WO2012014046A2

WO2012014046A2 - Supramolecular aggregates as drug carriers on cell expressing receptors for branched neurotensin

Info

Publication number: WO2012014046A2
Application number: PCT/IB2011/001731
Authority: WO
Inventors: Diego Tesauro; Antonella Accardo; Giancarlo Morelli; Carlo Pedone; Chiara Falciani; Alessandro Pini; Luisa Bracci
Original assignee: Diego Tesauro; Antonella Accardo; Giancarlo Morelli; Carlo Pedone; Chiara Falciani; Alessandro Pini; Luisa Bracci
Priority date: 2010-07-27
Filing date: 2011-07-27
Publication date: 2012-02-02
Also published as: WO2012014046A3

Abstract

The present invention relates to supramolecular aggregates (liposomes, vesicles, or micelles) for target selective drug delivery; more specifically it relates to supramolecular aggregates derivatized with Neurotensin peptide in a branched form and filled, in their inner compartment or in the phospholipid bilayer, with a pharmaceutical active principle such as a cytotoxic chemotherapeutic drug. The aggregates are selectively driven by the exposed branched bioactive peptides to specific receptors overexpressed in malignant cells.

Description

SUPRAMOLECULAR AGGREGATES AS DRUG CARRIERS ON CELL EXPRESSING RECEPTORS FOR BRANCHED NEUROTENSIN

The invention also concerns the preparation of the compositions, as well as injectable aggregates, their use and a kit for clinical use.

BACKGROUND OF THE INVENTION

Nanoparticles have attracted much attention for their potential application as in vivo carriers of active principles. The use of liposomes as drug carrier systems was proposed by Gregoriadis and Ryman in the early 70s.[l] These supramolecular aggregates are nontoxic, biodegradable, and non-immunogenic. Because of their size, which typically ranges in mean diameter from 50-300 nm, liposomes display unique pharmacokinetic properties. These include clearance via the reticuloendothelial system, which results in a relatively long systemic circulation time, as well as hepatic and splenic distribution. Furthermore, liposomes exhibit preferential extravasation and accumulation at the site of solid tumors due to the increased endothelial permeability and reduced lymphatic drainage in these tissues, which has been defined as the enhanced permeability and retention effect (EPR).[2-5] The hydrophobic core of micelles and inner cavity of liposomes are carrier compartments which are able to accommodate large amount of drugs, while the shell, consisting of brush-like protective corona, stabilizes them in physiological or serum conditions and reduces toxicity of the active principle in non-target organs. Thus, associating a drug with liposomes markedly changes its pharmacokinetic and pharmacodynamic properties and lowers systemic toxicity; furthermore, the drug is prevented from early degradation and/or inactivation following introduction to the target organism. [6-9] In systemic administration, micelles or liposomes should satisfy several base requirements: high drug loading, biodegradability, long blood circulation times, slow plasma clearance, and controllable drug release profiles. Many research efforts have been directed towards improving the safety profile of the cytotoxic anthracyclines doxorubicin, daunorubicin, and vincristine, which are associated with severe cardiotoxic side effects. For example, the alkylating agent doxorubicin acts by intercalating DNA and has been used in the liposomal