2.1.

Laboratory Animals

A group of 2-year-old female rats totally 20 animals were kept 14 days; besides the standard diet, the rats received 5 g each of nutrients such as sugar, dry milk, sunflower oil, and egg powder to induce alimentary obesity. Animals were divided into two groups of 10 rats each. Hair was removed within the region of ribs on the back, where treatment was carried out, by the depilation cream Veet (Reckitt Benckiser, France). All procedures with animals were done in accordance with the European Convention for the Protection of Animals used for experimental and other scientific purposes.⁶⁰ The experimental study was conducted in the Centre of Collective Use of Saratov State Medical University (Russia) and according to the guidelines of the University’s Animal Ethics Committee (Russia) and the relevant national agency regulating experiments with animals.

2.2.

Agents

2.2.2.

Capsule preparation and characterization

ICG (SIGMA-ALDRICH Co., Germany) was encapsulated into degradable polyelectrolyte microcapsules fabricated by a layer-by-layer assembly technique. There are a number of studies indicating that degradable polyelectrolyte microcapsules themselves are suitable for subcutaneous drug delivery, since they show a moderate tissue reaction after the injection in mice.^61–63

The ICG-loaded calcium carbonate (${\mathrm{CaCO}}_3$) microparticles were fabricated using earlier designed technology.⁵⁷ 1-ml volumes of 1 M ${\mathrm{Na}}_2{\mathrm{CO}}_3$ and 1 M ${\mathrm{CaCl}}_2$ water solutions were quickly added to 5 ml of $1\mathrm{mg}/\mathrm{ml}$ ICG water solution in a glass vessel at room temperature and then mixed using the magnetic stirrer Mini MR Standard (IKA, Germany) at 500 rpm. The synthesized ${\mathrm{CaCO}}_3$ particles were washed twice with water and then used as templates for microcapsule shell formation. Polyelectrolyte shells were prepared by a self-assembly method using biocompatible degradable polymers (chitosan and dextran sulfate sodium salt) adsorbed layer-by-layer on the templates. The polyelectrolytes were deposited from $1\mathrm{mg}/\mathrm{ml}$ solutions in 0.15 M NaCl. After each deposition, the suspension was centrifuged for 1 min at 5000 rpm to separate it into fractions and remove supernatant liquid and then was triply washed with water. After consequent deposition of the eight polyelectrolyte layers, ${\mathrm{CaCO}}_3$ templates were dissolved in 2 M ethylenediaminetetraacetic acid and the suspension of microcapsules were triply washed with water, centrifuged at 7000 rpm for 5 min, and re-suspended in 1 ml of saline. Thus, ICG-filled biocompatible degradable polyelectrolyte microcapsules were formed.

To study the morphology and microstructure, a drop of the microcapsule suspension was put on the silicon wafer, dried, sputtered with gold, and imaged with scanning electron microscopes (SEM), a MIRA II LMU (Tescan) at acceleration voltage of 20 kV and a Phillips XL 30 at 5 to 30 kV. Performed SEM images of the ICG-loaded polyelectrolyte microcapsules are shown in Fig. 1. The images demonstrate a spherical shape of the capsules and their narrow size distribution of $3.5\pm 0.5\mu \mathrm{m}$.

Fig. 1

Scanning electron microscopy images of ICG-loaded polyelectrolyte microcapsules at different magnifications. The microcapsules have a narrow size distribution of $3.5\pm 0.5\mu \mathrm{m}$.

To estimate the ICG concentration in polyelectrolyte containers, the supernatant was collected at each polyelectrolyte deposition and washing step, and the absorption spectrum was examined with a Lambda 950 spectrophotometer (Perkin Elmer). The total amount of loaded molecules was deduced by subtracting the measured amount of unloaded and washed-off molecules of the supernatant from the initial amount of $0.5\mathrm{mg}/\mathrm{ml}$ of ICG, which had been added to the system. As a measure of the amount of ICG in solution, its optical density spectrum was recorded in the spectral range of 300 to 1000 nm. A calibration curve was obtained from the measurements of known concentrations of ICG in saline.

The resulting concentration of ICG in the saline suspension of obtained microcapsules was found to equal $0.5\mathrm{mg}/\mathrm{ml}$, meaning 1 pg of ICG per one polyelectrolyte microcapsule.

2.3.

Protocol of Experiment

The skin sites for each rat with obesity were demarcated into four zones. Each sampling zone was about 1 cm in diameter. Zone 1 was kept as a control zone without any staining or radiation. For zone 2, subcutaneous dye injection (free ICG or encapsulated ICG, $0.5\mathrm{mg}/\mathrm{ml}$) was performed: the ICG solution was applied to the rats of the first group, while the encapsulated ICG was injected into the rats of the second group. Zone 3 was exposed to radiation from a diode NIR laser (wavelength 808 nm, power density of $8\mathrm{W}/{\mathrm{cm}}^2$, exposure of 1 min). For zone 4, free ICG or encapsulated ICG injection of the same volume and concentration was followed by light irradiation. Anesthesia of rats was carried out with Zoletil (Vibrac, France). After 1 h elapsed since the 1-min light exposure, the rats were decapitated. The design of the experiment was compiled on the basis of the Guide for the Care and Use of Laboratory Animals.⁶⁴

2.4.

