Method and apparatus for ion flux lipid nanoparticle manufacture

Abstract

A method for preparing nucleic acid-containing lipid nanoparticles (NALNP) from a nucleic acid-containing neutral pH ionic salt solution and a lipid solution is provided. The method includes combining the nucleic acid-containing neutral pH ionic salt solution and the lipid solution and forming a combined mixture. The method further includes introducing an aqueous solution of lower ionic concentration than that of the neutral pH ionic salt solution into the combined mixture, thereby creating an ionic flux and forming the nucleic acid-containing lipid nanoparticles (NALNP).

Description

[0001] METHOD AND APPARATUS FOR ION FLUX LIPID NANOPARTICLE MANUFACTURE

[0002] BACKGROUND

[0003] Lipid nanoparticles are accepted as a pharmaceutical carrier for, among other things, genetic elements to transform native or extracorporeal cells for vaccination, gene therapy, cancer treatment, and immune therapy.

[0004] Typical manufacture of a nucleic acid-containing lipid nanoparticle (NALNP) involves creation of a lipid nanoparticle (LNP) by combining two streams, one of nucleic acid in low pH buffer, and one of lipids in organic phase such as ethanol. Lipid nanoparticles are thus fabricated using a low pH buffer that is not compatible for direct injection and furthermore, the particle size distribution is wide. A downstream process such as a buffer exchange is needed to remove the low pH buffer. A simple illustration of the existing method of preparing LNP is shown generally in Fig. 1.

[0005] A more efficient method and apparatus for preparing LNP and / or NALNP is needed.

[0006] SUMMARY

[0007] Advantages of the invention include the ability to avoid or reduce processing of LNP and / or NALNP prior to injection, by avoiding the use of low pH buffer in the initial nucleic acid solution. Other advantages of the invention over conventional methods include, but are not limited to, significantly improved particle quality (size distribution), which leads to a higher yield of material in large scale manufacturing and the simplification of downstream processing of the particles by removing the need to do a buffer exchange.

[0008] In addition, in the instant invention, the optimal range of lipid / nucleic acid concentration ratio is larger than that used in conventional practice, which further improves manufacturability.

[0009] According to an aspect of the invention, a method for preparing nucleic acid-containing lipid nanoparticles (NALNP) from a nucleic acid-containing neutral pH ionic salt solution and a lipid solution is provided. The method includes combining the nucleic acid-containing neutral pH ionic salt solution and the lipid solution, and forming a combined mixture. The method further includes introducing an aqueous solution of lower ionic concentration than that of the neutral pH ionic salt solution into the combined mixture, thereby creating an ionic flux and forming the nucleic acid-containing lipid nanoparticles (NALNP).

[0010] In an embodiment, the combined mixture is formed in a first mixer having a first inlet and a second inlet and the nucleic acid-containing neutral pH ionic salt solution is passed into the first mixer via the first inlet and the lipid solution is passed into the first mixer via the second inlet.

[0011] In an embodiment, the method may further include closing the first inlet and second inlet and introducing the aqueous solution of lower ionic concentration into the combined mixture within the first mixer via a third inlet in the first mixer.

[0012] In an embodiment, the combined mixture may be formed in a first mixer and the method may further include passing the combined mixture from the first mixer into a second mixer wherein the aqueous solution of lower ionic concentration is introduced into the combined mixture.

[0013] In an embodiment, the method may include removing the NALNP via an output downstream of the second mixer.

[0014] In an embodiment, the first mixer includes a Dean vortex mixer, T-tube mixer, serpentine channel mixer, turbulent mixer, pipette mixer, agitation mixer, passive mixer, spiral channel mixer, herringbone channel mixer, flow focusing mixer, centrifugal channel mixer, or a combination thereof.

[0015] In an embodiment, the second mixer includes a Dean vortex mixer, T-tube mixer, serpentine channel mixer, turbulent mixer, pipette mixer, agitation mixer, passive mixer, spiral channel mixer, herringbone channel mixer, flow focusing mixer, centrifugal channel mixer, or a combination thereof.

[0016] In an embodiment, the time gap between after mixing of the neutral pH ionic salt solution and lipid solution to form the combined mixture, and before addition of the aqueous solution of lower ionic strength to the combined mixture is from about 0 to about 60 minutes.

[0017] In an embodiment, the pH of the neutral pH ionic salt and the aqueous solution is between a pH of about 6.0 and a pH of 7.4. In an embodiment, the pH of the neutral pH ionic salt solution and the aqueous solution is between a pH of 6.5 and a pH of 7.4.

[0018] In an embodiment, the pH of the neutral pH ionic salt solution and the aqueous solution is selected from a pH of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, and 7.4.

[0019] In an embodiment, the neutral pH ionic salt solution includes one or both of NaCl and KC1 salts to provide the ions.

[0020] In an embodiment, an ionic concentration of the ionic salt solution is in the range of about 10 mM to 600 mM.

[0021] In an embodiment, an ionic concentration of the combined mixture is at least 60 mM greater than an ionic concentration of the aqueous solution.

[0022] In an embodiment, the aqueous solution includes an ionic salt that is the same type of ionic salt as in the neutral pH ionic salt solution.

[0023] In an embodiment, substantially no nucleic acid-containing nanoparticles are formed in the combined mixture before introducing the aqueous solution.

[0024] In an embodiment, the nucleic acid-containing lipid nanoparticles (NALNP) formed is in a solution having a neutral pH.

[0025] In an embodiment, the nucleic acid-containing lipid nanoparticles (NALNP) formed is in a solution including an ionic concentration in a range of 0-300 mM.

[0026] In an embodiment, the nucleic acid-containing lipid nanoparticles (NALNP) formed is in a solution including 1-10% of ethanol.

[0027] According to another aspect of the invention, an apparatus for preparing nucleic acidcontaining lipid nanoparticles (NALNP) at neutral pH includes a first mixer downstream of a first inlet and a second inlet. The apparatus further includes a second mixer downstream of the first mixer, the second mixer being fluidly connected to the first mixer, the second mixer having a third inlet and an output for a NALNP product to exit the second mixer. Wherein one of first inlet and the second inlet into the first mixer provides an entry for a nucleic acid-containing neutral pH ionic salt solution and wherein the other one of the first inlet and the second inlet into the first mixer provides an entry for a lipid solution. The third inlet provides entry for an aqueous solution with a lower ionic concentration than that of the neutral pH ionic salt solution before the neutral pH ionic salt solution enters the first mixer.

[0028] In an embodiment, each of the first mixer and the second mixer is independently a Dean vortex mixer, T-tube mixer, serpentine channel mixer, turbulent mixer, pipette mixer, agitation mixer, passive mixer, spiral channel mixer, herringbone channel mixer, flow focusing mixer, centrifugal channel mixer, or a combination thereof.

[0029] In an embodiment, at least one of the first mixer and the second mixer are Dean vortex channel mixers.

[0030] In an embodiment, the third inlet provides passage of aqueous solution into the second mixer to create an ionic flux, thereby facilitating formation of the NALNP.

[0031] In an embodiment, the first mixer and the second mixer are arranged in tandem or are combined into a single mixer.

[0032] Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In drawings which illustrate embodiments of the invention,

[0035] Fig. 1 is an illustration of the prior art method of preparing nucleic acid-containing lipid nanoparticles (LNP).

[0036] Fig. 2A is a simplified illustration of a two-mixer apparatus layout according to embodiments of the invention.

[0037] Fig. 2B is a simplified illustration of a single mixer apparatus layout according to other embodiments of the invention.

[0038] Fig. 3 is a graphical representation of data showing the overall diameter of ENP and the size range of ENP for the prior art low pH buffer method (“current practice”) versus the method of the invention (“invention”). Fig. 4 is a bar graph representation of encapsulation efficiency for the prior art low pH buffer method (“current practice”) as well as the method of the invention (“invention”).

[0039] Fig. 5 is a graphical representation of the lipid nanoparticle size (nm) and polydispersity index (PDI) for both the conventional low pH buffer method (left, “current practice”) and the method of the invention (right, “invention”).

[0040] Fig. 6 is a line graph showing the effect of salt (X) concentration between 0 to 600 mM.

[0041] Fig. 7 is a line graph showing the effect of pH of the neutral pH ionic salt solution from about 6.2 to about 7.4.

[0042] Fig. 8 is a schematic of various embodiments of Mixer 1 and Mixer 2 combination used to demonstrate the effects of different mixer types on the quality of resulting NALNP.

[0043] Fig. 9 is a Cryo-TEM image of a dissected plan of the flash frozen lipid nanoparticles prepared according to the invention.

[0044] Fig. 10 is a line graph showing the effect of time delay between the fluid exiting an outlet of Mixer 1 and entering an inlet of Mixer 2 on percentage of dissolved materials (left y-axis, “Nucleic Acid” and “Ionizable Lipid” as legends) and encapsulation efficiency measured for the NALNP product collected from the outlet of Mixer 2 (right y-axis, “EE after flux” as legend). The time delay was measured between a time the combination mixture flowing out of an outlet of Mixer 1 and a time the combination mixture entering an inlet of Mixer 2 which is in fluidic communication with the outlet of Mixer 1.

