Stable compositions for storing and transporting single-stranded nucleic acid material

Abstract

The composition includes a plurality of biocompatible metal nanospheres, each having an outer surface connected to elemental carbon, and a plurality of single-stranded nucleic acids and a carrier medium, wherein at least a portion of each individual single-stranded nucleic acid is coordinately connected to the nanosphere.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 389,590, filed July 15, 2022, and U.S. Provisional Patent Application No. 63 / 413,533, filed October 5, 2022, each of which is incorporated by reference in its entirety.

[0002] Disclosed are compositions suitable for storing and / or transporting and / or delivering single-stranded nucleic acid material, such as ribonucleic acid (RNA) material. Also disclosed are methods for making such compositions and methods for storing / transporting / delivering RNA material. [Background technology]

[0003] Various types of single-stranded nucleic acids play important roles not only in research but also in medical treatment, disease prevention, and mediation. Messenger RNA (mRNA) is used as a vaccine or booster to fight diseases such as SARS-CoV-2.

[0004] One of the challenges in using nucleic acids, such as RNA, a type of single-stranded nucleic acid, in various biological and biomedical applications is the instability and / or reactivity of the nucleic acid strand. Long-term stable storage of this material has been a continuing challenge. To date, storage, transportation, and delivery of RNA material has been hampered by the extreme measures that must be taken to ensure that at least a portion of the stored material remains viable not only during storage, but also during transportation and delivery.

[0005] This challenge is evident in end uses, including but not limited to mRNA vaccine delivery. The challenge of obtaining viable single-stranded nucleic acid materials is that vaccines formulated with these materials must remain stable for days to weeks before administration. The ribonucleic acids, such as mRNA, used in these materials degrade easily and rapidly unless kept at low temperatures. Therefore, therapeutics based on ribonucleic acids, such as mRNA, or their precursors, must be transported and stored at low temperatures and warmed immediately before administration to maintain efficacy and potency. For example, for mRNA-based SARS-CoV-2 vaccines, the materials must be stored at temperatures between -20°C and -70°C, depending on the vaccine composition. These storage temperature requirements can pose challenges to the formulation, implementation, and use of RNA-based materials in therapeutics, especially when the therapeutic is administered routinely, e.g., daily.

[0006] There is a continuing need for better and more robust RNA storage and delivery methods, as well as compositions that facilitate them. To achieve this goal, the use of calcium, magnesium, or mixtures thereof as bioresorbable substrates has been explored. Both calcium and magnesium are present in high concentrations in the body and are easily degraded. For example, magnesium alloys have been explored for use as orthopedic screws.

[0007] The use of magnetic beads to isolate nucleic acids from biological samples has also been explored in analytical applications. Beads composed of materials such as the mineral oxide FeO have been used to isolate biomolecules, such as single-stranded nucleic acids, from mixtures. Additionally, nanoparticles with a magnetic FeO core coated with materials such as SiO have been used for nucleic acid isolation in research. In clinical applications, magnetic FeO nanoparticles coated with SiO have been used to isolate nucleic acids for disease diagnosis, prognosis, and safety testing. Summary of the Invention [Problem to be solved by the invention]

[0008] It would therefore be desirable to provide compositions that can be used to store and transport single-stranded nucleic acid materials that are capable of delivering large quantities of stored material, particularly storage that does not require extreme environmental storage conditions. It would also be desirable to provide compositions and methods for storing single-stranded nucleic acid materials that are highly biocompatible with biological materials and organisms. [Means for solving the problem]

[0009] The present disclosure is a composition comprising: a) a plurality of biocompatible nanospheres, each having an outer surface connected to elemental carbon; b) a plurality of single-stranded nucleic acids, at least a portion of each single-stranded nucleic acid being in a coordinated connection with the biocompatible nanosphere; and c) a carrier medium.

[0010] The present disclosure also provides a method for storing and / or transporting and / or delivering single-stranded nucleic acids, comprising forming biocompatible aggregates of nucleic acids and biocompatible nanospheres, wherein the biocompatible nanospheres have an outer surface connected to elemental carbon. In the formed biocompatible aggregates, the single-stranded nucleic acids are complexed with one or more nanospheres at one or more positions of the single-stranded nucleic acids to minimize the degrees of freedom of the single-stranded nucleic acids. In certain embodiments, the single-stranded nucleic acids present in the formed biocompatible aggregates are connected to at least two biocompatible nanospheres. [Effects of the Invention]

[0011] Disclosed herein are compositions suitable for efficient and economical storage and / or transport and / or delivery of single-stranded nucleic acids. In certain circumstances, the single-stranded nucleic acid may be a ribonucleic acid, such as a biologically active RNA involved in protein synthesis. Non-limiting examples of such single-stranded nucleic acids include messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (smRNA), and the like, and fragments thereof. It is also contemplated that the disclosed compositions can be used to facilitate one or more of the storage, transport, and / or delivery of various other types of RNA, including, but not limited to, RNA involved in processes such as post-transcriptional modification or DNA replication, and various regulatory RNAs, parasitic RNAs, and the like.

