Hybrid Silica-Coated PLGA Nanoparticles for Enhanced Enzyme-Based Therapeutics

Abstract

Some cancer cells rely heavily on non-essential biomolecules for survival, growth, and proliferation. Enzyme based therapeutics can eliminate these biomolecules, thus specifically targeting neoplastic cells; however, enzyme therapeutics are susceptible to immune clearance, exhibit short half-lives, and require frequent administration. Encapsulation of therapeutic cargo within biocompatible and biodegradable poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) is a strategy for controlled release. Unfortunately, PLGA NPs exhibit burst release of cargo shortly after delivery or upon introduction to aqueous environments where they decompose via hydrolysis. Here, we show the generation of hybrid silica-coated PLGA (SiLGA) NPs as viable drug delivery vehicles exhibiting sub-200 nm diameters, a metastable Zeta potential, and high loading efficiency and content. Compared to uncoated PLGA NPs, SiLGA NPs offer greater retention of enzymatic activity and slow the burst release of cargo. Thus, SiLGA encapsulation of therapeutic enzymes, such as asparaginase, could reduce frequency of administration, increase half-life, and improve efficacy for patients with a range of diseases.

Keywords: amino acid depletion; biocompatible; cancer; double emulsion; drug delivery; enzymes; nanoparticles; poly(lactic-co-glycolic acid); silica.

Figure 1

Synthesis of PLGA and SiLGA nanoparticles: Enzyme-encapsulated SiLGA NPs were synthesized using a water-oil-water double emulsion (PLGA NPs) and subsequent sol-gel polycondensation reaction of silicic acid to silica. Enzyme molecules (pink) were suspended in the first water phase (“W1”; blue). PLGA molecules (red and blue) were dissolved in an organic phase (“O”; yellow). The two phases were emulsified to form “Emulsion 1”, consisting of a suspension of aqueous droplets in oil. Emulsion 1 was then introduced to a second water phase (“W2”; blue) containing PVA molecules (green). A second emulsification produced “Emulsion 2”, which consisted of an aqueous suspension of oil droplets with an aqueous core. Subsequent evaporation of the oil phase solvent yielded an aqueous suspension of PLGA NPs (red) with an enzymatic core (pink) that was stabilized by PVA (green). Subsequent introduction of silicic acid (purple dots) initiated a polycondensation reaction, forming a porous silica layer (purple) at the surfaces of the PLGA NPs to form SiLGA NPs. A portion of this figure was created using Biorender.com (BioRender 2021), accessed on 18 October 2021.

Figure 2

DLS, NTA, and TEM show silica coating: Distributions of (a) Cumulant hydrodynamic diameters, (b) hydrated diameters, and (c) Zeta potential of PLGA (black) and SiLGA (red) NPs. Transmission electron micrographs of (d) PLGA NPs (scale bar is 200 nm) and (e) SiLGA NPs (scale bar is 100 nm).

Figure 3

Penicillinase loading and retention of activity after treatment with PK: (a) The percent retention of penicillinase activity for unencapsulated (“Bare”) and encapsulated (“PLGA”; “SiLGA”) formulations after treatment with the proteolytic enzyme, PK. Error bars represent one standard deviation of the mean across triplicate measurements. Representative curves shows the shift in optical absorbance when nitrocefin substrate is added to penicillinase in (b) unencapsulated, (c) PLGA, and (d) SiLGA formulations both before (black) and after (red) PK treatment. Each contour represents the average of technical duplicate measurements. (e) Standard curve for enzymatic activity as a function of penicillinase concentration (black diamonds; R² = 0.985). Measured penicillinase activity of wash buffers for PLGA (blue circles) and SiLGA (red squares) NPs were plotted to estimate respective penicillinase concentrations for loading efficiency and content calculations.

Figure 4

Silica slows burst release kinetics of BSA after intramuscular injection in mice: (a) Radiation efficiency of BSA-Cy7 PLGA NPs (top), BSA-Cy7 SiLGA NPs (middle), and unencapsulated BSA-Cy7 (bottom) in the hind legs of immunocompetent mice (B6a) as imaged over a five-day span; saline was injected into the negative control shown at Day 0. Normalized fluorescence levels of intramuscular BSA-Cy7 over a five-day span in (b) B6a and (c) NSG mice. Error bars represent one standard deviation above and below the mean normalized fluorescence level, calculated across six injection sites. (d) Normalized fluorescence levels of unencapsulated (“Bare”) PLGA, and SiLGA formulations of BSA-Cy7 after one day in (d) B6a and (e) NSG mice.