WO2008010000A1

WO2008010000A1 - Molecular dendritic transporters

Info

Publication number: WO2008010000A1
Application number: PCT/GR2007/000038
Authority: WO
Inventors: Constantinos Paleos; Oreozili Sideratou; Dimitrios Tsiourvas; Ioannis Tsogas; Theodosios Theodosiou
Original assignee: National Center For Scientific Research
Priority date: 2006-07-21
Filing date: 2007-07-23
Publication date: 2008-01-24
Also published as: GR1006666B; GR20060100424A

Abstract

The present invention provides molecular transporters which are based on dendritic polymers that exhibit adaptive solubility behaviour and complementarity for specific lipidic membrane receptors, including cell receptors. Specifically these dendritic polymers will be used as carriers for transporting bioactive molecules to liposomes and biological cells. More specifically, the bioactive molecules encapsulated inside the nanocavities of these dendritic polymers are transported together with their carriers through the membranes. For inducing the transport of bioactive molecules, these polymers are functionalized with the introduction of groups such as guanidinium, folate, or carbohydrate moieties, etc., which are complementary to membrane receptors. Alternatively, the bioactive molecules to be transported are covalently attached to dendritic polymers which exhibit the above mentioned properties. The same molecular transport characteristics are preserved when the above mentioned dendritic polymers are covalently or non-covalently attached to liposomes. In the latter case the so-called dendronized liposomes are obtained which incorporate bioactive molecules either in the liposomal or dendritic segments of the nanoparticles.

Description

MOLECULAR DENDRITIC TRANSPORTERS

Technical Field

The present invention provides molecular transporters based on dendritic polymers capable of exhibiting adaptive solubility behaviour and complementarity to lipidic membrane receptors, including those of cells. Furthermore, by employing dendritic polymers, it is possible to encapsulate bioactive molecules inside the nanocavities which are a feature of the above mentioned polymers. Alternatively, it is possible to covalently attach the bioactive molecules. Dendritic polymers exhibiting the above mentioned characteristics may also be covalently or non-covalently attached to liposomes conferring molecular transporting properties on these carriers.

Prior Art

The application of molecular transporters based primarily on peptides and proteins and more specifically on their arginine-rich derivatives for the intracellular delivery of bioactive molecules has been intensively investigated in recent years [1-16] . The translocational ability of arginine bearing peptides is attributed to the presence of the guanidinium moieties. These guanidinium moieties are capable of interacting through hydrogen-bonding and electrostatic forces [17-18] with the phosphate groups or other anionic moieties present in liposomal and cell membranes, thus triggering the process of molecular transporting through bilayer membrane. The number of L-arginine residues appears to play a crucial role [11,19] in the translocational ability of these peptides, since oligoarginines with 8-10 residues exhibit the best interaction effectiveness. Differences in molecular architecture or spacing between guanidinium groups and peptide backbone seems to have only minor significance. Also, a series of dendrimers functionalized with various groups have been employed [20-21] as translocating agents of bioactive molecules. These papers however, only fragmentarily deal with the subject of molecular transporting through membranes without providing a generalized strategy and rationalization of transport mechanism.

The properties of dendritic polymers i.e. their ability to encapsulate bioactive molecules in their nanocavities and their capacity for convenient surface functionalization or more appropriately multifunctionalization, render these polymers promising candidates for molecular transporting. The present invention describes a new application for further exploiting the functionalization strategy applied to dendritic polymers, which is primarily based on the introduction of groups at the surface of the dendritic polymers which are complementary to membrane receptors, including cell receptors.

In this manner, with the present invention are prepared recognizable dendritic polymers of various generations which exhibit higher translocating ability compared to the starting dendritic polymers. Thus, recognizable groups such as guanidinium, folate or carbohydrate moieties are introduced at the surface of dendritic polymers. Such dendritic polymers which may be used as basic and starting compounds for the preparation of the materials of the present invention are the commercially available dendrimers such as poly(amidoamine) (PAMAM), diaminobutane poly(propylene imine) (DAB), hyperbranched polymer polyethyleneimine (PEI), other dendritic based biodegradable polyesters or other tailor-made dendritic polymers which exhibit analogous structural features and which have the ability to be functionalized by appropriate recognizable groups and possibly other selected groups such as hydrophobic moieties. Further exploitation of these recognizable dendritic polymers is achieved by their incorporation in liposomal bilayers. In this manner the so-called dendronized liposomes are prepared.

Although there is no intention throughout the present specification to be bound by theory, present investigations have suggested that the molecular transporting properties of these dendritic polymers and dendronized liposomes may be due to the combined action and synergy of the following parameters: a. The diversity of complementary groups, which is also dependent on the dendrimers' generation, type and degree of functionalization. b. The polyvalent effects exhibited by dendritic polymers which secure enhanced binding to membrane receptors including those of cells. These effects are exercized due to the proximity of the recognizable groups at the relatively limited external surface area occupied by dendritic polymers. c. The adaptive solubility behaviour exhibited by dendritic polymers, i.e. their ability to be dissolved in both polar and non-polar solvents. This ability is due to the property of dendritic polymers to expose to the solvent either their hydrophilic or hydrophobic segment. d. The facile modification of the hydrophobicity of the surface of the dendritic polymers. e. The property of dendritic polymers to encapsulate bioactive molecules in their nanocavities; the latter are simultaneously transported together with their transporting agents through the membranes. f. The property of dendritic polymers, when they are properly functionalized with hydrophobic moieties, to be anchored inside the liposomal membrane or cell membrane. This leads to novel nanoparticles that exhibit molecular transporting properties and this is also an objective of the present invention.

Summary of the invention

In accordance with a first aspect, the present invention provides dendritic polymers, of symmetric or non-symmetric architecture, which are functionalized with moieties facilitating transport, which exhibit adaptive solubility and are capable of acting as molecular transporters, characterized in that they present complementarity with respect to membrane receptors including those of biological cells.

The present invention describes a new application in exploiting the functionalization strategy, which is based on the introduction, at the surface of the dendritic polymers, of groups which are complementary to membrane receptors including cell receptors.

In this manner, in the present invention, recognizable dendritic polymers are prepared which exhibit higher translocating ability compared to the starting dendritic polymers. Thus, complementary groups such as guanidinium, folate, carbohydrate moieties or transferrin etc. are introduced at the surface of dendrimeric polymers.

