Design of a Biocompatible Drug-Eluting Tracheal Stent in Mice with Laryngotracheal Stenosis

Summary

Laryngotracheal stenosis results from pathologic scar deposition that critically narrows the tracheal airway and lacks effective medical therapies. Using a PLLA-PCL (70% poly-L-lactide and 30% polycaprolactone) stent as a local drug delivery system, potential therapies aimed at decreasing scar proliferation in the trachea can be studied.

Abstract

Laryngotracheal stenosis (LTS) is a pathologic narrowing of the subglottis and trachea leading to extrathoracic obstruction and significant shortness of breath. LTS results from mucosal injury from a foreign body in the trachea, leading to tissue damage and a local inflammatory response that goes awry, leading to the deposition of pathologic scar tissue. Treatment for LTS is surgical due to the lack of effective medical therapies. The purpose of this method is to construct a biocompatible stent that can be miniaturized to place into mice with LTS. We demonstrated that a PLLA-PCL (70% poly-L-lactide and 30% polycaprolactone) construct had optimal biomechanical strength, was biocompatible, practicable for an in vivo placement stent, and capable of eluting drug. This method provides a drug delivery system for testing various immunomodulatory agents to locally inhibit inflammation and reduce airway fibrosis. Manufacturing the stents takes 28−30 h and can be reproduced easily, allowing for experiments with large cohorts. Here we incorporated the drug rapamycin within the stent to test its effectiveness in reducing fibrosis and collagen deposition. Results revealed that PLLA-PCL tents showed reliable rapamycin release, were mechanically stable in physiological conditions, and were biocompatible, inducing little inflammatory response in the trachea. Further, the rapamycin-eluting PLLA-PCL stents reduced scar formation in the trachea in vivo.

Introduction

Laryngotracheal stenosis (LTS) is a pathologic narrowing of the trachea most often due to iatrogenic post-intubation injury. The combination of bacterial colonization, foreign body response to a tracheostomy or endotracheal tube, and patient-specific factors lead to an aberrant inflammatory response. This maladaptive immune response leads to the deposition of collagen in the trachea, resulting in luminal narrowing of the trachea and subsequent stenosis^1,2. As current treatment for this disease is primarily surgical, developing an alternative medically-based treatment paradigm targeting the aberrant inflammatory and profibrotic pathways that lead to excessive collagen deposition has been studied. Rapamycin, which inhibits the mTOR signaling complex, has been shown to have immunosuppressive effects as well as a robust antifibroblast effect. However, when rapamycin is systemically administered, common side effects (e.g., hyperlipidemia, anemia, thrombocytopenia) can be pronounced³. The purpose of our methodology is to develop a vehicle for local drug delivery practicable for use in the airway that would lessen these systemic effects. Our assessments focus on investigating the local immune response to the drug delivery construct as well as its capacity to inhibit fibroblast function and alter the local immune microenvironment. Disease-specific outcomes include in vivo testing that evaluate markers of fibrosis.

Biodegradable drug-eluting stents have been used in animal models of disease in multiple organ systems, including the airway⁴. For the management of airway stenosis or collapse, previous investigations have used drug-coated silicone and nickel-based stents⁵. A PLLA-PCL construct was chosen for this particular method because of its drug elution profile and mechanical strength in physiological conditions over a period of 3 weeks, which has been demonstrated in previous published studies⁶. PLLA-PCL is also a biocompatible and biodegradable material already approved by the FDA⁴. Biocompatible stents eluting cisplatin and MMC have been studied in large animal models such as rabbits and dogs. However, in these animal models, stents were not placed in an animal model of disease and were implanted transcervically. This study provides a unique method for assessing a biocompatible drug-eluting stent placed transorally in a mouse model of airway injury and laryngotracheal stenosis. A biocompatible stent that elutes an immunomodulatory drug locally and can be miniaturized for study in a murine model is valuable for translational preclinical research. Previous attempts at stent utilization with other material constructs generated robust foreign body responses worsening the underlying inflammation that distinguishes LTS⁷. This methodology, to our knowledge, is the first of its kind to study the immunomodulatory and antifibrotic effects of a stent-based drug delivery system in a murine model of LTS. The murine model itself offers several advantages for studying the effects of an immunomodulatory drug on the trachea. Genetically modified mice and experimental cohorts of healthy and diseased mice can be studied, which can lead to experimental reproducibility and improve cost-effectiveness. Moreover, the delivery of the stent transorally into the mouse trachea mimics clinical delivery of such a stent in humans, which further highlights the translational advantage of this method. Finally, the relative ease with which the PLLA-PCL stent with the drug can be produced allows for modifications to deliver alternate drug therapies aimed at reducing scar formation in the trachea.

