Patent Application
20190240248
DNA molecules have been shown to be excellent platforms for the design and construction of mechanical molecular devices that sense, actuate and exert critical functions when exposed to external signals1. DNA-based robotics have been utilized as imaging probes2-4 and cargo delivery vehicles4-6 in cultured cells2, 5, multicellular organisms4 and insects7. However, robotic DNA machines serving as intelligent vehicles for in vivo targeting drug delivery and controlled release in mammals have not yet been described. In contrast to the strategies of killing tumor cells directly by cytotoxic anticancer drugs or by anti-angiogenic agents8, selective occlusion of tumor blood vessels, to deprive tumors of nutrients and oxygen and start an avalanche of tumor cell death, is an attractive strategy for combating cancer9-12. Vascular occlusion can exert its effects within hours following the rapid induction of thrombus formation in tumor vessels. This leads to much shorter treatment duration than many other therapies, and carries a decreased risk of resistance development. Moreover, vascular occlusion in tumors is a strategy that can be used for many types of cancer, since all solid tumor-feeding vessels are essentially the same. The coagulation protease, thrombin, regulates platelet aggregation by activating platelets and converting circulating fibrinogen to fibrin13, ultimately leading to obstructive thrombosis. Naked thrombin is short lived in the circulation and induces coagulation events indiscriminately, and thus has never been used as an injectable therapeutic vessel occluding agent in cancer treatment. A critical challenge for introducing thrombin as a potent antitumor therapeutic is the precise delivery of sufficient quantities of the active protease solely to tumor sites in a highly controlled manner to minimize its effects in healthy tissues.
Accordingly, safe and effective compositions and methods are needed to treat tumors.
As described herein, a DNA nanostructure nanorobot was constructed and its function as a molecular payload carrier has been demonstrated.
Certain embodiments of the invention provide a DNA nanostructure nanorobot comprising:
a single stranded DNA scaffold strand of about 5000 to 10,000 bases in length;
a plurality of staple strands of DNA, wherein each staple strand is about 20 to 40 bases in length, wherein each staple strand has a unique sequence and is hybridized to a specific position on the DNA scaffold strand, wherein the plurality of staple strands hybridized to the DNA scaffold form a sheet having a top surface and a bottom surface;
one or more fastener strands of DNA, wherein the one or more fastener strands of DNA is capable of fastening the sheet into an origami structure. As used herein, the term “origami structure” means a 3-dimentional structure. As used herein, a “fastener strand” is an oligonucleotide that operably links two strands of DNA to form an origami structure/shape. For example, a plurality of fastener strands can bind (“tie”) two edges of a rectangular DNA origami sheet to form a tube shape.
Certain embodiments of the invention provide DNA nanostructure nanorobot comprising:
a single stranded DNA scaffold strand comprising M13 phage DNA;
a plurality of staple strands 13-204 (as described herein) of DNA wherein the plurality of staple strands hybridized to the DNA scaffold to forms a rectangular sheet having a top surface, a bottom surface, and four corners;
at least six fastener strands of DNA, wherein each fastener strand of DNA is capable of fastening the rectangular sheet into a tube-shaped origami structure;
four DNA capture strands, wherein each capture strand is operably linked to a thrombin; and
at least four targeting strands, wherein each targeting strand is operably linked to an aptamer specific for nucleolin.
In certain embodimetns, the staple strands are selected from the following Staple strands pool (5′-3′):
In certain embodiments, the plurality of imaging strands comprise extended ssDNA sequences that hybridized to fluorescent dye-labeled ssDNA.
Certain embodiments of the invention provide a pharmaceutical composition comprising the DNA nanostructure nanorobot described herein.
Certain embodiments of the invention provide a method of treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of the DNA nanostructure nanorobot or pharmaceutical composition as described herein.
Certain embodiments of the invention provide a method of inhibiting tumor growth in a subject, comprising administering to the subject a therapeutically effective amount of the DNA nanostructure nanorobot or pharmaceutical composition as described herein.
Certain embodiments of the invention provide a use of the DNA nanostructure nanorobot or a composition as described herein for the manufacture of a medicament for inducing a tumor necrosis response in a subject (e.g., a mammal, such as a human).
Certain embodiments of the invention provide the DNA nanostructure nanorobot or a composition as described herein for the prophylactic or therapeutic treatment a disease or disorder.
Certain embodiments of the invention provide a kit comprising the DNA nanostructure nanorobot or a composition as described herein and instructions for administering the DNA nanostructure nanorobot/composition to a subject to induce an immune response or to treat a disease or disorder.
The invention also provides processes disclosed herein that are useful for preparing a DNA nanostructure nanorobot described herein.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
On the microthrombosis-related risk of the DNA nanorobot, it should be also noted that, while tumor blood vessels are mainly capillary-sized vessels which are easily occluded by microthrombi, in normal vessels even if a few microthrombi break off they will be quickly cleared in the bloodstream as reported previously [5, 6]. The low equivalent concentration of thrombin used in the nanorobot-Th therapy further reduced the potential risk of undesirable microthrombi causing damage to normal tissues.
The following formula was used to perform dose conversion between mice and pigs [7,8]: Dp=Dm×(Kmm/Kmp). Where Dp is the dose injected into pigs, Dm is the dose used in mice, Kmm is the Dose in mg/kg to Dose in mg/m2 conversion factor of mice and Kmp is the Dose in mg/kg to Dose in mg/m2 conversion factor of the pigs.
Robotic molecular systems have great potential as intelligent vehicles to enable the delivery of various potent molecules, which otherwise never could be used as therapeutics due to numerous limitations. Yet, achieving in vivo, precise molecular-level, and on-demand targeting and delivery has proven extremely challenging. An autonomous DNA robotic system was developed for targeted cancer therapy, programmed to transport molecular payloads and cause on-site tumor infarction.
In certain embodiments, a nanorobot, functionalized with tumor endothelium-specific DNA aptamers on its external surface, and the blood coagulation protease thrombin within its inner cavity, initiated tumor vessel occlusion and induces tumor necrosis. Due to the specific expression of nucleolin on the surface of tumor endothelial cells, nucleolin-targeting aptamers serve as both targeting and trigger molecules for the mechanical opening of the DNA nanorobot to expose thrombin molecules and activate coagulation at the tumor site. Using tumor-bearing mouse models of breast cancer and melanoma, it was demonstrated that intravenously injected DNA nanorobots delivered thrombin specifically to the tumor-associated vessels and induces intravascular thrombosis, resulting in tumor necrosis and inhibition of tumor growth. The nanorobot proved to be safe and immunologically inert for use in normal mice and Bama miniature pigs, eliciting no detectable changes in blood coagulation parameters and histological morphology in either model. Given its robust self-assembly behavior, exceptional designability, potent antitumor activity and minimal in vivo adversity, this DNA nanorobot represents a promising strategy for precise drug/therapeutic agent design for cancer therapeutics.
In certain embodiments, the DNA nanostructure nanorobot is comprised of one DNA scaffold strand, a plurality of staple strands, and functional strands of DNA, such as fasteners, and, optionally, a targeting, imaging or capture strands of DNA that are operably linked to the DNA scaffold. The different elements of the DNA nanobot are capable of self-assembling into a nanostructure. For example, in certain embodiments, the single stranded DNA molecule is M13 phage single stranded DNA and staple strand, as described herein. As described in the Example, this nanostructure may be used as a carrier for a molecular payload, including inducing anti-tumor vascularization effects.
In certain embodiments, the present invention provides a DNA nanostructure nanorobot comprising:
a single stranded DNA scaffold strand of about 5000 to 10,000 bases in length;
a plurality of staple strands of DNA of about 20 to 40 bases in length, wherein each staple strand has a unique sequence and is hybridized to a specific position on the DNA scaffold strand, wherein the plurality of staple strands hybridized to the DNA scaffold form a sheet having a top surface and a bottom surface, and having four corners;
one or more fastener strands of DNA, wherein the one or more fastener strands of DNA is capable of fastening the sheet into a tube-shaped origami structure.
In certain embodiments, the DNA nanostructure nanorobot further comprises DNA targeting strands, wherein each targeting strand is operably linked to a targeting moiety. In certain embodiments, the targeting moiety is an aptamer. In certain embodiments, the aptamer is specific for nucleolin.
In certain embodiments, the DNA nanostructure nanorobot further comprises DNA imaging strands, wherein each imaging strand is operably linked to an imaging agent. In certain embodiments, the imaging agent is fluorescent dye.
As used herein, the term “DNA nanostructure” refers to a nanoscale structure made of DNA, wherein the DNA acts both as a structural and function element. DNA nanostructures can also serve as a scaffold for the formation of other structures. DNA nanostructures may be prepared by methods known in the art using one or more nucleic acid oligonucleotides. For example, in certain embodiments, the DNA nanostructure is an DNA rectangle origami nanostructure, self-assembled from single-stranded DNA molecules using “staple strands.”
