Phosphorescent supramolecular systems for medicine anticounterfeiting

Domain: chemistry
Published: 2026-02-11
Editorial outcome: major_revision

Abstract
Realizing reliable medicine anticounterfeiting with safe and robust materials remains a challenge. We address this issue by preparing a class of edible phosphorescent supramolecules (VB10@α/β-CDs) based on easily available α/β-cyclodextrins (α/β-CDs) and vitamin B10 (VB10). Concisely grinding them with water or co-crystallization from aqueous solution, the resulting host−guest complexes VB10@α/β-CDs exhibit a long phosphorescence lifetime of up to 1.16 s and a high photoluminescent quantum yield of up to 86.5%, The encapsulation of α/β-CDs reverses the energy ordering of VB10’s excited singlet states, promotes the formation of the minimum energy crossing point (MECP) between singlet state and triplet state, and therefore boosts phosphorescence. VB10@α/β-CDs are attractive as phosphorescent inks for in-medicine anticounterfeiting because of the advantages of an edible nature, good moisture robustness, room-temperature phosphorescence and circularly polarized luminescence. Therefore, the present phosphorescent supramolecules as well as the elucidated MECP-involved mechanism would promote in-depth understanding of phosphorescence enhancement strategy.

Introduction
Counterfeiting medicines are a tremendous threat to patient safety and public health. More than 280,000 children in sub-Saharan Africa die every year because of counterfeiting and substandard medical products. The involved medicine is worth up to US$100 billion all over the world. Chemical analysis and chromatography/spectroscopy technologies for pharmaceutical ingredient detection, and anticounterfeit methods at the packaging level are commonly used to combat counterfeiting medicines. These methods work well with preconditions that expensive experiments/skilled personnel were equipped or that the drugs are not repackaged. In-medicine anticounterfeiting (or in-dose authentication) is a more advanced and effective method because every individual pill or capsule is verified independently of the packaging. Within this set, encrypting and printing information on the surface of pills and capsules by luminescent materials is particularly attractive due to low cost and intuitive detection. However, there are still no perfect luminescent materials for ink marking because of the strict requirements of in-medicine anticounterfeiting, such as non-toxic preferably edible, high signal-to-noise ratio of luminescence, and good robustness under ambient conditions.

Cyclodextrins (CDs) are widely used pharmaceutical excipients and are recognized as edible macrocyclic molecules. The hydrophilic surface and hydrophobic cavities make CDs excellent water-soluble macrocyclic hosts for encapsulating various organic guests. Pioneering works have demonstrated that the encapsulation can usually alter the physical and chemical properties of guests. Vitamin B10 (VB10) is an essential component of the folate, which is necessary for cellular growth and differentiation. It is sold as an essential nutrient when normal synthesis by intestinal bacteria is insufficient. Although VB10 exhibited a weak blue fluorescence, the theoretical calculations showed that it has multiple singlet states and triplet state. We postulated that it would be possible to regulate its excited state to induce room-temperature phosphorescence (RTP) and produce a good phosphorescent ink-marking material by encapsulating with CDs. RTP has a reputation of long persistent luminescence after ceasing irradiation, which endow RTP materials intrinsic advantages for anticounterfeiting. In particular, the well-developed constructing strategies for RTP materials provided enriched toolbox for designing and preparing RTP anticounterfeiting materials, such as crystallization engineering, doping into matrix, polymerization, self-assembly, host-guest interactions, and etc.

In addition, quantum chemistry calculations have been proven to be important tools for expounding on the underlying mechanisms of luminescent materials. The calculation of absorption spectra is well-established, and the HOMO/LUMO energy levels of the ground state are commonly used to infer the properties of excited states. However, revealing the luminescence mechanism through calculations, particularly for large organic molecular systems or even organic supramolecular systems, remains a significant challenge. Understanding the complete photophysical processes and the dominant decay pathways is essential for identifying the key factors that influence luminescence and for designing improved luminescent materials. The interactions between electron spin and orbital angular momentum, as well as between electrons and atomic nucleus, lead to complex low-lying excited-state crossings and multiple decay pathways for the excited molecules. Theoretically, while multi-reference methods offer higher accuracy, they make the quantitative calculation of photophysical processes for large systems expensive and complex, thus difficult to accomplish. Therefore, it is of great significance to develop a computational strategy that is suitable for large organic molecule/supramolecule system in the solid state.

Herein, two edible phosphorescent supramolecules, namely VB10@α-CD and VB10@β-CD, are facilely prepared by grinding VB10 and α/β-CD with water or co-crystallizing them from aqueous solution (Fig. 1). The resulting supramolecules show a photoluminescence quantum yield of up to 86.5%, a lifetime of up to 1.16 s, a visible afterglow lasting for 6 s, and good robustness in ambient conditions. The edible VB10@α-CD as phosphorescent ink-marking material is applied in the in-medicine anticounterfeiting. Moreover, we specifically develop a computational approach that combines single-reference time-dependent density functional theory (TDDFT) with the ONIOM model, with a focus on the minimum energy crossing point (MECP), to investigate the luminescence mechanism of VB10@α-CD. The computational results not only agree with the experimental data, but also offer clear insights into the distinct luminescent behavior of VB10@α-CD in both solution and the solid state. The crucial roles of non-radiative processes for phosphorescence are clearly revealed. The hydrogen bonds provided by CD regulate singlet states (Sn) of VB10, promote the formation of MECP between singlet state Sc and triplet state T1, and therefore boost phosphorescence. Thus, phosphorescent supramolecules VB10@α/β-CDs are suitable systems that combine edibility, low cost, high performance, robustness, and a fully elucidated solid-state phosphorescence mechanism. The phosphorescent supramolecular ensemble represents a paradigm for developing phosphorescent materials, which may find promising utility in high-security anticounterfeiting scenario within the pharmaceutical industry.

Results
The VB10 itself exhibited weak blue fluorescence (λmax = 404 nm) with a lifetime (τ) of 1.35 ns and a photoluminescence quantum yield (PLQY) of 1.8% (Supplementary Figs. 2 and 3). After grinding it with α-CD, which is known as a non-luminous macrocycle, the obtained VB10/α-CD displayed intense blue emission and a bright blue afterglow lasting up to 4 s with a lifetime of 210 ms and PLQY of 16.5% under ambient condition (Supplementary Figs. 7−10). This result indicated that α-CD could “turn on” the phosphorescence of VB10. After dissolving the VB10/α-CD into water and slowly evaporating for 6 days, the obtained single crystal of VB10/α-CD showed a longer blue afterglow (Fig. 2a and Supplementary Fig. 11). The photoluminescence (PL) spectra, delayed PL spectra, and time-resolved PL decay curves indicated that the afterglow is phosphorescence (λmax = 434 nm) with a lifetime of 977 ms, along with fluorescence peak at 340 nm (τ = 1.38 ns) (Figs. 2a, b and Supplementary Fig. 12). Notably, the PLQY of VB10@α-CD is up to 82.1%, indicated the great promotion of α-CD for the phosphorescence of VB10 (Fig. 2c and Supplementary Fig. 13). The longer lifetime and higher PLQY of crystals than that of grinded samples are probably because of better encapsulation of α-CD for VB10. The high-performance luminescence in ambient conditions proved its good robustness and provided an essential requirement for applications.

Since α-CD is a macrocycle possessing a chiral cavity, it is speculated that the chiral transfer between α-CD and VB10 may induce circular dichroism and circularly polarized luminescence (CPL) signals. The circular dichroism spectrum of solid VB10@α-CD features a stronger positive Cotton effect at 335 nm compared with that in aqueous solution (Supplementary Fig. 14). As shown in Supplementary Fig. 15, the VB10@α-CD exhibited positive distinct dual CPL including fluorescence (λmax = 340 nm) and phosphorescence (λmax = 440 nm), with |glum| values of 1.5×10-2 and 7.5×10-3, respectively. Considering the high PLQY, long lifetime, and relatively high |glum| values, the VB10@α-CD is among the high-performance organic RTP systems with CPL property (Supplementary Table 1). The phosphorescent CPL was further proven by the intense emission of CPL measurement in phosphorescence mode (Fig. 2d and Supplementary Fig. 16). These results verified that the chiral transfer from α-CD to VB10 could produce CPL. The relationship between the host−guest molar ratios and the phosphorescent properties of VB10/α-CD was further evaluated. When the molar ratios of VB10:α-CD increased from 1:1 to 1:1000, the afterglow remained and the PL spectra showed a slightly blue shift from 340 nm to 331 nm, which probably due to more completed encapsulation for VB10 (Supplementary Fig. 17a, b). The identical PL spectra of VB10@α-CD at 1:100 and 1:1000 revealed that VB10 was encapsulated by α-CD in these molar ratios. More α-CD probably translated more VB10 into the supramolecule VB10@α-CD, and therefore exhibited stronger emission. The phosphorescence lifetimes were at a range of 214−302 ms and PLQY increased from 16.5% for 1:1 to 33.2% for 1:1000, indicating that better encapsulation might occur by increasing the molar ratio of α-CD (Supplementary Figs. 17c, 17d and 18). It was hypothesized that this phosphorescence enhancement could be due to the encapsulation of α-CD for VB10.

