WO2018162676A1

WO2018162676A1 - DISINTEGRATABLE POROUS ORGANOSILICA OR ORGANOMETALOXIDE NANOPARTICLES AND USES THEREOF AS VEHICLE FOR CONTROLLED DELIVERY OF siRNA

Info

Publication number: WO2018162676A1
Application number: PCT/EP2018/055829
Authority: WO
Inventors: Luisa De Cola; Thomas Baumert; Laura Maggini; Valentina GIGLIO; Mike DENTINGER
Original assignee: Université De Strasbourg; Centre National De La Recherche Scientifique; Ihu Strasbourg - Institut Hospitalo-Universitaire De Strasbourg; Inserm - Institut National De La Santé Et De La Recherche Médicale -
Priority date: 2017-03-08
Filing date: 2018-03-08
Publication date: 2018-09-13

Abstract

The present invention relates to disintegratable porous organometaloxide (e.g., organosilica) nanoparticles for the delivery of nucleic acid-type biomolecules such as siRNA, a method for producing the same, and uses thereof including their use for prevention and treatment of human cancer.

Description

DISINTEGRATABLE POROUS ORGANOSILICA OR

ORGANOMETALOXIDE NANOPARTICLES AND USES THEREOF AS VEHICLE FOR CONTROLLED DELIVERY OF siRNA Related Patent Applications

This Application relates to European Patent Application n° EP 15700665.1 filed on 14 January 2015, and claims priority to provisional European Patent Application n° EP 17305249.9 filed on 8 March 2017; the entire contents of both of which are hereby incorporated by reference.

Field of the Invention

The present invention relates to disintegratable porous, preferably mixed- mesoporous, organometaloxide (e.g., organosilica) nanoparticles for the delivery of nucleic acid-type bio molecules such as siRNA, a method for producing the same, and uses thereof.

Background of the Invention

Conventional cancer treatments include surgery, radiotherapy and chemotherapy, all of which present significant drawbacks:

- Surgery: critical normal tissues invasion, ineffective for metastasis;

- Radiotherapy: damage normal cells, side effects;

- Chemotherapy: killing healthy cells, toxicity to the patient, drug resistance.

Furthermore, there is an unmet medical need for individualized therapies since each patient-specific cancer has a specific genetic make up.

The discovery of RNA interference - gene silencing technology by double stranded RNA in 1998 opened routes to gene therapy. However, several drawbacks associated with siRNA technology still exist, namely, the degradation by enzymes before reaching the target, fast renal clearance, and the inability to efficiently penetrate cell membranes.

Therefore, there remains a need for efficient technology that would enable to use siRNA technology without the above-mentioned drawbacks. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : a) Schematic representation of the synthetic process for LP-ssNPs b) SEM images of the LP-ssNPs. Scale bar = 500 nm. c) TEM images of the LP-ssNPs. Scale bar = 100 nm. d) Pore width distribution before (grey) and after pore expansion treatment (black), e) TEM analysis of a suspension of LP-ssNPs and f) LP-ssNPSs- APTES at 0.1 mg/mL, PBS, 37 °C undergoing GSH (10 mM) reduction (0-7 d). On the right side of the figure CTRL nanoparticles stirred in PBS for 7 days without GSH. Scale bar = 100 nm.

Figure 2 : Schematic representation of the different coating steps leading to the final material.

Figure 3 : a) FACS analysis after 3, 24 and 48 h incubation with r-LP-ssNPs at 50 and 100 μg·mL-l . b) Confocal images taken after 3, 24 and 48 h incubation with the r-LP-ssNPs (50 μg·mL^"1). Cell nuclei were stained with Hoechst 33342 (blue signal). The images on the right correspond to the merged signal, c) Z-stacking image of cells incubated for 24 h with r-LP-ssNPs (50 μgmL^"1). The cell nuclei were stained with DAPI (blue channel), cell membrane was stained with Alexa Fluor Phalloidin 647 (yellow channel). Excitation wavelengths are 355, 488, 594, and 633 nm for Hoechst 33342, Rhodamine B, and Alexa Fluor® 647 Phalloidin, respectively. Scale bars = 20 μιη.

Figure 4: a) Confocal images taken on Huh-7 cells after 24 h incubation with r-NH2- LP-ssNPs (50 μgmL^"1) . Colocalization experiments with Lysotracker® Blue DND- 22 revealed the sub- localization of particles in lysosomes area (overlap coefficient 0.62 ). Red channel: Rhodamine B; blue channel: Lysotracker®. ecxc = 405 and 488 nm for Lysotracker Blue DND-22 and Rhodamine B, respectively. Scale bars =

20 μιη. b) TEM images of Huh-7 cells incubated with LP-ssNPs (50 μg/mL) after 3, 24 and 48 h, as indicated on the images. The control image was taken on Huh-7 cells non exposed to the nanoparticles. Scale bars = 500 nm.

Figure 5: Cellular uptake of jp-PLKl@LP-ssNPs . Confocal images were taken after 3 h incubation with the particles. Cell nuclei were stained with Hoechst 33342 (blue) and the particles were doped with Rhodamine B (red). PLK1 siRNA were coupled to a Cyanine 5 dye (green) with a Cy5Label IT® siRNA Tracker Intracellular Localization Kit (Minis) and grafted on the particles. The right images correspond to the merge signal. Excitation wavelengths are 355, 488 and 633 nm. Scale bar = 20 μιη.

Figure 6 : Additional SEM images of the LP-ssNPs. Scale bar = 500 nm

Figure 7 : Additional TEM images of the sLP-ssNPs. Scale bar = 50 nm.

Figure 8 : N2 adsorption/desorption isotherms recorded on the ssNPs and LP-ssNPs. Figure 9 : SAXS pattern recorded on the LP-ssNPs.

Figure 10 : XPS survey spectra of LP-ssNPs.

Figure 11 : TGA thermogram recorded on the LP-ssNPs.

Figure 12 : a) UV-Vis spectra of different concentration of the PLK1 siRNA in MES buffer (pH 5), b) Calibration curve plot for the PLK1 siRNA in MES buffer pH 5. The equation given is y = 0.033 - 0.0115 with an R2 = 0.99943.

Figure 13 : Adsorption spectrum of the supernatants after incubation with the PLK1 siRNA with the following conditions: 0.1 mg/mL of NH₂-LP-ssNPs and NH₂-ssNPs were dispersed in 1 mL of a MES buffer at pH 5 (5 mM) in an Eppendorf tube. In another Eppendorf tube, the two strands of siRNA were mixed together in the same MES buffer pH 5 (1 mL in total) at a concentration of 60 μg/mL. The siRNA solution was shaken for 5 minutes before to add the dispersion of particles (total volume 2 mL). The particles were placed swirled o.n. at r.t. within a rotatory mixer and let overnight at room temperature. The particles were then centrifuged for 1 h at 14.5 krpm., The supernatant was removed and stored for further measurements. Absorbance spectra recorded with a UV- Visible Spectrophotometer. Reference of PLK1 siRNA at a concentration of 40 μg/mL (black curve), supernatant of jp- PLKl@NH2-ssNPs (blue) and supernatant of jp-PLKl@NH2-LP-ssNPs-PLKl

(red).

Figure 14 : Loading achieved after incubation of different concentration of siRNA with NH2-LP-ssNPs. Initial concentration of particles were 0.1 mg.mL

Figure 15 : Dynamic light scattering measurements proving the grafting of the different layer on the surface of the LP-ssNPs.

Figure 16 : Zeta Potential analysis after the different coating. Figure 17 : Metabolic activity of Huh-7 cells after incubation with different concentrations of LP-ssNPs.

Figure 18 : Excitation (solid lines) and emission spectra (dashed lines) of r-LP- ssNPs (red) andjp-PLKl@r-LP-ssNPs (blue). ). λ_θχε= 547 nm X_em= 580 nm. Figure 19 : TEM analysis of Huh-7 cells incubated with BPBPsLP-ssNPs at a concentration of 50 μg/mL for a) 3h, b) 24h and c) 48h. Scale bar = 500 nm.

Figure 20 : Biodistribution of the nanoparticles in vivo assessed by bio luminescence imaging. NMRI-nu mice were intravenously injected in the lateral tail vein with vehicle (n=2) or 9 mg/kg near-infrared fluorescent nanovector (n=3) and monitored for in vivo fluorescence for 33 days. Fluorescence (mean+/-SE) are expressed as average fluorescence efficiency. Fluorescence Imaging of 3 mice injected with nanovector and one control mouse injected with vehicle are shown for the time points lh, 3h, 5h and 24h.

Figure 21 : Schematic representation of the use of PLK1 -siR A loaded nanoparticles of the invention for the prevention and treatment of human cancer using human liver cancer/HCC as an example. The R A encoding polo-like kinase 1 protein was used as a target for an siR A-based prevention and treatment approach.

Figure 22: In vivo proof of concept for prevention and treatment of liver cancer: Anti-tumor activity of LP-ssNPs-PLKl in a human xenograft mouse model for liver cancer. NMRI-Nude mice bearing subcutaneous luciferase-expressing human Huh- 7 liver tumors were injected at the indicated time points (arrows) with vehicle, LP- ssNPs-Control or LP-ssNPs-PLKl nanoparticles. Tumor sizes shown as the median tumor volume and quantified by in vivo bio luminescence, are normalized at each time point to the initial value at day 0.

Figure 23: In vivo proof-of-concept for prevention and treatment of HCC using a mouse xenograft model with intrahepatic HCC (orthotopic model). To analyze the in vivo tumor suppression efficacy of nanoparticles-based siPLKl -delivery system in an orthotopic HCC model, we performed intra-tumoral injections (twice per week, 10μg of siRNA per mouse) of nanoparticles into intrahepatic tumors of Huh7- derived xenograft mouse model as described in the method section. (A) Approach:

Treatments with nanoparticle-PLKl were initiated when the average tumor volume reached 15- 100mm³ and the tumor growth was monitored at day 2, 5, 7, and 9 post treatments by ultrasound (see Methods). Results (mean ± s.e.m.) are reported as the tumor volume relative to the initial size. *p<0.05 Fisher's t-test. (B) Nanoparticles loaded with siRNA targeting PLK1 significantly reduced the tumor volume compared to the controls confirming that siPLKl was efficiently released from the particles targeting Huh7 cells. Abbreviation : BPBP - large pore redox-responsive mesoporous silica nanoparticles (also referred to as redox-responsive mixed- mesoporous organosilica nanoparticles). PLK1- polo-like kinase 1.

DEFINITIONS

To facilitate an understanding of the present invention, a number of terms and phrases are defined below:

In general, the term "substituted" whether preceded by the term "optionally" or not, and substituents contained in formulae of this invention, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds.

As used herein, the term "alkyl", refers to straight and branched alkyl groups. An analogous convention applies to other generic terms such as "alkenyl", "alkynyl" and the like. In certain embodiments, as used herein, "lower alkyl" is used to indicate those alkyl groups (substituted, unsubstituted, branched or unbranched) having about 1-6 carbon atoms. Illustrative alkyl groups include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, n-hexyl, sec-hexyl, moieties and the like, which again, may bear one or more substituents. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, l-methyl-2- buten-l-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl and the like.

The term "Ci-_Xalkylenyl", as used herein, refers to a linear or branched saturated divalent radical consisting solely of carbon and hydrogen atoms, having from one to x carbon atoms, having a free valence "-" at both ends of the radical. Likewise, the term "Ci-_xheteroalkylenyl", as used herein, refers to a linear or branched saturated divalent Ci-_Xalkylenyl radical as defined above, comprising at least one heteroatom selected from O, N, or S, and having a free valence "-" at both ends of the radical. When the Ci-_Xalkylenyl or Ci__xheteroalkylenyl is optionally substituted, at least one of the H atoms may be replaced by a substituent such as halogen or - OR where R may represent Cl-6alkyl.

The term "ethenylenyl", as used herein, refers to the divalent radical -CH=CH-. When the ethylenyl is optionally substituted, one or both the H atoms may be replaced by a substituent such as halogen or -OR where R may represent C 1 -6alkyl. In general, the term "aromatic moiety" or "aryl", as used herein, refers to stable substituted or unsubstituted unsaturated mono- or polycyclic hydrocarbon moieties having preferably 3-14 carbon atoms, comprising at least one ring satisfying the Hackle rule for aromaticity. Examples of aromatic moieties include, but are not limited to, phenyl, indanyl, indenyl, naphthyl, phenanthryl and anthracyl.

The term "halogen" as used herein refers to an atom selected from fluorine, chlorine, bromine and iodine.

As used herein, the term "independently" refers to the fact that the substituents, atoms or moieties to which these terms refer, are selected from the list of variables independently from each other (i.e., they may be identical or the same).

As used herein, the term "template" or "supramolecular template" refers to a self- aggregation of ionic or non-ionic molecules or polymers that have a structure directing function for another molecule or polymer.

As used herein, the term "and/or" means any one of the items, any combination of the items, or all of the items with which this term is associated.

As used herein, the term "about" can refer to a variation of ±5%, ±10%,±20%, or ±25%, of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term "about" can include one or two integers greater than and/or less than a recited integer. Unless indicated otherwise herein, the term "about" is intended to include values, e.g., weight percents, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment.

As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible subranges and combinations of subranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percents or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non- limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.

As will also be understood by one skilled in the art, all language such as "up to," "at least," "greater than," "less than," "more than," "or more," and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into subranges as discussed above. In the same manner, all ratios recited herein also include all subratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, as used in an explicit negative limitation.

An "effective amount" refers to an amount effective to treat a disease, disorder, and/or condition, or to bring about a recited effect. For example, an amount effective can be an amount effective to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is well within the capacity of persons skilled in the art. The term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, e.g., that is effective to treat or prevent a disease or disorder, or to treat the symptoms of the disease or disorder, in a host. Thus, an "effective amount" generally means an amount that provides the desired effect.

The terms "treating", "treat" and "treatment" as used herein, refer to partially or completely alleviating, inhibiting, delaying onset of, reducing the incidence of, ameliorating and/or relieving a disorder or condition, or one or more symptoms of the disorder, disease or condition. The terms include (i) inhibiting the disease, pathologic or medical condition or arresting its development; (ii) relieving the disease, pathologic or medical condition; and/or (iii) diminishing symptoms associated with the disease, pathologic or medical condition.

The term "prevent", "preventing" or "prevention" as used herein means that the compounds of the present invention are useful when administered to a patient who has not been diagnosed as possibly having the disease at the time of administration, but who would normally be expected to develop the disease or be at increased risk for the disease. The compounds of the invention will slow the development of disease symptoms, delay the onset of disease, or prevent the individual from developing the disease at all. Preventing also includes administration of the compounds of the invention to those individuals thought to be predisposed to the disease due to familial history, genetic or chromosomal abnormalities, and/or due to the presence of one or more biological markers for the disease. Thus, the terms "prevent", "preventing" or "prevention" refer to prophylaxis and/or prophylactic administration, as appropriate.

As used herein, and unless otherwise specified, the terms "therapeutically effective amount" and "effective amount" of a compound refer to an amount sufficient to provide a therapeutic benefit in the treatment, prevention and/or management of a disease, to delay or minimize one or more symptoms associated with the disease or disorder to be treated. The terms "therapeutically effective amount" and "effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease or disorder or enhances the therapeutic efficacy of another therapeutic agent.

The term "responsively disintegratable", when referring to the porous organometaloxide materials according to the invention, refers to the property of a material or particle that undergoes degradation (i.e., breakdown of the structural integrity of the material or particle) triggered by a particular signal. The signal can be, for example, a change in pH (either an increase or decrease), a change in redox potential, the presence of reducing or oxidizing agents, the presence of UV, visible or near infrared light, ultrasounds, electromagnetic radiation, an enzymatic cleavage, a change in temperature, etc. The term "responsively cleavable", when referring to a chemical bond, polymer fragment or linking group, refers to a covalent bond, polymer fragment or linking group that is cleaved upon application of one of the aforementioned particular inputs. Generally speaking, the presence of a responsively cleavable bond, polymer fragment or linker moiety within a porous organometaloxide material of the invention, confers to the material its disintegratable properties (the property of structurally breaking down upon application of a specific signal/stimulus, akin to "self-destructive" behavior).

As used herein, the term "organometaloxide" refers to organo-oxides of metals (e.g.,

Ti, Al, Zr) or organo-oxides of metalloids (e.g. Si). Likewise, as the term "metal" when used in reference to metal sites present in organometaloxides according to the invention, is meant to cover metals (e.g., Ti, Al, Zr) and metalloids (e.g. Si).

As used herein, the term "periodic mesoporous" refers to having an ordered arrangement of pores in terms of translation symmetry with a diameter between about 0.5 nm and about 50 nm.

As used herein, the term "mesoporous" refers to having pores with a diameter between about 0.5 nm and about 50 nm.

As used herein, the term "macroporous" refers to having pores with a diameter between about 50 and about 1 ,000 nm.

As used herein, the term "mesoporous-macroporous" refers to having two different kinds of pores one of which is between about 0.5 nm and 50 nm and the other of which is between about 50 nm and about 1,000 nm in the structure.

As used herein, the term "mixed mesoporous" refers to having two different kinds of pores one of which is of smaller size (e.g., pores having an average width in the size range of from about 0.5 to about 5 nm, preferably from 0.5 to 3 nm, more preferably from 0.5 to 2.5 nm, even more preferably from 0.5 to 2 nm), and another which is of larger size (e.g., pores having an average width in the size range of from about 1 to about 50 nm, preferably from 1 to 40 nm, more preferably from 1 to 30 nm, even more preferably from 1 to 20 nm, most preferably from 10 to 15 nm).

As used herein, the term "iR A" or "interfering R A" means any R A which is capable of down-regulating the expression of the targeted protein. It encompasses small interfering RNA (siRNA), double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), and short hairpin RNA (shRNA) molecules. RNA interference designates a phenomenon by which dsRNA specifically suppresses expression of a target gene at post-translational level. In normal conditions, RNA interference is initiated by double-stranded RNA molecules (dsRNA) of several thousands of base pair length. In vivo, dsRNA introduced into a cell is cleaved into a mixture of short dsRNA molecules called siRNA. The enzyme that catalyzes the cleavage, Dicer, is an endo-RNase that contains RNase III domains (Bernstein, Caudy et al. 2001

Nature). In mammalian cells, the siRNAs produced by Dicer are 21-23 bp in length, with a 19 or 20 nucleotides duplex sequence, two-nucleotide 3' overhangs and 5'- triphosphate extremities (Zamore, Tuschl et al. Cell; Elbashir, Lendeckel et al. Genes Dev; Elbashir, Martinez et al. EMBO J). According to the invention, iRNAs do not encompass microRNAs.

A number of patents and patent applications have described, in general terms, the use of siRNA molecules to inhibit gene expression, for example, WO 99/32619. siRNA or shRNA are usually designed against a region 50-100 nucleotides downstream the translation initiator codon, whereas 5'UTR (untranslated region) and 3'UTR are usually avoided. The chosen siRNA or shRNA target sequence should be subjected to a BLAST search against EST database to ensure that the only desired gene is targeted. Various products are commercially available to aid in the preparation and use of siRNA or shRNA.

In a preferred embodiment, the RNAi molecule is a siRNA of at least about 10-40 nucleotides in length, preferably about 15-30 base nucleotides.

siRNA or shRNA can comprise naturally occurring RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally- occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end of the molecule or to one or more internal nucleotides of the siRNA, including modifications that make the siRNA resistant to nuclease digestion.

siRNA or shRNA may be administered in free (naked) form or by the use of delivery systems that enhance stability and/or targeting, e.g., liposomes, or incorporated into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors, or in combination with a cationic peptide. They may also be administered in the form of their precursors or encoding DNAs. All these techniques are well known in the art.

In particular, the present invention also contemplates as inhibitor an expression vector encoding a siRNA or an shRNA, preferably a shRNA. Examples of vector include recombinant viral vectors, in particular an adenoviral, retroviral, adeno- associated viral, lentiviral, a herpes simplex viral or a sendaiviral vector. As used herein, the term "surfactant or block copolymer mesostructure" refers to an ordered supramolecular assembly of surfactant or block copolymer molecule micelles, with translation symmetry between about 2 and about 50 nm.

As used herein, the term "porous framework material" refers to a mesoporous or macroporous or mesoporous-macroporous or mixed-mesoporous material in which a (X3Mi)R¹-L-R²(M2X3)-type responsively cleavable linker is inserted.

As used herein, the term "linker" refers to a responsively cleavable

moiety '"'-R'-L-R²-*,

preferably a responsively cleavable moiety '"'-R'-L-R²-*, inserted into the organometaloxide framework b reaction of a -R¹-L-R²-M₂(X)3 precursor,

or

, preferably a

(X)3Mi-R¹-L-R²-M₂(X)3 precursor, by sol-gel chemistry (hydrolysis or condensation), with the linker being bound to the framework via two or more metal atoms in the framework. In other words, at least one X on each occurrence of Mi and M₂ on the precursor is hydrolyzed to lead to formation of the metaloxide framework.

