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NANOTECHNOLOGY FOR HEMATOLOGY ________________________________________________________________________

Dendrimer-based nanoparticles for cancer
James R. Baker Jr.1
Division of Allergy and Clinical Immunology, Michigan Nanotechnology Institute for Medicine and
Biological Sciences, Ann Arbor, MI

Recent work has suggested that nanoparticles in the form of dendrimers may be a keystone in the future
of therapeutics. The field of oncology could soon be revolutionized by novel strategies for diagnosis and
therapy employing dendrimer-based nanotherapeutics. Several aspects of cancer therapy would be
involved. Diagnosis using imaging techniques such as MRI will be improved by the incorporation of
dendrimers as advanced contrast agents. This might involve novel contrast agents targeted specifically to
cancer cells. Dendrimers can also be being applied to a variety of cancer therapies to improve their
safety and efficacy. A strategy, somewhat akin to the “Trojan horse,” involves targeting anti-metabolite
drugs via vitamins or hormones that tumors need for growth. Further applications of dendrimers in
photodynamic therapy, boron neutron capture therapy, and gene therapy for cancer are being examined.
This presentation will cover the fundamentals of research utilizing dendrimers for cancer diagnosis and
therapy. An evaluation of this new technologies will detail what advantage dendrimer based therapeutics
might have over conventional cancer drugs.

anotechnology has led to a remarkable conver- intervention for cancer. If this polymer-based approach is
gence of disparate fields including biology, successful in vivo, the targeting, sensing, and therapeutic
applied physics, optics, computational analysis, conjugates can be interchanged to address varied tumor
and modeling, as well as materials science. Because of this, types or different genetic or enzymatic alterations associ-
the application of nanoscale analytical, computational, and ated with different cancers. Thus, this approach would yield
synthetic approaches to understanding and manipulating common, interchangeable therapeutic platforms that
complex biological systems offers incredible potential for transcend any single tumor or cellular abnormality.
advances in the diagnosis and treatment of cancer.
Nanoparticle Cancer Therapeutics
Utilizing these advances, our interdisciplinary team at the Most cancer therapeutics are small drug molecules that
University of Michigan Nanotechnology Institute for Medi- after being ingested or injected into the bloodstream can
cine and Biological Sciences (M-NIMBS) has developed a easily diffuse through vascular pores and the extracellular
number of technical solutions that provide a scaffold strategy matrix to reach tumors. Complex therapeutics that involve
so that multifunctional combinatory therapeutics can be drug delivery mechanisms or imaging moieties have tended
designed and built. The scaffold is a dendritic polymer that is to be much larger. While the exact size of molecules that
uniquely suited to biomedical applications in that it is a can easily transverse vascular pores from the bloodstream
synthetic material that can be uniformly produced and yet has and reach tumor tissue is unclear, it is probably limited to
a diameter of only 5 nm. We have completed preliminary work the size of proteins (< 20 nm). Studies have documented
providing proof of concept1,2 and our team is now developing that molecules 100 nm in diameter do not effectively
several nanodevices that have shown success in animal trials. diffuse across the vascular endothelium,3 and even mol-
These designed, multifunctional devices will have applica- ecules 40 nm in diameter are problematic unless the
tions as targeted imaging and diagnostic agents for cancer at endothelium is traumatized by radiation or heating.4 The
the earliest stages as well as multifunctional devices that can vasculature in early neoplastic lesions may be more
deliver therapeutics directly to cancer cells. restrictive.5 Thus, designed nanoparticle-based therapeutics
require a complex, multi-functional device that is still
The technology we have developed will facilitate the small enough to exit the vasculature in order to intimately
concept of targeted, non-intrusive sensing, signaling, and interact with, and specifically eliminate cancer cells.

708 American Society of Hematology

Therapeutic agents will probably require several Before we initiated our research. working in parallel to individual dendrimers had been demonstrated for essen- prevent the development of resistant neoplasms. image the extent of the tumor and sense its signatures. extensive work has been completed with activated in normal cells. capil- changes in particular cells. good manufacturing processes (GMP) for biotechnology applications. will require liquid chromatography (HPLC). Different types of dendrimers can be synthesized based on the core structure that initiates the polymerization process.1. oligonucleotides. Identifying residual disease at the end of therapy (rather than after tumor re- growth) will facilitate eradication of the few remaining tumor cells. evidence of toxicity when administered intravenously. it tially every desired component required for a viable anti- will be important for the therapeutic agent to also monitor cancer nanostructure. Molecular weight and the number of terminal groups increase exponentially as a function of generation (the number of layers) of the polymer (Figure 2). Prior Work on Dendrimers by Other Groups The first technical result that underlies our proposal is the use of dendrimer. Dendrimer-based Nanoparticles for Cancer Treatment Two technical advances underlie our research. Thus.2 In this review. dendritic dendrimer molecules to be linked via complementary polymers. This is and are essential to monitor quality control of dendrimer difficult to accomplish since most chemotherapeutic agents manufacture for GMP applications and in vivo usage. Each generation increases the size.9 Poly(amidoamine) spherical dendrimers (PAMAM) with ethylene-diamine (EDA) as a tetravalent initiator core are used in our studies.7 Tumors often require higher PAMAM dendrimers and in vivo studies have shown no doses of therapeutics since they have developed mecha. This is the goal for our nanotherapeutic. A binary nanoparticle targeting a cancer cell. deliver a therapeutic. groups on the surface of the polymer. Hematology 2009 709 .8 The second is the development of a linking strategy that allows the Figure 2. high-performance on cancer cells in vivo. however. The analysis of cancer lary electrophoresis (CE).10. We have now produced 100-gram lots of this material under current Figure 1. an ideal therapeutic must have the ability to target cancer cells. lead to a tumor that is resistant to therapy. PAMAM dendrimers have been used the response to therapy by identifying residual disease as a scaffold for the attachment of several types of biologic immediately after treatment.6. This device. agents that can be efficiently delivered to specifically kill These tests assure the uniformity of the polymer population the abnormal cells without collateral damage. These polymers are synthesized as well- defined spherical structures ranging from 1 to 10 nanom- eters in diameter. Several generations of spherical. we will discuss these two molecular weight and number of primary amine advances in sequence. Dendrimers are characterized by electrospray- The nanoparticle therapeutic most often envisioned is a ionization mass spectroscopy (ES-MS). the performance of different mechanisms of action.11 nisms of evading anticancer drugs. and monitor cells for their response. This work has focused on the preparation of remaining cells may result in re-growth or worse. 13C nuclear mag- complex macromolecule that can identify specific targets netic resonance spectroscopy (NMR). This is crucial since a few materials. and gel permeation chromatogra- signature(s) must be coupled to one or more therapeutic phy (GPC) and a variety of gel electrophoresis techniques. will stop cell growth or induce apoptosis if inadvertently Importantly. These dendritic macromolecules are available commercially in kilogram quantities. size exclusion chromatog- more than the identification of specific pathophysiologic raphy (SEC) with multi-angle laser light scattering. Finally. such as molecular pumps. The first is the successful development of multifunctional nanodevices based on the dendritic polymer or dendrimer.

