WO2011119058A2 - F3-peptide targeted lipid-based nanoparticles useful for the treatment of angiogenesis-dependent diseases - Google Patents

Abstract

The present invention relates to F3 -peptide targeted lipid-based nanoparticles, adequate to encapsulate and delivery- one or more nucleic acids, comprising; a) one or more nucleic acids; b) one or more cationic lipids; c) one or more non- cationic lipids; d) one or more poly (ethylene glycol) - derivatized lipids; and e) one or more coupled targeting ligands which bind to the nucleolin receptor. The nanoparticles according to the invention have a high loading capacity, ability to protect the encapsulated nucleic acid, a size below 250 ran and a charge close to neutrality, which are adequate features for intravenous administration. The nanoparticles of the present inventon have the ability to selectively delivery a siRNA to cancer cells and/or endothelial cells from angiogenic blood vessels leading to an effective silencing of a target gene. These nanoparticles are used for the treatment of angiogenesis- dependent diseases, namely cancer, inflammation, an auto-immune disease or an ocular disorder.

Description

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DESCRIPTION

"F3-PEP IDE TARGETED LIPID-BASED NANOPARTICLES USEFUL FOR THE

TREATMENT OF ANGIOGENESIS-DEPENDENT DISEASES"

Field of the invention The present invention provides F3 peptide-mediated targeted lipid-based nanoparticles for the intravenous administration of nucleic acids. The developed targeted nanoparticles have the major advantage to be specifically internalized by cancer and/or endothelial cells from angiogenic blood vessels, leading to an effective and specific gene silencing .

The targeted nanoparticles of the present invention have an enormous potential to be applied in the treatment of different types of solid tumors, special in those with a strong angiogenic process, as well as in other angiogenic-dependent diseases .

Background information

Cancer is still a severe public health problem being one of the most deadliest disease in the western world [1]. The knowledge generated in the last decades in the oncobiology field has revealed cancer as a disease that involves several genetic alterations, which result in the deregulation of numerous signalling pathways. This deregulation enable tumor cells to acquire capabilities, like: self-sufficiency in growth signal, insensitivity to antigrowth signals, evading apoptosis, limitless replicative potential, sustained angiogenesis and tissue invasion and metastases [2]. Moreover, the classic reductionist view focused on the cancer cells has been gradually substituted by the idea that the interaction ("cross-talk") between different several types of cells existing in a tumor

(like the cancer cells themselves, cancer stem cells, endothelial cells, fibroblasts or immune cells) co-orchestrate for the tumor aggressiveness and progression. In this respect, endothelial cells assume a key role in the progression of solid tumors and metastasis formation [3] . In the adult mammals, the endothelial cells are in a quiescent state, which, upon facing a specific stimulus, can evolve to a state where they proliferate and migrate, leading to the formation of new tumor blood vessels

[4] . Consequently, angiogenesis inhibition constitutes one important strategy in the treatment of solid and metastatic tumors .

The limited effectiveness of conventional treatment strategies has urged the need for the development of new therapeutic approaches that preferentially target specifically, multiple altered signalling pathways in tumor and non-tumor cells existing in a tumor.

RNA interference (RNAi) is a natural process for silencing gene expression. In mammalian cells, RNAi could be achieved by short double stranded RNA of 21 to 23 nucleotides long, known as small-interfering RNA (siRNA) , which has the potential to inhibit the expression of any target gene through specific cleavage of perfectly complementary mRNA [5-6] . Therefore, siRNAs have an enormous potential to become a novel class of pharmaceutical drugs within different fields of medicine, since they can inhibit the expression of any pathological protein. Oncology is one of the medical areas that can benefit the most with this new therapeutic strategy, as it allows to modulate the expression of any oncogene such as, but not limited to, growth factors and their receptors, cell cycle regulators, anti-apoptotic proteins, and angiogenic factors [7- 9] . However, the translation of RNA interference technology from the bench to the clinic has been impaired by their limited cellular uptake, low biological stability and unfavorable pharmacokinetics. In general, siRNAs are easily degraded by blood nucleases and because of their negative charge and high molecular weight, their cellular internalization is impaired, and the extent of binding to serum proteins is increased. Overall, this leads to a rapid blood clearance [10- 12] . Such limitations emphasize the need for an efficient and safe system to mediate in vivo delivery of siRNA.

Different delivery systems such as, but not limited to poly (ethylene glycol) (PEG) -grafted cationic liposomes encapsulating nucleic acids such as "coated cationic liposomes" (CCD [13] , stabilized antisense lipid particles (SALP) [14] , or the related stabilized nucleic acid lipid particles encapsulating siRNA (SNALP) [15-18] have been developed in order to fulfill some of the previous-mentioned key requirements. The pegylated-derivatized lipid in the liposomal formulation strongly contributes for the formation of a hydrophilic cloud around the liposomes. Upon intravenous injection, such hydrophilic shell dramatically decreases the rate and the extent of electrostatic and hydrophobic interactions between the surface of liposomes and blood components that mediate liposomal blood clearance and/or disintegration [10, 19]. In addition, these systems are also characterized by high encapsulation efficiency of nucleic acids and their protection from serum nucleases as well, a small average size, and a net charge close to neutrality [13-18] .

The systemic use of the pegylated systems mentioned above has been explored with success to target liver-associated diseases, such as hypercholesterolemia [15] , ebola [16] , hepatitis B [17] and hepatic tumors [18] . This success is in part explained by the fact that the liver is a well -perfused organ with fenestrated endothelium, being the primary site of accumulation of foreign bodies. Although pegylated nanoparticles, with long blood circulation times, can exhibit some accumulation in solid tumors due to the large fenestrated endothelium (enhanced permeability and retention effect [20] ) , efficient systemic targeting of siRNA to solid tumors, still represents an enormous challenge that has not been successfully addressed by the previous- mentioned nanoparticles.

In this context, one of the most promising strategies related with molecularly guided pharmacology involves the covalent attachment of a targeting ligand, at the extremity of PEG chains grafted onto a delivery system, such as, but ^'not limited to liposomes, which will specifically interact with receptors overexpressed on the surface of tumor cells, leading to intracellular accumulation of the nucleic acid-containing particle [21] . The design of novel targeted anticancer strategies must take into account that the aggressiveness of a tumor does not rely only on the tumor cell, but rather on the cross-talk between the cancer cell and other cells from the tumor microenvironment like, for example, fibroblasts, macrophages and endothelial cells from angiogenic tumor blood vessels [22] . In this respect, the design of ligand-mediated targeted delivery systems containing nucleic acids like, but not limited to siRNA, which target angiogenesis , in addition to cancer cells, could be tremendously advantageous for the treatment of solid tumors as it compromises the access to oxygen and nutrients impairing tumor survival and proliferation. Moreover, vascular targeting has some additional advantages since endothelial cells are more accessible (than cancer cells) to the nanoparticle injected in the vascular compartment, and are less prone to acquire drug resistance. In addition, treatment selectivity can be achieved, as the formation of new blood vessels is restricted to some angiogenic-dependent diseases (inflammatory, auto-immune and ocular disorders) and to a few physiological processes such as wound healing, ovulation and pregnancy. Furthermore, formation of metastasis in distant organs is also angiogenesis-dependent and thus, metastization can be inhibited by anti -angiogenic therapies ([23]).

