WO2017050979A1 - Nanoparticules pour le diagnostic et l'administration de médicament - Google Patents

Nanoparticules pour le diagnostic et l'administration de médicament Download PDF

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Publication number
WO2017050979A1
WO2017050979A1 PCT/EP2016/072714 EP2016072714W WO2017050979A1 WO 2017050979 A1 WO2017050979 A1 WO 2017050979A1 EP 2016072714 W EP2016072714 W EP 2016072714W WO 2017050979 A1 WO2017050979 A1 WO 2017050979A1
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nanovector
cpt
nanoparticle
formula
moiety
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PCT/EP2016/072714
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English (en)
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Noureddine KHIAR
Ralf WELLINGER
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Consejo Superior De Investigaciones Científicas (Csic)
Universidad De Sevilla
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Publication of WO2017050979A1 publication Critical patent/WO2017050979A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the field of nanoparticles for diagnosis and therapeutical applications.
  • it relates to nanoparticles for delivery and release of pharmaceutically active agents, such as antitumoral agents, in their site of action.
  • nanometric-sized nanoparticles are being used as vehicles for selective and/or controlled release of cytotoxic drugs (Arvizo, et al. Chem. Soc. Rev. 2012, 41, 2943-2970)
  • nanoparticles as vehicles in medicine
  • the main advantages of using nanoparticles as vehicles in medicine include: (1 ) the ability to improve the pharmaceutical and pharmacological properties of drugs, without the need to alter the active ingredient, (2) increase the therapeutic efficacy by selective drug delivery to specific tissue or cell, (3) the delivery of drugs through a series of -epithelial and endothelial- biological barriers, (4) protection of a drug from premature degradation, (5) the ability to enhance intracellular penetration and (6) the ability to deliver a combination of therapeutic and contrast agents for real-time monitoring of therapeutic efficacy.
  • these nanovectors nano-sized vehicles comprising a pharmaceutically active compound
  • Doxil® which was the first nanocarrier approved in 1995 for the treatment of Kaposi syndrome associated with AIDS (Udhrain, A., et al. Int. J. Nanomedicine, 2007, vol. 2, p. 345).
  • Doxil® is formed by encapsulation of anti-carcinogenic doxorubicin (Dox) in PEGylated liposomes.
  • Doxil® showed enhanced pharmacokinetics and biodistribution of Dox, which greatly facilitated and increased the half-life of drug circulation, and therefore increased the cumulative dose of the drug within the tumor.
  • Doxil has a reduced cardiotoxicity unusually associated with the free drug as well as better efficacy against ovarian cancer resistant to taxane / platinum combination therapy.
  • CPT camptothecin
  • MPEG-SS-PCPT polyethylene glycol monomethyl ether-block-poly(2- methacryl ester hydroxyethyl disulfide-graft-CPT)
  • GSH glutathione
  • nanovectors described above are good examples of recent developments in drug delivery, but ample space is still present for improvement.
  • nanovectors should possess other characteristics such as biocompatibility, non-toxicity, water solubility and high loading capacity. Ease of internalization and/or release of the active compound in the target cells and its site of action is also an important requirement.
  • it may be of interest to provide some means for visualizing the nanovectors by non-invasive imaging techniques. This is useful to follow the targeting and delivery of the active compound for research purposes, as well as for diagnosis.
  • the present invention provides improved drug delivery systems.
  • Said systems comprise newly designed nanoparticles that efficiently deliver and release compounds of interest to their site of action.
  • a first aspect of the invention provides a nanoparticle having a size comprised from 2 to 300 nm comprising:
  • a core comprising on its surface a noble metal (M) selected from gold, silver and platinum, and
  • an amphiphilic linker that is optionally positively charged comprising:
  • hydrophilic moiety (B) that is optionally positively charged comprising:
  • P polyethylene glycol
  • PEG polychitosan
  • polydextran polydextran
  • copolymers thereof and a derivative thereof selected from poly(chitosan-g-lactic acid), poly(chitosan-N-isopropylacrylamide), phosphorylated chitosan and polycarboxymethyl dextran;
  • hydrophobic alkyl thiolate moiety (A) is attached to the noble metal on the core surface through M-S bond;
  • hydrophobic alkyl thiolate moiety (A) and the hydrophilic moiety (B) that is optionally positively charged are linked by a connecting diradical (CR) selected from the group consisting of the diradicals of formula ( ⁇ ), (II), (III), (IV), (V), (VI), (VII'), (VIII), (IX) and (X)
  • the hydrophilic moiety (B) is positively charged, being the positively charged group (G + ) within, and preferably in the middle of, the hydrophilic part of the linker. In these embodiments, this design has several advantages in terms of specificity and toxicity for tumoral cells, or in general, for negatively charged target sites, such as DNA or RNA.
  • the nanoparticles of the invention preferentially accumulate in tumoral sites, while the positively charged linker further increases the solubility in the physiological medium and the affinity for the negatively charged tumoral cells,.
  • the nanoparticles of the invention are then particularly well suited for the delivery of compounds of interest to cancer cells. Selective delivery of compounds of interest to other sites of action such as the cell nucleus is also enabled.
  • the nanoparticles also allow for a selective release of the compound of interest. This is achieved by GSH-mediated cleavage of the M-S bond between the linker and the M-containing core of the nanoparticle. While overexpressing GSH, tumoral cells are more likely to release the active- compound attached to the linker that normal cells, so that the compound of interest is preferentially released in the proximity or inside the target cancer cells, further improving specificity and reducing possible toxic effects.
  • the present nanoparticles are soluble in physiological fluids and resistant to opsonization, thus presenting long circulating times.
  • the NP of the invention may be useful by itself, for example by serving as contrast agent for imaging purposes, thanks to its specificity for the target site and the unique optical properties of the noble metals contained in the core.
  • another aspect of the invention provides nanoparticles as above for use as a contrast agent.
  • the nanoparticles of the invention are used as vehicles for the delivery of compounds of interest to their site of action.
  • the compound of interest may be a pharmaceutically active agent, for instance an antitumoral agent for cancer therapy, but also a contrast agent, mostly for imaging and diagnosis purposes.
  • the nanoparticle may be linked to a targeting agent to further enhance the specificity for the target cells.
  • a further aspect of the invention therefore provides a nanovector comprising the nanoparticle as defined above and a compound selected from a pharmaceutically active agent, a targeting agent, a contrast agent, and mixtures thereof, wherein said compound is attached to the optionally positively charged hydrophilic moiety of the nanoparticle complex through a connecting diradical (CR) selected from the group consisting of the diradicals of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) and (X)
  • a connecting diradical selected from the group consisting of the diradicals of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) and (X)
  • nanovectors of the invention are well suited to deliver, for example, antitumoral agents to cancer cells. Therefore another aspect provides for use of a nanoparticle or a nanovector of the invention for drug delivery.
  • the particular design and the interaction of the various features of the nanovector afford several advantages that ultimately enhance the antitumoral effect of said antitumoral agent while reducing its toxicity and side effects.
  • the particular design of the nanovector of the invention achieves prolonged release of the drug. Prolonged release may enable a more convenient administration regime in cancer treatment. Additionally, prolonged release of antitumoral agents greatly avoids the appearance of mechanisms that result in resistance to the drug, both improving the results of treatment of the primary tumor and, particularly, of metastatic cancers.
  • the conjugate formed by the anti-tumoral drug attached to the nanoparticle may be as active, or even more active, than the anti-tumoral drug on its own (i.e. the nanoparticle does not interfere, but rather enhances, the anti-tumoral activity).
  • nanovectors of the invention carrying antitumoral agents further enhance the pharmacologic profile of the drug by increasing retention time of the drug inside the cell. They also found that the nanovectors of the invention provide a mean to target different drug-sensitive organelles, namely, the mitochondria and the nucleus (Nucleic Acids Res. 2009 vol 4 e26). By leaking to both sites of action, the damage exerted by the drug is greater and the tumoral cells are more prone to cell death induced by apoptotic events.
  • nanovectors of the invention carrying antitumoral drugs seem to be more active in killing tumoral cells than healthy cells in vitro when compared to the free antitumoral drug, thus reducing side effects when the treatment is performed with the nanovector.
