Patent Application
20250073342
The Sequence Listing submitted Aug. 26, 2024, as a text file named “11073-003W01_ST26.txt” created on Aug. 26, 2024, and having a size of 4,560 bytes in size is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).
The disclosure relates to hyperbranched polymers, which can be used as transfection carrier of small interfering RNA (siRNA) for RNA interference (RNAi) mediated gene silencing therapy. The disclosure not only provides efficient siRNA delivery and excellent gene silencing, but also low cytotoxicity and biodegradable.
The listing or discussion of a document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.
RNA interference (RNAi) mediated gene silencing is a biological cellular pathway that targets the messenger RNAs to knockdown the RNA expression in a sequence-specific manner (Agrawal et al., Microbiol. Mol. Biol. Rev. 2003, 67, 657-85; Svoboda, Front Plant Sci. 2020, 11). This pathway can be externally induced by intracellular delivery of 21- to 23-nucleotide double stranded RNA, also known as small interfering RNA (siRNA), which can specifically recognize its homologous mRNAs sequence and direct the degradation of the mRNAs (Chi et al., Proc. Natl. Acad. Sci. 2003, 100, 6343-6346; Fire et al., Nature 1998, 391, 806-811). Targeted gene silencing via siRNA delivery holds great potential for the treatment of various diseases such as viral infections, cancer, and neurodegenerative disorders (Karagiannis & El-Osta, Cancer Gene Ther. 2005, 12, 787-795). However, the therapeutic approach based on siRNA delivery cannot be achieved without a safe and efficient delivery vector, which remains a central challenge to its therapeutic application (Whitehead, Langer, & Anderson, Nat. Rev. Drug Discov. 2009, 8, 129-138).
Non-viral gene vectors such as cationic polymers have been widely used as delivery carriers for siRNA delivery because of their safety profile, low-cost, large-scale manufacturing potential, stability, and capacity for larger nucleic acid payload (Ediriwickrema & Saltzman, ACS Biomater. Sci. Eng. 2015, 1, 64-78; Yin et al., Nat. Rev. Genet. 2014, 15, 541-555). Cationic polymers, such as polyethylenimine (PEI) (Helmschrodt et al., Mol. Ther. Nucleic Acids 2017, 9, 57-68; F. Liu et al., Int. J. Mol. Sci. 2022, 23, 5014; Lu, Morris, & Labhasetwar, J. Pharmacol. Exp. Ther. 2019, 370, 902-910; Malek, Czubayko, & Aigner, J. Drug Target. 2008, 16, 124-139), poly-L-lysine (PLL) (Alazzo et al., J. Mater. Chem. 2022, 10, 236-246; Inoue et al., J. Control. Release. 2008, 126, 59-66), and poly(2-dimethylaminoethyl methacrylate) (pDMAEMA) (Kong et al., J. Control. Release. 2009, 138 (2), 141-7; Li, Sharili, Connelly, & Gautrot, Biomacromolecules 2018, 19, 606-615) can spontaneously condense negatively charged RNA into complexes with sizes of around 50-200 nm and positive surface charge which in turn enhance the cellular uptake via charged-mediated endocytosis. Although showing good transfection efficiency, these early polymers are non-biodegradable which dramatically limits their application in the therapeutic field. Therefore, it is necessary to develop a biodegradable gene delivery system to ensure a good safety window.
Poly(β-amino esters) (pBAEs) is a class of biodegradable cationic polymers which have great potential and high transfection efficiency for DNA and siRNA delivery (Dosta et al., Cardiovasc. Eng. Technol. 2021, 12, 114-125; Dosta, Ramos, & Borrós, Mol. Syst. Des. Eng. 2018, 3, 677-689). A variety of linear pBAEs with different combinations of amine and diacrylate monomers has been developed for gene delivery applications with various cell types (Kamat et al., Mol. Cancer Ther. 2013, 12, 405-15; Mangraviti et al., ACS Nano 2015, 9, 1236-1249; Tzeng et al., Biomaterials 2011, 32, 5402-10; Tzeng, Higgins, Pomper, & Green, J. Biomed. Mater. Res.-A 2013, 101, 1837-45). However, linear structures contain only two terminal units and limited cationic charge center, hence limiting the chemical space for functionalization and development of gene delivery vector with different architectures. The transition from linear to highly branched pBAEs can significantly improve the transfection efficiency on a variety of cell lines, compared to their corresponding linear structure (Cutlar et al., Biomacromolecules 2015, 16, 2609-2617; Gao et al., J. Control. Release. 2020, 321, 654-668; S. Liu et al., Nat. Commun. 2019, 10, 3307; Zhou et al. Sci. Adv. 2016, 2, e1600102). Nevertheless, most of the hyperbranched pBAEs which have been shown to have excellent transfection efficiency were developed for DNA delivery using plasmid DNA. There is not much literature reporting on the development of hyperbranched pBAEs for efficient siRNA delivery and gene silencing.