[0045] Fig. 11 is a line graph illustrating the effects of payload concentration and the length of settling time after payload-containing LNP is collected from the outlet of the Mixer 2 on the encapsulation efficiency (EE) of payload-containing LNP products.

[0046] Fig. 12 is a line graph of the encapsulation efficiency (EE) versus the ratio of the ionic concentration between an ionic concentration of the salt in the aqueous solution and the ionic concentration of the salt in the neutral pH ionic salt solution. Fig. 13 is a line graph of the encapsulation efficiency (EE) vs. the ionic concentration of the salt in the aqueous solution as its concentration is varied while keeping the ionic concentration of the salt in the neutral pH ionic salt solution constant at 160 mM.

[0047] Fig. 14 is a line graph of the effect of flow rate ratio and cone, of the lipid in the organic solution on the method according to embodiments of the invention. Triangles represent data points for flow rate ratios of (4:1): N, where 4 is the relative flow rate of nucleic acid-containing neutral salt solution flowing into Mixer 1, 1 is the relative flow rate of lipids in organic solvent flowing into Mixer 1, and N is a variable represented in x-axis as the relative flow rate of the aqueous solution flowing into Mixer 2. The concentrations of the lipid in the organic solution used with the corresponding ratios are 16 mM, 8 mM, 16 mM, respectively.

[0048] Fig. 15 is a bar graph of the encapsulation efficiency (EE) versus the nucleic acid payload, according to embodiments of the invention. The nucleic acid payloads include 1.1 kilobase (kB) eGRP mRNA, 3.3kB pDNA, 12.8kB pDNA, and 11.5kB saRNA.

[0049] Fig. 16 is a bar graph of the size and poly dispersity index (PDI) versus the nucleic acid payload, according to embodiments of the invention. The nucleic acid payloads include l.lkB eGRP mRNA, 3.3kB pDNA, 12.8kB pDNA, and 11.5kB saRNA. Triangles represent data points for the PDI.

[0050] Fig. 17 is a line graph of the encapsulation efficiency (EE) versus the concentration (ug / mL) of nucleic acid payload in the NALNP sample collected at the outlet of Mixer 2. The nucleic acid payloads illustrated in Fig. 17 include 12.8kB pDNA and 3.3kB pDNA.

[0051] Fig. 18 is a bar graph of the encapsulation efficiency (EE) of neutral salts used in the ion flux process according to embodiments of the invention. The neutral salts illustrated in Fig. 18 include NaCl, KC1, MgSO4, MgCh, LiCl, and CaCl2.

[0052] Fig. 19 is a line graph of Mixer 1 and Mixer 2 size variation used to demonstrate the effects of different mixer types on the encapsulation efficiency (EE) of resulting NALNP. Fig. 20 is a line graph of a cell dose response curve of the percentage spike positive cells versus the dose of NALNP used.

[0053] Fig. 21 is a graph showing the expression of rhEPO using PBS as control (with no lipid nanoparticles or EPO mRNA), the NALNP sample collected directly after Mixer 2 and containing 20 ug / mL EPO mRNA and 6.6% ethanol (“Flux EtOH”), and an NALNP sample collected directly after Mixer 2 and then subjected to a downstream buffer exchange for removing ethanol, the NALNP sample containing 20 ug / mL EPO mRNA (“Flux Amicon”).

[0054] Fig. 22 is a line graph of the encapsulation efficiency (EE) versus Mixer 2 outlet concentration of the nucleic acid payload. The ionizable lipids tested were PNI516, PNI 550, PNI 560, PNI 660, PNI 723, PNI 728, PNI 734, and MC3 ionizable tested.

[0055] Fig. 23 is one embodiment of an apparatus for preparing NALNP at a neutral pH including Mixer 1 and Mixer 2.

[0056] DETAILED DESCRIPTION

[0057] In accordance with an embodiment of the invention, there is provided a method for preparing nucleic acid-containing lipid nanoparticles (NALNP) from a nucleic acid in neutral pH ionic salt solution and a lipid solution, wherein the nucleic acid in neutral pH ionic salt solution enters a first mixer (Mixer 1), optionally through a first inlet, and wherein the lipid solution enters Mixer 1, optionally through a second inlet, and wherein substantially no nucleic acid containing nanoparticles are formed in the combined mixture at that point, and wherein either the first and second inlet are closed or the combined mixture leaves Mixer 1 and enters a second mixer (Mixer 2). NALNP are formed only when an aqueous solution of lower ionic concentration than that of the nucleic acid in neutral pH ionic salt solution is introduced into the combined mixture which introduction creates an ionic flux facilitating the formation of NALNP.

[0058] In embodiments, and as described in greater detail herein, a single mixer may be utilized to carry out the aforementioned processes.

[0059] In a final step, the NALNP are removed or harvested via an output downstream of the inlets of the second mixer. Turning to Fig. 1, a schematic illustration of an existing method and apparatus of preparing NALNP is shown. In contrast to embodiments of the invention, prior art NALNP manufacturing processes 100 involve creation of a lipid nanoparticle by combining two streams, one stream 102 of nucleic acid in low pH buffer (e.g., pH = 4), and one stream 104 of lipids in an organic solvent, such as ethanol, in a mixer 106. Lipid nanoparticles exiting an output of mixer 106 includes low pH buffer that is not compatible for direct injection and furthermore, the particle size distribution is wide. The prior art methods require a buffer exchange 108 process that is necessary to remove the low pH buffer.

[0060] Referring to embodiments of the instant application, in some embodiments, a payload is encapsulated by an exemplary lipid nanoparticle composition. The payload may include a nucleic acid. A “nucleic acid” as used herein is any nucleic acid suitable for use in the invention. The nucleic acid is a substance intended to have a direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, or to have direct effect in restoring, correcting or modifying physiological functions, or to act as a research reagent. In some embodiments, examples of the nucleic acid include, but are not limited to, an siRNA, miRNA, a self-amplifying RNA (saRNA), self-amplifying messenger RNA (samRNA), a self-replicating DNA, an LNA, a DNA, a plasmid, a replicon, a messenger RNA (mRNA), a guide RNA, a transposon, a single gene, a complex of RNA and RNA- binding protein, or any combinations thereof. In some embodiments, the nucleic acid is referred to as a nucleic acid therapeutic or “NAT”. The nucleic acid can be single stranded, double stranded, or contain portions of both double stranded and single stranded sequence, as appropriate.

[0061] In some embodiments, the NAT is replaced by, or augmented by, a peptide or polypeptide. For the purposes of this invention, NAT may encompass these additional non-nucleic acids.

[0062] Neutral pH ionic salt solution has a pH of between 6.0 and 7.4 and an equilibrium of positive and negative ions derived from a salt.

[0063] The first stream includes a nucleic acid in an ionic solution. Suitable solutions are aqueous with neutral pH ionic salts. Non-limiting examples of neutral pH ionic salts include, but are not limited to, salts of alkali metals (e.g., NaCl, KC1, LiCl, CsCl, etc.) and / or alkaline earth metals (e,g., MgSCU, MgCh, CaSCU, CaCh, etc.). In some embodiments, the ionic salt includes NaCl, KC1, LiCl, MgSC , and / or MgCh. In certain embodiments, the ionic salt includes NaCl and / or KC1.

[0064] In embodiments, the nucleic acid in neutral pH ionic salt solution includes NaCl and / or KC1 salts to provide the ions. In embodiments, the ionic concentrations in the neutral pH ionic salt solution are from about 1 mM to about 600 mM, e.g., about 1 mM, about 10 mM, about 20 mM, about 40 mM, about 60 mM, about 80 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, about 325 mM, about 350 mM, about 375 mM, about 400 mM, about 425 mM, about 450 mM, about 475 mM, about 500 mM, about 525 mM, about 550 mM, about 575 mM, about 600 mM, or a concentration defined by a range of any two of the foregoing values. In embodiments, the ionic concentration in the aqueous solution neutral pH ionic salt solution are from about 40 mM to about 520 mM. In embodiments of the method, the aqueous solution has an ionic concentration of about 60 mM or more less than that of the combined mixture.

[0065] “Neutral pH,” as used throughout the current disclosure, refers to a pH of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or a pH defined by a range of any two of the foregoing pH values. For example, in some embodiments, the neutral pH is a pH in a range of about 6.0 to 7.4, in a range of about 6.3-7.4, about 6.5-7.4. In embodiments, the pH of nucleic acid solution and the aqueous solution is between a pH of about 6.0 and a pH of about 7.4. In embodiments, the pH of nucleic acid solution and the aqueous solution is between a pH of about 6.5 and a pH of about 7.4. In embodiments, the pH of nucleic acid solution and the aqueous solution is each independently selected from a pH of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or a pH defined by a range of any two of the foregoing pH values.