As used herein, the term "cleavable" refers both to the reversible/biodegradable

nature of the

or

linker, as defined herein, triggering the decomposition/disintegration of the bulk hybrid porous material. As such, the linker may contain a dynamic covalent bond. As used herein, the term "dynamic covalent bond" refers to any covalent chemical bond possessing the capacity to be formed and broken under equilibrium control. In this sense, they can be intended as "reversible" covalent bonds. [9]

As used herein, the term "biological polymer" or "biopolymer" refers to polymers produced by living organisms, or synthetic mimics of those. There are three main classes of biopolymers, classified according to the monomeric units used and the structure of the biopolymer formed: polynucleotides (R A and DNA), which are long polymers composed of 13 or more nucleotide monomers; polypeptides, which are short polymers of amino acids; and polysaccharides, which are often linear bonded polymeric carbohydrate structures.

As used herein, the term "biodegradable polymer" refers to synthetic polymers, which can undergo chemical dissolution by biological means (bacteria, enzymes, etc.)

As used herein the term "organometaloxide" refers to a compound, which contains at least a metal-carbon or metalloid-carbon bond.

As used herein the term "metaloxide" generally refers to metal oxide or metalloid oxide derivatives, such as silicon oxide derivatives but it could be generalized to other metal oxides, e.g. titanium oxide and zirconium oxide.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE INVENTION

As noted above, there has been increasing interest in recent years in the development of efficient siR A technology for medical purposes.

The interest of mesoporous silica nanoparticles (MSNs) as drug delivery system rises from their advantageous structural properties, biocompatibility, high loading capacities and the possibility to selectively functionalise the material both on the external surface and within the pores. However, MSNs, especially when they are functionalized to prevent aggregation, tends to accumulate into the different organs in the body hindering their commercialisation as a medical tool. To overcome this issue, stimulus responsive porous nanoparticles were developed that can deliver drugs and can break into small pieces after cell internalization. Amongst them, hybrid MSNs containing redox responsive disulfide (S-S) bridges (ss-MSNs) have been developed. These nanoparticles are able to break in small pieces upon their exposure to a reducing agent such as glutathione (GSH), a tripeptide present in high concentration into cancer cells, leading to an efficient exocytosis of the material but also in a faster delivery of the payload (Fig.1).

Provided herein is a solution making use of this material for the in-vivo delivery of siRNA into cells, notably fot he treatment of cancers.

In this context, there is provided herein novel porous organometaloxide materials, for example in the form of nanoparticles, whose framework contain metal (e.g., Ti, Zr, Al) or metalloid (e.g., Si) adjacent sites covalently bound via a responsively cleavable linker.

1) General Description of porous organometaloxide materials of the Invention

In one aspect, there is provided a porous organometaloxide material, preferably in the form of nanoparticles, comprising a porous three-dimensional framework of metal-oxygen (e.g., Ti-O, Zr-O, Al-0 bond) or metalloid-oxygen (e.g., Si-O) bonds, wherein at least a subset of metal (e.g., Ti, Zr, Al) or metalloid (e.g., Si) atoms in the material's framework are connected to at least another metal (e.g., Ti, Zr, Al) or metalloid atom (e.g., Si) in the framework through a linker having one of the following structures:

wherein:

each occurrence of * denotes a point of attachment to a metal atom in the material's framework;

A represents a monomer of a responsively cleavable fragment of biological/biodegradable polymer;

m is an integer from 2 to 10000 and m represents the number of monomers in the fragment of biological/biodegradable polymer;

L represents a responsively cleavable covalent bond or '"'-R'-L-R²-* independently represents a responsively cleavable moiety; and R¹ and R² independently represent an optionally substituted CI -20 alkylenyl moiety, an optionally substituted CI -20 heteroalkylenyl moiety, an optionally substituted ethylenyl moiety, -C≡C- or an optionally substituted phenyl moiety, wherein the CI -20 alkylenyl, CI -20 heteroalkylenyl or ethylenyl moiety may bear one or more substituents selected from halogen or -OR where R may represent H or CI -6 alkyl, and the phenyl moiety may bear one or more substituents independently selected from halogen, CI -6 alkyl, -N0₂, -CN, isocyano, -OR^p, - N(R^P)₂ wherein each occurrence of R^p independently represents H or CI -6 alkyl.

Advantageously, '"'-R'-Li-R²-* may independently comprise sugar derivatives such as mannose, hyaluronic acid derivatives, collagene, aminoacids or peptides.

Advantageously, the hyaluronic acid derivatives may be any suitable hyaluronic acid derivatives known to the person of ordinary skill in the art. It may be for example any commercially available hyaluronic acid derivatives, for example a hyaluronic acid derivative disclosed in Voigt J et al. "Hyaluronic acid derivatives and their healing effect on burns, epithelial surgical wounds, and chronic wounds: a systematic review and meta-analysis of randomized controlled trials." Wound Repair Regen. 2012 May- Jun;20(3):317-31 [23]. For example, a hyaluronic acid moiety may be introduced in the material via a hydrolysed version of the naturally occurring hyaluronic acid molecule (e.g., hydrolysis of -NHAc moiety into -NH₂).

Advantageously, the amino acids may be any suitable amino acid known to the person of ordinary skill in the art. It may be for example D or L amino acid. It may be for example amino acid selected from the group comprising alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, iso leucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. It may also be gamma aminobutyric acid.

Advantageously, the peptide moiety may be peptide moiety comprising for 3 to 20 amino acids, for example 3 to 5 amino acids.

Advantageously, the sugar moiety (carbohydrate moiety) may be any suitable sugar known to the person of ordinary skill in the art. It may be for example a sugar selected from the group comprising Arabinose, Fructose, Galactose, Glucose, Lactose, Inositol, Mannose, Ribose, Trehalose and Xylose, preferably glucose, lactose or mannose.

Advantageously, the porous organometaloxide material is a well-ordered crystalline material, and is preferably not amorphous.

Advantageously, when the porous organometaloxide material is in the form of nanoparticles, the outer surface of the porous organometaloxide nanoparticle may be functionalized with positively charged groups, such as quarternary amine salts (e.g., NH₃ ⁺), either directly or via a linker. This may be accomplished by grafting the outersurface of the nanoparticle with amine groups via a properly functionalized trialkoxysilane, such as 3-aminopropyl)triethoxysilane (APTES). This allows shifting the global charge of the material to a positive value, thus allowing an electrostatic interaction with negatively charged biomolecules, such as siRNA, for example PLK1 siRNA.

Advantageously, when the linker has the structure '"'-R'-L-R²-*, the subset of metal atoms in the material's framework that are connected to the linker *-R^!-L- R²-*, may represent at least 30% of the metal atoms present in the porous organometaloxide material of the invention. Such porous organometaloxide material will be said to be "at least 30% doped".

As used herein, "x" in the expression "x% doped" is calculated based on the % of metal centers in the porous organometaloxide material that comes from the starting material (X)₃Mi-R¹-L-R²-M2(X)₃ used to synthesize the organometaloxide material according to the invention. This % doping also reflects the contents of responsively cleavable covalent bond L in the organometaloxide material. The higher the % doping, the higher the content of linker L in the porous organometaloxide, and the greater the ability of the resulting organometaloxide material to undergo complete structural breakdown, suitable for the intended applications. Likewise, when the linker has the structure

or

^*"^^Rl" S^~h ^mR21^"* , as used herein, "x" in the expression "x% doped" is calculated based on the % of metal centers in the porous organometaloxide material

that comes from the starting material

or

used to synthesize the organometaloxide material according to the invention. This % doping also reflects the contents of

responsively cleavable fragment of bio logical/biodegradable polym

or

ganometaloxide material. The higher the %

doping, the higher the content

in the porous organometaloxide, and the greater the ability of the resulting organometaloxide material to undergo complete structural breakdown, suitable for the intended applications.

Advantageously, when the linker has the structure '"'-R'-L-R²-*, the subset of metal atoms in the material's framework that are connected to the linker *-R^!-L- R²-*, may range anywhere from 2% to 100% of the metal atoms present in the porous organometaloxide material of the invention. For example, the subset of metal atoms in the material's framework that are connected to the linker '"'-R'-L-R²- *, may range from 2% to 100%, from 5% to 90%, from 5% to 80%, from 5% to 70%, from 5% to 60%; from 5% to 50%, from 5% to 40%, of the metal atoms present in the porous organometaloxide material of the invention. For example, the subset of metal atoms in the material's framework that are connected to the linker *-R^!-L- R²-*, may represent at least 2%, at least 5%, at least 7%, at least 10%, at least 12%, at least 15%, at least 17%, at least 20%, at least 22%, at least 25%, at least 27%, at least 30%, of the metal atoms present in the porous organometaloxide material of the invention.

Advantageously, when the linker has the structure '"'-R'-L-R²-*, the subset of metal atoms in the material's framework that are connected to the linker *-R^!-L- R²-*, may range anywhere from 30% to 100% of the metal atoms present in the porous organometaloxide material of the invention. For example, the subset of metal atoms in the material's framework that are connected to the linker '"'-R'-L-R²- *, may range from 30% to 100%, from 30% to 90%, from 30% to 80%, from 30% to 70%, from 30% to 60%; from 30% to 50%, from 30% to 40%, of the metal atoms present in the porous organometaloxide material of the invention.

The final % doping of the porous organometaloxide material will depend on the respective molar ratios of starting materials (X)3Mi-R¹-L-R²-M2(X)3 and M(X^A)₄ used in the synthesis of the material (cf. section dealing with synthetic process, later in the present document). When no M(X^A)₄ is used in the preparation of the material, a doping of 100% will be reached (i.e., only (X)₃Mi-R¹-L-R²-M₂(X)3 is used as metal source).

Advantageously, for a slower and more controlled desintagrability/degradability of the porous organometaloxide material, the subset of metal atoms in the material's framework that are connected to the linker *-R^!-L- R²-*, may be in the lower % range; for example from 30% to 35% , from 30% to 40%), from 30% to 45%, from 30% to 50%, of the metal atoms present in the porous organometaloxide material of the invention. Advantageously, the subset of metal atoms in the material's framework that are connected to the linker '"'-R'-L-R²-*, may range from 30% to 40% , preferably about 30%.

Advantageously, for an even slower and more controlled desintagrability/degradability of the porous organometaloxide material, the subset of metal atoms in the material's framework that are connected to the linker *-R^!-L- R²-*, may be in a much lower % range; for example from 2% to 40% , from 5% to 35%, from 5% to 30%, from 5% to 25%, from 5% to 20%, from 5% to 15%, from 5% to 10%, of the metal atoms present in the porous organometaloxide material of the invention.

Advantageously, for a faster desintagrability/degradability of the porous organometaloxide material, the subset of metal atoms in the material's framework that are connected to the linker '"'-R'-L-R²-*, may be in the higher % range; for example from 55% to 60% , from 55% to 65%, from 55% to 70%, from 55% to 75%, from 55% to 80%, from 55% to 85%, from 55% to 90%, from 55% to 95%, from 55% to 100%, of the metal atoms present in the porous organometaloxide material of the invention.

Advantageously, when the linker has the structure

or

, the subset of metal atoms in the material's framework that are connected to said linker may represent 100% of the metal atoms present in the porous organometaloxide material of the invention. In other words, in that case, all the metal atoms in the porous organometaloxide material of the invention

originate from the starting materials

or d to prepare the organometaloxide material.

H

owever, this is not a requirement, and the contents of linker or

in the material's framework may be modulated same as described for the linker '"'-R'-L-R²-*, above. Thus, advantageously, when the linker has the structure

or

the subset of metal atoms in the material's framework

that are connected to the l

respectively, may range anywhere from 5% to 100% of the metal atoms present in the porous organometaloxide material of the invention. For example, the subset of metal atoms in the material's framework that are connected to the linker

, respectively, may range from 5% to

100%, from 5% to 90%, from 5% to 80%, from 5% to 70%, from 5% to 60%; from 5% to 50%, from 5% to 40%, from 5% to 30%, from 5% to 20%, from 5% to 10%, of the metal atoms present in the porous organometaloxide material of the invention. Advantageously, the subset of metal atoms in the material's framework that are

connected to the

or ^Rl½)^~h ^mR21^~* , respectively, may range from 10% to 100%, from 10% to 90%, from 10% to 80%, from 10% to 70%, from 10% to 60%; from 10% to 50%, from 10% to 40%, from 10% to 30%, from 10% to 20%, of the metal atoms present in the porous organometaloxide material of the invention. Advantageously, the subset of metal atoms in the

material's framework that are connected to the or

respectively, may range from 20% to 100%, from 20% to 90%, from 20% to 80%, from 20% to 70%, from 20% to 60%; from 20% to 50%, from 20% to 40%, from 20% to 30%, of the metal atoms present in the porous organometaloxide material of the invention. Advantageously, the subset of metal

atoms in the material's framework that are connected to the linker

^*"^^Rl" S^~h ^mR21^"* , respectively, may range from 30%> to 100%, from 30%> to 90%, from 30% to 80%, from 30% to 70%, from 30% to 60%; from 30% to 50%, from 30% to 40%, of the metal atoms present in the porous organometaloxide material of the invention. The final % doping of the porous organometaloxide material will depend on the respective molar ratios of starting materials

(X)₃M I— R¹- ®†_R2-M₂(X)3

_r ^{1 J} m respectively and M(X^A)₄ used in the synthesis of the material (cf. section dealing with synthetic process, later in the present document). When no M(X^A)₄ is used in the preparation of the material,

reached (i.e., only

or

is used as metal source).

Advantageously, for a slower and more controlled desintagrability/degradability of the porous organometaloxide material, the subset of metal atoms in the material's framework that are connected to the linker

, respectively, may be in the lower % range; for example from 5%> to 10%>, from 5%> to 15%>, from 5%> to 20%>, from 5%> to 25%, from 5% to 30%, from 5% to 35% , from 5% to 35% , from 5% to 40%, from 5% to 45%, from 5% to 50%, of the metal atoms present in the porous organometaloxide material of the invention. Advantageously, the subset of metal

atoms in the material's framework that are connected to the linker

or

^*"^^Rl" S^~h ^mR21^"* , respectively, may range from 30% to 35% , from 30% to 35% , from 30% to 40%, from 30% to 45%, from 30% to 50%; preferably from 30% to 40% , preferably about 30%.

Advantageously, for a faster desintagrability/degradability of the porous organometaloxide material, the subset of metal atoms in the material's framework

that are connected to the linker

or

respectively, may be in the higher % range; for example from 55% to 60% , from 55% to 65%, from 55% to 70%, from 55% to 75%, from 55% to 80%, from 55% to 85%, from 55% to 90%, from 55% to 95%, from 55% to 100%, of the metal atoms present in the porous organometaloxide material of the invention. Advantageously, the subset of metal atoms in the material's framework that are connected to the linker

respectively, may be 100%.

In all cases, the high content of linker *-R -L-R - ,

or

^*"^^Rl" S^~h ^mR21^"* in the porous organometaloxide material confers the resulting material the ability to undergo complete structural breakdown. As such, the resulting porous organometaloxide material exhibits enhanced biodegradability compared to other related materials known in the art upon application of a suitbale stimulus, thereby resulting in smaller, more easily hydrolysable, and consequently less harmful fragments.

Advantageously, the fragment of biological/biodegradable polymer may be an oligomer (i.e., m may range from 2 to 20), a medium sized fragment (i.e., m may range from 20 to 1000), or a large fragment (i.e., m may reach several thousands, for example it may range from 1000 to 10000).

Advantageously, in the linker '"'-R'-L-R²-*

, each occurrence of R¹ and R² may be identical.

Advantageously, in the linker

each occurrence of R¹ may be identical.

Advantageously, in the linker '"'-R'-L-R²-*, R¹ and R² may be any organic radical from any commercially available silylated derivative suitable for sol-gel chemistry. For example, R¹ and R² may independently represent -CH₂-, -(CH₂)₂-, - (CH₂)₃-, -(CH₂)₄-, or phenyl.

Advantageously, R¹ and R² may be identical and may each represent -CH₂-, - (CH₂)₂-, -(CH₂)₃-, -(CH₂)₄-, or phenyl.

Advantageously, the metal may be selected from Si-, Ti- or Zr, or mixture thereof, and the porous organometaloxide material according to the invention may be a Si-, Ti- and/or Zr-based porous organometaloxide material. The expression "and/or" in this context means that the porous organometaloxide material may: contain Si only as metal,

- contain Ti only as metal,

contain Zr only as metal, or Al

be a mixed-metal organometaloxide material containing any combination of at least two of Si, Ti, Al or Zr as metal(s) in the framework. Advantageously, the porous organometaloxide material according to the invention may:

contain 90.0-100% Si as metal (% based on the number of available metal sites in the framework), the remaining metal sites may be Ti, Al or Zr; contain 90.0-100% Ti as metal (% based on the number of available metal sites in the framework), the remaining metal sites may be Ti, Al or Zr; or contain 90.0-100% Zr as metal (% based on the number of available metal sites in the framework), the remaining metal sites may be Ti, Al or Zr. Advantageously, the porous organometaloxide material according to the invention may be a Si-Ti mixed-metal organometaloxide material containing 0.1- 50.0% Si and 0.1-50.0% Ti, the % sum of Si and Ti adding to 100% the number of available metal sites in the framework. For example, the porous organometaloxide material according to the invention may be a Si-based porous organometaloxide material doped with 0.1 to 10.0% Ti (% based on the number of available metal sites in the framework).

Advantageously, the porous organometaloxide material according to the invention may be a mixed metal, M-based porous organometaloxide material, where M may be Si or Ti,, containing at least 80.0%, preferably at least 85.0%, preferably at least 90.0%>, preferably at least 95.0%, preferably at least 95.5%, preferably at least 99.9% Si or Ti (% based on the number of available metal sites in the framework), the remaining metal sites being Si, Ti and/or Zr.

Advantageously, the substituent(s) on R¹ and R² may be suitably selected to facilitate the cleavage of the responsively cleavable linker L when the external signal/stimulus is applied (e.g., a change in pH (either an increase or decrease), a change in redox potential, the presence of reducing or oxidizing agents, the presence of UV, visible or near infrared light, ultrasounds, electromagnetic radiation, an enzymatic cleavage, a change in temperature, etc.). For example, the substituent(s) on R¹ and R² may be selected based on their electron-withdrawing or -donating properties, to facilitate the cleavage of the linker moiety. For example, for illustrative purposes, when L may be an imine bond and Ri and/or R₂ may be a phenyl group, the phenyl group may bear a nitro group to make the imine bond more reactive (i.e., more responsive to cleavage upon application of a suitable stimulus).

One advantageous aspect of this invention resides in the simple, yet compelling, underlying concept: namely a precursor having one of the following structures:

(X)₃MI-R¹-L-R²-M₂(X)3;

_or

, wherein A, L, R¹, R² and m are as defined above, and Mi and M₂ independently represent Si, Ti or Zr, is chemically inserted within the framework of the porous organometaloxide material via sol-gel chemistry. Advantageously, said precursor (X)₃Mi-R¹-L-R²-M₂(X)₃ is introduced in the the porous organometaloxide material framework so that the subset of metal atoms in the material's framework that are connected to the linker '"'-R'-L-R²-*, represent at least 30% of the metal atoms present in the porous organometaloxide material of the invention. In other words, at least 30% of the metal centers in the porous organometaloxide material framework originates from the precursor (X)₃Mi-R¹-L-R²-M₂(X)₃ (i.e., Mi and M₂ account for at least 30% of the metal center in the or anometaloxide material .-Advanta eousl

said precursor

introduced in the the porous organometaloxide material framework so that the subset of metal atoms in the material's framework that are connected to the linker

, respectively represent at least 5% of the metal atoms present in the porous organometaloxide material of the invention. In other words, at least 5% of the metal centers in the porous organometaloxide

material framework originates from the precursor

or (Χ)₃Μι A )+R²-M₂(X)3

m Mi and M₂ account for at least 5% of the metal center in the organometaloxide material).

In the above, X may represent a hydrolysable or nonhydrolyzable group, provided that on each occurrence of Mi and M₂, at least one occurrence of X represents a hydrolysable group.

When X represents a hydrolysable group, it may be selected from a CI -6 alkoxy, CI -6 acyloxy, halogen or amino moiety. Advantageously, when X represents a hydrolysable group, X may represent CI,

-OMe, -OEt, -OzPr or -OtBu.

