaminium dichloride, benzene-1,4diamine on the framework was replaced by 2,5dimercaptobenzene-1,4-diamine, thereby introducing sulydryl groups at the ortho position of the imine bond. The subsequent oxidation of the material with air at 85 C in N,N-

Review
Nanoscale Advances dimethylformamide/water by linkage transformation with neighboring-group participation, thiazole-linked COF-921 was generated. 345 Similarly, when using 4 equivalents of 2,5dihydroxybenzene-1,4-diaminium dichloride, oxazole-linked LZU-192 (as reported earlier) was generated. Monomer exchange can also occur between monomers with different types of functional groups. 346 Imine-linked COF TzBA and terephthaloyl dichloride were immersed in dioxane/ mesitylene (2 : 1, v/v) for 2 days at 4 C, and the [1,1 0biphenyl]-4,4 0 -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.
5.2.7 Interlayer reaction. 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. Via rational design, electrocyclization reactions between two adjacent layers of monomers become possible.
The rst example of a reversible COF interlayer reaction is the [4 + 4] cycloaddition reaction of anthracene-based COFs (Fig. 62A). 347 The boronate-ester-linked 2D COF of Ph-An-COF was obtained by the condensation of anthracene-2,3,6,7tetraol 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 3.4Å, which led to 9,10-photodimerization under light irradiation at 360 nm, disrupting the original conjugated system. As this conversion is reversible, the rearomatization 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. 348 Interlayer [2 + 2] cycloaddition of COFs has also been reported. Thomas 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 P 2 PV COF with a layer spacing of 3.4Å was synthesized (Fig. 62B). When P 2 PV COF was exposed to sunlight, vinylene underwent [2 + 2] cycloaddition, generating cyclobutane rings between the layer with a subsequent increase in the layer spacing to 4.9Å. The cycloaddition product was partially regenerated by heating at 200 C in mesitylene for 2 days.
5.2.8 Post-synthesis coordination modication. Transition metal complexes that cannot be incorporated pre-synthetically are possible to be incorporated into COF via post-SM. As compared to pre-synthesis coordination modication, the opportunities for post-synthesis coordination modication are much more plentiful. Metal coordination sites can either be a binding site on the monomer or linkages of COFs. 351 At present, monomers containing coordination sites can be categorized into four types (Fig. 63A): porphyrin, 352-355 2,2 0bipyridine, [356][357][358][359] catechol, 360,361 and 5,6,11,12,17,18- dehexahydrotribenzo[a,e,i] [12]annulene. 362 In general, COF metallization can be readily achieved by simply mixing COFs with metal salts or metal complexes to trigger metal coordination reactions or ligand exchange reactions. Therefore, even metal carbonyl complexes with poor stability can be xed in the COF pores. 357 On the other hand, even though there are no chelating coordination sites on the monomers, COF metallization can be performed via linkages as the ligands (Fig. 63B), particularly for hydrazine-based linkages with multiple heteroatoms. 363,364 For imine-linked COFs, only N atoms in the linkage can participate in the coordination process. However, the interlayer distance of $3-4Å of COFs makes the N atoms in the linkages come close to each other, which facilitates the insertion of metal ions between the layers and promotes the formation of coordination bonds. 64,356 In addition, it is benecial for imine-linked COFs to x metal ions by introducing hydroxyl groups, 365-367 carboxyl groups, 368 and pyridine 369 to the monomers, since additional coordination atoms are provided. For example, NiCl 2 and hydroxyl-containing RIO-12 COF were reuxed in ethanol under basic conditions, and Ni 2+ was coordinated to the N-salicylideneaniline unit in RIO-12 to form a catalyst for the Suzuki-Miyaura cross-coupling reaction. 365 Zhu et al. simply mixed the COFs containing the carboxyl group with the metal chloride solution to x Ca 2+ , Mn 2+ , and Sr 2+ on the framework, enhancing the ammonia uptake. 368 Besides, N,N 0 -bis(salicylidene)ethylenediamine (Salen) is an attractive structural unit for coordination chemistry due to its stable planar square structure aer chelating metal ions. [370][371][372] The post-synthesis coordination modication of COFs provides valuable opportunities to deposit metal or metal oxide nanoparticles in the COF pores. COFs coordinated with metal ions (M n+ @COF) can be further reduced or hydrolyzed to produce M@COF or M x O y @COF composite materials, which have been widely studied for a variety of heterogeneous catalytic applications. 373 379 Furthermore, another type of imidazolium-based cationic COF, namely, Im-COF-Br, can be used as an all-solid-state electrolyte for lithium-ion batteries by exchanging Br À in the framework with the bis(tri-uoromethylsulfonyl)imide (TFSI À ) ion. 380 In 2019, Zhang et al. reported anionic COFs as single-ionconducting COF solid electrolyte materials. 381 The treatment of benzimidazole-containing R-ImCOF (R ¼ -H, -CF 3 , and -CH 3 ) 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.
