Liposomal controlled release of PCR reaction reagents

Abstract

PCR methods and compositions are described for using a liposomal particle disclosed herein. In one embodiment, the liposomal particle comprises at least one lipid and at least one PCR reaction reagent, where the at least one lipid encapsulates the at least one PCR reaction reagent. The liposomal particles can be used to deliver specific PCR reaction reagents with precise accuracy using temperature as a regulator.

Description

LIPOSOMAL CONTROLLED RELEASE OF PCR REACTION REAGENTSRELATED APPLICATIONS

[0001] The present patent application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63 / 673,320, filed July 19, 2024, the contents of which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] Polymerase chain reaction (PCR) is a biochemical method used to amplify short segments of nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), to measurable quantities. PCR allows the nucleic acid sequences to be exponentially amplified, generating thousands to millions of copies of the particular nucleic acid segment being amplified.

[0003] Custom PCR reaction reagents with unique properties such as better single nucleotide polymorphisms (SNP) discrimination, faster & cleaner strand di spl acemen t / polymerase functionality, faster & cleaner reverse transcriptase with better thermal stability are constantly being designed. However, during the reaction, amplification products may compete with the PCR reaction reagents. For example, misamplification may originate from reverse transcriptasebased processes at low temperatures during thermal cycle ramping. Elimination of this misamplification may lead to PCR results with higher sensitivity. The ability to delay the introduction of PCR reaction reagents until the appropriate time in the PCR method may reduce this competition and increase the efficiency of the reaction.

[0004] In addition, new enzyme processes yet to be identified as commercially important for molecular diagnostic could also benefit from a delayed release of PCT reagents without identifying unique aptamer / antibody interactions. This technology could be especially useful for PCR (i.e. , gradient, accelerated PCR, isothermal, etc.) and highly multiplexed panel applications that utilize Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV), CM ReverseTranscriptase A- Enzyme (CMRT) and / or Ribonuclease Hl / 2 (RNAse Hl / 2) applications where the misamplification is originating from uncontrolled enzyme activity at lower temperatures. This would eliminate the need for development of antibody or aptamer-based silencing of new enzymes allowing for rapid product development of novel enzymes and PCR reaction reagents with highly tunable warm and hot start technologies using liposomes. As such a need exists for a delivery method, such as a thermosensitive delivery method, for the delayed release of PCR regents.BREIF SUMMARY

[0005] The present disclosure relates to a liposomal particle comprising at least one lipid and at least one PCR reaction reagent, wherein the at least one lipid encapsulates the at least one PCR reaction reagent.

[0006] In an aspect, the at least one lipid comprises one or more of an anionic lipid, a cationic lipid, a lysolipid, a neutral lipid, a neutral phospholipid, and a PEG-lipid conjugate.

[0007] In an aspect, the at least one lipid comprises one or more C12 to C40 tails.

[0008] In an aspect, the liposomal particle comprises the anionic lipid, wherein the anionic lipid is selected from DOPG, DMPG, DPPG, DSPG, DLPG, or DAPG. In an aspect, the liposomal particle comprises the lysolipid, wherein the lysolipid is selected from the group consisting of MPPC, Lyso-PC, Lyso-PE, Lyso-PG, Lyso-PI, and Lyso GA2. In an aspect, the liposomal particle comprises the PEG-lipid conjugate, wherein the PEG-lipid conjugate is selected from PEG-DMG, PEG-DPG, PEG-DSG, PEG-DAG, PEG-DAA, PEG- phospholipid, and PEG-ceramide.

[0009] In an aspect, the at least one lipid is present in an amount between about 0 mol% and about 100 mol%; alternatively between about 1 mol% and about 100 mol%; alternatively between about 2 mol% and about 100 mol%; alternatively between about 5 mol% and about 100 mol%; alternatively between about 10 mol% and about 100 mol%; alternatively between about 50 mol% and about 100 mol%; alternatively between about 60 mol% and about 100 mol%; alternatively between about 70 mol% and about 100 mol%; alternatively between about 80 mol% and about 100 mol%; alternatively between about 90 mol% and about 100 mol%; alternatively about 75 mol%; alternatively about 80 mol%; alternatively about 85 mol%; alternatively about 90 mol%; alternatively about 95% mol; alternatively about 96 mol%; alternatively about 97 mol%;alternatively about 98 mol%; alternatively about 99 mol%; or alternatively about 100 mol% based on the at least one lipid of the liposomal particle.

[0010] In an aspect, the liposomal particle comprises at least a first lipid and a second lipid.

[0011] In an aspect, the first lipid is present in an amount between about 50 mol% and about 100 mol%; alternatively between about 60 mol% and about 99 mol%; alternatively between about 70 mol% and about 99 mol%; alternatively between about 80 mol% and about 99 mol%; alternatively between about 85 mol% and about 99 mol%; alternatively between about 86 mol% and about 99 mol%; alternatively between about 87 mol% and about 99 mol%; alternatively between about 88 mol% and about 99 mol%; alternatively between about 89 mol% and about 99 mol%; alternatively between about 90 mol% and about 99 mol%; alternatively between about 91 mol% and about 99 mol%; alternatively between about 92 mol% and about 99 mol%; alternatively between about 93 mol% and about 99 mol%; alternatively between about 94 mol% and about 99 mol%; alternatively between about 95 mol% and about 99 mol%; alternatively between about 95 mol% and about 99 mol%; alternatively about 96 mol%; alternatively about 96.5 mol%; alternatively about 97 mol%; alternatively about 97.5 mol%; alternatively about 98 mol%; or alternatively about 98.5 mol% based on the first lipid of the liposomal particle; andthe second lipid is present in an amount between about 0 mol% and about 50 mol%; alternatively between about 1 mol% and about 40 mol%; alternatively between about 2 mol% and about 30 mol%; alternatively between about 3 mol% and about 20 mol%; alternatively between about 4 mol% and about 15 mol%; alternatively about 0.6 mol%, alternatively about 0.7 mol%, alternatively about 0.8 mol%, alternatively about 0.9 mol%, alternatively about 1.0 mol%, alternatively about 1.5 mol%, alternatively about 2 mol%, alternatively about 2.5 mol%, alternatively about 3 mol%, alternatively about 3.5 mol%, alternatively about 4 mol%, alternatively about 4.5 mol%, or alternatively about 5 mol%, based on the second lipid of the liposomal particle.

[0012] In an aspect, first lipid comprises the anionic lipid or the cationic lipid and the second lipid comprises the lysolipid or the PEG-lipid conjugate.

[0013] In an aspect, the liposomal particle further comprises a third lipid.

[0014] In an aspect, the third lipid is present in an amount between about 0.1 mol% and about 5 mol%, between about 0.5 mol% and about 5 mol%, between about 1 mol% and about 5 mol%, between about 0.1 mol% and about 4 mol%, between about 0.5 mol% and about 4 mol%, between about 1 mol% and about 4 mol%, between about 0.1 mol% and about 3 mol%, between about 1mol% and about 3 mol%, alternatively about 0.6 mol%, alternatively about 0.7 mol%, alternatively about 0.8 mol%, alternatively about 0.9 mol%, alternatively about 1.0 mol%, alternatively about 1.5 mol%, alternatively about 2 mol%, alternatively about 2.5 mol%, alternatively about 3 mol%, alternatively about 3.5 mol%, alternatively about 4 mol%, alternatively about 4.5 mol%, or alternatively about 5 mol%, based on the lipid of the liposomal particle. In an aspect, the first lipid comprises the anionic lipid or the cationic lipid, the second lipid comprises the lysolipid, and the third lipid comprises the PEG-lipid conjugate.

[0015] In an aspect, the at least one PCR reaction reagent comprises an enzyme, deoxyribonucleotide triphosphates, divalent metal cations, a forward primer, a reverse primer, a probe, an aptamer, a reducing agent, a RNase inhibitor, or a detergent, or a combination thereof.

[0016] In an aspect, the at least one PCR reaction reagent comprises an enzyme, and the liposomal particle has a lipid to enzyme charge ratio between about 2 and about 100.

