Zelin Lei
Rui Jia2- 1Lanzhou University First Hospital, Lanzhou, China
- 2Lanzhou University, Lanzhou, China
- 3Gansu University of Chinese Medicine, Lanzhou, China
Background: Mesenchymal Stem Cells (MSCs), derived from the mesoderm, are adult stem cells characterized by self-renewal, multipotency, and low immunogenicity, making them promising candidates for regenerative therapies. Their intrinsic capacity to migrate to sites of injury and differentiate into diverse cell types presents considerable therapeutic potential. Particularly for lung diseases such as Idiopathic Pulmonary Fibrosis (IPF)—a chronic, progressive, and fatal lung condition with limited treatment options. Despite the potential of MSCs therapy, key challenges remain, including poor homing efficiency and limited retention in target tissues, particularly after systemic administration. Current methods do not adequately address these limitations, resulting in suboptimal therapeutic outcomes in IPF treatment. Enhancing the homing and retention of MSCs in lung tissue is critical for maximizing their therapeutic efficacy, yet an effective strategy for overcoming this challenge is still lacking.
Methods: Here, the synthesis of SA2-PEG-Peptides and their analogs was conducted using solid-phase peptide synthesis (SPPS). Two distinct strategies were devised: the first based on metabolic glycoengineering with in vivo bioorthogonal copper-free click chemistry to modify functional molecules on the MSCs surface, and the second involving phospholipid-polyethylene glycol modification of MSCs, coupling lung-targeted peptides with phospholipids for surface modification. The efficacy of these strategies was evaluated by examining retention time on the cell membrane, cell viability, cytotoxicity, membrane integrity, hemolysis, and drug distribution in mice.
Results: While the Metabolic Glycoengineering (MGE) approach did not achieve the desired modification results, the co-modification strategy using SA2-PEG2000 and SA2-PEG2000-CAR significantly enhanced the homing and retention of MSCs in lung tissue. This modification also substantially improved the therapeutic efficacy of MSCs in treating IPF.
Conclusion: In this study, we developed a cellular modification strategy based on SA2-PEG-Peptides and PEGylation. Co-modifying MSCs with SA2-PEG2000 and SA2-PEG2000-CAR markedly improved their lung-targeting and retention capacity, resulting in enhanced therapeutic outcomes for IPF. This strategy offers a potential pathway for optimizing MSCs therapies for lung diseases and may be applied to enhance the efficacy of stem cell therapies across a variety of conditions.
1 Introduction
Idiopathic pulmonary fibrosis (IPF), the most common type of fibrotic interstitial lung disease, is a progressive, irreversible, and ultimately fatal disease (Glass et al., 2022; Gunatilaka et al., 2023; Kapoor et al., 2025; Q. Liu et al., 2023; Maher, 2024). It is characterized by aberrant differentiation of myofibroblasts and excessive accumulation of collagen-rich extracellular matrix (ECM) (Pei et al., 2022), leading to structural stiffening of the lungs, formation of fibrotic lesions, and irreversible loss of pulmonary function. Current clinical management strategies for IPF encompass both pharmacological and non-pharmacological approaches (D. H. Kim et al., 2025; Lamb, 2021; Oga et al., 2009). Mesenchymal stem cells (MSCs), pluripotent adult stem cells, can modulate the immune system and promote angiogenesis through differentiation and paracrine mechanisms (K. Liu et al., 2024; Melis et al., 2025; Németh et al., 2009). Their low MHC I expression and lack of MHC II (Hwang et al., 2015), along with the absence of costimulatory molecules like CD80, CD40, and CD86, endow them with immune-evasive properties and low immunogenicity (Chan et al., 2006). Notably, the lung may provide a unique niche for MSCs, making MSC-based therapies increasingly used for lung diseases (L.-T. Wang et al., 2016). Despite their broad therapeutic potential shown in numerous studies, challenges remain. As demonstrated in the study by Németh et al., intravenously administered murine MSCs accumulate in the lungs, with over half being rapidly taken up by pulmonary macrophages (Németh et al., 2009; Ouji-Sageshima et al., 2024). The surviving few move to organs like the liver and spleen before being cleared. This leads to inadequate homing and significant loss of MSCs reaching the target site, often necessitating higher doses for effective treatment (Jingxuan et al., 2023; Tian et al., 2021; Zhao et al., 2020).
To address this challenge, the present study engineered the surfaces of MSCs by conjugating targeting peptides to enhance their retention at pathological sites. Two distinct strategies for cell surface modification were employed: (1) covalent coupling via metabolic glycoengineering (MGE)-enabled bioorthogonal reactions (Kufleitner et al., 2023; D. Y. Lee et al., 2018; N. S. Liao et al., 2021), and (2) hydrophobic insertion mediated by lipid-anchored polyethylene glycol (lipid-PEG) derivatives (Kim and Kim, 2022). The MGE approach leverages the metabolic incorporation of azide-functionalized monosaccharides (e.g., Ac4ManNAz) into the cellular glycocalyx, thereby generating exogenous azide moieties that serve as bioorthogonal chemical handles (Gutmann et al., 2019; Luchini and Vitiello, 2021). Subsequent copper-free click chemistry between cell-surface azides and dibenzocyclooctyne (DBCO)-functionalized peptides enables site-specific labeling and covalent modification (Lim et al., 2021). The hydrophobic insertion strategy utilizes lipid-PEG conjugates containing terminal maleimide groups (Hulugalla et al., 2024; Takayama et al., 2023). These amphiphilic molecules spontaneously integrate into the lipid bilayer via their hydrophobic domains, while exposing reactive maleimide groups for thiol-mediated peptide conjugation. This membrane-anchoring approach preserves native cell membrane fluidity while providing stable peptide presentation (D. Y. Lee et al., 2018).
THA is a peptide capable of targeting human airway epithelial cells. Jost et al. screened a peptide sequence, H-Thr-His-Ala-Leu-Trp-His-Thr-OH (THALWHT, abbreviated as THA) through phage screening for its ability to target human airway epithelial cells (Jost et al., 2001). A complex comprising the THA sequence and cationic DNA demonstrated efficient gene delivery to human bronchial epithelial cell indicating that the THALWHT peptide can effectively mediate in vivo targeting of human airway epithelial cells. To identify peptides capable of homing to neovasculature in healing tissues, Järvinen et al. performed in vivo phage display screening using tendon wound models (Jarvinen and Ruoslahti, 2007). This approach led to the identification of a cyclic peptide, H-Cys-Ala-Arg-Ser-Lys-Asn-Lys-Asp-Cys-OH (CARSKNKDC, abbreviated as CAR), which exhibited distinct vascular homing properties (Toba et al., 2014). Intravenous administration of the CAR peptide resulted in significant tissue penetration and preferential accumulation within the granulation tissue of wound beds. Notably, systemic delivery of CAR showed dual targeting specificity, with significant accumulation in both healing wounds and pulmonary vasculature (Maldonado et al., 2023). In addition, modification of the delivery system using stearic acid (SA) enhances anchoring of the complexes on the surface of MSCs(Li et al., 2024).
This study aimed to enhance the homing and retention capabilities of MSCs in lung tissues following systemic administration via surface modification in mice. We developed a cellular modification strategy using SA2-PEG-peptides and PEGylation. Co-modification with SA2-PEG2000 and SA2-PEG2000-CAR significantly enhanced MSC homing and retention within lung tissue, thereby improving their therapeutic efficacy in IPF.
2 Materials and methods
2.1 Cell lines
C57BL/6 mouse bone marrow mesenchymal stem cells (abbreviated in the text as BMSCs) were sourced from Cyagen Biosciences Inc. (United States) and MSCs were grown in C57BL/6 mouse bone marrow mesenchymal stem cell medium (Cyagen Biosciences Inc., United States) supplemented with 10% fetal bovine serum (FBS) (Cyagen Biosciences Inc., United States) at 5% CO2 and 37 °C.
