Next Article in Journal
Multidrug Resistance-Associated Proteins 3 and 5 Play a Role in the Hepatic Transport of Mercuric Conjugates of Glutathione
Previous Article in Journal
Measurement of the Infection and Integrity of Monkeypox Virus: A New Method Using PMAxx-ddPCR
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 26
Issue 3
10.3390/ijms26031196
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Cetuximab-Immunoliposomes Loaded with TGF-β1 siRNA for the Targeting Therapy of NSCLC: Design, and In Vitro and In Vivo Evaluation

by
Yanan Shi
1,*,†,
Houqian Zhang
1,†,
Hao Chen
1,
Jianwei Guo
2,
Ranran Yuan
1,
Yu Tian
1,
Quanlin Xin
1,
Zhen Mu
1,
Yuping Tao
1,
Yongchao Chu
1,
Aiping Wang
1,
Zhiwen Zhang
3,
Jingwei Tian
1 and
Hongbo Wang
1,*
1
Key Laboratory of Molecular Pharmacology and Drug Evaluation, Ministry of Education, School of Pharmacy, Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China
2
Shandong Laboratory of Advanced Materials and Green Manufacturing at Yantai, Yantai 264006, China
3
School of Pharmacy, Fudan University, Shanghai 200437, China
*
Authors to whom correspondence should be addressed.
†
Yanan Shi and Houqian Zhang contributed equally to this work.
Int. J. Mol. Sci. 2025, 26(3), 1196; https://doi.org/10.3390/ijms26031196
Submission received: 3 January 2025 / Revised: 24 January 2025 / Accepted: 27 January 2025 / Published: 30 January 2025
(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)
Browse Figures
Versions Notes

Abstract

:
Transforming growth factor-β1 (TGF-β1) promotes the growth and metastasis of lung cancer cells. Therefore, TGF-β1 siRNA (siTGF-β1) gene therapy was introduced to inhibit the expression of TGF-β1 at the nucleic acid level to avert tumor growth and metastasis. However, the delivery of naked siRNA is typically restricted by a short half-life in vivo, difficulties in delivery in vivo, and safety issues. Using siTGF-β1 as a model drug, we established an actively targeted immunoliposome delivery system to investigate the role of siTGF-β1 in non-small-cell lung cancer (NSCLC). The results showed that the constructed immune liposomes were in a position to deliver siTGF-β1 to tumor cells, thus achieving a series of effects such as improving the poor stability and short half-life of naked siRNA. RNA interference of siTGF-β1 reduced the cell viability, growth, and migration potential of human non-small cell lung cancer cells (A549). Moreover, in an A549 tumor-bearing nude mouse model, siTGF-β1 transfection markedly reduced tumor growth and tumor volume. Inhibiting TGF-β1 diminished cancer cell viability and migration and promoted apoptosis in NSCLC, as confirmed by the findings of this study. Therefore, targeting siTGF-β1 with immunoliposomes may be a new therapeutic strategy for treating non-small-cell lung cancer.

1. Introduction

Non-small-cell lung cancer (NSCLC) is a common malignancy with high incidence [1] that is difficult to treat due to malignant migration and metastasis. In the past, the predominant lung cancer treatment avenues were chemotherapy and radiotherapy; however, traditional platinum-based chemotherapy schemes had limited benefits in terms of overall survival rates, and deteriorating physical conditions severely affected the quality of life of patients [2]. In 2004, mutations in the epidermal growth factor receptor (EGFR) were considered to be associated with NSCLC [3]. Approximately 10–30% of NSCLC neoplasms exhibit activating mutations in the tyrosine kinase structural domain of the EGFR gene, with an increased incidence of up to 60% in Asians patients [4]. In patients with metastable NSCLC with sensitized EGFR mutations, the first line of treatment is Osimertinib. Further first-line options approved by the FDA include Dacomitinib, Afatinib, Erlotinib, and Geftinib [5]. The two most common EGFR activation mutations are deletions in exon 19 and amino acid substitutions in exon 21, which make up 90% of the respective known mutations [6]. Therefore, EGFR mutations are biomarkers of lesions and therapy targets.
Transforming growth factor-β (TGF-β), particularly TGF-β1, is a key regulator of cell growth and is frequently upregulated in tumor cells, including those of non-small-cell lung cancer (NSCLC) [7]. TGF-β1 activation and Smad3 phosphorylation promote tumor growth and metastasis, highlighting the importance of targeting TGF-β1 for therapeutic interventions [7,8]. RNA interference using small interfering RNA (siRNA) offers a promising strategy to downregulate TGF-β1 expression. However, naked siRNA suffers from poor stability, a short half-life, and potential off-target effects, limiting its clinical application [9]. To address these challenges, various delivery systems have been developed, including viral vectors, which are highly efficient but limited by their toxicity and immunogenicity, and non-viral vectors such as cationic lipids and nanoparticles, which offer better biocompatibility and safety [10].
Regarding siRNA-loaded carriers, Polyethylene Glyco(PEG)-modified cationic liposomes are frequently used for siRNA transfection in vivo. PEG modification can protect siRNA liposomes from degradation by blood components such as macrophages, thus prolonging their retention time in blood circulation [11]. However, this approach exhibits negligible targeting effects. Tumor surface-associated antigens can be specifically bound by monoclonal antibodies, and subsequently, the drug or gene is translocated to the tumor cells with almost no effect on normal tissue. Cetuximab (CTX) is a monoclonal antibody chimeric with human/mouse derivative IgG1, which binds to the extracellular domain of EGFR with higher affinity than any endogenous ligand [12]. The use of CTX for NSCLC treatment has been widely proposed and has exhibited promising results [13,14,15]. Further, several studies have successfully linked CTX with nanocarriers to improve targeted delivery [15,16].
In this study, PEG cationic liposomes were modified with CTX to load TGF-β1 siRNA (siTGF-β1) for delivery to the tumor site, where the siRNA exerts gene silencing and inhibits TGF-β1 expression at the nucleic acid level. A further objective was to determine that the knockdown of TGF-β1 can achieve the purpose of inhibiting tumor growth and metastasis (Figure 1). After the successful construction of immunoliposomes, we assessed cytotoxicity, cell uptake, lysosome escape, cell migration and invasion in A549 cells. We further conducted tissue distribution and pharmacodynamic experiments in A549 tumor-bearing nude mice and evaluated treatment safety. We propose synthesizing siRNA delivery platforms for active targeted delivery, thus inspiring novel strategies for the treatment of NSCLC.

