John Garner

Maleimide-PEG-PLGA from PolySciTech used in fundamental research on thiol-maleimide conjugation for generating targeted nanoparticles

Blog Post created by John Garner on Mar 15, 2018

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A popular method for cancer treatment is to apply chemotherapeutic or other agents through nanoparticles that float through the patient’s bloodstream. Ideally, these particles are preferentially retained within the tumor site where they deliver their medicinal payload. To facilitate this, the nanoparticles are often covered with a specific targeting ligand. This ligand binds to a select site present on the cancer cells which is not found, or is less prevalent in, normal tissue. A popular chemical technique to create such a nanoparticle is to use PLGA-PEG-Maleimide as a precursor component for making nanoparticles covered with maleimide groups. These maleimides react readily with thiols (typically found in proteins as cysteine units) to bind the protein ligand to the outer surface of the nanoparticle. Despite this reaction’s popularity for use in generating targeted nanoparticles, little has been done in terms of understanding and optimizing the exact reaction kinetics involved with this reaction. Recently, researchers at Utrecht University (Netherlands) utilized reactive maleimide-PEG-PLGA (AI020) and inert methoxy-PEG-PLGA (AK037) from PolySciTech (www.polyscitech.com) to investigate the reaction kinetics and optimization parameters of thiol-maleimide conjugation as applied to nanoparticles. Notably, they found that the formed maleimide-coated nanoparticles should be used soon after manufacture as the maleimide unit itself can be affected by a hydrolysis reaction. This valuable research provides critical information for researchers looking to design targeted nanoparticles using this popular and robust chemistry. Read more: Martínez-Jothar, Lucía, Sofia Doulkeridou, Raymond M. Schiffelers, Javier Sastre Torano, Sabrina Oliveira, Cornelus F. van Nostrum, and Wim E. Hennink. "Insights into maleimide–thiol conjugation chemistry: conditions for efficient surface functionalization of nanoparticles for receptor targeting." Journal of Controlled Release (2018). https://www.sciencedirect.com/science/article/pii/S0168365918301238

“Abstract: Maleimide-thiol chemistry is widely used for the design and preparation of ligand-decorated drug delivery systems such as poly(lactide-co-glycolide) (PLGA) based nanoparticles (NPs). While many publications on nanocarriers functionalized exploiting this strategy are available in the literature, the conditions at which this reaction takes place vary among publications. This paper presents a comprehensive study on the conjugation of the peptide cRGDfK and the nanobody 11A4 (both containing a free thiol group) to maleimide functionalized PLGA NPs by means of the maleimide-thiol click reaction. The influence of different parameters, such as the nanoparticles preparation method and storage conditions as well as the molar ratio of maleimide to ligand used for conjugation, on the reaction efficiency has been evaluated. The NPs were prepared by a single or double emulsion method using different types and concentrations of surfactants and stored at 4 or 20 °C before reaction with the targeting moieties. Several maleimide to ligand molar ratios and different reaction times were studied and the conjugation efficiency was determined by quantification of the not-bound ligand by liquid chromatography. The kind of emulsion used to prepare the NPs as well as the type and concentration of surfactant used had no effect on the conjugation efficiency. Reaction between the maleimide groups present in the NPs and cRGDfK was optimal at a maleimide to thiol molar ratio of 2:1, reaching a conjugation efficiency of 84 ± 4% after 30 min at room temperature in 10 mM HEPES pH 7.0. For 11A4 nanobody the optimal reaction efficiency, 58 ± 12%, was achieved after 2 h of incubation at room temperature in PBS pH 7.4 using a 5:1 maleimide to protein molar ratio. Storage of the NPs at 4 °C for 7 days prior to their exposure to the ligands resulted in approximately 10% decrease in the reactivity of maleimide in contrast to storage at 20 °C which led to almost 40% of the maleimide being unreactive after the same storage time. Our findings demonstrate that optimization of this reaction, particularly in terms of reactant ratios, can represent a significant increase in the conjugation efficiency and prevent considerable waste of resources. Keywords: Nanoparticles; PLGA; Maleimide; Targeting; RGD; Nanobody”

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