WO2011108955A1 - Multi -targeting system comprising a nanocarrier, nucleic acid(s) and non-nucleic acid based drug(s) - Google Patents

Abstract

MuIti-component therapeutic strategies are frequently indicated for the treatment of heterogeneous diseases as cancer. However, the antitumor activity of drug combinations is extremely dependent of the molecular ratio of the combined drugs. This patent describes a multi -targeting system comprising a nanocarrier that was engineered in order to exhibit small size, high stability over time, high encapsulation yields of nucleic acids and the ability to specifically bind to receptors overexpressed at the surface of cancer cells. Further developments were performed in order to simultaneously encapsulate nucleic acids (gene silencing agents) and antitumural drugs (imatinib) in molar ratios allowing both molecules to be used in therapeutic doses. Therefore, with this product it is possible to mediate specific delivery to cancer cells and to address two specific molecular targets (a transcript and a protein) This unique properties thus render the system a great potential for cancer treatment, namely when resistance to chemotherapy is associated.

Description

DESCRIPTION

"MUL I -TARGETING SYSTEM COMPRISING A NANOCARRIER, NUCLEIC

ACID(S) AND NON-NUCLEIC ACID BASED DRUG(S)"

Field of the invention The present invention relates to the fields of therapy- arid diagnosis of human cancer and other diseases, including but not limited to inflammation, neurodegenerative diseases,

infectious diseases, and cardiovascular disorders; more

specifically to methods of selectively targeting therapeutic or diagnostic molecules to specific cells, combining cellular and molecular targeting.

Background information Many cancers are associated with abnormal expression of some genes. The specific silencing of these genes constitutes a targeted therapy leading to high specificity and therefore minimal toxicity. There are several strategies to inhibit gene expression. Not limiting examples are antisense oligonucleotides (asODN) ; RNA interference (RNAi) ; DNA enzymes (DNAzymes) ,

Ribozymes, DNA decoys and Aptamers . DNAzymes are catalytic DNA molecules, not naturally occurring, that bind to and cleave their target mRNA in a sequence specific manner (Bhindi et al . 2007; Kalota et al . 2004) . Ribozymes are naturally occurring catalytic RNA molecules, capable of sequence-specific cleavage of target mRNA, which catalytic activity is highly dependent on their structure (Bhindi et al . 2007; Rayburn and Zhang 2008) . Decoys are small double stranded DNA molecules that contain binding sites for a variety of protein targets, namely to transcription factors. Therefore, decoys compete with endogenous DNA for the transcription factors, which results in the decrease of the transcription rate of genes that are dependent of those transcription factors (Bhindi et al . 2007; Brennan et al . 2008; Kalota et al . 2004) . Aptamers are small stretches of RNA or DNA to which gene complementarity is not important because their activity is determined by their tertiary and quaternary

structures. In fact, aptamers have specific three dimensional structures that allow them to bind their target protein with high affinity and specificity, forming complexes and blocking their activity (Bhindi et al . 2007; Rayburn and Zhang 2008). asODN are stretches of usually 18-25 nucleotides in length that hybridize in a sequence specific manner to the target mRNA through Watson-Crick base pairing (Bhindi et al . 2007; Galderisi et al . 1999; Stahel and Zangemeister-Wittke

2003; Tamm 2006) . Inhibition of gene expression by antisense ODN is likely to occur by two mechanisms i) the formation of a hybrid complex mRNA:asODN that prevents the ribosomes from reading along the mRNA message by steric hindrance and ii) nuclease degradation of the target mRNA, mediated by the

endogenous enzyme RNase H, triggered by the recognition and selective destruction of the mRNA of the mRNA : asODN complex (Figure 1.2) (Bhindi et al . 2007; Galderisi et al . 1999; Mahato et al. 2005; Rayburn and Zhang 2008) . Other mechanisms were observed including interference with mRNA processing and

transport and, inhibition of gene transcription by forming DNA triple helices in a sequence specific manner (Rayburn and Zhang 2008; Stahel and Zangemeister-Wittke 2003) . RNAi is a naturally occurring and evolutionarily conserved mechanism (Chiu and Rana 2003; Kumar and Clarke 2007; Shrivastava and Sriyastava 2008) employed by cells to mediate gene regulation, protecting the genome from invading elements such as virus and transposons (Dykxhoorn et al . 2006; Shrivastava and Srivastava 2008). Thus, RNAi is thought to be evolved as a part of cell's innate

immunity, and mediates its activity through small double stranded RNA molecules, which complementarity to the target mRNA triggers the gene silencing by mRNA degradation, either by preventing translation or by silencing the chromatin (Dykxhoorn and Lieberman 2006; Dykxhoorn et al . 2006; Shrivastava and

Srivastava 2008) . Various types of small RNA molecules, such as small interfering RNA (siRNA) , microRNA (miRNA) , repeated- associated siRNA (rasiRNA) , short-hairpin RNA (shRNA) , small- modulatory RNA (smRNA) , tiny non-coding RNA (tncRNA) and piwi- interacting RNA (piRNA) were .identified as effectors of RNAi mechanism (Martin and Caplen 2007; Shrivastava and Srivastava 2008) . SiRNA are double stranded RNA molecules with 21-23 nucleotides (nts) in length, with a phosphate group at the 5' end and a hydroxyl group on the 3 ' end of each strand and with a two-nucleotide overhang on the 3' end of both strands (Chiu and Rana 2003; Huang et al . 2008; Martin and Caplen 2007), with a preference for uridine residues in the 3^' overhangs. It is very common to replace uridine residues for 2 ' -deoxythymidine to confer enhanced nuclease resistance (Kumar and Clarke 2007) . SiRNA molecules can be exogenously introduced into cell

cytoplasm. These synthetic siRNA are designed to mimic Dicer

(RNase I II -like enzyme) products enabling them to enter the RNAi machinery. Alternatively, siRNA can be endogenously produced from long dsRNA (-200 nts) which are processed by the Dicer into siRNA with 21-23 nts and 3 Overhangs (Huang et al . 2008; Martin and Caplen 2007; Rayburn and Zhang 2008) . Then, siRNA are incorporated into a multiprotein RNA induced silencing complex (RISC) that recognizes these dsRNA molecules and becomes active (Bantounas et al _r 2004; Huang et al . 2008; Lee and Sinko 2006; Martin and Caplen 2007; Rayburn and Zhang 2008) . Therefore, siRNA are unwinded through an ATP-dependent process by a helicase enzyme present in the RISC complex and the strand with lower thermodynamic stability at its 5' end remains in the complex and guide it to the complementary mRNA. Thus, the antisense strands (guide strand) remains in the RISC complex in opposition to the sense strand (passenger strand) which is eliminated from the RISC. The target mRNA is then cleaved by the nuclease Argonaute 2 protein of the RISC complex at a single site in the center of the duplex region, the phosphodiester bound of 10 nts from the 5' end of the siRNA (Bantounas et al . 2004; Dykxhoorn and Lieberman 2006; Lee and Sinko 2006) .

Consequently, the targeted mRNA is degraded and protein

expression is abolished or reduced.

In order to enhance the stability and improve the potency of the above mentioned gene silencing tools several structural modifications can be performed.

Even though a large number of clinical trials have been conducted with naked nucleic acids, it is well-known that the use of delivery agents have several advantages, such as nucleic acid protection from nuclease degradation and increased intracellular delivery.

Lipid-based gene delivery systems can be used to efficiently deliver gene silencing agents. Liposomes are micro or nanoparticles composed of one or more lipid bilayers, with an aqueous core (Drummond et al . 2008; Lasic 1998) . Liposomes were introduced as drug delivery vehicles in the 70s (Lasic 1998) , and their application in drug delivery depends on

physicochemical characteristics, such as composition, size, net charge, loading efficiency and stability (Drummond et al . 2008; Lasic 1998) . Therefore, the pharmacological profile of the drug entrapped in the liposomes is a function of the pharmacokinetic, biodistribution, and drug release characteristics of the

carrier .

For cancer gene silencing the access to metastasis disease sites, as well as primary tumors is vital. To fulfill these biodistribution demands, the lipid-based system must be designed considering a systemic application, thus being stable, exhibiting extended circulation life-times, and not interacting with blood components. On the other hand, these particles must be efficiently internalized and should have the ability to destabilize cell membranes promoting intracellular delivery of the carried nucleic acids (Leonetti et al . 2001; Wheeler et al . 1999) . The stabilized antisense lipid particles (SALP) or stabilized nucleic acids lipid particles (SNALP) lipid-based formulations incorporate some features that make them promising for systemic delivery applications, including minimal surface charge, small size and long circulation times, nucleic acid entrapment within lipid bilayers at high efficiencies,

protection of the nucleic acids and efficient delivery into tumor cells. Therefore, these lipid-based systems have been successfully used by others to mediate nucleic acid delivery (Leonetti et al . 2001; Mui et al . 2001; Semple et al . 2001;

Takasaki et al . 2006).

The only difference between SALP and SNALP is the nature of the entrapped nucleic acid. The SALP designation is used when the liposomes entrap asODN, whereas SNALP is used for liposomes entrapping siRNA. As both siRNA and asODN are nucleic acids, we decided to use indistinctively the term SNALP for this type of liposomes. SNALP are composed of lipid bilayers containing a mixture of cationic and fusogenic lipids coated with

polyethylene glycol-lipid (PEG-lipid) , which provides a

hydrophilic shield crucial for long circulation times in the blood stream and, that stabilizes the particles during their formation avoiding aggregation and fusion (Holland et al . 1996;

Morrissey et al . 2005; Semple et al . 2001). These particles are formed through a self-assembled process after addition of an ethanolic solution of lipids to an aqueous buffered solution of nucleic acids (Mui et al . 2001; Semple et al . 2001). Not

limiting examples of lipids used include cholesterol, a bilayer- forming lipid [such as 1,2- distearoyl -sn-glycero-3 - phosphatidylcholine (DSPC) ] , a protonable amino lipid [such as 1,2- d.ioleoyl-3-dimethylammonium-propane (DODAP) ] and a steric barrier lipid conjugate (PEG-lipid) . When the lipids are

injected into the acidic aqueous buffer, the protonable lipid becomes positively charged and complexes the negatively charged nucleic acids, resulting in liposomes entrapping the nucleic acids. Then, the external pH is raised to physiological values at which the protonable lipid turns to neutral (Leonetti et al . 2001; Mui et al . 2001; Semple et al . 2001).

The interaction of negatively charged nucleic acids with positively charged liposomes promotes membrane

destabilization which is enhanced by ethanol, a component of the SNALP preparation. This destabilizing effect induces the

formation of multilamellar liposomes with concentric bilayer shells from large unilamellar liposomes, thereby trapping nucleic acids between the lamellae of these multilamellar liposomes. Thus, concerning morphology, it was observed that unilamellar liposomes co-exist with bi- and multilamellar liposomes (Maurer et al . 2001) .

In order to reach all the requirements to cancer therapy, liposomes have to be engineered in a way to exhibit: i) prolonged circulation in the blood stream; ii) ability to specifically recognize and bind to target tissues or cells; iii) ability to provide an enhanced intracellular delivery of drugs and gene silencing tools, namely upon external or local stimulus (Torchilin 2009; Torchilin 2007) .

In order to increase circulation times of liposomes, the physicochemical properties of liposomes, such as charge, hydrophobicity, size, fluidity and packing of the lipid bilayer, influence their stability and biodistribution and have to be considered (Immordino et al . 2006) . Large, negative or positively charged liposomes have shorter half -life in the blood stream than small and neutral particles.