formulation known as Doxil for ovarian cancer treatment. Doxil exhibited increased circulation time and decreased cardiovascular-related toxicity as compared to free doxorubicin,[10] while encapsulated doxorubicin liposomes, combined with cyclophosphamide, showed high antitumor effects in an experimental pulmonary metastatic melanoma mouse model. [1 1] Labeling of nanoparticles with bioactive markers that are able to direct them toward specific biological target receptors has led to a new generation of delivery systems for active principles. [12] Peptides and antibodies are the bioactive markers commonly used to prepare target-selective supramolecular aggregates, such as micelles and liposomes. [ 13-15] In particular, low- molecular-weight peptides that remain stable in vivo, well-exposed on the aggregate surface, and in appropriate conformation for binding, could be promising tools to selectively deliver nanoparticles filled with active components to the cellular target. The peptides could act to target neovascularization sites in angiogenic processes or membrane receptors overexpressed in cancer cells. Receptors for various endogenous peptides are overexpressed in several human tumors and can be used as tumor antigens. [ 16] In the last decade, a number of different derivatives of somatostatin, luteinizing hormone-releasing hormone (LHRH), bombesin, [17] cholecystokinin,[18] neurotensin, and neuropeptide Y[19] have been used to target tumor cells. We have been studying the use of tetrabranched peptides (NT4) that contain the sequence of the human regulatory peptide neurotensin (NT) as tumor targeting agents. NT is a 13 amino acid peptide originally isolated from calf hypothalamus, the full amino acid sequence is QLYENKPRRPYIL, with the C-terminus is comprised of short active fragment 8-13 (RRPYIL). NT has the dual functions of neurotransmitter or neuromodulator in the nervous system and local hormone in the periphery. NT receptor type 1 (NTS 1) is overexpressed in severe malignancies such as small cell lung cancer and colon, pancreatic, and prostate carcinomas. [16, 19] NT has additional well-established targets on the cell surface: NT receptor 2, NT receptor 3 (NTR3, or Sortilin), and SorLA (LRU); these latter two membrane proteins belong to the novel Vps l Op-domain family. [20] Importantly, Sortilin has recently been described as having an important role in pancreatic ductal adenocarcinoma tumor cells. [21] It is well known that peptides synthesized in a branched form not only become resistant to proteases but also increase linear peptide biological activity through multivalent binding. Using branched NT4 fragment 8-13, conjugated to various functional units for tumor imaging and therapy,[22] we found that NT4 conjugated to methotrexate or 5-fluoro- deoxyuridine resulted in 60% and 50% reduction, respectively, [22-24] of tumor growth in xenografted mice. Additionally, branched NT peptides have been proven to discriminate between binding of tumor versus healthy tissue in human surgical samples, validating neurotensin receptors as highly promising tumor biomarkers.[23] Results obtained in the past for NT4 indicated that these branched peptides are promising, novel, multifunctional, cancer-targeting molecules. The flexibility of this synthetic approach suggested it would be possible to use the branched NT on liposomal surfaces for specific drug delivery into tumor cells.

