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. 2023 Jun 30;6(7):1006–1014. doi: 10.1021/acsptsci.3c00087

Delivery of Oleanolic Acid with Improved Antifibrosis Efficacy by a Cell Penetrating Peptide P10

Lidan Wang †,‡, Jingping Geng †,§, Hu Wang †,∥,*
PMCID: PMC10353059  PMID: 37470025

Abstract

graphic file with name pt3c00087_0005.jpg

Oleanolic acid (OA), a common pentacyclic triterpenoid found in plants, has several therapeutic uses, including the treatment of hepatopathy disorders. However, due to OA’s weak permeability and limited bioavailability, its therapeutic advantages are limited. Here, we showed that a short peptide known as p10 not only binds to OA but also rapidly enhances OA delivery into cultured hepatic stellate cells (HSCs), lowers their synthesis of fibrogenic proteins, and further reduces the HSC migration capacity. Our findings show that noncovalently conjugating short peptides to OA improves its pharmacological efficacy and permeability.

Keywords: Cell penetrating peptide, Drug delivery, Oleanolic acid, Fibrosis

Oleanolic acid (3-hydroxyolean-12-en-28-oic acid, OA) is a common pentacyclic triterpenoid found in fruits, vegetables, and medicinal herbs. It is well-known for its multiple pharmacological actions, including hepatoprotective, neuroprotective, cardioprotective, antimicrobial, anti-inflammatory, and antidiabetic properties.1 Although OA derivatives are commercially accessible and have been investigated as therapeutic possibilities in clinical trials for chronic renal disease, type 2 diabetes, and pulmonary hypertension in stages II and III.2,3 Because of its low water solubility and poor permeability, there is little indication of OA in clinical studies.4,5

Cell penetrating peptides (CPPs) are short peptides rich in positively charged residues that can boost their own and conjugated payload permeability across cell biological membranes, making them effective drug delivery vehicles.6−10 According to references, CPPs may transport payloads including as peptides, proteins, enzymes, antibodies, DNA/RNA, nanoparticles, and even organelles.6,7,9 Recently, our team discovered several human-originated CPPs protein sequences, hPP3,11 hPP10,12−14 Dot1l,15 P1,16 and P2,17 they have effectively transported a wide range of bioactive macromolecules into primary cultivated cells, cultured cell lines, and even in vivo tissues in animal models.

As we previously stated, positively charged CPPs may bind to the negatively charged phosphate backbone of DNA/RNA, allowing it to be delivered into several cultured cell lines, tissues, or organs. To improve the OA’s low permeability, we employed a novel CPP-P10 to deliver it into cultured HSC-T6 cells. The HSC-T6 cells were penetrated by stable peptide p10/OA complexes, which reduced the level of fibrogenic protein synthesis and migration. Our findings suggest that noncovalently short peptide conjugation can improve OA’s pharmacological effect while enhancing its permeability.

Materials and Methods

Peptide, Cell Line, and Cell Culture

Fluorescein isothiocyanate (FITC) conjugation at the N-terminus of peptide p10 (FITC-(Acp)-KKKKKRFSFKKSFKLSGFSFKKNKK, MW= 3080.8043) and nonsense NCO peptide commercially purchased from China Peptides (Shanghai, China) using F-moc solid-phase synthesis. Further examination was carried out using reversed-phase analytical high performance liquid chromatography with >96% purity. Lyophilized FITC-labeling peptides were dissolved in phosphate buffered saline (PBS) and kept at −20 °C for later usage. HSC-T6 rat hepatic stellate cell line was obtained from ATCC (American Type Culture Collection) and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin (100 U/mL)-streptomycin (0.1 mg/mL) at 37 °C and 5% CO2 humidified incubator.

Bioinformatic Analysis

CPP polarity and hydrophobicity were determined using R-code obtained from a previously published reference.18 To predict peptide p10 secondary structures, the PEP-FOLD3 online web (https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/) server was employed. PROCHECK19 and ERRAT20 were used to validate the three-dimensional (3D) model. The Molegro Molecular Viewer was used to see the expected 3D hydrophobicity, surface electrostatics, and energy map. MLCPP (https://balalab-skku.org/mLcpp2)21 predicted the peptide p10 uptake efficiency.

