DNA-based mechanosensitive delivery agents and uses thereof

Abstract

A nucleic acid-based nanostructure having a pre-defined geometrical shape is disclosed which encapsulates at least one active agent. The nanostructure comprises at least one force or pressure sensor, which, when activated, allows release of said active agent from said nanostructure.

Description

[0001] DNA-BASED MECHANOSENSITIVE DELIVERY AGENTS AND USES THEREOF

[0002] RELATED APPLICATION / S

[0003] This application claims the benefit of priority from US Patent Application No. 63 / 720,242, filed November 14, 2024, which is hereby incorporated by reference in its entirety.

[0004] SEQUENCE LISTING STATEMENT

[0005] The XML file, entitled SequenceListing.xml, created on November 12, 2025, comprising 479,260 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

[0006] FIELD AND BACKGROUND OF THE INVENTION

[0007] The present invention, in some embodiments thereof, relates to nucleic acid-based mechanosensitive delivery agents.

[0008] Drug delivery is a complex task that calls for particular solutions for treating different pathologies and diseases. Targeting of drugs and imaging agents may be based on utilizing abnormal features of disease state such as: elevated pH in tumor, enhanced blood vessel permeability in cancer, decreased oxygen level in hypoxic regions, up-regulated cell surface antigens or molecular affinity of targeting moieties to pathological tissue. Based on these characteristics, different drug delivery schemes have been developed.

[0009] Fluid shear stress is an important physiological feature of the blood circulation that is tightly regulated under normal physiological conditions. Shear stress has been shown to play a major role in regulating endothelial cell phenotype and gene expression, platelet and red blood cell (RBC) aggregation, arteriogenesis and hemodynamic properties. Stenosis, abnormal narrowing in blood vessels due to blockage, constriction or malformation, significantly alters the characteristics of local blood flow; differing this region from normal physiological conditions. For example, wall shear stress at atherosclerotic stenotic sites may be two orders of magnitude higher than normal physiological shear stress levels. These abnormal shear stresses induce platelet activation and facilitate thrombus formation.

[0010] Medical therapy has poor efficacy in treating aortic stenosis, due to different medical conditions and diseases such as thrombosis or vasospasm. These diseases are very common and leads to sever medical condition, especially in the modem western world. According to a report by the Global Market Insights, the global thrombosis drug market size was estimated at USD 26 billion in 2021 and is projected to grow at a Compound Annual Growth Rate (CAGR) of ~6% to reach USD 36 billion by 2027. While, according to a Data Bridge market research report, the vasospasm market is expected to grow at a rate of ~5% in the period of 2021 to 2028 and is expected to reach USD 2,856M by 2028. While thrombosis mainly increases due to unhealthy lifestyle, the increase of cerebral vasospasm resulted from traumatic brain injuries.

[0011] Background art includes US Patent Application No. 20140080198, PCT Application No. W02019109707, US Patent Application No. 20130224859 and EP Patent Application No.

[0012] 2611466B1. Additional Background art includes grantome(dot)com / grant / NIH / K99-HL 146945 -01.

[0013] SUMMARY OF THE INVENTION

[0014] According to an aspect of some embodiments of the present invention there is provided a nucleic acid-based nanostructure having a pre-defined geometrical shape, wherein the nanostructure encapsulates at least one active agent, wherein the nanostructure comprises at least one force or pressure sensor, which, when activated, allows release of the active agent from the nanostructure.

[0015] According to an aspect of some embodiments of the present invention there is provided a nucleic acid-based nanostructure having a pre-defined geometrical shape, wherein the nanostructure encapsulates at least one active agent, wherein the nanostructure comprises at least one force or pressure sensor, which, when activated, allows release of the active agent from the nanostructure, the at least one force or pressure sensor comprising a protrusion.

[0016] According to some embodiments of the invention, the sensor is activated by at least one of a shear stress, an elastic stress, a pressure gradient or an acceleration / velocity gradient.

[0017] According to some embodiments of the invention, the sensor is activated by shear stress. According to some embodiments of the invention, the at least one force or pressure sensor comprises at least two force or pressure sensors, each comprising a protrusion.

[0018] According to some embodiments of the invention, the protrusion extends outwardly from an external surface of the nanostructure by a distance of at least 1 nm and is configured to undergo a measurable deformation or displacement or rotation upon exposure to a mechanical stimulus.

[0019] According to some embodiments of the invention, the nanostructure comprises at least two elements connected to each other to form a hollow.

[0020] According to some embodiments of the invention, the sensor comprises double stranded DNA having a length between 2-200 nucleotides which connects a first element of the at least two elements to a second element of the at least two elements.

[0021] According to some embodiments of the invention, the nanostructure comprises:

[0022] a carrier portion which comprises an internal hollow and at least one opening to the hollow, the at least one active agent being comprised in the hollow; and wherein the at least one force or pressure sensor comprises a covering element which covers and protrudes over the at least one opening when the force or pressure sensor is non-activated, and which does not fully cover the at least one opening when the force or pressure sensor is activated so as to allow release of the active agent.

[0023] According to some embodiments of the invention, the nanostructure is resealable following activation of the force or pressure sensor.

[0024] According to some embodiments of the invention, the nanostructure is non-resealable following activation of the force or pressure sensor.

[0025] According to some embodiments of the invention, the force or pressure sensor further comprises a spring element which permanently connects the covering element to the carrier portion, wherein the spring temporarily extends when force or pressure is above a predetermined level so as to allow at least partial uncovering of the covering element and release of the active agent.

[0026] According to some embodiments of the invention, the nanostructure further comprises a hinge connecting element which permanently connects the covering element to the carrier portion when the force or pressure is above the predetermined level.

[0027] According to some embodiments of the invention, the spring element extends in length by at least 1.1 fold when force or pressure is above the predetermined level.

[0028] According to some embodiments of the invention, the spring element comprises a length between 5-10000 nm and elastic and an elastic force between of 1 femto-Newton - 1 microNewton.

[0029] According to some embodiments of the invention, the spring element extends in length when force or pressure is above the predetermined level to a greater extent than the hinge connecting element extends in length when the force or pressure is above the predetermined level.

[0030] According to some embodiments of the invention, the predetermined level is above about 100 dyne / cm2.

[0031] According to some embodiments of the invention, the spring and / or the hinge connecting element are fabricated from nucleic acids.

[0032] According to some embodiments of the invention, the spring connecting element is fabricated from a material capable of elastic deformation and reversible conformational change, the material being other than nucleic acid.

[0033] According to some embodiments of the invention, one end of the spring and the hinge connecting element are attached to an inner surface of the covering element and wherein the spring and the hinge connecting element are positioned on a distal and proximal sides of the covering element. According to some embodiments of the invention, another end of the spring and the hinge connecting element and attached to an inner surface of the carrier portion.

[0034] According to some embodiments of the invention, the carrier portion comprises a second opening to the hollow and wherein the nanostructure comprises a second force or pressure sensor which comprises a second covering element.

[0035] According to some embodiments of the invention, the second force or pressure sensor is identical to the first force or pressure sensor.

[0036] According to some embodiments of the invention, the second force or pressure sensor is nonidentical to the first force or pressure sensor.

[0037] According to some embodiments of the invention, the second force or pressure sensor comprises a second covering element which covers and protrudes over the second opening when the second force or pressure sensor is non-activated and which does not fully cover the second opening when the force or pressure sensor is activated.

[0038] According to some embodiments of the invention, the spring element comprises single stranded DNA, double stranded DNA or both.

[0039] According to some embodiments of the invention, the ratio of the lengths of the single stranded DNA: the double stranded DNA is in between 0.001 - 100.

[0040] According to some embodiments of the invention, the first end of the spring element comprises double stranded DNA and a second end of the spring element comprises double stranded DNA and a central portion of the spring element comprises single stranded RNA.

[0041] According to some embodiments of the invention, the targeting moiety or immobilizing agent is attached to an outer surface of the nanostructure.

[0042] According to some embodiments of the invention, the targeting moiety or immobilizing agent is selected from the group consisting of a protein, a nucleic acid and an aptamer.

[0043] According to some embodiments of the invention, the active agent is a therapeutic agent and / or a diagnostic agent.

[0044] According to some embodiments of the invention, the diagnostic agent is an imaging agent. According to some embodiments of the invention, the diagnostic agent is fluorescent or other detectable signal agent to be release in the blood and analyzed in a blood test.

[0045] According to some embodiments of the invention, the therapeutic agent is selected from the group consisting of an antithrombotic agent, a thrombolytic agent and a vasodilator.

[0046] According to some embodiments of the invention, the antithrombotic or thrombolytic agent is selected from the group consisting of anticoagulants, pro-coagulant antagonists, antiplatelet agents, thrombolytic agents, anti-thrombolytic agent antagonists, fibrinolytic enzymes, and any combinations thereof.

[0047] According to some embodiments of the invention, the thrombolytic agent is selected from the group consisting of tissue-type plasminogen activator (t-PA), streptokinase (SK), prourokinase, urokinase (uPA), alteplase, reteplase, tenecteplase, Streptase®, lanoteplase, monteplase, saruplase, staphylokinase, and anisoylated plasminogen-streptokinase activator complex and any combinations thereof.

[0048] According to some embodiments of the invention, the nanostructure is comprised of DNA. According to some embodiments of the invention, the nanostructure is comprised of RNA. According to some embodiments of the invention, the nanostructure is comprised of a combination of DNA and RNA.

[0049] According to an aspect of some embodiments of the present invention there is provided a method of releasing an agent from a nanostructure at a site of high shear stress comprising exposing the nucleic acid-based nanostructure described herein to a shear stress above a predetermined level, thereby releasing the agent.

[0050] According to some embodiments of the invention, the releasing is affected in vivo.

[0051] According to some embodiments of the invention, the releasing is affected in vitro.

[0052] According to some embodiments of the invention, the nucleic acid-based nanostructure is immobilized to a solid surface.

[0053] According to an aspect of some embodiments of the present invention there is provided a method of releasing a pharmaceutical agent from a carrier at a site of high force or stress comprising exposing a nucleic acid-based nanostructure which comprises the pharmaceutical agent, to an amount of shear stress greater than 100 dynes / cm2, thereby releasing the pharmaceutical agent.

[0054] According to some embodiments of the invention, the nucleic acid-based nanostructure is the nanostructure of claim 1 and the pharmaceutical agent is the active agent.

[0055] According to an aspect of some embodiments of the present invention there is provided a method of treating or preventing a disease or disorder associated with high shear stress in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising a nucleic acid-based nanostructure having a pre-defined geometrical shape, wherein the nanostructure encapsulates at least one pharmaceutical agent useful for treating the disease, wherein the nanostructure comprises at least one shear pressure sensor, which, when activated, allows release of the pharmaceutical agent from the nanostructure, thereby treating or preventing the disease.

[0056] According to some embodiments of the invention, the disease or disorder is associated with arterial stenosis and / or a stenotic lesion and / or an occlusive lesion. According to some embodiments of the invention, the nanostructure comprises: a carrier portion which comprises an internal hollow and at least one opening to the hollow, the at least one active agent being comprised in the hollow; and

[0057] wherein the at least one shear pressure sensor comprises a covering element which covers and protrudes over the at least one opening in a non-stenosed blood vessel and which does not fully cover the at least one opening at a stenosed blood vessel.

[0058] According to some embodiments of the invention, the pharmaceutical agent is selected from the group consisting of a vasodilator, a thrombolytic agent and antithrombotic agent.

[0059] According to some embodiments of the invention, the thrombolytic agent or antithrombotic agent is selected from the group consisting of anticoagulants, pro-coagulant antagonists, antiplatelet agents, thrombolytic agents, anti-thrombolytic agent antagonists, fibrinolytic enzymes, and any combinations thereof.

[0060] According to some embodiments of the invention, the thrombolytic agent is selected from the group consisting of tissue-type plasminogen activator (t-PA), streptokinase (SK), prourokinase, urokinase (uPA), alteplase, reteplase, tenecteplase, Streptase®, lanoteplase, monteplase, saruplase, staphylokinase, and anisoylated plasminogen-streptokinase activator complex and any combinations thereof.