5.2.10 Host-guest encapsulation. Host-guest encapsulation is a versatile and effective modication 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, 382-386 organic molecules, [387][388][389] and even biological macromolecules. [390][391][392][393] The structural basis of host-guest encapsulation is the pores of COFs, which have unique characteristics. 394,395 First, the pores of COFs have a precise polygonal reticular structure with well-dened 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 This journal is © The Royal Society of Chemistry 2020 Nanoscale Adv., 2020, 2, 3656-3733 | 3701

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Nanoscale Advances Review
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 signicantly important structural parameters to control the interactions between COFs and guest molecules. Host-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. 392,393 Meanwhile, the chemical modication of the pore walls of COFs can easily regulate the pore environment, thereby improving the interaction and compatibility of COFs with specic enzymes, as well as optimizing the orientation of the active sites of the enzyme, thereby further improving the catalytic activity of the enzymes. 391
6.1 Drug delivery and chemotherapy 6.1.1 Principle of chemotherapeutic drug delivery. Chemotherapy is one of the dominant means for clinical treatment of cancer. 416,417 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 (e.g., paclitaxel) 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 (e.g., cisplatin) toward the renal and digestive systems. 421,422 (iv) Drug resistance: the Review Nanoscale Advances proportion of drug-resistant cells that overexpress efflux transporter proteins increases during treatment, and cancer chemotherapy oen struggles to achieve the desired results. 423 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 in vivo circulation, which can be divided into three parts: phagocytosis escape, passive targeting, and active targeting.
First, 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. [424][425][426] Second, nanoparticles have an enhanced permeability and retention (EPR) effect 427 that increases the amount of accumulation at the tumor site, which is called passive targeting (Fig. 64). Although passive targeting is not yet described as selective and specic accumulation, it is a considerable improvement over small-molecule drugs. A common explanation for the EPR effect is that tumor cells secrete angiogenesisassociated growth factors, such as vascular endothelial growth factor in response to the rapid growth requirements that are highly dependent on tumor vessels for nutrients and oxygen. At this point, the newly generated tumor vessels are completely different in structure and morphology from normal blood vessels: large endothelial cell gap, missing smooth muscle layer of vessel wall, and decient angiotensin receptor function. In addition, the lack of lymphatic vessels in the tumor tissue results in lymphatic obstruction. Both situations allow the nanoparticles to accumulate in the tumor tissue and remain in the tumor tissue for a long time without being carried away by the lymphatic circulation. More importantly, the EPR effect can be further enhanced by some pathological and physiological factors, such as bradykinin, nitric oxide (NO), peroxynitrite (ONOO À ), prostaglandin, and tumor necrosis factor, which stimulate tumor vasodilation.
Finally, in order to further enhance the enrichment of nanoparticles at the tumor site, they can also be functionalized with targeting groups, 428 aptamers, 429,430 and antibodies 431 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][433][434] At the cellular and molecular levels, nanoparticles are taken up by the cells almost indiscriminately via endocytosis, thereby bypassing the limitations of the selective permeability of the cell membrane toward small-molecule drugs. 435 However, aer 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 À 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 specic organelles to achieve subcellular-targeting therapy. 439,440 This is benecial for minimizing drug doses, improving therapeutic benets, and reducing side-effects.
6.1.2 Model research on drug adsorption and release. As mentioned in the earlier section, the structural basis of COFrelated 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.
The adsorption and release behaviors of ibuprofen in other COFs have been extensively studied. Salonen et al. found that the equilibrium adsorption capacity of uorine-containing TpBD-(CF 3 ) 2 COF for ibuprofen in water was 119 mg g À1 , while the equilibrium adsorption capacity for the more hydrophilic acetaminophen and ampicillin was less than 20 mg g À1 . 441 Besides, MICOF@SiO 2 core-shell material not only exhibited superior adsorption capacity toward ibuprofen, but also showed excellent adsorption capacity for other nonsteroidal antiinammatory drugs such as ketoprofen, diclofenac, indomethacin, urbiprofen, and naproxen. 254 The adsorption of other model molecules in COFs has also been studied, such as sulfamerazine 389 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 conguration, and adsorption and desorption behaviors of the molecules in the COF pores.
