Aptamer-based sustained-release therapy for therapeutic agents.

Abstract

This disclosure provides a composition comprising an aptamer and a molecular payload, wherein the aptamer is a sustained-release carrier for the molecular payload. The molecular payload may be a therapeutic agent. The composition thus provides a means for controlled and extended delivery of a therapeutic agent. This disclosure further provides a kit comprising the composition, a method for treating or preventing a disease or disorder, and a method for preparing the composition.

Description

[Technical Field]

[0001] Related applications This application claims priority under 35 U.S. SC § 119(e) to U.S. Provisional Application No. USSN 63 / 461,525, filed on 24 April 2023, which is incorporated in its entirety by reference herein.

[0002] Research funded by the federal government This invention was made with government support under grant number GM131728, awarded by the National Institutes of Health. The government has certain rights to this invention. [Background technology]

[0003] background Aptamers can bind to small molecules with high affinity and specificity. Their affinity and selectivity to target molecules are comparable to those of antibodies, and aptamers do not exhibit apparent biological toxicity. Aptamers have also been shown to have limited or no immunogenicity. Furthermore, aptamers do not have sequence homology to any coding or non-coding gene sequences of any organism. These attributes have enabled the use of aptamers in a wide range of applications, including diagnostics, biosensor technologies, affinity isolation, biomarker discovery, and targeted therapies. Aptamers have been used as targeting ligands for various systemically delivered drug delivery systems. However, this approach is limited to drugs that can interact with aptamers through nonspecific interactions such as charge and hydrophobicity, and / or drugs in which the aptamer functions as a targeting moiety to position the drug at a specific biological target. [Overview of the Initiative]

[0004] overview This disclosure stems from the recognition that aptamers can function as sustained-release carriers for drug delivery and other applications by utilizing their binding to molecular payloads. Binding of aptamers to molecular payloads extends the duration of payload release (and therefore, the duration of effect) while reducing systemic toxicity associated with the molecular payload. For this reason, the disclosed compositions and methods provide a novel platform for the controlled delivery of therapeutic agents in various situations, as well as for the treatment of a wide range of diseases and / or conditions.

[0005] In one aspect, a composition is provided comprising an aptamer and a molecular payload, wherein the molecular payload is bound to the aptamer, and the aptamer is a sustained-release carrier for the molecular payload.

[0006] In another aspect, a method for treating a disease or condition is provided, the method comprising administering an effective amount of a composition.

[0007] In another aspect, a method is provided for preparing the composition, the method comprising: providing a molecular payload; identifying or preparing an aptamer to bind to the molecular payload (e.g., specifically binding); and combining the molecular payload and the aptamer in the composition.

[0008] In another aspect, a kit is provided which includes the composition and instructions for using the composition.

[0009] Details of specific embodiments of the present invention are described in the detailed descriptions of the specific embodiments, as set forth below. Other features, purposes, and advantages of the present invention will become apparent from the definitions, drawings, examples, and claims.

[0010] definition The terms "composition" and "formulation" are used interchangeably.

[0011] The “subjects” to which the administration is intended refers to humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, or adolescents), or adult subjects (e.g., young adults, middle-aged adults, or elderly adults)), or non-human animals. In certain embodiments, non-human animals are mammals (e.g., primates (e.g., crab-eating macaques or rhesus macaques), commercially relevant mammals (e.g., cattle, pigs, horses, sheep, goats, cats, or dogs), or birds (e.g., commercially relevant birds such as chickens, ducks, geese, or turkeys)). In certain embodiments, non-human animals are fish, reptiles, or amphibians. Non-human animals may be male or female at any developmental stage. Non-human animals may be transgenic animals or genetically modified animals. The term “patient” refers to a human subject requiring treatment for a disease or disorder.

[0012] The term “biological specimen” means any specimen that includes tissue specimens (such as tissue sections and needle biopsies); cell specimens (for example, cytological smears (such as Pap or blood smears), or specimens of cells obtained by microdissection); specimens of whole organisms (such as specimens of yeast or bacteria); or any specimen that includes cell fractions, fragments, or organelles (such as those obtained by lysing cells and separating their components by centrifugation or other means). Other examples of biological specimens include blood, serum, urine, semen, feces, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsy tissue (for example, obtained by surgical biopsy or needle biopsy), nipple aspirate, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material that contains biomolecules derived from the first biological specimen.

[0013] The terms “administer,” “dosing,” or “administer” refer to embedding, absorbing, ingesting, injecting, inhaling, or otherwise introducing any compound or composition described herein into or onto a subject.

[0014] The terms "treatment", "treating", and "treatment" refer to reversing, alleviating, delaying, or inhibiting the progression of a disease or disorder described herein. In some embodiments, treatment may be administered after or upon observation of the onset of one or more signs or symptoms of the disease or disorder. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a symptom history). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

[0015] The terms "prevent", "prevention", or "preventive" refer to prophylactic treatment of a subject who currently has no disease or disorder but is at risk of developing a disease or disorder, or who has had a disease or disorder in the past and currently has no disease or disorder but is at risk of relapse. In certain embodiments, the subject is at higher risk of developing a disease or disorder or of relapse than an average healthy member of the population of subjects.

[0016] The terms "condition", "disease", and "disorder" are used interchangeably.

[0017] An "effective amount" of a compound described herein refers to an amount sufficient to induce a desired biological response. The effective amount of a compound described herein may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health status of the subject. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactic treatment. In certain embodiments, the effective amount is the amount in a single dose of a compound described herein. In certain embodiments, the effective amount is a combined amount in multiple doses of a compound described herein.

[0018] The "therapeutically effective amount" of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. The therapeutically effective amount of a compound means the amount of a therapeutic agent that provides a therapeutic benefit in the treatment of a condition, either alone or in combination with other treatments. The term "therapeutically effective amount" can encompass an amount that improves overall treatment, reduces or avoids symptoms, signs, or causes of a condition, and / or enhances the therapeutic effectiveness of another therapeutic agent. In certain embodiments, the therapeutically effective amount is, by way of example, an amount sufficient to inhibit a biological target (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% inhibition of the target). In certain embodiments, the therapeutically effective amount is an amount sufficient to treat a disease or condition.

[0019] The "preventive effective amount" of a compound described herein is an amount sufficient to prevent a condition or one or more signs or symptoms associated with the condition or to prevent its recurrence. The preventive effective amount of a compound means the amount of a therapeutic agent that provides a preventive benefit in the prevention of a condition, either alone or in combination with other agents. The term "preventive effective amount" can encompass an amount that improves overall prevention or enhances the preventive effectiveness of another preventive agent. In certain embodiments, the preventive effective amount is an amount sufficient to inhibit the expression of a target nucleic acid. In certain embodiments, the preventive effective amount is an amount sufficient to treat a disease or disorder.

[0020] The term "molecular payload" refers to a molecule or species that functions to modulate and / or induce a biological outcome.

[0021] The term "nucleic acid" refers to biopolymers, macromolecules, that are essential to all known forms of living organisms. Nucleic acids include nucleotides, which are monomers composed of three components: a five-carbon sugar, a phosphate group, and a nitrogen-containing base. The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0022] The terms “nucleoside” and “nucleotide” refer to moieties that contain not only known purine and pyrimidine bases, but also other modified heterocyclic bases. In certain embodiments, nucleic acid bases are replaced by non-nucleic acid base moieties. Modified nucleosides or nucleotides can also include modifications on the sugar moiety, for example, one or more hydroxyl groups being replaced by hydrogen, halogens (e.g., fluoro), aliphatic groups, or functionalized as ethers or amines. The term “nucleotide unit” is intended to encompass nucleosides and nucleotides, as well as modified forms of both.

[0023] As used herein, the term “polynucleotide” encompasses polymers of deoxyribose nucleic acids (DNA) or ribose nucleic acids (RNA) having non-natural bases such as A, G, T, and C, which are standard nucleotide bases for DNA, or A, G, C, and U, in the case of RNA, a subset of standard nucleotide bases using fewer than four of the standard nucleotide bases, as well as 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds), pyrrole-2-carbaldehyde (Pa), 2-amino-8-(2-thienyl)purine)(s), f2-amino-6-(N,N-dimethylamino)purine(x), pyridine-2-one(y), 3-nitropyrrole, 5-nitroindole, and 4-[3-(6-aminohexaneamide)-1-propynyl]-2-nitropyrrole (Px) or other non-natural bases currently known or to be discovered later. The examples provided in this disclosure may refer to DNA or RNA, or any of the nucleotide bases A, G, T, C, and U. However, this should be understood as illustrative only and does not preclude implementations using RNA, DNA-RNA hybrids, and / or non-natural bases. In certain embodiments, polynucleotides may include chemically modified nucleotides.

[0024] The term "oligonucleotide" refers to nucleic acid compounds in the form of oligomers up to 200 nucleotides in length. Examples of oligonucleotides include, but are not limited to, DNA, RNA, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNA, gapmers, mixmers, phosphorodiamidite morpholino, peptide nucleic acids, aptamers, guide nucleic acids (e.g., Cas9 guide RNA), etc. Oligonucleotides may be single-stranded or double-stranded. In some embodiments, oligonucleotides may contain one or more modified nucleotides (e.g., 2'-O-methylglycosulfate, purine, or pyrimidine modification). In some embodiments, oligonucleotides may contain one or more modified internucleotide links. In some embodiments, oligonucleotides may contain one or more phosphorothioate links, which may be in the stereochemical configuration of Rp or Sp.

[0025] The terms "sequence" or "nucleotide sequence" refer to a sequence or order of nucleic acid bases or nucleotides, written as a sequence of letters using standard nucleotide nomenclature.

[0026] Where used herein, unless otherwise indicated, the term “complementary” means, when used to describe a first nucleotide sequence with respect to a second nucleotide sequence, the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize with the oligonucleotide or polynucleotide containing the second nucleotide sequence (forming base-hydrogen bonds under mammalian physiological conditions (or equivalent conditions in vitro)) and to form a double-stranded or double-helical structure under certain conditions. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimics, insofar as they satisfy the above requirement regarding the ability to hybridize.

[0027] As used herein, “perfectly complementary” or “completely complementary” means that all (100%) of the bases in the contiguous sequence of the first polynucleotide hybridize with the same number of bases in the contiguous sequence of the second polynucleotide. The contiguous sequence may consist of all or part of the first or second nucleotide sequence.

[0028] As used herein, “partially complementary” means that in a hybridized pair of nucleic acid base sequences, at least 70% (but not all) of the bases in the contiguous sequence of the first polynucleotide hybridize with the same number of bases in the contiguous sequence of the second polynucleotide.

[0029] As used herein, “substantially complementary” means that in a hybridized pair of nucleic acid sequences, at least 85% (but not all) of the bases in the sequence of the first polynucleotide hybridize with the same number of bases in the sequence of the second polynucleotide.

[0030] The terms “biologic,” “biologic drug,” and “biological product” refer to a broad range of products, including vaccines, blood and blood components, allergenics, somatic cells, gene therapies, tissues, nucleic acids, and proteins. Biologics may encompass sugars, proteins, nucleic acids, or complex combinations thereof, or they may be living organisms such as cells and tissues. Biologics may be isolated from various natural sources (e.g., humans, animals, microorganisms) or produced by biotechnological methods and other techniques.

[0031] The terms “small molecule” or “small molecule therapeutic” refer to molecules with relatively low molecular weight, whether naturally occurring or artificially created (e.g., via chemical synthesis). Typically, small molecules are organic compounds (i.e., they contain carbon). Small molecules may contain numerous carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyls, carbonyls, and heterocycles). In certain embodiments, the molecular weight of a small molecule may be about 1,000 g / mol or less, about 900 g / mol or less, about 800 g / mol or less, about 700 g / mol or less, about 600 g / mol or less, about 500 g / mol or less, about 400 g / mol or less, about 300 g / mol or less, about 200 g / mol or less, or about 100 g / mol or less. In certain embodiments, the molecular weight of the small molecule is at least about 100 g / mol, at least about 200 g / mol, at least about 300 g / mol, at least about 400 g / mol, at least about 500 g / mol, at least about 600 g / mol, at least about 700 g / mol, at least about 800 g / mol, at least about 900 g / mol, or at least about 1,000 g / mol. Combinations of the above ranges (for example, at least about 200 g / mol and less than or equal to about 500 g / mol) are also possible. In certain embodiments, the small molecule is a therapeutically active substance, such as a drug (for example, a molecule approved by the U.S. Food and Drug Administration as defined in the Code of Federal Regulations (CFR)). The small molecule may also be complexed with one or more metal atoms and / or metal ions. In this example, the small molecule is also referred to as a “small organometallic molecule”. Preferred small molecules are biologically active in that they produce a biological effect in animals, preferably mammals, and more preferably humans. Small molecules include, but are not limited to, radionuclides and contrast agents. In certain embodiments, small molecules are drugs. Preferably, but not necessarily, the drugs are those that have already been deemed safe and effective for use in humans or animals by the appropriate government or regulatory body.For example, drugs approved for use in humans are enumerated by the FDA under 21 CFR §§330.5, 331-361, and 440-460, which are incorporated herein by reference; drugs for veterinary use are enumerated by the FDA under 21 CFR §§500-589, which are incorporated herein by reference. All enumerated drugs are considered permissible for use in accordance with the present invention.

