Dendrimer-Based Drug Delivery Systems (From Theory to Practice) || Biological Properties of Phosphorus Dendrimers

4BIOLOGICAL PROPERTIESOF PHOSPHORUS DENDRIMERS

Anne-Marie Caminade and Jean-Pierre Majoral

4.1. INTRODUCTION

The aim of this chapter is to emphasize the unique role played by phosphorus-

containing dendrimers in the field of biology, with highlight on their ability for drug

delivery or as drugs by themselves. Phosphorus is a key element for all known forms of

life. In particular, various crucial roles are played by phosphates PO43�, which

constitutes the structural framework of DNA. They are also implied in nearly all

energetic cellular processes (as ATP, adenosine triphosphate), and are a crucial

component for stiffening the structure of bones. Furthermore, phosphorylation is a

key regulatory event in cells, and phospholipids are the main cellular components of

all cellular membranes. In view of all these key biological properties, it is not

surprising that many phosphorus chemicals are able to interfere with biological

systems, for theworse and for the best, from lethal nerve gases, eco-toxic insecticides,

detergents, and fertilizers, to various drugs, for instance against osteoporosis.

Thus, phosphorus-containing dendrimers, that are dendrimers having one phos-

phorus atom at each branching point, should play particular roles when interacting

with biological systems [1]. In this chapter, we will describe the main method of

synthesis of phosphorus-containing dendrimers, somemethods of functionalization

to render them water-soluble and biocompatible, their use for diverse biological

purposes, such as biological imaging, drug delivery (including transfection experi-

ments), and how can some of these compounds be considered as drugs by

themselves.

Dendrimer-BasedDrugDelivery Systems: FromTheory to Practice, First Edition. Edited byYiyun Cheng.� 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

139

4.2. SYNTHESIS AND FUNCTIONALIZATION

OF PHOSPHOROUS-CONTAINING DENDRIMERS

FOR BIOLOGICAL PURPOSES

There exist several methods of synthesis of phosphorus-containing dendrimers,

including some examples based on phosphates at each branching points [2], but

none of these compounds were used up to now for biological purposes. The most

widely used method of synthesis of phosphorus dendrimers necessitates two steps

to build one generation (one layer). The core is most generally trifunctional (from

P(S)Cl3) [3] or hexafunctional (from (N3P3)Cl6) [4]. The first step is the reaction of the

core with 4-hydroxybenzaldehyde in basic conditions (generally the sodium salt of

the phenol), and the second step is the condensation reaction of the aldehydes with the

phosphorhydrazide H2NNMeP(S)Cl2. Both reactions are quantitative and produce

only NaCl and H2O as by-products. The repetition of both steps allows the growing of

the dendrimers, andwas carried out up to generation 12 (PS)-G12 [5] from the P(S)Cl3core and up to generation 8 (N3P3)-G8 from the (N3P3)Cl6 core [4] (Fig. 4.1). It is well

known that the characterization of dendrimers is never trivial [6], but the presence of

phosphorus in these compounds allows their easy characterization by 31P-NMR,

which is an invaluable tool for assessing the completion of reactions at each step of the

synthesis, as well as the integrity of the whole structure [7].

These dendrimers have either P(S)Cl2 or aldehyde terminal functions, depending

on the step considered. These functions are among the most reactive and versatile in

phosphorus chemistry and organic chemistry, respectively. Numerous types of

reactions have already been carried out with such terminal groups, but we will focus

FIGURE 4.1 Chemical structure of the first and second generations built from P(S)Cl3((PS)-G1 and (PS)-G2, respectively), method of synthesis used, and chemical structure of the

first generation built from the (N3P3)Cl6 core ((N3P3)-G1).

