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Cancer Cell Targeted Drug Delivery - In Vivo

We are developing nanoparticle targeting of anticancer drugs into animal models of human cancers. This form of drug delivery improves the therapeutic response to anticancer drugs and allows the simultaneous monitoring of drug uptake by tumors. Modified PAMAM dendritic polymers (dendrimer) smaller than 5 nm in diameter are used as carriers.

One of the targets chosen for delivery is the high-affinity folate receptor for the vitamin folic acid, also known as the folate-binding protein. A therapeutic nanoparticle consists of acetylated dendrimer conjugated to folic acid as a targeting agent, later coupled to methotrexate as a drug and a fluorophor as an imaging agent. The conjugates are injected intravascularly into mice bearing human tumors that overexpress the folic acid receptor. In contrast to non-targeted dendrimer, a folate-conjugated nanoparticle concentrates in tumor tissue for over 4 four days after administration. The tumor tissue localization of the targeted nanoparticle can be attenuated by prior intravascular injection of free folic acid. Internalization of the drug nanoparticle into tumor cells can be confirmed by confocal microscopy by detecting the fluorophor that is delivered with the same dendrimer platform. Targeting methotrexate increases its antitumor activity and markedly decreases its toxicity, allowing therapeutic responses not possible with a free drug. The folate-targeted in vivo trials in mice were published in Cancer Research on June 15, 2005;65(12):5317-24.

To allow for detection of individual fluorescent cells using flow cytometry or fluorescent microscopy in animal efficacy studies of the therapeutic, the SKBR-3 cell line expressing the HER2 receptor and the KB cell line expressing the FA receptor were transfected with red and green plasmid, respectively. This will allow for monitoring the therapeutics functionalized with different targeting molecules, thus proving the targeting efficacy of multi-functional therapeutics.

Clinical applications of magnetic resonance imaging (MRI) require generation of tissue-specific contrast. We have synthesized a target-specific MRI contrast agent for tumor cells expressing high affinity folate receptor using generation five (G5) of polyamidoamine (PAMAM) dendrimer.  Surface modified dendrimer was functionalized for targeting with folic acid (FA) and the remaining terminal primary amines of the dendrimer were conjugated with the bifunctional NCS-DOTA chelator that forms stable complexes with gadolinium (Gd III).  Dendrimer-DOTA conjugates were then complexed with GdCl3, followed by ICP-OES as well as MRI measurement of their longitudinal relaxivity (T1 s-1mM-1) of water.  In xenograft tumors established in immunodeficient (SCID) mice with KB human epithelial cancer cells expressing folate receptor (FAR), the 3D MRI results showed specific and statistically significant signal enhancement in tumors generated with targeted Gd(III)-DOTA-G5-FA compared with signal generated by non-targeted Gd(III)-DOTA-G5 contrast nanoparticle.  The targeted dendrimer contrast nanoparticles infiltrated tumor and were retained in tumor cells up to 48 hours post-injection of targeted contrast nanoparticle.  The presence of folic acid on the dendrimer resulted in specific delivery of the nanoparticle to tissues and xenograft tumor cells expressing folate receptor in vivo.  We present the specificity of the dendrimer nanoparticles for targeted cancer imaging with the prolonged clearance time compared with the current clinically approved gadodiamide (OmniscanTM) contrast agent.  Potential application of this approach may include determination of the folate receptor status of tumors and monitoring of drug therapy.  (International Journal of Nanomedicine. 2008, 3(2), 1-10).

To allow for detection of individual fluorescent cells using flow cytometry or fluorescent microscopy in animal efficacy studies of the therapeutic, the SKBR-3 cell line expressing the HER2 receptor and the KB cell line expressing the FA receptor were transfected with red and green plasmid, respectively. This will allow for monitoring the therapeutics functionalized with different targeting molecules, thus proving the targeting efficacy of multi-functional therapeutics.

 

Mixed population of KB-GFP (green fluorescent) cells and SKBR-3-Red (red fluorescence) in bright (Left) and fluorescent (Right) light

Figure 1. Mixed population of KB-GFP (green fluorescent) cells and SKBR-3-Red (red fluorescence) in bright (Left) and fluorescent (Right) light.

We have demonstrated the in vitro and in vivo targeting of a generation 5 (G5) dendrimer-based multifunctional conjugate that contained folic acid (FA) as the targeting agent and methotrexate (MTX) as the chemotherapeutic drug.  In order to clinically apply the synthesized G5-FA-MTX nanotherapeutic, it is important that the anti-cancer conjugate elicits cytotoxicity specifically and consistently.  Toward this objective, we evaluated the large-scale synthesis of a G5-FA-MTX conjugate for its cytotoxic potential and specificity in vitro and in vivo.  The cytotoxicity and specificity were tested by using a co-culture assay in which FA receptor-expressing and non-expressing cells (KB and SK-BR-3 cells, respectively) were cultured together and preferential killing was examined.  The in vitro data were compared with the in vivo data obtained from artificial heterogenous tumor model (AHT).  The xenograft animal model of AHT showed that the nanotherapeutic was preferentially cytotoxic to KB cells.  Data obtained from the study showed preferential killing of targeted cells by the nanotherapeutic.  These findings confirm the specificity of the G5-FA-MTX nanotherapeutic and predict its clinical applicability as a potential drug for targeted anti-cancer therapy.  (Anti-Cancer Drugs, 2009, accepted).