[0032] The terms “therapeutic compound,” “therapeutic agent,” or “therapeutic part” refer to any substance having therapeutic properties that produce a desired, usually beneficial effect. For example, therapeutic compounds, therapeutic agents, and therapeutic parts may treat and / or induce remission of a disease or disorder. The therapeutic compounds, therapeutic agents, and therapeutic parts disclosed herein may be biological agents or small molecule therapeutics, or combinations thereof.

[0033] The term “sustained-release carrier” refers to an entity that can be associated with, interact with, chaperoning, and / or facilitate the delivery of a molecular payload (e.g., a therapeutic agent) to a desired location within the body of a subject. A sustained-release carrier may facilitate the local sustained release of the molecular payload. A sustained-release carrier may facilitate the systemic sustained release of the molecular payload. A sustained-release carrier may release the molecular payload slowly over a long period of time to allow for less frequent administration of the molecular payload. A sustained-release carrier may also provide a longer duration of therapeutic effect through slow absorption into the bloodstream and / or tissues.

[0034] The term "depot" typically refers to a composition or formulation containing a molecular payload that is placed in a single location within the body and slowly releases the molecular payload over an extended period. Depots are typically administered intramuscularly or subcutaneously to release the molecular payload to a local or systemic site.

[0035] The term “sustained release” (also called sustained release or controlled release) refers to the continuous or intermittent release of a molecular payload introduced into a subject's body over a period of time and at a therapeutic level sufficient to achieve the desired therapeutic effect throughout that period. The rate at which the molecular payload is released is slower than the rate at which the molecular payload is released when administered alone or in a non-sustained-release formulation. Sustained-release formulations may be prepared, for example, as films, plates, pellets, microparticles, microspheres, microcapsules, spheroids, shaped derivatives, and pastes. Formulations may also be in a form suitable for suspension in isotonic saline, physiological buffer, or other solutions acceptable for injection into a patient. Furthermore, formulations may be used in conjunction with any implantable, insertable, or injectable system that is useful in relation to the embodiments herein, including but not limited to parenteral formulations, microspheres, microcapsules, gels, pastes, implantable rods, pellets, plates, or fibers, as understood by those skilled in the art. [Brief explanation of the drawing]

[0036] Simple description of the drawing [Figure 1A-1C] Figures 1A-1E show aptamers for drug delivery. Figure 1A is a schematic diagram of aptamer construction, drug binding (site 1 sodium channel blocker (S1SCB) is used as an example), and subsequent in vivo release of the drug. Figure 1B shows the chemical structural units of the S1SCB-bound aptamer. PO: phosphodiester; PS: phosphorothioate. Figure 1C shows the chemical structures of tetrodotoxin (TTX), saxitoxin (STX), and bupivacaine hydrochloride (HCl). [Figure 1D-1E]Figure 1D shows microscale thermophoresis analysis of TTX binding affinity of TTX-binding aptamers (PO and PS) and aptamers with scrambled sequences (Scr-PS). The Y-axis shows the percentage of TTX bound to the aptamer. n=3 independent experiments. Figure 1E shows cumulative TTX release from aptamer / TTX complexes and controls at 12 hours. The TTX concentration for each group was 42 μM. Data are shown as mean ± sd, n=4 independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison test. NS, not statistically significant.

[0037] [Figure 2A-2B] Figures 2A-2H show peripheral nerve block by the PS / TTX complex. Figure 2A shows peripheral nerve block by 42 μM TTX, either free or complexed with aptamers (PO, PS, Scr-PS) (n=6 biologically independent animals for TTX and PS / TTX; n=4 biologically independent animals for PO / TTX and Scr-PS / TTX). Figure 2B shows peripheral nerve block by 22 μM STX alone or STX combined with a TTX-specific PS aptamer (n=4 biologically independent animals for STX and PS / STX). [Figure 2C-2D] Figure 2C shows peripheral nerve blockade by bupivacaine alone or in combination with a TTX-specific PS aptamer (n=6 biologically independent animals for bupivacaine and PS / bupivacaine). The bupivacaine concentration in each formulation was 15.4 mM. Figure 2D shows peripheral nerve blockade by PS / TTX complexes with varying molar ratios using 42 μM TTX. Data are mean ± sd. (n=6 biologically independent animals for TTX and PS / TTX 20:1; n=4 biologically independent animals for PS / TTX 1:1, 2:1, 5:1, 10:1 and 40:1). [Figure 2E-2F]Figure 2E shows sciatic nerve block with free TTX and PS / TTX (2:1) in the injected (left panel) and contralateral (right panel) hind limbs. Dagger symbols indicate 100% mortality. Data are mean ± sd, with n=6 biologically independent animals for TTX 42 μM and TTX 52 μM; n=4 biologically independent animals for TTX 31 μM, TTX 63 μM, and PS / TTX groups. Figure 2F shows the frequency of nerve block in the contralateral (uninjected) leg (n=6 biologically independent animals for TTX 42 μM and TTX 52 μM; n=4 biologically independent animals for TTX 31 μM and PS / TTX groups). [Figure 2G-2H] Figure 2G shows the mortality rate of animals after treatment with TTX and PS / TTX (2:1) (n=6 biologically independent animals for TTX 42 μM and TTX 52 μM; n=4 biologically independent animals for TTX 31 μM, TTX 63 μM, and PS / TTX groups). Figure 2H shows the effect of 55 μM epinephrine on the duration of sensory nerve blockade from PS / TTX (2:1, 73 μM, 84 μM, and 104 μM TTX). Data are mean ± sd, and n=4 rats per group. Statistical comparisons were performed using Student's t-test (two-tailed).

[0038] [Figure 3A-3B] Figures 3A–3C show the tissue distribution of PS aptamers. Figure 3A shows a representative time course for the retention of Cy5.5-labeled aptamers (PO or PS) or free Cy5.5 at the injection site, as monitored by IVIS. Colors represent fluorescence from Cy5.5. Figure 3B shows the quantification of fluorescence intensity over time (as a percentage of intensity at time = 0, immediately after sciatic nerve injection), derived from the data in Figure 3A. Data are mean ± sd, and n = 4 rats per group. Statistical comparisons were performed using Student's t-test (two-tailed). [Figure 3C]Figure 3C shows representative confocal images of frozen sections of rat sciatic nerve and surrounding tissue 4 hours after injection of Cy5.5 or Cy5.5-labeled aptamers (PO and PS) into the sciatic nerve. Each experiment was independently repeated three times to obtain similar results.

[0039] [Figure 4] Figure 4 shows tissue responses to free TTX, PS aptamer, and PS / TTX complex. H&E: Representative hematoxylin-eosin stained sections of muscle and adjacent loose connective tissue 4 and 14 days after sciatic nerve injection of TTX (52 μM), PS aptamer (146 μM), or PS / TTX (2:1, 73 μM TTX) in 0.3 mL of PBS. Toluidine blue: Representative toluidine blue stained sections of nerves 4 and 14 days after sciatic nerve injection of the above preparations. n = 4 animals in each group.

[0040] [Figure 5A-5B] Figures 5A-5B show the peripheral nerve blocking effect of STX-binding PS aptamer (PSAPSTX) / STX. Figure 5A shows the duration of sensory nerve block in the injected leg and the uninjected (contralateral) limb after sciatic nerve injection (n=4 biologically independent animals). Data are mean ± sd. Statistical comparisons were performed using Student's t-test (two-sided). Figure 5B shows the tissue response to STX (33 μM), PSAPSTX (90 μM), and the PSAPSTX / STX (2:1, 45 μM STX) complex at 4 and 14 days after administration.

[0041] [Figure 6A-6B] Figures 6A and 6B show a comparison of aptamer sizes. Figure 6A shows the size of aptamer / TTX conjugates at different molar ratios of aptamer to TTX, as measured by dynamic light scattering (DLS). The TTX concentration was fixed (42 μM). Data are mean ± sd, and n=3 independent experiments. Statistical comparisons were performed using Student's t-test (two-tailed). Figure 6B shows a representative number-mean size distribution of aptamer / TTX.

[0042] [Figures 7A-7B] Figures 7A-7B show the TTX release kinetics from aptamer / TTX conjugates and controls over 24 hours. Figure 7A shows free TTX vs. PO / TTX vs. PS / TTX. Figure 7B shows free TTX vs. Scr-PS / TTX. The TTX concentration for each group was 42 μM. Data are shown as mean ± sd, n=4 independent experiments. Statistical comparisons were performed using Student's t-test (two-tailed). NS, not statistically significant.

[0043] [Figure 8A-8B] Figures 8A-8B show the results of MTS cytotoxicity assays for myotoxicity (in C2C12 cells) and neurotoxicity (in PC12 cells) after 24-hour exposure to the following groups: free TTX, PO aptamer, PS aptamer, PO / TTX(2:1), or PS / TTX(2:1). The TTX concentration was 73 μM and the aptamer concentration was 146 μM. Data are mean ± sd, and n = 4 biological replicas per group.

[0044] [Figure 9A-9B] Figures 9A-9D show typical thermal latency time courses after sciatic nerve injection of aptamer formulations. All rats were injected with 42 μM TTX using 0.3 mL of PBS containing (Figure 9A) free TTX, (Figure 9B) PO / TTX (20:1), (Figure 9C) PS / TTX (20:1), or (Figure 9D) scrambled PS / TTX (20:1). Data are mean ± sd, and n = 4 biologically independent animals per group. [Figure 9C-9D]Figures 9A-9D show typical thermal latency time courses after sciatic nerve injection of aptamer formulations. All rats were injected with 42 μM TTX using 0.3 mL of PBS containing (Figure 9A) free TTX, (Figure 9B) PO / TTX (20:1), (Figure 9C) PS / TTX (20:1), or (Figure 9D) scrambled PS / TTX (20:1). Data are mean ± sd, and n = 4 biologically independent animals per group.

[0045] [Figure 10A-10B] Figures 10A-10B show typical time courses of thermal latency after sciatic nerve injection of (Figure 10A) PO aptamer or (Figure 10B) PS aptamer. All injections were administered in 0.3 mL of PBS at an aptamer concentration of 836 μM. Data are mean ± sd, and n = 4 biologically independent animals per group.

[0046] [Figure 11] Figure 11 shows the viscosity of PS / TTX (20:1) and Scr-PS / TTX (20:1). The TTX concentration was 42 μM. There was no statistically significant difference between these two groups (n=3, p>0.05). Data are mean ± sd, and n=3 independent experiments. Statistical comparisons were performed using Student's t-test (two-tailed).

[0047] [Figure 12] Figure 12 shows the viscosity of PS aptamer alone (84 μM) and PS aptamer at different molar ratios with TTX. The TTX concentration was 42 μM. There was no difference in the measured viscosity at the various ratios (n=3, all comparisons, p>0.05). Data are mean ± sd, n=3 independent experiments. Statistical comparisons were performed using Student's t-test (two-tailed).

[0048] [Figure 13]Figure 13 shows a representative comparison of the durations of sensory and motor block. Rats were injected with free TTX or PS / TTX (2:1) in 0.3 mL of PBS. The TTX concentration for each group is indicated in the figure. The P-value is the comparison of the duration of sensory block for each formulation to the duration of motor block. Data are mean ± sd; n = 4 biologically independent animals per group. Statistical comparisons were performed using Student's t-test (two-tailed).

[0049] [Figure 14] Figure 14 shows the typical time course of heat latency after sciatic nerve injection with PS / TTX (2:1, 73 μM TTX) or in combination with 55 μM epinephrine. Data are mean ± sd; n = 4 biologically independent animals per group.

[0050] [Figure 15] Figure 15 shows representative photographs of the severed sciatic nerve (white arrow) and surrounding tissue in rats 4 hours after sciatic nerve injection. The blue color is from Cy5.5.

[0051] [Figure 16] Figure 16 shows confocal images of frozen sections of rat sciatic nerve and surrounding tissue 4 hours after injection of Cy5.5-labeled PS aptamer into the sciatic nerve. Each experiment was independently repeated three times, yielding similar results.

[0052] [Figure 17] Figure 17 shows the cytotoxicity of STX, STX-binding PS aptamer (PSAPSTX), and PSAPSTX conjugates in C1C12 and PC12 cells. The STX concentration was 45 μM, and the PSAPSTX concentration was 90 μM. The molar ratio of PSAPSTX:STX was 2:1. Data are mean ± sd; n = 4 biological replicas per group.