140 BIOLOGICAL PROPERTIES OF PHOSPHORUS DENDRIMERS

here on those that have led to dendrimers suitable for biological purposes. The main

point is that these compoundsmust be soluble inwater [8]. In contrast to other types of

dendrimers that are “naturally” soluble in water, thanks to their rather hydrophilic

interior, and water-solubilizing terminal groups, these phosphorus dendrimers have

both a hydrophobic interior [9] and hydrophobic terminal groups in their “native”

form (aldehydes or P(S)Cl2). The only way to render them soluble in water [10] is to

modify their terminal functions, in particular by grafting functions bearing a charge

(positive or negative) [11]. Positively charged phosphorus dendrimers were first

obtained by reacting N,N-diethylethylene diamine directly with the terminal P(S)Cl2functions [12]. This reaction is shown in (Fig. 4.2) with the fourth generation

dendrimer 1-G4, but was carried out up to generation 8 [13].

Negatively charged terminal groups are also usable for inducing solubility in

water of phosphorus dendrimers. In a first attempt, carboxylic acids were grafted

starting from the aldehyde terminal groups, using a Doebner-like reaction with

malonic acid in pyridine and piperidine. This synthesis was carried out first with

dendrimers built from the P(S) core [14], then from (N3P3) core [13]. The second

generation 2-G2 was obtained as shown in (Fig. 4.3). The neutral form is generally

not soluble in water, contrarily to the carboxylate form, obtained for instance by

reaction with NaOH.

Phosphonic acid salts are also very hydrophilic. Theywere obtained in two steps as

terminal functions of dendrimers, starting from the P(S)Cl2 terminal groups. The first

step is the grafting of tyramine functionalized by two phosphonic esters; the second

step is the deprotection of the esters, to obtain azabisphosphonic acid salts, as shown

in (Fig. 4.4) for the first generation 3-G1 [15].

FIGURE 4.2 Synthesis of water-soluble dendrimers ended by ammonium groups.

FIGURE 4.3 Synthesis of dendrimers ended by carboxylic acids.

SYNTHESIS AND FUNCTIONALIZATION OF PHOSPHOROUS 141

4.3. CYTOTOXICITY ASSAYS OF PHOSPHORUS DENDRIMERS

Cytotoxicity of phosphorus dendrimers was measured in several cases, and was

found generally low, but dependent on the type of terminal groups. Positively charged

dendrimers were found generally more toxic than negatively charged dendrimers, and

dendrimerswith quaternary ammoniumgroupswere foundmore toxic than thosewith

tertiary ammonium groups [12]. Even in the case of tertiary ammoniums, the toxicity

depends on the type of terminal groups, but also on the type of cells. Figure 4.5

displays the cytotoxicity of three ammonium-ended fourth generation dendrimers

toward two types of human cells: one healthy cells strain (HUVEC, human umbilical

vein endothelial cells) and one cancerous cells line (HEK 293, human transformed

FIGURE 4.4 Synthesis of water-soluble dendrimers ended by azabisphosphonic acid salts.

FIGURE 4.5 Cytotoxicity of various ammonium dendrimers (1a-G4 (a and d), 1b-G4

(b and e), 1c-G4 (c and f)) toward HUVEC (upper row) and HEK 293 (lower row) cells, at

various concentrations (10, 20, and 50mg/mL) and for various times (24, 48, and 72 h)measured

by MTT assays in OPTI-MEM 1 cells culture medium. The black line corresponds to 100%

viability.


primary embryonal kidney). The amount of cell that survived after the experimentwas

determined byMTTassays. This test consists in the reduction of 3-(4,5-dimethylthia-

zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by the succinate dehydrogenase

intervening in the respiratory mitochondrial chain of viable cells. The water-soluble

yellow MTT is converted to the water-insoluble and purple formazan, which is later

dosed by spectrophotometry in an organic solvent [16]. The results ofMTTassays are

given in (Fig. 4.5); each value is the result of at least four concordant measurements.

