Hopping and crawling DNA-coated colloids.

Understanding the motion of particles with multivalent ligand-receptors is important for biomedical applications and material design. Yet, even among a single design, the prototypical DNA-coated colloids, seemingly similar micrometric particles hop or roll, depending on the study. We shed light on this problem by observing DNA-coated colloids diffusing near surfaces coated with complementary strands for a wide array of coating designs. We find colloids rapidly switch between 2 modes: They hop-with long and fast steps-and crawl-with short and slow steps. Both modes occur at all temperatures around the melting point and over various designs. The particles become increasingly subdiffusive as temperature decreases, in line with subsequent velocity steps becoming increasingly anticorrelated, corresponding to switchbacks in the trajectories. Overall, crawling (or hopping) phases are more predominant at low (or high) temperatures; crawling is also more efficient at low temperatures than hopping to cover large distances. We rationalize this behavior within a simple model: At lower temperatures, the number of bound strands increases, and detachment of all bonds is unlikely, hence, hopping is prevented and crawling favored. We thus reveal the mechanism behind a common design rule relying on increased strand density for long-range self-assembly: Dense strands on surfaces are required to enable crawling, possibly facilitating particle rearrangements.

Design and synthesis of multivalent drug delivery system with CA IX inhibitors as ligands.

A multivalent ligand delivery system holds tremendous potential in the field of tumor-targeted drug delivery. It addresses the challenges posed by the low affinity between small molecule ligand receptors and the rapid metabolism of small molecule drug conjugates (SMDCs) in vivo. Notably, existing multivalent ligand systems have demonstrated significant anti-tumor activity in various tumor models. In this study, we have developed a novel multivalent ligand delivery system for SN38, utilizing acetazolamide, a carbonic anhydrase IX (CA IX) inhibitor, as the target ligand. Our multivalent ligand delivery systems exhibited superior metabolic stability and enhanced targeting specificity compared to SMDC molecules. Furthermore, they demonstrated improved anti-proliferation activity, addressing the existing challenges associated with the low receptor affinity and rapid metabolism of SMDCs.

Synthesis, binding affinity, and inhibitory capacity of cyclodextrin-based multivalent glycan ligands for human galectin-3.

Human galectin 3 (Gal-3) has been implicated to play important roles in different biological recognition processes such as tumor growth and cancer metastasis. High-affinity Gal-3 ligands are desirable for functional studies and as inhibitors for potential therapeutic development. We report here a facile synthesis of ß-cyclodextrin (CD)-based Tn and TF antigen-containing multivalent ligands via a click reaction. Binding studies indicated that the synthetic multivalent glycan ligands demonstrated a clear clustering effect in binding to human Gal-3, with up to 153-fold enhanced relative affinity in comparison with the monomeric glycan ligand. The GalNAc (Tn antigen) containing heptavalent ligand showed the highest affinity for human Gal-3 among the synthetic ligands tested, with an EC50 of 1.4 µM in binding to human Gal-3. A cell-based assay revealed that the synthetic CD-based multivalent ligands could efficiently inhibit Gal-3 binding to human airway epithelial cells, with an inhibitory capacity consistent with their binding affinity measured by SPR. The synthetic cyclodextrin-based ligands described in this study should be valuable for functional studies of human Gal-3 and potentially for therapeutic applications.

Dendritic maleimide-thiol adducts carrying pendant glycosides as high-affinity ligands.

We synthesized N-acetylglucosamine-terminated hexavalent carbosilane dendrimers and investigated their binding to wheat germ agglutinin (WGA). The glycodendrimers were prepared by the conjugation of 3-mercaptopropyl, 4-mercaptobutyl, or 5­mercaptopentyl glycosides to maleimide-terminated hexavalent carbosilane dendrimers. Titration of WGA with the glycodendrimers yielded quenching of tryptophan fluorescence. All of the glycodendrimers exhibited high affinity with nanomolar dissociation constants (KD values). The best dendrimers were 1a and 1b with KD values of 6.5 ± 1.7 and 5.3 ± 1.7 nM, respectively. The magnitude of fluorescence quenching increased with decrease in the length of the thioalkyl spacer. Maleimide-pendant carbosilane dendrimers provide ready access to multivalent ligands with high-affinity potential.

Intrinsically cell-penetrating multivalent and multitargeting ligands for myotonic dystrophy type 1.

