Radiolabeled Cationic Peptides for Targeted Imaging of Infection

Aweda, Tolulope A.; Muftuler, Zumrut F. B.; Massicano, Adriana V. F.; Gadhia, Dhruval; McCarthy, Kelly A.; Queern, Stacy; Bandyopadhyay, Anupam; Gao, Jianmin; Lapi, Suzanne E.

doi:https://doi.org/10.1155/2019/3149249

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In Vivo Imaging of Inflammation and Infection 2019

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Research Article | Open Access

Volume 2019 | Article ID 3149249 | https://doi.org/10.1155/2019/3149249

Radiolabeled Cationic Peptides for Targeted Imaging of Infection

Tolulope A. Aweda,¹Zumrut F. B. Muftuler,²Adriana V. F. Massicano,¹Dhruval Gadhia,¹Kelly A. McCarthy,³Stacy Queern,¹Anupam Bandyopadhyay,⁴Jianmin Gao,³and Suzanne E. Lapi¹

Guest Editor: Xiang-Guo Li

Received23 Feb 2019

Revised20 May 2019

Accepted16 Jun 2019

Published29 Oct 2019

Abstract

Molecular probes targeting bacteria provide opportunities to target bacterial infections in vivo for both imaging and therapy. In the current study, we report the development of positron emission tomography (PET) probes for imaging of live bacterial infection based on the small molecules HLys-DOTA, a polycationic peptide synthesized as the D-isomer (RYWVAWRNRG) conjugated to 1, 4, 7, 10-tetraazacyclododecane-N′,N″,N‴,N-tetraacetic acid (DOTA) and AB1-HLys-DOTA, which includes an unnatural amino acid AB1 that preferentially binds to bacteria membrane lipids with amine groups via formation of iminoboronates. HLys-DOTA and AB1-HLys-DOTA peptides were radiolabeled with ⁶⁴Cu and investigated as PET imaging agents to track bacterial infection in vitro and in intramuscularly infected (IM) mice models. Cell uptake studies at 37°C in Staphylococcus aureus (SA) show higher uptake of ⁶⁴Cu-AB1-HLys-DOTA; 98.47 ± 3.54% vs ⁶⁴Cu-HLys-DOTA; 39.12 ± 3.27% at 24 h. Standard uptake values (SUV) analysis of the PET images resulted in mean SUV of 0.70 ± 0.08, 0.49 ± 0.04, and 0.31 ± 0.01 for ⁶⁴Cu-AB1-HLys-DOTA and 0.17 ± 0.06, 0.16 ± 0.02, and 0.13 ± 0.01 for ⁶⁴Cu-HLys-DOTA at 1, 4, and 24 h post injection, respectively, in the infected muscles. Similarly, in the biodistribution studies, dose uptake in the infected muscles was 4 times higher in the targeted ⁶⁴Cu-AB1-HLys-DOTA group than in the ⁶⁴Cu-HLys-DOTA group and 2‐3 times higher than in the PBS control group at 1, 4, and 24 h post injection. ⁶⁴Cu-AB1-HLys-DOTA was able to distinguish between SA-infected muscle and Pseudomonas aeruginosa (PA) infected muscle with lower mean SUV of 0.28 ± 0.10 at 1 h post injection. This illustrates the utility of the AB1 covalently targeting group in synergy with the HLys peptide, which noncovalently binds to bacterial membranes. These results suggest that ⁶⁴Cu-labeled AB1-HLys-DOTA peptide could be used as an imaging probe for detection of bacterial infection in vivo with specificity for Gram-positive bacteria.

1. Introduction

Infectious diseases are one of the top ten leading causes of death in the world and the number one cause of death in low-income developing countries. Current diagnostic methods for bacterial infections are time-consuming as they require samples from the patient to be cultured for hours to weeks before enough bacteria are isolated to run destructive diagnostic tests [1, 2]. As proper identification of the disease often requires long diagnostic processes and bacterial infections tend to progress rapidly, fast diagnostic techniques would allow physicians to identify what types of antimicrobial therapy may help a patient in a clinically relevant timeframe. The ability to image bacterial disease would permit rapid and specific diagnosis of infection. In addition, the ability to image infections would allow clinicians to determine whether a therapy is effective and to monitor patient response to therapy.

Recently, there has been an increase in research into noninvasive imaging of bacterial infection using PET/CT, SPECT, MRI, and other imaging modalities in preclinical studies of infectious diseases using natural and synthetic molecules [3]. The use of radionuclides for detecting and localizing infection has been employed for decades [4, 5]. Ideally, the radiopharmaceutical for detecting infection should be specific, sensitive, and clear from the body rapidly to facilitate early image acquisition and clear delineation of the infected area. These radionuclides have been utilized as salts or small molecules such as ^99mTc-methylene diphosphonate, ⁶⁷Ga-citrate, and ¹⁸F-FDG [6–10]. ¹¹¹In-oxine and ^99mTc-exametazime have also been used to label leukocytes for imaging in diagnosis of infection [7, 11, 12]. This approach, though valuable, has some drawbacks, requiring high levels of leukocytes, and detection of noninfectious inflammations and is thus not specific to the infection or bacteria [13, 14].

Another radioisotope of interest is ⁶⁸Ga, with a shorter half-life of 68 min compared to its counterpart ⁶⁷Ga: half-life of 78.3 h (used in SPECT), has undergone an increased utilization in preclinical and clinical PET imaging particularly in oncology. Although, [⁶⁸Ga]citrate PET-CT of bone and joint infection offered lower radiation dose, as well as earlier and shorter imaging times, and its sensitivity and specificity was found to be lower than [⁶⁷Ga ]citrate [15]. In other infection and inflammation imaging studies, ⁶⁸Ga has been used to radiolabel ligands, small molecules, and peptides to target a host of receptors, inhibitors, leukocytes, and pathways [16]. ⁶⁸Ga-apo-transferrin ([⁶⁸Ga]TF) has been used to image Staphylococcus aureus (SA) and found to detect the infected lesions better than [⁶⁸Ga]Cl₃; additionally, [⁶⁸Ga]TF was also found to detect the Gram-negative bacteria, Proteus mirabilis [17]. In other studies, the antibiotic ciprofloxacin was labeled with ⁶⁸Ga and used to distinguish inflamed muscle from SA-infected muscle [18].

