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Control the movement of magnetic iron oxide nanoparticles for targeted delivery of cytostatics
Author Toropova Y, Korolev D, Istomina M, Shulmeyster G, Petukhov A, Mishanin V, Gorshkov A, Podyacheva E, Gareev K, Bagrov A, Demidov O
Yana Toropova,1 Dmitry Korolev,1 Maria Istomina,1,2 Galina Shulmeyster,1 Alexey Petukhov,1,3 Vladimir Mishanin,1 Andrey Gorshkov,4 Ekaterina Podyacheva,1 Kamil Gareev,2 Alexei Bagrov,5 Oleg Demidov6,71Almazov National Medical Research Center of the Ministry of Health of the Russian Federation, St. Petersburg, 197341, Russian Federation; 2 St. Petersburg Electrotechnical University “LETI”, St. Petersburg, 197376, Russian Federation; 3 Center for Personalized Medicine, Almazov State Medical Research Center, Ministry of Health of the Russian Federation, St. Petersburg, 197341, Russia Federation; 4FSBI “Influenza Research Institute named after AA Smorodintsev” Ministry of Health of the Russian Federation, St. Petersburg, Russian Federation; 5 Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russian Federation; 6 RAS Institute of Cytology, St. Petersburg, 194064, Russian Federation; 7INSERM U1231, Faculty of Medicine and Pharmacy, Bourgogne-Franche Comté University of Dijon, France Communication: Yana ToropovaAlmazov National Medical Research Centre, Ministry of Health of the Russian Federation, Saint-Petersburg, 197341, Russian Federation Tel +7 981 95264800 4997069 Email [email protected] Background: A promising approach to the problem of cytostatic toxicity is the use of magnetic nanoparticles (MNP) for targeted drug delivery. Purpose: To use calculations to determine the best characteristics of the magnetic field that controls MNPs in vivo, and to evaluate the efficiency of magnetron delivery of MNPs to mouse tumors in vitro and in vivo. (MNPs-ICG) is used. In vivo luminescence intensity studies were performed in tumor mice, with and without a magnetic field at the site of interest. These studies were carried out on a hydrodynamic scaffold developed by the Institute of Experimental Medicine of the Almazov State Medical Research Center of the Russian Ministry of Health. Result: The use of neodymium magnets promoted the selective accumulation of MNP. One minute after administration of MNPs-ICG to tumor-bearing mice, MNPs-ICG mainly accumulates in the liver. In the absence and presence of a magnetic field, this indicates its metabolic pathway. Although an increase in the fluorescence in the tumor was observed in the presence of a magnetic field, the fluorescence intensity in the liver of the animal did not change over time. Conclusion: This type of MNP, combined with the calculated magnetic field strength, can be the basis for the development of magnetically controlled delivery of cytostatic drugs to tumor tissues. Keywords: fluorescence analysis, indocyanine, iron oxide nanoparticles, magnetron delivery of cytostatics, tumor targeting
Tumor diseases are one of the main causes of death worldwide. At the same time, the dynamics of increasing morbidity and mortality of tumor diseases still exist. 1 The chemotherapy used today is still one of the main treatments for different tumors. At the same time, the development of methods to reduce the systemic toxicity of cytostatics is still relevant. A promising method to solve its toxicity problem is to use nano-scale carriers to target drug delivery methods, which can provide local accumulation of drugs in tumor tissues without increasing their accumulation in healthy organs and tissues. concentration. 2 This method makes it possible to improve the efficiency and targeting of chemotherapeutic drugs on tumor tissues, while reducing their systemic toxicity.
