Thank you for visiting Nature.com.The browser version you are using has limited support for CSS.For the best experience, we recommend that you use an updated browser (or turn off compatibility mode in Internet Explorer).In the meantime, to ensure continued support, we will display the site without styles and JavaScript.
Gene vectors for the treatment of cystic fibrosis lung disease should target the conducting airways because peripheral lung transduction does not provide therapeutic benefit.Viral transduction efficiency is directly related to vector residence time.However, delivery fluids such as gene carriers naturally diffuse into the alveoli during inspiration, and therapeutic particles of any form are rapidly cleared by mucociliary transport.Prolonging the residence time of gene carriers in the airways is important but difficult to achieve.Gene carrier-conjugated magnetic particles that can be directed to the surface of the airways can improve regional targeting.Due to the challenges of in vivo visualization, 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 the in vivo motion of a series of magnetic particles in the trachea of anesthetized rats to examine the dynamics and patterns of individual and bulk particle behavior in vivo.We then also assessed whether the delivery of lentiviral magnetic particles in the presence of a magnetic field would increase transduction efficiency in the rat trachea.Synchrotron X-ray imaging reveals the behavior of magnetic particles in stationary and moving magnetic fields in vitro and in vivo.Particles cannot be easily dragged along the surface of the living airway with magnets, but during transport, the deposits are concentrated in the field of view where the magnetic field is strongest.Transduction efficiency was also increased sixfold when lentiviral magnetic particles were delivered in the presence of a magnetic field.Together, these results suggest that lentiviral magnetic particles and magnetic fields may be valuable approaches to improve gene vector targeting and increase transduction levels in conducting airways in vivo.
Cystic fibrosis (CF) is caused by variation 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 conducting airways, a major site of CF pathogenesis.CFTR defects lead to abnormal water transport, dehydrating the airway surface and reducing the depth of the airway surface liquid (ASL) layer.This also impairs the ability of the mucociliary transport (MCT) system to clear inhaled particles and pathogens from the airways.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 capable of measuring these parameters in vivo1.
LV vectors are one of the leading candidates for CF airway gene therapy, mainly because they can permanently integrate the therapeutic gene into the airway basal cells (airway stem cells).This is important because they can restore normal hydration and mucus clearance by differentiating into functional gene-corrected CF-associated airway surface cells, resulting in lifelong benefits.LV vectors should be directed against the conducting airway, as this is where CF lung disease begins.Delivery of the vector deeper into the lung may result in alveolar transduction, but this has no therapeutic benefit in CF.However, fluids such as gene carriers naturally migrate to the alveoli upon inspiration after delivery3,4 and therapeutic particles are rapidly cleared into the oral cavity by MCT.LV transduction efficiency is directly related to the length of time the vector remains next to target cells to allow cellular uptake – the “residence time”5 – which is easily reduced by typical regional airflow as well as coordinated particle mucus capture and MCT.For CF, the ability to prolong the residence time of the LV within the airway is important to achieve high levels of transduction in this region, but has so far been challenging.
To overcome this obstacle, we suggest that LV magnetic particles (MPs) may help in two complementary ways.First, they can be magnetically guided to the airway surface to improve targeting and help the gene carrier particles reside in the desired airway region; and ASL) to move to cell layer 6.MPs have been widely used as targeted drug delivery vehicles when they bind to antibodies, chemotherapeutic drugs, or other small molecules that attach to cell membranes or bind to relevant cell surface receptors and accumulate at tumor sites in the presence of static electricity. Magnetic Fields for Cancer Treatment 7. Other “hyperthermal” techniques aim to heat up MPs when they are exposed to oscillating magnetic fields, thereby destroying tumor cells.The principle of magnetic transfection, in which a magnetic field is used as a transfection agent to enhance the transfer of DNA to cells, is commonly used in vitro using a range of non-viral and viral gene vectors for difficult-to-transduce cell lines.The effectiveness of LV magnetotransfection has been established, with in vitro delivery of LV-MPs to a human bronchial epithelial cell line in the presence of a static magnetic field, increasing transduction efficiency by 186-fold compared to LV vector alone.LV-MP has also been applied to an in vitro CF model, where magnetic transfection increased LV transduction in air-liquid interface cultures by 20-fold in the presence of CF sputum10.However, in vivo magnetotransfection of organs has received relatively little attention and has only been evaluated in a few animal studies11,12,13,14,15, especially in the lungs16,17.Nonetheless, the opportunities for magnetic transfection in CF lung therapy are clear.Tan et al.(2020) stated that “a proof-of-concept study of efficient magnetic nanoparticle pulmonary delivery will pave the way for future CFTR inhalation strategies to improve clinical outcomes in CF patients”6.