[0012] The compositions of the present disclosure stably store and maintain desired single-stranded nucleic acids and allow for the delivery of large quantities of active, intact single-stranded nucleic acids for subsequent use. As used herein, the term "active, intact single-stranded nucleic acid" refers to a nucleic acid that is capable of performing its intended or desired coding function when delivered for its end use. Also disclosed are methods for storing and transporting single-stranded nucleic acid material so as to maintain and deliver large quantities of active, intact single-stranded nucleic acids for subsequent use. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 illustrates the interaction between an elemental carbon layer embedded in a biocompatible nanosphere disclosed herein and the heteroatom moiety of an associated single-stranded nucleic acid. [Figure 2] FIG. 2 is a diagram showing the relationship of theoretical binding energy to distance in the configuration of FIG. 1. [Figure 3] FIG. 1 is a theoretical schematic diagram of an exemplary embodiment of a biocompatible aggregate disclosed herein. [Figure 4] 1 is a photomicrograph showing one embodiment of a biocompatible aggregate disclosed herein. [Figure 5] Photographs show side-by-side unbound RNA samples and unbound RNA samples exposed to ribonuclease. [Figure 6] Photographs showing bound and unbound RNA samples. [Figure 7] FIG. 1 shows stability data for RNA present in urine samples. [Figure 8] 1 shows a diagram and a table showing the stability of RNA during transportation and storage. DETAILED DESCRIPTION OF THE INVENTION

[0014] Although the present disclosure will be described in connection with particular embodiments, it should be understood that the present disclosure is not limited to the disclosed embodiments, but is intended to encompass various modifications and equivalent arrangements within the scope of the appended claims, which scope should be accorded the broadest interpretation so as to encompass all such modifications and equivalent arrangements permitted under law.

[0015] The present disclosure is premised, at least in part, on the unexpected discovery that single-stranded nucleic acids can be attached to nanoparticle structures comprised of suitable biocompatible substrates in a manner that allows for safe storage and transport of the associated nucleic acid strand material over extended periods of time and / or at moderate temperatures. It is further believed that, in certain embodiments, single-stranded nucleic acids, when aggregated with nanoparticle structures, can be stably dispersed in an associated carrier fluid with little or no dispersing aid. It is also believed that, in certain embodiments, single-stranded nucleic acids, when aggregated with nanoparticle structures, result in compositions that are storage-stable at temperatures between 0°C and 25°C.

[0016] The nanoparticle structures present in the composition can be comprised of a matrix material having an outer surface to which elemental carbon is attached. If desired or required, the outer surface of the nanoparticle structure can include at least one rounded region. In certain embodiments, the matrix material can be configured in a suitable shape, such as a particle or bead. In certain embodiments, the matrix is configured as a bead or sphere. In certain embodiments, nanosphere-shaped substrates can be used.

[0017] The matrix material may be composed entirely or partially of biocompatible materials, such as silica and polymeric materials. In certain embodiments, the matrix material may be a biocompatible metallic material. Suitable biocompatible matrix materials may be biocompatible matrix materials that can be contacted with biological life forms, such as humans, without adversely affecting them. In certain embodiments, the biocompatible matrix material may be a biologically compatible transition metal, a biologically acceptable alkaline earth metal, or the like. In certain embodiments, the matrix material may be Fe, Co, Ni, Mg, Zn, or Ca, and mixtures of any of these. In certain embodiments, the biocompatible metallic material may be selected from the group consisting of Fe, Co, Ni, Mg, Zn, Ca, and mixtures thereof.

[0018] The substrate material can be nanoparticle-sized. In certain embodiments, the biologically compatible nanospheres can be 1-100,000 nm in diameter, and in certain embodiments, 20 nm-100,000 nm in diameter. It is also believed that the nanospheres can have an average particle size of 20-100,000 nm, and in certain embodiments, 50-200 nm.

[0019] In the disclosed compositions, at least a portion of the exterior surface of the substrate has elemental carbon in overlying relationship thereon and connections to the elemental carbon. The elemental carbon may be composed of or derived from any suitable source, including, but not limited to, pyrolytic carbon, graphite, graphene, and mixtures thereof. In certain embodiments, the elemental carbon can cover the substrate in a generally seamless conformal coating.

