A panoramic perspective of recent progress in 2D and 3D covalent organic frameworks for drug delivery

The development of efficient drug delivery systems is essential for improving the efficacy and safety of cancer drugs, particularly for aggressive and difficult‐to‐treat cancers. Covalent organic frameworks (COFs) are emerging as innovative porous nanomaterials in drug delivery systems (DDS), due to their unique properties, including the metal‐free organic skeleton, predetermined structures and pore geometries, high porosity, large surface area, facile surface modification potential, and good biocompatibility. These characteristics make COFs excellent candidates for improving drug delivery by enhancing drug loading capacity and enabling precise encapsulation. This review emphasizes the importance of donor‐acceptor‐based COFs, which provide channels for charge transportation, and we also explore how the π‐conjugated skeleton of COFs enhances its long‐acting fluorescent properties and facilitates drug uptake via cell endocytosis. While this review primarily focuses on recent advancements in COF‐based targeted DDS, it also acknowledges the challenges posed by the diverse pore geometries in porous materials and discusses potential solutions. Further, it underlines the potential of developing future drug carriers that can successfully and specifically target cancer cells, improving treatment efficiency while reducing adverse side effects.


INTRODUCTION
[3][4] Porous organic polymers are mainly recognized as different groups such as covalent organic frameworks (COFs), [5] covalent triazine frameworks (CTFs), [6] hydrogen-bonded organic frameworks, [7] conjugated microporous polymers, [8] and also porous aromatic frameworks. [9]The International Union of Pure and Applied Chemistry categorizes porous polymers into three groups microporous, mesoporous, and macroporous which feature pore sizes of less than 2, 2-50, and >50 nm, respectively. [10]he morphologies of these polymers directly influence their pore size and pore geometries throughout the structure, thereby endowing them with channels that enable efficient release and transportation.This makes them applicable in biomedicine, catalysis, energy, and absorption. [11,12]As a subgroup of porous organic polymers, COFs exhibit well-organized porous structure, superb binding sites, high chemical and thermal stability, large surface area, and low density due to their fully pre-designable structures containing covalent bonds involving light elements (C, B, O, and N). [13]Factors such as the size, linker's connection, and symmetry determine the geometry of COFs.Their structure depends on the building blocks containing multifunctional monomers, demonstrating defined symmetry.Hence, through the condensation of suitably chosen monomers, the geometry of the resultant COFs can be controlled while demonstrating intrapore chemical structure. [14]OF modification can be divided into pre-synthesis or postsynthesis stages.Pre-synthesis occurs when desired functional groups are introduced into the building blocks prior to the synthesis. [15]Post-synthesis involves the use of materials capable of interacting with COFs by direct interference in the crystallization step. [16,17]High porosity could be one of the most important advantages of COFs that offer post-synthetic modification after their synthesis. [5,18]Unlike linear and amorphous polymers, COFs formed in two or three dimensions are capable of displaying a high level of control and network tuning.The reversible nature of COFs provides them with a crystalline structure allowing for the rearrangement via bond reformation. [19]Several types of monomers such as imine, [20] imide, [21] hydrazine, [22] β-ketoenamine, [23] amine, [24] and azine [25] have been used to create new COFs.Among them, imine-based COFs are notably stable and can resist hydrolysis. [26]Jingya et al. synthesized an imine-based COF with an interspersed structure of multiple carbon-based wall nanotubes. [27]The main objective of these COFs was to enhance the electron transfer between the surfaces of electrodes with materials and prove their effectiveness for detecting synthetic phenolic antioxidants such as phenol and butylated hydroxyanis.Since the first report of COFs by Yaghi's group (2005), these COFs have found applications in numerous areas including photocatalysis, [28,29] controlled drug delivery, [30,31] energy storage, [32] absorption of organic pollutants, [33] optoelectronics, [34,35] and gas storage. [36]Their potential for chemical and structural modifications has accelerated their use in these domains as a robust platform.
Cancer is a devastating disease characterized by the uncontrolled growth of abnormal cells.[39] Traditional treatments in cancer like chemotherapy combined with surgical operation, biotherapy, hormone therapy, and radiotherapy, are normally based on the hypothesis that all somatic cells feature a similar malignant potential.These conventional strategies suffer a lack of specificity and multidrug resistance, which make them ineffective in giving long-lasting protection against cancer cells, followed by damaging healthy cells and severe side effects.Prescribed drugs in chemotherapy can circumvent the cancer cells from further division, and mitigate the stress caused by abnormal growth of cells. [40]Doxorubicin (DOX) remains the most prominent anticancer drug, and its therapeutic activity is usually accompanied by severe toxicities like cardiotoxicity and neurotoxicity, leading to limited longterm usage. [41]Another chemotherapy drug that poses major toxicities is cisplatin causing nausea, renal insufficiency, hepatotoxicity, etc. [42] In this regard, the restricted tumortargeting ability of chemotherapy has made researchers develop a potential drug delivery system (DDS). [43,44][47][48] Active-targeted carriers furnished with targeting ligands can interact with the surface receptors, thereby enhancing endocytosis.54] Recent advancements in drug delivery have been spurred by the design flexibility of COFs. [55,56]Although a wide range of drug carriers like micelles, [57] nanoparticles (NPs), [58] liposomes, [59] polymeric networks [60] have been tested, COFs have exhibited reliable encapsulation and drug release, due to their metal ion free structure and good biocompatibility. [61]Regardless of the capability of micellesbased DDS to solubilize hydrophobic or poor-water soluble drug molecules within their core, their low stability could be considered as their fundamental restriction in DDS, in particular during environmental changes.This is followed by the disintegration of micelles that lead to a burst release of encapsulated drug molecules into the bloodstream. [62]While COFs are less susceptible to change in physiological environments like phosphate-buffered saline (PBS) or slightly acidic intracellular parts. [63,64]On the other hand, other DDSs like liposomes suffer from inadequate drug loading along with their leakage, which limits the duration of impact and poses toxicity itself. [65]On the contrary, the ultra-porous structure of COFs gives them a sufficient drug-loading capacity in DDS. [66]Besides, the metal-free nature of COFs could entirely keep the potential toxicity away, following more biocompatibility in DDS. [67]n comparison to other DDS, not only can the size of COFs be reduced to nanoscale, but their morphology can also be optimized convincingly.Above and beyond all, the Nano-scaled COF networks possess high crystallization, small volume, significant biocompatibility, a robust skeleton, good dispersion, and ultra-high porosity with adoptable geometries.Recent advancements in COFs have led to more flexibilities in DDS, as the synthetic methods are to be improved and their porosity provides COFs with higher loading capacity.Other unique properties of COFs have garnered increasing interest in DDS, for instance, their donor-acceptor structure in starvation therapy, high near-infrared radiation absorption in targeted delivery of glucose oxidase (GOX), remodeling of extracellular matrix (ECM), and sequential pore wall of their functionalized structure for Camptothecin (CPT) drug release.70] In recent years, COF-based materials have emerged as an alternate strategy to circumvent the shortcomings of DDS (Table 1).Although nearly all COFs provide sustained release, pyridine-based COFs have a particularly sustained system releasing a large quantity of drug molecules over a typical time duration (4 days).Fluorine-functionalized COFs could rank second regarding the release of drugs over 3 days, whereas imine-linked COFs have displayed similar cumulative release in a more extended time.In comparison with redox-responsive COF which releases almost 90.00% of DOX during 2 days, amino-functionalized frameworks could release the same amount of drug in 5 days.COFs based on 1,3,5-tris(4-aminophenyl)benzene-2,5-dimethoxyterephthaldehyde have exhibited almost the slowest release system, and 8-hydroxyquinoline functionalized COFs have delivered approximately 60.00% of 5-FU for 2 days.
