Materials and Methods.



Manufacturer,Place of production


Human hepatocellular carcinoma cell line, HB-8065TM, ATCC

Cell Counting Kit



Trypsin-edta digestion solution(0.25%)


Niu-gene Biochemical Technology,Liaoning,China



Niu-gene Biochemical Technology,Liaoning,China

Trypan Blue Staining Solution(0.4%)


Labgic Technology Co., Ltd,Beijing,China



Niu-gene Biochemical Technology,Liaoning,China

Sodium Chloride Injection(0.9%)


Shijiazhuang No.4 Pharmaceutical Co., Ltd.,Shijiazhuang,China

75cm² Cell Culture Flask

Canted Neck

NEST Biotechnology Co.LTD.

 Centrifuge Tube


NEST Biotechnology Co.LTD.

1.5mL Microcentrifuge tube


Labgic Technology Co., Ltd,Beijing,China

Cell Culture Plate

96 Well

Greiner Bio-One International GmbH

Manual single channel adjustable pipette


DLAB Scientific,Beijing,China




Universal Microplate Reader



Malvern Zetasizer Nano ZS

Malvern Instruments,U.K.


Japan JOEL

JLU SKLSHM Magellan400

FEI Company

Preparation and Characterization

 Synthesis and Characterization of pTrp-pHis and pTrp-pHis-PLGLAG-PEG8

The synthesis of pTrp-pHis and pTrp-pHis-PLGLAG-PEG8 utilized in this study was outsourced to ChinaPeptides Co., Ltd. for custom production. The detailed synthetic procedure and specifications for the obtained peptide material can be found in our labbook, provided by the manufacturing company. This customized peptide material was subsequently employed in our experimental investigations.

 Preparation and Characterization of Polymeric Micelles

pTrp-pHis-PLGLAG-PEG  was dissolved in PBS (0.01M, pH 7.4) at a concentration of 141.1 mol/L and then subjected to 30 minutes of ultrasonication. After incubating overnight, the particle size, polydispersity, and zeta potential of the micelles were assessed using Dynamic Light Scattering (DLS) with a Zetasizer Nano. Transmission Electron Microscopy (TEM) images of the micelles were acquired on a JEOL JEM-2100F electron microscope at an operating voltage of 200 kV. To prepare the samples for TEM imaging, 10 microliter of the micellar solution was deposited onto a carbon-coated copper grid and air-dried completely. Similarly, to visualize the micelles by SEM (JLU SKLSHM Magellan400, America), the silicon wafer was cleaned with oxygen plasma for 1-3 minutes and then several drops of the micellar solution were deposited on its surface for 1.5 hours. The sample was placed in a vacuum dryer for about 1-2 hours to allow the droplets to dry completely.

 Exploration of Peptide Preparation Conditions

Effect of pH: Polymeric micelles were prepared using PBS with different pH values (0.01M, pH 7.4 and pH 6.5). Dynamic Light Scattering (DLS) was employed to assess particle size, polydispersity, and zeta potential.

Effect of Concentration: Polymeric micelles were prepared using PBS (0.01M, pH 7.4) with various concentrations of the polypeptide (ranging from 28.2 to 141.1 mol/L). DLS measurements were conducted to evaluate particle size, polydispersity, and zeta potential.

Molecular dynamics simulation of polypeptides monomers aggregation into nanoparticles

MD Simulations. All systems were solvated with water molecules, and Na+ and Cl− were added to obtain a salinity of roughly 0.1 M and maintain neutral total charge. There are in total ∼169,012 atoms in each simulation box with a size of 1.19 nm × 1.19 nm × 1.19 nm. Periodic boundary conditions were applied in all three dimensions. All systems were energy-minimized by 100000 steps by using the steepest descent method to make the system reach the initial equilibrium structure。


All simulations were performed with Gromacs 2021.5( Part of the input files for Gromacs were generated by the Charmm-gui online platform ( The Charmm36m force field is used to model the protein, ions and water. Water molecules are described by the TIP3P model. Each box is filled with an explicit solvent and filled with ions using a Monte Carlo method, ensuring that the protein has enough space in the box to perform Brownian motion. Temperature control is carried out by Langevin dynamics with a friction coefficient of 1 ps−1. The system is run in the NPT ensemble at 1 atm and 300 K. After stabilizing all thermodynamic properties, the molecular system is simulated at a time interval of 2 fs. The coordinates of all models are stored every 2 ps.

Cytotoxicity Assay

Cell Culture

Cancer cells (HepG2) was cultured in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin under 90% humidity and 5% CO2 at 37 ◦C.

Cell Viability Assay

The cytotoxicity of pTrp-pHis and pTrp-pHis-PLGLAG-PEG8 was evaluated by a CCK assay. All cells were seeded onto 96-well plates at a density of 2×103 cells in 100 µL of DMEM with 10% fetal bovine serum per well. After culturing for 24 h, the medium was replaced with fresh complete medium containing different concentrations of polypeptide ranging from 8.818 to 282.2 mol/L  at predetermined pH conditions( pH 7.4 and pH 6.5). Wells without polypeptide treatment and without cells were set as the positive control and negative control, respectively. After incubation for 6 h, the CCK solution was added into every well. Upon additional incubation for 2 h, the fluorescence intensity of each well was measured on an Infinite M200 microplate reader at an excitation wavelength of 450 nm. Cell viability was calculated by the following formula:

Cell viability (%) = [(Fluorescence polypeptide – Fluorescencene negative control)/(Fluorescence positive control – Fluorescence negative control)] × 100%

Enzyme (MMP-2) activation

Dilute rhMMP-2 to 100 mol/L in DEPC-Treated water. Activate rhMMP-2 by adding APMA to a final concentration of 1 mM.Then incubate the mixture at 37 °C for 1 hour. Frozen at minus 20 degrees.