Molecular Docking Studies

The p10 molecular structures were predicted by using PEP-FOLD3 as described above, and the three-dimensional structure file of the Oleanolic acid (Pubchem ID: CID 10494) was downloaded in SDF format from PubChem (http://pubchem.ncbi.nlm.nih.gov). Autodock Vina Chimera was used for the docking studies. Peptide p10 and OA files were converted from the PDB to PDBQT format for docking analysis, and hydrogen atoms were added. The grid box was centered to encompass the whole peptide p10 and allow for unrestricted molecular mobility. The grid box size was set to 30 Å × 30 Å × 30 Å with a grid point distance of 0.05 nm. The interacting modes were displayed using the UCSF Chimera 1.16 visualization tool.22

Fluorescence Microscopy, and Flow Cytometry Analysis

HSC-T6 cells were seeded on coverslips in a 24-well plate at a density of 1.6 × 105 cells per well for 24 h. After three washes with PBS, the cells were treated for 1 h with various doses of FITC-labeled p10 (2.5, 5, 7.5, and 10 μM). Cells were washed 3 times with PBS before being fixed with 300 μL of 4% PFA for 20 min, rinsed 3 times with PBS, and mounted on glass slide using antifade mounting solution containing DAPI. Images were acquired using a fluorescence microscope (Sunny, Hong Kong, China), or trypsin-detached single cell suspensions were directly analyzed by using EXFLOW flow cytometer (Dakewe).

To assess the cellular uptake of peptide p10 in the presence of various penetration enhancers (DMSO and Sucrose), or endocytosis inhibitors such as Chlorpromazine (30 μM), (5-(N-ethyl-N-isopropyl)-Amiloride (10.5 μM), heparin (50 μg/mL), NH4Cl (50 μM), Wortmannin (5 μM), and serum (10%). After 1 h of incubation with or without enhancers or inhibitors, images were acquired using a fluorescence microscope (Sunny, Hong Kong, China), or trypsin-detached single cell suspensions were directly analyzed by usingEXFLOW flow cytometer (Dakewe).

To assess the cellular uptake of the peptide p10/OA complex, various molar ratios of the p10/OA complex were introduced to each well of an HSC-T6 seeded 24-well plate for 1 h. After incubation, cells were rinsed three times with PBS before being fixed with 4% PFA for 20 min, washed three times more and mounted on glass slide using antifade mounting solution containing DAPI. Images were acquired using a fluorescence microscope (Sunny, Hong Kong, China), or trypsin-detached single cell suspensions were directly analyzed by using EXFLOW flow cytometer (Dakewe).

Gel Retardation Assay

The gel retardation experiment methodology from our previous paper23 was used to investigate the complex formation of p10 and OA. In brief, several molar ratios of p10 and OA (8:0, 8:4, 8:8, 8:12, 8:16, 8:24 and 8:32) were incubated at room temperature for 20 min in a 200 μL Eppendorf tube. Prior to nondenatured polyacrylamide gel electrophoresis (PAGE), a loading buffer (without sodium dodecyl sulfate (SDS)) was added to the p10/OA complex (8.13 mL of ddH2O, 1.67 mL of 30% acrylamide/bis(acrylamide), 0.5 mL of 10× TBE, 0.5 mL of 50% glycerol, 10 μL of TEMED, and 100 μL of 10% APS). After 1 h of pre-electrophoresis at 100 V voltage, electrophoresis with p10/OA samples was performed for 4 h at a fixed voltage (60 voltage). The imager captured images, and at least three replicates were performed.

To test the stability of the p10/OA complex, preformed p10/OA complexes were treated with bovine calf serum in a 1:1 ratio for 1 or 2 h at room temperature. The samples were then separated using nondenatured PAGE in loading buffer (without SDS). Images were captured, and at least three replicates were carried out.

