Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Summary

Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

Abstract

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally delivering bioactive compounds to the salivary glands, greater tissue concentrations can be safely achieved versus systemic administration. Furthermore, off target tissue effects from extra-glandular accumulation of material can be dramatically reduced. In this regard, retroductal injection is a widely used method for investigating both salivary gland biology and pathophysiology. Retroductal administration of growth factors, primary cells, adenoviral vectors, and small molecule drugs has been shown to support gland function in the setting of injury. We have previously shown the efficacy of a retroductally injected nanoparticle-siRNA strategy to maintain gland function following irradiation. Here, a highly effective and reproducible method to administer nanomaterials to the murine submandibular gland through Wharton's duct is detailed (Figure 1). We describe accessing the oral cavity and outline the steps necessary to cannulate Wharton's duct, with further observations serving as quality checks throughout the procedure.

Introduction

Salivary gland dysfunction has many etiologies, including Sjögren's syndrome, an autoimmune mediated loss of functional secretory tissue, and radiation induced hyposalivation (RIH), a common sequella of head and neck cancer radiotherapy¹. Loss of salivary function due to either condition predisposes individuals to oral and systemic infection, tooth decay, digestive and swallowing dysfunction, speech impairment, and major depression^1,2,3. As a result, quality of life significantly suffers, with interventions limited to palliation of symptoms ra....

Protocol

All in vivo procedures outlined below were approved by the University Committee on Animal Resources at the University of Rochester, Rochester, NY.

1. Preparation

1. Using 32G intracranial catheter tubing with wire inset, cut 3 cm of the tubing to form a beveled end, approximately 45° to the long axis. Confirm that the wire is at least 1 cm longer than the tubing.
2. Load 50 µL of PSMA nanoparticle solution (Figure 1), or other injection material, into a Hamilton syringe. To reduce the probability of barotrauma during injection, attach the catheter tubing, with the stylet ....

Results

Retroductal injection can be used to administer NPs to the murine SMG (Figure 1). Here, we deliver 50 µg PSMA NPs labeled with Texas Red fluorophore.

Proper placement of the mouse allows facile access and visualization of the floor of the mouth (Figure 2A-B). The submandibular papillae are identified as two fleshy protrusions beneath the tongue. Followi.......

Discussion

Retroductal injection is critical for localized drug delivery to the salivary gland. This technique has applications in screening therapeutic agents for conditions including Sjogren's syndrome and RIH^9,10,28. Direct drug delivery into the SMG via retroductal injection provides a key advantage over systemic administration in its potential to reduce off-target effects, including immune activation¹¹. The abi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Pilocarpine hydrochloride	Sigma Aldrich	P6503	Pilocarpine
Student Vannas Spring Scissors	Fine Science Tools	91500-9	Spring Scissors for Tracheostomy
Sterile Saline Solution	Medline	RDI30296H	Saline
Dumont #7 Forceps	Fine Science Tools	11274-20	Curved Forceps
Dumont #5 Forceps	Fine Science Tools	11251-10	Straight Forceps
Standard Pattern Forceps	Fine Science Tools	11000-12	Blunt Forceps
Fine Scissors- Tungsten Carbide	Fine Science Tools	14568-09	Dissection Scissors
Microhematocrit Heparinized Capillary Tubes	Fisher Scientific	22362566	Capillary tubes
Lubricant Eye Ointment	Refresh	N/A	Refresh Lacri-Lube
Goat polyclonal anti-Nkcc1	Santa Cruz Biotech	SC-21545	Nkcc1 Antibody
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)	Thermo Fisher Scientific	D1306	DAPI
GraphPad Prism	GraphPad	ver6.0	Statistical Software
Cotton tipped applicator	Medline	MDS202000	Applicator for eye ointment
0.5cc Insulin Syringe, 29G x 1/2"	BD	7629	Syringe for intraperitoneal injection