Among the various nanoparticles considered for targeted delivery of cytostatic agents, magnetic nanoparticles (MNPs) are of particular interest because of their unique chemical, biological, and magnetic properties, which ensure their versatility. Therefore, magnetic nanoparticles can be used as a heating system to treat tumors with hyperthermia (magnetic hyperthermia). They can also be used as diagnostic agents (magnetic resonance diagnosis). 3-5 Using these characteristics, combined with the possibility of MNP accumulation in a specific area, through the use of an external magnetic field, the delivery of targeted pharmaceutical preparations opens up the creation of a multifunctional magnetron system to target cytostatics to the tumor site Prospects. Such a system would include MNP and magnetic fields to control their movement in the body. In this case, both external magnetic fields and magnetic implants placed in the body area containing the tumor can be used as the source of the magnetic field. 6 The first method has serious shortcomings, including the need to use specialized equipment for magnetic targeting of drugs and the need to train personnel to perform surgery. In addition, this method is limited by high cost and is only suitable for “superficial” tumors close to the surface of the body. The alternative method of using magnetic implants expands the scope of application of this technology, facilitating its use on tumors located in different parts of the body. Both individual magnets and magnets integrated into the intraluminal stent can be used as implants for tumor damage in hollow organs to ensure their patency. However, according to our own unpublished research, these are not sufficiently magnetic to ensure the retention of MNP from the bloodstream.
The effectiveness of magnetron drug delivery depends on many factors: the characteristics of the magnetic carrier itself, and the characteristics of the magnetic field source (including the geometric parameters of permanent magnets and the strength of the magnetic field they generate). The development of successful magnetically guided cell inhibitor delivery technology should involve the development of appropriate magnetic nanoscale drug carriers, assessing their safety, and developing a visualization protocol that allows tracking their movements in the body.
In this study, we mathematically calculated the optimal magnetic field characteristics to control the magnetic nano-scale drug carrier in the body. The possibility of retaining MNP through the blood vessel wall under the influence of an applied magnetic field with these computational characteristics was also studied in isolated rat blood vessels. In addition, we synthesized conjugates of MNPs and fluorescent agents and developed a protocol for their visualization in vivo. Under in vivo conditions, in tumor model mice, the accumulation efficiency of MNPs in tumor tissues when administered systemically under the influence of a magnetic field was studied.
In the in vitro study, we used the reference MNP, and in the in vivo study, we used the MNP coated with lactic acid polyester (polylactic acid, PLA) containing a fluorescent agent (indolecyanine; ICG). MNP-ICG is included in In the case, use (MNP-PLA-EDA-ICG).
The synthesis and physical and chemical properties of MNP have been described in detail elsewhere. 7,8
In order to synthesize MNPs-ICG, PLA-ICG conjugates were first produced. A powder racemic mixture of PLA-D and PLA-L with a molecular weight of 60 kDa was used.
Since PLA and ICG are both acids, in order to synthesize PLA-ICG conjugates, first need to synthesize an amino-terminated spacer on PLA, which helps ICG chemisorb to the spacer. The spacer was synthesized using ethylene diamine (EDA), carbodiimide method and water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC). The PLA-EDA spacer is synthesized as follows. Add 20-fold molar excess of EDA and 20-fold molar excess of EDAC to 2 mL of 0.1 g/mL PLA chloroform solution. The synthesis was carried out in a 15 mL polypropylene test tube on a shaker at a speed of 300 min-1 for 2 hours. The synthesis scheme is shown in Figure 1. Repeat the synthesis with a 200-fold excess of reagents to optimize the synthesis scheme.
At the end of the synthesis, the solution was centrifuged at a speed of 3000 min-1 for 5 minutes to remove excess precipitated polyethylene derivatives. Then, 2 mL of a 0.5 mg/mL ICG solution in dimethyl sulfoxide (DMSO) was added to the 2 mL solution. The agitator is fixed at a stirring speed of 300 min-1 for 2 hours. The schematic diagram of the obtained conjugate is shown in Figure 2.
In 200 mg MNP, we added 4 mL PLA-EDA-ICG conjugate. Use an LS-220 shaker (LOIP, Russia) to stir the suspension for 30 minutes at a frequency of 300 min-1. Then, it was washed with isopropanol three times and subjected to magnetic separation. Use UZD-2 Ultrasonic Disperser (FSUE NII TVCH, Russia) to add IPA to the suspension for 5-10 minutes under continuous ultrasonic action. After the third IPA wash, the precipitate was washed with distilled water and resuspended in physiological saline at a concentration of 2 mg/mL.
