Soy phospholipids (SPC), DPPG, cholesterol (Chol) and DSPE-PEG2K were purchased from AVT (Shanghai) Pharmaceutical Technology Co., Ltd. DSPE-PEG2K-NHS was purchased from Xi'an Ruixi Biological Technology Co., Ltd. PNA was purchased from Medicago AB. CDDP and sulfo-Cyanine7 carboxylic acid (Cy7) were purchased from Dalian Meilun Biology Technology Co., Ltd.

The human lung cancer cell lines A549 (MUC1-high) and H460 (MUC1-low) were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. A549 cells were cultured in Dulbecco's modified Eagle's medium (Dalian Meilun Biology Technology Co., Ltd.) supplemented with 10% heat-inactivated foetal bovine serum (FBS; Biological Industries), 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere containing 5% CO₂ at 37°C. H460 cells were cultured in RPMI 1640 medium (Dalian Meilun Biology Technology Co., Ltd.) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. A total of 29 male BALB/C-nu mice (weight, 13-17 g; age, 4-6 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The Ethical Review Committee of Shandong Second Medical University (Weifang, China) approved the present study (approval no. 2022SDL448), which complied with institutional animal care guidelines.

The relationship between MUC1 expression and the clinicopathological characteristics of patients with lung cancer was statistically analysed using relevant clinical data from TCGA database (Project Name: Lung Adenocarcinoma; portal.gdc.cancer.gov/projects/TCGA-LUAD), and the figures were generated using GEPIA (gepia.cancer-pku.cn) and cbioportal (cbioportal.org) (24). Imaging data were obtained from the Human Protein Atlas (proteinatlas.org). The present study complies with the National Institutes of Health TCGA Human Guidelines for Subject Protection and Data Access Policy, as well as the proteinatlas.org citation regulations for images.

Due to the presence of trace amounts of sulphides, heavy metals and some UV-absorbing impurities in the dialysis bags, they need to be pretreated prior to use. Briefly, after the bags were cut to size, they were boiled for 10 min in 2% sodium bicarbonate and 1 mmol/l EDTA (pH 8.0), and 1 mmol/l EDTA (pH 8.0), respectively. The bags were then washed on the inside and outside surfaces with purified water and stored at 4°C.

A total of 10 mg PNA was weighed, completely dissolved in PBS (pH 7.4), reconstituted into a PNA solution with a concentration of 5 mg/ml and set aside. Subsequently, 100 μl DSPE-PEG2K-NHS in DMSO solution (100 mg/ml) was added to the PNA solution, and after 4 h of incubation at room temperature, the entire solution was transferred to treated dialysis bags. Dialysis was performed three times using ddH₂O (2 h each time), and DSPE-PEG2K-PNA was obtained after freeze-drying at -60°C for 24 h. PNA and DSPE-PEG2K-PNA (10 μg) then underwent SDS-PAGE on 12% gels. Subsequently, the gels were stained with 0.25% Coomassie brilliant blue staining solution. After the SDS-PAGE was decolorized with decolorization solution (cat. no. P0017C; Beyotime Institute of Biotechnology) until it was nearly colourless and the bands were clear, the SDS-PAGE mixture was stained with PEG dye to confirm the successful synthesis of DSPE-PEG2K-PNA. The infrared absorption spectra of PNA, DSPE-PEG2K and DSPE-PEG2K-PNA were examined using an infrared absorption spectrometer, and the characteristic peaks of PNA, DSPE-PEG2K and DSPE-PEG2K-PNA were compared to determine the successful synthesis of DSPE-PEG2K-PNA.

CDDP pretreatment was performed according to the methods reported by Zahednezhad et al (24). According to Le Chatelier's principle, CDDP undergoes a hydration reaction in water to produce Pt(NH₃)₂Cl₂ + 2 H₂O ⇆ [Pt(NH₃)₂(H₂O)₂]²⁺ + 2Cl⁻, and the CDDP generated using this derivative approach has a positive charge on its surface, enabling its active loading in liposomes. The product generated by this reaction is the form in which CDDP undergoes active intracellular action, and thus its antitumour effect is not altered (25). A pretreated CDDP solution was prepared by dissolving CDDP in pure water. The solution was then placed in a magnetic stirrer and set at a speed of 150 rpm. The mixture was incubated at 65°C for 6 h. Finally, the solution was stored in the dark at 4°C.

