A Planarity‐Hindrance Co‐Balance Strategy to Develop Antiparallel H‐Aggregates With Minimal Absorbance Blueshift for Type I Photodynamic Therapy

ABSTRACT

H‐aggregates offer intrinsic features for type I photodynamic therapy (PDT) by concurrently promoting triplet state formation and strengthening charge transfer ability. However, their exploitation remains limited by the inherently large absorption blueshift (usually >100 nm) arising from strong H‐type excitonic coupling in conventional parallel‐packed H‐aggregates, forcing short‐wavelength laser excitation with poor tissue penetration. Herein, this study reports a planarity‐hindrance co‐balance strategy to develop donor‐π‐acceptor‐based antiparallel‐packed H‐aggregates with minimal absorption blueshift for type I PDT. The results demonstrate that π‐bridge planarization drives H‐packing, while donor‐site steric tuning dictates the blueshift by modulating slipping angles and π‐π overlapping degree, and a steric threshold (Me/OMe) is identified beyond which blueshift becomes invariant. The optimized MTBSIC molecules form H‐aggregates with an exceptionally small blueshift of 15 nm over its monomers. MTBSIC H‐aggregates further display markedly enhanced type I ROS generation and improved photothermal conversion ability over their amorphous counterparts possessing similar monomeric photophysical properties. Mechanistic analyses reveal that H‐packing promotes both intersystem crossing and intermolecular charge transfer/separation, synergistically boosting type I ROS production. MTBSIC H‐aggregates further achieve potent tumor inhibition with high biocompatibility both in vitro and in vivo. This work establishes a generalizable molecular design paradigm for near‐monomer‐like H‐aggregates for high‐performance phototheranostics.

To circumvent the large absorption blueshift (often >100 nm) faced by traditional parallel‐packed H‐aggregates, this study introduces a planarity‐hindrance co‐balance strategy in donor‐π‐acceptor scaffolds to design antiparallel‐packed H‐aggregates with only a 15 nm blueshift. These H‐aggregates exhibit promoted intersystem crossing and charge transfer, synergistically boosting type I photodynamic and photothermal performance for effective tumor eradication.

1. Introduction

Photodynamic therapy (PDT) has emerged as a noninvasive cancer treatment modality with high spatiotemporal controllability and minimal systemic toxicity, which capitalizes on light and photosensitizers (PSs) to generate cytotoxic reactive oxygen species (ROS) that cause cancer cell death [1, 2, 3]. Compared to conventional type II PDT, the emerging type I PDT mainly generates radical species such as superoxide anion (O₂ ^•−), hydroxyl radical (⚫OH) in a less oxygen‐dependent manner, showing great potential in surmounting the hypoxia tumor dilemma [4, 5, 6]. The design of high‐performance type I PSs requires two key photophysical features: efficient intersystem crossing (ISC) to populate the triplet state (T₁) and strong charge transfer/separation capability to facilitate electron transfer [7, 8, 9]. Supramolecular assemblies, with ordered molecular packing and tailored intermolecular interactions, provide an ideal platform for regulating these crucial excited‐state decay pathways [10, 11, 12, 13, 14, 15, 16, 17]. Among them, H‐aggregates featuring a typically face‐to‐face packing mode represent a highly attractive class of supramolecular PSs. The characteristic H‐type exciton coupling suppresses radiative decay and is able to reroute excited‐state deactivation toward ISC, while the substantial π‐orbital overlap concomitantly promotes intermolecular transfer/separation [18, 19, 20, 21, 22, 23, 24]. The synergistic effects collectively highlight the advantages and great potential of H‐aggregates for type I PDT [25, 26, 27, 28, 29, 30].

Despite these advantages and recent advances, the rational construction of H‐aggregate‐based PSs is severely impeded by their intrinsically large absorption blueshift, often exceeding 100 nm relative to their monomers [26, 29, 31]. This drawback arises from the conventional H‐aggregate design principles that rely on highly symmetric and planar scaffolds, which favor strong parallel stacking with excessively large slip angles, leading to an overstrengthened H‐type excitonic coupling and hence large absorption blueshift (Scheme 1a) [32, 33]. This pronounced blueshift forces short‐wavelength laser excitation with poor tissue penetration and higher light scattering, limiting their in vivo applications for deeply seated tumors [34, 35, 36]. Moreover, the excessively strong H‐coupling predominantly promotes nonradiative decay pathways and leads to severe exciton quenching, thereby suppressing charge separation and type I ROS generation [14, 37, 38]. Therefore, overcoming the fundamental trade‐offs between the inherent advantages and spectral drawbacks of H‐aggregates remains appealing yet challenging to design effective type I PSs.

SCHEME 1.

(a) Schematic of parallel and antiparallel H‐aggregates with different absorption blueshift degrees. (b) Schematic of planarity‐hindrance co‐balance strategy in developing minimal‐blueshift H‐aggregates. (c) Schematic of H‐aggregation in boosting type I ROS generation and photothermal effect.