Therefore, nanoparticles targeted with ligands like the F3 peptide [24] , which is specifically internalized by the nucleolin receptor [25] , overexpressed in cancer and/or endothelial cells from tumor blood vessels, can have an enormous positive impact in the treatment of tumors and other angiogenesis-dependent diseases, as the therapeutic nucleic acid, encapsulated in the ligand-mediated targeted nanopar icle, will be delivered into both those cells. However, an improved cellular internalization (as compared to the non-targeted counterpart) is not necessarily synonymous of an efficient gene silencing. The work of Santos et al . [26] is one of such example. Antagonist G-targeted stabilized nucleic acid lipid particles (SNALP) , formed with 10 mol% of poly (ethylene glycol) and containing anti-BCL2 siRNA, presented a cellular uptake into small cell lung cancer cells that was 20-fold higher than the non-targeted counterpart, but failed to downregulate the target protein [26] .

With the proposed strategy, receptor-mediated endocytosis is the most common mechanism of cellular internalization of ligand-targeted liposomes [27] . Therefore, escape of the internalized material from the endocytotic pathway plays an important role to the global efficacy of the designed therapeutic strategy. It is well known that this process requires lipid mixing between the liposomal lipids and the endosomal membrane. It has been shown that this phenomenon is impaired by pegylated lipids since they decrease the contact between the liposomes and the endosomal membrane, inhibiting lipid exchange and the consequent membrane disruption [28] . Under these circumstances, the exposure of the encapsulated nucleic acid to the lysosomal environment might lead to its degradation. Therefore, any strategy that facilitates the escape of the nucleic acid from the endocytotic pathway might be of great benefit.

It has been suggested that endosomes are transported along microtubules to lysosomes . It is thus expected that upon interference with the microtubule dynamics it will be possible to inhibit this endocytotic trafficking and therefore avoid the siRNA degradation at the lysosomal stage. For this purpose, different strategies like, but not limited to microtubule- targeted drugs can be explored, as example but not limited to vinblastine, vincristine, vinorelbine, vinflunine, colchicines, paclitaxel, docetaxel, nocodazole, cytochalasin B and podophyllotoxin [29-31] . Moreover, liposomes containing a pH-sensitive disrupting agent, such as but not limited to peptides [32-33] and lipids [34] , are good candidates for nucleic acid delivery. Typically, pH-sensitive liposomes are composed of lipid mixtures containing 1 , 2 -dioleoyl - sn-glycero- 3 -phosphoethanolamine (DOPE) , a lipid that forms an inverted hexagonal (H_n) phase (cone-shape phase), and amphiphilic molecules, like cholesteryl hemisuccinate (CHEMS) . At neutral or basic pH, the negatively charged CHEMS attracts cations and the associated water molecules, increasing the volume of the headgroup, which thus has the ability to stabilize the cone-shape DOPE into a bilayer phase. Acidification, such as the one taking place at the endocytotic pathway, triggers protonation of the carboxylic groups of the amphiphiles, reducing their stabilizing effect and leading to liposomal destabilization and access of the nucleic acid to the cell cytosol [34-36] . The attachment of a targeting ligand onto pH-sensitive liposomes in order to target specific cells can further result in improved therapeutic efficacy. Although, in order to avoid unspecific cellular association, due to their capability to fuse with the cellular membrane, without compromising their capability to escape from endosomes, DOPE and CHEMS ratios have to be carefully selected.

Overall, the present invention provides F3 peptide- mediated targeted pegylated lipid-based nanoparticles adequate for the encapsulation, protection and systemic delivery of nucleic acids, such as but not limited to RNA interference molecules, such as siR As. The F3 peptide targets the nucleolin receptor, which is overexpressed on cancer and/or endothelial cells from angiogenic blood vessels [24] . The developed F3 peptide- targeted nanoparticles have therefore the major advantage to be specifically internalized by both those two different types of target cells, surprisingly leading to an effective gene silencing. This is something that Santos et al were not able to achieve with antagonist G-targeted nanoparticles, targeting small cell lung cancer cells [27] . Therefore, the nanoparticles of the present invention have a great potential to become a novel therapeutic approach with a major positive impact in the treatment of cancer, such as but not limited to prostate cancer and breast cancer, as well as other diseases involving angiogenic-dependent processes (such as, but not limited to inflammatory and auto- immune diseases and ocular disorders) .

Brief description of the drawings

Figure 1. Cellular association of non-pH sensitive liposomes with human cancer cell lines (MDA-MB-435S and MDA-MB- 231) , human microsvascular endothelial cells (HMEC-1) and human fibroblasts (BJ) , analyzed by flow cytometry. Different concentrations (0.2 - 0.6 mM of total lipid) of rhodamine- labelled F3 -targeted, targeted by a non-specific peptide (NS) and non-targeted (NT) liposomes, were incubated with 0.5x10^s cells, at A) 37^°C or B) 37^°C or 4^°C, during 1 h. After incubation, rhodamine signal was assessed by flow cytometry. Bars are the mean ± SD of 3 independent experiments. Two-way ANOVA analysis of variance with Tukey post test was used for multiple comparisons. ***p<0.001; **p<0.01; ns p>0.05.

Figure 2. Cellular association of pH sensitive liposomes, analyzed by flow cytometry in A) MDA-MB-435S and B) DA-MB-231. Rhodamine-labelled F3-targeted, targeted by a nonspecific peptide and non- targeted liposomes at 0.2, 0.4 or 0.6 mM total lipid/well were incubated with cells, at 37^°C for 1 h. After incubation, rhodamine signal was assessed by flow cytometry. Bars are the mean ± SD of 3 independent experiments. Two-way ANOVA analysis of variance with Tukey post test was used for multiple comparisons. ***p<0.001 ; **p<0.01; ns p>0.05.

Figure 3. Cellular internalization of non-pH sensitive liposomes by MDA-MB-435, MDA-MB-231, HMEC-1 and BJ cells, analyzed by confocal microscopy. Rhodamine-labelled (red) F3- targeted and non-targeted liposomes, encapsulating FITC-labelled siRNA (green) , at 0.2 mM of total lipid/well, were incubated with 2.5xl0⁴ cells, at 37^°C for 1 h. The nucleus was stained with DAPI (blue) . Cells were fixed in 4% paraformaldehyde, mounted in moviol mounting medium and visualized in a Zeiss LSM-510 point scanning confocal microscope.

Figure 4. Evaluation of eGFP silencing, in MDA-MB- 435S-eGFP and MDA-MB- 231 - eGFP , after treatment with non-pH sensitive liposomes. MDA-MB-435S-eGFP (A) and MDA-MB-231 -eGFP (B) cell lines were transfected with different concentrations of anti-eGFP siRNA encapsulated in F3-targeted or non-targeted liposomes, or of a non-specific siRNA encapsulated in the former, at 0 h and at 48 h. Alternatively, just one treatment at 0 h was performed. Ninety six hours after the beginning of the experiment, eGFP levels were evaluated by flow cytometry. Bars are the mean ± SEM of 3 independent experiments. Two-way ANOVA analysis of variance with Bonferroni post test was used for multiple comparisons. ***p<0.001; **p<0.01; *p<0.05.

