(F-205) Lysosome-directed delivery of theranostic polymersomes for GM1 gangliosidosis

Introduction:: Lysosomal storage disorders (LSDs), are caused by the dysfunction of a lysosomal enzyme causing eventual cell death. Neurodegenerative LSDs, like GM1 gangliosidosis (GM1) are especially devastating due to the essential nature of the brain paired with a lack of clinical management options. Enzyme replacement therapy (ERT) would be an effective treatment, as previously shown in non-neurodegenerative LSDs, if the challenges associated with delivery through the blood brain barrier (BBB) are met. We investigate hyaluronic acid (HA)-poly(lactic acid) (PLA) polymersomes as pathology-responsive drug carriers, integrating controlled drug delivery approaches with carrier mediated transport to allow for brain- and lysosome-targeted drug delivery. HA is recognized for internalization by CD44 receptors of the brain and is degraded by Hexosaminidase A (HexA), an enzyme that is upregulated in GM1, while PLA is degraded by acidic conditions like those of the lysosome. To monitor drug delivery as well as establish the first real-time diagnostic modality for GM1 (and ultimately any LSD), we are developing an acid-activated magnetic resonance imaging (MRI) contrast agent (CA). Due to their amphiphilic nature, HA-PLA polymersomes can be used to simultaneously encapsulate and deliver the missing enzyme in GM1 and a CA. Through polymeric modification of a T₁-dominant iron oxide nanoparticle, the CA will be muted by limiting direct tissue interaction until carrier release allows for pH-based polymeric degradation or conformational change. In totality, the loaded polymersome will yield a patient-centered, personalized theranostic tool.

Materials and Methods:: HA-PLA polymersomes made from various HA molecular weights (MW) (5 kDa, 7 kDa, and 9 kDa) were self-assembled via solvent-injection method and were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Fluorescein-bovine serum albumin (FITC-BSA) was used to assess polymersome loading and release performance; following dialysis, fluorescence intensity in water and in different buffers (neutral – blood, acidic – lysosome, or enzymatic/acidic – diseased lysosome), as determined by UV/vis spectroscopy, were used to calculate encapsulation efficiency (EE) and monitor release behavior. Release studies were performed to assess environmental release. Cell studies were performed using untreated cells as controls. Cellular uptake was analyzed both qualitatively and quantitatively by fluorescent microscopy and flow cytometry, respectively. An MTS assay was used to assess cell viability. Ultrasmall iron oxide nanoparticles (USPIONs, 3nm, purchased commercially) were PLA-coated as confirmed by DLS and TEM. The solvent-injection polymersome assembly protocol was modified slightly for polymersome loading with the dissolution of USPIONs into the organic phase. Colorimetric ferrozine assays measured EE based on iron content. CA performance evaluation was characterized via NMR.

Results, Conclusions, and Discussions:: Results: Regardless of HA MW, each polymersome formulation had attractive physical properties and in vitro performance for brain delivery. Lysosome-triggered release was supported by a biopathway release study showing limited release during the 6-hour incubation in neutral buffer, followed by burst release upon placement into an enzymatic buffer (Figure 1).

EE of β-galactosidase, the missing enzyme in GM1, can be related to both vesicle size and polymer MW; in our formulations, the greatest EE (44.51±14.87%) was achieved by the HA(5 kDa)-PLA formulation. Previous work demonstrated that HA-PLA polymersomes encapsulate β-galactosidase into their core. To maximize theranostic capacity, it is essential that CAs are loaded into the hydrophobic membrane instead. Therefore, USPIONs were PLA(15 kDa) coated; preliminary DLS data suggests successful coating with a size increase from 3 nm to ~25nm. In general, USPION loading increased polymersome diameter but the size was still less than 200 nm. EE decreased linearly with the mass of USPION dissolved. Longitudinal relaxation time for the uncoated and PLA-coated USPIONs as well as for loaded polymersome formulations will be measured by NMR.

Discussion: Ultimately, HA(5 kDa)-PLA polymersomes were chosen for further development based on high EE and most pronounced responsivity to pathological conditions.

For USPION performance, we seek low relaxivity in either PLA-coated or polymersome-encapsulated conditions and high relaxivity in the uncoated form. Therefore, polymersome release or subsequent PLA degradation will result in CA activation. With a pH-responsive CA in place, we hope to establish a correlation between MRI enhancement and disease severity based on relative lysosome concentration.

Conclusion: Encapsulation, release, and cellular uptake studies support these polymersomes as effective theranostic carriers for targeted delivery to the diseased lysosome. In further development for the integration of polymer-based activatable CA functionality, the HA(5 kDA)-PLA polymersome continues to show appropriate physical conditions now paired with effective USPION loading. In totality, this theranostic system will offer patient-focused advantages for the treatment and monitoring of GM1 gangliosidosis.


Acknowledgements (Optional): : This work was supported in part by the National Science Foundation CAREER program under NSF Award # 2047697.

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