6.1.3 COF-based drug delivery in vitro and in vivo. In 2016, Zhao et al. investigated the potential of imine-linked PI-3-COF and PI-2-COF (Fig. 66A) as drug delivery vehicles and their cytotoxicities in vitro. 396 The antitumor drug uorouracil (5-FU) was stirred with PI-3-COF and PI-2-COF in n-hexane for drug loading. As shown in Fig. 66B, cell inhibition experiments conrmed that PI-3-COF and PI-2-COF had good biocompatibility and no signicant inhibition on the proliferation of MCF-7 cells. However, 5-FU@PI-3-COF and 5-FU@PI-2-COF had obvious toxicity toward MCF-7 cells. Aer a total of 24 h of coincubation, the cell viability was reduced to about 40%. Although the cytotoxicity of 5-FU small molecules was stronger than that of 5-FU@PI-3-COF and 5-FU@PI-2-COF materials within 24 h, the COFs provided up to several days of continuous drug release ability, as per the drug release curve (Fig. 66C). This slow-release feature was not available in small-molecule drugs.

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Nanoscale Advances imine nitrogen reversibly anchored the quercetin guest molecules through noncovalent interactions (Fig. 67). Interestingly, although the SEM image of TTI-COF showed that the particle diameter of TTI-COF was even greater than 1 mm, quercetin@TTI-COF could efficiently deliver quercetin to the MDA-MB-231 cells, leading to cell apoptosis. COF-based quercetin delivery was not restricted by Bcrp1 overexpression, which is thought to be the primary cause that limits the cellular uptake of quercetin. Quercetin was unable to obtain its drug release prole due to its susceptibility to oxidation, but quercetin@TTI-COF consistently inhibited cell proliferation over the course of 4 day cell culture and was superior to quercetin small molecules with regard to cell inhibition, indicating slow drug release. In addition, the negligible effect of TTI-COF on cell proliferation was observed, suggesting the biocompatibility of TTI-COF. In addition to using the principle of host-object envelopment to load drugs into COFs via post-SM, Pang et al. demonstrated that DOX, a chemotherapeutic drug, could be directly loaded into the pores during COF formation via an in situ one-pot method. 221 Briey, DOX and 2,5-dimethoxyterephthalaldehyde were stirred for 1 h and then 1,3,5-tris(4-aminophenyl)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 xenogra model of H22 cells, an intratumoral injection of DOX@TAPB-DMTP-COF also showed signicant tumor-suppressive effects. Notably, the authors believed that the reaction of the amino in DOX with 2,5dimethoxyterephthalaldehyde resulted in a shi in the main diffraction peak of TAPB-DMTP-COF, possibly suggesting an alteration in the structure of COFs that requires further study.
The above examples demonstrate the feasibility of using COFs as drug delivery vehicles 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 modication of functional ingredients to ameliorate the deciency of bulk COFs becomes a possible solution.
Due to the strong chemical stability and abundant functionalization potential of COFs, it is feasible to use multistep post-SM for functionalization. The rst report in this regard discussed the preparation of folic acid (FA)-coupled TpASH-FA COF nanosheets for 5-FU-targeted drug delivery in vitro (Fig. 68) by the three-step post-SM of TpASH COF. 398 TpASH COF was prepared by mechanically grinding 2,4,6trihydroxybenzene-1,3,5-tricarbaldehyde and 4-amino-2hydroxybenzohydrazide as the monomers and 4-methylbenzenesulfonic acid as the catalyst. The hydroxyl groups on the TpASH COF monomer provided the reaction sites for post-SM. In the three-step post-SM processes, the rst step comprised the epoxide ring-opening reaction of glycidol to convert phenolic hydroxyl groups into alcoholic hydroxyl groups to yield TpASH-Glc; the second step involved the conversion of these surface alcoholic hydroxyl groups into amines in the presence of 3-(triethoxysilyl)propan-1-amine (APTES) to afford the aminefunctionalized 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 uorescent dye of rhodamine-B-isothiocyanate (RITC) to perform uorescent labeling. It should be noted that continuous post-SM led to the weakening of the p-p 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 mg mL À1 , 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 conrmed that the specic 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.
