Azido-modified D-alanine for click chemistry labeling

Characterization of Sulfo-DBCO-ICG Binding to 3-azido-d-alanine/D-AzAla-Bacteria [1]
Sulfo-DBCO-ICG was functionalized by conjugated with d-AzAla-bacteria. Therefore, we tested if sulfo-DBCO-ICG was effectively coated on 3-azido-d-alanine/d-AzAla-bacteria by confocal laser scanning microscope (CLSM), flow cytometry, and other experiments. As shown in Figure 2A, sulfo-DBCO-ICG-bacteria had ICG fluorescence detected by CLSM. The same result was confirmed by a flow cytometry assay. Strong ICG fluorescence signals of sulfo-DBCO-ICG-bacteria were tested with the flow cytometer, suggesting that ICG successfully reacted with d-AzAla-bacteria in contrast to only bacteria (without ICG fluorescence) (Figure 2B). As shown in Figure 2C, the UV-vis absorption spectra of sulfo-DBCO-ICG effectively coated on d-AzAla-bacteria exhibited a slightly red-shift compared with pristine ICG from 779 to 795 nm mainly attributed to the interactions between sulfo-DBCO-ICG and d-AzAla-bacteria. ICG can produce heat upon 808 nm NIR irradiation, which can make bacteria photothermal lysis. The photothermal performance of only bacteria and sulfo-DBCO-ICG-bacteria was investigated under an 808 nm laser. The maximum temperature of ICG-bacteria reached 57.2°C for 90 s irradiations, whereas the temperature of only bacteria was 26.7°C (Figure 2D). Next, we briefly verified whether sulfo-DBCO-ICG heat production could cause the effective release of ATP after bacterial lysis by an ATP detector. From Figure 2E, after NIR irradiation, the sulfo-DBCO-ICG coated on the d-AzAla-bacteria group released a large amount of ATP, while the other groups released almost low ATP due to ICG producing thermal-induced bacteria cracking under the 808 nm NIR irradiation. The morphological changes of ICG-bacteria under the 808 nm infrared laser irradiation were observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). As shown in Figures 2F and G, the morphology of sulfo-DBCO-ICG-bacteria was changed and lysed after irradiated 808 nm NIR Figures 2F and G (b). As shown in Figures 2F and G (a) bacteria were not bound to DBCO-ICG, and the morphology of the bacteria did not change with or without NIR irradiation. It also indirectly proved that the morphological change of bacteria is very slight or even below 808 nm infrared laser irradiation. All these abovementioned results suggest that sulfo-DBCO-ICG effectively coated on d-AzAla-bacteria and could produce heat to crack bacteria for releasing ATP under 808 nm NIR irradiation.

Then, evaluation of targeting ability was used in the in vivo imaging assays. In the verification of the target experiment, four groups were used to treat MRSA-infected mouse wounds: PBS, Free-ICG + 3-azido-d-alanine/d-AzAla, DBCO-ICG, and DBCO-ICG + d-AzAla. They were photographed in animal imagers at 0.5, 3, and 6 h after intravenous administration of several ICGs to observe the targeting impact of each treatment. As shown in Figures 5D and E, the DBCO-ICG + d-AzAla group markedly increased the distribution of encapsulated fluorophore in MRSA-infected skin compared with Free-ICG + d-AzAla group at each time point. In particular, the strongest fluorescence intensity could be observed in the DBCO-ICG + d-AzAla group at 3 h point (**p = 0.0036). We also discovered that Free-ICG in the body is not only untargeted but has a quick metabolism, which is consistent with the literature (Lim et al., 2014). On the contrary, DBCO-ICG fluorescence still appeared around the infected wound after 6 h, indicating that we added a DBCO group to Free-ICG, which not only gives ICG a click chemical reaction with d-AzAla but also prolongs ICG metabolism in the body. Next, we irradiated the infected wound of the MRSA-infected model with near-infrared after 3 h of administration to see if it could efficiently produce heat. As shown in Figure 5F, it was observed that after 180 s of near-infrared light in the DBCO-ICG + d-AzAla group, the temperature of the infected skin on the back of the mouse could reach 46°C, and bacteria could be effectively lysed at this temperature (Yang et al., 2019; Shen et al., 2020), but the temperature of other groups only increased to about 38°C. The above findings show that DBCO-ICG could effectively target the infection wound in the MRSA-infected model and could attain temperatures high enough to kill bacteria when exposed to 808 nm NIR irradiation. [1]