Figure 5. Evaluation of eGFP silencing, in MDA-MB- 435S-eGFP, after treatment with pH sensitive liposomes. MDA-MB- 435S-eGFP cells were transfected with different concentrations of anti-eGFP siRNA encapsulated in F3-targeted or in non- targeted liposomes, at 0 h and at 48 h. Ninety six hours later, eGFP levels were evaluated by flow cytometry. Bars are the mean + SEM of 3 independent experiments. Two-way ANOVA analysis of variance with Bonferroni post-test was used for multiple comparisons. ***p<0.001; **p<0.01; *p<0.05.

Figure 6. EGFP mRNA levels in MDA-MB-435S-eGFP after treatment with non-pH sensitive liposomes. Cells were transfected, at 0 h and at 48 h, with anti-eGFP siRNA encapsulated in F3-targeted and non-targeted liposomes, and with the control siRNA encapsulated in the former. EGFP mRNA levels were assessed 24 h after the second transfection by qRT-PCR and in comparison with the mRNA level of untreated cells. Bars are the mean ± SD of 3 independent experiments. Two-way ANOVA analysis was used for comparisons with untreated cells. **p<0.01, *p<0.05.

Figure 7. Impact on the viability of human prostate cancer cells (PC3 cells) , after treatment with pH sensitive liposomes. PC3 cells were transfected with different concentrations of anti-PLK-1 siRNA encapsulated in F3-targeted or non-targeted liposomes, or of a non-specific siRNA encapsulated in F3 -targeted liposomes, at 0 h and at 48 h. Ninety six hours after the beginning of the experiment, cell viability was accessed by the Resazurin reduction assay. Bars are the mean ± SD of 3 independent experiments. Two-way A OVA analysis of variance with Bonferroni post-test was used for multiple comparisons. ***p<0.001; **p<0.01; *p<0.05. Summary of the invention

The present invention provides a F3 peptide targeted lipid-based nanoparticles, characterized by a high encapsulation efficiency of one or more nucleic acids, with a size below 250 nm, a charge close to neutrality and capable of mediating specific internalization and intracellular delivery of nucleic acids into two cellular populations within a tumor (the cancer cells and the endothelial cells from angiogenic blood vessels) . The present invention provides F3 -peptide targeted lipid-based nanoparticles comprising one or more cationic lipids, one or more non-cationic lipids, one or more poly (ethylene glycol) - derivatized lipids, encapsulating one or more nucleic acids such as, but not limited to antisense oligonucleotide (asODN) , ribozyme, locked nucleic acid (LNA) , plasmids, immunostimulatory oligonucleotides, asymmetric interfering RNA (aiRNA) , microRNA (miRNA) , small interfering RNA (siRNA) , and covalently bound to one or more targeting ligands that provide selective targeting to tumor cells and endothelial cells from angiogenic tumor blood vessels. Besides cancer, other clinical applications for the novel delivery platform include other angiogenesis-dependent diseases such as, but not limited to inflammation (as example, but not limited to rheumatoid arthritis) auto-immune diseases (such as, but not limited to psoriasis) or ocular disorders (as example, but not limited to age-related macular degeneration, neovascular glaucoma and diabetic retinopathy) [37] .

The present invention provides a F3 -peptide targeted lipid-based nanoparticles, capable to be selectively recognized by the nucleolin receptor overexpressed on cancer and/or endothelial cells from angiogenic blood vessels and to delivery into these cells a single or a combination of the previous- mentioned (but not limited to) nucleic acids. The examples provided herein surprisingly demonstrated that the F3 peptide- targeted lipid-based nanoparticles presented in this invention are specifically internalized by cancer cells and/or endothelial cells from angiogenic tumor blood vessels and to an extent significantly higher than the non-targeted counterpart. In addition, the examples provide herein illustrate that F3 - targeted nanoparticles are capable to downregulate a target protein/mRNA in the target cells being the highest impact observed with the nanoparticles incorporating DOPE and CHEMS . Interestingly, treatment of a human prostate cancer cell line with such targeted nanoparticles resulted in a decrease on cell viability. Therefore, it is expected that the improved cell targeting herein presented by the developed F3 -targeted nanoparticles containing a nucleic acid, will lead to higher accumulation into the target organ, such as, but not limited to a tumor and further improved therapeutic activity of the former, as compared to the non-targeted counterpart or to the free nucleic acid.

Detailed description of the invention

The present invention provides a F3 peptide- targeted lipid-based nanoparticles, adequate to encapsulate and delivery nucleic acids (such as, but not limited to asODN, ribozymes, LNAs, plasmids, immunostimulatory oligonucleotides, aiRNAs, miRNAs, siRNAs) by intravenous administration. The nanoparticles are linked to a ligand that allows selective delivery of the encapsulated nucleic acids to cancer and/or endothelial cells from angiogenic blood vessels, in order to treat tumors as well as other angiogenesis-dependent diseases, such as, but not limited to, inflammation, auto-immune diseases or ocular disorders . The term "lipid-based nanoparticle" refers to particles composed of organic compounds insoluble in water, lipids, such as but not limited to fatty acids, phospholipids, sphingolipids , glycolipids and sterols, organized in an aqueous environment and that can be used to deliver one or more nucleic acids such as but not limited to siRNAs.

As used herein, the term "liposome" refers to a lipid formulation composed by a cationic lipid, one or more non- cationic lipids, a conjugated lipid that sterically stabilized the formulation, and an encapsulated nucleic acid. The term "pH- sensitive liposomes" refers to particles that destabilizes at mild pH (as take place in endosomes) leading to a more rapid and efficient content release.

The term "nucleic acid" means any molecule composed of deoxyribonucleot ides or ribonucleotides organized in single or double -stranded, such as asODN, ribozymes, LNAs, plasmids, immunostimulatory oligonucleotides, aiRNAs, miRNAs, siRNA.

The term "siRNA" refers to small double stranded RNA, of 19 to 23 nucleotides long, which can mediate degradation or translational inhibition of a target mRNA depending if the complementary is total or partial. Therefore, siRNAs are molecules capable of reduce or inhibit the expression of a target gene. SiRNAs can be chemically synthesized or generated from long double -stranded RNA after cleavage by Dicer enzyme [5- 6] . The term "modified siRNA" refers to siRNA with at least one chemical modification in one or more nucleotides of the sense and/or antisense strand. Several chemical modifications can be applied to nucleotides, at the ribose, phosphodiester bound and base level, as example but not limited to, 2 ' -O-Methylation, 2'- O-Methoxyethyl , phosphorothioate , among others. As used herein, the term "ligand" refers to any molecule linked to the developed lipid-based nanopart icles with the purpose to selectively interact with one or more target organs or one or more target tissues or one or more target cell populations in angiogenic -dependent diseases. The ligand can be a protein or a fragment thereof, a peptide, a peptide- like molecule, an antibody or a fragment thereof, which is specifically recognized by a receptor or other targeting molecule uniquely or just overexpressed in angiogenic blood vessels and/or tumor cells.

In the present invention, the term "peptide" refers to any small sequence of naturally and non-naturally occurring amino acids, with the ability to specifically interact with cancer cells and/or endothelial cells from angiogenic blood vessels such as KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO:l) , F3 peptide.

