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Gene vectors for the treatment of pulmonary cystic fibrosis must be targeted to the conductive airways, since peripheral lung transduction has no therapeutic effect. The efficiency of viral transduction is directly related to the residence time of the carrier. However, delivery fluids such as gene carriers naturally diffuse into the alveoli during inhalation, and therapeutic particles of any shape are rapidly removed by mucociliary transport. Extending the residence time of gene carriers in the respiratory tract is important but difficult to achieve. Carrier-conjugated magnetic particles that can be directed to the surface of the respiratory tract can improve regional targeting. Due to problems with in vivo imaging, the behavior of such small magnetic particles on the airway surface in the presence of an applied magnetic field is poorly understood. The aim of this study was to use synchrotron imaging to visualize in vivo the movement of a series of magnetic particles in the trachea of ​​anesthetized rats in order to study the dynamics and patterns of behavior of single and bulk particles in vivo. We then also assessed whether delivery of lentiviral magnetic particles in the presence of a magnetic field would increase the efficiency of transduction in the rat trachea. Synchrotron X-ray imaging shows the behavior of magnetic particles in stationary and moving magnetic fields in vitro and in vivo. Particles cannot be easily dragged across the surface of living airways using magnets, but during transport, deposits are concentrated in the field of view, where the magnetic field is strongest. Transduction efficiency was also increased six-fold when lentiviral magnetic particles were delivered in the presence of a magnetic field. Taken together, these results suggest that lentiviral magnetic particles and magnetic fields may be valuable approaches to improve gene vector targeting and transduction levels in the conductive airways in vivo.
Cystic fibrosis (CF) is caused by variations in a single gene called the CF transmembrane conductance regulator (CFTR). The CFTR protein is an ion channel that is present in many epithelial cells throughout the body, including the airways, a major site in the pathogenesis of cystic fibrosis. Defects in CFTR lead to abnormal water transport, dehydration of the airway surface, and decreased airway surface fluid layer (ASL) depth. It also impairs the ability of the mucociliary transport (MCT) system to clear the airways of inhaled particles and pathogens. Our goal is to develop a lentiviral (LV) gene therapy to deliver the correct copy of the CFTR gene and improve ASL, MCT, and lung health, and to continue developing new technologies that can measure these parameters in vivo1.
LV vectors are one of the leading candidates for cystic fibrosis gene therapy, mainly because they can permanently integrate the therapeutic gene into airway basal cells (airway stem cells). This is important because they can restore normal hydration and mucus clearance by differentiating into functional gene-corrected airway surface cells associated with cystic fibrosis, resulting in lifelong benefits. LV vectors must be directed against the conductive airways, as this is where lung involvement in CF begins. Delivery of the vector deeper into the lung may result in alveolar transduction, but this has no therapeutic effect in cystic fibrosis. However, fluids such as gene carriers naturally migrate into the alveoli when inhaled after childbirth3,4 and therapeutic particles are rapidly expelled into the oral cavity by MCTs. The efficiency of LV transduction is directly related to the length of time the vector remains close to the target cells to allow cellular uptake – “residence time” 5 which is easily shortened by typical regional airflow as well as coordinated uptake of mucus and MCT particles. For cystic fibrosis, the ability to prolong LV residence time in the airways is important to achieve high levels of transduction in this area, but has so far been challenging.
To overcome this hurdle, we propose that LV magnetic particles (MPs) can help in two complementary ways. First, they can be guided by a magnet to the airway surface to improve targeting and help gene carrier particles to be in the right area of ​​the airway; and ASL) move into cell layer 6. MPs are widely used as targeted drug delivery vehicles when they bind to antibodies, chemotherapy drugs, or other small molecules that attach to cell membranes or bind to their respective cell surface receptors and accumulate at tumor sites in presence of static electricity. Magnetic fields for cancer therapy 7. Other “hyperthermic” methods are aimed at killing tumor cells by heating MPs when exposed to oscillating magnetic fields. The principle of magnetic transfection, in which a magnetic field is used as a transfection agent to enhance the transfer of DNA into cells, is commonly used in vitro using a range of non-viral and viral gene vectors for difficult-to-transduce cell lines. . The efficiency of LV magnetotransfection with the delivery of LV MP in vitro into a cell line of human bronchial epithelium in the presence of a static magnetic field was established, increasing the efficiency of transduction by 186 times compared with the LV vector alone. LV MT has also been applied to an in vitro model of cystic fibrosis, where magnetic transfection increased LV transduction in air-liquid interface cultures by a factor of 20 in the presence of cystic fibrosis sputum10. However, in vivo organ magnetotransfection has received relatively little attention and has only been evaluated in a few animal studies11,12,13,14,15, especially in the lungs16,17. However, the possibilities of magnetic transfection in lung therapy in cystic fibrosis are clear. Tan et al. (2020) stated that “a validation study on effective pulmonary delivery of magnetic nanoparticles will pave the way for future CFTR inhalation strategies to improve clinical outcomes in patients with cystic fibrosis”6.
