WO2019129657A1

WO2019129657A1 - Actively targeted polymeric micelles for drug and gene delivery

Info

Publication number: WO2019129657A1
Application number: PCT/EP2018/086318
Authority: WO
Inventors: Diana FERNANDES DE SOUSA RAFAEL; Francesc MARTÍNEZ TRUCHARTE; Simón Schwartz Navarro; Fernanda DA SILVA ANDRADE
Original assignee: Fundació Hospital Universitari Vall D'hebron - Institut De Recerca; Consorcio Centro de Investigación Biomédica en Red, M.P.
Priority date: 2017-12-28
Filing date: 2018-12-20
Publication date: 2019-07-04

Abstract

The invention relates topolymeric micellesin aqueous media comprising amphiphilic block copolymer of polyoxyethylene and polyoxypropylene blocks; and gelatin. They are in particular for the delivery of oligonucleotides in targeted animal or plant cells. The invention also relates to pharmaceutical compositions comprising said polymeric micelles as well as to its use in the treatment of diseases, in particular by gene therapy.In particular, the polymeric micelles comprise carboxylated polyoxyethylene blocks allowing the coupling of a targeting moiety able to direct the micelles to particular animal cells.

Description

Actively targeted polymeric micelles for drug and gene delivery

This application claims the benefit of European Patent Application EP17382919.3 filed on 28^th December 2017.

Technical Field

This invention relates to a system for transporting drugs and other compounds into plant or animal cells. It also relates to the field of polymeric micelles as multifunctional delivery systems, in particular for the delivery of nucleic acids in a method for treating diseases by gene therapy.

Background Art

In recent years, nanomedicine, as an area of interdisciplinary research involving biology, chemistry, pharmaceutical engineering and medicine, has substantially evolved aiming to achieve great advances in diseases treatment and diagnosis. Among the wide range of nanometric vehicles used for drug delivery such as dendrimers, liposomes,

nanoemulsions, and inorganic nanoparticles (NPs), polymeric NPs composed of biodegradable polymers have been of great importance due to their ability to encapsulate drugs, peptides/proteins or nucleic acids, biocompatibility, size and surface control, controlled release properties, and easy functionalization by chemical surface modification.

Polymeric micelles are particular polymeric NPs formed by assembly of biodegradable block copolymers, said copolymers being polymer chains comprising both hydrophilic and hydrophobic blocks or portions. Polymeric micelles have been proved as efficient nano- sized (approximately 20 to 200 nm) polymeric self-assembly systems for encapsulating drugs. Examples of these are disclosed in patent documents, such as in EP1037611 B1 and EP1907444B1 ; as well as in other documents, such as in Wu et al.,“Peptide- mediated Tumor Targeting by a Degradable nano Gene Delivery Vector Based on

Pluronic-Modified Polyethyleneimine”, Nanoscale Research Letters- 2016, vol. no. 1 1 :122.

EP103761 1 B1 discloses biodegradable mixed polymeric micelles for gene delivery comprising (a) a mixture of amphiphilic polyester-polycation copolymer and an amphiphilic polyester-sugar copolymer comprising a sugar moiety, and (b) a nucleic acid complexed with the mixed polymeric micelle.

EP1907444B1 discloses drug-loaded micelles comprising a multiblock copolymer that comprises a polymeric hydrophilic block, a crosslinked poly(amino acid block), and a non- crosslinked poly(amino acid block), wherein the said micelle has a drug-loaded non- crosslinked core, a crosslinked outer core, and a hydrophilic shell. The micelles are proposed for the delivery of doxorobucin into several cancer cell lines (MCF-7, HeLa, and HepG2).

In the same way, Wu et al., (supra) disclose polymeric micelles in which DNA is complexed with modified Pluronic (Pluronic-PEI-DR5-TAT), a particular block copolymer modified with polyethylenimine and conjugated to a multifunctional peptide containing a cell-penetrating peptide (TAT) and a synthetic that binds to a receptor overexpressed in cancer cells (DR5). The complexes were highly specific for tumoral cells and showed a low toxicity to normal cells, and high gene transfection efficiency in tumor cells.

Oncology is the field that has benefit the most from the application of nanotechnology to drug and gene delivery. Nanotechnology-based vectors of any type easily reach extravascular spaces becoming concentrated preferentially inside tumor tissues by the well-known enhanced permeability and retention effect (EPR effect). This phenomenon consists in a passive targeting mechanism where nanoparticles take advantage of their lower size and the unique leaky properties of the tumor vasculature.

Gene therapy has appeared as a promising alternative for an effective and more specific treatment of cancer and other complex diseases with an important genetic background. Using the RNA interference (RNAi) technology, in concrete small interfering RNAs (siRNAs) it is possible to silence gene expression. However, the in vivo delivery of oligonucleotides (OGN) has precluded the clinical use of gene-based therapies mainly due to its vulnerability to enzymatic blood degradation, poor cellular uptake and rapid renal clearance. Therefore, the greatest challenge for a successful clinical application of gene therapy relies on the development of vectors able to condensate negatively charged OGN and to effectively deliver them into the cytoplasm and/or nucleus of target cells. Nowadays, viral vectors are still considered the most efficient, being the most commonly used for gene transfer in both pre-clinical and clinical research (see Kumar et al.,“Clinical development of gene therapy: results and lessons from recent successes”, Mol. Ther Methods Clin Dev.- 2016, vol. no. 3, pp.:16034). However, the well-known drawbacks related with viral-based vectors such as their immunogenicity, mutagenesis,

carcinogenesis, limited cargo loading, and time consuming/high cost procedures, boosted the development of safer vehicles using a wide range of lipids and polymers (non-viral vectors). Despite the efforts made for the design of drug delivery systems, there is still a need of alternative approaches that, in particular, may overcome said viral vector drawbacks for the delivery of the negatively charged OGN for gene therapy.

Summary of Invention

With the aim of designing alternative non-viral vectors for drug delivery (in particular for siRNA delivery) into cancer cells (e.g.: EGFR overexpressing breast cancer cells), inventors surprisingly found that polymeric micelles of simple composition were useful for the delivery of oligonucleotides (OGN) in an effective and safety mode into the cells (plant or animal cells). The polymeric micelles allowed the entrapment of high amounts of OGN and, in addition, the polymeric micelles once internalized into the cells were able to silence targeted genes. These polymeric micelles comprise biodegradable, biocompatible and low immunogenicity block copolymers (such as Pluronic^®); and a cationic component able to condense siRNA and improve its transfection efficiency and biological activity without toxic cell effects. Pluronic a trademark of a particular type of poloxamer is a block copolymer highly used in drug formulation. It is composed by both hydrophilic units of polyethylene glycol (PEG) forming to a block of polyoxyethylene, and hydrophobic units of polypropylene oxide (PPO) forming a block of polyoxypropylene, both block types disposed in sequential or grafted blocks (generally di- and triblock-copolymers).

Simultaneously, the search for a cationic component able to condense siRNA and improve its transfection efficiency and biological activity was also pursued.

Thus, a first aspect of the invention is a polymeric micelle in aqueous media comprising:

- an amphiphilic block copolymer of polyoxyethylene and polyoxypropylene blocks; and

- gelatin, wherein the weight ratio of amphiphilic block copolymer and of gelatin in the micelle is from 10:1 to 5:5.

As will be depicted in the examples below, a polymeric micelle of this type has spherical shape (Transmission Electron Microscopy-TEM) with a hydrodynamic diameter from 20 to 50 nm (measured by TEM), or from 150-300 nm (by Dynamic Light Scattering-DLS), with a homogeneous size distribution and with a charge from -30 to +30 mV, depending on the components of the micelle (block copolymer and gelatin) and the ratio between them. Thus, the polymeric micelles of the invention are nanoparticles able to deliver into cells compounds of different nature. In addition, gelatin, which is a non-toxic for cells cationic polymer, effectively buffers and complexes the negatively charged oligonucleotides (OGN) (due to phosphate functionalization of nucleosides), in such a way that the polymeric micelle is able to efficiently transfect these oligonucleotides into cells and therein to alter the expression of the targeted gene without causing toxicity in non-targeted cells. Other non-toxic cationic polymers could be used.

Structurally, the polymeric micelle comprises a shell or corona conformed by the polyoxyethylene blocks and the gelatin, which is also termed the outer side of the micelle; and a core conformed by the polyoxypropylene blocks, in which core a hydrophobic (i.e. not water friendly) space or centre of the micelle is defined by said polyoxypropylene blocks, and which is also called the inner side of the micelle. This conformation takes place in aqueous media. A polymeric micelle comprising the same amphiphilic block copolymer of polyoxyethylene and polyoxypropylene blocks; and the gelatin (in the proposed weight ratio) will adopt another conformation (or in other words the components will self-assemble differently) in a non-polar solvent.

More specifically and from the outer surface of the micelles facing the solvent (aqueous media) to their inner core, the polymeric micelles of the invention comprise: a layer or spatial zone comprising the polyoxyethylene blocks of the block copolymer and the gelatin; and an inner core comprising the polyoxypropylene blocks of the block copolymer.

This structure or supramolecular self-assembly is, in particular, obtained in polar solvents such as in water that optionally may comprise other compounds, such as buffered water solutions (phosphate buffered solution) or cell growth culture media including all nutrients for cell growth. In these media, the molecules adopt the above defined arrangement without guidance or management from an outside source, in such a way that they form an aggregate with the hydrophilic "head",“shell” or also called“corona” or“outer side” regions in contact with surrounding solvent, sequestering the hydrophobic regions in the micelle“centre” or“inner side” of the micelle.

Besides the capability of efficiently entrapping OGN, the polymeric micelles allow the entrapment and delivery of compounds for any other purpose, meanwhile other negatively charged compounds can be complexed with gelatin and efficiently transported within the polymeric micelle. In the same way, additional compounds that due to its size and nature assemble in such a way that they remain in the centre of the micelle, not necessarily complexed with gelatin, are effectively transported and delivered where required with the polymeric micelles of this first aspect. Proved below the efficacy and safety of the polymeric micelles in the delivery of OGN, another aspect of the invention is a drug-loaded polymeric micelle comprising within the polymeric micelle as defined in the first aspect, one or more therapeutic agents.

A fourth aspect of the invention is a pharmaceutical composition comprising in aqueous media a therapeutically effective amount of a drug-loaded polymeric micelle as defined above, together with pharmaceutically acceptable excipients and/or carriers.

The pharmaceutical composition is not to be limited to the use in gene therapy, since polymeric micelles are also able to entrap one or more therapeutic agents within its structure. Distribution of such therapeutic agents will be function of their chemical nature, and on how for thermodynamics all components conforming the micelle will be assembled aiming the low Gibbs free energy in the system, being the system constituted by the polymeric micelle with the therapeutic agent in the particular aqueous solvent. So that, some therapeutic agents will tend to be entrapped in the centre of the polymeric micelle due to its nature (more hydrophobic compounds or water-repellant compounds), while other drugs (more hydrophilic) will tend to be associated near the polyoxyethylene blocks and/or complexed with the gelatin as will do any negatively charged compound (e.g. oligonucleotides).

Both, the drug-loaded polymeric micelles as well as the pharmaceutical compositions as defined above are, in a fourth aspect of the invention, for use as a medicament.

As indicated before, the polymeric micelles of the first aspect allow the entrapment and delivery of several compounds and of several natures, which can be used for therapeutic and non-therapeutic purposes. Thus, yet another aspect of the invention is a

multifunctional delivery system for the delivery of one or more compounds to plant or animal cells, comprising a polymeric micelle as defined above in any of the first or second aspects.

As will be depicted below in examples, the multifunctional delivery systems of the invention allow the simultaneous treatment with two or more therapeutic agents, as well as the traceability of the delivered compounds and cells in case one of the delivered compounds is a cell marker (e.g. dye).