Protocol

NOTE: All methods described here were approved by the Johns Hopkins University Animal Care and Use Committee (MO12M354).

1. Preparation of rapamycin in PLLA-PCL

1. Prepare two glass vials (with caps) of 70:30 PLLA-PCL polymer solutions (inherent viscosity 1.3−1.8 DL/G; Table of Materials) solutions, with one vial containing 1.0% rapamycin and the other without rapamycin.
   1. Make a 1.0% rapamycin containing polymer solution by adding 6 mg of rapamycin to 600 mg of 70:30 PLLA-PCL in a glass vial.
   2. For polymer solution without rapamycin (control), only add 600 mg of 70:30 PLLA-PCL to the glass vial.
2. Under a fume hood, add 6 mL of dichloromethane to each glass vial.
   CAUTION: Dichloromethane is a corrosive material and only glass pipettes should be used. Appropriate safety precautions include personal eye protection and gloves.
3. Add 120 µL of glycerol to each glass vial (for 2% glycerol solution).
   NOTE: The addition of glycerol allows for increased flexibility and decreased stiffness in the stent construct.
4. Recap the glass vials and allow 70:30 PLLA-PCL with and without rapamycin to dissolve and homogenize for 6−12 h.
   NOTE: Glass vials can also be placed on a rotating shaking platform for faster dissolution.

2. Rapamycin elution testing

1. Pipette 1 mL of 70:30 PLLA-PCL solution containing 1% rapamycin into a glass Petri dish.
   NOTE: This volume of 70:30 PLLA-PCL solution containing 1% rapamycin will contain 120 µg of rapamycin and represents the total amount of rapamycin in each construct. Alternative volumes and concentrations can be used.
2. Allow 70:30 PLLA-PCL with 1% rapamycin to harden into a flat disk in the glass Petri dish.
3. Once hardened, place the disk in 2 mL of phosphate-buffered saline (PBS, pH 7.4) and incubate in a 37 °C chamber.
4. Collect and replace PBS (2 mL) every 24 h and use collected PBS for high-performance liquid chromatography (HPLC) analysis of rapamycin content.
5. Use an autosampler and a C₁₈ 4.6 cm x 250 mm HPLC column to run samples through the autosampler injector along with serial dilutions of rapamycin to create a calibrated standard curve. Run each sample in triplicate.
   NOTE: Serial dilutions of rapamycin to be tested include 10%, 1%, 0.1%, and 0.01%.
6. Use a mobile phase of HPLC grade water and acetonitrile with 10/90 volume/volume with a flow rate of 2.0 mL/min. Set absorbance during HPLC for rapamycin to 280 nm.
   NOTE: Figure 1 shows the elution of rapamycin over a 14 day period.

3. Creation of rapamycin-eluting PLLA-PCL murine airway stents

NOTE: Perform steps 3.2−3.9 using sterile materials and sterile technique to avoid contamination that would influence in vivo and in vitro applications.

