Preparation and Antitumor Activity Evaluation of Folic Acid-Modified Phospholipid–Gambogic Acid Nanocrystals

Abstract

Background: Liver cancer is a complex malignant tumor; gambogic acid (GA) has significant anti-cancer potential, but poor water solubility and low bioavailability limit its clinical application. In this paper, by integrating nanocrystal (NC) technology and an active targeting strategy, a new nanoagent—folic acid-modified phospholipid–gambogic acid nanocrystals (GA-NCs@FA)—was developed to improve the delivery efficiency and therapeutic effect of GA in the treatment of liver cancer. Methods: GA-NCs@FA was prepared by the CO₂-assisted precipitation method and the thin-film hydration method. The in vitro anti-tumor activity of GA-NCs@FA was evaluated by cytotoxicity, as well as a scratch and uptake test. A HepG2 tumor-bearing nude mouse model was established to investigate the in vivo distribution and tumor targeting of GA. The in vivo anti-tumor activity was evaluated by the tumor inhibition rate, and the pathological changes of organs in each group were observed by H&E staining. Results: GA-NCs@FA significantly reduced HepG2 cell viability (IC₅₀: 0.50 μg·mL⁻¹) and migration ability (48 h healing rate: 11.50%) and enhanced intracellular fluorescence intensity. In vivo analysis showed that GA-NCs@FA significantly increased the accumulation of drugs in tumor tissues by active targeting and achieved a tumor growth inhibition rate of 70.9%. Histopathology confirmed that GA-NCs@FA induced the most obvious nuclear pyknosis and necrosis in tumor tissues while maintaining good biosafety. Conclusions: GA-NCs@FA significantly prolongs the systemic circulation time of the drug and enhances intratumoral accumulation; therefore, it is a method that can be considered for active targeting and treatment of liver cancer.

1. Introduction

Liver cancer is one of the most common malignant tumors worldwide. Latest epidemiological statistics indicate that the incidence of liver cancer ranks sixth globally, while its mortality rate ranks third [1]. Hepatocellular carcinoma (HCC) is the most prevalent subtype of primary liver cancer, accounting for 75–85% of all cases [2]. Due to the insidious onset of HCC and the high compensatory capacity of the liver, most patients are diagnosed at an advanced stage, thereby missing the therapeutic window for curative surgical resection or liver transplantation [3]. Consequently, chemotherapy remains the cornerstone for prolonging patient survival and controlling disease progression. However, traditional chemotherapeutic agents lack the specificity required to recognize and target tumor tissues. While eliminating cancer cells, they inevitably induce severe adverse effects, such as gastrointestinal damage, hepatotoxicity, and nephrotoxicity, which ultimately compromise therapeutic outcomes [4,5]. Therefore, developing novel strategies to enhance drug efficacy while minimizing systemic toxicity remains a critical challenge to be addressed.

Gambogic acid (GA) is the major active ingredient extracted from the dried resin of Gamboge (Garcinia hanburyi). It exhibits various significant anti-tumor pharmacological activities, such as induction of apoptosis, inhibition of proliferation, and suppression of angiogenesis [6,7]. However, GA suffers from poor water solubility (0.013 mg·mL⁻¹), a short in vivo half-life, and systemic toxicity caused by non-specific distribution, all of which limit its clinical application [8]. Although attempts have been made to improve the pharmacokinetic behavior of GA by constructing various delivery strategies, such as the use of nanoparticles, liposomes, and micelles, these systems often face challenges such as poor stability, low drug loading, drug leakage, and potential excipient toxicity, making it difficult to maintain therapeutic dosages while ensuring biosafety. In contrast, Nanocrystal (NC) technology, with pure drugs as the core, has shown significant advantages in the delivery of poorly soluble drugs. NCs are typically defined as colloidal dispersion systems consisting of active pharmaceutical ingredients and a small amount of stabilizers, with an average particle size of less than 1000 nm; this can significantly reduce the safety risks associated with excessive use of excipients. In addition, the production of NCs by “bottom-up” or “top-down” methods is simple and reliable, making them conducive to quality control [9,10,11].

Nanoscale size is ideal for intravenous administration, particularly for tumor-targeted drug delivery [12]. Traditional NCs exhibit poor physical stability due to their high free surface energy, and they are easily recognized and cleared by the mononuclear phagocyte system. More importantly, while most existing studies on NCs focus on improving solubility through size reduction, research aimed at endowing NCs with “long circulation” and “active targeting” capabilities while ensuring “biosafety” remains relatively scarce. In the present study, amphiphilic materials (such as D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) and DSPE-mPEG) were employed for surface modification. These components not only enhance the stability of NCs through steric hindrance but also form a “hydration layer” to effectively reduce non-specific protein adsorption and prolong blood circulation time [13]. Furthermore, surface modification can endow GA-NCs with active targeting functions. For instance, by conjugating the folic acid (FA) of the targeting ligand to the distal end of the polymer, receptor-mediated endocytosis can be leveraged to enhance cellular uptake, increase the intracellular drug concentration, and achieve precise drug enrichment in folate receptor (FR)-overexpressing cancer cells. FA offers several advantages, including ease of production, low cost, and excellent biocompatibility [14,15,16].

The scientific significance of this study lies in the development of a GA-NCs delivery system that integrates physical stability, prolonged circulation, and active targeting. It not only significantly improves the solubility and in vitro dissolution rate of GA but also endows the system with specific recognition capabilities through surface modification. GA-NCs were prepared using the CO₂-assisted precipitation method combined with the thin-film hydration method. Compared with traditional preparation methods, such as high-pressure homogenization and media milling, this process is greener and more efficient, effectively avoiding organic solvent residues and impurities from mechanical wear, thereby ensuring the purity and biosafety of the preparation [17]. Subsequently, a folic acid-modified phospholipid–gambogic acid nanocrystal delivery system (GA-NCs@FA) was further constructed to achieve active targeted delivery to HepG2 liver cancer cells. The physicochemical properties of GA-NCs@FA were systematically characterized, and its advantages in active targeted delivery were evaluated at the cellular level (cytotoxicity, uptake, and scratch) and in vivo (tissue distribution, tumor inhibition rate, and safety) using tumor-bearing nude mice. This study aims to provide an efficient, low-toxicity, and precise delivery strategy for the clinical translation of GA and lay an experimental foundation for the future development of liver-targeted nano-preparations.

