DPPs (Scheme 1, Additional file 1: Table S1, S2 and Fig. S1–S3) were synthesized by ChinaPeptides Co., Ltd. (Suzhou, China). 2′-O-methyl (2′-OMe) modified antisense oligonucleotides (ASOs), 5′-fluorescein amidites (FAM)-labeled ASOs and primers were synthesized by Sangon Biotech (Shanghai, China) (Additional file 1: Fig. S3). 5′-Cyanine 5 (Cy5)-labeled ASOs were synthesized by Tsingke Biological Technology Co., Ltd (Xi’an, China). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol)-2000 (DSPE-mPEG2000) was purchased from Xi'an ruixi Biological Technology Co., Ltd. Trizol (Additional file 1: Fig. S3), FM4-64 dye, Lipofectamine 2000™ and Opti-MEM medium were purchased from Life Technologies (Invitrogen, CA, USA). Lysogeny broth and Mueller–Hinton (M–H) broth were purchased from Beijing Land Bridge Technology Co., Ltd (Beijing, China). Reversed enzyme and SYBER Green enzyme were purchased from Takara Bio Inc. (Kyoto, Japan).
Bacterial strains and cell line
Escherichia coli (E. coli, ATCC25922), extended spectrum β-lactamase-producing Escherichia coli (ESBLs-E. coli, ATCC35218), Klebsiella pneumonia (K. pneumonia, ATCC75293), multidrug resistant Pseudomonas aeruginosa (MDR-P. aeruginosa), Staphylococcus aureus (S. aureus, ATCC29213), Escherichia faecalis (E. faecalis, ATCC29212) and Bacillus subtilis (B. subtilis, ATCC23857) were stored in our laboratory, methicillin-resistant Staphylococcus epidermidis (MRSE), methicillin-resistant Staphylococcus aureus (MRSA), Multidrug-resistant Acinetobacter baumannii (MDR-A. baumannii XJ17014279) was isolated from Xijing Hospital, Fourth Military Medical University. E. coli (DH5α) were obtained from Beijing Beina Chuanglian Biotechnology Institute.
Nanoparticles (NPs) preparation
LF2000-NPs were prepared in strict accordance with the protocol described in our previous study . DPP storing solutions (Additional file 1: Table S3) were diluted 50 times to obtain the working solutions. DSPE-mPEG2000 decorated DPP/ASOs nanoparticles (DP-AD) were prepared in two steps. Firstly, the DPP working solution (3 μl) and ASOs (15 μl, 20 μM) dissolved in RNAse/DNAse free sterile water (0.02 mM) were diluted to 275 μl by RNAse/DNAse free sterile water and mixed at 2600 rpm for 1 min, followed by incubation at 37 °C for 30 min to form DPP/ASOs nanoparticles (ADs). Secondly, 7.5 μl DSPE-mPEG2000 solution (20 μM, 0.5-fold of ASOs) was added into the ADs solutions, and the mixture was mixed at 2600 rpm for 1 min and incubated at 37 °C for another 30 min to obtain the DP-AD solution (1 μM), whose concentration was calculated based on the concentration of ASOs. DP-AD with different DSPE-mPEG2000 ratios were prepared with the same procedure with different DSPE-mPEG2000 volumes. The linear DP-AD (L-DP-AD) were prepared with the same procedure used for DP-AD.
Agarose gel electrophoresis
The ASOs solution was incubated with DPPs at 37 °C for 30 min at geometric N/P molar ratios ranging from 0 to 16 to form ADs. The prepared ADs (10 μl, 40 μM) were analyzed by electrophoresis on agarose gel (1% wt/vol) and stained with ethidium bromide to obtain the images.
Transmission electronic microscope (TEM)
TEM was adopted to observe the morphology of the NPs. Briefly, a drop of NPs solution (30 μM) was added to slide-grids, followed by natural settling for 5 min in ambient conditions before the liquid was sucked away quickly. Then, the grids were dried in ambient conditions. Images were captured using JEM-1230 Electron Microscope (JEOL, Japan) at 80 kV.
Size and zeta potential measurement
Size and zeta potential of DP-AD were measured by zeta sizer (Marlvern Panalytical, UK). The NPs solution (1 ml) was added into a disposable cuvette with an optical path of 1 cm. The measurement conditions for size were as following: the dispersant was water; Mark-Houwink parameters were 0.428 (A parameter) and 7.67 × 10–5 (K parameter); measurement temperature was 25 °C; the measurement angle was 173°. The NPs solution (approximately 1 ml) was added into a disposable folded capillary cell, followed by the measurement of the zeta potential at 25 °C. The size of DP-AD in M–H broth were measured after 500 μl NPs solution were diluted with an equal volume of M–H broth. The analysis was performed in triplicate by Zetasizer software (version 7.13, Malvern).
Bacteria stored in 15% glycerin were streaked onto M–H agarose plate and cultured at 37 °C for 18 h. Next, a bacterial colony was transferred from the M–H agarose plate into Lysogeny broth (LB, 3 ml) in quartz tubes and cultured at 37 °C until reaching the logarithmic growth stage.