The liposome clearance mediated by the mononuclear phagocyte system {MPS ) is triggered by the binding of opsonins, which are serum proteins such as immunoglobulins, fibronectin, beta 2 -glycoprotein, C-reactive protein, beta 2 -macroglobulin and complement components (Immordino et al . 2006; Owens and Peppas 2006) . Indeed, the MPS does not recognize the liposomes themselves but, rather, recognize opsonins bound to the

liposomes. Opsonin proteins quickly bind to conventional non- stealth nanoparticles, allowing macrophages of the MPS to recognize and remove these particles before they can reach their target organ and exert their therapeutic function. This

sequestration in the MPS organs is very rapid, typically a matter of minutes, and usually is concentrated in the liver and spleen (Immordino et al . 2006; Owens and Peppas 2006) .

The most used method to mask or camouflage the liposomes from the MPS is the adsorption or grafting of

shielding groups which can block the attractive forces between the liposomes and the opsonins (Immordino et al . 2006; Owens and Peppas 2006) . These masked liposomes are named as stealth liposomes. The incorporation of hydrophilic carbohydrates or polymers on the liposome surface extends the liposome half-life from a few minutes (classical non-stealth liposomes) to several hours (stealth liposomes) (Immordino et al . 2006; Sapra and Allen 2003) . There are several biocompatible, soluble and hydrophilic polymers with a highly flexible main chain used to prepare long circulating liposomes, not limiting examples are poly (aer 1 amide), poly (vinyl pyrrolidone) , poly (acryloyl morpholine) , poly (2 -ethyl -2 -oxazoline) , poly (2 -methyl - oxazoline), phosphatidyl polyglycerols , polyvinyl alcohols, polysaccharides, PEG and PEG- containing copolymers (as

poloxamers and poloxamines) (Owens and Peppas 2006; Torchilin 2009; Torchilin 2006). Still, by far, the most successful and used approach is the covalent coupling of PEG chains to

lipososmes (PEGylation) . EG is a polyether diol which provides a very attractive combination of properties, such as solubility in aqueous and organic media, high flexibility of its polymer chain, very low toxicity, immunogenicity , and antigenicity

(Immordino et al . 2006; Torchilin 2006) . In addition, it

presents the lowest level of protein or cellular adsorption of any known polymer and has been FDA-approved for many injected biotech products. PEG is eliminated by a combination of hepatic and renal pathways (Immordino et al . 2006; Ryan et al . 2008). PEG molecular weight and structure can be freely modulated for specific purposes, and the process of lipid conjugation is easy and cheap (Immordino et al . 2006; Owens and Peppas 2006) . Not limiting examples of PEG- lipid conjugates to be used as stealth coatings for liposomes are: i) PEG-phosphatidylethanolamine ; ii) PEG-ceramide; iii) PEG-diacylglycerol ; iv) PEG- dialkyloxypropylamine and v) PEG- 1 -methyl -4 - (cis-9- dioleyl) methyl -pyridinium chloride ( PEG-SAINT) . The choice is dependent on the compromise between prolongation of the

circulation times, sufficient interaction of the carrier with the target and efficient drug delivery (Romberg et al . 2008) .

Targeting to specific sites or cell surface markers is performed by coupling cell surface-directing ligands in the targeted therapeutics (Baker et al . 2003; Torchilin 2006). The basic principle behind ligand- targeted therapeutics is that the delivery of drugs to cancer cells can be selectively enhanced by associating the drugs or the drug vehicles with molecules that specifically bind to antigens or receptors, which are either uniquely expressed or over-expressed on the target cells as compared to normal cells (Allen 2002; Baker et al . 2003; Sapra and Allen 2003) . Targeting moieties may include, but are not limited, antibodies, antibody fragments, naturally occurring or synthetic ligands like peptides, carbohydrates, glycoproteins, or receptor ligands, i.e. essentially a y molecule that selectively

recognizes and binds to target antigens or receptors (Sapra and Allen 2003; Torc ilin 2007). A classical target is the folate receptor, which has high affinity for the folic acid and is upregulated in many human cancers. The transferrin receptor, also over-expressed on the surface of many tumor cells, can be targeted with antibodies as well as transferrin (Torchilin

2007) .

Monoclonal antibodies or antibody fragments can be selected in order to exhibit a high degree of specificity for the target tissue. Some antibodies that bind to a specific surface receptor or antigen have intrinsic cytotoxicity, because they are able to interfere with cell proliferation and

differentiation. Thus, an added advantage of using these molecules for targeting purposes is the possibility of synergy interaction between the antibodies and the drug (Allen 2002; Sapra and Allen 2003) .

Transferrin (Trf) is a serum glycoprotein (80 KDa) responsible for iron transport (Baker et al . 2003; Li and Qian 2002) . The iron-linked transferrin is designated by holo- transferrin. The Trf receptor (TrfR) , also designated as CD71, is a membrane glycoprotein, a homodimer composed of two

identical transmembrane subunits. The Trf binding site is localized on the extracellular domain of the receptor, and each receptor subunit binds one transferrin molecule (Li and Qian 2002; Ponka and Lok 1999) . Trf attaches to the receptors on the cell surface, in a temperature- and energy- independent process (Ponka and Lok 1999) . Holo-Trf binds to TrfR, and the resulting complex undergoes endocytosis via clathrin-coated pits, by a temperature- and energy-dependent process. Upon endosomal maturation, the endosomal lumen is acidified to pH - 5.5. At this pH, the binding of iron to Trf is weakened leading to iron release from the protein. Although the observed acidification is not enough for efficient iron release, TrfR may play an

important role in this process presumably because the TrfR also changes its conformation at low pH and, thus forcing the

transferrin into an open conformation, facilitating release of iron. The free Fe³⁺ is reduced to Fe²⁺ on the endosomal membrane, which is subsequently transported out of endosome to the cytosol by the divalent metal transporter (D T1) to be used as a

cofactor or stored on ferritin. The resulting apo-Trf -TrfR complex is then recruited through exocytic vesicles back to the cell surface. At extracellular physiological pH, apo-Trf

dissociates from TrfR due to its low affinity at pH 7.4, being released into the circulation, and reutilized (Li and Qian 2002; Ponka and Lok 1999; Qian et al . 2002).

The TrfR is ubiquitously expressed in all nucleated cells in the body, however differs in levels of expression. It is highly expressed on rapidly dividing cells such as cells of the basal epidermis and intestinal epithelium and very low or frequently undetectable in non-proliferating cells. Various studies have shown raised TrfR expression on cancer cells when compared to their normal counterparts, this being attributed to the increased need of rapidly dividing cells for iron as a cofactor of the ribonucleotide reductase enzyme involved in DNA synthesis (Daniels et al . 2006; Li and Qian 2002; Qian et al . 2002) . Increased TrfR expression has been correlated with tumor grade and stage or prognosis (Daniels et al . 2006) .

Thus the elevated levels of TrfR in cancer cells, the extracellular accessibility of this molecule and its

constitutive internalization render this receptor with ideal properties for targeting therapeutics to cancer cells. Targeting TrfR can be mediated by coupling Trf or antibodies to the delivery vector, such as liposomes, lipoplexes or viral vectors (Nobs et al . 2004; Qian et al . 2002) .

The coupling of ligands to the liposome surface can be achieved by covalent or non-covalent bonds. Non-covalent methods have the great advantage of being easy to be carried out without the need of aggressive reagents, A simple method is to merely add the ligand to the phospholipids during the liposome

preparation. However, the coupling efficacy is relatively low (4-40%) and aggregation of the liposomes is frequently observed. Furthermore, the amount of ligand linked to the liposome is not easily controllable, and the correct orientation of the ligands is not ensured. Finally, detachment of the ligands in in vivo might occur (Nobs et al . 2004) . Covalent reactions are an effective way to irreversibly attach ligands to the carriers, because the linkage formed is much more stable and reproducible when compared to non-covalent methods (Nobs et al . 2004) .

However, covalent reactions require the need of chemical

reagents and when the coupling reaction is performed in preformed liposomes, the risk of altering the structure of the particles and in some cases of the encapsulated compounds must be considered. Therefore, it is essential to choose non- aggressive reagents and to work under mild, controlled

conditions (Nobs et al . 2004) . A way to overcome this limitation consists in coupling ligands to stealth liposomes by the "post- insertion" technique. In the "post-insertion" method ligands are coupled to end-functionalized groups in PEG micelles and then ligand-PEG conjugates are transferred in a simple incubation step into the outer monolayer of pre -formed, drug/nucleic acid loaded

liposomes (Allen et al . 2002; Ishida et al . 1999; Sapra and Allen 2003; Us r et al . 1996). Not limiting examples of end- functional ized derivatives of PEG have been synthesized for coupling ligands to the PEG-terminus are pyridyldiotiopropionoylamino (PDP) -PEG, hydrazide (Hz) -PEG and maleimide (Mai) -PEG . The reaction between thiol groups introduced in the ligands and the maleimide groups coupled to PEG is highly efficient leading to a stable thioether bond (Sapra and Allen 2003; Uster et al . 1996) . The combination therapy with multiple drugs or

multiple modalities is now a widespread practice in the

treatment of cancer with outcomes that would be unattainable with single drug treatments. The rationale for the use of multiple agents in chemotherapy relies on the heterogeneity of tumor cells and consequent differences in tumor cell sensitivity to individual drugs (Ishida et al . 1999; Saxon et al . 1999a; Zoli et al . 2001). Multi-target therapeutics can be more

efficacious and less vulnerable to adaptative resistance, because the biological system is less able to compensate for the action of two or more drugs simultaneously. Consequently, it is possible to better control complex diseases with less

probability to drug resistance development, accomplishing this way higher cytotoxicity and durable anticancer activity

(Zimmermann et al , 2007; Zoli et al . 2001).

The multi-target therapeutic strategies comprise different modalities: i) the components act on separate targets to create a combination effect; ii) one component alters the ability of the other to reach its target and therefore the modulation of one target facilitates action at a second target, iii) the components act at the same target to create a

combination effect and increase the pharmacological action

(Cullis et al . 1997) . Generally, the cellular targets and mechanisms of action are well-known for most drugs however their interference with each other when used in combination has not been

sufficiently investigated. It is well established that different drugs can interact with each other at the pharmacological level and modify the respective effects (Merlin 1994; Zoli et al .

2001) . The pharmacological interactions between the drugs can be classified as synergism, antagonism and additivity. A non- interactive or additive combina ion is observed when the

compounds act without any interaction among them and

consequently the observed effect is not statistically different from the expected, theoretically calculated from the response of every agent administered individually (Goldoni and Johansson 2007; Merlin 1994) . Synergism occurs when the observed effect is statistically higher than the calculated effect, and antagonism is observed when the obtained effect is statistically lower than the calculated effect (Chou 2006; Goldoni and Johansson 2007; Jia et al . 2009; Merlin 1994) .

Thus, the golden goal of combinatory therapy is to obtain synergistic drug combinations in order to achieve more favorable outcomes, such as enhanced efficacy, decreased dosage at equal level of efficacy, minimal or slower development of drug resistance, reduction of adverse effects (Chou 2006; Jia et al . 2009; Merlin 1994) . The dose reduction index (D I) is a measure of how many- fold the dose of a drug in a combination may be reduced to produce a given effect level compared with the dose of this drug used per se (Chou 2006) .

The antitumor activity of drug combinations can be significantly dependent on the molecular ratio of the combined drugs. For the same drug combination, some ratios can be

synergistic, whereas other ratios can be additive or even antagonistic. This highlighted the heed to control drug ratios being exposed to tumor cells (Mayer et al . 2006; Tardi et al . 2007) . In in vivo applications, in contrast to in vitro systems, where drug concentrations exposed to tumor cells can be tightly controlled, the individual agents of an anticancer drug combination will be distributed and eliminated independently of each other disrupting the original drug ratio. Therefore, uncoordinated pharmacokinetics of individual drugs utilized in the drug cocktails results in exposure to sub-optimal drug ratios with a concomitant loss in therapeutic activity (Mayer et al . 2006; Tardi et al . 2007).