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 nucleotides in length to about 10000 nucleotides in length.
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 al., Tet. Let. 27:4051-4054, 1986; Froehler et al., 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.
In certain embodiments, the DNA nanostructure has increased nuclease resistance (e.g., as compared to a control, such as an unfolded ssDNA molecule comprising the same nucleic acid sequence as the DNA nanostructure). In certain embodiments, nuclease resistance of the DNA nanostructure is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more than a control.
In certain embodiments, the DNA nanostructure is assembled using a single stranded DNA molecule as an initial scaffold. In certain embodiments, the DNA nanostructure comprises both single stranded and double stranded regions.
In certain embodiments, the present invention provides a DNA nanostructure nanorobot comprising:
a single stranded DNA scaffold strand of about 5000 to 10,000 bases in length;
a plurality of staple strands of DNA of about 32 bases in length, wherein each staple strand has a unique sequence and is hybridized to a specific position on the DNA scaffold strand, wherein the plurality of staple strands hybridized to the DNA scaffold form a rectangular sheet having a top surface and a bottom surface, and having four corners;
one or more fastener strands of DNA, wherein the one or more fastener strands of DNA is capable of fastening the rectangular sheet into a tube-shaped origami structure; and
one or more DNA capture strands, wherein each capture strand is operably linked to a therapeutic agent.
In certain embodiments, the DNA nanostructure nanorobot further comprises DNA targeting strands, wherein each targeting strand is operably linked to a targeting moiety. In certain embodiments, the targeting moiety is an aptamer.
In certain embodiments, the DNA nanostructure nanorobot further comprises DNA imaging strands, wherein each imaging strand is operably linked to an imaging agent. In certain embodiments, the imaging agent is fluorescent dye.
As used herein, “staple strands” are short single-stranded oligonucleotides of about 20 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. Exemplary staple strands are provided below as Staple Strands 13-204. In certain embodiments, the dimension of the rectangular sheet is about 90 nm×about 60 nm×2 nm.
In certain embodiments, the tube-shaped origami structure has a diameter of about about 19 nm.
In certain embodiments, the fastener strand is a Y-shaped structure. In certain embodiments, the Y-shaped structure comprises an F50 AS1411 aptamer sequence that specifically binds to nucleolin, and a Comp15 DNA strand partially complementary to the AS1411 sequence, wherein the F50 and the Comp15 sequences form a 14- to 16-base pair duplex.
In certain embodiments, the Y-shaped structure comprises 5′-FITC-labeled F50 and 3′-BHQ1-labeled Comp15; FITC-F50-48 and Comp15-48-Q; FITC-F50-73 and Comp15-73-Q; FITC-F50-97 and Comp15-97-Q; FITC-F50-120 and Comp15-120-Q; FITC-F50-144 and, Comp15-144-Q; or FITC-F50-169 and Comp15-169-Q.
In certain embodiments, the capture strand is extended with ssDNA comprising four binding sites to “capture” thrombin-DNA molecules.
In certain embodiments, DNA nanostructure robot further comprises one or more functional strand of DNA operably linked to an aptamer for targeting delivery of the nanorobot forming a targeting strand. {NINA]
In certain embodiments, the aptamer is specific for nucleolin.
In certain embodiments, one or more targeting strands are positioned at one or more corners of the rectangular sheet.
In certain embodiments, one or more capture strands is operably linked to a fluorescent dye to form an imaging strand.
In certain embodiments, the therapeutic agent is operably linked to the top surface of the rectangular sheet.
In certain embodiments, the therapeutic agent is operably linked to the bottom surface of the rectangular sheet.
In certain embodiments, the therapeutic agent is operably linked to an imaging agent. Imaging agents are well-known in the art and any can be operably linked to a therapeutic agent. In certain embodiments, the imaging agent is a fluorescent dye.
In certain embodiments, the therapeutic agent is a protein.
In certain embodiments, the therapeutic agent is thrombin.
In certain embodiments, the therapeutic agent is siRNA, a chemotherapeutic agent or a peptide therapeutic agent.
In certain embodiments, the thrombin is operably linked to the functional strand of DNA by means of a sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) as a bifunctional crosslinker.
In certain embodiments, the nanorobot comprises four thrombin molecules.
In certain embodiments, the target molecule is nucleolin.
In certain embodiments, the thrombin is operably linked to an imaging agent. In certain embodiments, the imaging agent is a fluorescent dye.
Certain embodiments of the invention provide a pharmaceutical composition comprising the DNA nanostructure nanorobot described herein.
In certain embodiments, the composition further comprises at least one therapeutic agent.
In certain embodiments, the at least one therapeutic agent is a chemotherapeutic drug (e.g., doxorubicin).
Certain embodiments of the invention provide a method of treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of the DNA nanostructure nanorobot or pharmaceutical composition as described herein.
In certain embodiments, the disease or disorder is cancer.
In certain embodiments, the cancer is breast cancer, ovarian cancer, melanoma or lung cancer.
Certain embodiments of the invention provide a use of the DNA nanostructure nanorobot or a composition as described herein for the manufacture of a medicament for inducing an tumor necrosis response in a subject (e.g., a mammal, such as a human).
Certain embodiments of the invention provide a DNA nanostructure nanorobot or a composition as described herein for the prophylactic or therapeutic treatment a disease or disorder.
Certain embodiments of the invention provide a kit comprising the DNA nanostructure nanorobot or a composition as described herein and instructions for administering the DNA nanostructure nanorobot/composition to a subject to induce an immune response or to treat a disease or disorder. In certain embodiments, the kit further comprises at least one therapeutic agent.
The invention also provides processes disclosed herein that are useful for preparing a DNA nanostructure nanorobot described herein.
In certain embodiments, one or more agents (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc.) may be operably linked to the DNA nanostructure, such as diagnostic agents or therapeutic agents. In certain embodiments, at least one diagnostic agent is operably linked to the DNA nanostructure. In certain embodiments, at least one therapeutic agent is operably linked to the DNA nanostructure. In certain embodiments, at least one diagnostic agent and at least one therapeutic agent are operably linked to the DNA nanostructure. Diagnostic agents are known in the art and include, e.g., fluorophores and radioisotopes, colorimetric indicator.
As used herein, the term “therapeutic agent” includes agents that provide a therapeutically desirable effect when administered to an animal (e.g., a mammal, such as a human). The agent may be of natural or synthetic origin. For example, it may be a nucleic acid, a polypeptide, a protein, a peptide, a radioisotope, saccharide or polysaccharide or an organic compound, such as a small molecule. The term “small molecule” includes organic molecules having a molecular weight of less than about, e.g., 1000 daltons. In one embodiment a small molecule can have a molecular weight of less than about 800 daltons. In another embodiment a small molecule can have a molecular weight of less than about 500 daltons.
In certain embodiments, the therapeutic agent is an immuno-stimulatory agent, a radioisotope, a chemotherapeutic drug (e.g., doxorubicin) or an immuno-therapy agent, such as antibody or an antibody fragment. In certain embodiments, the therapeutic agent is a vaccine, such as a cancer vaccine. In certain embodiments, the therapeutic agent is a tumor targeting agent, such as a monoclonal tumor-specific antibody or an aptamer. In certain embodiments, the therapeutic agent is an antibody (e.g., a monoclonal antibody, e.g., an anti-PD1 antibody). In certain embodiments, the therapeutic agent is an antigen (e.g., a tumor associated antigen or a tumor specific antigen). In certain embodiments, the therapeutic agent is a tumor antigen peptide(s). In certain embodiments, the therapeutic agent is thrombin.
As shown in
As shown in
As shown in
As shown in
As shown in
As shown in
As shown in
As shown in
Linkages
The linkage between the agent(s) and the DNA nanostructure is not critical, and may be any group that can connect the DNA nanostructure and the agent using known chemistry, provided that is does not interfere with the function of the agent or the DNA nanostructure. Chemistries that can be used to link the agent to an oligonucleotide are known in the art, such as disulfide linkages, amino linkages, covalent linkages, etc. In certain embodiments, aliphatic or ethylene glycol linkers that are well known to those with skill in the art can be used. In certain embodiments phosphodiester, phosphorothioate and/or other modified linkages are used. In certain embodiments, the linker is a binding pair. In certain embodiments, the “binding pair” refers to two molecules which interact with each other through any of a variety of molecular forces including, for example, ionic, covalent, hydrophobic, van der Waals, and hydrogen bonding, so that the pair have the property of binding specifically to each other. Specific binding means that the binding pair members exhibit binding to each other under conditions where they do not bind to another molecule. Examples of binding pairs are biotin-avidin, hormone-receptor, receptor-ligand, enzyme-substrate probe, IgG-protein A, antigen-antibody, aptamer-target and the like. In certain embodiments, a first member of the binding pair comprises avidin or streptavidin and a second member of the binding pair comprises biotin.
