Abstract
Oral squamous cell carcinoma (OSCC) is a significant public health
issue worldwide. Conventional chemotherapeutic agents do not adequately
meet the treatment demands because of their low efficacy and adverse
side effects. Toosendanin (TSN) is a natural extract with potential
anticarcinogenic activity. Nonetheless, its clinical application is
constrained by its poor water-solubility and limited bioavailability.
Therefore, we prepared TSN-loaded poly (lactic-co-glycolic acid)
nanoparticles (TSN-PLGA NPs) to improve the water-solubility of TSN and
potentially further enhance its bioavailability. TSN-PLGA NPs were
synthesized and characterized, and we showed their exceptional
properties for sustained release in vitro. TSN-PLGA NPs exhibited
cytotoxic effects against OSCC cells, potentially inhibiting
proliferation and promoting apoptosis by inducing cell-cycle arrest in
the S-phase at low concentrations. RNA-sequencing analysis revealed the
potential regulation of OSCC cell viability by TSN-PLGA NPs through
signaling pathways such as JAK/STAT and PI3K–Akt. Furthermore, animal
models provided evidence of the in vivo antitumor activity of TSN-PLGA
NPs, with no observable side effects in nude mice, which indicated
potential biocompatibility. Consequently, TSN-PLGA NPs may be a
promising chemotherapy candidate for OSCC treatment.
Supplementary Information
The online version contains supplementary material available at
10.1186/s12906-025-04957-0.
Keywords: Toosendanin, Oral squamous cell carcinoma, PLGA,
Nanomedicine, EPR effect
Introduction
Oral carcinoma is a prevalent malignant tumor of the head and neck
area, and ranks 16 th in terms of incidence among all cancers. Oral
squamous cell carcinoma (OSCC) is a predominant pathological subtype,
and comprises 90% of cases [[38]1, [39]2]. According to the estimation
provided by National Cancer Institute (NCI), lip and oral cavity cancer
accounts for approximately 54,540 new cases and 11,580 deaths in the
United States in 2023 [[40]3]. Moreover, due to the anatomical and
physiological characteristics of the oral and maxillofacial region,
which is rich in lymphatic tissue and blood supply and undergoes
frequent muscle movements, OSCC is prone to early lymph node metastasis
and late distant metastasis, thereby greatly compromising the prognosis
and reducing survival of patients [[41]4]. The 5-year overall survival
rate in OSCC patients is generally below 60%, which indicates an
unfavorable prognosis and presents a substantial obstacle for the
management of the disease [[42]5]. Currently, the primary treatment
approach for localized OSCC entails the surgical removal of the primary
tumor and affected lymph nodes. Adjuvant radiotherapy or chemotherapy
may follow, depending on the disease stage [[43]6]. Nevertheless, the
therapeutic efficacy of conventional chemotherapy drugs is limited, and
they are associated with notable adverse effects, including bone marrow
suppression, impairment of liver and kidney function, and neurotoxicity
[[44]7]. The current preventive and treatment strategies for oral
cancer are inadequate in addressing the public health demands.
Consequently, it is important to explore innovative and effective
methods for managing OSCC.
Natural herbs–derived substances, including bioactive elements, raw
extracts, and pure compounds, have been attracting growing attention of
researchers in the context of their potential contribution to cancer
treatment. Extensive research and data indicate that over 50% of
approved anticancer drugs are derived from natural compounds or herbal
derivatives. As an effective anticancer drug extracted from
plant-derived natural products, paclitaxel has been proven capable of
interfering with the signaling pathways of various cancers and
inhibiting the proliferation of cancer cells, and widely used in
clinical treatment [[45]8]. Inspired by this, a growing number of
compounds derived from herbs or traditional Chinese medicine are being
applied in clinical studies for cancer treatment. Toosendanin (TSN),
which is extracted from toosendan tree, is a compound that consists of
tetracyclic triterpenoids. Nowadays, with deep research, its
significant value in scientific research, clinical medicine, and
agriculture has been found [[46]9]. Previous studies have substantiated
its efficacy in alleviating pain, mitigating inflammation, countering
botulinum toxin, and combating bacterial infections [[47]10, [48]11].
TSN also possesses antiproliferative and apoptosis-inducing properties
in vitro against several human cancer cells, including gastric cancer,
colorectal cancer, liver cancer, and breast cancer [[49]12–[50]16].
However, the poor water solubility, low bioavailability, and
undesirable side effects have limited the clinical application of TSN.
Although TSN exhibits a fast oral absorption rate and clearance in
mouse plasma, its absolute bioavailability is reported to be only 9.9%
[[51]17]. Previous studies of TSN side effects have indicated that the
hepatic damage observed in mice as a result of TSN exposure may be
associated with a reduced level of glutathione, as well as compromised
mitochondrial function and abnormal lipid metabolism [[52]18].
Considering the potent anticancer activity of TSN, structural
modifications of TSN and the design of targeted drug delivery systems
to improve its water-solubility and bioavailability hold significant
clinical value.
Compared with conventional pharmaceuticals, nanomedicines offer several
advantages such as enhanced drug solubility, passive tumor targeting
and accumulation, controlled drug release, and reduced systemic side
effects [[53]19, [54]20]. In recent years, a variety of biodegradable
polymers, both natural and synthetic, have been employed biomedical
applications, especially in drug delivery and release systems [[55]21,
[56]22]. Interestingly, poly (lactic-co-glycolic acid) (PLGA) is a
biodegradable polymer material with good biocompatibility and
biodegradability and has widely been used in drug delivery systems
[[57]23]. In contrast to inorganic nano-delivery systems, which might
be often associated with systemic toxicity and instability, PLGA can
degrade into lactic acid and glycolic acid, which are ultimately
cleared as water and carbon dioxide. Furthermore, by encapsulating
chemotherapy drugs in PLGA, the drugs can be slowly released into tumor
tissues while avoiding rapid degradation in the blood or filtration by
the kidneys, which improves drug efficacy and reduces side effects. In
addition, PLGA nanoparticles possess good stability and prolonged
circulation time in blood vessels, which can extend the circulation
time of drugs in the body and facilitate their accumulation at the
tumor site, thereby enhancing the antitumor effect of the medications.
Overall, PLGA-encapsulated chemotherapy is a promising treatment
approach because it can improve drug targeting and efficacy while
reducing side effects [[58]24].