The behavior of small magnetic particles on the surface of the respiratory tract in the presence of an applied magnetic field is difficult to visualize and study, and therefore they are poorly understood. In other studies, we have developed a Synchrotron Propagation Based Phase Contrast X-Ray Imaging (PB-PCXI) method for non-invasive imaging and quantification of minute in vivo changes in ASL18 depth and MCT19 behavior,20 to directly measure gas channel surface hydration and is used as an early indicator treatment effectiveness. In addition, our MCT scoring method uses 10–35 µm diameter particles composed of alumina or high refractive index glass as MCT markers visible with PB-PCXI21. Both methods are suitable for imaging a range of particle types, including MPs.
Due to the high spatial and temporal resolution, our PB-PCXI-based ASL and MCT assays are well suited to study the dynamics and behavioral patterns of single and bulk particles in vivo to help us understand and optimize MP gene delivery methods. The approach we use here is based on our studies using the SPring-8 BL20B2 beamline, in which we visualized fluid movement following delivery of a dose of a dummy vector into the nasal and pulmonary airways of mice to help explain our heterogeneous gene expression patterns observed in our gene. animal studies with a carrier dose of 3.4 .
The aim of this study was to use the PB-PCXI synchrotron to visualize in vivo movements of a series of MPs in the trachea of ​​live rats. These PB-PCXI imaging studies were designed to test the MP series, magnetic field strength, and location to determine their effect on MP movement. We assumed that an external magnetic field would help the delivered MF stay or move to the target area. These studies also allowed us to determine magnet configurations that maximize the amount of particles left in the trachea after deposition. In a second series of studies, we aimed to use this optimal configuration to demonstrate the transduction pattern resulting from in vivo delivery of LV-MPs to the rat airways, on the assumption that delivery of LV-MPs in the context of airway targeting would result in increased LV transduction efficiency. .
All animal studies were conducted in accordance with protocols approved by the University of Adelaide (M-2019-060 and M-2020-022) and the SPring-8 Synchrotron Animal Ethics Committee. The experiments were carried out in accordance with the recommendations of ARRIVE.
All x-ray images were taken at the BL20XU beamline at the SPring-8 synchrotron in Japan using a setup similar to that described previously21,22. Briefly, the experimental box was located 245 m from the synchrotron storage ring. A sample-to-detector distance of 0.6 m is used for particle imaging studies and 0.3 m for in vivo imaging studies to create phase contrast effects. A monochromatic beam with an energy of 25 keV was used. The images were acquired using a high resolution X-ray transducer (SPring-8 BM3) coupled to a sCMOS detector. The transducer converts X-rays to visible light using a 10 µm thick scintillator (Gd3Al2Ga3O12), which is then directed to the sCMOS sensor using a ×10 (NA 0.3) microscope objective. The sCMOS detector was an Orca-Flash4.0 (Hamamatsu Photonics, Japan) with an array size of 2048 × 2048 pixels and a raw pixel size of 6.5 × 6.5 µm. This setting gives an effective isotropic pixel size of 0.51 µm and a field of view of approximately 1.1 mm × 1.1 mm. The exposure duration of 100 ms was chosen to maximize the signal-to-noise ratio of magnetic particles inside and outside the airways while minimizing motion artifacts caused by breathing. For in vivo studies, a fast X-ray shutter was placed in the X-ray path to limit the radiation dose by blocking the X-ray beam between exposures.
