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.

One of the important criteria for achieving efficient nanoparticle-based targeted drug delivery is that the drug is not prematurely released at off-target sites. Here we report the preclinical evaluation of a serum-stable dendrimer-based drug conjugate capable of actively targeting into prostate cancer (PC) cells, delivered through the prostate-specific membrane antigen (PSMA). Multiple molecules of PSMA-binding small molecule glutamate urea (GLA; targeting agent) and the drug methotrexate (MTX) were conjugated to generation 5 PAMAM dendrimer (G5) through Cu-free "click" chemistry. The GLA was conjugated through a stable amide bond, and the MTX was conjugated either through ester (Es)- or amide (Am)-coupling, to generate G5-GLA(m)-(Es)MTX(n) and G5-GLA(m)-(Am)MTX(n), respectively. In serum-containing medium, free MTX was slowly released from "G5-GLA(m)-(Es)MTX(n)", with ~8% MTX released from the dendrimer in 72 h, whereas the MTX on G5-GLA(m)-(Am)MTX(n) was completely stable. The G5-GLA(m)-(Am)MTX(n) bound and internalized into PSMA-expressing LNCaP cells, but not into PSMA-negative PC3 cells. The conjugate-inhibited recombinant dihydrofolate reductase and induced potent cytotoxicity in the LNCaP cells, but not in the PC3 cells. Similar to the action of free GLA, stable amide-linked dendrimer-GLA was capable of inhibiting the enzyme N-acetylated α-linked acidic dipeptidase (NAALADase) activity of PSMA. The G5-GLA(m)-MTX(n) may serve as a serum-stable nanoparticle conjugate to specifically and effectively target and treat PSMA-overexpressing prostate tumors.

Design of cancer-targeting nanotherapeutics relies on a pair of two functionally orthogonal molecules, one serving as a cancer cell-specific targeting ligand, and the other as a therapeutic cytotoxic agent. The present study investigates the validity of an alternative simplified strategy where a dual-acting molecule which bears both targeting and cytotoxic activity is conjugated to the nanoparticle as cancer-targeting nanotherapeutics. Herein we demonstrate that methotrexate is applicable for this dual-acting strategy due to its reasonable affinity to folic acid receptor (FAR) as a tumor biomarker, and cytotoxic inhibitory activity of cytosolic dihydrofolate reductase. This article describes design of new methotrexate-conjugated poly(amidoamine) (PAMAM) dendrimers, each carrying multiple copies of methotrexate attached through a stable amide linker. We evaluated their dual biological activities by performing surface plasmon resonance spectroscopy, a cell-free enzyme assay and cell-based experiments in FAR-overexpressing cells. This study identifies the combination of an optimal linker framework and multivalency as the two key design elements that contribute to achieving potent dual activity.

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A novel Schiff base obtained from curcumin and glycine was prepared and it was reacted with Co, Ni, Cu and Zn metals in order to form the stable metal complexes and characterized by elemental analysis, magnetic, molar conductance, IR, UV-Vis.,1H NMR and PXRD. The data shows that the complexes have the structure [M(II)-(cur-gly)H2O] system Electronic and magnetic data suggest a tetrahedral geometry for Co, Ni and Zn except Cu complex has a square planar geometry. The antimicrobial activity of cur-gly and its metal chelates were confirmed against the bacterial species as E. coli, P. aeruginosa, Enterococcus, B. cereus and S. aureus species. Antifungal activity was screened against C. albicans, C. parapsilosis and A. flavus. Metal chelates indicate excellent antimicrobial activity than their parent cur-gly and DNA photo cleavage activity shows that metal chelate effectively cleave the pUC 18 DNA.