Field of the invention

The present invention relates to the pharmaceutical field, in particular to specific drug delivery, more in particular to specific drug delivery to thyroid, more in particular for the treatment of thyroid cancer.

The present invention provides TSH-conjugated nanocarrier as a targeted drug delivery system against TSHR-expressing thyroid cancer cells.

Background of the invention

Thyroid carcinomas represent approximately 1.5% of malignant neoplasia and are the most frequent endocrine cancer. Mortality is estimated approximately 8-10% and may be higher, when considering subclasses of more aggressive tumours (Schlumberger M, N. Engl. J. Med. 338: 297-306 (1998)). The most important prognostic factor is represented by the ability of the tumour, primary or metastatic, in maintaining the iodide uptake capacity. This property allows by using the ¹³¹ I-scintiscan to identify and treat successfully recurrent or metastatic disease (Schlumberger M, Lacroix L, Russo D, et al. Nat. Clin. Pract. Endocrinol. Metab. 3:260-269 (2007)). Several studies have demonstrated that the majority of thyroid tumours present a reduction or loss of expression of the Sodium/Iodide symporter, responsible for reduced radioiodine uptake and the poor prognosis of these patients (Schlumberger M, Lacroix L, Russo D, et al, Nat. Clin. Pract. Endocrinol. Metab. 3:260-269 (2007); Filetti S, Bidart JM, Arturi F et al, Eur. J. Endocrinol. 141, 443 (1999)). Conversely, the expression of other thyroid specific markers of differentiation, especially the TSH receptor is often maintained even if at low levels (Brabant G, Maenhaut C, Kohrle J et al, Mol. Cell. Endocrinol. 82, R7-R12 (1991); Tanaka K, Otsuki T, Sonoo H et al, Eur. J. Endocrinol. 142, 340-346 (2000); Lazar V et al, J. Clin. Endocrinol. Metab. 84, 3228-3234 (1999); Durante C, Puxeddu E, Ferretti E, et al, J. Clin. Endocrinol. Metab. 92, 2840-2843, (2007)).

At present, the therapeutically approach for such tumours still represents a big issue. In fact, the use of antitumor drugs, able to destroy the tumor cells in vitro experiments or in animal models is strongly limited by dose-dependent toxic effects (Ain KB, Egorin MJ, DeSimone PA, Thyroid 10, 587 (2000)). Several studies are in progress to discover novel drugs provided with antitumoral activity acting against molecular targets expressed by thyroid tumor cells (i.e. inhibitors of tyrosine-kinase), but even such novel 'targeted' drugs present, when administrated 'in vivo', important side-effects (Woyach JA, Shah MH, Endocr. Rel. Cancer 16, 715-731 (2009)). A promising complementary therapeutic strategy being explored in cancer research is the attempt to enhance the delivery of the drug into tumour cells. An improvement of the intratumoral delivery will allow a reduction of the total dose of the drug and a decrease of the time of exposure of the whole organism to the drug. Moreover, the toxicity of an antitumoral drug will be further reduced by its specific targeting to particular neoplastic cells, obtainable by targeting an antigen selectively expressed on the surface of the tumor.

In the recent years, drug delivery systems have been investigated and prepared both to reduce the distribution volume of anticancer agents and to provide a selective distribution of drugs with a poor uptake in the sites which are not active.

Among the various drug delivery systems, liposomes are really promising as potential biological carriers of antitumoral drugs (Grant S, Dent P, Clin. Cancer Res. 16, 2305 (2004)). The liposomal carrier is a well-known very versatile delivery system for biologically active molecules. Furthermore, anticancer drug- loaded liposomes may allow an active targeting towards cancer cells by conjugation of the colloidal carrier with a certain antibody, thus achieving the so-called immuno liposome (Breitenlechner BC, et al, J. Mol. Biol. 353, 222

(2005) ).

It is well-known that the TSH receptor expression is maintained in the majority of thyroid differentiated carcinomas together with the possibility to enhance its expression even in the less differentiated cancer cells, as resulting by in vitro experiments (Wolber G., Langer T J, Chem. Inf. Model 45, 160 (2005); Aiello A, Pandini G, Frasca F et al, Endocrinology 147, 4463

(2006) ).

It has also been demonstrated that liposome entrapment of gemcitabine increases the antiproliferative action of this drug, due to a better delivery into the tumour cells, determining a cytotoxic effect on thyroid tumour cells at lower non-toxic dosages (Celano M, Calvagno MG, Bulotta S et al, BMC Cancer 4, 63 (2004); Vono M, et al TODD J (2010)). Such an enhanced delivery efficiency has been further confirmed even using in vivo experimental models (Celia C, Calvagno MG, Paolino D et al, J. Nanosci. Nanotechnol. 8, 2102-2113 (2008); Celano M, Schenone S, Cosco D, et al, Endocr. Rel. Cancer 15, 499-510 (2008)).

