WO2023028084A2 - Methods and compositions of nucleic acid encapsulated within external membranes - Google Patents

Abstract

Embodiments of the present disclosure relate to methods of manufacturing and generating nanoparticles comprising nucleic acids and an external membrane. Also provided are compositions comprising the nucleic acid nanoparticles, and methods of use the compositions for therapeutic and medical contexts.

Description

METHODS AND COMPOSITIONS OF NUCLEIC ACID ENCAPSULATED

WITHIN EXTERNAL MEMBRANES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Application No. 63/260,645, filed August 27, 2021 , which is hereby incorporated by reference in its entirety.

FIELD

[0002] The present disclosure relates to nanoparticles generated by encapsulating nucleic acid molecules, such as DNA or RNA, within external membranes, and therapeutic uses thereof.

BACKGROUND

[0003] Nucleic acid uptake within a tissue microenvironment may significantly affect the presentation, prognosis, and overall development of diseased or otherwise aberrant tissue. However, injection or delivery of nucleic acids into resident tissue is stymied by immune response and natural degradation while the nucleic acids are en route, necessitating larger doses of therapeutic nucleic acids, or a means of targeting and delivering nucleic acids within a tissue microenvironment As a result of immune clearance, some therapeutics rely on in-tumor injection, which relies on medical imaging and tissue accessibility of the site in order to successfully administer. Even with in-tumor injection, the threat of immune rejection may still be present.

