Protocol of Whey Protein Isolate–Based Microgel Targeted Delivery in Mouse Kidney

Materials and reagents

Reagents

1. Linseed oil OLEOS (Russia) (unrefined cold pressed linseed oil, refractive index n20/D 1.4795, density 0.93 g/mL at 25 °C, commercial use)

Note: It is possible to use linseed oil from another manufacturer. However, it is worth paying attention to its properties (the refractive index n20/D of 1.4795 and the density of 0.93 g/mL at 25 °C).

2. Whey protein isolate (WPI) California Gold Nutrition (USA), iHerb, https://www.californiagoldnutrition.com/products/california-gold-nutrition-sport-whey-protein-isolate-unflavored-5-lb-2-27-kg-76479 (27 g of protein, 6.2 g of BCAAs, 4.7 g of glutamic acid, low lactose; lactose is used as an excipient, which is not involved in the formation of emulsion microgels; no additives or flavor enhancers)

Note: It is important to pay attention to the content of additional proteins in the WPI composition (such as BCAAs and glutamic acid) since this can affect the properties of the resulting emulsion microgels. It is also worth avoiding the presence of flavor enhancers and additives in the WPI composition.

3. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number S9888)

4. Cyanine 7 NHS-ester (Cy7), analytical reagent grade; reagent for research and development. Quality control: NMR 1H, HPLC–MS (95%) (Lumiprobe, catalog number 65020)

5. Phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number P4417)

6. Dimethyl sulfoxide (DMSO) (EcoChemAnalyt, Russia, CAS: 67-68-5)

7. Sodium hydroxide (NaOH) (EcoChemAnalyt, Russia, State Standard 4328-77, CAS: 1310-73-2)

8. Nile red (Sigma-Aldrich, catalog number: 72485)

9. Milli-Q water (Millipore filtration system) (used in all experiments)

10. Zoletil (Virbac, catalog number: L042-00118-31/00014469)

11. Xylazine (Alfasan International, catalog number: 528-3-7.16-3256№PBI-3-8.0/03450)

Solutions

1. 5% WPI aqueous solution (see Recipes)

2. Saline, 0.9% NaCl solution (see Recipes)

3. 5 mg/mL Cy7 solution (see Recipes)

4. 1 M NaOH solution (see Recipes)

5. PBS solution (0.1 M, pH 8.3) (see Recipes)

Recipes

1. 5 % WPI (w/v) aqueous solution

Reagent	Final concentration	Amount
WPI	5 %	500 mg
Saline 0.9% NaCl solution (Recipe 2)	0.9% (w/v)	10 mL
Total	n/a	10 mL

Caution: The solution must be mixed thoroughly. Wait until the foam disappears before use.

Note: For the conjugation procedure, protein solutions are prepared in PBS (pH 8.3) in equivalent amounts.

2. Saline, 0.9% NaCl (w/v) solution

Reagent	Final concentration	Amount
NaCl	0.9% (w/v)	9 g
H2O	n/a	1 L
Total	n/a	1 L

3. 5 mg/mL Cy7 solution

Reagent	Final concentration	Amount
Cy7	5 mg/mL	5 mg
DMSO	n/a	1 mL
Total	n/a	1 mL

Note: Due to the difficulty in weighing a 5 mg sample, it is advisable to prepare a larger quantity of the Cy7, which necessitates a larger volume of DMSO to create the final solution with the desired Cy7 concentration of 5 mg/mL. To achieve this, you must proportionally increase the mass of the Cy7 and the volume of DMSO. For instance, weigh out 25 mg of Cy7 and then add DMSO until the volume reaches 5 mL. This will result in a final Cy7 solution of 5 mg/mL, in a volume of 5 mL.

4. 1 M NaOH solution (100 mL)

Reagent	Final concentration	Amount
NaOH	1 M	0.4 g
H2O	n/a	100 mL
Total	n/a	100 mL

5. PBS solution (0.1 M, pH 8.3)

Reagent	Final pH	Amount
PBS	pH 7.2	1 tablet
H2O	n/a	200 mL
Total	n/a	200 mL

Note: Adjust the pH of the resulting solution to 8.3 using NaOH 1 M.