When X represents a nonhydrolyzable group, it may be selected from an optionally substituted CI -20 alkyl, C2-20 alkenyl or C2-20 alkynyl moiety, an optionally substituted CI -20 heteroalkyl, C2-20 heteroalkynyl or C2-20 heteroalkynyl moiety, or an optionally substituted phenyl moiety, wherein the substituents on the phenyl, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl moieties may be independently selected from halogen, -N0₂, -CN, isocyano, CI -6 alkoxy, an oxirane/epoxyde moiety, -N(R)₂ wherein each occurrence of R is independently selected from H or CI -6 alkyl.

Advantageously, when X represents a nonhydrolyzable group, X may represent CI -6 alkyl or C2-6 alkenyl; preferably -Me, -Et or -CH=CH₂; most preferably -Me or -Et.

The insertion of the responsively cleavable linker within the framework of the porous metaloxide is performed during the synthesis of the porous organometaloxide material itself, no additional step is required, if not the

preparation of the required (X)₃Mi-R¹-L-R²-M₂(X)3;

or

precursor, which may also be carried out in situ. It is then important to choose the -R¹-L-R²-M₂(X)3; R -M₁ (X)_{3 or}

precursor in order to obtain the desired self-destructive behavior in the final operational environment.

In the case of organometaloxide material containing the linker '"'-R'-L-R²-*, the ratio (X)₃Mi-R¹-L-R²-M₂(X)₃/ M(X^A)₄ precursor used for the synthesis is also important, as it determines the % doping (and thus the ratio of responsively cleavable linker L within the material), and therefore the ability of the porous organometaloxide material to disintegrate upon application of a suitable stimulus. Advantageously, in the case of organometaloxide material containing the linker *- R'-L-R²-*, a minimum of 30 % of the whole metallic atoms present in the organometaloxide material should come from the (X)₃Mi-R¹-L-R²-M₂(X)₃ linker. Because (X)₃Mi-R¹-L-R²-M₂(X)₃ is bivalent (i.e., because this precursor contains two metal atoms per cleavable bond L), Ml and M2 represent 30% of the metal centers in the resulting organometaloxide material (i.e., 30% doping). This corresponds to 15% molar ratio if the responsively cleavable moiety of the linker is taken in account (L), as for each Mi and M₂ set only one L is associated. For a doping of 100%), (X)₃Mi-R¹-L-R²-M₂(X)₃ may be used as the exclusive source of metal (i.e., no M(X^A)₄ is used).

The following Table 1A describes exemplary ratios of equivalents (X)₃Mi- R^!-L-R²-M₂(X)₃/ M(X^A)₄ to reach the desired % doping:

Table 1A

M(X^A)₄ (Χ)₃Μι-^-Ι.- (X)₃Mi-R^!-L- % doping

R²-M₂(X)₃ R²-M₂(X)₃

0.95 eq. 0.05 eqi 0.025 eq. tt 5%

0.90 eq. 0.10 eqi 0.05 eq. tt 10% 0.80 eq. 0.20 eqi 0.10 eq.

20%

0.70 eq. 0.30 eqi 0.15 eq. 30%

0.60 eq. 0.40 eq. i 0.20 eq. tt 40%

0.50 eq. 0.50 eq. i 0.25 eq. tt 50%

0.40 eq. 0.60 eq. i 0.30 eq. tt 60%

0.30 eq. 0.70 eq. i 0.35 eq. tt 70%

0.20 eq. 0.80 eq. i 0.40 eq. tt 80%

0.10 eq. 0.90 eq. i 0.45 eq. tt 90%

- 1 eq. i 0.5 eq. tt 100%

+ equivalents expressed in terms of metal atoms (Mi and M₂) introduced by the bivalent starting material (X)3Mi-R¹-L-R²-M2(X)3 in the final organometaloxide material.

tt equivalents expressed in terms of responsively cleavable bond L introduced by the bivalent starting material (X)3Mi-R¹-L-R²-M2(X)3 in the final organometaloxide material.

The reaction conditions may be modulated, depending on the eq. ratios (X)₃Mi-R¹-L-R²-M₂(X)3 / M(X^A)₄ used. From the general knowledge in the field of organometaloxide chemistry, the practitioner will readily know how to adjust suitable reaction conditions, for example the type of solvent used to effect the reaction depending on the respective solubilities of the selected (X^Mi-R'-L-R²- M₂(X)₃ and M(X^A)₄.

(XfeM!— R¹-kj)- R²-M₂(X)3

Likewise for

or ^m , the following Tables IB and Table 1C describes exemplary ratios of equivalents

respectively, to reach the desired % doping (e.g., at least 5%): Table IB

+ equivalents expressed in terms of metal atoms (Mi) introduced by the

starting material final organometaloxide material.

Table 1C

M(X^A)₄

(XfeM!— R¹-k¾- R2-M₂(X)₃ %

^{1 J} m doping

0.90 eq. 0.10 eqi 10%

0.80 eq. 0.20 eqi 20%

0.70 eq. 0.30 eqi 30%

0.60 eq. 0.40 eq. i 40%

0.50 eq. 0.50 eq. + 50%

0.40 eq. 0.60 eq. i 60%

0.30 eq. 0.70 eq. + 70%

0.20 eq. 0.80 eq. i 80% 0.10 eq. 0.90 eq. i 90%

- 1 eq. i 100%

+ equivalents expressed in terms of metal atoms (Mi and M₂) introduced by the bivalent starting material

in the final organometaloxide material.

Advantageously, the porous organometaloxide material may be a hybrid material. The hybrid organic/inorganic nature of the material is naturally conferred

by the presence of the organic moiety '"'-R'-L-R²-*,

or

. However, other organic moieties may be introduced in the porous organometaloxide material by conventional sol-gel chemistry methods known in the art. For example, the use of a R³-M(R⁴)3 precursor, wherein M is Si, Ti or Zr; R³ is a nonhydrolyzable organic moiety bound to M via a carbon atom, and each occurrence of R⁴ is independently a hydro lysable group. By "nonhydrolyzable organic moiety" is meant an organic moiety that is not cleaved from the metal M during the sol-gel process leading to the porous organometaloxide framework material. Conversely, by "hydro lyzable group" is meant a radical that is hydrolyzed (cleaved from the metal M) during the sol-gel process leading to the porous organometaloxide framework material. Typically, R⁴ may be a CI -6 alkoxy, CI -6 acyloxy, halogen or amino group. R³ may be an optionally substituted CI -20 alkyl, C2-20 alkenyl or C2-20 alkynyl moiety, an optionally substituted CI -20 heteroalkyl, C2-20 heteroalkynyl or C2-20 heteroalkynyl moiety, or an optionally substituted phenyl moiety. Advantageously, R³ may bear a substituent that allows further functionalization of the organometaloxide material, or posses a functionality that imparts desired characteristics. For example, the substituents on the phenyl, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl moieties may be independently selected from halogen, -N0₂, -CN, isocyano, CI -6 alkoxy, an oxirane/epoxyde moiety, -N(R)₂ wherein each occurrence of R is independently selected from H or C 1 -6alkyl. Organic/inorganic hybrid Si-, Ti- and Zr-based porous organometaloxide framework materials are well-known in the literature, as well as methods for their preparation. (See for example, (a) for Si-based materials: [11]; (b) for Ti-based materials: [12] ; (c) for Zr-based materials: [13] . These methods may be readily adapted to the present hybrid porous organometaloxide materials, by using a (X)3Mi-R¹-L-R²-M₂(X)3 precursor as defined above and herein, in the sol-gel synthetic process.

The porous organometaloxide material according to the invention may be in any form known for conventional porous hybrid or purely inorganic Si-, Ti- or Zr-based metaloxide framework materials. For example, porous organometaloxide material according to the invention may be in the form of a monolith, a film (thin or thick film), powder, nanoparticles, or spherical, cubic, cylindrical or disc-like particles.

Advantageously, the porous organometaloxide material may be in the form of nanoparticles. For example, the porous material according to the invention may have a particle diameter from 1 to 1000 nanometers, preferably from 1 to 500 nm, preferably from 1 to 250 nm, preferably from 1 to 100 nm, from 1 to 50 nm preferably from 1 to 20 nm. Advantageously, the porous organometaloxide material may be in the form of nanoparticles ranging from 20-100 nm in particle diameter, for example about 20 nm, for example about 30 nm, for example about 40 nm, for example about 50 nm, for example about 60 nm, for example about 70 nm, for example about 80 nm, for example about 90 nm, for example about 100 nm, most preferably about 50-60 nm. The diameter particle may be modulated depending on reaction condition parameters, such as reaction time, temperature of reaction, base used (ammonium hydroxide, NaOH, ...), amount of organic solvent used (e.g.,

EtOH). The practitioner can adapt knowledge from general synthetic methods for mesoporous organometaloxide chemistry to fine tune the proper reaction conditions amenable to the desired particle diameter.

Advantageously, the porous organometaloxide material may be in the form of nanoparticles dispersed in a solvent. The solvent may be that used in the synthesis of the material. Advantageously, the porous organometaloxide material may be mesoporous, microporous, macroporous, mixed mesoporous-macroporous or mixed mesoporous; preferably mixed mesoporous.

Advantageously, the porous disintegratable organometaloxide nanoparticles of the invention preferably have pores having an average width in the size range of from about 1 to about 50 nm, preferably from 1 to 40 nm, more preferably from 1 to 30 nm, even more preferably from 1 to 20 nm. The pores may also be smaller: pores having an average width in the size range of from about 0.5 to about 5 nm, preferably from 0.5 to 3 nm, more preferably from 0.5 to 2.5 nm, even more preferably from 0.5 to 2 nm. Naturally, when the porous organometaloxide material is in the form of nanoparticles, as described above, the pore width size will be proportionally smaller than the particle size. These pores may be well-ordered and of uniform size (e.g., mesoporous structure). Advantageously, the pores may not be uniform in size. Advantageously, the porous disintegratable organometaloxide nanoparticles may have two populations of pore sizes: (i) pores having an average width in the size range of from about 1 to about 50 nm, preferably from 1 to 40 nm, more preferably from 5 to 30 nm, even more preferably from 5 to 20 nm, most preferably from 10 to 15 nm; and (ii) pores having an average width in the size range of from about 0.5 to about 5 nm, preferably from 0.5 to 3 nm, more preferably from 0.5 to 2.5 nm, even more preferably from 0.5 to 2 nm.

Advantageously, L may be any moiety that contains a responsively cleavable covalent bond, which can be cleaved upon exposure to a determined stimulus, or a responsively cleavable fragment of a biological compound (proteins, carbohydrates, etc.) or biodegradable synthetic polymer, able to undergo degradation {e.g., enzymatic) or a supramolecular assembly (non-covalent bond).

Advantageously, when^-R'-L-R²-* represents a responsively cleavable moiety, the substituent(s) on R¹ and R² may be suitably selected to facilitate the cleavage of the responsively cleavable linker L when an external signal/stimulus is applied (e.g., a change in pH (either an increase or decrease), a change in redox potential, the presence of reduction or oxidation agent, the presence of UV, visible or near infrared light, ultrasounds, electromagnetic radiation, an enzymatic cleavage, a change in temperature, etc.). For example, the substituent(s) on R¹ and R² may be selected based on their electron-withdrawing or -donating properties, to facilitate the cleavage of the linker moiety. For example, for illustrative purposes, when L may be an imine bond and Ri and/or R₂ may be a phenyl group, the phenyl group may bear a nitro group to make the imine bond more reactive (i.e., more responsive to cleavage upon application of a suitable stimulus).

Advantageously, '"'-R'-L-R²-* may be any moiety that contains a responsively cleavable covalent bond, which can be cleaved upon exposure to a determined stimulus. Advantageously, when the linker has the structure '"'-R'-L-R²-*, L may represent a responsively cleavable covalent bond selected from:

Disulfide Diselenide Anhydride Amide Imine

Acetal/ketal Urea Thiourea Hydrazone Oxyme Boronic acid derivatives

Carbamoyl Thioketal

Preferably, when the linker has the structure '"'-R'-L-R²-*, L may represent a responsively cleavable covalent bond selected from disulfide, imine, amide, ester, urea, or thiourea.

Advantageously, L may independently represent or comprise a disulfide, ester, imine or hydrazone bond, preferably a disulfide bond.

Advantageously, when the linker has the structure '"'-R'-L-R²-*, L may represent a disulfide responsively cleavable covalent bond.

Advantageously, when L represents an imine bond, '"'-R'-L-R²-* may preferably be a di-imine linker conjugated with an aromatic group such as phenyl. More preferably, '"'-R'-L-R²-* may comprise a para di-imino phenyl moiety. Such di-imine linkers may be cleaved in acidic conditions (e.g., at pH 5-6 for 24 hours, for example pH=5.2) thereby leading to disintegration of the organometaloxide material.

Advantageously, '"'-R'-L-R²-* may independently comprise sugar derivatives such as mannose, hyaluronic acid derivatives, collagene, aminoacids or peptides; all of which may serve as degradable crosslinker.

Advantageously, '"'-R'-L-R²-* may represent independently a responsively pH cleavable moiety of formula (III) :

(III)

wherein each occurrence of q independently represents an integer, for example q may be an integer from 1 to 6,

D independently represents for each occurrence a C1-C3 alkylenyl moiety, or

-N(Rz)- wherein Rz represents H or Cl-6alkyl. As such, '"'-R'-L-R²-* may contain more than one responsively cleavable covalent bond. In this case (linker of formula (III)), '"'-R'-L-R²-* contains two responsively pH cleavable covalent bond (two imine bonds).

Advantageously, '"'-R'-L-R²-* may represent independently a responsively pH cleavable moiety of formula Ilia, Ilia' or Illb :

(Illb).

Advantageously, '"'-R'-L-R²-* may represent independently a responsively cleavable moiety selected from:

or any one of formulae III, Ilia, Ilia', Illb.

Preferably, L may represent a responsively cleavable covalent bond selected from disulfide, diselenides, imine, amide, ester, urea, hydrazone or thiourea; preferably disulfide, imine (preferably #-R³-L-R⁴-# may comprise a para di-imino phenyl moiety), ester, or hydrazone; more preferably disulfide.

Advantageously, the linker may represent a responsively cleavable fragment of a biological/biodegradable polymer selected from polysaccharides, polypeptides (e.g., polylysine), polynucleotides (e.g., DNA or RNA fragment) and synthetic biodegradable polyethyleneglycol or polylactide polymers, and the linker may have

Advantageously, when the linker has the structure or

A may represent a carbohydrate monomer and the linker may be derived from a natural polysaccharide such as cellulose, amylose, dextran, etc. or a natural or synthetic oligosaccharide;

A may represent a peptide monomer (amino acid residue) and the linker may be derived from a naturally occurring protein or polypeptide (e.g., polylysine) or a synthetic polypeptide:

A may represent a polynucleotide and the linker may be derived from an RNA or DNA fragment. It is understood that the linker having the structure

or

A is identical), or a copolymer fragment (i.e., not all occurrences of A are identical).

In

of a block copolymer (i.e., ml adjacent occurrences of monomer Al, followed by m2 adjacent occurrences of monomer A2, etc.), or a polymer fragment where the different monomers are randomly distributed.

The linker

may be obtained by reaction of a reactive functional group present on the monomers A of a precursor polymer fragment

example, a hydroxyl, amino group, etc.) with an organosilane moiety X^B-R¹M¹(X)3, wherein A, m, M¹, R¹ and X are as defined above, and X^B represents a nonhydrolyzable group bearing a suitable functional group capable of forming a covalent bond with said reactive functional group present on A. Not all occurrences of A may end up being functionalized with, depending on the molar ratio X^B-R¹M¹(X)3:m. For example, when X^B-R¹M¹(X)3:m <1 (less than equimolar ratio), the monomers A may be randomly functionalized, the distribution of functionalized monomers A being controlled in part by the steric hindrance of X^B-

R¹M¹(X)₃, and the identity (type) of A whe

is made up of more than one type of monomer. When the monomers A are not all identical on the linker

(for example, it contains two types of monomers, Ai and A₂, it is possible to selectively functionalized one type of monomer over the other by suitably selecting the reactive functional group on X^B (for example, Ai may be selectively functionalized over A₂). For example, X^B may represent an optionally substituted C 1 -20 alkyl, C2-20 alkenyl or C2-20 alkynyl moiety, an optionally substituted CI -20 heteroalkyl, C2-20 heteroalkynyl or C2-20 heteroalkynyl moiety, or an optionally substituted phenyl moiety, wherein the phenyl, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl moieties may be independently bear at least one functional group capable of covalently reacting with a reactive functional group present on the monomers A of a precursor polymer fra

.

As non limiting examples:

when A represents a carbohydrate monomer: X^B may bear a reactive functional such as halogen, -CO2R, isocyano, or an oxirane/epoxyde moiety, wherein R is selected from H or CI -6 alkyl, which can react with an -OH group present on A. For example, X^B may represent -(CH₂)i-₆R^x, preferably

-(CH₂)₃R^X, wherein R^x represents halogen, -NCO, or

. when A represents a peptide monomer (amino acid residue): X^B may bear a reactive functional such as halogen, -CO2H, isocyano, or an oxirane/epoxyde moiety, which can react with an -OH or -NH₂ group present on A. For example, X^B may represent -( H₂)i-6R^x, preferably -(CH₂)3R^X, wherein

R^A represents halogen, -NCO, or

.

when A represents a nucleotide monomer (nucleotide base): X^B may bear a reactive functional such as halogen, -CO2H, isocyano, or an oxirane/epoxyde moiety, which can react with an -OH or -NH₂ group present on A. For example, X^B may represent -( H₂)i-₆R^x, preferably -(CH₂)₃R^X, wherein

R represents halogen, -NCO, or

Likewise,

may be obtained by reaction of reactive functional groups present at each extremity of a polymer precursor ^m (for example, a hydroxyl, amino group, etc.) with an organosilane moiety X^B-R¹M¹(X)₃, wherein A, m, M¹, R¹ and X are as defined above, and X^B represents a nonhydrolyzable group bearing a suitable functional group capable of forming a covalent bond with said reactive functional group present on each extremity

.

As non limiting examples:

sents a PEG fragment: X may bear a reactive functional such as halogen, -C0₂R, isocyano, or an oxirane/epoxyde moiety, wherein R is selected from H or C 1 -6 alkyl, which can react with the terminal PEG -OH groups. For example, X^B may represent -(CH₂)i-₆R^x, preferably -(CH₂)₃R^X, wherein R^x represents halogen, -NCO,

w

hen represents a polylactide fragment: X may bear a reactive functional such as halogen, -NH₂, -C0₂R, isocyano, or an oxirane/epoxyde moiety, wherein R is selected from H or CI -6 alkyl, which can react with the terminal -OH and -COOH groups of the polylactide fragment. Advantageously, the cleavage/degradation of the linker '"'-R'-L-R²-*,

triggered by any suitable means.

For example, it may be a change in pH (either an increase or a decrease), a change in redox potential, the presence of reduction or oxidation agent, application of UV, visible or near infrared light, ultrasounds, electromagnetic radiation, a change in temperature, enzymatic cleavage, DNA binding, etc... The following Table 2 gives examples of cleavage/degradation triggering means for each of the aforementioned types of responsively cleavable linkers :

Table 2

L Exemplary Triggers

Disulfide Reducing agents (e.g., NaBH₄, dithiothreitol (DTT), glutathione)

Diselenide Reducing agents (e.g. thiols, metal complexes)

Ester pH, enzymatic cleavage (e.g. esterase)

[14]

Amide Enzymatic cleavage (e.g. amidase) [15]

Imine pH

Acetal/ketal/thioketal pH

Anhydride pH

Urea/thiourea Enzymatic cleavage (e.g. urease) [16]

Hydrazone pH

Oxyme pH

Boronic acid (complexed with diols) pH, sugars

Boronic esters pH, reducing agents (e.g., LiAlH₄)

Carbohydrate pH, enzymatic cleavage (e.g.

glycosidases ) [17]

Peptide (e.g., polylysine) Enzymatic cleavage (e.g. protease) [18] Poly ethylenegly col (PEG) Enzymatic cleavage (e.g.

dehydrogenase) [19]

Polylactide Enzymatic cleavage (e.g. esterase) [20]

Polynucleotide (e.g., RNA or DNA) Enzymatic cleavage (e.g. nuclease, glycosidase) [21]

Advantageously, the porous organometaloxide material according to the invention may comprise in its pores or at its surface at least one compound depending on the intended use of the porous organometaloxide material.