Brief Description of Drawings)

FIG. 1 , related with example 1 , shows the in vitro cytotoxicity (by means of % of cell viability) of the different polymeric micelle (PM) formulations in MDA-MD-231 cells (FIG. 1 (A)), and the correspondent IC50 values (FIG. 1 (B)). Results are expressed as meaniSD (n=3). PM F127 designates polymeric micelles (PM) wherein the amphiphilic block copolymer is Pluronic® F127 (dark circles). PM F127:COOH designates PM of modified (carboxylated) Pluronic® F127; that is, fully functionalized with COOH groups in the terminal oxyethylene monomer units of the polyoxyethylene block (squares). PM F127:COOH 8:2 designates a polymeric micelle comprising a mixture of Pluronic® F-127 and carboxylated Pluronic® F127:COOH in a weight ratio (w/w) 8:2 (light circles). FIG. 2, related with example 1 , illustrates gene expression levels (NRQ) after transfection with PM entrapping a Serine/Threonine Kinase 2 (AKT2) silencing RNA siAKT2 (200 nM), relatively to the ones transfected with a control siC (FIG. 2(A)); and the in vitro cytotoxicity (by means of % of cell viability) of different formulations in MDA-MD-231 cells (FIG. 2(B)). PM F127:COOH 8:2:Gelatin designates a polymeric micelle comprising a mixture of Pluronic® F127 and carboxylated Pluronic® F127:COOH in a weight ratio (w/w) 8:2 and Gelatin (dark triangles). PM F127:COOH 8:2:Gelatin:Cet 0.1 mg/ml and PM F127:COOH 8:2:Gelatin:Cet designate, respectively, the polymeric micelles with the targeting moiety cetuximab (abbreviated Cet or Cetux) at 0.1 mg/ml (circles) or 1 mg/ml (squares) in final formulation.

FIG. 3, related with example 1 , shows internalization in MDA-MB-231 (FIG. 3(A)) and MCF7 (FIG. 3(B)) cells, of different types of PM labelled with 5-(4,6- dichlorotriazinyl)aminofluorescein (5-DTAF) (PM F127:COOH 8:2 and PM F127:COOH 8:2:Gelatin:Cet) and the percentage of cells emitting green fluorescence were quantified at different incubation time-points. Internalization is indicated with the percentage of fluorescent positive cells (%) along time (in hours).

FIG. 4, shows cell viability (in percentage %) in cells comprising PM of the invention with (PM F127:COOH 8:2:Gelatin; in triangles) and without (PM F127:COOH 8:2; in circles) gelatin.

FIG. 5 is a graphic schematically illustrating the structure of polymeric micelles in aqueous media according to the invention. TM means targeting moiety; PEO is the polyoxyethylene block of the block copolymer; PPO is the polyoxypropylene block of the block copolymer; OGN-gelatin designates the polyplexes of oligonucleotides (OGN) and gelatin.

FIG. 6, related with Example 2, shows the fluorescence intensity along time (for 4 hours) detected in two subpopulations of a line of human colon cancer, HCT1 16 cancer stem cells (CSC) and HCT1 16 non cancer stem cells (non-CSC) cells, that were incubated with PM labelled with 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF) and comprising or not Gelatin and one oligonucleotide (siRNA). For each sample, at least 10000 individual cells were collected and the presented value in FIG. 6 (A) and (B) corresponds to the mean fluorescence intensity. PM-COOH-Gelatin-siRNA CSC means Cancer Stem Cells incubated with labelled PM F127:COOH 8:2 including gelatin an the siRNA; PM CSC relates to PM F127:COOH 8:2 in CSC; the same legend applies to non-CSC.

Detailed description of the invention

All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition.

A“micelle” or micella (plural micelles or micellae, respectively) is an aggregate (or supramolecular assembly; a well-defined complex of molecules held together by noncovalent bonds) of surfactant molecules dispersed in a liquid colloid. A typical micelle in aqueous solution forms an aggregate with the hydrophilic "head" regions or outer side of the micelle in contact with surrounding solvent, sequestering the hydrophobic regions in the inner side of the micelle facing micelle centre. Micelles are approximately spherical in shape. Other phases, including shapes such as ellipsoids, cylinders, and bilayers, are also possible. The shape and size of a micelle are a function of the molecular geometry of its surfactant molecules and solution conditions such as surfactant concentration, temperature, pH, and ionic strength. The process of forming micelles is known as micellisation and forms part of the phase behaviour of many lipids according to their polymorphism. According to IUPAC a micelle is a particle of colloidal dimensions that exists in equilibrium with the molecules or ions in solution from which it is formed. Micelles form only when the concentration of surfactant is greater than the critical micelle concentration (CMC), and the temperature of the system is greater than the critical micelle temperature, or Krafft temperature. In colloidal and surface chemistry, the“critical micelle concentration (CMC)” is defined as the concentration of surfactants above which micelles form and all additional surfactants added to the system go to micelles. The value of the CMC for a given dispersant in a given medium depends on temperature, pressure, and (sometimes strongly) on the presence and concentration of other surface active substances and electrolytes. Micelles only form above critical micelle temperature. The concept of micelles was introduced to describe the“core-corona aggregates” of small surfactant molecules; however it has also extended to describe“aggregates of amphiphilic block copolymers in selective solvents”. These aggregates are also known as polymeric micelles. According to IUPAC a“polymeric micelle” or“micelle (polymer”) is an organized auto-assembly formed in a liquid and composed of amphiphilic macromolecules, in general amphiphilic di-or tri-block copolymers made of solvophilic and solvophobic blocks. An amphiphilic behavior can be observed for water and an organic solvent or between two organic solvents. Polymeric micelles have a much lower critical micellar concentration (CMC) than soap or surfactant micelles, but are nevertheless at equilibrium with isolated macromolecules called unimers. Therefore, micelle formation and stability are

concentration-dependent.“Block copolymers” are polymers that comprise two or more homopolymer subunits linked by covalent bonds. The union of the homopolymer subunits may require an intermediate non-repeating subunit, known as a junction block. Block copolymers with two or three distinct blocks are called diblock copolymers and triblock copolymers, respectively. These di- and triblock-copolymers, can be sequential di- and triblock-copolymers or grafted di- and triblock-copolymers. Schematically a sequential diblock copolymer has the following structure -(A)n-(B)m-; a sequential triblock copolymer corresponds to the structure -(A)n-(B)m-(A)p-, being n, m and p the number of monomers of A or B; a grafted polymers are segmented copolymers with a linear backbone of one composite and randomly distributed branches of another composite. Particular sequential diblock copolymers of polyoxyethylene (PEO) and polyoxypropylene (PPO) used in the micelles of the invention are schematically defined as (PEO)n-(PPO)m. Triblock copolymers are defined by (PEO)n-(PPO)m-(PEO)p; and -(PPO)m-(PEO)p-(PPO)n. In an aqueous media these di- or triblocks of PEO and PPO auto-assemble being the PEO block(s) facing the said aqueous media and the PPO block(s) conforming a core or inner face of the assembly (not in contact with the aqueous media). Different conformations will result from initial disposition of PEO and PPO blocks in the linear sequential copolymer. The monomers of oxyethylene or oxypropylene sited at the extremes of the corresponding end blocks are herewith termed“terminal monomer units”. Thus, PEO as an end block has oxyethylene terminal monomer units, and PPO as an end block has oxypropylene terminal monomer units. For“end block” is to be understood the blocks of polyoxyethylene and/or of polyoxypropylene that are sited at the right and left side of the linear polymeric copolymer structure. Thus, in a diblock (PEO)n-(PPO)m, both blocks are end blocks; meanwhile in a triblock (PEO)n-(PPO)m-(PEO)p only PEO blocks are end blocks. The terms "block copolymer" and "graft copolymer" are defined in accordance with the terminology used by the International Union of Pure and Applied Chemistry (IUPAC). "Block copolymer" refers to a copolymer containing a linear arrangement of blocks. The block is defined as a portion of a polymer molecule in which the monomer units have at least one constitutional or configurational feature absent from the adjacent portions. "Graft copolymer" refers to a polymer composed of macromolecules with one or more species of block connected to the main chain as side chains, these side chains having constitutional or configurational features that differ from those in the main chain. For“amphiphilic block copolymer” is to be understood a copolymer with a block or parts of a block possessing hydrophilic (water-loving, polar) properties, and a block or part of a block possessing lipophilic (fat-loving) or hydrophobic (no water-loving) properties, in such a way that the block copolymer possesses both properties and, as above exposed, in a determined solvent (for example in aqueous media) all the components of the copolymer assembled in a particular configuration which is that with a lower free Gibbs energy. Amphiphilicity of surfactants, in this description amphiphilicity of the block copolymers are determined by the hydrophilic-lipophilic balance (HLB), which is a measure of the degree to which it is hydrophilic or lipophilic, determined by calculating values for the different regions of the molecule. The skilled man is aware of the methods for determining HLB values. Particular HLB values of the block copolymers in the polymeric micelle of the invention are from 17 to 23; and greater than 24.

For“gelatin” is to be understood as a mixture of peptides and proteins produced by partial hydrolysis of collagen extracted from the skin, bones, and connective tissues of animals such as domesticated cattle, chicken, pigs, and fish. During hydrolysis, the natural molecular bonds between individual collagen strands are broken down into a form that rearranges more easily. Its chemical composition is, in many aspects, closely similar to that of its parent collagen. Gelatin is an irreversibly hydrolyzed form of collagen, wherein the hydrolysis results in the reduction of protein fibrils into smaller peptides, which will have broad molecular weight ranges associated with physical and chemical methods of denaturation, based on the process of hydrolysis. In the present invention pharmaceutical grade gelatins can be used (that is, those accepted by medical authorities (i.e.:FDA or EMA). Other gelatin sources include the non-pharmaceutical grade gelatins of Sigma Aldrich, references G2625, G6650, G9382, G1393, G9391 , G6144, G2500, G8150, G1890, G9136, G0411 , G7765, and G7041

(https://www.sigmaaldrich.com/content/dam/sigma- aldrich/docs/Sigma/Product_lnformation_Sheet/2/g2625pis.pdf, accesible on 27.12.2017). Although gelatin is 98-99% protein by dry weight, it has little additional nutritional value, varying according to the source of the raw material and processing technique. Amino acids present in gelatin are variable, due to varying sources and batches, but are approximately: Glycine 21 %; Proline 12%; Hydroxyproline 12%; Glutamic acid 10%;

Alanine 9%; Arginine 8%; Aspartic acid 6%; Other 22%. Isoelectric point (pi): The charge on a gelatin molecule and its isoelectric point are primarily due to the carboxyl, amino, and guanidino groups on the side chains. Type A gelatin has 78-80 millimoles of free carboxyl groups per 100 g of protein and a pi of 7.0-9.0; type B has 100-1 15 millimoles of free carboxyl groups per 100 g of protein and a pi of 4.7-5.2. The pH of a 1.5% solution at 25 ° C is 3.8-5.5 for Type A and 5.0-7.5 for Type B.

The term“therapeutic agent” relates to any compound or mixtures of compounds that are able to promote a therapeutic effect once administered in a“therapeutically effective” amount in an animal, particularly in a mammal, and more particularly in a human. The expression "therapeutically effective amount" as used herein, refers to the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed. The particular dose of compound administered according to this invention will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and the similar considerations.

The term "weight ratio”, expressed as“w/w" of a component in relation to another component refers to the amount in mass units (micrograms, grams) of one component relative to the amount in mass units of the other component. On the other side, the term "%w/w" or "percentage weight per weight" of a component refers to the amount of the single component relative to the total weight of the composition or, if specifically mentioned, of other component.

The term“volume ratio”, expressed as“v/v” of a component in relation to another component refers to the amount in volume units (ml, I, cm³) of one component relative to the amount in volume units of the other component. The term mass/volume ratio, expressed as“w/v” of a component in relation to another component refers to the amount in mass units (micrograms, grams) of one component relative to the amount in volume units (ml, I, cm³) of the whole solution or suspension wherein the component is.

For“hydrophobic compounds or hydrophobic therapeutic agents” is to be understood a compound with a partition coefficient (P) logarithm (logP) greater than 0 (logP>0). They are associated with or near the hydrophobic polyoxypropylene portion of the block copolymer conforming the polymeric micelle, or that are disposed in the inner side or centre of the micelle. That is, not facing the solvent or aqueous media. On the contrary, “hydrophlilic compounds or hydrophilic therapeutic agents”, are compounds with a partition coefficient logarithm lower than 0 (logP<0), and that in the polymeric micelle are associated near the polyoxyethylene portion of the block copolymer but inside the micelle. Hydrophilic compounds also include cationic and anionic compounds, the later associated with the gelatin of the micelle. Partition-coefficient ( P ) or distribution-coefficient (D) is the ratio of concentrations of a compound in a mixture of two immiscible phases at equilibrium. This ratio is therefore a measure of the difference in solubility of the compound in these two phases. The partition-coefficient generally refers to the

concentration ratio of un-ionized species of compound whereas the distribution-coefficient refers to the concentration ratio of all species of the compound (ionized plus un-ionized). The skilled man in the art will know how to calculate this parameter (see for example Comer J, Tam K (2001 ). "Lipophilicity Profiles: Theory and Measurement". In Testa B, van de Waterbed H, Folkers G, Guy R. Pharmacokinetic Optimization in Drug Research: Biological, Physicochemical, and Computational Strategies (secondary). Weinheim: Wiley-VCH. pp. 275-304.)

