Co-delivery of Sorafenib and Anti-miR-21 via a CD44-guided Nanocarrier for Dual-pathway Inhibition in HCC Cells

Abstract

Background/Aim

The clinical efficacy of sorafenib against hepatocellular carcinoma (HCC) is severely limited by limited efficacy and off-target toxicity. Overexpression of oncogenic miR-21 has been identified as a key driver of these limitations, as it downregulates the tumor suppressors PTEN and PDCD4, thereby aberrantly activating both the PI3K/Akt and STAT3 signaling pathways. To address the limitations of conventional sorafenib therapy, we aimed to construct a smart nanocarrier capable of simultaneously delivering sorafenib (SF) and anti-miR-21 specifically to HCC cells.

Materials and Methods

We designed a core-shell nanoparticle consisting of a PAMAM dendrimer/anti-miR-21 polyplex core and a hyaluronic acid-sorafenib conjugate shell. This architecture enables CD44-mediated active targeting and pH-responsive drug release in the acidic tumor microenvironment. The antitumor activity of the co-delivery system was evaluated in vitro using HepG2 cells, with assessments of cell proliferation, migration, apoptosis, and the underlying signaling pathways.

Results

The prepared nanoparticle exhibited potent anti-proliferative, anti-migratory, and pro-apoptotic effects in HepG2 cells. Mechanistic studies revealed that these benefits were associated with the reduced phosphorylation of key proteins in both the PI3K/Akt/mTOR and STAT3 cascades, effectively counteracting the therapy limitations that are normally fueled by miR-21 overexpression.

Conclusion

Our findings demonstrate that CD44-guided co-delivery of sorafenib and anti-miR-21 provides a promising nanotherapeutic strategy to counteract therapy limitations through coordinated blockade of two major oncogenic pathways in HCC cells.

Introduction

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality globally and is often diagnosed at advanced stages with limited effective treatments (1, 2). Sorafenib (SF), a conventional first-line multi-kinase inhibitor, is widely used as a first-line therapy for advanced HCC (3). However, its efficacy is frequently hampered by systemic toxicity, unfavorable pharmacokinetics, and drug therapy resistance (4). While immune checkpoint inhibitor-based combinations (like atezolizumab & bevacizumab and durvalumab & tremelimumab) are now preferred first-line options in current guidelines, sorafenib remains an important alternative when immunotherapy is contraindicated. There is a pressing need for innovative therapeutic strategies that can improve outcomes while reducing side effects.

Growing evidence highlights the role of microRNA (miRNA) dysregulation in HCC progression. In particular, miR-21 is significantly overexpressed in HCC tissues and is associated with poor prognosis and promotes tumorigenesis by suppressing key tumor-suppressor genes, including PTEN and PDCD4 (5-7). PTEN downregulation leads to constitutive activation of the PI3K/Akt/mTOR pathway, which supports cell survival and proliferation (8, 9). Similarly, PDCD4 inhibition enhances JAK/STAT3 signaling, contributing to inflammation, cell survival, and epithelial-mesenchymal transition (10). Thus, miR-21 simultaneously activates two major pathways –PI3K/Akt/mTOR and STAT3– both of which are implicated in poor therapeutic outcomes (11).

Dual inhibition of these pathways represents a promising strategy to overcome compensatory signaling and improve treatment response. However, delivering both a small-molecule drug and a nucleic-acid-based agent in a coordinated manner remains technically challenging (12). Nanotechnology-based co-delivery platforms offer a potential solution by enabling tumor-targeted delivery of multiple therapeutics within a single carrier, thereby enhancing efficacy and reducing off-target exposure (13, 14).

In this study, we developed a CD44-targeted core-shell nanoparticle for the combined delivery of sorafenib and anti-miR-21 (15, 16). The system consists of a PAMAM dendrimer core that complexes anti-miR-21 and is coated with a shell of hyaluronic acid (HA) conjugated to sorafenib via a pH-sensitive bond (17, 18). HA not only facilitates active targeting of CD44-overexpressing HCC cells but also shields the cationic core, improving biocompatibility and circulation time (19). Under acidic conditions, such as in the tumor microenvironment or within cellular endosomes, the bond is cleaved, resulting in the synchronized release of both therapeutic agents.

We hypothesized that this co-delivery system would exert combinational anti-tumor effects through dual suppression of the PI3K/Akt/mTOR and STAT3 pathways: sorafenib directly inhibits kinase activity, while anti-miR-21 restores PTEN and PDCD4 expression, thereby amplifying pathway inhibition. Here, we characterize the nanoparticle’s physicochemical properties, drug-release profile, and cellular uptake. We further demonstrate its superior ability to inhibit HCC cell proliferation, migration, and colony formation compared to single treatments or free drug combinations. Mechanistic studies confirm that the enhanced efficacy correlates with coordinated downregulation of p-Akt and p-STAT3 and induction of apoptosis. Together, these findings present a rational nanotherapeutic approach that integrates active targeting, microenvironment responsiveness, and multi-pathway inhibition for more effective HCC treatment.

