1. Materials
Dopamine hydrochloride 25g(Shanghai Macklin Biochemical Co.,Ltd);Temozolamide acid500mg(Shanghai Macklin Biochemical Co.Ltd);Tris-HCL 1000ml(Beijing leagene biotech.co.,ltd);Pep-1 10.2mg(Nanjing peptide biaotech Ltd);NH2-PEG-NH2(Huateng Pharmaceuticals);N,N-dimethylformamide:DMF;Fluorescent molecule coumarin CUM(Sigma,USA); DMEM(Jiangsu KeyGEN BioTECH Corp., Ltd);Fetal Bovine Serum (PBSJiangsu KeyGEN BioTECH Corp., Ltd KGB500);Cell Counting Kit-8(Japan DOJINDOLaboratariesCP736)Hoechst33342(Jiangsu-KeyGEN-BioTECHCorp.,Ltd);PBS(Jiangsu KeyGEN BioTECH Corp.,LtdKGB5001);The UV-vis absorption spectra were examined on a UV-3600 spectrophotometer(Shimadzu,Japan).Hitach HT7700 transmission electron microscope was used to collecte the transmission electron microscopy (TEM) images. The diameters of nanoparticles were tested by a Brookhaven Zeta PALS. The confocal laser scanning microscope (CLSM) images were obtained from an FV1000 scanning microscope (Olympus, Japan). The PA images and intensity were obtained from a photoacoustic/ultrasonic imaging system (Vevo LAZR). The Fotric 225 photothermal camera was used to record temperature and thermal images.
1.1 Cell Lines and Animals information
1.2 Cell Lines information
U87MG and C6 cells were provided by Jiangsu KeyGEN BioTECH Co.,Ltd.Complete medium was 90%DMEM + 10%FBS and grown in an incubator at 37℃, 5%CO2, with saturated humidity.
1.3 Animals information
Source, germ-line and strain: BABL / c nude mice, were purchased from Shanghai Slake Experimental Animal Co., Ltd.;Experimental Animal Production License: SCXK (Su) 2020-0009;Certificate No.:20220673; Laboratory Animal Use License: SYXK (Su) 2017-0040 ;Age: 4W;Gender: male;Number of animals: 5 animals in each group, 6 groups, and 30 animals.
Standard of feeding environmental conditions: national standard of People’s Republic of China GB14925-2010; Feeding environment control system: Shenzhen Haojin Obo Company automatically control the full fresh air central air conditioning system; Temperature: room temperature 20~26℃ (daily temperature difference 4℃); humidity: relative humidity 40~70%; Light: artificial lighting, 12 hours (on at 07:00, off at 19:00).
1.4 Model preparation
Cultured glioma cell U87 cell suspensions were collected,the concentration was 5×107/ml,Vaccination ulated at 0.1ml each in nude mice subcutaneously in the right axilla.
1.5 Group and administration
Transplantation tumors in nude mice were measured in diameter with vernier calipers, and the animals were randomized when the tumors grew to around 80mm3. Meanwhile, nude mice were started for drug administration in each group, and the dosing regimen was shown in the group and dosing regimen. After the experiment, the nude mice were killed and the tumors were surgically stripped and weighed.
2.0 methods
2.1 PDA synthesis
First, 2 mg/mL of dopamine hydrochloride solution (DA) was added to 10 mL of Tris–HCI (10 mM, pH value: 8.5) solution and was reacted at 25°C via shaking (500 rpm) for 8 h. The color of the mixture changed from clear bright to bright yellow and finally tan with the progression of reaction time. Subsequently, the mixture was centrifuged at 12000 rpm for 20 min and was washed several times with ultrapure water to collect the products (PDA nanoparticles).
2.2. TMZ@PDA synthesis
TMZA is the active metabolite of TMZ in vivo that has the same cytotoxicity as TMZ. 4 The carboxyl group of TMZA was condensed with the diamine linker arm, and the terminal amino group reacted with PDA. TMZA was modified to PDA via Schiff base reaction. In addition, TMZA was reacted with NH2-PEG-NH2 to obtain TMZ-PEG-NH2 .In brief, TMZA,NH2-PEG-NH2, and DCC were dissolved in anhydrous Dimethylformamide.Then, DMAP was added, and the mixture was reacted in a N2 atmosphere and stirred overnight at 25 °C. After this, the reaction solution was dialyzed against water for 2 days, which was then freeze-dried for further use.
Loading rate (LR) and encapsulation efficiency (EE) of the Pep-1@PDA-TMZA NPs The LR and EE were assayed by ultraviolet spectrophotometry. A certain amount of TMZ was dissolved in N,N-dimethylformamide DMF (to 10 µg/ml) and its absorption peak was measured by an ultraviolet spectrophotometer with DMF as a blank. Standard solutions of TMZ (4, 6, 8, 10 and 16 µg/ml) were prepared. The absorbance of different concentrations was measured with UV detection at 329 nm and a linear regression equation was obtained. An ultrafiltration tube (MWCO, 10 kDa) was used to separate free TMZ. LR and EE were defined as follows.52
LR = (W0 - Wf )/W0 x 100%
EE = (W0 - Wf )/Ws x 100%
where W0 and Wf are the weight of initial TMZ and free TMZ detected in solution, respectively, and Ws is the weight of the complex after lyophilization.