Histology

For histological studies, samples of skin with subcutaneous tissue of thickness 1.5 to 2.5 mm were taken by surgery from rats within the marked four-zones of the skin site. For histological examination of excised tissue samples, fixation was carried out by a 10% solution of formaldehyde. After fixation, the 5- to $7\text{-}\mu \mathrm{m}$-tissue slices across all layers of subcutaneous tissue were made, stained by hematoxylin-eosin using a standard technique, and then analyzed.⁶⁵ Morphological study was conducted independently by two experts in the field of pathological anatomy: one is a well-experienced postgraduate student and the other is a professor, doctor of medical sciences. Thus, the study was suited to a double-blind manner.

2.5.

Statistical Data Analysis

Statistical data analysis was performed using the “Statistics 6.0.” In the cases where the indication under study is not characterized by quantitative and qualitative variables, such as serial numbers, indices, signs “+” or “−” (binary scoring system), and so on, only nonparametric methods are possible. The scale of evaluation was dichotomous: any changes in subcutaneous adipose tissue were either present ($=1$) or not ($=0$) in the histological samples. The Cochran $Q$-test, a nonparametric test usually applied to the analysis of two-way randomized block designs where the response variable can take only two possible outcomes (coded as 0 and 1), was used.⁶⁶ The test provides a quick recognition of whether $k$ treatments have identical effects. It shows that the empirically found difference between the treatment alternatives is statistically significant.

3.1.

Histological Analysis of Control Samples

The samples were skin slices with a layer of subcutaneous adipose tissue. In the control samples (zone 1), no changes in the epidermis, dermis, and subcutaneous adipose tissue [Fig. 2(a)] were found. The epidermis is normally thin, and its cells do not have any degenerative changes. The dermis is represented by collagen fibers distributed in different areas and located close to each other. The contours of the fat cells are distinct. Muscle fibers with distinct cross striation are seen. Skin appendages are not changed.

Fig. 2

Histological (H&E staining) images of subcutaneous adipose tissue samples taken from in vivo treated rats (magnification $246.4\times$): (a) no treatment (control); (b) after injection of free-ICG solution ($0.5\mathrm{mg}/\mathrm{ml}$ in saline); (c) after injection of encapsulated ICG ($0.5\mathrm{mg}/\mathrm{ml}$ in saline); (d) hyperemia in the hypodermis in zone 3; (e) after 1-min 808-nm diode laser irradiation ($8\mathrm{W}/{\mathrm{cm}}^2$); (f) after free-ICG injection (ICG $0.5\mathrm{mg}/\mathrm{ml}$ in saline) and 1-min 808-nm diode laser irradiation ($8\mathrm{W}/{\mathrm{cm}}^2$); (g) edema with swelling of the certain muscle fibers in zone 4; (h) after encapsulated ICG injection (encapsulated ICG $0.5\mathrm{mg}/\mathrm{ml}$ in saline) and 1-min 808-nm-diode laser irradiation ($8\mathrm{W}/{\mathrm{cm}}^2$). Blue arrows—changed (thinned) membranes; red arrows—disrupted membranes.

3.2.

Histological Analysis of the Samples after the Injection of Agent

For the first group of rats, free ICG dissolved in saline was injected subcutaneously, while for the second group encapsulated ICG was applied. In the samples corresponding to zone 2, i.e., the subcutaneous injection of free-ICG [Fig. 2(b)] or encapsulated ICG [Fig. 2(c)], no essential morphological changes of epidermis and dermis were observed. No destruction of adipose tissue, even weakly expressed, was found. For the first group of rats, fat tissue was presented by cells with thinning and split boundaries of the external environment—the cell membrane-cytoplasm-fat drop. In the case of encapsulated ICG injection, no damage was noticed either. However, for some samples, delamination of cell membranes was found. This could be explained by the mechanical stress on cells caused by swelling.

According to the literature, a moderate tissue reaction is observed after subcutaneous injection of unloaded degradable polyelectrolyte microcapsules in mice.^61,62 An acute phase is characterized by recruitment of polymorphonuclear cells, and a more chronic phase is marked with environing of the injection site by fibroblasts during the microsphere phagocytosis.

3.3.

Histological Analysis of the Samples after Irradiation

For the samples corresponding to zone 3, after NIR diode laser irradiation (power density of $8\mathrm{W}/{\mathrm{cm}}^2$ and the exposure of 1 min), no significant changes were observed: just a little thinned out areas were found for the epidermis, while edema of the dermis was discovered. The irradiation leads to hyperemia in the hypodermis [Fig. 2(d)] of irradiated sites. Moreover, the signs of damage in adipose tissue were found: the boundary thinned and partly disrupted membranes [indicated by arrows in Fig. 2(e)] had a stretched shape, and membrane delamination was observed in some cases [Fig. 2(e)]. Changes of fat cells during 1 h of observation were characterized as a destruction process, which could potentially lead to lipolysis or necrosis.^67,68 NIR laser irradiation may stimulate an increase of permeability of fat cell membranes resulting in release of intracellular fat. The fatty triglycerides may flow out through the disrupted cell membranes into the interstitial space where they gradually pass through the lymphatic drainage system.⁶⁹

3.4.