[0061] According to some embodiments of the invention, the stenosis, stenotic, or occlusive lesion is selected from the group consisting of intermittent claudication (peripheral artery stenosis), angina (coronary artery stenosis) or myocardial infarction, carotid artery stenosis, aortic stenosis, buttonhole stenosis, calcific nodular stenosis, coronary ostial stenosis, double aortic stenosis, fish-mouth mitral stenosis, idiopathic hypertrophic subaortic stenosis, infundibular stenosis, mitral stenosis, muscular subaortic stenosis, pulmonary stenosis, pulmonary embolism, pulmonary hypertension, subaortic stenosis, subvalvar stenosis, supravalvar stenosis, tricuspid stenosis, renal artery stenosis, vascular hypertension, sickle cell anemia, and any combinations thereof; and / or results from trauma or injury, atherosclerosis, birth defects, diabetes, iatrogenic, infection, inflammation, ischemia, neoplasm, vasospasm, coronary vasospasm, Raynaud's phenomenon, stroke, blood clotting, Moyamoya disease, Takayasu's disease, polyarteritis nodosa, disseminated lupus erythematous, rheumatoid arthritis, tumors of the spine, Paget's disease of bone, fluorosis, hemodialysis, and any combinations thereof.

[0062] According to some embodiments of the invention, the disease is atherothrombosis.

[0063] According to some embodiments of the invention, the targeting moiety or immobilizing moiety is attached to an outer surface of the nanostructure.

[0064] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0065] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0066] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0067] In the drawings:

[0068] FIGs. 1A-D is a cartoon illustrating the principle behind mechano-responsive DNA origami structures for targeted drug delivery to narrowed blood vessels. It relies on shear forces generated in narrowed blood vessels that can stretch the DNA-based spring causing the capsule to open and releases its therapeutic load. (A) A three-dimensional scheme of the DNA origami structure. It consists of a hollow capsule enclosed by two large lids that are connected to the capsule via hinges and springs. (B) Under elevated shear force, the DNA spring stretches, and opens the capsule. Black circles mark the attachment points between the capsule and the upper lid. (C) Under normal physiological flow, shear forces are low, and the spring design ensures that the capsule stays closed. (D) In a narrowed vessel, the shear force increases significantly and triggers the opening of the capsule, thereby releasing the drug to the blood clot. Note: Capsules are not to scale, they are ~104times smaller than illustrated.

[0069] FIGs. 2A-F. Designed models of the DNA Origami capsule (DOC) and its AFM measurements. (A) Realistic simulation of the DNA Origami drug delivery structures created with SNUPI software (Figure s9) based upon our caDNAno design. The designed model consists of a hollow capsule (yellow) sealed by two opposing lids (turquoise). Each lid is connected by a joint on one edge and a spring that pulls it towards the capsule on the other (red). Springs are not shown in scale (they are much thinner). The small bumps on the lid's surface are designed from the short extra tail that remains from the scaffold and allows to avoid free ssDNA connecting to the capsule. Scale bars are 50 nm. (B, C) AFM images of the lids demonstrate high yield and a structure that aligns with our design. Scale bar 100 nm. (D, E) AFM images of the box with both lids. Scale bar 100 nm. The boxes are thicker, and they adhere to the surface so that its 3D structure cannot be analyzed. (F) A lid with intendent holes designed in order to demonstrate the position and size of the box that connects to the lid. Scale bar 50 nm. FIGs. 3A-I. Cryo-TEM results of the DOCs. Scale bars are 100 nm for A, C-H, and 50 nm for B. (A) Cryo-TEM image of the boxes without lids. (B) Slice from tomogram reconstruction of a capsule without lid. (C) Rendered segmentation of the capsule without lids from the tomogram in B. (D, E) Cryo-TEM image of DOCs (boxes with the lids). Note that Figure E clearly shows the edges of the parallel DNA-helixes of the lid. (F) A slice from the tomogram reconstruction of the full structure. (G) Cryo-TEM tomography of the capsule. Two of the box’s faces cannot be seen when the capsule is fully constructed, this is the missing wedge (see Methods and Figures s 16, s 18, s20 for reconstructed images. (H) Image of an open capsule where the lids are connected only from one edge of the capsule and there is no spring. (I) Gel electrophoresis of the open capsule structure. 1,7: 1 kb ladder, 2: only the box, 3: only the lid, 4,5: capsule with one lid only; 6: capsule with both lids; a -free boxes and lids, b - capsule with one lid only, c - the whole DOC structure (box with two lids), d, e - aggregates.

[0070] FIGs. 4A-C illustrate characterization of the ssDNA used as an elastic spring. A. Schematic representation of the stretching experiments. B-C. Optical tweezers force-extension results. The first pull is shown in black, and another pull in purple. Red and green dashed curves show WLC curves fitted the low and high force segments, corresponding to the full construct without stem-loop structures (red) and with them (green), respectively. The dwell time between each pull is 30 seconds.

[0071] FIGs. 5A-B illustrate analysis of the force-extension results. A. Boxplots of Lp,ss, L̄0and the end-to-end distance (related to L̄0) of the ssDNA springs. These distributions result from the different stem loops that can be formed. The averages are 0.78, 197 and 12 nm respectively. B. Force-extension curves showing the 70% confidence interval limits of the measured springs for the ssDNA length Loof 120 nm (purple) and 250 nm (blue). Forces below 1 pN correspond to healthy vessels (green background), whereas forces above 3 pN indicate pathological flow (red background). The gray dashed line marks an extension of 47 nm, the threshold at which the capsule opens.

[0072] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0073] The present invention, in some embodiments thereof, relates to nucleic acid-based mechanosensitive delivery agents.

[0074] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0075] DNA origami is a groundbreaking technique that utilizes the unique properties of DNA molecules to create intricate, nanoscale structures. The method relies on the ability of complementary DNA strands to hybridize and form double helices. Accordingly, it enables the precise folding and assembly of DNA into complex architectures and patterns. DNA origami nanostructures (DONs) are typically constructed of a long single-stranded DNA scaffold and a set of shorter complementary strands (staples), which fold the scaffold into precise 2D and 3D structures. DNA origami is particularly attractive for drug delivery due to its nanoscale dimensions, biocompatibility, and the ability to precisely control its biochemical and biophysical properties. These characteristics enable the design of highly targeted delivery systems, ensuring that therapeutics reach specific cells or tissues with high efficiency and minimal side effects.

[0076] Using DNA origami, the present inventors have now designed and assembled a unique mechano-responsive drug delivery capsule based on DNA origami and the elastic properties of DNA. The capsule is engineered to respond to changes in shear stress, enabling targeted drug release specifically at stenotic blood vessels, which pose an immediate risk for stroke, pulmonary embolism and heart attack. The present approach has the potential to improve clinical treatment by allowing the safe and effective use of potent drugs, such as tPA, at relatively high doses, as it prevents their release in healthy vessels where they could cause significant harm.

[0077] The disclosed device comprises a hollow capsule sealed by two large lids held together by a DNA-based spring which keeps the capsule closed under normal blood flow but stretches to open the capsule in response to the elevated shear forces found in narrowed blood vessels (see Figures 1A-D). This approach is broadly applicable to other conditions characterized by altered shear forces, such as vasospasm and other cardiovascular disease conditions.

[0078] Whilst reducing the present invention to practice, the present inventors successfully designed and synthesized the structure, as confirmed by both AFM (Figures 2A-F) and Cryo-TEM imaging (Figures 3A-H). Significant effort was dedicated to optimizing the properties of the DNA spring to meet the required response to shear stress. Optical tweezers measurements provided quantitative validation, demonstrating that the mechanical behavior of the system aligns with theoretical expectations and confirming its feasibility as a functional biomedical device (Figures 4B-C).

[0079] Both the structure and the DNA-based spring are highly adaptable, offering several degrees of freedom that allows one to fine-tune its functionality for different applications. Furthermore, the DNA origami structures can be integrated with various functional components, such as targeting ligands, biocompatible coatings, and different types of therapeutic payloads.

[0080] Thus, according to a first aspect of the present invention, there is provided a nucleic acid-based nanostructure having a pre-defined geometrical shape, wherein the nanostructure encapsulates at least one active agent, wherein the nanostructure comprises at least one shear pressure sensor, which, when activated, allows release of the active agent from the nanostructure. The term “nanostructure” refers to an assembly or construct having at least one dimension in the nanometer range (1–1000 nm), typically formed through self-assembly or directed synthesis of molecular building blocks. The nanostructure may have a pre-defined geometrical shape, including but not limited to polyhedral, tubular, spherical, cylindrical, or cage-like architectures. The nanostructure may be hollow or solid and may optionally include one or more internal compartments, channels, or openings. In certain embodiments, the nanostructure is designed to encapsulate, retain, or release one or more active agents.

[0081] According to a particular embodiment, the nanostructure is a cylindrical nanostructure.

[0082] According to another particular embodiment, the nanostructure is a box-shaped nanostructure. According to a particular embodiment, the cylindrical nanostructure has a diameter of about 10 nm-500 nm. According to another embodiment, the structure has a box shape having a length and width between 20-50 nm, or even 5-500 nm and a height between 10-500 nm.

[0083] As used herein, the term “nucleic acid nanostructure” refers to a nanoscale structure comprising at least one nucleic acid component (such as DNA or RNA), wherein the nucleic acid acts both as a structural and / or functional element (e.g. pressure sensor).

[0084] Nucleic acid nanostructures can also serve as a scaffold for the formation of other structures. Nucleic acid nanostructures may be prepared by methods known in the art using one or more nucleic acid oligonucleotides.

[0085] For example, in certain elements, the nucleic acid nanostructure is a DNA cylindrical origami nanostructure, self-assembled from single stranded DNA molecules using “staple strands”.

[0086] In another embodiment the nucleic acid nanostructure comprises at least two elements connected to one another such that they form a hollow within (i.e. a closed volume). On activation of the force or pressure sensor, the two elements detach, or open and the active agent is released. For example, the nucleic acid nanostructure may comprise a cylindrical body and at least one lid element which seals the cylindrical body on either side and prevents pharmaceutical agent encapsulated within from being released - see for example Figure 1 A. An exemplary single-stranded DN A sequence which can be used to fabricate the cylindrical body is set forth in SEQ ID NO: 3. An exemplary single-stranded DNA sequence which can be used fabricate the lid is set forth in SEQ ID NO: 4.

[0087] In still another embodiment, activation of the force or pressure sensor causes rupture of the structure.

[0088] 'The length of the single stranded DNA scaffold strand is variable and depends on for example, the type of nanostructure. In certain embodiments, the DNA scaffold strand is comprised of multiple oligonucleotide strands. In certain embodiments, the DNA scaffold strand Is comprised of a single oligonucleotide strand. In certain embodiments, the DNA scaffold strand is about 100 nucleotides in length to about 1,000 nucleotides in length. In certain embodiments, the DNA scaffold strand is about 1000 nucleotides in length to about 10,000 nucleotides in length or even larger.

[0089] For use in the present invention, the nucleic acids can be synthesized de novo using any of a number of procedures well known in the art. For example, the cyanoethyl phosphoramidite method (Beaucage, S. L., and Caruthers, M. H., Tet. Let. 22:1859,1981); nucleoside H-phosphonate method (Garegg et ah, Tet. Let 27:4051 4054,1986; Froehler et ah, Nucl. Acid. Res. 14:5399-5407,1986; Garegg et al., Tet. Let. 27: 4055-4058. 1986, Gaffney et al.,Tet. Let. 29: 2619-2622, 1988). These chemistries can be performed by a variety of automated oligonucleotide synthesizers available in the market, including the use of an in vitro transcription method.

[0090] In certain embodiments, the nucleic acid nanostructure is assembled using a single stranded DNA molecule as an initial scaffold.

[0091] In one embodiment, the nucleic acid structure single stranded DNA scaffold strand comprising M13 phage DNA and a plurality of staple strands, wherein the plurality of staple strands hybridize to the DNA scaffold to form a cylindrical nanostructure having at least one open end which is covered by a lid.