SUMMARY

[0004] The present disclosure relates to compositions that include nanoparticles that encapsulate nucleic acids. Also described are methods of making and using the compositions. The compositions and methods of making and using the compositions described herein can be used for targeted delivery of therapeutic nucleic acid molecules, to deliver specific molecules or gene therapies. The compositions described herein allow for targeted delivery by avoiding or delaying immune clearance by encapsulation of biological payloads with human cell membranes. [0005] Accordingly, provided herein are compositions of nanoparticles, as well as methods of generating, making, or producing nanoparticles that include an inner nucleic acid molecule, such as DNA or RNA, and external membrane. In some embodiments, compositions of nanoparticles, and methods of generating, making, or producing the nanoparticles that include an inner molecule, such as protein or virus species, and an external membrane are provided. Some embodiments provided herein allow one to overcome certain limitations in the prior art by providing efficient methods of generating nanoparticles including an inner nucleic acid molecule and an external membrane. In some embodiments, the external membrane is derived from erythrocytes. In some embodiments, the external membrane is derived from healthy tissue. In some embodiments, the external membrane is a lipid bilayer nanostructure. In some embodiments, the external membrane is an extracellular vesicle. In some embodiments, the external membrane is a cellular membrane. In some embodiments, the external membrane is derived from diseased tissue. In some embodiments, the nucleic acid molecules are circular plasmids. In some embodiments, methods of the present disclosure may provide for a method of manufacturing or generating said nanoparticles, for use in a variety of therapeutic contexts. [0006] Some embodiments provided herein relate to methods of manufacture of nanoparticles. In some embodiments, the methods include combining a nucleic acid molecule and an external membrane by extrusion, sonication, agitation, mixing, vortexing, or any combination thereof. In some embodiments, the external membrane is derived from erythrocytes. In some embodiments, the external membrane is a cellular membrane. In some embodiments, the external membrane is an extracellular vesicle. In some embodiments, the extracellular vesicle is a membranous lipid structure. In some embodiments, the nucleic acid molecule and the external membrane are processed simultaneously. In some embodiments, processing includes one or more of extrusion, sonication, agitation, mixing, vortexing, or any combination thereof. In some embodiments, the external membrane is sonicated in the presence of nucleic acids in order to encapsule the nucleic acids within the external membrane. In some embodiments, the extrusion process disassociates the external membrane. In some embodiments, the nucleic acid molecule is encapsulated by the external membrane following extrusion, sonication, or any combination of methods involving physical disruption. In some embodiments, the external membrane is generated by physical disruption of a parent cell. Physical disruption can include any one or more of extrusion, sonication, agitation, mixing, vortexing or other methods to apply physical forces to cells or cell membranes. In some embodiments, the physical disruption of the parent cell is achieved by hypotonic lysis, or physical separation. In some embodiments, the extrusion process includes passing the nucleic acid molecule with the external membrane through a porous membrane. In some embodiments, the porous membrane has pores ranging from 200 nm to 1,000 nm in diameter. In some embodiments, the external membrane is processed by tangential flow filtration. In some embodiments, the parent cell is isolated from whole blood. In some embodiments, the parent cell is comprised of red blood cells. In some embodiments, the parent cells are derived from T cells. In some embodiments, the parent cells are derived from fetal cells. In some embodiments, the parent cells are derived from diseased tissue cells. In some embodiments, the parent cells are derived from healthy tissue. In some embodiments, the parent cells are autologous. In some embodiments, the parent cells are allogenic. In some embodiments, the parent cells are xenogenic. In some embodiments, the nucleic acid molecule is comprised of circular or linear fragments. In some embodiments, the nucleic acid molecule is a plasmid. In some embodiments, the external membrane is stored in an aqueous solution. [0007] Some embodiments described herein relate to compositions that include a nanoparticle. In some embodiments, the nanoparticle includes an inner core and an outer sheath, or external membrane. In some embodiments, the outer sheath or external membrane includes a cellular membrane. In some embodiments, the outer sheath or external membrane includes an extracellular vesicle (“EV”). In some embodiments, the cellular membrane is derived from an erythrocyte. In some embodiments, the inner core includes a nucleic acid molecule. In some embodiments, the inner core and the outer sheath are combined through extrusion, sonication, agitation, mixing, vortexing, or any combination thereof. In some embodiments, the inner core and the external membrane are combined through extrusion, sonication, agitation, mixing, vortexing, or any combination thereof. In some embodiments, the inner core is encapsulated by the outer sheath. In some embodiments, the inner core is encapsulated by the external membrane. In some embodiments, vesicles may be generated by extrusion, and subsequently loaded with nucleic acids of interest by vortexing or other physical disruption. [0008] In some embodiments, the nanoparticle external membrane can shield a biological payload from immune system detection and clearance. The external membrane can refer to any membranous material that partially or completely envelops an inner core or other enveloped payload. In some embodiments, the external membrane refers to one or more of a cell membrane or an extracellular vesicle. In some embodiments, the external membrane is generated from the cell membrane surface. In some embodiments, the cell membrane surface contains self-recognition proteins, including but not limited, CD-47. In some embodiments, the external membrane can feature any number of targeting motifs, such that the nanoparticle is capable of targeting specific tissues or sites. In some embodiments, the external membrane can feature any number of targeting motifs and any number of self-recognition proteins. Any targeting motif and any self-recognition protein described in the art can be embedded, engineered, or present on the surface of the nanoparticle external membrane. [0009] In some embodiments, methods of treating a disease or disorder are provided. These methods can include intravenous drug administration, subcutaneous drug administration, timed-dose release administration, or other enteral or parenteral routes of medications known in the art. [0010] Additional embodiments are set forth in the following enumerated alternatives: [0011] 1. A method of manufacture of a nanoparticle comprising: providing a plurality of external membranes, wherein the external membranes are generated by membrane extrusion; providing a plurality of nucleic acid molecules, wherein the nucleic acid molecules are suspended in solution; combining the plurality of external membranes with the plurality of nucleic acid molecules; and encapsulating a portion of the plurality of nucleic acid molecules within a portion of the plurality of external membranes by physical disruption, wherein up to 15 kbp of nucleic acid are encapsulated within each external membrane. [0012] 2. The method of alternative 1, wherein the physical disruption comprises one or more of extrusion, sonication, agitation, mixing, or vortexing, [0013] 3. The method of any one of alternatives 1 or 2, wherein the plurality of external membranes are derived from erythrocytes. [0014] 4. The method of any one of alternatives 1-3, wherein the plurality of nucleic acid molecules and the plurality of external membranes are physically disrupted simultaneously. [0015] 5. The method of any one of alternatives 1-4, wherein membrane extrusion disassociates the plurality of external membranes. [0016] 6. The method of any one of alternatives 1-5, wherein the plurality of external membranes are filtered through tangential flow filtration. [0017] 7. The method of any one of alternatives 1-6, wherein the plurality of nucleic acid molecules are encapsulated by the external membrane following physical disruption. [0018] 8. The method of any one of alternatives 1-7, wherein the external membrane is generated by physical disruption of a parent cell. [0019] 9. The method of alternative 8, wherein the physical disruption of the parent cell is achieved by hypotonic lysis or physical separation. [0020] 10. The method of any one of alternatives 8-9, wherein the parent cell is isolated from whole blood. [0021] 11. The method of any one of alternatives 8-10, wherein parent cell is a red blood cell. [0022] 12. The method of any one of alternatives 1-11, wherein extrusion comprises passing the nucleic acid molecule and the external membrane through a porous membrane. [0023] 13. The method of alternative 12, wherein the porous membrane comprises pores having a diameter ranging from about 200 nm to about 1,000 nm. [0024] 14. A method of transfection using a plurality of nanoparticles comprising: providing a plurality of DNA nanoparticles, wherein the DNA nanoparticles individually comprise an inner core and an outer sheath, wherein the outer sheath is comprised of an external membrane, wherein the inner core comprises one or more nucleic acid molecules, wherein the inner core and the outer sheath are combined through physical disruption, wherein the inner core is encapsulated by the outer sheath; providing a plurality of cells, wherein the cells are able to uptake the nanoparticles; combining the plurality of DNA nanoparticles with the plurality of cells, wherein the cells are incubated in the presence of DNA nanoparticles for a period of time. [0025] 15. The method of alternative 14, wherein the period of time is up to 2 days. [0026] 16. The