Using UPLC–MS/MS and LC–MS/MS to Investigate the Pharmacokinetics in SD Rat Plasma and Tissue Distribution in KM Mice of Daphnoretin Nanomicelles

ABSTRACT

Daphnoretin (DAP) has various pharmacological activities, but its in vivo disposition after nanomicellar formulation remains unclear. This study compared the pharmacokinetics and tissue distribution of free DAP and two polymeric nanomicellar formulations, PP‐DAP and GA‐DAP, following intravenous administration. Plasma DAP concentrations in rats were determined by UPLC–MS/MS, and DAP concentrations in mouse tissues were determined by LC–MS/MS. Compared with free DAP, PP‐DAP and GA‐DAP showed higher systemic exposure, longer apparent elimination half‐lives, and lower apparent clearance. The AUC_0–∞ values of DAP, PP‐DAP, and GA‐DAP were 5474.14, 11,211.04, and 15,019.86 h · ng/mL, respectively, and the corresponding T _1/2 values were 6.84, 9.24, and 9.90 h. DAP was detected in the heart, liver, spleen, lung, and kidney, with the nanomicellar formulations showing altered tissue distribution and GA‐DAP exhibiting relatively sustained hepatic distribution. These findings suggest that nanomicellar formulation alters the in vivo disposition of DAP. However, free and micelle‐associated DAP were not separately quantified, precluding direct characterization of in vivo drug release, and the validation range of the tissue quantification method was limited. Further studies are warranted to evaluate the in vivo behavior and liver‐directed delivery potential of GA‐DAP.

1. Introduction

Hepatocellular carcinoma (HCC) is the predominant form of primary liver cancer and remains a major cause of cancer‐related mortality worldwide (Anwanwan et al. 2020; Llovet et al. 2021; Sung et al. 2021). The development and progression of HCC involve complex biological processes, among which tumor cell proliferation, migration, and metastasis are closely associated with disease progression and remain important concerns in HCC research and treatment (Jiang et al. 2021; Zuo et al. 2024). In recent years, naturally derived bioactive compounds have attracted considerable interest because of their structural diversity and broad biological activities, and several natural compounds have shown potential in experimental studies of HCC (Aghababaei and M. Hadidi 2023; Jiang et al. 2021).

Wikstroemia indica (L.) C.A. Mey is a traditional medicinal plant. Plants of the genus Wikstroemia contain a variety of natural constituents, including coumarins, flavonoids, and lignans, and exhibit diverse pharmacological activities (Huan et al. 2024). Daphnoretin (DAP) is a naturally occurring compound that has been isolated from W. indica (Shi et al. 2022). However, information on the pharmacokinetics and tissue distribution of DAP in different formulations remains limited. Comparison of DAP with its nanocarrier‐based formulations may therefore provide further insight into how formulation affects the in vivo disposition of DAP.

Polymeric micelles are nanoscale carriers formed by the self‐assembly of amphiphilic block copolymers, with a hydrophobic core capable of incorporating and delivering poorly water‐soluble drugs. Amphiphilic block copolymers such as PEG‐b‐PLA have been used to construct polymeric micelles for the delivery of poorly soluble drugs (Cho et al. 2016). Physicochemical properties of nanocarriers, including particle size, morphology, and surface characteristics, can affect their circulation, metabolic behavior, and biodistribution and may consequently influence the in vivo disposition of the incorporated drug (Cai et al. 2023; Di et al. 2021). In addition to these intrinsic properties, surface modification with functional ligands represents another approach to modulating the in vivo behavior of nanocarrier systems. Glycyrrhetinic acid (GA) has been used as a functional ligand in nanocarrier systems. A previous study using liposomes modified with both GA and a cell‐penetrating peptide reported altered pharmacokinetic behavior and greater drug distribution in the liver than that observed for the free drug (L. Li, Chen, et al. 2022). More recently, GA‐mediated self‐assembled micelles have been investigated with respect to their pharmacokinetic properties and liver‐directed delivery (Guan et al. 2024). In our previous study, PEG–PLA‐DAP nanomicelles (PP‐DAP) and GA‐modified GA‐PEG–PLA/PEG–PLA‐DAP nanomicelles (GA‐DAP) were developed and characterized (Zhu et al. 2022). Further investigation is therefore needed to characterize the pharmacokinetic and tissue distribution profiles of DAP in these different formulations, particularly the hepatic distribution of DAP following GA functionalization.

Plasma pharmacokinetic and tissue distribution studies provide complementary information on systemic exposure and tissue‐level disposition and can therefore be used together to evaluate the in vivo disposition of drugs administered in different formulations (Li et al. 2023). In the present study, UPLC–MS/MS and LC–MS/MS methods were used to investigate the plasma pharmacokinetics and major tissue distribution of DAP, PP‐DAP, and GA‐DAP following intravenous administration. The study aimed to compare the in vivo disposition of DAP among the three formulations and to provide experimental evidence for further investigation of DAP‐loaded nanomicelles and the potential of GA‐DAP for liver‐directed delivery.

2. Materials and Methods

2.1. Instruments

An ultra‐low‐temperature refrigerator (−80°C) with model DW‐86L626 was purchased from Qingdao Haier Special Electrical Appliance Co. Ltd. (Qingdao, China). A refrigerated centrifuge (model: Sorvall ST 8R) was obtained from Thermo Fisher Scientific Inc. (Massachusetts, United States). UPLC–MS/MS analysis was performed using an I‐Class ultra‐high performance liquid chromatograph coupled with a Xevo TQ‐S triple quadrupole mass spectrometer (Waters Corporation, Massachusetts, United States). LC‐MS/MS analysis was performed using a liquid chromatography system coupled with a Triple Quad 4500 triple quadrupole mass spectrometer (SCIEX, United States). An ultrasonic cleaner (model: BDA‐1002) was supplied by Shenzhen Boda Ultrasonic Equipment Co. Ltd. (Shenzhen, China). A thermostatic water bath (model: DK‐S22) was purchased from Shanghai Jinghong Electrical Appliance Co. Ltd. (Shanghai, China). An electronic balance (model: AUW120D) for accurate weighing was obtained from Shimadzu Corporation (Kyoto, Japan).

2.2. Drugs and Reagents

Formic acid (mass spectrometry grade, catalog number: 2096207) and acetonitrile (mass spectrometry grade, catalog number: 2192468) were purchased from Merck KGaA (Darmstadt, Germany). Methanol (high‐performance liquid chromatography grade, catalog number: 21020190) was supplied by TEDIA Company Inc. (Ohio, USA). β‐Glucosidase (catalog number: L9C0V80) was obtained from Solarbio Life Sciences (Beijing, China). Anhydrous sodium carbonate (catalog number: 180910), glacial acetic acid (catalog number: 190706), and sodium bicarbonate (catalog number: 180612) were purchased from Xilong Scientific Co. Ltd. (Shantou, China). Puerarin (purity ≥ 98%, catalog number: A410K021) was supplied by Solarbio Life Sciences (Beijing, China). Psoralen (purity ≥ 98%, catalog number: JB308390) and DAP (purity ≥ 98%, catalog number: M10HB177709) were purchased from Shanghai Yuanye Bio‐Technology Co. Ltd. (Shanghai, China).

