2.1 Materials and kits
Quercetin (Que, purity ≥ 98%) was obtained from North China Pharmaceutical Co., Ltd. Dipalmitoylphosphatidylcholine (DPPC)，mono-stearoyl-phosphatidyl-choline (MSPC), and DSPE-PEG2000-COOH were purchased from Shanghai AVT Pharmaceutical Technology Co., Ltd. (Shanghai, China). AS1411-NH2（5′-NH2-TTGGTG GTG GTG GTT GTG GTG GTG GTG G-3′）, FAM-AS1411-NH2（5′-NH2-TTGGTG GTG GTG GTT GTG GTG GTG GTG G-3′-FAM）and FAM-NC-NH2 (random DNA sequence, 5′-NH2-CCT CCT TCCTTC AAA ACA ACC AAC CACC-3′-FAM were obtained from Sangon Biotech. Co., Ltd. (Shanghai, China). sulforhodamine B (SRB) staining kits and Annexin V-FITC/PI Apoptosis Detection Kits were obtained from Beyotime Biotech. Co., Ltd. (Beijing, China).
2.2. Cell lines and animals
The human renal epithelial 293T cell line and human cervical cancer HeLa cell line was kindly gifted by Professor Chang at Tsinghua University. Mouse cervical cancer U14 cell line was provided by Tongpai (Shanghai) Biotechnology Co., Ltd. All cells were cultured in DMEM media (containing 10% FBS and 1% penicillin-streptomycin) at 37 °C in an incubator supplied with 5% CO2 (Thermo Scientific, Waltham, MA).
Kunming mice (female, 20±2 g, Lot No. 1100111911024294, License No. SCXK(Beijing) 2016-0011) were got from Beijing River Laboratory Animal Technology Co., Ltd. All the animals were raised under a standard SPF environmental conditions (12-hour-light/-dark cycle, 22 ± 2°C room temperature) with free access to food and water. All procedures with the experimental mice shall comply with the National Institutes of Health Guide for the Care and Use of Laboratory Animals strictly and was approved by the Committee of Animal Care and Use in Yanshan University of China (Ethics number: YD2019012)
2.3 Liposomes preparation
AQTSL was prepared by thin-film hydration . Briefly, 100 μL of DSPE-PEG2000-COOH (0.15 mg/mL ) and 100 μL activation solution (EDC/NHS) were mixed and sonicated in an ice-water bath for 30 min for further aptamer linking, then the AS1411-NH2 (5 μM) was added and sonicated in a water bath for 1 h, followed by reaction for 12 h to get AS1411-NH2-DSPE-PEG2000. Next, the products were separated with two kinds of ultrafiltration tubes (MWCO=4 KD and MWCO=10 KD, respectively) to remove the unreacted DSPE-PEG2000-COOH and AS1411-NH2. The upper layer of the second ultrafiltration tube was shaken by vertexing on an oscillator, and the liquid in the tube was transferred to a new EP tube and stored at 4°C.
DPPC, MSPC, and quercetin were weighed with a mass ratio of 9:1:1 and dissolved in a mixture of methanol and dichloromethane with a volume ratio of 2:1. The liquid mixture above was placed in a flask, sonicated for 2 min to dissolve the substances completely, followed by evaporating using a rotary evaporator. Next, the dried lipid film was hydrated with the AS1411-DSPE-PEG2000 coupling reagent to obtain the final lipid. Next, AQTSL were prepared by passing through 0.45 μm, 0.2 μm, and 0.1 μm membranes sequentially and extruding the resulting liposomes through 0.1 μm polycarbonate filter membrane 60 times at 60 °C using an extruder to make the particle size more uniform. Finally, the liposomes were packed into 14000 KD dialysis bags and dialyzed in neutral PBS solution for 2 days to remove the free quercetin from the system.
Thermosensitive liposomes (TSL), quercetin thermosensitive liposomes (QTSL), negative control (freedom DNA sequence) modified quercetin thermosensitive liposomes (NCQTSL), FAM decorated NCQTSL (FAM-NCQTSL), and FAM decorated AQTSL (FAM-AQTSL) were prepared according to the methods above with minor revisions respectively.
2.4. Characterizing of nanoparticles
A transmission electron microscopy (TEM, HT-7700, Hitachi, Japan.) was applied to observe the morphology of AQTSL, and a Malvern Zetasizer (Nano ZS, Malvern Instruments Ltd., U.K.) was used to measure the particle size and polydispersity index (PDI) of liposomes.
Measurement of the phase transition temperature was performed using a differential scanning calorimetry (DSC/DTA STA 449 F5 Jupiter, Netzsch, German). The samples were scanned with an average heating rate of 10℃/min from 10 ℃ to 70 ℃, and the nitrogen flow rate was 20~30 mL/min.
