Seok Ki Choi

Light-controlled release of caged doxorubicin from folate receptor-targeting PAMAM dendrimer nanoconjugate

We report the synthesis and in vitro evaluation of folate receptor-targeted nanoconjugate that releases its therapeutic payload via a photochemical mechanism.

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(n) 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-MTX(10) 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(n) conjugate may serve as a FR-targeted chemotherapeutic with potential for cancer therapy.

Dendrimer-Based Multivalent Vancomycin Nanoplatform for Targeting the Drug-Resistant Bacterial Surface

Vancomycin represents the preferred ligand for bacteria-targeting nanosystems. However, it is inefficient for emerging vancomycin-resistant species because of its poor affinity to the reprogrammed cell wall structure. This study demonstrates the use of a multivalent strategy as an effective way for overcoming such an affinity limitation in bacteria targeting. We designed a series of fifth generation (G5) poly(amidoamine) (PAMAM) dendrimers tethered with vancomycin at the C-terminus at different valencies. We performed surface plasmon resonance (SPR) studies to determine their binding avidity to two cell wall models, each made with either a vancomycin-susceptible (D)-Ala-(D)-Ala or vancomycin-resistant (D)-Ala-(D)-Lac cell wall precursor. These conjugates showed remarkable enhancement in avidity in the cell wall models tested, including the vancomycin-resistant model, which had an increase in avidity of four to five orders of magnitude greater than free vancomycin. The tight adsorption of the conjugate to the model surface corresponded with its ability to bind vancomycin-susceptible Staphylococcus aureus bacterial cells in vitro as imaged by confocal fluorescent microscopy. This vancomycin platform was then used to fabricate the surface of iron oxide nanoparticles by coating them with the dendrimer conjugates, and the resulting dendrimer-covered magnetic nanoparticles were demonstrated to rapidly sequester bacterial cells. In summary, this article investigates the biophysical basis of the tight, multivalent association of dendrimer-based vancomycin conjugates to the bacterial cell wall, and proposes a potential new use of this nanoplatform in targeting Gram-positive bacteria.

Dendrimer-based multivalent methotrexates as dual acting nanoconjugates for cancer cell targeting

Cancer-targeting drug delivery can be based on the rational design of a therapeutic platform. This approach is typically achieved by the functionalization of a nanoparticle with two distinct types of molecules, a targeting ligand specific for a cancer cell, and a cytotoxic molecule to kill the cell. The present study aims to evaluate the validity of an alternative simplified approach in the design of cancer-targeting nanotherapeutics: conjugating a single type of molecule with dual activities to nanoparticles, instead of coupling a pair of orthogonal molecules. Herein we investigate whether this strategy can be validated by its application to methotrexate, a dual-acting small molecule that shows cytotoxicity because of its potent inhibitory activity against dihydrofolate reductase and that binds folic acid receptor, a tumor biomarker frequently upregulated on the cancer cell surface. This article describes a series of dendrimer conjugates derived from a generation 5 polyamidoamine (G5 PAMAM) presenting a multivalent array of methotrexate and also demonstrates their dual biological activities by surface plasmon resonance spectroscopy, a cell-free enzyme assay, and cell-based experiments with KB cancer cells.

Design of riboflavin-presenting PAMAM dendrimers as a new nanoplatform for cancer-targeted delivery.

This communication describes the synthesis and in vitro biological evaluation of novel generation 5 PAMAM dendrimers conjugated with riboflavin as a targeting ligand. Cell-based experiments demonstrated that a dendrimer conjugated with riboflavin is able to undergo cellular binding and uptake in KB cells, and when the dendrimer is also conjugated with methotrexate, the riboflavin dendrimer conjugate can potently inhibit cell growth.