2.3. Laboratory Animals

Experimental animals: SPF grade male Sprague–Dawley (SD) rats and SPF grade male Kunming (KM) mice were purchased from the Animal Institute of Guizhou University of Traditional Chinese Medicine with the production license batch number. Eight‐week‐old male SD rats (200 ± 20 g) and KM mice (20 ± 2 g) were used. All animals were adaptively fed for 7 days, housed in a 12‐h light/dark cycle, at an indoor temperature of 25.0°C ± 2°C and humidity of 45.0%–60.0%, with free access to water and food. The animal experiments have been approved by the ethics committee of Guizhou University of Traditional Chinese Medicine, with the numbers 20251013002 and 20251013003, respectively.

2.4. Experimental Methods

2.4.1. Preparation of Drugs

• 1Preparation of DAP standard and internal standard solutions: The DAP standard was accurately weighed, dissolved in methanol according to the proportion, and sonicated to prepare DAP solutions at concentrations of 3856, 1928, 964, 482, 241, 120.5, 60.25, 30.13, 15.06, and 7.53 ng/mL.

Given the differences in sample matrices for pharmacokinetic and tissue distribution experiments, this study selected different internal standard substances for the two types of experiments respectively and optimized their concentrations to ensure the accuracy of quantitative analysis results. Specifically, in pharmacokinetics, puerarin is used as the internal standard. It was dissolved in methanol to prepare an internal standard solution with a final concentration of 280.40 ng/mL. Psoralen was selected as the internal standard for tissue distribution, and the internal standard solution with a final concentration of 259.00 ng/mL was prepared using methanol as the solvent.

• 2Preparation of DAP and nanomicelle formulations: DAP, PP‐DAP, and GA‐DAP lyophilized powders were accurately weighed and dissolved to prepare DAP solutions of specified concentrations for subsequent use. As demonstrated by the preliminary experiments of our research group, the characterization results of transmission electron microscopy (TEM) showed that both PP‐DAP and GA‐DAP nanomicelles presented quasi‐spherical structures, and the particle sizes were both less than 100 nm. In addition, the encapsulation efficiency of PP‐DAP is 76.04%, the drug loading is 11.13%, the particle size is 132 nm, the Zeta potential is approximately −12 mV, and the polydispersion index (PDI) is 0.196. In contrast, the encapsulation efficiency of GA‐DAP is 72.27%, the drug loading capacity is 8.89%, the particle size is 151.9 nm, the Zeta potential is approximately −14 mV, and the PDI is 0.173 (Zhu et al. 2022).

2.4.2. Sample Collection

Following a 7‐day adaptive feeding period, 18 SD rats were randomized into three groups (DAP, PP‐DAP, and GA‐DAP groups), whereas 48 KM mice were correspondingly randomized into three groups. DAP, PP‐DAP, and GA‐DAP solutions were delivered to the respective groups through tail vein injection in sequence. A total of 0.5 mL of blood was collected from the orbital venous plexus of SD rats, placed in centrifuge tubes coated with 1% heparin, centrifuged at 1006 ×g for 15 min, and the supernatant was aspirated for subsequent use. A total of 0.5 mL of blood was extracted from the orbit of KM mice at intervals of 0.5, 1.0, 6.0, and 12.0 h, then placed in centrifuge tubes lined with 1% heparin and spun at 1006 ×g for 15 min, and the supernatant was removed for future use. Following blood collection, the KM mice were euthanized, and about 0.2 g of the heart, liver, spleen, lung, and kidney were processed in centrifuge tubes, to which 600 μL of normal saline was added for homogenization.

2.4.3. Sample Preparation

2.4.3.1. Preparation of Plasma Samples

Blood samples (0.5 mL) were collected from SD rats via the orbital venous plexus into heparinized centrifuge tubes and centrifuged at 1006 ×g for 15 min to obtain plasma. An aliquot of plasma (100 μL) was mixed with 30 μL of β‐glucosidase solution (20,000 U/mL, pH 5.0), vortexed for 1 min, and incubated in a water bath at 37°C for 2 h. The enzymatic reaction was terminated in an ice‐water bath. Sodium bicarbonate buffer (30 μL, pH 9.0) was then added, and the sample was vortexed for 1 min. Subsequently, 50 μL of the internal standard solution (puerarin, 280.40 ng/mL) and 400 μL of methanol were added. The mixture was vortexed thoroughly and centrifuged at 16,099 ×g for 10 min. The supernatant was transferred to a clean centrifuge tube and evaporated to dryness under a stream of nitrogen. The residue was reconstituted in 200 μL of methanol, vortexed for 1 min, sonicated for 10 min, and centrifuged again at 12,000 rpm for 10 min. The resulting supernatant was subjected to UPLC–MS/MS analysis.

2.4.3.2. Preparation of Tissue Samples

Approximately 0.2 g of each tissue (heart, liver, spleen, lung, and kidney) was placed in a centrifuge tube and homogenized with 600 μL of normal saline. An aliquot of tissue homogenate (100 μL) was mixed with 30 μL of β‐glucosidase solution (20,000 U/mL, pH 5.0), vortexed for 1 min, and incubated in a water bath at 37°C for 2 h. The enzymatic reaction was terminated in an ice‐water bath. Sodium bicarbonate buffer (60 μL, pH 9.0) was then added, and the sample was vortexed for 1 min. Subsequently, 50 μL of the internal standard solution (psoralen, 280.40 ng/mL) and 600 μL of methanol were added. The mixture was vortexed thoroughly and centrifuged at 16,099 ×g for 10 min. The supernatant was transferred to a centrifuge tube and evaporated to dryness under a stream of nitrogen. The residue was reconstituted in 200 μL of methanol, vortexed for 1 min, sonicated for 10 min, and centrifuged again at 16,099 ×g for 10 min. The resulting supernatant was subjected to LC–MS/MS analysis.

2.5. Analytical Conditions

2.5.1. Analysis Conditions for Pharmacokinetic Study

1. Chromatographic conditions: Column: Waters BEH C18 (2.1 × 100 mm, 1.7 μm); guard column: Waters Van Guard BEH C18 (2.1 × 5 mm, 1.7 μm); column temperature: 40°C; autosampler temperature: 25°C; the mobile phase was composed of (A) 0.2% formic acid in water and (B) 0.2% formic acid in acetonitrile. A gradient elution program was set as follows: 0.0–1.0 min, 90%–28% A; 1.0–2.0 min, 28% A; 2.0–2.5 min, 28%–20% A; 2.5–3.0 min, 20% A; 3.0–3.2 min, 20%–10% A; 3.2–4.5 min, 10%–90% A; 4.5–5.0 min, 90% A. Injection volume: 2 μL.

2. Mass spectrometry setup: A positive ion mode electrospray ionization (ESI) source was employed. The capillary ionization voltage was set at 1.5 kV, and the ion source temperature was maintained at 150°C. The nebulizer and auxiliary gas used is N₂, with a desolvation gas flow rate of 1000 L/h and a temperature of 550°C. The multiple reaction monitoring (MRM) scan mode was used, with quantitative analysis parameters for DAP being m/z: 353.1 → m/z 179.1, a cone voltage of 15 V, and a collision voltage of 20 V. For the quantitative analysis of puerarin, the parameters were set as m/z: 417.0 → m/z 267.0, with a cone voltage of 50 V and a collision voltage of 30 V. Mass spectrometry data were acquired and processed using the MassLynx V4.1 workstation.