The encapsulation of α-CD for VB10 was verified by 1H NMR and 1H−1H NOESY NMR spectroscopy, isothermal titration calorimetry (ITC), and X-ray single crystal diffraction analysis. The protons Ha and Hb in VB10 showed downfield shifts of 0.01 and 0.18 ppm, respectively, after the addition of 2 equiv α-CD (Fig. 3a and Supplementary Fig. 19). This is a typical complexation-induced shift because of the hydrogen-bonding between CD and guests, proving the encapsulation and verifying that VB10/α-CD is the supramolecular complex VB10@α-CD. In the 1H−1H NOESY NMR spectra of VB10@α-CD, cross-peaks between Ha−H3,5 and Hb−H3,5,6 provided evidence for the close proximity between the phenyl group of the VB10 and the cavity of the α-CD, suggesting a deep encapsulation mode (Fig. 3b and Supplementary Fig. 20). ITC gave a binding constant (K) of (2.5±0.2)×103 M-1, accompanying with favorable enthalpy change and positive entropy contribution (Fig. 3c). Diffraction grade single crystals of VB10@α-CD were obtained as colorless block through the slow evaporation of an aqueous solution containing the α-CD and VB10 (1:1) at room temperature for 6 days. The resulting single-crystal structures were shown in Fig. 3d with associated data given in Supplementary Table 2. VB10 is deeply located into the cavity of α-CD, with COOH and NH2 groups pointing into the primary face and secondary face, respectively. The dihedral angle between the plane of the phenyl in VB10 and the cross-section formed by six glycosidic oxygen atoms in α-CD is 86.4°, revealing a vertical encapsulating manner for VB10 (Supplementary Fig. 21). The host−guest complexes stacked in register to form supramolecular structures. Furthermore, the COOH and NH2 groups formed multiple hydrogen bonds with its host α-CD and adjacent α-CD (1.68, 1.90, 2.00, 2.01, 2.07, 2.12, 2.17, 2.45, 2.54, and 2.91 Å) (Supplementary Fig. 22). Thus, the reason of robust phosphorescence in ambient conditions is clearer. Normally, the triplet states Tn (n≥1) of phosphor suffer serious quenching even by a very low concentration of oxygen, water, impurities, or aggregation-caused quenching (ACQ) because of triplet energy migration. In the case of supramolecule VB10@α-CD, host α-CD acted as a good shell to shelter the chromophoric core (VB10) by suppressing the above quenching and therefore stabilized the triplet states Tn (n≥1). Furthermore, every VB10 molecule in the VB10@α-CD supramolecule is in an individual micro-environment provided by α-CD, making each host−guest complex an independent phosphor unit. Possible defects (empty α-CD or free VB10) would not cause serious triplet energy migration and therefore had little effect on the whole phosphorescent performance. Collectively, these advantages promoted the robust phosphorescence of VB10@α-CD under ambient conditions.

To further elucidate the origin of phosphorescence in the solid state, we employed quantum mechanical calculations for VB10@α-CD (Supplementary Figs. 23−32). The ONIOM model was utilized, treating α-CD as the surrounding environment, to carry out the simulations using the PM6 semi-empirical method with D3 dispersion correction, while the core VB10 was calculated using the PBE0(GD3BJ)/6-31G* method. The detailed dynamics of the excited states were analyzed as follows: First, the ground state structure (S0min) was fully optimized. Subsequently, vertical excitation energies were computed using the TDA method, identifying the excited states corresponding to the main experimental absorption. Finally, the dynamics of the transition from the main excited states to the triplet state associated with phosphorescence was quantitatively described. The dominant electronic transitions of the three important low-lying excited singlet states are shown in Fig. 4a, and Supplementary Figs. 33−36, and Supplementary Data 1. Due to the very small energy differences among them and variable relative location, conventional naming rules such as S1, S2 and Sn are unsuitable. Therefore, we designated these states as singlet state A (SA, f=0.5364), singlet state B (SB, f=0.0025), and singlet state C (SC, f=0.0003) based on their oscillator strength values (f). Among these, SA corresponds to the main absorption band.

The non-radiative relaxation process of VB10@α-CD in the solid-phase ONIOM model is depicted in Fig. 4b, starting from the Frank-Condon point, passing through minimum energy crossing points (MECPSA/SB and MECPSB/SC), and crossing two very small energy barriers (1.2 kcal/mol between SA (118.6 kcal/mol) and MECPSA/SB (119.8 kcal/mol), and 0.4 kcal/mol between SB (113.4 kcal/mol) and MECPSB/SC (113.8 kcal/mol)). Based on the small barrier, we infer that VB10@α-CD is likely to reach the lowest-energy stationary point (SCmin, 100.1 kcal/mol) among all excited singlet states very quickly, and subsequently emit fluorescence at 375 nm, which is similar with the experimental data of 340 nm. Subsequently, VB10 can cross a very small energy barrier of 1.6 kcal/mol via MECPSC/TA (101.7 kcal/mol), reaching the stable minimum point of low-lying excited triplet state TA (TAmin, 73.2 kcal/mol) (Supplementary Fig. 37). Then, it relaxes from TA to S0min and emits phosphorescence at 426 nm, which is well matched with the experimental data of 434 nm. Figure 4b indicates that the presence of an MECPSC/TA is a key factor for phosphorescence in the solid state by facilitating the relaxation from the SC to TA. In contrast, in aqueous solution, SAmin is the lowest stationary point among all low-lying excited singlet states, and MECP is not obtained through our calculations (Fig. 4c and Supplementary Figs. 38−42). This is consistent with experimental result that no phosphorescence could be detected in the aqueous solution (Supplementary Fig. 43). This discrepancy can be clarified by two factors: First, SCmin is the lowest stationary point among all low-lying excited singlet states in the solid state, while SAmin is the lowest-energy excited singlet state in the aqueous solution. The reversed energy order of SC and SA in the solid-phase ONIOM model and solution phase can be understood by examining the structures of SA in both phases. In the solution phase, the orbital responsible for electron donation in SA excitation exhibits a pronounced antibonding characteristic in the C-N region (Supplementary Fig. 38), leading to a shortening of the C-N bond (1.35 Å) in SA compared to the ground state (S0min) (1.36 Å) (Supplementary Figs. 44 and 45). The structure of SAmin shows that the amino and phenyl groups are in a coplanar arrangement due to conjugation, which is energetically favorable compared to the distorted conformation of SCmin (Supplementary Fig. 46). Therefore, SAmin is the lowest stationary point among all low-lying excited singlet states in the solution phase. In the solid-phase ONIOM model, the amino group in the ground state (S0min) formed three hydrogen bonds with the α-CD (Supplementary Fig. 47). These hydrogen bonds inhibited the formation of a stable conjugated structure of SA, thereby increased its energy and reversed the energy order of SC and SA (Supplementary Figs. 48−51). Second, under the solid-phase ONIOM model, the SC can undergo relaxation to the TA via the MECPSC/TA pathway. However, in aqueous solution, our calculations show that the SAmin lacks any accessible low-energy MECP route, thereby preventing this relaxation process. Speculative analyses of the possible reasons why the MECPSA/TA point could not be located are provided in the Supplementary Information (Supplementary Figs. 41 and 42). It is evident that in the solution state, the main absorbing excited state SA is also the lowest stationary point among all low-lying excited singlet states, making the fluorescence process a more favorable process (the relative energy is 102.5 kcal/mol for SAmin and 110.7 kcal/mol for SCmin). In the solid state, the hydrogen bonds between the host α-CD and the guest VB10 raise the energy of SAmin, making the lower-energy SCmin more readily relax to the TA state via MECPSC/TA, thereby promoting the phosphorescence process.