The use of ⁶⁴Cu radioisotope for imaging purposes has increased over the years, and several studies have utilized this isotope to label compounds for detecting regions of infection [19]. In order to label these compounds, several suitable chelators for ⁶⁴Cu have been developed with significant progress over the years; these chelates range from acyclic to cage-like bifunctional chelators [20, 21]. ⁶⁴Cu, with its relatively long half-life (12.7 hours) compared to ¹⁸F or ^99mTc has been used to label peptides, antibody fragments, and whole antibodies for imaging [22]. For instance, necrotic pulmonary tuberculosis lesions in chronically infected mice were detected and demonstrated to be hypoxic using ⁶⁴Cu(II)-diacetyl-bis(N⁴-methyl-thiosemicarbazone), [⁶⁴Cu]ATSM [23]. In mice models of S. aureus endocarditis (heart valve infection), robust localization of [⁶⁴Cu]DTPA-prothrombin was used to noninvasively detect infection lesions as compared to the bacteria-free control mice, which had no accumulation at the site of endothelial trauma [24].

Radiolabeled molecular probes specifically targeting bacterial lipids provide opportunities to target bacterial infections in vivo for both imaging and therapy. In this study, synthetic peptides that preferentially bind to bacterial surfaces with amine-presenting lipids were radiolabeled and studied in vitro and in vivo. Two phospholipids abundant on Gram-positive bacteria, phosphatidylethanolamine (PE) and lysylphosphatidylglycerol (Lys-PG), were targeted for recognition by appropriately constructed short peptides used as low molecular weight probes [25]. For our studies, the D-isomer of a polycationic peptide HLys (RYWVAWRNRG), which binds noncovalently to bacteria surfaces, was conjugated to 1, 4, 7, 10-tetraazacyclododecane-N′, N″, N‴, N-tetraacetic acid (DOTA) to make HLys-DOTA. To improve the binding of the peptide to the bacterial surface, an unnatural amino acid AB1, which preferentially binds covalently to lipids with amine groups via formation of iminoboronates under physiological conditions, was conjugated to HLys-DOTA, as shown in Figure 1 [26]. By targeting PE and Lys-PG with AB1, the iminoboronate chemistry allows potent labeling of Gram-positive bacteria such as Staphylococcus aureus (SA) even in the presence of serum proteins, while bypassing mammalian cells and Gram-negative bacteria [26]. HLys-DOTA and AB1-HLys-DOTA peptide conjugates were radiolabeled with ⁶⁴Cu and studied as PET imaging agents to track bacterial infection in vitro and in intramuscularly infected (IM) mice models.

2. Materials and Methods

⁶⁴Cu was produced in-house on an ACSI TR-24 Cyclotron (Richmond, Canada) located in the UAB Cyclotron facility. 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG) was purchased from PETNET (Birmingham, AL). All solvents and reagents were obtained from Sigma-Aldrich Company unless otherwise specified. Staphylococcus aureus (SA), ATCC 6538, and Pseudomonas aeruginosa (PA), ATCC 47085, were purchased from ATCC (Manassas, VA), and Whatman 3 M silica gel thin-layer chromatography (TLC) plates were purchased from Fisher Scientific (Pittsburgh, PA). Diethylenetriaminepentaacetic acid (DTPA) was purchased from Sigma (St. Louis, MO). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS-ester) was purchased from Macrocyclics (Dallas, Texas). C18 SepPak cartridges were obtained from Waters Corporation (Milford. MA). Radiometric instant thin-layer chromatography measurements were accomplished using a TLC scanner (Bioscan AR-2000 Scanner). Reverse-phase chromatography analysis was performed on a C18 column (PA) with an infinity diode-array UV detector (Agilent, Lake Forest, CA) and a PMT/NaI remote radioactive detector (LabLogic Systems Ltd, Brandon, FL). Laura radiochromatography software (LabLogic Systems Ltd, Brandon, FL) was used to quantify chromatograms by integration. Radioactive samples were counted using a 2480 Wizard II automatic gamma counter (PerkinElmer, Downers Grove, IL). Imaging studies were done by using GNEXT PET-CT (Sofie BioSciences) [CT-80 Kvp, 5 min (approximately 100 μm), PET-Resolution ≤1 mm]. Acquired PET/CT images were analyzed with Inveon™ Research Workplace (IRW) (Siemens).

2.1. Synthesis and Structural Analysis of HLys-DOTA and AB1-HLys-DOTA Peptides

HLys-DOTA and AB1-HLys-DOTA were synthesized by following a previously described protocol for solid-phase peptide synthesis [26], and the DOTA moiety was conjugated to the N-terminus of peptides on resin using DOTA-NHS-ester as a precursor [27]. To ensure in vivo stability of the peptides, the D-isomer of all amino acids except AB1 was used for peptide synthesis. Literature reports have shown that the D-isomers of antimicrobial peptides behave similarly to their L-counterparts in bacterial binding [28]. The peptides were purified using HPLC, and their purity and integrity was confirmed by LC-MS (Supporting Information Figure 1).

2.2. Production of ⁶⁴Cu

⁶⁴Cu (t_1/2 = 12.7 h, β⁺ = 17%, β⁻ = 39%, EC = 43%, E_max = 0.656 MeV) was produced in-house at the University of Alabama at Birmingham Cyclotron facility via the ⁶⁴Ni(p,n)⁶⁴Cu nuclear reaction. Production and purification of ⁶⁴Cu was conducted using modified methods from literature [29, 30] via bombardment of enriched ⁶⁴Ni targets at 40 μA with an incident proton beam of 19 MeV degraded to 12 MeV with a 1 mm Al degrader.

