WO2020136647A1

WO2020136647A1 - Loaded nanoparticles having rna unlocking mechanism

Info

Publication number: WO2020136647A1
Application number: PCT/IL2019/051407
Authority: WO
Inventors: Itamar Willner; Rachel Nechushtai; Wei-hai CHEN; Yang-Sung SOHN; Guo-Feng Luo
Original assignee: Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
Priority date: 2018-12-25
Filing date: 2019-12-25
Publication date: 2020-07-02

Abstract

Nanoparticle loaded with pharmaceutically active agent(s) being locked within said nanoparticle by an RNA unlocking mechanism, compositions comprising said nanoparticles and uses thereof in the treatment of diseases and disorders.

Description

LOADED NANOPARTICLES HAVING RNA UNLOCKING MECHANISM

BACKGROUND OF THE INVENTION

[001] Recent research efforts are directed to the application of nucleic acid-capped drug-loaded micro/nano-carriers as stimuli-responsive materials for the triggered unlocking of the carriers and the release of the drugs.^{1 3} Nucleic acid-gated carriers, such as mesoporous S1O2 nanoparticles,^{4 6} liposomes,⁷ microcapsules^8,9 and hydrogels^{10 14} were reported as stimuli-responsive carriers for the release of anti-cancer drugs. Nucleic acid-gated drug-loaded micro/nano-carriers were unlocked by means of cancer cell-specific environmental conditions such as pH,^{15 16} by chemical agents such as ATP¹⁷ or VEGF,¹⁸ over-expressed in cancer cells, or by physical stimuli such as heat¹⁹ or light.^{20 22} Metal-organic framework nanoparticles (NMOFs) ^{23 25}, represent a class of highly porous materials that find broad applications in catalysis,^26,27 sensing,^{28 30} carriers for drug delivery,^{31 35} separation processes^{36 38} and proton conductivity materials for fuel cells.^39,40

[002] Specifically, nucleic acid-functionalized NMOFs attract recent research efforts as stimuli- responsive drug delivery materials. The unlocking of nucleic acid-modified NMOFs has been demonstrated using pH as triggers,⁴¹ dissociation of nucleic acid gates through the formation of ligand/aptamer complexes (ligand = ATP, VEGF),^42,43 and the catalytic cleavage of the nucleic acid capping units by DNAzymes.⁴¹ In addition, NMOFs were coated with stimuli-responsive DNA-based hydrogels and the triggered control of the hydrogel stiffness was used to stimulate the release of the drug encapsulated in the NMOFs.⁴⁴ The targeting of the drug-loaded NMOFs to cancer cells was reported and the selective biomarker-triggered cytotoxicity of the drug-loaded NMOFs toward cancer cells was demonstrated. Nonetheless, in the different systems biomarkers, such as pH, VEGF or ATP, that exist in non-cancerous, disease infected cells, were used to release the drugs, thus providing an undesired path to affect, also, normal cellular tissues.

[003] MicroRNAs (miRNAs) attract growing interest as biomarkers for the detection of the progression of diseases such as different cancers.^{45 48} While most of the research efforts are directed toward the selective and sensitive detection of miRN As, ^49-52 the use of miRNAs as a functional trigger to release anti-cancer drugs is undeveloped. Particularly, the low levels of miRNAs in blood or cellular containments provide a fundamental difficulty to use miRNAs as a functional trigger to unlock nano-sized or micro-sized drug carriers. SUMMARY OF THE INVENTION

[004] The invention provides a nanoparticle loaded with at least one pharmaceutically active agent, said at least one pharmaceutically active agent being locked within said nanoparticle by at least one first nucleic acid sequence partially hybridized with at least one second nucleic acid sequence, wherein said at least one first nucleic acid sequence is associated with said nanoparticle; and said at least one second nucleic acid sequence is fully based paired with at least one RNA biomarker from its 3’ and/or 5’ end.

[005] When referring to a nanoparticle of the invention being loaded with said at least one pharmaceutically active agent, it should be understood that said at least one pharmaceutically active agent is entrapped and held within said nanoparticle.

[006] Said at least one pharmaceutically active agent is locked within said nanoparticle due to the presence of gating at least one first and at least one second nucleic acid sequences on the surface of said nanoparticle.

[007] Said at least one first nucleic acid sequence is associated with said nanoparticle utilizing any type of bond between said at least one first nucleic acid sequence and said nanoparticle, including but not limited to a chemical bond, a sigma bond, a hydrogen bond, a coordination bond, a complex bond and so forth and any combinations thereof. In some embodiments, the moiety at the 5’ end of said at least one first nucleic acid sequence is used for said association. In other embodiments, said nanoparticle is modified to include a functional group for said association.

[008] Said at least one first nucleic acid sequence is partially hybridized with at least one second nucleic acid sequence, that is, at least a part of the nucleic acid sequence of each is hybridized and paired with at least a part of the other nucleic acid sequence. Said partial hybridization allows for the

3’ and/or 5’ ends of said second nucleic acid sequence to be non-hybridized thereby being single strand tethers and for the 3’ -end of said at least one first nucleic acid sequence to be non-hybridized thereby being single-strand tethers. Under these structural constraints, the duplex of said at least one first and at least one second nucleic acid sequence is not subjected to hydrolytic digestion by the exonucleases.

[009] Furthermore, said at least one second nucleic acid sequence further comprises a sequence which is based paired with at least one RNA biomarker from its 3’ and/or 5’ end.

[0010] Thus, upon pairing of said at least one second nucleic acid sequence with at least one RNA biomarker, said at least one second nucleic acid sequence is displaced from its pairing with said at least one first nucleic acid sequence, forming a new duplex with said at least one RNA biomarker and also unlocking the at least one pharmaceutically active agent from said nanoparticle and releasing them out of the nanoparticle.

