Co-delivery of icariside II and doxorubicin by self-assembled carrier-free nanofibers for anti-lung cancer therapy

Abstract

Icariside II (ICAII), a bioactive compound derived from Epimedii Folium, exhibits promising anti-tumor activity but encounters challenges in its clinical application due to its poor solubility and low bioavailability. Thus, this study developed a novel carrier-free co-delivery system of ICAII and doxorubicin (DOX) through their self-assembly into nanofibers. ICAII combined with DOX nanofibers (ICAII-DOX NFs), and ICAII-DOX/TPGS NFs (with TPGS as a stabilizer) were systematically characterized for their physicochemical properties, including size distribution, morphology, and molecular interactions. The synergistic anti-lung cancer effect of ICAII and DOX was evaluated in vitro and in vivo. The prepared ICAII-DOX NFs and ICAII-DOX/TPGS NFs showed mean sizes of 127 and 338 nm, respectively, with PDI values of 0.2–0.3 and drug loading contents of >48%. FTIR, fluorescence, NMR and X-ray powder diffraction analyses revealed that the formation of ICAII-DOX co-assembly was primarily driven by intermolecular hydrogen bonding between the two molecules. The nanofibers demonstrated controlled drug release profiles (cumulative release rate of DOX was 65.88% at 48 h, and cumulative release rate of ICAII was 74.29% at 48 h) and enhanced cellular uptake (1.8-fold more than that of the free DOX group). CCK-8 assay results showed that the IC₅₀ values (calculated in terms of DOX) of the ICAII-DOX mixture, ICAII-DOX NFs and ICAII-DOX/TPGS NFs were 0.67, 0.60 and 0.44 μM in A549 human lung carcinoma cells, respectively. In vivo studies using an A549 xenograft mouse model showed the improved therapeutic efficacy of the co-delivery system (the inhibition rate of the ICAII-DOX mixture and ICAII-DOX/TPGS NF groups was 29.90%) compared with single drug treatment (the inhibition rates of the DOX and ICAII groups were 8.70% and 17.72%, respectively). This study presents a self-assembled carrier-free co-delivery system, providing a potential strategy for treating lung cancer.

---

1. Introduction

Epimedii Folium (Yinyanghuo in Chinese) is the dried leaf of Epimedium brevicornu Maxim., Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium pubescens Maxim. or Epimedium koreanum Nakai.¹ It has been clinically used to treat cancers, including lung cancer and liver cancer, and as a traditional Chinese medicine in China for many years.² Icariside II (ICAII), also known as baohuoside I, is one of the main active ingredients of Epimedii Folium. It is reported to exhibit anti-tumor pharmacological activity against many types of cancer, such as lung cancer,³ liver cancer,⁴ melanoma,⁵ breast cancer⁶ and prostate cancer.⁷ However, the clinical use of ICAII for treating cancer is largely limited because of its insufficient therapeutic dose in tumor tissues owing to its poor solubility and low bioavailability. Poor solubility is a common problem of many active ingredients of many natural compounds such as curcumin, paclitaxel, and camptothecin. Therefore, improving its solubility and drug loading (DL) capacity is essential in drug delivery application.

One way to increase the solubility of ICAII is encapsulating it in micelles using amphiphilic copolymers as carriers.⁸ Yan et al. prepared mixed micelles consisting of D-α-tocopheryl polyethylene glycol succinate (TPGS) and Solutol HS 15 for the encapsulation and delivery of ICAII.⁹ Although the solubility of ICAII increased from 0.0124 g L⁻¹ to 2.176 g L⁻¹, the DL of the micelles was only 8.13%. The intravenous injection dosage of ICAII was 25 mg kg⁻¹ per day for mice through their tail vein, while the dosage of the carriers was 308 mg kg⁻¹ per day. Consequently, the high dose of these carriers raise safety concerns. Other reports of ICAII-loaded micelles were similar.^10,11 Recently, carrier-free drug delivery systems have been developed as a new pattern of nanomedicine.^12,13 In these drug delivery systems, hydrophobic drugs are assembled into nano-size systems without adding any carrier, or stabilized with a small amount of surfactant for good water dispersity and high biostability. They have high DL capacity, which can alleviate possible toxicity from carriers and problems associated with biodegradation.¹⁴ In preliminary studies, we discovered that ICAII and DOX used in combination showed better proliferation inhibition against human lung cancer A549 cells than used singly. More interestingly, we found that ICAII and DOX could self-assemble into nanofibers (NFs) in aqueous solution.

Doxorubicin (DOX) is an effective broad-spectrum chemotherapeutic agent for the treatment of a variety of cancers, such as lung cancer, breast cancer, and liver cancer. However, it is reported that DOX has severe side effects¹⁵ such as cardiotoxicity,¹⁶ DNA damage, and reactive oxygen species overproduction.¹⁷ Therefore, the clinical use of DOX is largely limited. To reduce the toxicity and side effects of DOX, as well as improve its efficacy, the combination of DOX with other anti-tumor drugs was studied in cancer therapy. It has been reported that docetaxel,¹⁸ paclitaxel,¹⁹ curcumin²⁰ and other drugs were used with DOX in cancer combination therapy.

In this study, a self-assembled co-delivery system was developed utilizing the complementary chemical features of ICAII and DOX. ICAII is a type of flavonol glycoside. It has two phenolic hydroxyl groups and a sugar moiety, which confer hydrophilicity, and a hydrophobic flavonol backbone with an isopentenyl group. This amphiphilic nature of ICAII implies that it may act as a surfactant-like compound that tends to self-associate in aqueous solution via non-covalent interactions.²¹ Indeed, prior studies demonstrated that many natural compounds can self-assemble into nanostructures due to these features,¹⁴ including flavonoids such as baicalin.²² Conversely, DOX is an amphiphilic anthracycline drug. It has a multi-ring aromatic aglycone (anthraquinone) that is hydrophobic, and an amino-sugar group, which is hydrophilic and protonatable. In aqueous solution, DOX can co-assembly with other natural compounds with non-covalent interactions to form nanodrugs.^23–25 ICAI has a pK_a of approximately 7.5 in its flavonoid nucleus 7-OH,²⁶ while the pK_a of –NH₂ of DOX is about 8.2.²⁷ Thus, we hypothesized that under physiological pH, DOX is predominantly protonated (–NH₃⁺), while the 7-OH group of ICAII remains largely neutral, forming a charge-assisted hydrogen bond that drives the co-assembly of ICAII-DOX nanostructures.

TPGS, a non-ionic surfactant approved by the United States Food and Drug Administration (FDA), is widely utilized in nanodrug delivery systems to improve their emulsification, solubility, stability, and penetration. It is safe, lacks toxicity and has good biocompatibility.²⁸ In addition, TPGS possesses antioxidant activity, which helps protect drugs from oxidative degradation during storage, thereby improving the stability of formulations. In this study, TPGS was used as an adjuvant to enhance the solubility and stability of the ICAII-DOX carrier-free co-delivery system.