[0053] [Figure 18A]Figures 18A–C show aptamers (Apt) for the sustained release of different drugs. Different drug-binding aptamers, including serotonin (Ser)-binding Apt (AptSer), kanamycin (Kan)-binding Apt (AptKan), and insulin (Ins)-binding Apt (AptIns), were used in this study. Figure 18A: In vitro drug release profile of Ser from the Apt-Ser complex (top) and cumulative release of AptSer from the corresponding Apt-Ins complex (bottom). Figure 18B: In vitro drug release profile of Kan from the Apt-Kan complex (top) and cumulative release of AptKan from the corresponding Apt-Ins complex (bottom). Figure 18C: In vitro drug release profile of Ins from the Apt-Ins complex (top) and cumulative release of AptIn from the corresponding Apt-Ins complex (bottom). n=4 independent experiments. Data are presented as mean ± sd. Statistical significance was assessed by one-way ANOVA with Tukey post-hoc tests in a, b, c, d, e, and f. *P<0.05, **P<0.01, ****P<0.0001. [Figure 18B]Figures 18A–C show aptamers (Apt) for the sustained release of different drugs. Different drug-binding aptamers, including serotonin (Ser)-binding Apt (AptSer), kanamycin (Kan)-binding Apt (AptKan), and insulin (Ins)-binding Apt (AptIns), were used in this study. Figure 18A: In vitro drug release profile of Ser from the Apt-Ser complex (top) and cumulative release of AptSer from the corresponding Apt-Ins complex (bottom). Figure 18B: In vitro drug release profile of Kan from the Apt-Kan complex (top) and cumulative release of AptKan from the corresponding Apt-Ins complex (bottom). Figure 18C: In vitro drug release profile of Ins from the Apt-Ins complex (top) and cumulative release of AptIn from the corresponding Apt-Ins complex (bottom). n=4 independent experiments. Data are presented as mean ± sd. Statistical significance was assessed by one-way ANOVA with Tukey post-hoc tests in a, b, c, d, e, and f. *P<0.05, **P<0.01, ****P<0.0001. [Figure 18C]Figures 18A–C show aptamers (Apt) for the sustained release of different drugs. Different drug-binding aptamers, including serotonin (Ser)-binding Apt (AptSer), kanamycin (Kan)-binding Apt (AptKan), and insulin (Ins)-binding Apt (AptIns), were used in this study. Figure 18A: In vitro drug release profile of Ser from the Apt-Ser complex (top) and cumulative release of AptSer from the corresponding Apt-Ins complex (bottom). Figure 18B: In vitro drug release profile of Kan from the Apt-Kan complex (top) and cumulative release of AptKan from the corresponding Apt-Ins complex (bottom). Figure 18C: In vitro drug release profile of Ins from the Apt-Ins complex (top) and cumulative release of AptIn from the corresponding Apt-Ins complex (bottom). n=4 independent experiments. Data are presented as mean ± sd. Statistical significance was assessed by one-way ANOVA with Tukey post-hoc tests in a, b, c, d, e, and f. *P<0.05, **P<0.01, ****P<0.0001. [Modes for carrying out the invention]

[0054] Detailed description of specific aspects In this specification, non-covalent complexing of molecular payloads (e.g., therapeutic agents) with aptamers has been demonstrated to significantly extend the duration of therapeutic effect while reducing systemic toxicity (e.g., local anesthesia from site 1 sodium channel blockers). This technique provides a drug delivery system for a wide range of drugs and indications.

[0055] In particular, aptamers provide sustained-release carriers for drug delivery where extended therapeutic effects are desirable and systemic toxicity from rapid drug release can be a concern. In addition to improving the duration of effect and limiting systemic toxicity, this new paradigm of drug delivery further provides means for delivering hydrophilic compounds that have historically been difficult to deliver physically via existing known methods of drug delivery (e.g., encapsulation).

[0056] In one aspect, a composition is provided comprising an aptamer and a molecular payload, wherein the molecular payload is bound to the aptamer, and the aptamer is a sustained-release carrier for the molecular payload. In a particular embodiment, the composition is a depot for the molecular payload.

[0057] Aptamer As described herein, aptamers are sustained-release carriers. In certain embodiments, sustained-release carriers promote local sustained-release of molecular payloads. In certain embodiments, sustained-release carriers promote systemic sustained-release of molecular payloads. In certain embodiments, aptamers specifically bind to molecular payloads.

[0058] In certain embodiments, the aptamer is an oligonucleotide. In certain embodiments, the aptamer is a single-stranded oligonucleotide. In certain embodiments, the aptamer is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In certain embodiments, the aptamer is deoxyribonucleic acid (DNA). In certain embodiments, the aptamer is ribonucleic acid (RNA).

[0059] The aptamer oligonucleotide may contain one or more modified nucleotides. In certain embodiments, the modified nucleotides include 2'-modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2' position of a five-membered sugar ring). Modified nucleotides include, but are not limited to, 2'-modified nucleotides, 2'-O-methylnucleotides (represented herein as lowercase "m" in the nucleotide sequence), 2'-deoxy-2'-fluoronucleotides (represented herein as lowercase "f" in the nucleotide sequence), 2'-deoxynucleotides, 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides, 2'-aminonucleotides, 2'-alkylnucleotides, 3'-to-3' linked (inverted) nucleotides, nucleotides containing non-natural bases, locked nucleotides, cross-linked nucleotides, peptide nucleic acids, 2',3'-seco nucleotide mimes (non-locked nucleic acid base analogs), locked nucleotides, 3'-O-methoxy (2' nucleotide linked) nucleotides, 2'-F-arabinonucleotides, morpholinonucleotides, vinylphosphonate deoxyribonucleotides, vinylphosphonate nucleotides, and debasalized nucleotides. It is not necessary for all positions in a given compound to be uniformly modified. Conversely, more than one modification may be incorporated into a single oligomer, or even into its single nucleotide. Oligonucleotides may be synthesized and / or modified by methods known in the art. Modifications in each nucleotide are independent of modifications in other nucleotides.

[0060] Modified nucleic acid bases include 5-substituted pyrimidines, 6-azapyrimidines, N-2-, N-6- and O-6-substituted purines (e.g., 2-aminopropyladenine), 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azo-uracil, 6-a This includes synthetic and native nucleic acid bases such as zo-cytosine, 6-azo-thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-substituted uracils and cytosines (e.g., 5-halouracil and cytosine (e.g., 5-bromouracil and 5-bromocytosine)), 5-trifluoromethyluracil, 5-trifluoromethylcytosine), 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0061] In certain embodiments, the aptamer is an oligonucleotide sequence of 10-200, 10-150, 10-100, 10-60, 15-60, 15-40, 15-35, 20-35, 25-35, 30-35, or 30-34 nucleotides. In certain embodiments, the aptamer is an oligonucleotide sequence of 15-60 nucleotides. In certain embodiments, the aptamer is an oligonucleotide sequence of 15-40 nucleotides. In certain embodiments, the aptamer is an oligonucleotide sequence of 25-35 nucleotides. In certain embodiments, the aptamer is an oligonucleotide sequence of 30-35 nucleotides.

[0062] In certain embodiments, the aptamer is an oligonucleotide sequence of approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides. In certain embodiments, the aptamer is an oligonucleotide sequence of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides. In certain embodiments, the aptamer is a 21-nucleotide oligonucleotide sequence. In certain embodiments, the aptamer is a 30-nucleotide oligonucleotide sequence. In certain embodiments, the aptamer is a 34-nucleotide oligonucleotide sequence. In certain embodiments, the aptamer is a 44-nucleotide oligonucleotide sequence.

[0063] For oligonucleotides, any nucleotide may be linked by phosphate-containing or non-phosphate-containing covalent nucleosides or nucleotide links. Modified nucleosides or nucleotide links or skeletons include phosphorothioate groups (represented herein as an asterisk (*) or lowercase "s" after a nucleotide, as in As, mUs, and fAx), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotryesters, aminoalkyl phosphotryesters, 3'-alkylene phosphonates, and methyl and other alkyl phosphonates including chiral phosphonates, phosphinates, and 3'-aminophosphoramides. This includes, but is not limited to, phosphoramidates containing phosphates and aminoalkylphosphorumidates, thionophosphorumidates, thionoalkyl-phosphonates, thionoalkylphosphotryesters, morpholino linkages, boranophosphates having the usual 3'-5' linkages, 2'-5' linked analogues of boranophosphates, and boranophosphates having inverted polarity where adjacent pairs of 3'-5' linkages of nucleoside units are 5'-3' linkages or 2'-5' linkages are 5'-2' linkages. In some embodiments, the modified internucleoside or internucleotide linkages or skeletons lack a phosphorus atom. The phosphorus-lacking modified internucleoside or internucleotide linkages include, but are not limited to, short-chain alkyl or cycloalkyl sugar linkages, mixed heteroatom and alkyl or cycloalkyl sugar linkages, or one or more short-chain heteroatom or heterocyclic sugar linkages. In some embodiments, the modified internucleoside or internucleotide skeletons include, but are not limited to, siloxane skeletons, sulfide skeletons, sulfoxide skeletons, sulfone skeletons, formacetyl and thioformacetyl skeletons, methyleneformacetyl and thioformacetyl skeletons, alkene-containing skeletons, sulfamate skeletons, methyleneimino and methylenehydrazino skeletons, sulfonate and sulfonamide skeletons, amide skeletons, and other skeletons having mixed N, O, S and CH2 components.

[0064] In certain embodiments, the aptamer is an oligonucleotide sequence having at least one phosphorothioate internucleotide linkage. In certain embodiments, the aptamer is an oligonucleotide sequence in which all internucleotide links are phosphorothioate internucleotide links. In certain embodiments, the aptamer is an oligonucleotide sequence having a complete phosphorothioate backbone.

[0065] In certain embodiments, the aptamer is a single-stranded deoxyribonucleic acid (DNA) having a complete phosphorothioate backbone. In certain embodiments, the aptamer is a single-stranded deoxyribonucleic acid (DNA) of 15 to 60 nucleotides having a complete phosphorothioate backbone. In certain embodiments, the aptamer is a single-stranded deoxyribonucleic acid (DNA) of 15 to 60 nucleotides having a complete phosphorothioate backbone. In certain embodiments, the aptamer is a single-stranded deoxyribonucleic acid (DNA) of 15 to 40 nucleotides having a complete phosphorothioate backbone. In certain embodiments, the aptamer is a single-stranded deoxyribonucleic acid (DNA) of 25 to 35 nucleotides having a complete phosphorothioate backbone. In certain embodiments, the aptamer is a single-stranded deoxyribonucleic acid (DNA) of 30 to 35 nucleotides having a complete phosphorothioate backbone. In certain embodiments, the aptamer is a single-stranded deoxyribonucleic acid (DNA) of approximately 30 nucleotides having a complete phosphorothioate backbone. In certain embodiments, the aptamer is a single-stranded deoxyribonucleic acid (DNA) of approximately 34 nucleotides with a complete phosphorothioate backbone.

[0066] In a particular embodiment, the nucleotide sequence of the aptamer has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0067] In a particular embodiment, the nucleotide sequence of the aptamer has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0068] In a particular embodiment, the nucleotide sequence of the aptamer has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 5.

[0069] In a particular embodiment, the nucleotide sequence of the aptamer has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 5.

[0070] In a particular embodiment, the nucleotide sequence of the aptamer has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the nucleotide sequence of SEQ ID NO: 1.

[0071] In a particular embodiment, the nucleotide sequence of the aptamer has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the nucleotide sequence of SEQ ID NO: 2.

[0072] In a particular embodiment, the nucleotide sequence of the aptamer has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the nucleotide sequence of SEQ ID NO: 3.

[0073] In a particular embodiment, the nucleotide sequence of the aptamer has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the nucleotide sequence of SEQ ID NO: 4.

[0074] In a particular embodiment, the nucleotide sequence of the aptamer has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the nucleotide sequence of SEQ ID NO: 5.

[0075] In a particular embodiment, the nucleotide sequence of the aptamer has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the nucleotide sequence of SEQ ID NO: 6.

[0076] In a particular embodiment, the nucleotide sequence of the aptamer has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the nucleotide sequence of SEQ ID NO: 7.

[0077] In a particular embodiment, the nucleotide sequence of the aptamer has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the nucleotide sequence of SEQ ID NO: 8.

[0078] In a particular embodiment, the nucleotide sequence of the aptamer has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the nucleotide sequence of SEQ ID NO: 9.

[0079] In a particular embodiment, the nucleotide sequence of the aptamer has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the nucleotide sequence of SEQ ID NO: 10.

[0080] In a particular embodiment, the nucleotide sequence of the aptamer has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the nucleotide sequence of SEQ ID NO: 11.

[0081] In certain embodiments, the aptamer is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. In certain embodiments, the aptamer is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In certain embodiments, the aptamer is SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 5. In certain embodiments, the aptamer is SEQ ID NO: 2 or SEQ ID NO: 5. In certain embodiments, the aptamer is SEQ ID NO: 1. In certain embodiments, the aptamer is SEQ ID NO: 2. In certain embodiments, the aptamer is SEQ ID NO: 3. In certain embodiments, the aptamer is SEQ ID NO: 4. In certain embodiments, the aptamer is SEQ ID NO: 5. In certain embodiments, the aptamer is SEQ ID NO: 6. In certain embodiments, the aptamer is SEQ ID NO: 7. In certain embodiments, the aptamer is SEQ ID NO: 8. In a particular embodiment, the aptamer is sequence number 9. In a particular embodiment, the aptamer is sequence number 10. In a particular embodiment, the aptamer is sequence number 11.

[0082] In certain embodiments, the aptamer is not a targeting portion (i.e., the aptamer does not bind to the biological target of interest). In certain embodiments, the aptamer is not a therapeutic agent. In certain embodiments, the aptamer is a therapeutic agent but does not function as one in the composition.

