Mice and Cell Lines
The BLAB/C mice, both male and female, aged 5–7 weeks and weighing 18–20 g, were raised under a specific pathogen-free humidity and temperature control environment, purchased from the Experimental Animal Center of the Chinese Academy of Sciences. SK-HEP-1, SW480 and normal liver cell line QSG7701 were purchased from the ATCC cell bank in the United States. This study was carried out in accordance with the recommendations of the Medical Animal Care and Welfare Committee of Shanghai Tianyou Hospital. The protocol was approved by the Medical Animal Care and Welfare Committee of Shanghai Tianyou Hospital. Influencing Factors on FA-CS-Bio.
Material Synthesis And Orthogonal Experiment Design
Taking the yield of FA-CS-Bio as the investigation index, the factors affecting the synthesis of FA-CS-Bio are the ratio of raw materials, the dosage of catalyst, the reaction time and the temperature during the experiment.
Product yield (PY) (%) = product amount (FA-CS-Bio)/total amount (Bio-CS + FA) 100%
1) Determining the best raw material ratio: Synthesize FA-CS-Bio with FA: Bio-CS in molar ratios of 24:1, 25:1, 26:1, 27:1, and 28:1, and study the effect of different ratios on the production of FA-CS-Bio. The best FA: Bio-CS with the highest PY was selected.
2) Establishment of the optimal catalyst usage ratio: Based on the best FA: Bio-CS, according to FA with 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC.HCl) in the molar ratio of 1:5, 1:6, 1:7, 1:8, and 1:9, FA-CS-Bio was synthesized, and its effect on the PY was observed. The highest best FA: EDC.HCl was selected.
3) Determining the best reaction time: Based on the optimal FA: Bio-CS and FA: EDC.HCl, the reaction times were 6 h, 12 h, 24 h, 48 h, and 72 h, respectively. The best reaction time was selected.
4) Determining the best reaction temperature: The reaction temperatures were room temperature, 45 ~ 55°C, 55 ~ 65°C, 65 ~ 75°C, and 75 ~ 85°C, respectively, based on the optimal FA: Bio-CS, FA: EDC.HCl, and reaction time. The effect of reaction temperature on PY was studied, and the best reaction temperature was selected.
5) The solvent's influence on PY: Based on the optimal FA: Bio-CS, FA: EDC.HCl, reaction time and temperature, the reaction solvents were divided into dimethylacetamide (DMAc), dimethylformamide (DMF), and dimethyl sulfoxide (DMSO), and the effect of different solvents on the PY was studied.
Taking the PY of FA-CS-Bio as the investigation index, the main factors affecting the synthesis (raw material ratio, catalyst usage, reaction time, and temperature) were analyzed. The orthogonal experiment of L9 (34) was designed according to three different levels, so as to obtain the optimal
Fourier Transform Infrared Spectroscopy
The samples of Bio, CS, FA, Bio-CS and FA-CS-Bio were recorded using a Nexus Fourier Transform Infrared Spectrometer (Nicolet™ Natus Medical Incorporated, San Carlos, CA, USA). The infrared spectrometer obtained infrared spectra of samples with five grades. Background readings were taken before each series of measurements. The spectra of the powder samples were scanned at 25°C with 64 scans, the background was removed, and the resolution of the scans was 4 cm− 1. The spectral range was from 4000 cm− 1 to 400 cm− 1.
Hydrogen Nuclear Magnetic Resonance Spectroscopy
The Bio, CS, FA, FA-CS, Bio-CS and FA-CS-Bio were tested by hydrogen− 1 nuclear magnetic resonance spectroscopy (1H-NMR, 600 MHz, Agilent Technologies, Santa Clara, CA, USA), and the chemical structures of these final products were observed. The sample was dissolved in a mixed solution of deuterated hydrochloric acid and deuterated water, using tetramethylsilane as the internal standard. The sample was dissolved in a mix of deuterated hydrochloric acid and deuterated water to form a mixed solution. The sample was measured at a frequency of 600 MHz, and the internal standard of the sample was tetramethylsilane. And according to the integral area of the characteristic proton peaks of FA and CS, the degree of substitution (DS) of FA was calculated as integrated area FA/integrated area CS×100%.