Preferably, transporting agents for the dendritic polymers of the invention may be a block of 8- 10 guanidinium groups, penetrating peptides, etc. In accordance with a second aspect of the present invention, there is provided dendritic polymers in accordance with the first aspect in further combination with bioactive molecules. The bioactive molecules are introduced in dendritic polymers through either covalent or non- covalent binding, in such a way, so as to be transported simultaneously with the dendritic polymers in lipidic membranes including those of biological cells. Furthermore, the bioactive molecules may be encapsulated inside the nanocavities of the dendritic polymers.

In accordance with a third aspect of the present invention there is provided a method of molecular transportation, the method comprising

(i) providing a dendritic polymer, of symmetric or non-symmetric architecture, exhibiting adaptive solubility, characterized in that it presents complementarity with respect to membrane receptors;

(ii) exposing the dendritic polymer to membrane receptors under conditions suitable to facilitate the dendritic polymer's incorporation in and/or transport through the membrane.

The particular membrane concerned may be, for example, a liposomic or a cell membrane.

When the membrane is a liposomic membrane, the dendritic polymer is incorporated into the liposome, either covalently or non-covalently, providing a dendronized liposome in accordance with a fourth aspect of the present invention. Furthermore, in accordance with a yet further aspect of the invention, the so-called dendronized liposomes can further incorporate bioactive molecules either in the liposomal or dendritic segments of the nanoparticles.

In accordance with a yet further aspect of the present invention, there is provided uses of the dendronized liposomes and dendritic polymers, including the embodiments in which they support bioactive molecules. The use of these molecular transporters in therapy, particularly in the preparation of medicament for the treatment of a condition in which direct and enhanced molecular transport of a bioactive molecule is optimal, is envisaged. Detailed description of the Invention

The present invention provides molecular transporters based on dendritic polymers which exhibit adaptive solubility behaviour. The dendritic polymers that are described in this invention exhibit complementarity to lipidic membrane receptors including those of biological cells. According to the present invention the bioactive molecules are encapsulated inside the nanocavities of dendritic polymers which bear functional groups complementary to cell receptors. For instance guanidinylated dendritic polymers, functional dendritic polymers with cell targeting moieties such as carbohydrate moieties or folic groups, exhibiting adaptive solubility behaviour are appropriate as molecular transporters.

Alternatively, bioactive molecules which are to be transported into liposomes or cells are, according to the invention, covalently linked to the above mentioned recognizable dendritic polymers. The same molecular transport characteristics are preserved or intensified when the above mentioned dendritic polymers are covalently or non-covalently attached to liposomes, hi the latter case the so-called dendronized liposomes are prepared, which incorporate bioactive molecules either in the liposomal or dendritic segments of the nanoparticles.

The present invention deals with the synthesis of molecular transporters based on dendritic polymers which exhibit adaptive solubility behaviour and complementarity to membrane receptors including those of cells. These polymers originate either from symmetric, commercially available dendrimers such as poly(amidoamine) (PAMAM) or diaminobutane poly(propylene imine) (DAB), or from non-symmetric hyperbanched polymers such as polyethyleneimine (PEI), or from other dendritic based biodegradable polyesters or other dendritic polymers exhibiting analogous structural features, which can be functionalized with appropriate recognizable groups or hydrophobicity modifiers . Furthermore, other tailor-made dendritic polymers which have the ability to be functionalized by appropriate recognizable groups may be as well used as starting structures.

The invention also deals with the synthesis of polymers resulting from the covalent interaction of bioactive molecules with dendritic polymers which exhibit molecular transporting properties. The covalently attached bioactive molecules are transported together with the dendritic polymers to lipidic membranes, including cell membranes. The bioactive molecules may alternatively be encapsulated inside the nanocavities of these dendritic polymers and are simultaneously transported together with these carriers to membranes bilayer, i.e. those of liposomes or cell membranes.

The same molecular transport characteristics are preserved or intensified when the above mentioned dendrimers are covalently or non-covalently attached to liposomes. In the latter case the so-called dendronized liposomes are prepared, which incorporate bioactive molecules either in the liposomal or dendritic segments of the nanoparticles.

When dendronized liposomes are prepared, in which the dendritic polymers exhibiting molecular transporting properties are covalently attached or non-covalently incorporated to liposomes, the molecular transport characteristics are preserved. The dendronized liposomes incorporate bioactive molecules either in the liposomal or dendritic segments of the nanoparticles. For instance, dendritic polymers with molecular transport properties are alkylated or functionalized with cholesterol in order their hydrophobic segments to be "anchored" inside the liposomal membrane. Alternatively, properly functionalized alkylated dendritic polymers (including dendrons) are employed for the formation of liposomes which encapsulate the bioactive molecules to be transported.

In the present invention, a mechanism of transporting bioactive molecules through liposomal membranes or cell membranes, is presented. According to this mechanism the recognizable moieties of dendritic polymers towards specific liposomal or cell membrane receptors, for instance the positively charged guanidinium groups are attached to membranes through their interaction with the negatively charged phosphate or carboxylate groups. The moieties are attached through combined electrostatic and hydrogen bonding interactions. In this case the guanidinylated dendrimers become less polar due to neutralization of their charges by anionic groups of the membranous surface and therefore, become more susceptible to enter the hydrophobic bilayer. At the same time, since the interfacial area of the membrane bilayer is less polar compared to bulk water, the interaction of guanidinium groups with phosphate or carboxylate groups or folate receptor or transferrin receptor is favoured. Synergistically to this behavior, as soon as the guanidinylated dendritic polymers are located in the vicinity of the liposomal or cell membrane bilayer, according to this invention, the adaptive solubility behaviour (chameleon behaviour) of the dendrimers is activated, rendering the dendritic polymers even more hydrophobic.

According to the present invention surface hydrophobicity of dendritic polymers can be modified by the introduction of appropriate groups, for instance through acetylation. A proper balance between hydrophobicity and molecular recognition ability, as obtained through appropriate functionalization according to the present invention, leads to effective transport through cell membranes. A schematic representation of a dendritic polymer with recognizable groups, that this invention describes, is shown in Figure 1.

Appropriate hydrophobicity of the surface of the dendritic polymers, obtained though functionalization, facilitates transport of the polymer through the membrane. This process is shown schematically in Figure 2. Crucial for this behavior is the presence of several guanidinium groups or other type of recognizable groups on the relatively limited dendritic surface, which facilitate interaction with complementary groups of the liposomes or cell membranes and subsequent transport through membrane bilayer.