The behavior of small magnetic particles on airway surfaces in the presence of an applied magnetic field is difficult to visualize and study, and thus poorly understood.In other studies, we developed a synchrotron-propagation-based phase-contrast X-ray imaging (PB-PCXI) method to noninvasively visualize and quantify minute in vivo changes in ASL depth18 and MCT behavior19,20 to directly measure gas Canal surface hydration and used as an early indicator of treatment efficacy.In addition, our MCT evaluation method uses 10–35 µm diameter particles composed of alumina or high refractive index glass as MCT markers visible using PB-PCXI21.Both techniques are suitable for visualization of a range of particle types, including MP.
Due to its high spatial and temporal resolution, our PB-PCXI-based ASL and MCT analysis techniques are well suited for examining the dynamics and patterns of single and bulk particle behavior in vivo to help us understand and optimize MP gene delivery techniques.The approach we employ here derives from our studies using the SPring-8 BL20B2 beamline, in which we visualized fluid movement following sham vector dose delivery into the nasal and pulmonary airways of mice to help explain our Non-uniform gene expression patterns observed in our gene carrier dose animal studies 3,4 .
The aim of this study was to use the synchrotron PB-PCXI to visualize the in vivo movements of a series of MPs in the trachea of living rats.These PB-PCXI imaging studies were designed to test a range of MPs, magnetic field strengths, and locations to determine their effect on MP motion.We hypothesized that an externally applied magnetic field would help the delivered MP stay or move to the target area.These studies also allowed us to identify magnet configurations that maximize the number of particles retained in the trachea after deposition.In a second series of studies, we sought to use this optimal configuration to demonstrate the transduction pattern resulting from in vivo delivery of LV-MPs to the rat airway, based on the assumption that the delivery of LV-MPs in the context of airway targeting would resulted in improved LV transduction efficiency.
All animal studies were performed according to protocols approved by the University of Adelaide (M-2019-060 and M-2020-022) and the SPring-8 Synchrotron Animal Ethics Committee.Experiments were performed according to ARRIVE guidelines.
All X-ray imaging was performed 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 generate phase contrast effects.A monochromatic beam energy of 25 keV was used.Images were captured using a high-resolution X-ray converter (SPring-8 BM3) coupled to a sCMOS detector.The converter converts X-rays to visible light using a 10 µm thick scintillator (Gd3Al2Ga3O12), which is then directed to a sCMOS sensor using a × 10 microscope objective (NA 0.3).The sCMOS detector was 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 setup yields an effective isotropic pixel size of 0.51 µm and a field of view of approximately 1.1 mm × 1.1 mm.An exposure length of 100 ms was chosen to maximize the signal-to-noise ratio of magnetic particles inside and outside the airway while minimizing breathing-induced motion artifacts.For in vivo studies, a fast X-ray shutter was placed in the X-ray path to limit radiation dose by blocking the X-ray beam between exposures.
The LV carrier 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 suppliers—covering a range of sizes, materials, iron concentrations, and applications—first to understand how magnetic fields affect MP motion within glass capillaries, and then in living airways. on the surface.MPs range in size from 0.25 to 18 μm and are made from a variety of materials (see Table 1), but the composition of each sample, including the size of the magnetic particles within the MP, is unknown.Based on our extensive MCT studies 19, 20, 21, 23, 24, we expect that MPs as small as 5 μm can be seen on the tracheal airway surface, for example by subtracting consecutive frames to see enhanced visibility of MP motion.A single 0.25 μm-sized MP is smaller than the resolution of the imaging device, but PB-PCXI is expected to detect their volume contrast and the motion of the surface fluid on which they are deposited after deposition.