SUMMARY OF THE INVENTION

The present invention concerns the preparation and the use of liposomes filled with a cytotoxic drug and functionalized on the external surface with a branched moiety containing four copies of the neurotensin (NT) peptide or its analogues. The new functionalized liposomes are obtained by co-aggregation of a phospholipid with a new synthetic amphiphilic molecule which contains a dendrimeric scaffold derivatized with_a. lipophilic moiety and the neurotensin peptide in a tetrabranched form. The neurotensin peptide belonging to the neurotensin family (endogenous neurotensin peptide, truncated forms, analogues with agonist or antagonist activity), in the tetrabranched form, are well known for their ability to bind the neurotensin receptors.

DESCRIPTION OF THE INVENTION

The present invention concerns the preparation and the use of supramolecular aggregates, preferably with a liposomal structure, obtained by co-aggregation of one or more phospholipids with a peptide containing monomer having general formula (I). The peptide containing monomer of formula (I) is present in the amount of 0.5- 15% (as molar ratio), preferably from 2 to 5%,sin the final liposomal composition. In the liposome composition other components may be included: for instance cholesterol, in order to stabilize the liposomal membrane or carbohydrates (i.e. trehalose or sucrose), in order to store the liposome integrity inhibiting fusion between liposomes during drying process.

The surfactant present in prevalent amount is a phospholipid or a mixtures of ionic and non-ionic surfactants, for example phosphatidylcholine linked to saturated or unsaturated fatty acids; preferably the phospholipid is selected from soy phosphatidylcholine (SPC), egg phosphatidylcholine (EPC) l ,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC), l,2-DipaImitoyl-,s?i- Glycero-3-Phosphocholine (DPPC), l,2-Disteroyl-src-Glycero-3- Phosphocholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), hydrogenated egg phosphatidylcholine (HEPC), phosphatidylglycerol (PG), 1 ,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-Dioleoyl-sn- glycero-3-Phospho-rac- l -glycerol phosphatidylglycerol (DOPG), l ,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-Dipalmitoyl- sn-glycero-3 Phospho-rac-1 -glycerol phosphatidylglycerol (DPPG), 1 ,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1 ,2-Distearoyl-sn- glycero-3 -Phospho-rac- 1 -glycerol phosphatidylglycerol (DSPG).

The peptide containing monomer having general formula (I) (see Figure 1) is formed by three units (P, L and H)

P-L-H (I)

wherein:

P is a peptide based system containing a branched skeleton and four copies of the neurotensin peptide (1-13 NT), or its truncated form (8-13 NT), or other NT analogues, as reported in the following table. The branched skeleton can be obtained linking through amide bonds three molecules containing two amine functions and a carboxylic function (i.e.: lysine, ornithine or diaminopropionic acid). Table. Peptide sequences of 1-13 NT, 8-13 NT, and other selected NT analogues:

* where pyroGlu can be substituted with Gin.

- L is a spacer containing polyoxoethylene moieties (Peg or analogs) or sequence of molecules containing polyoxoethylene functions with molecular weight between 600 and 5000 Dalton. The spacer could also contain one or more amino acid residues; one or more of them could be a residue containing three reactive functions (i.e.: lysine, ornithine or diaminopropionic acid, glutamic acid, aspartic acid).

- H is an hydrophobic moiety containing two lipophilic chains. The two lipophilic chains are aliphatic or aromatic hydrocarbons with a number of carbon atoms in the range 8-22 (preferably between 12-18). They may have the same or a different length, and may be saturated, unsaturated or polyunsaturated. The two chains are anchored to the L spacer through a bifunctional molecule.

The aggregates may be in form of vesicles or liposomes having a size ranging between 50 and 500 nm; lipidic bilayer or double strand aggregates, having a thickness ranging between 5 - 20 nm.

Mixed liposomes containing the phospholipid surfactant and the synthetic monomer having general formula (I) are prepared using known sonication and extrusion procedures. Liposomes can be also obtained by using the post-insertion method. In this case, liposomes based by the phospholipid surfactant are initially prepared by using standard procedures, and the monomer of formula (I) is inserted in liposomes by adding a solution of the NT-peptide containing monomer.

The obtained liposomes can be stored as lyophilized powder. In this case, carbohydrates (i.e. trehalose or sucrose) should be added to the final composition in order to store liposome integrity during the lyophilization process.

The liposomes may be used as carriers of drugs, particularly of antineoplastic agents such as doxorubicin, daunorubicin, epirubicin, esorubicin, idarubicin or an antitumor platinum complexes such as cis- platinum.

Said drugs may be loaded into the liposomes using the pH gradient method or the ammonium gradient method.

The invention also provides pharmaceutical compositions comprising said drug loaded aggregates and at least one carrier or excipient. The compositions of the invention may be successfully used for the therapy of tumors after enteral or parenteral administration at doses which will be easily determined by the skilled practitioner , taking into account the established dosage regimens of the drugs entrapped into the aggregates.

The following Examples further illustrate the invention.

EXAMPLE 1 :

Synthesis of a monomer having general formula (I).

