Alkali-resistant calcium phosphate / nucleic acid hybrid carrier for pest control and method for producing particles

Abstract

Alkali-resistant calcium phosphate / nucleic acid hybrid carriers for pest control and methods for producing particles are provided. A stable aqueous suspension includes a composition containing nanoparticles, wherein the nanoparticles include nucleic acids, which are at least partially encapsulated in a matrix containing calcium phosphate. The nanoparticle composition and suspension have high nucleic acid loading and high stability, and can be used to protect crops from pests and to downregulate the expression of target genes in pest species, thereby preventing and / or controlling pest infestations.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority under 35 U.S.C. §119 to U.S. Provisional Application Serial No. 62 / 945,780, filed on December 9, 2019, the contents of which are hereby relied upon and incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to compositions for delivering nucleic acids into cells or microorganisms. In particular, the present disclosure relates to delivering insecticidal RNA to pests and insects to protect plants and crops. Background Art

[0004] It is estimated that plant pests and pathogens reduce global crop yields by 30% to 40% annually. Meanwhile, chemical control (i.e., the use of pesticides), which has been used for decades and is considered to be the best choice for controlling pest populations at present, has now been regarded as a potential source of health risks, because it may cause many serious health effects and diseases, and can poison the environment permanently. The second problem caused by the overuse or abuse of chemical pesticides is the resistance that pests produce to these chemical agents. Pests with known short generation times can produce resistance faster, which makes pesticides unavailable quickly. Although the use of transgenic plants expressing insecticidal proteins has been considered, the deployment of transgenic plants faces public resistance and may not meet certain regulations in some countries. Therefore, the demand for new methods to protect crops is growing.

[0005] RNA interference (RNAi) has been used by researchers for many years in genetic studies, but it has recently been demonstrated that RNAi can be used to silence certain target genes in pests. RNAi is a process in which 19-30 small nucleic acid RNA molecules trigger the destruction or decay of complementary transcripts. RNAi-based pest control improves on traditional small molecule insecticides by providing high specificity for target species. This biological approach seems attractive because specific pests can be targeted and their populations controlled without affecting non-target species, which is not possible using chemical insecticides. For example, dsRNA introduced into mosquito larvae has been shown to induce RNAi-mediated knockdown of male fertility genes. RNA interference can be used to control Colorado potato beetle (CPB) populations simply by spraying the appropriate RNA on the leaves of potato plants.

[0006] It is now well known that if RNA interference is to be a promising option, methods of delivering RNA molecules to insects remain the main problem to be solved. Ideally, RNA needs to be delivered to pests such as insects by simple oral delivery. However, for oral treatment, dsRNA must survive ingestion and then must be taken up by epithelial cells and migrate to the appropriate tissues to activate the RNAi machinery. Another important problem to be solved comes from the low stability of RNA molecules in the environment. RNA is rapidly digested by RNases and self-cleaves in sunlight. RNases may be present in the digestive tract of the target insects and may also be present in the environment.

[0007] Some previous attempts to use RNAi to control pests involved simple topical application of naked RNA without any protection onto plant surfaces, such as leaves, but this technique requires large amounts of nucleic acids, as most of them degrade rapidly in the field, usually in less than a week, making the treatment economically unfeasible. It is estimated that such RNAi-based treatments would have to last at least a month to be economically beneficial, but for this to work the RNA would have to be protected from the outside world.

[0008] Another problem is that when the target pest is an insect, once the RNA reaches the target pest, it must resist the harsh environment observed in the insect gut lumen. For example, the gut of lepidopterans can exhibit a pH of 10-11, which is comparable to the pH of oven cleaners, which quickly cause hydrolysis of the most resistant molecules. In addition, there are many nucleases in the digestive tract that can also cause degradation of RNA. Yeast expressing RNA of interest for RNA interference can be used as a natural carrier for feeding target pests. However, yeast is a living organism, which complicates the use and storage of the product.

[0009] A simple method for protecting RNA and improving its delivery in target organism cells is to form a complex between negatively charged RNA and some positively charged polycations. The complex is called a polyplex, which is widely used in transfection for therapeutic applications. In effective polycations, synthetic PEI or chitosan derived from naturally occurring chitin and polymers containing guanidine are widely used for this purpose. In order to further improve the protection of nucleases, it is proposed to use polymers, such as dextran sulfate, which is known to be an effective inhibitor of nucleases, because most of sulfated polyanions are nuclease inhibitors (see, for example, U.S. Patent No. 8,841,272B2).

[0010] Recently, a clay composition for delivering double-stranded RNA to plants has been proposed, which protects plants from the influence of various organisms. In this method, RNA is simply adsorbed on a layered double hydroxide (LDH) clay complex. The composition can be in the form of a suspension that can be sprayed onto plants (see, for example, European Patent Application No. 3116312). LDH is a mixed hydroxide of divalent and trivalent metals with excess positive charge, which is balanced by internal ions and can absorb negatively charged nucleic acids. Although the combination of such nucleic acids and inorganic carriers seems to be easy to implement, the delivery of an appropriate amount of nucleic acids may not be so effective. Recently, improvements to clay-based technology have been reported in U.S. Patent No. 10,405,539, which is achieved by pre-treating clay particles with polycations as an additional step before contacting RNA to increase the electrostatic interaction between the clay and negatively charged nucleic acids so coated. When pre-treated with polyethyleneimine (PEI), the reported RNA load in a solid system is only 1% by weight. Such RNA loading is not high enough for application, particularly plant-based applications. However, by treating the clay with the synthetic cationic polymer poly(2-dimethylaminoethyl methacrylate) (PDMAEMA), they achieved RNA loadings of up to 25%. Although this modified method enabled higher RNA loadings, the method required an additional step of coating with a synthetic polymer.

[0011] These results show that the absorption of appropriate amounts of nucleic acids on the surface of inorganic carriers remains challenging and depends largely on the polycation used.

[0012] In the early 1970s, Graham and van der Eb (Virology; Issue 2, Vol. 52, April 1973, pp. 456-467) found that DNA was coprecipitated with calcium phosphate formed by the reaction between calcium chloride and phosphate buffer, and when exposed to cells, was adsorbed by cells. In the past few decades, this technology has been widely used for in vitro transfection of mammalian cells, and this calcium phosphate inorganic nanoparticle is considered to be a promising non-viral vector for gene therapy applications. However, the technology developed by Graham needs to use a large amount of excessive calcium to increase cell transport and therefore promote the internalization of nanoparticles, thereby promoting nucleic acid to penetrate into the cytoplasm. Usually, in Graham's scheme, the ratio of calcium to phosphate>200.

[0013] A large excess of calcium ions increases the ionic strength, which has a deleterious effect on the stability of electrostatically stabilized suspensions. This excess of calcium results in an unstable calcium phosphate / nucleic acid suspension that undergoes rapid aggregation and uncontrolled size increase. The resulting suspension flocculates and is not stable enough to be stored, and in fact they must be used for transfection immediately after preparation. Therefore, the methods reported by Graham and many other authors that rely on a high calcium to phosphorus ratio cannot be used to prepare a stable suspension that can meet the requirements of plant quarantine compositions for storage. In another example, M Jordan et al. reported in "Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation", 96-601 Nucleic Acids Research, 1996, No. 4, Vol. 24 that soluble DNA in the reaction mixture can be combined with calcium phosphate to form an insoluble complex in less than 1 minute, but extending the reaction time to 20 minutes will lead to uncontrolled aggregation and / or growth.

[0014] Therefore, there is a need for compositions and methods for preparing stable suspensions of CaP / nucleic acid hybrid nanoparticles with high nucleic acid loadings to avoid spraying large amounts of inorganic substances on crops to achieve the amount of RNA required to control pests through RNA interference, or to avoid giving animals too much medicine without being efficient enough. Summary of the invention

[0015] According to the first aspect, a composition comprises nanoparticles, wherein the nanoparticles comprise nucleic acids, which are at least partially encapsulated in a matrix comprising calcium phosphate. In some embodiments, the nucleic acids at least partially encapsulated in the calcium phosphate matrix comprise ribonucleic acid, deoxyribonucleic acid or derivatives thereof. In some embodiments, the composition also comprises a capping agent, which at least partially coats the nanoparticles or infiltrates the nanoparticles, or both coats the nanoparticles and infiltrates the nanoparticles.

[0016] In some embodiments, the capping agent comprises a polycation. In some embodiments, the polycation is selected from the group consisting of polyamines, polylysine, polyarginine, polyornithine, polyalkylene imine, chitosan, oligopeptides, cationized polysaccharides, cationized plant proteins or cationized animal proteins, protamine, diethylaminoethyl-dextran, polyamidoamines, poly(β-amino esters), aminopolysaccharides, polymethylaziridine, polyallylamine, polyvinylamine homopolymers or copolymers, poly(vinylpyridine) homopolymers or copolymers, poly(meth)acrylate homopolymers or copolymers, poly(meth)acrylamide homopolymers or copolymers and derivatives thereof. In some embodiments, the polycation is polyethyleneimine.

[0017] In some embodiments, the peak particle size of the nanoparticles is less than 500nm. In some embodiments, the peak particle size of the nanoparticles is in the range of 50nm to 500nm. In some embodiments, the weight ratio of calcium phosphate to nucleic acid in the nanoparticles is 100:1 to 1:1.5. In some embodiments, the weight ratio of calcium phosphate to nucleic acid to capping agent in the nanoparticles is 2:1:2 to 2:5:2. In some embodiments, the ratio of the number of positively charged nitrogen atoms in the polycation to the number of phosphorus atoms in the nucleic acid is greater than 1. In some embodiments, the nanoparticles are suspended in an aqueous dispersion with a pH of 6 to 11. In some embodiments, the nanoparticles are suspended in an aqueous dispersion with a pH of 7 to 8. In some embodiments, the composition is a stable aqueous suspension.