The major difference between DNA and siRNA is that siRNA has smaller size and higher rigidity than most plasmid DNA used for DNA delivery (Hagerman, Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 139-156; Kebbekus, Draper, & Hagerman, Biochem. 1995, 34, 4354-4357). As a result, there is decreased multivalency of electrostatic interactions between cationic polymers and siRNA, thus weakening the siRNA complexation ability. Meanwhile, the rigidity may affect the self-assembly process of cationic polymers and siRNA into nanoparticles (Kozielski, Tzeng, De Mendoza, & Green, ACS Nano 2014, 8, 3232-41). Therefore, it would be more difficult to develop a gene carrier for efficient siRNA delivery and gene silencing.
The present invention provides hyperbranched poly(β-amino esters) (pBAEs) polymers useful for delivery of small interfering RNA (siRNA) to cells. The polymers of formula (I) are linear structures. The polymers of formula (II) are hyperbranched structures. The aim of synthesizing polymers of formula (I) is to optimize their monomer composition for good siRNA complexation capability. The branching properties and degree of branching density are then introduced to synthesize polymers of formula (II) in order to further optimize the performance of gene vectors such as cytotoxicity and gene silencing efficiency.
WO2016/020474A1 describes branched poly(β-amino esters) with polymer backbone containing monomer composition of bisphenol A ethoxylate diacrylate, 4-amino-1-butanol and trimethylolpropane triacrylate which are useful for delivery of plasmid DNA to a broad spectrum of several cell types. Patent US20190125874A1 describes branched poly(β-amino esters) with polymer backbone containing monomer composition of bisphenol A glycerolate diacrylate, 3-morpholinopropylamine and N-methyl-1,3-diaminopropane, which are useful for delivery of mRNA to lung epithelial cells. The polymers of the present invention differ from these prior art polymers are that they are synthesized from different buildings blocks and the nucleic acid agent they carry. The present invention is optimized to have optimal monomer composition and branching density, that can efficiently deliver siRNA with low cytotoxicity and high gene silencing efficiency for RNA interference (RNAi) mediated gene silencing therapy. The siRNA delivery performance of the present invention has been verified in both cells that are easy to transfect and cells that are difficult to transfect.
U.S. Pat. No. 8,562,966 B2 describes end modified poly((β-amino esters) useful for a variety of medical applications. The end modified poly((β-amino esters) are prepared by addition of nucleophilic reagent (example amine) to an acrylate terminated poly (β-amino esters). The resulting end modified polymers are useful in drug delivery, particularly in the delivery of polynucleotides.
US 2017/0216455 A1 describes biodegradable, hyperbranched poly (β-amino esters) for gene therapy such as nucleic acid transfection agents. The invention describes improved transfection efficiencies that are both safe and non-toxic. This invention utilizes tri-acrylate monomers which react directly via Michael addition with amine monomers to give hyperbranched poly (β-amino esters).
Before the present methods and systems are disclosed and described, it is to be understood that the methods and systems are not limited to specific synthetic methods, specific components, or to particular compositions. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes, from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
“Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. “Exemplary” means “an example of” and is not intended to convey an indication of a preferred or ideal embodiment. “Such as” is not used in a restrictive sense, but for explanatory purposes.
Disclosed are components that can be used to perform the disclosed methods and systems. These and other components are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these components are disclosed that while specific reference of each various individual and collective combinations and permutation of these may not be explicitly disclosed, each is specifically contemplated and described herein, for all methods and systems. This applies to all aspects of this application including, but not limited to, steps in disclosed methods. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.