have produced a single device that has the different functions necessary for a targeted. MRI contrast agents. sensing apparatus for evaluation of physiologic changes within cells. This dendrimer-based agent has multiple dendrimer-antibody conjugates (for neutron capture functions and can be used as a testing platform to evaluate therapy). we Results from these studies have documented that. both acetamide and hydroxyl Figure 3. active sensing. there was non-specific uptake in developing nanomolecular. Multifunctional Single Dendrimer which have elevated levels of the high affinity folate Nanodevices: In Vitro Testing receptor. In contrast.12 for the production of dendrimer-chelant. a net overall negative charge (pre- of the molecule may be digested with either enzyme or dominantly carboxyl moieties) on the molecule surface was light-induced catalysis. Because differences in polymer surface charge were thought to be of paramount importance in the biological function of this scaffold. the dendrimer scaffold due to the loss of repulsion from 710 American Society of Hematology .16 targeting cells (through the high affinity folate receptor). it provided a direct means to evaluate structural fluorochromes and shown to enter cells.13-16 Some of these conjugates have also been folic acid was coupled to proteins or drugs as a means of employed in the magnetic resonance imaging of tumors. because we had experience with associated therapeutic agents to antigen-bearing tumors.dendrimer-antibody conjugates for use in in vitro diagnos. delivery (Figure 4). we could have attempted therapeutic agent on a single dendritic molecule (Figure 3). FITC to amino surface dendrimers as a means to initiate our studies. The carboxyl modification previously reported to achieve efficient delivery to cells did appear to improve accessibil- ity of folic acid molecules conjugated to the surface of the polymer. triggering devices.000 daltons. and cell detected within the cell in a manner compatible with internalization. tion steps that are required. They can then be aspects of the polymer scaffold. so we attempted to simply conjugate folate and trigger to release the therapeutic agents.17 Dendrimers have also been conjugated to once. When we placed this material in vitro with KB cells. multifunctional devices. the carboxyl surface molecules would interact with secondary and tertiary amines in the dendrimer and would cause aggregation of these molecules. we used computer modeling to evaluate several (MW 25. The techniques for this are described extensively by tics.20 While this was not an optimized system as encapsulated in dendrimer polymers has increased efficacy the FITC bleaches and the material could only be analyzed and is less toxic.18 Finally. antibodies can direct dendrimer. dendrimers have been constructed as Research had suggested that when coupling folic acid to differentiated block co-polymers where the outer portions proteins or polymers. diameter 5 nm). Because of prior reports where therapeutic. cell delivery. A single dendrimer can be used to produce a nanostructure by direct conjugation of multiple surface modifications resulted in more compact structures in moieties directly to different branches of the molecule.19 This would allow the controlled important for cellular targeting. we saw very poor uptake that required several Over the past several years. we have made great progress in hours. coupling FITC to proteins. many manipulations of the polymer surface or structure. Three different surface modifications were evaluated: carboxyl. PAMAM degradation of the polymer to release therapeutics at the dendrimers are synthesized with a surface of primary disease site and could provide a mechanism for an external amines. However. cells. deprotect steps are needed to produce such a multifunc- antibody constructs. However. we used FITC as a signaling Dendrimers also have been reported to enter tumors and device to follow the fate of the dendrimer complex within carry either chemotherapeutic agents or genetic therapeu. dendrimer. we first examined changes in the terminal groups on the polymer. modeling studies also suggested that at higher concentrations of nanostructure. In particular. molecules to the surface of a generation 5 dendritic polymer Instead. and for the development of boronated tional agent. We cell lines that lacked the high affinity folate receptor. when adopted folate as an initial targeting ligand to attach to the administered in vivo. In addition. current studies show that cisplatin Quintana et al.15. hydroxyl and acetamide substitutions (Figure 4). Several challenging protect- tic applications. More disconcerting. all This involves coupling of functional groups such as sensing of which would have been synthetically complex and units. each of these latter compounds is used as a cancer nanostructure and function. This has proven to be proposed polymer modifications that would improve an arduous synthetic endeavor given the multiple conjuga. and targeting would have required considerable time to accomplish. imaging To improve this performance.