method of any one of alternatives 14-15, wherein the plurality of cells are adherent. [0027] 17. The method of any one of alternatives 14-15, wherein the plurality of cells are in suspension. BRIEF DESCRIPTION OF THE DRAWINGS [0028] The foregoing and other features of the present disclosure will become more fully apparent from the following description, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only some embodiments in accordance with the disclosure and are therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings. [0029] FIG. 1 graphically illustrates flow cytometry (left) and fluorescent signal (right) for detection of nanoparticles as described in some embodiments herein in aqueous media. [0030] FIG. 2 graphically illustrates flow cytometry (left) and fluorescent signal (right) for detection of a liposomal formulation as described in some embodiments herein. [0031] FIG. 3 graphically illustrate flow cytometry (left) and fluorescent signal (right) for detection of nanoparticles with and without a nucleic acid molecule. [0032] FIG 4. graphically illustrate fluorescent signal detection of liposome or erythrocyte derived membrane (EDM) particles with and without a nucleic acid molecule. [0033] FIG. 5 graphically illustrates flow cytometry and fluorescent signal detection for aqueous media after storage. [0034] FIG.6 graphically illustrate flow cytometry and fluorescent signal detection for a liposomal formulation after storage. [0035] FIG.7 graphically illustrate flow cytometry and fluorescent signal detection for nanoparticles with and without a nucleic acid molecule after storage. [0036] FIG. 8 graphically illustrate fluorescent signal detection for particles with and without a nucleic acid molecule after storage. [0037] FIGs. 9A-9B graphically illustrates fluorescent imaging of Jurkat Cells transfected with GFP using a variety of DNA Nanoparticle populations. [0038] FIGs. 10A-10D illustrates transfection efficiency and fluorescent imaging of DNA nanoparticle, lipofectamine, and DNA only transfection DETAILED DESCRIPTION [0039] Embodiments of the present disclosure relate to compositions, methods of manufacture, methods of treating, and methods of transfecting using nanoparticles comprising nucleic acids encapsulated by an external membrane (which can be referred to, for example, as a nucleic acid nanoparticle). The nucleic acid nanoparticles can be used in a variety of therapeutic or medical contexts. [0040] The following description provides context and examples but should not be interpreted to limit the scope of the inventions covered by the claims that follow in this specification or in any other application that claims priority to this specification. No single component or collection of components is essential or indispensable. Any feature, structure, component, material, step, or method that is described and/or illustrated in any embodiment in this specification can be used with or instead of any feature, structure, component, material, step, or method that is described and/or illustrated in any other embodiment in this specification. [0041] It will be readily understood that the aspects of the present disclosure, as generally described herein, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein. [0042] The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. [0043] Generally, therapeutics and overall pharmacological formulations measure efficacy of any given formulation based on dose response and the amount of effective dose required to enact a physiological effect. Many different factors can affect how easily drugs are metabolized or delivered to certain tissues, and some therapeutic formulations may have acute, transient toxicity profiles, limiting the overall dose that may be introduced at any given time. Further, some pharmaceutical formulations may be susceptible to rapid clearing when in the bloodstream, thereby requiring frequent or high dose administrations, leading to increased therapeutic costs and complications relating to extended care. As such, it is advantageous to provide a composition comprising a variety of pharmaceutically active molecules which can target and penetrate certain tissues, as well as remain in circulation in the blood stream for extended periods of time. In some embodiments, the compositions provided herein have increased circulation half-life in the body and/or have increased drug efficacy. [0044] As described in literature, it is known that functional nanoparticles can be generated using existing cellular membranes from extant sources. These sources can include membranes derived from blood cells, (e.g., red blood cells (RBCs), white blood cells (WBCs), or platelets). See C-M. J. Hu et al., “Erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform,” Proc. Natl. Acad. Sci. EISA 2011, July 5; 108(27), 10980-10985. Attempts to encapsulate various biomolecules have been met with varying levels of success, depending on the method used to generate encapsulated nanoparticles. The cellular membranes derived from blood cells consist of the cellular lipid bilayers, and in the case of cellular membranes derived from red blood cells, are known as erythrocyte derived membranes (EDM). Due to the nature of blood groups and antigen detection by a host immune system, in some embodiments it is advantageous to use cellular membranes derived from universal donor blood type (e.g. Type O negative), thereby allowing for foreign payload delivery to any potential patient without concern(s) of nanoparticle compatibility or rejection. [0045] Methods to generate membranes derived from erythrocytes are described in literature. See Theranostics 2016, 6(7), 1004-1011. Whole blood is generally collected, and physical disruption is applied to a fraction of separated blood from the whole blood collected. When whole blood is collected, various anti-coagulating agents are added, and physical separation of a red blood fraction is achieved. To collect cellular membranes, procedures like hypotonic lysis of erythrocytes and physical separation of membranous fractions are utilized on the red blood fraction to generate erythrocyte derived membrane (EDM) fractions. [0046] Moreover, membranes from parent cells can be derived from a variety of cell types. In some embodiments, the parent cells are derived from T cells. In some embodiments, the parent cells are derived from fetal cells. In some embodiments, the parent cells are derived from diseased tissue cells. In some embodiments, the parent cells are derived from healthy tissue. In some embodiments, the parent cells are autologous. In some embodiments, the parent cells are allogenic. In some embodiments, the parent cells are xenogeneic. [0047] For example, in extrusion based encapsulation, by forcing external membranes through a porous membrane, with pores between 200 nm to 1,000 nm in diameter, spontaneous lipid membrane reformation occurs in a plurality of external membranes. The movement of external membranes, along with extra-membranous material across the porous membrane is otherwise known as extrusion. When EDM fractions undergo spontaneous lipid membrane reformation, any extra-membranous material in the solution may be encapsulated upon formation of the lipid membranes. In some embodiments, the extra-membranous material comprises DNA molecules. In some embodiments, the extra-membranous material comprises virus or viruses. The combination of DNA or any other contemplated molecules in a fraction containing EDMs, when extruded, generates nanoparticles as contemplated herein. [0048] In some embodiments, cellular membranes can be extracted from whole cells via extrusion, with a porous membrane comprising pores between 200 nm to 1,000 nm in diameter. The movement of cellular membrane, along with extra-membranous material across the porous membrane generates a vesicle population. In some embodiments, the vesicle population can be further purified, filtered, or refined to generate a purified vesicle population. [0049] Nanoparticles generated by the methods described herein have manifold advantages. One advantage is the protection of foreign molecules from detection by the host immune system. Another advantage would be to enhance the concentration or delivery, via tissue targeting or by preventing rapid clearance of the payload. Moreover, higher packing densities of the payload within a vesicle or membrane may be achieved. And further, high transfection efficiencies compared to gold standard transfection agents are observed. In accordance with the nanoparticles described herein, it is surprising that physical disruption or agitation of DNA molecules with an EDM fraction can generate a significant amount of encapsulated nanoparticles. Moreover, it was surprisingly discovered that the nanoparticles and methods of making the nanoparticles described herein can carry large molecular payloads with nucleic acids having multiple kilobases. [0050] For some conditions, nucleic acids may be applied therapeutically, and depending on the precise sequence, may serve to alter tissue protein expression and change a disease state (gene therapy). In some embodiments, the nucleic acid payload can further include additional cellular or molecular elements, including viruses. In some embodiments, the nucleic acid payload can be delivered as a virus, including therapeutic viruses (such as oncolytic viruses or AAVs). Due to the sequence of a nucleic acid payload, nucleic acid therapeutics, including direct gene delivery and vehicle mediated gene delivery have the potential to be specific, functionally diverse, and have limited toxicity in host tissues. However, nucleic acids are susceptible to breakdown in circulating blood streams when administered intravenously and may require targeted delivery to certain tissues or other vehicles to prolong survival while circulating in the blood stream. In some embodiments, cancer cells are targets of applied therapeutic nucleic acids as a gene therapy. In some embodiments, cells comprising harmful mutations, or otherwise expressing cellular disfunction, can be targets of the described applied therapeutic nucleic acids as a gene therapy. [0051] Multiple varieties of encapsulated molecules can be generated according to the methods described herein. In some embodiments, a nanoparticle generated according to any of the embodiments described herein may be generated