[0011] Furthermore, the duplex formed from said at least one second nucleic acid sequence and said at least one RNA biomarker is fully paired at the 3’ and or 5’ ends, thus being susceptible to exonuclease digestion, thereby releasing said at least one RNA from the duplex. The re-formation of said RNA marker is further paired with at least one second nucleic acid sequence on said nanoparticle. This allows for amplification of release in low concentrations of RNA.

[0012] When referring to RNA marker it should be understood to encompass any molecule that characterizes the cytoplasm of target cell population. The characterizing RNA marker is an RNA cytoplasmic sequence that is not universal to all cell types and is present in the cytoplasm and not the nucleolus. These may be coding RNA or non-coding RNA sequences, non-limiting examples are mRNA (coding for target cell specific proteins), microRNA, small interfering RNA (siRNA) , tRNA fragments and any additional coding or non-coding RNA that are specific the target cell population and any combinations thereof. [0013] In some embodiments, said nanoparticle is a porous nanoparticle and said at least one pharmaceutically active agent is loaded within its pores.

[0014] In other embodiments, said nanoparticle is a metal -organic framework nanoparticle.

[0015] In further embodiments, said partial hybridization of said at least one first nucleic acid sequence and at least one second nucleic acid sequence results in having the 3’- and 5’ -ends of said at least one second nucleic acid sequence free of hybridization with said at least one first nucleic acid sequence.

[0016] In other embodiments, said at least one RNA biomarker is a biomarker of at least one disease or disorder.

[0017] In other embodiments, said at least one RNA biomarker is selected from mRNA, microRNA

(miRNA), small interfering RNA (siRNA), tRNA and any combinations thereof.

[0018] In other embodiments, said loaded at least one pharmaceutically active agent is unlocked from said nanoparticle upon displacement of said at least one second nucleic acid sequence by said at least one RNA biomarker.

[0019] In further embodiments, hybridization of said at least one second nucleic acid said at least one

RNA biomarker is susceptible to digestion by at least one exonuclease.

[0020] In another aspect, the invention provides a composition comprising at least one nanoparticle of the invention. In a further aspect, said composition of the invention is for use as a medicament. In yet a further aspect a composition of the invention is for use in the treatment of at least one disease or disorder. In another aspect, a composition of the invention is for use in the treatment of cancer.

[0021] In another aspect the invention provides a method of treating a disease or disorder comprising administering to a patient in need thereof at least one nanoparticle loaded with at least one pharmaceutically active agent, said at least one pharmaceutically active agent being locked within said nanoparticle by at least one first nucleic acid sequence partially hybridized with at least one second nucleic acid sequence, wherein said at least one first nucleic acid sequence is associated with said nanoparticle; and said at least one second nucleic acid sequence is fully based paired with at least one RNA biomarker from its 3’ and/or 5’ end.

[0022] In some embodiments of the invention UiO-68 metal-organic framework nanoparticles (NMOFs) are loaded with a drug, such as for example doxorubicin and locked by means of structurally engineered duplex nucleic acid structures, where one strand is covalently linked to the NMOFs and the second strand is hybridized with the anchor strand. Besides the complementarity of the second strand to the anchor sequence, it includes the complementary sequence to the miRNA-21 or miRNA-221 that is specific miRNA biomarkers for MCF-7 breast cancer cells or OVCAR-3 ovarian cancer cells. In the presence of the respective miRNA biomarkers, the miRNA-induced displacement of the strand associated with the anchor strand proceeds, resulting in the release of DNA/miRNA duplexes. The released duplexes are, however, engineered to be digested, in the presence of exonuclease PI, Exo III, a process that recycles the miRNAs and provides the autonomous amplified unlocking of the NMOFs and the release of the doxorubicin load (or the fluorescent dye models) even at low concentrations of the miRNA. Preliminary cell experiments reveal that the respective NMOFs are unlocked by the miRNA-21 or miRNA-221, resulting in the selective cytotoxicity toward MCF-7 breast cancer cells or OVCAR-3 ovarian cancer cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

[0024] Figures 1(A)-1(C). 1(A) Schematic loading and locking of the doxorubicin anti-cancer drug in UiO-68 NMOFs and the subsequent amplified unlocking of the NMOFs in the presence of miRNA- 21 or miRNA-221 and exonuclease III. 1(B) SEM image of the nucleic acid (l)-modified UiO-68

NMOFs. 1(C) TEM image of the nucleic acid (l)-functionalized UiO-68 NMOFs.

[0025] Figures 2(A)-2(D). 2(A) Fluorescence spectra of the released Rhodamine 6G after a fixed time-interval of 60 minutes upon treatment of the loaded (l)/(2)-capped NMOFs in the presence of a fixed concentration of Exo III, 1 U/pL, and variable concentrations of miRNA-21 : (a) 0 mM. (b) 0.25 pM. (c) 0.5 pM. (d) 1 pM. (e) 2.5 pM. 2(B) Fluorescence spectra corresponding to the released

Rhodamine 6G after a fixed time-interval of 60 minutes upon subjecting the loaded (l)/(2)-gated

NMOFs to a fixed concentration of miRNA-21, 1 pM, and variable concentrations of Exo III: (a) 0

U/pL. (b) 0.1 U/pL. (c) 0.25 U/pL. (d) 0.5 U/pL. (e) 1 U/pL. (f) 2 U/pL. 2(C) Time-dependent release of Rhodamine 6G upon the treatment of the loaded (l)/(2)-locked NMOFs with: (a) No miRNA-21, no Exo III. (b) No miRNA-21 and added Exo III, 1 U/pL. (c) Added miRNA-21, 1 pM, without added

Exo PI. (d) Added miRNA-21, 1 pM, and Exo PI, 1 U/pL. 2(D) Fluorescence spectra corresponding to the release of Rhodamine 6G from the loaded (l)/(2)-functionalized NMOFs treated with Exo III,

1 U/pL, and different miRNA for a fixed time-interval of 60 minutes: (a) Added miRNA-21, 2.5 pM. (b) Added miRNA-221, 1 pM. (c) Added miRNA-145, 2.5 pM. (d) No added miRNA.