In summary, TSN may be a promising anticancer drug candidate, but its
therapeutic efficiency in OSCC requires further investigation. Herein,
we prepared TSN-loaded PLGA nanoparticles (TSN-PLGA NPs) and verified
the antitumor activity of TSN and TSN-PLGA NPs through in vitro
studies. Previous animal model studies have indicated that TSN may
mediate lysosomal dysfunction, thereby causing acute liver injury in
mice and zebrafish [[59]25–[60]27]. Consequently, we constructed a cell
line–derived xenograft (CDX) model to investigate the efficacy of the
drug after in vivo metabolism, while also assessing the in vivo safety
of TSN-PLGA NPs. The tumor microenvironment (TME) plays a pivotal role
in the growth and migration of tumors [[61]28]. In comparison to the
CDX model, a patient-derived xenograft (PDX) model will be able to
retain the original structure and molecular characteristics of the
primary tumor, accurately replicating the in vivo TME [[62]29].
Therefore, we extended our research from the CDX model to further
establish PDX model for in vivo studies. We anticipate that the PDX
model will improve the precision of predicting drug efficacy and its
anti-tumor effects by more accurately simulating in vivo tumor
microenvironment processes.
Materials and methods
Materials
TSN and 5-fluorouracil (5-FU) were purchased from Med Chem Express
(Shanghai, China). PLGA (MW 38–54 kDa), dimethyl sulfoxide (DMSO), and
polyvinyl alcohol (PVA, MW 145 kDa) were purchased from Sigma Aldrich
(St. Louis, MO, USA). Methanol (MeOH), ethyl alcohol, water (HPLC
grade), acetonitrile, and dichloromethane (DCM) were purchased from
Xilong Scientific (Guangzhou, China). A dialysis membrane (MWCO = 3.5
kDa) was purchased from Spectrum Laboratories Inc. (Rancho Dominguez,
CA, USA), and a cryoprotectant D-trehalose was purchased from BioFroxx
(Einhausen, Germany). Double-distilled water (ddH[2]O) was used for all
experiments.
Acquisition of tissue samples
Fresh tumor tissues of oral OSCC were obtained from the First
Affiliated Hospital of Fujian Medical University. The patients signed a
written informed consent. The study was approved by the Ethics
Committee of the Fujian Medical University.
Cell culture and mice
Normal human oral epithelial cells (HOEC) and OSCC cell lines (CAL27
and HN6) were obtained from the Fujian Key Laboratory of Oral Diseases.
The cells were cultured in dulbecco's modified eagle medium (DMEM,
Gibco, Shanghai, China) with 10% fetal bovine serum (FBS, SORFA,
Beijing, China), and kept in humidified incubators with 5% CO[2] at 37
°C.
BALB/C-nude mice were provided by SLAC Laboratory Animal Co., Ltd.
(Shanghai, China). The animal experimental protocols were approved by
the Ethics Committee of Fujian Medical University.
Preparation of TSN-PLGA NPs
The TSN-PLGA NPs were prepared by using a double emulsion-solvent
evaporation method [[63]30]. Briefly, TSN (10 mg) was dissolved in 700
μL of ethanol. Then, 800 μL of ddH[2]O was added. PLGA (300 mg) was
dissolved in 25 mL of DCM. Then, the TSN solution was added dropwise
into the PLGA solution, and the mixture was dispersed for another 20
min using an ultrasonic oscillator to form a W/O primary emulsion. PVA
(53 mg) was dissolved in 100 mL of ddH[2]O and stirred using a magnetic
stirrer at 800 rpm for 4 h to form a PVA solution (W2). The entire W/O
primary emulsion was transferred to a conical flask, and W2 was added
dropwise while being stirred with a mechanical stirrer at 1,800 rpm for
20 min. Then, the solution was treated with an ultrasonic processor
(20,000 Hz, 4 °C) for 15 min and stirred at room temperature to remove
the residual DCM. The supernatant was discarded by centrifugation (4
°C, 12,000 rpm, 45 min), and the precipitate was reconstituted in 20 mL
of ddH[2]O. The cryoprotectant D-trehalose was added. Finally, the NPs
were obtained after freeze-drying (Fevik TS8606, Shanghai, China).
Characterization of TSN-PLGA NPs
The structure of TSN-PLGA NPs was examined by scanning electron
microscopy (SEM, Frequency Electronics Quanta450, OR, USA) and
transmission electron microscopy (TEM, Frequency Electronics Tecnai G2,
OR, USA). TSN-PLGA NPs solution (containing TSN at a concentration of
200ug/ml) was prepared using ddH[2]O. After gold coating under vacuum
conditions, the samples were observed under SEM. In addition, after
staining with phosphotungstic acid, TSN-PLGA NPs were observed using
TEM.
The dynamic light scattering (DLS) method was employed to measure the
particle size distribution and polydispersity index (PDI) of TSN-PLGA
NPs, utilizing an Anton Paar Sur-PASS 3 instrument (Anton Paar,
Austria). Additionally, the zeta potential (ZP) of TSN-PLGA NPs was
determined using the same instrument.
The drug loading (DL) and encapsulation efficiency (EE) of TSN in
TSN-PLGA NPs were determined using high-performance liquid
chromatography (HPLC). The chromatographic column used was Agilent
ZORBAX Eclipse XDB-C18 (250 mm × 4.6 mm, 5 µm). The mobile phase
consisted of 0.1% formic acid and acetonitrile (40:60, v/v). The flow
rate was set at 1.0 mL/min. The UV detection wavelength was 210 nm. The
measurement volume was 10 µL. The following equations were used to
calculate DL and EE:
[MATH: DL%=Quantity of Loaded drug in
NanoparticlesQuantity of
Nanoparticles×100% :MATH]
1
[MATH: EE%=Quantity of Loaded drug in
NanoparticlesInitial Quantityof Drug
Added×100%<
/mrow> :MATH]
2
Protein adsorption assay
To investigate protein adsorption by prodrug-based nanoparticles,
bovine serum albumin (BSA) was chosen as the model protein. Free TSN
(150ug/ml), nanoparticles (containing TSN at a concentration of 150
µg/mL) were co-incubated with BSA (120 µg/mL) in PBS (pH 7.4) at 37 °C.
