Thommey P. Thomas

Nanoparticle Targeting of Anticancer Drug Improves Therapeutic Response in Animal Model of Human Epithelial Cancer

Prior studies suggested that nanoparticle drug delivery might improve the therapeutic response to anticancer drugs and allow the simultaneous monitoring of drug uptake by tumors. We employed modified PAMAM dendritic polymers <5 nm in diameter as carriers. Acetylated dendrimers were conjugated to folic acid as a targeting agent and then coupled to either methotrexate or tritium and either fluorescein or 6-carboxytetramethylrhodamine. These conjugates were injected i.v. into immunodeficient mice bearing human KB tumors that overexpress the folic acid receptor. In contrast to nontargeted polymer, folate-conjugated nanoparticles concentrated in the tumor and liver tissue over 4 days after administration. The tumor tissue localization of the folate-targeted polymer could be attenuated by prior i.v. injection of free folic acid. Confocal microscopy confirmed the internalization of the drug conjugates into the tumor cells. Targeting methotrexate increased its antitumor activity and markedly decreased its toxicity, allowing therapeutic responses not possible with a free drug.

DESIGN AND FUNCTION OF A DENDRIMER-BASED THERAPEUTIC NANODEVICE TARGETED TO TUMOR CELLS THROUGH THE FOLATE RECEPTOR

AbstractPurpose. We sought to develop nanoscale drug delivery materials that would allow targeted intracellular delivery while having an imaging capability for tracking uptake of the material. A complex nanodevice was designed and synthesized that targets tumor cells through the folate receptor. Methods. The device is based on an ethylenediamine core polyamidoamine dendrimer of generation 5. Folic acid, fluorescein, and methotrexate were covalently attached to the surface to provide targeting, imaging, and intracellular drug delivery capabilities. Molecular modeling determined the optimal dendrimer surface modification for the function of the device and suggested a surface modification that improved targeting. Results. Three nanodevices were synthesized. Experimental targeting data in KB cells confirmed the modeling predictions of specific and highly selective binding. Targeted delivery improved the cytotoxic response of the cells to methotrexate 100-fold over free drug. Conclusions. These results demonstrate the ability to design and produce polymer-based nanodevices for the intracellular targeting of drugs, imaging agents, and other materials.

The linked roles of nitric oxide, aldose reductase and, (Na+,K+)-ATPase in the slowing of nerve conduction in the streptozotocin diabetic rat.

Metabolic and vascular factors have been invoked in the pathogenesis of diabetic neuropathy but their interrelationships are poorly understood. Both aldose reductase inhibitors and vasodilators improve nerve conduction velocity, blood flow, and (Na+,K+)-ATPase activity in the streptozotocin diabetic rat, implying a metabolic-vascular interaction. NADPH is an obligate cofactor for both aldose reductase and nitric oxide synthase such that activation of aldose reductase by hyperglycemia could limit nitric oxide synthesis by cofactor competition, producing vasoconstriction, ischemia, and slowing of nerve conduction. In accordance with this construct, N-nitro-L-arginine methyl ester, a competitive inhibitor of nitric oxide synthase reversed the increased nerve conduction velocity afforded by aldose reductase inhibitor treatment in the acutely diabetic rat without affecting the attendant correction of nerve sorbitol and myo-inositol. With prolonged administration, N-nitro-L-arginine methyl ester fully reproduced the nerve conduction slowing and (Na+,K+)-ATPase impairment characteristic of diabetes. Thus the aldose reductase-inhibitor-sensitive component of conduction slowing and the reduced (Na+,K+)-ATPase activity in the diabetic rat may reflect in part impaired nitric oxide activity, thus comprising a dual metabolic-ischemic pathogenesis.

