WO2025217629A1

WO2025217629A1 - Nanoparticle dispersions and methods of manufacturing the same

Info

Publication number: WO2025217629A1
Application number: PCT/US2025/024532
Authority: WO
Inventors: Dustan Bonnin
Original assignee: Paretor LLC
Current assignee: Paretor LLC
Priority date: 2024-04-12
Filing date: 2025-04-14
Publication date: 2025-10-16
Anticipated expiration: 2026-10-12

Abstract

The present disclosure provides a cell-transfection agent including a composition including a de-watered functionalized polydisperse zwitterionic spinel nanoparticle dispersion, where each nanoparticle of the nanoparticle dispersion includes a core with a formula AB2O4 and is functionalized with a combination including free amine groups and thiol reactive groups, where A is a divalent cation and B is a trivalent cation, and where the composition is substantially free of irreversibly aggregated spinel nanoparticles. The present disclosure also provides therapeutic compositions including the cell-transfection agent, as well as methods of treatment and methods of manufacturing the cell-transfection agent and therapeutic compositions.

Description

NANOPARTICLE DISPERSIONSAND

METHODS OF MANUFACTURING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Patent Application No. 63/731,223, entitled “SPINEL NANOPARTICLE DISPERSIONS AND THEIR MANUFACTURE” filed on 12 April 2024, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present disclosure generally relates to a nanoparticle dispersion, and more particularly, to nanoparticle dispersions for delivering a payload molecule.

BACKGROUND

[0003] Research in the field of metallic nanoparticle dispersions for use in cellular transfection with payload molecules has shown promise. However, there is difficulty in the scalability of manufacture of nanoparticle dispersions for the purpose of cell-transfection for use in appropriate in-vitro or in-vivo applications. At present, there is significant difficulty in controlling and maintaining the uniformity of, for example, the poly dispersity of size, charge, magnetism, and functionalization of nanoparticles in the nanoparticle dispersion. These above- mentioned factors can impact the aggregation/agglomeration of the nanoparticles, which in turn impact manufacturing controls, the desired transfection profile, and any pharmacokinetic and/or pharmacodynamic characteristics required of the final product. There remains a need for cell-transfection agents useful for the delivery of payload molecules to desired cell types in-vitro and in-vivo for industrial, research, diagnostic, or therapeutic applications. There also remains a need for an efficient and automated method of manufacturing nanoparticle dispersions to be able to control and maintain characteristics of the nanoparticles such as the poly dispersity of size, charge, magnetism, and functionalization of nanoparticles in the nanoparticle dispersion.

SUMMARY

[0004] Disclosed herein are approaches for addressing various of the problems and shortcomings of the state of the art, as identified above. More particularly, disclosed herein are nanoparticle dispersions used for delivering payload molecules, e.g., to cells and/or biological tissues. [0005] In an aspect, the present disclosure provides a cell-transfection agent including a composition including a de-watered functionalized polydisperse zwitterionic spinel nanoparticle dispersion, where each nanoparticle of the nanoparticle dispersion includes a core with a formula AB2O4 and is functionalized with a combination including free amine groups and thiol reactive groups, where A is a divalent cation and B is a trivalent cation, and where the composition is substantially free of irreversibly aggregated spinel nanoparticles.

[0006] In an aspect, the present disclosure provides a method of manufacturing a celltransfection agent including a nanoparticle dispersion, including the steps of: generating a suspension includes the nanoparticle dispersion, where each nanoparticle of the nanoparticle dispersion includes a core with a formula AB2O4, and where A is a divalent cation, and B is a trivalent cation; coating the core of the each nanoparticle with at least one coating compound; and functionalizing each coated nanoparticle core with a combination including free amine groups and thiol reactive groups to produce the nanoparticle dispersion including a plurality of functionalized nanoparticles.

[0007] In an aspect, the present disclosure provides a cell-transfection agent including a nanoparticle dispersion manufactured by the steps of: generating a suspension including the nanoparticle dispersion having a plurality of nanoparticle cores, where each of the plurality of nanoparticle cores has a formula AB2O4, and where A is a divalent cation, and B is a trivalent cation; coating each of the plurality of nanoparticle cores in the suspension with at least one coating compound; functionalizing each of the plurality of coated nanoparticle cores with a combination including free amine groups and thiol reactive groups to produce a plurality of functionalized nanoparticles; and de-watering the suspension including the plurality of functionalized nanoparticles to output a de-watered composition including a substantially aggregation-free nanoparticle dispersion.

[0008] In an aspect, the present disclosure provides a cell-transfection agent for delivering an effective dose of a payload molecule to cells, the cell-transfection agent including: a composition including a de-watered functionalized polydisperse zwitterionic spinel nanoparticle dispersion, where each nanoparticle of the nanoparticle dispersion includes a core with a formula AB2O4 and functionalized with a combination including amine groups and thiol reactive groups, where A is a divalent cation, and B is a trivalent cation, where the composition is substantially free of irreversibly aggregated spinel nanoparticles, where a charge potential of the composition is configured according to the type of payload molecule; and where the composition is functionalized to target cells expressing exofacial thiols. [0009] In an aspect, the present disclosure provides a therapeutically effective composition including: a cell-transfection agent including: a composition including a de-watered functionalized polydisperse zwitterionic spinel nanoparticle dispersion, where each nanoparticle of the nanoparticle dispersion includes a core with a formula AB2O4 and functionalized with a combination including amine groups and thiol reactive groups, where A is a divalent cation and B is a trivalent cation, where the composition is substantially free of irreversibly aggregated spinel nanoparticles; and a therapeutically effective dose of a payload molecule, where a charge potential of the composition is configured according to the type of payload molecule.

[0010] In an aspect, the present disclosure provides a method of manufacturing a batch of a cell-transfection agent including the steps of: (a) generating a nanoparticle suspension by performing co-precipitation of a plurality input reagents exposed to air, where a total volume of the input reagents is less than 1.25L in a single automated reactor; (b) stabilizing the generated nanoparticle suspension by contacting the nanoparticle suspension with at least one coating agent; (c) purifying the stabilized nanoparticle suspension via a size exclusion column to generate a purified nanoparticle suspension including a plurality of nanoparticles, where each nanoparticle of the plurality of nanoparticles includes an un-agglomerated hydrodynamic diameter between 20nm and 70nm, where the size exclusion column includes a second coating agent and where the at least one coating agent is replaced by the second coating agent during the purification; (d) aminating the purified nanoparticle suspension to generate an aminated nanoparticle suspension configured for functionalization with a heterobifunctional linker; and (e) functionalizing the aminated nanoparticle suspension with the heterobifunctional linker to generate the batch of the cell-transfection agent including a functionalized aminated nanoparticle suspension, where the batch of the cell-transfection agent has a batch size of less than or equal to 1.25 grams.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1A shows a schematic of an exemplary aspect of a functionalized polydisperse zwitterionic spinel nanoparticle of a composition of a cell-transfection agent.

[0012] FIG. IB shows a schematic of an exemplary aspect of a functionalized polydisperse zwitterionic spinel nanoparticle of a composition of a cell-transfection agent.

[0013] FIG. 2 shows a schematic of an exemplary aspect of an automated reactor for manufacturing a functionalized polydisperse zwitterionic spinel nanoparticles of a composition of a cell-transfection agent as described in this specification.

[0014] FIG. 3 shows a schematic of an exemplary embodiment of a plurality of fluid vessels of a reactor as described in FIG. 2.

[0015] FIG. 4 shows a schematic of an exemplary embodiment of the atmospheric control system of a reactor as described in FIG. 2.

[0016] FIG. 5 shows a schematic of an exemplary embodiment of the plurality of fluidic implements of a reactor as described in FIG. 2.

[0017] FIG. 6 shows a schematic of an exemplary embodiment of the temperature regulation apparatus of a reactor as described in FIG. 2.

[0018] FIG. 7 shows a table comparing the exemplary method of manufacturing a batch of the cell-transfection agent via the automated reactor to a method of manufacturing a batch of the cell-transfection agent via a traditional manual method.

[0019] FIG. 8 shows an exemplary graphical representation of size and magnetism changes seen in four batches of a cell-transfection agent comprising the spinel nanoparticles produced using the exemplary method in an automated reactor as described in this specification.

DETAILED DESCRIPTION

[0020] The detailed description set forth below is intended as a description of various configurations of the subject technology and is not intended to represent the only configurations in which the subject technology may be practiced. The appended drawings are incorporated herein and constitute a part of the detailed description. The detailed description may include specific details for the purpose of providing a thorough understanding of the subject technology. However, the subject technology is not limited to the specific details set forth herein and may be practiced without these specific details. In some instances, structures and components are shown in block diagram form in order to avoid obscuring the concepts of the subject technology.

[0021] Described in this specification are spinel nanoparticle dispersions and their manufacture for in vitro and in vivo cell-transfection. Identical individual nanoparticles can “grow” into various sizes by combining together, complicating, for example, the manufacturing process diameter release criteria, transfection efficiency, or pharmacokinetic properties. For both manufacturing and cell-transfection functionality purposes, it is important that nanoparticle preparations are reproducibly agglomerated (where agglomeration is defined as weakly associated particles that can be dissociated with minimal to no disruption to the final product or its manufacturing process), with as little aggregation, (where aggregation is defined as more strongly associated particles that cannot be dissociated without disruption to the final product or its manufacturing process), as possible. The technologies described in this specification, including nanoparticles and methods of manufacture thereof, are designed to reduce the intermolecular attractions between individual nanoparticles thereby reducing the potential for aggregation, while leaving in place cellular transfection efficiencies in an in vitro context or with in vivo pharmacokinetic performance.

[0022] Without wishing to be bound by theory, the goal of manufacturing a functionally relevant aggregation-free nanoparticle concentrated and formulated for in vitro or in vivo celltransfection of cargo in physiologic medium runs contrary to Diffusion-Limited Colloidal Aggregation theory, which assumes that as single particles find each other they will stick together and are unlikely to become again a single unit in the suspension. There remains a need for the design and manufacture of substantially aggregation free commercially relevant nanoparticle suspensions for in vitro or in vivo cell-transfection of cargo for industrial applications, or diagnostic and therapeutic purposes.

[0023] Referring to the drawings, FIG. 1A shows a schematic of an exemplary aspect of a functionalized poly disperse zwitterionic spinel nanoparticle 100 of a composition of a cell- transfection agent as described in the present disclosure. In this aspect, the functionalized nanoparticle 100 comprises a core 101 surrounded by a coat 102 comprising at least one coating agent. In this aspect, the core 101 is further functionalized with a plurality of amine groups 103 and a plurality of thiol reactive groups 104.

[0024] With reference to FIG. 1 A, the core 101 is defined by a formula AB2O4, where A is a divalent cation and B is a trivalent cation. In an example aspect shown in FIG. 1A, the core 101 comprises magnetite (FesCU). In some aspects, the A can include one or more selected from a group consisting of iron, magnesium, zinc, manganese, and nickel, and B can include one or more selected from a group comprising aluminum, chromium, and iron. In some aspects, A can include one or more selected from a group consisting of iron, magnesium, zinc, manganese, and nickel, and B can be iron such that core 101 can have the formula AFe2O4.

[0025] In some aspects, the core 101 can be a spinel core 101 selected from a group including magnetite, maghemite, and other doped variants. In some aspects, the core 101 can be a defective spinel core 101. In some aspects comprising the defective spinel core 101, the defective spinel core 101 can be Mg-doped maghemite with the formula A[Fe³⁺]2O4 where A can be partially vacant or occupied by Fe³⁺ until occupied by Mg²⁺. In some aspects comprising the defective spinel core 101, the defective spinel core 101 can be maghemite with the formula AFe2O3 where A can be vacant or occupied by Fe³⁺.In some aspects, such as the aspect of FIG. 1 A, the spinel nanoparticle can be dewatered.

[0026] In other aspects, the core 101 can be defined by the formula MgFe2O4 (A=Magnesium and B = Iron), where the magnesium and iron in MgFe2C>4 are not in a stoichiometric ratio. In some aspects, the core 101 is defined by the formula MgFe2O4, where the MgFe2<)4 is defective. In other aspects, the core 101 can be or include defective Mg_xFe2O4, where x can be a deficiency factor between 0.05 and 0.5, or between 0.1 and 0.4, or between 0.15 and 0.3, or about 0.2. In some aspects, the core 101 can be or include defective (Mgi-_xFe_x)(Mg_x Fe2-x)O4, where x can be a deficiency factor between 0.05 and 0.5, or between 0.1 and 0.4, or between 0.15 and 0.3, or about 0.2. In some aspects, the core 101 can be or include MgFe2O4 doped with Zn.