2. Materials and Methods

2.1. Materials and Instruments

Gambogic acid (95%) was purchased from Nanjing Dausf Biotechnology Co., Ltd., Nanjing, Chian. D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), citric acid and coumarin 6 (C6) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China. Sodium bicarbonate (NaHCO₃), cholesterol and Paclitaxel (98%, PTX) were purchased from Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China. Soybean lecithin was purchased from Shanghai McLean Biochemical Technology Co., Ltd., Shanghai, China. Phosphate-buffered saline (PBS) and 4% paraformaldehyde fixative were purchased from Beijing Lanjieke Technology Co., Ltd., Beijing, China. DMEM high-glucose medium, CCK-8 cell proliferation assay kits and trypsin were purchased from Dalian Macklin Biotechnology Co., Ltd., Dalian, Chian. Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd., Zhejiang, Chian. DSPE-mPEG₂₀₀₀ and DSPE-PEG₂₀₀₀-FA were purchased from Shanghai Pengshuo Biotechnology Co., Ltd., Shanghai, China. Other chemicals (including methanol, anhydrous ethanol, chloroform, ether and dimethyl sulfoxide) were of analytical grade and used directly, without further purification. Sixty SPF female BALB/c nude mice with weights of 16–18 g were purchased from Jiangsu Jicu Yaokang Biotechnology Co., Ltd., Jiangsu, China. (production license number: SCXK (Su)-2023-0009; certificate number: B202409180338).

Agilent 1100 series high-performance liquid chromatography (HPLC) system, Santa Clara, CA, USA. FA2004N Analytical Balance, Shanghai Hengping Instrument Factory, Shanghai, China. UV-765 Ultraviolet Spectrophotometer, Shanghai Precision Scientific Instrument Co., Ltd., Shanghai, China. RE-52A rotary evaporator, Shanghai Yarong Biochemical Instrument Factory, Shanghai, China. Zetasizer Nano ZSE nanoparticle size potentiometer, Malvern Panalytical, Malvern, UK. TGL-20bR Desktop refrigerated Centrifuge, Shanghai Anting Scientific Instrument Factory, Shanghai, China. Talos F200S G2 transmission electron microscope (TEM), Thermo Fisher Instruments Co., Ltd., Eindhoven, Netherlands. SCIENTZ-950E Ultrasonic Cell Crusher, Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China. Fluorescence inverted microscope, Olympus, Tokyo, Japan. Envision Multifunctional Microplate Reader, BioTek Instruments, Winooski, VT, USA. Carbon dioxide incubator, Beijing Jiaxin Co., Ltd., Beijing, China. DL-CJ-2N cell culture Clean bench, Beijing Donglian Haer Instrument Manufacturing Co., Ltd., Beijing, China.

2.2. Preparation of GA-NCs

GA-NCs were prepared by the CO₂-assisted precipitation method and the thin-film hydration method. First, 3 mg GA, 6 mg TPGS, and 0.8 mg citric acid were dissolved in 3 mL methanol, then evaporated under reduced pressure in a water bath at 40 °C. After forming a uniform film, 5 mL of 0.2 mg·mL⁻¹ NaHCO₃ solution was added and hydrated in a rotary evaporator to obtain GA-NCs [18,19].

2.3. Quality Evaluation of GA-NCs

2.3.1. Physicochemical Characterization of GA-NCs

The morphology of GA-NCs was observed by TEM. The particle size, polydispersity index (PDI), and Zeta potential of GA-NCs were measured by a nanoparticle size potentiometer, and the average value was calculated based on three parallel measurements.

2.3.2. Encapsulation Efficiency and Drug Loading of GA-NCs

The 5 mL of GA-NCs was centrifuged in a 3000 MWCO ultrafiltration tube (6000 rpm, 5 min), and the filtrate was collected. The free GA content (C_free) was determined by HPLC, and three portions were measured in parallel. The encapsulation efficiency (EE) was calculated according to the initial dosage (W_total) [20].

The 5 mL of GA-NCs was lyophilized, and the total mass (W_nanocrystals) was recorded. A volume of 5 mL of methanol was added to dissolve the GA-NCs, and the mixture was centrifuged (13,000 rpm, 10 min). The supernatant was filtered through a 0.22 μm filter membrane and analyzed by HPLC to calculate the GA mass (W_entrapped). Drug loading (DL) was calculated in triplicate.

EE (%) = (1 − C_freeV/W_total) × 100% (1)

DL (%) = W_entrapped/W_nanocrystals × 100% (2)

2.3.3. Differential Scanning Calorimetry Analysis of GA-NCs

Appropriate amounts of GA, TPGS, physical mixture (GA + TPGS) and GA-NC lyophilized powder were placed on the DSC aluminum crucible, with nitrogen as the protective gas and the empty aluminum crucible as the reference. The scanning temperature ranged from 25 °C to 175 °C, and the heating rate was 10 °C·min⁻¹ for DSC analysis.

2.4. Preparation of GA-NCs@FA

Soybean lecithin (35.7 mg), cholesterol (10.5 mg), DSPE-mPEG₂₀₀₀ (3.2 mg) and DSPE-PEG₂₀₀₀-FA (1.4 mg) were dissolved in anhydrous ethanol and evaporated under reduced pressure at 40.8 °C to form a uniform film. The results of formula screening are shown in Tables S1–S3. The film was hydrated with a “2.2” GA-NC suspension and ultrasonically treated with a 120 W probe for 5 min. The resulting suspension was filtered through 0.45 μm and 0.22 μm microporous membranes in turn to obtain GA-NCs@FA. The preparation method of gambogic acid nanocrystals without folic acid modification (GA-NCs@0%FA) was similar, but DSPE-PEG₂₀₀₀-FA was replaced by equimolar DSPE-mPEG₂₀₀₀.