Liposomes can overcome the uncoordinated

pharmacokinetics of individual drugs utilized in the drug combinations, because a single liposomal formulation can entrap the combined drugs in the desired ratio, allowing in vivo tumor cells to be exposed to the optimal drug/drug ratio. It is important to keep in mind that the pharmacokinetic behavior of the co- formulated drugs will be dictated by the pharmacokinetics of the drug carrier used, and thus the plasma elimination and tissue distribution of the combined agents can be coordinated adequately.

There are two drug loading approaches, passive and active loading. In active loading, drug is loaded into preformed vesicles, in response to specific transmembrane gradients, such as pH gradient and transition metal ions gradient (Li et al . 2008) . The establishment of metal ions gradient across the liposomal membrane allows the efficient drug loading based on the formation of drug /metal complexes trapped inside liposomes (Abraham et al . 2004; Li et al . 2008) . On the other hand, the pH gradient method to encapsulate drugs is based on the fact that neutral forms of weak acids and weak bases can permeate through lipid bilayer membranes at much faster rates than the charged forms. Therefore, for drugs with protonable amines, as imatinib mesylate, the neutral form permeates the liposomal membrane and _ is subsequently protonated in the liposomal internal acidic buffer. As the charged (protonated) form of the drug permeates much less than the neutral form, the drug becomes trapped inside. The drug can also be precipitated by anions such as sulphate and citrate, thus increasing the drug loading (Cullis et al. 1997; Ishida et al. 1999; Li et al . 2008).

In the pH gradient method, not limiting examples of generation of a pH gradient are i) preparation of vesicles in acidic buffer and then exchange of external buffer or vice versa; ii) employment of a self-generating system such as ammonium sulphate gradient and, iii) using an ionophore (Abraham et al. 2004; Li et al . 2008; Mayer et al. 1986; Wang et al.

2005) .

The efficiency of drug trapping and the capability of the encapsulated drug to be retained by the liposomes are dependent on a variety of factors, such as stability of the pH gradient (which is dependent on the buffering capacity within the liposome and the amount of loaded drug) , the chemical characteristics of the drug (as the potential to form insoluble salt products) , and the permeability of the liposomal membrane (which is affected by lipid composition and temperature)

(Abraham et al. 2004; Cullis et al. 1997; Ishida et al. 1999; Saxon et al . 1999a) .

Brief description of the drawings

Figure 1. Encapsulation yields of imatinib and siRNA and siRNA/imatinib molar ratio in Trf-liposomes co-encapsulating both drugs. Different imatinib : total lipid molar ratios (1/3; 1/5; 1/8; 1/16; 1/32; 1/42) were incubated with SNALP liposomes loaded with an i-BC.R-.4I5I· siRNA as described in Materials and Methods. In addition, imatinib at 1/8 imatinib: total lipid ratio [referenced in the figure as 1/8 (imatj] was incubated with Trf- liposomes without siRNA. After liposome purification, the final siRNA and imatinib were quantified and the encapsulation yield of imatinib (A) and of siRNA (B) as well as the siRNA/ imatinib molar ratio (C) were assessed. No symbol p>0.05; *p<0.05;

**p<0.01 and ***p<0.001 when the comparison was established with the 1/3 formulation or between the conditions indicated by the lines (A and B) or with the 1/16 formulation (C) .

Figure 2- Extent of association of Trf-coupled

liposomes loaded with Cy3 - siRNA to LAMA- 84 cells. A) Cells were incubated at 37 ^bC during 4 h with different concentrations of Tr -coupled liposomes encapsulating 0.2-2.0 μΜ Gy3 - siRNA (0.2- 2.0 μΜ Trf), BSA-coupled liposomes encapsulating 0.5-2.0 μΜ Cy3 - siRNA (0.5-2.0 μΜ BSA) or non-targeted liposomes encapsulating 0.5-2.0 μΜ Cy3 - siRNA (0.5-2.0 μΜ NT) . B) Cells were incubated with different concentrations of Trf -liposomes loading Cy3 -siRNA at 37 ^DC in the presence (37 °C Trf RS) or absence (37 °C) of an excess of free Trf or incubated at 4 °C in the presence (4 °C Trf RS) or absence (4 °C) of free Trf. The fluorescence of the Cy3 -siRNA associated to LAMA- 84 cells was assessed by flow cytometry. A) One-way ANOVA analysis of variance combined with Tukey post test and B) regular two-way ANOVA analysis of

variance combined with Bonferroni post test for multiple

comparisons were used. ***p<0.001, **p<0.01, *p<0.05 and no symbol p>0.05 when the comparison was established between the conditions indicated by the lines (A) or when comparison Was established with cells treated at 37 °C in the absence of free Trf (B) . ###p<0.001 when the comparison was established with the corresponding concentration of the Trf-liposome formulation (A) .

Figure 3- Confocal microscopy images from studies performed in LAMA- 84 cells incubated with Trf-coupled liposomes loaded with Cy3 -siRNA. Cells were incubated for 4 h, at 37 °C with 1 μΜ of Cy3 -siRNA encapsulated in A,F) Trf -coupled

liposomes, B) BSA-coupled liposomes C) N -liposomes or D) Trf- coupled liposomes in the presence of an excess of free Trf . E) LAMA- 84 cells incubated with Trf-coupled liposomes at 4 °C.

Confocal images were acquired in a point scanning confocal microscope Zeiss LSM 510 Meta (Zeiss, Germany) , A-E) with a 4 Ox EC Plan-Neofluar . F) Differential interference contrast (DIG) images were taken with a 63x Plan-Apochromat oil immersion obj ectives .

Summary of the invention

The present invention provides a single drug delivery nanocarrier allowing triple targeting to human disease and disorders. Such a system co-encapsulates two or more molecules in well established therapeutic molar ratios. Long circulation times in the blood stream are conferred by hydrophilic polymers and the ability to target a specific cell population (cellular targeting) is conferred by coupling a targeting ligand to the surface of this nanocarrier. Multi-molecular targeting is attained through the therapeutic agents encapsulated or

incorporated into the nanocarrier. The nanocarrier is capable to transport the therapeutic agents to the target cells, avoiding degradation or biotransformation. Subsequently, it binds to the target cells, is internalized and delivers their content intracellulary .

Overall, as a major benefit, this invention provides, the possibility to target a cell population and two or more molecular targets simultaneously using one single nanocarrier, containing a specific molar ratio of two or more therapeutic agents. For example, for CML treatment, targeting ligands are coupled to the nanocarrier surface in order to target leukemia cells. Ant i - BCR-ABL siR A and imatinib are co-encapsulated into the targeted nanocarrier, which allows the simultaneous

knockdown of the BCR-ABL oncogene and the inhibition of the tyrosine kinase activity of the Bcr-Abl oncoprotein. The active agent or therapeutic agent is fully encapsulated within the lipid particle such that the active agent or therapeutic agent are protected from enzymatic degradation, e.g., by a nucleases or proteases.

Detailed description of the invention Introduction

The combination of different strategies directed to the same molecular target or to different molecular targets emerges as a promising therapeutic strategy for some diseases, allowing to reach outcomes unattainable with single therapeutic approaches. The rationale to the use of drug combinations for cancer treatment, is based on the heterogeneity of tumor cells and consequently on differences in tumor cell responses to individual drugs. One not limiting example of combined

strategies to achieve a more effective treatment for drug- sensitive or drug-resistant cancer cells is to combine imatinib mesylate with gene silencing tools, such as siRNA, targeting the oncogene BCR-ABL aiming at chronic myeloid leukemia (CML)

treatment. However, the antitumor activity of drug combinations can be significantly dependent on the molar ratio of the combined drugs. In fact, for the same drug combination some ratios can be synergistic, whereas other ratios can be additive or even antagonistic, which highlights the need to control drug ratios being exposed to tumor cells.

In in vitro cell culture systems, ratios of drug combinations exposed to tumor cells can be tightly controlled. This is something that upon systemic administration is extremely difficult to achieve, due to different pharmacokinetic profile of each one of the drugs entering the combination. Under the circumstances, tumor cells are therefore exposed to sub-optimal drug ratios with a concomitant loss in therapeutic activity. Such problem can be overcome upon incorporation/encapsulation of the drug combination into nanocarriers, like PEGylated

liposomes, able to maintain the drug ratio from the site of administration until it reaches the tumor cells.

Targeting the therapeutic agents to a specific cell population allows improvement of the therapeutic activity, while lowering the side effects promoted by these agents. Such

targeting is possible through coupling of specific targeting ligands to the therapeutic agents or to the nanocarriers that carry and mediate the intracellular delivery of the therapeutic agents .

The lipid particles of the present invention provides, the possibility to target a cell population and two or more molecular targets simultaneously using one single nanocarrier, containing a specific molar ratio of two or more therapeutic agents. The invention provides a single drug delivery

nanocarrier allowing triple targeting to human disease and disorders. The lipid particles and compositions of the present invention may be used for a variety of purposes, including the delivery of associated or encapsulated therapeutic agents to cells, both in vitro and in vivo. Accordingly, the present invention provides methods for treating human diseases or disorders, by treating the subject or the subject cells/tissues with the lipid particles described here containing one or more therapeutic agents.

Definitions

As used herein, the following terms have the meanings attributed to them unless specified otherwise.

The term "RNA interfering" or "RNAi" refers to single stranded RNA fe g mature micro RNA (miRNA) ) or double stranded RNA (e.g. siRMA) that is capable of reducing or inhibiting the expression of a target gene (e.g., by promoting the degradation or inhibiting the translation of the mRNA which are complementary to the siRNA/miRNA sequence) . RNA interference, thus refers to the single stranded RNA that is complementary to a target mRNA sequence, or to the double stranded RNA formed by two strands, with one of the strand complementary to the target mRNA. RNA interference may have complete complementarity to the target gene or sequence or may comprise a region of mismatch (i e, an uncomplementary motif) . RNA interference includes "small

interfering RNA" or "siRNA, " e g , siRNA of about 15-60 (duplex) nucleotides in length, more typically about 15-30 (duplex) nucleotides in length, and is preferably about 20-27 (duplex) nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-60 nucleotides in length, preferably about 20-27 nucleotides in length. siRNA duplexes may comprise 3' overhangs of about 1 to 4 nucleotides and 5' phosphate termini. Examples of siRNA include, but are not limited to, a double stranded polynucleotide molecule assembled from two separate stranded molecules, wherein one strand is the sense strand and the other is the complementary antisense strand.

SiRNA are chemically synthesized, or may be generated by

cleavage of longer dsRNA (e.g., dsRNA greater than about 25 nucleotides in length) with the endogenous enzyme Dicer, which process the dsRNA into biologically active siRNA, or siRNA may be encoded by a plasmid (e.g., transcribed from the plasmid

(circular dsDNA) encoded information as single nucleotide strand that automatically fold into duplexes with hairpin loops) .

The term "nucleic acid" as used herein refers to a polymer containing at least two deoxyribonucleotides or

ribonucleotides in either single or double stranded form, it includes both DNA and RNA molecules. Not limiting forms of DNA molecules are antisense molecules and plasmid DNA. Not limiting examples of RNA molecules are siRNA, asymmetrical interfering RNA (aiRNA) , microRNA. Nucleic acids may include nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring. Not limiting examples of such analogs are phosphorothioates , phosphoramidates , methyl phosphonates and peptide-nucleic acids (PNAs) .