[0027] Referring to FIG. 1 A, the core 101 is enveloped by the coat 102 comprising at least one coating agent. The coat 102 can allow for the functionalization of the spinel nanoparticle 100 and can affect the un-agglomeration and the un-aggregation of the spinel nanoparticles 100. In various aspects of the spinel nanoparticle 100, the coat 102 comprises at least one coating agent including dextran, dextran sulfate, citrate, or a combination thereof. In some aspects, the at least one coating agent can further comprise a transient coating compound. In the aspects of spinel nanoparticle 100 with the coat 102 comprising a combination of dextran, dextran sulfate, and citrate, the dextran sulfate and citrate is present in a ratio between 1 : 1 and 1 :20 or is about 1 : 1, or about 1 :2, or about 1 :3, or about 1 :4 or about 1 :5, or about 1 :6 or a about 1 :7 or about 1 :8, or about 1 :9, or about 1 : 10, or about 1 : 15, or about 1 :20, or about 1 :25. In further aspects, , the at least one coating agent can comprise a 99% dextran and 0.5% dextran sulfate comprises 1.2xl0⁵ free amines to create a zwitterionic state maintained at a Zeta potential of between - 30mV and +30mV, or between -20mV and +20mV in the spinel nanoparticle 100. In still further aspects, a spinel nanoparticle 100 can comprises a 10% magnesium core 101, a coat

102 comprising 0.5% dextran sulfate, 1.2xl0⁵ free amines to create a zwitterionic state maintained at a Zeta potential of between -30mV and +30mV, or between -20m V and +20mV. [0028] In some aspects, the spinel nanoparticle 100 with the core 101 and coat 102 can be dialyzed against a polymer solution to further add a polymer soft coat (not shown in FIG. 1 A). The polymer soft coat can further affect the effectiveness of the functionalization of the spinel nanoparticle 100 and affect the un-agglomeration and un-aggregation of the spinel nanoparticles 100. The polymer soft coat can cover the entire coat 102 or can cover only part of the coat 102. The polymer soft coat 102 can also be configured with an amount of polymer that is less than the amount necessary to induce micellization of spinel nanoparticles 100 that have been de-watered. In some aspects, the polymer soft coat can comprise at least one of a block copolymer of poly(ethylene oxide) and polypropylene oxide), poloxamers, pluronics, non-ionic surfactants, glucose polymer, poly(sarcosine) poly (2-oxazoline), or a low molecular weight dextran or dextran sulfate.

[0029] Referring to FIG. 1A, the spinel nanoparticle 100 has a certain core diameter, which measures a diameter of the core 101, and an un-agglom erated hydrodynamic diameter which measures a diameter of the core 101 and the coat 102 surrounding the core 101 combined. In various aspects, the spinel nanoparticle 100 can be configured to have an un-agglom erated hydrodynamic diameter between about 10 nm and 100 nm, e.g., of about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm, or about 45 nm, or about 50 nm, or about 55 nm, or about 60 nm, or about 65 nm, or about 70 nm. The coated spinel nanoparticles 100 can also have a poly dispersity of greater than about 0.03 and less than about 0.3, or about 0.05 or about 0.1 or about 0.15, or about 0.2, or about 0.25.

[0030] Referring to FIG. 1 A, the core 101 is functionalized with the plurality of amine groups

103 and the plurality of thiol reactive groups 104. In this aspect, the plurality of amine groups 103 and the plurality of thiol reactive groups 104 are disposed on the coat 102 comprising dextran, which has been cross-linked. In an example implementation, a cell-transfection agent is comprised of a spinel nanoparticle dispersion functionalized with zwitterionic material, for example, cysteine. In certain embodiments, the spinel nanoparticle dispersion is functionalized with amine groups by crosslinking dextran, for example, with epichlorohydrin. In some aspects, the spinel particle 100 is functionalized with the plurality of amine groups 103 and the plurality of thiol reactive groups 104 in a ratio of about 20: 1, or about 15: 1, or about 10: 1, or about 9: 1, or about 8: 1 or about 7: 1, or about 6: 1, or about 5: 1, or about 4: 1, or about 3: 1, or about 2: 1, or about 1 : 1, or about 1 :20, or about 1 : 15, or about 1 : 10, or about 1 :9, or about 1 :8, or about 1 :7, or about 1 :6, or about 1 :5, or about 1 :4, or about 1 :3, or about 1 :2. In other aspects, the plurality of amine groups 103 and the plurality of thiol reactive groups 104 have a bioavailability of about 10%, or about 25%, or about 50% or about 75% or about 90% of the amine groups, and about 10%, or about 25%, or about 50% or about 75% or about 90% thiol reactive groups.

[0031] The cell-transfection agent can comprise a composition comprising various aspects of the spinel nanoparticles 100, such as the aspects of the spinel nanoparticle 100 presented in FIGS. 1A and IB, and a suspension fluid. The composition can be configured to be substantially free of irreversibly aggregated spinel nanoparticles 100. The suspension fluid can be configured to be substantially RNAase-free. The cell-transfection agent can be configured to deliver an effective dose of one or more payload molecules to cells in vivo or in vitro. The payload molecule can be selected from a group including at least one of short polynucleotides, long polynucleotides, peptides, polypeptides, or small molecules. A charge potential of the composition can be configured according to the type of the payload molecule chosen to be delivered by the cell-transfection agent. In some aspects, the coat 102 of the spinel nanoparticles 100 can be configured to effect a change in the charge potential of the composition to configure the charge potential according to the type of the payload molecule chosen to be delivered by the cell-transfection agent. In further aspects, a spinel nanoparticle 100 can comprise a payload of between 10 and 20 short RNA of 20 nucleotides or less and between 200 and 400 free amines to create a zwitterionic state maintained at a Zeta potential of between -30mV and +30mV or between -20mV and +20mV. In still further aspects, a spinel nanoparticle can comprise a payload of between 1 and 5 short RNA of 120 nucleotides and between 120 to 600 free amines to create a zwitterionic state maintained at a Zeta potential of between -30mV and +30mV or between -20m V and +20mV.

[0032] The functionalized spinel nanoparticles 100 can be configured to target cells expressing exofacial thiols. The exofacial thiols can be selected from a group including CD36, CD206, PDI, CD71, or EGFR. In some aspects, the spinel nanoparticles 100 can be configured to target exofacial thiol expressing cells present in vitro. In some aspects, the spinel nanoparticles 100 can be configured to target exofacial thiol expressing cells present in reticuloendothelial tissue. In some aspects, the spinel nanoparticles 100 can be configured to target exofacial thiol expressing cells present in extra-hepatic tissue. In some aspects, the spinel nanoparticles 100 can be configured to target exofacial thiol expressing cells present in tumor tissue. The celltransfection agent can comprise the composition including the functionalized spinel nanoparticles 100 configured to deliver a payload molecule to cancer cells for a patient having cancer.

[0033] Described in this specification are cell-transfection agents comprised of a suspension of a functionalized polydisperse zwitterionic spinel nanoparticle dispersion of the formula AB2O4, wherein the spinel nanoparticle dispersion has a reduced magnetism, e.g., wherein the magnetic saturation of less than about 5 emu/g, or about 10 emu/g, or about 15 emu/g about 20 emu/g, or about 25 emu/g, or about 30 emu/g, or about 35 emu/g, or about 40 emu/g or about 60emu/g. In some implementations, a cell-transfection agent as described in this specification is comprised of a suspension of a functionalized polydisperse zwitterionic spinel nanoparticle dispersion of the formula AB2O4, wherein the spinel nanoparticle dispersion has reduced magnetism, e.g., wherein the magnetic coercion is less than about 5 Oc, or about 10 Oc, or about 15 Oc, or about 20 Oc, or about 25 Oc, or about 30 Oc, or about 35 Oc, or about 40 Oc, or about 45 Oc, or about 50 Oc or about 60 Oc. In certain implementations, a cell-transfection agent is comprised of a suspension of a functionalized polydisperse zwitterionic spinel nanoparticle dispersion of the formula AB2O4, wherein the spinel nanoparticle dispersion has reduced magnetism, e.g., wherein the magnetic remembrance of less than about 0.05 emu/g, or about 0.1 emu/g, or about 0.15 emu/g about 0.2 emu/g, or about 0.25 emu/g, or about 0.30 emu/g, or about 0.35 emu/g, or about 0.40 emu/g or about 0.6 emu/g.

[0034] Referring now to FIG. IB, FIG. IB shows a schematic of another exemplary aspect of a functionalized poly disperse zwitterionic spinel nanoparticle 100 of a composition of a maghemite based cell-transfection agent as described in the present disclosure. In this aspect, the functionalized nanoparticle 100 comprises a core 101 surrounded by a coat 102 comprising at least one coating agent. In this aspect, the core 101 is further functionalized with a plurality of amine groups 103 and a plurality of thiol reactive groups 104. Unlike the exemplary aspect of FIG. 1A, the spinel nanoparticle of FIG. IB further comprises a de-watering polymer soft coat 105 surrounding the spinel nanoparticle 100.

[0035] Referring now to FIG. 2, FIG. 2 presents an exemplary schematic of an exemplary aspect of an automated reactor 200 for manufacturing a functionalized polydisperse zwitterionic spinel nanoparticles 100 of the composition of the cell-transfection agent. FIG. 2 also presents the primary components of this aspect of the automated reactor 200, which include a plurality of fluid vessels 201, an atmospheric control system 202, a plurality of pumps 203, and a temperature regulation apparatus 204. In various aspects, the automated reactor 200 is configured to control the chemical reactions to manufacture a batch of the cell-transfection agent comprising the spinel nanoparticles 100 with user-defined characteristics.

[0036] In this aspect presented in FIG. 2, the plurality of fluid vessels 201 can be configured to support the plurality of reagents needed for the chemical reactions and the purification steps (described in this specification). The atmospheric control system 202 can be configured to maintain an inert or oxygenated atmosphere under which the chemical reactions take place and to assist in fluid movement of the reagents and the output fluids throughout the automated reactor 200. The plurality of pumps 203 can be configured move fluids without disrupting the stability of the spinel nanoparticles 100 via low-pressure and vacuum sealing. The temperature regulation apparatus 204 can be configured to regulate rapid and uniform temperature changes (heating and cooling) needed by the different chemical reactions in the automated reactor 200. [0037] Referring now to FIG. 3, FIG. 3 presents an exemplary schematic 300 of an exemplary embodiment of the plurality of fluid vessels 201 as described in FIG.2. As described in the sections above, the plurality of fluid vessels 201 can be configured support the chemical reactions in the automated reactor 200, the purification steps in the automated reactor 200, and to also provide sample and waste ports for some or all chemical reactions and purification steps. In this example embodiment, the primary components of the plurality of fluid vessels 201 include a plurality of reagent vessels 301 configured to hold and dispense the plurality of input reagents for the chemical reactions in the automated reactor 200, a plurality of diluent vessels 302 configured to hold and dispense a plurality of diluent fluids configured to dilute the chemical reactions in the automated reactor 200, and a plurality of receptacles 303 configured to receive the plurality of input reagents for the chemical reactions, provide turbulence in the reaction fluid (e.g., shaking, stirring, etc.), and purify the output of the chemical reactions in the automated reactor 200. In a further embodiment, the plurality of fluid vessels 201 can comprise ports used to determine the pH of the chemical reactions, determine the temperature of the chemical reactions, and determine the flow rate between the chemical reactions. In a further embodiment, the plurality of reagent vessels 301 can be configured to hold and dispense the plurality of input reagents. In a further embodiment, the plurality of dispensable input reagent vessels 301 are configured as a kit containing the plurality of input reagents.