The term“drug-loaded polymeric micelle” is to be understood as a polymeric micelle comprising one or more compounds that have a therapeutic effect. In particular, the said compounds with therapeutic effect are generally embedded in the inner side of the polymeric micelle, either associated with gelatin or with the hydrophobic zone conformed by the polyoxypropylene blocks.

The expression "pharmaceutically acceptable excipients or carriers" refers to

pharmaceutically acceptable materials, compositions or vehicles. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio.

The term“multifunctional delivery system” refers to a system for the delivery into plant or animal cells of more than one compound. Thus, a polymeric micelle comprising more than one type of therapeutic agents to be delivered, including oligonucleotides and other therapeutic agents as disclosed below. It encompasses also the concept of the multitarget delivery system, which means that two or more different targeting moieties are coupled to the polymeric micelle in order to deliver the one or more therapeutic agents or compounds to be delivered in different cell types that differentially recognize the two or more targeting moieties. Non limitative examples of the multifunctional delivery systems include a polymeric micelle comprising one or more oligonucleotides with therapeutic effect that are targeted by means of two different targeting moieties to two different cell types (breast cancer cells and colon cancer cells). Another example is a polymeric micelle with a cocktail of different therapeutic agents (i.e. a chemotherapeutic cocktail) targeted to a single group of tumour cells by means of a specific for the said cells targeting moiety (i.e. a ligand for a particular receptor only expressed in that cell type).

The polymeric micelles of the invention are formed in an aqueous media at neutral pH (pH= 6-7.5) and with an amount of amphiphilic block copolymer from 10 mg/ml to 100 mg/ml. Particular CMC values in the formation of the polymeric micelles will depend on the amphiphilic block copolymer of polyoxyethylene and polyoxypropylene blocks. In the present invention, there are used in a particular embodiment amphiphilic block copolymers of polyoxyethylene and polyoxypropylene blocks with a CMC from 2.0 x 10 ⁶ M to 5.0 x 10⁴ M.

The skilled man in the art will know particular amphiphilic block copolymers of

polyoxyethylene and polyoxypropylene blocks. Below there are listed particular ones with particular dispositions of the different blocks, as well as with particular repetition monomer units in each of the blocks. Synthesis of the block copolymers can be performed by several methods the skilled man will also know. As a way of a non-limitative example block copolymers are made up of blocks of different previously polymerized monomers.

On the other hand, commercial block copolymers of polyoxyethylene and

polyoxypropylene blocks are already available (see below), and they can be used as material for the synthesis of the polymeric micelles of the invention.

The polymeric micelle according to the first aspect of the invention, further comprises, in a particular embodiment, a modified amphiphilic block copolymer with one or more polyoxyethylene blocks and one or more polyoxypropylene blocks, wherein the

oxyethylene and/or oxypropylene terminal monomer units of the end blocks of

polyoxyethylene and/or polyoxypropylene are modified and comprise functional groups capable of forming an amide bond.

In a particular embodiment, the functional groups are selected from the group consisting of - COOR², being R² selected from H and -(CrC₈)-alkyl; -COR¹, wherein R¹ is a compound of formula -Z-COOR², being -Z- selected from (CH2)n with n an integer from 1 to 10, (CH=CH-CH)m with m an integer from 1 to 3, (CH2-CH=CH), and (CH=CH)rwith r an integer from 1 to 2; -NR³R⁵ being R³ and R⁵ independently selected from H and -(Cr C₈)-alkyl, or R³ and R⁵ conform together with the nitrogen atom a cyclic compound and wherein optionally one or more hydrogen atoms have been substituted by oxygen atoms; and combinations thereof. When R¹ is a compound of formula -Z-COOR² the terminal monomer units comprise functional groups derived from dicarboxylic acids.

Other functional groups can also be present in the terminal monomer units of the blocks.

In particular, those other functional groups allowing the formation, in particular, of covalent bond with proteins and peptides. For example, the functional groups may include -SR⁴, being R⁴ selected from H and -(CrC₈)-alkyl. These particular functional groups with sulphur atoms can form disulphide bonds with the cysteines of proteins.

Along the description the term“modified block copolymer” is to be understood as encompassing said block copolymers with one or more polyoxyethylene blocks and one or more polyoxypropylene blocks, wherein the oxyethylene and/or oxypropylene terminal monomer units of the end blocks are modified and comprise functional groups.

Throughout the description and claims, the term“-(Ci-C₈)-alkyl radical”, shall be construed as straight or branched. It includes, any of the radicals methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, n-butyl, sec-butyl (or 1-methylpropyl), isobutyl (or 2-methylpropyl), tert-butyl (or 1 ,1-dimethylethyl), n-pentyl, tert-pentyl (or 2-methylbutan-2-yl), neopentyl (or 2,2- dimethylpropyl), isopentyl (or 3-methylbutyl), sec-pentyl (or pentan-2-yl), 3-pentyl (or pental-3-yl), n-hexyl, isohexyl (or 4-methylpentyl), tert-hexyl, sec-hexyl, n-heptyl, tert- heptyl, isoheptyl, sec-heptyl, n-octyl, tert-octyl, sec-octyl, and isooctyl.

In a more particular embodiment, terminal monomer units of the polyoxyethylene block and/or polyoxypropylene block are modified and comprise the functional group -COOR², wherein R² is selected from H, and -(CrC₈)-alkyl.

In another particular embodiment terminal monomer units are modified and comprise functional groups that are selected from the group consisting of -COR¹, wherein R¹ is a compound of formula -Z-COOR², being -Z- selected from (CH2)n with n an integer from 1 to 10, (CH=CH-CH)m with m an integer from 1 to 3, (CH2-CH=CH), and (CH=CH)rwith r an integer from 1 to 2.

In a more particular embodiment, terminal monomer units of the polyoxyethylene and/or polyoxypropylene blocks are modified and comprise -C0₂CH=CH-C00R² groups, being R² more in particular hydrogen (H). In this particular embodiment the blocks are modified with maleic acid (CC>2H-CH=CH-COOH) and the block copolymer comprises carboxylic groups; thus, it is a carboxylated block copolymer.

In these particular cases where the block copolymer comprises carboxylic groups, the examples below show two possible methods for the functionalization of the copolymer. In particular, the two methods include Jones oxidation method (see Dual-functional c(RGDyK)-decorated Pluronic micelles designed for antiangiogenesis and the treatment of drug-resistant tumor. Int J Nanomedicine, 2015. 10: p. 4863-81 ); and the Maleic anhydride method (see Pluronic F127 nanomicelles engineered with nuclear localized functionality for targeted drug delivery. Mater Sci Eng C Mater Biol Appl, 2013. 33(5): p. 2698-707).

In these embodiment encompassing modified polyoxyethylene and/or polyoxypropylene units, the polymeric micelle comprises a mixture of a non-modified amphiphilic block copolymer of polyoxyethylene and polyoxypropylene blocks and a modified amphiphilic block copolymer of polyoxyethylene and polyoxypropylene blocks. The polymeric micelles comprising amphiphilic block copolymers of polyoxyethylene and polyoxypropylene blocks modified with the above indicated functional groups (-COOR²; - COR¹; -SR⁴; -NR³ R⁵ ) are, from the outer surface of the micelles facing the solvent (aqueous media) to their inner core, structurally defined by: an external or outer surface comprising the polyoxyethylene and/or polyoxypropylene blocks of the block copolymer, which terminal monomer units are functionalized and are optionally coupled to a targeting moiety; a layer or spatial zone comprising the non-modified polyoxyethylene blocks of the block copolymer and the gelatin; and an inner core comprising the polyoxypropylene and/or non-modified polyoxyethylene blocks of the block copolymer.

In a more particular embodiment, optionally in combination with any embodiment above or below, the weight ratio of non-modified block copolymer and modified block copolymer in the polymeric micelle is from 4:1 to 1 :1 w/w. More in particular is 4:1 w/w.

In a particular embodiment of the polymeric micelle comprising a mixture of a non- modified amphiphilic block copolymer and a modified amphiphilic block copolymer, the terminal units of the polyoxyethylene and/or polyoxypropylene blocks are modified and comprise the functional groups selected from: - COOR², being R² selected from H and - (CrC₈)-alkyl; -COR¹, wherein R¹ is a compound of formula -Z-COOR², being -Z- selected from (CH2)n with n an integer from 1 to 10, (CH=CH-CH)m with m an integer from 1 to 3, (CH2-CH=CH), and (CH=CH)rwith r an integer from 1 to 2; and wherein the weight ratio of non-modified block copolymer and modified block copolymer in the micelle is from 4:1 to 1 :1 w/w. More in particular is 4:1 w/w. In a more particular embodiment, the terminal units of the polyoxyethylene block are modified and comprise the functional group -COR¹, wherein R¹ is -Z-COOR², being -Z- (CH=CH)r and r is 1.

In the particular case of carboxylated block copolymers, certain cytotoxicity was previously reported by other authors, which made micelles with -COOH groups not usable for the delivery of drugs. However, inventors have surprisingly found that the polymeric micelles of the invention, comprising gelatin and the amphiphilic block copolymer, could further comprise certain amounts of carboxylated block copolymer (i.e. terminal carboxylated oxyethylene and/or oxypropylene monomer units in the polyoxyethylene or

polyoxypropylene chains or blocks) and the inherent cytotoxicity due to the additional carboxylic groups was undermined due to the presence of gelatin. These data are depicted in FIG. 4, wherein cell viability (in percentage %) is depicted for polymeric micelles comprising carboxylated and no-carboxylated block copolymers of

polyoxyethylene and polyoxypropylene blocks in a weight ratio of non- carboxylated:carboxylated copolymer of 8:2, said micelles comprising gelatin (PM

F127:COOH 8:2:Gelatin) and without gelatin (PM F127:COOH 8:2). This reduction of cytotoxicity makes the polymeric micelles able for use in any assay with cells or in the treatment of diseases, while avoiding by-side effects. A reduction of cytotoxicity in the presence of gelatin was not expected and surprising. Without being bound to any theory inventors propose that gelatin imbalance negative charge when disposed in between the polyoxyethylene blocks in the corona of the micelle, and this makes reactive -COOH groups not so accessible.

In another particular embodiment, optionally in combination with any embodiments above or below, the amphiphilic block copolymer is selected from sequential di- and triblock- copolymers, grafted di- and triblock-copolymers, and mixtures thereof.

In another particular embodiment, the molecular weight of the polyoxyethylene block in the block copolymer is from 100 to 7000 Daltons (Da). More in particular, it is from 1000 to 5000 Da.

In another particular embodiment, the molecular weight of the polyoxypropylene block in the block copolymer is from 950 to 4000 Daltons.

In a more particular embodiment of the first aspect, optionally in combination with any embodiment above or below, the polymeric micelle comprises an amphiphilic block copolymer, wherein the amphiphilic block copolymer is a sequential triblock-copolymer of formula (I):

wherein a is an integer from 2 to 150 and b is an integer from 15-67;

R^x is selected from H; COR¹, wherein R¹ is a compound of formula -Z-COOR², being R² selected from H and -(CrC₈)-alkyl, being -Z- selected from (CH2)n with n an integer from 1 to 10, (CH=CH-CH)m with m an integer from 1 to 3, (CH2-CH=CH), and (CH=CH)rwith r an integer from 1 to 2; and combinations thereof.

In these sequential triblock-copolymers of formula (I), the oxyethylene terminal monomer units of the polyoxyethylene blocks are, optionally, modified and comprise functional groups capable forming an amide bond due to the presence of carboxylic groups or of groups able to give free reactive carboxylic groups.

In another more particular embodiment of these polymeric micelles comprising a mixture of modified and non-modified terminal units of the polyoxyethylene block in the block copolymer of formula (I), the functional group is selected from: - COOR², being R² selected from H and -(Ci-C₈)-alkyl; -COR¹, wherein R¹ is a compound of formula -Z- COOR², being -Z- selected from (CH2)n with n an integer from 1 to 10, (CH=CH-CH)m with m an integer from 1 to 3, (CH2-CH=CH), and (CH=CH)rwith r an integer from 1 to 2; and wherein the weight ratio of non-modified block copolymer and modified block copolymer in the micelle is from 4:1 to 1 :1 w/w. More in particular is 4:1 w/w. In a more particular embodiment, the oxyethylene terminal monomer units of the polyoxyethylene block are modified and comprise the functional group -COR¹, wherein R¹ is -Z-COOR², being -Z- (CH=CH)r and r is 1.

In another particular embodiment of the polymeric micelles, optionally in combination with any embodiments above or below, the non-modified amphiphilic block copolymer of formula (I) and the modified amphiphilic block copolymer of formula (I) have identical respectively a and b values. That is, in both a is the same and it is an integer from 25-150 and b is the same and it is an integer from 30-60. More in particular, in both modified and non-modified copolymers of formula (I) a is 101 and b is 56.