Materials and Methods

Preparation of NPs. The HA-SF conjugate was synthesized via carbodiimide chemistry. Briefly, HA (924474, Sigma-Aldrich, St. Louis, MO, USA) (MW 15 kDa, 100 mg) was activated with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) (1.2 molar equivalents) and N-hydroxysuccinimide (NHS) (1.0 molar equivalent) in N,N-dimethylformamide (DMF) (10 ml) at room temperature (25 °C) for 3 h (pH 6.8), then reacted with sorafenib (HY-10201, MedChemExpress, Monmouth Junction, NJ, USA) (0.2 molar equivalents) in the presence of triethylamine. The product was purified by dialysis (MWCO 3.5 kDa) against deionized water for 72 h (water changed every 8 h) and lyophilization (freeze-drying at -50 °C under vacuum for 48 h) to obtain the HA-SF conjugate as a white powder. The degree of substitution was calculated to be approximately 8.5% (mol SF per mol HA disaccharide unit), corresponding to a drug loading of 9.2% (w/w), based on the feed molar ratio and confirmed by UV-Vis quantification of unreacted sorafenib in the dialysis supernatant. HA-SF/anti-miR-21 NPs were prepared by electrostatic self-assembly. First, a PAMAM (MW ~28 kDa) /anti-miR-21 (20 μM, Genepharma, Shanghai, PR China) polyplex core was formed at an N/P ratio of 6 (20, 21). Anti-miR-21 was purchased from Genepharma. This core was then coated by adding an HA-SF conjugate solution (2 mg/ml) at a volume ratio of 1:1 (HA-SF:polyplex) under vortexing, followed by incubation for another 30 min at room temperature to form the final HA-SF/anti-miR-21 NPs (final concentration 1 mg/ml). HA-anti-miR-21 NPs were prepared similarly using unmodified HA.

Physicochemical characterization. The hydrodynamic diameter, polydispersity index (PDI), and zeta potential were measured by dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern Panalytical, Malvern, UK) at a scattering angle of 173° with samples diluted in deionized water (1:10, v/v), equilibrated at 25 °C for 2 min prior to measurement; each measurement consisted of 3 technical replicates, and data were collected from three independently prepared batches, with results reported as intensity-weighted mean values. Morphology was observed by transmission electron microscope (TEM) (HT7800, Hitachi High-Tech, Tokyo, Japan) operating at an accelerating voltage of 80 kV, with samples negatively stained using 2% uranyl acetate; particle size distributions were quantified from TEM images by measuring over 200 randomly selected particles per sample using ImageJ. Complexation was analyzed by agarose gel electrophoresis. PAMAM/anti-miR-21 complexes were prepared and then run on a 2% agarose gel and visualized under ultraviolet (UV) light.

In vitro release. The pH-dependent release of SF and anti-miR-21 was investigated using a dialysis method. HA-SF/anti-miR-21 NPs were placed into a dialysis bag (MWCO 3,500Da) dialyzed against 30 mL of PBS (pH 7.4) and acetate buffer (pH 5.5) at 37 °C with gentle shaking (100 rpm). At predetermined intervals (0.5, 1, 2, 4, 8, 12, 24, 36, and 48 h), 1 ml of external medium were withdrawn and replaced with an equal volume of fresh medium. Released SF was detected by high performance liquid chromatography (HPLC) (LC-20A, Shimadzu, Kyoto, Japan), and anti-miR-21 was detected by a fluorescence spectrophotometer (F-7000, Hitachi High-Tech, Tokyo, Japan). The cumulative release was calculated as CR (%) = Cₜ × V / M_total × 100%, where Cₜ is the concentration of the receiver medium at the sampling time.

In vitro stability assessment. The stability of HA-SF/anti-miR-21 NPs was evaluated in PBS (pH 7.4) or PBS with 10% FBS. Briefly, HA-SF/anti-miR-21 NPs were kept in PBS (pH 7.4) or PBS with 10% FBS at 37°C (0.2 mg/ml). Samples were collected at predetermined time points (0, 2, 6, 12, 24, 48, and 72 h). The diameter and PDI of the NPs were immediately measured by DLS.

