A phenazine-linked π-conjugated covalent organic framework for conjugation-driven drug loading

Abstract

The rational design of π-conjugated covalent organic frameworks (COFs) represents a promising frontier in functional porous materials for drug delivery, particularly when conjugation–affinity correlations can be harnessed. Herein, we report the synthesis and characterization of a structurally unique phenazine-linked π-conjugated COF (TU-32) constructed from 2,7-di-tert-butylpyrene-4,5,9,10-tetraone and 9,10-dihydro-9,10-[1,2]benzenoanthracene-2,3,6,7,14,15-hexaamine hexahydrochloride. In contrast to conventional 2D COFs that exhibit π–π stacking, this COF adopts an atypical AB stacking mode along the c-axis, resulting in suppressed interlayer π-stacking and enhanced structural regularity. The incorporation of extended π-conjugation through phenazine linkages enables selective interactions with conjugated drug molecules. Among three drug molecules tested—5-fluorouracil, isoniazid, and captopril—the COF demonstrated the highest loading capacity (56 wt%) for 5-fluorouracil, which features a fully conjugated pyrimidine-like ring, followed by isoniazid (54 wt%), which contains a moderately conjugated pyridyl moiety. In contrast, captopril, which lacks significant π-conjugation, showed a lower loading (36 wt%). Our findings underscore the importance of molecular-level π–π interactions in drug encapsulation and highlight how precise framework engineering via π-conjugated building blocks enables conjugation-driven guest affinity, offering key insights and a design blueprint for next-generation conjugated porous frameworks for precision therapeutic delivery.

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New concepts

We introduce a new design principle—conjugation-driven drug encapsulation—in a structurally unique new phenazine-linked π-conjugated covalent organic framework (COF), TU-32. Unlike conventional COFs, TU-32 features an AB-stacked architecture that suppresses interlayer π–π interactions and exposes open, π-aligned channels. This enables selective and high-capacity loading of conjugated therapeutic molecules via π–π complementarity. Our study provides a unique demonstration of using extended π-electron systems in COFs to guide guest selectivity and release behavior, offering a new paradigm in the design of smart nanocarriers. These findings open avenues for π-system-engineered porous materials in drug delivery, molecular recognition, and responsive nanotechnology.

Introduction

Porous organic frameworks¹—particularly covalent organic frameworks (COFs)^2–16—have emerged as a rapidly advancing class of crystalline, porous materials that offer unprecedented modularity and designability at the molecular level. Among them, π-conjugated COFs have garnered growing attention due to their potential in diverse fields such as organic electronics, photocatalysis, and molecular separations.¹⁷ The integration of extended π-systems into the framework backbone allows for long-range charge delocalization, photophysical tunability, and chemical robustness. Seminal examples include early COFs based on planar aromatic aldehydes and amines that enabled the formation of 2D π-sheets such as the imine-linked architectures developed by Yaghi et al.,¹⁸ as well as conjugated frameworks from Dichtel's group exhibiting high capacitance and energy storage performance.¹⁹ More recently, strategies involving phenazine- and phenanthroline-based COFs have demonstrated enhanced redox activity and π–π interactions for catalytic and optoelectronic applications.^20,21 Phenazine units, with their electron-rich nitrogen heterocycles, not only extend conjugation but also serve as hydrogen-bond acceptors and redox-active centers, enabling fine-tuning of host–guest interactions. Through precise framework engineering using π-conjugated building blocks, it is now possible to access structurally robust COFs with tailor-made electronic properties and guest affinities, opening avenues for selective adsorption, catalysis, and most recently, conjugation-driven drug delivery.

The concept of conjugation-driven drug loading—whereby π–π interactions between the framework and aromatic or conjugated drug molecules dictate encapsulation efficiency—is rapidly gaining traction in precision therapeutic delivery. Traditional COF-based drug carriers^22–25 often rely on hydrogen bonding or electrostatic interactions;²⁶ however, these are typically non-directional and nonspecific. In contrast, π–π stacking and donor–acceptor interactions between the COF and aromatic drug molecules offer enhanced specificity, stability, and loading capacity. Notable work in this area includes a highly π-conjugated, fluorescent COF that enables direct visual monitoring of doxorubicin loading and achieves responsive release—demonstrating how extended conjugation within the framework can both encapsulate and signal drug uptake in real time.²⁷ Similarly, conjugated frameworks with nitrogen-rich structures and extended π-systems have been developed to enable high drug loading capacities, where the strong π-conjugation enhances fluorescence and facilitates robust interactions with therapeutic agents such as glucose oxidase and L-arginine,—supporting efficient cellular uptake and enabling synergistic starvation/gas cancer therapy with demonstrated in vivo efficacy.²⁸ Despite these advances, few studies have explicitly correlated the degree of π-conjugation within the framework with drug loading performance, nor explored the role of stacking geometries (e.g., AA vs. AB stacking) in governing guest affinity. There remains a critical knowledge gap in understanding how molecular-level conjugation can be harnessed as a design principle for intelligent drug delivery platforms.