Other suitable sequential triblock-copolymers of polyoxyethylene and polyoxypropylene blocks can be used for forming the polymeric micelles of the invention. Thus, in other particular embodiments, the polymeric micelle comprises an amphiphilic block copolymer, wherein the amphiphilic block copolymer is a sequential triblock-copolymer of formula (II):

(II),

wherein R^y is independently selected from NR³R⁵ being R³ and R⁵ independently selected from H and -(Ci-C₈)-alkyl, or R³ and R⁵ conform together with the nitrogen atom a cyclic compound, wherein optionally one or more hydrogen atoms have been substituted by oxygen atoms (i.e. N-hydroxysuccinimide); SR⁴, being R⁴ selected from H and -(CrC₈)- alkyl; and combinations thereof; being a’ and b integers as defined by a and b in compound of formula (I), or, when compound of formula (II) is obtainable from compound of formula (I) when R^x is H, the a’ have a value of a-1.

In yet another particular embodiment, the polymeric micelle comprises an amphiphilic block copolymer, wherein the amphiphilic block copolymer is a sequential triblock- copolymer of formula (III):

wherein R^z is OR⁶ being R⁶ selected from H, -(CrC₈)-alkyl; and combinations thereof; being a’ and b integers as defined by a and b in compound of formula (I), or, when compound of formula (II) is obtainable from compound of formula (I) when R^x is H, the a’ have a value of a-1. These sequential amphiphilic triblock-copolymers of formulas (I), (II) and (III) above are also known as poloxamers. As above exposed poloxamers are non-ionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene

(polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene

(poly(ethylene oxide)).

In another particular embodiment, optionally in combination with any embodiments above or below, a is an integer from 25-150 and b is an integer from 30-60. More in particular, a is 101 and b is 56. This particular triblock-copolymer of formula (I) with a = 101 and b = 56 is known also as poloxamer 407, also known by the commercial trade name Pluronic® F127, which has a CMC of 2.8 X 10 ⁶ M. This poloxamer has a HLB index from 18 to 23

(HLB=18-23).

In a more particular embodiment of the polymeric micelles of the invention, the poloxamers are selected from the group consisting of poloxamer 407, poloxamer 188, and poloxamer 338.

Poloxamer 188, also known by the commercial trade name Pluronic® F68 has a CMC of 4.8 X 10⁴ M. This poloxamer has a HLB higher than 24 (HLB>24). In poloxamer 188 a is 80 and b is 27, when referring to formula (I). Poloxamer 338 also known by the commercial trade name Pluronic® F108 has a CMC of 2.2 X 10⁵ M. This poloxamer has a HLB higher than 24 (HLB>24). In poloxamer 188 a is 141 and b is 44, when referring to formula (I).

Other commercial names of these poloxamers are Kolliphor, Lutrol, Pluracare, Antarox.

In a more particular embodiment, the polymeric micelles of the invention comprise:

- gelatin;

- an amphiphilic sequential triblock copolymer of formula (I), which is a non-modified triblock copolymer in which R^x is H and wherein a is an integer from 2 to150 and b is an integer from 15-67

(I),

- a modified amphiphilic triblock copolymer of formula (I), in which R^x is COR¹, wherein R¹ is a compound of formula -Z-COOR², being R² selected from H and -(CrC₈)-alkyl, being - Z- selected from (CH2)n with n an integer from 1 to 10, (CH=CH-CH)m with m an integer from 1 to 3, (CH2-CH=CH), and (CH=CH)rwith ran integer from 1 to 2;

- one or more therapeutic agents, in which at least one of the therapeutic agent is selected from the group consisting of small interfering RNA (siRNA), micro RNA (miRNA), short hairpin RNA (shRNA), and combinations thereof; and wherein:

- the weight ratio of non-modified tri block copolymer and modified triblock copolymer in the micelle is from 4:1 to 1 :1 w/w;

- the weight ratio of the total of modified and non-modified sequential triblock copolymer and of gelatin in the micelle is of 10:1 to 5:5 w/w;

- the polyoxypropylene blocks of the modified and non-modified triblock copolymers conform the inner side of the micelle, and the polyoxyethylene blocks of the modified and non-modified triblock copolymers and the gelatin conform the outer side of the micelle; and

- the modified block copolymer comprises one or more modified oxyethylene terminal monomer units coupled to an antibody that recognizes mammal epidermal growth factor receptor.

In a more particular embodiment of the polymeric micelles comprising a mixture of modified and non-modified amphiphilic block copolymers of formula (I), the modified amphiphilic triblock copolymer is of formula (I) in which R^x is COR¹ wherein R¹ is -Z- COOR², being -Z- (CH=CH)r and r is 1. In yet a more particular embodiment, integers a and b in the modified and non-modified amphiphilic block copolymers of formula (I) are identical. Identical integers a and b for the modified and non-modified amphiphilic block copolymers of formula (I) can be achieved, as a way of example, when optionally the modified block copolymer is obtainable from the non-modified copolymer of formula (I) in which R^x is H.

In a more particular embodiment of the polymeric micelles comprising a mixture of modified and non-modified amphiphilic block copolymers of formula (I), optionally in combination with any embodiment above or below, the weight ratio of non-modified block copolymer and modified block copolymer in the micelle is from 4:1 w/w to 1 :1 w/w. More in particular is 4:1 w/w.

The polymeric micelles according to this first aspect have, in a particular embodiment, a hydrodynamic diameter from 100 to 300 nm, more in particular from 110 to 250 nm.

In another particular embodiment of the first aspect of the invention, the polymeric micelles are obtainable by a method comprising the following steps:

(a) weighing the block copolymer(s) and dissolving it (them) in an organic solvent to obtain a solution with the block copolymer(s);

(b) removing the solvent by evaporation and/or by air-drying, to obtain a film of the block copolymer(s);

(c) hydrating the film of step (b) with an aqueous solution comprising gelatin to obtain the polymeric micelles in aqueous media.

Hydration of the film of step (b) gives rise to an aqueous composition comprising the self- assembled polymeric micelles of the invention.

In a particular embodiment, the organic solvent of step (a) is a mixture of organic solvents. More in particular, it is a mixture of two solvents selected from the group consisting of methanol, ethanol, dicloromethane, chloroform. More in particular is a mixture of methanol and ethanol in a volume ratio 1 :1 (v/v). In another particular embodiment of the first aspect of the invention, the modified block copolymer, when present in the polymeric micelle, comprises oxyethylene and/or oxypropylene terminal monomer units of the end blocks that are coupled to one or more targeting moiety capable of conducting the polymeric micelle to particular plant or animal cells and/or cell compartments (such as cell organelles), said cells able to recognize said targeting moiety. These polymeric micelles are referred in this description as targeted polymeric micelles. They are also an example of multifunctional delivery system. These particular targeted polymeric micelles with a targeting moiety, as disclosed above, are obtainable by the method comprising steps (a) to (c) as previously disclosed, and further comprising a step (d) of incubating said targeting moiety with the polymeric micelles of step (c). Particular incubation times will depend on the nature of the targeting moiety as well as on the desired degree of targeting moieties in the polymeric micelle.

Thus, the invention also relates to a targeted polymeric micelle obtainable by the above- mentioned method with steps (a) to (d).

In a particular embodiment of this method for obtaining targeted polymeric micelles, step (d) of incubating the targeting moiety with the polymeric micelles is carried out by:

(i) first activating the functional groups of the terminal units in the modified block copolymer to obtain an activated polymeric micelle; and

(ii) contacting the activated polymeric micelle with a targeting moiety to obtain a targeted polymeric micelle.

In the particular case when the functional group of the terminal monomer units of the polyoxyethylene and/or polyoxypropylene block is a -COOR², or a -COR¹ (i.e. -COOH), the step of (i) activating is carried out with a compound selected from carbodiimide or 1- ethyl-3-(3-dimethylaminopropyl)-carbodiimide, optionally accompanied with N- hydroxybenzotriazole or N-hydroxysuccinimide, to obtain an activated polymeric micelle; and (ii) contacting the activated polymeric micelle with a targeting moiety to obtain a targeted polymeric micelle, wherein the targeting moiety is coupled by an amide bond to the terminal units of the modified polyoxyethylene blocks of the polymeric micelle. All these methods can be performed, in a particular embodiment, in a one-pot process. The skilled man will know the protocols for obtaining amide and/or ester bonds using carbodiimide or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide.

In a particular embodiment of the targeted polymeric micelles, the one or more targeting moiety is selected from the group consisting of an antibody or fragment of said antibody, a peptide, an oligonucleotide and combinations thereof.

Particular embodiments include antibodies that are selected from monoclonal and polyclonal antibodies. For a fragment of an antibody, being from a polyclonal or from a monoclonal antibody, is to be understood any of the F(ab), F(ab^') and Fv fragments.

In another particular embodiment, the targeting moiety is an antibody or fragment of an antibody which recognizes a mammal, more in particular human, epidermal growth factor receptor (EGFR).

Even in a more particular embodiment, the targeting moiety is an antibody selected from the group consisting of cetuximab, panitumumab and fragments of any of them, said fragments capable of recognizing the EGFR.

The targeted polymeric micelles of the invention are spherical when observed by transmission electron microscopy, and have a mean hydrodynamic diameter when measured by dynamic light scattering from 200 to 300 nm.

In yet another particular embodiment, the polymeric micelle according to the first aspect of the invention, is a drug-loaded polymeric micelle comprising one or more therapeutic agents.

In a more particular embodiment, the therapeutic agent is a negatively charged (i.e.

anionic) therapeutic agent.

Even in a more particular embodiment, the negatively charged therapeutic agent is selected from the group consisting of small interfering RNA (siRNA), micro RNA (miRNA), short hairpin RNA (shRNA), a DNA plasmid, an oligopeptide, a protein, and combinations thereof.

These particular drug-loaded polymeric micelles are obtainable by the method comprising steps (a) to (c) as previously disclosed, optionally further comprising the step (d) of incubating a targeting moiety with the polymeric micelles of step (c), and wherein in step (c) of hydrating the film of the block copolymer, the aqueous solution comprising gelatin further comprises a negatively charged therapeutic agent complexed with the gelatin (for example an oligonucleotide); and/or a further step (e) in which the polymeric micelles in aqueous media are added to a dehydrated film of the drug; and/or a further step (f) in which a drug is dissolved in an organic solvent and added dropwise to the polymeric micelle solution in water of step (c), and the said organic solvent is evaporated and the drug is incorporated into the micelle during said organic solvent evaporation.

In another particular embodiment the negatively charged therapeutic agent is a protein selected from an enzyme, an antibody, a cytokine, and combinations thereof.

For“oligopeptide” or“peptide” (used herewith interchangeably) it is encompassed any amino acid polymer of two to twenty amino acids and that include dipeptides, tripeptides, tetrapeptides, and pentapeptides. For“protein” is understood as encompassing biomolecule consisting of one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in protein folding into a specific three-dimensional structure that determines its activity. A linear chain of amino acid residues is called a polypeptide. A protein contains at least one long polypeptide. Short polypeptides, containing less than 20-30 residues, are rarely considered to be proteins and are commonly called peptides, or sometimes oligopeptides as above indicated. Examples of proteins with multiple polypeptide chains include antibodies. For“antibody” (abbreviated Ab), also known as an immunoglobulin (Ig), is to be understood is a large, Y-shaped protein produced mainly by plasma cells that is used by the immune system to neutralize pathogens such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the pathogen, called an antigen, via the Fab's variable region. Each tip of the "Y" of an antibody contains a paratope (analogous to a lock) that is specific for one particular epitope (similarly analogous to a key) on an antigen, allowing these two structures to bind together with precision.

“Oligonucleotides” are short DNA or RNA molecules, oligomers, from 4 to 300 nucleotide units, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids can be manufactured as single-stranded molecules with any user-specified sequence, and so are vital for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, library construction and as molecular probes. According to this invention, the term includes small interfering RNA (siRNA), micro RNA (miRNA), short hairpin RNA (shRNA), and DNA primers. In nature, oligonucleotides are usually found as small RNA molecules that function in the regulation of gene expression (e.g. microRNA), or are degradation intermediates derived from the breakdown of larger nucleic acid molecules. “DNA plasmids (or simply plasmids)” are small DNA molecule within a cell that are physically separated from a chromosomal DNA and can replicate independently. They are most commonly found in bacteria as small circular, double-stranded DNA molecules; however, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids often carry genes that may benefit the survival of the organism, for example antibiotic resistance. While the chromosomes are big and contain all the essential genetic information for living under normal conditions, plasmids usually are very small and contain only additional genes that may be useful to the organism under certain situations or particular conditions. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. Artificially constructed plasmids may be used as vectors in genetic engineering. These plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to clone and amplify (make many copies of) or express particular genes. A wide variety of plasmids are commercially available for such uses. The gene to be replicated is normally inserted into a plasmid that typically contains a number of features for their use. These include a gene that confers resistance to particular antibiotics (ampicillin is most frequently used for bacterial strains), an origin of replication to allow the bacterial cells to replicate the plasmid DNA, and a suitable site for cloning (referred to as a multiple cloning site).