  • the nanovector of the invention is useful for medical purposes, and it is usually contained in a pharmaceutical composition that also includes appropriate excipients and carriers. Accordingly, another aspect of the invention is related to a pharmaceutical composition comprising a
  • a further aspect relates to a nanovector or a pharmaceutical composition comprising a nanovector as defined above, wherein the nanovector comprises a pharmaceutically active agent, for use as a medicament.
  • This aspect can also be formulated as the use of a nanovector or a pharmaceutical
  • composition comprising a nanovector as defined above, wherein the nanovector comprises a pharmaceutically active agent, for the manufacture of a medicament.
  • the present invention also relates to a method for the treatment or prevention of a disease, comprising administering a
  • a nanovector or a pharmaceutical composition comprising a nanovector as defined above, wherein the nanovector comprises a pharmaceutically active agent, together with pharmaceutically acceptable excipients or carriers, in a subject in need thereof, including a human.
  • the scope of the present invention generally refers to therapy in animals, including humans. Thus both medical and veterinary practices are included.
  • nanovectors of the invention are particularly suited for cancer therapy.
  • a nanovector or a pharmaceutical composition comprising a nanovector as defined above, wherein the nanovector comprises an anti-tumoral agent, for use in the prevention and/or treatment of cancer.
  • This aspect can also be formulated as the use of a nanovector or a
  • the present invention also relates to a method for the treatment and/or prevention of cancer, comprising administering a therapeutically effective amount of a nanovector or a pharmaceutical composition comprising a nanovector as defined above, wherein the nanovector comprises an anti-tumoral agent, together with pharmaceutically acceptable excipients or carriers, in a subject in need thereof, including a human.
  • the nanoparticles of the invention may by themselves act as contrast agents. They can also comprise an additional contrast agent or any other compound that is useful for diagnosis.
  • the nanovector is formulated together with appropriate additives.
  • the invention also provides a composition for diagnostic purposes comprising a diagnostic effective amount of a nanoparticle or a nanovector as defined above together with appropriate amounts of excipients and/or carriers acceptable for diagnosis.
  • the present invention also relates to a method for diagnosis, in particular for diagnosis of cancer, comprising administering a therapeutically effective amount of a nanoparticle as defined above, a nanovector as defined above or a composition for diagnosis comprising a nanoparticle or a nanovector as defined above, wherein the nanovector comprises a contrast agent or any other compound that is useful for diagnosis, together with excipients and/or carriers suitable for diagnosis, in a subject in need thereof, including a human.
  • FIG. 1 Comprehensive structure of a nanovector of the invention.
  • R active compound
  • CR connecting diradical
  • P hydrophilic polymer
  • G+ positively charged group
  • SG alkyl thiolate
  • @-Au core comprising Au on its surface
  • FIG. 2. Nanovector carrying camptothecin.
  • FIG. 3. Equilibrium between lactonic (A; active) and carboxylic forms of camptothecin (B; inactive)
  • FIG. 4. Synthesis of the therapeutic composition by place exchange reaction.
  • FIG. 5. Effect of the therapeutic composition on DNA Relaxation.
  • the addition of CPT or the Au-L-CPT to human Topoisomerase I (hTopl ) generates open circle plasmid DNA in the plasmid relaxation assay. Arrows indicate
  • FIG. 6 Analysis of DNA damage response upon acute, short term treatment of U2OS cells with either medium alone (control; white bar), camptothecin alone (CPT; light grey bar), the supernatant of CPT-containing nanovectors treated with glutathione (Linker-CPT; dashed bar), or Au-L-CPT (black bar).
  • A Schematic outline of the experimental conditions. 1 , seed cells; 2, Addition of medium supplemented with Linker-CPT or Au-L-CPT; 3, incubation in CPT- free medium; 4, 5, 6 and 7, analysis of DNA damage response.
  • B Schematic outline of the experimental conditions. 1 , seed cells; 2, Addition of medium supplemented with Linker-CPT or Au-L-CPT; 3, incubation in CPT- free medium; 4, 5, 6 and 7, analysis of DNA damage response.
  • B Schematic outline of the experimental conditions. 1 , seed cells; 2, Addition of medium supplemented with Linker-CPT or Au-L-CPT; 3, incubation in CPT
  • Y axis represents gamma- H2AX fluorescence as intensity per cell (x10 5 AU).
  • C Y axis represents micronuclei formation. Both gamma-H2AX fluorescence and micronuclei formation are indicators for DNA damage and/or apoptotic events. Note that the medium was supplement with 1 ⁇ CPT or Au-L-CPT that contained 1 ⁇ CPT.
  • FIG. 7 The survival of U2OS cells dramatically decreases after acute treatment with nanovectors of the invention.
  • A Scheme that outlines the conditions applied for chronic or acute treatments. Abobe: Acute clonogenic assay; 1 , seed cells; 2, DNA damaging agent; 3, PBS wash Fresh media. Below: Chronic clonogenic assay; 1 , seed cells; 2, DNA damaging agent.
  • B Representative images of acute (left) and chronic (right) treatments are shown.
  • C control.
  • C Percentage of survival after the acute (left) or chronic (right) treatment with Au-L-CPT or CPT.
  • Y axis represents % survival.
  • Non tumorogenic MCF10A cells are resistant to acute treatment with Au-L-CPT.
  • FIG. 7A Representative images of of MCF7 (tumorogenic; left) and MCFF10A (non-tumorogenic; right) mammary gland cells after the acute treatment with CPT or Au-L-CPT as outlined in figure 7A.
  • B Percentage of cell survival. As compared to MCF10A cells, cute treatment with Au-L-CPT but not CPT alone leads to a dramatic drop in MCF7 survival rate. The data represent mean ⁇ s.e.m. from 3 independent experiments.
  • FIG. 9 shows the tumor volume (in mm 3 ) over time (days of treatment) when the animals where treated with PBS, irinotecan (80 mg/Kg) and the
  • nanovector of the invention Au-L-CPT (Nano-Camp 24 mg/Kg). Irinotecan and the nanovector of the invention showed comparable antitumoral activity.
  • FIG. 10 shows the tumor volume (in mm 3 ) over time (days of treatment) when the animals where treated with PBS, irinotecan (80 mg/Kg) and the
  • nanovector of the invention Au-L-CPT (Nano-Camp 24 mg/Kg). where animals with tumors higher or lower than 2x mean ⁇ SEM in tumor volume and RTV (high dispersion) were excluded.
  • the nanovector of the invention Au-L-CPT showed better efficacy than irinotecan from day 10 after completion of the treatment.
  • FIG. 1 1 shows the variation of the mouse body weight (in g) over time (days of treatment).
  • Au-L- CPT Nano-Camp 24 mg/Kg
  • no body weight loss i.e, no toxicity
  • irinotecan considerable weight loss (toxicity) was observed.
  • FIG. 12 shows the H 1 -RMN spectrum of (CPT-L1 -SH) (18).
  • FIG. 13 shows the H 1 -RMN spectrum of (CPT-L2-SH) (23).
  • FIG. 14 shows the UV-Vis spectrum recorded of the NP of example 4 A) in dichloromethane.
  • FIG. 15 shows the H 1 -RMN spectrum of the NP of example 4 A).
  • FIG. 16 shows the IR spectrum of the NP of example 4 A).
  • FIG. 17 shows the TEM image of the NP of example 4 A).
  • FIG. 18 shows the UV-Vis spectrum recorded of the NP of example 4 B) (Au- L-CPT) in dichloromethane.
  • FIG. 19 shows the H 1 -RMN spectrum of the NP of example 4 B) (Au-L-CPT).
  • FIG. 20 shows the TEM image of the NP of example 4 B) (Au-L-CPT).
  • FIG. 21 shows the UV-Vis spectrum recorded of the NP of example 4 B) (Au- L1 -CPT) in dichloromethane.
  • FIG. 22 shows the H 1 -RMN spectrum of the NP of example 4 B) (Au-L1 -CPT).
  • FIG. 23 shows the UV-Vis spectrum recorded of the NP of example 4 B) (Au- L2-CPT) in dichloromethane.
  • FIG. 24 shows the H 1 -RMN spectrum of the NP of example 4 B) (Au-L2-CPT).
  • nanoparticle refers to a core-shell NP comprised by a core comprising a noble metal selected from gold, silver and platinum on its surface and a linker.
