Tea polyphenol-engineered hybrid cellular nanovesicles for cancer immunotherapy and androgen deprivation therapy | Journal of Nanobiotechnology

Materials

Flutamide, (-)-Epigallocatechin Gallate (EGCG), 3-Aminophenylboronic acid were purchased form Aladdin (Shanghai, China). DSPE-PEG-NHS was purchased from Xi’an Qi Yue Biotechnology Co., Ltd (Xi’an, China). Poly (D, L-lactide-co-glycolic acid) (PLGA 50:50 M = 24,000) was purchased from Macklin (Shanghai, China). The cell membrane mask Alexa647-conjugated wheat germ agglutinin (W32466) was purchased from ThermoFisher Scientific. 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich. Recombinant Murine IL-4 was purchased from ThermoFisher Scientific.

Cells and animals

RM-1 cells and RAW 264.7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen) supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics (penicillin/streptomycin) at 37 °C in a humidified 95% air/5% CO2 incubator (ThermoFisher Scientific, USA). C57BL/6 mice (male, 6–8 weeks) were bought from Viton Lever Laboratory Animal Technology Co., Ltd (Beijing, China), and the animal study was subject to approval by the Institutional Animal Care and Use Committee of Shenzhen Bay Laboratory.

Synthesis of PLGA-Flu

PLGA-Flu was prepared using the emulsion-diffusion-solvent evaporation method. 40 mg PLGA and 4 mg Flu were mixed in 2 mL of acetone: dichloromethane (1:4) mixture. Then, 4 mL of PVA aqueous solution (20 mg/mL) was combined with the mixture. The oil-in-water emulsion formed was further sonicated using a sonicator for 20 min. Following this, the homogeneous emulsion was centrifuged at 20,000 g for 40 min. The supernatant was then subjected to HPLC analysis to determine the free drug content, while the sediment underwent two washes with PBS. For the HPLC analysis, we used a mobile phase with water/methanol. Water phase: organic phase (40%/60% v/v) at a flow rate of 1.0 mL/min. The injection volume was 20 µL, and the detection wavelength was 230 nm. The Flu drug-loading capacity (DLC) and drug encapsulation efficiency (DEE) were evaluated by formula below: DLC% = (m total – m free) / m total × 100%; DEE% = (m total – m free) / m sediment × 100%. Here, m total represents the total amount of Flu, m free represents the free amount of Flu in supernatant, and m sediment represents the amount of sediment after free-drying.

Synthesis of DSPE-PEG-EGCG

The method used for synthesizing DSPE-PEG-EGCG was adapted from a previously documented procedure with slight adjustments. In brief, DSPE-PEG2000-NHS and 3-Aminophenylboronic acid (DSPE-PEG2000-NHS:3-aminophenylboronic acid: 2/1, mol/mol) were dissolved in 10 mL of N, N-Dimethylformamide (DMF). The reaction mixture was stirred gently at room temperature for 36 h. Then, the mixture was dialyzed extensively (dialysis bag Mw cutoff 2,000 Da) in distilled water for 48 h to eliminate any impurities and then lyophilized 48 h to produce DSPE-PEG-phenylboronic acid. After that, DSPE-PEG-phenylboronic acid and EGCG (DSPE-PEG-phenylboronic acid: EGCG: 2/1, mol/mol) were dissolved in 10 mL of anhydrous ethanol. The reaction mixture was stirred gently at room temperature for 36 h. Then, the mixture was dialyzed extensively (dialysis bag Mw cutoff 2,000 Da) in distilled water for 48 h to eliminate the remaining impurities and then lyophilized 48 h to obtain DSPE-PEG-EGCG. The characterization of the synthesized product is conducted through 1 H NMR analysis using a Bruker 400 MHz NMR spectrometer in DMSO-d6 solvent.