2.2 Animals
The animals used in this experiment were purchased from the Animal Experiment Center of Lanzhou University. The strains included 6–8-weeks-old Kunming mice and C57BL/6 mice. The mice were housed in a dedicated animal room under the following conditions: temperature of 22 °C ± 2 °C, a 12-h light/dark cycle, and were fed specialized breeding mouse food. The animal experiments were approved by the Ethics Committee of the School of Life Sciences, Lanzhou University, Lanzhou, China, under approval number EAF2020022.
2.3 Peptide synthesis and purification
The peptides and their analogs used in this study were synthesized step-by-step using the Fmoc-protected strategy and solid-phase peptide synthesis (SPPS) technique. The names and sequences of the targeted peptides synthesized in the experiment are shown in Supplementary Table S1. Additionally, the coupling of lipids to the target peptides was achieved by SPPS using either 2-chlorotrityl (2-Cl-Trt) resin or Wang resin, with 5-carboxyfluorescein (5-FAM) serving as a fluorescent probe attached to the peptide side chains. Purification: The peptide was loaded onto a C8 reverse-phase column (20 × 250 mm, 10 µm), operating on an HPLCONE. Samples were eluted with solvent A (40% isopropanol, 54% acetonitrile, and 6% H2O, supplemented with 0.1% TFA) and solvent B (H2O supplemented with 0.1% TFA) at a flow rate of 10 mL/min. The gradient was programmed as follows: 0–30 min, 10%–80% solvent A; 30–50 min, 80%–100% solvent A; 50–60 min, 100% solvent A. UV detection was performed at wavelengths of 220 nm and 254 nm.
Purity analysis: The peptide was loaded onto a C8 reverse-phase column (4.6 × 250 mm, 5 µm), analyzed using a Hanbon Sci. & Tech HPLC. Samples were eluted with solvent A (40% isopropanol, 54% acetonitrile, and 6% H2O, supplemented with 0.1% TFA) and solvent B (H2O supplemented with 0.1% TFA) at a flow rate of 1 mL/min. The gradient program was as follows: 0–10 min, 10% solvent A; 10–30 min, a linear increase from 10% to 100% solvent A; and 30–40 min, 100% solvent A. UV detection was performed at wavelengths of 220 nm and 254 nm.
2.4 Determination of the critical micelle concentration (CMC)
The master mix of lipid-peptide conjugates was diluted with PBS to final concentrations of 0, 1, 5, 10, 20, 30, 50, and 100 μM, followed by centrifugation with shaking and incubation at 37 °C for 2 h. Thereafter, 0.04 μM concentration of the ANS-Na fluorescent probe was then added to each sample group, followed by centrifugation with shaking and incubation at 37 °C for 20 min. The optical density (O.D.) of each sample in PBS buffer was measured using a fluorescence spectrophotometer at an excitation wavelength of 330 nm and an emission wavelength of 492 nm. Linear regression analysis was conducted on the plot of measured O.D. values against protein concentrations to determine the critical micelle concentration (CMC) of the lipid-peptide complexes.
2.5 Ac4ManNAz cell incubation and bioorthogonal reactions of DBCO analogs
Ac4ManNAz was diluted to experiment-specific concentrations using the medium supplemented with 10% fetal bovine serum. The original medium in the Petri dish was aspirated, followed by washing with D-PBS. The prepared Ac4ManNAz dilution was then added, mixed thoroughly, and incubated in a CO2 incubator at 37 °C with 5% CO2 and saturated humidity for 72 h. After incubation, the Ac4ManNAz solution was aspirated, and the cells were washed three times with D-PBS. The basal medium was then combined with the DBCO (Lu et al., 2025) analog at experiment-specific concentration, mixed thoroughly, and added to the Petri dishes for further incubation. After the specified incubation period, the medium was aspirated, the cells were washed three times with D-PBS, and the medium was replaced with complete medium.
2.6 Cell membrane co-localization
The lyophilized Lipid-PEG-Peptides powder was dissolved in a mixture of DMSO and water at a specified ratio to prepare a 1 mM stock solution. This stock solution was then diluted to various concentrations using culture medium, and the cells were co-incubated with the peptide drug for a predetermined duration. Subsequently, the cells were washed three times with D-PBS and incubated with 10 μg/mL of the cytosolic dye Hoechst 33342 (Beyotime, China) for 10 min at a density of 2.4 × 105 cells per well. Following incubation, the cells were washed three additional times with D-PBS to remove excess dye and imaged using a high-content imaging system (ImageXpress, Molecular Devices, United States) under culture medium conditions to assess peptide drug membrane incorporation.
2.7 Trypan blue (TB) staining assay
The drug master mix was diluted to various concentrations and co-incubated with 2.4 × 105 cells per well. After a specific incubation period, TB was added to quench the external fluorescence of the cell membrane. Subsequently, the dye was then washed out with PBS, and the average fluorescence intensity of the samples was measured using the FITC channel of a flow cytometer (NovoCyte Quanteon, Agilent, United States).
2.8 Cell counting kit-8 (CCK-8) assay
After seeding 5,000 cells suspended in 100 μL were seeded into each well of a 96-well plate, which was then pre-incubated for 24 h in a humidified incubator maintained at 37 °C with 5% CO2. Subsequently, 10 μL of various concentrations of the test substances were added to each well, and the plate was incubated for the designated duration (24, 48, 72, or 96 h). Following incubation, 10 μL of CCK-8 solution (Med Chem Express, United States) was carefully added to each well, avoiding bubble formation to prevent interference with the O.D. readings. The plate was then incubated for an additional 1–4 h before absorbance was measured at 450 nm using a microplate reader. Each experimental group included three replicate wells.
As: absorbance of experimental wells containing cells, culture medium, CCK-8 solution, and drug solution;
Ac: absorbance of the control well (containing cells, culture medium, and CCK-8 solution, but without drug solution);
Ab: absorbance of the blank control (containing cells and CCK-8 solution, but without the drug solution).
This procedure also detects cell proliferation.
2.9 Apoptosis assay
Cells were resuspended in 1 mL of the drug solution and incubated on a shaker at 37 °C and 300 rpm. After incubation, the cells were washed three times with PBS and then resuspended in 100 μL of 1× Binding Buffer. The cell suspension was transferred to flow cytometry tubes, and 5 μL of YF®488-Annexin V (Shanghai BioScience, China) and 5 μL of PI working solution were added to each tube. The samples were incubated at room temperature in the dark for 10–15 min. Subsequently, the cells were resuspended in 400 μL of 1× binding buffer per tube and analyzed using the FITC/PI channel of a flow cytometer.
Treatment group: 20 μM SA2-(PEG2)2-THA and 50 μM SA2-PEG2000-CAR.
Negative control group: untreated cell samples.
2.10 Measurement of cell membrane integrity via lactate dehydrogenase (LDH) assay
The master drug solution was diluted to final concentrations of 0, 1, 5, 10, 20, 30, 50, and 100 μM. Cells were then incubated with the drug at 37 °C for 2 h. Following incubation, the samples were collected and centrifuged. From each sample, 120 μL of the supernatant was carefully aspirated and mixed with 60 μL of the working solution from the LDH Cytotoxicity Assay Kit (Beyotime, China). The mixture was incubated on a shaking platform at room temperature, protected from light, for 30 min. O.D.490 was measured using a Thermo Varioskan Flash multifunctional enzyme reader (Thermo Scientific, United States). Each experimental group was set up with 3 duplicate wells, and the experiments were independently repeated three times.
