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Three positive charge nonapoptotic-induced photosensitizer with excellent water solubility for tumor therapy
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Zhe Li1, Ping-Zhao Liang1, Li Xu, Fei-Yu Yang, Tian-Bing Ren*, Lin Yuan, Xia Yin, Xiao-Bing Zhang*
Chinese Chemical Letters | 2024, 35(8) : 109190
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Chinese Chemical Letters | 2024, 35(8): 109190
• Communication •
Three positive charge nonapoptotic-induced photosensitizer with excellent water solubility for tumor therapy
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Zhe Li1, Ping-Zhao Liang1, Li Xu, Fei-Yu Yang, Tian-Bing Ren*, Lin Yuan, Xia Yin, Xiao-Bing Zhang*
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  • State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China
Published: 2024-08-15 doi: 10.1016/j.cclet.2023.109190
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Photodynamic therapy (PDT) has emerged as a significant cancer therapy option. Currently, cation-based organic small molecule aggregation-induced emission (AIE) photosensitizers (PSs) attract the wide attention of many scientists, due to improved reactive oxygen species (ROS) production after cationization. However, such PSs tend to localize only the mitochondria, limiting the death way of tumor cells (usually apoptosis) during PDT process, which may affect the therapeutic effect under some circumstances. Herein, we designed a novel water-soluble three positive charge PS, TPAN-18F, which could be distributed uniformly in cell cytoplasm and had distribution in different sub-organelles (mitochondria, endoplasmic reticulum, lysosome). The experimental results showed that TPAN-18F-based PDT process can not only disrupt mitochondrial functions (reducing ATP production and destroying mitochondrial membrane potential), but also elevate the intracellular lipid peroxides (LPOs) level, which evoke the non-apoptotic death manner of tumor cells. Further, in vivo studies showed that TPAN-18F-based PDT could effectively inhibit tumor growth. Accordingly, we believe that the construction of TPAN-18F is suggestive for tumor non-apoptotic therapy.

Water-soluble  /  Three positive charge  /  Photosensitizer  /  Nonapoptotic  /  Tumor therapy
Zhe Li, Ping-Zhao Liang, Li Xu, Fei-Yu Yang, Tian-Bing Ren, Lin Yuan, Xia Yin, Xiao-Bing Zhang. Three positive charge nonapoptotic-induced photosensitizer with excellent water solubility for tumor therapy[J]. Chinese Chemical Letters, 2024 , 35 (8) : 109190 - . DOI: 10.1016/j.cclet.2023.109190
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Malignant tumor is one of the diseases with the highest mortality rate in the 21st century and poses a serious threat to human health [1–4]. Therefore, the timely diagnosis and thorough treatment of malignant tumors are crucial for human health and safety. After decades of development, tumor-therapy methods (including surgical operation, phototherapy, chemotherapy and radiotherapy, etc.) [5–15] have made significant progress and been widely used in clinical treatment. Among various imaging techniques, fluorescence imaging has been considered as powerful tools for tumor diagnosis and therapy, due to high sensitivity, spatio-temporal resolution and low cost [16–23]. In addition, photodynamic therapy (PDT) as reliable and highly accurate tumor treatment could induce tumor cell death under light irradiation by activating photosensitizer (PS) to produce highly toxic reactive oxygen species (ROS), which has attracted increasing attention in tumor therapy [24,25]. Moreover, compared with chemotherapy, the photo-controllable characteristics of PDT could effectively reduce the side effects of the treatment process. Therefore, in recent years, PDT based on organic fluorescence molecules has emerged as a promising cancer treatment.
Phthalocyanines and porphyrins are the most widely investigated PSs, which have already been utilized in the clinic [26,27]. However, due to π-π stacking interactions from large aromatic ring system of molecular structure, these PSs usually aggregate in the water environment. This would cause low fluorescence intensity as well as terrible PDT efficiency, result from the aggregation-caused quenching (ACQ) effect [28]. In order to solve such problems, scientists have attempted to build PSs with the aggregation-induced emission (AIE) properties and improve their water solubility by introducing positive charges [29–34]. Delightingly, such cationization-based AIE PSs have evidently enhanced ROS generating ability (δEst is significantly reduced, which is conducive for ROS production) [28,35], and achieved impressive achievement in tumor therapy. Unfortunately, due to molecular structure with only single positive charge, such PSs could often only be localized in the mitochondria of tumor cells (Fig. 1a) [30,34–36], restricting the death way of tumor cells (usually apoptosis) during PDT process, which may affect the effectiveness of tumor treatment under some circumstances. As a result, it is still valuable for developing novel water-soluble AIE PSs to break through the limitations of mitochondrial localization.
