l. balogh, a. bielinska, j. d. eichman, r. valluzzi, i. lee, j. r. baker, t. s. lawrence and m. k....

8/3/2019 L. Balogh, A. Bielinska, J. D. Eichman, R. Valluzzi, I. Lee, J. R. Baker, T. S. Lawrence and M. K. Khan- Dendrimer Na

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Corresponding author: Lajos Balogh, The University of Michigan Center for BiologicNanotechnology, 4010 Kresge Research Building II, 200 Zina Pitcher Place, Ann Arbor, MI 48109-0533. Phone: (734) 615-0623, Fax: (734) 615-0621, [email protected]

DENDRIMER NANOCOMPOSITES INMEDICINE

L. Balogh,V A. Bielinska,V J. D. Eichman,V R. Valluzzi, I. Lee,VJ. R. Baker,VT. S. Lawrence

and M. K. Khan

VThe University of Michigan Center for Biologic Nanotechnology,Ann Arbor, MI 48109-0533Tufts Biotechnology Center

Department of Chemical Technology, Medford, MA 02155Department of Radiation Oncology,

University of Michigan, Ann Arbor, MI 48109

INTRODUCTION

Dendrimer based nanocomposites1-5 are novel organic-inorganic hybrid nanoparticles synthesizedby dispersing very small inorganic domains in nanoscopic size polymeric networks. Due to themolecular level mixing of their components they display unique material properties in addition toproperties represented by their individual components. Dendrimers are often used as templates inmaking these particles because they can be made uniform, and using these well-controlled anduniform templates leads to well controlled and uniform composite particles. Apart from obviousmaterials 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 injectingnanoparticle solutions directly into the tumor microvasculature.6 If a short-lived isotope is used, aprocedure like that may serve as an alternate method to direct irradiation. It has also been envisionedthat if the surface of such a nanoparticle had been equipped by appropriate targeting moieties thenanoscopic 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 imagedby transmission electron microscopy (TEM) both in vitro and in vivo. Silver/PAMAM complexesand different {Ag(0)n-PAMAM} dendrimer nanocomposites have also been tested againstStaphylococcus 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 orchloride ions.

Dendrimers. The term dendrimer refers only to an architectural motif and not a particularcompound. To date greater than 160 various polymers with dendritic structures are reported in theliterature. This term is used both for dendritic (macro)molecules and materials. Dendrimermolecules are monodisperse symmetric macromolecules built around a small molecule or a linearpolymer core7-10 using connectors and branching units. Interaction of dendrimer macromoleculeswith the molecular environment is dominantly controlled by their terminal groups. By modifying


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their termini, the interior of a dendrimer may be made hydrophilic while its exterior surface ishydrophobic, or vice versa.

Due to synthesis limitations, dendrimer materials always contain imperfect molecules that aremore similar to hyperbranched structures. There are other parameters to be taken into account:poly(amidoamine) (PAMAM) dendrimers,11for 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 surfacemodification. Fluorescent markers, such as fluorescein, or chelated metal ions, such as Gd 3+ forMRI are used to generate a measurable signal for imaging,17-19 Radiolabeled compounds, (3-H, 14-C, 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 monoclonalantibodies without loss of protein immunoreactivity. Nonetheless, conjugation of markers to thesurface of the macromolecule may dramatically change the solubility and other surface-relatedproperties.

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 DNAoligonucleotides or RNA. That particular function of PAMAM dendrimers relies on their ability tobind all forms of charged nucleic acids on the basis of electrostatic interactions. The resultingdendrimer/DNA complexes enhance DNA transfer, protect against intra- and extra-cellulardegradation and results in the transgene expression in a variety of mammalian cell lines in vitro aswell in variety of animal tissue in vivo.21-24Formation of DNA-dendrimer complexes seems to bebased entirely on charge interaction between negatively charged phosphate groups of the nucleicacid and protonated (positively charged) amino groups of the PAMAM polymers.24-25, Highlyefficient transfection of a broad range of eukaryotic cells and cell lines was achieved with minimalcytotoxicity 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-labeledpoly(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 investigatedby ESR. 26,27 The results show that although the structure of the complex depends on both thePAMAM generation and the length of the DNA strand, there is predominantly one binding modebetween DNA and PAMAM dendrimers. The structure or the stoichiometric complex and variousaggregates changes not only with surface charge differential but is dependent on the concentrationof both components.25