[0038] In the embodiment of FIG. 3, the plurality of fluid vessels 201 can further include a plurality of waste receptacles 304 configured to receive and hold waste products generated during the chemical reactions in the automated reactor 200, a plurality of intermediate vessels 305 configured to receive, hold, and/or dispense intermediate output products from the chemical reactions in the automated reactor 200, and a plurality of output vessels 306 configured to receive, hold, and/or dispense the nanoparticle dispersion comprising the spinel nanoparticles 100. In an exemplary embodiment, the plurality of fluid vessel can comprise 201 round or flat-bottomed vessels. In a further exemplary embodiment, the plurality of fluid vessels 201 can comprise two times, or four times, or six times the surface area used by the reaction fluid in the vessel, defined here as head space.

[0039] Referring now to FIG. 4, FIG. 4 presents an exemplary schematic 400 of an exemplary embodiment of the atmospheric control system 202 as described in FIG.2. As described in the sections above, the atmospheric control system 202 can be configured for regulating the atmospheric conditions under which the chemical reactions take place in the automated reactor 200. The atmospheric control system 202 can be configured to control the fluid movement in the automated reactor 200 via control of the atmospheric conditions.

[0040] In the embodiment presented in FIG. 4, The primary components of the atmospheric control system 202 includes a plurality of pinch valves 404 configured to control the movement of the fluid through the automated reactor 200, a plurality of tubing pathways 403 configured to apply, move, and release pressure and fluid are applied via the openings and closings of the plurality of pinch valves 404, a source of compressed gas 402 configured to generate the pressure to move the fluids in the plurality of tubing pathways 403, and a processor 401 electrically coupled to the plurality of pinch valves 404 and configured to open and close the plurality of pinch valves 404 to control the movement of fluids throughout the automated reactor 200. The fluidic system of the automated reactor 200 can be configured such that the pinch valves 404 do not directly contact fluid containing the nanoparticles 100 in order to eliminate or reduce fluidic shear stress on the particles. In some embodiments, the source of compressed gas 402 comprises compressed air or nitrogen gas.

[0041] Referring now to FIG. 5, FIG. 5 presents an exemplary schematic 500 of an exemplary embodiment of the plurality of pumps 203 as described in FIG. 2. As described in the sections above, the plurality of pumps 203 can be configured for propelling fluid movement in the automated reactor 200.

[0042] In the embodiment presented in FIG. 5, the primary components of the plurality of pumps 203 comprises at least one peristaltic pump 501 configured to generate peristaltic pressure to move diluents into a filtration system via reaction vessels in the plurality of fluid vessels 201, at least one vacuum pump 502 configured to generate vacuum pressure to move input reagents into the filtration system via the reaction vessels in the plurality of fluid vessels 201, a plurality of diluent reservoirs 504 configured to contain diluents, and a plurality of closed loop lines 502 configured to fluidically connect the at least peristaltic pump 501, the at least one vacuum pump 502, and the plurality of diluent reservoirs 504.

[0043] Referring now to FIG. 6, FIG. 6 presents an exemplary schematic 600 of an exemplary embodiment of the temperature regulation apparatus 204 as described in FIG.2. As described in the sections above, the temperature regulation apparatus 204 can be configured for controlling the temperature changes of the different chemical reactions in the automated reactor 200. The temperature regulation apparatus 204 can be configured to control both the heating and cooling via fluid exchange, e.g., to provide controlled temperature ramps and/or temperature holds. In this embodiment, the temperature regulation apparatus 204 comprises at least one jacketed reactor 603 configured to control the temperature of the chemical reactions in the automated reactor 200, at least one recirculating chiller 601 configured to cool the chemical reactions, and a plurality of fluid lines 602 comprising temperature-resistant fluid configured to fluidically connect the at least one jacketed reactor 603 to the at least one recirculating chiller 601.

[0044] A batch of cell-transfection agent comprising the composition including the spinel nanoparticles 100 can be manufactured via the automated reactor 200 described in FIGS. 2-6. An exemplary method of manufacturing the cell-transfection agent comprising the spinel nanoparticles 100 includes the steps of generating a nanoparticle suspension by performing coprecipitation of a plurality input reagents exposed to air, stabilizing the generated nanoparticle suspension by contacting the nanoparticle suspension with at least one coating agent, purifying the stabilized nanoparticle suspension via a size exclusion column to generate a purified nanoparticle suspension comprising a plurality of spinel nanoparticles 100, aminating the purified nanoparticle suspension to generate an aminated nanoparticle suspension configured for functionalization with a heterobifunctional linker, and functionalizing the aminated nanoparticle suspension with the heterobifunctional linker to generate the batch of the celltransfection agent comprising a functionalized aminated nanoparticle suspension. In this exemplary method, the total volume of the input reagents can be less than 1.25L with a single automated reactor 200 to produce a batch of the cell-transfection agent with a batch size of less than or equal to 5 grams. In other exemplary methods, the single automated reactor 200 has a total capacity of 1.25L, input reagents can be less than 1.25L to produce a batch of the celltransfection agent with a batch size of less than or equal to 1.25g. In other exemplary methods, the total volume of the input reagents can be more than 5L with more than one automated reactors 200 to produce more than one batch of the cell-transfection agent with each batch having a batch size of less than or equal to 5 grams. In other exemplary methods, the total volume of the input reagents can be more than 1.25L with more than one 5L total capacity automated reactors 200 to produce more than one batch of the cell-transfection agent with each batch having a batch size of less than or equal to 1.25 grams. In some exemplary methods, the method further comprises the step of quenching 100% or between 100% and 5% or between 80% and 20%, or between 60% and 40%, or 50% of the free amines after remaining functionalizing with the linker. In other exemplary methods, the reactor constantly recirculates a greater than 0.5ml of air per 1ml of reaction fluid using ambient or compressed air. In other exemplary methods, the reactor provides for more than one atmosphere in the different reaction vessels of the reactor. In further exemplary methods the reactor supplies air in vessels performing steps in the co-precipitation reaction and nitrogen in the cross-linking and amination reactions.

[0045] In this exemplary method described above, the co-precipitation of the plurality input reagents, comprising at least one divalent cation, can be performed under experimental conditions in the automated reactor 200 including a temperature range between 4°C and 65°C, temperature ramps occurring a rate equal to or greater than 1.5 °C or 2.0°C or 2.5°C per minute, and a time duration of the co-precipitation of no more than 150 minutes. The co-precipitation of the plurality of input reagents can be performed under experimental conditions in the automated reactor 200 including a temperature less than 65°C and at a pH of less than 8. The co-precipitation of the plurality of input reagents can comprise the co-precipitation of divalent and trivalent cations.

[0046] The plurality of input reagents of a process as described in this specification can comprise at least one of divalent cations, a stabilizing agent, and a precipitation agent. In this exemplary method, generating the suspension can comprise using a concentration equal to or less than 5 mg/ml, or 3mg/ml, or Img/ml, or 0.5mg/ml of the suspension comprising the plurality of functionalized spinel nanoparticles 100. The exemplary method can further include the step moving the suspension fluid without (direct) contact with a solid valve or pump element. For example, fluid containing the nanoparticles 100 can be moved (e.g., pushed) by a volume of diluent or other fluid in direct contact with a pump, e.g., a peristaltic pump, or valve. In a further exemplary method, the diluent and reaction fluid are pushed using compressed air. In a further exemplary method, the concentration of the input reagents prior to pushing is decreased two fold, or three fold or four fold. [0047] In this exemplary method described above, the at least one coating compound can comprise dextran, dextran sulfate, or citrate, or a combination thereof. The at least one coating compound can further comprise a transient coating compound. In this exemplary method described above where the at least one coating compound comprises a combination of dextran, dextran sulfate, and citrate, the dextran sulfate and the citrate are present in a ratio between 1 : 1 and 1 :25, or between 1 :5 and 1 : 15, or 1 : 10.

[0048] In this exemplary method described above, aminating the purified nanoparticle suspension comprising the spinel nanoparticles 100 can further comprise the steps of crosslinking purified nanoparticle suspension with epichlorohydrin, aminating the cross-linked and purified nanoparticle suspension with NH4OH, and purifying the aminated, cross-linked, and purified nanoparticle suspension to generate the aminated nanoparticle suspension.

[0049] In this exemplary method described above, the nanoparticle suspensions of each of the steps of the method can be moved throughout the automated reactor 200 via one or more of compressed air pressure sufficient to push a volume of diluent fluid, liquid pressure provided by peristaltic pumps pushing a volume of diluent fluid, and/or negative pressure provided by generation a vacuum pump.

[0050] In this exemplary method described above, the at least one peristaltic pump 501 is configured to be arranged to prevent any pinch points to avoid applying shear stress on the spinel nanoparticles 100 of the nanoparticle suspensions of each of the steps of the exemplary method. The steps of the exemplary method can be carried out under a pressure of 1.5 psi cm² , or 2.5 psi/cm² or 3.5 psi cm² , or 5 psi cm².

[0051] In this exemplary method described above, the batch of the cell-transfection agent is generated during a period of between 3 days and 9 days or between 4 and 8 days, or between 5 and 7 days. In the exemplary method described above, the volume of the batch of the celltransfection agent generated can be 2.5L, or 1.25L or 0.25L or .06L in 3 days, or 4, days, or 5 days. In the exemplary method described above, the size of the batch of the cell-transfection agent generated can be 2.5g, or 1.25g, or 0.25g, or 0.06g. In the exemplary method described above, the size of the batch of the cell-transfection agent generated can be 2.5g, or 1.25g, or 0.25g, or 0.06g, wherein the yield of the batch can be 10% or 15%, or 20%, or 25%, or 30%, or 35%, or 40%, or 45% or 50%.

[0052] In this exemplary method described above, the batch of the cell-transfection agent can be configured for delivering a payload molecule comprising at least one of short polynucleotides, long polynucleotides, peptides, polypeptides or small molecules. [0053] FIG. 7 presents a table comparing the exemplary method (described above) of manufacturing a batch of the cell-transfection agent via the automated reactor 200 to a method of manufacturing a batch of the cell-transfection agent via a traditional manual method. In the table presented in FIG. 7, the method of manufacturing using the automated reactor 200 disclosed in the present disclosure can be advantageous for a plurality of factors associated with manufacturing the batch including input reagents, particle identity, particle size control, surface chemistry, atmosphere control, fluidics, thermal tuning, purification, and/or scale.

[0054] FIG. 8 presents an exemplary graphical representation of size and magnetism changes seen in four batches of cell-transfection agent comprising the spinel nanoparticles 100 produced using the exemplary method in the automated reactor 200. The exemplary method for manufacturing the batches were designed to produce magnetite in a system capable of automation, and included keeping stable inert atmosphere, high concentration of colloids, saturation level of dextran, and high temperature. Experimental conditions were changed such as lowering the wash pH to 10.5 prior to purification and dextran cross-linking with epichlorohydrin, temperature ramping, and reaction hold time. FIG. 8 can be inferred to show that size and magnetism are highly dependent on the speed of temperature ramping and reaction hold time at 80 degrees C with trends of decreasing size on increase in temperature ramp and decrease in hold time.

[0055] The synthesis of metallic nanoparticles is rife with intentional compromises on scale, particle size, nucleation vs growth vs ripening, monodispersity, poly dispersity, surface chemistry, maximum and minimum temperature, temperature ramping speed, pressure, atmosphere, pH, coatings, magnetism, stability, yield, functionality, cargo-type, aggregation vs agglomeration. It then becomes a matter of priority. It can be argued that investigators making and using iron oxide nanoparticles for purposes of interacting with biologic systems have primarily prioritized the magnetism that enables their use in diagnostic imaging and therapeutic applications such as delivery of genetic medicines.

[0056] As presented in FIG. 7, and seen in Example 1, the requirements of scale through automation may conflict with magnetism as a priority. In the technologies of the current disclosure, scale through modular automation is prioritized. This simple change introduces constraints that alter the characteristics of the remaining intentional compromises. It is the iterations between these alterations that have led to unforeseen design and process changes as described in the current disclosure.

[0057] With the various aspects described above, the following examples are intended to further illustrate and not limit the invention. Example 1. Synthesis of functionalized superparamagnetic iron oxide nanoparticles.