[0026] Figures 3(A)-3(D). 3(A) Fluorescence spectra of the released anti-cancer drug doxorubicin after a fixed time-interval of 60 minutes upon treatment of the loaded (l)/(2)-capped NMOFs in the presence of a fixed concentration of Exo PI, 1 U/pL, and variable concentrations of miRNA-21 : (a)

0 pM. (b) 0.25 pM. (c) 0.5 pM. (d) 1 pM. (e) 2.5 pM. 3(B) Fluorescence spectra corresponding to the released doxorubicin after a fixed time-interval of 60 minutes upon subjecting the loaded (l)/(2)- capped NMOFs to a fixed concentration of miRNA-21, 1 pM, and variable concentrations of Exo III:

(a) 0 U/pL. (b) 0.1 U/pL. (c) 0.25 U/pL. (d) 0.5 U/pL. (e) 1 U/pL. (f) 2 U/pL. 3(C) Time-dependent release of doxorubicin upon the treatment of the loaded (l)/(2)-locked NMOFs with: (a) Added miRNA-21, 1 pM, and Exo III, 1 U/pL. (b) No miRNA-21, no Exo PI. (c) No miRNA-21 and added Exo III, 1 U/pL. (d) Added miRNA-21, 1 pM, without added Exo III. 3(D) Fluorescence spectra corresponding to the release of doxorubicin from the loaded (l)/(2)-functionalized NMOFs treated with Exo III, 1 U/pL, and different miRNA for a fixed time-interval of 60 minutes: (a) No added miRNA. (b) Added miRNA-221, 2.5 mM. (c) Added miRNA- 145, 2.5 pM. (d) Added miRNA-21,

2.5 pM.

[0027] Figures 4(A)-4(D). 4(A) Fluorescence spectra of the released anti-cancer drug doxorubicin after a fixed time-interval of 60 minutes upon treatment of the loaded (l’)/(2’)-capped NMOFs in the presence of a fixed concentration of Exo III, 1 U/pL, and variable concentrations of miRNA-221 : (a) 0 pM. (b) 0.25 pM. (c) 0.5 pM. (d) 1 pM. (e) 2.5 pM. 4(B) Fluorescence spectra corresponding to the released doxorubicin after a fixed time-interval of 60 minutes upon subjecting the loaded (l’)/(2’)- capped NMOFs to a fixed concentration of miRNA-221, 1 pM, and variable concentrations of Exo III: (a) 0 U/pL. (b) 0.1 U/pL. (c) 0.25 U/pL. (d) 0.5 U/pL. (e) 1 U/pL. (f) 2 U/pL. 4(C) Time- dependent release of doxorubicin upon the treatment of the loaded (l’)/(2’)-locked NMOFs with: (a) Added miRNA-221, 1 pM, and Exo IP, 1 U/pL. (b) No miRNA-221, no Exo III. (c) No miRNA-221 and added Exo III, 1 U/pL. (d) Added miRNA-221, 1 pM, without added Exo III. 4(D) Fluorescence spectra corresponding to the release of doxorubicin from the loaded ( r)/(2’)-functionalized NMOFs treated with Exo PI, 1 U/pL, and different miRNA for a fixed time-interval of 60 minutes: (a) No added miRNA. (b) Added miRNA-21, 2.5 pM. (c) Added miRNA-145, 2.5 pM. (d) Added miRNA- 221, 2.5 pM.

[0028] Figures 5(A)-5(D). Logic-gate release of two“output” fluorescent dyes from a mixture of Rhodamine 6G-loaded (l)/(2)-capped NMOFs and Methylene Blue-loaded (l’)/(2’)-gated NMOFs using miRNA-21 and/or miRNA-221 as the unlocking“inputs”. Panel I (5(A))-Response of the system in the absence of any input, output (0, 0). Panel P (5(B))-Subjecting the mixture to the miRNA-21 (1 pM) as input and in the presence of Exo III, 1 U/pL, output (1, 0). Panel PI (5(C))- Subjecting the mixture to miRNA-221 (1 pM) as input and in the presence of Exo PI, 1 U/pL, output (0, 1). Panel IV (5(D))-Treatment of the NMOFs mixture with miRNA-21 (1 pM) and miRNA-221 (1 mM) as inputs in the presence of Exo PI, 1 U/pL, output (1, 1). The output signals are recorded after subjecting the NMOFs mixture to the respective inputs for a time-interval of 60 minutes.

[0029] Figures 6(A)-6(B). Cytotoxicity of the anti-cancer drug doxorubicin-loaded (l)/(2)- functionalized NMOFs or the doxorubicin-loaded (l’)/(2’)-locked NMOFs towards MCF-7 breast cancer cells, OVCAR-3 ovarian cancer cells or MCF-IOA normal epithelial breast cells, and comparison to appropriate reference systems. Figure displays the cell viability upon subjecting the different cellular systems to the different loaded NMOFs and control NMOFs, after one day (Panel I (6(A))) and three days (Panel II (6(B))). The cell viabilities are presented as colored bars: Green- MCF-10A normal breast cells; Red-MCF-7 breast cancer cells; Blue-OVCAR-3 ovarian cancer cells (a) Non-treated cells (b) Treatment of the cells with unloaded (l)/(2)-modified NMOFs. (c) Treatment of the cells with unloaded (1’ )/(2’ )-fun cti on al i zed NMOFs. (d) Treatment of the cells with doxorubicin-loaded (l)/(2)-capped NMOFs. (e) Treatment of the cells with doxorubicin-loaded (G )/(2’ )-fun cti on al i zed NMOFs.

[0030] Figure 7. Synthesis of the UiO-68 NMOF particles and the sequent functionalization with nucleic acid (1) or (1’).