Crucially, the essential state model (ESM) predicts that the antiparallel‐packed H‐aggregates, formed by asymmetric molecules, are able to stabilize both ground and excited states, especially the latter, potentially narrowing the energy gap and mitigating absorption blueshifts [33]. Asymmetric scaffolds, Donor‐π‐acceptor (D‐π‐A) molecules, featuring electron‐rich aromatic donors (e.g., phenylamine) and electron‐deficient acceptors (e.g., dicyanovinyl) connected via π‐conjugated bridges, offer a modular platform to precisely modulate intramolecular charge transfer, dipole moments, and molecular packing [39, 40, 41]. Owing to their intrinsic permanent dipole moments, these D‐π‐A molecules facilitate antiparallel slipped stacking with tunable π‐π orbital overlap, enabling systematic regulation of intermolecular coupling in H‐aggregates [42, 43, 44]. Therefore, controlling slip geometry and effective π‐orbital overlap within D‐π‐A H‐aggregate systems represents a conceptually powerful strategy to mitigate H‐aggregate blueshifts while preserving the benefits of H‐packing mode [45, 46]. However, rational strategies to modulate the antiparallel packing of H‐aggregates to develop high‐performance PSs remain largely unexplored for type I PDT.

In this work, we report a planarity‐hindrance co‐balance strategy to develop antiparallel H‐aggregates with minimal absorption blueshift for hypoxia‐tolerant type I PDT (Scheme 1). This strategy is demonstrated with a twisted donor and a planar π‐acceptor to precisely regulate the intermolecular packing and slipping angles. With triphenylamine as a twisted donor and indanone‐derived malononitrile as a planar acceptor, H‐aggregates are first constructed in D‐π‐A molecules by extending the π‐bridge from thiophene (S) to thieno[3,2‐b]thiophene (BS) to strengthen the intermolecular π‐π interaction (Scheme 1b). Subsequently, fine‐tuning steric hindrance at the donor site systematically regulates π‐orbitals overlap and excitonic coupling, thereby suppressing excessive absorption blueshift while maintaining robust H‐packing. We find that the appropriate steric hindrance at the donor reduces overly planar overlapping and minimizes absorption blueshift in H‐aggregates, while the steric hindrance at the π‐bridge disfavors H‐packing. Through this design, the optimized molecule MTBSIC achieves an exceptionally small absorption blueshift of 15 nm upon H‐aggregation (610 nm versus 625 nm), representing a rare case of H‐aggregates with near‐monomer‐like absorption. Benefiting from the compact intermolecular interaction, the preferred H‐packing is also well preserved when co‐assembling with DSPE‐PEG₂₀₀₀ to form nanoparticles (NPs). In contrast, a structural analogue MTSIC, with a shorter π‐bridge shows nearly identical monomeric photophysics but forms amorphous aggregates, serving as an ideal control to evaluate the phototheranostic advantages of H‐aggregation. Under light irradiation, MTBSIC H‐aggregates show enhanced type I ROS generation compared to MTSIC amorphous aggregates (Scheme 1c). Mechanistic studies revealed that H‐aggregation efficiently promotes triplet generation and facilitates charge transfer and separation, thereby synergistically boosting type I ROS production. The resulting MTBSIC H‐packing NPs demonstrate superior type I PDT efficacy in tumor‐bearing mice, underscoring the translational potential of this molecular design strategy.

2. Results and Discussion

2.1. Molecular Design for H‐Aggregates

As aforementioned, D‐π‐A molecules are attractive scaffolds for constructing H‐aggregates with minimized absorption blueshift. To suppress excessive intermolecular π–π packing while enabling controllable supramolecular self‐assembly behaviors, a propeller‐like triphenylamine (T) unit was selected as the donor motif. To achieve planarity‐hindrance balance, planar thiophene (S) and 2‐(3‐oxo‐2,3‐dihydro‐1H‐inden‐1‐ylidene) malononitrile (IC) were chosen as the π‐bridge and electron acceptor [47]. Methoxy groups were introduced on triphenylamine (MT) to tune the steric hindrance, and π‐bridge was extended to thieno[3,2‐b]thiophene (BS) to enhance planarity and promote the intermolecular π‐π interaction for H‐packing. Based on this design principle, two pairs of planarity‐twist‐regulated molecules (TSIC/TBSIC, MTSIC/MTBSIC) were designed (Figure 1a; Figure S1). All four molecules were synthesized via a two‐step reaction (Figure S58), and their chemical structures were characterized by nuclear magnetic resonance (NMR) and mass spectrometry (MS) (Figures S62–S71).

FIGURE 1.

(a) Schematic of extending π‐bridge to design D‐π‐A molecular H‐aggregates. (b) Molecular configurations and (c) ESP distributions of TSIC, TBSIC, MTSIC, and MTBSIC. (d) Normalized absorption spectra and (e) emission spectra of two pairs of molecules, TSIC/TBSIC, MTSIC/MTBSIC, in THF. (f) Hydrodynamic sizes and (g) absorption peak changes of the two‐pair molecules in THF/water mixtures with different water fractions.