[0020] The elemental carbon coating attached to the substrate may have a thickness suitable for promoting interaction between the substrate structure and single-stranded nucleic acids. In certain embodiments, the thickness of the carbon coating layer may be 1 to 40 atomic layers. In some embodiments, the thickness of the elemental carbon attached to the biocompatible nanospheres is between 1 angstrom and 0.5 nm. If desired or necessary, the elemental carbon present in the carbon layer may be formed, in whole or in part, from one or more of graphene, graphite, or pyrolytic carbon. As used herein, graphene is a carbon allotrope formed in a two-dimensional hexagonal lattice, and graphite is a carbon allotrope formed from stacks of graphene. Pyrolytic carbon resembles graphite, but there are covalent bonds between the graphene sheets. More specifically, elemental carbon may be formed in the form of sheets similar to graphene or graphitic carbon. The elemental coating material may also contain a certain amount of graphite oxide. As used herein, graphite oxide is defined as one or more of the aforementioned elemental carbon materials exhibiting carboxylic acid and hydroxide groups formed at defects on the surface of the graphene sheets.

[0021] Biocompatible nanospheres can be formed by combining a biocompatible matrix material with elemental carbon. The amount of biocompatible matrix material present in a given biocompatible nanosphere is the amount necessary to support the carbon layer and impart the appropriate geometric contour to the biocompatible matrix material, resulting in nanoparticles with a suitable spherical or hemispherical contour. In the disclosed compositions, the amount of biocompatible matrix material present in the biocompatible nanospheres is sufficient to support the elemental carbon attached to the outer surface and maintain the resulting nucleic acid aggregates in suitable suspension in the relevant carrier medium. In the disclosed compositions, the biocompatible matrix can comprise at least 10% by weight of the biocompatible nanosphere. If desired or necessary, biocompatible nanospheres with attached elemental carbon can be composed of 10% to 99% by weight of the biocompatible matrix material, with the remainder being elemental carbon. In certain embodiments, the biocompatible matrix material can be present in an amount of 20% to 95%, 35% to 95%, 50% to 95%, or 75% to 95%.

[0022] Carbon-coated substrate materials can be prepared by any suitable method that applies a layer of elemental carbon to at least a portion of the outer surface of the associated substrate. In certain configurations, the elemental carbon applied to the outer surface of the substrate can have a thickness of only one atom. One non-limiting example of a method for preparing such materials is outlined in International Publication No. WO 2015 / 095398, incorporated herein by reference. Carbon-coated materials can be prepared by a pulsed laser with a wavelength between 200 nm and 1500 nm, a pulse repetition rate of at least 10 Hz, a pulse duration of greater than 1 ps, and an output of 10 J / cm. 2 It is believed that nanoparticles can be prepared by generating a laser pulse with a fluence exceeding 1000 rad / s and directing the laser at a target in a solvent. The laser beam can be scanned over the surface of the target, i.e., the desired core material (e.g., silica or a magnetic metal). The liquid in which the target is immersed is typically transparent to the laser radiation of the wavelength used and is usually a carbon-containing solvent such as xylene or toluene. The carbon shell is produced by laser processing. This process results in a seamless conformal coating of these particles with a carbon layer that has an affinity only for single-stranded nucleic acids.

[0023] The elemental carbon overlying and / or connected to the outer surface of the biocompatible matrix material can be spherical. The biocompatible nanospheres used in the compositions disclosed herein can have the elemental carbon conformally attached to the outer surface of the matrix material, with at least a portion of the benzene dimers present in the elemental carbon layer oriented in a planar lattice orientation relative to the matrix surface.

[0024] The biocompatible nanospheres obtained in the disclosed compositions can have an average diameter sufficient to maintain the biocompatible nanospheres in suspension in the resulting composition, as desired or necessary. In certain embodiments, the biocompatible nanospheres have an average diameter between 1 nm and 500 nm. In certain embodiments, the biocompatible nanospheres have an average diameter between 1 nm and 250 nm.

[0025] Without being bound by any theory, it is believed that this orientation promotes π-π stacking with one or more aromatic groups present in the single-stranded nucleic acid compound and the composition, particularly with one or more aromatic groups present in one or more base pairs present in the single-stranded nucleic acid or fragment thereof. The promoted π-π stacking may be of the sandwich type, T-shaped type, or translation type. Figure 1 shows a schematic diagram of the interaction between an elemental carbon layer and the heteroatom portion of an associated single-stranded nucleic acid. The theoretical binding energy versus distance relationship is shown in Figure 2.

[0026] In the compositions disclosed herein, individual single-stranded nucleic acids interact with individual biocompatible nanospheres to form nanoparticle aggregates with at least a portion of the single-stranded nucleic acids. In certain embodiments, the aggregates comprise individual single-stranded nucleic acids coordinated with one or more individual biocompatible nanospheres. In certain embodiments, the individual single-stranded nucleic acids coordinated with at least two nanospheres are oriented such that a portion of each individual single-stranded nucleic acid is located between the at least two nanospheres and is unattached. A theoretical schematic of the biocompatible aggregate is shown in Figure 3, and a micrograph of the material is shown in Figure 4.