The ZetaSizer Ultra equipment (Malvern Instruments, UK) was used to study the size distribution of the obtained MNP in the aqueous solution. A transmission electron microscope (TEM) with a JEM-1400 STEM field emission cathode (JEOL, Japan) was used to study the shape and size of the MNP.
In this study, we use cylindrical permanent magnets (N35 grade; with nickel protective coating) and the following standard sizes (long axis length × cylinder diameter): 0.5×2 mm, 2×2 mm, 3×2 mm and 5×2 mm.
The in vitro study of MNP transport in the model system was carried out on a hydrodynamic scaffold developed by the Institute of Experimental Medicine of the Almazov State Medical Research Center of the Russian Ministry of Health. The volume of the circulating liquid (distilled water or Krebs-Henseleit solution) is 225 mL. Axially magnetized cylindrical magnets are used as permanent magnets. Place the magnet on a holder 1.5 mm away from the inner wall of the central glass tube, with its end facing the direction of the tube (vertical). The fluid flow rate in the closed loop is 60 L/h (corresponding to a linear velocity of 0.225 m/s). Krebs-Henseleit solution is used as a circulating fluid because it is an analog of plasma. The dynamic viscosity coefficient of plasma is 1.1–1.3 mPa∙s. 9 The amount of MNP adsorbed in the magnetic field is determined by spectrophotometry from the concentration of iron in the circulating liquid after the experiment.
In addition, experimental studies have been carried out on an improved fluid mechanics table to determine the relative permeability of blood vessels. The main components of the hydrodynamic support are shown in Figure 3. The main components of the hydrodynamic stent are a closed loop that simulates the cross-section of the model vascular system and a storage tank. The movement of the model fluid along the contour of the blood vessel module is provided by a peristaltic pump. During the experiment, maintain the vaporization and required temperature range, and monitor the system parameters (temperature, pressure, liquid flow rate, and pH value).
Figure 3 Block diagram of the setup used to study the permeability of the carotid artery wall. 1-storage tank, 2-peristaltic pump, 3-mechanism for introducing suspension containing MNP into the loop, 4-flow meter, 5-pressure sensor in the loop, 6-heat exchanger, 7-chamber with container , 8-the source of the magnetic field, 9-the balloon with hydrocarbons.
The chamber containing the container consists of three containers: an outer large container and two small containers, through which the arms of the central circuit pass. The cannula is inserted into the small container, the container is stringed on the small container, and the tip of the cannula is tightly tied with a thin wire. The space between the large container and the small container is filled with distilled water, and the temperature remains constant due to the connection to the heat exchanger. The space in the small container is filled with Krebs-Henseleit solution to maintain the viability of blood vessel cells. The tank is also filled with Krebs-Henseleit solution. The gas (carbon) supply system is used to vaporize the solution in the small container in the storage tank and the chamber containing the container (Figure 4).
Figure 4 The chamber where the container is placed. 1-Cannula for lowering blood vessels, 2-Outer chamber, 3-Small chamber. The arrow indicates the direction of the model fluid.
To determine the relative permeability index of the vessel wall, the rat carotid artery was used.
The introduction of MNP suspension (0.5mL) into the system has the following characteristics: the total internal volume of the tank and connecting pipe in the loop is 20mL, and the internal volume of each chamber is 120mL. The external magnetic field source is a permanent magnet with a standard size of 2×3 mm. It is installed above one of the small chambers, 1 cm away from the container, with one end facing the container wall. The temperature is kept at 37°C. The power of the roller pump is set to 50%, which corresponds to a speed of 17 cm/s. As a control, samples were taken in a cell without permanent magnets.
One hour after the administration of a given concentration of MNP, a liquid sample was taken from the chamber. The particle concentration was measured by a spectrophotometer using Unico 2802S UV-Vis spectrophotometer (United Products & Instruments, USA). Taking into account the absorption spectrum of the MNP suspension, the measurement was performed at 450 nm.