As used herein "targeted nanoparticles" or "targeted liposomes" refer to the conjugation of liposomes encapsulating a siRNA with a ligand that is specifically recognized and internalized by cancer cells and/or endothelial cells from angiogenic blood vessels.

The terms "specifically recognized" and "specifically internalized" mean that the targeted nanopart icles interact with the target cells on a ligand- specific manner.

The term "tumor" refers to a complex tissue composed of uncontrolled growth cells, the tumors cells themselves, and other type of cells that co-orchestrate for the tumor aggressiveness and progression, such as the stromal cells and endothelial cells from the angiogenic blood vessels. As used herein, "normal cells" refers to cells with normal proliferation rate and no evidence of disease.

As used herein "%" refers to molar percentage in relation to the total lipid. The lipid-based targeted nanoparticles contain a single or a combination of unmodified or modified nucleic acids and comprise one or more cationic lipids, one or more non- cationic lipids, one or more lipids conjugated to hydrophilic polymers. Example, but not limited to, of the hydrophilic polymer could be poly (ethylene glycol) (PEG). Its presence avoids particles aggregation during preparation and confers long circulation time in blood, upon intravenous administration as it shields the nanoparticle from opsonization and uptake by the cells from the reticuloendothelial system.

Thus, it is a first object of the present invention F3 -peptide targeted lipid-based nanoparticles , adequate to encapsulate and delivery one or more nucleic acids, comprising: a) one or more nucleic acids; b) one or more cationic lipids; c) one or more non-cationic lipids; d) one or more poly (ethylene glycol) -derivatized lipids; and e) one or more coupled targeting ligands which bind to the nucleolin receptor.

Preferred nanoparticles of this invention have adequate features for, but not limited to intravenous administration, with a size below 250 nm, a charge close to neutrality, high loading capacity and ability to protect the encapsulated nucleic acid.

Preferably nanoparticles of the present invention encompass : one or more unmodified or modified nucleic acids, as example but not limited to, a RNA interference molecule such as siRNA, aiRNAs , miRNAs or mixtures thereof; one or more cationic lipid that can be any cationic lipid, as example, but not limited to, 1 , 2 -dioleoyl -3 - dimethylammonium-propane (DODAP) , 1 , 2 -dioleoyl -3 - dimethylammonium- chloride (DODAC) , 1 , 2-dioleoyl-3- trimethylammonium-propane (DOTAP) , 1 , 2 -di -O-octadecenyl - 3 - trimethylammonium propane (DOTMA) , 1 , 2 -dioleyloxy-N, N- dimethyl-3 -aminopropane (DODMA) , 1 , 2 -dilinoleyloxy-3 - (N, N- dimethyl) aminopropane (DinLMA) and 1 , 2 -dilinolenyloxy-N, N- dimethyl- 3 -aminopropane (DLenDMA) ; one or more non-cationic lipids that could be any anionic lipid or neutral lipids, as example, but not limited to, cholesterol (CHOL) and its derivates such as cholesteryl hemisuccinate (CHEMS) , a phospholipid such as, but not limited to distearoylphosphatidylcholine (DSPC) , hydrogenated soy phosphatidylcholine (HSPC) , 1 , 2 -dioleoyl - sn-glycero- 3 -phosphoethanolamine (DOPE) and 1 , 2 -distearoyl - s-n-glycero-3-phosphoethanolamine (DSPE) ; one or more pegylated lipids such as, but not limited to, PEG-ceramides (as example but not limited to C14-Ceramide mPEG₂ooo, C16-Ceramide mPEG₂ooo and C18-Ceramide mPEG₂₀oo) ; PEG- phospholipids (as example but not limited to 1 , 2 -distearoyl - sn-glycero-3 -phosphoethanolamine-N- [succinyl (polyethylene glycol) -2000] (mPEG₂₀ooDSPE) ) and PEG-diacyl glycerols (as example but not limited to PEG-succinoyl distearylglycerol (PEG-S-DSG) ) ; . a targeting ligand covalently coupled onto the nanoparticle surface, which binds the nucleolin receptor overexpressed at the tumor and/or endothelial cells from angiogenic tumor blood vessels and also with angiogenic vessels of other diseases like, but not limited to inflammatory and autoimmune diseases and ocular disorders, enabling the nanoparticle to be internalized into the target cell (s) .

The lipid-based nanoparticles of the present invention encompass a non-pH-sensitive nanoparticle, generally referred to herein as the "formulation Al", and a pH-sensitive nanoparticle referred to herein as the "formulation A2 " .

In a preferred embodiment, the non-pH-sensitive liposomes "formulation Al", typically comprise: a) one or more unmodified or modified RNA interference molecule such as, but not limited to, siRNA; b) from 10% to 70% of a ionizable lipid such as, DODAP or its derivates or a mixture thereof;

O

c.l) from 20% to 60% of CHOL or its derivates or a mixture thereof ;

c.2) from 5% to 50% of a phospholipid such as, DSPC or its derivates or a mixture thereof; d) . from 0.5% to 10% of a pegylated neutral lipid such as, ceramides (acyl chain length from C8 to C20) ; e) the F3 peptide (from 2 to 10 nmol of F3/^mol of total lipid) which is specifically recognized by nucleolin receptors overexpressed on cancer and/or endothelial cells from angiogenic blood vessels. A preferred "formulation Al " (described in Example I), comprises : a) siRNA; b) 30% of DODAP;

c.l) 45% of CHOL;

c.2) 23% of DSPC; d) 2% of CERC₁₆PEG₂₀oo; e) the F3 peptide (from 2 to 10 nmol of

of total lipid) . in another preferred embodiment, the pH-sensitive liposomes "formulation A2 " , typically comprise: a) one or more unmodified or modified RNA interference molecule such as, but not limited to, siRNA; b) from 10% to 70% of a cationic lipid such as, DOTAP or its derivates or a mixture thereof; c)

c.l) from 20% to 60% of CHOL or its derivates or a mixture thereof ;

c.2) from 5% to 60% of the amphiphilic lipid, CHEMS ;

c.3) from 5% to 60% of the neutral cone-shaped lipid, DOPE; c.4) from 5% to 50% of a phospholipid such as DSPC or its derivates or a mixture thereof; from 0.5% to 10% of a pegylated neutral lipid such as ceramides (acyl chain length from C8 to C20) ; the F3 peptide (from 2 to 10 nmol of F3//imol of total lipid) which is specifically recognized by nucleolin receptors overexpressed on cancer and/or endothelial cells from angiogenic blood vessels.

A preferred "formulation A2 " (described in Example I) , comprises: a) siRNA; b) 25% of DODAP; c)

c.l) 26% of CHOL;

C.2) 10% of CHEMS;

c.3) 25% of DOPE;

c.4 ) 10% of DSPC; d) 4% of CERC₁₆PEG₂₀oo; e) the F3 peptide (from 2 to 10 nmol of F3/μναοΐ of total lipid) .

Nanoparticles of the invention are specifically internalized by cancer cells and/or endothelial cells from angiogenic blood vessels leading to an effective silencing of a target gene.

It is a second object of the invention the above nanoparticles for the treatment of angiogenesis-dependent diseases . In a preferred embodiment the angiogenesis-dependent disease is a cancer, especially a solid tumor cancer. In another preferred embodiment the angiogenesis- dependent disease is inflammation, an auto- immune disease or an ocular disorder.