Herein, we designed a water-soluble three positive charge AIE PS (TPAN-18F) for tumor therapy. The experimental results showed that, TPAN-18F had good photosensitivity activity. Of note, TPAN-18F was distributed uniformly in cell cytoplasm, and had distribution in different sub-organelles (mitochondria, endoplasmic reticulum, lysosome). Due to such distribution characters, TPAN-18F-based PDT process can not only disrupt mitochondrial functions (reducing ATP production and destroying mitochondrial membrane potential), but also elevate the intracellular lipid peroxides (LPOs) level. On the basis, as expected, TPAN-18F could effectively initiate nonapoptotic cell death, as observed in cell morphology after light irradiation. Subsequent in vivo studies showed that the tumor growth was inhibited obviously by TPAN-18F-based PDT. Therefore, we are optimistic that such novel water-soluble three positive charge AIE PS are enlightening for tumor therapy.
Currently, triphenylamine-based organic AIE PSs had made many important achievements for cancer treatment (Fig. 1a). Among them, cationization-triphenylamine (with single positive charge) could effectively improve water solubility of organic compounds [29–34], further, it has been reported that appropriate introduction of positive charge could increase ROS production [28,35]. Considering that such PSs could often only be localized in the mitochondria of tumor cells [30,34–36], it would limit the death way of tumor cells (usually apoptosis) during PDT process, which may affect the effectiveness of tumor treatment under some circumstances [24]. On the basis, in order to break the single positive charge limitations and further increase water solubility, we tried to construct multi-charge PSs. As shown in Fig. 1b, by introducing ethyl to pyridine N, three-positive-charge molecule (TPAN-Et) can be obtained. Finally, to further increase the photosensitivity, we intend to modify TPAN-Et to form TPAN-18F by introduction six trifluoromethyl. According to previous literature, promoting intersystem crossing (ISC) from S1 to T1 is conducive to enhancing the photosensitization efficiency of PSs by increasing the spin-orbit coupling (SOC) and decreasing the δEST (the energy gap between S1 state and T1 state) based on the perturbation theory and Marcus semiclassical method [28,37]. In addition, the introduction of positive charge generally does not cause changes in SOC. Considering it, we speculated that the improvement of TPAN-18F was related to the change in δEST. To prove the feasibility of such option, we took advantage of density functional theory (DFT) to compute the orbital energy levels of TPAN, TPAN-Et and TPAN-18F (at the B3LYP/6–31G). As shown in Fig. S1 (Supporting information), the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of TPAN, are distributed on the whole molecule with the poor separation. On the contrary, TPAN-Et and TPAN-18F with positive charges, changes electronic cloud distribution evidently, which produces obvious separation. Generally, the significant HOMO-LUMO separation would result in small δEs1t1, thus TPAN-Et (δEs1t1 = 0.3702 eV) has superior photosensitivity compared to TPAN (δEs1t1 = 0.6907 eV). Moreover, due to addition of multi-trifluoromethyl, intramolecular charge transfer (ICT) effect of TPAN-18F is further enhanced, which improve photosensitivity activity of TPAN-18F (δEs1t1 = 0.3460 eV). Therefore, such calculation results validate the feasibility of our above modification strategy, and we subsequently synthesized TPAN, TPAN-Et and TPAN-18F. The corresponding synthesis steps were shown in Fig. 1c and Scheme S1 (Supporting information), respectively. The chemical structures were characterized by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), 1H, 13C nuclear magnetic resonance (NMR) and 19F NMR.
The spectroscopic properties were investigated by ultraviolet–visible (UV–vis) and photoluminescence (PL) spectroscopy. In order to ensure the complete dissolution of the above compounds, we first chose dimethyl sulfoxide (DMSO) as the solvent for testing. As shown in Figs. 2a and b, the absorption and emission peaks of TPAN were 360 and 460 nm, respectively. Further, as expected, wavelength of TPAN-Et (λab/λem = 430/600 nm) and TPAN-18F (λab/λem = 440/610 nm) redshifted to visible region, which was attributed to the significant increase in ICT effects after cationization and addition of multi-trifluoromethyl. Subsequently, in order to explore the water solubility of TPAN-Et and TPAN-18F, we tested the absorption spectrums in aqueous solutions with different DMSO content. As shown in Figs. 2c and d, TPAN-Et and TPAN-18F exhibited almost identical absorption spectra in different aqueous solutions, which indicated that they had excellent water solubility. Based on it, their photosensitive properties were studied in the water environment. By using 9′,10′-anthracenediyl-bis(methylene)-dimalonic acid (ABDA) as indicators, we first tested their 1O2 production capacity. As shown in Fig. 2e and Figs. S2a–d (Supporting information), companied with the progressive addition of multi charges and trifluoromethyls, TPAN derivates (TPAN, TPAN-Et and TPAN-18F) exhibited gradually enhanced 1O2 generation ability, and we calculated the singlet oxygen yield (ΦΔ) of TPAN-18F using Rose Bengal (RB) as a reference (ΦΔ = 75% in water), with values 57.4% (Fig. S3 in Supporting information). Moreover, we utilized 2′,7′-dichlorodihydrofluorescein (DCFH) as total ROS indicators to further confirm their photosensitivity activity (Fig. 2f and Figs. S2e–h in Supporting information). Consistent with previous results of 1O2 generation ability, ROS production capacity was becoming increasingly stronger with the increasing positive charge, in particular, TPAN-18F showed the highest performance of 1O2 generation (Fig. 2g), which also verified DFT calculation results.