using a Erythro-Nanosome Host- adapted Encapsulation System (ENHEnS). Some embodiments provided herein relate to methods of manufacture of a nanoparticle, comprising providing a plurality of external membranes. In some embodiments, the external membranes are a plurality of lipid bilayer nanostructures. In some embodiments, the external membranes are extracellular vesicles. In some embodiments, the external membrane are cellular membranes The methods may further include providing a plurality of nucleic acid molecules, wherein the nucleic acid molecules are suspended in solution. In some embodiments, the methods further include combining the plurality of external membranes with the plurality of nucleic acid molecules. In some embodiments, the methods include encapsulating a portion of the plurality of nucleic acid molecules within a portion of the plurality of external membranes by physical disruption, wherein up to 15 kilobase pairs (kbp) of nucleic acid are encapsulated within each external membrane. [0052] In some embodiments, physical disruption comprises one or more of extrusion, sonication, agitation, mixing, or vortexing. In some embodiments, the plurality of external membranes are derived from erythrocytes. In some embodiments, the plurality of nucleic acid molecules and the plurality of external membranes are physically disrupted simultaneously. In some embodiments, membrane extrusion disassociates the plurality of external membranes. In some embodiments, the plurality of external membranes are filtered through tangential flow filtration. In some embodiments, the plurality of nucleic acid molecules are encapsulated by the external membrane following physical disruption. In some embodiments, the external membrane is generated by physical disruption of a parent cell. In some embodiments, the physical disruption of the parent cell is achieved by hypotonic lysis or physical separation. In some embodiments, the parent cell is isolated from whole blood. In some embodiments, the parent cell is a red blood cell. In some embodiments, extrusion comprises passing the nucleic acid molecule and the external membrane through a porous membrane. In some embodiments, the porous membrane comprises pores having a diameter ranging from about 200 nm to about 1,000 nm. [0053] In some embodiments, methods of transfection using a plurality of nanoparticles are described. The methods include providing a plurality of DNA nanoparticles. In some embodiments, the DNA nanoparticles individually comprise an inner core and an outer sheath, wherein the outer sheath is comprised of either a cellular membrane or extracellular vesicle, wherein the inner core comprises one or more nucleic acid molecules, wherein the inner core and the outer sheath are combined through physical disruption, wherein the inner core is encapsulated by the outer sheath. In some embodiments, the methods further include providing a plurality of cells, wherein the cells are able to uptake the nanoparticles. In some embodiments, the methods further include combining the plurality of DNA nanoparticles with the plurality of cells, wherein the cells are incubated in the presence of DNA nanoparticles for a period of time. [0054] The foregoing description provides context and examples but should not be interpreted to limit the scope of the inventions covered by the claims that follow or in any other applications that claims priority to this specification. No single component or collection of components is essential or indispensable. Any feature, structure, component, material, step, or method that is described and/or illustrated in any embodiment in this specification can be used with or instead of any feature, structure, component, material, step, or method that is described and/or illustrated in any other embodiment in this specification. [0055] It will be readily understood that the aspects of the present disclosure, as generally described herein, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein. [0056] The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. [0057] Some embodiments provided herein relate to methods of making the compositions and formulations described herein. In some embodiments, the methods include physically disrupting a plurality of nucleic acid molecules with a plurality of cellular or external membranes, in amounts sufficient to prepare a plurality of nanoparticles. [0058] As used herein, the term “nanoparticle” has its ordinary meaning as understood in light of the specification and refers to a particle that is on the nano scale. For example, a nanoparticle is a particle that has a diameter ranging from about 0.1 nanometers to about 1000 nm, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nm, or a diameter within a range defined by any two of the aforementioned values. In some embodiments, a nanoparticle comprises an external membrane that encapsulates a nucleic acid molecule. As used herein the term “external membrane” has its ordinary meaning as understood in light of the specification, and refers to any membranous material that partially or completely envelops an inner core or other enveloped payload. In some embodiments, the external membrane is a cell membrane or an extracellular vesicle. [0059] As used herein, the term “extracellular vesicle” has its ordinary meaning as understood in light of the specification, and refers to a structure comprising a membranous material which separates the interior of the structure from the outside environment. Extracellular vesicles include, but are not limited to, varieties of exosomes, ectosomes, microparticles, microvesicles, oncosomes, exophers, migrasomes, enveloped viruses, exomeres, and apoptotic bodies, among other species. [0060] As used herein, the term “cellular membrane” has its ordinary meaning as understood in light of the specification and refers to a membranous material which separates the interior of a cell from the outside environment. In some embodiments, the cell membrane comprises a lipid bilayer that is semipermeable. In some embodiments, the cell membrane comprises outward facing receptors. In some embodiments, the outward facing receptors are antigens. In some embodiments, the cell membrane comprises transmembrane proteins. In some embodiments, the cell membrane is derived from an erythrocyte. In some embodiments, the cellular membrane may be disassociated from a progenitor cell. In some embodiments, the cellular membrane may encompass nucleic acids. [0061] As used herein, the term “nucleic acid” is intended to be consistent with its use in the art and includes naturally occurring nucleic acids or functional analogs thereof. Particularly useful functional analogs are capable of hybridizing to a nucleic acid in a sequence specific fashion or capable of being used as a template for replication of a particular nucleotide sequence. Naturally occurring nucleic acids generally have a backbone containing phosphodiester bonds. An analog structure can have an alternate backbone linkage including any of a variety of those known in the art. Naturally occurring nucleic acids generally have a deoxyribose sugar (e.g. found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g. found in ribonucleic acid (RNA)). A nucleic acid can contain any of a variety of analogs of these sugar moieties that are known in the art. A nucleic acid can include native or non-native bases. In this regard, a native deoxyribonucleic acid can have one or more bases selected from the group consisting of adenine, thymine, cytosine, or guanine and a ribonucleic acid can have one or more bases selected from the group consisting of uracil, adenine, cytosine, or guanine. Useful non-native bases that can be included in a nucleic acid are known in the art. The term “target,” when used in reference to a nucleic acid, is intended as a semantic identifier for the nucleic acid in the context of a method or composition set forth herein and does not necessarily limit the structure or function of the nucleic acid beyond what is otherwise explicitly indicated. Particular forms of nucleic acids may include all types of nucleic acids found in an organism as well as synthetic nucleic acids such as polynucleotides produced by chemical synthesis. Particular examples of nucleic acids that are applicable for encapsulation through extrusion or physical disruption with external membranes produced by methods as provided herein include genomic DNA (gDNA), expressed sequence tags (ESTs), DNA copied messenger RNA (cDNA), RNA copied messenger RNA (cRNA), mitochondrial DNA or genome, RNA, messenger RNA (mRNA) and/or other populations of RNA. Fragments and/or portions of these exemplary nucleic acids also are included within the meaning of the term as it is used herein. [0062] As used herein, the term “extrusion” refers to a process used to create objects of a fixed cross-sectional profile. A material is pushed through a die of the desired cross-section. The two main advantages of this process over other manufacturing processes are its ability to create very complex cross-sections, and to work materials that are brittle, because the material only encounters compressive and shear stresses. Extrusion as a process may be applied to cells and cellular material, by applying pressure to cells in media against a plurality of pores. [0063] As used herein, the term “physical disruption” refers to the application of physical forces to cells in order to generate a reaction or change in cell phenotype. In some embodiments, physical disruption may refer to hypotonic lysis, a process wherein cells placed into a hypotonic solution begin to lyse due to water moving into a cellular interior. Hypotonic solutions may consist of any aqueous solution wherein the concentration of certain solutes is less than the concentration of similar solutes within a cell. Hypotonic solutions are preferred because osmotic pressure differentials force water into a cell, eventually bursting said cell and lysing the external membrane. In some embodiments, physical disruption may refer to physical separation of cells, wherein forces applied to cells or cellular components serve to separate cell subpopulations based on physical characteristics, such as size, shape, density, and mass of various cell subpopulations. In some embodiments, application of certain forces may affect sedimentation, or separation of