Besides targeting groups, polyethylene glycol (PEG) derivatives have been modied onto the COF surface to enhance hydrophilicity and tumor accumulation. As shown in Fig. 69A, by the self-assembly of curcumin (CCM)-modied PEG and amine-functionalized APTES-COF-1@DOX, Jia et al. prepared a series of water-dispersible PEGylated COF nanodrugs of PEG X -CCM@APTES-COF-1@DOX (X ¼ 350, 1000, and 2000). A PEG X -CCM coating not only imparted uorescence 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 PEG 2000 -CCM@APTES-COF-1@DOX was 9.71 AE 0.13 wt% and the encapsulation efficiency was as high as 90.5 AE 4.1%. The in vitro experiments indicated that PEG X -CCM@APTES-COF-1@DOX was broken down in lysosomes, resulting in slow DOX release. Even at very low DOX concentrations (0.25 mg mL À1 ), PEG X -CCM@APTES-COF-1@DOX signicantly inhibited cell proliferation (Fig. 69B). The in vivo uorescence imaging on mice showed that the nanodrugs were mainly distributed in tumor tissues aer the injection of nanodrugs into the tumor-bearing mice via the tail vein for 24 h. Among them, PEG 2000 -CCM@APTES-COF-1@DOX exhibited the best tumor-targeting ability (Fig. 69C). These results were consistent with those of in vivo antitumor experiments (Fig. 69D).
Furthermore, the latest research conrms that PEG 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, PEG 350 -CCM@APTES-COF-1@pazopanib nanomedicine more signicantly inhibited angiogenesis and tumor growth, protected mice from systemic drug toxicity, and prolonged survival time.
In addition to PEG, Pluronic F68 can also achieve a similar result. For example, Zhang et al. prepared Pluronic F68-modied 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 0 -disulfanediyldianiline. The disulde bond enabled F68@SS-COF to decompose in the presence of glutathione, thereby releasing the encapsulated DOX. DOX-loaded F68@SS-COF was conrmed to have a signicant inhibitory effect on HepG2 cells.
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 coworkers. EDTFP-1, 402    PDT is a novel alternative method for cancer treatment that has shown superior potential for the minimally invasive treatments of various types of cancers, particularly supercial cancers. 443,444 As shown in Fig. 70A, PDT relies on a nontoxic photosensitizer (PS), e.g., porphyrin, 445 phthalocyanine, 446 BODIPY, 447 and cyanine, 448 to induce the formation of reactive oxygen species (ROS) under specic wavelengths of light, leading to cytotoxicity. 449 According to the photochemical mechanism of ROS generation (Fig. 70B), PDT can be divided into type I and type II mechanisms. 450,451 For the type I mechanism, the triplet excited PS interacts with biological substrates to generate free radicals by transferring electrons. These free radicals subsequently react with oxygen or water to form ROS such as hydrogen peroxide (H 2 O 2 ), hydroxyl radical (cOH), and superoxide anion (cO 2 À ). For the type II mechanism, the triplet excited PS directly transfers energy to oxygen to form singlet oxygen ( 1 O 2 ), which is considered to be the more common ROS in most cases. 452   The general procedures of PDT 453 are shown in Fig. 70C. The main advantages of PDT are as follows. (i) Since PS has no obvious toxicity under dark conditions, PDT is highly selective through local light and can kill tumor cells without damaging healthy organs. (ii) The toxicity of ROS to tumor cells is universal and no resistance has been observed; therefore, it can be treated multiple times at low doses to minimize adverse effects. (iii) PDT is a minimally invasive therapy option; even for visceral tumors, the required light can be directed to the affected area via ber optics and other means. 454 (iv) PDT can be easily combined with other treatments, such as chemotherapy and radiotherapy.
Similar to drug delivery, the improvement effect of nanotechnology on PDT can be mainly reected in the optimization of tumor accumulation. 455 Traditional small-molecule PSs are usually organic molecules with wide-range conjugated systems. They are poorly water-soluble and can aggregate easily. Aer 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 modi-cation of PSs on the nanoparticles prevents PS aggregation at the molecular level, thereby preventing uorescence quenching and improving the 1 O 2 quantum yield. 457 6.2.2 COFs for PDT. In 2019, COFs were used for cancer PDT for the rst time. 216 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 (Fig. 71), where the BOD-IPY contents were 0.136 and 0.155 mmol g À1 , respectively. SEM images showed that LZU-1, LZU-1-BODIPY-2I, and LZU-1-BODIPY-2H had a uniform size of about 110 nm. Due to the enhanced effect of iodine atoms on intersystem crossing (ISC), LZU-1-BODIPY-2I afforded higher 1 O 2 generation efficiency than that by LZU-1-BODIPY-2H. For in vitro PDT, under green LED irradiation, when the BODIPY concentration was 0.5 mM, 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 xenogra model, suggesting the signicant value of the heavy-atom effect at the animal level. Mechanistic studies conrmed 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).
It is also feasible to use the p-p interactions between COFs and PS to adsorb PSs onto the COF surface to prepare COFbased nano-PS. Yuan et 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 mg mL À1 . The PDT therapeutic effect of PcS@COF-1 has also been conrmed in CT26-tumorbearing mice.