Laboratory supplies

1. Centrifuge tubes with flat cap, 15 mL (JetBioFil, catalog number: CFT550150)

2. Centrifuge tubes with flat cap, 50 mL (JetBioFil, catalog number: CFT500500)

3. Microcentrifuge tubes, 1.5 mL (JetBioFil, catalog number: CFT000015)

4. Microcentrifuge tubes, 2.0 mL (JetBioFil, catalog number: CFT000020)

5. Laboratory glass jar 100 mL, with divisions, with screw lid, dark glass (MiniMed, catalog number: 10007205)

6. Vial 2 mL, dark glass (ALWSCI Technologies, catalog number: C0001177)

7. Vial 10 mL, clear glass, 22 mm × 52 mm (ALWSCI Technologies, catalog number: C0000053)

8. Dialysis bag M-Cel, pore diameter 14 kDa (Viscase, catalog number: 2141-1425)

9. Dialysis bag clamp (Scienova GmbH, catalog number: 40329)

10. Laboratory beaker, clear glass, 2 L (MiniMed, catalog number: 10003807)

11. Glass for microslides (MiniMed, catalog number: 12003421)

12. Cover glass for microslides (MiniMed, catalog number: 12003309)

13. Syringe 1 mL (BD Micro-Fine Plus, catalog number: 320935)

14. Pipette microtips, 2–20 μL (JetBioFil, catalog number: PPT100020)

15. Pipette microtips, 10–200 μL (JetBioFil, catalog number: PPT000200)

16. Pipette microtips, 100–1,000 μL (JetBioFil, catalog number: PPT000000)

Equipment

1. Bandelin Sonopuls HD 2070 homogenizer at a frequency of 20 kHz and a power density of 1 W/cm² (Germany)

2. Magnetic stirrer (IKA, model: 0025004132)

3. Multifunctional refrigerated centrifuge (Eppendorf, model: 5810R)

4. Inverted confocal microscope (Leica Microsystems, model: Leica TCS SP8 X)

5. Inverted microscope, 40× objective (Olympus, model: IX73)

6. Single-channel variable volume dispenser 100–1,000 mL, 10–100 mL, 20–200 mL, 5–50 mL, and 1,000–5,000 mL (Thermo Fisher Scientific)

7. IVIS SpectrumCT imaging system (PerkinElmer, Waltham, MA, USA)

8. Water purification system for ultrapure water (Merck Millipore, model: Milli-QTM Advantage A10TM)

Software and datasets

1. ImageJ software 1.51j8 (National Institutes of Health, USA) (the open-source version is available online for free for download)

2. OriginPro 2018 SR1 (OriginLab Corporation, USA)

3. Excel 2019 (Microsoft Corporation, USA)

4. LAS X Life Science Microscope Software (Leica Microsystems, Germany)

5. Living Image 4.7.3 software (PerkinElmer, USA)

Procedure

A. Conjugation of WPI by Cy7

1. Create a solution of 5% WPI in PBS (0.1 M, pH 8.3). Dissolve 1 g of WPI in 20 mL of PBS.

2. Prepare a 0.5 mL solution of Cy7 in DMSO (5 mg/mL) in a dark glass vial.

3. Combine the WPI and Cy7 solutions in a dark glass vial. Stir the resulting mixture at 900 rpm using an IKA magnetic stirrer for 24 h in the dark at 4 °C (Figure 1).

4. Place the prepared WPI-Cy7 solution in a dialysis bag (pore size 14 kD). Submerge the dialysis bag containing the WPI-Cy7 solution in 2 L of deionized water and stir at 300 rpm for 48 h in the dark at 4 °C.

5. Determine the Cy7 dye concentration in the freshly prepared WPI-Cy7 solution by the spectrophotometric method. To do this, measure the optical density of the WPI-Cy7 solution at a 671 nm wavelength. The amount of Cy7 is determined on the basis of the optical density value at 671 nm using the corresponding calibration curve for Cy7. See our previous protocol for detailed instructions for obtaining calibration curves for a fluorescent dye (DOI: 10.21769/BioProtoc.5027, Section E, p. 4).

6. Store 20.5 mL of the freshly prepared Cy7-conjugated WPI in a refrigerator, away from light.

Caution: Fluorescent dyes are light-sensitive, so any experiments involving proteins labeled with fluorescent markers must be conducted in dark containers or with aluminum foil covers. The Cy7 concentration should be optimized and controlled in experiments for consistent fluorescence intensity, particularly when different batches of fluorescent dye are used.

Figure 1. Scheme of labeling whey protein isolate with Cyanine 7 NHS ester produced by Lumiprobe

B. Formation of WPI-based emulsion microgels

1. Place 2 mL of 5% WPI-Cy7 solution in a 20 mL glass beaker.

2. Add 0.3 mL of linseed oil to the 2 mL of WPI-Cy7 solution. This corresponds to a ratio of WPI solution:oil (w:w) of 1:3.