(Arg-Arg-Pro-Tyr-Ile-Leu)₄-(Lys)₂-Lys-pAla-Lys-(AhOH)₂-(C18)₂ ([8- 13]NT₄Lys(C18)₂)

The monomer ([8-13]NT₄Lys(C18)₂), reported in Figure 2a, was synthesized on solid phase using Rink amide (MBHA) resin (0.54 mmolg^{" 1}; 0.048 mmol, 0.090 g) as polymeric support. After swelling of the resin in 2.0 rnL of Ν,Ν-dimethylformamide (DFM) for 1 h, the Fmoc protecting group was removed by a mixture of piperidine/DMF (30:70). The carboxylic group of Dde-Lys(Fmoc)-OH (0.100 mmol, 0.532 g) was activated by 1.0 equiv of benzotriazol-l-yl-oxy-tris(pyrrolidino) phosphonium (PyBop), 1- hydroxybenzotriazole (HOBt), and 2.0 equiv of N,N-diisopropylethylamine (DIPEA) in DMF. The solution was added to the resin, and the slurry suspension was stirred for 1 h. Coupling of the lysine residue was performed twice and checked by the Kaiser colorimetric test. The solution was filtered and the resin washed with three portions of DMF and three portions of CH₂C1₂. After the removal of the Fmoc protecting group from the lysine side chain, two molecules of Fmoc-Ahoh-OH were sequentially coupled according to previously described coupling and deprotection conditions. After Fmoc deprotection, Ν,Ν-dioctadecylsuccinamic acid (0.1 mmol, 0.62 g) was condensed, with 2.0 equiv dissolved in DMF/CH₂C1₂ (50:50). The lipophilic moiety was activated in situ by the standard HOBt/PyBop/DIPEA procedure, and the coupling reaction proceeded for 1 h. The resin was washed three times with DMF, then, l-(4,4-dimethyl-2,6-dioxo-cyclohexylidene) 3-methylbutyl (Dde) was removed from the N-terminal amine functionality of the lysine residue by treatment with DMF/hydrazine (98:2). The peptide-resin was stirred with 3.0 mL of this solution for 10 min. The treatment was repeated twice, and the deprotection reaction was monitored by the qualitative Kaiser test and UV spectroscopy. The following amino acid derivatives were coupled sequentially to the free N-terminal amine functionality using the previous described coupling and deprotection conditions: Fmoc- DAIa-OH, Fmoc- Lys(Fmoc)-OH, Fmoc-Lys(Frnoc)-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc- Tyr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH. Finally, the N-terminal Fmoc protecting group was removed, and the amphiphilic peptide was cleaved with TFA containing triisopropylsylane (2.5 %), and water (2.0%) over a period of 24 h. The peptide product was precipitated by adding water dropwise at 0°C, purified by HPLC, and lyophilized. The final product ([8- 13]NT4Lys(C18)₂) was obtained in 25% yield and analyzed by HPLC and MALDI-TOF mass spectroscopy: HPLC: t_R=39.2 min; MALDI-TOF: MW=5068 Da.

EXAMPLE 2:

Liposome formulation and characterization.

Mixed liposomes containing the phospholipid surfactant DOPC and the synthetic amphiphilic monomer having general formula (I), [8- 13]NT4Lys(C18)₂, in a 95:5 molar ratio were prepared using known sonication and extrusion procedures. Briefly, monomers were dissolved in a chloroform/methanol mixture, and the solvent was subsequently evaporated. The resulting film was hydrated in 0.1M buffered solution (pH 7.4) at room temperature. Aggregation was successfully achieved by sonicating for 30 min and subsequent extrusion. Complete [8- 13]NT4Lys(C18)₂ incorporation into DOPC liposomes was verified by analyzing a small amount of the liposomal solution using a Sephadex column. Presence in the first gel filtration fractions of a peak at 275 nm, with a UV absorbance corresponding to the Tyr residue in [8-13]NT4Lys(C18)₂, confirms the presence of an NT fragment on the liposome shell. Self-assembled DOPC liposomes were also prepared and characterized for the purpose of comparison. Dynamic light scattering measurements were taken for pure DOPC and mixed DOPC-[8- 13]NT4Lys(C18)₂ aggregates in 10 mM phosphate buffer at pH 7.4. Both aggregate systems show monomodal distribution, due to the translational diffusion process, with apparent translational diffusion coefficients D. The hydrodynamic radius (RH) found for DOPC and DOPC-[8- 13]NT4Lys(C 18)₂ liposomes were 90.0±8.1 and 88.3±4.4 nm, respectively.