[0031] Figure 8. The X-ray diffraction pattern simulated from CIF file of UiO-68 MOF (black) and the X-ray diffraction pattern spectrum of nucleic acid (l)-modified NMOFs (blue). The X-ray diffraction spectrum of the nucleic acid (l)-modified NMOFs is identical to the reported spectrum⁵³ and corresponds to the space group Fm3m. The X-ray diffraction spectral bands of the nucleic acid (l)-modified NMOFs reveal band at similar diffraction angles as the simulated UiO-68 MOF, implying that the NMOFs retain their crystallize structure.

[0032] Figures 9(A)-9(D). 9(A) Fluorescence spectra of the released Methylene Blue after a fixed time-interval of 60 minutes upon treatment of the loaded (l’)/(2’)-capped NMOFs in the presence of a fixed concentration of Exo III, 1 U/pL, and variable concentrations of miRNA-221 : (a) 0 mM. (b) 0.25 pM. (c) 0.5 pM. (d) 1 pM. (e) 2.5 pM. 9(B) Fluorescence spectra corresponding to the released Methylene Blue after a fixed time-interval of 60 minutes upon subjecting the loaded (l’)/(2’)-capped

NMOFs to a fixed concentration of miRNA-221, 1 mM, and variable concentrations of Exo PI: (a) 0

U/pL. (b) 0.1 U/pL. (c) 0.25 U/pL. (d) 0.5 U/pL. (e) 1 U/pL. (f) 2 U/pL. 9(C) Time-dependent release of Methylene Blue upon the treatment of the loaded (l^,)/(2’)-locked NMOFs with: (a) Added miRNA-221, 1 pM, and Exo III, 1 U/pL. (b) No miRNA-221, no Exo PI. (c) No miRNA-221 and added Exo III, 1 U/pL. (d) Added miRNA-221, 1 pM, without added Exo III. 9(D) Fluorescence spectra corresponding to the release of Methylene Blue from the loaded (G )/(2’ )-fun cti on al i zed

NMOFs treated with Exo III, 1 U/pL, and different miRNA for a fixed time-interval of 60 minutes:

(a) No added miRNA. (b) Added miRNA- 145, 2.5 pM. (c) Added miRNA-21, 2.5 pM. (d) Added miRNA-221, 2.5 pM.

[0033] Figure 10. Time-dependent release of Rhodamine 6G upon the treatment of the loaded (l)/(2)-locked NMOFs with a fixed concentration of Exo III, 1 U/pL, and variable concentrations of miRNA-21 : (a) 0 pM. (b) 0.05 pM. (c) 0.1 pM. (d) 0.5 pM. (e) 1 pM.

[0034] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0035] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well- known methods, procedures, and components have not been described in detail so as not to obscure the present invention. [0036] The inventors describe the synthesis of miRNA-responsive drug-loaded NMOFs and their selective unlocking by miRNA-21, a specific biomarker for MCF-7 breast cancer cells,^54,55 or by miRNA-221, a specific biomarker for ovarian cancer cells.⁵⁶ hi addition, the inventors present a means to amplify the miRNA biomarkers by the biocatalytic regeneration of the miRNA by exonuclease present in the respective cells. The miRNA-responsive doxorubicin-loaded NMOFs reveal selective cytotoxicity toward the respective cancer cells. Besides the miRNA-specific release of anti-cancer drug, the inventors demonstrate that a mixture of miRNA-21- and miRNA-22wel- responsive drug-loaded NMOFs can be used as a dual therapeutic nanocarrier for treating breast cancer and ovarian cancer that concomitantly appear.

[0037] Figure 1(A) depicts the concept of miRNA-stimulated amplified unlocking of the drug- loaded NMOFs, that results in the release of anti-cancer drug. The UiO-68 NMOFs are functionalized with the nucleic acid (1) or (1’), and further are loaded with the respective drug model or anti-cancer drug (doxorubicin) and the loads are locked by hybridization of the nucleic acid, (2) or (2’), with (1) or (1’), respectively. The nucleic acid (2) or (2’) includes the base sequence complementary to the miRNA-21 or miRNA-221. In the presence of the miRNA-21 or the miRNA-221, the formation of the miRNA-21/(2) or miRNA-221/(2’) unlocks the NMOFs, resulting in the release of the loads. Realizing, however, that the concentration of the miRNA is low, the unlocking process and the subsequent release of the loads are inefficient. To overcome this limitation, the inventors have further engineered the released strands (2) and (2’) to allow the effective unlocking of the NMOF s even under the constrained low concentration conditions of the respective miRNAs. This is accomplished by the application of exonuclease-type catalysts, which present in the MCF-7 cancer cells⁵⁷ or the OVCAR- 3 ovarian cancer cells.⁵⁸ That is, the strands (1) and (1’) are designed to include non-hybridized single strand tethers at their 3’ -end, and the miRNA sensing strand (2) and (2’) include at their 3’- and 5’- ends single-strand tethers that do not participate in the hybridization of (l)/(2) or (G)/(2’). Under these structural constraints, the duplexes (l)/(2) or (l’)/(2’) are not subjected to hydrolytic digestion by the exonucleases. The strands (2) or (2’) are designed, however, in such a way that in their displacement by the miRNA-21 or the miRNA-221 yields duplex structures miRNA-21/(2) or miRNA-221/(2’) duplexes, where the 3’ -ends of (2) or (2’) in the resulting duplexes are fully base-paired with the miRNA duplexes (The miRNA include single-strand tethers at their 3’ -end). Under these conditions, the exonucleases present in the two types of cancer cells digest from the 3’ -end of (2) or (2’) in the miRNA-21/(2) or miRNA-221/(2’) duplexes, resulting in the release of the miRNAs for the cyclic and continuous unlocking of the capping units associated with the NMOFs. That is, the exonuclease- stimulated regeneration of the miRNA-21 or miRNA-221 increases the effective concentration of the miRNA for unlocking the NMOFs and the release of the loads.