“Cytokines” are a broad and loose category of small proteins (-5-20 kDa) that are important in cell signalling. Their release has an effect on the behaviour of cells around them. It can be said that cytokines are involved in autocrine signalling, paracrine signalling and endocrine signalling as immunomodulating agents. Their definite distinction from hormones is still part of ongoing research. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumour necrosis factors but generally not hormones or growth factors (despite some overlap in the terminology). Cytokines are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells; a given cytokine may be produced by more than one type of cell. They act through receptors, and are especially important in the immune system; cytokines modulate the balance between humoral and cell-based immune responses, and they regulate the maturation, growth, and responsiveness of particular cell populations. Some cytokines enhance or inhibit the action of other cytokines in complex ways.

In the particular case of oligonucleotides, the polymeric micelles of the invention comprise the said oligonucleotides associated with gelatin. Interaction of both is mainly performed by means of ionic bonds, although other forces such as hydrogen bonds and van der Waals forces do also take place. The polymeric micelles of the invention with one or more therapeutic agents comprise, in another particular embodiment, one or more therapeutic agents in the inner side or core of said polymeric micelle, said core conformed by the polyoxypropylene blocks of the copolymer .

In a particular embodiment, the therapeutic agent is selected from analgesics, anti- inflammatory agents, anthelminthic, anti-arrhythmic agents, anti-bacterial agents, anti-viral agents, anti-coagulants, anti-depressants, anti-diabetics, anti-epileptics, anti-fungal agents, anti-gout agents, anti-hypertensive agents, anti-malarials, anti-migraine agents, anti-muscarinic agents, anti-neoplastic agents, erectile dysfunction improvement agents, immunosuppressants, anti-protozoal agents, anti-thyroid agents, anxiolytic agents, sedatives, hypnotics, neuroleptics, b-blockers, cardiac inotropic agents, corticosteroids, diuretics, anti-parkinsonian agents, gastro-intestinal agents, histamine receptor antagonists, keratolyptics, lipid regulating agents, anti-anginal agents, Cox-2 inhibitors, leukotriene inhibitors, macrolides, muscle relaxants, nutritional agents, opiod analgesics, protease inhibitors, sex hormones, stimulants, muscle relaxants, anti-osteoporosis agents, anti-obesity agents, cognition enhancers, anti-urinary incontinence agents, anti-benign prostate hypertrophy agents, essential fatty acids, non-essential fatty acids, and mixtures thereof. In a more particular embodiment, the hydrophobic therapeutic agent is selected from acetretin, albendazole, albuterol, aminoglutethimide, amiodarone, amlodipine, amphetamine, amphotericin B, atorvastatin, atovaquone, azithromycin, baclofen, beclomethasone, benezepril, benzonatate, betamethasone, bicalutanide, budesonide, bupropion, busulfan, butenafine, calcifediol, calcipotriene, calcitriol, camptothecin, candesartan, capsaicin, carbamezepine, carotenes, celecoxib, cerivastatin, cetirizine, chlorpheniramine, cholecalciferol, cilostazol, cimetidine, cinnarizine, ciprofloxacin, cisapride, clarithromycin, clemastine, clomiphene, clomipramine, clopidogrel, codeine, coenzyme Q10, cyclobenzaprine, cyclosporin, danazol, dantrolene, dexchlorpheniramine, diclofenac, dicoumarol, digoxin, dehydroepiandrosterone, dihydroergotamine,

dihydrotachysterol, dirithromycin, donezepil, efavirenz, eprosartan, ergocalciferol, ergotamine, essential fatty acid sources, etodolac, etoposide, famotidine, fenofibrate, fentanyl, fexofenadine, finasteride, fluconazole, flurbiprofen, fluvastatin, fosphenytoin, frovatriptan, furazolidone, gabapentin, gemfibrozil, glibenclamide, glipizide, glyburide, glimepiride, griseofulvin, halofantrine, ibuprofen, irbesartan, irinotecan, isosorbide dinitrate, isotretinoin, itraconazole, ivermectin, ketoconazole, ketorolac, lamotrigine, lansoprazole, leflunomide, lisinopril, loperamide, loratadine, lovastatin, L-thryroxine, lutein, lycopene, medroxyprogesterone, mifepristone, mefloquine, megestrol acetate,

methadone, methoxsalen, metronidazole, miconazole, midazolam, miglitol, minoxidil, mitoxantrone, montelukast, nabumetone, nalbuphine, naratriptan, nelfinavir, nifedipine, nilsolidipine, nilutanide, nitrofurantoin, nizatidine, omeprazole, oprevelkin, oestradiol, oxaprozin, paclitaxel, paracalcitol, paroxetine, pentazocine, pioglitazone, pizofetin, pravastatin, prednisolone, probucol, progesterone, pseudoephedrine, pyridostigmine, rabeprazole, raloxifene, rofecoxib, repaglinide, rifabutine, rifapentine, rimexolone, ritanovir, rizatriptan, rosiglitazone, saquinavir, sertraline, sibutramine, sildenafil citrate, simvastatin, sirolimus, spironolactone, sumatriptan, tacrine, tacrolimus, tamoxifen, tamsulosin, targretin, tazarotene, telmisartan, teniposide, terbinafine, terazosin, tetrahydrocannabinol, tiagabine, ticlopidine, tirofibran, tizanidine, topiramate, topotecan, toremitfene, tramadol, tretinoin, troglitazone, trovafloxacin, ubidecarenone, valsartan, venlafaxine, verteporfin, vigabatrin, vitamin A, vitamin D, vitamin E, vitamin K, zafirlukast, zileuton, zolmitriptan, zolpidem, zopiclone, pharmaceutically acceptable salts, isomers, and derivatives thereof, and mixtures thereof.

In yet a more particular embodiment the drug-loaded polymeric micelle of the invention comprises one or more antiproliferative or chemotherapeutic agents selected from

Abarelix, aldesleukin, Aldesleukin, Alemtuzumab, Alitretinoin, Allopurinol, Altretamine, Amifostine, Anastrozole, Arsenic trioxide, Asparaginase, Azacitidine, BCG Live,

Bevacuzimab, Avastin, Fluorouracil, Bexarotene, Bleomycin, Bortezomib, Busulfan, Calusterone, Capecitabine, Camptothecin, Carboplatin, Carmustine, Celecoxib,

Cetuximab, Chlorambucil, Cisplatin, Cladribine, Clofarabine, Cyclophosphamide,

Cytarabine, Dactinomycin, Darbepoetin alfa, Daunorubicin, Denileukin, Dexrazoxane, Docetaxel, Doxorubicin (neutral), Doxorubicin hydrochloride, Dromostanolone Propionate, Epirubicin, Epoetin alfa, Erlotinib, Estramustine, Etoposide Phosphate, Etoposide, Exemestane, Filgrastim, floxuridine fludarabine, Fulvestrant, Gefitinib, Gemcitabine, Gemtuzumab, Goserelin Acetate, Histrelin Acetate, Hydroxyurea, Ibritumomab, Idarubicin, Ifosfamide, Imatinib Mesylate, Interferon Alfa-2a, Interferon Alfa-2b, Irinotecan,

Lenalidomide, Letrozole, Leucovorin, Leuprolide Acetate, Levamisole, Lomustine, Megestrol Acetate, Melphalan, Mercaptopurine, 6-MP, Mesna, Methotrexate,

Methoxsalen, Mitomycin C, Mitotane, Mitoxantrone, Nandrolone, Nelarabine,

Nofetumomab, Oprelvekin, Oxaliplatin, Paclitaxel, Palifermin, Pamidronate, Pegademase, Pegaspargase, Pegfilgrastim, Pemetrexed Disodium, Pentostatin, Pipobroman,

Plicamycin, Porfimer Sodium, Procarbazine, Quinacrine, Rasburicase, Rituximab, Sargramostim, Sorafenib, Streptozocin, Sunitinib Maleate, Talc, Tamoxifen,

Temozolomide, Teniposide, VM-26, Testolactone, Thioguanine, 6-TG, Thiotepa,

Topotecan, Toremifene, Tositumomab, Trastuzumab, Tretinoin, ATRA, Uracil Mustard, Valrubicin, Vinblastine, Vincristine Vinorelbine, Zoledronate, or Zoledronic acid.

The polymeric micelles of the invention can be used for encapsulating and delivering any therapeutic agent, independently of its molecular size, since according to its nature they will assemble within the polymeric micelles by means of hydrophobic-hydrophilic interactions, electrostatic interactions, or by covalent binding to the block copolymer chemically modified (e-g. functionalized as above indicated). Indeed, in the particular case when the targeting moiety is an antibody capable of recognizing EGFR, even the targeting moiety is acting as therapeutic agent.

Particular drug-loaded polymeric micelles of the invention are polymeric micelles comprising:

- gelatin;

- an amphiphilic sequential triblock copolymer of formula (I), which is a non-modified triblock copolymer in which R^x is H and wherein a is an integer from 2 to150 and b is an integer from 15-67;

(I),

- a modified amphiphilic triblock copolymer of formula (I), in which R^x is COR¹, wherein R¹ is a compound of formula -Z-COOR², being R² selected from H and -(Ci-C₈)-alkyl, being - Z- selected from (CH2)n with n an integer from 1 to 10, (CH=CH-CH)m with m an integer from 1 to 3, (CH2-CH=CH), and (CH=CH)rwith ran integer from 1 to 2;

- the polyoxypropylene blocks of the modified and non-modified triblock copolymers conform the inner side of the micelle, and the polyoxyethylene blocks of the modified and non-modified triblock copolymers and the gelatin conform the outer side of the micelle; and - the modified block copolymer comprises modified oxyethylene terminal monomer units coupled to an antibody that recognizes mammal epidermal growth factor receptor.

In a more particular embodiment of the drug-loaded polymeric micelles comprising a mixture of modified and non-modified amphiphilic block copolymers of formula (I), the modified amphiphilic triblock copolymer is of formula (I) in which R^x is COR¹ wherein R¹ is -Z-COOR², being -Z- (CH=CH)r and r is 1 .

In another particular embodiment of the drug-loaded micelle, optionally in combination with any embodiments above or below, a is an integer from 25-150 and b is an integer from 30-60. More in particular, a is 101 and b is 56. In yet a more particular embodiment, the non-modified amphiphilic block copolymer of formula (I) and the modified amphiphilic block copolymer obtainable from that of formula (I) have identical respectively a and b values. That is, in both a is the same and it is an integer from 25-150 and b is the same and it is an integer from 30-60. More in particular, in both modified and non-modified copolymers of formula (I) a is 101 and b is 56. Identical integers a and b for the modified and non-modified amphiphilic block copolymers of formula (I) can be achieved when optionally the modified block copolymer is obtainable from the non-modified copolymer of formula (I) in which R^x is H.

The pharmaceutical compositions according to the invention are obtained in a process in which a therapeutically effective amount of a drug-loaded polymeric micelle as defined above is mixed with the rest of the excipients and/or carriers.

In a more particular embodiment, the pharmaceutical composition comprises a polymeric micelle, wherein the micelle is a drug-loaded micelle and comprises:

- gelatin;

(I),

- the modified block copolymer comprises modified oxyethylene terminal monomer units coupled to an antibody that recognizes mammal epidermal growth factor receptor. In yet a more particular embodiment of pharmaceutical composition of the invention, the drug-loaded polymeric micelles comprising a mixture of modified and non-modified amphiphilic block copolymers of formula (I), comprises a modified amphiphilic triblock copolymer of formula (I), in which R^x is COR¹ wherein R¹ is -Z-COOR², being -Z- (CH=CH)r and r is 1. In another particular embodiment of the pharmaceutical composition, optionally in combination with any embodiments above or below, a is an integer from 25- ISO and b is an integer from 30-60. More in particular, a is 101 and b is 56.

In another particular embodiment, the non-modified amphiphilic block copolymer of formula (I) and the modified amphiphilic block copolymer of formula (I) have identical respectively a and b values. That is, in both a is the same and it is an integer from 25-150 and b is the same and it is an integer from 30-60. More in particular, in both modified and non-modified copolymers of formula (I) a is 101 and b is 56.