[0027] When the disclosed biocompatible nanospheres interact to form biocompatible nanoparticle aggregates, the biocompatible nanoparticle aggregates can be large enough to remain dispersed in the associated carrier medium. In certain embodiments, the size of the biocompatible nanoparticle aggregates can be between 50 and 750 nm, and in certain embodiments, between 50 and 500 nm.

[0028] The carrier medium used in the compositions disclosed herein is compatible with nucleic acid compounds and can facilitate long-term storage of the single-stranded nucleic acid and nanosphere aggregates. The carrier medium can be a fluid or liquid carrier medium that allows for the dispersion and / or suspension of the single-stranded nucleic acid and nanosphere aggregates in whole or in part in the carrier medium.

[0029] The selected carrier medium may be any suitable fluid material, such as a biocompatible liquid. Suitable biocompatible liquids include water and / or biocompatible organic fluids, and mixtures thereof. In certain embodiments, the carrier medium may include a suitable biocompatible suspending aid, etc.

[0030] The carrier medium can be any medium capable of maintaining the single-stranded nucleic acid present in the biocompatible nanoparticles in a substantially intact state, stabilized against RNase activity. The carrier medium can be composed of suitable aqueous fluids, organic fluids, and mixtures thereof. If desired or necessary, the carrier can be composed of water and, optionally, one or more of natural or synthetic lipids, buffers, and electrolytes. Within the scope of the present disclosure, additive materials can be varied depending on specific storage and transportation needs.

[0031] Non-limiting examples of suitable lipids include compounds such as ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), polyethylene glycol [PEG] 2000 dimyristoyl glycerol, and 1,2-distearoyl-sn-glycero-3-phosphocholine.

[0032] The carrier medium can include biologically acceptable group II ions. These ions can be obtained from biologically suitable group II ionic materials. In certain embodiments, the biologically suitable group II ionic material is present in an amount sufficient to enhance binding between the single-stranded nucleic acid and the biocompatible nanospheres. In certain embodiments, the biologically suitable group II ionic material can be present in an amount of 10 mM to 1000 mM; 50 mM to 1000 mM; 100 mM to 1000 mM; 200 mM to 1000 mM; 500 mM to 1000 mM; 700 mM to 1000 mM; 10 mM to 500 mM; 50 mM to 500 mM; 100 mM to 500 mM; 200 mM to 500 mM; 250 mM to 500 mM; 300 mM to The Group II ionic material may be present at a concentration of 500 mM; 400 mM to 500 mM; 10 mM to 400 mM; 50 mM to 400 mM; 100 mM to 400 mM; 150 mM to 400 mM; 200 mM to 400 mM; 250 mM to 400 mM; 300 mM to 400 mM; 350 mM to 400 mM; 10 mM to 250 mM; 50 mM to 250 mM; 100 mM to 250 mM; 200 mM to 250 mM. Without being bound by any theory, it is believed that at least a portion of the Group II ionic material may be derived from the osmolarity adjusting buffer.

[0033] If desired or necessary, the Group II ionic material may be Ca 2+ , Mg 2+ and mixtures thereof. In certain applications, such materials may be derived, at least in part, from an appropriate lysis buffer. Non-limiting examples of lysis buffers include calcium ion lysis buffers containing calcium ions derived from calcium chloride, magnesium ion lysis buffers containing magnesium ions derived from MgCl2, and mixtures thereof. Without being bound by any theory, it is believed that the biologically suitable Group II ionic material unexpectedly functions to strengthen the bond between the single-stranded nucleic acid and the biocompatible nanospheres.

[0034] In certain embodiments, the carrier medium may include CaCl2 at a concentration of 50 mM to 500 mM as a functional component to enhance binding.

[0035] The carrier medium may contain ethanol or may be heated, as single-stranded nucleic acids are known to complex with carbon more quickly and these components denature nucleic acids to single strands. The carrier medium may also contain chaotropic salts or detergents as needed to free the nucleic acids for binding to the beads. For example, in many applications, such as diagnostic urine testing, nucleic acids may be present within cells, and it is necessary to separate the material from these cells before binding can occur.

[0036] Non-limiting examples of such buffers include materials such as guanidinium thiocyanate, ethylenediaminetetraacetic acid (EDTA), tris(hydroxymethyl)aminomethane (Tris), dithiothreitol (DTT), and Triton X. In certain embodiments, the binding buffer may be guanidinium thiocyanate, ethylenediaminetetraacetic acid (EDTA), tris(hydroxymethyl)aminomethane (Tris), dithiothreitol (DTT), and Triton X, and the solution may be adjusted to pH 6.5. EDTA and DTT may optionally be present to inactivate proteins and may be present at concentrations of 1 mmol to 100 mmol. Tris may optionally be present as a buffer to maintain the pH at 6.5. The pH may range from 4 to 9. The concentration of the Tris buffer is 20 to 200 mmol. Triton X is a detergent and may optionally be used to homogenize the solution. The concentration of Triton X is 1 to 50 mmol. In certain embodiments, a suitable binding buffer may require only 1 M to 7 M guanidinium ions.