Cellular uptake and targeting assay. Human hepatocellular carcinoma HepG2 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. HepG2 cells were seeded into a Nunc™ glass-bottom Petri dish (150680, Thermo Fisher Scientific, Waltham, MA, USA). For the CD44 blocking experiment, cells were pre-incubated with 10 μg/ml CD44 antibody (397502, BioLegend, San Diego, CA, USA) for 1 h at 37°C (22). Cells were then incubated with free Cy5-anti-miR-21 or HA/Cy5-anti-miR-21 NPs (50 nM Cy5-anti-miR-21) for 4 h. Cells were washed with PBS and stained with iF488-Wheat Germ Agglutinin and DAPI. Images were captured using LSM 880 confocal microscope (Carl Zeiss Microscopy, Jena, Germany).

Lysosomal escape assay. HepG2 cells were incubated with FITC-labeled HA-SF/anti-miR-21 NPs for 2 h or 6 h. During the last 30 min, LysoTracker Red (75 nM) (C1046, Beyotime Biotechnology, Shanghai, PR China) was added to the dishes. Cells were collected and washed twice. Cell nuclei were labeled with DAPI. Cell imaging was performed using a confocal microscope.

In vitro cytotoxicity assay. For the CCK-8 assay, HepG2 cells were seeded in 96-well plates. After 24 h, the medium was replaced with fresh medium containing PBS, free SF, HA-anti-miR-21 NPs, or HA-SF/anti-miR-21 NPs (SF, 5 μM; anti-miR-21, 50 nM). After incubation for 24, 48, and 72 h, 10 μl of CCK-8 (C0038, Beyotime Biotechnology) was added to each well. After 1 h of incubation, the absorbance at 450 nm was measured using a Synergy H1 microplate reader (BioTek Instruments, Winooski, VT, USA). Cell viability was calculated. Then the cytotoxicity of the blank HA-PAMAM nanoparticles was assessed to evaluate carrier safety. HepG2 cells were seeded in 96-well plates. After 24 h, the culture medium was replaced with fresh medium containing PBS or three concentrations of blank NPs (low, medium, and high concentrations). After incubation for 24 or 48 h, cell viability was quantified using the CCK-8 assay as before. LO2 cells were treated with formulations for 48 h and viability was assessed by CCK-8 assay, as described for HepG2 cells.

Cell apoptosis by flow cytometry. HepG2 cells were seeded in 6-well plates and treated with PBS, free SF, HA-anti-miR-21 NPs, or HA-SF/anti-miR-21 NPs (SF, 5 μM; anti-miR-21, 50 nM) for 48 h. Cells were then collected, washed with PBS, and resuspended in 1× Annexin V binding buffer. Subsequently, cells were stained with Annexin V-FITC and PI using the Annexin V-FITC Apoptosis Detection Kit (C1062L, Beyotime Biotechnology). After 15 min of incubation in the dark at room temperature (RT), samples were immediately analyzed by a flow cytometer (Fortessa, BD Biosciences, San Jose, USA). Data were analyzed with FlowJo v10.8.1 (FlowJo LLC, Ashland, OR, USA).

Wound healing assay. HepG2 cells were seeded in 24-well plates and grown to 90% confluence. A uniform scratch wound was created across the cell monolayer using a sterile 200 μl pipette tip. The cells were washed with PBS, and fresh medium containing the respective treatments (SF 2.5 μM) was added. Images were captured at 0 h and 24 h using an inverted microscope. The wound area was measured using ImageJ software, and the percentage of wound closure was calculated.

Colony formation assay. HepG2 cells were seeded in 6-well plates with 500 cells/well and allowed to attach overnight. Cells were then treated with the indicated formulations at low concentrations (SF 1 μM) for 48 h. Afterward, the medium was replaced with fresh complete medium, and cells were cultured for an additional 14 days, with medium changes every 3 days. Subsequently, colonies were fixed with methanol, stained with 0.1% crystal violet (C0121-100ml, Beyotime Biotechnology), and manually counted. Only colonies containing more than 50 cells were counted. The plating efficiency and surviving fraction were calculated.

Quantitative real-time PCR (qRT-PCR). Total RNA was extracted from treated HepG2 cells using TRIzol reagent (T3809, Sigma-Aldrich, St. Louis, MO, USA). For miR-21 analysis, cDNA was synthesized using a miRNA-specific stem-loop primer and reverse transcriptase. For mRNA analysis of PTEN and PDCD4, cDNA was synthesized using random primers (23-25). qPCR was performed on a QuantStudio 3 system using SYBR Green PCR Master Mix. The expression of miR-21 was normalized to U6 small nuclear RNA, and mRNA levels were normalized to GAPDH (26). The 2^ΔΔCt method was used to calculate relative expression levels. Statistical analysis was performed on ΔCt values using one-way ANOVA with Tukey’s post hoc test. Primer sequences are listed in Supplementary Table I.

