Dual-Ligand Mitochondria-Targeted Nanosystem Achieves 85.23% Tumor Suppression in Hepatocellular Carcinoma
核心洞察
Researchers developed IR820 (搜索)/EVO@MPDA-TPP/cRGD (搜索), a mitochondria-targeted nanosystem co-delivering evodiamine and IR820 for synergistic chemo-photothermal therapy of hepatocellular carcinoma (搜索).
The dual-ligand design (cRGD (搜索) for tumor homing, TPP for mitochondrial targeting) achieved a 1.47-fold increase in cellular uptake and the highest photothermal conversion efficiency of 42.06%.
Combined laser treatment produced a tumor suppression rate of 85.23% in a HepG2 xenograft model, with near-complete mitochondrial membrane potential collapse (92.5% reduction).
A research team has engineered a mitochondria-targeted, dual-ligand nanosystem that combines chemotherapy and photothermal therapy to achieve potent tumor suppression in hepatocellular carcinoma (搜索) (HCC). The platform, designated IR820 (搜索)/EVO@MPDA-TPP/cRGD (搜索), co-delivers the natural alkaloid evodiamine (EVO) and the near-infrared dye IR820 within mesoporous polydopamine (搜索) (MPDA) nanoparticles, functionalized with cRGD peptides for tumor homing and triphenylphosphonium (搜索) (TPP) for mitochondrial localization.
Hepatocellular carcinoma (搜索) represents a major global health burden with particularly high prevalence in China and other regions of Asia. The disease is characterized by insidious onset, aggressive progression, frequent metastasis, and high recurrence rates. The absence of distinct early-stage symptoms often results in delayed diagnosis, with most patients presenting at advanced stages where curative surgical intervention is no longer feasible. Conventional treatment modalities, including radiotherapy and chemotherapy, provide limited efficacy, while responses to emerging options such as targeted agents and immunotherapy are often variable and inconsistent.
A "Membrane-to-Mitochondria Sequential Relay Targeting" Design
The novelty of this platform lies in the strategic exploitation of the spatial synergy between cRGD (搜索) and TPP to engineer what the authors term a "Membrane-to-Mitochondria Sequential Relay Targeting" modality. Surface conjugation of cRGD facilitates evasion of the first-pass effect to achieve robust intratumoral retention within HepG2 xenografts, while the subsequent exposure of TPP moieties directs the nanoparticles to bypass lysosomal sequestration and anchor precisely at the mitochondrial outer membrane, a site exquisitely sensitive to thermal stress.
This design constitutes more than a physical admixture of established ligands. It leverages the temporally programmed exposure of cryptic TPP cationic sites, thereby mitigating the non-specific scavenging by the hepatic reticuloendothelial system that frequently compromises the efficacy of conventional TPP-only modifications.
Physicochemical Characterization and Photothermal Performance
The MPDA nanoparticles exhibited a well-defined spherical morphology with a clear mesoporous structure, an average hydrodynamic diameter of 209.52 ± 3.9 nm, and a zeta potential of −19.34 ± 0.85 mV. BET analysis confirmed mesoporosity with a pore size of 18.89 nm and a surface area of 5.58 m²/g. Following drug loading and dual-ligand functionalization, the final EVO-3 formulation showed a diameter of 218.53 ± 3.39 nm and a zeta potential of −21.63 ± 1.5 mV.
IR820 (搜索) and EVO were efficiently encapsulated into the mesoporous channels of MPDA via hydrophobic interactions, achieving loading capacities of 5.0% and 4.6%, respectively. Upon 808 nm laser irradiation at 1 W/cm², the photothermal conversion efficiency (η) of EVO-3 reached 42.06%, the highest among all formulations tested, indicating that co-loading with IR820 and dual-ligand functionalization synergistically enhance light-to-heat conversion.