[0018] According to the second aspect, a stable aqueous suspension comprises: a composition comprising nanoparticles, wherein the nanoparticles include nucleic acids, which are at least partially encapsulated in a matrix comprising calcium phosphate. In certain embodiments of the stable aqueous suspension, the composition further comprises a capping agent, which at least partially coats the nanoparticles or permeates the nanoparticles, or both coats and permeates the nanoparticles. In certain embodiments, the nucleic acid loading is about 0.1 wt % to about 50 wt % relative to the total mass of the nanoparticles. In certain embodiments, the concentration of calcium phosphate in the aqueous suspension is less than 100 μg / ml. In certain embodiments, the molar ratio of calcium to phosphorus in the calcium phosphate suspension is 1.5:1 to 5:1. In certain embodiments, the suspension is alkali-resistant. In certain embodiments, the stable aqueous suspension further comprises an additive selected from the group consisting of a nuclease inhibitor, a surfactant, a wetting agent, a UV absorber, an antioxidant, an antifreeze agent, a preservative, a colorant, an insecticide, a fungicide, an attractant, a repellent, and a rheology modifier.

[0019] According to the third aspect, a method for protecting crops from insects, the method comprising: administering a composition to the crop, wherein the composition comprises nanoparticles, wherein the nanoparticles comprise nucleic acids, and the nucleic acids are at least partially encapsulated in a matrix comprising calcium phosphate. In certain embodiments, the nucleic acids at least partially encapsulated in the calcium phosphate matrix comprise ribonucleic acids, deoxyribonucleic acids, or derivatives thereof. In certain embodiments, the composition further comprises a capping agent that at least partially coats the nanoparticles or permeates the nanoparticles, or both coats and permeates the nanoparticles. In certain embodiments, the composition is administered to the crop by spraying, dripping, or irrigating. In certain embodiments, the nucleic acids in the composition comprise insecticidal double-stranded ribonucleic acids. In certain embodiments, the method comprises: protecting the crop from hemiptera, coleoptera, siphonaptera, dichyoptera, lepidoptera, orthoptera, and diptera.

[0020] According to the fourth aspect, a method for producing hybrid nanoparticles comprises: adding a nucleic acid to one of a calcium salt solution or a phosphate solution in a reactor; adjusting the pH of the calcium salt solution and the phosphate solution to about 6 to about 9; and mixing the calcium salt solution with the phosphate solution to form calcium phosphate-nucleic acid hybrid nanoparticles. In some embodiments, the method further comprises: adding a polycation solution to the mixed solution. In some embodiments, the calcium salt is selected from the group consisting of calcium chloride, calcium nitrate, calcium acetate, and calcium lactate. In some embodiments, the phosphate is selected from the group consisting of trisodium phosphate, tripotassium phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, ammonium phosphate, and ammonium hydrogen phosphate. In some embodiments, the nucleic acid is at least partially encapsulated in the calcium phosphate nanoparticles. In some embodiments, the peak particle size of the hybrid nanoparticles is in the range of 50 nm to 500 nm. In some embodiments, the pH of the calcium salt solution is about 7.5. In some embodiments, the pH of the phosphate solution is about 7.5. In certain embodiments of the method, the reactor is selected from the group consisting of a batch reactor, a semi-batch reactor, a continuous flow reactor, an advanced flow reactor, a fluidized bed reactor, and combinations thereof.

[0021] According to the fifth aspect, a method for preparing a suspension of hybrid nanoparticles in a continuous flow reactor comprises: adding nucleic acid to one of a calcium salt solution or a phosphate solution; flowing the calcium salt solution into the first inlet of the flow reactor; flowing the phosphate solution into the second inlet of the flow reactor so that the phosphate solution stream mixes with the calcium salt solution stream and produces a calcium phosphate-nucleic acid hybrid nanoparticle stream flowing from the reactor; collecting and processing the suspension containing calcium phosphate-nucleic acid hybrid nanoparticles from the reactor. In some embodiments, the method further comprises: flowing the capping polymer solution into the third inlet of the flow reactor so that the capping polymer stream mixes with the calcium phosphate stream to produce a polymer-capped calcium phosphate stream encapsulated with nucleic acid flowing from the reactor. In some embodiments, the method further comprises: collecting and processing the polymer-capped calcium phosphate encapsulated with nucleic acid from the reactor. In some embodiments, the ζ (zeta) potential of the nanoparticles is -50 to 50 millivolts. In some embodiments, the median particle size of the nanoparticles is in the range of 30nm to 250nm. In some embodiments, the nanoparticles are suspended in a liquid medium. In certain embodiments, the liquid medium is selected from the group consisting of water, alcohols, hydrocarbons, and oils.

[0022] According to the 6th aspect, a water-in-oil emulsion (W / O emulsion) includes a composition comprising nanoparticles, wherein the nanoparticles include nucleic acids, which are at least partially encapsulated in a matrix comprising calcium phosphate. In some embodiments, the nucleic acids at least partially encapsulated in the calcium phosphate matrix include ribonucleic acid, deoxyribonucleic acid or derivatives thereof. In some embodiments, the composition also includes a capping agent, which at least partially coats the nanoparticles or permeates the nanoparticles, or both coats the nanoparticles and permeates the nanoparticles.

[0023] According to the 7th aspect, a water-in-oil-in-water double emulsion (W / O / W emulsion) includes a composition comprising nanoparticles, wherein the nanoparticles include nucleic acids, which are at least partially encapsulated in a matrix comprising calcium phosphate. In some embodiments, the nucleic acids at least partially encapsulated in the calcium phosphate matrix include ribonucleic acid, deoxyribonucleic acid or derivatives thereof. In some embodiments, the composition also includes a capping agent, which at least partially coats the nanoparticles or permeates the nanoparticles, or both coats the nanoparticles and permeates the nanoparticles.

[0024] According to an eighth aspect, a glass container contains an aqueous suspension containing nanoparticles and a composition, wherein the nanoparticles include nucleic acids at least partially encapsulated in a matrix comprising calcium phosphate. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings and the following description set forth details of one or more embodiments of the subject matter described in this specification. Other features, aspects, and advantages of the subject matter of this application will become apparent from the description, drawings, and claims.

[0026] Figure 1 Illustrated is a schematic flow diagram of a basic method for preparing nucleic acid loaded calcium phosphate nanoparticles (without polycation) and a perspective view of the resulting hybrid nanoparticles according to one or more embodiments shown and described herein.

[0027] Figure 2 Illustrated is a schematic flow diagram of a basic method for preparing nucleic acid-loaded calcium phosphate nanoparticles with polycationic capping and a perspective view of the resulting capped hybrid nanoparticles in accordance with one or more embodiments shown and described herein.

[0028] Figure 3 A graph showing particle DLS size of calcium phosphate-DNA nanoparticles is illustrated in accordance with one or more embodiments shown and described herein.

[0029] Figure 4 According to one or more embodiments shown and described herein, bar graphs of the zeta potential of calcium phosphate (CaP) particles without any nucleic acid loading, calcium phosphate loaded with DNA (CaP / DNA) particles, and calcium phosphate / nucleic acid (CaP / DNA / PEI) particles capped with PEI polycations are illustrated.

[0030] Figure 5 A graph showing the sizes of nucleic acid carrier particles CaP / DNA [2:1] at acidic pH, neutral pH, and alkaline pH, wherein the nucleic acid carrier particles CaP / DNA [2:1] are made of CaP prepared according to the method described in Example 1 and nucleic acid.

[0031] Figure 6 Illustrated are graphs showing the sizes of nucleic acid carrier particles CaP / DNA / PEI[2:1:2] at acidic pH, neutral pH, and alkaline pH, wherein the nucleic acid carrier particles CaP / DNA / PEI[2:1:2] are made of a PEI outer layer prepared according to the method described in Example 3, CaP, and nucleic acid.

[0032] Figure 7 Graphs showing the particle size evolution of diluted and undiluted CaP / DNA [2:1] and CaP / DNA / PEI [2:1:2] are illustrated.

[0033] Figure 8 A schematic diagram showing the apparatus of a tubular flow reactor for preparing a suspension in continuous mode (flow) according to Example 4 is illustrated.

[0034] Fig. 9 Illustrated is a schematic diagram showing a hypothetical AFR reactor setup for preparing a suspension in a continuous mode according to one or more embodiments shown and described herein.

[0035] Fig. 10A and 10B Fluorescence images of negative control and cells transfected with GFP plasmid are shown, respectively, according to one or more embodiments shown and described herein.

[0036] Fig.11 According to one or more embodiments shown and described herein, electrophoretic mobility of DNA profiles in various nanoparticle samples is shown.

[0037] Fig.12 SEM micrographs showing the morphology of nanoparticles prepared from dsDNA / calcium phosphate / PEI [2:4:0] prepared according to the method described in Example 13 (dry state) are shown.

[0038] Fig.13A and 13B Shown are SEM micrographs at two magnifications showing the morphology and size (dry state) of nanoparticles prepared from dsDNA / calcium phosphate / PEI [2:1:2].

[0039] Fig.14 are images showing gel retardation experiments (0.8% agarose) performed on two concentrations of dsRNA / calcium phosphate / PEI [2:1:2] nanoparticle suspensions prepared according to the method described in Example 10.

[0040] Like reference numbers and symbols in different drawings indicate like elements. DETAILED DESCRIPTION

[0041] The various embodiments shown in the accompanying drawings will now be described in detail. Whenever possible, the same reference numerals are used in the accompanying drawings to represent the same or similar parts. The components in the accompanying drawings are not necessarily drawn to scale, but rather focus on illustrating the principles of the exemplary embodiments.