  • the "linker” is a molecule comprising the features defined above, basically, a hydrophobic alkylthiolate and an amphiphilic moiety that is optionally positively charged comprising: at least one polymeric chain selected from PEG, polychitosan, polydextran, copolymers thereof, and a derivative thereof.
  • a compound of interest such as a pharmaceutically active agent, a contrast agent or targeting agent
  • these nanovectors are, as mentioned above, vehicles for the delivery of said compound of interest.
  • the NP of the invention has at least two dimensions at the nanoscale, particularly with all three dimensions at the nanoscale, where the nanoscale is the range about 1 nm to about 500 nm. Particularly, when the NP is
  • nanoparticle refers to a particle with at least two dimensions at the nanoscale, these two dimensions being the cross-section of the NP.
  • the core of the NP of the invention contains on its surface a noble metal (M) selected from Au, Ag and Pt. These metals show a high affinity towards sulfur groups (including SH groups), such as the sulfur groups of alkyl thiols present in the linker. Thus, a free SH group has a high tendency to spontaneously react with the metallic NP to form a pseudo-covalent bond M-S.
  • the strong binding between the linker and the NP is needed to avoid desorption of the linker molecule.
  • the noble metal on the surface of the NPs core is Au.
  • the inorganic NP is a good antenna for electromagnetic fields including for example gamma ray, X-ray, Near Infrared (NIR) or UV-Vis and microwaves).
  • electromagnetic fields including for example gamma ray, X-ray, Near Infrared (NIR) or UV-Vis and microwaves.
  • the core is made of gold.
  • Au NPs are particularly suitable because of their excellent physico-chemical properties. Firstly, they are not susceptible to photobleaching, biocompatible and noncytotoxic. Additionally, Au NPs are easily synthesized, form
  • the NPs per se may also be successfully used for photothermal therapy in cancer treatment.
  • the Au-containing NPs emit an intense heat when they are stimulated with the correct frequency of laser light or other heat source (microwave, radio frequency, ultrasound etc.).
  • a group of small NPs of the invention which preferentially locate in tumoral sites, can locally warm an area a thousand times their size, acting as nanoscopic heaters activated by light, inducing death by hyperthermia of the tumoral cells in which they are internalized.
  • Au NPs have a strong surface plasmon enhanced absorption and scattering, making them ideal as imaging labels and contrast agents.
  • the NPs of the invention have nanometric size and may have different shapes and sizes, for example spheres, rods, discs, tubes and hemispheres.
  • the NPs are spherical or quasi-spherical.
  • the NPs are nanospheres comprising a core made of gold.
  • the size of the NP must be such that allows prolonged plasma life, i.e. the conjugate remains in the systemic circulation until it encounters
  • the term "size” refers to a characteristic physical dimension.
  • the size of the NP corresponds to the diameter of the NP.
  • the size is defined in terms of the sphere, inscribed inside the nanocube or the nanoprism, which has the maximum diameter possible.
  • the size of the NP corresponds to the diameter of the cross-section of the NP.
  • a size of a set of NPs can refer to a mode of a distribution of sizes, such as a peak size of the distribution of sizes.
  • the NPs are spherical or substantially spherical, thus the term "nanosphere” is also used herein.
  • the nanospheres of the invention have a diameter comprised in the range from 2 to 300 nm, particularly in the range from 5 to 200 nm, more particularly from 10 to 100 nm, even more particularly from 10 to 50.
  • the diameter of the nanoparticle, in particular of the nanosphere refers to the diameter of the NP with the linker, and does not relates to the diameter of the core.
  • the nanospheres of the invention have a core diameter comprised in the range from 2 to 20 nm, more preferably from 2 to 10 nm, even more preferably 2 nm. In both cases, the diameter of said sphere is comprised in the general and particular ranges defined above for spherical NPs.
  • the nanoparticles are nanospheres
  • the hydrophilic moiety comprised in the amphiphilic linker is positively charged
  • the distance from the external surface of the nanoparticle exposed to the environmental fluids to the positively charged group (G + ) is preferably from 15 to 50 atoms.
  • Z is a compound selected from an active agent, a targeting agent, or a contrast agent, that can be linked through an ester bond
  • the distance between the ligand at the surface and the charged group (G+) is of 20 atoms. It is thought that the fact that the positively charged group is placed significantly far away from the surface exposed to environmental fluids is beneficial for the activity of the nanoparticle, avoiding unspecific interaction with negatively charged entities, by mere electrostatic interactions.
  • the NPs of the invention are nanospheres having a core made of gold and a diameter from 10 to 50 nm, more preferably from 2 to 20 nm, and more preferably from 2 to 10 nm.
  • the size of the NPs (and nanovectors) of the invention may be determined by dynamic light scattering (DLS) or by Transmission electron microscopy (TEM), both techniques being well known in the state of the art. Other well-known techniques for measuring NP size may also be employed to determine size for the present NPs.
  • the NPs core comprises a first material covered by a second material, wherein the first material is metal, (such as iron, silver, platinum, gadolinium), metal oxide (such as silica or
  • the second material is a noble metal selected from Au, Ag and Pt.
  • the liposomes and micelles are generally comprised by phospholipids and may additionally encapsulate other compounds of interests such as drugs or contrast agents.
  • the noble metal is generally coating the first material, i.e. it completely covers the surface of the core. However, in particular embodiments it may happen that the noble metal is partially covering the core surface.
  • the NPs of the invention comprise an amphiphilic linker, which is optionally positively charged, bound to the core's metal surface, which preferably contains Au.
  • the amphiphilic linker of the NP of the invention comprises a hydrophobic Ci- C 2 0 alkyl thiolate moiety (A), and a hydrophilic moiety (B) that are linked by a connecting diradical (CR) selected from the group consisting of the diradicals of formula ( ⁇ '), (II), (III), (IV), (V), (VI), (VII'), (VIII), (IX) and (X) as previously defined.
  • a connecting diradical (CR) selected from the group consisting of the diradicals of formula ( ⁇ '), (II), (III), (IV), (V), (VI), (VII'), (VIII), (IX) and (X) as previously defined.
  • the connecting diradical (CR) is of formula ( ⁇ ) it is preferably attached to the hydrophobic alkyl thiolate moiety (A) through the carbonyl group, i.e.
  • the connecting diradical (CR) when the connecting diradical (CR) is of formula (III), it is preferably attached to the hydrophobic alkyl thiolate moiety (A) through the carbon atom, i.e. thereby forming the imine fragment (Ilia); when the connecting diradical (CR) of formula (IV), it is preferably attached to the hydrophobic alkyl thiolate moiety (A) through the carbonyl group, i.e.
  • connecting diradical (CR) when the connecting diradical (CR) is of formula (V), it is preferably attached to the hydrophobic alkyl thiolate moiety (A) through the sulfonyl group, i.e. thereby forming the sulfonamide fragment (Va); when the connecting diradical (CR) is of formula (VIII), it is preferably attached to the hydrophobic alkyl thiolate moiety (A) through the carbonyl group, i.e.
  • the invention relates to the nanoparticle as previously defined, wherein the hydrophobic alkyl thiolate moiety (A) and the hydrophilic moiety (B) are linked by a connecting diradical (CR) of formula ( ⁇ ), wherein X is NH, which is attached to the alkyl thiolate moiety (A) through the carbonyl group, and to the hydrophilic moiety (B) through the nitrogen atom, i.e. thereby forming the fragment (laa):
  • the invention relates to the nanoparticle as previously defined, wherein the hydrophilic moiety comprises the triazol moiety of formula (XXII):
  • the invention relates to the nanoparticle as previously defined, wherein the hydrophilic moiety comprises the triazol moiety of formula (XXIIa):
  • the invention relates to the nanoparticle as previously defined, wherein the hydrophilic moiety (B) comprises the biradical of formula (XXIII) or the biradical of formula (XXIV)
  • m is a value from 2 to 100, and wherein the biradical of formula (XXIII) or formula (XXIV) is attached to the connecting radical linking the hydrophobic alkyl thiolate moiety (A) and the hydrophilic moiety (B) optionally positively charged through a carbon atom. More particularly, in the biradical of formula (XXIII) or the biradical of formula (XXIV) m is a value from 2 to 30 or from 2 to 20.