[0017] In an aspect, the at least one PCR reaction reagent comprises an enzyme selected from the group consisting of a nucleic acid polymerase, a reverse transcriptase enzyme, an RNase H, HiDi, strand displacement enzymes, and a combination thereof. In an aspect, the RNase H is selected from the group consisting of RNase Hl and RNase H2.

[0018] In an aspect, the at least one PCR reaction reagent comprises the forward primer, the reverse primer, and / or the probe, wherein the forward primer, the reverse primer, and / or the probe are specific for a target nucleic acid. In an aspect, the forward primer, the reverse primer, and / or the probe are specific for the same target nucleic acid.

[0019] In an aspect, the encapsulation inhibits activity of the at least one PCR reaction reagent.

[0020] In an aspect, when the liposomal particle is thermally disrupted, the at least one PCR reaction reagent is released and / or the at least one PCR reaction reagent is activated.

[0021] In an aspect, the liposomal particle is thermally disrupted when the liposomal particle is subjected to a temperature of between about 40°C or about 100°C, between about 40°C or about 80°C, between about 40°C or about 70°C, between about 40°C or about 60°C, between about 45°C or about 75°C, between about 45°C or about 70°C, between about 45°C or about 60°C, between about 50°C or about 70°C, between about 50°C or about 60°C, alternatively greater than 50°C, alternatively greater than 55°C, alternatively greater than 60°C, alternatively greater than 65°C, alternatively greater than 70°C, alternatively greater than 75°C, alternatively greater than 85°C, alternatively greater than 90°C, or alternatively greater than 95 °C.

[0022] In an aspect, the liposomal particle is about 50 nm to about 150 nm in size. In an aspect, the liposomal particle has a PDI less than 0.5, or alternatively between about 0.15 and about 0.50.

[0023] One aspect of the disclosure is a composition comprising any one of the disclosed liposomal particles. In an aspect, the composition is a dried composition. In an aspect, the composition is a liquid or aqueous composition. In an aspect, the composition has a pH of between about 3 to about 7, alternatively about 3, alternatively about 4, alternatively about 5, alternatively about 6, or alternatively about 7.

[0024] In an aspect, the PCR reaction reagent comprises an enzyme selected from the group consisting of a nucleic acid polymerase, a reverse transcriptase enzyme, an RNase H, HiDi, strand displacement enzymes, and a combination thereof.

[0025] In an aspect, the composition further comprises a nucleic acid polymerase, deoxyribonucleotide triphosphates, a divalent metal cation, an aqueous buffer, a forward primer, a reverse primer, and / or a reverse transcriptase.

[0026] In an aspect, the composition is contained within a cartridge device.

[0027] One aspect of the disclosure is a method for performing an amplification reaction, wherein the method comprises using any one of the disclosed liposomal particles or any one of the disclosed compositions.

[0028] In an aspect, the amplification reaction is a real-time PCR reaction.

[0029] In an aspect, the PCR reaction reagent is released from the at least one lipid when the amplification reaction reaches a temperature between about 40°C or about 100°C; alternatively between about 40°C or about 80°C; alternatively between about 40°C or about 70°C; alternatively between about 40°C or about 60°C; alternatively between about 45 °C or about 75 °C; alternatively between about 45 °C or about 70°C; alternatively between about 45 °C or about 60°C; alternatively between about 50°C or about 70°C; alternatively between about 50°C or about 60°C, alternatively greater than 50°C, alternatively greater than 55°C, alternatively greater than 60°C, alternatively greater than 65°C, alternatively greater than 70°C, alternatively greater than 75°C, alternatively greater than 85°C, alternatively greater than 90°C, or alternatively greater than 95°C.

[0030] In an aspect, the method further comprises receiving, in a module having a receiving bay, a cartridge device comprising any one of the disclosed compositions, wherein the module includes one or more mechanisms within the receiving bay for manipulating a fluid sample within thecartridge device; and interfacing with a reaction vessel with a memory having programmable instructions for operating the module recorded thereon.

[0031] In an aspect, the method results in at least one of the following characteristics: reduced misamplification during reverse transcriptase steps in the real-time PCR reaction, reduced enzyme activation at lower temperatures, or greater sensitivity.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] An understanding of the features and advantages of the present invention will he obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0033] FIGs. 1A and IB depict schematics for producing and designing thermosensitive liposomes that inhibit activity of PCR reaction reagents.

[0034] FIG. 2 is a graph generated using a method according to an aspect of this disclosure showing the signal (relative fluorescence units, RFU) of unencapsulated CMRT compared with exemplary liposomal particles according to an aspect of this disclosure.

[0035] FIG. 3 is a graph generated using a method according to an aspect of this disclosure showing the signal (relative fluorescence units, RFU) from hgDNA PCR using encapsulated CMRT and exemplary liposomal particles according to an aspect of this disclosure.

[0036] FIG. 4 is a graph generated using a method according to an aspect of this disclosure showing the signal (relative fluorescence units, RFU) of exemplary liposomal particles according to an aspect of this disclosure using RT substrate at 27 °C with variable charge ratios and pH values.

[0037] FIG. 5 is a graph generated using a method according to an aspect of this disclosure showing the signal (relative fluorescence units, RFU) of exemplary liposomal particles according to an aspect of this disclosure using RT substrate at 55°C with variable charge ratios and pH values.

[0038] FIG. 6 is a graph generated using a method according to an aspect of this disclosure showing the signal (relative fluorescence units, RFU) of exemplary liposomal particles according to an aspect of this disclosure using 0.01% Brij detergent at 27°C.

[0039] FIG. 7 is a graph generated using a method according to an aspect of this disclosure showing the signal (relative fluorescence units, RFU) of exemplary liposomal particles according to an aspect of this disclosure using 0.01% Brij detergent at 55°C.

[0040] FIG. 8 is a graph generated using a method according to an aspect of this disclosure showing the signal (relative fluorescence units, RFU) of exemplary liposomal particles according to an aspect of this disclosure at 27°C.

[0041] FIG. 9 is a graph generated using a method according to an aspect of this disclosure showing the signal (relative fluorescence units, RFU) of exemplary liposomal particles according to an aspect of this disclosure at 40°C.

[0042] FIG. 10 is a graph generated using a method according to an aspect of this disclosure showing the signal (relative fluorescence units, RFU) of exemplary liposomal particles according to an aspect of this disclosure compared with C&G aptamer formulated CMRT (thermosensitive aptamer formulated CMRT) at 27°C.

[0043] FIG. 11 is a graph generated using a method according to an aspect of this disclosure showing the signal (relative fluorescence units, RFU) of exemplary liposomal particles according to an aspect of this disclosure compared with C&G aptamer formulated CMRT (thermosensitive aptamer formulated CMRT) at 40°C.

[0044] FIG. 12 is a graph generated using a method according to an aspect of this disclosure showing the signal (relative fluorescence units, RFU) of exemplary liposomal particles according to an aspect of this disclosure compared with C&G aptamer formulated CMRT (thermosensitive aptamer formulated CMRT) at 55°C.

[0045] FIG. 13 depicts the cycle thresholds (Cts) of exemplary liposomal particles according to an aspect of this disclosure generated using a real-time PCR protocol.

[0046] FIGs. 14A - 14D depict the amplification plot of thermosensitive liposomes, unencapsulated CMRT, or both used in TSR bead (5-plex) comprising CoV RNA (FIGs. 14A and 14B) or a no template control, NTC (FIGs. 14C and 14D) in real-time PCR protocol at 2 mins RT and 10 mins RT.

[0047] FIGs. 15A - 15D depict the post PCR melt of thermosensitive liposomes, unencapsulated CMRT, or both used in TSR bead (5-plex) comprising CoV RNA (FIGs. 15A and 15B) or a no template control, NTC (FIGs. 15C and 15D) in real-time PCR protocol at 2 mins RT and 10 mins RT.DETAILED DESCRIPTION

[0048] Liposomal particles are formations with varying sizes and characterized by one or more hydrophobic lipid bilayers and a hydrophobic core. The liposomal particles are typically spherical but may take other forms. The lipid membrane shields internal cargo from degradation and allows for controlled release making these particles a common carrier for various potentially active molecules. Depending upon the ratio of active molecule to lipid and the nature of the particular lipid employed, the rate of active molecules release can be controlled. Liposomal particles of the present disclosure are configured to release active molecules at a certain temperature. These thermosensitive lipid-based particles have previously been developed as nanocarrier systems for various medical uses such delivery vehicles that release the active pharmaceutical ingredient in a targeted way.