LV media was not used in any SPring-8 PB-PCXI imaging studies because the BL20XU imaging chamber is not Biosafety Level 2 certified. Instead, we selected a range of well-characterized MPs from two commercial vendors covering a range of sizes, materials, iron concentrations, and applications , — first in order to understand how magnetic fields affect the movement of MPs in glass capillaries, and then in living airways. surface. The size of the MP varies from 0.25 to 18 µm and is made from various materials (see Table 1), but the composition of each sample, including the size of the magnetic particles in the MP, is unknown. Based on our extensive MCT studies 19, 20, 21, 23, 24, we expect that MPs down to 5 µm can be seen on the tracheal airway surface, for example, by subtracting consecutive frames to see improved visibility of MP movement. A single MP of 0.25 µm is smaller than the resolution of the imaging device, but PB-PCXI is expected to detect their volumetric contrast and the movement of the surface liquid on which they are deposited after being deposited.
Samples for each MP in the table. 1 was prepared in 20 μl glass capillaries (Drummond Microcaps, PA, USA) with an internal diameter of 0.63 mm. Corpuscular particles are available in water, while CombiMag particles are available in the manufacturer’s proprietary liquid. Each tube is half filled with liquid (approximately 11 µl) and placed on the sample holder (see Figure 1). The glass capillaries were placed horizontally on the stage in the imaging chamber, respectively, and positioned at the edges of the liquid. A 19 mm diameter (28 mm long) nickel-shell magnet made of rare earth, neodymium, iron and boron (NdFeB) (N35, cat. no. LM1652, Jaycar Electronics, Australia) with a remanence of 1.17 T was attached to a separate transfer table to achieve Remotely change your position during rendering. X-ray imaging begins when the magnet is positioned approximately 30 mm above the sample and images are acquired at 4 frames per second. During imaging, the magnet was brought close to the glass capillary tube (at a distance of about 1 mm) and then moved along the tube to assess the effect of field strength and position.
An in vitro imaging setup containing MP samples in glass capillaries at the stage of translation of the xy sample. The path of the X-ray beam is marked with a red dotted line.
Once the in vitro visibility of MPs was established, a subset of them was tested in vivo on wild-type female Wistar albino rats (~12 weeks old, ~200 g). Medetomidine 0.24 mg/kg (Domitor®, Zenoaq, Japan), midazolam 3.2 mg/kg (Dormicum®, Astellas Pharma, Japan) and butorphanol 4 mg/kg (Vetorphale®, Meiji Seika). Rats were anesthetized with Pharma (Japan) mixture by intraperitoneal injection. After anesthesia, they were prepared for imaging by removing the fur around the trachea, inserting an endotracheal tube (ET; 16 Ga intravenous cannula, Terumo BCT), and immobilizing them in the supine position on a custom-made imaging plate containing a thermal bag to maintain body temperature. 22. The imaging plate was then attached to the sample stage in the imaging box at a slight angle to align the trachea horizontally on the x-ray image as shown in Figure 2a.
(a) In vivo imaging setup in the SPring-8 imaging unit, X-ray beam path marked with red dotted line. (b,c) Tracheal magnet localization was performed remotely using two orthogonally mounted IP cameras. On the left side of the image on the screen, you can see the wire loop holding the head and the delivery cannula installed inside the ET tube.
A remote controlled syringe pump system (UMP2, World Precision Instruments, Sarasota, FL) using a 100 µl glass syringe was connected to a PE10 tubing (0.61 mm OD, 0.28 mm ID) using a 30 Ga needle. Mark the tube to ensure that the tip is in the correct position in the trachea when inserting the endotracheal tube. Using a micropump, the syringe plunger was removed and the tip of the tube was immersed in the MP sample to be delivered. The loaded delivery tube was then inserted into the endotracheal tube, placing the tip at the strongest part of our expected applied magnetic field. Image acquisition was controlled using a breath detector connected to our Arduino-based timing box, and all signals (e.g., temperature, respiration, shutter open/close, and image acquisition) were recorded using Powerlab and LabChart (AD Instruments, Sydney, Australia) 22 When Imaging When the housing was unavailable, two IP cameras (Panasonic BB-SC382) were positioned at approximately 90° to each other and used to control the position of the magnet relative to the trachea during imaging (Figure 2b, c). To minimize motion artifacts, one image per breath was acquired during the terminal respiratory flow plateau.
The magnet is attached to the second stage, which may be located remotely on the outside of the imaging body. Various positions and configurations of the magnet were tested, including: placed at an angle of approximately 30° above the trachea (configurations are shown in Figures 2a and 3a); one magnet above the animal and the other below, with the poles set for attraction (Figure 3b). , one magnet above the animal and one below, with the poles set for repulsion (Figure 3c), and one magnet above and perpendicular to the trachea (Figure 3d). After setting up the animal and magnet and loading the MP under test into the syringe pump, deliver a dose of 50 µl at a rate of 4 µl/sec upon acquisition of images. The magnet is then moved back and forth along or across the trachea while continuing to acquire images.