1. Prepare PLLA-PCL solutions containing rapamycin as described in section 1.
2. Using a glass Pasteur pipette with a rubber balloon, apply 1 mL of PLLA-PCL solution containing 1% rapamycin onto the venous cannula of a 22 G fluorinated ethylene propylene based angiocatheter (Table of Materials). Hold the angiocatheter in one hand and use the glass pipette to drop the PLLA-PCL solution onto the angiocatheter. Slowly rotate the angiocatheter to ensure a homogenous coverage of the angiocatheter with the PLLA-PCL solution.
   NOTE: At first, the PLLA-PCL solution will be thin, but as the dichloromethane evaporates during the application onto the angiocatheter, the solution will become more viscous to mold onto the angiocatheter. Continuously turning the angiocatheter during the application is beneficial. This step must be done slowly and meticulously to ensure the consistent thickness of the stent.
3. Prop the molded angiocatheter with the tip of the angiocatheter facing downward and the hilt on the edge of a glass Petri dish for drying.
4. Allow stents to dry for 24 h in a vacuum hood at room temperature (Figure 2A).
5. Remove the stent construct from the underlying angiocatheter by gently twisting the underlying catheter free and sliding it out of the molded stent (Figure 2B).
6. Check the stent circumferentially for any defects that may have resulted during the casting process. If a defect in the casted stent is present, discard it.
7. Trim each end of the casted stent with fine straight scissors so that the edges are axial.
8. Using fine straight scissors, cut 3 mm axial segments of the cast stent for use in a mouse model of LTS (Figure 2C).
   NOTE: Approximately 8 stents can be made from one casted angiocatheter, with each 3 mm stent containing 120 µg of rapamycin.
9. Load the 3 mm stent onto a new 22 venous catheter for use in a mouse model of LTS (Figure 1).

4. Laryngotracheal stenosis induction in mice

1. Prior to conducting in vivo mouse studies obtain approval from the Animal Care and Use Committee.
2. Anesthetize a 9-week old male C57BL/6 by giving an intraperitoneal injection of ketamine (80−100 mg/kg) and xylazine (5−10 mg/kg).
   NOTE: Other strains of mice can be substituted, but it is recommended that the mice weight be between 20−27 g.
3. Randomize mice into an experimental group (chemomechanical injury with a placement of a 1% rapamycin-containing PLLA-PCL stent) and two control groups: 1) chemomechanical injury with the placement of a PLLA-PCL stent, 2) chemomechanical injury without the placement of a stent.
4. Place a mouse on a surgical platform in a supine position. Use a small loop of thread affixed to the top of the platform to extend the cervical spine by looping it around the central incisors.
5. Affix the hands and legs of the mouse to the table using 2 inch pieces of tape. Pinch mouse paw to ensure it has had adequate anesthesia.
6. The surgical site is then prepped. The overlying fur should be removed and the skin should be cleaned (alternating iodine or chlorhexidine scrub with alcohol or diluted skin disinfectant) three times. The area should be draped and sterile gloves be worn. The sterilized instruments should be laid on a sterile surface when not in use.
7. Make a 1.5 cm midline vertical incision in the neck of the mouse using fine curved iris scissors. Divide the overlying thymus and lateralize the two resulting lobes to visualize the trachea. Divide the overlying sternohyoid and sternothyroid (strap) muscles at the superior attachment bilaterally.
   NOTE: The entire laryngotracheal complex should be completely exposed after this.
8. Pass a 22 G angiocatheter transorally through the larynx into the trachea. Use small forceps to apply pressure to the anterior larynx of the mouse to assist in the correct placement of the angiocatheter. Visualize the white angiocatheter through the trachea to ensure correct placement (non-esophageal).
   NOTE: The mouse trachea is extremely thin and the white angiocatheter will be visualized through the translucent trachea.
9. Pass a bleomycin-coated wire brush through the inserted angiocatheter.
10. Slowly withdraw the angiocatheter so that only the wire brush remains in the trachea.
11. Apply counter pressure using fine forceps on the trachea to mechanically disrupt the tracheal lumen with the wire brush.
12. Reinsert the angiocatheter into the trachea over the wire brush.
13. Remove the wire brush and reapply bleomycin.
14. Repeat steps 4.8−4.12 a total of 5x. Apply bleomycin to the brush between each application.
    NOTE: Validation and description of this model can be found in Hillel et al.⁸.
15. Remove the angiocatheter from the mouse trachea.
    NOTE: If no stent is to be placed, the incision can be closed with tissue glue.