Blank control: cell-free medium;
Control: cell wells without drug;
Cell maximum enzyme activity: cell wells with 10% volume of LDH release reagent (added 1 h before the assay and mixed thoroughly by repeated pipetting).
Treated: cell wells with prepared samples of different concentrations.
Aa: absorbance of blank;
Ab: absorbance of control;
Ac: absorbance of cell maximum enzyme activity;
Ad: absorbance of treatment.
2.11 Hemolytic activity assay
Prepare a 2 mg/mL solution of heparin sodium (Sigma-Aldrich, Germany) in PBS. Add 150 μL of the prepared heparin sodium solution to a 1.5 mL centrifuge tube. Select Kunming mice weighing 18–25 g and collect approximately 1 mL of fresh blood from the eyeball, then add it to the pre-cooled centrifuge tube containing sodium heparin. Mix the blood and sodium heparin by gently inverting the tube to prevent clotting. Centrifuge at 4 °C, 800 g for 10 min and discard the supernatant. Resuspend the cells by adding 1 mL of PBS, centrifuge again, discard the supernatant, and repeat this washing step three times. The resulting erythrocytes are considered as 100% and diluted to 8% of the total volume with PBS. Add 100 μL of the diluted erythrocytes to each well of a 96-well plate. Prepare the test samples as dilutions of 0, 1, 5, 10, 20, 30, 50, and 100 μM in basal medium. The samples were incubated at 37 °C for 1 h. After incubation, centrifuge the plate at 4 °C at 1,000 g for 15 min. Carefully transfer 150 μL of the supernatant from each well to a new 96-well plate. Measure the absorbance of each well at 490 nm using a Thermo multi-functional enzyme marker. Each experimental group was set up in triplicate wells, and three independent replicate experiments were performed in total.
Treat: cell wells with prepared samples of different concentrations;
Negative control: cell wells with 100 μL of PBS buffer;
Positive control: cell wells with 100 μL of PBS buffer containing 4% Triton X-100.
As: absorbance of treatment;
An: absorbance of negative control;
Ap: absorbance of positive control.
2.12 Cell adhesion assay
Cell adhesion assays were performed in 24- plates coated with collagen I (CI) at a concentration of 5 μg/cm2 (Ji et al., 2024). The wells were washed 3 times with PBS and blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at 37 °C. MSCs were preincubated with SA2-PEG2000-CAR and SA2-(PEG2)2-THA at 50 μM for 30 min. Cell-free medium served as a blank, and unmodified MSCs were used as a control. Then 3 × 104 MSCs were seeded into each well and incubated for 30 min at 37 °C. The MSCs were subsequently washed three times with PBS and resuspended in serum-free medium (Honig and Shapiro, 2020). The number of adherent cells was determined using the Cell Counting Kit-8 (Med Chem Express, United States), with n = 4.
2.13 Construction and administration of bleomycin (BLM) mouse lung fibrosis model
C57BL/6 mice weighing 20–25 g were selected for the lung fibrosis model. The mice were anesthetized with 1.25% Tribromoethanol (sigma) at a dosage of 250 mg/kg. Following sterilization of the neck area with 75% alcohol, the mice were placed in a supine position on a mouse board. A 1 cm incision was made in the neck, and the trachea was exposed via blunt dissection. Using a 1 mL syringe, BLM was administered via tracheal instillation at a dose of 3 mg/kg (Chen et al., 2024) in a total volume of 100 μL. After completing the administration, the incision was closed. For the control group of the IPF model, 100 μL of PBS buffer was injected into the trachea after anesthesia.
Seven days following the administration of bleomycin via tracheal drip, tail vein injections of PBS, MSCs, CAR-MSCs, and PCAR-MSCs were initiated. MSCs for each experimental group were resuspended in a 1:1 ratio of 1 mg/mL sodium heparin solution and PBS buffer, making up a total volume of 200 μL. MSCs were administered every 7 days, with 1 × 106 cells in each injection, for a total of three administrations. After 28 days, the mice were sacrificed, and lung tissue was collected for Masson staining and H&E staining.
2.14 In vivo targeting assay
Male Kunming mice weighing 22–25 g were selected for in vivo targeting experiments. Cells were labeled with the lipophilic fluorescent dye (DID, Solarbio, China), which is suitable for in vivo tracer studies. MSCs were incubated with 30 μM DID for 2 h. After three washes with PBS, the cells were digested with trypsin. SA2-PEG2000-CAR solution was prepared in basal medium and added to the MSCs separately, ensuring the cells were gently agitated to allow thorough contact with the solution. The cells were incubated on a shaker for 15, 30, and 30 min, respectively. Following incubation, the cells were centrifuged at 1,500 rpm for 5 min, and the supernatant was discarded. MSCs, CAR-PEG2000-MSCs, and PCAR-PEG2000-MSCs were obtained. The cells were resuspended in 1 mL of PBS, centrifuged, and the supernatant was discarded. This process was repeated three times. After washing, the cells were counted and resuspended in PBS containing 0.5 mg/mL sodium heparin, followed by gentle agitation. A single-cell suspension containing 1 × 106 cells per 200 μL of PBS was prepared. According to the time gradient, 200 μL of the peptide-modified MSCs suspension was injected into the tail vein of different groups of mice at 0.5, 6, 12, 24, 48, 72, and 96 h before the assay. Non-drug-treated MSCs served as the control group, and PBS alone was injected as the blank control. After injection, the mice were housed in cages for further observation.
The mice in the 0.5, 6, 12, 24, 48, 72, and 96 h post-injection groups were euthanized, and major organs (heart, liver, spleen, lungs, and kidneys) were harvested. The distribution of MSCs was observed and quantitatively analyzed using a Bioin vivo imaging system (Aniview100, Guangzhou Biolight Biotechnology Co. Ltd., China) at Ex/Em: 644/663 nm.
2.15 Quantitative reverse transcription PCR
Total RNA was extracted from mouse lung tissue using the SteadyPure Universal RNA Extraction Kit II (Accurate Biotechnology, China). 1 μg of total RNA was used for the reverse transcription of RNA into cDNA in a reaction using the Evo M-MLV RT Mix Kit (Accurate Biotechnology, China). SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, China) was used, and relative gene expression quantitation was determined using the 2−ΔΔCT method and normalized to the GAPDH gene. There were three replicates per group. The PCR primers are listed in Supplementary Table S3 (Beijing Tsingke Biotech, China). Fluorescence signals were detected by Mx3005P Real-Time PCR System (Agilent Technologies Inc., United States).
3 Results
3.1 FAM-Lys (DBCO)-THA modified BMSCs via MGE and copper-free orthogonal click chemistry techniques
Initially, SPPS was used to synthesize the DBCO-targeted peptide conjugate, followed by fluorescent labeling with the dye 5-FAM, yielding FAM-K (DBCO)-THA. Both RT-HPLC analysis and ESI-MS confirmed that the purity of FAM-K (DBCO)-THA exceeded 95% (Supplementary Table S2). Additionally, laser confocal microscopy demonstrated successful stem cell modification based on MGE and bioorthogonal click chemistry (Supplementary Figure S1). Furthermore, we demonstrated that azide groups generated on the cell surface were saturated at a 20 μM Ac4ManNAz incubation, and a concentration-dependent reaction occurred between sulfo-Cy5.5-DBCO and the azide groups (Supplementary Figures S2A,B). Moreover, incubation time had no significant effect on either experimental set (data not shown).
