(118f) Biohybrid Polymersome-Mediated Enzyme Replacement Therapy for GM1 Gangliosidosis

Background: Lysosomal storage disorders (LSDs) are a group of 50 rare metabolic disorders caused by deficiency or absence of the lysosomal enzymes and accumulation of their substrate molecules in lysosomes. Enzyme replacement therapy (ERT) is one of the currently available treatments of LSDs. It involves intravenous delivery of the missing enzyme from an external source to compensate for the deficiency. GM1 gangliosidosis is an autosomal recessive LSD caused by the mutations in the GLB1 gene, which encodes the production of the lysosomal enzyme β-galactosidase (β-gal). Subsequently, substrate molecules of β-gal, primarily GM1 ganglioside, and others like keratan sulfate and a few oligosaccharides with terminal β -galactosyl groups build-up in the lysosomes¹. GM1 gangliosidosis has a prevalence of one in 100,000 to 200,000². Due to the CNS involvement in GM1 gangliosidosis, the blood-brain barrier (BBB) presents a challenge for treatment using ERT. The presence of BBB necessitates the use of a drug delivery system that can encapsulate and transport the deficient enzyme across the BBB through receptor-mediated transcytosis (RMT). Previous work in our lab³ demonstrates the effectiveness of a pH-responsive polymer-based system to encapsulate and deliver β-gal to GM1 gangliosidosis-affected cells. However, the payload release is limited to ~35%.

Motivation: A lysosomal enzyme β-hexosaminidase A (HexA) is previously reported to be upregulated in GM1 gangliosidosis-affected felines, with HexA reaching normal levels after treatment with a viral vector⁴. To utilize this phenomenon, hyaluronic acid-b-poly(lactic acid) (HA-PLA) nanoparticles are used to encapsulate and deliver β-gal. HexA degrades hyaluronic acid⁵, and PLA undergoes acidic hydrolysis, thereby rendering the nanoparticles dual-responsive. Due to the presence of hydrophilic HA and hydrophobic PLA, the amphiphilic HA-PLA copolymer readily self-assembles into nanoparticles in the aqueous phase. The hydrophilic fraction (f) of a polymer is the ratio of the mass of hydrophilic block to the total mass. It dictates the structure of the self-assembled nanoparticles. The enzymatic nature of the cargo requires the use of vesicular nanoparticles called polymersomes. Functionalizing the surface of these nanoparticles with apolipoprotein E (ApoE), a ligand that binds to the low-density lipoprotein receptors expressed on the BBB, can facilitate the RMT across BBB^6,7.

Methods: The HA-PLA polymer is synthesized through a two-step polymer conjugation process⁸. Briefly, hyaluronic acid (mol. wt. 5000 Da) is terminally functionalized with 1,4-diaminobutane to introduce an amine group at the reducing end. The aminated HA is then reacted with N-Hydroxysuccinimide (NHS) activated PLA in the presence of N,N-diisopropylethylamine. The reaction mixture is dialyzed against DI water to remove solvents and unreacted hyaluronic acid, followed by lyophilization to obtain the polymer HA-PLA. The polymer is subsequently used to synthesize nanoparticles using the solvent-injection method. The particle size and structure are analyzed using the dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The enzyme-based degradation of nanoparticles is determined using both DLS and TEM.

Results: The conjugated polymer is analyzed using Attenuated total reflectance-Fourier-transform infrared (ATR-FTIR) spectroscopy. The resultant spectrograph analysis demonstrated peaks corresponding to hyaluronic acid and poly(lactic acid) polymer blocks, confirming the conjugation. To prepare nanoparticles, HA-PLA is dissolved in dimethyl sulfoxide and injected into aqueous phase at controlled rate. DLS analysis of nanoparticles after filtration resulted in a peak diameter of 199.4 ± 42.6 nm. The TEM imaging of the nanoparticles revealed a vesicular structure. To determine the enzyme-based degradation of the polymersomes, they were incubated separately with hyaluronidase (HYAL) (cognate enzyme) and β-gal (non-cognate enzyme). HexA and HYAL have similar functionality with regards to the degradation of hyaluronic acid⁵. After incubating with the enzymes for 24 hours, particles incubated with HYAL presented a peak diameter (in particle size distribution of DLS analysis) at a smaller size indicating degradation. The peak diameter of the particles incubated with β-gal remained in the same range as before incubation indicating little to no degradation. For release study, polymersomes loaded with the dye Fluorescein Isothiocyanate-Dextran (FITC-D) as model encapsulant were incubated with HYAL and β-gal, and the fluorescence signal is monitored for hours. A higher release of FITC-D upon incubation with HYAL was observed compared to the release in β-gal incubation.

Conclusions: These results corroborate the conjugation of the hyaluronic acid and poly(lactic acid) blocks. The ability of the amphiphilic HA-PLA polymer to self-assemble into vesicular nanoparticles has been proven with the help of TEM. DLS analysis and TEM imaging show that the resultant nanoparticles can undergo enzymatic degradation in the presence of hyaluronidase. Release study showed the selective degradation of the nanoparticles in the presence of cognate enzyme (HYAL).

References:

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