Western blot analysis. After treatment, cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (Epizyme Biotech, Shanghai, PR China). Protein concentrations were determined using a BCA assay (P0009, Beyotime Biotechnology). Equal amounts of 20 μg of protein were separated by SDS-PAGE and transferred onto PVDF membranes (Epizyme Biotech). Membranes were blocked and then incubated overnight at 4°C with primary antibodies against p-Akt (Ser473, 1:1,000) (9271, Cell Signaling Technology, Danvers, MA, USA), p-STAT3 (Tyr705, 1:1,000) (9145, Cell Signaling Technology), Cleaved Caspase-3 (1:1,000) (9661, Cell Signaling Technology), Cleaved PARP (1:1,000) (9541, Cell Signaling Technology), and GAPDH (1:5,000) (2118, Cell Signaling Technology). Next, membranes were incubated with HRP-conjugated secondary antibodies (7074, Cell Signaling Technology). Proteins were then visualized using an enhanced chemiluminescence (ECL) substrate and imaged with a ChemiDoc imaging system (Bio-Rad Laboratories, Hercules, CA, USA).

Statistics. All experiments were performed in at least three independent replicates. Data are presented as mean±standard deviation (SD). Statistical comparisons between two groups were conducted using a two-tailed Student’s t-test. For comparisons among multiple groups, one-way analysis of variance (ANOVA) was performed, followed by Tukey’s post hoc test. All statistical analyses were carried out using GraphPad Prism software (version 8.0, GraphPad Software, San Diego, CA, USA).

Results

Synthesis and characterization of the HA-SF/anti-miR-21 NPs. To develop a combined treatment strategy for HCC, we constructed a core-shell NP designed for the co-delivery of SF and anti-miR-21. The NPs were prepared through a two-step self-assembly process (Figure 1A). First, the cationic PAMAM dendrimer was electrostatically complexed with anionic anti-miR-21, forming a positively charged polyplex core. This core was then coated with hyaluronic acid-sorafenib (HA-SF) conjugate via charge-mediated assembly, forming HA-SF/anti-miR-21 NPs. Here, HA serves both as a targeting ligand for CD44 receptors overexpressed on HCC cells and as a pH-responsive carrier – SF is conjugated to HA through an acid-labile ester bond that cleaves under the acidic conditions typical of the tumor microenvironment. Based on the mechanistic pathway in Figure 1B, HA-SF/anti-miR-21 NPs are designed to enter HCC cells via CD44-mediated endocytosis. Once internalized, the acidic environment of endosomes/lysosomes triggers the release of both sorafenib and anti-miR-21. This co-release is intended to jointly inhibit the PI3K/Akt/mTOR and STAT3 signaling pathways, thereby suppressing tumor growth and promoting cell death.

Figure 1.

Fabrication and characterization of the HA‑SF/anti‑miR‑21 co‑delivery nanoparticles. (A) Schematic of the two‑step self‑assembly process used to prepare the core‑shell nanoparticles (NPs). (B) Proposed mechanism of action: Following CD44‑mediated endocytosis into hepatocellular carcinoma (HCC) cells, the NPs release sorafenib (SF) and anti‑miR‑21 in response to the acidic endo‑/lysosomal environment. The two agents act combinational to suppress phosphatidylinositol 3‑kinase (PI3K) /Akt/mammalian target of rapamycin (mTOR) and signal transducer and activator of transcription 3 (STAT3) signaling, thereby inhibiting proliferation and migration while promoting apoptosis. (C) Size distribution and a representative TEM image (inset) showing spherical NPs with a uniform diameter of approximately 190 nm. Scale bar: 200 nm. (D) Zeta‑potential measurements of the poly(amidoamine) (PAMAM)/anti‑miR‑21 polyplex core and the final HA‑SF/anti‑miR‑21 NPs. The reversal from positive to negative surface charge confirms successful coating with the HA‑SF shell. (E) Cumulative release profiles of sorafenib and anti‑miR‑21 from the NPs in phosphate‑buffered saline (PBS, pH 7.4) and acetate buffer (pH 5.5). Data are presented as mean ± standard deviation (SD) (n=3).

The physicochemical properties were characterized. Dynamic light scattering (DLS) revealed a uniform hydrodynamic diameter of 192.3±3.5 nm with a polydispersity index (PDI) of 0.12±0.02, indicating good monodispersity (Figure 1C). Transmission electron microscopy (TEM) further confirmed a well-defined spherical morphology. Zeta-potential measurements showed a clear surface-charge reversal from +26.10±1.6 mV for the PAMAM/anti-miR-21 core to −20.0±1.5 mV for the final NPs, confirming successful coating with the negatively charged HA-SF shell (Figure 1D). Efficient complexation of anti-miR-21 within the PAMAM core was verified, and anti-miR-21 was completely retained in the loading well, whereas free miRNA migrated freely at an N/P ratio of 6 (Supplementary Figure 1).