[0049] In the present disclosure, liposomal particles may be designed to deliver PCR components, such as PCR reaction reagents. In certain embodiments, the liposomal particles of the current invention are used as a delivery system that releases PCR reaction reagents in a targeted way at a specific temperature during the PCR cycle. In some aspects, at a specific, or thermal, temperature the polymer solution undergoes separation from one phase to two phases, releasing the PCR reaction reagent. The liposomal particles are configured to release a PCR reaction reagent at discrete, defined, or predetermined temperature by thermolytic disruption of the liposome. Use of appropriate combinations of lipids can allow for reactivation at nearly any desired target release temperature with a narrow thermal release range allowing for fine-tuned thermal activation of any PCR reaction reagent of choice. One of the challenges in PCR is nonspecific or misamplification amplification which can occur when primers bind to unintended sequences. The disclosed liposomal particles are able to overcome some of the challenges of PCR by selectively releasing the components at specific temperatures. This allows for universal formulations to thermally control the activation of any PCR reaction reagent at any desired activation temperature for better control of PCR processes.II. Definitions

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. Any reference to standard methods refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0051] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0052] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0053] The words “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0054] The term “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0055] By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0056] The singular form “a,” “an”, and “the” include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term “or” is generally employed in its usual sense including “and / or” unless the content clearly dictates otherwise. The term “and / or” means any one or more of the items in the list joined by “and / or.” As an example, “x and / or y” means any element of the three-element set {(x), (y), (x, y)}. In other words, “x and / or y” means “one or both of x and y.” As another example, ”x, y, and / or z” means any element of the seven-element set { (x), (y), (z), (x, y), (x, z), (y, z), (x, y, z) } . In other words, “x, y and / or z” means “one or more of x, y and z.”

[0057] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that arc expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, “up to” a number (for example, up to 50) includes the number (for example, 50). The term “in the range” or “within a range” (and similar statements) includes the endpoints of the stated range.

[0058] Reference throughout this specification to “one aspect”, “an aspect”, “certain aspects”, or “some aspects”, etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0059] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” As used herein in connection with a measured quantity, the term “about” refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term “about” as used in connection with a numerical value throughout the specification and the claims denotesan interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / -10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0060] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0061] The term “exemplary” means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms “e.g.,” and “for example” set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0062] As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, “substantially” may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0063] The term “real-time polymerase chain reaction” or “real-time PCR” is a molecular biology technique based on PCR. Unlike conventional PCR, it monitors the amplification of a targeted DNA molecule during the PCR process, allowing for real-time observation. Real-time PCR can be used both quantitatively and semi-quantitatively (i.e., to determine if the amount of DNA molecules is above or below a certain threshold).

[0064] Two common methods for detecting PCR products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence- specific DNAprobes, which are oligonucleotides labeled with a fluorescent reporter, allowing detection only after the probe hybridizes with its complementary sequence.

[0065] The term “qPCR” is used herein to refer to quantitative real-time polymerase chain reaction (PCR), which is also known as “real-time PCR” or “kinetic polymerase chain reaction;” all terms refer to PCR with real-time signal detection.

[0066] As used herein, the terms “nucleic acid” refers to a polymer comprised of nucleotides. The term “nucleic acid” includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and analogs thereof. Non-limiting examples of nucleic acid molecules include DNA (e.g., genomic DNA, cDNA, etc.), RNA molecules (e.g., mRNA, rRNA, cRNA, tRNA, SiRNA, SaRNA, etc.), and chimeras thereof. A nucleic acid molecule can be obtained by cloning. Nitrogenous bases may be conventional bases (adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U)), known analogs thereof (e.g., inosine), known derivatives of purine or pyrimidine bases, or “abasic” residues in which the backbone includes no nitrogenous base for one or more residues. A nucleic acid may comprise only conventional sugars, bases, and linkages, as found in RNA and DNA, or may include both conventional components and substitutions (e.g., conventional bases linked via a methoxy backbone, or a nucleic acid including conventional bases and one or more base analogs).

[0067] The term “oligonucleotide” is used to refer to a nucleic acid that is relatively short, generally shorter than 200 nucleotides, more particularly, shorter than 100 nucleotides, most particularly, shorter than 50 nucleotides. Typically, oligonucleotides are single-stranded DNA molecules.

[0068] As used herein, the term “complementary” refers to the capacity for precise pairing between two nucleotides; i.e., if a nucleotide at a given position of a nucleic acid is capable of hydrogen bonding with a nucleotide of another nucleic acid to form a canonical base pair, then the two nucleic acids are considered to be complementary to one another at that position. Complementarity between two single stranded nucleic acid molecules may be “partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single-stranded molecules. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[0069] “Substantially complementary,” as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary. Substantially complementary sequences arc able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template.

[0070] “Specific hybridization” refers to the binding of a nucleic acid to a target nucleotide sequence in the absence of substantial binding to other nucleotide sequences present in the hybridization mixture under defined stringency conditions.

[0071] The term “deoxynucleotide triphosphate (dNTP),” as used herein, refers is a monomeric unit of DNA and formed by reducing ribonucleotides with the enzyme ribonucleotide reductase (RNR). It contains a nitrogen base, bound to deoxyribose sugar, and three phosphate groups attached to its 5' carbon.

[0072] The term “divalent metal cations,” as used herein, refer are positively charged ions that form when a neutral atom loses two electrons in the process of oxidation.

[0073] The term “primer,” as used herein, refers to an oligonucleotide capable of acting as a point of initiation for DNA synthesis under suitable conditions. Suitable conditions include those in which hybridization of the oligonucleotide to a template nucleic acid occurs, and synthesis or amplification of the target sequence occurs, in the presence of four different nucleoside triphosphates and an agent for extension (e.g., a DNA polymerase) in an appropriate buffer and at a suitable temperature. Primers for use in PCR arc designed to have regions that arc substantially complementary to regions of the DNA to be used as a template for the PCR.

[0074] “Forward primers” are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified. “Upstream” is used herein to refer to a location 5’ to the DNA sequence to be amplified relative to the coding strand.

[0075] “Reverse primers” are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified. “Downstream” is used herein to refer to a location 3' to the DNA sequence to be amplified relative to the coding strand.

[0076] The term “aptamer” as used herein refers to a nucleic acid that has a specific binding affinity for a target molecule. Like all nucleic acids, a particular' nucleic acid ligand may be described by a linear sequence of nucleotides (A, U, T, C, and G), typically 30-75 nucleotides long.

[0077] The term “probe,” such as a DNA or RNA probe, refers to a probe which allows detection of specific, complementary, nucleic acid sequences.

[0078] The term “reducing agent” or “reducing agents” refers to compounds which may be used to disrupt, or reduce, disulfide bonds in nucleic acids.

[0079] The term “Ribonuclease inhibitors” or “RNase inhibitors” refer to recombinant enzymes used to inhibit RNase.

[0080] The term “detergents” refers to any detergent suitable for PCR use. Non-limiting examples include non-ionic detergents such as Tween 20, NP-40, and Triton X-100.

[0081] The term “RNase H” used herein refers to the Ribonuclease H family of non-sequence- spccific endonuclease enzymes.

[0082] The term “HiDi” or “HiDi Taq DNA polymerase” as used herein refers to a highly selective DNA polymerase variant used for assays associated with High Discrimination.

[0083] The term “strand displacement enzymes” refers to a DNA polymerase able to facilitate strand displacement amplification such as a helicase.