[0092] In certain embodiments, the nucleic acid structure comprises both single stranded and double stranded regions.

[0093] As used herein, the term '‘staple strands” refers to short single stranded oligonucleotides of about 10 to about 40 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, or 40 nucleotides in length, wherein one end of the staple strand hybridizes with a region of the scaffold strand, and the second end of the staple strand hybridizes with another region of the scaffold strand, thereby “stapling” the two regions of the scaffold strand.

[0094] Exemplary staple sequences which can be used to generate a 3D nanostructure front the scaffold sequence of SEQ ID NO: 3 are provided in SEQ ID NOs: 5-257.

[0095] Exemplary staple sequences which can be used to generate a 3D structure from the lid sequence of SEQ ID NO: 4 are provided in SEQ ID Nos: 258-517.

[0096] In some embodiments, the scaffold strand defines the overall geometry of the nanostructure, for example a tube, cube, tetrahedron, capsule, or other polyhedral shape. The folding pattern of the scaffold strand is determined by the spatial arrangement of the staple strands, which direct local hybridization and define crossovers, helices, and junctions. The scaffold can therefore be designed computationally using existing DNA origami software or by manual sequence design to achieve a desired topology, wall thickness, or internal cavity volume. The open ends or defined apertures in such scaffolded structures provide natural sites for attachment of lid or covering elements that participate in shear-responsive behavior. In certain embodiments, the scaffold is not limited to a single continuous strand but may include two or more partially overlapping scaffold strands that co-assemble to form a larger composite structure. This configuration allows the construction of nanostructures larger than those that can be achieved with a single M13-derived strand and also facilitates the incorporation of distinct functional domains, for example, a force-responsive region, a targeting region, and a structural support region. The multiple scaffold strands may be linked covalently or through complementary hybridization, and may optionally include synthetic linkers, peptide connectors, or modified nucleotides.

[0097] The scaffold strand can be chemically modified to impart additional functionality or stability. Examples include incorporation of 5' or 3' amino or thiol groups for conjugation, biotinylation for avidin coupling, or phosphorothioate modifications for nuclease resistance. In some embodiments, selected bases along the scaffold strand are replaced with non-natural nucleotides, such as 2'-O-methyl RNA, peptide nucleic acid (PNA), or locked nucleic acid (LNA) residues, to modulate flexibility and hybridization strength. These modifications can be used to fine-tune the mechanical rigidity of the scaffold or to introduce localized responsive regions that contribute to the overall activation of the shear-pressure sensor.

[0098] In other embodiments, the scaffold comprises both nucleic acid and non-nucleic-acid segments, forming a hybrid backbone that integrates mechanical support from proteins, peptides, lipids, or polymers. For example, a central portion of the scaffold may be composed of a biopolymer filament, such as collagen or actin, to increase tensile strength, while peripheral regions remain nucleic-acid-based to enable programmable folding through staple hybridization. Such hybrid scaffolds extend the durability of the nanostructure and may improve stability in physiological environments where purely nucleic acid structures would be prone to degradation.

[0099] The scaffold strand may also include predetermined weak points or responsive sequences that facilitate controlled rupture or rearrangement upon activation. For instance, inclusion of short doublestranded domains with defined melting forces allows the structure to undergo selective strand separation under a specified shear threshold. Rupture of these sites can lead to partial or complete disassembly of the nanostructure, allowing rapid release of the encapsulated agent. The number, length, and placement of such responsive domains can be chosen to modulate the release profile and degree of structural opening.

[0100] Assembly of the scaffold and staple strands can be achieved by gradual annealing, for example heating the mixture to 90 °C and cooling slowly to 20 °C over several hours, or by isothermal folding using magnesium-containing buffers. The folding process may be carried out in aqueous buffers containing 1–20 mM MgCl₂ or other divalent cations to promote correct hybridization. Following assembly, the nanostructures can be purified by gel electrophoresis, filtration, ultracentrifugation, or size-exclusion chromatography to remove excess staple strands and to obtain monodisperse products.

[0101] In certain embodiments, the scaffold strand is extended with overhangs or connector domains that permit attachment of auxiliary modules, such as springs, hinges, targeting ligands, or fluorescent markers. These connector domains can be complementary to short bridging oligonucleotides that serve to integrate the mechanical or sensing components with the primary scaffold. In embodiments where the shear-pressure sensor forms part of the scaffold itself, the responsive double-stranded region may be incorporated directly into the scaffold backbone, such that rupture of the scaffold under shear stress corresponds to activation of the sensor.

[0102] The DNA scaffold may further serve as an anchoring platform for additional molecules or nanoparticles. For example, gold nanoparticles, quantum dots, or magnetic beads can be attached to specific staple sites to provide optical or magnetic responsiveness. Incorporation of such components may enhance the mechanical sensitivity of the nanostructure or allow external monitoring of its deformation under shear forces.

[0103] The overall dimensions of the scaffolded nanostructure can be adjusted by varying the number of helical turns, the spacing between crossovers, or the total length of the scaffold strand. In one embodiment, the resulting structure has an overall diameter between about 10 nm and about 1 µm, and an internal cavity volume sufficient to contain one or more molecules of the active agent. Larger scaffolds can be achieved by concatenating or interlinking multiple DNA origami units through sticky ends or bridging staples, thereby forming modular assemblies that respond cooperatively to mechanical stress.

[0104] In still further embodiments, scaffold strands derived from RNA or from hybrid DNA-RNA sequences are employed. RNA scaffolds may offer enhanced flexibility and can be generated in large quantities by in vitro transcription, followed by annealing with complementary DNA staples. The use of RNA or hybrid scaffolds also enables direct coupling with ribozymes or aptamer domains that provide additional functional responsiveness.

[0105] Through selection of scaffold composition, length, and folding pattern, the mechanical and geometric properties of the nanostructure can be precisely controlled. The scaffold thus forms the fundamental framework of the nucleic acid nanostructure, defining its size, shape, and mechanical behaviour, and serving as the structural basis for integration of shear-responsive, targeting, or releasecontrolling elements.

[0106] In certain embodiments, a targeting moiety or immobilizing agent is associated with the nanostructure to promote localization or retention at a selected site, such as a vascular lesion, thrombus, or diagnostic surface. The targeting moiety may be covalently or non-covalently attached to any portion of the nanostructure, including the outer surface of the covering element, the carrier portion, or directly to the nucleic acid scaffold itself. The targeting moiety may comprise, for example, an antibody or antibody fragment, a peptide ligand, an aptamer, a nucleic acid sequence, a receptorbinding protein, a carbohydrate, a lipid, or a small molecule with specific affinity for a cellular or extracellular target.

[0107] In some embodiments, the targeting moiety binds directly to the scaffold strand or to exposed regions of the scaffold without the use of separate targeting or connector strands. Direct attachment can be achieved through chemical modification of the scaffold, such as the introduction of reactive functional groups (for example, amino, thiol, azide, or alkyne residues) at predetermined positions along the scaffold backbone. These groups allow site-specific conjugation of the targeting moiety via amide coupling, thiol-maleimide linkage, or click chemistry. In other embodiments, direct binding occurs through affinity interactions between the targeting moiety and naturally occurring features of the scaffold, such as base-specific hydrogen bonding, electrostatic attraction, or hydrophobic stacking. For instance, certain peptides or proteins that recognize DNA or RNA sequences can be engineered to bind directly to exposed scaffold regions without the need for an intermediary oligonucleotide.

[0108] Direct attachment of the targeting moiety to the scaffold provides a simplified and robust architecture that reduces the number of auxiliary strands required for assembly. This configuration minimizes potential interference with the mechanical responsiveness of the nanostructure and allows more predictable positioning of the targeting domain relative to the scaffold surface. The density and spatial distribution of the directly attached targeting moieties can be controlled by the number and placement of modification sites on the scaffold strand, enabling precise tuning of binding avidity and multivalency.

[0109] In alternative embodiments, targeting moieties are attached indirectly via bridging or “targeting” strands that hybridize to complementary regions of the scaffold or of the covering element. This approach permits reversible attachment or exchange of targeting ligands, and allows modular adaptation of the nanostructure to different biological targets. Both direct and indirect attachment strategies may be employed within a single nanostructure, providing regions of stable anchoring and regions of dynamic, replaceable targeting.

[0110] The targeting moiety may recognize a variety of biological structures, such as cell-surface receptors, extracellular matrix proteins, membrane lipids, or clot-associated fibrin. For example, antibodies or peptides that bind to integrins, selectins, collagen, or fibrin may be used to guide the nanostructure to regions of vascular injury or thrombosis. In diagnostic applications, nucleic-acid aptamers or small molecules that recognize biomarkers can be directly linked to the scaffold to enable selective detection of circulating analytes. Exemplary molecules which can be targeted include those that are upregulated in stenotic regions. These include endothelial cell adhesion molecules such as VCAM-1, ICAM-1 and E-selectin, agents that bind integrin (e.g. RGD peptides).

[0111] In certain embodiments, platelet glycoprotein lb (GPIb), also known as the von Willebrand factor receptor or CD42b is targeted. GPIb is a transmembrane glycoprotein expressed on the surface of platelets and megakaryocytes, and it plays a critical role in the initial adhesion of platelets to damaged vascular endothelium. The binding of GPIb to von Willebrand factor (vWF) occurs predominantly under conditions of elevated shear stress, such as those found in arterial circulation or at sites of vascular stenosis.

[0112] Targeting the nanostructure to GPIb therefore provides dual functional advantage: first, it localizes the nanostructure to regions of platelet accumulation and thrombus formation; second, these are precisely the microenvironments characterized by high shear forces that can activate the shearpressure sensor. The combination of shear-responsive activation and GPIb-mediated binding thus produces site- specific, mechanically triggered release of the encapsulated therapeutic or diagnostic agent.

[0113] The GPIb-targeting moiety may be selected from peptides, antibody fragments, aptamers, or small molecules known to interact with the extracellular domain of GPIb. For example, peptides derived from the Al domain of von Willebrand factor or engineered antibodies against GPIba may be used. Such moieties can be conjugated directly to the scaffold or to the outer surface of the nanostructure as described herein, using either covalent coupling or affinity-based attachment.

[0114] The targeting moieties may be IgG antibodies, F(ab) and scFv fragments which can be linked to the DNA origami. These entities can be used in order to bind the nanostructures to T-cells [Klaus F. Wagenbauer et al., Programmable multispecific DNA-origami-based T-cell engagers, Nature Nanotechnology 18, 1319-1326 (2023)]

[0115] In certain embodiments, the direct attachment of targeting moieties to the scaffold enhances mechanical coupling between the biological target and the nanostructure, thereby influencing the local shear environment experienced by the device. Binding to a stationary surface, such as a thrombus or vessel wall, may increase local fluid shear over the nanostructure and facilitate activation of the shearpressure sensor, thereby producing a self-triggered, site- specific release of the active agent.

[0116] The targeting moiety or immobilizing agent may be a protein, a nucleic acid or an aptamer. The term “shear-pressure sensor” (also referred to herein as a “force or pressure sensor”) refers to an element, portion, or domain of the nanostructure configured to respond to shear-induced mechanical stress exerted by a surrounding fluid or by deformation of the nanostructure itself. When exposed to shear stress or shear-derived pressure gradients, the sensor undergoes a conformational or positional change that triggers partial or complete release of the encapsulated active agent.

[0117] The shear-pressure sensor typically comprises one or more of the following:

[0118] a protrusion that transduces tangential flow forces into mechanical strain;

[0119] a spring element that elongates in response to shear-derived tension; and / or

[0120] a hinge connecting element that undergoes bending or angular motion under shear stress Activation of the shear-pressure sensor occurs when the local shear stress (e.g., from blood flow, microvascular constriction, or fluid acceleration) exceeds a predetermined threshold, typically a pressure that is above about 100 dyne / cm2or force that is larger than 3 pico-Newton. Upon activation, the protrusion is displaced to expose an opening in the carrier portion, thereby allowing the release of the active agent.

[0121] In one embodiment, the force or pressure sensor comprises a double stranded DNA (e.g. between 2-200 nucleotides, or 2-20 nucleotides). The double stranded DNA remains intact up to a threshold force (e.g. shear force) and above that force, the double stranded DNA opens (e.g. unravels).