Compounds disclosed herein may be provided in the form of acceptable salts, for example pharmaceutically acceptable salts. Examples of such salts are acid addition salts formed with inorganic acids, for example, hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acids and the like; salts formed with organic acids such as acetic, oxalic, tartaric, succinic, maleic, fumaric, gluconic, citric, malic, methanesulfonic, p-toluenesulfonic, napthalenesulfonic, and polygalacturonic acids, and the like; salts formed from elemental anions such as chloride, bromide, and iodide; salts formed from metal hydroxides, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide, and magnesium hydroxide; salts formed from metal carbonates, for example, sodium carbonate, potassium carbonate, calcium carbonate, and magnesium carbonate; salts formed from metal bicarbonates, for example, sodium bicarbonate and potassium bicarbonate; salts formed from metal sulfates, for example, sodium sulfate and potassium sulfate; and salts formed from metal nitrates, for example, sodium nitrate and potassium nitrate.
The term “alkyl” refers to a radical of a straight-chain or branched hydrocarbon group having a specified range of carbon atoms (e.g., a “C1-16 alkyl” can have from 1 to 16 carbon atoms). An alkyl group can be a saturated alkyl group or an unsaturated alkyl group, i.e., an alkyl group having one or more carbon-carbon double/triple bonds, i.e., an alkenyl or alkynyl group. Unless specified to the contrary, an “alkyl” group includes both saturated alkyl groups and unsaturated alkyl groups.
The term “aliphatic” as used herein refers to an organic system composed of one or more alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups in any permissible configuration. An aliphatic group may include any permissible number of substituents or degrees of unsaturation. An aliphatic group may be defined by the number of carbon atoms present, but unless specified to the contrary may include any number of heteroatoms as well. By way of example, a C2aliphatic group includes —CH2CH2—, —CH2OCH2—, —CH2C(═O)—, oxiranyl, and —CF2CF2—.
The term “heteroalkyl” refers to an alkyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. By way of example, a heteroC1-6alkyl (which may also be designated a C1-6heteroalkyl) group includes, but is not limited to, the following structures:
The term “heteroalkyl” preceded by a separate heteroatom refers to a heteroalkyl group bonded through the specified heteroatom. By way of example, a OC1-6heteroalkyl group includes, but it not limited to, the following structures:
When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example, “C1-6 alkyl” is intended to encompass C1, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6 alkyl.
Affixing the suffix “-ene” to a group indicates the group is a polyvalent moiety, e.g., boned to two or more groups. Alkylene is the polyvalent moiety of alkyl, alkenylene is the divalent moiety of alkenyl, alkynylene is the divalent moiety of alkynyl, heteroalkylene is the divalent moiety of heteroalkyl, heteroalkenylene is the divalent moiety of heteroalkenyl, heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclylene is the divalent moiety of carbocyclyl, heterocyclylene is the divalent moiety of heterocyclyl, arylene is the divalent moiety of aryl, and heteroarylene is the divalent moiety of heteroaryl (each of which parent groups as defined herein).
The term “alkoxy” refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.
The term “aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 T electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-14 aryl”). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents.
“Aralkyl” is a subset of “alkyl” and refers to an alkyl group substituted by an aryl group, wherein the point of attachment is on the alkyl moiety.
The term “heteroaryl” refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-14 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl).
Exemplary heteroaryl and heterocyclyl rings include: benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyL cirrnolinyl, decahydroquinolinyl, 2H,6H′ 1,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, IH-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl.
Unless specified to the contrary, the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups defined herein (and the “ene” versions of said groups) may be substituted or unsubstituted. A substituted group includes a non-hydrogen substituent at a position where in the unsubstituted version a hydrogen atom would be found. Substituents include, but are not limited to, halogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, —NRaRb, —NRaC(═O)Rb, —NRaC(═O)NRaNRb, —NRaC(═O)ORb, —NRaSO2Rb, —C(═O)Ra, —C(═O)ORa, —C(═O)NRaRb, —OC(═O)NRaRb, —ORa, —SRa, —SORa, —S(═O)2Ra, —OS(═O)2Ra and —S(═O)2ORa. Ra and Rb in this context can be the same or different and independently hydrogen, halogen, hydroxyl, alkyl, alkoxy, alkyl, amino, alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl.