At various time points, 200 µL aliquots of each sample were centrifuged
(14,000 g, 15 min) to facilitate the precipitation of adsorbed protein
aggregates. The BSA standard curve was then established using a BCA
protein assay kit (Beyotime Biotechnology Shanghai, China) following
the manufacturer's instructions. The concentration of unadsorbed
protein was measured using a microplate reader (Thermo Scientific, USA)
under the same conditions. The proportion of adsorbed protein was
subsequently calculated at each time point.
In vitro release study
The drug release curves of TSN-PLGA NPs in vitro over a period of 36 h
were assessed using the dialysis bag method [[64]31]. In total, 150 mg
of TSN-PLGA NPs was dissolved in PBS buffer (pH 5.4 or 7.4, 1.5 mL).
The solution was quickly transferred into a prepared dialysis bag (MWCO
3.5 kDa), immersed in PBS buffer (pH 5.4 or 7.4, 2 mL) solution, and
placed in a 37 °C water bath shaker. At specific time points (0.5, 1,
2, 3, 4, 6, 8, 12, 24, and 36 h), 200 μL was removed from the
surrounding solution and replaced with an equivalent amount of fresh
buffer. HPLC was utilized to detect the content of released TSN from
the TSN-PLGA NPs. The size and charge of TSN-PLGA nanoparticles were
measured using dynamic light scattering (DLS) both before incubation
and after 36 h of incubation. The DLS data were analyzed with Prism
software to compare changes in particle size and charge under different
pH conditions.
In vitro cytotoxicity
Cytotoxic efficacy of TSN and TSN-PLGA NPs was assessed using the CCK-8
method. CAL27 and HN6 cells were seeded in 96-well plates at a density
of 3,000 cells per well, with 15 replicates per group. For each time
point (0, 24, 48, 72, and 120 h), three replicates per group were
tested (replicates were not reused). Different concentrations of TSN
and TSN-PLGA NPs (ranging from 0.001 to 10 μM) were prepared in the
cell culture medium, with TSN dissolved in DMSO. After 24 h, 100 μL of
the drug solution was added to each well. At the designated time
points, 100 μL of the culture medium containing 10% CCK-8 reagent was
introduced, and cells were incubated for one hour. Absorbance at 450 nm
was measured to determine cell viability (%).
EdU assay
The proliferation ability was evaluated with an EdU assay kit (Uelandy,
Shanghai, China, Cat. No C6015S) in accordance with the manufacturer’s
instructions. After seeding the cells in six-well plates (3 × 10^6
cells per well), DMSO, 5 nM TSN, 5 nM TSN-PLGA, 10 nM TSN, and 10 nM
TSN-PLGA were added to the respective wells. Images were taken under a
fluorescence microscope, and we determined the proportion of cells that
tested positive for EdU.
Measurement of apoptosis by Annexin V-FITC/PI Assay
CAL27 and HN6 cells (3 × 10^6 cells per well) were seeded in six-well
plates and exposed to TSN and TSN-PLGA NPs (5 nM or 10 nM,
TSN-equivalent concentrations) for another 48 h, while
phosphate-buffered saline (PBS) was set as control. The cells were then
stained with Annexin V-FITC/PI fluorescence detection kit (Uelandy,
Shanghai, China, Cat. No F6012L) for 15 min and analyzed by flow
cytometry (Becton, Dickinson and Company, LSRFortessaX-20, NJ, USA).
Cell cycle assay
The effect of TSN-PLGA NPs on cell cycle was assessed using a cell
cycle and apoptosis kit (Uelandy, Shanghai, China, Cat. No C6031S). HN6
cells were placed in six-well plates (3 × 10^6 cells per well) and
treated with TSN-PLGA NPs (5 nM or 10 nM) for 48 h. Ultimately, the
cells were dyed in accordance with the provided guidelines. Flow
cytometry was utilized to analyze the distribution of cells in
different phases of cell cycle, employing an emission wavelength
greater than 535 nm. The distribution of cells in the G0/G1, S, or G2/M
phases was assessed by measuring the respective regions.
RNA sequencing analysis
After treating CAL27 and HN6 cell lines with DMSO and TSN for 48 h, RNA
was extracted using the Trizol method and transferred to RNA-free EP
tubes for storage at − 80 °C. Library preparation, sequencing, and data
analysis were performed at OE Biotech (Shanghai, China). The remaining
samples were kept at − 80 °C for subsequent qRT-PCR analysis.
Quantitative reverse-transcription PCR
In accordance with the kit instructions, the extracted RNA was
reverse-transcribed. The synthesized cDNA was then diluted tenfold. The
following genes were selected for quantitative reverse-transcription
PCR reaction: JUN, PCSK9, INHBA, NR4 A1, CDKN1 A, and PLAGL1. The total
reaction volume was 20 μL, including 10 μL SYBR Premix Ex Taq II, 1 μL
of each forward and reverse primer, 2 μL cDNA, and 7 μL DEPC-H[2]O. The
PCR program was as follows: 95 °C for 5 min, 98 °C for 10 s, 40 cycles
of 60 °C for 30 s, and 72 °C for 10 min. The 2 − ΔΔCt method was
employed to calculate the relative expression level, with GAPDH serving
as the reference gene. Primers used for qPCR are listed in
Supplementary Table 1.
Construction of the CDX model
The CDX model was established by injecting an HN6 cell suspension (100
μL) subcutaneously into the right flank of nude mice. Tumor dimensions,
namely the length (L) and width (S), were documented every 2 days.
Tumor volume was calculated as follows:
[MATH: Tumor
volume=0.5×L×S2 :MATH]
3
Upon reaching an average tumor volume of 180 mm^3, the nude mice were
randomly divided into four groups: the NS group (normal saline), the
5-FU group (5 mg/kg 5-Fluorouracil), the TSN group (5 mg/kg free
Toosendanin), and the TSN-PLGA group (TSN-PLGA nanoparticles,
delivering an effective dose of 5 mg/kg TSN). All treatments were
administered via intraperitoneal injection. The treatment was given
once a week for 2 weeks. 5-FU is a widely used antimetabolite drug in
cancer chemotherapy and is also frequently used in chemotherapy for
OSCC [[65]32]. Therefore, we chose the 5-FU group as the positive
control group in this study.