Caution: It is crucial to adhere to the precise proportions of the WPI and oil phases, as the size of the resulting microgels will be significantly influenced by this ratio.

Note: For fluorescent visualization of emulsion microgels, Nile red was dissolved with linseed oil to a final concentration of 1 mg/mL.

a. Immerse the probe of the ultrasonic homogenizer into the resulting mixture of WPI solution and oil. Apply ultrasound at a frequency of 20 kHz and a power density of 1 W/cm² for 1 min to obtain the emulsion microgels (Figure 2A).

b. Transfer the resulting emulsion microgels to 2 mL Eppendorf tubes and centrifuge at 10,500× g for 5 min.

c. Carefully collect the infusion fluid using a syringe.

d. Then, wash emulsion microgels with saline solution twice according to sections D and E.

e. Store the prepared emulsion microgels in 2 mL of saline solution in a refrigerator at 4 °C.

Figure 2. Method for synthesizing emulsion microgels and characterizing them using microscopy. (A) Scheme of the synthesis of emulsion microgels using whey protein isolate (WPI) labeled with Cy7 fluorescent dye and linseed oil containing Nile Red dye. (B) Visual representations of Cy7-WPI and linseed oil containing Nile Red solution before and after ultrasonic mixing. (С) Split channel images of emulsion droplets, demonstrating oil core stained with Nile Red (red color staining) and Cy7-WPI (green color staining), located at the oil–water interface and indicating WPI stabilization of emulsions. (D) Optical image of emulsion microgels. Scale bar, 50 μm.

Note: The reproducibility of the results depends on the amount of encapsulated substances and the size of the resulting microgel. The loading capacity of the microgel can be determined by a spectrophotometric method by maximum absorption in accordance with the protocol [10.21769/BioProtoc.5027].

C. Confocal laser scanning microscopy

1. Dilute the initial emulsion microgel sample 1,000× with saline (0.15 M NaCl). Place the 5 μL diluted emulsion microgel on a glass slide and cover with a coverslip.

2. Observe the fluorescent properties of the prepared emulsion samples using a Leica TCS SP8 X inverted confocal microscope equipped with diode-pumped solid-state lasers (561 nm and 671 nm) focused through a 20×/0.70 N.A. objective (Figure 2B).

3. The Cy7 fluorescence is stimulated by a laser with a wavelength of 671 nm, and the resulting emission is measured in the range of 730–795 nm (green channel, Figure 2B).

4. The Nile Red fluorescence is activated by a laser with a wavelength of 561 nm, and the resulting emission is measured in the range of 580–650 nm (red channel, Figure 2B).

5. Use the LAS X Life Science Microscope Software for image processing.

D. Optical microscopy and determination of emulsion microgel sizes

1. Measure the diameters of the emulsion microgels using the optical images of the samples.

2. For the sample preparation process for optical microscopy, follow a similar procedure to that of scanning laser microscopy, as described in section C1.

Obtain optical images of emulsion microgels with the Olympus I × 73 inverted microscope and a 40× objective.

4. Measure the diameters of the emulsion microgel particles with the optical images obtained in the previous step using measurement tools of the ImageJ software.

5. For the calculation of the average diameter of the emulsion microgel, at least 100 measurements and 10 images for each sample are analyzed. Present the average diameter of particles as mean ± standard deviation.

6. In our case, the microgels were predominantly composed of particles with an average diameter of 2.2 ± 0.3 μm.

E. Preparing mice for intravenous injection

1. Administer general anesthesia to a mouse by intraperitoneal injection using a mixture of Zoletil (40 mg/kg) and 2% Xylazine (10 mg/kg). We carried out this experiment with Balb/c mice (age 6–8 weeks, weight 20–25 g).

Note: Alternatively, 2% Rometar can be used instead of Xylazine.

2. After 5–7 min, check for the reflex response of the hind limbs. If there is no hind limb reflex, proceed with manipulation. If the reflex is still present, wait a few more minutes before continuing.

3. Place the mouse on the desk and use a trimmer to shave off all the fur from its body, except for the head.

4. Apply a depilatory cream designed for sensitive skin. Carefully remove the cream using cotton pads soaked in saline or water.

Note: Be sure not to leave the cream on the skin for more than 30–60 s to avoid any potential harm.