Therapeutic Agents to be Administered
In certain embodiments, the therapeutic agent is thrombin.
Certain embodiments of the invention also provide a composition comprising a DNA nanostructure nanorobot described herein and a carrier. In certain embodiments, the composition comprises a plurality of DNA nanostructure nanorobots.
In certain embodiments, the composition further comprises at least one therapeutic agent known in the art. In certain embodiments, the composition is pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.
The present invention further provides kits for practicing the present methods. Accordingly, certain embodiments of the invention provide a kit comprising an DNA nanostructure described herein and instructions for administering the DNA nanostructure nanorobot to induce an immune response (e.g., anti-tumor immunity) or to treat a disease or condition. In certain embodiments, the kit further comprises a therapeutic agent described herein and instructions for administering the therapeutic agent in combination (e.g., simultaneously or sequentially) with the DNA nanostructure.
As described in the Example, a DNA nanostructure nanorobot described herein may be used to induce vascular occlusion, slow the increase of or reduce the tumor burden, slow the increase of or reduce tumor size, reduce or block blood circulation in a tumor, slow the increase or reduce tumor cell metastasis, reduce or inhibit proliferation of a tumor, or induce a tumor necrosis.
Accordingly, certain embodiments of the invention provide a method of inducing an immune response in a subject, comprising administering to the subject an effective amount of a DNA nanostructure nanorobot or composition as described herein.
In certain embodiments, the administration increases an immune response by at least about, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more (e.g., as compared to a control). Methods of measuring an immune response are known in the art, for example using an assay described in the Example. The phrase “inducing an immune response” refers to the activation of an immune cell. Methods of measuring an immune response are known in the art, for example using an assay described in the Example.
Certain embodiments of the invention also provide a method of treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a DNA nanostructure nanorobot or a composition as described herein.
As used herein, the term “disease or disorder” refers to any disease or disorder that would benefit from induction of an immune response, vascular occlusion, a slowing in the increase of or reduction in tumor burden, a slowing in the increase of or reduction tumor size, reduction or blocking of blood circulation in a tumor, a slowing in the increase or reduction of tumor cell metastasis, reduction or inhibition tumor proliferation, or induction of a tumor necrosis response and include cancer.
In certain embodiments, a method of the invention further comprises administering at least one therapeutic agent to the subject.
The at least one therapeutic that can be administered is any therapeutic agent that can be used in the treatment of the disease or disorder of interest and include the therapeutic agents described herein.
The at least one therapeutic agent may be administered in combination with the DNA nanostructure. As used herein, the phrase “in combination” refers to the simultaneous or sequential administration of the DNA nanostructure and the at least one therapeutic agent. For simultaneous administration, the DNA nanostructure and the at least one therapeutic agent may be present in a single composition or may be separate (e.g., may be administered by the same or different routes).
Certain embodiments of the invention provide a DNA nanostructure nanorobot or a composition as described herein for use in medical therapy.
Certain embodiments of the invention provide the use of a DNA nanostructure nanorobot or a composition as described herein for the manufacture of a medicament for inducing an immune response in a subject.
Certain embodiments of the invention provide the use of a DNA nanostructure nanorobot or a composition as described herein for the manufacture of a medicament for inducing an immune response in a subject, in combination with at least one therapeutic agent.
Certain embodiments of the invention provide a DNA nanostructure nanorobot or a composition as described herein for inducing an immune response.
Certain embodiments of the invention provide a DNA nanostructure nanorobot or a composition as described herein for inducing an immune response, in combination with at least one therapeutic agent.
Certain embodiments of the invention provide the use of a DNA nanostructure nanorobot or a composition as described herein for the manufacture of a medicament for treating a disease or disorder in a subject.
Certain embodiments of the invention provide the use of a DNA nanostructure nanorobot or a composition as described herein for the manufacture of a medicament for treating a disease or disorder in a subject, in combination with at least one therapeutic agent.
Certain embodiments of the invention provide a DNA nanostructure nanorobot or a composition as described herein for the prophylactic or therapeutic treatment a disease or disorder.
Certain embodiments of the invention provide a DNA nanostructure nanorobot or a composition as described herein for the prophylactic or therapeutic treatment of a disease or disorder, in combination with at least one therapeutic agent.
In certain embodiments, the cancer is breast cancer, melanoma, ovarian cancer, lung cancer, carcinoma, lymphoma, blastoma, sarcoma, or leukemia. In certain embodiments, the cancer is a solid tumor cancer.
In certain embodiments, the cancer is squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, renal cell carcinoma, gastrointestinal cancer, gastric cancer, esophageal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (e.g., endocrine resistant breast cancer), colon cancer, rectal cancer, lung cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, melanoma, leukemia, or head and neck cancer. In certain embodiments, the cancer is breast cancer.
As described herein, methods of the invention comprise administering a DNA nanostructure described herein, and optionally, a therapeutic agent to a subject. Such compounds (i.e., a DNA nanostructure and/or therapeutic agent) may be formulated as a pharmaceutical composition and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, intraperitoneal or topical or subcutaneous routes.
Thus, the compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained. The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.
The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
Examples of useful dermatological compositions which can be used to deliver a compound to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
Useful dosages of compounds can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
The compound may be conveniently formulated in unit dosage form. In one embodiment, the invention provides a composition comprising a compound formulated in such a unit dosage form. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
As used herein, the term “about” means ±10%.
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
“Operably-linked” refers to the association two chemical moieties so that the function of one is affected by the other, e.g., an arrangement of elements wherein the components so described are configured so as to perform their usual function.
The term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, made of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues.
The terms “nucleotide sequence” and “nucleic acid sequence” refer to a sequence of bases (purines and/or pyrimidines) in a polymer of DNA or RNA, which can be single-stranded or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers, and/or backbone modifications (e.g., a modified oligomer, such as a morpholino oligomer, phosphorodiamate morpholino oligomer or vivo-mopholino). The terms “oligo”, “oligonucleotide” and “oligomer” may be used interchangeably and refer to such sequences of purines and/or pyrimidines. The terms “modified oligos”, “modified oligonucleotides” or “modified oligomers” may be similarly used interchangeably, and refer to such sequences that contain synthetic, non-natural or altered bases and/or backbone modifications (e.g., chemical modifications to the internucleotide phosphate linkages and/or to the backbone sugar).
Modified nucleotides are known in the art and include, by example and not by way of limitation, alkylated purines and/or pyrimidines; acylated purines and/or pyrimidines; or other heterocycles. These classes of pyrimidines and purines are known in the art and include, pseudoisocytosine; N4, N4-ethanocytosine; 8-hydroxy-N6-methyladenine; 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil; 5-fluorouracil; 5-bromouracil; 5-carboxymethylaminomethyl-2-thiouracil; 5-carboxymethylaminomethyl uracil; dihydrouracil; inosine; N6-isopentyl-adenine; 1-methyladenine; 1-methylpseudouracil; 1-methylguanine; 2,2-dimethylguanine; 2-methyladenine; 2-methylguanine; 3-methylcytosine; 5-methylcytosine; N6-methyladenine; 7-methylguanine; 5-methylaminomethyl uracil; 5-methoxy amino methyl-2-thiouracil; β-D-mannosylqueosine; 5-methoxycarbonylmethyluracil; 5-methoxyuracil; 2-methylthio-N6-isopentenyladenine; uracil-5-oxyacetic acid methyl ester; psueouracil; 2-thiocytosine; 5-methyl-2 thiouracil, 2-thiouracil; 4-thiouracil; 5-methyluracil; N-uracil-5-oxyacetic acid methylester; uracil 5-oxyacetic acid; queosine; 2-thiocytosine; 5-propyluracil; 5-propylcytosine; 5-ethyluracil; 5-ethylcytosine; 5-butyluracil; 5-pentyluracil; 5-pentylcytosine; and 2,6,-diaminopurine; methylpsuedouracil; 1-methylguanine; 1-methyl cytosine. Backbone modifications are similarly known in the art, and include, chemical modifications to the phosphate linkage (e.g., phosphorodiamidate, phosphorothioate (PS), N3′phosphoramidate (NP), boranophosphate, 2′, 5′phosphodiester, amide-linked, phosphonoacetate (PACE), morpholino, peptide nucleic acid (PNA) and inverted linkages (5′-5′ and 3′-3′ linkages)) and sugar modifications (e.g., 2′-O-Me, UNA, LNA).
The oligonucleotides described herein may be synthesized using standard solid or solution phase synthesis techniques that are known in the art. In certain embodiments, the oligonucleotides are synthesized using solid-phase phosphoramidite chemistry (U.S. Pat. No. 6,773,885) with automated synthesizers. Chemical synthesis of nucleic acids allows for the production of various forms of the nucleic acids with modified linkages, chimeric compositions, and nonstandard bases or modifying groups attached in chosen places through the nucleic acid's entire length.