The synergistic effect of ICAII and DOX was studied. Its physical characteristics including size, PDI, zeta potential were characterized. The intermolecular interaction of ICAII and DOX in NFs was studied via FTIR, fluorescence spectroscopy, NMR and X-ray powder diffraction (XPRD). The in vitro drug release and cellular uptake of the NFs were examined. Then, in vivo studies were performed in an A549 xenograft mouse model. BALB/c nude mice were chosen due to their immunodeficient status, allowing the engraftment of human tumor cells without immune rejection. Male mice were selected to minimize hormonal variability, which could influence tumor growth and drug metabolism. Mice instead rats were used due to their advantages in terms of cost, ease of handling, and the availability of established tumor models.

In summary, we developed a carrier-free co-delivery system of ICAII and DOX for synergistic lung cancer therapy, aiming to maximize DL, minimize the use of excipients, and enable dual-drug delivery. This strategy offers a promising platform for efficient combination chemotherapy with reduced formulation complexity.

2. Experimental

2.1. Materials

Doxorubicin hydrochloride was purchased from Dalian Meilun Biotech Co., Ltd (Dalian, China). ICAII was prepared according to our previous report.²⁹ Cell counting kit-8 (CCK-8) was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Annexin V-APC/Syto X apoptosis detection kit was purchased from Keygen Biotech Co., Ltd (Nanjing, China). D-α-Tocopherol polyethylene glycol succinate (TPGS) was purchased from Aladdin Biochemical Technology Co., Ltd (Shanghai, China).

2.2. Cell culture

A549 human lung carcinoma cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM with high glucose, supplemented with 10% FBS, 100 U mL⁻¹ penicillin and 100 mg mL⁻¹ streptomycin. The cells were maintained at 37 °C in a humidified incubator with 5% CO₂. The culture medium was changed every two days.

2.3. Cell viability and proliferative assays

The cytotoxicity of ICAII-DOX NFs and ICAII-DOX/TPGS NFs in A549 cells was evaluated using the CCK-8 assay. The cells were plated in a 96-well plate (5000–8000 cells per well) and incubated at 37 °C in an atmosphere containing 5% CO₂ overnight. Then, the cell culture media were replaced with fresh media containing DOX, ICAII, ICAII-DOX NFs and ICAII-DOX/TPGS NFs with different concentrations and incubated for 48 h. The media were replaced by a mixture of 100 μL fresh media and 10 μL CCK-8. After incubation for 0.5–1 h, the absorbance was determined at 450 nm using a microplate reader (Eon, BioTek Instruments, Inc., USA). All experiments were repeated in triplicate. The cell viability was estimated and IC₅₀ values were calculated using the GraphPad Prism software (GraphPad Software Inc., USA).

2.4. Cell apoptosis assay

A549 cells (5 × 10⁵ per well) in 6-well plates were treated with DOX (2 μM), ICAII (10 μM) and ICAII-DOX mixture (at a DOX concentration of 5 μM DOX and an ICAII concentration of 10 μM) of agents for 4 h, and then harvested and washed twice in ice cold PBS. The cell apoptosis assay was conducted using the Annexin V-APC/Syto X apoptosis detection kit according to the manufacture's instruction and analyzed on a flow cytometer (FACS Calibur, BD Biosciences, USA). The data were analyzed using FlowJo software V10 (Tree Star, USA).

2.5. Preparation and characterization of ICAII-DOX NFs and ICAII-DOX/TPGS NFs

Briefly, to prepare ICAII-DOX NFs, ICA II and DOX were dissolved in 25 μL DMSO, 2 mL double distilled water was added, and then sonicated at 250 W for 10 min. To prepare ICAII-DOX/TPGS NFs, ICA II and DOX were dissolved in 25 μL DMSO, 2 mL TPGS solution (1 mg mL⁻¹) was added, and then sonicated at 250 W for 10 min. ICAII-DOX NFs and ICAII-DOX/TPGS NFs were dialyzed against distilled water for 4 h to remove DMSO and free DOX, and then the above-mentioned solution was collected.

After diluting 20 times with double distilled water, the particle size, polydispersity index (PDI) and zeta potential of ICAII-DOX NFs were determined using a particle size and zeta potential analyzer (Zetasizer Nano ZS90, Malvern Instruments, UK). The morphology of ICAII-DOX NFs and ICAII-DOX/TPGS NFs was observed via transmission electron microscopy (TEM, JEM 2100, JEOL Ltd, Japan). The samples were prepared by dripping the solutions onto mesh copper grids with a carbon film, and then staining with 1% phosphotungstic acid and drying under vacuum for 30 min.

To measure DL, ICAII-DOX NFs and ICAII-DOX/TPGS NFs were diluted with methanol and sonicated to destroy the structure of NFs. The amount of ICAII was determined using an Agilent 1200 HPLC Systems (Agilent Technologies, USA) equipped with a Diamonsil C₁₈ chromatographic column (5 μm, 4.6 mm × 250 mm, Dikma, China).²⁹ Also, the amount of DOX was determined using a UV-Vis spectrophotometer (UV, 8454, Agilent Technologies, USA) at the wavelength of 480 nm. DL (%) = weight of loaded DOX − (weight of DOX-loaded micelles) × 100%.

2.6. Molecular interaction determination

ICAII-DOX NFs were freeze-dried, and a small amount of dried powder was taken to record their infrared spectrum using an FTIR spectrometer (4000–450 cm⁻¹) equipped with an ATR annex (iS10, Thermo Fisher Scientific Inc., USA). ¹H-NMR, ¹³C-NMR, HMBC and NOESY spectra of ICAII-DOX NFs in DMSO-D₆ were recorded using a 400 MHz NMR spectrometer (AVANCE NEO, Bruker Corporation, Switzerland). XRPD analysis of ICAII-DOX NFs was performed using a Bruker D8 Advance X-ray diffractometer operating at a tube voltage of 40 kV and current of 20 mA. Fluorescence spectra of DOX and ICAII-DOX NFs were recorded at 480 nm excitation using a fluorescence spectrophotometer (Cary Eclipse, Agilent Technologies, USA).

2.7. Stability of ICAII-DOX NFs and ICAII-DOX/TPGS NFs

The stability of ICAII-DOX NFs and ICAII-DOX/TPGS NFs was investigated. Briefly, the NFs were stored at 25 °C and 4 °C, respectively. The size of the NFs was monitored at predetermined intervals using a particle size and zeta potential analyzer.