[0058] In the attempt to maintain the dual priority of magnetism and scale through automation, four batches of iron oxide nanoparticles were synthesized. The procedure was adapted for automation potential from a publication (Medarova Z. et al. Controlling RNA Expression in Cancer Using Iron Oxide Nanoparticles Detectable by MRI and In Vivo Optical Imaging. Methods Mol Biol. 372: 163-179. 2016.) and briefly summarized below. Superparamagnetic iron oxide nanoparticles of the formula Fe3O4 (magnetite) were synthesized via under an inert atmosphere using the co-precipitation method, wherein the input reagents Fe²⁺, Fe³⁺ and excess dextran were precipitated using NH4OH at high temperatures and purified using PES membrane filtration. After cross-linking the dextran core with epichlorohydrin the nanoparticles are conjugated to the hetero-bifunctional linker N-succinimidyl 3-[2- pyridyldithio]-propionate (SPDP; Thermo Scientific Co., Rockford, IL) by dissolving SPDP in anhydrous DMSO.

[0059] The automation capable reactor used to synthesize the nanoparticles was a stand-alone semi -automated pneumatic laminar pressure-driven reactor with balanced fluid forces and temperature controlled hard piped glass reaction vessels. Each vessel is connected to another with silicon tubing having numbered pinch clamps that control atmospheric pressure input and release, diluent, and reaction fluid movement. To maintain the particle design and synthesis procedure required for magnetism using an apparatus capable of full-automation iterative changes were made in nucleation vs growth vs ripening, monodispersity, poly dispersity, surface chemistry, maximum and minimum temperature, temperature ramping speed, pressure, atmosphere, pH, coatings, stability, and yield.

Example 2. Synthesis of zwitterionic functionalized defective spinel nanoparticles.

[0060] In the attempt to prioritize scale through automation in Example 1, magnetism was deprioritized. Using the automation capable reactor reduced magnetism maghemite nanoparticles as depicted in FIG. 8 were synthesized using a co-precipitation reaction at low-temperature (maximum 60°C), low pH (between 7.5 and 8.0), in an air environment recirculating a greater than 0.5ml of air per 1ml of reaction fluid with temperature ramps of 2.5°C per minute. Input reagents were 60mg of 90% Fe²⁺ and 10% Mg+ in a ratio of 1 : 1 grams to milliliters with water, lOmM citrate. 28% NH4OH was dropwise added in a volume that did not exceed 3ml and stirred at 300 rpm with no vortex. After resting for 30 minutes at 4°C output fluid was PES 0.22 filtered then run over a 1x46cm 4% cross-linked agarose column loaded with 50mg of dextran T10 then cross-linked with 7ml epichlorohydrin for 16 hours at 35°C, and aminated with 4M of 28% NH4OH. Particles were then functionalized with the heterobifunctional linker SPDP, purified, iron levels determined and subsequently dialyzed against a % micelle forming concentration of Pluronic F68 and 0.01% dextran sulfate.

[0061] The automation capable reactor used to synthesize the nanoparticles was the standalone semi-automated pneumatic laminar pressure-driven reactor with balanced fluid forces and temperature controlled hard piped glass reaction vessels used in Example 1, with changes to include the size exclusion column for the first reaction. Each vessel is connected to another with silicon tubing having numbered pinch clamps that control atmospheric pressure input and release, diluent, and reaction fluid movement. Using the apparatus capable of full-automation required iterative changes in reaction volume, input reagents, the quantity input reagents, nucleation vs growth vs ripening, monodispersity, poly dispersity, surface chemistry, maximum and minimum temperature, temperature ramping speed, pressure, atmosphere, pH, coatings, stability, and yield.

Itemized Implementations

Example Products (Items A)

[0062] Al. A cell-transfection agent comprising: a composition comprising a de-watered functionalized polydisperse zwitterionic spinel nanoparticle dispersion, wherein each nanoparticle of the nanoparticle dispersion comprises a core with a formula AB2O4 and is functionalized with a combination comprising free amine groups and thiol reactive groups, wherein A is a divalent cation and B is a trivalent cation, and wherein the composition is substantially free of irreversibly aggregated spinel nanoparticles.

[0063] A2. The cell-transfection agent of item Al, wherein A is one or more selected from a group consisting of iron, magnesium, zinc, manganese, and nickel, and B is one or more of a group consisting of aluminum, chromium, and iron.

[0064] A3. The cell-transfection agent of item Al, wherein A is one or more selected from a group consisting of iron, magnesium, zinc, manganese, and nickel, and B is iron such that each nanoparticle of the nanoparticle dispersion has a core with the formula AFe2O4.

[0065] A4. The cell-transfection agent of item Al, wherein the core is a spinel core selected from a group consisting of magnetite, maghemite, and doped variants.

[0066] A5. The cell-transfection agent of item A4, wherein the spinel core is a defective spinel core.

[0067] A6. The cell-transfection agent of any of the preceding items A, wherein each nanoparticle of the nanoparticle dispersion is configured to have a magnetic saturation of less than 60 emu/g. [0068] A7. The cell-transfection agent of any of the preceding items A, wherein each nanoparticle of the nanoparticle dispersion is configured to have a magnetic coercion of less than 60 Oc.

[0069] A8. The cell transfection agent of any of the preceding items A, wherein each nanoparticle of the nanoparticle dispersion is configured to have a magnetic remembrance of less than 0.6 emu/g.

[0070] A9. The cell transfection agent of any of the preceding items A, wherein the core comprises MgFe2O4, wherein the magnesium and iron in MgFe2O4 are not present in a stoichiometric ratio.

[0071] A10. The cell transfection agent of any one of the preceding items A, wherein the core comprises defective MgFe2C>4.

[0072] Al l. The cell transfection agent of any one of the preceding items, wherein the core comprises defective Mg_xFe2O4 , wherein x is a deficiency factor between 0.05 and 0.5.

[0073] A12. The cell transfection agent of any one of the preceding items A, wherein the core comprises defective (Mgi._xFe_x)(Mg_x Fe2-_x)O4, wherein x is a deficiency factor between 0.05 and 0.5.

[0074] Al 3. The cell transfection agent of any of the preceding items A, wherein the core comprises MgFe2O4 doped with Zn.

[0075] A14. The cell transfection agent of any of the preceding items A, wherein the core has un-agglomerated hydrodynamic diameter of between 5 nm and 50 nm.

[0076] Al 5. The cell transfection agent of any of the preceding items A, wherein the core has a poly dispersity of between 0.03 and 0.3.

[0077] Al 6. The cell transfection agent of any of the preceding items A, wherein the core is coated with at least one coating compound.

[0078] Al 7. The cell transfection agent of item Al 6, wherein the at least one coating compound comprises dextran, dextran sulfate, or citrate, or a combination thereof.

[0079] A18. The cell transfection agent of any of items A16-17, wherein the at least one coating compound comprises a transient coating compound.

[0080] Al 9. The cell transfection agent of item Al 7, wherein the at least one coating compound comprises a combination of dextran, dextran sulfate, and citrate, wherein the dextran sulfate and the citrate are present in a ratio between 1 : 1 and 1 :25.

[0081] A20. The cell transfection agent of any one of the preceding items A, wherein the coated core has an un-agglomerated hydrodynamic diameter between 20 nm and 70 nm. [0082] A21. The cell transfection agent of any of the preceding items A, wherein the coated core is further coated with a polymer soft coat.

[0083] A22. The cell transfection agent of item A21, wherein the polymer soft coat comprises at least one of: block copolymers of polyethylene oxide) and polypropylene oxide), poloxamers, pluronics, non-ionic surfactants, glucose polymer, poly(sarcosine) poly (2- oxazoline), a low molecular weight dextran or a low molecular weight dextran sulfate.

[0084] A23. The cell transfection agent of any one of items A21-22, wherein the core is soft coated with an amount of polymer that is less than the amount necessary to induce micellization of the de-watered cell transfection agent.

[0085] A24. The cell transfection agent of any of the preceding items, wherein each nanoparticle of the nanoparticle dispersion is functionalized with free amine groups and thiol reactive groups in a ratio between 1 : 1 and 20: 1.

[0086] A25. The cell transfection agent of item A24, wherein between 10% and 90% of the amine groups are bioavailable.

[0087] A26. The cell transfection agent of any one of the preceding items A, wherein the composition is functionalized to target cells expressing exofacial thiols.

[0088] A27. The cell transfection agent of any one of the preceding items A, wherein the composition is functionalized to target exofacial expressing cells present in extra-hepatic tissues.

[0089] A28. The cell transfection agent of any one of the preceding items, wherein the composition is functionalized to target exofacial thiol expressing cells present in reticuloendothelial tissue.

[0090] A29. The cell transfection agent of any one of the preceding items A, wherein the composition is functionalized to target exofacial thiol expressing cells present in tumor tissue. [0091] A30. The cell transfection agent of any one of items A26-29, wherein the exofacial thiols comprise a group including CD36, PDI, CD71, or EGFR.

[0092] A31. The cell transfection agent of any of the preceding items A, comprising the composition and a suspension fluid.

[0093] A32. The cell transfection agent of item A31, wherein the suspension fluid is substantially RNAase-free.

Example Methods of Manufacturing (Items B)

[0094] Bl. A method of manufacturing a cell-transfection agent comprising a nanoparticle dispersion, comprising the steps of: generating a suspension comprising the nanoparticle dispersion, wherein each nanoparticle of the nanoparticle dispersion comprises a core with a formula AB2O4, and wherein A is a divalent cation, and B is a trivalent cation; coating the core of each nanoparticle with at least one coating compound; and functionalizing each coated nanoparticle core with a combination comprising free amine groups and thiol reactive groups to produce the nanoparticle dispersion comprising a plurality of functionalized nanoparticles.

[0095] B2. The method of item Bl, wherein A is one or more selected from a group consisting of iron, magnesium, zinc, manganese, and nickel, and B is one or more of a group consisting of aluminum, chromium, and iron.

[0096] B3. The method of item Bl, wherein A is one or more selected from a group consisting of iron, magnesium, zinc, manganese, and nickel, and B is iron such that each nanoparticle of the nanoparticle dispersion has a core with the formula AFe2O4.

[0097] B4. The method of item Bl, wherein the core is a spinel core or a defective spinel core selected from the group consisting of magnetite, maghemite, and doped variants.

[0098] B5. The method of item B4, wherein the spinel core is a defective spinel core.

[0099] B6. The method of any of the preceding items B, wherein each nanoparticle of the nanoparticle dispersion is configured to have a magnetic saturation of less than 60 emu/g.

[0100] B7. The method of any of the preceding items B, wherein each nanoparticle of the nanoparticle dispersion is configured to have a magnetic coercion of less than 60 Oc.

[0101] B8. The method of any of the preceding items B, wherein each nanoparticle of the nanoparticle dispersion is configured to have a magnetic remembrance of less than 0.6 emu/g. [0102] B9. The method of any of the preceding items B, wherein the core comprises MgFe2O4, wherein the magnesium and iron in MgFe2O4 are not present in a stoichiometric ratio.

[0103] B10. The method of any one of the preceding items B, wherein the core comprises defective MgFe2O4.

[0104] Bl l. The method of any one of the preceding items B, wherein the core comprises defective Mg_xFe2O4 , wherein x is a deficiency factor between 0.05 and 0.5.

[0105] B12. The method of any one of the preceding items B, wherein the core comprises defective (Mgi-_xFe_x)(Mg_xFe2-x)O4, wherein x is a deficiency factor between 0.05 and 0.5.

[0106] B13. The method of any of the preceding items B, wherein the core comprises MgFe2O4 doped with Zn.

[0107] B14. The method of any of the preceding items B, wherein the core has unagglomerated hydrodynamic diameter of between 5 nm and 50 nm.

[0108] Bl 5. The method of any of the preceding items B, wherein the core has a poly dispersity of between 0.03 and 0.3. [0109] Bl 6. The method of any of the preceding items B, wherein the core is coated with at least one coating compound.

[0110] Bl 7. The method of item Bl 6, wherein the at least one coating compound comprises dextran, dextran sulfate, or citrate, or a combination thereof.

[OHl] Bl 8. The method of any of items Bl 6- 17, wherein the at least one coating compound comprises a transient coating compound.

[0112] B19. The method of item B17, wherein the at least one coating compound comprises a combination of dextran, dextran sulfate, and citrate, wherein the dextran sulfate and the citrate are present in a ratio between 1 : 1 and 1 :25.

[0113] B20. The method of any one of the preceding items B, wherein the coated core has an un-agglomerated hydrodynamic diameter between 20 nm and 70 nm.

