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C-reactive protein (CRP) is widely used as biomarkers of infection and inflammation. It has a well-described ability to bind phosphocholine (PC), as well as PC-clusters from compromised and inflamed cell membranes and tissues. The binding of PC-clusters to CRP is of interest as this binding determines subsequent innate immune activity. We investigated PC-decorated dendrimers as mimics for PC-clusters. Five generations of poly(propylene imine) (PPI) dendrimers were modified with PC surface groups via a three-step synthetic sequence obtaining the PC-decorated dendrimers in high purity. The dendrimers were analyzed by NMR and infrared spectroscopy as well as HPLC. We developed immunoassays to show that dendrimer-PC binding to CRP was Ca 2+ -dependent with an apparent overall K d of 11.9 nM for first generation (G1) PPI-PC, while G2-PPI-PC and G3-PPI-PC had slightly higher affinities, and G4-PPI-PC and G5-PPI-PC had slightly lower affinities. For all PC-dendrimers, the affinity was orders of magnitude higher than the affinity of free phosphocholine (PC), indicating a PC-cluster effect. Next, we investigated the binding of CRP:PPI-PC complexes to complement component C1q. C1q binding to CRP was dependent on the generation of PPI-PC bound to CRP, with second and third generation PPI-PCs leading to the highest affinity. The dendrimer-based approach to PC-cluster mimics and the simple binding assays presented here hold promise as tools to screen PC-compounds for their abilities to tune the innate immune activity of CRP.

Synucleinopathies are neurodegenerative pathologies in which disease progression is closely correlated to brain accumulation of insoluble α-synuclein, a small protein abundantly expressed in neural tissue. Here, two types of modified polypropyleneimine (PPI) dendrimers having either urea or methylthiourea (MTU) surface functional groups were investigated in a cellular model of synucleinopathy. Dendrimers are synthetic macromolecules that may be produced in a range of well-defined molecular sizes. Using cellomics array scan high-content screening, we show that both types of dendrimers are able to significantly reduce intracellular levels of α-synuclein aggregates dependent on the concentration, the type and molecular size of the dendrimer with the bigger size MTU-dendrimers having the highest potency. The intracellular clearance of α-synuclein aggregates by dendrimers was achieved at noncytotoxic concentrations.

Amino-terminated dendrimers are well-defined synthetic hyperbranched polymers and have previously been shown to destabilize aggregates of the misfolded, pathogenic, and partially protease-resistant form of the prion protein (PrPSc), transforming it into a partially dissociated, protease-sensitive form with strongly reduced infectivity. The mechanism behind this is not known, but a low pH, creating multiple positively charged primary amines on the dendrimer surface, increases the efficiency of the reaction. In the present study, surface amines of the dendrimers were modified to yield either guanidino surface groups (being positively charged at neutral pH) or urea groups (uncharged). The ability of several generations of modified dendrimers and unmodified amino-terminated dendrimers to deplete PrPSc from persistently PrPSc-infected cells in culture (SMB cells) was studied. It was found that destabilization correlated with both the generation number of the dendrimer, with higher generations being more efficient, and the charge density of the surface groups. Urea-decorated dendrimers having an uncharged surface were less efficient than positively charged unmodified- (amino) and guanidino-modified dendrimers. The most efficient dendrimers (generation 4 (G4) and G5-unmodified and guanidino dendrimers) cleared PrPSc completely by incubation for 4 days at less than 50 nM. In contrast to both unmodified and guanidine-modified dendrimers, the uncharged urea dendrimers showed much lower cytotoxicity toward noninfected SMB cells. Therapeutic uses of modified dendrimers are indicated by the low concentrations of dendrimers needed.