The preparation procedure was adapted from Semple et al, for the encapsulation of antisense oligonucleotides (US 2005/6858225 B2 ) . Briefly, the lipid mixture in absolute ethanol is added, slowly and under strong vortex agitation, to an aqueous solution with an acidic pH containing the nucleic acid, followed by extrusion through polycarbonate membranes.

The present invention provides lipid-based nanoparticles targeted to a specific population of cells, upon covalent coupling of the F3 peptide. The ligand is coupled to the nanoparticle surface in a way that allows its specific interaction with nucleolin receptors overexpressed on the surface of cancer cells and endothelial cells from angiogenic tumor blood vessels.

Nucleolin is a ubiquitous protein with several functions such as, ribosome biogenesis, chromatin decondensation, cytokinesis, among others. It is exclusively nuclear in non-dividing cells but is highly express at the surface of actively growing cells. Therefore, nucleolin expression is associated with high cellular proliferative rates common to, but not limited, to cancer cells and endothelial cells from angiogenic tumor blood vessels [25] .

In the present invention, the targeting ligand can be, but it is not limited to a peptide, a peptidomimetic , an aptamer, a nanobody, a protein, an antibody or an antigen- binding fragment that specifically binds the nucleolin receptor overexpressed on cancer cells and endothelial cells from angiogenic tumor blood vessels. In a preferred embodiment, the ligand comprises a 31 aminoacid peptide derived from the protein group HMGN2 , with the following amino acid sequence, KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO:l) (F3 peptide) [24]. In the present invention, Traut's reagent (2 - iminothiolane hydrochloride) is used to introduce thiol groups onto primary amines (N-terminus and/or side chain of lysines) of the peptide. Then, the thiols groups react with the cross-linker reactive group, maleimide, present in PEG-derivatized lipid, forming a stable thioether bond. The pegylated lipid conjugated to maleimide can be for example, but not limited to, 1,2- Distearoyl - sn-Glycero- 3 -Phospatidylethanolamine-N-[Maleimide (Polyethylene Glycol ) ₂ooo] ammonium salt (DSPE-PEG-MAL).

In another embodiment, the thiolated peptide could be directly coupled to preformed liposomes comprising DSPE-PEG-MAL (direct coupling method) or to micelles of the conjugate DSPE- PEG-MAL. In the latter case, F3 peptide covalently coupled to DSPE-PEG-MAL conjugates (DSPE-PEG-MAL-F3 ) could be further inserted onto preformed liposomes (post - insertion method) . In the lipid-based nanoparticle described in the present invention, micelles of, but not limited to DSPE-PEG-MAL-F3 are incubated with preformed liposomes at 0.5 mol% to 10 mol%, of conjugate, relative to total lipid, from about 30 min to about 24 h, at a temperature, from about 35^°C to about 60^°C. As example, but not limited to, micelles of DSPE- PEG-MAL-F3 , at 2 mol%, relative to total lipid, are incubated with preformed liposomes comprising one or more cationic lipids, one or more non-cationic lipids, one or more pegylated-lipid conjugates, encapsulating one or more nucleic acids. In preferred embodiment, the incubation takes place during 1 h at 50^°C. As shown in example II (table 1) , for non-pH and pH- sensitive-liposomes , the siRNA encapsulation efficiencies are close to 100%. Moreover, siRNA molecules are fully encapsulated/protected, as can be concluded by the reduced interaction between the probe Quant-iT Ribogreen and siRNAs, which further indicates a high degree of nuclease resistance. The final charge is close to neutrality, between about -5mV to about +5mV, preventing the interaction with serum proteins and further blood clearance by the reticuloendothelial system. The mean diameter of the F3-targeted liposomes is small enough to enable passively passage through fenestrations gaps at the level of tumor blood vessels. Overall, the developed F3 -targeted liposomes exhibit adequate features for in vivo use, namely for intravenous administration of nucleic acids.

In addition, and as showed in examples III, IV and V, both non-pH and pH-sensitive F3-targeted liposomes have the advantage to be selectively internalized by cancer cells and/or endothelial cells from angiogenic tumor blood vessels, in an extent significantly higher than the non-targeted counterpart. Moreover, from the example III and IV, it is also possible to observe that the internalization is almost abolished at 4^°C (a non-permissive temperature for endocytosis) indicating that the cellular internalization is temperature-dependent. Therefore, it can be concluded that the cellular internalization of the targeted nanoparticle provided in the present invention is mediated by receptor-mediated endocytosis. The ability of the targeted nanoparticle, to downregulate a protein, was assessed in human cancer cell lines (MDA-MB-435 and MDA-MB-231) expressing a reporter gene, enhanced green fluorescent protein (eGFP) , as it allows to evaluate gene silencing in a more direct and easy way. It is important to clarify that the ability of the present F3 -targeted nanoparticle to silence eGFP expression can be extrapolated to any gene, as the developed nanoparticle is adequate for the encapsulation and delivery of any siR A sequence. Therefore, it is expected that the use of F3-targeted liposomes to encapsulate a siRNA against a therapeutic target (anti-tumor and/or anti -angiogenesis) , instead of eGFP, will improve the treatment of solid tumors, such as but not limited to prostate and breast cancer. Results from example V, surprisingly demonstrate (and in contrast with the work of Santos et al . , [26]) that the improvements obtained in the extent of cellular internalization, were correlated with an effective gene silencing, as eGFP downregulation was just achieved in the cells treated with the targeted liposomes. This result evidences the importance of the F3 peptide as a targeting ligand. Moreover, example VI confirms that the developed F3-targeted liposomes interfere at the eGFP mRNA level. The new insights into the different signalling pathways of cancer cells and others within a tumor, had allowed the identification of numerous targets whose downregulation could result in cell death and/or proliferation inhibition and/or angiogenesis inhibition. Several anti -tumor targets are described in the literature and commonly consist in genes/proteins that are involved in tumor cell growth, cell cycle division, metastasis formation, evasion to cell death and formation of new blood vessels. In this regard, silencing a proto-oncogene like, but not limited to PLK-1 with anti-PL l siRNA could encompass a new and valuable therapeutic approach for the treatment of tumors such as, but not limited to prostate cancer. PLK-1 is a serine/threonine kinase that regulates mitosis entry and progression. It is undetectable in normal tissues but is overexpressed in tumors and is often correlated with poor prognosis. Plk-1 overexpression in cancer cells overrides the cell cycle checkpoints, thus contributing for the capability of cancer cells to uncontrolled proliferation [38] .

Downregulation of Plk-1 resulted in growth inhibition, mitosis arrest, and induction of apoptosis in cancer cell lines from different histological origins- [18, 39-42] and sensitization to cancer drugs [43-44]. The development of a safe and efficient ligand-mediated targeted nanoparticle for the specific intracellular delivery of, but not limited to anti -PLK1 nucleic acids (such as, but not limited to siR As) to cancer cells and/or endothelial cells of the tumor vasculature, can have a tremendous positive impact in the treatment of tumors as well as, but not limited to inflammatory and auto-immune diseases and ocular disorders. In fact, results from example VII confirms the positive impact of the F3-targeted pH-sensitive- liposomes, encapsulating an anti-PLKl siRNA, on the viability of a prostate cancer cell line, indicating that the strategy presented herein may have a truthfully potential in the treatment of tumors .