US2009/0186364, to Mie University, discloses recombinant proteoliposome prepared by fusion of budded virus particles of a recombinant baculovirus expressing a target membrane receptor, for example TSH receptor, with liposomes. These proteo liposomes have improved binding capability to an autoantibody and are used for diagnostic kits, for example for Grave's disease. However, an efficient system for targeting drugs to cells expressing TSH receptor is not yet available from the state of the art. Summary of the invention

It has now been found that a liposome comprising a PEG-PDP-TSH component solves the technical problem to specifically and efficiently deliver a variety of drugs into cells expressing TSH receptor. To this purpose, the present invention provides a TSH-nanocarrier.

In the context of the present invention, PEG means polyethylene glycol, PDP means (pyridyldithio)propionate and TSH means thyreo stimulating hormone.

A first object of the present invention is a liposome comprising a PEG-PDP-TSH component. A second object of the present invention is the above liposome loaded with a drug to be intracellularly delivered to a cell expressing a TSH receptor.

Another object of the present invention is a process for the preparation of the above liposome.

Still another object of the present invention is the above liposome as a carrier for drugs.

A further object of the present invention is the above liposome loaded with a drug to be intracellularly delivered to a cell expressing a TSH receptor for use as a medicament.

The invention presents many advantages with respect to the prior art. Among these advantages, the liposomes of the present invention allow to specifically deliver a drug to cells expressing

TSH receptor in an efficient way. To the purposes of the present invention "efficient way" is intended as the specific delivery of the drug to the intended target at therapeutically effective dosages and with reduced, even absent, occurrence of side effects. This is also intended by those skilled in the art of pharmaceuticals as a good therapeutic index.

These and other objects of the present invention will be disclosed in detail in the foregoing sections also by means of Figures and Examples.

In the Figures:

Figures la and lb show in vitro cytotoxicity of gemcitabine (black dots), gemcitabine loaded PEGylated liposomes (empty dots) and gemcitabine loaded TSH-PEGylated liposomes (black triangles) on CHO wild type (CHO-W) and TSH receptor- transfected cells (CHO-T) as a function of drug concentration and exposition time. Data are expressed as percentage of cellular viability as evaluated by MTT test. Results are the mean of six different experiments ± standard deviation. Error bars, if not shown, are within symbols.

Figure 2 shows in vitro interaction between [ ³ H]CHE radiolabeled PEGylated and TSH- PEGylated liposomes with CHO-W (panel a) and CHO-T (panel b) cells as a function of incubation time in the presence or not of free TSH. The experiments were carried out at 37°C. Each bar represents the mean value of five different experiments ± standard deviation.

Figure 3 shows intracellular uptake of gemcitabine loaded PEGylated liposomes (Lip) or TSH- PEGylated liposomes (Lip-TSH) within CHO-W and CHO-T as a function of drug exposure time. The experiments were carried out at 37°C at a final drug concentration of 1 μΜ. Each bar represents the mean value of five different experiments ± standard deviation.

Figure 4 shows the biodistribution of [ ³ H]CHE radiolabeled unilamellar TSH-PEGylated and

PEGylated liposomes in Wistar rats after 3 h administration. Each bar represents the mean value of five different experiments ± standard deviation.

Detailed description of the invention

The present invention provides a liposome formulation useful as a selective drug carrier for the intracellular delivery to cells expressing the TSH receptor.

The liposome of the present invention is characterized by having a component of PEG-PDP- TSH.

The liposome of the present invention is useful to specific delivery of drugs to thyroid gland. There is no virtual limitation to the kind of liposome formulation for the carrying out of the present invention. The suitable liposome formulation will be designed by the person of ordinary skill in the art taking into account the nature and kind of drug to be delivered to cells expressing THS receptor, in particular thyroid cells.

Liposomes are closed vesicles with lipid bilayer containing phospholipids and internal aqueous environment. Their use have been known since long time as vehicles for the delivery of active ingredients.

Liposomes are classified into multilamellar vesicles (MLV), wherein several lipid bilayers are onion-like layered, and unilamellar vesicles (UV) with only one lipid bilayer. UV are classified as small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV). SUV are applied in this invention.

Phospholipids are esters of phosphoric acid with lipids. Among others, there are glycerophospho lipids and sphyngophospho lipids. Non limiting examples of components suitable for the formulation of the liposome of the invention are phosphatidylcholine (PC), phospha- idylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI); glycerophospholipids, such as phosphatidylglycerol, diphosphatidylglycerol (cardiolipin), phosphatidic acid (PA), sphyn-gomyelin; dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), di-stearoylphosphatidylcholine (DSPC), palmitoyl-oleoylphosphatidylcholine (POPC), dioleoylpho-sphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidyl-ethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), cholesterolhemisuccinate (CHEMS), cholesterol-(3-imidazol-l-ylpropyl)carbamate (CHIM), dimethyldioctadecylammonium bromide (DDAB), (l,2-dioleoxypropyl)-N,N,N,- trimethylammonium salt (DOTAP), dioleoylpho-sphatidylserine (DOPS), dioleoylphosphatidylglycerol (DOPG), cholesterol sulphate (Chol-SC^), 4-(2-aminoethyl)- morpholino-cholesterolhemisuccinate (MoChol), histaminylcholesterolhemisuccina-te (HisChol), l,2-dipalmitoylglycerol-3-hemisuccinate (DGSucc), l,2-distearoylglycerol-3-hemi- succinate 1 ,2-dimiristoylglycerol-3-hemisuccinate, 1 ,2-dioleoylgly-cerol-3-hemisuccinate, palmito-yl-oleolylglycerol-3-hemisuccinate. These components can be used in any combination available to the skilled person.

