Influenza A virus mimetic nanoparticles trigger selective cell uptake.

Poor target cell specificity is currently a major shortcoming of nanoparticles (NPs) used for biomedical applications. It causes significant material loss to off-target sites and poor availability at the intended delivery site. To overcome this limitation, we designed particles that identify cells in a virus-like manner. As a blueprint, we chose a mechanism typical of influenza A virus particles in which ectoenzymatic hemagglutinin activation by target cells is a mandatory prerequisite for binding to a secondary target structure that finally confirms cell identity and allows for uptake of the virus. We developed NPs that probe mesangial cells for the presence of angiotensin-converting enzyme on their surface using angiotensin I (Ang-I) as a proligand. This initial interaction enzymatically transforms Ang-I to a secondary ligand angiotensin II (Ang-II) that has the potential to bind in a second stage to Ang-II type-1 receptor (AT1R). The presence of the receptor confirms the target cell identity and triggers NP uptake via endocytosis. Our virus-mimetic NPs showed outstanding target-cell affinity with picomolar avidities and were able to selectively identify these cells in the presence of 90% off-target cells that carried only the AT1R. Our results demonstrate that the design of virus-mimetic cell interactive NPs is a valuable strategy to enhance NP specificity for therapeutic and diagnostic applications. Our set of primary and secondary targets is particularly suited for the identification of mesangial cells that play a pivotal role in diabetic nephropathy, one of the leading causes of renal failure, for which currently no treatment exists.

Branched Polymer-Drug Conjugates for Multivalent Blockade of Angiotensin II Receptors.

The use of angiotensin receptor blockers (ARBs) for treatment of ocular diseases associated with neovascularizations, such as proliferative diabetic retinopathy, shows tremendous promise but is presently limited due to short intravitreal half-life. Conjugation of ARB molecules to branched polymers could vastly augment their therapeutic efficacy. EXP3174, a potent non-peptide ARB, was conjugated to branched poly(ethylene glycol) (PEG) and poly(amido amine) (PAMAM) dendrimers: 7.8 ligand molecules were tethered to each 40 kDa PEG molecule whereas 16.7 ligand molecules were linked to each PAMAM generation 5 dendrimer. The multivalent PEG and PAMAM conjugates blocked AT1R signaling with an IC50 of 224 and 36.3 nM, respectively. The 6-fold higher affinity of the multivalent ligand-conjugated PAMAM dendrimers was due to their unique microarchitecture and ability to suppress polymer-drug interactions. Remarkably, both polymer-drug conjugates exhibited no cytotoxicity, in stark contrast to plain PAMAM dendrimers. With sufficiently long vitreous half-lives, both synthesized polymer-ARB conjugates have the potential to pave a new path for the therapy of ocular diseases accompanied by retinal neovascularizations.

Nanoparticle multivalency counterbalances the ligand affinity loss upon PEGylation.

The conjugation of receptor ligands to shielded nanoparticles is a widely used strategy to precisely control nanoparticle-cell interactions. However, it is often overlooked that a ligand's affinity can be severely impaired by its attachment to the polyethylene glycol (PEG) chains that are frequently used to protect colloids from serum protein adsorption. Using the model ligand EXP3174, a small-molecule antagonist for the angiotensin II receptor type 1 (AT1R), we investigated the ligand's affinity before and after its PEGylation and when attached to PEGylated nanoparticles. The PEGylated ligand displayed a 580-fold decreased receptor affinity compared to the native ligand. Due to their multivalency, the nanoparticles regained a low nanomolar receptor affinity, which is in the range of the affinity of the native ligand. Moreover, a four orders of magnitude higher concentration of free ligand was required to displace PEGylated nanoparticles carrying EXP3174 from the receptor. On average, one nanoparticle was decorated with 11.2 ligand molecules, which led to a multivalent enhancement factor of 22.5 compared to the monovalent PEGylated ligand. The targeted nanoparticles specifically bound the AT1R and showed no interaction to receptor negative cells. Our study shows that the attachment of a small-molecule ligand to a PEG chain can severely affect its receptor affinity. Concomitantly, when the ligand is tethered to nanoparticles, the immense avidity greatly increases the ligand-receptor interaction. Based on our results, we highly recommend the affinity testing of receptor ligands before and after PEGylation to identify potent molecules for active nanoparticle targeting.

Structural properties of model phosphatidylcholine flippases.

Lipid translocation from one lipid bilayer leaflet to the other, termed flip-flop, is required for the distribution of newly synthesized phospholipids during membrane biogenesis. However, a dedicated biogenic lipid flippase has not yet been identified. Here, we show that the efficiency by which model transmembrane peptides facilitate flip of reporter lipids with different headgroups critically depends on their content of helix-destabilizing residues, the charge state of polar flanking residues, and the composition of the host membrane. In particular, increased backbone dynamics of the transmembrane helix relates to its increased ability to flip lipids with phosphatidylcholine and phosphatidylserine headgroups, whereas a more rigid helix favors phosphatidylethanolamine flip. Further, the transmembrane domains of many SNARE protein subtypes share essential features with the dynamic model peptides. Indeed, recombinant SNAREs possess significant lipid flippase activity.