2.5. Quality Evaluation of GA-NCs@FA

2.5.1. Physicochemical Characterization of GA-NCs@FA

The method was the same as that described in Section 2.3.1, and the mean value was calculated based on three measurements conducted in parallel.

2.5.2. Encapsulation Efficiency and Drug Loading of GA-NCs@FA

A volume of 1 mL of GA-NCs@FA and a certain amount of lyophilized GA-NCs@FA were accurately measured, and each was diluted with methanol to 10 mL for demulsification and allow to stand. Then, they were subjected to 0.22 μm microporous membrane filtration, the filtrate was analyzed by HPLC, the GA content was determined, the EE and DL were calculated.

EE (%) = W_actual/W_input × 100% (3)

DL (%) = W_actual/W_total × 100% (4)

where W_actual is the actual amount of drugs in nanocrystals, W_input is the amount of drug input, and W_total is the total mass of nano-preparations.

2.5.3. X-Ray Diffraction Analysis of GA-NCs@FA

Appropriate amounts of lyophilized GA, TPGS, GA-NCs, GA-NCs@0%FA, and GA-NCs@FA samples were taken for XRD analysis. The specific measurement parameters were: a scanning range of 5°–50° (2θ), a scanning speed of 5°·min⁻¹, a tube voltage of 40 kV, and a tube current of 100 mA. The whole experiment was carried out at a temperature of 25 °C and a relative humidity of 55.2%.

2.5.4. Differential Scanning Calorimetry Analysis of GA-NCs@FA

Appropriate amounts of lyophilized GA, blank excipients (soybean lecithin, cholesterol, DSPE-mPEG₂₀₀₀, and DSPE-PEG₂₀₀₀-FA), physical mixture (blank excipients and GA), GA-NCs@0%FA and GA-NCs@FA samples were accurately weighed. The DSC analysis was carried out with nitrogen as the protective gas and the empty aluminum crucible as the reference. The scanning temperature ranged from 25 °C to 150 °C, and the heating rate was 10 °C·min⁻¹.

2.5.5. Stability Investigation

Storage Stability

GA-NCs, GA-NCs@0%FA and GA-NCs@FA were placed at 4 °C and 25 °C for 7 d. Equal portions (200 μL) were taken out at 0, 1, 3, 5 and 7 d, and the storage stability was evaluated using particle size as an indicator.

Plasma Stability

The above three nano-preparations were diluted with PBS (pH 7.4, 10% serum) and incubated at 37 °C. Then, 4 mL of each sample was mixed with 8 mL of the incubation medium. At predetermined time intervals (0, 1, 3, 5 and 7 d), aliquots of 200 μL were withdrawn to monitor changes in particle size as an indicator of stability.

2.5.6. Hemolytic Investigation

Seven EP tubes were prepared and mixed uniformly according to the ratios specified in Table 1, with different volumes of GA-NCs@FA solution added. Tube Nos. 6 and 7 served as the negative control and positive control, respectively. After thorough mixing, the samples were incubated at 37.0 ± 0.5 °C for 3 h, followed by centrifugation at 4 °C and 5000 rpm for 5 min. Hemolysis and hemagglutination were evaluated by observing the color of the supernatant and the amount of residual red blood cells at the bottom. Subsequently,100 μL of the supernatant from each sample was transferred to 96-well plates (in triplicate), and the absorbance was measured at a wavelength of 540 nm using a microplate reader to calculate the hemolysis rate [21].

Hemolysis Rate (%) = (A_sample − A_{negative control})/(A_{positive control} − A_{negative control}) × 100% (5)

Table 1.

Experimental design for the hemolysis assay of GA-NCs@FA.

Test Tube	2% RBC Suspension (mL)	GA-NCs@FA (mL)	Normal Saline (mL)	Deionized Water (mL)	Total Volume (mL)
1	2.5	0.5	2.0	0.0	5.0
2	2.5	0.4	2.1	0.0	5.0
3	2.5	0.3	2.2	0.0	5.0
4	2.5	0.2	2.3	0.0	5.0
5	2.5	0.1	2.4	0.0	5.0
6	2.5	0	2.5	0.0	5.0
7	2.5	0	0	2.5	5.0

2.5.7. In Vitro Release Study

The in vitro release behavior of GA preparation in PBS buffer (pH 7.4) was investigated by the dialysis bag method. Equal volumes (3 mL) of GA, GA-NCs, GA-NCs@0%FA and GA-NCs@FA (all at a GA concentration of 0.6 mg·mL⁻¹) were placed in dialysis bags (MWCO:14000 Da), sealed in 150 mL PBS release medium, and shaken at 37 °C and 100 rpm. At the predetermined time points (0, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72 and 96 h), 1 mL of sample was taken, and the same amount of fresh release medium was supplemented. The concentration of GA was quantified by HPLC to calculate the cumulative release percentage.

2.6. In Vitro Antitumor Activity Study

2.6.1. Biocompatibility Evaluation of Blank Carrier

Following the cultivation of HepG2 cells, those in the logarithmic growth phase were seeded into 96-well plates at a density of 1 × 10⁴ cells per well. After cell adherence, each well was treated with different final concentrations (8.75, 17.5, 35, 70 and140 μg·mL⁻¹) of the blank carrier for 24 h. Then, 100 μL of CCK-8 solution (diluted 1:10) was added into each well and incubated for 3 h. After shaking for 3 min to ensure uniformity, the absorbance at 450 nm was measured using a microplate reader, and the cell viability was subsequently calculated.

