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Emerging Technologies

Photo-controlled Mechanism of Drug Release

The targeted delivery of therapeutic and imaging agents using nanoconjugates is a burgeoning field. Strategies to develop cancer cell specific nanoconjugates vary, but all attempts to selectively deliver therapeutics to cells using nanoscale carriers that include targeting and therapeutic agents. We design dendrimer-based targeted nanoconjugates such that the therapeutic agents are released, and therefore active, only under particular conditions. The release mechanisms we are currently exploring are a photochemical-based approach. In this scenario, the targeted drug conjugate is first placed on a surface, such as skin, lung, gastrointestinal tract or bone marrow. After the exposure, the nanoconjugate drug is specifically taken up by the tumor or other targeted 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. We have successfully demonstrated this release mechanism for antitumor agents such as doxorubicin[1] and methotrexate[2,3], and recently developed a tamoxifen photoprobe[4] for Cre-ER controlled gene modification (in collaboration with Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine). This strategy can be broadly applied to cell targeting systems, particularly those that require time- and tissue-dependent control of drug activation.

Publications: (1) Choi, S. K.; Thomas, T.; Li, M.; Kotlyar, A.; Desai, A.; Baker Jr, J. R. Chem. Commun. (Cambridge, U. K.) 2010, 46, 2632; (2) Choi, S. K.; Thomas, T. P.; Li, M.-H.; Desai, A.; Kotlyar, A.; Baker, J. R. Photochem. Photobiol. Sci. 2012, 11, 653; (3) Choi, S. K.; Verma, M.; Silpe, J.; Moody, R. E.; Tang, K.; Hanson, J. J.; Baker Jr, J. R. Bioorg. Med. Chem. 2012, 20, 1281; (4) Inlay, M. A.; Choe, V.; Bharathi, S.; Fernhoff, N. B.; Baker, J. J. R.; Weissman, I.; Choi, S. K. Chem. Commun. 2013, 49, in press. DOI: 10.1039/C3CC42179A.




Dendrimer-Based Nanoplatform for Targeting Bacteria Cells. Gram-positive bacterial infections cause numerous serious medical conditions including sepsis, bacteremia, pneumonia and endocarditis. Such infections can be life-threatening, especially when associated with drug-resistant bacteria. Despite growing concerns about serious infectious diseases, current technology is not able to fully and accurately detect, enumerate, or treat those causative bacterial cells inhabiting the blood and other vital organs. For example, vancomycin represents the preferred ligand for bacteria-targeting nanosystems. However, it is inefficient for emerging vancomycin-resistant species because of its poor affinity to the reprogrammed cell wall structure. We are interested in employing a multivalent strategy as an effective way for overcoming such an affinity limitation in Text Box:  bacteria targeting. We designed vancomycin-presenting PAMAM dendrimers and investigated to determine whether it serves a new platform that enables detection and isolation of bacterial pathogens[1]. These conjugates showed remarkable enhancement in avidity in the cell wall models tested, including the vancomycin-resistant model, which had an increase in avidity of four to five orders of magnitude greater than free vancomycin (Figure A). The tight adsorption of the conjugate to the model surface corresponded with its ability to bind vancomycin-susceptible Staphylococcus aureus bacterial cells in vitro as imaged by confocal fluorescent microscopy (Figure A). This vancomycin platform was then used to fabricate the surface of iron oxide nanoparticles by coating them with the dendrimer conjugates, and the resulting dendrimer-covered magnetic nanoparticles were demonstrated to rapidly sequester bacterial cells (Figure B).

Publication: (1) Choi, S. K.; Myc, A.; Silpe, J. E.; Sumit, M.; Wong, P. T.; McCarthy, K.; Desai, A. M.; Thomas, T. P.; Kotlyar, A.; Holl, M. M. B.; Orr, B. G.; Baker, J. R. ACS Nano 2013, 7, 214.


Riboflavin Receptor-targeting Dendrimers for Targeted Drug Delivery.

Water soluble riboflavin (RF) is an essential vitamin (B2) necessary for the biosynthesis of flavin-based redox cofactors, and its cellular availability is mediated by riboflavin receptors that facilitate the uptake of the riboflavin molecules with high efficiency. We have recently begun to explore RF as a ligand for targeting cancer therapy because riboflavin receptors are over-expressed in certain human cell lines from breast and prostate cancers, potentially making this family of proteins a type of tumor biomarker. We successfully demonstrated receptor-mediated RF-targeted drug delivery to cancer cells in vitro [1]. In this study RF-conjugated dendrimer nanoparticles delivered the anti-cancer drug methotrexate (MTX) to kill KB cells via receptor-mediated internalization (Figure A). This work suggested a general route for the selective delivery of anticancer drug molecules to the cancer cells that overexpress riboflavin receptors.