Liposomes can be also obtained by using the post-insertion method. In this case DOPC liposome are initially prepared by using standard procedures and the monomer of formula (I) is inserted in liposomes by adding a solution of the peptide containing monomer.

The obtained liposomes can be stored as lyophilized powder. In this case, carbohydrates (i.e. trehalose or sucrose) should be added to the final composition in order to store liposome integrity during the liophilization process.

EXAMPLE 3:

Doxorubicin loading.

DOPC-Doxo and DOPC-[8- 13]NT4Lys(C18)₂-Doxo liposomal formulations were prepared by loading doxorubicin HCl into DOPC and DOPC-[8- 13]NT4Lys(C 18)₂ supramolecular aggregates, respectively. Doxo was loaded using the pH gradient method with free Doxo removed by gel filtration or by ultracentrifugation. Briefly, the liposomal solution was prepared as reported above at pH 4.0 using 0.1M citrate-phosphate buffer. The pH was adjusted from 4.0 to 7.4 by dropwise addition of a l .OM stock solution of NaOH. Next, 210 xL of 2.36 10^"3 M Doxo solution in 2.5 mM phosphate buffer were added to 100 μΐ. of liposomal solution. This suspension was stirred for 30 min at room temperature. The Doxo concentration in all experiments was determined by spectroscopic measurements (UV or fluorescence) using calibration curves obtained by measuring absorbance at 480 nm or fluorescence emission at 590 nm. Emission spectra were recorded at room temperature. Equal excitation and emission bandwidths were used throughout experiments, with a recording speed of 125 nm min^"1 and automatic selection of the time constant. Subsequently, unloaded Doxo was removed using a Sephadex G50 column pre-equilibrated with 2.5 mm phosphate buffer at pH 7.4. The Doxo loading content (DLC, defined as the weight ratio of encapsulated Doxo versus the amphiphilic moieties) was quantified by subtraction of the amount of Doxo removed from the total amount of Doxo loaded.

EXAMPLE 4:

Cell internalization of DOPC-[8-13]NT4Lys(C18)₂-Doxo liposomes. The selective internalization of NT4-derivatized liposomes, compared to nude liposomes, was tested in HT29 human colon adenocarcinoma and TE671 human rhabdomyosarcoma. The HT29 cell line expresses two NT receptors, NTR1 and NTR3, while TE671 only expresses NTR3, as confirmed by RT-PCR (Figure 3a and 3b). Internalization was studied by confocal microscopy following the red fluorescence signal of Doxo (Figure 3c and 3d). Cells were incubated at a number of temperatures (4, 25, and 37°C) for various time intervals (from 30 min-3 h). The concentrations of liposomes, (200 nm, 400 nm, and 1 mm) were calculated as the molarity of Doxo. Figure 3 is representative and shows images taken after 2 h incubation with 200 nm liposomes at 37°C. At lower temperatures and all concentrations, the internalization of both types of liposomes is slightly lower, as expected. The internalization process is near completion by 30 min at all temperatures and concentrations. The intracellular red fluorescence was very strong for cells incubated with DOPC-[8-13]NT4Lys(C18)₂-Doxo liposomes, whereas unfunctionalized liposomes gave very faint signals under the same experimental conditions. A comparison between functionalized and nude liposomes suggests an important net advantage obtained through conjugation of the particles with branched NT.