[0114] B21. The method of any of the preceding items B, wherein the coated core is further coated with a polymer soft coat.

[0115] B22. The method of item B21, wherein the polymer soft coat comprises at least one of: block copolymers of poly(ethylene oxide) and polypropylene oxide), poloxamers, pluronics, non-ionic surfactants, glucose polymer, poly(sarcosine) poly (2-oxazoline), a low molecular weight dextran or a low molecular weight dextran sulfate.

[0116] B23. The method of any one of items B21-22, wherein the core is soft coated with an amount of polymer that is less than the amount necessary to induce micellization of the dewatered cell transfection agent.

[0117] B24. The method of any of the preceding items B, wherein each nanoparticle of the nanoparticle dispersion is functionalized with free amine groups and thiol reactive groups in a ratio between 1 : 1 and 20: 1.

[0118] B25. The method of item B24, wherein between 10% and 90% of the amine groups are bioavailable.

[0119] B26. The method of any one of the preceding items B, wherein the composition is functionalized to target cells expressing exofacial thiols.

[0120] B27. The method of any one of the preceding items B, wherein the composition is functionalized to target exofacial thiol expressing cells present in extra-hepatic tissues.

[0121] B28. The method of any one of the preceding items B, wherein the composition is functionalized to target exofacial thiol expressing cells present in reticuloendothelial tissue.

[0122] B29. The method of any one of the preceding items B, wherein the composition is functionalized to target exofacial expressing cells present in tumor tissue. [0123] B30. The method of any one of items B26-29, wherein the exofacial thiols comprise a group including CD36, PDI, CD71, or EGFR.

[0124] B31. The method of any of the preceding items B, comprising the composition and a suspension fluid.

[0125] B32. The method of item B31, wherein the suspension fluid is substantially RNAase- free.

[0126] B33. The method of any of the preceding items B, wherein the steps of the method are carried out within a closed and automated system.

[0127] B34. The method of any of the preceding items B, further comprising moving the suspension fluid without contact with a solid valve or pump element.

[0128] B35. The method of any of the preceding items B, wherein generating the suspension comprising the nanoparticle dispersion further comprises the co-precipitation of divalent and trivalent cations.

[0129] B36. The method of item B35, wherein the co-precipitation occurs under experimental conditions including: a pH of less than 8; and a temperature less than 65°C.

[0130] B37. The method of any of the preceding items B, wherein generating the suspension comprises using a concentration equal to or less than 5 milligrams per milliliter of the suspension comprising the plurality of functionalized nanoparticles.

[0131] B38. The method of any of items B35-37, wherein temperature ramps during the co- precipitation occur at a rate equal to or greater than 2.0°C /minute.

Example Transfection Agents (Items C)

[0132] Cl. A cell-transfection agent comprising a nanoparticle dispersion manufactured by the steps of: generating a suspension comprising the nanoparticle dispersion having a plurality of nanoparticle cores, wherein each of the plurality of nanoparticle cores has a formula AB2O4, and wherein A is a divalent cation, and B is a trivalent cation; coating each of the plurality of nanoparticle cores in the suspension with at least one coating compound; functionalizing each of the plurality of coated nanoparticle cores with a combination comprising free amine groups and thiol reactive groups to produce a plurality of functionalized nanoparticles; and dewatering the suspension comprising the plurality of functionalized nanoparticles to output a dewatered composition comprising a substantially aggregation-free nanoparticle dispersion.

[0133] C2. The cell-transfection agent of item Cl, wherein A is one or more selected from a group consisting of iron, magnesium, zinc, manganese, and nickel, and B is one or more of a group consisting of aluminum, chromium, and iron. [0134] C3. The cell-transfection agent of item Cl, wherein A is one or more selected from a group consisting of iron, magnesium, zinc, manganese, and nickel, and B is iron such that each nanoparticle of the nanoparticle dispersion has a core with the formula AFe2O4.

[0135] C4. The cell-transfection agent of item Cl, wherein the core is a spinel core or a defective spinel core selected from the group consisting of magnetite, maghemite, and doped variants.

[0136] C5. The cell-transfection agent of item C4, wherein the spinel core is a defective spinel core.

[0137] C6. The cell-transfection agent of any of the preceding items C, wherein each nanoparticle of the nanoparticle dispersion is configured to have a magnetic saturation of less than 60 emu/g.

[0138] C7. The cell-transfection agent of any of the preceding items C, wherein each nanoparticle of the nanoparticle dispersion is configured to have a magnetic coercion of less than 60 Oc.

[0139] C8. The cell transfection agent of any of the preceding items C, wherein each nanoparticle of the nanoparticle dispersion is configured to have a magnetic remembrance of less than 0.6 emu/g.

[0140] C9. The cell transfection agent of any of the preceding items C, wherein the core comprises MgFe2O4 , wherein the magnesium and iron in MgFe2O4 are not present in a stoichiometric ratio.

[0141] CIO. The cell transfection agent of any one of the preceding items C, wherein the core comprises defective MgFe2C>4.

[0142] Cl 1. The cell transfection agent of any one of the preceding items C , wherein the core comprises defective Mg_xFe2O4, wherein x is a deficiency factor between 0.05 and 0.5.

[0143] C12. The cell transfection agent of any one of the preceding items C, wherein the core comprises defective (Mgi-_xFe_x)(Mg_x Fe2-x)O4„ wherein x is a deficiency factor between 0.05 and 0.5.

[0144] C13. The cell transfection agent of any of the preceding items C, wherein the core comprises MgFe2O4 doped with Zn.

[0145] C14. The cell transfection agent of any of the preceding items C, wherein the core has un-agglomerated hydrodynamic diameter of between 5 nm and 50 nm.

[0146] Cl 5. The cell transfection agent of any of the preceding items C, wherein the core has a poly dispersity of between 0.03 and 0.3. [0147] Cl 6. The cell transfection agent of any of the preceding items C, wherein the core is coated with at least one coating compound.

[0148] Cl 7. The cell transfection agent of item Cl 6, wherein the at least one coating compound comprises dextran, dextran sulfate, or citrate, or a combination thereof.

[0149] Cl 8. The cell transfection agent of any of items Cl 6- 17, wherein the at least one coating compound comprises a transient coating compound.

[0150] Cl 9. The cell transfection agent of item Cl 7, wherein the at least one coating compound comprises a combination of dextran, dextran sulfate, and citrate, wherein the dextran sulfate and the citrate are present in a ratio between 1 : 1 and 1 :25.

[0151] C20. The cell transfection agent of any one of the preceding items C, wherein the coated core has an un-agglomerated hydrodynamic diameter between 20 nm and 70 nm.

[0152] C21. The cell transfection agent of any of the preceding items C, wherein the coated core is further coated with a polymer soft coat.

[0153] C22. The cell transfection agent of item C21, wherein the polymer soft coat comprises at least one of: block copolymers of polyethylene oxide) and polypropylene oxide), poloxamers, pluronics, non-ionic surfactants, glucose polymer, poly(sarcosine) poly (2- oxazoline), a low molecular weight dextran or a low molecular weight dextran sulfate.

[0154] C23. The cell transfection agent of any one of items C21-22, wherein the core is soft coated with an amount of polymer that is less than the amount necessary to induce micellization of the de-watered cell transfection agent.

[0155] C24. The cell transfection agent of any of the preceding items C, wherein each nanoparticle of the nanoparticle dispersion is functionalized with free amine groups and thiol reactive groups in a ratio between 1 : 1 and 20: 1.

[0156] C25. The cell transfection agent of item C24, wherein between 10% and 90% of the amine groups are bioavailable.

[0157] C26. The cell transfection agent of any one of the preceding items C, wherein the composition is functionalized to target cells expressing exofacial thiols.

[0158] C27. The cell transfection agent of any one of the preceding items C, wherein the composition is functionalized to target exofacial thiol expressing cells present in extra-hepatic tissues.

[0159] C28. The cell transfection agent of any one of the preceding items C, wherein the composition is functionalized to target exofacial expressing thiol cells present in reticuloendothelial tissue. [0160] C29. The cell transfection agent of any one of the preceding items C, wherein the composition is functionalized to target exofacial thiol expressing cells present in tumor tissue. [0161] C30. The cell transfection agent of any one of items C26-29, wherein the exofacial thiols comprise a group including CD36, PDI, CD71, or EGFR.

[0162] C31. The cell transfection agent of any of the preceding items C, comprising the composition and a suspension fluid.

[0163] C32. The cell transfection agent of item C31, wherein the suspension fluid is substantially RNAase-free.

Therapeutic Example 1 (Items D)

[0164] DI. A cell-transfection agent for delivering an effective dose of a payload molecule to cells, the cell-transfection agent comprising: a composition comprising a de-watered functionalized polydisperse zwitterionic spinel nanoparticle dispersion, wherein each nanoparticle of the nanoparticle dispersion comprises a core with a formula AB2O4 and functionalized with a combination comprising amine groups and thiol reactive groups, wherein A is a divalent cation, and B is a trivalent cation, wherein the composition is substantially free of irreversibly aggregated spinel nanoparticles, wherein a charge potential of the composition is configured according to the type of payload molecule; and wherein the composition is functionalized to target cells expressing exofacial thiols.

[0165] D2. The cell transfection agent of item DI, wherein the core is coated with at least one coating compound.

[0166] D3. The cell transfection agent of item D2, wherein the at least one coating compound comprises dextran, dextran sulfate, or citrate, or a combination thereof

[0167] D4. The cell transfection agent of any of items D2-3, wherein the at least one coating compound comprises a transient coating compound.

[0168] D5. The cell transfection agent of item D3, wherein the at least one coating compound comprises a combination of dextran, dextran sulfate, and citrate, wherein the dextran sulfate and the citrate are present in a ratio between 1 : 1 and 1 :25.

[0169] D6. The cell transfection agent of any one of the preceding items D, wherein the coated core has an un-agglomerated hydrodynamic diameter between 20 nm and 70 nm.

[0170] D7. The cell transfection agent of any of the preceding items D, wherein the coated core is further coated with a polymer soft coat.

[0171] D8. The cell transfection agent of item D7, wherein the polymer soft coat comprises at least one of: block copolymers of polyethylene oxide) and polypropylene oxide), poloxamers, pluronics, non-ionic surfactants, glucose polymer, poly(sarcosine) poly (2-oxazoline), a low molecular weight dextran or a low molecular weight dextran sulfate.

[0172] D9. The cell-transfection agent of any of the preceding items D , wherein the coating of the core of each nanoparticle is configured to effect the charge potential.

[0173] DIO. The cell transfection agent of any of the preceding items D , wherein the exofacial thiols comprise a group including CD36, PDI, CD71, or EGFR.

[0174] Dl l. The cell transfection agent of any of the preceding items D , wherein the payload molecule comprises at least one of short polynucleotides, long polynucleotides, peptides, polypeptides, or small molecules.

[0175] D12. The cell transfection agent of any one of the preceding items D , wherein the composition is functionalized to target cells expressing exofacial thiols.

[0176] D13. The cell transfection agent of any one of the preceding items D , wherein the composition is functionalized to target exofacial thiol expressing cells present in extra-hepatic tissues.

[0177] D14. The cell transfection agent of any one of the preceding items D , wherein the composition is functionalized to target exofacial thiol expressing cells present in reticuloendothelial tissue.

[0178] DI 5. The cell transfection agent of any one of the preceding items D , wherein the composition is functionalized to target exofacial thiol expressing cells present in tumor tissue. [0179] DI 6. The cell transfection agent of any of the preceding items D, wherein the celltransfection agent comprises a therapeutically effective amount of the payload for delivery to a patient.

[0180] DI 7. The cell transfection agent of item DI 6, wherein the patient is a patient having cancer.

Therapeutic Example 2 (Items E)

[0181] El. A therapeutically effective composition comprising: a cell-transfection agent comprising: a composition comprising a de-watered functionalized polydisperse zwitterionic spinel nanoparticle dispersion, wherein each nanoparticle of the nanoparticle dispersion comprises a core with a formula AB2O4 and functionalized with a combination comprising amine groups and thiol reactive groups, wherein A is a divalent cation and B is a trivalent cation, wherein the composition is substantially free of irreversibly aggregated spinel nanoparticles; and a therapeutically effective dose of a payload molecule, wherein a charge potential of the composition is configured according to the type of payload molecule. [0182] E2. The therapeutically effective composition of item El, wherein the core is coated with at least one coating compound.