[0083] Molecular payload In certain embodiments, the molecular payload is a therapeutic portion or therapeutic agent. In certain embodiments, the molecular payload is a therapeutic agent. In certain embodiments, the molecular payload is a biological agent or a small molecule. In certain embodiments, the molecular payload is a combination of a biological agent and a small molecule (e.g., and an antibody-drug conjugate). In certain embodiments, the molecular payload is a polynucleotide, polypeptide, or small molecule. In certain embodiments, the molecular payload is a small molecule. In certain embodiments, the molecular payload is a biological agent.

[0084] In certain embodiments, the molecular payload may be an anesthetic, antibiotic, or neurotransmitter.

[0085] In certain embodiments, the molecular payload is an anesthetic. In certain embodiments, the molecular payload is a site 1 sodium channel blocker. In certain embodiments, the molecular payload is tetrodotoxin or saxitoxin. In certain embodiments, the molecular payload is tetrodotoxin. In certain embodiments, the molecular payload is saxitoxin.

[0086] In certain embodiments, the molecular payload is an antibiotic. In certain embodiments, the molecular payload is an aminoglycoside antibiotic. In certain embodiments, the molecular payload is kanamycin. In certain embodiments, the molecular payload is a kanamycin analog.

[0087] In certain embodiments, the molecular payload is a neurotransmitter. In certain embodiments, the molecular payload is serotonin. In certain embodiments, the molecular payload is a serotonin analog.

[0088] In certain embodiments, the molecular payload is a polypeptide. In certain embodiments, the molecular payload is a peptide hormone. In certain embodiments, the molecular payload is insulin. In certain embodiments, the molecular payload is synthetic insulin. In certain embodiments, the molecular payload is an insulin analog.

[0089] In certain embodiments, the molecular payload is not covalently bound to the aptamer.

[0090] In certain embodiments, the molecular payload is hydrophilic. In certain embodiments, the molecular payload is hydrophobic. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than 0. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than 0.5. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than 1. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than 1.5. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than 2. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than 2.5. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than 3. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than 3. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than 3.5. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than 4. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than 4.5. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than 5. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than -0.5. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than -1. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than -1. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than -1.5. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than -2. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than -2.5. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than -3.In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than -3.5. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than -4. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than -4.5. In certain embodiments, the molecular payload has a LogD or LogP that is equal to or less than -5. In certain embodiments, the molecular payload has a LogD or LogP that is between -5 and 0. In certain embodiments, the molecular payload has a LogD or LogP that is between 0 and 5.

[0091] In certain embodiments, the composition contains an effective amount of molecular payload. In certain embodiments, the composition contains a therapeutically effective amount of molecular payload. In certain embodiments, the composition contains a preventively effective amount of molecular payload. In certain embodiments, the effective amount is an effective amount for treating a disease or disorder in which administration of the therapeutic agent would be beneficial in a subject requiring the administration of the therapeutic agent. In certain embodiments, the effective amount is an effective amount for preventing a disease or disorder in which administration of the therapeutic agent would be beneficial in a subject requiring the administration of the therapeutic agent. In certain embodiments, the effective amount is an effective amount for reducing the risk of developing a disease or disorder in which administration of the therapeutic agent would be beneficial in a subject requiring the administration of the therapeutic agent.

[0092] Forms of composition In certain embodiments, the molar ratio of the aptamer to the molecular payload is greater than 1.0. In certain embodiments, the molar ratio of the aptamer to the molecular payload is greater than 1.0, greater than 2.0, greater than 3.0, greater than 4.0, greater than 5.0, greater than 10.0, greater than 15.0, greater than 20.0, greater than 25.0, greater than 30.0, greater than 35.0, or greater than 40.0.

[0093] In certain embodiments, the molar ratio of the aptamer to the molecular payload is at least 1.0, at least 2.0, at least 3.0, at least 4.0, at least 5.0, at least 10.0, at least 15.0, at least 20.0, at least 25.0, at least 30.0, at least 35.0, or at least 40.0. In certain embodiments, the molar ratio of the aptamer to the molecular payload is at least 2.0, at least 3.0, at least 4.0, at least 5.0, at least 10.0, at least 15.0, at least 20.0, at least 25.0, at least 30.0, at least 35.0, or at least 40.0. In certain embodiments, the molar ratio of the aptamer to the molecular payload is at least 2.0. In certain embodiments, the molar ratio of the aptamer to the molecular payload is at least 3.0. In certain embodiments, the molar ratio of aptamer to molecular payload is at least 4.0. In certain embodiments, the molar ratio of aptamer to molecular payload is at least 5.0. In certain embodiments, the molar ratio of aptamer to molecular payload is at least 10.0. In certain embodiments, the molar ratio of aptamer to molecular payload is at least 15.0. In certain embodiments, the molar ratio of aptamer to molecular payload is at least 20.0. In certain embodiments, the molar ratio of aptamer to molecular payload is at least 25.0. In certain embodiments, the molar ratio of aptamer to molecular payload is at least 30.0. In certain embodiments, the molar ratio of aptamer to molecular payload is at least 35.0. In certain embodiments, the molar ratio of aptamer to molecular payload is at least 40.0.

[0094] In certain embodiments, the molar ratio of aptamer to molecular payload is about 2.0, about 3.0, about 4.0, about 5.0, about 10.0, about 15.0, about 20.0, about 25.0, about 30.0, about 35.0, or about 40.0. In certain embodiments, the molar ratio of aptamer to molecular payload is about 2.0. In certain embodiments, the molar ratio of aptamer to molecular payload is about 3.0. In certain embodiments, the molar ratio of aptamer to molecular payload is about 4.0. In certain embodiments, the molar ratio of aptamer to molecular payload is about 5.0. In certain embodiments, the molar ratio of aptamer to molecular payload is about 10.0. In certain embodiments, the molar ratio of aptamer to molecular payload is about 15.0. In certain embodiments, the molar ratio of aptamer to molecular payload is approximately 20.0. In certain embodiments, the molar ratio of aptamer to molecular payload is approximately 25.0. In certain embodiments, the molar ratio of aptamer to molecular payload is approximately 30.0. In certain embodiments, the molar ratio of aptamer to molecular payload is approximately 35.0. In certain embodiments, the molar ratio of aptamer to molecular payload is approximately 40.0.

[0095] In certain embodiments, the molar ratio of aptamer to molecular payload is 1.0–20.0, 1.0–40.0, 1.1–20.0, 1.1–40.0, 2.0–20.0, or 2.0–40.0. In certain embodiments, the molar ratio of aptamer to molecular payload is 1.0–20.0. In certain embodiments, the molar ratio of aptamer to molecular payload is 1.0–40.0. In certain embodiments, the molar ratio of aptamer to molecular payload is 1.1–20.0. In certain embodiments, the molar ratio of aptamer to molecular payload is 1.1–40.0. In certain embodiments, the molar ratio of aptamer to molecular payload is 2.0–20.0. In certain embodiments, the molar ratio of aptamer to molecular payload is 2.0–40.0.

[0096] In certain embodiments, the composition is characterized in that, when tested in vitro by placing the composition in a medium, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload is released from the composition 48 hours after the composition is placed in the medium. In certain embodiments, the composition is characterized in that, when tested in vitro by placing the composition in a medium, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload is released from the composition 24 hours after the composition is placed in the medium. In a particular embodiment, the composition is characterized in that, when tested in vitro by placing the composition in a medium, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload is released from the composition 12 hours after the composition is placed in the medium. In a particular embodiment, the composition is characterized in that, when tested in vitro by placing the composition in a medium, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload is released from the composition 6 hours after the composition is placed in the medium. In certain embodiments, the composition is characterized in that, when tested in vitro by placing the composition in a medium, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload is released from the composition 3 hours after the composition is placed in the medium. In certain embodiments, the medium is phosphate-buffered saline (PBS) having a pH of about 7.4.

[0097] In certain embodiments, the composition is characterized in that, when administered to a subject, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload is released from the composition 48 hours after administration. In certain embodiments, the composition is characterized in that, when administered to a subject, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload is released from the composition 24 hours after administration. In certain embodiments, the composition is characterized in that, when administered to a subject, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload is released from the composition 12 hours after administration. In certain embodiments, the composition is characterized in that, when administered to a subject, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload is released from the composition 6 hours after administration. In a particular embodiment, the composition is characterized in that, when administered to a subject, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload is released from the composition 3 hours after administration.

[0098] In certain embodiments, the composition, when administered to a subject, is characterized in that the duration of the therapeutic effect is extended by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or at least 200% compared to the duration of the therapeutic effect when administered with the molecular payload alone or with the composition without the aptamer. In certain embodiments, the therapeutic effect is a nerve block and / or reduced pain. In certain embodiments, the therapeutic effect is a nerve block. In certain embodiments, the therapeutic effect is reduced pain.

[0099] This disclosure also provides methods for preparing the compositions disclosed herein. In certain embodiments, the methods include providing a molecular payload; identifying or preparing an aptamer to bind to the molecular payload; and combining the molecular payload and the aptamer in a composition. In certain embodiments, the molecular payload is any molecular payload as defined herein. In certain embodiments, the aptamer is any aptamer as defined herein. The aptamer and the molecular payload may be combined in any manner known to those skilled in the art.

[0100] Pharmaceutical compositions, kits, and administrations This disclosure provides compositions comprising aptamers and molecular payloads. In certain embodiments, the compositions are pharmaceutical compositions. In certain embodiments, the compositions further comprise pharmaceutically acceptable excipients.

[0101] In certain embodiments, the pharmaceutical composition contains an effective amount of molecular payload. In certain embodiments, the effective amount is a therapeutic effective amount. In certain embodiments, the effective amount is a preventive effective amount. In certain embodiments, the effective amount is an effective amount for treating a disease or condition in a subject that requires treatment of the disease or condition. In certain embodiments, the effective amount is an effective amount for preventing a disease or condition in a subject that requires prevention of the disease or condition. In certain embodiments, the effective amount is an effective amount for reducing the risk of developing a disease or condition in a subject that requires reduction of the risk of developing the disease or condition. In certain embodiments, the molecular payload is a therapeutic agent.

[0102] The pharmaceutical compositions described herein may be prepared by any method known in the field of pharmacology. Generally, such preparation methods involve the step of combining a composition containing a molecular payload (e.g., a therapeutic agent) with an aptamer. In certain embodiments, the preparation further includes adding and / or one or more other auxiliary components, and then, if necessary and / or desirable, shaping and / or packaging the product into desired single or multiple dose units.

[0103] Pharmaceutical compositions may be prepared, packaged, and / or sold in bulk as single unit doses and / or as multiple single unit doses. As used herein, “unit dose” is a discrete amount of a pharmaceutical composition containing a given amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient that would be administered to a subject, and / or a convenient fraction of such a dose, such as half or one-third of such a dose.

[0104] The compositions provided herein may be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, percutaneous, interdermal, rectal, vaginal, intraperitoneal, topical, mucosal, nasal, buccal, sublingual; by intratracheal drip infusion, intrabronchial drip infusion, and / or inhalation; and / or as oral spray, nasal spray, and / or aerosol.

[0105] The compositions described herein may be administered in combination with one or more additional agents (e.g., pharmaceuticals, e.g., therapeutically and / or prophylactically active agents). The compositions may be administered in combination with additional pharmaceuticals that improve the activity of the composition (e.g., activity in treating disease in subjects that require treatment of disease, in preventing disease in subjects that require prevention of disease, and / or reducing the risk of developing disease in subjects that require reduction of the risk of developing disease (e.g., potency and / or effectiveness)), improve bioavailability, improve the ability of the composition to cross the blood-brain barrier, improve safety, reduce drug resistance, reduce and / or modify metabolism, inhibit excretion, and / or modify distribution in subjects or cells. It will also be understood that the treatments used may achieve the desired effect for the same disorder, and / or different effects. In certain embodiments, the pharmaceutical compositions described herein that include the therapeutic agent and additional agents described herein exhibit synergistic effects that are not present in pharmaceutical compositions that include either the therapeutic agent or the additional agent, but not both. In a particular embodiment, the additional agent is a therapeutic agent. In a particular embodiment, the additional agent is epinephrine.

[0106] The composition may be administered concurrently with, or prior to, one or more additional pharmaceuticals, which may be useful as, for example, combination therapy. Pharmaceuticals include therapeutic activators. Pharmaceuticals also include prophylactic activators. Pharmaceuticals include drug compounds (for example, compounds approved by the U.S. Food and Drug Administration for human or veterinary use as defined in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNA, RNA, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and small organic molecules such as cells. Each additional pharmaceutical may be administered in a dose and / or time schedule determined for that pharmaceutical. Additional pharmaceuticals may also be administered in single doses with each other and / or with the compounds or compositions described herein, or separately in different doses. The specific combinations used in a regimen take into account the compatibility of the compounds described herein with additional pharmaceuticals, and / or the desired therapeutic and / or preventive effects to be achieved. In general, the additional pharmaceuticals combined are expected to be used at levels not exceeding those used individually. In some embodiments, the combined level is lower than the level used individually.