Construction of the PDX model
To establish the PDX model, fresh tumor tissues were collected from the
patient with OSCC of the tongue. The tumor tissues were collected
within 30 min of surgical resection, focusing on tumor edges with
minimal necrosis. The tissues were then soaked in DMEM culture medium
containing 10% FBS and 0.005% penicillin–streptomycin at 4 °C. The
tumor tissues were trimmed into 2–3 mm^3 fragments using tissue
scissors. The mice were anesthetized with 1% sodium pentobarbital
solution (80 mg/kg) via intraperitoneal injection. The trimmed tumor
tissues were inserted into the subcutaneous space on the back of the
mice (F1). When the volume of the transplanted tumors reached
approximately 100 mm^3, the tumor tissues were trimmed into 2–3 mm^3
fragments and transplanted into new BALB/c nude mice (F2). The
third-generation nude mice (F3) were constructed using the same method.
Hematoxylin–eosin (HE) staining and immunohistochemical staining were
performed on the mouse tumor tissues from the PDX model and the
original patient tumor tissues to evaluate their homogeneity. When the
average tumor volume of the F3 nude mice reached 180 mm^3, they were
randomly divided into four treatment groups: the NS group (normal
saline), the 5-FU group (5 mg/kg 5-Fluorouracil), the TSN group (5
mg/kg free Toosendanin), and the TSN-PLGA group (TSN-PLGA
nanoparticles, containing an effective dose of 5 mg/kg TSN). All
treatments were administered via intraperitoneal injection.The
treatment was administered once a week for 2 weeks. The mice were
observed daily for diet, behavior, and activity. Tumor volume was
measured with calipers every 2 days, and the weight and tumor volume of
each nude mouse were recorded. A tumor growth curve was plotted based
on the data.
Two weeks after the first injection, the nude mice were euthanized by
intraperitoneal injection of an excess amount of 1% sodium
pentobarbital solution. The tumors were dissected, and their volume was
measured. Tumor weight was recorded to calculate the percentage of
tumor growth inhibition (TGI) using the following equation:
[MATH: TGI=1-average tumor weight of the
treatment groupaverage tumor weight of the
NS
group×100%<
/mrow> :MATH]
4
The tumor samples were immersed in a 4% paraformaldehyde solution,
embedded in paraffin, and sectioned for subsequent immunohistochemical
and TUNEL staining. Additionally, the heart, liver, kidney, and jejunum
of the nude mice were collected for HE staining.
HE staining
To assess organ toxicity, we performed HE staining on the tumor tissues
and examined the heart, liver, kidney, and jejunum of the nude mice. In
short, the tissues were fixed in 4% paraformaldehyde for 24 h, embedded
in paraffin, and sectioned. The sections were treated with HE, sealed
using neutral resin, observed through an optical microscope, and
captured in photographs.
Immunohistochemical staining
The tumor tissues were sectioned and deparaffinized following routine
procedures. Antigen retrieval was performed using citrate buffer, and
blocking was carried out using goat serum. After an overnight
incubation with the primary antibody at 4 °C, the samples were stained
with DAB. They were observed and photographed using an optical
microscope.
TUNEL staining
Terminal deoxynucleotidyl transferase-mediated nick end labeling
(TUNEL) staining was performed on the mouse tumor tissues to provide
information about tumor cell apoptosis. TUNEL staining was performed in
accordance with the instructions. DAPI staining was subsequently
performed, and the slides were finally mounted using an anti-fade
mounting medium. The slides were observed and photographed under a
fluorescence confocal microscope.
Statistical analysis
Statistical analysis was conducted using GraphPad Prism 9 software
(GraphPad, San Diego, CA, USA), employing Student's t-test and one-way
analysis of variance (ANOVA). The data are expressed as the mean
± standard error of the mean (SEM). p < 0.05 was considered
statistically significant.
Results
Preparation and characterization of TSN-PLGA NPs
Figure [66]1A and B display the chemical structures of TSN and PLGA,
respectively. We employed the double emulsion-solvent evaporation
method to prepare the TSN-PLGA NPs. The NPs were subsequently
freeze-dried for 24 h, which resulted in the formation of a white,
loose powder (Fig. [67]1C). Examination under SEM and TEM
(Fig. [68]1D,E) revealed that the TSN-PLGA NPs exhibited a
predominantly spherical particle morphology. The particles demonstrated
a relatively uniform size distribution and uniform dispersion, with a
size range of approximately 100–200 nm within the observed field.
Moreover, we found that the PLGA outer membrane was intact, ensuring
complete encapsulation of TSN.
Fig. 1.
[69]Fig. 1
[70]Open in a new tab
Characterization and the in vitro drug release profile of TSN-PLGA NPs.
A The chemical structure of TSN. B The chemical structure of PLGA. C
Appearance of TSN-PLGA NPs lyophilized powder. D SEM image of TSN-PLGA
NPs. E TEM image of TSN-PLGA NPs. F Particle size distribution of
TSN-PLGA NPs (n = 3). G Zeta potential distribution of TSN-PLGA NPs
(n = 3). (H) The cumulated release curve of TSN from TSN-PLGA NPs in
PBS (pH 5.4 or 7.4, n = 3). (I) Zeta potential distribution of TSN-PLGA
NPs at 0 h (n = 3). (J-K) Zeta potential distribution of TSN-PLGA NPs
at 36 h (PBS, pH = 5.4 or 7.4, n = 3). (L-M) Particle size distribution
of TSN-PLGA NPs at 0 h and TSN-PLGA NPs at 36 h (PBS, pH = 7.4 or 5.4,
n = 3). (N) Protein adsorption of nanoparticles and free TSN was
measured after incubation at 37 °C (pH 7.4) for different time
intervals, with BSA as the standard (mean ± SD, n = 3)
We utilized an Anton Paar particle size and ZP analyzer to investigate
the particle size, distribution, and ZP of TSN-PLGA NPs. Particle size
was measured by DLS technique. As shown in Fig. [71]1F, the NPs
exhibited a unimodal normal distribution with an average particle size
of 135.48 ± 0.37 nm and PDI of (0.05 ± 0.04) %. ZP was − 18.33 ± 1.33
mV (Fig. [72]1G), suggesting that the particles possessed a negative
surface charge—a characteristic that promotes their stability in the
bloodstream and potentially extends the duration of drug circulation
within the blood. EE and DL of TSN-loaded PLGA-NPs were 88.23% and
1.08%, respectively, as measured using HPLC.