[0183] E3. The therapeutically effective composition of item E2, wherein the at least one coating compound comprises dextran, dextran sulfate, or citrate, or a combination thereof [0184] E4. The therapeutically effective composition of any of items E2-3, wherein the at least one coating compound comprises a transient coating compound.

[0185] E5. The therapeutically effective composition of item E3, wherein the at least one coating compound comprises a combination of dextran, dextran sulfate, and citrate, wherein the dextran sulfate and the citrate are present in a ratio between 1 : 1 and 1 :25.

[0186] E6. The therapeutically effective composition of any one of the preceding items E, wherein the coated core has an un-agglomerated hydrodynamic diameter between 20 nm and 70 nm.

[0187] E7. The therapeutically effective composition of any of the preceding items E, wherein the coated core is further coated with a polymer soft coat.

[0188] E8. The therapeutically effective composition of item E7, wherein the polymer soft coat comprises at least one of: block copolymers of poly(ethylene oxide) and polypropylene oxide), poloxamers, pluronics, non-ionic surfactants, glucose polymer, poly(sarcosine) poly (2- oxazoline), a low molecular weight dextran or a low molecular weight dextran sulfate.

[0189] E9. The therapeutically effective composition of any of the preceding items E, wherein the coating of the core of each nanoparticle is configured to effect the charge potential.

[0190] E10. The therapeutically effective composition of any of the preceding items E, wherein the exofacial thiols comprise a group including CD36, PDI, CD71, or EGFR.

[0191] El 1. The therapeutically effective composition of any of the preceding items E, wherein the payload molecule comprises at least one of short polynucleotides, long polynucleotides, peptides, polypeptides, or small molecules.

Example Methods of Manufacturing a Batch of the Cell-Transfection Agent (Items F)

[0192] Fl . A method of manufacturing a batch of a cell-transfection agent comprising the steps of: (a) generating a nanoparticle suspension by performing co-precipitation of a plurality input reagents exposed to air, wherein a total volume of the input reagents is less than 1.25L in a single automated reactor; (b) stabilizing the generated nanoparticle suspension by contacting the nanoparticle suspension with at least one coating agent; (c) purifying the stabilized nanoparticle suspension via a size exclusion column to generate a purified nanoparticle suspension comprising a plurality of nanoparticles, wherein each nanoparticle of the plurality of nanoparticles comprises an un-agglomerated hydrodynamic diameter between 20nm and 70nm, wherein the size exclusion column comprises a second coating agent and wherein the at least one coating agent is replaced by the second coating agent during the purification; (d) aminating the purified nanoparticle suspension to generate an aminated nanoparticle suspension configured for functionalization with a heterobifunctional linker; and (e) functionalizing the aminated nanoparticle suspension with the heterobifunctional linker to generate the batch of the cell-transfection agent comprising a functionalized aminated nanoparticle suspension, wherein the batch of the cell-transfection agent has a batch size of less than or equal to 1.25 grams.

[0193] F2. The method of item Fl, wherein the co-precipitation of the plurality of input reagents comprises at least one divalent cation and is performed in a chemical reaction vessel under experimental conditions including: a head space of four times surface area used by the plurality of input reagents in the in the chemical reaction vessel; a temperature range between 4°C and 60°C; a rate of change of temperature no less than 2.0°C per minute; constantly recirculation of air at a rate greater than 0.5ml per 1ml of the plurality of input reagents; and a time duration of the co-precipitation no more than 150 minutes.

[0194] F3. The method of any of the preceding items F, wherein the at least one coating compound comprises dextran, dextran sulfate, or citrate, or a combination thereof.

[0195] F4. The method of any of the preceding items F, wherein the at least one coating compound comprises a transient coating compound.

[0196] F5. The method of item F3, wherein the at least one coating compound comprises a combination of dextran, dextran sulfate, and citrate, wherein the dextran sulfate and the citrate are present in a ratio between 1 : 1 and 1 :25.

[0197] F6. The method of any of the preceding items F, wherein aminating the purified nanoparticle suspension further comprises the steps of cross-linking purified nanoparticle suspension with epichlorohydrin; aminating the cross-linked and purified nanoparticle suspension with NH4OH; and purifying the aminated, cross-linked, and purified nanoparticle suspension to generate the aminated nanoparticle suspension configured for functionalization with the heterobifunctional linker.

[0198] F7. The method of any of the preceding items F, wherein the plurality of input reagents of step (a) comprises at least one of divalent cations, a stabilizing agent, and a precipitation agent.

[0199] F8. The method of any of the preceding items F, wherein the heterobifunctional linker is SPDP. [0200] F9. The method of any of the preceding items F, wherein the nanoparticle suspensions of each of the steps of the method are moved through one or more conduits via one or more of: compressed air pressure sufficient to push a volume of diluent fluid; liquid pressure provided by peristaltic pumps pushing the volume of diluent fluid; and negative pressure provided by generation a vacuum pump.

[0201] F10. The method of item F9, wherein the peristaltic pumps are configured to be arranged to prevent any pinch points to avoid applying shear stress on the nanoparticle suspensions of each of the steps of the methods.

[0202] Fl 1. The method of any of the preceding items F, wherein the steps of the method are carried out under a pressure in a range between 1.5 psi/cm² to 5 psi/cm².

[0203] F12. The method of any of the preceding items F, wherein the batch of the celltransfection agent is generated in between 3 days and 9 days.

[0204] F13. The method of any of the preceding items F, wherein the batch size of the batch of the cell-transfection agent generated is 5 grams and the batch of the cell-transfection agent is generated in 3 days.

[0205] F14. The method of any of the preceding items F, wherein the batch of the celltransfection agent is configured for delivering a payload molecule comprising at least one of short polynucleotides, long polynucleotides, peptides, polypeptides or small molecules.

[0206] Fl 5. The method of any of the preceding items F, wherein more than one automated reactor is used to generate more than one batch of the cell-transfection agent to generate a total batch size larger than 1.25 grams.

[0207] Fl 6. The method of any of the preceding items F, wherein the steps (b) and (c) are configured to occur simultaneously.

[0208] Fl 7. The method of any of the preceding items F, wherein the second coating agent is selected from a group comprising dextran or dextran sulfate.

[0209] Fl 8. The method of any of the preceding items F, further comprising the step of: quenching between 100% and 5% of the free amines after remaining functionalizing with the linker.

[0210] A reference to an element in the singular is not intended to mean one and only one unless specifically so stated, but rather one or more. For example, “a” module may refer to one or more modules. An element proceeded by “a,” “an,” “the,” or “said” does not, without further constraints, preclude the existence of additional same elements.

[0211] Phrases such as an aspect, the aspect, another aspect, some aspects, one or more aspects, an implementation, the implementation, another implementation, some implementations, one or more implementations, an embodiment, the embodiment, another embodiment, some embodiments, one or more embodiments, a configuration, the configuration, another configuration, some configurations, one or more configurations, the subject technology, the disclosure, the present disclosure, other variations thereof and alike are for convenience and do not imply that a disclosure relating to such phrase(s) is essential to the subject technology or that such disclosure applies to all configurations of the subject technology. A disclosure relating to such phrase(s) may apply to all configurations, or one or more configurations. A disclosure relating to such phrase(s) may provide one or more examples. A phrase such as an aspect or some aspects may refer to one or more aspects and vice versa, and this applies similarly to other foregoing phrases.

[0212] It is understood that the specific order or hierarchy of steps, operations, or processes disclosed is an illustration of exemplary approaches. Unless explicitly stated otherwise, it is understood that the specific order or hierarchy of steps, operations, or processes may be performed in different order. Some of the steps, operations, or processes may be performed simultaneously. The accompanying method claims, if any, present elements of the various steps, operations or processes in a sample order, and are not meant to be limited to the specific order or hierarchy presented. These may be performed in serial, linearly, in parallel or in different order. It may be understood that the described instructions, operations, and systems can generally be integrated together in a single software/hardware product or packaged into multiple software/hardware products.

[0213] The disclosure is provided to enable any person skilled in the art to practice the various aspects described herein. In some instances, well-known structures and components are shown in block diagram form in order to avoid obscuring the concepts of the subject technology. The disclosure provides various examples of the subject technology, and the subject technology is not limited to these examples. Various modifications to these aspects will be readily apparent to those skilled in the art, and the principles described herein may be applied to other aspects. [0214] All structural and functional equivalents to the elements of the various aspects described throughout the disclosure that are known or later come to be known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the claims. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0215] The various illustrative logical blocks, modules, circuits, and algorithm steps described in connection with the embodiments disclosed herein may be implemented as electronic hardware, computer software, or combinations of both. To clearly illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules, circuits, and steps have been described above generally in terms of their functionality. Whether such functionality is implemented as hardware or software depends upon the particular application and design constraints imposed on the overall system. Skilled artisans may implement the described functionality in varying ways for each particular application, but such implementation decisions should not be interpreted as causing a departure from the scope of the present invention.

[0216] The hardware used to implement the various illustrative logics, logical blocks, modules, and circuits described in connection with the aspects disclosed herein may be implemented or performed with a general purpose processor, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. A general -purpose processor may be a microprocessor, but, in the alternative, the processor may be any conventional processor, controller, microcontroller, or state machine. A processor may also be implemented as a combination of computing devices, e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration. Alternatively, some steps or methods may be performed by circuitry that is specific to a given function.

[0217] In one or more exemplary aspects, the functions described may be implemented in hardware, software, firmware, or any combination thereof. If implemented in software, the functions may be stored as one or more instructions or code on a non-transitory computer- readable medium or non-transitory processor-readable medium. The steps of a method or algorithm disclosed herein may be embodied in a processor-executable software module which may reside on a non-transitory computer-readable or processor-readable storage medium. Non- transitory computer-readable or processor-readable storage media may be any storage media that may be accessed by a computer or a processor. By way of example but not limitation, such non-transitory computer-readable or processor-readable media may include RAM, ROM, EEPROM, FLASH memory, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium that may be used to store desired program code in the form of instructions or data structures and that may be accessed by a computer. Disk and disc, as used herein, includes compact disc (CD), laser disc, optical disc, digital versatile disc (DVD), floppy disk, and Blu-ray disc where disks usually reproduce data magnetically, while discs reproduce data optically with lasers. Combinations of the above are also included within the scope of non-transitory computer-readable and processor-readable media. Additionally, the operations of a method or algorithm may reside as one or any combination or set of codes and/or instructions on a non-transitory processor-readable medium and/or computer-readable medium, which may be incorporated into a computer program product.

[0218] The title, background, brief description of the drawings, abstract, and drawings are hereby incorporated into the disclosure and are provided as illustrative examples of the disclosure, not as restrictive descriptions. It is submitted with the understanding that they will not be used to limit the scope or meaning of the claims. In addition, in the detailed description, it can be seen that the description provides illustrative examples and the various features are grouped together in various implementations for the purpose of streamlining the disclosure. The method of disclosure is not to be interpreted as reflecting an intention that the claimed subject matter requires more features than are expressly recited in each claim.

Claims

CLAIMS What is claimed is:

1. A cell-transfection agent comprising: a composition comprising a de-watered functionalized polydisperse zwitterionic spinel nanoparticle dispersion, wherein each nanoparticle of the nanoparticle dispersion comprises a core with a formula AB2O4 and is functionalized with a combination comprising free amine groups and thiol reactive groups, wherein A is a divalent cation and B is a trivalent cation, and wherein the composition is substantially free of irreversibly aggregated spinel nanoparticles.

2. The cell-transfection agent of claim 1, wherein A is one or more selected from a group consisting of iron, magnesium, zinc, manganese, and nickel, and B is one or more of a group consisting of aluminum, chromium, and iron.

3. The cell-transfection agent of claim 1, wherein A is one or more selected from a group consisting of iron, magnesium, zinc, manganese, and nickel, and B is iron such that each nanoparticle of the nanoparticle dispersion has a core with the formula AFe2O4.

4. The cell-transfection agent of claim 1, wherein the core is a spinel core selected from a group consisting of magnetite, maghemite, and doped variants.