F. Tail vein injection of emulsion microgel suspension and lifetime bio-visualization

1. To prepare the tail for injection, it is important to submerge it in water at 42 °C for approximately 20–30 s. After that, the tail should be dried with a paper towel to remove any excess moisture.

Note: This will help dilate the blood vessels in the tail.

Caution: Locate the tail vein by following the middle dorsal side of the tail. The left and right tail veins are located on either side of the artery. These veins are the targets for administration. However, pigmentation on the tail may obscure some blood vessels, so it is important to be careful when locating them.

2. After locating the vein, slowly insert the needle of the syringe (30 G) at a slight angle, about 5 cm from the tip of the tail (at an angle of 10–15°).

3. Slowly press down on the plunger of the syringe, injecting 50 μL of microgel suspended in saline solution at a concentration of 5 × 10⁷ microgels. Inject at a constant rate of about 30 μL/s to prevent damage to the blood vessels. A graphical representation of the in vivo experiment is shown in Figure 3A and Video 1.

4. Place the animal, positioned on a dark background, in the IVIS SpectrumCT imaging system, using an excitation/emission wavelength of 745/800 nm. Observe each mouse before and at specific times after the administration (10 min, 1 h, 5 h).

5. Analyze the results using the Living Image 4.7.3 software (Figure 3B).

Figure 3. Administration of the suspension into the tail vein of mice. (A) Schematic process of the biodistribution of the fluorescent signal from the WPI-Cy7 dye, which is immobilized in emulsion microgel, after intravenous injection into the tail vein of a mouse. (B) Microgel administration into the mouse’s tail vein at a 10–15° angle. (C) Dynamics of biodistribution of microgel suspensions stabilized with WPI-Cy7 after intravenous administration in vivo in the dorsal (right) and ventral (left) positions for each time point.

Data analysis

Calculation of droplet size of emulsion microgels

To determine the mean size of emulsion droplets, at least 100 measurements were taken with the use of 10 images per sample. ImageJ software was used for image processing and data analysis. First, an optical image of the emulsion microgel was imported into the ImageJ program. Next, the image scale was calibrated in terms of length. Then, using the linear measurement tools available within the program, the diameter of each emulsion droplet was measured directly on the image. Based on the 100 measurements, the average particle diameter and standard deviation were calculated using statistical methods in Excel. Data should be presented as the mean ± standard deviation. Considering the 5% WPI concentration and the 1:3 WPI solution to oil ratio used in the microgels synthesis, we expect the average size of the microgels to be approximately 3.5 ± 0.2 μm. To compare the average particle sizes of different types of emulsion microgels, ANOVA can be used. If the p-value is less than 0.05, the differences are considered statistically significant. To ensure the reproducibility of data analysis and to make it more transparent, we have identified outliers using Grubbs' test statistical method.

Semi-quantitative measurement of the quantity of Cy-WPI following intravenous injection ex vivo

The Living Image 4.7.3 software was used to analyze the distribution of the fluorescent signal in the organs of mice (n = 3) after intravenous administration of microgels. The total and average radiation efficiency values were determined for each region of interest (ROI), which represents the boundaries of the organs (bladder, left and right kidney, lungs, heart, spleen, liver, stomach, intestines, and appendix) ex vivo (Figure 4A).

The total radiation efficiency is the total number of photons per second emitted from a selected area and is normalized by the intensity of illumination. The average radiation efficiency is the number of photons per square centimeter that leave the tissue and are emitted at a solid angle of one steradian, again normalized by the incident radiation intensity. For each organ, the results are presented as an average radiation efficiency value, from which the control group's average values have been subtracted (Figure 4B). Organ autofluorescence indices were analyzed similarly using control (intact) animals (n = 3) without the introduction of fluorescent microgels.

Figure 4. Analysis of the distribution of microgels in various organs and the dynamics of their biodistribution. (A) Representative image of the ex vivo biodistribution analysis process, with regions of interest (ROIs) allocated for each organ: bladder, left kidney, right kidney, lungs, heart, spleen, liver, stomach, intestines, and appendix. (B) The ROI Measurements tab displays data for all ROIs created in images or sequences during the session (one ROI per row). The table provides a convenient way to review and export ROI data. (C) Dynamics of biodistribution of microgel suspensions stabilized by WPI-Cy7 at 5 h after intravenous administration ex vivo. Fluorescent images of the kidneys of experimental animals 5 h after administration of the microgel suspension. * The unit of epi-fluorescence is [p/s]/[μWcm²]. All data are presented as the mean ± SD, n = 3.