Nevertheless, some intrinsic limitations like poor cellmembrane permeability and non-specific targeting can restrain COFs application in biomedical fields, which is keeping their research in a preliminary stage.Severe synthesis conditions, single function, and poor solubility could be further taken into account as difficulties in COFs-based DDS. [78]Another well-known challenge during drug release from COFs is the strong interaction between them and guest drug molecules, which may give rise to difficulties under biological conditions for a limited time.Also, large aggregations in a concurrent pathway around the drug-loaded site are likely to limit drug delivery. [79]So, one question that might arise is whether modification of COFs, such as functionalizing or other methods overcomes these limitations.The biodegradable COFs could be highly desirable so that their removal problem after use would be solved.One possibility might be coating COFs with biopolymers or modifying their surfaces to enhance their stability, dispersibility, flexibility, and targeting ability. [80]Despite devoted studies uncovering the outstanding properties of COFs in drug release, certain aspects need attention when designing drug carriers.The most important factor is their biocapacity; the cytotoxicity of COF-based drug carriers must be minimized.For effective cellular uptake in in-vivo drug release, the optimal particle size should be below 200 nm.Notably, the limited-release strategies for COFs polymer suggest that their controlled and targeted drug delivery falls short of expectations but is highly desirable. [81,82]Currently, most studies concerning COFs are focused on layered eclipsed two-dimensional (2D) structures.However, only a few examples of three-dimensional (3D) COFs have been studied so far for drug delivery.As reported, 2D COFs could be obtained by multidirectional 2D building blocks, in which the lattice structure is organized with discrete pores.On the other side, the synthesis of 3D COFs requires at least one unit featuring either T d or orthogonal geometry, pushing the polymer skeleton into a 3D network. [83]n this review, we aim to offer a comprehensive overview perspective on recent advancements in COF-based structures as carriers in DDS, primarily within the past six years.These newly-emerged materials are promising porous networks with exceptional qualities such as biocompatibility, tunable functionalities, flexible active sites, well-organized channels, and predesignable structure.Tailoring COFs down to the nanoscale, followed by designing nanoplatforms with excellent properties in drug delivery has garnered a great deal of interest.3D COFs tend to be arranged in a 3D covalently bonded framework, which provides more active sites, vast surface area, and organized pores.Particular pairings like C1, C2, C3, and C4, incorporated with a T d knot are required to fabricate 3D COFs, making them a specific platform for a higher amount of guest encapsulation in DDS.On the other hand, the donor-acceptor structure of 2D COFs endows them with regulated charge transportation, leading to enhanced efficacy of anti-cancer treatment.This work assimilates dispersed information from global research groups, centering on 2-and 3D COF networks.We discuss the correlation between well-connected pore structures of COFs with loading capacity, followed by sustained release relating reported findings in recently published studies.We also outline the synthesis evolution of COFs since their discovery and how they can be used to target cancerous tissues.Furthermore, we will delve into the contentious topic of the many reactive sites and pore channels that still pose challenges in the use of COFs in DDS.Towards the end, the emerging and breakthrough studies of COF-based DDS, which are employed in the cutting-edge drug release field are summarized.This work is arranged in a thought sequence to address challenges during the usage of COFs in DDS.By reviewing the latest developments, we hope to illuminate potential plans for developing new COF-based nanomaterials.Considering the growing interest of newcomers to the COFs field, the innovative methods proposed by worldwide researchers may serve as valuable resources for future investigations.

THE UTILIZATION OF 3D COF-BASED MATERIALS IN DRUG RELEASE
In general, COFs can grow in 2D or 3D, playing a central role in using covalent bonds to control chain conformation.Though nearly all 3D COFs possess a microporous structure, they typically exhibit lower porosity due to multi-fold interpenetration occupying space.Consequently, managing both folding and interpenetration presents an ongoing challenge during 3D COFs synthesis. [84]However, their unique structures, including abundant active sites, large surface areas, and pore channels, have marked them as promising nanomaterials.These qualities provide ideal conditions for numerous applications such as efficient transportation of guest molecules in DDS. [85,86]Unlike 2D COFs, which require planar units for synthesis, 3D COFs need one unit with T d geometry to direct the polymeric backbone into a covalent-based 3D structure.To achieve 3D COFs, various combinations, such as C1, C2, C3, and C4, can be merged with the T d knot.To date, eight distinct 3D networks in the Reticular Chemistry Structure Resources have been identified, including dia, ctn, rra, ffc, lon, srs, bor, and pts, and these networks determine the topology of 3D COFs.
F I G U R E 1 Topology diagrams representing a general basis for covalent organic frameworks (COFs) design and construction of three-dimensional (3D) COFs. Figure reproduced [26] with permission.Copyright 2021 Elsevier.
To cite an example, the combination of [T d + C 3 ] would form ctn or bor, in which there will be no interpenetration demonstrating high surface area (Figure 1). [26,87,88]Notably, organic molecules typically present numerous reactive sites, arranged to facilitate extended interconnections.This aspect presents the primary challenge in advancing the functionality of porous 3D structures. [89]erein, two new 3D COFs with pts topology were synthesized by Negishi et al. [71] The T d -symmetric tetrahedral-like shape (1,3,5,7-tetrakis(4-formylphenyl)adamantine) interacted by the usage of two C 2 -symmetric rectangular linkers (1,3,6,8-tetrakis(4-aminophenyl)pyrene and 1,1,2,2tetrakis(4-aminophenyl)ethane) resulted in two different COFs (named COF-1 and COF-2), featuring two-and threefold interpenetrated topology, respectively.These 3D COFs demonstrated a crystalline porous structure, as well as the Brunauer-Emmett-Teller (BET) specific surface area of 1320 and 940 m 2 /g.Drugs 5-FU and cytarabine were loaded onto these COFs, and drug release studies were conducted using the impregnation method.Surprisingly, after an accurate analysis of the synthesized 3D COFs crystal structure, the pore network of COF-1 revealed better connectivity compared with COF-2, providing drug molecules with more binding sites.It is worth mentioning that both cytarabineloaded COFs showed a sustained drug release pattern after a 14-day time.On the other side, the high loading capacity (near 18.00 wt%) and a slow-release rate of 88.40% were observed for COF-1 with 5-FU loaded on.Besides, the 5-FU-loaded COF-2 system displayed a prolonged release rate of 33.00% after 14 days.Generally, the interconnected pore channels of these COFs could endow drug molecules with efficient loading, which makes them a suitable nanocarrier for chemotherapeutic drugs.
Researchers have found that 3D COFs have unique interconnected pore channels, which make them highly versatile for applications like drug delivery and catalysis. [90]In one study, Negishi and colleagues successfully synthesized a 3D non-interpenetrated COFs with stp topology and a considerable pore size of 4.7 nm. [91]They loaded this COF with different drugs such as captopril (CAP), ibuprofen (IBU), isoniazid, 5-FU, and brimonidine tartrate.The results showed that the COFs had a high drug-loading capacity and exhibited sustained release due to their well-connected pore structure.The 5-FU-loaded COFs showed a release rate of 12.00% after 9 days, and a high entrapment capacity of 17.34 wt%.It is clearly observed that surface area plays a critical role in drug loading efficiency.What would make COFs a more promising candidate was their specific surface area which was carried out after drug release studies.According to nitrogen absorption isotherms of drug-loaded COFs, the increased specific surface area was observed.
Considered COFs as porous materials be capable of improving controlled drug release systems, a novel pyridine-3-boronic acid-based COFs was synthesized under a selfassembly process by Salehi et al. [72] In addition, to study the drug release pattern of the synthesized COF, an antibiotic drug named Trimethoprim (TMP) was loaded on it.As could be observed in the drug release curve, TMP@COF exhibited a rate release of 95.00% after 4 days.Since COF could modify the formulation of antibiotic drugs, the antibacterial characteristics of TMP@COF were studied against both gram-negative (Escherichia coli) and gram-positive (Staphylococcus aureus) bacteria.The minimum inhibitory concentration values of TMP encapsulated by COF were noticeably decreased compared to COF and TMP.That is to say, fewer concentration of TMP@COF was needed to kill bacteria.It means that a porous network can enable COF to tackle solubility limitations, followed by enhancing antibacterial activity.