Certain embodiments of the invention encompass isolated or substantially purified nucleic acid compositions. In the context of the present invention, an “isolated” or “purified” DNA molecule or RNA molecule is a DNA molecule or RNA molecule that exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or RNA molecule may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. For example, an “isolated” or “purified” nucleic acid molecule is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an “isolated” nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
The term “complementary” as used herein refers to the broad concept of complementary base pairing between two nucleic acids aligned in an antisense position in relation to each other. When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are substantially complementary to each other when at least about 50%, at least about 60% and or at least about 80% of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T (A:U for RNA) and G:C nucleotide pairs).
The term “subject” as used herein refers to humans, higher non-human primates, rodents, domestic, cows, horses, pigs, sheep, dogs and cats. In one embodiment, the subject is a human.
The term “therapeutically effective amount,” in reference to treating a disease state/condition, refers to an amount of a therapeutic agent that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease state/condition when administered as a single dose or in multiple doses. Such effect need not be absolute to be beneficial.
The terms “treat” and “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or decrease an undesired physiological change or disorder. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
In certain embodiments, the present invention provides a DNA nanostructure nanorobot comprising:
a single stranded DNA scaffold strand of about 5,000 to 10,000 bases in length;
a plurality of staple strands of DNA, wherein each staple strands are about 20 to 40 bases in length, wherein each staple strand has a unique sequence and is hybridized to a specific position on the DNA scaffold strand, wherein the plurality of staple strands hybridized to the DNA scaffold form a sheet having a top surface and a bottom surface; and
one or more fastener strands of DNA, wherein the one or more fastener strands of DNA is capable of fastening the sheet into an origami structure.
In certain embodiments, the DNA nanostructure nanorobot further comprises one or more DNA targeting strands, wherein each targeting strand is operably linked to a targeting moiety.
In certain embodiments, the targeting moiety is an aptamer that specifically binds a target molecule.
In certain embodiments, the aptamer is specific for nucleolin.
In certain embodiments, the targeting strand comprises a domain for attaching to the single stranded DNA scaffold strand.
In certain embodiments, the DNA nanostructure nanorobot, further comprises DNA imaging strands, wherein each imaging strand is operably linked to an imaging agent.
In certain embodiments, the imaging agent is a fluorescent dye.
In certain embodiments, the sheet is a rectangle having four corners and it shaped into a tube-shape.
In certain embodiments, the dimension of the rectangular sheet is about 90 nm×about 60 nm×about 2 nm.
In certain embodiments, the one or more targeting strands are positioned at one or more corners of the rectangular sheet.
In certain embodiments, the tube-shaped origami structure has a diameter of about 19 nm.
In certain embodiments, each of the fastener stands of DNA comprise a first and a second strand of DNA.
In certain embodiments, the first and second strand of DNA form a Y-shaped structure.
In certain embodiments, the second strand of DNA comprises a domain partially complementary to the first strand.
In certain embodiments, the first and second strands hybridize to form a 14- to 16-base pair duplex.
In certain embodiments, the first strand of DNA comprises an aptamer that specifically binds a target molecule and a domain partially complementary to the second strand.
In certain embodiments, the aptamer specifically binds nucleolin.
In certain embodiments, the aptamer that specifically binds nucleolin is an F50 AS1411 aptamer sequence.
In certain embodiments, the oligonucleotide partially complementary to the aptamer comprises a Comp15 DNA sequence.
In certain embodiments, the first or second strand comprises a quencher moiety.
In certain embodiments, the other of the first or second strand comprises a fluorophore moiety.
In certain embodiments, the Y-shaped structure comprises:
5′-FITC-labeled F50 and 3′-BHQ1-labeled Comp15;
FITC-F50-48 and Comp15-48-Q;
FITC-F50-73 and Comp15-73-Q;
FITC-F50-97 and Comp15-97-Q;
FITC-F50-120 and Comp15-120-Q;
FITC-F50-144 and, Comp15-144-Q; or
FITC-F50-169 and Comp15-169-Q.
In certain embodiments, the nanorobot further comprises from one to four capture strands.
In certain embodiments, the one or more capture strand binds to a poly(A) region in the DNA scaffold strand.
In certain embodiments, the one or more capture strand is positioned on the top surface of the sheet.
In certain embodiments, the capture strand is positioned on the bottom surface sheet.
In certain embodiments, one or more capture strands is operably linked to a therapeutic agent.
In certain embodiments, the one or more capture strand comprises poly(T).
In certain embodiments, the one or more capture strand comprises an imaging agent.
In certain embodiments, the imaging agent is a fluorescent dye.
In certain embodiments, the therapeutic agent is a protein.
In certain embodiments, the therapeutic agent is thrombin.
In certain embodiments, the thrombin is conjugated to the functional strand of DNA by means of a sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) as a bifunctional crosslinker.
In certain embodiments, the nanorobot comprises from one to four thrombin molecules.
In certain embodiments, the nanorobot comprises four thrombin molecules.
In certain embodiments, the thrombin is operably linked to a fluorescent dye.
In certain embodiments, each staple strand is about 25 to 35 bases in length.
In certain embodiments, each staple strand is about 32 bases in length.
In certain embodiments, the present invention provides a DNA nanostructure nanorobot comprising:
a single stranded DNA scaffold strand comprising M13 phage DNA;
a plurality of staple strands 13-204 of DNA, wherein the plurality of staple strands hybridized to the DNA scaffold forms a rectangular sheet having a top surface and a bottom surface, and four corners;
at least six fastener strands of DNA, wherein each fastener strand of DNA is capable of fastening the rectangular sheet into a tube-shaped origami structure;
four DNA capture strands, wherein each capture strand is operably linked to a thrombin; and
at least four targeting strands, wherein each targeting strand is operably linked to an aptamer specific for nucleolin;
a plurality of imaging strands comprising extended ssDNA sequences that hybridized to fluorescent dye-labeled ssDNA.
In certain embodiments, the present invention provides a pharmaceutical composition comprising the DNA nanostructure nanorobot as described herein and a pharmaceutically acceptable carrier
In certain embodiments, the pharmaceutical composition further comprises at least one therapeutic agent.
In certain embodiments, the at least one therapeutic agent is a chemotherapeutic agent.
In certain embodiments, the present invention provides a method of treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of the DNA nanostructure nanorobot or a composition as described herein.
In certain embodiments, the disease or disorder is cancer.
In certain embodiments, the cancer is breast cancer, ovarian cancer, melanoma or lung cancer.
In certain embodiments, the present invention provides a method of inhibiting tumor growth in a subject, comprising administering to the subject a therapeutically effective amount of the DNA nanostructure nanorobot or a composition as described herein.
In certain embodiments, the present invention provides a use of the DNA nanostructure nanorobot or a composition as described herein for the manufacture of a medicament for inducing a tumor necrosis response in a subject.
In certain embodiments, the present invention provides a use of the DNA nanostructure nanorobot or a composition as described herein for inducing a tumor necrosis response.
In certain embodiments, the present invention provides a use of the DNA nanostructure nanorobot or a composition as described herein for the manufacture of a medicament for treating a disease or disorder in a subject.
In certain embodiments, the present invention provides a use of the DNA nanostructure nanorobot or a composition as described herein for the prophylactic or therapeutic treatment a disease or disorder.
In certain embodiments, the present invention provides a kit comprising the DNA nanostructure nanorobot or a composition as described herein and instructions for administering the DNA nanostructure nanorobot/composition to a subject to induce an immune response or to treat a disease or disorder.
In certain embodiments, the kit further comprises at least one therapeutic agent.
The invention will now be illustrated by the following non-limiting Examples.
DNA origami is a method that enables the rational design and production of DNA nanostructures with controlled size, shape and spatial addressability14-19, producing functional platforms for biological applications3, 5, 7, 20-23. Inspired by these advances, a DNA nanorobotic system was constructed to protect thrombin until triggered only when localized in tumor vessels. This was accomplished by designing a method to create thrombin-DNA co-assembling nanostructures with multiple functional elements. Using the thrombin-loaded nanorobot, on-site tumor blood vessel infarction and targeted cancer treatment was demonstrated in vivo.