Film dispersion was used to create blank liposomes (Lip) (26,27). In a clean round-bottomed flask, the phospholipids needed to make liposomes were measured in accordance with a mass ratio of SPC:DPPG:Chol:DSPE-PEG2K=15:4:15:4 (SPC:DPPG:Chol:DSPE-PEG2K:DSPE-PEG2K-PNA=15:4:15:4:3 for the PNA-modified liposomes). An ultrasonic cell washer was used to sonicate (20 kHz; 20% power at 0°C; duration, 2 sec) the aforementioned phospholipids to thoroughly dissolve and combine them; a chloroform-methanol ratio of 2:1 was used for this purpose. The chloroform-methanol mixture with dissolved lipid material was then transferred to an eggplant flask and linked to a rotary evaporator (Yamato Scientific Shanghai Corp.). The rotary evaporator speed, water bath temperature and pressure were adjusted so that all the organic solvents were evaporated slowly and uniformly until a homogeneous lipid film was visible to the naked eye, after which the rotary evaporator pressure was adjusted to the maximum and maintained for >30 min to ensure that all the organic reagents were removed 5 ml ddH₂O was then added to the eggplant bottle, which was submerged in the ultrasonic cell washer for 5 min to ensure that all the lipid films were eluted; the liposomes were hydrated using the rotary evaporator; and after 1 h, the hydrated liposomes were sonicated (20 kHz; 20% power at 0°C) for 5 min using the probe and filtered three times using 0.45 and 0.22-μm filters. The dialysis bags were removed and completely washed with ethanol on the surface and inside with pure water, and all of the filtered liposomes were then transferred to the washed bags for dialysis. The liposomes were dialyzed using ddH₂O for 6 h to eliminate any phospholipids that did not form liposomes. The water was replaced every 2 h to ensure thorough dialysis. Finally, the liposomes were collected from the dialysis bag and stored at 4°C.

The method used for the preparation of CDDP-Lip was essentially the same as that aforementioned. Briefly, the formed lipid film was subsequently hydrated with the pretreated CDDP solution at a ratio of 10:1 total phospholipid mass to drug mass, and the hydration time was suitably extended to allow the pretreated CDDP to be well encapsulated by the liposomes, followed by sonication (20 kHz; 20% power at 0°C; duration, 2 sec) of the CDDP-Lip for 10 min using a probe. CDDP-Lip were obtained by dialysis with ddH₂O three times (2 h each time) and were stored at 4°C.

Liposomes were labelled with Rhodamine B (RhB) and Cy7 fluorescent probes to study the cellular absorption and biodistribution of the preparations in vitro (RhB) and in vivo (Cy7) (28-30). The method used for the preparation of Cy7-loaded liposomes (Cy7-Lip) or RhB-loaded liposomes (RhB-Lip) was essentially the same as that aforementioned. Notably, the total mass of phospholipids relative to the mass of the fluorescent marker was 50:1 during hydration and when the fluorescent marker was added and hydrated.

Several types of liposomes (CDDP-PNA-Lip for electron microscopy; Lip, CDDP-Lip, PNA-Lip and CDDP-PNA-Lip for characterisation measurements) were diluted 10 times with ddH₂O. Subsequently, a transmission electron microscope (TEM; JEOL, Ltd.) was used to examine the samples. Samples were prepared by negative staining with phosphotungstic acid. Briefly, the sample was added dropwise directly onto a copper grid and left to stand at room temperature for 3 min before excess liquid was aspirated. After waiting for the sample to dry completely, 2% phosphotungstic acid was added dropwise, and after 2 min of staining at room temperature, the excess liquid was aspirated, and the sample was left to dry naturally. Using a Malvern Zetasizer Nano ZS 90 (Malvern Panalytical, Ltd.), the ζ potentials and particle size distributions were measured.

High-performance liquid chromatography (HPLC) was used to quantify drug loading (DL) and entrapment efficiency (EE). Briefly, the HPLC was performed using an Agilent 1260 Infinity II system (Agilent Technologies, Inc.), with a column size of 4.6×200 mm packed with Hypersil ODS2 (filler particle size, 5 μm; Dalian Elite Analytical Instruments Co., Ltd.) on a sample size of 20 μl. The mobile phase consisted of a mixture of methanol and water (v/v=75:25) at a flow rate of 1 ml/min, and the column temperature was maintained at 25°C. The standard was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (cat. no. B24462), and the peak areas of 100, 75 and 50 μg/ml were measured to determine the standard curve (31). Triton X-100 (0.1%) was applied to quickly release the internal CDDP solution and destroy the CDDP-Lip. After mixing sodium diethyldithiocarbamate (DDTC) and the sample, the mixture was subsequently incubated in a water bath at 37°C. Afterward, the sample was thoroughly mixed with chloroform, and the complex formed by DDTC and CDDP was extracted and detected. The EE and DL of the CDDP-Lip were calculated using the following formulas: EE ( % ) = Mass of the CDDP loaded/Total mass of the CDDP added x100 DL ( % ) =  Mass of the CDDP loaded/Total mass of liposomes x100

The stabilities of the liposomes, PNA-liposomes, CDDP-Lip and CDDP-PNA-Lip were investigated by removing liposomes stored in ddH₂O at 4°C every 2 days for 14 days, and measuring their particle sizes and ζ potentials.