2.5.2. Tissue Distribution Analysis Conditions

Chromatographic conditions: Separation was achieved on a Waters HSS T3 column (2.1 × 150 mm, 1.8 μm) equipped with a Waters BEH C18 guard column (2.1 × 5 mm, 1.7 μm). The column temperature was maintained at 40°C. The mobile phases consisted of 0.2% formic acid in water (A) and 0.2% formic acid in acetonitrile (B). The gradient program was as follows: 0–0.1 min, 70% B; 0.1–4.9 min, 70%–65% B. The run was terminated at 5.0 min. The flow rate was 0.3 mL/min, and the injection volume was 3 μL.

Mass spectrometric conditions: Mass spectrometric detection was performed using an AB SCIEX Triple Quad 4500 triple‐quadrupole mass spectrometer equipped with a Turbo Spray ESI source and operated in positive‐ion mode. The ion spray voltage was set at 5500 V, and the source temperature was set at 500°C. The curtain gas (CUR), ion source gas 1 (GS1), and ion source gas 2 (GS2) were set at 35, 45, and 45, respectively, and the collision gas (CAD) was set at 9. Quantification was performed in MRM mode using the transitions m/z 353.1 → 179.0 for DAP and m/z 187.0 → 131.1 for psoralen. The declustering potential (DP) and collision energy (CE) were 120 and 37 V for DAP and 81 and 35 V for psoralen, respectively. Data acquisition and processing were performed using the SCIEX software.

2.6. Bioanalytical Method Evaluation

2.6.1. Method Evaluation for Plasma Pharmacokinetic Analysis

The plasma method used for the pharmacokinetic analysis of DAP was evaluated in terms of selectivity, calibration curve, precision, accuracy, extraction recovery, matrix effect, and stability.

• 1Selectivity

Blank rat plasma, blank plasma spiked with DAP and the internal standard puerarin, and plasma samples collected after administration were analyzed to assess potential interference from endogenous matrix components with the determination of DAP and the internal standard.

• 2Calibration Curve

Plasma calibration samples were prepared by spiking blank rat plasma with a series of DAP standard working solutions and the corresponding internal standard solution. Calibration curves were constructed by plotting the peak‐area ratio of DAP to the internal standard (Y) against the nominal DAP concentration (X).

• 3Precision and Accuracy

Precision and accuracy were evaluated using plasma quality control (QC) samples at low, medium, and high concentration levels. Precision was expressed as the relative standard deviation (RSD) of replicate measurements, and accuracy was expressed as the relative error (RE) between the measured and nominal concentrations.

$$\mathit{RSD}\left(\%\right)=\left(S/\overline{x}\right)\times 100$$ (1)

$$\mathit{RE}\left(\%\right)=\left(x_m-x_t\right)/x_t\times 100$$ (2)

where x _m denotes the measured concentration and x _t denotes the theoretical concentration.

• 4Extraction Recovery and Matrix Effect

The extraction recovery and matrix effect of DAP were evaluated at low, medium, and high QC concentration levels.

For extraction recovery, 100 μL of blank rat plasma was spiked with DAP QC samples at the corresponding concentration levels and the internal standard solution before sample preparation. The samples were then processed according to the plasma sample preparation procedure, and the resulting DAP peak area was recorded as A. In parallel, blank rat plasma was first processed using the same sample preparation procedure and then spiked with DAP QC samples at the corresponding concentration levels and the internal standard solution. The resulting DAP peak area was recorded as B.

For evaluation of the matrix effect, the DAP peak area obtained from post‐extraction spiked matrix samples was recorded as B, whereas that obtained from non‐matrix standard solutions at the same nominal concentrations was recorded as C.

$$\mathit{\text{Extraction recovery}}\text{rate}\left(\%\right)=A/B\times 100$$ (3)

$$\mathit{\text{Matrix effect}}\left(\%\right)=B/C\times 100$$ (4)

• 5Stability

The stability of DAP in plasma was evaluated using QC samples at low, medium, and high concentration levels under the following conditions: storage at room temperature (25°C) for 4 h, storage at −20°C for 3 days, and three freeze–thaw cycles at −20°C.

2.6.2. Method Evaluation for Tissue Distribution Analysis

The method for determining DAP in tissue samples was evaluated using blank homogenates of the heart, liver, spleen, lung, and kidney in terms of selectivity, calibration curve, precision, accuracy, extraction recovery, matrix effect, and stability. Because different calibration ranges were used for the individual tissue matrices, while the method evaluation was performed at the same three predefined concentrations of 15.06, 482.00, and 1928.00 ng/mL, some of these concentrations fell outside the corresponding tissue‐specific calibration ranges. Therefore, these concentrations were treated as predefined method‐evaluation levels and were not uniformly designated as low, medium, and high‐concentration QC levels across all tissue matrices.

• 1Selectivity

Blank tissue homogenates, blank tissue homogenates spiked with DAP and the internal standard psoralen, and tissue samples collected after administration were analyzed to assess potential interference from endogenous matrix components with the determination of DAP and the internal standard.

• 2Calibration Curve

Calibration samples were prepared separately in blank heart, liver, spleen, lung, and kidney homogenates by spiking each matrix with a series of DAP standard working solutions and the corresponding internal standard solution. Calibration curves were constructed for each tissue matrix by plotting the peak‐area ratio of DAP to the internal standard against the nominal DAP concentration.

• 3Precision and Accuracy

Precision and accuracy were evaluated in each tissue matrix at the predefined concentrations of 15.06, 482.00, and 1928.00 ng/mL. Precision was expressed as the RSD of replicate measurements, and accuracy was expressed as the RE between the measured and nominal concentrations.

• 4Extraction Recovery and Matrix Effect

Extraction recovery and matrix effect of DAP were evaluated separately in blank heart, liver, spleen, lung, and kidney homogenates at three predefined concentrations of 15.06, 482.00, and 1928.00 ng/mL. Blank tissue homogenates were prepared by homogenizing approximately 0.2 g of each tissue with 600 μL of normal saline. A 100‐μL aliquot of each blank tissue homogenate was used for the extraction recovery and matrix effect experiments.

For extraction recovery, blank tissue homogenates were spiked with DAP standard solution and internal standard solution before extraction and then processed according to the established sample preparation procedure. The resulting DAP peak area was designated as A. For the post‐extraction samples, blank tissue homogenates were first processed using the same extraction procedure and then spiked with DAP standard solution at the same nominal concentration and an equal amount of internal standard solution. The resulting DAP peak area was designated as B. DAP standard solutions at the same nominal concentrations together with the internal standard solution were also analyzed directly without tissue matrix, and the resulting DAP peak area was designated as C. Extraction recovery and matrix effect were calculated as follows:

$$\mathit{\text{Extraction recovery rate}}\left(\%\right)=A/B\times 100$$ (5)

$$\mathit{\text{Matrix effect}}\left(\%\right)=B/C\times 100$$ (6)

• 5Stability

Stability was evaluated in the corresponding tissue matrices at the predefined concentrations of 15.06, 482.00, and 1928.00 ng/mL under the following conditions: storage at room temperature (25°C) for 4 h, storage at −20°C for 3 days, and three freeze–thaw cycles at −20°C.