Results
Design and Synthesis of Thrombin-Loaded DNA Nanorobot
To deliver active thrombin solely to tumor sites in a highly controlled way, a DNA nanorobotic system was developed based on a self-assembled origami nanotube with multiple functional elements (
The present hollow tube-shaped DNA nanorobot, with a diameter of ˜19 nm and a length of 90 nm (
DNA Nanorobot-Triggered Activation and Endothelial Cell Targeting In Vitro
It was hypothesized that when the fastener strands recognize their targets, e.g., nucleolin proteins selectively expressed on the surface of actively proliferating tumor vascular endothelial cells25, the hybridized duplexes would dissociate to induce reconfiguration of the nanorobot to expose the contained cargo. To test this hypothesis, flow cytometry was used to examine the nucleolin recognition and dissociation of the DNA fastener strands. The Y-shaped fastener structures (
Next, in vitro blood coagulation was investigated to determine whether reconfiguration of the nanorobot occurs in response to the target protein. When mouse plasma was mixed with the thrombin-loaded nanorobot (nanorobot-Th) and HUVECs, it coagulated rapidly, with a fibrin formation time of 82 s, as compared to 176 s when cells were absent (
The ability of the DNA nanorobot to target surface nucleolin-positive cells was next examined. To ensure a maximal targeting effect in vitro, we decorated the staple strands of the origami structures with additional targeting aptamer sequences at both ends of the tubes (
In Vivo Tumor Targeting and Biodistribution of DNA Nanorobots
Fluorescence imaging was performed on orthotopic tumor-bearing mice to investigate tumor vessel targeting of the nanorobot in vivo. Human breast cancer cells (MDA-MB-231) were injected into the mammary fat pads of BALB/c nude mice for the establishment of a tumor model. Mice displaying tumor volumes around 100 mm3 were intravenously injected (i.v.) with Cy5.5-labled DNA nanorobots. The nanorobots progressively accumulated in the tumor, reaching a maximal accumulation at 8 h post-injection (
The time-dependent organ distribution and clearance of DNA nanorobots and other controls after intravenous injection was also examined (
Nanorobots Induce On-Site Tumor Vessel Occlusion and Necrosis
After targeting delivery to tumor-associated blood vessels, the tubular shaped nanorobot undergoes a structural reconfiguration, triggered by nucleolin-mediated unfastening, to expose the loaded thrombin. The thrombin proteins localized to tumor vessels induce thrombosis by activating platelets and inducing the generation of fibrin strands, resulting in vessel infarction and tumor necrosis (
To investigate the therapeutic potential of nanorobot-Th, MDA-MB231 tumor-bearing nude mice were randomly sorted and each group was treated through tail vein injection as follows: saline, free thrombin, targeted empty nanorobot, nontargeted nanotube-Th, targeted nanotube-Th or nanorobot-Th. Injections were carried out 6 times at intervals of 3 days. Mice treated with saline, free thrombin, empty nanorobot or nontargeted nanotube-Th formed fast growing tumors (
In order to further explore the role of targeted delivery, its antitumor activity in less vascularized tumors was investigated. Mice bearing subcutaneous xenografts of human ovarian cancer cells SK-OV3, reported to be poorly vascularized28 and exhibited relatively low permeability and retention for Evans blue (
Safety Assessment for Thrombin-Loaded DNA Nanorobots
Finally, comprehensive in vitro and in vivo safety assessments of the DNA nanorobotic system were carried out. It was first found that doses of free thrombin equivalent to those used in vivo in the nanorobot system elicited no observable thrombi in the cerebral microcirculation in non-tumor bearing healthy mice. However, transient, reversible microthrombi were apparent in much higher concentrations (
The safety of the DNA nanorobots was further characterized in normal Bama miniature pigs, which exhibit high similarity to humans in anatomy and physiology32. Intravenous injections of free thrombin at 150 U/pig, a dose equivalent to the accumulated therapeutic dose of thrombin in the mouse model treated with nanorobot, had no effect on blood coagulation parameters and did not induce thrombosis in major organs (Fig. S28a-c). However, activated partial thromboplastin time (APTT) prolongation occurred but was still under 43 s after single or multiple injections of thrombin at up to 350 U/pig; D-dimer content also increased after injection of this dose, but disappeared with a final measurement of normal levels 3 d after the last treatment, indicating a transient and reversible blood coagulation system activation with no visible thrombotic formation in major tissues. Treatment with nanorobot-Th system did not lead to any significant variations in the blood coagulation parameters (Fig. S28d,e) or histological morphology (Fig. S28f) when compared to the control group, demonstrating that the nanorobot-Th is decidedly safe in the normal tissues of large animals.
Directed coagulation of tumor vasculature has enormous potential to eradicate solid tumors, yet specifically targeting tumor blood supply via systemic therapeutic agent administration appears an impossible mission. Although intra-arterial embolization, such as transhepatic arterial embolization (TAE) and transhepatic arterial chemoembolization (TACE), is routine practice for the effective treatment of a variety of hepatic malignancies, assisted by image-guided therapy33, 34, vascular occlusion-based cancer therapies remain challenging due to the lack of safe and effective vessel occluding agents administered systemically. A novel nanorobotic system was developed for the intelligent delivery of therapeutic thrombin in vivo to tumor-associated blood vessels, to elicit highly efficient blockage of tumor blood supply and inhibition of tumor growth. With tumor-targeted delivery, recognition of tumor microenvironmental signals, triggered nanostructural changes and payload exposure, the thrombin delivery DNA nanorobot constitutes a major advance in the application of DNA nanotechnology for cancer therapy. In a melanoma mouse model, the nanorobot not only affected the primary tumor, but also prevented the formation of metastasis, showing promising therapeutic potential.
Methods
Materials and Reagents.
Oligonucleotides (origami staple strands and functional strands) were purchased from Invitrogen (Shanghai, China) and used without further purification. The dye labeled DNA strands were further purified for use by denaturing polyacrylamide gel electrophoresis (PAGE). For flow cytometry and immunohistochemistry, the following antibodies35-36 were used for nucleolin (Sigma-Aldrich, St. Louis, Mo., catalog No. N2662); mouse platelet CD41 (BD Pharmingen, San Diego, Calif., catalog No. 553847); P-selectin (BD Pharmingen, San Diego, Calif., catalog No. 553744); CD34 (Sigma-Aldrich, St. Louis, Mo., catalog No. WH0000947M1); fibrin IIβ chain (Accurate Chemical & Scientific, Westbury, N.Y., catalog No. NYBT2G1P); thrombin (MyBioSource, San Diego, Calif., catalog No. MBS2001306); FITC-conjugated goat anti-rabbit IgG (BD Pharmingen, San Diego, Calif., catalog No. 554020); FITC-conjugated goat anti-mouse IgG (BD Pharmingen, San Diego, Calif., catalog No. 5540001) and Alexa Fluor 488 AffiniPure Donkey anti-rat IgG (Yeasen Biotech, Shanghai, China, catalog No. 34406ES60). Cell culture media, fetal bovine serum, hemacytometers, and cell culture supplies were purchased from Fisher Scientific. Tris-base, acetic acid, sodium chloride, ethylenediaminetetraacetic acid disodium salt dehydrate and other common chemical reagents were purchased from Sigma-Aldrich.
DNA origami design details. Production of M13 bacteriophage single-stranded DNA was according to Douglas et al.'s methods37. In detail, JM109 E. coli were cultured overnight in LB medium, 5 ml were diluted in 2×YT medium with 5 mM MgCl2 and placed in a 37° C. shaker. When the optical density (OD 600) reached 0.5, p7249 M13 phages were mixed with the bacteria, and the culture incubated in a 37° C. shaker. After a 5 h incubation, the culture was collected and centrifuged at 6,000 g for 30 min to remove the bacteria pellets. NaCl (30 g/l) and PEG (40 g/l) were added to the supernatant containing phages and the mixture was incubated on ice for 1 h. After 30 min centrifugation at 10,000 g, the phage pellet was collected and suspended in Tris-Cl (10 mM, pH 8.5). After adding 0.2 M NaOH and 1% SDS, the phage solution was mixed and incubated at 25° C. for 3 min. After the addition of potassium acetate (3 M, pH5.5) for neutralization, the solution was incubated on ice for 10 min and centrifuged (12,000 g, 30 min). The ssDNA (7249 nt) containing supernatant was collected and precipitated in ethanol (70%) on ice for 2 h. After centrifuging at 12,000 g for 30 min, the DNA pellet was collected and washed in ethanol (70%) and then resuspended in Tris-Cl (10 mM, pH 8.5). The concentrations of ssDNA were determined by UV-Vis spectrometry (Shimadzu Corp. Kyoto, Japan).
The original design of the rectangular DNA origami structure shown in
Rectangular DNA Nanosheets.
Rectangular DNA nanostructures were made by mixing a long single stranded scaffold strand (7249 DNA derived from M13 bacteriophage) with short strands (including staple strands and functional strands) in 1×TAE-Mg buffer (40 mM Tris, 20 mM acetic acid, pH 8.0, 2 mM EDTA, 12.5 mM Mg(CH3COOH)2). The final concentrations of scaffold DNA and basic staple strands were 10 nM and 80 nM, respectively. The mixture was heated to 65° C., then annealed by cooling to 25° C. at a rate of 10 min/° C. using an Eppendorf thermal cycler (Eppendorf Mastercycler ep Gradient S, Hamburg, Germany). Unless otherwise stated, all DNA strands or aptamers were purchased from the oligonucleotide synthesis service of Invitrogen (Invitrogen, Carlsbad, Calif.).