cellular subpopulations based on density. In some embodiments, physical disruption includes methods of agitation, including but not limited to extrusion, sonication, agitation, mixing, vortexing, or any combination thereof. Differences in cellular density allows sedimentation into fractions when centrifugal force is applied, the fractions being concentrations of various cellular subpopulations. [0064] As used herein, the term “sonication” refers to the act of applying sound energy to agitate particles or compounds within a sample. In some instances, sonication may specifically refer to the disruption of external membranes by the application of high frequency sound waves. In some embodiments, sonication can allow for the partial disruption of external membranes for encapsulating payloads within. [0065] As used herein, the term “tangential-flow filtration” refers to a process in which the fluid mixture containing the components to be separated by filtration is recirculated at high velocities tangential to the plane of the membrane to increase the mass-transfer coefficient for back diffusion. In such filtrations a pressure differential is applied along the length of the membrane to cause the fluid and filterable solutes to flow through the filter. This filtration is suitably conducted as a batch process as well as a continuous-flow process. For example, the solution may be passed repeatedly over the membrane while that fluid which passes through the filter is continually drawn off into a separate unit or the solution is passed once over the membrane and the fluid passing through the filter is continually processed downstream. [0066] The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. One skilled in the art will appreciate readily that the present disclosure is well adapted to carry out the objectives and obtains the ends and advantages mentioned, as well as those objectives, ends and advantages inherent herein. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art. [0067] In some embodiments, provided herein are methods of manufacture of nanoparticles, the methods including combining a nucleic acid molecule and an external membrane by extrusion or other physical disruption. In some embodiments, the external membrane is derived from erythrocytes. In some embodiments, the nucleic acid molecule and the external membrane are physically disrupted simultaneously. In some embodiments, the physical disruption process disassociates the external membrane. In some embodiments, the nucleic acid molecule is encapsulated by the external membrane following physical disruption. In some embodiments, the external membrane is generated by physical disruption of a parent cell. In some embodiments, the physical disruption of the parent cell is achieved by hypotonic lysis, or physical separation. [0068] In some embodiments, the extrusion process comprises passing the nucleic acid molecule with the external membrane through a porous membrane, the porous membrane having pores ranging from about 200 nm to about 1,000 nm in diameter, such as 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 nm, or a diameter within a range defined by any two of the aforementioned values. [0069] In some embodiments, the ratio of nucleic acid molecules to external membranes prior to combination is 1:100 by volume. In some embodiments, the ratio of nucleic acid molecules to external membranes prior to combination is 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:200, 1:300, 1:400, 1:500, 1:1000 or any value in between by volume or mass. In some embodiments, the ratio of external membranes to nucleic acid molecules prior to combination is 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:200, 1:300, 1:400, 1:500, 1:1000 or any value in between by volume or mass. In some embodiments, the ratio of nucleic acid molecules to external membranes can exceed 1000:1 by mass. In some embodiments, the cell membrane is processed by tangential flow filtration. In some embodiments, the parent cell is isolated from whole blood. In some embodiments, the parent cells include red blood cells. In some embodiments, the nucleic acid molecule is comprised of circular or linear fragments. In some embodiments, the nucleic acid molecule is a plasmid. In some embodiments, the external membrane is stored in an aqueous solution. [0070] In some embodiments, nucleic acids are encapsulated within erythrocyte derived membranes (EDMs). In some embodiments, an amount of nucleic acid encapsulated within or loaded into EDMs is in an amount of 375 ^g/mL. In some embodiments, the loading amount of nucleic acids into EDMs can be as high as 1000 ^g/mL. In some embodiments, the loading amount of nucleic acids into EDMs can be 100, 150, 200, 250, 300, 350, 400, 450, 500 ^g/mL, 1000 ^g/mL or any value in between. In some embodiments, up to 12 kbp of nucleic acids can be encapsulated in an EDM. In some embodiments, up to 20 kbp of nucleic acids can be encapsulated in an EDM. In some embodiments, up to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 kbp or any value in between of nucleic acid can be encapsulated in an EDM. In some embodiments, up to 2, 3, 4, 5, 6, 7, 8, 910, 15, 20 kbp or any value in between of nucleic acid can be encapsulated in an EDM. [0071] In some embodiments, the nucleic acid encapsulated within or loaded into the EDM is DNA or RNA. [0072] In some embodiments, the nanoparticle external membrane can shield a biological payload from immune system detection and clearance. In some embodiments, the nanoparticle external membrane can be engineered to preferentially target specific tissues. In some embodiments, the external membrane surface contains self-recognition proteins, including but not limited, CD-47. In some embodiments, the external membrane surface can feature any number of targeting motifs, such that the nanoparticle is capable of targeting specific tissues or sites. In some embodiments, the targeting motifs can include antibodies and antibody species, including, but not limited to, bispecific antibodies and antibodies with lipid anchors. In some embodiments, the external membrane surface can feature any number of targeting motifs and any number of self-recognition proteins. Any targeting motif and any self- recognition protein described in the art can be embedded, engineered, or present on the surface of an external membrane. [0073] In some embodiments, the methods provided herein may have a transfection efficiency of at least about 10%. In some embodiments, the methods provided herein may have a transfection efficiency of at least about 20%. In some embodiments, the methods provided herein may have a transfection efficiency greater than 10%. In some embodiments, the methods provided herein may have a transfection efficiency greater than 20%. In some embodiments, the methods provided herein may have a transfection efficiency of at least about 50%. In some embodiments, the methods provided herein may have a transfection efficiency greater than 50%. In some embodiments, the method or system may have a transfection efficiency of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or greater than 50%. [0074] In some embodiments, provided herein are methods of manufacture of nanoparticles, the methods including combining a virus and an external membrane by extrusion or other physical disruption. In some embodiments, the external membrane is derived from erythrocytes. In some embodiments, the virus and the external membrane are physically disrupted simultaneously. In some embodiments, the physical disruption process disassociates the external membrane. In some embodiments, the virus is encapsulated by the external membrane following physical disruption. In some embodiments, the external membrane is generated by physical disruption of a parent cell. In some embodiments, the physical disruption of the parent cell is achieved by hypotonic lysis, or physical separation. In some embodiments, the extrusion process comprises passing the virus with the external membrane through a porous membrane, the porous membrane having pores ranging from about 200 nm to about 1,000 nm in diameter, such as 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 nm, or a diameter within a range defined by any two of the aforementioned values. In some embodiments, the ratio of virus to external membranes prior to combination is 1:100 by volume. In some embodiments, the ratio of virus to external membranes prior to combination is 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:200, 1:300, 1:400, or any value in between by volume or mass. In some embodiments, the ratio of external membranes to virus prior to combination is 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:200, 1:300, 1:400, or any value in between by volume or mass. In some embodiments, the cell membrane is processed by tangential flow filtration. In some embodiments, the parent cell is isolated from whole blood. In some embodiments, the parent cells include red blood cells. In some embodiments, the virus is comprised of circular or linear fragments. In some embodiments, the virus is a plasmid. In some embodiments, the external membrane is stored in an aqueous solution. [0075] In some embodiments, provided herein are methods of transfection. In some embodiments, the methods include adding DNA nanoparticles to a population of cells in media. In some embodiments, the cells may be suspended or adherent. In some embodiments, the methods further include incubating the cells. In some embodiments, incubation may be for a period of time including 1 hour, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 66 hours, 72 hours or any value in between. [0076] In some embodiments, described herein are compositions of a nanoparticle, comprising an inner core and an outer sheath, wherein the outer sheath is comprised of a cellular membrane derived from erythrocytes, wherein the inner core is comprised of a plurality of nucleic acid molecules, and wherein the inner core and the outer sheath are combined through physical disruption, wherein the inner core is encapsulated by the outer sheath. EXAMPLES Example 1: Method of Confirming DNA Nanoparticle Generation [0077] To test whether resulting extruded EDM (erythrocyte derived membranes) encapsulated DNA molecules, a variety of comparative experiments were run to see whether a fluorescence signal and recognizable nanoparticle population as determined by flow cytometry were detectable. Generally, replicates to be run through a Becton Dickenson CytoFlex flow cytometer consisted of either: 1. Aqueous solution with no cellular membranes; 2. Commercially available liposome formulations (Avanti Polar Lipids, #300202S- 1EA) extruded in the presence of DNA; 3. Commercially available liposome formulations (Avanti Polar Lipids, #300202S- 1EA) extruded in the absence of DNA; 4. EDM fractions extruded in the presence of DNA; or 5. EDM fractions extruded in the absence of DNA. [0078] The EDM fraction was generated from collected whole blood mixed with anti-coagulating agents. A Red Blood Cell portion was isolated from the whole blood sample, and a series of steps to achieve RBC lysis (hypotonic lysis), followed by tangential flow filtration were run on the red blood cell portion to yield an EDM fraction. The EDM fraction was then stored in an aqueous solution. [0079] DNA molecules that were introduced to the various liposome or EDM fractions were plasmids of a known length (~2,680 base pairs). The DNA molecules were detectable by labeling the plasmid with nucleic acid stain SYBR GREEN I (CAS 163795-75- 3). 