[0066] A “lipid solution,” as used herein, is lipids in an organic carrier or organic solvent. In embodiments, the organic solvent is miscible with the first solvent.

[0067] In some embodiments, a lipid solution comprises a stabilizing agent or stabilizer. Any suitable stabilizing agent or stabilizer can be used in embodiments of the present invention. In some embodiments, the stabilizing agent is chosen from polysorbates (Tweens), Brij™ S20 (polyoxyethylene (20) stearyl ether), Brij™35 (Polyoxyethylene lauryl ether, Polyethyleneglycol lauryl ether), Brij™S10 (Polyethylene glycol octadecyl ether, Polyoxyethylene (10) stearyl ether), Myrj™52 (polyoxyethylene (40) stearate), PEG-DMG, PEG-DMG 2000, Triolein, Tridecyl-D-maltoside, Tween 20, Polysorbate 80, Lipid H, TPGS1000, polyoxyethylene (4) lauryl ether, DiD, or any combinations thereof. Certain combinations of stabilizing agents used in some embodiments include, but are not limited to, polysorbate and maltoside, Alkyl polyglycosides (TBD), PEG-conjugated lipids or other polymer conjugated lipids. In some embodiments, the lipid solution comprises more than one stabilizing agent or stabilizers. For example, in some embodiments, the lipid solution comprises one or more, two or more, three or more, or four or more stabilizing agents or stabilizers. In some embodiments, stabilizing agent or stabilizer is PEG free.

[0068] In some embodiments, a lipid solution comprises an ionizable lipid. Any suitable ionizable lipid can be used in embodiments of the present invention. An ionizable lipid is a lipid that is cationic or becomes ionizable (protonated) as the pH is lowered below the pKa of the ionizable group of the lipid but is more neutral at higher pH values. The pKa value of an ionizable lipid is said to be when 50% if the lipid is positively charged (ionized), and 50% of the lipid is neutral (not ionized). At pH values below the pKa, the majority of lipid is then able to associate with negatively charged nucleic acids (e.g., oligonucleotides). Examples of suitable ionizable lipids are disclosed, for example, in PCT Publication Nos. WO2020252589 and W02021000041.

[0069] In some embodiments, the ionizable lipid is DODMA (l,2-dioleyloxy-3- dimethylaminopropane), DLin-MC3-DMA (O-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen- 19-yl)-4-(N,N-dimethylamino)), DLin-KC2-DMA (2-dilinoleyl-4-dimethylaminoethyl- [l,3]-dioxolane), butanoic acid, BOCHD-C3-DMA (4-(dimethylamino)-,9-(2- octylcyclopropyl)-l-[8-(2 octylcyclopropyl) octyl]nonyl ester), or C12-200. In some embodiments, the ionizable lipid includes, but is not limited to, (Z)-3-(2-((l,17-bis(2- octylcyclopropyl)heptadecan-9-yl)oxy)-2-oxoethyl)-2-(pent-2-en-l-yl)cyclopentyl 4- (dimethylamino)butanoate (referred to as PNI 516) , 3-(2-((l,17-bis(2- octylcyclopropyl)heptadecan-9-yl)oxy)-2-oxoethyl)cyclopentyl 4- (dimethylamino)butanoate (PNI 550), (Z)-3-(2-((l,17-bis(2- octylcyclopropyl)heptadecan-9-yl)oxy)-2-oxoethyl)-2-(pent-2-en-l-yl)cyclopentyl 1,4- dimethylpiperidine-4-carboxylate (PNI 560), or (2R,3S,4R)-2-(((l,4-dimethylpiperidine- 4-carbonyl)oxy)methyl)tetrahydrofuran-3,4-diyl (9E,9'E,12E,12'E)-bis(octadeca-9,12- dienoate) (referred to as PNI 127), ((2R,3R,4S)-3,4-bis((2- hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl 1 ,4-dimethylpiperidine-4-carboxylate (PNI 660), ((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl 4- (diethylamino)butanoate (PNI 723), or any combinations thereof. In some embodiments, a lipid solution comprises a structural lipid. A structural lipid may also be known as a helper lipid or neutral lipid. Any suitable structural lipid can be used in the embodiments of the present invention. Suitable structural lipids support the formation of particles during manufacture. Structural lipids refer to any one of a number of lipid species that exist in either an anionic, uncharged or neutral zwitterionic form at physiological pH. Representative structural lipids suitable for use in the present invention include, but are not limited to, diacylphosphatidylcholines, diacylphosphatidylethanolamines, diacylphosphatidylglycerols, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, cerebrosides, or any combinations thereof.

[0070] Exemplary structural lipids include, but are not limited to, zwitterionic lipids, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), l-stearoyl-2-oleoyl-sn-glycero-3- phosphocholine (SOPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl -phosphatidylethanolamine (DSPE), 16-0-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl- phosphatidy ethanol amine (SOPE), l,2-dielaidoyl-sn-glycero-3-phophoethanolamine (trans DOPE), or any combinations thereof. In one embodiment, the structural lipid is distearoylphosphatidylcholine (DSPC) .

[0071] In some embodiments, the lipid solution includes a sterol. Any suitable sterol can be used. In some embodiments, the sterol includes cholesterol, beta-sitosterol, 20-alpha- hydroxysterol, or phytosterol, or any combinations thereof. In a specific embodiment, the sterol is cholesterol.

[0072] The lipid composition of the present invention can include any suitable combinations of components including ionizable lipid, structural lipid, sterol, and / or stabilizing agent. In some non-limiting embodiments, the lipid composition comprises 47.5 mol% ionizable lipid, 12.5 mol% structural lipid, 38.5 mol% sterol, and 1.5 mol% stabilizing agent. In some embodiments, the lipid composition comprises 40 mol% ionizable lipid, 20 mol% structural lipid, 37.5 mol% sterol, and 2.5 mol% stabilizing agent. It is to be understood that the present invention is not limited to the compositions described herein, rather, various lipid compositions including combinations of components (e.g., ionizable lipid, structural lipid or helper lipid, sterol, and / or stabilizing agent) in various molar ratios can be used with the present invention.

[0073] An organic carrier is described below.

[0074] The second stream includes lipid composition materials which are soluble in an organic carrier. Suitable organic carriers include solvents in which the lipids according to embodiments of the invention are soluble, and that are miscible with the first solvent. Suitable second solvents include, but are not limited to, 1,4-dioxane, tetrahydrofuran, acetone, ethanol, benzene, tert-butyl, methanol, chloroform, acetonitrile, dimethyl sulfoxide, dimethylformamide, acids, or alcohols, or any combinations thereof. In embodiments, second solvent includes about 90% ethanol or anhydrous ethanol.

[0075] In embodiments, the mixer is a channel mixer wherein two or more fluids are brought into contact with each other with a certain amount of energy. In some embodiments, turbulence is created in a volume where two fluids meet, accelerating diffusion.

[0076] In one embodiment of the invention, a suitable mixing geometry for one or both the first mixer (Mixer 1) and the second mixer (Mixer 2) includes one or more microchannels (i.e., a channel having its greatest dimension less than 1 millimeter) or one or more macrochannels (i.e., a channel having it greatest dimension less than 5 mm). In one example, the microchannel has a diameter from about 20 to about 5000 pm. In examples, at least one region of the microchannel has a principal flow direction and one or more surfaces having at least one groove or protrusion defined therein, the groove or protrusion having an orientation that forms an angle with the principal direction (e.g., a staggered herringbone mixer), as described in U.S. Pat. No. 9,943,846, or a bifurcating toroidal flow as described in U.S. Pat. No. 10,076,730, both of which are incorporated by reference in their entireties, including, but not limited to, Figs. 1 - 6, 10 - 16, 20 - 23 and columns 2 - 24 of U.S. Pat. No. 10,076,730, and Figs. 1, 7A - 9, 12 - 14 and columns 1 - 24 of U.S. Pat. No. 9,943,846. To achieve maximal mixing rates, it is advantageous to avoid undue fluidic resistance prior to the mixing region. Thus, one example of a device has non-microfluidic channels having dimensions greater than 1000 pm, to deliver the fluids to a single mixing channel.

[0077] Less automated mixing methods and apparatus such as those disclosed in Zhang, S. H et al., Chemical Eng. J. 144(2): 324-328 (2008), and Stroock, A. et al., U.S. Published Patent Application US20040262223, and Jeffs, LB et al., Pharm. Resch., 22(3): 362-372 (2005), all of which are incorporated by reference in their entireties, may be used to form the first or second mixer (Mixer 1 or Mixer 2) or both according to the invention, in some embodiments.

