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Research - Targeted Therapeutics Studies Introduction

Current chemotherapeutic treatments for cancer are often less effective due to the inability of the drug to distinguish between cancerous and normal cells, limiting its therapeutic index. The toxicity of chemotherapeutics can be overcome to a large extent if the drugs can be targeted more specifically to the tumor tissue rather than the entire host. In addition, a targeted therapy utilizing a specific receptor-mediated cellular internalization process may also be effective in decreasing clinical failures due to drug resistance. Multifunctional therapeutic nanodevices have been designed and synthesized using G5-dendrimer produced at the MNIMBS under good manufacturing processes (GMP) and have been tested extensively. Nanoparticle therapeutics offer tremendous potential to improve the therapeutic index of many types of

drugs, especially cytotoxic molecules, in the treatment of many diseases.  We have produced polymer-based nanodevices for the intracellular targeting of (chemo-) therapeutic drugs, imaging agents, and other materials.

Figure 1:  Computer generated model of G5-PAMAM dendrimer cancer therapeutic with surface-conjugated molecules such as Folate (targeting agent), fluorescein (imaging agent), and Docetaxel (chemotherapeutic).

The development of these (targeted) macromolecules also allows for safer and more effective treatments for diseases from arthritis to inflammatory bowel disease that would improve outcomes for patients with these diseases.  Importantly, intracellular targeting of therapeutics can reduce the cost of healthcare by preventing costly side effects and chronic care requirements.  We report on several of our research projects utilizing this technology.

Biomedical Sciences Research Building:

Dendrimer biology work

Chemistry Building:

Dendrimer chemistry work

A target-specific, dendrimer-based nanoparticle contrast agent for tumor cells expressing the folate receptor is under development. In tumors that express the folate receptor, magnetic resonance imaging confirmed that the present of folic acid on the dendrimer nanoparticle resulted in specific delivery of the nanoparticle to cancer tissues and tumor cells that express the folate receptor. This research shows great promise for improved targeted cancer imaging, determination of the folate receptor status of tumors, and monitoring of drug therapy.

Swanson S.D., Kukowska-Latallo J.F., Patri A.K., Chen C., Ge S., Cao Z., Kotlyar A., East A.T., and Baker Jr. J.R. Targeted Gadolinium-Loaded Dendrimer Nanoparticles for Tumor-Specific Magnetic Resonance Contrast Enhancement, Int J Nanomedicine 3(2), 201-210, 2008.

 

Targeted Therapeutics Studies: Project

Cancer Nanotechnology Platform Partnerships: DNA-linked Dendrimer Nanoparticle Systems for Cancer Diagnosis and Treatment.

NIH-NCI: 1R01CA119409

Key Investigators

James R. Baker Jr., MD, Principal Investigator, Professor of Internal Medicine and Biomedical Engineering, Director, MNIMBS

Istvan Majoros, PhD, Research Assistant Professor of Internal Medicine, MNIMBS

Yuehua Zhang, PhD, Research Assistant Professor of Internal Medicine, MNIMBS

Seok-Ki Choi, PhD, Research Assistant Professor of Internal Medicine, MNIMBS

Thommey P. Thomas, PhD, Research Assistant Professor of Internal Medicine, MNIMBS

Jolanta Kukowska-Latallo, PhD, Research Assistant Professor of Internal Medicine, MNIMBS

Mark M. Banaszak Holl, PhD, Professor of Chemistry and Macromolecular Science and Engineering, MNIMBS

Bradford G. Orr, PhD, Professor of Physics, Chair of Department of Physics, Associate Director, MNIMBS

Theodore Norris, PhD, Professor of Electrical Engineering & Computer Science, MNIMBS

Abstract

M-NIMBS has developed a technical solution that provides a scaffold and linking strategy so that multifunctional combinatory therapeutics can be designed and built.  The scaffold is a dendritic polymer that is uniquely suited to biomedical applications in that it is a synthetic material that can be uniformly produced and yet has a diameter of only five nanometers. Originally our linker mechanism utilized complementary DNA oligonucleotides conjugated to the dendrimer clusters (Figure 1). We have also successfully utilized “click” chemistry as a synthetic strategy to chemically link two monofunctional dendrimer molecules.  The goal of this project is to take development of several nanodevices through small animal trials.  These designed multifunctional devices will have applications as targeted

Figure 1. Complementary DNA oligonucleotides conjugated to the dendrimer clusters.

imaging and diagnostic agents for cancer at the earliest stages as well as multifunctional devices that can deliver multiple therapeutics directly to cancer cells.  The technology will facilitate the concept of targeted, non-intrusive sensing, signaling, and intervention for cancer.  If this linked polymer cluster approach is successful in vivo, the targeting, sensing, and therapeutic conjugates can be interchanged to address varied tumor types or different genetic or enzymatic alterations associated with different cancers.  Thus, the linked cluster approach would yield common, interchangeable therapeutic platforms that transcend any single tumor or cellular abnormality.  We are now starting the fifth year of performance for this project.