5. Transoral PLLA-PCL stent placement in mice

1. Load one 3 mm casted stent onto an empty 22 G angiocatheter (Figure 3A).
   NOTE: The stent should rest 5 mm from the tip of the angiocatheter.
2. Draw a thin black vertical line on the 3 mm casted stent.
   NOTE: This line allows for improved visualization of the stent in the trachea.
3. Transorally intubate the mouse with the angiocatheter that has been preloaded with the stent.
4. Visualize the stent on the angiocatheter in the trachea through the transcervical incision (Figure 3B).
   NOTE: The trachea is very thin, allowing the black dye on the stent to be visualized through the trachea. Use this to confirm correct tracheal placement.
5. Hold the stent in place by gripping the trachea through the transcervical incision with fine forceps.
6. Remove the angiocatheter transorally while maintaining a grip on the stent with the fine forceps.
   NOTE: It is extremely important not to apply too much force to the stent to not crush the lumen when the angiocatheter is removed. However, enough force is required to ensure the stent is not removed with the angiocatheter. A 0.8 mm sialendoscope can be used to visualize the location and placement of the stent in the trachea.
7. Close the transcervical incision with tissue glue.
8. Allow each mouse to recover to its original activity level before placing it back in its cage.

6. Histologic preparation of samples

1. Anesthetize mice with inhaled anesthetic and sacrifice mice with cervical dislocation per animal protocol after 7, 14, or 21 days.
   NOTE: Based on the chronicity of the study, different time intervals can be used (e.g., 28, 30 days, etc.).
2. Position a mouse on the surgical platform as described in steps 4.4 and 4.5.
3. Reopen the neck incision on the mouse using fine curved iris scissors.
4. Expose the trachea per step 4.6.
5. Divide the distal trachea below the level of the stent using fine curved iris scissors.
6. Divide the proximal trachea below the larynx and above the stent using fine curved iris scissors.
   NOTE: It is important to divide the distal trachea first to prevent the trachea from retracting into the thorax.
7. Separate the divided trachea from the esophagus posteriorly and remove trachea from the mouse.
   NOTE: Stents will still be retained within the mouse trachea.
8. Remove stent from the trachea and fix in 10% formalin for 24 h.
9. Embed formalin-fixed mouse trachea specimens in paraffin with the distal end oriented superiorly so that axial cuts of the trachea can be made in the next step.
10. Cut 5 μm sections of the paraffin-embedded mouse trachea using a microtome.
    NOTE: Specimens should be cut axially starting from the distal trachea.
11. Obtain a 5 μm representative section every 250 μm along the length of the specimen.
12. Complete H&E stain on each representative section.
    1. Deparaffinize the slides by placing in xylene 2x per slide for 3 min.
    2. Place the slides in 100% ethanol for 2 min, 95% ethanol for 2 min, and 70% ethanol for 2 min to rehydrate.
    3. Wash the slides with running tap water for 2 min.
    4. Stain the slides in hematoxylin for 3−5 min.
    5. Wash the slides with running tap water for 5 min.
    6. Place the slides in 1% acid alcohol for 1 min.
    7. Wash the slides with running tap water for 1 min.
    8. Rinse in 0.2% ammonia water for 30 s.
    9. Rinse the slides in running tap water for 5 min. Dip in 95% ethanol 10x.
    10. Eosin stain by placing slide in eosin phloxine for 30 s.
    11. Dehydrate the slides by placing in 95% ethanol for 5 min, followed by absolute ethanol 2x for 5 min.
    12. Place in xylene 2x for 5 min.
    13. Mount the slides with xylene-based mounting medium.
13. Measure the thickness of the lamina propria as described in Hillel et al.⁸.

