DENDRIMER NANOCOMPOSITES IN MEDICINE

L. Balogh,V A. Bielinska, V J. D. Eichman, V R. Valluzzi, I. Lee, V J. R. Baker, V T. S. Lawrence and M. K. Khan

V

The University of Michigan Center for Biologic Nanotechnology, Ann Arbor, MI 48109-0533 Tufts Biotechnology Center Department of Chemical Technology, Medford, MA 02155 Department of Radiation Oncology, University of Michigan, Ann Arbor, MI 48109

INTRODUCTION Dendrimer based nanocomposites1-5 are novel organic-inorganic hybrid nanoparticles synthesized by dispersing very small inorganic domains in nanoscopic size polymeric networks. Due to the molecular level mixing of their components they display unique material properties in addition to properties represented by their individual components. Dendrimers are often used as templates in making these particles because they can be made uniform, and using these well-controlled and uniform templates leads to well controlled and uniform composite particles. Apart from obvious materials science applications, such as catalysis and photonic materials, dendrimer nanocomposites (DNC) have a great potential in biomedical applications due to their controlled composition, predetermined size, shape and optional surface functionalities. They may be used as drug delivery vehicles to deliver bioactive guests such as silver or for an incorporated radioactive isotope. Radioactive dendrimer nanocomposites can be delivered directly to the tumor by injecting nanoparticle solutions directly into the tumor microvasculature.6 If a short-lived isotope is used, a procedure like that may serve as an alternate method to direct irradiation. It has also been envisioned that if the surface of such a nanoparticle had been equipped by appropriate targeting moieties the nanoscopic device could find its way selectively to cancerous cells anywhere in the body. In this paper, gold and silver poly(amidoamine) (PAMAM) dendrimer nanocomposites, {Au(0)n PAMAM} and {Ag(0)n -PAMAM} have been selected for demonstration of concept.. {Au(0)n PAMAM} gold dendrimer nanocomposites with a well-defined size were synthesized and imaged by transmission electron microscopy (TEM) both in vitro and in vivo. Silver/PAMAM complexes and different {Ag(0)n -PAMAM} dendrimer nanocomposites have also been tested against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. Both the [(Ag+)n PAMAM] silver complexes and the {Ag(0)n -PAMAM} nanocomposites displayed considerable antimicrobial activity without the loss of solubility and activity even in the presence of sulfate or chloride ions. Dendrimers. The term dendrimer refers only to an architectural motif and not a particular compound. To date greater than 160 various polymers with dendritic structures are reported in the literature. This term is used both for dendritic (macro)molecules and materials. Dendrimer molecules are monodisperse symmetric macromolecules built around a small molecule or a linear polymer core7-10 using connectors and branching units. Interaction of dendrimer macromolecules with the molecular environment is dominantly controlled by their terminal groups. By modifying

Corresponding author: Lajos Balogh, The University of Michigan Center for Biologic Nanotechnology, 4010 Kresge Research Building II, 200 Zina Pitcher Place, Ann Arbor, MI 481090533. Phone: (734) 615-0623, Fax: (734) 615-0621, baloghl@umich.edu