10,000X SYBR stock was diluted to 2X in compatible aqueous media and added to DNA to achieve 1X staining SYBR concentration (DNA concentration was ~454 ng/μL). Extrusion apparatus was assembled per the manufacturer’s instructions, utilizing a 1000 nm (1 μm) pore size membrane. [0080] For each replicate, aqueous solutions containing 500 μL of Liposome or 500 μL of human EDM (hEDM) were aspirated into 1 mL glass syringes, as well as one syringe containing aqueous solution without either liposome or hEDM present. Additionally, aqueous solutions of 500 μL of Liposome or 500 μL of human EDM (hEDM), each with 8 μL of SYBR stained DNA (3,636 ng of DNA) were prepared in syringes as well. [0081] For each run, a syringe was attached to the filter housing unit with an identical syringe on the opposite side of the filter housing. The mixture was extruded through the filter 8 times completely, once back-and-forth each for 8 times total. Additionally, all samples were reserved and stored at 4°C until analysis. Analysis was assessed on the day of sample preparation and 24 hours post-preparation on samples stored at 4°C. [0082] Sample preparations were analyzed for fluorescent signal intensity detected with the “green” (FITC) channel on a Becton Dickinson CytoFlex flow cytometer. The raw data values were stored as “.fcs” file types and analyzed with FCSalyzer software (Version 0.9.21-alpha). [0083] A compatible aqueous media with 1X SYBR stain was first run through the instrument to collect data for any non-specific fluorescent signal. No detectable signal was recorded (Figure 1). The Liposomal formulation was readily detected (see top left panel, FSC vs SSC) yet no fluorescent signal intensity was recorded (top right panel) (Figure 2). Similarly, it was observed that Liposomal formulation did not interact with stained DNA in any discernable or substantial way. While Liposome extruded in the presence of SYBR stained DNA had a similar physical detection to that without DNA (see bottom left panel), as with no DNA sample no fluorescent signal intensity was registered (see bottom right panel) (Figure 2). The hEDM was similarly readily detected (see top left panel) and showed no fluorescence of substance when analyzed alone (see top right panel) (Figure 3). While hEDM extruded in the presence of SYBR stained DNA had a similar physical detection to that without DNA (see bottom left panel), the hEDM extruded with DNA registered substantial fluorescent signal intensity (see bottom right panel) (Figure 3). When the signal intensity of each condition (Liposome alone or with DNA and hEDM alone or with DNA) were overlaid the increase in fluorescent signal intensity, because of the specific interaction with SYBR stained DNA, is evident (left and right panels, respectively) (Figure 4). [0084] The results recapitulate after 24 hours storage at 4°C. There was no difference observed in when the staining buffer was analyzed (Figure 5). Liposome, whether analyzed as the solution directly as is, extruded, or with SYBR labeled DNA rendered no discernable signal (Figure 6). Neither did hEDM as is or extruded produce any fluorescent signal; hEDM extruded with SYBR labeled DNA again shows intense signal intensity indicating the direct, preserved interaction with fluorescently labeled DNA (Figure 7). The difference in signal intensity if observed when plots of the fluorescent signal are overlaid, as shown in Figure 8. In Figure 8, top panel is Liposome overly, with liposome extruded in back, liposome as is in middle, and liposome extruded with SYBR labeled DNA in front; bottom panel is hEDM with hEDM extruded with SYBR labeled DNA in the back, hEDM extruded in the middle, and hEDM as is in front. Example 2: Generation of DNA Nanoparticles [0085] Preparation of DNA nanoparticles were accomplished by collecting fractions of EDM (erythrocyte derived membranes) from live cells with nucleic acids. EDM fractions (1010 ^g/mL) were combined with Vector DNA encoding GFP (1073 ng/^L) using a variety of methods to encapsulate nucleic acids, including extrusion, sonication, and mixing. [0086] For encapsulated DNA by extrusion, hEDM fractions were combined with Vector DNA encoding GFP (Green Fluorescent Protein) at a ratio of 1:100 and extruded with 1 ^m PC filters. For encapsulated DNA by sonication, hEDM fractions were combined with Vector DNA encoding GFP at a ratio of 1:100 and sonicated using a probe sonicator. Lastly, for encapsulated DNA by mixing, hEDM fractions were combined with Vector DNA encoding GFP and mixed thoroughly with a pipette. Example 3: DNA Nanoparticle Delivery to Cell Populations [0087] To determine the suitability of encapsulated DNA for transfection, a population of encapsulated DNA nanoparticles were generated and overall transfection efficiency was assessed. Results were compared to industry standard lipofectamine transfection. EDM (Erythrocyte derived membranes) encapsulated DNA molecules (DNA Nanoparticles) and controls were generated according to the following populations: 1. Jurkat cells transfected with EDM+DNA Nanoparticles (Extrusion) 2. Jurkat cells transfected with EDM+DNA Nanoparticles (Sonication) 3. Jurkat cells transfected with EDM+DNA Nanoparticles (Mixed) 4. Jurkat Cells with Lipofectamine + DNA [0088] For encapsulated DNA by extrusion, hEDM fractions were combined with Vector DNA encoding GFP (Green Fluorescent Protein) at a ratio of 1:100 and extruded with 1 ^m PC filters. For encapsulated DNA by sonication, hEDM fractions were combined with Vector DNA encoding GFP at a ratio of 1:100 and sonicated using a probe sonicator. Lastly, for encapsulated DNA by mixing, hEDM fractions were combined with Vector DNA encoding GFP and mixed thoroughly with a pipette. [0089] Populations of extruded encapsulated DNA, sonicated encapsulated DNA, and mixed encapsulated DNA were then centrifuged with 3 aliquots of Jurkat cells, respectively. A fourth aliquot of Jurkat cells was transfected using Lipofectamine. Images were captured after 48 hours. [0090] Figure 9A shows GFP transfection and subsequent GFP signal from Jurkat cells through fluorescent imaging using DNA nanoparticles generated through mixing. Figure 9B likewise demonstrates GFP signal observed in Jurkat cells through fluorescent imaging using DNA nanoparticles generated through sonication. DNA nanoparticles generated through sonication, and DNA nanoparticles generated through mixing exhibited similar levels of successful DNA transfection. The process of sonication and mixing in combination resulted in unexpectedly superior results of efficiently produced nucleic acid nanoparticles. These results were surprising and unexpectedly superior to products produced using other methods. [0091] The results indicated significantly higher observed GFP signal for the DNA nanoparticles generated using mixing and sonication compared to Lipofectamine. Further, it was unexpected and surprising that mixed and/or sonicated nanoparticle populations exhibited higher GFP signal than extrusion generated DNA nanoparticles. The results were unexpected and surprising due to potential charge bias; Lipofectamine is positively charged and supposedly should integrate DNA more efficiently than negatively charged EDM coatings. Example 4: DNA Nanoparticle Delivery to Cell Populations [0092] Further experiments on using EDMs as carriers for nucleic acid payloads compared to lipofectamine were run. Replicates on the following populations were run: Bare DNA, Lipofectamine+DNA, Nanoparticles (EDMs+DNA) and transfection efficiency was assessed against Jurkat cells, which are CD4 positive T-Cells. Additionally, Lipofectamine is a positively charged liposomal formulation that allows for mixing and incubation to accomplish transfection. Around 1 ^g of DNA was used for all experimental groups, and each formulation was added to separate suspension cultures of 150,000 Jurkat cells in 0.5 mL of media and incubated for 2 days prior to imaging. Figures 10A-10D demonstrate an efficiency plot and fluorescence imagery of GFP transfection and overall observed signal. Figure 10A shows greater than 20% transfection efficiency (ENHEnS + DNA) compared to Lipofectamine which demonstrated around 3-10% transfection efficiency. Figure 10D represents DNA control in which no GFP signal was observed. Figures 10B and 10C represent ENHEnS and lipofectamine mediated transfected cells, respectively. As shown in Figure 10A, greater transfection efficiency was observed for ENHEnS + DNA mediated transfected cells compared to lipofectamine. [0093] The results were unexpected and surprising given the high transfection efficiency of the generated DNA nanoparticle populations compared to lipofectamine, which exhibited anywhere from 6-fold to 2-fold less efficiency compared to nanoparticle transfection. Example 5: Assessing DNA loading into EDMs [0094] 240 nm empty vesicles (EDMs) were generated by extrusion methods and loaded with DNA using a vortexer. DNA loading amounts into 2 mg/mL of EDMs can be as high as 375 ^g/mL (2.7 kbp). Particle sizes (Zavg) of the final DNA nanoparticle and overall polydispersity (PDI) to determine overall aggregation were then assessed. TABLE 1: DNA Loading