No significant cytotoxicity is detected for dendrimers 1a-G4 and 1b-G4, even if the

noncancerous HUVEC cells are more sensitive to the presence of the dendrimers than

the cancerous cells (HEK 293). On the other hand, dendrimer 1c-G4 (methylpiper-

azinium extremities) is the most toxic against HUVEC cells and seems to increase the

percentage of cancerous cells [17].

A surprising influence of water-soluble charged phosphorus dendrimers on the

growth of neuronal cells on surfaces was observed. Glass substrates were covered by

multilayer films obtained by layer-by-layer (LbL) deposition of negatively (2-G4) and

positively (1-G4) charged dendrimers. Fetal cortical rat neurons were cultured on the

dendrimer films in order to investigate the influence of the surface charge of the

outermost layer on their adhesion and maturation. It was found that neurons attached

preferentially and matured slightly faster on film surfaces terminated with positively

charged dendrimers (1-G4) than on negatively charged surfaces (2-G4) [18]. These

experiments have to be correlated to previous experiments using polymers for coating

surface, for which the behavior of cells was shown to depend on a complex

combination of several parameters, including the molecular architecture and chem-

ical nature of polymers in terms of rigidity, functionality, surface charge, surface free

energy, roughness, hydrophilicity, and so on [19].

4.4. PHOSPHORUS DENDRIMERS FOR BIOLOGICAL IMAGING

New water-soluble fluorescent labels are always needed in biology, for trying to

understand biological events not only at the molecular level but also at the level of the

whole body. Fluorescent water-soluble dendrimers might afford versatile tools in this

field [20]. Fluorescent phosphorus dendrimers were synthesized for monitoring

the first steps of transfection experiments, and of the activation of the human immune

system; they will be shown in the parts 4.5.2 and 4.6.2, respectively. Here we will

focus on the use of phosphorus dendrimers for in vivo imaging. For this purpose,

dendrimers having two-photon absorption properties appears as the most suitable.

Indeed, two-photon excited fluorescence is of particular interest for the biological

community, due to a highly spatially confined excitation and an intrinsic three-

dimensional resolution [21], and an increased penetration depth in tissues and organs

with reduced photo-damages thanks to the excitation with a wavelength in the near-

infra-red region instead of the UV region. Such technique appears as particularly

promising for biological imaging of living animals. Quantum dots (inorganic nano-

crystals) have been found suitable for such purpose, but they suffer from several

drawbacks, in particular, an important toxicity that necessitate their coating, and a

PHOSPHORUS DENDRIMERS FOR BIOLOGICAL IMAGING 143

blinking phenomenon that diminishes their fluorescence properties. Thus, the syn-

thesis of a fully organic alternative to these quantum dots, which will not have such

defects, is of major importance.

In a first attempt, specially engineered fluorophores possessing two-photon

absorption (TPA) properties were grafted as terminal groups of phosphorus dendri-

mers. An additive behavior was observed for the fluorescence intensity, depending on

the generation of the dendrimer, hence on the number of fluorophores; generation four

has the same two-photon absorption efficiency than the best quantum dots [22].

Furthermore, TPAcooperative enhancement between thefluorophore terminal groups

was observed [23]. The high modularity of these systems allowed the synthesis of

other series of TPA dendrimers in which a blue fluorophore is used as core and the

terminal groups are ammonium derivatives to ensure the solubility in water. Such

architecture should prevent the quenching offluorescence often induced bywater. The

second generation 4a-G2was injected intravenously to a rat and allowed two-photon

imaging of the vessel of the dorsal part of its olfactory bulb at a depth of about

200 mm [24]. Analogously the dendrimer 4b-G2 having a green emitter as core was

used for the three-dimensional imaging of the blood vessel of themuscle of a xenopus

tadpole after intra-cardiac injection [25] (Fig. 4.6).