2.3. Radiolabeling of HLys-DOTA and AB1-HLys-DOTA Peptides

The two peptides, HLys-DOTA and AB1-HLys-DOTA, were radiolabeled with ⁶⁴CuCl₂ buffered in 0.1 M NH₄OAc, pH 6, at 56°C according to modified previously published methods [31, 32]. Briefly, radiolabeling of the peptides was achieved by adding 50 μg of HLys-DOTA or AB1-HLys-DOTA into 120–130 MBq (3.2–3.5 mCi) of ⁶⁴CuCl₂ in 1450 μL of 0.1 M NH₄OAc, pH 6. The reactions were incubated on a mixer with 800 rpm agitation at 56°C for 45 min, and radiolabeling was also attempted at lower temperature. Radiolabeling yield and radiochemical purity were assessed using silica gel plates developed in 50 : 50 of methanol: 1 M ammonium acetate and high-performance liquid chromatography, respectively, with gradient: 0–12 min: 95% A to 20% A (A = water, 0.1% TFA, B = acetonitrile, 0.1% TFA), 12–15 min: 20% A to 95% A. ⁶⁴Cu-AB1-HLys-DOTA was purified according to the Jacobson method [33]. A SepPak C18-cartridge was activated with 5 mL of ethanol and 10 mL of water. The reaction mixture of ⁶⁴Cu-AB1-HLys-DOTA was diluted with 5 mL of water and loaded slowly onto the activated SepPak C18-cartridge using a syringe. The cartridge was washed with 10 mL of water followed by elution of the desired labeled peptide with 1 mL of 10 mM HCl in ethanol. The ethanol was evaporated, and the radiolabeled peptide was reformulated with saline.

2.4. Serum Stability Studies

An aliquot of 10 μL of 3.3 MBq (∼90 μCi) of ⁶⁴Cu-labeled AB1-HLys-DOTA compound was added to 90 mL of PBS, human serum (HSA), or mouse serum and incubated at 37°C with agitation (500 rpm) separately. Aliquots were removed at time points 0, 0.17, 1, 2, 4, and 24 h and analyzed using radio-TLC. All reactions were conducted in triplicate.

2.5. In Vitro Studies

2.5.1. Bacterial Cell Culture

Bacterial in vitro uptake and imaging experiments were performed using Gram-positive bacteria, Staphylococcus aureus (SA), and Gram-negative bacteria, Pseudomonas aeruginosa (PA), as control. Bacterial cells from a single colony were grown overnight in LB broth at 37°C with agitation until the cells reached the mid logarithmic phase (OD₆₀₀∼0.6–0.7). For the in vitro experiments, 750–1000 μL of the bacterial cell culture was spun down at 7000 rpm for 7 min in a 1.5 mL centrifuge tube.

2.5.2. Uptake Studies of ⁶⁴Cu-Labeled Peptides in Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA)

Bacteria cell uptake studies of ⁶⁴Cu-HLys-DOTA and ⁶⁴Cu-AB1-HLys-DOTA in SA and PA bacteria were performed at 37°C. LB broth was inoculated with SA or PA bacteria and after 18–22 hours, cells were harvested by spinning down and washing twice in 750–1000 μL of PBS (50 mM sodium phosphate, pH = 7.4), and the OD₆₀₀, OD₂₃₀, and OD₂₆₀ determined. 750 μL of the PBS suspended bacteria (0.9–1.3 × 10⁷ CFU/mL) was incubated with 0.925–0.999 MBq (25–27 μCi) of ⁶⁴Cu-HLys-DOTA and ⁶⁴Cu-AB1-HLys-DOTA at 37°C in triplicates at different time points: 5, 40, 60, 240, and 1440 min. At each time point, the mixture was spun down, washed twice with PBS, and the final bacterial pellets were measured using a gamma counter to determine the percent of associated radiolabeled peptide. Mammalian cell uptake was verified by following a similar procedure in a nonbacterial cell, SKBR3 breast cancer cell line.

2.5.3. Small Animal Imaging and Biodistribution Studies

All animal experiments were performed according to animal use protocols approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee (IACUC). Animals were housed under controlled conditions with a natural light-dark cycle. They were allowed to adapt to the housing environment for at least 48 h prior to study. Biodistribution and PET imaging studies with ⁶⁴Cu-HLys-DOTA and ⁶⁴Cu-AB1-HLys-DOTA were conducted in mice intramuscularly (IM) infected in the thigh muscle with SA and PBS as a control. Thirty-six male mice (25–30 g, 4–5 weeks old) infected with SA were used for animal studies. For each peptide, 4 mice were used for small animal PET imaging at 1, 4, and 24 h, and then sacrificed at 24 h for a post-PET biodistribution. Another set of 4 mice each was used for biodistribution studies at 1 and 4 h. A similar set of mice was used for blocking studies with ⁶⁴Cu-AB1-HLys-DOTA peptide. In order to check the specificity of ⁶⁴Cu-AB1-HLys-DOTA for Gram-positive bacteria, imaging and post-PET biodistribution studies were conducted in another set of mice (four) intramuscularly (IM) infected in the thigh muscle with PA and PBS.

2.5.4. Infection Animal Model

Mice for infection studies were anesthetized prior to the intramuscular infection of SA or PA. Animals were immobilized with isoflurane. Bacteria cells concentrated in sterile PBS were injected into the thigh muscle and the site of injection pinched to minimize bleeding. SA or PA suspension (50 μL, 2.6–2.8 × 10⁷ CFU per mouse) was intramuscularly injected into the right thigh muscle, and sterile PBS (50 μL) was injected into the left thigh muscle as control. Mice were placed back into the cages and allowed to recover with frequent monitoring to identify any sign of extreme distress. The infection was allowed to develop for 3–5 days before intravenous injection of the radiolabeled probe. The muscles on the left and right thighs were harvested, homogenized, and plated on mannitol agar plates or cultured in LB broth to verify the presence of infection. In addition, [¹⁸F]FDG imaging was also performed in infected animal models as described in the PET imaging section below to further confirm the existence of infection in the right thigh and the absence thereof in the left thigh.

2.5.5. Biodistribution Studies

For biodistribution studies, 3.3–3.7 MBq (90–100 μCi) of ⁶⁴Cu-HLys-DOTA or ⁶⁴Cu-AB1-HLys-DOTA was injected into prewarmed tail vein of the animals under anesthesia. Animals were euthanized at 1 and 4 h after radiotracer administration (n = 4 for each tracer at each time point). At 24 h after small animal PET imaging, the mice were also euthanized for a post-PET biodistribution. In a similar set of experiment, excess of the unlabeled peptide, 150 nmol, was coinjected with the radiolabeled peptides per mouse. The organs of interest were collected, weighed, and measured for radioactivity content using a gamma counter. The data were background-corrected and decay-corrected. The biodistribution data were expressed as percentage of injected radioactive dose per gram of tissue (%ID/g) for selected organs as the mean value of four mice.