Density functional theory (DFT) theoretical calculations suggested very similar optimized ground‐state (S₀) geometries for all four molecules (Figure 1b). The dihedral angles between the π‐bridge and IC acceptor were all smaller than 1°, suggesting that the intramolecular S···O═C lock significantly extends the π‐conjugation planarity [48, 49]. Methoxy substitution slightly increased twisting in MTSIC and MTBSIC, with larger dihedral angles ranging from ∼43° to ∼47° in the donor part. Moreover, the BS extension of the π‐bridge had negligible impact on their frontier orbital distributions (Figure S2). The highest occupied molecular orbital (HOMO) is mainly distributed at the donor and π‐bridge, while the lowest unoccupied molecular orbital (LUMO) is mainly located at the acceptor unit, with similar HOMO‐LUMO energy gaps. In contrast, the BS extension obviously enhanced the positive electrostatic potential (ESP) (red) on the π‐bridge as compared to the S unit, while the negative ESP (blue) remains at the cyano group of the acceptor (Figure 1c). This suggests a much stronger electrostatic interaction between acceptor and π‐bridge in TBSIC/MTBSIC over TSIC/MTSIC, facilitating the antiparallel H‐packing.

2.2. Photophysical Properties of Monomer and Aggregates

The photophysical properties of the monomers were investigated in tetrahydrofuran (THF), while aggregation was induced by gradually increasing the water fraction (f_w ) in THF/water mixtures. At the monomer state (i.e., in pure THF solution), TSIC and TBSIC showed nearly identical absorption (centred at 590 vs. 592 nm) and emission (centred at 906 vs. 910 nm) spectra (Figure 1d,e). A similar trend was also observed for MTSIC/MTBSIC. These results confirm that extending the π‐bridge from S to BS minimally affects their monomeric photophysical properties. The aggregation behavior was then investigated in the THF/water mixtures, with dynamic light scattering (DLS) to monitor aggregate formation (Figure 1f), where the aggregation was induced by gradually increasing f_w . All four molecules remained monomerized below f_w = 50%, but initially formed aggregates at f_w of 50% or 60%. Further increasing f_w led to a transformation from loosely packed aggregates into more compact aggregates with decreased hydrodynamic sizes.

Before reaching the aggregation threshold, increasing f_w caused slightly redshifted absorption maxima (Abs_max ) for all four molecules (Figure 1g; Figure S3), attributed to the intramolecular charge transfer (ICT) feature in D‐π‐A skeleton and the increased solvent polarity [50]. However, their behaviour diverged sharply after aggregation. Bearing the shorter S bridge, TSIC and MTSIC only exhibited a slightly blue‐shifted absorption (e.g., Abs_max changes from 598 to 587 nm for TSIC) upon increasing f_w to 90%, attributed to the relatively less polar microenvironment created by surrounding TSIC/MTSIC molecules (Figure 1g; Figure S3a,c). The nearly identical absorption spectra before and after aggregation with only minor broadening, also suggested their amorphous aggregation nature (Figure S4). In sharp contrast, TBSIC and MTBSIC displayed characteristic H‐packing spectral features at high f_w values, where the compact aggregates of TBSIC (Abs_max = 540 nm at f_w = 90%) exhibited an absorption blueshift of 58 nm and a increased vibronic coupling ratio (A_0‐1/A_0‐0 ), compared to its loose aggregates (Abs_max = 598 nm at f_w = 50%) (Figure 1g; Figure S3b,d). Similar distinct spectral changes induced by H‐aggregation were also observed for MTBSIC but with a smaller blueshift (from 631 nm at f_w = 50% to 610 nm at f_w = 90%). These results indicate that π‐bridge extension promotes H‐aggregation, while donor‐site steric tuning modulates the H‐coupling magnitude.

Fluorescence evolution during the aggregation process also confirmed their distinct aggregation modes. As f_w increased, all molecules initially showed decreased fluorescence emission due to the ICT‐quenched fluorescence in polar solvents (Figures S5 and S6) [51]. Further increasing f_w to induce aggregation enhanced their emission, derived from their aggregation‐induced emission (AIE) features. However, both TBSIC and MTBSIC aggregates showed much weaker fluorescence than their counterparts, indicating a non‐negligible contribution from H‐packing to fluorescence quenching. Notably, it is quite rare and intriguing to simultaneously observe the antagonistic interplay between the AIE effect and the H‐aggregation quenching effect. Considering the blueshift absorption and suppressed emission during the aggregation process, it is reasonable to speculate on the H‐aggregation tendency of TBSIC and MTBSIC.

2.3. Single‐Crystal Analysis of H‐Aggregation

To elucidate the molecular basis of their distinct aggregation modes, single‐crystal structures of MTSIC and MTBSIC were obtained (Figures S7 and S8 and Table S1). Both molecules adopted similar twisted‐planar conformations in their crystals (Figure 2a,b), consistent with the DFT calculation. Notably, with the extended π‐bridge, MTBSIC shows smaller dihedral angles (7.27° and 2.30°) between donor/acceptor and π‐bridge as compared to MTSIC (11.69° and 5.95°). This extended planarity consequently leads to extensive π–π planar overlap (face‐to‐face) within the well‐defined H‐dimers of the MTBSIC crystal (Figure 2b). In contrast, the extracted amorphous‐dimer (A‐dimer) in MTSIC shows mainly C─H⋯O and C─H⋯S interactions of 2.50 Å and 2.54 Å (Figure 2a). Moreover, the extracted representative MTBSIC tetramer shows strong π–π packing interaction with distances of 3.38–3.44 Å (Figure 2c), whereas the MTSIC tetramer only shows the intermolecular interactions with more hydrogen bonds and weaker π–π packing interactions, with distances of 3.31–3.52 Å (Figure 2d; Figure S9). Although MTSIC showed partial J‐packing tendency in a single crystal, its short π‐planar length weakens the crystallization ability, giving rise to the formation of amorphous aggregates, consistent with our previous findings that MTSIC requires external energy input to crystallize [47]. In comparison, MTBSIC exhibits predominantly H‑aggregation‑guided crystal packing (Figure 2e). ESP distribution and intermolecular interactions analysis further confirmed both the electrostatic complementarity and the dispersion‐dominated moderate interactions across the π‐stacking interface (large green IGMH isosurface) in MTBSIC H‐dimers (Figure 2f; Figure S10). In contrast, MTSIC A‐dimer was mainly stabilized by moderate C─H⋯S and C─H⋯O interactions.