FIGURES

Figure 1 is a schematic repesentation of a dendritic polymer with recognizable groups.

Figure 2 is a schematic representation of the translocation mechanism of dendrimers through liposomal membranes.

Figures 3A, 3B, 3C, and 3D show the fluorescence loading of A549 human lung carcinoma cells with DABi₆(Ac)₈G₈ and DAB₃₂(Ac)i₈Gi₄ at 20 μM versus incubation time.

Figures 3A and 3B show: Cells loaded with DAB₁₆(Ac)₈G₈ and DAB₃₂(Ac)_I8Gu respectively, in the absence of foetal bovine serum (FBS).

Figures 3C and 3D show: Cells treated with DAB₁₆(Ac)₈G₈ and DAB₃₂(Ac) _I8G₁₄ in 10% FBS.

Fluorescence was in all cases registered from: intact cells in PBS ( * ), cell supernatant

C ^'"), supernatant of cells treated with 5mM digitonin for 5 minutes for selective permeabilisation of their plasma membrane (^""*^"") and cells subsequently treated with triton

X-100 for complete internal membrane rupture (^{~τ^}).

Figures 4A, 4B, 4C and 4D show fluorescence microscopy images of A549 cells incubated for

4h with DABi₆(Ac)₈G₈ (A, C) and DAB₃₂(Ac)₁₈Gi₄ (B, D) at 20 μM dendrimer concentration. Figure 4A and Figure 4B were acquired following dendrimer incubation in the absence of FBS while Figure 4C and Figure 4D were taken following dendrimer incubation in the presence of 10% FBS.

Examples

Specific embodiments of the various aspects of the present invention will now be illustrated with reference to the following examples, which are not intended to limit the scope of the invention in any way.

Examples of the synthesis of dendritic polymers with molecular transporting properties are described below as well as experiments illustrating transporting properties. For performing molecular transporting experiments properly selected liposomes are used as cell models since these nanoparticles are structurally the closest analogs of biological cells. Procedures for the synthesis of a guanidinylated dendrimeric derivatives, originating from diaminobutane poly(propylene imine) dendrimers (DAB-n, where n number of external amino groups), are described as well as experiments illustrating molecular transporting through properly functionalized liposomes. Human lung carcinoma cell line A549 was also employed for assessing molecular transporting properties of selected dendrimeric derivatives that were previously prepared and used for liposomal transport. These examples are described for the illustration of the invention only and in no way should it be considered that the object of the invention should be restricted to those.

For the preparation of molecular transporters based on dendrimeric or hyperbranched polymers (collectively named dendritic polymers) which is the object of the present invention, commercially available dendritic polymers such as poly(amidoamine) (PAMAM), and diaminobutane poly(propylene imine) (DAB), dendrimers of various generations or poly(ethyleneimine) hyperbranched polymer (PEI) and selected dendritic hyperbranched polyesters of various molecular weights or other tailor-made dendritic polymers which exhibit analogous structural features were subjected to functionalization and in certain cases to multifunctionalization. EXAMPLE 1. Fully Guanidilylated Dendritic Polymers

In dimethylformamide solution in which 8 parts of lH-pyrazole-1-carboxamidine hydrochloride (99%) and 8 parts of iV,N-diisopropylethylamine are dissolved, 1 part of poly(propylene imine) dendrimer of the fifth generation (DAB-64), dissolved in the same solvent, is added under an inert atmosphere. The solution is allowed, under stirring, at room temperature for 24 hours. The product of the reaction is precipitated with diethyl ether and isolated by centrifugation. The product obtained is purified from traces of the solvent and subsequently it is dissolved in water. The aqueous solution is purified by dialysis in order all the low molecular weight compounds to be removed from the solution. The introduction of guanidinium group and the percentage of substitution is determined by ΝMR.

IH ΝMR: (250 MHz, DMSO-dό) δ= 7.85 (broad s, NH), 7.20 (broad s,NH2+), 3.15 (m, NCH2CH2CH2NH), 2.95 (t, NCH2CH2CH2NH), 2.70 (t,CH2NH2), 2.50 (m, NCH2CH2CH2N, NCH2CH2CH2CH2N, NCH2CH2CH2NH2), 1.55 (m, NCH2CH2CH2N, NCH2CH2CH2CH2N, NCH2CH2CH2NH), 1.40 (broad s, NH2).

Completely guanidinylated dendrimers of the third generation (DAB- 16) and fourth generation (DAB-32) were also obtained with the same method. Dendrimers bearing L-arginine moieties at their surface were also prepared.

EXAMPLES 2. Partially Guanidinylated and Partially Acetylated Dendritic

Polymers

A series of partially guanidinylated DAB-32 dendrimers with 6, 12, 18 and 24 guanidinium groups was prepared. The remaining primary amino groups were reacted with propylene oxide. Partially acetylated dendrimers were also prepared by reaction of distilled acetic anhydride with DAB- 16 and DAB-32 in the presence of DIPEA in freshly distilled methanol. DAB-16(Ac)₈ and DAB-32(Ac)₁₈ with 8 and 18 acetyl groups were prepared and the remaining amino groups were guanidinylated as above (shown in Scheme I). Fully guanidinylated DAB- 16 and DAB-32 were also prepared.

DAB_n(SpG) , n = 16, 32

Scheme I

Example 2.1 Partially Acetylated Dendritic Polymers

Partially acetylated dendrimers were obtained by reacting poly(propylene imine) dendrimers of third (DAB- 16) or fourth (DAB-32) generation with acetic anhydride in the presence of triethylamine in anhydrous methanol (Scheme I). Thus for DAB- 16, 0.5 g of the dendrimer (4.74 mmol of surface amino groups) were dissolved in dry methanol followed by the addition of 10% molar excess of triethylamine with respect to the amino groups (5.2 mmol) and 2.61 mmol of acetic anhydride (55% molar excess with respect to the amino groups of the dendrimer), affording DAB_n(Ac)_n-x. The structure of partially acetylated dendrimers was established by ¹H and ¹³C NMR.