In addition to post-SM, the modularity advantage of COFs allows PS as the monomer to get directly involved in the construction of COF scaffolds. The photosensitizing properties of some porphyrin-based COFs have been reported; 283,[458][459][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 (Fig. 72A) with the renal-clearable property for in vitro and in vivo PDT. 163 First, 4,4 0 ,4 00 ,4 000 -(porphyrin-5,10,15,20-tetrayl)tetraaniline and 2,5-dihydroxyterephthalaldehyde were used to synthesize bulk TphDha COF in Pyrex tubes; subsequently, ultrasonic exfoliation, surface modication using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG), and ltering 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 cOH and 1 O 2 production at the same time via the type I and type II mechanisms (Fig. 72B). In the dark, the nanodots exhibited marginal toxicities toward HeLa, MDA-MB-231, RAW 264.7, and L929 cells, even at concentrations of up to 200 mg mL À1 . However, the nanodots obviously inhibited HeLa cell proliferation in a concentration-dependent manner under laser irradiation at 638 nm for 5 min (Fig. 72C and D). Antitumor experiments conducted in H22-tumor-bearing mice further conrmed the excellent PDT effect of these nanodots. Subsequently, biodistribution and pharmacokinetic assays were performed in healthy mice. Aer intravenous injection of these nanodots, the blood circulation half-lives of distribution and clearance phases were calculated to be 0.27 and 4.36 h, respectively, based on a two-compartment model of the blood circulation curve (Fig. 72E). Further, the nanodots were mainly distributed in the liver and kidneys of healthy mice. Over time, the nanodot concentration in the main organs gradually decreased (Fig. 72F). Research on excretions conrmed that the nanodots mainly existed in urine rather than feces (Fig. 72G); therefore, it was speculated that the nanodots were excreted through the kidneys, which was closely related to the ultrasmall size of the nanodots. Such easily metabolized and excreted nanodots are meaningful for reducing the long-term toxicity of materials.
Very recently, Tang and co-workers developed a TphDha-COF-based theranostic nanoplatform (Fig. 73A) by integrating tetramethylrhodamine-labeled survivin antisense strand onto TphDha COF for simultaneous cancer diagnosis and PDT. 405 The uorophore was quenched by TphDha COF due to its large plane p-electron system. However, once it penetrated into the cancer cells, in the presence of survivin mRNA as the cancer biomarker, more stable RNA duplexes were formed and divorced from the TphDha COF surface, recovering the uorescence signal of tetramethylrhodamine and enabling tumor-specic imaging. Furthermore, TphDha COF was irradiated with red light, generating toxic 1 O 2 in cancer cells to induce oxidation stress and trigger cell apoptosis through PDT. Therefore, highly tumor-selective PDT became feasible by reasonably combining uorescence imaging and PDT.
In situ growth of TphDha COF on the surface of upconversion nanoparticles (UCNPs) enables PDT excited by near-infrared (NIR) light at 980 nm (Fig. 73B). 261 Oleic-acid-capped NaYF 4 :-Yb,Er,Tm UCNPs (OA-UCNPs) 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   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 p-electron delocalization system in the TPAPC-COF layer. When excited by a 635 nm laser, TPAPC-COF exhibited stronger photosensitivity properties than the H 3 TPAPC monomer and induced more efficient 1 O 2 production, which may be related to the reduced uorescence emission and enhanced ISC of TPAPC-COF. For in vitro experiments, DSPE-PEG-coated TPAPC-COF effectively inhibited MCF-7 proliferation through PDT.