Samples for each MP in Table 1 were prepared in 20 μl glass capillaries (Drummond Microcaps, PA, USA) with an inner diameter of 0.63 mm.Corpuscular particles are available in water, while CombiMag particles are available in the manufacturer’s proprietary fluid.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 sample stage in the imaging box, respectively, and positioned the edges of the fluid.A 19 mm diameter (28 mm long) nickel shell rare earth neodymium iron boron (NdFeB) magnet (N35, cat. no. LM1652, Jaycar Electronics, Australia) with a residual magnetization of 1.17 Tesla was attached to a separate translation stage to achieve Change its position remotely during imaging.X-ray image acquisition begins when the magnet is positioned approximately 30 mm above the sample, and images are acquired at a rate of 4 frames per second.During imaging, the magnet was brought close to the glass capillary tube (about 1 mm away) and then translated along the tube to assess the effects of field strength and position.
In vitro imaging setup containing MP samples in glass capillaries on the sample xy translation stage.The path of the X-ray beam is marked with a red dashed line.
Once the in vitro visibility of MPs was established, a subset of them were tested in vivo in wild-type female albino Wistar rats (~12 weeks old, ~200 g).0.24 mg/kg medetomidine (Domitor®, Zenoaq, Japan), 3.2 mg/kg midazolam (Dormicum®, Astellas Pharma, Japan) and 4 mg/kg butorphanol (Vetorphale®, Meiji Seika) Rats were anesthetized with a mixture of Pharma), Japan) by intraperitoneal injection.After anesthesia, they were prepared for imaging by removing the fur around the trachea, inserting an endotracheal tube (ET; 16 Ga iv cannula, Terumo BCT) and immobilizing them supine on a custom-made imaging plate containing a thermal bag to maintain body temperature 22 .The imaging plate was then attached to the sample translation stage in the imaging box at a slight angle to align the trachea horizontally in the X-ray image, as shown in Figure 2a.
(a) In vivo imaging setup in the SPring-8 imaging box, the path of the X-ray beam is marked with a red dashed line.(b,c) Magnet localization on the trachea was performed remotely using two orthogonally mounted IP cameras.On the left side of the screen image, the wire loop holding the head can be seen, and the delivery cannula in place within the ET tube.
A remote-controlled syringe pump system (UMP2, World Precision Instruments, Sarasota, FL) using a 100 μl glass syringe was connected to PE10 tubing (OD 0.61 mm, ID 0.28 mm) via a 30 Ga needle.Mark the tube to ensure that the tip is in the correct position in the trachea when inserting the ET tube.Using the micropump, the syringe plunger was withdrawn while 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 within the strongest part of our expected applied magnetic field.Image acquisition was controlled using a respiration detector connected to our Arduino based timing box, and all signals (eg temperature, respiration, shutter opening/closing and image acquisition) were recorded using Powerlab and LabChart (AD Instruments, Sydney, Australia) 22. When imaging When the enclosure was inaccessible, two IP cameras (Panasonic BB-SC382) were positioned at approximately 90° to each other and were used to monitor the position of the magnet relative to the trachea during imaging (Fig. 2b,c).To minimize motion artifacts, one image was acquired per breath during the end-tidal flow plateau.
A magnet is attached to a second stage that can be located remotely from outside the imaging housing.Various magnet positions and configurations were tested, including: Mounted at an angle of approximately 30° above the trachea (configurations shown in Figures 2a and 3a); one magnet above the animal and the other below, with poles set to attract (Figure 3b ); one magnet above the animal and the other below, with the poles set to repel (Figure 3c); and one magnet above and perpendicular to the trachea (Figure 3d).Once the animal and magnet are configured and the MP to be tested is loaded into the syringe pump, deliver a 50 μl dose at a rate of 4 μl/sec while acquiring images.The magnet is then moved back and forth along or laterally across the trachea while continuing to acquire images.
Magnet configuration for in vivo imaging (a) a single magnet above the trachea at an angle of approximately 30°, (b) two magnets set to attract, (c) two magnets set to repel, (d) a single magnet above and perpendicular in the trachea.The observer looked down from the mouth to the lungs through the trachea, and the X-ray beam passed through the rat’s left side 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 confounding breathing and cardiac motion.Therefore, at the end of the imaging period, animals were humanely killed for pentobarbital overdose (Somnopentil, Pitman-Moore, Washington Crossing, USA; ~65 mg/kg ip).Some animals were left on the imaging platform, and once breathing and heartbeat stopped, the imaging process was repeated, adding an additional dose of MP if no MP was visible on the airway surface.