Preparation Of Fa-cs-bio/5-fu Nanoparticles
The FA-CS-Bio/5-FU mass ratio was set to 1:4, and the FA-CS-Bio was placed in a centrifuge tube with an appropriate amount of deionized water and glacial acetic acid (HAC) before being placed in a 50°C water bath. The HAC, deionized water, and 50 mM NaAC were added after heating for more than 10 min to dissolve, and the water bath was placed at 45 ~ 50°C for 5 ~ 10 min to obtain the FA-CS-Bio solution. The 5-FU was heated to 45 ~ 55°C for 5 ~ 10 min before being poured into the Bio-GC solution, immediately vortexed (2000 rpm) for 30 s, the sample was lifted for more than 30 min and centrifuged at about 4000 rpm, the residue was washed, dispersed, centrifuged and washed to remove NaOH and 5-FU, etc., and then was redispersed in deionized water, freeze-dried, and saved for later use.
Particle Size Analysis And Zeta Potential Determination
The nanoparticle suspension with a 5 mL syringe was sucked and injected into Zeta Potential measurements were taken with a Zetasizer HS3000 (Malvern Instruments, Malvern, UK) in a potential conduit, and particle shapes were determined using a TECNA10 transmission electron microscope (Philips Company, Philips, the Netherlands).
Preparation Of The 5-fu Standard Curve
The 5-FU standard substance was accurately weighed; the 5-FU concentration with simulated body fluid into a series of standard solutions with concentrations of 0.1, 0.2, 0.5, 1.0, 5.0, 10 and 20 g/mL was prepared, respectively; and 20 mL of the above-mentioned solutions was accurately drawn and injected into a liquid chromatograph (Shimadzu Corp., Kyoto, Japan), and the peak area was measured and recorded according to the chromatographic conditions. The regression was performed with the concentration of 5-FU (x) and the corresponding peak area (y).
Conditions used for chromatography: As the stationary phase, an octadecylsilane (C18) column with a particle size of 3.5 µm, an inner diameter of 3.0 mm, and a length of 100 mm was used at 30°C. The mobile phase consisted of acetonitrile, MeOH, and 20 mM ammonium acetate in a volume ratio of 54:36:10 and was used at a flow rate of 1.0 mL/min, with injections limited to 20 L per injection.
In vitro release, encapsulation efficiency, and drug loading assay
20 mg of FA-CS-Bio/5-FU nanoparticles and 5-FU were weighed respectively and put into a dialysis bag. 30 mL of simulated body fluid (SBF) was added to form a suspension (pH 7.4) and shaken at 37°C at a constant temperature. Dynamic dialysis was carried out in the device (frequency of 60 r/min); after 0, 20 min, 40 min, 60 min,... (that is, 6 hours), 1 d, 2 d, 3 d,... 10 d, the dialysate was taken out, and 30 mL of fresh SBF was added at the same time. The original volume was kept unchanged, and the absorbance value was measured. The amount of 5-FU released by nanoparticles at different times was calculated according to the standard absorption curve. The concentration was calculated according to the standard curve equation, and the average value of three experiments was performed. Cumulative drug release (%) = (5-FU released from samples) / (total amount of 5-FU) ×100%
The nanoparticles were destroyed with 1% HCL, SBF was added to the volume, the SBF was used as a blank control, and its absorbance at 254 nm was measured. The encapsulation efficiency and drug-loading capacity of FA-CS-Bio/5-FU nanoparticles were calculated according to the following formula compared to the standard curve of 5-FU in SBF.
Encapsulation efficiency (%) = Drug amount of drug-loaded nanoparticles/Dosage amount×100%
Drug loading (%) = Drug amount of drug-loaded nanoparticles/Total amount of drug-loaded nanoparticles×100%
Cell Proliferation Assay
SW480 and SK-HEP-1 were subcultured in DMEM medium containing 10% fetal bovine serum and dual antibodies (100 U/mL each of penicillin and streptomycin) at 37°C in a 5% carbon dioxide incubator, the tumor cells was collected, centrifuged at 1000 rpm for 5 min, the cell density was adjusted with DMEM medium, and cells in a 96-well plate at 1000 cells/100µL/well was seeded. Each drug (control group, 5-FU, CS/5-FU, Bio-CS/5-FU, FA-CS/5-FU and FA-CS-Bio/5-FU) was fully cultured with DMEM, and formulated into 10, 3, 1, 0.3, 0.1, 0.03, 0.01 µg/mL. Three action time points of 24, 48 and 72 h were selected for each concentration group, and the absorbance was measured at 450 nm with an M5 multifunctional microplate reader (Bio-Rad, Hercules, CA, USA), and each group was detected three times. At the same time, the final concentration of 5-FU was 0.3 µg/mL, the cells treated with various treatments, the absorbance was detected 3 times at each time point of 1d, 2d, 3d, 4d, 5d, 6d and 7d. Tumor inhibition rate = (Control A450-Experiment A450)/Control A450×100%.