Magnet configuration for in vivo imaging (a) one magnet above the trachea at an angle of approximately 30°, (b) two magnets configured for attraction, (c) two magnets configured for repulsion, (d) one magnet above and perpendicular to the trachea. The observer looked down from the mouth to the lungs through the trachea and the X-ray beam passed through the left side of the rat and exited the right side. The magnet is either moved along the length of the airway or left and right above the trachea in the direction of the X-ray beam.
We also sought to determine the visibility and behavior of particles in the airways in the absence of mixing of respiration and heart rate. Therefore, at the end of the imaging period, animals were humanely euthanized due to pentobarbital overdose (Somnopentyl, Pitman-Moore, Washington Crossing, USA; ~65 mg/kg ip). Some animals were left on the imaging platform, and after the cessation of breathing and heartbeat, the imaging process was repeated, adding an additional dose of MP if no MP was visible on the airway surface.
The resulting images were corrected for flat and dark field and then assembled into a movie (20 frames per second; 15–25 × normal speed depending on respiration rate) using a custom script written in MATLAB (R2020a, The Mathworks).
All studies on LV gene vector delivery were conducted at the University of Adelaide Laboratory Animal Research Center and aimed to use the results of the SPring-8 experiment to assess whether LV-MP delivery in the presence of a magnetic field could enhance gene transfer in vivo. To evaluate the effects of MF and magnetic field, two groups of animals were treated: one group was injected with LV MF with magnet placement, and the other group was injected with a control group with LV MF without magnet.
LV gene vectors have been generated using previously described methods 25, 26 . The LacZ vector expresses a nuclear localized beta-galactosidase gene driven by the MPSV constitutive promoter (LV-LacZ), which produces a blue reaction product in transduced cells, visible on fronts and sections of lung tissue. Titration was performed in cell cultures by manually counting the number of LacZ-positive cells using a hemocytometer to calculate the titer in TU/ml. Carriers are cryopreserved at -80°C, thawed prior to use, and bound to CombiMag by mixing 1:1 and incubating on ice for at least 30 minutes prior to delivery.
Normal Sprague Dawley rats (n = 3/group, ~2-3 anesthetized ip with a mixture of 0.4mg/kg medetomidine (Domitor, Ilium, Australia) and 60mg/kg ketamine (Ilium, Australia) at 1 month of age) ip) injection and non-surgical oral cannulation with a 16 Ga intravenous cannula. To ensure that tracheal airway tissue receives LV transduction, it was conditioned using our previously described mechanical perturbation protocol in which the tracheal airway surface was rubbed axially with a wire basket (N-Circle, nitinol stone extractor without tip NTSE-022115 ) -UDH , Cook Medical, USA) 30 p28. Then, about 10 minutes after the perturbation in the biosafety cabinet, tracheal administration of LV-MP was performed.
The magnetic field used in this experiment was configured similarly to an in vivo x-ray study, with the same magnets held over the trachea with distillation stent clamps (Figure 4). A 50 µl volume (2 x 25 µl aliquots) of LV-MP was delivered to the trachea (n = 3 animals) using a gel-tipped pipette as described previously. The control group (n = 3 animals) received the same LV-MP without the use of a magnet. After completion of the infusion, the cannula is removed from the endotracheal tube and the animal is extubated. The magnet remains in place for 10 minutes before being removed. Rats were dosed subcutaneously with meloxicam (1 ml/kg) (Ilium, Australia) followed by anesthesia withdrawal by intraperitoneal injection of 1 mg/kg atipamazole hydrochloride (Antisedan, Zoetis, Australia). Rats were kept warm and observed until complete recovery from anesthesia.
LV-MP delivery device in a biological safety cabinet. You can see that the light gray Luer-lock sleeve of the ET tube protrudes from the mouth, and the gel pipette tip shown in the figure is inserted through the ET tube to the desired depth into the trachea.