Since VB10 (G1) possessed an electron-donating group of amino group and an electron-withdrawing group of carboxyl group, we evaluated other guests (G2−G10) to further reveal the relationship between structures and phosphorescence properties (Fig. 5a). After replacing the carboxyl group by carboxylic ester (G2) and sulfo acid (G3), the resulted G2/α-CD showed a dual emission of fluorescence (λmax = 335 nm, τ = 0.90 ns) and phosphorescence (λmax = 420 nm, τ = 96.3 ms) and a PLQY of 34.6%, which is inferior to the VB10@α-CD both in lifetime and quantum yield (Supplementary Figs. 52−54). For G3/α-CD, no phosphorescence was observed (Fig. 5b and Supplementary Figs. 55 and 56). These results revealed that carboxyl group is crucial for the phosphorescence of supramolecules. We then replaced amino group by hydroxyl (G4), carboxyl (G5), and removed it (G6). Both G4/α-CD and G5/α-CD showed obvious phosphorescence (λmax = 414 nm, τ = 324 ms, PLQY = 20.8% for G4/α-CD; λmax = 417 nm, τ = 95.6 ms, PLQY = 9.2% for G5/α-CD) (Supplementary Figs. 57−62). While G6/α-CD have no phosphorescence (Supplementary Figs. 63 and 64). These results indicated that amino group can be replaced by other groups but cannot be removed. We further investigated the influence of positional isomerization of functional groups by comparing m-aminobenzoic acid (G7) with VB10 (p-aminobenzoic acid) and comparing o/m/p-benzenedicarboxylic acid (G8, G9 and G5) with each other. It should be pointed out that o-aminobenzoic acid is unavailable because of its one of role as drug raw materials. Different from the intense phosphorescence of VB10@α-CD, the G7/α-CD have no phosphorescence (Supplementary Figs. 65 and 66). This also occurred in the G8, G9 and G5. Both G8/α-CD and G9/α-CD have no phosphorescence, while G5/α-CD exhibited blue phosphorescence (Supplementary Figs. 67−70). These results verified that positional isomerization of functional groups have great influence on the phosphorescence of supramolecules, and the para-position is the best. As a comparison, the photophysical properties of guests (G2−G9) were also investigated and all of them showed weak emission without any phosphorescence excepted G4 (Supplementary Figs. 71−87 and Supplementary Table 3).

The origin of these differences in phosphorescence properties caused by replacing substituents or changing positions were further clarified by 1H NMR and ITC methods. After titrating with α-CD, guests G2, G4−G6 and G9 showed obvious chemical shifts and K range of (0.68−5.2)×103 M-1, indicated the formation of host−guest complexes guests@α-CD (G2@α-CD, G4@α-CD, G5@α-CD, G6@α-CD, and G9@α-CD) (Supplementary Figs. 88−97 and Supplementary Table 4). For G3, G7 and G8, no thermal change was observed in the ITC measurements although there were tiny chemical shifts in the 1H NMR spectra, revealing the non-bonding between these guests and α-CD (Supplementary Figs. 98−103 and Supplementary Table 4). Interestingly, only guests (G1−2, G4−G5 and G9) that can be encapsulated by α-CD showed phosphorescence, confirming that the formation of supramolecule with α-CD is the essential requirement for the turning on the phosphorescence (Fig. 5c).

α-CD was replaced by larger hosts of β-CD and γ-CD to evaluate the influence of size effect for the phosphorescence of supramolecules. In the solid state, VB10/β-CD exhibited a bright blue luminescence and displayed a persistent blue afterglow up to 6 s after ceasing the 254 nm UV irradiation (Supplementary Fig. 104). The PL spectra, delayed PL spectra, and time-resolved PL decay curves proved the dual emission of phosphorescence (λmax = 434 nm, τ = 1.16 s) and fluorescence (λmax = 335 nm, τ = 1.17 ns), with a PLQY of 86.5% (Supplementary Figs. 105−107). The identical emission peaks, and similar lifetime and quantum yields revealed that the larger host β-CD could also provide similar circumstance for VB10 with that of α-CD. The circular dichroism spectra of VB10/β-CD showed opposite Cotton effect both in the solid state and in aqueous solution (Supplementary Fig. 108). Moreover, VB10/β-CD exhibited positive CPL, with a |glum| value of 3.6×10-3 (Supplementary Fig. 109). For γ-CD/VB10, no phosphorescence could be observed, possible because of unfavorable complexation (Supplementary Figs. 110 and 111). These hypotheses were further proven by 1H NMR spectra and ITC experiments. β-CD could encapsulate VB10 to form a supramolecule VB10@β-CD and showed a K of (7.5±0.4)×102 M-1 (Supplementary Figs. 112 and 113). No detectable thermal change was observed after titrating VB10 with γ-CD, although there were chemical shifts in 1H NMR spectra, indicating the weak interaction between them (Supplementary Figs. 114 and 115). These results verified the importance of size matching between hosts and VB10 and further confirmed the requirement of host−guest complexation for turning on phosphorescence.

The obtained edible phosphorescent supramolecules VB10@α/β-CD have the advantages of high performance, low cost, and good robustness. Therefore, we verified their applications in medicine anticounterfeiting. As aforementioned, encrypting and printing information on or into pills and capsules is an advanced and effective method because medicines can be authenticated independent of the packaging. We explored three methods of surface coating, doping into medicine, and spray display to verify medicine anticounterfeiting. For surface coating, aqueous solution of VB10@β-CD was utilized as a phosphorescent ink to paint on the surface of pills, and no information could be found under daylight. Under the irradiation of 254 nm, labeled “2025” and “LR” appeared (Fig. 6a). They are clearer after ceasing irradiation because background autofluorescence interference from medicine no longer exists. Similarly, this phosphorescent ink can also be used for labeling capsules. Printing the “AM” and “CA” on the surface of amoxicillin (AM) and cephalosporin (CA) capsules by VB10@β-CD, the authenticity of the medicines can only be identified after ceasing irradiation and neither under daylight nor UV lamps (Fig. 6a and Supplementary Fig. 116a). In addition, doping VB10@β-CD into capsule or pills is another method for medicine anticounterfeiting. After mixing small amount (2 mg) of VB10@β-CD powder into medicine AM or MTZ, the obtained capsule and pill displayed persistent blue afterglow after removing the UV irradiation, while the unlabeled contrasts have no such afterglow (Fig. 6b). Interestingly, the afterglow can only be excited by UV wavelength below 320 nm but not long wavelength such as 365 nm, which provided additional guarantee for anticounterfeiting (Supplementary Fig. 116b). Even the pill was chopped, each part of the pill emitted a persistent blue light (Fig. 6b and Supplementary Fig. 116c). The third method is spray display on medicine. Since the encapsulation is the requirement for turning on RTP, β-CD as one of components can be premixed into the pills and the prepared medicine cannot be detected with luminescence except the autofluorescence. Then the medicine authenticity can only be identified by spraying aqueous solution of another component (VB10) which can be co-packaged with medicine (Fig. 6c). This anticounterfeiting cannot be cloned by other methods such as premixing phosphorescent materials into medicine, therefore ensuring high safety. Moreover, the good CPL phosphorescence qualified the supramolecules as useful systems for chiral anticounterfeiting.

Methods
Materials
All reagents and solvents were obtained commercially and used without further purification, unless otherwise noted.

Measurements
1H NMR spectra were recorded on Bruker Avance III 400 MHz instrument. Single crystal X-ray data were collected by direct methods using a Bruker SMART APEX II diffractometer. Photoluminescence spectra and lifetime were obtained on FLS1000 and HAMAMATSU C16361-01. Fluorescence and phosphorescence quantum yields were measured on HAMAMATSU Quantaurus-QY plus. ITC experiments were performed on MicroCal PEAQ-ITC instrument. CPL spectra were measured on JASCO CPL-300 spectrophotometer. CD spectra were measured on JASCO J-815 spectrophotometer.

