A number of “biological barriers” protect the body against foreign materials, including injected therapeutic and contrast agents, keeping carriers from reaching their intended destinations [34
]. These barriers can restrict the function of carriers by blocking their movement, causing physical changes to them, or by inducing a negative host response by using biochemical signaling [35
]. Upon intravascular administration, carriers immediately encounter blood, a high-ionic-strength, heterogenous solution that can induce carrier agglomeration, altering their magnetic properties and inducing particle sequestration. Additionally, carriers can nonspecifically interact with plasma proteins (which can trigger the adaptive immune system), extracellular matrices, and nontargeted cell surfaces while in the bloodstream [36
]. In each case, the carriers are in danger of prematurely binding to or being taken up by cells before reaching its target tissue. In addition to coping with the vascular environment, carriers must overcome various anatomical size restrictions, which limit carrier access to target tissue (e.g., extravasation of lymph-targeting carriers from the blood vessels) [34
]. The organ distribution of carriers depends on their size and biochemical and physical properties [37
3.2.2. In Vivo Fluorescence Lifetime Imaging
Using an IVIS live imaging system, we next examined carrier biodistribution under the influence of an external magnetic field applied to a mouse paw.
The organ distribution of MNPS
(BSA-Cy7-TA) carriers after administration at different time intervals (Figure 4
a) (0 min [control, before carrier administration], immediately after administration [5 min], 1 h, and 48 h). The relative fluorescence intensity was calculated with account taken of the initial amount of fluorescence intensity for the mice before the carrier was inserted. The magnet was removed at 60 min after carrier administration (dotted black line in Figure 4
The carriers were nonfluorescent at the time of administration (Figure 4
). Since the introduction of the carrier, the signal in the left paw with the magnet increased and was higher than in the intact right paw until the magnet was removed. The level of fluorescence in the paw with the magnet was increasing from the moment of administration, and at the point of 0.5–1 h, it was already possible to see a noticeable signal in the images of the whole body (Figure 4
a), which was most likely due to the gradual release of the dye and the natural destruction of the carriers. The fluorescence intensity in the intact right paw increased only slightly after injection.
After the magnet was removed, the level of fluorescence in the paw to which a magnet had been applied started to decrease, approaching the level in the intact paw. This was due to the gradual elution of the particles from the vessels in the paw. Yet after 1 h the fluorescence level of the whole body of the mouse increased significantly, peaking after 24 h, which according to previous in vitro studies indicates the gradual destruction of the shell and the MNP–Cy7 complex. Thus, the results obtained in vivo mirror the in vitro data on the stability of carrier fluorescence in various biological fluids (Figure 1
d). The strongest intensity came from the liver area after 24 h of intravenous injection (Figure 4
3.2.3. Histological Studies and Magnetometry
For a better understanding of carrier distribution, mice were killed 1 h after the magnet had been removed and the MNP concentration in the collected organs was measured by magnetometry. Analysis of the tissue distribution of MNPs (BSA–Cy7/TA) shows that the carrier predominantly accumulated in the lung, liver, and spleen (Figure 5
The accumulation of nano- and micro-particles in the liver and spleen is a well-known fact and has been described for many objects, such as polymer-coated microcarriers and liposomes [1
]. The effect of size on carrier biodistribution is organ-specific and nonlinear [41
]. This is due in part to the organ-specific physical and physiological barriers that systemically administered carriers encounter [42
]. Studies on liposomes have shown that splenic sequestration of particles decreases linearly with decreasing of particle size [43
]. For the liver, the dependence on size is also nonlinear. While larger carriers are sequestered in the liver (consistent with observations in the spleen), very small carriers (less than 70 nm) can pass through the sinusoidal fenestrations in the liver and be entrapped by the underlying parenchymal cells [11
]. For polymer microcarriers obtained by LBL technology, significant accumulation in the liver and spleen has also been observed [10
]. The absorption of nanoparticles by macrophages is usually considered the main mechanism of accumulation of foreign materials in the liver [41
]. The tissue distribution of the magnetite-containing microcarriers is shown in Figure 5
There is much less information in the literature about the accumulation of particles in the lungs. Such accumulation was described for silica-coated magnetic nanoparticles [43
], titanium dioxide nanoparticles [44
] and other similar structures [46
]. Absorption by tissue macrophages is considered the main mechanism of particle accumulation in the lungs, similarly to what is observed in the liver [44
]. However, in our case, the concentration of carriers in the lungs was very high (about four times higher than that in the liver and five times higher than that in the spleen). We believe that this phenomenon cannot be explained only by the absorption of particles by macrophages, because the main pool of phagocytes in the lungs is represented by alveolar macrophages with no direct contact with the blood.
The most probable cause for the intrapulmonary accumulation of particles is the features of their movement through the lung capillaries. It is known that the lungs function as a microfilter and retain most of the blood cell aggregates and other foreign objects [11
]. Although the diameter of the carriers is smaller than that of the capillaries, even unstable aggregates consisting of two or three particles can embolize the pulmonary capillary. Furthermore, the distribution of particles in the bloodstream may be important. The blood flow velocity is maximal in the center and minimal or equal to zero at the periphery of the capillary [12
]. Particles trapped in the parietal area of the bloodstream slow down their movement significantly or even completely stop.
This process can be reversible, and eventually, the concentration of “parietal” particles decreases. However, when the particles adhere to the endothelial cells more strongly, they may accumulate long-term in the lungs. If the particles activate platelets and/or plasma clotting factors, adhesion to the endothelium may lead to capillary thrombosis with the subsequent organ dysfunction. In our case, there was no evidence of pulmonary capillary thrombosis, because no subsequent lung dysfunction was observed.
It should also be noted that when the particles accumulate in the capillaries through the “hemodynamics mechanism,” this process should be more pronounced in pulmonary tissue owing to the “first pass effect,” because 100% of the substance administered intravenously passes through the lung capillaries.
Our analysis of the accumulation of carriers in the kidneys, heart, and intestines shows that their concentrations in these organs are proportional to the degree of their vascularization. This fact indicates the mainly intravascular character of carrier circulation, especially early after administration.
The concentration of the carriers in muscle tissue is to be analyzed separately, because in this study, the hip muscle was the point of targeting of magnetically controlled objects. The results are shown in the inset of Figure 5
. The choice of the hip muscle was primarily due to the anatomical availability of this area for the application of the magnetic field. Although the blood flow in muscles without physical activity is small, using this tissue has an undisputable advantage, i.e., the possibility of comparing the concentration of carriers in the intact muscle and in the muscle placed in a magnetic field gradient in the same animal at the same time.
One can see that the concentration of carriers in the muscles was lower, as compared to that in the other organs. However, in the paw placed in a magnetic field, the concentration of the particles was higher by 70% than it was in the intact paw. Probably, the mechanism of accumulation is based on the displacement of carriers by the magnetic field gradient from the axial region of the capillary to its periphery, where, as already mentioned, the blood flow velocity is lower.
The histological analysis of the paws with and without the magnet showed that the muscle fibers were located in parallel and that the transverse striation was retained. Throughout the thickness of the slice, in the muscle tissue of the paw to which the magnet was attached, we observed diffusely located aggregates of magnetite (Figure 5