One week after the LV-MP administration procedure, animals were humanely sacrificed by inhalation of 100% CO2 and LacZ expression was assessed using our standard X-gal treatment. The three most caudal cartilage rings were removed to ensure that any mechanical damage or fluid retention due to endotracheal tube placement would not be included in the analysis. Each trachea was cut lengthwise to obtain two halves for analysis and placed in a cup containing silicone rubber (Sylgard, Dow Inc) using a Minutien needle (Fine Science Tools) to visualize the luminal surface. The distribution and character of the transduced cells were confirmed by frontal photography using a Nikon microscope (SMZ1500) with a DigiLite camera and TCapture software (Tucsen Photonics, China). Images were acquired at 20x magnification (including the maximum setting for the full width of the trachea), with the entire length of the trachea displayed step by step, providing enough overlap between each image to allow images to be “stitched”. The images from each trachea were then combined into a single composite image using Composite Image Editor version 2.0.3 (Microsoft Research) using the planar motion algorithm. The area of LacZ expression within the tracheal composite images from each animal were quantified using an automated MATLAB script (R2020a, MathWorks) as previously described28, using settings of 0.35 < Hue < 0.58, Saturation > 0.15, and Value < 0.7. The area of ​​LacZ expression within the tracheal composite images from each animal were quantified using an automated MATLAB script (R2020a, MathWorks) as previously described28, using settings of 0.35 < Hue < 0.58, Saturation > 0.15, and Value < 0.7. Площадь экспрессии LacZ в составных изображениях трахеи от каждого животного была количественно определена с использованием автоматизированного сценария MATLAB (R2020a, MathWorks), как описано ранее28, с использованием настроек 0,35 <оттенок <0,58, насыщенность> 0,15 и значение <0,7. The area of ​​LacZ expression in composite tracheal images from each animal was quantified using an automated MATLAB script (R2020a, MathWorks) as previously described28 using settings of 0.35<hue<0.58, saturation=”">0.15 and value<0 .7.如前所述，使用自动MATLAB 脚本（R2020a，MathWorks）对来自每只动物的气管复合图像中的LacZ 表达区域进行量化，使用0.35 < 色调< 0.58、饱和度> 0.15 和值< 0.7 的设置。如 前所 述 ， 自动 自动 Matlab 脚本 （（r2020a ， Mathworks） 来自 每 只 的 气管 复合 图像 的 的 的 的 表达 量化 ， 使用 使用 使用 0.35 <色调 <0.58 、> 0.15 和值 <0.7 的。。。。。。。。。。。。。。。。。。。。。。。。。 HIP Области экспрессии LacZ на составных изображениях трахеи каждого животного количественно определяли с использованием автоматизированного сценария MATLAB (R2020a, MathWorks), как описано ранее, с использованием настроек 0,35 <оттенок <0,58, насыщенность> 0,15 и значение <0,7. Areas of LacZ expression on composite images of the trachea of ​​each animal were quantified using an automated MATLAB script (R2020a, MathWorks) as previously described using settings of 0.35 < hue < 0.58, saturation > 0.15 and value < 0.7 . By tracking tissue contours in GIMP v2.10.24, a mask was manually created for each composite image to identify the tissue area and prevent any false detections outside the tracheal tissue. The stained areas from all composite images from each animal were summed to give the total stained area for that animal. The painted area was then divided by the total area of ​​the mask to obtain a normalized area.
Each trachea was embedded in paraffin and sectioned 5 µm thick. Sections were counterstained with neutral fast red for 5 minutes and images were acquired using a Nikon Eclipse E400 microscope, DS-Fi3 camera and NIS element capture software (version 5.20.00).
All statistical analyzes were performed in GraphPad Prism v9 (GraphPad Software, Inc.). Statistical significance was set at p ≤ 0.05. Normality was tested using the Shapiro-Wilk test and differences in LacZ staining were assessed using an unpaired t-test.
The six MPs described in Table 1 were examined by PCXI, and the visibility is described in Table 2. Two polystyrene MPs (MP1 and MP2; 18 µm and 0.25 µm, respectively) were not visible by PCXI, but the remaining samples could be identified (examples are shown in Figure 5). MP3 and MP4 are weakly visible (10-15% Fe3O4; 0.25 µm and 0.9 µm, respectively). Although MP5 (98% Fe3O4; 0.25 µm) contained some of the smallest particles tested, it was the most pronounced. The CombiMag MP6 product is difficult to distinguish. In all cases, our ability to detect MFs was greatly improved by moving the magnet back and forth parallel to the capillary. As the magnets moved away from the capillary, the particles were pulled out in long chains, but as the magnets approached and the magnetic field strength increased, the particle chains shortened as the particles migrated towards the upper surface of the capillary (see Supplemental Video S1: MP4), increasing the particle density at the surface. Conversely, when the magnet is removed from the capillary, the field strength decreases and the MPs rearrange into long chains extending from the upper surface of the capillary (see Supplementary Video S2: MP4). After the magnet stops moving, the particles continue to move for some time after reaching the equilibrium position. As the MP moves towards and away from the upper surface of the capillary, the magnetic particles tend to draw debris through the liquid.