2.7. Statistical Analysis

Statistical analyses were performed using SPSS version 27.0. Data are presented as the mean ± standard deviation (SD). Differences among groups were evaluated by one‐way analysis of variance (ANOVA). When the overall ANOVA indicated a significant difference, pairwise comparisons were performed using the least significant difference (LSD) post hoc test. A two‐sided p value < 0.05 was considered statistically significant.

3. Results

3.1. Pharmacokinetic Study of DAP Nanomicelles

3.1.1. Specificity Assessment

Blank plasma from rats, blank plasma spiked with DAP and puerarin, and plasma samples collected 2 h after administration were determined using the mass spectrometry conditions outlined in Section 2.5.1. The findings are illustrated in Figure 1. The results show that DAP was distinctly separated from the internal standard, with no notable chromatographic peaks appearing at the analyte's retention time in any samples, indicating the method's high specificity.

FIGURE 1.

Specificity for the pharmacokinetic study of daphnoretin nanomicelles in plasma. (A) Blank rat plasma. (B) Daphnoretin standard with internal standard. (C) Plasma collected 2 h after administration.

3.1.2. Establishment of the Standard Curve and Lower Limit of Quantification

Blank plasma from rats was spiked with a series of standard solutions at different concentrations along with the internal standard solution and analyzed using the chromatographic conditions outlined in Section 2.5.1. A linear regression equation was created with the ratio of the analyte peak area to the internal standard on the Y‐axis and the standard concentration on the X‐axis. The findings are presented in Table 1.

TABLE 1.

Regression equations and linear ranges of daphnoretin nanomicelles in plasma.

Sample	Linear equation	Correlation coefficient (R 2)	Linear range (ng/mL)	Limit of quantification (ng/mL)
Plasma	Y = 0.0193X + 0.5609	0.9987	7.53–3856.00	7.53

As shown in Table 1, DAP has a correlation coefficient of 0.9987. This coefficient applies to the concentration range of 7.53 to 3856.00 ng/mL. It indicates a strong linear relationship within this interval. The lower limit of quantification is 7.53 ng/mL.

3.1.3. Precision and Accuracy

Standard solutions at concentrations of 15.06, 482, and 1928 ng/mL were each injected six times within a single day under the specified chromatographic conditions, and the procedure was repeated over three consecutive days. The precision and accuracy within a single day and between different days were evaluated by determining the RSD and RE.

The results are presented in Table 2. As shown in Table 2, the validation results demonstrated that the intra‐day and inter‐day precision and accuracy for DAP were acceptable, with all values within the ± 15% criterion, indicating good instrument precision and method accuracy.

TABLE 2.

Intra‐day and inter‐day precision and accuracy of daphnoretin nanomicelles in plasma.

Theoretical concentration (ng/mL)	Measured concentration (ng/mL)	Intra‐day		Measured concentration (ng/mL)	Inter‐day
		Precision (RSD%)	Accuracy (RE%)		Precision (RSD%)	Accuracy (RE%)
15.06	17.00 ± 1.94	11.44	12.88	16.59 ± 2.13	12.84	9.20
482.00	447.26 ± 11.25	2.51	−7.21	452.28 ± 5.97	1.32	−6.57
1928.00	1882.80 ± 31.35	1.67	−2.34	1837.26 ± 78.68	4.28	−4.71

3.1.4. Extraction Recovery and Matrix Effect

A 100‐μL portion of blank rat plasma was mixed with QC samples at low, medium, and high concentrations, along with an internal standard solution, and then processed following the plasma sample preparation method. The resulting peak area was recorded as A. Another 100 μL of blank rat plasma was first processed using the same preparation method, followed by the addition of low, medium, and high concentration QC samples and internal standard solution; the resulting peak area was recorded as B. Additionally, low, medium, and high concentration QC samples and internal standard solution were directly injected, and the resulting peak area was recorded as C. The results can be seen in Table 3.

TABLE 3.

Extraction recovery and matrix effects of daphnoretin nanomicelles in plasma.

Theoretical concentration (ng/mL)	Extraction recovery rate (%)	RSD (%)	Matrix effect (%)	RSD (%)
15.06	86.25 ± 11.98	13.89	100.10 ± 14.37	14.35
482.00	106.23 ± 2.30	2.17	87.34 ± 1.89	2.16
1928.00	104.25 ± 1.99	1.91	87.16 ± 1.67	1.91

As shown in Table 3, the extraction recovery and matrix effect of DAP were evaluated at its low, medium, and high concentration levels, all within a deviation range of ± 15%, indicating that under these conditions, the compound demonstrates good extraction recovery and no significant matrix effect.

3.1.5. Stability Evaluation

To evaluate the analyte's stability, QC samples at low, medium, and high levels were made using blank rat plasma and stored under these conditions: 25°C for 4 h and −20°C for 3 days. As shown in Table 4, the RSD and RE values of DAP at all three concentrations remained within ± 15%, indicating that the analyte was relatively stable under the tested conditions.

TABLE 4.

Stability assessment of daphnoretin nanomicelles in plasma.

Examination conditions	Theoretical concentration (ng/mL)	Measured concentration mean value (ng/mL)	RSD (%)	RE (%)
25°C at room temperature for 4 h	15.06	15.45 ± 2.01	12.98	2.57
	482.00	418.73 ± 16.12	3.85	−13.13
	1928.00	1780.75 ± 113.82	6.39	−7.64
Storage −20°C 3 d	15.06	16.71 ± 2.42	14.50	10.96
	482.00	427.03 ± 22.79	5.34	−11.40
	1928.00	1755.83 ± 80.82	4.60	−8.93
Three freeze–thaw cycles at −20°C	15.06	15.72 ± 0.99	6.32	4.38
	482.00	434.94 ± 4.66	1.07	−9.76
	1928.00	1829.57 ± 64.40	3.52	−5.11

3.1.6. Pharmacokinetics Study

Non‐compartmental analysis (NCA) using the WinNonLin 8.2 software (Phoenix, Pharsight Corporation, United States) was performed to calculate the pharmacokinetic parameters. The mean plasma concentration‐time curves of DAP nanomicelles are shown in Figure 2, and the pharmacokinetic parameters are summarized in Table 5.

FIGURE 2.

Concentration‐time curves of daphnoretin nanomicelles in plasma.

TABLE 5.

Pharmacokinetic parameters of daphnoretin nanomicelles in plasma ($\overline{x}$ ± s, n = 6).

Pharmacokinetic parameters	DAP	PP‐DAP	GA‐DAP
T 1/2(h)	6.84 ± 1.43	9.24 ± 0.95	9.90 ± 3.06#
T max(h)	0.083 ± 0.00	0.083 ± 0.00	0.083 ± 0.00
C max (ng/mL)	2336.37 ± 804.46	3168.24 ± 847.84	3306.04 ± 557.67#
AUC0–t(h • ng/mL)	4630.55 ± 420.21	10,126.99 ± 6224.08#	12,050.04 ± 2700.22##
AUC0–∞(h • ng/mL)	5474.14 ± 594.62	11,211.04 ± 6238.88#	15,019.86 ± 2219.12##
Vd(L/kg)	26.82 ± 5.40	14.26 ± 7.98##	10.02 ± 3.54##
CL(L/kg/h)	1.85 ± 0.20	1.13 ± 0.47##	0.71 ± 0.12##*
MRT0–t(h)	6.43 ± 0.38	6.14 ± 0.37	7.08 ± 0.48#
MRT0–∞(h)	11.47 ± 2.16	10.24 ± 1.54	13.02 ± 5.85

Note: # indicates a comparison with DAP; * indicates a comparison with PP‐DAP.