[0078] An “inlet” as used herein is an opening in a mixer (Mixer 1 or Mixer 2, for example) to allow the entry of a fluid from outside the mixer. In embodiments, the channels of the inlets, Mixer 1 and Mixer 2 and outputs are less than 1 mm in diameter. In other embodiments, the channels of the inlets, Mixer 1 and Mixer 2 and outputs are greater than 1 mm. In still other embodiments, the channels of the inlets, Mixer 1 and Mixer 2 and outputs are greater than 2 mm. In still other embodiments, the channels of the inlets, Mixer 1 and Mixer 2 and outputs are greater than 3 mm. In still other embodiments, the channels of the inlets, Mixer 1 and Mixer 2 and outputs are greater than 4 mm. The channels, inlets and Mixer channels do not need to be consistently sized but may vary along these measurements from one to the other. Inlets are reversibly or irreversibly shut after they perform, to retain mixed fluid in Mixer 1 or Mixer 2. The shape of the channels leading to the inlets, and inlets is usually round or oval to minimize friction, but may be rectilinear.

[0079] An output is the port of egress for fluids in a mixer (Mixer 1 or Mixer 2 for example) to allow departure of fluid from the mixer. The physical parameters of the output are similar as for inlets described above, but in some embodiments, the output may have an additional sterile filtration component. Opening and closing of the output as well as the inlets will be governed by computer program or user interface controls, or a combination of the two.

[0080] Ionic concentration is the molar concentration of ions in the solution.

[0081] An ionic solution contains ions, which are atoms that have gained or lost electrons to acquire a positive (lost electron) or negative (gained electron) charge. Often, ions exist as part of an ionic compound where two ions are bound together because of their opposite charges attracting each other.

[0082] Ionic strength is a way of describing the ionic charge combined with concentration. A lower concentration of ions would have a lower ionic strength.

[0083] In embodiments of the invention, ionic salt or a solution including an ionic salt is mixed with a payload (e.g., nucleic acid) or a solution including the payload to form a combined solution, such as, nucleic acid in a neutral pH ionic salt solution. The pH of the combined solution may be further adjusted to reach a predetermined pH value within a range of neutral pH. Then the nucleic acid in neutral pH ionic salt solution and a lipid solution in an organic solvent are mixed to form a combined mixture. In embodiments, substantially no nucleic acid-containing nanoparticles are formed in the combined mixture. An aqueous solution of lower ionic concentration than that of the neutral pH ionic salt solution is then added into the combined mixture, which causes local ionic concentrations of the ionic salt in the combined mixture to reduce as the ions diffuse out of the combined mixture, causing local “electromagnetic” fields to trigger self-assembly of the negatively charged nucleic acids and positively charged ionizable lipid as well as the associated lipid components (for example, structural lipids, sterol, and / or stabilizers described above). In embodiments, the ionic salt includes alkali metal salts including, but is not limited to, NaCl, KC1, LiCl, or CsCl, or a combination thereof. In embodiments, the ionic salt includes alkaline earth metal salts including, but is not limited to, MgCh, CaCh, or MgSCL, CaSO4, or a combination thereof. In embodiments, the aqueous solution includes the same type of ionic salt yet a lower ionic concentration than that of the neutral pH ionic salt solution. In embodiments, the aqueous solution includes a different type of ionic salt and with a lower ionic concentration than that of the neutral pH ionic salt solution. The aqueous solution of lower ionic concentration is added into the combined mixture, which causes the local ionic concentration (e.g., Na+or K+and Cl’ ion concentration) of the ionic salt in the combined mixture to reduce as the ions diffuse out of the combined mixture, causing “electromagnetic” fields to trigger / facilitate selfassembly of the lipid nanoparticles (LNP).

[0084] In the process of mixing the aqueous solution of lower ionic concentration with the combined mixture of the nucleic acid in neutral pH ionic salt solution and the lipid solution, ions in the combined mixture diffuse towards the aqueous solution (e.g., “ion flux”) faster than the nucleic acid and lipids can.

[0085] The faster movement of ions (e.g., Na+or K+and Cl’) in the combined mixture relative to the nucleic acid and lipids will generate “electromagnetic” fields at the nanoscale. The electromagnetic fields combined with reduction of the ions between the nucleic acid and lipids start and / or facilitate self-assembly of the lipid nanoparticles (LNP).

[0086] Lower ionic strength is generally lower ionic concentration.

[0087] Combined mixture is the fluid resulting from the nucleic acid in neutral pH ionic salt and lipid solution coming together. In embodiments, the combined mixture is formed in the first mixer (e.g., Mixer 1). Nucleic acid-containing lipid nanoparticles (NALNP) are lipid nanoparticles containing a nucleic acid pay load. Lipid nanoparticles (LNP) are generally assemblies of lipids (including, for example, ionizable lipids and structural lipids), sterols, and stabilizing agents. Lipid nanoparticles (LNP) are generally spherical but may take other forms. Positive and negative charges, ratios, as well as hydrophilicity and hydrophobicity dictate the physical structure of the lipid particles in terms of size and orientation of components. The structural organization of these lipid may lead to an aqueous interior with a minimum bilayer as in liposomes or it may have a solid interior as in a solid nucleic acid lipid nanoparticle. There may be phospholipid monolayers or bilayers in single or multiple forms. Lipid particles are generally between 1 and 1000 nanometers in size.

[0088] The lipid nanoparticle of the present invention may include a pay load. In embodiments, the payload may include, but is not limited to, nucleic acid, protein and / or other active pharmaceutical ingredients. The nucleic acid-containing lipid nanoparticle (NALNP) comprises a nucleic acid. Any suitable nucleic acid can be used in the lipid nanoparticle. The nucleic acid is a substance intended to have a direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, or to have direct effect in restoring, correcting or modifying physiological functions, or to act as a research reagent. In some embodiments, the nucleic acid is a siRNA, miRNA, a self-amplifying RNA (saRNA), self-amplifying messenger RNA (samRNA), a self-replicating DNA, an LNA, a DNA, replicon, a messenger RNA (mRNA), a guide RNA, a transposon, or a single gene. In some embodiments, the nucleic acid is referred to as a nucleic acid therapeutic or NAT.

[0089] In some embodiments, the NAT is replaced by, or augmented by, a peptide or polypeptide. For the purposes of this invention, NAT may encompass these additional non-nucleic acids.

[0090] Downstream is toward the direction of fluid flow, either from higher elevation to lower, or from higher pressure to lower.

[0091] Upstream is the term referring to something that is earlier in the fluid path than downstream.

[0092] An “aqueous solution” as used herein includes water or water with a lower concentration of ions than that of the combined mixture or the neutral pH ionic salt solution.

[0093] Ionic flux refers to a force caused by a change in ion concentration at one locale of a volume, driving a movement of ions and charge to re-equilibrate. When the fluid from an outlet of Mixer 1 mixes with the aqueous solution of lower ionic concentration in Mixer 2, there is a short amount of time that the two fluids are close together, but diffusion has yet to occur to fully mix the fluids. It is at this moment that the ions diffuse from a high concentration to a low concentration in the aqueous solution, settling at an equilibrium. This movement of ions cause local electromagnetic fields that attract the positively charged ionized lipids to the negatively charged nucleic acid, and self-assembly has begun. The result is lipid nanoparticles containing nucleic acid at the outlet of Mixer 2.

[0094] In embodiments, the first mixer, e.g., Mixer 1, is selected from Dean vortex mixers, T- tube mixers, serpentine channel mixers, turbulent mixers, pipette mixers, agitation mixers, passive mixers, spiral channel mixers, herringbone channel mixers, flow focusing mixers, and centrifugal channel mixers. In embodiments, the second mixer, e.g., Mixer 2, is selected from Dean vortex mixers, T-tube mixers, serpentine channel mixers, turbulent mixers, pipette mixers, agitation mixers, passive mixers, spiral channel mixers, herringbone channel mixers, flow focusing mixers, and centrifugal channel mixers. In embodiments the mixer types may be in tandem or combined.

[0095] In some embodiments, the time between mixing of the nucleic acid solution and lipid solution to form a combined mixture, and addition of the aqueous solution of lower ionic strength to the combined mixture is from 0 to 60 minutes.

[0096] In embodiments, the pH of nucleic acid solution, the lipid solution and the aqueous solution is between a pH of about 6.0 and a pH of 7.4. In embodiments, the pH of nucleic acid solution, the lipid solution and the aqueous solution is selected from a pH of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or a range including any of the pH value disclosed here. In embodiments, the pH is in a range of between about 6.5 and about 7.4.

[0097] In some embodiments of the method, instead of there being two mixers, the first mixer, e.g., Mixer 1, is converted to a second mixer, e.g., Mixer 2, by closing the first two inlets and opening a third inlet in the first mixer.