The visibility of MP under PCXI varies considerably between samples. (a) MP3, (b) MP4, (c) MP5 and (d) MP6. All images shown here were taken with a magnet positioned approximately 10 mm directly above the capillary. The apparent large circles are air bubbles trapped in the capillaries, clearly showing the black and white edge features of the phase contrast image. The red box indicates the magnification that enhances the contrast. Note that the diameters of the magnet circuits in all figures are not to scale and are approximately 100 times larger than shown.
As the magnet moves left and right along the top of the capillary, the angle of the MP string changes to align with the magnet (see Figure 6), thus delineating the magnetic field lines. For MP3-5, after the chord reaches the threshold angle, the particles drag along the upper surface of the capillary. This often results in MPs clustering into larger groups near where the magnetic field is strongest (see Supplementary Video S3: MP5). This is also especially evident when imaging close to the end of the capillary, which causes the MP to aggregate and concentrate at the liquid-air interface. The particles in the MP6, which were harder to distinguish than those in the MP3-5, did not drag when the magnet moved along the capillary, but the MP strings dissociated, leaving the particles in view (see Supplementary Video S4: MP6). In some cases, when the applied magnetic field was reduced by moving the magnet a long distance from the imaging site, any remaining MPs slowly descended to the bottom surface of the tube by gravity, remaining in the string (see Supplementary Video S5: MP3).
The angle of the MP string changes as the magnet moves to the right above the capillary. (a) MP3, (b) MP4, (c) MP5 and (d) MP6. The red box indicates the magnification that enhances the contrast. Please note that the additional videos are for informational purposes as they reveal important particle structure and dynamic information that cannot be visualized in these static images.
Our tests have shown that moving the magnet back and forth slowly along the trachea facilitates the visualization of the MF in the context of complex movement in vivo. No in vivo tests were performed because the polystyrene beads (MP1 and MP2) were not visible in the capillary. Each of the remaining four MFs was tested in vivo with the long axis of the magnet positioned over the trachea at an angle of about 30° to the vertical (see Figures 2b and 3a), as this resulted in longer MF chains and was more effective than a magnet. . configuration terminated. MP3, MP4 and MP6 have not been found in the trachea of ​​any live animals. When visualizing the respiratory tract of rats after humanely killing the animals, the particles remained invisible even when additional volume was added using a syringe pump. MP5 had the highest iron oxide content and was the only visible particle, so it was used to evaluate and characterize MP behavior in vivo.
Placement of the magnet over the trachea during MF insertion resulted in many, but not all, MFs being concentrated in the field of view. Tracheal entry of particles is best observed in humanely euthanized animals. Figure 7 and Supplementary Video S6: MP5 shows rapid magnetic capture and alignment of particles on the surface of the ventral trachea, indicating that MPs can be targeted to desired areas of the trachea. When searching more distally along the trachea after MF delivery, some MFs were found closer to the carina, which indicates insufficient magnetic field strength to collect and hold all MFs, since they were delivered through the region of maximum magnetic field strength during fluid administration. process. However, postnatal MP concentrations were higher around the image area, suggesting that many MPs remained in airway regions where the applied magnetic field strength was highest.
Images of (a) before and (b) after delivery of MP5 into the trachea of ​​a recently euthanized rat with a magnet placed just above the imaging area. The depicted area is located between two cartilaginous rings. There is some fluid in the airways before the MP is delivered. The red box indicates the magnification that enhances the contrast. These images are taken from the video featured in S6: MP5 Supplementary Video.