A new multifunctional nanocarrier based on copper sulfide NPs along with COF (CuS@COFs) was obtained by Li and co-workers [54] aiming at developing targeted chemotherapy and photothermal therapy and chemo dynamic therapy (CDT).A promising invasive photothermal therapy and CDT were successfully provided by the presence of CuS NPs due to their high photothermal effect, and good Fentonlike activity.Moreover, by using near-infrared light and an acidic tumor microenvironment, the release of DOX was stimulated, which caused a rise in the H 2 O 2 level through cancer cells.It could lead to combat against the endogenous H 2 O 2 deficiency itself, followed by improving the CDT of CuS@COF.Eventually, bovine serum albumin-folic acid (BSA-FA) as a targeted molecule was used to functionalize CuS@COF.Existing folate receptors on tumor cells, BSA-FA decorating could regulate the uptake of CuS@COF, and also enhance their biocompatibility and physiological stability.In the effort to evaluate the pH-responsive potential of the prepared nanocomposite, the release of DOX from CuS@COFs-BSA-FA was monitored at the acidic pH (4.0), with the cumulative release of 24.20% was observed after the first 4 h.It is suggested that the significant amount of DOX releasing may have been related to the protonation of nitrogen atoms in CuS@COF causing an increase in release.In addition to this, the applied near-infrared light irradiation increases with the release of DOX, confirming the effect of irradiation on controlled release in biomedical applications.In the light of the evaluation of in vitro therapeutic efficiency, 4T1 cells with CuS@COFs-BSA-FA were incubated for 24 h.Results exhibited a cell viability of above 90%, indicating the improved uptake of CuS@COFs in cell lines.
Admittedly, mitochondrion as the most crucial subcellular organ has drawn attention due to its main role in metabolism.One of the practical ways to cause tumor cell apoptosis is by targeting mitochondrial oxidation based on generating reactive oxygen species (ROS).This is ultimately followed by controlling damages in the function of the mitochondrion and leading to mitochondria-assisted cell death. [92]COFs as a new generation of porous materials represent high porosity and great specific surface area that make them be employed in the co-loading of drugs like CPT and DOX.Besides, COFs have generally been found to be more stable in physiological conditions due to their covalent bonding. [93]Encouraged by it, a mitochondria-targeted co-DDS was developed by utilizing methoxylated COF as a carrier in Wang's group. [94]n this regard, CPT which can play an important role in mitochondrial oxidative damage was co-loaded by using DOX-lipid drug.The double drug loading of CPT/DOX was capable of raising the amount of active singlet oxygen species and improving anti-tumor activity.With the aim of achieving a pH-sensitive drug release, cholesterol succinate monoester was used during the process of lipid bilayer preparation, destabilizing the phospholipid bilayer at acidic pH.As a consequence, the cumulative release of CPT/DOX@ methoxylated COF reached 40.40% at pH = 5.0.More importantly, CPT/DOX@ methoxylated COF showed a sustained release profile, which can extend the release time of in vivo studies.Also, it is worth mentioning that the extracellular area of tumors features a relatively acidic pH of about 6.5-6.9, owing to a higher amount of glycolysis existing in cancerous cells, which supply energy for survival.Therefore, if NPs are pH-sensitive, the intracellular acidic parts (pH = 5.5 and 5.0 for endosome and lysosome, respectively) could be employed to activate drug release. [95]n recent years, researchers have shown increasing interest in GOX-assisted starvation therapy, a new green approach for treating various types of cancer.The idea is to use GOX, an enzyme that interacts with glucose metabolism, to consume glucose and subsequently block the energy supply of tumor cells, causing them to die.However, GOX alone cannot stop the cancer cells from gaining nutrients and oxygen.Hence, the necessity of designing a targeted DDS for efficient cancer treatment. [96,97]In this context, Liu and his team introduced a new system that uses a donor-acceptor COF to release GOX. [11]This system has remarkable properties, such as long-term stability in water and a unique ability to target cancer cells.The donor-acceptor structure, integral to the system, creates channels for charge transport, enhancing COF's functionality to amplify the effect of starvation therapy.To make the COF even more effective, a layer of polydopamine (PDA) and folic acid (FA) was put over its surface to improve biocompatibility (Figure 2).To closely observe how the drug gets released inside the cells, a laser confocal scanning microscope was used.As a part of this study, the COF-PDA-FA sample, combined with a fluorescent dye (Rhodamine B, RhB), was put together with HeLa cells-the first line of human cells to be successfully grown in a lab for an hour.After this short incubation, the cells were observed to show a red fluorescence, which only got stronger after a 5-h incubation.This finding is crucial as it indicates that the cells absorb COF-PDA-FA, and more of the RhB dye was released into cells.This suggests that COF-PDA-FA offers excellent cell uptake, which is a significant indicator of successful drug delivery.
Several types of side effects such as gastrointestinal disturbances, thrombocytopenia, nausea, and hyperpigmentation of the nails stemming from DOX used as an anti-cancer drug have been observed thus far.As a traditional method, triethylamine used to be utilized to convert hydrophilic DOX into a hydrophobic drug through the removal process of hydrochloric acid, which this approach could alter the drug activity itself. [98]Hence, to overcome such problems, nanosized DDS as promising alternatives can be used to enhance drug release.Nowadays, porous materials like COFs showing responsive-release properties with high drug loading capacity have significantly attracted attention.Regarding, an iminelinked COF was synthesized by Dinari et al. [99] by means of an autoclave, and used as a pH-responsive carrier for DOX.The in vitro drug delivery profile of DOX showed a notable increase in cumulative release as pH decreased from 7.4 (32.00%) to 5.4 (54.00%) after 72 h.This result may be related to the interaction of DOX with the synthesized COFs confirming no remarkable change under physiological conditions.In addition, the electrostatic interactions were triggered between DOX and COFs owing to the feasible protonation of imine bonds, which led to enhanced drug release.As a whole, the pH-responsive DOX@COFs provided non-cancer cells protection against DOX side effects.Thus, a noticeable amount of drug molecules can remain in the COF structure, and be released when reach the cancerous region (pH = 5.4).To explore the cytotoxicity of COFs F I G U R E 2 Schematic illustration of (A) synthesis and modification processes and (B) its synergistic therapy and potential mechanism in tumor treatment.(p53: tumor protein p53; Bcl-2: B-cell lymphoma 2; Bax: Bcl-2 Associated X; Caspase 3: cysteine-aspartic acid protease; Cyt C: Cytochrome c). Figure reproduced [11] with permission.Copyright © 2022 Elsevier.against non-tumorigenic epithelial breast (MCF10) and breast cancer (MDA-MB-231) cell lines, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was carried out for 3 days.Accordingly, COFs did not have an effect on the proliferation of both MCF10 and MDA-MB-231 cells at a concentration below 250 μg/mL.Whereas, DOX@COFs inhibited their proliferation at a concentration of about 25 μg/mL, indicating the high viability of DOX@COFs during breast cancer treatment.As a consequence, the faster release may be due to the acidic pH inside the endosome and/or lysosome.
Zhai et al. [73] could synthesize two new imine-based COF functionalized by fluorine, through the condensationbased reaction between 2,5-difluoro terephthalaldehyde with 4,4′,4′′-(1,3,5-triazine-2,4,6-triyl) trianiline or 1,3,5-tri-(4aminophenyl) benzene, which resulted COF-8 and COF-9, respectively.5-FU and CAP were chosen to load on the synthesized COFs as these drugs contain sulfhydryl, amide, and carboxyl groups enabling them to form Fluorine-Hydrogen bonds with COFs.As a result of this, drug molecules would be more easily absorbed by COFs.On the other side, COFs with high porosity structures endow drug molecules with better loading through non-covalent bindings.As could be seen in the drug release profiles of prepared samples, the majority of 5-FU was released from both COFs, especially, COF-9 (up to 80.00%) after 3 days.It might have been related to the larger pore size of COF-9 making it absorb 5-FU in an improved way, and release drug molecules more easily.Hence, these COFs NPs are capable of serving suitable drugs as carriers.