[0098] Referring to Fig. 2A and Fig. 2B, according to embodiments of the invention, an apparatus 200 for preparing nucleic acid-containing lipid nanoparticles (NALNP) at neutral pH, the apparatus comprising a first mixer 210 (e.g., Mixer 1) downstream of (e.g., fluidly connected to) a first inlet 206 / 208 and a second inlet 208 / 206, a second mixer 220 (e.g., Mixer 2) downstream of the first mixer 210 (Mixer 1), the second mixer 220 / Mixer 2 having a connector (e.g. tubing) from the first mixer 210 / Mixer 1, and a third inlet 216, and an output 224 for a NALNP product to exit, and wherein one of the first of the two inlets 206, 208 into the first mixer 210 / Mixer 1 (e.g., the first inlet 206) allows nucleic acid in neutral pH ionic salt solution to be passed into the first mixer and wherein the second of the first two inlets (e.g. the second inlet 208) allows the lipid solution to be passed into the first mixer and wherein the third inlet provides passage of an aqueous solution with a lower ionic concentration than that of the neutral pH ionic salt solution before the nucleic acid in neutral pH ionic salt solution enters the first mixer / Mixer 1. It is noted that while the first inlet 206 and the second inlet 208 are illustrated in Fig. 2A to allow the nucleic acid in neutral pH ionic salt solution and the lipid solution to be passed into the first mixer, respectively, it is to be understood that the first inlet 206 and the second inlet 208 can also be configured to allow the lipid solution and the nucleic acid in neutral pH ionic salt solution to be passed into the first mixer, respectively. Similarly, the first inlet 306 and the second inlet 308 as shown in Fig. 2B can be arranged to allow the lipid solution and the nucleic acid in neutral pH ionic salt solution to be passed into Mixer 2, respectively.

[0099] In embodiments the first and second mixers 210, 220 (Mixer 1 and Mixer 2) are independently chosen from a group of Dean vortex mixers, T-tube mixers, serpentine channel mixers, turbulent mixers, pipette mixers, agitation mixers, passive mixers, spiral channel mixers, herringbone channel mixers, flow focusing mixers, and centrifugal channel mixers.

[0100] In some embodiments, at least one of the first and second mixers 210, 220 (Mixer 1 and Mixer 2) are Dean vortex channel mixers.

[0101] In certain embodiments, the third inlet 216 allows aqueous solution to be passed into the second mixer 220 (Mixer 2) to create an ionic flux, which flux creates NALNP.

[0102] In some embodiments, the first and second mixers 210, 220 (e.g., Mixer 1 and Mixer 2) are one volume (e.g., a single mixer) with three inlets.

[0103] Suitable methods of the physical components of the mixing process can be used to form the lipid nanoparticles according to the invention. The first and second mixers according to embodiments of the invention can be independently Dean vortex mixers, T-tube mixers, serpentine channel mixers, turbulent mixers, pipette mixers, agitation mixers, passive mixers, spiral channel mixers, herringbone channel mixers, flow focusing mixers, and centrifugal channel mixers, in tandem or combined with another mixing technique for pre or post ionic flux. Operation

[0104] As mentioned, in an embodiment, the first mixer 210 (Mixer 1) is used to combine the ionic nucleic acid 206 and organic lipid solution 208. The combined solution 211 can optionally move to another vessel (e.g., a second mixer 220 / Mixer 2) to undergo a second step of mixing (Fig. 2A). Alternately, as shown in Fig 2B, an aqueous solution 316 may be introduced through a channel / inlet into a single mixer 310 (here the second mixer / Mixer 2) at from 0 to 60 minutes post combining the ionic nucleic acid 306 and organic lipid solution 308. As will be appreciated, in a single mixer embodiment, the apparatus 300 is not limited to using a second mixer, e.g., “Mixer 2,” and a “first” (or sole) mixer may be used for combining the ionic nucleic acid and organic lipid solution, as well as for the introduction of the aqueous solution 316.

[0105] An example of one embodiment of the apparatus 200 is shown in simplified schematic form in Fig. 2B. Another example of an apparatus 200 is shown in Fig. 2A, wherein there is a combining vessel (e.g., a first mixer 210 / Mixer 1) preceding a second mixer 220 / Mixer 2, into which aqueous solution 216 is introduced to create the ionic flux.

[0106] In one embodiment, the outlet parameters of the second mixer 220 / Mixer 2 shown in Fig. 2A were calculated and measured by controlling the materials in the nucleic acid in the neutral pH ionic salt solution, the lipid solution, and the aqueous solution. In embodiments, the nucleic acid in neutral pH ionic salt solution is a nucleic acid in a water solution containing ionic salts. The water may be deionized (DI) water. The lipid solution includes an organic solvent. The aqueous solution is water or a water solution including a lower ionic concentration than that of the neutral pH ionic salt solution. These solutions are combined, as shown in Fig. 2A, to create nucleic acid-containing lipid nanoparticles. In a non-limiting example, the neutral pH ionic salt solution 160 mM NaCl in water, the organic solvent is ethanol, the aqueous solution is DI water, and the resulting nucleic acid-containing lipid nanoparticles is in an 11 mM NaCl, DI water, 6.6% ethanol solution at a measured pH of 7.2 at the outlet of the Mixer 2. In embodiments, the lipid nanoparticles discharged from the outlet may include salt concentrations between 0-300 mM, e.g., about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 60 mM, about 80 mM, about 100 mM, about 120 mM, about 140 mM, about 160 mM, about 180 mM, about 200 mM, about 220 mM, about 240 mM, about 260 mM, about 280 mM, about 300 mM, or a concentration defined by a range of any two of the foregoing values. In embodiments, the ethanol percentage in the nucleic acid-containing lipid nanoparticles is between 0-20%, e.g., 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, or a percentage defined by a range of any two of the foregoing values. In embodiments, the ethanol percentage in the nucleic acid-containing lipid nanoparticles is between 0-20%, e.g., 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, or a percentage defined by a range of any two of the foregoing values.

[0107] In other examples, the lipid nanoparticles discharged from the outlet may include salt concentrations between 0-200 mM and ethanol percentages between 0-10%.

[0108] The inlet parameters and mixing parameters can be easily adjusted through flow rate ratios and starting concentrations to create lipid nanoparticles in a neutral pH with salt concentrations between 0-200 mM and ethanol percentage between 0-10%.

[0109] Having two mixers, Mixer 1 and Mixer 2 enables the method to be continuous rather than batchwise.

[0110] The inventive method for preparing nucleic acid containing lipid nanoparticles (NALNP), comprises a mixing method with an ionic flux resulting in NALNP with better size distribution and no need for buffer removal.

[0111] The inventive method includes mixing or a series of mixings, in one embodiment, one or more of those mixings are provided by bifurcating toroidal mixers. Mixing preferably includes one mixer or two mixers in series.

[0112] The step between Mixer 1 and Mixer 2 can be immediate or delayed by up to about 60 minutes. After that time, the quality of the nanoparticles is compromised. The combined mixture in Mixer 1 is flowed into Mixer 2, wherein there is an influx of aqueous solution whereby the ionic concentration was reduced.

[0113] Examples

[0114] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0115] All solvents and reagents were commercial products and used as such unless noted otherwise. Temperatures are given in degrees Celsius. The following abbreviations are used with respect to the Examples: NxGen™ mixer type is a toroidal mixing geometry disclosed in, for example, US patent no.10,076,730 and sold by Precision NanoSystems ULC (Vancouver, Canada) in a variety of sizes and formats. saRNA: Self-amplified mRNA expressing A5: SARS-Cov-2 spike protein using V101 vector as disclosed in PCT publication WO23057979 Al by Abraham, S; Chemmannur S; Geall A et al. This is the nucleic acid therapeutic used as a non-limiting representative.

[0116] IL: Ionizable lipid. One non-limiting example is PNI 516: (Z)-3-(2-((l,17-bis(2- octylcyclopropyl)heptadecan-9-yl)oxy)-2-oxoethyl)-2-(pent-2-en-l-yl)cyclopentyl 4- (dimethylamino)butanoate (as disclosed in WO 2020 / 252589).

[0117] V02 composition: A lipid composition comprising 47.5 mol% IL, 12.5 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% PEG-DMG. There are many alternatives for each component in the lipid composition, as listed in the description of the current disclosure. Furthermore, the lipid composition is not limited to the specific ratios described here and can include lipid compositions disclosed in WO23057979 Al and WO 2020 / 252589.

[0118] Methods a. Techniques for measuring the size, polydispersity index (PDI) and encapsulation efficiency (EE) of LNPs.

[0119] Size and PDI of the LNPs were measured by Dynamic Light Scattering (DLS) using a ZetaSizer™ Nano ZS™ (Malvern Instruments). He / Ne laser of 633 nm wavelength was used as the light source. Data were measured from the scattered intensity data conducted in backscattering detection mode (measurement angle = 173 Angstrom). 0.5 to 2 pL of the sample was placed in a cuvette and diluted with PBS (0.3 mL). Measurements were an average of 10 runs of two cycles each per sample. Z -Average size was reported as the particle size and is defined as the harmonic intensity averaged particle diameter. EE of the LNPs was measured by Quant- iT™ RiboGreen® RNA reagent (Invitrogen) according to manufacturer’s directions. These LNP characteristics, as well as the results of the encapsulation efficiency (EE) of the LNP encapsulating the nucleic acid are described in the following examples.