Advantageously, the porous organometaloxide nanoparticle according to the invention may comprise in its pores and/or at its surface, preferably at its surface, at least one nucleic acid-type biomolecule such as short interfering RNA ("siRNA") molecule: the porous organometaloxide nanoparticle is said to be "loaded" with at least one nucleic acid-type biomolecule such as short interfering RNA ("siRNA") molecule. Preferably, the nanoparticule has a mixed mesoporous porosity, meaning that it has two different kinds of pore populations; one of which is of smaller size (e.g., pores having an average width in the size range of from about 0.5 to about 5 nm, preferably from 0.5 to 3 nm, more preferably from 0.5 to 2.5 nm, even more preferably from 0.5 to 2 nm), and another which is of larger size (e.g., pores having an average width in the size range of from about 1 to about 50 nm, preferably from 1 to 40 nm, more preferably from 1 to 30 nm, even more preferably from 1 to 20 nm, most preferably from 10 to 15 nm). For example, the siRNA may be directed to a nucleic acid encoding one or both subunits of PLK1 (polo like kinase protein 1), herein referred to as "PLK1 -siRNA". Advantageously, the mesoporous organometaloxide nanoparticle loaded with at least one nucleic acid-type biomolecule such as short interfering RNA ("siRNA") molecule is further covered with a linear polyethylenimine, which helps prevent the degradation of the siRNA and enhance the cellular uptake of the cargo into the cells. For example, a linear polyethylenimine such as jetPEI® commercialized by Polyp lus© may be used. The polymer (polyethylenimine), thanks to its positive charge, is able to bind electrostatically the siR A and create a positive charge on the surface of the nanoparticles.

Advantageously, the compound may be a marker and/or cosmetically or pharmaceutically active principle. Advantageously, the marker may be selected from a contrast agent, a tracer, a radioactive marker, a fluorescent marker , a phosphorescent marker, a magnetic resonance imaging agent or a positron emission tomography agent, such as pyrene, rhodamine, IR783, Gd-EDTA or ⁶⁴Cu-EDTA.

Advantageously, the marker may be any commercial dye. For example it may be a fluorescent molecule selected from rhodamines, fluorescein, luciferase, pyrene-containing markers, aminopyrrolidino-7-nitrobenzofurazan, or indocyanine green (ICG) for NIR emission.

2) Synthetic Overview:

In yet another aspect, there is provided a method for producing a new class of nanocomposite materials so called disintegratable hybrid porous organometaloxides, disintegratable hybrid microporous and macroporous organometaloxides, or disintegratable hybrid mesoporous-macroporous metaloxides (collectively, "DHMOs"). This new class of materials includes porous organometaloxide framework systems in whose framework a

precursor having one of the following structures:

, wherein A, L, R¹, R² and m are as defined above, and Mi and M₂ independently represent Si, Ti or Zr, has been chemically inserted via conventional sol-gel chemistry. The unusual combination of inorganic and organic chemical structure with this scale of porosity and surfaces, together with the enhanced degradability, suggest a myriad of uses for DHMOs, such as but not limited to the controlled release and uptake of chemicals and drugs, ink, their use for sensing, diagnostics, bioassays, cosmetics, catalysis, and any use of porous Si-, Ti- and/or Zr-based organometaloxide materials known in the art. Thus, in one aspect, there is provided a method of synthesizing disintegratable hybrid mesoporous organometaloxide materials (DHMOs) by covalently introducing a preselected precursor (general structure

(X)₃Mi-R¹-L-R²-M₂(X)3

) with a responsively cleavable linker, as defined herein, in the framework of the porous material itself. As such, the resulting DHMOs have a porous framework and present controlled self-destructive behavior in the environment where it is intended to perform its activity. The controlled self-destructive behavior is a property that provides numerous avenues of important applications for such porous systems, ranging from medical to cosmetics to catalysis and purification.

The practitioner has a well-established literature of porous organometaloxide materials chemistry to draw upon, in combination with the information contained herein, for guidance on synthetic strategies, protecting groups, and other materials and methods useful for the synthesis of the disintegratable materials of this invention.

General synthetic methods

Advantageously, the method (method 1) may comprise steps of:

a) Producing a supramolecular template by mixing a suitable surfactant and an aqueous solvent;

b) Adding a mixture of a precursor M(X^A)₄ and a selected precursor

having the structure: (X)₃Mi-R¹-L-R²-M₂(X)_3;

in an aqueous solvent under alkaline conditions; thereby coating the supramolecular template with an organometaloxide sol-gel mixture obtained by hydrolysis-condensation of metal alkoxide; and Removing the supramolecular template; thereby producing a porous organometaloxide material comprising a porous three-dimensional framework of metal-oxygen bonds, wherein at least a subset of metal atoms in the material's framework are connected to at least another metal atom in the framework through a linker having one of the following structures:

M and each occurrence of Mi and M₂ independently represents a metal selected from Si, Ti and Zr;

each occurrence of X and X^A independently represents a hydrolysable or nonhydrolyzable group, provided that on each occurrence of Mi and M₂, at least one occurrence of X represents a hydrolysable group and at least two occurrences of X^A in the precursor M(X^A)₄ independently represent a hydrolysable group; wherein (i) when X or X^A represents a nonhydrolyzable group, it may be selected from an optionally substituted CI -20 alkyl, C2-20 alkenyl or C2-20 alkynyl moiety, an optionally substituted CI -20 heteroalkyl, C2-20 heteroalkynyl or C2-20 heteroalkynyl moiety, or an optionally substituted phenyl moiety, wherein the substituents on the phenyl, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl moieties may be independently selected from halogen, -NO2, -CN, isocyano, CI -6 alkoxy, an oxirane/epoxyde moiety, - N(R)₂ wherein each occurrence of R is independently selected from H or CI -6 alkyl; and (ii) when X or X^A represents a hydro lysable group, it may be selected from a CI -6 alkoxy, CI -6 acyloxy, halogen or amino moiety;

L represents a responsively cleavable covalent bond; and R¹ and R² independently represent an optionally substituted Cl-20alkylenyl moiety, an optionally substituted Cl- 20heteroalkylenyl moiety, an optionally substituted ethylenyl moiety, -C≡C- or an optionally substituted phenyl moiety, wherein the Cl-20alkylenyl, Cl-20heteroalkylenyl or ethylenyl moiety may bear one or more substituents selected from halogen or -OR where R may represent H or C l-6alkyl, and the phenyl moiety may bear one or more substituents independently selected from halogen, Cl- 6alkyl, -NO2, -CN, isocyano, -OR^p, -N(R^P)₂ wherein each occurrence of R^p independently represents H or Cl-6alkyl.

Advantageously, a minimum of 5% molar ratio (based on the metal centers) of (X)₃Mi-R¹-L-R²-M₂(X)₃ to 70% of M(X^A)₄ precursor may be used. Advantageously, a minimum of 30% molar ratio (based on the metal centers) of (X)₃Mi-R^!-L-R²-M₂(X)₃ to 70% of M(X^A)₄ precursor may be used. Because (X)₃Mi-R¹-L-R²-M₂(X)₃ is bivalent (i.e., because this precursor contains two metal atoms per cleavable bond L), the ratio 0.15 eq (X)₃Mi-R¹-L-R²-M₂(X)₃ / 0.70 eq M(X^A)₄ means that Mi and M₂ will represent 30% of the metal centers in the resulting organometaloxide material (i.e., 30%> doping). For a doping of 100%, (X)₃Mi-R¹-L-R²-M₂(X)₃ may be used as the only source of metal (i.e., no M(X^A)₄ is used). For exemplary ratios of equivalents (X)₃Mi-R¹-L-R²-M₂(X)₃ / M(X^A)₄ to reach a variety of %> doping >30%>, see Table 1A. Advantageously, a minimum of 5% molar ratio (based on the metal centers)

(XbM_!— R¹-k¾- R2-M₂(X)₃

^m is to 95% of M(X^A)₄ precursor may be used. See Tables IB and 1C.

Advantageously, for 100% doped porous organometaloxide materials, the method (method 2) may comprise steps of:

b) Adding a selected precursor having the structure:

in an aqueous solvent under alkaline conditions; thereby coating the supramolecular template with an organometaloxide sol-gel mixture obtained by hydrolysis-condensation of metal alkoxide; and c) Removing the supramolecular template; thereby producing a porous organometaloxide material comprising a porous three-dimensional framework of metal-oxygen bonds, wherein at least a subset of metal atoms in the material's framework are connected to at least another metal atom in the framework through a linker having one of the following structures:

wherein:

each occurrence of * denotes a point of attachment to a metal atom in the material's framework; A represents a monomer of a responsively cleavable fragment of biological/biodegradable polymer;

m is an integer from 2 to 10000 and m represents the number of monomers in the fragment of biological/biodegradable polymer; each occurrence of Mi and M₂ independently represents a metal selected from Si, Ti and Zr;

each occurrence of X independently represents a hydrolysable or nonhydrolyzable group, provided that on each occurrence of Mi and M₂, at least one occurrence of X represents a hydrolysable group; wherein (i) when X represents a nonhydrolyzable group, it may be selected from an optionally substituted CI -20 alkyl, C2-20 alkenyl or C2-20 alkynyl moiety, an optionally substituted CI -20 heteroalkyl, C2-20 heteroalkynyl or C2-20 heteroalkynyl moiety, or an optionally substituted phenyl moiety, wherein the substituents on the phenyl, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl moieties may be independently selected from halogen, -N0₂, -CN, isocyano, CI -6 alkoxy, an oxirane/epoxyde moiety, -N(R)₂ wherein each occurrence of R is independently selected from H or CI -6 alkyl; and (ii) when X represents a hydrolysable group, it may be selected from a CI -6 alkoxy, CI -6 acyloxy, halogen or amino moiety;

L represents a responsively cleavable covalent bond; and

R¹ and R² independently represent an optionally substituted Cl-20alkylenyl moiety, an optionally substituted Cl- 20heteroalkylenyl moiety, an optionally substituted ethylenyl moiety, -C≡C- or an optionally substituted phenyl moiety, wherein the Cl-20alkylenyl, Cl-20heteroalkylenyl or ethylenyl moiety may bear one or more substituents selected from halogen or -OR where R may represent H or Cl-6alkyl, and the phenyl moiety may bear one or more substituents independently selected from halogen, Cl- 6alkyl, -N0₂, -CN, isocyano, -OR^p, -N(R^P)₂ wherein each occurrence of R^p independently represents H or Cl-6alkyl. Advantageously, for both methods 1 and 2, the supramolecular template may be made up of any suitable surfactant known in the art to be used in the preparation of porous organometaloxide materials. For example, in a non-limiting exemplary embodiment, the supramolecular template may be formed of self-aggregated rod- shaped micelles of suitable ionic or non-ionic surfactant molecules.

In an effort to avoid unnecessary repetitions, and for concision purposes, every single variant and embodiments described above in section 1) with respect to variables M, Ml, M2, A, m, Rl, R2, L, X, the precursors (X)₃Mi-R¹-L-R²-M₂(X)3;

(XfeM_!— R¹"kA )- R²-M₂(X)3

and ^m , as well as the linkers *-

Rⁱ-L-R²-*,

or , are applicable mutatis mutandis to the synthetic method described immediately above.

It will be appreciated that the exemplary responsively cleavable linkers described herein are for purposes of illustrating and are not in any way meant to limit the scope of the present invention. Other responsively cleavable linkers based on the same concept may also be used. The reader will know how to adapt the teachings described herein, and the Examples for suitable synthetic approaches for these other linkers.

For the biological/biodegradable polymer strategy, two different a roaches

can be pursued: either each of the units of the extremities of the polymeric fragment (

be equipped with a linking function, of the general formula [(X)₃MR-], with X being as defined herein (preferably X may be CI, Me, OMe, OEt, OPr, OBu) and R having the same definition as R¹ and R² as defined herein. Advantageously, for both methods 1 and 2, in step b) a pH adjusting agent may be used to modulate the pH to the desired value. As the pH-adjusting agent, there can be mentioned, for example, acids such as sulfuric acid, hydrochloric acid and the like; and alkalis such as sodium hydroxide, ammonia and the like. Advantageously, in the case of disintegratable mesoporous silicon oxide materials according to the present invention, the pH of the reaction system may be preferably adjusted to 0 to 5, most preferably 1 to 5, when an acid agent is used, and to 8 to 14, most preferably, 8 to 13, when an alkaline agent is used.

Advantageously, for both methods 1 and 2, the removing step c) may be carried out using various methodologies depending on the type of responsively cleavable linker :

thermal removal usually means heating in air or oxygen to oxidatively remove said template containing organic- functionalized porous materials from the material obtained in step b), under conditions that do not destroy said terminal organic function.

Photochemical removal usually means irradiating said template containing organic- functionalized porous materials with ultraviolet light in air or oxygen to photooxidatively remove said template from the material obtained in step b), under irradiation conditions that do not destroy said terminal organic function.

Chemical removal usually means reacting said template containing organic- functionalized porous materials with a reagent that serves to chemically remove said template from the material obtained in step b), under conditions that do not destroy said terminal organic function.

Advantageously, refluxing in a solvent in which the template is soluble allows to remove said template, for example by extraction. For example, a solvent like ethanol, methanol, toluene or any other suitable solvent may be used to remove the template.

Embodiments related to organosilica, and particularly mesoporous organosilica, as the porous framework material but it will be described in more details. This is by no means meant to limit the invention to mesoporous organosilica porous frameworks. Similar embodiments related to other metal oxides will be readily apparent to the skilled artisan base don extensive knowledge on the field of sol-gel synthesis of organometaloxides materials (porous or non-porous) and from reading the contents of the present disclosure. As such, the skilled practitioner will know how to adapt the teachings herein to the preparation of disintegratable porous organometaloxides materials other than organosilica, and will be bale to practice the present invention in its full scope.

Disintegratable porous silicon oxide materials

What follows deals with specific embodiments drawn to disintegratable porous organosilane materials, but the teachings are readily applicable to Ti- and Zr- based materials according to the present invention.

Advantageously, the metal (M, Mi or M₂, as defined above) may be Si. Thus, there is provided a synthetic strategy for creating a new class of disintegratable hybrid mesoporous, macroporous, or mesoporous-macroporous organometaloxide materials, exemplified but not limited to hybrid mesoporous organosilicas. In one aspect, this strategy involves the incorporation of either responsively cleavable covalent bonds or responsively cleavable fragments of biological/biodegradable polymers; directly in the porous framework of the material, as shown as exemplary embodiment in Figure 1.

The resulting materials, which may be preferably in the form of nanoparticles, are hence able to respond to a specific trigger (e.g., chemical, physical or enzymatic stimulation), by undergoing a structural breakdown. This property leads to an improved porous material with potential for multiple types of application, ranging from controlled release and uptake of chemicals and drugs, or bioassays, cosmetics, catalysis to name a few. Indeed, the unusual behavior of the materials according to the invention confers them an enhanced biodegradability, reducing larger particles into smaller, more easily hydrolysable, and consequently less harmful fragments. This in turn reduces the persistence phenomenon of the materials in their working environment, consequently reducing accumulation risks, and purification/removal costs. Advantageously, the metal M may be Si and M(X^A)₄ may represent any Si source suitable for carrying out sol-gel silicon oxide framework synthesis, for example, colloidal silica, sodium silicate, silicon alkoxides, tetramethylammonium silicate and tetraethylorthosilicate (TEOS) and the like. Advantageously, M(X^A)₄ may represent a tetraalkoxysilanes such as tetramethoxysilane, tetraethoxysilane and tetrapropoxysilane, preferably tetraethoxysilane (TEOS).

Advantageously, the silane precursor M(X^A)₄ may preferably contain an alkoxysilane having an organic functional group; in other word, at least one occurrence of X^A may be substituted with a substituent bearing an organic functional group, such that it allows further functionalization. Using the alkoxysilane, it is possible to form a silica framework out of alkoxysilyl groups while disposing organic functional groups on the surfaces of the materials. It is further possible to give suitable properties to the mesoporous silica particles by chemically modifying the organic functional group with other organic molecules or the like.

Functionalized organosilane chemistry is well known, and the reader may refer to the following citations for illustrative synthetic guidance that may be readily adapted in the context of the present invention: [6]

Advantageously, the surfactant may be a cationic surfactant, an anionic surfactant, a non-ionic surfactant; preferably a cationic surfactant such as octadecyl trimethyl ammonium bromide, hexadecyl trimethyl ammonium bromide, tetradecyl trimethyl ammonium bromide, dodecyl trimethyl ammonium bromide, decyl trimethyl ammonium bromide, octyl trimethyl ammonium bromide, hexyl trimethyl ammonium bromide and other quaternary ammonium salt-type cationic surfactants. There can also be mentioned, for example, cetyltrimethylammonium bromide (CTAB), cetyltrimethylphosphonium, octadecyltrimethylphosphonium, benzyltrimethylammonium, cetylpyridinium, myristyltrimethylammonium, decyltrimethylammonium, dodecyltrimethylammonium, dimethyldidodecylammonium, pore swelling agents like 1,3,5-trimethylbenzene (TMB) and the like.

Because certain silane precursors Si(X^A)₄ like TEOS are not soluble in water alone, a co-solvent, preferably ethanol, may be added. Other solvent such as methanol or DMF can be used. Advantageously, the aqueous solvent comprises an alcohol, such as methanol or ethanol.

Advantageously, the silane precursor M(X^A)₄ may be TEOS and the surfactant/TEOS mole ratio can be varied to control the pore-volume fraction in the final material and to vary the pore structure. Also, it will be recognized by those skilled in the art that a much wider range of surfactant sizes and amounts in the family of small polyoxy ethylene ethers may be possible with different solvent amounts.

The size and shape of the pores may be controlled by varying the type, the quantity and concentration of surfactant in step a). It will be appreciated that the technique of adding a hydrophobic additive to enlarge mesopores when preparing a mesoporous material is disclosed in prior documents. [10] Thus, in certain embodiments, the method may comprise adding a hydrophobic additive to control the size of the mesopores.

The mixing ratio of the silica precursors M(X^A)₄ and (X)3Si-R¹-L-R²-Si(X)3 to the surfactant is not particularly limited, but a molar ratio of 3 : 1 is preferred. If the amount of the surfactant is outside this range relative to the silica precursors, the structure of the product may be less regular, and it may be difficult to obtain mesoporous silica particles with a regular arrangement of mesopores. It is possible to easily obtain mesoporous silica particles with a regular arrangement of mesopores particularly when the molar percentage of linker is in the range but not limited to 10 and 50 % of overall silicon source.

As described above, advantageously, a minimum of 5% molar ratio (based on the metal centers) of (X)₃Mi-R¹-L-R²-M₂(X)3 to 70% of M(X^A)₄ precursor may be used, to effect a minimum of 5% doping of the resulting organosilica material.

As described above, advantageously, a minimum of 30%> molar ratio (based on the metal centers) of (X)₃Mi-R¹-L-R²-M₂(X)3 to 70% of M(X^A)₄ precursor may be used, to effect a minimum of 30% doping of the resulting organosilica material. For a doping of 100%, (X)3Si-R¹-L-R²-Si(X)3 may be used as the only source of metal (i.e., no Si(X^A)₄ is used). For exemplary ratios of equivalents (X^Si-R'-L-R²-

Si(X)3 / Si(X^A)₄ to reach a variety of % doping, see Table 1 A. Likewise, advantageously, a minimum of 5% molar ratio (based on the metal

(X)₃M

centers) of ^{r1 M} I (^X)3 or — ^R1~H£H^~R2_M2(X); is to 95% of M(X^A)4 precursor may be used. See Tables IB and 1C. Advantageously, the linker may comprise a disulfide bond (-S-S-), a peptide bond, an imine bond (-N=CH-) or a carbohydrate moiety, as responsively cleavable bond or moiety. Advantageously, the linker may comprise a disulfide, ester, imine or hydrazone bond, preferably a disulfide bond. Advantageously, the precursor having the structure (X)3M-R¹-L-R²-M(X)3 may produced in situ. For example, a general synthetic approach for in situ generation of the precursor is depicted in Scheme 1 below:

Table 2: Exemplary synthetic conditions for preparing porous organosiliconoxide materials according to the invention

(e.g.

HSi(OEt)₃). A

similar

strategy may

be used for

thioketal- containing

linkers.

Synthesis of O O

II II

an anhydride

nⁿ + HSi(OEt)₃ functionalized

with 2

Suitable surfactant, TEOS,

functional

auxiliary solvent (e.g., L=- groups (e.g.

EtOH, DMF, etc.) Anhydride

double bonds)

(X)₃Mi-R¹-L-R²-M₂(X)₃

reactable with

a silane source

(e.g.

HSi(OEt)₃).

Condensation (EtO)₃Si^-\^ NCO ₊ of an

isocyanate

(e.g. 3-

Suitable surfactant, TEOS,

(Triethoxysilyl

auxiliary solvent (e.g., L=- )- propyl

EtOH, DMF, etc.) Urea/thiourea

isocyanate)

(X)₃Mi-R¹-L-R²-M₂(X)₃

and an amino- derivatized

silane (e.g.