Synthesis, characterization, and intracellular uptake of carboxyl-terminated poly(amidoamine) dendrimer-stabilized iron oxide nanoparticles

We report the synthesis and characterization of a group of carboxyl-functionalized poly(amidoamine) (PAMAM) dendrimers of generation 3 (G3) that were used for the stabilization of superparamagnetic iron oxide (Fe(3)O(4)) nanoparticles (NPs). Folic acid (FA) molecules were conjugated onto the dendrimer surfaces in an attempt to achieve specific targeted imaging of tumor cells that overexpress FA receptors using dendrimer-stabilized Fe(3)O(4) NPs. Fe(3)O(4) NPs were synthesized using controlled co-precipitation of Fe(ii) and Fe(iii) ions and the formed dendrimer-stabilized Fe(3)O(4) NPs were characterized using transmission electron microscopy (TEM) and polyacrylamide gel electrophoresis (PAGE). The intracellular uptake of dendrimer-stabilized Fe(3)O(4) NPs was tested in vitro using KB cells (a human epithelial carcinoma cell line) that overexpress FA receptors. It appears that carboxyl-terminated PAMAM dendrimer-stabilized Fe(3)O(4) NPs can be uptaken by KB cells regardless of the repelling force between the negatively charged cells and the negatively charged particles. In the presence of a large amount of carboxyl terminal groups on the dendrimer surface, the receptor-mediated endocytosis of Fe(3)O(4) NPs stabilized by FA-modified dendrimers was not facilitated. It implies that the surface charge of dendrimer-stabilized magnetic iron oxide NPs in biological medium is an important factor influencing their biological performance.

Synthesis, characterization, and in vitro testing of superparamagnetic iron oxide nanoparticles targeted using folic Acid-conjugated dendrimers.

Organic-coated superparamagnetic iron oxide nanoparticles (OC-SPIONs) were synthesized and characterized by transmission electron microscopy and X-ray photoelectron spectroscopy. OC-SPIONs were transferred from organic media into water using poly(amidoamine) dendrimers modified with 6-TAMRA fluorescent dye and folic acid molecules. The saturation magnetization of the resulting dendrimer-coated SPIONs (DC-SPIONs) was determined, using a superconducting quantum interference device, to be 60 emu/g Fe versus 90 emu/g Fe for bulk magnetite. Selective targeting of the DC-SPIONs to KB cancer cells in vitro was demonstrated and quantified using two distinct and complementary imaging modalities: UV-visible and X-ray fluorescence; confocal microscopy confirmed internalization. The results were consistent between the uptake distribution quantified by flow cytometry using 6-TAMRA UV-visible fluorescence intensity and the cellular iron content determined using X-ray fluorescence microscopy.

Folate-targeted nanoparticles show efficacy in the treatment of inflammatory arthritis

OBJECTIVE To investigate the uptake of a poly(amidoamine) dendrimer (generation 5 [G5]) nanoparticle covalently conjugated to polyvalent folic acid (FA) as the targeting ligand into macrophages, and to investigate the activity of an FA- and methotrexate (MTX)-conjugated dendrimer (G5-FA-MTX) as a therapeutic for the inflammatory disease of arthritis. METHODS In vitro studies were performed in macrophage cell lines and in isolated mouse macrophages to check the cellular uptake of fluorescence-tagged G5-FA nanoparticles, using flow cytometry and confocal microscopy. In vivo studies were conducted in a rat model of collagen-induced arthritis to evaluate the therapeutic potential of G5-FA-MTX. RESULTS Folate-targeted dendrimer bound and internalized in a receptor-specific manner into both folate receptor β-expressing macrophage cell lines and primary mouse macrophages. The conjugate G5-FA-MTX acted as a potent antiinflammatory agent and reduced arthritis-induced parameters of inflammation such as ankle swelling, paw volume, cartilage damage, bone resorption, and body weight decrease. CONCLUSION The use of folate-targeted nanoparticles to specifically target MTX into macrophages may provide an effective clinical approach for antiinflammatory therapy in rheumatoid arthritis.

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.

Enhanced two-photon biosensing with double-clad photonic crystal fibers

A double-clad photonic crystal fiber was used to improve detection efficiency over a standard single-mode fiber in a two-photon fluorescence detection scheme in which the dye was excited and the fluorescence was detected back through the same fiber.

Tumor angiogenic vasculature targeting with PAMAM dendrimer–RGD conjugates

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.

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.