As used herein, the designation of a polyvalent moiety without specifying the specific order of attachment is intended to cover all possible arrangements. By way of example, a compound represented by the formula:
A-X—B,
wherein X is NHC(═O) embraces both:
As used herein, a chemical bond depicted represents either a single, double, or triple bond, valency permitting. By way of example,
Some compounds disclosed herein may exist as one or more tautomers. Tautomers are interconvertible structural isomers that differ in the position of one or more protons or other labile atom. By way of example:
Unless stated to the contrary, a substituent drawn without explicitly specifying the point of attachment indicates that the substituent may be attached at any possible atom. For example, in a benzofuran depicted:
the substituent may be present at any one of the six possible carbon atoms.
As used herein, the term “null,” when referring to a possible identity of a chemical moiety, indicates that the group is absent, and the two adjacent groups are directly bonded to one another. By way of example, for a genus of compounds having the formula CH3—X—CH3, if X is null, then the resulting compound has the formula CH3—CH3. A group having the subscript ‘0’ is understood to represent a null group as well. By way of example, in the compound CH3—(X)z—CH3, if X is CH2 and z is 0, then the compound has the formula CH3—CH3.
A bracketed group with no numeric subscript should be understood to include any number of repeating groups:
includes
etc. . . .
A bracketed functional group with a subscripted variable or numeral should be understood to denote the number of repeated bracketed groups present. For example:
In certain instances, two or more variable groups may together form a ring. It is understood that any depicted atoms separated the identified groups will themselves form part of the ring:
When the variable groups are substituted on an aromatic system the new ring will be a fused ring, and unless specified to the contrary may be either aromatic or non-aromatic, carbocyclic or heterocyclic:
The ring may further be defined by the number of carbon atoms in the specific ring formed by the variable groups, which includes the atoms separating the variable groups:
Each of the above results when R1 and R2 together form a six membered (or six atom) ring. Other rings, including 3, 4, 5, 7, and 8-member rings may also be formed, and may be further limited by a specified number of carbon atoms. Although the singular “a ring” may be used to define the group, unless specified to the contrary both monocyclic and polycyclic rings are possible:
Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer, diastereomer, and meso compound, and a mixture of isomers, such as a racemic or scalemic mixture. Unless stated to the contrary, a formula depicting one or more stereochemical features does not exclude the presence of other isomers.
Some compounds disclosed herein may exist as one or more tautomers. Tautomers are interconvertible structural isomers that differ in the position of one or more protons or other labile atom. By way of example:
The prevalence of one tautomeric form over another will depend on the specific chemical compound as well as its local chemical environment. Unless specified to the contrary, the depiction of one tautomeric form is inclusive of all possible tautomeric forms.
Disclosed herein are polymer compositions. In some implementations, the polymer has the following structure:
In certain implementations, R1 and R2 are not the same.
In certain implementations the polymer can have the formula:
wherein Ra is H, CH3, or CH2CH3.
In some implementations, Rx can be N, phenyl, pyridinyl, or C1-4alkyl, for example a C1alkyl having the formula: C—Ra, wherein Ra is H, CH3, or CH2CH3.
In some implementations, R1 is a C4-8heterocyclyl group, optionally substituted one or more times by halo, hydroxy, COOH, NH2, NH(C1-5alkyl), N(C1-5alkyl)2, or O(C1-5alkyl). In some implementations, R1 is an unsubstituted C4-8heterocyclyl group. In certain implementations, R1 can include at least one secondary nitrogen atom (i.e., a nitrogen atom bearing two non-hydrogen substituents), present either in the C4-8heterocyclyl group itself or as a substituent.
In various implementations, R1 is a C4-8heterocyclyl group having the formula:
In certain implementations, x is 2, 3, or 4, preferably 2. In some implementations, x is 2 and R1 is:
In certain implementations, X1 is O.
L1 can be any suitable divalent linking group, for example a C2-10aliphatic group. In certain implementations L1 can be a C2-8alkylene group or C2-8heteroalkylene group. In some implementations L1 can be an unsubstituted C2-8alkylene group or an unsubstituted C2-8heteroalkylene group. In other implementations, L1 can be a C2-8alkylene group or C2-8heteroalkylene group, substituted one or more times by halo, hydroxy, COOH, NH2, NH(C1-5alkyl), N(C1-5alkyl)2, or O(C1-5alkyl). In some implementations L1 can have the formula:
wherein n is 0-8, preferably 0, 1, or 2, and wavy line 1 represents the point of connection to X1 and wavy line 2 represents the point of connection to Rc.