All the COF-based PDT systems mentioned above are based on well-known PSs. However, an impressive study showed that COF-Trif-Benz and COF-SDU1 with photodynamic properties could be fabricated from monomers that did not have photosensitive properties at all, although the detailed mechanism has not yet been elucidated. 462 In 2019, Deng et al. synthesized COF-808 and COF-909 with photodynamic properties using inactive 5 0 ,5 000 -bis(4-formylphenyl)-[1,1 0 :3 0 ,1 00 :4 00 ,1 000 :3 000 ,1 0000 -quinquephenyl]-4,4 0000 -dicarbaldehyde (L-3C) and 4 0 ,4 0000 -(1,4-phenylene) bis(([2,2 0 :6 0 ,2 00 -terpyridine]-5,5 00 -dicarbaldehyde)) (L-3N) monomers (Fig. 74A). 407 Although the frontier molecular orbitals of the monomers did not match with those of the superoxide anion, the bandgap of the resulting COFs was precisely narrowed down to provide suitable overlap, which considerably promoted the photodynamic property. In particular, spectroscopic measurements showed that COF-909 had a gap between the frontier orbitals of 1.96 eV and absorbed visible light at 630 nm. However, the bandgap of L-3N was 2.79 eV, making it difficult to excite at the same wavelength (Fig. 74B). When compared with L-3N, COF-909 afforded longer excited state lifetimes, higher separation efficiencies of electrons and holes, and lower charge recombination rates. Therefore, ROS could be easily generated through electron transfer from COF-909 to dissolved oxygen (Fig. 74C). The quantitative determination of ROS showed that the ROS generation ability of COF-909 was even better than that of porphyrin-based MOF PCN-224 (Fig. 74D). At the cellular level, when 630 nm laser was irradiated, COF-909 effectively generated ROS in CT26 cells (Fig. 74E) and caused signicant cell death (Fig. 74F). At the animal level, by the intratumoral injection of COF-909 in CT26-tumor-bearing mice, the PDT efficacy was further conrmed (Fig. 74G). Furthermore, in 2020, Qiu et al. reported NDA-TN-AO COFs based on non-photosensitive monomers. 463 6.3 PTT 6.3.1 Principle of PTT. PTT is another potential phototherapy method. 12 It utilizes photothermal agents (PTAs) 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, 11,465 e.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 p-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.
6.3.2 COFs for PTT. Till now, COF-based PTT mainly includes two implementation methods. The rst one is to combine PTA with COFs and the COF material, in our case, is used as a PTA carrier. 232 The second is to design and synthesize COFs with light-heat conversion capabilities, e.g., copper(II) tetraphenylporphyrin-based COFs. 69,469 In 2019, Pang et al. synthesized LZU-1 nanoparticles loaded with CuSe 223 and Ag 2 Se. 408 Under an 808 nm laser (1.5 W cm À2 ), the photothermal conversion efficiency of CuS-loaded LZU-1 (200 mg mL À1 ) was 26.3%, while that of Ag 2 Se-loaded LZU-1 (500 mg mL À1 ) was 37.9%. The authors also reported that the photodynamic properties could be attributed to LZU-1, but further cautious conrmation is necessary. Pang et al. also prepared micron-sized ower-like HCOF linked by b-ketoenamine. 409 Aer metalation with Fe 3+ , the photothermal conversion efficiency of Fe-HCOF (800 mg mL À1 ) was 13.9% under an 808 nm laser (1.9 W cm À2 ).
Recently, COFs containing free radical cations have been explored for in vivo photoacoustic imaging and PTT. 410 By performing two sequential post-SM processes of quaternization and one-electron reduction, 2,2 0 -bipyridine-based Py-BPy-COF was converted into cationic-radical-containing Py-Bpy + c-COF (Fig. 75A). Its AA-stacking structure enabled the overlap of redox centers with each other in the COF layers, thereby promoting intercharge transfer through p-coupling multilayers and eventually inducing enhanced NIR absorption and signicant photothermal conversion by promoting nonradiative transitions. The absorption spectrum data showed that PEG-functionalized Py-BPy + c-COF dispersion exhibited a broad featureless absorption band in the range of $600-1300 nm, where the absorbance was remarkably higher than those of Py-BPy-COF/PEG and Py-BPy 2+ -COF/PEG under the same concentration (Fig. 75B). Under laser irradiation at 808 nm, the photothermal conversion efficiencies were 19.3, 47.2, and 63.8% for Py-BPy-COF/PEG, Py-BPy 2+ -COF/PEG, and Py-BPy + c-COF/PEG, respectively (Fig. 75C). Further, for laser irradiation at 1064 nm, the photothermal conversion efficiencies were 10.4, 40.1, and 55.2% for Py-BPy-COF/PEG, Py-BPy 2+ -COF/PEG, and Py-BPy + c-COF/PEG, respectively (Fig. 75D). Because of the remarkable NIR absorption and photothermal conversion properties, the potential of Py-BPy + c-COF/PEG as a PTA has been conrmed in antitumor experiments in vitro and in vivo using lasers at 808 and 1064 nm (Fig. 75E-I).

