Evaluating Binding Avidities of Populations of Heterogeneous Multivalent Ligand-Functionalized Nanoparticles

Ligand-functionalized, multivalent nanoparticles have been extensively studied as targeted carriers in biomedical applications for drug delivery and imaging. The chemical synthesis method used, however, generates nanoparticles that are heterogeneous with respect to the number of ligands on each nanoparticle. This article examines the role this heterogeneity in ligand number plays in multivalent interactions between nanoparticle ligands and targeted receptors. We designed and synthesized a model heterogeneous multivalent nanoparticle system and developed a unique kinetic analysis to quantify the avidity interactions. This system used mono-dispersed poly(amidoamine) (PAMAM) dendrimers that were then chemically functionalized with ssDNA oligonucleotides as to yield the heterogeneous nanoparticle platform (ligand valencies <i>n</i> = 1.7, 3.1, 6), and employed complementary oligonucleotides as targeted receptors on a surface plasmon resonance (SPR) biosensor to evaluate the multivalent binding of the nanoparticle population. Kinetic analysis of both parallel initial rate and dual-Langmuir analyses of SPR binding curves was performed to assess avidity distributions. We found that batches of multivalent nanoparticles contain both fast- and slow-dissociation subpopulations, which can be characterized as having “weak” and “strong” surface interactions (“binding”), respectively. Furthermore, we found that the proportion of “strong” binders increased as a function of the mean oligonucleotide valence of the nanoparticle population. These analyses allowed an assessment of how avidity distributions are modulated by the number of functionalized ligands and suggested that there are threshold valences that differentiated fast- and slow-dissociation nanoparticles

Ligand Characteristics Important to Avidity Interactions of Multivalent Nanoparticles

Multivalent interactions involve the engagement of multiple ligand–receptor pairs and are important in synthetic biology as design paradigms for targeted nanoparticles (NPs). However, little is known about the specific ligand parameters important to multivalent interactions. We employed a series of oligonucleotides as ligands conjugated to dendrimers as nanoparticles, and used complementary oligonucleotides on a functionalized SPR surface to measure binding. We compared the effect of ligand affinity to ligand number on the avidity characteristics of functionalized NPs. Changing the ligand affinity, either by changing the temperature of the system or by substitution noncomplementary base pairs into the oligonucleotides, had little effect on multivalent interaction; the overall avidity, number of ligands required for avidity per particle, and the number of particles showing avidity did not significantly change. We then made NP conjugates with the same oligonucleotide using an efficient copper-free click chemistry that resulted in essentially all of the NPs in the population exceeding the threshold ligand value. The particles exceeding the threshold ligand number again demonstrated high avidity interactions. This work validates the concept of a threshold ligand valence and suggests that the number of ligands per nanoparticle is the defining factor in achieving high avidity interactions

Multivalent Dendrimer Vectors with DNA Intercalation Motifs for Gene Delivery

Poly­(amido amine) (PAMAM) dendrimers constitute an important class of nonviral, cationic vectors in gene delivery. Here we report on a new concept for dendrimer vector design based on the incorporation of dual binding motifs: DNA intercalation, and receptor recognition for targeted delivery. We prepared a series of dendrimer conjugates derived from a fifth generation (G5) PAMAM dendrimer, each conjugated with multiple folate (FA) or riboflavin (RF) ligands for cell receptor targeting, and with 3,8-diamino-6-phenylphenanthridinium (“DAPP”)-derived ligands for anchoring a DNA payload. Polyplexes of each dendrimer with calf thymus dsDNA were made and characterized by surface plasmon resonance (SPR) spectroscopy, dynamic light scattering (DLS) and zeta potential measurement. These studies provided evidence supporting polyplex formation based on the observation of tight DNA-dendrimer adhesion, and changes in particle size and surface charge upon coincubation. Further SPR studies to investigate the adhesion of the polyplex to a model surface immobilized with folate binding protein (FBP), demonstrated that the DNA payload has only a minimal effect on the receptor binding activity of the polyplex: <i>K</i>_D = 0.22 nM for G5­(FA)­(DAPP) versus 0.98 nM for its polyplex. Finally, we performed in vitro transfection assays to determine the efficiency of conjugate mediated delivery of a luciferase-encoding plasmid into the KB cancer cell line and showed that RF-conjugated dendrimers were 1 to 2 orders of magnitude more effective in enhancing luciferase gene transfection than a plasmid only control. In summary, this study serves as a proof of concept for DNA-ligand intercalation as a motif in the design of multivalent dendrimer vectors for targeted gene delivery

Specific and Cooperative Interactions between Oximes and PAMAM Dendrimers As Demonstrated by ¹H NMR Study

Oximes are important in the treatment of organophosphate (OP) poisoning, but have limited biological half-lives. Complexing these drugs with a macromolecule, such as a dendrimer, could improve their pharmacokinetics. The present study investigates the intermolecular interactions that drive the complexation of oxime-based drug molecules with fifth generation poly­(amidoamine) (PAMAM) dendrimers. We performed steady-state binding studies of two molecules, pralidoxime and obidoxime, employing multiple NMR methods, including 1D titration, ¹H–¹H 2D spectroscopy (COSY, NOESY), and ¹H diffusion-ordered spectroscopy (DOSY). Several important insights were gained in understanding the host–guest interactions occurring between the drug molecules and the polymer. First, the guest molecules bind to the dendrimer macromolecule through a specific interaction rather than through random, hydrophobic encapsulation. Second, this specificity is driven primarily by the electrostatic or H-bond interaction of the oxime at a dendrimer amine site. Also, the average strength for each drug and dendrimer interaction is affected by the surface modification of the polymer. Third, individual binding events between oximes and a dendrimer have a negative cooperative effect on subsequent oxime binding. In summary, this report provides a novel perspective important for designing host systems for drug delivery