Intracellular Drug Concentration Of Hepatocellular Carcinoma By Fa-cs-bio
SK-HEP-1 and QSG 7701 cells were cultured. Actively proliferating cells were collected, counted, centrifuged at 1000 rpm for about 5 min, and seeded into culture wells at a concentration of 1000 cells/100 µL. The various drugs (5-FU, CS/5-FU, Bio-CS/5-FU, FA-CS/5-FU, and FA-CS-Bio/5-FU) were prepared in complete medium at 1 µg/mL, 2 µg/mL, 3 µg/mL, 6 µg/mL, and 12 µg/mL incubation time points for each drug concentration. At the same time, each drug group was treated for 1 h, 2 h, 4 h, 6 h, and 8 h with a final concentration of 3 µg/mL of 5-FU. The cells were washed three times, digested with enzymes, centrifuged, the supernatant was removed, 100 µL methanol was added, the cells were freeze-thawed, disrupted, and centrifuged with a high-speed centrifuge, 20 µL of the supernatant was collected, and the intracellular 5-FU concentration was measured by high-performance liquid chromatography according to the pretreatment method, three times in each group. The intracellular drug concentration was calculated from the standard curve. The ratio of the 5-FU concentration in liver cancer cells and hepatocytes reflects the targeting ability of the material to a certain extent. In this study, the ratio of SK-HEP-1/QSG 7701 (S/Q) was used.
Cell Migration Assay
50 µL of 5 µg/mL Fibronectin gel (ProSpec-Tany TechnoGene Ltd., Ness-Ziona, Israel) was dropped onto a Transwell chamber (Corning Inc., Corning, NY, USA), and air-dried overnight in a biological safety cabinet. SK-HEP-1 cells were subcultured in DMEM containing 10% FBS at 37°C in a 5% carbon dioxide incubator. SK-HEP-1 cells were collected, counted, centrifuged at 1000 rpm for 5 min, and adjusted the cell density with DMEM; 2000 cells/100 µL/well, seeded cells in the upper chamber, without serum. For the lower chamber, 500 µL of SK-HEP-1 cells containing 10% FBS was collected. In the upper chamber, five drugs (5-FU, CS/5-FU, Bio-CS/5-FU, FA-CS/5-FU and FA-CS-Bio/5-FU) were performed with 0.3 µg/mL of 5-FU. After 48 h of incubation, 50 µL of 0.5% crystal violet solution was added to the upper chamber and photographed. The cells were digested under the chamber with trypsin to make 20 µL of cell suspension. A small amount of the suspension was drawn to fill the counting plate pool, and 5 fields of view under the microscope (the upper, middle, lower, left, and right) were tokenized at 200 magnification and counted through the chamber. The number of cells that passed through the membrane was used to evaluate the migration ability, and each group of results was repeated 3 times.
Animal Model Establishment
After the mouse model was successfully established by inoculating H22 cells into BLAB/C mice subcutaneously, when the tumor grew to 2 ~ 3 cm, the mice were sacrificed, the tumor tissue was dissected out, and the vigorous and fresh tumor tissue was selected to make 6 × 107 cells/mL of tumor cell suspension. After intraperitoneal anesthesia with 20% Ulatan, a median incision was made, and 50 µL of tumor cell suspension was injected into the left hepatic lobe capsule with a 1 mL syringe.
Drug Concentration Of Fa-cs-bio/5-fu Nanoparticles In Various Tissues
After the orthotopic liver cancer transplantation model was established, they were randomly divided into 5 groups with 3 animal models in each group and were injected into the tail vein with the 5-FU, CS/5-FU, FA-CS/5-FU, Bio-CS/5-FU and FA-CS-Bio /5-FU at doses of 0.5 µg/g, and the mice were sacrificed 24 hours later, and the liver, spleen, kidney, lung, and heart tissues of the animal model were washed with normal saline (blood removed), blotted dry with filter paper, then 0.1 g of tissue was placed in a centrifuge tube, and 1 mL of 50% methanol was added. The cells were crushed into a homogenate with an ultrasonic cell crusher and centrifuged at 8000 r/min for 10 min. 20 µL of the supernatant was aspirated, and the concentration of 5-FU in each tissue was measured by high performance liquid chromatography.