  • the invention relates to the nanoparticle as previously defined, wherein the linker has the general formula (XXV)
  • n is a value from 1 to 20; n', m and m' are independently a value from 1 -200; x is 0 or 1 ; y is 0 or 1 , and each Ri is independently (Ci-C2o)alkyl.
  • the linker is selected from the group consisting of L, L1 , and L2, wherein L has the general formula (XXV) wherein n is 8, m is 2, and m' is 3, x is 1 , y is 0 and each Ri is methyl; L2 has the general formula (XXV) wherein n is 8, m is 18, m' is 19, x is 1 , y is 1 , n' is 3, and each Ri is methyl; and L3 has the general formula (XXV) wherein n is 8, m and m' are 19, x is 0, y is 1 , n' is 3, and each Ri is methyl.
  • the nanoparticle is preferably a nanosphere having a core diameter preferably from 2-20 nm, more preferably 2-10 nm, even more preferably 2 nm.
  • the invention relates to the nanoparticle as previously defined, wherein the hydrophobic alkyl thiolate moiety is a C5-C15 alkyl thiolate moiety.
  • the invention relates to a NP that comprises a positively charged amphiphilic linker which comprises: a) a hydrophobic C1-C20 alkyl thiolate moiety A), and b) a positively charged hydrophilic moiety (B) comprising: i) at least one polymeric chain (P) selected from polyethylene glycol (PEG), polychitosan, polydextran, copolymers thereof, and a derivative thereof selected from poly(chitosan-y-lactic acid), poly(chitosan-N-isopropyl- acrylamide), phosphorylated chitosan and polycarboxymethyl dextran; and ii) at least one positively charged group (G + ).
  • PEG polyethylene glycol
  • PEG polychitosan
  • polydextran polydextran
  • copolymers thereof and a derivative thereof selected from poly(chitosan-y-lactic acid), poly(chitosan-N-isopropyl- acrylamide), phosphoryl
  • G + is selected from a quaternary ammonium salt and quaternary sulfonium salt. More particularly G + is a diracdical selected from - N + Ri Ri- or -S + Ri Rr, wherein each Ri is independently (Ci-C2o)alkyl, even more particularly, G + is -N + Ri Rr, wherein each Ri is independently
  • each Ri is methyl.
  • the hydrophilic, positively charged moiety generally has a structure according to formula XI: - ([P1 ]a-[CR1 ]a-[G1 + ]a-[CR2] a -[P2] a -[CR3]a-[G2 + ] a -[CR4]a-[P3]a)m -
  • P1 , P2 and P3 are each a polymeric chain of molecular weight comprised from 300-30000 Da independently selected from PEG, polychitosan, polydextran, copolymers and a derivative thereof selected from poly(chitosan- g-lactic acid), poly(chitosan-N-isopropylacrylamide), phosphorylated chitosan and polycarboxymethyl dextran;
  • G1 + and G2 + are each a positively charged group independently selected from quaternary ammonium salts and quaternary sulfonium salts;
  • CR1 , CR2, CR3 and CR4 are each a connecting diradical independently selected from the group consisting of the diradicals of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) and (X) as defined above; a is 1 or 0, with the proviso that the hydrophilic positively charged group comprises at least one P and one G +
  • connecting diradicals are not exhaustive. The skilled person synthesizing the linker will know that other connecting radicals may result from the particular synthesis procedure. Furthermore, the choice of connecting diradicals is not usually important for the functionality of the NP of the invention as long as it contains the other features defined above, i.e., a size comprised from 3 to 300 nm, an alkyl thiolate and a hydrophilic moiety comprising one of the polymeric chains which is optionally positively charged as defined above.
  • the hydrophilic, optionally positively charged moiety comprises at least one PEG chain. In other embodiments, it comprises two PEG chains.
  • PEG, polychitosan, polydextran and their derivatives provide water-solubility to the NP. Said polymers also achieve a "masking" effect, e.g. they protect the molecule against drug transporter opsonization and
  • the hydrophilic, positively charged moiety comprises at least one PEG and a quaternary ammonium salt.
  • the hydrophilic, positively charged moiety comprises a positively charged polychitosan.
  • the nanoparticle or nanovector of the invention comprises a plurality of linkers as herein defined which all have the same size.
  • the structure of the linker may vary as long as it comprises the above- mentioned features and the NP does not exceed a size within the range from 3 to 300 nm, preferably from 10 to 50 nm.
  • the NP does not exceed a size within the range from 3 to 300 nm, preferably from 10 to 50 nm.
  • the linker has a structure according to formula XII, wherein, n is 1 -20, m is 2-100, n" is 2-100 and R1 is C1-C20 alkyl.
  • n 5-10
  • m 2-10
  • n' 2-10
  • R1 is C1-C10 alkyl.
  • R1 is C1-C5 alkyl, for example C1 , C2, C3, C4 or C5 alkyl.
  • the NP of the invention may be bound to a compound of interest selected from a pharmaceutically active agent, a targeting agent, a contrast agent, and mixtures thereof, thus constituting a "nanovector" of the invention.
  • pharmaceutically active agent herein also referred to as “drug” generally refers to a molecule that provides an activity or other effect in the cure, mitigation, treatment, or prevention of a disease.
  • drug generally refers to a molecule that provides an activity or other effect in the cure, mitigation, treatment, or prevention of a disease.
  • active agents in veterinary and in medicine, thus generally referring to prevention, mitigation and/or treatment of animals, in particular mammals, and more particularly in humans.
  • pharmaceutical composition refers to compositions for administration to human and non-human animals. This term thus includes veterinary
  • the compound of interest is attached to the linker through a connecting diradical selected from the diradicals of formula (I) to (X) as defined above.
  • a connecting diradical selected from the diradicals of formula (I) to (X) as defined above.
  • the list of diradicals is not exhaustive.
  • the diradical connecting the linker and the compound of interest is cleavable, for example, in the presence of glutathione, hydrolytic enzymes or in acidic environment.
  • the connecting diradical between the linker and the compound of interest is selected from the diradicals of formula (I), (II), (III), (IV), (V), (VI), (VIII), (IX) and (X).
  • the compound of interest may be a small molecule, DNA, RNA or a protein.
  • these compounds are protected from degradation and transported through the cell membrane.
  • the nanovector comprises a pharmaceutically active agent selected from anti-tumoral agents, antibiotics, antioxidants, anti-inflammatory agents and a gene therapy agent.
  • said pharmaceutically active agents are attached to the linker through a cleavable connecting diradical selected from those with formula (I), (II), (III), (IV), (V), (VI), (VIII), (IX) and (X).
  • the nanovectors of the invention comprise a gene therapy agent. Preferential delivery of said gene therapy agent to the cell nucleus is achieved by using the nanovector of the invention, thus enhancing the effect of the gene therapy.
  • gene therapy agent refers to compounds that modify the expression of a target gene.
  • the gene therapy agent may be another gene, or an oligonucleotide, whose function is to overexpress or underexpress the target gene, for example by inhibiting or enhancing regulatory elements that control the transcription of said gene.
  • the gene therapy agent may be an "inhibitory oligonucleotide", which generally refers to an oligonucleotide that may reduce transcription of a target gene.
  • Said inhibitory oligonucleotides are usually small single stranded RNA, small hairpin RNA (shRNA) or a small interference RNA (siRNA).
  • shRNA small hairpin RNA
  • siRNA small interference RNA
  • the nanovectors of the invention comprise an anti-tumoral agent and are useful for cancer prevention and/or treatment.
  • Any type of anti- tumoral agent may be delivered by a nanovector of the invention.
  • the anti-tumoral drug belongs to the group of cytotoxic, DNA-damaging anti- tumoral agents, which kill rapidly dividing cancer cells, such as alkylating agents (e.g. cis-platin), antimetabolites (e.g. methotrexate, pemetrexed, capecitabine), anti-microtubule agents (e.g. taxanes, vinca alkaloids), topoisomerase inhibitors (e.g. camptothecin, SN-38 (7-Ethyl-10-hydroxy- camptothecin), irinotecan, topotecan) and cytotoxic antibiotics (e.g.