2.5. Drug encapsulation efficiency (EE) and drug release study of AQTSL in vitro
Firstly, the standard curve for quercetin of gradient concentrations vs their absorbances at 370 nm was drawn. Next, the obtained QTSL or AQTSL (without dialysis) was then placed in an ultrafiltration tube (10 KD, Millipore) and followed by centrifuging to separate the non-encapsulated quercetin from QTSL or AQTSL (5000 rpm, 15min). Subsequently, the concentration of resulted free quercetin solution was calculated by the above regression curve. The formulas used for the computation of EE and drug loading (DL) of QTSL or AQTSL were expressed as follows:
EE%=（1-Mf/Ms) × 100% (Equation 1)
DL%=（Ms-Mf）/Mp × 100% (Equation 2)
Where Mf, Ms, and Mp represented the mass of free quercetin that unencapsulated in liposomes after ultrafiltration, the total mass of the supplemented quercetin, and the total mass of the ingredients of the prescription for AQTSL respectively.
The in vitro drug release profiles of AQTSL under different temperatures were obtained by the dialysis method as we reported previously . 100 mL of PBS (pH7.4) was placed in a 250 mL beaker. After 2 mL of AQTSL suspension was loaded into a dialysis bag and hung in the beaker containing dialysis buffer, the contents of the beaker were stirred at a speed of 300 rpm by a magnetic stirrer under 37 ℃ or 42 ℃ to promote drug release. 2mL of sample was taken from the beaker at specific time points and the same volume of dialysis buffer was supplemented at the same time. The concentrations of quercetin in the taken samples were determined according to the standard curve of above.
2.6. Cell proliferation inhibitory effects of AQTSL in vitro
To assess the targeting inhibitory effects mediated by AS1411 of AQTSL on cancer cells in vitro, HeLa cells were placed in a 96-well culture plates (1×104 cells/well) and further cultured for 24 hours (37℃, 5% CO2 ). Next, QTSL or AQTSL with quercetin final concentration of 50 μg/mL were added into the culture plates, and culture medium was taken as blank control. After 48 h of treatment, the proliferation inhibition activity was determined by SRB staining method . The absorbance of blank control cells was considered as 100%, cell viabilities of different nanoparticle treatments were calculated according to the following formula.
CV % = (A540T/A540 BC) ×100% (Equation 3)
Where CV represented the cell viabilities of treatment group, A540T and A540 BC were the absorbance values of treatment group cells and the blank control cells at 540 nm.
2.7. Cytotoxicity of temperature dependent-drug release of AQTSL on HeLa cells
To explore the thermosensitive release of AQTSL in vitro, we obtained the 30 min quercetin release after incubation under 37℃ and 42℃ respectively, followed by an SRB assay to detect the in vitro cytotoxicity of different concentrations of quercetin release from AQTSL on HeLa cells. Briefly, AQTSL was incubated in a water bath at 37 ℃ or 42 ℃ respectively for 30 min to release quercetin. After drug releasing, ultrafiltration and centrifugation were applied to separate the released quercetin, and then the released quercetin was diluted to 10%, 20%, 40%, 60%, and 80% of the initial concentration with DMEM. 200 μL HeLa cells were seeded in 96-well culture plates at a density of 5×104 cells/mL and further cultured for 24 hours (37 ℃, 5% CO2). Then 100 μL quercetin diluted solution of above was added to each well and an SRB staining assay was conducted as the method of 2.6.
2.8. Cell Apoptosis Assay
The Annexin V-FITC/PI Apoptosis Detection Kit was used to detect the apoptosis inducing ability of different nanoparticles in vitro. After being treated with different nanoparticles, the cells in 6-well plate were washed three times by precooled PBS and then digested by trypsin without EDTA. The collected cells were resuspended with 500 μL binding buffer and incubated with annexin V-FITC for 15 min and PI for 5 min respectively in darkness, and then analyzed by flow cytometry (Becton Dickinson, FacsCalibur, USA).
2.9. Flow cytometry
To clarify the targeting ability of AQTSL, flow cytometry was conducted further. HeLa cells were seeded in a 6-well plate (1.0×105 cells per well) and digested by trypsin after reaching a 90% fusion rate to 1.5 mL centrifuge tubes. Then 300 μL of FAM-NCQTSL or FAM-AQTSL and 200 μL of binding buffer (4.5 mg/mL glucose、5 mM MgCl2、0.1 mg/L tRNA、0.1 mg/L Salmon sperm DNA、25mg/mL CaCl2、2 mg/L BSA in PBS) were added to each tube and incubated at 37 °C for 1h. After washing three times using washing buffer (4.5mg/mL glucose, 1mg/mL MgCl2 in PBS), cells resuspended in PBS were subjected to flow cytometry analysis, and the FL1 detection channel was selected for detection at an excitation wavelength of 488 nm. The affinity between different nanoparticles and different cells was represented by the fluorescence intensity (I). The fluorescence intensity was calculated by the formula as follow.