¹H NMR (500 MHz, D₂O) δ = 3.10 (t, CH₂NHCOCH₃), 2.60 (t, CH₂NH₂), 2.30-2.50 (m, NCH₂CH₂CH₂N, NCH₂CH₂CH₂CH₂N, NCH₂CH₂CH₂NH₂, NCH₂CH₂CH₂NH), 1.80 (s, NHCOCH₃), 1.45-1.70 (m, NCH₂CH₂CH₂N, NCH₂CH₂CH₂NH, NCH₂CH₂CH₂NH₂), 1.45 (m, NCH₂CH₂CH₂CH₂N). ¹³C NMR (62.9 MHz, D₂O) δ = 177 (NHCOCH₃), 53-54 (NCH₂CH₂CH₂N, NCH₂CH₂CH₂CH₂N, NCH₂CH₂CH₂NH), 42 (CH₂NHCOCH₃), 40.5 (CH₂NH₂), 30 (NCH₂CH₂CH₂NH), 27 (NCH₂CH₂CH₂N), 23-24 (NCH₂CH₂CH₂CH₂N, CH₃).

Example 2.2 Guanidinylation of Partially Acetylated Dendritic Polymers

The remaining primary amino groups of the dendrimers were subsequently guanidinylated using a 10% molar excess of lH-pyrazole-1-carboxamidine hydrochloride as already reported

10 in the literature affording the corresponding dendrimeric derivatives, DAB_n(Ac)_n-xG_x. Their structure was established by ¹H and ¹³C NMR.

¹H NMR (500 MHz, D₂O and DMSOd₆) δ = 7.80-8.00 (broad s, NH of guanidinium group and NHCOCH₃), 6.90-7.6 (broad d, NH₂ ⁺), 3.0-3.20 (m, CH₂NHCOCH₃ and NCH₂CH₂CH₂NHC(NH₂ ⁺):.), 2,35-2,70 (m, NCH₂CH₂CH₂N, NCH₂CH₂CH₂CH₂N, NCH₂CH₂CH₂NH), 1.85 (s, NHCOCH₃), 1.50-1.75 (m, NCH₂CH₂CH₂N, NCH₂CH₂CH₂NH), 1.45 (m, NCH₂CH₂CH₂CH₂N). ¹³C NMR (62.9 MHz, D₂O) δ = 177 (NHCOCH₃), 159 (NHC(NH₂ ⁺)₂), 52-54 (NCH₂CH₂CH₂N, NCH₂CH₂CH₂CH₂N, NCH₂CH₂CH₂NH), 42 (CH₂NHCOCH₃), 40 (CH₂NHC(NH₂ ⁺)₂), 27 (NCH₂CH₂CH₂NH, NCH₂CH₂CH₂N), 23-24 (NCH₂CH₂CH₂CH₂N, CH₃).

Example 2.3 Spacer- Functionalized Guanidinylated Dendritic Polymers

Furthermore, two derivatives having a spacer between the amino group of the dendrimer and the guanidinium moiety were prepared (Scheme I) by the reaction of DAB₁₆ or DAB₃₂ with N-Boc-4-isothiocyanatobutyl amine. For the preparation of the DAB₃₂ derivative, 0.5 g of the dendrimer (4.74 mmol of surface amino groups) were dissolved in dry DMF and 1.2 g of jV-Boc-4-isothiocyanatobutyl amine (10% molar excess with respect to the dendrimer amino groups) were added in the presence of triethylamine. Removal of the Boc group was performed with 95% TFA for 1 hour. The product was precipitated and redissolved in water containing triethylamine to deprotonate the amino groups. After dialysis for the removal of by-products and lyophilization, the product was dissolved in methanol and the primary amines were guanidinylated, as described above, affording the final products DAB_π(spG).

The structure of DAB_n(spG) was established by ¹H and ¹³C NMR. ¹H NMR (500 MHz, D₂O and DMSO-d₆) δ = 7.80-8.00 (broad s, NH of guanidinium group and NHCSNH), 6.90- 7.6 (broad d, NH₂ ⁺), 3.25-3.65 (m, CH₂NHCSNHCH₂), 3.00-3.20 (m, CH₂NHC(NH₂ ⁺)I), 2.5- 2.9 (m, NCH₂CH₂CH₂N, NCH₂CH₂CH₂CH₂N, NCH₂CH₂CH₂NH), 1.65-1.95 (m, NCH₂CH₂CH₂N, NCH₂CH₂CH₂NH) 1.40-1.65 (m, CSNHCH₂CH₂CH₂CH₂NH, NCH₂CH₂CH₂CH₂N). ¹³C NMR (62.9 MHz, D₂O) δ = 182 (NHCSNH), 159 (NHC(NH₂ ⁺)₂), 55.4-52.8 (NCH₂CH₂CH₂N, NCH₂CH₂CH₂CH₂N, NCH₂CH₂CH₂NH), 46.5

11 (CH₂NHCSNHCH₂), 43 (CH₂NHC(NH₂ ^{^})₂), 28 (NHCH₂CH₂CH₂CH₂NH), 26.5 (NCH₂CH₂CH₂NH, NCH₂CH₂CH₂N), 23 (NCH₂CH₂CH₂CH₂N).

3. Monitoring Transport Properties of Dendritic Polymers

Before monitoring the transport properties of dendritic polymers through cell membranes the same polymers were applied to liposomes which are considered as the closest analogs of biological cells, hi addition the dendritic polymers were labelled with fluorescein isothiocyanate for probing with fluorescence spectroscopy the translocation of the dendritic polymers from the external to the internal site of liposomal membrane. Before describing these experiments, liposomes' preparation is described followed by the results of characterization related to their size, stability and fusion.

Example 3.1 Preparation and characterization of Liposomes.

Small unilamellar liposomes of 100 run diameter were prepared using the extrusion method. The lipids were dissolved in a 2:1 butanol/chloroform mixture in a round-bottom flask. The solvents were evaporated and the remaining film was dried under vacuum for 24 hours. The film was hydrated with phosphate buffer (1OmM, pH 7,4) and the sample was extruded from two-stacked polycarbonate membranes with pore diameter of 100 nm. In a typical experiment, for the preparation of a 4 ml dispersion, 2.19 mg of DHP, 60,8 mg of PC and 14,71 mg of Choi were used.

The mean diameter of these liposomes was in the range of 70-100 nm. It should be noted that molecular transport experiments were performed under conditions that liposomes were non-leaky, i.e. guanidinylated dendrimers having non-disruptive effect for the liposomal bilayer and non-fusing to large aggregates.