The term "lipid" refers to a group of organic water insoluble compounds which are the basic components of biological membranes. Lipids are a broad group of molecules which includes fats, waxes, sterols, phospholipids. Lipids may be broadly defined as hydrophobic or amphiphilic molecules; the amphiphilic nature of some lipids allows them to form structures such as vesicles, liposomes, or membranes in an aqueous environment. A "lipid particle" or "liposomes" are micro or nanoparticles composed of one or more lipid bilayers, with an aqueous core, which is used herein to refer to a lipid formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., a siRNA) , to a target site of interest. In the liposomes of the invention, which are formed from a cationic lipid, a neutral lipid, and a conjugated lipid (e.g., PEG-lipid) that prevents aggregation of the liposomes and increases blood stream circulation times, the therapeutic agents and/or diagnosis agent may be encapsulated in the liposomes, thereby protecting the agents from enzymatic degradation and improving the pharmacokinetics features. As used herein, the term "SALP" refers to stabilized antisense lipid particles and "SNALP" refers to a stabilized nucleic acid lipid particle. SALP is the term used to refer to a nucleic acid-lipid particle encapsulating asODN within the lipid particles. SNALP is the term used to refer to a nucleic acid-lipid particle

encapsulating siRNA within the lipid particles. SALP/SNALP represent particles made from lipids (e.g., a cationic lipid, a neutral lipid, and a conjugated lipid that prevents aggregation and increases blood stream circulation times) , encapsulating nucleic acids (e.g., siRNA, aiRNA, mlRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA) , dsRNA, or a plasmid, including plasmids from which an interfering RNA is transcribed) . As used herein, the term "SNALP" includes SPLP (stabilized plasmid particles; whenever the lipid particles encapsulate plasmid molecules) ; SNALP and SALP . SNALP can exhibit extended

circulation lifetimes following intravenous (I.V.) injection, they can accumulate at distal sites (e.g., sites physically separated from the administration site) , they can mediate expression of the transfected gene or silencing of target gene expression at these distal sites, they can mediate the efficient delivery of the non- nucleic acid-based therapeutic agents co- encapsulated with the nucleic acids.

The term "fusogenic" refers to the ability of a liposome, or other drug delivery system to fuse with membranes of a cell .

The term "cationic lipid" refers to any lipid that carries a net positive charge at a selected pH. Not limiting examples of cationic lipids are 1 , 2 -dioleoyl-3 -dimethylammonium- propane (DODAP) , 1 , 2 -dilinoleyloxy- 3 - (2 -N, - dimethylamino) ethoxypropane (DLin-EG-D A) , N, -dioleyl -N, - dimethylammonium chloride (DODAC) , 1 , 2 -dioleyloxy-N, N-dimethyl - 3 -aminopropane (DODMA) , 1 , 2 -distearyloxy-N, N-dimethyl -3 - aminopropane (DSDMA) , N- (1- (2 , 3 -dioleyloxy) propyl) -N, N, - trimethylammonium chloride (DOTMA) , N-(l-(2,3- dioleoyloxy) propyl ) -N, N, N-trimethylammonium chloride (DOTAP) , 3- (N- ( ' , ' -dimethylaminoethane) -carbamoyl ) cholesterol (DC-Choi) , N- (1, 2-dimyristyloxyprop-3-yl) -N, -dimethyl -N-hydroxyethyl ammonium bromide (D RIE) , 2 , 3 -dioleyloxy-N- [2 (spermine- carboxamido) ethyl] - , -dimethyl - 1 -propanaminium trifluoroacetate (DOSPA) , dioctadecylamidoglycylspermine (DOGS) , N, N-dimethyl - 3 , 4-didleyloxybenzylamme (DMOBA) , 1 , 2 -N, N ' -dioleylcarbamyl -3 - dimethylaminopropane (DOcarbDAP) , 1 , 2 -N, N ' -dilmoleylcarbamyl - 3 - dimethylaminopropane (DLincarbDAP) , 1 , 2 -dilinoleyloxy-N, N- dimethylaminopropane (DLinDMA) , 1 , 2 -dilinolenyloxy-N, N- dimethylaminopropane (DLenDMA) , 2 , 2 -dilinoleyl -4 - (2 - dimethylaminoethyl ) - [1,3] -dioxolane (DLin-K C2 -DMA) , 2,2- dilinoleyl -4 - (3 -dimethylarninopropyl ) - [1,3] -dioxolane (DLin-K- C3 -DMA) , 2 , 2 -dilinoleyl -4 - (4 -dimethylaminobutyl ) - [1,3] -dioxolane (DLin-K-C -DMA) , 2 , 2 -dilinoleyl - -dimethylaminomethyl - [1,3] - dioxolane (DLin-K-DMA) _; 1 , 2 -dilinoleylcarbamoyioxy-3 - dimethylaminopropane (DLin-C-DAP) , 1 , 2 -dilinoleyoxy- (dimethylamino) acetoxypropane (DLin-DAC) , 1 , 2 -dilinoleyoxy-3 - morpholinopropane (DLin-MA) , 1 , 2 -dilinoleoyl -3 - dimethylaminopropane (DLinDAP) , 1 , 2-dilinoleylthio-3- dimethylaminopropane (DLin-S-DMA) , l-linoleoyl-2 -linoleyloxy-3 - dimethylaminopropane (DLin-2 -DMAP) , 1 , 2 -dilinoleyloxy-3 - trimethylaminopropane chloride salt (DLin-TMA. Cl ) , 1,2- dil inoleoyl -3 - trimethylaminopropane chloride salt (DLin-TAP . Cl ) , 1 , 2 -dilinoleyloxy-3 - (N-methylpiperazino) propane (DLin-MPZ) , 3- (N, N-dilinoleylamino) -1 , 2 -propanediol (DLinAP) , or mixtures thereof .

The term "neutral lipid" refers to any lipid that exist either in an uncharged or neutral z itterionic form at a selected pH. The neutral lipid components may be cholesterol or a derivative, phospholipids, or a mixture of phospholipids and cholesterol or a derivative. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone , cholestenone , coprostanol, cholesteryl-2¹ -hydroxyethyl ether, cholesteryl -4¹ - hydroxybutyl ether, and mixtures thereof.

Examples of neutral lipids include but are not limited to dipalmitoylphosphatidylcholine (DPPC) ,

distearoylphosphatidylcholine (DSPC) ,

dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl - phosphatidylcholine (POPC) , palmitoyloleoyl - phosphatidylethanolamine (POPE) , palmitoyloleyol - phosphatidylglycerol (POPG) , dipalmitoyl- phosphatidylethanolamine (DPPE) , dimyristoyl- phosphatidylethanolamine (DMPE) , distearoyl- phosphatidylethanolamine (DSPE) , monomethyl- phosphatidylethanolamine, dimethyl -phosphatidylethanolamine , dielaidoyl- phosphatidylethanolamine (DEPE) , stearoyloleoyl- phosphatidylethanolamine (SOPE) , egg phosphatidylcholine (EPC) , and mixtures thereof .

"Increased blood stream circulation," as used herein, refers to a broad biodistribution of a therapeutic agent such as siRNA within an organism through the improvement of the time in circulation of the delivery vehicle. The enhancement of the amount of the active agent available to be exposed to most parts of the body is generally achieved by decreasing degradation and/or blood clearance (such as by first pass organs (liver, spleen, lung, etc.) and nonspecific cell binding.

The term "cancer" is the designation adopted for a group of more than 100 human diseases that have in common the uncontrolled cell growth (division beyond the normal cell control), invasion (intrusion of adjacent tissues), and

sometimes metastasis (spread to other locations in the body) . Cancers are caused by abnormalities in the genetic material of the transformed cells. These abnormalities may be due to the effects of carcinogens, such as tobacco smoke, radiation, chemicals, or infectious agents. Cancer promoting genetic abnormalities may be caused by random errors in DNA replication, or are inherited. The term "leukemia" refers to a broad group of

diseases characterized by accumulation of malignant white cells in the bone marrow and blood that do not tend to form solid masses of cells (Hoff^'brand et al . 2006a; Ottensmeier 2001). This accumulation of abnormal white cells leads to bone marrow failure and infiltration of leukemia cells in different organs (e.g. liver, brain, skin, spleen) {Hoff-brand et al . 2006a) .

Leukemias can be classified in four main types, the acute and chronic leukemias, which are subdivided into lymphoid and myeloid leukemias. Chronic leukemias are distinguished from acute by their slower progression in opposition to the

aggressive character Of the acute leukemias. Myeloid and

lymphoid leukemias differ in the hematopoietic lineage affected by the malignant transformation; myeloid leukemias are

originated in the myeloid lineage and lymphoid leukemias in the lymphoid lineage (Hoffbrand et al . 2006a) .

Chronic myeloid leukemia is a myeloproliferative disease originated from hematopoietic stem cells, and was the first human malignancy to be linked with a dominant acquired genetic mutation. Chronic myeloid leukemia is caused by a translocation between chromosomes 9 and 22 which create the oncogene BCR-ABL and an abnormal 22 chromosome (Philadelphia chromosome; Ph) . There are no known predisposing factors, except the exposure to ionizing radiation, since the incidence of CML was greatly increased in survivors of the atomic bomb exposures in Japan. No inheritable factors have been identified, although it is assumed that an acquired genetically chromosomal

instability might be the precondition for the translocation (Hoffbrand et al . 2006b; Pasternak et al . 19.98) .

The terms "silencing", "knockdown the expression of " and "down -regulation the expression of," herein refer to the at least partial suppression of the expression of the target gene as assessed by the reduction of the amount of the target mRNA.

The term "lipid conjugate" refers to a lipid conjugated to a shielding group that inhibits aggregation of nucleic acid lipid particles and increase blood stream

circulation times. The incorporation of hydrophilic

carbohydrates or polymers, as shielding groups, on the liposome surface extends the liposome half-life from a few minutes

(classical non-stealth liposomes) to several hours (stealth liposomes) (Immordino et al . 2006; Sapra and Allen 2003) . Such lipid conjugates include, but are not limited to,

poly (acrylamide) , poly (vinylpyrrolidone) ,

poly (aeryloylmorpholine) , pol (2-ethyl-2-oxazoline) , poly (2- methyl -oxazoline) , phosphatidyl polyglycerols , polyvinyl

alcohols, polysaccharides, PEG and PEG-containing copolymers (as poloxamers and poloxamines) (Owens and Peppas 2006; Torchilin 2009; Torchilin 2006), and mixtures thereof. PEG is a polyether diol which provides a very attractive combination of properties, such as solubility in aqueous and organic media, high

lity of its polymer chain, very low toxicity,

immunogenicity, and antigenicity (Immordino et al . 2006;

Torchilin 2006) . In addition, it presents the lowest level of protein or cellular adsorption of any known polymer and has been FDA-approved for many injected biotech products. Not limiting examples of PEG- lipid conjugates are: i) PEG- phosphatidylethanolamine ; ii) PEG-ceramide ; iii) PEG- diacylglycerol ; iv) PEG-dialkyloxypropylamine and v) PEG-1- methyl-4- (cis-9-dioleyl) methyl -pyridinium chloride (PEG-SAINT) (Romberg et al . 2008).

The term "post insertion" refers to a 1 igand coupling method in which 1 igands attached to a conjugated lipid are introduced into preformed liposomes.

The term "micelles" refers to an aggregate of surfactant molecules dispersed in a liquid. Generally, micelles in aqueous solution form an aggregate with the hydrophilic regions "head" in contact with the surrounding solvent (water) , whereas, the hydrophobic single tail regions are in the micelle cent e .