EXAMPLE 5:

Cytotoxicity of DOPC-[8-13]NT4Lys(C18)₂-Doxo liposomes

HT29 and TE671 cells were incubated with various concentrations, from 8 nm-25 mm, of DOPC-[8- 13]NT4Lys(C 18)₂-Doxo liposomes and DOPC-Doxo liposomes. After 8 h incubation, cells were washed and incubated for six days. Washing was performed to avoid diffusion of free Doxo from the liposomes during the six day incubation period. As reported in Figure 4, DOPC-[8-13]NT4Lys(C 18)₂-Doxo liposomes showed an EC₅₀ value of 1.30 mm in HT29, whereas the EC₅₀ value of DOPC-Doxo liposomes was 5.48 mm; therefore, the labeling of liposomes with a tumor selective moiety produced a four-fold increase in activity (p<0.05). The same increase in cytotoxicity was observed in TE671.

EXAMPLE 6:

FACS analysis

HT29 and TE671 cells were incubated with various concentrations

(from 500 nm-10 mm) of either DOPC-[8- 13]NT4Lys(C 18)₂-Doxo liposomes or DOPC-Doxo liposomes (Figure 5). At the highest concentration, the difference between the two types of liposomes was maximized. In HT29, DOPC-[8- 13]NT4Lys(C18)₂-Doxo liposomes gave an increase in fluorescent signal of 40% compared to the analogous DOPC-Doxo liposomes. In TE671 the NT4-derivatized liposomes showed a 20% increase in fluorescence with respect to the nude analogues.

EXAMPLE 7:

Synthesis of a monomer having general formula (I).

(H-Gln-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu)₄- ((AP)₂Gly)₂-(AP)₂Gly-Lys-(AhOH)₂-(C18)₂ ([l-13]NT₄Lys(C18)₂)

The monomer ([l-13]NT₄Lys(C18)₂), schematized in Figure 2b, was synthesized on solid phase using Rink amide (MBHA) resin (0.54 mmolg^"1; 0.048 mmol, 0.090 g) as polymeric support. The synthesis in the first steps was carried out in the same way of the previous monomer obtaining the H- Lys-(AhOH)₂-(C18)₂ linked to the resin. The following amino acid derivatives were coupled sequentially to the free N-terminal amine functionality using the previous described coupling and deprotection conditions: (Fmoc)₂(N,N-bis-3- aminopropyl-Gly-OH [(Fmoc₂-(AP)₂Gly)], (Fmoc)₂(N,N-bis-3-aminopropyl- Gly-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn-OH, Fmoc-Glu(OBut)₂-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln-OH. Finally, the N-terminal Fmoc protecting group was removed. N-terminal glutamine residues can spontaneously cyclize to become pyroglutamate. The amphiphilic peptide was cleaved with TFA containing triisopropylsylane (2.5 %), and water (2.0%) over a period of 24 h. The peptide product was precipitated by adding water dropwise at 0°C, purified by HPLC, and lyophilized. The final product ([l-13]NT4Lys(C18)₂) was obtained in 25% yield and analyzed by HPLC and MALDI-TOF mass spectroscopy: HPLC: t_R=39.2 min; MALDI-TOF: MW= 8617.5 Da.

EXAMPLE 8:

Liposome formulation and characterization.

Mixed liposomes containing the phospholipid surfactant DOPC and the synthetic amphiphilic monomer having general formula (I), [ 1- 13]NT4Lys(C18)₂, in a 95:5 molar ratio were prepared using known sonication and extrusion procedures, as reported in Example 2. The hydrodynamic radius (RH) found for DOPC-[ l- 13]NT4Lys(C18)₂ liposomes were 90.0±15.0. Also in this case mixed DOPC-[l- 13]NT4Lys(C18)₂ liposomes could be prepared according the post-insertion method.

The obtained liposomes can be stored as lyophilized powder. In this case, carbohydrates (i.e. trehalose or sucrose) should be added to the final composition in order to store liposome integrity during the Iiophilization process.

EXAMPLE 9:

Doxorubicin loading.

DOPC-[l- 13]NT4Lys(C 18)₂-Doxo liposomal formulations were prepared by loading doxorubicin-HCl into DOPC-[l- 13]NT4Lys(C 18)₂ liposomes. Doxo was loaded using the pH gradient method, and free Doxo removed by gel filtration, as reported in Example 3.