The drug release profiles of CDDP, CDDP-Lip and CDDP-PNA-Lip were measured using in vitro dialysis. The CDDP concentrations of the different groups were adjusted to the same level using PBS and then added to the dialysis bags, which were subsequently placed into conical flasks filled with 100 ml PBS. The samples were incubated at 37°C in a constant temperature shaker at 100 rpm, and the PBS in the conical flask was removed at different time points and replenished to 100 ml with fresh PBS. The cumulative in vitro release curves of CDDP were plotted using HPLC, as aforementioned, to determine the CDDP concentration in the samples at different time points, with time as the horizontal coordinate and the cumulative percentage release of CDDP as the vertical coordinate.

MUC1-positive cells were detected via western blotting. Briefly, the A549 and H460 cell lines were inoculated into culture dishes and cultured for 48 h in complete medium supplemented with 10% FBS. After the cells were washed with PBS, the proteins were extracted from the remaining cells with protein extraction buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 10 mM MgCl₂, 1% Triton X-100 (v/v), 1% sodium deoxycholate (v/v), 0.1% SDS (v/v), 5 mg/ml sodium orthovanadate]. Protein quantification was performed using a BCA assay. Subsequently, proteins (10 μg) were separated by SDS-PAGE (10% resolving gel and 4% stacking gel). The proteins were then transferred to BioTrace nitrocellulose membranes, which were blocked with 5% non-fat dry milk at 37°C for 1 h. The membranes were then incubated with the MUC1 (D9O8K) XP^® Rabbit mAb (1:1,000; cat. no. 14161; Cell Signaling Technology, Inc.) and β-actin antibody as a loading control (1:1,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.) overnight at 4°C, followed by incubation with anti-rabbit IgG and anti-mouse IgG, HRP-linked secondary antibody (1:2,000; cat. nos. 7074S and 7076S; Cell Signaling Technology, Inc.) for 1 h at room temperature. The blots were visualized using WesternBright ECL, (cat. no. 230329-20; Advantsa Inc.) and underwent densitometric analysis using ImageJ (version 2.9 011.53t; National Institutes of Health). Subsequently, MUC1 expression in the cell lines was detected by western blotting to distinguish between MUC1-positive cell lines and MUC1-negative cell lines.

The cellular uptake of CDDP-PNA-Lip was simulated by observing the in vitro uptake of RhB fluorescent liposomes by A549 and H460 cells through laser confocal microscopy. The targeting efficiency of CDDP-PNA-Lip was evaluated. A549 and H460 cells were seeded at a density of 5×10⁵ cells/dish in Petri dishes suitable for laser confocal microscopy using complete medium supplemented with 10% FBS. The cells were incubated at 37°C for 24 h. Subsequently, different concentrations of RhB, RhB-Lip and RhB-loaded PNA-modified liposomes (RhB-PNA-Lip) were added to the medium (at a final RhB concentration of 50 μg/ml), and the mixture was incubated for 6 h at 37°C. Afterwards, the medium was removed, and the cell surface was cleaned using precooled PBS at 4°C. Subsequently, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature and washed 3-5 times with PBS to remove residual paraformaldehyde and unfixed cells. Next, the cells were treated with 0.1% Triton X-100 for 10 min at room temperature to permeabilize the cell and nuclear membranes, and were washed 3-5 times with PBS until the PBS was clear and foam free. The nuclei were then stained with DAPI staining solution for 10 min at room temperature and washed 3-5 times with PBS to remove any residual DAPI and the samples were finally observed using a laser confocal microscope.

The cytotoxicity of various drugs was assessed using the MTS assay, and the reaction product was dissolved in DMEM). Briefly, A549 and H460 cells were inoculated into 96-well plates at a density of 5,000 cells/well and were cultured in complete medium for 24 h. Once cell adhesion and extension were observed, various concentrations of CDDP and hydrated CDDP (concentration gradient, 50.00, 25.00, 12.50, 6.25, 3.12, 1.56, 0.78 and 0.39 μg/ml) were added. After incubation for 48 h, the optical density (OD) values were measured using a microplate reader (PerkinElmer, Inc.) at 490 nm. The rate of cell viability was calculated using the following equation: Cell viability (%)=(ODsample-ODblank)/(ODcontrol-OD-blank) ×100.