Enhanced Cellular Uptake and Mitochondrial Targeting
Confocal microscopy revealed minimal cellular uptake of both free IR820 (搜索) and untargeted IR820/EVO@MPDA (EVO-1). The cRGD (搜索)-modified formulation (EVO-2) primarily accumulated at the cell membrane with limited internalization. In contrast, HepG2 cells extensively internalized the dual-ligand EVO-3 nanoparticles, which exhibited prominent perimitochondrial distribution. Quantitative HPLC analysis confirmed that EVO-3 uptake was 1.47-fold higher than that of EVO-1 after 24 hours of incubation.
The half-maximal inhibitory concentration (IC50) values for HepG2 cells were 29.65 μg/mL for free EVO, 28.87 μg/mL for EVO-1, 23.31 μg/mL for EVO-2, and 17.75 μg/mL for EVO-3, demonstrating progressively enhanced cytotoxicity with each functionalization step.
Mitochondrial Dysfunction and Cell Death Mechanisms
The dual-ligand nanosystem induced profound mitochondrial dysfunction. EVO-3 elicited a substantial increase in reactive oxygen species (ROS) even in the absence of laser treatment, with the highest ROS levels achieved in the EVO-3 + Laser group. Mitochondrial membrane potential (ΔΨm) analysis revealed that EVO-3 induced an approximately 9-fold greater reduction in ΔΨm compared to EVO-2. Laser irradiation further intensified this effect, with EVO-3 + Laser resulting in near-complete membrane potential collapse, a reduction of 92.5%.
Transmission electron microscopy confirmed progressive structural damage, with the EVO-3 + Laser group displaying the most extensive injury, including complete loss of cristae architecture. Western blot analysis demonstrated upregulation of pro-apoptotic proteins (Bax, Cytochrome C, and Cleaved Caspase-3) and downregulation of the anti-apoptotic protein Bcl-2, alongside activation of the PINK1/Parkin-mediated mitophagy pathway as evidenced by substantial upregulation of LC3-II and PINK1.
In Vivo Antitumor Efficacy
In a HepG2 cell-derived xenograft (HCDX) model, the EVO-3 + Laser group demonstrated the most potent antitumor effect, achieving a tumor suppression rate (TSR) of 85.23%. Free EVO exhibited moderate antitumor effects with a tumor growth inhibition rate of 40.07%. The average tumor weight in the EVO-3 + Laser group was significantly lower than that in the free EVO and non-irradiated EVO-3 groups, highlighting the strong synergistic effect between photothermal therapy and chemotherapy.
Fluorescence imaging confirmed that EVO-3 achieved efficient tumor-specific enrichment, with fluorescence intensity at the tumor site peaking at 24 hours post-injection. Immunohistochemical analysis showed a significant reduction in Ki67-positive cells and markedly elevated expression of UCP2, a marker of mitochondrial dysfunction, in the EVO-3 + Laser group.
Biosafety Profile
Regarding biosafety, no statistically significant differences in body weight were detected across groups during the treatment period. Serum biochemistry showed no significant elevation in key biomarkers, including ALT, AST, BUN, and Cr, across any of the nanoparticle-treated groups. Importantly, mice treated with free EVO exhibited a marked increase in ALT levels, implying potential drug-induced hepatotoxicity at the equivalent dose. In contrast, all MPDA-based nanoformulations effectively reduced this adverse effect and demonstrated improved biocompatibility.
Limitations and Future Directions
The authors acknowledge several limitations. In vivo antitumor evaluation was conducted solely in a subcutaneous HepG2 xenograft model, which does not fully recapitulate the complex tumor microenvironment, stromal interactions, and immune landscape of clinically relevant orthotopic or metastatic HCC. Long-term toxicity assessments, including chronic organ damage, immunogenicity, and reproductive toxicity, were not performed. Future studies will focus on validating antitumor efficacy in orthotopic and patient-derived xenograft (PDX) models, incorporating immune-competent animal studies, and conducting systematic long-term toxicological evaluations to rigorously assess translational potential.