I=log (x-mode)×340 (Equation 4)
2.10. Antitumor activity of AQTSL in vivo
The density of U14 cells was adjusted to 1×107/mL, and 0.2 mL cell suspension was injected subcutaneously into the armpit of the left upper limb of each mouse to establish a cervical cancer-bearing mouse model. Then the model mice were randomly divided into 9 groups with 6 mice in each group (Day 0): 1) physical saline (Saline), 2) free DOX (DOX, positive control), 3) blank thermosensitive liposomes (BTSL), 4) blank thermosensitive liposomes (+ 42 °C heating) (BTSL+H), 5) free quercetin (free-Que), 6) quercetin thermosensitive liposomes (QTSL), 7) quercetin thermosensitive liposomes (+ 42 °C heating) (QTSL+H), 8) aptamer-modified quercetin thermosensitive liposomes (AQTSL) and 9) aptamer-modified quercetin thermosensitive liposomes (+ 42 °C heating) (AQTSL+H) and the corresponding treatment in each group was carried out according to the experimental design, the doses of different formulae required by each group (DOX=5 mg/kg.bw, quercetin=5 mg/kg.bw, the quercetin doses in QTSL and AQTSL=5 mg/kg.bw) was based on our preliminary experiment. After 24 h of inoculation (Day 1), the tail vein injection was conducted every two days for a total of 7 times, and the left upper limb axilla of mice in 42°C administration groups were soaked into 42°C water bath after tail vein injection 5min for 5min. The groups administrated with Saline, BTSL and free DOX were chosen as the negative control, blank carrier control and the positive control, respectively. Throughout the experiment, the experimental animals were free to eat and water. The bodyweights of mice were weighed every day, and the health indicators such as activities and feeding status were monitored. After the formation of solid tumor, the maximum diameters and the minimum diameters of tumors were measured and recorded every two days, and the tumor volumes were calculated according to the following formula.
V (mm3) = 0.5×D (max) ×D (min)2 (Equation 5)
Where V represented the volume of the tumor, D (max) stands for the maximum diameter and D (min) stood for the minimum diameter of the measured tumor .
On the 16th day of the tumor implantation (Day 15), all the experimental mice were sacrificed under ether anesthesia. The heart, liver, kidney, spleen, thymus and tumor tissues were dissected carefully, weighed accurately, and stained by H.E for pathological observation respectively. The tumor inhibition rate was calculated by the formula as follow.
TIR (%) =(1-Wa/Wn)×100% (Equation 6)
TIR was the tumor inhibition rate, Wa represented the tumor weight of the different administration group, and Wn stood for the tumor weight of the negative control (normal saline) group.
The organ coefficients were expressed by the following formula.
OC (mg/10g) = OW (mg)/BW (g) ×10 (Equation 7)
OC represented the organ coefficient of different organs in mice, OW and BW stood for the organ weight and the body weight of mice respectively.
The blood of experimental animals was collected and the serum was separated for the determination of liver function and renal function indexes (ALT, AST, BUN and CRE) to evaluate the toxic effects of different nanoparticles further.
2.11. Hemolysis experiment
Different concentrations of AQTSL (62.5, 125, 250, and 500 µg/mL, respectively), physical saline (negative control) and distilled water (positive control) were added to chicken erythrocyte to evaluate the adverse effects of AQTSL. Next, the samples were evenly mixed and incubated at 37 ℃ for 30 minutes, further centrifuged (5000 rpm, 5 min) and separated to obtain the supernatants. Finally, a UV-Vis spectrophotometer (UV-2550, Shimadzu Ltd., Japan) was applied to determine the absorbance of each sample supernatant at 570 nm wavelength. The hemolysis rate was calculated according to the following formula.
HR(%)=[(As-An)/(Ap-An) ]×100% (Equation 8)
Where HR stood for the hemolysis rate for different concentrations of AQTSL administrated samples. As, An and Ap represented the absorbances of AQTSL treated samples, negative control (physical saline) treated sample and positive control (distilled water) treated samples, respectively.
2.12. Statistical analysis
All the experiment obtained data were expressed as mean±SD with three independent experiments. The data analysis was performed using Graphpad Prism 8 software (GraphPad Software Inc., La Jolla, and CA. USA). Multiple comparisons analyzed by variance analysis and considered p <0.05 significant.