There are several other targeting ligands that can be placed on the surface of the dendrimer that are currently being developed for in vivo applications, such as aptamers, peptides, antibodies, and antibody fragments that interact with specific target molecules on tumor cells. Conjugation of different, clinically approved drugs such as taxol or doxorubicine is being developed for alternative nanoparticle conjugates. The lack of high enough affinities of the ligands to achieve targeted delivery in vivo will be improved by attaching multiple copies of each molecule to a dendrimer. Prior work from our group with sialic acid-conjugated dendrimers documents the co-operative binding of dendrimers functionalized with multiple ligands to influenza virus, while molecular modeling and in vitro experiments suggest that folate-targeted dendrimers have cooperative polyvalent binding to cells

(Reuter et al., Bioconjug Chem 1999; 10: 271-278; Quintana et al., Pharm Res 2002; 19: 1310-1316). 

Tumor-targeted drug delivery can enhance the effectiveness of chemotherapeutics while decreasing their systemic toxicity.  The use of monoclonal antibodies (mAbs) with anti-tumor specificity and activity is an established strategy in cancer therapy.  Monoclonal antibodies have been used to deliver chemotherapeutic drugs, plant and bacterial toxins, and radionuclides to tumors.  To enhance carrier capacity of antibodies a drug reservoir can be attached such as nanoparticle, liposome or polymer.  This allows delivery of a higher payload of drugs specifically targeted into tumor cells.  The human epidermal growth factor receptor-2 (HER2) present in breast and ovarian cancers, and associated with a poor clinical outcome is one of the most desired targets in cancer immunotherapy.  The recombinant humanized monoclonal anti-HER2 antibody (Trastuzumab/Herceptin®) has been approved for clinical use and is an ideal high-affinity ligand for HER2.  Our group has conjugated Herceptin to PAMAM dendrimer to increase the utility of the conjugate as a targeted drug delivery carrier.  The new carrier demonstrated rapid cellular uptake, efficient internalization and different trafficking in HER2-expressing cells compared with free Herceptin antibody without alterations in specificity of targeting.  In vivo studies demonstrated targeting of the conjugate in HER2–expressing tumors.  Our prior studies demonstrated utility of the antibody-drug conjugates in targeting prostate-specific membrane antigen (PSMA) and CD14 antigen. (Shukla et al., Bioconjug Chem 2006; 17: 1109-1115; Patri et al., Bioconjug Chem 2004; 15: 1174-1181; Thomas et al., Biomacromolecules 2004; 5: 2269-2274).

We are pursuing the development of specific targeting molecules for diagnostic and therapeutic applications that could enhance the treatment of a wide range of conditions such as inflammation, infectious diseases, and cancer.  The most common molecule currently employed and FDA-approved for numerous applications are monoclonal antibodies or immunoconjugates of these antibodies, which bind with high specific binding affinities with antigens.  Unlike antibodies there are many small molecules that are not immunogenic, and therefore cannot be identified by antibodies.  To present alternative synthetic molecule that can substitute for antibodies we synthesized and evaluated the biological activity for a targeted, binary PAMAM dendron avidity platform synthesized through a unique alkyne group at the dendron focal point.  The alkyne is reacted with an azide-functionalized dye in a copper-catalyzed 1,3-dipolar cycloaddition, commonly referred to as “click chemistry”. This strategy is attractive because it provides high versatility and yields, mild reaction conditions and no alteration to neighboring functional groups that eliminates the need for protecting groups.  The platform itself gives precise 1:1 ratio of the attachment of multiple functionalities, preventing aggregate formation.  A dendron, functionalized with multiple RGD on the surface and a single Alexa Fluor 488 dye molecule at the focal point, can specifically target αVβ3 integrin expressing human umbilical vein endothelial cells (HUVEC) and human glioblastoma cells (U87MG), displaying the biological utility of the platform.  Thus, the ability to conjugate specific and varied effector sites to the dendron periphery to generate a high avidity binding agent and use orthogonal reaction chemistry at the focal point to add additional functionalities makes these “dendro–bodies” an attractive alternative to antibodies for targeting applications. (Hong et al. Bioconjug Chem September 2009, 20(10), 1853–1859).

 

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