As well known, triphenylamine group is a kind of classical AIE active units. Therefore, the effect of TPAN-18F on the AIE properties were subsequently studied. As shown in Figs. S4b, d, f, h and j (Supporting information), as the organic phase (EtOH and THF) content increased, the fluorescence of TPAN-18F was significantly enhanced, resulting from restricted molecular rotation, which was typical AIE properties. As a comparative experiment, we also tested the AIE properties of TPAN. The experimental results showed that, TPAN had similar properties to TPAN-18F (Figs. S4a, c, e, g and i in Supporting information). Besides, we found that TPAN and TPAN-18F bright solid-state fluorescence (Figs. S4i and j). And we tested the particle size of TPAN-18F in different solvents. The experimental results showed that TPAN-18F can be well dissolved in the water and no nanoparticle can be detected. But in the organic phase (EtOH and THF), significant particle sizes of TPAN-18F were observed. These results indicated that TPAN-18F indeed displayed a AIE performance (Fig. S5 in Supporting information). According to the above experimental results, we then selected TPAN-18F to perform following experiments.
In order to investigate the intracellular properties, we first explored the intracellular distribution of TPAN-18F. The distribution of TPAN-18F in cells could be conveniently visualized by confocal laser scanning microscopy. Various sub-organelle co-localization indicators, including Hoechst 33258 (for cell nuclear), Mito-Deep Red (for mitochondria), Lyso-Red (for lysosome) and ER-Blue (for endoplasmic reticulum), were utilized for imaging after co-incubation with TPAN-18F in cells. As shown in Fig. 3a, TPAN-18F entered the living cells obviously. According to overlap images, TPAN-18F had a certain distribution in sub-organelles (mitochondria, lysosomes, endoplasmic reticulum) in the cytoplasm except for the cell nucleus, which meant that TPAN-18F had the potential to affect many different intracellular physiological processes. Different from cationization-triphenylamine PSs with single positive charge, TPAN-18F did not localized in the mitochondria, which may be attributed to further improvements in water solubility form addition of two positive charges. Subsequently, to assess the potential of TPAN-18F for tumor therapy, we tested the capability to produce ROS in intracellular environment by utilizing DCFH diacetate (DCFH-DA) as indicators. As observed in Fig. 3b, the green channel of DCFH-DA displayed palpable enhancement after white light irradiation for TPAN-18F treated cells, which indicated good ROS generation capacity of TPAN-18F in cells.
And we further evaluated the ability of TPAN-18F to kill tumor cells under light irradiation by live/dead staining experiments based on calcein AM/propidium iodide (PI). As observed in Fig. 3d, green fluorescence from calcein AM was shown in cells for only TPAN-18F group as well as only light irradiation group, which indicated their outstanding cell viability. And TPAN-18F treated cells with light irradiation exhibited evident red fluorescence, which confirmed that the presence of TPAN-18F-based PDT would kill tumor cells. In addition, we also conducted MTT assays to further evaluate cell viability (Fig. S6 in Supporting information), and the experimental results showed that TPAN-18F had negligible dark toxicity, which was consistent with the above results. Of note, the TPAN-18F incubated cells with light irradiation showed obvious bubbles on the cell membrane (Fig. 3c), which was significantly distinct from apoptosis (e.g., nuclear fragmentation or cell shrinkage) [33].