their termini, the interior of a dendrimer may be made hydrophilic while its exterior surface is hydrophobic, or vice versa. Due to synthesis limitations, dendrimer materials always contain imperfect molecules that are more similar to hyperbranched structures. There are other parameters to be taken into account: poly(amidoamine) (PAMAM) dendrimers,11 for instance, undergo all kinds of changes in size, shape and flexibility (from being a small branching molecule at generation zero to behaving as a hard sphere at generation seven) as a function of increasing generations.12-15 Examples of using PAMAM dendrimers in medicine Imaging. PAMAM dendrimers have been used for imaging but only after appropriate surface modification. Fluorescent markers, such as fluorescein, or chelated metal ions, such as Gd3+ for MRI are used to generate a measurable signal for imaging,17-19 Radiolabeled compounds, (3-H, 14C, 90-Y, 125-I, 153-Gd and 212-Bi) were also used in biodistribution studies.20 Various metal chelators were coupled to PAMAM dendrimers as well as to monoclonal antibodies without loss of protein immunoreactivity. Nonetheless, conjugation of markers to the surface of the macromolecule may dramatically change the solubility and other surface-related properties. Gene transfer. It has been reported that PAMAM dendrimers can be effective carriers for the introduction of various types of nucleic acids e.g. the expression plasmids, single stranded DNA oligonucleotides or RNA. That particular function of PAMAM dendrimers relies on their ability to bind all forms of charged nucleic acids on the basis of electrostatic interactions. The resulting dendrimer/DNA complexes enhance DNA transfer, protect against intra- and extra-cellular degradation and results in the transgene expression in a variety of mammalian cell lines in vitro as well in variety of animal tissue in vivo.21-24Formation of DNA-dendrimer complexes seems to be based entirely on charge interaction between negatively charged phosphate groups of the nucleic acid and protonated (positively charged) amino groups of the PAMAM polymers.24-25, Highly efficient transfection of a broad range of eukaryotic cells and cell lines was achieved with minimal cytotoxicity using these DNA/dendrimer complexes. In the majority of cells the transfection can be also enhanced with the addition of chloroquine. This indicating involvement of endosomes in trafficking of the complexes. To clarify the structure of DNA-PAMAM complex interactions of nitroxide-labeled poly(amidoamine) dendrimers (EDA core, amine terminated, generation 2 and 6, i.e., PAMAM_E2.NH2 and PAMAM_E6.NH2 ) to various DNA molecules have also been investigated by ESR. 26,27 The results show that although the structure of the complex depends on both the PAMAM generation and the length of the DNA strand, there is predominantly one binding mode between DNA and PAMAM dendrimers. The structure or the stoichiometric complex and various aggregates changes not only with surface charge differential but is dependent on the concentration of both components.25 The simplest structural model proposes that DNA binding with dendrimers can be divided into tightly bound DNA regions and linker DNA regions. The binding between DNA and dendrimers also effected by ionic strength and pH, which is consistent with the importance of the electrostatic interactions in the DNA-dendrimer binding. Targeting and drug delivery. Targeting of tumor cells may be performed by either non-specific or specific ways as it is described in the related literature. Receptor specific targeting and optimization of surface properties are also possible. Targeting groups can be conjugated to the host dendrimers surface29 to allow the imaging agent to bind selectively to specific sites such as receptors on tumor cells to improve detection. For example, there are a number of cell surface receptors that are known to be overexpressed on prostatic cancer cells, including epidermal growth factor receptor (EGFR) and the laminin receptor VLA-6. 30 For a non-specific targeting, it has been shownthat appropriately sized dendrimers and dendrimer-based metal complexes (e.g., cisplatinum) become trapped in tumor vasculature due to the enhanced permeability and retention (EPR) effect.31 2

Composites and nanocomposites. Composites are physical mixtures or two or more components which display improved properties in one or more areas as compared to their individual bulk constituents. They are widely used in many diverse industries and form the basics of engineering plastics, structural adhesives and matrices. Nanocomposites can be defined as having at least one component with at least one of their dimension less than 100 nm. Thus, nanocomposite properties will be strongly influenced by extensive interfacial interactions between the components and the rule of mixtures fails to provide estimates of nanocomposite properties. RESULTS AND DISCUSSION Dendrimer nanocomposites. According to the general concept of reactive encapsulation, dendrimer nanocomposites,1 , 2 are made by preorganizing small precursors by an appropriately selected dendrimer. This pre-organization is followed by in-situ chemical reaction(s) or physical treatment (irradiation, etc.,) that generates reaction products immobilized in a polymeric network. Figure 1. This procedure yields dispersed small domains of guest molecules that are integrated with the dendrimer molecule(s) without creating covalent bonds between the dendrimer and the topologically entrapped matter. Details of experimental procedures and analytical techniques can be found in previous studies.4,38 Some properties of PAMAM nanocomposites used are summarized in Table I. Table I. Silver as an antimicrobial agent. Healing of burn wounds are of primary interest for military, firemen and people caught in accidents. The major reason of fatalities is the sepsis after the thermal injury. Infections are caused mostly by Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli.32 The topical treatment is Silver sulfadiazine (AgSD), which acts through AgCl formation.33,34 Using AgSD both silver sensitivity and sulfadiazine resistance may develop in patients. The search for alternative treatments includes the use of other antibiotics and different ways of providing silver. It has been also indicated that silver nylon dressings may be a valuable antimicrobial burn wound covering device.35 Silver nylon dressing of wounds was found protective either with or without an applied current.36 Despite major advances in burn wound management and other supportive care regimens, infection remains the leading cause of morbidity in the thermally injured patient, and search for different treatments and new ideas is continuing. Antimicrobial activity of silver nanocomposites Silver nanocomposite solutions have been prepared from various dendrimers and with different silver content in and have been tested against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli were collected using the standard agar overlay method by measuring the area of inhibition. Silver nanocomposites were stable and remained soluble in the test media. In dialysis experiments guest and host diffused together as if they were simple macromolecules. In the absence of dendrimers silver ions quickly precipitated in the form of insoluble silver salts when contacted with chloride and sulfate ion containing solutions. While all silver nitrate solutions displayed approximately the same activity, an increased antimicrobial activity was observed with dendrimer carboxylate salts (Figure 2). Figure 2 We attributed this effect to the very high local concentration of silver (256 carboxylate groups around a 54 diameter sphere that could retain many silver ions) in the nanoscopic size composite particles. These silver domains - in any form of Ag+, Ag(0), or any other compound, such as AgCl 3