Fluorescent Intensity Measurement

To test the capacity of p10 and OA to form complexes, 10 μL volumes of varied molar ratios (8:0, 8:4, 8:8, 8:12, 8:16, 8:24 and 8:32) of p10 and OA were incubated at room temperature for 20 min in the dark. Supernatant and precipitation were transferred to 96-well plate containing 100 μL of PBS after centrifugation at 12000 rpm for 15 min. Multimode spectrophotometry (TECAN, Mannedorf, Switzerland) was used to measure the fluorescence intensity of supernatant and precipitation in PBS. At least three replicates were carried out.

Ultraviolet Spectrophotometry

Different molar ratios of p10 and OA (8:0, 8:4, 8:8, 8:12, 8:16, 8:24, and 8:32) were incubated at room temperature for 20 min in the dark before being transferred to a 96 well plate containing 100 μL of PBS. The absorption spectra of p10/OA encapsulation were recorded at 470–524 nm using a UV Multiskan Spectrum (Thermo Fisher Scientific, Waltham, MA, USA) reader.

Immunofluorescence Staining

In a 24-well plate, HSC-T6 cells were seeded on a coverslip at a density of 1.6 × 105 cells/well. After 24 h of culture, cells were washed three times with PBS before being treated for 24 h with preformed p10/OA complex at molar ratios of 8:16 and 8:20., Cells were fixed in 4% PFA for 20 min after being washed three times with PBS. The cells were then washed 3 times with PBS, before being incubated with rabbit polyclonal α-SMA (Wanleibio; 1:1000) and COL1A1 primary antibody (Wanleibio; 1:1000) overnight at 4 °C. After TBST washes 3 times , cells were incubated for 1 h with Cy3 (Servicebio; 1:500) or FITC-labeled (Biosharp; 1:500) goat anti-rabbit or FITC labeled secondary antibody. Images were captured using a fluorescence microscope (Sunny, Hong Kong, China) after staining with 4’,6-diamino-2-phenylindole (DAPI).

Western Blot Assay

HSC-T6 cells were plated in a six-well plate at a density of 5.0 × 105 cells per well and cultured overnight in an incubator. For 24 h, cells were treated with various molar ratios (8:16 and 8:20) of preformed p10/OA complex. After three times washing with cold PBS, cells were lysed using RIPA lysis buffer containing the protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Equal quantities of protein (approximately 10–20 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% nonfat milk in TBST for 1 h, the PVDF membrane was incubated overnight at 4 °C with rabbit polyclonal α-SMA (Wanleibio; 1:1000) and mouse monoclonal COL1α2 primary antibody (Servicebio; 1:1000). Following TBST washes 3 times, the PVDF membrane was incubated for 1 h at room temperature with horseradish peroxidase conjugate secondary antibody (Santa Cruz biotechnology; 1:3000). The Clinx ChemiScope 3000 mini enhanced chemiluminescence (ECL) detection reagent was used to detect a chemical reaction light signal.

MTT Assay

HSC-T6 cells were seeded at a density of 8000 cells per well in a 96-well culture plate and grown overnight. After washing with 100 μL of PBS, each well received a distinct molar ratio of preformed p10/OA complexes. Cells were cultured for 24 or 48 h in a 5% CO2 incubator at 37 °C. After thoroughly washing with PBS, each well received 20 μL of MTT (5 mg/mL). Finally, the supernatant was discarded, and the formazan crystals were dissolved in 150 μL of DMSO. Each well’s absorbance was measured at 490 nm with a Multiskan Spectrum reader (Thermo Fisher Scientific, Waltham, MA).

Scratch Test

In a six-well plate, HSC-T6 cells were seeded and incubated overnight. After ensuring that a monolayer culture had established, the HSC-T6 cell monolayer was scratched with lines with a plastic tip. To eliminate any debris, the wells were washed three times with a DMEM medium. In a 6-well plate, different molar ratios of preformed p10/OA complex were added and cultivated in an incubator at 37 °C. Images were acquired at various time intervals using a microscope (Sunny, Hong Kong, China). The cells’ migration distance was calculated using the ImageJ program.