The simplest structural model proposes that DNA binding with dendrimers can be divided intotightly bound DNA regions and linker DNA regions. The binding between DNA and dendrimersalso effected by ionic strength and pH, which is consistent with the importance of the electrostaticinteractions 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 andoptimization of surface properties are also possible. Targeting groups can be conjugated to the hostdendrimers surface29 to allow the imaging agent to bind selectively to specific sites such asreceptors on tumor cells to improve detection. For example, there are a number of cell surfacereceptors that are known to be overexpressed on prostatic cancer cells, including epidermal growthfactor receptor (EGFR) and the laminin receptor VLA-6. 30 For a non-specific targeting, it has beenshownthat appropriately sized dendrimers and dendrimer-based metal complexes (e.g., cis-platinum) become trapped in tumor vasculature due to the enhanced permeability and retention(EPR) effect.31


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Composites and nanocomposites. Composites are physical mixtures or two or morecomponents which display improved properties in one or more areas as compared to their individualbulk constituents. They are widely used in many diverse industries and form the basics ofengineering plastics, structural adhesives and matrices. Nanocomposites can be defined as having atleast 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 componentsand 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 appropriatelyselected dendrimer. This pre-organization is followed by in-situ chemical reaction(s) or physicaltreatment (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 thedendrimer molecule(s) without creating covalent bonds between the dendrimer and the topologically

entrapped matter. Details of experimental procedures and analytical techniques can be found inprevious 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 causedmostly by Staphylococcus aureus, Pseudomonas aeruginosa, andEscherichia coli.32

The topical treatment is Silver sulfadiazine (AgSD), which acts through AgCl formation.33,34Using AgSD both silver sensitivity and sulfadiazine resistance may develop in patients. The searchfor 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 woundcovering device.35 Silver nylon dressing of wounds was found protective either with or without anapplied 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 fordifferent treatments and new ideas is continuing.

Antimicrobial activity of silver nanocompositesSilver nanocomposite solutions have been prepared from various dendrimers and with differentsilver 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 areaof inhibition. Silver nanocomposites were stable and remained soluble in the test media. In dialysisexperiments 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 contactedwith 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 groupsaround a 54 diameter sphere that could retain many silver ions) in the nanoscopic size compositeparticles. These silver domains - in any form of Ag+, Ag(0), or any other compound, such as AgCl


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or Ag2SO4 - are bound in the dendrimer network with a calculated specific surface area of severalthousand 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-dendrimercomplexes and the silver nanocomposites displayed antimicrobial activity comparable or better tothose of silver nitrate solutions.37

Gold nanocomposites. Gold/PAMAM nanocomposites usually are fabricated by UV-irradiation3 or by chemical reduction4.5 of tetrachloroaurate salts of PAMAM dendrimers. Based onthe locality of the guest atoms three different types of single nanocomposite architectures have beenidentified, 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 thenumber and character of the surface groups on the dendrimer used. Gold nanocomposites are easyto observe in cells and in tissue because they are very much differ by contrast, size and shape. Inimaging, gold nanocomposites offer the high electron density of the immobilized gold atoms, A fewgold atoms per dendrimer already renders the visualization of the amorphous single and multiplenanocomposite particles possible by electron microscopy.

Figure 3:

Due to the synthetic procedure used {(Au(0)n}m clusters are almost always present in the solutionof {(Au(0)n} nanocomposites.

4 Of course, visibility of the larger clusters are much better than thesingle units, which often appear as small grayish round objects due to their low and homogenouslydispersed gold content. This characteristic size, shape and contrast make the observation of goldnanocomposites simple by electron microscopy. Furthermore, no other objects can be found intissues 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 ispossible to use gold nanocomposites as selective stains. DNCs can have a specific charge on theirhost 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 whichthen were examined on a Philips CM100 electron microscope.

In Figure 6 lipid bilayer structures are shown that were treated with single unit {Au(0)}+ goldnanocomposites. The positively charged {Au(0)}+ nanoparticles have entered the polar region of thebilayers 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 dendrimersDue 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)10PAMAM_E5.NH2} were formed in double distilled H2O at a 10:1 ({Au}:pDNA) chargeratios, which represent the typical condition of complex formation. Gene transfections were


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performed by incubating the complexes with Cos-1 cells (monkey kidney fibroblasts; 1 x 105cells/cm3) for three hours at 37C. Cells were washed and grown overnight (24 hrs, 37C) ingrowth medium supplemented with 10% FBS.

Gold/PAMAM nanocomposites displayed gene transfection efficiency similar to their hostdendrimer,40 The luciferase expression was found comparable to that in the control transfectionsperformed 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 andtranslation of the introduced DNA. We have not observed any increase in toxicity due to the metalcontent of gold nanocomposites indicating that toxicity of these nanomaterials is determined by thedendrimer 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 thesecomplexes were found attached to the cell surface (A). Then, they were subsequently endocytosedand 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 thepDNA-{Au} complexes. However, images taken at a later time indicate the movement of the darknanoparticles toward the internal surface of the lysozomal vesicles cell indicating flow characteristicof 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 increasedpermeability and the tendency to express unique proteins suitable for targeting Known differencesin permeability between tumor and normal vasculature can be exploited to produce selectivedeposition of isotope containing nanoparticles in tumor.