Example 4. Preparation of Fluorescein Isothiocyanate (FITC)-labelled dendrimers:

To monitor the translocation of dendrimers as they interact with liposomes, FITC-labelled dendrimers were prepared. Thus, for instance, guanidinylated DAB-32 was dissolved in dry methanol and a methanolic solution of fluorescein isothiocyanate was added. When L-arginine modified dendrimers were employed the reaction was carried out in carbonate buffer pH=9.5. The mixture was allowed to react for 24 hours at room temperature, under constant stirring in Argon atmosphere. To avoid photobleaching of the fluorescent probe, the reaction was carried out in the dark. Unreacted FITC was removed by dialysis against water. H NMR established that one FITC molecule reacted with one molecule of modified dendrimer. The reaction scheme is given below (Scheme II).

DAB-Gn DAB-FGn

Scheme II

Example 5. Transport of Guanidinylated Dendrimers through Liposomal Membranes Experiment 1. Transport of Fully and Partially Guadinylated Dendrimers

Fluorescence studies were employed to investigate the ability of the guanidinylated dendrimers to penetrate the liposomal membrane. Quenching experiments were performed using the FITC-labeled dendrimeric derivatives and a 20% (w/v) NaI solution. Thus, 100 \L of the liposomal dispersion was diluted to 2.5 ml with phosphate buffer and FITC-labeled guanidinylated dendrimers were added to a 50% guanidinium/phosphate molar ratio. Samples were incubated for 20 minutes at 25 ⁰C and 65 ⁰C. For comparison, FITC-labeled guanidinylated dendrimers were also titrated with NaI. Excitation was performed at 490 run and emission was monitored at 520 nm. The quenching constant Ksv was determined by linear regression of the Stern- Volmer equation

F₀/F = l + Ksv[Q] where F and F₀ are the fluorescence intensities in the presence and in the absence of the quencher respectively and [Q] is the molar concentration of the quencher.

Experiments were also performed using Nal-loaded liposomes. Loading was performed by hydration of the lipid film with phosphate buffer containing NaI 0.142 M. Removal of the non-encapsulated NaI was performed by column chromatography using SEPHADEX G-50 (coarse). To avoid leakage of NaI due to osmotic effects, phosphate buffer

13 containing NaCl at the same concentration was used as eluent. To the resulting suspensions, FITC-labeled modified dendrimers were added to a guanidinium/phosphate molar ratio of 0.05. Fluorescence intensity was measured prior to mixing (F₀), 20 minutes after mixing and after incubating the sample at 65 C for 20 minutes. The fraction Fo/F was used to evaluate the percentage of dendrimer penetrating the bilayer, where F₀ is the intensity of the sample immediately after mixing and F is the intensity after the incubation period. Blank experiments were also carried out in order to determine NaI leakage from the interior of liposomes, which would contribute to the fluorescence quenching. Thus, liposome suspensions were incubated at 65 ⁰C for 20 minutes, then FITC-labeled modified dendrimers were added and the fluorescence intensity was recorded.

For finding the location where the dendrimeric derivatives reside after their interaction and incubation with the liposomal bilayer, two different fluorescence techniques were used. Quenching experiments upon addition of I^" in the bulk aqueous phase were employed to determine the solvent accessibility of the fluorescein attached on the surface of dendrimeric derivatives following their interaction with liposomes. If the dendrimeric derivatives were located either inside the bilayer or in the aqueous core of the liposomes, a reduction in the apparent quenching constant, Ksv, would have to be observed since I^" ions cannot quantitatively penetrate the bilayer.

The Ksv values were determined after incubation of the suspensions at 25 or 65 ⁰C for 20 min and compared with the corresponding values observed in the absence of liposomes. After incubation at 25 ⁰C even at 5% molar ratios the Ksv values obtained are within experimental error equal to the values obtained in the isotropic media suggesting that fluorescein is primarily located in the bulk phase. From the results presented in Table 1 it becomes evident that when the guanidinium/phosphate molar ratio employed is 50% the Ksv values obtained after incubating at 25 or 65 ⁰C have only minor differences. Therefore, at these high molar ratios, in which formation of large aggregates predominates, this technique can not provide conclusive information about the location of dendrimers. However, of significant interest are the processes taking place at low guanidinium/phosphate molar ratios, i.e. at ratios that do not lead to considerable aggregation and also at ratios that are anticipated in in-vivo experiments. Thus, when the guanidinium/phosphate molar ratio employed was 5%, the Ksv values after incubation of the samples at 65 ⁰C are considerably smaller than those determined at 25 ⁰C for

14 the dendrimeric derivatives bearing 6, 12 or 18 guanidinium groups. Thus, the dendrimeric derivatives are less accessible to iodine anions suggesting that these derivatives are located either in the lipid bilayer or in the interior aqueous pool. On the contrary, the derivatives with high end-group functionalization, i.e. DAB-G24 and DAB-G32, or the one with no guanidinium groups, DAB-GO, do not show significant decrease of the Ksv values.

Based on the above results, the penetration and localization of the dendrimeric derivatives inside the aqueous core was established by fluorescence spectroscopy employing NaI loaded liposomes and fluorescein labelled dendrimers (DAB-FGn) at 5% guanidinium/phosphate molar ratio. Fluorescence quenching would be observed only if the dendrimeric derivative translocates across the bilayer, where I^" can access and therefore quench the fluorescein moiety. Thus, for the highly guanidinylated dendrimeric derivatives (DAB-FG32 and DAB- FG24) no decrease in the fluorescence intensity was recorded, suggesting that they are unable to penetrate the liposomal bilayer (Table 2). The high hydrophilicity and surface charge of these derivatives is apparently a restrictive factor for translocation across the hydrophobic membrane. Instead they are positioned at the bilayer as the previous fluorescence experiments suggest. Dendrimers bearing 6, 12 and 18 guanidinium groups at their periphery are able to penetrate the bilayer and translocate in the core with the partially guanidinylated dendrimer DAB-FGi₈ showing the higher translocation efficiency at 25 ⁰C, while DAB-FG₁₂ is the more efficient when the bilayer is in the liquid-crystalline phase.

Table 1. Calculated Λ^v of FITC -labeled dendrimers following their interaction with liposomes at 5 and 50% guanidinium/phosphate ratio after incubation at the corresponding temperature for 20 minutes.