Crystal structure of VB10@α-CD
A mixed aqueous solution (2 mL) of VB10 (5×10-2 mol/L) and α-CD (5×10-2 mol/L) was passed through a 0.22 μm filter. After a slow evaporation of the solution for 6 days, colorless block crystals were obtained.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Wu et al. report concise phosphorescent supramolecular systems for medicine anticounterfeiting. The\nedible phosphorescent supramolecules VB10@α/β-CD can be facilely prepared by grinding two edible components of VB10\nand α/β-CD with water. The supramolecules showed high quantum yields of 82.1% and 86.5%, respectively, with a visible\nafterglow last for 6 s, and good robustness in ambient conditions. Authors also demonstrated the ingenious applications of\nthe supramolecules in the in-medicine anticounterfeiting. Both VB10 and α/β-CD are common compounds, and it is\nparticularly commendable that their host-guest encapsulation—achieved through a simple process—yields remarkable\nphosphorescent enhancement. The low cost of the materials also paves the way for further practical applications.\nFor luminescence mechanism study, the authors employ a TD-DFT combined with ONIOM multi-layer computational\napproach, realizing a well-considered balance between computational accuracy and resource efficiency for large\nsupramolecular systems. The theoretical results clearly demonstrate that host–guest interactions—particularly hydrogen\nbonding networks formed by cyclodextrins—play a critical role in modulating the ordering of VB10 excited states. This\nprovides valuable mechanistic insight into how the supramolecular environment influences photophysical behavior. A\nminimum energy crossing point (MECP) was successfully located and analyzed, offering strong theoretical support for the\nproposed intersystem crossing (ISC) pathway and the subsequent population of the triplet state. This is a notable strength of\nthe study.\nOn the whole, I think this is an interesting, thoroughly-conducted and well-written manuscript and I strongly support its\npublication in Nat. Commun. after some minor changes:\n1. A NOESY experiment of VB10/β-CD can give more details for the binding mode in aqueous solution, please provide.\n2. Authors stated that no phosphorescence could be observed for the γ-CD/VB10, but there are lifetimes in Table S3, please\ncheck. Moreover, the row height of Table 3 should be adjusted for clearer.\n3. Chemical shifts that move upfield should be denoted with negative values, as exemplified in Figures S85, S87, S97, and\nS100.\n4. As the MECPSC/TA plays a crucial role in explaining phosphorescence formation, it would be appropriate to mention key\ngeometric features (e.g., H-bond stabilization) briefly in the main manuscript instead of relying solely on the SI.\n5. Natural transition orbital (NTO) analysis for the key excited states (SA, SC, TA) would enhance the reader’s\nunderstanding of the underlying photophysical processes.\n6. Beyond locating a single MECP point, scanning the potential energy surfaces (PES) along excited-state relaxation\ncoordinates (e.g., FC→ SC，SC → TA) would provide further mechanistic insight. Use of relaxed scans, or IRC-like paths\nfor excited states could reveal whether there are competing minima or energy barriers affecting radiative or nonradiative\ndecay.", "reviewer_2": "In this paper, Li and Zhao et al. reported a class of edible phosphorescent supramolecular complexes based on α/β-\ncyclodextrins (α/β-CDs) and vitamin B10 (VB10) (VB10@α/β-CDs). The host-guest complex VB10@α/β-CDs exhibits long\nphosphorescence lifetimes, high photoluminescence quantum yields and detectable dual circularly polarized luminescence\n(CPL) in both fluorescence and phosphorescence. Theoretical calculations elucidated the phosphorescence mechanism.\nThe phosphorescent material was further applied to medicine anticounterfeiting, with three anticounterfeiting methods\nsuccessfully validated for both pills and capsules.\nAlthough merging edible drugs and RTP shows promise as an innovative concept, this research needs further refinement to\nreach the publication level of Nature Communications.\nThe phosphorescence of cyclodextrin (CD) complexes with p-aminobenzoic acid (PABA) has been previously reported\n(Room-Temperature Luminescence Properties of p-Aminobenzoic Acid and 4-Phenylphenol Adsorbed on α-\nCyclodextrin/NaCl Mixtures; Phosphorescence Properties of p-Aminobenzoic Acid Immobilized on a Nylon Membrane). The\nauthors must highlight the innovative aspects of their work compared to existing literature.\nSufficient experimental data in the main text is necessary to support the claims. The main teat is too preliminary. Some of the\ncritical characterization data currently adhere to the supporting Information should be moved into the main text.\nThere appears to be an obvious error in Figure 1d and S13a. The labels of CPL and DC assignments are reversed, which\nmust be revised.\nThe CPL spectra of VB10@β-CD have not been studied. Additionally, it is hoped to provide the CD spectra of the solution\nand solid states of VB10@α/β-CDs systems.\nIn the primary anticounterfeiting test, PL is the used. It is not necessary that the coating system showed CPL. It is unclear\nwhy CPL of the complex system is investigated.\nIn Figure 5, α-cyclodextrin (α-CD) is employed as the primary anticounterfeiting substrate. However, it should be noted that\nα-CD has not yet been approved for food applications in several countries. The safety needs to be confirmed.", "reviewer_3": "In this work, the authors propose to use vitamin B10 encapsulated by cyclodextrin as a marker for printing information on the\nsurface of pills and capsules. This would be useful for in-medicine anticounterfeiting, since each pill/capsule can be verified\nindependently. Regarding the photophysics, the idea is to regulate the excited states of vitamin B10 to induce room-\ntemperature phosphorescence, that can be later used to identify the individual capsules/pills.\nTo this end, the authors investigated the photophysical properties of this system and other derivatives, and identified the\nrelevant processes that enable room temperature phosphorescence. Experimentally, they also investigated how the\ncomplex would behave as a marker in pills and capsules. This work shows a thorough investigation of the use of vitamin\nB10 encapsulated by cyclodextrin and its potential use as an anti-counterfeiting medicine\nmarker. This work will likely have a significance on the field and societal impact. Given my expertise, I particularly focused on the computational investigation presented by the authors, where the data provided support the authors’ conclusions. However, I list below some suggestions\nto improve the clarity and precision of the text.\n• Line 89: Please clarify to which type of quantum yield the authors refer to, for instance,\nfluorescence, phosphorescence…?\n• Line 131: There is a floating “This”, please remove.\n• Line 193, Figure 3a, S21-S24: Please, be more precise on what is being shown as “electronic transitions”. Are these the\nmolecular orbitals of the respective states or the ground state? What is the DeltaE shown, HOMO-LUMO gaps or actual\nexcitation energies? The number of decimals on the DeltaE value is also unphysical; the precision is usually around two\ndecimals only.\n• The authors refer to the ONIOM model they created as “solid phase” which is not an accurate terminology. Please,\ncorrect.\n• Lines 209-210: The authors mention two barriers being crossed between Sa (Sb) state and the corresponding crossing\npoint Sa/Sb (Sb/Sc). As it is written, it implies the existence of an intermediate barrier between the two points. At the same\ntime, no information is given about what is the energy difference between the Sa_min and the MECP Sa/Sb. Please clarify\nwhich barrier is referred to in the text.\nIn case the referred barrier is an extra intermediate barrier between Sa_min and the MECP Sa/Sb, please add more\ninformation about how these barriers were computed. Were they obtained through rigid or relaxed scans between the Sa min\n(Sb min) and the corresponding MECP? Please, add more information to the ESI and refer to the info in the main manuscript.\n• Line 212: The authors state that the Sc_min is reached very quickly. Although there is an argument that due to the small\nbarrier connecting the states, Sc would likely be reached quickly, it is not possible to categorically affirm that as it is written\nwithout computing the rate constants of these transitions. Please correct.\n• Line 238: It is not clear what is the reasoning behind the argument that, since Sa and Ta would have similar molecular\nstructures at their respective minima, that would lead an absence of low-energy MECP Sa/Ta. Please clarify."}, "author_response": "Response to Reviewer #1\nComments: In this manuscript, Wu et al. report concise phosphorescent supramolecular\nsystems for medicine anticounterfeiting. The edible phosphorescent supramolecules\nVB10@α/β-CD can be facilely prepared by grinding two edible components of VB10 and α/β-\nCD with water. The supramolecules showed high quantum yields of 82.1% and 86.5%,\nrespectively, with a visible afterglow last for 6 s, and good robustness in ambient conditions.