[0038] The synthesis of nucleic acid (1) or (l’)-functionalized NMOFs was displayed in Figure 7.

The nucleic acid (1) or (l’)-modified NMOFs were characterized by microscopy and spectroscopic means. The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of the nucleic acid (l)-functionalized particles are shown in Figure 1(B) and 1(C).

Bipyramidal particles ca. 250-350 nm in diameter are observed. The X-ray diffraction pattern of the NMOFs are shown in Figure 8, and it is consistent with the reported data.⁵⁹ The average coverage of the NMOFs with nucleic acid (1) or (1’) was evaluated spectroscopically and it corresponds to ca.

10.5 nmol and 9.8 nmol per milligram NMOFs, respectively.

[0039] In the first step, the nucleic acid (l)-modified NMOFs were loaded with the fluorescent Rhodamine 6G as drug model, and the NMOFs were locked by the hybridization of nucleic acid (2) with the loaded nucleic acid (l)-modified NMOFs. Figure 2(A) shows the fluorescence spectra of the released Rhodamine 6G load upon subjecting the loaded NMOFs to variable concentrations of miRNA-21 and a constant concentration of Exo PI, 1 U/pL, for a fixed time-interval of 60 minutes. In the absence of miRNA-21, the release of the load is inefficient, and the release of the load increases as the concentration of miRNA-21 is higher. Figure 2(B) shows the fluorescence spectra of the released load upon subjecting the NMOFs to a fixed concentration of miRNA-21, 1 mM, in the presence of variable concentrations of Exo III. Even though the concentration of miRNA-21 is high, the unlocking of the NMOFs is very inefficient in the absence of Exo PI. The release of the load is, however, enhanced as the concentration of Exo III increases, indicating that the biocatalytic regeneration of the miRNA-21 is, indeed, important to amplify the unlocking process, and to stimulate the release of the load. Figure 2(C) shows the time-dependent fluorescence changes upon releasing the load. Evidently, the release of the load is effective at a miRNA-21 concentration of 1 mM and Exo III concentration of 1 U/pL. The release process reaches a saturation value after ca. 180 minutes, implying full release of the Rhodamine 6G load. Using an appropriate calibration curve, and knowing the content of the NMOFs, the inventors derived the loading of the fluorophore in the NMOFs to be 45.6 nmol/mg. The unlocking process is specific to the miRNA-21/Exo III and other miRNAs do not unlock the NMOFs. For example, Figure 2(D) shows the fluorescence spectra of the released Rhodamine 6G load from the (l)/(2)-locked NMOFs with the trigger of the miRNA-21/Exo III, curve (a), in comparison to the release of the load in the presence of foreign miRNA-221 and miRNA-145 in the presence of Exo III, curve (b) and curve (c), respectively. Only in the presence of miRNA- 21/Exo III, the NMOFs are unlocked, leading to the release of the load.

[0040] Similar experiments were performed where the anti-cancer drug doxorubicin was loaded in the (l)/(2)-locked NMOFs, Figure 3. The fluorescence spectra of the released doxorubicin after a fixed time-interval of 60 minutes in the presence of variable concentrations of miRNA-21, and a fixed concentration of Exo III, 1 U/pL, are shown in Figure 3A. As the concentration of miRNA-21 increases, the release of the drug is enhanced. In addition, the release of doxorubicin is controlled by the concentrations of the amplifying miRNA-21 regeneration biocatalyst, Exo III. Figure 3(B) shows the doxorubicin fluorescence spectra released from the (l)/(2)-gated drug-loaded NMOFs, upon treatment of the nanocarriers with a fixed concentration of miRNA-21, 1 pM, and variable concentrations of Exo PI for a fixed time-internal of 60 minutes. As the concentration of Exo III increases, the release of the drug is more effective, consistent with the enhanced unlocking of the NMOF carriers by the regenerated miRNA-21. Figure 3(C) shows the time-dependent release of the doxorubicin load from the NMOFs subjected to the miRNA-21 and Exo III, curve (a). For comparison, the time-dependent release of the load from the NMOFs, in the absence of miRNA-21 and the Exo III, in the presence of Exo IP only or in the presence of miRNA-21 only are shown in curves (b), (c) and (d), respectively. Evidently, the release of the drug in the presence of only the miRNA-21 is very inefficient. The residual inefficient release of the drug from the NMOFs in the absence of miRNA-21 and Exo III, or only in the presence of Exo III, is attributed to the leakage of the drug from“defective” incompletely (l)/(2)-capped pores. Figure 3(D) shows the selective miRNA-21 -stimulated unlocking of the doxorubicin-loaded NMOFs gated by the (l)/(2) capping units. The (l)/(2)-gated drug-loaded NMOFs subjected to foreign miRNAs, e.g., miRNA-145 or miRNA-221, do not lead to the release of doxorubicin and only the residual leakage of the drug from the incompletely gated pores is observed.

[0041] As stated, analogous loaded NMOFs that are unlocked by miRNA-221 specific to OVCAR-3 ovarian cancer cells were prepared. Toward this goal, the UiO-68 NMOFs were functionalized with the nucleic acid, (1’), loaded with the drug model Methylene Blue and capped by hybridization of the nucleic acid (2’) that recognizes miRNA-221, (Cf. Figure 1). As for the previous NMOFs system, the NMOFs are unlocked by the hybridization with miRNA-221, and the resulting released duplex miRNA-221/(2’) is engineered to allow the Exo ni-stimulated regeneration of the miRNA-221 for the amplified release of the loads. Figures 9(A)-9(D), shows the detailed characterization of the Methylene Blue-loaded miRNA-221 -responsive (l’)/(2’)-capped NMOF s, and the miRNA-221 -triggered release of Methylene Blue from the NMOFs. The following conclusions are derived from these experiments: (i) The NMOFs are unlocked by miRNA-221, but the release rate is inefficient (ii) In the presence of miRNA-221 and Exo PI, the effective release of the load is observed. That is, the Exo Ill-induced regeneration of the miRNA-221 provides an amplification path for the effective unlocking of the (G)/(2’) capping units, resulting in the efficient release of the load. (iii) The release of the Methylene Blue load by miRNA-221 is selective and foreign miRNAs, e.g., miRNA-21 or miRNA-145, do not unlock the NMOFs.