In this work, we report a structurally distinctive phenazine-linked π-conjugated COF, TU-32 (TU = Tohoku University) synthesized from 2,7-di-tert-butylpyrene-4,5,9,10-tetraone (DTPT) and 9,10-dihydro-9,10-[1,2]benzenoanthracene-2,3,6,7,14,15-hexaamine hexahydrochloride (DBAH). Unlike conventional 2D COFs that predominantly adopt eclipsed AA stacking to maximize interlayer π–π overlap, TU-32 features a slipped AB stacking arrangement along the c-axis. This configuration not only reduces direct interlayer π-stacking interactions but also creates expanded diffusion pathways and alleviates steric hindrance at pore entrances, thereby improving molecular accessibility for guest loading. While AB or staggered stacking motifs have been reported in other COFs,²⁹ such arrangements remain relatively uncommon, particularly in the context of functional applications like drug delivery, where their influence on host–guest chemistry remains underexplored. In TU-32, this combination of structural openness and extended π-conjugation introduced via phenazine linkages enhances guest–host interactions with conjugated drug molecules, as demonstrated by the preferential loading of 5-fluorouracil (5-FU) over non-conjugated drugs (Fig. 1). Comparative drug loading experiments reveal that this conjugation-enabled affinity directly governs selective encapsulation, with TU-32 showing superior capacity and sustained release performance for conjugated therapeutics. This study not only introduces a new structurally and electronically distinctive COF, but also offers fundamental insights into conjugation-driven host–guest chemistry in porous frameworks for targeted drug delivery applications.

Fig. 1 Schematic illustration of the construction of the phenazine-linked π-conjugated COF, TU-32, via polycondensation between DTPT and DBAH, and its selective adsorption behavior toward conjugated drug molecules, as demonstrated by significantly higher uptake of 5-FU and isoniazid, in contrast to the lower affinity observed for the non-conjugated drug captopril.

Experimental

Synthesis of TU-32

A mixture of DTPT (16.84 mg, 0.045 mmol) and DBAH (16.89 mg, 0.030 mmol) was transferred to a Pyrex tube (inner diameter: 8 mm; outer diameter: 10 mm). To this, a 1 : 1 (v/v) solvent mixture of anhydrous mesitylene (0.5 mL) and 1,4-dioxane (0.5 mL) was added. The suspension was subjected to ultrasonication for 15 min to ensure thorough dispersion of the monomers. Afterward, 0.2 mL of 12 M aqueous acetic acid was introduced, and the mixture was further sonicated for an additional 15 min. The reaction tube was flash-frozen in a liquid nitrogen bath (77 K), evacuated to an internal pressure below 0.2 mbar, and flame-sealed under vacuum. The sealed tube (reduced to ∼9 cm in length) was allowed to warm to ambient temperature and then heated at 120 °C for 3 days. After the reaction period, the tube was cooled to ambient temperature. The resulting solid was recovered by centrifugation, thoroughly washed with THF until the supernatant was clear, and purified further via 24 h Soxhlet extraction in THF. After initial air-drying, the solid was dried under vacuum at room temperature for 5 h and then activated at 100 °C under high vacuum overnight, yielding TU-32 as a pale brown powder (76% yield). Anal. calcd for C₂₂₄H₁₈₄N₂₄: C: 84.32; H: 5.14; N: 10.54. Found: C: 67.38; H: 5.60; N: 10.97.

Drug loading

Activated TU-32 (60.0 mg) was immersed in 30.0 mL of a 0.05 M aqueous solution of 5-FU or isoniazid (0.1 M for captopril), and the mixture was gently stirred at ambient temperature for 4 h. Drug-encapsulated COF samples were recovered by vacuum filtration, washed extensively with DI water, and dried under reduced pressure.

Drug release

Drug release studies were performed by immersing semipermeable dialysis membranes containing 30.0 mg of drug-loaded COF into 10.0 mL of phosphate-buffered saline (pH 7.4, mimicking physiological conditions) and incubated at 37 °C in a thermostated water bath. Periodically, 10.0 mL of the medium was withdrawn and substituted with an equal volume of fresh buffer. The concentration of released drug was quantified using UV-vis spectroscopy based on standard calibration plots. The results are expressed as the average of three independent measurements ± standard deviation.