A photochemical approach for controlled drug release in targeted drug delivery

Photochemistry provides a unique mechanism that enables the active control of drug release in cancer-targeting drug delivery. This study investigates the light-mediated release of methotrexate, an anticancer drug, using a photocleavable linker strategy based on o-nitrobenzyl protection. We evaluated two types of the o-nitrobenzyl-linked methotrexate for the drug release study and further extended the study to a fifth-generation poly(amidoamine) dendrimer carrier covalently conjugated with methotrexate via the o-nitrobenzyl linker. We performed the drug release studies by using a combination of three standard analytical methods that include UV/vis spectrometry, (1)H NMR spectroscopy, and anal. HPLC. This article reports that methotrexate is released by the photochemical mechanism in an actively controlled manner. The rate of the drug release varies in response to multiple control parameters, including linker design, light wavelength, exposure time, and the pH of the medium where the drug release occurs.

Avidity Modulation of Folate-Targeted Multivalent Dendrimers for Evaluating Biophysical Models of Cancer Targeting Nanoparticles

We investigated two types of generation 5 polyamidoamine (PAMAM) dendrimers, each conjugated stochastically with a mean number of 5 or 10 methotrexate (MTX) ligands per dendrimer (G5-MTX5, G5-MTX10), for their binding to surface-immobilized folate binding protein (FBP) as a function of receptor density. The binding study was performed under flow by surface plasmon resonance spectroscopy. Two multivalent models were examined to simulate binding of the dendrimer to the receptor surface, showing that at relatively high receptor density, both dendrimer conjugates exhibit high avidity. However, upon reducing the receptor density by a factor of 3 and 13 relative to the high density level, the avidity of the lower-valent G5-MTX5 decreases by up to several orders of magnitude (KD = nM to μM), whereas the avidity of G5-MTX10 remains largely unaffected regardless of the density variation. Notably, on the 13-fold reduced FBP surface, G5-MTX5 displays binding kinetics similar to that of monovalent methotrexate, which is patently different from the still tight binding of the higher-valent G5-MTX10. Thus, the binding analysis demonstrates that avidity displayed by multivalent MTX conjugates varies in response to the receptor density and can be modulated for achieving tighter, more specific binding to the higher receptor density by modulation of ligand valency. We believe this study provides experimental evidence supportive of the mechanistic hypothesis of multivalent NP uptake to a cancer cell over a healthy cell where the diseased cell expresses the folate receptor at higher density.

Avidity Mechanism of Dendrimer–Folic Acid Conjugates

Multivalent conjugation of folic acid has been employed to target cells overexpressing folate receptors. Such polymer conjugates have been previously demonstrated to have high avidity to folate binding protein. However, the lack of a monovalent folic acid–polymer material has prevented a full binding analysis of these conjugates, as multivalent binding mechanisms and polymer-mass mechanisms are convoluted in samples with broad distributions of folic acid-to-dendrimer ratios. In this work, the synthesis of a monovalent folic acid–dendrimer conjugate allowed the elucidation of the mechanism for increased binding between the folic acid–polymer conjugate and a folate binding protein surface. The increased avidity is due to a folate-keyed interaction between the dendrimer and protein surfaces that fits into the general framework of slow-onset, tight-binding mechanisms of ligand/protein interactions.

Photochemical release of methotrexate from folate receptor-targeting PAMAM dendrimer nanoconjugate

Nanoparticle (NP)-based targeted drug delivery involves cell-specific targeting followed by a subsequent therapeutic action from the therapeutic carried by the NP system. NPs conjugated with methotrexate (MTX), a potent inhibitor of dihydrofolate reductase (DHFR) localized in cytosol, have been under investigation as a delivery system to target cancer cells to enhance the therapeutic index of methotrexate, which is otherwise non-selectively cytotoxic. Despite improved therapeutic activity from MTX-conjugated NPs in vitro and in vivo, the therapeutic action of these conjugates following cellular entry is poorly understood; in particular it is unclear whether the therapeutic activity requires release of the MTX. This study investigates whether MTX must be released from a nanoparticle in order to achieve the therapeutic activity. We report herein light-controlled release of methotrexate from a dendrimer-based conjugate and provide evidence suggesting that MTX still attached to the nanoconjugate system is fully able to inhibit the activity of its enzyme target and the growth of cancer cells.

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 K(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.