Preferred components included in the previous list are: l,2-dipalmitoyl-sn-glycero-3- phosphocholine monohydrate (DPPC), l ,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

According to the present invention, the liposome must comprise a PEG-PDP-TSH component. A preferred essential component is l ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [PDP(methoxypoly ethylene glycol-2000)] (DSPE-mPEG 2000-PDP).

Molar ratios of the different components the liposome are determined by the person of ordinary skill in the art of liposome formulation.

Other components can be present, such as helper lipids and other conventional ingredients. Cholesterol (Choi) is a preferred helper lipid.

A first preferred embodiment of the present invention is the liposome DPPC/Chol/DSPE- mPEG2000/DSPE-mPEG2000-PDP (6:3:0.6:0.4 molar ratio), conjugated with TSH. Said liposome is a further object of the present invention.

Another object of the present invention is a process for the preparation of said liposomes.

The process is carried out according to the general knowledge in this field.

A lipid mixture of liposome components is dissolved in a suitable solvent. Generally, such a solvent is an organic, lipophilic solvent, for example an halogenated hydrocarbon, such as dichloromethane or chloroform. Conveniently, the dissolution of the lipid mixture can be carried out in an organic solvent mixture, for example a chloroform/methanol mixture. A preferred solvent system is chloroform/methanol 3:1 v/v.

The lipid solution is then treated to form a lipid film. Any technique for forming lipid film can be used. Vacuum evaporation of the solvent is a convenient method, for example is a vacuum rotating evaporator or other equivalent means.

The lipid film is subsequently processed according to conventional techniques for making liposomes. For example, the lipid film is taken up in an appropriate medium, for example saline solution and processed to provide an MLV. One embodiment of MLV formation is warming and vortexing repeatedly. For example, a number, such as three, cycles (typically 3 min) of warming (about 58°C) and vortexing (typically 700 rpm) can be used. According to the present invention, disulfide reduction can be carried out by means of suitable chemical reaction. One embodiment of the present invention provides incubation with a dithiotreitol (DTT). An appropriate concentration of DTT (for example 50 mM DTT solution, 1 :2 v/v respectively) is used for incubation. A typical running time is 30 min.

The liposome is then separated from incubation medium. A convenient separation technique is centrifugation (for example 20,000 g for 60 minutes at 4 °C), but any other known technique, not affecting vesicles integrity can be used.

Liposomes devoid of any excess of disulfide bond reducing agent are successively resuspended with in a suitable medium.

In a preferred embodiment of the present invention, liposomes are resuspended in an ammonium sulfate solution, typically 250 mM.

The suspension is then submitted to a number (typically ten) of freezing cycles (for example with liquid nitrogen) and thawing (for example with a water bath at 40 °C), thus achieving a pH gradient with a homogenous acid environment in the intraliposomal aqueous compartments. Multilamellar vesicles are then submitted to a process for obtaining small unilamellar vesicles. Small unilamellar colloidal vesicles are obtained by conventional techniques and it is essential that unilamellar vesicles have a diameter lower than 1 μιη.

Preferably, small unilamellar vesicles have a diameter lower than 500 nm, more preferably lower than 400 nm, much more preferably lower than 200 nm, even more preferably lower than 150 nm and most preferably lower than 100 nm. Systemic administration, in particular intravenous, of a liposomal administration provides that the average size of colloidal vesicles is < 150 nm in order to reduce uptake by macrophages of reticuloendotelial system, allowing the vesicles to remain longer in the circulating system and concentrate in the target organs. The most preferred embodiment of the present invention provides liposomes of nanometric dimensions (nano liposomes), typically lower than 100 nm.

Mean size and size distribution of the nanoparticle can be determined by appropriate instrumentation. For example hydrodynamic size can be measured with light scattering techniques, for example a commercially available Zetasizer Nano ZS (Malvern Instruments Ltd., Worchestershire, United Kingdom), a dynamic light scattering spectrophotometer, by applying the third-order cumulant fitting correlation function. A 4.5 mW laser diode operating at 670 nm was used as a light source for size analysis and the back scattered photons were detected at 173°. The real and imaginary refractive indexes were set at 1.59 and 0.0, respectively. The medium refractive index (1.330), medium viscosity (1.0 mPa x s), and dielectric constant (80.4) were set before the experiments. Quartz cuvettes were used for the analysis. Typically, submicrometric, preferably nanometric vesicles are obtained by a conventional extrusion apparatus.