Results and discussion

Preparation and characterization of TU-32

The strategic combination of C₂-symmetric DTPT and D_3h-symmetric DBAH linkers enables a [3+2] topological ring fusion reaction, wherein each tritopic DBAH unit connects to three ditopic DTPT monomers through orthogonal condensation at the ortho-diketone and ortho-diamine positions, generating extended two-dimensional π-conjugated networks via phenazine linkages. The use of DTPT, a rigid planar pyrene core functionalized with electron-deficient carbonyl moieties, facilitates the formation of highly conjugated phenazine bridges upon condensation with DBAH's electron-rich amine functionalities. The tert-butyl substituents at the 2,7-positions of DTPT were purposefully incorporated to improve the solubility of the monomer in nonpolar organic solvents and mitigate excessive aggregation during polymerization, thus promoting uniform nucleation and crystal growth. Additionally, these steric bulks may serve to subtly modulate the interlayer stacking by introducing spatial hindrance, thereby favoring an offset (AB-type) stacking geometry and contributing to the structural order and accessibility of the porous channels. Fourier-transform infrared (FT-IR) spectroscopy provided key evidence for the successful formation of phenazine linkages in TU-32. The spectrum of the final COF (Fig. 2a, sky-blue curve) displayed characteristic vibrational bands at 1566, 1454, and 1366 cm⁻¹, which are consistent with C=N and C–N stretching modes typical of the extended phenazine core, confirming the cyclocondensation between the carbonyl and amine functionalities. In contrast, the N–H stretching bands of DBAH at 3404, 3346, and 3221 cm⁻¹ (red curve) were markedly reduced, indicating the consumption of terminal –NH₂ groups during framework formation. Likewise, the C=O stretch of DTPT at 1677 cm⁻¹ (blue curve) was also suppressed, signifying effective incorporation of the diketone into the fused aromatic system. Notably, the vibrational bands at 2961, 2905, and 2868 cm⁻¹, attributed to the methyl stretching modes of the tert-butyl substituents on the DTPT unit, remained intact, suggesting that these peripheral alkyl groups were preserved during polymerization and did not participate in the condensation reaction. As shown in the solid-state ¹³C cross-polarization magic angle spinning (CP/MAS) NMR spectrum of TU-32 (Fig. 2b), the resonances at 30.1 and 34.7 ppm are attributed to the methyl and quaternary carbon atoms of the tert-butyl groups in the DTPT units, while the signal at 52.7 ppm corresponds to the bridgehead aliphatic carbon of the bicyclo[2.2.2]octane moiety in the DBAH linker. The aromatic region features distinct peaks at 109.2, 128.1, 143.1, and 151.8 ppm, corresponding to various sp²-hybridized carbon environments across the extended π-system of the fused aromatic backbone. Notably, the signal at 128.1 ppm is characteristic of quinone-derived carbon atoms involved in phenazine formation, where conjugation with adjacent nitrogen atoms deshields the carbon nuclei, shifting them downfield. The additional peaks at 109.2, 143.1, and 151.8 ppm can be attributed to internal aromatic carbons within the newly formed polycyclic phenazine network. Importantly, the spectrum lacks signals in the 190–200 ppm range, characteristic of unreacted carbonyl carbons from DTPT, as well as in the 40–60 ppm range typical of α-carbons adjacent to free amines in DBAH, confirming complete consumption of the monomers and successful construction of the covalently fused aromatic framework. The calculated elemental composition assumes a fully condensed, guest-free framework (C₂₂₄H₁₈₄N₂₄), whereas the lower experimental carbon content (67.38% vs. 84.32%) and slightly higher hydrogen and nitrogen values likely result from the use of DBAH in its hexahydrochloride salt form, where residual chloride counterions may remain associated with the framework or trapped in the pores. Additionally, the presence of partially condensed or protonated amines, as well as retained solvents may also contribute to the observed discrepancy. The morphology of TU-32, as observed by scanning electron microscopy (SEM) (Fig. 2c), reveals a lamellar, plate-like architecture composed of stacked nanosheets with well-defined edges and relatively uniform lateral dimensions. High-resolution transmission electron microscopy (HRTEM) images of TU-32 (Fig. 2d, e and Fig. S1) reveal distinct and periodic lattice fringes, indicative of long-range structural order within the crystalline framework, while the corresponding fast Fourier transform (FFT) patterns display well-defined diffraction spots arranged periodically, further corroborating the high crystallinity and structural fidelity of the COF. The thermogravimetric analysis (TGA) profile of TU-32, pre-activated at 100 °C, reveals a three-step weight loss pattern indicative of distinct thermal events associated with the COF structure (Fig. S2). An initial ∼3% weight loss from room temperature to 100 °C is attributed to the desorption of physisorbed water and residual solvents trapped within the porous framework, facilitated by polar interactions with nitrogen-rich phenazine units. A second weight loss of ∼10% occurring between 100 °C and 350 °C is likely due to the thermal cleavage of tert-butyl side groups on the DTPT linker. The final major weight loss of ∼30% from 350 °C to 605 °C marks the onset of structural decomposition of the extended π-conjugated backbone, including the phenazine, pyrene, and anthracene units, affirming the high thermal stability of the crystalline COF architecture. Besides, TU-32 demonstrated notable chemical stability, as evidenced by the retention of its crystallinity following 24-hour immersion in a range of organic solvents (Fig. S3).

Fig. 2 (a) FT-IR spectra of TU-32 and the linkers DBAH and DTPT. (b) Solid-state ¹³C CP/MAS NMR spectrum of TU-32. (c) SEM image of TU-32. (d) and (e) HRTEM micrographs of TU-32 with FFT insets derived from regions highlighted by red boxes.