[0042] Nucleic acid (l’)-modified NMOFs were loaded with the doxorubicin anti-cancer drug and locked the NMOFs with the duplex (l’)/(2’) miRNA-221 -responsive capping units. The unlocking features of the doxorubicin-loaded NMOFs by the miRNA-221 are presented in Figure 4. The fluorescence spectra of the doxorubicin released from the NMOFs, subjected to different concentrations of miRNA-221, and a fixed concentration of Exo PI, 1 U/pL, for a fixed time-interval of 60 minutes are shown in Figure 4(A). As the concentration of miRNA-221 increases, the release of the drug is enhanced, consistent to the improved unlocking of the NMOF carriers. Figure 4(B) depicts the fluorescence spectra of the released doxorubicin upon interacting the NMOFs with a fixed concentration of miRNA-221, 1 mM, and variable concentrations of Exo PI, for a fixed time-interval of 60 minutes. As the concentration of Exo III increases, the release of the drug is enhanced, consistent with the improved unlocking of the gating units by the Exo III. Figure 4(C), curve (a), depicts the time-dependent release of the drug from the (r)/(2’)-locked NMOFs in the presence of miRNA-221 and Exo IP. For comparison, the background leakage of the drug from the NMOFs in the absence of miRNA-221 and Exo IP is shown in curve (b). In addition, curves (c) and (d) show the time- dependent release of doxorubicin in the presence of Exo III only and in the presence of miRNA-221 only, respectively. Inefficient release of the drug is observed, implying that the effective release of the drug requires the cooperative, amplified unlocking of the NMOFs by miRNA-221 and Exo PI. The release of the drug from the NMOFs is selective, and it is driven only by miRNA-221, Figure 4(D). In the presence of other miRNAs (miRNA-21 or miRNA-145) only the background leakage of the drug proceeds.

[0043] The successful selective release of the load triggered by the miRNA-21 and the miRNA-221 suggests that the (l)/(2)- and (r)/(2’)-gated NMOFs could act as a mixture of therapeutic carriers that realize the miRNA-guided release of drugs against MCF-7 or OVCAR-3 ovarian cancer cells, or the parallel release of the drugs against the two types of cancer cells. In other words, the mixture of the (l)/(2)- and (l’)/(2’)-gated NMOFs may act as a“logic gate” (“OR”-gate) system for releasing drugs against the two types of cancer. This is exemplified in Figure 5 using the Rhodamine 6G- and Methylene Blue-loaded NMOF carriers gated by (l)/(2) and (G)/(2’) locks, respectively. (In these systems, the two fluorescent dyes represent models for two different drugs). The miRNA-21 and/or miRNA-221 act as inputs for the guided, logic release of the respective loads. In the absence of miRNA-21 and miRNA-221 (input 0, 0), only the background leakage of the loads is observed, panel I. Subjecting the mixture to miRNA-21 (input 1, 0) or miRNA-221 (input 0, 1) leads to the selective unlocking of the (l)/(2)- or (l’)/(2’)-gated NMOFs and to the release of the Rhodamine 6G or the Methylene Blue fluorescent loads, panel II and III, respectively. Treatment of the mixture of NMOFs with the both miRNA-21 and miRNA-221 (input 1, 1) leads to the unlocking of the two kinds of NMOFs and to the release of the two different fluorescent loads, panel IV. This set of experiments supports the basic idea that two different miRNAs could selectively release two different drugs for the same type of cancer cells or for two different types of cancer cells.

[0044] In the next step, the inventors attempted to probe the cytotoxicity of the miRNA-21- and miRNA-221 -responsive NMOFs towards MCF-7 breast cancer cells and OVCAR-3 ovarian cancer cells, respectively. Applying the miRNAs in the mM range concentrations are far higher than the concentrations of miRNAs in cancer cells or the blood stream. Furthermore, in order to effectively unlock the NMOFs, it was essential to couple the unlocking process to the Exo III enzyme as amplifying, miRNA regeneration catalyst to stimulate the opening of the miRNA-responsive carriers. Thus, the presence of Exo Ill-type enzymes in the respective cancer cells were essential. It was reported^60,61 that MCF-7 cancer cells include 20000 miRNA-21 copies per cell and that the OVCAR- 3 ovarian cancer cells include the Exo Ill-type enzyme too⁵⁷ (although the content of the miRNA-221 were not specified). Accordingly, the inventors examined the release of the Rhodamine 6G-loaded (l)/(2)-capped NMOFs in the presence of different concentrations of miRNA-21 in the presence of 1 U/pL of the Exo PI catalyst, see Figure 10. It was found that as the concentration of miRNA decreases the unlocking process is less effective. Nonetheless, it was found that at a miRNA concentration of

50 nM, a concentration comparable to the number of miRNA-21 copies available in the MCF-7 cancer cells, the controlled release of the load is detected. That is, the result suggests that the miRNA- responsive carrier could be unlocked in the respective cancer cells, and eventually reveal selective cytotoxicity. Figure 6 shows the cytotoxicity of the (l)/(2)-gated doxorubicin-loaded NMOFs