Patent US 2005/6858225 B2 describes the methodology used herein to prepare the lipid-based nanoparticles . Although we had used the same process to prepare the nanoparticles , the lipids and their ratios, as well as the encapsulated nucleic acid were different. Moreover, the application of these lipid- based nanoparticles for the specific delivery of siRNAs to cancer and endothelial cells from angiogenic blood vessels is herein assessed for the first time.

Patent WO 2003/087124 describes the F3 peptide and its ability to simultaneously target tumor and endothelial cells from the angiogenic blood vessels. However, this document does not include the description of lipid-based nanoparticles for the encapsulation, protection and systemic delivery of nucleic acids .

Santos et al . [26], Mendon a et al . [45], and Yang et al . [46] have developed liposomes similar to the non-pH- sensitive liposomes described herein. Antagonist G, transferrin and folate were the targeting ligands used by those authors, respectively, and are an important difference from the targeting approach described herein, as none of the previous mentioned targeting ligand successfully targeted cancer and/or endothelial cells from angiogenic tumor blood vessels. Moreover, in the work of Santos et al . , [26], gene silencing was not achieved despite the improvements obtained at the level of cellular internalization .

The ability of the F3 peptide to target cancer and/or endothelial cells from angiogenic blood vessels has been explored in different therapeutic strategies such as, nanoparticles for the delivery of photosensitizers [47-49] and cisplatin [50] , quantum dots for the siRNA delivery [51] , directly coupled to the a-emitter ²¹³Bi [52] , or to asODN [53] . However, these strategies neither included the use of lipid- based nanoparticles for the intravenous administration of nucleic acids nor the use of fusogenic lipids like DOPE, which facilitated the access of the nucleic acid to its molecular target (mRNA) located in the cytoplasm. Although patent WO 2009/142525 describes F3-targeted pH-sensitive liposomes, they do not present the characteristics required for the encapsulation, protection and delivery of nucleic acids. Examples

The following examples intended to be illustrative and not to limit the invention.

Example I

Preparation of liposomes Non-pH sensitive liposomes (formulation Al) were composed of DODAP : DSPC : CHOL : CERCi₆PEG₂₀oo (30:23:45:2 % of total lipid) while pH-sensitive liposomes (formulation A2) were composed of DOTAP : DSPC : DOPE : CHOL : CHEMS : CERC₁₆PEG₂₀₀o

(25:10:25:26:10:4 % of total lipid) .

In both cases, the lipid mixture was prepared in absolute ethanol and the anti-GFP siRNA or control siRNA in 20 mM citrate buffer. After heating at 60°C, lipid mixtures were added, slowly and under strong agitation, to the respective siRNA solution. Afterwards, the resulting particles were extruded 21 times through polycarbonate membranes of 100 nm pore diameter, using a LipoFast mini extruder. The liposomes were then run through a Sepharose CL-4B column equilibrated with

HEPES buffered saline (HBS) (20 mM HEPES, 145 mM NaCl ) , pH 7.4,

I

in order to remove the excess of ethanol and the non- encapsulated siRNA.

To prepare the DSPE-PEG-MAL-F3 conjugate, the F3 peptide was thiolated upon reaction with 2 - iminothiolane hydrochloride in HBS buffer, pH 8, during 1 h at room temperature. The thiolated peptide was then incubated with DSPE- PEG-MAL micelles in MES buffered saline (MBS) (20mM MES, 20mM HEPES) , pH 6.5, overnight at room temperature. Finally, 2 mol% of DSPE- PEG-MAL-F3 conjugate (relative to the total lipid) was incubated with the preformed liposomes (post -insertion method) during 1 h at 50^°C. For the non-targeted lipid particles, post - insertion was performed only with DS PEG-MAL micelles without ligand.

Free maleimide groups were quenched upon incubation with an excess of 2 -mercaptoethanol for 30 min at room temperature. The liposomes were then run through a Sepharose CL- 4B column equilibrated with HBS , pH 7.4, in order to remove uncoupled DSPE- PEG-MAL -F3 conjugates. Example II

Characterization of the liposomes

The final total ^'lipid concentration was inferred from the cholesterol concentration that was determined using the

Infinity™ liquid stable reagent (Thermo Scientific) . The quantification of the siRNA that was encapsulated into the liposomes was determined using the Quanti-iT ™ Ribogreen reagent

(Invitrogen) in the presence of octaethylene glycol monododecyl ether (Ci₂E₈) detergent. The encapsulation efficiency was calculated using the formula [ (siRNA/total lipid) _fi_nai molar ratio/ (siR A/total lipid) i_nitiai molar ratio] xl00. In order to assess if the siRNA was fully encapsulated and protected by the lipid nanoparticle , the ability of the probe Quant-iT Ribogreen to intercalate the siRNA without the detergent C₁₂E₈ was evaluated.

The mean diameter of the resulting liposomes was determined by

Photon Correlation Spectroscopy, using a N5 submicron particle size analyser (Beckman Coulter) . The zeta potential was assessed using a Particle Size Analyzer 90 Plus (Brookhaven) .

To determine the amount of DSPE- PEG-MAL- F3 conjugate that was trans ferred onto the preformed liposomes, the F3 peptide was quantified using the CBQCA protein quantitification Kit (Invitrogen, Molecular Probes) . Table 1. Physico-chemical characterization of targeted or non- targeted liposomes containing nucleic acid, either non-pH or pH- sensitive liposomes. Values are the mean + SD of at least 3 independent experiments.

For both, non-pH and pH-sensit ive liposomes, the encapsulation efficiency of the targeted and non-targeted liposomes was close to 100%, which indicated that the post- insertion of the conjugates DSPE-PEG-F3 did not interfere with the loading of nucleic acids in both formulations. Moreover, in the absence of the liposomes-disrupting detergent C₁₂E₈, the probe Ribogreen was not able to intercalate with the siRNA, thus indicating that the siRNAs were fully encapsulated inside the liposomes and therefore, protected from the nucleases, being the levels of protection close to 100%.

The measurement of the surface charge revealed a zeta potential close to neutrality for both targeted and non-targeted liposomes. This is important as the absence of surface charges contributes to reduce the interaction with serum proteins and therefore, to decrease the extent of blood clearance following systemic administration.

Targeted and non- targeted liposomes were homogeneous in size (polydispersion index below 0.3) and, as expected, the mean size of F3-targeted liposomes is higher than the non- targeted counterpart. The amount of DSPE-PEG-MAL-F3 conjugate transferred onto the preformed liposomes was assessed through the quantification of the F3 peptide. Similar amounts were observed for both non-pH and pH-sensitive targeted liposomes (4.30 + 0.66 and 4.39 ± 0.36 nmol ligand / πιοΐ TL) , which were further correlated with similar extent of cellular internalization . Overall, the developed F3-targeted, non-pH and pH- sensitive, liposomes presented adequate features for intravenous administration, with a high nucleic acid loading capacity, ability to protect the encapsulated siRNA and a surface charge close to neutrality.