Yao et al. [100] reported two new 3D carbonbased COFs with the sp 2 hybridization, by the interaction between tetrakis(4-formylphenyl)silane with 2,2′-(1,4-phenylene)diacetonitrile and 2,2′-(biphenyl-4,4′-diyl)diacetonitrile (denoted as COF-580 and COF-581, respectively), under the reaction of double carbon-based bonds (also named as olefin-linked COFs).The obtained results could confirm some prominent properties of COFs such as high chemical stability, large pores, and permanent porosity.In the meantime, their potential as a fluorescent tracer in drug delivery studies was assessed by loading one of the most commonly used drugs, cisplatin, on the prepared COFs.To confirm the successful loading, various techniques were employed X-ray diffraction, transmission electron microscopy, and inductively coupled plasma optical emission spectrometry.The structural integrity was approved as X-ray diffraction and transmission electron microscopy results were in agreement with the starting materials.In addition, the spectroscopy method demonstrated a significant amount of loading on COF-581 (20.00 wt%).Importantly, the green fluorescence of COF-581 was seen in human prostate cancer cell lines (PC-3) after the 4-h incubation.To put it in other words, cisplatin@COF-581 can stain cell debris of PC-3 in comparison to 1,1′-dioctadecyl-3,3,3′,3′tetramethylindocarbocyanine perchlorate as a commercial dye, indicating the noticeable cellular staining capability of COF-581.The resultant COFs not only be considered good drug carriers for sustained release, but according to fluorescence imaging, they will offer a long-term fluorescent tracer in drug transportation.
Pang and his team were inspired by the impressive qualities of COFs such as their high porosity and their potential for being used in DDS, therefore, they created a novel 3D COF with good crystallinity through a condensation reaction. [74]o test the release behavior of DOX, an anti-cancer drug, they used a one-pot method to trap drug molecules in the COF structure.The method they used didn't just impact how long the drug took to be absorbed; it also showed a high level of drug-loading entrapment.The resulting DOX-loaded COF had a high drug entrapment of about 30%, along with promising pH-responsive drug release qualities.During the in vitro tests in the lab, they noticed that DOX was released quickly at first within 2 h at normal body pH (7.4).However, under acidic conditions at a pH of 5.0-6.5, most of the drug molecules were released after 2 h and released completely after a day.This initial burst of DOX could be due to its instability in the test solution (PBS).This fits with studies of the physical appearance of the DOX-loaded COF, which showed a significant change from spherical to irregular shapes under normal body pH.This could have been because of the breaking down of the imine groups in DOX-loaded COF when mixed with PBS.In this study, COF as an efficient platform for DOX loading and release has shown great potential with high drug loading capacity.
Enzyme prodrug therapy (EPT) is capable of converting compounds into pharmacological drugs showing no or even low cytotoxicity.Among all the widely investigated enzymes, the mixture of natural horseradish peroxidase (HRP) with indole-3-acetic acid (IAA) could be considered one of the noticeable strategies for cancer oxidation therapy.Indeed, having catalyzed by the means of HRP, one electron of IAA is oxidized, followed by ROS production. [101]However, the HRP-IAA system displays some insurmountable challenges, owing to its reaction mechanism.Some enzymes like HRP are susceptible to losing their activity while ROS are generated during IAA oxidation, diminishing the EPT efficiency. [102]o address this limitation, porous materials like COFs could aid HPR in achieving a controlled release pattern.In this regard, an enzyme nanocapsule based on a novel 3D COF was prepared by Tang et al. in order to assess the long-term properties of EPT. [103]According to the release studies, the resultant COF-based nanocapsules were able to deliver HRP constantly demonstrating its prolonged time activity during the EPT method.Subsequently, the prepared nanocapsules showed an effective strategy for highly efficient EPT features.
Recently reported literature has shown that COFs with high porosity and structural stability can immobilize the drug molecules in their structure.However, releasing these drugs can be tricky.Also, the intracellular release of photosensitizer might encounter difficulty as the diffusion distance of singlet oxygen is relatively short during photodynamic therapy (PDT). [104,105]On the other side, hypoxia as one of the common pathological properties of almost all solid tumors would be aggravated if PDT consumes oxygen molecules to activate oxygen species. [106,107]To combat these issues, a hypoxia-responsive COF was synthesized by Jiang et al., by incorporating azobenzene groups into the COF framework. [108]This was followed by the co-loading of tirapazamine as a hypoxia-activated prodrug along with Chlorin e6 as the photodynamic agents on COF.Then, near-infrared light was irradiated to make Ce6 use oxygen molecules, which led to generating activated oxygen species, and ultimately elevated hypoxia.Consequently, this approach stimulates the deintegration of COF resulting in the activation of tirapazamine, followed by its release.Altogether, aside from that this work made use of the porous structure of COF as a promising material, but also a new approach towards hypoxia responsiveness was successfully employed in DDS.
In a separate study, Li and his team developed a new type of 3D-COF-COF-HQ, which is functionalized with 8-hydroxyquinoline.This COF has excellent porosity and disperses well in biological fluids, making it suitable for application in the field of nanomedicine.They tested how the cancer drug 5-FU was released from the COF and found enhanced loading capacity, owing to quinoline groups in the COF. [75]The drug release profile of 5-FU-loaded drug on the resultant COF-HQ was assessed, through which an enhanced loading capacity was observed, owing to the presence of quinoline groups.Accordingly, 5-FU@COF-HQ at different pHs represented a controlled drug release, due to the pH sensitivity related to Nitrogen atoms in quinoline and C═N groups.As a matter of fact, hydrogen bonding, hydrophobic, electrostatic interactions, π-π stacking, and a highly conjugated system of quinoline may have played a crucial role in boosting drug loading on COF-HQ.Encouraged by the excellent biocompatibility and anti-tumor activity of the obtained COF, in vivo anti-cancer activity was also carried out on mice (Figure 3).On the basis of the results, a remarkable inhibition of tumor growth in relation to 5-FU@COF-HQ was observed compared to free 5-FU indicating the positive effect of COF polymer as a drug carrier.Generally, the synthesized COF-HQ in this work would provide new approaches as an applicable drug carrier for cancer chemotherapy showing efficient tumor inhibition.
Lots of newly created drugs cannot be applied to clinical trials mainly due to their lack of target selectivity, which means that they could inadvertently harm healthy cells and cause toxicity.As a result, there is a need for better DDS that can effectively target specific cells.Some targeting ligands like peptides and FA have drawn attention in nanomedicine, as they can specialize their targets. [109]egarding this, the synthesis of folate receptor-based COF, using 4,4′,4′′-(1,3,5-triazine-2,4,6-triyl)trianiline-2,5dihydroxyterephthalaldehyde (FADT-COF), through making folate conjugate to the pores of COF structure was pioneered by Yu et al. [110] The obtained COF could display high affinity to folate receptor positive cancer cell lines because of its active targeting capability.To further evaluate the drug release profile, Withaferin A was loaded on FADT COF, followed by conducting in vitro drug delivery.It showed boosted killing activity towards folate receptor-positive cancer cells when they were assessed in vitro.This work can open up a new way to decorate COF porous nanomaterials with ligand receptors for targeted drug delivery.This research marks an innovative step towards using COFs in drug targeting.