Unentrapped ammonium sulfate solution is then removed, for example by centrifugation.

Other methods of preparation of liposomes are well-known in the art and the invention is not limited to the process disclosed above. Any process available in the art can be used. Examples can be found in US 2009/186364 and US 2007/104775.

At this point, the liposome according to the present invention can be loaded with the drug of interest and subsequently derivatized with TSH or vice versa.

Drug loading techniques are absolutely conventional and do not require any special instruction here. Loading technique will depend on the drug kind and nature and instructions can be found in the literature, see for example Crosasso P, Ceruti M, Brusa P et al, J. Control. Release 63 : 19-30

(2000); Calvagno MG, Celia C, Paolino D et al, Curr. Drug. Deliv. 4:89-101 (2007).

An essential step of the process according to the present invention is conjugation of the liposome

(in case pre-loaded with the desired drug) with TSH. A simple incubation of the liposome with

TSH is sufficient. For example, overnight incubation in order to allow the bioconjugation with reduced PDP chains is sufficient. Incubation temperature is generally room temperature.

As said before, there is no virtual limitation to the kind of drug which can be delivered intracellularly to cells expressing TSH receptor.

In a preferred embodiment of the present invention, it is of interest to use the liposome for targeting drugs used for the treatment of diseases affecting thyroid gland. All chemotherapeutic drugs can be used. A non limiting list is gemcitabine, doxorubicin, cisplatin, paclitaxel, capecitabine, tirosine-kinase inhibitors such as axitinib, vandetanib, motesanib, sunitinib and sorafenib, other drugs such as romidepsin, decitabine, bortezomib and lenalidomide.

The present liposomes can also be used as co-carriers for more drugs at the same time.

The liposome of the present invention loaded with gemcitabine is a preferred embodiment.

In particular, DPPC/Chol/DSPEmPEG2000/DSPE-mPEG2000-PDP (6:3:0.6:0.4 molar ratio), conjugated with TSH and loaded with gemcitabine (GEM) is a particularly preferred object of the present invention. According to this embodiment, small unilamellar colloidal vesicles obtained by the above described process are suspended in an isotonic solution of GEM- hydrochloride and kept at room temperature, for example for 3 h.

Quite advantageously, the present invention, and this embodiment especially, provides a novel treatment of thyroid diseases and especially those thyroid neoplasia not responsive to the current therapies. In fact, the TSH receptor represents an ideal target for this kind of treatment for two main reasons: a) its expression is maintained in a large number of thyroid differentiated carcinomas (Brabant G, Maenhaut C, Kohrle J et al, Mol. Cell. Endocrinol. 82, R7-R12 (1991); Tanaka K, Otsuki T, Sonoo H et al, Eur. J. Endocrinol. 142, 340-346 (2000); Lazar V et al, J. Clin. Endocrinol. Metab. 84, 3228-3234 (1999); Durante C, Puxeddu E, Ferretti E, et al, J. Clin. Endocrinol. Metab. 92, 2840-2843, (2007)), and it has also been explored the possibility to enhance its expression even in the less differentiated cancer cells, as resulting by in vitro experiments (Aiello A, Pandini G, Frasca F et al, Endocrinology 147, 4463 (2006); Landriscina M, Fabiano A, Altamura S et al, J. Clin. Endocrinol. Metab. 90, 5663 (2005)); b) eventual damages of thyroid normal cells are to be excluded since such a novel therapy will be reserved to patients with recurrent or metastatic cancers, who had already undergone total thyroidectomy, in accordance with the current guidelines of thyroid malignant nodules management (Gharib H, Papini E, Paschke R. Eur. J. Endocrinol. 159, 493-505 (2008)).

According to the present invention, the liposomes conjugated with TSH are useful as carrier for drugs to be delivered into cells expressing TSH receptor.

Said liposomes, when loaded with at least one drug are used as medicaments.

A preferred embodiment of the present invention provides the use of liposomes loaded with at least one anticancer drug for the treatment of thyroid tumors. A specific embodiment of the invention provides the use of said loaded liposomes for the treatment of thyroid carcinoma, in particular aggressive subclasses of this tumor. According to the present invention both primary and metastatic thyroid tumors can be treated with the liposomes herein disclosed. According to the present invention, thyroid neoplasia not responsive to the current therapies can be treated. According to the present invention, by the terms "treatment" or "treating", it is intended the administration of the liposomes according to the present invention loaded with at least one drug, in particular an anticancer drug, with the aim to induce regression of tumor. The above terms also include reduction of tumor or block of tumor growth. The above terms also comprise multiple therapy with other drugs, also anticancer drugs, according to the therapeutical protocols provided for the treatment of thyroid tumors.