The pH-dependent release profile of the NPs, a key feature for tumor-targeted delivery, was evaluated under simulated physiological (pH 7.4) and acidic (pH 5.5) conditions (Figure 1E). At pH 7.4, both SF and anti-miR-21 were released slowly and incompletely over 48 h. In contrast, at pH 5.5 –mimicking the tumor microenvironment and the lysosomal environment– the release of both agents was significantly accelerated. SF, linked via the acid-labile bond, exhibited rapid release with over 80% cumulative release within 4 h. Anti-miR-21 release followed a similar pH-sensitive trend, although slightly delayed, consistent with its location in the core. This differential release profile confirms the stimulus-responsive capability of the nanoplatform.

Finally, the stability of the HA-SF/anti-miR-21 NPs was assessed in PBS and in PBS with 10% FBS over 72 h at 37 °C (Supplementary Figure 2). The NPs maintained consistent size and dispersity under both conditions, demonstrating sufficient stability for subsequent cellular experiments.

Figure 2.

CD44‑targeted cellular uptake and intracellular trafficking of HA‑SF/anti‑miR‑21 nanoparticles (NPs). (A) Confocal microscopy images showing CD44‑dependent cellular uptake in HepG2 cells. Cells were treated with free Cy5‑anti‑miR‑21, HA/Cy5‑anti‑miR‑21 NPs, or HA/Cy5‑anti‑miR‑21 NPs after pre‑blocking CD44 receptors with CD44 antibody (+CD44 Ab). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue) and the cell membrane with Alexa Fluor 488‑conjugated wheat germ agglutinin (iF488‑WGA) (green). Scale bar: 20 μm. (B) Intracellular trafficking and lysosomal escape visualized by confocal microscopy. HepG2 cells were incubated with fluorescein isothiocyanate (FITC)‑labeled NPs (green) and LysoTracker Red (lysosomal marker) for 2 h or 6 h. Yellow signal in merged images indicates colocalization. At 2 h, NPs were largely localized within lysosomes; at 6 h, green fluorescence dispersed throughout the cytoplasm, demonstrating successful lysosomal escape. Scale bar: 10 μm.

Cellular uptake, CD44-mediated targeting, and endosomal escape of HA-SF/anti-miR-21 NPs. The targeting ability of the nanoparticle system was systematically examined. To assess CD44-dependent cellular uptake, HepG2 cells (CD44-high) were incubated with free Cy5-anti-miR-21 or HA/Cy5-anti-miR-21 NPs. As shown in Figure 2A, minimal fluorescence was observed in cells treated with Cy5-anti-miR-21, confirming the poor membrane permeability of naked nucleic acids. In contrast, treatment with the NPs resulted in strong fluorescence of Cy5 that overlapped with the cytoskeletal stain (iF488-WGA), indicating efficient intracellular delivery. Notably, when cells were pre-blocked with CD44 antibody, HA/Cy5-anti-miR-21 NP uptake was dramatically reduced, confirming that internalization occurs via a CD44-mediated mechanism.

We further investigated the intracellular fate of the NPs, focusing on endosomal escape, a crucial step for cytosolic delivery of nucleic acids. HepG2 cells were incubated with FITC-labeled HA-SF/anti-miR-21 NPs and LysoTracker Red. After 2 h of incubation, strong colocalization (yellow in merged images) was observed, indicating that most NPs were trapped in acidic lysosomes (Figure 2B). However, the green NP fluorescence dispersed widely throughout the cytoplasm and separated clearly from the punctate lysosomal signal, demonstrating successful lysosomal escape at 6 h. This effect is likely attributable to the proton-sponge capacity of the PAMAM dendrimer core, which promotes endosomal rupture and facilitates the timely release of anti-miR-21 into the cytosol for gene-silencing activity.

Combined anti-proliferative and pro-apoptotic effects of HA-SF/anti-miR-21 NPs in vitro. The anticancer efficacy of the co-delivery system was first evaluated by its effect on HCC cell proliferation. Based on the results of the CCK-8 assay, both SF and HA-anti-miR-21 NP groups showed moderate, time-dependent inhibition of HepG2 cell viability over 72 h (Figure 3A). However, HA-SF/anti-miR-21 NPs exhibited significantly reduced cell viability at all time points. After 48 h, cell viability in the co-delivery group was reduced to about 30.4%, markedly lower than that of cells in the free SF group or HA-anti-miR-21 NPs alone, indicating a significant combination benefit.

Figure 3.

