Formulation and evaluation of a two-stage targeted liposome coated with hyaluronic acid for improving lung cancer chemotherapy and overcoming multidrug resistance

Abstract Multidrug resistance (MDR) has emerged as a prominent challenge contributing to the ineffectiveness of chemotherapy in treating non-small cell lung cancer (NSCLC) patients. Currently, mitochondria of cancer cells are identified as a promising target for overcoming MDR due to their crucial role in intrinsic apoptosis pathway and energy supply centers. Here, a two-stage targeted liposome (HA/TT LP/PTX) was successfully developed via a two-step process: PTX-loaded cationic liposome (TT LP/PTX) were formulated by lipid film hydration & ultrasound technique, followed by further coating with natural anionic polysaccharide hyaluronic acid (HA). TT, an amphipathic polymer conjugate of triphenylphosphine (TPP)-tocopheryl polyethylene glycol succinate (TPGS), was used to modify the liposomes for mitochondrial targeting. The average particle size, zeta potential and encapsulation efficiency (EE%) of HA/TT LP/PTX were found to be 153 nm, −30.3 mV and 92.1% based on the optimal prescription of HA/TT LP/PTX. Compared to cationic liposome, HA-coated liposomes showed improved stability and safety, including biological stability in serum, cytocompatibility, and lower hemolysis percentage. In drug-resistant A549/T cells, HA was shown to improve the cellular uptake of PTX through CD44 receptor-mediated endocytosis and subsequent degradation by hyaluronidase (HAase) in endosomes. Following this, the exposure of TT polymer facilitated the accumulation of PTX within the mitochondria. As a result, the function of mitochondria in A549/T cells was disturbed, leading to an increased ROS level, decreased ATP level, dissipated MMP, and increased G2/M phase arrest. This resulted in a higher apoptotic rate and stronger anticancer efficacy.


Introduction
Lung cancer is a prevalent malignant tumor, with a five-year overall survival rate of less than 20% [1].NSCLC is the primary histological subtype that accounts for over 80% of all lung cancer cases [2].Paclitaxel (PTX), a monomeric compound extracted from the bark of Taxus chinensis, is a first-line chemotherapeutic agent used to treat NSCLC, ovarian, and breast cancer [3].However, clinical studies indicate that the marketed formulation Taxol® is associated with severe PTX resistance, which can be observed in most newly diagnosed patients and almost all patients with recurrence [4].Furthermore, intravenous administration of Taxol® has been reported to cause toxic and side effects, including allergic reactions, nephrotoxicity, and neurotoxicity, resulting from adjuvant Cremophor EL [5].
Chemotherapy is commonly associated with two major challenges.The first is the lack of selective accumulation, leading to damage to normal organs and tissues [6][7][8].The second challenge is the emergence of MDR, which is a leading cause of chemotherapy failure [9,10].MDR is typically divided into two types: inherent resistance, which occurs before treatment, and acquired resistance, which occurs after treatment [11,12].P-glycoprotein (P-gp) is one of the ATP-binding cassette transporters that is overexpressed in MDR cells, and the subsequent efflux of intracellular chemotherapeutics, an energy-dependent process, is the most critical mechanism for drug resistance [13].
Nanoparticles have become increasingly popular for their potential to improve the selectivity of drugs in tumor sites and to overcome MDR in cancer therapy.They are considered an effective drug delivery system.Liposomes, in particular, have been extensively investigated due to their high biocompatibility and clinical utility.Drugs such as Doxil®, Daunoxome®, and Ambisome® are all based on liposomal formulations [14][15][16].Non-ionic surfactants or polymers such as D-alphatocopheryl poly (ethylene glycol 1000) succinate (TPGS), Tween80, and Pluronic have been used to inhibit P-gp and overcome MDR.These agents enhance the therapeutic efficacy of drugs such as PTX by increasing the intracellular concentration of the drug [17][18][19].TPGS, which is approved by the FDA, is a safe adjuvant that can be used as a P-gp ATPase inhibitor.
Mitochondria are crucial subcellular organelles that have been widely investigated for their role as decisive regulators in intrinsic apoptosis pathway and energy supply centers [20].The mitochondrial membrane potential (MMP) of tumor cells is more negative (ΔΨm = −220 mV) compared to that of normal cells (ΔΨm = −140 mV), making mitochondrial targeted therapy feasible [21,22].Delocalized lipophilic cationic groups such as triphenylphosphine (TPP) and dequalinium (DQA) can be used to target the mitochondrial matrix through electrostatic adsorption between positive and negative charges.Therefore, mitochondrial-targeted drug delivery systems have been successfully developed by incorporating TPP or DQA moieties [23][24][25].TT, which stands for triphenylphosphine (TPP)-tocopheryl polyethylene glycol succinate (TPGS) conjugate, is an amphipathic polymer that can modify liposomes for mitochondrial targeting.The polymer TT is capable of improving the efficacy of revers-ingMDR by targeting and disrupting mitochondria [26,27].However, the lack of selectivity for tumor cells and stability in serum limits the clinical application of cationic nanoparticles modified with TT.To address this limitation, we hypothesize that nanoparticles further modified with HA via electrostatic interaction with TT could provide improved biological stability and biocompatibility due to their negatively charged surface.Moreover, this modification could confer active targeting ability through specific recognition between HA and overexpressed CD44 receptors.
Here, a two-stage targeted PTX-loaded liposomes modified with TT and HA (HA/TT LP/PTX) was prepared using lipid film hydration and ultrasound technique for the treatment of MDR lung cancer cells A549/T, as demonstrated in Figure 1.To optimize the formulation, an orthogonal experiment was performed based on the results of single-factor experiments.Subsequently, the physicochemical properties of the liposomes, including morphology, particle size, stability, biocompatibility, degradation of HA, and sustained-drug release behavior, were systematically characterized.Additionally, the study explored the mechanism and efficacy of the formulation in reversing MDR, including its active-targeting and mitochondria-targeting abilities, cytotoxicity, intracellular level of reactive oxygen species (ROS) and adenosine triphosphate (ATP), MMP, pro-apoptotic effects, and cell cycle arrest in G 2 /M. and trypsin were purchased from Gibco, US.Thiazole blue (MTT) was purchased from Bomei Biotechnology Co., China.Other reagents are analytical pure and purchased from Sinopharm Chemical Reagent Co., China.