[0122] The term "protrusion" refers to a structural feature of the nanostructure that extends outwardly from a surface or plane of the nanostructure body. A protrusion may be integral with, or attached to, the surrounding material, and may have any suitable geometry including rod-like, dome-like, pillarlike, hook-like, or flap-like configurations.

[0123] In certain embodiments, the protrusion itself constitutes the covering or lid element of the nanostructure, extending over an opening that leads to an internal hollow. In this configuration, the protrusion both covers and protrudes over the hollow when the shear-pressure sensor is in a nonactivated state, and moves or flexes under shear stress to at least partially uncover the opening, thereby allowing release of the encapsulated active agent.

[0124] In the context of a shear-pressure sensor, the protrusion functions as a mechanotransducing element that converts external shear forces acting tangentially on the nanostructure into localized mechanical deformation of another component such as a spring or hinge connecting element. The protrusion may thereby serve as the initiating feature that senses the applied shear stress and triggers extension or bending of a spring element, resulting in opening of the covering element and release of the active agent.

[0125] Protrusions may be fabricated from nucleic acids, proteins, lipids, synthetic polymers, metallic or inorganic nanorods, or combinations thereof. The protrusion may be rigid or flexible, may vary in length (for example, 1-500 nm), and may be oriented normal or oblique to the nanostructure surface. In some embodiments, the protrusion itself carries a sensing or binding moiety that enhances responsiveness or specificity toward a target analyte or shear environment.

[0126] The term “spring element” refers to an elastically deformable connector that links two portions of the nanostructure (for example, a carrier portion and a covering element. The spring element is designed to deform elastically under a mechanical force, for example, in response to shear-induced stress, and to return substantially to its original length and configuration once the applied force is removed or reduced. The elongation of the spring under stress causes transient displacement of the covering element and permits the release of the encapsulated active agent.

[0127] Design of DNA springs is disclosed in Gerland et al. who measured the extension of RNA in response to a force that is pulling it in both ends [U. Gerland, R. Bundschuh and T. Hwa, Force-Induced Denaturation of RNA, Biophysical Journal 81, 1324-1332 (2001). DNA was also measured in a similar manner [J. M. Huguet, C. V. Bizarro, N. Forns, S. B. Smith, C. Bustamante and F. Ritort, Single-molecule derivation of salt dependent base-pair free energies in DNA, Proceedings of the National Academy of Sciences USA 107, 15431-15436 (2010)], the contents of which are incorporated herein.

[0128] The spring element may be composed of nucleic acids, non-nucleic-acid materials, or a combination thereof. In certain embodiments, the spring comprises a nucleic acid strand or hybrid strand that includes regions of single-stranded DNA, double-stranded DNA, RNA, or combinations of these. The presence of both single-stranded and double- stranded regions allows fine control of the spring’s stiffness and elasticity. Single- stranded nucleic acid segments impart flexibility and a high degree of stretchability, whereas double-stranded segments confer structural integrity and defined mechanical resistance.

[0129] The total contour length of the spring element may vary depending on the desired mechanical response. In various embodiments, the spring has a length between about 2 nanometres and about 50 micrometres, for example between about 5 nanometres and about 10 micrometres. Shorter springs are suitable for highly compact nanostructures, whereas longer springs are appropriate for structures intended to deform over larger distances or to respond to lower shear thresholds. The elastic force generated by the spring may range from approximately 0.1 femtonewton to 10 micronewtons, for example between 1 femtonewton and 1 micronewton, depending on its length, composition, and degree of hydration.

[0130] The spring element extends in length by at least 1.1, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold when force or pressure is above said predetermined level.

[0131] According to an exemplary embodiment, the spring comprises the sequences SEQ ID NOs: 518, 519 and 520. The relative proportions of single- stranded and double- stranded nucleic acid within the spring can be selected to achieve the desired elastic behavior. In some embodiments, the ratio of the length of the single-stranded portion to that of the double- stranded portion is between about 0.001 and about 1000, more typically between 0.001 and about 100, more typically between about 0.01 and about 100. Ratios near unity yield balanced mechanical properties, while ratios substantially above or below unity favor flexibility or rigidity, respectively. This ratio may be adjusted by design of the nucleic acid sequence, by introducing hairpin loops, mismatches, bulges, or other structural features, or by the controlled hybridization of complementary strands.

[0132] In one exemplary configuration, the spring element comprises a first and a second terminal region that are double- stranded, and a central region that is single-stranded. The double- stranded regions provide stable anchoring points for attachment to the carrier portion and the covering element, while the central single-stranded region serves as the flexible hinge that undergoes reversible extension. The central portion may comprise RNA or a mixture of RNA and DNA to adjust the stiffness and conformational responsiveness of the spring. RNA bases provide slightly increased flexibility and torsional mobility relative to DNA, which may be advantageous for sensors that respond to subtle shear gradients.

[0133] In other embodiments, the spring element may include alternating single-stranded and doublestranded segments along its length, thereby forming a repeating pattern of flexible and rigid domains. This configuration enables fine tuning of the elastic modulus and permits stepwise extension under gradually increasing mechanical load.

[0134] The spring element may also be formed from non-nucleic-acid materials having comparable elastic and conformational characteristics, such as peptide chains, protein filaments (for example actin, elastin, or collagen derivatives), lipid chains, or synthetic polymers including polyethylene glycol, polyacrylamide, or polyurethane. Hybrid springs incorporating both nucleic acid and non-nucleic-acid regions are also contemplated, for instance by covalent or non-covalent linkage of a nucleic acid domain to a polymeric or peptide domain.

[0135] The spring element can be attached to the carrier portion and to the covering element through complementary base pairing, covalent linkage, or affinity interactions such as biotin-streptavidin, thiol-gold, or click-chemistry reactions. The attachment points may be positioned on inner surfaces of the respective elements so that the spring is contained within the nanostructure cavity, or on outer surfaces if the deformation occurs externally. The positioning may be such that one end of the spring is located on a distal side of the covering element and the other on a proximal side, providing directional tension and controlled opening under shear stress. In operation, when the nanostructure is exposed to fluid shear forces above a predetermined level, the spring element elongates in proportion to the applied stress. The extension of the spring displaces the covering element away from the opening of the carrier portion, thereby creating a passage for the release of the active agent. When the shear force decreases below the threshold level, the inherent elasticity of the spring causes it to recoil, pulling the covering element back toward the opening. Depending on the specific design, this recoil may result in resealing of the opening or may lead to partial closure that still permits residual diffusion of the active agent.

[0136] The spring element may optionally be chemically or physically modified to tailor its mechanical or environmental responsiveness. Examples include crosslinking to enhance rigidity, partial methylation or phosphorothioate substitution to improve resistance to nuclease degradation, or the incorporation of responsive moieties such as photo- switchable bases or pH- sensitive linkers. These modifications allow external control over the activation behavior in addition to the shear-based trigger.

[0137] The described design thus provides a versatile and tunable spring component that governs the mechanical responsiveness of the nanostructure. By varying the length, composition, and ratio of single- stranded to double-stranded regions, the spring can be engineered to deform at specific shear thresholds and to provide either reversible or irreversible opening of the nanostructure. The modular nature of the spring element allows its integration into a wide range of nanoscale devices intended for controlled release, sensing, or mechanical actuation.

[0138] The hinge connecting element provides a permanent connection between two structural components of the nanostructure, such as the carrier portion and the covering or lid element. The hinge serves primarily as a mechanical link or pivot point, maintaining connectivity during activation of the shear-pressure sensor and during movement of the covering element relative to the carrier portion.

[0139] The hinge may be present in combination with a spring element or may function independently as the sole connecting feature between the covering element and the carrier portion. In embodiments that include both elements, the hinge provides a stable attachment point while the spring undergoes elastic deformation in response to shear stress. In alternative embodiments, the hinge alone provides the mechanical linkage; in such cases, the covering element may pivot, rotate, or translate relative to the carrier portion without requiring elastic extension of any component.

[0140] The hinge connecting element need not be flexible. In certain embodiments, it is rigid or semirigid, providing a fixed axis or limited range of motion that constrains the direction and amplitude of movement of the covering element. Such rigidity may be advantageous where precise alignment of the opening and covering element is required, or where release is intended to occur only under abrupt or high-magnitude shear events. In other embodiments, the hinge may permit limited bending or rotation, for example where partial flexibility is desired to accommodate the motion of the covering element during opening. The extent of movement can be defined by the material properties of the hinge, its geometry, or the nature of its attachment to the adjoining elements.

[0141] The hinge connecting element can be fabricated from a wide variety of materials, including nucleic acids, proteins, peptides, lipids, synthetic polymers, metallic nanowires, or combinations thereof. When formed of nucleic acids, the hinge may comprise one or more double- stranded segments, optionally joined to single-stranded linkers or junctions that provide rotational freedom. In a non-nucleic-acid embodiment, the hinge may consist of a short peptide or polymer chain, a rigid rod, or a planar plate-like connector joined to both the covering and carrier portions by covalent or non-covalent bonds.

[0142] The length of the hinge may range from about 1 nanometer to about 50 micrometers, more typically between about 5 and 1000 nanometers, depending on the overall dimensions of the nanostructure. The hinge may have a defined cross-sectional area, thickness, or width selected to achieve a desired mechanical response. For example, a thin plate-like hinge may allow limited bending, while a thicker or shorter hinge may remain effectively rigid under the same loading conditions.

[0143] The hinge connecting element may be permanently attached to the covering and carrier portions through base-pairing, chemical conjugation, or other stable linkages such as click chemistry, disulphide bridges, or metal coordination bonds. The connection is preferably permanent throughout activation of the nanostructure, ensuring that the covering element remains tethered and does not detach completely upon exposure to shear stress.

[0144] In certain embodiments, the hinge is positioned on a proximal side of the covering element, opposite a spring element that may be located on a distal side. This configuration allows the covering element to pivot or tilt around the hinge axis while being pulled or released by the spring. In embodiments that lack a spring, the hinge alone may define the motion path of the covering element; for example, a single rigid hinge may allow the lid to swing open when an external force acts on the protrusion or on the covering element itself.

[0145] The hinge can also serve a purely structural role without providing significant motion. For instance, a rigid hinge may maintain the covering element in a fixed orientation relative to the carrier portion while other components, such as a deformable wall or shear-responsive membrane, mediate the release of the active agent. In this context, the hinge acts as a reinforcement or scaffold rather than a flexible connector. The material of the hinge may be selected based on the intended application environment. Nucleic-acid hinges may be designed to self-assemble through complementary hybridization or multiarm junctions, while protein-based or polymeric hinges may be formed by molecular folding, templating, or microfabrication. Metal-based hinges, for example those containing gold or titanium nanowires, may provide added strength and conductivity, enabling potential integration with electromechanical sensing modalities.

[0146] The mechanical behavior of the hinge can be characterized by its torsional stiffness, rotational freedom, or bending modulus. In embodiments where the hinge is designed to move, the angular displacement during activation may range from about 1° to about 90°, preferably between about 5° and 45°. In embodiments where the hinge is intended to be rigid, the angular displacement may be less than about 1°, effectively maintaining a fixed geometry.

[0147] In use, when the nanostructure is subjected to shear stress or a shear-derived pressure gradient, the hinge connecting element maintains the physical linkage between the covering element and the carrier portion. If a spring is present, the hinge provides the pivot axis while the spring extends and retracts. If the hinge alone is present, the covering element moves about the hinge or around its fixed attachment point in response to the applied force. The result in both cases is the uncovering of the opening to allow release of the active agent, followed by either resealing or permanent opening depending on the structural configuration.

[0148] The hinge connecting element thus ensures mechanical integrity of the nanostructure under repeated or sustained exposure to mechanical forces while enabling controlled and directional movement of the covering element.

[0149] In some embodiments, the shear-pressure sensor is reversible (resealable), such that removal or reduction of the shear stress allows the spring element to recoil and restore closure of the covering element.

[0150] In other embodiments, activation results in irreversible (non-resealable) release.