[0042] As described above, the technology of the present disclosure generally relates to compositions and methods for delivering desired materials, such as nucleic acids, to cells and tissues. In various embodiments, compositions and systems comprising particles, such as nanoparticles, are provided herein for delivering therapeutic agents to cells or tissues of plants, animals and insects, as well as methods for making and using the same.

[0043] definition

[0044] Definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the meaning commonly understood by one of ordinary skill in the art to which the technology of this disclosure belongs.

[0045] definition

[0046] As used in this specification and claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a" component includes aspects having two or more such components, unless the context clearly indicates otherwise. For example, reference to "a cell" includes combinations of two or more cells, etc. Furthermore, when the word "or" is used without the word "either" (or other similar language indicating that "or" is explicitly exclusive - for example, only one of x or y, etc.), it should be interpreted as inclusive (for example, "x or y" means one or both of x or y). In general, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry described below are those well known and commonly used in the art.

[0047] The term "and / or" should also be interpreted as inclusive (e.g., "x and / or y" means one or both of x or y). Where "and / or" or "or" is used as a conjunction of a group of three or more items, the group should be interpreted as including only one item, all items together, or any combination or quantity of such items. In addition, terms such as having, possessing, covering, and containing used in the specification and claims should be understood as synonymous with the terms comprising and including. Other elements other than those specifically identified by the "and / or" clause may optionally be present, whether or not related to those specifically identified elements. As a non-limiting example, a reference to "X and / or Y" may refer to only X (optionally including elements other than Y) in one embodiment; may refer to only Y (optionally including elements other than X) in some embodiments; and may refer to both X and Y (optionally including other elements) in some embodiments.

[0048] As used herein, the term "about" with respect to numbers is generally considered to include numbers that fall within 1%, 5%, or 10% in either direction (greater than or less than) of that number unless otherwise stated or obvious from the context (unless the number is less than 0% of the possible value or greater than 100% of the possible value).

[0049] Unless otherwise stated, "particle size" as used herein refers to the diameter of the particle. Particle size is, for example, expressed as a peak particle size, which represents the hydrodynamic diameter of the particle obtained by dynamic light scattering (DLS) technology. Particle size is also expressed as a median or average particle size.

[0050] The present technology relates to stable aqueous suspensions prepared from nucleic acids and precipitated alkali-stable calcium phosphates in combination with at least one cationic polymer for delivering nucleic acids to harmful organisms, and to methods for producing the aqueous suspensions. The methods of the present technology relate to spontaneous formation of calcium phosphate nanoparticles from calcium salts and phosphates in the presence of nucleic acids to be loaded, thereby obtaining stable calcium phosphate / nucleic acid nanoparticle suspensions. Additional methods relate to external coating of nucleic acid / calcium phosphate particles with polycations to provide stability in variable pH conditions, including in highly alkaline conditions.

[0051] Composition

[0052] Aspects of the present technology relate to compositions and suspensions comprising nanoparticles. In various embodiments, the nanoparticles include a matrix that includes calcium phosphate (CaP). In various embodiments, CaP represents a family of minerals containing calcium cations and phosphate anions. Exemplary minerals may include, but are not limited to, hydroxyapatite (Ca 10 (PO4)6(OH)2) or a suitable precursor thereof, monophasic or biphasic calcium phosphate, dicalcium phosphate, tricalcium phosphate, β-tricalcium phosphate (β-Ca3PO4), a complex of calcium sulfate and hydroxyapatite, a complex of hydroxyapatite or a combination thereof. Suitable nanoparticle matrix materials include CaP biomaterials that can be used in various pharmaceutical, biomedical, food and cosmetic applications.

[0053] Nanoparticles with calcium phosphate matrix can also include nucleic acid, which is at least partially encapsulated in the matrix. The nucleic acid can be encapsulated in or incorporated into the CaP matrix, can be coated or located on the CaP matrix surface, or can be both encapsulated in and coated on the nanoparticle surface. Suitable nucleic acid includes any ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), which can be unmodified or modified. In various embodiments, nucleic acid includes but is not limited to single-stranded and double-stranded RNA, single-stranded and double-stranded DNA, single-stranded region and double-stranded region mixed RNA, single-stranded region and double-stranded region mixed DNA, and hybrid molecules comprising DNA and RNA, which DNA and RNA can be single-stranded, or more generally, can be double-stranded or single-stranded region and double-stranded region mixed. In addition, nucleic acid refers to comprising RNA or DNA or both, DNA or RNA comprising one or more modified bases, and triple-stranded regions of DNA or RNA modified for stability or other reasons of the skeleton. In some embodiments, nanoparticles can include other therapeutic agents except nucleic acid. Suitable nucleic acids and therapeutic agents can be selected from the group consisting of genes, shRNA, siRNA, microRNA, DNA fragments, RNA fragments, plasmids, and combinations thereof. In some embodiments, the nucleic acid comprises ribonucleic acid, deoxyribonucleic acid, or a derivative thereof. In some embodiments, the nucleic acid comprises ribonucleic acid or a derivative thereof. In other embodiments, the nucleic acid comprises deoxyribonucleic acid or a derivative thereof. In some embodiments, the nucleic acid comprises an interfering ribonucleic acid molecule. In some embodiments, the nucleic acid comprises a small interfering RNA (siRNA).

[0054] The nanoparticles may include a capping agent that at least partially coats or permeates the nanoparticles, or both coats and permeates the nanoparticles. In various embodiments, the capping agent may include a compound or composition that helps deliver nucleic acids to cells. For example, the capping agent may include a cationic compound that is a compound having a polar group that is positively charged at or near physiological pH. Since these compounds have the ability to neutralize the charge of nucleic acids, it is speculated that these compounds help deliver nucleic acids to cells. Exemplary cationic compounds include, but are not limited to, polycations, cationic lipids, cationic polymers, or mixtures thereof. In certain embodiments, the capping agent comprises a polycation. The polycation to be used as a capping agent may be a synthetic polymer, a naturally occurring polymer, or a chemically modified naturally occurring polymer. The polycation may suitably have a plurality of positively charged groups at a pH between 5 and 11. The charged group may be selected from alkylamines (including primary amines, secondary amines, tertiary amines, and quaternary amines), imines, guanidine salts, and aromatic amines. The polycation may be branched or linear. For example, in the case of PEI, linear polyethyleneimine (PEI) contains all secondary amines, while branched PEI contains primary, secondary and tertiary amino groups. The pKa of positively charged amino groups can be greater than 7.5, for example, greater than 8, greater than 9 or greater than 10. For some applications, net cationic charge is maintained under alkaline conditions. As a result, nucleic acid remains bound to polycations, which provides certain protection to prevent degradation by nucleases (known polycation / nucleic acid complexes show better stability than non-complex forms). Polycations can contain one or more types of amino groups with different pKa values. Polycations can be homopolymers or copolymers. When the positively charged polymer is a copolymer, a comonomer to be copolymerized with a monomer containing a charged amino group is selected to maintain a sufficient level of water solubility for the copolymer. Other water-soluble polycations can be selected in chitosan and its derivatives (e.g., trimethyl chitosan, guanidinized chitosan), cationized polysaccharides (e.g., DEAE dextran) and cationized plant proteins or cationized animal proteins.

[0055] Suitable polycations include, but are not limited to, polyamines, polylysine, polyarginine, polyornithine, polyalkyleneimines, chitosan, oligopeptides, cationized polysaccharides, cationized plant proteins or cationized animal proteins, protamine, activated dendrimers, diethylaminoethyl dextran, polyamidoamines, poly(β-amino esters), aminopolysaccharides, polymethylaziridine, polyallylamine, polyvinylamine homopolymers or copolymers, poly(vinylpyridine) homopolymers or copolymers, poly(meth)acrylate homopolymers or copolymers, poly(meth)acrylamide homopolymers or copolymers and derivatives thereof, cationic compounds comprising peptides and protein fragments, or combinations thereof. Preferred examples are polyethyleneimine (PEI), hexadimethrine bromide [available under the trade name polybrene], polyhexamethyleneguanidine (PHMG), polyhexamethylenebiguanide, polyallylamine, guanylated poly(allylamine), polyaminopropylmethacrylamide, poly-[N-(3-guanidinopropyl)methacrylamide], polyaminoethyl(meth)acrylate, polydimethylaminoethylmethacrylate (PDMAEMA). In various embodiments, the polycation comprises polyethyleneimine.

[0056] A sufficient amount of polycation may be added to convert the negative charge of the CaP / nucleic acid parent particles to positively charged particles with a surface charge of at least +10 mV, such as at least +20 mV, thereby providing acceptable colloidal stability.

[0057] In another aspect, provided herein are suspensions comprising nanoparticle compositions described herein. Stable suspensions can include calcium phosphate-nucleic acid nanoparticles suspended in a suitable solvent. Exemplary solvents include water and water-soluble organic solvents. Exemplary solvents include water, alcohols, hydrocarbons, and oils. In various embodiments, stable aqueous suspensions include compositions that can be stable aqueous suspensions comprising calcium phosphate nanoparticles described herein. In various embodiments, the suspensions can include CaP-nucleic acid nanoparticles suspended in an aqueous dispersion. The stable aqueous suspensions can be prepared from nucleic acids and precipitated alkali-stable calcium phosphates.

[0058] The aqueous dispersion of the suspension can suitably have a near neutral or alkaline pH. For example, the suspension can include nanoparticles suspended in an aqueous dispersion having a pH of about 6 to about 11. This includes pHs of about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6 to about 7, about 7 to about 11, about 7 to about 10, about 7 to about 9, about 7 to about 8, about 8 to about 11, about 8 to about 10, about 8 to about 9, about 9 to about 11, about 9 to about 10, or about 10 to about 11, or any two of these values ​​and / or any range between these two values. In various embodiments, the suspension can include nanoparticles suspended in an aqueous dispersion having a pH of about 7 to about 8.