The resulting rectangular DNA origami sheets were separated from excess staple strands using Amicon Ultra-0.5 ml 100 kD centrifugal filters (Millipore Corporation, Bedford, USA). The initial filtration was performed by adding 350 μl 1×TAE-Mg buffer to 100 μl DNA sheets, and centrifuging for 3 min at 2,075 g. Then two wash steps were performed by adding 350 μl 1×TAE-Mg buffer and centrifuging for 3 min each at 2,075 g. The remaining solution was collected and characterized using 1% agarose gel electrophoresis and atomic force microscopy (AFM).
Preparation of Thrombin-DNA Conjugates and Thrombin Activity Analysis.
Thrombin-DNA conjugates were prepared using sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC, Sigma-Aldrich, St. Louis, Mo.) as a bifunctional crosslinker between thrombin and DNA. In a typical synthesis, 200 μl 3 μM thrombin (Sigma-Aldrich, St. Louis, Mo., catalog No. T4648) was mixed with 9 μl 15 mM sulfo-SMCC at room temperature for 1 h. Excess sulfo-SMCC was removed using an Amicon Ultra-0.5 ml 30 kD centrifugal filter and washing three times by centrifugation. The initial washing was conducted by adding 250 μl PBS to 200 μl of the mixture of sulfo-SMCC and thrombin, and centrifuging for 3 min at 5,534 g. The subsequent two washing steps were performed by adding 350 μl PBS and centrifuging for 3 min at 5,534 g. The residual solution was collected and a 15-fold excess of thiolated polyT DNA was added. The mixture was incubated at 4° C. overnight and the final thrombin-DNA conjugates were purified using 30 kD centrifugal filters. Conjugation was verified by 4-12% SDS-polyacrylamide gel electrophoresis.
Thrombin activity was assayed using tosyl-glycyl-prolyl-arginine-4-nitroanilide acetate (chromozym TH) as a substrate, following the manufacturer's protocol (Boehringer Mannheim, Indianapolis, Ind.). In brief, reactions were conducted at 37° C. for 5 min, 10 min, 30 min, 1 h or 1.5 h, with free thrombin as a positive control and a blank DNA sheet as a negative control. Thrombin-DNA conjugates and DNA sheets loaded with thrombin were assayed at concentrations equivalent to 90 nM thrombin and at a substrate concentration of 2 mg/ml. Reactions were carried out in a volume of 100 μl. The increase in absorbance at 405 nm was monitored on a Beckman DU-30 spectrophotometer.
Thrombin-Loaded Tubular-DNA Nanorobot.
Thrombin-DNA conjugates were mixed with the rectangular DNA origami structures (containing thrombin capture strands) at a molar ratio of 10:1 in 1×TAE-Mg buffer. The mixture was heated to 45° C., then cooled to 25° C. at a rate of 10 min/° C. to facilitate annealing. The thrombin-rectangle-origami assemblies were purified using 100 kD centrifugal filters to remove excess thrombin-DNA conjugates. Tube origami structures were then constructed by adding a 20-fold molar excess of fasteners to the thrombin-rectangle-origami, and then a 5-fold molar excess of additional targeting strands were added to the mixture. To facilitate assembly, the mixture was heated to 37° C., then cooled to 15° C. at a rate of 10 min/° C.
Platelet Aggregation.
Fresh blood from healthy volunteers (informed consent was obtained from all subjects) was collected using ACD (2.5% trisodium citrate, 2.0% D-glucose, 1.5% citric acid) as the anticoagulant. Platelets were washed with CGS buffer (0.123 M NaCl, 0.033 M D-glucose and 0.013 M trisodium citrate, pH 6.5), and resuspended in modified Tyrode's buffer (2.5 mM Hepes, 150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO3, 5.5 mM D-glucose, pH 7.4), supplemented with 1 mM CaCl2 and 1 mM MgCl2, to a final concentration of 3×108/ml. After incubation at 22° C. for 2 h, platelet aggregation was assessed using a turbidometric platelet aggregometer (Xinpusen, Chengdu, China) by adding 0.3 ml of washed platelets and free thrombin, DNA origami sheets, DNA-origami sheets with conjugated thrombin or DNA-origami tubes with conjugated thrombin (at equivalent thrombin concentrations where appropriate) into glass aggregometer cuvettes at 37° C. under stirring.
DNA Nanorobot Preparation for In Vitro and In Vivo Imaging.
Thrombin-loaded rectangular DNA nanostructures with imaging strands were mixed with fluorescent dye-conjugated DNA in 1×TAE-Mg buffer. The molar ratio of dye-modified DNA to each imaging strand was 3:1. The mixtures were heated to 45° C., then cooled to 25° C. at a steady rate of 10 min/° C. using an Eppendorf Mastercycler. Excess dye-modified DNA was removed using 100 kD centrifugal filters. A 20-fold molar excess of fasteners and a 5-fold molar excess of targeting strands, including the AS1411 sequence, were subsequently added. The mixture was heated to 37° C., then cooled to 15° C. at a rate of 10 min/° C. to promote assembly.
Cell Culture.
All cell lines were purchased from the American Type Culture Collection (Manassas, Va., USA) unless stated otherwise. The human breast cancer cell line MDA-MB-231, and human umbilical vein endothelial cells (HUVECs) were maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin. The human ovarian cancer cell line SK-OV3 (Shanghai Institutes for Biological Sciences, Shanghai, China) was maintained in McCoy's 5A medium supplemented with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin. The murine melanoma cell line B16-F10 was grown in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin and streptomycin. Mouse brain endothelial cells (bEnd.3) were maintained in high glucose DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin. Cell line authentication was performed by short tandem repeat DNA profiling and comparison with a reference database. The cells were cultured at 37° C., 5% CO2 and were routinely tested for mycoplasma contamination.
Cell Surface Expression of Nucleolin.
The expression of nucleolin on the cell surface was assessed with antibodies specific to human nucleolin using a Beckman Coulter CyAn ADP flow cytometer (Fullerton, Calif., USA). HUVECs were trypsinized, washed twice in PBS, resuspended in 10% goat serum in PBS, and incubated at 4° C. for 30 min. Cells were then pelleted and resuspended in 2% goat serum in PBS containing 20 μg/ml anti-nucleolin antibody. After one hour incubation on ice, cells were washed three times with PBS and incubated with a 1:500 dilution of FITC-conjugated goat anti-rabbit IgG in PBS for 30 min on ice. Cells were washed with PBS twice and analyzed by flow cytometry.
Cell Surface Binding of AS1411 Aptamer-Containing DNA Strands.
HUVECs were prepared and blocked with goat serum. FITC-labeled F50-containing the AS1411 aptamer sequence (5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGG TCTAAAGTTTTGTCGTGAATTGCG-3′, the region of AS1411 aptamer is in bold font) that can recognize nucleolin on the surface of HUVECs was used. Cells were incubated with 2 μM FITC-labeled F50 strands (Invitrogen, Carlsbad, Calif.) or 2 μM random DNA sequence (5′-GAGAACCTGAGTCAGTATTGCGGAGATCTAAAGTTTTGTCGTGAATTGCG-3′) as a control for 2 h at 37° C. Cells were washed twice with PBS and assayed by flow cytometry using a BD Biosciences BD Accuri C6 flow cytometer (San Jose, Calif., USA).
Aptamer Competition Assays.
To determine whether cell surface binding of the F50 DNA strand was specific for nucleolin, HUVECs were pretreated with antibodies against human nucleolin at 45 μg/ml before addition of the aptamer. Cells were then incubated with 2 μM FITC-labeled F50 for 2 h at 37° C. Next, the cells were washed with PBS twice as described above and analyzed by flow cytometry.
Binding of F50 and Fastener Strands.
HUVECs were prepared and treated with goat serum. Cells were then incubated with 2 μM FITC-labeled F50, which are Y-shaped DNA structures with either a 15 base pair duplex (F50+C15) or a 26 base pair duplex (F50+C26) that was annealed in 1×TAE/Mg buffer, for different durations. Cells were washed with PBS twice as detailed above and analyzed by flow cytometry.
The DNA sequences used are shown below:
F50+C15, mixture of fastener:
F50+C26, mixture of fully complementary AS1411 portion structures:
Fastener Duplex Opening by Recombinant Nucleolin.
20 μM mixtures of the Y-shaped structures in 1×TAE/Mg buffer were heated to 95° C. and then annealed by cooling to 25° C. at a rate of 3 min/° C. using an Eppendorf thermal cycler. The DNA sequences used are shown below:
F50+C15, mixture of fastener:
F50+C26, mixture of fully complementary AS1411 portion structures:
A 10-fold molar excess of recombinant nucleolin (RPC242Hu01, Cloud-Clone Corp.) was added and incubated with the Y-shaped DNA structures (F50+C15 and F50+C26, 2 μM) at 37° C. Fluorescence intensity measurements were performed with a fluorescence spectrometer (LS55, Perkin Elmer).