2.8. Drug release

The release of ICAII and DOX from ICAII-DOX NFs and ICAII-DOX/TPGS NFs was studied via a dialysis method in PBS (10 mM, pH 7.4) with 0.5% Tween-80. Briefly, 3 mL ICAII-DOX NFs or ICAII-DOX/TPGS NFs was transferred to a dialysis bag (MWCO 3500 Da, Spectrum Laboratories Inc., USA), and then dialyzed against 40 mL release medium in a tube. The tubes were placed in a shaking incubator at 37 °C (100 rpm). 5 mL media was taken out and an equivalent volume of fresh media was added at predetermined intervals. The amount of released ICAII was determined using an Agilent 1200 HPLC Systems. DOX was determined using a UV-Vis spectrophotometer at the wavelength of 480 nm.

2.9. Cellular uptake

Laser scanning confocal microscopy (CLSM) was used to study the cellular uptake and intracellular distribution of ICAII-DOX NFs and ICAII-DOX/TPGS NFs in A549 cells. Briefly, the cells were seeded at a density of 2 × 10⁴ per well in glass bottom cell culture dishes (NEST Biotechnology Co. LTD, China) and incubated overnight at 37 °C. Then, 100 μL DOX, ICAII, ICAII-DOX NFs and ICAII-DOX/TPGS NFs (at a final DOX concentration of 10 μM) were added and incubated for 24 h. The cells were washed twice with ice-cold PBS (pH 7.4) and fixed with 4% paraformaldehyde for 30 min, followed by washing with PBS three times. The cell nuclei were stained with 100 μL DAPI (1 μg mL⁻¹) for 10 min and washed with PBS four times. Fluorescence images were captured using CLSM (TCS SP2, Leica Microsystems, Germany). The fluorescent intensity of DOX in the cells was analyzed using the LAS AF software (Leica Microsystems, Germany).

A flow cytometer was used to verify the cellular uptake of ICAII-DOX NFs and ICAII-DOX/TPGS NFs in A549 cells. A549 cells (1 × 10⁶ per well) in 6-well plates were incubated with 1 mL DOX, ICAII, ICAII-DOX NFs and ICAII-DOX/TPGS NFs (at a final DOX concentration of 10 μM) in a cell incubator for 24 h. Then the cells were washed thrice with cold PBS and harvested. After aspiration of the supernatant, the cells were resuspended in 0.5 mL PBS and analyzed on a flow cytometer (FACS Calibur, BD Biosciences, USA). The data were analyzed using FlowJo software V10 (Tree Star, USA).

2.10. In vivo anti-tumor effect

Male BALB/c nude mice (Xipuer Bikai Co., Ltd, Shanghai, China), weighing 18–20 g, were quarantined in specific pathogen-free animal laboratory with controlled humidity and temperature. A549 cells (8 × 10⁶ per mouse) were subcutaneously injected into the right back (near the armpit) of the mice. When the tumor size reached 40–50 mm³, the mice were randomly divided into control, DOX, ICAII, ICAII-DOX Mixture, and ICAII-DOX/TPGS NFs groups, with 5 mice in each group. The mice were intravenously injected with saline (control), DOX, ICAII, ICAII-DOX mixture, and ICAII-DOX/TPGS NFs in the corresponding groups every 3 days 7 times. The dose of DOX and ICAII of each group was 3.0 and 15.0 mg kg⁻¹, respectively. The tumor volumes were monitored using calipers (volume = width² × length × 0.5) and the body weight was record at predetermined intervals. The mice were sacrificed on day 23. The tumors were collected and weighed. The inhibition rate of tumor = (1 − average tumor weight of each drug group − control group) × l00%.

All animal experiments adhered to the Guide for the Care and Use of Laboratory Animals. Animal protocols were approved by the Ethics Committee of Shanghai University of Traditional Chinese Medicine (Approval Protocol Number: 2018004003).

2.11. Histological analysis

All tissues were fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and sliced at 5 μm thickness. The sections were stained with haematoxylin and eosin (H&E), and examined by light microscopy (CKX53, Olympus Corporation, Japan).

2.12. Statistical analysis

The experimental data are presented as mean ± standard deviation (SD). Differences between groups were analyzed by student's t-test or ANOVA. Differences were considered statistically significant when the P-values were less than 0.05.

3. Results and discussion

3.1. Combination therapy of ICAII and DOX against A549 cells

The in vitro cytotoxicity of the combination of ICAII and DOX on A549 cells was evaluated using the CCK-8 assay and analyzed by the CalcuSyn 2.0 software (Biosoft, Cambridge, UK). The results (Table S1, ESI†) showed that when the weight ratio of ICAII : DOX = 5 : 1, the D_m value of DOX was lower than the other ratios, at 0.39 μM, while the D_m of ICAII was 1.97 μM, indicating the enhanced synergistic anti-tumor activity at this proportion. The lowest D_m of ICAII was 1.57 μM in the ratio of 2.5 : 1. Considering that it has been reported that DOX has many side effects such as cardiotoxicity, the ratio of 5 : 1 was chosen for combination treatment of ICAII and DOX to minimize the dose of DOX.

The combination index (CI) was used to estimate the synergistic effect of ICAII and DOX. As shown in Table S2 (ESI†), the CI of ICAII and DOX was less than 1, which indicated that these two drugs have a synergistic effect. When the concentration of DOX was lower than 0.5 μM, the synergistic effect was strong as the CI was between 0.2–0.4 μM.³⁰

3.2. Cell apoptosis assay

The apoptosis-inducing effect of free and combination drugs on A549 cells was measured through an Annexin V-APC/Syto X apoptosis detection kit. As shown in Fig. 1, the induced rate of early stage (Annexin V⁺/Syto X⁻) and late stage (Annexin V⁺/Syto X⁺) apoptosis by the combo drugs was much higher than that by the single drugs after incubation for 4 h. This indicated that the combination of DOX and ICAII significantly promoted the apoptosis of A549 cells.

Fig. 1 (A) Dotted plot showing apoptosis of A549 cells induced by DOX (2 μM), ICAII (10 μM) and ICAII-DOX mixture (at a DOX concentration of 5 μM DOX and ICAII concentration of 10 μM) for 4 h. The experiments were repeated three times and representative plots are shown. (B) Apoptosis rate of A549 cells. Early apoptotic cells are defined as Annexin V⁺/Syto X⁻ and late apoptotic/necrotic cells are Annexin V⁺/Syto X⁺. * for p < 0.05, ** for p < 0.01, *** for p < 0.001.

3.3. Preparation and characterization of ICAII-DOX NFs

ICAII-DOX NFs were prepared without any excipient. The aqueous dispersion of ICAII-DOX NFs was transparent and precipitate free, and its color was red (Fig. 2(A)). Upon exposure to laser radiation, an apparent Tyndall effect could be observed in the aqueous dispersion of ICAII-DOX NFs (Fig. 2(B)).