FIGURE 2.

The monomer and dimer structures for (a) MTSIC and (b) MTBSIC extracted from their single‐crystal structures. The representative tetramer structures of (c) MTBSIC and (d) MTSIC. (e) The H‐packing structures in the MTBSIC single crystal. (f) The IGMH analysis results of intermolecular interactions in MTSIC A‐dimer and MTBSIC H‐dimers (the δ_inter in the graph was 0.004, top view and side view, calculated by Multiwfn software).

Collectively, these results demonstrate that π‐bridge extension and donor steric tuning effectively program H‐aggregation within D‐π‐A systems. While TSIC/MTSIC and TBSIC/MTBSIC share similar monomeric photophysics, only the BS‐containing molecules are oriented to H‐packing. Notably, MTBSIC achieves an exceptionally small 15 nm absorption blueshift after H‐aggregation, significantly smaller than those of previously developed H‐aggregate phototheranostic agents (Figure S11), which should be attributed to the asymmetric D‐π‐A skeleton and the regulated donor steric hindrance. The tunable H‐aggregation of TBSIC/MTBSIC illustrates that H‐aggregate blueshifts are not intrinsic and can be molecularly engineered, paving the way toward designing H‐aggregate photo‐functional materials with minimal spectral penalties.

2.4. Molecular Engineering Regulated H‐Aggregation

According to exciton coupling theory, the absorption blueshift in H‐aggregates increases with the exciton coupling strength, which is highly sensitive to the slipping angles between adjacent packing molecules [43, 52]. A rational strategy to modulate the absorption blueshift is, therefore, to tune the accessible π‐overlap via molecular steric engineering. To further validate the universality of our strategy, the peripheral donor of the D‐π‐A scaffold was modified to regulate the H‐aggregation behaviours and absorption blueshift (Figure 3a). Two peripheral phenyl rings of triphenylamine were either replaced with ethyl groups to afford DE‐BSIC with reduced steric hindrance, or linked with a carbon single bond (i.e., carbazole) to afford Cz‐BSIC with enhanced steric hindrance (Figures S59 and S72–S75). DFT calculation confirmed the ethyl groups lie in‐plane in DE‐BSIC, while carbazole significantly twists out‐of‐plane in Cz‐BSIC, imposing a much larger steric hindrance effect than triphenylamine in TBSIC (Figure 3b; Figure S12). ESP maps showed enhanced positive ESP at the donor site of DE‐BSIC, indicating the much stronger donor/acceptor interaction that favors ideal antiparallel H‐packing with a large π‐π orbital overlap (Figure S13). In contrast, the positive ESP shifts toward the π‐bridge in Cz‐BSIC, hinting at decreased π‐plane overlap.

FIGURE 3.

(a) Schematic of modifying the steric hindrance on the donor site to regulate the H‐aggregation tendency. (b) The optimized molecular configurations of Cz‐BSIC and DE‐BSIC from the top and side view (c, d) The absorption spectrum changes of (c) DE‐BSIC and (d) Cz‐BSIC in THF/water mixtures with different water fractions. (e) Absorption peak changes of DE‐BSIC, Cz‐BSIC, and TBSIC in THF/water mixtures with different water fractions. (f) Schematic of peripheral steric regulation at the donor part in TBSIC. (g) Absorption peak changes of Me‐BSIC, n‐Bu‐BSIC, and t‐Bu‐BSIC in THF/water mixtures with different water fractions. (h) Schematic of H‐aggregate destruction via alkyl group modification. (i) Absorption spectra of C8‐MTBSIC and MT‐C8‐BSIC in molecular or aggregate states.