Fastener Duplex Opening by Cell Surface-Expressed Nucleolin.
The 20 μM mixtures of Y-shaped structures in 1×TAE/Mg buffer were heated to 95° C. and then annealed by cooling to 25° C. at a rate of 3 min/° C. using an Eppendorf thermal cycler.
The DNA sequences used are shown below:
F50+C15, mixture of fastener:
F50+C26, mixture of fully complementary AS1411 portion structures:
FC+CC, mixture of no AS1411 structures:
HUVECs were prepared and blocked with goat serum as detailed above. Cells were then incubated with the Y-shaped DNA structures (F50+C15, F50+C26 and FC+CC) at a concentration of 2 μM for 2 h at 37° C. Cells were washed twice with PBS and assayed using flow cytometry.
DNA Nanorobot Opening by Recombinant Nucleolin.
Six pairs of fasteners were designed containing 5′-FITC-labeled F50 and 3′-BHQ1-labeled Comp15: FITC-F50-48, Comp15-48-Q; FITC-F50-73, Comp15-73-Q; FITC-F50-97, Comp15-97-Q; FITC-F50-120, Comp15-120-Q; FITC-F50-144, Comp15-144-Q; and FITC-F50-169, Comp15-169-Q. The fluorophore-quencher pairs served as switchable fluorescent beacons. 20 μM mixtures of fluorophore-quencher pairs in 1×TAE/Mg buffer were heated to 95° C. and then annealed by cooling to 25° C. at a rate of 3 min/° C. using an Eppendorf thermal cycler. A 20-fold molar excess of fluorophore-quencher fasteners was added to the DNA sheets. The mixture was heated to 37° C., then cooled to 15° C. at a rate of 10 min/° C. to promote assembly. Extra fastened pairs were removed using filtration devices.
The DNA sequences used are shown below:
A 20-fold molar excess of recombinant nucleolin was added and incubated with DNA nanorobots labeled by fluorophore-quencher fasteners at 37° C. Fluorescence intensity measurements were performed with a fluorescence spectrometer (LS55, Perkin Elmer).
Cell Surface Nucleolin Triggering of DNA Nanorobot Reconfiguration.
20 μM mixtures of FRET pairs in 1×TAE/Mg buffer were heated to 95° C. and then annealed by cooling to 25° C. at a rate of 3 min/° C. using an Eppendorf thermal cycler. A 20-fold molar excess of FRET fasteners was added to the DNA sheets. The mixture was heated to 37° C., then cooled to 15° C. at a rate of 10 min/° C. to promote assembly.
HUVECs were prepared and blocked with goat serum as described above. Serum-starved HUVECs where surface nucleolin expression was downregulated were also used. Non-starved and serum-starved cells were incubated with DNA nanorobots labeled with fluorophore-quencher fasteners for 2 h at 37° C. Cells were washed twice with PBS and assayed using flow cytometry.
Cell Binding of Nanorobot.
HUVECs were trypsinized, washed twice with 1 ml PBS, seeded onto Lab-Tek Chamber Slides (Nunc) and cultured overnight. Cells were then incubated with either 15 μM Alexa 594-labeled AS1411 aptamer or Alexa 594-labeled nanorobots or nanotubes at a concentration equivalent to 15 μM F50 at 37° C. for different time periods. To determine whether binding of the nanorobot was dependent on surface nucleolin, the cells were pretreated with anti-nucleolin antibodies (45 μg/ml). After treatment, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 30 min. Then the cells were stained with DIO (plasma membrane) and DAPI (nucleus) (Sigma-Aldrich, St. Louis, Mo.) and imaged with a Nikon Ti-e microscope equipped with an UltraVIEW Vox confocal attachment (Perkin Elmer, Boston, Mass., USA).
Animal Studies.
All animal studies were performed in accordance with ARRIVE guidelines, with the approval of the Ethics of Animal Experiments of the Health Science Center of Peking University Committee. Six to eight-week-old female nude mice (nu/nu) and C57BL/6J mice were obtained from Vital River Laboratory Animal Technology Co. Ltd (Beijing, China) and housed with a 12 h light/dark cycle, at 22° C. and food and water ad libitum. Bama miniature pigs (Sus scrofa domestica) weighing 20 to 25 kg (8-10 months old, half male and half female) were also used in the study. All pigs were healthy and maintained in the animal facility at the Farm Animal Research Center (Beijing, China) under standard conditions prescribed by the Institutional Guidelines. The study protocol was approved by the Institutional Animal Care and Use Committee of the Institute of Zoology, Chinese Academy of Sciences.
In Vivo Targeting.
MDA-MB-231 cells (2.0×106) mixed with an equal volume of Matrigel (BD Pharmingen, San Diego, Calif.), were injected subcutaneously into the mammary fat pads of female nude mice. When the tumor size reached ˜100 mm3, as determined using digital calipers, the mice received tail-vein injections of Cy5.5-labeled DNA nanorobot and were examined using an in vivo optical imaging system (Maestro, CRi Inc., Woburn, Mass., USA) at various time points thereafter (n=3). For biodistribution analysis, the mice were sacrificed at the indicated time points post-injection and the tumors and major organs were harvested (n=3).
Staining of Tissues.
For histological examination, tumors and major organs were collected from MDA-MB-231-bearing nude mice at the indicated time points post-administration of DNA nanorobot-Th and various controls. Tissue samples were fixed in 4% paraformaldehyde, immunostained with anti-CD41 antibody for thrombosis or anti-CD34 antibody for endothelial cells. For evaluating tissue necrosis, sections were stained with hematoxylin and eosin (H&E).
In Vivo Therapeutic Efficacy.
To assess the in vivo efficacy of the nanorobots, nude mice bearing ˜100 mm3 MDA-MB-231 tumors were randomly divided into six groups of eight mice per treatment group and treated with saline, free thrombin, empty nanorobot, nontargetd nanotube-Th, targeted nanotube-Th or nanorobot-Th (˜1.5 U accumulated thrombin/mouse), by tail vein injection every 3 d for a total of 6 treatments. The day of the first injection was designated day 0. Tumors were measured with calipers in three dimensions. The following formula was used to calculate tumor volume: Volume=length×width2/2.
To confirm antitumor efficacy, a syngeneic B16-F10 melanoma tumor model was established by subcutaneous injection of 5×106 murine B16-F10 cells into the right posterior flank of C57BL/6J mice. When the tumors reached a size of ˜150 mm3, the mice (eight mice per group) were treated intravenously with saline, free thrombin, empty nanorobot, nontargeted nanotube-Th, targeted nanotube-Th or nanorobot-Th every other day for 14 d. Tumor volume was determined as described above. The animals were euthanized 2 d after the last treatment, and the livers were excised and weighed. Liver sections were stained with H&E for metastasis analysis.
Two other tumor models, an ovarian cancer SK-OV3 xenograft model and an inducible KRASG12D lung tumor model were used to investigate the versatility of nanorobot-Th. For the SK-OV3 model, nude mice bearing ˜100 mm3 SK-OV3 xenografts (eight mice per group) were treated intravenously with saline, free thrombin, empty nanorobot, a scrambled aptamer control, nontargeted nanotube-Th, targeted nanotube-Th or nanorobot-Th every 3 d for a total of 6 treatments (˜1.5 U accumulated thrombin/mouse).
The inducible KRASG12D model was established using transgenic TetO-KRASG12D mice as described method29. Mice were fed with doxycycline diet since the 6th week after birth to induce primary lung adenomas. After being induced for two weeks, mice with tumors were randomly divided into four groups (three animals per group) and treated with saline, free thrombin, nontargeted nanotube-Th or nanorobot-Th, respectively, by intravenous injection once every 3 d. The progress of lung tumors was monitored by MRI imaging one week and two weeks after treatment started.
In Vivo MRI Imaging.
TetO-KRASG12D transgenic mice were imaged using a 7.0 T Bruker Biospec animal MRI instrument (Germany). The imaging parameters were set as follows: FOV (field of view)=30×30 cm2, MTX (matrix size)=256×256, slice thickness=1 mm, TE=61.2 ms, TR=2320 ms, and NEX=4. The mice were anesthetized with 1.5% isoflurane delivered via nose cone before and during the imaging sessions.
Cell Viability Assay.
The cytotoxicity of DNA nanorobot was assessed in murine endothelial bEnd3 cells. Cells (2000 cells/well) were added to the wells of a 96-well plate (Corning, Woburn, Mass., USA). After culturing at 37° C. for 4 h, the cells were incubated with DNA nanorobot-Th at either 3.3 nM or 6.6 nM (in PBS) for a further 24, 48 or 72 h. The proportion of viable cells was evaluated using a CCK-8 kit (Sigma-Aldrich, St. Louis, Mo., catalog No. 96992). Blank wells only with culture media and PBS-treated wells were used to define 0 and 100% viability, respectively.