Fig. 2 Characterization of ICAII-DOX NFs and ICAII-DOX/TPGS NFs. (A) Photos of ICAII-DOX NFs and ICAII-DOX/TPGS NFs and (B) their Tyndall phenomenon (illuminated by a laser light). (C) Size distribution and TEM micrograph of ICAII-DOX NFs and ICAII-DOX/TPGS NFs.

To improve the stability of the NFs, ICAII-DOX/TPGS NFs were prepared by adding TPGS (1 mg mL⁻¹) as a stabilizer. The aqueous dispersion of ICAII-DOX/TPGS NFs was emulsion-like and precipitate free, and its color was red (Fig. 2(A)). Upon exposure to laser radiation, an apparent Tyndall effect could also be observed (Fig. 2(B)).

The size, PDI, and zeta potential of ICAII-DOX NFs and ICAII-DOX/TPGS NFs are shown in Table 1. The size of ICAII-DOX NFs was smaller than ICAII-DOX/TPGS NFs, and the PDI of ICAII-DOX/TPGS NFs is small (0.200), which indicated that it had narrow size distribution. The zeta potential results demonstrated that ICAII-DOX NFs exhibit a moderately positive surface charge of +24.43 ± 1.00 mV, indicating that the –NH₂ groups in DOX are mainly protonated as –NH₃⁺. DOX has two pK_a values of approximately 8.2 (−NH₂) and 9.5 (phenolic 11-OH). Pyne et al. reported that the co-assembly behavior of DOX can be disrupted by the deprotonation of the –NH₃⁺ moiety at pH 8.9.²⁷ Consequently, DOX remains predominantly protonated as –NH₃⁺ at physiological pH. This protonated state significantly enhances its ability to participate in non-covalent interactions (e.g. hydrogen-bonding and electrostatic interactions), thereby reinforcing the stability of the self-assembled nanostructure.³¹ It has been reported that NFs with a smaller size have more chance to escape liver and kidney filtration, thus enhancing their EPR effect.^32,33 However, the positive potential of the NFs may make them easy to be eliminated by MPS in vivo. In this case, decoration with the amphiphilic excipient TPGS reduced the potential of ICAII-DOX/TPGS NFs to a nearly neutral value of +0.07 ± 0.15 mV, thus extending their in vivo circulation time. Nanoparticles with neutral surface charge tend to exhibit prolonged blood circulation in vivo, potentially enhancing their therapeutic efficacy by allowing more time for the drug to reach the target tissues.³⁴ High zeta potentials (20–40 mV) ensure colloidal stability by decreasing aggregation and increasing polydispersity due to high charge repulsion.³⁵ Conversely, the near-zero zeta potential of ICAII-DOX/TPGS NFs would normally promote aggregation. However, the densely grafted PEG1000 chains of TPGS provide a formidable steric and hydration barrier that compensates for the reduced charge repulsion, preserving their dispersion stability.³⁶ Vuddanda et al. demonstrated that TPGS-coated nanoparticles remained stable without agglomeration even at neutral zeta potential due to the presence of a steric stabilization layer.

Table 1 Size and zeta potential ICAII-DOX NFs and ICAII-DOX/TPGS NFs

Sample	Size (nm)	PDI	Zeta potential (mV)
ICAII-DOX NFs	127.13 ± 2.63	0.319 ± 0.036	+24.43 ± 1.00
ICAII-DOX/TPGS NFs	338.17 ± 5.04	0.200 ± 0.008	+0.07 ± 0.15

---

Notably, the DL reached 100% in ICAII-DOX NFs given that no carrier or stabilizer was added, whereas ICAII-DOX/TPGS NFs achieved a substantial DL of 48.80%. The DL of these two NFs was significantly higher compared to traditional delivery systems such as liposomes and micelles. This enhanced DL minimizes the amount of excipients, thereby reducing the risk of excipient-related side effects. By accommodating higher concentrations of active drugs, these NFs enable efficient delivery with a smaller formulation dose, optimizing both the safety and efficacy of the treatment.³⁷

The particle morphology of ICAII-DOX NFs and ICAII-DOX/TPGS NFs was observed by TEM (Fig. 2(C)). The TEM micrograph demonstrated that the two NFs were spiral filamentous in shape. To the best of our knowledge, most of the reported micelles formed using TPGS as a carrier are spherical or near spherical in shape,^38,39 which is obviously different from the NFs prepared in this study. Thus, the results suggest that ICAII-DOX NFs are a novel nano-formulation.

3.4. Self-assembly mechanism of ICAII-DOX NFs

The functional groups present in ICAII-DOX NFs were determined by FTIR. As shown in Fig. 3(A), the O–H and N–H stretching vibration band of ICAII-DOX NFs appeared at 3162 cm⁻¹, which is broader than the O–H stretching vibration band of ICAII at 3111 cm⁻¹, as well as the O–H and N–H stretching vibration band of DOX at about 3527–3319 cm⁻¹. These results indicated that the hydroxyl and amino group were in an association state, suggesting that ICAII and DOX may form molecular association through their hydroxyl and amino groups.

Fig. 3 (A) FTIR spectra of ICAII, DOX and ICAII-DOX NFs. (B) ¹H-NMR and (C) HMBC spectra of phenolic –OH groups in ICAII-DOX NFs. (D) NOESY spectrum showing the interaction between ICAII 7-OH and the DOX sugar moiety. (E) XRPD patterns and (F) fluorescence spectra of DOX, ICAII and ICAII-DOX NFs.

The ¹H-NMR spectrum (Fig. 3(B)) showed that the phenolic –OH signals of the ICAII-DOX NFs resonated between 10.87 and 14.04 ppm.²⁹ In the HMBC spectrum (Fig. 3(C)) of ICAII-DOX NFs, the ICAII 5-OH proton (12.53 ppm) displayed clear long-range correlations with the adjacent carbons, indicating intact intramolecular ³J_CH coupling. Conversely, the absence of cross-peaks between the 7-OH proton (10.87 ppm) and neighboring carbons suggests strong hydrogen bonding, presumably with the amino or other electron-rich sites of DOX. These hydrogen bonds can accelerate proton exchange, causing peak broadening or complete loss of correlations in the HMBC spectra, and underline the critical role of the ICAII 7-OH group in intermolecular interactions that drive the self-assembly of the ICAII-DOX nanostructure. The NOESY spectrum (Fig. 3(D)) revealed clear spatial proximity between the phenolic 7-OH group of ICAII and the sugar moiety of DOX near the amino group (4′-OH at 5.46 ppm, H-1′ at 4.88 ppm, H-2′ at 4.11 ppm, and H-5′ at 3.57 ppm),⁴⁰ indicating a strong non-covalent association between the two molecules. Overall, these results indicate that the amino group of DOX forms a strong hydrogen bond with the 7-OH group of ICAII, leading to the formation of an ICAII–DOX complex. This interaction drives the co-assembly of ICAII and DOX into stable NFs, as previously speculated in Section 3.3.

