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Drug Delivery

Drug Delivery - Poster Session E

Poster X16 - Theranostic Polymersomes for T2-weighted Magnetic Resonance Imaging of Enzyme Delivery as Therapy for GM1 Gangliosidosis

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  GM1 Gangliosidosis is a fatal lysosomal disorder caused by a genetic mutation that leads to the dysfunction of the β-galactosidase (β-gal) enzyme. Enzyme Replacement Therapy (ERT) is a potential treatment method that involves replacing the β-gal enzyme through intravenous injection. However, the blood-brain barrier (BBB) prevents β-gal from reaching the central nervous system, making ERT an ineffective treatment method.
Polymersomes (PSs) are hollow nanoparticles that can be loaded with hydrophilic drugs, such as β-gal. The nanoscale size of PSs makes them small enough to cross the BBB with the help of receptor-mediated transcytosis. In this project, we first tuned the molecular weight (MW) of our amphiphilic polymer to optimize enzyme-responsive polymersomes for degradation in response to upregulated hexosaminidase A, which is a hallmark of GM1 gangliosidosis, as well as to acidic conditions, which are characteristic of lysosomes. Polymersomes can also be co-encapsulated with hydrophobic entities within their membrane, enabling the development of theranostic system.
Contrast Agents (CAs) allow specific regions to be highlighted ion Magnetic Resonance Imaging (MRI) by enhancing surrounding contrast. Ultra Small Superparamagnetic Iron-Oxide Nanoparticles (USPIONs) are CAs that are small enough for membrane loading but too small to act with much strength when freely dispersed. By co-loading alongside β-gal into our stimuli-responsive PSs, the USPIONs associate to act as one large, strong CA up until the time of release, effectively allowing for drug tracking and brain delivery verification.

Materials and

Methods:  Hyaluronic acid (HA)-b-poly(lactic acid) (PLA) was synthesized using various MW HA blocks (5, 7, and 9 kDa), with a PLA block of 15 kDa. HA-b-PLA PSs were then synthesized using the solvent injection method. The PSs were fully characterized through dynamic light scattering and transmission electron microscopy before evaluating encapsulation efficiency towards hydrophilic proteins as modeled with fluorescein isothiocyanate bovine serum albumin (FITC-BSA) and quantified using UV-vis. Ultimately, release studies, also using UV-vis and FITC-BSA, were the most informative for differentiating the formulations on the basis of their propensity to responsively release.
Having identified HA(5)-PLA(15) to best suit our desired applications, we loaded slightly hydrophobic USPIONs into the membrane by incorporating them into the organic phase of synthesis (termed “PS-USPIONS”). CA loading was verified through TEM imaging and ferrozine assay which allowed for quantification. We also assessed co-loading capacity again using FITC-BSA.
MRI phantom images were taken on a 3.0 T scanner to assess PS-USPION behavior under different physiological conditions, similar to those examined in the aforementioned release studies. Next, the system was evaluated in SHSY-5Y cells using an MTS assay for cell viability. Having revealed no cytotoxicity concerns, the PS-USPIONs were assessed in animal studies. Postmortem imaging following an intramuscular injection was used to verify the imageability of the CA. Finally, pharmacokinetics were monitored in fluorescently co-loaded PS-USPIONs through blood draws at selected time points over 24 hours after tail vein injection to confirm the pathology responsive nature of the developed system.

Results, Conclusions, and Discussions: PS formation was confirmed through imaging that was consistent with the sub-100 nm diameters determined for each formulation via DLS. Each polymer yielded PSs suitable for brain and lysosomal delivery based on their small size and negative zetapotential which is stabilizing for cellular uptake. Hydrophilic loading capacity decreased slightly with increasing HA MW. Ultimately, the release studies served as the most differentiating factor – while all PSs showed increased release in an acidic/enzymatic environment over a neutral one, the sensitivity of PS degradation was greater at lower HA MW (Figure 1A-C). For our purposes of preferentially delivering therapeutics to the GM1 lysosome, the HA(5)-PLA(15) PSs showed the most promise.

CA loading was confirmed through TEM imaging consistent with the DLS-determined diameter (Figure 1D). MRI phantom images showed an increasing T2-weighted darkening effect with increasing USPION concentration, with contrast lost upon incubation in a pathophysiological environment (Figure 1E-F). This is consistent with prior release studies, suggesting that the MRI response is due to PS degradation. Altogether, this shows that USPIONs released in much the same way as hydrophilic payloads which would establish a visual basis to track drug course in the body. The MTS assay on SHSY-5Y cells showed above 80% viability for PS-USPION (Figure 1G) dosages without a significant difference between treatment times of 4 hours and 24 hours.

100 µL of 225 mg PS-UPSIONs/mL PBS injected intramuscularly showed a brightening effect on MRI in the injected muscular tissues, indicating the detectability of the polymersomes in situ (Figure 1H). Finally, UV-vis analysis on blood draws and IVIS imaging following PS-USPION tail vein injection showed increased fluorescence at all time points in β-gal knockout mice, with fluorescence detected at 2 orders of magnitude greater than in wild-type mice (Figure 1I). This data demonstrates the pathology-responsive nature of PS-USPIONs in vivo.

In conclusion, HA(5)-PLA(15) PSs can be effectively co-loaded with proteins and MRI CAs, making them a responsive foundation towards MRI trackable ERT for GM1 gangliosidosis. Simultaneously, PS-USPIONs degrade more rapidly in a pathology environment of GM1-affected β-gal-/- mice, causing delivery of the payload and of the CA to a diseased area.

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