APTES).

Functionalizati o o

on of an

Suitable surfactant, TEOS, ⁿ + NH2NH2 -7

hydrazone

auxiliary solvent (e.g., L=-N- H₂N. _{N N}. NH₂

with a silane

EtOH, DMF, etc.) N=CH- derivative

(Χ)₃Μι-^-Ε^²-Μ₂(Χ)₃

(e.g. 3-

(EtOkSi^^-^ NCO

(triethoxysilyl)

Scheme 1: Exem lary synthesis of a pH responsive linker (M = Si).

In this aspect of the invention the step of preparing a porous framework material may include synthesizing the porous framework material by mixing the precursors ((X3Si)Rl-L-R2(SiX3); and Si(X^A)₄) of the framework material with a suitable supramolecular template under conditions suitable for self-assembly of the particulate constituent to form the framework material, and subsequent removing the supramolecular template. The framework material being an organometaloxide, which may be mesoporous, macroporous, or combined mesoporous-macroporous having a porosity containing both size regimes of pores. The metaloxide may be silica.

The method for producing disintegratable porous silica materials of the present invention is not particularly limited, but the method preferably includes the following steps. The first step is a "surfactant micellar assembly step" wherein the surfactant serve as template for the porous organosiliconoxide material (step a)). The next step is a "organosiliconoxide covering step" comprising:

Method 1 : adding a silica source Si(X^A)₄ to ether

with a suitable precursor (X)₃Mi-R¹-L-R²- or

M=Ml=M2=Si), to thereby cover the surface (periphery) of the surfactant template with organosiliconmetaloxide (step b)); or

i-R¹-L-R²-M₂(X)_3;

Ml=M2=Si), to the surfactant template, to thereby cover the surface (periphery) of the surfactant template with organosiliconmetaloxide (step b)).

As discussed before, for method 1, advantageously, the mixing ratio of the

or

respectively, may be adjusted to control the desired % doping in the resulting porous organosiliconmetaloxide material. For exemplary ratios of equivalents (X)₃Si-R¹-L-R²-Si(X)₃ / Si(X^A)₄ to reach a variety of % doping >30%, see Table 1 A. See also Tables IB and 1C for the polymer A version.

The final step is a "removal step" of removing the surfactant template (step c)). The final step may be performed or not depending on the type/use of the surfactant .

Advantageously, the following may be used as (X)₃Si-R¹-L-R²-Si(X)₃ pre

wherein q and D are as as defined generally and in any variant above;

wherein each occurrence of R may independently represent Me, Et, z^'Pr or tBu. Advantageously, the following may be used as (X)3Si-R¹-L-R²-Si(X)3 precursor:

wherein q and D are as as defined generally and in any variant above; Si(OR)₃

(RO)₃Si

(RO)₃Si " ^'— ^' "S'

wherein each occurrence of R may independently represent Me, Et, z^'Pr or tBu.

Advantageously, the surface of the porous silica material according to the invention may be functionalized with a surface agent, for example by using a functional group-containing trialkoxysilane, such as a PEG group linked to a trialkoxysilane or 3-aminopropyl)triethoxysilane (APTES). Advantageously, the functionalized trialkoxysilane is 3-aminopropyl)triethoxysilane (APTES), which allows shifting the global charge of the material to a positive value, thus allowing an electrostatic interaction with negatively charged biomolecules, such as siRNA.

Likewise, marking of the porous silica material (for example for medical purposes) may be achieved by condensation of a marker-containing trialkoxysilane. The marker may be selected from a contrast agent, a tracer, a radioactive marker, any commercial dye, such as a fluorescent marker or a phosphorescent marker, a magnetic resonance imaging agent or a positron emission tomography agent, such as pyrene, rhodamine, IR783, Gd-EDTA or ⁶⁴Cu-EDTA. The marker may be a fluorescent molecule selected from rhodamines, fluorescein, luciferase, pyrene- containing markers, aminopyrrolidino-7-nitrobenzofurazan, or indocyanine green (ICG) for NIR emission.

As used herein, the term "surface agent" refers to a molecule that partly or totally covers the surface of the porous material, allowing the surface properties of the material to be modified, for example:

- changing its outer electric charge for better interaction/binding with biomolecules of interest; and/or

- modifying its biodistribution, for example to avoid its recognition by the reticulo-endothelial system ("furtiveness"), and/or

- giving it advantageous bioadhesion properties during oral, ocular or nasal administration, and/or

- enabling it to specifically target certain sick organs/tissues, etc.

According to the invention, several surface agents may be used to combine the abovementioned properties. For example, a surface agent combining at least two of the abovementioned properties may be used. For example, the organic surface agent may be chosen from:

an oligosaccharide, for instance cyclodextrins,

a polysaccharide, for instance chitosan, dextran, fucoidan, alginate,

, amylose, starch, cellulose or xylan,

a glycosaminoglycan, for instance hyaluronic acid or heparin, a polymer, for instance polyethylene glycol (PEG), polyvinyl alcohol or polyethyleneimine,

a surfactant, such as pluronic or lecithin,

vitamins, such as biotin,

coenzymes, such as lipoic acid,

antibodies or antibody fragments,

amino acids or peptides.

In another example, the organic surface agent may be chosen from: - poly(ethylene glycol) (PEG),

- polyethylenimine (PEI),

- 3-(trihydroxysilyl)propyl methylphosphonate (THPMP),

- N-(trimethoxysilylpropyl)ethylenediamine triacetic acid (EDTAS), - N-[3-(trimethoxysilyl)propyl]ethylenediamine,

- N-[3-(trimethoxysilyl)propyl]-N,N,N-trimethylamrnonium (TA- trimethoxysilane),

- (3-mercatopropyl)trimethoxysilane (MPTMS),

- tumor-targeting ligand modification

Scheme 2: Exemplary synthesis of a redox responsive linker (M = Si).

-

TEOS BTSPD

3) Compositions and uses

The porous organometaloxide framework materials of the invention are useful for any known use of porous organometaloxide framework materials known in the art. The porous organometaloxide framework materials of the invention are particularly adapted for uses of this type of materials where the self-destructive behavior that characterizes the organometaloxide of the invention provides an advantage. In particular, in contrast to conventional porous organometaloxide framework materials known in the art, the materials of the invention have the unexpected property of completely losing their structural integrity (disintegration) upon application of a suitable stimuli. Owing to the intrinsic porosity combined to their disintegratable properties, the materials of the invention prove much more efficient in releasing and delivering compounds that they might be loaded with (e.g., therapeutically and/or cosmetically active principles, or other chemicals). In other words, release of the compounds trapped/encapsulated in the materials' porous framework occurs much more efficiently than with conventional porous organometaloxides known in the art. For biomedical applications (e.g., when the framework metal is Si), this means less bio-accumulation, better elimination, and less toxicity.

These uses include:

Biomedical applications, including controlled drug release and uptake, and their use in sensing, diagnostics and bioassays. See for example WO2005087369, WO2011124739, WO2009024635 , us2013195963, us20100278931

Cosmetics WO 2010030252, JP 2002348380, WO 2010030252

Catalysis K 2013113770

Photovoltaics WO 2013154964, US 20130269782

Inks/ Paints additive WO2011119265 Al , US4877451 A

Optical coating WO 2012022983 Al

Anti-microbial WO2006120135 Al

Accordingly, there is provided compositions comprising a disintegratable porous organometaloxide framework material according to the invention and any compound and/or additive suitable for any one or more of the material's intended use describe above.

Thus, for applications that involve a disintegratable porous organometaloxide framework material according to the invention loaded with one or more compounds (for medical or cosmetic uses for example), the process for preparing the porous organometaloxide materials according to the invention may further comprise a step (d) of introducing, into the pores and/or at the surface of the porous organometaloxide materials, at least one molecule of interest, which may be a biomolecule, a pharmaceutically active principle and/or a marker. Any method known to those skilled in the art may be used to that end. The molecule of interest may be introduced, for example, into the porous organometaloxide materials of the present invention:

via impregnation, by immersing the material in a solution of the molecule of interest;

by sublimation of the molecule of interest, and the gas is then adsorbed by the material; or via rotary roll milling, which consists in mechanically mixing the material and the molecule of interest.

The form of the porous material of the invention may be adapted to fit the intended use. For example, for applications in catalysis, the disintegratable porous organometaloxide framework material according to the invention may be in the form of a monolith or fragments. For ink, paint, biomedical or cosmetic applications, the disintegratable porous organometaloxide framework material according to the invention may be in the form of nanoparticles. For separation/purification and catalysis applications, the disintegratable porous organometaloxide framework material according to the invention may be in the form of thin or thick films.

In one aspect, for medical applications, a composition according to the invention may comprise a disintegratable porous organometaloxide nanoparticle according to the invention loaded with a pharmaceutically active principle and/or a marker, for example in its pores or at its surface.

In one preferred variant, a composition according to the invention may comprise a disintegratable porous organometaloxide nanoparticle, the outer surface of which may be functionalized with positively charged groups, such as quarternary amine salts (e.g., NH₃ ⁺), either directly or via a linker (for example by grafting the outersurface of the nanoparticle with amine groups via a properly functionalized trialkoxysilane, such as 3-aminopropyl)triethoxysilane (APTES)), further loaded at its surface and/or in its pores with at least one nucleic acid-type biomolecule such as short interfering RNA ("siRNA") molecule. The siRNA loaded nanoparticle may be further covered with a linear polyethylenimine, which helps prevent the degradation of the siRNA and enhance the cellular uptake of the cargo into the cells. For example, a linear polyethylenimine such as jetPEI® commercialized by Polyplus© may be used. The polymer (polyethylenimine), thanks to its positive charge, is able to bind electrostatically the siRNA and create a positive charge on the surface of the nanoparticles. Advantageously, the siRNA may be PLK1 -siRNA. In another aspect of the present invention, pharmaceutically acceptable compositions are provided, wherein these compositions comprise any of the porous organometaloxide materials as described herein, and optionally comprise a pharmaceutically acceptable carrier, adjuvant or vehicle. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents.

The use of carriers and vectors for molecules of interest, especially molecules with a therapeutic effect or markers, has become a major issue for the development of novel diagnostic methods or novel medicaments. Specifically, the molecules of interest have characteristics that have an influence on the pharmacokinetics and biodistribution of these molecules and that are not always favorable or adaptable to the medium into which they are introduced. They are, for example, physicochemical characteristics, such as instability, a strong tendency toward crystallization, poor water solubility and/or biological characteristics such as toxicity, biodegradability, etc.

The porous organometaloxide materials according to the invention may be used for that purpose, namely for drug delivery, and for improving the pharmacokinetic profile of drugs, for example in terms of escaping the immune system and/or uptake by certain organs, for example the liver or the kidney, thus avoiding their accumulation in these organs.

There is thus provided porous organometaloxide materials (for example in the form of nanoparticles) for use as medicament and/or drug delivery/controlled release. For example, the porous organometaloxide materials may comprise in their pores or at their surface at least one pharmaceutically active principle. There is also provided porous organometaloxide materials (for example in the form of nanoparticles), for use in medical imaging. For example, the porous organometaloxide materials may comprise in their pores or at their surface at least one marker. There is also provided a method for treating a condition or disease comprising administering to a subject in need thereof a disintegratable porous organometaloxide material according to the present invention, appropriately loaded on its surface or in its pore with a drug moiety adapted for such treatment. For example, the nanoparticles according to the invention may be loaded with PLKl-siRNA in its pores and/or at its surfaces, preferably at its surface, and may find applications in the prevention and/or treatment of cancer, for example hepatocellular carcinoma. Accordingly, there is provided a method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a porous, preferably mixed mesoporous, organometaloxide material according to the invention, preferably in form of nanoparticles. Preferably, the treatment method uses a porous, preferably mixed mesoporous, organometaloxide material according to the invention, preferably in form of nanoparticles, wherein PLKl-siRNA is loaded on the outersurface and/or in the pores of the nanoparticle, preferably at its surface. Advantageously, the subject may be a mammal, preferably a human subject. In an exemplary embodiment, the method is for treating hepatocellular carcinoma.

Likewise, for cosmetic applications, a composition according to the invention may comprise a disintegratable porous organometaloxide framework material according to the invention loaded with a cosmetically active principle, for example in its pores.

In another aspect, the invention provides the use of disintegratable porous organometaloxide framework material according to the invention in a cosmetic composition.

In another aspect, the invention provides the use of disintegratable porous organometaloxide framework material according to the invention in catalysis. In another aspect, the invention provides the use of disintegratable porous organometaloxide framework material according to the invention in photovoltaics.

The disintegratable porous organometaloxide materials according to the invention therefore can find applications in in vitro and in vivo diagnostics, therapy, in cosmetics, in drug delivery, and in any other application where a release can be envisaged.

Other advantages may also emerge to those skilled in the art upon reading the examples below, with reference to the attached figures, which are provided as nonlimiting illustrations. EQUIVALENTS

The representative examples that follow are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples which follow and the references to the scientific and patent literature cited herein. It should further be appreciated that the contents of those cited references are incorporated herein by reference to help illustrate the state of the art.

The following examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and the equivalents thereof.

EXEMPLIFICATION

The compounds of this invention and their preparation can be understood further by the examples that illustrate some of the processes by which these compounds are prepared or used. It will be appreciated, however, that these examples do not limit the invention. Variations of the invention, now known or further developed, are considered to fall within the scope of the present invention as described herein and as hereinafter claimed.

The present invention will now be exemplified using mesoporous organosilica as the porous framework material but it will be understood this is not meant to limit the invention to mesoporous organosilica porous frameworks.

The tested particles will be tested in their triggered degradation ability, and evidence of the breakdown of the material will be given by demonstrating the structural/morphological transformations occurring in the responsively disintegratable particles during the triggered breakdown process.

Abbreviations:

LP-ssNPs : large pore disulfide-doped mixed-mesoporous organosilica nanoparticles (also referred to as BPBP); i.e., disulfide-doped mesoporous organosilica nanoparticles in which larger pores have been created according to the teachings of the present invention, thereby forming disulfide-doped mixed- mesoporous organosilica nanoparticles.

ssNPs : small pore disulfide-doped mesoporous organosilica nanoparticles

NH2-LP-ssNPs : LP-ssNPs grafted with 3-aminopropyl groups

NH₂-ssNPs : ssNPs grafted with 3-aminopropyl groups

PLKl@NH₂-LP-ssNPs: NH₂-LP-ssNPs particles loaded with a double stranded PLK1 specific siRNA

PLK1 : polo like kinase protein 1

siRNA@NH₂-LP-ssNPs: NH₂-LP-ssNPs loaded with a double stranded siRNA non-specific for PLK1

PLK1@NH₂- ssNPs : small pore ssNPs loaded with PLK1 siRNA

r-LP-ssNPs or r-NH₂-LP-ssNPs : NH₂-LP-ssNPs functionalized on the surface with Rhodamine B isothiocyanate

jp-PLKl@LP-ssNPs: NH₂-LP-ssNPs loaded with a double stranded siRNA nonspecific for PLK1 and coated with jetPEI®

BPBP : (large pore) redox-responsive mixed-mesoporous silica nanoparticles (also referred to as LP-ssNPs).

PLK1: polo-like kinase 1.

Example 1 - Introducing an S-S bond in the framework of mesoporous silica nanoparticles (SNPs)

Synthesis

• Synthesis of 20 nm average diameter S-S doped SNPs: CTAB (145 mg) was added to a mixture of 72 ml of H₂0, 3 ml of EtOH, and 0,6 ml of a 28 wt% ammonia solution. The reaction mixture was stirred at 50°C for 1 h before the addition of 1,25 ml of a 0.88 M ethanolic solution of: the disulfide silane (bis(3- triethoxysilylpropyl)disulfide, 30% in molar ratio with respect to the Si centers), phosphonated silane (3-(trihydroxysiiyl)propyi methylphosphonate monosodium salt, 3%); and Tetraethyi orthosilicate (TEOS) (67% in molar ratio with respect to the Si centers). The above reaction mixture was continuously stirred for 24 h at

70°C. The Cetyltrimethyiammonium bromide (CTAB) mesoporous template was then removed by stirring the sample in acidic ethanol (50 ml) at 90 °C for 12 h. The resulting solid was recovered by centrifugation, washed with water and ethanol several times, and finally preserved in water as a suspension. · Fluorescein-tagging of SS doped SNPs (SS-NPs): the S-S doped SNPs were dispersed in 20 ml of toluene, before adding to the suspension a solution of fluorescein isothiocyanate (FITC) and (3-aminopropyi)-triethoxysiiane (A PTES) in ethanol, characterized by a concentration of 0.5 mmol per mg of NPs to be functionalized. The suspension was then heated at reflux for 14 h. The resulting solid was recovered by centrifugation, washed with toluene, water and ethanol several times, and finally preserved in water.

• Chemical biodegradability test: reduction of the S-S bond was performed with NaBH₄, being an efficient and irreversible reducing agent, not forming residual by-products, which might hamper visual evaluation of the efficacy of the reduction. In a typical reduction experiment, NaBH₄ (1 mg) was added to a stable suspension of the luminescent SS-NPs in MeOH (1 mg/ml). After 6 hours, the reaction was quenched by addition of a small amount of water (0.1 ml) to deactivate unreacted NaBH₄.

Analysis and results

· Characterization of the composition of the nanoparticle was firstly performed by means of electron diffraction X-ray spectroscopy (EDX) and thermogravimetric analysis (TGA), in order to confirm the presence of the linker in the structure of the particle. The EDX spectrum and EDX map reports the presence of the signal of S in the material, clearly confirming the introduction of the disulfide bridge in the SS-NPs' structure.

• The presence of the organic fragments was further sustained by thermogravimetric analysis. From the TGA plot it resulted possible to detect the content of organic material in the NPs, characterized by a lower decomposition temperature compared to the silica framework. Indeed, the pristine SS doped NPs are characterized by a percentage weight loss of 23% at 350 °C. This data corresponds to a presence of disulfide bridging ligand. For the fluorescein functionalized SS-NPs a weight loss of 30% was instead recorded at 350 °C, due to the further functionalization with the dye.

• Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and high resolution Transmission Electron Microscopy (HRTEM) were used to confirm the nanoparticle morphology of the synthesized responsively disintegratable material, as well as the presence of the porous framework. It was hence determined that the average diameter of the particles results around 20 nm. After the chemical degradation, the porous structure was lost, and only small particle fragments, or amorphous silica was detected in the sample.

· Important evidence of the degradation in solution came from Dynamic light scattering (DLS) analysis. As can be clearly seen the initial size of the nanoparticles (19 nm ± 10) is completely changed after the reduction reaction and the particles size is reduced to 5 nm ± 3.

• Absorption (UV-Vis) and emission properties of the SS-NPs were evaluated before and after their reduction. Regarding the absorption properties, after the reduction, the broken particles' suspension presents a much lower degree of scattering, due to the reduced size of the particles. Also the luminescence of the reduced material increases considerably, indicating a lower quenching occurring from the particles, again attributable to the smaller size of the reduced material. · Small angle X-ray scattering (SAXS) analysis confirmed the HRTEM results, indicating that the synthesized responsively disintegratable SS-NPs indeed present a mesoporous network, which is partially lost after the breaking experiment. To prove that these pores system can be used in biomedicine and that the degradation can occur also in biological systems, where for biological systems is intended cells, bacteria, virus, cell experiment were done.

• The cell up-take of SS-NPs was tested with Hela cells. The results show that the NPs are internalized by the cells. The NPs have been labeled with a green fluorescent dye and therefore the localization of the porous particles and the dye will be the same if the particles are not degraded. Indeed this is the case for the first 4 hours. However after longer time incubation the fluorescein seems to diffuse inside the cell, suggesting the occurring decomposition of the particles in the cellular environment and the spreading of the small resulting pieces all over the cytoplasm. To prove that the fragments are not toxic viability tests have been performed on the cell monitoring their death after 4, 24 and 48 hours. The results do not show any mortality of the cells.

Example 2 - Introducing a small peptide in the framework of mesoporous silica nanoparticles

Synthesis

• Synthesis of trilysine-doped mesoporous silica nanoparticles (Pep-SNPs): CTAB was dissolved in a mixture of H₂0 and 28 wt% ammonia solution (amount depending on the requested morphology). The reaction mixture was stirred at 50 °C for 1 h before the addition of a solution of Tri- lysine (LLL), containing 6 mg of the peptide, 27 μΐ of 3-(Triethoxysiiyi)propyi isocyanate (NCO-PTES), 15 μΐ of TEA in 1 ml of DMF. The above described reaction mixture was hence stirred for an additional 2 h at 50 °C. The CTAB mesoporous template was removed by stirring the sample in ethanol (50 ml) at 50 °C for 6 h. The resulting solid was recovered by centrifugation, washed with water and ethanol several times, and finally preserved in water as a suspension.