It occurred within minutes. correct in studies with intact cells expressing the receptor. In addition. The modeling of the amine surface molecule (data not shown. the polymer followed by the sequential folate conjugates demonstrated no binding. which corresponds to folate receptor recycling. This suggested that the molecules were aggregating and no longer available for binding to the cell surface receptor. it appeared that all of the folic acids on the surface of the molecule were accessible for binding with cellular receptors in the acetamide molecule. the uptake appeared to stop. however. see Quintana et al20) suggested that none of the folic acid molecules were externalized where they could bind to cellular receptors. approximately 4 to 6 hours. Panel A demonstrates increases of cell-associated fluorescence after incubation with 30 nm The next step was to develop a more of acetamide. The cellular uptake of the acetamide and hydroxyl surface polymers was very rapid and very efficient. The activity of these molecules in targeting predicted to have the folate in a surface position that was likely the folate receptor on KB cells in vitro was then evaluated to interact with receptors on cells. This was proven to be and compared to that predicted in the molecular modeling.20 All panels except Panel D demonstrate experiments performed at 37 ºC. This was accomplished Significant background fluorescence was not observed until the concentration of non- by first acetylating approximately 80% targeted complex reached 300 nm. documents progressive increases of cell fluorescence after incubation with increasing folate and drug were conjugated on a concentrations of acetamide-surfaced fluorescent dendrimer-folate conjugate. nanostructure. the carboxyl surface mol- ecules initially had rapid uptake. fluorescein. and Reprinted with permission from Quintana et al. confirming the modeling prediction that there were more surface folate groups available for binding. but non-specific interactions between surface amines and cell dendrimers (middle panel) and substituted with identical numbers of folic acid and lacked non-specific interactions. peaking at approximately 20 or 30 minutes and appeared to resume after another 30 minutes. The results were dramatic (Figure 5). Also in accordance with the modeling. Amine surfaced dendrimer. but as the concentration of the nanostructure was increased. Uptake was faster for the acetamide surface rather than the hydroxyl surface. Cell-associated fluorescence as a function of dendrimer-folate conjugate concentration is presented for each of the three types of of the primary amines on the surface of dendrimer. hydroxyl. adjacent surface charged molecules (Figure 4). This provided confidence that folic acid coupled polymer with neutraliz- ing surface modifications might be able to efficiently target Figure 4. Binding of fluorescent dendrimer-folate conjugates to KB cells confocal microscopy to internalize after after 30 min incubation. while approximately two thirds of the targeting moieties in the hydroxyl molecule were available for binding. attachment of folate. the acetimide was FITC moieties. or carboxyl-surfaced dendrimer-folate conjugates. The acetamide surface polymer continued to accumulate within the cells over time and appeared by Figure 5.20 one of two different drugs—either Taxol Hematology 2009 711 . at 37 ºC in Panel C and at 4 ºC in Panel D. Modeling of folate-conjugated dendrimer cells through the high affinity folate receptor. single polymer. Folate is exposed on the surface of the amine- Polymers with the three modified surfaces were synthesized surfaced polymer (left panel). Panel B complex device in which fluorescein.

21 The free FA level receptor interactions and that the data suggest that targeting achieved in the serum of the mice prior to the experiment this drug to resistant cancer cells might be more effective approximated human serum levels. conjugates were produced and tested in the KB cells. with both members of each pair containing the same tracer. the level of G5-3H-FA Reprinted with permission from Quintana et al. while the amide binding stability on the cell surface. Panel B shows that at 1000 uM only the targeted G5-3H in the kidney decreased conjugate where methotrexate is linked by an ester is acutely cytotoxic to the tumor rapidly and was maintained at a moderate cells.22. one pair without the antineoplastic drug MTX—while the other conjugate lacked FA and served as a non-targeted control with or without the drug. This suggested that the ester-linked drug was active. In contrast. In addition. Data in this sub-section docu- a MTT assay) and by clonagenic assay. whereas the amide-linked drug was not. These multiple receptor-folate interactions. agent. the cumula- tive clearance of the targeted G5-3H-FA over the first 4 days was lower than that of G5-3H. Panels C and D compare the efficiency in killing tumor cells and the acute level over the next several days (Figure cytotoxicity (respectively) of ester-conjugated drug and free methotrexate. because the Biodistribution of Tritiated Dendrimers drug was released approximately 4 to 6 hours after internal. possibly through linkage should retain drug and serve as a control. Full details of the study can be found in Kukowska- Latallo et al. Panel A shows that the devices are readily taken FR on its tubules. Mice were evaluated at various time points (5 minutes to 7 days) following intra-venous administration of the conjugates. While the kidney is the major clearance organ for these dendrimers. it is Figure 6. 7B). The drugs were attached through nanostructure remained after acidic wash at 0 oC. we initiated material was tested for the ability to induce cytotoxicity in animals trials to examine distribution and efficacy of the the KB cells. while free two linker mechanisms. 7A). We first examined the biodistribution and elimination of ization within the cell. tritiated G5-3H-FA to test its ability to target the FR-positive delivered MTX that induced cytotoxicity was compared to human KB tumor xenografts established in immunodefi- free methotroxate. which may reflect retention of G5- 3 H-FA within tissues expressing folate receptors. One member of each pair contained FA—one pair with.20 increased slightly over the first 24 hours. the polymers with the drug were Multifunctional Single Dendrimer internalized as efficiently as polymers that had only Nanodevices: In Vivo Testing fluorescein on their surface. 50% of bound particles were synthesized. Studies examining intracellular delivery of methotrexate with also known to express high levels of the a folate/FITC nanostructure. The level of non- into KB cells regardless of drug conjugate.23 Four pairs of nano- than using free drug.8 To summarize. In this culture system. The ester linkage should hydrolyze once the that the folate-dendrimer conjugates have increased device internalized within the KB cell.or methotrexate (MTX). 712 American Society of Hematology . this targeted With the completion of the in vitro studies. As shown in Figure 6. the folate-targeted cient nude mice. This suggested amide linkage. The polymer delivered drug is 5 to 10 times more efficient at killing the KB cells. We believe that this might be due to polyvalent mize the circulating levels of FA. In addition. Two groups of mice received either control non-targeted tritiated G5-3H dendrimer or targeted tritiated G5-3H-FA conjugate (Figures 7A. by both an assay of mitochondrial activity (in targeted nanotherapeutic. one an ester linkage the other an folate is easily removed by this treatment. The mice were maintained on a folate- material was 5-to 10-fold more active than equipotent ?? deficient diet for the duration of the experiment to mini- free drug. The concentration of dendrimer. The ester-linked ment tumor cell-targeted delivery and therapeutic effect of drug delivered with the folate was active as a cytotoxic drug-dendrimer conjugates in vivo.