  • alkylating agents e.g. cis-platin
  • antimetabolites e.g. methotrexate, pemetrexed, cap
  • the nanovectors of the invention may also deliver anti- tumoral agents for targeted cancer therapy, such as tyrosine kinase inhibitors, serine/threonine kinase inhibitors and monoclonal antibodies. These tumoral agents for targeted cancer therapy block the growth of cancer cells by interfering with specific targeted molecules needed for carcinogenesis and tumor growth.
  • the nanovectors of the invention achieve efficient and preferential delivery of the antitumoral drug to the cancer cell. As mentioned above, resistance to opsonization and EPR effect maintains the nanovector circulating around the tumoral cells for a longer time. Targeting of said tumoral site is also enhanced by the positive charge of the linker. Additionally, the nanovector's
  • the configuration allows for its effective uptake and facilitates delivery of the antitumoral agent to the cell nucleus, which is one preferential site of action when the drug is a DNA-damaging anti-tumoral.
  • the M-S (preferably Au- S) bond between the linker and the NPs core is cleavable in the presence of glutathione, which is overexpressed in tumoral cells.
  • GSH- mediated preferential release of the linker in the interior or the proximity of tumoral cells further increases the selectivity of the treatment and minimizes side effects by reducing toxicity to non-tumoral cells.
  • the nanovector of the invention comprises an antitumoral agent and is for use in the prevention and/or treatment of metastatic and/or drug-resistant cancer.
  • the nanovectors of the invention enhance the activity of the anti-tumoral drugs forming part of the nanovector, in particular, the activity against tumoral cells (when compared with non-tumoral cells).
  • the anti-tumoral agent is camptothecin, SN-38, irinotecan, topotecan, paclitaxel, docetaxel or doxorubicin.
  • the antitumoral agent is camptothecin.
  • Camptothecin also called "CPT” is a cytotoxic quinoline alkaloid which inhibits the DNA enzyme topoisomerase I (topo I).
  • CPT has very high cytotoxic activity but its clinical use is limited by its low solubility and adverse side effects.
  • Using the nanovectors of the inventions to deliver CPT enhances its solubility and generally improved its pharmacological profile while reducing side effects as explained above.
  • the nanovector of the invention further increases the activity of the drug by maintaining the "closed" lactone form. This feature is very important, since it is known that, in physiological conditions where the pH is close to pH 7, the lactone ring of the CPT molecule is opened resulting in the carboxylate form (Figure 3), which is inactive because its negative charge impedes the binding of CPT to - and its diffusion across - the cell membrane. Also, the carboxylate form of CPT was shown to have a lower activity against Topo I.
  • the invention relates to a nanovector wherein the pharmaceutically active agent is selected from camptothecin, irinotecan, SN-38 and topotecan.
  • the pharmaceutically active agent is attached to the amphiphilic linker through the X atom of a connecting diradical (CR) of formula (I), wherein X is O.
  • the nanovector has general formula (XXVI):
  • n 8
  • m 2
  • m' 3
  • x 1
  • R is OH
  • R 2 is H
  • R3 is ethyl
  • n 8
  • m 2
  • m' 3
  • x 1
  • R 4 is OH
  • n 8
  • m 2
  • m' 3
  • x 1
  • R 4 is 4-(piperidin-1 -yl)piperidine-1 -carbonyloxy, R2 is H and R3 is ethyl.
  • n 8
  • m 18
  • m' 19
  • x 1
  • y 1
  • n' 3
  • each Ri is methyl
  • R 4 is OH
  • R 2 is H
  • R 3 is ethyl
  • n 8
  • m 18
  • m' 19
  • x 1
  • y 1
  • n' 3
  • each Ri is methyl
  • R is OH
  • R 2 is dimethylaminomethyl
  • R3 H.
  • n 8
  • m 18
  • m' 19
  • x 1
  • y 1
  • n' 3
  • each Ri is methyl
  • R 4-(piperidin-1 -yl)piperidine-1 -carbonyloxy
  • R 2 is H and R3 is ethyl.
  • n 8
  • m and m' are 19, x is 0, y is 1
  • n' 3
  • each Ri is methyl
  • R is OH
  • R 2 is H
  • R3 is ethyl
  • n 8
  • m and m' are 19, x is 0, y is 1
  • n' 3
  • each Ri is methyl
  • R is OH
  • R 2 is dimethylaminomethyl
  • R3 H.
  • the nanoparticle is preferably a nanosphere having a core diameter preferably from 2-20 nm, more preferably 2-10 nm, even more preferably 2 nm.
  • the nanovector of the invention has general formula XIII:
  • n 1 -20, m is 2-100, n' is 2-100 and R1 is C1-C20 alkyl .
  • n 5-10, m is 2-10, n' is 2-10 and R1 is C1-C10 alkyl.
  • the nanovector of the invention comprises a
  • pharmaceutically active agent in particular an antitumoral agent that is substantially inactive and insoluble in water in physiologic conditions. Said active agents then become soluble in water and chemically stable when forming part of the nanovector.
  • the nanovector of the invention may comprise a targeting agent or a contrast agent.
  • targeting agent refers to compounds that target a particular cell, organ, and /or tissue for which treatment is desired.
  • the targeting agents may be small molecules, antibodies, RNA, DNA, and aptamers.
  • the targeting agent is folic acid.
  • contrast agent refers to a substance used to enhance the contrast of structures or fluids within the body in medical imaging.
  • the NP of the invention per se may be a contrast agent thanks to the optical properties of the core's noble metal. For instance, when the core's surface comprises gold, surface enhanced Raman spectrometry (SERS), as well as interaction of light in the visible and near-infrared wavelengths with electrons on the gold NPs surface, can be used to visualize the NPs of the invention, rendering them useful tools for cancer diagnosis.
  • SERS surface enhanced Raman spectrometry
  • the core may be comprised by a first material coated by a noble metal, preferably gold, said first material also possessing optical properties, which widens the versatility of the NPs for imaging purposes.
  • a first material coated by a noble metal preferably gold
  • said first material also possessing optical properties, which widens the versatility of the NPs for imaging purposes.
  • FRET Forster resonance energy transfer
  • the core may comprise a liposome or a micelle which may encapsulate a contrast agent.
  • the invention refers to a nanovector comprising a contrast agent, or a targeting agent, wherein the contrast agent, or the targeting agent, is directly attached to the linker through a connecting diradical as defined above.
  • a pharmaceutically active agent is not required for imaging applications or even for research purposes.
  • particular embodiments provide a nanovector comprising a pharmaceutically active agent and a contrast agent, such as a small fluorescent molecule.
  • nanovector comprising a pharmaceutically active agent and a targeting agent, such as folic acid.
  • a targeting agent such as folic acid.
  • the nanovector comprises CPT and folic acid.
  • the presence of a targeting agent may further enhance selectivity of the nanovector by
  • nanovector comprising a pharmaceutically active agent, a contrast agent and a targeting agent.
  • the NPs or the nanovectors of the invention are generally formulated as a pharmaceutical composition (for therapeutic purposes) or as a composition for diagnosis (for diagnosis applications), and therefore also contain appropriate excipients and carriers.
  • the formulations may be liquid or solid and can be administered to subjects by various methods known to persons of ordinary skill in the art.
  • the therapeutic compositions of the present invention can be administered by oral
  • the therapeutic compositions can be administered by topical application (e.g. transdermal, ointments, creams, slaves, eye drops, and the like). Additional modes of administration can also be envisioned by persons of ordinary skill in the art.
  • the administration of the pharmaceutical composition is intramuscular, intravenous, intraperitoneal or intratumoral.
  • suitable formulations include aqueous and non- aqueous, isotonic sterile injection solutions which have suitable pH and stability, which can contain for instance anti-oxidant agents, buffers and bacteriostatic agents; and aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the nanovectors of the invention may be useful for the treatment of cancer in a patient suffering therefrom, especially in cases of resistant or metastatic cancers.
  • cancers of interest include, but are not limited to head, neck and lung tissue; gastrointestinal tract and pancreas, such as gastric carcinoma, colorectal adenoma, colorectal carcinoma and pancreatic carcinoma; hepatic tissue, such as hepatocellular carcinoma; Kidney and urinary tract, such as bladder carcinoma, renal carcinoma; breast tissue, such as breast carcinoma; neural tissue, such as neuroblastoma and meningioma malignant; skin tissue, such as melanoma; and hematological tissues, such as lymphoma and leukemia.