---

In Vivo Antibacterial and Biocompatibility [1]
After confirming drug targeting and thermogenesis in vivo, we began to evaluate the antibacterial efficacy of DBCO-ICG in MRSA-infected mice wounds. Dorsal wounds induced on the back of mice were topically treated with PBS + NIR, DBCO-ICG (D-ICG) + NIR, Free-ICG + 3-azido-d-alanine/d-AzAla (F-ICG + d) + NIR, and DBCO-ICG + d-AzAla (D-ICG + d) + NIR, respectively. The cut injury of mice and the subsequent MRSA incubation were conducted as shown in Figure 6A. We evaluated the antibacterial therapeutic effect of the drugs in each group based on the wound healing and the number of bacteria on the back skin wounds after 2 weeks. As shown in Figures 6B and C, we could see that the back wounds of all kinds of mice healed after treatment, but the degree of healing was not consistent. The D-ICG + d treated group noticeably had less scar, which was much better than other groups, suggesting that the D-ICG + d treated group could distinctly accelerate wound healing and had the best bactericidal effect. Then, we also could see that there was no difference between the treatment effects of groups F-ICG + d and D-ICG, and the effect was not as good as the D-ICG + d group. This phenomenon suggests that Free-ICG was not targeted in vivo, could not form an azide reaction with amino acids on the surface of bacteria, and also reflects the poor antibacterial effect of DBCO-ICG alone. It must allow bacteria to wear ICG clothes and fluorescence double detections and PTAT for bacterial infection. DBCO-ICG reacted with d-AzAla modified bacteria by copper-free click chemistry to achieve ICG-coated bacteria. Meanwhile, on day 14, we conducted another investigation utilizing the agar plate dilution method to detect the number of bacteria on the wound skin tissues. There is no doubt that D-ICG + d treated group had exceptional antibacterial activity; the D-ICG + d group was able to destroy around 95% of MRSA (***p < 0.001) (Figures 6D and E). In addition, the wound that had been treated differently were stained with H&E and photographed. Figure 6F shows images of the four groups stained with H&E, and large inflammatory cells (neutrophils, red arrow) appeared in the others group. However, inflammatory cells were scarce in the D-ICG + d treated group. This result was consistent with our observations from H&E of normal skin tissue, which shows that the D-ICG + d group has excellent antibacterial properties and can promote wound healing. Finally, the systemic biosafety of the DBCO-ICG was assessed in vivo. Compared with the PBS group, the H&E staining of the main organs treated with Free-ICG + d-AzAla, DBCO-ICG, and DBCO-ICG + d-AzAla had no noticeable tissue damage and changes in morphology, indicating that the DBCO-ICG above had no obvious biological toxicity (Supplementary Figure S3). These results demonstrate that DBCO-ICG showed an effective antibacterial effect in the MRSA-infected model, and no appreciable toxic side effects observed were induced by the DBCO-ICG treatments.

Confocal Laser Scanning Microscope and Flow Cytometry of Sulfo-DBCO-ICG-bacteria [1]
Bacterial cell suspensions were diluted to obtain cell samples containing 1 × 106 to 1 × 107 colony-forming units (CFU) mL−1. In 1.5 ml microcentrifuge tubes, we mixed 250 μl of bacterium suspensions with 40 μl of 1 mg ml−1 DBCO-ICG and 100 μl of 3-azido-d-alanine/d-AzAla. Then, the mixture (DBCO-ICG-bacteria) was incubated in an incubator in the dark at 37°C for 1 h. Ten microliters of the stained bacterial suspension were trapped on an 18 mm square coverslip. We observed them in confocal laser scanning microscopy. In the same method, DBCO-ICG-bacteria were fixed in 4% paraformaldehyde. All samples were run on LSRFortessa, and the data were analyzed using FlowJo v9.9.8. The excitation/emission of the dyes was 780–810 nm for the ICG.

---

ATP Bioluminescence-Based Bacterial Detection Following Photothermal Lysis [1]
Bacteria (200 μl) prepared were mixed with 3-azido-d-alanine/d-AzAla and DBCO-ICG successively and then incubated at room temperature for 30 min. Thereafter, solutions were irradiated for 90 s using an 808 nm laser (1.0 W cm−2). The resulting solution was added to a luciferin−luciferase solution, and bioluminescence was immediately measured using a microplate reader. The bioluminescent signal was measured in less than 30 s.

Establishment of MRSA-Infected Mouse Models [1]
On day 4 before the infection, the mice were administered one dose of CTX. 150 mg CTX per kg mouse body weight (150 mg kg−1) was injected i. p. This treatment fostered a more vulnerable environment in the mice to infection. The mice were anesthetized using a standard anesthesia procedure, and then the dorsal region of the mice was shaved to prepare for surgery. Then one round wound of 80 mm in diameter was made using a puncher on the left back of mice weighing 30–40 g with 5 ICR male mice in each group. After 6 h, 50 μl of MRSA (1 × 108 CFU ml−1) was slowly added to each wound.