Crystal structure and stacking analysis

The crystalline structure of TU-32 was elucidated through a combination of experimental PXRD analysis, structural modeling, and refinement protocols (Fig. 3). Geometry optimization of the proposed framework was performed using the Forcite module in Materials Studio 7.0,³⁰ applying a force-field-based energy minimization approach to derive a thermodynamically favorable configuration. Among the different stacking sequences tested, the AB stacking model within the P1 space group provided the best agreement with the experimental data, yielding optimized unit cell parameters of a = 27.1141 Å, b = 27.3224 Å, c = 22.4717 Å, and angles α = 90°, β = 90°, γ = 119.46° (Table S2). The simulated PXRD pattern based on this AB-stacked structure (black trace) showed strong correlation with the experimental PXRD data (red trace), as shown in Fig. S5. The experimental PXRD profile displayed distinct Bragg reflections at 3.64°, 5.41°, 6.69°, 7.18°, 7.53°, 8.13°, 8.72°, and 10.04°, which were indexed to the (010), (101), (110), (020), (200), (021), (012), and (120) crystal planes, respectively. Pawley refinement of the experimental PXRD pattern yielded refined unit cell parameters of a = 27.1194 Å, b = 27.3289 Å, c = 22.4736 Å, α = 90°, β = 90°, γ = 119.98°, with low agreement residuals (R_p = 1.77%, R_wp = 2.83%), further validating the structural model. Simulations based on alternative AA and ABC stacking arrangements were also explored using identical symmetry constraints (Fig. S8, S10 and Tables S4, S5), but the corresponding PXRD profiles exhibited significant mismatch with experimental data (Fig. S7 and S9), ruling them out as plausible configurations. Taken together, the evidence supports a predominantly AB-like stacking mode with possible local disorder, which likely arises from interlayer slippage or turbostratic misalignment—common in 2D COFs. This arrangement yields an interlayer distance of 0.59 nm and reflects a slipped-layer configuration that reduces π–π overlap while maintaining overall crystallinity and periodic order (Fig. S6).

Fig. 3 XRD patterns of TU-32 showing experimental data (red dots), Pawley-refined fit (black), simulated pattern (pink), and the difference curve (blue) between the experimental and refined profiles. Green vertical markers indicate the Bragg reflection positions.

Gas uptake behavior and porosity evaluation

The porosity of TU-32 was systematically characterized via nitrogen adsorption–desorption isotherm measurements at 77 K across a relative pressure (P/P₀) range of 0 to 1, following thorough degassing at 120 °C for 8 hours to remove residual solvents (Fig. 4a). The resulting isotherm displayed a typical type I profile, characterized by a steep increase in nitrogen uptake at very low P/P₀ values (<0.01), indicative of permanent microporosity. This was followed by a plateau region, consistent with saturation of the micropores, a hallmark of well-defined, rigid microporous frameworks. Multi-point Brunauer–Emmett–Teller (BET) analysis of the adsorption branch yielded a specific surface area of 531 m² g⁻¹ (Fig. S4), reflecting the high internal surface area of the phenazine-linked framework. Nonlocal density functional theory (NLDFT) calculations, based on a cylindrical pore model, revealed a bimodal pore-size distribution with two prominent peaks at approximately 1.0 and 1.7 nm (Fig. 4b). These values are in good agreement with the simulated pore diameter of ∼1.5 nm derived from the AB-stacked structural model of TU-32, constructed using energy-minimized geometry. The slight deviations between the simulated and experimentally derived pore sizes can be rationalized by considering the inherent flexibility of the phenazine linkage and the steric effects introduced by the bulky tert-butyl substituents on the pyrene units. These features may cause minor framework distortions or local deviations from idealized stacking in the solid state, slightly altering the pore environment.

Fig. 4 (a) Nitrogen adsorption–desorption isotherms of TU-32 and (b) corresponding pore size distribution profile derived from the adsorption branch using NLDFT.

Drug encapsulation and affinity trends

To evaluate the drug-carrying potential of the phenazine-linked COF TU-32, we selected three clinically relevant small-molecule drugs—5-FU, isoniazid, and captopril—for encapsulation studies. These molecules were chosen based on their molecular dimensions, clinical utility, and electronic structure, particularly their degree of π-conjugation, which plays a critical role in modulating host–guest interactions with the π-conjugated backbone of TU-32.

The calculated pore diameter of TU-32 is 1.5 nm, providing sufficient internal free volume to accommodate small drug molecules via pore encapsulation. The molecular dimensions of 5-fluorouracil (∼0.53 nm × 0.49 nm),³¹ isoniazid (∼0.72 nm × 0.41 nm),³² and captopril (∼0.86 nm × 0.54 nm),³³ as estimated from crystallographic and computational studies, are all smaller than the pore aperture of TU-32, making them amenable to diffusion and entrapment within the COF's microporous channels.

Therapeutically, 5-FU is an antimetabolite used in the treatment of various cancers,³⁴ while isoniazid is a first-line antibiotic against tuberculosis,³⁵ and captopril is an angiotensin-converting enzyme (ACE) inhibitor used to treat hypertension and heart failure.³⁶ Encapsulation of these drugs into COFs is particularly advantageous for improving their stability, solubility, and controlled release, which are major challenges in clinical delivery.

A central goal of this study was to uncover the role of π–π interactions in driving drug loading efficiencies in TU-32. Structurally, TU-32 features an extended π-conjugated phenazine linkage and an AB-stacked layer configuration that effectively suppresses interlayer π–π eclipsing. This unique stacking arrangement, achieved through precise reticular design, introduces interlayer offsets that preserve open and accessible pore channels along the framework. These channels promote directional host–guest interactions and reduce steric hindrance, thereby enabling strong supramolecular affinity—particularly with aromatic or conjugated drug molecules. Importantly, this AB stacking motif facilitates efficient guest diffusion and stable encapsulation within the pores, eliminating the need for post-synthetic functionalization or additional activation protocols.