[0151] According to a particular embodiment, the nanostructure is made up entirely of nucleic acids. In another embodiment, the nanostructure is a hybrid nanostructure comprising both nucleic acids and one or more non-nucleic acid elements (e.g., protein, lipid, filament, polysaccharides or synthetic polymer), which together provide structural and / or functional properties.

[0152] Typically, the hybrid nanostructure retains at least one nucleic acid element that functions as a structural or sensing component.

[0153] In one embodiment, the non-nucleic acid element serves as a functional element (e.g. a spring or hinge, as further described herein below). As mentioned, the nanostructures disclosed herein are designed to release a payload under different types of stress - including shear stress, elastic stress, pressure gradients and acceleration of velocity gradients.

[0154] As used herein, the term "shear stress" refers to the part of the stress (force / area) that is applied in parallel to the surface of a body. A fluid that flows in parallel to the surface of a body will apply a shear stress on it. Normal stress refers to the case where force is applied in perpendicular to the surface of a body. When fluid flows through a channel that its cross-section area is reduced at a certain point along the flow, the fluid velocity is increased, resulting in a velocity gradient along the fluid stream. The fluid velocity also increases as distance from the wall increases. The differences in fluid velocity, as indicated by the velocity gradient, result in a shear and normal stress being applied on cells and particles flowing in the fluid stream. The size of the stress is also a function of vessel radius, fluid viscosity and flow velocity, and changes in the flow cross section area.

[0155] As used herein, the term “elastic stress” refers to the internal restoring force generated within a deformable particle or within biological tissue when subjected to strain (i.e., deformation). Elastic stress arises from the stretching, compression, or bending of an elastic or viscoelastic material, such as a polymer shell or hydrogel matrix. Unlike shear stress, which is caused by fluid flow parallel to a surface, elastic stress results from a direct mechanical deformation of the particle structure itself. For instance, when a compressive force acts radially on a soft spherical capsule (such as during passage through a constricted vessel or under pulsatile pressure), the capsule may deform elastically and store mechanical energy. An elastically responsive particle may exploit this deformation to actuate a release event — such as by opening pores, breaking crosslinks, or mechanically expelling encapsulated material once the elastic strain exceeds a threshold (e.g., 1-20% deformation).

[0156] As used herein, the term “pressure gradient” refers to a difference in hydrostatic or hydrodynamic pressure across a defined spatial distance or across the particle itself. In a vessel, pressure gradients (AP) arise from flow restriction or occlusion, and are typically expressed as AP = Pi - P2, where Pi and P2 are pressures at two points along the flow path. In stenotic regions, local pressure drops can exceed 1-100 mmHg relative to normal segments. This is equal to a pressure of (0.13 - 13)X10-3pN / nm2.

[0157] Pressure gradient sensitivity is distinct from shear responsiveness because it depends on normal forces acting perpendicularly across the particle, rather than tangential forces along its surface.

[0158] As used herein, the term “acceleration or velocity gradient” refers to the rate of change of flow velocity either spatially or temporally within the fluid domain. Acceleration may be linear or rotational and can arise from sudden expansions, bifurcations, pulsatile flow, or turbulence.

[0159] Structures responsive to acceleration or velocity gradients may include internal components of differing densities or viscosities. When exposed to rapid changes in flow velocity, these components experience relative displacement, shear, or compression, which can be transduced into a release event - for example, by breaking an internal membrane, causing cavitation, or exposing a reactive surface.

[0160] As used herein, the term "shear stress conditions" refers to conditions under which a shearing stress is applied by a fluid. ’The shear stress generated by the flowing fluid can be transferred or applied to molecules, particles and aggregates that may be present in the flowing fluid. These shear stress conditions can occur in a fluid having generally laminar or turbulent flow characteristics. The size of the shear stress on a particle is also a function of its size and geometry,

[0161] Generally, in normal blood vessels the wall shear stress is well below 70 dyn / cm2(7 Pa) while at the stenosis site shear stress exceeds 70dyn / cm2(AM Malek, S. A. & S. Izumo " Hemodyamic shear stress and its role in atherosclerosis." JAMA, 1999, 282: 2035-2042). Accordingly, the shear stress under which the particle opens is 5 to 3000dyn / cm2. In some embodiments, the shear stress under which the particles described herein are opened is > 5dyn / cm2, > 6dyn / cm2, > 7dyn / cm2, > 8dyn / cm2, > 9dyn / cm2, > 10dyn / cm2, > 11 dyn / cm2, > 12dyn / cm2, > 13dyn / cm2, > 14dyn / cm2, > 15dyn / cm2> 20dyn / cm2, > 30dyn / cm2, > 40dyn / cm2, > 50dyn / cm2, > 60dyn / cm2, > 70dyn / cm2, or > SOdyn / cm2.

[0162] The particles described herein can release the drug by at least 5%, at least 10%. at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, orl00% under shear stress conditions (e.g., a stenosis site shear stress) as compared to a control shear condition (e.g., normal blood vessel shear stress).

[0163] Therapeutic and diagnostic agents which can be encapsulated in the nanostructures described herein include those disclosed in European Patent No. 2611466B1, the contents of which are incorporated herein by reference.

[0164] For example, in some embodiments of this and other aspects of the invention, the therapeutic agent is a thrombolytic agent. As used herein, the term "thrombolytic agent" refers to any agent capable of inducing reperfusion by dissolving, dislodging or otherwise breaking up a clot, e.g., by either dissolving a fibrin-platelet clot, or inhibiting the formation of such a clot. Reperfusion occurs when the clot is dissolved and blood flow is restored. Exemplary thrombolytic agents include, but are not limited to, tissue-type plasminogen activator (t-PA), streptokinase (SK), prourokinase, urokinase (uPA), alteplase (also known as Activase®, Genentech, Inc.), reteplase (also known as r-PA or retavase®, Centocor, Inc.), tenecteplase (also known as TNKTM, Genentech, Inc.), Streptase® (AstraZeneca, LP), lanoteplase (Bristol-Myers Squibb Company), monteplase (Eisai Company, Ltd,), saruplase (also known as r-scu-PA andrescupaseTM, Grunenthal GmbH, Corp.), staphylokinase, and anisoylated plasminogen- streptokinase activator complex (also known as APS AC, Anistreplase and Eminase®. SmithKline Beecham Corp.). Thrombolytic agents also include other genetically engineered plasminogen activators. The invention can additionally employ hybrids, physiologically active fragments or mutant forms of the above thrombolytic agents. The term "tissue-type plasminogen activator" as used herein is intended to include such hybrids, fragments and mutants, as well as both naturally derived and recombinantly derived tissue-type plasminogen activator.

[0165] The term "anticoagulant" as used herein, refer to any agent capable of prolonging the prothrombin and partial thromboplastin time tests and reducing the levels of prothrombin and factors VII, IX and X. Anticoagulants typically include cormarin derivatives and heparin as well as aspirin, which may also be referred to as an antiplatelet agent.

[0166] In some embodiments of this disclosure, the pharmaceutically active agent include those agents known in the art for treatment of inflammation or inflammation associated disorders, or infections. Exemplary anti-inflammatory agents include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs - such as aspirin, ibuprofen, or naproxen), coricosteroids (such as presnisone), anti- malarial medication (such as hydrochloroquine), methotrexrate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamise, mycophenolate, dexamethasone, rosiglitazone, prednisolone, corticosterone, budesonide, estrogen, estrodiol, sulfasalazine, fenfibrate, provastatin, simvastatin, proglitazone, acetylsalicylic acid, mycophenolic acid, mesalamine, hydroxyurea, and analogs, derivatives, prodrugs, and pharmaceutically acceptable salts thereof.

[0167] In some embodiments of this and other aspects of the invention, the pharmaceutically active agent is a vasodilator. A vasodilator can be selected from the group consisting of alpha-adrenoceptor antagonists (alpha-blockers), agiotensin converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), beta2-adrenoceptor agonists (p2-agonists), calcium-channel blockers (CCBs), centrally acting sympatholytics, direct acting vasodilators, endothelin receptor antagonists, ganglionic blockers, nitrodilators, phosphodiesterase inhibitors, potas slum-channel openers, renin inhibitors, and any combinations thereof. Exemplary vasodilator include, but are not limited to, prazosin, terazosin, doxazosin, trimazosin, phentolamine, phenoxybenzamine, benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, quinapril, ramipril, candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, valsartan, Epinephrine, Norepinephrine, Dopamine, Dobutamine, Isoproterenol, amlodipine, felodipine, isradipine, nicardipine, nifedipine, nimodipine, nitrendipine, clonidine, guanabenz, guanfacine, a-methyldopa, hydralazine, Bosentan. trimethaphan camsylate, isosorbide dinitrate, sosorbide mononitrate, nitroglycerin, erythrityl tetranitrate, pentaerythritol tetranitrate, sodium nitroprusside, milrinone, inamrinone (formerly amrinone), cilostazol, sildenafil, tadalafil, minoxidil, aliskiren, and analogs, derivatives, prodrugs, and pharmaceutically acceptable salts thereof.

[0168] In some embodiments, the vasoactive agent is a substance derived or extracted from a herbal source, selected from the group including ephedra sinica (ma huang), polygonum bistorta (bistort root), hamamelis virginiana. (witch hazel), hydrastis canadensis (goldenseal), lycopus virginicus (bugleweed), aspidosperma quebracho (quebracho bianco), cytisus scoparius (scotch broom), cypress and salts, isomers, analogs and derivatives thereof.

[0169] In some embodiments of this disclosure, the therapeutic agent is selected from the group consisting of aspirin, wafarin (coumadin), acenocoumarol, ancrod, anisindione, bromindione, clorindione, coumetarol, cyclocumarol, dextran, dextran sulfate sodium, dicumarol, diphenadione, ethyl biscoumacetate, ethylidene dicoumarol, fluindione, heparin, hirudin, lyapolate sodium, oxazidione, pentosan polysulfate, phenindione, phenprocoumon, phosvitin, picotamide, tioclomarol. dipyridamole (persantin), sulfinpyranone (anturane), ticlopidine (ticlid), tissue plasminogen activator (activase), plasmin, pro-urokinase, urokinase (abboklnase) streptokinase (streptase), and anistreplase / APSAC (eminase), and analogs, derivatives, prodrugs, and pharmaceutically acceptable salts thereof.

[0170] Alternatively, and / or additionally, the nanostructures described herein can comprise an imaging agent. As used herein, the tenn "imaging agent" refers to an element or functional group in a molecule that allows for the detection, imaging, and / or monitoring of the presence and / or progression of a condition(s), pathological disorder(s), and / or disease(s). The imaging agent may be an echogenic substance (either liquid or gas), non-nretallic isotope, an optical reporter, a boron neutron absorber, a paramagnetic metal ion, a ferromagnetic metal, a gamma-emitting radioisotope, a positron-emitting radioisotope, or an x-ray absorber.

[0171] Suitable optical reporters include, but are not limited to, fluorescent reporters and chemiluminescent groups. A wide variety of fluorescent reporter dyes are known in the art. Typically, the fluorophore is an aromatic or heteroaromatic compound and can be a pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine, salicylate, anthranilate, coumarin, fluorescein, rhodamine or other like compound. Suitable fluorescent reporters include xanthene dyes, such as fluorescein or rhodamine dyes, including, but not limited to, Alexa Fluor® dyes (InvhtrogenCorp.; Carlsbad, Calif), fluorescein, fluorescein isothiocyanate (FITC), Oregon GreenTM, rhodamine, Texas red, tetrarhodamine isothiocynate (TRITC), 5-carboxyfluoreseein (FAM), 2’7'-dimethoxy-4’5'-dichloro-6- carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N, N, N, N'- tetramethyl- 6 -carboxyrhodamine (TAMRA), 6-carboxy-X -rhodamine (ROX). Suitable fluorescent reporters also include the naphthylamine dyes that have an amino group in the alpha or beta position. For example, naphthylamino compounds include 1 -dimethylamino- naphthyl- 5-sulfonate, 1 -anilino- 8-naphthalene sulfonate, 2-p-toluidiny1-6-naphthalene sulfonate, and 5-(2’-ammoethyljaminonaphthalene- 1 -sulfonic acid (EDANS). Other fluorescent reporter dyes include coumarins, such as 3-phenyl-7-isocyanatocoumarin; acridines, such as 9-isothiocyanatoacridine and acridine orange; N-(p(2-benzoxazoly I (phenyl Jmaleimide; cyanines, such as Cy2, indodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5), indodicarbocyanine 5.5 (Cy5.5), 3-(-carboxy-pentyl)-3'ethyl-5,5:-dimethyloxacarbocyanine (CyA); 1 H,5H,11H, 15H-Xantheno[2,3,4-ij:5,6,7-i'j']diquinolizin-l 8- ium. 9-[2(or 4)-[[[6-[2,5-dioxo-l-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or 2)- sulfophenyl]-2,3, 6, 7,12,13,16,17octahydro-inner salt (TR or Texas Red); BODIPYTM dyes; benzoxadiazoles; stilbenes; pyrenes; and the like. Many suitable forms of these fluorescent compounds are available and can be used.

