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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