2.5.6. PET/CT Imaging Studies

Small animal PET/CT imaging studies were conducted in SA-infected and PA-infected animal models. Each radiolabeled peptide, 3.3–3.7 MBq (90–100 μCi), was administrated via tail vein injection of prewarmed mice under anesthesia. The inflammatory response caused by the infection was confirmed by intravenous injection of [¹⁸F]FDG, 3.7 MBq (100 μCi), into mice infected on the right thigh muscle with SA or PA and on the left with PBS as control. For this small animal PET imaging, 1 and 4 h post-injection static scans were collected. Following imaging studies, the mice were sacrificed for biodistribution. Mice were allowed to recover from anesthesia until the time of imaging. Mice were anesthetized with 2‐3% isoflurane/oxygen and imaged on the small animal PET/CT scanner. Static images were collected at 1, 4, and 24 h for 15, 20, and 30 min, respectively. PET images were coregistered with CT image for anatomical colocalization. Regions of interest (ROI) were manually drawn over organs of interest with CT anatomical guidelines, and the associated radioactivity was measured using Inveon Research Workstation software. Standard uptake values (SUV) were calculated as nCi/cc × animal weight/injected dose, and comparisons in pharmacokinetics of radiolabeled peptides were assessed.

2.6. Statistical Analysis

Statistical calculations were carried out using Prism 7 (GraphPad Software) and expressed as mean ± SD. One-way analysis of standard deviation at 95% confidence level () were considered statistically significant.

3. Results

3.1. HLys-DOTA and AB1-HLys-DOTA Peptide Synthesis, Characterization, and Stability

The analysis of HLys-DOTA and AB1-HLys-DOTA peptides was performed using LC-MS in positive mode [M+H] allowing detection of their corresponding molecular ion at m/z 1691.92 and m/z 2074.15, respectively [26]. HLys-DOTA and AB1-HLys-DOTA peptides were radiolabeled with ⁶⁴Cu at molar activities of 3873 ± 398 and 3809 ± 943 MBq/μmol (105 ± 11 and 103 ± 25 mCi/μmol), respectively, at 56°C. Incubations at lower temperature resulted in lower yields (See Supporting Information Table 1 for radiolabeling kinetics). Radiochemical and labeling yield of ⁶⁴Cu-HLys-DOTA was ≥95% after TLC and HPLC analysis but ⁶⁴Cu-AB1-HLys-DOTA required purification through a C18 SepPak to achieve ≥95% radiochemical purity. The need for extra purification of ⁶⁴Cu-AB1-HLys-DOTA in order to achieve ≥95% radiochemical purity could be due to the extra benzyl group and the hydrophobic portion of AB1 preventing good complexation of radiocopper ions.

3.2. Stability and In Vitro Cell Uptake Studies

Excellent stability of ⁶⁴Cu-AB1-HLys-DOTA was observed in the PBS, human serum, and LB broth with >98% intact peptide observed at 24 h post incubation in all solutions, as shown in Figure 2(a). Bacteria cell uptake of ⁶⁴Cu-AB1-HLys-DOTA conjugate at 37°C in Staphylococcus aureus (SA) was significantly higher than that of ⁶⁴Cu-HLys-DOTA at all time points: 0.08, 0.67, 1, 4, and 24 h; 98.5 ± 3.5% vs 39.1 ± 3.3% at 24 h, Figure 2(b). Specific uptake in Gram-positive bacteria SA was confirmed by the lower uptake of the peptides in Pseudomonas aeruginosa (PA), a Gram-negative bacterium. At 5 min after incubation, the uptake of ⁶⁴Cu-AB1-HLys-DOTA and ⁶⁴Cu-HLys-DOTA showed 9.8 ± 4.4% and 1.9 ± 0.4% binding to PA, respectively, which increased slightly to 13.3 ± 1.1% and 5.2 ± 0.3%, respectively, at 24 h after incubation. This indicates a 5–10 times lower uptake of the peptides in PA as compared to SA confirming the specificity of AB1 for Gram-positive bacteria. Specific bacterial cell uptake was confirmed by the low uptake (9.4 ± 2.3%) of ⁶⁴Cu-AB1-HLys-DOTA in mammalian SKBR3 breast cancer cells at 24 h post incubation.

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3.3. Confirmation of Infection in Animal Model

The development of infection was evident by presence of a palpable mass filled with abscess observed during dissection and harvesting. The infected muscle and control muscle were harvested, homogenized, and the CFU of bacteria counted. In the infected muscle, 4.8 ± 2.5 × 10⁶ cfu/g of bacteria was found and only one PBS-control muscle was positive with colony at 1.8 × 10⁵ cfu/g cfu. [¹⁸F]FDG in vivo characterization of the infection site verified high uptake in the SA-infected muscle versus the control muscle at 1 and 4 h post injection, .