In vitro drug release curve of TSN-PLGA NPs
TSN-PLGA nanoparticles were incubated in PBS buffer at pH 5.4 or 7.4 to
simulate drug release, with pH 5.4 mimicking the acidic environment of
lysosomes in tumor cells and pH 7.4 reflecting the physiological
conditions of the human body. As shown in Fig. [73]1H, the drug
exhibited rapid release within the first 6 h, followed by stabilization
of the release curve after 12 h. At 36 h, approximately 85.00% of TSN
was released in the pH 5.4 group, while about 40.26% was released in
the pH 7.4 group. These results demonstrate that TSN-PLGA nanomaterials
exhibit excellent drug release performance in an acidic environment.
Therefore, it can be anticipated that once these TSN-PLGA NPs enter
tumor cells, they can achieve efficient and controlled drug release in
the acidic environment of tumor cell lysosomes. As shown in
Fig. [74]1I-M, the DLS results of TSN-PLGA nanoparticles before and
after 36 h of incubation are presented. Under the pH 7.4 condition, the
charge and particle size of the nanoparticles did not show significant
changes, indicating that TSN-PLGA nanoparticles maintain good stability
under physiological pH conditions. In contrast, at pH 5.4, both the
absolute value of the charge and the particle size of TSN-PLGA
nanoparticles significantly decreased, suggesting that the
nanoparticles underwent degradation in the acidic environment. Based on
these findings, we conclude that the drug release from TSN-PLGA
nanoparticles may result from a combined mechanism involving both
degradation and charge alteration.
Protein adsorption assay
PLGA enhances protein solubility and plasma stability [[75]33–[76]35].
To evaluate the interaction between nanoparticles and proteins, we used
BSA as a model plasma protein. As shown in Fig. [77]1N, under
physiological conditions (pH 7.4), nanoparticles exhibited minimal
protein adsorption after 2 h of incubation, with only slight adsorption
observed after 4 h. After 36 h under the same conditions, prodrug-based
nanoparticles continued to show low protein adsorption, while free TSN
exhibited strong protein adsorption. Compared to free TSN, the
nanoparticles demonstrated reduced nonspecific protein adsorption,
suggesting that nanoparticles may prolong blood circulation time.
Therefore, TSN-PLGA nanoparticles represent a promising drug delivery
system with enhanced plasma stability.
Analysis of the viability of OSCC cells in vitro
The CCK-8 assay was used to evaluate the viability of CAL27, HN6, and
HOEC cells after 48 h of treatment with TSN and TSN-PLGA NPs. The
dose–response curves were fitted, and the IC50 values were calculated.
As shown in Fig. [78]2A, the IC50 values of TSN against CAL27 and HN6
cells were 25.7 nM and 16.3 nM, respectively. For TSN-PLGA NPs, the
IC50 values against CAL27 and HN6 cells were 8.1 nM and 13.1 nM,
respectively. Based on the determined IC50 values for the two drugs,
5 nM and 10 nM concentrations were chosen for the subsequent
experiments. As shown in Figure S1, the cell viability of HOEC cells
treated with TSN and TSN-PLGA NPs for 48 h was not significantly
different from that of the control. Preliminarily, this finding
indicated negligible toxicity of the drugs on HOEC cells.
Fig. 2.
[79]Fig. 2
[80]Open in a new tab
CCK-8 assay for cell viability. A Fitting curves of cell viability
after 48 h of treatment with TSN and TSN-PLGA NPs. B CCK-8 assay to
detect changes in cell proliferation ability of CAL27 and HN6 cells
treated with drugs (n = 3). * p < 0.05, **** p < 0.0001
To investigate the effects of TSN and TSN-PLGA NPs on OSCC cell
proliferation, we performed CCK-8 assays on the cells treated with two
different concentrations (5 nM and 10 nM) of TSN and TSN-PLGA NPs. We
found significant disparities in cell proliferation after 3 days
between the treatment groups and the DMSO group. Significant variations
in cell proliferation were also noticed between the treatment groups
and the DMSO group (Fig. [81]2B) following a 5-day culture period. The
proliferation activity of OSCC cells decreased with time, and the
concentration of TSN and TSN-PLGA NPs increased within a certain range.
In both CAL27 and HN6 cells, the suppressive effect of TSN-PLGA NPs on
OSCC cell growth surpassed that of TSN, with 5 nM TSN-PLGA NPs
exhibiting a stronger inhibitory effect than 10 nM TSN. These findings
preliminarily indicate the drug release properties of TSN-PLGA NPs.
EdU assay
The EdU assay is used to directly detect the proliferation of live
cells. This method enables rapid detection of cell proliferation and
provides a more accurate reflection of the cell proliferation status.
The results (Fig. [82]3) demonstrated a significant decrease in cell
proliferation capacity of CAL27 and HN6 cells after the treatment with
TSN and TSN-PLGA NPs. The inhibitory effect increased with the increase
in drug concentration. The cell proliferation capacity of OSCC cells
treated with 10 nM TSN-PLGA NPs was significantly weaker than that of
the cells treated with 10 nM TSN (CAL27: p < 0.05; HN6: p < 0.01).
These preliminary findings suggest that both TSN and TSN-PLGA NPs have
the ability to inhibit OSCC cell proliferation, but TSN-PLGA NPs
exhibit stronger inhibitory effects than TSN.
Fig. 3.
[83]Fig. 3
[84]Open in a new tab
EdU assay to detect changes in cell proliferation ability of CAL27 and
HN6 cells treated with drugs (scale bar = 50 μm). * p < 0.05, ** p <
0.01
Induction of S-phase cell cycle arrest and apoptosis in OSCC
Apoptosis is one of the most common mechanisms of cancer cell death. To
investigate whether TSN and TSN-PLGA NPs can induce apoptosis in OSCC
cells, different concentrations of TSN and TSN-PLGA NPs were used to
treat CAL27 and HN6 cells for 48 h, followed by Annexin V-PI staining
and flow cytometry analysis. Figure [85]4A showed that the level of
apoptosis of CAL27and HN6 cells increased to varying degrees after the
treatment with TSN and TSN-PLGA NPs, but TSN-PLGA NPs exhibited a
stronger ability to promote apoptosis at the same concentration.
Fig. 4.