[0107] In certain embodiments, the subject is an animal. The animal may be of either sex and at any stage of development. In certain embodiments, the subject described herein is a human. In certain embodiments, the subject is a non-human animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject is a domestic animal such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal such as a dog or cat. In certain embodiments, the subject is a domestic animal such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a zoo animal. In another embodiment, the subject is a research animal such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate. In certain embodiments, the animal is a genetically modified animal. In certain embodiments, the animal is a transgenic animal (e.g., transgenic mouse and transgenic pig). In certain embodiments, the subject is a fish or a reptile.

[0108] This disclosure also covers kits (e.g., pharmaceutical packs). The kits provided may include the pharmaceutical compositions described herein and containers (e.g., vials, ampoules, bottles, syringes, and / or dispenser packages, or other suitable containers). In some embodiments, the kits provided may optionally further include a second container containing pharmaceutical excipients for dilution or suspension of the compositions described herein. In some embodiments, the compositions described herein, provided in the first and second containers, are combined to form a single unit dosage form.

[0109] Accordingly, in one aspect, a kit is provided comprising a first container containing the composition described herein. In certain embodiments, the kit is useful for treating a disease or condition (e.g., pain) in a subject that requires treatment of the disease or condition (e.g., pain). In certain embodiments, the kit is useful for preventing a disease or condition (e.g., pain) in a subject that requires prevention of the disease or condition (e.g., pain). In certain embodiments, the kit is useful for reducing the risk of developing a disease or condition (e.g., pain) in a subject that requires reduction of the risk of developing the disease or condition (e.g., pain).

[0110] In certain embodiments, the kits described herein further include instructions for using the kit. The kits described herein may also include information as required by regulatory agencies such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kit is prescription information. In certain embodiments, the kits described herein may also include one or more additional medicinal products described herein as separate compositions.

[0111] How to use This disclosure also provides a method for treating a disease or condition in a subject that requires treatment of the disease or condition, the method comprising administering a provided composition to the subject. This disclosure also provides a method for preventing a disease or condition in a subject that requires prevention of the disease or condition, the method comprising administering a provided composition to the subject. This disclosure also provides a method for reducing the risk of developing a disease or condition in a subject that requires reduction of the risk of developing the disease or condition.

[0112] In certain embodiments, the disclosed compositions may be used to treat subjects (e.g., humans) having a disease or condition in which administration of a molecular payload (e.g., a therapeutic agent) would be beneficial. In certain embodiments, the subject is administered an effective amount of one or more of the disclosed compositions. In certain embodiments, the subject is administered a therapeutically effective amount of one or more of the disclosed compositions. In certain embodiments, the subject is administered a prophylactically effective amount of one or more of the disclosed compositions. In certain embodiments, the subject is an animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject is a human aged 18 years or older. In certain embodiments, the subject is a human aged 12 to 18 years (excluding both ends). In certain embodiments, the subject is a human aged 2 to 12 years (including both ends). In certain embodiments, the subject is a human under 2 years of age. In certain embodiments, the subject is a non-human animal. In certain embodiments, the subject is a non-human mammal.

[0113] In certain embodiments, the disease or condition being treated is related to a specific biological target. In certain embodiments, the target is a voltage-opening sodium channel. In certain embodiments, the target is a site-1 sodium channel. In certain embodiments, the disease or condition is pain.

[0114] In certain embodiments, the disease or condition is an infectious disease. In certain embodiments, the disease or condition is a bacterial infection. In certain embodiments, the disease or condition is tuberculosis. In certain embodiments, the disease or condition is diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes).

[0115] In certain embodiments, a method for treating a disease or condition involves administering a composition such that a molecular payload (e.g., a therapeutic agent) is delivered to a biological target at a slower rate than if the molecular payload were administered alone. In such embodiments, the molecular payload is delivered via sustained release. In certain embodiments, the administration is local (i.e., a local administration for a local effect, e.g., a subcutaneous administration) such that the sustained release is a local sustained release. In certain embodiments, the administration is systemic (i.e., a systemic administration for a dispersed or systemic effect, e.g., a parenteral administration) such that the sustained release is a systemic sustained release.

[0116] In certain embodiments, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload (e.g., a therapeutic agent) is released from the composition 48 hours after administration to a subject. In certain embodiments, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload (e.g., a therapeutic agent) is released from the composition 24 hours after administration to a subject. In certain embodiments, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload (e.g., a therapeutic agent) is released from the composition 12 hours after administration to a subject. In certain embodiments, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload (e.g., a therapeutic agent) is released from the composition 6 hours after administration to a subject. In certain embodiments, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload (e.g., a therapeutic agent) is released from the composition 3 hours after administration to the subject. In certain embodiments, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload (e.g., a therapeutic agent) is released from the aptamer 48 hours after administration to the subject. In certain embodiments, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload (e.g., a therapeutic agent) is released from the aptamer 24 hours after administration of the composition to the subject.In certain embodiments, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload (e.g., a therapeutic agent) is released from the aptamer 12 hours after administration of the composition to the subject. In certain embodiments, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload (e.g., a therapeutic agent) is released from the aptamer 6 hours after administration of the composition to the subject. In certain embodiments, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload (e.g., a therapeutic agent) is released from the aptamer 3 hours after administration of the composition to the subject.

[0117] In certain embodiments, the duration of the therapeutic effect is extended by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000%, or at least 10,000% compared to the duration of the therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer. In certain embodiments, the duration of the therapeutic effect is extended by at least 20% compared to the duration of the therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer. In certain embodiments, the duration of the therapeutic effect is extended by at least 30% compared to the duration of the therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer. In certain embodiments, the duration of the therapeutic effect is extended by at least 40% compared to the duration of the therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer. In certain embodiments, the duration of the therapeutic effect is extended by at least 50% compared to the duration of the therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer. In certain embodiments, the duration of the therapeutic effect is extended by at least 60% compared to the duration of the therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer. In certain embodiments, the duration of therapeutic effect is extended by at least 70% compared to the duration of therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without the aptamer.In certain embodiments, the duration of the therapeutic effect is extended by at least 80% compared to the duration of the therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer. In certain embodiments, the duration of the therapeutic effect is extended by at least 90% compared to the duration of the therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer. In certain embodiments, the duration of the therapeutic effect is extended by at least 100% compared to the duration of the therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer. In certain embodiments, the duration of the therapeutic effect is extended by at least 200% compared to the duration of the therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer. In certain embodiments, the duration of the therapeutic effect is extended by at least 300% compared to the duration of the therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer. In certain embodiments, the duration of the therapeutic effect is extended by at least 400% compared to the duration of the therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer. In certain embodiments, the duration of the therapeutic effect is extended by at least 500% compared to the duration of the therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer. In certain embodiments, the duration of the therapeutic effect is extended by at least 600% compared to the duration of the therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer. In certain embodiments, the duration of therapeutic effect is extended by at least 700% compared to the duration of therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer. In certain embodiments, the duration of therapeutic effect is extended by at least 800% compared to the duration of therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer.In certain embodiments, the duration of the therapeutic effect is extended by at least 900% compared to the duration of the therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer. In certain embodiments, the duration of the therapeutic effect is extended by at least 1,000% compared to the duration of the therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer. In certain embodiments, the duration of the therapeutic effect is extended by at least 10,000% compared to the duration of the therapeutic effect when the molecular payload (e.g., the therapeutic agent) is administered alone or in a composition without an aptamer.

[0118] In certain embodiments, the therapeutic effect is a nerve block and / or reduced pain. In certain embodiments, the therapeutic effect is a nerve block or reduced pain. In certain embodiments, the therapeutic effect is a nerve block and reduced pain. In certain embodiments, the therapeutic effect is a nerve block. In certain embodiments, the therapeutic effect is reduced pain. In certain embodiments, the therapeutic effect is a reduction of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of pain compared to the baseline level of pain prior to administration.

[0119] This disclosure also provides the use of the compositions provided in the manner described herein. This disclosure also provides the use of the pharmaceutical compositions provided in the manner described herein. This disclosure also provides compositions provided for use in the manner described herein. This disclosure also provides pharmaceutical compositions provided for use in the manner described herein.

[0120] example Examples are provided below to allow for a more complete understanding of the inventions described herein. The examples described herein are provided to illustrate the compounds, pharmaceutical compositions, and methods provided herein and should not be construed as limiting their scope.

[0121] Example 1. Design of chemically modified aptamers for TTX binding and controlled release. To create a non-covalent complex with TTX, a high-affinity TTX-binding aptamer (5'-AAAAATTTCACACGGGTGCCTCGGCTGTCC-3' (SEQ ID NO: 1)) was prepared (Figure 1A and Table 1). To enhance the aptamer's resistance to the nuclease, the phosphodiester (PO) skeleton was chemically modified with phosphorothioate (PS) (Figure 1B and Table 1).

[0122] To assess the binding affinity of TTX to PS aptamers (Figure 1C), the interaction between TTX (in PBS) and Cy5-labeled aptamers (Table 1) was studied by microscale thermophoresis (MST), and K d This enabled the determination of the value. PS modification increased the binding affinity of the aptamer to TTX by 29.5 ± 2.1 μM compared to the unmodified PO aptamer. d The concentration improved twofold, from 14.3 ± 1.7 μM (Figure 1D). The PS aptamer containing the scrambled sequence (Scr-PS) (Table 1) showed reduced binding to TTX (K d (=3.84mM).

[0123] To test whether this binding affinity could be the basis for a sustained-release system, TTX was complexed with an aptamer (PO or PS) in a molar ratio of 2:1 or 20:1 (aptamer:TTX) by simple mixing, and the release kinetics of TTX from these formulations were investigated. These samples exhibited very similar average diameters of approximately 3 nm, as measured by dynamic light scattering (DLS) (Figure 6A-6B), indicating that association with TTX did not cause aptamer aggregation. The release kinetics of TTX were studied by dialyzing 200 μL of aptamer / TTX in 14 mL of PBS (the TTX concentration was 42 μM; the aptamer concentration varied). The release of TTX was quantified by enzyme-linked immunosorbent assay (ELISA). Both PO / TTX and PS / TTX complexes (2:1 or 20:1) increased the duration of TTX release compared to free TTX (p<0.0001 at 24 hours, Figure 1E and Figures 7A-7B). Release from PS / TTX was statistically significantly slower than release from PO / TTX at both 12 hours (p<0.0001) and 24 hours (p=0.026). There was no statistically significant difference in TTX release between the two molar ratios tested (2:1, 20:1) for PO / TTX or PS / TTX (p>0.05). To verify that this sequence is essential for aptamer interaction with TTX, the release kinetics of non-selective scrambled PS aptamers (Scr-PS; Table 1) attached to TTX at two different molar ratios, 2:1 and 20:1 (Scr-PS / TTX), were evaluated. Release of TTX from Scr-PS / TTX was more rapid than from PS / TTX, with 92.8±3.6% (2:1) and 94.4±3.8% (20:1) of TTX released in 12 hours, which was similar to the release rate of TTX without the aptamer (p>0.05 for all comparisons). These data demonstrate that the interaction between the aptamer and TTX is sequence-specific and that PS modification delayed release compared to PO. The slower release from PS / TTX is thought to be due to improved binding affinity between the PS aptamer and TTX.

[0124] The cytotoxicity of aptamers / TTX was tested in two cell lines associated with local anesthesia-associated tissue injury: the myoblast C2C12 cell line was used to assess potential myotoxicity, and the pheochromocytoma PC12 cell line was used to assess potential neurotoxicity. Cells were incubated for 24 hours with free TTX, PO aptamer, PS aptamer, PO / TTX(2:1) complex, or PS / TTX(2:1) complex at the same TTX concentration of 73 μM and / or 146 μM aptamer concentration, and cell viability was measured using the 3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)(MTS) assay. All groups in both cell types maintained cell viability of over 90% compared to untreated cells (Figure 8A-8B).

[0125] Example 2. Comparing the efficacy of free TTX and aptamer / TTX complex for rat sciatic nerve blockade in vivo. To explore the feasibility of aptamers as drug delivery systems in vivo, the ability of aptamers to extend the duration of rat sciatic nerve block was investigated. 0.3 mL of TTX, either alone or in combination with an aptamer (PO, PS, or Scr-PS) / TTX, was injected into the left sciatic nerve of male Sprague-Dawley rats (n=4 or 6 per group). 4 μg (42 μM) of free TTX in PBS provided nerve block without lethality in rats, and this was set as the initial TTX dose for all formulations. Initially, aptamer (PO or PS) / TTX complexes were tested with a fixed aptamer:TTX molar ratio of 20:1. After injection, rats underwent neurobehavioral examination to determine the duration of functional deficits (sensory and motor nerve blockade) in both hind limbs. Heat latency (the time expressed in seconds from when the rat lifted its hind limbs off the hot plate) was the primary evaluation criterion for sensory nerve block. A sensory nerve block was considered successful if the latency was longer than 7 seconds. The duration of the sensory nerve block was defined as the time it took for the thermal latency to return to 7 seconds (the midpoint between the baseline (2 seconds) and the maximum latency (12 seconds)). Deficiencies in the left (ipsilateral) hind limb reflected a nerve block, while defects in the right (contralateral) hind limb reflected systemic drug distribution (i.e., systemic toxicity).