According to another object of the present invention, the liposomes herein disclosed are administered in the form of a pharmaceutical composition. Typically, the composition comprises a therapeutically effective dose of the liposome loaded with at least one drug in admixture with at least one pharmaceutically acceptable vehicle and/or excipients. The therapeutic dosage will be determined by the person of ordinary skill in the art with standard procedures of dose finding in the field of pharmacology, in particular for tumor diseases. Elements to be taken into consideration are, for example, the severity of the disease, the general state of the subject to be treated, other concomitant therapies. A general guideline can be found in Remington 's Pharmaceutical Handbook, last edition. Any well-known administration form for liposomes can be used in the practice of the present invention. A particularly preferred embodiment of the invention provides intravenous administration. To this end, injectable formulations can be prepared according to well-known techniques. Examples of such formulations can be found in US 5,653,998, WO 2006/105155, US 5,626,832, For example liposome suspensions in pharmaceutically acceptable vehicle are well known in the art. Intranasal administration is also a suitable embodiment of the invention.

The following examples further illustrate the present invention.

Materials

l,2-dipalmitoyl-sn-glycero-3-phosphocholine monohydrate (DPPC) and N-(carbonyl- methoxypoly- ethylene glyco 1-2000)- 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE- MPEG 2000), were purchased from Genzyme (Suffolk, UK). l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[PDP(methoxypolyethylene glycol-2000)] (DSPE-mPEG2000-PDP) was obtained by Avanti Polar (Alabaster, Alabama, USA). Thyroid stimulating hormone (TSH, from human pituitary), N-(fluorescein-5-tiocarbamoyl)-l ,2-dihexadecanoyl-sn-glycero-3- phosphoethanolamine triethylam-monium salt (fluorescein-DHPE), 3-[4,5-dimethylthiazol-2-yl]- 3,5-diphenyltetrazolium bromide salt (tetrazolium salt), DL-dithiothreitol were purchased from Sigma Chemicals Co. (St. Louis, USA). F12-medium, minimum essential medium (MEM) with glutamine, trypsin/EDTA (l x) solution, fetal bovine serum and penicillin-streptomicin solution were obtained by Gibco (Invitrogen Corporation, UK). [ ³ H]cholesteryl hexadecyl ether ([ ³ H]CHE, 40 Ci/mmol) was obtained from Perkin Elmer-Italia (Monza, Italy).

Example 1

Liposome preparation

Liposomes were made up of DPPC/Chol/DSPEmPEG2000/DSPE-mPEG2000-PDP (6:3:0.6:0.4 molar ratio). The lipid mixture (20 mg) was dissolved in a round-bottomed flask by using a chloroform/methanol (3 : 1 v/v) solvent mixture, which was removed by means of a rotary evaporator (Bvichi R-210 Switzerland) and by an overnight storage at room temperature in a Buchi T51 glass drying oven connected to a vacuum pump, thus allowing the formation of a thin layer lipid film. When required, fluorescent labeled liposomes were prepared by co-dissolving fluorescein-DHPE (0.1% molar) with the lipids. The lipid film was hydrated with 1 ml of saline solution (NaCl 0.9% w/v).

Multilamellar liposomes (MLVs) were achieved by submitting the lipid/aqueous phase mixtures to three alternate cycles (3 min each) of warming at 58 °C (thermostated water bath) and vortexing at 700 rpm. To reduce the disulfide bond, the formulation was incubated with a 50 mM DTT solution (1 :2 v/v respectively) for 30 min. Then, it was centrifuged at 20,000 g for 60 minutes at 4 °C with a Beckman Coulter Allegra 64R with the aim of removing the excess of DTT. Successively, the pellet was resuspended with a 250 mM ammonium sulfate solution (1 ml) and then submitted to ten cycles of freezing (with liquid nitrogen) and thawing (with a water bath at 40 °C), thus achieving a pH gradient with a homogenous acid environment in the intraliposomal aqueous compartments. Multilamellar vesicles were submitted to extrusion through 400, 200 and 100 nm pore size two stacked polycarbonate filters (Costar, Corning Incorporated, NY, USA) by using a stainless steel extrusion device (Lipex Biomembranes, Vancouver, BC, USA) and un-entrapped ammonium sulfate solution was removed by centrifugation. Small unilamellar colloidal vesicles were suspended in an isotonic solution (1 ml) of GEM-hydrochloride (1 mM) and kept at room temperature for 3 h. Finally, 2.5 μg of TSH were incubated overnight with the formulation in order to allow the bioconjugation with reduced PDP chains.

Example 2

Cytotoxic activity

Cytotoxic effects of free or liposome entrapped gemcitabine were evaluated by MTT-test (cell viability). The cultured cells were plated in 96-well culture dishes (5x 10 ³ cells/0.2 ml) and incubated for 24 h at 37 °C to promote their adhesion to the plate. The culture medium was then removed, replaced with the different formulations (i.e. free GEM, GEM loaded PEGylated liposomes and GEM loaded TSH-PEGylated liposomes) and incubated for 24, 48 or 72 h.