(miRNA-21 -responsive) and of the (l’)/(2’)-gated doxorubicin-loaded NMOFs (miRNA-221- resopnsive) on MCF-7 and OVCAR-3 cancer cells. For comparison, the cytotoxicity of the doxorubicin-loaded miRNA-responsive NMOFs on epithelial MCF-IOA normal breast cells was examined. Additional control experiments examined the cytotoxicity of drug-unloaded miRNA-21 - and miRNA-221-reponsive NMOFs. The cytotoxicities of the different NMOFs were examined after one day and three days of interaction with the respective cells. The results show that the empty miRNA-21 -responsive NMOFs and the miRNA-221 -responsive NMOFs have no effect on the viability of the MCF-7, OVCAR-3 cancer cells and epithelial MCF-IOA normal breast cells after one or three days. Treatment of the different kinds of cells with the doxorubicin-loaded miRNA-21- responsive NMOFs decreased the cell viability of the MCF-7 cancer cells to 75% after one day and to 60% after a time-interval of three days. The normal MCF-IOA cells and the OVCAR-3 cancer cells revealed, however, cell viabilities of 90% and 85% after one and three days of interaction with the miRNA-21 -responsive carrier. That is (l)/(2)-capped doxorubicin-loaded NMOFs revealed a significantly enhanced selective toxicity toward the MCF-7 breast cancer cells, consistent with the presence of the miRNA-21 as a biomarker in the MCF-7 cancer cells that unlocks the carrier and releases the drug. In addition, treatment of the different cells with the miRNA-221 -responsive doxorubicin-loaded NMOFs after one day and three days results in OVCAR-3 cancer cells’ viabilities corresponding to 80% and 60% while the viability of the MCF-7 cancer cells and MCF-IOA normal breast cells corresponded to > 90% after these time-intervals. The results indicate selective cytotoxicity for the MCF-7 breast cancer cells by the (l)/(2)-capped miRNA-21 -responsive doxorubicin-loaded NMOFs, and selective cytotoxicity for the OVCAR-3 ovarian cancer cells by the

(l’)/(2’)-capped doxorubicin-loaded miRNA-221 -responsive NMOFs. The two doxorubicin-loaded miRNA-responsive NMOFs were not toxic toward the MCF-IOA normal breast cells.

[0045] In conclusion, the inventors have introduced miRNA-responsive NMOFs for the selective release of the anti-cancer drug doxorubicin into two types of cancer cells (breast cancer cells and ovarian cancer cells). The miRNA-stimulated unlocking of the drug-loaded NMOFs represents a versatile autonomous sense-and-treat therapeutic approach. The method can be broadened to include miRNAs characteristic of other diseases, and particularly for other types of cancers, for the controlled release of drugs. In addition, many other drugs can be loaded in the miRNA-responsive carriers. The advantages of the miRNA-responsive carriers include: (i) Selective unlocking of the carriers in the cancer cells that include the specific biomarker. This minimizes harmful cytotoxicity towards normal cells (ii) Dual miRNA-responsive drug therapeutic carriers may be envisaged, where mixtures of miRNA induce the unlocking of different drug-loaded NMOF carriers for the treatment of two related cancer cells or cancer cells/metastatic cells (iii) The miRNA induced release of the therapeutic loads provides a means for controlled release.

EXPERIMENTAL SECTION

[0046] Materials: Doxorubicin, Rhodamine 6G, Methylene Blue, dibenzocyclooctyne-sulfo-N- hydroxysuccinimidyl ester (DBCO-sulfo-NHS), tert-butyl nitrite (tBuONO), and azidotrimethylsilane (TMSN3) were purchased from Sigma-Aldrich. Exo III was purchased from New England Biolabs Inc. (Beverly, MA, USA). Other reagents and solvents were purchased from Sigma-Aldrich and used directly. Ultrapure water was obtained by aNANOpure Diamond instrument (Bamstead International, Dubuque, IA, USA). All oligonucleotides and miRNAs were synthesized, standard desalting purified, and freeze-dried by Integrated DNA Technologies, Inc.

[0047] The detailed sequences of the nucleic acid used are: ^■ (1) 5 ' -Nth - AGC TT AT C AGATT -3 ' ;

^■ (2) 5'-TTAAAACATCAGTCTGATAAGCTA-3';

^■ (1’) 5'-NH₂-GCTACATTGTCAA-3';

^■ (2’) 5'-TTTTAACCCAGCAGACAATGTAGCT-3';

^■ miRNA-21 : 5'-UAGCUUAUCAGACUGAUGUUGA-3';

^■ miRNA-221 : 5'-AGCUACAUUGUCUGCUGGGUUUC-3';

^■ miRNA-145: 5'-GUCCAGUUUUCCCAGGAAUCCCU-3'.

[0048] Synthesis of nucleic acid (1) and (l’)-modified NMOFs: The nucleic acid (1) and (1’)- modified NMOFs were synthesized according to Chen, W. H.; Yu, X.; Liao, W. C.; Sohn, Y. S.; Cecconello, A.; Kozell, A.; Nechushtai, R.; Willner, I. Adv. Funct. Mater. 2017, 27, 1702102. The simple synthetic route was displayed in Figure 7.

[0049] Loading of the nucleic acid (1) or (l’)-modified NMOFs with drug models or chemotherapeutic drug: The nucleic acid (1) or (l’)-modified NMOFs (1.5 mg/mL) were dispersed in HEPES buffer solution and incubated with Rhodamine 6G (0.2 mg/mL), Methylene Blue (0.2 mg/mL) or the anti-cancer drug doxorubicin (0.5 mg/mL) overnight, respectively. Then, the NMOFs were transferred to HEPES buffer (10 mM, pH 7.4, containing 50 mM NaCl) solution and hybridized with nucleic acid (2) or (2’), respectively, leading to the locked state of the loaded, duplex DNA-gated NMOFs. After 12 h, the NMOFs were washed several times to remove the excess and nonspecifically bound Rhodamine 6G, Methylene Blue or doxorubicin.