In THF/water mixtures (f_w > 50%), both DE‐BSIC and Cz‐BSIC formed H‐aggregates, evidenced by clear absorption blueshifts (Figure 3c,d; Figure S14). Critically, upon H‐aggregation, the more twisted Cz‐BSIC exhibited a smaller blueshift (40 nm) than TBSIC (58 nm), whereas the relatively planar DE‐BSIC showed a larger blueshift (71 nm) (Figure 3e). Thus, increasing donor twist reduces effective face‐to‐face overlap and coupling strength, favoring small absorption blueshift in H‐aggregates. Conversely, increasing planarity promotes extensive π‐orbital overlaps and leads to a large blueshift in absorption. Notably, DE‐BSIC displayed a slight absorption redshift upon increasing f_w from 70% to 90% (Figure 3e; Figure S14a). This phenomenon can be rationalized by its dynamic structural relaxation from a kinetically trapped H‐packing state to a more thermodynamically favored H‐packing state, with the latter exhibiting slightly reduced slip angles. Fluorescence changes also corroborated these trends. Although H‐packing suppresses radiative decay, more twisted Cz‐BSIC exhibits a predominant AIE effect with an AIE factor (α_AIE) of 3.8, while relatively more planar DE‐BSIC behaves as a typical ACQ fluorophore with continuously decreased emission as the f_w increased, in line with its ideal H‐packing (Figure S15). The distinct aggregation behaviors of Cz‐BSIC and DE‐BSIC clearly confirm that donor steric twist could finely regulate the H‐packing and absorption blueshift degree.

Steric hindrance effect at the periphery of the triphenylamine site in TBSIC was further investigated by methyl (Me‐BSIC), t‐butyl (t‐Bu‐BSIC), and n‐butyl (n‐Bu‐BSIC) group incorporation (Figure 3f; Figures S60 and S76–S82). All the molecules maintained their H‐aggregation tendency with clear absorption blueshift during the aggregation process (Figure 3g; Figure S16). Notably, introducing peripheral methyl groups reduced the absorption blueshift degree to 26 nm for Me‐BSIC (f_w = 90% versus 70%), much smaller than that of TBSIC (58 nm). However, changing the peripheral group to bulkier n‐butyl or t‐butyl groups didn't further reduce the absorption blueshift upon H‐aggregation, indicating a steric saturation point beyond which the H‐packing conformation becomes relatively invariant. The methyl (Me‐BSIC) substitution appears to be sufficient to reach this plateau point (blueshift of 19 nm, from monomer at 607 nm to H‐aggregates at 588 nm, Figure S17). Moreover, all three alkylated derivatives showed similar AIE titration profiles (Figures S18 and S19), and the lower fluorescence of n‐Bu‐BSIC and t‐Bu‐BSIC compared to Me‐BSIC after aggregation should be caused by their enhanced vibrational and rotational relaxation in the excited states, rather than by differences in the aggregation types.

To further clarify the steric‐planarity design principle in H‐packing modulation, steric n‐octyl groups were introduced either on the triphenylamine donor or on the π‐bridge, yielding C8‐MTBSIC and MT‐C8‐BSIC, respectively (Figure 3h; Figures S61 and S83–S86). Extending the alkyl group from methyl to n‐octyl groups on the donor periphery minimally affected the H‐aggregation tendency of MTBSIC derivatives, with C8‐MTBSIC absorption blue‐shifted from 625 to 606 nm and greatly diminished fluorescence upon aggregation (Figure 3i; Figure S20). However, MT‐C8‐BSIC exhibited red‐shifted absorption upon aggregation, hinting that steric hindrance at the π‐bridge disrupts H‐aggregation tendency. This was further corroborated by the pronounced AIE feature of MT‐C8‐BSIC (α_AIE = 6.7) (Figure S21). In addition, sonication during the assembly process caused minimal influence on MTBSIC aggregation at f_w = 80% and 90%, but induced a metastable, more blue‐shifted H‐aggregate of MTBSIC (580 nm) at f_w = 70% that is inaccessible through spontaneous assembly (Figure S22), further validating the steric hindrance strategy in suppressing such undesired large spectral shifts. Collectively, strong π‐π interactions drive H‐packing in D‐π‐A scaffolds, while peripheral steric design allows precise control of slip angles and effective π‐overlaps, thereby tuning absorption blueshifts. Importantly, modest donor‐steric (Me/OMe‐substituted triphenylamine) yields minimal‐blueshift H‐aggregates without destroying packing order, providing a generalizable design rule for near‐monomer‐like, NIR‐absorbing H‐aggregates as high‐performance phototherapeutic agents.

2.5. Characterization of H‐Aggregates NPs

The effect of H‐aggregation on type I ROS generation was further evaluated with TBSIC/MTBSIC H‐aggregates against their counterparts, TSIC/MTSIC amorphous aggregates, since TBSIC/TSIC and MTBSIC/MTSIC exhibited similar monomeric photophysical properties (Figure 4a). These molecules were fabricated into colloidally stable NPs with an amphiphilic DSPE‐PEG₂₀₀₀ matrix, yielding similar hydrodynamic sizes of ∼35 nm and similar size distributions for all NPs (Figure 4b, Figures S23 and S24). MTBSIC NPs exhibited minimally changed sizes upon storage in DMEM for 7 days (Figure S25), indicating their excellent colloidal stability. These NPs also exhibited consistent absorption spectra, resembling their bare aggregates, indicating that both H‐type and amorphous aggregation are retained upon polymeric encapsulation (Figure 4c; Figure S26). Specifically, TBSIC NPs and MTBSIC NPs exhibited absorption blueshifts of 50 nm and 16 nm, relative to their monomers in THF. Their emission behaviours were also consistent with their aggregates (Figure 4d; Figure S27). Moreover, MTBSIC NPs exhibited a sharp powder X‐ray diffraction (PXRD) pattern with pronounced π–π stacking peaks similar to the single crystal (Figure S28). The slight shifts of these peaks shall be attributed to their interaction with DSPE‐PEG₂₀₀₀ polymers, which leads to slightly increased stacking distances. Nevertheless, the similar absorbance blueshift in MTBSIC aggregates and NPs suggests their similar H‐packing structure.

