Lung cancer is a highly aggressive malignancy; non-small cell lung cancer (NSCLC) constitutes the majority (85%) of cases. The advanced stage diagnosis (stage IV) in 30-40% of NSCLC cases contributes to a poor prognosis [1-3]. The standard treatment for advanced NSCLC involves combination therapy with multiple drugs, which exhibits synergistic antitumor effects and targets various tumorigenesis mechanisms. However, challenges such as poor solubility, non-specific organ toxicity, and limited efficacy necessitate the development of novel therapeutic drug delivery strategies.

Drug delivery systems have garnered significant attention as multifunctional carriers that offer advantages such as improved drug bioavailability, enhanced cellular uptake, targeted delivery, and controlled release to minimize side effects [4]. Tumor tissues exhibit unique microenvironments characterized by low pH (about 4.5-6.5) and overexpression of glutathione (GSH) levels (2-10 mM) [5,6]. The role of oxidative stress in cancer development and progression suggests that antioxidant treatments hold potential as anticancer strategies.

Mitochondria, the primary organelles in oxygen consumption, are major reactive oxygen species (ROS) sources [7]. While the precise mechanisms remain unclear, increased ROS production resulting from mitochondrial dysfunction can regulate autophagy in tumors, contributing to cancer progression [8]. Consequently, mitochondria-targeted drug delivery holds immense promise in cancer therapy, with previous research highlighting the close association between mitochondrial dysfunction and non-small cell lung cancer (NSCLC) [9].

Autophagy is a cellular degradation process involving autophagosome formation, fusion with lysosomes, and subsequent degradation [10]. Mitophagy, a selective form of autophagy, specifically removes damaged or dysfunctional mitochondria in response to metabolic stressors [11]. This process aids in alleviating oxidative stress and preventing carcinogenesis, suggesting that the induction of mitophagy can have detrimental effects on cancer cells.

In this study, our focus was on enhancing the targeted therapeutic efficacy of NSCLC by designing a mitochondria-targeted drug delivery system. We aimed to induce mitophagy by localizing the released PTX prodrugs within NSCLC cell mitochondria. To achieve this, we encapsulated the PTX prodrug in HGC nanoparticles containing disulfide bonds that could be triggered for release by intracellular glutathione (GSH), a reducing agent. The findings from this investigation contribute to the understanding of the behavior of aGC-SS-proPTX NPs and provide the essential groundwork for further studies exploring their therapeutic potential in NSCLC treatment.

2.1. Material

Paclitaxel (PTX) was purchased from LC Laboratories (USA). Acetyl chloride, Glycol chitosan (GC), 5β-Cholanic acid, 3,3’-dithiodipropionic acid (DTPA), N, N-dimethylformamide (DMF), 4-Dimethylaminopyridine (DMAP), N, N'-Dicyclohexylcarbodiimide (DCC), and Triphenylphosphine (TPP) were purchased from Sigma. MitoTracker® Red CMXRos was purchased from Invitrogen. Dimethyl sulfoxide (DMSO), Methyl alcohol (MeOH), D-PBS(-), and 4% Paraformaldehyde (PFA) were purchased from Fujifilm Wako Pure Chemical Corp. (Osaka, Japan). Cell Counting Kit-8 (CCK) was purchased from DOJINDO Laboratories. (Kumamoto, Japan). Hoechst 33258, RPMI 1640, Fetal Bovine Serum, 0.25% Trypsin EDTA, was purchased from Thermo Scientific.

2.2. Preparation and analysis of redox-sensitive aGC

Acetyl chloride was added to DTPA to avoid the non-specific reaction and refluxed at 65°C for 2 h [3,12]. The final product was cooled to RT and the solvent was fully evaporated. The residue was precipitated in pre-chilled diethyl ether and lyophilized (3, 3’-Dithiopropionic anhydride). An amphiphilic GC (aGC, 250 kDa) was prepared by a covalent chemical reaction between GC and 5β-cholanic acid. EDC and NHS initiated the chemical reaction and the resulting solutions were dialyzed against distilled water for 3 days (MWCO 12-14 kDa) [13,14]. The aGC (200 μM) was dissolved in water and MeOH (1 : 1, v/v), and the 3, 3’-Dithiopropionic anhydride (20 μM) dropped wisely added into the aGC. After a 16 h reaction at RT, the obtained product was purified by dialysis against distilled water (MWCO 12-14kDa) for 2 days and lyophilized. The conjugation was confirmed by proton Nuclear Magnetic Resonance (1H NMR, JEOL). The product was denoted as aGC-SS polymer.

2.3. Preparation and analysis of PTX-TPP pro-drug

TPP (20 mM) was added into PTX (20 mM) and vigorously stirred overnight with DCC and DMAP. The obtained product was precipitate by diethyl ether and lyophilized. The conjugation was confirmed by proton Nuclear Magnetic Resonance (1H NMR, JEOL).