Laser Confocal Detection
SK-HEP-1 cells and QSG 7701 cells were seeded in 6-well plates, incubated for 24 h, and then fresh medium containing different FITC-labeled CS, FA-CS, Bio-CS, and FA-CS-Bio nanoparticles was incubated for 4 h. Thereafter, after fixing cells with 4% paraformaldehyde for 20 min at room temperature, the nuclei were stained with the fluorescent dye Hoechst 33258 (Biyuntian Biotechnology Co., Ltd., Shanghai, China), and washed three times with 0.01 M PBS. The coverslip was taken out with small tweezers and placed on a glass slide. After mounting with glycerol buffer solution, the fluorescence images of the samples were observed by a laser co-polymerization microscope (Olympus FV-1000, Tokyo, Japan). Laser excitation wavelength during scanning: FITC was 488 nm, Hoechst 33258 was 405 nm, Alexa was 633 nm, and the captured images were superimposed with NIS element imaging software.
Dynamic imaging of FA-CS-Bio nanoparticles in vivo
In order to ensure that the fluorescence signal intensity of different modified nanoparticles was consistent, some amino groups on the nano-framework CS with Rhodamine B Isothiocyanate (RBITC) were reacted to obtain the same amount of isothiocyanate. Rhodamine B-labeled nanomaterials RBITC-CS, and then synthesized RBITC-Bio-CS, RBITC-FA-CS, and RBITC-FA-CS-Bio. On the fifth day after the orthotopic liver cancer transplantation model in mice, RBITC-CS, RBITC-Bio-CS, RBITC-FA-CS, and RBITC-FA-CS-Bio nanoparticles were injected from the tail vein at a dose of 0.4 mg/20 g. In mice, the dynamic distribution of CS, Bio-CS, FA-CS, and FA-CS-Bio in mice was dynamically observed by a small animal in vivo imaging system (CRi Maestro™, USA). The observation time points were 2 h and 4 h, 8 h, 12 h, and 24 h, respectively. Three mice in each group were sacrificed at each time point, and the fluorescence intensities in liver, kidney, spleen, and brain tissues were detected, representing the amount of nanoparticles entering the site. After the mice were sacrificed at the time point of 24 h, the number of fluorescent photons in the liver and liver cancer areas was detected by the CRi Maestro™ imaging system, and the fluorescence ratio of liver cancer (C)/liver (L) was calculated.
Survival Analysis Of Fa-cs-bio/5-fu Nanoparticles In The Mouse Orthotopic Liver Cancer Transplantation Model
On the 5th day after the mouse orthotopic liver cancer transplantation model was established, the liver cancer tissue was about 4 ~ 6 mm, and the experimental model was divided into 6 groups: control, FA-CS-Bio, 5-FU, CS/5-FU, FA-CS/5-FU, Bio-CS/5-FU and FA-CS-Bio/5-FU groups. The control group received an intravenous injection of normal saline. The dose was 200µL. FA-CS-Bio group: 200 µL was intravenously injected with FA-CS-Bio. 5-FU, CS/5-FU, FA-CS/5-FU, Bio-CS/5-FU, and FA-CS-Bio/5-FU groups were respectively given corresponding drugs in 200 µL (containing 0.371 mg of 5-FU). Beginning on the 6th day after molding, administration through the tail vein continued for 5 days. Nine experimental model mice in each group were used for survival analysis.
Blood Biochemical Analysis
After 10 days of treatment, the serum samples of the mice in each group were taken, and the blood biochemical indexes alanine aminotransferase (ALT) and aspartate aminotransferase were detected by the DA 3500 Discrete Analyzer automatic chemical analyzer (Fuji Medical System Co., Ltd., Tokyo). The white blood cells (WBC), red blood cells (RBC), hemoglobin (Hgb), and platelets (PLT) were detected by the Sysmex XS-800i automatic blood cell analyzer (Sysmex Shanghai Ltd, Shanghai, China).
Statistical Analysis
Normally distributed data are expressed as mean ± standard deviation, A t-test was used to compare the two groups; an one-way analysis of variance (ANOVA) was used to compare multiple groups; and the least significant difference method (LSD-t) was used for pairwise comparison between groups. Survival time was analyzed by the Kaplan-Meier method, and p < 0.05 was regarded as statistically significant.