The term "antisense oligonucleotides" or "asODN" are single stranded molecules of RNA or DNA usually with 18-25 nucleotides in length that hybridize in a sequence specific manner to the target mRNA/DNA and down regulate the target gene, Description of the embodiments

The lipid particles of the present invention provides, the possibility to target a cell population and two or more molecular targets simultaneously using one single nanocarrier. Thus, the drug delivery nanocarrier is targeted for specific organ, tissue or cells by coupling of targeting ligands to its surface. Not limiting examples of targeting moieties include peptides, polypeptides, antibodies, polyclonal antibodies, monoclonal antibodies, antibody fragments, humanized antibodies, recombinant antibodies, recombinant human antibodies, proteins and cell surface ligands. The ligands are linked to the surface of the nanocarrier in a way that it is able to interact with a specific molecule, protein, glycoprotein and/or cell surface receptor that is overexpressed or specifically expressed on the cell surface of a specific cellular target. An appropriate spacer can be positioned between the nanocarrier and the ligand to avoid hindrance on the interaction between the ligand and its target. The nanocarrier can allow multivalent cellular

targeting, presenting more than one homing ligand that

selectively homes the delivery agent to specific molecules on the target cells.

The nanocarrier should have a diameter comprised between 100 and 200 nm to travel in the blood stream circulation without occluding circulation and without being rapidly removed from circulation by first passage organs; being available to specifically interact with the target cells in therapeutic dosages .

The drug delivery nanocarrier of the present invention provides a method for simultaneous encapsulation, adsorption or complexation of two or more gene silencing agents (nucleic acids) and two or more non-nucleic acid based active agents (therapeutic drugs) . The gene silencing agents (nucleic acids) may comprise, but not limited to plasrnids, antisense

oligonucleotides (asODN) , siRNA, mi NA, shRNA, aiRNA, DNA enzymes (DNAzymes) , Ribozymes, DNA decoys, Aptamers and mixtures thereof. The gene silencing agents may comprise modified nucleotides including, but not limited to phosphorothioate linkages, 2 ' -O-methyi (2'OMe) nucleotides, 2 ' -deoxy-2 ' - fluoro (2'F) nucleotides, 2¹ -deoxy nucleotides, 2 ' -0- (2 -methoxyethyl ) (MOB) nucleotides, locked nucleic acid (LNA) nucleotides, and mixtures thereof. The gene silencing agents may comprise at least one or a cocktail (e.g., at least two, three, four, five, six, seven or more) of unmodified and/or modified gene silencing agents. The cocktail of gene silencing agents may comprise sequences which are directed to the same region or domain and/or to different regions or domains of one or more target genes. The gene silencing may be performed by any viral vector capable of accepting the coding sequences for the gene silencing agent, including but not limited to retrovirus, herpes virus,

adenovirus and adenoassociated virus.

Non-nucleic acid based active agents (therapeutic drugs) may include, but not limited to chemotherapy drugs, hormonal therapeutic agents, immunotherapeutic agents, antiviral drugs, anti - inflammatory compounds, antidepressants, stimulants, analgesics, antibiotics, antipyretics, vasodilators, an i -angiogenics, c o ascular agents, signal transduction inhibitors, anti -arrhythmic agents, hormones, vasoconstrictors, and steroids .

Non-limiting examples of chemotherapy drugs include platinum-based drugs (e.g. cisplatin, carboplat in, etc) ;

alkylating agents (e.g., cyclophosphamide, chlorambucil, busulfan, melphalan, lomustine, carmustine, estramust ine , treosulfan, thiotepa, mitobronitol , etc); anti-metabolites (e.g., 5-fluorouracil , methotrexate, capecitabine, cytarabine, fludarabine, gemcitabine, cladribine, raltitrexed,

mercaptopurine, etc); plant alkaloids (e.g., , vincristine, vinblastine, vindesine, paclitaxel, docetaxel, etc) ,

topoisomerase inhibitors (e.g., irinotecan, topotecan,

etoposide , etc); cytotoxic antibiotics (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone , aclarubicin, idarubicin, dactinomycin, etc ), taxanes (e.g., docetaxel, paclitaxel); tyrosine kinase inhibitors (e.g., gefitinib, sunitinib, erlotinib, lapatinib, canertinib, semaxinib, vatalanib, sorafenib, imatinib,

dasatinib, leflunomide, vandetanib, pharmaceutically acceptable salts thereof, stereoisomers thereof, analogs thereof,

derivatives thereof, and mixtures thereof; anti-inflamatory agents such as but not limited to ibuprofen, aceclofenac, acemetacin, acid acetilsalicilic, azapropazone , celecoxib, diclofenac sodium, diflunisal, cetodolac, fenbufen, fenoprofen, flubiprofen, indomethacin, acetaminocin, piroxicam, rofecoxib, sulindac, tenoxicam,- antiangiogenic agents or angiolytic agents such as but not limited to angiostatin (plasminogen fragment) , antiangiogenic antithrombin III, vasculostatin, vasostatin and mixtures thereof.

The nanocarrier is composed, but not limited, by liposomes and/or polymers. Not limiting examples of liposomes are stabilized nucleic acid lipid particles (SNALP) . SNALP liposomes comprise one or more cat ionic lipids, one or more neutral lipids and one or more conjugated lipid that inhibits aggregation of particles and provide long circulation times to the liposomes. The cationic lipids may comprise from 10 to 60 mol%, the neutral lipid may comprise from 10 to 70 mol%, the conjugated lipid that inhibits aggregation and provides long circulation times may comprise from 1 to 10 mol%. Not limiting examples of cationic lipids are 1,2- dioleoyl-3-dimethylammonium-propane (DODAP) , 1 , 2 -dilinoleyloxy- 3-(2-N,N- dimethylamino) ethoxypropane (DLin-EG-DMA) , N,N- dioleyl-N,N-dimethylammonium chloride (DODAC) , 1 , 2 -dioleyloxy- N, -dimethyl -3 -aminopropane (DODMA) , 1 , 2 -distearyloxy-N, N- dimethyl -3 -aminopropane (DSDMA) , N- (1 -{2,3 -dioleyloxy) propyl) - Ν,Ν,Ν- trimethyl ammonium chloride (DOTMA) , N-(l-(2,3- dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTAP) , 3- (N- (Ν' ,Ν¹ -dimethylaminoethane) -carbamoyl ) cholesterol (DC-Choi) , N- (1 , 2-dimyristyloxyprop-3-yl) -N, N-dimethyl -N- hydroxyethylammonium bromide (DMRIE) , 2 , 3 -dioleyloxy-N- [2 (spennine-carboxamido) ethyl] -N, -dimethyl - 1 - propanaminiumtrifluoroacetate (DOSPA) ,

dioctadecylamidoglycylspermine (DOGS), N, -dimethyl -3 , - dioleyloxybenzylamme (DMOBA) , 1 , 2 -N, ¹ -dioleylcarbamyl -3 - dimethylammopropane (DOcarbDAP) , 1 , 2-N, ' -dilmoleylcarbamyl-3 - dimethylaminopropane (DLincarbDAP) , 1 , 2 -dilinoleyloxy-N, N- dimethylaminopropane (DLinDMA) , 1 , 2 -dilinolenyloxy-N, N- dimethylaminopropane (DLenDMA) , 2 , 2-dilinoleyl-4- (2- dimethylaminoethyl) - [1, 3] -dioxolane (DLin-K-C2 -DMA) , 2,2- dilinoleyl-4- (3-dimethylarninopropyl) - [1,3] -dioxolane (DLin-K- C3 -DMA) , 2 , 2 -dilinoleyl -4- (4 -dimethylaminobutyl ) - [1,3] -dioxolane (DLin-K-C4 -DMA) , 2 , 2 -dilinoleyl -4 -dimethylaminomethyl - [1,3] - dioxolane (DLin- -DMA) , 1 , 2 -dilinoleylcarbamoyloxy-3 - dimethylaminopropane (DLin-C-DAP) , 1 , 2 -dilinoleyoxy-

(dimethylamino) acetoxypropane (DLin-DAC) , 1 , 2 -dilinoleyoxy-3 - morpholinopropane (DLin-MA) , 1 , 2 -dilinoleoyl -3 - dimethylaminopropane (DLinDAP) , 1 , 2 -dilinoleylthio-3 - dimethylaminopropane (DLin-S- DMA), 1-linoleoyl -2 -linoleyloxy-3- dimethylaminopropane (DLin-2 -DMAP) ., 1 , 2 -dilinoleyloxy-3 - trimethylaminopropane chloride salt (DLin-TMA. CI) , 1,2- dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP . Cl ) , 1, 2-dilinoleylbxy-3- (N-methylpiperazino) propane (DLin-MPZ) , 3- (N,N-dilinoleylamino) -1 , 2 -propanediol (DLinAP) , or mixtures thereof. The neutral lipid components may be cholesterol or a derivative; phospholipids; or a mixture of phospholipids and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone , coprostanol, cholesteryl -2 ' - hydroxyethyl ether, cholesteryl-4¹ - hydroxybutyl ether, and mixtures thereof. Examples of neutral lipids include but are not limited to dipalmitoylphosphatidylchol ine (DPPC) ,

distearoylphosphatidylcholine (DSPC) ,

dioleoylphosphatidylethanolamine (DOPE) , palmitoyloleoyl - phosphatidylcholine (POPC) , palmitoyloleoyl - phosphatidylethanolamine (POPE) , palmitoyloleyol- phosphatidylglycerol (POPG) , dipalmitoyl- phosphatidylethanolamine (DPPE) , dimyristoyl- phosphatidylethanolamine (DMPE) , distearoyl- phosphat idylethanolamine (DSPE) , monomethyl- phosphatidylethanolamine , dimethyl -phosphatidylethanolamine , dielaidoyl- phosphatidylethanolamine (DEPE) , stearoyloleoyl - phosphatidylethanolamine (SOPE) , egg phosphatidylcholine (EPC) , and mixtures thereof.

Not limiting examples of biocompatible, soluble and hydrophilic polymers with a highly flexible main chain used to prepare long circulating liposomes and avoid particles

aggregation during preparation are poiy (acryl amide), poly (vinyl pyrrol idone ) , poly (acryloyl morpholine) , poly (2 -ethyl -2 - oxazoline) , poly (2 -methyl -oxazoline) , phosphatidyl

polyglycerols, polyvinyl alcohols, polysaccharides, PEG and PEG- containing copolymers (as poloxamers and poloxamines) , or mixtures therefore. Examples of PEG-lipid particles include but are not limited to PEG-diacylglycerol (DAG) , PEG

dialkyloxypropyl (DAA) , PEG-ceramide (Cer) , PEG- phosphatidylethanolami e , PEG- 1 -methyl -4 - ( cis - 9 -dioleyl ) methyl - pyridinium chloride (PEG-SAINT) or mixtures thereof. Examples of PEG-Cer conjugate include but are not limited to PEG- dilauryloxypropyl (C 12), a PEG- dimyristyloxypropyl (C 14), a PEG-dipalmityloxypropyl (C 16) , a PEG-distearyloxypropyl (C 18) , or mixtures thereof. The PEG moiety of the PEG-lipid conjugates described herein may compose an average molecular weight ranging from about 550 da1tons to about 10 000 daltons.

The present invention will be described in detail through specific examples. Examples are presented for

illustrative purposes, and do not limit the invention in any manner, since some noncritical parameters can be modified to yield essentially the same results.