The acquired images were flat-field and dark-field corrected and then assembled into a movie (20 frames per second; 15-25 × normal speed depending on respiratory rate) using a custom script written in MATLAB (R2020a, The Mathworks).
All LV gene vector delivery studies were conducted at the Laboratory Animal Research Facility at the University of Adelaide 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 assess the effects of MP and magnetic field, two groups of animals were treated: one group was given LV-MP with a magnet placed, and the other group received a control group with LV-MP without a magnet.
LV gene vectors were generated using previously described methods 25, 26 .The LacZ vector expresses the nuclear-localized beta-galactosidase gene driven by the constitutive MPSV promoter (LV-LacZ), which produces a blue reaction product in transduced cells, visible in lung tissue fronts and tissue sections.Titration was performed in cell cultures by manually counting the number of LacZ positive cells with 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 at a 1:1 ratio and incubating on ice for at least 30 minutes prior to delivery.
Normal Sprague Dawley rats (n = 3/group, ~2-3 were anesthetized intraperitoneally with a mixture of 0.4 mg/kg medetomidine (Domitor, Ilium, Australia) and 60 mg/kg ketamine (Ilium, Australia) month-old) ip) injection and non-surgical oral cannulation with a 16 Ga iv cannula.To ensure that the 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 Tipless Stone Extractor NTSE-022115) -UDH, Cook Medical, USA) 30 s28.Tracheal administration of LV-MP was then performed in a biological safety cabinet approximately 10 minutes after perturbation.
The magnetic field used in this experiment was configured in a similar manner to the in vivo X-ray imaging study, with the same magnets held above the trachea using distillation stent clips (Figure 4).A 50 μl volume (2 × 25 μl aliquots) of LV-MP was delivered into the trachea (n = 3 animals) using a pipette containing a gel tip as previously described.A control group (n = 3 animals) received the same LV-MPs without the use of a magnet.After the infusion is complete, the cannula is removed from the ET tube and the animal is extubated.The magnet remains in place for 10 minutes, then it is removed.Rats received a subcutaneous dose of meloxicam (1 ml/kg) (Ilium, Australia) followed by reversal of anesthesia by ip injection of 1 mg/kg atipamazole hydrochloride (Antisedan, Zoetis, Australia).Rats were kept warm and monitored until full recovery from anesthesia.
LV-MP delivery device in a biological safety cabinet.The light grey Luer hub of the ET tube can be seen protruding from the mouth and the gel tip of the pipette shown in the picture is inserted through the ET tube to the desired depth into the trachea.
One week after the LV-MP dosing procedure, animals were humanely killed by 100% CO2 inhalation and LacZ expression was assessed using our standard X-gal treatment.The three caudal most cartilaginous rings were removed to ensure that any mechanical damage or fluid retention from endotracheal tube placement was not included in the analysis.Each trachea was cut longitudinally to create two halves for analysis, and they were mounted in a dish containing silicone rubber (Sylgard, Dow Inc) using a Minutien needle (Fine Science Tools) to visualize the luminal surface.The distribution and pattern of 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 highest setting for the full width of the trachea), with the entire length of the trachea imaged step-by-step, ensuring sufficient overlap between each image to allow for image “stitching”.Images from each trachea were then assembled into a single composite image using the Image Composite Editor v2.0.3 (Microsoft Research) utilizing a planar motion algorithm.LacZ expression areas in composite images of the trachea from 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 tracing the contours of the tissue, a mask was manually generated in GIMP v2.10.24 for each composite image in order to identify the tissue area and prevent any false detections from outside the tracheal tissue.The stained areas from all composite images from each animal were summed to generate the total stained area for that animal.The stained area was then divided by the total mask area to generate the normalized area.
Each trachea was embedded in paraffin and 5 μm sections were cut.Sections were counterstained with neutral fast red for 5 min and images were acquired using a Nikon Eclipse E400 microscope, DS-Fi3 camera and NIS element capture software (version 5.20.00).