[0084] The invention is defined in the claims. However, below is a non-exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.III. Liposomal particle

[0085] One aspect of the disclosure is a liposomal particle comprising at least one lipid and at least one PCR reaction reagent, wherein the at least one lipid encapsulates the at least one PCR reaction reagent.

[0086] Liposomal particle refers to a liposomal based delivery system that responds to external stimuli such as temperature. The term “liposome,” “liposomal”, “liposomal particle” as used herein, refers to a lipid vesicle composed of one or more lipid bilayers. These vesicles can be used to encapsulate and carry various products. As used herein, “delivery system” or “delivery vehicle” refers to any substance that facilitates, at least in part, the delivery of a PCR component, such as a PCR reaction reagent. In some aspects, the liposomal particle may be a lipid nanoparticle (LNP).

[0087] In some aspects, the liposomal particle is about 50 nm to about 150 nm in size; alternatively about 55 nm in size; alternatively about 60 nm in size; alternatively about 65 nm in size; alternatively about 70 nm in size; alternatively about 75 nm in size; alternatively about 80 nm in size; alternatively about 90 nm in size; alternatively about 95 nm in size; alternatively about 100 nm in size; alternatively about 110 nm in size; alternatively about 120 nm in size; alternatively about 130 nm in size; or alternatively about 140 nm in size. In some aspects, the liposomal particle has a lipid to enzyme charge ratio between about 2 and about 100; alternatively about 5 and about 95; alternatively between about 10 and about 90; alternatively between about 20 and about 80; or alternatively between about 30 and about 70. The charge ratios are calculated according to the number of positive charges represented by lipid- specific head groups (one positive charge per head group) and the number of negative charges represented by the enzyme. In some embodiments, the liposomal particle can have a polydispersity index (PDI) less than 0.5, or alternatively between about 0.15 and about 0.50. However, the versatility of the liposomal particle lies in the fact that its biophysical characteristics, e.g., vesicle size, lamellarity, surface charge, and / or membrane fluidity, can be modified by the lipid composition and / or preparation method used.

[0088] In some aspect, the lipid is or comprises one or more of an anionic lipid, a cationic lipid, a lysolipid, a neutral lipid, a neutral phospholipid, and a PEG-lipid conjugate. The term “lipid” as used herein, refers to the natural or synthetic structures used to assemble liposomes. Structurally, phospholipids can be used to form a lipid bilayer membrane of a liposome. Thebilayer phospholipid membrane can have a hydrophobic tail and a hydrophilic head that leads to the formation of an amphiphilic structure. Lipid composition can affect characteristics such as particle size, stability, and electric charge. Liposomes may be referred to as anionic, cationic and neutral based on the surface charge. The lipid may further include one or more C12 to C40 tails - the lipid hydrocarbon tails that make up membrane of lipid membrane.[0089J Cationic lipids carry a positive charge while anionic lipids carry a negative charge. Nonlimiting examples of anionic lipids include, l,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), l,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dipalmitoyl-sn-glycero- 3 -phosphoglycerol (DPPG), l,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1,2- dilauroyl-sn-glycero-3-phosphoglycerol (DLPG), or 2,4-diacetylphloroglucinol (DAPG).

[0090] In some aspects, the at least one lipid is present in an amount between about 0 mol% and about 100 mol%; alternatively between about 1 mol% and about 100 mol%; alternatively between about 2 mol% and about 100 mol%; alternatively between about 5 mol% and about 100 mol%; alternatively between about 10 mol% and about 100 mol%; alternatively between about 50 mol% and about 100 mol%; alternatively between about 60 mol% and about 100 mol%; alternatively between about 70 mol% and about 100 mol%; alternatively between about 80 mol% and about 100 mol%; alternatively between about 90 mol% and about 100 mol%; alternatively about 75 mol%; alternatively about 80 mol%; alternatively about 85 mol%; alternatively about 90 mol%; alternatively about 95% mol; alternatively about 96 mol%; alternatively about 97 mol%; alternatively about 98 mol%; alternatively about 99 mol%; or alternatively about 100 mol% based on the at least one lipid of the liposomal particle.

[0091] In other aspects, the liposomal particle comprises at least two lipids, alternatively at least three lipids, or alternatively at least four or more lipids. In an embodiment, the liposomal particle comprises at least a first lipid and a second lipid.

[0092] In certain embodiments, the first lipid is present in an amount between about 50 mol% and about 100 mol%; alternatively between about 60 mol% and about 99 mol%; alternatively between about 70 mol% and about 99 mol%; alternatively between about 80 mol% and about 99 mol%; alternatively between about 85 mol% and about 99 mol%; alternatively between about 86 mol% and about 99 mol%; alternatively between about 87 mol% and about 99 mol%;alternatively between about 88 mol% and about 99 mol%; alternatively between about 89 mol% and about 99 mol%; alternatively between about 90 mol% and about 99 mol%; alternatively between about 91 mol% and about 99 mol%; alternatively between about 92 mol% and about 99 mol%; alternatively between about 93 mol% and about 99 mol%; alternatively between about 94 mol% and about 99 mol%; alternatively between about 95 mol% and about 99 mol%; alternatively between about 95 mol% and about 99 mol%; alternatively about 96 mol%; alternatively about 96.5 mol%; alternatively about 97 mol%; alternatively about 97.5 mol%; alternatively about 98 mol%; or alternatively about 98.5 mol% based on the lipid of the liposomal particle.

[0093] In certain embodiments, the second lipid is present in an amount between about 0 mol% and about 50 mol%; alternatively between about 1 mol% and about 40 mol%; alternatively between about 2 mol% and about 30 mol%; alternatively between about 3 mol% and about 20 mol%; alternatively between about 4 mol% and about 15 mol%; alternatively about 0.6 mol%, alternatively about 0.7 mol%, alternatively about 0.8 mol%, alternatively about 0.9 mol%, alternatively about 1.0 mol%, alternatively about 1.5 mol%, alternatively about 2 mol%, alternatively about 2.5 mol%, alternatively about 3 mol%, alternatively about 3.5 mol%, alternatively about 4 mol%, alternatively about 4.5 mol%, or alternatively about 5 mol%, based on the lipid of the liposomal particle.

[0094] In some embodiments, the first lipid may comprise an anionic lipid or a cationic lipid. In some embodiments the second lipid may comprise a lysolipid or PEG-lipid conjugate. However, any combination of first and second lipids may be used in the liposomal particle.

[0095] Lysolipids are lipid derivatives in which one or both acyl derivatives have been removed by hydrolysis. Non-limiting examples include l-palmitoyl-2-hydroxy-sn-glycero-3- phosphocholine (MPPC), lysophosphatidylcholine (Lyso-PC), l-octadecanoyl-sn-glycero-3- phospho-(l'racglycerol) (Lyso-PG), lysophospholipids (Lyso-PI), and Lyso GA2.

[0096] In certain aspects, the lysolipid is present in an amount of about 15 mol% or less, alternatively about 14 mol% or less, alternatively about 13 mol% or less, alternatively about 12 mol% or less, alternatively about 10 mol% or less, alternatively about 8 mol% or less,alternatively about 5 mol% or less, alternatively about 3 mol% or less, alternatively about 2 mol% or less, or alternatively about 1 mol% or less, based the lipid of the liposomal particle.

[0097] In certain embodiments, the liposomal particle comprises at least a first lipid, a second lipid, and a third lipid. In some embodiments, the third lipid is present in an amount between about 0.1 mol% and about 5 mol%, between about 0.5 mol% and about 5 mol%, between about 1 mol% and about 5 mol%, between about 0.1 mol% and about 4 mol%, between about 0.5 mol% and about 4 mol%, between about 1 mol% and about 4 mol%, between about 0.1 mol% and about 3 mol%, between about 1 mol% and about 3 mol%, alternatively about 0.6 mol%, alternatively about 0.7 mol%, alternatively about 0.8 mol%, alternatively about 0.9 mol%, alternatively about 1.0 mol%, alternatively about 1.5 mol%, alternatively about 2 mol%, alternatively about 2.5 mol%, alternatively about 3 mol%, alternatively about 3.5 mol%, alternatively about 4 mol%, alternatively about 4.5 mol%, or alternatively about 5 mol%, based on the lipid of the liposomal particle.