Histological Analysis of the Samples after the Dye Subcutaneous Injection and Irradiation

In the samples with subcutaneous injection of the ICG solution and subsequent laser irradiation, definite signs of damage in adipose tissue were observed [Fig. 2(f)]. The epidermis was in a normal state, while a local hemorrhage in the dermis and a moderate infiltration of neutrophils and eosinophils in the hypodermis took place. Edema with swelling of certain muscle fibers was observed in the muscle tissue [Fig. 2(g)]. Adipose tissue had pronounced signs of damage seen as strongly stretched cells. Disrupted membranes [indicated by red arrows in Fig. 2(f)] were also found. The ICG tissue staining via subcutaneous injection and subsequent laser irradiation at a physiological constant temperature lead to the generation of oxygen radicals (superoxide and hydroxyl radicals) that causes a modification of fat cell phospholipid membranes and transmembrane cell lipolysis coming out as cell shape transformations.

In the case of encapsulated ICG-injection, we observed similar changes, but with weakly expressed cell destruction [Fig. 2(h)]. In the course of the photochemical/photothermal reaction, in addition to direct cell lipolysis, cell apoptosis can be induced when the microdamages of the cell membrane reach a critical value or strength.^70,71 Encapsulated ICG tissue treatment rendered a smaller damaging ability. However, such damages could also lead to cell lipolysis and/or apoptosis.^72,73

It is important to note that, for the samples used to demonstrate posteffects 2-weeks after laser irradiation of the stained tissue sites, significant changes were observed. Intact adipocytes were not found at the site. The fat tissue layer was completely destroyed, partly eliminated with some remaining necrotic masses, and replaced with connective tissue.

In our previous work, we investigated photochemical treatment using an ICG solution with a concentration of $1\mathrm{mg}/\mathrm{ml}$ dissolved in alcohol and 808-nm laser irradiation (power density of $16\mathrm{W}/{\mathrm{cm}}^2$ and the exposure time 1 min), which caused the pronounced changes in ex vivo adipose tissue accompanied by cell apoptosis and/or necrosis depending on the level of tissue damage.⁷⁴ It was established that application of the free ICG-water solution ($1\mathrm{mg}/\mathrm{ml}$) or encapsulated ICG-water solution ($2\mathrm{mg}/\mathrm{ml}$) as a photochemical dye in the ex vivo experiment increased the cell membrane permeability leading to lipolysis.⁷⁵ For tissue stained by the ICG-water solution, a total lipolysis was seen after 295 min of observation, while for tissue stained by an encapsulated ICG it happened much earlier, after 165-min elapsed.⁷⁵

In our previous work, we found that photochemical treatment in rats in vivo at subcutaneous injection of the ICG solution ($0.5\mathrm{mg}/\mathrm{ml}$ in saline or in alcohol mixture) and follow-up 808-nm-diode laser irradiation ($16\mathrm{W}/{\mathrm{cm}}^2$ and 1-min exposure) caused the development of pronounced changes in tissue morphology.⁷⁶

In the current study, the biopsy specimens for histology were taken 60 min after light exposure when only the first signs of cell damage and lipolysis occurred. This experiment rendered the encapsulated form of ICG more effective (see Table 1) and promising due to the ability to control cell lipolysis more smoothly and to avoid the unwanted effects of necrosis.

Table 1

Statistics for the modification appearance in subcutaneous adipose tissue determined from histological studies of rat skin sites at different treatments in vivo.

The Cochran $Q$ test can be used to evaluate the relation between two variables that are measured on a nominal scale. One of the variables may consist of only two possible values (dichotomous scale). In our case, six different treatments for 10 laboratory animals were evaluated. The scale of evaluation was dichotomous: any changes in subcutaneous adipose tissue either were present ($=1$) or not ($=0$) in the histological samples.

The value of $Q$ becomes greater if there is a statistical association between the variables. If there is no association and only chance is operating, $Q$ reaches exactly the same values as Chi squared (${\chi }^2$-distribution). The degree of freedom equals ($k-1$), where $k$ is the number of cases or treatments; thus, for our study we have the $\text{degree of freedom}=5$. On the basis of data presented in Table 1, the $Q$ value of 27.6 was calculated using the “Statistics 6.0” program. It is known that results would be significant when $Q$ has a higher value than 15.09 for a significance level $p=0.01$.⁶⁶ Therefore, the Cochran $Q$ test shows that the empirically found difference between the treatment alternatives is statistically significant because in our case $Q>15.09$.

Thus, the combined treatment with the encapsulated ICG dye and the 808-nm laser (last column in the Table 1) is more effective in comparison with the other treatments. The level of significance of such a conclusion is rather high, $p=0.01$.