2. Results

2.1. Characterization of Liposomes

PEGylated siTGF-β1 cationic liposomes (siTGF-β1@LP) was composed of DOTAP/DOPE/CH/CLS-PEG2000/CLS-PEG2000-Mal (molar ratio 1:3:1.5:0.12:0.03) and was then functionalized with cetuximab. Liposomes with CLS-PEG2000-Mal were used to couple thiolated cetuximab onto the distal end of the PEG polymer, forming immunoliposomes (siTGF-β1@ILP) with a connection efficiency of 62.02% ± 5.12%. DLS analysis showed that the average particle size of siTGF-β1@LP was 151.0 ± 1.3 nm, the polydispersity index (PDI) was 0.176 ± 0.018, and the potential was 1.2 ± 0.1 mV. After the functionalization of cetuximab, the average particle size of siTGF-β1@ILP increased to 159.2 ± 0.8 nm, the average value of PDI was 0.214 ± 0.012, and the potential was 4.9 ± 0.5 mV (Figure 2A,B). The morphology of siTGF-β1@LP and siTGF-β1@ILP as observed by TEM is shown in Figure 2C,D. After drug loading or functionalizing liposomes with cetuximab, siTGF-β1@LP and siTGF-β1@ILP retained their spherical shape and uniform particle size.
Agarose gel block tests showed that the liposomes and siTGF-β1 were completely complexed with a lipid:siTGF-β1 mass ratio of approximately 32:1 or higher, because all siRNA oligomers were loaded into liposomes to form high-molecular-weight complexes without any strip of free siRNA (Figure 3A). The relatively low weight ratio may offer the possibility of loading more drugs with reduced toxicity [17]. Therefore, the siRNA complex with a mass ratio of 32:1 was used for further experiments (Figure 3B).
Further, we examined the serum stability and enzyme stability of siTGF-β1@ILP. One of the obstacles to siRNA delivery is the lack of stability during in vivo transmission; thus, siRNA protection of siTGF-β1@ILP was determined through gel block tests. The band of bare siTGF-β1 disappeared completely after 1 h of incubation with Ribonuclease A (RNase A) or serum. However, siRNA bands in siTGF-β1@ILP remained detectable for up to 8 h (Figure 3C,D). The results showed that siTGF-β1@ILP has excellent stability, can resist nuclease attack, and remains stable in serum, which ensures the activity of siRNA in vivo and the ability to increase the amount of siRNA delivered to tumor sites, resulting in pronounced gene silencing effects.

2.2. The Primary Source of In Vitro Cytotoxicity Is siTGF-β1

An MTT assay was used to assess the cytotoxicity of naked siTGF-β1, siTGF-β1@LP, and siTGF-β1@ILP on A549 cells. As shown in Figure 4A,B, blank LP and blank ILP decreased cell viability in the range of 0.11 μg/mL to 11.20 μg/mL of siRNA concentration, because the high positive charge density of liposomes would indeed have increased cytotoxicity, and blank ILP also had a certain toxicity due to external CTX. In the same cell, all formulations, naked siTGF-β1, siTGF-β1@LP, and siTGF-β1@ILP, showed dose-dependent cytotoxicity, and their Half Maximal Inhibitory Concentration (IC50) values at 24 h were 274.4, 8.961, and 5.277 μg/mL, respectively. These results indicate that naked siRNA has no marked inhibitory effect on cells, which leads to the high survival rate of cancer cells during transfection. siTGF-β1@ILP showed stronger inhibition than siTGF-β1@LP, which confirmed that CTX attached to LP was active, enabling the functionalized liposome to have an active targeting effect, and was better able to restrain the growth and proliferation of cancer cells.

2.3. Cellular Uptake Study

To evaluate the uptake of siTGF-β1@LP and siTGF-β1@ILP by cells, flow cytometry was used for analysis after 4 h of treatment in the medium containing the drug, with free Fluorescein(FAM)-siRNA as a negative control. Flow cytometry showed a similar uptake of naked siRNA as in the blank control (Figure 4C). The uptake efficiency of the siTGF-β1@LP and siTGF-β1@ILP groups was higher than that of the naked siRNA group. These results were mainly because naked siRNA was degraded by intracellular enzymes without visible uptake, which also confirmed the protective effect of siTGF-β1@LP and siTGF-β1@ILP. When the cells were incubated with siTGF-β1@ILP, the concentration of FAM-positive A549 cells was markedly higher than when the cells were incubated with siTGF-β1@LP. The underlying reason is that in the presence of CTX modification, liposomes actively target tumor cells and internalize them, after which siRNA exerts gene silencing in the cytoplasm. This shows that the functionalized liposomes provide an ideal stage for siRNA entry into tumor cells.

2.4. DOPE and DOTAP: Enabling siTGF-β1 to Achieve Endosomal Escape

In the process of siRNA transfection, the internalized RNA is readily degraded by lysosome enzymes (such as acid RNA enzyme and exonuclease), resulting in reduced therapeutic efficacy [18]. Therefore, the endosome escape ability of siRNA complexes is crucial for siRNA to enter the cytoplasm to achieve gene silencing effects. DOPE has a pH responsive structural transition, which can destroy the stability of endosomal membrane in the acidic environment of endosomes, induce the fusion of liposome membrane and endosomal membrane, and promote drug release. DOTAP may also form ion pairs with endosome lipids, thereby disrupting the stability of the endosomes. Consequently, it is speculated that DOPE and DOTAP in the constructed liposomes ensure the endosomal escape of siRNA.
The escape ability from endosomes was tested using Confocal laser scanning microscope (CLSM). In the CLSM images (Figure 4D), the yellow signal on the overlapping area of red fluorescence (lysosomal red) and green fluorescence (siTGF-β1 labeled with green fluorescent FAM) indicates that siTGF-β1 was trapped within the endosome. After siTGF-β1 escaped from the endosome, bright green fluorescence was observed, and separation of green fluorescence and the red area was also detected. In the naked siTGF-β1 group, siTGF-β1 was degraded by intracellular enzymes at 4 h; thus, no substantial fluorescence occurred, and the small amount of fluorescence present was mainly in the endosome. In the siTGF-β1@LP and siTGF-β1@ILP groups, fast and effective endosome escape was observed even after 4 h, as pronounced green signals were observed, indicating that siRNA diffused from the endosome. Compared with the siTGF-β1@LP group, the red and green signals in the siTGF-β1@ILP group were separated more strongly. The siTGF-β1@LP group also showed some red/green fluorescence separation; however, it had more yellow signals. The results showed that the delivery system constructed could stably protect siRNA and promote the endosomes escape of siRNA, and the escape effect of the siTGF-β1@ILP group was stronger than that of the siTGF-β1@LP group. That is, when cetuximab was modified, liposomes could actively target tumor cells and escape from the endosomes, thus exerting the gene silencing effect.

2.5. Liposomes Inhibit the Invasion and Migration of A549 Cells

The cell scratch test showed that the cell migration ability of the siTGF-β1@LP and siTGF-β1@ILP groups was lower than that of the naked siTGF-β1 group, and the cell migration ability of the siTGF-β1@ILP group was lower than that of the siTGF-β1@LP group (Figure 5A,B). In the transwell migration experiment, compared with the naked siTGF-β1 group, the amount of of cell invasion in the siTGF-β1@LP and siTGF-β1@ILP groups was markedly reduced (Figure 5C). These results confirm the successful delivery and efficient expression of siRNA in cells and show that the inhibition of TGF-β1 can effectively inhibit the growth and migration of A549 cells.

2.6. In Vivo Biodistribution of Liposomes in a Cell Line Xenograft Model

Three hours after injection, free FAM-siRNA displayed faint fluorescence intensity at the tumor site, suggesting the instability and rapid clearance of naked siRNA in vivo, as shown in Figure 6A. By contrast, siTGF-β1@ILP containing the same amount of FAM-siRNA revealed distinct fluorescence at the tumor site, confirming stability in vivo and the successful delivery of siRNA to the tumor region via siTGF-β1@ILP, which was attributed to the active targeting and binding ability of CTX to EGFR. At the same time, siTGF-β1@ILP can prolong the complex circulation time (up to 12 h or even longer), thus increasing accumulation in the tumor. In the siTGF-β1@LP group, only a small fluorescence signal occurred at the tumor site, while a stronger signal intensity was observed in other parts of the body and was readily cleared by the reticuloendothelial system due to the positive charge.

