Measuring the Adhesion Forces for the Multivalent Binding of Vancomycin-Conjugated Dendrimer to Bacterial Cell-Wall Peptide

Multivalent ligand–receptor interaction provides the fundamental basis for the hypothetical notion that high binding avidity relates to the strong force of adhesion. Despite its increasing importance in the design of targeted nanoconjugates, an understanding of the physical forces underlying the multivalent interaction remains a subject of urgent investigation. In this study, we designed three vancomycin (Van)-conjugated dendrimers G5­(Van)_<i>n</i> (<i>n</i> = mean valency = 0, 1, 4) for bacterial targeting with generation 5 (G5) poly­(amidoamine) dendrimer as a multivalent scaffold and evaluated both their binding avidity and physical force of adhesion to a bacterial model surface by employing surface plasmon resonance (SPR) spectroscopy and atomic force microscopy. The SPR experiment for these conjugates was performed in a biosensor chip surface immobilized with a bacterial cell-wall peptide Lys-d-Ala-d-Ala. Of these, G5­(Van)₄ bound most tightly with a <i>K</i>_D of 0.34 nM, which represents an increase in avidity by 2 or 3 orders of magnitude relative to a monovalent conjugate G5­(Van)₁ or free vancomycin, respectively. By single-molecule force spectroscopy, we measured the adhesion force between G5­(Van)_<i>n</i> and the same cell-wall peptide immobilized on the surface. The distribution of adhesion forces increased in proportion to vancomycin valency with the mean force of 134 pN at <i>n</i> = 4 greater than 96 pN at <i>n</i> = 1 at a loading rate of 5200 pN/s. In summary, our results are strongly supportive of the positive correlation between the avidity and adhesion force in the multivalent interaction of vancomycin nanoconjugates

Control of an Unusual Photo-Claisen Rearrangement in Coumarin Caged Tamoxifen through an Extended Spacer

The use of coumarin caged molecules has been well documented in numerous photocaging applications including for the spatiotemporal control of Cre-estrogen receptor (Cre-ERT2) recombinase activity. In this article, we report that 4-hydroxytamoxifen (4OHT) caged with coumarin <i>via</i> a conventional ether linkage led to an unexpected photo-Claisen rearrangement which significantly competed with the release of free 4OHT. The basis for this unwanted reaction appears to be related to the coumarin structure and its radical-based mechanism of uncaging, as it did not occur in <i>ortho</i>-nitrobenzyl (ONB) caged 4OHT that was otherwise linked in the same manner. In an effort to perform design optimization, we introduced a self-immolative linker longer than the ether linkage and identified an optimal linker which allowed rapid 4OHT release by both single-photon and two-photon absorption mechanisms. The ability of this construct to actively control Cre-ERT2 mediated gene modifications was investigated in mouse embryonic fibroblasts (MEFs) in which the expression of a green fluorescent protein (GFP) reporter dependent gene recombination was controlled by 4OHT release and measured by confocal fluorescence microscopy and flow cytometry. In summary, we report the implications of this photo-Claisen rearrangement in coumarin caged compounds and demonstrate a rational linker strategy for addressing this unwanted side reaction

Photocontrolled Release of Doxorubicin Conjugated through a Thioacetal Photocage in Folate-Targeted Nanodelivery Systems

Despite their proven ability for precise and targeted release, nanoplatform systems for photocontrolled delivery often face formidable synthetic challenges, in part due to the paucity of advanced linker strategies. Here, we report on a novel linker strategy using a thioacetal <i>ortho</i>-nitrobenzaldehyde (TNB) cage, demonstrating its application for delivery of doxorubicin (Dox) in two nanoscale systems. This photocleavable linker, TNB­(OH), which presents two identical arms, each terminated with a hydroxyl functionality, was prepared in a single step from 6-nitroveratraldehyde. TNB­(OH) was used to cross-link Dox to a folate receptor (FAR)-targeting poly­(amidoamine) dendrimer conjugate G5­(FA)_<i>n</i>=5.4(Dox)_<i>m</i>=5.1, and also used to prepare an upconversion nanocrystal (UCN) conjugate, UCN–PPIX@(Dox)­(G5FA), a larger core/shell nanostructure. In this core/shell nanostructure, the UCN core emits UV and visible light luminescence upon near-infrared (NIR) excitation, allowing for the photocleavage of the TNB linker as well as the photostimulation of protoporphyrin IX (PPIX) coupled as a cytotoxic photosensitizer. Drug-release experiments performed in aqueous solutions with long-wavelength ultraviolet A (UVA) light showed that Dox release occurred rapidly from its TNB linked form or from its dendrimer conjugated form with comparable decay kinetics. Cellular toxicity studies in FAR-overexpressing KB carcinoma cells demonstrated that each nanoconjugate lacked intrinsic cytotoxicity until exposed to UVA or NIR (980 nm) (for the UCN nanoconjugate), which resulted in induction of potent cytotoxicity. In summary, this new TNB strategy offers synthetic convenience in drug conjugation chemistry with the ability for the temporal control of drug activation at the delivery site