Transdermal delivery of amphotericin B using deep eutectic solvents for antifungal therapy

Transdermal delivery of amphotericin B using deep eutectic solvents for antifungal therapy

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Bing Xie^a^,¹, Qi Jiang^a^,¹, Fang Zhu^b^,¹, Yaoyao Lai^a, Yueming Zhao^*^,^b, Wei He^*^,^a^,^c, Pei Yang^*^,^d

Chinese Chemical Letters | 2025, 36(5) : 110508

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Chinese Chemical Letters | 2025, 36(5): 110508

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Transdermal delivery of amphotericin B using deep eutectic solvents for antifungal therapy

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Bing Xie^a^,¹, Qi Jiang^a^,¹, Fang Zhu^b^,¹, Yaoyao Lai^a, Yueming Zhao^*^,^b, Wei He^*^,^a^,^c, Pei Yang^*^,^d

Affiliations

^aSchool of Pharmacy, China Pharmaceutical University, Nanjing 211198, China

^bDepartment of Respiratory and Critical Medicine, Nanjing First Hospital, Nanjing Medical University, Nanjing 210000, China

^cKey Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Chinese Medicine, Nanchang 330004, China

^dSchool of Science, China Pharmaceutical University, Nanjing 211198, China

Published: 2025-05-15 doi: 10.1016/j.cclet.2024.110508

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Abstract

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Candida albicans is one of the most common pathogens causing invasive fungal infections, with a mortality rate of up to 20%–50%. Amphotericin B (AmB), a biopharmaceutics classification system (BCS) IV drug, significantly inhibits Candida albicans. AmB is primarily administered via oral and intravenous infusion, but severe infusion adverse effects, nephrotoxicity, and potential hepatotoxicity limit its clinical application. Deep eutectic solvents (DESs), with excellent solubilization ability and skin permeability, are attractive for transdermal delivery. Herein, we used DESs to deliver AmB for antifungal therapy transdermally. We first prepared and characterized DESs with different stoichiometric ratios of choline (Ch) and geranate (Ge). DESs increased the solubility of AmB by a thousand-fold. In vitro and in vivo, skin permeation studies indicated that DES_1:2 (Ch and Ge in 1:2 ratio) had the most outstanding penetration and delivered fluorescence dye to the dermis layer. Then, DES_1:2-AmB was prepared and in vitro antifungal tests demonstrated that DES_1:2-AmB had superior antifungal effects compared to AmB and DES_1:2. Furthermore, DES_1:2-AmB was skin-irritating and biocompatible. In conclusion, DES-AmB provides a new and effective therapeutic solution for fungal infections.

Key words

Amphotericin B / Deep eutectic solvents / Transdermal delivery / Candida albicans / Fungal infections

Cite this Article

Bing Xie, Qi Jiang, Fang Zhu, Yaoyao Lai, Yueming Zhao, Wei He, Pei Yang. Transdermal delivery of amphotericin B using deep eutectic solvents for antifungal therapy[J]. Chinese Chemical Letters, 2025 , 36 (5) : 110508 - . DOI: 10.1016/j.cclet.2024.110508

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About 6.5 million people worldwide suffer from invasive fungal infections each year, of which about 2.5 million die, posing a severe threat to human health [1]. Invasive fungal infections occur in the subcutaneous tissues and internal organs. Candida, especially Candida albicans, is one of the most common pathogens causing invasive fungal infections [2]. According to the list of fungal priority pathogens issued by the World Health Organization, Candida albicans belongs to the critical priority group, and its mortality rate of invasive infections is up to 20%–50% [3, 4].

Amphotericin B (AmB) is a polyene antifungal antibiotic from the biopharmaceutics classification system (BCS) IV drug, exerting antifungal effects by disrupting the permeability of cell membranes and forming extramembranous aggregates [5]. Due to its broad-spectrum antifungal activity and low resistance, AmB is the gold standard drug for invasive fungal infections [6, 7]. AmB is administered via oral and intravenous infusion, however, severe infusion adverse effects, nephrotoxicity, hemolysis, and poor oral absorption limit its clinical application [8, 9]. For instance, AmBisome®, an approved liposomal formulation, can significantly reduce toxicity but suffers from high treatment costs and potential hepatotoxicity [10-12]. Therefore, developing a safe and low-cost strategy for the efficient delivery of AmB is urgent.