Preparation and characterization of hNVs@Flu-EGCG

(i) RM-1 cells were collected and then suspended in the PBS buffer. The cells were then disrupted 5 min at 40% power using the ultrasonic disruptor. After fragmentation, the supernatant was gathered through centrifugation at 4,000 g for a duration of 40 min, while the membranes were isolated via centrifugation at 100,000 g for a period of 1 h on the Beckman Coulter Optima XE-90 ultracentrifuge to obtain cell membrane particles. The cellular membrane pellets were gathered and resuspended in PBS buffer. Following this, they were extruded through polycarbonate porous membranes of varying sizes (800 nm, 400 nm, and 200 nm) utilizing a mini extruder provided by Avanti Polar Lipids, allowing for the isolation of NVs. (ii) In order to isolate EcN OMVs, bacteria were introduced to 400 mL of LB broth and incubated at 37 °C with shaking (200 rpm) for a period of 48 h. Subsequently, the bacterial cells were removed from the culture via centrifugation at 5,000 g for 20 min at 4 °C, and the resulting supernatant was filtered using a 0.45 μm filter. The resulting concentrated solution underwent further centrifugation at 150,000 g for 2 h at 4 °C using a Beckman Coulter Optima XE-90 ultracentrifuge to harvest the OMVs. The OMVs were then retrieved and suspended in PBS buffer. Following this, the NVs and OMVs were combined at a protein weight ratio of 1:2 and extruded through 200-nm pores on a mini extruder in order to produce hNVs. PLGA-Flu and hNVs were mixed (PLGA-Flu mass weight: hNVs protein weight:1:1) and treated with a water-bath ultrasound for 30 min to obtain hNVs@Flu. After that, DSPE-PEG-EGCG and hNVs@Flu (DSPE-PEG-EGCG mass weight: hNVs@Flu protein weight:1:5) were co-inoculated at 37 °C for 30 min to obtain hNVs@Flu-EGCG. (iii) The nanoparticles’ sizes and zeta potentials were measured utilizing Dynamic light scattering (DLS; Zetasizer Nano ZS, Malvern Instruments) and NanoSight NS300 (Malvern Instruments). Additionally, the nanoparticles’ morphology was examined with transmission electron microscopy (JEM-2100).

SDS-PAGE

For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), OMVs, NVs hNVs and hNVs@Flu-EGCG were added into the protein extraction buffer, and followed by protein concentration measurement using a bicinchoninic acid (BCA) protein quantification kit (KeyGEN BioTECH, China), and the protein was denatured by heating the samples at 100 °C for 5 min Subsequently, equal amounts of proteins were loaded into a 10% SDS-polyacrylamide gel and separated based on the specific parameters (80 V for 30 min and 120 V for 90 min). After electrophoresis, the gel was stained with Commassie blue (Solarbio, China) for 30 min, washed with DI water and decolorized three times before the observation.

Cell toxicity assays

The CCK8 assay was conducted to do the cytotoxicity test. Initially, Plating RM-1 cells onto 96-well polystyrene plates at a density of 10,000 cells per 100 µL of medium per well. Following a 12 h incubation at 37 °C, the cells were exposed to varying concentrations of blank PLGA, Flu, PLGA-Flu, OMVs, hNVs, hNVs@Flu, hNVs-EGCG, and hNVs@Flu-EGCG in the medium for 24 h. Subsequently, the cells were incubated with 100 µL of CCK8 solution at 37 °C for 2 h. Cell viability was assessed by measuring the absorbance at 450 nm using a microplate reader.

Live/dead cell staining assay

To conduct the live/dead cell staining experiment, RM-1 cells were plated in 6-well plates at a density of 10,000 cells per well. They were then treated with PBS, PLGA-Flu, hNVs, hNVs-EGCG, and hNVs@Flu-EGCG (Flu at a concentration of 20 µg/mL) for 24 h. Following treatment, the cells were incubated with the Calcein AM/PI assay working solution in the absence of light for 30 min, and subsequently imaged using CLSM.

Cell colony formation assay

RM-1 cells were seeded in 6-well plates at 5,000 cells per well and cultured for 96 h, and then incubated with PBS, PLGA-Flu, hNVs, hNVs-EGCG and hNVs@Flu-EGCG (Flu, 20 µg/mL) for other 72 h. After replacing fresh medium, cells were continued to be cultured until a sufficiently large colony was formed, and then the colony formation was analyzed by calculating after fixing with paraformaldehyde and staining with crystal violet reagent (Beyotime, Shanghai, China).