[0050] miRNA-triggered unlocking of the NMOFs and the release of the encapsulated loads: The duplex DNA-capped, drug model- (Rhodamine 6G or Methylene Blue) or doxorubicin-loaded NMOFs, at a concentration corresponding to 1 mg/mL, were subjected to the respective miRNAs (miRNA-21 or miRNA-221) to unlock the NMOFs and release the loads. The NMOF solutions were treated with different concentrations of miRNAs and a fixed concentration of Exo III (1 U/pL) or interacted with a fixed concentration of miRNAs (1 mM) in the presence of variable concentrations of Exo III. At regular time intervals, the respective sample solutions were centrifuged to precipitate the NMOFs (10000 rpm for 10 minutes), and the fluorescence of the released loads in the supernatant solutions were measured using a Cary Eclipse Fluorescence Spectrophotometer (Varian Inc.).

[0051] Operation of the dictated“logic gate” system for releasing the loads: The“logic gate” system was performed in a NMOF solution consisting of the Rhodamine 6G-loaded, (l)/(2)-gated, miRNA-

21-responsive NMOFs (1 mg/mL) and the Methylene Blue-loaded, (l’)/(2’)-capped, miRNA-221- responsive NMOFs (1 mg/mL) in HEPES buffer (10 mM, pH 7.4, containing 50 mM NaCl). The mixture solution was treated with a constant concentration of Exo III, 1 U/pL, and different combinations of the miRNA-21 and the miRNA-221 as inputs, such as (miRNA-21, miRNA-221): (0, 0), (1, 0), (0, 1), and (1, 1). Digital“1” represents that the concentration of miRNA-21 and miRNA-

221 was 1 pM and 1 pM, respectively, and the digital“0” represents that no miRNA was added. After

60 minutes incubation, the NMOF mixtures were centrifuged at 10000 rpm for 10 minutes to precipitate the residual NMOFs, and the fluorescence spectra of the released Rhodamine 6G and/or

Methylene Blue in the supernatant solution were measured separately using a Cary Eclipse Fluorescence Spectrophotometer (Varian Inc.).

[0052] Cell culture: Human breast cancer cells (MCF-7) were grown in 5% CO2 RPMI-1640 medium supplemented with 10% FCS, _L-glutamine, and antibiotics (Biological Industries). Human ovarian cancer cells (OVCAR-3) were cultured in RPMI-1640 medium supplemented with 10% FCS,

0.01 mg/mL bovine insulin and penicillin/streptomycin (1 unit/mL). Normal breast cells (MCF-IOA) were maintained in complete growth medium consisting of 1 : 1 mixture of Dulbecco’s modified

Eagle’s medium and Ham’s F12 medium supplemented with horse serum (5%), epidermal growth factor (20 ng/mL), cholera toxin (CT, 0.1 pg/mg), insulin (10 pg/mL), hydrocortisone (500 ng/mL), and penicillin/streptomycin (1 unit/mL). Cells were plated one day prior to the experiment on 96-well plates for cell viability. [0053] Cell viability experiments: Cell viability was assayed after incubation of the anti-cancer drug (doxorubicin) unloaded or loaded NMOFs with MCF-IOA, MCF-7 and OVCAR-3 cells planted at a density of 1.0 ^c 10⁴ cells per well in 96-well plates. After seeding the cells overnight, the cells were incubated with the drug-unloaded NMOFs or doxorubicin-loaded NMOFs for 6 h, respectively. Following intensive washing, the cells were further incubated for 3 days with growth medium and the cell viability was determined with the fluorescent redox probe, Alamar Blue. The fluorescence of Alamar Blue was recorded on a plate-reader (Tecan Safire) after 1 h of incubation at 37 °C (M_s = 560 nm; _em = 590 nm).

[0054] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

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Claims

CLAIMS What is claimed is:

1. A nanoparticle loaded with at least one pharmaceutically active agent, said at least one pharmaceutically active agent being locked within said nanoparticle by at least one first nucleic acid sequence partially hybridized with at least one second nucleic acid sequence, wherein said at least one first nucleic acid sequence is associated with said nanoparticle; and said at least one second nucleic acid sequence is fully based paired with at least one RNA biomarker from its 3’ and/or 5’ end.

2. A nanoparticle according to claim 1, wherein said nanoparticle is a porous nanoparticle and said at least one pharmaceutically active agent is loaded within its pores.

3. A nanoparticle according to claims 1 or 2, wherein said nanoparticle is a metal-organic framework nanoparticle.

4. A nanoparticle according to any one of the preceding claims, wherein said partial hybridization of said at least one first nucleic acid sequence and at least one second nucleic acid sequence results in having the 3’- and 5’ -ends of said at least one second nucleic acid sequence free of hybridization with said at least one first nucleic acid sequence.

5. A nanoparticle according to any one of the preceding claims, wherein said at least one RNA biomarker is a biomarker of at least one disease or disorder.

6. A nanoparticle according to any one of the preceding claims, wherein said at least one RNA biomarker is selected from mRNA, microRNA (miRNA), small interfering RNA (siRNA) , tRNA and any combinations thereof.

7. A nanoparticle according to any one of the preceding claims, wherein said loaded at least one pharmaceutically active agent is unlocked from said nanoparticle upon displacement of said at least one second nucleic acid sequence by said at least one RNA biomarker.

8. A nanoparticle according to any one of the preceding claims, wherein hybridization of said at least one second nucleic acid said at least one RNA biomarker is susceptible to digestion by at least one exonuclease.

9. A composition comprising at least one nanoparticle according to any one of claims 1 to 7.

10. A composition according to claim 9, for use as a medicament.

11. A composition according to claims 9 or 10, for use in the treatment of at least one disease or disorder.

12. A composition according to any one of claims 9 to 11, for use in the treatment of cancer.

13. A method of treating a disease or disorder comprising administering to a patient in need thereof at least one nanoparticle loaded with at least one pharmaceutically active agent, said at least one pharmaceutically active agent being locked within said nanoparticle by at least one first nucleic acid sequence partially hybridized with at least one second nucleic acid sequence, wherein said at least one first nucleic acid sequence is associated with said nanoparticle; and said at least one second nucleic acid sequence is fully based paired with at least one RNA biomarker from its 3’ and/or 5’ end.