To probe this conjugation-driven selectivity, we carried out comparative loading studies using the above-mentioned drug molecules, which differ in their aromatic character: 5-FU contains a fully conjugated pyrimidine-like ring; isoniazid includes a moderately conjugated pyridyl ring; and captopril is largely non-conjugated. Drug loading was performed via the impregnation method, where activated TU-32 was suspended in aqueous drug solutions under stirring for 4 hours to allow diffusion-driven uptake. Post-encapsulation characterization using N₂ sorption revealed significant decreases in BET surface area from 531 m² g⁻¹ (pristine TU-32) to 384, 245, and 261 m² g⁻¹ for 5-FU-, isoniazid-, and captopril-loaded COFs, respectively (Fig. S15–S17), indicating pore occupancy by the drug molecules. Further characterization by PXRD confirmed that the crystallinity of the COF framework was retained after drug loading, as evidenced by unaltered Bragg peak positions (Fig. S11). SEM micrographs (Fig. S12–S14) also mirrored the morphology of the parent COF, ruling out significant surface adsorption and confirming that drug molecules were primarily hosted within the internal pore channels.

Quantitative analysis of drug loading via UV-vis spectroscopy yielded loading capacities of 56.46 wt% for 5-FU, 54.91 wt% for isoniazid, and 36.67 wt% for captopril (Fig. S18–S23). These results underscore the key role of π–π stacking interactions between the COF framework and aromatic drug molecules. The highest loading was observed for 5-FU, likely due to its strong electronic complementarity with the phenazine-based pore walls. Isoniazid also showed efficient encapsulation due to its aromatic pyridine ring, albeit slightly lower than 5-FU. In contrast, captopril, which lacks a π-conjugated system, showed substantially lower uptake, highlighting the importance of conjugation-assisted host–guest affinity.

To gain deeper insight into the drug–framework interactions and elucidate the underlying factors driving selective drug encapsulation, we performed density functional theory (DFT) calculations to model the binding of representative drug molecules—5-fluorouracil and isoniazid—within the TU-32 COF framework. The computed adsorption energies are summarized in Table S1. The more negative the adsorption energy, the stronger the binding affinity between the drug and the COF. Our calculations indicate that TU-32 exhibits a stronger interaction with 5-fluorouracil (−0.77066 eV) compared to isoniazid (−0.67347 eV), consistent with the experimentally observed preferential uptake behavior. These results support the notion that conjugation-driven host–guest interactions, in concert with the electronic structure and geometry of the COF, contribute significantly to selective drug loading. The optimized structures of the COF–drug complexes are presented in Fig. S24 and S25, providing visual evidence of the molecular binding conformations.

Collectively, these findings reveal that the extended π-surface area of TU-32, coupled with its open AB-stacked topology, facilitates conjugation-directed drug uptake via π–π interactions and spatial compatibility. This study establishes TU-32 as a promising platform for selective loading of conjugated therapeutics, offering insights for future COF-based drug delivery systems that leverage supramolecular recognition mechanisms.

Release profiles and temporal drug delivery behavior

Drug release studies were carried out using dialysis-based diffusion experiments under physiological conditions (pH 7.4, 37 °C), and the temporal release of each drug from TU-32 was monitored by UV-vis spectroscopy over time using established calibration curves (Fig. 5b, e and h). Sustained drug release is a critical feature for modern therapeutics, as it helps maintain effective plasma drug concentrations over extended periods, reduces dosing frequency, minimizes systemic toxicity, and enhances patient compliance.^37,38 This is especially important for chemotherapeutic agents like 5-FU, which suffer from rapid metabolism and short half-life.³⁹ Prolonged and controlled release of 5-FU allows continuous therapeutic exposure to tumor cells, improving efficacy while reducing the side effects associated with bolus administration.

Fig. 5 UV-vis absorption spectra of (a) 5-FU, (d) isoniazid, and (g) captopril in simulated body fluid (pH 7.4, phosphate buffer solution) at various concentrations. Corresponding linear calibration plots for (b) 5-FU, (e) isoniazid, and (h) captopril. Drug release profiles of (c) 5-FU, (f) isoniazid, and (i) captopril from TU-32, along with fitting curves. Data represent mean ± SD from three independent trials.

In our study, TU-32 enabled remarkably sustained release of 5-FU, with only 9.72 wt% released after 30 days, even with a high initial loading of 56.46 wt%. This performance significantly surpasses those of previously reported porous carriers. For instance, TpASH-FACOF nanosheets, with a loading capacity of 12 wt%, released 50 wt% of 5-FU within 3 days.⁴⁰ Cage-COF-TT, with a loading of 21.40 wt%, showed a rapid release of 93 wt% within 52 hours,⁴¹ while PI-2-COF and PI-3-COF, which hosted 30 wt% drug, released 85 wt% over about 5 days.⁴² The superior release profile of TU-32 is attributable to its high surface area, π-conjugated phenazine backbone, and AB-aligned layered structure, which collectively facilitate strong π–π interactions and tight host–guest binding that retards premature drug diffusion.