[0095] No significant change was observed in zeta potential, suggesting that the less negative charge (-16 mV) did not mask the surface of the particles (otherwise the zeta potential would be going toward zero). DNA up to 375 ^g/mL was observed to be encapsulated by EDMs with negligible changes to PDI and Zeta potential. [0096] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions, and modifications may be made to the methods, compositions, kits, and uses described herein without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims. [0097] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. [0098] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. Thus, the term “a cellular membrane” may refer, for example to a plurality of cellular membranes. Likewise, the term “an external membrane” may refer to a plurality of external membranes. Similarly, the term “a nucleic acid molecule”, “a DNA molecule”, or “a parental cell” may refer to a plurality of such molecules or cells. [0099] In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.” [0100] 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. [0101] As will be understood by one of skill in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth. [0102] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those of skill in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

WHAT IS CLAIMED IS: 1. A method of manufacture of a nanoparticle comprising: providing a plurality of external membranes, wherein the external membranes are generated by membrane extrusion; providing a plurality of nucleic acid molecules, wherein the nucleic acid molecules are suspended in solution; combining the plurality of external membranes with the plurality of nucleic acid molecules; and encapsulating a portion of the plurality of nucleic acid molecules within a portion of the plurality of external membranes by physical disruption, wherein up to 15 kbp of nucleic acid are encapsulated per encapsulating external membrane.