3.2 Optimisation of conditions for FAM-THA modification
The targeted peptide THA was conjugated to DBCO, forming THA-DBCO. Co-localization results (Supplementary Figure S3A) indicated that the red fluorescence of sulfo-Cy5.5-DBCO overlapped with the green fluorescence of FAM carried by FAM- THA, producing distinct yellow fluorescence spots, confirming successful co-localization. The group treated with free THA did not display visible FAM fluorescence, suggesting that FAM-THA effectively modified MSCs through the reaction between DBCO in the coupler and azide groups generated on the cell membrane. To optimize stem cell modification efficiency using metabolic glycan engineering and bioorthogonal click chemistry, the incubation time and concentration of FAM-THA were optimized. As shown in Supplementary Figures S3B,C, incubating with 50 μM FAM--THA for 1 h was optimal for targeting cell membranes.
3.3 Insufficient retention time of FAM-Lys (DBCO)-THA modification on the cell membrane
The dynamics of membrane flow and flipping may have led to the internalization or detachment of FAM-Lys (DBCO)-THA from the cell membrane. MSCs pretreated with 20 μM Ac4ManNAz for 72 h were subsequently incubated with 50 μM Sulfo-Cy5.5-DBCO for 1 h, and fluorescence changed on the cells were observed using microscopic imaging. As shown in Supplementary Figure S4, fluorescence on the cells decreased rapidly over time, with a significant reduction at 12 h and complete loss of detectability at 24 h. These results suggest that metabolic glycan-based engineering and bioconjugation of MSCs are applicable for MSC treatments. However, the cellular modification approach using metabolic glycan engineering and bioorthogonal click chemistry exhibited insufficient retention time to effectively enhance the lung-targeting ability of MSCs.
3.4 SA2-FAM modified MSCs and the effect of drug incubation duration on internalization
Previous studies on MGE-based stem cell targeting modifications did not show the expected results. Therefore, this study attempted to explore alternative cell surface modification to conjugate targeted peptides onto stem cells (Kim and Kim, 2022; Zhong et al., 2021). SPPS was used to synthesize SA2-PEG-Peptides, and their quality was assessed by RT-HPLC (Supplementary Table S2). The results demonstrated that the purity of the targeted peptides and their analogs exceeded 95%, and mass spectrometry results aligned with the calculated theoretical values.
We utilized a stearic acid double chain (SA2) resembling lipid and incorporated it into the mesenchymal stem cell membrane through hydrophobic interactions, subsequently enabling targeted delivery of MSCs with the SA2 targeting peptide. Therefore, the SA2 modification needed to be retained in the cell membrane to expose the attached targeting peptide on the cell surface. MSCs were incubated with the cell membrane fluorescent probe DID and SA2-FAM. As shown in Figure 1A, both DID and FAM fluorescence were distributed around the MSCs, and the green fluorescence (SA2-FAM) overlapped with the red fluorescence (DID), producing a distinct yellow fluorescent signal. This indicates that SA2-FAM successfully modified the MSC cell membrane.
Figure 1. SA2-FAM co-localized with cell membranes, modification time and cell internalization. (A) SA2-FAM (10 μM) was incubated for 1 h, followed by washing and staining with the cell membrane dye DID (10 μM) for 1 h. Fluorescence co-localization was observed by laser confocal, red: DID dye; green: SA2-FAM; blue: Hoechst 33342. Scale bar: 20 μm. (B) 10 μM SA2-FAM was incubated for 15, 20, and 30 min 1 mg/mL of Trypan blue (TB) was added to quench the fluorescence on the cell membranes, and the cells were washed after 2 min of incubation. High-content cell imager observation. Green: SA2-FAM; blue: Hoechst 33342. Scale bar: 20 μm.
Due to the motility of the cell membrane, SA2-FAM, which was inserted on the membrane surface, was endocytosed by the cell, preventing the drug from remaining exclusively on the membrane surface. To prolong the retention of SA2-FAM on the cell membrane, we observed the effects of drug-cell co-incubation at various time points. The fluorescence on the cells gradually increased as the incubation time extended (Figure 1B, top), indicating that the amount of SA2-FAM inserted into the MSC membranes increased with longer incubation. As shown in Figure 1B (bottom), fluorescence was largely quenched after 15 and 20 min, suggesting that the majority of SA2-FAM remained on the cell surface, with only a minor fraction internalized. When the incubation time was extended to 30 min, some fluorescence was still retained in the MSCs, indicating that SA2-FAM was inserted into the cell membrane and underwent cellular internalization. Thus, by controlling the incubation time, it is possible to achieve high-efficiency membrane surface modification with the drug.
3.5 Construction of SA2-PEG-peptide complexes for the modification of stem cell membranes
The strong hydrophobicity of SA combined with the hydrophilicity of PEG-conjugated targeting peptides significantly reduces the internalization of SA2-PEG-Peptides during the modification process. To enhance cell membrane modification efficiency and targeting capability, various Lipid-PEG-Peptides were synthesized and differentially modified based on three targeting peptides, including THA and CAR, in this study. As illustrated in Figure 2A, SA were conjugated to either the N- or C-terminus of each targeting peptide to improve the insertion efficiency of the compounds into the cell membrane. Additionally, PEG chains of varying lengths were introduced between the SA and targeting peptides to modulate cellular internalization. However, owing to the amphiphilic properties of these analogs, micelle formation occurred at certain concentrations, which adversely affected the efficiency of SA insertion into MSC membranes. Hydrophobic fluorescent probes (ANS-Na) were used to determine the critical micelle concentration (CMC) of each group of modified peptides. As illustrated in Figures 2D–G, the peptides SA2-(PEG2)2-THA, SA2-PEG2000-THA, SA2-(PEG2)2-CAR and SA2-PEG2000- CAR exhibited distinct CMC values; however, none formed significant micelles at higher concentrations, indicating that micelle formation did not compromise membrane modification efficiency. Further modifications of the SA2-PEG peptides involved variations in the SA linkage site and PEG chain length. These analogs were labeled with the fluorescent dye 5-FAM to facilitate comparative analysis. The membrane insertion efficiencies of the different analogs were assessed through quantitative flow cytometry following treatment with Trypan blue to evaluate fluorescence differences among groups. As shown in Figure 2B, after incubation at 20 μM for 15 min of incubation, the fluorescence intensities corresponding to SA2-(PEG2)2-THA and SA2-PEG2000-THA accounted for 55.28% and 55.73%, respectively, of the total cellular fluorescence. Similarly, fluorescence associated with SA2-(PEG2)2-CAR and SA2-PEG2000-CAR cell membranes represented 55.87% and 58.16%, respectively (Figure 2C). These results indicate that SA2-PEG2000-CAR exhibits superior cell membrane insertion efficiency. Consequently, subsequent experiments were performed using SA2-(PEG2)2-THA and SA2-PEG2000-CAR.
Figure 2. Efficiency of cell membrane modification by SA2-(PEG)n-Peptides and their analogs. (A) Schematic design of SA2-(PEG)n-Targeted peptide analogs. The phospholipid portion is more hydrophobic and the targeting peptide and modified polyethylene glycol portion are more hydrophilic. (B) THA analogs: 5-FAM-labeled SA2-(PEG)n -THA analogs were added to MSCs for incubation. SA2-(PEG2)2-THA, SA2-PEG2000-THA 20 μM incubation for 15 min (C) CAR analogs: SA2-(PEG2)2-CAR, SA2-PEG2000-CAR 20 μM incubation for 15 min Quenching group: 1 mg/mL Trypan blue was added to quench the extracellular fluorescence for 2 min. Flow cytometry was used to detect the total fluorescence intensity on the cells and the fluorescence intensity that was internalized by the cells. (D–G) The critical micelle concentration of each phospholipid-targeting peptide was calculated by taking the absorbance curve with concentration as the variable and finding the regression equation of the curve. SA2-(PEG2)2-THA, SA2-PEG2000-THA, SA2-(PEG2)2-CAR, SA2-PEG2000-CAR, had critical micelle concentrations of 70, 181, 45 and 113.1 μM, respectively. n = 3. *p < 0.05; **p < 0.01; ****p < 0.0001 indicating that this experimental group showed a Statistical difference.