3.4. Biodistribution Studies

The biodistribution and pharmacokinetics of the peptides ⁶⁴Cu-AB1-HLys-DOTA and ⁶⁴Cu-HLys-DOTA were assessed in mice intramuscularly infected with S. aureus on the right thigh muscle and PBS injected in the left thigh muscle as control. The biodistribution showed that the uptake of both peptides in the infected muscle was significantly higher than the PBS-injected control muscle of the same mouse at all time points: 1, 4, and 24 h post injection, as shown in Figure 3. The uptake of ⁶⁴Cu-AB1-HLys-DOTA conjugate in the infected thigh was 2‐3 times higher than the control muscle at later time points, although no significant difference was observed at 1 h (4.8 ± 2.5 %ID/g vs. 2.5 ± 0.5 %ID/g). At 4 h, the dose uptake of ⁶⁴Cu-AB1-HLys-DOTA in the infected muscle was 3.6 ± 0.5 %ID/g vs. 1.1 ± 0.4 %ID/g in the control muscle, . Similarly, at 24 h, the uptake of ⁶⁴Cu-AB1-HLys-DOTA in the infected muscle was 2.6 ± 0.6 %ID/g vs. 0.8 ± 0.1 %ID/g in the control muscle, . In contrast, the dose accumulation of ⁶⁴Cu-HLys-DOTA in the infected muscle was only 1- or 2-fold higher than the control muscle in the same mouse, except at 24 h where no significant difference was observed between them. At 1 and 4 h, the uptake of ⁶⁴Cu-HLys-DOTA in the infected muscle vs. control muscle was 1.3 ± 0.5 %ID/g vs. 0.6 ± 0.1 %ID/g, , and 0.8 ± 0.1 %ID/g vs. 0.4 ± 0.1 %ID/g, , respectively. We also observed that uptake in the infected muscle was 4 times higher in the targeted ⁶⁴Cu-AB1-HLys-DOTA group compared to that in the ⁶⁴Cu-HLys-DOTA group, as shown in Figure 3(c). The uptake of ⁶⁴Cu-AB1-HLys-DOTA in the infected muscle was 4.8 ± 2.5, 3.6 ± 0.5, and 2.6 ± 0.6 %ID/g vs. 1.3 ± 0.5, 0.8 ± 0.1, and 0.6 ± 0.1 %ID/g for ⁶⁴Cu-HLys-DOTA, respectively, at 1, 4, and 24 h post injection, at 4 and 24 h. The highest accumulation of the radiolabeled peptides was observed in the liver and kidney with ⁶⁴Cu-AB1-HLys-DOTA also showing some uptake in the lungs. ⁶⁴Cu-AB1-HLys-DOTA also showed higher retention in the blood and heart than ⁶⁴Cu-HLys-DOTA at 1 and 4 h post injection but at 24 h, both peptides had similar activity in the blood. A similar experiment using an excess of the unlabeled peptides, 150 nmol, did not show a statistical difference (data not shown) in the uptake of the peptides at the infection site. This may indicate that the binding sites at the bacterial surface cannot be saturated.

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3.5. PET/CT Imaging

The uptake of ⁶⁴Cu-AB1-HLys-DOTA in the infected muscle was visibly observed via small animal PET/CT imaging as early as 1 and 4 h post injection, Figure 4. The uptake in the infected muscle was higher in the PET images of ⁶⁴Cu-AB1-HLys-DOTA than in the images of ⁶⁴Cu-HLys-DOTA at 1 and 4 h post injection in agreement with the biodistribution data. SUV analysis of the PET images showed 2.4 to 4-fold increase in accumulation of ⁶⁴Cu-AB1-HLys-DOTA vs. ⁶⁴Cu-HLys-DOTA at the infection. The mean SUV of ⁶⁴Cu-AB1-HLys-DOTA and ⁶⁴Cu-HLys-DOTA in the infected muscle was 0.70 ± 0.08 vs. 0.17 ± 0.06, , 0.49 ± 0.04 vs. 0.16 ± 0.02, , and 0.31 ± 0.01 vs. 0.13 ± 0.01, at 1, 4, and 24 h post injection, respectively. There was no statistical difference between mean SUV uptake of ⁶⁴Cu-HLys-DOTA in the infected muscle and ⁶⁴Cu-AB1-HLys-DOTA uptake in the control muscles at all time points. This indicates ⁶⁴Cu-HLys-DOTA uptake in the infected muscle is as low as the background uptake of ⁶⁴Cu-AB1-HLys-DOTA observed in the control muscle. Mean SUV analysis of the heart supports the trend in the biodistribution study with blood retention of ⁶⁴Cu-AB1-HLys-DOTA at 0.65 ± 0.08 vs. 0.25 ± 0.05 for ⁶⁴Cu-HLys-DOTA, , at 1 h. At 24 h post injection, both peptides had similar dose retention in the heart: 0.26 ± 0.04 and 0.21 ± 0.04 for ⁶⁴Cu-AB1-HLys-DOTA and ⁶⁴Cu-HLys-DOTA, respectively. The images also showed high signal in the liver and kidney possibly due to the fast clearance of the cationic peptides through these organs.

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Specificity of ⁶⁴Cu-AB1-HLys-DOTA peptide for Gram-positive bacteria was confirmed by low uptake in the Gram-negative bacteria Pseudomonas aeruginosa (PA) as observed in the PET images and the 4 h post-PET biodistribution, shown in Supporting Information Figure 3. ⁶⁴Cu-AB1-HLys-DOTA uptake in the SA-infected muscle at 1 and 4 h was significantly higher than that in the PA-infected muscle: 0.70 ± 0.08 vs. 0.28 ± 0.10, , and 0.49 ± 0.04 vs. 0.19 ± 0.10, , respectively. A similar trend was observed in the 24 h post-PET biodistribution with percent uptake of ⁶⁴Cu-AB1-HLys-DOTA in the SA-infected muscle at 2.64 ± 0.57 %ID/g vs. 0.49 ± 0.11 %ID/g in the PA-infected muscle, .

[¹⁸F]FDG uptake in the SA-infected muscle was significantly higher than that in the control muscle at 4 h post injection with mean SUV of 1.96 ± 0.19 vs. 0.41 ± 0.09, , which confirmed the presence of the SA infection [34–36], as shown in Figures 5(a) and 5(b). The higher uptake of [¹⁸F]FDG in the SA-infected muscles was also confirmed with post-PET imaging biodistribution data at 4 h (5.4 ± 1.3 %ID/g vs. 1.0 ± 0.1 %ID/g, ), as shown in Figure 5(c).

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4. Discussion

Antimicrobial peptides produced by phagocytes and other cell types in the body are part of the innate immunity systems against infection resulting from pathogens. The cationic domains of these peptides interact with the negatively charged surface of the microorganisms eliciting an antimicrobial reaction when the peptide is inserted into the microbial membranes [37]. Antimicrobial peptides, due to their diversity, elicit antimicrobial activity through other mechanisms such as the Shai-Matsuzaki-Huang, albeit at micromolar concentration [38]. These peptides and other synthetic peptide can be radiolabeled with specificity for detecting localized infections using a variety of chemistries [39]. In this study, by targeting the membrane lipids enriched in bacterial cells, namely PE and Lys-PG, the iminoboronate chemistry allows selective labeling of bacterial cells over mammalian cells [26]. Radiolabeling of AB1-HLys-DOTA and HLys-DOTA peptides with ⁶⁴Cu allowed monitoring of the infection up to 24 h and observation of their biological clearance. This was evident in the superior uptake of ⁶⁴Cu-AB1-HLys-DOTA conjugate in Staphylococcus aureus bacteria in comparison to ⁶⁴Cu-HLys-DOTA. Specific bacterial cell uptake of ⁶⁴Cu-AB1-HLys-DOTA was also confirmed by the low uptake (less than 10%) in mammalian SKBR3 breast cancer cells, while specificity in Gram-positive bacteria was confirmed with lower uptake in Pseudomonas aeruginosa. Previous studies have shown specific uptake of ⁶⁸Ga-radiolabeled probes at sites of infection [17]. Similarly, in this study, the biodistribution numbers show that ⁶⁴Cu-AB1-HLys-DOTA has higher affinity to the infection and persists within the infection longer than ⁶⁴Cu-HLys-DOTA that lacks the AB1 group.

