Optimization of Mucus Penetrating Particles and Vehicle Composition for Improved Mucosal Surface Drug Delivery

Introduction

Local mucosal drug delivery has many applications, including treatment of various cancers, inflammatory conditions such as inflammatory bowel disease and chronic obstructive pulmonary disease, and prevention of sexually transmitted infections. However, achieving effective drug delivery to mucosal epithelia can be challenging. Epithelial surfaces of the female reproductive tract, gastrointestinal tract, and lung have numerous folds and/or topography, making significant portions of the epithelial surface difficult to access. Additionally, mucosal epithelia are highly absorptive and permeable, which can limit the duration of delivery of soluble drug systems. Nanoparticles can be used as controlled-release delivery systems to slow the absorption of drugs, but the viscoelastic, adhesive mucus layers lining mucosal epithelia trap conventional nanoparticles (CP), limiting distribution and facilitating rapid clearance. We recently discovered that densely coating the surface of nanoparticles with low molecular weight polyethylene glycol (PEG) effectively shields the nanoparticle core from adhesive interactions with human cervicovaginal mucus (CVM) and human respiratory mucus (1,2). These so-called mucus penetrating particles (MPP) rapidly diffuse through the fluid-filled pores in the mucus mesh, whereas CP are adhesively immobilized. However, diffusion alone may not be rapid enough for MPP to distribute throughout mucosal surfaces, including penetrating the mucus blanket. Thus, we have found that using osmotic gradients to induce fluid absorption and subsequent advective delivery to the epithelial surface is very effective; MPP delivered in a hypotonic vehicle rapidly distributed throughout the vaginal tract of mice, including reaching into the deep folds within 10 minutes (3). By reaching the more slowly cleared mucus layers in the rugae, MPP were well-retained in the vagina, whereas mucoadhesive CP were rapidly cleared with the luminal mucus layers (3). We anticipate that similar improvements in distribution and retention with hypotonically-administered MPP can be achieved at various mucosal surfaces, potentially improving drug delivery for a wide range of conditions. Here, we optimize nanoparticle surface PEG density and vehicle tonicity for enhanced distribution, facilitating effective drug delivery.

    

Materials and Methods

Non-biodegradable probe MPP for testing various vehicles, capable of diffusing through human CVM, were prepared as previously reported (4). Biodegradable nanoparticles composed of poly(lactic-co-glycolic acid) (PLGA) and PEG copolymer (PLGA-PEG) were synthesized with various PEG surface densities using a self-assembly emulsification method and varying ratios of PLGA:PLGA-PEG. PEG surface density was quantified using two NMR methods: the first method involved dissolving the nanoparticles and determining total PEG content, while the second method measured surface PEG content of intact particles. The PEG density (Γ) was calculated as the number of PEG molecules on the nanoparticle surface per 100 nm². The number of unconstrained molecules per 100 nm² (Γ*) was calculated by assuming PEG behaves as a flexible polymer in a random-walk conformation. The ratio Γ/Γ* represents the relative steric constraint on the surface PEG; as Γ/Γ* increases, the PEG conformation progresses from the mushroom to the brush regime (5). Transport rates of MPP and CP were quantified using multiple particle tracking (6). Vehicles of varying osmolality were prepared with various amounts of water and saline. Vaginal distribution was assessed by qualitatively (cross-sectional images) and quantitatively (surface area covered on flattened tissue).

Results

We found that increasing PEG content more effectively shielded the nanoparticle core, reducing mucoadhesion and increasing transport rates in human mucus. Increasing PEG content reflected an increase in the relative ratio of PEG chains per surface area compared to the number of unconstrained PEG chains that would occupy a given surface area, indicating a more constrained surface conformation and close packing in the “brush” regime. Improvement in mucus penetration correlated well with improved vaginal distribution and surface coverage in mice. Administering MPP in hypotonic vehicles led to rapid fluid absorption and distribution throughout the vaginal tract. We found that even minimally hypotonic vehicles (220 mOsm/kg) facilitated favorable distribution over the entire epithelial surface, including the deep folds. In contrast, MPP administered in an isotonic vehicle were found diffusely spread throughout the vaginal lumen, rather than forming the uniform coating on the vaginal epithelium seen with hypotonically-administered MPP.

Discussion and Conclusion

Many conventional vehicles for vaginal and rectal delivery are hypertonic, which causes fluid secretion that facilitates clearance (7). Hypertonicity has also been demonstrated to cause inflammation that increases susceptibility to sexually transmitted infections (8,9). Although purely hypotonic vehicles appear to be safe after daily vaginal administration for 1 week (3), we anticipate that minimally hypotonic vehicles are less likely to cause any toxicity or epithelial distress, particularly with repetitive administration. As such, we demonstrated here that improved epithelial distribution can be achieved with a minimally hypotonic vehicle. Although osmotically-induced fluid absorption appears to be advantageous for nanoparticle delivery at mucosal surfaces, it is vital that the nanoparticle be sufficiently mucoinert to penetrate through the mucus barrier. Although we previously demonstrated that nanoparticles sufficiently coated with PEG can rapidly penetrate human CVM, here we more thoroughly characterize what is a “sufficient” coating. We found that increasing PEG content, such that the conformation was in the “brush” regime, correlated with increased diffusion rates in human CVM and improved distribution in the mouse vagina. We anticipate that the PEG surface density can be similarly optimized for penetration in mucus at other mucosal surfaces, including the GI tract and the lungs. Additionally, mucosal surfaces such as the GI tract and the lung are also absorptive, such that hypotonic vehicles will also be beneficial for distribution and retention. Combining MPP with and hypotonic vehicles has promise for more efficacious drug and gene delivery to various mucosal epithelia, potentially improving prevention and treatment of a wide array of mucosal diseases and conditions.

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