Dendrimeric [guanidinium]/[phosphate] =0.5 [guanidinium]/[phosphate] =0.05 derivative ^_SV (25 ⁰C ) Ksv (65 ⁰C ) ^S_V (25 ⁰C ) ^SV (65 ⁰C )

DAB-FG₃₂ 15.3 13.0 4.25 6.0

DAB-FG₂₄ 22.5 22.1 21.0 17.4

DAB-FG₁₈ 9.7 7.8 20.2 11.1

DAB-FG₁₂ 10.5 8.6 21.9 16.6

DAB-FG₆ 9.2 7.9 17.6 11.5 Table 2. Calculated penetration of modified FITC-labeled dendrimers following interaction with Nal-loaded liposomes after incubation at the corresponding temperatures for 20 minutes.

Dendrimeric penetration (%) penetration (%) derivative at 25 ⁰C at 65 ⁰C

DAB-FG32 0 0

DAB-FG24 0 0

DAB-FG 18 7.9 12.3

DAB-FG 12 1.6 23.1

DAB-FG6 2.1 18.1

DAB-FGO 0 0

Experiment 2. Transport of Partially Guadinylated-Acetylated Dendrimers and Spacer Functionalized Derivatives

In this experiment also quenching experiments employing FITC -modified dendrimers and I^" as a quencher were performed for the determination of Ksv values during their interaction with liposomes following incubation at 25 ⁰C and 65 ⁰C for 20 minutes at guanidinium/phosphate molar ratios of 5% and 50% (Table 3). When interaction is taking place at 25 ⁰C, the Ksv values are very close to the Ks v values determined for the corresponding lipo some-free dendrimeric solutions. Thus, although interaction between dendrimers and liposomal membranes is ensuing, the dendrimer is still accessible to the I^" ions, i.e. it is basically located at the liposomal-water interface. When the interactions are occurring at 65 ⁰C, at 5% molar concentration, the calculated Ksv values are lower than those at 25 ⁰C reflecting that in this case, the fluorescent label is less accessible to iodine ions. The observed reduction of the quenching constants leads to the assumption that a portion of the dendrimeric derivatives are either incorporated in the liposomal bilayer or have penetrated it reaching the aqueous liposomal core. On the contrary when 50% molar ratios were employed no significant differences in Ksv values were observed between the two different temperatures. This suggests that when high dendrimer concentrations are employed most of the dendrimers are also localized on the outer surface of the liposomes. Translocation of the dendrimeric derivatives across the lipid bilayer was determined using Nal-loaded liposomes. Reduction in the fluorescence intensity would be observed only if the dendrimers reached the aqueous liposomal core. Measurements were performed at low guanidinium/phosphate molar ratios (5%) where aggregation and leakage is negligible, as shown by Dynamic Light Scattering and calcein fluorescence measurements, and where the ATsv values differ considerably from those determined in the isotropic media. The results are presented in Table 4. It is evident that dendrimer translocational ability is negligible at the lower temperature, as also expected from the ATsv values, but becomes significant when the bilayer is in the liquid crystalline phase.

The acetylated derivatives show an enhanced translocation ability in contrast to the DAB_n(spG) derivatives which exhibited either low penetrability, for the third generation derivative, or even negligible for the fourth generation one. Acetylated derivatives, following interaction and charge neutralization of their guanidinium moieties by the liposomal phosphate groups, are less hydrophilic compared to non-acetylated derivatives and therefore it is possible to penetrate the liposomal bilayer more efficiently, in consistence with the mechanism presented in previous work.^p4'^39] With regard to DAB_n(spG), the third generation derivative proved more efficient. The lower number of surface groups along with the spacer flexibility provided effective charge neutralization of the guanidinium moieties by the liposomal phosphate groups, leading to a non polar complex able to penetrate the liposomal bilayer. In the case of the fourth generation derivative, however, the size and dense surface functionalization inhibited effective internalization. Therefore, appropriate balance between the number of guanidinium groups interacting with the phosphate groups and the degree of hydrophobicity is favoring efficient translocation.

17 Table 3. Calculated K_sv of FITC-labeled dendrimers following their interaction with liposomes at 5% and 50% guanidinium/phosphate molar ratio after incubation at the corresponding temperature for 20 minutes.

[Guanidinium]/[phosphate]=0.5 [Guanidinium]/[phosphate]=0.05

Dendrimeric

Ksv at 25 ⁰C £sv at 65 ⁰C Ksv at 25 ⁰C £sv at 65 ⁰C derivative

DAB₁₆(Ac)₈G₈ 28.2 24.0 13.6 10.0

DAB₃₂(Ac)I₈G₁₄ 10.9 10.7 13.0 11.0

DAB₁₆(SpG) 8.0 8.2 12.8 6.4

DAB₃₂(SpG) 5.9 6.0 6.9 7.0

Table 4. Calculated penetration of FITC-labeled dendrimeric derivatives following interaction with Nal-loaded liposomes at 5% guanidinium/phosphate molar ratio after incubation at the corresponding temperatures for 20 minutes.

dendrimeric penetration (%) at 25 ⁰C penetration (%) at 65 ⁰C derivative

DAB₁₆(Ac)₈G₈ 1.0 UA

DAB₃₂(Ac)_I8G₁₄ 4.4 20.0

DAB₁₆(SpG) 1.4 7.5

DAB₃₂(SpG) 1.3 2.0

Example 6: Transport of Guanidinylated Dendrimers through Cellular Membranes Cell culture: The human lung carcinoma cell line A549 was used which was grown in RPMI 1640 with 10% FBS, penicillin/streptomycin at 37 ⁰C in a 5% CO₂ atmosphere. Cells were inoculated into either 96-well plates (2^χl O⁴ cells/ lOOμl media per well) or 35mm dishes with 2cm radius round glass coverslips (5 ^χ 10⁴ cells/2 mL media per well per dish) 24 h before experiments.

Fluorescence Microscopy in cells: Cells inoculated on coverslips in 35mm Petri dishes, as elaborated above, were incubated for 4h with 20μM FITC-DAB₁₆(Ac)₈G₈ and 20μM FITC-

18 DAB₃₂(Ac)₁₈Gi₄. These concentrations were found to be subtoxic by standard XTT assays, performed both immediately following incubation, as well as 24h later.

Fluorescence time-course experiments: Dendrimer loading of A549 cells was studied through FITC fluorescence. DABi₆(Ac)₈G₈ and DAB₃₂(Ac) _I8G₁₄ coupled to FITC were added to cell media with/without 10% FBS at a 20 μM final concentration. The cells were washed twice with PBS and fluorescence was registered versus incubation time from 0 to 5 hours, in a Fluostar Galaxy plate reader (BMG Labtechnologies) with the excitation wavelength set to 492 run and emission set to 520 nm.

