Bacteria-driven hypoxia targeting delivery of chemotherapeutic drug proving outcome of breast cancer | Journal of Nanobiotechnology

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Reagents, animals and bacteria

BSA was purchased from Meilun Biological Technology Co. Ltd. (Dalian, China). Adriamycin hydrochloride was purchased from Macklin Biochemical Co. Ltd. (Shanghai, China). Female Blab/c mice weighting 16–18 g (6 weeks of age) were purchased from Tengxin Biological Technology Co. Ltd. (Chongqing, China). All animal experimental procedures were approved by the Ethics and Science Committee of the Animal Care and Treatment Committee of Southwest Medical University. Balb/c mice were purchased from Tengxin Biotechnology Co. Ltd., Chongqing, China). The mice were housed under specific pathogen-free conditions at 24 °C and relative humidity of 50–60% under a 12-h-light/12-h-dark schedule, with ad libitum access to standard rodent food and tap water. All the mice were healthy and had no infection during the experimental period. B. infantis (GIMI.207) was purchased from the Strains Preservation Center of Guangzhou Institute of Microbiology (Guangdong, China), and incubated anaerobically on agar plates at 37 °C for 48 h. The colonies were collected in sterile dry centrifuge tubes and centrifuged at 2000 rpm for 5 min to obtain single cell suspensions.

Synthesis and characterization of DOX-NPs and biohybrid Bif@DOX-NPs

DOX and BSA were dissolved in double-distilled water and stirred for 2 h at room temperature, and the pH was adjusted to 7 ~ 10. Ethanol was added dropwise at the rate of 0.5 mL/min and the solution was stirred continuously. The components were cross-linked by adding 20 μL 8% glutaraldehyde at room temperature for 12–24 h. The organic solvent was then removed using a rotary evaporator (RE, R201, Shenshun Biotechnology Co., Shanghai, China) and the resulting NPs were harvested. The morphology of the DOX-NPs was observed by transmission electron microscopy (TEM, Tecnai G2 F20, FEI, USA). Particle size and zeta potential were measured using dynamic light scattering (DLS, NanoBrook90 plus Zeta, Brookhaven, NY) at 25 °C. In vitro stability of DOX-NPs in PBS, DMEM and distilled water (DW) was monitored by measuring particle size. Drug loading (DL) and encapsulation efficiency (EE) were determined by UV spectrophotometry (UV-5800PC, Shanghai Metash Instruments Co., Ltd., Shanghai, China) at 25 °C. Briefly, the samples were dissolved in double-distilled water and OD at 485 nm was measured. DL and EE were calculated using the following equations.

$$\mathrm{DL}=\frac{Drug}{(BSA+Drug)}\times 100\mathrm{\%}$$

$$\mathrm{EE}=\frac{Actual \, DL}{Theoretical \, DL}\times 100\mathrm{\%}$$

To construct Bif@DOX-NPs biohybrids, Bif was incubated anaerobically in agar medium at 37 °C for 48 h. The bacterial colonies were resuspended, washed thrice, and the Bif suspension (2.0 × 107 cfu/mL) was incubated anaerobically at 37 °C with DOX-NPs (40ug/mL) for 4 h. The solution was centrifuged at 2000 rpm for 5 min, and the supernatant was removed and washed twice to obtain the Bif@DOX-NPs biohybrids. The surface morphology of Bif@DOX-NPs was imaged with a field emission scanning electron microscope (SEM, Hitachi S3400N, Japan). Bif@HSA-NPs and Bif@KER-NPs were similarly prepared using human serum albumin (HAS) and keratin (KER) instead of BSA. To ascertain the co-localization of Bif and the protein NPs, the bacterial cells were labelled with FITC prior to constructing the biohybrids, which were then observed by confocal laser scanning microscopy (Zeiss LSM 880, Carl Zeiss, Germany). The fluorescence absorption of Bif@DOX-NPs was measured by fluorescence spectrophotometry (Perkin-Elmer LS-55, Smeo Analytical Instruments Co, China). Flow cytometry (BD FACSVerse, Piscataway, NJ) was used to quantify the fluorescence intensity of adriamycin in Bif@DOX-NPs. To determine whether the loading of DOX-NPs on the surface of Bif affected its activity, equal amounts of Bif@DOX-NPs and Bif were inoculated onto agar plates and the colonies were counted after 24 h. The binding stability of Bif@DOX-NPs was also evaluated. The Bif@DOX-NPs were resuspended in PBS (pH = 7.4). The matrix metalloproteinase 2 (MMP-2) was added to the Bif@DOX-NPs solution and incubated for 24 h. The solution without MMP-2 was used as control. Then the solutions were centrifuged and the color change of supernatant was photoed. The fluorescence intensity of the supernatant was measured and used to calculate the dissociation rate of DOX-NPs from Bif@DOX-NPs biohybrids.