[0126] Nerve block with 42 μM free TTX lasted for 0.9 ± 0.6 hours. Delivery as PO / TTX doubled the block duration to 1.9 ± 0.8 hours, and delivery as PS / TTX increased the block duration 7.7 times, to 7.1 ± 2.4 hours (Figures 2A and 9A-9D). Block from PS / TTX was statistically significantly longer than block from TTX (p=0.0001) or PO / TTX (p=0.0033).

[0127] To assess whether the nerve block was due to intrinsic aptamer properties (rather than TTX), PO or PS aptamers (83.6 μM or 836 μM) without TTX were injected into the left sciatic nerve of rats. The nerve block was not detected over a 6-hour period (Figures 10A–10B and Table 2).

[0128] Example 3. Specificity of PS aptamer / TTX in vivo To determine the specificity of the TTX-binding PS aptamer, we further examined its selectivity for TTX in vivo using three control groups. First, the non-selective scrambled PS aptamer described above was complexed with TTX at a fixed molar ratio of 20:1 (Scr-PS / TTX). Scr-PS / TTX (42 μM TTX) showed a 2.3-fold improvement in nerve block duration compared to free TTX (to 2.1 ± 0.8 hours, p = 0.0207), which is likely due to nonspecific interactions between Scr-PS and TTX, but is lower than that of the more selective PS / TTX portion (7.7-fold improvement, 7.1 ± 2.4 hours, p = 0.0043) (Figure 2A and Figures 9A-9D). Notably, both PS / TTX(20:1) and Scr-PS / TTX(20:1) had remarkably similar and low viscosities (<0.002 Pa s), as determined by rheometry, which were not involved in the difference in nerve block duration (Figure 11). Secondly, the selectivity of the PS aptamer for TTX was confirmed by complexing the PS aptamer with STX, another S1SCB that acts on the same sodium channel site as TTX, has a similar molecular weight and guanidinium group, but is otherwise structurally very different (Figure 1C). 2 μg (22 μM) of STX provided sensory nerve blockade lasting 2.0 ± 0.4 hours, whereas nerve block from the same concentration of STX in a 20:1 molar ratio with TTX-specific PS (PS / STX) remained unchanged for 2.2 ± 0.4 hours (p=0.49), indicating that the TTX-binding aptamer did not prolong nerve blockade from STX (Figure 2B). Thirdly, bupivacaine, an aminoamide local anesthetic, was combined with the TTX-binding aptamer. Bupivacaine binds to the same sodium channel as TTX, but at a different site on the inner surface of the cell membrane, and is structurally very different (Figure 1C). The combination of 836 μM PS aptamer and 15.4 mM bupivacaine hydrochloride did not significantly prolong the duration of nerve block (3.9 ± 0.4 hours with PS / bupivacaine vs. 3.4 ± 0.4 hours with free bupivacaine; p = 0.0611) (Figure 2C). These results confirm a specific interaction between TTX and TTX-binding PS aptamer.

[0129] Example 4. Ratio of PS aptamer to TTX to enhance nerve blockade. The effects of PS / TTX ratios (1:1, 2:1, 5:1, 10:1, 20:1, and 40:1) on nerve block duration were investigated at a constant TTX concentration of 42 μM. These formulations were injected into the sciatic nerve site. PS / TTX with a 1:1 molar ratio induced a sensory nerve block lasting 0.3 ± 0.3 hours, which was similar in duration to the block from free TTX at the same concentration (Figure 2D). In the PS / TTX ratio range from 2:1 to 20:1, the block was significantly prolonged to 5.8 ± 1.5 hours (2:1), 5.4 ± 0.3 hours (5:1), 5.6 ± 0.8 hours (10:1), and 7.1 ± 2.4 hours (20:1). Across this range of molar ratios, there were no statistically significant differences in the observed latency on the injected or contralateral side. However, when the PS / TTX ratio was increased to 40:1, the duration of nerve block decreased to 2.6 ± 0.4 hours. As a result, a PS / TTX with a molar ratio of 2:1, which provided the longest nerve block duration at the lowest aptamer concentration, was selected for subsequent studies.

[0130] All PS / TTX complexes exhibited extremely low viscosity of less than 0.003 Pa s in the tested angular frequency range (Figure 12). Since there was no difference in the measured viscosity of the complexes at different ratios (n=3, p>0.05 for all comparisons), differences in viscosity did not cause differences in nerve block.

[0131] Example 5. Dose response using aptamer / TTX Dose-response studies were conducted in rat left sciatic nerve block models (n=4 or 6) using free TTX drug or PS / TTX (2:1) complex. Nerve block on the injected side was significantly longer with PS / TTX than with free TTX at all doses used (Figure 2E and Table 2). The duration of nerve block increased with increasing PS / TTX dose (1.9 ± 0.9 hours with 31 μM TTX, 12.0 ± 0.4 hours with 73 μM TTX). PS enhanced the performance of low-dose TTX: 31 μM (3 μg) of free TTX did not induce nerve block, whereas the same dose with PS / TTX resulted in a median duration of sensory nerve block of 1.9 ± 0.9 hours, with successful nerve block in 100% of animals. In addition, PS / TTX improved the safety of TTX, as demonstrated by the absence or reduction of systemic toxicity from TTX (i.e., loss in the contralateral limb; Figure 2F and Table 2). For example, 63 μM (6 μg) of free TTX was uniformly lethal, whereas rats did not die at any of the PS / TTX doses tested (Figure 2G and Table 2). Contralateral latency was dose-dependent with both TTX and PS / TTX, but was significantly greater with TTX than with PS / TTX. Motor block was 1.3 times longer than sensory block in PS / TTX-treated rats (p<0.05) (Figure 13).

[0132] Example 6. Enhancement of the local anesthetic activity of PS / TTX by adding epinephrine. The extension of nerve block with PS is due to the control of TTX release from the injection site. This control enabled the delivery of doses of TTX that would otherwise be lethal. It was hypothesized that the delivered dose, and therefore the duration of effect, could be further increased by co-injection of epinephrine, an active agent whose release from the injection site can be delayed by pharmacological means. Co-injection of PS / TTX (2:1) and 55 μM epinephrine in the sciatic nerve enabled the delivery of TTX concentrations as high as 104 μM without death (Figure 2H). This is the dose at which TTX was uniformly lethal in the absence of the aptamer, and even with epinephrine administration, there was a 67% mortality rate. With 104 μM PS / TTX with epinephrine, a duration of block of 22.0 ± 2.8 hours could be achieved, which was almost twice the longest duration achievable with PS / TTX without epinephrine and without animal death. The peak-contralateral latency was significantly reduced by epinephrine, for example, 12 seconds for 73 μM PS / TTX and 2 seconds for PS / TTX with epinephrine (p<0.0001) (Figure 14).

[0133] Example 7. Tissue retention of PS / TTX To assess the local retention of aptamers in tissue, PO and PS aptamers (Cy5.5-PO / PS, Table 1), covalently modified at the 5' end with the near-infrared fluorescent dye Cy5.5, were injected into the left sciatic nerve of rats. Fluorescence intensity at the injection site was monitored at predetermined intervals using an in vivo imaging system (IVIS). Free Cy5.5 was rapidly eliminated, with less than 20% remaining after 4 hours, while approximately 63% of PO aptamers were eliminated within 10 hours (Figure 3A). For PS aptamers, ~50% of peak fluorescence persisted after 24 hours. The fluorescence intensity of PS-Cy5.5 was statistically significantly stronger than that of Cy5.5 and PO-Cy5.5 at all time points tested (Figure 3B). These data confirm that the PS aptamer enabled longer tissue retention.

[0134] Four hours after injection, the sciatic nerve and surrounding tissue were collected, processed for histological examination, stained with Hoechst 33342 (nucleus staining), and imaged by laser scanning confocal microscopy. In rats injected with PS aptamer, fluorescence from Cy5.5 was detected in the tissue surrounding the sciatic nerve at approximately 4 times higher levels than in animals injected with PO aptamer (Figures 3C and 15). Aptamer fluorescence was observed in the connective tissue between muscle and nerve (i.e., at the injection site near the nerve), but not within the sciatic nerve itself (Figures 3C and 16).

[0135] The presence of TTX in tissues at pharmacologically reasonable concentrations is revealed by neurobehavioral bioassays: TTX is present while the nerve block persists; and when the nerve block is resolved, TTX / STX essentially disappears.

[0136] Example 8. Tissue reaction On days 4 and 14 after sciatic nerve injection, sciatic nerve tissue with surrounding tissue was harvested and prepared for histological examination (n=4 for all groups; Figure 4; Table 3). Muscle tissue was stained with hematoxylin-eosin (H&E). Free TTX induced very mild inflammation (score = 0 or 1 in all animals), which is consistent with previous reports. A mixed inflammatory response (score = 2 or 3 in all animals) was observed at the injection site on day 4 in the PS aptamer and PS / TTX groups. Inflammation subsided by day 14 (score = 1 or 2 in all animals) (n=4, p<0.05 compared to day 4). No myotoxicity was observed at any time point in either the treated group (score = 0) or the untreated rats (Table 3). To investigate neurotoxicity, the sciatic nerve was embedded in Epon and stained with toluidine blue (H&E staining is relatively insensitive to identifying nerve damage). Neither the PS aptamer nor the PS / TTX complex caused any nerve damage at any point in time (Figure 4).

[0137] Example 9: STX-bound PS aptamer (PSAP)STX ) In vivo nerve block of the / STX complex To test the generality of the use of aptamers as drug delivery systems, a PS aptamer specific for STX (PSAP STX , Table 1) was used to investigate STX-induced peripheral nerve block. STX has approximately twice the potency of TTX in vivo with respect to rat sciatic nerve block. Injection of 33 μM free STX in PBS caused a nerve block that lasted 2.6 ± 0.5 hours (Figure 5A and Table 4), with contralateral deficits in all animals (duration of 2.3 ± 0.9 hours). In contrast, 33 μM STX in PSAP STX / STX (molar ratio 2:1) increased the nerve block in the injected limb 2.6-fold, to 6.9 ± 0.8 hours (p<0.0001), and animals had no increase in contralateral latency (Figure 5A). 45 μM STX in a certain PSAP STX / STX (2:1) provided a sensory nerve block that lasted 11.3 ± 0.7 hours and there was an increase in contralateral latency, but animals did not die, whereas injection of free STX at the same dose was uniformly lethal.

[0138] PSAP STX and PSAP STX / STX had no cytotoxicity in C2C12 and PC12 cell lines by the MTS assay (Figure 17). To assess tissue reaction, rats were euthanized 4 and 14 days after injection (n = 4 at each time point). The sciatic nerve and surrounding tissue were harvested and processed for histological examination by H&E staining (for inflammation and myotoxicity) or toluidine blue staining (for neurotoxicity). Inflammation was similar to that seen with PS / TTX. Similarly, no myotoxicity or neurotoxicity was present on day 4 and day 14 in any animals treated with free PSAP STX or the PSAP STX / STX complex (Figure 5B and Table 5).

[0139] Example 10. Discussion of Examples 1 - 9 The use of aptamers to create highly effective depot-type drug delivery systems is demonstrated herein. Control of drug release is achieved by forming aptamer / drug non-covalent complexes, and in vivo results demonstrate a significant enhancement of the efficacy and therapeutic index of small molecule drugs (TTX and STX) in a rat sciatic nerve block model.

[0140] This method has been effective with two classes of molecules that are difficult to encapsulate in many traditional drug delivery systems: small, hydrophilic molecules. Furthermore, the incorporation of such molecules in many traditional drug delivery systems can result in significant burst release, potentially causing toxicity. To improve their incorporation, many methods have been developed that rely on techniques such as electrostatic interactions with charged compounds or covalent anchoring to polymers. These methods differ considerably in terms of their effectiveness and complexity. The method disclosed herein is simple and effective.

[0141] The aptamer DDS described herein offers several advantages over existing systems. Aptamers are easy to synthesize and chemically modify. These aptamers are synthesized through solid-phase technology and phosphoramidite chemistry, which allows for the addition of multiple functional moieties at specified positions. These synthesis techniques are simple, efficient, easily scaled up, and inexpensive. Importantly, the drug filling process is a simple one-step mixing, which offers a clear advantage over many other drug filling processes, which are often multi-step and have relatively low efficiency.

[0142] Drug-aptamer interactions are the basis for the controlled-release functionality of this system. As a result, this technique can be used for a wide range of therapeutic agents. Here, local anesthesia was used as an in vivo model to demonstrate efficacy and safety. However, this technique can also be used for the treatment of many other types of diseases for which a depot drug delivery system is desirable, whether for local or systemic effects (as in this case), at least based on these results. In systemic administration (i.e., intravenous injection of drug-aptamer complexes), the use of specific interactions of aptamers for drug binding would likely mean that the aptamer could not be used to target specific tissues.

[0143] PS chemical modification improves the in vivo stability of aptamers against nuclease degradation. PS modification can increase the binding affinity between aptamers and drugs, improving drug release kinetics. The enhanced affinity of PS aptamers to TTX may be due to altered electrostatic and hydrophobic interactions between them, resulting from the substitution of oxygen to sulfur in the DNA backbone. This was observed with TTX (Figures 1D and 1E), resulting in prolonged effect and reduced toxicity (Figure 2E). PS aptamers also demonstrated much longer tissue retention than PO aptamers (Figure 3). Two possible explanations are that the PS backbone confers resistance to nuclease digestion to the aptamers, and that PS has a high tendency to nonspecifically bind to various proteins in vivo, which enhances tissue retention. The time frame for nerve block was much shorter than the time frame for tissue retention, but it is unclear to what extent the enhanced tissue retention was beneficial in prolonging nerve block from TTX, as this may be important for drugs with longer durations of effect.