Every plate had 8 wells with untreated cells as the control and 8 wells with cells treated with empty liposomes as the blank. After each incubation period, 10 μΐ of tetrazolium salt solubilized in PBS solution (5 mg/ml) were added to every well and the plates were incubated again for 3 h. The medium was removed and the formazan salts (precipitated on the well bottom after oxidation) were dissolved with 200 μΐ of a mixture of DMSO/ethanol (1 : 1, v/v), by shaking the plates for 20 min at 230 rpm (IKA ^® KS 130 Control, IKA ^® WERKE GMBH & Co., Staufen, Germany). The solubilised formazan was quantified with a microplate spectrophotometer (Multiskan MS 6.0, Labsystems) at a wavelength of 540 nm with reference at a wavelength of 690 nm. The percentage of cell viability was calculated according to the following equation: Cell viability (%)= Abs _T /Abs _c x 100

where Absi is the absorbance of treated cells and Absc is the absorbance of control (untreated) cells. The formazan concentration is directly proportional to the cell viability that was reported as the mean of six different experiments ± standard deviation.

Example 3 Liposome/cells interaction

In order to correlate the interaction rate between the different liposomal formulations and the different cell lines (CHO-W and CHO-T) in function of the time, [ ³ H]CHE (0.003% w/w corresponding to 3 nmol of [ ³ H]CHE) radiolabeled vesicles were used. In particular, cells were plated in 6-well culture dishes (5>< 10 ⁵ cells/ml) and successively treated with 100 μΐ of tritiated formulation. Free TSH (0.1-1000 μΙΙ/ml) was used in order to evaluate the possible competition with hormone receptors. After 3 h incubation, cells were centrifuged (1200 rpm at room temperature for 10 min) in order to eliminate the culture medium, washed two times with PBS and transferred in polypropylene liquid scintillation vials (20 mL-size) (Sigma- Aldrich Chemie, GmbH, Steinheim, Germany) and dissolved in a quaternary ammonium hydroxide solution (2 ml) (BTS-450, Beckman Instruments, Inc., Fullerton, Netherlands) under continuous shaking for 1 h at 60 °C using an incubator shaker (Innova™ 4300, New Brunswick Scientific, Edison, NJ, USA). At the end of the incubation time, the liquid scintillation cocktail (7 ml) (Ready Organic™, Beckman Coulter Inc., Fullerton, USA) was added, and the samples were vigorously mixed and analyzed using a Wallac Win Spectral™ 1414 liquid scintillation counter (PerkinElmer Life and Analytical Sciences, Inc. Waltham, MA, USA). A 1414 Win Spectral Wallac LCS Software was used for data analysis.

Confocal Laser Scanning Microscopy (CLSM)

The interaction between the CHO cells and fluorescein-labelled liposomes (with or without TSH molecule) was evaluated by CLSM studies. For these experiments, cells (4>< 10 ⁵ cells/ml) were placed in 6-well culture plates with culture medium and a sterile glass slide was positioned in each well. Plates were incubated for 24 h and then cells were treated with fluoresceinated vesicles for different incubation times (3 and 6 h). After incubation, each well was washed with PBS (3 times) to remove the excess of colloids and cells were fixed on the sterile glass slides by using 1 ml of an ethanol solution (70% v/v). Each slide was washed again with PBS three times and PBS (2 ml) was added to each well. Plates were stored at 4 °C up to the CLSM analysis. Before analysis, cover-slides were positioned over glass slides using a glycerol solution (70%> v/v) to remove enclosed air and hence were fixed by a transparent glue. The analysis was carried out using a Leika TCS SP2 MP CLSM at =496 nm e _em =5 l9 nm. A scan resolution up to 4096 ^χ 4096 pixels with an Ar/Kr laser beam of 75 mW, equipped with a fluorescein analyzer filter, was used for experimental investigations. Samples were recorded by a macro developer software package having multi-dimensional series acquisition and direct-access digital control knobs. An immersion oil lens lOOx was used.

Example 4 Intracellular uptake of gemcitabine

CHO W and CHO-T were seeded (3 x 10 ⁴ cells/ml) in 12-well plastic culture dishes and incubated with free or liposomally entrapped gemcitabine (1 ml) at a final concentration of 1 μΜ. At different incubation times, the cells were scraped from the wells, collected together in a sample, and centrifuged (1200 rpm) at 22 °C for 10 minutes. Cellular pellets were separated from the supernatant solution, resuspended in PBS buffer solution (1 ml), and disrupted by sonication (SONOPOLUS GM 70, Bandelin Electronic, Berlin, Germany) at 50 cycles per second for 3 minutes. The intracellular amount of gemcitabine was determined by an HPLC method. Each value was the average of five different experiments.

Each sample was prepared for HPLC analysis by adding a 2% (w/v) zinc sulfate solution (1.4 ml) in a methanol/water (30:70 v/v) mixture. The resulting mixture was vortex-mixed for 5 minutes and then centrifuged at 6000 rpm with an Eppendorf Megafuge centrifuge (BJB Labcare Ltd., Buckinghamshire, United Kingdom). The supernatant was filtered through a 0.22-μιη pore size nylon membrane (Whatman Inc.), lyophilized, solubilized in 100 of the mobile phase and submitted to HPLC analysis.