2.7. In Vivo Anticancer Activity and Safety Evaluation

To assess the inhibition of tumor growth in vivo, an A549 tumor model was established using BALB/c nude mice. Various formulations, including free siTGF-β1, siTGF-β1@LP, and siTGF-β1@ILP, were injected through the tail vein once every 2 d. The normal saline group did not show inhibited tumor growth during the administration period and reached the maximum tumor volume (Figure 6B,D). The group administered free siRNA showed larger tumors at the end of treatment. The tumors in the siTGF-β1@LP group and siTGF-β1@ILP groups were smaller than those in the free siRNA- and saline-treated groups, whereas tumors in the siTGF-β1@ILP group were smaller than those in the siTGF-β1@LP group, with a substantial difference between them (p < 0.05). These data indicated that siTGF-β1@ILP has better selectivity and apparent growth inhibition effects on tumors, and siRNA embedded in siTGF-β1@ILP can achieve enhanced tumor inhibition and provide improved therapeutic effects, indicating that siTGF-β1@ILP may be a promising drug delivery system for NSCLC treatment.
Hematoxylin and Eosin staining (HE) showed histological changes in tumor tissues after treatment in each group (Figure 7A). The cytoplasm in normal tumor cells and in blood vessels was visible. After treatment with siTGF-β1@LP and siTGF-β1@ILP, tumor cell abundance decreased and the intercellular space increased, which was a typical characteristic of necrosis. The results showed that siTGF-β1@ILP was able to damage tumor tissue and inhibit tumor growth, and its effect was stronger than that of siTGF-β1@LP.
Considering that the preparation may cause immune reactions and systemic toxicity after intravenous injection, body weight changes in mice were recorded once every 2 d to evaluate the systemic toxicity of the vehicle in vivo (Figure 6C). None of the groups exhibited weight loss during the experiment, thus confirming the safety of siTGF-β1@LP and siTGF-β1@ILP. HE staining indicated that each group of preparations does not cause visible toxic pathological changes in the heart, liver, spleen, lung, or kidney (Figure 7B), which suggested the low toxicity of the constructed liposome.

2.8. Immunohistochemistry (IHC)

IHC results of tumor tissue showed no significant treatment effect in the siTGF-β1 group, whereas the siTGF-β1@ILP group showed stronger effects than the siTGF-β1@LP group. The expression levels of TGF-β1 and p-Smad3 were markedly reduced (Figure 8); thus, the growth and metastasis of A549 cells were prevented by inhibiting TGF-β/Smad signaling.

3. Discussion

Non-small-cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for approximately 85% of all lung cancer cases [19]. As a malignant tumor originating in the lungs, NSCLC is characterized by high incidence and mortality rates and is often diagnosed at advanced stages, making it difficult to treat and resulting in poor prognosis [20]. TGF-β1 is a key member of the TGF-β superfamily and plays a complex and multifaceted role in the tumor microenvironment. TGF-β1 not only promotes the proliferation and survival of cancer cells but also induces epithelial–mesenchymal transition (EMT), thereby enhancing the invasiveness and metastatic potential of tumor cells. This makes TGF-β1 closely related to the development and progression of NSCLC [21,22,23]. Therefore, inhibiting the expression of TGF-β1 has become an important strategy for treating NSCLC.
siRNA technology, based on the principle of RNA interference (RNAi), is a powerful tool for gene silencing that can specifically degrade target mRNA, thereby inhibiting the expression of specific genes [24]. In recent years, siRNA technology has garnered significant attention for its potential applications in cancer therapy. Previous studies have shown that delivering siTGF-β1 via liposomes can effectively inhibit the expression of TGF-β1 in pulmonary epithelial cells and fibroblasts, thereby alleviating the symptoms of idiopathic pulmonary fibrosis (IPF) [25]. Additionally, research using bovine milk exosomes to deliver siTGF-β1 has demonstrated that this method can effectively transport siRNA to the lungs, inhibiting inflammatory responses, EMT, and the production of fibrosis-related molecules [26]. These studies provide a theoretical basis and practical references for utilizing siRNA technology to treat non-small-cell lung cancer (NSCLC).
Liposomes, as nanoscale vesicles composed of a phospholipid bilayer, are capable of encapsulating and protecting RNA molecules, ensuring their stability in the body and facilitating delivery to target cells [27]. However, traditional liposomes have several limitations, such as susceptibility to degradation and poor targeting ability. The positively charged character of DOTAP lipids allowed the negatively charged siRNA to easily compound with liposomes, and the cholesterol portion provided stability for the phospholipid bilayer, making it difficult for siRNA to escape from the liposome during delivery to the target sites [28,29,30]. During endocytosis, DOPE, a neutral auxiliary lipid, enables liposomes to destroy the endosomal membrane and release the loaded drug into the cytoplasm [29,31]. By contrast, grafting CLS-PEG2000 to the liposome surface was expected to potentially reduce blood circulation by avoiding cationic liposome aggregation and avoiding non-specific uptake in the reticuloendothelial system [32].
In this study, we selected PEGylated cationic liposomes as the carrier for siRNA and modified them with cetuximab to enhance their targeting ability and gene silencing activity. Cetuximab is a monoclonal antibody targeting the extracellular domain of the epidermal growth factor receptor (EGFR) and inhibiting its activation [14]. Overexpression of EGFR is common in NSCLC [33]; therefore, liposomes modified with cetuximab can specifically target tumor cells and improve the delivery efficiency of siRNA [15,34]. Experimental results showed that these modified liposomes could effectively bind siRNA and maintain stability in serum, protecting siRNA from RNase degradation.
Cytotoxicity assays revealed that naked siTGF-β1 had limited inhibitory effects on A549 cells, whereas siTGF-β1@LP and siTGF-β1@ILP, encapsulated in liposomes, significantly enhanced the inhibitory effects on A549 cells. Notably, cetuximab (CTX)-modified siTGF-β1@ILP exhibited stronger cytotoxicity, indicating that its active targeting ability enabled more effective siRNA delivery to tumor cells, thereby achieving better gene silencing effects. Wound healing assays further validated the inhibitory effects of siTGF-β1@LP and siTGF-β1@ILP on the migratory capacity of A549 cells, with siTGF-β1@ILP showing more pronounced effects. These results provide strong experimental evidence for TGF-β1 as a therapeutic target for non-small-cell lung cancer (NSCLC).
In in vivo experiments, siTGF-β1@ILP effectively delivered siRNA to tumor tissues through the targeting action of cetuximab, which is consistent with the stability of siRNA and cellular uptake observed in vitro. Pharmacodynamic tests demonstrated that siTGF-β1@ILP exhibited excellent activity in inhibiting tumor growth and did not cause significant systemic toxicity during the experimental period. Hematoxylin and eosin (HE) staining results showed that neither siTGF-β1@LP nor siTGF-β1@ILP induced apparent toxic pathological changes in major organs, confirming their good biocompatibility. In patients with non-small-cell lung cancer (NSCLC), plasma levels of TGF-β are significantly elevated compared to individuals without lung cancer, and this elevation is closely associated with low survival rates [35]. TGF-β1 significantly promotes the proliferation, migration, and invasion of tumor cells by activating the Smad3 signaling pathway [36]. IHC results indicated that siTGF-β1@ILP, modified with cetuximab, achieved active targeting, significantly enhanced the enrichment and gene silencing effects of siRNA in tumor cells, and exhibited strong inhibitory effects on TGF-β1 and p-Smad3 protein expression. The reduced expression levels of TGF-β1 and p-Smad3 proteins further confirmed that siTGF-β1@ILP effectively inhibited tumor cell proliferation and migration by suppressing the TGF-β/Smad signaling pathway, thereby exerting significant antitumor effects. Overall, as a targeted delivery system, siTGF-β1@ILP demonstrated excellent antitumor activity and safety in vivo and holds potential as a therapeutic strategy for non-small-cell lung cancer (NSCLC).
Despite the positive progress achieved in this study, there is still room for further optimization. First, TGF-β1 is only one factor in the TGF-β signaling pathway, and whether other members of the TGF-β family also have potential therapeutic effects on NSCLC requires further validation. Second, the low drug loading capacity of siRNA has always been a challenge for this technology, and how to increase the drug loading and delivery efficiency of liposomes will be a key focus for future research. In summary, by encapsulating siTGF-β1 in liposomes and modifying them with PEG and cetuximab (CTX), we successfully developed a targeted delivery system that effectively inhibits the expression of TGF-β1. This system not only demonstrated excellent antitumor activity and biocompatibility in both in vitro and in vivo experiments but also provided new insights and strategies for the treatment of NSCLC.

