Istvan J. Majoros

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.

Dendritic polymer macromolecular carriers for drug delivery

Dendrimers are synthetic, highly branched, mono-disperse macromolecules of nanometer dimensions. Started in the mid-1980s, the research investigations into the synthetic methodology, physical and chemical properties of these macromolecules are increasing exponentially with growing interest in this field. Potential applications for dendrimers are now forthcoming. Properties associated with these dendrimers such as uniform size, water solubility, modifiable surface functionality and available internal cavities make them attractive for biological and drug-delivery applications.

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.

Current Dendrimer Applications in Cancer Diagnosis and Therapy

In recent years, medicinal chemists have begun to realize that dendrimers may be a keystone in the future of medicine. The field of oncology will soon be revolutionized by novel strategies for diagnosis and therapy employing dendrimer-based nanodevices. In the near future, cancer diagnosis via MRI will be improved by the incorporation of dendrimers as advanced contrast agents. Novel dendrimer-based contrast agents can not only be targeted specifically to cancer cells but may also incorporate a cytotoxic function to induce apoptosis on said cells as well. Dendrimers are being applied to a variety of cancer therapies to improve the safety and effectiveness of many common therapeutics. Investigations into the applicability of dendrimers in photodynamic therapy, boron neutron capture therapy, and gene transfection are also being undertaken. This review will cover the fundamentals of cutting-edge research utilizing dendrimers for cancer diagnosis and therapy. An objective review of these new technologies will detail how dendrimer-based nanodevices are advantageous over conventional medicine.

Cationic Poly(amidoamine) Dendrimer Induces Lysosomal Apoptotic Pathway at Therapeutically Relevant Concentrations

Poly(amidoamine) (PAMAM) dendrimers carrying different amounts of surface amino groups were synthesized and tested for their effects on cellular cytotoxicity, lysosomal pH, and mitochondria-dependent apoptosis. In KB cells, the PAMAM dendrimers were taken up into the lysosomal compartment, and they increased the lysosomal pH and cytotoxicity as a function of the number of surface amino groups on the dendrimer. PAMAM dendrimers that were surface-neutralized by acetylation of >80% of the surface amino groups failed to show any cytotoxicity. The positively charged, amine-terminated PAMAM dendrimer induced cellular apoptosis, as demonstrated by mitochondrial membrane potential changes and caspase activity measurements. These results suggest that PAMAM dendrimers are endocytosed into the KB cells through a lysosomal pathway, leading to lysosomal alkalinization and induction of mitochondria-mediated apoptosis.

Interaction of Dendrimers (Artificial Proteins) with Biological Hydroxyapatite Crystals

This investigation sets out to mimic protein-crystal interaction during biomineralization with the use of artificial proteins (dendrimers). It is hypothesized that these interactions depend on the surface charge of hydroxyapatite crystals. This was investigated with the use of dendrimers with capped surfaces of different charges to probe the surface. We used AFM images of crystal-bound dendrimers to determine the distribution of the surface charge, and its magnitude was correlated to the binding capacity of the dendrimers to the surface. The binding capacity of the dendrimers in ascending order at pH 7.4 was: acetamide-capped, -NHC(O)CH3, neutral charge; carboxylic-acid-capped, -COOH, negative charge; and amine-capped, -NH2, positive charge. AFM images of the crystals showed dendrimers spaced equally along the crystal. The results suggest that the crystal surface has alternating bands of positive and negative charge or a differential charge array, i.e., alternating bands of either more or less positive or negative charge.