[0037] When the disclosed biocompatible nanospheres interact to form biocompatible nanoparticle aggregates, the resulting biocompatible nanoparticle aggregates can be large enough to remain dispersed in the associated carrier medium. In certain embodiments, the biocompatible nanoparticle aggregates can have a size between 50 nm and 1000 nm, and in certain embodiments, between 50 nm and 500 nm; between 50 nm and 400 nm; or between 50 nm and 300 nm.

[0038] Without being bound by any theory, it is believed that biocompatible aggregates consisting of single-stranded nucleic acids and biocompatible nanospheres exhibit certain specific binding patterns when present in the compositions disclosed herein. The interaction between the single-stranded nucleic acids and biocompatible nanoparticles disclosed herein limits the ability of the associated nucleic acid strand to react with other regions on the nucleic acid strand or other reactive compounds that may be present or introduced into the composition.

[0039] Without being bound by any theory, it is believed that at least a portion of the binding or attraction between biocompatible nanospheres and single-stranded nucleic acids occurs between the lattice structure of benzene and / or pyridine moieties of the elemental carbon layers present in the nucleic acid structure evidenced in π-π bonds.

[0040] It is also believed that the size of the biocompatible nanospheres is such that, for many types of single-stranded nucleic acid lengths, two or more biocompatible nanospheres can be bound or associated with a given nucleic acid strand, and further restrict the free movement of the associated nucleic acid strand.

[0041] The resulting carrier medium composition, comprising aggregates of single-stranded nucleic acids and one or more biocompatible nanospheres having an outer surface with elemental carbon in coating relationship thereon, provides a stable storage medium for the associated single-stranded nucleic acid material.

[0042] Quite unexpectedly, it has been found that single-stranded nucleic acids, such as messenger RNA (mRNA), when present in the disclosed compositions are storage stable as suspensions at temperatures between -20°C and 25°C, and when required for administration or use, the biocompatible aggregates can be used directly or the single-stranded nucleic acid material can be separated from contact with the biocompatible nanospheres by any suitable means.

[0043] In addition to being temperature stable, the compositions disclosed herein are believed to be shock resistant, providing a means of storing and transporting single-stranded nucleic acid material in a manner that maintains the activity of the associated RNA and minimizes strand breaks and deletions.

[0044] Also disclosed are methods for storing and / or transporting single-stranded nucleic acid material. The disclosed methods can be used to maintain single-stranded nucleic acid material in an archivally stable or substantially stable form for storage and transport between locations. Single-stranded nucleic acid material suitable for sub-storage methods includes, but is not limited to, ribonucleic acid (RNA). In certain embodiments, the RNA to be stored may be messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (smRNA), etc., and fragments thereof.

[0045] In the disclosed method, biocompatible aggregates are formed between single-stranded nucleic acid material and biocompatible particles, such as nanospheres, such that the freedom of movement of individual nucleic acid strands is restricted and each strand of the single-stranded nucleic acid material is connected to at least one biocompatible nanosphere. At least a portion of the single-stranded nucleic acid material is believed to be associated with two or more nanospheres. The formed biocompatible aggregates can be maintained in a suitable suspension or dispersion composed of one or more carriers.

[0046] The biocompatible nanoparticles, such as nanospheres, used each have an outer surface composed of a suitable biocompatible material and having elemental carbon attached thereto. The surface layer of elemental carbon can be composed of a material having a thickness and structure sufficient to induce and support π-π bonding between the aromatic ring structure present in the nucleic acid strand material and the carbon lattice structure present in the elemental carbon layer. In certain embodiments, the elemental carbon material in the layer is composed, in whole or in part, of one or more compounds, such as graphene, graphite, pyrolytic carbon, etc. In certain embodiments, the elemental carbon layer is graphene.

[0047] The biocompatible matrix material may be any suitable biocompatible material capable of bonding or otherwise connecting elemental carbon. In certain embodiments, the biocompatible matrix material may be a biologically compatible transition metal, a biologically acceptable alkaline earth metal, or the like. In certain embodiments, the matrix material may be Fe, Co, Ni, Mg, Zn, and mixtures of any thereof. In certain embodiments, the biocompatible metallic material may be selected from the group consisting of Fe, Co, Ni, Mg, Zn, and mixtures thereof.

[0048] The elemental carbon layer can be attached to the biocompatible substrate material by any suitable method. Non-limiting examples of such attachment methods include bonding and mechanical vapor deposition. The elemental carbon layer can have a thickness suitable for promoting interaction between the lattice structure present in the carbon layer and the specific aromatic functional groups present in the nucleic acid strand material. In certain embodiments, the elemental carbon is present in a layer thickness of 1 to 40 atomic layers.