2.4. Synthesis of PTX pro-drug loaded redox-sensitive NPs

aGC-SH polymer (5 mg/mL) was prepared in MeOH and deionized water (1 : 1, v/v). The solution was probe-type sonication for 10 min and added 0.5 mg/mL of PTX-TPP pro-drug. After sonication, the free PTX-TPP pro-drug was removed by dialysis against distilled water (MWCO 12-14 kDa) for 6 h and lyophilized. The product was denoted as aGC-SS-proPTX NPs.

2.5. Characterization of aGC-SS-proPTX NPs

The aGC-SS-proPTX NPs size distribution and zeta potential were measured using Dynamic Light Scattering (DLS). All measurements were repeated five times. To confirm the redox-sensitive capability, aGC-SS-proPTX NPs were resuspended in deionized water and 5 mM DTT, respectively.

The size stability was evaluated for 2 days, kept at 37°C.

To estimate the encapsulation and loading efficacy of PTX, aGC-SS-proPTX NPs were dissolved in DMSO and measured by reversed-phase high liquid chromatography (RP-HPLC) at 235 nm wavelength. The efficacy was calculated as follows:

2.6. Cell culture

Human non-small cell lung cancer, A549, was purchased from the JCRB cell bank (Japan). The A549 cells were cultured in RPMI 1640 including 10% FBS and 1% penicillin/streptomycin at 37°C incubator 5% CO₂.

2.7. Cellular toxicity

A549 cells were seeded at 5 × 10³ cells/well in a 96-well plate and incubated for 24 h. Cells were treated with ten different concentrations (0 - 5 μM) of aGC-SS-proPTX NPs, PTX-TPP pro drug, and PTX for 48 h (n = 6). The CCK-8 absorbance was measured at 450nm using a spectrophotometer (Infinite 2000, Tecan) and GraphPad Prism calculated the IC₅₀ values.

2.8. ATP assay

A549 cells were seeded at 5 × 10³ cells/well in a 96-well plate and incubated for 24 h. Cells were treated with PTX, PTX-TPP pro drug, and aGC-SS-proPTX NPs (50 nM) for 48 h (n = 5). An ATP assay kit was used following the manufacturer’s instructions. A spectrophotometer measured the luminescence signal.

2.9. Cellular imaging of mitophagy

The cellular mitophagy reaction was analyzed by confocal microscopy. A549 cells were seeded at 1 × 10⁴ cells on a 35 mm2 glass-bottom dish and incubated for 48 h at 37°C under 5% CO₂ condition. The cells were treated with 500 nM of PTX, PTX-TPP, and aGC-SS-proPTX NPs. After 6 h incubation, the cells were stained with MitoTracker Deep Red TM as manufacturer’s instruction and fixation the cells. The fixed cells were stained with anti-LC-3B antibody and counterstained with Hoechst 33342. Images were obtained by confocal laser scanning microscopy with a 64X oil immersion lens (Leica).

2.10. Western blot

The protein levels of active Caspase-3 and LC-3B were examined through western blot analysis to assess autophagic reactivity and cell death. A549 cells were seeded at a density of 1 × 10⁵ cells per well and treated with 500 nM of PTX, PTX-TPP, and aGC-SS-proPTX NPs for 48 hours. The proteins were extracted using RIPA buffer, and their concentrations were determined using a BCA assay (Thermo Scientific). The samples (10 μg/Lane) were electrophoresed on a 4-20% MINI-Protean precast gel (Bio-Rad) and transferred onto nitrocellulose membranes. These membranes were then incubated with anti-cleaved Caspase-3 (19 and 17 kDa) and anti-LC-3B (18 and 16 kDa) antibodies. The resulting signals were visualized using SuperSignal® West Pico (Thermo Scientific) and captured using the Fusion Solo Chemidoc system. GAPDH (37 kDa) was employed as an internal control.

3.1. Preparation of aGC-SS polymer and PTX-TPP

We designed aGC-SS-proPTX NPs to inhibit the mitophagy metabolite under high GSH conditions and induced mitophagy for NSCLC cell death (Scheme 1). After internalized into the cancer cell, the NPs opened in the cytosol due to the high level of GSH and released PTX-TPP prodrug [15,16]. TPP is widely recognized as a positively charged chemical substrate with the ability to accumulate within the mitochondrial matrix due to the negative membrane potential of the mitochondrial inner membrane [5]. Based on this knowledge, we hypothesize that the prodrug form of PTX-TPP can effectively reach the mitochondria and inhibit the targeted metabolites.