[86]Fig. 4
[87]Open in a new tab
TSN and TSN-PLGA NPs induce S-phase cell cycle arrest and promote
apoptosis in OSCC cells. A The apoptotic activity of CAL27 and HN6
cells after treatment with drugs for 48 h. The cells were stained by
AnnexinV and PI and analyzed by flow cytometry. B The cell cycle
distribution of HN6 cells after treatment with TSN-PLGA NPs for 48 h
(n = 3). * p < 0.05 compared with the control group
It has been suggested that TSN may participate in the regulation of
crucial processes such as cell cycle, thereby affecting tumor
occurrence and development [[88]14]. Therefore, to examine the effects
of TSN-PLGA NPs on cell cycle of OSCC cells and the differences among
the groups, we used propidium iodide DNA staining and flow cytometry to
detect cell cycle arrest. As shown in Fig. [89]4B, in HN6 cells, the
percentage of cells in S phase after the treatment with 5 nM and 10 nM
TSN-PLGA NPs was(26.3 ± 0.7)%, and (27.2 ± 1.95)%, respectively, which
was significantly higher than that in the control group (19.2 ± 2.97)%
(p < 0.05). These results indicate that TSN-PLGA NPs might induce
apoptosis of HN6 cells by slowing down the progression of cells through
S phase.
RNA sequencing analysis and validation
To investigate the inhibitory mechanism of TSN on the proliferation of
OSCC cells, we performed a 48-h treatment of OSCC cells with TSN.
Subsequently, the Trizol method was employed to extract total RNA for
RNA-sequencing analysis. Through differential gene enrichment analysis,
the HN6 cell line showed a total of 965 differentially expressed genes
(DEGs), namely 573 upregulated and 392 downregulated genes. The CAL27
cell line had a total of 3124 DEGs, namely 1563 upregulated and 1561
downregulated genes (Fig. [90]5A). Figure [91]5B shows the shared and
unique DEGs between the HN6 and CAL27 cell lines. In total, 487 DEGs
were shared between the cell lines. Additionally, a volcano plot was
generated for all genes that met the criteria of p < 0.05 and
|log2(fold change)|> 1 to visualize the differential gene expression
between the two groups. As shown in Fig. [92]5C, the volcano plot
depicts the distribution of DEGs between the TSN group and the DMSO
group, with red and blue indicating significantly upregulated and
downregulated DEGs, respectively. We next conducted KEGG pathway
enrichment analysis of the identified DEGs. Figure [93]5D shows the 20
most significantly enriched pathways. Specifically, the DEGs were
highly enriched in pathways such as JAK–STAT, MAPK, and PI3 K–Akt, with
a relatively high number of genes, indicating their potential
involvement in the mechanisms affected by TSN treatment.
Fig. 5.
[94]Fig. 5
[95]Open in a new tab
RNA-sequencing analysis and validation of qPCR. A Total number of DEGs.
B Shared and unique expressed genes between CAL27 and HN6 cell lines. C
DEGs volcano plot. D Bubble plot of KEGG enrichment analysis. E GSEA
plot. F Heat map of DEGs. G Validation of genes expression after DMSO
and TSN treatment in CAL27 cells. ** p < 0.01, *** p < 0.001 compared
with the DMSO group
We used gene set enrichment analysis (GSEA) to evaluate whether
predefined gene sets showed statistically significant differences
between the DMSO and TSN groups, so as to identify important pathways
from the overall gene expression matrix. As shown in Fig. [96]5E, there
was a dense distribution of core genes in the gene set of the JAK/STAT
pathway of interest, displaying a consistently high expression trend.
Thus, we identified a gene set with consistently different expression
patterns in this pathway, providing a research direction for
investigating the mechanism of TSN's effect on OSCC cells.
The DEGs identified through RNA sequencing are shown in Fig. [97]5F as
a heat map. We performed qPCR validation of these genes in CAL27 cells
after DMSO and TSN treatment, as shown in Fig. [98]5G. In the TSN
group, the expression levels of JUN, PCSK9, INHBA, NR4 A1, CDKN1 A, and
PLAGL genes were significantly higher than those in the DMSO group. The
qPCR results were consistent with the RNA-sequencing results,
indicating the high reliability of the RNA-sequencing analysis.
Moreover, JUN and PCSK9 genes are key genes that promote cell
apoptosis, while INHBA, NR4 A1, CDKN1 A, and PLAGL1 genes are closely
associated with cell cycle arrest. These experimental results further
suggest that TSN may inhibit tumor cell growth by upregulating
apoptosis-related genes and interfering with cell cycle regulation.
TSN and TSN-PLGA NPs regress tumor growth of OSCC in the CDX model
The aforementioned in vitro experimental results indicate that both TSN
and TSN-PLGA NPs possess the capability to hinder the proliferation and
promote apoptosis of OSCC cells. Hence, to further investigate the
effectiveness and safety of TSN and TSN-PLGA NPs in vivo, the CDX tumor
model was established.
In the CDX model, there were no deaths, infections, or signs of
toxicity during the treatment period in the different groups of nude
mice. There were no obvious changes in the body weight of the nude mice
among the three treatment groups compared with the NS group
(Fig. [99]6B). As shown in Fig. [100]6C, both the TSN group and
TSN-PLGA NPs group exhibited superior tumor-suppression effects
relative to the 5-FU group (p < 0.001), but the TSN-PLGA NPs group
demonstrated a more pronounced tumor-inhibitory effect than the TSN
group (p < 0.05).
Fig. 6.
[101]Fig. 6
[102]Open in a new tab
TSN and TSN-PLGA NPs regress tumor growth of OSCC in the CDX model. A
Comparison of tumor tissue in nude mice (n = 6). B Body weight change
curves of nude mice. C Tumor volume growth curves of nude mice. D
Comparison of tumor weight in nude mice. E–F HE staining and effect of
drug on the expression of Ki-67 in tumor of nude mice (scale bar = 100
μm). G– H TUNEL staining of tumor tissues in nude mice (scale bar = 100
μm). * p < 0.05, ** p < 0.01, *** p < 0.001
After the completion of the experimental period, the tumors were
extracted and weighed (Fig. [103]6A and D). Then, TGI was calculated,
and the percentage of TGI was 41.17%, 58.82%, and 64.70% for the 5-FU,
TSN, and TSN-PLGA groups, respectively. These results were consistent
with the tumor volume findings, collectively indicating that TSN and
TSN-PLGA NPs have a greater ability to inhibit tumor growth than 5-FU.