5. The cell-transfection agent of claim 4, wherein the spinel core is a defective spinel core.

6. The cell-transfection agent of any of the preceding claims, wherein each nanoparticle of the nanoparticle dispersion is configured to have a magnetic saturation of less than 60 emu/g.

7. The cell-transfection agent of any of the preceding claims, wherein each nanoparticle of the nanoparticle dispersion is configured to have a magnetic coercion of less than 60 Oc.

8. The cell transfection agent of any of the preceding claims, wherein each nanoparticle of the nanoparticle dispersion is configured to have a magnetic remembrance of less than 0.6 emu/g.

9. The cell transfection agent of any of the preceding claims, wherein the core comprises MgFe2C>4, wherein the magnesium and iron in MgFe2O4 are not present in a stoichiometric ratio.

10. The cell transfection agent of any one of the preceding claims, wherein the core comprises defective MgFe2O4.

11. The cell transfection agent of any one of the preceding claims, wherein the core comprises defective Mg_xFe2O4 , wherein x is a deficiency factor between 0.05 and 0.5.

12. The cell transfection agent of any one of the preceding claims, wherein the core comprises defective (Mgi-_xFe_x)(Mg_xFe2-x)O4, wherein x is a deficiency factor between 0.05 and 0.5.

13. The cell transfection agent of any of the preceding claims, wherein the core comprises MgFe2C>4 doped with Zn.

14. The cell transfection agent of any of the preceding claims, wherein the core has unagglomerated hydrodynamic diameter of between 5 nm and 50 nm.

15. The cell transfection agent of any of the preceding claims, wherein the core has a poly dispersity of between 0.03 and 0.3.

16. The cell transfection agent of any of the preceding claims, wherein the core is coated with at least one coating compound.

17. The cell transfection agent of claim 16, wherein the at least one coating compound comprises dextran, dextran sulfate, or citrate, or a combination thereof

18. The cell transfection agent of any of claims 16-17, wherein the at least one coating compound comprises a transient coating compound.

19. The cell transfection agent of claim 17, wherein the at least one coating compound comprises a combination of dextran, dextran sulfate, and citrate, wherein the dextran sulfate and the citrate are present in a ratio between 1 : 1 and 1 :25.

20. The cell transfection agent of any one of the preceding claims, wherein the coated core has an un-agglomerated hydrodynamic diameter between 20 nm and 70 nm.

21. The cell transfection agent of any of the preceding claims, wherein the coated core is further coated with a polymer soft coat.

22. The cell transfection agent of claim 21, wherein the polymer soft coat comprises at least one of: block copolymers of polyethylene oxide) and polypropylene oxide), poloxamers, pluronics, non-ionic surfactants, glucose polymer, poly(sarcosine) poly (2-oxazoline), a low molecular weight dextran or a low molecular weight dextran sulfate.

23. The cell transfection agent of any one of claims 21-22, wherein the core is soft coated with an amount of polymer that is less than the amount necessary to induce micellization of the dewatered cell transfection agent.

24. The cell transfection agent of any of the preceding claims, wherein each nanoparticle of the nanoparticle dispersion is functionalized with free amine groups and thiol reactive groups in a ratio between 1 : 1 and 20: 1.

25. The cell transfection agent of claim 24, wherein between 10% and 90% of the amine groups are bioavailable.

26. The cell transfection agent of any one of the preceding claims, wherein the composition is functionalized to target cells expressing exofacial thiols.

27. The cell transfection agent of any one of the preceding claims, wherein the composition is functionalized to target exofacial expressing cells present in extra-hepatic tissues.

28. The cell transfection agent of any one of the preceding claims, wherein the composition is functionalized to target exofacial thiol expressing cells present in reticuloendothelial tissue.

29. The cell transfection agent of any one of the preceding claims, wherein the composition is functionalized to target exofacial thiol expressing cells present in tumor tissue.

30. The cell transfection agent of any one of claims 26-29, wherein the exofacial thiols comprise a group including CD36, PDI, CD71, or EGFR.

31. The cell transfection agent of any of the preceding claims, comprising the composition and a suspension fluid.

32. The cell transfection agent of claim 31, wherein the suspension fluid is substantially RNAase-free.

33. A method of manufacturing a cell-transfection agent comprising a nanoparticle dispersion, comprising the steps of: generating a suspension comprising the nanoparticle dispersion, wherein each nanoparticle of the nanoparticle dispersion comprises a core with a formula AB2O4, and wherein A is a divalent cation, and B is a trivalent cation; coating the core of each nanoparticle with at least one coating compound; and functionalizing each coated nanoparticle core with a combination comprising free amine groups and thiol reactive groups to produce the nanoparticle dispersion comprising a plurality of functionalized nanoparticles.

34. The method of claim 33, wherein A is one or more selected from a group consisting of iron, magnesium, zinc, manganese, and nickel, and B is one or more of a group consisting of aluminum, chromium, and iron.

35. The method of claim 33, wherein A is one or more selected from a group consisting of iron, magnesium, zinc, manganese, and nickel, and B is iron such that each nanoparticle of the nanoparticle dispersion has a core with the formula AFe2O4.

36. The method of claim 35, wherein the core is a spinel core or a defective spinel core selected from the group consisting of magnetite, maghemite, and doped variants.

37. The method of claim 36, wherein the spinel core is a defective spinel core.

38. The method of any one of claims 33-37, wherein each nanoparticle of the nanoparticle dispersion is configured to have a magnetic saturation of less than 60 emu/g.

39. The method of any one of claims 33-38, wherein each nanoparticle of the nanoparticle dispersion is configured to have a magnetic coercion of less than 60 Oc.

40. The method of any one of claims 33-39, wherein each nanoparticle of the nanoparticle dispersion is configured to have a magnetic remembrance of less than 0.6 emu/g.

41. The method of any one of claims 33-40, wherein the core comprises MgFe2O4, wherein the magnesium and iron in MgFe2O4 are not present in a stoichiometric ratio.

42. The method of any one of claims 33-41, wherein the core comprises defective MgFe2O4.

43. The method of any one of claims 33-43, wherein the core comprises defective Mg_xFe2O4 , wherein x is a deficiency factor between 0.05 and 0.5.

44. The method of any one of claims 33-44, wherein the core comprises defective (Mgi- _xFe_x)(Mg_xFe2-x)O4, wherein x is a deficiency factor between 0.05 and 0.5.

45. The method of any one of claims 33-44, wherein the core comprises MgFe2C>4 doped with Zn.

46. The method of any one of claims 33-45, wherein the core has un-agglomerated hydrodynamic diameter of between 5 nm and 50 nm.

47. The method of any one of claims 33-46, wherein the core has a poly dispersity of between 0.03 and 0.3.

48. The method of any one of claims 33-47, wherein the core is coated with at least one coating compound.

49. The method of claim 48, wherein the at least one coating compound comprises dextran, dextran sulfate, or citrate, or a combination thereof

50. The method of any one of claims 48-49, wherein the at least one coating compound comprises a transient coating compound.

51. The method of claim 49, wherein the at least one coating compound comprises a combination of dextran, dextran sulfate, and citrate, wherein the dextran sulfate and the citrate are present in a ratio between 1 : 1 and 1 :25.

52. The method of any one of claims 33-51, wherein the coated core has an un-agglomerated hydrodynamic diameter between 20 nm and 70 nm.

53. The method of any one of claims 33-52, wherein the coated core is further coated with a polymer soft coat.

54. The method of claim 53, wherein the polymer soft coat comprises at least one of: block copolymers of poly(ethylene oxide) and polypropylene oxide), poloxamers, pluronics, nonionic surfactants, glucose polymer, poly(sarcosine) poly (2-oxazoline), a low molecular weight dextran or a low molecular weight dextran sulfate.

55. The method of any one of claims 53-54, wherein the core is soft coated with an amount of polymer that is less than the amount necessary to induce micellization of the de-watered cell transfection agent.

56. The method of any one of claims 33-55, wherein each nanoparticle of the nanoparticle dispersion is functionalized with free amine groups and thiol reactive groups in a ratio between 1 : 1 and 20: 1.

57. The method of claim 56, wherein between 10% and 90% of the amine groups are bioavailable.

58. The method of any one of claims 33-57, wherein the composition is functionalized to target cells expressing exofacial thiols.

59. The method of any one of claims 33-58, wherein the composition is functionalized to target exofacial thiol expressing cells present in extra-hepatic tissues.

60. The method of any one of claims 33-59, wherein the composition is functionalized to target exofacial thiol expressing cells present in reticuloendothelial tissue.

61. The method of any one of claims 33-60, wherein the composition is functionalized to target exofacial expressing cells present in tumor tissue.

62. The method of any one of claims 58-61, wherein the exofacial thiols comprise a group including CD36, PDI, CD71, or EGFR.

63. The method of any one of claims 33-62, comprising the composition and a suspension fluid.

64. The The method of claim 63, wherein the suspension fluid is substantially RNAase-free.

65. The method of any one of claims 33-64, wherein the steps of the method are carried out within a closed and automated system.

66. The method of any one of claims 33-65, further comprising moving the suspension fluid without contact with a solid valve or pump element.

67. The method of any one of claims 33-66, wherein generating the suspension comprising the nanoparticle dispersion further comprises the co-precipitation of divalent and trivalent cations.

68. The method of claim 67, wherein the co-precipitation occurs under experimental conditions including: a pH of less than 8; and a temperature less than 65°C.

69. The method of any one of claims 33-68, wherein generating the suspension comprises using a concentration equal to or less than 5 milligrams per milliliter of the suspension comprising the plurality of functionalized nanoparticles.

70. The method of any one of claims 67-69, wherein temperature ramps during the co- precipitation occur at a rate equal to or greater than 2.0°C /minute.

71. A cell-transfection agent comprising a nanoparticle dispersion manufactured by the steps of: generating a suspension comprising the nanoparticle dispersion having a plurality of nanoparticle cores, wherein each of the plurality of nanoparticle cores has a formula AB2O4, and wherein A is a divalent cation, and B is a trivalent cation; coating each of the plurality of nanoparticle cores in the suspension with at least one coating compound; functionalizing each of the plurality of coated nanoparticle cores with a combination comprising free amine groups and thiol reactive groups to produce a plurality of functionalized nanoparticles; and de-watering the suspension comprising the plurality of functionalized nanoparticles to output a de-watered composition comprising a substantially aggregation-free nanoparticle dispersion.

72. The cell-transfection agent of claim 71, wherein A is one or more selected from a group consisting of iron, magnesium, zinc, manganese, and nickel, and B is one or more of a group consisting of aluminum, chromium, and iron.

73. The cell-transfection agent of claim 71, wherein A is one or more selected from a group consisting of iron, magnesium, zinc, manganese, and nickel, and B is iron such that each nanoparticle of the nanoparticle dispersion has a core with the formula AFe2O4.

74. The cell-transfection agent of claim 71, wherein the core is a spinel core or a defective spinel core selected from the group consisting of magnetite, maghemite, and doped variants.

75. The cell-transfection agent of claim 74, wherein the spinel core is a defective spinel core.

76. The cell-transfection agent of any one of claims 71-75, wherein each nanoparticle of the nanoparticle dispersion is configured to have a magnetic saturation of less than 60 emu/g.

77. The cell-transfection agent of any one of claims 71-76, wherein each nanoparticle of the nanoparticle dispersion is configured to have a magnetic coercion of less than 60 Oc.

78. The cell transfection agent of any one of claims 71-77, wherein each nanoparticle of the nanoparticle dispersion is configured to have a magnetic remembrance of less than 0.6 emu/g.

79. The cell transfection agent of any one of claims 71-78, wherein the core comprises MgFe2O4, wherein the magnesium and iron in MgFe2O4 are not present in a stoichiometric ratio.

80. The cell transfection agent of any one of claims 71-79, wherein the core comprises defective MgFe2C>4.

81. The cell transfection agent of any one of claims 71 -80, wherein the core comprises defective Mg_xFe2O4 , wherein x is a deficiency factor between 0.05 and 0.5.

82. The cell transfection agent of any one of claims 71-81, wherein the core comprises defective (Mgi-_xFe_x)(Mg_xFe2-x)O4, wherein x is a deficiency factor between 0.05 and 0.5.

83. The cell transfection agent of any one of claims 71-82, wherein the core comprises MgFe2C>4 doped with Zn.

84. The cell transfection agent of any one of claims 71-83, wherein the core has unagglomerated hydrodynamic diameter of between 5 nm and 50 nm.

