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 inflammator....

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.......

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