Flow cytometry analysis
To measure the delivery efficiency, FAM-labeled DP-AD were added to bacterial cultures (approximately 5 × 106 CFU in 300 μl M–H broth) and incubated at 37 °C for 1 h away from light. The bacterial solutions were centrifuged at 2500 g at 4 °C for 5 min and washed twice with phosphate buffered saline (PBS) before analyzing them using the BL1 (green) channel in flow cytometry (Novocyte, Acer, USA). Data were analyzed using Flowjo software (version 10.0.7, Tree Star, Ashland, OR, USA). Free FAM-labeled ASOs and LF2000-NPs were used as the negative and positive control, respectively.
To measure the delivery rate of DP-AD7 in different types of bacteria, the bacteria (approximately 5 × 106 CFU in 300 μl M–H broth) were incubated with FAM-labeled DP-AD7 for 5, 10, 30 or 60 min at 37 °C or at 4 °C for 60 min in dark, then the bacterial solutions were processed, measured and analyzed as described above.
E. coli (DH5α), an engineering bacterial strain expressing EGFP, was adopted to measure the antisense efficacy of DP-AD in vitro. 3 × 106 CFU bacteria in 300 μl M–H broth were treated with 300 μl amphipathic DP-AD2, 3, 7 or 8 solutions (1 μM) at 37 °C for 1 h. Then, fluorescence intensity was measured by flow cytometry and analyzed as described above.
The FAM positive rates of ESBLs-E. coli (ATCC35218), K. pneumoniae (ATCC75293), MDR-P. aeruginosa, E. faecalis (ATCC29212), B. subtilis (ATCC23857), MRSE, MRSA and MDR-A. baumannii (XJ17014279) were measured as following: 3 × 106 CFU bacteria in 300 μl M–H broth were incubated with 300 μl DP-AD7 at 37 °C for 1 h. Then, fluorescence intensity was measured by flow cytometry and analyzed as described above.
Confocal laser scanning microscope (CLSM)
Firstly, cy5-labeled DP-AD7 solution (300 μl, 2 μM) was incubated with ESBLs-E. coli or B. subtilis (107 CFU in 300 μl M–H broth) for 1 h at 37 °C away from light. Secondly, the bacterial solutions were centrifuged at 2500 g for 5 min to discard the supernatant, and washed twice with 500 μl PBS, followed by resuspension of the bacteria with 20 μl PBS. Then, 1 μl FM4-64 dye was added into the bacterial solutions to stain the plasma membrane at ice for 1 min, then, the solutions were centrifuged at 2500 g for 5 min, discarded the supernatant, and resuspended the bacteria with 20 μl PBS. Then, several drops of these solutions were added onto a 0.5-cm cover glass and dried at 37 °C, followed by the addition of a drop of glycerin 50% to fix the bacteria. Lastly, CLSM was adopted to measure bacterial fluorescence. Images were captured and analyzed by Olympus Fluoview Viewer (version 3.0, Olympus Corp., Japan).
Serum stability of DP-AD
500 μl FAM-labeled DP-AD (1 μM) was mixed with equal volume of 10% fetal bovine serum (FBS, DMEM as the medium) for 1 h at 37 °C, the DLS was used to measure the size of DP-AD. Then, the nanoparticles were centrifuged at 13,000 g for 30 min, then the fluorescence intensity of the supernatant from each DP-AD was measured. And the release of the ASOs from nanoparticles was measured. The DP-AD treated with equal volume of DMEM without FBS was used as the control. 1 ml FAM-labeled DP-AD (1 μM) was mixed with equal volume of 10% FBS for 6 h at 37 °C, then the size and PDI were measured as described above.
Minimum inhibitory concentration (MIC)
DPP bacterial toxicity was evaluated by MIC assay of DPPs in E. coli, ESBLs-E. coli, S. aureus and MRSA. Briefly, DPP (50 μl, with a geometric concentration ranging from 64 μg ml−1 to 0.5 μg ml−1 dissolved in M–H broth) was added into 96-well plates, followed by the addition of an equal volume of the tested bacterial solutions (approximately 106 CFU). Then, the mixture was incubated at 37 °C for 24 h and the optical density was measured using Bio-Rad 680 microplate reader (BioRad Corporate, Hercules, California, USA).
1 ml blood (Drawn from Zhou Chen) was centrifuged with 1000 rpm for 5 min to pellet the red blood cells, which was washed twice with 1 ml PBS. Then, the cells were resuspended with 1 ml PBS, followed by dilute by PBS to get 4% red blood cells solution. Then, 200 μl cell solution was mixed with the equal volume of DP-AD with the concentration ranging from 0.25 μM to 2 μM in 1.5 ml tube. After 1 h of incubation at 37 °C, the solutions were centrifuged at 1000 rpm for 5 to pellet the unlysed cells, 100 μl liquid from each tube was transferred to a clean 96-well plate. The optical density of 540 nm was measured. Finally, the percent hemolysis in each assay was calculated. PBS or 1% triton was used as negative and positive control, respectively.