Examples ^Materials and Methods

Example 1- Preparation of targeted nanocarrxers co-encapsulating siRNA and imati ib

Preparation of Trf -coupled or BSA-coupled micelles:

Coupling of Trf or BSA to PEG₂ooo~DSPE micelles was performed accordingly to Ishida et al (Ishida et al . 1999) . Briefly, Trf or BSA protein was modified with the addition of thiol groups through reaction with 2 -iminothiolane hydrochloride (2-IT) . For this purpose, Trf or BSA and 2-IT freshly dissolved in HEPES buffer (20 mM HEPES, 145 mM NaCl , pH 8) were mixed in a

protein/2-IT molar ratio of 1/10 and gently stirred for 1 h, in the dark at room temperature.

A lipid film of DSPE-PEG-MAL was prepared by solvent evaporation under a mild stream of N₂ and further dried under vacuum for 2 h. This dried lipid film was then hydrated with MES buffer (20 mM HEPES, 20 mM MES, pH 6.5), at a concentration above 2.3 μΜ, the critical micellar concentration of the lipid (Ishida et al . 1999) . Micelles were formed by strong vortex followed by 15 s heating in a water bath at 38 °C, followed by a second vortex shaking. Then, the freshly thiolated protein was coupled to the freshly prepared DSPE-PEG-MAL micelles by a thioesther linkage (protein to micelles molar ratio of 1:1) . The coupling reaction was performed overnight, ih the dark at room temperature with gentle stirring. The remaining free MAL groups in the micelles were quenched by the addition of β

mercaptoethanol at a maleimide : β mercaptoethanol molar ratio Of 1:5, under stirring for 30 min at room temperature. Encapsulation of siRNA into the nanocarriers : a solution containing 13 μπιοΐ of total lipid composed of Choi:

DSPC:DODAP: mPEG 2000 Ci₆Ceramide (45:22:25:8, mol%) in 200 μΐ of absolute ethanol , and a solution of 0.041 μτηοΐ of siRNA in 300 μΐ of 20 mM citrate buffer, pH 4, were heated at 60 °C. The lipids were then slowly added under strong vortex to the siRNA solution. In some experiments, empty liposomes were used. In this case, lipids were added to 300 μΐ of citrate buffer under similar conditions as those used for siRNA-encapsulating

liposomes preparation. Upon their formation the liposomes were extruded, 21 times, in a LipoFast mini extruder (Lipofast,

Avestin, Toronto, Canada) through 100 nm diameter polycarbonate filters (Avestin, Toronto, Canada) . Then, a dialysis was

performed in HBS, pH 7.4, through regenerated cellulose tubular membrane with MWCO 6000-8000 (Cellu Sep T2 , Membrane Filtration Products, Inc Seguin, TX, USA) during 3 h at room temperature to remove ethanol and raise the external pH. Subsequently, the total lipid concentration was assessed by cholesterol

quantification. For this purpose, samples were added to absolute ethanol (1/6, v/v) and Infinity™ Cholesterol Liquid Stable

Reagent (Thermo Electron; Melbourne, Australia) . Absorbance was measured at 500 nm in a spectrophotometer and the concentration assessed against a cholesterol standard curve. The cholesterol quantification allowed the determination of the total lipid that remained at this stage and consequently the determination of the amount of imatinib and micelles to be added. Encapsulation of imatinib in siRNA-containing liposomes and post-insertion of Trf at the liposome surface:

Immediately after the dialysis of the liposomes containing siRNA (performed as described previously) , imatinib was encapsulated into the liposomes, by addition of imatinib to the siRNA- containing liposomes at different imatinib/total lipid molar ratios (1/3, 1/8, 1/16, 1/32, 1/42; initial imatinib/total lipid) and incubation for 1 h, at 60 °C, in a water bath. The liposomes were then allowed to reach the room temperature and 4 mol % of Trf -micelles was added and incubated for 17 h, at 38 -C, in a water bath under dark.

Purification of liposomes: After incubation with micelles, Trf -liposomes were purified by size exclusion

chromatography on a Sepharose CL-4B column, using HBS , pH 7.4, as running buffer to remove external siRNA and imatinib as well as chemical reagents used during the liposomal preparation.

SiRNA quantification: The amount of siRNA entrapped inside liposomes was assessed by the Quant -iT RiboGreen RNA Assay (Molecular Probes, Invitrogen, Karlsruhe, Germany) against a siRNA standard curve. Liposomes were dissolved upon addition of 0.6 m of octaethylene glycol monododecyl ether (C₁₂E₈) and the RiboGreen fluorescence (Xex 485 nm, Xem 530 nm, cut off 515 nm) was measured using a Spectra Max Gemini EM plate reader fluorimeter (Molecular Devices, Sunnyvale, CA) .

Imatinib quantification: The method for imatinib quantification was developed by adaptation of the Dharmacon RNA Technologies (Lafayette, CO, USA) protocol for siRNA

precipitation. In microfuge tubes, 0.1 μτηοΐ of total lipid was added to the precipitation reagent (400 μΐ destilled water, 40 μΐ of 10 M ammonium acetate, pH 7 and 1.5 ml absolute ethanol) up to 800 μΐ , samples were then submitted to 30 s of strong vortex and transferred to -80 °C/2 h or -20 °C/overnight .

Subsequently, frozen samples were slightly thawed at room temperature and centrifuged at 18 000 g for 20 min at 4 °C.

Imatinib concentration was determined in the supernatant by measuring the absorbance at 259 nm against a standard curve of imat inib. This quantification method was optimized to eliminate any interference by the other components of the formulation.

Example 2- Cell viability of targeted nanocarriers co- encapsulating siRNA and imatinib Cell lines: Two human chronic myeloid leukemia cell lines in blast crisis, positive for BCR-ABL oncogene, with the b3a2 translocation (K562 and LAMA- 84 cells) purchased from DSMZ (Braunschweig, Germany) were maintained in culture at 37 °C, 5% C0₂ under humidified atmosphere in RPMI-1640 ιηεάίμΓη supplemented with 10% (v/v) heat -inactivated fetal bovine serum (FBS) (Gibco, invitrogeil, California, USA) , penicillin (100 U/ml) and

streptomycin (100 μg/ml) (Cambrex, NJ, USA) .

Development of imatinib-resistant cell line: K562 cells maintained in culture as previously described, were incubated with increasing concentrations of imatinib, starting at 0,05 μΜ and with 0.05 μΜ increments every 4 days of culture, until cells acquired the ability to grow at 1 μΜ. At this time point, drug resistance was assessed and cells were designated as IRK562. The new cell line was maintained continuously in culture in the presence of 1 μΜ of imatinib and was washed with drug- free medium before all experimental procedures.

Cell transfection: K562 and LAMA- 8 cells (20 000 cells/well) and IRK562 cells (25 000 cells/well) in RPMI-1640 culture medium supplemented with 10% FBS and antibiotics were seeded in 96-round well plates. Cells were transfected with Trf- associated liposomes co-encapsulating siRNA and imatinib at different molar ratios at 37 °C for 4 h. After incubation with liposomes, the medium was replaced with fresh medium and cells further incubated for 44 h. Cell viability: Cell viability was evaluated by the resazurin reduction assay (O'Brien et al . 2000). The assay measures the chemical reduction of the resazurin dye resulting from cellular metabolic activity, and allows the determination of viability over the culture period without harming the cells. Briefly, the culture medium was replaced with 10% (v/v)

resazurin dye in RPMI-1640 medium without serum and antibiotics, which was added to each well. After 2.5 h of incubation at 37 °C, the absorbance at 540 nm (reduced form) and 630 nm (oxidized form) was measured in a microplate reader Multiskan Ex (Thermo Labsystems, Vantaa, Finland) . Cell viability was calculated as percentage of control cells using the equation:

[ (A₅4o-A₆₃o) treated cells xlOO) / (A₅₄₀-A₆3o) control cells]

Assessment of imatinib IC₅₀ and DRI : The required drug concentration to promote reduction of 50% in cell viability (IC₅₀) and dose reduction index (DRI), which is the magnitude of dose reduction allowed for a drug when given in a drug

combination (IC₅₀ of the drug when administered alone/ IC_S0 of the drug when administered in the combination) were assessed. For IC₅₀ determination non-linear curve fit assuming sigmoidal dose- response was performed. Example 3- Cell association and cell internalization of Trf - or BSA- associated nanocarriers

Flow Cytometry studies: To evaluate the extent of cell association of Trf- or BSA-associa ed liposomes or NT-liposomes , cells were transfected with fluorescently labelled siRNA (Cy3- siRNA) encapsulated in each one of the mentioned liposomes.

Cells were seeded in RPMI-1640 culture medium (200 000

cells/well) in 48-well plates. To demonstrate that the receptor- mediated internalization is temperature -dependent , cells were incubated 1 h at 4 ° or at 37 °C, prior to liposome addition. To demonstrate that the internalization of Trf -liposomes is mediated by TrfR, this receptor was saturated by incubating the cells during 30 min with 125 μΜ of free holo-transferrin

(dissolved in HBS, pH 7.4), as performed by others (Cardoso et al. 2008; Chiu et al . 2006) , After these pre -treatments , cells were incubated with the formulations under study for h at 37 ^CC or 4 °C. Subsequently, cells were collected in conic tubes (BD, Biosciences) , washed twice with cold PBS and re-suspended in 500 L of cold PBS. All samples were immediately analyzed in a FAGS Calibur flow cytometer (BD, Biosciences) . Cy3

fluorescence was evaluated in the FL-2 channel. The data were then analyzed in the Cell Quest software (BD) . Cell association was only assessed in viable cells, these being gated based on the morphological features and on Trypan Blue staining (FL-3 channel) . The cell association efficiency of each formulation was assessed by comparing the relative fluorescence units (RFU) increase relative to non-treated cells (RFU of cells treated with the formulation under study/RFU of non-treated cells) . Confocal microscopy studies: In order to assess the extent of cellular internalization of the developed liposomes, confocal microscopy studies on transfected cells were performed. Cells were transfected with the formulations under study as described in the previous section. Then, cells were collected in conic tubes (BD, Biosciences) , washed with cold PBS, fixed with

4% paraformaldehyde for 20 min in the dark, at room temperature, washed with cold PBS, stained with the fluorescent DNA-binding dye Hoechst 33342 (Molecular Probes, Oregon, USA) (1 /xg/mL) for 5 min, in the dark, washed with cold PBS, and mounted in Mowiol mounting medium (Fluka, Sigma) . Confocal images were acquired in a point scanning confocal microscope Zeiss LSM 510 Meta (Zeiss, Germany) , with a 40x EC Plan-Neofluar or with a 63x Plan- Apochromat oil immersion objectives, and an argon (488nm) , a DPSS (561 nm) , a diode (405 nm) and a helium-neon (633 nm) lasers. Preparations were excited at 561 nm for the Cy3 (em: >560 nm) and at 405 nm for Hoechst 33342 (em: 420-480 nm) . Differential interference contrast (DIC) images were obtained using the helium-neon (633 nm) laser. Digital images were acquired using the LS 510 Meta software. All instrumental parameters pertaining to fluorescence detection and image analyses were held constant to allow sample comparison.

Results :

As described in Example 1, after the encapsulation of siRNA in SNALP liposomes, a transmembrane pH gradient between the aqueous content of the liposome (citrate buffer, pH 4) and the external liposome milieu (HBS, pH 7.4) was generated, which is the driving force for the active encapsulation of the imatinib. Trf-PEG-DSPE conjugates were then inserted onto the pre-formed liposomes by the post - insertion method. Using this methodology, the effect of imatinib on siRNA encapsulation yields and the effect of siRNA on imatinib encapsulation was assessed upon incubation with different imatinib : lipid ratios (1/3; 1/8; l/l6; 1/32; 1/42). As illustrated in Figure 1A, the encapsulation yields of imatinib increase with decreasing of imatinib : total lipid ratios, being 11.88 ± 2.09% for the 1/3 ratio and of 19.8 ± 2.32% for the 1/8 ratio. For ratios above 1/16, the encapsulation yields were very similar, being around 25%. As can be observed in Table I, for ratios exhibiting the same yield, the higher loading of imatinib is obtained with increasing imatinib/lipid ratios (i.e. 1/16 > 1/32 > 1/42). It is important to notice that the presence of siRNA significantly enhances the imatinib encapsulation yields. For example, at the ratio of 1/8 the encapsulation yield of imatinib increase from 5.96 ± 2.39%, in liposomes without siRNA, to 19.80 ± 2.32% when imatinib is co-encapsulated with siRNA.

