Background

The prevalence of chronic kidney disease continues to steadily increase each year [1,2]. Delayed initiation of adequate therapy can lead to kidney failure [3,4]. Often, intensive systemic therapy causes complications in other weakened organs and systems, highlighting the need for targeted drug delivery directly to the affected organ to minimize systemic side effects of treatment. The method of administering drugs through the renal artery has the potential to concentrate therapeutic agents in the cargo, which could greatly enhance the treatment of chronic kidney disorders. However, this approach carries risks such as vascular damage, aortic blockage, and the need for highly trained personnel.

To address this issue and to reduce the necessary therapeutic doses, alternative addressing methods are being developed, such as micro-sized targeted delivery systems [5,6]. These systems can effectively regulate the distribution of encapsulated drugs in the organs, opening new horizons in the treatment of chronic kidney disease.

Currently, emulsion systems are actively being studied as a promising treatment for diseases of the genitourinary system [7]. One of the main advantages of these systems for targeted drug delivery is their ability to provide long-term release of both hydrophobic and hydrophilic drugs [7]. This is due to the presence of both aqueous and oil phases in the system.

Modification of emulsions with mucoadhesive components allows an increase in targeting of the drug delivery system, their retention on the mucous surface of organs, and, at the same time, the localization, safety, and effectiveness of the administered drug. The use of mucoadhesive emulsions is gaining popularity due to their unique combination of properties that make them ideal for delivering drugs directly to the mucosal surfaces. In our study, we use the naturally derived mucoadhesive component whey protein isolate (WPI). WPI, along with its efficient mucosal specificity, provides the formation of the hydrogel shell at the oil surfaces upon the ultrasonically stimulated denaturation. In this way, the emulsion microgel system is formed.

Recently, we described the protocol for synthesizing emulsion microgels with high mucosal adhesion properties to the urothelium of the bladder provided by the mucoadhesive component, WPI [8]. We provided precautions and nuances that should be paid attention to at each stage of the experiment. During the study, the main physico-chemical parameters of microgels, such as filling capacity and drug release rate, were studied. The processes of drug release, both from the oil core of microgels (hydrophobic drug) and from the hydrogel protein shell (hydrophilic drug), strongly depend on the ionic strength of the medium in which microgels are incubated [8–10]. It has been shown that the ionic strength affects the degradation rate of protein hydrogel shells and, thus, the overall stability of microgels. This, in turn, affects the drug release behavior. The high ionic strength of the medium causes the high hydrophobicity of the protein molecules in the shell, which, accordingly, increases the stability of the microgels. The stability of microgels in artificial urine (0.3 M) is higher than in saline solution (0.15 M). Correspondingly, in artificial urine, the drug release from microgels demonstrates a more prolonged behavior, as compared with saline 0.15 M NaCl. The amount of hydrophilic drug released from the protein shell reaches 45% at 72 h of incubation in artificial urine [8]. The biodistribution of emulsion microgels in vivo after intravesical administration showed that local delivery ensures effective accumulation of carriers in the bladder due to the high adhesive properties of the protein on the surface of the microgels.

Due to the possibility of varying the synthesis conditions to produce microgels with precisely specified physical and chemical properties, such as particle size, surface area, zeta potential, and drug loading efficiency, emulsion microgels have been studied as a potential drug delivery system for the kidneys [11]. Intravenous delivery of these microgels resulted in a long-term accumulation (at least 5 days) of these particles in the liver and kidneys, indicating a high efficiency of biodistribution and targeting to the target organ. The experimental conditions for synthesizing these microgels allow for the creation of particles with exactly the desired characteristics. The developed microgels provide a customized therapeutic approach to the treatment of kidney diseases, with reduced side effects. This opens up new possibilities for the systemic treatment of the urinary system.

This protocol provides detailed instructions on the synthesis, preparation, and research of fluorescently labeled emulsion microgels based on whey protein isolate. It allows researchers to accurately replicate these procedures and achieve the desired results.

The protocol includes the following steps:

1. Synthesis of fluorescently labeled microgels: This involves the preparation of a mixture of whey protein isolate and a fluorescent dye, which is then processed to create the microgel structure.

2. Preparation of samples for confocal microscopy: The microgels are prepared for analysis by confocal microscopy, which helps to visualize their structure and distribution.

3. Optical microscopy analysis: The stability of the microgels is assessed using optical microscopy, specifically focusing on the size and distribution of the emulsion droplets within the microgel.

4. In vivo imaging: A suspension of the microgels is injected into the tail vein of a mouse, allowing for the visualization of the distribution of the fluorescent dye signal in the animal's body using fluorescence imaging.

Overall, this protocol provides a comprehensive guide for researchers interested in studying fluorescently labeled emulsion microgels using whey protein as a basis. It ensures accurate and efficient sample preparation, making it a valuable tool for further research in this field.