[0049] It is contemplated that the biocompatible matrix material may be maintained in a suitable carrier medium, typically a fluid carrier medium such as water, an organic fluid, or a mixture thereof, which may facilitate the uptake and stable storage of the single-stranded nucleic acid material and facilitate the dispersion of the biocompatible nanoparticles.

[0050] The biocompatible aggregates formed during the forming step can be produced by a process in which single-stranded nucleic acid material is contacted with biocompatible nanospheres. The single-stranded nucleic acid material can be produced and / or purified by any suitable means or method. It is contemplated that the introduced single-stranded nucleic acid material can be homogeneous or essentially homogeneous, as desired or necessary.

[0051] The introduction of single-stranded nucleic acids into the carrier can be carried out by any suitable method and can proceed so as to achieve aggregation. In certain embodiments, it is believed that the formation process can occur under specific solution conditions that promote such incorporation.

[0052] The method can also include maintaining the biocompatible aggregates of single-stranded nucleic acids and biocompatible nanospheres at a temperature that maintains the viability and activity of the single-stranded nucleic acids present in the biocompatible aggregates. In certain embodiments, the temperature during storage can be between -20°C and 30°C, and in certain embodiments, the temperature can be between 0°C and 20°C.

[0053] The biocompatible aggregates thus formed can be transported in an aggregated state from location to location and can be subjected to additional manipulation after storage, if desired or required.

[0054] The method may include, if desired or necessary, delivering the biocompatible aggregates to a biological destination after a storage period. In certain embodiments, the biocompatible aggregates can be delivered directly to the intended destination. In other embodiments, the single-stranded nucleic acids may be separated from the nanospheres and then delivered to the intended site or destination.

[0055] The following examples are used to illustrate the present invention but are not intended to limit the scope of the invention. All parts and percentages are by weight unless otherwise specified.

[0056] Example 1 Biocompatible nanospheres composed of one of a variety of biocompatible metals with average diameters ranging from 5 nm to 250 nm and a layer of elemental carbon ranging in thickness from 1 to 40 carbon atoms are mixed in an aqueous solution. Five biocompatible matrix materials were evaluated: Fe, Co, Ni, Mg, Ca, Zn, and mixtures thereof. The most exceptional example was Fe. 65 Co 35 and Ca. Fe 65 Co 35 The core material is used in diagnostic applications because it can be separated from the solution matrix using a magnet. Another exceptional example is Ca, which is a material that can be absorbed by the human body and is most useful for therapeutic applications.

[0057] A 20 mg quantity of each biocompatible nanosphere was mixed with 18 ml of aqueous carrier solution at 25°C. 70 μl of the nanosphere solution was introduced to an aliquot of single-stranded nucleic acid. The occurrence of binding was confirmed by visual inspection, where binding is visualized by the formation of particle clusters, as shown in Figure 5, or by quantification using an instrument such as the Denovix Nanodrop, which can analyze nucleic acid aliquot fragments by light absorption at a wavelength of 260 nm for the presence of nucleic acid. In the original nucleic acid solution, significant light absorption at 260 nm indicates the presence of nucleic acid. After binding, this signal disappears as the nucleic acid is no longer in solution, indicating the formation of biocompatible aggregates.

[0058] Example 2 The stability of biocompatible aggregates formed by the process outlined in Example 2 in a carrier medium consisting of RNase-free water at pH 10 was investigated by exposing 2 ml of the carrier medium with the biocompatible aggregates. A control sample was also prepared containing a single-stranded nucleic acid at a concentration of 5 μg / ml. Sample materials were maintained at 25°C.

[0059] Samples were mixed thoroughly by shaking and analyzed by capillary gel electrophoresis after 1, 5, and 25 days to determine RNA integrity and the associated RNA integrity number (RIN).

[0060] Figure 5 shows side-by-side photographs of unbound RNA in RNase-free water and RNA bound to biocompatible aggregates. The visible aggregation of the RNA-binding material is evidence of efficient RNA binding. The RIN and associated gel electrophoresis chart are shown in Figure 6.

[0061] Samples of biocompatible aggregate material at days 1 and 5, and unbound material at days 1 and 5, each had an RIN greater than 8. Variation in the data at day 25 occurred, with the biocompatible aggregate material achieving an RIN greater than 8, while the unbound RNA not contained in the biocompatible aggregate had an RIN of 3.2, demonstrating the long-term storage capabilities of the compositions and methods disclosed herein. An example of how this is relevant industrially is the distribution of mRNA vaccines. Because RNA degrades after 25 days, even in pure water, refrigerated storage is typically required to maintain RNA integrity. Biocompatible aggregates maintain their integrity for at least 25 days at room temperature.