Next, we performed HE staining and Ki-67 immunohistochemical staining
of the tumor tissues from the CDX model (Fig. [104]6E). In the NS
group, most tumor cells exhibited nuclear pleomorphism, indicating a
significant degree of dedifferentiation. The NS group had the highest
percentage of Ki-67–positive cells, suggesting that the tumor cells
were in a rapidly proliferating stage. As shown in Fig. [105]6F, the
TSN-PLGA NPs group exhibited a lower proportion of Ki-67–positive cells
than did the other groups. Our results suggest that both TSN and
TSN-PLGA NPs exhibit antitumor properties, but TSN-PLGA NPs demonstrate
superior efficacy.
TUNEL expression level was examined to determine the degree of
apoptosis in OSCC cells. As shown in Fig. [106]6G, the TSN-PLGA NPs
group displayed extensive areas with intense red fluorescence signals,
possibly reflecting the presence of clustered apoptotic cells. As shown
in Fig. [107]6H, the TSN-PLGA NPs group showed a higher proportion of
cells undergoing apoptosis than did the other groups. These results
imply that both TSN and TSN-PLGA NPs possess the capacity to induce
apoptosis in tumor cells to a certain extent.
There were no significant changes in cardiac myocytes, liver sinusoids,
renal glomeruli, intestinal glands, and other morphological structures
(Figure S2). These findings demonstrated that TSN and TSN-PLGA NPs at a
dose of 5 mg/kg did not exhibit significant toxic side effects on the
vital organs of the mice, demonstrating potential biological safety.
Further investigation of the inhibitory effects of TSN and TSN-PLGA NPs on
OSCC in the PDX model
Based on the aforementioned in vivo studies via the CDX model, we
further constructed the PDX model—which better simulates the tumor
microenvironment—to further validate the therapeutic effects of the two
drugs on OSCC. The original tumor tissue was obtained from a
58-year-old man diagnosed with high-grade squamous cell carcinoma of
the tongue (T[3]N[0]M[0]). The fresh surgical specimen of the primary
lesion was trimmed into 2–3 mm^3 tissue fragments and implanted into
the first-generation nude mice (F1). The tumor formation time in the F1
mice was 45 days. Subsequently, the tumor tissue from F1 was
transplanted into other nude mice (F2), and the tumor formation time in
the F2 mice was 30 days. Finally, the tumor tissue from F2 was
transplanted into the third-generation nude mice (F3), and the tumor
formation time in the F3 mice was approximately 28 days (Fig. [108]7A).
We performed HE staining and immunohistochemical (IHC) staining for
CK56, Ki-67, P16, and P53 on both the patient's primary tumor tissue
and the tumor tissue from the F3 nude mice to confirm the homogeneity
of the xenograft model (Figure S3). The results showed a high degree of
consistency between the constructed PDX model and the primary tumor
derived from the patient.
Fig. 7.
[109]Fig. 7
[110]Open in a new tab
TSN and TSN-PLGA NPs regress tumor growth of OSCC in the PDX model. A
The PDX model construction method. B Comparison of tumor tissue in nude
mice (n = 4). C Body weight change curves of nude mice n = 4. D Tumor
volume growth curves of nude mice (n = 4). E Comparison of tumor weight
in nude mice (n = 4). F– H HE staining and effect of drug on the
expression of Ki-67 and p-STAT3 in tumors of nude mice (n = 4, scale
bar = 100 μm). I–J TUNEL staining and expression of tumor tissues in
nude mice (n = 4,scale bar = 100 μm). * p < 0.05, ** p < 0.01, *** p <
0.001
Similarly, there were no notable changes in body weight among the
treatment groups during the entire dosing period relative to the NS
group (Fig. [111]7C). After a 2-week period of intraperitoneal
injections, the tumor tissues were dissected (Fig. [112]7B). According
to the tumor volume growth curves (Fig. [113]7D), all treatment groups
exhibited inhibitory effects on OSCC growth, but TSN-PLGA NPs showed
the strongest effect. The percentage of TGI in the 5-FU, TSN, and
TSN-PLGA groups was 30.48%, 50.34%, and 66.10%, respectively.
Consistent conclusions were drawn regarding tumor weight
(Fig. [114]7E). The results of IHC staining (Fig. [115]7F) showed that
the TSN-PLGA NPs group exhibited lower Ki-67 expression than did the
other groups (p < 0.05) (Fig. [116]7G). Aberrant activation of STAT3,
indicated by increased levels of p-STAT3, is often observed in OSCC
tissues. As shown in Fig. [117]7H, there was no difference between the
NS group and the 5-FU group in p-STAT3 expression. However, the TSN
group and the TSN-PLGA NPs group exhibited notably reduced levels of
p-STAT3 expression in comparison to the 5-FU group (p < 0.01). Our
results revealed that TSN and TSN-PLGA NPs have the potential to
greatly decrease the levels of p-STAT3 in OSCC tissues. Furthermore, as
shown in the TUNEL cell apoptosis assay (Fig. [118]7I), both TSN and
TSN-PLGA NPs have the ability to promote tumor cell apoptosis.
Additionally, TSN-PLGA NPs may have a stronger effect in promoting OSCC
apoptosis than TSN alone (Fig. [119]7J). HE staining of vital organs in
all of the groups of nude mice showed no significant organ changes,
suggesting that the drugs administered at a dosage of 5 mg/kg exhibited
a considerable degree of safety (Figure S4).
Discussion
OSCC has become a significant public health issue due to its aggressive
nature and high metastatic potential. It is associated with negative
effects on patients'survival time and quality of life. Patients
diagnosed with advanced OSCC have a survival rate below 30% [[120]36].
Therefore, it is crucial to explore novel and effective treatment
strategies to complement current therapies for OSCC. The utilization of
natural bioactive compounds found in herbs is a promising option for
improving treatment efficiency and reducing adverse effects. As a
promising anticancer agent, herb-extracted TSN has been demonstrated to
exhibit antiproliferative activity against various tumor cell lines in
vitro [[121]37–[122]39]. However, TSN itself has low water-solubility
and bioavailability, and exhibits certain hepatotoxicity [[123]40]. In
previous studies, numerous drugs derived from natural herbs, such as
atropine, scopolamine, paclitaxel, and camptothecin, have been used.