The Role of Ganglioside GM1 in Cellular Internalization Mechanisms of Poly(amidoamine) Dendrimers

Generation 7 (G7) poly(amidoamine) (PAMAM) dendrimers with amine, acetamide, and carboxylate end groups were prepared to investigate polymer/cell membrane interactions in vitro. G7 PAMAM dendrimers were used in this study because higher-generation of dendrimers are more effective in permeabilization of cell plasma membranes and in the formation of nanoscale holes in supported lipid bilayers than smaller, lower-generation dendrimers. Dendrimer-based conjugates were characterized by (1)H NMR, UV/vis spectroscopy, GPC, HPLC, and CE. Positively charged amine-terminated G7 dendrimers (G7-NH(2)) were observed to internalize into KB, Rat2, and C6 cells at a 200 nM concentration. By way of contrast, neither negatively charged G7 carboxylate-terminated dendrimers (G7-COOH) nor neutral acetamide-terminated G7 dendrimers (G7-Ac) associated with the cell plasma membrane or internalized under similar conditions. A series of in vitro experiments employing endocytic markers cholera toxin subunit B (CTB), transferrin, and GM(1)-pyrene were performed to further investigate mechanisms of dendrimer internalization into cells. G7-NH(2) dendrimers colocalized with CTB; however, experiments with C6 cells indicated that internalization of G7-NH(2) was not ganglioside GM(1) dependent. The G7/CTB colocalization was thus ascribed to an artifact of direct interaction between the two species. The presence of GM(1) in the membrane also had no effect upon XTT assays of cell viability or lactate dehydrogenase (LDH) assays of membrane permeability.

Molecular heterogeneity analysis of poly(amidoamine) dendrimer-based mono- and multifunctional nanodevices by capillary electrophoresis.

Poly(amidoamine) (PAMAM) dendrimer-based nanodevices are of recent interest in targeted cancer therapy. Characterization of mono- and multifunctional PAMAM-based nanodevices remains a great challenge because of their molecular complexity. In this work, various mono- and multifunctional nanodevices based on PAMAM G5 (generation 5) dendrimer were characterized by UV-Vis spectrometry, (1)H NMR, size exclusion chromatography (SEC), and capillary electrophoresis (CE). CE was extensively utilized to measure the molecular heterogeneity of these PAMAM-based nanodevices. G5-FA (FA denotes folic acid) conjugates (synthesized from amine-terminated G5.NH(2) dendrimer, approach 1) with acetamide and amine termini exhibit bimodal or multi-modal distributions. In contrast, G5-FA and bifunctional G5-FA-MTX (MTX denotes methotrexate) conjugates with hydroxyl termini display a single modal distribution. Multifunctional G5.Ac(n)-FI-FA, G5.Ac(n)-FA-OH-MTX, and G5.Ac(n)-FI-FA-OH-MTX (Ac denotes acetamide; FI denotes fluorescein) nanodevices (synthesized from partially acetylated G5 dendrimer, approach 2) exhibit a monodisperse distribution. It indicates that the molecular distribution of PAMAM conjugates largely depends on the homogeneity of starting materials, the synthetic approaches, and the final functionalization steps. Hydroxylation functionalization of dendrimers masks the dispersity of the final PAMAM nanodevices in both synthetic approaches. The applied CE analysis of mono- and multifunctional PAMAM-based nanodevices provides a powerful tool to evaluate the molecular heterogeneity of complex dendrimer conjugate nanodevices for targeted cancer therapeutics.

Methotrexate delivery via folate targeted dendrimer-based nanotherapeutic platform.

This paper provides a synopsis of the advancements made in advancing a dendrimer-based nanomedicine towards human clinical trials by the Michigan Nanotechnology Institute for Medicine and Biological Sciences. A brief description of the synthesis and characterization of a targeted multifunctional therapeutic will demonstrate the simple yet delicate task of producing novel chemotherapeutic agents. The results obtained from in vitro and in vivo studies not only authenticate the potential of using nanoparticles to target therapeutics but also provide valuable insight towards the future directions of this technology. A fundamental, cross-disciplinary collaboration was necessary to achieve the synthesis and testing of this technology, and was the keystone to establishing this innovative invention. Throughout this paper, we will stress that the unique collaboration that facilitated the evolution of this technology is vital to the success of future developments in nanomedicine.