For isoniazid, TU-32 also demonstrated high encapsulation (54.91 wt%) along with favorable sustained release behavior, achieving only 14 wt% release over 10 days. In contrast, MIL-100(Fe), a representative metal–organic framework (MOF), showed a much lower loading capacity of 12.85 wt% and a rapid release of 72.22 wt% within just 24 hours.⁴³ Similarly, TUS-64 COF demonstrated lower drug loading (17.37 wt%) and faster release kinetics, with 22 wt% released over the same 10-day period.⁴⁴ This reinforces that TU-32's extended conjugation and tailored pore architecture enable more effective binding and prolonged delivery of moderately conjugated drugs like isoniazid.

Even for captopril, a non-conjugated drug with limited π–π interactions, TU-32 achieved a high loading of 36.67 wt%—substantially higher than Cage-COF-TT (22.30 wt%),⁴¹ TUS-84 COF (16.00 wt%),⁴⁵ and TUS-64 COF (14.71 wt%).⁴⁴ While captopril release from TU-32 reached 89 wt% after 10 days, this was still slower than the rates reported for these other frameworks: 94 wt% in 52 hours for Cage-COF-TT, 98 wt% in 5 days for TUS-84, and 95 wt% in 6 days for TUS-64. These observations indicate that while conjugation plays a key role, even non-conjugated molecules can benefit from TU-32's steric confinement and diffusion-limiting channel architecture, enabling moderated release kinetics. Structural integrity and porosity of TU-32 following drug release were evaluated by PXRD and nitrogen adsorption measurements. As shown in Fig. S26, the PXRD patterns of TU-32 after drug release retained the characteristic diffraction peaks of the pristine COF, indicating preservation of long-range crystallinity. Furthermore, nitrogen adsorption isotherms (Fig. S27–S29) demonstrated a marked increase in BET specific surface areas compared to the corresponding drug-loaded samples, confirming successful release of the encapsulated molecules and restoration of accessible porosity within the framework. A comparative summary of recently reported drug nanocarriers is presented in Table S2, highlighting the performance of TU-32 relative to other state-of-the-art porous materials in terms of drug loading capacity and release kinetics.

In conclusion, TU-32 stands out as a multifunctional drug delivery platform offering: (a) exceptional loading capacities for diverse drugs, including highly conjugated (5-FU), moderately conjugated (isoniazid), and non-conjugated (captopril) agents; (b) sustained and delayed release profiles across all tested drugs, outperforming most state-of-the-art COFs and MOFs; and (c) a structure-driven release mechanism, whereby π–π interactions and framework topology synergistically modulate release rates. This highlights TU-32's potential as a next-generation drug delivery material for controlled and long-term therapeutic applications.

Conclusions

In this study, we have demonstrated the successful synthesis and structural elucidation of TU-32, a phenazine-linked π-conjugated COF that features a distinct AB stacking geometry and a highly ordered pore architecture. The integration of extended π-conjugation via phenazine linkages not only endows TU-32 with structural robustness but also imparts selective affinity toward conjugated drug molecules, as exemplified by the markedly higher loading of 5-fluorouracil and isoniazid compared to non-conjugated captopril. The suppression of interlayer π-stacking in favor of an AB-aligned framework appears to play a critical role in facilitating accessible pore channels and promoting directional host–guest interactions. This structure–function relationship was further validated by drug loading and release experiments, which demonstrated not only higher encapsulation efficiencies for conjugated drugs but also significantly prolonged release profiles compared to state-of-the-art porous carriers. These results offer compelling evidence that rational electronic design and stacking control in COFs can be leveraged to optimize molecular recognition and encapsulation performance. This research marks a conceptual shift in drug delivery design—offering a π-electronic complementarity–based strategy that serves as a springboard for developing next-generation porous carriers with far-reaching implications across therapeutics, diagnostics, and responsive materials.

Author contributions

S. D. and Y. N. designed the study and supervised the project. K. S. and T. I. carried out the synthesis and material characterization. M. N. contributed to the characterization experiments. T. K. performed the material characterization. S. D. drafted the manuscript with feedback and revisions from all co-authors. All authors reviewed and approved the final version of the manuscript.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the SI. See DOI: https://doi.org/10.1039/d5nh00470e

Acknowledgements

The authors sincerely thank Prof. Y. Idemoto and Dr T. Ichihashi of Tokyo University of Science for their valuable assistance with the TEM measurements. This study was financially supported by the Scientific Research on Innovative Areas “Aquatic Functional Materials” (grant no. 22H04562), JSPS KAKENHI (grant no. 23H00289 and 23KK0098), the Yazaki Memorial Foundation for Science and Technology, and the FUSO Innovative Technology Fund.