[0172] Examples of fluorescent proteins suitable for use as imaging agents include, but are not limited to, green fluorescent protein, red fluorescent protein (e.g., DsRed), yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, and variants thereof (see, e.g., U. S. Pat. Nos. 6,403, 374, 6,800,733, and 7,157,566 ). Specific examples of GFP variants include, but are not limited to. enhanced GFP (EGFP), destabilized EGFP, the GFP variants described in Doan et al. Mol. Microbiol, 55:1767-1781 (2005), the GFP variant described in Crameri et al, Nat. Biotechnol., 14:315319 (1996), the cerulean fluorescent proteins described in Rizzo et al, Nat. Biotechnol, 22:445 (2004) and Tsien, Annu. Rev. Biochem., 67:509 (1998), and the yellow fluorescent protein described in Nagal et al, Nat. Biotechnol., 20:87-90 (2002). DsRed variants are described in, e.g., Shaner et al, Nat. Biotechnol., 22:1567-1572 (2004), and include mStrawberry, mCherry, mOrange, mBanana, mHoneydew, and niTangerine. Additional DsRed variants are described in, e.g., Wang et al, Proc. Natl. Acad. Sci. U. S. A., 101: 16745-16749 (2004) and include mRaspberry and mPlum. Further examples of DsRed variants include mRFPmars described in Fischer et al. FEBS Lett., 577:227-232 ( 2004 ) and mRFPruby described in Fischer et al, FEBS Lett, 580:2495-2502 (2006). (Suitable echogenic gases include, but are not limited to, a sulfur hexafluoride or perfluorocarbon gas, such as perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluorocyclobutane, perfluropentane, or perfluorohexane.

[0173] Suitable non-metallic Isotopes include, but are not limited to, “C,i4C,i

[0174]

[0175] 3N,18F,i2JI,iz4I, and125I Suitable radioisotopes include, but are not limited to, "mTc,9

[0176]

[0177] 5Tc,r!!In,62Cu,6’Cu, Ga,68Ga, andi5jGd. Suitable paramagnetic metal ions include, but are not limited to, Gd(III), Dy(III), Fe(III), and Mn(II).

[0178] Suitable X-ray absorbers include, but are not limited to, Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd, La,. Au, Au, Yb, Dy, Cu, Rh, Ag, and Ir.

[0179] In some embodiments, the radionuclide is bound to a chelating agent or chelating agent-linker attached to the nanostructure. Suitable radionuclides for direct conjugation include, without limitation,18F,

[0180]

[0181] 12”I,lz5I,B1I, and mixtures thereof. Suitable radionuclides for use with a chelating agent include, without limitation.47Sc,&’Cu,67Cu,89Sr,86Y,87Y,90Y.3O5Rh,inAg,113In,il7mSn,i49Pm,353Sm,166Ho,177Lu,i86Re,l88Re,233At,2!2Bi, and mixtures thereof. Suitable chelating agents include, but are not limited to, DOTA, BAD, TETA. DTPA, EDTA, NTA, HDTA, their phosphonate analogs, and mixtures thereof. One of skill in the art will be familiar with methods for attaching radionuclides, chelating agents, and chelating agent-linkers to the nanoparticles.

[0182] In one embodiment, the nanostructure can be designed such that it can be used for the slow release of therapeutic or diagnostic agent.

[0183] As well as for drug delivery, the nanostructure may be used as a force-indicator. In one embodiment, force-indicator structure comprises at least two elements attached to each other, where there are two fluorochromes attached to the two elements, so that when the structure is closed, it emits a first signal and when the elements are separated from each other, the fluorescence color, or its intensity are emit a second signal. The two entities can be two fluorochromes, for fluorescence resonance energy transfer (FRET), or a fluorochrome and a quencher.

[0184] The nanostructures disclosed herein may be used to release agents at a site of high shear stress. Since many diseases are associated with high shear stress, it is contemplated that the nanostructures may be used to treat or prevent diseases associated with high shear stress.

[0185] Thus, according to another aspect of the invention, there is provided a method of treating or preventing a disease or disorder associated with high shear stress in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising a nucleic acid-based nanostructure having a pre-defined geometrical shape, wherein the nanostructure encapsulates at least one pharmaceutical agent useful for treating the disease, wherein the nanostructure comprises at least one shear pressure sensor, which, when activated, allows release of said pharmaceutical agent from said nanostructure, said at least one shear pressure sensor comprising a protrusion, thereby treating or preventing the disease.

[0186] According to a particular embodiment, the disease or disorder is associated with arterial stenosis and / or a stenotic lesion and / or an occlusive lesion. It will be appreciated for treatment of such diseases, the nanostructure comprises: a carrier portion which comprises an internal hollow and at least one opening to the hollow, and at least one active agent being comprised in the hollow; and

[0187] wherein said at least one shear pressure sensor comprises a covering element which covers and protrudes over said at least one opening in a non-stenosed blood vessel and which does not fully cover said at least one opening at a stenosed blood vessel.

[0188] As used herein, the term "stenosis" refers to narrowing or stricture of a hollow passage (e.g., a duct or canal) in the body. The term "vascular stenosis" refers to occlusion or narrowing of a canal or lumen of the circulatory system. Vascular stenosis often results from fatty deposit (as in the case of atherosclerosis), excessive migration and proliferation of vascular smooth muscle cells and endothelial cells, acute narrowing due to clot formation, or as a result of vascular malformation. As used herein, the term "vascular stenosis" includes occlusive lesions. Arteries are particularly susceptible to stenosis. The term “stenosis" as used herein specifically includes initial stenosis and restenosis. Typical examples of blockages within a canal or lumen include in situ or embollzed atheromatous material or plaques, aggregations of blood components, such as platelets, fibrin and / or other cellular components. In clots resulting from disease or injur)' or at the site of wound healing. Clot-forming conditions include thrombosis, embolisms and in an extreme case, abnormal coagulation states. Other vascular blockages include blockages resulting from an infection by a microorganism or macroorganism within the circulatory system, such as fungal or heartworm infections. Sickle cell disease also can result in vessel obstruction as a result of RBC sickling and stacking into structures that are larger than the lumen of the microvessel. Thus, during sickle cell crisis, RBC change shape / stiffness and can occlude blood vessel. This phenomena is also present during crisis stages of malaria.

[0189] The term “restenosis" refers to recurrence of stenosis after treatment of initial stenosis with apparent success, For example, "restenosis" in the context of vascular stenosis, refers to the reoccurrence of vascular stenosis after it has been treated with apparent success, e.g. by removal of fatty deposit by balloon angioplasty. One of the contributing factors in restenosis is intimal hyperplasia. The term "intimal hyperplasia", used interchangeably with "neointimal hyperplasia" and "neointimal formation", refers to thickening of the inner most layer of blood vessels, intimal, as a consequence of excessi ve proliferation and migration of vascular smooth muscle cells and endothelial cells. The various changes taking place during restenosis are often collectively referred to as "vascular wall remodeling. "

[0190] The terms "balloon angioplasty" and "percutaneous transluminal coronary angioplasty" (PTCA) are often used interchangeably, and refer to a non-surgical catheter-based treatment for removal of plaque from the coronary artery. Stenosis or restenosis often lead to hypertension as a result of increased resistance to blood flow.

[0191] The term "hypertension" refers to abnormally high blood pressure, i.e. beyond the upper value of the normal range.

[0192] Some exemplary causes of stenosis and / or stenotic lesion include, but are not limited to, trauma or injury, atherosclerosis, birth defects, diabetes, iatrogenic, infection, inflammation, ischemia, neoplasm, vasospasm, coronary.' vasospasm, Raynaud’s phenomenon, stroke, blood clotting, Moyamoya disease, Takayasu's disease, polyarteritis nodosa, disseminated lupus erythematous, rheumatoid arthritis, tumors of the spine, Paget's disease of bone, fluorosis, extracorporeal devices (e.g., hemodialysis, blood pumps, etc.), thrombotic and / or embolic disorders, Sickle Cell Disease, and any combinations thereof.

[0193] As used herein, the term "thrombotic and / or embolic disorders" means acute or chronic pathological states or conditions resulting from occlusion or partial occlusion of a blood vessel due to thrombus or embolus. Similarly, the term "thrombotic or embolic occlusion" means occlusion or partial occlusion of a blood vessel due to thrombus or embolus. Examples of thrombotic and embolic disorders include, but are not limited to cerebral thrombotic and embolic disorders such as cerebral infarct (stroke), transient ischemic attack and vascular dementia; thrombotic and embolic disorders of the heart such as myocardial infarct, acute coronary syndrome, unstable angina and ischemic sudden death: pulmonary embolism; pulmonary or renal infarcts, peripheral circulatory disorders and deep vein thrombosis.

[0194] In some embodiments of this and other aspects of the invention, stenosis or stenotic lesion is selected from the group consisting of intermittent claudication (peripheral artery stenosis), angina (coronary artery stenosis), carotid artery stenosis (leads to strokes and transient ischaemic episodes), aortic stenosis, buttonhole stenosis, calcific nodular stenosis, coronary ostial stenosis, double aortic stenosis, fish-mouth mitral stenosis, idiopathic hypertrophic subaortic stenosis, infundibular stenosis, mitral stenosis, muscular subaortic stenosis, pulmonary stenosis, subaortic stenosis, subvalvar stenosis, supravalvar stenosis, tricuspid stenosis, renal artery stenosis, pyloric stenosis (gastric outflow obstruction), obstructive jaundice (biliary tract stenosis), bowel obstruction, phimosis, hydrocephalus, stenosing tenosynovitis, spinal stenosis, subglottic stenosis (SGS), and any combinations thereof.

[0195] As used herein the term “about” refers to ± 10 %.

[0196] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0197] The term “consisting of’ means “including and limited to”. The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0198] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0199] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0200] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0201] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0202] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0203] When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0204] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0205] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0206] EXAMPLES

[0207] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0208] EXAMPLE 1

[0209] Creating and Folding DNA origami structures:

[0210] A. Sequence Design

[0211] The design of the folded scaffold which will serve as the backbone of the structure ensures the functionality of the structure. It is based on the understanding of the rules that govern the structure of DNA strands that are connected to each other to form double stranded DNA, and the geometry that results from the internal forces applied by the single strands on each other. Initially, a scaffold is selected, which is a long and continuous single-strand DNA molecule. Selection of the scaffold length (in number of nucleotides) requires to start with an initial folding design according to the required structure geometry. Ideally - the structure uses the whole scaffold, with no leftover nucleotides. Scaffolds can be purchased, as an example, from different companies, such as Tilibit nanosystems (Munich, Germany). Scaffolds are usually extracted from viruses, which single-strand plasmid is their native genomic code.