or Ag2 SO 4 - are bound in the dendrimer network with a calculated specific surface area of several thousand m2 /g. The antimicrobial activity was smaller when internal silver complexes were applied, indicating, that the accessibility of the silver is an important factor. Both the silver-dendrimer complexes and the silver nanocomposites displayed antimicrobial activity comparable or better to those of silver nitrate solutions.37 Gold nanocomposites. Gold/PAMAM nanocomposites usually are fabricated by UVirradiation3 or by chemical reduction4.5 of tetrachloroaurate salts of PAMAM dendrimers. Based on the locality of the guest atoms three different types of single nanocomposite architectures have been identified, such as internal (I), external (E) and mixed (M) type nanocomposites.4 Structure of these nanocomposites was found to be the function of the host as well as the guest, and the chemistry involved.4 Imaging of {Au} gold nanocomposites in vitro and in vivo. For imaging, gold nanocomposites offer the high electron density of the immobilized gold atoms. Interaction of the nanoparticles with the biological environment can be controlled by changing the number and character of the surface groups on the dendrimer used. Gold nanocomposites are easy to observe in cells and in tissue because they are very much differ by contrast, size and shape. In imaging, gold nanocomposites offer the high electron density of the immobilized gold atoms, A few gold atoms per dendrimer already renders the visualization of the amorphous single and multiple nanocomposite particles possible by electron microscopy. Figure 3: Due to the synthetic procedure used {(Au(0)n }m clusters are almost always present in the solution of {(Au(0) n } nanocomposites.4 Of course, visibility of the larger clusters are much better than the single units, which often appear as small grayish round objects due to their low and homogenously dispersed gold content. This characteristic size, shape and contrast make the observation of gold nanocomposites simple by electron microscopy. Furthermore, no other objects can be found in tissues or cells that are similar in shape, size and contrast to the gold nanocomposite clusters. Due to the variable surface charge and adherence of the gold/PAMAM nanocomposites, it is possible to use gold nanocomposites as selective stains. DNCs can have a specific charge on their host polymer and therefore may be used to map certain cell compartments and cellular structures. Interacting cellular structures can be made visible this way, as charged and hydrophilic {Au} nanocomposites does not stain the hydrophobic epoxy resin of the specimen. In this demonstration, {Au(0)n -E5.NH2 } was used to stain macrophage cell monolayers which then were examined on a Philips CM100 electron microscope. In Figure 6 lipid bilayer structures are shown that were treated with single unit {Au(0)}+ gold nanocomposites. The positively charged {Au(0)}+ nanoparticles have entered the polar region of the bilayers and appear on the TEM image as individual dots, similar to the raster on computer screens. Measured thickness of one bilayer is 8 nm, in good agreement with commonly observed values (Figure 4). Figure 4. Transfection and visualization of intracellular trafficking of dendrimers Due to the metal content, the cellular uptake and trafficking of {Au} nanocomposites and their DNA complexes can be simultaneously observed using electron microscopy. We have used reporter plasmid DNA and positively charged gold {Au(0)n -PAMAM} nanocomposites to visualize the intracellular trafficking of DNA/PAMAM dendrimer complexes. Complexes of plasmid DNA (pCF1/LUC; 5.5 kb) and gold nanocomposites of {Au(0)10 PAMAM_E5.NH2 } were formed in double distilled H2 O at a 10:1 ({Au}:pDNA) charge ratios, which represent the typical condition of complex formation. Gene transfections were 4