Hemolysis test

Blood was collected from the healthy male C57BL/6 mice and placed in anticoagulant tubes containing EDTA for 1 h. Then, the blood underwent centrifugation at 1,200 rpm for 5 min and was then suspended in PBS buffer a total of three times until the color of the supernatant was no longer visible. Following this, the blood cells were exposed to varying levels of hNVs@Flu-EGCG for a duration of 5 h, with distilled water serving as a positive control and PBS as the negative control. Subsequently, the supernatant was retrieved through centrifugation at 15,000 rpm for 15 min, and its absorbance at 570 nm was measured using a microplate reader. The hemolysis ratio (HR) was calculated by the following formula: HR = (sample absorbance – negative absorbance) / (positive absorbance − negative absorbance) × 100%.

Apoptosis assay

The Annexin V-FITC/PI Apoptosis Detection Kit (Dojindo, Japan) was utilized to perform the apoptosis assay and data analysis was done by FACS flow cytometry. Cells were initially plated in 6-well plates with 5 × 10⁵ cells per well and cultured for 12 h. Subsequently, different treatments including PBS, PLGA-Flu, hNVs, hNVs-EGCG, and hNVs@Flu-EGCG (with Flu at 20 µg/mL) were administered to the cells for a period of 24 h. After the incubation, the cells were collected and stained with Annexin-V FITC and PI as per the provided guidelines. Flow cytometry was employed to analyze the stained cells, and the resultant data was processed using FlowJo software.

In vitro cellular uptake of hNVs@Flu-EGCG

Flow cytometry (CytoFLEX LX, Beckman Coulter) and LSM900 (Zeiss) were used to measure the cellular uptake of hNVs@Flu-EGCG. hNVs@Flu-EGCG were labeled with fluorescent dyes 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO). For flow cytometry analysis, RM-1 cells were seeded in 6-well plates and incubated with DiO-labeled hNVs@Flu-EGCG for 0, 1, 2, 4, 8, 12 and 24 h. After that, the cells underwent a series of preparatory steps which included being collected, washed thrice with PBS, and suspended in 300 µL of FACS buffer for flow cytometry analysis. In the context of fluorescence imaging analysis, RM-1 cells were first seeded in the confocal dish and allowed to adhere for a period of 12 h. Subsequently, DiO-labelled hNVs@Flu-EGCG in fresh DMEM were added to the cells and incubated for the same duration as described earlier. The cells were then fixed using 4% paraformaldehyde for 15 min and subjected to another round of triple PBS washes. Following this, the nucleus was stained with DAPI and the cell membrane was stained with Alexa647-conjugated wheat germ agglutinin (WGA) before observation using LSM900.

Western blot

After 24 h of culturing RM-1 cells in 10 cm dishes, the medium was replaced with the fresh ones containing PBS, PLGA-Flu, hNVs, hNVs-EGCG, and hNVs@Flu-EGCG (Flu, 20 µg/mL), and they were cultured for an additional 24 h. Subsequently, the collected cells were subjected to total protein analysis. The denatured samples were loaded onto a 10% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes, which were then blocked with 5% nonfat milk at room temperature for an hour, followed by incubation with primary antibodies: GAPDH (5174, 1:1000), Caspase-9 (9508, 1:1000), Caspase-3 (14,220, 1:1000), Cleaved caspase-3 (9664, 1:1000), Bcl-2 (ab182858, 1:1000), Bax (ab32503, 1:1000), PSMA (ab314142, 1:1000), androgen receptor (A19611, 1:1000), p-65 (ab32536, 1:1000), pho-p65 (ab76302, 1:1000) at 4 °C overnight. Following the completion of the initial steps, the PVDF membranes were washed thrice with Tris-buffered saline with Tween 20 (1× TBST). Subsequently, they were incubated with a secondary IgG-horseradish peroxidase (HRP) antibody for a duration of 2 h at room temperature. This was followed by another round of washing with 1× TBST. The blots were developed by using a chemiluminescence detection system (Tanon 5200 Multi, Shanghai, China).

In vivo biotoxicity evaluation

Healthy C57BL/6 mice (6–8 weeks old) were divided into five groups at random and received intravenous injections of PBS (as the control), PLGA-Flu, hNVs, hNVs-EGCG, and hNVs@Flu-EGCG (Flu, 10 mg/kg). On day 7 post-injection, all mice were euthanized. The major organs, including heart, liver, spleen, lung, and kidney were collected, fixed in 4% paraformaldehyde, sectioned, and then stained with hematoxylin-eosin (H&E) for histopathological examination (Servicebio, Wuhan, China). Additionally, blood samples were obtained from the mice for the purpose of performing both blood biochemistry and blood routine analysis including alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelets (PLT).