Tsai et al. successfully synthesized a new thioetherterminated triazole bridge based on COF (TCOF) via uncomplicated click chemistry using C≡C and − N═N + ═N − monomers with the aim of double-sensitive [pH and glutathione] system in DDS. [111]The synthesized TCOF revealed a pore size of about 30 nm, based on the results of both Powder X-ray diffraction and BET.In the next step, polyethylene glycol (PEG) modification was done due to the flexible nature of TCOF.The drug release evaluation of DOXloaded TCOF/PEG was conducted at both lysosomal-like pH 5.0 and physiological pH 7.4, as well as the glutathione environment.As evident, just a small amount of DOX was released at physiological conditions (20.00%), suggesting a stable drug release system.In contrast, nearly 70.00% to 80.00% of loaded DOX was released from TCOF/PEG at the other two environments after 3 days.It can be put down to the fact that glutathione molecules would attack the triazole ring F I G U R E 3 Schematic diagram of covalent organic framework (COF)-HQ for drug delivery and drug release in vivo.Figure reproduced [75] with permission.Copyright 2020 Elsevier.making changes in the conformation of DOX@TCOF/PEG, and releasing more DOX-loaded drugs.On the other side, DOX-loaded COF with no triazole ring did not demonstrate a remarkable release, confirming the efficient function of the triazole bridge.Overall, the newly synthesized TCOF as an effective carrier paves the way in therapeutic applications owing to its biocompatibility.
Besides cancer cells, the environment of tumors also includes non-cancer cells and blood vessels that sit in a network called the ECM.This ECM plays a key role in tumor growth, drug resistance, and metastasis.It can also hinder drug and oxygen supply to cancer tissues. [112,113]To bypass this limitation, Zhang and his colleagues developed another special type of COF that was designed to remodel the ECM for better photodynamic therapy efficacy. [114]They loaded this COF with an anti-fibrotic drug pirfenidone (PFD) and upon testing, found that it was released slowly and effectively in a controlled manner.An added bonus was that it was found to accumulate in tumor environments enabling the efficient delivery of the drug and subsequently reducing tumor growth.This suggests an exciting potential application of COFs in cancer treatment (Figure 4).
Zhang et al. developed a new process for making a COF that reacts to oxidation and reduction, acting as a nanocarrier for DOX. [76]It was prepared through the interaction between Pluronic F68 with a disulfide under the condensation reaction (F68@SS-COF).Interestingly, this COF interacted with DOX through both hydrophobic (water-repelling) and π-π stacking processes, and it quickly released DOX when exposed to lutathione, in the release environment.The drug release profile was studied in three different media, including PBS pH 7.4, PBS pH 5.0, and PBS pH 5.0 with lutathione.The cumulative release in the former was very low (9.40%) after 48 h, which could be related to the low solubility of DOX at this pH.Whereas, in the PBS pH 5.0 (with or without lutathione), a higher release rate was observed owing to acid-induced protonation of the drug leading to an increase in the solubility of DOX, and drug diffusion rate.The highest release rate (90% after just 24 h) was observed in the acidic environment with lutathione.This was likely due to the resulting environment being very similar to that found inside tumor cells.So, these findings suggest that F68@SS-COF could offer great potential for targeted drug delivery in cancer treatment.
On the other hand, Qiu et al. created two completely new 3D polyimide-based COFs that showed impressive thermal stability, large pore size, and a surface area of 2400 m 2 /g. [115]hey achieved this by reacting a linear linking molecule called pyromellitic dianhydride with two other molecules.These reactions formed two types of COF (named PI-COF-4 and PI-COF-5), which they then tested for drug delivery with the medication IBU.They found that PI-COF-5 released the drug at a slightly slower rate than PI-COF-4 after 12 h (49% released compared to 60%), an observation that indicates the role of pore size and geometry in the COFs' drug delivery mechanism.They also tested the delivery of two other drugs, CAP and caffeine, both of which exhibited similar release patterns to IBU.These results demonstrate the usefulness of PI-COF-4 and PI-COF-5 in pharmaceutical applications.

APPLICATION OF 2D COFs IN DRUG DELIVERY
When it comes to 2D COFs, an important group of layeredstacked porous materials presenting a periodic π-arrays structure of building linkages along with a rigid conjugated system would come to mind.This structure provides an applicable way, by which COFs could have a well-organized network including covalent, hydrogen, or/and Van der Waals bonds.Their column-like layers usually stack face-to-face along with quite strong interactions.In other words, it would be feasible for charge transporters to convey more effortlessly, suggesting that this type of COFs might be utilized in some  [114] with permission.Copyright 2020 Elsevier.
other applications like optoelectronics as they create a novel π-bond electronic. [116]Obviously, by combining the different building units and linkages, a 2D structure with various skeletons and pores can be synthesized. [117]Besides, 2D COFs with predictable topologies such as trigonal, hexagonal, and tetragonal could offer tunable functionalities for various applications such as gas separation, [118] biomedicine, [119] and sensors [120] by using appropriate monomers, owing to their ultra-high and rich porous structure. [121,122]The different types of combinations between monomers lead to the generalization of various organized structures of COFs.To make an analogy, the mixture of [C 3 + C 2 ] creates a hexagonal structure, while [C 3 + C 3 ] would also generate a hexagonal network with varied pore size (Figure 5). [26,123]n comparison to 3D COFs that can be mainly applied to gas storage fields because of their high specific surface area, 2D COFs are exceptional candidates for biomedical exploration such as drug carriers.This could be put down to their 2D extended sheets, which are stacked in layers, and are able to generate periodic π-arrays. [124]Moreover, a huge effort has been recently devoted to morphological management for developing nanosheets of 2D COFs as they can be easily organized into nanosheets.As evident, 2D COFs possessing high surface area provide greater absorption of drug molecules, and their thinness makes them be employed in bioimaging. [125]What is more, the high tenability of 2D COFs is considered their major advantage in nanomedicine applications.It means that their porosity could be tuned regarding their size and shape by changing building blocks leading to further investigation in drug release. [119]Considered one of the efficient drug platforms, a new 2D COF with a spherical structure for loading 5-FU was synthesized by Namazi et al. [126] to study its potential in colon cancer treat-ment.Although its imine bonds showed low stability in harshacidic conditions, the high surface area (230.58 m 2 /g) made it capable of loading 5-Fu with a capacity of ∼93.33%.To overcome the aforementioned issue, carboxymethyl starchgelatin (CMS-Gel) was employed in order to cap 5-FU@COF (Figure 6).Based on the in vitro drug delivery results, the percentage of 69.60% of 5-FU@COF was released after 2 h, whereas it fell to a low of 14.10% in the case of 5-FU@COF/CMS-Gel.The fast-release pattern of the former could be attributed to its lower stability in relation to the imine bonds in an acidic environment.Another major reason for this observation might have been related to the protonation of nitrogen atoms in COF, due to the existence of a huge amount of H + that could hinder the interaction of COF with protonated 5-FU.Interestingly, nearly 80.00% of 5-FU was released from COF/CMS-Gel during 8 h, indicating that the usage of CMS-Gel as a coating agent affected the release rate of 5-FU.This result could be related to the tardy diffusion of the drug through the CMS-Gel matrix into the release media.As a whole, the observations in this work would shed new light on COF synthesis, and its application in DDS studies.
The arrangement of recently developed drug carriers is due to secure them from any destruction during the release of drug molecules.Herein, a newly emerged class of porous structure named CTFs could be utilized for this purpose in therapeutic applications as they normally exhibit magnificent properties like low density, excellent chemical and thermal stability, and also high specific surface area.More importantly, possessing aromatic rings in their structure presents a new approach to drug release by playing a hosting role for charged and neutral drugs. [127]Dinari and co-workers [128] prepared a new CTP based on polybenzimidazole under the solvothermal reaction.Then, piroxicam (PRX) and mefenamic acid (MFA) were chosen to load on the resultant CTP through applied F I G U R E 5 Topology diagrams representing a general basis for covalent organic frameworks (COFs) design and construction of two-dimensional (2D) COFs. Figure reproduced [26] with permission.Copyright 2021, Elsevier.