  • the invention also provides a method for preparing the NPs and nanovectors of the invention.
  • Said method comprises first forming a NP core comprising a noble metal (M), preferably gold, on its surface [Core-M] (step 1 ).
  • M noble metal
  • Several methods to prepare NPs are known in the art (Boisselier,ef al, Chem Soc. Rev. 2009, vol. 38, p. 1759-1782; Dreaden, et al, Chem. Soc. Rev. 2012, vol. 41 , p.
  • Non- metalic NPs may also be prepared and further coated with a noble metal, preferably gold, but also Ag or Pt, by methods known in the art (Jin, et al, ACC. Chem. Res. 2014, vol. 47, p. 138-148).
  • Non-metallic NPs for instance, magnetic iron oxide, silica, carbon or polymeric NPs, but also liposomes or micelles with nanometric size, may be likewise prepared by known methods (Ho, et al, Acc. Chem. Res. 201 1 , vol. 44, p. 875-882).
  • the linker is prepared by conventional organic chemistry synthesis methods, as illustrated in the Examples.
  • said ligand comprises al alkylthiolate in one end [HS-(CH 2 ) n -Linker, wherein n is 1 to 20]
  • a third step (step 3) the NP of the invention is formed by carrying out an exchange reaction of the nanoparticulated core carrying a noble metal, preferably gold, on its surface with a large excess of the prepared HS-(CH 3 ) n - Linker.
  • This reaction is known as the Murray place-exchange reaction
  • the above preparation sequence may vary, for example, by preparing first the linker or by preparing the linker and the NP core at the same time. It is also possible to prepare a NP core and perform the place exchange reaction with a linker precursor, so long as said linker precursor contains an alkylthiolate, and later complete the synthesis of the linker.
  • the compound of interest i.e. a pharmaceutically active agent, contrast agent or targeting agent
  • step 1 ' the compound of interest
  • a pharmaceutically active agent i.e. a pharmaceutically active agent, contrast agent or targeting agent
  • Comp.lnt.-RG+RG'-Linker-(CH 2 )n-S-PG ⁇ Comp.lnt.-CR-Linker-(CH 2 ) n -S-PG wherein CR is one of the diradicals of formula (l)-(X) defined above, preferably a cleavable diradical of formula (I), (II), (III), (IV), (V), (VI), (VIII), (IX), and (X).
  • Non-limiting RG and RG' are -OH, -CO 2 H, -OP(O)(OR') 2 , -CHO, -NR-SO 2 H, - NR-CHO, -NH 2 , -SO 2 H and -SH where R' is H or (Ci-C 6 )alkyl .
  • a second step an exchange reaction is performed with the nanoparticulated core carrying a noble metal and the de-protected product of step 1 '.
  • the above preparation sequence may present several modifications.
  • the Comp.lnt. may be first bound to a linker precursor (step 1 'a). This step is performed in the same conditions as defined for step 1 ' above. The synthesis of the linker is then completed by conventional organic synthesis methods. Finally, the de- protected compound of interest bound to the linker [Comp.lnt.-CR-Linker- (CH 2 )n-SH] is attached to the nanoparticulated core carrying a noble metal by place exchange reaction.
  • An exemplary procedure to prepare a nanovector according to this embodiment of the invention is provided below. A still more detailed procedure according to this embodiment is provided in the examples.
  • the hydrophilic moiety (B) comprises the triazol moiety of formula (XXII):
  • n is a value from 1 to 20; n', m and m' are independently a value from 1 -200; x is 0 or 1 , y is 0 or 1 , and each Ri is independently (Ci-C2o)alkyl, and wherein Z is a compound selected from an active agent, a targeting agent, or a contrast agent that can be linked through an ester bond, wherein the process comprises the following steps:
  • n, n', m, m', x, y, Ri and Z are as previously defined and R is a thiol protective group PG, preferably trityl, or alternatively, when in the nanovector of fornnula (XXVIa) x is 0, carrying out a cycloaddition reaction of a compound of formula (XXX) and a compound of formula (XXIX)
  • Z is a compound selected from SN-38, camptothecin and irinotecan, which are attached as shown in the nanovector of formula (XXVI) as herein defined.
  • thiol protective groups can be used.
  • Protective and deprotective conditions are described for example in T. W. Green and P. G. M. Wuts, Protective Groups in Organic Chemistry (Wiley, 3rd ed. 1999, Chapter 6, pp. 454-493).
  • Thioacid (XXXV) can be prepared by protecting the thiol group of the corresponding commercial thioacid.
  • Aminopolyethyleneglycol (XXXIV) can be prepared by the process shown below:
  • compound of formula (XLV) it can be obtained by esterification of the hydroxyl group at C20 of the CPT derivative with the bifunctional spacer of formula (XLIV), which can be obtained from polyethylene glycol in 4 steps consisting of (i) mesylation, (ii) reaction in an SN2 manner with metal azide, and (iii) reaction of the obtained metal alkoxide with haloacetic acid.
  • y is 1 (i.e. compound of formula (XLVI)), it can be obtained from compound (XLV) and acid (XLIV).
  • NMR spectra were recorded with following spectrometers: Bruker DRX-300, DPX-400, and DRX-500 using CDCI 3 as solvent. Chemical shift values ( ⁇ ) are reported in ppm and referred to tetramethylsilane (TMS), utilized as internal reference; coupling constants are reported in Hz.
  • TMS tetramethylsilane
  • Example 1 Synthesis of amhiphilic positively charged linker-tethered camptothecin (CPT-L-SH) Synthesis of 11-mesyl-3,6,9-trioxaundecan-1 -ol (1). HO " v ⁇ ' ⁇ " v v OMs
  • N-hydroxysuccinimide 165.12 mg, 1 .43 mmol
  • EDC 286 mg, 1 .49 mmol
  • the reaction was stirred during 12 hours.
  • the reaction was stopped with H 2 O (10 mL) and the CH2CI2 was extracted.
  • the organic extracts were dried using anhydrous Na2SO 4 and the solvent was evaporated.
  • the product obtained (610 mg, 1 .12 mmol) was added directly to a reaction with 1 1 -amino-3,6,9,trioxaundecan-1 - ol, 4 (310.07 mg, 1 .12 mmol) in the presence of Et 3 N (156 ⁇ _, 1 .12 mmol) and CH 2 Cl2 (20 mL) during 14 hours.
  • Et 3 N 156 ⁇ _, 1 .12 mmol
  • CH 2 Cl2 20 mL
  • the solvent was evaporated and the product was purified by flash column chromatography, eluting with a mixture CH2CI2 / MeOH (13:1 ), to give 6 (150 mg, 20%) as an uncoloured syrup.
  • a tetraoctylammonium bromide solution TOAB (2.1 g / 150 ml toluene; 3.8 mmol; 1 .5 eq) were added upon a gold salt solution HAuCI 4 (1 g/ 150 ml H 2 0; 2,5mmol; 1 eq; Carbosynth 50%) for being stirred for 15 min.
  • pentanethiol (0,7 ml; 5 mmol; 2 eq) was dropped during 15 minutes above the previously prepared solution changing the color of the solution from orange to white.
  • Gold salt reduction was performed by addiction of sodium borohydride (2g / 10 ml H 2 O; 50 mmol; 20 eq) and the reaction was left to react during 5 hours under strong magnetic stirring. A separation funnel was used and organic phase was evaporated before adding ethanol (600 ml). Nanoparticles in the ethanol were freeze-stored during two days leading to precipitation of NP at the bottom. Ethanol was removed and changed for fresh ethanol for 5 times. When purification was completed, 500 mg of black solid NP protected with pentanethiol were obtained.
