Water‐dispersible and soluble porous organic polymers for biomedical applications

Porous organic polymers (POPs) have become an emerging class of advanced porous organic materials owing to their structural diversity and tailored functions in solid state and organic media. Creating water‐soluble and related water‐dispersible POPs is still very challenging in the research area of porous organic materials. Their porosity‐based functions with diverse topological architectures in aqueous media offer promising platforms in bio‐related fields. This review highlights recent progress on water soluble or dispersible POPs for biomedical applications including bioimaging and biosensing, nanocarriers for drug delivery and tumor targeting, phototherapeutics, protein and gene delivery, biomacromolecule encapsulation and discrimination, and anti‐microbial activity.


INTRODUCTION
The porous organic polymers (POPs) have drawn extensive attention as a result of their structure diversity and broad applications over the past decades. Traditional POPs usually have porous structures in solid state and organic media, as represented by several typical subclasses including intrinsic microporosity (PIMs), [1] conjugated microporous polymer, [2] covalent triazine polymers, [3] porous aromatic frameworks, [4] and covalent organic frameworks (COFs). [5] These classic POPs in the solid state have been widely used in bio-related areas due to their tunable pore size, convenient functionalization, high loading capacity of drugs, relatively low cytotoxicity, and long blood circulation properties. Several recent reviews have comprehensively summarized their biomedical applications. [6][7][8][9][10] However, their heterogeneous feature may lead to potential phase separation in a heterogeneous manner, which is unfavorable for biomedical applications, especially for in vivo therapeutics. To tackle this issue, the creation of homogeneous porous materials in aqueous solutions is exceptionally desirable but remains a challenge synthetically. As highlighted in this article, many research groups demonstrate that the introduction of ionic backbones or hydrophilic side chains could endow POPs with water dispersibility or solubility. This has spawned a particular branch of POPs, the so-called water-soluble or dispersible POPs (ws-POPs), which enable the extension of porosity-based functions/applications to aqueous scenarios. [11] Compared to the POPs existed in solid state and organic media, ws-POPs hold great promise to display unique properties and advantages from their aqueous porous structures. This may attribute to their specific physicochemical features such as high surface area, well-defined pore size and pore engineering, stimuliresponsive absorption and release of guest molecules, negligible toxicity, variable post-synthesis modification strategies, as well as probably good biocompatibility, which are all important for biomedical applications. The ws-POPs have extended the concept of conventional POPs that are recognized as porous solid materials. In contrast to several recent reviews, which focused on the bio-applications of POPs in a heterogonous manner, this review highlights water-dispersible and water-soluble POPs that display their porosity-based functions in a more homogeneous way in aqueous solutions and offer new insights for their unique features for biomedical applications.
Water is the only medium for life, thus the water soluble porous materials are expected to display better biocompatibility than insoluble porous materials. To explore the potential bio-related applications is, of course, the most relevant motivation for developing ws-POPs. To endow the porous structures with water solubility and processability, two main strategies have been developed. For POPs that are not intrinsically soluble in water, the most widely used method is postmodification, [12] for example: introducing hydrophilic chains or functional groups to surface/edges of the materials. The generated porous structures in this way are usually dispersible in water; a long-term stability in aqueous media is also needed for achieving required functions. The second one is using water-soluble subunits for "bottom-up" fabrication to introduce hydrophilicity, [13] that is, using molecular building blocks with positive/negative charges and/or introducing supramolecular interactions. The resulted POPs in this way generally exhibited "intrinsic" water-solubility. This also gives the kind of organic porous materials with a "soft" or "flexible" property that generally exhibit versatile functions and applications in aqueous solutions or pure water. The construction strategies are based on two main types: (i) covalent or dynamic covalent bonds, (ii) noncovalent bonds, that is, coordination, host-guest interactions, and hydrophobicity. This review will focus on fabrication and biomedical applications of ws-POPs by utilizing their water soluble/dispersible porosity for bioimaging, drug delivery, tumor targeting and therapy, photothermal therapy (PTT), protein and gene delivery, biomacromolecule encapsulation, and discrimination, as well as antibacterial activity. In this review, the description of ws-POPs was divided into two sections, water-dispersible, and water-soluble POPs, and then related porous materials were classified into sub-sections based on their morphologies and synthesis/assemble strategies. We also summarize the representative structures and their preparation strategies and bio-related applications in Table 1. Last but not least, in the conclusion and outlook section, some remaining challenges in this rapidly growing field will be discussed. We anticipate that the compiled construction principles for ws-POPs in this review will inspire the future design of novel porous materials in aqueous phase with controllable structures and tailored biomedical applications.

