2.1 Preparation of HPAA derivative
2.1.1 Materials
CBA was synthesized based on our previous studies[17, 18]. 1-(2-Aminoethyl) piperazine (AEPZ) and dicyclohexylcarbodiimide (DCC) were obtained from Aladdin Industrial Corporation (Shanghai, China). α-Malemidyl-ω-N-hydroxysuccinimidyl polyethyleneglycol (MAL-PEG
-NHS, MW:5000) was purchased from Xi’an ruixi Biological Technology Co. Ltd. (Xi’an, China). The branched polyethylenimines (bPEIs) were provided by Sigma-Aldrich. The solvents were provided by Chemical Reagent Factory (Guangzhou, China). A pLenti lentivirus was utilized for transfection and the generation of the LNCaP cell line to construct PSMA-expressing metastatic tumors animal models (OBiO, Shanghai, China). miRNA-133a-3p, negative control-miRNA and the A10-3.2 anti-PSMA aptamer modified with infrared dye Cy5 at the 5’ terminus were purchased from RiboBio (Guangzhou, China).
2.1.2 Preparation of HPAA-PEG and HPAA-PEG-APT
HPAA was synthesized as described previously[17, 18]. First, 200 mg of NHS-PEG-MAL was dissolved in phosphate-buffered solution (PBS, pH 7.4) for HPAA and NHS-PEG-MAL conjugation. Next, a 10:1 dilution of HPAA and NHS-PEG-MAL was conjugated through a specific reaction between the main amino groups of HPAA and NHS groups of the PEG derivative. The synthesis was performed in PBS at ambient temperature in the dark for 2 h. Next, the HPAA-PEG conjugate was purified by ultrafiltration and then was freeze-dried and kept at 4 °C until ready for use.
To conjugate the anti-PSMA aptamer with HPAA-PEG, 100 mg of HPAA-PEG was dissolved in PBS (pH 6.5–7.0). Next, 2 ml of DMSO containing 1 mg of APT was added dropwise to the HPAA-PEG and stirred in 30 min, and the compound was reacted at ambient temperature in the dark for 48 h. The HPAA-PEG-APT conjugate was also purified by ultrafiltration, followed by lyophilization and then storage at 4 °C.
2.2 Characterization
2.2.1 NMR
The HPAA derivatives were characterized by nuclear magnetic resonance (NMR). Two milligrams each of HPAA, HPAA-PEG, NHS-PEG-MAL and HPAA-PEG-APT was dissolved in D2O and NMR. The results were compared using MestReNova software.
2.2.2 Size and zeta potential
The changes in the particle size and zeta potential of HPAA derivatives (with or without 10 mM GSH) were characterized by dynamic light scattering (DLS; Zetasizer ZS90, Malvern). One milligram of samples was solubilized in high-purity water and tested at 25 °C. The morphology was visualized using a JEM-2010HR transmission electron microscope.
2.2.3 UV characterization
The amount of APT in HPAA-PEG-APT was measured using UV-vis spectrophotometry, and the absorbance of APT was quantified at 260 nm using free APT as the reference standard.
2.3 Gel electrophoresis
To prepare HPAA-PEG-APT/miRNA complexes, the miRNA-133a-3p gene was mixed with HPAA-PEG-APT buffer at various HPAA-PEG-APT/miRNA mass ratios. For HPAA-PEG-APT, the gene-binding ability was tested by agarose gel electrophoresis. The result was visualized using a UV transilluminator (Bio-Rad, USA).
2.4 Cell lines and culture
The human PCa cell line LNCaP was provided by The Third Affiliated Hospital of Sun Yat-sen University. HUVECs were purchased from Shanghai Chinese Academy of Sciences cell bank (China). LNCaP and HUVECs were cultured as described previously[12].
2.5 Specificity of the A 10 − 3.2 aptamer
The colocalization of Cy5 -labeled aptamer and anti-PSMA monoclonal antibodies (Abcam, Cambridge, MA, USA) in LNCaP cells was detected by LSCM.
2.6 In vitro transfection
LNCaP cells were cultured overnight before transfection in complete DMEM on 24-well plates (5⊆104 cells/per well). Next, the cells were supplemented with Opti-MEM (without serum) containing HPAA or HPAA-PEG-APT complexes incubated with FAM-labeled miRNA (miRNA:1 µg/well) at different mass ratios (60, 80, 100, 120:1). Thereafter, all the groups were supplemented with fresh DMEM for 6 h, followed by culture for another 24–48 h. Next, the cells were washed with PBS and digested, following by centrifugation and resuspension using moderate PBS. The transfection efficiency was measured by flow cytometry (Accuri C6), and PEI-25 k/miRNA was used as the positive control.
2.7 Anticancer effect in vitro
The inhibitory effects of LNCaP cells were observed using the CCK-8 Kit (CCK-8; Beyotime, Shanghai, China). LNCaP cells were incubated in 96-microwell plates at 1 × 104 cells/well for 24 h at 37 °C. The cells were mixed with different concentrations (0.02–50 µM) of complexes: HPAA-PEG-APT/miRNA, HPAA/miRNA and HPAA-PEG-APT/NCmiRNA for 72 h. Subsequently, 10 µL of CCK-8 was applied to all wells, followed by incubation for 4 h. The absorbance of different wells was measured using a microplate reader (MULTISKAN MK3), a standard curve was plotted based on the absorbance and cell count, and each sample was measured three times. The IC50 value was counted using GraphPad 8.0 PRISM®[11].
2.8 Bcl2, EGFR and MET protein expression
In total, 5 ⊆104 LNCaP cells were cultured in 6-well plates and incubated in 5% CO2 at 37 °C for 24 h, reaching 80–90% confluence. Different samples (HPAA-PEG-APT/NCmiRNA, HPAA/miRNA and HPAA-PEG-APT/miR-133a-3p, PBS) were mixed with the cells and reacted for 72 h (for protein extraction). Western blotting was performed using a previously described standard method24. Antibodies against Bcl2, EGFR and MET were purchased from Abcam (Cambridge, USA), and EGFR and MET were provided by Cell Signaling Technology.
2.9 Cytotoxicity of HPAA derivatives for LNCaP cells
The cytotoxicity of HPAA derivatives was observed using the CCK-8 assay in LNCaP and HUVEC lines. In total, 1 × 104 cells per well of 96-well plates were seeded in DMEM. After 24 h of incubation, fresh media containing HPAA, HPAA-PEG and HPAA-PEG-APT at different concentrations (from 10 to 500 µg/mL) were applied. Next, 100 µL of fresh medium containing 10% CCK-8 was added to all wells, followed by washing with PBS. The absorbance was measured using a microplate reader at a 450-nm wavelength, and the cell viability was defined as the ratio of the treated groups to the control group. Cell viability (%) = Absorbance of the treated group − Absorbance of the control group/Absorbance of the negative control group − Absorbance of the control group ⊆100%.
2.10 Blood compatibility
One-milliliter PBS solutions containing HPAA, HPAA-PEG, HPAA-PEG-APT, or PEI at different concentrations (0.01 mg/mL, 0.5 mg/mL,1 mg/mL) were prepared, while pure water and the PBS group were used as the positive control and negative control, respectively. Next, 16% hematocrit (v/v) red blood cells (RBCs) suspended in PBS were mixed with 5 mL of PBS containing different concentrations of HPAA derivatives. The positive controls (100% hemolysis caused by PBS containing 5 mL of 0.1% NaCO3 solution) and negative controls (0% hemolysis, only PBS) were prepared. The samples were incubated at 37 °Cfor 1 h, 3 h, 6 h, and 12 h, followed by centrifugation for 5 min at 1000 ⊆ g and measurement of the absorbance of the supernatant at 540 nm using a microplate reader. Finally, the ratio of hemolysis was determined by calculating the optical density (OD): Hemolysis (%)=[(OD of the treated sample – OD of negative control) ⊆100]/OD of positive control. Each sample was tested in triplicate.
2.11 Intraosseous model of prostate cancer and treatments
All animal procedures were approved by The Institutional Animal Care and Use Committee of Sun Yat-sen University (No. L102012016110D). The bone metastasis animal model was induced by injecting LNCaP-luc cells expressing the luciferase gene into the tibia4. Briefly, 5⊆105 LNCaP-luc cells were collected from 25-cm cell culture dishes (Corning, USA) and were resuspended in 20 µL of PBS. The cell suspension was administered into the lumen of the tibia using a 0.1-mL insulin needle. Tumor induction and its progression were investigated by estimating the bioluminescence signal intensity (p/s/cm2/sr). After 5-min intraperitoneal injection of luciferin (200 mg/kg; VivoGlo™ Luciferin; Promega, Madison, WI), the mice were imaged using IVIS® Lumina II (PerkinElmer, Waltham, MA). Two weeks following LNCaP cell injection, the mice were separated into 3 groups randomly with 5 mice per group. Next, 0.2-mL mixtures containing PBS (as control), HPAA-PEG-APT/miRNA-133a-3p, and HPAA/miRNA-133a-3p (HPAA derivatives: miRNA = 67 µg: 33 µg) were administered via tail vein twice every 4 weeks. Tumor growth was observed by bioluminescence signal intensity and was monitored. Additionally, hematoxylin and eosin (H&E) staining of the major organs (heart, lung, spleen, liver, kidney and prostate) was performed.
The criteria for the euthanasia of mice were as follows: 20% weight loss; movement difficulty because of tumor burden; tumor ulceration; necrosis; or bleeding. If the mice were also euthanized for signs of poor health, such as dehydration, lethargy, and cachexia, these mice were considered dead in the survival data.
2.12 Statistical analysis
All the data were expressed as means ± standard deviation. Analyses of the difference between groups were performed by one-way analysis of variance (ANOVA) using GraphPad Prism 8). P < 0.05 was defined as the significance level, * for P < 0.05, ** for P < 0.01. All statistical tests were two-tailed.