According to the Rus-LASA-FELASA guidelines, all animals are raised and raised in specific pathogen-free facilities. This study complies with all relevant ethical regulations for animal experiments and research, and has obtained ethical approval from the Almazov National Medical Research Center (IACUC). The animals drank water ad libitum and fed regularly.
The study was conducted on 10 anesthetized 12-week-old male immunodeficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/Szj, Jackson Laboratory, USA) 10, weighing 22 g ± 10%. Since the immunity of immunodeficiency mice is suppressed, the immunodeficiency mice of this line allow transplantation of human cells and tissues without transplant rejection. The littermates from different cages were randomly assigned to the experimental group, and they were co-bred or systematically exposed to the bedding of other groups to ensure equal exposure to the common microbiota.
The HeLa human cancer cell line is used to establish a xenograft model. The cells were cultured in DMEM containing glutamine (PanEco, Russia), supplemented with 10% fetal bovine serum (Hyclone, USA), 100 CFU/mL penicillin, and 100 μg/mL streptomycin. The cell line was kindly provided by the Gene Expression Regulation Laboratory of the Institute of Cell Research of the Russian Academy of Sciences. Before injection, HeLa cells were removed from the culture plastic with a 1:1 trypsin:Versene solution (Biolot, Russia). After washing, the cells were suspended in complete medium to a concentration of 5×106 cells per 200 μL, and diluted with basement membrane matrix (LDEV-FREE, MATRIGEL® CORNING®) (1:1, on ice). The prepared cell suspension was injected subcutaneously into the skin of the mouse thigh. Use electronic calipers to monitor tumor growth every 3 days.
When the tumor reached 500 mm3, a permanent magnet was implanted into the muscle tissue of the experimental animal near the tumor. In the experimental group (MNPs-ICG + tumour-M), 0.1 mL of MNP suspension was injected and exposed to a magnetic field. Untreated whole animals were used as controls (background). In addition, animals injected with 0.1 mL of MNP but not implanted with magnets (MNPs-ICG + tumor-BM) were used.
The fluorescence visualization of in vivo and in vitro samples was performed on the IVIS Lumina LT series III bioimager (PerkinElmer Inc., USA). For in vitro visualization, a volume of 1 mL of synthetic PLA-EDA-ICG and MNP-PLA-EDA-ICG conjugate was added to the plate wells. Taking into account the fluorescence characteristics of the ICG dye, the best filter used to determine the luminous intensity of the sample is selected: the maximum excitation wavelength is 745 nm, and the emission wavelength is 815 nm. The Living Image 4.5.5 software (PerkinElmer Inc.) was used to quantitatively measure the fluorescence intensity of the wells containing the conjugate.
The fluorescence intensity and accumulation of the MNP-PLA-EDA-ICG conjugate were measured in in vivo tumor model mice, without the presence and application of a magnetic field at the site of interest. The mice were anesthetized with isoflurane, and then 0.1 mL of MNP-PLA-EDA-ICG conjugate was injected through the tail vein. Untreated mice were used as a negative control to obtain a fluorescent background. After administering the conjugate intravenously, place the animal on a heating stage (37°C) in the chamber of the IVIS Lumina LT series III fluorescence imager (PerkinElmer Inc.) while maintaining inhalation with 2% isoflurane anaesthetization. Use ICG’s built-in filter (745–815 nm) for signal detection 1 minute and 15 minutes after the introduction of MNP.
To assess the accumulation of conjugate in the tumor, the peritoneal area of ​​the animal was covered with paper, which made it possible to eliminate the bright fluorescence associated with the accumulation of particles in the liver. After studying the biodistribution of MNP-PLA-EDA-ICG, the animals were humanely euthanized by an overdose of isoflurane anesthesia for subsequent separation of tumor areas and quantitative assessment of fluorescence radiation. Use Living Image 4.5.5 software (PerkinElmer Inc.) to manually process the signal analysis from the selected region of interest. Three measurements were taken for each animal (n = 9).