[0059] Described nano-particle is unique, because nucleic acid is embedded in particle, and is adsorbed on particle surface.Therefore, nucleic acid load is significantly higher and can be compatible with multiple applications.The amount of nucleic acid loaded on nano-particle can be higher than the amount that uses traditional adsorption method to obtain.In some embodiments, relative to the gross mass of nano-particle, the amount of nucleic acid loaded on nano-particle can be greater than about 0.1 weight %.In other embodiments, relative to the gross mass of nano-particle, the amount of nucleic acid loaded on nano-particle can be greater than about 10 weight %.In some embodiments, relative to the gross mass of nano-particle, the amount of nucleic acid loaded on nano-particle can be greater than about 20 weight %.In each embodiment, relative to the gross mass of nano-particle, the nucleic acid load in nano-particle can be in the scope of about 0.1 weight % to about 55 weight %. For example, relative to the gross mass of nanoparticle, the nucleic acid load therein can be about 0.1 % by weight to about 50 % by weight, about 1 % by weight to about 45 % by weight, about 5 % by weight to about 40 % by weight, about 10 % by weight to about 35 % by weight, about 15 % by weight to about 30 % by weight, or about 20 % by weight to about 20 % by weight, or include and / or any scope between any two of these numerical values. In various embodiments, relative to the gross mass of nanoparticle, the nucleic acid load in the nanoparticle is about 0.1 % by weight to about 50 % by weight.

[0060] Although the calcium phosphate / nucleic acid nanoparticle suspension of part flocculation may be used in some applications, for most industrial applications, it is desirable to avoid the stable suspension of aggregation.Preferably stable suspension is to avoid the blocking of delivery equipment and ensure uniform deposition and delivery.Therefore, in order to avoid flocculation, the calcium phosphate concentration in the final suspension can be less than 200 μ g / ml.For example, the calcium phosphate concentration in the suspension can be less than about 150 μ g / ml, less than about 120 μ g / ml, less than about 100 μ g / ml, less than about 90 μ g / ml, less than about 80 μ g / ml, less than about 70 μ g / ml, less than about 60 μ g / ml, or less than about 50 μ g / ml. The calcium phosphate concentration in the suspension can be about 1 μ g / ml to about 150 μ g / ml, about 10 μ g / ml to about 100 μ g / ml, about 20 μ g / ml to about 90 μ g / ml, about 30 μ g / ml to about 80 μ g / ml, about 40 μ g / ml to about 70 μ g / ml, or about 50 μ g / ml to about 60 μ g / ml, or include and / or any scope between any two numerical values ​​in these numerical values. In each embodiment, the calcium phosphate concentration in the suspension is less than 100 μ g / ml. In each embodiment, the calcium phosphate concentration in the suspension is less than 50 μ g / ml.

[0061] Calcium can affect the stability of nanoparticles relative to the content of total phosphate ions (that is, the phosphate of phosphate adds the phosphate from nucleic acid). Calcium is slightly excessive relative to phosphate ions for producing stable nanoparticle suspensions is desirable. However, excessive calcium may cause nanoparticles to assemble rapidly in suspension. The mol ratio (Ca / P) of calcium and phosphorus in the suspension can be in the range of about 1.5:1 to 5:1, for example, about 1.6:1 to 4.5:1, about 1.7:1 to 4:1, about 1.8:1 to 3.5:1, about 1.9:1 to 3:1 or about 2:1 to 2.5:1, or include and / or any scope between any two numerical values ​​in these numerical values. In each embodiment, the mol ratio of calcium and phosphorus in the calcium phosphate suspension is 1.5:1 to 5:1. In each embodiment, the mol ratio of calcium and phosphorus in the suspension is about 1.7:1.

[0062] The suspension of the present technology not only shows high stability, but they are also alkali resistant. The polycation / nucleic acid complex of the nanoparticles of the encapsulation may be stabilized to a pKa value as high as the polycation. Higher than the pKa, the complex may dissociate and release the composite nucleic acid. Alkali resistance is important for the application of nanoparticles delivered to the digestive system of insects, and the digestive system of known insects is highly alkaline (e.g., pH>9.0), and it is still challenging to make nucleic acids withstand this harsh alkaline environment. Suspension as herein described advantageously has alkali resistance.

[0063] The stable aqueous suspension may also include additives known in the art and listed herein. Suitable additives include, but are not limited to, nuclease inhibitors, surfactants, wetting agents, UV absorbers, antioxidants, antifreeze agents, preservatives, colorants, insecticides, fungicides, attractants, repellents, and rheology modifiers. Unless otherwise mentioned, the amount of the additive is based on the total weight of the nanoparticles in the suspension. When present, the suspension may include about 0.01% by weight to about 5% by weight of the additive.

[0064] The hybrid nanoparticles of the composition have narrow particle size distribution and optimal hydrodynamic diameter. Narrower particle size range corresponds to more uniform particle size distribution. Hydrodynamic diameter is generally slightly larger than the geometric diameter of the particle, because it includes both the particle size itself and the solvation shell surrounding the particle. The peak particle size of the nanoparticle is less than about 500nm, for example, less than about 450nm, less than about 400nm, less than about 350nm, less than about 300nm, less than about 250nm, less than about 200nm, less than about 150nm, less than about 100nm, or less than about 50nm. The peak particle size of the nanoparticle can be in the range of about 20nm to about 500nm, for example, about 50nm to about 500nm, about 100nm to about 400nm, about 150nm to about 300nm or about 200nm to about 250nm, or include and / or any range between any two of these numerical values. In each embodiment, the peak particle size of the nanoparticle is in the range of 50nm to 500nm. In various embodiments, the peak particle size of the nanoparticles is in the range of 50 nm to 100 nm.

[0065] Used CaP and nucleic acid ratio can affect the degree of combination or encapsulation of nucleic acid and the stability of nanoparticle suspension.In the situation that nucleic acid is greatly excessive, it is believed that some nucleic acids remain uncombined, so the stability of nanoparticle is reduced.The weight ratio of calcium phosphate in nanoparticle and nucleic acid is in the scope of about 100:1 to 1:1.5, includes but not limited to about 80:1 to 1:1.2, about 50:1 to 1:1, about 20:1 to 2:1, or about 10:1 to 5:1, about 2:1 to 1:1, or include and / or any scope between any two numerical values ​​in these numerical values.In each embodiment, the weight ratio of CaP and nucleic acid is about 2:1.When described ratio, calcium phosphate / nucleic acid shows high stability, and almost all given nucleic acids can be captured in nanoparticle.

[0066] For CaP / nucleic acid / capping agent hybrid nanoparticles, the mass loading ratio (payload) expressed by the ratio of these three components is such that all nucleic acids are associated with CaP or capping agents. Therefore, in order to maximize the binding of nucleic acids, the weight ratio of calcium phosphate to nucleic acid to capping agent in the nanoparticle is in the range of about 2:1:2 to about 2:5:2, including but not limited to about 2:1.5:2 to about 2:4.5:2, about 2:2:2 to about 2:4:2, about 2:2.5:2 to about 2:3.5:2, or about 2:2.7:2 to about 2:3:2, or any range including and / or between any two of these numerical values. In various embodiments, the weight ratio of calcium phosphate to nucleic acid to capping agent in the nanoparticle is about 2:1:2 to about 2:5:2.

[0067] In order to provide the particles with a sufficiently high positive charge to confer acceptable colloidal stability to the suspension, the N / P ratio (where N is the number of positively charged nitrogen atoms in the polycation and P is the number of phosphorus atoms in the nucleic acid) may be above 1, e.g., above 5 or even above 10. This stability may be achieved when the zeta potential at the final pH is above +10 mV, e.g., above +20 mV. In addition to having a better suspension stability, the positive charge enables the particles to adhere to negatively charged cell membranes, a prerequisite for the internalization process (endocytosis). In various embodiments, the ratio of the number of positively charged nitrogen atoms in the polycation to the number of phosphorus atoms in the nucleic acid is greater than 1.

[0068] Although the aqueous suspension described herein can be used as is, it can also be advantageously combined in an emulsion or multiple emulsions. The emulsion can be a water-in-oil emulsion or a multiple emulsion. Examples of multiple emulsions include double emulsions, such as water-in-oil-in-water (W / O / W) emulsions. Other single or multiple emulsions can also be used. W / O / W emulsions represent double emulsions in which oil (O) droplets encapsulating water (W) droplets are dispersed in water (W). W / O / W emulsions are easily diluted with water to deliver a suitable amount of nucleic acid. Without wishing to be bound by theory, it is speculated that the oil droplets encapsulating the aqueous suspension can provide additional protection to the nucleic acid from the influence of nucleases. Such double emulsions are widely used in biopesticide applications. The final external phase is water, and the product can be simply diluted by adding more water as a diluent. The internal phase containing the particle suspension is protected by an intermediate oil phase. The oil droplets containing the aqueous suspension of nanoparticles adhere to hydrophobic surfaces, for example, leaf surfaces in plant or crop applications.

[0069] Although embodiments described herein are directed to nucleic acids, the nanoparticles may be used to contain other suitable therapeutic agents or medicaments, which may include any compound or substance for treating or preventing any given disease or condition or for regulating the physiological condition in a human or animal subject. In some embodiments, the therapeutic agent may include an antibiotic, an antifungal, a peptide, a protein, a polymer, or a combination thereof. In some embodiments, the nanoparticle may include a therapeutic agent except the nucleic acid contained therein.

[0070] method

[0071] Various methods can be used to produce nanoparticles, compositions, suspensions and emulsions as described herein. For example, the present technology provides simple methods to prepare the suspension of calcium phosphate / nucleic acid nanoparticles of alkali resistance, and relative to the gross mass of the particle, the nanoparticles show extremely high nucleic acid loading (e.g., 0.1 to 50 wt %). The preparation of the suspension depends on a single-step process, wherein, during the calcium phosphate precipitation, a large amount of nucleic acids are at least partially encapsulated in the nanoparticle inner side.