Milestones

Milestone 1: Complete scale-synthesis of click-chemistry linked devices to produce quantities necessary for animal studies (20-30 mg).

Milestone 2: Complete in vitro analysis of click-chemistry linked devices produced above.

Milestone 3:  Initiate large-scale animal studies of click-chemistry linked two-function therapeutic and two-function imaging agent produced above.

Milestone 4: Initiate bio-distribution studies in animals of the click-chemistry linked two-function imaging agent.

Milestone 5: Continue efficacy and distribution studies of targeted shell-crosslinked iron nanoparticles (SCIO NPs).

 

Studies and Results

Milestone 1: Complete scale-synthesis of click-chemistry linked devices to produce quantities necessary for animal studies (20-30 mg).

Click-chemistry was successfully employed to generate a two-function dendrimer-based imaging agent for use in a large-scale animal study.   In addition, we have successfully isolated dendrimer with exact numbers of alkyne molecules which dramatically increases our ability to generate dendrimer-based platforms with exceptional purity.  Click chemistry was also employed to synthesize bifunctional dendron devices.  This study marks the first time two functionalized PAMAM dendrons have been coupled together.

 

Synthesis of binary dendrimers (FA and 6TAMRA) using Click chemistry

The two-function imaging agent was sythesized by coupling together two dendrimer-based modules.  The first module was composed of a folic acid conjugated dendrimer.  39.2 mg of this module was generated.  In addition to the folic acid molecules conjugated to the dendrimer, this dendrimer module also posessed a linker molecule with a terminal alkyne.  The second module was a dendrimer with 6TAMRA.  26.3 mg of the 6TAMRA conjugated dendrimer was generated.  This dendrimer also posessed a linker molecule with a terminal azide.  The two modules were coupled via the 1,3-dipolar cycloaddition reaction (‘click’ chemistry) between the alkyne moiety on the first dendrimer and the azide moiety on the second dendrimer.  24.6 mg of this compound was generated. 

The FA-6TAMRA dendrimer-based modular platform was evaluated in vitro with a human epithelial cancer cell line (KB) and found to specifically target the over-expressed folic acid receptor.  This material is currently being used for an animal study.

 

Isolation of dendrimer with exact numbers of functional groups

Methods that are commonly used to functionalize dendrimer with targeting, imaging, and theraputic molecules result in a distribution of dendrimer species, each with a different number of conjugated groups.  This population distribution is, in-fact, common for many different types of nanoparticles that are being developed for theraputic use.  Recently, using semi-prep HPLC, a dendrimer conjugate with an average of 0.45 azide linker molecules was separated and isolated into the individual dendrimer species that give rise to the average number for the material.  1H NMR was then used to confirm the number of linker molecules per dendrimer for each of the isolated species.


Figure 2. Reaction scheme for creating click-conjugated dendrons employed in the in vitro studies.

This is the first time that dendrimer with exact numbers of functional groups have been isolated and dramatically enhances our ability to control the synthesis of functional dendrimer platforms.  Because the isolated dendrimer samples have exact numbers of ‘click’ functional groups (alkyne or azide), a number of different targeting, drug, dye, dendrimers, and dendrons can be conjugated to the isolated dendrimer.  This development has the ability to bring substantial increases to the biological activity of the dendrimer platform due to the material purity that can now be achieved.

Synthesis of bifunctional dendrons (RGD and Methotrexate) using Click chemistry

The targeted dendron, alkyne-G3(COOH)(RGD), was used as a scaffold for the attachment of an additional functional moiety (Figure 2).  The functionalized PAMAM dendrons maintain the binding specificity and multivalency of previous dendritic models while creating an orthogonally coupled scaffold more suitable for personalized medicine and applications.  Binary devices were synthesized through a unique alkyne group at the dendron focal point.  The alkyne is reacted with an azide-functionalized dye, biotin, therapeutic molecule or a second dye-conjugated dendron in a copper-catalyzed 1,3-dipolar cycloaddition.  This marks the first time two functionalized PAMAM dendrons have been coupled together.