#p < 0.05.

##p < 0.01.

^* p < 0.05.

As shown in Table 5, the T _1/2 values for DAP, PP‐DAP, and GA‐DAP were 6.84, 9.24, and 9.90 h, respectively. Compared with DAP, the half‐life was extended by 1.35‐fold in the PP‐DAP group and by 1.45‐fold in the GA‐DAP group. Both formulation groups (PP‐DAP and GA‐DAP) had an extended half‐life (T _1/2). This was compared with the free drug group (DAP). The GA‐DAP group showed a statistically significant difference from the DAP group. The difference met the statistical criterion (p < 0.05). The area under the curve AUC_0−∞ (h • ng/mL) was 5474.14 for the DAP group, 11,211.04 ± 6238.875 for the PP‐DAP group, and 15,019.86 ± 2219.12 for the GA‐DAP group. Statistically significant differences emerged between the PP‐DAP and DAP groups (p < 0.05), and the GA‐DAP and DAP groups showed a meaningfully crucial difference (p < 0.01). Compared with DAP, the plasma clearance rates (CL) of the PP‐DAP and GA‐DAP groups were reduced by 1.64‐fold and 2.61‐fold, respectively, indicating a relative decrease in the total amount of drug eliminated per unit time in the formulation groups. These results indicate that nanomicellar formulations increased DAP‐related plasma exposure, reduced apparent clearance, and prolonged the apparent elimination half‐life, suggesting that micellar formulation altered the overall in vivo disposition of DAP.

3.2. Study on the Tissue Distribution of DAP Nanomicelles

3.2.1. Specificity Assessment

Blank tissue homogenate, blank tissue homogenate spiked with DAP and internal standard, and homogenate from the administration group with internal standard were determined employing the chromatographic conditions detailed in Section 2.5.2. The results are illustrated in Figures S1‐S5. It was found that endogenous substances in the tissues did not interfere with either the internal standard or the analyte, indicating good specificity.

3.2.2. Calibration Curves

Calibration samples were prepared by spiking blank heart, liver, spleen, lung, and kidney homogenates with a series of DAP standard working solutions and the internal standard. Calibration curves were constructed by plotting the peak‐area ratio of DAP to the internal standard against the nominal DAP concentration. The regression equations, coefficients of determination (R ²), and calibration ranges for the five tissue matrices are summarized in Table 6. Good linearity was observed over the respective calibration ranges, with R ² values ranging from 0.9905 to 0.9993.

TABLE 6.

Regression equations and calibration ranges of daphnoretin in different tissues.

Tissue	Regression equation	R 2	Calibration range (ng/mL)
Heart	Y = 699.42X − 9222.4	0.9912	22.50–548.50
Liver	Y = 1165.7X − 31,516	0.9929	62.50–2320.00
Spleen	Y = 1304.5X − 45,054	0.9929	42.00–570.00
Lung	Y = 2104.3X − 64,318	0.9905	39.00–600.00
Kidney	Y = 978.84X − 7843.3	0.9993	26.25–685.00

3.2.3. Precision and Accuracy

The precision and accuracy of DAP determination in each tissue matrix were evaluated at three predefined concentrations of 15.06, 482.00, and 1928.00 ng/mL. Intra‐day precision and accuracy were assessed by replicate analyses within the same analytical run, whereas inter‐day precision and accuracy were evaluated over three consecutive days. Precision was expressed as RSD and accuracy as RE. The results are summarized in Table 7. At the concentrations tested, all RSD values were ≤ 15%, and all RE values were within ± 15%.

TABLE 7.

Intra‐day and inter‐day precision and accuracy of DAP in different tissue matrices at the tested concentrations.

Tissue	Tested concentration (ng/mL)	Measured concentration (ng/mL)	Intra‐day		Measured concentration (ng/mL)	Inter‐day
			RSD (%)	RE (%)		RSD (%)	RE (%)
Heart	15.06	15.37 ± 0.67	4.36	2.04	15.30 ± 0.52	4.46	2.59
	482	495.67 ± 32.23	7.55	2.84	436.75 ± 53.15	12.17	−9.39
	1928	2108.17 ± 235.13	11.15	9.34	2185.83 ± 99.86	4.57	13.37
Liver	15.06	15.35 ± 1.68	11.91	0.13	15.67 ± 1.90	12.1	4.07
	482	358.46 ± 17.53	12.38	−11.32	445.80 ± 50.53	11.33	−7.51
	1928	1879.50 ± 127.83	6.8	−2.52	1801.25 ± 184.07	10.22	−6.57
Spleen	15.06	16.81 ± 1.71	9.91	11.66	17.08 ± 1.50	8.79	13.41
	482	538.33 ± 51.33	8.7	11.77	550.85 ± 55.54	10.08	14.29
	1928	2041.17 ± 74.75	3.66	5.87	2044.75 ± 88.80	4.34	6.06
Lung	15.06	16.69 ± 2.03	12.17	10.79	16.65 ± 2.41	14.45	10.57
	482	490.00 ± 23.66	4.83	1.66	501.33 ± 26.96	5.38	4.01
	1928	1931.67 ± 183.02	9.47	0.19	1930.00 ± 209.28	10.84	0.1
Kidney	15.06	14.84 ± 1.32	8.9	−1.45	15.62 ± 1.69	10.83	3.73
	482	475.33 ± 51.72	10.88	−1.38	455.83 ± 54.07	11.86	−5.42
	1928	1982.00 ± 59.40	2.99	2.8	2003.88 ± 100.85	4.61	4.49

3.2.4. Extraction Recovery and Matrix Effect

The extraction recovery and matrix effect of DAP in heart, liver, spleen, lung, and kidney homogenates were evaluated at the predefined concentrations of 15.06, 482.00, and 1928.00 ng/mL, and the results are summarized in Table 8. The mean extraction recovery ranged from 85.17% to 108.99%, whereas the mean matrix effect ranged from 85.24% to 107.42% across the five tissue matrices. The corresponding RSDs were 3.56%–12.93% for extraction recovery and 5.75%–13.39% for matrix effect.

TABLE 8.

Extraction recovery and matrix effect of DAP in different tissue matrices at the tested concentrations.