The cytotoxicity of CDDP, CDDP-Lip and CDDP-PNA-Lip was assessed using the MTS assay, and the reaction product was dissolved in DMEM. Briefly, A549 and H460 cells were inoculated into 96-well plates at a density of 5,000 cells/well and were cultured in complete medium for 24 h. Once cell adhesion and extension were observed, various concentrations of drugs (concentration gradient, 50.00, 25.00, 12.50, 6.25, 3.12, 1.56, 0.78 and 0.39 μg/ml) were added. After incubation for 48 and 24 h, the OD values were measured using a microplate reader (PerkinElmer, Inc.) at 490 nm. The rate of cell viability was calculated using the following equation: Cell viability (%)=(ODsample-ODblank)/(ODcontrol-OD-blank) ×100.

The percentage of NSCLC cells that underwent apoptosis after various drug treatments was analysed using the FITC Annexin V Apoptosis Assay kit (cat. no. 556547; BD Biosciences). A549 and H460 cells were inoculated into 6-well plates at a density of 3×10⁵ cells/well, cultured in complete medium for 24 h, and were treated with 2.5 μg/ml CDDP, CDDP-Lip or CDDP-PNA-Lip for 48 h at 37°C. Subsequently, all of the cells were collected and washed twice with precooled PBS at 4°C. The cells were diluted to 1×10⁶ cells/ml using 1X binding buffer, and were then co-incubated with FITC-conjugated Annexin V and PI (binding buffer:FITC-conjugated Annexin V/PI=20:1, v/v) staining solution for 15 min at room temperature in the dark. Subsequently, the incubation was terminated by the addition of 1X binding buffer, and the cells were analysed using the BD Accuri C6 Plus Flow Cytometer (BD Biosciences) and BD Accuri C6 Plus software (version 1.0.34.1; BD Biosciences).

The effect of CDDP, CDDP-Lip and CDDP-PNA-Lip treatments on the lateral migration ability of NSCLC cells was examined using a cell scratch wound healing assay. A549 and H460 cells in the logarithmic growth phase were inoculated into separate 6-well plates. Once the cells had grown to cover the entire well, various drugs were added to each well (2.5 μg/ml, with normal saline used as a control) and the medium was replaced with medium containing 1% FBS. The cell surface (100% confluence) was then scratched using a sterile 200-μl pipette tip and the cells were incubated at 37°C for 24 h. Each scratch was imaged at 0 and 24 h using an inverted light microscope (Nikon Corporation). The rate of cell migration was measured using ImageJ Plus (version 2.9 011.53t; National Institutes of Health).

Scratch wound healing rate (%)=(initial scratch width-scratch width at observation)/initial scratch width ×100.

Transwell assays (6.5 mm Transwell^® with 0.4 μm pore polycarbonate membrane insert; cat. no. 3413; Corning, Inc.) were used to measure the longitudinal migration capacity of NSCLC cells treated with different formulations. A total of 2,000 A549 and H460 cells were separately inoculated into Transwell chambers containing medium supplemented with 1% FBS. Complete medium containing 10% FBS was added to the lower chamber. After 24 h incubation at 37°C, the dead cells were washed and removed from the inside of the Transwell chambers. Subsequently, DAPI staining was performed as aforementioned. The cells were then observed and images were captured using an inverted fluorescence microscope (Motic Incorporation, Ltd.). The number of cells was calculated using ImageJ Plus.