HPLC quantification

Analysis was carried out using an HPLC system (Varian Inc., Palo Alto, CA, USA) composed of a 200-2031 Metachem online degasser, a M210 binary pump, a ProStar 410 autosampler, a G1316A thermostated column compartment, and a 25 μΐ CSL20 Cheminert Sample Loop injector. Data were acquired and processed with a Galaxie ^® chromatography manager software (Varian Inc.). Chromatographic separation was carried out at room temperature using a GraceSmart RP CI 8 column (4.6x250 mm, 5μιη particle size, Alltech Grom GmbH, Rottenburg- Hailfmgen, Germany). The mobile phase was water/acetonitrile (95:5 v/v). The flow rate was 1 ml/min and UV detection was performed at 269 nm.

The chromatographic method provided a suitable separation of the peaks of GEM and 2 ^I ,2 ^I - difluorodeoxyuridine, which showed a retention time of 6.00 and 8.70 min, respectively. GEM quantification was carried out using an external standard curve in the linear concentration range between 0.1 μg/ml and 10 μg/ml. A standard solution of GEM (1 mg/ml) was used for the construction of the standard curve. The amounts of GEM were determined using the standard curve according to the following equation:

AUC=0.60112x + 0.02840

where x is the drug concentration ^g/ml) and AUC the area under the curve (mAuxmin). GEM amounts were expressed as μg/ml. Experimental data are the mean of three different experiments. Example 5

In Vivo Experiments

The experiments were carried out in agreement with the principles and procedures outlined by the local Ethical Committee and in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). The animals were maintained at standard conditions of both temperature (20±2 °C) and humidity (65%) with 12 h light/ 12 h dark cycle (light on 8:00 a.m.) and food and water ad libitum.

Biodistribution

Biodistribution experiments were carried out in Wistar rats. [ ³ H]CHE (0.003% w/w corresponding to 3 nmo 1 of [ ³ H]CHE) radiolabeled liposomes (200 μΐ) were injected into mice through the tail vein. After 3 h, the rats were sacrificed by cervical dislocation and the thyroids were collected. Complete organs were transferred in polypropylene liquid scintillation vials (20 mL-size) (Sigma-Aldrich Chemie, GmbH, Steinheim, Germany) and mixed with a quaternary ammonium hydroxide solution (2 ml) (BTS-450, Beckman Instruments, Inc., Fullerton, Netherlands) under continuous shaking for 4 h at 60 °C using an incubator shaker (Innova ^™ 4300, New Brunswick Scientific, Edison, NJ, USA) to allow complete dissolution. At the end of the incubation time, various samples were decolorized with 2 ml of 24% (v/v) H ₂ 0 ₂ at room temperature for 5 min, the liquid scintillation cocktail (7 ml) (Ready Organic ^™ , Beckman Coulter Inc., Fullerton, USA) was added, these mixtures were vigorously mixed and analyzed using a Wallac Win Spectral ^™ 1414 liquid scintillation counter (PerkinElmer Life and Analytical Sciences, Inc. Waltham, MA, USA). A 1414 Win Spectral Wallac LCS Software was used for data analysis. To eliminate the blood radioactivity contained in the organ samples, derived from the contribution of liposomes in the vascular space, as well as the tissue parenchyma (including the macrophages and the capillary endothelial cells) ( tissue), a correction was made according to the following equation:

where, _OTg an represented levels of radioactivity recovered from the various organ samples; V0 was the total volume of the vascular space and interstitial fluid, as determined by the radioactivity level in the whole organ samples divided by the blood concentration 10 min after the i.v. injection of the [ ³ H]CHE-liposomes and C(t) was the blood concentration at the indicated time. A further quenching correction factor was obtained by measuring the radioactivity of blank tissues from non-injected rats spiked with known amounts of [ ³ H]CHE (0.030 μθι/μιηοΐ lipids).

Example 6

Anticancer activity The in vitro anticancer activity was evaluated in terms of cytotoxicity by using the cell viability MTT test. Cytotoxic effect was evaluated as a function of both the incubation time (24, 48 or 72 h) and the drug concentration (from 0.01 to 10 μΜ) to define the time-exposition and the dose- response effects, respectively. The experiment were carried out both on CHO wild type (CHO- W) and TSH receptor-transfected cells (CHO-T), (Figures la and lb).

After 24 h of incubation it is possible to observe that no remarkable cytotoxic effects were observed at a drug concentration of 0.01 μΜ and 0.1 μΜ of free gemcitabine both in CHO-W and CHO-T. An improvement of the gemcitabine cytotoxicity was observed at a concentration of 1 μΜ and 10 μΜ of free drug both in CHO-W and CHO-T (respectively -10% and 15% of cell vitality reduction for both cell lines). No remarkable differences between two cell lines were observed also after 48 h and 72 h of incubation at all the concentrations of free drug tested.