[0062] Example 3 To further analyze the stability of the compositions and methods disclosed herein, urinary RNA is stabilized at room temperature using the methods described herein, and glyceraldehyde-3-phosphate dehydrogenase (GADPH) activity 702, β-actin activity 704, and 18s rRNA 706 a6s are monitored on days 0, 2, 4, and 6. The data are presented graphically and tabularly in Figure 7. The stability of RNA collected from urine is important for applications such as at-home kidney transplant rejection testing and prostate cancer screening.

[0063] Example 4 The composition disclosed in Example 1 was used to collect and transport RNA from urine.

[0064] To evaluate the storage and transport capabilities of the compositions disclosed herein, urine samples were introduced into sample vials along with the appropriate enzymes, and biocompatible nanospheres with an elemental carbon coating on an Fe substrate were added and dissolved. The vials were then placed in a magnetic holder to remove the liquid. Ethanol was then added to the vials, which were then sealed and shipped. The materials were kept at room temperature.

[0065] The stored RNA was tested for activity against Pumillo homolog 1 (PUM1)802, actin804, 18S ribosomal RNA (18s rRNA)806, and TATA-bonding protein (TBP)808 at the start, 5 days, 10 days, 15 days, and 25 days. Activity values did not change over the test period, indicating that the single-stranded RNA remained bound to biocompatible aggregates. The results are summarized in Figure 8.

[0066] While the present invention has been described in connection with particular embodiments, it is to be understood that the invention is not limited to the disclosed embodiments but is intended to cover various modifications and equivalent arrangements within the scope of the appended claims, which scope is to be accorded the broadest interpretation so as to encompass all such modifications and equivalent arrangements permitted under law.

Claims

1. a plurality of biocompatible nanospheres; a plurality of individual single-stranded nucleic acids; A carrier medium; Including, the biocompatible nanospheres each have an outer surface having elemental carbon attached thereto; The composition, wherein at least a portion of the plurality of individual single-stranded nucleic acids are coordinately linked to the biocompatible nanosphere.

2. the individual single-stranded nucleic acids and the individual biocompatible nanospheres form nanoparticle aggregates; The composition of claim 1 , wherein at least a portion of the nanoparticle aggregates comprise individual single-stranded nucleic acids coordinately linked to at least two individual biocompatible nanospheres.

3. The composition of claim 2, wherein the individual single-stranded nucleic acids coordinately linked to at least two biocompatible nanospheres are oriented such that a portion of the individual single-stranded nucleic acid is located between at least two individual biocompatible nanospheres that are in an unbound state.

4. The composition of claim 1 , wherein at least one of the plurality of individual single-stranded nucleic acids is RNA.

5. the biocompatible nanospheres are composed of a biocompatible matrix material; 10. The composition of claim 1, wherein the biocompatible matrix material is selected from the group consisting of Fe, Co, Ni, Mg, Zn, and mixtures thereof.

6. The composition of claim 5 , wherein the biocompatible matrix material is present in the biocompatible nanospheres in an amount of at least 10% by weight.

7. 10. The composition of claim 1, wherein the elemental carbon bound to the biocompatible nanospheres is present at a thickness of between 1 Angstrom and 0.5 nm.

8. The composition of claim 1, wherein the biocompatible nanospheres have an average diameter of 1 nm to 500 nm.

9. The composition of claim 1, wherein the biocompatible nanospheres have an average diameter of 5 nm to 250 nm.

10. The composition of claim 1 , wherein the elemental carbon is present as a conformal coating.

11. The composition of claim 10, wherein the elemental carbon has a thickness of 1 to 40 atomic layers.

12. 11. The composition of claim 10, wherein the elemental carbon is formed into sheets similar to graphene or graphitic carbon.

13. The composition of claim 2, wherein the nanoparticle aggregates have a size of 50 to 750 nm.

14. The composition of claim 1 , wherein the carrier medium is a liquid.

15. The composition of claim 1 wherein the carrier medium is water.

16. The composition of any one of claims 1 to 15, further comprising a biologically acceptable group II ion.

17. 17. The composition of claim 16, wherein the biologically acceptable Group II ions are selected from the group consisting of calcium ions, magnesium ions, and mixtures thereof.

18. 17. The composition of claim 16, wherein the biologically acceptable group II ion is present in an amount of 50 mM to 500 mM.

19. The composition of claim 1, wherein the coordination connection between the single-stranded nucleic acid and the elemental carbon comprises a π-π bond.

20. 1. A composition suitable for storing RNA, comprising: a plurality of biocompatible nanospheres; a liquid carrier medium; a biologically acceptable group II ion present in a concentration sufficient to enhance binding between the RNA and the biocompatible nanosphere; Including, the biocompatible nanospheres each have an outer surface connected to elemental carbon; the biocompatible nanospheres have an average diameter of 1 nm to 500 nm; The composition, wherein the elemental carbon is present as a conformal coating.