Survival Rate (%) = (OD_experimental – OD_blank)/(OD_nc – OD_blank) × 100% (6)

2.6.2. Cytotoxicity Test

HepG2 cells in the logarithmic growth phase were harvested using trypsin and seeded into 96-well plates at a density of 1 × 10⁴ cells per well. After incubation at 37 ℃, 5% CO₂, and 95% RH for 24 h to allow for cell attachment, the culture medium was replaced with 100 μL of fresh medium containing various concentrations of GA, GA-NCs, GA-NCs@0%FA and GA-NCs@FA (0.4, 0.8, 1.6, 3.2 and 6.4 μg·mL⁻¹; n = 6). After 24 h of treatment, 100 μL of 10-fold diluted CCK-8 solution was added to each well and incubated for an additional 3 h. The absorbance at 450 nm was measured using a microplate reader after shaking for 3 min, and the cell viability was subsequently calculated.

2.6.3. Cell Scratch Test

HepG2 cells in the logarithmic growth phase were seeded into 6-well plates at a density of 1 × 10⁶ cells·mL⁻¹ (2 mL per well). After the formation of a cell monolayer, scratches were prepared using a standard tip, and localization markers were performed. The old medium was aspirated and washed three times with PBS buffer to remove the residual cells. Subsequently, solutions of GA, GA-NCs, GA-NCs@0%FA, and GA-NCs@FA diluted with DMEM medium containing 2% FBS were added, while the control group was given an equal volume of drug-free medium. At 0, 24, and 48 h post treatment, three fields were randomly selected to capture images of the scratched areas using a microscope. The scratch area was measured using ImageJ software (Version 1.54t, National Institutes of Health, Bethesda, MD, USA), and the scratch healing rate was calculated according to the following formula:

Scratch healing Rate (%) = (Area_{0 h} – Area_th)/Area_{0 h} × 100% (7)

Area_{0 h}: The scratch area at 0 h; Area_th: The scratch area at a designated time point.

2.6.4. Cell Uptake Test

The cellular uptake of different drug groups by HepG2 cells was investigated. Due to the lack of fluorescence characteristics of GA, it could not be directly detected. The fat-soluble fluorescent C6 probe (C₂₀H₁₈N₂O₂S) was used as a surrogate marker. Four groups of C6 solution, C6-NCs, C6-NCs@0%FA, and C6-NCs@FA were set up. After HepG2 cells were cultured in 6-well plates for 24 h, the medium was removed and replaced with an equal C6 concentration of drug-containing medium for incubation for 4 h. The cells were washed three times with PBS, fixed with 4% paraformaldehyde for 20 min, and washed three times with PBS; then, the nuclei were stained with DAPI for 15 min and washed. Finally, cells were observed under a fluorescence microscope.

2.7. Tissue Distribution

2.7.1. Animal Model Construction and Group Administration

The tumor-bearing nude mouse model was established by subcutaneous injection of 0.2 mL HepG2 cells (2 × 10⁷ cells·mL⁻¹) into the right armpit of nude mice [20]. When the tumor volume reached approximately 100 mm³, 36 mice with good tumor formation were selected and divided into 4 treatment groups by the random number table method (n = 9): GA, GA-NCs, GA-NCs@0%FA, and GA-NCs@FA groups. Each group received tail-vein injections at an equivalent GA dose of 8 mg·kg⁻¹ in a volume of 100 μL per mouse, and the blank group of tumor-bearing nude mice received no treatment. The nude mice in the experimental group were sacrificed at 2, 4, and 8 h post administration (n = 3 per time point for each group). The heart, liver, spleen, lung, kidney and tumor tissues were collected. Following homogenization and centrifugation, the resulting supernatants were collected and stored at −80 °C for further analysis. The blank control group was processed following the same procedure.

2.7.2. Tissue Distribution of GA-NCs@FA

The tissue distribution experiment was performed according to the protocol described in Section 2.7.1. The organs (heart, liver, spleen, lungs, and kidneys) and tumor tissues were weighed, and the drug content was quantified by UPLC-MS/MS after treatment. The drug concentration in each tissue homogenate was then calculated.

C_tissue = C_homogenate × V_homogenate/W_tissue (8)

2.8. In Vivo Antitumor Efficacy and Safety Evaluation

2.8.1. In Vivo Antitumor Efficacy Assay

A total of 24 tumor-bearing nude mice were established. When the tumor volume reached approximately 100 mm³, the mice were randomly assigned to six groups (n = 4): GA, GA-NCs, GA-NCs@0%FA, GA-NCs@FA, PTX and saline control. Formulations were administered intravenously every two days for a total of six doses. The injection volume was 0.1 mL per mouse at an equivalent GA (or PTX) dose of 8 mg·kg⁻¹. The body weight and tumor volume were measured on days 0, 2, 4, 6, 8, 10 and 12. The main organs (heart, liver, spleen, lungs and kidneys) were collected following a period of fasting for 24 h after the last administration, and the tumor growth inhibition rate (IR) was calculated.

IR (%) =(m₁ − m₂)/m₁ × 100% (9)

where m₁ is the average tumor mass of mice in the control group and m₂ is the average tumor mass of mice in the administration group.

2.8.2. Histopathological Observation of Tumor Tissues

Tumor tissues fixed in 4% paraformaldehyde were dehydrated and embedded in paraffin for sectioning. The sections were stained with hematoxylin and eosin (H&E) and observed under an optical microscope to evaluate pathological changes.

2.8.3. Biological Safety Evaluation

The collected heart, liver, spleen, lung and kidney tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and subjected to H&E staining. Histological morphology was examined to assess the potential systemic toxicity of the formulations.

This study strictly followed the 3R (Replacement, Reduction, and Refinement) principle of experimental animal ethics and minimized the number of experimental animals under the premise of ensuring reliable and repeatable experimental results. All animals were fed in an SPF animal laboratory under 12/12 h alternating light and dark. The temperature was (25 ± 2) °C, and the humidity was 50–60%. Animals could drink and eat freely. During the study, all experimental animals met the criteria, and no data elimination occurred.