WATER-DISPERSIBLE POPS
Traditional POPs, produced by discrete covalent or dynamic covalent coupling strategies, generally display inherent hydrophobicity. Usually, when mixed with water, this kind of POPs only forms a dispersion in a heterogeneous manner instead of a fully dissolved status. In this section, waterdispersible POPs are defined as POPs that are stably suspended in water, usually have nano-scale sizes, and do not aggregate or precipitate in water or aqueous media with a water content of >50%. [11] For achieving water solubility, covalent organic polymers with intrinsic porosity have to overcome the inherent hydrophobicity, which usually requires the modification with hydrophilic groups to their backbones, surface, or framework edges. Three practical approaches are commonly used to realize this aim including: (i) physical/chemical exfoliations, (ii) postmodification with hydrophilic groups, and (iii) size-controlled synthesis. These formed dispersible porous organic nanoparticles should have sufficient colloidal stability for further studies of their biomedical applications in aqueous media. In the following subsections, several representative works are highlighted and discussed.

Organic polymer nanosheets
The organic polymer nanosheets are one type of twodimensional (2D) covalent organic polymers, which hold great possibilities for bioapplications including bio-imaging, drug delivery, and photodynamic therapy (PDT), etc. [14][15][16] The postmodification methods have been used for construction of water dispersible 2D organic frameworks and nanosheets. In this section, we will discuss water-dispersible polymer nanosheets that have no confirmed periodicity. In 2011, Ying and coworkers synthesized a porous polyisocyanurate (PICU-A) 2D frameworks by the cyclotrimerization reaction of the 3,3′-dimethoxy-4,4′-biphenylene diisocyanate building blocks with an N-heterocyclic carbine catalyst. The yielded PICU material is amorphous with many modifiable terminal groups. [14] To improve the water solubility, they introduced the D-glucosamine moiety to form the modified nanosheets NS-Ac ( Figure 1A). The generated nanosheetacylation (NS-Ac) nanosheets could be used as nano-carriers for a hydrophobic dye molecule Nile red. And the delivery of Nile red into live cells has been demonstrated for bioimaging application ( Figure 1B), which was possibly driven by the noncovalent interactions and hydrophobicity. They also assessed the cytotoxicity of the nanosheets on HepG2 and KB cell lines using the methyl thiazolyl tetrazolium assay ( Figure 1B). As a special kind of 2D porous polymer nanosheets, porous boron nitrides and graphite carbon nitrides have been vigorously investigated. [16] However, the main challenge for biological applications results from their extremely poor solubility and poor processability in water. [17] In 2014, Golberg and coworkers reported a water-soluble, porous boron nitride material via postsubstitution on carbon atoms in graphitic carbon nitrides (g-C3N4) by boric acids. [18] The high degree of hydroxylation existed in the boron nitride materials enhanced the water solubility. The postmodified boron nitride materials exhibited good water solubility and low cytotoxicity, which were further used to load anti-tumor agent doxorubicin (DOX). The drug-loaded nanocarriers exhibited better viability reducing ability of prostate cancer cells than free drugs. Similarly, the other important approach for postmodification and functionalization of porous graphene materials was also reported by Wang et al. They found that the prepared porous graphene derivatives could form a stable black dispersions in aqueous solution via free radical oxidation. [19] No agglomeration was observed after 3 days of storage, which indicated that the dispersion was stable. The dispersed system was thus be further used for the separation of biomacromolecules
their versatile functions and applications. [5,10] However, the poor water solubility and limited aqueous-phase processability of this kind of periodic materials have considerably restricted their potentials for biomedical applications. For 2D COFs, exfoliation has been developed to afford few-layer and nano-sized COFs in water. Further, hydrophilic polymers or lipophilic surfactants were modified on the as-exfoliated COF layers to obtain water dispersions due to the avoidance of π-π stacking and hydrophobic aggregation. In 2017, Banerjee group used a three-step sequentially postsynthetic modification method to afford a simultaneously delaminated and functionalized nanosheets-like triphenoltrialdehyde-aminosalicylic