Tissue	Tested concentration (ng/mL)	Extraction recovery (%)	RSD (%)	Matrix effect (%)	RSD (%)
Heart	15.06	87.00 ± 20.03	12.93	85.24 ± 9.61	11.04
	482	93.03 ± 0.67	4.24	107.42 ± 7.74	5.88
	1928	102.90 ± 16.55	12.15	97.60 ± 12.66	5.98
Liver	15.06	108.99 ± 9.47	11.27	93.13 ± 20.76	10.80
	482	99.37 ± 9.64	5.25	88.08 ± 14.60	13.39
	1928	99.32 ± 27.99	12.53	106.30 ± 12.85	6.35
Spleen	15.06	89.01 ± 11.59	9.18	104.08 ± 4.36	6.30
	482	99.27 ± 11.30	6.16	87.97 ± 2.93	7.84
	1928	101.02 ± 9.86	4.03	85.38 ± 0.60	9.70
Lung	15.06	86.43 ± 11.67	10.47	96.03 ± 12.40	5.94
	482	99.47 ± 13.85	6.77	89.94 ± 14.96	9.45
	1928	107.61 ± 18.05	9.14	86.04 ± 0.81	9.00
Kidney	15.06	104.77 ± 17.95	8.78	97.19 ± 16.50	8.74
	482	85.17 ± 8.20	12.19	101.06 ± 18.26	7.78
	1928	103.95 ± 2.38	3.56	104.35 ± 3.80	5.75

3.2.5. Stability Evaluation

The stability of DAP in heart, liver, spleen, lung, and kidney homogenates was evaluated at the predefined concentrations of 15.06, 482.00, and 1928.00 ng/mL after storage at 25°C for 4 h, storage at −20°C for 3 days, and three freeze–thaw cycles at −20°C. The results are summarized in Table 9. At the tested concentrations, all RSD values were ≤ 15%, and all RE values were within ± 15% under the evaluated conditions, indicating that DAP was stable under the specific conditions tested.

TABLE 9.

Stability of DAP in different tissue matrices at the tested concentrations.

Tissue	Examination conditions	Tested concentration (ng/mL)	Measured concentration (ng/mL)	RSD (%)	RE (%)
Heart	25°C at room temperature for 4 h	15.06	15.36 ± 1.01	6.61	1.99
		482	551.00 ± 64.13	11.64	14.32
		1928	2016.67 ± 251.06	12.45	4.60
	Storage −20°C 3 d	15.06	15.70 ± 1.44	9.19	4.25
		482	463.67 ± 62.00	13.37	−3.80
		1928	1967.33 ± 87.64	4.45	2.04
	Three freeze–thaw cycles at −20°C	15.06	14.04 ± 1.67	11.86	−6.77
		482	413.33 ± 10.70	2.59	−14.25
		1928	2033.63 ± 189.03	9.30	5.46
Liver	25°C at room temperature for 4 h	15.06	15.36 ± 1.02	6.61	1.99
		482	500.67 ± 19.01	3.80	3.87
		1928	1960.00 ± 72.11	3.68	1.6
	Storage −20°C 3 d	15.06	14.70 ± 0.53	3.60	−2.39
		482	437.00 ± 56.67	12.97	−9.34
		1928	1897.38 ± 121.33	6.4	−1.6
	Three freeze–thaw cycles at −20°C	15.06	13.70 ± 1.20	8.74	−8.99
		482	416.67 ± 11.15	2.68	−13.56
		1928	2093.34 ± 236.85	11.8	8.58
Spleen	25°C at room temperature for 4 h	15.06	15.57 ± 0.94	6.07	3.36
		482	524.33 ± 67.57	12.89	8.78
		1928	2066.06 ± 267.64	12.95	7.19
	Storage −20°C 3 d	15.06	14.73 ± 0.40	2.74	−2.17
		482	463.46 ± 62.00	13.37	−3.8
		1928	2067.13 ± 288.59	13.96	7.23
	Three freeze–thaw cycles at −20°C	15.06	13.71 ± 1.32	9.67	−8.99
		482	436.67 ± 33.08	7.58	−9.41
		1928	1977.33 ± 151.39	7.66	2.56
Lung	25°C at room temperature for 4 h	15.06	16.20 ± 1.47	9.09	7.57
		482	511.12 ± 35.54	6.95	6.02
		1928	2160.00 ± 43.59	2.02	12.03
	Storage −20°C 3 d	15.06	17.07 ± 2.50	14.66	13.32
		482	473.56 ± 46.81	9.90	−1.87
		1928	2000.71 ± 80.51	4.02	3.77
	Three freeze–thaw cycles at −20°C	15.06	15.71 ± 2.18	13.87	4.29
		482	436.67 ± 24.42	5.59	−9.41
		1928	1844.02 ± 196.95	10.68	−4.36
Kidney	25°C at room temperature for 4 h	15.06	17.24 ± 1.17	6.55	14.21
		482	497.67 ± 34.00	11.98	0.14
		1928	2107.33 ± 331.45	13.23	13.76
	Storage −20°C 3 d	15.06	15.84 ± 1.92	1.97	−2.61
		482	481.69 ± 73.11	14.53	2.28
		1928	2084.67 ± 143.25	4.73	9.46
	Three freeze–thaw cycles at −20°C	15.06	15.26 ± 2.41	6.17	−4.56
		482	452.67 ± 28.73	7.34	−9.34
		1928	1984.33 ± 110.58	13.16	−2.30

3.2.6. Tissue Distribution Study

After intravenous administration of free DAP, DAP was rapidly distributed to the examined tissues. At 0.5 h post‐dose, tissue concentrations followed the order liver > heart > spleen > lung > kidney, with the highest concentration observed in the liver. Thereafter, DAP concentrations varied among tissues and sampling time points and tended to decrease at later time points in several tissues. The results are shown in Table 10 and Figure 3A.

TABLE 10.

Distribution of concentrations of DAP, PP‐DAP, and GA‐DAP across various tissues (n = 4).

		Concentration (ng/g)
		0.5 h	1 h	6 h	12 h
Heart	DAP	64.53 ± 3.57	51.81 ± 5.70	54.65 ± 5.63	39.49 ± 6.42
	PP‐DAP	67.34 ± 14.21	65.31 ± 5.80	83.38 ± 3.20	52.41 ± 11.16
	GA‐DAP	85.28 ± 3.58	76.20 ± 3.18	76.30 ± 2.34	77.30 ± 1.60
Liver	DAP	83.73 ± 11.28	65.97 ± 10.75	72.14 ± 12.36	82.62 ± 13.13
	PP‐DAP	151.54 ± 3.40	128.12 ± 6.46	112.17 ± 8.05	80.51 ± 7.84
	GA‐DAP	167.59 ± 3.54	140.80 ± 9.58	124.11 ± 12.65	111.35 ± 3.96
Spleen	DAP	63.43 ± 7.69	52.20 ± 4.65	55.28 ± 2.26	51.68 ± 3.87
	PP‐DAP	71.43 ± 14.80	77.19 ± 12.44	71.92 ± 3.36	50.98 ± 4.78
	GA‐DAP	94.42 ± 4.92	87.75 ± 4.33	86.35 ± 11.24	79.42 ± 2.83
Lung	DAP	42.48 ± 2.22	39.91 ± 3.23	43.03 ± 2.34	40.72 ± 5.30
	PP‐DAP	83.89 ± 15.71	75.31 ± 9.40	67.78 ± 9.32	58.59 ± 7.96
	GA‐DAP	87.69 ± 12.21	82.26 ± 7.81	76.89 ± 11.52	73.59 ± 5.37
Kidney	DAP	39.19 ± 4.32	44.00 ± 5.50	37.04 ± 6.22	37.74 ± 1.72
	PP‐DAP	59.73 ± 8.56	47.66 ± 8.17	60.30 ± 9.31	51.11 ± 4.36
	GA‐DAP	82.81 ± 5.49	73.80 ± 8.58	82.01 ± 5.04	74.14 ± 4.46

FIGURE 3.