The difference in accumulation of the peptides shows that the noncovalent attraction of HLys for bacterial cell surface was greatly enhanced by the covalent binding of AB1 to the lipids on the surface. In the biodistribution and PET images, clear uptake of ⁶⁴Cu-AB1-HLys-DOTA was observed at 1 h post injection, which may be due in part to its higher bioavailability (higher %ID/g in the blood) and slower clearance.

⁶⁴Cu-AB1-HLys-DOTA was able to distinguish between Gram-positive bacterial infection and Gram-negative bacterial infection. This class of peptides offers selective synthetic targets for bacterial lipids, which may give rise to new imaging methods of bacterial infection [26]. Although we are yet to ascertain its utility in distinguishing infection from sterile inflammation, but as reported by Sellmyer et al., it is possible to distinguish infection from background and other noninfection inflammation sites. ¹⁸F-labeled small-molecule antibiotic trimethoprim [¹⁸F]FPTMP showed high uptake at the infection and low background signal in normal tissues and other noninfection inflammation sites but did not differentiate between Gram-negative and Gram-positive strains [40]. Thus, future effort in work would focus on using the peptide to distinguish sterile inflammation from bacterial infection. Additionally, optimizing the peptides with functional groups that can modulate their clearance rates and therefore increase uptake at the infection sites would be implemented in future work.

5. Conclusion

The purpose of this study was to investigate the potential of AB1-HLys-DOTA peptide to image and distinguish infection due to Gram-positive bacteria from Gram-negative bacteria with higher specificity than HLys-DOTA peptide. ⁶⁴Cu-AB1-HLys-DOTA showed higher uptake in S. aureus bacteria cells in vitro and improved accumulation at the infection site of SA-inoculated mice compared with the noncovalently targeting ⁶⁴Cu-HLys-DOTA. In the small-animal PET images, the dose uptake of ⁶⁴Cu-AB1-HLys-DOTA at the infected site was distinguishable as early as 1 h after administration, indicating its potential for fast detection of infection. These results illustrate that the ⁶⁴Cu-labeled AB1-HLys-DOTA peptide could be used as imaging probe for detection of bacterial infection in vivo with specificity for Gram-positive bacterial infection.

Data Availability

The cell uptake, biodistribution, and small animal PET/CT data used to support the findings of this study are included within the article.

Conflicts of Interest

The authors do not have any conflicts of interest to declare.

Acknowledgments

Funding for the project was provided by UAB Department of Radiology and UAB Comprehensive Cancer Center’s Preclinical Imaging shared facility grant (P30CA013148). Funding for Dr. Gao and coworkers was provided by the US National Institutes of Health grant (GM102735). Funding support for postdoctoral fellowship (BIDEP-2219) of Dr. F. Zumrut B. Muftuler was provided by Scientific and Technological Research Council of Turkey (TUBITAK). The authors would like to acknowledge Solana Fernandez, Sheila Bright, and Caroline Baker for animal imaging and dissection and Jennifer Bartels and Lauren Radford for ⁶⁴Cu production.

Supplementary Materials

Supporting Information Figure 1: (a) HPLC chromatogram of HLys-DOTA and (b) LC-MS of HLys-DOTA with m/z values at 423.7, m/z+ = 4; 846.4, m/z+ = 2; 1692.8, m/z+ = 1; (c) HPLC chromatogram of AB1-HLys-DOTA and (d) LC-MS of AB1-HLys-DOTA with m/z values at 519.0, m/z+ = 4; 691.6, m/z+ = 3; 1037.0, m/z+ = 2. Supporting Information Table 1: radiolabeling kinetics of AB1-HLys-DOTA with ⁶⁴Cu at different temperature and labeling ratio. Supporting Information Figure 2: HPLC chromatogram showing the radioactive peak of (a) ⁶⁴Cu-AB1-HLys-DOTA and (b) ⁶⁴Cu-HLys-DOTA and UV absorbance at 280 nm of (c) ⁶⁴Cu-AB1-HLys-DOTA and (d) ⁶⁴Cu-HLys-DOTA. Supporting Information Figure 3: small-animal PET/CT images of (a) ⁶⁴Cu-AB1-HLys-DOTA in mice infected on the right thigh muscle with Pseudomonas aeruginosa, PA, (b) mean SUV values calculated from PA infected and PBS control muscle, and (c) post-PET biodistribution of ⁶⁴Cu-AB1-HLys-DOTA at 24 h after i.v. injection. Dashed arrow indicates the PBS-control muscle, and solid arrow indicates the infected right muscle (n = 4). (Supplementary Materials)

