Jowairia Chaudhry

Prof. Huixin He. Department of Chemistry, Rutgers The State University of New Jersey, 73 Warren St., Newark, NJ, 07102-

1811, Jowairia.ch@gmail.com

Fabrication of Highly Stable Third Generation Poly(Propylene Imine) Dendrimer-Encapsulated

Silver Nanoparticles and Their Application to Develop Multifunctional DNA Delivery Agents

Fabrication of highly stable and multifunctional generation-three (G3)

Poly-propyleneimine (PPI) dendrimer-encapsulated Ag nanocomposites

with narrow size distribution has been done.

This was followed by the condensation of plasmid DNA using the resultant

Ag nanoparticles as the condensing agents to produce multifunction DNA

delivery vehicles.

The fabrication method employed was a one-step one-pot method by which

silver nanoparticles were yielded directly by heating the aqueous solutions

of G3 PPI dendrimer and AgNO3 at 80°C for 90 minutes.

Synopsis:

No additional reducing agent, surfactant or a protecting group was added

because the G3 PPI dendrimers eloquently satisfied all those functions.

The G3 PPI dendrimer-encapsulated nanoparticles have been analyzed

via Atomic Force Microscopy (AFM), Transmission Electron

Microscopy (TEM) and UV-vis Spectroscopy and their characterization

has been reported in this presentation.

However, their application in the compaction of DNA into highly

multifunctional DNA delivery agents will be studied in the near future

via TEM, AFM and Surface-Enhanced Raman Spectroscopy (SERS).

Synopsis: (cont.)

Dendrimers: e.g., G3 PAMAM dendrimer

Structure: Dendrimers are monodisperse macromolecules with regular and highly branched 3-D

architecture and exceedingly well surface functionality. They are basically composed of a core molecule and branches, which regularly extend from the core to the terminal group. Each new layer originating from the focal point of branching creates a new generation, with double the number of active sites (end-groups) and approximately double the molecular weight of the previous generation.

Often, if not always, dendrimers are depicted as spherical molecules, which can be misleading.

Dendrimers generally have globular shape in the presence of a good solvent or when the end groups are so bulky that they get in the way of each other’s ability to move around, making their structure rigid (steric hindrance).

Bare dendrimers are very flexible. They have the ability to completely change their conformation to for lipid-like structures or layers if secondary interactions, such as hydrophobic or any other surface interactions, are introduced based on the affinity of their end groups. They can transform their shape to almost flat, if such interactions are adequate enough.

An Introduction:

The dendrimer is grown in stepwise manner from the central core to periphery, that is, it follows the bottom-up approach.

It has been reported that this method provides a high yield. However, it is necessary to employ a purification process in order to remove unreacted

dendrons or defected dendrons after the reaction (e.g., size exclusion chromatography). The presence of small number of defects can be avoided, if and only if, the substitution is

perfect in every step.

2. Convergent Pathway:

The synthesis starts from the periphery and ends at the core. In this method, a constant and low number of reaction sites are present in every reaction step

throughout the synthesis. And hence, only a small number of side products are formed in each reaction and the dendrimers synthesized are relatively defect-free.

There are two methods to synthesize dendrimers:

1. Divergent Pathway: (commercial method for PPI, PAMAM)

Properties: Template: Since dendrimers contain a large number of regularly spaced internal and

external functional groups, they can operate as template to grow inorganic crystals in their internal or external regions.

For instance, dendrimer-encapsulated metal nanocomposites can be created when metal ions interact with the surface functional groups of dendrimers. Here, dendrimers act as template, and the complex formation takes place due to ligand-metal ion interactions and acid-base interactions etc.

Surface Functionality: Low generation dendrimers tend to exit in the relatively open forms which provide reactive sites at the periphery of dendrimer, while higher generations have a spherical 3-D structure in which the interior sites act as reactive sites for the inorganic molecules.

For instance, PAMAM and PPI have interior tertiary amine groups (PPI do not contain amide groups) that act as reaction sites for the metal molecules such as Ag, Au, Cu, etc.

Multipurpose: Dendrimers fulfill the role of reducing agent, surfactant and protective group to

nanoparticles. It has been reported that these dendrimer-encapsulated nanoparticles are really

composites with no covalent interactions between their components. The interactions between the metal atoms and metal-solvent are substituted with metal dendrimer and dendrimer-solvent interactions. This is the reason why dendrimer nanocomposite solutions are stable for a very long time.