In certain implementations, some or all of the Rc groups can include a bioerodible moiety, Bioerodible moieties include disulfides, phosphonates, acetals, thioketals [e.g., R—S—CH2—S—R, R—S—C(CH3)2—S—R], oxalate esters, boronic esters, and Schiff bases/imines. In certain implementations, some, but not all, of the Rc units include a bioerodible moiety, preferably a disulfide. In certain implementations, from 1-50%, from 1-25%, from 1-15%, from 1-10%, from 1-5%, from 1-2.5%, from 2.5-5%, from 5-10%, from 10-25%, from 15-30%, from 20-40%, or from 30-50% of the Rc groups include a bioerodible moiety.
In certain implementations, Rc is independently chosen from:
wherein Z is null, CH2, C(CH3)2, O, or NH, Re is a bioerodible group, for example S—S, S—CH2—S, S—C(CH3)2—S, or O—C(═O)—C(═O)—O, and Rs is a C2-6aliphatic group. In some implementations Rs is in each case CH2CH2, C(CH3)2CH2, CH(CH3)CH2, CH2CH2CH2, C(CH3)2CH2CH2, or CH(CH3)CH2CH2, preferably Rs is in each case CH2CH2.
In some implementations, each instance of Rc in the polymer is the same moiety. In other implementations, the polymer may include multiple different Rc groups. For example, in some implementations Rc is independently:
In some implementations, the ratio of [moiety A]:[moiety B] is from 1:0.01-0.5, from 1:0.01-0.25, from 1:0.01-0.1, from 1:0.1-0.2, from 1:0.2-0.3, from 1:0.3-0.4, from 1:0.4-0.5, from 1:0.1-0.25, or from 1:0.25-0.5. In certain implementations Z is CH2 or C(CH3)2, or O.
In some implementations Rc is independently:
In some implementations, the ratio of [moiety A]:[moiety B] is from 1:0.01-0.5, from 1:0.01-0.25, from 1:0.01-0.1, from 1:0.1-0.2, from 1:0.2-0.3, from 1:0.3-0.4, from 1:0.4-0.5, from 1:0.1-0.25, or from 1:0.25-0.5. In certain implementations Z is CH2.
In certain implementations, R2 is H, OH, NH2, NHCH3, N(CH3)2, C4-8heterocyclyl, or C3-6heteroaryl. When R2 is a C4-8heterocyclyl or C3-6heteroaryl, R2 can be substituted one or more times by halo, hydroxy, COOH, NH2, NH(C1-5alkyl), N(C1-5alkyl)2, or O(C1-5alkyl). In certain implementations, R2 does not include any primary or secondary amine groups.
In some implementations R2 is a C4-8heterocyclyl or C3-6heteroaryl having the formula:
In some implementations y is 2, 3, or 4. In further implementations R1 is OH or N-morpholinyl.
In certain implementations, Rx is N or a group having the formula:
wherein Ra is H, CH3, or CH2CH3.
In some implementations Rx is a group having the formula:
wherein Ra is CH2CH3 and X2 is CH2.
The disclosed polymers may be obtained by (a) synthesizing an uncapped polymer by a providing a mixture including a compound of Formula (1):
R2—(CH2)y—NH2 [Formula (1)], and
a compound of Formula (2)
and
(b) reacting the uncapped polymer with a compound of Formula (4):
R1—(CH2)x—NH2 [Formula (4)],
The uncapped polymer can have the following formula:
In certain implementations of the compound of Formula (1) R2 is H, OH, NH2, NHCH3, N(CH3)2, C4-8heterocyclyl, or C3-6heteroaryl. When R2 is a C4-8heterocyclyl or C3-6heteroaryl, R2 can be substituted one or more times by halo, hydroxy, COOH, NH2, NH(C1-5alkyl), N(C1-5alkyl)2, or O(C1-5alkyl). In certain implementations, R2 does not include any primary or secondary amine groups.