3.6 SA2-PEG-peptide complexes exhibit limited cellular internalization
It was hypothesized that the incorporation of hydrophilic targeting peptides and PEG could effectively inhibit cellular internalization, thereby enhancing the targeting of MSCs. To evaluate this hypothesis, 5-FAM-labeled SA2-(PEG2)2-THA and SA2-PEG2000-CAR were co-incubated with MSCs for 30 min at a concentration of 20 μM. Following incubation, cell membrane fluorescence was quenched using 1 mg/mL Trypan blue, and fluorescence changes were observed through microscopy and flow cytometry. Strong fluorescence signals were observed on MSCs in all groups prior to quenching. However, post-quenching, the SA2-(PEG2)2-THA and SA2-PEG2000-CAR groups demonstrated a significant reduction in fluorescence intensity (Figure 3), indicating successful modification of the cell membrane. In contrast, the two free targeting peptides (THA and CAR) exhibited negligible fluorescence, suggesting that cellular modification by the SA2-PEG-Peptides was predominantly mediated through hydrophobic interactions between the SA moiety and the cell membrane (Figures 3B,D).
Figure 3. Cell internalization assay. (A) SA2-(PEG2)2-THA group: 20 μM SA2-(PEG2)2-THA was incubated for 30 min. Fluorescence on the cell membrane was quenched by the addition of 1 mg/mL of Trypan blue, and the cells were incubated for 2 min and visualized by a high-intensity cell imager. Free THA was used as a control. (B) Flow quantification of cell internalization; Green: SA2-(PEG2)2-THA; Blue: Hoechst 33342. Scale bar: 20 μm. (C) SA2-PEG2000-CAR group: 20 μM SA2-PEG2000-CAR was incubated for 30 min 1 mg/mL Trypan blue was added to quench the fluorescence on the cell membrane, and the cells were incubated for 2 min and observed by a high content cell imager. Free CAR was used as a control. (D) Flow quantitative analysis of cell internalization; Green: SA2-PEG2000-CAR; Blue: Hoechst 33342. Scale bar: 20 μm **p < 0.01; ***p < 0.001; indicated that this experimental group exhibited statistical differences compared with the control group.
3.7 Prolonged retention of lipid hydrophobic modification modalities within cell membranes
The targeting efficiency of SA2-PEG-Peptides towards MSCs is influenced by the targeting capabilities of the modified peptide, the quantity of peptides successfully conjugated to the cell membrane, and the duration of these modifications on the membrane. To optimize incubation conditions, MSCs were incubated with varying concentrations (5, 10, 20, 30, and 50 μM) of SA2-(PEG2)2-THA and SA2-PEG2000-CAR for 15 min. Following incubation, peptide fluorescence was quenched using Trypan blue, and the difference in fluorescence before and after quenching was measured to identify the optimal incubation concentration. As shown in Figures 4A,B, the membrane insertion efficiencies of SA2-(PEG2)2-THA were 55.3%, 54.8%, 65.5%, 55.4%, and 45.6% at concentrations of 5, 10, 20, 30, and 50 μM, respectively. These results indicate that 20 μM SA2-(PEG2)2-THA achieved the highest membrane modification efficiency with minimal cellular internalization. A similar analysis of SA2-PEG2000-CAR revealed insertion efficiencies of 73.84%, 75.04%, 85.55%, 89.62%, and 91.27% at 5, 10, 20, 30, and 50 μM, respectively (Figures 4C,D), suggesting that 50 μM is the optimal concentration for SA2-PEG2000-CAR surface modification.
Figure 4. Cell membrane retention time of SA2-(PEG2)2-THA and SA2-PEG2000-CAR with correct incubation concentration and incubation time. SA2-(PEG2)2-THA group: (A) Microscopic imaging of SA2-(PEG2)2-THA incubated for 15 min at 5, 10, 20, 30, and 50 μM. The effect of incubation time on the internalization of SA2-(PEG2)2-THA was detected by quenching the fluorescence on the cell membrane with Trypan blue. (B) Results of flow quantitative analysis of cell internalization. Green: SA2-PEG2000-CAR; blue: Hoechst 33342. n = 3. Scale bar: 50 μm. SA2-(PEG2)2-THA group: (C) Microscopic imaging of 5, 10, 20, and 30 μM SA2-PEG2000-CAR incubated for 30 min. And Trypan blue was added to quench the fluorescence on the cell membrane to detect the effect of incubation concentration on the internalization of SA2-PEG2000-CAR. (D) Results of flow quantitative analysis of cell internalization. Green: SA2-PEG2000-CAR; blue: Hoechst 33342. n = 3. Scale bar: 50 μm SA2-(PEG2)2-THA group: (E) Microscopic imaging of 20 μM SA2-(PEG2)2-THA incubated for 15, 30, 45, and 60 min. The effect of incubation time on SA2-(PEG2)2-THA cell internalization was detected after Trypan blue quenching. (F) Results of flow quantitative analysis of cell internalization. Green: SA2-(PEG2)2-THA; blue: Hoechst 33342. n = 3. Scale bar: 50 μm. SA2-(PEG2)2-THA group: (G) Microscopic imaging of 20 μM SA2-(PEG2)2-THA incubated for 15, 30, 45, 60 min. The effect of incubation time on SA2-(PEG2)2-THA cell internalization was detected after Trypan blue quenching. (H) Results of flow quantitative analysis of cell internalization. Green: SA2-(PEG2)2-THA; blue: Hoechst 33342. n = 3. Scale bar: 50 μm. (I) 20 μM SA2-(PEG2)2-THA was incubated for 15 min and quantified by flow cytometry. (J) 50 μM SA2-PEG2000-CAR was incubated for 30 min and quantitatively analyzed by flow cytometry. Quenching group: 1 mg/mL Typan blue quenching for 2 min n = 3.
Optimal incubation times were evaluated by applying time gradients of 15, 30, 45, and 60 min for each group. The results indicated that the most effective conditions for inhibiting cell membrane internalization20 μM SA2-(PEG2)2-THA with a 15-min incubation and 50 μM SA2-PEG2000-CAR with a 30-min incubation (Figures 4E–H).
Using these conditions, the retention times of 5-FAM labeled SA2-(PEG2)2-THA and SA2-PEG2000-CAR on the membranes of MSCs were evaluated via flow cytometry. Over a period of 24 h, the fluorescence intensity of SA2-(PEG2)2-THA gradually diminished (Figure 4I). In contrast, the fluorescence of SA2-PEG2000-CAR on the cell membranes persisted for up to 72 h without significant reduction, with 66.14% of the fluorescence retained (Figure 4J). These findings suggest that lipid hydrophobic modification, which remains on the cell membrane for an extended duration, may enhance the targeting capability of MSCs relative to MGE modifications.