[0098] PEG-lipid conjugates are PEG derivatives containing lipid molecules. Non-limiting examples include l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG- DMG), l,2-dipalmitoyl-rac-glycero-3-methylpolyoxyethylene (PEG-DPG), 1, 2-distearoyl-sn- glycero-3-phosphoethanolamine-poly(ethylene glycol) (PEG-DSG), diacylglycerolpolyethyleneglycols (PEG-DAG), polyethyleneglycol dialkyloxypropyl (PEG-DAA), polyethyleneglycol-phospholipids (PEG-phospholipid), and PEG-ceramide.

[0099] In some embodiments, the first lipid may comprise an anionic lipid or a cationic lipid. In some embodiments, the second lipid may comprise a lysolipid. In some embodiments, the third lipid may comprise a PEG-lipid conjugate. However, any combination of first, second, and third lipids may be used in the liposomal particle.

[0100] In certain embodiments, the at least one lipid encapsulates at least one PCR reaction reagent. The term “encapsulation” or “encapsulates” as used herein, refers to technique of entrapping one substance within another material such as a liposome. Encapsulation can inhibit the activity of the substance within liposome. In some aspects, the lipid partially encapsulates the at least one PCR reaction reagent. In other aspects, the lipid fully encapsulates the at least one PCR reaction reagent.

[0101] A PCR reaction reagent refers to a substance used during a PCR that can be within a liposomal encapsulation. In certain aspects, the PCR reaction reagent may be an enzyme. The enzyme encapsulated by the lipid includes enzymes used for a polymerase chain reaction. Nonlimiting examples include nucleic acid polymerase, a reverse transcriptase, an RNase H, HiDi, strand displacement enzymes, DNA polymerase I enzyme, and an isolate from Thermus aquaticus, known as Taq DNA. In some aspects, the RNase H is RNase Hl. In other aspects, the RNase H is RNase H2.

[0102] In certain aspects, the PCR reaction reagent may be deoxyribonucleotide triphosphates, divalent metal cations, a forward primer, a reverse primer, a probe, an aptamer, a reducing agent, a RNase inhibitor, or a detergent, or a combination thereof (including a combination with enzymes).

[0103] In some embodiments, the encapsulation of the at least one PCR reaction reagent by the lipid inhibits the activity of the PCR reaction reagent. In these embodiments, when the liposomal particle is thermally disrupted, the at least one PCR reaction reagent is released and / or the at least one PCR reaction reagent required for PCR is activated when the liposomal particle is subjected to a thermal temperature.

[0104] The thermal temperature, also referred to as the critical temperature or separation temperature, may be between about 40°C or about 100°C, between about 40°C or about 80°C, between about 40°C or about 70°C, between about 40°C or about 60°C, between about 45°C or about 75 °C, between about 45 °C or about 70°C, between about 45 °C or about 60°C, between about 50°C or about 70°C, between about 50°C or about 60°C, alternatively greater than 50°C, alternatively greater than 55°C, alternatively greater than 60°C, alternatively greater than 65°C, alternatively greater than 70°C, alternatively greater than 75°C, alternatively greater than 85°C, alternatively greater than 90°C, or alternatively greater than 95°C. Depending on the PCR reaction reagent(s) encapsulated, the thermal temperature may be lower than about 40°C or greater than about 100°C.

[0105] In some embodiments, the liposomal particle may be configured as a composition. The composition may further include buffers. The buffer components may play a factor in the performance of the reaction. In non-limiting aspects, the buffers may be water-based or aqueousbuffer solutions. The liposomal particle may be combined with buffers and other components via simple mixing of the components by pipetting, or microfluidic mixing processes such as those involving T-mixers, vortex mixers, or other chaotic mixing structures.

[0106] It is to be understood that the particular process conditions for preparing liposomal particle compositions described herein may be adjusted or selected accordingly to provide the desired physical properties. For example, parameters for mixing the components that may influence the final compositions may include, but are not limited to, order of mixing, temperature of mixing, mixing speed / rate, flow rate, physical dimensions of the mixing structure, concentrations of starting solutions, molar ratio of components, and solvents used.

[0107] In some aspects, the composition may be dried or lyophilized. In other aspects, the composition may be a liquid or aqueous composition. In some embodiments, the composition has a pH of between about 3 to about 7, alternatively about 3, alternatively about 4, alternatively about 5, alternatively about 6, or alternatively about 7.

[0108] In some aspects, the PCR reaction reagent is an enzyme and the composition includes the liposomal particle, deoxyribonucleotide triphosphates, a divalent metal cation, an aqueous buffer, a forward primer, a reverse primer, and / or a reverse transcriptase.

[0109] The composition could be stored in a compact apparatus (e.g., in the form of a small cartridge) which facilitates automated molding and assembly. This system can advantageously include dilution and mixing capability, intermediate wash capability, and positive pressurization capability. The fluid paths inside the system could be normally closed to minimize contamination and facilitate containment, control of fluids within the system, and allow the control of temperature. The reaction vessel could be conveniently detachable from the system.

[0110] Sample extraction, amplification, and detection may all be carried out within a self- contained cartridge. The GENEXPERT® utilizes a self-contained, single use cartridge. See e.g., U.S. Pat. Nos. 5,958,349, 6,403,037, 6,440,725, 6,783,736, 6,818,185; each of which is herein incorporated by reference in its entirety.

[0111] Components of the cartridge include, but are not limited to, processing chambers containing reagents, filters, and capture technologies useful to extract, purify, and amplify target nucleic acids. A rotatable valve enables fluid transfer from chamber to chamber and containsnucleic acids lysis and filtration components. An optical window enables real-time optical detection. A reaction tube extending from the body of the cartridge enables very rapid thermal cycling.

[0112] Each module may include optical and thermal components for amplification and detection, along with mechanical components for sample preparation and controlling fluidic movements in the cartridge.IV. Methods of Use

[0113] In some embodiments, the liposomal particle or a composition containing the liposomal particle is used in a method for performing an amplification reaction. In certain aspects, the amplification reaction is a real-time PCR reaction. In some aspects, the method results in reduced misamplification during reverse transcriptase steps in the real-time PCR reaction, reduced enzyme activation at lower temperatures, and / or greater sensitivity.

[0114] PCR is an enzymatic reaction in which DNA fragments are synthesized and amplified from a substrate DNA in vitro. The chain reaction generally consists of a series of 10 to 40 cycles. Standard PCR can be conducted in a machine called a thermal cycler at specific temperatures. In each cycle, the reaction is heated separate, or denature, the DNA strands. After cooling the reaction, annealing between the primers and their complementary sequences on the single- stranded template to form double- stranded structures along complementary nucleotide sequences. The extension of the new strands of DNA by Taq polymerase from the primers occurs after the reaction temperatures is raised. PCR reactions require at least one PCR reaction reagent; alternatively at least two PCR reaction reagents; alternatively at least three PCR reaction reagents; alternatively at least four PCR reaction reagents; or alternatively at least five PCR reaction reagents.

[0115] Amplification, according to the present teachings, encompasses any means by which at least a pail of at least one target nucleic acid is reproduced, typically in a template-dependent manner, including without limitation, a broad range of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Illustrative means for performing an amplifying step include PCR, nucleic acid strand-based amplification (NASBA), two-step multiplexed amplifications, rolling circle amplification (RCA), and the like, including multiplex versionsand combinations thereof, for example but not limited to, OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as combined chain reaction — CCR), helicase-dependent amplification (HD A), and the like.

[0116] In each cycle, the substrate DNA is first denatured at high temperature. After cooling down, synthetic primers which arc present in vast excess hybridize to the substrate DNA to form double-stranded structures along complementary nucleotide sequences. The primer- substrate DNA complexes will then serve as initiation sites for a DNA synthesis reaction catalyzed by a DNA polymerase, resulting in the synthesis of a new DNA strand complementary to the substrate DNA strand.

[0117] In some aspects, the PCR method is hot-start PCR. Assays for the detection of RNA viruses arc often highly complex (high quantity of different oligonucleotides) due to low sequence conservation of the RNA genome. In general, mispriming occurs at temperatures below the specific annealing temperature of the oligonucleotides. One of the biggest challenges in real-time PCR is the formation of non-specific products due to elongation of misprimed oligonucleotides by the enzyme at temperatures beneath the specific annealing temperature. The formation of non-specific products can be reduced by using hot start variants of the enzymes, which are inactive at low temperatures and activated at higher temperatures, appropriate for specific primer annealing to the target nucleic acid. Hot-start PCR methods use enzyme modifiers like antibodies, affibodies, aptamers, or chemical modifications to inhibit PCR reaction reagent activity (such as enzymes) at room temperature. In some embodiments, the disclosed liposomal particles are used to inhibit the activity of one or more PCR reaction reagents. For example, the liposomal particles can be used for the reversible inactivation of reverse transcriptase. This inhibition prevents nonspecific amplification caused by primers binding to template sequences with low homology (mispriming) and primers binding to each other (primer-dimers) during reaction setup. In other examples, by blocking DNA polymerase activity at room temperature, the hot-start approach allows for the convenient setup of multiple reactions at ambient temperature (as in high-throughput experiments) without significantly compromising specificity and amplification.

[0118] In an aspect, the liposomal particle or a composition containing the liposomal particle is added to a PCR reaction mixture. At room or ambient temperature (e.g., about 20°C or about25°C) the liposomal particle (in particular the lipid encapsulation) blocks the activity of the PCR reaction reagent. During the initial heating step, the PCR reaction reagent is released from the lipid encapsulation, resulting in a functional PCR reaction reagent.

[0119] In this method, the PCR reaction reagent is released from the lipid encapsulation when the thermal temperature of the reaction mixture reaches between about 40°C or about 100°C, between about 40°C or about 80°C, between about 40°C or about 70°C, between about 40°C or about 60°C, between about 45°C or about 75°C, between about 45°C or about 70°C, between about 45°C or about 60°C, between about 50°C or about 70°C, between about 50°C or about 60°C, alternatively greater than 50°C, alternatively greater than 55 °C, alternatively greater than 60°C, alternatively greater than 65°C, alternatively greater than 70°C, alternatively greater than 75°C, alternatively greater than 85°C, alternatively greater than 90°C, or alternatively greater than 95°C.

[0120] In an aspect, the method may also include a module having a receiving bay for receiving a cartridge device comprising the composition containing the liposomal particle. In certain embodiments, the module includes one or more mechanisms within the receiving bay for manipulating a fluid sample within the cartridge device; and an instrument that interfaces with a reaction vessel with a memory having programmable instructions recorded thereon, that are configured to operate the module. In one example, components of the cartridge include, but arc not limited to, processing chambers containing reagents, filters, and capture technologies useful to extract, purify, and amplify target nucleic acids. A valve enables fluid transfer from chamber to chamber and contains nucleic acids lysis and filtration components. An optical window enables real-time optical detection. A reaction tube enables very rapid thermal cycling.

[0121] In some embodiments, the GENEXPERT® system includes a plurality of modules for scalability. Each module includes a plurality of cartridges, along with sample handling and analysis components.

[0122] After the sample is added to the cartridge, the sample is contacted with lysis buffer and released nucleic acid (NA) is bound to an NA-binding substrate such as a silica or glass substrate. The sample supernatant is then removed, and the NA eluted in an elution buffer such as a Tris / EDTA buffer. The eluate may then be processed in the cartridge to detect target genesas described herein. In some embodiments, the eluate is used to reconstitute at least some of the PCR reagents, which can be present in the cartridge as lyophilized particles.

[0123] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specific embodiments of the present technology. By providing these specific examples, it is not intended to limit the scope and spirit of the present technology. It will be understood by those skilled in the art that the full scope of the presently described technology encompasses the subject matter defined by the claims appending this specification, and any alterations, modifications, or equivalents of those claims.EXAMPLES

[0124] Example 1: Generation of exemplary liposomal particles.

[0125] As shown in FIG. 1A and FIG. IB, liposomal particles were formulated under various conditions to encapsulate payload (CMRT (CM Reverse Transcriptase A- Enzyme)) and was disrupted when heated.

[0126] Table 1 details the formulation of the liposomal particles at pH 4 at varied overall lipid concentrations. The CMRT concentration for each formulation was kept constant at 100 pg / mL. Table 2 details the post-centrifugation sizing data of liposomal particles of Table 1.

[0127] DOPG, also known as l,2-diolcoyl-sn-glyccro-3-phosphoglyccrol, is a phospholipid containing oleic acid (18:1) inserted at the sn-1 and sn-2 positions, was used as the main thermosensitive lipid and provided the liposomal particle with the required structural support. MPPC, also known as PC(14:0 / 16:0) or l-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine, is an asymmetrical phosphatidylcholine containing a myristic acid (14:0) at the sn-1 position and a palmitic acid (16:0) at the sn-2 position, was used to provide thermolabile properties to the liposomal particle. DMG-PEG, a synthetic lipid formed by the PEGylation of myristoyl diglyceride, was used to prevent aggregation in the formulation.

[0128] Table 1: Formulation of exemplary liposomal particles at pH 4 using various lipid concentrations.

[0129] Table 2: Sizing data of exemplary liposomal particles post centrifugation.

[0130] Example 2: Activity investigation of exemplary liposomal particles.

[0131] Exemplary liposomal particles, referred to as LNP, were formulated with CMRT. These LNPs showed no signal in activity substrates suggesting that lipids that form liposome could be PCR inhibitors. Spike-ins of various concentrations of LNP to different reactions were tested to measure potential inhibition of the enzyme. LNP stocks were quantitated by BCA assay and used as ug / mL relative to CMRT.

[0132] As shown in FIG. 2, a blank control, containing kinetic substrate with no enzymes, was used to establish a baseline for 0% activity. As expected, the CR 12 sample containing onlyLNP, matched this 0% activity baseline. A CMRT control sample was also analyzed. The resulting trace correlates to 100% activity (i.e., no inhibition). The remaining concentrations of CMRT and LNP are listed in Table 3.

[0133] Table 3. Exemplary LNPs

[0134] The encapsulation of CMRT by the LNP resulted in decreased enzymatic activity as compared to the CMRT control, indicating the LNP is suitable for use as a delayed release or liposomal particle.

[0135] To further evaluate the efficiency of the exemplary LNPs, hgDNA was amplified using PCR. In this example, a PCR reaction mixture was prepared containing all the components needed for the PCR reaction. After mixing, aliquots of the PCR reaction mixture were placed into six different PCR tubes. The 0.3U / pL of CATA was added to each sample tube. One PCR tube served as a blank, with no CMRT or LNP added and one served as a control with only CMRT added. The remaining concentrations of LNP are listed in Table 4.

[0136] Table 4. Exemplary LNP formulations for hgDNA PCR

[0137] The PCR tubes were placed into a GENEXPERT® and samples underwent 45 cycles at 95 °C for 8 sec and 60 °C for 30 sec.

[0138] As shown in FIG 3, the LNP spike-ins show small dose dependent delay in cycle threshold (Ct). The CMRT control samples showed a larger delay in Ct, indicating the LNP is suitable for use as a delayed release or liposomal particle.

[0139] Example 3: Generation of exemplary liposomal particles at neutral pHs.

[0140] Formulations of exemplary liposomal particles at pH 5, pH 6, and pH 7 using various lipid concentrations were generated and evaluated (Table 5).

[0141] Table 5. Formulation of exemplary liposomal particles at pH 5, 6, and 7 using various lipid concentrations.

[0142] These liposomal particles, made with milder conditions at different charge ratios and neutral pH, were evaluated at equal ug / mL input. In this example, a PCR reaction mixture wasprepared containing all the components needed for a PCR reaction, including 0.1 % detergent (Brij). After mixing, aliquots of the PCR reaction mixture were placed into different PCR tubes. A blank (as described above), a control sample (as described above) and the exemplary liposomal particles disclosed in Table 5 were analyzed using standard hot start PCR protocol and RT substrate.[0143J FIG. 4 and FIG. 5 detailed the signal of the liposomal particles at 27°C and 55°C, respectively. The signal generated by these liposomal particles at a more neutral pH showed improvement in liposomal activity. Particularly, liposome activity was delayed by about 60s at 27°C, and about 30s at 55°C. Liposomal particles formulations at lower charge ratios and pH 6- 7 had faster kinetic rates once activity begins.

[0144] Example 4: Detergent effects.

[0145] Several of the exemplary liposomal particles from Table 5 including a blank and control sample were used to evaluate the effect of the use of detergent in the PCR reaction. Enzymes can become more difficult to solubilize as temperature increases. As such, there is a need to find a balance of detergent % to have minimal impact on PCR efficiency while keeping solubility of the enzyme.

[0146] The PCR methods to evaluate the detergent effects were similar to those of Example 3, but 0.01% Brij was used in the PCR reaction mixture. As shown in FIG. 6 and FIG. 7, which are graphs that detail the signal of thermosensitive liposomes with the use of detergent at 27°C and 55°C, respectively, reduction in Brij (detergent) from 0.1% (FIGs. 4 and 5) to 0.01% (FIGs. 6 and 7) delays the start of reaction further but moderately reduces PCR efficiency. As shown in FIG. 7, for example, the exemplary liposomal particles had a reduction in activity as compared to the control sample. Additionally, Brij was shown to be needed for solubility especially at high temperatures.

[0147] Example 5: Generation of exemplary liposomal particles at pH 6.

[0148] Formulations of exemplary liposomal particles at pH 6 using various lipid concentrations were generated and evaluated (Table 6). These exemplary liposomal particles has increased DOPG and reduced MPPC for greater thermal stability. In the examples shown, MPPC, whichis a thermolabile component, was reduced from 10% to 0%, while DOPG was increased from 88.5% to 98.5%.

[0149] Table 6. Formulation of exemplary liposomal particles at pH 6 using various lipid concentrations.

[0150] FIG. 8 and FIG. 9 are graphs showing the resulting signals of these exemplary liposomal particles at 27°C and 40°C, respectively. Testing the PCR at 40°C allows for curves to reach endpoint of cycles (that is, RFU consistent with the control, unencapsulated CMRT, at end of PCR cycles). Some of the exemplary liposomal particles were shown to have a delayed release of enzyme at 40°C.

[0151] Example 6: Evaluation of exemplary liposomal particles formulations using RNA aptamers and hot staid PCR

[0152] In this example, an RNA aptamer, referred to as C&G aptamer, was used in a hot start protocol. The C&G aptamer was described in a paper by Chen and Gold in 1994 (Biochemistry 1994, 33, 29, 8746-8756). The PCR methods to evaluate the aptamer effects were similar to those of Example 3, but the C&G aptamer was used in the PCR reaction mixture. Ablank and control sample were evaluated, along with C&G formulated CMRT and two exemplary liposomal particles formulations (MPPC 10% and MPPC 0%).

[0153] As shown in FIG. 10, FIG. 11, and FIG. 12, which are graphs showing the resulting signals of these exemplary liposomal particles at 27°C, 40°C, and 55°C, respectively, the aptamer appears to be less effective at inhibiting enzyme activity at 27 °C, but more readily releases to full enzyme activity at 40°C and 55 °C.

[0154] Example 7 -Real Time- PCR using exemplary liposomal particles formulations.

[0155] A real-time PCR method was carried out to detect RNA-dependent RNA polymerase (RdRp). A singleplex PCR reaction mixture stock solution containing a forward primer, a reverse primer, a probe, CoV RNA, dNTP, CAT A, RNase inhibitor, buffers, detergents, and salts was prepared. The stock solution was divided into three batches and different exemplary liposomal particle formulations were added at a concentration of 0.909 pg / mL to each batch as shown in Table 7. Each batch was diluted prior to performing real-time amplification.

[0156] Table 7. Exemplary liposomal particles formulations for real-time PCR

[0157] Real-time amplification was performed with a GENEXPERT®. The real-time PCR conditions were as follows: reverse transcription was conducted at 40°C for 2-15 mins, denatured for 95°C for 8s, and annealed at 72°C. FIG. 13 detailed the cycle thresholds (Cts) of the different enzyme conditions used within real-time PCR protocol. As shown, the lipid particles required up to 15 mins of reverse transcription for more complete disruption. Forexample, as can be seen from FIG. 13, as the reverse transcription time increases, the exemplary liposomal particles present lower and more consistent Cts.

[0158] FIGs. 14A - 14D depict the amplification plot of exemplary liposomal particles formulations, unencapsulated CMRT, or both used in TSR bead (5-plex) comprising CoV RNA (FIGs. 14A and 14B) or a no template control, NTC (FIGs. 14C and 14D) real-time PCR protocol at 2 mins RT and 10 mins RT.

[0159] FIGs. 15A - 15D depict the post PCR melt of thermosensitive liposomes, unencapsulated CMRT, or both used in TSR bead (5-plcx) comprising CoV RNA (FIGs. 15A and 15B) or a no template control, NTC (FIGs. 15C and 15D) real-time PCR protocol at 2 mins RT and 10 mins RT.

[0160] All features disclosed in the specification, including the claims, abstracts, and drawings, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0161] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMS1. A liposomal particle comprising at least one lipid and at least one PCR reaction reagent, wherein the at least one lipid encapsulates the at least one PCR reaction reagent.

2. The liposomal particle of claim 1, wherein the at least one lipid comprises one or more of an anionic lipid, a cationic lipid, a lysolipid, a neutral lipid, a neutral phospholipid, and a PEG- lipid conjugate.

3. The liposomal particle of claim 2, wherein the at least one lipid comprises one or more Cn to C40 tails.

4. The liposomal particle of claim 2, comprising the anionic lipid, wherein the anionic lipid is selected from DOPG, DMPG, DPPG, DSPG, DLPG, or DAPG.

5. The liposomal particle of claim 2, comprising the lysolipid, wherein the lysolipid is selected from the group consisting of MPPC, Lyso-PC, Lyso-PE, Lyso-PG, Lyso-PI, and Lyso GA2.

6. The liposomal particle of claim 2, comprising the PEG-lipid conjugate, wherein the PEG- lipid conjugate is selected from PEG-DMG, PEG-DPG, PEG-DSG, PEG-DAG, PEG-DAA, PEG- phospholipid, and PEG-ceramide.

7. The liposomal particle of any one of the preceding claims, wherein the at least one lipid is present in an amount between about 0 mol% and about 100 mol%; alternatively between about 1 mol% and about 100 mol%; alternatively between about 2 mol% and about 100 mol%; alternatively between about 5 mol% and about 100 mol%; alternatively between about 10 mol% and about 100 mol%; alternatively between about 50 mol% and about 100 mol%; alternatively between about 60 mol% and about 100 mol%; alternatively between about 70 mol% and about 100 mol%; alternatively between about 80 mol% and about 100 mol%; alternatively between about 90 mol% and about 100 mol%; alternatively about 75 mol%; alternatively about 80 mol%; alternatively about 85 mol%; alternatively about 90 mol%; alternatively about 95% mol; alternatively about 96 mol%; alternatively about 97 mol%; alternatively about 98 mol%; alternatively about 99 mol%; or alternatively about 100 mol% based on the at least one lipid of the liposomal particle.

8. The liposomal particle of claim 7, wherein the liposomal particle comprises at least a first lipid and a second lipid.

9. The liposomal particle of claim 8, wherein the first lipid is present in an amount between about 50 mol% and about 100 mol%; alternatively between about 60 mol% and about 99 mol%; alternatively between about 70 mol% and about 99 mol%; alternatively between about 80 mol% and about 99 mol%; alternatively between about 85 mol% and about 99 mol%; alternatively between about 86 mol% and about 99 mol%; alternatively between about 87 mol% and about 99 mol%; alternatively between about 88 mol% and about 99 mol%; alternatively between about 89 mol% and about 99 mol%; alternatively between about 90 mol% and about 99 mol%; alternatively between about 91 mol% and about 99 mol%; alternatively between about 92 mol% and about 99 mol%; alternatively between about 93 mol% and about 99 mol%; alternatively between about 94 mol% and about 99 mol%; alternatively between about 95 mol% and about 99 mol%; alternatively between about 95 mol% and about 99 mol%; alternatively about 96 mol%; alternatively about 96.5 mol%; alternatively about 97 mol%; alternatively about 97.5 mol%; alternatively about 98 mol%; or alternatively about 98.5 mol% based on the first lipid of the liposomal particle; and the second lipid is present in an amount between about 0 mol% and about 50 mol%; alternatively between about 1 mol% and about 40 mol%; alternatively between about 2 mol% and about 30 mol%; alternatively between about 3 mol% and about 20 mol%; alternatively between about 4 mol% and about 15 mol%; alternatively about 0.6 mol%, alternatively about 0.7 mol%, alternatively about 0.8 mol%, alternatively about 0.9 mol%, alternatively about 1.0 mol%, alternatively about 1.5 mol%, alternatively about 2 mol%, alternatively about 2.5 mol%, alternatively about 3 mol%, alternatively about 3.5 mol%, alternatively about 4 mol%, alternatively about 4.5 mol%, or alternatively about 5 mol%, based on the second lipid of the liposomal particle.

10. The liposomal particle of claim 8 or claim 9, wherein the first lipid comprises the anionic lipid or the cationic lipid and the second lipid comprises the lysolipid or the PEG-lipid conjugate.

11. The liposomal particle of any one of claims 8 to 10, wherein the liposomal particle further comprises a third lipid.

12. The liposomal particle of claim 1 1 , wherein the third lipid is present in an amount between about 0.1 mol% and about 5 mol%; alternatively between about 0.5 mol% and about 5 mol%; alternatively between about 1 mol% and about 5 mol%; alternatively between about 0.1 mol% and about 4 mol%; alternatively between about 0.5 mol% and about 4 mol%; alternatively between about 1 mol% and about 4 mol%; alternatively between about 0.1 mol% and about 3 mol%; alternatively between about 1 mol% and about 3 mol%; alternatively about 0.6 mol%; alternatively about 0.7 mol%; alternatively about 0.8 mol%; alternatively about 0.9 mol%; alternatively about 1.0 mol%; alternatively about 1.5 mol%; alternatively about 2 mol%; alternatively about 2.5 mol%; alternatively about 3 mol%; alternatively about 3.5 mol%; alternatively about 4 mol%; alternatively about 4.5 mol%; or alternatively about 5 mol% based on the third lipid of the liposomal particle.

13. The liposomal particle of claim 11 or claim 12, wherein the first lipid comprises the anionic lipid or the cationic lipid, the second lipid comprises the lysolipid, and the third lipid comprises the PEG-lipid conjugate.

14. The liposomal particle of any one of claims 1 to 13, wherein the at least one PCR reaction reagent comprises an enzyme, deoxyribonucleotide triphosphates, divalent metal cations, a forward primer, a reverse primer, a probe, an aptamer, a reducing agent, a RNase inhibitor, or a detergent, or a combination thereof.

15. The liposomal particle of claim 14, wherein the at least one PCR reaction reagent comprises the enzyme, and the liposomal particle has a lipid to enzyme charge ratio between about 2 and about 100.

16. The liposomal particle of claim 14 or claim 15, wherein the at least one PCR reaction reagent comprises the enzyme selected from the group consisting of a nucleic acid polymerase, a reverse transcriptase enzyme, an RNase H, HiDi, strand displacement enzymes, and a combination thereof.

17. The liposomal particle of claim 16, wherein the RNase H is selected from the group consisting of RNase Hl and RNase H2.

18. The liposomal particle of any one of claims 14 to 17, wherein the at least one PCR reaction reagent comprises the forward primer, the reverse primer, and / or the probe, wherein the forward primer, the reverse primer, and / or the probe are specific for a target nucleic acid.

19. The liposomal particle of claim 18, wherein the forward primer, the reverse primer, and / or the probe are specific for the same target nucleic acid.

20. The liposomal particle of any one of claims 1 to 19, wherein the encapsulation inhibits activity of the at least one PCR reaction reagent.

21. The liposomal particle of any one of claims 1 to 20, wherein when the liposomal particle is thermally disrupted, the at least one PCR reaction reagent is released and / or the at least one PCR reaction reagent is activated.

22. The liposomal particle of claim 21, wherein the liposomal particle is thermally disrupted when the liposomal particle is subjected to a temperature of between about 40 °C or about 100°C, between about 40°C or about 80°C, between about 40°C or about 70°C, between about 40°C or about 60°C, between about 45°C or about 75 °C, between about 45 °C or about 70°C, between about 45°C or about 60°C, between about 50°C or about 70°C, between about 50°C or about 60°C, alternatively greater than 50°C, alternatively greater than 55°C, alternatively greater than 60°C, alternatively greater than 65°C, alternatively greater than 70°C, alternatively greater than 75 °C, alternatively greater than 85°C, alternatively greater than 90°C, or alternatively greater than 95°C.

23. The liposomal particle of any one of claims 1 to 22, wherein the liposomal particle is about 50 nm to about 150 nm in size.

24. The liposomal particle of any one of claims 1 to 23, wherein the liposomal particle has a PDI less than 0.5, or alternatively between about 0.15 and about 0.50.

25. A composition comprising a liposomal particle according to any one of claims 1 to 24.

26. The composition of claim 25, wherein the composition is a dried composition.

27. The composition of claim 25, wherein the composition is a liquid or aqueous composition.

28. The composition of any one of claims 25 to 27, wherein the composition has a pH of between about 3 to about 7, alternatively about 3, alternatively about 4, alternatively about 5, alternatively about 6, or alternatively about 7.

29. The composition of any one of claims 25 to 28, wherein the PCR reaction reagent comprises an enzyme selected from the group consisting of a nucleic acid polymerase, a reverse transcriptase enzyme, an RNase H, HiDi, strand displacement enzymes, and a combination thereof.

30. The composition of any one of claims 25 to 29, wherein the composition further comprises a nucleic acid polymerase, deoxyribonucleotide triphosphates, a divalent metal cation, an aqueous buffer, a forward primer, a reverse primer, and / or a reverse transcriptase.

31. The composition of any one of claims 25 to 30, wherein the composition is contained within a cartridge device.

32. A method for performing an amplification reaction, wherein the method comprises using the liposomal particle of any one of claims 1 to 24 or the composition of any one of claims 25 to 31.

33. The method of claim 32, wherein the amplification reaction is a real-time PCR reaction.

34. The method of claim 32 or claim 33, wherein the PCR reaction reagent is released from the at least one lipid when the amplification reaction reaches a temperature between about 40°C or about 100°C; alternatively between about 40°C or about 80°C; alternatively between about 40°C or about 70°C; alternatively between about 40°C or about 60°C; alternatively between about 45 °C or about 75°C; alternatively between about 45°C or about 70°C; alternatively between about 45°C or about 60°C; alternatively between about 50°C or about 70°C; alternatively between about 50°C or about 60°C, alternatively greater than 50°C, alternatively greater than 55°C, alternatively greater than 60°C, alternatively greater than 65°C, alternatively greater than 70°C, alternatively greater than 75°C, alternatively greater than 85°C, alternatively greater than 90°C, or alternatively greater than 95 °C.

35. The method of any one of claims 32 to 34, wherein the method further comprises:receiving, in a module having a receiving bay, a cartridge device comprising the composition of any one of claims 25 to 31, wherein the module includes one or more mechanisms within the receiving bay for manipulating a fluid sample within the cartridge device; and interfacing with a reaction vessel with a memory having programmable instructions for operating the module recorded thereon.

36. The method of any one of claims 32 to 35, wherein the method results in at least one of the following characteristics: reduced misamplification during reverse transcriptase steps in the realtime PCR reaction, reduced enzyme activation at lower temperatures, or greater sensitivity.

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