In this demonstrative experiment amine surface gold-PAMAM nanocomposites were used tovisualize {Au(0)}+ in tumor tissue by TEM. One million B16F10 melanoma cells were injectedsubcutaneously and then grown on the dorsal surface of a C57Bl6/J mouse for two weeks theninjected with nanocomposite solution . The tumor and other organs (liver and lung tissue) wereisolated 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 indicatesthat 1.25 hours after the direct injection only a few larger gold nanocomposite clusters are presentin the interstitium. Most of the smaller clusters were found in the nucleus and cytoplasm. Duringthe internalization of gold nanocomposites the diffusion through the cell wall appears to be thedominant 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. Theyreadily penetrate into cells and are easy to observe by transmission electron microscopy and opticalmethods.

Gold/PAMAM DNCs can be used for imaging by electron microscopy under both in vitro and invivo experimental conditions. Based on these observations we could identify the following steps inthis gene transfection process: first, large clusters containing many DNA molecules and manydendrimers form. These complexes bind to the cell surface then undergo endocytosis

In case of lipid bilayers, positively charged gold/dendrimer single nanocomposite units easilypenetrated into the polar zone of the bilayer structure. Using radioactive gold as a guest, the


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nanoscopic agent can be directly injected into the tumor microvasculature where the positivelycharged 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 silversulfadiazine, 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 sulfateions 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 Institutesof Health, under Contract No. NO1-CO-97111 and in part by the Defense Advanced ResearchProjects Agency, award MDA972-97-1-0007, entitled "Nanomolecule Based Agents for PathogenCounter Measure. Antimicrobial silver nanocomposite research was sponsored by the ArmyResearch Laboratory under the contract of DAAL-01-1996-02-0044. LB thanks to D. Sorensonand C. Edwards of the Microscope and Imaging Laboratory at the University of Michigan for theirtechnical support.

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1. L. Balogh, D. R. Swanson, R. Spindler and D. A. Tomalia: Formation and Characterizationof Dendrimer-Based Water Soluble Inorganic Nanocomposites; Proceedings of ACSPMSE, 1997 (77), 118.

2. Nora Beck Tan, L. Balogh and S. Trevino: Structure of Metallo-Organic NanocompositesProduced from Dendrimer Complexes; Proceedings of ACS PMSE,1997 (77), 120.

3. Esumi, K., Suzuki, A., Aihara, N., Usui, K., Torigoe, K.,Langmuir1998, 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, August1984, 65

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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.

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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.


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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 & MedicinalChemistry 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), 733-74220 . 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. NucleicAcids 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. BioconjugateChem.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, 813-82 528.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) 100633.Fuller, F.W., Parrish, M., Nance, F.C.,J. Burn. Care Rehabil.,1994, 15(3), 21334.Harrison, N. H.,Arch. Surg.,1979, 114(3), 28135.Chu, C.S., McManus, A.T., Pruitt, B.A. Jr., Mason, A.D. Jr.,J Trauma1988, 28(10), 148836.Greenfield, E., McManus, A.T.,Nurs. Clin. North. Am.,1997, 32(2), 29737.

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.


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APPENDIX

Nomenclature. To describe the complex structure of these nanoscopic complexes andnanocomposites we use the following convention to describe the composition of the synthesizednanomaterials: brackets denote complexes as it is common while braces represent nanocompositestructures. 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 dendrimersfollowed by terms used for dendrimer identification, i.e., core, generation and surface. Naming of ageneral 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 whicha 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 surfacePAMAM containing seven Cu(II) ions and seven silver ions per dendrimer. The above schemeprovides 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.


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Table I. Properties of some PAMAM Dendrimer Nanocomposites

No. of surfacegroups

Surface charge of thehost

Mn

{(Au(0)10.82-PAMAM_E5.CH2CH2COO-} 512 negative 55,044

{(Au(0)10.18-PAMAM_E5.NH2} 128 positive 30,830

{(Ag(0)59.18-PAMAM_E4. CH2CH2COO-} 256 negative 59,297

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


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

Dendrimer

D [(MY)n-D] {(MX)n-D}

+X+ MY

- Y

Complex Nanocomposite


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0.0

5.0

10.0

15.0

20.0

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 particlediameters. Separate maxima can be observed for dimers, trimers and tetramers and pentamers ofalmost uniform single particles.


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


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


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(A) (B)Figure 5: Visualization of gene transfection with plasmid DNA-gold/PAMAM nanocompositecomplexes. Unstained specimen. A: Cell surface attachment, B: Complex just undergoingendocytosis.


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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 thenucleus are preferred for {Au}.

l. balogh, a. bielinska, j. d. eichman, r. valluzzi, i. lee, j. r. baker, t. s. lawrence and m. k....

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