  • the nanoparticles were characterized by UV-Vis spectroscopy (see FIG. 14, UV-Vis spectrum recorded in dichloromethane show a width surface plasmon band centered at 500 nm characteristic of small gold NP); NMR spectroscopy (see FIG. 15: Gold NP were purified from TOAB and effectively thiol protected because of pentanethiol bands were solely observed in the RMN spectrum, but not the TOAB bands); IR spectroscopy (see FIG. 16: As compared with pure pentanehiol, IR spectrum of gold NP only shows bands from this thiol); TEM analysis (see FIG. 17: synthesized gold NP have an average core diameter of 2 nm as shown in TEM image)
  • the nanoparticles Au-L-CPT were characterized by UV-vis spectroscopy (see FIG. 18, UV-Vis spectrum recorded in dichloromethane. Comparing with the previous step, UV-Vis spectrum of Au-L-CPT shows the two characteristical CPT bands (355 and 380 nm), and due to functional ization plasmon band was shifted to longer wavelengths; from 500 to 530 nm); DLS (an average diameter of 20 nm was obtained for gold NP functionalized with the short organic ligand in an aqueous dispersion. Zeta potential in water is positive (25 mV).
  • the nanoparticles Au-L1 -CPT were characterized by UV-vis spectroscopy (see FIG. 21 : UV-vis spectra of Au-L1 -CPT shows the two typical CPT bands. Plasmon band is shifted to 540 nm and sharper compared to previous step without Ligand); DLS (The average size of the Au-L1 -CPT in solution was measured by dynamic light scattering. The measured diameter is 50 nm in water and 30 nm in PBS (20 mM in potassium phosphate and 100 mM in NaCI). In both cases a slight proportion of bigger NP were also observed); NMR spectroscopy (see FIG.
  • the nanoparticles Au-L2-CPT were characterized by UV-vis spectroscopy (see FIG. 23: The UV-vis spectrum of functionalized NP shows CPT signal as in the previous cases. The plasmon band becomes sharper and shift to 524nm in water and at 532 nm in PBS); DLS (The DLS study of an aqueous solution of Au-L2-CPT gives an average diameter of 30 nm and 7 nm in PBS. If DMSO was added to the aqueous dispersion, average diameter becomes both smaller and uniform (4,8 nm). Zeta potential values ranges from -0 and 1 mV); NMR spectroscopy (see FIG.
  • Linker-CPT was efficiently released from Au- L-CPT in the presence of GSH.
  • the rate of release was proportional to GSH concentration.
  • H2AX is a variant of the H2A protein family, which is a component of the histone octomer in nucleosomes. It is phosphorylated by kinases such as ataxia telangiectasia mutated (ATM) and ATM-Rad3-related (ATR) in the PI3K pathway. This newly phosphorylated protein, gamma-H2AX, is the first step in recruiting and localizing DNA repair proteins.
  • ATM ataxia telangiectasia mutated
  • ATR ATM-Rad3-related
  • DSBs can be induced by mechanisms such as ionizing radiation or cytotoxic agents and subsequently, gamma-H2AX foci quickly form. These foci represent the DSBs in a 1 :1 manner and can be used as a biomarker for damage.
  • An antibody can be raised against gamma-H2AX which can therefore be visualized by immunofluorescence through secondary antibodies.
  • the detection and visualization of gamma-H2AX by flow cytometry allow the assessment of DNA damage, related DNA damage proteins and DNA repair.
  • Gamma-H2AX also has other applications in the detection of genomic damage caused by cytotoxic chemical agents and environmental and physical damage, especially in the context of cancer treatment and therapy (In Vivo, 2008, vol. 3, p.
  • U2OS cells (ATCC number HTB-96) were cultured in Dulbecco ' s modified Eagle ' s medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma), 2 mM L-Glutamine (Sigma) and standard antibiotics.
  • DMEM Dulbecco ' s modified Eagle ' s medium
  • FBS fetal bovine serum
  • FBS 2 mM L-Glutamine
  • U2OS cells (5 x 10 4 cells) were seeded in 2 cm 2 wells with coverslips and incubated for 16 hours with 5% CO2 at 37°C. Different treatments were carried out for 30 minutes and 150 minutes in the presence of the nucleotide analogue EdU (10 ⁇ ): DMSO (Sigma), CPT (2 ⁇ , Sigma), Au-C5, and Au- L-CPT (equivalent to 2 ⁇ of CPT). After the incubation period, cells were fixed in the wells for 10 minutes with 4% para-formaldehyde (Sigma).
  • the cell samples were permeabilized during 2 hours with 0.2% Triton in PBS, washed in PBS, and then blocked in PBS with 5% BSA for 30 minutes.
  • Cells were incubated in the wells, with coverslips, with anti-YH2AX-ms primary antibody (Millipore) at a dilution of 1 :1000 for 30 minutes.
  • Cells were washed three times with PBS-Tween 0.1 % for 5 minutes each wash.
  • the cells were incubated with goat anti-mouse Alexa Fluor 488 secondary antibody (Invitrogen) at a dilution of 1 :1000 for 30 minutes.
  • Cells were washed three times with PBS-Tween 0.1 % for 5 minutes each wash.
  • VectaShield mounting media Vector Laboratories
  • samples were analysed by a Leica DM-6000B microscope equipped with a 100x/1 .40 NA oil immersion objective lens using the corresponding filters for DAPI detection, EdU and ⁇ 2 ⁇ detection.
  • Images were taken with a digital charge-coupled device camera (DFC350; Leica) and LAS AF (Leica) software and quantified using MetaMorph Microscopy automation and imaging software (Molecular Devices).
  • Linker- CPT and Au-L-CPT will damage all tumor cells that undergo DNA synthesis and proliferation within 4-5 days after acute treatment.
  • cytostatic drugs such as MX, topotecan (TPT), doxorubicin, and daunorubicin.
  • U2OS (ATCC number HTB-96)human osteosarcoma cells were cultured in Dulbecco ' s modified Eagle ' s medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma), 2 mM L-Glutamine (Sigma) and standard antibiotics.
  • MCF7 (ATCC number HTB-22) human mammary gland, adenocarcinoma breast cells were cultured in RPMI-1640 medium (RPMI-1640 Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma), 2 mM L-Glutamine (Sigma) and standard antibiotics.
  • MCF10A (ATCC number CRL-1031 ) human mammary gland, fibrocystic breast cells were cultured in DMEM-F12 (Sigma) supplemented with 5% inactivated horse serum (HS, Gibco), 10mg/mL insulin (Sigma), 20 ng/mL epithelial growth factor (EGF PeproTech), 10ng/ml_ cholera toxin (Sigma) 500 ng/mL hydrocortisone (Sigma), 2 mM L-Glutamine (Sigma) and standard antibiotics.
  • HS horse serum
  • EGF PeproTech epithelial growth factor
  • 10ng/ml_ cholera toxin (Sigma) 500 ng/mL hydrocortisone (Sigma)
  • 2 mM L-Glutamine (Sigma) and standard antibiotics.
  • the data for the survival curve for the U2OS cell line were generated by clonogenic assays.
  • the cells were treated with CPT (10 ⁇ , 1 ⁇ , and 0.1 ⁇ ) and Au-L-CPT (equivalent to 1 ⁇ , 0.5 ⁇ , 0.2 ⁇ , 0.1 ⁇ and 0.02 mM of CPT) for the intense treatment, or for the chronic treatment, CPT (100 nM, 10 nM and 1 nM) and Au-L-CPT (equivalent to 100 nM, 10 nM and 1 nM of CPT) were added to the cells.
  • the intense treatment cells were treated for 150 minutes. Cells were then washed with PBS and fresh media was added. The cells were incubated for 12-14 days at 37°C to allow the growth of colonies. After this period, the colonies were stained with 0.5% crystal violet / 20% ethanol and counted. For the intense treatment, the treatment was prolonged for 12-14 days at 37°C, before the colonies were stained and counted.
  • the data for the survival curve for the MCF7 cell line were generated by clonogenic assays.
  • Cells were submitted to an intense treatment with CPT (1 ⁇ , 0.2 ⁇ and 0.04 ⁇ ) and Au-L-CPT (equivalent to 0.2 ⁇ , 0.04 ⁇ and 0.0082 ⁇ of CPT), using 0.001 % DMSO and Au-C5 at the same
  • the cells were treated for 150 minutes, then washed with PBS and fresh media was added. The cells were incubated for 12-14 days at 37°C to allow the growth of colonies. After this period, the colonies were stained with 0.5% crystal violet / 20% ethanol and counted. The data for the survival curve for the MCF10A cell line were generated by clonogenic assays.