acylhydrazide coupling (TpASH)-COF. The COF nanosheets were able to form dispersion in water and further be used to deliver a potent antitumor drug, 5-fluorouracil, to breast cancer cells ( Figure 2). [20] The drug-loaded COF nanosheets were further used to target human breast cancer cells, and the release of 5-fluorouracil could be achieved to reduce the viability of the cancer cells through a receptormediated endocytosis pathway. The Banerjee group also reported an example of self-exfoliated ionic covalent organic nanosheets (iCONs) with guanidinium groups. The intrinsic ionic character of the guanidinium segments contributed the iCONs with not only good water-dispersibility via selfexfoliation but also the antibacterial activity against Grampositive and -negative bacteria. [21] Noncovalent postmodification method to introduce hydrophilic groups was also used to construct waterdispersible COFs. In 2018, Jia et al. reported that an example of polyethylene glycol (PEG)-coated COF nanocomposites, which could be used as nanocarriers for drug delivery in vivo ( Figure 3). [22] The water-dispersible polymer-modified COF layers (PEG-CCM@APTES-COF-1) were fabricated via hydrophobicity-driven self-assembly between the aminefunctionalized COF (APTES-COF-1) and polyethyleneglycol modified curcumin derivatives (PEG-CCM). The pharmacokinetic modifier PEG-monofunctional curcumin (CCM) derivates functioned as a surfactant, which allows its hydrophobic part to attach on the surface of 3-aminopropyl triethoxysilane (APTES)-COF-1. They further observed that the biocompatibility was enhanced, and the blood circulation time was extended in the polymer-COF layers. The assembled PEG 2000 -CCM@APTES-COF-1 was able to load up to 9.7 wt% DOX. The ex vivo fluorescence imaging further evidenced that the PEG2000-CCM@APTES-COF-1 displayed a high drug accumulation on tumor site. For a brain tumor model, compared to the control groups, the drug-loaded PEG-CCM@APTES-COF-1 exhibited better tumor inhibition due to a higher tumor penetration and longer retention on tumor sites.
Water-dispersible COFs could also be achieved via a mechanical exfoliation combined with postmodification strategy and be applied for biomedical applications. An example of COF nanocomposites were prepared by Tian group through this method by first exfoliating COFs via ultrasound then followed with loading IR783 dye via hydrophobicity induced self-assembly. [23] Compared to the bulky COFs, the nanocomposites could be well dispersed in water with a decent solubility. The coassembly of the IR783 anionic dye with the COF nanocomposites not only endowed the COF nanocomposites with PTT but also improved the blood circulation and permeability. The The sequential postmodifications of TpASH-covalent organic framework (COF) to afford functionalized targeted covalent organic nanosheets (TpASH-CON). (B) Schematic representation of targeted drug delivery by the functionalized CONs. Reproduced with permission. [20] Copyright 2017, American Chemical Society The scheme illustration of the preparation of doxorubicin (DOX)-loaded polyethylene glycol (PEG)-CCM@APTES-covalent organic framework (COF)-1. Reproduced with permission. [22] Copyright 2018, Nature Publishing Group anticancer cis-aconityl-DOX prodrug could also be loaded to afford COF@IR783@CAD. The combination therapy realized by the COF@IR783@CAD displayed better tumor ablation effect than PTT or chemotherapy alone.

Covalent polymer nanospheres
Apart from the 2D morphologies, porous polymer nanospheres with intrinsic porosity and water dispersibility could also be used for biomedical applications. [24,25] In 2012, Wang group reported an surfactant-free onestep process to prepare water-dispersible porous polymer nanospheres (PPSs) via emulsion polymerization by Suzuki reaction. [26] The micelle-like nanospheres were formed by oil/water emulsion polymerization at the early stage via a dilute Suzuki reaction between tris(4-bromophenyl)amine and a diboronic acid monomer. During the reaction, the ∼10 nm nanoparticles were first precipitated in droplets and merged into aqueous media. Further aggregation and connection of these small individual nanoparticles gave rise to the formation of ∼200 nm porous nanoparticles ( Figure 4). Reproduced with permission. [26] Copyright 2014, American Chemical Society The uniform self-assembly of individual nanoparticles in water led to controlled secondary porous structures. The generated PPS was found to have a low cytotoxicity and could be used as a fluorescence probe by modification with dibromo-benzothiadiazole for bioimaging of L929 cells.