In vitro anti‑proliferative and pro‑apoptotic effects of HA‑SF/anti‑miR‑21 nanoparticles (NPs) in HepG2 cells. (A) Cell viability measured by Cell Counting Kit‑8 (CCK‑8) assay after 24, 48, and 72 h of treatment with phosphate‑buffered saline (PBS), free SF, HA‑anti‑miR‑21 NPs, or HA‑SF/anti‑miR‑21 NPs (SF, 5 μM; anti-miR-21, 50 nM). Results are expressed as a percentage relative to the control. (B) Quantitative analysis of total apoptosis (early + late apoptotic cells) based on Annexin V‑fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. (C) Representative flow cytometry dot plots after 48 h of treatment. Quadrants indicate viable (Annexin V⁻/PI⁻), early apoptotic (Annexin V⁺/PI⁻), late apoptotic (Annexin V⁺/PI⁺), and necrotic (Annexin V⁻/PI⁺) populations. All data are shown as mean ± standard deviation (SD) (n=3). Data are presented as mean ± standard deviation (SD). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. ***p<0.001.

To determine whether growth inhibition involved apoptosis, Annexin V-FITC/PI staining and flow cytometry analysis were performed. The quantitative analysis of total apoptotic cells is shown in Figure 3B, with representative dot plots in Figure 3C. Free SF and HA-anti-miR-21 NPs induced modest apoptosis, while HA-SF/anti-miR-21 NPs triggered a much stronger apoptotic response.

To confirm that the observed effects were due to the loaded therapeutic agents SF and anti-miR-21 and not carrier toxicity, blank HA-PAMAM NPs were tested at varying concentrations (Low, Medium, High: 100, 300, and 1,000 µg/ml) for 24 h and 48 h. As shown in Supplementary Figure 3, cell viability remained about 100% even at the highest concentration after 48 h, with no significant difference from the PBS control, demonstrating excellent biocompatibility of the nanocarrier.

The selectivity of the platform was further examined in normal LO2 hepatocytes (Supplementary Figure 4). Free SF showed considerable toxicity to LO2 cells, whereas both HA-anti-miR-21 NPs and HA-SF/anti-miR-21 NPs maintained cell viability above 85%, highlighting the targeting advantage conferred by HA-mediated CD44 recognition in cells with low receptor expression.

Figure 4.

Suppression of cell migration and clonogenic survival by HA‑SF/anti‑miR‑21 nanoparticles (NPs). (A) Representative images from a wound‑healing assay at 0 h and 24 h after treatment. (B) Representative images of crystal violet‑stained colonies following 10-14 days of low‑dose treatment. (C) Quantitative analysis of wound closure, expressed as percentage of the initial scratch area. (D) Quantitative analysis of clonogenic survival. Colonies containing >50 cells were counted and normalized to the phosphate-buffered saline (PBS) control group. Data are presented as mean±standard deviation (SD) (n=3 measurements per group). Data are presented as mean±standard deviation (SD). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. **p<0.01 and ***p<0.001.

In summary, these results confirm that the co-delivery system efficiently inhibits HCC cell proliferation and potently induces apoptosis, while showing favorable biocompatibility and selectivity toward cancer cells.

HA-SF/anti-miR-21 NPs potently inhibit HCC cell migration and long-term clonogenic survival. In addition to apoptosis induction, we examined whether the nanoplatform could inhibit metastasis-related cell migration and long-term clonogenic survival–key features of tumor aggressiveness. The anti-migratory effect was first assessed using a wound-healing assay. As shown in Figure 4A and quantified in Figure 4C, free sorafenib or HA-anti-miR-21 NPs alone moderately slowed wound closure relative to the PBS control after 24 h. In contrast, cells treated with HA-SF/anti-miR-21 NPs showed nearly complete suppression of migration, with wound areas remaining largely open, indicating a combined blockade of cell motility.

The long-term impact on tumor-initiating capacity was evaluated via a clonogenic survival assay. Representative images of crystal violet-stained colonies are presented in Figure 4B, and the quantitative analysis is shown in Figure 4D. While treatment with free SF or HA-anti-miR-21 NPs reduced colony number and size, the HA-SF/anti-miR-21 NPs exhibited the strongest inhibitory effect, almost completely preventing colony formation. The relative clonogenic survival rate in the co-delivery group of HA-SF/anti-miR-21 NPs was significantly lower than that of free SF or HA-anti-miR-21 NPs. These results suggest that the nanoplatform not only eliminates bulk tumor cells but also effectively targets the self-renewing subpopulation often associated with tumor recurrence and therapy limitations.

Combined effect of dual pathway inhibition with apoptosis induction. To elucidate the molecular mechanism underlying the observed combination, we first examined changes in gene expression. Quantitative RT-PCR confirmed effective delivery of anti-miR-21 by the nanocarrier (primer sequences are listed in Supplementary Table I). As shown in Figure 5A, both HA-anti-miR-21 NPs and HA-SF/anti-miR-21 NPs significantly decreased miR-21 levels compared with the PBS group or the free SF group. This reduction led to significant upregulation of miR-21 target tumor-suppressor genes PTEN (Figure 5B) and PDCD4 (Figure 5C), with the strongest effect observed in the HA-SF/anti-miR-21 NPs group, indicating the successful restoration of key signaling brakes.