Gemcitabine loaded PEGylated liposomes caused an increase of toxicity with respect to free drug at all concentrations and exposition time not related to the type of cells (CHO-W and CHO- T). The cell vitality reduction increased at the increase of the drug concentration (from -20% for a drug concentration of 0.01 μΜ and 24 h of treatment, up to 70% for a drug concentration of 10 μΜ and 72 h of treatment for the CHO-W; from 10% at a drug concentration of 0.0 ΙμΜ and 24 h of incubation up to 60% for 10 μΜ and 72 h of treatment). No significative differences were observed between wild type and transfected cells.

A significative difference in term of cell mortality was observed for gemcitabine loaded TSH- PEGylated liposomes at all the concentrations and incubation times. After 24 h of incubation, for example, at a drug concentration of 0.01 microM, this formulation showed a decrease of cell viability of about 20% on CHO-T cells, while no cytotoxic difference was obtained on CHO-W cells with respect to the liposomal formulation without TSH. This trend remained after 48 and 72 h incubation, furnishing a reduction of 90% of CHO-T cell vitality at a drug concentration 10 microM in the case of TSH-liposomal formulation.

Example 7

To correlate the interaction rate between the different liposomal formulations and the different cell lines in function of the time, [ ³ H]CHE radiolabeled vesicles were used, and free TSH was used to evaluate the possible competition against the hormone receptors. Results are reported in Figure 2. The interaction of pegylated liposomes with CHO-W and CHO-T is not significantly different. The use of TSH-Pegylated liposomes gives an interaction twice higher in case of CHO- T with respect to the CHO-W cells. This result is a clear evidence of the active targeting of our nanocarrier. Moreover, while in CHO-W cells the co-incubation of TSH-Pegylated liposomes with TSH gives an interaction not significantly different (no competition with hormone occurs), in CHO-T cells the interaction is affected by the presence of TSH in an inversely proportion (increasing the TSH concentration decreases the interaction between TSH-Pegylated liposomes and CHO-T). These results are a clear evidence that our nanocarrier acts with a selective target. The cellular interaction between the CHO cells and fluorescein-labelled liposomes (with or without TSH molecule) was also evaluated by CLSM studies. The experiments were carried out for 3 h and 6. Micrographs clearly reported an evident green fluorescence in all the panels, but significantly higher when CHO-T cells were treated with gemcitabine loaded TSH-PEGylated liposomes for either 3 or 6 hours. These results further demonstrate that the targeting of investigated liposomes is active.

We investigated the ability of our carrier to selectively deliver a drug into target cells. The intracellular uptake of gemcitabine prompted by gemcitabine loaded PEGylated liposomes or TSH-PEGylated liposomes was investigated as a function of time. As reported in Figure 3 the use of gemcitabine loaded TSH-PEGylated liposomes causes a significant increase of the intracellular uptake in CHO-T, but not in CHO-W, during all the exposition times.

Example 8

In order to confirm the in vitro data, the bio distribution profile of [ ³ H]CHE radiolabeled liposomes in rats was investigated after i.v. administration. In particular, as possible to observe in Figure 4, the presence of TSH molecule on the liposomal surface allowed to increase the colloidal concentration in the thyroid tessue. More in detail, after 3 h administration, TSH- PEGylated liposomes provided a thyroid accumulation value 3-fold higher than that of the colloidal formulation without bioconjugate.

Our data show the effectiveness of our construct to increase the specific targeting of TSH- Pegylated liposomes toward TSHR-expressing cells. In fact the use of gemcitabine loaded TSH- Pegylated liposomes was able to ensure in TSH receptor expressing cells, an increase of the interaction rate between the liposomal formulations and the cells resulting in enhanced drug delivery and cytotoxic effect at lower dose and exposure time.

Moreover, the same behaviour was observed in vivo: even in rat thyroid tissue, in fact, binding of labelled TSH-pegylated liposomes occurred more efficaciously with respect to untargeted pegylated liposomes, also thanks to a reduced competition with the native TSH, obtained by administration of thyroid hormones.

Our findings demonstrate the benefit of the use of our nanocarriers to selectively deliver a cytotoxic drug against TSH receptor expressing cancer cells obtaining valid effects with lower dosage presumably associated with less systemic cytotoxicity. Moreover, the availability of a carrier with a specific activity of binding the TSH receptor offers additional opportunities of uses other than against thyroid cancer cells. As an example, thyroid hyperfunctioning adenomas require, before surgical or radioiodine treatment, a medical therapy with antithyroid drugs which is not side-effect free. They are very well differentiated benign lesions which express normal levels of constitutively active TSH receptor. In addition, serum levels of endogenous TSH are low or undetectable for the inhibitory feedback exerted by the high levels of thyroid hormone produced by the hyperfunctioning nodule. Again, the availability of a selective anti-TSHR carrier allows to target more selectively the thyroid cells, reducing the doses of the antithyroid drug and its systemic extra-thyroid action.

Finally, the proposed strategy offers an important possibility not only for our target molecule, but also for other molecules that can be conjugated to the carrier surface to deliver the drug against a particular target. Moreover, our strategy may be adapted also to other colloidal carriers such as polymeric micro- and nanoparticles, lipidic colloidal carriers, and any carrier able to be chemically functionalised.