Statistical Analysis

All of the control and experimental data presented here are expressed as means ± standard error of the mean (SEM). GraphPad software Prism 7.0 (GraphPad Software, San Diego, CA, USA) was used for significance analysis; statistical analyses were performed using one-way ANOVA followed by Turkey’s multiple comparison test, and p < 0.05 was considered as statistically significant difference.

Results

Penetrating Property of Peptide p10

The effector domain (residues 151–175) of the recently discovered myristoylated alanine-rich C kinase substrate (MARCKS) is the source of a novel CPP-P1 capable of successfully delivering noncovalently conjugated plasmid DNA into cultured cells.16 Previous studies revealed that the aromatic phenylalanine (Phe) residues in the basic effector domain’s may influence the structure or binding of membranes in the lipid bilayer model,24 and Watkins and colleagues found that the N-terminal Phe of the Nur-77-derived D-isoform sequence fsrslhsll, which targets Bcl-2, collaborates with the cell-penetrating moiety to boost peptide absorption at low harmless levels.25 These findings raise the question of whether the Phe residue might affect CPP-P1’s penetrating efficacy and conjugated cargo transport capabilities in a cultured cell model, which is yet unknown. MARCKS-(151–175), also known as peptide P1, has 13 basic residues and 5 Phe residues, as we discovered.

As shown in Figure S1, absorption efficiency decreased when Phe residues (from a single to all five Phe residues) were converted to alanine as well as when Phe was absent in various places. This allowed us to see if the Phe residue affected P1 penetration effectiveness. Surprisingly, one Phe residue may have a better absorption efficiency than two, three, or even all of the Phe residues. To test this theory, we designed a peptide named p10 that lacked the last Phe residue at the C-terminus of peptide P1. After that, the FITC-labeled peptide p10 was incubated for 1 h with HSC-T6 cells at the proper dose. Under a microscope, fluorescence can be shown to be well-distributed throughout the cytoplasm (Figure S2). Compared with a non-CPP we reported,26−29 fluorescence microscopy demonstrated that when peptide p10 concentration rose, the number of green positive cells and fluorescence intensity increased (Figure S2A). Flow cytometry analysis revealed equivalent results (Figure S2B). Furthermore, the efficiency of peptide p10 uptake in the presence of several penetration enhancer26,27 (Figure S3), as expected, the peptide p10s penetrating capacity was enhanced by DMSO and sugar. These findings demonstrated that the Phe-free peptide P10 (which preserves Phe at the C-terminus of P1) had comparatively high penetrating characteristics.

The effectiveness of peptide p10 uptake was then examined in the presence of a variety of cellular internalization inhibitors, such as CPZ (chlorpromazine), EIPA (5-(N-ethyl-N-isopropyl)-amiloride), heparin (a soluble analogue of heparin sulfate proteoglycans that inhibits endocytosis), NH4Cl (lysosomal pH (receptor-mediated endocytosis inhibitor of blocking PI-3 kinase).16Figure S4 reveals that serum can significantly diminish this efficiency as expected due to peptide’s potential to bind to the proteins in serum, as was discovered in prior studies,2,16 even if endocytosis-related inhibitors failed to reduce the penetration efficacy of peptide p10. The penetrating capacity of peptide p10 can only be decreased by serum, according to a flow cytometry assay, and the other endocytosis inhibitors stated earlier did not substantially differ from peptide p10 incubation alone. These results suggested that the penetration of peptide p10 may involve direct translocation rather than endocytosis.