Ability Bind with DNA and low toxicity: Dendrimers have the ability to bind with DNA and shuttle the resultant complex to the target the cells bearing restricted toxicity. And hence , are being keenly being studied as potential gene-therapy agents.

For example: Cationic dendrimers, like amine terminated PPI and PAMAM dendrimers, have been reported to give rise to cationic groups near physiological pH. And their toxicity has been reported by various research workers to be directly proportional to their generation, that is , it increases with increasing generation of the dendrimer.

In view of these facts, we employed low generation(G3) amine terminated PPI dendrimers to fabricate PPI-encapsulated Ag nanocomposites, and utilized the embedded DNA condensation properties of the ensuing nanoparticles to compact DNA into highly multifunctional DNA delivery agents.

In a Nutshell: Dendrimers are remarkable nanoparticles with numerous applications. They have

attracted attention because of their well-defined structure and chemical versatility. And hence , are widely used in the fields of medicinal chemistry, biotechnology, biophysics and electronics etc.

Dendrimers are particularly compatible for hosting a wide range of nanoparticles because of their uniform composition and structure.

Various nanoparticles have been stabilized, protected, reduced and encapsulated by dendrimers (driven primarily by non-covalent interactions).

Dendrimers branches are being used as selective gates to control access to molecules to encapsulated nanoparticles(drug-delivery).

Their active sites are being used to interact with a range of different nanostructures such as metal nanocomposites and DNA (gene therapy, catalysis and so on).

Dendrimers based techniques such as gene therapy and drug delivery are growing very rapidly but more research needs to be done to gain further control over their toxicity and ability to trigger the release of DNA or drugs, prior to conducting any clinical trials.

Goal: To fabricate highly stable G3 PPI dendrimer-encapsulated Ag nanoparticles to develop multifunctional DNA delivery

vehicles.One Step Preparation &Characterization of PPI Dendrimer-Protected Ag Nanoclusters. Macromolecules, 2004, 37, 7105-7108.

Experiment: 1 January 31, 2005

Fabrication of G3 PPI dendrimer-encapsulated Ag nanoparticles:

A 0.0168M and 0.0463M aqueous solutions of AgNO3 and G3 PPI dendrimers were prepared.

Then aliquots of both of these solutions were mixed into a properly cleaned flask with a 1:10 v/v ratio of G3 dendrimers to AgNO3.

This G3 PPI/AgNO3 mixture was heated at 80°C for 90 minutes.

This is because the fabrication method employed was a one-step one-pot method.

The Ag nanoparticles were yielded directly by heating the aqueous solutions of G3 PPI dendrimer and AgNO3 at 80°C for 90 min.

No additional reducing agent, surfactant or a protecting agent was used because the G3 PPI dendrimers eloquently satisfied all those functions.

Why our method of fabrication of these colloidal particles significant?

Characterization:

The characterization of G3 PPI dendrimer-encapsulated Ag nanoparticles have been analyzed via:

UV-Vis Spectroscopy

Atomic Force Microscopy (AFM)

Transmission Electron Microscopy

(TEM)

However their application in the

compaction of DNA into highly functional DNA delivery

agents have been studied by:

Atomic Force Microscopy (AFM

Upon the completion of the 90 minutes of heating the G3 PPI/ AgNO3

aqueous solution at 80°C, the color of the resultant colloidal solution changed from colorless to yellow.

This is a characteristic color transition observed when the reduction of Ag+ ion to Ag° takes place. This confirmed the formation of Ag nanoclusters

By UV-vis Spectroscopy (Varian Cary 500 Scan UV-VIS-NIR Spectrophotometer):

A characteristic plasmon absorptions was observed at 415 nm.

This substantiated the encapsulation of the Ag nanoparticles by G3 PPI dendrimers.

Characterization of G3 PPI Dendrimer-Encapsulated Ag nanoparticles:

TEM (courtesy of NJIT):Silver displays natural contrast in TEM, therefore, the encapsulation of Ag nanoparticles by G3 PPI dendrimers was further confirmed by conducting TEM.

Generally speaking, TEM allows the determination of the internal structure of various materials. This is done by irradiating an electron beam onto the analyte. Because the wavelength of electrons is much more smaller than light, the optimal resolution of TEM images is many times higher than in resolution than from light microscope or even AFM.

