(229h) pH-Mediated Mucus Penetration of Zwitterionic Polydopamine-Modified Silica Nanoparticles

The mucus lining on the mucosal surface works as a natural barrier against the invasion of pathogens. However, the existence of mucus hinders the trans-mucus delivery of medications, resulting in low administration efficiency and poor bioavailability. Zwitterionic polymers have emerged as promising trans-mucus nanocarriers due to their superior anti-fouling properties. However, the different behaviors caused by various pH environments have been observed in certain zwitterionic polymers. When delivered to various mucosal tissues, the pH-sensitive behaviors of zwitterionic polymers were noted due to a wide range pH micro-environment of mucus. The mucus layer is approximately neutral in airway (pH 7.1 ± 0.1) and eyes (pH ~7.26) of human. Along the gastrointestinal tract, the mucus pH increases from harsh acidic (pH 1-3) in stomach to slightly acidic or neutral in small intestine (pH 5.0-7.5). Additionally, the mucus pH of cervix ranges from slightly acidic to basic conditions (pH 5.4-8.2) with the menstrual cycle. Thus, the pH-sensitive zwitterionic materials could exhibit the desired mucus penetrability in the targeted mucus microenvironment that further determines their applicability as trans-mucus delivery vehicles. The systematic study on the effect of pH on the mucus penetrability of pH-sensitive zwitterionic NPs and the underlying mechanism is currently missing.

Herein, we prepared a library of zwitterionic polydopamine-modified silica nanoparticles (SiNPs-PDA). With the increase of the mass of PDA grafted on SiNPs, a library of SiNPs-PDA with different Si:DA ratios was prepared, and they were noted as SiNPs-PDA-11, SiNPs-PDA-12, and SiNPs-PDA-15, respectively. The Transmission electron microscopy (TEM) images and dynamic light scattering (DLS) analysis showed that pristine SiNPs were spherical-shaped with diameters of around 300 nm, and the coating became thicker and more compact with the increase of PDA. The Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), and acid orange analysis were used to determine the surface groups, which increased with the increase of DA:Si ratio. Besides, the ζ potentials of SiNPs-PDA were about 20 mV, 0 mV and -15 mV at pH values of 3.0, 5.6, and 7.0, respectively, and the isoelectric point (IEP) was around 5.6.

Using the library of pH-sensitive PDA-modified NPs, we further investigated their diffusion behavior in both in vitro and in vivo mucus models. The pH of reconstituted mucus was adjusted from acidic to neutral (pH= 3.0, 5.6, and 7.0) to mimic the pH microenvironment in different mucosal tissues, and multiple particle tracking (MPT) was used to determine the motility behavior of particles. The movement of SiNPs-PDA was displayed visually by representative trajectories and movies. It was shown that the SiNPs-PDA diffused freely at pH 5.6 and were restricted significantly as the mucus shifted to acidic (pH = 3.0) or neutral (pH = 7.0) condition. The ensemble mean squared displacement (<MSD>) was calculated to evaluate the diffusion ability of NPs. Under different modification densities, the SiNPs-PDA all exhibited the maximum <MSD> values in mucus with pH value of 5.6. It indicated that the balanced charges on SiNPs-PDA, even in a small amount, provided particles with good penetration ability. When mucus pH was changed to 3.0 or 7.0, the <MSD> values were reduced by different degrees. This reduction was augmented with the increase of PDA density, reaching 2.8 and 2.5 times for SiNP-PDA-15 at pH values of 3.0 and 7.0, respectively. It suggested that the pH-mediated mucus penetrability of SiNPs-PDA became more significant with the increase of unbalanced positive and negative charges on the particle surface. The logarithms of the effective diffusion coefficient (D_eff) and anomalous exponent (α) were calculated to further confirm the unhindered behavior of SiNPs-PDA at pH value of 5.6. Additionally, the mucosal tissues of mouse with different pH micro-environments were selected to further study the particle distribution in vivo. SiNPs-PDA-15 were diffused deeper into the intestinal villi (slightly acidic), and evenly distributed along the mucus layer. In contrast, SiNPs-PDA-15 were sparsely and discontinuously accumulated on the surface of the stomach (acidic) and airway (neutral), respectively. Together with the in vitro results, it further demonstrated that the SiNPs-PDA-15 tended to diffuse better in slightly acidic mucus but were trapped or removed in acidic and neutral mucus.

To understand the pH-mediated diffusion behavior of SiNPs-PDA in mucus, a series of biophysical characterizations were used to analyze this process. As the major protein in mucus, the mucin entangles and forms a dense fiber mesh against the diffusion of foreign particles. The particle-mucin interaction has been demonstrated as a key parameter in the filtering effect of mucus mesh, determining the mucus penetrability of particles. The interaction between SiNPs-PDA and mucin was systematically analyzed from the aspect of surface properties of NPs. For NPs in mucus, the highly hydrophobic surface could typically lead to a trapped state by mucin due to hydrophobic interaction. Thus, the hydrophobicity of SiNPs-PDA-15 at different pH values was determined. The pyrene assay was used for the hydrophobicity measurement, which indicated higher hydrophobicity of NPs at pH of 3.0 than that at pH of 5.6 and 7.0, respectively. Additionally, the Circular Dichroism (CD) spectrum showed that the α-helix in mucin was weakened and widened with the environmental pH values decreasing, caused by the change in stability of salt bridges between -COOH and -NH₂. With the unfolding of the glycosyl side chain, the hydrophobic regions were gradually exposed and the hydrophobicity of mucin was increased. Altogether, the higher hydrophobicity of both mucin and SiNP-PDA-15 might lead to the strong hydrophobic interaction at the pH of 3.0, while the hydrophobic interaction was weakened when the hydrophobicity of the mucin or NPs decreased at pH values of 5.6 and 7.0. Additionally, the interaction on the molecular level was investigated using isothermal titration calorimetry (ITC) analysis. The exothermic and entropy loss processes indicated that the interaction was dominated by the formation of non-covalent bonds, e.g., electrostatic interactions and hydrogen bonds. Notably, the association constants (K_a) value at the pH value of 5.6 was around 7.7 and 1.3 times lower than that at pH values of 3.0 and 7.0, respectively, and the number of adsorption sites (n) at pH of 5.6 was smaller than that at pH value of 3.0 and 7.0. It suggested that the mucin-SiNPs-PDA-15 interaction was weaker at pH of 5.6 than that at 3.0 and 7.0, which was consistent with MPT analysis. Additionally, the hydrogen bond formation between SiNPs-PDA-15 and mucin was confirmed by the blue shift of the mucin peak in the amide I region after adsorption on particles using the FTIR analysis.

In this paper, we demonstrated the pH-mediated mucus penetration capability of zwitterionic polydopamine-coated silica nanoparticles. When the IEP of NPs was equal to the pH value of the mucus microenvironment, the NPs exhibited superior diffusional transportation in both in vitro and in vivo models. Further biophysical analysis found that the trans-mucus behavior of SiNPs-PDA was mediated by combined hydrophobic, electrostatic, and hydrogen bonding interactions between mucin and the particles. This study reveals the significance of matching the charge balance state with the mucus pH microenvironment for efficient trans-mucus delivery of zwitterionic NPs, which provides rational design strategies for applications of zwitterionic polymers in various mucus microenvironments.