Determination of Platelet Surface P-Selectin, Plasma Fibrin and Thrombin Levels and Platelet Counts.
Nude mice bearing MDA-MB-231 tumors were injected intravenously with DNA nanorobot-Th every two days for a total of six injections (n=6 mice). Mouse whole blood was then collected retro-orbitally into a 3.8% sodium citrate solution in blood collection tubes at the indicated time points. For platelet activity studies, the blood was mixed with an equal volume of 2% paraformaldehyde for 30 min at RT and centrifuged to obtain platelet-rich plasma (PRP). The PRP was incubated with FITC-conjugated P-selectin-specific monoclonal antibodies and analyzed by flow cytometry. Fibrin or thrombin levels in the PRP were quantified by enzyme-linked immunosorbent assay (ELISA) with an antibody specific for mouse fibrin (Abeam, ab157527) or thrombin (Abeam ab108844). Platelet numbers were counted manually with a hemocytometer using optical microscopy.
ELISA for Serum Cytokines.
Non-tumor-bearing C57BL/6J mice were injected intravenously with DNA nanorobot-Th every two days for a total of six injections (n=3 mice). Mouse serum was obtained by centrifuging whole blood taken by retro-orbital venous puncture at different time points. Serum cytokine concentrations including IL-6, IP-10, TNFα (R&D Systems, China, Shanghai, China, SM6000B, SMCX100, SMTA00B) and IFNα (ThermoFisher Scientific, Shanghai, China, KMC4011) were measured by ELISA as per the manufacturer's protocol using 50 μl serum38-41.
In Vivo Thrombotic Risk Assessment in Bama Miniature Pigs.
To evaluate the in vivo safety of DNA nanorobot-Th, Bama minipigs were randomly divided into three groups of three pigs per treatment group as follows: group 1, saline; group 2, free thrombin at a dose of 150 U thrombin/pig (equivalent to ˜1.5 U accumulated thrombin/mouse); group 3, nanorobot-Th. The animals were injected via marginal ear vein every other day for a total of three injections. Day 0 marks the first day of injection. Blood was collected from the marginal ear vein into sodium citrate (3.8% final concentration) at different time points (0 h, 2 h, 24 h, 3 d and 5 d) and immediately sent to the Clinical Laboratory, Changping hospital, Beijing, China, for measurements of coagulation parameters. At this institute, normal values for these parameters are as followings: PT: 11.5-15 sec; APTT: 28-43 sec; TT: 13-21 sec; Fibrinogen: 2-4 g/1; D-dimer: 0-0.5 μg/ml. The animals were killed 3 d after the last treatment and the major organs were excised and stained with H&E for histological examination. The following formula was used to perform dose conversion between mice and pigs:
Dp=Dm×(Kmm/Kmp)
where Dp is the dose injected into pigs, Dm is the dose used in mice, Kmm is the Dose in mg/kg to Dose in mg/m2 conversion factor of mice and Kmp is the Dose in mg/kg to Dose in mg/m2 conversion factor of the pigs.
Blinding:
All experimental procedures and quantification of results, including injections, isolation of the tumors or organs, tissue histological analysis and FACS, were done by two independent researchers.
Statistical Analysis.
Quantitative data are presented as mean±s.d. Tumor volumes were compared using a Kruskal-Wallis test followed by a Mann-Whitney test. The differences in mean volumes between treatment groups were compared using one-way analysis of variance (ANOVA) with repeated measures followed by Tukey's HSD test. Cumulative survival curves in various groups were compared using Kaplan-Meier curves followed by the Log rank test. Two-sided P values less than 0.05 were considered statistically significant. Statistics were performed using SPSS 18.0 software.
Supplementary Materials
DNA Origami Design
DNA origami is constructed in a honeycomb lattice or a square lattice, in which the rule of DNA helicity enables customized orientation of the free ends of staple strands [1]. In the present study, the rectangular DNA origami sheet is assembled into a square lattice, which means the backbone of the DNA strand rotates 270° at 8 bp intervals. This enables the free ends of the staple strands, 32 nt in length, to all lay on the same side of the rectangular DNA sheet surface. As shown in
Characterization of Free Thrombin, Thrombin-DNA Conjugates and Thrombin-Bounded DNA Sheet.
Cy5-modified oligonucleotides were conjugated to thrombin molecules via the 5′-thiol using the sulfo-SMCC chemistry described above. It was estimated that polyT DNA-labeled thrombin had an average DNA-to-protein ratio of 2.5±0.8; we use this average value of 2.5 for further calculations. Quantification of Cy5-DNA-labeled enzyme-bound origami sheet was carried out by UV-Vis spectrometry. The DNA origami concentration was quantified by measuring the absorbance at 260 nm (A260) using an extinction coefficient of 1.09×108M-1cm-1. The Cy5-S15-Thrombin was quantified by measuring the absorbance at 650 nm (A650) using an extinction coefficient of 2.5×105 M-1cm-1 [2]. The average number of thrombins on the DNA origami was calculated as follows:
Ratio(thrombin/origami)=CCy5-S15-thrombin/Corigami=(CCy5-S15/2.5)/Corigami=3.8±0.4
Additional Characterization of DNA Robots
Additional characterization of DNA robots is shown in
Thrombin molecules can be loaded on the top or the bottom surface of origami sheets. After fastening of the rectangular sheet into a tube, the top surface will be preferentially rolled inside of the tube due to curvature driving forces. Thus, thrombin can be loaded on the inside or the outside surface of origami tubes. Only thrombin loaded inside tubes can be protected and shielded before delivery to the target location in vivo. We estimated the topography through a platelet aggregation assay. For the rectangular DNA sheet structures, the long single stranded scaffold strand 7249 nt M13 phage DNA and short strands, including basic staple strands (gray strands in
The thrombin-loaded rectangular and tubular DNA origami nanostructures were applied to the platelet aggregation assay mentioned above. Additionally, nanorobot-Th and ctrl-tube-DNA origami-Th were pretreated with proteinase K for 15 min at room temperature to remove the thrombin molecules on the outside surface of the tubes. Next, the proteinase-treated nanostructures were degraded with 20 U/ml DNase I (Invitrogen, Carlsbad, Calif., USA) at 37° C. for 30 min to expose the thrombin molecules inside the tubes. The considerably low platelet aggregation using nanorobot-Thor ctrl-tube-DNA origami-Th structures without such treatment indicates that only a small amount of thrombin molecules loaded onto the outside surface of the tubes. On the other hand, aggregation results of tube structures after proteinase and DNase I treatment elicited potent platelet aggregation, reflecting the vast majority of thrombin loaded inside the tubes. The results of these experiments were used to estimate the percentage of thrombin loaded on the inside and outside surfaces of nanorobot-Th and ctrl-tube-DNA origami-Th. The data in
Supplementary characterization of the nanorobot functions are shown in
Characterization of in vivo nanorobot targeting and activity is shown in
Safety assessment of the thrombin-loaded nanorobot is shown in
§Thrombin was intravenously injected to estimate the severity of microthrombus formation. The hind-limb paralysis rate or death rate was expressed as the ratio of paralyzed or dead mice to the total animals used in each group. All mice injected with more than 15.0 U of thrombin died of acute thromboembolism. Figures in parentheses indicate total number of mice used.
†PT prothrombin time (institutional normal range: 11.5-15), APTT activated partial thromboplastin time (institutional normal range: 28-43), TT thrombin time (institutional normal range: 13-21), Fibrinogen (institutional normal range: 2-4), D-dimer (institutional normal range: 0-0.5).
§Different doses of free thrombin were injected into pigs via marginal ear vein once or three times. At the indicated time points, blood was drawn from the marginal ear vein and plasma was prepared. Plasma PT, APTT and TT and levels of fibrinogen and D-dimer were measured. Compared to controls, there were no differences in any plasma coagulation parameter after single or multiple administrations of thrombin at 150 U thrombin/pig. All pigs injected with more than 1,540 U/pig (equivalent to ~15.0 U/mouse) died of acute thromboembolism, consistent with the lethal dose obtained from mice. 0 h, before injection.
Although the foregoing specification and examples fully disclose and enable the present invention, they are not intended to limit the scope of the invention, which is defined by the claims appended hereto.
All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.
Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2017/115004 | Dec 2017 | CN | national |
| PCT/CN2018/106742 | Sep 2018 | CN | national |
This application claims priority to International Application Number PCT/CN2017/115004, filed Dec. 7, 2017 and International Application Number PCT/CN2018/106742, filed Sep. 20, 2018; this application also claims priority to U.S. Provisional Application No. 62/616,131, filed Jan. 11, 2018. The entire content of the applications referenced above are hereby incorporated by reference herein.
This invention was made with government support under R01 GM104960 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62616131 | Jan 2018 | US |