Moving the magnet along the trachea in vivo resulted in a change in the angle of the MP chain on the airway surface, similar to that observed in capillaries (see Figure 8 and Supplementary Video S7: MP5). However, in our study, MPs could not be dragged along the surface of living respiratory tracts, as capillaries could do. In some cases, the MP chain lengthens as the magnet moves left and right. Interestingly, we also found that the particle chain changes the depth of the surface layer of the fluid when the magnet is moved longitudinally along the trachea, and expands when the magnet is moved directly overhead and the particle chain is rotated to a vertical position (see Supplementary Video S7). : MP5 at 0:09, bottom right). The characteristic movement pattern changed when the magnet was moved laterally across the top of the trachea (i.e., to the left or right of the animal, rather than along the length of the trachea). The particles were still clearly visible during their movement, but when the magnet was removed from the trachea, the tips of the particle strings became visible (see Supplementary Video S8: MP5, starting at 0:08). This agrees with the observed behavior of the magnetic field under the action of an applied magnetic field in a glass capillary.
Sample images showing MP5 in the trachea of ​​a live anesthetized rat. (a) The magnet is used to acquire images above and to the left of the trachea, then (b) after moving the magnet to the right. The red box indicates the magnification that enhances the contrast. These images are from the video featured in S7′s Supplementary Video: MP5.
When the two poles were tuned in a north-south orientation above and below the trachea (i.e., attracting; Fig. 3b), the MP chords appeared longer and were located on the lateral wall of the trachea rather than on the dorsal surface of the trachea (see Appendix). Video S9:MP5). However, high concentrations of particles at one site (i.e., the dorsal surface of the trachea) were not detected after fluid administration using a dual magnet device, which usually occurs with a single magnet device. Then, when one magnet was configured to repel opposite poles (Figure 3c), the number of particles visible in the field of view did not increase after delivery. Setting up both two magnet configurations is challenging due to the high magnetic field strength which attracts or pushes the magnets respectively. The setup was then changed to a single magnet parallel to the airways but passing through the airways at a 90 degree angle so that the lines of force crossed the tracheal wall orthogonally (Figure 3d), an orientation intended to determine the possibility of particle aggregation on the lateral wall. be observed. However, in this configuration, there was no identifiable MF accumulation movement or magnet movement. Based on all these results, a configuration with a single magnet and a 30-degree orientation was chosen for in vivo studies of gene carriers (Fig. 3a).
When the animal was imaged multiple times immediately after being humanely sacrificed, the absence of interfering tissue motion meant that finer, shorter particle lines could be discerned in the clear intercartilaginous field, ‘swaying’ in accordance with the translational motion of the magnet. clearly see the presence and movement of MP6 particles.
The titer of LV-LacZ was 1.8 x 108 IU/mL, and after mixing 1:1 with CombiMag MP (MP6), animals were injected with 50 µl of a tracheal dose of 9 x 107 IU/ml of LV vehicle (i.e. 4.5 x 106 TU/rat). ).). In these studies, instead of moving the magnet during labor, we fixed the magnet in one position to determine if LV transduction could (a) be improved compared to vector delivery in the absence of a magnetic field, and (b) if the airway could be focused. The cells being transduced in the magnetic target areas of the upper respiratory tract.
The presence of magnets and the use of CombiMag in combination with LV vectors did not appear to adversely affect animal health, as did our standard LV vector delivery protocol. Frontal images of the tracheal region subjected to mechanical perturbation (Supplementary Fig. 1) showed that the LV-MP treated group had significantly higher levels of transduction in the presence of a magnet (Fig. 9a). Only a small amount of blue LacZ staining was present in the control group (Figure 9b). Quantification of X-Gal-stained normalized regions showed that administration of LV-MP in the presence of a magnetic field resulted in an approximately 6-fold improvement (Fig. 9c).
Example of composite images showing tracheal transduction with LV-MP (a) in the presence of a magnetic field and (b) in the absence of a magnet. (c) Statistically significant improvement in the normalized area of ​​LacZ transduction in the trachea with the use of a magnet (*p = 0.029, t-test, n = 3 per group, mean ± standard error of the mean).
Neutral fast red-stained sections (example shown in Supplementary Fig. 2) indicated that LacZ-stained cells were present in the same sample and in the same location as previously reported.
The key challenge in airway gene therapy remains the precise localization of carrier particles in areas of interest and the achievement of a high level of transduction efficiency in the mobile lung in the presence of airflow and active mucus clearance. For LV carriers intended for the treatment of respiratory diseases in cystic fibrosis, increasing the residence time of the carrier particles in the conductive airways has hitherto been an unattainable goal. As pointed out by Castellani et al., the use of magnetic fields to enhance transduction has advantages over other gene delivery methods such as electroporation because it can combine simplicity, economy, localized delivery, increased efficiency, and shorter incubation time. and possibly a lower dose of vehicle10. However, in vivo deposition and behavior of magnetic particles in the airways under the influence of external magnetic forces has never been described, and in fact the ability of this method to increase gene expression levels in intact living airways has not been demonstrated in vivo.