[0120] Example 1 Method for Preparing Lipid Nanoparticles (LNP) According to Embodiments of the Invention

[0121] Components of lipid compositions include ionizable lipid, structural lipid or helper lipid, sterol, and stabilizing agent. In the lipid composition, various components are combined in various molar ratios. Stabilizing agent may include PEG-DMG or stabilizing agents as defined throughout the description under that category. Lipid compositions were prepared in an organic solvent by combining prescribed amounts of components in a mixture of ionizable lipid, structural lipid, sterol and stabilizing agent from individual component stocks in the organic solvent. In embodiments, the organic solvent includes, but is not limited to, ethanol. Suitable organic solvents may include, but are not limited to, 1,4- dioxane, tetrahydrofuran, acetone, ethanol, benzene, tert-butyl, methanol, chloroform, acetonitrile, dimethyl sulfoxide, dimethylformamide, acids, or alcohols, or any combinations thereof. In embodiments, the organic solvent includes 90% ethanol or anhydrous ethanol. In the Examples, the prescribed amounts corresponded to V02 described above. It is to be understood that the present invention is not limited to V02 composition described above, rather, many other lipid compositions including different components (e.g., ionizable lipid, structural lipid or helper lipid, sterol, and / or stabilizing agent) in various molar ratios can be used with the present invention.

[0122] In embodiments, the mixing of nucleic acid therapeutics (NAT) and lipids occurred as follows. Initially, lipid solution in an organic solvent was combined with a neutral pH ionic salt solution including a payload such as the nucleic acid in Mixer 1. Ionic salts that provide neutral pH when dissolved in water including, but not limited to, NaCl or KC1 can be used to form the neutral pH ionic salt solution in which the pay load is dissolved. Through testing using sterile filters of increasing sizes, DLS, and Ribogreen assays, we determined that the lipid components in the lipid solution remained in the combined mixture and do not encapsule the payload yet. This further demonstrates that substantially no nucleic acid-containing nanoparticles are formed in the combined mixture at this stage.

[0123] The combined mixture in Mixer 1 was flowed into Mixer 2 and an aqueous solution of lower ionic concentration is added to Mixer 2. The influx of the aqueous solution triggers ionic flux and the ionic concentrations of the ionic salt in the combined mixture was reduced as the ions diffused out of the combined mixture. The self-assembly of the nucleic acid-containing lipid nanoparticles started in response to the flux flow of the ion. The NALNP formed in the Mixer 2 was harvested and tested. Testing of the contents collected from the outlet of Mixer 2 through DLS and Ribogreen assays confirmed the formation of nucleic acid-containing lipid nanoparticles (NALNP).

[0124] In a non-limiting example, self-assembling mRNA in 80 mM NaCl was added as one stream into Mixer 1 of a double microfluidic mixer as illustrated in Fig. 2A, with 16 mM ionizable lipid (in this case PNI 516) added via the second inlet stream. The nucleic acidcontaining lipid nanoparticles (NALNP) formulation collected from the output of Mixer 2 may have a (1 :4): 15 ratio (1 is lipids in organics, 4 is nucleic acid in neutral salt solution, and 15 is water).

[0125] The comparison of average diameter of LNP and the particle diameter dispersity of LNP for the prior art method versus the method of the invention is shown in Fig. 3. There is clear advantage for the method and apparatus of the invention versus the industry standard of one step mixing in acidic buffer, with the current invention achieving LNP with a lower size dispersity. This represents improved quality by most standards.

[0126] The comparison of encapsulation efficiency (EE) for the control (acidic buffer with low pH) versus the method of the invention is shown in Fig. 4. The EE accomplished in the method of the invention is closer to 100% and is better than the EE of the conventional method.

[0127] Fig. 5 shows the comparison of the size and size variation (PDI) of saRNA NALNP made by (left) the control method using low pH buffer and (right) by the method of the present invention. More significantly, the size consistency (PCI) of the new method NALNP is much better (white bar within the larger striped bar). The average size of the NALNP was under 100 nm and within the targeted size range for the NALNP.

[0128] To put these results in context, for a composition comprising a population of LNPs with different diameters, it is preferred that at least 80% by number of the LNPs have diameters in the range of 60-180 nm, e.g., in the range of 80-160 nm, and the average diameter (by intensity, e.g. Z-average measure by DLS) of the population is ideally in the range of 60-180 nm, e.g., in the range of 80-160 nm; and / or the diameters within the plurality have a polydispersity index <0.2.

[0129] Example 2

[0130] Encapsulation Efficiency as a Result of Salt type and Salt Concentration in Mixer 1

[0131] Figs. 6 and 18 display graphs of the encapsulation efficiency and the salt type / salt concentration. In Fig. 6, NaCl and KC1 were used as non-limiting examples but it is to be understood that other salts disclosed in the instant application could be used. More specifically, Fig. 6 displays the encapsulation efficiency (EE, %) on the y-axis and the salt concentration on the x-axis. The concentration of NaCl or KC1 in the neutral pH ionic salt solution used for dissolving the payload and entering into Mixer 1 (at neutral pH) was varied from 0 to 514 mM to understand the impact of salt concentration. To achieve the ionic flux effect, going into Mixer 2 requires a salt concentration difference between the neutral pH ionic salt solution and the aqueous solution is required (i.e., salt concentration going from high to low from the first mixer (e.g., Mixer 1) to the second mixer (e.g., Mixer 2) in order to achieve the principal flux of ions. In embodiments, significantly better results were found when the salt concentration difference between the fluid entering Mixer 1 and Mixer 2 was greater than 60 mM. This test was accomplished by adding neutral salt (X) in water to Mixer 2 and testing the resulting LNP.

[0132] NaCl and KC1 showed similar results over the tested concentration range.

[0133] Additional neutral salts were tested for the ionic flux mechanism, including Magnesium sulfate (MgSCE), Magnesium dichloride (MgCh), Lithium Chloride (LiCl), and Calcium Chloride (CaCh). These salts were chosen as they are all neutral salts, meaning they do not lower or raise the pH of the solution. All salts created lipid nanoparticles of sizes ranging from 69-87 nm and had a PDI below 0.07. All salts caused encapsulation of the nucleic acid above 80% EE as shown in Fig. 17. This test further demonstrates that the ionic flux method of creating particles in a universal effect for all salts.

[0134] Example 3

[0135] Encapsulation v. pH in Mixer 1

[0136] The pH in the first step in Mixer 1 were varied from about pH 6.2 to about pH 7.4 to understand the effect of pH on the method. The pH was adjusted by adding small amounts of low concentrations of sodium citrate and PBS to lower and raise the pH.

[0137] The flux mechanism worked for the range of pH tested with a decreasing performance near pH 7.4. This reduced performance near pH 7.4 is believed to be the effect of the pH on the ionizable lipid used. The pKa of the ionizable lipid used (PNI 516) is around pH 6.4, and at a 1 pH unit above an ionizable lipid pKa, very little to none of the ionizable lipid is ionized and therefore NALNP will not form (nucleic acid having negative charges which associate it with the ionizable lipid).

[0138] EE is show in Fig. 7 for pH from about 6.1 to a little under 7.4. Example 4

[0139] The Effect of Different Types of Mixer 1 and Mixer 2 on the Quality of The LNP Product

[0140] It was found that the LNP only formed after entering Mixer 2 (see Example 6), but even so, the type and quality of Mixer 1 made a greater difference to the quality of the final product.

[0141] Referring generally to Figs. 8, 23, and Table 1 below, NxGen™ toroidal mixers 400, some versions of which are sold by Precision Nanosystems, performed best for the first mixer prototypes regardless of the geometry of the second mixer. The quality of the LNP were determined by EE, diameter and PDI (smaller PDI demonstrates more consistent LNP size). Figs. 8 and 23 illustrate Mixer 1 and Mixer 2 combinations and Table 1 shows the resulting LNP characteristics. Mixer 1 and Mixer 2 may have the same channel size leading into the mixers. However, in another embodiment, Mixer 1 and Mixer 2 may have different channel sizes leading into the mixers. For example, Mixer 2 may have a larger channel than Mixer 1. The larger channel may reduce pressure since there is more fluid going through Mixer 2 than Mixer 1. As depicted, NxGen™ toroidal mixers 400 were tested along with Inline / T mixers 402, and pipette mixing 404, in various combinations. The use of NxGen™ toroidal mixers 400 for both the first mixer (Mixer 1) and the second mixer (Mixer 2) provided the best results. Test results for the various combinations is summarized below in Table 1.

[0142] Table 1. LNP characteristics for different mixing designs Additionally, the sizes of the mixers may be varied, as shown in Fig. 19. Different Mixer 1 and Mixer 2 mixer types and sizes were tested using a 1.1 kB eGFP mRNA payload. Mixer 1 was a NxGen mixer and two sizes (a 160 um channel width and a 900 um channel width) were tested and compared. Mixer 2 was either a NexGen mixer or a Tee mixer with combinations shown in Fig. 19. All mixer types and combinations created lipid nanoparticles with a high EE up to a Mixer 2 outlet concentration of 42 ug / mL. It also shows that Mixer 2 outlet concentration EE values were dependent on the Mixer 1 and 2 combinations.