In the XRPD patterns (Fig. 3(E)), compared to DOX and ICAII, the ICAII-DOX NFs exhibited fewer diffraction peaks with broader and more diffuse shapes. The results imply that after the formation of ICAII-DOX NFs, DOX and ICAII changed from a crystalline morphology to amorphous morphology. The fluorescence spectrum (Fig. 3(F)) of free DOX exhibited a strong fluorescence emission at approximately 593 nm. Upon co-assembly with ICAII, the emission intensity of ICAII-DOX NFs decreased by approximately 5%, while the peak position remained unchanged at 593 nm, indicating that the electronic environment of the anthraquinone chromophore remained largely unchanged upon self-assembly. The observed fluorescence quenching indicates the formation of a self-assembled structure, primarily driven by intermolecular interactions between DOX and ICAII. Lee et al. reported that the fluorescence spectra of HA/DOX nanoaggregates had lower fluorescence intensity than free DOX (Ex = 480 nm) due to self-quenching within the aggregates.⁴¹ Therefore, the formation of ICAII-DOX NFs is likely driven by non-covalent interactions (e.g. hydrogen bonding and electrostatic interaction) at the side chains (e.g. daunosamine moiety), rather than involving the anthraquinone chromophore, which typically leads to fluorescence quenching or a red-shift.

In summary, the combined results of the FTIR, NMR, XRPD and fluorescence analyses consistently indicate that ICAII-DOX NFs were formed primarily through intermolecular hydrogen bonding interactions between the protonated amino group of DOX and the phenolic hydroxyl group of ICAII. These interactions stabilized the complex structure, facilitating its orderly self-assembly into NFs without significantly perturbing the π-conjugated system of DOX. Furthermore, the co-assembly approach used here can, in principle, be adapted for other drug combinations. The key is finding molecules that can form stable assemblies via non-covalent interactions such as hydrogen bonding, electrostatic interaction and π–π stacking interactions.⁴² For example, drugs that can interact with DOX through non-covalent interactions, such as hydrogen bonding, π–π stacking and hydrophobic interactions, might form a self-assembled nanodrug with it. Many studies have reported the co-assembly of DOX with other therapeutic agents to form carrier-free nanodrugs.^23–25 For instance, Wang et al. co-assembled DOX with rhein into carrier-free nanoparticles via π–π stacking and hydrogen bonds.²³ Zhang et al. co-assembled DOX with atovaquone and IR820 to create carrier-free nanomedicine driven primarily by hydrophobic interactions and π–π stacking.²⁵ DOX and indomethacin can be self-assembled into nanoparticles.²⁴ However, research on the self-assembly of ICAII has not been reported to date. If DOX is replaced with another drug, it must be ensured that the substitute can interact with ICAII by non-covalent interactions to form stable nanostructures. Many natural compounds and chemotherapeutics have been successfully co-assembled into carrier-free nanodrugs, suggesting that our platform is adaptable.¹⁴ Other drugs can potentially be delivered using a similar strategy, provided they have structural features that complement ICAII or DOX (or another self-assembling drug).

3.5. Drug release

To investigate the sustained-release behavior of the NFs, the cumulative release of DOX and ICAII from ICAII-DOX NFs and ICAII-DOX/TPGS NFs was investigated at 37 °C in PBS (pH 7.4). Fig. 4(A) and (B) show that all the samples had an initial burst release within 4 h, followed by a delayed release, respectively. Both ICAII-DOX NFs and ICAII-DOX/TPGS NFs showed an apparently sustained-release effect. The ICAII-DOX NFs exhibited significantly higher cumulative release for both ICAII and DOX over a 48 h period compared to ICAII-DOX/TPGS NFs. Specifically, at 48 h, ICAII-DOX NFs released 77.22% of DOX and 79.77% of ICAII, whereas ICAII-DOX/TPGS NFs released 65.88% of DOX and 74.29% of ICAII. These results indicate that the presence of TPGS effectively decreased and prolonged the drug release rates, suggesting the stronger retention of ICAII and DOX in the ICAII-DOX/TPGS NFs compared with ICAII-DOX NFs.

Fig. 4 (A) In vitro release of ICAII and (B) DOX from ICAII-DOX NFs and ICAII-DOX/TPGS NFs at 37 °C in PBS (pH 7.4). (C) Size curves of ICAII-DOX NFs and ICAII-DOX/TPGS NFs at 25 °C and 4 °C. ** for p < 0.01, *** for p < 0.001.

3.6. Stability of ICAII-DOX NFs and ICAII-DOX/TPGS NFs

The change in the size of ICAII-DOX NFs and ICAII-DOX/TPGS NFs was investigated at 25 °C or 4 °C within 23 days. As shown in Fig. 4(C), the change in the size of ICAII-DOX NFs was significant at both 25 °C and 4 °C, demonstrating their poor stability. The size of ICAII-DOX/TPGS NFs increased by 26.92% after storage at 4 °C for 23 days. However, the size of ICAII-DOX/TPGS NFs increased by 50.24% after storage at 25 °C for only 1 day, and the size change was significant and irregular within 3–23 days. This result indicates that the stability of ICAII-DOX NFs was obviously enhanced by adding TPGS as a stabilizer. In addition, ICAII-DOX/TPGS NFs showed much better stability at 4 °C than at 25 °C.

3.7. Cellular uptake study

The cellular uptake of DOX in A549 cells was studied using flow cytometry. As shown in Fig. 5, the DOX uptake in the ICAII-DOX NF group was 1.8-fold more than that in the free DOX group. However, there was no significant difference in DOX uptake between the ICAII-DOX NF group and ICAII-DOX/TPGS NF group. The results demonstrated that ICAII-DOX NFs enhanced the cellular uptake of DOX in A549 cells, and TPGS had no effect on the uptake of DOX.

Fig. 5 Cellular uptake study of DOX in A549 cells using flow cytometry. Flow cytometry (A) histogram and (B) data. ** for p < 0.01, *** for p < 0.001.

CLSM was used to further study the uptake and intracellular release of DOX in A549 cells. Given that DOX can inhibit DNA replication and RNA synthesis, its main active location is in the nucleus.⁴³ As shown in Fig. 6, it is beneficial that the DOX in all the groups was mainly distributed in the cell nucleus, and there were no obvious differences among free DOX, ICAII-DOX mixture, ICAII-DOX NFs and ICAII-DOX/TPGS NFs. The formation of ICAII-DOX NFs and the addition of TPGS did not obviously affect the intracellular distribution of DOX in the A549 cells. Moreover, the DOX fluorescence in the ICAII-DOX NF group and ICAII-DOX/TPGS NF group was significantly stronger than that in the free DOX group (2.3-fold stronger) and ICAII-DOX mixture group (1.5-fold stronger). This result indicates that the formation of ICAII-DOX NFs and ICAII-DOX/TPGS NFs could increase the uptake of DOX in A549 cells, while TPGS had no obvious effect on the uptake of DOX, which is in accordance with the flow cytometry study. Previous research demonstrated that the cellular uptake of high aspect ratio nanoparticles (e.g., worm-like and rod-like nanoparticles) is higher compared to spherical nanoparticles, which is probably because of their enhanced adhesion to the cell membrane, resulting from their greater surface contact area.⁴⁴ Thus, the enhanced cellular uptake can be primarily attributed to the shape of ICAII-DOX NFs, which possessed a larger surface area for interaction with the cell membrane.