3. Results

3.1. Physicochemical Characterization

Upon storage at room temperature for a specified period, GA-NCs@FA maintained a transparent and uniform yellow colloidal dispersion, and the prominent Tyndall effect further validated its superior physical stability. GA-NCs, GA-NCs@0%FA, and GA-NCs@FA were all spherical, with a symmetrical unimodal size distribution and narrow PDI, confirming the high uniformity of the system and effectively reducing the risk of particle aggregation. With progressive surface modification, the particle size gradually increased from 126.63 ± 0.42 nm to 183.07 ± 0.55 nm. Notably, the absolute value of the Zeta potential increased significantly after FA coupling (from −10.60 mV to −17.70 mV). This is attributed to the fact that deprotonation of carboxyl groups in the FA structure increases the surface negative charge density and that the absolute value of the potential is increased to enhance the electrostatic repulsion between particles, which helps to improve the stability of the system, in addition to confirming the successful modification of FA (Figure 1A–K).The EE of GA-NCs was 95.25 ± 0.39%, and the DL was 20.46 ± 0.42%. Due to phospholipid and FA modification, the EE of GA-NCs@FA was 84.64 ± 0.57%, and the DL decreased to 4.33 ± 0.07%. Although the DL decreased after modification, its high EE ensured a sufficient drug payload, which provided a basis for its subsequent targeted anti-tumor effect.

Figure 1.

Characterization of GA-NCs, GA-NCs@0%FA and GA-NCs@FA. (A) Macroscopic view of GA-NCs@FA. (B) GA-NCs@FA Tyndall effect. (C) TEM images of GA-NCs. (D) TEM images of GA-NCs@0%FA. (E) TEM images of GA-NCs@FA. (F) Particle size distribution of GA-NCs. (G) Particle size distribution of GA-NCs@0%FA. (H) Particle size distribution of GA-NCs@FA. (I) Zeta potential of GA-NCs. (J) Zeta potential of GA-NCs0%FA. (K) Zeta potential of GA-NCs@FA. (L) XRD curve analysis of GA, TPGS, GA-NCs, GA-NCs@0%FA and GA-NCs@FA. (M) DSC curve analysis of GA, TPGS, physical mixture, and GA-NCs. (N) DSC curve analysis of GA, blank excipients, physical mixtures, GA-NCs@0%FA, and GA-NCs@FA.

The XRD pattern of Figure 1L shows that GA exhibits characteristic crystallization peaks at 34° and 46° (2θ), confirming its crystalline nature. However, after the preparation of nanocrystalline preparations, these characteristic peaks disappeared and were replaced by a broad diffuse halo, indicating that the drug and lipid materials were effectively integrated. DSC further confirmed this phenomenon (Figure 1M,N): GA showed a significant endothermic melting peak at 74 °C, while this peak disappeared completely in all nano-formulations, which only retained the thermal characteristics of the excipients. Clear GA characteristic peaks can still be observed in the physical mixture, which proves that the phase of GA in GA-NCs, GA-NCs@0%FA, and GA-NCs@FA changes and is encapsulated in lipid materials.

3.2. Results of Stability Investigation

The physical stability of the preparation in different environments was investigated. The dynamic changes in the particle size of GA-NCs, GA-NCs@0%FA, and GA-NCs@FA under the storage temperature and simulated physiological conditions were evaluated.

3.2.1. Storage Stability

After storage at 4 °C and 25 °C for 7 d, the particle size of GA-NCs increased slightly but remained within a narrow distribution range. In contrast, no significant fluctuations were observed in the particle sizes of GA-NCs@0%FA and GA-NCs@FA at either temperature (Figure 2A,B), demonstrating excellent physical stability. This indicates that the phospholipid coating, acting as a physical barrier, effectively prevents Ostwald ripening and particle aggregation of nanocrystals during storage.

Figure 2.

The stability of GA-NCs@FA was investigated. The particle size of GA-NCs, GA-NCs@0%FA and GA-NCs@FA stored at 4 °C for 7 d (A) and at 25 °C for 7 d (B) (n = 3). (C) Stability of GA-NCs, GA-NCs@0%FA and GA-NCs@FA in PBS (pH 7.4, containing 10% serum) (n = 3). (D) Hemolysis phenomenon of GA-NCs@FA (n = 3). (E) Hemolysis rate of GA-NCs@FA (n = 3). (F) In vitro release curves (n = 3).

3.2.2. Plasma Stability

PBS containing 10% serum (pH 7.4) was used to simulate physiological conditions, and the stability of the preparations was monitored (Figure 2C). The particle sizes of GA-NCs@FA and GA-NCs@0%FA remained stable over a 7 d period in this medium, showing good resistance to non-specific adsorption of plasma proteins. In contrast, the particle size of GA-NCs without phospholipid modification increased significantly over time, and obvious aggregation occurred.

The difference in stability is primarily attributed to the introduction of DSPE-PEG segments, which form a highly hydrated protective layer on the surface of the nanocrystals, significantly improving the physical stability of the system through a potent steric stabilization effect. In addition, the particle size stability data of GA-NCs@FA and GA-NCs@0%FA are highly consistent, which confirms that the conjugation of FA does not compromise the physical stability of the system. GA-NCs@FA exhibits excellent storage stability and physiological adaptability, providing a solid basis for its subsequent in vivo pharmacodynamics studies.

3.3. Hemolytic Evaluation

The blood compatibility and intravenous safety of GA-NCs@FA were evaluated via a hemolysis test (Figure 2D,E). According to macroscopic observation, the positive control group showed a uniform and transparent red hemolysate due to the complete rupture of erythrocytes, while the GA-NCs@FA treatment groups across all concentrations were consistent with the negative control group. The erythrocytes were completely sedimented at the bottom of the tubes, leaving the supernatant clear and colorless. No obvious hemolysis or erythrocyte agglutination was observed. Furthermore, the absorbance was measured by a microplate reader to calculate the hemolysis rate. The results indicated that the hemolysis rate of each group was below the 5% safety threshold. This is mainly attributed to the phospholipid modification on the surface of GA-NCs@FA, which significantly reduces the non-specific interaction between the nanoparticles and the erythrocyte surfaces, thereby avoiding physical damage or chemical disruption of the cell membrane caused by direct contact with the drug.