Our in vitro experiments on the PCXI synchrotron showed that all of the particles we tested, with the exception of the MP polystyrene, were visible in the imaging setup we used. In the presence of a magnetic field, magnetic fields form strings, the length of which is related to the type of particles and the strength of the magnetic field (i.e., the proximity and movement of the magnet). As shown in Figure 10, the strings we observe are formed as each individual particle becomes magnetized and induces its own local magnetic field. These separate fields cause other similar particles to collect and connect with group string motions due to local forces from the local forces of attraction and repulsion of other particles.
Diagram showing (a, b) chains of particles forming inside fluid-filled capillaries and (c, d) an air-filled trachea. Note that the capillaries and trachea are not drawn to scale. Panel (a) also contains a description of the MF containing Fe3O4 particles arranged in chains.
When the magnet moved over the capillary, the angle of the particle string reached the critical threshold for MP3-5 containing Fe3O4, after which the particle string no longer remained in its original position, but moved along the surface to a new position. magnet. This effect likely occurs because the surface of the glass capillary is smooth enough to allow this movement to occur. Interestingly, MP6 (CombiMag) did not behave this way, perhaps because the particles were smaller, had a different coating or surface charge, or the proprietary carrier fluid affected their ability to move. The contrast in the CombiMag particle image is also weaker, suggesting that the liquid and particles may have the same density and therefore cannot easily move towards each other. Particles can also get stuck if the magnet moves too fast, indicating that the magnetic field strength cannot always overcome the friction between particles in the fluid, suggesting that the magnetic field strength and the distance between the magnet and the target area should not come as a surprise. important. These results also indicate that although magnets can capture many microparticles flowing through the target area, it is unlikely that magnets can be relied upon to move CombiMag particles along the surface of the trachea. Thus, we concluded that in vivo LV MF studies should use static magnetic fields to physically target specific areas of the airway tree.
Once the particles are delivered into the body, they are difficult to identify in the context of the complex moving tissue of the body, but their detection capability has been improved by moving the magnet horizontally over the trachea to “wiggle” the MP strings. While real-time imaging is possible, it is easier to discern particle movement after the animal has been humanely killed. MP concentrations were usually highest at this location when the magnet was positioned over the imaging area, although some particles were usually found further down the trachea. Unlike in vitro studies, particles cannot be dragged down the trachea by the movement of a magnet. This finding is consistent with how the mucus that covers the surface of the trachea typically processes inhaled particles, trapping them in the mucus and subsequently clearing them through the muco-ciliary clearance mechanism.
We hypothesized that using magnets above and below the trachea for attraction (Fig. 3b) could result in a more uniform magnetic field, rather than a magnetic field that is highly concentrated at one point, potentially resulting in a more uniform distribution of particles. . However, our preliminary study did not find clear evidence to support this hypothesis. Similarly, setting a pair of magnets to repulse (Fig. 3c) did not result in more particle settling in the image area. These two findings demonstrate that the dual-magnet setup does not significantly improve the local control of MP pointing, and that the resulting strong magnetic forces are difficult to tune, making this approach less practical. Similarly, orienting the magnet above and across the trachea (Figure 3d) also did not increase the number of particles remaining in the imaged area. Some of these alternative configurations may not be successful as they result in a reduction in the magnetic field strength in the deposition zone. Thus, the single magnet configuration at 30 degrees (Fig. 3a) is considered the simplest and most efficient in vivo testing method.
The LV-MP study showed that when LV vectors were combined with CombiMag and delivered after being physically disturbed in the presence of a magnetic field, transduction levels increased significantly in the trachea compared to controls. Based on synchrotron imaging studies and LacZ results, the magnetic field appeared to be able to keep the LV in the trachea and reduce the number of vector particles that immediately penetrated deep into the lung. Such targeting improvements can lead to higher efficiency while reducing delivered titers, non-targeted transduction, inflammatory and immune side effects, and gene transfer costs. Importantly, according to the manufacturer, CombiMag can be used in combination with other gene transfer methods, including other viral vectors (such as AAV) and nucleic acids.