The fluorescence time-course uptake study was performed in intact cells, their supernatant as well as the supernatant following selective plasma membrane permeabilization with 5μVI digitonin (5min) and subsequently complete disruption of intracellular membranes with Triton X-100 (0.25%). Blank values measured in control cells (not incubated with dendrimers) were subtracted in each case.

Fluorescence uptake experiments.

In order to investigate the cellular uptake of the dendrimeric derivatives those derivatives found to exhibit the most efficient translocation ability for liposomes, namely DABj₆(Ac)8G8 and DAB32(Ac) _I8Gj₄, were selected. The cellular uptake of these dendrimeric derivatives was studied versus incubation time in the presence and absence of Foetal Bovine Serum (FBS) through fluorescence as described above. The results shown in Figure 3, indicate that cell loading is much more efficient in the absence of FBS (Figures 3A, 3B), probably due to complexation of the dendrimers with FBS causing the formation of precipitates, as verified by optical microscopy. In all cases DAB₃₂(Ac)₁₈G₁₄ (Figures 3B, 3D) was found to permeate cells more efficiently than DAB₁₆(Ac)₈Gg (Figures 3 A, 3C). This can be attributed to the higher guanidinylation of the former, resulting in more efficient interaction with the available anionic polar groups located on the outer cell membrane. The intact cell fluorescence measurements showed that in absence of FBS DAB₃₂(Ac)₁₈G₁₄ has at least a 7fold higher cell internalization capability than DAB₁₆(Ac)₈G₈ (Figures 3A, 3B), while in the presence of FBS (10%) this ratio drops to 3-4. The same patterns are followed by fluorescence registration from the cell supernatant, indicating a dynamic equilibrium of dendrimer influx-efflux. Digitonin selective permeabilisation of the plasma membrane, allows the cytosolic fraction of the cells to be released into the surrounding media. Fluorescence measurements of this supernatant containing the cytosolic contents, showed a marked cytoplasmic localization of the dendrimer in the case of DAB32(Ac)i₈Gi₄ while the corresponding fluorescence registration from DAB₁₆(Ac)₈G₈ was practically background. Finally, complete cellular membrane rupture with Triton X-100 showed that a considerable part of internalized dendrimer was accumulated in subcellular organelles and mainly the nucleus as shown by fluorescence microscopy (vide infra). In fact, in the case of DAB₁₆(Ac)₈G₈ the majority of internalized dendrimer was found to localize in subcellular organelles since as explained above the cytosolic fluorescence was negligible. With regard to DAB₃₂(Ac) _I8G₁₄ the cytosolic localisation seems to be equivalent to that in inner organelles as can be seen from the appropriate traces in Figures 3B, 3D.

Fluorescence Microscopy

The subcellular localisation of selected dendrimeric derivatives was investigated employing fluorescence microscopy. Thus, A549 cells seeded on 22mm round coverslips were incubated with subtoxic concentrations of the FITC conjugated dendrimeric derivatives with/without FBS, namely 20μM DABi₆(Ac)₈G₈ and DAB₃₂(AC)_I8G_H and imaged according to the protocol described earlier. The results are shown in Figures 4A - 4D. Specifically, internalization of DABi₆(Ac)₈G₈ in absence of FBS is shown in Figure 4A with a corresponding image for DAB₃₂(Ac)_I8Gi₄ appearing in Figure 4B. From these images it is evident that the fluorescence resulting from incubation of the cells in the absence of FBS with 20 μM Of DABi₆(Ac)₈G₈ is mainly nuclear while in the case of DAB₃₂(Ac)i₈Gi₄ there is also a substantial cytosolic localization in addition to the distinct nuclear localisation. This is in good agreement with our results from the fluorescence uptake studies. Internalisation of both DABi₆(Ac)8G8 and DAB32(AC)_{I 8}G_H in the presence of FBS (20 μM 4h incubation, 10% FBS) is shown in Figures 4C, 4D respectively and it has to be noted that a very small number of cells were found to have internalized either of the compounds in contrast to the case of FBS absence.

REFERENCES

[1] R. B. Kim Transporters and Drug Discovery: Why, When, and How, MoI. Pharmaceutics, 3 (2006) 26 - 32. [2] L. Zhang, J. M. Strong, W. Qiu, L. J. LesKo, S.-M. Huang, Scientific Perspectives on Drug Transporters and Their Role in Drug Interactions, MoI. Pharmaceutics, 3 (2006) 62 - 69. [3] J. B. Rothbard, T. C. Jessop, P A. Wender, Adaptive translocation: the role of hydrogen bonding and membrane potential in the uptake of guanidinium-rich transporters into cells, Adv. Drug Delivery Rev., 57 (2005) 495-504.

[4] S. Futaki, Membrane-permeable arginine-rich peptides and the translocation mechanisms, Adv. Drug Delivery Rev., 57 (2005) 547-558.

[5] S. Futaki, S. Goto, Y. Sugiura, Membrane permeability commonly shared among arginine- rich peptides, J. MoI. Recognit., 16 (2003) 260-264.

[6] J. S. Wadia, S. F. Dowdy, Modulation of Cellular Function by TAT Mediated Transduction of Full Length Proteins, Curr. Protein. Pept. ScL, 4 (2003) 97-104.

[7] L.R. Wright, J.B. Rothbard, P.A. Wender, Guanidinium rich peptide transporters and drug delivery, Curr. Protein. Pept. Sci. 4 (2003) 105-124.

[8] A. Prochiantz, Messenger proteins: homeoproteins, TAT and others, Curr. Opin. Cell Biol. 12 (2000) 400^106.

[9] U. Langel, Cell-Penetrating Peptides: Processes and Applications, CRC Press, Boca Raton, FL, 2002.

[10] E. Vives, P. Brodin, B. Lebleu, A truncated HTV-I Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus, J. Biol. Chem. 272 (1997) 16010- 16017.

[11] S. Futaki, T. Suzuki, W. Ohashi, T. Yagami, S. Tanaka, K. Ueda, Y. Sugiura, Arginine- rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery, J. Biol. Chem. 276 (2001) 5836- 5840. [12] P.A. Wender, D.J. Mitchell, K. Pattabiraman, E.T. Pelkey, L. Steinman, J.B. Rothbard, The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 13003- 13008. [13] Kxeider, P.L. McGrane, P.A. Wender, P.A. Khavari, Conjugation of arginine oligomers to cyclosporin A facilitates topical delivery and inhibition of inflammation, Nat. Med. 6 (2000) 1253-1257.