3.4. In Vitro Release Results

GA exhibited a significant burst release effect, with the cumulative release rate reaching approximately 90% at 12 h (Figure 2F). The nano-preparations exhibited significant sustained-release characteristics. At 96 h, the cumulative release rate of GA-NCs was 83.25 ± 3.30%; the release rates of GA-NCs@0%FA and GA-NCs@FA were further retarded, reaching 73.64 ± 2.56% and 73.56 ± 1.69%, respectively. This is mainly attributed to the introduction of DSPE-mPEG₂₀₀₀ into the lipid material to form a dense phospholipid layer on the surface of the nanocrystals, which increases the diffusion resistance of the drug molecules to the external medium, thereby achieving a more stable drug release process.

The release curves of GA-NCs@0%FA and GA-NCs@FA were highly consistent, and there was no significant difference in the release rate, which confirmed that the conjugation of the FA ligand did not change the internal lipid structure. This long-term release feature is conducive to maintaining a stable blood concentration in the body, reducing the frequency of administration, and mitigating systemic toxicity by minimizing peak-to-trough fluctuations, providing a solid basis for its efficient anti-tumor effect.

To address the physicochemical properties of GA, folic acid-modified phospholipid–gambogic acid nanocrystals were constructed, representing the primary novelty of this study. The excellent physicochemical properties of GA-NCs@FA establish a robust foundation for evaluating subsequent biological effects.

3.5. The Results of In Vitro Antitumor Activity Study

3.5.1. Biocompatibility Evaluation Results of Blank Carrier

Figure 3A shows the in vitro cytotoxicity results of the blank carrier on HepG2 cells. After incubation with different concentrations of blank vector for 24 h, no significant effect on cell viability was observed. When the mass concentration was as high as 140 μg·mL⁻¹, the cell viability remained above 90%, indicating that the blank carrier itself had very low cytotoxicity.

Figure 3.

The results of the in vitro antitumor activity study. (A) Effect of blank vector on the survival rate of HepG2 cells (n = 5). (B) Effect of different drug groups on the survival rate of HepG2 cells (n = 5) (GA group, *** p < 0.001; GA-NCs group, ^### p < 0.001; GA-NCs@0%FA group, ⁺⁺⁺ p < 0.001, ⁺ p < 0.05). (C) Effect of different treatment groups on the scratch healing rate of HepG2 cells (n = 3) (*** p < 0.001, ** p < 0.01). (D) Effect of different treatment groups on the migration ability of HepG2 cells (n = 3). (E) HepG2 cell uptake of different drug groups.

3.5.2. Cytotoxicity Test Results

The cytotoxicity results are shown in Figure 3B. Each treatment group exhibited a significant concentration-dependent inhibitory effect on HepG2 cell growth; specifically, cell viability decreased as the drug concentration increased. The IC₅₀ values of GA, GA-NCs, GA-NCs@0%FA, and GA-NCs@FA against HepG2 cells were 2.45, 1.12, 0.89 and 0.50 μg·mL⁻¹,respectively. GA-NCs exhibited low cytotoxicity, which may be due to the poor stability of unmodified nanocrystals in the medium, leading to aggregation and subsequently reduced cellular uptake. The toxicity of GA-NCs@0%FA was higher than that of GA-NCs, possibly because PEG modification enhanced the stability of GA-NCs in the culture medium, facilitating passive uptake by HepG2 cells. The cytotoxicity of GA-NCs@FA was significantly stronger than that of GA and other nano-preparation groups (p < 0.001), with a cytotoxic potency approximately five-fold higher than that of GA. This further confirms the advantage of FA modification: through specific recognition and high-affinity binding between FA and the FR overexpressed on HepG2 cells, receptor-mediated endocytosis was triggered, significantly increasing intracellular drug accumulation.

3.5.3. Cell Scratch Test Results

As shown in Figure 3C,D compared with the blank control group, both GA and its nano-preparations could significantly inhibit the migration of HepG2 cells. The GA-NCs@FA group showed the strongest inhibitory effect, and its scratch healing rates were 8.52% and 11.50% at 24 h and 48 h (*** p < 0.001), respectively. This enhanced and sustained inhibitory effect indicates that GA-NCs@FA can continuously release drugs to enhance efficacy.

3.5.4. Cell Uptake Test Results

The cellular uptake of each preparation by HepG2 cells was C6 < GA-NCs < GA-NCs@0%FA < GA-NCs@FA (Figure 3E). Only weak green fluorescence signals were observed in the C6 and GA-NCs groups. Their lack of stability in the physiological environment, coupled with particle aggregation and a lack of specific recognition, hindered efficient endocytosis. In contrast, the fluorescence intensity of the GA-NCs@0%FA group was enhanced, which was mainly attributed to the phospholipid layer improving the physicochemical stability of the nanocrystals and effectively preventing aggregation. This coating provides superior biocompatibility and facilitates drug uptake through non-specific endocytosis pathways. The fluorescence intensity of the GA-NCs@FA group was the most pronounced, strongly confirming that FA specifically recognizes and binds to FR highly expressed on the surface of HepG2 cells, triggering efficient receptor-mediated endocytosis. This active transport mechanism allows more nanocrystals to enter the cells by bypassing the limitations of simple diffusion. These findings are highly consistent with the cytotoxicity results, where GA-NCs@FA exhibited the lowest IC₅₀ value, further clarifying the mechanism of the formulation’s high anti-tumor activity at the cellular level.

3.6. Tissue Distribution Results

The distribution of GA in the organs and tumor tissues of mice at different time points was investigated via vein injection (Figure 4). Free GA was widely distributed and exhibited high accumulation in the liver and kidney, suggesting that the free drug could cause potential systemic toxicity to normal tissues. Compared with the GA group, all nano-preparation groups exhibited significantly reduced GA content in the liver and kidneys (* p < 0.05) and showed significant enrichment in tumor tissues (*** p < 0.001), effectively achieving tissue redistribution of the drug. The drug concentration in the GA-NCs@FA group at the tumor site was significantly higher than that in the GA-NCs@0%FA group (^# p < 0.05). This was attributed to the fact that while GA-NCs and GA-NCs@0%FA mainly relied on the enhanced permeability and retention (EPR) effect for passive targeting, GA-NCs@FA could specifically recognize FR via surface folic acid modification, triggering a receptor-mediated active targeting mechanism. This dual “active + passive” targeting strategy allowed drugs to accumulate accurately at the target site, significantly reducing exposure in non-target tissues, thereby increasing tumor accumulation while mitigating organ toxicity.