3.8 Biocompatibility assessment of SA2-(PEG2)2-THA and SA2-PEG2000-CAR demonstrates high compatibility
Heterozygous, homozygous, and injured MSCs, particularly those exhibiting surface markers such as phosphatidylserine, may trigger physiological tissue clearance mechanisms, leading to immunosuppression or tolerance (Galipeau and Sensebe, 2018). Apoptotic MSCs, which exhibit functional impairment, are cleared in vivo, thereby diminishing therapeutic efficacy. To assess whether the hydrophobic insertion of SA2-PEG-Peptides induces programmed cell death in MSCs, flow cytometric analysis was performed using the YF®488-Annexin V/PI Apoptosis Kit (Beyotime, China). MSCs were incubated with SA2-(PEG2)2-THA and DSPE-PEG2000-CAR at 37 °C under optimal conditions, with untreated MSCs serving as a control. The flow cytometry data were divided into four quadrants: Q1 (necrotic cells), Q2 (late apoptotic cells), Q3 (early apoptotic cells), and Q4 (normal cells). Apoptotic cells were quantified as the sum of Q2 and Q3. As shown in Figure 5A, the control group exhibited 7.59% apoptotic cells, whereas treatment with 50 μM SA2-(PEG2)2-THA for 15 min resulted in 4.97% apoptosis, indicating no significant induction of apoptosis. Conversely, MSCs treated with 50 μM SA2-PEG2000-CAR for 30 min demonstrated an apoptosis rate of 9.34%. These findings suggest that modifications with both SA2-(PEG2)2-THA and SA2-PEG2000-CAR maintain high cellular safety.
Figure 5. Biosafety Testing of SA2-(PEG2)2-THA and SA2-PEG2000-CAR. (A) Flow cytometry assays were performed using the YF®488-Annexin V/PI Apoptosis Kit. Apoptosis of SA2-(PEG2)2-THA and SA2-PEG2000-CAR at their respective optimal concentrations was detected by flow cytometry. Left: apoptotic scatter plot of normal cells; Middle: apoptotic scatter plot of SA2-(PEG2)2-THA; Right: apoptotic scatter plot of SA2-PEG2000-CAR. (B) The cell proliferation of three SA2-PEG-Peptides: SA2-(PEG2)2-THA (20 μM), SA2-PEG2000-CAR (50 μM) was determined by CCK-8 kit after incubation for 0, 24, 48, 72, 96 h. The cell proliferation was detected by multifunctional enzyme marker with the OD value of cells at 450 nm, n = 3. (C) Cytotoxicity assay. Cytotoxicity of SA2-(PEG2)2-THA and SA2-PEG2000-CARincubated for 24 h at different concentrations (5, 10, 20, 30, 50, 75, 100 μM). Blade azurite was added and incubated for 2 h. Multifunctional zymography was used to detect the uptake value at 570/590 and calculate the cell survival rate, n = 3. (D) LDH assay for cell membrane integrity. Using LDH Cytotoxicty Assay Kit, SA2-(PEG2)2-THA and SA2-PEG2000-CAR were incubated with MSCs at different concentrations for 2 h. TritonX-100-treated erythrocytes served as a positive control, and cells were detected by multifunctional zymography at OD value at 490 nm and calculated the LDH release rate, n = 3. (E) Determination of hemolytic activity of SA2-(PEG2)2-THA and SA2-PEG2000-CAR erythrocytes. Different concentrations of peptides (10, 25, 50, 100 μM) were incubated with erythrocytes at 37 °C for 1 h. TritonX-100-treated erythrocytes were used as a positive control, and multifunctional enzyme labeling instrument was used to detect the absorption value at 490 and calculate the hemolytic activity at each concentration, n = 3.
To evaluate the impact of SA2-PEG-Peptides hydrophobic insertion on the proliferation of MSCs, a CCK-8 assay was performed. MSCs were incubated with SA2-(PEG2)2-THA (20 μM for 15 min) and SA2-PEG2000-CAR (50 μM for 30 min), and their proliferation was monitored over a 96-h period. Figure 5B provides a clear representation of the absorbance values measured at 450 nm for both control and treated groups. Absorbance increased over time in all groups, indicating normal cell proliferation. No significant decrease in absorbance values was observed in any treated group compared to the control within the 96-h timeframe, suggesting that the hydrophobic modification of MSC membranes by the three SA2-PEG-Peptides did not adversely affect cell proliferation.
The safety of peptide molecular was assessed through multiple assays. The cytotoxicity of SA2-(PEG2)2-THA and SA2-PEG2000-CAR on MSCs was tested using a blade azurophilic assay at concentrations ranging from 5 to 100 μM. As illustrated in Figure 5C, cell viability remained close to 100% at the optimal peptide concentrations (20 μM for SA2-(PEG2)2-THA and 50 μM for SA2-PEG2000-CAR), indicating the absence of significant cytotoxicity.
To evaluate whether the hydrophobic insertion of SA2-PEG-Peptides disrupts cell membrane integrity, an LDH release assay was performed. As shown in Figure 5D, the LDH release rates for SA2-(PEG2)2-THA and SA2-PEG2000-CAR were below 2% after a 2-h incubation with MSCs at various concentrations. These results indicate that the hydrophobic insertion of the lipid-targeting peptides does not disrupt the membrane integrity of MSCs.
To evaluate the potential hemolytic effects of SA2-PEG-Peptides in blood circulation, hemolytic activity assays were conducted using erythrocytes derived from male Kunming mice. The erythrocytes were incubated for 1 h with varying concentrations of SA2-(PEG2)2-THA and SA2-PEG2000-CAR, while erythrocytes treated with Triton X-100 served as positive control. As illustrated in Figure 5E, both SA2-(PEG2)2-THA and SA2-PEG2000-CAR exhibited no significant hemolytic activity at a high concentration of 100 μM, with hemolysis rates remaining below 2%. These findings suggest that the SA2-PEG-Peptides possess favorable blood compatibility.
3.9 CAR-MSCs and PCAR-MSCs improve the homing and retention of MSCs in the lungs
Modification of the cell surface by peptide drugs alters the adhesion capacity between cells and the extracellular matrix, which may result in intravenously infused MSCs becoming entrapped in the lungs and forming lethal microemboli (Gao et al., 2001; R. H. Lee et al., 2009). Therefore, assessing changes in cell adhesion capacity following incubation is of critical importance. As illustrated in Figure 6A, MSCs modified with SA2-PEG2000-CAR or SA2-(PEG2)2-THA were incubated at 37 °C for 30 min, followed by co-incubation with CI for an additional 30 min to evaluate their adhesion to CI. The results demonstrated that the adhesion capacity of MSCs modified with SA2-PEG2000-CAR was significantly reduced, thereby decreasing the probability of vascular embolism induced by the modified cells in this group.
Figure 6. Cell adhesive assay and fluorescence distribution in various organs after tail vein injection of MSCs in mice. (A) Peptide-preincubated MSCs were seeded on collagen I (5 μg/cm2) monolayers and incubated for 30 min, adherent cells were then calculated (n = 4). (B) Fluorescence distribution maps of major organs (heart, liver, spleen, lung and kidney) observed by BioLive Imager of MSCs, CAR-MSCs and PCAR-MSCs at 0.5, 6, 12, 24, 48, 72 and 96 h after tail vein injection. Control group: PBS; MSCs group: simple MSCs not co-incubated with any drugs; CAR-MSCs group: 50 μM SA2-PEG2000-CAR incubated with MSCs for 30 min; PCAR-MSCs group: 50 μM SA2-PEG2000-CAR and 10 μM SA2-PEG2000 co-incubated for 30 min. (C) Quantitative analysis of ROI of fluorescence signals in each major organ at 0.5, 6, 12, 24, 48, 72, and 96 h after tail vein injection of MSCs, CAR-MSCs, and PCAR-MSCs, n = 3. He, heart; Li, liver; Sp, spleen; Lu, lung; Ki, kidney.
The primary challenge in retaining MSCs following systemic administration arises from their phagocytosis and subsequent clearance by the reticuloendothelial system. Modification with PEG has been demonstrated to facilitate evasion of clearance by the monocyte-macrophage system. Prior to experimentation, screening of SA2-PEG2000-CAR was performed to evaluate its targeting efficiency and therapeutic potential in vivo.