4.5. PHOSPHORUS DENDRIMERS AS NANO-CARRIERS

Various types of potential uses of dendrimers for drug delivery [26] are shown in

(Fig. 4.7). Covalent association that necessitates the cleavage for the delivery (A),

encapsulation inside dendrimers (B), and electrostatic interactions with the terminal

FIGURE 4.6 Two examples of dendrimers having a two-photon absorption fluorophore as

core and used for the imaging of the blood vessels of living animals.


groups of dendrimers represent the main types of uses. In the case of electrostatic

interactions, depending on the size of the object to be delivered, either several

objects cover the surface of the dendrimers (C) or several dendrimers wrap around the

object (D).

In the case of phosphorus dendrimers, we have mainly studied the case of

electrostatic interactions (cases C and D), preferably reinforced in the case C with

other effects such as lipophilic interactions.

4.5.1. Electrostatic Interactions with One Drug per Terminal Function

The dendrimers ended by carboxylic acids of type 2-Gn were reacted with an

aminolactitol elaborated for being an analog of the amphiphilic galactosylceramide

(galb1cer). Galb1cer is present on the surface of cells. It is known that in one of the

first events of infection, it acts through its highly specific affinity for the V3 loop

region of the gp120 viral envelope protein of HIV-1 [27]. The interaction of the

aminolactitol with the dendrimer occurs by proton transfer from the acid of the

dendrimer to the amine of the aminolactitol. The idea here was to obtain a chimera of

galb1cer, able to interact strongly with gp120, thus inhibiting the action of galb1cerand preventing the infection of cells. Several types of saline dendrimers, built either

from the trifunctional core [28] or the hexafunctional core [29] were synthesized, and

the same experiments were carried out alsowith dendrimers ended by various types of

phosphonates [30] bearing an alkyl chain of variable length [31]. Some examples are

shown in (Fig. 4.8). The influence of the core functionality and number of branches of

the dendrimerswas clearly identified for the series built from the trifunctional (5a-Gn)

and hexafunctional (5b-Gn) cores. Surprisingly, the bioactivity was found core-

dependent, but not generation dependent [29]. In the case of the phosphonate

derivatives (such as 6-Gn), the inhibitory assays indicate that the length of the alkyl

chain influences the efficiency of these inhibitors [30].

The same concept of saline interaction of phosphorus dendrimers with a drug was

applied for the ocular delivery of carteolol (an antihypertensive drug to

treat glaucoma) into rabbits’ eyes. For in vivo experiments, the biocompatibility

of the drug delivery system is particularly relevant in the case of eyes, which have a

quasi-impermeable corneal surface epithelium, necessitating a long residence time

for increasing the efficiency of the drug delivery. The most common method consists

FIGURE 4.7 Schematization of various types of interactions of dendrimers with active

substances, suitable for drug delivery.

PHOSPHORUS DENDRIMERS AS NANO-CARRIERS 145

in increasing the viscosity of the drug delivery system, but this may induce a disturbed

vision. The series of dendrimers 7-Gn was elaborated to fulfill two criteria: the first

one was the interaction of the terminal groups with carteolol, the second one was the

limitation of the number of chemical entities in the formulation; for this purpose the

dendrimer was built from an ammonium core, in order to replace the benzalkonium

derivative generally used as preservatives in formulations. The saline species 7-G0 is

fairly soluble in water, but 7-G1 and 7-G2 are poorly soluble (Fig. 4.8). The series of

dendrimers 7-Gn dissolved in water was instilled into the eyes of rabbits. No irritation

was observed whatever the generation used was and even after several hours.

Measurement of the quantity of carteolol having penetrated inside eyes shows

practically no difference between carteolol alone and carteolol interacting with

7-G0. Due to the very low solubility of 7-G2, the quantity of carteolol that penetrates

inside the eye is low, but higher than expected when compared with carteolol alone at

the same initial concentration (2.5 times larger) [32]. This observation highlights the

potential usefulness of this type of approach for drug delivery, even if the solubility in

water has to be increased.