In vitro drug release, cell uptake and cytotoxicity assays

The samples were put into dialysis bags (molecular weight cutoff 3500 Da) that were then placed into 40 mL PBS (pH = 7.4, 6.0, 5.0) containing 1% Tween 80 (v/v) in a water bath shaker (37 ± 0.5 °C). At the stipulated time points, 3 ml media was collected for analysis and replaced with the equal amount of PBS. The amount of the drug in the buffer was measured by UV–Vis spectrophotometry (UV-5800PC, Shanghai Metash Instruments Co., Ltd., Shanghai, China).

To measure cellular uptake of the NPs, 4T1 cells were seeded in 6-well plates at the density of 2 × 105 cells/mL and incubated with normal saline (NS), free DOX or DOX-NPs for 2 h. The cells were then stained with DAPI for 5–10 min, washed thrice with PBS, and imaged by a fluorescence microscope (OLYMPUS, IX73, Japan). The fluorescence intensity of the internalized DOX was quantified by flow cytometry (BD FACSVerse, Piscataway, NJ) [52]. For the cytotoxicity assay, 4T1 cells were seeded into 96-well plates and treated with DOX, DOX-NPs or Bif@DOX-NPs for 24 h, and the MTT solution was then added to each well, after incubating the cells for another 4 h, 100 μL dimethyl sulfoxide was added to each well, and the OD was measured. To investigate the effect of Bif on normal LO2 cells and BEAS-2B cells, the two kinds of cells were seeded into 6-well plates (5 × 104/well) and incubated with Bif for 24 h. Then the cells were collected and resuspended by adding Annexin V-mCherry Binding Buffer, then stained with Annexin V-mCherry and SYTOX Green for 10–20 min, finally apoptosis was detected by flow cytometry (BD FACSVerse, Piscataway, NJ).

In vitro apoptosis and wound healing assays

The 4T1 cells were seeded into 6-well plates (5 × 104/well) and incubated with NS, Bif, DOX, DOX-NPs, Bif + DOX or Bif@DOX-NP for 24 h. The cells were harvested, resuspended in Annexin V-mCherry Binding Buffer and then stained with Annexin V-mCherry and SYTOX Green for 10–20 min. The percentage of apoptotic cells were detected by flow cytometry (BD FACSVerse, Piscataway, NJ). The effect of Bif on the apoptosis rates of normal hepatopulmonary cells (LO2 cells and BEAS-2B cells) as well as hepatocellular carcinoma cell lines was evaluated by the same procedure. For the in vitro wound healing assay, 4T1 cells were cultured till confluent and the monolayer was scratched using a sterile pipette tip. After washing off the dislodged cells, fresh DMEM containing Bif, DOX, DOX-NPs, Bif + DOX or Bif@DOX-NPs was added. The wound coverage area was photographed 6, 12 and 24 h and the migration rate was calculated.

Evaluation of the anaerobic targeting of bacterial/NPs biohybrids

The top compartment of Transwell insert was filled with Bif@NPs (200 μL, 5 × 107 cfu/mL) and the bottom with 0.4 mL mixture of glucose (0.4 mg/mL), glucose oxidase (0.5 kU) and catalase (0.5 kU). The oxidation of glucose by glucose oxidase depleted the oxygen, and the hydrogen peroxide produced during this reaction was quenched by catalase, resulting in a hypoxic environment. In the control group, the bottom compartment was filled with 0.4 mL glucose solution. After 2 h of incubation, the Bif@NPS in the bottom compartment were harvested and the number of cells was counted. Bif@DOX-NPs were injected into tumor-bearing mice through the tail vein, and tumor tissues were harvested 24 h later. The tissue sections were stained with Cy3-labeled anti-Bif antibody (Cy3@Ab) and Alexa Fluor 488-labeled anaerobic induction factor (488@HIF-1α) to demarcate the hypoxic zone, and the nuclei were counterstained with DAPI. The distribution of Bif in the hypoxic sites was observed under a fluorescence microscope and counted. In another experiment, tumor-bearing mice were injected with Bif@DOX-NPs or NS and euthanized 1-, 4-, 7- and 14 days post-injection. The major organs (heart, liver, spleen, lung and kidney) and tumor tissues were harvested, and homogenized in sterile water containing 0.1% Triton X-100. The tissue homogenates were serially diluted and incubated on solid LB agar plates at 37 °C for 24 h. The colonies were counted and photographed. The organs with high bacterial colonization (liver and kidney) and the tumor tissues were embedded in paraffin, cut into sections, and stained as described above. The fluorescence intensity in each tissue was observed by fluorescence microscopy and statistically analyzed.