---

In Vivo Antimicrobial Assay [1]
After the establishment of MRSA-infected mouse models, the infection sites were treated with drugs and the same volume of PBS via the tail vein. PTAT treatment was conducted by irradiating the infection sites with laser irradiation (808 nm, 1.0 W cm−2) for 180 s. The infection sites were treated with PTAT solutions every 2 days. The regeneration process of wounds was studied through wound area monitoring and histomorphological determination. In wound size measurement, the mice in each group were anesthetized, and the wound size was determined by tracing the boundaries of wounds on days 3, 7, 11, and 14. For histomorphological evaluation, wound tissue of day 14 was collected for biochemical analysis. The samples were made into 0.5 cm2 square shape and immersed in standard formalin solution. Then tissue samples were conducted with H&E staining and prepared into a wax section for observation.

[1]. Bacteria Wear ICG Clothes for Rapid Detection of Intracranial Infection in Patients After Neurosurgery and Photothermal Antibacterial Therapy Against Streptococcus Mutans. Front Bioeng Biotechnol. 2022 Jul 6;10:932915.

Bacterial infection is one of the most serious physiological conditions threatening human health. There is an increasing demand for more effective bacterial diagnosis and treatment through non-invasive approaches. Among current antibacterial strategies of non-invasive approaches, photothermal antibacterial therapy (PTAT) has pronounced advantages with properties of minor damage to normal tissue and little chance to trigger antimicrobial resistance. Therefore, we developed a fast and simple strategy that integrated the sensitive detection and photothermal therapy of bacteria by measuring adenosine triphosphate (ATP) bioluminescence following targeted photothermal lysis. First, 3-azido-d-alanine (d-AzAla) is selectively integrated into the cell walls of bacteria, photosensitizer dibenzocyclooctyne, and double sulfonic acid-modified indocyanine green (sulfo-DBCO-ICG) are subsequently designed to react with the modified bacteria through in vivo click chemistry. Next, the sulfo-DBCO-ICG modified bacteria under irradiation of 808 nm near-infrared laser was immediately detected by ATP bioluminescence following targeted photothermal lysis and even the number of bacteria on the infected tissue can be significantly reduced through PTAT. This method has demonstrated the ability to detect the presence of the bacteria for ATP value in 32 clinical samples. As a result, the ATP value over of 100 confirmed the presence of bacteria in clinical samples for 22 patients undergoing craniotomy and ten otitis media patients. Overall, this study paves a brand new avenue to facile diagnosis and a treatment platform for clinical bacterial infections.[1]

---

In summary, our study developed a simultaneous detection and antibacterial platform for bacterial infection. It is a fast and simple strategy for the sensitive detection of bacteria by measuring ATP bioluminescence following targeted photothermal lysis. Sulfo-DBCO-ICG reacted with 3-azido-d-alanine/d-AzAla modified bacteria by copper-free click chemistry to complete a precise and rapid target. The sulfo-DBCO-ICG effectively coated on d-AzAla-bacteria and could produce heat to crack bacteria for releasing ATP below 808 nm NIR irradiation in vitro. The sulfo-DBCO-ICG could rapidly detect intracranial infections in patients after neurosurgery through ATP detection and flow cytometry. The sulfo-DBCO-ICG has targeting ability, good antibacterial effect, and biocompatibility in the MRSA-infected model. This method is faster than the clinical microbiological culture process, does not cause any drug-resistance as caused by antibiotics, and is not toxic/harmful to healthy cells. It may provide a good idea for the clinical rapid detection of bacteria.[1]

C3H6N4O2

130.10533952713

130.049

105928-88-9

3-Azido-D-alanine hydrochloride; 1379690-01-3

55785

Typically exists as solid at room temperature

-2.5

2

5

3

9

150

0

OC(C(CN=[N+]=[N-])N)=O

CIFCKCQAKQRJFC-UHFFFAOYSA-N

InChI=1S/C3H6N4O2/c4-2(3(8)9)1-6-7-5/h2H,1,4H2,(H,8,9)

2-amino-3-azidopropanoic acid

D-Alanine, 3-azido-; 2-amino-3-azidopropanoic acid; D-Azidoalanine; DL-Azidoalanine; 3-Azidoalanine; 105928-88-9; 88192-18-1; 108342-09-2;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)