Developing highly active, multivalent ligands as therapeutic agents is challenging because of delivery issues, limited cell permeability, and toxicity. Here, we report intrinsically cell-penetrating multivalent ligands that target the trinucleotide repeat DNA and RNA in myotonic dystrophy type 1 (DM1), interrupting the disease progression in two ways. The oligomeric ligands are designed based on the repetitive structure of the target with recognition moieties alternating with bisamidinium groove binders to provide an amphiphilic and polycationic structure, mimicking cell-penetrating peptides. Multiple biological studies suggested the success of our multivalency strategy. The designed oligomers maintained cell permeability and exhibited no apparent toxicity both in cells and in mice at working concentrations. Furthermore, the oligomers showed important activities in DM1 cells and in a DM1 liver mouse model, reducing or eliminating prominent DM1 features. Phenotypic recovery of the climbing defect in adult DM1 Drosophila was also observed. This design strategy should be applicable to other repeat expansion diseases and more generally to DNA/RNA-targeted therapeutics.

Combined Tumor Environment Triggered Self-Assembling Peptide Nanofibers and Inducible Multivalent Ligand Display for Cancer Cell Targeting with Enhanced Sensitivity and Specificity.

Many new technologies, such as cancer microenvironment-induced nanoparticle targeting and multivalent ligand approach for cell surface receptors, are developed for active targeting in cancer therapy. While the principle of each technology is well illustrated, most systems suffer from low targeting specificity and sensitivity. To fill the gap, this work demonstrates a successful attempt to combine both technologies to simultaneously improve cancer cell targeting sensitivity and specificity. Specifically, the main component is a targeting ligand conjugated self-assembling monomer precursor (SAM-P), which, at the tumor site, undergoes tumor-triggered cleavage to release the active form of self-assembling monomer capable of forming supramolecular nanostructures. Biophysical characterization confirms the chemical and physical transformation of SAM-P from unimers or oligomers with low ligand valency to supramolecular assemblies with high ligand valency under a tumor-mimicking reductive microenvironment. The in vitro fluorescence assay shows the importance of supramolecular morphology in mediating ligand-receptor interactions and targeting sensitivity. Enhanced targeting specificity and sensitivity can be achieved via tumor-triggered supramolecular assembly and induces multivalent ligand presentation toward cell surface receptors, respectively. The results support this combined tumor microenvironment-induced cell targeting and multivalent ligand display approach, and have great potential for use as cell-specific molecular imaging and therapeutic agents with high sensitivity and specificity.

Ligand-receptor-mediated attachment of lipid vesicles to a supported lipid bilayer.

The interaction of exosomes (cell-secreted [Formula: see text]100 nm-sized extracellular vesicles) or membrane-enveloped virions with cellular lipid membranes is often mediated by relatively weak ligand-receptor bonds. Interactions of this type can be studied using vesicles and observing their attachment to receptors located in a lipid bilayer formed at a solid surface. The contact region between a vesicle and the supported lipid bilayer and accordingly the number of ligand-receptor pairs there can be increased by deforming a vesicle. Herein, I (i) estimate theoretically the corresponding deformation energy assuming a disk-like or elongated shape of vesicles, (ii) present the equations allowing one to track such deformations by employing total internal reflection fluorescence microscopy and surface plasmon resonance, and (iii) briefly discuss some related experimental studies.

Synthesis and bioactivity of MSH4 oligomers prepared by an A2 + B2 strategy.

Oligomers incorporating the tetrapeptide MSH4, the minimum active sequence of melanocyte stimulating hormone, were synthesized by an A2 + B2 strategy involving microwave-assisted copper-catalyzed azide-alkyne cycloaddition. A2 contained an MSH4 core while B2 contained a (Pro-Gly)3 spacer. Soluble mixtures containing compounds with up to eight MSH4 units were obtained from oligomerizations at high monomer concentrations. The avidities of several oligomeric mixtures were evaluated by means of a competitive binding assay using HEK293 cells engineered to overexpress the melanocortin 4 receptor. When based on total MSH4 concentrations, avidities were only minimally enhanced compared with a monovalent control. The lack of variation in the effect of ligands on probe binding is consistent with high off rates for MSH4 in both monovalent and oligomeric constructs relative to that of the competing probe.

Synthesis of a cluster-forming sialylthio-D-galactose fullerene conjugate and evaluation of its interaction with influenza virus hemagglutinin and neuraminidase.

In order to obtain self assembling, multivalent ligand for influenza virus hemagglutinin α-N-acetylneuraminyl-(2-6)-D-galactopyranose has been synthesized and bonded to a water soluble fullerene derivative using 1,3-dipolar cycloaddition click reaction. The aggregating amphiphilic compound did not inhibit the influenza virus hemagglutinin, but it proved to be an inhibitor of its neuraminidase with a 50% inhibitory concentration of 81 µM.