In some implementations of the compound of Formula (1) R2 is a C4-8heterocyclyl or C3-6heteroaryl having the formula:
In some implementations of the compound of Formula (1), y is 2, 3, or 4. In further implementations of the compound of Formula (1), R1 is OH or N-morpholinyl.
In some implementations of the compound of Formula (4), R1 is a C4-8heterocyclyl group, optionally substituted one or more times by halo, hydroxy, COOH, NH2, NH(C1-5alkyl), N(C1-5 alkyl)2, or O(C1-5alkyl). In some implementations, R1 is an unsubstituted C4-8heterocyclyl group. In certain implementations, R1 can include at least one secondary nitrogen atom (i.e., a nitrogen atom bearing two non-hydrogen substituents), present either in the C4-8heterocyclyl group itself or as a substituent. In certain implementations, x is 2 or 3, preferably 2.
In some implementations of the compound of Formula (4), R1 has the formula:
In some implementations of the compound of Formula (4), R1 is:
and x is 2.
In some implementations of the compound of Formula (2) X1 is O, and L1 can be any suitable divalent linking group, for example a C2-10aliphatic group. In certain implementations L1 can be a C2-8alkylene group or C2-8heteroalkylene group. In some implementations L1 can be an unsubstituted C2-8alkylene group or an unsubstituted C2-8heteroalkylene group. In other implementations, L1 can be a C2-8alkylene group or C2-8heteroalkylene group, substituted one or more times by halo, hydroxy, COOH, NH2, NH(C1-5alkyl), N(C1-5alkyl)2, or O(C1-5alkyl). In some implementations, L1 can have the formula:
wherein n is 0-8, preferably 0, 1, or 2, and wavy line 1 represents the point of connection to X1 and wavy line 2 represents the point of connection to Rc.
In some implementations of the compound of Formula (2) Rc is independently chosen from:
In some implementations Rs is in each case CH2CH2, C(CH3)2CH2, CH(CH3)CH2, CH2CH2CH2, C(CH3)2CH2CH2, or CH(CH3)CH2CH2, preferably Rs is in each case CH2CH2.
In certain preferred implementations of the compound of Formula (2), Rc has the formula:
wherein Z is null, CH2, C(CH3)2, O, or NH, preferably CH2.
In certain implementations, the compound of Formula (2) is a compound of Formula (2a):
In certain implementations of the compound of Formula (2a), L1 can be a C2-8alkylene group or C2-8heteroalkylene group. In some implementations L1 can be an unsubstituted C2-8alkylene group or an unsubstituted C2-8heteroalkylene group. In other implementations, L1 can be a C2-8alkylene group or C2-8heteroalkylene group, substituted one or more times by halo, hydroxy, COOH, NH2, NH(C1-5alkyl), N(C1-5alkyl)2, or O(C1-5alkyl). In some implementations, L1 can have the formula:
wherein n is 0-8, preferably 0, 1, or 2, and wavy line 1 represents the point of connection to the acrylate group and wavy line 2 represents the point of connection to the phenyl ring.
In certain implementations, the mixture can further include a crosslinking compound. In some implementations, the crosslinking compound is a tri-acrylate or a tetra-acrylate. In some implementations, the crosslinker can be a compound of Formula (3):
In some implementations, Rx is phenyl (for example, the X2 groups may be attached at the 1, 3, and 5 positions), pyridinyl, or C—Ra, wherein Ra is H, CH3, or CH2CH3.
In some implementations, the compound of Formula (3) is selected from:
In some implementations, the mixture further includes a bioerodible compound. Bioerodible compounds include disulfides, phosphonates, acetals, thioketals [e.g., R—S—CH2—S—R, R—S—C(CH3)2—S—R], oxalate esters, boronic esters, and Schiff bases/imines. In some implementations the bioerodible compound can be a compound of Formula (5):
wherein Rs is a C2-6aliphatic group and Re is a bioerodible group. In some implementations Re is S—S, S—CH2—S, S—C(CH3)2—S—, or O—C(═O)—C(═O)—O. In some implementations, the bioerodible compound is a compound of Formula (5a):
In some implementations, Rs is in each case CH2CH2, C(CH3)2CH2, CH(CH3)CH2, CH2CH2CH2, C(CH3)2CH2CH2, or CH(CH3)CH2CH2, preferably Rs is in each case CH2CH2.
