Localized Delivery of Pilocarpine to Hypofunctional Salivary Glands through Electrospun Nanofiber Mats: An Ex Vivo and In Vivo Study

Abstract

Dry mouth or xerostomia is a frequent medical condition among the polymedicated elderly population. Systemic pilocarpine is included in the first line of pharmacological therapies for xerostomia. However, the efficacy of existing pilocarpine formulations is limited due to its adverse side effects and multiple daily dosages. To overcome these drawbacks, a localized formulation of pilocarpine targeting the salivary glands (SG) was developed in the current study. The proposed formulation consisted of pilocarpine-loaded Poly(lactic-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) nanofiber mats via an electrospinning technique. The nanofiber mats were fully characterized for their size, mesh porosity, drug encapsulation efficiency, and in vitro drug release. Mat biocompatibility and efficacy was evaluated in the SG organ ex vivo, and the expression of proliferation and pro-apoptotic markers at the cellular level was determined. In vivo short-term studies were performed to evaluate the saliva secretion after acute SG treatment with pilocarpine-loaded nanofiber mats, and after systemic pilocarpine for comparison purposes. The outcomes demonstrated that the pilocarpine-loaded mats were uniformly distributed (diameter: 384 ± 124 nm) in a highly porous mesh, and possessed a high encapsulation efficiency (~81%). Drug release studies showed an initial pilocarpine release of 26% (4.5 h), followed by a gradual increase (~46%) over 15 d. Pilocarpine-loaded nanofiber mats supported SG growth with negligible cytotoxicity and normal cellular proliferation and homeostasis. Salivary secretion was significantly increased 4.5 h after intradermal SG treatment with drug-loaded nanofibers in vivo. Overall, this study highlights the strengths of PLGA/PEG nanofiber mats for the localized daily delivery of pilocarpine and reveals its potential for future clinical translation in patients with xerostomia.

Epigallocatechin-3-Gallate Protects Pro-Acinar Epithelia Against Salivary Gland Radiation Injury

“…Thus, our future studies will use novel nanocarriers to improve the oral bioavailability of EGCG towards the protection of SG secretory epithelia from radiation injury. One of such nanocarriers that we are exploring is hydrogel-based nanofiber mats that can promote agents’ local delivery to the salivary glands [ 40 ]. Hence, local delivery of EGCG would promote a more effective oral bioavailability of this agent and confer protection against IR-induced apoptosis and enrich the pro-acinar epithelial cell populations leading towards an improved acinar salivary secretion.…”

“…The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Biosafety Committee of the Chulalongkorn University Faculty of Dentistry (DENT CU-IBC 006/2019 on March 2019 and DENT CU-IBC 006/2020 on March 2020). Fetal submandibular glands were surgically dissected from ICR mouse embryos at embryonic day E13 under a stereomicroscope (SZH10 model from Olympus, Tokyo, Japan or SMZ1270i model from Nikon Corporation, Tokyo, Japan), as previously described [ 13 , 40 ]. All reagents and plasticware were purchased from Thermo Fisher Scientific (Waltham, MA, USA) or Sigma-Aldrich (Merck, St. Louis, MO, USA) and its subsidiaries unless stated otherwise.…”

“…All reagents and plasticware were purchased from Thermo Fisher Scientific (Waltham, MA, USA) or Sigma-Aldrich (Merck, St. Louis, MO, USA) and its subsidiaries unless stated otherwise. After dissections, glands were cultured on porous polycarbonate membranes (Whatman TM Nucleopore) and placed at the air/medium interface at the center well of 50 mm dishes as previously described [ 13 , 40 ]. These membranes were kept floating on growth media (GM) composed of phenol-free DMEM/F12, 1% penicillin/streptomycin, 150 µg/mL ascorbic acid and 100 µg/mL human holo-transferrin.…”

“…Salivary glands were observed under bright-field and phase-contrast microscopy at baseline, 24, 40, 48 and 72 h, and images were acquired at 5–10× magnification with a Leica DMi1 light microscope (Leica Microsystems, Wetzlar, Germany). The epithelial growth during the SG branching morphogenesis process was determined by counting the number of epithelial buds at every time point on a blinded approach using ImageJ (NIH, Bethesda, MD, USA) as per our previous studies [ 13 , 40 ]. Epithelial growth ratio was quantified at each time point by dividing the end bud numbers at each time point with the end bud number at baseline.…”

“…Untreated and treated SG with EGCG at 7.5 µg/mL (optimized concentration) were fixed after 72 h of culture using 4% paraformaldehyde (PFA) for 20 min at room temperature (RT). The immunohistochemistry protocol has been published elsewhere [ 40 , 41 ]. Briefly, SMGs were permeabilized with 0.1% Triton X for 15 min and washed using 1× phosphate-buffered saline (PBS) three times.…”