\nAuthors also demonstrated the ingenious applications of the supramolecules in the in-\nmedicine anticounterfeiting. Both VB10 and α/β-CD are common compounds, and it is\nparticularly commendable that their host-guest encapsulation—achieved through a simple\nprocess—yields remarkable phosphorescent enhancement. The low cost of the materials also\npaves the way for further practical applications.\nFor luminescence mechanism study, the authors employ a TD-DFT combined with ONIOM\nmulti-layer computational approach, realizing a well-considered balance between\ncomputational accuracy and resource efficiency for large supramolecular systems. The\ntheoretical results clearly demonstrate that host–guest interactions—particularly hydrogen\nbonding networks formed by cyclodextrins—play a critical role in modulating the ordering of\nVB10 excited states. This provides valuable mechanistic insight into how the supramolecular\nenvironment influences photophysical behavior. A minimum energy crossing point (MECP)\nwas successfully located and analyzed, offering strong theoretical support for the proposed\nintersystem crossing (ISC) pathway and the subsequent population of the triplet state. This is\na notable strength of the study.\nOn the whole, I think this is an interesting, thoroughly-conducted and well-written manuscript\nand I strongly support its publication in Nat. Commun. after some minor changes:\nResponse: We sincerely appreciate your positive comments. The detailed responses are as\nfollows.\n1. A NOESY experiment of VB10/β-CD can give more details for the binding mode in aqueous\nsolution, please provide.\nResponse: NOESY spectroscopy is indeed one of the most powerful techniques for elucidating\nthe binding mode in host–guest systems. In accordance with your suggestion, we have\nperformed detailed NOESY experiments on the VB10/β-cyclodextrin system in D O solution\n2\n(Figure R1). Regrettably, no clear NOE correlations between VB10 and β-cyclodextrin were\nobserved in the resulting spectra after changing mixing time, concentration, and acquisition\ntime. We attribute this outcome to a relatively weak binding interaction between VB10 and\nβ-cyclodextrin. In such a binding mode, the residence time of VB10 within the cyclodextrin\ncavity is too short to allow the accumulation of detectable NOE signals. This interpretation is\nconsistent with the moderate binding affinity (K ≈ (7.5 ± 0.4) × 102 M⁻¹) measured by ITC,\na\nwhich falls near the detection limit of conventional NOESY experiments.\nFigure R1. 1H,1H-NOESY NMR spectrum (600 MHz, D O, 298 K) of VB10@β-CD ([β-CD] =2[VB10]\n2\n= 2 × 10-3 mol/L). Mixing time: 1.3 s.\n2. Authors stated that no phosphorescence could be observed for the γ-CD/VB10, but there\nare lifetimes in Table S3, please check. Moreover, the row height of Table 3 should be adjusted\nfor clearer.\nResponse: We sincerely thank you for this careful observation. As shown in Figure S109, no\nRTP was observed for the γ-CD/VB10 complex. We have corrected this data in Table S3.\nFigure R2 (S109). (a) Excitation spectra of VB10/γ-CD in solid state. (b) PL spectra of VB10/γ-\nCD in solid state. (c) Time-resolved PL decay curves of VB10/γ-CD in solid state at 298 K @ 350\nnm.\n3. Chemical shifts that move upfield should be denoted with negative values, as exemplified\nin Figures S85, S87, S97, and S100.\nResponse: We are grateful to you for pointing out this important convention in NMR\nspectroscopy. We have corrected the representation of chemical shifts throughout the\nSupplementary Information as suggested. The upfield shifts are now properly denoted with\nnegative values in all relevant figures, including Figures S97, S99, S111 and S114.\nFigure R3 (S97). 1H NMR spectra of G3/α-CD 1:2 complexes (400 MHz, D O, 1.00 × 10-3 mol/L,\n2\n298K). Changes in the chemical shift are marked with blue for the guest.\nFigure R4 (S99). 1H NMR spectra of G7/α-CD 1:2 complexes (400 MHz, D O, 1.00 × 10-3 mol/L,\n2\n298K). Changes in the chemical shift are marked with blue for the guest.\nFigure R5 (S111). 1H NMR spectra of VB10/β-CD 1:2 complexes (400 MHz, D O, 1.00 × 10-3\n2\nmol/L, 298K). Changes in the chemical shift are marked with blue for the guest.\nFigure R6 (S114). 1H NMR spectra of VB10/γ-CD 1:2 complexes (400 MHz, D O, 1.00 × 10-3\n2\nmol/L, 298K). Changes in the chemical shift are marked with blue for the guest.\n4. As the MECPSC/TA plays a crucial role in explaining phosphorescence formation, it would\nbe appropriate to mention key geometric features (e.g., H-bond stabilization) briefly in the\nmain manuscript instead of relying solely on the SI.\nResponse: Thank you for this suggestion, we have mentioned the key geometric features of\nMECPSC/TA in the main text as Figure S36 and revised the description as:\n“Subsequently, VB10 can cross a very small energy barrier of 1.6 kcal/mol via MECP\nSC/TA\n(101.7 kcal/mol), reaching the stable minimum point of low-lying excited triplet state T (T ,\nA A min\n73.2 kcal/mol) (Figure S36)”.\nFigure R7 (S36). MECP structure: The C–N bond length, one N–H hydrogen bond, and two\nSC/TA\nO–H hydrogen bond lengths are indicated. The non-frozen region is highlighted in red.\n5. Natural transition orbital (NTO) analysis for the key excited states (SA, SC, TA) would\nenhance the reader’s understanding of the underlying photophysical processes.\nResponse: We thank you for this excellent suggestion. We agree that Natural Transition Orbital\n(NTO) analysis will provide deeper insight into the nature of the key excited states (S , S , and\nA C\nT ). We have performed the NTO analysis (Figures R8–R14) and added the results in the\nA\nSupporting Information as Figures S32–S35 and S37–S39. Moreover, the NTO analysis method\nhas been included in the Computational Methods section of the Supporting Information.\nFigure R8 (S32). S electronic structure of low-lying excited states and ONIOM high-layer NTO\nA\nanalysis in the relaxation processes of VB10@α-CD, computed using TDA-DFT.\nFigure R9 (S33). S electronic structure of low-lying excited states and ONIOM high-layer NTO\nB\nanalysis in the relaxation processes of VB10@α-CD, computed using TDA-DFT.\nFigure R10 (S34). S electronic structure of low-lying excited states and ONIOM high-layer NTO\nC\nanalysis in the relaxation processes of VB10@α-CD, computed using TDA-DFT.\nFigure R11 (S35). T electronic structure of low-lying excited states and ONIOM high-layer NTO\nA\nanalysis in the relaxation processes of VB10@α-CD, computed using TDA-DFT.\nFigure R12 (S37). Electronic structures and ONIOM high-layer NTO analysis of low-lying\nexcited state (S ) of VB10@α-CD in aqueous solution, computed using TDA-DFT.\nA\nFigure R13 (S38). Electronic structures and ONIOM high-layer NTO analysis of low-lying\nexcited state (S ) of VB10@α-CD in aqueous solution, computed using TDA-DFT.\nB\nFigure R14 (S39). Electronic structures and ONIOM high-layer NTO analysis of low-lying\nexcited state (S ) of VB10@α-CD in aqueous solution, computed using TDA-DFT.\nC\n6. Beyond locating a single MECP point, scanning the potential energy surfaces (PES) along\nexcited-state relaxation coordinates (e.g., FC→ SC，SC → TA) would provide further\nmechanistic insight. Use of relaxed scans, or IRC-like paths for excited states could reveal\nwhether there are competing minima or energy barriers affecting radiative or nonradiative\ndecay.\nResponse: We thank you for this insightful suggestion. We fully agree that mapping the\npotential energy surface (PES) along the relaxation coordinates would provide valuable\nmechanistic depth. While the ONIOM methodology in Gaussian presents technical constraints\nfor performing standard IRC calculations on these excited states, we have sought to address\nthe core of your suggestion by tracking the steepest descent principle from the Franck–\nCondon point to describe the relaxation pathways of the nuclei across different electronic\npotential energy surfaces and to evaluate the structural changes of the molecule during the\nrelaxation process.\nThe results, now included as Figures S22–S31 (Figures R15–R24) in the Supporting\nInformation, clearly illustrate the continuous energy profile and structural changes along the\nFC→TA pathway, complemented by corresponding NTO analyses. We believe this analysis\nsuccessfully captures the essential excited-state dynamics suggested by the reviewer.\nFigure R15 (S22). Steepest descent energy paths from the Franck–Condon points on the S\nA\npotential energy surface in aqueous solution, with characteristic bond lengths (Å) labeled; the\ncorresponding NTO analysis is also presented.\nFigure R16 (S23). Steepest descent energy path of VB10@α-CD on the potential energy\nsurface from the Franck–Condon point to T\nA min\nFigure R17 (S24). Franck–Condon point structure.\nFigure R18 (S25). S structure and ONIOM high-layer NTO analysis.\nA min\nFigure R19 (S26). MECP structure.\nSA/SB\nFigure R20 (S27). S structure and ONIOM high-layer NTO analysis.\nB min\nFigure R21 (S28). MECP structure.\nSB/SC\nFigure R22 (S29). S structure and ONIOM high-layer NTO analysis.\nC min\nFigure R23 (S30). MECP structure.