Notes and references

1. S. Das, P. Heasman, T. Ben and S. Qiu, Porous Organic Materials: Strategic Design and Structure–Function Correlation, Chem. Rev., 2017, 117, 1515–1563.
2. O. M. Yaghi, M. J. Kalmutzki and C. S. Diercks, Introduction to Reticular Chemistry: Metal-Organic Frameworks and Covalent Organic Frameworks, Wiley-VCH, Weinheim, Germany, 2019, ch. 7–11, pp. 177–283.
3. F. Haase and B. V. Lotsch, Solving the COF trilemma: towards crystalline, stable and functional covalent organic frameworks, Chem. Soc. Rev., 2020, 49, 8469–8500.
4. K. T. Tan, S. Ghosh, Z. Wang, F. Wen, D. Rodríguez-San-Miguel, J. Feng, N. Huang, W. Wang, F. Zamora, X. Feng, A. Thomas and D. Jiang, Covalent organic frameworks, Nat. Rev. Methods Primers, 2023, 3(1), 1.
5. S.-Y. Ding and W. Wang, Covalent organic frameworks (COFs): from design to applications, Chem. Soc. Rev., 2013, 42, 548–568.
6. X. Zhao, P. Pachfule and A. Thomas, Covalent organic frameworks (COFs) for electrochemical applications, Chem. Soc. Rev., 2021, 50, 6871–6913.
7. M. S. Lohse and T. Bein, Covalent Organic Frameworks: Structures, Synthesis, and Applications, Adv. Funct. Mater., 2018, 28, 1705553.
8. Y. Song, Q. Sun, B. Aguila and S. Ma, Opportunities of Covalent Organic Frameworks for Advanced Applications, Adv. Sci., 2019, 6, 1801410.
9. S. Kandambeth, K. Dey and R. Banerjee, Covalent Organic Frameworks: Chemistry beyond the Structure, J. Am. Chem. Soc., 2019, 141, 1807–1822.
10. X. Li, P. Yadav and K. P. Loh, Function-oriented synthesis of two-dimensional (2D) covalent organic frameworks – from 3D solids to 2D sheets, Chem. Soc. Rev., 2020, 49, 4835–4866.
11. Y. Li, W. Chen, G. Xing, D. Jiang and L. Chen, New synthetic strategies toward covalent organic frameworks, Chem. Soc. Rev., 2020, 49, 2852–2868.
12. X. Han, C. Yuan, B. Hou, L. Liu, H. Li, Y. Liu and Y. Cui, Chiral covalent organic frameworks: design, synthesis and property, Chem. Soc. Rev., 2020, 49, 6248–6272.
13. J. L. Segura, M. J. Mancheño and F. Zamora, Covalent organic frameworks based on Schiff-base chemistry: synthesis, properties and potential applications, Chem. Soc. Rev., 2016, 45, 5635–5671.
14. H. L. Nguyen, Reticular design and crystal structure determination of covalent organic frameworks, Chem. Sci., 2021, 12, 8632–8647.
15. X. Guan, F. Chen, Q. Fang and S. Qiu, Design and applications of three dimensional covalent organic frameworks, Chem. Soc. Rev., 2020, 49, 1357–1384.
16. T. Irie, S. Das, Q. Fang and Y. Negishi, The Importance and Discovery of Highly Connected Covalent Organic Framework Net Topologies, J. Am. Chem. Soc., 2025, 147, 1367–1380.
17. H. V. Babu, M. G. M. Bai and M. R. Rao, Functional π-Conjugated Two-Dimensional Covalent Organic Frameworks, ACS Appl. Mater. Interfaces, 2019, 11, 11029–11060.
18. A. P. Cote, A. I. Benin, N. W. Ockwig, M. O’Keeffe, A. J. Matzger and O. M. Yaghi, Porous, Crystalline, Covalent Organic Frameworks, Science, 2005, 310, 1166–1170.
19. C. R. DeBlase, K. E. Silberstein, T.-T. Truong, H. D. Abruña and W. R. Dichtel, β-Ketoenamine-Linked Covalent Organic Frameworks Capable of Pseudocapacitive Energy Storage, J. Am. Chem. Soc., 2013, 135, 16821–16824.
20. J. Guo, Y. Xu, S. Jin, L. Chen, T. Kaji, Y. Honsho, M. A. Addicoat, J. Kim, A. Saeki, H. Ihee, S. Seki, S. Irle, M. Hiramoto, J. Gao and D. Jiang, Conjugated organic framework with three-dimensionally ordered stable structure and delocalized π clouds, Nat. Commun., 2013, 4, 2736.
21. H. Sun, H. Ji, D. Qiao, Y. Xu, X. Qu, Y. Qi, Z. Feng, X. Zhang, F. Zhang, R. Wang and B. Dong, Vinylene-linked covalent organic frameworks based on phenanthroline for visible-light-driven bifunctional photocatalytic water splitting, Chem. Eng. J., 2025, 507, 160448.
22. M. Moharramnejad, R. E. Malekshah, Z. Salariyeh, H. Saremi, M. Shahi and A. Ehsani, The synthetic strategies of COFs, for drug delivery, photo/sono-dynamic, photo/microwave thermal and combined therapy, Inorg. Chem. Commun., 2023, 153, 110888.
23. B. Wang, X. Liu, P. Gong, X. Ge, Z. Liu and J. You, Fluorescent COFs with a highly conjugated structure for visual drug loading and responsive release, Chem. Commun., 2020, 56, 519–522.
24. W. Zhang, L. Yang, J. Zou, D. Xu, G. Liu and Z. Lu, Covalent organic frameworks: Prospects and potential in tumor diagnosis and therapy, Chem. Eng. J., 2025, 519, 165302.