performed by incubating the complexes with Cos-1 cells (monkey kidney fibroblasts; 1 x 105 cells/cm3 ) for three hours at 37C. Cells were washed and grown overnight (24 hrs, 37C) in growth medium supplemented with 10% FBS. Gold/PAMAM nanocomposites displayed gene transfection efficiency similar to their host dendrimer,40 The luciferase expression was found comparable to that in the control transfections performed by using regular PAMAM_E5.NH2 . This indicated that presence of metal component in the {Au} nanocomposite does not inhibit processes of transfer, subsequent transcription and translation of the introduced DNA. We have not observed any increase in toxicity due to the metal content of gold nanocomposites indicating that toxicity of these nanomaterials is determined by the dendrimer used. {Au}n clusters appear as 25-30 nm dark spots while the {Au}+ positively charged single gold/dendrimer nanocomposites stain the nanocoposite/pDNA complex gray. Initially these complexes were found attached to the cell surface (A). Then, they were subsequently endocytosed and they entered lysosomal compartment of cells. Figure 5 illustrates these three steps. Interestingly, the original distribution of the highly visible {Au}n clusters is clearly random in the pDNA-{Au} complexes. However, images taken at a later time indicate the movement of the dark nanoparticles toward the internal surface of the lysozomal vesicles cell indicating flow characteristic of a living cell (Figure 5). Figure 5: Visualization of gold/PAMAM nanocomposite uptake in tumor cells Substantial literature demonstrates that tumor vasculature is characterized by increased permeability and the tendency to express unique proteins suitable for targeting Known differences in permeability between tumor and normal vasculature can be exploited to produce selective deposition of isotope containing nanoparticles in tumor. In this demonstrative experiment amine surface gold-PAMAM nanocomposites were used to visualize {Au(0)}+ in tumor tissue by TEM. One million B16F10 melanoma cells were injected subcutaneously and then grown on the dorsal surface of a C57Bl6/J mouse for two weeks then injected with nanocomposite solution . The tumor and other organs (liver and lung tissue) were isolated Ultrathin sections were examined on a Philips CM100 electron microscope. Figure 6 In Figure 6, a TEM image of one of these sections with three cells is shown. The image indicates that 1.25 hours after the direct injection only a few larger gold nanocomposite clusters are present in the interstitium. Most of the smaller clusters were found in the nucleus and cytoplasm. During the internalization of gold nanocomposites the diffusion through the cell wall appears to be the dominant mechanism, although transfer through a nuclear pore has also been observed. SUMMARY Monodisperse dendrimer nanocomposites can be synthesized in various predetermined sizes and their interaction with biological objects may be adjusted by modifying their surface properties. They readily penetrate into cells and are easy to observe by transmission electron microscopy and optical methods. Gold/PAMAM DNCs can be used for imaging by electron microscopy under both in vitro and in vivo experimental conditions. Based on these observations we could identify the following steps in this gene transfection process: first, large clusters containing many DNA molecules and many dendrimers form. These complexes bind to the cell surface then undergo endocytosis In case of lipid bilayers, positively charged gold/dendrimer single nanocomposite units easily penetrated into the polar zone of the bilayer structure. Using radioactive gold as a guest, the 5