All statistical analyses were performed in GraphPad Prism v9 (GraphPad Software, Inc.).Statistical significance was set at p ≤ 0.05.Normality was verified using the Shapiro-Wilk test, and differences in LacZ staining were assessed using the unpaired t-test.
The six MPs described in Table 1 were examined using 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 under PCXI, but the rest of the samples were identifiable (examples are shown in Figure 5).MP3 and MP4 (10-15% Fe3O4; 0.25 μm and 0.9 μm, respectively) are faintly visible.Although containing some of the smallest particles tested, MP5 (98% Fe3O4; 0.25 μm) was the most pronounced.The CombiMag product MP6 is hard to spot.In all cases, our ability to detect MP was significantly enhanced by translating the magnet back and forth parallel to the capillary.When the magnets moved away from the capillary, the particles extended in long strings, but as the magnets got closer and the magnetic field strength increased, the particle strings shortened as the particles migrated toward the top surface of the capillary (see Supplementary Video S1: MP4), increasing The particle density of the surface.Conversely, when the magnet is removed from the capillary, the field strength decreases and the MPs rearrange into long strings extending from the upper surface of the capillary (see Supplementary Video S2:MP4).After the magnet stops moving, the particles continue to move for a short time after reaching the equilibrium position.As the MP moves toward and away from the top surface of the capillary, the magnetic particles typically drag the debris through the fluid.
The visibility of MP under PCXI varies significantly between samples.(a) MP3, (b) MP4, (c) MP5 and (d) MP6.All images shown here were taken with a magnet located 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 phase contrast imaging.The red box contains the contrast-enhancing magnification.Note that the diameters of the magnet schematics in all figures are not to scale and are approximately 100 times larger than shown.
As the magnet is translated 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 a threshold angle, the particles are dragged along the top surface of the capillary.This often results in MPs clustering into larger groups close to where the magnetic field is strongest (see Supplementary Video S3:MP5).This is also particularly evident when imaging close to the capillary end, which causes MPs to aggregate and concentrate at the fluid-air interface.Particles in MP6, which were more difficult to discern than MP3-5, were not dragged as the magnet moved along the capillary, but the MP strings dissociated, leaving the particles in the field of view (see Supplementary Video S4:MP6).In some cases, when the applied magnetic field was reduced by moving the magnet a large distance from the imaging location, any remaining MPs slowly descended to the bottom surface of the tube by gravity while remaining in the string (see Supplementary Video S5: MP3).
The angle of the MP string changes as the magnet is translated to the right above the capillary.(a) MP3, (b) MP4, (c) MP5 and (d) MP6.The red box contains the contrast-enhancing magnification.Note that the supplementary videos are informative as they reveal important particle structure and dynamic information that cannot be visualized in these static images.
Our tests showed that moving the magnet slowly back and forth along the trachea facilitates the visualization of MP in the context of complex movement in vivo.In vivo testing was not performed as polystyrene beads (MP1 and MP2) were not visible in the capillary.Each of the remaining four MPs were tested in vivo with the magnet long axis configured above the trachea at an angle of about 30° to vertical (see Figures 2b and 3a), as this resulted in longer MP chains and was more effective than the magnet configuration terminated.MP3, MP4 and MP6 were not detected in the trachea of any live animals.When the rat airways were imaged after the animals were humanely killed, 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, and was therefore used to assess and characterize the in vivo behavior of MP.
Placing the magnet over the trachea during MP delivery resulted in many, but not all, MPs being concentrated in the field of view.Particles entering the trachea are best observed in humanely sacrificed 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 directed to desired regions of the trachea.When searching more distally along the trachea after MP delivery, some MPs were found closer to the carina, suggesting that the magnetic field strength was insufficient to collect and retain all MPs, as they were delivered through the region of maximum magnetic field strength during the fluid process.Nonetheless, postpartum MP concentrations were higher around the imaged area, suggesting that many MPs remained in the airway regions where the applied magnetic field strength was highest.
Images from (a) before and (b) after delivery of MP5 into the trachea of a recently euthanized rat with the magnet positioned directly above the imaging area.The imaged area is located between the two cartilage rings.Before MP delivery, there is some fluid in the airway.The red box contains the contrast-enhancing magnification.These images are from the video shown in Supplementary Video S6:MP5.