In order to figure out the intrinsic reasons of this morphology, we analyzed the content of some intracellular parameters. In our cell experiments, companied with increasing concentration, TPAN-18F-based PDT was found to decrease greatly in ATP levels (Fig. 3g). According to reported literature, one of the significant features of mitochondrial damage is reducing intracellular ATP production [38]. Therefore, the mitochondrial membrane potential (MMP) during PDT was further monitored by utilizing JC-10. As shown in Figs. 3e and f, TPAN-18F-based PDT could severely rise mitochondrial depolarization with enhanced green channel, which further confirmed mitochondrial damage. Considering the widespread distribution of TPAN-18F in the cytoplasm, which may affect the function of membranes of sub-organelles, the probe Liperfluo was utilized to analyze the intracellular LPOs level. The results showed that, the green signal surrounded the nuclei indicating the increased LPOs level in cells (Figs. 3h and i), which is the important bio-marker of ferroptosis [39,40]. Therefore, based on the above results, TPAN-18F-based PDT could initiate non-apoptotic death manner of tumor cells.
TPAN-18F-mediated PDT was further conducted in 4T1-tumor-bearing mice. Therefore, 4T1 cancer cells were inoculated in the right leg of mice to construct the mouse tumor models (Fig. S7 in Supporting information). These 4T1-tumor-bearing mice were divided into 3 groups (that is, blank group, TPAN-18F group and TPAN-18F + light group), and monitored them every day after light irradiation.
Before the treatment, intravenous injection was conducted with 4T1 tumor-bearing mice. However, the fluorescence signal of PDT agents was not observed at the tumor region. Accordingly, in order to obtain accurate treatment results and avoid the side effects on other healthy tissues (or organs) in clinical use, in situ injection of PSs was selected to confirm the treatment efficiency of TPAN-18F. Subsequently, tumor phototherapy in vivo was performed by intratumorally injecting TPAN-18F before light irradiation. All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Hunan University, and experiments were approved by the Animal Ethics Committee of the College of Biology (Hunan University). Tumor growth was monitored every day after treatment to evaluate tumor efficacy (Fig. 4a). The results showed that tumors treated with TPAN-18F and light irradiation were suppressed compared to blank group (Figs. 4c and d). In contrast, tumor growth in the TPAN-18F group only showed finite inhibited. Such results indicated that the TPAN-18F-mediated PDT achieved therapeutic effects. However, perhaps due to the shallow penetration of white light, the tumor did not completely ablate. In addition, to investigate the biological safety of TPAN-18F, experimental mice were monitored for body weight changes and processed for histological analysis of major organs. Experimental mice pre-treated with TPAN-18F remain stable their body weight with or without irradiation during the 7-day experimental period (Fig. 4b). According to histological analysis (hematoxylin-eosin (H.E.) results), no significant tissue damage in heart, liver, spleen, lung and kidney (Fig. 4e). Therefore, TPAN-18F-mediated PDT had promising biocompatibility and biosafety.
In summary, we have designed a novel water-soluble three positive charge AIE PS, TPAN-18F. Of note, TPAN-18F had excellent water solubility, whose absorption spectrum in water was coincided with DMSO. And TPAN-18F could enter different sub-organelles (mitochondria, lysosomes, endoplasmic reticulum) in the cytoplasm which ensured that PDT could be aimed to disrupt tumor cell across the board. More importantly, TPAN-18F-based PDT process could not only disrupt mitochondrial functions (reducing ATP production and destroying mitochondrial membrane potential), but also elevate the intracellular LPOs level, which evoke the non-apoptotic death manner of tumor cells. This work showed that, multi-charge-based PDT reagents with huge potential, are very promising applications in tumor treatment.
Declaration of competing interest
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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
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This work is supported by the National Science Foundation of China (No. 21890744), and the National Key R&D Program of China (No. 2019YFA0210100).
Supplementary materials
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Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2023.109190.
References
Less
[1]
H.W. Liu, L. Chen, C. Xu, et al., Chem. Soc. Rev. 47 (2018) 7140–7180.
[2]
Z. Hu, C. Fang, B. Li, Z. Zhang, et al., Nat. Biomed. Eng. 4 (2019) 259–271.
[3]
C. Wang, W. Fan, Z. Zhang, et al., Adv. Mater. 31 (2019) 1904329.
[4]
C. Li, Y. Pang, Y. Xu, et al., Chem. Soc. Rev. 52 (2023) 4392–4442.
[5]
A.B. Chagpar, R.C. Martin, L.J. Hagendoorn, C. Chao, K.M. McMasters, Am. J. Surg. 188 (2004) 399–402.
[6]
M.C. Smitt, K. Horst, Ann. Surg. Oncol. 14 (2007) 1040–1044.
[7]
S. Gioux, H.S. Choi, J.V. Frangioni, Mol. Imaging 9 (2010) 237–255.
[8]
M. Rizzo, R. Iyengar, S.G. Gabram, et al., Ann. Surg. Oncol. 17 (2010) 228–234.
[9]
A.L. Vahrmeijer, M. Hutteman, J.R. van der Vorst, C.J. van de Velde, J.V. Frangioni, Nat. Rev. Clin. Oncol. 10 (2013) 507–518.