\nSC/TA\nFigure R24 (S31). T structure and ONIOM high-layer NTO analysis.\nA min\nResponse to Reviewer #2\nComments: In this paper, Li and Zhao et al. reported a class of edible phosphorescent\nsupramolecular complexes based on α/β-cyclodextrins (α/β-CDs) and vitamin B10 (VB10)\n(VB10@α/β-CDs). The host-guest complex VB10@α/β-CDs exhibits long phosphorescence\nlifetimes, high photoluminescence quantum yields and detectable dual circularly polarized\nluminescence (CPL) in both fluorescence and phosphorescence. Theoretical calculations\nelucidated the phosphorescence mechanism. The phosphorescent material was further\napplied to medicine anticounterfeiting, with three anticounterfeiting methods successfully\nvalidated for both pills and capsules.\nAlthough merging edible drugs and RTP shows promise as an innovative concept, this research\nneeds further refinement to reach the publication level of Nature Communications.\nResponse: We sincerely appreciate for your comments. The detailed responses are shown as\nfollows.\nThe phosphorescence of cyclodextrin (CD) complexes with p-aminobenzoic acid (PABA) has\nbeen previously reported (Room-Temperature Luminescence Properties of p-Aminobenzoic\nAcid and 4-Phenylphenol Adsorbed on α-Cyclodextrin/NaCl Mixtures; Phosphorescence\nProperties of p-Aminobenzoic Acid Immobilized on a Nylon Membrane). The authors must\nhighlight the innovative aspects of their work compared to existing literature.\nResponse: Thank you for your comment on clarifying the novelty of our work. We have\ncarefully read the literatures (Studies of Cyclodextrin-Enhanced Room-Temperature\nPhosphorescence. Analytica Chimica Acta. 1989, 217, 171-176, Ref. R1; Phosphorescence\nProperties of p-Aminobenzoic Acid Immobilized on a Nylon Membrane. Applied Spectroscopy.\n2004, 58, 836-842, Ref. R2). After a thorough comparison with these systems, we have\nsummarized their key photophysical properties alongside those of our work in Table R1.\nIndeed, all the phosphorescent systems are based on the phosphor PABA. But Ref. R1\nconcluded that β-cyclodextrin enhanced the heavy-atom effect for all four compounds, except\nfor the effect of iodide and chloride on PABA emission. In the system, PABA, heavy atom, and\nfilter paper are essential conditions. The addition of CD can enhance the RTP of anthracene,\ndibenzofuran, and phenanthrene, but decrease the RTP of PABA. For Ref. R2, PABA showed\nphosphorescence when adsorbed on a nylon membrane and in the presence of α-cyclodextrin\nand heavy atoms.\nIn our work, concisely grinding α/β-cyclodextrins (α/β-CDs) and vitamin B10 (VB10) with\nwater or co-crystallization from aqueous solution, the resulted host−guest complexes\nVB10@α/β-CDs can exhibit a long phosphorescence lifetime of up to 1.16 s, a high\nphotoluminescent quantum yield of up to 86.5%, and a dual circularly polarized luminescence.\nThe VB10@α/β-CDs developed in our work serve as a purely organic RTP system and\ndemonstrate excellent phosphorescent performance. We provide direct evidence of the host-\nguest inclusion structure through single-crystal X-ray diffraction. Moreover, the crucial roles\nof non-radiative process for the phosphorescence of organic supramolecule in the solid state\nare clearly revealed through complete theoretical calculations. The good moisture robustness\nand room-temperature phosphorescence endow VB10@β-CD excellent edible\nphosphorescent inks for in-medicine anticounterfeiting.\nTable R1. Summary of relative RTP systems with PABA@α/β-CD and our work.\nPhotophysical properties\nEmitter References\nτ (ms) Ф (%) |g |\np PL lum\nAnalytica Chimica\nPABA/KI/β-CD/filter paper Null Null Null Acta. 1989, 217, 171-\n4.6 Null Null\nMembrane 2004, 58, 836-842.\nVB10 (PABA) @α-CD 977 82.1 25 × 10−3 Our work\nVB10 (PABA) @β-CD 1160 86.5 6.0 × 10−4 Our work\nSufficient experimental data in the main text is necessary to support the claims. The main teat\nis too preliminary. Some of the critical characterization data currently adhere to the\nsupporting Information should be moved into the main text.\nResponse: Thanks for your constructive suggestion, we have thoroughly reorganized the\nmanuscript. Figure S19 has now been incorporated into the main text as Figure 2b (Figure\nR25). We are grateful for this suggestion, which has undoubtedly improved the clarity and\nimpact of our work.\nFigure R25 (Figure 2). Host−guest properties of VB10@α-CD. (a) 1H NMR (400 MHz, D O, 298\n1H-1H NOESY NMR spectrum (400 MHz, D O, 298 K) of VB10@α-CD ([α-CD] =2[VB10] = 2 × 10-\n2\n2 mol/L). (c) ITC titration data of α-CD titrated into VB10 ([VB10] = 3.5 × 10-4 mol/L, [α-CD] =\n8.0 × 10-3 mol/L). (d) Single-crystal structures of VB10@α-CD (Front view and top view). α-CD\nand VB10 are illustrated as stick and space-filling representations, respectively.\nThere appears to be an obvious error in Figure 1d and S13a. The labels of CPL and DC\nassignments are reversed, which must be revised.\nResponse: We greatly appreciate you identifying this labeling error. The incorrect labels for\n\"CPL\" and \"DC\" in Figures 1d and S14a (Figures R26–R27) have now been corrected.\nFigure R26 (Figure 1). Photophysical properties of VB10@α-CD in crystal state. (a)\nPhotoluminescence (PL) spectra (black) and delayed PL spectra (blue) of VB10@α-CD (Inset:\nluminescence photographs of VB10@α-CD crystals under 254 nm and after ceasing\nirradiation). (b) Time-resolved PL decay curves of VB10@α-CD at 298 K. (c) Measured lifetimes\nand quantum yields of VB10, α-CD, and VB10@α-CD. (d) CPL spectra of VB10@α-CD at 298 K.\nFigure R27 (S14). (a) CPL spectra and (b) |g | of VB10/α-CD in crystal state in PL mode (λ :\nlum ex\n300 nm).\nThe CPL spectra of VB10@β-CD have not been studied. Additionally, it is hoped to provide the\nCD spectra of the solution and solid states of VB10@α/β-CDs systems.\nResponse: We thank you for this valuable suggestion. We have obtained CPL spectra of\nVB10@β-CD and the CD spectra of VB10@α/β-CD in the aqueous solution and in the solid\nstate (Figures R28-R30). All the spectra have been added to the Supporting Information as\nFigures S13, S107, and S108. Related discussion has been added in main text:\nPage 8, “The CD spectrum of solid VB10@α-CD features a stronger positive Cotton effect\nat 335 nm compared with that in aqueous solution (Figure S13).”\nPage 17, “The CD spectra of VB10@β-CD showed negative Cotton effect both in solid\nstate and in aqueous solution (Figure S107). Moreover, the VB10@α-CD exhibited positive CPL\n(λ = 346 nm), with |g | values of 6×10-4 (Figure S108).”\nmax lum\nFigure R28 (S13). (a) UV-Vis absorption and (b) CD spectrum of VB10/α-CD in solution. (c) UV-\nVis absorption and (d) CD spectrum of VB10/α-CD in solid-state.\nFigure R29 (S107). (a) UV-Vis absorption and (b) CD spectrum of VB10/β-CD in solution. (c)\nUV-Vis absorption and (d) CD spectrum of VB10/β-CD in solid-state.\nFigure R30 (S108). CPL spectra and (b) |g | of VB10/β-CD in crystal state in PL mode (λ :\nlum ex\n300 nm).\nIn the primary anticounterfeiting test, PL is the used. It is not necessary that the coating\nsystem showed CPL. It is unclear why CPL of the complex system is investigated.\nResponse: We thank you for your valuable comments. Indeed, the encryption application\npresented in our work relies solely on PL rather than CPL. At the outset of the design, our goal\nwas to achieve CPL encryption. Therefore, we have thoroughly characterized the\nphotophysical properties of the complex regarding CPL. In the actual CPL encryption\nexperiments, we were unable to achieve the anticipated encryption results. Therefore, we did\nnot present these negative encryption cases in the manuscript. Considering that CPL is a\ncrucial component of photophysical properties, we retained this fundamental data.\nIn Figure 5, α-cyclodextrin (α-CD) is employed as the primary anticounterfeiting substrate.\nHowever, it should be noted that α-CD has not yet been approved for food applications in\nseveral countries. The safety needs to be confirmed.\nResponse: Thank you for your valuable reminder. After thorough investigation, we have\nconfirmed that α-CD is indeed not yet approved for food applications in some countries. We\ngreatly appreciate your important correction, which enabled us to promptly avoid this issue.\nβ-CD possesses excellent safety profiles and is approved for use across multiple\ninternational regulatory frameworks. It is recognized as Generally Recognized as Safe (GRAS)\nby the U.S. FDA under 21 CFR § 184.1277 and is listed in the United States Pharmacopeia (USP,\nProduct No. 1154569) as a pharmaceutical excipient (https://www.ecfr.gov/current/title-\n21/chapter-I/subchapter-B/part-184#184.1277;\nhttps://www.sigmaaldrich.com/HK/zh/product/usp/1154569 ). In the European Union, it is\napproved as a food additive (E459, product code B0950000)\n(https://www.sigmaaldrich.com/HK/zh/product/sial/b0950000). In China, it is listed in the\nNational Food Safety Standard for Food Additives (GB 2760-2024, code CNS 20.024) and\nincluded as an oral pharmaceutical excipient in the Chinese Pharmacopoeia\n(https://2760.foodmate.net/addtives.html).