Figure 4.

Distribution of gambogic acid in various tissues at different time points after tail-vein injection. (A) Tissue distribution after 2 h of administration (n = 3). (B) Tissue distribution after 4 h of administration (n = 3). (C) Tissue distribution after 8 h of administration (n = 3). (GA group, *** p < 0.001, ** p < 0.01,* p < 0.05; GA-NCs@0%FA group ^## p < 0.01, ^# p < 0.05).

3.7. Results of In Vivo Antitumor Efficacy and Safety Evaluation

3.7.1. In Vivo Antitumor Activity

The body-weight change curve of mice in the control group showed a trend of early growth (0–8 d) and late decline (8–12 d), which may be related to activity and feeding restriction caused by an increased tumor load (Figure 5A). The body weight of mice in the GA group decreased significantly (about 13.5%, * p < 0.05). In contrast, the body weight of mice in the nano-preparation group remained stable, without significant fluctuations. This fully demonstrates that encapsulating GA in the dosage form significantly reduces its toxicity and proves the good safety profile of the carrier material.

Figure 5.

The results of antitumor activity in vivo. (A) Weight-change curve of mice (n = 4) (compared with the GA-NCs@FA group, * p < 0.05). (B) Change curve of tumor volume during 12 d drug intervention (n = 4) (control group, * p < 0.05, *** p < 0.001, **** p < 0.0001, GA-NCs@0%FA group, ^## p < 0.01). (C) Tumor appearance after 12 d of drug treatment in each group. (D) Changes in tumor weight in mice (n = 4) (control group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (E) Tumor suppression efficiency in different groups (n = 4) (PTX group, *** p < 0.001, **** p < 0.0001; GA-NCs@0%FA group, ^# p < 0.05). Statistical significance was analyzed by one-way ANOVA.

The results of in vivo pharmacodynamic experiments (Figure 5B–E) showed that compared with the control group with rapid tumor growth, each treatment group displayed a significant anti-tumor effect. Among them, the inhibitory effect of the GA-NCs@FA group was the most prominent, resulting in the lowest tumor volumes and weights. The tumor inhibition rate was not only significantly higher than that of the non-targeted GA-NCs@0%FA group (^# p < 0.05) but also significantly superior to that of the PTX group (*** p < 0.001). This excellent efficacy indicates that FA modification achieves more efficient tumor suppression than free drugs and ordinary nano-preparations, providing strong experimental support for the clinical translation of GA-NCs@FA.

3.7.2. Histopathological Evaluation of Tumors

The in vivo anti-tumor activity of each preparation was further verified from a microscopic pathological perspective, and H&E staining analysis was performed on the harvested tumor tissues (Figure 6A). In the control group, cancer cells were densely packed and clustered, with no obvious necrosis observed. The tumor tissues in the PTX, GA, GA-NCs, GA-NCs@0%FA, and GA-NCs@FA groups exhibited cell atrophy, fragmentation or dissolution, and the intercellular spacing was significantly increased compared to the control group. The severity of tissue damage followed the order of GA-NCs@FA > GA-NCs@0%FA > GA-NCs > GA > PTX. Among them, the necrotic areas observed in the GA-NCs@FA group were the most extensive and diffuse, and the tumor-disrupting effect of this formulation was significantly greater than that of the GA-NCs@0%FA and PTX groups.

Figure 6.

Histopathological analysis. (A) Pathological section of tumor-bearing nude mice. (B) Microscopic images of important organs and tissues (heart, liver, spleen, lungs, and kidneys) of mice in each group. The scale is 100 μm.

3.7.3. Biological Safety Evaluation Results

To evaluate the biosafety of each preparation at the histological level, the major organs of mice were analyzed via H&E staining (Figure 6B). The GA and GA-NCs groups showed the most severe systemic toxicity. Specifically, the heart tissue showed myocardial fiber disorganization and enlarged interstitial spaces. The liver displayed significant sinusoidal dilation and nuclear shrinkage. The spleen exhibited an obscured demarcation between the red and white pulp. The lungs presented significant peribronchial inflammatory cell infiltration, and the kidneys exhibited tubular congestion with narrowed lumens. Damage in the PTX group was mainly confined to the heart (interstitial space enlargement) and spleen (obscured red and white pulp boundary). In contrast, no obvious pathological changes or organ toxicities were observed in the GA-NCs@0%FA and GA-NCs@FA groups, whose organ morphologies were comparable to that of the control group. These results indicate that GA-NCs@FA significantly mitigates the inherent toxic side effects of GA while achieving potent tumor suppression, demonstrating excellent biosafety.

4. Discussion

The clinical application of GA has long been limited by its complex, rigid, hydrophobic structure, which leads to low water solubility, poor bioavailability, and dose-limiting toxicity [22]. To address this bottleneck, this paper abandons the traditional carrier loading strategy and innovatively uses the CO₂-assisted precipitation method combined with the thin-film hydration method to construct a GA-NCs@FA delivery system with sustained-release characteristics and active targeting capabilities.