4.5.2. Electrostatic Interactions for Transfection Experiments

The interactions of dendrimers with DNA [33], and particularly their use as synthetic

vectors in transfection experiments were recognized very early [34].Most of thework

in this field is carried out with PAMAM (polyamidoamine) dendrimers and their

derivatives [35], but cationic phosphorus dendrimers are also usable for such purpose.

The first experiments were carried out with dendrimers 1-Gn (n¼ 1–5) and with the

analogous series 8-Gn in which the proton on the terminal nitrogen atoms is replaced

by a methyl group. Both the series were used for the transfection of 3T3 cells with the

plasmid luciferase. The efficiency of these dendrimers depends on the generation (the

size and number of charges), and the nature of the terminal groups. The series

terminated by the tertiary ammonium groups (1-Gn) is nontoxic toward these cells,

and more efficient when the generation increases. On the contrary, the series with

FIGURE 4.8 Some examples of dendrimers having anti-HIV properties (5a,b-Gn–6-Gn),

and drug delivery system for carteolol (7-Gn), all obtained by saline interactions.


quaternary ammonium terminal groups (8-Gn) is toxic and the efficiency of the

transfection decreases when the generation increases, as shown in (Fig. 4.9).

This might be due to the high, stable, positive charge density, which may disrupt

the cell membrane, leading to cell death with the series 8-Gn. In the case of the series

1-Gn, the charge density can be modulated by the microenvironmental modification

of the pH valueswhen approaching the cell membrane. Thismight be also a key factor

for the release of the luciferase gene within endosome. It must be noted that the most

efficient dendrimers in this series (1-G3–1-G5) are as efficient as linear PEI (poly-

ethyleneimine) used at its optimal conditions [12]. The same dendrimers 1-Gn were

used for the efficient delivery of fluorescein-labeled oligodeoxyribonucleotide into

HeLa cells (human epithelioid cervical carcinoma cell line), and also of the DNA

plasmid containing the functional gene of enhanced green fluorescent protein

(EGFP) [36]. A fluorescent analogue of dendrimer 1-G2 was also synthesized in

an attempt to better understand the phenomenon implied in transfection experiments

with dendrimers [37].

Other types of phosphorus dendrimers ended by various types of ammonium

groups (Fig. 4.10) were also used for transfection experiments of single- and double-

stranded DNA into three cell strains (HeLa, HEK 293, and HUVEC). The dendrimer

with pyrrolidinium terminal groups 1b-G4 after protonation was found to be the most

efficient in this series, and also one of the less toxic as was seen in (Fig. 4.5) [17].

4.6. PHOSPHORUS DENDRIMERS AS DRUGS BY THEMSELVES

There exist numerous examples inwhich a known drugwas grafted as terminal groups

of dendrimers, in most cases of PAMAM dendrimers. On the contrary, in the case of

phosphorus dendrimers, no such attempt was made up to now, but in several cases,

FIGURE4.9 Structure of a dendrimer terminated by quaternary ammonium salts, and results

of transfection experiments of 3T3 cells with dendrimers of series 1-Gn (tertiary ammonium

groups) and 8-Gn, and the plasmid of luciferase.

PHOSPHORUS DENDRIMERS AS DRUGS BY THEMSELVES 147

phosphorus dendrimers possess biological properties that are not observed when the

monomeric analogue of the terminal group is tested alone. This fact is illustrated in

mainly two cases up to now, for antiprion activity and for stimulation of the human

immune system.

4.6.1. Antiprion Activity of Phosphorus Dendrimers

Transmissible spongiform encephalopathies are characterized by the accumulation of

the abnormal scrapie isoformof the prion protein (PrPSc) in the brain [38]. They are fatal

neurodegenerative diseases that include Creutzfeldt–Jakob disease in humans, scrapie

in sheep and goats aswell as bovine spongiform encephalopathy (BSE) [39]. The use of

dendrimers for such diseases was first proposed for PAMAM and PPI dendrimers [40].