References

R. P. Peters, M. A. van Agtmael, S. A. Danner, P. H. Savelkoul, and C. M. Vandenbroucke-Grauls, “New developments in the diagnosis of bloodstream infections,” The Lancet Infectious Diseases, vol. 4, no. 12, pp. 751–760, 2004.
View at: Publisher Site | Google Scholar
S. Mignard and J. P. Flandrois, “16S rRNA sequencing in routine bacterial identification: a 30-month experiment,” Journal of Microbiological Methods, vol. 67, no. 3, pp. 574–581, 2006.
View at: Publisher Site | Google Scholar
L. A. Jelicks, M. P. Lisanti, F. S. Machado, L. M. Weiss, H. B. Tanowitz, and M. S. Desruisseaux, “Imaging of small-animal models of infectious diseases,” The American Journal of Pathology, vol. 182, no. 2, pp. 296–304, 2013.
View at: Publisher Site | Google Scholar
R. Kumar, S. Basu, D. Torigian, V. Anand, H. Zhuang, and A. Alavi, “Role of modern imaging techniques for diagnosis of infection in the era of 18F-fluorodeoxyglucose positron emission tomography,” Clinical Microbiology Reviews, vol. 21, no. 1, pp. 209–224, 2008.
View at: Publisher Site | Google Scholar
S. J. Goldsmith and S. Vallabhajosula, “Clinically proven radiopharmaceuticals for infection imaging: mechanisms and applications,” Seminars in Nuclear Medicine, vol. 39, no. 1, pp. 2–10, 2009.
View at: Publisher Site | Google Scholar
C. Bleeker-Rovers, F. Vos, F. Corstens, and W. Oyen, “Imaging of infectious diseases using [18F] fluorodeoxyglucose PET,” The Quarterly Journal of Nuclear Medicine and Molecular Imaging, vol. 52, 2008.
View at: Google Scholar
D. K. Hughes, “Nuclear medicine and infection detection: the relative effectiveness of imaging with 111In-oxine-, 99mTc-HMPAO-, and 99mTc-stannous fluoride colloid-labeled leukocytes and with 67Ga-citrate,” Journal of Nuclear Medicine Technology, vol. 31, no. 4, pp. 196–201, 2003.
View at: Google Scholar
J. Malamitsi, H. Giamarellou, K. Kanellakopoulou et al., “Infecton: a 99mTc-ciprofloxacin radiopharmaceutical for the detection of bone infection,” Clinical Microbiology and Infection, vol. 9, no. 2, pp. 101–109, 2003.
View at: Publisher Site | Google Scholar
R. Weiner, P. B. Hoffer, and M. L. Thakur, “Lactoferrin: its role as a Ga-67-binding protein in polymorphonuclear leukocytes,” Journal of Nuclear Medicine, vol. 22, no. 1, pp. 32–37, 1981.
View at: Google Scholar
C. P. Bleeker-Rovers, F. J. Vos, and F. H. M. Corstens, “Scintigraphic detection of infection and inflammation,” in Clinical Nuclear Medicine, pp. 347–359, Springer, Berlin, Germany, 2007.
View at: Publisher Site | Google Scholar
M. Charron, J. F. del Rosario, and S. Kocoshis, “Use of technetium-tagged white blood cells in patients with Crohn’s disease and ulcerative colitis: is differential diagnosis possible?” Pediatric Radiology, vol. 28, no. 11, pp. 871–877, 1998.
View at: Publisher Site | Google Scholar
C. Love and C. J. Palestro, “Radionuclide imaging of infection,” Journal of Nuclear Medicine Technology, vol. 32, no. 2, pp. 47–57, 2004.
View at: Google Scholar
C. J. Palestro, “Radionuclide imaging of infection: in search of the grail,” Journal of Nuclear Medicine, vol. 50, no. 5, pp. 671–673, 2009.
View at: Publisher Site | Google Scholar
Y. Sugawara, T. D. Gutowski, S. J. Fisher, R. S. Brown, and R. L. Wahl, “Uptake of positron emission tomography tracers in experimental bacterial infections: a comparative biodistribution study of radiolabeled FDG, thymidine, l-methionine, 67 Ga-citrate, and 125 I-HSA,” European Journal of Nuclear Medicine and Molecular Imaging, vol. 26, no. 4, pp. 333–341, 1999.
View at: Publisher Site | Google Scholar
T. Segard, L. M. J. A. Morandeau, M. L. Dunne et al., “Comparison between gallium‐68 citrate PET‐CT and gallium‐67 citrate scintigraphy for infection imaging,” Internal Medicine Journal, vol. 49, no. 8, pp. 1016–1022, 2019.
View at: Publisher Site | Google Scholar
I. Velikyan, “Prospective of 68Ga radionuclide contribution to the development of imaging agents for infection and inflammation,” Contrast Media & Molecular Imaging, vol. 2018, Article ID 9713691, 24 pages, 2018.
View at: Publisher Site | Google Scholar
V. Kumar, D. K. Boddeti, S. G. Evans, F. Roesch, and R. Howman-Giles, “Potential use of 68Ga-apo-transferrin as a PET imaging agent for detecting Staphylococcus aureus infection,” Nuclear Medicine and Biology, vol. 38, no. 3, pp. 393–398, 2011.
View at: Publisher Site | Google Scholar
D. Satpati, C. Arjun, R. Krishnamohan, G. Samuel, and S. Banerjee, “68Ga-labeled ciprofloxacin conjugates as radiotracers for targeting bacterial infection,” Chemical Biology & Drug Design, vol. 87, no. 5, pp. 680–686, 2016.
View at: Publisher Site | Google Scholar
H. Eggleston and P. Panizzi, “Molecular imaging of bacterial infections in vivo: the discrimination between infection and inflammation,” Informatics, vol. 1, no. 1, pp. 72–99, 2014.
View at: Publisher Site | Google Scholar
Z. Cai and C. J. Anderson, “Chelators for copper radionuclides in positron emission tomography radiopharmaceuticals,” Journal of Labelled Compounds and Radiopharmaceuticals, vol. 57, no. 4, pp. 224–230, 2014.
View at: Publisher Site | Google Scholar
E. W. Price and C. Orvig, “Matching chelators to radiometals for radiopharmaceuticals,” Chemical Society Reviews, vol. 43, no. 1, pp. 260–290, 2014.
View at: Publisher Site | Google Scholar
P. J. Blower, J. S. Lewis, and J. Zweit, “Copper radionuclides and radiopharmaceuticals in nuclear medicine,” Nuclear Medicine and Biology, vol. 23, no. 8, pp. 957–980, 1996.
View at: Publisher Site | Google Scholar
J. Harper, C. Skerry, S. L. Davis et al., “Mouse model of necrotic tuberculosis granulomas develops hypoxic lesions,” The Journal of Infectious Diseases, vol. 205, no. 4, pp. 595–602, 2011.
View at: Publisher Site | Google Scholar
P. Panizzi, M. Nahrendorf, J.-L. Figueiredo et al., “In vivo detection of Staphylococcus aureus endocarditis by targeting pathogen-specific prothrombin activation,” Nature Medicine, vol. 17, no. 9, pp. 1142–1146, 2011.
View at: Publisher Site | Google Scholar
J. Gao and H. Zheng, “Illuminating the lipidome to advance biomedical research: peptide-based probes of membrane lipids,” Future Medicinal Chemistry, vol. 5, no. 8, pp. 947–959, 2013.
View at: Publisher Site | Google Scholar
A. Bandyopadhyay, K. A. McCarthy, M. A. Kelly, and J. Gao, “Targeting bacteria via iminoboronate chemistry of amine-presenting lipids,” Nature Communications, vol. 6, no. 1, p. 6561, 2015.
View at: Publisher Site | Google Scholar
L. M. De León-Rodríguez and Z. Kovacs, “The synthesis and chelation chemistry of DOTA−peptide conjugates,” Bioconjugate Chemistry, vol. 19, no. 2, pp. 391–402, 2008.
View at: Publisher Site | Google Scholar
D. Wade, A. Boman, B. Wahlin et al., “All-D amino acid-containing channel-forming antibiotic peptides,” Proceedings of the National Academy of Sciences, vol. 87, no. 12, pp. 4761–4765, 1990.
View at: Publisher Site | Google Scholar
D. W. McCarthy, R. E. Shefer, R. E. Klinkowstein et al., “Efficient production of high specific activity 64Cu using a biomedical cyclotron,” Nuclear Medicine and Biology, vol. 24, no. 1, pp. 35–43, 1997.
View at: Publisher Site | Google Scholar
M. A. Avila-Rodriguez, J. A. Nye, and R. J. Nickles, “Simultaneous production of high specific activity 64Cu and 61Co with 11.4MeV protons on enriched 64Ni nuclei,” Applied Radiation and Isotopes, vol. 65, no. 10, pp. 1115–1120, 2007.
View at: Publisher Site | Google Scholar
B. V. Marquez, O. F. Ikotun, J. J. Parry, B. E. Rogers, C. F. Meares, and S. E. Lapi, “Development of a radiolabeled irreversible peptide ligand for PET imaging of vascular endothelial growth factor,” Journal of Nuclear Medicine, vol. 55, no. 6, pp. 1029–1034, 2014.
View at: Publisher Site | Google Scholar
N. Bandara, A. Zheleznyak, K. Cherukuri et al., “Evaluation of Cu-64 and Ga-68 radiolabeled glucagon-like peptide-1 receptor agonists as PET tracers for pancreatic β cell imaging,” Molecular Imaging and Biology, vol. 18, no. 1, pp. 90–98, 2016.
View at: Publisher Site | Google Scholar
O. Jacobson, I. D. Weiss, L. P. Szajek et al., “PET imaging of CXCR4 using copper-64 labeled peptide antagonist,” Theranostics, vol. 1, pp. 251–262, 2011.
View at: Publisher Site | Google Scholar
C. Love, M. B. Tomas, G. G. Tronco, and C. J. Palestro, “FDG PET of infection and inflammation,” Radiographics, vol. 25, no. 5, pp. 1357–1368, 2005.
View at: Publisher Site | Google Scholar
M. J. Kim, C.-H. Lee, Y. Lee et al., “Glucose-6-phosphatase expression–mediated [18F] FDG efflux in murine inflammation and cancer models,” Molecular Imaging and Biology, vol. 21, no. 5, pp. 917–925, 2019.
View at: Publisher Site | Google Scholar
D. Pellegrino, A. A. Bonab, S. C. Dragotakes, J. T. Pitman, G. Mariani, and E. A. Carter, “Inflammation and infection: imaging properties of 18F-FDG–labeled white blood cells versus 18F-FDG,” Journal of Nuclear Medicine, vol. 46, no. 9, pp. 1522–1530, 2005.
View at: Google Scholar
R. M. Epand and H. J. Vogel, “Diversity of antimicrobial peptides and their mechanisms of action,” Biochimica et Biophysica Acta (BBA)-Biomembranes, vol. 1462, no. 1-2, pp. 11–28, 1999.
View at: Publisher Site | Google Scholar
M. Zasloff, “Antimicrobial peptides of multicellular organisms,” Nature, vol. 415, no. 6870, pp. 389–395, 2002.
View at: Publisher Site | Google Scholar
M. M. Welling, P. H. Nibbering, A. Paulusma-Annema, P. S. Hiemstra, E. K. Pauwels, and W. Calame, “Imaging of bacterial infections with 99 mTc-labeled human neutrophil peptide-1,” Journal of Nuclear Medicine, vol. 40, no. 12, pp. 2073–2080, 1999.
View at: Google Scholar
M. A. Sellmyer, I. Lee, C. Hou et al., “Bacterial infection imaging with [18F]fluoropropyl-trimethoprim,” Proceedings of the National Academy of Sciences, vol. 114, no. 31, pp. 8372–8377, 2017.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2019 Tolulope A. Aweda et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Contrast Media & Molecular Imaging