Recently, Ma and coworkers also reported the construction of water-dispersible pillar[n]arene-based nanospheres via a supra-amphiphilic template method. [27] The size of host molecule-based nanospheres was controlled by supraamphiphilic template method. Two dye molecules acridine orange and indocyanine green and two antitumor drugs DOX hydrochloride and mitoxantrone were used as guest molecules. Both the dyes and drugs could be encapsulated by host-guest interaction to the cavity of water-soluble pillar [6]arene as a host. The DOX-loaded nanospheres could overcome multidrug resistant (MDR) cancer cells (MCF-7/ADR) with a nine-fold reduction in the IC50 value in comparison with free drugs. The robust supramolecular encapsulation of antitumor drugs in water-dispersible nanospheres played a significant role to avoid drug efflux for overcoming MDR cancer cells as confirmed by a mechanistic study.

WATER SOLUBLE POPS
Creation of homogeneous porous organic materials in aqueous solutions is highly desirable but remains a great challenge synthetically. In contrast to water-dispersible POPs, watersoluble POPs were defined as homogeneous POPs that dissolve in water to form transparent solutions and do not display Tyndall effect. This means that the POPs do not exist as densely aggregating particles with detectable nanoscale sizes, which is expected to facilitate the explorations of their "designed" properties or functions. [11] One promising way to achieve this goal is creating supramolecular POPs, which are prepared through the self-assembly of molecular building blocks by reversible noncovalent interactions with high directionality. [28][29][30][31] This also provides a very powerful way to generate 2D mono-layer porous organic structures, which usually exhibit outstanding water solubility and solution processability. On the other hand, water-soluble threedimensional (3D) POPs represent another promising platform for bio-applications in aqueous media. The accessibility of such kind POPs could be achieved by using ionic backbones and/or hydrophilic side chains. The ionic character of polymers with intrinsic electrostatic repulsion also avoids their structural interpenetration. There water-soluble porous materials could be further used as biocompatible nanocarriers for the inclusion and delivery of drugs and bioactive molecules.
Several representative examples of the water-soluble POPs and their biomedical applications will be discussed in following sections. Additionally, some porous structures constructed via platinum coordination bonds were also found to have water solubility and could be further used to develop theranostic platforms and biomedicines. [32][33][34][35][36] However, they were generally regarded as organic-inorganic hybrid materials that are beyond the scope of this review.

Monolayer supramolecular polymers
As an emerging class of supramolecular polymers, the watersoluble porous supramolecular monolayers also demonstrated great potentials for biomedical applications. Recently,

S C H E M E 1
The structure of compounds 1-6 encapsulation of aromatic dimers into a host molecule cucurbit [8]uril (CB [8]) driven by hydrophobicity has been demonstrated as the most efficient binding motif for the formation of the so-called supramolecular organic frameworks (SOFs). [13,37,38] In this way, the 2D SOFs can be assembled in water, [39][40][41] which displayed great advantage for creating water-soluble porous monolayers. These self-assembled frameworks, assembled from rigid and preorganized precursors with CB [8], generally display good porosity with regularity or periodicity in aqueous media. The first example of water-soluble 2D monolayer SOF through self-assembly of CB [8] and compound 1, was reported by Li and coworkers in 2013 (Scheme 1). [42] Similarly, the donor-acceptor interaction, enhanced by the encapsulation of CB [8], could also be used for creation of 2D monolayer SOFs in water. [43] One example was reported by Li group that the donor-acceptor interaction between the naphthelene units of 3 or 4 and BIPY 2+ units of 2 could be significantly enhanced by encapsulation within the cavity of CB[8] (Scheme 1). As a result, the strategy provides a novel way to integrate three-component into one 2D SOF system. [44] Although 3 and 4 have a conformational flexibility, the regularity of the two 2D SOFs was maintained in aqueous solution, which was evidenced by synchrotron solution-phase small angle X-ray scattering (SAXS). Notably, both 2D SOFs displayed antibacterial activity against methicillin-resistant Staphylococcus aureus, which could be attributed to the enrichment of cationic groups and the increase of affinity to the bacteria cell surface.
For the development of water-soluble 2D SOF with aggregation induced emission (AIE) effect, Li and coworkers prepared a tritopic compound 5 (Scheme 1) with three pyridiniumethylene substituted arms encapsulated in the cavity of CB [8]. [45] This 2D SOF was also found biocompatible and exhibited an enhanced AIE effect in water. With the addition of DNA, the photoluminescence of the 2D SOF could be significantly enhanced, which could be used for intracellular DNA imaging of HeLa cells.