This was followed by a decrease over the next several days as the compound was cleared by the kidney (Figure 7B). The highest hours after injection.0 mg/kg accumulated in 10 to 15 injections. Confocal microscopy images were obtained of tumor samples at 15 hours following intrave- nous injection of the targeted G5-6T-FA and the non- targeted G5-6T conjugates (Figure 8). Twice a week. attached to and internalized by organ (% ID/g ± SD). The above and below it by 25% of its thickness. As the plane of view is moved from min. and 5. 21. Hematology 2009 713 . Saline and the conjugate without MTX (G5-FI-FA) were used as controls. the top to the bottom of the tissue slide. and at 1. dendrimers conjugated with 6-TAMRA (6T) were employed. Six groups of immunodefi- cient SCID mice with 5 mice in each group were injected subcutaneously on one flank with 5 × 106 KB cells in 200 μL PBS suspension.0 sections. and finally their basal section. Figure 7. a conjugate without MTX. free MTX.B- 17 mice bearing subcutaneous human KB xenografts and was compared with equivalent and higher doses of free MTX. Biodistribution of radio-labeled non- targeted (A) and targeted (B) conjugate in nu/nu mice Cellular Internalization of Dendrimer Conjugate bearing KB xenograft tumor depicted as percentage of Confocal microscopy also demonstrated that the conjugate injected dose of dendrimer recovered per gram of is present in the tumors.3 mg/kg. harvested 15 saline per 20 g of mouse. starting on day 4 after tumor implantation. and each section overlaps the optical section mg/kg total cumulative dose of free MTX (Figure 9). based on mouse survival. A given image typically appears in approximately 40 optical peutic used equals 55. the animals received via the tail vein an injection of either a conjugate containing MTX. and 7 days after delivery are the means ± SD of 3 to 5 mice. cells are seen at their apical. The medial therapeutic dose was compared with three different (cumu- sections of tumor cells show fluorescence throughout the lative) doses of free MTX equivalent to 33.0 mg/kg and is equivalent to a 5.most likely due to FR present on the kidney tubules. 2 h. The tumor tissue demonstrated a significant number of fluorescent cells with targeted-dye-conjugated dendrimer G5-6T-FA (Figure 8C) compared with those with non-targeted dendrimer (Figure 8B). medial.7 cytosol from the 6-T of the conjugate. 4. Fluorescence images of mouse KB cell tumor. Flow cytometry analysis of a single-cell suspension isolated from the same tumors showed higher mean channel fluorescence for tumor cells from mice receiving G5-6T-FA. The compounds were delivered in a 200 μL volume of Figure 8. The values for different organs at 5 many of the tumor cells. targeted material by the tumor. or saline as a control. The body weights of Targeted Drug Delivery to Tumor Cells Through the mice were monitored throughout the experiment as an the Folate Receptor The efficacy of different doses of conjugates was tested on SCID C. with the cell and mg/kg. There is greatly enhanced uptake of the folic acid total dose of G5-FI-FA-MTX thera. nucleus boundary clearly visible (data not shown). Biodistribution of Fluorescent Dendrimer Conjugate To further confirm and localize the dendrimer nanoparticles within tumor tissue.

tumor growth based on the end- point volume of 4 cm3 can be delayed by at least 30 days. Toxicity of Dendrimer Conjugates lent dose of MTX delivered with both targeted conjugates All mice were observed for the duration of these studies for to the surviving mice was higher than the dose of free MTX signs of dehydration. respectively (data not shown). the therapeutic dose of conjugate that was delivered drug (open circles) was equal to the 21. inability to eat or drink. Drug-induced necrosis of the tumor on the flank At the termination of the trial. No gross toxicity. ). Figure 10 shows the mice dosed During a second 99-day trial.7 mg/ kg in 13 injections).indication of adverse effects of the drug. there was a statistically with MXT and those with MTX conjugated to targeted significant (P < .0 mg/kg of free MTX Figure 9. histopathology analysis of the liver revealed advanced liver lesions. whereas the free drug at this concentra- tion had no effect on tumor growth (Figure 9. no adverse effects from the chemotherapy. of the mouse in the upper right corner. For the highest cumulative doses of free MTX used.05) slower growth of tumors that were dendrimer. hair and appear not palpable for the next 20 sick. The survival of mice from groups receiving chronically up to 99 days was observed regardless of G5-FI-FA-MTX or G5-FA-MTX conjugate indicates that whether or not the dendrimer conjugate contained MTX. either acutely or 66 of the trial. effective as the second highest dose of free MTX (21. We have achieved a complete cure in one mouse treated with G5-FA-MTX conjugate at day 39 of the trial. The equiva. This value indicates the anti- tumor effectiveness of the conjugate because it mimics clinical end-points and requires observation of the mice throughout the progression of the disease. without FITC. compared with those treated with free MTX. The remaining experimental groups had very uniform body weight fluctuations non-indicative of toxicity when compared with control groups with saline or conjugate without MTX (data not shown). The tumor in this mouse was Figure 10. or because all of the mice receiving free MTX had died by day change in activity level. Mice on right dosed with dendrimer-transported folate-targeted methotrexate exhibit days up to day 60 of the trial. Total MTX dose of the polymer- Importantly. saline or non-targeted G5-FI-MTX conjugate. and peri-portal inflamma- tion. 714 American Society of Hematology . weakness. Mice on left dosed with methotrexate lose weight.7 mg/kg. O). Although the two doses of free drug were affecting tumor growth.3 mg/kg and 21. In contrast. treatment regiments. The there were 3 (out of 8) survivors receiving G5-FA-MTX and conjugate without MTX also had no therapeutic effect 2 (out of 8) survivors receiving G5-FI-Fa-MTX.7 mg/kg dose equal to the lowest dose of free MTX used was as equally of free drug. collections of inflammatory cells. no mice surviving in the group receiving free MTX or in any other control group. Tumor volume (mm3) versus time of various nor free MTX at the same dose were toxic (Figure 9. There were when compared with injections of saline (Figure 9. The effective dose of conjugate was not toxic treated with G5-FI-FA-MTX or G5-FA-MTX conjugate based on weight change and histopathology examination. and the changes of body weight demonstrated acute and chronic toxicity in the highest and in the second highest cumulative doses of free MTX equal to 33. both became lethal by days 32 to 36 of the trial (Figure 9). ). neither the total accumulated dose of therapeutic conjugate equivalent to 5.