In this study, we did not quantify the successful loading of ICG on MNPs-ICG. In addition, we did not compare the retention efficiency of nanoparticles under the influence of permanent magnets of different shapes. In addition, we did not evaluate the long-term effect of the magnetic field on the retention of nanoparticles in tumor tissues.
Nanoparticles dominate, with an average size of 195.4 nm. In addition, the suspension contained agglomerates with an average size of 1176.0 nm (Figure 5A). Subsequently, the portion was filtered through a centrifugal filter. The zeta potential of the particles is -15.69 mV (Figure 5B).
Figure 5 The physical properties of the suspension: (A) particle size distribution; (B) particle distribution at zeta potential; (C) TEM photograph of nanoparticles.
The particle size is basically 200 nm (Figure 5C), composed of a single MNP with a size of 20 nm, and a PLA-EDA-ICG conjugated organic shell with a lower electron density. The formation of agglomerates in aqueous solutions can be explained by the relatively low modulus of the electromotive force of individual nanoparticles.
For permanent magnets, when the magnetization is concentrated in the volume V, the integral expression is divided into two integrals, namely the volume and the surface:
In the case of a sample with a constant magnetization, the current density is zero. Then, the expression of the magnetic induction vector will take the following form:
Use MATLAB program (MathWorks, Inc., USA) for numerical calculation, ETU “LETI” academic license number 40502181.
As shown in Figure 7 Figure 8 Figure 9 Figure-10, the strongest magnetic field is generated by a magnet oriented axially from the end of the cylinder. The effective radius of action is equivalent to the geometry of the magnet. In cylindrical magnets with a cylinder whose length is greater than its diameter, the strongest magnetic field is observed in the axial-radial direction (for the corresponding component); therefore, a pair of cylinders with a larger aspect ratio (diameter and length) MNP adsorption is the most effective.
Fig. 7 The component of the magnetic induction intensity Bz along the Oz axis of the magnet; the standard size of the magnet: black line 0.5×2mm, blue line 2×2mm, green line 3×2mm, red line 5×2mm.
Figure 8 The magnetic induction component Br is perpendicular to the magnet axis Oz; the standard size of the magnet: black line 0.5×2mm, blue line 2×2mm, green line 3×2mm, red line 5×2mm.
Figure 9 The magnetic induction intensity Bz component at the distance r from the end axis of the magnet (z=0); the standard size of the magnet: black line 0.5×2mm, blue line 2×2mm, green line 3×2mm, red line 5×2mm.
Figure 10 Magnetic induction component along the radial direction; standard magnet size: black line 0.5×2mm, blue line 2×2mm, green line 3×2mm, red line 5×2mm.
Special hydrodynamic models can be used to study the method of MNP delivery to tumor tissues, concentrate nanoparticles in the target area, and determine the behavior of nanoparticles under hydrodynamic conditions in the circulatory system. Permanent magnets can be used as external magnetic fields. If we ignore the magnetostatic interaction between the nanoparticles and do not consider the magnetic fluid model, it is sufficient to estimate the interaction between the magnet and a single nanoparticle with a dipole-dipole approximation.
Where m is the magnetic moment of the magnet, r is the radius vector of the point where the nanoparticle is located, and k is the system factor. In the dipole approximation, the field of the magnet has a similar configuration (Figure 11).
In a uniform magnetic field, the nanoparticles only rotate along the lines of force. In a non-uniform magnetic field, force acts on it:
Where is the derivative of a given direction l. In addition, the force pulls the nanoparticles into the most uneven areas of the field, that is, the curvature and density of the lines of force increase.
Therefore, it is desirable to use a sufficiently strong magnet (or magnet chain) with obvious axial anisotropy in the area where the particles are located.
Table 1 shows the ability of a single magnet as a sufficient magnetic field source to capture and retain MNP in the vascular bed of the application field.