Synthesis of Pyridyl Disulfide-Functionalized Nanoparticles for Conjugating Thiol-Containing Small Molecules, Peptides, and Proteins

Abstract

Previously we reported emulsion polymerization of propylene sulfide with Pluronic F127 as an emulsifier, yielding nanoparticles (NPs) in the 25 nm size range. Immunologically functional NPs were prepared by adding an antigen-Pluronic conjugate to the polymerization mixture ( Reddy , S. T. , et al. ( 2007 ) Nat. Biotechnol. 25, 1159 ). We sought a more flexible scheme for conjugation of antigens and other biomolecules to the NP surfaces that would allow for milder reaction conditions than achievable during the polymerization step. Here, we present the synthesis of such functionalizable NPs in the form of NPs that carry thiol-reactive groups, to which thiol-containing antigens (peptide or protein) or other biomolecules can be conjugated under mild conditions to yield immunofunctional NPs. The Pluronic-stabilized poly(propylene sulfide) (PPS) NPs with thiol-reactive pyridyl disulfide groups are prepared in two steps by (1) emulsion polymerization of propylene sulfide in the presence of a carboxylate-Pluronic and (2) reaction of the carboxylic acid groups on the NP surface with cysteamine pyridyl disulfide and a water-soluble carbodiimide reagent. We choose pyridyl disulfide groups to have a reduction-sensitive disulfide bond linking the antigen to the NP surface, allowing efficient release of antigen inside the cell in response to the reductive conditions within the endosome. The functionalizable NPs are characterized by proton NMR, dynamic light scattering (DLS), UV/vis spectroscopy, and transmission electron microscopy (TEM). Conjugation of small molecules and protein to the NP surface is presented.

Nanoparticle conjugation of antigen enhances cytotoxic T-cell responses in pulmonary vaccination

“…For antigen conjugation, ova and NPs were incubated for 6 h at room temperature in the presence of guanidinium hydrochloride (Sigma-Aldrich) to expose by partial unfolding the free thiols on the protein. The extent of ova conjugation was monitored by pyridine-2-thione release at 340 nm (7). Free antigen and guanidinium hydrochloride were removed at the end of the reaction as described earlier; NP-ova solution was eluted and stored in PBS solution at room temperature.…”

Enhancing Efficacy of Anticancer Vaccines by Targeted Delivery to Tumor-Draining Lymph Nodes

“…Nanoparticles developed by our group target skin-draining LNs and resident antigen-presenting cells (APC) upon intradermal administration (20,21). To determine whether nanoparticles could be targeted to the tdLN harnessing lymphatic drainage, we polarized one side of the mouse as being tumordraining by inoculating 10 6 E.G7-OVA tumor cells intradermally on one side of the back of mice, thus defining an ipsi tumor-draining side and the contra non-tumor-draining side ( Supplementary Fig.…”

“…Strategies based on delivering adjuvants or antigens carried by nanoparticles or liposomes have led to improved targeting of LNs, activation of LN-resident DCs, and enhanced induction of antigen-specific CD8 þ T cells, hence better protection in tumor challenge studies (14)(15)(16)(17)(18)(19). Ultrasmall, functionalizable nanoparticles (NP) developed in our laboratory effectively target DCs in skin-draining LNs upon intradermal delivery (20,21). These nanoparticles lead to antigen crosspresentation by DCs and to enhanced cytotoxic antigenspecific CD8 þ T-cell immunity when coupled with an antigen or an adjuvant (22)(23)(24).…”

Intracellular Cleavable CpG Oligodeoxynucleotide-Antigen Conjugate Enhances Anti-tumor Immunity

“…These include particulate and soluble formulations where either the antigen or adjuvant was conjugated with a reversible linker onto either a nanoparticle or a polymer carrier. 8,10,[22][23][24][25][26][27][28] The redoxresponsive disulphide bond was selected as a conjugate linker to achieve triggered release of the antigen from the adjuvant once the soluble conjugate was delivered inside the cell. The disulphide bond can be cleaved through the thiol-disulphide exchange reactions with a redox molecule such as glutathione (GSH), which is found at millimolar concentrations (0.5-10 mM) in intracellular compartments, while its concentration is much lower in extracellular milieu (2-40 mM).…”