Example III

Cell culture The human cancer cell lines, MDA-MB-435S and MDA-MB-

231, were cultured in RPMI 1640 medium (Sigma) and human fibroblasts cells, BJ, were cultured in DMEM medium (sigma) . Both media were supplemented with 10% (v/v) heat - inactivated foetal bovine serum (Invitrogen) , and 100 U/ml penicillin, 100 g/ml streptomycin (Invitrogen) . Human microvascular endothelial cells, HMEC-1, were culture in RPMI 1640 supplemented with 10 ng/ml of mouse epidermal growth factor (mEGF) and 1 g/ml hydrocortisone (Sigma) . Cells were maintained at 37°C, in a 90% humidified atmosphere, containing 5% C0₂. Assessment of cellular association by flow cytometry

Cellular association studies were performed by flow cytometry. Half million of tumor, fibroblast and endothelial cells were seeded in 48-well plate. Twenty four hours later, cells were incubated with rhodamine- labelled targeted liposomes, targeted by a non-specific peptide (ARALPSQRSR (SEQ ID NO : 2 ) ) or non- targeted, at 0.2, 0.4 or 0.6 mM of total lipid, at 37^°C or 4^°C, during 1 h. Afterwards, cells were washed three times with phosphate buffer saline (PBS), pH 7.4, detached with dissociation buffer and immediately analyzed by flow cytometry using a FACS Calibur flow cytometer (BD, Biosciences) . Rhodamine fluorescence was evaluated in the FL2 channel and a total of 20.000 events were collected. Data were analyzed with the Cell Quest Pro software.

At 37^°C, the level of cellular association of the F3- targeted liposomes non-pH sensitive liposomes was significantly higher than the one observed for non-targeted liposomes or liposomes targeted by a non-specific peptide. These results indicated that the presence of the F3 peptide at the liposome's surface brings an important gain, as more liposomes are recognized and internalized into the cancer (MDA-MB-435S or MDA- MB-231 cells) and human microvascular endothelial (HMEC-1) cells. As an example, when cells were incubated with 0.4 mM of total lipid, it was observed a 1 -fold increase in the rhodamine signal for the MDA-MB-435S cell line and a 12-fold increase for both MDA-MB-231 and HMEC-1 cells. The interaction of the developed F3 -targeted liposomes revealed to be peptide-specific as it was pointed out by the low level of cellular association observed with the liposomes targeted by a non-specific peptide (Figure 1A) . A similar trend was observed in the experiments performed with F3 -targeted pH-sensitive liposomes (Figures 2A and 2B) . In similar experiments performed with a non- transformed cell line, BJ fibroblasts, the previous- -mentioned differences between targeted and non-targeted liposomes were dissipated, thus indicating that the interaction of the former with the target cells was also cell-specific (Figure 1A) .

Incubation of F3 -targeted liposomes at 4^°C, a temperature non-permissive for endocytosis, strongly inhibited cellular association when compared to incubations at 37 ^°C, a condition where both binding and endocytosis take place. These results suggested that a receptor-mediated endocytotic process was involved in the uptake of F3-targeted liposomes (Figure IB).

Example IV

Assessment of cellular internalization by confocal fluorescence microscopy

In order to confirm the previous results, cellular association was further assessed by confocal microscopy on the cell lines used in the previous experiments.

Two hundred and fifty thousand cells were seeded on glass cover slips in 12-well plate and further incubated at 37 or 4°C, for 1 h, with rhodamine-labelled liposomes (targeted or non-targeted) encapsulating a FITC- labelled siRNA. After washing three times with PBS, cells were fixed with 4% paraformaldeyde , and the nucleus stained with DAPI , wash with PBS, and finally, mounted in mowiol mounting medium. Confocal images were acquired in a Zeiss LSM-510 point scanning confocal microscope (Zeiss, Germany) , using a diode (405 nm) , an argon (488 nm) and a DSPP excitations lasers for DAPI, FITC and Rhodamine, respectively and a 63x oil immersion objective. Images were acquired and analyzed using the LSM 510 Meta software. All instrumental parameters pertaining to fluorescence detection and images analyses were held constant to allow sample comparison.

By confocal microscopy, it was observed that after 1 h of incubation, F3 -targeted liposomes were localized in the cytoplasm of tumor and endothelial cells (as can be observed by the intense red and green fluorescence) , but not in the fibroblasts or when cells were incubated with non-targeted liposomes. In addition, when MDA-MB-435S cells were incubated with F3-targeted liposomes, at 4^°C, it was not observed any significant internalization. Overall, these findings corroborate the previous results observed by flow cytometry.

Example V

Gene silencing evaluation

Human cancer cell lines expressing enhanced green fluorescence protein (eGFP) , MDA-MB-435S-eGFP and MDA-MB-231- eGFP, were used to evaluate the potential of the F3-targeted liposomes to downregulate a target protein. In this specific example, eGFP was used as target since the measurement of fluorescence could be easily and directly assessed by flow cytometry .

In order to evaluate the downregulation of eGFP, 30,000 cells were seeded in 48-well plates. Twenty-four hours later, cells were transfected, at 37^°C during 4 h, with different concentrations of F3-targeted liposomes or non-targeted liposomes containing an anti-eGFP siRNA, or F3-targeted liposomes containing a control siRNA. Afterwards, the medium was replaced with fresh medium and a second transfection was performed 48 h after the beginning of the experiment, with the same formulations and concentrations used in the first transfection. Alternatively, in another set of experiments, just one treatment at 0 h was performed. Ninety six hours after the begining of the experiment, cells were detached and eGFP levels were evaluated by flow cytometry using a FACS Calibur flow cytometer (BD, Biosciences) . EGFP fluorescence was evaluated in the FL1 channel and a total of 20,000 events were collected. Data were then analyzed with the Cell Quest Pro software. The eGFP silencing was expressed in percentage of the ratio eGFP signal treated cells/eGFP signal untreated cells.

For non-pH-sensitive liposomes, total absence of eGFP silencing was registered when cells were treated with non- targeted liposomes encapsulating anti-eGFP siRNA, whereas with the F3-targeted counterpart, a significant eGFP silencing (from 19.9 to 42.7 %, at concentrations ranging from 250 nM to 2 μΜ) was observed (Figure 4 ) . For pH-sensitive liposomes, 2 to 3 fold increase on eGFP silencing (from 27.5 to 65.7 % when concentrations from 250 nM to 2 μΜ were applied) was obtained with the F3-targeted liposomes. These results evidenced the major positive impact of both targeted formulations in the effective delivery of siRNAs and silencing of a target protein. The results obtained with non-pH- sensitive liposomes, in MDA-MB- 435-eGFP and MDA-MB-231 -eGFP, demonstrated that the extent of eGPF silencing differs in the two cell lines, being the highest levels observed in the MDA-MB-435S-eGFP cells probably because of its high level of cellular association.