Nevertheless, although these drugs initially displayed some side
effects, they have found extensive use in clinical treatment following
chemo- or nano-modification, which served as a source of inspiration
for us [[124]41]. Hence, considering the potent anticancer capabilities
of TSN and aiming to address these limitations, we employed PLGA
encapsulation (TSN-PLGA NPs) to improve the water-solubility and
efficacy of the TSN.
It has been shown that nanomedicines with particle sizes ranging from
100 to 200 nm exhibit optimal enhanced permeability and retention (EPR)
effect [[125]42]. The EPR effect can effectively utilize the
physiological characteristics and structural differences of tumor
tissues, allowing large molecular substances to be efficiently targeted
and distributed in tumor tissues, thereby improving the treatment
efficacy and reducing adverse reactions [[126]43]. TSN-PLGA NPs had a
particle size of 135.48 ± 0.37 nm, which could enable drug accumulation
in tumor tissues via EPR effect. The characterization of TSN-PLGA NPs
demonstrated that the nanocarrier particles prepared in our study had
appropriate particle size, uniform dispersion, system stability, and
sustained release behavior.
Our study showed that both TSN and TSN-PLGA NPs exhibit anticancer
effects by inhibiting the survival and proliferation of OSCC cells.
Additionally, they both induce apoptosis and cause S-phase cell cycle
arrest. These results are in accordance with a previous study, which
showed that TSN dose-dependently induced colorectal cancer cell cycle
arrest in S phase [[127]15]. TSN and TSN-PLGA NPs exhibit selective
cytotoxicity against OSCC, as they can inhibit the growth of OSCC cells
at lower concentrations without affecting HOEC viability.
In addition, we found that TSN and TSN-PLGA NPs had superior antitumor
effects than 5-FU after absorption and metabolism in both the CDX and
PDX models. The results were consistent with the in vitro experiments,
as no significant adverse side effects were found in the mice after
treatment. Histological examination of the liver tissue revealed no
apparent pathological changes in the nude mice. According to
experimental work in mice by Yang et al. [[128]37], administering 10
mg/kg TSN by intraperitoneal injection can cause severe liver damage,
with elevated Alanine Aminotransferase/Aspartate Transaminase (ALT/AST)
activity and typical histopathological changes in the liver. This
provides a reference basis for our animal experiments. In our study,
the dosage of intraperitoneally injected TSN (5 mg/kg) was relatively
safe. Furthermore, the modification of TSN with PLGA resulted in
TSN-PLGA NPs that possessed passive tumor-targeting capabilities and
exhibited enhanced biosafety.
From the analysis of RNA-sequencing data, we may infer that TSN hinders
the progression of OSCC and promotes apoptosis by activating signaling
pathways such as JAK/STAT and PI3 K–Akt. The JAK/STAT and PI3 K–Akt
signaling pathways play a crucial role in tumor formation, engaging in
diverse biological activities such as cellular proliferation,
metabolism, survival, and apoptosis [[129]44, [130]45]. The STAT family
is a group of signal transducers and transcription activators that can
bind to DNA. Phosphorylation of STAT3 can lead to significant
activation of the JAK/STAT3 pathway, thereby promoting cell
proliferation. It has been reported that STAT3 is highly expressed in
tumor tissues of OSCC and has a significant effect on invasion
[[131]46]. The study by Yang et al. demonstrated that TSN inhibited
liver cancer metastasis by activating the tumor suppressor gene WWOX
and suppressing the JAK/STAT3 signaling pathway [[132]47]. Furthermore,
Zhang et al. [[133]48] demonstrated that TSN directly bound to the SH2
domain of STAT3, inhibiting the phosphorylation of STAT3.
The activation of the PI3 K/Akt pathway can increase the proliferation
of tumor cells by regulating cell cycle regulatory factors and
promoting protein synthesis. Additionally, Akt activation enables tumor
cells to evade apoptosis [[134]49]. The study conducted by Zhang et al.
[[135]50] observed that TSN effectively suppressed the phosphorylation
of PI3 K, Akt, and mTOR proteins in glioma cells. Furthermore, the
inhibitory effect of TSN on glioblastoma was reversed by PI3 K
activators. Additionally, another study has shown that TSN can reverse
the resistance of breast cancer cells to doxorubicin by inhibiting
doxorubicin-induced Akt phosphorylation [[136]16]. Therefore, TSN may
have great potential as a PI3 K and STAT3 phosphorylation inhibitor for
the treatment of OSCC.
Conclusions
In this study, we prepared TSN-PLGA NPs that met the requirements of
EPR effect, with appropriate particle size and ZP. The modified
TSN-PLGA NPs exhibited significantly enhanced effects on OSCC cells
compared with free TSN. The findings from the experiments conducted in
vitro and in vivo demonstrated that both TSN and TSN-PLGA NPs
effectively inhibited the proliferation of OSCC cells, promoted
apoptosis, and induced cell cycle arrest in S phase. RNA sequencing
analysis showed that TSN might regulate OSCC cells through the JAK/STAT
and PI3 K/Akt signaling pathways. TSN and TSN-PLGA NPs significantly
inhibited the growth of OSCC at a dose of 5 mg/kg, showing no apparent
toxicity in nude mice and demonstrating potential biocompatibility. In
conclusion, TSN delivered and encapsulated by PLGA might serve as an
effective anticancer agent for OSCC. Nevertheless, this study does have
certain limitations, and further research is warranted to delve into
the mechanisms of TSN in OSCC treatment.
Supplementary Information
[137]12906_2025_4957_MOESM1_ESM.docx^ (4.2MB, docx)
Supplementary Material 1: Table S1. Primer sequences for JUN, PCSK9,
INHBA, NR4 A1, CDKN1 A, PLAGL1; Figure S1. Cell viability after 48
hours of drug treatment of Human normal oral epithelial cells (HOEC);
Figure S2. HE staining results of nude mouse heart, liver, kidney,
spleen, lung and jejunum tissue sections in CDX model (scale
bar=100μm); Figure S3. Immunohistochemical (IHC) and HE results of the
primary tumor and the PDX tumor(scale bar=100μm); Figure S4. HE
staining results of nude mouse heart, liver, kidney, spleen, lung and
jejunum tissue sections in PDX model(scale bar=100μm); Figure S5.
Figure S5. Standard curve of TSN; Figure S6. The HPLC Spectrum of TSN.
Acknowledgements