\nBased on the above results, we have repeated the medicine anticounterfeiting by using\nVB10@β-CD and updated Figure 5 (Figure R31) and Figure S115 (Figure R32) and text\ndescriptions accordingly.\nFigure R31 (Figure 5). Medicine anticounterfeiting by three methods of surface coating,\ndoping into medicine, and spray display. Surface coating: coat information onto the surface of\npills by aqueous solution of VB10@β-CD. Doping into medicine: dope powder of VB10@β-CD\ninto capsule or pills. Spray display: dope β-CD into pills and then spray aqueous solution of\nVB10).\nFigure R32 (S115). (a) Medicine anticounterfeiting by surface coating on capsule. (b) Medicine\nanticounterfeiting by doping into medicine under 365 nm irradiation. (c) Medicine\nanticounterfeiting by doping into medicine under 254 and 365 nm irradiation.\nResponse to Reviewer #3\nComments: In this work, the authors propose to use vitamin B10 encapsulated by cyclodextrin\nas a marker for printing information on the surface of pills and capsules. This would be useful\nfor in-medicine anticounterfeiting, since each pill/capsule can be verified independently.\nRegarding the photophysics, the idea is to regulate the excited states of vitamin B10 to induce\nroom-temperature phosphorescence, that can be later used to identify the individual\ncapsules/pills.\nTo this end, the authors investigated the photophysical properties of this system and other\nderivatives, and identified the relevant processes that enable room temperature\nphosphorescence. Experimentally, they also investigated how the complex would behave as a\nmarker in pills and capsules. This work shows a thorough investigation of the use of vitamin\nB10 encapsulated by cyclodextrin and its potential use as an anti-counterfeiting medicine\nmarker. This work will likely have a significance on the field and societal impact. Given my\nexpertise, I particularly focused on the computational investigation presented by the authors,\nwhere the data provided support the authors’ conclusions. However, I list below some\nsuggestions to improve the clarity and precision of the text.\nResponse: We appreciate for the reviewer’s positive comments sincerely. The detailed\nresponses are as follows.\n• Line 89: Please clarify to which type of quantum yield the authors refer to, for instance,\nfluorescence, phosphorescence…?\nResponse: Thank you for raising this point for clarification. The \"quantum yield\" mentioned in\nline 89 (and throughout the text) specifically refers to the photoluminescence quantum yield\n(PLQY). Although a clear phosphorescence emission peak can be observed in Figure 1a, there\nis currently no standardized methodology or software that can reliably distinguish the\nrespective contributions of fluorescence and phosphorescence to the total luminescence.\nTherefore, we have consistently reported the total photoluminescence quantum yield in our\nstudy.\n• Line 131: There is a floating “This”, please remove.\nResponse: We thank the reviewer for this observation. The floating \"This\" has been removed.\n• Line 193, Figure 3a, S21-S24: Please, be more precise on what is being shown as “electronic\ntransitions”. Are these the molecular orbitals of the respective states or the ground state?\nWhat is the DeltaE shown, HOMO-LUMO gaps or actual excitation energies? The number of\ndecimals on the DeltaE value is also unphysical; the precision is usually around two\ndecimals only.\nResponse: We thank the reviewer for raising this point for clarification. The term \"electronic\ntransitions\" refers to the dominant electronic transitions of the respective excited states,\nobtained from TDA-DFT calculations. The molecular orbitals correspond to the dominant\nelectronic transitions of the respective excited states obtained from TDA-DFT calculations. The\nΔE values represent the excitation energies calculated using TDA-DFT, rather than the HOMO-\nLUMO gaps. To provide a clearer interpretation, we have added the NTO analysis in the\nSupporting Information (Section S32–S35 and S37-S39) and included the NTO analysis method\nin the Computational Methods section of the Supporting Information. Furthermore, we have\nadjusted the numerical precision of the ΔE values in accordance with the reviewer’s\ncomments.\nFigure R8 (S32). S electronic structure of low-lying excited states and ONIOM high-layer NTO\nA\nanalysis in the relaxation processes of VB10@α-CD, computed using TDA-DFT.\nFigure R9 (S33). S electronic structure of low-lying excited states and ONIOM high-layer NTO\nB\nanalysis in the relaxation processes of VB10@α-CD, computed using TDA-DFT.\nFigure R10 (S34). S electronic structure of low-lying excited states and ONIOM high-layer NTO\nC\nanalysis in the relaxation processes of VB10@α-CD, computed using TDA-DFT.\nFigure R11 (S35). T electronic structure of low-lying excited states and ONIOM high-layer NTO\nA\nanalysis in the relaxation processes of VB10@α-CD, computed using TDA-DFT.\nFigure R12 (S37). Electronic structures and ONIOM high-layer NTO analysis of low-lying\nexcited state (S ) of VB10@α-CD in aqueous solution, computed using TDA-DFT.\nA\nFigure R13 (S38). Electronic structures and ONIOM high-layer NTO analysis of low-lying\nexcited state (S ) of VB10@α-CD in aqueous solution, computed using TDA-DFT.\nB\nFigure R14 (S39). Electronic structures and ONIOM high-layer NTO analysis of low-lying\nexcited state (S ) of VB10@α-CD in aqueous solution, computed using TDA-DFT.\nC\n• The authors refer to the ONIOM model they created as “solid phase” which is not an\naccurate terminology. Please, correct.\nResponse: We thank the reviewer for the valuable suggestion regarding the terminology. To\nimprove accuracy and clarity, we have revised the expression “solid phase” to “solid-phase\nONIOM model.” This correction has been implemented consistently throughout the\nmanuscript.\n• Lines 209-210: The authors mention two barriers being crossed between Sa (Sb) state and\nthe corresponding crossing point Sa/Sb (Sb/Sc). As it is written, it implies the existence of an\nintermediate barrier between the two points. At the same time, no information is given about\nwhat is the energy difference between the Sa_min and the MECP Sa/Sb. Please clarify which\nbarrier is referred to in the text.\nResponse: We thank the reviewer for raising this point for clarification. The values given in\nparentheses in Lines 209–210 (“1.2 kcal/mol between S and MECP , and 0.4 kcal/mol\nA min SA/SB\nbetween S min and MECP ”) indeed refer to the energy barriers from S to S via the\nB SB/SC A min B min\nMECP . Specifically, 1.2 kcal/mol represents the energy difference between S and\nSA/SB A min\nMECP , while 0.4 kcal/mol corresponds to the barrier from S to S , as also illustrated\nSA/SB B min C min\nin Figure 3b. These barriers were obtained through relaxed scans. The procedure was as\nfollows: once the MECP was located, steepest descent paths were traced on both electronic\nstates starting from the MECP, thereby confirming that the MECP indeed connects the\ncorresponding minima of the two electronic states. We are grateful to the reviewer for the\ninsightful comments on the computational details, and we have added the relevant\ninformation in the Supporting Information and cited it in the main manuscript.\nIn case the referred barrier is an extra intermediate barrier between Sa_min and the MECP\nSa/Sb, please add more information about how these barriers were computed. Were they\nobtained through rigid or relaxed scans between the Sa min (Sb min) and the corresponding\nMECP? Please, add more information to the ESI and refer to the info in the main manuscript.\nResponse: We thank the reviewer for this astute observation and for prompting us to clarify\nthis important point. Since the ONIOM method in Gaussian does not support IRC calculations,\nwe instead follow the steepest descent principle from the Franck–Condon point to describe\nthe relaxation pathways of the nuclei across different electronic potential energy surfaces and\nto evaluate the structural changes of the molecule during the relaxation process. The\ncomputational details are as described in the previous comment: once the MECP was located,\nsteepest descent paths were traced on both electronic states starting from the MECP, thereby\nconfirming that the MECP indeed connects the corresponding minima of the two electronic\nstates.\nFigure R15 (S22). Steepest descent energy paths from the Franck–Condon points on the S\nA\npotential energy surface in aqueous solution, with characteristic bond lengths (Å) labeled; the\ncorresponding NTO analysis is also presented.\nFigure R16 (S23). Steepest descent energy path of VB10@α-CD on the potential energy\nsurface from the Franck–Condon point to T\nA min\nFigure R17 (S24). Franck–Condon point structure.\nFigure R18 (S25). S structure and ONIOM high-layer NTO analysis.\nA min\nFigure R19 (S26). MECP structure.\nSA/SB\nFigure R20 (S27). S structure and ONIOM high-layer NTO analysis.\nB min\nFigure R21 (S28). MECP structure.\nSB/SC\nFigure R22 (S29). S structure and ONIOM high-layer NTO analysis.\nC min\nFigure R23 (S30). MECP structure.\nSC/TA\nFigure R24 (S31). T structure and ONIOM high-layer NTO analysis.