Development of a photocleavable linker system for the controlled release of therapeutics.  We designed and synthesized a photocleavable linker system for the controlled release of therapeutics (Figure 3).  Photocaging, a technique that refers to the temporary inactivation of an active molecule using a photolabile group, has found numerous applications including spatiotemporal control of biological processes and light-triggered payload release from nanomaterials.  We have performed initial studies applying the photocaging strategy to selectively release free methotrexate.  To accomplish this, methotrexate was modified to include a photocleavable linker, thereby forming a methotrexate prodrug.  Note that this conjugation step is amenable to nearly all therapeutics.  The prodrug was then attached to a FA-targeted dendrimer to form the dual-targeted therapeutic nanoparticle.  We were able to show that methotrexate is selectively released from pro-drug incubated with serum following exposure to UV light.  Because we have shown that methotrexate is active even while on the nanoparticle, we also synthesized a photocleavable-based therapeutic using doxorubicin. Doxorubicin, a fluorescent anthracycline anti-biotic cancer therapeutic that prevents the DNA replication process, is ideal for this system because like many therapeutics we could imagine using with

Figure 3.  Design and structure of a photocleavable linker for the controlled release of Doxorubicin and Methotrexate.  This linker system may be used to enhance the specificity of the binary dendrimer system described above. 

our click-chemis-try linked devi-ces, it is inactive prior to photo-cleavage. There-fore, the develop-ment of this strategy will be useful for broa-dening the thera-peutic application of click-chemistry linked devices.

 

Milestone 2: Complete in vitro analysis of click-chemistry linked devices produced above.

Cellular uptake of binary dendrimers (FA and 6TAMRA) at different concentrations (30 nM, 100 nM, 300 nM, and 1000 nM of was documented in KB cells that express a high cellular membrane concentration of folic acid receptor (FAR).  Cellular uptake and activity of bifunctionalized dendrons (RGD and Methotrexate & RGD and AlexaFluor) was documented in HUVEC cells that express a high cellular membrane concentration of the αVβ3 integrin.

In vitro analysis of binary dendrimers (FA and 6TAMRA)

Cellular uptake of the folic acid targeted dendrimer system at four different concentrations (30 nM, 100 nM, 300 nM, and 1000 nM) was measured in KB cells that express a high cellular membrane concentration of folic acid receptor (FAR).  Fluorescence uptake was quantified by flow cytometry.  As seen in Figure 4a and 4-blue, a dose dependent uptake was observed with saturation occurring at 100 nM.  This binding affinity is consistent with previous studies on single dendrimer platforms possessing multiple FITC and multiple FA molecules.

A series of control experiments were performed in order to ensure that uptake of the folic acid targeted dendrimer system was occurring via receptor-mediated endocytosis and not non-specific membrane interactions.  The first set of controls measured uptake of single dendrimers possessing the azide linker and multiple FITC at 30 nM, 100 nM, 300 nM, and 1000 nM (Figure 4b and 4f-purple).  No uptake was observed for this sample above the background level.  The second control sample contained a non-conjugated (un-clicked) mixture of the two dendrimers functionalized with either FITC or folic acid.  Uptake of this mixture was quantified at 30 nM, 100 nM, and 300 nM (Figure 4c and 4f-teal). 

At all three concentrations, no florescence uptake was observed.  This control eliminates the possibility that the dendrimer modules could form a non-covalently linked complex that would be internalized.  A third control sample was composed of an untargeted dendrimer module coupled to the FITC conjugated imaging module.  The un-targeted dual module platform was assembled under the same conditions used to form the folic acid targeted platform.

 

Figure 4: Binding and uptake of the fluorescent modular targeted dendrimer platform in KB cells as measured by flow cytometrya) Uptake FA3.5-G5-Ac107-L-G5Ac106-FITC3.2 (15). b) Uptake of G5-Ac106-Azide2.5-FITC3.2 (14) is not observed for 30 nM, 100 nM, and 300 nM. Very minimal uptake of this un-targeted module is observed at 1000 nM.  c) Similarly, no uptake is observed for an uncoupled mixture of G5-Ac106-Azide2.5-FITC3.2 (14) and G5-Ac107-Alkyne1.6-FA3.5 (13).  d) Uptake of FA3.5-G5-Ac107-L-G5Ac106-FITC3.2 (15) is successfully blocked using a 20 fold excess of free folic acid.  e) Uptake of FA3.5-G5-Ac107-L-G5Ac106-FITC3.2 (15) is also successfully blocked using a 20 fold excess (with respect to the folic acid content) of G5-Ac107-Alkyne1.6-FA3.5 (13).  f) Summary of mean fluorescence values for a-e.  Uptake of FA3.5-G5-Ac107-L-G5Ac106-FITC3.2 (15) is displayed in blue.  Uptake of the targeted platform (15) blocked by a 20 fold excess of G5-Ac107-Alkyne1.6-FA3.5 (13) is shown in orange.  Uptake of the targeted platform (15) blocked by a 20 fold excess of free folic acid can be found in green.  Uptake of a mixture of G5-Ac106-Azide2.5-FITC3.2 (14) and G5-Ac106-Alkyne1.6-FA3.5 (13) can be found in teal.  Finally, uptake of G5-Ac106-Azide2.5-FITC3.2 (14) can be found in purple.  Error bars indicate standard deviation as computed from half-peak coefficient of variation (HPCV) values.

 

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