F I G U R E 6
Schematic pathway of the covalent organic framework (COF) fabrication, 5-FU loading, and COF/5-FU coating with carboxymethyl starchgelatin (CMS-Gel) in the hydrogel bead form.Figure reproduced [126] with permission.Copyright 2023 Elsevier.ultrasonicate irradiation.A noteworthy loading efficiency was observed for both drugs, which may be related to the π-π stacking forces existing in the aromatic rings of CTFs structure (PRX = 53.00%and MFA = 49.00%).According to the carried out release study of PRX@CTP and MFA@CTP hybrids, it was found that 81.00% and 92.00% were released, respectively.Although using CTFs in biomedicine is still in its infancy stage, this work could give new insights into CTFs-based DDS.
Another novel CTF based on polyimine was synthesized by Dinari's group [129] to assess the drug release of sorafenib (Sor).This drug is defined as a multi-tyrosine kinase inhibitor demonstrating anti-proliferation effects, which has been tried in the treatment of some solid tumors, specifically hepatocellular carcinoma.It is approved that using Sor therapy in phase III trials can cure patients diagnosed with late-stage hepatocellular carcinoma. [130]Herein, the in vitro Sor release was evaluated in PBS at both acidic and physiological pH conditions (5.3 and 7.4, respectively).The cumulative release of Sor was significantly increased when pH changed from 7.4 (48.00%) to 5.3 (66.00%).It might be related to the protonation of nitrogen atoms of CTFs that at pH = 5.3 waken hydrogen bindings leading to faster release of Sor.In general, the sustained and passive-targeted release of Sor from the resultant CTFs might profit DDS by decreasing the required drug doses as well as reducing side effects.To evaluate the cytotoxicity of free Sor and Sor@CTFs against prostate cancer (LNCaP), in vitro MTT assay was performed for 3 days.Both encapsulated and free Sor could inhibit the proliferation of LNCaP in time-and dose-dependent patterns.Besides, free CTFs were not toxic against LNCaP cells with a cell viability of approximately 70% at higher concentrations.Results also showed that CTFs not only had no toxicity against normal fibroblast (L929) cells but could improve their proliferation, indicating the biocompatibility of CTFs as nanocarriers for in vivo antitumor studies.
Asadi's research group synthesized a new vanillin-based CTF that can act as a good drug carrier for biomedicine. [131]matinib, a drug, was loaded on these CTFs and had a high loading capacity, that is, 82.00%.The release of Imatinib was significantly affected by different pH levels, increasing from 48.00% at pH 7.4 to 73.00% at pH 5.3.This shows that these CTFs are very sensitive to changes in pH levels when releasing drugs.Their biocompatibility was confirmed by conducting an assay against certain cell lines.It was found that these CTFs do not affect healthy cells while reducing the viability of certain cancer cells.
Microscopic machines known as microrobots are becoming increasingly popular in biomedicine due to their ability to interact with cells and release drug molecules. [132]They must be designed to encapsulate these drugs and release them under specific conditions.Porous materials can be used for this purpose as they have inner spaces that allow for drug loading.However, an overabundance of these pores can cause difficulties in controlling the drug release. [133]Another research group, headed by Podjaski, created two types of COF nanospheres to achieve controlled drug release. [134]hese COFs were then tested as carriers in cellular media and in an aqueous environment.Two drugs, DOX and insulin, were used to evaluate their effectiveness.It was found that DOX was released more quickly in the first 60 min at a pH of 7.2 from COF-10, while the release from COF-11 was more stable.When the pH was lowered to 3.5, more DOX was released.On the other hand, insulin molecules were released more efficiently, especially in more acidic environments.Therefore, these drug-loaded COFs could serve as effective structures for therapeutic uses.
In yet another study, by Sohrabnezhad and collaborators, they succeeded in loading antibiotics into a COF-based nanostructure carrier for the first time. [135]A probe in a biological environment found that nearly 70% of the loaded antibiotic was released after 50 h.There were two phases of release: an initial fast one and a subsequent slower one.This might be because of the distribution of the antibiotic within the nanocarrier.This COF-based nanohybrid carrier showed no toxicity against breast cancer cells, making it a potential candidate for future drug delivery studies.
The 2D COFs with hollow-like spherical morphology based on 2,3,6,7,10,11-hexakis(4-aminophenyl) triphenylene as a precursor were designed and synthesized by Chen and co-workers [136] using the self-templated method and solvothermal condition.In this regard, the precursor interacted with terephthaldehyde, tris(4-formylphenyl) benzene, or tris(4-formylphenyl) amine resulting in COF-3, COF-4, and COF-5, respectively.Based on transmission electron microscopy results, the obtained COFs were composed of crystalline nanosheets along with hexagonal single crystals.Moreover, the pore volume of up to 1.947 cm 3 g −1 for these COFs was observed, which made them superior nanocarriers for drug release.Hence, IBU was loaded on the obtained COFs and showed a sustained release without any burst effect.After 7 h, almost half of IBU was released, and the cumulative release after 5 days was 91.70%.This was due to the fact that IBU had entered a one-dimensional channel of 2D COFs, and their small pore size affected the drug release.In general, the synthesized COF-3 displayed a significant drug loading capacity compared to other reported 2D COFs to date.With the aim of reasonably evaluation of the COFs' potential as DDS, their cytotoxicity effect was assessed against MDA-MB-231 cell lines using an MTT assay for 2 days.Notably, the cell viability of above 95% was observed after a two-day incubation.These COFs not only showed non-toxicity, but excellent biocompatibility against cells.Therefore, the COFs can be safe for human use in further in vivo studies.
Weng and co-workers [137] were one of the first to report a facile interface design method to synthesize COF NPs through Michael's addition of p-benzoquinone and 4-[1,2,2tris(4-aminophenyl) ethenyl] aniline.To evaluate the drug release profile of COF NPs, CPT was firstly loaded on it, and then in vitro drug release study was done at different pH environments.Evidently, CPT was released in an acidic condition more quickly and thoroughly, which implies the successful function of COF NPs in tumor surroundings.Besides, to further study of drug delivery effect, in vivo experiment was conducted using 4T1 tumor-bearing mice.In comparison to control groups, CPT@COF NPs revealed a higher inhibition effect towards tumor growth, confirming the good biocompatibility of COF NPs as a drug platform in therapeutic applications.
Gong's group [138] proposed the great potential of novel core-shell-based COFs, and magnetic iron oxides (Fe 3 O 4 ) as the core for drug delivery application (Fe 3 O 4 @COF).In this regard, having synthesized COF by using a facile sonication method, it was then modified BSA that had been linked with FA (BSA-FA).The modification endowed the COF structure with long-time stability and targeting ability in a cancerous environment.In addition, the well-dispersed Fe 3 O 4 @COF could create a good condition for high drug loading of DOX through both π-π interactions and hydrogen binding.Gong and co-workers further visually assessed the drug release of DOX/Fe 3 O 4 @COF/BSA/FA by using a laser scanning confocal microscope.After the incubation of DOX/Fe 3 O 4 @COF/BSA/FA with HeLa cell lines, the red fluorescence of DOX could be detected in cells.According to the obtained results, it was found that DOX might have been released more and more throughout the cells, followed by the cleaving of the deoxyribonucleic acid strands, which would ultimately result in cell death.In general, the magnetic Fe 3 O 4 @COF could be considered a promising candidate in magnetic resonance imaging applications, and its great photothermal performance would make it to be used in combined photothermal and chemotherapy.