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

Force Spectroscopy of Multivalent Binding of Riboflavin-Conjugated Dendrimers to Riboflavin Binding Protein

Putative riboflavin receptors are considered as biomarkers due to their overexpression in breast and prostate cancers. Hence, these receptors can be potentially exploited for use in targeted drug delivery systems where dendrimer nanoparticles with multivalent ligand attachments can lead to greater specificity in cellular interactions. In this study, the single molecule force spectroscopy technique was used to assess the physical strength of multivalent interactions by employing a riboflavin (RF)-conjugated generation 5 PAMAM dendrimer G5­(RF)_<i>n</i> nanoparticle. By varying the average RF ligand valency (<i>n</i> = 0, 3, 5), the rupture force was measured between G5­(RF)_<i>n</i> and the riboflavin binding protein (RFBP). The rupture force increased when the valency of RF increased. We observed at the higher valency (<i>n</i> = 5) three binding events that increased in rupture force with increasing loading rate. Assuming a single energy barrier, the Bell–Evans model was used to determine the kinetic off-rate and barrier width for all binding interactions. The analysis of our results appears to indicate that multivalent interactions are resulting in changes to rupture force and kinetic off-rates

Atomic Force Microscopy Probing of Receptor–Nanoparticle Interactions for Riboflavin Receptor Targeted Gold–Dendrimer Nanocomposites

Riboflavin receptors are overexpressed in malignant cells from certain human breast and prostate cancers, and they constitute a group of potential surface markers important for cancer targeted delivery of therapeutic agents and imaging molecules. Here we report on the fabrication and atomic force microscopy (AFM) characterization of a core–shell nanocomposite consisting of a gold nanoparticle (AuNP) coated with riboflavin receptor-targeting poly­(amido amine) dendrimer. We designed this nanocomposite for potential applications such as a cancer targeted imaging material based on its surface plasmon resonance properties conferred by AuNP. We employed AFM as a technique for probing the binding interaction between the nanocomposite and riboflavin binding protein (RfBP) in solution. AFM enabled precise measurement of the AuNP height distribution before (13.5 nm) and after chemisorption of riboflavin-conjugated dendrimer (AuNP–dendrimer; 20.5 nm). Binding of RfBP to the AuNP–dendrimer caused a height increase to 26.7 nm, which decreased to 22.8 nm when coincubated with riboflavin as a competitive ligand, supporting interaction of AuNP–dendrimer and its target protein. In summary, physical determination of size distribution by AFM imaging can serve as a quantitative approach to monitor and characterize the nanoscale interaction between a dendrimer-covered AuNP and target protein molecules in vitro

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

Biophysical Characterization of a Riboflavin-Conjugated Dendrimer Platform for Targeted Drug Delivery

The present study describes the biophysical characterization of generation-five poly­(amidoamine) (PAMAM) dendrimers conjugated with riboflavin (RF) as a cancer-targeting platform. Two new series of dendrimers were designed, each presenting the riboflavin ligand attached at a different site (isoalloxazine at N-3 and d-ribose at N-10) and at varying ligand valency. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) were used to determine the binding activity for riboflavin binding protein (RfBP) in a cell-free solution. The ITC data shows dendrimer conjugates have <i>K</i>_D values of ≥465 nM on a riboflavin basis, an affinity ∼93-fold lower than that of free riboflavin. The N-3 series showed greater binding affinity in comparison with the N-10 series. Notably, the affinity is inversely correlated with ligand valency. These findings are also corroborated by DSC, where greater protein–conjugate stability is achieved with the N-3 series and at lower ligand valency

Polyvalent Dendrimer-Methotrexate as a Folate Receptor-Targeted Cancer Therapeutic

Our previous studies have demonstrated that a generation 5 dendrimer (G5) conjugated with both folic acid (FA) and methotrexate (MTX) has a higher chemotherapeutic index than MTX alone. Despite this, batch-to-batch inconsistencies in the number of FA and MTX molecules linked to each dendrimer led to conjugate batches with varying biological activity, especially when scaleup synthesis was attempted. Since the MTX is conjugated through an ester linkage, there were concerns that biological inconsistency could also result from serum esterase activity and differential bioavailability of the targeted conjugate. In order to resolve these problems, we undertook a novel approach to synthesize a polyvalent G5–MTX_<i>n</i> conjugate through click chemistry, attaching the MTX to the dendrimer through an esterase-stable amide linkage. Surface plasmon resonance binding studies show that a G5–MTX₁₀ conjugate synthesized in this manner binds to the FA receptor (FR) through polyvalent interaction showing 4300-fold higher affinity than free MTX. The conjugate inhibits dihydrofolate reductase, and induces cytotoxicity in FR-expressing KB cells through FR-specific cellular internalization. Thus, the polyvalent MTX on the dendrimer serves the dual role as a targeting molecule as well as a chemotherapeutic drug. The newly synthesized G5–MTX_<i>n</i> conjugate may serve as a FR-targeted chemotherapeutic with potential for cancer therapy