Since the largest subunit acetic anhydride to the terminal. wanted to test the ability of this novel dendrimer platform to target to cancer cells over-expressing folate receptor by conjugating a targeting (FA) and an imaging (FITC) molecule to two different dendrimers. We expect the oligos to be susceptible to nuclease dimer formation and retro-Michael reaction in digestion. This allows us to place each component of the nanostructure on a different polymer and assemble them Figure 11. a star topology generation 3 dendrimers with specific functional groups. imaging and targeting agents. The acetylation reaction of the amine-termi.9. Each specific device must be produced individually. We conditions. which are then linked with complementary oligonucleotides (34 bp). is 11 nm and the center-to-center distance of the two functional nated dendrimer is very efficient and stoichiometrically dendrimers is estimated to be 20 nm.25. The degree of acetylation was the cluster could be controlled allowing for construction of measured by 1H NMR. units. Prediction of overall dimension of the The entire synthesis of the cluster agent consists of three target DNA-linked functional dendrimer cluster agent conjugation reactions and one hybridization reaction by computer modeling (Insight II®). This result is attached to each dendrimer (Figure 11). using the specific acetyl proton peak combinatorial libraries of dendrimer-linked anticancer at 1. the fragments would be eliminated the dendrimer in the presence of a triethylamine base through the kidney.752 g/mol. an alternative approach uses a structure to assemble dendritic polymer components into a single molecule. The yield of this reaction was Development of a multifunctional nanostructure that does 85% by weight (782 mg) and the calculated molecular not require all of the synthetic chemistry involved to be weight of the final product was 28. Finally. resulted in partial conversion of the primary amine groups mentary oligonucleotides. using different sets of comple. is clustering dendrimers with different compared to the theoretical value of 128) by potentiometric functions using complementary oligonucleotides that are titration and SEC-MALLS measurement.24 To determine the schematically shown on the periphery of the dendrimer. There are a number of cluster agent. While we clearly have been able to produce this type of agent (as described in the description above). The DNA linker (34bp) (Scheme 1). Hematology 2009 715 . The proto- type DNA-assembled nanocluster of the FA-conjugated dendrimer and FITC-conjugated dendrimer would then be characterized and evaluated in vitro to test tumor cell– specific binding and internalization. the number of the terminal primary amine groups The second enabling technology. which we developed over on the dendrimers was first determined to be 110 (as the past few years. intramolecular cyclization. as the clusters are self-assembling problems in the divergent synthesis of the dendrimer without additional chemical alterations that could harm the causing: missing repeating unit. supramolecular assembly. The increasing intensity of this signal indicated therapeutics. Figure 12. Schematic diagram of an oligonucleotide as a single. hybridization and the stability of the duplex at physiologic nucleotides in a dumbell configuration (Figure 12).9 ppm. generation 5 dendrimer. primary amine groups of will be a G5 (25 kDa). This approach consistent with other findings explaining the inherent allows greater flexibility than using a single dendrimer with structural defects from incomplete reactions or other multiple functions. performed on a single molecule is attractive for several reasons. We initiated our studies with the simplest conjugation Oligonucleotides are designed to assure the specificity of the strategy: two dendrimers linked via complimenary oligo. the ratios of different subunits in to acetamide moieties. Different units are then combined in physiologic conditions and aggregate into a device.26 The addition of 82 molar equivalents of after it is delivered to the cells. Oligonucleotides are covalently conjugated to possible architectures for such an assembly. Chemical modifications of a single polymer required for coupling different components can damage other components already on the device. Complementary oligonucleotides are coupled to a core is shown in Figure 11. FITC and FA are controllable as reported by Majoros et al. that an average of 80 acetyl groups were added to each dendrimer (G5-Ac80-(NH2)30).Prior Work on Complementary sub-stoichiometric equivalent of acetic anhydride to the Oligonucleotide Linkages by M-NIMBS dendrimer. which would allow the breakdown of the device dendrimers.