In 2019, Liu et al. reported that a monolayer polypseudoroxatane-type nanostructure could be formed by assembly from CB [8] with a triphenylamine derivative 6 (Scheme 1) in water. [46] The styrylpyridinium units of 6 could further undergo quantitative [2+2] photodimerization in the cavity of CB [8] to form a covalent-bond-linked network with good water solubility. The polypseudoroxatanetype supramolecular monolayer could be further used as a template to form water-soluble porous covalent organic monolayer via quantitative [2+2] photodimerization. With a pore diameter of 3.4 nm, this porous monolayer could trap C 60 in water. The resulting composites exhibited an excellent PDT effect that could cleave the DNA of cells by reactive oxygen species generated by photo-irradiation ( Figure 5).
The tetraphenylethene (TPE) has been widely used as a building block to create aromatic scaffold with AIE effect. [47] In 2018, Cao et al. prepared two TPE derivatives 7a and 7b and investigated their coassembly with CB [8] in water (Scheme 2 and Figure 6). [48] Both 7a and 7b mixtures with CB [8] led to the formation of monolayer 2D SOFs in water. One of them was characterized to be planar, whereas the other one was characterized to be curved due to the structural flexibility of the arms of 7b. Remarkably, compared to their monomers, the two SOFs exhibited a significant red-shifting of their emission peaks and displayed a stimuli-responsive turn-off/turn-on property of fluorescence, which could be further applied for cellular imaging. The same group also reported a preparation of compound 8 with four coumarin units (Scheme 2). [49] As linking units, two coumarin units could be encapsulated in the cavity of CB [8] to afford 2D SOF. And the SOF was further demonstrated for peptide/protein discrimination with the modification of L-/D-phenylalanine to the edge of the organic framework. Anticlockwise-typed M-SOF or clockwise-typed P-SOF-1 with circular dichroism (CD) signals and circularly polarized luminescence could be achieved form the adaptive chirality of the 2D SOF in water, which may attribute to the dynamic TPE conformation of compound 8. By inducing different biomolecules to display characteristic CD spectra, the 2D SOF could discriminate different chiral biomolecules such as dipeptide sequences, that is, Phe-Ala and Ala-Phe, and polypeptides and proteins, that is, somatostatin and human insulin.

SOFs
Water-soluble 3D POPs also hold great potentials as biocompatible carriers for biomedical applications, for example, they could be efficient platforms to load and deliver drugs and biomacromolecules. Their water-soluble homogeneous porosity could provide several typical unique features, such as reversible guest molecule adsorption and release, avoidance of structural interpenetration and channel blockage, rapid realization of thermodynamic equilibrium, uniform distribution of adsorbates, and versatile pore structures. Additionally, porous materials with good solubility also hold great promise for decent biocompatibility due to the absence of in vivo phase separation and aggregation. As a class of supramolecular polymers, SOFs feature themselves with regular or periodic porosity in 2D or 3D dimension. Because SOFs are constructed from organic molecules through  [8], and C60@(TPA-SP PD ⊂CB [8]) with or without white light irradiation to demonstrate their photodynamic therapy (PDT) activity. Reproduced with permission. [46] Copyright 2019, Royal Society of Chemistry noncovalent bonds, conceptually we may regard SOFs as extension of conventional POPs, [11] which allows for the investigations of porosity-based functions or applications of homogeneous organic polymers in water. As a typical representative, the so-called water-soluble 3D SOFs with regular porosity have been demonstrated to display remarkable advantages as ideal platforms for bio-applications.
The solution-phase self-assembly process and their ionic character are also helpful to avoid the interpenetration of the networks. [50] The first example of water-soluble 3D SOF via selfassembly of 1:2 mixture of tetrahedral compound 9a and CB [8] in water was reported by Li group in 2014 (Scheme 3). [51] The porosity periodicity of the 3D SOF F I G U R E 6 (A) Formation of planar and curved 2D supramolecular organic frameworks (SOFs) by 7a and 7b and cucurbit [8]uril (CB [8]) (1:2) in water. (B) The illustration of stimuli-responsive turn-off of fluorescence by the coassembly of 7b and CB [8] to form 2D SOF and the turn-on by the addition of 3,5-dimethyl-1-adamantylamine•HCl (Me2ADA) to the 2D SOF. Reproduced with permission. [48] Copyright 2018, Wiley−VCH

S C H E M E 2
The structure of compounds 7-8 could be evidenced by various solution-phase techniques, such as XRD and SAXS. Based on the experimental data, the computational modeling analysis also displayed that this 3D SOF may have a diamondoid topology with defined 2.1nm aperture of pores. As a special kind of 3D supramolecular cationic polyelectrolyte, this periodic SOF could adsorb anionic dyes, drugs, oligopeptides, oligonucleotides, and dendrimers as an ionic sponge in water. Using anionic drugs as models, Li group further reported that the 3D SOFs could release adsorbed drugs as triggered by an acidic atmosphere in water. Particularly, the time-independent absorption for all guest molecules further rationally reflected the homogeneity of the water-soluble 3D SOFs.