nanoscopic agent can be directly injected into the tumor microvasculature where the positively charged DNCs rapidly accumulated in the nucleus of the tumor cell. The diffusion of various dendrimer nanocomposite is controlled by the polymer host. Antimicrobial silver nanocomposites delivered the same active component than silver nitrate or silver sulfadiazine, but the polymer host prevented the aggregation and subsequent precipitation of silver. These silver nanocomposites were able to prevent bacteremia of burn wounds. It was found that the silver immobilized in the nanocomposite was protected against deactivation by chloride or sulfate ions and by light. Acknowledgments These projects have been funded with Federal funds in part from the U.S. Department of Energy, under Award No. FG01-00NE22943, in part from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-97111 and in part by the Defense Advanced Research Projects Agency, award MDA972-97-1-0007, entitled "Nanomolecule Based Agents for Pathogen Counter Measure. Antimicrobial silver nanocomposite research was sponsored by the Army Research Laboratory under the contract of DAAL-01-1996-02-0044. LB thanks to D. Sorenson and C. Edwards of the Microscope and Imaging Laboratory at the University of Michigan for their technical support. REFERENCES 1. L. Balogh, D. R. Swanson, R. Spindler and D. A. Tomalia: Formation and Characterization of Dendrimer-Based Water Soluble Inorganic Nanocomposites; Proceedings of ACS PMSE, 1997 (77), 118. 2. Nora Beck Tan, L. Balogh and S. Trevino: Structure of Metallo-Organic Nanocomposites Produced from Dendrimer Complexes; Proceedings of ACS PMSE, 1997 (77), 120. 3. Esumi, K., Suzuki, A., Aihara, N., Usui, K., Torigoe, K., Langmuir 1998, 14(12), 3157. 4. Balogh, L., Valluzzi, R., Laverdure, K. S., Gido, S. P.; Hagnauer, G. L., Tomalia, D. A., J. Nanopart. Res. 1999, 1(3), 353. 5. Garcia, M. E., Baker, L. A., Crooks, R. M., Anal. Chem., 1999, 71, 256. 6. Bielinska, J. D. Eichman, I. Lee, J. R. Baker, Jr., and L. Balogh, (submitted to J. of Nanoparticle Res.) 7. Tomalia, D. A.; Dewald, J. R.; Hall, M. J.; Martin, S. J.; Smith, P. B. First SPSJ Int. Polym. Conference, Kyoto, Japan, August 1984, 65 8. Tomalia, D.A.; Baker, H.; Dewald, J.; Hall, M.; Kallos, M.; Martin, S.; Roeck, J.; Ryder,J.; Smith, P. Polym. J. (Tokyo) 1985, 17, 117-132. 9. Newkome, G. R.; Yao, Z-Q; Baker, G. R.; Gupta, V. K., J. Org. Chem. 1985, 50, 2003. 10. Hawker, C. J.; Frechet, J. M. J., J. Am. Chem. Soc. 1990, 112, 7638. 11. Tomalia, D. A.; Dvornic, P., Polymeric Materials Encyclopedia, Ed.: J. C. Salamone, 1996, Volume 3, D-E, CRC Press, 1814. 12. Tomalia, D. A., Naylor, A. M.; Goddard, W. A. III, Angew. Chem. Int. Ed. Engl. 1990, 29, 138. 13. de Gennes, P. G.; Hervet, H., J. de Physique Lett. (Paris) 1983, 44, 9, 351. 14. Mansfield, M. L.; Klushin, L. I., Macromolecules 1993, 26, 4262. 6

15. Bhalgat, Mahesh K.; Roberts, Jeanette C., Eur. Polym. J. 2000, 36(3), 647-651. 16. He, J.-A., Valluzzi, R., Yang, K., Dolukhanyan, T., Sung, C. M., Kumar, J., Tripathy, S. K., Samuelson, L., Balogh, L., Tomalia, D. A., Chem. Mater. 1999, 11, 3268. 17. Wu, C.; Brechbiel, M. W.; Kozak, R. W.; Gansow, O. A. Bioorganic & Medicinal Chemistry Letters, 1994, 4(3), 449. 18. Margerum, L. D.; Campion, B. K.; Koo, M.; Shargill, N.; Lai, J.; Marumoto, A.; Sontum, P. C. J. Alloys Compd. 1997, 249 (1-2), 185. 19. Kim, Yoonkyung; Zimmerman, Steven C. Curr. Opin. Chem. Biol. 1998, 2(6), 733-742 20. H. Kobayashi, N. Sato, S. Kawamoto, T. Saga, A. Hiraga, T. L. Haque, T. Ishimori, J. Konishi, K.Togashi, M. W. Brechbiel, Bioconjugate Chem. 2001, 12, 100-107 21. Bielinska, A.; Kukowska-Latallo, J. F.; Johnson, J.; Tomalia, D. A.; Baker, J. R., Jr. Nucleic Acids Res., 1996, 24, 2176. 22. Kukowska-Latallo, J. F.; Bielinska, A. U.; Johnson, J.; Spindler, R.;Tomalia, D. A.; Baker, J. R., Jr. Proc. Natl. Acad. Sci. 1996, 93, 4897. 23. Qin, L.; Pahud, D. R.; Ding, Y.; Bielinska, A. U.; Kukowska-Latallo, J. F.; Baker, J. R., Jr.; Bromberg, J. S. Hum. Gene Ther. 1998, 9(4), 553-560 24. Bielinska, A. U.; Kukowska-Latallo, J. F.; Baker, J. R., Jr. Biochim. Biophys. Acta 1997, 1353, 2, 180. 25. Bielinska, Anna U.; Chen, Chunling; Johnson, Jennifer; Baker, James R., Jr. Bioconjugate Chem. 1999, 10, 843-850 26. Chen, W., Turro, N..J., Tomalia, D.A., Langmuir, 2000, 16,15. 27. D. S. Wilbur at al., Bioconjugate Chem., 1998, 9, 813-82 5 28. Bielinska, Anna U.; Chen, Chunling; Johnson, Jennifer; Baker, James R., Jr. Bioconjugate Chem. 1999, 10, 843-850 29. Bonkhoff, H. et al., Hum. Pathol. 1993, 24, 243-8). 30. Duncan, R. Malik, N., Richardson, S., Ferruti, P., Polym. Prepr., Am. Chem. Soc. Div. Polym. Chem. 1998, 39, 180 31. Muggia, FM., Clin. Cancer Res., 1999, 5, 7. 32. Mozingo, D.W., McManus, A.T., Kim, S.H., Pruitt, B. A. Jr., J. Trauma. 1997, 42(6) 1006 33. Fuller, F.W., Parrish, M., Nance, F.C., J. Burn. Care Rehabil., 1994, 15(3), 213 34. Harrison, N. H., Arch. Surg., 1979, 114(3), 281 35. Chu, C.S., McManus, A.T., Pruitt, B.A. Jr., Mason, A.D. Jr., J Trauma 1988, 28(10), 1488 36. Greenfield, E., McManus, A.T., Nurs. Clin. North. Am., 1997, 32(2), 297 37. L. Balogh, D. R. Swanson, D. A. Tomalia, G. L. Hagnauer, A. T. McManus, Nano Letters, 2001, 1(1) 18-21 38. Jackson, Catheryn L.; Chanzy, Henri D.; Booy, Frank P.; Drake, Bartholomew J.; Tomalia, Donald A.; Bauer, Barry J.; Amis, Eric J., Macromolecules 1998, 31, 6259-6265.