We employed phosphoramidate chemistry developed by This was consistent with 1H NMR spectroscopy evalua. the number of FA molecules was calculated to be 2 ± 0. has been studied extensively. three dendrimers. From the calibration curve of shown in Figure 13.5 M LiCl. which is close to that expected in a dimeric dendrimer cluster.0) consistent with the ones expected for individual DNA- in 0. charge acetylated dendrimer was conducted in the presence of interaction must be prevented to avoid insoluble complex water-soluble carbodiimide (1-ethy-3-(3-dimethylamino. The distances between the linked FITC molecules per G5 dendrimer was determined by UV particles are consistently approximately 20 nm. However.6.72). The A at 502 nm shows a linear good agreement with the theoretical length of the hybrid- relationship with the conjugate concentration. dumbbell-shaped clusters whose dimensions are DMF:DMSO (3:1. then cooled to room 80 nm3) of the imaged objects suggests assemblies of two or temperature over 3 h.4.27-29 Recently. dendrimer to covalently attach a synthetic oligonucleotide DNA to the dendrimer surface. From the integral ratio of aromatic protons in FA molecules (8. Given the potential for The condensation reaction between the active ester of folic interaction of the positively charged amine-terminated acid and the remaining primary amines on the partially dendrimer and the negatively charged DNA. the partial capping of the surface primary propyl) carbodiimide. 16h. The UV spectrum of the amine groups with acetamide groups was also necessary to conjugate in water at a concentration of 2 nM showed a minimize electrostatic interactions at physiological pH. such as proteins and metallic nanoparticles. 6. v/v). In 716 American Society of Hematology . 7. maximum peak at 280 nm and a broad shoulder at 370 nm.7 ppm) to the signal at 1.1M EDC/0. (4) 0. there are a number of (1) triethylamine.31 Figure 13 shows a representative example of AFM images of the DNA-linked dendrimer nanoclusters adsorbed to a mica substrate in dry state. The approximate number of conjugated assembled into a cluster. (5) 10 mM phosphate buffer (pH 7. which showed that FITC convolution effects in the volume measurement. From the calibration curve of the free FA.33 Thus. covalent attachment of oligonucleotide to either the core or the periphery of the dendron of various dendrimers has been reported. and/or dendrimer clusters. extinction ized cON50-sON50 oligonucleotides as schematically coefficient = 246600 M-1cm-1. Synthetic DNA has been used as a tool to self-assemble nanoscale objects27-29 and the attachment of DNA oligonucleotides to organic or inorganic polymers. Measuring the average volume (286 ± NaCl.0. MeOH. Chu et al32 between the 5′ phosphate group of single- tion using the sum of the integrals of the aromatic stranded DNA and the terminal amine group of the PAMAM protons of FITC. (3) EDC in smaller. 150 mM linked dendrimers. Some of the larger objects Scheme I. EDC).4). annealed at 90°C for 10 min.8 ppm.1 M Imidazole (pH 6. the free dye (data not shown). which corresponds to the methyl protons in the acetamide groups on the surface. Note that the very low aspect FITC was qualitatively documented to be conjugated to the ratio of these topographical features helps to minimize tip dendrimer by TLC analysis.5 molecules of FA residue per dendrimer. the average number of FA moiety in the conjugate was calculated to be 2. where SH- modified oligonucleotides or SH-core dendrons are coupled to a linker maleimide group or oligonucleotide to produce the conjugates.30. formation. the particles have larger volumes indicating that more than while non-conjugated free FITC migrated at the solvent two dendrimers with a few oligonucleotides attached have front (Rf = 0. (2) DMSO. when five molar equivalents of FA to the dendrimer were used in the conjugation reaction.26 Some of conjugated to dendrimer was being retained at the origin. the average number of FITC molecules per dendrimer was calculated to be 5. which is in and 1H NMR spectroscopy. which agrees with the UV result. 8. Synthetic scheme for the preparation of a appeared to be aggregates of many individual dendrimers DNA-linked cluster of G5-FITC and G5-FA dendrimers.6.

uterus.20 increase of cell-associated fluorescence after incubation with This binding capacity of the cluster allowed for a 200% increasing concentrations of DNA-linked G5-FITC and G5-FA increase in the mean fluorescence measured at 500 nm after dendrimer cluster. on average. Since the affinity of folate conjugates for the cell surface folate recep- tors is high (KD~ 10-10M). (b) a three-dimensional image of one single separation method allows for separation of the target dimeric cluster arrowed in (a). The intra-dendrimer distance was theoretically calculated to be 20 nm and observed to be 21 ± 2 nm dendrimer components. and (c) a line scan nanostructure from the unlinked globular FITC-conjugated analysis of the cluster according to the white line in or FA-conjugated dendrimers or unhybridized DNA- (a). Thus. the use of molar excesses of either dendrimer or DNA in conjunction with salt (LiCl) prevented complex formation. including cancers of the ovary. we used a five molar excess of dendrimer to DNA so that the number of DNA conjugated to the dendrimer would be limited to approximately a single strand per dendrimer. especially when grown in low folic acid medium. which retains the desired DNA-linked functional dendrimer nanocluster. the folic acid modification of dendrimers allows the specific delivery of diagnostic and therapeutic agents to cancer cells. Binding of DNA-linked functional occurred at 40 nM. FA is known to be internalized into cells through a high affinity. Since the cluster is Figure 13. In addition. As we have reported earlier1 and suggested by DeMattei et al. brain. which is comparable to the binding dendrimers to KB cells after 1 h incubation. testis. addition. The sigmoidal profile of the dose-dependent curve suggests that the uptake of the dendrimer cluster is saturable.36-39 The KB cells we employed for this study are a human epidermoid carcinoma type cell that over-expresses folate receptors.34 minimizing non-specific interaction of DNA and dendrimer and limiting the number of DNA on each dendrimer are crucial to constructing dendrimer components into a nanostructure with defined size and shape. colon. receptor-mediated process. indicating the binding of the cluster to the KB cells over-expressing folate receptors with an apparent affinity of at least 15 nM to achieve 50% of maximal binding. (a) A representative image of DNA-linked assumed to have a dumbbell-like structure with a theoreti- G7-G5 dendrimer nanocluster on mica (500 nm × 500 cal molecular weight of approximately 86 kDa. (a) Gradual capacity (200 nM) of the single dendrimer nanodevices. nM. increases in cell fluorescence from the Hematology 2009 717 . In this study. (b) The concentration-dependent saturation curve indicates specific binding of the DNA-linked cluster to KB incubation for 1 hour at 37°C with a concentration of 40 cells over-expressing folate receptor. lung and myelocytic blood cells. any un-reacted dendrimer was removed after the hybridization reaction of the DNA- conjugates by centrifugal filtration using a 100 kDa MWCO membrane. dendrimer folic acid conjugates linked to either a single drug molecule or assem- bly of molecules can bind to and enter receptor-expressing tumor cells via folate-mediated endocytosis. kidney. The saturation of the binding curve Figure 14.40 The binding of the cluster nanodevice to KB cells showed concentration-dependent uptake with saturation occurring at around 40 nM (Figure 14A). In this way. this nm).35 The high affinity receptor for folic acid is over-expressed on a number of human tumors.20.