In vivo tumor targeting

PLGA-Flu, OMVs@Flu and hNVs@Flu were labelled with fluorescent dyes 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanine iodide (DiR) to investigate the in vivo fluorescence imaging. The RM-1 cells (5 × 10⁵ cells) were injected subcutaneously into the right thighs of C57BL/6 mice (6–8 weeks old) to establish a mouse model of subcutaneous prostate cancer. Once the tumor volume reached around 100 mm³, DiR-labelled PLGA-Flu, OMVs@Flu, and hNVs@Flu were administered to RM-1 tumor-bearing mice via intravenous injection. Following this, imaging was conducted at specific time points (0, 2, 4, 8, 12, 24, and 48 h) using the IVIS imaging system (PerkinElmer, USA) to evaluate the distribution within the body. Post imaging, the mice were humanely euthanized, and key organs, including the heart, liver, spleen, lungs, kidney, and tumor were harvested for subsequent in vitro imaging analysis after 48 h. Furthermore, the organs and tumor tissues were processed, sliced, and stained to facilitate additional fluorescence imaging analysis in vitro.

In vivo antitumor capabilities

The RM-1 cells (5 × 10⁵ cells) were injected subcutaneously into the right thighs of C57BL/6 mice (6–8 weeks old). Once the tumor volume of RM-1 tumor-bearing mice grew to 100 mm³-200 mm³, the mice were categorized into five groups at random and were intravenously injected with PBS (as control), PLGA-Flu, hNVs, hNVs-EGCG and hNVs@Flu-EGCG (Flu, 10 mg/kg) once every other day for five times. Monitoring of tumor size and body weight of the mice occurred every other day. The part of the mice was sacrificed, and the tumors were extracted, fixed using 4% paraformaldehyde, sectioned, and stained with Ki-67 and TUNEL to investigate the impact of the different treatments on tumor cell proliferation and apoptosis (Servicebio, Wuhan, China). In order to assess the antitumor capabilities of nanoparticles, tumors were taken from each group to be photographed on the last day after treatment (n = 5 per group). At the same time, mice blood samples were collected to detect PSA level using a Prostate Specific Antigen ELISA kit (nenbioscience, China). For the survival study (n = 5 per group), mice were considered dead once the tumor volume exceeded 1500 mm³. Tumor inhibition rate (TIR) was worked out by the formula beneath. TIR (%) = (Vc − Vt) /Vc × 100%. Here, Vc represents the mean tumor volume of control group; Vi represents the mean tumor volume of different treatment groups.

In vivo tumor immune microenvironment analysis

The tumors were processed following the sacrifice of the RM-1 tumor-bearing mice. The tumors were excised, cut into small pieces, and then subjected to an incubation process with RPMI 1640 media containing collagenase IV (0.2 mg/mL), DNase I (250 U mg/mL) and hyaluronidase (0.1 mg/mL) for a duration of 40 min at 37 ℃. Subsequently, the homogenates underwent filtration through a 70 μm nylon mesh to obtain single-cell suspensions which were then washed with PBS. The cells were further treated with fluorescence-labelled antibodies: CD45 (APC-Cy7; BD, USA), CD11b (FITC; BD, USA), F4/80 (PE-Cy7; BD, USA), CD80 (Pacific Blue; BD, USA), CD206 (Alexa Fluor 647; BD, USA). All antibodies were diluted appropriately and incubated with the cells under specific temperature conditions as per the provided instructions. Subsequently, the samples obtained were assessed through flow cytometry methods. Figure S11 (Supporting Information) provides comprehensive details on flow cytometric gating and sorting approaches. For ELISA assay, RM-1 tumor tissues were harvested to detect cytokine levels. The intratumor levels of TNF-α, IL-6, and IL-10 were detected with ELISA kits (Servicebio, Wuhan, China). For immunofluorescence staining, Tumor tissues were harvested and fabricated into the paraffin section and then were stained with the anti-CD206 antibody (red), anti-CD80 antibody (green), and DAPI (blue) (Servicebio, Wuhan, China).

Statistical analysis

All data are presented as mean ± standard deviation (S.D.) unless otherwise noted. Statistical significance was analyzed using one-way (or 2way) ANOVA with a Tukey’s test (or with a Dunnett’s test). GraphPad Prism software was used to analyze the data. Statistical significance was defined as P < 0.05.