Translating the magnet along the trachea in vivo caused the MP chain to change angle within the airway surface in a manner similar to that seen in capillaries (see Figure 8 and Supplementary Video S7:MP5).However, in our study, MPs could not be dragged along the surface of the living airway as they could with capillaries.In some cases, the MP chain will get longer as the magnet moves left and right.Interestingly, we also found that the particle string appears to change the depth of the surface fluid layer when the magnet is moved longitudinally along the trachea, and expands when the magnet is moved directly overhead and the particle string is rotated to a vertical position (see Supplementary Video S7). : MP5 at 0:09, bottom right).The characteristic pattern of motion changed when the magnet was translated across the top of the trachea laterally (that is, to the left or right of the animal rather than along the length of the trachea).The particles were still clearly visible as they moved, 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 is consistent with the MP behavior we observed under an applied magnetic field in a glass capillary.
Example 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 the magnet is moved to the right.The red box contains the contrast-enhancing magnification.These images are from the video shown in Supplementary Video S7:MP5.
When the two poles were configured 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 sidewall of the trachea rather than on the dorsal tracheal surface (see Supplementary Video S9:MP5).However, high concentrations of particles at a single location (ie, the dorsal surface of the trachea) were not detected after fluid delivery when a dual-magnet device was used, which typically occurs when a single-magnet device is used.Then when one magnet was configured to repel the poles reversed (Fig. 3c), the number of particles visible in the field of view did not appear to increase after delivery.The setup of both dual-magnet configurations is challenging due to the high magnetic field strengths that pull or push the magnets, respectively.The setup was then changed to a single magnet parallel to the airway but passing through the airway at 90 degrees so that the field lines crossed the tracheal wall orthogonally (Fig. 3d), an orientation designed to determine if particle aggregation on the side wall could be observed .However, in this configuration, there was no identifiable movement of MP accumulation or magnet movement.Based on all these results, a single-magnet, 30-degree orientation configuration (Figure 3a) was chosen for in vivo gene carrier studies.
When the animal was repeatedly imaged immediately after humane killing, the absence of confounding tissue motion meant that finer and shorter particle lines could be discerned in the clear interchondral field, “wobbly” in line with the translational motion of the magnet. Nonetheless , still cannot clearly see the presence and motion of MP6 particles.
The LV-LacZ titer was 1.8 × 108 TU/ml, and after 1:1 mixing with CombiMag MP (MP6), animals received a 50 μl tracheal dose of 9 × 107 TU/ml LV vehicle (i.e. 4.5 × 106 TU/rat). ).In these studies, instead of translating the magnet during labor, we fixed the magnet in one position to determine whether LV transduction (a) could be improved compared to vector delivery in the absence of a magnetic field, and (b) could be focused Airway cells are transduced to magnetic target regions of the upper airway.
The presence of magnets and the use of CombiMag combined with LV vectors did not appear to have adverse effects on animal health, as did our standard LV vector delivery protocol.Frontal images of the tracheal region subjected to mechanical perturbation (Supplementary Fig. 1) indicated that there were significantly higher levels of transduction in the group of animals treated with LV-MP when the magnet was present (Fig. 9a).Only a small amount of blue LacZ staining was present in the control group (Fig. 9b).Quantification of normalized X-Gal stained areas showed that administration of LV-MP in the presence of a magnetic field produced an approximately 6-fold improvement (Fig. 9c).
Example composite images showing tracheal transduction by LV-MP (a) in the presence of a magnetic field and (b) in the absence of a magnet.(c) Statistically significant improvement in normalized LacZ transduction area within the trachea when using the magnet (*p = 0.029, t-test, n = 3 per group, mean ± SEM).
Neutral fast red-stained sections (example shown in Supplementary Fig. 2) showed LacZ-stained cells present in a similar pattern and location as previously reported.
A key challenge for airway gene therapy remains the accurate localization of carrier particles to regions of interest and achieving high levels of transduction efficiency in the moving lung in the presence of airflow and active mucus clearance.For LV carriers designed to treat CF airway disease, increasing the residence time of carrier particles within the conducting airways has been a hitherto elusive goal.As pointed out by Castellani et al., the use of magnetic fields to improve transduction has advantages compared to other gene delivery methods such as electroporation, as it can combine simplicity, cost-effectiveness, delivery localization, increased efficiency, and shorter incubation times , and possibly a smaller carrier dose10.However, the in vivo deposition and behavior of magnetic particles in the airways under the influence of external magnetic forces has never been described, nor has actually the feasibility of this method been demonstrated in vivo to enhance gene expression levels in intact living airways.