Phosphorus dendrimers endingwith ammoniumgroups (1-Gn, n¼ 3–5) have also such

properties, and were found efficient even in vivo. These compounds decrease both the

quantity of PrPSc and infectivity in scrapie infected cells at nontoxic doses with an IC50

in the nM range (Fig. 4.11). These compounds are effective against preexisting PrPSc

FIGURE 4.10 Examples of dendrimers usable for transfection experiments after

protonation.

FIGURE 4.11 Antiprion activity of the series of dendrimers 1-Gn (n ¼ 3–5).


aswas observedwith brain homogenates infectedwith different prion strains, including

BSE. The fourth generation 1-G4was found to be the most efficient, thus it was chosen

for in vivo experiments with mice infected with the scrapie form of prion issued from

brain homogenates derived from terminally ill mice. A group of the infected mice

received 50 or 100mg of dendrimer 1-G4 per mouse every 2 days for 1month by

intraperitoneal injection (i.p.). The mice were sacrificed 30 days after infection.

Analysis of mouse spleens revealed that treatment with 50 or 100mg of dendrimers

inhibited PrPSc accumulation significantly by up to 66 or 88%, respectively. These

molecules have a high bioavailability and therefore, exhibit relevant potential for prion

therapeutics for at least postexposure prophylaxis [41].

These first experiments were followed by several assays to better understand the

influence of this dendrimer. It was shown that 1-G4 was able to interfere with

the aggregation behavior of the prion peptide PrP 185–208 by both slowing down the

formation of aggregates and decreasing the final amount of amyloid fibril, which is a

common hallmark of conformational diseases [42]. This processmight imply heparin,

which is able to accelerate or inhibit fibrilogenesis, depending on its concentration;

the dendrimer 1-G4 was shown to interact with heparin [43]. Furthermore, the same

dendrimer is able to interact with the Ab 1–28 peptide involved in Alzheimer

diseases [44]. It was shown by EPR analyses that the dendrimers interfere with the

nucleation phase of this peptide [45].

4.6.2. Stimulation of the Human Immune System

Peripheral blood mononuclear cells (PBMCs) are a very important component of the

immune system found with the circulating pool of blood, able to fight against

infections and intruders. It was shown previously that pamidronate (an amino

bisphosphonate, PCP linkage) can activate and/or multiply the TCRgdþ subset of

T lymphocytes [46] that is at the borderline between adaptive and innate immunity.

Aminodiphosphonate (PCNCP linkages) used as terminal functions of phosphorus

dendrimers, such as 3-G1, and the corresponding fluorescent analogue 3-G1-FITC, inwhich one terminal function (statistically) is replaced by FITC (fluorescein isothio-

cyanate) were used to determine if such compounds could interfere with the human

immune system.

The FITC-derived phosphorus dendrimer 3-G1-FITC (20 mM) incubated for

30minwith human PBMCs freshly isolated from a healthy donor induced exclusively

the labeling of monocytes (white blood mononuclear cells), which are a pivotal cell

population of innate immunity. Images of the interaction filmed by confocal video

microscopy showed that dendrimer 3-G1-FITC rapidly boundwithin a few seconds to

themonocyte surface andwas progressively internalizedwithin a fewminutes and for

hours. Within 3–6 days, monocytes in culture with dendrimer 3-G1 underwent

morphological changes indicating that they were activated by the dendrimer. Impor-

tantly, they remained viable over longer periods than control monocytes [15]. The

analysis of the gene expression of monocytes activated by dendrimer 3-G1 by

comparison with untreated monocytes showed that 78 genes were upregulated,

whereas 62 genes were downregulated. Analysis of these genes led to the hypothesis

PHOSPHORUS DENDRIMERS AS DRUGS BY THEMSELVES 149

of an alternative-like, anti-inflammatory activation of human monocytes [47]. Begin-

ning the study of the structure/activity relationship, it was shown that the correspond-

ingmonomeric azadiphosphonic derivative, analogous to the terminal groups of 3-G1,

displayed no activity. Furthermore, varying the number of terminal phosphonate

functions by using a core-controlled strategy for the selective functionalization of

1–6 Cl of (N3P3)Cl6 core shows that the activation of human monocytes depends on

the number of phosphonic groups, with a neat decrease of the efficiency for

compounds with less than six aminodiphosphonate groups per dendrimer [48].