Fig. 6 Cellular uptake study of DOX in A549 cells using CLSM. (A) CLSM images of A549 cells incubated with DOX, ICAII-DOX mixture, ICAII-DOX NFs and ICAII-DOX/TPGS NFs for 4 h. (B) Mean fluorescence intensity of DOX in A549 cells incubated with DOX, ICAII-DOX Mixture, ICAII-DOX NFs or ICAII-DOX/TPGS NFs for 4 h. **p < 0.01 and ***p < 0.001.

3.8. In vitro cytotoxicity tests

The in vitro cytotoxicity of the ICAII-DOX mixture, ICAII-DOX NFs and ICAII-DOX/TPGS NFs on A549 cells was evaluated using the CCK-8 assay. The results showed that ICAII-DOX NFs and ICAII-DOX/TPGS NFs effectively inhibited the A549 cells, and the inhibition was dependent on the drug concentration. Moreover, the inhibition by the two NFs was stronger than ICAII-DOX mixture (Fig. 7). The IC₅₀ (calculated in terms of DOX) of the ICAII-DOX mixture, ICAII-DOX NFs and ICAII-DOX/TPGS NFs was 0.67, 0.60 and 0.44 μM in A549 cells, respectively. These results indicate that the formation of NFs could enhance the inhibition effect of ICAII and DOX, and the inhibition effect could be further enhanced by adding TPGS.

Fig. 7 In vitro cytotoxicity of ICAII-DOX mixture, ICAII-DOX NFs and ICAII-DOX NFs/TPGS NFs to A549 cells. ICAII : DOX = 5 : 1 (m/m).

3.9. In vivo anti-tumor effect

The anti-tumor effect of DOX, ICAII, ICAII-DOX mixture, and ICAII-DOX/TPGS NFs was investigated in A549 tumor-bearing nude mice in vivo. The A549 tumor-bearing nude mouse model was established by subcutaneous injection into the right back of male BALB/c nude mice. The result showed that (Fig. 8) DOX and ICAII had an inhibition effect on the A549 tumor, and the inhibition rate was 8.70% and 17.72%, respectively. The inhibition effect of ICAII and DOX was not strong given that their dose was low. Alternatively, the inhibition effect was improved by the combination of ICAII and DOX (ICAII-DOX mixture group), and its inhibition rate was 29.90%. These results indicate that the combination of ICAII and DOX was highly effective, likely due to their synergistic effect on the tumor cells, as demonstrated in Section 3.1. Among the groups, the ICAII-DOX/TPGS NF group had the strongest inhibition effect, with the inhibition rate of 56.74%, which was 3.2-fold and 6.5-fold of the DOX group and ICAII group, respectively, and 1.9-fold of the ICAII-DOX mixture group. This result demonstrated that the formation of ICAII-DOX/TPGS NFs could significantly enhance the anti-tumor activity of ICAII and DOX. The reason for this might be that the NFs could improve the tumor-targeting ability of the drugs by the EPR effect,^32,33 and their in vivo circulation time could be increased by adding TPGS. As a safe pharmaceutical excipient approved by the FDA, TPGS has a PEG long-chain, which can prolong the in vivo circulation time of drugs,³³ and thus improve their bioavailability. In addition, the formation of NFs enhanced the cellular uptake of the drugs in vitro, as demonstrated in Section 3.7. Consequently, the anti-tumor efficacy of ICAII and DOX may be improved due to their increased intracellular accumulation in vivo.

Fig. 8 In vivo anti-tumor effect study of DOX, ICAII, ICAII-DOX mixture, and ICAII-DOX/TPGS NFs in A549 tumor-bearing nude mice. (A) Tumor volume curves during treatment. (B) Tumor weight and (C) tumor photos after treatment for 23 days. (D) Body weight curve of A549 tumor-bearing nude mice during treatment. * for p < 0.05 and ** for p < 0.01.

The body weight of the A549 tumor-bearing nude mice was recorded and shown in Fig. 7(D), where it can be observed that no obvious weight loss occurred during the treatment. The commonly used doses of DOX and ICAII in previous studies were reported to be 2–6 mg kg^−145–47 and 25–30 mg kg⁻¹, respectively.⁴⁸ However, due to the severe side effects of DOX, significant weight loss was observed in mice at doses exceeding 4 mg kg⁻¹ after they were intravenously injected with DOX.^47,49 Our preliminary experiments demonstrated that a 3 mg kg⁻¹ dose of DOX effectively inhibited tumor growth with minimal adverse effects in the treated nude mice. Therefore, 3 mg kg⁻¹ was selected as the DOX dose in this study. The ICAII dose was set at 15 mg kg⁻¹ based on the in vitro cytotoxicity results (Section 3.1) from combined treatment with ICAII and DOX, which indicated that a 5 : 1 weight ratio of ICAII to DOX was optimal for minimizing the D_m value of DOX. In this study, no significant weight loss was observed in the nude mice following intravenous injection of either DOX or ICAII-DOX mixture at a DOX dose of 3 mg kg⁻¹, suggesting the low toxicity and favorable safety at this dosage.