AFM(DI, Veeco):These samples were prepared by immobilizing aliquots of 10X diluted

original solution on unmodified muscovite mica.

We used Tapping-Mode AFM to probe the topology of the resulting PPI dendrimer-encapsulated Ag nanoparticles by slightly tapping the sample surface with an oscillating etched-silicon tip.

Operation Mechanism of AFM (particularly Tapping Mode) :

http://etheses.nottingham.ac.uk/archive/00000077/01/Hosam.pdf

Fig. 3 A sharp probe on a flexible cantilever is scanning across the surface at constant force, its position monitored by reflecting a laser beam off the back of the cantilever onto a split photodiode. A piezoelectric scanner controls this scanning motion. The subsequent bending of the lever due to probe-sample interaction reveals an image at nanometer resolution.

The force between the tip and the sample is very small ( ~ 10-9 N). The detection system does not measure force directly, rather it senses the

deflection of the cantilever. A diode laser is focused on the cantilever. The back side of the cantilever reflects the laser beam onto a dual element

(split) photodetector . Even a small deflection of the cantilever will tilt the reflected beam and

change the position of the beam onto the photodetector. The photodetector measures the difference in the light intensities between

its two elements , and converts them into voltage. The feedback from this voltage signal enables the tip to maintain contain

height and force above sample (through a computer software control).

AFM:

In the tapping mode, the tip is alternatively placed in contact with the surface and then lifted off the surface, ensuring high resolution and preventing the tip from dragging across the sample surface.

It is implemented in ambient air by oscillating the cantilever at or near its resonating frequency via piezoelectric crystal.

The piezoelectric motion causes the cantilever to oscillate with a high amplitude (~ 20nm) when the tip is not in touch with the surface. The oscillating tip is moved toward the surface until it lightly touches the surface.

Energy is lost when the tip touches the sample surface. As a result oscillation is reduced and this reduction in amplitude is used to identify and measure the surface features.

Tapping Mode AFM:

The oscillation amplitude and frequency are then maintained constant for the rest of operation by a feedback loop .

The force on the sample is automatically set and maintained to a lowest possible level.

When the tip passes over a depression, the cantilever has more room to oscillate and the amplitude of oscillation increases.

On the other hand, when the tip over a bump, the cantilever has less room to oscillate and the amplitude of oscillation decreases.

Tapping Mode AFM:

Taping Mode AFM gets rid of the major problems associated with friction, adhesion, electrostatic forces and other tip-sample related difficulties experience via other modes of AFM.

Tapping mode inherently prevents the tip from sticking to the surface and damaging it.

The oscillation amplitude is sufficient enough to overcome the adhesive forces between the tip and sample.

The applied force is vertical, so the sample surface does not get pulled sideways due to shear forces, preventing defects in the scans.

Since, the samples for dendrimers-encapsulated Ag nanoparticles are highly soft/delicate, we used TM-AFM to get their high resolution topological image without scratching their surface.

Why did we use Taping Mode AFM?

AgN

N N

N

N

NH2

NH2

NH2

NH2

N

N N

H2N H2N NH2

NH2

N

NN

H2NH2NH2N

H2N

NN

N

H2N

H2N

H2NH2N

NN

N N

N

NH2

NH2

NH2NH2

N

N

N

NH2

NH2

NH2

NH2N

N

N

H2N

H2N

H2N

H2N N

NN

H2NH2N

NH2

NH2

NN

NN

N

H2N

H2N

H2N H2N

N

N

N

H2N

H2N

H2N

H2NN

N

N

NH2

NH2

NH2

NH2N

NN

NH2NH2

H2NH2N

N

N

N

N N

NH2 NH2NH2

NH2

NN

N

H2N

H2N

NH2 NH2

NN

NNH2

NH2

H2NH2N

N

N

N

H2N

H2N

H2N

H2NN

N

NN

NH2N

H2NNH2

NH2

NN

N

H2N

H2N

H2N

H2N

NN

N

NH2

NH2

NH2

NH2

N

NN

NH2NH2H2N

H2N

N N

N N

N

NH2

NH2

NH2NH2

N

N

N

NH2

NH2

NH2

NH2

N

N

N

H2N

H2N

H2N

H2NN

NNH2N

H2N

NH2NH2

Fig. 4 Possible structure of G3PPI dendrimer-encapsulated Ag nanoparticles.

