Optimizing Mannose “Click” Conjugation to Polymeric Nanoparticles for Targeted siRNA Delivery to Human and Murine Macrophages

Abstract

“Smart”, dual pH-responsive, and endosomolytic polymeric nanoparticles have demonstrated great potential for localized drug delivery, especially for siRNA delivery to the cytoplasm of cells. However, targeted delivery to a specific cell phenotype requires an additional level of functionality. Copper-catalyzed azide–alkyne cycloaddition (CuAAC) is a highly selective bioconjugation reaction that can be performed in conjunction with other polymerization techniques without adversely affecting reaction kinetics, but there exists some concern for residual copper causing cytotoxicity. To alleviate these concerns, we evaluated conjugation efficiency, residual copper content, and cell viability in relation to copper catalyst concentration. Our results demonstrated an optimal range for minimizing cytotoxicity while maintaining high levels of conjugation efficiency, and these conditions produced polymers with increased targeting to M2-polarized macrophages, as well as successful delivery of therapeutic siRNA that reprogrammed the macrophages to a proinflammatory phenotype.

Increased canonical NF-kappaB signaling specifically in macrophages is sufficient to limit tumor progression in syngeneic murine models of ovarian cancer

“…Previous studies from our group have shown that increasing canonical NF-κB activity in macrophages is associated with a shift away from the M2 pro-tumor phenotype [23,24]. We therefore used immunofluorescence analysis to examine whether the anti-tumor effects observed in IKFM mice were accompanied by reduced M2 polarization of TAMs.…”

“…Although one study found that inhibition of canonical NF-κB signaling in TAMs leads to the anti-tumorigenic M1 phenotype [22], an increasing body of literature demonstrates the opposite, finding that increased NF-κB activity in macrophages polarizes them towards an M1-like anti-tumorigenic phenotype. We have shown that knockdown of cytoplasmic IκBα, inhibitor of NF-κB, promotes a shift towards the M1 phenotype and induces cytotoxicity towards tumor cells in cultured macrophages [23,24]. Moreover, complementary in vivo studies in transgenic mice overexpressing a constitutively activated form of cytoplasmic IKK2 specifically in macrophages, termed IKFM, show reduced growth and metastatic seeding of injected melanoma and mammary tumor cells [19,21].…”

“…Accumulating evidence from our group and others indicates that a key regulator of macrophage behavior is canonical nuclear factor-kappaB (NF-κB) signaling, via nuclear translocation and binding of p65/p50 transcription factors [18][19][20][21][22][23][24]. Although one study found that inhibition of canonical NF-κB signaling in TAMs leads to the anti-tumorigenic M1 phenotype [22], an increasing body of literature demonstrates the opposite, finding that increased NF-κB activity in macrophages polarizes them towards an M1-like anti-tumorigenic phenotype.…”

Refined construction of antibody-targeted nanoparticles leads to superior antigen binding and enhanced delivery of an entrapped payload to pancreatic cancer cells

“…These reaction conditions bypass potential toxicity associated with copper catalysis, which has been employed in many similar efforts to functionalise nanoparticles via alkyne-azide cycloaddition. [17][18][19] Characterisation of these nanoparticles revealed a marginal increase in diameter versus the nude azide NP control and a protein content of 16.1 ± 1.5 µg mg −1 polymer, indicating that conjugation had been achieved (Table 1).…”

Mannosylated Cationic Copolymers for Gene Delivery to Macrophages

“…[ 10 ] It is shown to bind various carbohydrates and oligosaccharides, in particular, mannose either in a free form or conjugated to polymer scaffolds. [ 11 ] As of today, there is an extensive body of work using mannose as a targeting moiety to mannose macrophage receptor (MMR), in which various nanocarriers were employed for the delivery of small molecules, [ 12–16 ] proteins, [ 17,18 ] nucleic acids, [ 19–23 ] and structures like nanoparticles of different types [ 24–26 ] or supramolecular aggregates. [ 27 ] As far as the delivery of the plasmid DNA (pDNA) is concerned the most suitable vehicles are either lipoplexes or polyplexes.…”