2.3 Synthesis parameters of Pep-1
NH2-CGEMGWVRC-SH;Amino acid residue base:9
Sequence:Cys-Gly-Glu-Met-Gly-Trp-Val-Arg-Cys
Molecular weight:1040.26g/mol;Isoelectric points:6.2;;Electrostatic charge:-0.1(pH=7 )
Average hydrophilicity:-0.2;Proportion of hydrophilic residues:22%
2.4 Pep-1@PDA-TMZA NP synthesis
The Pep-1@PDA-TMZA NPs were obtained by modifying Pep-1 into TMZ@PDA using the sulfhydryl group in the side chain of the Pep-1 structure according to the abovementioned reaction steps. Finally, transmission electron microscopy, scanning electron microscopy, Fourier transform infrared, and ultraviolet spectra were used for evaluation and validation.
2.5 Stability of Pep-1@PDA-TMZA NPs
To evaluate the solubility and biostability of nanoparticles, water aqua and fetal bovine serum (FBS, 100%) were used to dissolve the nanoparticles, respectively. After 7 days, the transparency and homogeneity of nanoparticles in the water and FBS were evaluated independently.
2.6 Responsive release of the chemotherapeutic agent TMZA
The Pep-1@PDA-TMZA NPs were uniformly dispersed in acidic (pH = 5.0) and near-neutral (pH = 7.4) PBS buffer and continuously shaken at room temperature. The old PBS buffer was replaced with a new one every 2 h, and the total solution volume did not change. Subsequently, the absorbance of the supernatant was measured using ultraviolet-visible (UV-Vis) spectrophotometry. Finally, the characteristic absorption values of TMZA were used to calculate and plot the release curve of the chemotherapeutic drug TMZA using the following equation: 13
CC:The corrected concentration at time t,μg/mL
Ct:The concentration of the t moment,μg/mL
V:bulk volume,mL
v:The volume removed at each time,mL
2.7 Photothermal effect analysis
To investigate the photothermal effect of Pep-1@PDA-TMZA NPs, the temperature variations of Pep-1@PDA-TMZA NP solutions at different concentrations (20, 40, 60, 80, and 100 μg mL-1) were evaluated under near-infrared (NIR) light irradiation (808 nm, 1.0 W cm-2). In addition, the temperature variations were validated at different time points (0, 15, 30, 60, 90, 120, 180, 240, 300, 360, 420, 480, 540, and 600 s) via infrared thermography, with ultrapure water as the control group. Finally, the correlation between the temperature variations of Pep-1@PDA-TMZA NPs and the laser power density was verified using the same method.
2.8 Cellular uptake analysis
Human malignant glioma U87MG and C6 cell lines were selected to investigate the uptake of Pep-1@PDA-TMZA NPs. The cell inoculation procedure was as follows: cells were digested, counted (cell suspension configured at a concentration of 5×104 cells/mL), and added to 96-well cell culture plates (100 μL cell suspension added per well). Then, these plates were incubated at 37℃ in an incubator containing 5% CO2 for 24 h for wall attachment. Drugs were diluted with complete medium to the desired concentration, and 100 μL TMZ@ PDA was added to each wall. In addition, the Pep-1@PDA-TMZA NP medium was used, and a negative control group was set up. Thereafter, 96-well cell culture plates were incubated at 37°C in an incubator containing 5% CO2 for 48 h. In the NIR irradiation group, cells and materials were subjected to NIR irradiation for 10 min after co-incubation for 12 h and then left until 48 h. The 96-well plates were stained with CCK-8. Finally, the cellular uptake behavior of Pep-1@PDA-TMZA NPs was observed via confocal laser scanning microscopy.
2.9 Cytotoxicity analysis
Cytotoxicity was detected using the CCK-8 assay. Then, 96-well cell plates were stained with CCK-8 (λ=450 nm), and OD values were evaluated. In addition, 10 μL of CCK-8 was added to each well, and incubation was continued for 3 h in the incubator and gently mixed with a shaker for 10 min (λ=450 nm). The OD values of each well were assessed using a microplate reader to calculate the inhibition rate of each group:
2.10 In vivo antitumor targeting
2.11 Establishment of the tumor nude mouse model
Data collection: The day of administration was set as day 0. The body weight and tumor volume of nude mice in each group were counted every other day. The tumor volume was calculated using the following equation:
where a is the long diameter of the tumor and b is the short diameter of the tumor (mm).
2.12 In vivo antitumor activity
In total, 30 U87 tumor-bearing nude mice were divided into six groups (n = 5 in each group). Coumarins at a concentration of 200 μg/mL (150 μL) were injected into the caudal vein of mice in the Pep-1@PDA NPs (laser- and laser+) group, Pep-1@PDA-TMZA NPs (laser- and laser+) group, and TMZ@PDA NPs (laser- and laser+) group. After 12 h, the tumors in the corresponding (laser+) groups were irradiated with an 808-nm laser (1 W/cm2) for 10 min to record the temperature and photothermal maps of the tumor sites.
After corresponding treatment for 24 h, tumor sections were made and stained with hematoxylin &eosin (H&E) to obtain stained images of the tumor sites.
The changes in the body weight of nude mice in each group were recorded every other day. H&E-stained images of the main organs of nude mice were collected separately after 2 weeks of treatment.
Blood tests were performed on nude mice before and after treatment to assess the safety of the nanoparticles.
3.0 Statistical Analysis
Means were represented as X ± SD, the intergroup analysis was statistically processed with a t-test, and the results were statistically analyzed using SPSS (Staffstical Package for the Social Science) 17.0. P values <0.05 represented statistically significant.