N

N

NN

NH2N

H2NNH2

NH2

NN

N

H2N

H2N

H2N

H2N

NN

N

NH2

NH2

NH2

NH2

N

NN

NH2NH2H2N

H2N

Fig.3 G3 PPI dendrimer with 16 end groups. Size= ~ 2.4 nm

Proposed Structure and Mechanism:

G3 PPI dendrimers are very small in size and are barely 2.4nm across. Yet they have a highly regular and branched spherical architecture with a very high surface functionality.

Each of the G3 PPI dendrimer has a stem core to which branches are attached that further branch out bearing 16 end groups.

Every nitrogen (N) is a branching point to which 2 branches are attached. Thereby, the number of branches and branching points are doubled per generation growth.

Every branching of the nitrogen atoms represents the formation of a new generation.

G3 PPI Dendrimer:

Proposed Mechanism:

Keeping in view the size of the G3 PI dendrimer, it can easily be understood that the silver nanoclusters can not form inside the G3 PPI dendrimer- the size of the Ag particles is much larger than G3 PPI dendrimer itself.

It has been suggested by Prof. He that probably the dendrimers adsorb onto the silver nanoclusters in order to protect them, thus stabilizing them by attaching themselves around individual silver metal nanocomposites.

Concisely, it can be said that upon the mixing of Ag ions with PPI dendrimers in solution, complex formation occurs when dendrimer molecules adsorb on the surface of Ag nanoparticles by attaching multiple dendrimers to a single Agº particle.

.

Proposed Mechanism:

The silver cations obviously interact with surface functional of the dendrimers so that the resulting nanoparticles reside on the surface of the dendrimers.

Probably, ligand-metal interactions and acid-base interactions etc occur between the surface functional groups of the G3 PPI dendrimers and the Ag colloidal particles, thus giving them structural stability.

Since G3 PPI dendrimers are cationic dendrimers (amine terminated), they interact with the phosphate groups of DNA to give rise to G3 PPI-encapsulated Ag/DNA condensates (experiment 2).

Fig. 1 UV-vis spectrum of 10x diluted G3 PPI dendrimer-encapsulated Ag colloidal solution prepared by heating G3 PPI/ AgNO3 aqueous solution at 80 °C for 90 min.

Fig. 2 TEM image of 10X diluted colloidal solution of G3 PPI dendrimer encapsulated nanoparticles.

Diameter of the largest one = 40nm , Average diameter = ~35.5 nm

Fig.5 Tapping Mode AFM image of 10X diluted colloidal solution of G3 PPI dendrimer encapsulated nanoparticles after 12 days of their fabrication. Avg. height = 33.630+ 8.37 nm, Avg. diameter = 230 + 40 nm, number of particles = 35

Fig. 6 Tapping Mode AFM image of 10X diluted colloidal solution of G3 PPI dendrimer encapsulated nanoparticles after 42 days of their fabrication.

Avg. height = 28.11 0 + 3.128nm, Avg. diameter = 62.42+ 7.786nm,

Number of particles = 6

10 15 20 25 30 35 40 450

50

100

150

200

250

230

62.42

33.6328.11

Height(nm) and Diameter(nm) of G3 PPI dendrimer-encapsulated nanoparticles Vs.

Time (days)

Height (nm)Linear (Height (nm))Diameter(nm)Linear (Diameter(nm))

days

nm

Key Points:

The formation of smaller sized nanoparticles is kinetically favorable whereas the

formation of larger ones is thermodynamically favorable. Therefore, small particles

nucleate readily at low temperatures. Since these particles are high in energy, they are

extremely unstable and have a tendency to grow into larger structures to reach their

energetically favorable state.

Since we heated our reaction mixture at 80 C, which is quite high, the resultant G3 PPI

dendrimer-encapsulated nanoparticles were large in size and hence, extremely stable. Our

reaction mixture remained free from any flocculation for about 5 months. (01/31/05-

05/23/05)

To further gain control over the size of the resultant PPI-encapsulated, we repeated the

reduction reaction at 100 °C and condensed plasmid DNA with the resultant nanoparticles.