5. 2001. There is also hope that We also used several control samples to show that the cell. Correspondence James R. Bilbao R. Baker JR. 2005. MI 48109–5648. analysis documented the presence of the cell-associated fluorescence of the DNA-linked dendrimer cluster both in Summary the periphery of the cell membrane and at the cytoplasm of In summary. These control experiments document that tion of the DNA-linked cluster agent (80 nM) as well as non-specific associations of the DNA-conjugated functional autofluorescence of the control cells in the measured dendrimers with the cell surface is not the cause of the cell emission wavelength (515 nm). of the cells. 2004. suggesting that the clusters had been internalized and oligonucleotide linked dendrimers appear to be able to across the membrane into the cell. delivering drug by this approach could overcome drug associated FITC fluorescence originated from the DNA. Michigan Nanotechnology Institute for Medicine and Biological Sciences. with controls (G5-FITC-cON34 and the mixture Res. The mixture of the DNA-conjugated dendrimer. Baker JR. (b) The cluster failed specific targeting. Cancer Res.60:4440-4445. Synthesis and functional evaluation of DNA-assembled Figure 15. Division of Allergy and Clinical Immunology. Kong G.4:391-397. Hyperthermia did not occur in vitro without in situ DNA hybridization enables tumor-specific nanoparticle delivery: effect of reaction. The DNA-conjugated G5-FITC dendrimer 15) was further confirmed in confocal microscopic images (G5-FITC-cON34) alone did not show significant binding. 3: the effect of hyperthermia on nanoparticle extravasa- mixture of G5-FA-sON34 and G5-FITC-cON34 with no tion from tumor vasculature. Dewhirst MW. and JTM Research. Holl MMB. the confocal binding and uptake we observed. of the DNA-dendrimer conjugates.binding of the cluster nanodevice were effectively blocked dendrimers (without regard to the presence of a duplex DNA by the addition of free FA in the medium. Cancer free FA. Dewhirst MW. Kong G. 2000. Ann Arbor. DNA-directed synthesis of generation 7 and 5 PAMAM dendrimer nanoclusters. et al. Baker.61:3027-3032. Alzuguren P. Braun RD. to bind to the folate receptor down-regulated KB cells 3. suggesting a clustering of dendrimer-DNA conjugates 4. LLC. (a) Competitive inhibition of the cluster polyamidoamine dendrimer clusters for cancer cell- agent (40 nM) by free FA (5 mM). e-mail: jbakerjr@umich. 1150 West Medical Center Drive. Hyperthermia enables tumor-specific intensity of the cluster and the cluster antagonized by nanoparticle delivery: effect of particle size. MD. Islam MT. annealing process) showed no non-specific binding. Off-label drug use: None disclosed. Mecke A. Thomas T. improve the therapeutic index of cytotoxic drugs by direct delivery of the drugs to cancer cells. Further toxicological studies and GMP synthesis of this material are underway to allow the initiation of clinical trials. 9220 MSRB III. which did not undergo the annealing process. Choi YS. Disclosures Conflict-of-interest disclosure: The author is a consultant and major equity holder in NanoBio. Fax: 734-936-2990. nanoparticle therapeutics based on dendrimers the cell. is not significant. Braun RD. Avidimer Therapeu- tics. bound poorly The binding observed in the flow cytometry studies (Figure to the KB cells. Orr BG.12:35-43. Bustos M. A significant difference of fluorescence from suggesting the non-specific binding of the G5-FITC-cON34 the PBS control was not obtained due to the low concentra. References 1. (d) Comparison of the mean fluorescence particle size. A blood-tumor 718 American Society of Hematology . Nano Lett. Phone: 734-647-2777. Kotlyar A. resistance in tumor cells via bypassing p-glycoprotein linked FITC-dendrimer and FA-dendrimer cluster rather pumps that would normally export drugs that must diffuse than a non-specific aggregate of the two functional into cells. Characterization of (FAR-) (c) Control groups (2: G5-FITC-cON34. Chem Biol. linkage). Choi YS. However.