FIGURE 4.

(a) Schematic of H‐aggregates for enhanced type I ROS generation under light irradiation. (b) Hydrodynamic diameters of TSIC NPs, TBSIC NPs, MTSIC NPs, and MTBSIC NPs, detected by DLS. (c) Absorption spectra and (d) emission spectra of TSIC NPs, TBSIC NPs, MTSIC NPs, and MTBSIC NPs. The relative fluorescence intensity changes (I/I₀ ) of (e) DCFH, (f) DHR123 and HPF in the presence of MTSIC NPs, MTBSIC NPs or Ce6 under 660 nm irradiation. The relative fluorescence intensity changes (I/I₀ ) of g) DCFH, (h) DHR123, and HPF in the presence of TSIC NPs, TBSIC NPs or Ce6 under white light irradiation. (i) The calculated excited‐state energy distributions, SOC(ξ), and (j) hole and electron distribution of MTSIC A‐dimer and MTBSIC H‐dimer‐1. (k) The photocurrent test of MTBSIC and MTSIC aggregates.

The crucial ROS generation ability was further investigated with DCFH, DHR123, HPF, and ABDA as total ROS, O₂ ^•−, ⚫OH, and ¹O₂ detection probes, respectively [53]. Based on their different absorption windows, MTSIC NPs and MTBSIC NPs were excited with a 660 nm laser, while TSIC NPs and TBSIC NPs were excited with white light. Total ROS generation was reflected by DCFH fluorescence enhancement factors (Figure 4e; Figure S29). MTBSIC NPs exhibited a remarkably high DCFH fluorescence enhancement factor of 115.33 within 90 sec of 660 nm laser irradiation, enhanced by 2.04 times over amorphous‐packed MTSIC NPs. Additionally, MTBSIC NPs also showed much better O₂ ^•− and ⚫OH generation ability over MTSIC NPs, as illustrated by the fluorescence enhancement of DHR123 (135.39 versus 103.29) and HPF (26.79 vs 19.30) (Figure 4f; Figures S30 and S31). The quantum yield of O₂ ^•−, the dominant ROS species generated by MTBSIC NPs, was estimated to be 6.5% under 520 nm laser irradiation, using Rose Bengal as the reference standard (Figure S32) [54, 55, 56]. However, both MTSIC NPs and MTBSIC NPs exhibited minimal ABDA absorption decline and negligible ¹O₂ generation (Figure S33) with a negligible quantum yield of 0.28% for MTBSIC NPs [57], indicating their pure type I ROS generation ability. In contrast, the commercial PS Ce6 only exhibited a moderate and ¹O₂‐predominant ROS generation ability. The different types of ROS species generated by MTBSIC NPs were further confirmed by electron spin resonance (ESR) analysis (Figure S34), consistent with the fluorescence and absorbance probe‐based assays. Similarly, TBSIC NPs also exhibited much better type I ROS generation, as compared to TSIC NPs (Figure 4g,h; Figures S35–S38). The ROS comparisons were summarized in Figure S39, which clearly proves that H‐aggregation specifically promotes type I ROS generation.

In addition, H‐aggregation also conferred operational robustness. H‐packed TBSIC NPs and MTBSIC NPs exhibited enhanced photostability over amorphous‐packed TSIC NPs and MTSIC NPs (Figures S40 and S41). Moreover, MTBSIC NPs delivered excellent photothermal conversion ability, causing a rapid temperature elevation of 39.0°C within 10 min of irradiation (100 µm) with a photothermal conversion efficiency (PCE) of 71.94%, while MTSIC NPs only elevated the temperature by 30.9°C with a PCE value of 49.97% (Figure S42). MTBSIC NPs also showed concentration‐ and power‐dependent photothermal effects and excellent photothermal stability, with nearly unchanged temperature elevation ability in five successive laser on/off cycles (Figure S43). The high photothermal effect and superior ROS generation of MTBSIC NPs shall be attributed to the unique advantages of the appropriate H‐type exciton coupling, which suppresses the radiative fluorescence pathway and redirects the excited‐state energy dissipation pathways towards internal conversion and ISC for robust heat and ROS generation (Figure 4a) [26, 28, 29]. Notably, both amorphous MTSIC NPs and H‐packed MTBSIC NPs predominantly generate type I ROS, which shall be attributed to their asymmetric D‐π‐A scaffold that favors the electron‐transfer pathway. The further enhanced type I ROS generation in MTBSIC NPs shall be contributed by the suppressed radiative decay and further promoted electron transfer capability in H‑aggregates [25]. Leveraging these photophysical advantages, MTBSIC delivers amplified type I ROS generation, high photostability, and synergistic photothermal effects, making it a favorable candidate for multimodal phototheranostics.