Experiment: 2 March 06, 2005

Goal: Condensation of DNA via G3 PPI Dendrimer-Encapsulated Ag Nanoparticles.

Some important points: The DNA used in this experiment was plasmid DNA (PGL-3) with a

concentration of 0.07 OD. This plasmid DNA is double stranded and comprises of 5256 base-pairs

(bp). 1 OD double strand DNA corresponds to a concentration of 50 μg/ml and

0.01 OD is equivalent to 1.5 μ M .

For this experiment:

Target concentration = 1.5 μ M

This concentration was attained by diluting the 0.07OD PGL-3 DNA 2X with 10mM tris/HCL buffer (pH 7) and then by mixing it with 2X diluted original solution of the G3 PPI-encapsulated Ag nanoparticles (centrifuged & redispersed 2X, detail on next slide) with a 1:1 ratio.

Concentration of DNA:

An aliquot of the stock solution of the dendrimer-encapsulated Ag

nanoparticles was centrifuged at a frequency of 8500 rpm for 3.5min at

25 °C using a Beckman Coulter/Avanti Centrifuge (J-20XP, rotor ID: JA 25.50) .

The supernant was removed and secured and the precipitate was re-

dispersed in DI water. This procedure was repeated twice.

Then, the Plasmid DNA was dissolved in 10mM tris/HCL buffer (pH 7)

so that its concentration becomes 0.035 OD (i.e., 2X diluted).

Condensation of plasmid DNA to get complex DNA condensates:

• Subsequently, both of the prepared solutions were mixed together

with a 1:1 ratio as described in the previous slide (i.e. final DNA

conc.= 1.5μM; 0.01OD).

• The color of the redispersed G3 PPI-encapsulated Ag nanoparticle

solution changed from yellow dirty green (slightly brownish green).

Fig. 7 Tapping Mode AFM image of 10X diluted solution of DNA condensates after 30-45 min. after condensation . For large condensates: Avg. height = 25 + 1.719 nm, Avg. diameter= 178.39 + 21.958 nm For small condensates: Avg. height= 1.977 + 0.677nm, Avg. diameter= 78.092 + 45.418 nm

* For each time interval, the particles were categorized as large or small to execute better particle analysis.

Fig. 8 Tapping Mode AFM image of 10X diluted solution of DNA condensates after 30-45 min. after condensation. Three different types of particles formed:For small condensates: Avg. height= 1.932 + 2.23 nm, For large condensates: Avg. height= 42.231 + 5.649nm

Goal: Condensation of DNA via G3 PPI Dendrimer-Encapsulated Ag Nanoparticles… a repetition of experiment 2.

•For each interval, the particles were categorized as large or small to execute better particle analysis.

Experiment: 3 March 20, 2005

Condensation of DNA via G3 PPI Dendrimer-Encapsulated Ag Nanoparticles

For Large Particles:

Time Passed (hrs)

Min. Height (nm)

Max. Height (nm)

Avg. Height (nm)

Min. Diameter (nm)

Max. Diameter(nm)

Avg. Diameter (nm)

0.25(15 min)

10.097 13.404 11.905+ 1.009 80.331 110.85 100.10+ 9.297

1 4.961 10.942 8.919+ 1.388 58.398 137.70 93.006+ 12.772

3 7.582 11.811 8.370+ 2.071 123.23 287.57 174.37+ 59.853

4 7.280 18.756 11.702 + 2.066 73.192 175.51 121.67+ 24.105

5 5.228 11.157 6.864+ 1.292 88.155 170.35 122.37+ 23.382

336(2 weeks)

16.165 29.289 21.862+ 3.225 408.90 762.49 504.88+ 78.427

* For each time interval, the particles were categorized as large or small to execute better particle analysis.

0 50 100 150 200 250 300 350 4000

100

200

300

400

500

600

100.1

93.006

174.37121.67 122.37

504.88

11.905

8.9198.37 11.702

6.86399999999999

21.862

Average Height(nm) and Diameter(nm) of LARGE G3 PPI dendrimer-encapsulated nanoparticles Vs.