[14] T. A. Kirschberg, CL. VanDeusen, J.B. Rothbard, M. Yang, P.A. Wender, Arginine-based molecular transporters: the synthesis and chemical evaluation of releasable taxol-transporter conjugates, Org. Lett. (2003) 3459- 3462. [15] S. Futaki, M. Niwa, I. Nakase, A. Tadokoro, Y. Zhang, M. Nagaoka, N. Wakako, Y. Sugiura, Arginine carrier peptide bearing Ni(II) chelator to promote cellular uptake of histidine tagged proteins, Bioconjug. Chem. 15 (2004) 475- 481.

[16] V.P. Torchilin, R. Rammohan, V. Weissig, T.S. Levchenko, TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 8786- 8791.

[17] M. Onda, K. Yoshihara, H. Koyano, K. Ariga, T. Kunitake, Molecular Recognition of Nucleotides by the Guanidinium Unit at the Surface of Aqueous Micelles and Bilayers. A Comparison of Microscopic and Macroscopic Interfaces, J. Am. Chem. Soc. 118 (1996) 8524- 8530.

[18] K. Ariga, T. Kunitake, Molecular Recognition at Air- Water and Related Interfaces: Complementary Hydrogen Bonding and Multisite Interaction, Ace. Chem. Res. 31 (1998) 371- 378.

[19] P. A. Wender, D. J. Mitchell, K. Pattabiraman, E. T. Pelkey, L. Steinman, J. B. Rothbard, The design, synthesis and evaluation of molecules that enable or enhance cellular uptake: Peptoid molecular transporters, Proc. Natl. Acad. Sci. U.S.A., 97 (2000) 13003-13008. [20] P. Kolhe, E. Misra, R. M. Kannan, S. Kannan and M. Lieh-Lai, Drug complexation, in vitro release and cellular entry of dendrimers and hyperbranched polymers, Int. J. Pharm. 259 (2003) 143-160.

[21] M. El-Sayed, CA. Rhodes, M. Ginski, H. Ghandehari, Transport mechanism(s) of poly (amidoamine) dendrimers across Caco-2 cell monolayers, Int. J. Pharm. 265 (2003) 151-157. [22] M. Mammen, S.K. Choi, G.M. Whitesides, Polyvalent interactions in biological systems: Implications for design and use of multivalent ligands and inhibitors, Angew. Chem., Int. Ed. 37 (1998) 2755-2794.

[23] P.I. Kitov, D.R. Bundle, On the Nature of the Multivalency Effect: A Thermodynamic Model, J. Am. Chem. Soc. 125 (2003) 16271-16284.

22

Claims

CLAIMS 23

1. Dendritic polymers, of symmetric or non-symmetric architecture, which are capable of acting as molecular transporters, which exhibit adaptive solubility and are functionalized with moieties facilitating transport, characterized in that they present complementarity with respect to membrane receptors including those of biological cells.

2. Dendritic polymers according to claim 1 which have a symmetric architecture and are based on polymers selected from the group consisting of poly(amidoamino) (PAMAM) dendrimers or -diaminobutane poly(propyleno imino) (DAB) dendrimers.

3. Dendritic polymers according to claim 1 which have a non-symmetric architecture and are based on polymers selected from the group consisting of hyperbranched polymer poly(ethyleneimine) (PEI).

4. Dendritic polymers according to any one of claims 1 to 3 wherein transporting agents are a block of 8-10 guanidium groups or penetrating peptides.

5. Dendritic polymers according to claim 1 which are based on polymers selected from the group consisting of dendritic based biodegradable polyesters.

6. Dendritic polymers according to any one of claims 1 to 5 wherein the complementarity with respect to membrane receptors is by virtue of functionalization with groups selected from guanidinium, folate, carbohydrate moieties or transferrin.

7. Dendritic polymers according to claim 6 wherein the complementarity with respect to membrane receptors is by virtue of functionalization by guanidinium groups capable of interacting with membrane receptors through electrostatic forces and / or hydrogen bonds.

8. Dendritic polymers according to claim 7 wherein the membrane receptors comprise phosphate or carboxylate groups, folate receptor or transferrin receptor.

9. DDeennddrriittiicc ppoollyymmeerrss aaccccoorrddiinngg ttoo a annyy oonnee ooff ccll;aims 1 to 8 wherein the polymers further comprise or support one or more bioactive molecules.

10. Dendritic polymers according to claim 9 wherein the bioactive molecules are supported through either covalent or non-covalent bonding.

11. Dendritic polymers according to claim 9 or claim 10 wherein the bioactive molecules are encapsulated inside the nanocavities of the dendritic polymers.

12. A method of molecular transportation, the method comprising

13. A method according to claim 12 wherein the membrane comprises a lipidic cell membrane.

14. A method according to claim 12 wherein the membrane comprises a liposomal membrane.

15. A method according to claim 14 wherein the dendritic polymer is incorporated covalently into the liposome.

16. A method according to claim 14 wherein the dendritic polymer is incorporated non- covalently into the liposome.

17. A method according to any of claims 14 to 16 wherein the dendritic polymer presents complementarity with respect to the liposome by virtue of functionalization.

18. A method according to any of claims 14 to 16 wherein the dendritic polymer presents complementarity with respect to the liposome by virtue of appropriate functionalization.

19. A dendronized liposome obtainable by any one of the methods of claims 12 to 18.

20. A dendronized liposome according to claim 19 which further comprises or supports one or more bioactive molecules.

21. A dendronized liposome according to claim 20 wherein the one or more bioactive molecules is located in the liposomal segment of the nanoparticle.

22. A dendronized liposome according to claim 20 wherein the one or more bioactive molecules is located in the dendritic segment of the nanoparticle.

23. A method of treatment which comprises administering to a subject in need a bioactive molecule supported by a dentritic polymer as defined in any one of claims 1 to 18.

24. A method of treatment which comprises administering to a subject in need a bioactive molecule supported by a dendronised liposome as defined in any one of claims 19 to 22.

25. Use of a dendritic polymer as defined in any one of claims 1 to 18 in therapy.

26. Use of a dendronized liposome as defined in any one of claims 19 to 22 in therapy

25