[0072] Aspects of the present technology relate to methods for producing nanoparticles of calcium phosphate and nucleic acid without a capping agent. In various embodiments, the method for preparing nanoparticles comprises: coprecipitating calcium phosphate and nucleic acid. Figure 1 , which illustrates an exemplary schematic flow chart of the basic process for preparing a CaP / nucleic acid suspension. As shown, calcium phosphate can be prepared using calcium salts and phosphates known in the art. In various embodiments, the method includes: adding nucleic acid to a calcium salt solution or a phosphate solution, adjusting the pH of the salt solution or mixed solution to a suitable value, and mixing the calcium salt solution with the phosphate solution to form calcium phosphate-nucleic acid nanoparticles.

[0073] The formation of calcium phosphate nanoparticles and the kinetics of CaP particle growth are driven by the concentrations of calcium and phosphate salts, temperature, and pH. Figure 1 As shown, before mixing, the pH of the calcium salt solution and / or the phosphate solution can be independently adjusted using a buffer solution, at least one of the calcium salt solution and the phosphate solution having nucleic acid. Alternatively, the salt solution, at least one of which has nucleic acid, can be mixed first, and then the pH can be adjusted to the desired value. Accordingly, the coprecipitation process is carried out at a pH higher than 6 and lower than 9, for example, at a pH between 7 and 8. If the pH is lower than 6, only a few particles can be obtained, and when the pH exceeds 9, it may be difficult to control the growth of the particles. In various embodiments, the pH of the calcium salt solution is about 7.5. In various embodiments, the pH of the phosphate solution is about 7.5.

[0074] The temperature during mixing can be about 1 ℃ to 70 ℃, including but not limited to about 1 ℃ to 70 ℃, about 5 ℃ to 60 ℃, about 10 ℃ to 50 ℃, about 15 ℃ to 40 ℃, or about 20 ℃ to 30 ℃, or any scope including and / or between any two of these numerical values. The time required to form nanoparticles at room temperature can be a few seconds to a few minutes. Suitable reaction or mixing time can be about 5s to about 10 minutes, including but not limited to about 8s to about 5 minutes, about 10s to about 1 minute, about 20s to about 2 minutes, or about 30s to about 1 minute, or any scope including and / or between any two of these numerical values.

[0075] Therefore described method provides in the presence of nucleic acid to be loaded, spontaneously forms calcium phosphate nanoparticles by calcium salt and phosphate, thereby obtains highly stable calcium phosphate / nucleic acid nanoparticle suspension.Through single-step process, the nucleic acid existing during the formation of calcium phosphate becomes encapsulated in the CaP particle.On the one hand, described method provides a kind of suspension, it comprises aqueous medium, is suspended with nanoparticle therein, and described nanoparticle is made by the nucleic acid of interest (for example RNA) embedded in calcium phosphate.Due to the negative surface charge of the particle that nucleic acid provides, this suspension made by the CaP / nucleic acid nanoparticle without any outer coating shows high stability, and can deliver or be applied to plant, animal or insect as is.

[0076] Another aspect of the present technology relates to a method for producing hybrid nanoparticles of calcium phosphate and nucleic acid in the presence of a capping agent. In various embodiments, the method for preparing the nanoparticles comprises: coprecipitating calcium phosphate and nucleic acid, and capping the CaP / nucleic acid particles with a suitable capping agent. Figure 2 , which illustrates an exemplary schematic flow chart of a method for preparing nucleic acid-loaded nanoparticles with a polycationic coating (outer layer) and an idealized view of the resulting hybrid nanoparticles. As shown in the figure, the suspension of CaP / nucleic acid particles can be referred to above Figure 1 The preparation can be carried out, and the particles obtained can be further coated with a suitable coating agent. For example, a solution of a coating agent (or its salt) can be slowly added to a CaP / DNA particle suspension. The suspension can be mixed and further diluted with a suitable solvent (e.g., water). In various embodiments, the method comprises: adding nucleic acid to a calcium salt solution or a phosphate solution, adjusting the pH of the salt solution or the mixed solution to a suitable value, mixing the calcium salt solution with the phosphate solution to form calcium phosphate-nucleic acid nanoparticles, and adding a coating agent solution to the nanoparticles to show a mixed CaP / nucleic acid / covering agent nanoparticle suspension. In various embodiments, due to the combination with the coating agent, the coating method enables the nucleic acid to be at least partially encapsulated in the calcium phosphate nanoparticles and / or coated on the surface of the nanoparticles.

[0077] Suitable calcium salts and phosphates for preparing calcium phosphate are known in the art and can be used in the methods of the present technology. Exemplary calcium salts include, but are not limited to, calcium chloride, calcium nitrate, calcium acetate, and calcium lactate. Exemplary phosphates include, but are not limited to, trisodium phosphate, tripotassium phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, ammonium phosphate, and ammonium hydrogen phosphate.

[0078] Based on fast generation kinetics, flow reactor can be used to advantageously prepare the suspension of nucleic acid / calcium phosphate nanoparticles on a large scale and at low cost to meet market demand. Exemplary reactors that can be used for this technology method include but are not limited to batch reactors, semi-batch reactors, continuous flow reactors, advanced flow reactors, fluidized bed reactors and combinations thereof. Figure 8 and 9 Examples of suitable reactors are depicted, such as flow reactors and advanced flow reactors, respectively.

[0079] In another aspect, the present invention provides a method for preparing a suspension of hybrid nanoparticles in a continuous flow reactor. The method includes: adding nucleic acid to one of a calcium salt solution or a phosphate solution; allowing the calcium salt solution to flow into a first inlet of the flow reactor; allowing the phosphate solution to flow into a second inlet of the flow reactor so that the phosphate solution stream mixes with the calcium salt solution stream and produces a calcium phosphate-nucleic acid hybrid nanoparticle stream flowing from the reactor; and collecting and processing a suspension containing calcium phosphate-nucleic acid nanoparticles from the reactor. In some aspects, the method may also include: allowing the capping polymer solution to flow into a third inlet of the flow reactor so that the capping polymer stream mixes with the calcium phosphate stream to produce a polymer-capped calcium phosphate stream encapsulated with nucleic acids flowing from the reactor.

[0080] The zeta potential change of the nanoparticle surface can be utilized to study the combination of nucleic acid on the nanoparticle surface or the combination of a capping agent on the nanoparticle surface. The change of the zeta potential indicates that the ligand is bound to the surface or surface coating. The zeta potential is a measure of the surface charge on the nanoparticle, and can affect the stability and aggregation of the particles in the suspension. In various embodiments, the nanoparticle is constructed to have a certain material, and when being in a pH level that effectively binds nucleic acid to the inner side of the CaP matrix, the material has a low zeta potential. For particles with a capping agent, the nanoparticle shows a high zeta potential due to the coating on the nanoparticle surface. For example, CaP / nucleic acid particles can show a negative charge, and CaP / nucleic acid / capping agent can show a positive charge. In various embodiments, the zeta potential of the nanoparticles is from about -50 to about 50 millivolts (mV), including -50 mV to about 0 mV, -40 mV to about 0 mV, about -30 mV to about 0 mV, about -20 mV to about 0 mV, about -10 mV to about 0 mV, about 0 mV to about 10 mV, 0 mV to about 20 mV, about 0 mV to about 30 mV, about 0 mV to about 40 mV, about 0 mV to about 50 mV, about -10 mV to about 10 mV, -15 mV to about 15 mV, about -20 mV to about 20 mV, about -25 mV to about 25 mV, about -30 mV to about 30 mV, or any range including and / or between any two of these values.

[0081] The nanoparticles formed by the method of the present technology are suspended in a liquid medium, such as water, alcohol, hydrocarbon and oil. The average or median particle size of the nanoparticles can be in the range of about 30nm to about 250nm, including about 40nm to about 200nm, about 50nm to about 150nm, about 60nm to about 100nm, about 70nm to about 80nm, or any range including and / or between any two of these numerical values.

[0082] Nanoparticle compositions and suspensions described herein can be delivered to various types of cells. Any type of cell that allows nucleic acid delivery can be used as a conversion target for the present technology. Examples include, but are not limited to, insect cells, animal cells and embryos, fungi, fish, yeast, and plant cells. Stable aqueous suspensions can be used as part of plant quarantine compositions for the prevention and control of harmful organisms. For example, stable suspensions can be used to protect crops from insects, in which case the nucleic acid is an insecticidal double-stranded RNA. Using interfering ribonucleic acid (RNA) molecules, the compositions and suspensions can be used to prevent and / or control plant pests. Compositions and combinations comprising interfering RNA molecules of the present technology are topically applied in the form of, for example, insecticides.

[0083] Aspects of the present technology relate to methods for protecting crops from insects, comprising administering to the crops a composition or suspension comprising nanoparticles as described herein. In various embodiments, the method comprises protecting the crops from hemiptera, coleoptera, siphonaptera, dichyoptera, lepidoptera, orthoptera, and diptera. The composition can be administered to the crops using various methods known in the art, for example, by spraying, dripping, or irrigating. In these methods, the nucleic acid in the composition may comprise an insecticidal double-stranded ribonucleic acid.

[0084] Another aspect of the present technology relates to a method for down-regulating the expression of a target gene in a pest species to prevent and / or control pest infestation, the method comprising contacting the pest species with an effective amount of a composition or suspension of hybrid nanoparticles as described herein, the nanoparticles comprising at least one interfering ribonucleic acid (RNA). Suitable interfering RNAs are known in the art, for example, as described in PCT Publication No. WO2012143542A1, which is incorporated herein by reference in its entirety. In certain embodiments, the present technology relates to a method for controlling pests using a nanoparticle composition containing RNAi, which involves simply topically applying naked RNA without any protection on a plant surface, for example, on a leaf surface.