Physicochemical Properties, Structure Prediction, and Binding of Peptide p10 with OA

The physical, chemical, and structural properties of peptide p10 were then investigated by using bioinformatic techniques. First, we assessed the hydrophobicity and polarity of peptide p10 and compared them to those of our previously characterized CPPs. It nevertheless has a medium prevalence of CPPs peptide, like P1 and Dot1l, despite leaving the green zone with a high CPPs prevalence (Figure S5A). I-TASSER’s structural prediction of peptide p10 has a Ramachandran plot assay accuracy of only 57.1% and an overall quality factor 31.25 from the ERRAT assay (data was not shown). We then performed a structural prediction using a variety of techniques. However, PEP-FOLD3 was employed to predict the structure of the peptide p10 (Figure S5B). Given that most residues in the Ramachandran plot (95.2%) are in permitted conformation red zones (Figure S5C) and that the overall quality factor from ERRAT validation is 100 (Figure S5D), this predicted structure is appropriate for the upcoming docking experiments. The three-dimensional hydrophobicity, surface electrostatics, and energy map depiction of peptide p10 are shown in Figure S5E. To further evaluate the probable binding pattern of the peptide p10/OA complex, the molecular surface electrostatic potential of peptide p10 was first examined. A positive charge outside of the pocket and a pocket in the center of the peptide p10 surface, as shown in Figure 1A, may provide the possibility for peptide p10 and OA binding. Autodock Vina in Chimera was then utilized for docking. The combination of peptide p10 and OA has a docking score of −7.749, as shown in Figure 1B. This implies that peptide p10 and OA may form a stable complex in the pocket shown in Figure 1B.

Figure 1.

Figure 1

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Molecular docking of peptide p10/OA complex. (A) Depiction of the peptide p10’s molecular surface electrostatic potential. The stick diagram of peptide p10 was buried within the 30% transparency of electrostatic surface maps, and charge intensity was colored according to scale at the bottom right of the image (negative in red, positive in blue). Charge distribution features across the peptide p10 surface with x-axis and y-axis rotation are colored according to the electrostatic intensity. (B) Molecular docking of the p10/OA complex at the molecular level. The right panel displays a cartoon image of the p10/OA complex with various x- and y-axis rotations and various orientations as predicted by software UCSF Chimera 1.16. The color of OA is purple, whereas the color of peptide p10 is golden.

Peptide p10 Noncovalently Interacts with OA and Forms a Stable p10/OA Complex

We first carried out an electrophoresis in native-PAGE to determine the noncovalent interaction between peptide p10 and OA (Figure 2A and B) before we determined how peptide p10 affects OA delivery. The mobility of peptide p10 and OA is shown in Figure 2A at various mole ratios. The peptide p10/OA complex band’s gray analysis, which got more pronounced as the mole ratio of p10 to OA increased, is quantified in Figure 2B. We subsequently determined the peptide p10/OA complex’s fluorescence intensity in the pellet (Figure 2C) and supernatant (Figure 2D) after 20 min of incubation. The fluorescence intensity of the peptide/OA complex pellets rose as the mole ratio increased (Figure 2C), but that of the supernatant decreased (Figure 2D). These results indicate that the peptide p10/OA can form a stable complex. The absorbance of the peptide/OA was then measured by using UV light with a wavelength ranging from 474 to 522 nm. The absorbance rose with increasing peptide p10/OA mole ratios, but no OA absorbance was observed (Figure 2E). We evaluated the stability of the peptide p10/OA complex in 50% serum using electrophoresis in native-PAGE, and 50% serum disrupted around 30% of the peptide p10/OA complex (Figure 2F). We concluded that peptide p10 and OA may interact noncovalently to form a stable peptide p10/OA complex.

Figure 2.

Figure 2

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Preformed peptide p10/OA complex characterization. (A) Illustrations of the peptide p10/OA complex native PAGE electrophoresis from mol ratios of 8:4 to 8:32. (B) Densitometric evaluation of three separate tests for native PAGE electrophoresis of the peptide p10/OA complex from mol ratios of 8:4 to 8:32. (C) Pellet fluorescence intensity measurement after 20 min of incubation with various molar ratios of peptide p10/OA. (D) Fluorescence intensity measurement of the supernatant following a 20 min incubation with various molar ratios of the peptide p10/OA. (E) The peptide p10/OA complex’s absorption spectrum. F. Densitometric analysis of three separate experiments that examined the peptide p10/OA complex’s native PAGE electrophoresis in the presence or absence of 50% serum (v/v).