In vivo distribution of DAP (A), PP‐DAP (B), and GA‐DAP (C).

Following intravenous administration of PP‐DAP, relatively high concentrations were also detected in the examined tissues at 0.5 h, following the order liver > lung > spleen > heart > kidney. The highest concentrations in the liver and lung were observed at 0.5 h, whereas the highest concentrations in the spleen and heart occurred at 1 and 6 h, respectively. Kidney concentrations at 0.5 and 6 h were similar. The results are shown in Table 10 and Figure 3B.

For GA‐DAP, tissue concentrations at 0.5 h followed the order liver > spleen > lung > heart > kidney, with the highest concentration observed in the liver. Thereafter, GA‐DAP concentrations generally decreased, although relatively high concentrations were maintained in several tissues at later sampling time points. At 12 h, GA‐DAP concentrations remained higher than those of free DAP in all examined tissues. Notably, relatively high liver concentrations were maintained throughout the observation period. The results are shown in Table 10 and Figure 3C.

Overall, PP‐DAP and GA‐DAP showed higher tissue concentrations than free DAP in several tissues and at multiple sampling time points, indicating tissue distribution profiles different from that of free DAP. GA‐DAP also maintained relatively high concentrations in several tissues at later time points. Among the examined tissues, the liver showed a pronounced and sustained distribution of GA‐DAP throughout the observation period.

4. Discussion

DAP is an important bioactive constituent of Wikstroemia indica (L.) C.A. Mey and has been reported to exhibit antitumor activity (Wang et al. 2021). In the present study, the pharmacokinetic and tissue distribution profiles of DAP, PP‐DAP, and GA‐DAP were compared following intravenous administration. Compared with DAP, PP‐DAP and GA‐DAP showed higher plasma exposure, lower apparent clearance, and longer apparent elimination half‐lives. The AUC_0–∞ values of DAP, PP‐DAP, and GA‐DAP were 5474.14, 11,211.04, and 15,019.86 h · ng/mL, respectively; the corresponding T _1/2 values were 6.84, 9.24, and 9.90 h, and the CL values were 1.85, 1.13, and 0.71 L/kg/h, respectively. Previous studies have shown that the circulation, clearance, and tissue distribution of polymeric micelles in vivo are influenced by both the physicochemical properties of the carrier and its interactions with the biological environment (Y. Cai et al. 2022). Thus, the increased AUC, decreased CL, and prolonged T _1/2 observed in this study indicate that nanomicellar formulation altered the overall in vivo disposition of DAP.

However, interpretation of these pharmacokinetic findings should take into account the ability of the analytical method used in this study to distinguish between different forms of DAP. Following administration of PP‐DAP and GA‐DAP, plasma may contain both DAP released from the nanomicelles and DAP that remains associated with the micelles. Because these two fractions were not separated before sample extraction, the measured plasma concentrations are more appropriately regarded as DAP‐related concentrations obtained under the specified sample preparation and analytical conditions, rather than as separate measurements of released and micelle‐associated DAP. Accordingly, the current method does not selectively measure released DAP, nor was it validated to quantify total DAP as the sum of released and micelle‐associated fractions. Previous bioanalytical studies have indicated that separate determination of released and carrier‐associated drug can provide a more accurate interpretation of the pharmacokinetic behavior of different drug fractions in nanocarrier formulations (S. Li, Zeng, et al. 2022). Therefore, the higher C _max and AUC values observed for PP‐DAP and GA‐DAP support an increase in DAP‐related plasma exposure following nanomicelle administration. However, because the different forms of DAP were not quantified separately, the present data cannot be used to directly determine the actual release process or in vivo release kinetics of DAP from the nanomicelles.

The interpretation of the early pharmacokinetic phase is also limited by the sampling schedule. The observed T _max was 0.083 h (5 min after intravenous administration) in all three groups, which was also the earliest blood sampling time in this study. Therefore, the identical observed T _max only indicates that the highest measured plasma concentration in each group occurred at the first sampling point and does not demonstrate that the three formulations had the same early in vivo disposition. In particular, the higher C _max values of PP‐DAP and GA‐DAP, together with an observed T _max identical to that of DAP, should not be interpreted simply as evidence of rapid DAP release from the nanomicelles or similar release behavior among the formulations. Therefore, the present data can neither confirm nor exclude delayed release of DAP from PP‐DAP and GA‐DAP. Future studies with additional sampling points during the early phase after intravenous administration, combined with separate quantification of different forms of DAP, would help to further characterize the early in vivo behavior of the nanomicellar formulations.

Sample preparation is another important factor to consider when interpreting the present findings. Both plasma and tissue samples were treated with β‐glucosidase before extraction. β‐Glucosidase catalyzes the hydrolysis of β‐D‐glucosidic linkages and may therefore affect components in the samples that are susceptible to enzymatic hydrolysis (Ouyang et al. 2023). It should be noted that β‐glucosidase treatment and determination of plasma protein binding are separate issues. Enzymatic treatment itself cannot distinguish protein‐bound from unbound DAP; the unbound fraction is generally determined using equilibrium dialysis, ultrafiltration, or other appropriate separation methods (Courville et al. 2024; Di 2021). Previous bioanalytical studies have shown that enzymatic hydrolysis before analysis may influence subsequent LC–MS/MS quantification of a compound in its parent form. Comparison of hydrolyzed and non‐hydrolyzed samples can therefore help clarify the contribution of enzymatic treatment to the measured concentration (Matsuo et al. 2024). Because hydrolyzed and non‐hydrolyzed samples were not analyzed in parallel in the present study, and potentially hydrolysable related components were not independently identified or quantified, the specific contribution of β‐glucosidase treatment to the final measured DAP concentrations cannot currently be determined. Given this methodological limitation, the plasma and tissue concentrations reported in this study should be understood as DAP‐related concentrations obtained under the specified β‐glucosidase treatment and extraction conditions and should not be interpreted as measurements of unbound DAP or used to assess the plasma protein‐binding status of DAP.

The same analytical considerations are also relevant to the interpretation of the tissue distribution data. The tissue distribution results showed that, under the analytical conditions used in this study, DAP was detectable in the heart, liver, spleen, lung, and kidney following intravenous administration of DAP, PP‐DAP, and GA‐DAP. Tissue concentrations in the DAP group generally decreased over time, whereas PP‐DAP and GA‐DAP showed different concentration–time patterns across several tissues, suggesting that nanomicellar formulation affected not only the plasma pharmacokinetics of DAP but also its tissue distribution. However, as with the plasma analysis, analysis of tissue homogenates could not distinguish between different forms of DAP. The observed differences in tissue concentrations are therefore more appropriately interpreted as differences in DAP‐related tissue exposure and overall distribution among the formulations, rather than as direct evidence that the nanomicelles enhanced cellular uptake of DAP. The measured DAP‐related concentrations in tissues may also have been influenced by carrier distribution, the state of DAP within the tissues, and sample preparation. The present data are therefore more suitable for comparing overall tissue distribution patterns among the formulations.