Scheme 1. Schematic illustration of the aGC-SS-proPTX NPs. The nanoparticles loaded prodrug and internalized into the cancer cells. Following endosomal destruction and the presence of cytosolic glutathione (GSH), the prodrug is released from the nanoparticles into the cytosol. Due to its lipophilic cationic properties, the prodrug accumulates in the mitochondrial matrix. Once inside the mitochondria, the prodrug is activated, leading to mitophagy-induced apoptosis in NSCLC cells.

The GC was covalently modified with 5β-cholanic acid and DTPA (Fig. 1(A)). Due to the possibilities of crosslinking copolymerization among GC, DTPA was dehydrated and then reacted with the amino group of GC. The 1H NMR spectra indicated that the peak ranging from 0.6 to 2.4 ppm belongs to aGC, and the peak at 2.4 to 2.9 ppm belongs to DTPA (Fig. 1(B)). The peaks of aGC and DTPA spectra were all observed in aGC-SS polymer, which confirmed that aGC-SH polymer was successfully synthesized.

Figure 1. Chemical structure and 1H NMR spectrum of aGC-SS polymer. A. Chemical structure of aGC-SS polymer. The amphiphilic glycol chitosan is prepared by conjugating 5β-cholanic acid. B. The 1H NMR spectrum of aGC-SS polymer.

The PTX and TPP were chemically conjugated using DCC/DMAP reaction under anhydrous conditions (Fig. 2(A)). The PTX-TPP conjugation was confirmed with 1H NMR. The characteristic peaks at 7.5 to 8.0 ppm belong to protons of TPP and PTX phenyl rings (Fig. 2(B)) [17]. Furthermore, the non-reactive PTX and TPP signal was not detected. Altogether, the aGC-SS polymer and PTX-TPP were successfully prepared.

Figure 2. Chemical structure and 1H NMR spectrum of PTX-TPP prodrug. A. Chemical structure of PTX-TPP. The TPP was covalently conjugated to PTX. B. The 1H NMR spectrum of PTX-TPP.

3.2. Fabrication and characterization of aGC-SS-proPTX NPs

The aGC-SS-proPTX NPs were prepared by the nanoemulsion method. The specimen size and polydispersity index (PdI) were measured by dynamic light scattering (DLS). aGC-SH-proPTX NPs showed 340.0 ± 23.1 nm diameter with 0.240 ± 0.002 of PdI (Fig. 3(A)). Furthermore, TEM images demonstrated a spherical shape with a diameter of 300-350 nm, corresponding to the result of DLS analysis (Fig. 3(B)). The PTX encapsulation and loading efficacy were evaluated by HPLC. The drug encapsulation and loading efficacy were 26.83% and 5.63%, respectively (Fig. 3(C)).

Figure 3. Characterization of aGC-SS-proPTX NPs. A. Size distribution in aqueous solutions. B. Transmission emission microscopy (TEM) images of the aGC-SS-proPTX NPs. Scale bar: 0.5 μm C. Drug encapsulation and loading efficacy of the aGC-SS-proPTX NPs. D. Stability tests at 37°C in water and 5 mM DTT.

To investigate whether the disulfide bonds of aGC-SS-proPTX NPs degraded under an intracellular reductive environment, size of aGC-SS-proPTX NPs was evaluated in response to 5 mM DTT for 72 h. As a result, the size increased over 440 nm in 24 h and sharply decreased up to 72 h in 5 mM DTT (Fig. 3(D)). However, there were no significant size changes in the water. These results suggest that the aGC-SS-proPTX NPs highly efficient in the controlled release under the redox high expressed environment representative in the tumor.

3.3. Evaluation of In vitro Cytotoxicity and cellular ATP

The anti-cancer activity of PTX, PTX-TPP, and aGC-SS-proPTX NPs was evaluated in the human NSCLC cell line, A549, for 48 h. The IC₅₀ values of PTX, PTX-TPP, and aGC-SS-proPTX NPs were determined as 0.7451 μM, 0.2093 μM, and 0.918 μM, respectively (Fig. 4(A)). These results clearly indicated that PTX-TPP exhibits the most potent toxic effect among the treatment. It revealed that the antimetabolite characteristics are enhanced through TPP modification, resulting in the induction of cell apoptosis. The aGC-SS-proPTX NPs exhibited a higher IC₅₀ compared to PTX and PTX-TPP. This difference can be attributed to the controlled release of PTX-TPP from the aGC-SS NPs over time. The sustained release profile of PTX-TPP from the aGC-SS NPs may contribute to a slower and prolonged drug release, resulting in a relatively higher IC₅₀ value for the aGC-SS-proPTX NPs compared to the free PTX and PTX-TPP.