4. Materials and Methods

4.1. Cell Culture and Materials

A549 cells were acquired from Luye Pharmaceutical Co., Ltd. (Yantai, China) and were incubated in DMEM medium (Procell Life Science & Technology Co., Ltd., Wuhan, China) with 10% (v/v) FBS, and 1% (v/v) penicillin–streptomycin. The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. FBS was obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).
(2,3-Dioleoyloxy-propyl)-trimethylammonium-chloride (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (CH), Cholesterol-PEG2000 (CLS-PEG2000), and Cholesterol polyethylene glycol maleimide (CLS-PEG2000-Mal) were acquired from RVT Pharmaceutical Technology Co., Ltd. (Shanghai, China). Cetuximab was sourced from Weisman Bioengineering Co., Ltd. (Wuhan, China). All siRNA oligos including FAM-labeled siRNA were acquired from Gene Pharma (Shanghai, China), and the sense and antisense sequences of siTGF-β1 were 5′-GCAACAACGCCAUCUAUGATT-3′ and 5′-UCAUAGAUGGCGUUGUUGCTT-3′, respectively. All other chemicals were of analytical or chromatographic grade.

4.2. Preparation of Liposomes

siTGF-β1 cationic liposomes were prepared using a modified ethanol injection method [37]. The lipid mixture composed of DOTAP, DOPE, and CH (molar ratio 1:3:1.5) was dissolved in an appropriate amount of absolute ethanol; the product was then transferred it to a glass bottle which was immediately placed in a water bath at 65 °C, and the liquid was stirred until the ethanol was volatilized. To load siTGF-β1, we added 5 mL diethyl pyrocarbonate water containing 280 μg siTGF-β1 to hydrate the dry lipid membrane. We used an ultrasonic cell disruptor for treatment in an ice water bath with 150 W for 15 min. The resulting siTGF-β1 liposomes were consecutively passed through microporous filter membranes of a 0.80, 0.45, and 0.22 µm mesh. Finally, polyethylene glycol liposomes were prepared using a post-insertion method, and CLS-PEG2000 and CLS-PEG2000-Mal (molar ratio 0.12:0.03) were slowly added to the liposomes [32].
After siTGF-β1@LP were prepared, cetuximab was used to functionalize the liposomes to obtain siTGF-β1@ILP [38]. Cetuximab and Traut’s reagent were sulfhydrylated at a molar ratio of 1:20 [15]. Thiolated cetuximab was incubated with the liposomes for 20 h at room temperature in the dark to bind the thiolated antibody to the liposome. Unbound cetuximab was removed by ultrafiltration tube centrifugation (molecular weight cut-off (MWCO) = 300 K; Amicon, Burlington, MA, USA), followed by disinfection by filtration through a sterile filter (0.22 µm) to obtain siTGF-β1@ILP. The product was stored at 4 °C. The conjugation efficacy of antibodies on liposomes was evaluated with the following equation: (Mt − Mu)/Mt (Mt: the mass of total antibodies; Mu represents the mass of unconjugated antibodies, and the antibodies were measured by the BCA Protein Assay Kit). The process for the preparation of FAM-siRNA-loaded liposomes was identical to that of siTGF-β1 liposomes.

4.3. Physicochemical Characterization of Liposomes

4.3.1. Size and Zeta Potential Measurements

The particle size and zeta potential of siTGF-β1@LP and siTGF-β1@ILP were analyzed using a Malvern Instrument (Zetasizer Nano ZS, Malvern, UK). The morphology of siTGF-β1@LP and siTGF-β1@ILP was assessed using transmission electron microscopy (TEM; JEM-1400 Plus, Tokyo, Japan). Liposomes were negatively stained with a droplet of 2% phosphotungstic acid negative staining solution. Data were collected at an accelerating voltage of 80 kV.

4.3.2. Gel Electrophoresis

Gel-blocking assays were used to detect the formation of siRNA–liposome complexes [39]. The complexes were prepared with different siRNA concentrations, and samples with different mass ratios (10 μL) were analyzed through 1.5% agarose gel electrophoresis (100 V, 30 min). Then, the gel was stained with Fluorescent Deoxyribonucleic Acid (DNA) loading buffer, and the stained bands were visualized under ultraviolet light.

4.3.3. siRNA Protection

For assessing the serum stability and protection of siRNA in RNAase, free siTGF-β1 or equivalent siTGF-β1@ILP was mixed with equal volumes of FBS (50% serum concentration) or RNase A (1 mg/mL) at 37 °C. Samples collected at various time points were stored at −80 °C. Before gel electrophoresis, 10% heparin sodium solution was added to the samples to extract siRNA; gel electrophoresis was performed as described above. Untreated free siRNA was used as a control.

4.4. In Vitro Cytotoxicity

The cytotoxicity of naked siTGF-β1, siTGF-β1@LP, and siTGF-β1@ILP (n = 3, each) was assessed using an MTT assay [40]. A549 cells were trypsinized and were seeded into 96-well plates at 5 × 103 cells per well. After 24 h, different concentrations of naked siTGF-β1 and siTGF-β1 loaded liposomes (equivalent to an siTGF-β1 of 0.11, 0.56, 1.12, 5.6, and 11.2 µg/mL) were spiked into each well of treated cells. Then, the cells were cultured for 24 h. Thereafter, 20 μL of MTT solution (5 mg/mL) was added to each well, followed by incubation for 4 h. The supernatant was removed, 200 μL of dimethyl sulfoxide was added, and the culture was shaken for 10 min to dissolve formazan crystals. A microplate reader (Model 550, Bio-Rad, Hercules, CA, USA) was used to assess absorbance at 570 nm for each well, and the untreated group acted as a control to calculate cell viability. Blank liposomes and blank immunoliposome were also evaluated using the same protocol.