• Rhodamine-tagging of the Pep-SNPs: the peptide doped NPs were dispersed in 50 ml of EtOH, before adding to the suspension a solution of rhodamine B isothiocyanate (RITC) in EtOH, characterized by a concentration of 0.5 mmol per mg of NPs to be functionalized. The suspension was then heated at reflux for 14 h. The resulting solid was recovered by centrifugation, washed with ethanol and water several times, and finally conserved in water.

· Biodegradability test: destruction of the Rho-tagged Pep-SNPs was performed upon exposure of the particles to an enzyme (trypsin), able to attack and destroy the peptide fragment introduced in the framework of the material. Specifically, in a typical experiment, trypsin (50 μΐ) was added to a stable suspension of the luminescent Pep-SNPs (0.1 mg/ml) in a 1 w% dispersion of poly- lysine in water, and the mixture incubated at 37.5 °C for 3 days.

Analysis and results To prove that the degradation can also enzymatically triggered a small peptide was introduced in the framework of mesoporous silica nanoparticles. The results clearly indicated that also in this case a full breaking of the silica is observed and the supported experiments are reported below.

· Scanning electron microscopy (SEM) images of Pep-SNPs before and after exposure to the trypsin enzyme highlight a difference in the morphology of the material. In fact, the typical annular morphology of the particles is lost, and mainly amorphous silica is detected.

• The cell up-take of Rho-tagged Pep-SNPs was also tested with Hela cells. Preliminary results show that the NPs are up-taken by the cells (average viability of

95%). Also, even more relevantly for the biodegradability issue, whilst at the beginning the NPs enter the cell as small agglomerates, with time the rhodamine seems to diffuse, suggesting, once again, the occurring decomposition of the NPs. Example 3 - Mixed Si-Ti mesoporous nanoparticles

Synthesis

Synthesis of mixed Si-Ti mesoporous nanoparticles (STNPs): 1 g of Pluronic PI 23 surfactant was dissolved in 25g of EtOH. The mixture was stirred at 40°C for 1 hour before the addition of 5 mL ethanol solution of 1.04 g of TEOS and 2.37 g of bis(triethoxysilyl-propyl)disulfide and 1.42 g of titanium isopropoxyde (TIPO). After several minutes, 1.89 g of concentrated HC1 was added to the solution. The above reaction mixture was continuously stirred for an additional 1 h and transferred to petri dish. The mother gel was then kept in the oven for 4 days at 35°C. The surfactant template was removed by Soxhlet extraction techniques.

Analysis and results

· Characterization of the composition of the materials was firstly performed by means of small angle x-ray scattering (SAXS), electron diffraction X-ray spectroscopy (EDX) and thermogravimetric analysis (TGA), in order to confirm the presence of the linker in the structure of the materials.

• The presence of the organic fragments was further sustained by thermogravimetric analysis. From the TGA plot it resulted possible to detect the content of organic material in the materials, characterized by a lower decomposition temperature compared to the inorganic framework.

• Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and high resolution Transmission Electron Microscopy (HRTEM) were used to confirm the morphology of the synthesized responsively disintegratable material, as well as the presence of the porous framework.

Example 4 - Synthesis of 100 nm average diameter SS-doped SNPs (SS-doped mesoporous silica nanoparticles) and tagging with Rhodamine

Synthesis of 100 nm SS-doped SNPs

Synthesis of SS-doped SNPs (100 nm average diameter): CTAB (250 mg) was dissolved in a solution of distilled water (110 mL), EtOH (10 mL) and NaOH (2M, 0.875 mL) that was heated to 80 °C and stirred vigorously. After complete solubilization of CTAB, TEOS (0.875 mL) and Bis-(triethoxy-silyl-propyl)- disulfide ("BTSPD", 0.390 mL) were added under vigorous stirring. After 6 h the solution was cooled to room temperature and the particles, recovered by centrifugation (20 minutes at 40 krcf), were then purified through a sequence of sonication/centrifugation cycles in EtOH. To remove the surfactants from the pores, the particles were dissolved in acidic EtOH (100 mL, 30 mL of HCl) and refluxed o.n. The particles were hence purified by a sequence of sonication/centrifugation cycles in EtOH and finally dried under vacuum. The material was finally thoroughly characterized by means of XPS, TGA, SAXS, N2 adsorption, SEM and TEM analysis techniques.

Synthesis of Rhodamine tagged SS-doped SNPs

Synthesis of S-S doped SNPs (100 nm average diameter): 2.5 mg of rhodamine B isothiocyanate (RITC) were dissolved in EtOH (5 mL) before adding APTES (6 mL). In another flask CTAB (250 mg) was dissolved in a solution of distilled water (110 mL), EtOH (10 mL) and NaOH (2M, 0.875 mL) that was heated to 80°C and stirred vigorously. The RITC/ APTES solution was stirred for 30 minutes before adding TEOS (0.875 mL) and Bis-(triethoxy-silyl-propyl)-disulfide ("BTSPD", 0.390 mL). Once the temperature of the CTAB solution had stabilized, the solution containing the silane sources was added. After 6 h the solution was cooled to room temperature and the particles, recovered by centrifugation (20 minutes at 40 krcf where "rcf ' stands for "relative centrifugal force"), were then purified through a sequence of sonication/centrifugation cycles in EtOH. To remove the surfactants from the pores, the particles were dissolved in acidic EtOH (100 mL, 30 mL of HC1) and refluxed o.n. The particles were hence purified by a sequence of sonication/centrifugation cycles in EtOH and finally dried under vacuum. The material was finally thoroughly characterized by means of XPS, TGA, SAXS, N2 adsorption, SEM and TEM analysis techniques.

Example 5 - Synthesis of ICG-tagged SS-doped SNPs (SS-doped SNPs tagged with fluorescent tag indocyanin green)

Functionalization with ICG-NHS

Synthesis of Rhodamine B doped SS-doped SNPs functionalized with Indocyanine Green N-succinimidyl ester (ICG-ss-NPs): 20 mg of ss-NPs were dispersed by sonication in DMSO (1 mL) and the solution diluted in toluene (4 mL). In a separate flask, ICG-NHS (0.1 mg) and 3-aminopropyldimethylmethoxysilane (3 μί) were stirred for 2 h in DMSO (1 mL) at r.t..[22, Lu et al, Small 2010, 16, 1794] This solution was hence added to the NPs suspension and the whole kept stirring at r.t. for further 20 h. The mixture was then centrifuged (20 minutes at 40 krcf) and the supernatant removed, before adding to the pelleted particles fresh EtOH (10 mL). The particles were hence purified by a sequence of sonication/centrifugation cycles in EtOH and finally dried under vacuum.

Biodistribution:

Mice were injected with 0.5 mg of ssNPs (tagged with ICG and dispersed in 200 uL of PBS) by intravenous retro-orbital injection. After 3 h from the injection biodistribution was performed. Excretion was monitored up to 48 h, when no more emission was observed in the animals. The biodistribution results show that from the examination of the organs the particle seems to accumulate mainly in the lungs and in the liver.

The excretion results show the absence of specific emission already after 2 days.

Example 6 - Synthesis of PLKl-siRNA loaded SS-doped SNPs

Breakable mesoporous silica nanoparticles were synthetized by a modified Stober process. Basically, the surfactant cetyltrimethylammonium bromide (CTAB) was dissolved in a mixture of water/ethanol to form the micellar template. Then, tetraethyl orthosilicate and bis[3-(triethoxysilyl)propyl]disulphide (70/30 wt%) were added to the CTAB solution under basic conditions to form breakable silica nanoparticles around 100 nm with a pore size of 2.4 nm (Fig 1, a, b and c). In order to attract more siR A into the particles, the ss-MSNs were treated for several days with mesytilene, a swelling agent able to expand the pores of the particles. As shown in Fig Id, the pore size increases having finally a width between 10-15nm. The swelling effect was also proven by transmission electron microscopy (TEM) (Fig lc).

Once the material fully characterized, the negatively charged particles were grafted with (3-aminopropyl)triethoxysilane (APTES) shifting the global charge of the material to a positive value. Thus allowing an electrostatic interaction with the negatively charged siRNA. This functionalization was optimized in order to have the highest charge without compromising the breakability of the material.

The 55-MSNs were then incubated with different amount of siRNA in a MES buffer at pH 5 in order to reach the highest loading possible. A plateau was reached for a concentration of 66 μg of siRNA/mg of ss-MSNs. The siRNA loading was also confirmed by Dynamic Light Scattering (DLS) and HR-TEM, showing a slight increase of 10 nm after incubation with the small molecule. Moreover, the surface charge of the material shifted from a positive value (+26mV for ss-APTES) to a negative value (-25mV for ss-PLKl proving the efficient grafting of the gene. To prevent the degradation of the siRNA and to enhance the cellular uptake of the cargo into the cells, a linear polyethylenimine (jetPEI®) given by Polyplus© was used to cover the siRNA. The polymer, thanks to its positive charge, was able to bind electrostatically the siRNA and create a positive charge on the surface of the particles. The presence of the layer was confirmed by DLS and Zeta Potential, giving a particle size of 160 nm and a positive charge surface of +7 mV in a PBS solution.

Material and Methods

• All commercial solvents and reagents were used as received from, without further purification : Tetraethyl orthosilicate (TEOS), (3- aminopropyl)triethoxysilane (APTES), glutathione (GSH), rhodamine B isothiocyanate (RITC, mixed isomers), triethylamine (TEA), paraformaldehyde (PFA), glutaraldehyde 25 wt.%, Triton X-100, Bovine Serum Albumine (BSA), Fluoromont acqueous mounting medium, diethyl pyrocarbonate (DEPC) and trimethylbenzene (TMB) 2-(4- morpholino)ethanesulfonic acid hydrate (MES), Kaiser test kit and all solvents were purchased from Sigma Aldrich and used as such. Bis(triethoxysilyl-propyl)disulfide (BTSPD, 95%) was purchased from Fluorochem and Cetyltrimethylammonium bromide (CTAB) were purchased from Acros Organics. The jetPEI® was kindly provided by Polyplus-transfection SA. iScript cDNA synthesis kit and sso SYBR mix were purchased from Biorad. Trizol Reagent was purchased from Ambion. Control and PLK1 siRNAs were purchased from Eurogentec and the GAPDH and PLK1 primers were bought from eurofms. Alamar Blue was purchased from Thermo Fisher. Dulbecco's Modified Eagle's Medium (DMEM), Phosphate Buffered Saline 7.4 (PBS), Fetal Bovine Serum (FBS), penicillin, streptomycin and L-glutamine 200mM were purchased from Gibco (Life Technologies). Hoechst 33342, Lysotracker Blue-DND 22 and Alexa Fluor® 647 Phalloidin were purchased from Invitrogen. Huh-7 cells were obtained from ATCC/LGC Standards GmbH (Wesel, Germany) and cultivated according to the provider's protocol. Table 3: siR A sequences

Table 4: Primers sequences

• Synthesis of the breakable organosilica nanoparticles : In a flask, CTAB (250 mg) was dissolved in a solution of distilled water (110 mL), EtOH (10 mL) and NaOH (2M, 0.875 mL) that was heated to 80 °C and stirred vigorously. In another flask, TEOS (0.875 mL) and BTSPD (0.390 mL) were dissolved in 5 mL of EtOH. Once the temperature of the CTAB solution had stabilized, the solution containing the silane sources was added dropwise. After 6 h, the solution was cooled to r.t. and the particles, recovered by centrifugation (20 min at 40 krcf). • Pore expansion treatment (LP-ssNPs): 100 mg of ssNPs previously synthesized were dispersed in EtOH by sonication for 30min, followed by the addition of 20 mL of a 1 :1 mixture (v/v) of H₂0 and TMB. The mixture was placed in the oven, and kept at 160 °C for 3 days without stirring. The resulting white powder was washed with ethanol and water four times each. Finally, the organic surfactant was removed by means of extraction in a mixture HCl/EtOH (5% v/v) under refiux overnight. LP-ssNPs were then centrifuged, washed thoroughly with ethanol several times and finally dried under vacuum. The material was characterized by means of: SEM, TEM, XPS, TGA, SAXS, DLS and ζ- potential. Grafting of 3-(aminopropyl)triethoxysilane (NH2-LP-ssNPs): 20 mg of LP-ssNPs were dispersed in toluene by sonication for 10 min, then 5 of APTES and 3 of TEA were added and the mixture was stirred overnight at rt. NH2-LP- ssNPs were then collected by centrifuging for 20 min at 14.5 krpm. The precipitate was finally re-dispersed by sonication in toluene and centrifuged five times to remove unreacted silane. The material was recovered and dried under vacuum before being characterized by means of ζ-potential. Quantification of functional primary amino groups were performed with a Kaiser test kit following the providers protocol. (Kim, M.-H.; Na, H.-K.; Kim, Y.-K.; Ryoo, S.-R.; Cho, H. S.; Lee, K. E.; Jeon, H.; Ryoo, R.; Min, D.-H. ACS Nano 2011, 5 (5), 3568-3576 [31] and Mizutani, M.; Yamada, Y.; Nakamura, T.; Yano, K., Chem Mater. 2008, 20, 4777- 4782 [32]).

• Grafting of Rhodamine B isothiocyanate for confocal imaging (LP-ssNPs): 10 mg of NH2-LP-ssNPs were dispersed in 2 mL of EtOH and sonicated for 30 min. Then, 0.1 mg of RITC were then added and stirred for 3 h. The particles were then washed several times by sequences of sonication/centrifugation cycles until a clear supernatant was obtained. The RhodB-grafted particles were then dried under vacuum.

• siRNA loading and jetPEI® coating (jp-siRNA@LP-ssNPsBPBPs-PLKl and BPBPs-Ctrl): 1 mg of NH2-BPBPsLP-ssNPs were dispersed in 1 mL of a MES buffer at pH 5 (5 mM) in an Eppendorf tube. In another Eppendorf tube, the two strands of siRNA were mixed together in the same MES buffer pH 5 (1 mL in total) at the desired concentration. The siR A solution was shaken for 5 minutes before to add the dispersion of particles (total volume 2 mL). The particles were placed swirled o.n. at r.t. within a rotatory mixer and let overnight at room temperature. beforeThe particles were then centrifuged for 1 h at 14.5 krpm., The supernatant was removed and stored for further measurements. The particles were then redispersed in 1.89 mL of MES buffer before to add 110 of a jetPEI® solution (0,2 mg/mL). After 1 h, Finally the material was centrifuged for 1 h at 14.5 krpm, the supernatant removed and the particles were redispersed in 1 mL of a PBS pH 7.4 solution. To determine the loading of the siRNA onto the BPBP-APTESNH2-LP-ssNPs, the supernatant containing the siRNA was then measured by UV-VIS spectroscopy at 260 nm.

Example 7 : In vitro experiments Material and Methods :

• Cell culture: Huh-7 cells were cultured in culture medium (CM) containing 88% Dulbecco's Modified Eagle Medium (D-MEM), 10% Fetal Bovine Serum (FBS), 1% Penicillin-Streptomycin and 1% L-Glutamine 200 mM at 37°C under 5% of C02 atmosphere.

· Flow cytometry: for FACS analysis, Huh-7 cells were seeded in a 24 well plate (30000 per wells) and allowed to adhere and grow for 24 h. The cells were then incubated with r-LP-ssNPs, jp-PLKl @r-LP-ssNPs in CM (50 and 100 μg/mL). After 3 h, 24 h and 48 h, the cells were washed 5 times with PBS, trypsinated and centrifuged for 3 min at 1 krpm. The pellets were resuspended in 500 μΐ of PBS and centrifuged again for 3 min before to be resuspended in 500 μΐ of PFA (2 %> in PBS) for FACS measurements.

• Confocal microscopy: 30 000 Huh-7 cells were seeded onto glass cover slips in a 24 well plates and allowed to adhere and grow for 24 h. The cells were then incubated with r-LP-ssNPs in CM (40 μg/mL). After 3 h, 24 h and 48 h, the cells were washed 5 times with PBS and fixed with 4 % PFA for 15 min. The cells were then washed again 3 times with PBS. In order to visualize the nuclear region, the samples were then stained with Hoechst 33342 and washed 3 times with PBS. The glass cover slips were mounted and fixed on a glass microscope slide with Fluoromont acqueous mounting medium for confocal microscope analysis. For Z- stacking experiments, cells (Huh-7) were prepared as previously explained for the cellular uptake and incubated with r-LP-ssNPs under the same conditions. After 24 h of incubation, cells were washed 5 times with PBS and fixed with 4 wt. % PFA for 15 min. Cells were then washed with PBS and kept in Triton X-100 (0.1 % in PBS) for 10 minutes and afterwards in 1% bovine serum albumin (BSA) in PBS for 20 min. The cell layer on glass cover slip was stained with Phalloidin Alexa Fluor® 647 for F-actin/membrane staining, for 20 min in the dark at room temperature, and washed twice with PBS. The nuclear region was stained with Hoechst 33342 for 5 minutes and washed 3 times with PBS. The cover slips were mounted onto glass slides for confocal microscopy measurements. The excitation wavelength for Hoechst 33342 and PJTC (grafted on the particles surface) were 355 and 488 nm respectively, while with Alexa Fluor® 647 Phalloidin was excited at 650 nm. For co-localization experiments, Huh-7 cells (30000 cells) were seeded onto glass bottom dishes (MatTek) and allowed to grow for 24 h. After this time, the culture media was removed and fresh media containing LP-ssNPs-Rhod at a concentration of 50 μg/mL was added to the cells and incubated for 24 h. Cells were then washed 5 times with PBS and incubated for 2 h with a solution of 75 nM of Lysotr acker®

Blue DND-22 in culture media. The cells were washed three times with PBS and fresh culture media was added before live cell imaging with the confocal microscope. The excitation wavelength for Lysotracker® Blue DND-22 was 405 nm.

• siRNA labelling and cellular uptake: 40 μg of PLK1 siRNA were labelled using a Cy5Label IT® siRNA Tracker Intracellular Localization Kit (Minis). The siRNA was incubated for 1 h at 37 °C with the labelling kit (total volume 100 μί). Then, 10 μΐ, of 4 M NaCl and 250.5 μΐ, of ice cold 100% ethanol were added. The solution was then placed at -20 °C for lh before centrifuging at full speed (14 krpm) in a refrigerated microcentrifuge for 30 min. The pellets were then washed with 500 μΐ_^ room temperature 70% ethanol and centrifuged again for 30 min at 4 °C. Finally, the siRNA was resuspended in 20 μΐ of siRNA buffer solution. In another Eppendorf, 200 μg of r-LP-ssNPs were dispersed in 200 μΐ, of MES buffer pH5 before adding the siRNA solution. The suspension was shaken overnight at room temperature. The siRNA labelled particles were centrifuged (1 h, 14.5 krpm) and resuspended in 200 μΐ, of MES buffer pH 5 + 5 of jetPEI (2 mg/mL) and incubated 1 h at room temperature. Finally, the particles were centrifuged (1 h, 14,5 krpm) and suspended in 200 μΐ, of PBS 7,4. The particles were then incubated with 30 000 cells at a concentration of 50 μg/mL for 3h. Then, the samples were washed 5 times with PBS, fixed with PFA (4%) for 15 min and washed again 3 times. Finally, the cells were stained with Hoechst 33342 for 10 min and washed again 3 times before mounting on microscope glass slides for confocal imaging. Excitation wavelengths were 355, 488 and 633 nm for Hoechst 33342, LP-ssNPs-Rhod and PLK1 siRNA respectively.

• Cell viability: 1.5x 104 Huh-7 cells were seeded in 24 well plates and allowed to grow for 24h. The cells were then incubated with LP-ssNPs in CM (5, 10, 20, 40, 60, 80 and 100 μg/mL). After 3, 24 and 48 h, 100 μΕ of Alamar Blue were added in each well plate and let incubate for 2 to 4 hours. Then the culture media were transferred to a 96 well plates and the absorbance of each well plates was measured at 570 nm and 600 nm with a microplate reader. Each samples were triplicate.

• mRNA expression by rtPCR and qPCR: Huh 7 cells (50 000 cells) were seeded in a 24 well plates and allowed to grow 24 h. The cells were then incubated with sLP-ssNPs, PLK1 siRNA and LP-ssNPs-PLKl (5 μg/mL of siRNA) for 24 h.

RNA was then isolated using a TRIzol® Reagent Kit and cDNA was synthetized using a iScriptTM cDNA Synthesis Kit. Quantitative Real-Time PCR (qPCR) was performed in triplicate using a SsoAdvancedTM Universal SYBR® Green Supermix using CFX96TM Real Time System (Bio-Rad) and the following cycling condition: 95 °C for 3 min followed by 40 cycles of 95 C for 10 s then 55 °C for 30 s. Primer sequences for PLK1 and GAPDH (eurofms) are provided in Table 4. Relative mRNA of each gene of interest was calculated using the AACT method.