In Vivo Imaging of Inflammation and Infection 2019

Radiolabeled Cationic Peptides for Targeted Imaging of Infection

Abstract

1. Introduction

2. Materials and Methods

2.1. Synthesis and Structural Analysis of HLys-DOTA and AB1-HLys-DOTA Peptides

2.2. Production of 64Cu

2.3. Radiolabeling of HLys-DOTA and AB1-HLys-DOTA Peptides

2.4. Serum Stability Studies

2.5. In Vitro Studies

2.5.1. Bacterial Cell Culture

2.5.2. Uptake Studies of 64Cu-Labeled Peptides in Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA)

2.5.3. Small Animal Imaging and Biodistribution Studies

2.5.4. Infection Animal Model

2.5.5. Biodistribution Studies

2.5.6. PET/CT Imaging Studies

2.6. Statistical Analysis

3. Results

3.1. HLys-DOTA and AB1-HLys-DOTA Peptide Synthesis, Characterization, and Stability

3.2. Stability and In Vitro Cell Uptake Studies

3.3. Confirmation of Infection in Animal Model

3.4. Biodistribution Studies

3.5. PET/CT Imaging

4. Discussion

5. Conclusion

Data Availability

Conflicts of Interest

Acknowledgments

Supplementary Materials

References

Copyright

2.2. Production of ⁶⁴Cu

2.5.2. Uptake Studies of ⁶⁴Cu-Labeled Peptides in Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA)