Vitrified reagents for enhanced transfection and processes thereof

Abstract

Provided herein are transfection compositions that include a transfection agent wherein the transfection composition is in a glassy state with a water content of 10 weight percent or less. The transfection composition may include a vitrification agent and the transfection agent, optionally wherein a ratio of the transfection agent to the vitrification medium is greater than 1:10, optionally wherein a ratio of the transfection agent to the vitrification medium is less than or equal to 1:200. Also provided are processes of transfecting a cell or tissue with a transfection composition as provided herein. Also provided are processes of forming a transfection composition as provided herein.

Description

VITRIFIED REAGENTS FOR ENHANCED TRANSFECTION AND PROCESSES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application depends from and claims priority to U.S. Provisional Application No: 63 / 621,297 filed January 16, 2024, the entire contents of which are incorporated herein by reference.FIELD

[0002] The present disclosure concerns compositions and methods for transfection of cells or tissues with one or more biological materials.BACKGROUND

[0003] Expression of non-native protein or nucleic acid sequences or alternative levels of a native protein or nucleic acid sequence in a cell or tissue involve introducing a system into that cell or tissue enabling the expression of the desired sequence, or alternatively altering the natural expression levels of the desired sequence. Many techniques exist for the introduction of materials into a cell or tissue, yet liposome-mediated transfection (lipofection) remains one of the most robust and safest means of introducing materials into a cell.

[0004] Lipofection involves the use of liposome forming lipids, such as cationic lipids or non-lipid polymers, alone or with one more helper molecules to fuse to a cell wall and allow penetration and delivery of a cargo molecule. This method has been used for the delivery of many types of molecules including but not limited to nucleic acids, proteins, cytotoxic drugs, among many others demonstrating that this technique is versatile and robust.

[0005] Liposome-mediated transfection of cells offers several advantages over other transfection methods. For example, liposome-mediated delivery can achieve relatively high efficiency of nucleic acid transfer into cells. This method is particularly suited to cell types, such as primary cells that are very sensitive to toxic effects of other traditional chemical transfer reagents such as calcium phosphate or DEAE-dextran. Further, not only can this technique be used for the delivery of a cargo molecule in vitro, but lipofection has been successfully employed for cargo delivery in vivo, which is in contrast to other delivery methods such as chemical methods or electroporation, both of which are unsuitable for in vivo delivery.

[0006] Liposomes designed for gene transfer are generally prepared in an aqueous solvent and form bilayer structures. Unilamellar vesicles are generally prepared by sonication, and the resultant liposome vesicles typically have sizes ranging from 50 to 100 nm. Multilamellarvesicles, 300-700 nm in diameter, can be prepared by vortex action of lipids in an aqueous solvent. Unilamellar and multilamellar vesicles have both successfully been used as gene delivery reagents. Selecting the appropriate transfection reagent to achieve optimum transfection efficiency is important for the best results. An ideal reagent ideally has low cytotoxicity, high transfection efficiency for the required cell types, and thermal stability. Typically, the reagents are stored at +2 °C to +8 °C. While transfection efficiency for existing agents is high, it is desirable to improve transfection efficiency so as to either be able to reduce dosing of a particular agent and reduce the amount of reagents used and therefore cost. Further, the requirement for cold chain storage makes shipping of reagents difficult and requires specific storage requirements, for which some delivery areas are not suitable.

[0007] As such, new reagents and processes are needed for improving transfection efficiency and delivery of a cargo to a cell or tissue.SUMMARY

[0008] The following summary is provided to facilitate an understanding of some of the innovative features unique to the present disclosure and is not intended to be a full description. A full appreciation of the various aspects of the disclosure can be gained by taking the entire specification, claims, drawings, and abstract as a whole.

[0009] Provided herein are transfection compositions that include a transfection agent, wherein the transfection composition is in a glassy state with a water content of 10 weight percent or less. Optionally, a ratio of the transfection agent to the vitrification medium is greater than 1 : 10. Optionally, a ratio of the transfection agent to the vitrification medium is less than or equal to 1 :200, optionally 1 : 150, optionally 1 : 100, optionally 1 :90, optionally 1 :80, optionally 1 :70, optionally 1 :60. In some aspects, the transfection composition further includes a biological material, optionally a biological material comprising one or more polynucleotide chains. Optionally, a polynucleotide chain comprises an expression vector. In addition or alternatively, a biological material may include an enzyme, an antibody, a protein, polydeoxyribonucleotide chain, a polyribonucleotide chain, siRNA, shRNA, Cas9 or a Cas9 expression plasmid, a cell or cell fragment, or a genome or portion thereof. Optionally, a transfection agent comprises one or more cationic polymers, a lipid, a virus, a cell penetrating peptide, a dendrimer, or a polynucleotide-binding protein. Optionally, a cationic polymer comprises one or more of DEAE-dextran, poly-amino acids (optionally poly-L-lysine),poly(amidoamine) (PAMAM) dendrimers, PPI (poly-(propylenimine)), PEI (polyethylenimine), PDMAEMA (polymethacrylic acid N,N-dimethylaminoethyl ester), poly(P-amino ester), PHP (Poly(4-hydroxy-L-proline ester)), PAGA (poly[a-(4-aminobutyl)- 1-glycolic acid]), PVL poly(S-valerolactone), aminated PAHA (aminated poly(a-hydroxy acids)), PPE (polyphosphoester), polylactide (poly(lactic acid), PLA), N,N- diethylethylenediamine-polyurethane (DEDA-PU), poly(p-phenyleneethynylene), polythiophene, poly(fluorine-co-phenylene), poly(p-phenylenevinylene), protamine, or a combination thereof. Optionally, a lipid includes DOSPA (2,3 -di oleoyloxy -N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propaniminium trifluoroacetate), 1,2- Dioleoyl-sn-glycerophosphoethanolamine (DOPE), DOTMA (l,2-Dioleoyl-3- trimethylammonium propane), cholesterol, polyethylene glycol (PEG)-lipid conjugate, 3060iio (tetrakis(8-methylnonyl) 3,3',3",3"'-(((methylazanediyl) bis(propane-3,l diyl))bis (azanetriyl))tetrapropionate; 9A1P9, decyl (2-(dioctylammonio)ethyl) phosphate); A2-Iso5- 2DC 18 (ethyl 5, 5-di((Z)-heptadec-8-en- 1 -y 1)- 1 -(3 -(pyrrolidin- 1 -yl)propyl)-2, 5 -dihydro- 1H- imidazole-2-carboxylate); ALC-0315 (2-hexyl-decanoic acid, l,l’-[[(4- hy droxybutyl)imino]di-6, 1 -hexanediyl] ester); ALC-0159 (a-[2~(ditetradecylamino)-2- oxoethyl] -©-methoxy -poly (oxy- 1 ,2-ethanediyl)); P-sitosterol ((3 S, 85, 9S, 1 OR, 137?, 145, 177?)- 17-((27?,57?)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl- 2,3,4,7,8,9,10,11, 12, 13, 14, 15, 16,17-tetradecahydro-177-cy cl openta[a]phenanthren-3-ol); BAME-O16B (2-(dodecyldisulfanyl)ethyl 3-[2-[2-[bis[3-[2-(dodecyldisulfanyl)ethoxy]-3- oxopropyl]amino]ethyl-methylamino]ethylamino]propanoate); BHEM-Cholesterol ((N,N- bis(2-hydroxyethyl)-N-methyl-N-(2-cholesteryloxy carbonyl aminoethyl) ammonium bromide); cKK-E12 (3, 6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione); DC-Cholesterol (3P-[ V-( V', V'-dimethylaminoethane)-carbamoyl]cholesterol); DLin-MC3- DMA ((6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino) butanoate); DOPE ([(2R)-3-[2-aminoethoxy(hydroxy)phosphoryl]oxy-2-[(Z)-octadec-9-enoyl]oxypropyl] (Z)-octadec-9-enoate); DO SPA (2- [2, 5 -bi s(3 -aminopropylamino)pentanoylamino] ethyl- [2, 3 - bis[(Z)-octadec-9-enoxy]propyl]-dimethylazanium;chloride;tetrahydrochloride); DOTAP (2,3-bis[[(Z)-octadec-9-enoyl]oxy]propyl-trimethylazanium); DOTMA (2,3-bis[(Z)-octadec- 9-enoxy]propyl-trimethylazanium;chloride); DSPC ([(2R)-2,3-di(octadecanoyloxy)propyl] 2- (trimethylazaniumyl)ethyl phosphate); ePC (ethylphosphatidylcholine); FTT5 (octan-4-yl 9- [3-[[3,5-bis[3-[bis(9-octan-4-yloxy-9- oxononyl)amino]propylcarbamoyl]benzoyl]amino]propyl-(9-octan-4-yloxy-9- oxononyl)amino]nonanoate); Lipid H (SM-102) (heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate); OF-Deg-Lin (2-[4-[5-[4-[bis[2-[(9Z,12Z)-octadeca-9,12- dienoyl]oxyethyl]amino]butyl]-3,6-dioxopiperazin-2-yl]butyl-[2-[(9Z,12Z)-octadeca-9,12- dienoyl]oxyethyl]amino]ethyl (9Z,12Z)-octadeca-9,12-di enoate); PEG2000-DMG (1,2- Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000); TT3 (N1, N3, N5-tri s(3-(didodecylamino)propyl)benzene-l,3,5-tricarboxamide), or combinations thereof.

[0010] In any of the foregoing paragraphs of this section in any combination, the transfection agent and said biological material are intermixed or said biological material is housed within the transfection agent. Optionally, the transfection agent and said biological material are layered in or on a substrate. Optionally, the transfection composition is associated with a substrate comprising a capillary network, optionally wherein the capillary network is provided by contours along a surface of the substrate. A substrate may be or include a wall of a desiccation chamber or is associated with a wall of the desiccation chamber. Optionally, the capillary network is contacted by an underlying solid support substrate.

[0011] In any of the foregoing paragraphs of this section in any combination, the vitrification agent may be trehalose and one or more of glycerol, betine, and / or choline. Optionally, a total volume of transfection composition is from 0.1 pL to 10 pL. Optionally, the transfection composition is present in a vitrification mixture comprising said vitrification agent, at 1 pg or less. Optionally, the transfection composition is of a total mass of 5 pg or less, optionally less than 1 pg, optionally a total mass of 0.1 pg to 1 pg.

[0012] Also provided are processes of transfecting a cell or a tissue including contacting a cell or tissue with the transfection composition of any of the foregoing paragraphs of this section in any combination. Optionally, a transfection efficiency of the transfecting is greater than a control transfection efficiency of a control transfection composition not subjected to said glassy state and / or wherein a transfection efficiency of the transfecting is double or greater, optionally triple, optionally four-fold, than a control transfection efficiency of a control transfection composition not subjected to said glassy state. A process optionally further includes eluting the transfection composition in an elution buffer prior to contacting with the cell or tissue. An elution buffer optionally includes a buffering agent, water and optionally one or more salts. A cell is optionally a eukaryotic cell or a prokaryotic cell.

[0013] Also provided are processes of forming a transfection composition of any of the foregoing paragraphs of this section in any combination, optionally including: overlaying a vitrification mixture including the transfection agent and a vitrification medium on a substrateincluding a capillary network, the substrate in a desiccation chamber, wherein the vitrification medium comprises the vitrification agent; lowering an atmospheric pressure within the desiccation chamber; providing a heat energy from the surface to the vitrification mixture, wherein the heat energy is sufficient to prevent the vitrification mixture from experiencing a freezing condition; and desiccating the vitrification mixture by capillary action until the vitrification mixture enters the glassy state; wherein the providing is optionally simultaneous with or prior to initiating the desiccating. Optionally, the transfection agent is layered onto or into the substrate. Optionally, the transfection agent is intermixed with the biological material. Optionally, the biological material is layered onto the transfection agent onto or into the substrate. Optionally, the biological material is layered onto the transfection agent prior to the step of desiccating. Optionally, the biological material is layered onto the transfection agent following the step of desiccating, and further comprising subjecting the biological material to a second desiccating step. In any of the foregoing in any combination, capillary network is optionally provided by contours along the surface of the substrate. Optionally, the substrate is a wall of the desiccation chamber or is associated with a wall of the desiccation chamber. In some aspects, the capillary network within the desiccation chamber is contacted by an underlying solid support substrate. Optionally, vitrification of the vitrification mixture occurs in less than 30 minutes, optionally less than 10 minutes, optionally less than 1 minute. In some aspects, the heat energy is provided by heating the vitrification mixture. Optionally, in the processes, the atmospheric pressure is lowered to a value of from about 0.9 atm to about 0.005 atm, optionally lowered to about 0.004 atm. Optionally, the heat energy provided is sufficient to prevent crystallization within the vitrification mixture during vitrification. Optionally, the provided heat energy is sufficient to keep the biological material at a temperature of from about 0 °C to about 40 °C during the vitrifying. Optionally, the vitrification medium includes trehalose and one or more of glycerol, betine, and / or choline. In some aspects, the capillary network is hydrophilic. Optionally, the capillary network includes capillary channels with a continuous wall from a first side of the substrate to a second side of the substrate.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The aspects set forth in the drawings are illustrative and exemplary in nature and not intended to limit the subject matter defined by the claims. The following detailed description of the illustrative aspects can be understood when read in conjunction with the following drawings.

[0015] FIG. 1 illustrates transfection of CH0-K1 cells using a transfection composition with a vector encoding GFP wherein (A) illustrates fluorescence of the cells following transfection and (B) illustrates quantification of the GFP expression in the cells.

[0016] FIG. 2 illustrates that the relative amount of vitrification agent relative to transfection agent dramatically effects the resulting transfection efficiency wherein (A) illustrates fluorescence of the cells following transfection and (B) illustrates quantification of the GFP expression in the cells at various ratios of transfection agent:vitrification medium, and (C) and (D) illustrate that a fast transition was observed at a ratio of 1 : 18 from 1 : 17 where suddenly the transfection efficiency doubled.

[0017] FIG. 3 illustrates transfection of CHO-K1 cells using a transfection composition with a vector encoding GFP wherein (A) illustrates fluorescence of the cells following transfection and (B) illustrates quantification of the GFP expression in the cells and demonstrating that while ordering of reagents all improve transfection efficiency, combining the transfection agent with the biological material then vitrifying the combination resulted in the greatest improvement of transfection efficiency relative to the control non-vitrified material.

[0018] FIG. 4 illustrates that using LNPs, that transfection of HEK-293 cells demonstrated that when the vitrification buffer is added to the cells with the LNP the cells show improved transfection in each cell (A) and successful transfection of more cells (B).

[0019] FIG. 5 illustrates that titrating the volume ratio of LNP to vitrification medium is able to illustrate improved transfection wherein the LNP (PC) was either held constant and the vitrification medium was titrated (A) or the vitrification medium (VB) was held constant and the LNP titrated (B).DETAILED DESCRIPTION

[0020] As required, detailed aspects of the present disclosure are disclosed herein; however, it is to be understood that the disclosed aspects are merely exemplary of the disclosure that may be embodied in various and alternative forms.

[0021] Provided herein are reagents and methods that dramatically improve transfection efficiency of materials suitable for use in liposome-mediated, including lipid nanoparticle, transfection of cells or tissues. It was unexpectedly found that by incorporating transfection agents in an amorphous glass then reconstituting them in a desired buffer or water that theability of these agents to promote transfection is dramatically increased thereby radically improving transfection efficiency relative to materials that were not subjected to incorporation in an amorphous glass.

[0022] The formation of an amorphous glass with respect to biological molecules and agents is termed herein as vitrification. Vitrification is the process of direct transition from a liquid to an amorphous glassy state. The process of vitrification is often applied to preserve biological materials and historically involves cooling the biological materials to cryogenic temperatures either intentionally or due to exposure to reduced atmospheric pressures. At cryogenic temperatures, vitrification techniques may lead to exposure to some of the damaging effects that normally occur with cryogenic storage due to ice cr stal formation, which are known to form during conventional cryopreservation. To help address this issue, cryogenic protectants (CPAs) are used often at high concentrations to avoid ice nucleation. Lower concentrations of CPAs are desired but in order to achieve preservation at these lower concentrations, ultra-fast heat transfer is required.

[0023] Anhydrous vitrification at ambient temperatures is an alternative strategy for preserving biological materials. In nature, a wide variety of organisms can survive extreme dehydration, which correlates in many cases with the accumulation of large amounts (as much as 20% of their dry weight) of glass forming sugars such as trehalose and sucrose in intracellular space. Such “glass forming” sugars historically need to be present on both sides of the plasma membrane to provide protection against the damaging effects of desiccation. Desiccation techniques dramatically limit or arrest the material’s biochemical processes in a glassy matrix.

[0024] Prior techniques for desiccation have applied a vacuum to rapidly dry materials. However, the application of the vacuum can significantly lower the temperature of the vitrification mixture such that the transfection agent or biological material experiences ice crystal formation, which can lead to damage. Furthermore, the atmospheric change provided by a vacuum also affects the boiling point of materials therein, providing a further risk to damaging the materials. It was recently found that by preventing exposure of the materials to cryogenic temperatures by particular techniques such as those described in WO 2022 / 109315, further improvements in both the rate of vitrification and the stable storage characteristics of the vitrified materials could be achieved. The fast desiccation methods of WO 2022 / 109315 produce improved viability of vitrified biological material through avoidance of freezing within the sample and without solution boiling to as to significantly facilitate long term storage ofbiological materials at non-cryogenic temperatures as well as overcome the challenges associated with prior cryogenic vitrification and storage technologies.

[0025] By forming an amorphous glass using either vitrification or lyophilization techniques in the presence of a glass forming agent (vitrification agent), it was surprisingly found that transfection agents such as cationic lipids and non-lipid polymers are far more readily able to promote transfection of a cell. As such, provided herein are reagents for cell or tissue transfection and delivery of one or more cargo molecules (e.g. biological materials) to the intracellular space, and related processes of production and use thereof.

[0026] The following terms or phrases used herein have the exemplary meanings listed below in connection with at least one aspect:

[0027] As used herein “cryogenic” temperature or temperatures for “cryogenesis” or similar refer to a temperature at which a transfection agent and / or the biological material is exposed to freezing conditions. It will be understood in some aspects that the cryogenic temperature may include a freezing temperature of the transfection agent, biological material and / or vitrification medium. It should further be understood that a cryogenic temperature is not bound by a particular threshold or range of values of temperatures in either Fahrenheit or Celsius, but instead can be determined by the relationship between temperature, pressure and molecular energy for the vitrification mixture of interest. It is further to be understood that as used herein, while certainly possible within the definition as set forth, “cryogenesis” and similar derivatives thereof are not limited to temperatures associated with liquid nitrogen at 1 atm (atmosphere) or of about -80 °C.

[0028] “Above cryogenic temperature,” as used herein, accordingly refers to a temperature above the freezing point of a vitrification mixture. A point “above cryogenic temperature” may further include temperature values wherein relation to the surrounding atmosphere and the molecular energy, a freezing condition is absent. Room temperature, as used herein, refers to a temperature of about 25 °C.

[0029] As used herein, “boiling” may refer to a point at which a material transitions to a vapor, often marked by the formation of vapor bubbles within the material that can escape into a surrounding atmosphere and dissipate therein.

[0030] “ Glass transition temperature” or “Tg” means the temperature above which material behaves like liquid and below which material behaves in a manner similar to that of a solid phase and enters into amorphous / glassy state. This is not a fixed point in temperature, but isinstead variable dependent on characteristics of the vitrification mixture of interest. In some aspects, glassy state may refer to the state the vitrification mixture enters upon dropping below its glass transition temperature.

[0031] “Amorphous” or “glass” refers to a non-crystalline material in which there is no long-range order of the positions of the atoms referring to an order parameter of 0.3 or less. Solidification of a vitreous solid occurs at the glass transition temperature Tg. In some aspects, the vitrification mixture may be in the form of an amorphous material.

[0032] “Crystal” means a three-dimensional atomic, ionic, or molecular structure consisting of one specific orderly geometrical array, periodically repeated and termed lattice or unit cell.

[0033] “Crystalline” means that form of a substance that is comprised of constituents arranged in an ordered structure at the atomic level, as opposed to glassy or amorphous. Solidification of a crystalline solid occurs at the crystallization temperature Tc.

[0034] “Vitrification” as used herein, is a process of converting a material into an amorphous solid material. The amorphous solid may be free of any crystalline structure.

[0035] “Vitrification mixture” as used herein, means a heterogeneous mixture of transfection agent(s) and a vitrification medium containing one or more vitrification agents and optionally other materials.

[0036] “Biological material” as used herein, refers to materials that may be isolated or derived from living organisms or can encode a material that is expressible or functional within a cell or tissue. Examples of biological materials include, but are not limited to, proteins, nucleic acids such as DNA, RNA, siRNA, shRNA, etc., cells, tissues, organs, cell-based constructs, fragments thereof, or combinations thereof. In some aspects, biological material may refer to a nucleic acid. In other aspects, biological material may refer to proteins, enzymes, or active fragments thereof, or combinations thereof. In some aspects, biological material may refer to reproductive cells including sperm cells, spermatocytes, oocytes, ovum, blastocysts, embryos, germinal vesicles, or combinations thereof. Optionally, however, biological material may exclude reproductive cells including sperm cells, spermatocytes, oocytes, ovum, blastocysts, embryos, germinal vesicles, or combinations thereof. In other aspects, biological material may refer to whole blood, red blood cells, white blood cells, platelets, viruses, bacteria, algae, fungi, or combinations thereof.

[0037] “Vitrification agent” as used herein, is a material that forms an amorphous structure, or that suppress the formation of crystals in other material(s), as the mixture of the vitrificationagent and other material(s) cools and desiccates. The vitrification agent(s) may also provide osmotic protection or otherwise enable cell survival during dehydration. In some aspects, the vitrification agent(s) may be any water soluble solution that yields a suitable amorphous structure of a transfection agent or biological material.

[0038] “ Storable or storage” as used herein, refers to a biological material’s ability to be preserved and remain viable for use at a later time.

[0039] “Hydrophilic” as used herein, means attracting or associating preferentially with water molecules. Hydrophilic materials with a special affinity for water, maximize contact with water and have smaller contact angles with water relative to hydrophobic materials.

[0040] “Hydrophobic” as used herein, means lacking affinity for water. Materials that are hydrophobic naturally repel water, causing droplets to form, and have large contact angles with water.

[0041] “Capillary” as used herein, pertains to or occurring in or as if in a tube of fine bore having a cross sectional area of about 2000 m2or less.

[0042] “Cryopreservation” typically refers to rapid cooling of a material, often through the use of liquid nitrogen due to its low temperature which will rapidly cool a liquid material, or small volume of biological materials by direct immersion. The rate of cooling reduces the mobility of the material’s molecules before they can pack into a more thermodynamically favorable crystalline state. Over a more prolonged period, the molecules can arrange to crystallize which can produce damaging results, particularly in biological materials. Water is a significant concern in biological materials as it can crystallize quickly, and its abundance in living tissues can prove to be significantly damaging the more it is allowed to crystallize. Protective additives, often referred to as cryoprotectants, that interfere with the primary constituent’s ability to crystallize may produce amorphous / vitrified material.

[0043] Provided herein are transfection compositions that include one or more transfection agents and one more vitrification agents wherein the transfection composition is in a glassy state with a water content of 10 weight percent or less. Such compositions may, in some aspects, be prepared in such a manner that the composition may be free or substantially free of crystal structure. In some aspects, the relative ratio of the transfection agent to the vitrification agent promotes vastly superior transfection efficiency. Optionally, the ratio of the transfection agent to amount of vitrification medium housing the vitrification agent was found todramatically improve transfection efficiency relative to other ratios that do not appreciably alter transfection efficiency.

[0044] As such, in some aspects, the ratio of the transfection agent (pg) to a vitrification medium (pl (1200 mM vitrification agent)) is about 1 :0.5, optionally about 1.1, optionally about 1 :3, optionally about 1 : 10 or greater vitrification medium. It was found that an unexpected increase in transfection efficiency is found when using transfection agents that were vitrified with a vitrification medium at a ratio of about 1 : 18 or greater relative amounts of vitrification medium. “About” is defined with respect to the ratio of transfection agent to vitrification medium to vary by as much as 30% based on experimental variability. As such, in some aspects, a ratio of transfection agent to vitrification medium (as defined above relative to an amount of vitrification agent) is optionally about 1 :0.5 or greater amount of vitrification medium t, optionally about 1 : 1 or greater amount of vitrification medium, optionally about 1 :2 or greater amount of vitrification medium, about 1 :3 or greater amount of vitrification medium, about 1 :4 or greater amount of vitrification medium, about 1 :5 or greater amount of vitrification medium, about 1 :6 or greater amount of vitrification medium, about 1 :7 or greater amount of vitrification medium, about 1 :8 or greater amount of vitrification medium, about 1 :9 or greater amount of vitrification medium, about 1 : 10 or greater amount of vitrification medium, about 1 : 11 or greater amount of vitrification medium, about 1 : 12 or greater amount of vitrification medium, optionally about 1 : 13 or greater amount of vitrification medium, optionally about 1 : 14 or greater amount of vitrification medium, optionally about 1 : 15 or greater amount of vitrification medium, optionally about 1 : 16 or greater amount of vitrification medium, optionally about 1 :17 or greater amount of vitrification medium, optionally about 1 : 18 or greater amount of vitrification medium, optionally about 1 : 19 or greater amount of vitrification medium, optionally about 1 :20 or greater amount of vitrification medium, optionally about 1 :21 or greater amount of vitrification medium, optionally about 1 :22 or greater amount of vitrification medium, optionally about 1 :23 or greater amount of vitrification medium, optionally about 1 :24 or greater amount of vitrification medium, optionally about 1 :25 or greater amount of vitrification medium.

[0045] In addition, it was found that transfection efficiency of a transfection agent was reduced when the ratio (as defined above) of transfection agent to vitrification medium exceeded a relative vitrification medium volume of about 100 pl (at 1200 mM vitrification agent)) relative to 1 pg transfection agent. It is not believed that the reduction in transfection efficiency at higher vitrification agent amounts is due to reduced amounts of transfection agentvitrified as the power of the vitrification protocol is that wide variability in initial volumes may be vitrified and the entire amount of vitrification mixture is loaded on to the scaffold for vitrification such that the same amount of transfection agent is vitrified, yet at higher relative amounts of vitrification medium (e.g. vitrification medium amounts producing a ratio of 1 :39 or greater amounts of vitrification medium) produce reduced levels eventual transfection efficiency. As such, a maximum ratio of transfection agent to vitrification medium where increasing or maximum is based on increased relative amounts of vitrification medium may be about 1 :40, optionally about 1 :45, optionally about 1 :45, optionally about 1 :50, optionally about 1 :55, optionally about 1 :60, optionally about 1 :70, optionally about 1 :80, optionally about 1 :85, optionally about 1 :90, optionally about 1 :95, optionally about 1 : 100.

[0046] In some aspects, a ratio of transfection agent (pg) to a vitrification medium (pl (1200 mM vitrification agent)) is optionally about 1 : 1 to about 1 : 100, optionally about 1 : 1 to about 1 :50, optionally about 1 :3 to about 1 : 100, optionally about 1 :3 to about 1 :50, optionally about 1 : 1 to about 1 :25, optionally about 1 :3 to about 1 :25, optionally about 1 : 1 to about 1 :20, optionally about 1 :3 to about 1 :20, or any value or range therebetween.

[0047] Alternatively, a mass ratio of transfection agent to vitrification agent dramatically effects the transfection efficiency. Optionally, a mass ratio (pg: pg) of transfection agent to vitrification agent is 1 : 1644 or greater relative amount of vitrification agent. Optionally, the mass ratio is about 1 :3699 or greater relative amount of vitrification agent. Optionally, the mass ratio is about 1 :6987 or greater relative amount of vitrification agent. Optionally, the mass ratio is about 1 :7398 or greater relative amount of vitrification agent. Optionally, the mass ratio is about 1 :7809 or greater relative amount of vitrification agent. Optionally, the mass ratio is about 1 : 16029 or greater relative amount of vitrification agent. Optionally, the mass ratio is about 1 :24249 or greater relative amount of vitrification agent. Optionally, the mass ratio of transfection agent to vitrification agent is about 1 :41100 or less relative amount of vitrification agent. Optionally, the mass ratio of transfection agent to vitrification agent is about 1 :39045 or less relative amount of vitrification agent. Optionally, the mass ratio of transfection agent to vitrification agent is about 1 :36990 or less relative amount of vitrification agent. Optionally, the mass ratio of transfection agent to vitrification agent is about 1 :34935 or less relative amount of vitrification agent. Optionally, the mass ratio of transfection agent to vitrification agent is about 1 :32880 or less relative amount of vitrification agent. Optionally, the mass ratio of transfection agent to vitrification agent is about 1 :30825 or less relative amount of vitrification agent. Optionally, the mass ratio of transfection agent to vitrification agent is about1 :28770 or less relative amount of vitrification agent. Optionally, the mass ratio of transfection agent to vitrification agent is about 1 :26715 or less relative amount of vitrification agent. Optionally, the mass ratio of transfection agent to vitrification agent is about 1 :24660 or less relative amount of vitrification agent. Optionally, the mass ratio of transfection agent to vitrification agent is about 1 : 24249 or less relative amount of vitrification agent. It is appreciated that in some aspects, the mass ratio of transfection agent to vitrification agent is optionally about 1 :4932 to about 1 :41100 or any value or range therebetween, optionally about 1 :7398 to about 1 :41110 or any value or range therebetween, optionally about 1 :7398 to about 1 :24249 or any value or range therebetween.

[0048] In some aspects, a ratio of transfection agent (pg) to vitrification medium pl (1200 mM vitrification agent)) is optionally about 1 : 12 or greater amount of vitrification medium, optionally 1 : 13 or greater amount of vitrification medium, optionally 1 : 14 or greater amount of vitrification medium, optionally 1: 15 or greater amount of vitrification medium, optionally 1 : 16 or greater amount of vitrification medium, optionally 1 : 17 or greater amount of vitrification medium, optionally 1 : 18 or greater amount of vitrification medium, optionally 1 : 19 or greater amount of vitrification medium, optionally 1 :20 or greater amount of vitrification medium, optionally 1 :21 or greater amount of vitrification medium, optionally 1 :22 or greater amount of vitrification medium, optionally 1 :23 or greater amount of vitrification medium, optionally 1 :24 or greater amount of vitrification medium, optionally 1 :25 or greater amount of vitrification medium, and a maximum ratio of transfection agent to vitrification medium where increasing or maximum is based on increased relative amounts of vitrification medium is about 1 :40, optionally about 1 :45, optionally about 1 :45, optionally about 1 :50, optionally about 1 :55, optionally about 1 :60, optionally about 1 :70, optionally about 1 :80, optionally about 1 :85, optionally about 1 :90, optionally about 1 :95, optionally about 1 :100.

[0049] A transfection agent as used herein is optionally includes one or more cationic polymers, lipids, viruses or portions thereof, cell penetrating peptides, dendrimers, polynucleotide-binding proteins, or any combination thereof. In particular aspects, a transfection agent is a cationic polymer or a lipid. Optionally, the cationic polymer may be one or more of DEAE-dextran, poly-amino acids (optionally poly-L-lysine), poly(amidoamine) (PAMAM) dendrimers, PPI (poly-(propylenimine)), PEI (polyethylenimine), PDMAEMA (polymethacrylic acid N,N-dimethylaminoethyl ester), poly(P-amino ester), PHP (Poly(4- hydroxy-L-proline ester)), PAGA (poly[a-(4-aminobutyl)-l-glycolic acid]), PVL (poly(5-valerolactone), aminated PAHA (aminated poly(a-hydroxy acids)), PPE (polyphosphoester), polylactide (poly(lactic acid), PLA), N,N-diethylethylenediamine-polyurethane (DEDA-PU), poly(p-phenyleneethynylene), polythiophene, poly(fluorine-co-phenylene), poly(p- phenylenevinylene), protamine, or a combination thereof.

[0050] Optionally, a lipid used in the system as described herein may be: DOSPA (2,3- dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propaniminium trifluoroacetate), 1,2-Dioleoyl-sn-glycerophosphoethanolamine (DOPE), DOTMA (1,2- Dioleoyl-3 -trimethylammonium propane), cholesterol, polyethylene glycol (PEG)-lipid conjugate, 3060iio (tetrakis(8-methylnonyl) 3,3',3",3"'-(((methylazanediyl) bis(propane-3,l diyl))bis (azanetriyl))tetrapropionate; 9A1P9, decyl (2-(dioctylammonio)ethyl) phosphate); A2-Iso5 -2DC 18 (ethyl 5 , 5 -di((Z)-heptadec-8-en- 1 -y 1 ) - 1 -(3 -(pyrrolidin- 1 -yl)propyl)-2, 5 - dihydro- l / Z-imidazole-2-carboxylate); ALC-0315 (2-hexyl-decanoic acid, l,1'-[[(4- hydroxybutyl)imino]di-6, 1 -hexanediyl] ester); ALC-0159 (a-[2-(ditetradecylamino)-2- oxoethyl]-G)-methoxy-poly(oxy- 1 ,2-ethanediyl)); P-sitosterol ((3 S, 85, 9S, 1 OR, 135, 145, 175)- 17-((25,55)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11, 12, 13, 14, 15, 16,17-tetradecahydro-U / -cy cl openta[a]phenanthren-3-ol);BAME-016B (2-(dodecyldisulfanyl)ethyl 3-[2-[2-[bis[3-[2-(dodecyldisulfanyl)ethoxy]-3- oxopropyl]amino]ethyl-methylamino]ethylamino]propanoate); BHEM-Cholesterol ((N,N- bis(2-hydroxyethyl)-N-methyl-N-(2-cholesteryloxy carbonyl aminoethyl) ammonium bromide); cKK-E12 (3, 6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione); DC-Cholesterol (3P-[5-(5f'Af'-dimethylaminoethane)-carbamoyl]cholesterol); DLin-MC3- DMA ((6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino) butanoate); DOPE ([(2R)-3-[2-aminoethoxy(hydroxy)phosphoryl]oxy-2-[(Z)-octadec-9-enoyl]oxypropyl] (Z)-octadec-9-enoate); DO SPA (2- [2, 5 -bi s(3 -aminopropylamino)pentanoylamino] ethyl- [2, 3 - bis[(Z)-octadec-9-enoxy]propyl]-dimethylazanium;chloride;tetrahydrochloride); DOTAP (2,3-bis[[(Z)-octadec-9-enoyl]oxy]propyl-trimethylazanium); DOTMA (2,3-bis[(Z)-octadec- 9-enoxy]propyl-trimethylazanium;chloride); DSPC ([(2R)-2,3-di(octadecanoyloxy)propyl] 2- (trimethylazaniumyl)ethyl phosphate); ePC (ethylphosphatidylcholine); FTT5 (octan-4-yl 9- [3-[[3,5-bis[3-[bis(9-octan-4-yloxy-9- oxononyl)amino]propylcarbamoyl]benzoyl]amino]propyl-(9-octan-4-yloxy-9- oxononyl)amino]nonanoate); Lipid H (SM-102) (heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo- 6-undecoxyhexyl)amino]octanoate); OF-Deg-Lin (2-[4-[5-[4-[bis[2-[(9Z, 12Z)-octadeca-9, 12- dienoyl]oxyethyl]amino]butyl]-3,6-dioxopiperazin-2-yl]butyl-[2-[(9Z,12Z)-octadeca-9,12-dienoyl]oxyethyl]amino]ethyl (9Z,12Z)-octadeca-9,12-di enoate); PEG2000-DMG (1,2- Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000); TT3 (N1, N3, N5-tri s(3- (didodecylamino)propyl)benzene-l,3,5-tricarboxamide), or combinations thereof.

[0051] Commercially available transfection agents or systems may be used as a transfection agent in the present system. Illustratively, a transfection agent may be Lipofectamine based such as 2000 (Lipofectamine MessengerMAX) LTX, Plus, or oligofectamine from Invitrogen (Rockford, IL) siPORT from ThermoFisher Scientific, Waltham, MA, DharmaFECT, 1, 2, 3, and 4 by Dharmacon, Cambridge UK, HiPerfect from Qiagen (Valencia, CA), Endofecitne@Max by CeneCopeia (Rockville, MD), Nanofectamine from GE Healthcare, Escor VI liposome by Sigma-Aldrich (St. Louis, MO).

[0052] Illustrative examples of mixed lipid and non-lipid materials that are commercially available as transfection agents include but are not limited to Arrest-In by Dharmacon (Cambridge, UK), TurboFect by ThermoFisher Scientific (Huntsville, AL), Effectane, Attractene, POlyFect, and SuperFect by Qiagen (Valencia, CA), ExpressFect by Thomas Scientific (Metuchen, NJ), ExGen 500 by Fermentas International Inc (Burlington ON), GeneJammer by Strategene (La Jolla, CA), ViFect, FuGENE 6 and FuGENE HD by Promega (Madison, WI), JetPEI, JetPrime, and INTERFERin by Polyplus transfection (New York, NY), Nanofectin by System Biosciences (Shanghai, China), among many others as are recognized in the art.

[0053] In some aspects, a transfection agent is a dendrimer. Dendrimers are typically used to enter a cell by non-specific endocytosis and are then transported via lysosomes. The biological material is then able to function following release from the dendrimer. Illustrative examples of a dendrimer include, but are not limited to PAMAM dendrimers as described by Denning, Top Curr Chem. 2003;228:227-36. Other dendrimers are as described by Shah, et al., Ini. J. Pharm., 2000; 208:41-48.

[0054] In some aspects, a transfection agent is a virus or portion thereof such as a viral envelope or portion thereof. Viral vectors such as adenoviral, adeno associated viruses, oncoretroviral, herpes virus, and lentiviral vectors or portions thereof may be used as a transfection agent. A viral vector may be inactivated or otherwise unable to further replicate or may not include all or a portion of the viral genetic material.

[0055] In some aspects, a cationic polymer and a lipid are both used in a transfection agent wherein the cationic polymer is any one or more of the above and a lipid may be one or more of the above.

[0056] A transfection composition optionally includes an arrangement wherein the transfection agent and the biological material are intermixed or the biological material is housed within the transfection agent. Optionally, the transfection agent and the biological material are intermixed wherein the transfection agent may surround partially or otherwise contact all or a portion of the biological material or the two are merely components of a heterogeneous mixture. Optionally, the biological material is housed within the transfection agent such as when the transfection agent is in the form of a particle or other that is capable of surrounding a volume or space that includes the biological material. For example, in some aspects, a transfection agent is in the form of a single layer or bilayer particle that houses within the particle one or more biological materials alone or in the presence of a vitrification agent that may or may not be also present within the particle.

[0057] A transfection composition is present as or was previously present at an amorphous glass (“glass state”) with a water content of about 10 weight percent or less. In some aspects, the water content is about 9 weight percent or less, optionally about 8 weight percent or less, optionally about 7 weight percent or less, optionally about 6 weight percent or less, optionally about 5 weight percent or less, optionally about 4 weight percent or less, optionally about 3 weight percent or less, optionally about 2 weight percent or less, optionally about 1 weight percent or less, optionally about 0.5 weight percent or less, optionally about 0.1 weight percent or less, optionally about 0.01 weight percent or less.

[0058] A transfection composition is optionally produced by combining a transfection agent and a vitrification agent in a vitrification buffer and subjecting the material to a process of vitrification optionally as described in WO 2022 / 109315 or US Patent No: 10,433,540 or to lyophilization. Optionally, the process of vitrification includes overlaying a vitrification mixture comprising a transfection agent, optionally a biological material, and a vitrification medium including the vitrification agent on a substrate comprising a capillary network, said substrate in a desiccation chamber; lowering the atmospheric pressure within the desiccation chamber; optionally providing a heat energy from the surface to the vitrification mixture, and optionally preventing the vitrification mixture from experiencing a freezing condition or producing crystal within the vitrification medium; and desiccating the vitrification mixture by capillary action until the vitrification mixture enters a glassy state. The use of tailored temperature and pressure during vitrification enhances vitrification and stability of transfection agent while reducing or eliminating the risk of exposure to freezing conditions or boiling, either of which could reduce the viability of the transfection agent(s).

[0059] In some aspects, a biological material is included with a transfection agent and a vitrification agent. The relative amount of biological material is dictated by the art recognized amount relative to transfection agent in prior sues of such material. In some aspects, a transfection agent in a vitrification medium is combined with the biological material prior to vitrification. Optionally, a transfection agent in a vitrification medium is layered on a substrate and the biological material in vitrification medium is layered on top or below the transfection agent and then the overall material is subjected to vitrification. Optionally, a transfection agent or biological material is vitrified first after which the other of the transfection agent or biological material is layered on top and subjected to a second vitrification process.

[0060] A biological material may include a cell, a collection of cells, a tissue sample, a cell fragment, an isolated and / or recombinant protein, an isolated or synthetic nucleic acid sequence (including RNA, DNA, single stands and double strands thereof), an expression vector, a bodily fluid, a hormone, a steroid, a cell receptor, a virion, a prokaryote, a simple eukaryote, a phospholipid, and / or a cell organelle, and combinations thereof. Optionally, a biological material is an enzyme, an antibody, a protein, polydeoxyribonucleotide chain, a polyribonucleotide chain, siRNA, shRNA, Cas9 or a Cas9 expression plasmid, a cell or cell fragment, or a genome or portion thereof. In particular aspects, a biological material is a polydeoxyribonucleotide chain, a polyribonucleotide chain, siRNA, shRNA, Cas9 or a Cas9 expression plasmid, and a transfection agent is a cationic polymer or a lipid. Optionally, a biological material is or is housed in an expression vector. An expression vector is as is recognized in the art as a polynucleotide chain that either has functionality itself or encodes a functional protein such as an enzyme as well as any required promoters, optional origin of replication, optional selection marker (e.g. label, encodes green fluorescent protein, antibiotic resistance gene), a ribosome binding site (optionally, the Shine-Dalgarno sequence), a transcriptional terminator for the CDS, and optionally other genetic elements like operators, multiple cloning sites and / or riboswitches if needed or desired. In other aspects, a biological material is an siRNA that when transfected into a cell has the desired outcome such as reducing or eliminating the expression or one or more other biological materials such as proteins.

[0061] A transfection composition is optionally formed by combining a transfection agent and a vitrification agent in water or other vitrification buffer to form a vitrification medium and subjecting the vitrification medium vacuum to desiccate the transfection agent under particular conditions to produce an amorphous glass with 10 weight percent or less water. The vitrification agent is optionally a glass forming agent. In the presence of appropriate glassforming agent(s), it is possible to store transfection agents in a vitrified matrix above cryogenic temperatures with vitrification achieved by dehydration. Some animals and numerous plants are capable of surviving complete dehydration. This ability to survive in a dry state (anhydrobiosis) depends on several complex intracellular physiochemical and genetic mechanisms. Among these mechanisms is the intracellular accumulation of sugars (e.g., saccharides, disaccharides, oligosaccharides) that act as a protectant during desiccation. Trehalose is one example of a disaccharide naturally produced in desiccation tolerant organisms.

[0062] Sugars like trehalose may offer protection to desiccation tolerant organisms in several different ways. A trehalose molecule may effectively replace a hydrogen-bounded water molecule from the surface of a folded protein without changing its conformational geometry and folding due to the unique placement of the hydroxyl groups on a trehalose molecule. A sugar molecule may also prevent cytoplasmic leakage during rehydration by binding with the phospholipid heads of the lipid bilayer. Furthermore, many sugars have a high glass transition temperature, allowing them to form an above cryogenic temperature or a room temperature glass at low water content. The highly viscous ‘glassy’ state reduces the molecular mobility, which in turn prevents degradative biochemical reactions that lead to deterioration of cell function and death. Vitrification of biological materials by dehydration in the presence of glass forming sugar trehalose has been disclosed (see, N Chakraborty, et al., Biopreservation and Biobanking, 2010, 8 (2), 107-114).

[0063] The transfection agent is optionally moved into an amorphous glass by a vitrification process that combines low atmospheric pressure and heat energy to achieve even and rapid vitrification of a transfection agent in a vitrification mixture. In some aspects, application of heat energy to a vitrification mixture is used whereby vitrification occurs under reduced atmospheric pressure. In some aspects, heat energy is applied to a vitrification mixture to prevent the crystallization of the vitrification mixture or exposure to a freezing condition.

[0064] During vitrification of the transfection agent, the temperature of the vitrification mixture is optionally controlled during desiccation and / or vitrification. For example, a vitrification mixture may be placed within a desiccation chamber and heat energy is applied to the vitrification mixture to restrict or prevent the vitrification mixture from experiencing a freezing condition. In some aspects, heat energy is transferred to the vitrification mixture to prevent crystallization therein.

[0065] In some aspects, the temperature of the vitrification mixture is controlled within an applied vacuum or reduction in atmospheric pressure around the vitrification mixture. As is discussed herein, application of a low atmospheric pressure can significantly lower the temperature of the vitrification mixture causing the vitrification mixture to crystallize and / or go into cryogenesis. If a biological material that may be combined with a transfection agent enters a freezing condition, irrevocable damage can occur therein which can negatively impact any desired activity or use when reconstituted. As is also identified herein, reduction in atmospheric pressure around the vitrification mixture can alter the molecular activity within the vitrification mixture, such that the boiling point is reduced. Similar to freezing, boiling the biological material and / or vitrification mixture or overheating can be detrimental. Boiling of a vitrification mixture can lead to biological material or transfection agent loss of tertiary structure, crosslinking and degradation of the components therein, including proteins, fatty acids and nucleic acids and the like, rendering any activity upon reconstitution compromised. In certain aspects, a vitrification mixture is maintained at a temperature above a cryogenic temperature while in low atmospheric pressure such as a vacuum, partial vacuum or in a generally reduced pressure atmosphere.

[0066] In certain aspects, the vitrification mixture including the transfection agent, optional biological material, and the vitrification medium including the vitrification agent may be heated directly to control the temperature of such during desiccation. In other aspects, the vitrification mixture may have the temperature of such controlled by conduction, convection and / or radiation means. In other aspects, the vitrification mixture may have its temperature controlled by controlling the temperature outside of the desiccation chamber and relying on conduction through the desiccation chamber or portion thereof to control the temperature of the vitrification mixture. In such instances, it will be appreciated that the physical properties of the walls of the desiccation chamber will need to be taken into consideration. For example, a poorly thermal conducting material of the desiccation chamber may require an applied temperature different from that required by the vitrification mixture in order to allow the vitrification mixture to receive the appropriate heat energy. Such necessary adaptations will be readily appreciated by those in the art. In some aspects, heat may be applied through a heating pad, a heated bath, a flame, a heated bed, such as glass bead, a heated block and similar. In some cases, the heat energy may be from an electric source of generated heat and / or a heat energy released by combustion and / or a heat energy generated by electrical resistance.

[0067] In some aspects, heat energy may be provided to the vitrification mixture through an underlying support substrate. While a porous material of a capillary network may also provide heat energy to the vitrification mixture, in some instances the porous material is of a poor thermally conducting material, such as glass or a polymer. However, the underlying substrate may be of a metal or similarly efficient conducting material and easily connected to a heat source outside of the desiccation chamber or an electrical source and provide heat by resistance created therein. The application of heat energy from the solid support may further provide a temperature gradient to assist in capillary evaporation.

[0068] In some aspects, the vitrification mixture is maintained at a temperature above its cryogenic temperature during vitrification under low atmospheric pressure. In some aspects, the vitrification mixture is preheated prior to desiccation under low atmospheric pressure. In other aspects, the vitrification mixture is heated during vitrification under low atmospheric pressure. In other aspects, heat is applied at or around the time vitrification commences. It will be appreciated that the amount of heat energy applied to the vitrification mixture may be constant or may vary during vitrification under low atmospheric pressure process. In some aspects, the introduction of low atmospheric pressure within the desiccation chamber can cause a rapid drop in temperature of the vitrification mixture. In such aspects, having the vitrification mixture ready to receive or already receiving heat energy can increase the recovery rate from the drop in temperature.

[0069] In certain aspects, a constant temperature is applied to the vitrification mixture, such that the vitrification mixture is maintained at a temperature of from about Tgof the vitrification mixture in °C to about 40 °C, including about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, and 39 °C. In certain aspects, a higher temperature may be applied to the desiccation chamber or the porous material to provide the necessary heat energy to the vitrification mixture. Such applied temperatures may illustratively be of from about 15 °C to about 70 °C, depending on the size of the desiccation chamber and the conductive means available to transfer effectively to the transfection agent and / or vitrification mixture.

[0070] In some aspects, the desiccation may occur in a desiccation chamber, whereby the vitrification mixture may be placed therein so as to be exposed to low atmospheric pressure. Such a desiccation chamber may be connected to a vacuum source to apply a low atmospheric pressure to the vitrification mixture. As set forth herein, a vitrification mixture can be prepared with a vitrification medium or a glass forming agent such as trehalose and subjected to lowatmospheric pressure, such as through application of a vacuum. In some aspects, the low atmospheric pressure is from about 0.9 atm to about 0.005 atm, including 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.255, 0.25, 0245, 0.24, 0.235, 0.23, 0.225, 0.22, 0.215, 0.21, 0.205, 0.2, 0.195, 0.19, 0.185, 0.18, 0.175, 0.17, 0.165, 0.16, 0.155, 0.15, 0.145, 0.14, 0.135, 0.13, 0.125, 0.12, 0.115, 0.11, 0.105, 0.1, 0.095, 0.09, 0.085, 0.08, 0.075, 0.07, 0.065, 0.06, 0.055, 0.05, 0.045, 0.04, 0.035, 0.03, 0.025, 0.02, 0.015, and 0.01 atm.

[0071] Optionally, the pressure within the desiccation chamber is lowered to a point above the triple point of the vitrification mixture. In other aspects, the pressure is lowered to a point above the triple point of water, such as greater than 0.006 atm. As set forth herein, lowered atmospheric pressure lowers the temperature of the vitrification mixture while also reducing its boiling point. In some aspects, the pressure within the desiccation chamber is lowered to about 0.04 atm or about 29 mmHg.

[0072] Optionally, the vitrification mixture is placed in a vacuum or partial vacuum at an elevated temperature or maintained at a temperature above the cryogenic temperature of the vitrification mixture at the atmospheric pressure applied, such that the vitrification mixture does not experience a freezing condition during the rapid decrease in atmospheric pressure. In further aspects, the temperature of the vitrification mixture will fall below the Tgof the vitrification medium to allow the vitrification of the transfection agent and any optional biological material(s).

[0073] Maintaining the low atmospheric pressure can require containing the vitrification mixture in a sealed enclosure, such as a desiccation chamber. It will be appreciated by those skilled in the art that providing and / or maintaining a low atmospheric pressure around the vitrification mixture will typically require that the desiccation chamber be capable of withstanding the low pressure therein. Such can be of any suitable or desired shape and / or material, being constrained by a requirement to maintain a low atmospheric pressure therein, requiring a sufficient seal and sufficient wall strength. The desiccation chamber can be operably connected to a vacuum source to lower the atmospheric pressure therein, while further allowing air to return upon vitrification completion. The desiccation chamber may be sufficiently sealed or closed so as to allow for an applied vacuum to effectively lower the atmospheric pressure in the desiccation chamber to the desired range.

[0074] In some aspects, the vitrification mixture is placed on or in a capillary network to enhance vitrification of the vitrification mixture. In further aspects, a capillary network canprevent the vitrification mixture from boiling under a reduced atmospheric pressure. The principles of capillary assisted evaporation are described in US 10,568,318.

[0075] In some aspects, the capillary network is of sufficient thickness to restrict liquid or fluid from accumulating on the surface thereof. To realize the capillary effect the liquid must be accommodated within the pores of the capillary substrate forming a meniscus. The liquid fraction ( ) at the capillary interface, i.e., the volume occupied by the liquid, is a parameter for consideration for capillary evaporation. Capillary driven evaporation occurs when the viscous pressure drop in the liquid surpasses the maximum capillary pressure at the liquid-vapor interface. The liquid fraction 5, is related to the overall pressure drop from the bulk to the liquidvapor interface. Under atmospheric pressure and no applied heat flux the liquid covers large area, leading to a liquid fraction, >1. Under these conditions, the capillary driven evaporation rate is minimal. Reducing the ambient pressure reduces 5, and in turn increases the evaporation rate. However, beyond certain threshold pressure drop, nucleation boiling can occur which is undesirable. An applied heat flux Q can also enhance the evaporation rate, but the risk of film boiling exists, which is also undesirable. Applying the heat flux from the surface of the capillary meniscus eliminates the risk of film boiling. Under large AP and Q applied in a counter gradient fashion leads to the liquid meniscus confined to the pores, i.e., the liquid fraction « 1 (-0.25), resulting in highest evaporation rate while avoiding boiling (^= — - 4p where p is distance between ridges or height of the capillary substrate and d the diameter of the circle formed by the shape of the liquid meniscus). Therefore, maintaining a temperature gradient between the surface and the bulk liquid leads to capillary evaporation where the fast evaporation can be achieved. As the liquid level recedes into the capillary substrate, capillary evaporation phenomena is still realized as long as the pressure gradient and temperature gradients are maintained. In some aspects, a capillary network under the mRNA or compositions thereof may assist in the evaporative processes during desiccation.

[0076] In some aspects, the heat energy is applied to a vitrification mixture as it undergoes vitrification on a capillary network. In some aspects, an underlying capillary network can allow for even and complete vitrification of a vitrification mixture receiving heat energy while protecting the vitrification mixture from boiling. The capillary network can be a contiguous network of capillaries meaning that there is no branching between a first end of a capillary and a second end of a capillary. In some instances, the capillary substrate network can be provided by an underlying porous material, such as a membrane, or an underlying contoured or ridgedsurface wherein the troughs and peaks thereof provide a bed sufficient to subject a liquid vitrification mixture to capillary action during vitrification.

[0077] Desiccation of larger volume of liquid can be conveniently achieved under vacuum by deploying a porous material of a network of capillaries to facilitate capillary evaporation, such as through the introduction of a substrate of contiguous capillary channels. When the liquid accumulates on the surface of the capillary substrate, however, boiling still can occur in the accumulated liquid which is as described herein can be undesirable. The presence of a temperature gradient between the surface and the bulk liquid allows for capillary evaporation where the fast evaporation can be achieved. Accordingly, in some aspects of the present disclosure, the volume of fluid present in the vitrification mixture can be established such that fluid can fill the capillary network without overflowing or pooling on the surface.

[0078] Optionally, the desiccation chamber or capillary substrate contained therein may be suitably patterned such that the walls of the chamber or capillary substrate provide a capillary network to the vitrification mixture when placed therein. For example, a contour or ridge can line the walls of a chamber to provide an underlying capillary network. In other aspects, a porous material of a contiguous capillary network with a vitrification mixture therein is provided into a sealable desiccation chamber. In certain aspects, a porous material may include a membrane of a plurality of contiguous capillary channels.

[0079] A capillary substrate may itself be heated to avoid the vitrification mixture from experiencing a temperature below the Tg. In some aspects, a support scaffold may be included between the capillary substrate and the heating block or side of any chamber in which the capillary substrate is located to separate the capillary substrate from the surface from which heat is produced. This prevents direct heating from beneath the sample to substantially allow for desiccation from two directions or avoiding the greater heat exposed to the troughs of the membrane material, in some aspects.

[0080] A heating element may be instead or in addition introduced within the chamber allowing for directed heat application to the surface of the vitrification capillary substrate thereby promoting effective vitrification at the desired locations within / on the membrane to promote excellent capillary action and prevent boiling of the vitrification mixture during vitrification.

[0081] In some aspects, heat is not provided or the transfection composition is made by traditional lyophilization processes such as cold temperature vacuum drying in whichdesiccation is promoted by removal of water in the form of ice by sublimation. Either vitrification or lyophilization may be used in some aspects. Alternatively, a transfection composition is made by vitrification, not lyophilization.

[0082] The presence of contours and / or ridges on a substrate provides capillary ridges to enhance vitrification. The presence of a vitrification mixture over the surface allows for fast evaporation from the peaks and by drawing the vitrification mixture toward the peaks by capillary action. The presence of a contiguous capillary network further allows the fluid volume of the vitrification mixture to evenly evaporate and prevent boiling while also preventing excess fluid build-up, which can also experience damaging boiling. Similarly, a porous material such as a membrane or substrate of contiguous capillary channels, may provide an underlying capillary network. In such aspects, a porous material, such as a membrane or substrate, is housing the vitrification mixture and the capillary action therein provides for enhanced vitrification. Accordingly, in some aspects, the vitrification mixture is placed on a contiguous capillary network. In further aspects, the vitrification mixture is placed on a patterned and / or ridged and / or contoured optionally porous material. In further aspects, the contiguous capillary network is formed by patterns and / or ridges and / or contours within walls of the desiccation chamber. In other aspects, the capillary network is provided by a porous material including a plurality of contiguous capillary channels.

[0083] The desiccation chamber should further be capable of or arranged to house the vitrification mixture therein. In some aspects, the desiccation chamber should be capable of housing the vitrification mixture on a porous material and / or a supporting substrate. In some aspects, the vitrification mixture is prepared for vitrification by placement upon a substrate. In some aspects, the substrate may be a porous material, such as a membrane and / or a bed of arranged capillaries. In further aspects, the walls of the desiccation chamber serve as a supporting substrate and are ridged and / or patterned and / or contoured to provide a capillary network therein.

[0084] In further aspects, a supporting substrate may be utilized to provide and / or transfer heat energy to the vitrification mixture. It will be appreciated by those skilled in the art that to provide heat energy effectively to the vitrification mixture, the supporting substrate may in some aspects be of a good conducting material, such as a metal. In other aspects, a porous material to provide a contiguous network of capillaries may be located between the vitrification mixture and the underlying solid support substrate.

[0085] The substrate can provide an interface for rapid evaporation. The capillary network formed from either an underlying patterned ridged support or of a porous material such as a membrane may be made of a material that is not toxic and not reactive to the transfection agent or biological materials and does not react chemically or physically with the vitrification medium. The material can be of a suitable polymer, metal, ceramic, glass, or a combination thereof. In some aspects, a capillary network is formed from a material of polydimethylsiloxane (PDMS), polycarbonate, polyurethane, polyethersulphone (PES), polyester (e.g. polyethylene terephthalate), among others. Illustrative examples of a capillary channel containing membrane suitable as a surface in the devices and processes provided herein include hydrophilic filtration membranes such as those sold by EMD Millipore, Billerica, MA or US425N-MA and Ultrasorb-MA foams sold by Foamtec Medical, Waco, TX. In certain aspects, the porous material does not substantially bind, alter, or otherwise produce a chemical or physical association with a component of a transfection agent, a biological material and / or vitrification medium. The porous material is optionally not derivitized. Optionally, capillary channels may be formed in a substrate (e.g. desiccation chamber walls) of desired material and thickness by PDMS formation techniques, laser drilling, or other bore forming technique as is known in the art.

[0086] In some aspects, the capillary network is of sufficient thickness to restrict liquid or fluid from accumulating on the surface thereof. Increased fluid or liquid accumulation on the surface can lead to detrimental or damaging boiling. Increasing the thickness and / or layers of the capillary network can provide an increased space to handle increased fluid within the capillary channel or within the troughs. The liquid fraction is determined by the relationship between the area of the circle formed by the meniscus and the height or distance between the ridges: ^=— . Optimally, 5, is of about 0.25, with an upper limit of about 1. At values below 0.25, the system may begin to dry up prior to desired vitrification.

[0087] In some aspects, a capillary network may assist in the evaporative processes during desiccation. As described herein, capillaries may be provided by patterning or contouring the walls of a desiccation chamber to effectively provide an underlying capillary bed or by providing a porous material of a contiguous capillary network, such as with a membrane. In some aspects, the capillary network provided by a porous material and / or a patterned and / or contoured surface features pores of about 20 pm or less, such that the pores provide underlying capillaries to assist in vitrification. In some aspects, the pores or peak to peak distance may beof an average opening of from about 20 pm to about 0.1 pm, including about 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, and 0.2 pm. A capillary channel may have a length optionally defined by the thickness of a substrate that forms the channels or by one or a plurality of individual channels themselves. A capillary channel length is optionally about one millimeter or less, but is not to be interpreted as limited to such dimensions. Optionally, a capillary channel length is of about 0.1 microns to about 1000 microns, or any value or range therebetween. Optionally, a capillary channel length is of about 5 to about 100 microns, optionally of about 1 to about 200 microns, and / or optionally of about 1 to about 100 microns. A capillary channel length is optionally about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 microns. In some aspects, the length of the capillary channels varies throughout a plurality of capillary channels, optionally in a non-uniform variation.

[0088] The cross-sectional area of the capillary channel(s) may be of about 2000 pm2or less. Optionally a cross-sectional area is of about 0.01 pm2to about 2000 pm2, optionally of about 100 pm2to about 2000 pm2, or any value or range therebetween. Optionally, a cross- sectional area of the capillary channel(s) is of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 pm2or less.

[0089] Capillary assisted evaporation rate may be affected by both atmospheric demand (humidity, temperature and velocity of air / gas at the evaporating surface), and (i) the characteristics of the capillary channels that generate the driving capillary force, (ii) the liquid meniscus depth, and (iii) the viscous resistance to flow through the capillary. Consequently, complex and highly dynamic interactions between capillary properties, transport processes, and boundary conditions result in wide range of evaporation behaviors. For fast drying the key parameters may include: (1) the conditions that support formation and sustain a liquid network at the evaporating surface and (2) the characteristics that promote formation of capillary pressure that induce sufficient flow to supply water at the evaporating surface.

[0090] In some aspects, the porous material may be ridged and / or contoured or placed upon a ridged and / or contoured underlying support substrate, such that the porous material adopts a similar shape when placed or pressed thereon. The contours and / or ridges of a patterned material may increase surface area to provide for increased exposure for evaporation.

[0091] In some aspects, the capillary network is of a hydrophilic material. In other aspects, the capillary network may be of a hydrophobic material and further treated to be hydrophilicor more hydrophilic in nature, such as through exposure to a plasma. An originally hydrophobic membrane may be treated with cold plasma to render it more hydrophilic.

[0092] In some aspects, the transfection agent, and the optional biological material, is placed or covered or mixed in a vitrification medium including a vitrification agent to form a vitrification mixture. The presence of appropriate vitrification agents in a vitrification medium can be essential as the transfection agent desiccates under the surrounding conditions as set forth herein. Fast desiccation methods as set forth herein by itself does not necessarily assure success in the viability of the cells or other vitrified biological material following desiccation. A vitrification medium that forms glass and / or that suppresses the formation of crystals in other materials may be required. A vitrification medium may also provide osmotic protection or otherwise enable transfection agent survival during dehydration. Illustrative examples of vitrification agents to include in a vitrification medium may include one or more of the following: dimethyl sulfoxide, glycerol, sugars, polyalcohols, methylamines, betines, antifreeze proteins, synthetic anti-nucleating agents, polyvinyl alcohol, cyclohexanetriols, cyclohexanediols, inorganic salts, organic salts, ionic liquids, or combinations thereof. In some aspects, a vitrification medium optionally contains 1, 2, 3, 4, or more vitrification agents.

[0093] In some aspects, a vitrification medium may include a vitrification agent at a concentration that is dependent on the identity of the vitrification agent. Optionally, the concentration of the vitrification agent is at a concentration that is below that which will be toxic to the transfection agent and / or the biological material being vitrified where toxic is such that functional or biological viability is not achieved upon subsequent sample use. The concentration of a vitrification agent is optionally of about 500 pM (micromolar) to about 6 M (molar), or any value or range therebetween, including about 1, 2, 3, 4, or 5 M. For the vitrification agent trehalose, the concentration is optionally of about 1 M to about 6 M, including 2, 3, 4, or 5 M. Optionally, the total concentration of all vitrification agents when combined is optionally of about 1 M to about 6 M, including 2, 3, 4, or 5 M.

[0094] Trehalose, a glass forming sugar, has been employed in anhydrous vitrification and may provide desiccation tolerance in several ways. However, vitrified 1.8 moles / liter (M) trehalose in water has a glass transition temperature of -15 to 43 °C. To achieve vitrification above 0 °C, higher concentrations (6 8 M) are required which could be damaging to the transfection agent and / or the biological materials. Alternatively, the vitrification medium may include buffering agents and / or salts to increase the Tgvalue of the vitrification medium. In some aspects, a vitrification medium may optionally include water or a solvent and / or abuffering agent and / or one or more salts and / or other components. A buffering agent may be any agent with a pKa of about 6 to about 8.5 at 25 °C. Illustrative examples of buffering agents may include HEPES, TRIS, PIPES, MOPS, among others. A buffering agent may be provided at a concentration suitable to stabilize the pH of the vitrification medium to a desired level.

[0095] A vitrified medium including 1.8 M trehalose, 20 mM HEPES, 120 mM ChCl, and 60 mM betine provides a glass transition temperature of +9 °C. An exemplary vitrification medium for the capillary assisted vitrification method disclosed herein may include trehalose, and one or more buffering agents containing large organic ions (>120 kDa) such as choline or betine or HEPES as well as buffering agent(s) containing small ions such as K or Na or Cl. In some aspects, a vitrification medium includes 1200 mM trehalose and 5 wt% glycerol in phosphate buffered saline.

[0096] In some aspects, the transfection agent, and the biological material if present, is coated or immersed in a vitrification medium and placed on a support substrate to retain the vitrification mixture during the steps of vitrification as set forth herein. In certain aspects, the capillary network absorbs some of the vitrification mixture while allowing a thin layer of fluid to remain.

[0097] It is appreciated that a transfection composition as provided herein may include at least one transfection agent, optionally 2, 3, 4, or more transfection agents. The methods include preparing a vitrification mixture through the assembly of the parts described herein. For example, a vitrification mixture of a transfection agent, optional biological material, and a vitrification medium is placed on or in contact with a solid support substrate. In some aspects, the underlying solid support is contoured and / or ridged to provide an underlying capillary network. In some aspects, the underlying support is part of a desiccation chamber, such as a wall thereof. In other aspects, a porous membrane can be placed between the vitrification mixture and a solid support. In some aspects, a contiguous capillary network supports the vitrification mixture and draws in fluid therefrom. The capillary network and / or porous material is to be of a sufficient thickness or quantity so as to avoid the presence and / or pooling of liquid above the surface of the capillary network.

[0098] The vitrification mixture with the substrate may be placed in a desiccation chamber, the desiccation chamber being operably connected to a vacuum or other means for reducing the atmospheric pressure therein. In certain aspects, the vitrification mixture is held in place on a porous or contoured material within the desiccation chamber. In some aspects, thevitrification mixture is placed on part of the desiccation chamber, wherein the part is patterned and / or contoured so as to providing an underlying capillary network. In some aspects, a solid support substrate, a porous material, such as a membrane, and the vitrification mixture are placed in the desiccation chamber.

[0099] The transfection agent may optionally be coated and / or mixed with a vitrification medium in the desiccation chamber. In other aspects, the transfection agent may be prepared with a vitrification medium prior to placement within the desiccation chamber.

[0100] Once assembled, the methods of the present disclosure may include reducing atmospheric pressure around the vitrification mixture, providing capillary-assisted evaporation to the vitrification mixture and / or applying heat energy to the vitrification mixture / capillary network without inducing boiling therein so as to produce the transfection composition as provided herein in a glassy state and with the water content at 10 weight percent or less. As described herein application of all three can provide for rapid and even vitrification and desiccation of the vitrification mixture, while avoiding experiencing a cryogenic temperature and avoiding boiling, thereby significantly reducing any damage to the transfection agent during the process.

[0101] In certain aspects, the methods include exposing the vitrification mixture to a low or reduced atmospheric pressure. In some aspects, the exposure to low atmospheric pressure occurs by operating a vacuum connected to the desiccation chamber. In some aspects, the atmospheric pressure is lowered to a point above the triple point of the vitrification mixture. In other aspects, the pressure is lowered to about 0.04 atm or 29.5 mmHg.

[0102] Once the vitrification mixture is placed within the desiccation chamber, the atmospheric pressure therein is lowered. In some aspects, the atmospheric pressure is lowered to a point above the triple point of the vitrification medium or vitrification mixture therein. In other aspects, the atmospheric pressure is lowered to a point above the triple point of water. In further aspects, the pressure is lowered within the desiccation chamber to about 0.04 atm.

[0103] In some aspects, the transfection composition may be formed by desiccation for a desiccation time. A desiccation time is a time sufficient to promote suitable drying to vitrify the vitrification mixture. A desiccation time is optionally from about 1 second to about 1 hour, including but not exceeding about 10 s, 30 s, 1 min, 5 min, 10 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min and 55 min. Optionally, a desiccation time is of from about 1 second to about 30 min, optionally of from about 5 seconds to about 10 min.

[0104] The duration a transfection agent may remain viable in vitrified state during storage above cryogenic temperature may vary from one sample material to the next. In some aspects, a transfection agent may remain viable while in storage above cryogenic temperature for 2-20 days. In other aspects, a transfection agent may remain viable while in storage above cryogenic temperature for 10 weeks. In other aspects, a transfection agent may remain viable in storage above cryogenic temperature for up to one year. In other aspects, a transfection agent may remain viable while in storage above cryogenic temperature for up to 10 years.