[10]
S. Liu, L. Luo, L. Zhao, et al., Nat. Commun. 12 (2021) 4014.
[11]
C. Yang, G. Song, H. Yuan, et al., CCS Chem. 3 (2021) 1116–1128.
[12]
Y. Xu, C. Li, J. An, et al., Sci. China Chem. 66 (2022) 155–163.
[13]
J. Yuan, Q.H. Zhou, S. Xu, et al., Angew. Chem. Int. Ed. 61 (2022) e202206169.
[14]
Z. Li, P.Z. Liang, L. Xu, et al., Nat. Commun. 14 (2023) 1843.
[15]
H. Zhu, K. Ma, R. Ruan, et al., Chin. Chem. Lett. 35 (2024) 108536.
[16]
J. Mei, N.L.C. Leung, R.T.K. Kwok, J.W.Y. Lam, B.Z. Tang, Chem. Rev. 115 (2015) 11718–11940.
[17]
G. Hong, A.L. Antaris, H. Dai, Nat. Biomed. Eng. 1 (2017) 0010.
[18]
P. Lu, X. Zhang, T. Ren, L. Yuan, Chin. Chem. Lett. 31 (2020) 2980–2984.
[19]
S. Wang, W.X. Ren, J.T. Hou, et al., Chem. Soc. Rev. 50 (2021) 8887–8902.
[20]
X. Zhang, T. Ren, F. Yang, L. Yuan, Chin. Chem. Lett. 32 (2021) 3890–3894.
[21]
H. Li, H. Kim, F. Xu, et al., Chem. Soc. Rev. 51 (2022) 1795–1835.
[22]
Z. Li, P.Z. Liang, T.B. Ren, L. Yuan, X.B. Zhang, Angew. Chem. Int. Ed. 62 (2023) e202305742.
[23]
X. Zhang, F. Yang, T. Ren, et al., Chin. Chem. Lett. 34 (2023) 107835.
[24]
W. Fan, P. Huang, X. Chen, Chem. Soc. Rev. 45 (2016) 6488–6519.
[25]
X. Li, S. Lee, J. Yoon, Chem. Soc. Rev. 47 (2018) 1174–1188.
[26]
J. Kou, D. Dou, L. Yang, Oncotarget 8 (2017) 81591–81603.
[27]
R.C.H. Wong, P.C. Lo, D.K.P. Ng, Coord. Chem. Rev. 379 (2019) 30–46.
[28]
Z. Liu, H. Zou, Z. Zhao, et al., ACS Nano 13 (2019) 11283–11293.
[29]
C. Gui, E. Zhao, R.T.K. Kwok, et al., Chem. Sci. 8 (2017) 1822–1830.
[30]
D. Wang, M.M.S. Lee, G. Shan, et al., Adv. Mater. 30 (2018) e1802105.
[31]
D. Wang, H. Su, R.T.K. Kwok, et al., Chem. Sci. 9 (2018) 3685–3693.
[32]
Y. Gao, X. Wang, X. He, et al., Adv. Funct. Mater. 29 (2019) 1902673.
[33]
T. Zhang, Y. Li, Z. Zheng, et al., J. Am. Chem. Soc. 141 (2019) 5612–5616.
[34]
W. Xu, M.M.S. Lee, J.J. Nie, et al., Angew. Chem. Int. Ed. 59 (2020) 9610–9616.
[35]
Y. Yu, S. Wu, L. Zhang, et al., Biomaterials 280 (2022) 121255.
[36]
Z. Zheng, H. Liu, S. Zhai, et al., Chem. Sci. 11 (2020) 2494–2503.
[37]
S. Xu, Y. Yuan, X. Cai, et al., Chem. Sci. 6 (2015) 5824–5830.
[38]
C. Jiang, X. Li, F. Pan, et al., Adv. Funct. Mater. 33 (2023) 2211698.
[39]
Z. Dong, P. Liang, G. Guan, et al., Angew. Chem. Int. Ed. 61 (2022) e202206074.
[40]
W. Li, S. Yin, Y. Shen, et al., J. Am. Chem. Soc. 145 (2023) 3736–3747.
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Year 2024 volume 35 Issue 8
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doi: 10.1016/j.cclet.2023.109190
  • Receive Date：2023-08-09
  • Online Date：2025-11-21
  • Published：2024-08-15
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  • Received：2023-08-09
  • Revised：2023-09-28
  • Accepted：2023-10-07
Affiliations
    State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China
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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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