Shi's team developed a novel 2D cage-like material using molecular cages containing triammonia and a compound called terephthalaldehyde. [70]The MTT results of COFs in this study showed no noticeable toxicity against L929 and human hepatocyte cells (L-02).Thus, the applicability of COFs as a carrier was subsequently studied through in vitro drug release.In this regard, having loaded COFs with three drugs (IBU, 5-FU, and CAP), their drug release was studied in different conditions.Impressively, after about 2 days, over 90% of each drug had been released from this 2D material into lab-simulated body fluid, showing promise for timed drug release.The insertion of functional groups into the structure of COFs plays a crucial role, as it helps postsynthetic modification of these porous materials.Another research team led by Botella incorporated functional groups into similar 2D materials to improve their post-building modification. [139]Despite some challenges, they were able to create a new 2D material that incorporated primary amino groups.They tested the drug loading and release ability of this new material using an anti-cancer drug called camptothecin (CPT).Astonishingly, within fewer than 8 h, over 80% of CPT was released quickly.
Akyuz's group synthesized a 2D material using a chemical reaction and tested it with carboplatin, an anti-cancer drug. [140]They found that the rate of drug release was higher in an acidic environment (pH 5.0) than in a natural body environment (pH 7.4).This could be due to weaker interactions between the drug and our material in an acidic environment.A notable burst effect was observed between 6 and 12 h, followed by a sustained phase over 5 days.Tian's team tackled the problem of COFs' low dispersibility and stability in water by synthesizing a 2D water-dispersible material using cyanines as stabilizers. [141]This new material showed good stability in water, which could be beneficial for intravenous injections.They loaded this material with an anti-cancer drug and found that almost half of the drug was released after only one day.Xi's group created various water-dispersible materials using different chemicals and tested drug release with a popular anti-cancer drug, DOX. [81]hey found significant improvement in drug loading capacity using one of their synthesized materials and concluded that these new materials could serve as efficient carriers for enhancing drug release and endocytosis.
Covalent organic polymers (COPs), a type of structurally crosslinked porous material, have promising applications in drug delivery, particularly pH-responsive COPs that react to the slightly acidic conditions of tumors. [142]Liu's team developed a new COP that responded to pH levels by using specific compounds. [143]They also incorporated a substance called PEG to complete the reaction and form a protective shell.They used this COP to load a drug called DOX within the porous structure and tested it at two different pH levels.An interesting fact was that the particles increased in size at a pH of 6.0 (compared to a neutral pH), leading to faster drug release.This suggested that their COP could be used for tar-geted delivery of anti-cancer drugs.The anticancer potential of COP was evaluated against 4T1 (breast cancer) cells via cellular experiments, by which the improved cellular uptake of PEG/DOX@COP was observed.It simply illustrated the possible release of DOX from the COP network at acidic cellular lysosomes.
Another type of 2D COFs, known as covalent organic nanosheets (CONs), has received attention due to their chemical stability and easily accessible surface.Banerjee's team developed two different types of CONs and loaded them with 5-FU, a common medicine used in cancer treatment. [78]The amount of drug loaded was significantly higher than that of other substances.When tested, three-quarters of the drug was released after 3 days at a pH of 5.0, showing a sustained drug release profile that could lower the potential side effects of targeted drug delivery.
Undoubtedly, the π-conjugated skeleton of COFs plays an essential role in promoting fluorescent properties. [144]lthough a majority of recent works mainly concentrate on the forming of various bonds between different monomers, little attention is devoted to developing the conjugated systems by the usage of pre-conjugated monomers.To tackle these challenges, a novel DDS based on excellent fluorescent COFs was reported by Gong and co-workers. [145]They synthesized an activated monomer and added benzidine to it.By doing this, they enhanced the fluorescent properties while also making the whole system more effective in absorbing drugs.They tested this by loading this COF with DOX, a popular fluorescent-spectrum drug for cancer treatment.They noticed significant changes in the fluorescence resonance energy transfer with different amounts of the loaded drug, which allowed them to evaluate the drug loading process under ultraviolet and natural light.When tested with cells, a significant amount of DOX was released into the nucleus of the cell, showing that this system can effectively deliver drugs through the cell endocytosis process.Since DOX represents a bright peak of fluorescence spectra under various excitation wavelengths, it can be utilized as a useful fluorophore to validate drug release. [146]Having incubated by lung carcinoma epithelial cells (1 h), the green fluorescent signal related to DOX was mainly distributed through the cytoplasm, while just a small part could enter the nucleus.However, after 4-h incubation, a significant amount of DOX was released into the nucleus.Also, the flow cytometry study was carried out for further assessment, in which the DOX@COF system displayed noticeable fluorescence signals.Importantly, more amount of DOX was released as its concentration was increased, confirming easier taking up of DOX@COF via cell endocytosis.
In an effort to enhance the efficiency of colon cancer treatment, an imine-based porous COF with a pore diameter of 8.5058 nm was designed by Namazi et al. [147] through a condensation reaction.Then, by considering the co-drug system advantages in cancer treatment, 5-FU and CUR were loaded on synthesized COF.In addition to this, to improve the structural stability in simulated stomach media, a co-drug system of 5-FU/CUR@COF was coated by using two different polysaccharides named alginate (Alg) and carboxymethyl starch (CMS) (Figure 7).The drug release pattern of Alg/CMS/5-FU/CUR@COF in an acidic medium was carried out via the gastrointestinal tract and simulated gastric fluid.Interestingly, in the latter the cumulative F I G U R E 7 Schematic representation of covalent organic framework (COF)-OH synthesis, co-drug loading, and bio-coating with Alg/CMS. Figure reproduced [147] with permission.Copyright 2023 Elsevier.
release was lower than that of the former, indicating the successful usage of polysaccharides.The biocompatibility of Alg/CMS/5-FU/CUR@COF was assessed against human colon adenocarcinoma (HT 29) cell lines using an MTT assay.Based on it, more cell viability could explain the sustained release pattern of 5-FU/CUR from COFs, which led to the low-active drug molecules concentration in the treating media.Also, Alg/CMS@COF did not have toxicity on cell lines, while could kill cells after 5-FU/CUR loading.Generally, the rate of hemolysis (less than 5.00%), and high cell viability (about 80.00%) could be considered as two positive approaches in this study, which made the Alg/CMS/5-FU/CUR@COF system a more sensitive carrier to use in the colon cancer treatment.
Developing a carrier, which features a stable binding affinity for insulin molecules has remained discouraged as they can be charged either positively or negatively based on the isoelectric points.Thus, it is worth making an effort to explore a creative approach for the delivery of insulin molecules without any disorder in insulin activity. [148]Keeping this in mind, Gao et al. [149] managed to utilize an effective strategy for the synthesis of a COF-based DDS including glucose and pH dual-responsive insulin.At first, both insulin and GOX were encapsulated into the COF structures (denoted as COF-6 and COF-7, respectively), and then functionalized by polyethylene glycolatedisothiocyanate.Using in vitro drug delivery studies, the synthesized COFs polymer composites showed a slow release of insulin molecules.More intriguing, the COF-7 composite exhibited more well-organized drug release, which may be related to more dissociation of COF-7 compared to COF-6.Generally, it could be concluded that such a facile strategy is capable of regulating the level of blood sugar with no need for medication.
A new method involves decorating a hypoxia-responsive COF activated by laser using tetra(4-hydroxyphenyl) porphine as a cross-linker, and singlet oxygen-cleavable thioketal as a linker was presented by Xue's group. [150]By taking advantage of external OH groups, PEG was used for biochemical modification of tetra(4-hydroxyphenyl) porphine/thioketal via esterification.Furthermore, prodrug banoxantrone along with porphyrin were loaded on the surface of tetra(4-hydroxyphenyl) porphine/singlet oxygencleavable thioketal-PEG NPs so that the prodrug would be protected from being degraded through blood circulation.Exposed to laser irradiation, the synthesized COF was capable of generating cytotoxic singlet oxygen for PDT while consuming oxygen at the tumor area.Thus, owing to the presence of a thioketal linker, COF could selectively fall apart at the tumor location, and banoxantrone was released in a controlled way.In general, an ultrasensitive drug release with effective therapeutic application can be achieved by this method.