Our in vitro synchrotron PCXI experiments showed that all particles we tested, with the exception of polystyrene MP, were visible in the imaging setup we used.In the presence of a magnetic field, MPs form strings whose lengths are related to particle type and magnetic field strength (i.e. proximity and motion of the magnet).As shown in Figure 10, the strings we observe are formed due to each individual particle being magnetized and inducing its own local magnetic field.These separate fields cause other similar particles to aggregate and connect, with group string-like motions due to local forces from the local attractive and repulsive forces of other particles.
Schematic showing (a,b) particle trains generated inside fluid-filled capillaries and (c,d) air-filled trachea.Note that the capillaries and trachea are not drawn to scale.Panel (a) also contains a description of the MP, which contains Fe3O4 particles arranged in strings.
When the magnet was moved above the capillary, the angle of the particle string reached a critical threshold for MP3-5 containing Fe3O4, after which the particle string no longer stayed in the original position, but moved along the surface to a new position.magnet.This effect is likely to occur because the glass capillary surface is smooth enough to allow this movement to occur.Interestingly, MP6 (CombiMag) did not behave this way, possibly because the particles were smaller, had different coatings or surface charges, or a proprietary carrier fluid affected their ability to move.The image contrast of CombiMag particles is also weaker, suggesting that the fluid and particles may have similar densities and therefore not easily move toward 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 perhaps it is not surprising that the magnetic field strength and the distance between the magnet and the target area Very important.Taken together, these results also suggest that, while magnets can capture many MPs that flow through the target area, it is unlikely that magnets can be relied on to move CombiMag particles along the surface of the trachea.Therefore, we conclude that in vivo LV-MP studies should utilize static magnetic fields to physically target specific regions of the airway tree.
When particles are delivered in the body, they are difficult to identify in the context of complex moving body tissue, but the ability to detect them was enhanced by translating the magnet horizontally above the trachea to “wiggle” the MP strings.Although live imaging is possible, it is easier to discern particle motion once the animal has been humanely killed.MP concentrations were generally highest at this location when the magnet was positioned above the imaging area, although some particles were usually found further along the trachea.In contrast to in vitro studies, particles cannot be dragged along the trachea by translating the magnet.This finding is consistent with how the mucus that coats the surface of the trachea typically processes inhaled particles, trapping them in the mucus and subsequently cleared by the mucociliary clearance mechanism.
We hypothesized that the use of magnets for attraction above and below the trachea (Fig. 3b) might result in a more uniform magnetic field, rather than a magnetic field that is highly concentrated at one point, potentially leading to a more uniform distribution of particles.However, our preliminary study did not find clear evidence to support this hypothesis.Likewise, configuring a pair of magnets to repel (Fig. 3c) did not result in more particle deposition in the imaged area.These two findings demonstrate that the dual-magnet setup does not significantly improve local control of MP targeting, and that the resulting strong magnetic forces are difficult to configure, making this approach less practical.Similarly, orienting the magnet above and through the trachea (Fig. 3d) also did not increase the number of particles retained in the imaged area.Some of these alternative configurations may not be successful because they result in lower magnetic field strengths within the deposition area.Therefore, the single 30-degree angle magnet configuration (Figure 3a) is considered the easiest and most efficient method for in vivo testing.
The LV-MP study showed that when LV vectors were combined with CombiMag and delivered after physical perturbation in the presence of a magnetic field, transduction levels were significantly increased in the trachea compared to controls.Based on the synchrotron imaging studies and LacZ results, the magnetic field was apparently able to preserve the LV within the trachea and reduce the number of vector particles that immediately penetrated deep into the lung.Such targeting improvements may lead to higher efficacy while reducing delivered titers, off-target transduction, inflammatory and immune side effects, and gene carrier costs.Importantly, according to the manufacturer, CombiMag can be used in conjunction with other gene transfer methods, including with other viral vectors (such as AAV) and nucleic acids.