[0105] Alternatively, after vitrification above cryogenic temperatures employing the teachings and devices of the current disclosure, the transfection composition can be stored at cryogenic temperatures and / or in liquid nitrogen for very long periods without risk of crystal formation within the material. For many biological materials, this is a preferred approach to avoid cryoinjury that commonly occurs during direct vitrification at cryogenic temperatures. A preferred approach in one aspect is to vitrify the transection agent alone or with one or more biological materials at room temperatures utilizing low concentrations of vitrification agents (e.g. <2 M trehalose) and then immediately store at cryogenic temperatures. Therefore, the said device is optionally made out of materials storable at a temperature between -196 °C to 60 °C following the vitrification according to the teachings of the current disclosure.

[0106] In some aspects, a smaller volumes of a vitrification mixture can also be successfully vitrified and provide full or exceptionally high performance of the transfection agent upon reconstitution. In some aspects, a volume of 10 pL or less can be vitrified through the processes as set forth herein and retain full ability upon reconstitution. In some aspects, the processes of vitrification as set forth herein can vitrify samples of from about 0.1 pL to 10 pL or greater, including about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, and 9 pL of sample. For example, as set forth in the working examples herein, providing 3 pL of ribonucleic acids to the vitrification mixture and storing the resulting transfection composition for more than 3 months at temperatures well above room temperature resulted in less than 3 ng of lost material from a starting 3 pg when reconstituted.

[0107] In some aspects, the present disclosure provides processes for vitrification that allow for excellent recovery of low massed samples. In some aspects, the processes herein allow for excellent recovery of samples with a mass of 5 pg of transfection agent or less, including about 4, 3, 2, and 1 pg of transfection agent. In some aspects, the mass of the transfection agent can be below 1 pg, including masses of 100 ng or less. In some aspects, the mass of the transfectionagent to be vitrified is of from about 0.1 to 1 jug, including 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 jug.

[0108] In some aspects of the present disclosure, the processes of vitrification can be applied to more than one sample or material for successful storage and preservation. In some aspects, multiple samples can be vitrified within the same vitrification mixture. It will be apparent that some samples may require isolation from other materials prior to a desired end use. In such aspects, the separated materials can be vitrified in separate vitrification mixtures on the same capillary substrate, utilizing physical space to isolate. In other aspects, sequential vitrifications or lyophilizations may be utilized such that one material is vitrified upon a second previously vitrified material and so on.

[0109] In some aspects, the present disclosure concerns two or more transfection agents, and or one or more transfection agents and one or more biological materials vitrified within the same area of a capillary substrate. Optionally, 2, 3, 4, 5, or more components (e.g. transfection agent, biological material) may be vitrified into a single capillary substrate. Such may be achieved by co-vitrification or by successive vitrification steps, or combinations thereof.

[0110] A transfection composition as provided herein optionally includes one or more other reagents that may be suitable for subsequent transfection of a cell or tissue. Reagents may include buffers, salts, substrates, enzyme substrates, antigens, chemical compounds, peptides, and small nucleic acids such as primers or siRNA. A transfection composition may be provided as a component of a kit. It will be apparent that in some aspects, a kit may require or optionally include one or more non-vitrified materials, such as liquid solutions for reconstitution, non- perishable or oxidation-susceptible chemicals or salts, buffers, cell media, anti-microbiotics, or similar. It will further be appreciated that not all materials need to be vitrified for a kit for single use. For example, a user may supply a test material or sample with which the vitrified transfection compositions may be used.

[0111] In some aspects, the present disclosure concerns kits that include one or more transfection compositions alone or combined with one or more other components. Such may include one or more transfection compositions and / or one or more accompanying solutions. In some aspects, the kit may include only one or more transfection compositions. In some aspects, the kit may include at least one transfection composition and at least one biological material. In some aspects, the kit may include at least one biological materials co-vitrified with another transfection composition(s). In some aspects, all or some components of the kit may be vitrifiedindependently, either on separate regions or areas of a shared underlying substrate or on individual substrates, or combinations thereof.

[0112] The kits may include one or more vitrified nucleic acids, proteins, peptides, polynucleotide chains, DNA, RNA, mRNA, tRNA, antibodies, buffers, salts, enzymes, enzyme-substrates, lipids, cationic lipids, ionizable lipids, sterols, steroids, therapeutics, adjuvants, carriers, excipients, substrates, catalysts, amino acids, deoxyribonucleotide triphosphate (dNTPs), combinations thereof, or similar.

[0113] Also provided herein are process of transfecting a cell or tissue wherein the cell or tissue is contacted with a transfection composition as provided herein. Optionally, a transfection composition is eluted from the glass state to a liquid state prior to contact with a cell or tissue. Optionally, an elution buffer is used to elute the transfection composition prior to contacting the cell or tissue. An elution buffer may be water alone or in combination with one more other reagents such as but no limited to buffering agents (e.g. HEPES, TRIS, bicarbonate buffers, phosphate buffers, etc.), salts (NaCl, sodium pyruvate, KC1, etc.), cell media components (e.g. glucose, glutamine), amino acids, vitamins, biological material as provided herein, or other desired components. In some aspects, an elution buffer is or includes water. Optionally, an elution buffer is or includes a cell culture reagent (e.g. DMEM or other), or an injectable composition (e.g. TRIS buffer or other, osmolytes, cryoprotectant, etc.). The transfection composition may be eluted in any desired amount of elution buffer, but is optionally eluted in 1-100 fold volume of elution buffer relative to transfection composition.

[0114] Optionally, a transfection composition is eluted into an elution buffer that includes one or more biological materials. For example, a transfection composition (that may or may not include a biological material) is may be eluted in an elution buffer that also includes one more biological materials that may then associate with the eluted transfection composition and be transfected into a cell or tissue upon contact with the transfection agent. Alternatively, or in addition, a transfection composition itself includes one or more biological materials such that the eluted material is immediately able to be used to transfect a cell or tissue with the biological material.

[0115] It was unexpectedly found that the transfection compositions as provided herein have vastly superior transfection efficiency relative to control transfection agents that were not transitioned into a glassy state composition. Transfection efficiency is defined as amount of transfected biological material or expression of the transfected biological material in a cell or tissue following transfection relative to a control. In some aspects, transfection efficiency is10% or more than control, optionally 20% or more than control. In some aspects, transfection efficiency is doubled or more than doubled relative to that of a control. In some aspects, transfection efficiency is tripled or more than tripled relative to that of a control. In some aspects, transfection efficiency is four or more fold greater relative to that of a control. Optionally, the transfection efficacy of the transfection process as used herein or other use of the transfection compositions as provided herein are 110% or greater relative to control, optionally 150% or greater relative to control, optionally 175% or greater relative to control, optionally 190% or greater relative to control, optionally 200% or greater relative to control, optionally 225% or greater relative to control, optionally 250% or greater relative to control, optionally 275% or greater relative to control, optionally 300% or greater relative to control, optionally 325% or greater relative to control, optionally 350% or greater relative to control, optionally 375% or greater relative to control, optionally 400% or greater relative to control, optionally 450% or greater relative to control.

[0116] A process optionally includes transfection of a cell or a tissue. Optionally, a cell is transfected. A cell is optionally any cell desired to be transfected. Optionally, a cell is a eukaryotic cell. Optionally, a cell is a prokaryotic cell. Optionally, a cell is housed within an organism or is to be subsequently transplanted into an organism. Optionally, a process of transfection is performed ex vivo in a cell or tissue that is taken from a subject and may optionally be reintroduced into the subject following transfection with a transfection composition as provided herein.

[0117] Various aspects of the present invention are illustrated by the following non-limiting examples. The examples are for illustrative purposes and are not a limitation on any practice of the present invention. It will be understood that variations and modifications can be made without departing from the spirit and scope of the disclosure.EXAMPLESExample 1 : Transfection compositions enhance transgene expression

[0118] Initial examples were arranged to assess the role of applying heat to the desiccation process under low atmospheric pressure and the effect of capillary substrate size on the ability to rebound the sample temperature following application of the vacuum.

[0119] Vitrification scaffolds of 10 mm diameter circular polyurethane foam (US425N-MA or Ultrasorb-MA foam sold by Foamtec Medical, Waco, TX) were prepared. A vitrification medium was prepared containing 1200 mM trehalose and 5 wt% glycerol in 0.01 M phosphatebuffered saline (0.138 M NaCl; 0.0027 M KC1; pH 7.4). For each sample, 4 pL transfection agent (1 pg / pl solution) (e.g. Lipofectamine MessengerMAX (ThermoFisher Scientific, Waltham MA) or a similar agent with 1 :3 molar ratio of cationic lipid, e.g. DOSPA:DOPE) was mixed with 76 pL vitrification medium (1200 mM trehalose), and incubated for 10 minutes at room temperature. Following incubation, 80 pL vitrification mixture was loaded onto the scaffold. The loaded scaffolds were placed into a custom vacuum chamber with heated shelves set to a temperature of approximately 37 °C and dried under vacuum for 30 minutes. Following vitrification, the samples were packaged in a resealable silver Mylar pouch with one CaCh desiccant, stored for 28 days at room temperature or at 37 °C.

[0120] After storage, each of the samples were eluted using 80 pL warmed incomplete Fluorobrite Dulbecco's Modified Eagle Medium (DMEM) in 1.5 mL spin column tube.

[0121] Reference aliquoted identical lipid controls were stored at 4 °C (positive control), and 50 pL aliquoted lipid controls were stored at room temperature or 37 °C (negative controls).

[0122] For each of the test samples and the controls, 20 pL of each were mixed in 19 pL incomplete Fluorobrite DMEM, added in a tube. In a separate tube 1 pL (Ipg) DasherGFP(r) mRNA added into 19 pL incomplete Fluorobrite DMEM. After 10-minute incubation, diluted the transfection agent or control reagents were mixed with mRNA and incubated for 5 minutes to form mRNA-transfection agent complexes.

[0123] Prior to transfection, confluent CHO-K1 cells were pelleted and resuspended using Fluorobrite DMEM with 10% FBS and GlutaMAX. Cells were diluted to 0.12 x 106cells / 200 pL and added to each well of a cell culture plate, kept at 37 °C overnight for attachment, and used for the transfection.

[0124] For transfection, 10 pL mRNA-transfection agent complex was added to each well. After 24 h incubation, cell transgene expression was visualized using Leica Fluorescence Microscope, and the signal was detected using Synergy Hl Plate Reader. The experimental samples percentage of relative fluorescence unit (RFU) were calculated respective to the liquid control stored at 4 °C. Results are as in the FIG. 1.

[0125] As is observed in FIG. 1, following storage by forming a glassy state the resulting eluted transfection agent enhances transgene expression by more than double as observed by an increase in fluorescence of about 280% after storage at both 25 °C and 37 °C for 28 days. The liquid controls were substantially identical to the material stored at 4 °C independent of whether the control material was stored at 25 °C and 37 °C for the same time period. Theseresults confirm prior studies that the tested commercially available transfection agent is storage stable, but also demonstrate that by using that transfection agent in forming a transfection composition as provided herein produces a material that at equimolar concentrations enhances transfection efficiency dramatically.Example 2:

[0126] Test and control materials were made as in Example 1, but the relative amount of transfection agent to vitrification medium was altered. Briefly, 1 pL stock transfection agent (1 pg / pl solution) was mixed with either 4, 9, 17, 18, 19, 39, or 59 pL vitrification medium, incubated for 10 minutes at room temperature and loaded onto the scaffold. The remainder of the studies was identical as Example 1, but samples were eluted within a day of vitrification or immediately upon making for liquid control samples.

[0127] As illustrated in FIG. 2 (depicting ratio of pg transfection agent (1 pg / pl solution): pl of vitrification medium), the relative amount of vitrification agent relative to transfection agent dramatically effects the resulting transfection efficiency. At low relative amounts of transfection agent to vitrification agent (1:4 and 1 :9) a decrease in resulting transfection efficiency was observed (A and B). A fast transition was observed at a ratio of 1 : 18 from 1 : 17 where suddenly the transfection efficiency doubled (FIG. 2 C and D). These data demonstrate that vitrification radically enhances transfection efficiency and the relative ratio of transfection agent to vitrification agent affects how well the resulting material functions.Example 3 : Order of transfection complex formation

[0128] To test whether the order of combining the biological material with the transfection agent has any role in eventual transfection efficiency, we tested three different combinations: 1) the transfection agent and biological material are combined prior to vitrification; 2) the transfection agent is first layered on a substrate followed by layering of the mRNA followed by vitrification; and 3) the transfection agent is layered and vitrified followed by layering of the mRNA and vitrifying as a second layer. For 1) pL Lipofectamine MessengerMAX reagent was incubated with 19 pL vitrification medium for 10 minutes at room temperature and mixed with 1 pL (Ipg) DasherGFP(r) mRNA in 19 pL incomplete Fluorobrite Dulbecco's Modified Eagle Medium (DMEM) and the 40 pL complex was loaded onto the scaffold and subjected to vitrification. For 2): The 1 pL Lipofectamine MessengerMAX reagent was incubated with 19 pL vitrification medium for 10 minutes at room temperature and loaded on the scaffold. Further, 1 pL (Ipg) DasherGFP(r) mRNA mixed with 1 pL vitrification medium was alsoloaded onto the same scaffold then the two were simultaneously vitrified. For 3): The 1 pL Lipofectamine MessengerMAX reagent was incubated with 19 pL vitrification medium for 10 minutes at room temperature, loaded on the scaffold and vitrified. The 1 pL (Ipg) DasherGFP(r) mRNA was mixed with 1 pL vitrification medium, loaded onto the vitrified scaffold and vitrified again. The reminder of the processes of Example 1 are the same.

[0129] As is observed from FIG. 3, the order of addition did have an effect on final vitrification efficiency. Combining the transfection agent with the biological material then vitrifying the combination resulted in 463% the activity of the control non-vitrified material. Lower, but still very high transfection efficiencies that are similar to the transfection efficiencies observed in Examples 1 and 2 where the transfection agent was vitrified alone then reconstituted prior to combination with the mRNA.Example 4: LNP transfection agent

[0130] The studies of Example 1 were repeated using lipid nanoparticle transfection agents instead of the transfection agents therein. A vitrification medium was prepared containing 1200 mM trehalose and 5 wt% glycerol in 0.01 M phosphate buffered saline (0.138 M NaCl; 0.0027 M KC1; pH 7.4). For each sample, 4 pL lipid nanoparticles (1.2 pg / pl with about 50 pg / ml GFP mRNA housed therein; ProMab, Richmond, CA) was mixed with 76 pL vitrification medium, and incubated for 10 minutes at room temperature. Samples were vitrified as in Example 1.