2.6. Mechanism Study of H‐Aggregation‐Enhanced Type I ROS Generation

To unravel the type I ROS enhancement mechanism, time‐dependent (TD)‐DFT calculations were performed on the extracted MTSIC amorphous dimer (A‐dimer) and MTBSIC H‐dimers. MTBSIC H‐dimers exhibited dominant electron transitions to high‐lying S₄ singlet state with oscillator strength (f) > 2.00, while MTSIC dimer favors low‐lying S₀ → S₁ (f = 1.23) and S₀ → S₂ (f = 1.66), proving that the stronger excitonic coupling in H‐packing blocks low‐lying transitions (Figure S44 and Table S2), consistent with the experimental absorption blueshifts in MTBSIC H‐aggregates. The ISC landscape also changes with its distinct packing. At the monomer level, MTSIC (ξ_S1‐T3/4/5> 0.50 cm⁻¹) shows more apparent ISC channels and larger spin‐orbit coupling (SOC) values than MTBSIC (ξ_S1‐T2 = 0.20 cm⁻¹, ξ_S1‐T3 = 0.35 cm⁻¹) (Figure S45). However, the trend inverts at the dimer levels (Figure 4i; Figure S46), where MTBSIC H‐dimers exhibited multiple ISC channels to neighboring triplet states (E_ST < 0.3 eV) with larger SOC values (e.g., ξ_S1‐T4 = 0.45 cm⁻¹, ξ_S1‐T5 = 0.26 cm⁻¹ in H‐dimer‐1), while the MTSIC A‐dimer yielded fewer and less effective ISC channels. Thus, H‐packing mode rather than molecular composition alone governs ISC efficiencies (Table S3), explaining the higher triplet population and ROS generation in H‐aggregates.

Frontier molecular orbitals revealed delocalized HOMO and LUMO distributions across the whole MTBSIC H‐dimers, conducive to intermolecular charge transfer (Figure 4j; Figure S47). In contrast, the MTSIC A‐dimer exhibited intramolecular HOMO and LUMO distributions, constrained to their monomers. Hole‐electron analysis further showed spatially separated hole‐electron distributions across the whole MTBSIC H‐dimers (Figure S48). In contrast, MTSIC A‐dimer remained intramolecularly distributed electrons and holes, hinting at its limited charge transfer and separation ability. These theoretical findings were further corroborated by photocurrent measurements, where the MTBSIC aggregates generated a significantly stronger photocurrent than the MTSIC aggregates, confirming more efficient transfer/separation in H‐aggregates (Figure 4k). In addition, MTBSIC also exhibited enhanced ROS generation upon increasing f_w in THF/water mixtures (Figure S49), further confirming the H‐aggregation‐enhanced ROS production. In summary, H‐aggregation not only enhances triplet population by facilitating multiple and effective ISC channels, but also promotes intermolecular charge transfer and separation, which underlies the significant boost in type I ROS generation.

2.7. In Vitro Antitumor PDT of H‐Aggregates

Given the predominant type I ROS generation of H‐packed MTBSIC NPs, their phototherapeutic effect was further evaluated at the cellular level, with 4T1 mouse breast cancer cells (RRID: CVCL_0125) as the model cell line. After 24 h incubation and laser irradiation, intracellular ROS generation was visualized with 2,7‐dichlorodihydrofluorescein diacetate (DCFH‐DA) probe (Figure 5a). NPs only or laser only groups exhibited negligible fluorescence. MTBSIC NPs + L (L refers to 660 nm laser irradiation) group showed a markedly brighter green fluorescence than the MTSIC NPs + L group under normoxic conditions, indicating more efficient intracellular ROS generation by H‐packed MTBSIC NPs. Notably, MTBSIC NPs still delivered higher DCFH fluorescence than MTSIC NPs under hypoxic conditions, which retained 86% of the value under normoxic conditions (Figure S50), indicative of their effective anti‐hypoxia type I ROS generation feature.

FIGURE 5.

(a) The confocal images of 4T1 cells stained with DCFH‐DA within different groups, scale bar = 50 µm. Cell viabilities of 4T1 cells incubated with (b) MTBSIC NPs and (c) MTSIC NPs with or without 660 nm laser irradiation (0.2 W/cm², 10 min). (d) The live/dead staining confocal images and (e) flow cytometry analysis of 4T1 cells within different groups, scale bar = 200 µm.

The tumoricidal efficacy was further evaluated using the methylthiazolyldiphenyl‐tetrazolium bromide (MTT) assay, where both NPs showed negligible dark toxicity (Figure 5b). Similar to their intracellular ROS generation, MTBSIC NPs exhibited significantly higher phototoxicity than MTSIC NPs (Figure 5c). Moreover, MTBSIC NPs retained their effective phototoxicity with an IC₅₀ of 8.81 µm under hypoxic conditions (Figure S51). Selective inhibition MTT assays confirmed that ROS‐mediated PDT (∼65%) is the predominant mechanism of tumor cell killing (Figure S52). Live/dead cell staining assay using calcein‐acetoxymethyl ester and propidium iodide also corroborated these trends, with predominant dead‐cell‐indicative red fluorescence across the whole observation window for MTBSIC NPs + L group under both normoxic and hypoxic conditions (Figure 5d). Flow cytometry analysis further revealed 87.55% of apoptotic cells in the MTBSIC NPs + L group with 63.22% in early apoptosis and 24.33% in late apoptosis, exceeding the MTSIC NPs + L group (65.60% of apoptotic cells) (Figure 5e). Minimal apoptosis was observed in the NPs only or laser‐only groups, supporting the photo‐triggered killing efficacy and excellent dark biocompatibility of MTBSIC NPs.