The drug-loaded polymeric micelles or any pharmaceutical composition comprising them can be used as medicaments. In a particular embodiment, the medicament is for use in the treatment of cancer. More in particular is for the treatment of epithelial cancers in which mutations, amplifications or misregulations of EGFR or family members are implicated; Non-limitative examples of cancer are selected from breast cancer, colon cancer, including metastatic colon cancer, squamous-cell carcinoma of the lung, anal cancers, glioblastoma and head and neck cancer.

Inventors also detected that the polymeric micelles of the invention were able to pass through the blood-brain barrier (BBB), thus they can be targeted to cerebral tumours of any aetiology.

The invention encompasses also as a multifunctional delivery system for the delivery of one or more compounds to plant or animal cells, comprising a polymeric micelle as defined above in any of the first or second aspects.

In a particular embodiment, the multifunctional (multicompound) delivery system is a nucleic acid delivery system for the delivery of nucleic acids to plant or animal cells, and comprises a polymeric micelle as defined in any of the aspects and in any of the particular embodiments above, wherein the polymeric micelle further comprises an effective amount of a nucleic acid associated with the gelatin of the polymeric micelle.

As above indicated the nucleic acid, in particular oligonucleotides, are complexed with the gelatin due to the opposed ionic charges they have (i.e. cationic charge of the gelatin and anionic charge of the nucleic acid).

Inventors have detected that the polymeric micelles according to the invention are internalized in high amounts by cancer stem cells. Thus, drug-loaded polymeric micelles are, in another particular embodiment for use in treating cancer, wherein the treatment comprises targeting said cancer stem cells of any tumour type.

Cancer stem cells (CSCs) are cancer cells (found within tumors or hematological cancers) that possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample. CSCs are therefore tumorigenic (tumor-forming), perhaps in contrast to other non-tumorigenic cancer cells. CSCs may generate tumors through the stem cell processes of self-renewal and differentiation into multiple cell types. Such cells are hypothesized to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Therefore, development of specific therapies targeted at CSCs holds hope for

improvement of survival and quality of life of cancer patients, especially for patients with metastatic disease. The efficacy of cancer treatments is, in the initial stages of testing, often measured by the ablation fraction of tumor mass (fractional kill). As CSCs form a small proportion of the tumor, this may not necessarily select for drugs that act specifically on the stem cells. The theory suggests that conventional chemotherapies kill differentiated or differentiating cells, which form the bulk of the tumor but do not generate new cells. A population of CSCs, which gave rise to it, could remain untouched and cause relapse.

Thus, in a particular embodiment the drug-loaded polymeric micelles according to the invention are for use in treating cancer, said cancer treatment comprising targeting cancer stem cells of a tumour. The drug-loaded polymeric micelles are, therefore, for use in the treatment of cancers in which cancer stem cells are involved in the metastatic processes of the said cancer.

In a more particular embodiment the drug-loaded polymeric micelles are for use in the treatment of cancer, wherein the cancer stem cells are cancer stem cells of a colon tumour (or, in other words, colonic cancer stem cells).

In yet a more particular embodiment, the drug-loaded polymeric micelles for use in the treatment of cancers by targeting cancer stem cells, the drug-loaded polymeric micelle comprises:

- gelatin;

(I),

- a modified amphiphilic triblock copolymer of formula (I), in which R^x is COR¹, wherein R¹ is a compound of formula -Z-COOR², being R² selected from H and -(CrC₈)-alkyl, being - Z- selected from (CH2)n with n an integer from 1 to 10, (CH=CH-CH)m with m an integer from 1 to 3, (CH2-CH=CH), and (CH=CH)rwith ran integer from 1 to 2; - one or more therapeutic agents, in which at least one of the therapeutic agent is selected from the group consisting of small interfering RNA (siRNA), micro RNA (miRNA), short hairpin RNA (shRNA), and combinations thereof; and wherein:

- the weight ratio of non-modified triblock copolymer and modified triblock copolymer in the micelle is from 4:1 to 1 :1 w/w;

- optionally, the modified block copolymer comprises modified oxyethylene terminal monomer units coupled to a targeting moiety that recognizes a CSC-surface marker.

In another particular embodiment of the drug-loaded polymeric micelles for use in the treatment of cancers by targeting cancer stem cells, optionally in combination with any embodiments above or below, a is an integer from 25-150 and b is an integer from 30-60. More in particular, a is 101 and b is 56. In yet a more particular embodiment, the non- modified amphiphilic block copolymer of formula (I) and the modified amphiphilic block copolymer obtainable from that of formula (I) have identical respectively a and b values. That is, in both a is the same and it is an integer from 25-150 and b is the same and it is an integer from 30-60. More in particular, in both modified and non-modified copolymers of formula (I) a is 101 and b is 56. This particular triblock-copolymer of formula (I) with a = 101 and b = 56 is known also as poloxamer 407, also known by the commercial trade name Pluronic® F127, which has a CMC of 2.8 X 10 ⁶ M. This poloxamer has a HLB index from 18 to 23 (HLB=18-23). Identical integers a and b for the modified and non-modified amphiphilic block copolymers of formula (I) can be achieved when optionally the modified block copolymer is obtainable from the non-modified copolymer of formula (I) in which R^x is H. Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps.

Furthermore, the word“comprise” encompasses the case of“consisting of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

Examples

Example 1. Process for obtaining polymeric micelles of Pluronic® F127 (abbreviated F127) and gelatin associated with siRNA and with cetuximab (Cet) as targeting moiety.

1. Materials and Methods

1.1. Materials

Pluronic® F127 was kindly provided by BASF (Ludwigshafen, Germany). Gelatin, protamine, histamine, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) and 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF) were purchased from Sigma- Aldrich (Madrid, Spain), while PEI was purchased from Alfa Aesar (Madrid, Spain). RPMI medium, phosphate buffered saline (PBS), and fetal bovine serum (FBS) were purchase from Lonza (Barcelona, Spain). L-glutamine, non-essential amino acids, 10000 U/mL penicillin and 10000 g/ml_ streptomycin and 0.25% Trypsin-EDTA, were purchased from Gibco (Life Technologies Ltd., Madrid, Spain). Lipofectamine® 2000, Cell Mask® Red DND-99, DAPI, SYBR Green, and ProLong® Gold Antifade Mountant were purchased from Life Technologies Ltd., Madrid, Spain. siRNAs were designed by Shanghai Gene Pharma (Shanghai, China); sense siAKT2 sequence used was 5’- GCUCCUUCAUUGGGUACAATT-3’ (SEQ ID NO: 1 ), while a non-specific sequence 5’- UUCUCCGAACGUGUCACGUTT-3’ (SEQ ID NO: 2) was used as control (siC). Organic solvents were obtained from Panreac (Madrid, Spain). Other reagents such as Cr03, (1- ethyl-3-(3-dimethylaminopropyl)-carbodiimide) (EDC), maleic anhydride, sulfuric acid, sodium hydroxide, and hydrochloric acid, dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (Madrid, Spain). For all the experiments it was used Type 1 ultrapure water (18.2 MO.cm at 25 °C, Milli-Q®, Billerica, MA, USA).

1.2. Methods

1.2.1. Jones oxidation method for modification of Pluronic® F127 (F127)

F127 was dissolved in acetone under magnetic stirring at room temperature (RT) for 1 h. For the Jones oxidation method, Jones Reagent (JR) was prepared with H2S04 and Cr03 (0.02M) a fixed Cr03/H2S04 molar ratio of 0.5 at desired volume according to the literature (see Chen, Y., et al., Dual-functional c(RGDyK)-decorated Pluronic micelles designed for antiangiogenesis and the treatment of drug-resistant tumor. Int J

Nanomedicine, 2015. 10: p. 4863-81 ). The calculated volume of JR was added to the F127 solution and the reaction was kept under magnetic stirring at RT overnight. The reaction was finished by the addition of the calculated volume of isopropyl alcohol. Then, isopropyl alcohol and acetone were evaporated and the aqueous product was extracted by CHCI3-diethyl ether extraction. The carboxyl-terminated product (F127:COOH) was obtained after filtration under vacuum followed by drying for 48 hours.

1.2.2. Maleic anhydride method for modification of Pluronic® F127 (F127)

The correct stoichiometry for this reaction was calculated from the literature (see Li, Y,Y., et al. Pluronic F127 nanomicelles engineered with nuclear localized functionality for targeted drug delivery. Mater Sci Eng C Mater Biol Appl, 2013. 33(5): p. 2698-707), being 11 the ratio of maleic anhydride over polymer. F127 and maleic anhydride were dissolved in distilled CHCI3 and the solution was allowed to react for 24h under stirring at 70 °C in a condensation system to avoid any loss of solvent. The solution was concentrated and poured twice into an excess amount of iced cold diethyl ether to precipitate the reaction product. Then, F127-COOH was dried under a vacuum dehydration and collected as white powder.

1.2.3. Polymers characterization

After synthesis each batch of polymers were characterized through Fourier Transform- Infrarred (FT-IR) and Proton Nuclear Magnetic Ressonance (¹H-NMR) analysis. FT-IR was carried out in Characterization of Soft-Materials Services at Institut de Ciencia de Materials de Barcelona (ICMAB-CSIC) using a spectrometer Perkin-Elmer Spectrum One (energy range: 450-4000 cm ¹) equipped with a Universal Attenuated Total Reflectance accessory (U-ATR).

¹H-NMR analyses have been carried out in Nuclear magnetic resonance service (SeRMN) of the Universitat Autonoma de Barcelona (UAB) using a spectrometer

Avancelll-400nb.

1.2.4. Production of polymeric micelles (PM)

PM were prepared using the film hydration technique. Briefly, the polymer was individually weighted and dissolved in a mixture of methanokethanol (1 :1 ). Then, the solvent was removed under vacuum in a rotary evaporator and the formed film was left to dry overnight at RT to eliminate any remaining solvent. The film was then hydrated with H₂0 or the previously prepared polyplexes formed between gelatin and a siRNA to prepare empty and loaded micelles, respectively, and vortexed during 5 minutes. For the PM functionalization with cetuximab (Cet) (Erbitux®), an adequate amount of EDC was incubated with the formulation. After 30 minutes of incubation at RT, the Cet solution was added and incubated under stirring 2 hours at RT. Samples were freeze-dried for long- term storage and characterization using a VirTis BenchTop Freeze-Dryer from SP Scientific.

1.2.5. Micelles physicochemical characterization

Particles mean hydrodynamic diameter (md) and polydispersity index (Pdi) were measured by dynamic light scattering (DLS) and zeta potential was assessed by laser doppler micro-electrophoresis using a NanoZS measurement range of 0.3 nm-10.0 microns and sensitivity of 0.1 mg/ml_ (Malvern Instruments, UK). For each formulation, at least three batches were produced and analyzed. Particle shape and morphology were observed by transmission electron microscopy (TEM) analyses performed at the Electron Microscopy Service at ICMAB, Barcelona using the 120 kV JEOL 1210 TEM, which have a resolution point of 3.2. Gatan software was used to process information and get measures form TEM images.

1.2.6. Entrapment efficiency

The non-associated siRNA present in the aqueous phase of the PM was separated by centrifugation with filtration (10,000 rpm, 10 minutes, 4°C) using centrifugal devices with a 100K membrane (Nanosep® Centrifugal Devices, Millipore, USA) and measured by a spectrophotometry method (Nanodrop NP-1000, Thermo Scientific, USA). The association efficiency was calculated according to Equation 1 :

AE = [(total amount of siRNA-free siRNA in filtrate) / (total amount of siRNA)] x 100

(Equation 1 )

1.2.7. Cell culture conditions

MDA-MB-231 (ATCC number HTB-26) and MCF-7 (ATCC number HTB-22) breast cancer cell lines were obtained from American Type Culture Collection (ATTC, LGC Standards, Barcelona, Spain). The cells were cultured in RPMI medium supplemented with 10% FBS, 1% penicillin-streptomycin, 1% L-glutamine, 1 % non-essential amino acids and 1 % of sodium pyruvate. The cells were maintained at 37°C under 5% C02 saturated atmosphere. The medium was changed every other day and, upon confluence, cells were harvested from plates with 0.25% trypsin-EDTA to be passed to other plates to continue expansion, be frozen or used in in vitro studies.

1.2.8. Cell Toxicity assays

In order to assess the effect of formulations on the viability of breast cancer cell lines, 5000 cells/well were seeded in 96-well plates and left to attach for 24 hours. Then, cells were incubated with increasing concentrations of different formulation complexes for 48 hours. Complete medium was used as negative control and 50% DMSO as positive control of toxicity. Cell viability was measured using the Presto Blue® (Thermo Fisher, Spain) reagent accordingly the manufacturer instructions. The absorbance of each well was read on an absorbance microplate reader ELx800 (BioTek, Germany), at 590 nm. The results of cell viability were used for the determination of IC50 index by nonlinear regression of the concentration-effect curve fit using Prism 6.02 software (GraphPad Software, Inc., CA, USA).