A total volume of 200 μL, containing 1 × 106 cells, resuspended in a 1:1 mixture of 1 mg/mL sodium heparin and PBS, was administered via tail vein injection in mice. The control group received injections of PBS alone. In the CAR-MSCs group, MSCs, pre-labeled with 30 μM DID for 2 h, were co-incubated with 50 μM SA2-PEG2000-CAR for 30 min prior to injection. For the PCAR-MSCs group, MSCs were co-incubated under identical conditions with both 50 μM SA2-PEG2000-CAR and 10 μM SA2-PEG2000 (DID-labeled). The MSCs group consisted solely of MSCs labeled with 30 μM DID.
As illustrated in Figures 6B,C, fluorescence signals following tail vein injections were predominantly localized in the lungs and liver from 0.5 to 96 h post-injection, with minimal fluorescence observed in the spleen and negligible signals observed in the heart and kidneys. These findings indicate that MSCs in the CAR-MSCs, PCAR-MSCs, and control groups were primarily distributed within the lungs. These findings indicate that MSCs in the CAR-MSCs, PCAR-MSCs, and control groups were primarily distributed within the lungs.
To further evaluate lung homing and retention, fluorescence intensity within lung tissues was analyzed (Figure 7). Compared to the MSCs group, both CAR-MSCs and PCAR-MSCs exhibited significantly enhanced lung fluorescence signals up to 72 h post-injection, with PCAR-MSCs showing the most pronounced increase. These results propose that SA2-PEG2000-CAR modification improves MSCs homing to the lungs, while co-modification with SA2-PEG2000 further enhances retention. This dual modification strategy may augment the therapeutic efficacy of MSC-based treatments for IPF.
Figure 7. Fluorescence signal distribution maps and quantitative analysis of lung tissues after tail vein injection of MSCs in mice. (A) Fluorescence signals in the lungs of MSCs, CAR-MSCs, and PCAR-MSCs at 0.5, 6, 12, 24, 48, 72, and 96 h after tail vein injection were observed by Bioinvivo Imaging. Control group: PBS; MSCs group: simple MSCs not co-incubated with any drugs; CAR-MSCs group: 50 μM SA2-PEG2000-CAR incubated with MSCs for 30 min; PCAR-MSCs group: 50 μM SA2-PEG2000-CAR and 10 μM SA2-PEG2000 co-incubated for 30 min. (B) Fluorescence signals in the lungs of MSCs, CAR-MSCs, and PCAR-MSCs at 0.5, 6, 12, 24, 48, 72, and 96 h after tail vein injection ROI quantitative analysis, n = 3. *p < 0.05, indicating that this experimental group exhibited statistical differences compared to the MSCs group.
3.10 PCAR-MSCs enhance IPF treatment-efficiency
Compared to unmodified MSCs, PCAR-MSCs exhibited enhanced lung homing and retention capabilities. To investigate whether CAR-MSCs and PCAR-MSCs could enhance therapeutic efficacy in an IPF mouse model, MSC-based treatments were administered. IPF was experimentally induced in mice through the tracheal administration of BLM. Seven days following induction, the mice were administered tail vein injections of PBS, MSCs, CAR-MSCs, or PCAR-MSCs at a dosage of 1 × 106 cells per injection, delivered once weekly for a total of three doses.
After 4 weeks, lung tissues were collected and analyzed using H&E and Masson staining (Figure 8B). In H&E-stained tissues, the BLM group exhibited characteristic IPF pathology, including alveolar wall thickening, infiltration of inflammatory cells, and disruption of alveolar architecture. In contrast, lung tissues from the CAR-MSCs and PCAR-MSCs groups demonstrated marked histological improvement. Notably, the PCAR-MSCs group exhibited tissue morphology comparable to that of the healthy control group, characterized by reduced inflammatory infiltration, diminished fibrosis, and preserved alveolar walls.
Figure 8. In vivo therapeutic capacity of IPF for lung-targeted MSCs. (A) Schematic diagram of IPF modeling and MSCs administration. (B) BLM and H&E and Masson staining of lung tissues of MSCs, CAR-MSCs, and PCAR-MSCs treatment groups. Analysis of morphological changes in lung tissues of mice, n = 3. Scale bar: 100 μm. Control group: PBS; BLM group: bleomycin; MSCs group: simple MSCs not co-incubated with any drugs; CAR-MSCs group: 50 μM SA2-PEG2000-CAR incubated with MSCs for 30 min; PCAR-MSCs group: 50 μM SA2-PEG2000-CAR and 10 μM SA2-PEG2000 co-incubated for 30 min. (C) Relative expression levels of TGF-β 1. (D) Relative expression levels of Collagen 1. The transcription level of the ctrl was defined as 1.0. Experiments were performed in biological triplicates, and error bars indicate the standard deviations. Asterisks indicate statistically significant differences compared to the WT: **P < 0.01; ***P < 0.001; ****P ≤ 0.0001.
Masson staining further confirmed that both CAR-MSCs and PCAR-MSCs significantly reduced fibrotic areas, with PCAR-MSCs exhibiting the most pronounced improvement. These results indicate that CAR modification effectively enhances the homing of MSCs to lung tissues, whereas PEG modification increases the retention of MSCs at the disease site. This combined strategy enhances the therapeutic efficacy of MSCs in treating IPF by prolonging their functional presence within the lungs.
As illustrated in Figures 7C,D, the expression levels of IPF-related genes, specifically TGF-β1 and collagen 1, were reduced in the group treated with MSCs compared to the BLM-treated group, approaching those observed in the control (Ctrl) group. These results suggest that stem cell treatment exerts a reparative effect on pulmonary fibrosis. Notably, the expression levels in the CAR and PCAR groups were closer to those in the Ctrl group, with the PCAR group demonstrating the lowest expression levels.
In conclusion, the co-modification of MSCs with SA2-PEG2000-CAR and SA2-PEG2000 represents an effective strategy to enhance MSC homing and retention within the lungs, thereby improving therapeutic outcomes for IPF.
4 Discussion
MSCs therapy has increasingly demonstrated considerable potential in the treatment of various diseases and clinical applications, presenting a promising alternative for conditions lacking effective pharmacological interventions (Le Blanc et al., 2004). Nevertheless, numerous studies and clinical trials have indicated that transplanted MSCs exhibit inadequate homing to pathological sites and possess limited retention capacity in vivo (Fang et al., 2025; Fu et al., 2023; Yu et al., 2023). These limitations result in rapid clearance by the reticuloendothelial system, thereby reducing therapeutic efficacy (Goldman et al., 2021). Cell surface modification, which entails the attachment of functional molecules to the cell membrane, can regulate critical cellular processes including differentiation, proliferation, adhesion, and interactions with neighboring cells and biomolecules (Fang et al., 2025; H. Han et al., 2025; M.-M. Han et al., 2023; J. Liao et al., 2025; Sagaradze et al., 2020; H. Wang et al., 2020; Wu et al., 2025). Consequently, cell surface modification of MSCs may provide an effective strategy to enhance their homing ability and retention at disease sites, ultimately improving the therapeutic outcomes of MSC-based treatments (Lemos and Duffield, 2018; El Agha et al., 2017; Lin et al., 2011; Rabelink and Little, 2013; Toonkel et al., 2013).
Our experiments demonstrated that Lipid-PEG-Peptides effectively modified the surfaces of MSCs via hydrophobic insertion, thereby facilitating the efficient incorporation of lung-targeting peptides into the MSC membrane and thereby enhancing the cells’ homing ability to lung tissue. PEG component imparts hydrophilicity and modulates the internalization of SA2-PEG-Peptides, resulting in increased peptide retention on the cell membrane. Additionally, the long PEG chains provide sufficient spatial flexibility, enabling lung-targeting peptides lung-targeting peptides to fully exert their targeting functions. Although systemically administered MSCs accumulate within the alveolar capillary network, they are typically rapidly cleared by macrophage-mediated phagocytosis. Surface modification of MSCs with SA2-PEG forms a protective PEGylated shell that evades clearance by the monocyte-macrophage system. Specifically, SA2-PEG2000 modification inhibits macrophage-mediated phagocytosis and prolongs MSC retention in lung tissues. The combined modification strategy, employing both SA2-PEG-Peptides and SA2-PEG2000, significantly enhances MSC homing and retention in lung tissues following systemic administration (Figure 9, comprehensive overview is drawn by GDP - Generic Diagramming Platform).