Mammalian cellular toxicity
The cytotoxicity of DPPs and DP-AD were investigated on HIEC cells. A total of 60,000 cells plated in 96-well plates the day before transfection were incubated with 1 μM of DP-AD and equivalent DPPs solutions for 24 h. Cytotoxicity was measured by colorimetric MTT assay (Sigma, Germany). Cell culture medium was removed and replaced with PBS containing 2.5 mg/ml of MTT for 4 h.
Bacterial solutions (approximately 3 × 107 CFU in 300 μl M–H broth) were incubated with DP-AD (300 μl, 3 μM) at 37 °C for 3 h. Bacterial total RNA was extracted using Trizol (Invitrogen, CA, USA). RNA was reversely transcribed to cDNA using HiScript™ Reverse Transcriptase (Vazyme Biotech co., Ltd) and mRNA was quantified using SYBR green detection kit following the manufacturer’s instructions with a Real-Time Q-PCR System (Mx3005p, Agilent Technologies StrataGene, La Jolla, CA, USA). The antisense sequences were 5′-cttcgatagtg-3′ for acpP [37, 41], 5′-acagctcctcgcccttcg-3′ for egfp in E. coli and ESBLs-E. coli, while 5′-tttctcgtca-3′ for rpoD in S. aureus and MRSA. The primers used were the following: E. coli 16 s ribosomal RNA (rRNA) forward 5′-cggacgggtgagtaatgt-3′ and reverse 5′-gtgcttcttctgcgggta-3′; acpP forward 5′-gagaattcatgagcactatcgaagaac-3′ and reverse 5′-agttaagcttgaccgcctggagatgttc-3′. S. aurues 16 s rRNA forward 5′-cgtggataacctacctataagact-3′ and reverse 5′-gattccctactgctgcctc-3′, rpoD forward 5′-cagatactgacgagaaa-3′ and 5′- gaataacataccacgac-3′. The PCR results in the tested strains were normalized using 16 s rRNA as the housekeeping gene. Results were presented as the average fold change relative to the untreated control group by 2−ΔΔCt method.
Bacterial growth curve
E. coli solution in LB was centrifuged at 2500 g for 5 min at 4 °C and resuspended in M–H broth. Bacterial solutions (106 CFU in 300 μl M–H broth) were mixed with 300 μl DP-ADanti-acpP (E. coli and ESBLs-E. coli) or DP-ADanti-rpoD (S. aureus or MRSA) solutions. The mixtures were divided into three wells on a culture plate and incubated into BioScreen C analyser at 37 °C for 18 h to measure the optical density per hour. Data were plotted and analyzed by Graphpad Prism (version 5.00, GraphPad Software, lnc., La Jolla, CA, USA).
In vivo fluorescent image acquisition
400 μl DP-AD7 was administered into male Balb/c mice intraperitoneally, and the fluorescent images of the mice were acquired after 2 h. Then, the mice were sacrificed and the fluorescent images of the organs (heart, liver, spleen, lung and kidney) were acquired by an in vivo imaging system.
Survival assay of DP-AD7 on infected animals
Male Balb/c mice (6–8 weeks old and weighing 18–22 g) were used in this experiment. The experimental and animal care procedures were approved by the Animal Care and Use Committee of Fourth Military Medical University. All methods were carried out strictly in accordance with the approved guidelines. 4 × 105 CFU ESBLs-E. coli in 400 μl M–H broth were administrated intraperitoneally into the mice to construct a sepsis model. Then, the mice were randomly divided to ten groups, which were administered intraperitoneally with 400 μL solutions containing 0.5 (based on ASOs), 1 or 1.5 mg kg−1 DP-AD7anti-acpP and the counterpart DP-AD7mismatch, 1.5 mg kg−1 L-DP-AD7anti-acpP, 1.5 mg kg−1 LF2000-NPsanti-acpP, 4 mg kg−1 ceftazidime or PBS at 0.5 and 6 h after infection. The surviving mice in each group were monitored every 6 h for the first day, afterwards, monitored every 12 h for 7 days day after infection, and the survival ratio was analyzed by Kaplan–Meier estimator.
Two mice in each group were sacrificed 10 h after infection to study the bacterial clearance. The liver and kidney were harvested aseptically, one kidney and one lobe of liver of each mouse were weighed, and homogenized in 500 μl sterile saline solution, then 50 μl homogenate sample of each mice was performed serial tenfold dilutions to a 10–5 dilution. Carefully spread 100 μL of each dilution onto a M–H agarose plate using a glass spreader. Incubate plates at 37 °C overnight and calculate the bacterial load. Colony counts were expressed as CFU g−1 of tissue.
The other kidney and liver lobe of each mouse were stored in 4% paraformaldehyde, paraffin embedding and hematoxylin and eosin staining were performed to observe the tissue lesions. The staining processes of tissues were carried out by the technicians in Department of Pathology, Fourth Military Medical University. The images were capture on Olympus BX15 with DP controller (version 18.104.22.1687, Olympus Corp., Japan).
Results are expressed as mean values (± SE). Statistical analyses were performed with SPSS (version 20.0.0, IBM Corp., Armonk, NY, USA). Differences between two groups were compared using t tests, and groups of two or more were compared to the control group using Dunnett t tests.