Time (hours)

Avg. Height (nm) Linear (Avg. Height (nm) )Avg. Diameter (nm) Linear (Avg. Diameter (nm) )

hrs

nm

Time Passed (hrs)

Min. Height (nm)

Max. Height (nm)

Avg. Height (nm)

Min. Diameter (nm)

Max. Diameter(nm)

Avg. Diameter (nm)

0.25(15 min)

1.044 9.786 3.766+ 1.387 22.039 100.99 38.959+ 12.326

1 0.998 8.314 3.428+ 1.878 6.233 88.155 34.853+ 20.934

3 1.515 6.102 3.197+ 0.716 51.650 162.07 71.852+ 14.512

4 2.279 6.834 3.720 + 0.880 51.754 100.22 59.131+ 10.540

5 2.361 4.362 3.224+ 0.799 55.623 78.663 66.458+ 6.758

336(2 weeks)

5.299 15.839 10.681+ 2.109 114.57 330.57 247.55+ 48.472

For Small Particles:

Note: The sample for the time interval of 2 hrs was defective. Upon scanning it with TM-AFM only particle was found (Height = 3.489 nm; Diameter= 106.24 nm

0 50 100 150 200 250 300 350 4000

50

100

150

200

250

300

38.95934.853

71.852

59.13166.458

247.55

3.766

3.428

3.197

3.72

3.224 10.681

Average Height(nm) and Diameter(nm) of SMALL G3 PPI dendrimer-encapsulated nanoparticles Vs.

Time (hours)

Avg. Height (nm) Linear (Avg. Height (nm) )Avg. Diameter (nm) Linear (Avg. Diameter (nm) )

hrs

nm

The sample for the time interval of 2 hrs was defective. Upon scanning it with TM-AFM only particle was found (Height = 3.489 nm; Diameter= 106.24 nm).

Special Case: The second sample for time interval of 3 hrs contained the following structures:

A long Jumble:

Length = 1.484 nm

Height = 37.7 nm

Diameter = 351 . 56 nm

A Toroid:

Diameter = 238.28 nm, 199.22 nm & 207.03 nm (from all three directions)

A Solid round ball:

Height = 111 . 40 nm

Diameter = 683.59 nm

Note:

In view of these facts, we have employed low generation (G3) amine terminated PPI

dendrimers to encapsulated Ag nanocomposites and have utilized the embedded DNA

condensation properties of the ensuing nanoparticles to compact DNA into highly

multifunctional DNA delivery agents.

We chose to work with Ag nanoparticles as potential condensing agents due to their

natural high contrast in TEM. In the near future, this property of Ag nanoparticles will not

only allow us to visualize the condensing agent in the final condensing products, but it

will also allow us to study the interaction between DNA and its condensing agent at a

molecular structural level. Into the bargain, silver nanoparticles are also known to play an

important role as substrates in the study of Surface-enhanced Raman Scattering and have

the ability to significantly enhance Raman signals. Therefore, we have also planned to

develop techniques to fabricate larger complex DNA condensates to conduct Surface-

Enhanced Raman Scattering analyses.

Conclusion:

Our primary incentive behind conducting this research was to develop efficient

DNA delivery agents. Non-viral gene therapy is a potential treatment to breast

cancer. To fulfill this promise plasmid DNA must be delivered to mutated target

cells. In the past retroviruses and lipid-based systems have been used as potential

DNA delivery vehicles but their toxicity on repeated dosing proved to be daunting

problem.

Dendrimers are being studied zealously now days as potential gene-therapy agents

due to their ability to bind with DNA and shuttle the resultant complex to target the

cells bearing restricted toxicity.

Cationic dendrimers, like amine terminated PPI and PAMAM dendrimers, have

been reported to give rise to cationic groups near physiological pH. And their

toxicity has been reported by various research workers to be directly proportional

to their generation, that is, it increases with the increasing generation of the

dendrimer.

Conclusion:

To conduct TEM analysis of the DNA condensates in order to visualize the

Ag nanoparticles in the final condensate at a molecular structural level and

to understand the mechanism of condensation.

Develop techniques to fabricate larger complex DNA condensates to

conduct Surface-enhance Raman Spectroscopic analysis.

To further conduct topological analysis of the complex particles via AFM.

Finally, introduce the DNA condensates to the cell to test their efficiency as

DNA delivery vehicles.

Intended Future Plans:

Acknowledgements:

Dr. Huixin HeOleh TartulaAlex ChenShah AliYufung Maand Jaiming Zhange

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[8] Dendrimers: Dream Molecules Approach Real Applications. Science vol. 267, 27 January, 1995.

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