The cellular uptake and cytotoxicity of an engineered multifunctional dendritic nanodevice containing folic acid (FA) as the targeting molecule, methotrexate (MTX) as the chemotherapeutic drug, and fluorescein (FI) as the detecting agent were studied in vitro. FI and FA were conjugated to the generation 5 poly(amidoamine) (G5) dendrimer carrier through a thiourea and amide linkage and MTX was conjugated through an ester linkage to the carrier to generate the trifunctional dendritic device, G5-FI-FA-MTX. This trifunctional dendrimer-drug conjugate bound to FA receptor-expressing KB cells in a dose-dependent and saturable manner. Confocal microscopic analysis demonstrated cellular internalization of the conjugate. G5-FI-FA-MTX induced a time- and dose-dependent inhibition of cell growth in KB cells. The targeted dendrimer conjugates G5-FI-FA-MTX and G5-FA-MTX inhibited cell growth in KB cells, whereas the nontargeted G5-MTX failed to induce growth inhibition. These studies show the potential of G5-FI-FA-MTX or G5-FA-MTX for targeting and growth suppression of tumor cells that overexpress FA-receptors.

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 ...

PAMAM dendrimer-RGD-4C peptide conjugate was synthesized and in vitro targeting efficacy to integrin receptor expressing cells was studied by flow cytometry and confocal microscopy.

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–MTXn 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–MTXn conjugate may serve as a FR-targeted chemotherapeutic with potential for cancer therapy.

We sought to produce dendrimers conjugated to different biofunctional moieties (fluorescein [FITC] and folic acid [FA]), and then link them together using complementary DNA oligonucleotides to produce clustered molecules that target cancer cells that overexpress the high-affinity folate receptor. Amine-terminated, generation 5 polyamidoamine (G5 PAMAM) dendrimers are first partially acetylated and then conjugated with FITC or FA, followed by the covalent attachment of complementary, 5'-phosphate-modified 34-base-long oligonucleotides. Hybridization of these oligonucleotide conjugates led to the self-assembly of the FITC- and FA-conjugated dendrimers. In vitro studies of the DNA-linked dendrimer clusters indicated specific binding to KB cells expressing the folate receptor. Confocal microscopy also showed the internalization of the dendrimer cluster. These results demonstrate the ability to design and produce supramolecular arrays of dendrimers using oligonucleotide bridges. This will also allow for further development of DNA-linked dendrimer clusters as imaging agents and therapeutics.