[0143] Example 5

[0144] LNP Cyro-Transmission Electron Microscopy

[0145] Samples were prepared by applying 2 uL of PBS containing LNP at 20-40 mg / mL total lipid to a standard electron microscopy grid with a perforated carbon film (Protochips, Inc., Raleigh NJ). Excess liquid was removed by blotting, then the grid was plunge frozen in liquid ethane with a Vitrobot™ system (FEI, Hillsboro, OR) and then plunge-freezing the LNP suspension in liquid ethane to rapidly freeze the vesicles in a thin film of amorphous ice. Images were taken under cryogenic conditions at a magnification of 29K with a Gatan Ultrascan 1000XP-P 2k X 2k CCD camera. Samples were loaded with a Gatan Cryo-TEM (626) holder in an FEI Tecnai Osiris 200kV S / TEM under low dose conditions with an underfocus of 5-8 microns to enhance image contrast. The LNP prepared with NxGen as Mixer 1 and NxGen as Mixer 2 are shown in Fig. 9.

[0146] Example 6

[0147] Timing of Events

[0148] The effect of increasing the time delay between the time the combined mixture of ionic nucleic acid solution and lipid in organic phase elutes from Mixer 1 and the time the combination mixture enters Mixer 2 on LNP property and nucleic acid encapsulation efficiency was investigated. Mixer 1 was an adapted NxGen toroidal mixer (Precision NanoSystems) and Mixer 2 was manual pipette mixing. The state of the combined mixture of the ionic nucleic acid solution and lipid organic phase was tested with a Zetasizer at various points in time to observe rates of precipitation of the components (ionizable lipids in ethanol, nucleic acid in neutral pH ionic salt solution) out of the solution, and how the time delays affected the NALNP eluting after Mixer 2 (EE after flux). It was found that the majority of lipids and nucleic acid remained dissolved in the combined solution up to 30 minutes after being combined in Mixer 1, with 80-90% remaining dissolved at 2 minutes after Mixer 1, and 50-60% remaining dissolved at 30 minutes after Mixer 1.

[0149] At 60 minutes after Mixer 1, less than 20% of lipids and nucleic acid remained dissolved, with the remaining precipitating out of solution.

[0150] The left vertical axis of Fig 10 shows the % dissolved of ionizable lipid and nucleic acid, and other lipid components in the lipid composition (e.g., cholesterol, DSPC, PEG DMG) precipitated out of solution at a similar rate to the ionizable lipid and are not shown here. The encapsulation efficiency (EE, %) of the LNP after the time delay up to 60 minutes is shown by the right vertical axis of Fig 10.

[0151] The nucleic acid encapsulation efficiency showed a downward trend with increasing time delay.

[0152] The time delay can be created and / or adjusted in various ways. In embodiments, physical distance between Mixer 1 and 2 may be changed to adjust the time delay in a continuous flow through Mixer 1 and 2. The time delay can also be introduced by changing dimensions and / or lengths of the microfluidic channels of the Mixer 1 and / or 2. In embodiments, the time delay may be created by collecting the mixture flowing out of an outlet of Mixer 1 and after a predetermined time delay, allowing the flow of the mixture to enter an inlet of Mixer 2.

[0153] This data indicates that the ionic flux mechanism works on material that remains dissolved in solution since the % of dissolved material and the % of encapsulated material have similar downward trends with time. Material that precipitated out of solution before Mixer 2 created particles with uncontrolled size containing no nucleic acid.

[0154] It is preferrable to minimize the time delay between the fluid passing between the Mixer

[0155] 1 and Mixer 2 to maximize the quality of NALNP. In embodiments, a time delay of less than 2 minutes could be implemented. The EE after Flux could be tested as soon as 20 seconds after Mixer 2, and this time point showed the highest EE of the time delay data. It is therefore expected that the dissolved material trend would continue to follow the EE trend below time delays that were illustrated, for example, the time delay can be less than

[0156] 2 minutes. Example 7

[0157] The time it takes for LNP particles to stabilize was tested for two different nucleic acid payload concentrations. The payload concentrations of 5.6 and 14.1 ug / mL were tested at the output of Mixer 2. The encapsulation data shows a high encapsulated efficiency for a pay load concentration of 5.6 ug / mL at the shortest time of testing of 10 minutes. The higher payload concentration of 14.1 ug / mL took 180 minutes before the encapsulated efficiency reached 90%.

[0158] Example 8

[0159] The effect of the ratio between the salt concentration of the aqueous solution and the salt concentration of the neutral pH ionic salt solution (“salt ratio”) on the encapsulation efficiency of LNP is illustrated in Fig. 12. Salts such as NaCl, KC1, and other salts described in the current invention can be used. It is shown that when the salt ratio is adjusted to be within 0-0.5, a significantly higher EE is achieved. This also shows that a concentration differential is important for the ionic flux mechanism. When the ratio is controlled to be below 0.5, the salt concentration of the neutral pH ionic salt solution is much higher than that of the salt concentration of the aqueous solution, providing a driving force for the ionic flux to occur. When the salt concentration of the aqueous solution is higher than that of the salt concentration of the neutral pH ionic salt solution, the ionic flux significantly slows down, and substantially stopped when the ratio reached 2.

[0160] Example 9

[0161] The effect of ion concentration difference between the two inlets of Mixer 2 on the encapsulation efficiency of LNP was tested. The first inlet of Mixer 2 is configured to receive the combination mixture of the payload in neutral pH ionic salt solution and the lipid in organic solvent that elutes from the outlet of Mixer 1. The second inlet of Mixer 2 was configured to receive the aqueous solution having a lower ion concentration than that of the combined mixture. The payload in neutral pH ionic salt solution having a salt concentration (e.g., NaCl) of 160 mM was added into the payload inlet of Mixer 1, and the salt concentration (X (mM)) of the aqueous solution that enters the aqueous inlet of Mixer 2 was varied from 0 to 320 mM. As shown in Fig. 13, encapsulation efficiency was above 80% for salt concentrations (X) between 0 and 40 mM, and was reduced to 40% as the salt concentration increased to 160 mM. When the salt concentration of the aqueous solution was increased to 320 mM, EE went to zero. This data shows that there can be ions in both inlets of Mixer 2, but the payload inlet that is fluidically coupled to the outlet of Mixer 1 must have a higher ion concentration than the second inlet of Mixer 2.

[0162] Example 10

[0163] The Effect of Flow Rate Ratios on Ion Flux Mixing

[0164] The effect of the flow rate ratio of the various incoming streams were investigated. The flow rate ratio of the aqueous solutions (with lower ionic strength than that of the neutral pH ionic salt solution) was about 7 to about 15 times the flow rate of the lipids in organic solvent. Water or other aqueous solution including neutral salts as described herein may be used as the aqueous solution.

[0165] Triangles on the graph in Fig. 14 represent data points for 16 mM lipid concentration and flow rate ratios of (1:4): N, where 1 is the relative flow rate of lipids in organics flowing into Mixer 1, 4 is the relative flow rate of nucleic acid in neutral salt solution flowing into Mixer 1, and N is the relative flow rate of the aqueous solution flowing into Mixer 2. EE reached about 90% as N increased from 7 to 11, and then remained constant as N increased further.

[0166] Squares on the graph represent data points for 8 mM lipid concentration and flow rate ratios (1:4): N where 1 is the relative flow rate of lipids in organics, 4 is the relative flow rate of nucleic acid-containing neutral pH salt solution, and N is the relative flow rate of the aqueous solution flowing into Mixer 2. N was varied from 1 to 10. Data shows an increasing of EE as N increases from 1 to 2, and then remained constant just above 90% as N increased further.

[0167] Circles represent data points for 16 mM lipid concentration and flow rate ratios (1:9): N where 1 is the relative flow rate of lipids in organics, 9 is the relative flow rate of nucleic acid-containing neutral salt solution, and N is the relative flow rate of the aqueous solution flowing into Mixer 2. N was varied from 0.5 to 3. Data for this set of variables showed increasing EE as N increased from 0.5 to 1, and then remained constant just above 90% as N increased further.