Figure 4. Cellular cytotoxicity and ATP generation by aGC-SS-proPTX NPs. A. A549 cells were treated with various concentrations of PTX, PTX-TPP, and aGC-SS-proPTX NPs, respectively. After 48 h incubation, the IC50 was calculated by CCK-8 (n = 6). B. The ATP synthase activity level in A549 cells was confirmed after the treatment of PTX, PTX-TPP, AGC-SS NPS (w/o PTX-TPP) and aGC-SS-proPTX NPs (n = 5).

Mitochondria serve as crucial energy sources, generating ATP to fuel a wide range of cellular activities [18,19]. However, induction of mitophagy and subsequent mitochondrial damage can disrupt the balance of ATP supply within the cell. This impairment of mitochondrial function can have significant consequences, affecting cellular energy homeostasis and compromising various cellular processes reliant on ATP [20,21]. Intracellular ATP measurements revealed a meaningful decline in ATP activity following drug treatment. A549 cells treated with PTX-TPP and aGC-SS-proPTX NPs exhibited a significant reduction in ATP activity compared to the control and aGC-SS NPs (excluding PTX-TPP). Notably, aGC-SSproPTX NPs exhibited the lowest ATP levels among the tested groups (Fig. 4(B)). These findings clearly demonstrate the capacity of PTX-TPP and aGC-SS-proPTX NPs to suppress ATP levels effectively. Moreover, utilizing a nanocarrier for the delivery of PTX-TPP enhances its impact on ATP depletion, highlighting the superior effectiveness of this approach in targeting and reducing ATP activity.

3.4. Mitophagy imaging and cell death protein

Because mitophagy can degrade dysfunctional mitochondria and limit ROS production, its function has been associated with tumor suppression. Therefore, mitophagy modulation could be beneficial for metastasis prevention and improve therapeutic efficacy.

To assess the cellular mitophagy response, confocal microscopy was employed in this study. A549 cells were treated with PTX, PTX-TPP, and aGC-SS-proPTX NPs for 6 h. After the treatment, the cells were stained with mitoTracker to visualize mitochondria and anti-LC-3B antibodies to detect autophagy-related signaling [22]. The microscopy analysis revealed a correlation between the presence of mitochondria and the expression of the LC-3B autophagy signal (Fig. 5(A)). Notably, PTX-TPP and aGC-SS-proPTX NPs exhibited a more pronounced LC-3B signal originating from the mitochondria, suggesting an enhanced mitophagy response induced by these nanoparticle treatments. These findings provide direct visual evidence of the mitophagy-promoting effects of PTX-TPP and aGC-SS-proPTX NPs, indicating their potential as modulators of cellular autophagy for targeted cancer therapies. The evaluation of cellular autophagy and apoptotic proteins revealed evidence of increased activity of LC-3B and active caspase-3 in response to PTX-TPP and aGC-SS-proPTX NPs (Fig. 5(B)). This finding indicates that the treatment effectively induces cellular autophagy, subsequently activating apoptotic pathways.

Figure 5. Intracellular activation of mitophagy by aGC-SS-proPTX NPs. A. Representative images of A549 cells treated with PTX, PTX-TPP, and aGC-SS-proPTX NPs for 6 h. Mitochondria were stained with MitoTracker Deep Red (red), and autophagy was visualized using an antibody against LC-3B (green). (n = 5, Scale bar: 10 μm) B. Protein expression levels of LC-3B and cleaved caspase-3 in A549 cells following treatment with PTX, PTX-TPP, and aGC-SS-proPTX NPs.

The obtained results clearly demonstrate that both PTX-TPP and aGC-SS-proPTX NPs facilitate the process of autophagosome-lysosome fusion and induce mitophagy, ultimately leading to apoptosis. The promotion of autophagy and mitophagy reactions by PTX-TPP and aGC-SS-proPTX NPs highlights their potential as promising therapeutic agents in targeting cancer cells and inducing apoptosis.

In this study, we developed a novel approach for developing antimetabolite chemotherapeutics targeting non-small cell lung cancer (NSCLC). Our focus centered on utilizing PTX prodrug-loaded redox-sensitive nanocarriers, aGC-SS-proPTX NPs. These nanocarriers exhibited a remarkable sensitivity to the intracellular glutathione environment, resulting in their degradation and subsequent release of the PTX-TPP prodrug. Importantly, our research demonstrated that the aGC-SS-proPTX NPs possessed ATP-inhibitory effects and induced cell death through mitophagy. The in vitro experiments conducted in this study strongly support the potential of aGC-SS-proPTX NPs as a promising strategy to suppress metabolic activity and facilitate effective cancer treatment.

This work was financially supported by the Brain Korea 21 Four Program through the National Research Foundation of Korea (NRF) under the Ministry of Education (MSIT) (2022M3C1A309202212) and the Brain Pool program funded by the Ministry of Science and ICT through the National Research Foundation of Korea (No. 2022H1D3A2A01096503).