As can be observed in Figure IB, the siRNA encapsulation yield is also significantly affected by the presence of imatinib, namely by the imatinib : total lipid ratio used in the co-encapsulation process. Thus, the formulations prepared with higher amounts of imatinib (higher imatinib : total lipid ratios, e.g. 1/3) resulted in lower siRNA encapsulation yields, as compared to the formulations prepared with lower amounts of imatinib (lower imatinib : total lipid ratios, e.g. 1/42} . When siRNA is encapsulated alone, under the same

conditions as those in this co-encapsulating process, the encapsulation yield is 92.17 + 1.39%, (Mendonca et al . 2010). Overall, these results demonstrate that when imatinib is co- encapsulated with siRNA, the siRNA encapsulation yield decreases in a manner dependent on the imatinib/total lipid ratio pre- incubated with the liposomes. However, for ratios lower than 1/16, no significant difference in the siRNA loading of the formulations prepared was observed (Table I) . Nevertheless, the imatinib loading is smaller for the lower ratios (Table I), allowing to reach higher siRNA/imatinib ratios (Figure 1C) .

Thus, the lowest imatinib/lipid ratio tested (1/42) resulted in the highest siRNA/imatinib ratio (0.63). At this ratio it is therefore possible to obtain therapeutic concentrations of both imatinib and siRNA inside the same liposome. In contrast, the 1/3 ratio at which the imatinib loading is too high as compared to that of siRNA, did not allow to reach therapeutic

concentrations for both agents, in fact, with this formulation the achievement of therapeutic concentrations for siRNA will lead to extremely cytotoxic imatinib concentration, which would block the possible therapeutic contribution from the siRNA molecules. Trf-liposomes co-encapsulating imatinib and siRNA molecules prepared from imatinib : total lipid ratios of 1/16, 1/32 and 1/42 (1/16; 1/32 and 1/42 formulations) , resulted in 0.15, 0.35 and 0.63 siRNA/imatinib molar ratio, respectively (formulations are codenamed by the resulting siRNA/imatinib molar ratios) . In what concerns to the role of imatinib in the siRNA entrapment yield, a remarkable decrease in the siRNA yield of encapsulation was observed for the highest ratios of

imatinib/total lipid, whereas no impact was observed for the lower imatinib/lipid ratios, as compared to the encapsulation of siRNA in the absence of imatinib. Thus, as observed by others, we have shown that the inclusion of a second drug may induce leakage of the first encapsulated drug (Saxon et al . 1999b;

Waterhouse et al . 2001), in a drug amount -dependent manner. In opposition, the presence of siRNA enhances the imatinib

encapsulation yields. This can be explained by interactions between the negatively charged siRNA and the positively charged imatinib (at the intra-liposomal milieu) , which may increase the amount of imatinib entrapped inside the liposomes. As described in Example 2, the formulations that allow to obtain therapeutic concentrations for both imatinib and siRNA within the same liposome formulation were tested against cell lines sensitive to imatinib ( 562 and LAMA-84) as well as against the imatinib-resistant IR 562 cell line. As can be observed in Table II, for all tested cell lines the formulation with higher amount of anti - BCR -ABL siRNA (0.63) led to higher imatinib IC_S0 reduction and, consequently, to higher dose reduction index (DRI) . IRK562 cells were more sensitive to the increment of the siRNA dose in the combination of siRNA and imatinib than the imatinib sensitive cell line LAMA-84, since for the 1/16 and 1/42 formulations the DRI was of 1.16 and 3.84, respectively, whereas for LAMA-84 the difference between the DRI obtained for the different formulations is not so evident. In fact, lowe siRNA/ imatinib ratios were required to reach the same DRI in LAMA-8 cells as compared to K562 and IRK562 cells, indicating that cell lines with higher BCR-ABL oncogene levels are more dependent on the gene silencing agent concentration (levels of BCR -ABL mRNA : IR 562 > K562 >LAMA-84, as assessed by qRT-PCR, data not shown) . For all tested cell lines, the 1/42 formulation (highest relative siRNA contribution in the drug combination) led to the highest imatinib IC_So reduction,

demonstrating the importance of achieving a level for both molecules within the range of therapeutic concentrations.

Results also revealed that the imatinib-resistant cell line IRK562 required higher siRNA/imatinib ratios, as compared to non-resistant cell line LAMA- 8 . A correlation between the cellular response and the expression of Trf receptor and BCR-ABL mRNA levels could be established. In fact, the cell line with higher Trf receptor expression and lower BCR-ABL mRNA levels ( LAMA-84) demonstrated higher response to the tested

formulations. Therefore, even considering that other cellular features may play relevant roles in the response to targeted therapy, our results strongly suggest that both the levels of Trf receptors and BCR-ABL mRNA can be used as biomarkers to predict the efficacy of the developed therapies. Cytotoxicity of encapsulated and free imatinib was also tested. LAMA- 84 cells were also treated with the combination of different

concentrations of free imatinib with 1 μΜ of siRNA encapsulated in Trf -liposomes (Table II) . Such treatments require higher siRNA/ imatinib ratios than that required by the strategy in which both molecules are co-encapsulated in the same liposome. In fact, for the combination of imatinib and encapsulated siRNA, the siRNA/ imatinib ratios used were 2.5 to 200, which led to a 2.85 DRI, whereas with the 1/42 formulation, in which

siRNA/ imatinib ratio is 0.63, a higher DRI (3.43) was obtained. Thus, upon co-encapsulation of siRNA and imatinib in the same liposomal particle, lower siRNA/ imatinib ratios can be employed to induce a certain degree of cytotoxicity as compared to the co- treatment of the cells with encapsulated siRNA and free imatinib. Both free imatinib and imatinib encapsulated in Trf- liposomes promoted similar cytotoxicity, suggesting that these Trf -coupled liposomes loading imatinib allow efficient

intracellular drug delivery (Table II) . As described in Example 3, the extent of association of liposomes bearing Trf or BSA attached at the end of PEG chains or of NT- liposomes , encapsulating Cy3 -labelled siRNA, to LAMA- 84 cells was assessed by flow cytometry. As can be observed in Figure 2A, the presence of Trf attached to the end of PEG- grafted lipids is the main responsible for the observed extent of iiposome-cell association. Cells incubated with BSA- liposomes or NT-liposomes revealed no significant cell association, illustrated by the low increase of the fluorescence intensity of the transfected cells as compared to non-treated cells. As can be observed, the RFU fold increase for cells treated with 2.0 μΜ siRNA encapsulated in Trf-liposomes (2.0 μΜ siRNA Trf) was 7.20 ± 0.40, in contrast to 3.20 ± 0.07 and 1.57 ± 0.22 fold increase observed upon treatment of the cells with BSA-liposomes and NT- liposomes, respectively. Our results also indicate that the extent of cell association of Trf-coupled liposomes containing

Cy3 - siRNA is dependent on the lipid concentration incubated with the cells up to 0.34 mM of total lipid which is correspondent to 1.0 μΜ siRNA, since no significant difference on the RFU of cells treated with 1.0 μΜ or 2.0 μΜ siRNA encapsulated in Trf- liposomes was observed.

To clearly demonstrate that the ligand Trf coupled at the surface of the liposomes trigger their cell internalization through theTrfR, competitive inhibition studies were performed. Cells were incubated with 125 μΜ of free human holo-Trf to block the TrfR. As illustrated in Figure 2B, a drastic decrease of cell association of Trf -liposomes for all tested concentrations was observed. At 1.0 μΜ siRNA and incubation at 37 °C, the observed RFU was 7.84 ± 0.54, whereas with pre -saturation of the Trf receptor, the RFU decreased to 2.85 + 0.54. The effect of the temperature on the extent of liposome-cell association was examined through the incubation of tumor cells with Trf- liposomes at 4 °C and 37 °C. As expected, incubation at 4 °C resulted in a significant reduction of the extent of liposome- cell association as compared to the incubation at 37 °C (at 1.0 μΜ siRNA, the RFU was reduced from 7.84 ± 0.54 to 3.72 ± 1.25). It was also observed that Trf receptor saturation performed at 4°C, resulted in the highest reduction observed for the extent of cellular association, with a RFU of 1.48 ± 0.15, indicating that binding of the liposomes to the cell surface is also affected by the saturation of TrfR. As illustrated in Figure 3, confocal microscopy studies were performed to assess the transferrin-receptor dependency of cell internalization of the Trf -associated nanocarriers . Upon incubation of the cells with liposomes, at 37 °C, cell internalization of Cy3-siR A was only detected for the cells incubated with Trf -liposomes (Figure 3A and F) . For BSA- (Figure 3B) or NT- liposomes (Figure 3G) no Cy3- siRNA inside the cells was observed. This same observation was registered upon saturation of the Trf receptor at 37 °C (Figure 3D) or upon treatment of the cells at 4 °C (Figure 3E) . Thus, the cell association and internalization is temperature- and TrfR- dependent.

Tables Table I. Imatinib and siRNA loading parameters of Trf-liposomes co -encapsulating imatinib and siRNA

imatiiiib: total lipid ratio imatinib (nmol/anol TL) siRNA. (nmol/μποΐ TU)

1/16 20.91 ± 7.0 3.19 ± 1.49

1/32 9.70 + 4.42 3.38 + 1.5

1/42 5.99 + 0.82 3.90 + 2.02

Table II. Effect of Trf-liposomes loading different

siRNA/imatinib molar ratios on imatinib IC₅₀ and DRI,

imatinib-resistant and non-resistant leukemia cells

siRNA/ijj-itinib free free imatinib 0.15 0.35 0.63 imtinib + imtinib encaps

Cell line

imatinib/lipid ~ ~~ ^{' "}siRNA

(1/16) (1/32) ^' (1/42) eneaps

IRKS62 ICso ίμΜ) 3 .03 ± 0.91 ±

1.49 ± 0 .19 3 .49 ± 0 .32 n.a .

0 .95 0.09

DRI 1.16 2.34 3^' 84

K562 IC¾₀ (nM) 85.90 ± 138 .0 + 122 .0 ±

60.40 + 4.60 n.a . n.a.

6.78 34 .0 14.0

DRI 1.61 2.28

LSMA-84 ICjo (nM) 50.40 + 35.60 ± 54 . 10 ±

36 . 80 ± 2 .50 84 .0 ± 8 .0 70.0 ± 4 .3.0

4 .2 16.20

DRI 2 .42 .32 3 .43 2.85 References :

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Claims

1. A multi-targeting system comprising a nanocarrier in which are included one or more nucleic acids and one or more non-nucleic acid based drugs.

2. The multi-targeting system according to claim wherein the inclusion are performed by simultaneous

encapsulation, adsorption or complexation of said one or more nucleic acids and one or more non-nucleic acid based drugs.

3. The multi-targeting system according to claim 1 or 2, wherein the multi-targeting system further comprises one or more ligands coupled to the nanocarrier surface.

