We report the synthesis and in vitro evaluation of folate receptor-targeted nanoconjugate that releases its therapeutic payload via a photochemical mechanism.The targeted delivery of therapeutic and imaging agents using nanoconjugates is a burgeoning field. [1][2][3][4] Strategies to develop cancer-cell specific nanoconjugates vary, but all attempts to selectively deliver therapeutics to cells use nanoscale carriers such as dendritic macromolecules, 2 liposomes, 5 polymers, 6 metal nanoparticles 3 or viruses 7 that include targeting and therapeutic agents. The desired result is less side toxicity in normal cells and more effective tumoricidal activity. Nanoconjugates also can be designed such that the therapeutic agents are released, and therefore active, only under particular conditions. The release mechanisms currently being explored are based primarily on reactions catalyzed by endogenous physiological factors such as reduction, 1 low pH, 3 and hydrolytic enzymes. 4 This communication describes a photochemical-based approach to release targeted drugs after delivery. In this scenario, the targeted drug conjugate is first placed on a surface, such as skin, or lung/gastrointestinal tract epithelium. After the exposure, the nanoconjugate drug is specifically taken up by the tumor cells and is washed away from the normal tissue; light is then applied from a laser device attached to an endoscope to specifically target the cancer cells. The strategy presented may be broadly applied to other cell targeting systems, particularly those that require time-and tissue-dependent control of drug activation.Photocaging refers to the temporary inactivation of a biologically active molecule using a protective photocleavable group. Upon UV irradiation of the photocleavable group, the active form of the caged molecule is irreversibly released. 8 Photocaging has been frequently applied in vitro towards the spatiotemporal control of biological processes 9-11 and the light-triggered payload release from nanoscale materials. 12,13 However, it has only been rarely applied in in vivo experiments 14,15 because of the low level tissue penetration and phototoxicity associated with short wavelength UV light.Recent advances in two-photon excitation 14,15 and optical fiber technology, however, have made it possible to cleave photocaged compounds by irradiation in the near-IR (720-800 nm 14 ). Because of this potential for higher level tissue penetration, we have applied the † Electronic supplementary information (ESI) available: Experimental details for synthesis and characterization of 1-9; details for photocleavage experiments of 3 and 7. See DOI: 10.1039/b927215cFax: (734) 615-0621; Tel: (734) 615-0618. NIH Public Access Author ManuscriptChem Commun (Camb). Author manuscript; available in PMC 2010 July 12. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript photocaging approach towards the targeted delivery of doxorubicin, 16 an anticancer drug that inhibits DNA replication through intercalation (Fig. 1).In ...

No abstract

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.

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.

Our group previously developed a multifunctional, targeted cancer therapeutic based on Generation 5 (G5) polyamidoamine (PAMAM) dendrimers. In those studies we conjugated the targeting molecule folic acid (FA) and the chemotherapeutic drug methotrexate (MTX) sequentially. This complex macromolecule was shown to selectively bind and kill KB tumor cells that overexpress folate receptor (FR) in vitro and in vivo. However, the multistep conjugation strategy employed in the synthesis of the molecule resulted in heterogeneous populations having differing numbers and ratios of the functionally antagonistic FA and MTX. This led to inconsistent and sometimes biologically inactive batches of molecules, especially during large-scale synthesis. We here resolved this issue by using a novel triazine scaffold approach that reduces the number of dendrimer conjugation steps required and allows for the synthesis of G5 conjugates with defined ratios of FA and MTX. Although an unoccupied γ-glutamyl carboxylate of FA has been previously suggested to be nonessential for FR binding, the functional requirement of an open α-carboxylate still remains unclear. In an attempt to also address this question, we have synthesized isomeric FA dendrimer conjugates (α-carboxyl or γ-carboxyl linked). Competitive binding studies revealed that both linkages have virtually identical affinity toward FR on KB cells. Our studies show that a novel bifunctional triazine-based conjugate G5-Triazine-γMTX-αFA with identical numbers of FA and MTX binds to FR through a polyvalent interaction and induces cytotoxicity in KB cells through FR-mediated cellular internalization, inducing higher toxicity as compared to conjugates synthesized by the multistep strategy. This work serves as a proof of concept for the development of bifunctional dendrimer conjugates that require a defined ratio of two functional molecules.

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 n = 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.

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.

The present study screened riboflavin mimicking small molecules to determine their binding activity for the riboflavin binding protein. We performed thermodynamic and kinetic binding studies of these molecules using a combination of two analytical approaches; isothermal titration calorimetry and surface plasmon resonance spectroscopy. Screening of a biased set of non-riboflavin based small molecules by microcalorimetry led to the discovery of two known drug molecules, quinacrine and chloroquine, as favorable ligands for the riboflavin receptor with KD value of 264, and 2100 nM, respectively. We further demonstrated that quinacrine is a competitive ligand for the receptor as measured by surface plasmon resonance. Thus this study describes the identification of a novel class of dual acting riboflavin antagonists that target riboflavin receptor for cellular uptake and display multifunctional activities upon cellular entry.