Combination therapy
Nanomaterial-based drug delivery and phototherapy have achieved certain success in preclinical tumor treatments. However, due to the inherent shortcomings of monotherapy (e.g., chemotherapy resistance mechanisms, insufficient nanomaterial accumulation at the tumors, and supercial penetration depth of light) 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  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 et al. used stepwise BDF and host-guest encapsulation to modify the PS of 5-(4-aminophenyl)-10,15,20triphenylporphyrin (Por) and the PTA of vanadyl 2,11,20,29tetra(tert-butyl)-2,3-naphthalocyanine (VONc) into TPB-DMTP-COF NCOF to obtain PDT/PTT dual-functional VONc@COF-Por nanomedicine (Fig. 76A). 220 VONc@COF-Por maintained the nanoscale spherical morphology of TPB-DMTP-COF NCOF, where the contents of Por and VONc were 0.091 and 0.256 mmol mg À1 , respectively. When exposed to a red LED, VONc@COF-Por effectively induced 1 O 2 production (Fig. 76B). When exposed to an 808 nm laser, the photothermal conversion efficiency of VONc@COF-Por was as high as 55.9% (Fig. 76C). In vitro antitumor experiments (Fig. 76D) showed that the IC 50 value of the combination therapy was 42 mg mL À1 , which was signicantly lower than that of PDT (131 mg mL À1 ) or PTT (93 mg mL À1 ) 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 signicant change in the intracellular 1 O 2 level when the temperature increased. In vivo experiments conducted in MCF-7 xenogra 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.
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 (Fig. 77A)  min). Photoacoustic imaging in vivo showed that NCOF-366 spread to the entire tumor within 1.5 h aer an intratumoral injection. At this time, irradiating the tumor with a laser at 635 nm (1.5 W cm À2 ) for 5 min resulted in almost completely inhibiting tumor growth within 14 days.
Chen et al. also reported the donor-acceptor TP-Por COF nanosheets (Fig. 77B) for a combination of type I PDT with PTT to overcome the limited efficacy caused by hypoxia in solid tumors. 162 Upon laser irradiation at 635 nm, the lamellar structure of TP-Por COF nanosheets was conducive to efficient charge carrier separation and transportation. Subsequently, the electrons reduced oxygen to form cO 2 À and the holes oxidized water to generate cOH. Both were highly toxic ROS, leading to cell apoptosis and cell necrosis via type I PDT. Furthermore, the energy loss due to the inevitable nonradiative attenuation caused a rise in temperature, achieving efficient photothermal conversion for use in PTT. As expected, even under hypoxia, the combination therapy induced by TP-Por COF nanosheets still effectively inhibited the proliferation of HeLa cells. In vivo experiments also conrmed this excellent antitumor effect. 6.4.2 Combined drug delivery and PTT. The TP-Por COF mentioned above was also used as a carrier for PTA and chemotherapeutic drug. 161 COF@IR783 nanosheets (diameter: 200 nm; thickness: 9.5 nm) were prepared by adding IR783 during the ultrasonic exfoliation of TP-Por (Fig. 78). Herein, IR783 not only acted as an organic PTA for PTT, but also as a stabilizer for the nanosheets. Under laser irradiation at 808 nm, COF@IR783 did not produce ROS, but displayed photothermal conversion efficiency of 15.5%. Further loading cisaconityldoxorubicin (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.
6.4.3 Combined drug delivery and PDT. The COF-based combination therapy of drug delivery and PDT was also achieved by a more exquisite strategy. 412 COF TTA-DHTA with a poly(lactic-coglycolic-acid)-poly(ethylene glycol) (PLGA-PEG) amphiphilic polymer coating was used to deliver the anti-brotic drug of pirfenidone (PFD) to the tumor site (Fig. 79). PFD@COF TTA-DHTA @PLGA-PEG reduced the content of collagen I and hyaluronic acid in the extracellular matrix, abated solid stress of the tumor, restored vascular function, improved tumor oxygen supply, and ultimately enhanced PDT induced by protoporphyrin-IX-conjugated peptide nanomicelles (NM-PPIX). This is a landmark study that combines tumor physiology with nanomedicine for the rst time, providing a promising and readily scalable anticancer strategy for targeting the extracellular matrix.