\nA min\n• Line 212: The authors state that the Sc_min is reached very quickly. Although there is an\nargument that due to the small barrier connecting the states, Sc would likely be reached\nquickly, it is not possible to categorically affirm that as it is written without computing the rate\nconstants of these transitions. Please correct.\nResponse: We thank the reviewer for this rigorous comment. In the revised text, we have\nmodified the statement to avoid categorical affirmation. Specifically, we now write: “Based on\nthe small barrier, we infer that VB10@αCD is likely to reach the lowest-energy stationary point\n(S , 100.1 kcal/mol) among all excited singlet states very quickly, and subsequently emits\nC min\nfluorescence at 375 nm.”\n• Line 238: It is not clear what is the reasoning behind the argument that, since Sa and Ta\nwould have similar molecular structures at their respective minima, that would lead an\nabsence of low-energy MECP Sa/Ta. Please clarify.\nResponse: We thank the reviewer for this astute observation and for prompting us to clarify\nthis important point. In the solution phase, our calculations did not identify any low-energy\nMECP , and experimentally we also did not observe phosphorescence. Thus, theory and\nSA/TA\nexperiment are consistent. In Line 238 of the main text, our intention was to provide a\nreasonable qualitative hypothesis for the absence of low-energy MECP .\nSA/TA\nSince it is not feasible to exhaustively compute all possible crossing regions, it is\nimpossible to provide rigorous proof for the absence of MECP in the low-energy region.\nSA/TA\nThis is the reason we aimed to propose a qualitative rationale instead. Our reasoning is as\nfollows:\n1. Although the spin of one electron differs, S and T share very similar electronic\nA A\nstructures, as shown in Figure S41 (a, b).\n2. Such similarity in electronic structure gives rise to certain analogous properties. For\nexample, in terms of conjugation between the amino group and phenyl ring (Figure S41c),\ncompared with the ground state, both S and T show electron depletion from the strongly π*\nA A\nantibonding orbital, strengthening the C–C bond and enhancing amino–phenyl conjugation,\nleading to a more planar geometry for the S and T structures.\nA min A min\n3. When molecular vibrations distort the geometry, the similar electronic structures of S\nA\nand T cause their energy profiles to evolve in parallel, making it difficult for a crossing to occur\nA\nin the low-energy region. As an example, Figure S41(d) illustrates the C–N rotation between\nthe amino and phenyl groups. Both S and T increase sharply in energy upon C–N\nA min A min\nrotation, which prevents low-energy crossings. In contrast, the conjugation between the\namino and phenyl groups is much weaker in S , and C–N rotation has little effect on its\nC min\nenergy. Thus, S can cross with T in the low-energy region through C–N rotation.\nC A\nThis serves as a clarification of the statement in the main text: “Second, under the solid-\nphase ONIOM model, the S can undergo relaxation to the T via the MECP pathway.\nC min A min SC/TA\nHowever, in aqueous solution, our calculations show that the S lacks any accessible low-\nA min\nenergy MECP route, thereby preventing this relaxation process. Speculative analyses of\nSA/TA\nthe possible reasons why the MECP point could not be located are provided in the\nSA/TA\nSupplementary Information.”\nWe realize that our original descriptions in both the main text and the Supporting\nInformation were insufficiently clear. We greatly appreciate the reviewer for pointing this out.\nFollowing the reviewer’s suggestion, we have revised the statements in both the main text\nand the Supporting Information to improve clarity. As emphasized, the text in Line 238 was\nnot intended as conclusive evidence but rather as a qualitative hypothesis, which is why we\nused “may be.”\nFigure R33 (S41). (a, b) Identical electronic structures of low-lying excited states S and T of\nA A\nVB10@α-CD in aqueous solution. (c) Dihedral angel of phenyl and NH of VB10@α-CD in\n2\naqueous solution. (d) Relationship between relative energy and dihedral angel (phenyl and\nNH ) of VB10@α-CD in aqueous solution. S and T have the same excited orbitals (Figure S41a\nand S41b), resulting in similar stable structures in the excited state (Figure S41c shows the\ndihedral angle between the amino and phenyl groups). The energy variation trend induced by\nmolecular distortion is also expected to be similar, making it difficult for the molecule to\nequalize the energies of S and T through its own distortion. This may explain why we cannot\nA A\ndetect the MECP , while the MECP is easily found, as shown in Figure S41d.\nSA/TA SC/TA"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The manuscript has been properly revised and can be accepted for publication now.", "reviewer_2": "This revision answered most of the questions. However, there are still minor points which should be clarified before publication. The main doubt is from the spectra noted as below.\nIn Figure R29C, the absorption spectra look strange. There is an absorption minimum lower than the baseline. The spectrum should be remeasured. In Figure R29a, the absorption appears also at 500 nm. However, the florescence is just around 350 nm.\nIn Figure R 30b, there is a spectrum between g and wavelength. It is seen there are two CPL peaks in the spectrum. However, the system emitted only in one band. Thus, the peak around 425 nm is meaningless. The glum spectra cannot be simply calculated by intensity over the DC. It is necessary to understand the real meaning of the g value. In addition, the g maximum is in different wavelength. The peaks positions are quite different. These, these data are doubtful.", "reviewer_3": "The authors have successfully addressed my comments. This work address an important problem of counterfeiting drugs, it is thorough, well-written, and the data provided ensures the reproducibility of the work. Therefore, I suggest this paper for publication."}, "author_response": "Reply to Reviewer #1\nComments: The manuscript has been properly revised and can be accepted for publication\nnow.\nResponse: We thank the reviewer for the recommendation of publication.\nReply to Reviewer #2\nComments: This revision answered most of the questions. However, there are still minor\npoints which should be clarified before publication. The main doubt is from the spectra noted\nas below.\nResponse: We sincerely appreciate the useful comments from the reviewer. The detailed\nresponses are as follows.\nIn Figure R29C, the absorption spectra look strange. There is an absorption minimum lower\nthan the baseline. The spectrum should be remeasured. In Figure R29a, the absorption\nappears also at 500 nm. However, the florescence is just around 350 nm.\nResponse: As suggested, we have remeasured the UV and CD spectra of VB10/β-CD both in\naqueous solution and in the solid state (Figure R1). This time, we have minimized background\ninterference and optimized the baseline correction as much as possible. We have updated\nthese figures accordingly.\nFigure R1 (S107). (a) UV-Vis absorption and (b) CD spectra of VB10/β-CD in solution. (c) UV-\nVis absorption and (d) CD spectra of VB10/β-CD in the solid state.\nIn Figure R 30b, there is a spectrum between g and wavelength. It is seen there are two CPL\npeaks in the spectrum. However, the system emitted only in one band. Thus, the peak around\n425 nm is meaningless. The glum spectra cannot be simply calculated by intensity over the\nDC. It is necessary to understand the real meaning of the g value. In addition, the g maximum\nis in different wavelength. The peaks positions are quite different. These, these data are\ndoubtful.\nResponse: Thank you for pointing out these issues. We have remeasured the CPL spectra by\nusing higher-quality single crystals of VB10@β-CD (Figure R2). The g value was calculated\nlum\naccording to g =2ΔI / I = 2(I - I ) / (I + I ), where I and I are the intensities of the left and\nlum L R L R L R\nright circularly polarized emission, respectively. We have updated these figures, and the\nrelated discussion has been revised in the main text.\nPage 16, “Moreover, VB10@β-CD exhibited positive CPL, with a |g | value of 3.6×10-3\nlum\n(Figure S108).”\nFigure R2 (S108). (a) CPL spectra and (b) |g | of VB10/β-CD in crystal state in PL mode (λ :\nlum ex\n300 nm).\nReply to Reviewer #3\nComments: The authors have successfully addressed my comments. This work address an\nimportant problem of counterfeiting drugs, it is thorough, well-written, and the data provided\nensures the reproducibility of the work. Therefore, I suggest this paper for publication.\nResponse: We thank the reviewer for the recommendation of publication."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The revision has remeasured the sepctra, which become reasonable now. I support the publication of the paper in its current form."}, "author_response": "Reply to Reviewer #2\nComments: The revision has remeasured the sepctra, which become reasonable now. I\nsupport the publication of the paper in its current form.\nResponse: We thank the reviewer for the recommendation of publication."}]

Decision letter