In the present study, the construction of a xenograft mouse model was performed by subcutaneous injection. A total of 29 male nude mice (age, 4-6 weeks; weight, 15±2 g) were used in the present study; 9 mice underwent this experiment and 20 mice underwent the subsequent experiment. The mice were maintained in a specific pathogen-free environment at a temperature of 20-26°C and humidity of 40-70%. The mice were provided free access to sterile food and water and were maintained under a 12-h light-dark cycle. The humane endpoints were as follows: Tumour volume reaching 10% of body weight of the mouse; the tumour severely affected the mobility or quality of life of the mouse; or if the mouse showed signs of significant pain and distress. Each nude mouse was subcutaneously injected with 5×10⁶ A549 cells diluted with 0.2 ml PBS in the left hind limb. After the tumours were considered palpable, the tumour volume was measured every 2 days. The subcutaneous tumour volume was close to 200 mm³ when mice with comparable tumour volumes and body weights were selected and randomly divided into three groups [Cy7, Cy7-Lip, Cy7-loaded PNA-modified liposomes (Cy7-PNA-Lip); n=3/group]. Cy7 instead of CDDP was encapsulated into liposomes and the position of Cy7 was reflected by small animal imaging. Cy7-Lip and Cy7-PNA-Lip were prepared to mimic the distribution of CDDP in tumour-bearing nude mice. The mice were injected with 10 mg/kg Cy7, Cy7-Lip or Cy7-PNA-Lip via tail vein within 15 min. Fluorescence images of Cy7 in tumour-bearing nude mice were acquired and analysed using the IVIS Spectrum (PerkinElmer, Inc.) after mice were anaesthetized by inhalation using isoflurane at 2, 4, 8, 12, 24 and 48 h post-injection. During the induction phase, the isoflurane concentration was set at 4%, and during maintenance anaesthesia, the isoflurane concentration was reduced to 2% to maintain stable anaesthesia while reducing the effects on the physiological functions of the mice. The anaesthetic gas was dispersed through oxygen to ensure that the mice received an adequate supply of oxygen throughout the process. All experimental animals were euthanized by CO₂ inhalation 48 h after injection (volumetric emission rate of 4 l/min; ~40% container volume/min) to detect the distribution of Cy7 in major organs and tumour sites in nude mice. Fluorescence images of Cy7 were then acquired and analysed using the IVIS Spectrum Small Animal Live Imaging System and the accompanying software Living Image (version 4.7.2.20319; PerkinElmer, Inc.).

The present study investigated the in vivo antitumour activity of CDDP-PNA-Lip by treating nude mice with subcutaneous tumours. An animal model of subcutaneous tumour-bearing nude mice was established using male BALB/C-nu nude mice (age, 4-6 weeks; weight, 15±2 g). The nude mice were housed for 1 week in a specific pathogen-free grade environment at a temperature of 20-26°C and, humidity of 40-70%. The mice were provided free access to sterile food and water and were maintained under a 12-h light-dark cycle. Each nude mouse was injected subcutaneously with 5×10⁶ A549 cells diluted with 0.2 ml PBS in the left hind limb, and the tumour volume and body weight of the mice were measured every 2 days. A total of 14 days after the subcutaneous injection, the subcutaneous tumour volume in the mice approached 100 mm³. The mice were then randomly divided into four groups (n=5/group) and administered the following solutions: Saline, CDDP saline solution, CDDP-Lip saline solution or CDDP-PNA-Lip saline solution via intraperitoneal injection. All four solutions were administered at a dose of 2 mg/kg every 4 days for a total of 24 days. Tumour volume and body weight were measured every 2 days. After the length and width of the tumour were measured using digital Vernier callipers, the tumour volume was estimated using the following formula: Tumour volume=(tumour length) × (tumour width)² ×0.5. Tumour growth inhibition was measured using the following formula: Tumour growth inhibition value (%)=[1-RTV (experimental group)/RTV (control group)] ×100, where RTV indicates relative tumour volume.

After 24 days, the animals were euthanized by CO₂ inhalation, blood was collected from the heart, and the tumours, liver, kidney, heart, spleen and lungs were also collected. The tissues were weighed, fixed in 4% paraformaldehyde at room temperature for 6 h and processed. The blood was treated with sodium heparin and centrifuged at 500 × g and 4°C for 10 min to extract the serum. All organs and tumour tissues were embedded in paraffin for haematoxylin and eosin (H&E) staining. Sections (6 μm) were stained using haematoxylin for 7 min at room temperature and eosin for 1 min at room temperature and were observed under a light microscope, while the serum was analysed for liver and kidney function indicators. Alanine transaminase (ALT; cat. no. H001), alkaline phosphatase (ALP; cat. no. H004), blood urea nitrogen (BUN; cat. no. H044) and creatinine (Cr; cat. no. H047) test kits were obtained from Weifang Kanghua Biotechnology Co., Ltd.