The mixture may further include a solvent, for example a polar aprotic solvent, a polar protic solvent, an apolar solvent, or a combination thereof. Exemplary solvents include DMSO, acetone, DMF, water, methanol, ethanol, ethyl acetate, and combinations thereof.
In certain implementations, the compound of Formula (1) and Formula (2) are present in the mixture in a molar ratio from 1.00-2.00:1, from 1.00-1.5:1, from 1.50-2.00:1, from 1.50-1.75:1, from 1.75-2.00:1, from 1.00-1.75:1, from 1.00-1.25:1, from 1.00-1.10:1, from 1.05-1.25:1, from 1.05-1.15:1, from 1.10-1.25:1, from 1.15-1.30:1, from 1.20-1.35:1, from 1.25-1.40:1, from 1.30-1.45:1, from 1.35-1.50:1, from 1.40-1.55:1, from 1.45-1.60:1, or from 1.50-1.65:1, preferably from 1.50-1.75:1.
In certain implementations, the compound of Formula (2) and Formula (3) are present in the mixture in a molar ratio from 1:0.1-0.75, from 1:0.5-0.75, from 1:0.25-0.75, from 1:0.25-0.50, or from 1:0.1-0.25.
In certain implementations, the compound of Formula (2) and Formula (5) are present in the mixture in a molar ratio from 1:0.01-0.50, from 1:0.01-0.35, from 1:0.01-0.2, from 1:0.01-0.1, from 1:0.1-0.50, from 1:0.1-0.35, from 1:0.1-0.2, from 1:0.2-0.3, from 1:0.3-0.4, or from 1:0.4-0.5.
In some implementations, the compound of Formula (4) may be employed in a suitable amount to ensure complete capping of the terminal acrylate groups in the uncapped polymer.
Also disclosed herein are compositions including the polymer and a nucleic acid. The polymer and nucleic acid may be present in a weight ratio (polymer:nucleic acid) from 5-500:1, from 5-250:1, from 5-100:1, from 5-50:1, from 5-25:1, from 10-100:1, from 10-50:1, from 10-25:1, from 20-40:1, from 25-50:1, from 25-75:1, from 40:60:1, from 50-100:1, from 50-75:1, or from 75-100:1.
The polymer/nucleic acid compositions may be provided as nanoparticle aggregates of the polymer and nucleic acid. In some implementations the nanoparticle can have a particle size (determined by DLS) from 10-1,000 nm, from 10-500 nm, from 10-250 nm, from 10-100 nm, from 10-50 nm, from 10-25 nm, from 25-100 nm, from 25-250 nm, from 50-100 nm, from 50-150 nm, from 50-250 nm, from 100-200 nm, from 100-250 nm, from 250-500 nm, or from 500-1,000 nm.
In some implementations, the nanoparticles can be characterized by Zeta potential. In certain implementations, the nanoparticles can have a Zeta potential (determined by DLS) from 2-100 mV, from 2-50 mV, from 2-25 mV, from 2-10 mV, from 5-25 mV, from 5-15 mV, from 10-25 mV, from 15-30 mV, from 25-50 mV, from 25-75 mV, from 50-75 mV, or from 75-100 mV.
A variety of different nucleic acids may be combined with the disclosed polymers. In implementations the nucleic acid can be long noncoding (Inc) ribonucleic acid (RNA) (lncRNA), small non-coding (snc) RNA (sncRNA), Piwi-interacting RNA (piRNA), interfering RNA (RNAi), small interfering RNA (siRNA), repeat associated small interfering RNA (rasiRNA), microRNA (miRNA), and/or antisense oligonucleotide. In some implementations the polymer may be combined with a two or more different types of nucleic acids.
The disclosed compositions may be made by mixing the polymer (or combinations of polymers) and a nucleic acid. The polymer and nucleic acid aggregate due to electrostatic interactions. The mixing may take place in an aqueous solvent, for example an aqueous buffer having a pH from 6-8, 6-7, 6.5-7, 6.5-7.5, 7-7.5, 7-8, or 7.5-8.
Also disclosed herein are methods of administering nucleic acids to a subject in need thereof using the disclosed compositions. Also disclosed herein are methods of providing gene therapy to a subject in need thereof using the disclosed compositions. Also disclosed herein are methods of silencing genes to a subject in need thereof using the disclosed compositions.