Transdermal delivery provides a non-invasive, highly compliant, and safe treatment option [13-15]. However, the stratum corneum (SC) comprises keratinocytes and intercellular lipids; only drugs with specific chemical properties can pass through it [16-18]. Deep eutectic solvents (DESs) are liquid mixtures that are primarily formed by hydrogen bonding between hydrogen bond acceptors and donors [19, 20], also referred to as ionic liquids, as the two have a molar ratio of 1:1 [21, 22]. DESs significantly increase the solubility and enhance the skin permeability of insoluble drugs, and have been widely used in the transdermal delivery of insoluble drugs, biomolecules, and rigid particles [23-26]. In particular, choline (Ch) and geranate (Ge)-based DESs exhibit superior biocompatibility and biodegradability and are attractive options for transdermal delivery [27].

In this study, we prepared Ch-Ge-based DESs-AmB to treat fungal infections via transdermal administration. We first prepared and characterized various DESs with different Ch-Ge stoichiometric ratios. DESs significantly improved AmB's solubility and skin permeability. More significantly, DESs-AmB exhibited excellent anti-Candida albicans activity without skin irritation. Therefore, DES-AmB is a promising strategy for the transdermal treatment of fungal infections.

First, DESs with different Ch-Ge stoichiometric ratios (4:1, 2:1, 1.5:1, 1:1, 1:2, and 1:4 ratios) were prepared by salt metathesis reaction, expressed as DES_4:1, DES_2:1, DES_1.5:1, DES_1:1, DES_1:2, and DES_1:4, respectively. DESs were transparent and clear liquids (Fig. S1 in Supporting information). Then, we characterized the DESs using proton nuclear magnetic resonance (¹H NMR) and Fourier-transform infrared spectroscopy (FTIR) (Fig. S2 in Supporting information). In the ¹H NMR spectroscopy, an upfield shift of H in the Ge-related fragment indicated the disruption of the original intermolecular hydrogen bonding and the formation of new hydrogen bonding in DESs. In the FTIR spectra, the absorption peaks of O–H and C=O in choline bicarbonate appeared at 3389.9 and 1624.1 cm⁻¹, respectively. The absorption peaks of O–H and C=O in Ge appeared at 2969.6 and 1691.8 cm⁻¹, respectively. The shifts of O–H and C=O absorption bands in DESs changed with the molar ratio of Ch and Ge. The O–H and C=O peaks in DESs shifted at 2967.4–3418.5 cm⁻¹ and 1621.4–1692.1 cm⁻¹. The results confirmed the formation of DESs.

Next, we studied the DES's ability to solubilize AmB and transdermally deliver the payloads. DESs demonstrated significant solubility enhancement of the drug. The AmB solubility in DESs was 110–5585 times higher than in water (Fig. 1A and Table S1 in Supporting information). Then, we tested the skin penetration of DESs-1, 1′-dioctadecyl-3, 3, 3′, 3′-tetramethylindotricarbocyanine iodide (DiR) and -fluorescein isothiocyanate (FITC). The cumulative DiR-penetration amounts from DESs (DES_4:1, DES_2:1, DES_1.5:1, DES_1:1, DES_1:2, and DES_1:4) were 23.13 ± 0.40, 24.46 ± 0.32, 25.96 ± 1.34, 26.30 ± 2.50, 28.74 ± 1.05, and 26.93 ± 1.48 ng/cm², respectively (Fig. 1B). The results indicated that DESs improved the skin penetration of DiR compared to DiR solution, and DES_1:2 had the highest penetration ability. Furthermore, the penetration depth of DESs-FITC in vitro was observed using fluorescence microscope (Fig. 1C). DESs-FITC passed through the SC and diffused into the epidermis and dermis layers. Again, DES_1:2-FITC showed higher penetration than other DESs (Fig. 1D). This enhanced skin permeability may be attributed to DESs' lipid extraction [28]. Moreover, increasing Ch in DESs compromised the skin permeability due to high viscosity and high hydrophilicity [29]. As a result, the penetration elevated as the Ch-Ge stoichiometric ratio reduced from 4:1 to 1:2.

To characterize the drug-loading DES, we prepared DES-AmB (with 1 mg/mL AmB) by vortexing and sonication. DES-AmB exhibited a clarified and transparent appearance (Fig. S3 in Supporting information). To verify the interaction between AmB and DES, we performed the test of FTIR and ¹H NMR of DES_1:2-AmB. The ¹H NMR peaks at δ 6.41–5.99 and 1.23–0.91 ppm in AmB disappeared in DES_1:2-AmB, indicating the interactions between AmB and DES_1:2 (Fig. 2A). As shown in Fig. 2B, the O–H stretching vibration peak in AmB shifted from 3383.9 cm⁻¹ to 3396.7 cm⁻¹. The C=O stretching vibration peak in AmB shifted from 1689.5 cm⁻¹ to 1645.3 cm⁻¹, demonstrating the hydrogen bonding in DES_1:2-AmB.