The experiments were independently conducted three times. After the data were entered into GraphPad Prism (version 8.0; Dotmatics), statistical analysis was performed using an independent sample t-test for two groups of data or one-way ANOVA followed by Dunnett's multiple comparisons test for three or more groups of data. Data are presented as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

The bioinformatics analysis of NSCLC tissues revealed that, compared with in normal tissues from patients with lung adenocarcinoma, there was a trend towards elevated expression levels of MUC1 in NSCLC tissues; however, this difference was not significant (Fig. 1B). In addition, >15% of patients with NSCLC exhibited upregulated MUC1 expression (Fig. 1A). These findings suggested that the development of therapeutic regimens targeting MUC1 may be important for patients with NSCLC and has significant clinical implications. Moreover, immunohistochemical staining results from the Human Protein Atlas database revealed that MUC1 was widely and intensively expressed in malignant tumours (Fig. 1C and D), which was consistent with the results of the bioinformatics analysis, further indicating that the present study has good clinical application potential. The results revealed that cells with abnormally elevated tumour MUC1 expression were mainly distributed on the tumour surface, which facilitated the targeting of liposomes for more precise localization at the tumour site.

PNA-modified DSPE-PEG2K was synthesized by reacting the active ester (-NHS) of DSPE-PEG2K-NHS with an amino group (-NH₂) on PNA to form an amide bond (-CO-NH-). The successful synthesis of DSPE-PEG2K-PNA was verified by the characteristic peaks of DSPE-PEG2K-PNA detected using infrared spectroscopy and SDS-PAGE (Fig. 2A and B). Images of PEG distribution using PEG dye (Fig. 2Ba) and Coomassie brilliant blue staining of SDS-PAGE gels (Fig. 2Bb) are shown.

As shown in Table I and Fig. 3A, the prepared CDDP-PNA-Lip were heterogeneous and stable, and could effectively encapsulate CDDP. The particle sizes of Lip and PNA-Lip were ~80 nm, while the particle size of the liposomes increased to ~120 nm after encapsulating CDDP. This increase in size is likely due to the electrostatic force of some of the CDDP molecules on the surface of the liposomes. The polydispersity index of each liposome was ~0.2, which represented good lipid homogeneity. The ζ potential of CDDP-Lip was ~−30 mV, and the encapsulation of CDDP did not result in a change in the surface charge. This finding is consistent with the findings of other protocols for DL that rely on electrostatic forces of adsorption (24). The EE was >35%, and the DL capacity was greater than 3%. This DL capacity was greater than that of reported CDDP-Lip (32), which achieved only a 1% DL capacity. Since the DL of CDDP was achieved through electrostatic adsorption of liposomes, it led to an increase in the particle size of liposomes encapsulating CDDP compared to those that were not encapsulated. The morphology of the CDDP-PNA-Lip was observed using a TEM. The results indicated that the PNA-CDDP-Lip were uniform three-dimensional spheres with smooth surfaces and particle sizes (Fig. 3E).

The in vitro dialysis experiments were performed using dialysis bags, and the in vitro release profile of CDDP-Lip is shown in Fig. 3B. Depending on the release rate, the cumulative release of CDDP from unmodified liposomes exhibited two distinct phases, i.e., a rapid release phase and a sustained release phase. The rapid release phase lasted for ~2 h, during which ~20% of the CDDP was released from the liposomes; this phase was followed by a sustained release phase, during which CDDP continued to be slowly released; the cumulative release of the drug was ~35% after 48 h of the experiment. Although the cumulative release of CDDP from the modified liposomes also showed two phases, its release profile was smoother than that of unmodified liposomes, and it had a better slow-release effect than that of unmodified liposomes within 12 h. Notably, ~20% of CDDP was released from CDDP-PNA-Lip in the first 4 h, followed by a sustained release phase at a slower rate; the first 20% seemed to be more quickly released from CDDP-PNA-Lip than from CDDP-Lip within 4 h. It was hypothesized that this result may be due to the addition of phospholipids containing PEG components within the liposomes, which allowed CDDP-Lip and CDDP-PNA-Lip to exhibit a sustained and stable release during the experiment. The results suggested that encapsulating CDDP with liposomes may prolong the release time of the drug, which is beneficial for achieving sustained release at the tumour target (Fig. 3B).

All the liposomes were diluted 10-fold with ddH₂O and stored at 4°C in the dark. The particle size and potential of the liposomes were monitored every 2 days, and the corresponding change curves are presented in Fig. 3C and D. The particle size of all the liposomes fluctuated within a range of ±10 nm and did not change significantly across 15 days. Similarly, the potential did not vary significantly, indicating that the liposomes prepared in the present study were stable.

Western blotting was used to detect the expression levels of MUC1 in the A549 and H460 cell lines. The grayscale analysis revealed that MUC1 was expressed at significantly higher levels in A549 cells than in H460 cells (Fig. 4A and B).