Rosenberg SA.10:180. Leamon CP. Wang S. Chu BCF.39:1579-1585. Control Rel. 32. Elghanian R. Mathias CJ. Subbi J. Metal. Anal Chem. Zera RT. Synthesis and characterization of saturated shell 18. Wiener EC. Soloway AH. 33. Pfeiffer CM. Piehler L. 1983. 1994. McLean ME. Evaluation Structural deviations in poly(amidoamine) dendrimers: of the effects of intravascular MR contrast media a MALDI-TOF MS analysis. Low PS. Tomalia DA. Low PS.6:305.8:1165-1179. Nano Lett. 15. proteins. 14. 7. Belz S. Li J. B. Barth RF. Alivisatos P. Hedstrand DM. Clinical applications of gene therapy for Med.31:1. J. 1991. 27. Mirkin CA. The immunotherapy and gene therapy Solid-phase extraction-electrospray ionization mass of cancer. et al. Magnin RL. Uppuluri S. 1996. Bull T. Culver KW. Proc Natl Acad Sci U S A. living cells: a method that exploits folate receptor 1993. 35. Margerun L. Adv Mater. Lu YJ. bio-bar codes for the ultrasensitive detection of 13. Science. 2004. Barth RF.88:5572- 20. Horn T. 1994. Zhou 1996. Lopp M. et al. et al. Curr Top Med Chem. immunoconjugates. Macromol- to macroscopic matter. 25. Synthesis and chelate-dendrimer-antibody constructs for use in characterization of covalently linked singe-stranded radioimmunotherapy and imaging. 1996. resonance angiography of the body. 2003. 22. erythrocytes. Lin SH. Hagnauer 17. Anal Biochem. Molecular weight determination of a 11. spectrometry for the quantification of folate in human 8. Bioconj Lett.23:105. Bioorg Med Chem DNA oligonucleotide-dendrom conjugates.12:796-800. 2003. Rapid Comm Mass StarburstTM dendrimers.39:33-42. Kukowska-Latallo J. Candido K. J Mag Res Imag. Magn Reson Med. Tomalia DA. Turek JJ. Delivery of 1997. 4. 24. Baker JR Jr. Science. Starburst 1998.7:1824. optical properties of gold nanoparticles. Design and 5576. Dendrimer development. endocytosis. Low PS. Alam F. Nelson CP. Goddard WA. Bhalgat MK. Selective colorimetric detection of system for neutron capture therapy. 1990. GL. (gadolinium dendrimer) on 3D time of flight magnetic 2003. dendrimers: molecular-level control of size. Darby MV. plasma or serum. Quintana A. 2005. Naylor AM. the effect of vasoactive compounds. Drug Pharmaceutical Res. Bell SA. topology. Williams CR.277:1078-1081. ecules.4:771-777. coupled with a microbiological assay. Delivery of macromolecules into 19. Kopelman R. 1994. Shortreed MR. DeMattei CR. 2002. Thaxton CS. Raczka E. Green MA.65:5317.40:510. boron-10 for neutron capture therapy by means of 30. 1991. Tomalia DA. Science.36:5526-5529. Cancer Res. J Nucl 6. Adv. Barker SL.40:1845. et al. Wu C. Folate-mediated delivery of macromo- targeted to tumor cells through the folate receptor. 2002.54:675-693. Duncan R. Mucic RC. Nau H. 2004. Tomalia DA. Woehler S. Piehler LT.5:383-386. Majoros IJ. function of a dendrimer-based therapeutic nanodevice 36. Determination of folate patterns in therapeutic response in animal model of human mouse plasma. Swanson DR. Eur Polymer J.261:534. J Biomedl Mat Res. 1998. Utilization of models. Acetyla- surface chemistry. Soloway AH. Dendrimer. Boronated starburst dendrimer-monoclonal antibody 29.265:157-166. 2008. Spectrom. et al. Majoros IK. Chem. Waters DJ. Urdea MS. Alam F. 10. Wahl GM. Clin Chem. The use of nanocrystals in biological 12. Peterson J. Nelson BC.14:488-493. Clin Chem. 31. Bioact Mater. Oh SK. 26. StarburstTM detection. dendrimers: enhanced performance and flexibility for 28. polyamidoamine starburst polymer by electrospray cal evaluation of polyamidoamine (PAMAM) ionization mass spectrometry. nance imaging contrast agents. cancer. Hematology 2009 719 . Preliminary biologi. J Clin Oncol. shape. Cao ZY. Bioconj Chem. 1992.19:1310-1316. Adams DM. 22:47-52.11:6513-6529. 2000. Margolis SA. Hylton N. Plenum Press: New York. Anal Biochem. 16. Huang BH. Nam JM.301:1884-1886. Kozak RW. Baker JR Jr. Brechbiel MW. Roberts JC. Core-shell tecto(dendrimers): 1. 2003. et al.29:138-175. Current monoclonal antibody-Starburst dendrimer dendrimer application in cancer diagnosis and therapy. and flexibility from atoms tion of poly(amidoamine) dendrimers. Angew Chem Int Ed Engl.5:58. Gene Therapy. 9. folate-receptor-targeted radiopharmaceutical. lecular anticancer therapeutic agents. 449. immunoconjugates: evaluation as a potential delivery Mirkin CA. Storhoff JJ. 1994. Nat Biotechnol. Derivatization of based metal chelates: a new class of magnetic reso. Bourne MW. Allikmaa V. Nanoparticle-based immunoassays. 1992. Orgel LE. 2004. lipophilic ionic additivies in liquid polymer film 34. Delivery Rev. Adams DM. Letsinger RL. Gansow OA. Kallos GJ. barrier limits gene transfer to experimental liver cancer: 21. 1997. Pehk T. Indium-111-DTPA-folate as a potential 2000. Gansow OA.30:53. Malik N. Nanoparticle targeting of anticancer drug improves 23. Singh P. polynucleotides based on the distance-dependent 1994. optodes for selective anion activity measurements.325:41-51. Moll F III. Nucleic Acids Res. unprotected polynucleotides. and embryos by HPLC epithelial cancer. Lewis S.69:990.