2. The method of claim 1, wherein the physical disruption comprises one or more of extrusion, sonication, agitation, mixing, or vortexing,

3. The method of claim 2, wherein the plurality of external membranes are derived from erythrocytes.

4. The method of claim 2, wherein the plurality of nucleic acid molecules and the plurality of external membranes are physically disrupted simultaneously.

5. The method of claim 4, wherein membrane extrusion disassociates the plurality of external membranes.

6. The method of claim 4, wherein the plurality of external membranes are filtered through tangential flow filtration.

7. The method of claim 2, wherein the plurality of nucleic acid molecules are encapsulated by the external membrane following physical disruption.

8. The method of claim 2, wherein the external membrane is generated by physical disruption of a parent cell.

9. The method of claim 8, wherein the physical disruption of the parent cell is achieved by hypotonic lysis or physical separation.

10. The method of claim 8, wherein the parent cell is isolated from whole blood.

11. The method of claim 8, wherein parent cell is a red blood cell.

12. The method of claim 2, wherein extrusion comprises passing the nucleic acid molecule and the external membrane through a porous membrane.

13. The method of claim 12, wherein the porous membrane comprises pores having a diameter ranging from about 200 nm to about 1,000 nm.

14. A method of transfection using a plurality of nanoparticles comprising: providing a plurality of DNA nanoparticles, wherein the DNA nanoparticles individually comprise an inner core and an outer sheath, wherein the outer sheath is comprised of an external membrane, wherein the inner core comprises one or more nucleic acid molecules, wherein the inner core and the outer sheath are combined through physical disruption, wherein the inner core is encapsulated by the outer sheath; providing a plurality of cells, wherein the cells are able to uptake the nanoparticles; combining the plurality of DNA nanoparticles with the plurality of cells, wherein the cells are incubated in the presence of DNA nanoparticles for a period of time.

15. The method of claim 14, wherein the period of time is up to 2 days.

16. The method of claim 14, wherein the plurality of cells are adherent.

17. The method of claim 14, wherein the plurality of cells are suspended.