Figure 5.

Mechanistic investigation of combined activity: dual‑pathway inhibition and apoptosis induction. (A‑C) Quantitative real‑time polymerase chain reaction (qRT‑PCR) analysis of miR‑21 and its target genes in HepG2 cells after 48 h of treatment. Relative expression of (A) miR‑21 (normalized to U6), (B) PTEN, and (C) PDCD4 [both normalized to glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH)]. Data are expressed relative to the phosphate‑buffered saline (PBS) control (set as 1) and shown as mean ± standard deviation (SD) (n=3). Data are presented as mean±standard deviation (SD). Statistical analysis was performed on ΔCt values using one‑way ANOVA with Tukey’s post hoc test. ***p<0.001; ns: not significant. Relative expression levels are shown as fold change (2⁻ΔΔCt) normalized to the respective reference gene (U6 for miR‑21; GAPDH for PTEN and PDCD4). (D) Western blot analysis of key signaling and apoptosis‑related proteins. Representative blots show phosphorylated Akt (p‑Akt), phosphorylated STAT3 (p‑STAT3), cleaved caspase‑3, cleaved poly (ADP‑ribose) polymerase (PARP), and GAPDH (loading control). Lane 4 (HA‑SF/anti‑miR‑21 NPs) shows the most pronounced reduction in p‑Akt and p‑STAT3 levels, accompanied by increased cleavage of caspase‑3 and PARP.

We then evaluated corresponding changes at the protein level by Western blotting (Figure 5D). In line with the transcriptional data, the HA-SF/anti-miR-21 NPs group showed markedly reduced p-Akt (Ser473) and p-STAT3 (Tyr705) levels (27, 28). In addition, increased cleavage of caspase-3 and PARP was observed in this group, consistent with apoptotic cell death. The expression levels in the free SF and HA-anti-miR-21 NPs groups contrasted sharply with the intense signals in the co-delivery group of HA-SF/anti-miR-21 NPs, visually underscoring the mechanistic enhanced efficiency associated with coordinated downregulation of PI3K/Akt and STAT3 signaling.

Discussion

In this study, we successfully constructed a CD44-targeted, pH-responsive core-shell nanoplatform (HA-SF/anti-miR-21 NPs) for the co-delivery of sorafenib and anti-miR-21 and demonstrated its combined therapeutic efficacy against HCC through coordinated dual-pathway inhibition. The rational design of this nanocarrier addresses several key challenges currently limiting HCC therapy, including therapeutic problems, lack of tumor specificity, and suboptimal pharmacokinetics of combination regimens. In this context, Kitaoka et al. demonstrated that epigallocatechin gallate could overcome molecular therapy limitations in HCC, supporting the notion that combination strategies represent a viable avenue to improve therapeutic outcomes (29).

The HA-based shell serves a dual function: it provides active targeting via CD44 receptor recognition, which is overexpressed on HCC cells, while simultaneously shielding the cationic PAMAM core to reduce non-specific interactions and improve biocompatibility. This targeting strategy was validated by the significantly reduced cytotoxicity observed in normal hepatocytes, which express low levels of CD44, compared to HCC cells. Importantly, the pH-responsive release profile, facilitated by the acid-labile ester bond linking sorafenib to HA, ensures that both therapeutic cargos are preferentially released within the acidic tumor microenvironment and endo/lysosomal compartments. This feature not only enhances local drug concentrations at the target site but also minimizes systemic exposure and off-target toxicity. The successful construction of the core-shell nanostructure was supported by both TEM imaging and zeta potential measurements. TEM directly revealed an electron-dense PAMAM core surrounded by a lighter polymeric shell, while the charge reversal from positive to negative confirmed the deposition of the HA-SF layer onto the cationic core. These complementary results collectively confirm the formation of the intended nanoplatform architecture.