The effect of ligand modification on improving the liposomal delivery efficiency was explored by performing in vitro cellular uptake assays (Fig. 5). The MUC1-negative NSCLC cell line used in the experiment was H460, and the MUC1-positive NSCLC cell line was A549. The impact of the ligand modification on enhancing the efficiency of liposomal drug delivery was studied by examining the cellular uptake of each liposome type using confocal laser scanning microscopy (CLSM). The uptake of each type of RhB liposome was assessed in A549 and H460 cells after 6 h. As shown in Fig. 5A and B, CLSM revealed the strongest red fluorescence in the A549 cell line treated with RhB-PNA-Lip. The quantitative analysis showed that the intensities of red fluorescence in the RhB-PNA-Lip group were 1.53 and 1.24-fold greater than those in the RhB and RhB-Lip groups, respectively. These results indicated that the PNA modification enhanced the uptake of liposomes by A549 cells. By contrast, RhB-PNA-Lip did not exhibit a stronger fluorescence intensity in the H460 cell line. These findings suggested that the modification of liposomes with PNA significantly improved their targeting effect on MUC1-positive NSCLC cells, such as A549 cells.

Investigations into the cytotoxic efficiency of ligand-modified liposomes were conducted using the MTS assay to determine the viability of A549 and H460 cells treated with CDDP-Lip. The MTS assay results showed no significant difference in cytotoxicity between CDDP and hydrated CDDP in the two cell lines (Fig. 6A and B), which was previously described by Lippard (31), where hydrated CDDP regenerated CDDP in the presence of Cl⁻, which did not affect its antitumour activity. After treating A549 and H460 cells with CDDP, CDDP-Lip and CDDP-PNA-Lip for different durations, it was revealed that CDDP had a dose-dependent antitumour effect. After 48 h of drug treatment, the A549 cells in the CDDP-PNA-Lip treatment group exhibited the lowest cellular viability, which was not observed in the three treatment groups of H460 cells (Fig. 6C-F). This finding suggested that the ligand-modified liposomes may have greater delivery efficiency and stronger cytotoxicity against MUC1-positive NSCLC cell lines.

The results of the MTS experiments prompted the evaluation of the apoptosis of A549 and H460 cells treated for 48 h with 2.5 μg/ml CDDP and liposomes. The levels of Annexin V on the cell surface were detected using flow cytometry. The results revealed no significant difference in the effects of CDDP-Lip or CDDP-PNA-Lip on early apoptosis in MUC1-negative H460 cells (Fig. 6G). For MUC1-positive A549 cells, the percentage of apoptotic cells in the CDDP-PNA-Lip group was 1.5 times higher than that in the CDDP-Lip group (Fig. 6G). These findings suggested that modification of CDDP-Lip with PNA may induce the apoptosis of MUC1-positive NSCLC cells.

Notably, the utilization of liposomes to encapsulate CDDP seems to diminish its antitumour efficacy on H460 cells. It may be hypothesized that this is because liposomes exhibit a sustained-release effect on the drug, and cells uptake liposomes with varying efficiency. This phenomenon was also observed by Diao et al (33).

Cell scratch wound assays and Transwell assays were used to verify the lateral and longitudinal migration abilities of the different PNA-modified liposomes. For the cell scratch wound assay, normal saline, free CDDP, CDDP-Lip or CDDP-PNA-Lip were added to the scratched cells in 6-well plates, which were subsequently cocultured with the cells for 24 h. Images of the cells were captured at 0 and 24 h. The scratch wound healing rate of the A549 cells in the normal saline group was 19.32±2.93%, whereas that in the CDDP-PNA-Lip group was the smallest, with a scratch wound healing rate of 9.08±2.03% (Fig. 7A and C). These rates were significantly lower than those in the CDDP group and CDDP-Lip group (14.38±1.48 and 14.59±2.51%, respectively). The healing rate of the saline group (13.77±1.89%) was notably higher than that of the free CDDP, CDDP-Lip and CDDP-PNA-Lip groups (8.36±2.81, 8.26±2.83 and 9.00±2.58%, respectively) in A549 cells; however, no significant differences were observed among the drug treatment groups in H460 cells (Fig. 7A and C).

In the Transwell assay, the cells were cultured for 24 h, stained with DAPI and images were captured using an inverted fluorescence microscope. The number of A549 cells treated with CDDP-PNA-Lip was the lowest in the field of view and was significantly lower than that in the other groups; however, no significant differences were observed in H460 cells (Fig. 7B and D). These results indicated that the ability of liposomes to inhibit tumour cell migration was enhanced when they were modified with PNA.

The effects of the ligand modification on enhancing target acquisition in vivo were investigated by monitoring animals with near-infrared fluorophore imaging. Cy7 was encapsulated in liposomes to mimic the circulation of CDDP in nude mice. Cy7, Cy7-Lip and Cy7-PNA-Lip were then injected into the nude mice via the tail vein. A total of 4 h after injection, Cy7-PNA-Lip began to distribute predominantly to the tumour site in nude mice and was most evident at 8 h. Over time, Cy7 was metabolized and excreted from the nude mice. The fluorescent signal in the bodies of the mice gradually weakened, and all residual fluorescence disappeared completely after 24 h (Fig. 8A). Observations of mice injected with Cy7-PNA-Lip revealed that the fluorescent signal at the tumour site was markedly stronger than that in mice injected with Cy7 or Cy7-Lip. This high signal intensity persisted for 48 h. Before the nude mice were euthanized, the mice injected with Cy7-PNA-Lip still exhibited notably greater fluorescent signals at the tumour site than the other two groups (Fig. 8A). Subsequently, the mice were euthanized, fluorescence imaging was conducted, and an analysis of their major organs and tumour sites was performed. The results revealed that the tumours of nude mice injected with Cy7-PNA-Lip still exhibited strong fluorescent signals even after 48 h (Fig. 8B and C). Furthermore, due to the absence of an epidermal barrier, the fluorescence intensity at the tumour sites was significantly higher in the Cy7-PNA-Lip-injected nude mice than in the other two groups. These results indicated that the in vivo targeting ability of PNA-Lip aligns with the results of cellular uptake. Furthermore, the PNA modification of liposomes may enable them to actively target tumours. In addition, based on the change in the fluorescence intensity of Cy7 within the tumours of Balb/c-nu mice, the modification of liposomes with PNA extends the duration of drug presence in tumours and enhances drug accumulation at the tumour site. It was thus hypothesized that the utilization of PEG and PNA may enhance the active targeting ability of a drug and prolong its presence at the tumour site. These characteristics, in turn, could reduce the required drug dosage and frequency of administration.

The in vivo antitumour efficacy of CDDP-PNA-Lip was evaluated by treating nude mice bearing subcutaneous tumours with saline, CDDP, CDDP-Lip or CDDP-PNA-Lip. By monitoring and analysing the body weights of the animals, no significant weight loss was observed in any of the four groups after drug administration (Fig. 9B). This finding suggested that the drug treatment had few toxic side effects. After the initial treatment (day 0; Fig. 9A), the growth of the tumour tissue stopped in all groups of subcutaneous tumour-bearing nude mice. However, as the number of doses increased (from day 4; Fig. 9A), the tumour tissue in the subcutaneous tumour-bearing nude mice treated with CDDP and CDDP-Lip continued to grow, whereas that in the mice treated with CDDP-PNA-Lip showed sustained tumour suppression. The results indicated that CDDP-PNA-Lip exerted the strongest antitumour effect, with a tumour inhibition rate of 83.1%. The tumour volume of the mice treated with CDDP was 2.65 times larger than that of the mice treated with CDDP-PNA-Lip, while the tumour volume of the mice treated with CDDP-Lip was 1.63 times larger than that of the mice treated with CDDP-PNA-Lip. At the end of the treatment, all mice were sacrificed and the tumours were weighed, the results showed that mice treated with CDDP-PNA-Lip had the smallest tumour weights, which were significantly different compared with the other groups (Fig. 9C-E). H&E staining revealed that the tumour tissues in the saline control group were densely packed, whereas those in the CDDP-PNA-Lip group exhibited the highest levels of apoptosis and necrosis. A large number of vacuoles appeared between the tissues, indicating the significant antitumour efficacy of CDDP-PNA-Lip (Fig. 10A). The modification of liposomes with PNA significantly enhanced the antitumour efficacy of the liposomes against NSCLC tissues with high MUC1 expression.

The biosafety and antitumour effects of CDDP-PNA-Lip were evaluated using H&E staining. After histological sectioning and staining of the heart, liver, spleen, lungs and kidneys, no histopathological abnormalities were found in any of the four groups of animals (Fig. 10A). The effect of CDDP-PNA-Lip on liver and kidney function in nude mice was evaluated by measuring physiological indicators of liver and kidney function in serum (Fig. 10B). The results showed that biochemical indices, such as Cr, ALT, ALP and BUN, were not significantly altered in any of the groups. These results indicated that liposomes modified with PNA and containing CDDP colloids do not cause significant systemic toxicity in experimental mice.