Li group further reported that compounds 9b-9e could also be assembled with CB [8] to afford water-soluble 3D with similar diamondoid structures. [52,53] At physiological pH of 7.4, these porous SOFs could instantaneously adsorb the anticancer drug DOX with a 13-17 wt% loading capacity in water driven by hydrophobicity. [52] The SOFs could overcome the multidrug resistance and deliver DOX into human breast cancer cells (MCF-7/Adr). The acidic microenvironment of lysosome in cells and cancer tissues could trigger the release of DOX from the SOFs. The drug-loaded SOFs significantly improved the cytotoxicity of DOX for the MCF-7/Adr cells and tumors as evidenced by in vitro and in vivo experiments. Compared to naked DOX, the DOX-loaded SOF nanocar-riers displayed a higher efficiency for cancer cell killings with a 13-19-fold IC 50 decrease. In 2017, Li and coworkers also reported that a clinically used chemotherapeutic agent dianionic pemetrexed (PMX) could be absorbed to the SOF drug delivery systems with a 20-23 wt% loading capacity driven by both electrostatic interactions and hydrophobicity in water. [53] The as prepared drug-loading SOFs were highly stable in water, which could maintain a significant amount of drugs in the interior of the SOFs during plasma circulation. Remarkably, they found that the PMX@SOFs could enter the cancer cells and further be used to overcome multidrug resistance of a breast cancer MCF-7/Adr cells. The release of drug in cancer cells could be triggered by the acidic microenvironment of cancer cells with a considerable improvement of the efficacy of PMX as evidenced by 6-12-fold IC 50 decrease compared with individual PMX. This new strategy for drug loading and delivery exhibited an outstanding advantage to omit the complicated preloading processes required by many reported and clinically used drug delivery systems based on nanomaterial platforms. Thus, it holds promising potentials for future creation of low-cost drug delivery systems.
In 2019, Li and coworkers also reported that 3D SOFs formed by 9a-9f and CB [8] were able to include oligonucleotides of 21, 23 or 58 nt with 12-70 wt% loading capacity and effectively deliver them into normal and cancer cells. [54] Among the 126 delivery tests, the delivery efficiencies of 98 cases surpassed that of Lipo2000, one famous and commercially used standard for delivery system. The mechanism of the inclusion of DNA molecules into SOFs could be rationally explained by the electrostatic attraction and hydrophobicity in aqueous media. An endocytosis mechanism was proposed, which involved diffusion-caused intracellular DNA delivery and SOF degradation-caused DNA release (Figure 7). [54] A compound 10 with four quinoline groups as appended aromatics could also assemble with CB [8] to produce another 3D SOF. [54] These 3D SOFs have a pore size of 2.0-2.2 nm, which is comparable with that of dsDNA (∼2.0 nm). [51] The fact that the SOFs could include and deliver dsDNA may be attributed to the dynamic feature of the supramolecular frameworks.
More recently, Ma and coworkers reported that the watersoluble 3D SOFs formed by 9a, b, g, and CB [8] could coassemble with a hydrophobic PDT agent temoporfin to form uniform nanoparticles in water. [55] The self-assembly structures led to an alleviation of the aggregation-caused quenching effect of temoporfin as evidenced by monitoring the generation of singlet oxygen. The SOF-based systems S C H E M E 3 The structure of compounds 9 and 10 F I G U R E 7 (A) Schematic representation of the in situ loading and delivery of DNA by polycationic 3D supramolecular organic frameworks (SOFs) formed by compounds 9a-9f and cucurbit [8]uril (CB [8]) (1:2) in water. (B) Confocal laser scanning microscopy (CLSM) images of 293A cells after incubation with Cy5-ssDNA-21, Cy5-ssDNA-21⊂Lipo2000, and Cy5-ssDNA-21⊂SOF. Reproduced with permission. [54] Copyright 2019, Chinese Chemical Society were found to have enhanced PDT effect both in vitro and in vivo and display good biocompatibility.