[0144] When administered topically to the rat sciatic nerve, the tissue response to PS aptamers was mild at the tested doses. Furthermore, aptamers have a proven track record in clinical use. Therefore, safety concerns are unlikely to hinder the transfer of aptamers into the drug delivery system.

[0145] Thus, the use of drug-specific DNA aptamers as a drug delivery system is presented herein. This method provides a simple and safe delivery method for molecular payloads such as small molecule drugs.

[0146] Example 11. The method used in Examples 1-10 material Phosphoramidites and supplies for aptamer synthesis were purchased from Glen Research Co., USA. Mouse C2C12 myoblast (CRL-1772) and rat PC12 pheochromocytoma (CRL-1721) cell lines were purchased from American Type Culture Collection (Rockville, MD, USA). Sulfocyanine 5.5 amine (Cy5.5, 95.0%) was obtained from Lumiprobe Corporation (Hunt Valley, Maryland, USA). Tetrodotoxin (TTX) was purchased from Abcam (Waltham, MA, USA). The TTX ELISA kit was purchased from Reagen LLC (Moorestown, NJ, USA). Dulbecco's Minimum Essential Medium (DMEM), horse-bovine serum (HBS), fetal bovine serum (FBS), and penicillin streptomycin were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). STX was obtained from the U.S. Food and Drug Administration (FDA). Sodium fluorescein and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich Co. (MO, USA). MALDI-TOF MS measurements were performed using a Bruker Microflex LT mass spectrometer (Bruker Daltonics Inc., MA, USA). Reverse-phase HPLC was performed using a Waters (Waters Co., MA, USA) Breeze 2 HPLC system coupled with a SunFire C18 5 μm, 10 × 100 mm reverse-phase column and a 2998 PDA detector, with TEAA buffer (0.1 M) and HPLC-grade acetonitrile as the mobile phase. DLS data were recorded using a Malvern Zetasizer Pro. Histological studies were conducted at the Kock Institute Swanson Biotechnology Center and iHisto Inc., Massachusetts Institute of Technology.

[0147] Aptamer synthesis The synthesis of DNA aptamers (both phosphodiester [PO] and phosphorothioate [PS] versions) was performed using a Model 391 DNA synthesizer (Applied Biosystems, Inc., CA, USA) with a standard solid-phase phosphoramidite methodology. Deprotection of the aptamers was carried out at room temperature for 24 hours using ammonium hydroxide (28% NH3 in H2O). The crude product was purified by reverse-phase HPLC liquid chromatography. The purified aptamers were then treated with 20% acetic acid in H2O for 1 hour to remove the dimethoxytrityl (DMT) protecting group, followed by three extractions with ethyl acetate in aqueous solution. The resulting aptamers were quantified using a NanoDrop® OneC microvolume UV-vis spectrophotometer and stored at -20°C after lyophilization. To synthesize dye-labeled aptamers, cyanine 5 (Cy5) or cyanine 5.5 (Cy5.5) was incorporated into the 5' end by using Cy5 phosphoramidite and Cy5.5 phosphoramidite, respectively. The success of all aptamer synthesis in this work was confirmed by MALDI-TOF MS.

[0148] Preparation of aptamer / TTX and aptamer / STX complexes In short, a TTX-conjugated aptamer (PO or PS) was dissolved in 1×PBS (154 mM NaCl, 5.6 mM Na2HPO4, 1.1 mM KH2PO4; pH 7.4) in a microcentrifuge tube, heated to 95°C for 5 minutes, and then slowly cooled to room temperature. After the annealing process, TTX dissolved in citrate buffer (5 mg / mL) was diluted with PBS (200 μg / mL) and then added to the aptamer solution in a predetermined molar ratio. For further testing, the resulting solution was gently shaken overnight at room temperature. Similarly, aptamer / STX complexes were prepared using the same method.

[0149] Microscale thermophoresis (MST) analysis of binding interactions MST without immobilization is a rapid and accurate method for studying small molecule-aptamer interactions in solution. MST monitors the thermophoretic motion of different molecular ratios between target molecules and ligands through a μm-sized temperature gradient. To establish these ratios, a constant amount of Cy5-labeled aptamer is mixed with different amounts of ligand. Serial dilutions of TTX in PBS were performed to provide solutions with concentration ranges of 152.6 nM to 5 mM. Each 5 μl solution was mixed with 5 μl of Cy5-labeled aptamer maintained at a constant concentration of 16 nM. The final concentrations of TTX in each capillary ranged from 76.3 nM to 2.5 mM. Each sample was analyzed at 25°C using Monolith NT. Automated (NanoTemper Technologies, Munich, Germany) with 40% LED power and 80% laser power. The data was fitted using MO.Affinity Analysis software (version 2.3, NanoTemper Technologies) and the K of the aptamer d To determine the value, the MST on time was set to 1.5 seconds.

[0150] in vitro TTX release The TTX release kinetics from the aptamer / TTX complex were determined by placing 200 μL of these complexes (TTX, 42 μM) into a Slide-A-Lyzer MINI dialyzer with a 3500 MW cutoff, further dialyzing with 14 mL of PBS, and incubation at 37°C on a platform shaker. At predetermined intervals, the dialysate was replaced with fresh, pre-warmed PBS. The concentration of released TTX in the medium was quantified by enzyme-linked immunosorbent assay (ELISA).

[0151] Viscosity determination The rheological properties of aptamers and aptamer / TTX were measured using an AR2000 rheometer (TA Instruments, New Castle, DE, USA) with a parallel plate configuration and temperature control. Parallel plates with a diameter of 20 mm were used, with a gap distance of 0.3 mm. Dynamic properties were measured at room temperature (0.01–100 rads). -1 In frequency sweep tests (at frequencies in the range of ), a constant shear stress of 0.1 Pa and 0.01 rads -1 The vibration frequency was tracked as a function of time.

[0152] cell culture C2C12 mouse myoblasts were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum (FBS) and 1% penicillin streptomycin. To induce differentiation into myotubes, C2C12 cells (8.0 × 10⁶) were cultured. 3 The cells were seeded in 24-well plates and incubated in DMEM with 2% horse serum and 1% penicillin streptomycin for 7–10 days. The differentiation medium was changed every 2–3 days. PC12 rat adrenal pheochromocytoma cells were grown in DMEM with 5% FBS, 5% horse serum and 1% penicillin streptomycin. For neuronal induction, PC12 cells were divided into 1.0 × 10⁶ wells. 4 Cells were seeded in 24-well plates at a cell / well density and cultured for 10–14 days in DMEM containing 5% FBS, 5% horse serum, and 50 ng / mL nerve growth factor (NGF).

[0153] MTS cytotoxicity assay The cytotoxicity of aptamers, TTX, and aptamer / TTX complexes was evaluated using an MTS colorimetric assay. C2C12 and PC12 cells were treated with varying doses of free TTX, aptamers, or aptamer / TTX complexes. Cells incubated with vehicle (PBS) were set as controls. After 24 hours of incubation, 40 μL of MTS-based CellTiter 96® AQ was administered. ueousOne Solution Reagent was added to each well. Cells were incubated for a further 4 hours, and absorbance (490 nm) was measured on a BioTek® Synergy® Mx microplate reader (BioTek Inc., VT, USA).

[0154] animal research The animal study was approved by the Boston Children's Hospital Animal Care and Use Committee and conducted according to protocols following the guidelines of the International Association for the Study of Pain. Adult male Sprague-Dawley rats weighing 400-500g (Charles River Laboratories, Wilmington, MA, USA) were housed in groups under a 12-hour / 12-hour light / dark cycle, with the lights turned on at 6:00 a.m.

[0155] Sciatic nerve block and neurobehavioral examination Rats were randomly assigned to each group and injected with 300 μL of each formulation into the left sciatic nerve under short-term isoflurane-oxygen anesthesia. A 23G needle was introduced posteromedially to the greater trochanter, pointing anteromedially. The drug was injected onto the sciatic nerve upon contact with the bone. The rats were then subjected to neurobehavioral examinations at predetermined intervals.

[0156] Sensory nerve blocks were assessed using the modified hot plate test described above. Briefly, the plantar surface of a rat's hind limb was placed on a preheated hot plate (Model 39D Hot Plate Analgesia Meter; IITC) at 56°C. The time it took for the rat to retract its foot (heat latency) was recorded using a stopwatch. If the rat did not retract its foot after 12 seconds, the limb was removed from the hot plate to avoid injury. This test was repeated three times at different time intervals (with a 10-second break between tests). A heat latency of 7 seconds or more indicated a successful nerve block for the purpose of calculating the duration of the nerve block.

[0157] Motor nerve block was assessed using a weight-bearing test to determine the motor intensity of the rat's hind limb, as previously reported. Briefly, the rat was placed on a digital balance by one hind limb and required to support its own body weight. The maximum weight the rat could support without its ankle touching the balance was recorded. The duration of motor blockage was defined as the time it took for the weight-bearing load to return from maximum block to half of normal, as previously described.

[0158] In vivo imaging system (IVIS) imaging Sprague-Dawley rats were partially shaved and injected into the left sciatic nerve with 0.3 mL of free Cy5.5 or Cy5.5-labeled aptamer (Cy5.5, 4.64 μM) under isoflurane-oxygen anesthesia. They were scanned at 0, 1, 4, 10, 24, 48, and 72 hours post-injection using the IVIS 200 imaging system (Caliper Life Sciences, Inc. MA, USA). Quantitative analysis will be performed using IVIS Live Imaging software. The tissue retention half-life is the time required for a 50% decrease in fluorescence intensity after injection and was calculated based on fluorescence intensity.

[0159] Use confocal imaging to track the location of aptamers in tissue. Rats were injected into the left sciatic nerve with 0.3 mL of PBS containing Cy5.5 or Cy5.5-labeled aptamer (84 μM) under isoflurane-oxygen anesthesia. Four hours after injection, the rats were euthanized with carbon dioxide. The sciatic nerve and surrounding tissue were collected, embedded in OCT compound (Fisher Scientific Inc., USA), and stored at -20°C. Using a cryostat microtome, the frozen tissue was sectioned into 8 μm thick sections and placed on glass slides. The slides were then fixed with 4% paraformaldehyde, stained with Hoechst 33342, and imaged on an LSM-880 confocal laser scanning microscope (Carl Zeiss Ltd., Cambridge, UK).

[0160] Histological examination Sprague-Dawley rats (n=4) were treated with 0.3 mL of aptamer (146 μM), free TTX (52 μM), or aptamer / TTX (146 μM / 73 μM) at the left sciatic nerve, and then euthanized to assess acute (day 4) and chronic (day 14) inflammation and tissue injury, respectively. The sciatic nerve and surrounding tissue were collected, fixed in 10% neutral buffered formalin, and subjected to standard processing for H&E stained slides. The slides were analyzed and scored for the presence of inflammation (0–4) and myotoxicity (0–6) by an independent, certified pathologist (Matthew Gregory Torre), who was not informed of the nature of the individual samples. The inflammation score is a subjective quantification of severity, where 0 is normal and 4 is severe inflammation (0: no inflammation, 1: peripheral inflammation, 2: deep inflammation, 3: semifascicular inflammation of the muscle, 4: total fascicular inflammation of the muscle). Myotoxicity scores are determined by the internal migration and regeneration of muscle cells. Internal migration is characterized by muscle cells with nuclei located away from their normal position at the cell periphery. Regeneration is characterized by the presence of contracted muscle cells with basophilic cytoplasm. The scoring scale is as follows: 0 = Normal; 1 = Perifascicular internal migration; 2 = Deep internal migration (more than 5 cell layers); 3 = Perifascicular regeneration; 4 = Deep tissue regeneration (more than 5 cell layers); 5 = Semifascicular regeneration; 6 = Total fascicular regeneration.

[0161] To access the neurotoxicity of the aptamer formulations, sciatic nerves were fixed with Karnovsky's KII solution (1.25% formaldehyde, 2.5% glutaraldehyde, and 0.03% picric acid in 0.1 M sodium cacodylate buffer, pH 7.4). The fixed tissue was washed with 0.1 M sodium cacodylate buffer and post-fixed in 1% osmium tetroxide / 1.5% potassium ferrocyanide (in H2O) for 2 hours. The samples were then washed in maleic acid buffer and post-fixed in 1% uranyl acetate in maleic acid buffer for 1 hour. The tissue was then rinsed in ddH2O and dehydrated through a series of ethanols (50%, 70%, 95%, (2×)100%) for 15 minutes per solution. Dehydrated tissue was placed in propylene oxide for 5 minutes before being immersed overnight at 4°C in an epone mixture of propylene oxide and propylene oxide. The samples were polymerized in the epone resin for 48 hours in an oven at 60°C. The samples were then sectioned into 500 nm thin sections, which were stained with toluidine blue and imaged on a high-resolution optical microscope.