6.4.4 Photoimmunotherapy. Cancer immunotherapy is one of the most advanced clinical cancer treatment methods. 472 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 This journal is © The Royal Society of Chemistry 2020 Nanoscale Adv., 2020, 2, 3656-3733 | 3715

Review
Nanoscale Advances activation, proliferation, and differentiation. A large number of clinical trials and clinical practices have conrmed the effectiveness of this method for specic populations, although the side-effects are not less than those of traditional chemotherapy to a certain extent. [475][476][477] In fact, the effectiveness of immunotherapy is largely restricted by the limited activation of the immune system and nonspecic 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][480][481] Phototherapy can efficiently activate antitumor host immunity, realizing the combination of phototherapy and immunotherapy. This combination therapy is also known as photoimmunotherapy. 482,483 Photoimmunotherapy has been proven in different types of nanomaterials. 484 However, research on COF-based photoimmunotherapy is still in its infancy. ICG-loaded COF-1 nanosheets were surface-modied with polydopamine (PDA) and PEG to afford ICG@COF-1@PDA nanosheets with a size of 170 nm (Fig. 80A). Under laser irradiation at 808 nm, ICG@COF-1@PDA-induced PDT and PTT combination caused immunogenic cell death (ICD) of the tumor cells by triggering oxidative stress and endoplasmic reticulum stress. In the CT26 colorectal tumor model, the combination phototherapy almost completely ablated mice tumors, and there was no recurrence during the 14  day observation period (Fig. 80B). At the end of the treatment, the four cured mice were inoculated with tumor cells again. Aer additional observation for 18 days, 2 mice remained tumor-free (Fig. 80C), which indicated that ICG@COF-1@PDAinduced combination phototherapy activated systemic antitumor immunity. In addition, antitumor experiments conducted in the bilateral colorectal tumor model showed that this combination phototherapy induced by ICG@COF-1@PDA exhibited the abscopal effect (Fig. 80D). In terms of mechanism, this combination phototherapy upregulated the damageassociated molecular patterns (DAMPs) including HSP70 and HMGB1, promoted dendritic cell maturation, subsequently induced CD8 + T cells to inltrate into distant tumors, upregulated IFN-g in the distal tumor, and nally slowed the growth of untreated distal tumors. More importantly, in a triple-negative breast cancer metastasis model, ICG@COF-1@PDA-induced combination phototherapy even suppressed lung metastasis and liver metastasis (Fig. 80E).
Combining phototherapy with immune checkpoint inhibitors can further enhance the activation of the immune system, enhancing antitumor therapy. In 2020, Pang et al. reported the combination of PDT, PTT, and a-PD-L1 checkpoint blockade therapy. 414 Using p-p interactions, ICG was adsorbed in TAPB-BTCA-COF, and then chicken ovalbumin (OVA) 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 a-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.
6.4.5 Combined PDT and ion-interference therapy. Some metal and nonmetal ions (e.g., Na + , K + , Ca 2+ , Zn 2+ , Mg 2+ , Fe 2+ , Cl À , H 2 PO 4 À , and HCO 3 À ) 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][490][491][492][493] Very recently, Dong et al. reported a TPB-DMTP-COF-based nanoagent, namely, CaCO 3 @COF-BODIPY-2I@GAG, 415 which comprised BODIPY-2I PS, CaCO 3 nanoparticle, and glycosaminoglycan (GAG) CD44-target coating (Fig. 81). Under green LED irradiation, the surface-decorated BODIPY-2I could not only generate 1 O 2 to directly kill the tumor cells, but also destroy the ability of mitochondria to regulate Ca 2+ . Under these precarious circumstances, Ca 2+ released into the cytoplasm due to CaCO 3 decomposition in the lysosomes irreversibly caused This journal is © The Royal Society of Chemistry 2020 Nanoscale Adv., 2020, 2, 3656-3733 | 3717

Review
Nanoscale Advances intracellular Ca 2+ overload. As a result, enhanced antitumor efficiency could be achieved via the synergistic action of PDT and Ca 2+ overload. On the other hand, as a specic targeting agent for CD44 receptors on tumor cells in the digestive tract, the GAG coating signicantly promoted nanoagent uptake in the HCT-116 cells, consequently achieving more effective antitumor activity against colorectal carcinoma along with a weaker side-effect on normal tissues. Hopefully, the COF-based ioninterference therapy discussed here can be incorporated with other treatment methods to realize efficient synergistic cancer therapy.

Summary and outlook
In this review, we have provided a detailed summary of the recent advances made in COFs as multifunctional therapeutic platforms in oncology, including the preparation of COFs, reduction in the size of COFs to the nanoscale, introduction of the desired functional groups into COFs, and existing COFbased therapeutics. COFs exhibit several signicant advantages in the biomedical elds, such as high bioaffinity and biocompatibility, ordered framework structures, adjustable and open pore structures, and easily modiable surface and pore walls. These distinct properties are highly advantageous for realizing biomedical applications in vivo. Although COF-based therapeutic systems are still in their infancy, their fascinating properties and promising potential for oncology have inspired an increased number of researchers to dedicate their efforts to this promising eld. However, the current challenges and limitations faced by COFs cannot be ignored, which may become obstacles to clinical translation.  (i) 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 eld of nanomedicine. 494 (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 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.
(iii) The biological safety of COFs has not been studied in detail. Until now, there are only some preliminary 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.
(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 (such as Fe, Ga, Mn, and I). 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 uorescence imaging. 498 (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.
(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. 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.
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 eld 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.

Conflicts of interest
There are no conicts to declare.