4.5. Cellular Uptake

A549 cells were inoculated in six-well plates at a density of 2 × 105 cells per well and were cultivated at 37 °C overnight for cell uptake analysis. The original substrate was then substituted with new DMEM containing naked siTGF-β1, siTGF-β1@LP, and siTGF-β1@ILP (siTGF-β1 labeled with FAM). The ultimate concentration of FAM-labeled siTGF-β1 was 100 nM [41]. After incubation at 37 °C for 4 h, the cells were washed using PBS. For the quantification of cellular uptake, cells were trypsinized, centrifuged, and resuspended in 200 µL PBS, after which they were assayed for fluorescence intensity using a flow cytometer (BD Biosciences, San Jose, CA, USA). The experiment was performed in triplicate (n = 3).

4.6. Endosomal Escape

A549 cells were plated at 2 × 105 cells/well in a six-well plate and were placed in an incubator at 37 °C overnight. Then, the medium was replaced with 2 mL new medium containing naked siTGF-β1, siTGF-β1@LP, and siTGF-β1@ILP (siTGF-β1 labeled with FAM). The medium containing the complex was aspirated after 4 h of culture, and cells were washed twice with PBS and were then incubated with a lysosomal red fluorescent probe at 37 °C for 1 h to stain the lysosomes. The cells were then immobilized with 4% paraformaldehyde, and nuclei were 4′,6-Diamidino-2-phenylindole-stained (DAPI-stained) for 30 min, followed by examination and photography using CLSM. The experiment was performed in triplicate (n = 3).

4.7. Cell Migration Assay

4.7.1. Wound Healing Assay

A549 cells were cultured in six-well plates at a density of 2 × 105 cells for 24 h to achieve 90% monolayer fusion. A sterile pipette tip (200 μL volume) was then used to scratch the monolayer, followed by washing twice with PBS [42]. The cells were treated with naked siTGF-β1, siTGF-β1@LP, and siTGF-β1@ILP, and were cultured in DMEM containing 10% FBS for 48 h. The untreated group was used as a control. Wound width was measured using an inverted microscope (TS2R-FL, Tokyo, Japan) at 0, 12, 24, and 48 h. The cell migration rate was calculated as follows: (0 h scratch width − 48 h scratch width)/0 h scratch width × 100%. The experiment was performed in triplicate (n = 3).

4.7.2. Transwell Migration Assay

For the transwell migration test, A549 cells were resuspended in serum-free medium at a concentration of 3 × 104 cells/mL and were inoculated onto the upper surface of the transwell cavity [43]. Then, 10% FBS medium containing naked siTGF-β1, siTGF-β1@LP, and siTGF-β1@ILP (n = 3, each) was placed on the bottom of the chamber. After 24 h of cultivation, cells invading the lower surface of the chamber were immobilized with 4% paraformaldehyde and were stained using crystal violet solution. Then, the stained cells were washed using PBS and were recorded using an inverted microscope.

4.8. In Vivo Biodistribution of Liposomes in the Cell Line Xenograft Model

The A549 cell suspension (2 × 106 cells/100 μL) was subcutaneously injected into the flanks of BALB/c nude mice (five weeks old, male, 18–20 g). After tumor volumes reached approximately 200 mm3, the A549 tumor-bearing mice were injected via the tail vein with naked FAM-siRNA or FAM-siRNA@LP/FAM-siRNA@ILP (siRNA concentration was 100 nM, 100 μL). Whole-body fluorescence images in fluorescence channels of FAM were measured 3, 6, and 12 h after injection using IVIS spectroscopy (Caliper Life Science, Hopkinton, MA, USA) and IVIS in vivo imaging software (Version 4.4). The measurement was conducted in triplicate (n = 3).

4.9. In Vivo Antitumor Effect and Biosafety

When the xenografted tumors grew to approximately 200 mm3, the nude mice were randomly assigned to four groups (n = 5, each), which were treated with saline, naked siTGF-β1, siTGF-β1@LP, or siTGF-β1@ILP via tail vein injection (100 nM, 100 μL) every 2nd day until day 18. Tumor volumes were measured to monitor the treatment effects in each group. Furthermore, the weights of A549 tumor-bearing mice were recorded to evaluate the systemic toxicity of the tested complexes. The mice were killed on day 18, and tumors and major organs including the heart, liver, spleen, lungs, and kidneys were harvested. The organ tissue was embedded in paraffin and was sliced into 5 μm thick sections for HE staining. A light microscope was employed to capture images to examine the anti-tumor effects and in vivo safety of the various siRNA preparations. The experiment was performed in triplicate (n = 3–5).

4.10. IHC of Tumor Tissue

The resected tumor tissue was fixed with 4% paraformaldehyde, embedded and sectioned with conventional paraffin, dewaxed, rehydrated, repaired with heat-induced epitopes, incubated with the respective primary antibodies at 4 °C overnight, and was dripped with the secondary antibody at room temperature for 1 h. Diaminobenzidine coloration, hematoxylin double-dyeing, and dehydration were performed. The staining results were observed using a light microscope. The experiment was performed in triplicate (n = 3–5).

4.11. Statistical Analyses

The data are shown as the means ± standard deviation (SD) from independent experiments. Differences between groups in continuous variables were analyzed using Student’s t-test. Differences between multiple groups were analyzed using a one-way ANOVA. Statistical significance is reported at p < 0.05.

5. Conclusions

In conclusion, these assays strongly suggest that the prepared siTGF-β1@ILP based on cetuximab-functionalization was an excellent system to deliver TGF-β1 siRNA to A549 cells, which could enhance the gene silencing effect of siRNA by promoting tumor-targeting effects, so as to achieve the purpose of treating NSCLC. After functionalization with cetuximab, the delivery system effectively delivered siRNA to A549 cells in vitro and successfully directed the delivery system to tumor sites in vivo. Our results thus show a new drug delivery system with visible gene delivery capability, and the delivery of TGF-β1 siRNA by immunoliposomes will be a promising therapeutic method for NSCLC.

Author Contributions

Y.S. and H.Z. performed the conception and design of the study. Y.S., H.Z., H.C., J.G., R.Y., Y.T. (Yu Tian), Q.X., Z.M., Y.T. (Yuping Tao), Y.C., J.T., A.W., Z.Z. and H.W. performed the material preparation, in vitro and in vivo experiments, and statistical analysis. H.Z., J.G. and R.Y. performed image analyses. H.Z. and Y.T. (Yuping Tao) wrote the manuscript. Y.C., J.T., A.W., Z.Z., H.W. and Y.S. supervised the entire study, made scientific guides, and provided the financial support. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Shandong Laboratory Program [SYS202205], Yantai University Doctoral Program [No. SM20B35], Natural Science Foundation of Shandong Province [No. ZR2021MH395], Development of engineered exosomes for nucleic acid drug delivery in the treatment of pulmonary fibrosis [No. SK22KH114] and Research on the development of multiple methods to increase exosome production [No. SK22KH304], Science Fund of Shandong Laboratory of Advanced Materials and Green Manufacturing at Yantai [AMGM2024A07], National Natural Science Foundation of China [No. 82404552].

Institutional Review Board Statement

All animal experiments were approved by the Experimental Animal Ethics Committee of the College of Pharmacy, Yantai University (Approval number, YTDX 24 January 2018).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Acknowledgments

We appreciate the support from the laboratory of the School of Pharmacy at Yantai University.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Herbst, R.S.; Morgensztern, D.; Boshoff, C. The biology and management of non-small cell lung cancer. Nature 2018, 553, 446–454. [Google Scholar] [CrossRef] [PubMed]
  2. Alexander, M.; Kim, S.Y.; Cheng, H. Update 2020: Management of Non-Small Cell Lung Cancer. Lung 2020, 198, 897–907. [Google Scholar] [CrossRef] [PubMed]
  3. Vecchiarelli, S.; Bennati, C. Oncogene addicted non-small-cell lung cancer: Current standard and hot topics. Future Oncol. 2018, 14, 3–17. [Google Scholar] [CrossRef] [PubMed]
  4. Midha, A.; Dearden, S.; McCormack, R. EGFR mutation incidence in non-small-cell lung cancer of adenocarcinoma histology: A systematic review and global map by ethnicity (mutMapII). Am. J. Cancer Res. 2015, 5, 2892–2911. [Google Scholar] [PubMed]
  5. Rolfo, C.; Caglevic, C.; Santarpia, M.; Araujo, A.; Giovannetti, E.; Gallardo, C.D.; Pauwels, P.; Mahave, M. Immunotherapy in NSCLC: A Promising and Revolutionary Weapon. Adv. Exp. Med. Biol. 2017, 995, 97–125. [Google Scholar]
  6. Harrison, P.T.; Vyse, S.; Huang, P.H. Rare epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer. Semin. Cancer Biol. 2020, 61, 167–179. [Google Scholar] [CrossRef]
  7. Sun, M.; Zhuang, X.; Lv, G.; Lin, Z.; Huang, X.; Zhao, J.; Lin, H.; Wang, Y.; Ansari, I.A. Ginsenoside CK Inhibits TGF-β-Induced Epithelial-Mesenchymal Transition in A549 Cell via SIRT1. BioMed Res. Int. 2021, 2021, 9140191. [Google Scholar] [CrossRef]
  8. Li, L.; Lv, Y.; Yan, D. Inhibition of Ep3 attenuates migration and promotes apoptosis of non-small cell lung cancer cells via suppression of TGF-β/Smad signaling. Oncol. Lett. 2018, 16, 5645–5654. [Google Scholar] [CrossRef]
  9. Lundstrom, K. Are Viral Vectors Any Good for RNAi Antiviral Therapy? Viruses 2020, 12, 1189. [Google Scholar] [CrossRef]
  10. Yan, Y.; Liu, X.-Y.; Lu, A.; Wang, X.-Y.; Jiang, L.-X.; Wang, J.-C. Non-viral vectors for RNA delivery. J. Control. Release 2022, 342, 241–279. [Google Scholar] [CrossRef]
  11. Hattori, Y.; Tamaki, K.; Sakasai, S.; Ozaki, K.I.; Onishi, H. Effects of PEG anchors in PEGylated siRNA lipoplexes on in vitro gene-silencing effects and siRNA biodistribution in mice. Mol. Med. Rep. 2022, 22, 4183–4196. [Google Scholar] [CrossRef] [PubMed]
  12. Humble, Y. Cetuximab: An IgG(1) monoclonal antibody for the treatment of epidermal growth factor receptor-expressing tumours. Expert. Opin. Pharmacother. 2004, 5, 1621–1633. [Google Scholar] [CrossRef] [PubMed]
  13. Mazzarell, L.; Guida, A. Cetuximab for treating non-small cell lung cancer. Expert. Opin. Biol. Ther. 2018, 18, 483–493. [Google Scholar] [CrossRef] [PubMed]
  14. Vincenzi, B.; Schiavon, G.; Silletta, M.; Santini, D.; Tonini, G. The biological properties of cetuximab. Crit. Rev. Oncol./Hematol. 2008, 68, 93–106. [Google Scholar] [CrossRef]
  15. Lu, X.; Liu, S.; Han, M.; Yang, X.; Sun, K.; Wang, H.; Mu, H.; Du, Y.; Wang, A.; Ni, L.; et al. Afatinib-loaded immunoliposomes functionalized with cetuximab: A novel strategy targeting the epidermal growth factor receptor for treatment of non-small-cell lung cancer. Int. J. Pharm. 2019, 560, 126–135. [Google Scholar] [CrossRef]
  16. Yang, X.; Wang, Y.; Chen, S.; Zhang, S.; Cui, C. Cetuximab-Modified Human Serum Albumin Nanoparticles Co-Loaded with Doxorubicin and MDR1 siRNA for the Treatment of Drug-Resistant Breast Tumors. Int. J. Nanomed. 2021, 16, 7051–7069. [Google Scholar] [CrossRef]
  17. Yao, Y.; Su, Z.; Liang, Y.; Zhang, N. pH-Sensitive carboxymethyl chitosan-modified cationic liposomes for sorafenib and siRNA co-delivery. Int. J. Nanomed. 2015, 10, 6185–6197. [Google Scholar]
  18. Degors, I.M.S.; Wang, C.; Rehman, Z.U.; Zuhorn, I.S. Carriers Break Barriers in Drug Delivery: Endocytosis and Endosomal Escape of Gene Delivery Vectors. Acc. Chem. Res. 2019, 52, 1750–1760. [Google Scholar] [CrossRef]
  19. Relli, V.; Trerotola, M.; Guerra, E.; Alberti, S. Abandoning the Notion of Non-Small Cell Lung Cancer. Trends Mol. Med. 2019, 25, 585–594. [Google Scholar] [CrossRef]
  20. Skribek, M.; Rounis, K.; Tsakonas, G.; Ekman, S. Complications following novel therapies for non-small cell lung cancer. J. Intern. Med. 2022, 291, 732–754. [Google Scholar] [CrossRef]
  21. Akhurst, R.J. Targeting TGF-β Signaling for Therapeutic Gain. Cold Spring Harb. Perspect. Biol. 2017, 9, a022301. [Google Scholar] [CrossRef] [PubMed]
  22. Thiery, J.P.; Acloque, H.; Huang, R.Y.J.; Nieto, M.A. Epithelial-Mesenchymal Transitions in Development and Disease. Cell 2009, 139, 871–890. [Google Scholar] [CrossRef] [PubMed]
  23. Wang, L.; Yang, H.; Lei, Z.; Zhao, J.; Chen, Y.; Chen, P.; Li, C.; Zeng, Y.; Liu, Z.; Liu, X.; et al. Repression of TIF1γ by SOX2 promotes TGF-β-induced epithelial–mesenchymal transition in non-small-cell lung cancer. Oncogene 2015, 35, 867–877. [Google Scholar] [CrossRef] [PubMed]
  24. Chalbatani, G.M.; Dana, H.; Gharagozlo, E.; Mahmoodzad, H.; Zeinalinia, E.; Rezaeian, O.; Pilvar, P.; Ardaneh, M.; Meghdadi, S.; Memari, F.; et al. Microrna a New Gate in Cancer and Human Disease: A Review. J. Biol. Sci. 2017, 17, 247–254. [Google Scholar] [CrossRef]
  25. Yu, C.; Xu, Q.; Cao, X.; Cheng, S.; Zhang, Z.; Huang, J. siTGF-β1 and pirfenidone contained Ionizable-Liposomal nanodrug for enhanced treatment of Idiopathic pulmonary fibrosis. Chem. Eng. J. 2024, 497, 154850. [Google Scholar] [CrossRef]
  26. Qiu, C.; Zhao, Z.; Xu, C.; Yuan, R.; Ha, Y.; Tu, Q.; Zhang, H.; Mu, Z.; Xin, Q.; Tian, Y.; et al. Nebulized milk exosomes loaded with siTGF-β1 ameliorate pulmonary fibrosis by inhibiting EMT pathway and enhancing collagen permeability. J. Nanobiotechnol. 2024, 22, 434. [Google Scholar] [CrossRef]
  27. Sanati, M.; Afshari, A.R.; Ahmadi, S.S.; Kesharwani, P.; Sahebkar, A. Advances in liposome-based delivery of RNA therapeutics for cancer treatment. Prog. Mol. Biol. Transl. Sci. 2024, 204, 177–218. [Google Scholar]
  28. Lee, J.; Cho, Y.J.; Lee, J.-W.; Ahn, H.J. KSP siRNA/paclitaxel-loaded PEGylated cationic liposomes for overcoming resistance to KSP inhibitors: Synergistic antitumor effects in drug-resistant ovarian cancer. J. Control. Release 2020, 321, 184–197. [Google Scholar] [CrossRef]
  29. Lin, Q.; Chen, J.; Zhang, Z.; Zheng, G. Lipid-based nanoparticles in the systemic delivery of siRNA. Nanomedicine 2013, 9, 105–120. [Google Scholar] [CrossRef]
  30. Lu, H.; Liu, X.; Zhang, M.; Bera, H.; Xu, W.; Jiang, H.; Zhao, X.; Wu, L.; Cun, D.; Yang, M. Pulmonary fibroblast-specific delivery of siRNA exploiting exosomes-based nanoscaffolds for IPF treatment. Asian J. Pharm. Sci. 2024, 19, 100929. [Google Scholar] [CrossRef]
  31. Mochizuki, S.; Kanegae, N.; Nishina, K.; Kamikawa, Y.; Koiwai, K.; Masunaga, H.; Sakurai, K. The role of the helper lipid dioleoylphosphatidylethanolamine (DOPE) for DNA transfection cooperating with a cationic lipid bearing ethylenediamine. Biochim. Biophys. Acta (BBA)-Biomembr. 2013, 1828, 412–418. [Google Scholar] [CrossRef] [PubMed]
  32. Nosova, A.S.; Koloskova, O.O.; Nikonova, A.A.; Simonova, V.A.; Smirnov, V.V.; Kudlay, D.; Khaitov, M.R. Diversity of PEGylation methods of liposomes and their influence on RNA delivery. MedChemComm 2019, 10, 369–377. [Google Scholar] [CrossRef] [PubMed]
  33. Kobayashi, S.; Boggon, T.J.; Dayaram, T.; Jänne, P.A.; Kocher, O.; Meyerson, M.; Johnson, B.E.; Eck, M.J.; Tenen, D.G.; Halmos, B.; et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N. Engl. J. Med. 2005, 352, 786–792. [Google Scholar] [CrossRef] [PubMed]
  34. Setia, A.; Kumari, P.; Vikas; Kumar Mehata, A.; Kumar Malik, A.; Kumar Mahto, S.; Muthu, M.S. Cetuximab decorated redox sensitive D-alpha-tocopheryl- polyethyleneglycol-1000-succinate based nanoparticles for cabazitaxel delivery: Formulation, lung targeting and enhanced anti-cancer effects. Int. J. Pharm. 2024, 653, 123881. [Google Scholar] [CrossRef]
  35. Barthelemy-Brichant, N.; David, J.L.; Bosquée, L.; Bury, T.; Seidel, L.; Albert, A.; Bartsch, P.; Baugnet-Mahieu, L.; Deneufbourg, J.M. Increased TGFbeta1 plasma level in patients with lung cancer: Potential mechanisms. Eur. J. Clin. Investig. 2022, 32, 193–198. [Google Scholar] [CrossRef]
  36. Yang, X.-Y.; Liao, J.-J.; Xue, W.-R. FMNL1 down-regulation suppresses bone metastasis through reducing TGF-β1 expression in non-small cell lung cancer (NSCLC). Biomed. Pharmacother. 2019, 117, 109126. [Google Scholar] [CrossRef]
  37. Wang, F.; Zhang, G.; Zang, C.; Pan, H.; Ma, L.; Li, C.; Hou, H.; Su, P.; Gao, Y.; Sun, J.; et al. Preparation and In Vitro/Vivo Evaluation of Nano-Liposomal Form of Febrifugine Hydrochloride. J. Nanosci. Nanotechnol. 2020, 20, 2558–2566. [Google Scholar] [CrossRef]
  38. Yang, F.; Zheng, Z.; Zheng, L.; Qin, J.; Li, H.; Xue, X.; Gao, J.; Fang, G. SATB1 siRNA-encapsulated immunoliposomes conjugated with CD44 antibodies target and eliminate gastric cancer-initiating cells. OncoTargets Ther. 2018, 11, 6811–6825. [Google Scholar] [CrossRef]
  39. Sun, J.; Zhou, Y.; Jin, G.; Jin, Y.; Quan, J. Preparation and Preliminary Evaluation of Dual-functional Nanoparticles for MRI and siRNA Delivery. Iranian J. Pharm. Res. 2021, 20, 265–277. [Google Scholar]
  40. Li, F.F.; Zhang, H.; Li, J.J.; Cao, Y.N.; Dong, X.; Gao, C. Interaction with adipocytes induces lung adenocarcinoma A549 cell migration and tumor growth. Mol. Med. Rep. 2018, 18, 1973–1980. [Google Scholar] [CrossRef]
  41. Chen, D.; Zhang, P.; Li, M.; Li, C.; Lu, X.; Sun, Y.; Sun, K. Hyaluronic acid-modified redox-sensitive hybrid nanocomplex loading with siRNA for non-small-cell lung carcinoma therapy. Drug Deliv. 2022, 29, 574–587. [Google Scholar] [CrossRef] [PubMed]
  42. Li, J.; Wu, D.-M.; Han, R.; Yu, Y.; Deng, S.-H.; Liu, T.; Zhang, T.; Xu, Y. Low-Dose Radiation Promotes Invasion and Migration of A549 Cells by Activating the CXCL1/NF-κB Signaling Pathway. OncoTargets Ther. 2020, 13, 3619–3629. [Google Scholar] [CrossRef] [PubMed]
  43. Wang, Y.; Zhai, W.; Wang, H.; Xia, X.; Zhang, C. Benzo(a)pyrene promotes A549 cell migration and invasion through up-regulating Twist. Arch. Toxicol. 2014, 89, 451–458. [Google Scholar] [CrossRef] [PubMed]
Ijms 26 01196 g001
Figure 1. Schematic of the construction of immunoliposomes encapsulating siRNA, and intracellular transport of the delivery system, including endocytosis into endosomes (i), endosome escape (ii), liposome breakdown and siRNA release (iii), downregulation of TGF-β1 mRNA expression (iv), and RNAi-induced increase in apoptosis and decrease in proliferation (v).
Figure 1. Schematic of the construction of immunoliposomes encapsulating siRNA, and intracellular transport of the delivery system, including endocytosis into endosomes (i), endosome escape (ii), liposome breakdown and siRNA release (iii), downregulation of TGF-β1 mRNA expression (iv), and RNAi-induced increase in apoptosis and decrease in proliferation (v).
Ijms 26 01196 g001
Ijms 26 01196 g002
Figure 2. Transmission electron microscopy (TEM) images, particle size, and zeta potential of siTGF-β1@LP (A,C) and siTGF-β1@ILP (B,D) (n = 3).
Figure 2. Transmission electron microscopy (TEM) images, particle size, and zeta potential of siTGF-β1@LP (A,C) and siTGF-β1@ILP (B,D) (n = 3).
Ijms 26 01196 g002
Ijms 26 01196 g003
Figure 3. (A) Complexation of siRNA with siTGF-β1@ILP in agarose gel at various lipid/siRNA ratios (n = 3). (B) Particle sizes and zeta potentials of siTGF-β1@ILP formed at different lipid/siRNA ratios. Shown are the means ± SD (n = 3). Stability of naked siTGF-β1 and siTGF-β1@ILP after treatment with RNase (C) or serum (D) at different times (n = 3).
Figure 3. (A) Complexation of siRNA with siTGF-β1@ILP in agarose gel at various lipid/siRNA ratios (n = 3). (B) Particle sizes and zeta potentials of siTGF-β1@ILP formed at different lipid/siRNA ratios. Shown are the means ± SD (n = 3). Stability of naked siTGF-β1 and siTGF-β1@ILP after treatment with RNase (C) or serum (D) at different times (n = 3).
Ijms 26 01196 g003
Ijms 26 01196 g004
Figure 4. (A) In vitro cytotoxicity of blank LP and blank ILP at 24 h. Shown are the means ± SD (n = 3). (B) In vitro cytotoxicity of various liposomal formulations against A549 cells following 24 h incubation. Shown are the means ± SD (n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (C) In vitro uptake study. Flow cytometric histogram profiles of fluorescence intensity of different formulations in A549 cells following 4 h of incubation at 37 °C (n = 3) are also shown. (D) Endosomal escape ability of siTGF-β1@LP and siTGF-β1@ILP in A549 cells. The blue fluorescence signal indicates the cell nucleus, red fluorescence indicates the stained lysosome, and the green fluorescence indicates siTGF-β1 labeled with FAM. Scale bar: 20 μm (n = 3).
Figure 4. (A) In vitro cytotoxicity of blank LP and blank ILP at 24 h. Shown are the means ± SD (n = 3). (B) In vitro cytotoxicity of various liposomal formulations against A549 cells following 24 h incubation. Shown are the means ± SD (n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (C) In vitro uptake study. Flow cytometric histogram profiles of fluorescence intensity of different formulations in A549 cells following 4 h of incubation at 37 °C (n = 3) are also shown. (D) Endosomal escape ability of siTGF-β1@LP and siTGF-β1@ILP in A549 cells. The blue fluorescence signal indicates the cell nucleus, red fluorescence indicates the stained lysosome, and the green fluorescence indicates siTGF-β1 labeled with FAM. Scale bar: 20 μm (n = 3).
Ijms 26 01196 g004
Ijms 26 01196 g005
Figure 5. (A) Wound healing assay. Scale bar: 100 μm. (B) Migration rates of different complexes (n = 3) (** p < 0.01, **** p < 0.0001). (C) Transwell migration assay. Scale bar: 100 μm (n = 3).
Figure 5. (A) Wound healing assay. Scale bar: 100 μm. (B) Migration rates of different complexes (n = 3) (** p < 0.01, **** p < 0.0001). (C) Transwell migration assay. Scale bar: 100 μm (n = 3).
Ijms 26 01196 g005
Ijms 26 01196 g006
Figure 6. (A) In vivo distribution and imaging of siTGF-β1, siTGF-β1@LP, and siTGF-β1@ILP in tumors-bearing nude mice. (B) Tumor volume (mm3) of different groups. (C) Body weight changes of different groups. (D) Average tumor mass isolated from the mice of each experimental group. Shown are the means ± SD (n = 3–5); * p < 0.05, **** p < 0.0001.
Figure 6. (A) In vivo distribution and imaging of siTGF-β1, siTGF-β1@LP, and siTGF-β1@ILP in tumors-bearing nude mice. (B) Tumor volume (mm3) of different groups. (C) Body weight changes of different groups. (D) Average tumor mass isolated from the mice of each experimental group. Shown are the means ± SD (n = 3–5); * p < 0.05, **** p < 0.0001.
Ijms 26 01196 g006
Ijms 26 01196 g007
Figure 7. (A) Histological changes in tumor tissues after treatments, on day 18 (n = 3–5). (B) HE staining of major organs after treatment. Scale bar: 50 μm (n = 3–5).
Figure 7. (A) Histological changes in tumor tissues after treatments, on day 18 (n = 3–5). (B) HE staining of major organs after treatment. Scale bar: 50 μm (n = 3–5).
Ijms 26 01196 g007
Ijms 26 01196 g008
Figure 8. Immunohistochemistry (IHC) staining of tumor sections was used to detect the protein levels of TGF-β1 and p-Smad3. Scale bar: 50 μm (n = 3–5).
Figure 8. Immunohistochemistry (IHC) staining of tumor sections was used to detect the protein levels of TGF-β1 and p-Smad3. Scale bar: 50 μm (n = 3–5).
Ijms 26 01196 g008
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Shi, Y.; Zhang, H.; Chen, H.; Guo, J.; Yuan, R.; Tian, Y.; Xin, Q.; Mu, Z.; Tao, Y.; Chu, Y.; et al. Cetuximab-Immunoliposomes Loaded with TGF-β1 siRNA for the Targeting Therapy of NSCLC: Design, and In Vitro and In Vivo Evaluation. Int. J. Mol. Sci. 2025, 26, 1196. https://doi.org/10.3390/ijms26031196

AMA Style

Shi Y, Zhang H, Chen H, Guo J, Yuan R, Tian Y, Xin Q, Mu Z, Tao Y, Chu Y, et al. Cetuximab-Immunoliposomes Loaded with TGF-β1 siRNA for the Targeting Therapy of NSCLC: Design, and In Vitro and In Vivo Evaluation. International Journal of Molecular Sciences. 2025; 26(3):1196. https://doi.org/10.3390/ijms26031196

Chicago/Turabian Style

Shi, Yanan, Houqian Zhang, Hao Chen, Jianwei Guo, Ranran Yuan, Yu Tian, Quanlin Xin, Zhen Mu, Yuping Tao, Yongchao Chu, and et al. 2025. "Cetuximab-Immunoliposomes Loaded with TGF-β1 siRNA for the Targeting Therapy of NSCLC: Design, and In Vitro and In Vivo Evaluation" International Journal of Molecular Sciences 26, no. 3: 1196. https://doi.org/10.3390/ijms26031196

APA Style

Shi, Y., Zhang, H., Chen, H., Guo, J., Yuan, R., Tian, Y., Xin, Q., Mu, Z., Tao, Y., Chu, Y., Wang, A., Zhang, Z., Tian, J., & Wang, H. (2025). Cetuximab-Immunoliposomes Loaded with TGF-β1 siRNA for the Targeting Therapy of NSCLC: Design, and In Vitro and In Vivo Evaluation. International Journal of Molecular Sciences, 26(3), 1196. https://doi.org/10.3390/ijms26031196

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop