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. 2018 Feb;2(2):95-103.
doi: 10.1038/s41551-017-0187-5. Epub 2018 Jan 22.

Antibiotic-loaded nanoparticles targeted to the site of infection enhance antibacterial efficacy

Sazid Hussain  1 , Jinmyoung Joo  2   3   4 , Jinyoung Kang  5 , Byungji Kim  6 , Gary B Braun  1   7 , Zhi-Gang She  1   8 , Dokyoung Kim  2 , Aman P Mann  1 , Tarmo Mölder  9 , Tambet Teesalu  1   9   10 , Santina Carnazza  11 , Salvatore Guglielmino  11 , Michael J Sailor  2   5   6 , Erkki Ruoslahti  12   13
Affiliations

Antibiotic-loaded nanoparticles targeted to the site of infection enhance antibacterial efficacy

Sazid Hussain et al. Nat Biomed Eng. 2018 Feb.

Abstract

Bacterial resistance to antibiotics has made it necessary to resort to antibiotics that have considerable toxicities. Here, we show that the cyclic 9-amino acid peptide CARGGLKSC (CARG), identified via phage display on Staphylococcus aureus (S. aureus) bacteria and through in vivo screening in mice with S. aureus-induced lung infections, increases the antibacterial activity of CARG-conjugated vancomycin-loaded nanoparticles in S. aureus-infected tissues and reduces the needed overall systemic dose, minimizing side effects. CARG binds specifically to S. aureus bacteria but not Pseudomonas bacteria in vitro, selectively accumulates in S. aureus-infected lungs and skin of mice but not in non-infected tissue and Pseudomonas-infected tissue, and significantly enhances the accumulation of intravenously injected vancomycin-loaded porous silicon nanoparticles bearing the peptide in S. aureus-infected mouse lung tissue. The targeted nanoparticles more effectively suppress staphylococcal infections in vivo relative to equivalent doses of untargeted vancomycin nanoparticles or of free vancomycin. The therapeutic delivery of antibiotic-carrying nanoparticles bearing peptides targeting infected tissue may help combat difficult-to-treat infections.

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Conflict of interest statement

Competing financial interests M.J.S is a scientific founder of Spinnaker Biosciences, Inc., and has an equity interest in the company. Although the R01 AI132413-01 grant has been identified for conflict of interest management based on the overall scope of the project and its potential benefit to Spinnaker Biosciences, Inc., the research findings included in this particular publication may not necessarily relate to the interests of Spinnaker Biosciences, Inc. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. All the other authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Schematic representation of the peptide library screening by in vivo phage display in S. aureus-induced pneumonia model
Lung infection was generated by direct inoculation of S. aureus into the lungs by intratracheal surgery. Forty-eight −72 h after the bacterial inoculation, a CX7C-peptide library was screened by in vivo phage display. After 3 in vivo rounds, the enriched phage pool was subjected to in vitro biopanning on cultured S. aureus. The in vitro phage pool was further screened in vivo in infected animals. The graph shows the enrichment obtained in 4 rounds of in vivo biopanning. The recovered phage pools were subjected to high throughput sequencing and the data were analyzed for dominant binding motifs.
Figure 2
Figure 2. CARG peptide shows selective binding to cultured S. aureus in vitro and homes to infected lungs in vivo
(a) Binding of FAM-labeled CARG peptide to cultured S. aureus in vitro (top row). The control peptide (FAM-Ctl) shows no bacterial binding (bottom row). Scale bar: 5 μm. (b) Super-resolution microscopy images of S. aureus after incubation with FAM-CARG peptide (green). Staphylococci visualized with an antibody are red, and bacterial nucleoid is stained with DAPI (blue). Scale bar: 3 μm. (c) S. aureus-induced lung infection was generated by intratracheal inoculation of S. aureus into the lungs in surgery. At 48–72 h post-infection, FAM-CARG was intravenously injected via the tail vein and allowed to circulate for 30 min. Lungs and other organs were harvested after perfusion and fixed for histological analysis. Sham-operated control mice underwent the same surgical procedure without S. aureus inoculation. Immunofluorescence microscopy of lung sections from mice with (top) or without (bottom) S. aureus infection. FAM-CARG (green), S. aureus (red) and cell nuclei stained with DAPI (blue). Images representative of at least five sections from each lung (n = 3–5 mice per group) are shown. Scale bar: 50 μm. (d) Immunohistochemical staining for the FAM label reveals intense signal (brown) from FAM-CARG in S. aureus-infected lungs as compared to healthy lungs. Sections were counterstained with DAPI (blue). Scale bar: 200 μm. (e) FAM integrated intensity was quantified using ImageJ (mean ± S.D., P < 0.001, two-tailed Student’s t test, n = 3; A. U. is arbitrary units).
Figure 3
Figure 3. CARG is highly specific in targeting S. aureus infections
(a–b) Skin infection in mice was generated by subcutaneous inoculation of S. aureus bacterial suspension. The sham-infected control mice received subcutaneous injection of PBS. At 72 h post-infection, FAM-CARG was intravenously injected via the tail vein and allowed to circulate for 30 min to home to the skin abscesses. (a) Immunofluorescence microscopy of skin sections from mice with (top) or without (bottom) S. aureus infection at low (20X) magnification. High (40X) magnification of CARG homing to infected skin section (Dashed square site). FAM-CARG (green), S. aureus (red) and cell nuclei stained with DAPI (blue). Representative images of at least five skin sections from each group (n = 3 mice per group) are shown. Scale bar: 50 μm (low magnification); 20 μm (high magnification). (b) Imunohistochemical staining for the FAM label reveals intense signal (brown) from FAM-CARG in S. aureus-infected skin as compared to healthy skin. Sections were counterstained with DAPI (blue). Scale bar: 200 μm.
Figure 4
Figure 4. CARG peptide zeroes in on S. aureus bacteria at sites of infection and co-localizes with host phagocytic cells containing intracellular bacteria
(a) The homing of FAM-labeled CARG to S. aureus infection was studied as described in Fig. 2. The FAM signal (green) shows strong homing and uptake in regions of Staphylococcus-infection (red). Cell nuclei are stained with DAPI (blue). The merged confocal image shows co-localization of the CARG peptide with bacteria at the infection site (white arrows). Scale bar: 20 μm. (b) Infected lung sections stained for CD11b (red, top row), CD45 (red, bottom row), S. aureus (magenta, pseudocolor), FAM (green) and nuclei (blue). The CARG peptide co-localizes with CD45-expressing leukocytes. Scale bar: 10 μm. (c) Infected lung sections stained for the neutrophil marker Ly-6G (red), S. aureus (magenta; pseudocolor), FAM (green) and nuclei (blue). The high magnification confocal image shows uptake and co-localization of the CARG peptide in neutrophils with engulfed bacteria (white arrowheads). Scale bar: 20 μm Representative images of at least five sections from infected lungs (n = 3–5 mice) are shown.
Figure 5
Figure 5. Targeted in vivo delivery of nanoparticles to S. aureus-infected lungs
(a) A schematic illustration of therapeutic nanoparticle system consisting of pSiNP, chemotherapeutic agent, and homing peptide. (b) Transmission electron microscope (TEM) image of vancomycin-loaded pSiNP prepared by self-sealing chemistry. (c) Hydrodynamic size distribution of the nanoparticles measured by dynamic light scattering. Data are presented as mean ± standard deviation (n=3). (d) Release profile of vancomycin payload from the nanoparticles in phosphate-buffered saline at 37°C. (e) Time-gated luminescence images of pSiNPs in major organs harvested from mice after 1 hr of circulation (λex: 500 nm). Nanoparticles were intravenously injected to infected mice 24 h after intratracheally introduced S. aureus infection. White dashed line designates the outer boundary of each organ. Control nanoparticles were pSiNPs grafted with polyethylene glycol only (no targeting peptide). Corresponding volume (50 μL) of phosphate-buffered saline (PBS) was injected to mice intravenously as another negative control. Inset: White light photograph of lung tissues corresponding to the time-gated luminescence image. (f) Biodistribution of nanoparticles obtained from the luminescence intensity in each organ (n = 4 mice per group; * p < 0.01; n.s. means not significant, from two-tailed Student’s t test; Data are presented as mean ± standard deviation; a.u. is arbitrary units). Note that there is significantly greater accumulation of CARG-pSiNPs compared with control pSiNPs in infected lung tissue, but not in other organs. (g) Confocal fluorescence microscope images of infected lung tissue of mice injected with CARG-pSiNP. Green channel, CARG peptide labeled with FAM; red, intrinsic photoluminescence of pSiNPs; and blue, DAPI nuclear stain. Scale bar: 20 μm.
Figure 6
Figure 6. Targeted, infection-homing nanoparticles enhance antibiotic therapy
(a) Survival rate (n = 9) of mice after intratracheal inoculation with 5 × 107 colony forming units (CFU) of S. aureus. Groups of mice received the following therapeutics intravenously 24 hours after the bacterial inoculation: free vancomycin, CARG-pSiNP-vancomycin, and control-pSiNP-vancomycin. The therapeutic compounds were pre-suspended in PBS prior to injection into the tail vein. The amount of vancomycin administered was adjusted to be the same in each therapeutic (3 ± 0.2 mg/kg). (b) Hematoxylin and eosin (H&E)-stained tissue sections from infected mice, showing that intratracheal injection of S. aureus only causes lung infection, whereas other organs show no detectable changes. Note that the infected lung lesions are resolved by treatment with vancomycin-loaded CARG-pSiNP (n=3; representative images are shown). Scale bar: 50 μm.
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