APPENDIX Nomenclature. To describe the complex structure of these nanoscopic complexes and nanocomposites we use the following convention to describe the composition of the synthesized nanomaterials: brackets denote complexes as it is common while braces represent nanocomposite structures. Within the brackets or braces, first the complexed/encapsulated components are listed (with an index of their average number per dendrimer molecule) then the family of dendrimers followed by terms used for dendrimer identification, i.e., core, generation and surface. Naming of a general dendrimer nanocomposite then would follow this pattern: {(component#1)i(component#2)j(component#3)k -FAMILY_CoreGeneration.Terminal group}. Thus, the formula of {(Ag0 )14 -PAMAM_E4.A} denotes a silver dendrimer nanocomposite in which a generation four amine terminated ethylenediamine (EDA or E for short) core poly(amidoamine) (PAMAM) dendrimer contains fourteen zerovalent silver atoms and [(Cu2+ )7 {(Ag+)7 PAMAM_E4.T}] denotes the copper complex of an EDA core generation four TRIS surface PAMAM containing seven Cu(II) ions and seven silver ions per dendrimer. The above scheme provides a relatively simple but consistent way to identify materials with a complicated structure. Here, as only PAMAM dendrimers were used, we usually omitted naming the dendrimer. This way, {(Au(0))10 -PAMAM_E5.NH2 } and {(Au(0))10 -E5.NH2 } are considered to be equivalent names.

Table I. Properties of some PAMAM Dendrimer Nanocomposites No. of surface groups {(Au(0)10.82 -PAMAM_E5.CH2 CH2 COO -} {(Au(0)10.18 -PAMAM_E5.NH2 } {(Ag(0)59.18 -PAMAM_E4. CH2 CH2 COO -} 512 128 256 Surface charge of the host negative positive negative

Mn 55,044 30,830 59,297

*Calculated as the average mass of nanocomposite units composed of the template and the encapsulated gold atoms

+ MY D [(MY) n-D]

+X {(MX) n-D} -Y

Dendrimer

Complex

Nanocomposite

Figure 1: General scheme of dendrimer nanocomposite formation through complex formation and reactive encapsulation.

10

20.0

15.0

10.0

5.0

0.0 0 10 20 30 40

diameter nm
(A) (B) Figure 2: (A) TEM image of {Ag(0)97 -PAMAM_E5.COOAg} nanoparticles. Magnification:150,000. (B) Particle size distribution curve: relative frequency of various particle diameters. Separate maxima can be observed for dimers, trimers and tetramers and pentamers of almost uniform single particles.

11

Figure 3: Comparison of single {(Au(0)n -E5.NH2 } particles and nanocomposite clusters

12

Figure 4: Single {Au} nanocomposite particles inside a lipid bilayer structure.

13

(A)

(B)

Figure 5: Visualization of gene transfection with plasmid DNA-gold/PAMAM nanocomposite complexes. Unstained specimen. A: Cell surface attachment, B: Complex just undergoing endocytosis.

14

Figure 6: Electron microscope image of mouse tumor tissue 1,25 hours after {Au} injection. Unstained specimen, black dots represent gold nanocomposite clusters, dark areas within the nucleus are preferred for {Au}.

15