Flexible organic frameworks
Taking the advantage of flexible linkers and preorganized tetrahedral nodes, the desired water-solubility could also be achieved for the resulting organic frameworks. Recently, Li group successfully constructed the so-called flexible organic frameworks (FOFs) through the utilization of preorganized tetrahedral building blocks linked by dynamic covalent bonds. [56][57][58] The FOFs constructed in aqueous phase, differ from traditional crystalline COFs that formed in solid-state, generally display intrinsic homogeneous porosity in water with good solution processability. The FOF1a-d were synthesized through the coupling of the aldehyde group of 11a and the hydrazine group of 12a-d to form hydrazone in water ( Figure 8). [56] The coupling reactions underwent a nearly quantitatively (≥97%) process as the diagnostic proton signal of the formyl group of 11a vanished completely in 1 H NMR. These FOFs have a dynamic range of hydrodynamic diameters from 50 to 120 nm as evidenced by dynamic light scattering (DLS) experiments. Molecular modeling suggested a 3D diamondoid-styled porous structure with an aperture size of 5.3 or 6.7 nm. The large pores of the FOFs with both ionic and hydrophobic inner domains were found to efficiently accommodate proteins, such as green and orange fluorescent proteins, bovine serum albumin, and deliver them into normal and tumor cells, with an efficiency value up to 99.8%. Li group further reported that the coupling of 11b with 12a-d could form another series of FOFs as FOF2a-d ( Figure 8). [57] The hydrodynamic diameters of these FOFs range from 68 to 167 nm in aqueous media as evidenced by DLS data. These FOFs displayed an aperture of ca. 4 nm in diameter. Fluorescence imaging and flow cytometry analysis further showed that FOF2a-d could in situ efficiently load single and double strand DNA of 21 nucleotides. The FOFs displayed an efficient intracellular delivery for normal and tumor cells up to a percentage of 99.5%. Both of them exhibited relatively low cytotoxicity and decent biocompatibility. Using the similar strategy, the same group further reported that water-soluble FOFs could be used as platforms for creating FOF-conjugated prodrugs. [58] The new FOF3e was synthesized through a reaction of compound 11b with an citric acid derivated trihydrazine linker 12e with an 2:1 molar ratio in aqueous solution. Once prepared, the FOF3e displayed a significant water-solubility as no precipitates were observed after 2 weeks for the FOF solution. As monitored by 1 H NMR, 96% of the aldehyde group on 11b was converted into hydrazine, thus the FOFs would still allow for ca. 4% of its acylhydrazine units to be conjugated with drug molecules. Four antitumor anthracyclines, DOX, daunorubicin (DNR), epirubicin (EPI), and pirarubicin (THP), were then conjugated to the free acylhydrazine group of FOF3e via a hydrazine bond located at C-13 keto position of anthracyclines. The prodrug-bearing FOFs were further studied on their therapy effect for overcoming the multidrug resistance of MCF-7/ADR tumor cells, and they exhibited an important increase of inhibitory performance for killing MCF-7/ADR tumor in-vivo compared with individual drugs. This approach also provided a promising platform for loading and delivery of other antitumor drugs to generate FOF-prodrugs with the further discovery of novel applications by exploration of more FOF structures. Very recently, Li group reported that FOF4 and FOF5 could be prepared from tetratopic precursors 13, 14, and ditopic precursor 12a through the quantitative formation of the hydrazone bond at room temperature ( Figure 9A). [59] These two FOFs were found to adsorb bacterial lipopolysaccharides (LPS, endotoxins) with a deactivation efficacy of up to ca. 80% as evidenced by chromogenic limulus amebocyte lysate experiments ( Figure 9B). The detoxification effect of FOFs was further assessed, both FOF4 and FOF5 could reduce the toxicity of LPS as shown in cytokine inhibition experiments with RAW264.7 cells (Figure 9C,D). The in vitro and in vivo experiments demonstrated that the FOFs have good biocompatibility and can detoxify the LPS through efficient encapsulation in water. This provides a novel sequestration strategy to neutralize the toxicity of LPS based on the water-soluble POPs with cationic backbones.