EXAMPLE 10:

Cell internalization of DOPC-[l-13]NT₄Lys(C18)₂-Doxo liposomes.

Internalization was studied by confocal microscopy following the red fluorescence signal of Doxo, similarly to the experiment performed with DOPC-[8-13]NT₄Lys(C 18)₂-Doxo liposomes. TE671 and HT29 cells (Figure 6a and 6b, respectively) were incubated at 25°C for 30 min with DOPC-[l- 13]NT₄Lys(C18)₂-PE-NBD-Doxo liposomes (DOPC / [l -13]NT₄Lys(C18)₂ / PE-NBD 94/5/1 molar ratio). PE-NBD is a green fluorophore that highlights liposomes fusion with cell membrane. The concentrations of liposomes were calculated as the molarity of Doxo (5microM). The intracellular red fluorescence was very strong for cells incubated with DOPC-[l- 13]NT4Lys(C18)₂-Doxo liposomes (Figure 6a and 6b, right, arrow; grey in the black and white figure), whereas unfunctionalized liposomes gave very faint signals under the same experimental conditions (Figure 6a and 6b, left). The green signal from PE-NBD (white in black and white figures) is predominantly localized on the outer cell membrane, showing that in both types of cells the liposome fuses with the cell membrane. The green fluorescence pervades uniformly the membrane of HT29 whereas it concentrates in clots in TE671 membrane.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1: Schematic representation of the monomer having general formula (I).

FIGURE 2: 2a) Structural formula of [8-13]NT₄Lys(C18)₂, 2b) Structural formula of [l- 13]NT₄Lys(C18)₂.

FIGURE 3. RT-PCR expression of NT receptors in a) HT29 and b) TE671 cells, lane 0=DNA standard, lane 1 = NT 1, lane 2 = NTR2, lane 3 = NTR3, lane 4 = beta 2 microglobulin. Confocal microscopy: c) HT29 and d) TE671 cells were incubated with DOPC-[8- 13]NT₄Lys(C18)₂-Doxo liposomes (200 nm, right) and with DOPC-Doxo liposomes (200 nm, left) for 2 h at 37°C. Plasma membrane was stained with lectin-FITC. In black and white figures red of Doxo is gray (arrow) and green of FITC is white.

FIGURE 4: Cytotoxicity: a) HT29 and b) TE671 cells were incubated with various concentrations, from 8 nm-25 mm, of DOPC-[8- 13]NT₄Lys(C18)₂-Doxo liposomes (a) and DOPC-Doxo liposomes (o). After 8 h incubation, cells were washed and incubated for six days. Percentage of cell survival is calculated in comparison to untreated controls. EC₅₀ values: DOPC-[8-13]NT₄Lys(C18)₂-Doxo liposomes EC₅₀=1.81 mm (HT29), EC₅₀=1.53 mm (TE671); DOPC-Doxo liposomes EC₅₀=5.48 mm (HT29), EC₅₀=6.72 mm (TE671).

FIGURE 5: FACS. HT29 cells and TE671 were incubated with [8-

13]NT₄Lys(C18)₂ -doxo liposomes (squares) and with doxo liposomes (circles). 100000 cells/well were fixed for lOmin at room temperature with 4% PFA-TBS and then incubated in 96well U-bottom plates for 1 and ½ hr at room temperature with different concentrations of liposomes (from 10μΜ to 500nM) in TBS-EDTA 5mM-BSA 0. 5%. Flow cytometry was obtained analysing 10000 events with a BD FACSCanto II (BD, NJ. USA). Results were analyzed by nonlinear regression analysis using GraphPad Prism 3.02 software.