Peptide p10/OA Complex Efficiently Enters Cultured HSC-T6 Cells

The uptake effectiveness of the peptide p10/OA complex in grown HSC-T6 cells was then assessed. Under a microscope, HSC-T6 cells displayed fluorescence as was to be expected, as shown in Figure 3A. In Figure 3B, the findings of a flow cytometry analysis are shown. Fluorescent signaling is present in 18% to 53% of the cells with a mole ratio of 8:0 to 8:32. As shown in Figure S6A (top panel) and S6B (bottom panel), we added OA and peptide p10 into each well without first forming a complex to rule out the possibility that OA would increase the penetration efficiency of peptide p10. We also investigated whether the amount of DMSO used as the solvent to dissolve the OA could increase the penetration efficiency of peptide p10 and discovered that there was no difference between different mole ratio groups, as shown in Figure S6A (bottom panel) and S6B (right panel). These results demonstrate that the peptide p10/OA complex can efficiently enter the cytoplasm. Several cellular internalization inhibitors, such as CPZ, EIPA, heparin, NH4Cl, and Wortmannin, were then used to evaluate the efficiency of peptide p10/OA complex absorption. Figure S7 shows that although heparin and EIPA could decrease the penetration effectiveness of the peptide p10/OA complex at mole ratios of 8:16 and 8:20, endocytosis-related inhibitors (Wortmannin, NH4Cl, and CPZ) were unable to do so.

Figure 3.

Figure 3

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Cellular uptake of FITC-labeled peptide p10/OA complex in HSC-T6 cells. (A) Fluorescence microscopy of peptide p10/OA complex cellular uptake at various molar ratios in HSC-T6 cells. (B) Analysis of p10/OA complex uptake by HSC-T6 cells using flow cytometry. The proportion of the flow cytometry-analyzed cell population is shown in the bottom panel.

Peptide p10/OA Complex Efficiently Inhibits HSC-T6 Activation

Since activated HSCs play a crucial role in the development of hepatic fibrosis,30 it is important to investigate potential therapies that target or downregulate activated HSCs. OA is frequently found in medicinal plans as a pentacyclic triterpenoid,1 despite the fact that it possesses hepatoprotective potential for acute liver damage, chronic liver fibrosis, and cirrhosis. Its limited application is due to its poor permeability and low water solubility.4,5 Considering the peptide p10/OA complex’s ability to permeate cultured HSC-T6 cells, we next looked at its effect on preventing fibrosis. Following the time point of earlier research set,12,31 the cell viability of HSC-T6 treated with peptide p10/OA complex was assessed after 24 or 48 h at mole ratios of 8:4, 8:20, and 8:32 (Figure 4A). The cell viability of HSC-T6 in the 8:32 group was, however, significantly lower after 48 h than it was at 24 h. Regardless of whether it was 24 or 48 h, we were unable to detect a difference in the level of cell vitality between the 8:4 and 8:20 group (Figure 4A). Then, we examined fibrogenic protein α-SMA and COL1α2 levels by Western blot (Figure 4B), and assessed α-SMA and COL1A1 protein levels by immunofluorescence staining (Figure 4C and D). The peptide p10/OA complex considerably outperformed the OA group in terms of lowering α-SMA, COL1α2 and COL1A1 levels at the mole ratios of 8:16 and 8:20. A wound healing test was performed to determine this activity since HSC-T6 cell migration represents the liver’s wound-healing response to repeated injury (Figure 4E and F). The findings showed that as compared with OA alone, the peptide p10/OA complex significantly reduced HSC-T6 cell migration. As a result, the aforementioned information above shows that the peptide p10/OA complex effectively enters cultivated HSC-T6 cells and affects crucial cellular processes such as the fibrogenic protein level and migratory capacity.

Figure 4.