[0085] Another aspect of the present technology relates to a glass container containing the composition and aqueous suspension described herein. Any glass container suitable for holding liquid materials and inert or non-reactive to the composition and suspension can be used. Exemplary glass containers include glass bottles, glass vials, glass barrels, glass capsules, glass cylinders, glass jars, glass balls, etc.

[0086] The composition and suspension of the present technology advantageously provide calcium phosphate / nucleic acid nanoparticles, wherein the inorganic material surrounding the nucleic acid is calcium phosphate, the particles are stable at neutral and alkaline pH, and rapidly dissolve at acidic pH. Therefore, the particles are stable in the alkaline medium present in the environment and in the insect intestine, thereby protecting the nucleic acid to a certain extent, but rapidly dissolve at endosomal pH, thereby releasing the nucleic acid in the cells of the target insect. Submicron particles carrying nucleic acids (e.g., insecticidal RNA) are easily absorbed by the cells of the digestive tract of the target pest. In the absence of any polycations, the amount of nucleic acid loaded in the mixed calcium phosphate / nucleic acid nanoparticles (also referred to as effective load) can be higher than 30% by weight, which is significantly higher than the amount provided by methods known in the art. Therefore, the amount of inorganic carriers to be sprayed on the field can be minimized, because a lower amount of carriers will be able to deliver an effective amount of insecticide. Compared to the simple surface adsorption described in the prior art, the coprecipitation method for the present technology can achieve significantly higher nucleic acid loads. In fact, the amount of nucleic acid adsorbed on the carrier surface is still limited, for example, less than 1% by weight, even when using submicron-sized particles. In the case of the coprecipitation method used by the present technology, nucleic acid is captured in large quantities in the inner side of the composite structure, rather than only on the surface of the carrier. The inventor found that more than 90% of the nucleic acid was captured during particle formation, which provides a high encapsulation yield and avoids wasting expensive nucleic acid. When polycations (such as PEI) are applied to the surface of nucleic acid / calcium phosphate particles, the yield almost reaches 100%, because during the coprecipitation process, polycations can capture unbound nucleic acid. The calcium phosphate material forming the carrier is completely biocompatible, biodegradable, nontoxic and non-immunogenic, and is prepared by safe and cheap reagents (such as calcium salts and phosphates), so production is simplified and production costs are reduced. When used for crop protection, in addition to its harmful biological control effect, nucleic acid / calcium phosphate particles can be considered as potential fertilizers because they are phosphorus sources. The submicron size of the particle promotes rapid dissolution in an acidic environment, and is therefore conducive to the availability of phosphorus. The method of the present technology can prepare a stable suspension (i.e., free from aggregation) for use in plant quarantine compositions. The stable suspension may contain at least 20 μg / ml of double-stranded nucleic acids (e.g., RNA), which is typically required for crop protection using RNA interference. The loaded particles and their suspensions can be stable for several weeks and can be sprayed as is or included in an emulsion for spraying onto plants.

[0087] It will be apparent to those skilled in the art that various modifications and variations may be made without departing from the spirit or scope of the subject matter claimed. Therefore, the subject matter claimed is not limited by any content other than the attached claims and their equivalents.

[0088] Example

[0089] Various embodiments will be further illustrated by the following examples, which are in no way intended to limit the present disclosure thereto.

[0090] Example 1: Preparation of Stable Nucleic Acid / CaP Nanoparticle Suspension

[0091] 718.6 μl of pure H2O, 500 μl of HEPES (50 mM, pH 7.5) solution containing 7.5 mM Na3PO4, and 31.4 μl of 10 mg / ml sheared salmon sperm ds-DNA were added to a glass vial. Then, a second mixture of 10 mM Tris HCl and 100 μl of 62.5 mM CaCl2 containing 1150 μl of pH 7.5 in ultrapure (UP) water was added dropwise under vigorous stirring. The total volume was 2500 μl. The particle suspension was immediately diluted by adding 2,500 μl of the prepared particle suspension to 10,000 μl of ultrapure water. The final volume was 12,500 μl. Based on the feed, the solution theoretically contained 628 μg of calcium phosphate and 314 μg of DNA. Therefore, the theoretical nucleic acid load was about 33% by weight. The final nucleic acid concentration in the suspension was about 24 μg / ml and the theoretical concentration of calcium phosphate was 48 μg / ml. The resulting stable suspension was made of particles with an average hydrodynamic diameter of 49 nm and an average zeta potential of -11 mV.

[0092] Particle size characterization of aqueous suspensions was provided using a Malvern Zetasizer Nano analyzer which determines the hydrodynamic diameter of the particles by dynamic light scattering (DLS) technology. Figure 3 Shown is a DLS chart showing the size of CaP / DNA particles prepared by coprecipitation without any polycation, the size of the polyplex obtained by complexation of DNA and PEI polycation without any CaP, and finally the size of particles prepared by coprecipitation but with PEI coating. The chart clearly shows that the CaP / DNA / PEI particles have the largest hydrodynamic diameter and are significantly larger than simple DNA / PEI polyplexes. The chart also shows that a narrow particle size distribution can be achieved. Figure 4 Is a bar graph showing the zeta potential of calcium phosphate (CaP) particles without any nucleic acid loading, calcium phosphate (CaP / DNA) particles loaded with DNA, and calcium phosphate / nucleic acid (CaP / DNA / PEI) particles capped with PEI polycations. These particles exhibit neutral, negative, and positive charges, respectively.

[0093] The suspension exhibited limited stability over time. Figure 5The illustrated graphs show the size of nucleic acid carrier particles CaP / DNA [2:1] made of nucleic acid and CaP at acidic pH, neutral pH and alkaline pH, and the following Table 2 summarizes the change in particle size over time under various pH conditions. The data show that the nucleic acid carrier dissolves rapidly at acidic pH, but does not dissolve at neutral and alkaline pH.

[0094] Table 2:

[0095]

[0096] As a comparative example, the same protocol was repeated, but the suspension was not diluted after particle formation. The resulting suspension contained 125 μg / ml nucleic acid and 215 μg / ml calcium phosphate. It was observed that the obtained stable suspension was made of particles with an average hydrodynamic diameter of 220 nm and an average zeta potential of -26 mV. Despite the high zeta potential, the suspension showed a rapid increase in particle size, exceeding 10 μm, as shown in Figure 2. Figure 7 shown.

[0097] Example 2: Scale-up preparation of stable nucleic acid / CaP nanoparticle suspension

[0098] Example 1 was repeated, but all quantities were multiplied by 5 to show that the formation of nanoparticles can be scaled up without significant impact on the particle size or surface charge obtained. A stable suspension was obtained and the particles had the same size and the same surface charge.

[0099] Example 3: Preparation of Stable PEI-Capped Nucleic Acid / CaP Nanoparticle Suspension

[0100] In a glass vial, 718.6 μl of pure H2O, 500 μl of a solution HEPES (50 mM, pH 7.5) containing 7.5 mM trisodium phosphate Na3PO4, and 31.4 μl of 10 mg / ml sheared salmon sperm ds-DNA as a ds-RNA substitute were added. Then, under vigorous stirring, a second mixture containing 1150 μl of 10 mM Tris HCl at pH 7.5 and 100 μl of 62.5 mM CaCl2 in ultrapure (UP) water was added dropwise to the previous solution. Next, 628 μl of 1 μg / μl linear PEI hydrochloride (prepared from PEI with Mw 25,000, catalog number 23966-1 and converted to its chloride salt according to the supplier's protocol) purchased from Polysciences, Inc. (Warrington, PA, USA) in water at pH 7 was added dropwise to the CaP / DNA particle suspension to obtain a total volume of 3,128 μl.

[0101] The particle suspension was immediately diluted by adding 3,128 μl of the prepared particle suspension to 12,512 μl of ultrapure water under stirring. The final volume was 15,640 μl. The solution contained 628 μg of calcium phosphate and 314 μg of DNA and 628 μg of PEI. The CaP / DNA / PEI ratio of the particle composition was observed to be 2:1:2. Therefore, the theoretical nucleic acid load in the particle was 20% by weight. The nucleic acid concentration in the final suspension was about 20 μg / ml, the concentration of PEI was about 40 μg / ml, and the theoretical concentration of calcium phosphate was about 40 g / ml. It was observed that the obtained stable suspension was made of particles having a hydrodynamic diameter of about 155 nm and a zeta potential of +22 mV. The suspension exhibited extremely high stability over time and did not show any signs of particle growth after at least 20 days.

[0102] As a comparative example, the same protocol was repeated, but the suspension was not diluted immediately after particle formation. The resulting suspension contained 100 μg / ml nucleic acid, 200 μg / ml calcium phosphate and 200 μg / ml PEI.

[0103] Figure 6 The illustrated graphs show the size of nucleic acid carrier particles CaP / DNA / PEI [2:1:2] made of nucleic acid, CaP and PEI outer layer at acidic pH, neutral pH and alkaline pH, and Table 3 below summarizes the change in particle size over time under various pH conditions.

[0104] Table 3:

[0105]

[0106]

[0107] The suspension showed a rapid increase in particle size exceeding 10 μm, as Figure 7 The obtained stable suspension was observed to be made of particles with a hydrodynamic diameter of 164 nm and a zeta potential of +22 mV. Despite the high zeta potential, the suspension showed a rapid increase in particle size exceeding 10 μm after storage at RT for 5 days, as shown in FIG. Figure 7 Table 4 below summarizes the particle size, zeta potential and suspension stability of the undiluted and diluted compositions prepared according to Examples 1 and 3.