Preparation of PTX-loaded liposomes
Liposomes were fabricated via the lipid film hydration and ultrasound method.Initially, a predetermined amount of SPC, Chol, TT, and PTX was added to a pear-shaped flask, followed by chloroform to facilitate dissolution.Subsequently, the solvent was evaporated using a rotary vacuum evaporator to produce a thin lipid film along the flask's interior walls.Then, the lipid film was hydrated with 10 mL of 5% glucose solution and incubated at 37 °C for 1 h, followed by ultrasound treatment using an ultrasound probe in an ice bath for several minutes (TT LP/PTX).After that, a measured quantity of HA solution was added, stirred, and sonicated for 5 min (HA/TT LP/PTX).Additionally, the LP/PTX (excluding TT and HA), three blank liposomes (without PTX), and three C6-loaded liposomes (where PTX was replaced with C6) were prepared utilizing the same approach mentioned above.

HPLC analysis
The PTX concentration in the liposomes was determined by HPLC using an Liquid Chromatograph (Dionex U3000) and a ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm).The mobile phase was composed of a 50:50 mixture of water and acetonitrile, which was pumped at a flow rate of 1.0 mL/min while maintaining the column temperature at 25 °C using a column oven.The injection volume was set to 20 μL, and the UV wavelength of detection was set at 227 nm.A typical retention time for PTX was observed at 12.63 min.A calibration curve was constructed for PTX within a concentration range of 1 ∼ 50 µg/mL.

Determination of entrapment efficiency (EE%) of liposome
To determine the EE%, the unencapsulated PTX or C6 in the liposomes was removed through a filtration membrane method.Specifically, 1800 μL of methanol was added to 200 μL of drug-loaded liposomes (before or after passing through a 0.22 μm microporous filter) for demulsification.The PTX content before or after passing the filter was labeled as M1 and M2, respectively.The EE% of the liposomes was then calculated according to the following equation:

Orthogonal experiment
The formulation of liposomes was optimized through an orthogonal design (with 3 factors and 3 levels), based on the results of single-factor experiments (Table S1).
The three factors considered were the mass ratio of Chol to SPC (factor A), the mass ratio of PTX to SPC (factor B), and the mass concentration of SPC (factor C).The EE% was chosen as the optimization index in the orthogonal experiments.Nine different formulations were tested based on the L9(33) orthogonal table (Table S2).

The morphology, particle sizes and zeta potential
The morphology of the liposomes was analyzed through the Transmission Electron Microscopy (TEM, JEM-1400, JEOL Co., Japan) after staining with 1% uranyl acetate.Additionally, the particle size and zeta potential of the different liposomes were determined using a Nano-ZS90 Zetasizer (Malvern instruments, Ltd, UK).

Hemolysis
Red blood cells (RBCs) were collected from the heparinized blood of male Balb/C mice through centrifugation cycles and washed with phosphate-buffered saline (PBS) until the supernatant became clear.A volume of 200 μL of RBC suspension was added to 800 μL of distilled water (positive control), PBS (negative control), or varying concentrations of liposomes, and incubated at 37 °C for 2 h.The suspensions were then centrifuged at 1500 rpm for 10 min, and the supernatants were collected and analyzed by measuring the absorbance at 570 nm using a microplate reader.Hemolysis percentage = [(OD test −OD neg )/(OD pos −OD neg )] × 100%, Where OD test , OD neg , and OD pos represent absorbance values at 570 nm of the samples, negative control and positive control, respectively.

Stability
The change in particle size and EE% of the liposomes was investigated to assess their long-term storage stability in distilled water at 4 °C for 10 days.Furthermore, the biological stability of drug-loaded liposomes was evaluated in the presence of 50% FBS.Briefly, the liposome solution mixed with FBS in a ratio of 1:1 (v/v) and subsequently incubated at 37 °C with shaking for 24 h.At predetermined intervals, the size and EE% of the samples were collected and analyzed.

Degradation of HA
The pH-dependent and HAase-mediated degradation of HA in HA/TT LP/PTX was evaluated by monitoring the changes in particle size and zeta potential.HA/TT LP/ PTX was treated at different pH values (pH 7.4, pH 6.8, and pH 5.0) without HAase or with HAase, and then incubated in a water bath at 37 °C with shaking for 4 h.
The particle size and zeta potential of the samples were measured at predetermined time points to assess the degradation of HA.