The morphology of GA-NCs@FA was spherical, and the particle size distribution curve showed a symmetrical monomodal peak with a narrow distribution range, indicating that the prepared nanocrystals were uniform in size. The significant decrease in Zeta potential compared with that of GA-NCs confirmed the successful modification of negatively charged phospholipids and FA. The deprotonation of the FA terminal carboxyl group in a weakly alkaline environment increased the negative surface charge density. The enhancement of electrostatic repulsion not only improved the physical stability of the preparation but also provided the necessary charge characteristics for subsequent active-targeting recognition [16,23]. Compared with traditional preparation processes, which often suffer from high energy consumption, potential disruption of the drug’s crystalline structure, and media contamination, the CO₂-assisted precipitation method selected in this study is a technology that induces the rapid precipitation of poorly soluble drugs into nanosized particles by generating gas in situ at the liquid-phase interface. When the organic film containing citric acid contacts the NaHCO₃ solution during hydration, a large number of microbubbles released by the acid–base neutralization reaction act as a dynamic disturbance and a spatial barrier. This process not only provides a strong instantaneous shear force to inhibit crystal growth and aggregation, effectively reducing the particle size, but also disrupts the long-range order of drug molecules, inducing the stable precipitation of drugs in a highly dispersed state and fundamentally overcoming the solubility limits of poorly soluble drugs such as GA [19].

Although albumin and chitosan have been widely used to stabilize nanocrystals, chitosan exhibits poor solubility in neutral and basic media, limiting its use in such conditions [24]. While albumin exhibits excellent biocompatibility, it is sensitive to organic solvents and thermal stress, which can readily lead to denaturation [25]. Neither of these stabilizers satisfies the specific requirements of this study regarding the preparation process and targeting. In contrast, phospholipids were selected as stabilizers based on their natural biomimetic properties and efficient binding affinity for hydrophobic drugs [26]. The steric hindrance provided by the phospholipids inhibited crystal growth and significantly improved the stability (Figure 2A–C) and sustained-release performance (Figure 2F) of GA-NCs@FA. Furthermore, the ease of functionalizing phospholipids facilitates the introduction of DSPE-PEG-FA to achieve active targeting. When the surface of GA-NCs@0%FA was modified with FA, GA-NCs@FA exhibited stronger growth-inhibitory activity against cells and enhanced in vivo anti-tumor efficacy. This is primarily attributed to the overexpression of FRs on the surface of tumor cells, which facilitates the active accumulation of GA-NCs@FA in tumor tissues through receptor-mediated endocytosis, thereby significantly optimizing the in vivo biodistribution of the drug [16]. Tissue distribution studies (Figure 4) showed that GA-NCs@FA effectively reduced the non-specific accumulation of GA in normal liver and kidney tissues and achieved efficient enrichment at tumor sites. These findings are highly consistent with the observation of no obvious pathological damage in major organs via H&E staining (Figure 6B).There was no significant difference in the in vitro release behavior between GA-NCs@FA and GA-NCs@0% FA (Figure 2F), which indicates that the covalent conjugation of FA occurred primarily at the distal end of the PEG chain and did not disrupt the dense arrangement of the phospholipid layer. This structural integrity is crucial to ensure the ‘low leakage’ of drugs in blood circulation.

5. Conclusions

In this study, the construction of GA-NCs@FA and its superior efficacy against hepatocellular carcinoma (HCC) are reported and characterized for the first time. A stable system with an average particle size of 183.07 ± 0.55 nm and a Zeta potential of −17.70 ± 0.17 mV was successfully prepared, exhibiting prolonged sustained-release behavior (a 96 h cumulative release rate of approximately 73.6%). Regarding biological evaluation, GA-NCs@FA demonstrated significant synergistic efficacy and reduced toxicity: the toxicity against HepG2 cells was as low as 0.50 μg·mL⁻¹, which is approximately five-fold lower than that of free GA, showing a strong migration-inhibitory capacity. In the HepG2 tumor-bearing nude mouse model, facilitated by the FA-mediated active targeting mechanism, the system achieved a tumor inhibition rate of up to 70.9%, which is significantly higher than that of GA-NCs@0%FA (^# p < 0.05), overcoming the limitations of non-specific biodistribution of GA in vivo. Furthermore, H&E staining and organ index analysis confirmed that the system exhibited no significant toxic side effects on major organs, demonstrating excellent biocompatibility. The GA-NCs@FA system prepared in this study exhibits multiple advantages, including high stability, sustained-release characteristics, and active targeting capabilities. It not only provides an efficient and low-toxicity candidate for the precise treatment of HCC but also serves as a promising technical platform and theoretical basis for the clinical translation of other poorly soluble natural active ingredients with complex structures.

Abbreviations

The following abbreviations are used in this manuscript:

HCC	Hepatocellular carcinoma
GA-NCs	Gambogic acid nanocrystals
GA-NCs@FA	Folic acid-modified and phospholipid-coated gambogic acid nanocrystals
GA-NCs@0%FA	Folic acid-free phospholipid-coated gambogic acid nanocrystals

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pharmaceutics18020253/s1, Table S1: Effect of drug dosage on particle size and PDI of GA-NCs ($\overline{x}\pm s$, n = 3); Table S2: Effect of lipid-drug ratio on particle size and encapsulation efficiency of GA-NCs@FA ($\overline{x}\pm s$, n = 3); Table S3: Effect of DSPE-PEG₂₀₀₀-FA modification ratio on particle size and encapsulation efficiency of GA-NCs@FA ($\overline{x}\pm s$, n = 3).

Author Contributions

X.Z.: validation, data curation, and investigation. S.L.: writing (original draft) and writing (review and editing). Q.H.: methodology and data curation. Y.L.: visualization and investigation. J.S. (Jingqi Sun): data curation and software. Y.P.: funding acquisition and resources. J.S. (Jin Su): funding acquisition, resources, and supervision. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The animal study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of Institutional Animal Care and Use Committee of Jiamusi University (protocol code JDYXY-2024001), 26 September 2024.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be made available upon request.

Conflicts of Interest

The authors declare no competing interests. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding Statement

This research was funded by the North Medicine and Functional Food Characteristic Subject Project in Heilongjiang Province (HLJTSXK-2022-03); Supported by Heilongjiang Provincial Natural Science Foundation of China (LH2024H003); The Basic Research Support Program for Excellent Young Teachers in Heilongjiang Province (YQJH2023216); National College Student Innovation and Entrepreneurship Training Program (202510222166); Heilongjiang Provincial Key Laboratory Fund of New Drug Development and Evaluation of Toxicology (kfkt 2023-05).