1.2.9. Assessment of polycations buffer capacity

To analyze the buffer capacity of the cationic components, including gelatin and predict their proton sponge effect and endosomal release capacity, the variation in the pH of the components solution in response to acid addition was determined. For that, the pH of cationic polymer aqueous solutions (10 mg/ml) were individually measured and set to values superior to 10 with NaOH 0.1 M. Then, the pH was measured after the addition drop by drop of HCI 0.1 M up to 2 ml_.

1.2.10. Silencing efficacy assays

The different PM-siRNA were transfected into MDA-MB-231 cells accordingly. 2x10⁵ cells were seeded in complete medium in 6 well plates for 24 hours to allow adhesion Different PM-siRNA formulations were transfected to MDA-MB-231 cells in order to obtain a final siRNA concentration in the well of 200 nM. The medium was changed after 4 hours of incubation with the PM. Cells were harvested 72 hours after the transfection.

Lipofectamine® 2000 (Life Technologies, Madrid, Spain) were used as a transfection positive control accordingly the supplier protocol. Cationic polymers were used at concentrations between 1 and 10 mg/ml in final formulation.

1.2.11. RNA Extraction and Quantitative RT-PCR (qRT-PCR) Total RNA was extracted from cells using the RNeasy Micro Kit (Qiagen, Madrid, Spain) and the obtained RNA was reverse transcribed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Madrid, Spain) accordingly to the manufacturer instructions. The cDNA reverse transcription product was amplified with specific primers for AKT2 (hAKT2 F: 5’ CAA GGA TGA AGT CGC TCA CAC A 3’ (SEQ ID NO: 3); hAKT2 R: 5’ GAA CGG GTG CCT GGT GTT C 3’ (SEQ ID NO: 4)) GAD PH (hGADPH F: 5’ ACC CAC TCC TCC ACCGAC 3’ (SEQ ID NO: 5); hGADPH R: 5’ CAT ACC AGG AAA TGA GCT TGA CAA 3’ (SEQ ID NO: 6) and Actin (hActin F: 5’ CAT CCA CGA AAC TAC CTT CAA CTC C 3’ (SEQ ID NO: 7); hActin R: 5’ GAG CCG CCG ATC CAC AC 3’ (SEQ ID NO: 8)) by qPCR using the SYBR Green method. The reaction was performed in triplicate on a 7500 Real time PCR system (Applied Biosystems, Madrid, Spain). Actin and S18 were used as endogenous controls. The relative mRNA levels were calculated using the comparative Ct method (2e- AACt). hAKT2 (human AKT2) sequence is that of UniProt KB database accession number P31751 version 2 of sequence of November 1 , 1995 and version 206 of entry in database.

1.2.12. Conjugation of F127 with 5-(4,6-Dichlorotriazinyl) Aminofluorescein (5-DTAF)

F127 was fluorescently conjugated with 5-DTAF in an aqueous medium via nucleophilic aromatic substitution by an addition-elimination pathway as previously described (see Andrade, F., et al., Biological assessment of self-assembled polymeric micelles for pulmonary administration of insulin. Nanomedicine, 2015. 11 (7): p. 1621-31 ). Briefly, a stock solution of 20 g/L 5-DTAF in DMSO was diluted in 0.1 M sodium

bicarbonate (pH 9.3) and added to a 6 % (w/v) F127 solution in 0.1 M sodium bicarbonate (pH 9.3) to a final molar ratio of 1 :2 (F127:5-DTAF). The reaction proceeded overnight in the dark at RT. The labeled polymer was purified from the excess of unreacted 5-DTAF by dialysis (12,000-14,000 MWCO Spectra/Por® membrane from Spectrum Europe BV, The Netherlands) against Type I ultrapure water. The dialyzed polymer solutions were lyophilized and stored in closed containers protected from light.

1.2.13. Micelles Internalization

Flow cytometry and confocal microscopy were used to verify the internalization of 5- DTAF-fluorescently labeled PM in MDA-MB-231 and MCF-7 parental breast cancer cells. For the quantitative fluorescence-activated cell sorting (FACS), 2x104 cells were seeded in complete medium in 96 well plates for 24 hours to allow adhesion. Micelles were added to cells at different time points: 0.5, 1 , 2, 4 and 6 hours. Then, cells were washed with 1x PBS, detached with 0.25% trypsin-EDTA, and re-suspended in PBS supplemented with 10% FBS and DAPI (1 g/ml_) used for vital staining. The plate was analyzed in a cytometer Fortessa (BD Biosciences, USA). Data was analyzed with FCS Express 4 Flow research edition software (De Novo Software, Los Angeles, USA). Forward and side scatter gating removed contaminants. For each sample, at least 10000 individual cells were collected and the mean fluorescence intensity was evaluated. For the qualitative confocal microscopy assay, cells were cultured in 0.1 % gelatin-treated coverslips at a density of 2.5x105 cells per well in 6 well plates. After 24 hours, 5’-DTAF-fluorescently labeled PM were added to cells and incubated during 1 hour and further incubated for 30 minutes with the Cell Mask® Red. Subsequently cells were fixed to the coverslips using 4%

paraformaldehyde the nucleus was stained with DAPI (0.2 mg/mL) for 5 minutes in the dark and the cells visualized with a Spectral Confocal Microscope MFV1000 (Olympus, USA)

1.2.14. Statistical Analysis

At least three batches of each PM were produced and characterized and the results expressed as the mean ± SD. For biological studies, at least 3 replicates, each involving at least two technical replicates were involved in final results expressed as the mean SD. Statistical analysis was performed with GraphPad Prism® version 6.0 using the ANOVA. Differences were regarded as statistically significant when p-value were smaller than 0.05.

2. Results

2.1. Polymer functionalization: synthesis and characterization

In order to functionalize PM with Cet it was necessary the formation of an amide bond between the amine groups of Cet and the carboxylic groups of the polymer. F127 presents free alcohol groups in each end of the chain. With the purpose to conjugate Cet onto PM surface, a terminal carboxyl group was required. Two approaches were carried out for polymer carboxylation: the Jones oxidation method and the maleic anhydride method. The obtainment of the desired chemical modifications was assessed by FT-IR. For the Jones oxidation method, a Cr0₃/polymer (C/P) molar ratio from 1.5 to 4.5 was tested. The change of color solution from orange (given by jones reagent) to green-blue indicates the reduction of Cr(VI) to Cr(IV), and it is a useful indicator of alcohol oxidation. A C/P ratio of 4.5 has shown to be too high since a green precipitate appeared on the bottom of the beaker, while C/P ratios of 1.5 and 3 allowed a homogeneous green-blue solution. The FTIR spectra (data not shown) of F127 and carboxylated F127 at C/P ratios of 1.5 and 3 gave stretching vibrations typical of carbonyl group belonging to carboxylic acid at 1733 and 1724 cm ¹ for C/P ratios of 1.5 and 3 respectively, indicates the successful carboxylation of F127 (F127:COOH). The formation of F127:COOH for both C/P ratios is further confirmed by the appearance of a broad peak at 3400 cm ¹, which is associated to the -OH of carboxyl group. The yield of the reaction for both C/P ratios was around 78%.

Regarding the maleic anhydride method, the appearance of a peak at 1725 cm ¹ indicated the formation of carbonyl group, which belongs to the carboxylic acid of modified F127. The yield of the reaction was around 86%. Because the F127:COOH obtained by the Jones oxidation reaction has shown a higher in vitro cytotoxicity compared to the one obtained with the maleic anhydride, the last one was chosen for the production of the PM.

2.2. Design and synthesis of carboxylated PM

PM composed by non-modified F127 (PM F127) presented a hydrodynamic diameter around 28 nm, with a homogenous size distribution and a near neutral charge. These results were further corroborated by TEM that revealed also the spherical shape of PM. These data are depicted in Table 1.

Table 1

In order to conjugate Cet onto PM surface, PM formulation was reformulated to include the previously synthesized F127:COOH. Two different formulations of PM were tested: the first one composed exclusively by F127:COOH (PM F127:COOH) and the second one with a mixture of F127 and F127:COOH at a ratio of 8:2 (PM F127:COOH

8:2). Both formulations were characterized regarding size, surface charge and cytotoxicity with the purpose to select the one presenting the more appropriate characteristics for clinical application. In former Table 1 are depicted the characterization of diameters, Pdi, zeta potential, morphology and cytotoxic profile for PM F127, PM F127:COOH and PM F127:COOH 8:2. The hydrodynamic diameter of PM F127:COOH and PM F127:COOH 8:2 was increased while the zeta potential decreased in comparison to PM F127. Contrary to the DLS results, the geometric diameter measured by TEM showed a similar size distribution between all the formulations (data not shown). Moreover, no morphological differences among the different formulations were denoted in the TEM analysis. The cytotoxicity profile of the systems is presented in FIG. 1 , wherein in pannel (A) there is depicted the in vitro cytotoxicity of the different PM formulations in MDA-MD-231 cells, and in (B) the correspondent IC50 values, obtained by interpolation of y=50 from the dose-effect curves fitting using the real concentrations used for each sample. Results are expressed as meaniSD (n=3). PM F127:COOH showed to be more cytotoxic than PM F127 and PM F127:COOH 8:2 formulations.

FIG. 5 schematically illustrates said PM F127:COOH 8:2, wherein the polyoxypropylene (PPO) central block conforms the core of the micelle, and the two lateral polyoxyethylene (PEO) blocks of the copolymer are disposed conforming the outer side or corona of the micelle. Some of the terminal oxyethylene units in the polyoxyethylene blocks are carboxylated. Oligonucleotides (OGN) complexes with the gelatin (OGN-gelatin) are also conforming the corona. Besides, TG is the targeting moiety (and as will be disclosed in more detail below is in this particular case the monoclonal antibody cetuximab (Cet, which is coupled using amide bonds (-N-C(O)-) to the polymeric micelle.

2.3. Selection of a cationic polymer to complex the siRNA

Once PM was already carboxylated and prepared for the conjugation of Cet, the next step was the incorporation of a cationic polymer in the formulation in order to complex the negatively charged siRNA molecules by electrostatic interactions. Different polycations were candidates to formulate the PM: PEI 10K, Histamine, Protamine, and Gelatin. For each one it was assessed their buffering capacity and predicted proton sponge effect ability, and silencing efficacy, taking AKT2 as model reference gene. Further, cytotoxicity of different polycations was also assessed in MDA-MB-231 using siC. PEI and histamine displayed the higher buffer capacity and expected higher ability to promote the proton sponge effect (data not shown). Gelatin presented some buffer capacity while protamine didn’t show any ability to promote proton sponge effect. Despite the differences regarding the buffer capacity, all polymers formulated in the PM F127 were able to efficiently transfect siRNA and promote a silencing efficacy similar to the gold standard

Lipofectamine® 2000. Furthermore, it was assessed the cytotoxicity for each formulation loaded with siC using MDA-MB-231 cells, and their IC50 values determined. Histamine and gelatin were the polymers presenting higher IC50 values. Gelatin was the selected to complex the siRNA of interest. Obtained IC50 values are depicted in Table 2. Table 2

2.4. PM F127:COOH (8:2) functionalization with Cetuximab

The carboxylic group of PM F127:COOH was activated by EDC before conjugation with Cet. Different concentrations of Cet were tested in order to determine differences between them as chemical changes of PM surface or cell internalization. Three different concentrations were tested: 1 mg/ml, 0,1 mg/ml and 0,01 mg/ml (concentrations in final formulation). FTIR spectra was recorded with the objective of make sure that amide bond was successfully accomplished and to monitor chemical changes in function of the concentration. Successful conjugation of Cet with F127:COOH through the formation of an amide bond, originating PM F127:COOH:Cet was confirmed by the peak at 1725 cm-1 corresponding to the -C=0 of carboxyl group shifted to 1715 cm-1 and a peak

corresponding to the -C=0 of amide group appeared at 1641 cm-1 (data not shown).

