Nanoparticles (NP) interact with complex protein milieus in biological fluids, and these interactions have profound effects on NP physicochemical properties and function. Surprisingly, most studies neglect the impact of these interactions, especially with respect to NP-mediated siRNA delivery. Here, the effects of serum on colloidal stability and siRNA delivery of a pH-responsive micellar NP delivery system were characterized. Results show cationic NP-siRNA complexes aggregate in ≥ 2% serum in buffer, but are stable in serum-free media. Furthermore, non-aggregated NP-siRNA delivered in serum-free media result in 4-fold greater siRNA uptake in vitro, compared to aggregated NP-siRNA. Interestingly, pH-responsive membrane lysis behavior, which is required for endosomal escape, and NP-siRNA dissociation, necessary for mRNA knockdown, are significantly reduced in serum. Consistent with these data, non-aggregated NP-siRNA in serum-free conditions result in highly efficient gene silencing, even at doses as low as 5 nM siRNA. NP-siRNA diameter was measured at albumin and IgG levels mimicking biological fluids. Neither albumin nor IgG alone induces NP-siRNA aggregation, implicating other serum proteins in NP colloidal instability. Finally, as a proof-of-principle that stability is maintained in established in vivo models, transmission electron microscopy reveals NP-siRNA are taken up by ductal epithelial cells in a non-aggregated state when injected retroductally into mouse salivary glands in vivo. Overall, this study shows serum-induced NP-siRNA aggregation significantly diminishes efficiency of siRNA delivery by reducing uptake, pH-responsive membrane lysis activity, and NP-siRNA dissociation. Moreover, these results highlight the importance of local NP-mediated drug delivery, and are broadly applicable to other drug delivery systems.

Radiotherapy for head and neck cancers commonly causes damage to salivary gland tissue, resulting in xerostomia (dry mouth) and numerous adverse medical and quality-of-life issues. Amifostine is the only Food and Drug Administration-approved radioprotective drug used clinically to prevent xerostomia. However, systemic administration of amifostine is limited by severe side effects, including rapid decrease in blood pressure (hypotension), nausea, and a narrow therapeutic window. In this study, we demonstrate that retroductal delivery of amifostine and its active metabolite, WR-1065, to murine submandibular glands prior to a single radiation dose of 15 Gy maintained gland function and significantly increased acinar cell survival. Furthermore, in vivo stimulated saliva secretion was maintained in retrograde-treated groups at levels significantly higher than irradiated-only and systemically treated groups. In contrast to intravenous injections, retroductal delivery of WR-1065 or amifostine significantly attenuated hypotension. We conclude that localized delivery to salivary glands markedly improves radioprotection at the cellular level, as well as mitigates the adverse side effects associated with systemic administration. These results support the further development of a localized delivery system that would be compatible with the fractionated dose regimen used clinically.

Hyposalivation is commonly observed in the autoimmune reaction of Sjögren's syndrome or following radiation injury to the major salivary glands. In these cases, questions remain regarding disease pathogenesis and effective interventions. An optimized technique that allows functional assessment of the salivary glands is invaluable for investigating exocrine gland biology, dysfunction, and therapeutics. Here, we present a step by step approach to performing pilocarpine stimulated saliva secretion, including tracheostomy and the dissection of the three major murine salivary glands. We also detail the appropriate murine head and neck anatomy accessed during these techniques. This approach is scalable, allowing for multiple mice to be processed simultaneously, thus improving the efficiency of the work flow. We aim to improve the reproducibility of these methods, each of which has further applications within the field. In addition to saliva collection, we discuss metrics for quantifying and normalizing functional capacity of these tissues. Representative data are included from submandibular glands with depressed salivary gland function 2 weeks following fractionated radiation (4 doses of 6.85 Gy).

SHORT ABSTRACT We present a detailed approach to performing saliva collection, including murine tracheostomy and the isolation of three major salivary glands. LONG ABSTRACT Hyposalivation is commonly observed in the autoimmune reaction of Sjögren’s syndrome or following radiation injury to the major salivary glands. In these cases, questions remain regarding disease pathogenesis and effective interventions. An optimized technique that allows functional assessment of the salivary glands is invaluable for investigating exocrine gland biology, dysfunction, and therapeutics. Here, we present a step by step approach to performing pilocarpine stimulated saliva secretion, including tracheostomy and the dissection of the three major murine salivary glands. We also detail the appropriate murine head and neck anatomy accessed during these techniques. This approach is scalable, allowing for multiple mice to be processed simultaneously, thus improving the efficiency of the work flow. We aim to improve the reproducibility of these methods, each of which has further applications within the field. In addition to saliva collection, we discuss metrics for quantifying and normalizing functional capacity of these tissues. Representative data are included from submandibular glands with depressed salivary gland function 2 weeks following fractionated radiation (4 doses of 6.85 Gy).