• In vitro breakability test by means of TEM: for the preparation of biological TEM samples 1.106 Huh-7 cells were seeded on glass cover slips and allowed to grow for 24 h. After this time the media was removed and fresh media containing sLP-ssNPs in CM (50 μg/mL) was added to the cells and incubated for 3 h, 24 h and 48 h at 37oC in a humidified atmosphere with 5% C02. Subsequently, cells were washed with PBS five times fixed with glutaraldehyde (2.5 wt. %). The cells were post fixed with 0.5% osmium tetroxyde (EMS) in H20 and dehydrated through immersion in different solutions, where the content of EtOH in the mixture H20/EtOH was varied from 50 to 100 %, before being embedded in epoxy resin, Embed 812 (EMS). The resin was cut with an ultramicrotome, Leica EM UC6 (Leica) and the ultrathin sections were counterstained with uranyl acetate before TEM analysis.

Example 8 : Results and discussion :

Synthesis and characterization of LP-ssNPs :

The small pores disulfide-doped silica nanoparticles (ssNPs) were synthetized by a modified Stober process that we recently described (Maggini, L.; Cabrera, I.; Ruiz- Carretero, A.; Prasetyanto, E. A.; Robinet, E.; Cola, L. D. Nanoscale 2016, 8 (13), 7240-7247 [33]). The ssNPs were therefore synthesized through co-condensation of tetraethyl orthosilicate (TEOS) and bis(triethoxysilyl-propyl)disulfide (BTSPD) in 70:30 molar ratio in the presence of cetyltrimethylammonium bromide (CTAB) as template and aq. NaOH as catalyst. The enlargement of the pores was then performed following a recently described procedure(Mizutani, M.; Yamada, Y.; Nakamura, T.; Yano, K., Chem Mater. 2008, 20, 4777-4782 [32] and Kim, M.-H.; Na, H.-K.; Kim, Y.-K.; Ryoo, S.-R.; Cho, H. S.; Lee, K. E.; Jeon, H.; Ryoo, R.; Min, D.-H. ACS Nano 2011, 5 (5), 3568-3576 [31]) by treating the ssNPs at high temperature (160 °C) in the presence of the swelling agent TMB in a H20:EtOH mixture (50/50, v/v). The morphological characterization of the organosilica material was first performed by scanning and transmission electron microscopy (SEM and TEM). The SEM images reported in Fig. la and Fig. 7 display homogeneous spherical particles characterized by an average diameter of 9± 24 nm . TEM images (Fig. lb and 8) revealed an enlarged mesoporous structure, as suggested by the variation of the contrast within the particles, and a rougher particle surface. The surface etching process occurred to a certain extent, due to the dissolution of small pieces of silica in the water/ethanol mixture during the solvothermal treatment. (Chem Mater, 2008, 20, 4777-4782, [24]). Conversely, the ssNPs show a higher contrast and a smoother surface, and no evidence of an ordered array of pores. The porosity was assessed by nitrogen adsorption measurements performed on both the particles before and after the enlargement process and that clearly showed a 6-fold pore enlargement. (Fig. lc). In fact, the analysis of the adsorption/desorption isotherms allowed to calculate for ssNPs a BET surface area of 684 m² g-1, total pore volume of 0.67 cm³ g-1 and an average pore size of 2.2 nm. The LP-ssNPs show instead a smaller BET surface area of 430 m² g-1 as a result of the presence of larger pores and related decreased wall thickness, the total pore volume at p/p0=0.99 resulted 1.07 cm³ g-land the increase can be explained by the increase of interstitial voids due the presence of non- smooth particles surfaces. The data analysis gave a broader pore width distribution centered at 12 nm (Fig. lc and 9) with the presence, to a lesser extent though, of micropores (1.7 nm) and smaller mesopores (2.7 nm). The small angle X-ray scattering (SAXS, Fig. 10) pattern recorded on the LP-ssNPs did not show the presence of Bragg peaks, thus revealing the lack of an ordered array of mesopores in accordance with TEM images (Fig. 1).

The presence of the cleavable linker within the particles was confirmed by elemental analysis of the material conducted by X-ray photoelectron spectroscopy (XPS; Table 5 and Fig. 9) that detected the signal of S in high atomic percentage. Thermogravimetric analysis (TGA; Fig. 12) showed a significant organic doping of the material, by detecting a weight loss of 23% attributable exclusively to the organic components of the particle (i.e. S-S linker). The ζ potential value of the LP- ssNPs was found to be -22.4 ± 1.3 mV (Fig. 11), comparable to the values generally obtained for pristine particles.

Table 5. XPS results

Table 6. N₂ adsorption analysis results for LP-ssNPs

As mentioned above, attractive electrostatic interactions can favor the loading of siR A, for this reason, primary amine groups were introduced on the interior and exterior of LP-ssNPs by using 3-aminopropyltriethoxysilane (APTES) through a post-synthetic grafting (NH2-LP-ssNPs). The introduction of the amino groups was confirmed by ζ-potential measurements with a positive shift to 26.6 ± 3.2 mV (Fig. 12) and by Ka^'iser test , a colorimetric assay widely- used for the quantification of primary amino groups. Upon reaction with a primary amine, ninhydrin converts into an adduct (2-[(3-hydroxy-l-oxo-lH-inden-2-yl)imino]-lH-indene-l,3(2H) characterized by a characteristic absorbance centered at 570 nm .(Analytical Biochemistry, 1970, 34, 595-598, Langmuir, 2012, 28, 5562-5569, [25]) Measuring the absorption of a dispersion of NH2-LP-ssNPs reacted with ninhydrin (Fig. 13) we were able to estimate the amount of amino groups on the particles as 26 μιηοΐ/mg STEM investigations on LP-ssNPs grafted with different amounts of APTES for comparison (X, Y and Z%) showed that such functionalization degree appeared The best compromise for an efficient grafting of the oligonucleotide without preventing the breakability properties of the material. In fact it is possible to observe that for particles grafted using higher quantities of APTES the breakdown upon exposure to GSH occurred.

Once the particles had been fully characterized, their response to reduced glutathione (GSH) was investigated in order to verify whether breakdown upon exposure to reducing agents, allowing for a selective disintegration of the particles within cancerous cells. GSH is in fact a thiol-containing tripeptide able to reduce disulfide bonds, present in the cytosol of tumor cells at a concentration (2-10 mM) which is significantly higher than the one in the plasma (1-2 μΜ). A dispersion of LP-ssNPs, and also NH₂-LP-ssNPs to demonstrate that the grafting did not modify the breakability of the particles in PBS (0.1 mg ml/¹, pH 7.4) was therefore stirred at 37 °C in the presence of GSH (10 mM) and aliquots of suspension taken at several time points (up to 7 days) were analyzed by TEM. The images reported in Fig. 1 clearly show for both type of particles that the exposure to the reducing agent leads to the degradation of the nanoparticles with their persistent exposure to GSH. Already after 3 days it was possible to observe a significant structural breakdown leading to loss of spherical morphology and presence of small fragments after 7 days. As a control experiment, the same analysis was performed in the absence of GSH and after 7 days the particles mostly retained their morphology and only a few particles seemed to start degrading suggesting that silica hydrolysis known to occur in PBS (to a rate that generally depends many and diverse parameters) (Croissant, J.G.; Fatieiev, Y.; Khashab; N.M., Adv. Mater. 2017, 29, 1604634 [34]) on starts to take place.

Once the degradation of NH2-LP-ssNPs triggered by GSH had been proven, the particles were loaded with a double stranded PLK1 specific siRNA (PLK1@NH2- LP-ssNPs). As mentioned above, the choice of this particular siRNA was dictated by its ability to silence the gene expressing the PLK1 protein, which is an interesting target for cancer therapy playing a key role during cell mitosis and being overexpressed in hepatocellular carcinoma cells. In order clearly assess both the efficacy of the anti-PLKl siRNA and the beneficial effect of using large pore particles, we decided to perform for our biological investigations control experiments using also NH2-LP-ssNPs loaded with a double stranded siRNA nonspecific for PLK1 and showing no silencing activity on genes present in hepatocellular carcinoma cells and (siRNA@NH2-LP-ssNPs) and small pore ssNPs loaded with PLK1 siRNA (PLK1@NH2- ssNPs). The loading experiments were performed swirling a dispersion of nanoparticles (c = 0.1 mgmL-1) in MES buffer

(pH = 5.0) overnight in the presence of siRNA (concentration range from 10 to 200 μ§.ηιΙ.-1). MES buffer at pH 5.0 was chosen in order to ensure the protonation of the amino groups and therefore a favored adsorption of siR A to the carrier. The nanoparticles were then recovered by centrifugation and washed with MES buffer solution (how many times) for the removal of the non physisorbed siRNA. The oligonucleotide loading was hence quantified by means of UV-vis spectroscopy. The absorbance value at 260 nm was measured on the collected supernatant, the loaded siRNA was calculated to reach a maximum of 182 μg.mg-l for NH2-LP- ssNPs and only 25 μg of siRNA per mg of NH2-ssNPs (Fig 15). The procedure for the calculation of the concentration of the oligonucleotide on the particles is detailed herein.

The efficient adsorption of siRNA, was also proven by Dynamic Light Scattering (DLS) measurements on the loaded particles showing an increase of NH2--LP- ssNLPs hydrodynamic diameter (Dh) of 10 nm (Fig 16), consistent with the loading of siRNA that possess a large molecule structure of 2 x 8 nm (Nanoscale, 2016, 8, 4007-4019, [26]). Further confirmation was provided by the drop of surface charge down to -24.0 ± 3.9 mV determined by ζ-potential after the incubation (Fig 17). The last step in the preparation of our carrier was the coating with jetPEI® in order to prevent nuclease degradation of the oligonucleotide and to enhance the cellular uptake of the cargo into the cells . In addition, it has been reported that, once internalized into the endosomes, the polycation is able to induce a series of cellular events that leads to the opening of the polymeric network and release of the siRNA. In fact, once in the endosomes the jetPEI® acts as a proton sponge, altering the osmolarity of the vesicles and inhibiting the lysosomal nucleases. The accumulation of protons within the endosomes induces an influx of chloride anions, resulting in an osmotic swelling of the vesicles, and to the protonation of the jetPEI®, creating an internal charge repulsion, opening the polymeric network. These combined effects reduce the endosomal life and allow for the release of the siRNA into the cytoplasm.. After incubation with the siRNA and centrifugation, the particles were dispersed in 890 μΐ, of MES buffer pH 5 before the addition of 1 10 μΙ, οΪ Ά jetPEI solution (0.2mg.mL-l). After lh incubation, the particles were finally centrifuged and resuspended in PBS pH 7.4. The presence of jetPEI® around the loaded nanoparticles was proven both by the increase of Dh of ca. 40 nm for the coated nanoparticles (163 ± 30 nm, Fig. 15) revealed by DLS and by the positive surface charge corresponding to 8.15 ± 3.32 mV as determined by ζ-potential measurements (Fig. 17). A schematic representation of the final material is depicted in Fig. 2. Cellular uptake, cytotoxicity and exocytosis of NH2-LP-ssNPs

First of all the cytotoxicity and cellular uptake of LP-ssNPs were evaluated to prove their suitability for biological applications. The hepatocellular carcinoma Huh-7 cells were incubated with LP-ssNPs at 8 different concentrations (5, 10, 20, 40, 60, 80 and 100 μgmL^"1) and the cell activity was evaluated by an Alamar Blue^® assay after 3, 24 and 48 h and no significant decrease of the metabolic activity was observed in the entire range of concentrations tested as shown in Fig 18. The cellular association of the NH₂-LP-ssNPs was quantified by fluorescence-activated cell sorting (FACS) analysis. The NH₂-LP-ssNPs were functionalized on the surface with Rhodamine B isothiocyanate (r-LP-ssNPs, see SI for details), the particles were stirred for 3 h at r.t. in the dark and thoroughly rinsed to remove the unreacted dye. The fluorescence spectrum recorded on the particles shows the occurred functionalization. The Huh-7 cells were then incubated with 50 μg and 100 μg·mL^" ^J) suspensions of r-LP-ssNPs in a Dulbecco complete culture medium for 3, 24 and 48 h. FACS analysis showed an efficient cellular uptake after 3h increasing overtime. As shown in Fig. 3a. the mean fluorescence intensity (MFI) of the Rhodamine B grafted on the particles increased significantly with the increasing incubation time (3h to 48h) and concentration (50 μg and 100 μg·mL^"1).

Confocal laser scanning microscopy (CLSM) analysis was performed on the cancerous cells incubated with r-NH₂-LP-ssNPs (50 μg.mL^"1) to prove the occurred internalization (Fig. 3b). In agreement with FACS data, a good uptake is shown already after the first 3 h incubation, clearly increasing over time. The Z-stacking analysis on cells incubated for 24 h (Fig. 3c) and recorded after staining the membrane with Alexa Fluor® 647 Phalloidin demonstrated that the nanoparticles are internalised inside the cells. Colocalization studies using the lysosome-specific fluorescent marker Lysotracker® Blue DND-22 were also carried out to gain insight on the localization of the internalized particles within the cells. As shown in Fig. 4a after 24h incubation the r-NH₂-LP-ssNPs were mostly localized into lysosomes, indicating that the internalization of the nanoparticles occurs through a classical endocytosis process. To confirm this and to investigate the fate and degradation of the nanoparticles within the tumor cells, TEM analysis was conducted on cells incubated with the nanoparticles (Fig. 4b and 18).

After 3 h incubation, the particles were mainly localized into early endosomes, first step of clathrin-dependent endocytosis (Journal of controlled release, 190 (2014) 485-499; Journal of controlled release, 145 (2010) 182-195, [27]). Whereas after 24 h, the nanoparticles were mostly present within lysosomes and their partial degradation already occurred, most probably triggered by the high intracellular concentration of GSH as we recently demonstrated for ssNPs. (Maggini, L.; Cabrera, I.; Ruiz-Carretero, A.; Prasetyanto, E. A.; Robinet, E.; Cola, L. D. Nanoscale 2016, 8 (13), 7240-7247 [33]). The decrease of contrast in the imaged nanoparticles and the loss of spherical morphology for those in a more advanced degradation phase clearly demonstrated the occurring dissolution. After 48 h incubation a more extensive degradation could be imaged, indicating that already after 48 h an efficient release of the PLK1 siRNA may be achieved.

Delivery and release of SiRNA

The delivery and release of PLK1 siRNA were investigated by CLSM analysis. Cyanine-5 labelled PLK1 siRNA was used in order to track the oligonucleotide within the cell. The labelling was performed by using Cy5Label IT® siRNA Tracker Intracellular Localization Kit. The cells were then incubated with the r-NH₂-LP- ssNPs loaded with Cy5-labelled PLK1 siRNA and coated with jetPEI^® for 3h and then washed with PBS (40μg/mL). The confocal analysis on the cells (Fig. 5) showed that the particles were internalized within the cells. Moreover, the green signal, corresponding to the labelled PLK1 siRNA showed a signal that is broader compared to the r-NH₂-LP-ssNPs, probably due to the release of the siRNA in the cytoplasm. Example 9: In vivo proof-of concept for prevention and treatment of cancer in two state-of-the-art mouse models for human liver cancer/HCC.

To prepare the in vivo proof-of-concept studies for prevention and treatment of HCC, we first analysed the in vivo biodistribution of nanoparticles in a mouse model using in vivo fluorescence imaging. The method of in vivo fluorescence imaging is described in (Thirunavukkarasu Devarasu et al. J. Mater. Chem. B, 2013,1, 4692- 4700, [28]). As shown in Figure 20 following intravenous injection the nanoparticles were found in several organs including the liver within a time period of 24 hours. To demonstrate in vivo proof-of-concept for the use of nanovectors for prevention and treatment of human liver cancer/HCC we applied two state-of-the-art human xenograft mouse models for HCC and nanoparticles loaded with siR A targeting the polo-like kinase protein 1, a protein involved in cell division and over expressed in many cancers (Fig. 21). In the first model shown in Fig. 22 human liver tumor/cancer cells of the Huh7 hepatoma cell line are growing subcoutanously in the back of the mouse (Lebouef C et al. Mol Ther. 2014 Mar;22(3):634-644. doi: 10.1038/mt.2013.277. Epub 2013 Dec 6, [29]), in the second model shown in Fig. 23 human tumor/cancer cells of the Huh7 cell line are growing within the mouse liver (orthotopic model). The state-of-the-art models are described in detail in (Wu T Sci Rep. 2016 Oct 14;6:35230. doi: 10.1038/srep35230, [30]).

As shown in Figures 22-23, the intratumoral injection of nanovectors loaded with siRNA targeting PLK1 resulted in a delayed onset of tumor growth and to a marked, significant and specific inhibition of tumor growth in all models and experimental approaches. As an example shown in Fig. 22, the efficacy of LP- ssNPs-PLKl nanoparticles was evaluated in NMRI-Nude mice bearing subcutaneous Huh-7-Luc tumors. Six intra-tumoral injections of LP-ssNPs-PLKl were performed at dO, 1, 3, 6, 8 and 10 and the tumor growth was monitored by bio luminescence imaging. The median tumor size showed a 2.4-fold increase in the vehicle-injected control group. A non-specific decrease of the median tumor size (32% of decrease, as compared with the initial median size) was observed in LP- ssNPs-control siRNA-treated group while > 95% decrease of median tumor size was observed in the LP-ssNPs-PLKl -treated group (Fig 22), indicating that LP-ssNPs- PLK1 were able to efficiently provide a potent anti-tumor effect. Similar results were observed in the orthotopic model (Fig. 23). The anti-cancer effect was specific for nanoparticles loaded with PLK1 -specific siR A, since control particles loaded with a non-targeting control siRNA did not show a detectable anti-cancer effect (Fig. 23). These in vivo proof-of-concept studies performed in two different and independent mouse human xenotraft cancer models demonstrate that nanovectors loaded with siRNA targeting a specific cancer cell protein are suitable for prevention and treatment of human cancer including liver cancer.

Chemo therapeutic approaches using nanovectors to deliver potentially toxic anticancer drugs requires a well-controlled transport and release of the molecules avoiding their premature release that could have detrimental impact on non- tumorous cells. We show here an effective delivery of the nanovectors to human organs including the liver in vivo following intravenous administration. Intratumoral injection resulted in a marked effect on tumor cell growth without detectable adverse effects. These in vivo proof-of-concept results unravel the nanovectors as an optimal delivery system for cancer prevention and treatment. Furthermore, we could envision to enhance the therapeutic efficacy and intracellular concentration of our anticancer drugs by generating antibody fragment-armed nanovectors against glypican-3, a highly expressed cell surface protein on tumor cells. All together, our data support the concept of using our newly designed nanovectors to deliver anticancer molecules targeting specifically tumor cells. By enabling to design siRNAs for patient-specific cancer drivers this approach will enable precision medicine for cancer.

Materials and Methods:

Animal experimentation

Animal experimentations were performed in accordance with European recommendations (Directive 2010/63/UE, September 22nd, 2010) and French regulations (Edict 2013-118, February 1st, 2013) and received the approval of the local ethical committee (Comite Regional d'Ethique en Matiere d'Experimentation Animale de Strasbourg, approval n° 03111). NMRI-nu (Rj:NMRI- Foxnlnu/Foxnlnu) female mice purchased from Janvier Labs (Le Genest Saint Isle, France) were used for experimentation.

Biodistribution:

NMRI-nu mice were intravenously injected in the lateral tail vein with vehicle (n=2) or 9 mg/kg near-infrared fluorescent nanovector (n=3) and monitored for in vivo fluorescence as described in Thirunavukkarasu Devarasu et al. J. Mater. Chem. B, 2013,1, 4692-4700, [28]).

Cell-derived xenograft tumor models.