In vivo evaluation of the antitumor effect of Bif@DOX -NPs

A breast cancer xenograft model was established by inoculating 1.0 × 106 4T1 cells (107 cells/mL) into the right leg of Balb/c mice. Once the mean tumor volume reached approximately 90 mm3, the mice were randomly divided into the following groups (n = 10 per group): (1) normal saline (NS), (2) Bif, (3) Bif@BSA-NPs, (4) Bif + DOX, (5) DOX-NPs and (6) Bif@DOX-NPs. The respective drugs were injected via the tail vein once every three days for a total of three times. The weight and tumor volume of mice were also monitored every other day. Three days after the last drug administration, the heart, liver, spleen, lungs, kidneys and tumor tissues were harvested and embedded in paraffin for further analysis. H&E staining was performed for histopathological analysis, and fibrosis was measured by Masson’s Trichome staining. Apoptosis was evaluated using terminal-deoxynucleotidyl transferase (TUNEL) labelling. The in-situ expression of the hypoxia inducible factor (HIF-1α) and SPARC in tumor tissues were analyzed by immunohistochemistry.

To assess early treatment response, Micro-PET/CT scans (Siemens, Germany) were performed 48 h after the last dose. Briefly, the mice were fasted at least 6 h prior to the scan, and injected with 150–250 μCi FDG. Thirty minutes later, the mice were anesthetized via isoflurane inhalation, and whole-body PET/CT scans were performed in two-dimensional mode (10 min per location emission scan) using the parameters of 80 kV, 500 mA and 1.5 mm slice collimation. The PET/CT images were analyzed by two nuclear medicine physicians. The regions of interest (ROIs) on the tumor images were drawn manually and randomly, and the maximum normalized uptake value (SUVmax) and mean uptake value (SUVmean) were calculated using the hottest individual pixel within the tumor.

To investigate the in vivo drug biodistribution, Bif@DOX-NPs were injected through the tail vein, and the mice were euthanized 24 h later. The heart, liver, spleen, lungs, kidneys and tumors were harvested and homogenized, and the homogenates were centrifuged at 10,000 rpm for 10 min. The supernatant was transferred to the colorimetric cup and the fluorescence intensity was measured using a spectrophotometer (Perkin- Elmer LS-55, Smeo Analytical Instruments Co, China).

In vitro hemolysis analysis and in vivo toxicity evaluation of bacterial nanohybrids

One milliliter erythrocyte suspension (0.2%, v/v) was mixed with 1 mL saline containing Bif, BSA-NPs or Bif@BSA-NPs, and incubated at 37 °C for 4 h. The positive control was hemolyzed in double-distilled water and saline was included as the negative control. All samples were centrifuged at 4 °C and 3000 rpm for 6 min, and the OD of the supernatant was measured at 540 nm using a UV–Vis spectrophotometer (UV-5800PC, Shanghai Metash Instruments Co., Ltd., Shanghai, China). The hemolysis rate was calculated according to the following equation

$$\mathrm{Hemolysis \, Rate }(\mathrm{\%}) = \frac{OD \, value \, of \, experimental\, group – OD \, value \, of \, saline\, group}{OD \, value \, of \, positive \, control \, group – OD \, value \, of \, saline \, group}\times 100\mathrm{\%}$$

To study the in vivo toxicity of Bif@DOX-NPs, 12 healthy SD rats were randomly divided into the NS, Bif, Bif + DOX and Bif@DOX-NPs groups (n = 3), and injected intravenously with the respective drugs every three days, three times in total. Blood was collected via the retroorbital route for measuring red blood cells (RBC), white blood cells (WBC), platelets (PLT), hemoglobin (HGB), mean hemoglobin (MCH), mean blood cell volume (MCV), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), blood urea nitrogen (BUN) and creatine kinase (CK).

Statistical analysis

All data were expressed as mean ± standard deviation (SD). Two groups were compared using the Student’s t-test, and one-way analysis of variance (ANOVA) was used for multiple group comparison. Survival curves were plotted using the Kaplan–Meier method, and survival times and 95% confidence intervals were compared using the log-rank test. Statistical analyses were performed using GraphPad Prism version 6.07 (GraphPad Software, Inc). P values < 0.05 were considered statistically significant.

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