Fluorescent Labeling and Imaging of IL-22 mRNA-Loaded Lipid Nanoparticles

Materials and reagents

Biological materials

1. C57BL/6J mice (Jackson Laboratory, female, 6–7 weeks of age)

Reagents

1. Curved feeding needles (Kent Scientific, catalog number: FNC-20-1.5-2)

2. 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide (DiR') (Thermo Scientific, Invitrogen^TM, catalog number: D12731)

3. Dimethyl sulfoxide (DMSO) (Fisher Scientific, catalog number: BP231-100)

4. Phosphate-buffered saline (PBS) (Corning, catalog number: 21-040-CV)

Laboratory supplies

1. 15 mL conical centrifuge tube (Thermo Scientific, Nunc^TM 15 mL, catalog number: 339650)

2. Amicon^® Ultra 15 mL centrifugal filter (Millipore, Ultracel^®-100k, catalog number: UFC910024)

3. 5 mL Eppendorf tube (Eppendorf, catalog number: 0030119401)

4. 1 mL syringe PP/PE without needle (Sigma-Aldrich, catalog number: Z683531-100A)

5. Petri dish (CELLTREAT, catalog number: 229620)

Equipment

1. Micropipettes, 10–100 µL (Eppendorf, catalog number: 13-684-251)

2. Orbital shaker (CORNING, model: LSE^TM)

3. Centrifuge (Thermo Scientific, model: SORVALL ST-16R)

4. In vivo imaging system (PerkinElmer, model: IVIS Spectrum CT)

Software and datasets

1. Living Image software (PerkinElmer, IVIS^® version: 4.7.4)

Procedure

1. Fluorescent labeling of IL-22 mRNA-loaded LNPs

1. Prepare 5 mL of IL-22 mRNA-loaded LNPs in a 15 mL conical centrifuge tube following the previously published protocol [5].

2. Dissolve 5 mg of DiR' powder in 1 mL of DMSO to make a DiR'/DMSO solution at a concentration of 5 mg/mL.

3. Add 10 µL of DiR'/DMSO solution to 5 mL of IL-22/LNPs in a 15 mL conical centrifuge tube (Figure 1).

   
   

   
   Figure 1. Spiking a 10 µL DiR' solution into the IL-22/LNPs suspension




4. Cover the tube with aluminum foil and incubate the suspension mix at room temperature (RT) for 15 min (Figure 2, left) shaking at 100 rpm on an orbital shaker.

   
   

   
   Figure 2. Incubating the lipid nanoparticles (LNPs) DiR' mix on an orbital shaker




5. Transfer the LNP suspension to 15 mL centrifugal filters, each containing 2.5 mL of LNPs (Figure 3).

   
   

   
   Figure 3. Transfer the lipid nanoparticles (LNP) suspension to a centrifugal filter




6. Centrifuge at 4,696× g for 10 min at 10 °C.

7. Take out the filter (Figure 4).

   
   

   
   Figure 4. DiR'-labeled lipid nanoparticles (LNPs) in the filter after centrifugation




8. Add 1 mL of PBS to each filter and reconstitute the suspension by repeated pipetting (> 100 times). Then, transfer the suspension to a 5 mL Eppendorf tube.




2. Oral gavaging of DiR'-labeled IL-22/LNPs

   1. Take healthy mice and divide them into two groups (Control group and Treated group).

   2. Fast the mice for 4 h before gavage.

   3. Fill DiR'-labeled IL-22/LNPs suspension into a 1 mL syringe equipped with an animal feeding needle and remove all air bubbles (Figure 5).

      
      

      
      Figure 5. Filling of DiR'-labeled IL-22/LNPs suspension into syringe

   
   

   4. Restrain each mouse by manually grasping it and carefully insert the feeding needle into its mouth and esophagus to administer 200 µL of DiR'-labeled IL-22/LNPs suspension directly into the stomach of the treated group. To the control group, administer 200 µL of free LNPs suspension (without labeling) (Figure 6).

      
      

      
      Figure 6. Gavaging of DiR'-labeled IL-22/LNPs suspension to mouse

      
      

      Note: If any resistance is experienced, it may indicate incorrect positioning of the feeding needle; in such cases, retract the feeding needle and reposition it.

   5. After gavage, fast the mice for 1 h.

   6. Euthanize mice 24 h after being gavaged with mRNA-loaded LNPs.

   7. Dissect the mice and collect their GI tract.




3. Imaging of DiR-labeled IL-22/LNPs biodistribution in mouse GI tract

   1. Turn on the IVIS Spectrum CT instrument 45 min prior to the test.

   2. Start Living Image software, click the initializing button, and wait until the temperature button turns green (Figure 7; CCD camera reaches -90 °C).

   3. Place the GI tract (with and without DiR-labeled IL-22/LNPs) in the imaging chamber.

   4. Set the imaging mode to fluorescence, excitation filter to 745 nm, and emission filter to 800 nm in the IVIS acquisition control panel (Figure 7).

   5. Take imaging by selecting Acquire in the IVIS acquisition control panel and save the acquired pictures to the appropriate path (Figure 8).

      
      

      
      Figure 7. Parameter for acquiring imaging of fluorescently labeled IL-22/LNPs

      
      

      
      Figure 8. Living Image software. Left side: GI tract of the control mouse without IL-22/LNPs treatment. Right side: DiR-labeled IL-22/LNPs deposition in the gastrointestinal (GI) tract of the treated mouse.

Validation of protocol

1. Sung et al. [4]. Oral delivery of IL-22 mRNA-loaded lipid nanoparticles targeting the injured intestinal mucosa: A novel therapeutic solution to treat ulcerative colitis. Biomaterials (Supplementary Figure 10, panel A).

Acknowledgments

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (RO1-DK-116306 to D.M.) and the Department of Veterans Affairs (Merit Award BX002526 to D.M.). D.M. is a recipient of a Senior Research Career Scientist Award (BX004476) from the Department of Veterans Affairs.

Competing interests

The authors declare no conflicts of interest within the work.

References

1. Yan, J., Yu, J., Liu, K., Liu, Y., Mao, C. and Gao, W. (2021). The Pathogenic Roles of IL-22 in Colitis: Its Transcription Regulation by Musculin in T Helper Subsets and Innate Lymphoid Cells. Front Immunol. 12: e758730.
2. Lindner, J. R. and Link, J. (2018). Molecular Imaging in Drug Discovery and Development. Circ: Cardiovasc Imaging. 11(2): e005355.
3. Abdel-Mottaleb, M. M., Beduneau, A., Pellequer, Y. and Lamprecht, A. (2015). Stability of fluorescent labels in PLGA polymeric nanoparticles: Quantum dots versus organic dyes. Int J Pharm. 494(1): 471–478.
4. Sung, J., Alghoul, Z., Long, D., Yang, C. and Merlin, D. (2022). Oral delivery of IL-22 mRNA-loaded lipid nanoparticles targeting the injured intestinal mucosa: A novel therapeutic solution to treat ulcerative colitis. Biomaterials. 288: 121707.
5. Alghoul, Z., Sung, J., Wu, K., Alpini, G., Glaser, S., Yang, C. and Merlin, D. (2023). Preparation and Characterization of IL-22 mRNA-Loaded Lipid Nanoparticles. Bio Protoc. 13(7): e4647.