Figure 9. Lung-targeting peptides and pegylated surface modifications enhance homing and retention of MSCs in lung tissue: a comprehensive overview.
This study aimed to improve the homing and retention of MSCs in lung tissues following systemic administration via cell surface modification using lung-targeted peptides. Two primary cell surface engineering strategies were employed: metabolic glycan engineering combined with copper-free orthogonal click chemistry, and lipid-PEG hydrophobic modification. Microscopic imaging analyses confirmed the successful conjugation of 5-FAM fluorescently labeled DBCO and SA2 to the MSC surface, thereby validating the applicability of both strategies to MSCs.
Further experiments investigated the concentration dependence of Ac4ManNAz and DBCO incubation using microscopic imaging and flow cytometry. To assess the cellular retention of the first strategy, flow cytometry was used to measure the retention time on MSCs surfaces. Results indicated that the metabolic glycan engineering strategy, when combined with copper-free orthogonal click chemistry, resulted in a retention time of less than 24 h. Due to its inefficiency in MSCs-targeting and the complexity of the procedure, focus shifted to the second strategy, Lipid -PEG hydrophobic modification. Subsequent investigations revealed that SA2-PEG2000-CAR, applied in the Lipid-PEG modification, conferred enhanced retention on the MSC surface.
The vascular homing peptide CAR exhibits penetrative capacity penetrate cells and tissues, including granulation tissue at wound sites and remodeled pulmonary vascular walls in rats with pulmonary arterial hypertension (PAH) (Urakami et al., 2011). CAR induces syndecan-4-dependent activation of the small GTPase ARF6 via the guanine nucleotide exchange factor celladon-2, and promoting keratinocyte migration mediated by syndecan-4, ARF6, and cytohesin-2. Thereby, systemically administered CAR peptides selectively target pulmonary tissue (Jost et al., 2001; Maldonado et al., 2023).
The incorporation of hydrophilic PEG chains was identified as a critical factor in inhibiting cellular internalization, while the length of the PEG chains modulated the efficiency of cellular modification. We synthesized SA2-PEG-Peptides and their analogs with varying PEG chain lengths, coupling positions, and quantities. Comparative analyses of these analogues demonstrated that PEG chain length, lipid coupling position, and the number of lipid moieties significantly affect cellular internalization. Targeting peptides with N-terminal lipid attachment and PEG doping typically exhibited lower cellular internalization.
To achieve optimal modification efficiency, the effects of incubation concentration and time were assessed for SA2-(PEG2)2-THA and SA2-PEG2000-CAR. Flow cytometry and microscopic imaging results indicated that these couplers exhibited varying degrees of internalization based on concentration and incubation time. Further examination of cytotoxicity, membrane integrity, and hemolytic activity identified the optimal conditions: 50 μM SA2-(PEG2)2-THA for 15 min and 50 μM SA2-PEG2000-CAR for 30 min. Modification efficiencies were 69.35% and 75.15%, respectively. Consequently, SA2-PEG2000-CAR was selected for further experiments.
In vivo targeting efficacy was evaluated using bioimaging, where exhibited a marked increase in lung tissue fluorescence at 72 h post-injection. Numerous studies have leveraged the hydrophilicity of PEG to self-assemble it into nanoparticles for drug delivery (M.-M. Han et al., 2023; He et al., 2024; Nguyen et al., 2024; Nguyen et al., 2023; Yu et al., 2023), or to prepare liposomes by incorporating it as a membrane component (Fang et al., 2025; H. Han et al., 2025). Given this, we propose directly modifying it onto the surface of stem cell membranes (Takayama et al., 2023). The long PEG chains reduce endocytosis of surface-modified peptides due to membrane fluidity, forming a protective shell on the stem cell surface. Treating MSCs as the delivered drug, targeted peptides enable precise delivery to the lesion site. Leveraging the stem cells’ self-repair capabilities facilitates tissue repair at the diseased site. In the IPF model, the group treated with PCAR-MSCs demonstrated superior therapeutic outcomes, characterized by diminished lung structural damage, preservation of alveolar walls, and reduced fibrosis. These findings suggest that CAR peptide modification effectively enhances MSC homing to lung tissues, thereby decreasing the required transplantation dose while increasing therapeutic efficacy. Furthermore, PEGylation of MSCs further improved their retention at disease sites, resulting in sustained therapeutic benefits over time.
In conclusion, surface modification with SA2-PEG2000-CAR and SA2-PEG2000 significantly improved MSCs lung targeting and retention. Lung-targeted PCAR-MSCs demonstrated improved therapeutic efficacy in the treatment of IPF, highlighting the potential of the SA2-PEG-peptide strategy for broader applications in cell modification and disease therapy.
5 Conclusion
This study develops a co-modification strategy using SA2-PEG2000-CAR and SA2-PEG2000 SA2-PEG2000-CAR and SA2-PEG2000 to enhance the therapeutic potential of MSCs for IPF. The optimized PEG configuration improved cell surface modification, leading to significantly increased lung homing and retention of MSCs after systemic administration. As a result, MSCs modified with PCAR exhibited significantly enhanced effectiveness in addressing IPF, presenting a promising and versatile framework for cutting-edge stem cell therapies.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.
Ethics statement
The animal study was approved by the Ethics Committee of the School of Life Sciences, Lanzhou University, Lanzhou, China, under approval number EAF2020022. The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
ZL: Conceptualization, Funding acquisition, Methodology, Resources, Visualization, Writing – review and editing, Project administration, Supervision, Writing – original draft. RJ: Formal Analysis, Investigation, Methodology, Writing – review and editing, Data curation, Visualization. YR: Formal Analysis, Investigation, Writing – original draft, Data curation, Methodology, Visualization. YZ: Formal Analysis, Investigation, Writing – review and editing, Data curation, Visualization. YW: Investigation, Writing – review and editing. WC: Investigation, Writing – review and editing. XB: Supervision, Visualization, Writing – review and editing. YP: Conceptualization, Supervision, Visualization, Writing – review and editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This study was supported by the Funds for Natural Science Foundation of Gansu Province [No. 23JRRA1593] and the National Natural Science Foundation of China [No. 81960017].
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2025.1710750/full#supplementary-material
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Keywords: mesenchymal stem cells, lung targeted peptide, cell surface modification, stem cell therapy, idiopathic pulmonary fibrosis
Citation: Lei Z, Jia R, Ren Y, Zhao Y, Wang Y, Cao W, Bai X and Peng Y (2025) Targeted peptide modification of mesenchymal stem cells enhances their therapeutic efficacy in the treatment of idiopathic pulmonary fibrosis. Front. Cell Dev. Biol. 13:1710750. doi: 10.3389/fcell.2025.1710750
Received: 22 September 2025; Accepted: 02 December 2025;
Published: 18 December 2025.
Edited by:
Joaquim Vives, Banc de Sang i Teixits, SpainReviewed by:
Maria Tsoumakidou, National and Kapodistrian University of Athens, GreeceFurong Yan, Fujian Medical University, China
Copyright © 2025 Lei, Jia, Ren, Zhao, Wang, Cao, Bai and Peng. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Zelin Lei, bGVpemxAbHp1LmVkdS5jbg==