In vitro drug release
The in vitro drug release approach was utilized to assess the release rate of PTX from four formulations.This was accomplished by subjecting the formulations to dialysis against various release media.Specifically, a volume of 2 mL suspension of each formulation was encapsulated in a dialysis bag (MWCO: 8000-14000 Da) and subsequently submerged in 20 mL of PBS supplemented with 1% Tween80 (w/v).These suspensions were then subjected to different pH conditions, either in the absence or presence of HAase at a concentration of 1 mg/mL.At the specified time (0.5, 1, 2, 4, 8, 10, 12, 24, 48 h), 1 mL of the release medium was taken out for analysis and replenished with an equal volume of fresh medium.The amount of PTX present in the release medium was quantified by HPLC as the established methodology in '2.3' .

Cell cultures
The human lung adenocarcinoma A549 cells (drug-sensitive) and A549/T cells (drug-resistant) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin 100 U/mL and streptomycin 100 μg/mL).The cultures were maintained under optimal conditions of 5% CO 2 at a temperature of 37 °C.

Endocytosis pathway and cellular uptake
The aim of this study is to investigate the uptake pathway and efficiency of liposomes in entering A549/T cells.Initially, the effect of temperature on the uptake of liposomes by the cells was investigated.A549/T cells were seeded in 6-well cell culture plates at a density of 5 × 10 5 cells/well and cultured for 24 h to allow the cells to adhere.Then the cells were incubated at either 4 °C or 37 °C for 1 h before the addition of HA/TT LP/C6 (2 μg/mL C6), followed by another 4 h incubation at the same temperature.After that, the cells were washed with PBS, collected by centrifugation after trypsinization, and resuspended in PBS.The fluorescence intensity in the cells was quantified utilizing flow cytometry (Agilent, NovoCyte D2060R).Subsequently, the impact of endocytosis inhibitors on the uptake of liposomes was examined.After cultured for 24 h, the cells were treated with endocytosis inhibitors, including indomethacin (110 μg/mL), chlorpromazine hydrochloride (50 μg/ mL), amiloride hydrochloride (50 μg/mL), or HA (100 μg/mL) for a duration of 1 h.Then, the HA/TT LP/C6 was added to the cells and the cells were cultured for an additional 4 h.The fluorescence intensity in the cells was detected in the same manner as before.
Finally, the cellular uptake efficiency of three different liposomes by A549/T cells was measured.After cultured for 24 h, the cells were treated with three various C6-loaded liposomes for 4 h, respectively.Then the fluorescence intensity in the cells was detected using the same method.

Mitochondrial localization
Confocal laser scanning microscope (CLSM) was employed to observe the mitochondrial co-localization of diverse liposomes in A549/T cells.Specifically, A549/T cells were seeded in confocal microscopy dishes at a density of 1 × 10 4 cells per dish for a duration of 24 h, and subsequently cultured with C6-loaded liposomes (2 μg/ mL C6) for an additional 4 h.After the incubation, the cells were washed with PBS and sequentially stained with MitoTracker-Red (50 nM) for 30 min and Hoechst 33342 (10 μg/mL) for 10 min.After each staining, the cells were washed with PBS and observed using CLSM.The value of Pearson's coefficient was calculated using Image J software.

Cell viability assay
The MTT assay was employed to assess the cytotoxicity of blank liposomes on 293 T cells and A549/T cells, as well as the four formulations on A549 cells and A549/T cells.To elaborate, cells were seeded at a density of 5 × 10 3 cells/well in a 96-well plate and allowed to adhere for 24 h.Subsequently, the cells were treated with blank liposomes (at concentrations ranging from 16.2 to 1000 μg/mL) or the four formulations (with final PTX concentrations ranging from 1.6 to 100 μg/mL) and then incubated for an additional 48 h.Following incubation, the cells were washed with PBS, and 25 μL of 5% MTT solution was added to each well, and incubated for 4 h.
The formazan crystals generated was dissolved in 150 μL of DMSO solvent, and the absorbance intensity of each sample was measured at 570 nm using a microplate reader (xMark, Bio-Rad, USA).

Cell viability OD
The half-maximal inhibitory concentration (IC 50 ) value was calculated by GraphPad Prism software.

Intracellular ROS level
DCFH-DA fluorescent probe was utilized to determine the intracellular ROS level of A549/T cells.Initially, A549/T cells were seeded in 6-well plates at a density of 5 × 10 5 cells/well and cultured for 24 h.Subsequently, the cells were treated with four formulations for 24 h.For qualitative detection, the cells were washed and stained with DCFH-DA for 20 min at 37 °C.Following staining, the cells were observed using a fluorescent microscope.For quantitative detection, the cells were washed, collected by centrifugation after trypsinization, and stained with DCFH-DA for 20 min.The cells were then washed with serum-free medium, resuspended, and evaluated via flow cytometry.Excitation and emission wavenumbers were set to 488 and 525 nm, respectively.

MMP depolarization
JC-1 was used as a fluorescent probe to measure the change of MMP (ΔΨm) in A549/T cells.In detail, A549/T cells were seeded in a 6-well plate at a density of 5 × 10 5 cells/well and cultured for 24 h and treated with four formulations for 24 h (PTX 30 µg/mL).For qualitative detection, the cells were washed with PBS, stained with JC-1 for 20 min at 37 °C, and washed again.The red or green fluorescence intensity in cells was observed using a fluorescent microscope (BX53F, Olympus, Japan).For quantitative detection, the cells were collected by centrifugation after trypsinization, stained with JC-1 for 20 min, washed, resuspended, and detected using flow cytometry.The excitation and emission wavelengths of green fluorescence and red fluorescence were 490/530 and 525/590 nm, respectively.

ATP level
The A549/T cells were seeded in 6 cm cell culture dishes at a density of 1 × 10 6 cells/dish and cultured for 24 h.After that, the cells were treated with four different formulations (PTX 30 μg/mL) for a duration of 24 h.Following treatment, the cells were harvested via trypsin digestion and subsequently processed according to the protocol outlined in the ATP assay kit instructions.Ultimately, the ATP level was calculated using the formula provided in the assay kit.

Cell cycle distribution assay
The cell cycle distribution was assessed using a cell cycle and apoptosis analysis kit through flow cytometry.Initially, A549/T cells were seeded in a 6-well plate at a density of 5 × 10 5 cells/well for 24 h and then treated with the four formulations for 24 h.Subsequently, the cells were collected after trypsin digestion and fixed overnight in 70% cold ethanol at 4 °C.Afterwards, the cells were washed with PBS, treated with 100 μg/ mL RNase enzyme at 37 °C for 15 min and stained with 50 μg/mL PI at 4 °C for 30 min.
Finally, the analysis of cell cycle distribution was performed by flow cytometer.

Cell apoptosis assay
The apoptosis of A549/T cells was assessed using the Annexin V-FITC/PI double staining assay kit.Concisely, A549/T cells were seeded in 6-well plates at a density of 5 × 10 5 cells/well for 24 h, followed by exposure to four distinct formulations for 24 h.Subsequently, the cells were washed, collected, and resuspended in binding buffer.The cells were then stained with Annexin V-FITC for 15 min and finally with PI for 5 min before the flow cytometry analysis.

Western blot
The expression levels of mitochondria apoptosis-related proteins, including Bcl-2, Bax, cleaved caspase 3, and cleaved caspase 9, in A549/T cells were determined using Western blot analysis.After treatment with free PTX, LP/PTX, and HA/TT LP/PTX, the A549/T cells were washed with cold PBS and lysed in RIPA buffer on ice for 30 min.The supernatant protein concentrations were determined using a BCA protein assay kit after centrifugation at 12,000 rpm/min for 15 min at 4 °C.Equal amounts of protein were subjected to electrophoresis on an SDS-polyacrylamide (8-12%) gel and transferred onto nitrocellulose membranes.The membranes were blocked with 5% non-fat milk in TBST buffer for 1.5 h and then incubated with primary antibodies against Bcl-2, Bax, cleaved caspase-3, and cleaved caspase-9 overnight at 4 °C.After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature.Finally, the protein signals were detected using an enhanced chemiluminescence (ECL) detection kit.

Statistical analysis
All experiments were repeated at least three times.Statistical significance difference was analyzed by student's t-test or one-way analysis of variance (ANOVA) using the Graphpad Prism 8 software.p < 0.05 was considered as statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001).

Singe factor experiments
The EE% of the liposome is a critical quality inspection indicator and a key parameter for evaluating drug carrier quality and formulation screening.To optimize the EE% of TT LP/PTX, five individual factors were tested.The variation trends of the EE% with each factor were presented in Figure S1.It was evident from Figure S1A-C that factor A (the ratio of Chol to SPC), factor B (the ratio of drug to lipid), and factor C (the mass concentration of SPC) significantly influenced the EE% within the experimental range.
The EE% reached the maximum value (91.6%) when the ratio of Chol to SPC was adjusted to 1:10.Chol is a lipid that mainly affects membrane fluidity.If the membrane of liposome becomes too fluid, it can lose its structural integrity.Furthermore, the peak value of EE% was 92.8% when the mass ratio of PTX to SPC was 1:15, after which the trend of EE% slightly decreased.Additionally, the highest level of EE% (92.0%) was observed when the SPC mass concentration was 5 mg/mL.Excessively high concentrations of SPC can lead to phospholipid aggregation and decreased EE%.The fourth and fifth influential factors had minimal effects on EE% within the range of experiments.Consequently, the optimal mass ratio of TT to SPC was determined to be 1:10 and the optimal ultrasound time was 9 min based on EE%.As a result, three mainly effective factors (i.e.factor A, factor B, and factor C) were selected for the subsequent orthogonal test.

Analysis of orthogonal experiments
Based on the results of the single-factor experiments, the liposome prescription was optimized using an L9(3 3 ) orthogonal array.EE% was used as the evaluation index, and the range value represented the degree of impact of each factor on EE%.Analysis of the results in Table S2 indicated that the order of influence on EE% was factor B > factor A > factor C, i.e. the factor A and factor B had a significant effect on EE%.Consequently, the final optimal liposome prescription was determined to be A 3 B 2 C 2 .

Optimal ratio of HA to TT
Due to the positive charge, TT LP/PTX liposomes were more likely to undergo electrostatic interaction with negatively charged cell membranes or serum proteins.This can lead to poor biocompatibility and stability during in vivo circulation.However, the negatively charged polysaccharide HA, which is a specific ligand of CD44 receptors, can bind to the surface of TT LP/PTX to shield the positive charge and enhance cellular internalization.Nonetheless, excessive HA in the delivery system could reduce the cellular uptake of liposomes coated with HA through competitive interaction.Consequently, the size and zeta potential of the liposomes were used as evaluation index to screen the optimal ratio of HA to TT.
In Figure S2A, it can be observed that when the mass ratio of HA to TT increased from 0:1 to 1:1, the zeta potential of liposomes decreased significantly from 38.3 mV to −30.9 mV.However, further increasing the ratio of HA to TT resulted in negligible changes in zeta potential, indicating HA used for neutralizing positive charge was excessive.The size of the liposomes also increased with an increase in the ratio of HA to TT, which may be due to the electrostatic force between the liposomes and the viscosity of the mixed solution resulted from HA [28,29].To verify the optimal ratio of HA to TT, HA/TT LP/C6 with different ratios of HA to TT were prepared to assess the targeting efficiency to cancer cells, as depicted in Figure S2B-D.The fluorescence in the control group was negligible, while the highest fluorescence intensity was observed when the mass ratio of HA to TT was set at 1:1.Therefore, the optimal mass ratio of HA to TT was confirmed to be 1:1.
In summary, the optimal formulation composition of the HA/TT LP/PTX was determined as follows: the mass ratio of Chol to SPC was 1:10, the mass ratio of PTX to SPC was 1:15, the SPC mass concentration was 5 mg/mL, the mass ratio of TT to SPC was 1:10, the ultrasound time was 9 min, and the mass ratio of HA to TT was 1:1.Confirmatory tests were conducted by preparing three batches of HA/ TT LP/PTX using the optimal formulation.The average EE% was found to be 92.1% with a relative standard deviation (RSD) value of 1.4%, indicating the EE% of liposomes prepared under optimal conditions was high and the reproducibility of the fabrication method was well.

The morphology, particle size and zeta potential of liposomes
Three types of PTX-loaded liposomes were prepared using the lipid film hydration and ultrasound technique.As shown in Figure 2A, the solutions of the three liposomes were translucent with a blue opalescence, and there were no visible insoluble impurities or lumps.Upon irradiation with a laser pointer, the Tyndall effect was observed perpendicular to the direction of the laser light.
In Figure 2B, the morphology of LP/PTX, TT LP/PTX, and HA/TT LP/PTX exhibited regularly shaped spherical and uniform distribution, with average sizes of 107, 92, and 153 nm, respectively.The size of liposomes is a critical determinant of their efficacy in drug delivery, influencing both the blood circulating time and the enhanced permeability and retention (EPR) effect [30,31].Nanoparticles with a size smaller than 10 nm are rapidly eliminated by the renal system, while those larger than 10 nm are mainly cleared by the mononuclear phagocytic or reticuloendothelial systems.The EPR effect results from the characteristic pathological properties of cancer that involve blood vessel leakiness and dysfunctional lymphatic drainage.Nanoparticles with a size below 400 nm could extravasate into tumors, and those smaller than 200 nm could do so more effectively.Moreover, the underdeveloped lymphatic vessels would extend the retention of the leaked nanoparticles in tumors.Therefore, the sizes of the liposomes in this study are suitable for permeating into tumor tissues through the EPR effect.
In Figure 2C, the zeta potential of LP was measured to be 5.5 mV.Following the introduction of TT into LP, the zeta potential value of TT LP/PTX underwent a significant shift from 5.5 to 39.7 mV.This modification suggests that the addition of TT to LP alters its surface charge, potentially affecting its biological properties.Upon coating with an HA shell via electrostatic interaction, the resulting HA TT LP/PTX complex exhibited a negative zeta potential of −30.3 mV.This electrostatic repulsion between the anionic HA shell and negatively charged serum proteins is expected to enhance the stability of the liposomes in serum.

Safety and biocompatibility of liposomes
As a drug delivery system administered through intravenous injection, it is crucial to investigate the biocompatibility and safety of liposomes.To this end, we first employed the MTT assay to evaluate the cytotoxicity of blank liposomes on A549/T and 293 T cells.Figure 3A-B demonstrated that the three types of blank liposomes showed no significant toxicity within the experimental concentration range on A549/T cells (cell viability ≥ 85%).However, it was observed that blank TT LP exhibited slight cytotoxicity towards 293 T cells at high concentration (cell viability ≥ 77%).This toxicity was probably attributed to the strong electrostatic interaction between cationic liposomes and the cell surface, which could impair cell membrane functions.
Then the hemolysis percentage (%) was measured to assess the safety of the liposomes against RBCs.As shown in Figure 3C-D, both blank and drug-loaded liposomes, including LP, LP/PTX, HA/TT LP, and HA/TT LP/PTX, exhibited hemolysis percentages lower than 5%, except for TT LP and TT LP/PTX.These findings indicated the excellent biocompatibility of normal and HA-coated liposomes.Notably, cationic liposomes at high concentrations may potentially cause hemolysis.

Stability of liposomes
The stability of liposomes is important for their clinical applications, both in terms of their long-term storage at low temperatures and their biological stability under physiological conditions.As illustrated in Figure 4A-B LP/PTX demonstrated no significant changes in particle size and EE% within 10 days, indicating their excellent storage stability at 4 °C.
The biological stability of liposomes under physiological conditions is a vital prerequisite for their in vivo applications.To evaluate the biological stability of the two-stage targeted liposomes, particle size and EE% were determined in the presence of 50% FBS (Figure 4E-F).For TT LP/PTX, the variation in particle size and EE% was significantly larger than that observed for non-cationic liposomes.In contrast, the changes in particle size and EE% were minimal for LP/PTX and HA/TT LP/ PTX under the same conditions.Ran et al. have previously reported that cationic liposomes coated with PEGylated HA showed good stability in FBS due to their anionic negative charge [32].The size alteration of the liposomal siRNA delivery system (naked NP) was substantial in the presence of 50% FBS, while the changes were minor for the HA-NP and PEG-HA-NP under similar conditions.Similar findings were also reported by Yang et al. [33].These findings suggested that the instability of TT LP/PTX may be due to the binding of positively charged liposomes and negatively charged plasma proteins, resulting in increased particle size and drug leakage.In contrast, the multifunctional HA/TT LP/PTX was found to be more stable in serum.

Degradation of HA
The degradation of the HA shell was investigated by monitoring the particle size and zeta potential of HA/TT LP/PTX after incubation at different pH values in the presence or absence of HAase.As depicted in Figure 5A-B, in the absence of HAase, HA/TT LP/PTX did not exhibit a change in charge at pH 7.4 and 6.8.However, at pH 5.0, the zeta potential changed from −30.3 mV to 3.8 mV after 4 h, indicating the pH-dependent degradation of HA.This observation was consistent with the particle size reduction from 152.5 nm to 121.1 nm at pH 5.0, which was the most substantial change of size observed.Upon incubation with HAase, HA/TT LP/PTX exhibited charge conversion at all three pH conditions.Particularly, the zeta potential changed remarkably from −30.3 mV to 37.2 mV after 4 h at pH 5.0.Additionally, the particle size decreased from 152.5 nm to 90.1 nm under the same condition.The zeta variation of HA/TT LP/PTX was similar to the findings reported by Chen et al. [34].Based on the results, it can be inferred that the degradation of HA exhibited pH-dependent and HAase-mediated characteristics, and it was reasonable to assume that HA/TT LP/PTX could achieve charge conversion in the endosomes of cancer cells.

In vitro drug release behavior
The drug release behavior of the four formulations is presented in Figure 5C-D.The cumulative drug release of the four PTX formulations after 48 h in a PBS solution at pH 7.4 containing 1% Tween-80 was 90.3%, 42.1%, 39.1%, and 34.7%, respectively.This slower release rate indicated that the liposomes possessed good sustained-release properties.Furthermore, the release rate from HA/TT LP/PTX was slower compared to the other two liposomes.This phenomenon was potentially attributed to the coating of HA, which blocked the drug release channels.
Subsequently, the accumulative release rate of HA/TT LP/PTX was evaluated in different release media.Overall, the drug release behavior exhibited pH-dependent and HAase-mediated characteristics.Following immersion in the release medium for 48 h, the accumulative release rates were 34.7%, 46.4%, and 54.7% at pH 7.4 without HAase, pH 5.0 without HAase, and pH 5.0 with HAase, respectively.This drug release profile was consistent with the degradation pattern of HA.Likewise, Lei et al. reported similar release behavior of paclitaxel and sorafenib from a cationic liposome modified with polylysine-deoxycholic acid copolymer and coated with HA [35].The slow release of HA/TT LP/PTX under physiological conditions contributed to minimizing drug leakage in systemic circulation, while the rapid release in the acidic environment in the presence of HAase facilitated drug release in endosomes or lysosomes of cancer cells.

Endocytosis pathway and cellular uptake
To investigate the cellular uptake pathways and efficiency of HA/TT LP/C6, C6-loaded liposomes were used for qualitative and quantitative detection.Initially, low temperatures and specific inhibitors were employed to assess the cellular uptake pathways of HA/TT LP/C6 in A549/T cells.We employed low temperatures and specific inhibitors to evaluate the cellular uptake pathways of HA/TT LP/C6.Our results demonstrated that cancer cells primarily take up HA/TT LP/C6 through endocytosis, as the intracellular fluorescence intensity decreased to 50% at 4 °C compared to the control temperature of 37 °C (Figure 6A).
Additionally, the endocytosis of HA/TT LP/C6 was assessed using several inhibitors, including indomethacin, chlorpromazine, amiloride and HA.Specifically, the selection of indomethacin, chlorpromazine, and amiloride was based on their known effects on the caveolin-dependent, clathrin-dependent, and macropinocytosis pathways of endocytosis.HA is a specific ligand of CD44 receptors, which are overexpressed on the cell membrane of many solid cancer cells, including NSCLC.The interaction between HA and CD44 receptors has been investigated as a potential target for drug delivery to cancer cells.As shown in Figure 6A, the factors that most influenced intracellular fluorescence intensity were HA, chlorpromazine, indomethacin, and amiloride in that order, indicating that the endocytic pathways of HA/TT LP/C6 were primarily mediated by CD44 receptors, followed by clathrin-mediated, caveolin-mediated, and macropinocytosis pathways.
To further evaluate the targeted delivery of HA/TT LP/C6 to A549/T cells, the cellular uptake efficiency of the three various liposomes was quantitatively measured by flow cytometry.As depicted in Figure 6B, the intracellular fluorescence intensity of TT LP/C6 and HA/TT LP/C6 was significantly higher compared to LP/C6.The improved cellular uptake of TT LP/C6 may be attributed to the inhibition of P-gp by TPGS in TT.Moreover, the intracellular fluorescence intensity in the HA/TT LP/C6 group was 4.2-times higher than that in the LP/C6 group.Luo et al. demonstrated that a lipid-albumin nanosystem (TLA/PTX@CS) modified with TPGS and chondroitin sulfate (CS) was endocytosed through caveolae and clathrin dual-mediated pathways, and showed active targeting and MDR reversing ability to MCF-7/MDR cells [36].Therefore, it was reasonable to conclude that HA/TT LP/C6 could significantly increase cellular uptake through two-stage targeting and MDR reversal.

Mitochondrial localization
The mitochondrial-targeted ability of TT-modified liposomes was evaluated using confocal laser scanning microscopy (CLSM).Mito-tracker Red, a commercial dye, was used to stain the mitochondria in A549/T cells for this purpose.As shown in Figure 6C, the intracellular red mitochondria were uniformly distributed in different groups, and the yellow region represented the colocalization of liposomes with mitochondria.Compared with LP/C6, both TT LP/C6 and HA/TT LP/C6 showed better colocalization after 4 h of incubation.Additionally, a quantitative analysis was carried out by calculating the Pearson correlation coefficient (P) to assess the degree of colocalization (Figure 6D).The P value of TT LP/C6 and HA/TT LP/C6 was found to be 2.4-and 2.5-fold higher than that of LP/C6, respectively.These results indicated that the TT moiety of the liposomes had the ability to deliver the cargoes to the mitochondria.

Cytotoxicity
MTT assay was performed to investigate the effects of free PTX and liposomes on reversing MDR.As shown in Figure 7, the inhibitory effects of the four formulations on A549 and A549/T cells were demonstrated.The IC 50 of free PTX in A549/T cells (75.2 μg/mL) was 3.1-fold higher than that in A549 cells (24.3 μg/mL), indicating that A549/T cells met the requirement of a PTX-resistant cell line.The liposomes were found to be more effective than the free drug in terms of antiproliferative activity.The IC 50 values of LP/PTX, TT LP/PTX, and HA/TT LP/PTX in A549 cells were 17.4, 8.9, and 7.1 μg/mL, respectively, while the IC 50 values in A549/T cells were 57.7, 29.4, and 28.3 μg/mL, respectively.The reversal factor (RF), which represents the ability of liposomes to reverse MDR, was calculated.The RF values of LP/PTX, TT LP/PTX, and HA/TT LP/PTX were 1.3, 2.6, and 2.7, respectively.The highest RF value demonstrated that HA/TT LP/PTX could directly disturb mitochondrial function via increased intracellular uptake and mitochondrial targeting, thereby overcoming MDR.

Intracellular ROS level
The intracellular ROS level is an essential parameter for evaluating the antitumor efficacy of drugs, as excessive ROS is related to various cellular processes, including apoptosis and cell signaling.To explore the effect of different drug formulations on intracellular ROS levels, DCFH-DA was used as a membrane-permeable oxidative stress indicator.As shown in Figure 8A-C, while free PTX and LP/PTX treatments increased intracellular ROS slightly compared to the control group, the ROS levels in cells treated with TT LP/PTX and HA/TT LP/PTX were found to be 1.5-times and 1.6-times more effective than LP/PTX, respectively.This enhanced ROS production can be attributed to the combined effect of increased cellular uptake due to active targeting by HA, effective mitochondrial delivery by TT, and the ROS-promoting ability of α-tocopheryl succinate (α-TOS) present in TT.Previous studies had shown that tumor cells were more sensitive to high levels of ROS than normal cells, and enhanced intracellular ROS levels could induce cell death by destroying the mitochondrial membrane [37].Therefore, the results of ROS measurements in different treatment groups were consistent with the results of cytotoxicity assays.

MMP
The production of ROS is known to trigger the opening of the mitochondrial permeability transition pore (MPTP), leading to the collapse of the MMP.In turn, this plays a critical role in regulating apoptotic signaling pathways.To assess whether the drug could accumulate in the mitochondria, the changes in MMP were evaluated using JC-1, a fluorescent probe that selectively accumulated in the mitochondrial matrix.When MMP is depolarized, JC-1 shifts from red aggregates to green monomers.As demonstrated in Figure 8E-G, A549/T cells treated with all formulations exhibited a dissipated MMP compared to the control group.Of these formulations, the most significant loss of Δψm was caused by HA/TT LP/PTX.The dissipation of MMP is usually accompanied by the release of cytochrome c, which further activates downstream caspase-9 and caspase-3.Additionally, the depolarized MMP is associated with the inhibition of ATP, which is the energy required to maintain the efflux pump of P-gp [38].

Intracellular ATP level
The MMP is a critical parameter that reflects mitochondrial function, cellular activities, and ATP production.Following confirmation of MMP dissipation induced by HA/TT LP/PTX, we examined the effect of the four formulations on intracellular ATP levels in A549/T cells.As shown in Figure 8D, no significant differences were observed between the cells in the control group and those treated with free PTX.However, the three types of liposomes, were capable of reducing intracellular ATP levels, with HA/TT LP/PTX exhibiting the most potent inhibitory effect.Wang et al. reported that increased ROS levels could oxidize nicotinamide adenine dinucleotide (NADH) to NAD + and decrease ATP levels used for efflux pumps when photodynamic therapy was applied to mitochondria [39].Chen et al. reported that PTX-loaded micelles prepared using an amphiphilic TPGS-indomethacin polymer could decrease intracellular ATP levels and depolarize the MMP of mitochondria by interfering with the formation of the MPTP [40].Furthermore, Singh et al. demonstrated that the ATPase activity of P-gp could be inhibited by the long polyethylene glycol residue of TPGS.Therefore, polymers with polyethylene backbones, including TPP-TPGS, had the potential to decrease ATP levels [41].Collectively, the ATP inhibition observed with HA/TT LP/PTX was likely due to a combination of these factors.

Cell cycle
An established anticancer mechanism of PTX is its involvement in mitotic inhibition, G 2 /M phase cell cycle arrest, and apoptosis [5].In light of this, the effect of the four formulations on the cell cycle was investigated using flow cytometry.As shown in Figure 9A-B  by 1.4-, 1.5-, 1.7-, and 2.0-fold, respectively.Therefore, the enhanced G 2 /M phase block indicated that HA/TT LP/PTX was more effective than the other PTX formulations.Therefore, the increase in G 2 /M phase block suggested that HA/TT LP/ PTX was more effective than other PTX formulations.

Apoptosis
To certify the apoptosis-induced ability of the four formulations, an Annexin V-FITC/ PI staining assay was performed.As depicted in Figure 9C-D, the apoptotic percentage of the control group and four formulations were 3.1%, 6.6%, 14.2%, 16.1%, and 21.7%, respectively.It was reported that TPP-TPGS could interfere with the CII enzyme of the respiratory chain, leading to an enhancement of intracellular ROS in breast cancer 4T1 cells and thus greater apoptosis when the cells were exposed to TPP-TPGS [40].HA/TT LP/PTX induced more apoptosis than other formulations, which could be attributed to the mitochondria-targeted delivery of PTX and the ROS promotion of TT.
Apoptosis, a form of programmed cell death, is crucial for preventing cancer development, and inducing apoptosis has become a popular target for cancer treatment [42].There are two major apoptotic pathways: the exogenous (mitochondrial pathway) and endogenous pathways (death receptor pathway), both of which aim to initiate cell death.The Bcl-2 protein family plays a key role in apoptosis regulation, including anti-apoptotic/pro-survival proteins such as BCL-2, BCL-XL, as well as pro-apoptotic proteins such as Bax and Bak.In the mitochondrial apoptosis pathway, activated Bax forms multimeric pores in the mitochondrial membrane, leading to a decrease in MMP and an increase in membrane permeability [43].Subsequently, a series of apoptotic factors are released from mitochondria into the cytoplasm.The apoptosis initiator protease caspase-9 is then activated, followed by downstream effector caspase-3 activation, ultimately inducing apotosis.As shown in Figure 9E-F, after treatment with HA/TT LP/PTX, the expression of the anti-apoptotic protein Bcl-2 was downregulated, while the expression of Bax, cleaved caspase 3, and cleaved caspase 9 was upregulated, indicating the induction of apoptosis.

Conclusion
In summary, this study reports on the successful construction and preparation of a multifunctional two-stage targeted liposome aimed at improving the delivery of PTX to cells and overcoming MDR.The resulting HA/TT LP/PTX showed a dispersed spherical shape with uniform particle size, as well as good stability and safety.The use of HA in the liposome allowed for specific recognition with CD44 receptors, resulting in superior internalization in A549/T cells.Furthermore, the exposure of TT following the degradation of HA, which was triggered by the high HAase level in endosomes, facilitated the drug's delivery to the mitochondria.As a result of the accumulation of PTX in mitochondria, the function of these organelles was disturbed.This was accompanied by increased levels of ROS, decreased ATP levels, dissipated MMP, and increased G 2 /M phase arrest, ultimately leading to a higher apoptotic rate and stronger anticancer efficacy.In future work, it will be necessary to evaluate

Figure 1 .
Figure 1. the schematic illustration of two-stage targeted liposomes (Ha/tt lP/PtX) and their mitochondrial-targeting strategy.

Figure 3 .
Figure 3. the cell cytocompatibility of blank liposomes including lP, tt lP and Ha/tt lP by mtt cell viability assay in 293 t (a) and a549/t cell line (B).Hemolysis percentage of blank liposomes (c) and drug-loaded liposomes (d) with distilled water as positive control.

Figure 4 .
Figure 4. the changes of particle size (a) and ee% (B) of three kinds of liposomes stored for 10 days at 4 °c. the changes of particle size (c) and ee% (d) of three kinds of liposomes stored for 24 h in the presence of 50% fBs at 37 °c.

Figure 5 .
Figure 5. the changes of particle size (a) and zeta potential (B) of Ha/tt lP/PtX after storage at different pH values without Haase or with Haase within 4 h.(c) the accumulative drug release rate of free PtX, lP/PtX, tt lP/PtX and Ha/tt lP/PtX at pH 7.4 at 37 °c within 48 h.(d) the accumulative drug release rate of Ha/tt lP/PtX in different pH without Haase or in different pH with Haase at 37 °c within 48 h.

Figure 6 .
Figure 6.(a) relative cellular uptake of Ha/tt lP/c6 in a549/t cells at 4 °c or in the presence of specific endocytosis inhibitors.(B) Quantification of cellular uptake in a549/t cells treated with lP/c6, tt lP/c6 and Ha/tt lP/c6 for 4 h by flow cytometry.(c) confocal images of cellular uptake of c6-loaded liposomes in a549/t cells after incubation for 4 h, respectively.the mitochondria and nuclei were stained with mitotracker red and Hoechst33342, respectively.yellow spots in the merged picture denoted the co-localization of the liposomes and mitochondria.scale bars: 10 μm.***p < 0.001, Vs. control, ### p < 0.001, Vs. lP/c6.

Figure 7 .
Figure 7. Viability of the a549 cells and a549/t cells (c) after incubation with different PtX formulations for 48 h. the ic 50 of different PtX formulations in a549 cells (B) and a549/t cells (d).
, compared to the control group (G 2 /M, 22.5%), the G 2 /M phase of cells treated with free PTX, LP/PTX, TT LP/PTX, and HA/TT LP/PTX increased