[0212] A typical staple can have a size of 32 nucleotides, but it can also have sizes in between 16-64 nucleotides. When designing the structure, normally a size of 32 nucleotides is used, except for edges, special points in the structure and other areas where a different size is selected. Complementary short DNA strands, called staple strands, are then designed to bind to specific regions along the scaffold strand, in order to create and hold the designed folds together. These staple strands act like molecular staples, guiding the scaffold into the desired shape. The design can be done manually, or make use of developed software tools such as caDNAno or caDNAnoSQ. After the design is ready, the exact sequence of all the staples is defined. These staples have to be synthesized, for example, by following chemical synthesis according to known protocols. It can also be ordered from different companies such as Integrated DNA Technologies (IDT). Once the scaffold and staples are available according to the design, the structures can be fabricated.

[0213] B. Mixing and Annealing

[0214] The scaffold and staples are mixed together in a tube under controlled conditions, including temperature, pH and salt concentration. Also, one should choose adequate ratio between the number of staples and scaffold in the solution. Normally, the concentration of staples should be 5-10 times higher than the scaffold’s concentration. This mixture is then heated and gradually cooled in a PCR machine that allows to control the temperature as a function of time, this process is known as annealing. During annealing, the staple strands bind to the scaffold strand in a specific order, forming the desired shape.

[0215] C. Verification and Characterization

[0216] After the annealing process, the sample should be purified from excess staples that are leftover in the solution. Then, the resulting DNA origami structures must be carefully verified and characterized. Gel electrophoresis can be used for verifying the size of the nano structure. By using atomic force microscopy (AFM), the actual shape and dimension of the structure can be verified. Electron microscopy can also be used instead of AFM. One can also use fluorescence labelling to visualize the structures under a microscope, which is valuable in some cases.

[0217] D. Functionalization

[0218] To make DNA origami structures useful for various applications, they can also be functionalized by attaching other molecules, such as nanoparticles, proteins, or drugs to specific sites on the structure. This functionalization should be designed in advance, for example by designing some of the staples so that only part of it is attached to a required site on the structure, while the other part is loosen. The loosen edge can be used for attaching either a protein, fluorochrome or any other entity.

[0219] E. Particle assembly

[0220] Finally, the structure should be assembled by connecting the different parts and filling the hollow volume with the drug. This can be done by immersing the particles in a saturated solution of the drug and maintain the relevant environmental conditions for the particle to assemble, including temperature, pH level and ratio concentration. EXAMPLE 2

[0221] Design and characterization of the capsule

[0222] The boxes and lids were initially designed using caDNAno (Douglas, S. M. et al. Nucleic Acids Res. 37, 5001-5006 (2009)) and their shape and curvature were examined using SNUPI software (Lee et al., ACS Nano 15, 1002-1015 (2021)) (Figure 2A). The structures were subsequently refined through several iterations to achieve sufficiently flat lids, thereby ensuring full closure of the capsules. In DNA origami design, curvature often arises from intrinsic mechanical stress within the structure. This can be mitigated by selectively shortening a subset of staples at specific sites to relieve tension and improve planarity. The dimensions of the boxes were designed to be 32.5 X 32.5 X 50 nm3so that its volume can accommodate at least 10 drug molecules. If necessary, larger structures can be synthesized to increase payload capacity. The lids were designed with a rectangular geometry exceeding the capsule's cross-section area, enabling the shear force generated by blood flow to act over a broad surface. Specifically, the lid dimensions are 76 X 90 nm2.

[0223] To visualize the structures, atomic force microscopy (AFM) was used using peak-force tapping mode (see methods). Figure 2B, C shows AFM images of lids and Figure 2D, E shows image of boxes with lids. The lids are rather flat while the boxes are thicker, but its 3D structure cannot be discerned as they tend to adhere to the mica surface in the solution that contains MgCl2aimed for sticking the structures to the surface. The AFM data confirms the structure architecture and high yield of the structures which means that high concentration of structures can be synthesized for the application (Figure 2B, D). Figure 2F shows a special structure that was synthesized in order to demonstrate the size difference between the box and the lid, where the box size is shown by the holes designed in this structure.

[0224] To assess the three-dimensional shape and scale of the whole DOC structure, Cryo-TEM (Figures 3A-H) was used. A few different samples were prepared: 1. Only the boxes (Figures 3 A, B, C), 2. The full DNA origami capsule (DOC) (Figures 3D-G), and 3. The DOC without the spring where the lids are connected only along one edge of the capsule (Figure 3H).

[0225] The dimensions of the structures, as measured by AFM and TEM, closely match the design specifications predicted by caDNAno. Based on AFM measurements, the dimensions of the lids were found to be 90+4 nm long and 73+3 nm wide and the capsules were found to be 65+4 nm long and 53+1 nm wide. According to the measurements of four cryo-TEM tomogram, the lid dimensions are 91+8 nm long and 62+8 nm wide, and the capsule height is 62+2 nm, and its width is 36+1 nm. The complete structure (Figure 3D-G) also aligns well with the design in both shape and dimensions, resembling a hollow, square-like box sealed on both sides by rectangular lids. These observations further confirm the effectiveness of the hinges and springs in connecting the lids to the capsules and maintaining closure. Finally, the structures were analyzed using gel electrophoresis (Figure 31). The results confirm the successful formation of the designed structures and demonstrate a high yield of uniformly assembled capsules. Notably, the data also highlight the high efficiency of the hinges and springs connections, supporting the reliability of the overall assembly process.

[0226] Design of the spring

[0227] The ability of the springs to keep the capsules securely closed under normal blood flow, yet open in response to elevated shear forces within narrowed vessels, is critical for the intended drug delivery application. To estimate the order of magnitude of the shear forces acting on the DOCs while flowing through a stenotic artery compared to an unconstricted artery, the well-known dumbbell model was used, which consists of two spheres (radius a) separated by the box and a spring of length d. This model has been widely applied in molecular mechanics, including studies on Von Willebrand Factor, which is known to unfold under high shear conditions.

[0228] Under low Reynolds number conditions, the tensile forces produced by shear flow on the DOC near the vessel wall can be estimated using Stokes’ law: F = αμGa2, where F is the normal force, a is a force coefficient that depends on the particle, dumbbell geometry and orientation, is the fluid viscosity, G is the shear rate and a is the particle dimensions. Thus, for a DOC near the wall, the tensile force will be linearly dependent on the wall shear rate, which in stenotic vessels can be one to two orders of magnitude higher than in normal circulation. Assuming one side of the DOC is static near the wall and applying Stokes’ law to the lid, simplified as a sphere at a distance d from the wall, we obtain the drag force: F = 6πμ(Gd)a. For a rectangular structure that is perpendicular to the flow, the factor

[0229]

[0230] is replaced by 16 / zr. For normal blood (-45% hematocrit) with a = 50 nm and d = 75 nm, the estimated force in a stenotic artery with a wall shear rate of 104s-1is 3–4 picoNewton (pN), whereas in a normal human artery with a wall shear rate of 250 s-1, the force is -40 times smaller (0.08 pN). Additionally, elongational flow occurring away from the wall at the leading edge of the stenosis can also contribute to elevated tensile forces in DOCs flowing through narrowed arteries.

[0231] The spring in the present design consists of a long single-stranded DNA (ssDNA) segment, anchored to the box and lids by forming short dsDNA regions at each end. Using ssDNA offers two key advantages. First, its significantly shorter persistence length compared to dsDNA provides the flexibility needed to respond mechanically at the -50 nm scale, allowing substantial increase in its extension under pathological shear forces to facilitate release of the encapsulated molecules. In addition, ssDNA can form stem-loop structures that shorten its effective length and alter its elastic properties. This feature facilitates self-assembly of the spring, and ensures it is tensed even in the absence of flow, helping to prevent leakage of the encapsulated molecules due to flow fluctuations that are inherent even in physiological normal flow. To achieve this, the box, spring, and lids, which are synthesized separately, are combined and incubated under a limited thermal gradient. Initially, many of the stem-loop regions remain open, enabling the long ssDNA strand to hybridize efficiently to both the box and the lid. As the mixture cools, the stem-loops gradually re-form, reducing the effective length of the ssDNA and increasing its stiffness to the desired levels.

[0232] The spring’s mechanical properties can be tuned by several parameters, including the length and sequence of the ssDNA and the length of the flanking dsDNA segments at each end. The design of the ssDNA and dsDNA mechanical properties is based on its Worm-like Chain (WLC) forceextension dependence33:

[0233] F·Lp / kBT = (1 / 4)(1 - x / L0)-2- 1 / 4 + x / L0

[0234]

[0235] Here F is the force applied on the DNA, Lpis its persistence length of either the ssDNA or dsDNA, kBis Boltzmann constant, T is the temperature, LQ is the ssDNA or dsDNA nominal contour length, and x is the extension.

[0236] Based on initial calculations that take into account the required elastic properties of the spring and the DNA properties, a 2,049 nt ssDNA segment amplified from Lambda DNA was used. 25 nts at each end forms dsDNA for anchoring, leaving 1,999 nts ssDNA (Figure 4A). The full contour length of this ssDNA is approximately 1,199 nm (assuming 0.6 nm per nucleotide). However, the effective contour length is shorter due to the formation of stem- loop structures. These secondary structures can be predicted computationally using mfold, which suggests an ensemble of possible folding configurations. Based on these results, only 158+ 54 nt are left unpaired. Nevertheless, it is assumed that only a fraction of the possible stem loops adopts a closed conformation at the range of relevant forces for our device, a fraction that may be as small as 50%, and can be tested experimentally. An approximate calculation of the spring's average end-to-end distance yields a value of 37 nm, which is shorter than the 47 nm spacing between its anchoring points. This indicates that the spring is under constant tension, thereby ensuring that the capsule stays closed.

[0237] Measurements of the spring elastic properties

[0238] In order to directly assess the spring’s force-extension properties, single-molecule measurements were performed using a high-resolution dual-trap optical tweezers (OT) setup (Figure 4A). The construct consisted of the 2049 nt ssDNA spring flanked by two 2000 bp dsDNA handles. Each handle was modified at its free end with either double digoxigenin or biotin, enabling specific attachment to anti-digoxigenin and streptavidin-coated microspheres, respectively. A typical force-extension curve (Figure 4B, C, black trace) begins with a gradual increase in both force and extension, reflecting the stretching of the dsDNA handles and the initially unpaired ssDNA regions. This is followed by a series of abrupt force drops, each accompanied by an increase in extension. These discrete events occur repeatedly along the pulling trajectory and correspond to the sequential unfolding of stem-loop structures, whose position and size vary between experiments (Figure 4B, C). Once the ssDNA is fully unfolded, the force increases above 15 pN with further extension, consistent with the elastic response of a fully extended single-stranded construct flanked by the dsDNA handles. Upon reaching ~20 pN, the trap movement is reversed, reducing the trap distance at a constant rate to relax the applied force. This stretch-relax cycle is repeated multiple times for each molecule. Notably, subsequent stretching curves differ from the initial one (Figure 4C, black vs. purple), indicating the formation of different stem-loop structures during relaxation. After ~10 cycles, the structures no longer reform within the experimental timeframe, possibly due to trap-induced oxidative DNA damage.

[0239] The high-force, post-unfolding segment of each force-extension curve was modeled using three serially connected polymers (the fully unfolded ssDNA segment flanked by two dsDNA handles), with each component described by the Worm-Like Chain (WLC) model (Equation 1).