  • Cells were submitted to an intense treatment with CPT (1 ⁇ , 0.2 ⁇ and 0.04 ⁇ ) and Au-L-CPT (equivalent to 1 ⁇ , 0.2 ⁇ , and 0.04 ⁇ of CPT), using 0.001 % DMSO and Au-C5 at the same concentration as the highest concentration of Au-L-CPT as controls for the CPT and Au-L- CPT, respectively.
  • the cells were treated for 150 minutes, then washed with PBS and fresh media was added. The cells were incubated for 12-14 days at 37°C to allow the growth of colonies. After this period, the colonies were stained with 0.5% crystal violet / 20% ethanol and counted.
  • results are shown if figures 7 and 8. It was observed that cell growth of tumoral U2OS and MCF7 cells is inhibited by Au-L-CPT to a greater extend when compared with CPT alone (figure 7B and C, and figure 8A and B). This indicates that the anti-tumoral agent does not lose its activity when forming part of a nanovector of the invention. Indeed, the results show that the nanovector of the invention even enhances activity of CPT. On the other side it was shown that the nanovector of the invention comprising CPT is relatively less damaging to non-cancerous cells. As observed in figure 8A and B, Au-L- CPT did not inhibit cell growth of non-tumoral MCF10A cells as much as CPT alone. This indicates that the nanovector of the invention comprising anti- tumoral CPT can reduce the toxicity of the drug for normal cells, thus reducing un-desired side effects of the treatment.
  • Example 9 Antitumor eficacy study of CPT coated nanoparticles in a subcutaneous tumor model of the colorectal HT29 cell line To evaluate the ability of CPT-coated nanopartilces in vivo for neutralizing the tumor growth athymic mice bearing subcutaneous tumors [Athymic Nude- Foxnl nu (females, 6-7 weeks old, Harlan Laboratories)] were used.
  • Tumors were allowed to grow during 16 days until reaching a mean tumor volume of approximately 140 mm 3 . Then, animals were treated by
  • the nanovector of the invention Au-L-CPT (administered at doses of 24 mg/kg) was as effective as irinotecan (administered at doses of 80 mg/kg) both during the treatment and during the following 10 days after completing the treatment (see FIG. 9).
  • a core comprising on its surface a noble metal (M) selected from gold, silver and platinum, and
  • a positively charged amphiphilic linker comprising:
  • a hydrophilic positively charged moiety comprising: at least one polymeric chain (P) selected from polyethylene glycol (PEG), polychitosan, polydextran, copolymers thereof, and a derivative thereof selected from poly(chitosan-g-lactic acid), poly(chitosan-N- isopropylacrylamide), phosphorylated chitosan and polycarboxymethyl dextran; and at least one positively charged group (G + );
  • PEG polyethylene glycol
  • PEG polychitosan
  • polydextran polydextran
  • copolymers thereof and a derivative thereof selected from poly(chitosan-g-lactic acid), poly(chitosan-N- isopropylacrylamide), phosphorylated chitosan and polycarboxymethyl dextran
  • G + at least one positively charged group
  • the hydrophobic alkyl thiolate moiety is attached to the noble metal (M) on the core surface through M-S bond;
  • a connecting diradical selected from the group consisting of the diradicals of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) and (X)
  • P1 , P2 and P3 are each a polymeric chain of molecular weight comprised from 300-30000 Da independently selected from PEG, polychitosan, polydextran, copolymers and a derivative thereof selected from poly(chitosan- g-lactic acid), poly(chitosan-N-isopropylacrylamide), phosphorylated chitosan and polycarboxymethyl dextran;
  • G1 + and G2 + are each a positively charged group independently selected from quaternary ammonium salts and quaternary sulfonium salts;
  • CR1 , CR2, CR3 and CR4 are each a connecting diradical independently selected from the group consisting of the diradicals of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) and (X) as defined in clause 1 ;
  • a is 1 or 0, with the provisio that the hydrophilic positively charged group comprises at least one P and one G + ;
  • n is comprised from 1 to 5.
  • Clause 4 The nanoparticle according to anyone of clauses 1 -3, wherein the core comprises a first material covered by a second material, wherein the first material is selected from metal, metal oxide, metal organic framework (MOF), carbon, polymer, liposome and micelle and the second material is a noble metal selected from Au, Ag and Pt.
  • first material is selected from metal, metal oxide, metal organic framework (MOF), carbon, polymer, liposome and micelle
  • MOF metal organic framework
  • the second material is a noble metal selected from Au, Ag and Pt.
  • a nanovector comprising the nanoparticle as defined in anyone of clauses 1 -6, and a compound selected from a pharmaceutically active agent, a targeting agent, a contrast agent, and mixtures thereof, wherein said compound is attached to the hydrophilic positively charged moiety of the nanoparticle complex through a connecting diradical (CR) selected from the group consisting of the diradicals of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) and (X) as defined in clause 1 .
  • CR connecting diradical
  • Clause 8 The nanovector according to clause 7, which comprises a pharmaceutically active agent selected from anti-tumoral agents, antibiotics, antioxidants, anti-inflammatory agents and a gene therapy agent.
  • Clause 1 1 A pharmaceutical composition or a composition for diagnostic purposes comprising a therapeutically or diagnostic effective amount of a nanopartide as defined in any of the clauses 1 -6 or a nanovector as defined in any of the clauses 7-10, together with appropriate amounts of
  • composition comprising a nanovector as defined in clause 1 1 , wherein the nanovector comprises a pharmaceutically active agent, for use as a medicament.
  • composition comprising a nanovector as defined in clause 1 1 , wherein the nanovector comprises an anti-tumoral agent, for use in the prevention and/or treatment of cancer.
  • Clause 14 A nanopartide as defined in anyone of clauses 1 -6, a nanovector as defined in clause 7 or a composition for diagnosis comprising a
  • nanoparticle or a nanovector as defined in clause 1 1 wherein the nanovector comprises a contrast agent, for use in diagnosis.
  • Clause 15. Use of a nanoparticle as defined in anyone of clauses 1 -6 for drug delivery
  • Bailly C DNA relaxation and cleavage assays to study topoisomerase I inhibitors. Methods Enzymol. 2001 ;340:610-623

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Abstract

La présente invention concerne une nanoparticule présentant une taille comprise entre 2 et 300 nm, comprenant : (i) un noyau comprenant sur sa surface un métal noble sélectionné parmi l'or, l'argent ou le platine; et (ii) un lieur amphiphile chargé éventuellement positivement comprenant une fraction d'alkyle thiolate en C1-C20 hydrophobe et une fraction hydrophile qui est éventuellement chargée positivement et hydrophile. L'invention concerne également des nanovecteurs comprenant les nanoparticules de l'invention et un composé d'intérêt, tel qu'un agent pharmaceutiquement actif, en particulier un agent anti-tumoral. La présente invention concerne également des nanoparticules et des nanovecteurs tels que définis ci-dessus pour le diagnostic ou pour la prévention et/ou le traitement d'une maladie, en particulier le cancer.
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CN109125266A (zh) * 2018-09-03 2019-01-04 南京大学 脂质体包裹有机金属骨架纳米递药系统的制备方法及应用
CN114848846A (zh) * 2022-05-26 2022-08-05 深圳市世格赛思医疗科技有限公司 一种药物递送系统及其制备方法与应用
WO2023154740A1 (fr) * 2022-02-08 2023-08-17 Indiana University Research And Technology Corporation Nanoparticule amphiphile antibiotique et ses procédés d'utilisation contre des bactéries gram négatif et/ou gram positif

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108864324A (zh) * 2018-06-12 2018-11-23 中国科学院烟台海岸带研究所 一种三氮唑基壳聚糖双季铵盐及其制备方法和应用
CN108864324B (zh) * 2018-06-12 2020-08-04 中国科学院烟台海岸带研究所 一种三氮唑基壳聚糖双季铵盐及其制备方法和应用
CN109125266A (zh) * 2018-09-03 2019-01-04 南京大学 脂质体包裹有机金属骨架纳米递药系统的制备方法及应用
WO2023154740A1 (fr) * 2022-02-08 2023-08-17 Indiana University Research And Technology Corporation Nanoparticule amphiphile antibiotique et ses procédés d'utilisation contre des bactéries gram négatif et/ou gram positif
CN114848846A (zh) * 2022-05-26 2022-08-05 深圳市世格赛思医疗科技有限公司 一种药物递送系统及其制备方法与应用

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