Pathology-responsive polymersomes towards theranostic management of GM1 gangliosidosis

Introduction: GM1 gangliosidosis (GM1) is a fatal neurodegenerative disorder characterized by insufficiency in the lysosomal enzyme, β-galactosidase (β-gal), without which GM1 gangliosides accumulate. Eventually, substrate overload causes cell death, which occurs everywhere in the body but has the most severe effect in the brain. Despite the efficacy of enzyme replacement therapy (ERT) in non-neuropathic lysosomal storage disorders, this method has never been applied to GM1 due to challenges associated with brain delivery, primarily the blood-brain barrier. We optimized hyaluronic acid (HA)-poly(lactic acid) (PLA) polymersomes (PSs) to deliver β-gal with release triggered by pathophysiological conditions distinct for GM1-affected lysosomes. Through PS coupling with an endothelial receptor, PSs will be directed to neural lysosomes and experience release-triggering conditions therein. Due to the dual loading capabilities of PSs, they can release and deliver more than one payload, enabling their application in theranostics.
Diagnostics represent another inadequacy in GM1 management. Magnetic resonance imaging (MRI) is an obvious choice for soft tissue imaging, but contrast agents (CAs) are often necessary. Advanced functionalities for CAs can be especially helpful when the target condition is chemical rather than structural. Therefore, to further capitalize on the intrinsic responsivity and targetability of our PSs, we membraneously co-loaded IONPs to enable drug delivery and disease progression tracking. Pathology- and lysosome-driven degradation would not only trigger release of β-gal but also the IONP, causing a measurable change in relaxivity. In situ contrast changes can then be used to verify delivery and track lysosome density as a biomarker for disease progression

Materials and

Methods: HA-PLA PSs were optimized by varying molecular weight (MW) of HA to stably encapsulate hydrophilic proteins until exposure to acidic and enzymatic environments. Encapsulation efficiency (EE) and release studies were performed using fluorescein isothiocyanate bovine serum albumin (FITC-BSA). Release was compared in a neutral buffer and in an acidic/enzymatic buffer under various conditions –constant incubation, buffer transfer modeling the pathway following intravenous administration, and with different enzymatic concentrations (Figure 1).
HA(5 kDa)-b-PLA(15 kDa) PSs were loaded during self-assembly with IONPs of differing MRI orientations (PH160s [T1]) or USPIONs [T2]).Transmission electron microscopy (TEM) and ferrozine assays were used to visualize and quantify iron content. Visual responsivity of the PS-IONPs was evaluated by comparing MRI signals over extended incubation in a neutral environment, representing the blood, or an acidic and hyaluronidase-rich environment, representing the pathological site of delivery. Loading and release studies with FITC-BSA were repeated to assess co-loading with IONPs. PSs were tagged with Apolipoprotein E (ApoE) through a covalent surface reactionand verified by dynamic light scattering (DLS) and Lowry assay. MRI studies were repeated with fully functionalized PSs to verify the maintenance of contrast activity, confirmed both in phantom and in vitro MRI images. Cellular uptake in GM1-affected and normal fibroblasts was analyzed by flow cytometry. An ATP-sensing bioluminescent assay was used to assess cell viability. Finally, enzyme assays probed the efficacy of the PSs in restoring appropriate enzyme activity.

Results, Conclusions, and Discussions: HA MW impacted physical characteristics and loading performance of the HA-PLA PSs (Figure 1A). Hydrophilic loading content decreased with increasing MW. Low MW HA-PLA PSs degrade at a higher rate when exposed to acidic/enzymatic conditions as compared to neutral ones (Figure 1B-D). Decreasing HA MW increases responsivity to enzymatic conditions (Figure 1E). Additionally, in HA(5kDa)-PLA PSs, the release rate was directly related to enzyme activity (Figure 1F).
Hydrodynamic diameters (HD) of PSs increased following IONP loading (Figure 2F). On TEM, dark rings evidenced the membrane localization of the IONPs. While the PS-PH160s exhibited a single dark ring consistent with a standard PS (Figure 2A), PS-USPIONs clearly possessed multiple membranes (Figure 2B). Multilamellarity is thought to be a consequence of the bulky PLA coating on the USPIONs (Figure 2D), which increases the overall hydrophobicity of the system.
Both PS-IONP formulations had high relaxivities in resting conditions but exhibited significant weakening following incubation with disease conditions. The r1 of PS-PH160s decreased from 4.90 to 3.51 mM-1s-1 immediately in the acidic/enzymatic buffer. The r2/r1 ratio is the most suitable variable to indicate overall performance changes since T2 was secondarily affected. While r2/r1 stayed consistent in neutral buffer, it spiked initially in the GM1 environment, indicating the system weakened as a T1 agent (Figure 2C) althoughthe loss of T1 strength was recovered by 24 hours. Under normal conditions, PS-USPIONs exhibited an exceptionally high r2 of 211.14 mM-1s-1. The initially high r2 of PS- dropped to 14.371 mM-1s-1 (over a 90% reduction) after only 6 hours in the GM1 environment (3.0 T). The lower relaxivity was maintained over five days but phantom images supported relaxivity decrease as being linked with release CA release (Figure 2F).
UV-vis studies with FITC-BSA confirmed therapeutic co-loading (Figure 2F) and multifunctionality of the HA-PLA PS towards GM1 management.

Conclusion: HA-PLA PSs degrade tunably to pH and enzymatic conditions, creating the basis for not only responsive drug release, but also responsive imaging. The versatility of the PS system could apply to various diseases, but ultimately offer a brain-permeable option for addressing lysosomal enzyme deficiency and inadequate diagnostics in GM1 Gangliosidosis.

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References: 1. Brunetti-Pierri, N; Scaglia, F. Mol. Genet. Metab., 2008. 2. Foster et al. Nanomedicine, 2024. 3. Paruchuri et al. Front. Chem. Eng., 2022. 4. Kelly et al. Nanomedicine, 2017. 5. Foster, D; Larsen, J. ACS Biomater. Sci. Eng., 2023.