CONCLUSION AND OUTLOOK
This review summarizes the ws-POPs with intrinsic watersoluble or water-dispersible internal porosity for biomedical Reproduced with permission. [56] Copyright 2020, American Chemical Society applications. Both covalent and noncovalent construction strategies could be used for the production of ws-POPs, among which several typical postmodification and assembly methods also strongly facilitate their structural diversity and thus enable the versatile functions for biomedical applications. For example, the intrinsic hydrophilic building blocks with positive charges and supramolecular assembly with CB [8] were utilized to generate homogeneous water-soluble porous structures in water. For their bio-related functions and biomedical applications, many examples have been demonstrated, which include bioimaging, drug delivery, tumor targeting and therapy, PTT, protein and gene delivery, biomacromolecule encapsulation, and discrimination, as well as antibacterial activity. The bio-related functions are expected to be further explored and expanded for the future development of ws-POPs. A couple of points need to be taken into consideration for future research. For the development of novel bioimaging platforms, the ws-POPs may hold a favorable place. We expect that various bioimaging probes can be readily absorbed into the interior of designed or embedded via rationally covalent postmodification. In principle, this water-soluble porous POP-based strategy may overcome the stacking or aggregation of organic dye-derived imaging reagents. The preliminary established approaches for construction and characterization of the ws-POPs have paved the way for the achievement of the above potentials, but more advanced methods need to be explored to offer a deeper and broader understanding of the structure-function relationship for bio-applications in vivo or in vitro. The inclusion and delivery of 3D POPs, particularly SOFs, has also been demonstrated. As porous nanocarriers for drug delivery, the reported water-soluble SOFs feature an in situ loading process, which remarkably simplifies complicated drug loading or entrapment required for clinically used liposome nanocarriers. However, the controlled release of bioactive loaded components, that is, drugs and biomacromolecules, is still a challenge. Introduction of well-established stimuliresponsive strategies, including photo, pH, and in vivo enzyme, may find a way to address this issue. The dynamic covalent bonding, such as imine, hydrazone, and disulfide, also provide attractive solutions. In particular, the release of drugs from the ws-POPs could be achieved by framework decomposition via S-S bond exchange with high level of glutathione in the tumor microenvironment. The in vivo metabolism of ws-POPs, for example, the blood circulation and organ clearance, play a critical role for future exploration of practical biomedical applications. Current studies suggest that the ws-POP nanoparticles could display a long blood circulation time and stimulus-responsive properties to micro-environments of cancer cells. The water-soluble POPs featured with homogeneous pores may have a lower agglomeration chance and quicker transportation in vivo than bulky POPs. As constructed via noncovalent bonds, the SOFs are expected to have a more effective dissolution or degradation pathway for organ clearance than covalent polymers. F I G U R E 9 (A) Synthesis of two types of hydrazone-based flexible organic frameworks (FOFs) in water. (B) The absorption of lipopolysaccharides (LPS) by FOFs quantified by chromogenic limulus amebocyte lysate assay in water. Suppression of (C) LPS-induced tumor necrosis factor-α (TNF-α) and (D) interleukin-6 (IL-6) expression in RAW264.7 cells by FOFs. Reproduced with permission. [59] Copyright 2022, American Chemical Society However, the systematical studies for the in vivo metabolism of ws-POPs are still very rare. The development of standard procedures for in situ, real-time, and quantitative analysis, and further reveal of the structure-function relationship for ws-POPs will pave the way for future in vivo metabolism studies. The constructions strategies of water-soluble POPs, which display homogeneous porosity in aqueous media, are still very limited. For example, the CB [8] intermolecular encapsulation is still the only workable way for the construction of SOFs. More host molecules with excellent biocompatibility and water-solubility should be taken into consideration. The γ−cyclodextrin (CD) and its derivatives, as natural products, may be another kind of candidate for achieving the encapsulation-enhanced interactions and open more opportunities to extend the structural diversity of supramolecular frameworks. The porous properties of ws-POPs in aqueous solution are significantly different from those in solid state as seen in many cases. [36,42,51] Their porous structures may suffer from collapse after the removal of solvents or occupation of counterions. Thus, the parameters measured by Brunauer-Emmett-Teller analysis are usually not applicable to reflect porous properties in solution. As there is not yet a wellestablished method to characterize the porosity in aqueous solution, the development of universal standards for this aim is highly needed in future research.
In summary, the research interest in ws-POPs is rapidly growing, which will continuously unlock their full potentials, the biomedical and other bio-related applications. Biomolecule-conjugated ws-POPs will in theory have the best opportunity for development of future physiologically compatible nanomedicines. As this research area is still in their infant stage, biocompatibility and toxicological risk assessment should be systematically tested for using ws-POPs as nano-theranostics. The potential capacity for using ws-POPs as practical bio-materials needs to be further explored in future clinical trials and finally by the market.

A C K N O W L E D G M E N T S
The authors thank the National Natural Science Foundation of China (grant numbers: 21921003, 21890732, and 21890730) for financial support of this work.