Further research into PDT-based multimodal hepatocellular carcinoma therapy led to the synthesis of a porphyrinbased COF nanoplatform, COF-366, in Liu's group. [151]It was functionalized with N-acetyl-galactosamine and RhB and called GR-COF-366 (Figure 8).N-acetyl-galactosamine aids in targeting the nanoplatform towards asialoglycoprotein receptors in liver cells and tissue.They loaded Sor onto GR-COF-366 and evaluated its release profile under physiological and acidic conditions (pH 7.4 and 5.6, respectively).A significant amount of Sor was released at acidic pH after 16 h (83.2 ± 3.8%), while only 12.8 ± 1.7% was released at pH 7.4.This release control could be attributed to the acid-sensitivity of Sor@GR-COF-366′s Schiff base linkage.Thus, this COF-based carrier shows promise in PDT for hepatocellular carcinoma treatment.
In another study, Kumar and his team synthesized an imine -based COF (RT-COF-1) using 1,3,5-benzenetricarbaldehyde and 1,3,5-tris(4-aminophenyl) benzene, to assess the release of IBU, a model drug.Images showed hexagonal flakes measuring about 50 to 70 nm (Figure 9) . [152]Upon loading IBU into the porous structure of the COF, it was found that the F I G U R E 8 Schematic illustration of the preparation of the Sor@GR-COF-366 and mechanism of Sor@GR-COF-366 for targeted synergetic PDT/chemotherapy with the aid of minimally invasive intervention (HCC: hepatocellular carcinoma; DMSO: dimethylsulfoxide; ASGPR: asialoglycoprotein receptor; GalNAc: N-acetyl-galactosamine). Figure reproduced [151] with permission.Copyright 2022 Elsevier.rate of drug release increased with time, reaching 33%.After a certain interval, the absorbance leveled out, confirming full IBU release.This COF may serve as a basis for additional DDS research for critical diseases.
Zhao and his team synthesized two novel COFs, each with an average diameter of 50 nm, to explore their drug release characteristics and biocompatibility. [77]hey created PI-3-COF and PI-2-COF via a condensation reaction between benzene-1,3,5-tricarbaldehyde and either 2,4,6-Tri(4-pyridyl)−1,3,5-triazine or benzidine, respectively.Afterward, they loaded 5-FU onto these COFs.Both of them exhibited high drug loading capacity and efficient F I G U R E 9 Schematic representation of RT-COF-1 synthesis and ibuprofen (IBU) tagging with RT-COF-1.Figure reproduced [152] with permission.Copyright 2022 Elsevier.dispersibility in water.PI-2-COF, owing to its larger pore size, showed slightly higher drug loading capacity as it could enclose more 5-FU.In drug delivery tests, both PI-3-COF and PI-2-COF released 5-FU at similar rates, with about 85% cumulative release after 72 h.Overall, this research successfully demonstrated the potential of these new COFs for therapeutic applications.

CONCLUSION AND PERSPECTIVE
The flourishing research into COF-based porous materials over the past decade has introduced numerous novel applications in various fields, including biomedical.These innovative structures possess remarkable qualities like high-tunable porosity, biocompatibility, large surface area, organized channels, and predetermined structure, and allow a vast selection of building blocks.The majority of COF studies in recent years have focused on 2D structures, though developing biocompatible 3D COFs still remains a significant challenge.This article reviews various techniques and the latest breakthroughs in the use of different categories of COFs, particularly in drug delivery and fighting cancer: i) 3D COF-based materials in drug release These nanomaterials, due to their potent adsorptive features, vast surface areas, and channelized pores, hold promise in various applications.One key potential is the delivery of guest molecules in DDS.Fabrication of 3D COFs demands specific pairings, like C1, C2, C3, and C4, incorporated with a T d knot.Significantly, combining [T d + C3] tends to yield 3D COFs with large surface areas, making them suitable for higher amounts of guest encapsulation for drug delivery.
ii) 2D COFs-based networks These layered porous materials represent a periodic πarrays structure and predictable topologies, offering tunable functionalities for biomedical applications due to high porosity.Owing to their unique physiochemical properties, COFs have demonstrated potential advantages in anti-cancer therapy such as drug delivery and phototherapy.Their immense interconnected pores and broad surface area allow COFs to entrap drug particles proficiently, while also showing impressive antibacterial activity.Certain studies hint that COFs, when combined with NPs or even receptors, have the capacity to precisely target cancerous tissues.Moreover, COF-based systems that carry multiple drugs could potentially elevate anti-cancer activity by boosting the quantity of active oxygen species.Most notably, the donor-acceptor structure of COFs, which aids charge transportation, can further boost the efficacy of anti-cancer treatment.
Though these prospects appear promising, deploying COFs in biomedical applications is still in its infancy.Challenges to surmount include controlling drug loading due to COFs' porous structure and tackling issues like poor cellmembrane permeability and non-specific targeting.Strategies such as biopolymer coating or nanoparticle utilization may offer solutions to these challenges.To address tissue-specific ailments, precisely targeted delivery is imperative.Multipurpose platforms can release specific agents for target-oriented treatments, sparing non-targeted tissues.Utilizing COFs as targeted delivery systems involves labeling them with surface modifiers, such as key receptors, ensuring selective release for desired responses.Consequently, significant efforts have been dedicated to enhancing surface modification, biofunctionality, crystallinity, and the development of targeted COFs-drug delivery systems.Certainly, the size and structure of COFs-whether they are 2D or 3D-significantly affect how well they work.We look forward to seeing advances in both 2D and 3D types, which could greatly enhance their usefulness in fields like medicine.There are some exciting possible developments that we hope to see getting realized.(i) The high degree of tailorability of COFs opens new avenues for designing more efficient drug delivery systems.The ability to modify and control COFs' structures and pore geometries will allow researchers to engineer targeted solutions for differing therapeutic needs.(ii) The focus thus far has been predominantly on cancer therapy; however, the application of COFs could be extended to other medical conditions.Diseases requiring long-term medication or targeted intervention could greatly benefit from COFs' capability for sustained and controlled drug release.(iii) Investigations into improving biocompatibility and decreasing any potential toxic effect in COFs' application are anticipated to surge.This will enhance safety profiles, making them more suitable for human use (iv) The advancement in COF technology might herald the creation of multifunctional drug delivery systems.These would incorporate diagnostic and therapeutic functions in one system, possibly transforming the way diseases are treated and managed.Given the exciting progress made so far, it is anticipated that there will be abundant research in COF-based materials in the years to come.
Ongoing research and innovative explorations are anticipated to unveil the full capabilities of nanoplatforms based on COFs, enabling more sustained processes.We envision significant breakthroughs in the use of COFs as drug carriers, particularly with dual-and multimodal approaches.These advancements are poised to make their mark in clinical applications, exerting a profound impact on personalized treatments in the near future.

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Schematic illustration of PCPP-mediated tumor extracellular matrix (ECM) degradation to enhance tumor photodynamic therapy (PDT) effect.(A) Restricted tumor PDT effect due to the insufficient oxygen supply as well as the limited uptake of NM-PPIX in the tumor.(B) Selective delivery and release of pirfenidone (PFD) in tumor tissues by PCPP, PFD-mediated tumor ECM depletion, promotion of tumor vasculature functionality, and alleviation of the hypoxic state of the tumor.(C) Enhanced reactive oxygen species (ROS) generation and tumor PDT effect due to the improved oxygen supply and tumor uptake of NM-PPIX.(NM-PPIX: protoporphyrinl IX (PPIX)-conjugated peptide formed nanomicelles.Figure reproduced Overview of the recent covalent organic framework (COF)-based nanomaterials in drug release studies.
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