DESs have self-assembling behavior in water and form colloidal systems, such as micelles or emulsions, due to the hydrophilic of Ch and hydrophobic tails of Ge [30-32]. In addition, the micellar structure can increase skin permeability through diffusion and fusion during transdermal drug delivery [33]. Here, we diluted DES-AmB to 30% (v/v) using water and measured the particle size (Fig. 2C and Table S2 in Supporting information). The formulation of 30% DES_1:2-AmB demonstrated the most minor diameter (14.08 ± 1.90 nm, PDI 0.282 ± 0.012). In contrast, other formulations of 30% DES-AmB displayed a significant increase in particle size and PDI, likely due to the compromising interaction between the drug and DESs and the ratios of Ch-Ge [29, 34]. Transmission electron microscopy (TEM) examination displayed that the 30% DES_1:2-AmB nanocomplexes were sub-spherical (Fig. 2D). In the differential scanning calorimetry (DSC) spectra (Fig. 2E), AmB and DES_1:2 showed heat-absorption peaks at 205 and 241 ℃, respectively. DES_1:2-AmB had a heat-absorption peak at 239 ℃ with a similar peak to DES_1:2, indicating that AmB was miscible with DES_1:2.

As shown in Fig. S4 (Supporting information), storage stability indicated that the AmB content in DES_1:2-AmB remained constant after one-month storage at 4 ℃ (Figs. S4A and B). However, long-term testing indicated that the drug content in 30% DES_1:2-AmB decreased significantly under similar storage conditions (Fig. S4C). AmB in 30% DES_1:2-AmB was almost completely degraded under high temperature, high humidity and intense light due to the sensitivity of AmB to water, light, and temperature (Figs. S4D–G). The results indicated that DES_1:2-AmB instead of 30% DES_1:2-AmB should be stored at low temperature and low humidity and protected from light. We also found that the drug stability was related to the Ch-Ge stoichiometric ratios in DES.

The antifungal effects of DESs and AmB were studied by measuring the inhibition zone diameters (IZDs) (Figs. 3A–C). DESs with different stoichiometric ratios significantly inhibited Candida albicans, and the antifungal effect of DES_1:2 was comparable to that of other DESs except for DES_1:4 (Fig. 3D). DES_1:2 and AmB enhanced antifungal activity in a dose-dependent manner (Figs. 3E and F). Considering the convenience and applicability, 30% DES_1:2-AmB with appropriate flowability was selected for the following study. When the concentration of AmB was 0.125, 1, and 8 µg/mL, the IZDs of DES_1:2-AmB were more significant than that of AmB (Figs. 3G–I). In particular, when the concentration of AmB was 8 µg/mL, the IZDs of DES_1:2-AmB were 1.2 and 3.4 times higher than those of DES_1:2 and AmB, respectively, and the significant difference indicated the synergistic antifungal effects between DES_1:2 and AmB (Fig. 3I).

The minimum inhibitory concentration (MIC₉₀) of AmB and DES_1:2-AmB was further performed to evaluate the antifungal ability. The MIC₉₀ of AmB and DES_1:2-AmB were 0.25 and 0.125 µg/mL, respectively (Fig. 3J). The antifungal effect of DES_1:2-AmB may be attributed to several antifungal mechanisms. AmB binds to ergosterol on fungal cell membranes, creating micropores and killing the fungus [35]. Recent studies have shown that AmB extracts ergosterol and forms extramembranous aggregates to exert antifungal effects [36, 37]. DESs may extract or fluidize microbial membrane lipids [38, 39]. The results indicated that combining DESs and antifungal drugs could be an effective strategy against fungal infections.

Finally, safety was assessed by studying the cytotoxicity and skin irritation. The viability of human immortalized epidermal (HaCaT) cells decreased with the increase of DES_1:2 concentration. When the concentration of DES_1:2 reached 16 mg/mL, the survival rate of HaCaT cells was still higher than 80%, indicating that DES_1:2 had a comprehensive safety range (Fig. 4A). In addition, histopathological analysis revealed that the treatment using DES_1:2-AmB showed a tight connection between the SC, epidermis layer, and dermis layer and allowed little inflammatory cell infiltration and necrosis compared to saline administration (Fig. 4B), indicating biocompatibility. All the animal experimental were performed according to the protocols approved by China Pharmaceutical University Institutional Animal Care and Use Committee (No. 202409016).