4. The multi-targeting system according to any one of claims 1 to 3, wherein the nucleic acids are selected from plasmid, antisense oligonucleotides (asODN) , siRNA, miRNA, shRN ) aiRNA, DNA enzymes (DNAzymes) , Ribozymes, DNA decoys, A amers and mixtures thereof .

5. The multi -targeting system according to claim 4, wherein the nucleic acid comprises from about 15 to about 60 nucleotides.

6. The multi- targeting system according to claim 5 wherein the nucleic acid comprises at least one modified nucleotide ,

7. The multi -targeting system according to any one of claims 1 to 6 , wherein the non-nucleic acid based drugs are selected from chemotherapy drugs, hormonal therapeutic agents, immunotherapeutic agents, anti -viral drugs, anti-inflammatory compounds, antidepressants, stimulants, analgesics, antibiotic antipyretics, vasodilators, anti-angiogenics , cytovascular agents, signal transduction inhibitors, tyrosine kinase

inhibitors, ant i-arrhythmic agents, hormones, vasoconstrictors, and steroids .

8. The multi- targeting system according to claim 7, wherein the chemotherapy drugs include platinum-based drugs (e.g. cisplatin, carboplatin, etc.); alkylating agents (e.g., cyclophosphamide, chlorambucil, busulfan, melphalan, lomustine, carmustine, estramustine , treosulfan, thiotepa, mitobronitol , etc.); anti -metabolites (e.g., 5 - fluofouracil , methotrexate, capecitabine , cytarabine, fludarabine, gemcitabine, cladribine, raltitrexed, mercaptopurine , etc.); plant alkaloids (e.g., vincristine, vinblastine, vindesine, paclitaxel, docetaxel, etc), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, etc.); cytotoxic antibiotics (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone , aclarubicin, idarubicin, dactinomycin, etc.), taxanes (e.g., docetaxel, paclitaxel); tyrosine kinase inhibitors (e.g.; gefitinib, sunitinib, erlotinib, lapatinib, canertinib, semaxinib, vatalanib, sorafenib, imatinib,

dasatinib, leflunomide , vandetanib, salts thereof, stereoisomers thereof, analogs thereof, derivatives thereof, and mixtures thereof; the anti- inflamatory agents include ibuprofen,

aceclofenac, acemetacin, acetilsalicilic acid, azapropazone , celecoxib, diclofenac sodium, diflunisal, cetodolac, fenbufen, fenoprofen, flubiprofen, indomethacin, acetaminocin, piroxicam, rofecoxib, sulindac, tenoxicam; the antiangiogenic agents or angiolytic agents include angiostatin (plasminogen fragment) , antiangiogenic antithrombin III, vasculostatin, vasostatin and mixtures thereof .

9. The multi- targeting system according to any one of claims 3 to 8 comprising one or more ligands linked to the rianocarrier , wherein each one of the ligands specifically bind to an overexpressed or to a specifically expressed molecule or receptor or protein of the targeted cell population.

10. The mul i. - targeting system according to claim 9, wherein the ligand comprise peptides, polypeptides, antibodies, polyclonal antibodies, monoclonal antibodies, antibody

fragments, humanized antibodies, recombinant antibodies,

recombinant human antibodies, nanobodies, aptamers, proteins and cell surface ligands.

11. The multi -targeting system of claim 10, wherein the ligands are linked to the surface of the nanocarrier in a way that it is able to interact with a specific molecule, protein, glycoprotein and/or cell surface receptor that is overexpressed or specifically expressed on the cell surface of a specific cellular target, wherein an appropriate spacer can be positioned between the nanocarrier and the ligand to avoid hindrance on the interaction between the ligand and its target.

12. The multi- targeting system according to any one of claims 1 to 11, wherein the nanocarrier presents more than one homing ligand that selectively homes the delivery agent to specific molecules on the target cells in order to allow

multivalent cellular targeting.

13. The multi-targeting system according to any one of claims 2 to 12, wherein the molecular ratios of the co- encapsuiated/adsorbed/complexed molecules remains unaltered during storage and upon in vivo administration.

14. The multi-targeting system according to any one of claims 1 to 13, wherein the nanocarrier is composed by lipids and/or polymers .

15. The multi-targeting system according to claim 14, wherein the nanocarrier is composed by stabilized nucleic acid lipid particles (SNALP) .

16. The multi-targeting system according to claim 15, wherein SNALP liposomes comprise one or more cationic lipids, one or more neutral lipids and one or more conjugated lipid that inhibits aggregation of particles and provide long circulation times to the liposomes.

17. The multi -targeting system according to claim 16, wherein the cationic lipids may comprise from 10 to 60 mol%.

18. The multi-targeting system according to claim 16, wherein the neutral lipid, may comprise from 10 to 70 mol%.

19. The multi-targeting system according to claim 16, wherein the conjugated lipid that inhibits aggregation and provides long circulation times may comprise from 1 to 10 mol%.

20. The multi-targeting system according to claim 16 or 17, wherein the cationic lipids are 1 , 2 -dioleoyl -3 - dimethylammonium-propane (DODAP) , 1 , 2 -dilinoleyloxy-3 - (2 -N, - dimethylamino) ethoxypropane (DLin-EG-DMA) , N, N-dioleyl -N, N- dimethylammonium chloride (DODAC) , 1 , 2-dioleyloxy-N, N-dimethyl - 3 -arninopropane (DODMA) , 1 , 2 -distearyloxy-N, N-dimethyl -3 - aminopropane (DSDMA) , N- ( 1- (2 , 3 -dioleyloxy) propyl ) -N, , - rimethylammonium chloride (DOTMA) , N-(l-(2,3- dioleoyloxy) propyl ) -N, , -trimethylammonium chloride (DOTAP) , 3- (N- (N' , ¹ -dimethylaminoethane) -carbamoyl ) cholesterol (DC-Choi) , N- (1, 2-dimyristyloxyprop-3-yl) -N, -dimethyl -N- hydroxyethylammonium bromide (DMRIE) , 2 , 3 -dioleyloxy-N- [2 (spennine-carboxarnido) ethyl] N, N-dime hyl 1 - propanaminiumtrifluoroacetate (DOSPA) , dioctadecylamidoglycyl spermine (DOGS), N, N-dimethyl -3 , 4 -dioleyloxybenzylamme (DMOBA) , 1 , 2 -N, ' -dioleylcarbamyl -3 -dimethylammopropane (DOcarbDAP) , 1,2- Ν,Ν' -dilmoleylcarbamyl-S-dimethylaminopropahe (DLi c bDAP) , 1, 2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) , 1,2- dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA) , 2,2- dilinoleyl -4 - (2-dimethylaminoethyl) - [1,3] -dioxolane (DLin-K-C2- DMA), 2 , 2 -dilinoleyl -4 - (3-dimethylarninopropyl) - [1, 3] - dioxolane

(DLin-K-C3 -DMA) , 2 , 2 -dilinoleyl -4 - (4 -dimethylaminobutyl ) -[1,3]- dioxolane (DLin-K-C4 -DMA) , 2 , 2 -dilinoleyl -4 -dimethylaminomethyl -

[1 , 3 ] -dioxolane (DLin-K-DMA) , 1 , 2 -dilinoleylcarbamoyioxy-3 - dimethylaminopropane (DLin-C-DAP) , 1,2· dilinoleyoxy- (dimethylamino) acetoxypropane (DLin-DAC) , 1 , 2 -dilinoleyoxy-3 - morpholinopropane (DLin-MA) , 1 , 2 -dilinoleoyl -3 - dimethylaminopropane (DLinDAP) , 1 , 2 -dilinoleylthio- 3 - dimethylaminopropane (DLin-S-DMA) , 1 - linoleoyl -2 - linoleyloxy-3 - dimethylaminopropane (DLin-2 -DMAP) , 1 , 2 -dil inoleyloxy-3 - trimethylaminopropane chloride salt (DLin-TMA. CI ) , 1,2^L

dilinoleoyl -3 -trimethylaminopropane chloride salt (DLin-TAP . Cl ) , 1 , 2 -dilinoleyloxy-3 - (N-methylpiperazino) propane (DLin-MPZ) , 3-

(N, N-dilinoleylamino) -1, 2 -propanediol (DLinAP) , or mixtures thereof .

21. The multi-targeting system according to claim 16 or 18, wherein the neutral lipid components may be cholesterol or a derivative thereof; phospholipids; or a mixture of

phospholipids and cholesterol or a derivative thereof. Examples of cholesterol derivatives include cholestanol, cholestanone , cholestenone , coprostanol, cholesteryl -2 ' -hydroxyethyl ether, cholesteryl -4 ' - hydroxybutyl ether, and mixtures thereof.

Examples of neutral lipids include but are not limited to dipalmitoylphosphatidylcholine (DPPG) ,

distearoylphosphatidylcholine (DSPC) ,

dioleoylphosphatidylethanolamine (DOPE) , palmitoyloleoyl - phosphatidylcholine (POPC) , palmitoyloleoyl - phosphatidylethanolamine (POPE) , palmitoyloleyol - phosphatidylglycerol (POPG) , dipalmitoyl- phosphatidylethanolamine (DPPE) , dimyristoyl- phosphatidyiethanolamine (D PE) , distearoyl- phosphatidylethanolamine (DSPE) , monomethyl- phosphatidylethanolamine, dimethyl -phosphatidyiethanolamine , dielaidoyl- phosphatidyiethanolamine (DEPE) , stearoyloleoyl - phosphatidyiethanolamine (SOPE) , egg phosphatidylcholine (EPC) , and mixtures thereof .

22. The multi-targeting system according to claim 16 or 19, wherein conjugated lipids are prepared using

biocompatible, soluble and hydrophilic polymers with a highly flexible main chain selected from poly(acryl amide), poly(vinyl pyrrolidone) , poly (acryloyl morpholine) , poly (2 -ethyl -2 - oxazoline), poly (2 -methyl -oxazoline) , phosphatidyl

polyglycerols , polyvinyl alcohols, polysaccharides, PEG and PEG containing copolymers (as poloxamers and poloxamines) , or mixtures therefore.

23. The multi -targeting system according to claim 22 wherein PEG polymer is used in the formulation as PEG-lipids such as PEG-diacylglycerol (DAG) , PEG dialkyloxypropyl (DAA) , PEG-ceramide (Cer) , PEG-phosphatidylethanolamine , PEG- 1 -methyl - 4-(cis-9- dioleyl) methyl -pyridinium chloride ( PEG-SAINT) or mixtures thereof. Examples of PEG-Cer conjugate include but are not limited to PEG-dilauryloxypropyl (C 12) , a PEG- dimyristyloxypropyl (C 14), a PEG-dipalmityloxypropyl (C 16), a PEG-distearyloxypropyl (C 18), or mixtures thereof.

23. The multi- targeting system of claim 22, wherein the PEG moiety of the PEG-lipid conjugates described herein may compose an average molecular weight ranging from about 550 daltons to about 10 000 daltons.

24. The multi-targeting system according to claim 23 wherein the PEG moiety of the PEG-lipid conjugates described herein may compose an average molecular weight ranging from about 550 daltons to about 10 000 daltons.

25. A method of selectively targeting therapeutic or diagnostic molecules to specific cells, combining cellular and molecular targeting, using the the mult i- arge ing system of any one of claims 1 to 2.4.

26. A pharmaceutically acceptable Composition

comprising the multi- targeting system of any one of claims 1 tc 25 and a pharmaceuticall acceptable carrier for in vivo

administration.

27. The multi-targeting system of any one of claims 1 to 2 or a pharmaceutica11y acceptable composition of claim 26, for the treatment, diagnosis and prevention of human cancer and other : diseases , including inflammation, neurodegenerative diseases, infect ious diseases , and cardiovascular disorders .