[0240] The fitting procedure included only two free parameters: an extension offset (to account for variability in bead size and attachment position) and the ssDNA persistence length, Lpss. All other parameters were held constant: the persistence length of dsDNA, Lpds = 50 nm; the contour lengths of dsDNA (0.34 nm / bp) and ssDNA (0.6 nm / nt). Examples of fitted curves are shown as red dashed lines in Figure 4B, C. The distribution of fitted Lpssvalues is presented in Figure 5A (left panel), with a mean value Lpss= 0.79 ± 0.08 nm. Next, using the extracted Lpssvalue, we fitted the initial preunfolding segment of the force-extension curve, which includes the dsDNA handles and the ssDNA segment with an unknown number of nucleotides, n, that are not engaged in stem-loop structures. In this fit, the only free parameter was the effective ssDNA contour length, which may vary between molecules due to differences in stem-loop structures formation. Two representative fits are shown as dashed green lines in Figure 4B, C, and the resulting distribution of L̄0is shown in the boxplot (Figure 5 A), demonstrating a significant degree of variability. Notably, these values of L̄0correspond to n = 328 ± 161 nt, suggesting that about half of the nucleotides in the loops predicted by mFold are in an unpaired configuration. Note that in some traces the force increases more gradually, without distinct steps. These likely correspond to the progressive unfolding of small stem-loop structures and were excluded from the analysis. The end-to-end distance distribution (Figure 5C) demonstrates the spring's ability to keep the lids sealed, as all values in the distribution are shorter than the 47 nm spacing between its anchoring points. Based on these results, it is now possible to evaluate the expected performance of the spring in the context of the full mechano-responsive DOC (Figure 5B). To address the measured variability in L̄0, we used equation 1 to calculate force-extension curves for two extreme cases L̄0= 120 nm (Figure 5B, red) and L̄0=250 nm (blue), spanning -70% of the measured springs (one standard deviation). At force values below 1 pN, which corresponds to up to 10 times the physiologically normal flow as calculated above, the two curves show an extension below 47 nm (the size of the capsule), ensuring the capsule remains closed. Given that the spring is partially stretched and constrained to an extension 47 nm in the closed DOC, this means that the spring will exert a tension of 1-2.5 pN opposing opening and preventing opening of the DOC by normal flow fluctuations. At forces exceeding 3 pN, which is typical of pathological flow in stenotic sites, the elastic response of ssDNA predicts an extension of 50-90 nm, indicating that the capsule will fully open under such conditions. Notably, the length of ssDNA, as well as the length of the flanking dsDNA, can be further adjusted according to the range of forces in the blood stream to fine-tune the capsule's performance under real physiological conditions.

[0241] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0242] It is the intent of the applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

Claims

1. WHAT IS CLAIMED IS:

1. A nucleic acid-based nanostructure having a pre-defined geometrical shape, wherein the nanostructure encapsulates at least one active agent, wherein the nanostructure comprises at least one force or pressure sensor, which, when activated, allows release of said active agent from said nanostructure.

2. A nucleic acid-based nanostructure having a pre-defined geometrical shape, wherein the nanostructure encapsulates at least one active agent, wherein the nanostructure comprises at least one force or pressure sensor, which, when activated, allows release of said active agent from said nanostructure, said at least one force or pressure sensor comprising a protrusion.

3. The nanostructure of claims 1 or 2, wherein said sensor is activated by at least one of a shear stress, an elastic stress, a pressure gradient or an acceleration / velocity gradient.

4. The nanostructure of any one of claims 1-3, wherein said sensor is activated by shear stress.

5. The nanostructure of any one of claims 1-4, wherein said at least one force or pressure sensor comprises at least two force or pressure sensors, each comprising a protrusion.

6. The nanostructure of any one of claims 2-5, wherein said protrusion extends outwardly from an external surface of the nanostructure by a distance of at least 1 nm and is configured to undergo a measurable deformation or displacement or rotation upon exposure to a mechanical stimulus.

7. The nanostructure of any one of claims 1-5, comprising at least two elements connected to each other to form a hollow.

8. The nanostructure of claim 7, wherein said sensor comprises double stranded DNA having a length between 2-200 nucleotides which connects a first element of said at least two elements to a second element of said at least two elements.

9. The nanostructure of any one of claims 1-8, comprising:a carrier portion which comprises an internal hollow and at least one opening to said hollow, said at least one active agent being comprised in said hollow; and11.wherein said at least one force or pressure sensor comprises a covering element which covers and protrudes over said at least one opening when said force or pressure sensor is non-activated, and which does not fully cover said at least one opening when said force or pressure sensor is activated so as to allow release of said active agent.

10. The nanostructure of claim 9, being resealable following activation of said force or pressure sensor.

11. The nanostructure of claim 9, being non-resealable following activation of said force or pressure sensor.

12. The nanostructure of claims 9 or 10, wherein said force or pressure sensor further comprises a spring element which permanently connects said covering element to said carrier portion, wherein said spring temporarily extends when force or pressure is above a predetermined level so as to allow at least partial uncovering of said covering element and release of said active agent.

13. The nanostructure of any one of claims 9-12, wherein said nanostructure further comprises a hinge connecting element which permanently connects said covering element to said carrier portion when said force or pressure is above said predetermined level.

14. The nanostructure of claim 13, wherein said spring element extends in length by at least 1.1 fold when force or pressure is above said predetermined level.

15. The nanostructure of claim 13, wherein said spring element comprises a length between 5-10000 nm and elastic and an elastic force between of 1 femto-Newton - 1 microNewton.

16. The nanostructure of claim 13, wherein said spring element extends in length when force or pressure is above said predetermined level to a greater extent than said hinge connecting element extends in length when said force or pressure is above said predetermined level.

17. The nanostructure of any one of claims 9-16, wherein said predetermined level is above about 100 dyne / cm2.

18. The nanostructure of claim 13, wherein said spring and / or said hinge connecting element are fabricated from nucleic acids.

19. The nanostructure of any one of claims 12-18, wherein said spring connecting element is fabricated from a material capable of elastic deformation and reversible conformational change, the material being other than nucleic acid.

20. The nanostructure of any one of claims 13-19, wherein one end of said spring and said hinge connecting element are attached to an inner surface of said covering element and wherein said spring and said hinge connecting element are positioned on a distal and proximal sides of said covering element.

21. The nanostructure of claim 20, wherein another end of said spring and said hinge connecting element and attached to an inner surface of said carrier portion.

22. The nanostructure of any one of claims 9-20, wherein said carrier portion comprises a second opening to said hollow and wherein the nanostructure comprises a second force or pressure sensor which comprises a second covering element.

23. The nanostructure of claim 22, wherein said second force or pressure sensor is identical to said first force or pressure sensor.

24. The nanostructure of claim 22, wherein said second force or pressure sensor is nonidentical to said first force or pressure sensor.

25. The nanostructure of claim 22, wherein said second force or pressure sensor comprises a second covering element which covers and protrudes over said second opening when said second force or pressure sensor is non-activated and which does not fully cover said second opening when said force or pressure sensor is activated.

26. The nanostructure of any one of claims 12-25, wherein said spring element comprises single stranded DNA, double stranded DNA or both.

27. The nanostructure of claim 26, wherein the ratio of the lengths of said single stranded DNA: said double stranded DNA is in between 0.001 - 100.

28. The nanostructure of claim 26, wherein a first end of said spring element comprises double stranded DNA and a second end of said spring element comprises double stranded DNA and a central portion of said spring element comprises single stranded RNA.

29. The nanostructure of any one of claims 1-28, wherein a targeting moiety or immobilizing agent is attached to an outer surface of said nanostructure.

30. The nanostructure of claim 29, wherein said targeting moiety or immobilizing agent is selected from the group consisting of a protein, a nucleic acid and an aptamer.

31. The nanostructure of any one of claims 1-30, wherein said active agent is a therapeutic agent and / or a diagnostic agent.

32. The nanostructure of claim 31, wherein said diagnostic agent is an imaging agent.

33. The nanostructure of claim 31 or 32, wherein said diagnostic agent is fluorescent or other detectable signal agent to be release in the blood and analyzed in a blood test.

34. The nanostructure of claim 31, wherein said therapeutic agent is selected from the group consisting of an antithrombotic agent, a thrombolytic agent and a vasodilator.

35. The nanostructure of claim 34, wherein said antithrombotic or thrombolytic agent is selected from the group consisting of anticoagulants, pro-coagulant antagonists, antiplatelet agents, thrombolytic agents, anti-thrombolytic agent antagonists, fibrinolytic enzymes, and any combinations thereof.

36. The nanostructure of claim 34, wherein said thrombolytic agent is selected from the group consisting of tissue-type plasminogen activator (t-PA), streptokinase (SK), prourokinase, urokinase (uPA), alteplase, reteplase, tenecteplase, Streptase®, lanoteplase, monteplase, saruplase, staphylokinase, and anisoylated plasminogen-streptokinase activator complex and any combinations thereof.

37. The nanostructure of any one of claims 1-36, comprised of DNA.

38. The nanostructure of any one of claims 1-36, comprised of RNA.

39. The nanostructure of any one of claims 1-36, comprised of a combination of DNA and RNA.

40. A method of releasing an agent from a nanostructure at a site of high shear stress comprising exposing the nucleic acid-based nanostructure of any one of claims 1-39 to a shear stress above a predetermined level, thereby releasing the agent.

41. The method of claim 40, wherein said releasing is affected in vivo.

42. The method of claim 40, wherein said releasing is affected in vitro.

43. The method of claim 40, wherein said nucleic acid-based nanostructure is immobilized to a solid surface.

44. A method of releasing a pharmaceutical agent from a carrier at a site of high force or stress comprising exposing a nucleic acid-based nanostructure which comprises said pharmaceutical agent, to an amount of shear stress greater than 100 dynes / cm2, thereby releasing the pharmaceutical agent.

45. The method of claim 44, wherein said nucleic acid-based nanostructure is the nanostructure of claim 1 and the pharmaceutical agent is the active agent.

46. A method of treating or preventing a disease or disorder associated with high shear stress in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising a nucleic acid-based nanostructure having a pre-defined geometrical shape, wherein the nanostructure encapsulates at least one pharmaceutical agent useful for treating the disease, wherein the nanostructure comprises at least one shear pressure sensor, which, when activated, allows release of said pharmaceutical agent from said nanostructure, thereby treating or preventing the disease.

47. The method of claim 46, wherein the disease or disorder is associated with arterial stenosis and / or a stenotic lesion and / or an occlusive lesion.

48. The method of claim 46, wherein said nanostructure comprises:47.a carrier portion which comprises an internal hollow and at least one opening to said hollow, said at least one active agent being comprised in said hollow; and48.wherein said at least one shear pressure sensor comprises a covering element which covers and protrudes over said at least one opening in a non-stenosed blood vessel and which does not fully cover said at least one opening at a stenosed blood vessel.

49. The method of any one of claims 46-48, wherein said pharmaceutical agent is selected from the group consisting of a vasodilator, a thrombolytic agent and antithrombotic agent.

50. The method of claim 49, wherein said thrombolytic agent or antithrombotic agent is selected from the group consisting of anticoagulants, pro-coagulant antagonists, antiplatelet agents, thrombolytic agents, anti-thrombolytic agent antagonists, fibrinolytic enzymes, and any combinations thereof.

51. The method of claim 49, wherein said thrombolytic agent is selected from the group consisting of tissue-type plasminogen activator (t-PA), streptokinase (SK), prourokinase, urokinase (uPA), alteplase, reteplase, tenecteplase, Streptase®, lanoteplase, monteplase, saruplase, staphylokinase, and anisoylated plasminogen-streptokinase activator complex and any combinations thereof.

52. The method of claim 47, wherein the stenosis, stenotic, or occlusive lesion is selected from the group consisting of intermittent claudication (peripheral artery stenosis), angina (coronary artery stenosis) or myocardial infarction, carotid artery stenosis, aortic stenosis, buttonhole stenosis, calcific nodular stenosis, coronary ostial stenosis, double aortic stenosis, fish-mouth mitral stenosis, idiopathic hypertrophic subaortic stenosis, infundibular stenosis, mitral stenosis, muscular subaortic stenosis, pulmonary stenosis, pulmonary embolism, pulmonary hypertension, subaortic stenosis, subvalvar stenosis, supravalvar stenosis, tricuspid stenosis, renal artery stenosis, vascular hypertension, sickle cell anemia, and any combinations thereof; and / or results from trauma or injury, atherosclerosis, birth defects, diabetes, iatrogenic, infection, inflammation, ischemia, neoplasm,vasospasm, coronary vasospasm, Raynaud's phenomenon, stroke, blood clotting, Moyamoya disease, Takayasu's disease, polyarteritis nodosa, disseminated lupus erythematous, rheumatoid arthritis, tumors of the spine, Paget's disease of bone, fluorosis, hemodialysis, and any combinations thereof.

53. The method of any one of claims 46-49, wherein said disease is atherothrombosis.

54. The method of any one of claims 46-51, wherein a targeting moiety or immobilizing moiety is attached to an outer surface of said nanostructure.

Images