To further confirm the carboxylation of F127 and the conjugation of Cet with F127:COOH, ¹H NMR was carried out. The solvent chemical shift (d) of deuterated chloroform (CDCI3) was found at 7.28 ppm. The characteristic peaks at 1.13-1.15 and 3.38-3.83 ppm were attributed to protons of PPO (-CH3) and PPO/PEO (-(CH2-CH(CH3)- 0)-/-(CH2-CH2-0)-) in F127 and F127:COOH. The introduction of carboxyl groups onto F127 structure involved the appearance of a signal at 4.36-4.38 ppm (-CH-COOH) while the signal at 2.10 ppm coming from protons of -CH2-OH disappeared. Furthermore in F127:COOH spectra appeared two chemical shift peaks at 6.21-6.24 and 6.38-6.41 ppm which revealed the existence of an olefin structure indicating the presence of maleic acid ensuring even greater polymer carboxylation. Regarding the Cet conjugation, the presence of chemical shift peaks at 2.70 and 2.83 ppm ascribed to protons of Cet indicates the existence of Cet in PM F127:COOH 8:2:Cet.

2.5. Development and characterization of PM F127:COOH 8:2:Gelatin:Cet

DLS and TEM characterization were carried out in the functionalized PM in order to assess possible changes in its characteristics. As it was expected, an increase of almost 100 nm in the hydrodynamic diameter of PM after conjugation with Cet was observed. Nevertheless, TEM images only denoted a slight increase in the size of PM F127:COOH 8:2:Cet compared to PM F127:COOH 8:2. The spherical shape of PM was maintained after conjugation. An increase in the surface charge of PM after Cet conjugation was also observed (around 8 mV vs -3 mV prior functionalization) indicating the presence of Cet at the surface of PM. The incorporation of gelatin to the system in the form of polyplexes with siRNA promoted a slight increase in the size determined both by DLS and TEM. A significant increase in the surface charge from 8 mV to 30 mV was observed due to the cationic charges of gelatin. The obtained PM F127:COOH

8:2:gelatin:Cet were able to efficiently complex the siRNA with an AE around 85%. All these data are depicted in Table 3 that follows: Table 3

Regarding the biological assessment of the systems, the presence of Cet increased the cytotoxicity of PM as observed in FIG. 2, showing gene expression levels after

transfection with PM entrapping siAKT2 (200 nM), relatively to the ones transfected with siC; and the in vitro cytotoxicity of different formulation in MDA-MD-231 cells. Cell cytotoxicity could be explained by the cytostatic/cytotoxic effect of Cet. Most importantly, the PM F127:COOH 8:2:gelatin:Cet were able to efficiently transfect the siRNA against AKT2 into MDA-MB-231 cells and silence the expression of the AKT2 gene (FIG. 2 (A)). Moreover, at PM concentrations lower than 10 mg/ml, PM F127:COOH

8:2:gelatin:Cet(0.1 mg/ml) showed the lower toxicity of all nanoconjugates (FIG. 2(B)) and good siRNA gene silencing.

2.6. Internalization of PM F127:COOH 8:2:gelatin:Cet into EGFR

overexpressing cells

Fluorescently labeled PM F127:COOH 8:2:gelatin and PM F127:COOH 8:2:gelatin:Cet (1 mg/ml and 0,1 mg/ml of Cet) were incubated with MDA-MB-231 and MCF-7 breast cancer cells at different time-points (0, 0,5, 1 , 2, 4 and 6 h). MDA-MB-231 were used as EGFR overexpressing cells, while MCF-7 as a control of low levels of these receptors.

The fluorescent intensity of the cells that internalize labeled-PM was quantified by flow cytometry. As expected, for EGFR overexpressing cells, a significant difference in the uptake rate of targeted-PM versus non-targeted-PM was observed, especially at 30 min, 1 and 2h time-points (FIG. 3(A)). This difference was also confirmed by the confocal microscopy analysis (not shown), where after 1 hour of incubation was possible to observe a higher number of cells that taken up Cet-functionalized PM in comparison with non-targeted PM. Moreover, the fluorescence intensity seems higher for the targeted PM. Additionally, the orthogonal slices put in evidence the localization of targeted PM inside the cells and near the nucleus, while for the non-functionalized PM, particles at the surface of the cells membrane were also observed. On the other hand, no significant differences in the internalization pattern of PM were observed for MCF-7 cells, emphasizing the role of Cet in the targeting and internalization of therapeutic delivery systems for EGFR overexpressing cancer cells (Fig. 3(B)). This FIG. 3 shows

internalization in MDA-MB-231 (A) and MCF7 (B) cells, incubated with the 5-DTAF labeled PM F127:COOH(8:2):Gelatin:Cet and the percentage of cells emitting green fluorescence were quantified at different incubation time-points.

3. Conclusions

All these data allowed the development of PM for the targeted delivery of siRNAs into breast cancer cells through the chemical modification of F127 and surface

functionalization with Cetuximab. The maleic anhydride reaction was selected to efficiently achieve the F127 carboxylation. After polymer carboxylation and activation, PM were efficiently modified with Cet and their ability to actively target cancer cells assessed. The functionalization of PM with Cet significantly improved its internalization rate into EGFR overexpressing breast cancer cells. In addition to an efficient active targeting, the proposed system seems to gather the requirements for an efficient and safe transport of siRNA in terms of their physicochemical characteristics, biological efficacy and toxicity profile. The biocompatibility and biodegradability of the used polymers as well as the simple preparation method, make these PM an excellent alternative to the complex and often toxic proposed gene delivery systems. These results makes these new PM a technological platform for the development of systems to encapsulate different siRNA, as well as other types of genetic material, biomolecules or different types of drugs. The enormous flexibility endorsed by the proposed PM of the invention to formulate different types of active substances provides an important contribution for the nanomedicine field applied to different diseases. This is mainly due the possibility to construct an effective targeted multifunctional delivery system, on the way to the so increasingly required personalized medicine and combined therapies. Example 2. Preferential internalization of polymeric micelles (PM) into Cancer Stem Cells (CSC): The internalization of fluorescent-labeled micelles was assessed quantitatively by flow cytometry.

HCT1 16 (human colon cancer cells) CSC and non-CSC cells subpopulations were incubated with 5-DTAF labeled PM at different time-points. Fluorescence intensity, related with the number of PM inside each cell, was detected. Fluorescence intensity was higher in CSC than in non-CSC subpopulation. For each sample, at least 10000 individual cells were collected and the presented value in FIG. 6 below corresponds to the mean fluorescence intensity. These results demonstrate a tendency of the PM to be internalized by CSC, even without the need of an active targeting. Data are depicted in FIG. 6(A) and (B). As can be deduced from FIG. 6 (A), the presence of gelatin and of an oligonucleotide

(siRNA) aids in the internalization of the PM in relation with the PM free of -COOH, gelatin and oligonucleotide.

Tested PM where produced as indicated in Example 1 , subparts 1.2.4 but without the functionalization with cetuximab, and 1.2.12 for the conjugation with 5-DTAF.

All these data allow concluding that the polymeric micelles of the invention are useful tools in treating cancer types by targeting cancer stem cells of a tumour

Citation List

Patent Literature

- EP1037611 B1

- EP1907444B1

Non Patent Literature

- Wu et al.,“Peptide-mediated Tumor Targeting by a Degradable nano Gene

Delivery Vector Based on Pluronic-Modified Polyethyleneimine”, Nanoscale Research Letters- 2016, vol. no. 1 1 :122

Kumar et al.,“Clinical development of gene therapy: results and lessons from recent successes”, Mol. Ther Methods Clin Dev.- 2016, vol. no. 3, pp.:16034

Chen, Y., et al., Dual-functional c(RGDyK)-decorated Pluronic micelles designed for antiangiogenesis and the treatment of drug-resistant tumor. Int J

Nanomedicine, 2015. 10: p. 4863-81.

Li, Y,Y., et al. Pluronic F127 nanomicelles engineered with nuclear localized functionality for targeted drug delivery. Mater Sci Eng C Mater Biol Appl, 2013. 33(5): p. 2698-707

- Andrade, F., et al., Biological assessment of self-assembled polymeric micelles for pulmonary administration of insulin. Nanomedicine, 2015. 1 1 (7): p. 1621-31 - Comer J, Tam K (2001 ). "Lipophilicity Profiles: Theory and Measurement". In Testa

B, van de Waterbed H, Folkers G, Guy R. Pharmacokinetic Optimization in Drug Research: Biological, Physicochemical, and Computational Strategies.

(secondary). Weinheim: Wiley-VCH. pp. 275-304

Claims

1.- A polymeric micelle in aqueous media comprising:

2.- The polymeric micelle according to claim 1 , wherein the polyoxypropylene blocks of the copolymer conform the inner side of the micelle and the gelatin and polyoxyethylene blocks of the copolymer conform the outer side of the micelle.

3.- The polymeric micelle according to any of claims 1-2, further comprising a modified amphiphilic block copolymer with one or more polyoxyethylene blocks and one or more polyoxypropylene blocks, wherein the oxyethylene and/or oxypropylene terminal monomer units of end blocks of polyoxyethylene and/or polyoxypropylene are modified and comprise functional groups capable of forming an amide bond.

4.- The polymeric micelle according to claim 3, wherein the functional groups are selected from the group consisting of - COOR², being R² selected from H and -(CrC₈)-alkyl; -

COR¹, wherein R¹ is a compound of formula -Z-COOR², being -Z- selected from (CH2)n with n an integer from 1 to 10, (CH=CH-CH)m with m an integer from 1 to 3, (CH2- CH=CH), and (CH=CH)rwith ran integer from 1 to 2; -NR³R⁵ being R³ and R⁵

independently selected from H and -(CrC₈)-alkyl, or R³ and R⁵ conform together with the nitrogen atom a cyclic compound; and combinations thereof.

5.- The polymeric micelle according to any of claims 1-4, wherein the amphiphilic block copolymer is selected from sequential di- and triblock-copolymers, grafted di- and triblock- copolymers, and mixtures thereof.

6.- The polymeric micelle according to any of claims 1-5, wherein the amphiphilic block copolymer is a sequential triblock-copolymer of formula (I):

wherein a is an integer from 2 to 150 and b is an integer from 15-67;

R^x is selected from H; COR¹, wherein R¹ is a compound of formula -Z-COOR², being R² selected from H and -(Ci-C₈)-alkyl, and being -Z- selected from (CH2)n with n an integer from 1 to 10, (CH=CH-CH)m with m an integer from 1 to 3, (CH2-CH=CH), and (CH=CH)r with r an integer from 1 to 2; and combinations thereof.

7.- The polymeric micelle according to claim 6 wherein a is an integer from 25-150 and b is an integer from 30-60.

8.- The polymeric micelle according to any of claims 3-7, wherein the modified block copolymer comprises oxyethylene and/or oxypropylene terminal monomer units of the end blocks that are coupled to a targeting moiety capable of conducting the polymeric micelle to particular plant or animal cells and/or cell compartments, said cells able to recognize said targeting moiety.

9.- The polymeric micelle according to claim 8, wherein the targeting moiety is selected from the group consisting of an antibody or fragment of said antibody, a peptide, an oligonucleotide and combinations thereof.

10.- The polymeric micelle according to any of claims 8-9, wherein the targeting moiety is an antibody or fragment of an antibody which recognizes a mammal epidermal growth factor receptor.

1 1.- The polymeric micelle according to any of claims 1 -10, which is a drug-loaded polymeric micelle comprising one or more therapeutic agents.

12.- The polymeric micelle according to claim 1 1 , wherein the therapeutic agent is a negatively charged therapeutic agent.

13.- The polymeric micelle according to claim 12, wherein the negatively charged therapeutic agent is selected from the group consisting of small interfering RNA (siRNA), micro RNA (miRNA), short hairpin RNA (shRNA), a DNA plasmid, an oligopeptide, a protein, and combinations thereof.

14.- A pharmaceutical composition comprising in aqueous media a therapeutically effective amount of a polymeric micelle as defined in any of claims 11-13, together with pharmaceutically acceptable excipients and/or carriers.

15.- The pharmaceutical composition according to claim 14, comprising a polymeric micelle, wherein the micelle is a drug-loaded micelle and comprises:

- gelatin;

(I),

- the modified block copolymer comprises modified oxyethylene terminal monomer units coupled to an antibody that recognizes mammal epidermal growth factor receptor.

16.- A drug-loaded polymeric micelle as defined in any of claims 1 1 -13, or a

pharmaceutical composition as defined in any of claims 14-15, for use as a medicament.

17.- The drug-loaded polymeric micelles for use according to claim 16, which are for use in treating cancer, and wherein the treatment comprises targeting cancer stem cells of a tumour.

18.- The drug-loaded polymeric micelles for use according to claim 17, wherein the cancer stem cells are cancer stem cells of colon tumour.

19.- The drug-loaded polymeric micelles for use according to any of claims 17-18, wherein the drug-loaded polymeric micelle comprises:

- gelatin;

(I),

20.- A multifunctional delivery system for the delivery of one or more compounds to plant or animal cells, comprising a polymeric micelle as defined in any of claims 1-13.