FIGURE 6. Confocal microscopy: a) HT29 and b) TE671 cells were incubated with DOPC-[l- 13]NT₄Lys(C18)₂-Doxo liposomes (5microM, right) and with DOPC-Doxo liposomes (same concentration, left) for 30 min at 25°C. Plasma membrane was stained with lectin-FITC. In black and white figures red of Doxo is gray (arrow) and green of FITC is white. REFERENCES

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Claims

1. A supramolecular aggregate consisting of at least two constituents, a phospholipid or a mixtures of ionic and non-ionic surfactants, and a peptide containing monomer of general formula:

P-L-H

wherein:

P is a peptide based system containing a branched skeleton and four copies of the neurotensin peptide (1-13 NT), or its truncated form (8-13 NT), or other NT analogues;

L is a spacer containing polyoxoethylene moieties (Peg or analogs) or sequences of molecules containing polyoxoethylene functions with molecular weight between 600 and 5000 Dalton, possibly containing one or more amino acid residues;

H is an hydrophobic moiety containing two lipophilic tails.

2. The aggregate of claim wherein the branched skeleton is obtained by linking through amide bonds three molecules containing two amine functions and a carboxylic function.

3. The aggregate of claim 2 wherein the molecules containing two amine functions and a carboxylic function are selected from lysine, ornithine or diaminopropionic acid.

4. The aggregate of any one of claims 1-3, wherein the phospholipid is selected from Soy phosphatidylcholine (SPC), Egg Phosphatidylcholine (EPC) l,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC), 1,2-DipalmitoyI-sn- Glycero-3-Phosphocholine (DPPC), l,2-Disteroyl-sn-Glycero-3-

Phosphocholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), hydrogenated egg phosphatidylcholine (HEPC), phosphatidylglycerol (PG), 1 ,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-Dioleoyl-sn- glycero-3 Phospho-rac- 1 -glycerol phosphatidylglycerol (DOPG), 1,2- Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-Dipalmitoyl-sn- glycero-3-Phospho-rac-l-glycerol phosphatidylglycerol (DPPG), 1 ,2- Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-Distearoyl- sn- glycero-3 Phospho-rac- 1 -glycerol phosphatidylglycerol (DSPG).

5. The aggregate of any one of claims 1-4, wherein the two lipophilic chains, in the moiety H, are saturated, unsaturated or polyunsaturated aliphatic or aromatic hydrocarbons with a number of carbon atoms in the range 8-22, (preferably between 12-18), said chains having the same or a different length.

6. The aggregate of any one of claims 1-5, wherein the bioactive peptide, in the moiety P, is 1-13 NT having the sequence SEQID 1 (pyroGlu-Leu-Tyr- Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH), 8-13 NT having the sequence SEQID 2 (Arg-Arg-Pro-Tyr-Ile-Leu-OH) or NT analogs having the sequences SEQID 3 and SEQID4. (pyroGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Lys- Lys-Pro-Tyr-Ile-Leu-OH or Lys-Lys-Pro-Tyr-Ile-Leu-OH).

7. The aggregate of any one of claims 1-6 in form vesicles or liposomes having a size ranging between 50 and 500 nm; lipidic bilayers, double strand aggregates, having a thickness ranging between 5 - 20 nm.

8. The aggregate of claim 7 further containing cholesterol or other stabilizing compounds.

9. The aggregate of claim 7 further containing trehalose or other carbohydrates able to preserve the liposome structure during the lyophilization process.

10. The aggregate of any one of claims 1-9, wherein the number ratio of the monomer of general formula (I) and the phospholipid is comprised between

0.5 and 15% (molar ratio), preferably between 2 and 5%.

1 1. Pharmaceutical compositions comprising the aggregates of claims 1- 10 and a suitable excipient.

12. Pharmaceutical compositions according to claim 1 1 wherein the aggregates are loaded with antineoplastic agents.

13. Pharmaceutical compositions according to claim 1 1 wherein the antineoplastic agent is selected from doxorubicin, daunorubicin, epirubicin, esorubicin and idarubicin.

14. Pharmaceutical compositions according to claim 12 wherein the antineoplastic is cis platinum or an antitumor platinum complex.