[0168] All data sets show that the minimum flow rate ratio of Mixer 2 varied due to changes of formulation parameters, and that EE is high (but plateaus) as this ratio is increased. Example 11

[0169] Payloads

[0170] The ion flux method of creating particles works for all nucleic acid payloads tested. saRNA, pDNA, and mRNA nucleic acid payloads were tested under identical formulation conditions. A non-limiting example of the formulation parameters includes: a Mixer 1 comprising a NxGen mixer where the Mixer 1 flow rate ratio is 4: 1 (4 for the nucleic acid-containing neutral pH salt solution and 1 for the lipid in organic solvent). The second mixer comprising a NxGen mixer where the Mixer 2 flow rate ratio was 2: 1 (2 for the aqueous solution and 1 for the combined mixture including the nucleic acidcontaining neutral pH salt solution and the lipid in organics, wherein the combined mixture elutes from an outlet of Mixer 1 and enters an inlet of Mixer 2). In certain embodiments, the total flow rate was 36 mL / min with an N / P ratio of 6 and a lipid concentration of 1.2 mM which outputs a 2.1 ug / mL of the nucleic acid in lipid nanoparticles. It is to be emphasized that the parameters listed here are for illustration purposes only and not to be taken as limiting the scope of the invention.

[0171] The EE for all nucleic acids is high at above 90%, as shown in Fig. 15. Fig. 16 shows the size of the particles vary with nucleic acid and a correlation between nucleic acid length kilobase (kB) and particle size was observed. Larger payloads create larger particles (yet still within desirable size ranges). PDI for all nucleic acids were extremely low and below 0.03.

[0172] Fig. 17 shows data for the 3.3 kB and 12.8 kB pDNA. The smaller 3.3 kB pDNA has a high encapsulation efficiency over a larger payload concentration range than the larger 12.8 kB pDNA. A similar trend was observed with the larger 11.5 kB saRNA having a reduced EE at 28 ug / mL Mixer 2 outlet concentration, and the 1.1 kB eGFP mRNA having a reduced EE at 81 ug / mL Mixer 2 outlet concentration. This data suggested that smaller nucleic acid payloads may lead to a larger payload concentration range with a higher EE then that of larger nucleic acid payloads.

[0173] Example 12

[0174] In vivo and In vitro Data

[0175] An assessment of in vitro potency was conducted on cells with saRNA-containing lipid nanoparticles prepared with the ionic flux method of the invention (“Ion Flux lipid nanoparticles”). A dose response curve is shown in Fig. 20 which had a 50 % response with a 175 ug / mL dose and a R2of 0.996.

[0176] Referring now to Fig. 21, an in vivo study was conducted on mice with Ion Flux lipid nanoparticles containing EPO mRNA. In particular, three sets of data are shown in Fig. 21, a PBS control containing no lipid nanoparticles or EPO mRNA (“PBS”), a NALNP sample collected directly from an outlet of Mixer 2 containing 20 ug / mL EPO mRNA and 6.6 % ethanol (“Flux EtOH”), and a NALNP sample that was collected from the outlet of Mixer 2 and subjected to an additional downstream buffer exchange for removing ethanol, where the NALNP contains 20 ug / mL EPO mRNA (“Flux Amicon”). The mice were injected at a concentration of 0.25 mg / kg. The results showed that the NALNP produced with the method of the current invention had a high response at 6 hours after injection. The process of the current invention provides additional benefits including, but are not limited to, eliminating the additional downstream buffer exchange.

[0177] Example 13

[0178] Ionizable Lipids

[0179] Referring to Fig. 22, the Ion Flux method of the current invention worked for a variety of classes of ionizable lipids. As illustrated as non-limiting examples, PNI 516, PNI 550, PNI 560, PNI 660, PNI 723, PNI 728, PNI 734, and MC3 ionizable were tested where the lipid composition comprises 47.5 % of the ionizable lipid, 38.5 % cholesterol, 12.5 % DSPC, and 1.5 % PEG DMG (2000). The nucleic acid for this test was eGFP mRNA. All ionizable lipids encapsulated the nucleic acid payload with a high efficiency.

[0180] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0181] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0182] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest. Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / -10%. Thus, "about" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.1 %, 0.05%, 0.01 %, or 0.001 % greater or less than the stated value. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0183] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0184] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. And, as appropriate, any combination of two or more steps may be conducted simultaneously. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0185] Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

What is claimed is:

1. A method for preparing nucleic acid-containing lipid nanoparticles (NALNP) from a nucleic acid-containing neutral pH ionic salt solution and a lipid solution, the method comprising: combining the nucleic acid-containing neutral pH ionic salt solution and the lipid solution, and forming a combined mixture; and introducing an aqueous solution of lower ionic concentration than that of the neutral pH ionic salt solution into the combined mixture, thereby creating an ionic flux and forming the nucleic acid-containing lipid nanoparticles (NALNP).

2. The method of claim 1, wherein the combined mixture is formed in a first mixer having a first inlet and a second inlet; wherein the nucleic acid-containing neutral pH ionic salt solution is passed into the first mixer via the first inlet and the lipid solution is passed into the first mixer via the second inlet.

3. The method of claim 2, further comprising: closing the first inlet and second inlet; and introducing the aqueous solution of lower ionic concentration into the combined mixture within the first mixer via a third inlet in the first mixer.

4. The method of claim 1, wherein the combined mixture is formed in a first mixer and the method further comprises: passing the combined mixture from the first mixer into a second mixer wherein the aqueous solution of lower ionic concentration is introduced into the combined mixture.

5. The method of claim 4, including removing the NALNP via an output downstream of the second mixer.

6. The method of claims 2 or 4, wherein the first mixer includes a Dean vortex mixer, T-tube mixer, serpentine channel mixer, turbulent mixer, pipette mixer, agitation mixer, passive mixer, spiral channel mixer, herringbone channel mixer, flow focusing mixer, centrifugal channel mixer, or a combination thereof.

7. The method of claims 4 or 5, wherein the second mixer includes a Dean vortex mixer, T-tube mixer, serpentine channel mixer, turbulent mixer, pipette mixer, agitation mixer, passive mixer, spiral channel mixer, herringbone channel mixer, flow focusing mixer, centrifugal channel mixer, or a combination thereof.

8. The method of claims 1 - 4, wherein a time gap between after mixing of the neutral pH ionic salt solution and the lipid solution to form the combined mixture, and before addition of the aqueous solution of lower ionic strength to the combined mixture is from about 0 to about 60 minutes.

9. The method of claims 1 - 4, wherein a pH of the neutral pH ionic salt solution and the aqueous solution is between a pH of about 6.0 and a pH of 7.4.

10. The method of claims 1 - 4, wherein a pH of the neutral pH ionic salt solution and the aqueous solution is between a pH of 6.5 and a pH of 7.4.

11. The method of claims 1 - 4, wherein a pH of the neutral pH ionic salt solution and the aqueous solution is selected from a pH of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, and 7.4.

12. The method of claim 1 - 4, wherein the neutral pH ionic salt solution includes one or both of NaCl and KC1 salts to provide ions.

13. The method of claims 1 - 4, wherein an ionic concentration of the ionic salt solution is in a range of about 10 mM to 600 mM.

14. The method of claim 1 - 4, wherein an ionic concentration of the combined mixture is at least 60 mM greater than an ionic concentration of the aqueous solution.

15. The method of claim 1, wherein the aqueous solution includes an ionic salt that is the same type of ionic salt as in the neutral pH ionic salt solution.

16. The method of claim 1, wherein substantially no nucleic acid-containing nanoparticles are formed in the combined mixture before introducing the aqueous solution.

17. The method of claim 1, wherein the nucleic acid-containing lipid nanoparticles (NALNP) formed is in a solution having a neutral pH.

18. The method of claim 1, wherein the nucleic acid-containing lipid nanoparticles (NALNP) formed is in a solution including an ionic concentration in a range of 0-300 mM.

19. The method of claim 1, wherein the nucleic acid-containing lipid nanoparticles (NALNP) formed is in a solution including 1-10% of ethanol.

20. An apparatus for preparing nucleic acid-containing lipid nanoparticles (NALNP) at neutral pH, the apparatus comprising: a first mixer downstream of a first inlet and a second inlet; a second mixer downstream of the first mixer, the second mixer being fluidly connected to the first mixer, the second mixer having a third inlet and an output for a NALNP product to exit the second mixer; wherein one of the first inlet and the second inlet into the first mixer provides an entry for a nucleic acid-containing neutral pH ionic salt solution; wherein the other one of the first inlet and the second inlet into the first mixer provides an entry for a lipid solution; and wherein the third inlet provides entry for an aqueous solution with a lower ionic concentration than that of the neutral pH ionic salt solution before the neutral pH ionic salt solution enters the first mixer.

21. The apparatus of claim 20, wherein each of the first mixer and the second mixer is independently a Dean vortex mixer, T-tube mixer, serpentine channel mixer, turbulent mixer, pipette mixer, agitation mixer, passive mixer, spiral channel mixer, herringbone channel mixer, flow focusing mixer, centrifugal channel mixer, or a combination thereof.

22. The apparatus of claim 21, wherein at least one of the first mixer and the second mixer are Dean vortex channel mixers.

23. The apparatus of claim 20, wherein the third inlet provides passage of aqueous solution into the second mixer to create an ionic flux, thereby facilitating formation of the NALNP.

24. The apparatus of claim 20, wherein the first mixer and the second mixer are arranged in tandem or are combined into a single mixer.

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