85. The cell transfection agent of any one of claims 71-84wherein the core has a poly dispersity of between 0.03 and 0.3.

86. The cell transfection agent of any one of claims 71-85, wherein the core is coated with at least one coating compound.

87. The cell transfection agent of claim 86, wherein the at least one coating compound comprises dextran, dextran sulfate, or citrate, or a combination thereof

88. The cell transfection agent of any one of claims 86-87, wherein the at least one coating compound comprises a transient coating compound.

89. The cell transfection agent of claim 87, wherein the at least one coating compound comprises a combination of dextran, dextran sulfate, and citrate, wherein the dextran sulfate and the citrate are present in a ratio between 1 : 1 and 1 :25.

90. The cell transfection agent of any one of claims 71-89, wherein the coated core has an unagglomerated hydrodynamic diameter between 20 nm and 70 nm.

91. The cell transfection agent of any one of claims 71-90, wherein the coated core is further coated with a polymer soft coat.

92. The cell transfection agent of claim 91, wherein the polymer soft coat comprises at least one of: block copolymers of polyethylene oxide) and polypropylene oxide), poloxamers, pluronics, non-ionic surfactants, glucose polymer, poly(sarcosine) poly (2-oxazoline), a low molecular weight dextran or a low molecular weight dextran sulfate.

93. The cell transfection agent of any one of claims 91-92, wherein the core is soft coated with an amount of polymer that is less than the amount necessary to induce micellization of the dewatered cell transfection agent.

94. The cell transfection agent of any one of claims 71-93, wherein each nanoparticle of the nanoparticle dispersion is functionalized with free amine groups and thiol reactive groups in a ratio between 1 : 1 and 20: 1.

95. The cell transfection agent of claim 94, wherein between 10% and 90% of the amine groups are bioavailable.

96. The cell transfection agent of any one of claims 71-95, wherein the composition is functionalized to target cells expressing exofacial thiols.

97. The cell transfection agent of any one of claims 71-96, wherein the composition is functionalized to target exofacial thiol expressing cells present in extra-hepatic tissues.

98. The cell transfection agent of any one of claims 71-97, wherein the composition is functionalized to target exofacial expressing thiol cells present in reticuloendothelial tissue.

99. The cell transfection agent of any one of claims 71-98, wherein the composition is functionalized to target exofacial thiol expressing cells present in tumor tissue.

100. The cell transfection agent of any one of claims 96-99, wherein the exofacial thiols comprise a group including CD36, PDI, CD71, or EGFR.

101. The cell transfection agent of any one of claims 71-100, comprising the composition and a suspension fluid.

102. The cell transfection agent of claim 101, wherein the suspension fluid is substantially RNAase-free.

103. A cell-transfection agent for delivering an effective dose of a payload molecule to cells, the cell-transfection agent comprising: a composition comprising a de-watered functionalized polydisperse zwitterionic spinel nanoparticle dispersion, wherein each nanoparticle of the nanoparticle dispersion comprises a core with a formula AB2O4 and functionalized with a combination comprising amine groups and thiol reactive groups, wherein A is a divalent cation, and B is a trivalent cation, wherein the composition is substantially free of irreversibly aggregated spinel nanoparticles, wherein a charge potential of the composition is configured according to the type of payload molecule; and wherein the composition is functionalized to target cells expressing exofacial thiols.

104. The cell transfection agent of claim 103, wherein the core is coated with at least one coating compound.

105. The cell transfection agent of claim 104, wherein the at least one coating compound comprises dextran, dextran sulfate, or citrate, or a combination thereof

106. The cell transfection agent of any one of claims 104-105, wherein the at least one coating compound comprises a transient coating compound.

107. The cell transfection agent of claim 105, wherein the at least one coating compound comprises a combination of dextran, dextran sulfate, and citrate, wherein the dextran sulfate and the citrate are present in a ratio between 1 : 1 and 1 :25.

108. The cell transfection agent of any one of claims 103-107, wherein the coated core has an un-agglomerated hydrodynamic diameter between 20 nm and 70 nm.

109. The cell transfection agent of any one of claims 103-108, wherein the coated core is further coated with a polymer soft coat.

110. The cell transfection agent of claim 109, wherein the polymer soft coat comprises at least one of: block copolymers of polyethylene oxide) and polypropylene oxide), poloxamers, pluronics, non-ionic surfactants, glucose polymer, poly(sarcosine) poly (2-oxazoline), a low molecular weight dextran or a low molecular weight dextran sulfate.

111. The cell-transfection agent of any one of claims 103-110, wherein the coating of the core of each nanoparticle is configured to effect the charge potential.

112. The cell transfection agent of any one of claims 103-111, wherein the exofacial thiols comprise a group including CD36, PDI, CD71, or EGFR.

113. The cell transfection agent of any one of claims 103-112, wherein the payload molecule comprises at least one of short polynucleotides, long polynucleotides, peptides, polypeptides, or small molecules.

114. The cell transfection agent of any one of claims 103-113, wherein the composition is functionalized to target cells expressing exofacial thiols.

115. The cell transfection agent of any one of claims 103-114, wherein the composition is functionalized to target exofacial thiol expressing cells present in extra-hepatic tissues.

116. The cell transfection agent of any one of claims 103-115, wherein the composition is functionalized to target exofacial thiol expressing cells present in reticuloendothelial tissue.

117. The cell transfection agent of any one of claims 103-116, wherein the composition is functionalized to target exofacial thiol expressing cells present in tumor tissue.

118. The cell transfection agent of any one of claims 103-117, wherein the cell-transfection agent comprises a therapeutically effective amount of the payload for delivery to a patient.

119. The cell transfection agent of claim 118, wherein the patient is a patient having cancer.

120. A therapeutically effective composition comprising: a cell-transfection agent comprising: a composition comprising a de-watered functionalized polydisperse zwitterionic spinel nanoparticle dispersion, wherein each nanoparticle of the nanoparticle dispersion comprises a core with a formula AB2O4 and functionalized with a combination comprising amine groups and thiol reactive groups, wherein A is a divalent cation and B is a trivalent cation, wherein the composition is substantially free of irreversibly aggregated spinel nanoparticles; and a therapeutically effective dose of a payload molecule, wherein a charge potential of the composition is configured according to the type of payload molecule.

121. The therapeutically effective composition of claim 120, wherein the core is coated with at least one coating compound.

122. The therapeutically effective composition of claim 121, wherein the at least one coating compound comprises dextran, dextran sulfate, or citrate, or a combination thereof

123. The therapeutically effective composition of any of claims 121-122, wherein the at least one coating compound comprises a transient coating compound.

124. The therapeutically effective composition of claim 122, wherein the at least one coating compound comprises a combination of dextran, dextran sulfate, and citrate, wherein the dextran sulfate and the citrate are present in a ratio between 1 : 1 and 1 :25.

125. The therapeutically effective composition of any one of claims 120-124, wherein the coated core has an un-agglomerated hydrodynamic diameter between 20 nm and 70 nm.

126. The therapeutically effective composition of any one of claims 120-125, wherein the coated core is further coated with a polymer soft coat.

127. The therapeutically effective composition of claim 126, wherein the polymer soft coat comprises at least one of: block copolymers of poly(ethylene oxide) and polypropylene oxide), poloxamers, pluronics, non-ionic surfactants, glucose polymer, poly(sarcosine) poly (2- oxazoline), a low molecular weight dextran or a low molecular weight dextran sulfate.

128. The therapeutically effective composition of any one of claims 120-127, wherein the coating of the core of each nanoparticle is configured to effect the charge potential.

129. The therapeutically effective composition of any one of claims 120-128wherein the exofacial thiols comprise a group including CD36, PDI, CD71, or EGFR.

130. The therapeutically effective composition of any one of claims 120-129, wherein the payload molecule comprises at least one of short polynucleotides, long polynucleotides, peptides, polypeptides, or small molecules.

131. A method of manufacturing a batch of a cell-transfection agent comprising the steps of:

(a) generating a nanoparticle suspension by performing co-precipitation of a plurality input reagents exposed to air, wherein a total volume of the input reagents is less than 1.25L in a single automated reactor;

(b) stabilizing the generated nanoparticle suspension by contacting the nanoparticle suspension with at least one coating agent;

(c) purifying the stabilized nanoparticle suspension via a size exclusion column to generate a purified nanoparticle suspension comprising a plurality of nanoparticles, wherein each nanoparticle of the plurality of nanoparticles comprises an un-agglomerated hydrodynamic diameter between 20nm and 70nm, wherein the size exclusion column comprises a second coating agent and wherein the at least one coating agent is replaced by the second coating agent during the purification;

(d) aminating the purified nanoparticle suspension to generate an aminated nanoparticle suspension configured for functionalization with a heterobifunctional linker; and

(e) functionalizing the aminated nanoparticle suspension with the heterobifunctional linker to generate the batch of the cell-transfection agent comprising a functionalized aminated nanoparticle suspension, wherein the batch of the cell-transfection agent has a batch size of less than or equal to 1.25 grams.

132. The method of claim 131, wherein the co-precipitation of the plurality of input reagents comprises at least one divalent cation and is performed in a chemical reaction vessel under experimental conditions including: a head space of four times surface area used by the plurality of input reagents in the in the chemical reaction vessel; a temperature range between 4°C and 60°C; a rate of change of temperature no less than 2.0°C per minute; constantly recirculation of air at a rate greater than 0.5ml per 1ml of the plurality of input reagents and a time duration of the co-precipitation no more than 150 minutes.

133. The method of any one of claims 131-132132, wherein the at least one coating compound comprises dextran, dextran sulfate, or citrate, or a combination thereof

134. The method of any one of claims 131-133any claims 131-133, wherein the at least one coating compound comprises a transient coating compound.

135. The method of claim 133, wherein the at least one coating compound comprises a combination of dextran, dextran sulfate, and citrate, wherein the dextran sulfate and the citrate are present in a ratio between 1 : 1 and 1 :25.

136. The method of any one of claims 131-135, wherein aminating the purified nanoparticle suspension further comprises the steps of cross-linking purified nanoparticle suspension with epichlorohydrin; aminating the cross-linked and purified nanoparticle suspension with NH4OH; and purifying the aminated, cross-linked, and purified nanoparticle suspension to generate the aminated nanoparticle suspension configured for functionalization with the heterobifunctional linker.

137. The method of any one of claims 131-136, wherein the plurality of input reagents of step (a) comprises at least one of divalent cations, a stabilizing agent, and a precipitation agent.

138. The method of any one of claims 131-137, wherein the heterobifunctional linker is SPDP.

139. The method of any one claims 131-138, wherein the nanoparticle suspensions of each of the steps of the method are moved through one or more conduits via one or more of compressed air pressure sufficient to push a volume of diluent fluid; liquid pressure provided by peristaltic pumps pushing the volume of diluent fluid; and negative pressure provided by generation a vacuum pump.

140. The method of claim 139, wherein the peristaltic pumps are configured to be arranged to prevent any pinch points to avoid applying shear stress on the nanoparticle suspensions of each of the steps of the methods.

141. The method of any one of claims 131-140, wherein the steps of the method are carried out under a pressure in a range between 1.5 psi/cm² to 5 psi/cm².

142. The method of any one of claims 131-141, wherein the batch of the cell-transfection agent is generated in between 3 days and 9 days.

143. The method of any one of claims 131-142, wherein the batch size of the batch of the cell-transfection agent generated is 5 grams and the batch of the cell-transfection agent is generated in 3 days.

144. The method of any one of claims 131-143, wherein the batch of the cell-transfection agent is configured for delivering a payload molecule comprising at least one of short polynucleotides, long polynucleotides, peptides, polypeptides or small molecules.

145. The method of any one of claims 131-144, wherein more than one automated reactor is used to generate more than one batch of the cell-transfection agent to generate a total batch size larger than 1.25 grams.

146. The method of any one of claims 131-145, wherein the steps (b) and (c) are configured to occur simultaneously.

147. The method of any one of claims 131-146, wherein the second coating agent is selected from a group comprising dextran or dextran sulfate.

148. The method of any one of claims 131-147, further comprising the step of: quenching between 100% and 5% of the free amines after remaining functionalizing with the linker.