[0162] Statistics and Reproducibility All quantitative measurements (i.e., TTX binding affinity analysis, TTX release, and fluorescence intensity quantification) have at least three independent replicates. Sample sizes in Examples 1–10 were calculated using power analysis. Previous animal studies, or small pilot studies where necessary, served as a basis for calculating the expected mean and deviation used to determine power, with experimental studies set to a value of 0.8 for power. This typically resulted in experimental group sizes of n=4–6, depending on the experiment. Sample sizes are explicitly stated for each experimental group for each individual experiment in the brief descriptions of the figures and in Examples 1–10. No data was excluded from the analysis. Origin 2022b and GraphPad Prism 9 were used for plotting. ImageJ (version 1.53t) was used for image processing. MO.Affinity Analysis (version 2.3) software was used for binding affinity analysis. Statistical comparisons were performed using GraphPad Prism 9. Statistical comparisons were performed using Student's t-test (two-tailed) unless otherwise specified. Heat latency, inflammation, and myotoxicity scores are reported as medians and quartiles due to their ordinal or non-Gaussian properties. Data are presented as mean + / - standard deviation. Statistical significance was set at p<0.05. Example 12. Tables referenced in Examples 1-11 [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

[0163] Additional aptamer-drug conjugates The feasibility of aptamers for sustained release of various types of therapeutic drugs other than local anesthetics was investigated. For this purpose, aptamers known to conjugate the neurotransmitter serotonin (Ser), the antibiotic kanamycin (Kan), and the antidiabetic drug insulin (Ins) were selected (Table 6). The sustained release of small molecule drugs, specifically Ser, from aptamer systems was investigated first. Ser-conjugating aptamer (Apt Ser PS-Apt was mixed with Ser in a 2:1 molar ratio and incubated overnight at 4°C. The release kinetics of the resulting Apt-Ser complex were evaluated by dialyzing 250 μL of the resulting Apt-Ser complex against 25 mL of PBS. Ser -Ser initially showed slower ser release than free ser, but became similar after 24 hours (P=0.9577, Figure 18A). In contrast, PO-Apt Ser -Ser showed dramatically slower ser release than free ser, with a low cumulative ser release of 3.31 ± 1.61% over 24 hours. PS-Apt Ser The insufficient sustained release of -Ser may be due to disruption induced by PS modification of the tertiary structure of the aptamer in this particular sequence. Then, using a similar experimental setup, Kan-binding aptamer (Apt Kan The sustained release of another small molecule drug from the PS-Apt system, Kan, was investigated. Kan -Kan and PO-Apt Kan -Kan showed similar Kan release over 24 hours (P=0.8530, Figure 18B), but both had slower release compared to free Kan (P=0.0141 and P=0.0.063).

[0164] Having demonstrated that aptamers can act as sustained-release systems for the small molecule drugs Ser and Kan, we used a similar experimental setup to introduce an Ins-binding aptamer (Apt) for the protein drug Ins. Ins The feasibility of mediated sustained release was further investigated. PS-Apt Ins-Ins release was significantly less from Ins than release from free Ins over 24 hours (P=0.0117, Figure 18C). On the other hand, PO-Apt Ins -The release of Ins from Ins and free Ins did not differ significantly over 24 hours (P=0.1217). Compared to conventional release systems, the aptamer release system was relatively small, with a molecular weight of approximately 10 kDa. Therefore, it was necessary to ensure that the aptamer, as a drug loading and release platform, was not released from the dialysis machine. Indeed, aptamer release from the dialysis machine was not observed for any of the aptamer systems (Figure 18A-C). Thus, controlled release of all three drugs was achieved by aptamers. [Table 6]

[0165] The serotonin ELISA kit was obtained from ImmuSmol (BA-E-5900R, France). The kanamycin ELISA kit (502370) was obtained from Cayman Chemical (Ann Arbor, MI). The insulin ELISA kit was obtained from Cystal Chem (90095, Downers Grove). The Quant-iT OliGreen ssDNA Assay Kit (O11492) and insulin (RP-10935) were obtained from Thermo Fisher Scientific Inc. (Waltham, MA).

[0166] The release kinetics of serotonin, kanamycin, and insulin from aptamer / drug conjugates (molar ratio, 2:1) were evaluated by placing 250 μL of these conjugates (serotonin 94 μM; kanamycin 69 μM; insulin 7 μM) into a dialyzer (cutoff of 3.5–5 kDa for serotonin and kanamycin, and 25 kDa for insulin). The dialyzer was then dialyzed against 25 mL of PBS on a shaker at 37°C. At predetermined intervals, the dialyzed solution was collected and fresh PBS was added to the system. The concentrations of serotonin, kanamycin, and insulin in the released medium were quantified by the corresponding ELISA kits. The concentration of aptamers in the released medium was quantified by the Quant-iT OliGreen ssDNA Assay kit.

[0167] Equivalents and range Unless otherwise indicated or otherwise obvious from the context, articles such as “a,” “an,” and “the” in a claim may mean one or more. Unless otherwise indicated or otherwise obvious from the context, a claim or statement containing “or” among one or more members of a group is considered satisfied if one member, more than one member, or all members of that group are present in, adopted into, or otherwise related to a given product or process. The present invention encompasses embodiments in which exactly one member of that group is present in, adopted into, or otherwise related to a given product or process. The present invention encompasses embodiments in which more than one or all of the members of that group are present in, adopted into, or otherwise related to a given product or process.

[0168] Furthermore, the present invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim dependent on another claim may be modified to include one or more limitations found in any other claim dependent on the same basic claim. Where elements are presented, for example, as enumerated in Markush group form, each subgroup of the element is also disclosed, and any element(s) may be removed from the group. In general, where the present invention or an aspect of the present invention is considered to include certain elements and / or features, certain aspects of the present invention or certain aspects of the present invention should be understood to consist of, or substantially consist of, such elements and / or features. For the sake of brevity, those aspects are not specifically defined in this specification in haec verba. Note also that the terms “comprising” and “containing” are intended to be open and allow for the inclusion of additional elements or processes. Where a range is given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise obvious from the context and the understanding of those skilled in the art, a value expressed as a range may be assumed to be any specific value or a subrange of the range described in different aspects of the invention, up to one-tenth of the lower limit of the range, unless the context explicitly indicates otherwise.

[0169] This application references various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of any conflict between any of the incorporated references and this specification, this specification shall prevail. In addition, any particular aspect of the Invention that falls within the scope of prior art may be expressly excluded from any one or more of the claims. Such aspects may be excluded even if the exclusion is not expressly stated herein, as they would be considered known to those skilled in the art. Any particular aspect of the Invention may be excluded from any of the claims for any reason, whether or not it relates to the existence of prior art.

[0170] Those skilled in the art will recognize many equivalents to the specific embodiments described herein, or at best, can verify them using routine experiments. The scope of the embodiments described herein is not intended to be limited to the foregoing, but rather as described in the appended claims. Those skilled in the art will understand that various modifications and alterations to this description may be made without departing from the spirit or scope of the invention, as defined in the following claims.

Claims

1. A composition comprising an aptamer and a molecular payload, wherein the molecular payload is bound to the aptamer, and the aptamer is a sustained-release carrier for the molecular payload.

2. The composition according to claim 1, wherein the aptamer is an oligonucleotide.

3. The composition according to claim 1 or 2, wherein the aptamer is a single-stranded oligonucleotide.

4. The composition according to any one of claims 1 to 3, wherein the aptamer is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

5. The composition according to any one of claims 1 to 4, wherein the aptamer is single-stranded deoxyribonucleic acid (DNA).

6. The composition according to any one of claims 1 to 5, wherein the aptamer is an oligonucleotide sequence of 15 to 60 nucleotides.

7. The composition according to any one of claims 1 to 6, wherein the aptamer is an oligonucleotide sequence of 30 to 35 nucleotides.

8. The composition according to any one of claims 1 to 7, wherein the aptamer is an oligonucleotide sequence having at least one phosphorothioate nucleotide linkage.

9. The composition according to any one of claims 1 to 8, wherein the aptamer is an oligonucleotide sequence having a complete phosphorothioate skeleton.

10. The composition according to any one of claims 1 to 9, wherein the aptamer is a single-stranded deoxyribonucleic acid (DNA) having a complete phosphorothioate skeleton.

11. The composition according to any one of claims 1 to 10, wherein the aptamer is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:

11.

12. The composition according to any one of claims 1 to 11, wherein the molar ratio of the aptamer to the molecular payload is greater than 1.

0.

13. The composition according to any one of claims 1 to 12, wherein the molar ratio of the aptamer to the molecular payload is at least 2.

0.

14. The composition according to any one of claims 1 to 13, wherein the molar ratio of the aptamer to the molecular payload is at least 2.0, at least 3.0, at least 4.0, at least 5.0, at least 10.0, at least 15.0, at least 20.0, at least 25.0, at least 30.0, at least 35.0, or at least 40.

0.

15. The composition according to any one of claims 1 to 14, wherein the molar ratio of the aptamer to the molecular payload is 1.0 to 20.0, 1.0 to 40.0, 1.1 to 20.0, 1.1 to 40.0, 2.0 to 20.0, or 2.0 to 40.

0.

16. The composition according to any one of claims 1 to 15, wherein the molecular payload is not covalently bonded to the aptamer.

17. The composition according to any one of claims 1 to 16, wherein the aptamer is not the targeting portion.

18. The composition according to any one of claims 1 to 17, wherein the aptamer is not a therapeutic agent.

19. The composition according to any one of claims 1 to 18, wherein the molecular payload is hydrophilic (for example, LogD or LogP < 0).

20. The composition according to any one of claims 1 to 18, wherein the molecular payload is a therapeutic agent.

21. The composition according to any one of claims 1 to 20, wherein the molecular payload is a small molecule or a biological preparation.

22. The composition according to any one of claims 1 to 21, wherein the molecular payload is a small molecule.

23. The composition according to any one of claims 1 to 22, wherein the molecular payload is an anesthetic, an antibiotic, or a neurotransmitter.

24. The composition according to any one of claims 1 to 23, wherein the molecular payload is an anesthetic.

25. The composition according to any one of claims 1 to 24, wherein the molecular payload is a site 1 sodium channel blocker.

26. The composition according to any one of claims 1 to 25, wherein the molecular payload is tetrodotoxin or saxitoxin.

27. The composition according to any one of claims 1 to 23, wherein the molecular payload is an antibiotic.

28. The composition according to any one of claims 1 to 23, wherein the molecular payload is kanamycin.

29. The composition according to any one of claims 1 to 23, wherein the molecular payload is a neurotransmitter.

30. The composition according to any one of claims 1 to 23, wherein the molecular payload is serotonin.

31. The composition according to any one of claims 1 to 21, wherein the molecular payload is a polypeptide.

32. The composition according to any one of claims 1 to 21, wherein the molecular payload is insulin.

33. A composition according to any one of claims 1 to 32, comprising an effective amount of molecular payload.

34. A composition according to any one of claims 1 to 33, comprising a therapeutically effective amount of molecular payload.

35. The composition according to any one of claims 1 to 34, further comprising a pharmaceutically acceptable excipient.

36. The composition according to any one of claims 1 to 35, further comprising a therapeutic agent (e.g., epinephrine).

37. The composition according to any one of claims 1 to 36, characterized in that when the composition is tested in vitro by placing the composition in a medium, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload is released from the composition 24 hours after the composition is placed in the medium.

38. The composition according to any one of claims 1 to 37, characterized in that when the composition is administered to a subject, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload is released from the composition 24 hours after administration of the composition.

39. A composition according to any one of claims 1 to 38, characterized in that, when the composition is administered to a subject, the duration of the therapeutic effect is extended by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or at least 200% compared to the duration of the therapeutic effect when the molecular payload is administered alone or in the composition without an aptamer.

40. The composition according to claim 39, wherein the therapeutic effect is nerve block and / or reduced pain.

41. The composition according to any one of claims 1 to 40, wherein the composition is a depot for a molecular payload.

42. A method for treating a disease or condition, the method comprising administering an effective amount of a composition according to any one of claims 1 to 41 to a subject in need thereof.

43. The method according to claim 42, wherein less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the molecular payload is released from the composition 24 hours after administration of the composition to a subject.

44. The method according to claim 42 or 43, wherein the duration of therapeutic effect is extended by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000%, or at least 10,000% compared to the duration of therapeutic effect when the molecular payload is administered alone or in a composition without an aptamer.

45. The method according to claim 44, wherein the therapeutic effect is nerve block and / or reduced pain.

46. The method according to any one of claims 42 to 45, wherein the disease or condition is pain.

47. The method according to any one of claims 42 to 44, wherein the disease or condition is an infectious disease, tuberculosis, or diabetes.

48. The method according to any one of claims 42 to 47, wherein the subject is a mammal.

49. The method according to any one of claims 42 to 48, wherein the subject is a human.

50. A method for preparing a composition according to any one of claims 1 to 41, comprising: providing a molecular payload; identifying or preparing an aptamer to bind to the molecular payload; and combining the molecular payload and the aptamer in the composition.

51. A kit comprising the composition according to any one of claims 1 to 41, and instructions for use.

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