(T1030-02-09) Development and Characterization of Highly Muco-penetrative PLGA Nanoparticles for theTreatment of Inflammation in Chronic Lung Disease

Purpose: A wide array of chronic and debilitating respiratory diseases, including cystic fibrosis, chronic obstructive pulmonary disorder and various pulmonary fibroses are characterized by overproduction of highly viscous airway mucus [1, 2]. This serves to trap both inhaled pathogens (leading to infection and inducing inflammation in the airways), as well as drugs administered through the inhalational route (which are then cleared either by mucociliary action, macrophages or enzymes within the mucus matrix), thereby reducing their efficacy [3]. The conjugation of short (≤5 kDa) chain polyethylene glycol (PEG) has been shown to enhance the penetration and movement of nanoparticles through the mucus mesh [4, 5]. Concomitantly, the chronic respiratory diseases outlined previously are also characterized by chronic inflammation of the airways, either as a result of recurring pathogenic infections, dysregulated immune response, or underlying disease pathology (oxidative stress, genetic factors etc.) [6]. This in turn can have a range of adverse lung-specific and systemic downstream effects such as airway remodelling, increased risk of cardiovascular disease, gastroesophageal reflux disease and more [7]. To address these interrelated pathological features, we propose a biodegradable, cytocompatible polymeric nanoparticle (NP) platform encapsulating the anti-inflammatory agent all-trans retinoic acid (ATRA), able to move rapidly through highly viscous mucus by virtue of nanoparticle surface modification with low-molecular weight polyethylene glycol and n-acetylcysteine (NAC). These are envisioned to improve on existing mucopenetrative strategies by increasing the rate of particle penetration through mucus and a higher fraction of administered nanoparticles reaching the airway epithelium.

Methods: ATRA-encapsulated PLGA NPs were synthesized via a single-emulsion method using maleimide-terminated PLGA, chloroform and 5% polyvinyl alcohol. Encapsulation efficiency and release kinetics of ATRA from the NPs was assessed using UV-vis absorbance spectrophotometry. Conjugation of thiol and amine terminated polyethylene glycol (SH-PEG-NH₂, 2 kDa) to the surface of the NPs was achieved using thiol-maleimide click chemistry, with conjugation efficiency determined by way of Draggendorff reagent. NAC was conjugated to the amine terminals of the PEG using carbodiimide chemistry, and efficiency quantified by means of UV-vis absorbance spectrophotometry. General particle characterization was carried out by way of dynamic light scattering/zeta potentiometry, scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy FT-IR) and thermogravimetric analysis (TGA). To assess mucopenetrative properties of the NPs, diseased mucus was developed by means of a previously published method by Alp and colleagues. The rheological properties of the mucus were confirmed using frequency and flow sweeps on a TA Instruments HR30 rheometer. The mucopenetrative properties of bare, PEG- and PEG+NAC-conjugated were evaluated using particle tracking, while the efficacy of the NAC in reducing the viscosity of synthetic mucus was assessed using rheometry by way of cone-and-plate geometry.

Results: ATRA-encapsulated PLGA NPs (≤200 nm) with uniform, spherical morphologies were successfully synthesized (Figure 1) ATRA encapsulation was assessed at two drug:polymer ratios, 1:5 and 1:10, with the 1:5 formulation yielding a 99.5% encapsulation of the anti-inflammatory drug. Release was assessed over 21 days in SLF, with a burst release phase observed within the initial 24-48 hours. The conjugation of both PEG and NAC were confirmed using FT-IR spectroscopy. The diseased mucus synthesized by us had a G’ of 16.02 ± 4.0 Pa, a G” of 8.79 ± 1.5 Pa and a complex viscosity of 18.28 ± 4.3 Pa.s (ω= 1 rad/s) (Figure 2).

Conclusion: We have successfully synthesized ATRA-encapsulated nanoparticles with a very high encapsulation efficiency and have demonstrated its controlled release at physiological conditions. Work is ongoing to more fully characterize the NAC-induced changes in the rheological properties in our synthetic mucus model, as well as their uptake by the pulmonary epithelium.

References: [1] R.C. Boucher, Airway Surface Dehydration in Cystic Fibrosis: Pathogenesis and Therapy, Annu. Rev. Med. 58 (2007) 157–170. https://doi.org/10.1146/annurev.med.58.071905.105316.
[2] G. Alp, N. Aydogan, Lipid-based mucus penetrating nanoparticles and their biophysical interactions with pulmonary mucus layer, Eur. J. Pharm. Biopharm. 149 (2020) 45–57. https://doi.org/10.1016/j.ejpb.2020.01.017.
[3] B. Button, L.-H. Cai, C. Ehre, M. Kesimer, D.B. Hill, J.K. Sheehan, R.C. Boucher, M. Rubinstein, A Periciliary Brush Promotes the Lung Health by Separating the Mucus Layer from Airway Epithelia, Science (80-. ). 337 (2012) 937–941. https://doi.org/10.1126/science.1223012.
[4] Y. Cu, W.M. Saltzman, Controlled Surface Modification with Poly(ethylene)glycol Enhances Diffusion of PLGA Nanoparticles in Human Cervical Mucus, Mol. Pharm. 6 (2009) 173–181. https://doi.org/10.1021/mp8001254.
[5] J.T. Huckaby, S.K. Lai, PEGylation for enhancing nanoparticle diffusion in mucus, Adv. Drug Deliv. Rev. 124 (2018) 125–139. https://doi.org/10.1016/j.addr.2017.08.010.
[6] F.M. Carlier, C. de Fays, C. Pilette, Epithelial Barrier Dysfunction in Chronic Respiratory Diseases, Front. Physiol. 12 (2021). https://doi.org/10.3389/fphys.2021.691227.
[7] A. Agustí, R. Faner, Systemic Inflammation and Comorbidities in Chronic Obstructive Pulmonary Disease, Proc. Am. Thorac. Soc. 9 (2012) 43–46. https://doi.org/10.1513/pats.201108-050MS.

Acknowledgements: We would like to acknowledge the Rhode Island IDeA Network of Biomedical Research Excellence (RI-INBRE) for use of core equipment.

Scanning electron micrographs of bare and ATRA-encapsulated NPs (Scale bar= 200 nm), showing particles of approximately 150-200 nm in size. Table displays a summary of particle characterization data, including zeta potentials. A higher drug to polymer ratio was correlated with higher particle sizes and zeta potentials.

Summary of rheological characterization data of untreated synthetic diseased mucus model: A) Amplitude sweep (forward and reverse) showing bounds of linear viscoelastic region (highlighted); B) Elastic and viscous moduli plotted alongside complex viscosity. Table displays a summary of this data.