Significant differences in the level of eGFP silencing were not observed when just one treatment was applied to cells. This result reflects the ability of siRNAs to be recycled along time leading to the degradation of several mRNA molecules

The results presented herein demonstrate that the F3- targeted liposomes have the ability to effectively deliver a siRNA, in a specific manner, to human cancer cells, resulting in the downregulation of a target protein. Example VII

Impact on cell viability upon treatment with F3- targeted pH- sensitive-liposomes

In order to evaluate the impact on cell viability of the F3-targeted liposomes described herein, 25.000 PC3 cells, a human prostate cancer cell line, were seeded in 24 -well plates. Twenty-four hours later, cells were transfected, at 37^°C during 8 h, with different concentrations of F3-targeted pH-sensitive- liposomes or non- targeted pH- sensitive liposomes containing an anti-PLK-1 siRNA, or F3 -targeted pH-sensitive liposomes containing a control siRNA. Afterwards, the medium was replaced with fresh medium and a second transfection was performed 48 h after the beginning of the experiment, with the same liposomes and concentrations used in the first transfection. Ninety-six hours after the beginning of the experiment, cell viability was evaluated using the Resazurin reduction - method . Briefly, cells were incubated, at 37 C during 2 h, with medium containing 10% of resazurin dye. Afterwards, observances at 540 and 630 nm were measured in a microplate fluorimeter Microdevices SpectraMax Gemini EM. Cell viability was extrapolated from the resazurin reduction using the following equation: [ (£_0xi eso * A540 - £_oxi 540 * A630) treated cells/ (£_{oxi 630} * A540 - £_{oxi 540} * A630) untreated cells] *100.

As can be observed in figure 7, non-targeted pH- sensitive- 1 iposomes containing the anti-PLK-1 siRNA and the F3 - targeted pH-sensitive-liposomes containing a non-specific siRNA, did not have significant effects on cell viability of PC3 cells. In contrast, F3 -targeted pH-sensitive-liposomes, encapsulating the anti-PLK-1 siRNA, led to a decrease on cell viability that was dependent on the dose. As an example, when cells were treated with 2 μΜ of anti-PLK-1 siRNA, a decrease of 47.3 ± 4.07 % was observed. This result demonstrated that the presence of the F3 peptide on the liposomes 'surface strongly contributes to improve the cytotoxicity of a siRNA against a validated antitumor target .

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Claims

1. F3 -peptide targeted lipid-based nanoparticles , adequate to encapsulate and delivery one or more nucleic acids, comprising : a) one or more nucleic acids; b) one or more cationic lipids; c) one or more non-cationic lipids; d) one or more poly (ethylene glycol ) -derivatized lipids; and e) one or more coupled targeting ligands which bind to the nucleolin receptor.

2. Nanoparticles according to claim 1, having adequate features for intravenous administration, with a size below 250 nm, a charge close to neutrality, high loading capacity and ability to protect the encapsulated nucleic acid.

3. Nanoparticles according to claim 1 or 2, comprising: a) one or more unmodified or modified nucleic acids such as a RNA interference molecule selected from siRNA, aiRNAs, miRNAs or mixtures thereof; b) one or more cationic lipid selected from 1 , 2 -dioleoyl -3- dimethylammonium-propane (DODAP) , 1 , 2 -dioleoyl - 3 - dimethylammonium-chloride (DODAC) , 1 , 2 -dioleoyl -3 - trimethylammonium-propane (DOTAP) , 1 , 2 -di -O-octadecenyl - 3 - trimethylammonium propane (DOTMA) , 1 , 2 -dioleyloxy-JV, N- dimethyl -3 -aminopropane (DODMA) _; 1 , 2 -dilinoleyloxy- 3 - (N, N- dimethyl ) aminopropane (DinLMA) and 1 , 2 -dilinolenyloxy-N, N- dimethyl- 3 -aminopropane (DLenDMA) ;

) one or more non-cationic lipids selected from cholesterol (CHOL) and its derivatives such as cholesteryl hemisuccinate (CHEMS) , a phospholipid such as distearoylphosphatidylcholine (DSPC) , hydrogenated soy phosphatidylcholine (HSPC) , 1 , 2 -dioleoyl - s.n-glycero-3 - phosphoethanolamine (DOPE) and 1 , 2 -distearoyl - s.n-glycero-3 - phosphoethanolamine (DSPE) ;

) one or more pegylated lipids selected from PEG-ceramides such as C1 -Ceramide mPEG₂₀oo, C16-Ceramide mPEG₂ooo and C18- Ceramide mPEG₂ooo; PEG-phospholipids , such as 1 , 2 -distearoyl - sn-glycero-3 -phosphoethanolamine-N- [succinyl (polyethylene glycol) -2000] (mPEG₂₀ooDSPE) and PEG-diacyl glycerols, such as PEG-succinoyl distearylglycerol (PEG-S-DSG) ; and

) a targeting ligand covalently coupled onto the nanoparticle surface which bind to the nucleolin receptor, enabling the nanoparticle to be internalized into the target cell(s) .

4. Nanoparticles according to claim 3, comprising:

) one or more unmodified or modified RNA interference molecule such as siRNA;

) from 10% to 70% of a ionizable lipid such as DODAP or its derivatives or a mixture thereof;

)

c.l) from 20% to 60% of CHOL or its derivatives or a mixture thereof ; c.2) from 5% to 50% of a phospholipid such as DSPC or derivatives or a mixture thereof; from 0.5% to 10% of a pegylated neutral lipid such as ceramides (acyl chain length from C8 to C20) ; and the F3 peptide (from 2 to 10 nmol of Ρ3/μπιο1 of total lipid) which is specifically recognized by nucleolin receptors overexpressed on cancer and/or endothelial cells from angiogenic blood vessels.

5. Nanoparticles according to claim 4, comprising: siRNA;

30% of DODAP;

c .1) 45% of CHOL;

C.2) 23% of DSPC;

2% of CERC16PEG2000 /' the F3 peptide (from 2 to 10 nmol of Ρ3/μηηο1 of total lipid) .

6. Nanoparticles according to claim 3, comprising: one or more unmodified or modified RNA interference molecule such as siRNA; from 10% to 70% of a cationic lipid such as DOTAP or its derivatives or a mixture thereof; c) c.l) from 20% to 60% of CHOL or its derivatives or a mixture thereof ;

c.2) from 5% to 60% of the amphiphilic lipid, CHEMS;

c.3) from 5% to 60% of the neutral cone-shaped lipid, DOPE; c.4) from 5% to 50% of a phospholipid such as DSPC or its derivatives or a mixture thereof; from 0.5% to 10% of a pegylated neutral lipid such as ceramides (acyl chain length from C8 to C20); and the F3 peptide (from 2 to 10 nmol of Ε3/μτηο1 of total lipid) which is specifically recognized by nucleolin receptors overexpressed on cancer and/or endothelial cells from angiogenic blood vessels.

7. Nanoparticles according to claim 6, comprising: a) siRNA; b) 25% of DODAP;

C.l) 26% of CHOL;

c.2) 10% of CHEMS;

c.3) 25% of DOPE;

c.4) 10% of DSPC; d) 4% of CERCi₆PEG₂ooo; e) the F3 peptide (from 2 to 10 nmol of

of total lipid) .

8. Nanoparticles according to any one of claims 1 to 7, which are specifically internalized by cancer cells and/or endothelial cells from angiogenic blood vessels leading to an effective silencing of a target gene.

9. Nanoparticles according to any one of claims 1 to 8, for the treatment of angiogenesis-dependent diseases.

10. Nanoparticles according to claim 9, wherein the angiogenesis-dependent disease is a cancer.

11. Nanoparticles according to claim 10, wherein the cancer is a solid tumor cancer.

12. Nanoparticles according to claim 9, wherein the angiogenesis-dependent disease is inflammation, an auto-immune disease or an ocular disorder.