(38f) Development of Folate-PAMAM and Azithromycin-PAMAM Nanodevices for Biodistribution, Imaging, Cellular Uptake, and Targeted Delivery to Chlamydial Infections

Chlamydia trachomatis is an intracellular pathogen that causes major sexually transmitted diseases. Chlamydiae are very immunogenic bacteria and can cause inflammatory reactions in different parts of the body (1). Reactive arthritis (ReA) is one such example of an inflammatory reaction caused by chlamydial infection; ReA develops after dissemination of infected cells from the original site of infection (usually genital tract) (2). Since Chlamydiae can go into a persistent infection form, treatment with antibiotics such as azithromycin is a challenge. Dendrimers are emerging as a new class of nanoscale drug delivery vehicle, because of their well-defined structure, tailored surface properties, and ability to deliver drugs intracellularly (3). Since targeting ligands can be used to target dendrimers to the sites of inflammation it can be an effective delivery vehicle. This study seeks to develop, characterize and evaluate (in vitro and in vivo both) dendrimer-based targeted drug delivery nanodevices for therapy of chlamydial infections including targeting chlamydia-induced arthritic inflammation in a murine model (4).

It has been suggested that folic-acid conjugated nanoparticles can target inflammation in vivo. Therefore, we expect dendrimer-folic acid conjugates could also target inflammation. To explore this concept, we have synthesized two nanodevices for in vivo animal imaging experiments. We conjugated Cy5.5, a near IR imaging agent to the PAMAM dendrimer (PAMAM-Cy5.5) as a control, and a folic acid-PAMAM conjugate (PAMAM-Cy5.5-FA), by conjugating folic acid to the dendrimer and labeled it with Cy5.5. PAMAM-Cy5.5 was prepared by conjugating Cy5.5 NHS ester to the PAMAM dendrimer alone (G4-NH2). The purity of the nanodevices was determined by reverse phase HPLC. Folate mediated nanodevice was developed by reacting folic acid with PAMAM dendrimer (G4-NH2) under coupling reaction condition (5, 6). The resulting compound was purified by dialysis (MW cutoff 1000 Da) and lyophilized to get PAMAM-FA and characterized by proton NMR and MALDI TOF mass. The resulting compound has a comparatively stable amide bond and a multiplet at 6.6 ppm represents aromatic protons of folic acid and indicates the formation of compound. The loading of folic acid to the surface of dendrimer was calculated by proton NMR techniques by comparing amidic protons of dendrimer and aromatic protons of folic acid. Cy5.5 NHS ester was reacted to the above intermediate under basic conditions (7) to get folate mediated nanodevice, PAMAM-Cy5.5-FA.

The biodistribution of the nanodevices was imaged at the Cy5.5 wavelength (Ex 625 nm; Em 700 nm), using a Kodak whole animal imager 2h to 120h after injection at 7-14 dpi. The paws (major sites of inflammatory response), and the various organs were harvested for quantification of nanodevice uptake. PAMAM-Cy5.5-FA was taken up into inflamed paws at significantly higher levels than PAMAM-Cy5.5. All infected mice showed similar internal pattern of Cy5.5 distribution i.e., paws, genital tract, thymus, spleen, and lung in both cases. We did not see any significant localization in the liver, heart or the kidney. These results indicate that dendrimers are certainly playing a role in the delivery to the sites of inflammation, but folate-targeting (PAMAM-Cy5.5-FA) enabled higher accumulation and retention at the sites of inflammation (up to 120h). This lengthy time of detection further suggests that FA facilitates the retention of the dendrimer in the circulation and may be targeting to specific sites containing inflammatory cells which bear FA receptors. Analyses were performed with ImagePro Plus software (Media Cybernetics, Inc).

The therapeutic nanodevices based on PAMAM dendrimer conjugated to Azithromycin (PAMAM-AZ) were evaluated in vitro in C trachomatis infected cells. First we have reacted Azithromycin with glutaric acid under coupling reaction condition to get an acid functionality intermediate. A multiplet at 2.0 ppm represents methylene groups of glutaric acid and confirms the formation of the intermediate. This intermediate was further reacted with hydroxy group of PAMAM dendrimer under same reaction condition to get our therapeutic nanodevice (PAMAM-AZ). The reaction mixture was purified by dialysis and lyophilized to get PAMAM-AZ and characterized by proton NMR and MALDI TOF mass. The loading of Azithromycin to the surface of the dendrimer was calculated by comparing amidic proton of the dendrimer to aliphatic protons of the Azithromycin in between 4.0-5.0 ppm in NMR spectra and we conclude that 6.6 molecules were tagged per molecule of dendrimer. Chlamydia trachomatis-infected mammalian HEp2 cells were treated with either free AZ (control) or PAMAM-AZ (at equivalent AZ concentrations) at either the time of infection (t=0) or 24 hours post infection (t=24). PAMAM-AZ added at the time of infection (t=0) completely prevented infection of the cells, as measured by the total absence of Chlamydial inclusions. When PAMAM-AZ was added 24 hours after infection, it reduced the inclusion count by more than 30%. The therapeutic nanodevice delivers the drug in a more efficient way in a shorter span of time and maintains its effect for a longer time frame as seen by reduced inclusion size (area). This suggests that dendrimers transport the drug into the cells, and release it in a successful manner to reduce overall infectivity and infectious load within infected cells (size of inclusion).

In both imaging and biodistribution and in vitro cell entry experiments, we found a considerable level of targeting of the nanodevices to the sites of infection. The whole animal imaging data suggest a significantly higher uptake of folic acid mediated nanodevice compared to nanodevice having no folic acid. Based on the results of our experiments, we will design therapeutic nanodevices with folic acid as ligand which would target inflammation at sites of chlamydial infection and thereby significantly improve drug delivery to the infected cells. This is a noble step towards diagnosis and treatment of Chlamydia related infections and associated inflammatory diseases. It represents the first report of dendrimer-FA targeting of chlamydia-associated arthritic inflammation to the best of our knowledge.

References:

1. Bavoil, P.M. and Wyrick, P.B., eds., Chlamydia Genomics and Pathogenesis, eds Horizon Bioscience, Norfolk, UK, 2006.

2. Whittum-Hudson, J.A., Gérard, H.C., Schumacher, H.R. Jr., and Hudson A.P. In: Chlamydia Genomics and Pathogenesis, eds Bavoil, P.M., Wyrick, P.B. (Horizon Bioscience, Norfolk, UK) 2006, 475-504.

3. Lee, C.C., MacKay, J.A., Fréchet, J.M.J. and Szoka F.C.; Nature Biotechnology, 2005, 23, 1517-1526.

4. Whittum-Hudson, J.A., Gérard, H.C., Clayburne, G., Schumacher, H.R., and Hudson, A.P.; Revu de Rheum (English), 66 (1, Suppl), 1999, 50S-56S.

5. Shukla, S., Wu, G., Chatterjee, M.,Yang, W., Sekido, M., Diop, L. A., Muller, R., Sudimack, J. J., Lee, R. J., Barth, R. F. and Tjarks, W.; Biocojugate Chem., 2003, 14, 158-167.

6. Kano, K., Liu, M. and Frechet, M. J.; Biocojugate Chem., 1999, 10, 1115-1121.

7. Chen, Z., Levi, J., Xiong, Z., Gheysens, O., Keren, S., Chen, X., and Gambhir, S. S.; Bioconjugate Chem., 2006, 17, 662-669.