[0131] Prior to transfection, confluent HEK-293 cells were pelleted and resuspended using Fluorobrite DMEM with 10% FBS and GlutaMAX. Cells were diluted and 100,000 cells were added to each well of a cell culture plate, kept at 37 °C overnight for attachment, and used for the transfection.

[0132] For transfection, 10 pL LNP-mRNA complex was added to each well. After 24 h incubation, cells were trypsinized and stained for visualization using far red LD stain and washed. Cell transgene expression was visualized using a flow cytometer detecting 10,000 cells (gates applied for detection of single cells using both FSC and SSC, live cells, and GFP positive) and results analyzed using Flojo software. Results are as in the FIG. 4. Sample replicates (n=3) show consistent results and excellent repeatability. When the vitrification buffer is added to the cells with the LNP the cells show improved transfection in each cell (A) and successful transfection of more cells (B).

[0133] Titration of various amounts of LNP and vitrification medium were also performed using different ratios of the LNP or the vitrification medium as illustrated in FIG. 5. As a control the LNP (PC) was either held constant and the vitrification medium was titrated (A) or the vitrification medium (VB) was held constant (B). The results show that increasing the vitrification buffer to a relative volume ratio (LNP to VB) of 1 : 1 to 1 :3 improves transfection signal. Note that the ratio of transfection agent in pg to vitrification medium volume in pl at 1200 mM vitrification agent is readily calculated from the information provided herein. Similarly, when VM was held constant and LNP was increased to a relative ratio of 1 :0.3 to 1 :3 (VB to LNP) signal was also improved. However, maximal signal was achieved using 3- fold less LNP when the vitrification medium was present in excess. Quantitative results are illustrated in Table 1.Table 1: titration of LNPs or vitrification medium on HEK-293 cell transfection in volume to volume ratios.Ratio 1 to 0.3 1 to 3 1 to 1 0.3 to 1 3 to 1LNP (pl) 2.5 2.5 2.5 0.75 7.5 VB (pl) 0.75 7.5 2.5 2.5 2.5 MFI avg 16508 29289 18664 7515 23353 RSD 5.1 8.7 3.3 44.0 13.0

[0134] Overall, these results demonstrate that by creating a transfection composition as provided herein, either in the presence of a biological material or by combining the biological material with reconstituted transfection composition, a dramatic increase in transfection efficiency is produced.

[0135] Additional exemplary aspects1. A transfection composition including: a transfection agent and a vitrification agent; and wherein the transfection composition is in a glassy state optionally with a water content of 10 weight percent or less.2. The transfection composition of aspect 1, wherein a ratio of the transfection agent (in micrograms) to the vitrification medium (in microliters (at 1200 mM vitrification agent)) is equal to or greater than 1 :0.5.3. The transfection composition of aspect 2, wherein a ratio of said transfection agent to said vitrification medium is less than or equal to 1:200, optionally 1:150, optionally 1:100, optionally 1:90, optionally 1:80, optionally 1:70, optionally 1:60.4. The transfection composition of any one of aspects 1-3 further including a biological material.5. The transfection composition of aspect 4, wherein said biological material comprises one or more polynucleotide chains.6. The transfection composition of aspect 5, wherein the polynucleotide chain comprises an expression vector.7. The transfection composition of aspect 4, wherein said biological material comprises an enzyme, an antibody, a protein, polydeoxyribonucleotide chain, a polyribonucleotide chain, siRNA, shRNA, Cas9 or a Cas9 expression plasmid, a cell or cell fragment, or a genome or portion thereof.8. The transfection composition of any of aspects 1-6, wherein said transfection agent comprises one or more cationic polymers, a lipid, a virus, a cell penetrating peptide, a dendrimer, or a polynucleotide-binding protein.9. The transfection composition of aspect 8, wherein said cationic polymer comprises one or more of DEAE-dextran, poly-amino acids (optionally poly-L-lysine), poly(amidoamine) (PAMAM) dendrimers, PPI (poly-(propylenimine)), PEI (polyethylenimine), PDMAEMA (polymethacrylic acid N,N-dimethylaminoethyl ester), poly(P-amino ester), PHP (Poly(4-hydroxy-L-proline ester)), PAGA (poly[a-(4-aminobutyl)- 1-glycolic acid]), PVL poly(S-valerolactone), aminated PAHA (aminated poly(a-hydroxy acids)), PPE (polyphosphoester), polylactide (poly(lactic acid), PLA), N,N- diethylethylenediamine-polyurethane (DEDA-PU), poly(p-phenyleneethynylene), polythiophene, poly(fluorine-co-phenylene), poly(p-phenylenevinylene), protamine, or a combination thereof.10. The transfection composition of aspect 7, wherein said lipid comprises DOSPA (2,3- dioleoyloxy-N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propaniminium trifluoroacetate), 1,2-Dioleoyl-sn-glycerophosphoethanolamine (DOPE), DOTMA (1,2- Dioleoyl-3 -trimethylammonium propane), cholesterol, polyethylene glycol (PEG)-lipid conjugate, 3060iio (tetrakis(8-methylnonyl) 3,3',3",3"'-(((methylazanediyl) bis(propane-3,ldiyl))bis (azanetriyl))tetrapropionate; 9A1P9, decyl (2-(dioctylammonio)ethyl) phosphate); A2-Iso5 -2DC 18 (ethyl 5 , 5 -di((Z)-heptadec-8-en- 1 -yl)- 1 -(3 -(pyrrolidin- 1 -yl)propyl)-2, 5 - dihydro- lJ / -imidazole-2-carboxylate); ALC-0315 (2 -hexyl-decanoic acid, 1, lf-[[(4- hydroxybutyl)imino]di-6, 1 -hexanediyl] ester); ALC-0159 (a-[2-(ditetradecylamino)-2- oxoethyl]-co-methoxy-poly(oxy- 1 ,2-ethanediyl)); P-sitosterol ((35,85,95, 1 OR, 135, 145, 175)- 17-((25,55)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl- 2,3,4,7,8,9,10,11, 12, 13, 14, 15, 16,17-tetradecahydro-17 / -cy cl openta[a]phenanthren-3-ol); BAME-016B (2-(dodecyldisulfanyl)ethyl 3-[2-[2-[bis[3-[2-(dodecyldisulfanyl)ethoxy]-3- oxopropyl]amino]ethyl-methylamino]ethylamino]propanoate); BHEM-Cholesterol ((N,N- bis(2-hydroxyethyl)-N-methyl-N-(2-cholesteryloxy carbonyl aminoethyl) ammonium bromide); cKK-E12 (3, 6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione); DC-Cholesterol (3P-[A-(A',A'-dimethylaminoethane)-carbamoyl]cholesterol); DLin-MC3- DMA ((6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino) butanoate); DOPE ([(2R)-3-[2-aminoethoxy(hydroxy)phosphoryl]oxy-2-[(Z)-octadec-9- enoyl]oxypropyl] (Z)-octadec-9-enoate); DOSPA (2-[2,5-bis(3- aminopropylamino)pentanoylamino]ethyl-[2,3-bis[(Z)-octadec-9-enoxy]propyl]- dimethylazanium;chloride;tetrahydrochloride); DOTAP (2,3-bis[[(Z)-octadec-9- enoyl]oxy]propyl-trimethylazanium); DOTMA (2,3-bis[(Z)-octadec-9-enoxy]propyl- trimethylazanium;chloride); DSPC ([(2R)-2,3-di(octadecanoyloxy)propyl] 2- (trimethylazaniumyl)ethyl phosphate); ePC (ethylphosphatidylcholine); FTT5 (octan-4-yl 9- [3-[[3,5-bis[3-[bis(9-octan-4-yloxy-9- oxononyl)amino]propylcarbamoyl]benzoyl]amino]propyl-(9-octan-4-yloxy-9- oxononyl)amino]nonanoate); Lipid H (SM-102) (heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo- 6-undecoxyhexyl)amino]octanoate); OF-Deg-Lin (2-[4-[5-[4-[bis[2-[(9Z,12Z)-octadeca-9,12- dienoyl]oxyethyl]amino]butyl]-3,6-dioxopiperazin-2-yl]butyl-[2-[(9Z,12Z)-octadeca-9,12- dienoyl]oxyethyl]amino]ethyl (9Z,12Z)-octadeca-9,12-di enoate); PEG2000-DMG (1,2- Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000); TT3 (A1,A3,A5-tris(3- (didodecylamino)propyl)benzene-l,3,5-tricarboxamide), or combinations thereof.11. The transfection composition of any one of aspects 1-10, wherein said transfection agent and said biological material are intermixed or said biological material is housed within said transfection agent.12. The transfection composition of any one of aspects 1-10, wherein said transfection agent and said biological material are layered in or on a substrate.13. The transfection composition of any one of aspects 1-12, wherein said composition is associated with a substrate including a capillary network.14. The transfection composition of aspect 13, wherein the capillary network is provided by contours along a surface of the substrate.15. The transfection composition of aspect 13, wherein the substrate is a wall of a desiccation chamber or is associated with a wall of the desiccation chamber.16. The transfection composition of aspect 13, wherein the capillary network is contacted by an underlying solid support substrate.17. The transfection composition of any of aspects 1-16, wherein said vitrification agent is trehalose and one or more of glycerol, betine, and / or choline.18. The transfection composition of aspect 17, wherein said vitrification agent is trehalose.19. The transfection composition of any of aspects 1-18, wherein a total volume of transfection composition is from 0.1 pL to 10 pL.20. The transfection composition of any of aspects 1-18, wherein said transfection composition is present in a vitrification mixture including said vitrification agent, at 1 pg or less.21. The transfection composition of any of aspects 1-18, wherein said transfection composition is of a total mass of 5 pg or less, optionally less than 1 pg.22. The transfection composition of any of aspects 1-18, wherein said transfection composition is of a total mass of 0.1 pg to 1 pg.23. A process of transfecting a cell or tissue, including contacting said cell or tissue with the transfection composition of any one of aspects 1-22.24. The process of aspect 23, wherein a transfection efficiency of said transfecting is greater than a control transfection efficiency of a control transfection composition not subjected to said glassy state.25. The process of aspect 23, wherein a transfection efficiency of said transfecting is double or greater than a control transfection efficiency of a control transfection composition not subjected to said glassy state.26. The process of aspect 23, wherein a transfection efficiency of said transfecting is triple or greater than a control transfection efficiency of a control transfection composition not subjected to said glassy state.27. The process of aspect 23, wherein a transfection efficiency of said transfecting is fourfold or greater than a control transfection efficiency of a control transfection composition not subjected to said glassy state.28. The process of aspect 23 further including eluting said transfection composition in an elution buffer prior to said contacting.29. The process of any of aspects 23-28 wherein said cell is a eukaryotic cell.30. The process of any of aspects 23-28 wherein said cell is a prokaryotic cell.31. The process of aspect 28 wherein said elution buffer comprises a buffering agent, water and optionally one or more salts.32. A process of forming a transfection composition of any of aspects 1-22 including: a) overlaying a vitrification mixture including the transfection agent and a vitrification medium on a substrate including a capillary network, said substrate in a desiccation chamber, wherein said vitrification medium comprises said vitrification agent; b) lowering an atmospheric pressure within the desiccation chamber; c) providing a heat energy from the surface to the vitrification mixture, wherein the heat energy is sufficient to prevent the vitrification mixture from experiencing a freezing condition; and d) desiccating the vitrification mixture by capillary action until the vitrification mixture enters the glassy state, wherein said providing is optionally simultaneous with or prior to initiating said desiccating.33. The process of aspect 32, wherein said transfection agent is layered onto or into said substrate.34. The process of aspect 33, wherein said transfection agent is intermixed with said biological material.35. The process of aspect 33, wherein said biological material is layered onto said transfection agent onto or into said substrate.36. The process of aspect 33, wherein said biological material is layered onto said transfection agent prior to said step of desiccating.37. The process of aspect 33, wherein said biological material is layered onto said transfection agent following said step of desiccating, and further including subjecting said biological material to a second desiccating step.38. The process of any of aspects 32-37, wherein the capillary network is provided by contours along a surface of the substrate.39. The process of any of aspects 32-37, wherein the substrate is a wall of the desiccation chamber or is associated with a wall of the desiccation chamber.40. The process of any of aspects 32-37, wherein the capillary network within the desiccation chamber is contacted by an underlying solid support substrate.41. The process of any of aspects 32-37, wherein vitrification of the vitrification mixture occurs in less than 30 minutes.42. The process of aspect 41, wherein vitrification of the vitrification mixture occurs in less than 10 minutes.43. The process of any of aspects 32-37, wherein the heat energy is provided by heating the vitrification mixture.44. The process of any of aspects 32-37, wherein the atmospheric pressure is lowered to a value of from about 0.9 atm to about 0.005 atm.45. The process of any of aspects 32-37, wherein the atmospheric pressure is lowered to about 0.004 atm.46. The process of any of aspects 32-37, wherein the heat energy provided is sufficient to prevent crystallization within the vitrification mixture during vitrification.47. The process of any of aspects 32-37, wherein the provided heat energy is sufficient to keep the biological material at a temperature of from about 0 °C to about 40 °C during said vitrifying.48. The process of any of aspects 32-37, wherein said vitrification medium comprises trehalose and one or more of glycerol, betine, and / or choline.49. The process of any of aspects 32-37, wherein the capillary network is hydrophilic.50. The process of any of aspects 32-37, wherein the capillary network comprises capillary channels with a continuous wall from a first side of said substrate to a second side of said substrate.

[0136] While aspects of the disclosure have been illustrated and described, it is not intended that these aspects illustrate and describe all possible forms of the disclosure. Rather, the words used in the specification are words of description rather than limitation, and it is understood that various modifications and substitutions may be made thereto without departing from the spirit and scope of the disclosure.