Liver-targeting of primaquine-(poly-γ-glutamic acid) and its degradation in rat hepatocytes.

We have synthesized poly-γ-glutamic acid (PGA) modified with a synthetic trivalent glyco-ligand (TriGalNAc) for the hepatocyte asialoglycoprotein receptor (ASGP-R). We investigated in vivo distribution of unmodified PGA and TriGalNAc-modified PGA (TriGalNAc-PGA) in mice after intravenous injection. Most of unmodified PGA administered was transported to the bladder over 20-80min, suggesting a rapid excretion of unmodified PGA into urine. In contrast, TriGalNAc-PGA was found exclusively in the liver over the same period of time. We further synthesized TriGalNAc-PGA-primaquine conjugate (TriGalNAc-PGA-PQ), and investigated binding, uptake, and catabolism of the conjugate by rat hepatocytes. Our studies indicated that approximately 250ng per million cells of the conjugate bound to one million rat hepatocytes at 0°C, and approximately 2µg per million cells of the conjugate was taken up over 7h incubation at 37°C. Furthermore, our results suggested that TriGalNAc-PGA-PQ was almost completely degraded over 24h, and small degradation products were secreted into cell culture medium. The results described in this report suggest that the TriGalNAc ligand can serve as an excellent targeting device for delivery of PGA-conjugates to the liver hepatocytes, and rat hepatocytes possess sufficient capacity to digest PGA even modified with other substituents.

Construction of a Linear Cell Cross-Linker with Multivalent Glycyrrhetinic Acid Ligands for Rapid Formation of Hepatocyte Spheroids.

We report herein the development of a novel cellular cross-linker further employed in the construction of hepatocellular spheroids based on multivalent strategy. The linker structure consists of a glycyrrhetinic acid derivative (GA-N(CH3)2) modified alginate (ALG). GA-N(CH3)2 is selected as the recognition ligand due to its high affinity toward hepatocyte, whereas ALG represents the linear backbone accounting for its good biocompatibility and flexible structure. These features endow the cellular cross-linker with the ability to flexibly interact with the cell through multiple binding sites, providing superior binding force between the cell and cross-linker, and subsequently rapidly combining multiple cells into cellular spheroids.

The study of angiogenesis stimulated by multivalent peptide ligand-modified alginate.

Enhancing the affinity of scaffolds for endothelial cell (EC) is a crucial procedure for promoting angiogenesis in tissue engineering. In this work, to achieve stronger binding affinity with ECs, the peptide sequence REDV was conjugated onto gold nanoparticles (AuNPs) to construct a multivalent ligand (cREDV). Then, the EC adhesion and proliferation were studied to determine ligand affinity for ECs in vitro. The results show that the cREDV-modified alginate (cREDV-ALG) surface exhibited a selective adhesion to human umbilical vein endothelial cells (HUVECs) compared with NIH 3T3 cells. The average area of individual HUVEC that adhered to cREDV-ALG was approximately 2.27-fold higher than that adhered to the monovalent REDV-modified alginate (REDV-ALG) surface. Additionally, a superior ability to promote the proliferation of HUVECs compared to the REDV-ALG surface was demonstrated. In vivo angiogenic assays were also carried out to assess the effect of multivalent strategy on the vascularization of scaffolds. The results illustrated that cREDV-ALG could stimulate new vessel formation in the scaffold, and the blood vessel density was at least 20% higher than that observed with monovalent REDV-ALG with a similar degree of ligand substitution. These results demonstrated that a multivalent ligand strategy was beneficial for the vascularization of engineered tissues.

Chemically activatable viral capsid functionalized for cancer targeting.

AIM: To design a theranostic capsule using the virus-like nanoparticle of the hepatitis E virus modified to display breast cancer cell targeting functional group (LXY30). METHODS: Five surface-exposed residues were mutated to cysteine to allow conjugation to maleimide-linked chemical groups via thiol-selective linkages. Engineered virus-like nanoparticles were then covalently conjugated to a breast cancer recognized ligand, LXY30 and an amine-coupled near-infrared fluorescence dye. RESULTS: LXY30-HEV VLP was checked for its binding and entry to a breast cancer cell line and for tumor targeting in vivo to breast cancer tissue in mice. The engineered virus-like nanoparticle not only targeted cancer cells, but also appeared immune silent to native hepatitis E virus antibodies due to epitope disruption at the antibody-binding site. CONCLUSION: These results demonstrate the production of a theranostic capsule suitable for cancer diagnostics and therapeutics based on surface modification of a highly stable virus-like nanoparticle.