Figure 4

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Enhanced delivery of OA mediated by peptide p10 in HSC-T6 cells. (A) The MTT test was used to investigate the p10/OA complexes’ cell viability. (B) α-SMA and COL1α2 Western blot examination, with or without peptide/OA complex treatment. (C) α-SMA and COL1A1 immunofluorescence staining with or without peptide/OA complex treatment. (D) Measuring the relative fluorescence intensity of COL1A1 and α-SMA with or without treatment with a peptide/OA combination. (E) Illustrations of scratch wound healing in HSC-T6 cells at various time periods following treatment with the peptide p10/OA combination. (F) Measuring the scratch open width in HSC-T6 cells at various time periods after treatment with the peptide p10/OA combination.

Discussion

Through covalent conjugations, CPPs have been used to deliver a range of functional macromolecules, including full-length proteins, peptides, and liposomes.6,7 Although noncovalently conjugated nucleic acids can be delivered by CPPs in vitro cultured cells, there is presently no evidence that pentacyclic triterpenoid can be delivered by CPPs. Here, our initial finding illustrates how noncovalent conjugations between CPP and OA affect OA interaction and subsequent OA delivery by enhancing OA permeability. Additionally, our findings might help us to better understand how CPPs-based payloads are delivered.

There is growing evidence that liver fibrosis and liver failure, which can cause fatal liver damage and permanent liver damage,32 can develop from significant liver fibrosis. Chronic harm to hepatocytes causes the synthesis and release of soluble substances that stimulate HSCs to become activated myofibroblasts, which is the basic process of fibrosis. Extracellular matrix (ECM) is overproduced in and around inflamed or injured tissue.12 Because HSC activation is a crucial step in the onset of liver fibrosis, HSCs are recognized as the primary target cells for the development of drugs to treat and prevent hepatic fibrosis.

Traditional medicine has made substantial use of the naturally occurring triterpenoid known as OA, which is widely present in plants and herbs, as an antibacterial, an antifungal, and an anti-inflammatory medication.1 Recent studies have shown that OA is a potent antioxidant that can be used to treat hepatopathy disorder.33−35 Despite this, OA has a low water solubility and poor permeability,36 which restrict its bioavailability. Since of our research interest in CPP-based delivery, we used CPP as a delivery vector to deliver OA to an in vitro cultured cell model.

In this study, we described a new CPP-p10 that is devoid of a Phe in the C-terminal of the peptide, p1, which was previously found. Peptide p10 displays great penetration efficiency in HSC-T6 cell culture via a nonendocytic pathway, Additionally, we found that the peptide p10/OA complex may enter the cytoplasm and downregulate the level of fibrogenic protein as well as their migratory capacity. Most importantly, the peptide p10-mediated OA delivery group has a higher ability to deactivate HSC-T6 than that of OA alone. These results demonstrated that in addition to transporting protein, peptide, and nucleic acids, CPP also can deliver natural products like OA into cultured cells. However, it is not clear if CPP can help with OA delivery in vivo. Therefore, a greater in vivo investigation into the function of peptide p10 in OA delivery is required in the future.

In conclusion, the newly discovered CPP peptide p10 not only has the capacity to enter cultured HSC-T6 cells on its own but also has the capacity to bind to OA and form the p10/OA complex, which enhances the delivery of OA, reduces the level of fibrogenic protein, and inhibits the migration of cultured HSC-T6 cells.

Data Availability Statement

The data generated during this work can be available upon request from the corresponding author H.W.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.3c00087.

  • Uptake efficiency prediction; cellular uptake assay; penetration enhancer treatment; cellular uptake mechanism of peptide p10; physicochemical properties; uptake efficiency of p10/OA complex, and cellular uptake mechanism of p10/OA complex (PDF)

Author Contributions

# L.W. and J.G. contributed equally to this study. L.W., J.G. performed the investigation, formal analysis, data curation, and writing–original draft. H.W. carried out the conceptualization, methodology, project administration, supervision, writing–review and editing, and funding acquisition.

This work was supported by the Science Foundation of CTGU (KJ2014B066).

The authors declare no competing financial interest.

Supplementary Material

pt3c00087_si_001.pdf (1.8MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

pt3c00087_si_001.pdf (1.8MB, pdf)

Data Availability Statement

The data generated during this work can be available upon request from the corresponding author H.W.

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