21. 21. The composition of claim 20, wherein the biologically acceptable Group II ions are selected from the group consisting of calcium ions, magnesium ions, and mixtures thereof.

22. 21. The composition of claim 20, wherein the biologically acceptable group II ion is present in an amount of 50 mM to 500 mM.

23. 21. The composition of claim 20, wherein the carrier medium is water.

24. 21. The composition of claim 20, wherein the biocompatible nanospheres have an average diameter of 5 nm to 250 nm.

25. 25. The composition of claim 20, wherein the elemental carbon has a thickness of 1 to 40 atomic layers and is formed into sheets similar to graphene or graphite carbon.

26. 26. The composition of claim 25, wherein the elemental carbon bound to the biocompatible nanospheres is present at a thickness of between 1 Angstrom and 0.5 nm.

27. a plurality of biocompatible nanospheres; a plurality of individual single-stranded nucleic acids; a liquid carrier medium; a biologically acceptable group II ion present in a concentration sufficient to enhance binding between one individual single-stranded nucleic acid and one biocompatible nanosphere; Including, the plurality of biocompatible nanospheres each having an outer surface connected to elemental carbon; the plurality of biocompatible nanospheres have an average diameter of 1 nm to 500 nm; the elemental carbon is present as a conformal coating; At least a portion of each of the single-stranded nucleic acids is in coordinated connection with at least two biocompatible nanospheres; at least one of said individual single-stranded nucleic acids is RNA; A composition wherein the individual single-stranded nucleic acids that are coordinately connected to at least two of the biocompatible nanospheres are oriented so that portions of the individual single-stranded nucleic acids are located between the at least two biocompatible nanospheres.

28. 28. The composition of claim 27, wherein the biologically acceptable Group II ions are selected from the group consisting of calcium ions, magnesium ions, and mixtures thereof.

29. 28. The composition of claim 27, wherein the biologically acceptable group II ion is present in an amount of 50 mM to 500 mM.

30. 28. The composition of claim 27, wherein the liquid carrier medium is water.

31. 28. The composition of claim 27, wherein the biocompatible nanospheres have an average diameter of 5 nm to 250 nm.

32. 28. The composition of claim 27, wherein the elemental carbon has a thickness of 1 to 40 atomic layers and is formed into sheets similar to graphene or graphitic carbon.

33. 33. The composition of claim 32, wherein the elemental carbon attached to the biocompatible nanospheres is present at a thickness of between 1 Angstrom and 0.5 nm.

34. the biocompatible nanospheres are made of a biocompatible metallic material; 28. The composition of claim 27, wherein the biocompatible metallic material is selected from the group consisting of Fe, Co, Ni, Mg, Zn, and mixtures thereof.

35. 28. The composition of claim 27, wherein the coordination connection between the single-stranded nucleic acid and the elemental carbon comprises a π-π bond.

36. the individual single-stranded nucleic acids and the individual biocompatible nanospheres interact to form nanoparticle aggregates; 36. The composition of any one of claims 27-35, wherein at least a portion of the aggregates comprise one individual single-stranded nucleic acid coordinated with at least two individual nanospheres.

37. 1. A method for storing and transporting single-stranded nucleic acid material, comprising: forming biocompatible aggregates of single-stranded nucleic acids and biocompatible nanospheres; A method for storing and transporting single-stranded nucleic acid material, wherein the biocompatible aggregates comprise biocompatible nanospheres each having an outer surface connected by elemental carbon and at least two individual nucleic acid strands connected to the biocompatible nanospheres.

38. 38. The method of claim 37, further comprising maintaining the biocompatible aggregates of single-stranded nucleic acids and biocompatible nanospheres at a temperature between 0°C and 30°C during storage.

39. 39. The method of claim 38, further comprising the step of delivering the biocompatible aggregate to an in vivo destination after a storage period.

40. 39. The method of claim 38, further comprising separating the single-stranded nucleic acid from contact with the biocompatible nanosphere after a storage period.

41. 38. The method of claim 37, wherein the biocompatible nanospheres have an average diameter of 1 nm to 500 nm and the elemental carbon is present as a conformal coating.

42. 38. The method of claim 37, wherein the elemental carbon has a thickness of 1 to 40 atomic layers and is formed in a sheet form similar to graphene or graphitic carbon.

43. 43. The method of claim 42, wherein the elemental carbon bound to the biocompatible nanospheres is present at a thickness of between 1 Angstrom and 0.5 nm.

44. 42. The method of claim 37 or claim 41, wherein the formation of the biocompatible aggregates proceeds within a composition comprising the biocompatible nanospheres and the single-stranded nucleic acid, and further comprising a liquid carrier medium and a biologically acceptable group II ion selected from the group consisting of calcium ions, magnesium ions, and mixtures thereof.

45. 45. The method of claim 44, wherein the liquid carrier medium is water.

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