The activation of monocytes occurs in short time cultures of PBMCs (maximum

6 days, and generally 1 day). Very surprisingly, a totally different behavior was

observed for longer times of culture. First, an important increase in the number of

PBMCs was observed (proliferation index 5.5 in 2week old cultures). Second,

phenotyping of the cells multiplied in cultures with 3-G1 revealed the prominence

of natural killer (NK) cells, which are part of the innate immunity. Experiments with

PBMCs obtained from six healthy donors revealed in all cases an important increase

in both the percentage and the number of NK cells. A mean multiplication of the

number of NK cells by a factor of 105 was achieved in medium supplemented with

10-G1 versus a mean multiplication only by a factor of 7.5 without it, after 4 weeks in

culture. These large-scale prototype cultures of PBMCs comprised 1million NK cells

on average at the beginning; multiplications over 500-fold were obtained with some

donors (Fig. 4.12) [49].

The multiplication of NK cells observed up to 500-fold in certain cases is

unprecedented, thus the biological properties of these NK cells were assayed. The

bioactivity of the NK cells generated in the presence of dendrimers is not modified.

Cultures with these dendrimers neither induce activation or inhibition of the NK cells

lytic response nor compromise direct toxicity for their target cells and preserve

autologous lymphocytes. This dendrimer constitutes a new tool in nanomedicine,

having in mind that the proliferation of NK cells was extremely tedious to

achieve [50]. In view of this important result, several variations of the initial structure

were synthesized in this case also. In particular, a new series of phosphorus-

FIGURE4.12 Number (A) and percentage (B) ofNK cells obtained from4week old cultures

of PBMCs without (gray dots) and with (black squares) 20mMof 3-G1. Data obtained from the

blood of six donors (a–f).


containing dendrimers capped with nonsymmetrical azadiphosphonic acids was

synthesized. Their ability to activate human monocytes of healthy individuals was

assessed. All of them were found active, but none of them displayed a higher activity

than 3-G1 [51]. Themechanism of action of this dendrimer is very complex; only part

of it is elucidated to date. As shown in the previous paragraph, the first step is the

activation of monocytes. It was also found that phosphonate-capped dendrimers

inhibit the activation, and therefore the proliferation of CD4þ T lymphocytes,

without affecting their viability. This allows a rapid enrichment of NK cells and

further expansion [52].

4.7. CONCLUSIONS

Dendrimers in general and phosphorus-containing dendrimers in particular constitute

a versatile platform whose intrinsic parameters can be controlled and modified on

demand. Such properties open the way to nano-medicine, with great promises as

nano-vehicles for drug delivery, and also as drugs by themselves, generally correlated

to their multivalency effects [53]. We are still working in all the fields evoked in this

paper, and exciting new results have been published recently [54], opening new

perspectives for biological uses of phosphorus dendrimers. The real challenge now is

to bridge the gap between fundamental researches and market applications. This

milestone has been reached recently for a topic related to those displayed in this

chapter, which concerns the elaboration of biosensors using phosphorus dendrimers.

They were found particularly useful for improving the sensitivity. Indeed, the

detection sensitivity of these devices was found 10- to 100-fold higher than arrays

made with most other functionalized glass slides [55]. Furthermore, the stability and

the reusability of these devices elaborated with phosphorus dendrimers were found

excellent, as well as the possibility to detect mutations [56]. This work has led to the

creation of a start-up last year (DendrisTD).

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Dendrimer-Based Drug Delivery Systems (From Theory to Practice) || Biological Properties of Phosphorus Dendrimers