Text Box:  We performed the biophysical characterization of G5 PAMAM dendrimers conjugated with riboflavin (RF) as a cancer targeting platform[2,3]. We designed two new series of dendrimers, each presenting the riboflavin ligand attached at a different site (isoalloxazine at N-3, and D-ribose at N-10) and at varying ligand valency. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) were used to determine the binding activity for riboflavin binding protein (RfBP) in a cell free solution. The ITC data shows dendrimer conjugates have KD values of ≥568 nM on a riboflavin basis, an affinity ~113-fold lower than that of free riboflavin (Figure B). The N-3 series showed greater binding affinity in comparison to the N-10 series. Notably, the affinity is inversely correlated with ligand valency. These findings are also corroborated by DSC where greater protein-conjugate stability is achieved with the N-3 series, and at lower ligand valency.

In the follow up study [4], we focused on the identification and characterization of novel, dual-acting RF antagonists that have structural similarity to RF. Use of the dual acting RF antagonists provide potential benefits for targeted delivery in cancer because of their ability to bind to the RF receptor for targeted uptake of nanoconjugates. In addition, unlike native RF that have positive trophic effects on cancer, these molecules can kill cancer cells by interfering with the cellular functions of flavin cofactors such as FMN and FAD, which are essential for cellular maintenance. We screened riboflavin-mimics, small molecules that have potential binding activity for the riboflavin binding protein by performing isothermal titration calorimetry and surface plasmon resonance spectroscopy. This study led to the discovery of two known drug molecules, quinacrine and chloroquine, as favorable ligands for the riboflavin receptor withvalue of 250, and 2252 nM, respectively (Figure C).

Publications. (1) Thomas, T. P.; Choi, S. K.; Li, M.-H.; Kotlyar, A.; Baker Jr, J. R. Bioorg. Med. Chem. Lett. 2010, 20, 5191; (2) Plantinga, A.; Witte, A.; Choi, S.-K.; Sinniah, K. Biophys. J. 2011, 100, 553a; (3) Witte, A. B.; Timmer, C. M.; Gam, J. J.; Choi, S. K.; Banaszak Holl, M. M.; Orr, B. G.; Baker, J. R.; Sinniah, K. Biomacromolecules 2012, 13, 507; (4) Plantinga, A.; Witte, A.; Li, M.-H.; Harmon, A.; Choi, S. K.; Banaszak Holl, M. M.; Orr, B. G.; Baker Jr, J. R.; Sinniah, K. ACS Med. Chem. Lett. 2011, 2, 363.



Text Box:  Mechanism of Dendrimer-Oxime Drug Complexation.

We investigated a G5 PAMAM dendrimer as a multivalent carrier for the extended release of drug molecules [1,2]. We performed binding studies of the dendrimer (unmodified, and surface modified) with oxime-based antidote molecules (pralidoxime, obidoxime). Pralidoxime (2-PAM) and obidoxime belong to a class of oxime antidotes developed for the treatment of organophosphate poisoning.

Both drugs have short durations of action that could, in principle, be extended by complexing the drugs to nanocarriers for increasing their circulation half lives. We performed extensive NMR experiments including 1D titration experiments, 2D 1H-1H COSY/NOESY and 1H Diffusion-Ordered Spectroscopy. We performed quantitative analysis of the molecular interaction between the dendrimer and the oxime drug molecules in the nanoscale architecture. The guest molecules bind through the formation of specific interactions (H-bond, electrostatic) on the surface rather than random encapsulation (Figure A). Furthermore, individual binding events contributing to the complexation at a global level occur in a negatively cooperative manner (Figure B), pointing to the significant role of steric interactions within the dendrimer framework. These studies provided a useful new insight in host-guest interactions based on the macromolecular multivalent receptor.

Publications. (1) Choi, S. K.; Leroueil, P.; Li, M.-H.; Desai, A.; Zong, H.; Van Der Spek, A. F. L.; Baker Jr, J. R. Macromolecules 2011, 44, 4026; (2) Choi, S. K.; Thomas, T. P.; Leroueil, P. R.; Kotlyar, A.; Van Der Spek, A. F. L.; Baker, J. R. J. Phys. Chem. B 2012, 116, 10387.



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