To demonstrate in vivo proof-of-concept for the use of nanovectors for prevention and treatment of human liver cancer/HCC we applied two state-of-the-art human xenograft mouse models for HCC and nanoparticles loaded with siRNA targeting the polo-like kinase protein 1, a protein involved in cell division and over expressed in many cancers (Fig. 21). In the first model human liver tumor/cancer cells of the Huh7 hepatoma cell line are injected subcoutanously in the back of the mouse (Lebouef C et al. Mol Ther. 2014 Mar;22(3):634-644. doi: 10.1038/mt.2013.277. Epub 2013 Dec 6, [29]), in the second model human tumor/cancer cells of the Huh7 cell line are injected intrasplenically with subsequent growth the mouse liver (orthotopic model). The state-of-the-art models are described in detail in (Wu T Sci Rep. 2016 Oct 14;6:35230. doi: 10.1038/srep35230, [30]). In brief, in the orthotopic model 10⁶ luciferase-expressing Huh-7 (Huh-7-Luc) human hepatoma cells were orthotopically transplanted by echo-guided intrahepatic injection and monitored by ultrasound imaging (USI) and monitored by bio luminescence imaging (BLI) as previously described (Wu T Sci Rep. 2016 Oct 14;6:35230. doi: 10.1038/srep35230,

[30]). All surgical procedures were performed under 1 to 3% isoflurane anesthesia (Axience Laboratories, Pantin, France) with 2 to 3 L/mn air flow rate, with or without 0.2 L/mn 02 flow rate. Analgesia was performed at initiation of the procedures by administration, directly in the abdominal cavity, of buprenorphine (Buprecare®, Axience Laboratories) at a dose of 0.1 mg/kg. Intraperitoneal injections of buprenorphine at the same dose were performed eight hours later and, if required, the following days. Paracetamol (Doliprane, Sanofi-Aventis, Paris) was given at a dose of 1 mg/ml in the drinking water until the end of the experimentation.

Experimental protocols:

Subcutanous human cancer xenograft mouse model:

Huh-7-Luc cells were subcutaneously injected in the back of NMRI-nu mice and tumor growth was monitored by bio luminescence imaging. Once the tumor volume reached 10.5 p/s/cm², nanovector-siControl, vehicle or nanovector-siPLKl were intra-tumorally injected at days 0, 1, 3, 6, 8, and 10 (Fig. 22). The tumor size was measured before nano vector injections at days and at day 13 similar as described previuously in (Lebouef C et al. Mol Ther. 2014 Mar;22(3):634-644. doi: 10.1038/mt.2013.277. Epub 2013 Dec 6, [29]).

Orthotopic human liver cancer xenograft mouse model:

Three weeks after Huh-7-Luc transplantation (procedure described in Wu T Sci Rep. 2016 Oct 14;6:35230. doi: 10.1038/srep35230, [30]), i.e. at day 0 of treatment, mice were analysed for tumor surface and randomly allocated to the experimental groups. The median tumor surface of each experimental group was then calculated and the randomization was considered as valid when the coefficient of variation of the median values was below 5%. US-guided intratumoral injections of nanovectors (incorporating 10 ug of siRNA per injection) were performed at DO, D2, D5, D7, and D9. At each time points after treatment, data were expressed as the relative tumor growth, calculated for each tumor at the indicated time point, normalized to the tumor volume at DO of treatment.

Ultrasound guided- imaging for injection of cancer cells and treatment approach. Ultrasound imaging was performed as previously described (Wu T Sci Rep. 2016

Oct 14;6:35230. doi: 10.1038/srep35230, [30]) and was used for the percutaneous intrahepatic injection of Huh- 7 cells, the intratumoral injection of nanovectors and the monitoring of tumor growth. B-Mode, or brightness mode, imaging was used to acquire two dimensional images of an area of interest and for identification of anatomical structures using Vevo 2100 high-resolution imaging system (Visualsonics, Tonroto, Ontario, Canada).

While we have described a number of embodiments of this invention, it is apparent that our basic examples may be altered to provide other embodiments that utilize the catalysts and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example.

List of References

[1] a) M. E. Davis, Nature, 2002, 417, 813; b) U. Ciesla, F. Schuth, Micropor.

Mesopor. Mat. 1999, 27, 131.

[2] D. Zhao, J. Feng, Q. Huo, N. Melosh, G. H. Fredrickson, B. F. Chmelka, G. D. Stucky, Science, 1998, 279, 548.

[3] T. Linssen, K. Cassiers, P. Cool, E. F. Vansant, Adv. Coll. Interf. Sci. 2003, 103, 121.

[4] T. Asefa, G. A. Ozin, H. Grondey, M. Kruk, M. Jaroniec, Studies Surf. Sci.

Catal. 2002, 141, 1.

[5] a) D. S. Shephard, W. Zhou, T. Maschmeyer, J. M. Matters, C. L. Roper, S. Parsons, B. F. G. Johnson, M. J. Duer, Angew. Chem., Int. Ed. 1998, 37, 2719; b) F. de Juan, E. Ruiz-Hitzky, Adv. Mater. 2000, 12, 430; c) K. Cheng, C. C. Landry, J. Am. Chem. Soc. 2007, 129, 9674.

[6] K. J. Shea, D. A. Loy, Chem. Mater. 2001, 13, 3306.

[7] S. Inagaki, S. Guan, T. Ohsuna, O. Terasaki, Nature, 2002, 416, 304.

[8] V. Valtchev, L. Tosheva, Chem. Rev., 2013, 113, 6734.

[9] S. J. Rowan, S. J. Cantrill, G. R. L. Cousins, J. K. M. Sanders, J. F. Stoddart, Angew. Chem. Int. Ed., 2002, 41, 898.

[10] M. Kruk, Acc. Chem. Res., 2012, 45, 1678.

[11] F. Hoffmann, M. Cornelius, J. Morell, M. Froba, Angew. Chem. Int. Ed.

2006, 45, 3216 - 3251.

[12] S. V. M. de Moraes, J. B. Passos, P. Schossler, E. B. Caramao, C. C. Moro, T. M. H. Costa, E. V. Benvenutti, Talanta, 2003, 59, 1039.

[13] M. He, J. Bu, X. Yuan, Integrated Optics: Devices, Materials, and Technologies X. Edited by Sidorin, Yakov; Waechter, Christoph A.

Proceedings of the SPIE, 2006, 6123, 130.

[14] M. Graffner-Nordberg, K. Sjodin, A. Tunek, A. Hallberg, Chem. Pharm.

Bull, 1998, 46, 591.

[15] M. Kobayashi, Y. Fujiwara, M. Goda, H. Komeda, S. Shimizu, PNAS, 1997, 94, 11986.

[16] C. Lopreore, L. D. Byers, Arch. Biochem. Biophys., 1998, 349, 299. [17] E. Khalikova, P. Susi, T. Korpela, Microbiol. Mol. Biol. Rev., 2005, 69, 306.

[18] R. Weissleder, C.-H. Tung, U. Mahmood, A. Bogdanov Jr., Nat. Biotech., 1999, 17, 375.

[19] M. Yamashita, A. Tani, F. Kawai, Appl. Microbiol. BiotechnoL, 2004, 66, 174.

[20] S. H. Lee, W. S. Song, Text. Res. J., 2013, 83, 229.

[21] P. D. Hsu, D. A. Scott, J. Weinstein, F. A. Ran, S. Konermann, V.

Agarwala, Y. Li, E. J. Fine, X. Wu, O. Shalem, T. J Cradick, L. A. Marraffmi, G. Bao, F. Zhang, Nat. Biotech., 2013, 31, 827; M. Furutani, K.

Ito, Y. Oku, Y. Takeda, K. Igarashi, Microbiol. Immunol, 1990, 34, 387.

[22] Lu et al, Small 2010, 16, 1794.

[23] Wound Repair Regen. 2012 May-Jun; 20(3):317-31.

[24] Chem Mater, 2008, 20, 4777-4782.

[25] Analytical Biochemistry, 1970, 34, 595-598, Langmuir, 2012, 28, 5562- 5569.

[26] Nanoscale, 2016, 8, 4007-4019.

[27] (a) Journal of controlled release, 190 (2014) 485-499; (b) Journal of controlled release, 145 (2010) 182-195.

[28] Thirunavukkarasu Devarasu et al. J. Mater. Chem. B, 2013,1, 4692-4700.

[29] Lebouef C et al. Mol Ther. 2014 Mar;22(3):634-644. doi:

10.1038/mt.2013.277. Epub 2013 Dec 6.

[30] Wu T Sci Rep. 2016 Oct 14;6:35230. doi: 10.1038/srep35230.

[31] Kim, M.-H.; Na, H.-K.; Kim, Y.-K; Ryoo, S.-R.; Cho, H. S.; Lee, K. E.;

Jeon, H.; Ryoo, R.; Min, D.-H. ACS Nano 2011, 5 (5), 3568-3576.

[32] Mizutani, M.; Yamada, Y.; Nakamura, T.; Yano, K., Chem Mater. 2008, 20, 4777-4782.

[33] Maggini, L.; Cabrera, I.; Ruiz-Carretero, A.; Prasetyanto, E. A.; Robinet, E.; Cola, L. D. Nanoscale 2016, 8 (13), 7240-7247.

[34] Croissant, J.G.; Fatieiev, Y.; Khashab; N.M., Adv. Mater. 2017, 29,

1604634

Claims

Disintegratable porous organometaloxide nanoparticles comprising a porous three-dimensional framework of metal-oxygen or metalloid-oxygen bonds, wherein at least a subset of metal or metalloid atoms in the material's framework are connected to at least another metal atom in the framework through a linker having one of the following structures:

^-L-R²-*,

wherein :

L represents a disulfide bond; or '"'-R'-L-R²-* independently represents a disulfide-containing responsively cleavable moiety; and

R¹ and R² independently represent an optionally substituted Cl-20alkylenyl moiety, an optionally substituted Cl-20heteroalkylenyl moiety, an optionally substituted ethylenyl moiety, -C≡C- or an optionally substituted phenyl moiety, wherein the Cl-20alkylenyl, Cl-20heteroalkylenyl or ethylenyl moiety may bear one or more substituents selected from halogen or -OR where R may represent H or Cl-6alkyl, and the phenyl moiety may bear one or more substituents independently selected from halogen, Cl-6alkyl, -NO2, - CN, isocyano, -OR^p, -N(R^P)2 wherein each occurrence of R^p independently represents H or Cl-6alkyl;

wherein the nanoparticle is preferably mixed mesoporous and has two populations of pore sizes: (i) pores having an average width in the size range of from about 1 to about 50 nm, preferably from 1 to 40 nm, more preferably from 5 to 30 nm, even more preferably from 5 to 20 nm, most preferably from 10 to 15 nm; and (ii) pores having an average width in the size range of from about 0.5 to about 5 nm, preferably from 0.5 to 3 nm, more preferably from 0.5 to 2.5 nm, even more preferably from 0.5 to 2 nm;

wherein the nanoparticle is optionally further functionalized at its outersurface with positively charged groups, such as quarternary amine salts (e.g., NH₃ ⁺), either directly or via a linker, for example via a properly functionalized trialkoxysilane, such as 3-aminopropyl)triethoxysilane (APTES);

wherein the nanoparticle is further loaded in its pores and/or at its surface, preferably at its surface, with at least one nucleic acid-type bio molecule such as short interfering RNA ("siRNA") molecule, preferably PLKl-siRNA; wherein the nanoparticle is optionally further covered with a linear polyethylenimine such as such as jetPEI®.

The material of claim 1, wherein the three-dimensional framework of metal- oxygen bonds is mixed mesoporous.

The material of claim 1 or 2, wherein the metal is selected from Si, Ti or Zr, or any combination of at least two of these metals.

The material of any one of claims 1 to 3, wherein the material contains 90.0- 100% Si, 90.0-100% Ti or 90.0-100% Zr as metal, wherein the % are based on the number of available metal sites in the framework.

The material of any one of claims 1 to 4, wherein the material is a Si-Ti mixed- metal organometaloxide material containing 0.1-50.0% Si and 0.1-50.0% Ti, the % sum of Si and Ti adding to 100% the number of available metal sites in the framework.

The material of any one of claims 1 to 4, wherein in the linker represents *- R'-L-R²-*, R¹ and R² are identical, and each represent -CH₂-, -(CH₂)₂-, - (CH₂)₃-, -(CH₂)₄-, or phenyl.

The material of any one of claims 1 to 6, said material comprising in its pores or at its surface at least one marker and/or cosmetically or pharmaceutically active principle.

The material of claim 7, wherein the marker is selected from a contrast agent, a tracer, a radioactive marker, a fluorescent marker, a phosphorescent marker, a magnetic resonance imaging agent or a positron emission tomography agent. The material of any one of claims 1 to 8, wherein the material is in the form of a monolith, a thin or thick film, a powder, nanoparticles, or spherical, cubic, cylindrical or disc-like particles, preferably nanoparticles. A method for preparing a material of any one of claims 1 to 9, preferably in the form of nanoparticles, comprising steps of:

b) Adding a mixture of a precursor M(X^A)₄ and a selected precursor having the structure:

(X)₃Mi-R¹-L-R²-M₂(X)3;

in an aqueous solvent under alkaline conditions; thereby coating the supramolecular template with an organometaloxide sol-gel mixture obtained by hydrolysis-condensation of metal alkoxide; and

c) Removing the supramolecular template; thereby producing a porous organometaloxide nanoparticles comprising a porous three- dimensional framework of metal-oxygen bonds, wherein at least a subset of metal atoms in the material's framework are connected to at least another metal atom in the framework through a linker having one of the following structures:

^-L-R²-*, wherein:

each occurrence of X and X^A independently represents a hydrolysable or nonhydrolyzable group, provided that on each occurrence of Mi and M₂, at least one occurrence of X represents a hydrolysable group and at least two occurrences of X^A in the precursor M(X^A)₄ independently represent a hydrolysable group; wherein (i) when X or X^A represents a nonhydrolyzable group, it may be selected from an optionally substituted Cl-20alkyl, C2- 20alkenyl or C2-20alkynyl moiety, an optionally substituted Cl- 20heteroalkyl, C2-20heteroalkynyl or C2-20heteroalkynyl moiety, or an optionally substituted phenyl moiety, wherein the substituents on the phenyl, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl moieties may be independently selected from halogen, -NO2, -CN, isocyano, Cl- 6alkoxy, an oxirane/epoxyde moiety, -N(R)₂ wherein each occurrence of R is independently selected from H or Cl-6alkyl; and (ii) when X or X^A represents a hydro lysable group, it may be selected from a Cl-6alkoxy, Cl-6acyloxy, halogen or amino moiety;

L represents a disulfide bond; and

R¹ and R² independently represent an optionally substituted Cl-20alkylenyl moiety, an optionally substituted Cl-20heteroalkylenyl moiety, an optionally substituted ethylenyl moiety, -C≡C- or an optionally substituted phenyl moiety, wherein the Cl-20alkylenyl, Cl-20heteroalkylenyl or ethylenyl moiety may bear one or more substituents selected from halogen or -OR where R may represent H or Cl-6alkyl, and the phenyl moiety may bear one or more substituents independently selected from halogen, Cl-6alkyl, -NO2, - CN, isocyano, -OR^p, -N(R^P)₂ wherein each occurrence of R^p independently represents H or Cl-6alkyl;

d) functionalizing the outersurface of the nanoparticle with positively charged groups, such as quarternary amine salts (e.g., NH₃ ⁺), either directly or via a linker, for example via a properly functionalized trialkoxysilane, such as 3- aminopropy l)triethoxy silane ( APTE S) ;

further loading the pores and/or the surface of the nanoparticle, preferably at its surface, with at least one nucleic acid-type bio molecule such as short interfering RNA ("siRNA") molecule, preferably PLKl-siRNA;

optionally further covering the nanoparticle with a linear polyethylenimine such as such as jetPEI®.

The method of claim 10, wherein the ratio of equivalents (X)₃Mi-R¹-L-R²- M₂(X)₃ /M(X^A)₄ is one listed in the table below:

M(X^A)₄ (X)₃Mi-R¹-L- (X)₃Mi-R^!-L- % doping

R²-M₂(X)₃ R²-M₂(X)₃

0.95 eq. 0.05 eqi 0.025 eq. tt 5%

0.90 eq. 0.10 eqi 0.05 eq. tt 10%

0.80 eq. 0.20 eqi 0.10 eq. 20%

0.70 eq. 0.30 eqi 0.15 eq. 30%

0.60 eq. 0.40 eq. i 0.20 eq. tt 40%

0.50 eq. 0.50 eq. i 0.25 eq. tt 50%

0.40 eq. 0.60 eq. i 0.30 eq. tt 60%

0.30 eq. 0.70 eq. i 0.35 eq. tt 70%

0.20 eq. 0.80 eq. i 0.40 eq. tt 80%

0.10 eq. 0.90 eq. i 0.45 eq. tt 90%

- 1 eq. i 0.5 eq. tt 100%

+ equivalents expressed in terms of metal atoms (Mi and M₂) introduced by the bivalent starting material (X)3Mi-R¹-L-R²-M₂(X)3 in the final organometaloxide material.

tt equivalents expressed in terms of responsively cleavable bond L introduced by the bivalent starting material (X)₃Mi-R¹-L-R²-M₂(X)3 in the final organometaloxide material.

12. A method for preparing a material of any one of claims 1 to 9 being 100% doped, preferably in the form of nanoparticles, comprising steps of:

b) Adding a selected precursor having the structure:

(X)₃Mi-R¹-L-R²-M₂(X)3;

^-L-R²-*, wherein:

each occurrence of Mi and M₂ independently represents a metal selected from Si, Ti and Zr;

each occurrence of X independently represents a hydrolysable or nonhydrolyzable group, provided that on each occurrence of Mi and M₂, at least one occurrence of X represents a hydrolysable group; wherein (i) when X represents a nonhydrolyzable group, it may be selected from an optionally substituted CI -20 alkyl, C2-20 alkenyl or C2-20 alkynyl moiety, an optionally substituted CI -20 heteroalkyl, C2-20 heteroalkynyl or C2-20 heteroalkynyl moiety, or an optionally substituted phenyl moiety, wherein the substituents on the phenyl, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl moieties may be independently selected from halogen, -N0₂, -CN, isocyano, CI -6 alkoxy, an oxirane/epoxyde moiety, -N(R)₂ wherein each occurrence of R is independently selected from H or CI -6 alkyl; and (ii) when X represents a hydrolysable group, it may be selected from a CI -6 alkoxy, C I -6 acyloxy, halogen or amino moiety;

L represents a disulfide bond; and

R¹ and R² independently represent an optionally substituted Cl-20alkylenyl moiety, an optionally substituted Cl- 20heteroalkylenyl moiety, an optionally substituted ethylenyl moiety, -C≡C- or an optionally substituted phenyl moiety, wherein the Cl-20alkylenyl, Cl-20heteroalkylenyl or ethylenyl moiety may bear one or more substituents selected from halogen or -OR where R may represent H or C l-6alkyl, and the phenyl moiety may bear one or more substituents independently selected from halogen, Cl- 6alkyl, -NO2, -CN, isocyano, -OR^p, -N(R^P)2 wherein each occurrence of R^p independently represents H or Cl-6alkyl;

optionally further functionalizing the outersurface of the nanoparticle with positively charged groups, such as quartemary amine salts (e.g., NH₃ ⁺), either directly or via a linker, for example via a properly functionalized trialkoxysilane, such as 3-aminopropyl)triethoxysilane (APTES);

further loading the pores and/or the surface of the nanoparticle, preferably at its surface, with at least one nucleic acid-type bio molecule such as short interfering RNA ("siRNA") molecule, preferably PLK1 -siRNA;

13. The method of any one of claims 10 to 12, wherein the metal is Si.

14. The method of any one of claims 10 to 11, wherein the metal is Si and M(X^A)₄ represents a tetraalkoxysilane such as tetramethoxysilane, tetraethoxysilane and tetrapropoxysilane, preferably tetraethoxysilane (TEOS).

15. The method of any one of claims 10 to 14, wherein the surfactant is a cationic surfactant, an anionic surfactant, a non-ionic surfactant; preferably a cationic surfactant such as octadecyl trimethyl ammonium bromide, hexadecyl trimethyl ammonium bromide, tetradecyl trimethyl ammonium bromide, dodecyl trimethyl ammonium bromide, decyl trimethyl ammonium bromide, octyl trimethyl ammonium bromide, hexyl trimethyl ammonium bromide and other quaternary ammonium salt-type cationic surfactants.

16. The method of any one of claims 12 to 15, wherein the aqueous solvent comprises an alcohol, such as methanol or ethanol.

17. The method of any one of claims 10, 11 and 13 to 16, wherein the precursor having the structure (X)₃M-R¹-L-R²-M(X)₃ is produced in situ.

18. Porous organometaloxide material obtainable by a method of any one of claims 10 to 17, preferably in the form of nanoparticles, preferably mixed mesoporous.

19. Porous organometaloxide material of any one of claims 1 to 9, preferably in the form of nanoparticles, for use as medicament, for example in the prevention and treatment of cancer.

20. Porous organometaloxide material for use according to claim 19, wherein the organometaloxide material is organosilica material, preferably mixed- mesoporous.

21. Porous organometaloxide material for use according to claim 19 or 20, wherein the biomolecule loaded on the outersurface and/or in the pores of the porous organometaloxide material , preferably in the form of nanoparticle, is PLKl-siRNA.

22. Porous organometaloxide material for use according to claim 21 in the prevention and treatment of hepatocellular carcinoma.