The histological analysis of liver tissues showed slight hepatocyte swelling and cytoplasm rarefaction in the DOX-treated mice (Fig. 9). These changes are consistent with the known DOX-associated hepatotoxicity. In contrast, there was no significant hepatocyte swelling in the ICAII-, ICAII-DOX mixture- and ICAII-DOX/TPGS-treated mice. This indicates that combination treatment of ICAII and DOX, as well as ICAII-DOX/TPGS NFs showed no obvious damage or lesions in the liver. In the heart tissues, there was slight cellular disorganization in the DOX-treated mice, suggesting mild cardiotoxicity. In contrast, there was no significant cardiac damage in the ICAII-, ICAII-DOX mixture- and ICAII-DOX/TPGS NF-treated mice, with their cardiac muscle fibers maintaining the normal arrangement. This indicates that the combination treatment of ICAII and DOX, as well as ICAII-DOX/TPGS NFs alleviated the cardiotoxicity induced by DOX. DOX-induced cardiac damage involves oxidative stress, autophagy dysregulation, mitochondrial dysfunction, and apoptosis.⁵⁰ Although direct evidence is lacking, ICAII might alleviate DOX-induced cardiotoxicity through antioxidant⁵¹ and autophagy-regulating effects.⁷ It has been reported that ICAII reduces cardiac injury and oxidative stress via the AMPK/PGC-1α/SIRT3⁵¹ and Nrf2/SIRT3 pathways,⁵² and improves cardiac function by activating the PI3K/AKT pathway.⁵³ It also protects against cardiac hypertrophy and fibrosis through the AMPK/mTORC pathway.⁵⁴ Additionally, its precursor, icariin, reduces DOX-induced cardiotoxicity by inhibiting oxidative stress and enhancing protective autophagy via caveolin-1 regulation.⁵⁵ Thus, co-delivery of ICAII with DOX may reduce DOX-associated cardiotoxicity. The histological analysis of the tumor tissues demonstrated moderate tumor cell death in the DOX-treated mice. Alternatively, enhanced tumor cell necrosis was observed in the ICAII-DOX mixture group, while the ICAII-DOX/TPGS NF group showed the most extensive tumor destruction with significant necrotic areas. This indicates that ICAII-DOX/TPGS NFs exhibited superior anti-tumor efficacy compared with the free DOX and ICAII-DOX mixture. The ICAII-DOX/TPGS NFs particularly showed enhanced efficacy in the tumor tissue, likely improving the therapeutic effect of DOX, while mitigating its systemic toxicity.

Fig. 9 Representative H&E staining images of the heart, liver and tumor sections from saline-, DOX-, ICAII-, ICAII-DOX mixture- and ICAII-DOX/TPGS NF-treated mice.

4. Conclusions

In this study, we explored the possibility of using ICAII in combination with DOX for the treatment of lung cancer. The results showed that the combination of these two drugs could improve the anti-tumor effects of ICAII and DOX through synergistic interaction, while reducing the drug dose. In addition, we discovered that ICAII and DOX could self-assemble into NFs (ICAII-DOX NFs) via hydrogen bonds without any carrier. Furthermore, TPGS was used as an adjuvant to form ICAII-DOX/TPGS NFs, which enhanced the solubility and stability of the ICAII-DOX NFs. Animal experiments showed that co-administration of ICAII and DOX via carrier-free NFs could improve the anti-tumor effects in vivo, and avoid possible toxicity from carriers and problems associated with biodegradation.

In future studies, we plan to investigate the therapeutic mechanism of ICAII-DOX NFs in lung cancer treatment, as well as the potential cardioprotective effects of ICAII against DOX-induced cardiac injury. In addition, the EPR effect promoted the tumor targeting of the NFs, but the EPR effect was variable in vivo, and thus we will study the tumor targeting effect of the NFs, and also consider adding active targeting molecules in future work. Moreover, our present work evaluated acute toxicity indicators (e.g., body weight, general health, and histopathology of the liver and heart) in the treated mice. However, we did not perform specialized toxicological assays such as mutagenicity, carcinogenic and neurotoxicity studies. These aspects are important for clinical translation and will be addressed in future studies. Furthermore, we plan to explore the co-assembly of ICAII with other therapeutic agents to develop a broader range of carrier-free nanodrugs, thereby evaluating the adaptability of this self-assembled nanoplatform.

Author contributions

Yishun Yang: conceptualization, methodology, data curation, validation, investigation, and writing – original draft. Yue Ding: methodology, resources, and writing – review and editing. Tong Zhang: conceptualization, methodology, funding acquisition, supervision, and writing – review and editing.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting the findings of this study are available within the article and the ESI.†

Acknowledgements

This research was funded by the Program of Shanghai Leading Talents (grant number 2019100).