[0108] Table 4:

[0109]

[0110] Example 4: Preparation of stable PEI-capped nucleic acid / calcium phosphate nanoparticle suspensions in continuous mode

[0111] Use as Figure 8 A stable PEI-coated nucleic acid / calcium phosphate nanoparticle suspension was prepared in a continuous mode using the reactor apparatus shown in the scheme of . Briefly, a small tubular flow reactor was prepared using a dual syringe infusion pump and the pump was connected to a Y-connector made of PEEK via two PFA 1 / 16" OD tubes. The outlet of the Y-connector was connected to a PFA 1 / 16" OD tube to collect the suspension. The third syringe was placed downstream and connected via a second Y-connector. The first syringe (total volume capacity = 1.5 ml) was filled with a solution containing 718.6 μl of pure H2O, 500 μl of a solution of HEPES (50 mM, pH 7.5) containing 7.5 mM Na3PO4, and 31.4 μl of 10 mg / ml sheared salmon sperm ds-DNA. The second syringe was filled with 1150 μl of 10 mM Tris HCl at pH 7.5 and 100 μl of 62.5 mM CaCl2 in ultrapure water. The third syringe was filled with 628 μl of 1 μg / μl of linear PEI hydrochloride with a Mw of 25,000. The reagents were delivered at an appropriate rate to give nanoparticles with a size of 150-200 nm.

[0112] Alternatively, other types of microchannel reactors, such as Corning Inc.'s Advanced Flow Reactor (AFR), can also be used to prepare stable PEI-capped nucleic acid / calcium phosphate nanoparticle suspensions in a continuous mode, such as Fig. 9 shown.

[0113] Example 5: Nucleic Acid Capture Measurements

[0114] The nucleic acid capture efficiency during the coprecipitation process was estimated by measuring the optical density of the supernatant at 260nm after centrifuging the suspension at 13000rpm / 4℃ for 60 minutes. 100μl of 880μg / ml CaCl2 was added to each test sample to destabilize the suspension (proving that the addition of CaCl2 does not induce any precipitation of naked nucleic acids). The blank is DNA only without any CaP. A calibration curve was prepared using various DNA concentrations from 0 to 125μg / ml. Assuming that the precursor generates Ca5(PO4)3(OH), the weight % of nucleic acid loaded in the hybrid nanoparticles is indirectly estimated by using the theoretical amount of CaP formed calculated based on the salt precursor concentration and the amount of captured nucleic acid determined based on the OD value, i.e., the effective load. In the case of PEI-coated particles, the total mass of the carrier is calculated by adding the amount of PEI to the amount of CaP and nucleic acid. Table 5 below summarizes the concentration of nucleic acids (DNA) in the samples before and after coprecipitation. The results show that most nucleic acids were captured during calcium phosphate precipitation.

[0115] Table 5:

[0116]

[0117] The mass loading ratio (payable loading) of CaP / dsDNA / PEI was determined using different mass ratios, e.g., 0:1:0, 2:1:0, 2:1:2.

[0118] Example 6: Measurement of cellular uptake of hybrid CaP / DNA / PEI nanoparticles

[0119] To show that the nucleic acid loaded in the novel hybrid CaP / DNA / PEI nanoparticles can be internalized by cells and that the nucleic acid is still functional, CaP / DNA / PEI [2: 1: 2] particles were prepared according to Example 3, but 10% by weight of salmon sperm DNA, i.e., 31.4 μg, was replaced with the same amount of GFP plasmid encoding green fluorescent protein (GFP). 500K cells were inoculated into each well of a 6W cell binding plate in 4 ml of culture medium (RPMI+10% FBS+PS+Glu) and allowed to attach and grow for one day. The culture medium was then replaced, and a suspension volume equivalent to 5.1 μg total DNA and 0.51 μg plasmid was added to the well. One day after the particle suspension was added to the cell monolayer, the cells were harvested and the expression of green fluorescent protein (GFP) was determined by flow cytometry. Figures 10a and 10b show the fluorescence of cells transfected with GFP plasmid using nanoparticle carriers and the fluorescence of negative controls with HEK 293T cells without any transfection agent added, respectively. The results showed that more than 65% of the cells expressed GFP, which proved that the nucleic acid-loaded hybrid particles of the present technology were internalized and transfection occurred, while the negative control showed no fluorescence.

[0120] Example 7: Preparation of Stable PEI-Capped Nucleic Acid / Calcium Phosphate Nanoparticle Suspension

[0121] The process described in Example 3 was repeated, but the amount of DNA was adapted to achieve a Cap / DNA / PEI mass ratio of [2:2:2]. It was observed that the obtained stable suspension was made of particles with a hydrodynamic diameter of about 197 nm. The suspension exhibited excellent stability over time and did not show any signs of flocculation.

[0122] Example 8: Preparation of stable PEI-capped nucleic acid / calcium phosphate nanoparticle suspensions with different DNA concentrations

[0123] The process described in Example 3 was repeated, but the Cap / DNA / PEI mass ratio was varied to 2:1:2, 2:2:2, 2:3:2, 2:4:2, 2:5:2, 2:7.5:2, 2:10:2, and 2:20:2.

[0124] The resulting mixture was subjected to gel electrophoresis using 0.8% agarose gel. Fig.11 As shown, as the amount of nucleic acid increases relative to CaP and PEI, the intensity of the bottom band for free DNA becomes stronger, indicating that some DNA is not bound. Nucleic acid is observed to be fully bound to CaP / PEI at 2:1:2 to 2:5:2. This ratio corresponds to a DNA load of 20 wt % to 55 wt %. At these ratios, all nucleic acids associate with CaP / PEI, so free DNA is not left to migrate downward along the gel.

[0125] Example 9: Preparation of Stable dsRNA / Calcium Phosphate [2:1] Nanoparticle Suspension

[0126] The process of Example 1 was repeated, but dsRNA was used instead of dsDNA. The buffer was prepared by nuclease-free water and autoclaved before use. Good laboratory practice was followed to prevent nuclease contamination. In order to demonstrate that dsRNA of various sizes can be incorporated into particles, annealed double-stranded RNA fragments with 21 to 500 base pairs were used. The theoretical dsRNA load of the prepared hybrid particles was about 33% by weight. The nucleic acid concentration in the final suspension was about 24 μg / ml, and the theoretical concentration of calcium phosphate was 48 μg / ml. It was observed that the obtained stable suspension was made of particles with a hydrodynamic diameter of 120 nm and a zeta potential of -18 mV.

[0127] Example 10 Preparation of a stable dsDNA / calcium phosphate nanoparticle suspension with a weight ratio of [2:4]

[0128] Example 1 was repeated, but the weight ratio of CaP to DNA was 2:4. Fig.12 The SEM micrographs shown depict the morphology of nanoparticles (in dry state) prepared from dsDNA / calcium phosphate / PEI [2:4:0].

[0129] Example 11 Preparation of a stable dsDNA / calcium phosphate / polycation [2:1:2] nanoparticle suspension using poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) instead of PEI.

[0130] The process of Example 3 was repeated, but using PDMAEMA [Mn 33.000, purchased from PolymerSource Inc.] instead of linear PEI. Prior to the coprecipitation process, the pH of the 1 μg / μl PDMAEMA solution was adjusted to 7.2 using hydrochloric acid. The particles obtained exhibited a hydrodynamic diameter of 195 nm when measured by DLS, and a zeta potential of +21 mV.

[0131] Example 12 Preparation of Stable dsDNA / Calcium Phosphate / Polycationic [2:1:2] Nanoparticle Suspension Using Branched PEI

[0132] The procedure of Example 3 was repeated, but using branched PEI [average Mw -25,000 by LS, average Mn -10,000 by GPC, purchased from Sigma Aldrich] instead of linear PEI. The particles exhibited a hydrodynamic diameter of 156 nm and a zeta potential of +28 mV. Fig.13A and 13B SEM micrographs at two magnifications are shown showing the morphology and size (dry state) of nanoparticles prepared from dsDNA / calcium phosphate / PEI [2:1:2]. The average size of the particles is 52 nm, and they have a uniform size distribution.

[0133] Example 13 Preparation of Stable dsRNA / Calcium Phosphate / PEI [2:1:2] Nanoparticle Suspension

[0134] The process of Example 3 was repeated, but similar to that described in Example 9, dsRNA was used instead of dsDNA. For the diluted solution, the theoretical nucleic acid load in the particles at the [2:1:2] ratio was therefore 20% by weight. The dsRNA concentration in the final suspension was about 20 μg / ml, the concentration of PEI was about 40 μg / ml, and the theoretical concentration of calcium phosphate was about 40 g / ml. It was observed that the stable suspension obtained was made of particles with a hydrodynamic diameter of about 188 nm and a zeta potential of +24 mV.

[0135] The suspension was also prepared as described in Example 10, but without dilution (Note: "conc" stands for concentrated). To verify that all dsRNA was bound, the obtained suspension was subjected to gel electrophoresis using 0.8% agarose gel. Fig.14 As shown, for the 2:1:2 ratio, the dsRNA is shown to be completely bound to the CaP / PEI. At these ratios, all nucleic acids are associated with the CaP / PEI, so no free DNA is left to migrate down the gel. This ratio corresponds to a 20% by weight dsRNA loading.

[0136] Unless otherwise expressly stated, any method described herein should not be construed as requiring that its steps be performed in a specific order, or requiring that any device have a particular orientation. Thus, if a method claim does not actually recite the order in which its steps are to be followed, or if any device claim does not actually recite the order or orientation of the components, or if the claims or specification do not otherwise specifically state that the steps are limited to a specific order, or if a specific order or orientation of the device components is not recited, then no order or orientation should be inferred in any respect. This applies to any possible non-expressive basis for interpretation, including: logical issues involving the arrangement of steps, operational flow, order of components, or orientation of components; issues of obvious meaning derived from grammatical organization or punctuation; and issues of the number or type of embodiments described in the specification.