7. Stent biocompatibility in vivo

1. Prepare tissue sections as in steps 6.1−6.4. Do not remove stent from these tracheal sections in order to visualize the changes in inflammation relative to the location of the stent within the lumen of the trachea.
   1. To determine the acute foreign body response to the stent, observe macrophage and T cell activity using CD3 and F4/80 markers.
      NOTE: Other markers can also be used to determine acute inflammatory reaction to the stent.
   2. Obtain 5 μm cut sections of the paraffin-embedded trachea on commercially available hydrophilic plus slides (Table of Materials). Place two sections on each slide.
      NOTE: These sections should be from an area of the trachea where the PLLA-PCL stent was placed.
2. Place the slides in xylene 2x for 5 min each.
3. Place the slides in 100% ethanol 2x for 3 min each.
4. Place the slides in 95% ethanol for 1 min.
5. Place the slides in a histology staining rack so that they are immersed in antigen retrieval buffer (Table of Materials) and then place in a vegetable steamer with boiling water for 20 min.
6. Remove the staining racks from the steamer and discard the antigen retrieval buffer.
7. Replace the buffer with 1 mL of PBS.
8. Place the slides in a wet staining box for 1 min.
9. Discard the PBS and incubate the slides with Dulbecco's modified eagle medium (DMEM) with 10% FBS for 30 min.
10. Discard the DMEM and add a mixture of two primary antibodies raised in different species. Cover the slides with paraffin film and incubate overnight at 4 °C in a dark room.
    NOTE: In this protocol rabbit anti-CD3 and rat anti-F4/80 (Table of Materials) were used.
11. Wash the slides in PBS 3x for 5 min.
12. Incubate the slides with secondary antibodies specific to the primary antibody species (Table of Materials) diluted in DMEM with 10% FBS for 0.5−1 h at room temperature in the dark.
13. Wash the slides in PBS 3x for 5 min in the dark.
14. Mount the slides on coverslips with a drop of mounting medium with 4'6-diamidino-2-phenylindole (DAPI). Store the slides in the dark at either 20 °C or 4 °C.
15. Observe the stained slides on a laser scan confocal microscope and photograph.
16. Quantify the total number of cells staining with DAPI and antibodies of interest for comparison to uninjured trachea.

8. Mouse trachea quantitative gene expression analysis

1. Collect mouse trachea as described in steps 6.1−6.7 and remove stent.
   NOTE: Harvested mouse tracheas can be stored in a -80 °C freezer for up to 2 years.
2. Desiccate the tracheal tissue harvested in step 8.1 using fine scissors and further homogenize using a bead mill homogenizer with 1.4 mm ceramic beads (Table of Materials) as part of a commercially available column-based RNA extraction kit (Table of Materials).
   NOTE: Bead mill homogenizer settings = one 40 s cycle at 6 m/s.
3. Extract RNA from tracheal lysate using a column-based extraction and purification kit (Table of Materials) following manufacturer's instructions.
4. Quantify RNA from each sample using a spectrophotometer (Table of Materials).
   NOTE: RNA purity is assessed using the 260/230 nm ratio with a pure RNA sample reading 2.0.
5. Create complementary DNA from extracted RNA using reverse transcriptase (Table of Materials) and a thermocycler with the following settings: 5 min at 25 °C (priming), 46 °C for 30 min (reverse transcription), and then 95 °C for 1 min (inactivation of reverse transcriptase).
6. Dilute complementary DNA samples with RNase free water to a concentration of 10−25 ng/mL for use in quantitative real-time PCR.
7. Mix 1 µL of the complementary DNA (10−25 ng/mL) with 10 µL of a PCR mastermix (Table of Materials), 8 µL of ddH₂O and 0.5 μL of 10 μM forward and reverse primers for the gene of interest in one well of a 96-well PCR plate (Table of Materials).
   NOTE: The total volume in each well should be 20 μL. Each well on the 96 well plate should contain only one sample and one gene of interest.
8. Repeat steps 8.1–8.7 for all genes of interest and run each sample for each gene in triplicate.
   NOTE: The gene-specific forward and reverse primers (Table of Materials) for collagen 1, collagen 3, and the reference gene β-actin are commonly used to investigate markers of fibrosis.
9. Place the 96 well plate on the real-time quantitative PCR machine and initiate the following protocol: denature at 95 °C for 15 s and anneal and extend at 60 °C for 60 s for 40 cycles.
10. Normalize the cycle threshold value (CT) for gene product detection of each gene of interest (GOI) to the CT value for the reference gene β-actin for all samples to obtain a ΔCT for each GOI. Then, compare the ΔCT values for each GOI for treated and untreated samples to generate a ΔΔCT.
    NOTE: The cycle threshold is the point on the amplification curve when a predetermined amount of gene product is present (ΔRn). The ΔRn value for each reaction should be determined for each gene of interest and should fall on the linear portion of the amplification curve.
    NOTE: ΔCT = CT (GOI) – CT (reference gene); ΔΔCT = ΔCT (treated) – ΔCT (untreated).
11. Calculate the relative change in gene expression between treated and untreated samples as an expression of fold change by calculating 2^ΔΔCT.

