Amphipathic dendritic poly-peptides carrier to deliver antisense oligonucleotides against multi-drug resistant bacteria in vitro and in vivo

Abstract

Background: Outbreaks of infection due to multidrug-resistant (MDR) bacteria, especially Gram-negative bacteria, have become a global health issue in both hospitals and communities. Antisense oligonucleotides (ASOs) based therapeutics hold a great promise for treating infections caused by MDR bacteria. However, ASOs therapeutics are strangled because of its low cell penetration efficiency caused by the high molecular weight and hydrophilicity.

Results: Here, we designed a series of dendritic poly-peptides (DPP1 to DPP12) to encapsulate ASOs to form DSPE-mPEG2000 decorated ASOs/DPP nanoparticles (DP-AD1 to DP-AD12) and observed that amphipathic DP-AD2, 3, 7 or 8 with a positive charge ≥ 8 showed great efficiency to deliver ASOs into bacteria, but only the two histidine residues contained DP-AD7 and DP-AD8 significantly inhibited the bacterial growth and the targeted gene expression of tested bacteria in vitro. DP-AD7_anti-acpP remarkably increased the survival rate of septic mice infected by ESBLs-E. coli, exhibiting strong antibacterial effects in vivo.

Conclusions: For the first time, we designed DPP as a potent carrier to deliver ASOs for combating MDR bacteria and demonstrated the essential features, namely, amphipathicity, 8-10 positive charges, and 2 histidine residues, that are required for efficient DPP based delivery, and provide a novel approach for the development and research of the antisense antibacterial strategy.

Keywords: Antibacterial strategy; Antisense; Delivery; Dendritic poly-peptides; Multidrug-resistant bacteria; Nanoparticles; Oligonucleotide.

Scheme 1

DPP design. The positive charges in hydrophilic DPP1 were averaged to obtain DPP2. Trp and Leu in DPP1 and DPP6 were substituted with hydroxyl containing Ser and Thr to obtain DPP4 and DPP5, respectively. The positive charges in amphipathic DPP2 were aggregated on one side to obtain amphipathic DPP3, and two Lys residues in the positive arms were substituted with two His residues to yield DPP7. The positive charge was reduced to obtain DPP8 and DPP10. The number of His residues in DPP7, 8 and 10 were increased to four to obtain DPP11, DPP9 and DPP12, respectively. In addition, a linear DPP (L-DPP) with the same sequence as DPP7 was also synthesized as controls

Fig. 1

Preparation and characteristics of DP-AD. a Schematic of the two-step preparation of DP-AD. b Screening of the best N/P molar ratios ranging from 1 to 16 by 1% agarose gel electrophoresis. The upper and lower images indicated hydrophilic DP-AD1 and amphipathic DP-AD7, respectively. c TEM images of AD1 and AD7. d The size of ADs in dd H₂O (white columns) and diluted with equal volume of M–H broth (black columns). e TEM images of DP-AD1 and DP-AD7. f The size of DP-AD in dd H₂O (white columns) and diluted with equal volume of M–H broth (black columns), and zeta potential (blue line) of DP-AD in dd H₂O determined by DLS. Bar = 200 nm

Fig. 2

Screening the DP-AD by delivery efficiency and antisense efficacy in vitro. a FAM-positive ratio of ESBLs-E. coli (left) and MRSA (right) were tested by flow cytometry after incubation with FAM-labeled DP-AD for 1 h in dark at 37 °C. Free FAM-labeled ASOs (red) and LF2000-NPs (light blue) were used as negative and positive controls, respectively. b GFP fluorescence intensity of E. coli (DH5α) expressing the GFP measured by flow-cytometry after incubation with 1 μM DP-AD_anti-egfp for 3 h. c Growth curves of ESBLs-E. coli treated with different amphipathic DP-AD_anti-acpP (1 μM), OD_600nm, the optical density at 600 nm

Fig. 3

Uptake profiles of DP-AD7. a Fluorescence positive ratios of ESBLs-E. coli, K. pneumoniae, MDR-A. baumannii, MDR-P. aeruginosa, MRSA, B. subtilis, MRSE and E. faecalis measured by flow-cytometry after incubation with 1 μM FAM labeled DP-AD7 for 1 h in dark. b The FAM positive rate of the tested bacterial strains measured by flow cytometry after incubation with FAM-labeled DP-AD7 in the dark at 37 °C for 5, 10, 30 and 60 min. c CLSM images of ESBLs-E. coli (upper panel) and B. subtilis (lower panel) after incubation with cy5-labeled DP-AD7 for 1 h in dark at 37 °C. FM4-64 was used to stain the bacterial cell membrane. Bar = 2 μm

Fig. 4

Antibacterial activity of DP-AD7 in vitro. a Growth inhibition of DP-AD7_anti-acpP on E. coli (left) and ESBLs-E. coli (right) or b DP-AD7_anti-rpoD on S. aureus (left) and MRSA (right) were measured. Ceftazidime and LF2000-NPs_anti-acpP were used as positive control, and free ASOs and L-DP-AD were used as negative control. OD_600nm, the optical density at 600 nm

Fig. 5

Antibacterial activity of DP-AD7_anti-acpP against ESBLs-E. coli in the sepsis model. a, b Biodistribtution of DP-AD7_anti-acpP in mice determined by in vivo imaging system. Mice were intraperitoneally administrated with 400 μl cy5-labeled DP-AD7. Fluorescent signals were detected in the live mice (a) or the collected organs of the mice (b) 2 h after injection. sp1 and sp2 were two independent samples. c The diagram of treatment and analysis procedure of the in vivo experiment. d Survival rate of BalB/c mice treated with DP-AD7_anti-acpP (1.5, 1 and 0.5 mg/kg), DP-AD7_mismatch (1.5, 1 or 0.5 mg/kg), or L-DP-AD_anti-acpP (1.5 mg/kg) (n = 10). LF2000-NPs_anti-acpP (1.5 mg/kg) and ceftazidime (4 mg/kg) were used as positive controls. e, f Colonization of ESBLs-E.coli inocula in liver (e) and kidney (f). Data represent the mean ± SE (n = 6). *p < 0.05