(T1230-06-32) Plasmon-Waveguide Resonance Spectroscopy to Evaluate Mucin and Phage-like Particle (PLP) Interactions

Purpose: A major challenge faced in mucosal drug delivery is the inability to effectively deliver large molecule drugs and nanoparticles through the mucousal layer present on several epithelial linings of the body. Mucus, an integral component of the innate immune defense, acts as a viscous, rapidly clearing, and adhesive gel, safeguarding surfaces of organs like the eye, intestines, and lungs, among others. Mucous linings can retain and remove foreign particles within a few seconds to hours. This rapid clearance mechanism often poses difficulties in delivering macromolecules and nanoparticles through mucus. With respect to the eye surface where there is rapid drainage of an applied eye drop, we hypothesize that while tight binding and lack of binding diminish nanoparticle delivery, an intermediate binding affinity might be superior for delivery. Thus, it is beneficial to develop novel techniques to examine the binding properties of nanoparticles with the mucousal barrier. Plasmon-waveguide resonance (PWR) is a highly sensitive, label-free optical waveguide spectroscopic technology that captures real-time interactions between a ligand and a substrate by monitoring the refractive index, mass density, and conformational changes within a range of 100 nm from the hydrophilic surface of the PWR prism.  By detecting differences in mass distribution, PWR spectroscopy provides insights into structural changes in immobilized samples, utilizing both p- and s-polarization modes. Mucin, a key component of the mucous layer, serves as a model for mucous membranes, aiding in understanding drug delivery challenges. By closely mimicking the natural properties of mucus, including composition and adhesion, mucin helps evaluate drug delivery systems' ability to penetrate or adhere to mucousal surfaces. This study investigated mucin's interaction with the PWR sensor surface and nanoparticles. Phage-like particles (PLPs) from bacteriophage lambda are promising candidates for mucosal drug delivery because of their tunable binding affinities to various biological surfaces, which in turn may overcome the challenges of rapid clearance and poor permeability via mucousal barriers. Engineered to possess both mucoadhesive and mucopenetrative properties, these lambda PLPs aim to transiently adhere to the mucous layer while efficiently diffusing through it towards the epithelial tissues. Preliminary studies modifying PLPs' surface proteins enhanced their functionality, which might be beneficial in optimizing their interaction with mucin and improving drug delivery efficacy. Using PWR spectroscopy, biological events like adsorption and binding were monitored to explore PLPs' interplay with mucin. Characterizing mucin interactions with the PWR sensor surface, this study advances understanding of mucosal drug delivery challenges, aiming for optimized formulations with enhanced therapeutic outcomes.

Methods: The Emanant PWR Spectroscopy System (Figure 1), coupled with BEAM software (Mainline Scientific LLC), was utilized in this study.  Mucin (porcine gastric mucin type III (Sigma)) solutions (0.05, 0.1, 0.2, 0.5, 1, and 2% w/v) were prepared in PBS (pH 7.4) and incubated overnight at 4 °C for solubilization. Phage-like particles (PLPs) (designed and prepared by Catalano Lab) were diluted to 1 nM in PBS before use. Mucin and PLP preparations were equilibrated to room temperature for at least 30 min prior to the study. Reference scans in the 0–50,000 millidegree range were obtained for air, water, and PBS (pH 7.4) using p- and s-polarization modes before each experiment. Pipeline conditioning with distilled water at 5 mL/min and a volume of 5700 µL was conducted. Baseline measurements with water and PBS were made by pumping solutions through the PWR pipeline at 5 mL/min and volumes of 2850 µL for water and 5700 µL for PBS. Mucin immobilization involved injecting 100 µL of mucin directly into the reaction cells with a needle. Continuous scans were taken between p- and s-polarization modes, ensuring the undisturbed state of the reaction cell. Once mucin equilibrated on the prism surface, PLPs (20 µL) were injected similarly, and scans performed.

Results: Preliminary analysis of the angle of incidence unveiled a distinct angle for mucin relative to references such as air, water, and PBS, with the angles increasing in the order air < water < PBS < mucin for both p- and s-polarization modes (Figures 2 and 3). The angles for water and PBS fell within similar ranges, yet PBS consistently exhibited a higher angle of incidence compared to water, regardless of equilibration time. Introducing PLPs resulted in a rapid shift in the angle compared to the angle observed during mucin-prism interaction. These studies need to be repeated with various concentrations of PLPs to better understand mucin-PLP interactions.

Conclusion: PWR spectroscopy effectively detected molecular-level changes in mucin-sensor surface interaction, characterizing mucin adsorption with sensitivity to peak shifts in the millidegree range. Mucin adsorbed on the PWR sensor surface may serve as a valuable tool in evaluating nanoparticle interactions with mucin.

References: 1) Hruby VJ, Alves I, Cowell S, Salamon Z, Tollin G. Use of plasmon waveguide resonance (PWR) spectroscopy for examining binding, signaling and lipid domain partitioning of membrane proteins. Life Sci. 2010 Apr 10;86(15-16):569-74. doi: 10.1016/j.lfs.2009.02.027. Epub 2009 Mar 20. PMID: 19281827.
2) Zhang H, Orosz KS, Takahashi H, Saavedra SS. Broadband plasmon waveguide resonance spectroscopy for probing biological thin films. Appl Spectrosc. 2009 Sep;63(9):1062-7. doi: 10.1366/000370209789379295. PMID: 19796490; PMCID: PMC2912159.
3) Hruby VJ, Tollin G. Plasmon-waveguide resonance (PWR) spectroscopy for directly viewing rates of GPCR/G-protein interactions and quantifying affinities. Curr Opin Pharmacol. 2007 Oct;7(5):507-14. doi: 10.1016/j.coph.2007.08.001. Epub 2007 Sep 14. PMID: 17869585; PMCID: PMC2151673.
4) Ensign LM, Schneider C, Suk JS, Cone R, Hanes J. Mucus penetrating nanoparticles: biophysical tool and method of drug and gene delivery. Adv Mater. 2012 Jul 24;24(28):3887-94. doi: 10.1002/adma.201201800. PMID: 22988559; PMCID: PMC3710133.

Acknowledgements:

Funding: Supported by an unrestricted research gift from Mainline Scientific, Inc. Also funded in part by ABNexus collaborative seed grant award by University of Colorado.
Conflict of Interest Statements: HZ is an employee of Mainline Scientific, Inc.


(A) Depicts the schematic diagram of the Emanant PWR Spectroscopy Principle, while (B) outlines the experimental process. This involves the application of mucin onto the substrate (PWR sensor surface) and the subsequent interaction of PLPs with the immobilized mucin.


PWR curves showing the interaction of (A-B) varying concentrations of mucin on the PWR prism; (C-D) 0.1% Mucin with 1nM PLP over 30 minutes


Time course study conducted to observe the interaction of immobilized mucin when exposed to PLP. (A-B) 0.05% Mucin with 1nM PLP (C-D) 0.1% Mucin with 1nM PLP