Optimization of Protein Precipitation for High-Loading Drug Delivery Systems for Immunotherapeutics

Abstract

Cancer is the second leading cause of death in the world and is often untreatable. Protein-based therapeutics, such as immunotherapeutics, show promising results in the fight against cancer, resulting in their market share increasing every year. Unfortunately, most protein-based therapeutics suffer from fast degradation in the blood, making effective treatment expensive, causing more off-target effects (due to the high doses necessary), and often require repeated injections to stay within the correct therapeutic range. Encapsulation of these proteins inside nanocarriers is prompted to overcome these problems by enhancing targeted drug delivery and, thus, leading to a less frequent administration and lower required dose. However, most current protein encapsulation methods show very low loading capacities (LC). This leads to even more expensive treatments and might pose a further risk for the patient caused by systemic toxicity against high concentrations of the carrier material. We investigated and optimized protein nanoprecipitation as a method to obtain a high protein LC and encapsulation efficiency (EE) inside poly(lactic-co-glycolic acid; PLGA) nanoparticles via a simple two-step process. In this work, we used model proteins to investigate the influence of various parameters such as precipitation solvent, addition speed, and protein concentration on protein activity. Our work is a critical step towards the high-loading encapsulation of immunotherapeutics.

Polymeric nanomaterial strategies to encapsulate and deliver biological drugs: points to consider between methods

“…88 Notably, the precipitation method that Liao et al 88 used to compare them was a one-step nanoprecipitation that did not precipitate catalase in NP form before mixing the catalase with the PLGA–PEG/acetone solution. Combined with the conclusion from Nelemans et al , 99 the acetone solution in PLGA may impact both the distribution and the activity of catalase.…”

“…Nelemans et al 99 recently optimised a drug delivery system to encapsulate BSA and amylase with PLGA. This study first showed that only acetonitrile could precipitate BSA with a homogeneous particle distribution while acetone and ethanol caused a heterogeneous particle distribution.…”

“…The authors eventually encapsulated amylase with PLGA and 1% Pluronic F68 (diameter: 147.7 ± 4.3 nm, including a 14 nm PLGA shell). However, Nelemans et al 99 only encapsulated about 24 ± 11.2% of the amylase into the PLGA NPs. They tested the initially precipitated proteins for activity by directly dissolving the proteins in PBS.…”

Preparation of protein-loaded nanoparticles based on poly(succinimide)-oleylamine for sustained protein release: a two-step nanoprecipitation method

“…Data analyses were performed using GraphPad Prism (GraphPad Software Inc.) ACN to BSA solution under stirring (figure 2(A)). Previous studies demonstrated the fewest protein aggregates with smaller particle sizes and uniform distribution when ACN was utilised as the non-solvent compared with acetone and ethanol [38]. Thus, ACN was selected as the non-solvent for BSA.…”

“…We modified the protocol of Morales-Cruz et al [37] and Nelemans et al [38]. BSA was first precipitated into small spherical NPs.…”