[0137] PATENT DOCUMENT REFERENCES

[0138] 6,808,651 Bl 10 / 2004 Katagiri, et al.

[0139] 7,883,664 B2 2 / 2011 G. Elliott; N. Chakraborty.

[0140] 8,349,252 B2 1 / 2013 G. Elliott; N. Chakraborty.

[0141] 10,568,318 B2 2 / 2020 P.S. Mohanty, N. Chakraborty

[0142] US 2013 / 0157250 Al 6 / 2013 Gutierrez et al.

[0143] US 2013 / 0260452 Al 10 / 2013 Toner et al.

[0144] NON-PATENT REFERENCES

[0145] Chakraborty N, Menze MA, Malsam J, Aksan A, Hand SC, et al. (2011) Cryopreservation of Spin-Dried Mammalian Cells, PLoS ONE 6(9): e24916.

[0146] Chakraborty N, Biswas D, Elliott GD (2010) A Simple Mechanistic Way to Increase the Survival of Mammalian Cells During Processing for Dry Storage, Biopreservation and Biobanking, 8 (2), 107-114.

[0147] Various modifications of the present disclosure, in addition to those shown and described herein, will be apparent to those skilled in the art of the above description. Such modifications are also intended to fall within the scope of the appended claims.

[0148] It is appreciated that all reagents are obtainable by sources known in the art unless otherwise specified.

[0149] It is also to be understood that this disclosure is not limited to the specific aspects and methods described herein, as specific components and / or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing particular aspects of the present disclosure and is not intended to be limiting in any way. It will be also understood that, although the terms “first,” “second,” “third” etc. may be used herein to describe various elements, components, regions, layers, and / or sections, these elements, components, regions, layers, and / or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. Thus, “a first element,” “component,” “region,” “layer,” or “section” discussed below could be termed a second (or other) element, component, region, layer, or section without departing from the teachings herein. Similarly, as used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms, including “at least one,” unless the content clearly indicates otherwise. “Or” means “and / or.” As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. It will be further understood that the terms “comprises” and / or “comprising,” or “includes” and / or “including” when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof. The term “or a combination thereof’ means a combination including at least one of the foregoing elements.

[0150] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0151] Reference is made in detail to exemplary compositions, aspects and methods of the present disclosure, which constitute the best modes of practicing the disclosure presently known to the inventors. The Figures are not necessarily to scale. However, it is to be understood that the disclosed aspects are merely exemplary of the disclosure that may be embodied in various and alternative forms. Therefore, specific details disclosed herein are notto be interpreted as limiting, but merely as a representative basis for any aspect of the disclosure and / or as a representative basis for teaching one skilled in the art to variously employ the present disclosure.

[0152] Patents, publications, and applications mentioned in the specification are indicative of the levels of those skilled in the art to which the disclosure pertains. These patents, publications, and applications are incorporated herein by reference to the same extent as if each individual patent, publication, or application was specifically and individually incorporated herein by reference.

[0153] The foregoing description is illustrative of particular embodiments of the disclosure, but is not meant to be a limitation upon the practice thereof. The following claims, including all equivalents thereof, are intended to define the scope of the disclosure.

Claims

CLAIMS1. A transfection composition comprising: a transfection agent and a vitrification agent; and wherein the transfection composition is in a glassy state.

2. The transfection composition of claim 1, wherein a ratio of the transfection agent (in micrograms) to the vitrification medium (in microliters (at 1200 mM vitrification agent)) is equal to or greater than 1:0.5.

3. The transfection composition of claim 2, wherein a ratio of the transfection agent to the vitrification medium is less than or equal to 1 :200, optionally 1 : 150, optionally 1:100, optionally 1:90, optionally 1:80, optionally 1:70, optionally 1:60.

4. The transfection composition of any one of claims 1-3 further comprising a biological material.

5. The transfection composition of claim 4, wherein the biological material comprises one or more polynucleotide chains.

6. The transfection composition of claim 5, wherein the polynucleotide chain comprises an expression vector.

7. The transfection composition of claim 4, wherein the biological material comprises an enzyme, an antibody, a protein, polydeoxyribonucleotide chain, a polyribonucleotide chain, siRNA, shRNA, Cas9 or a Cas9 expression plasmid, a cell or cell fragment, or a genome or portion thereof.

8. The transfection composition of any of claims 1-3, wherein the transfection agent comprises one or more cationic polymers, a lipid, a virus, a cell penetrating peptide, a dendrimer, or a polynucleotide-binding protein.

9. The transfection composition of claim 8, wherein the cationic polymer comprises one or more of DEAE-dextran, poly-amino acids (optionally poly-L-lysine), poly(amidoamine) (PAMAM) dendrimers, PPI (poly-(propylenimine)), PEI (polyethylenimine), PDMAEMA (polymethacrylic acid N,N-dimethylaminoethyl ester), poly(P-amino ester), PHP (Poly(4-hydroxy-L-proline ester)), PAGA (poly[a-(4-aminobutyl)- 1-glycolic acid]), PVL poly(S-valerolactone), aminated PAHA (aminated poly(a-hydroxy acids)), PPE (polyphosphoester), polylactide (poly(lactic acid), PLA), N,N- diethylethylenediamine-polyurethane (DEDA-PU), poly(p-phenyleneethynylene), polythiophene, poly(fluorine-co-phenylene), poly(p-phenylenevinylene), protamine, or a combination thereof.

10. The transfection composition of claim 7, wherein the lipid comprises DOSPA (2,3 -di oleoyloxy -N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propaniminium trifluoroacetate), 1,2-Dioleoyl-sn-glycerophosphoethanolamine (DOPE), DOTMA (1,2- Dioleoyl-3 -trimethylammonium propane), cholesterol, polyethylene glycol (PEG)-lipid conjugate, 3060iio (tetrakis(8-methylnonyl) 3,3',3",3"'-(((methylazanediyl) bis(propane-3,l diyl))bis (azanetriyl))tetrapropionate; 9A1P9, decyl (2-(dioctylammonio)ethyl) phosphate); A2-Iso5 -2DC 18 (ethyl 5 , 5 -di((Z)-heptadec-8-en- 1 -y 1) - 1 -(3 -(pyrrolidin- 1 -yl)propyl)-2, 5 - dihydro- U / -imidazole-2-carboxylate); ALC-0315 (2 -hexyl-decanoic acid, 1, l'-[[(4- hydroxybutyl )imino]di-6, 1 -hexanediyl] ester); ALC-0159 (a-[2-(ditetradecylamino)-2- oxoethyl]-co~methoxy~poly(oxy- 1 ,2-ethanediyl)); P-sitosterol ((35, 85,95, 1 OR, 135, 145, 175)- 17-((25,55)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11, 12, 13, 14, 15, 16,17-tetradecahydro-U / -cy cl openta[a]phenanthren-3-ol); BAME-O16B (2-(dodecyldisulfanyl)ethyl 3-[2-[2-[bis[3-[2-(dodecyldisulfanyl)ethoxy]-3- oxopropyl]amino]ethyl-methylamino]ethylamino]propanoate); BHEM-Cholesterol ((N,N- bis(2-hydroxyethyl)-N-methyl-N-(2-cholesteryloxy carbonyl aminoethyl) ammonium bromide); cKK-E12 (3, 6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione); DC-Cholesterol (3P-[7V-(A',A7-dimethylaminoethane)-carbamoyl]cholesterol); DLin-MC3-DMA ((6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino) butanoate); DOPE ([(2R)-3-[2-aminoethoxy(hydroxy)phosphoryl]oxy-2-[(Z)-octadec-9- enoyl]oxypropyl] (Z)-octadec-9-enoate); DOSPA (2-[2,5-bis(3- aminopropylamino)pentanoylamino]ethyl-[2,3-bis[(Z)-octadec-9-enoxy]propyl]- dimethylazanium;chloride;tetrahydrochloride); DOTAP (2,3-bis[[(Z)-octadec-9- enoyl]oxy]propyl-trimethylazanium); DOTMA (2,3-bis[(Z)-octadec-9-enoxy]propyl- trimethylazanium;chloride); DSPC ([(2R)-2,3-di(octadecanoyloxy)propyl] 2- (trimethylazaniumyl)ethyl phosphate); ePC (ethylphosphatidylcholine); FTT5 (octan-4-yl 9- [3-[[3,5-bis[3-[bis(9-octan-4-yloxy-9- oxononyl)amino]propylcarbamoyl]benzoyl]amino]propyl-(9-octan-4-yloxy-9- oxononyl)amino]nonanoate); Lipid H (SM-102) (heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo- 6-undecoxyhexyl)amino]octanoate); OF-Deg-Lin (2-[4-[5-[4-[bis[2-[(9Z,12Z)-octadeca-9,12- dienoyl]oxyethyl]amino]butyl]-3,6-dioxopiperazin-2-yl]butyl-[2-[(9Z,12Z)-octadeca-9,12- dienoyl]oxyethyl]amino]ethyl (9Z,12Z)-octadeca-9,12-di enoate); PEG2000-DMG (1,2- Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000); TT3 (A1,A3,A5-tris(3- (didodecylamino)propyl)benzene-l,3,5-tricarboxamide), or combinations thereof.

11. The transfection composition of any one of claims 1-3, wherein the transfection agent and the biological material are intermixed or the biological material is housed within the transfection agent.

12. The transfection composition of any one of claims 1-3, wherein the transfection agent and the biological material are layered in or on a substrate.

13. The transfection composition of any one of claims 1-3, wherein the composition is associated with a substrate comprising a capillary network.

14. The transfection composition of claim 13, wherein the capillary network is provided by contours along a surface of the substrate.

15. The transfection composition of claim 13, wherein the substrate is a wall of a desiccation chamber or is associated with a wall of the desiccation chamber.

16. The transfection composition of claim 13, wherein the capillary network is contacted by an underlying solid support substrate.

17. The transfection composition of any of claims 1-3, wherein the vitrification agent is trehalose and one or more of glycerol, betine, and / or choline.

18. The transfection composition of claim 17, wherein the vitrification agent is trehalose.

19. The transfection composition of any of claims 1-3, wherein a total volume of transfection composition is from 0.1 pL to 10 pL.

20. The transfection composition of any of claims 1-3, wherein the transfection composition is present in a vitrification mixture comprising the vitrification agent, at 1 pg or less.

21. The transfection composition of any of claims 1-3, wherein the transfection composition is of a total mass of 5 pg or less, optionally less than 1 pg.

22. The transfection composition of any of claims 1-3, wherein the transfection composition is of a total mass of 0.1 pg to 1 pg.

23. A process of transfecting a cell or tissue, comprising contacting the cell or tissue with the transfection composition of any one of claims 1-3.

24. The process of claim 23, wherein a transfection efficiency of the transfecting is greater than a control transfection efficiency of a control transfection composition not subjected to the glassy state.

25. The process of claim 23, wherein a transfection efficiency of the transfecting is double or greater than a control transfection efficiency of a control transfection composition not subjected to the glassy state.

26. The process of claim 23, wherein a transfection efficiency of the transfecting is triple or greater than a control transfection efficiency of a control transfection composition not subjected to the glassy state.

27. The process of claim 23, wherein a transfection efficiency of the transfecting is four-fold or greater than a control transfection efficiency of a control transfection composition not subjected to the glassy state.

28. The process of claim 23 further comprising eluting the transfection composition in an elution buffer prior to the contacting.

29. The process of claims 23, wherein the cell is a eukaryotic cell.

30. The process of claims 23, wherein the cell is a prokaryotic cell.

31. The process of claim 28, wherein the elution buffer comprises a buffering agent, water and optionally one or more salts.

32. A process of forming a transfection composition of any of claims 1-3 comprising: a) overlaying a vitrification mixture comprising the transfection agent and a vitrification medium on a substrate comprising a capillary network, the substrate in a desiccation chamber, wherein the vitrification medium comprises the vitrification agent; b) lowering an atmospheric pressure within the desiccation chamber; c) providing a heat energy from the surface to the vitrification mixture, wherein the heat energy is sufficient to prevent the vitrification mixture from experiencing a freezing condition; and d) desiccating the vitrification mixture by capillary action until the vitrification mixture enters the glassy state, wherein the providing is optionally simultaneous with or prior to initiating the desiccating.

33. The process of claim 32, wherein the transfection agent is layered onto or into the substrate.

34. The process of claim 33, wherein the transfection agent is intermixed with the biological material.

35. The process of claim 33, wherein the biological material is layered onto the transfection agent onto or into the substrate.

36. The process of claim 33, wherein the biological material is layered onto the transfection agent prior to the step of desiccating.

37. The process of claim 33, wherein the biological material is layered onto the transfection agent following the step of desiccating, and further comprising subjecting the biological material to a second desiccating step.

38. The process of claim 32, wherein the capillary network is provided by contours along a surface of the substrate.

39. The process of claim 32, wherein the substrate is a wall of the desiccation chamber or is associated with a wall of the desiccation chamber.

40. The process of claim 32, wherein the capillary network within the desiccation chamber is contacted by an underlying solid support substrate.

41. The process of claim 32, wherein vitrification of the vitrification mixture occurs in less than 30 minutes.

42. The process of claim 41, wherein vitrification of the vitrification mixture occurs in less than 10 minutes.

43. The process of claim 32, wherein the heat energy is provided by heating the vitrification mixture.

44. The process of claim 32, wherein the atmospheric pressure is lowered to a value of from about 0.9 atm to about 0.005 atm.

45. The process of claim 32, wherein the atmospheric pressure is lowered to about0.004 atm.

46. The process of claim 32, wherein the heat energy provided is sufficient to prevent crystallization within the vitrification mixture during vitrification.

47. The process of claim 32, wherein the provided heat energy is sufficient to keep the biological material at a temperature of from about 0 °C to about 40 °C during the vitrifying.

48. The process of claim 32, wherein the vitrification medium comprises trehalose and one or more of glycerol, betine, and / or choline.

49. The process of claim 32, wherein the capillary network is hydrophilic.

50. The process of claim 32, wherein the capillary network comprises capillary channels with a continuous wall from a first side of the substrate to a second side of the substrate.

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