25. P. Ghosh and P. Banerjee, Drug delivery using biocompatible covalent organic frameworks (COFs) towards a therapeutic approach, Chem. Commun., 2023, 59, 12527–12547.
26. V. S. Vyas, M. Vishwakarma, I. Moudrakovski, F. Haase, G. Savasci, C. Ochsenfeld, J. P. Spatz and B. V. Lotsch, Exploiting Noncovalent Interactions in an Imine-Based Covalent Organic Framework for Quercetin Delivery, Adv. Mater., 2016, 28, 8749–8754.
27. P. Chen, Y. Li, Y. Dai, Z. Wang, Y. Zhou, Y. Wang and G. Li, Porphyrin-based covalent organic frameworks as doxorubicin delivery system for chemo-photodynamic synergistic therapy of tumors, Photodiagn. Photodyn. Ther., 2024, 46, 104063.
28. P. Gong, K. Zhao, X. Liu, C. Li, B. Liu, L. Hu, D. Shen, D. Wang and Z. Liu, Fluorescent COFs with a Highly Conjugated Structure for Combined Starvation and Gas Therapy, ACS Appl. Mater. Interfaces, 2022, 14, 46201–46211.
29. J. Wang, X. Zhang, R. Shen, Q. Yuan and Y. Yang, Staggered-Stacking Two-Dimensional Covalent Organic Framework Membranes for Molecular and Ionic Sieving, ACS Nano, 2024, 18, 34698–34707.
30. Materials Studio, ver. 7.0, Accelrys Inc., San Diego, CA.
31. R. A. Al-Thawabeia and H. A. Hodali, Use of Zeolite ZSM-5 for Loading and Release of 5-Fluorouracil, J. Chem., 2015, 2015, 403597.
32. A. Lemmerer, Covalent assistance to supramolecular synthesis: modifying the drug functionality of the antituberculosis API isoniazid in situ during co-crystallization with GRAS and API compounds, CrystEngComm, 2012, 14, 2465–2478.
33. V. Castelletto, J. Seitsonen, J. Ruokolainen, S. A. Barnett, C. Sandu and I. W. Hamley, Self-Assembly of Angiotensin-Converting Enzyme Inhibitors Captopril and Lisinopril and Their Crystal Structures, Langmuir, 2021, 37, 9170–9178.
34. D. B. Longley, D. P. Harkin and P. G. Johnston, 5-Fluorouracil: mechanisms of action and clinical strategies, Nat. Rev. Cancer, 2003, 3, 330–338.
35. C. Vilcheze and W. R. Jacobs, Jr., The Mechanism of Isoniazid Killing: Clarity Through the Scope of Genetics, Annu. Rev. Microbiol., 2007, 61, 35–50.
36. F. Fyhrquist, Clinical Pharmacology of the ACE Inhibitors, Drugs, 1986, 32(Suppl. 5), 33–39.
37. S. Adepu and S. Ramakrishna, Controlled Drug Delivery Systems: Current Status and Future Directions, Molecules, 2021, 26, 5905.
38. E. Mutschler and H. Knauf, Current Status of Sustained Release Formulations in the Treatment of Hypertension, Clin. Pharmacokinet., 1999, 37(Suppl. 1), 1–6.
39. A. A. Valencia-Lazcano, D. Hassan, M. Pourmadadi, A. shamsabadipour, R. Behzadmehr, A. Rahdar, D. I. Medina and A. M. Díez-Pascual, 5-Fluorouracil nano-delivery systems as a cutting-edge for cancer therapy, Eur. J. Med. Chem., 2023, 246, 114995.
40. S. Mitra, H. S. Sasmal, T. Kundu, S. Kandambeth, K. Illath, D. Díaz Díaz and R. Banerjee, Targeted Drug Delivery in Covalent Organic Nanosheets (CONs) via Sequential Postsynthetic Modification, J. Am. Chem. Soc., 2017, 139, 4513–4520.
41. M. Li, Y. Peng, F. Yan, C. Li, Y. He, Y. Lou, D. Ma, Y. Li, Z. Shi and S. Feng, A cage-based covalent organic framework for drug delivery, New J. Chem., 2021, 45, 3343–3348.
42. L. Bai, S. Z. F. Phua, W. Q. Lim, A. Jana, Z. Luo, H. P. Tham, L. Zhao, Q. Gao and Y. Zhao, Nanoscale covalent organic frameworks as smart carriers for drug delivery, Chem. Commun., 2016, 52, 4128–4131.
43. M. A. Simon, E. Anggraeni, F. E. Soetaredjo, S. P. Santoso, W. Irawaty, T. C. Thanh, S. B. Hartono, M. Yuliana and S. Ismadji, Hydrothermal Synthesize of HF-Free MIL-100(Fe) for Isoniazid-Drug Delivery, Sci. Rep., 2019, 9, 16907.
44. Y. Zhao, S. Das, T. Sekine, H. Mabuchi, T. Irie, J. Sakai, D. Wen, W. Zhu, T. Ben and Y. Negishi, Record Ultralarge-Pores, Low Density Three-Dimensional Covalent Organic Framework for Controlled Drug Delivery, Angew. Chem., Int. Ed., 2023, 62, e202300172.
45. S. Das, T. Sekine, H. Mabuchi, T. Irie, J. Sakai, Y. Zhao, Q. Fang and Y. Negishi, Three-Dimensional Covalent Organic Framework with scu-c Topology for Drug Delivery, ACS Appl. Mater. Interfaces, 2022, 14, 48045–48051.

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Footnote

† These authors contributed equally.

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