2.8. In Vivo Tumoricidal Performance of H‐Aggregates

The in vivo tumoricidal effect was further evaluated in 4T1‐tumor‐bearing BALB/c mice (with approval No. 2024D038 from the Animal Ethics Committee of South China Agricultural University). When tumor volumes reached around 100 mm³, the mice were randomly divided into 6 groups (n = 5 mice per group): PBS, MTSIC NPs, MTBSIC NPs, PBS + L (L refers to 660 nm laser irradiation), MTSIC NPs + L and MTBSIC NPs + L. At 2 h post‐intratumoral injection of PBS, MTSIC NPs or MTBSIC NPs, selected mice were irradiated with a 660 nm laser for 10 min. The photothermal imaging revealed that the tumor temperature in the MTBSIC group reached ∼48°C upon laser irradiation, higher than that of the MTSIC‐treated group (Figure S53), consistent with the superior photothermal effect of MTBSIC. The tumor volumes and the body weights of the mice were then monitored for 14 days. Dark groups and laser‐only groups showed minimal tumor inhibition effect, with tumor volume increasing over 7‐fold on day 14. In contrast, the MTBSIC NPs + L group showed the best tumor suppression performance with tumor volume increasing only by ∼62% over 14 days, while the MTSIC NPs + L group delivered a moderate tumor inhibition ability with a 2.05‐fold increase in tumor volume (Figure 6a; Figure S54).

FIGURE 6.

(a) The relative tumor volume growth curves of mice from different groups; L refers to 660 nm (0.4 W/cm², 10 min). (b) The photographs and (c) weights of tumors from different groups, extracted on day 14. (d) The body weight changes of mice within 14 days. (e) H&E, Ki‐67, and TUNEL staining images of tumor tissues from different groups, scale bar = 100 µm.

The best tumor inhibition effect of MTBSIC NPs + L group was also evidenced by the photographs and weights of tumors extracted on day 14 (Figure 6b,c). Moreover, no significant variation in mice's body weights was observed within 14 days (Figure 6d), indicating the excellent biosafety of NPs and laser treatment. Histological and immunohistochemical analyses were performed with hematoxylin and eosin (H&E) staining, Ki‐67 staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. H&E images showed more severe nucleus shrinkage in the MTBSIC NPs + L group than in the MTSIC NPs + L group, whereas no obvious cell damage was seen in other groups (Figure S55). Ki‐67 staining showed the weakest green fluorescence in the MTBSIC NPs + L group, indicating the highest loss of the proliferative ability of tumor cells. The TUNEL images also suggested the most serious apoptotic level of tumor cells in the MTBSIC NPs + L group (Figure 6e).

The biosafety and biocompatibility of these NPs were further evaluated in healthy mice by intravenous injection with PBS, MTSIC NPs, and MTBSIC NPs. The hematological parameters were all within the normal ranges, suggesting no systemic inflammation was caused (Figure S56). The serum hepatic and renal function parameters in mice injected with NPs were similar to those in the PBS control group, indicating the high safety of these NPs (Figure S57). Collectively, the prepared MTBSIC NPs showed the strongest tumor inhibition effect and good biocompatibility, serving as an excellent phototherapeutic agent for cancer treatment.

3. Conclusion

In summary, we established a rational molecular engineering paradigm by balancing the planarity and steric hindrance in D‐π‐A scaffolds to program excitonic coupling in antiparallel H‐type supramolecular aggregates for minimal absorption blueshift. By first enhancing planarity through π‐bridge engineering (from TSIC to TBSIC) to strengthen π‐orbital overlap, antiparallel packed H‐aggregates are fabricated for BSIC derivatives. Subsequent donor steric control dictates the slipping angles and π‐π overlap, allowing regulation of the absorption blueshift for these H‐aggregates. Based on π‐bridge engineered H‐aggregation, the structure exploration reveals a clear structure‐packing‐photophysical property relationship: a more twisted donor favors a smaller absorption blueshift by enforcing a larger slip angle (e.g., Cz‐BSIC), whereas a more planar donor leads to a larger absorption blueshift (e.g., DE‐BSIC). We also identify a donor steric threshold (methyl/methoxy substitution) beyond which blueshift becomes invariant. With these design rules, we achieved an optimized D‐π‐A candidate MTBSIC, with an exceptionally small absorption blueshift of 15 nm upon H‐aggregation, preserving NIR absorption while maintaining H‐packing. The antiparallel packed H‐aggregates further enhance ISC and intermolecular transfer/separation, delivering a dual mechanistic advantage for type I PSs design. Consequently, the developed H‐packed MTBSIC NPs effectively and predominantly generate type I ROS under laser irradiation and show excellent antitumor effects and good biocompatibility both in vitro and in vivo. This planarity‐hindrance co‐balance strategy not only offers a unified route to achieve nearly non‐hypsochromic shift H‐aggregates for high‐performance NIR‐absorbing materials, but also provides compelling design principles for tunable photo‐functional π–π packing systems, a key objective of the supramolecular assembly field.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: adma73868‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.