[0137] The disclosed ranges should be understood to include any and all subranges or any and all individual values ​​contained in each range, and provide support for the claims describing these subranges or individual values. For example, the stated range of 1 to 10 should be understood to be any and all subranges between a minimum value of 1 and a maximum value of 10 or individual values ​​between them (including and / or excluding endpoints), and provide support for the claims describing these subranges or individual values; that is, all subranges starting with a minimum value of 1 or greater and ending with a maximum value of 10 or less (e.g., 5.5 to 10, 2.34 to 3.56, etc.) or any value from 1 to 10 (e.g., 3, 5.8, 9.9994, etc.). Any listed range can undoubtedly be considered to be fully described, and the same range can be decomposed into at least equal halves, one-third, one-quarter, one-fifth, one-tenth, etc. As a non-limiting example, each range discussed herein can be easily decomposed into a lower third, a middle third, and an upper third, etc. It will also be understood by those skilled in the art that all language such as "up to," "at least," "greater than," "less than," and similar language include the stated numbers and refer to ranges that can subsequently be broken down into sub-ranges as described above. Finally, as will be understood by those skilled in the art, ranges include each individual member. Thus, for example, a group having 1-3 layers refers to groups having 1, 2, or 3 layers. Similarly, a group having 1-5 layers refers to groups having 1, 2, 3, 4, or 5 layers, and so on.

[0138] The drawings should be interpreted as illustrating one or more embodiments that are drawn to scale and / or one or more embodiments that are not drawn to scale. This means that the drawings may be interpreted as showing, for example: (a) everything is drawn to scale, (b) everything is not drawn to scale, or (c) one or more features are drawn to scale and one or more features are not drawn to scale. Therefore, the drawings may provide support for describing the size, proportion and / or other dimensions of any illustrated feature, either alone or in combination with each other. In addition, all such sizes, proportions and / or other dimensions should be understood to vary from 0-100% in either direction and therefore provide support for claims describing these values ​​or any and all ranges or sub-ranges that may be formed from these values.

[0139] Terms described in the claims should be determined by reference to relevant entries in widely used general dictionaries and / or dictionaries of relevant technology, meanings commonly understood by persons skilled in the art, etc., and should be understood to have the broadest meaning given by any one or combination of these sources (e.g., entries of two or more relevant dictionaries should be combined to provide the broadest meaning of the combination of the entries, etc.) subject only to the following exceptions: (a) if a term is used in a manner broader than its ordinary and customary meaning, the term should have its ordinary and customary meaning plus an additional extended meaning, or (b) if a term has been expressly defined to have a different meaning by following the term with the phrase "a term used in this document shall mean" or similar language (e.g., "the term means," "the term is defined," "for the purposes of this disclosure, the term refers to," etc.) to describe the term. The use of "i.e.," the word "technology," etc. when referring to specific examples is not intended to invoke exception (b) or otherwise limit the scope of the claimed terms. Except where exception (b) applies, nothing in this document shall be deemed to waive or deny the scope of the claims.

[0140] Unless otherwise defined, the meaning of all terms (including scientific and technological terms) used herein is the same as that commonly understood by those of ordinary skill in the art to which the technology belongs. It should also be understood that terms, such as those defined in common dictionaries, should be interpreted as having a meaning consistent with their meaning in the context of the present application and related art, and should not be interpreted in an idealized or overly formal sense unless clearly defined herein. Although not clearly defined below, such terms should be understood according to their common meaning.

[0141] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0142] It should be specifically noted that, unless otherwise stated, the various features of the present technology described herein can be used in any combination. In addition, the present disclosure also contemplates that in some embodiments, any feature or combination of features shown herein can be excluded or omitted. For example, if the specification states that the composite comprises components A, B, and C, it is specifically intended to indicate that any one or combination of A, B, or C can be omitted and excluded individually or in any combination.

[0143] Unless expressly stated otherwise, all specific embodiments, features, and terms are intended to include those embodiments, features, or terms and their biological equivalents.

[0144] All patents, patent applications, provisional applications, and publications cited or referenced herein, including all figures and tables, are hereby incorporated by reference in their entirety to the extent they are not inconsistent with the explicit teachings of this specification.

Claims

1. A composition comprising nanoparticles, wherein: The nanoparticles include a nucleic acid at least partially encapsulated in a matrix comprising calcium phosphate, The composition further comprises a capping agent, wherein the weight ratio of calcium phosphate to nucleic acid to the capping agent in the nanoparticles is 2:1:2 to 2:5:2, and Wherein, the capping agent comprises a polycation.

2. The composition according to claim 1, wherein The nucleic acid comprises RNA or DNA.

3. The composition according to claim 1, wherein The capping agent at least partially coats the nanoparticles or permeates the nanoparticles, or both coats the nanoparticles and permeates the nanoparticles.

4. The composition according to claim 1, wherein The polycation is selected from the group consisting of polyamines, polylysine, polyarginine, polyornithine, polyalkylene imine, chitosan, oligopeptides, cationized polysaccharides, cationized plant proteins or cationized animal proteins, protamine, diethylaminoethyl-dextran, polyamidoamines, poly(β-amino esters), aminopolysaccharides, polymethylaziridine, polyallylamine, polyvinylamine homopolymers or copolymers, poly(vinylpyridine) homopolymers or copolymers, poly(meth)acrylate homopolymers or copolymers, poly(meth)acrylamide homopolymers or copolymers, and derivatives thereof.

5. The composition according to claim 4, wherein The polycation is polyethyleneimine.

6. The composition according to claim 1, wherein The peak particle size of nanoparticles is less than 500 nm.

7. The composition according to claim 6, wherein The peak particle size of the nanoparticles is in the range of 50 nm to 500 nm.

8. The composition of claim 1, wherein The ratio of the number of positively charged nitrogen atoms of the polycation to the number of phosphorus atoms of the nucleic acid is greater than one.

9. The composition of claim 1, wherein The nanoparticles are suspended in an aqueous dispersion with a pH range of 6 to 11.

10. A stable aqueous suspension comprising the composition of claim 1.

11. The stable aqueous suspension of claim 10, wherein The nucleic acid loading is from about 0.1 wt % to about 50 wt % relative to the total mass of the nanoparticle.

12. The stable aqueous suspension of claim 10, wherein: (i) the concentration of calcium phosphate in the suspension is less than 100 μg / ml; or (ii) The molar ratio of calcium to phosphorus in the calcium phosphate suspension is 1.5:1 to 5:

1.

13. The stable aqueous suspension of claim 11, further comprising an additive selected from the group consisting of nuclease inhibitors, surfactants, wetting agents, UV absorbers, antioxidants, antifreeze agents, preservatives, colorants, insecticides, fungicides, attractants, repellents, and rheology modifiers.

14. A method for protecting crops from pests, the method comprising administering the composition of claim 1 to the crops.

15. The method of claim 14, wherein: The nucleic acid in the composition comprises insecticidal double-stranded ribonucleic acid.

16. The method of claim 14, wherein: The method comprises protecting crops from Hemiptera, Coleoptera, Siphonaptera, Dictyoptera, Lepidoptera, Orthoptera and Diptera.

17. A method for producing hybrid nanoparticles, the method comprising: adding the nucleic acid to one of the calcium salt solution or the phosphate salt solution in the reactor; adjusting the pH of the calcium salt solution and the phosphate solution to about 6 to about 9; as well as mixing the calcium salt solution with the phosphate solution to form calcium phosphate-nucleic acid hybrid nanoparticles, The method further comprises: adding a polycation solution to the mixed solution, wherein the weight ratio of calcium phosphate in the nanoparticles to the nucleic acid to the polycation solution is 2:1:2 to 2:5:

2.

18. The method of claim 17, wherein: (i) a calcium salt selected from the group consisting of calcium chloride, calcium nitrate, calcium acetate and calcium lactate; or (ii) a phosphate selected from the group consisting of trisodium phosphate, tripotassium phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, ammonium phosphate and ammonium hydrogen phosphate.

19. The method of claim 17, wherein: The nucleic acid is at least partially encapsulated in calcium phosphate nanoparticles.

20. The method of claim 17, wherein: The peak particle size of the hybrid nanoparticles is in the range of 50 nm to 500 nm.

21. The method of claim 17, wherein: The pH of the calcium salt solution or phosphate solution is about 7.

5.

22. A method for preparing a suspension of hybrid nanoparticles in a continuous flow reactor, the method comprising: adding the nucleic acid to one of a calcium salt solution or a phosphate salt solution; allowing a calcium salt solution to flow into a first inlet of a flow reactor; flowing a phosphate solution into a second inlet of the flow reactor to mix the phosphate solution stream with the calcium salt solution stream and produce a calcium phosphate-nucleic acid hybrid nanoparticle stream flowing from the reactor; collecting and processing the suspension containing calcium phosphate-nucleic acid hybrid nanoparticles from the reactor, The method further comprises: flowing the capping polymer solution into a third inlet of the flow reactor so that the capping polymer stream mixes with the calcium phosphate stream to produce a polymer-capped, nucleic acid-encapsulated calcium phosphate stream flowing from the reactor, wherein the weight ratio of calcium phosphate in the nanoparticles to nucleic acid to the capping polymer solution is 2:1:2 to 2:5:2, and Wherein, the capping polymer comprises a polycation.

23. The method of claim 22, further comprising: The polymer-capped, nucleic acid-encapsulated calcium phosphate is collected from the reactor and processed.

24. The method of claim 22, wherein: The zeta potential of the nanoparticles ranged from -50 to 50 mV.

25. The method of claim 22, wherein: The median particle size of the nanoparticles ranges from 30 nm to 250 nm.

26. The method of claim 22, wherein: The nanoparticles are suspended in a liquid medium.

27. A water-in-oil (W / O) emulsion comprising the composition of claim 1.

28. A water-in-oil-in-water (W / O / W) double emulsion comprising the composition according to claim 1.

29. A glass container comprising the composition of claim 1, or a stable aqueous suspension containing the composition of claim 1.

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