Results

The biodegradable PLLA-PCL stent construct loaded with rapamycin used in this study was capable of eluting rapamycin in a consistent and predictable fashion in physiological conditions (Figure 1). Figure 2 shows the PLLA-PCL stent casted around a 22 G angiocatheter for use in a murine model of LTS. To determine if the effects of rapamycin elution in the trachea is efficacious in attenuating fibrosis, measured changes in fibrosis-related gene expression and marke...

Discussion

The most critical steps for successfully constructing and using a drug-eluting stent in vivo are 1) determining the optimal PLLA-PCL ratio for the desirable drug elution rate, 2) determining the appropriate concentration of drug to be eluted, 3) molding the stents around the angiocatheter for in vivo use, and 4) transorally delivering the stent into the mice after LTS induction without causing fatal airway obstruction.

While there are several methods for drug delivery using stents in animal mo...

Materials

Name	Company	Catalog Number	Comments
1. For stent
22-gauge angiocatheter	Jelco	4050
Dichloromethane	Sigma Aldrich	270997-100ML
Glycerol	Fisher Scientific	56-81-5	Available from other vendors as well.
PDLGA	Sigma Aldrich	739955-5G
PLLA-PCL (70 : 30)	Evonik Industries AG	65053
Rapamycin	LC Laboratories	R-5000
2. Animal surgery
Wire brush	Mill-Rose Company	320101
3. For immunohistochemistry staining
Antigen retrival buffer	Abcam	ab93678	Available from other vendors as well; acidic pH needed
DAPI	Cell Signaling	8961S
DMEM	ThermoFisher Scientific	11965-092	Available from other vendors as well.
FBS (Fetal Bovine Serum)	MilliporeSigma	F4135-500ML
Goat anti-rabbit-488 antibody	Lif technology	a11008
Goat anti-rat-633 antibody	Lif technology	a21094
Hydrophilic plus slide	BSB7028
PBS	ThermoFisher Scientific	100-10023	Available from other vendors as well.
Rabbit anti-CD3 antibody	Abcam	ab5690
Rat antiF4/80 antibody	Biolengend	123101
Zeiss LSM 510 Meta Confocal Microscope	Zeiss
4. For quantative PCR
0.5mm glass beads	OMNI International	19-645
Bead Mill Homoginizer	OMNI International
Gene Specific Forward/Reverse Primers	Genomic Resources Core Facility
Nanodrop 2000 spectrophotometer	Thermo Scientific
Power SYBR Green Mastermix	Life Technologies	4367659
RNeasy mini kit	Qiagen	80404
StepOnePlus Real Time PCR system	Life Technologies