Among these distribution differences, the hepatic distribution of GA‐DAP was particularly noteworthy. During the observation period, GA‐DAP maintained relatively high DAP‐related concentrations in the liver, showing a pronounced and sustained hepatic distribution pattern. Previous studies have reported that GA‐functionalized nanocarriers can improve targeted drug delivery to HCC (Wu et al. 2020), and preferential hepatic distribution has also been observed with GA‐modified nanomicelles (Zhou et al. 2020). The hepatic distribution pattern of GA‐DAP observed in the present study is consistent with the general trend reported in these studies, suggesting that GA modification may be one of the factors affecting the hepatic distribution of DAP.

Nevertheless, the higher DAP‐related concentrations observed in the liver do not directly demonstrate that GA‐DAP provides HCC‐specific active targeting. First, the tissue distribution study was conducted in healthy mice rather than in an HCC‐bearing model, making it impossible to distinguish general hepatic distribution from true tumor‐specific targeting. Second, the hepatic distribution of nanocarriers may also be influenced by their physicochemical properties, circulation behavior, and uptake and clearance mediated by the mononuclear phagocyte system. The present findings are therefore more appropriately interpreted as evidence of a pronounced hepatic distribution pattern of GA‐DAP rather than proof of HCC‐specific active targeting. Further studies using appropriate HCC models should compare drug distribution between tumor tissue and normal liver tissue and incorporate cellular‐level or imaging‐based localization studies to better characterize the tumor‐directed delivery of GA‐DAP.

Beyond these interpretative considerations, the tissue distribution study also had limitations related to bioanalytical method validation. Interpretation of the tissue distribution results should also take into account the validation range of the tissue bioanalytical method. Different calibration ranges were established for the individual tissue matrices, whereas method evaluation was performed uniformly at concentrations of 15.06, 482.00, and 1928.00 ng/mL. Some of these concentrations fell outside the corresponding calibration ranges for certain tissues. Therefore, these three concentrations cannot uniformly be regarded as fully validated low, medium, and high QC levels for all tissue matrices, and the available validation data do not fully characterize quantitative performance across the entire calibration range of each tissue. In addition, the lowest calibration standard for each tissue was not independently and fully validated as the LLOQ with respect to precision and accuracy. Quantitative performance at the lower end of the calibration curves therefore requires further confirmation. These limitations do not preclude comparison of the overall tissue distribution patterns observed under the present analytical conditions, but they warrant caution when interpreting concentrations close to the lower end of the calibration range and when making precise quantitative comparisons among tissues or formulations. Future studies should establish appropriate QC levels according to the actual calibration range of each tissue and independently validate the corresponding LLOQ to improve the reliability of tissue quantification.

Sample stability should also be considered when interpreting the present results. In this study, stability was evaluated after storage at 25°C for 4 h, at −20°C for 3 days, and after three freeze–thaw cycles at −20°C. However, some plasma and tissue samples were stored at −80°C for longer than 3 days before analysis. Therefore, the available stability data do not fully cover the actual storage duration of all study samples, and long‐term stability at the actual storage temperature has not yet been fully validated. Future studies should include long‐term stability assessments at −80°C for a duration covering at least the maximum storage period of the study samples.

Overall, polymeric nanomicellar formulation altered the in vivo disposition and tissue distribution of DAP, as reflected by increased DAP‐related plasma exposure, decreased apparent clearance, and prolonged apparent elimination half‐life. Among the formulations, GA‐DAP showed a relatively pronounced and sustained hepatic distribution pattern. Because the current analytical method did not distinguish released DAP from micelle‐associated DAP and the samples were treated with β‐glucosidase before extraction, the present findings cannot directly characterize the in vivo release kinetics of DAP from the nanomicelles, nor can the measured concentrations be regarded as concentrations of unbound DAP. In addition, the use of healthy animals and the limitations in the validation range of the tissue bioanalytical method warrant caution when interpreting HCC‐specific targeting and some of the low‐concentration tissue data. Future studies should establish methods for the separate quantification of different forms of DAP, clarify the influence of β‐glucosidase treatment on the measured concentrations, improve tissue‐specific bioanalytical method validation, and further evaluate the in vivo distribution and tumor‐directed delivery of GA‐DAP in appropriate HCC models.

5. Conclusion

The present study showed that DAP, PP‐DAP, and GA‐DAP exhibited different pharmacokinetic and tissue distribution profiles following intravenous administration. Compared with DAP, PP‐DAP and GA‐DAP showed higher plasma AUCs, lower apparent clearance, and longer apparent elimination half‐lives. GA‐DAP showed a more sustained distribution in the liver, suggesting that GA functionalization may be associated with changes in the hepatic distribution of DAP. Because free DAP and micelle‐associated DAP were not quantified separately in this study, the pharmacokinetic data obtained using the current analytical method cannot directly characterize the in vivo release kinetics of DAP from the nanomicelles. In addition, the quantitative method used for the tissue distribution study had certain limitations in its validation range, which should be taken into account when interpreting the tissue distribution data. Overall, these findings provide a basis for further understanding the in vivo disposition of DAP following nanomicellar formulation and for evaluating the potential of GA‐DAP for liver‐directed delivery.

Author Contributions

Zhi‐min Lv: conceptualization, data curation, formal analysis, writing – original draft, methodology. Ju Zhang: project research, data organization and analysis. Guo Feng: data curation, funding acquisition, project administration, resources, writing – review and editing. Xiang‐Ling Qu: visualization. Ting‐ting Liu: investigation. Xin‐ping Wei: supervision. Li Zhang: supervision. Shi‐qiong Qin: data analysis. Yun‐li Wei: data analysis. Gang Liu: visualization. Ke‐xin Ma: investigation. Jin‐xin Hou: investigation. Wei Li: resources, validation. Yi Sui: visualization.

Funding

This study was supported by the Guizhou Province Science and Technology Foundation Project (grant no. Guizhou Scientific Basis ZK[2022] General 461), the National and Provincial Science and Technology Innovation Talent Team Cultivation Project of Guizhou University of Traditional Chinese Medicine (No. Guizhong Medicine TD He Zi [2024] 001), the National Natural Sciences Foundation of China (No. 82060767, 81760766), the “Thousand” level Innovative Talents Project in Guizhou Province (grant no. Qrlf [2020]4) the Guizhou University of Traditional Chinese Medicine (“Thousand level” Talent) Research Project (No. ZQ2018001), the Guizhou Traditional Chinese Medicine Processing Technology Inheritance Base Construction Project (Project No. Qian Traditional Chinese Medicine Letter ([2024]No.22), the Central‐level major increase or decrease expenditure projects (No. 222060302), the 2024 Guizhou Provincial Enterprise ‘Science and Technology Deputy Positions’ and ‘Science and Technology Commissioners’ Project (No. Qian Ke He Ren Cai KJZY [2025] 060), and the Guizhou Provincial Health Commission Science and Technology Fund Project (No. gzwkj2024‐061).

Ethics Statement

All animals received humane care according to the Guide for the Care and Use of Laboratory Animals. The procedures for all animal experiments detailed were approved by the Institutional Animal Care and Use Committee of Guizhou University of Traditional Chinese Medicine.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

FIGURE S1: Specificity assessment in heart tissue.

FIGURE S2: Specificity assessment in liver tissue.

FIGURE S3: Specificity assessment in spleen tissue.

FIGURE S4: Specificity assessment in lung tissue.

FIGURE S5: Specificity assessment in kidney tissue.