In summary, the Ch-Ge-based DESs could dramatically solubilize AmB and improve transdermal delivery. Notably, DES_1:2 possessed potent antifungal effects, and its combined application with AmB exhibited excellent inhibition of Candida albicans without significant skin irritation. DES_1:2-AmB is promising to treat dermatologic conditions caused by fungal infections and has the potential for clinical translation. In addition, due to simple composition and preparation, DES_1:2-AmB could be adapted to other antifungal and antibacterial drugs, reducing resistance and improving therapeutic efficacy.

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.

CRediT authorship contribution statement

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Author contributions

Bing Xie: Writing – original draft, Methodology, Investigation. Qi Jiang: Methodology, Investigation, Formal analysis, Data curation. Fang Zhu: . Yaoyao Lai: Methodology, Formal analysis, Data curation. Yueming Zhao: Writing – review & editing, Validation, Supervision, Investigation, Data curation, Conceptualization. Wei He: Writing – review & editing, Supervision, Project administration, Funding acquisition, Formal analysis, Data curation, Conceptualization. Pei Yang: Writing – review & editing, Supervision, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

Acknowledgments

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This study was supported by the National Natural Science Foundation of China (Nos. 81872823, 82073782, and 82241002) and the Key R & D Plan of Ganjiang New District of Jiangxi (No. 2023010).

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.2024.110508.

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Year 2025 volume 36 Issue 5

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doi: 10.1016/j.cclet.2024.110508

Receive Date：2024-08-02
Online Date：2025-10-29
Published：2025-05-15

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History

Received：2024-08-02
Revised：2024-09-23
Accepted：2024-09-25

Affiliations

^aSchool of Pharmacy, China Pharmaceutical University, Nanjing 211198, China

^bDepartment of Respiratory and Critical Medicine, Nanjing First Hospital, Nanjing Medical University, Nanjing 210000, China

^cKey Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Chinese Medicine, Nanchang 330004, China

^dSchool of Science, China Pharmaceutical University, Nanjing 211198, China

Corresponding:

^*E-mail addresses: sabrina-zym@163.com (Y. Zhao), weihe@cpu.edu.cn (W. He), yangpei@cpu.edu.cn (P. Yang).

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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 species (%)
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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Fig. 1 Solubilization and permeation enhancement. (A) Equilibrium solubility of AmB in DESs with different stoichiometry. (B) The cumulative penetration amount of DESs-DiR in vitro. (C) In vivo DESs-FITC permeation into rat skin (SC, stratum corneum). Scale bar: 250 µm. (D) Semi-quantitative analysis of FITC fluorescence intensity (mean ± SD, n = 20). ***P < 0.001 vs. the control group; ^|||P < 0.001 vs. DES_1:2-FITC group. ns, no significance.

Fig. 2 Preparation and characterization of DESs-AmB. (A) ¹H NMR and (B) FTIR spectra of AmB, DES_1:2-AmB and DES_1:2. (C) Particle size and PDI of 30% DESs-AmB. (D) TEM image of 30% DES_1:2-AmB (scale bar: 200 nm). (E) DSC thermograms of AmB, DES_1:2-AmB and DES_1:2.

Fig. 3 Antifungal activity in vitro. (A) The inhibitory circle of DESs. (B) The inhibitory circle of DES_1:2 with different concentrations. (C) The inhibitory circle of AmB and DES_1:2-AmB. (D) The IDZs of DESs (***P < 0.001). (E) The IDZs of AmB with different concentrations (^|P < 0.05, ^||P < 0.01 vs. 0.125 µg/mL). (F) The IDZs of DES_1:2 with various concentrations (***P < 0.001 vs. the 30% DES_1:2; ^|P < 0.05). (G-I) The IDZs of AmB, DES_1:2 and DES_1:2-AmB. The concentration of AmB was 0.125, 1, and 8 µg/mL (***P < 0.001, ****P < 0.0001 vs. the AmB group; ^|P < 0.05). (J) The minimum inhibitory concentration of AmB and DES_1:2-AmB. Data are presented as mean ± SD, n = 3.

Fig. 4 Safety evaluation. (A) In vitro cytotoxicity of DES_1:2 (mean ± SD, n = 6). (B) Histopathological evaluation of rats' skin sections treated with saline, DES_1:2, AmB and DES_1:2-AmB (scale bar: 100 µm).

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