References

1. Y. Lu, Q. Luo, X. Jia, J. P. Tam, H. Yang, Y. Shen and X. Li, J. Pharm. Anal., 2023, 13, 239–254.
2. J. Ding, C. Li, G. Wang, Y. Yang and J. Li, Nutr. Cancer, 2024, 76, 885–901.
3. J. Song, L. Shu, Z. H. Zhang, X. B. Tan, E. Sun, X. Jin, Y. Chen and X. B. Jia, Chem.-Biol. Interact., 2012, 199, 9–17.
4. Y. Sun, B. Pang, Y. Wang, J. Xiao and D. Jiang, Chem. Biodiversity, 2021, 18, e2100063.
5. Y. G. Peng and L. Zhang, Pharm. Biol., 2018, 56, 43–50.
6. M. Hou, H. Li, T. He, S. Hui, W. Dai, X. Hou, J. Zhao, J. Zhao, J. Wen, W. Kan, X. Xiao, X. Zhan and Z. Bai, J. Pharm. Pharmacol., 2024, 76, 499–513.
7. S. Li, Y. Zhan, Y. Xie, Y. Wang and Y. Liu, Drug Des., Dev. Ther., 2020, 14, 4169–4178.
8. J. Song, H. Huang, Z. Xia, Y. Wei, N. Yao, L. Zhang, H. Yan, X. Jia and Z. Zhang, Integr. Cancer Ther., 2016, 15, 390–399.
9. H. Yan, Z. Zhang, X. Jia and J. Song, Int. J. Nanomed., 2016, 11, 4563–4571.
10. H. Yan, J. Song, X. Jia and Z. Zhang, Drug Delivery, 2017, 24, 30–39.
11. H.-M. Yan, J. Song, Z.-H. Zhang and X.-B. Jia, Drug Delivery, 2016, 23, 2911–2918.
12. Y. Xu, Z. Chen, W. Hao, Z. Yang, M. Farag, C. T. Vong, Y. Wang and S. Wang, J. Nanobiotechnol., 2024, 22, 538.
13. M. Zhang, Y. Zhao, B. Lv, H. Jiang, Z. Li and J. Cao, ACS Appl. Mater. Interfaces, 2024, 16, 47270–47283.
14. X. Xu, X. Xu, Y. Wang, Z. Li and C. Han, J. Explor. Res. Pharmacol., 2022, 7, 223–233.
15. H. I. Ammar, S. Saba, R. I. Ammar, L. A. Elsayed, W. B. Ghaly and S. Dhingra, Am. J. Physiol. Heart Circ. Physiol., 2011, 301, 2413–2421.
16. H. S. Shoukry, H. I. Ammar, L. A. Rashed, M. B. Zikri, A. A. Shamaa, S. G. Abou elfadl, E. A.-A. Rub, S. Saravanan and S. Dhingra, PLoS One, 2017, 12, e0181535.
17. K. Li, Y. Zhang, M. Chen, Y. Hu, W. Jiang, L. Zhou, S. Li, M. Xu, Q. Zhao and R. Wan, Int. J. Nanomed., 2018, 13, 19.
18. M. T. Sheu, H. J. Jhan, C. Y. Su, L. C. Chen, C. E. Chang, D. Z. Liu and H. O. Ho, Colloids Surf., B, 2016, 143, 260–270.
19. M. Zhou, W. Wei, X. Chen, X. Xu, X. Zhang and X. Zhang, Nanomedicine, 2019, 20, 102008.
20. W. Ma, Q. Guo, Y. Li, X. Wang, J. Wang and P. Tu, Eur. J. Pharm. Biopharm., 2017, 112, 209–223.
21. X. Lin, X. Huang, X. Tian, Z. Yuan, J. Lu, X. Nie, P. Wang, H. Lei and P. Wang, ACS Omega, 2022, 7, 43510–43521.
22. P. Pang, W. Liu, S. Ma, J. Liu, S. Wu, W. Xue, S. Zhang, J. Zhang and X. Ji, Chem. Eng. J., 2025, 506, 160171.
23. R. Wang, Y. Yang, M. Yang, D. Yuan, J. Huang, R. Chen, H. Wang, L. Hu, L. Di and J. Li, J. Nanobiotechnol., 2020, 18, 116.
24. C. Zhang, Y. Yuan, Q. Xia, J. Wang, K. Xu, Z. Gong, J. Lou, G. Li, L. Wang, L. Zhou, Z. Liu, K. Luo and X. Zhou, Adv. Sci., 2025, 12, e2415902.
25. R. Zhang, L. Guo, Q. Li, Y. Liang, Y. Liao, H. Xu, C. Liu, G. Zhou, L. Wang, S. Xu and M. Yuan, ACS Nano, 2025, 8, 4884–4897.
26. I. Matei, C. Tablet, S. Ionescu and M. Hillebrand, Rev. Roum. Chim., 2014, 59, 401–405.
27. A. Pyne, S. Kundu, P. Banerjee and N. Sarkar, Langmuir, 2018, 34, 3296–3306.
28. M. A. Farooq and N. L. Trevaskis, Pharm. Res., 2023, 40, 245–263.
29. T. Cheng, J. Yang, T. Zhang, Y.-S. Yang and Y. Ding, BioMed Res. Int., 2016, 2016, 10.
30. G. Juli and M. Oliverio, Cancers, 2019, 11, 990.
31. W. M. David and J. S. Brodbelt, J. Am. Soc. Mass Spectrom., 2003, 14, 383–392.
32. S. Zheng, Z. Jin, J. Han, S. Cho, V. D. Nguyen, S. Y. Ko, J. O. Park and S. Park, Colloids Surf., B, 2016, 143, 27–36.
33. E. Blanco, H. Shen and M. Ferrari, Nat. Biotechnol., 2015, 33, 941.
34. H. Cabral, J. Li, K. Miyata and K. Kataoka, Nat. Rev. Bioeng., 2024, 2, 214–232.
35. Z. Németh, I. Csóka, R. Semnani Jazani, B. Sipos, H. Haspel, G. Kozma, Z. Kónya and D. G. Dobó, Pharmaceutics, 2022, 14, 1798.
36. J. Schubert and M. Chanana, Curr. Med. Chem., 2019, 25, 4553–4586.
37. Y. Yao, Z. Xu, H. Ding, S. Yang, B. Chen, M. Zhou, Y. Zhu, A. Yang, X. Yan, C. Liang, X. Kou, B. Chen, W. Huang and Y. Li, J. Nanobiotechnol., 2025, 23, 108.
38. S. S. Katiyar, R. Patil, R. Ghadi, K. Kuche, V. Kushwah, C. P. Dora and S. Jain, AAPS PharmSciTech, 2022, 23, 238.
39. C. Huang, F. Chen, L. Zhang, Y. Yang, X. Yang and W. Pan, Int. J. Nanomed., 2020, 15, 2987–2998.
40. R. R. Reddy, J. Subramanian and B. V. N. Phani Kumar, J. Phys. Chem. B, 2022, 126, 10237–10248.
41. Y. G. Lim, H. G. Park and K. Park, Biomimetics, 2025, 10, 91.
42. X. Zhang, S. Hu, L. Huang, X. Chen, X. Wang, Y. N. Fu, H. Sun, G. Li and X. Wang, Molecules, 2023, 28, 7065.
43. C. F. Thorn, C. Oshiro, S. Marsh, T. Hernandezboussard, H. Mcleod, T. E. Klein and R. B. Altman, Pharmacogenet. Genomics, 2010, 21, 440.
44. B. Karagoz, L. Esser, H. T. Duong, J. S. Basuki, C. Boyer and T. P. Davis, Polym. Chem., 2014, 5, 350–355.
45. Y. Choi, H. Han, S. Jeon, H. Y. Yoon, H. Kim, I. C. Kwon and K. Kim, Pharmaceutics, 2020, 12, 974.
46. Y. Zou, J. Wei, Y. Xia, F. Meng, J. Yuan and Z. Zhong, Signal Transduction Targeted Ther., 2018, 3, 32.
47. I. Y. Abdelgawad, M. K. O. Grant, F. E. Popescu, D. A. Largaespada and B. N. Zordoky, Int. J. Mol. Sci., 2021, 22, 9023.
48. F. Xu, Q. Wu, L. Li, J. Gong, R. Huo and W. Cui, Front. Pharmacol., 2021, 12, 663776.
49. X. Xu, L. Lian, Z. Zhou, S. Wei and H. Yuan, Int. J. Pharm., 2016, 507, 50–60.
50. P. S. Rawat, A. Jaiswal, A. Khurana, J. S. Bhatti and U. Navik, Biomed. Pharmacother., 2021, 139, 111708.
51. Y. Li, L. Feng, D. Xie, M. Lin, Y. Li, N. Chen, D. Yang, J. Gao, Y. Zhu and Q. Gong, Antioxidants, 2022, 11, 1465.
52. Y. Li, L. Feng, D. Xie, Y. Luo, M. Lin, J. Gao, Y. Zhang, Z. He, Y. Z. Zhu and Q. Gong, Eur. J. Pharmacol., 2023, 956, 175987.
53. B. Guan, X. Dai, Q. Huang, D. Zhao, J. Shi, C. Chen, Y. Zhu and F. Ai, Mol. Med. Rep., 2020, 22, 3151–3160.
54. X. Liu, H. Liao, H. Feng, N. Zhang, J. Yang, W. Li, S. Chen, W. Deng and Q. Tang, J. Pharmacol. Sci., 2018, 138, 38–45.
55. M. Scicchitano, C. Carresi, S. Nucera, S. Ruga, J. Maiuolo, R. Macrì, F. Scarano, F. Bosco, R. Mollace, A. Cardamone, A. R. Coppoletta, L. Guarnieri, M. C. Zito, I. Bava, L. Cariati, M. Greco, D. P. Foti, E. Palma, M. Gliozzi, V. Musolino and V. Mollace, Nutrients, 2021, 13, 4070.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5tb00245a

---