Mechanistically, the enhanced antitumor efficacy of the co-delivery system was associated with concurrent modulation of two major oncogenic signaling cascades. The reduced p-Akt and p-STAT3 levels observed in the co-delivery group suggest modulation of these signaling cascades in this cellular model. We acknowledge that total Akt and total STAT3 protein levels were not examined in the current study; therefore, these findings should be interpreted as correlative evidence of pathway modulation rather than definitive proof of direct kinase inhibition. The concurrent restoration of PTEN and PDCD4 expression at the mRNA level, together with the observed increase in cleaved caspase-3 and PARP, supports the interpretation that the co-delivery system promotes apoptosis. This coordinated action, as evidenced by the pronounced reduction in p-Akt and p-STAT3 levels observed exclusively in the co-delivery group, is consistent with the mitigation of the compensatory activation often seen with single-agent treatments. The consequent robust activation of the caspase-3/PARP apoptotic cascade provides a mechanistic explanation for the observed superior anti-proliferative and anti-migratory effects. Recent single-cell omics studies, such as that by Tan et al., have further elucidated the heterogeneity of HCC cancer stem cells, revealing that multi-pathway targeting may be essential to eliminate therapy-resistant subpopulations (30). Similarly, Yue et al., through integrated proteomics, identified ALDH1A1 as an oncogenic driver and regulatory hub in HCC, underscoring the importance of targeting key regulatory nodes to achieve effective tumor suppression (31). Our co-delivery system, by concurrently suppressing PI3K/Akt and STAT3 pathways, may therefore offer a broader and more durable therapeutic effect.

Compared to previously reported nanocarriers for HCC therapy, our system offers several distinct advantages. Notably, unlike the use of natural products like amentoflavone as sensitizers to enhance the activity of molecular targeted agents (32), our nanocarrier achieves combined co-delivery through a targeted, programmable release system. First, the covalent conjugation of sorafenib to HA via a pH-sensitive bond ensures synchronized release with the nucleic acid cargo, unlike simple physical encapsulation approaches that often suffer from premature leakage. Second, the selection of anti-miR-21 as the gene therapeutic agent is particularly strategic, as it targets a master regulator that simultaneously controls two major survival pathways, thereby amplifying the combination benefit. Third, the use of the clinically compatible materials (HA and PAMAM) and the straightforward self-assembly fabrication process suggest good potential for scalability and clinical translation.

Study limitations. Despite these promising results, some limitations should be acknowledged. The current study is confined to in vitro evaluations, and the behavior of these nanoparticles in the complex in vivo environment –including biodistribution, tumor penetration, and potential immunogenicity– remains to be systematically investigated. In addition, the formation of a protein corona in serum-containing biological fluids, as indicated by the slight size increase observed in our stability study, may affect targeting efficiency and pharmacokinetic profiles in vivo. Furthermore, while the HA-CD44 interaction confers tumor selectivity, CD44 is also expressed on certain normal tissues, which might contribute to undesired accumulation. These aspects warrant thorough in vivo investigation in future studies. We further acknowledge that the Western blot data presented in this study are limited to phosphorylated Akt and STAT3, without corresponding total protein levels. We were also unable to examine PTEN and PDCD4 expression at the protein level. Therefore, the pathway-related conclusions are drawn from correlative evidence. Future studies incorporating total protein normalization and additional protein targets will be necessary to fully validate the proposed signaling mechanism.

In summary, the HA-SF/anti-miR-21 NPs represent a promising nanotherapeutic strategy for enhancing sorafenib efficacy in HCC through coordinated multi-pathway blockade. The integration of tumor-targeting, pH-responsiveness, and combinational gene-chemotherapy within a single nanoplatform provides a compelling approach to enhance efficacy while reducing off-target toxicity. Further validation in appropriate animal models and long-term safety assessments will be essential to advance this platform toward potential clinical application.

Conclusion

In conclusion, we developed a CD44-targeted, pH-responsive core-shell nanoplatform for the co-delivery of sorafenib and anti-miR-21, with this effect associated with concurrent downregulation of PI3K/Akt/mTOR and STAT3 pathways. This rationally designed system offers a promising strategy to improve therapeutic outcomes, with future work focused on in vivo validation to support its clinical translation.

Supplementary Material

The Supplementary Figures and Tables associated with this manuscript have been deposited in Figshare at the following DOI: 10.6084/m9.figshare.33069332

Conflicts of Interest

The Authors declare that there are no conflicts of interest regarding the research conducted, authorship, or publication of this article.

Authors’ Contributions

Lipeng Xu conceptualized and designed the study, performed the experiments and data analysis, interpreted the results, and drafted and revised the manuscript. He also contributed to funding acquisition and visualization of the data. Ling Qin participated in the methodology development, conducted the experiments, performed data curation and validation, and revised the manuscript critically for important intellectual content. Wenying Zhao supervised the project, provided overall conceptual guidance, administered the research activities, secured resources and funding, and revised the manuscript. All Authors have read and approved the final version of the manuscript. All Authors have read and approved the final version of the manuscript.

Acknowledgements

The Authors would like to thank all members of our laboratory for their helpful discussions and technical assistance during this work.

Artificial Intelligence (AI) Disclosure

During manuscript preparation, the authors used DeepSeek-V3 solely for language polishing. No AI tools were used for data analysis, figure generation, or content creation. The authors take full responsibility for the final manuscript.