(370c) Award Submission: Mucus Penetration of Surface-Engineered Nanoparticles in Various pH Microenvironments.

The inhaled medications delivered by nano- or micro-sized carriers are widely used for the prevention and treatment of lung diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), asthma, and COVID-19. During the transport of the drug carriers in the airway, the mucus lines the surface of the airway and hinders the effective delivery of active ingredients. The mucus layer is composed of 98% water, 1.6% mucin, and 0.4% of the remainder, e.g., cellular debris, salts, DNA, lipids, globular proteins, etc. Mucin form a three-dimensional network to restrain the movement of particles via the size exclusion and interaction filtration effect. It has been demonstrated that proper control of physicochemical properties of carriers could improve the transport and fate of medications.

The neutral charge and hydrophilic surface have been identified as critical requirements for the effective trans-mucus drug carriers. Poly (ethylene glycol) (PEG) has been used as a stealth polymer that repels protein adsorption to assist the drug carriers to escape the binding of mucin. Their molecular weight, chain length, and content have been shown to affect mucus penetration performance. The zwitterionic design mimics the surface characteristics of the virus surface, and further exhibits the equal densities of positive and negative charges without the presence of hydrophobic patches. The surface modification by zwitterionic polymer and density adjustment enables enhanced mucus penetration of particles. Furthermore, the particle surface modification by block copolymer (F-127), peptides and protein were potentially alternative strategies to improve penetration ability of particles. Although PEGylation is often considered as a “gold standard” to assess mucus penetration, and various surface modification strategies have been proposed. the mechanistic understandings of particle surface property-mediated mucus penetration are currently missing.

The mucus microenvironment changes according to the physiological and pathological states. In asthma exacerbation, healthy, and airway infection or inflammation states, the pH values of mucus is altered. However, the airway mucus penetration model in current study is mainly derived from commercial porcine gastric mucin. Regardless of the mucin suspension or the artificial mucus, the pH value of the system is acidic (pH=3-5), which is different from the natural airway health neutral mucus environment. Although sputum taken from patients such as cystic fibrosis (CF) as a surrogate is similar to the mucus environment experienced by drug carriers after airway administration, factors such as the causes (endogenous or exogenous exposure) and states of diseases (mild or severe) were not considered. Acid respiration occurs in exacerbation of CF (mucus pH=5.3) and asthma (pH=5.2), but the pH of mucus is increased in mild asthma (pH=7.6) and stable CF (pH=5.9). Moreover, the mucus is an alkaline environment (pH=7.8-8.5) in inflammatory diseases. Therefore, mucus pH microenvironment should be taken into consideration during inhalation delivery.

In this study, we prepared a comprehensive library of engineered silica nanoparticles (SNPs) with controlled surface ligand type and density. According to the size exclusion mechanism of mucus, nanoparticles with a size range of 200-500 nm in diameter are easier to penetrate the mucus barrier. Therefore, the 300 nm SNPs were selected to study their mucus penetration ability. The pristine SNPs were synthesized using the Stöber method, and SNPs were doped with fluorescein isothiocyanate (FITC) for particle tracking. Then, SNPs were further modified with silane coupling agents to prepare a library of engineered SNPs with controlled functional groups and ligand densities. Then, particles were dispersed in the airway mucus with mimetic physiological and pathological pH values to evaluate particles movement behavior by using multiple-particle tracking (MPT). PEGylated and amine SNPs showed pH-independent mucus penetration and entrapment, the carboxyl-SNPs exhibited movement enhancement only in weakly alkaline environments. The mechanisms were further systematically analyzed from three aspects, i.e., mucus properties, SNPs surface properties, and mucus-SNPs interactions, respectively.

A recent study has revealed that mucin undergoes a sol-gel transition in acidic-neutral environments, and the cross-linking properties of mucus are stronger in acidic than that in neutral condition. In our study, it was demonstrated that the pH-dependent changes in mucus macro-rheology and micro-structure were the main reasons for the observed variations of particle motion behavior. From the perspective of mucin micro-structure, the changes in the macro-crosslink properties of mucus at different pH was due to the changes in the microstructure of mucin. During the acidification of mucus, carboxylates on mucin were protonated. As a result, the electrostatic interaction between carboxylate and amine groups were compromised, and hydrophobic domains hidden in salt bridge folded structure were exposed, promoting the cross-linking of mucus gel. In addition to the influence of the hydrophobic domain, electrostatic interactions due to the exposure of mucin glycosyl side chains also play an essential role. In generally, mucin is negatively charge due to the sialic and sulfate components in the glycosyl side chain. In the present study, this pH-dependent conformation and charge transition were observed. The circular dichroism (CD) analysis revealed that mucin was dominated with the random coil conformation, and the pH-dependent intensity and width changes at 206 nm was observed, suggesting the conformation undergoes a transition from isotropic random coil to anisotropic extended random coil with the decrease of pH values. Additionally, the mucin fiber in acidic environment exhibited increased zeta potential, thus enhancing electrostatic interactions between mucin fibers, increasing the viscosity of the mucus and inhibiting the movement of the particles.

Furthermore, isothermal titration calorimetry (ITC) reveals that the independent of the functional groups and mucus pH, the interaction of mucin with SNPs were exothermic and entropy loss process, indicating an enthalpy-driven interaction and the formation of non-covalent bonds was reason why particles were trapped in mucus, while the dense brush structure formed by PEG modification shielded the interaction between particles and mucin to achieve mucus penetration. Furthermore, to study the effect of mucus pH, the amine-modified SNPs were selected to titrate with mucin solutions. The association constant (K_a) of SNPs in acidic (pH 5.2) environment was an order of magnitude higher than those in neutral (pH 7.0) and slightly alkaline (pH 7.7) ones. The stronger SNP-mucin interaction was correlated with the fact that particles being trapped in the in acidic mucus. Therefore, this study demonstrated that the fate of drug carrier particles in airway mucus was pH microenvironment, particle surface ligand type, and density-dependent, and provides an engineered approach to control the mucus penetrating capabilities of drug delivery carrier for the personalized treatment and prevention of pulmonary diseases.