The compositions may be administered enterally, parenterally, intranasally, vaginally, by inhalation, or a combination thereof. When the composition is administered enterally, it may be administered by oral administration, sublingual administration, buccal administration, rectal administration, or a combination thereof. When the composition is administered parenterally, it may be administered by intramuscular injection, intravenous injection, subcutaneous injection, or a combination thereof.
In certain implementations, the composition is an aqeuous composition that may be administered by intravenous injection.
The compositions may be used to treat a variety of disease states in subjects. In some implementations, the compositions can be used to treat infectious disease in a subject in need thereof. Exemplary infectious diseases include viral infections, for example HIV, hepatitis A, hepatitis B, hepatitis C, HPV, or West Nile virus.
In some implementations, the compositions can be used to treat cancer in a subject in need thereof. In some implementations the compositions can be used to treat leukemia.
In some implementations, the compositions can be used to treat respiratory disease in a subject in need thereof. In some implementations the compositions can be used to treat asthma, COPD, or cystic fibrosis.
In some implementations, the compositions can be used to treat neurodegenerative disorders in a subject in need thereof. In some implementations the compositions can be used to treat ALS or Huntington's disease.
The following examples are for the purpose of illustration of the invention only and are not intended to limit the scope of the present invention in any manner whatsoever.
Trimethylolpropane triacrylate (TMPTA), bisphenol A ethoxylate diacrylate, bisphenol A glycerolate (1 glycerol/phenol) diacrylate, 4-(2-aminoethyl) morpholine, 4-amino-1-butanol, 1-(2-aminoethyl)piperazine, acryloyl chloride, triethylamine (TEA), bis(2-hydroxylethyl) disulfide, anhydrous dimethyl sulfoxide (DMSO) and anhydrous tetrahydrofuran (THF) were purchased from Sigma-Aldrich. Lipofectamine 2000 (Lipo2k), Alexa Fluor® Red siRNA, Silencer™ GFP siRNA and negative control siRNA were purchased from Life Technologies for transfection studies.
DNase 1 type IV, poly-D-lysine (PDL), Bovine Serum Albumin (BSA, A9647-100G), N-acetyl cysteine NAC Sigma A8199, D-biotin Sigma B4639 ITS solution (Sigma 13146), all the components of SATO (Putrescine Sigma P-5780, L-Thyroxine Sigma T-1775, Tri-iodothyroxine Sigma T-6397, Progesterone Sigma P-8783, Bovine Serum Albumin fraction V Sigma A-4919), Cytarabine (PHR1787), and Triton X-100 were purchased from Sigma-Aldrich. Mounting Medium (SP15-500) was purchased from Fisher Scientific. Alexa-Fluor Goat anti-Rabbit 488, Alexa-Fluor Donkey anti-Rat 555, Alexa-Fluor Donkey anti-Mouse 555, DAPI (4′,6-diamidino-2-phenylindole), minimum essential media (MEM), DMEM high glucose, neurobasal medium (NB), fetal bovine serum (FBS, 10270106), Glutamax (35050061), B-27 supplement (17504044), N2 supplement (17502048), phosphate buffered saline (PBS, 10010023), Dulbecco's phosphate-buffered saline (DPBS, 14040133) and penicillin/streptomycin (Pen/Strep) were purchased from Life Technologies. Rat anti-MBP (aa82-87) was purchased from Bio-Rad. Mouse anti-Tuj1 (#MMS-435P) was purchased from BioLegend. Papain suspension was purchased from Worthington. 4% paraformaldehyde solution (sc-281692), 10× phosphate buffered saline (PBS) were purchased from Axil Scientific. RNA extraction kit was purchased from Qiagen (Singapore). Reversal transcript kit (NEB #E3010) and qPCR kit (NEB #M3003) were purchased from New England Biolabs Inc. TransIT-TKO (MIR2150) was purchased from MirusBio. All the primers and siRNA are pre-designed sequences that were purchased from Integrated DNA Technologies.
The polymers were dissolved in deuterated DMSO and characterized by 1H NMR. The proton NMR spectra were recorded on a Bruker Avance 400 MHz NMR spectrometer with at least 8 scans. Chemical shifts were quoted relative to the solvent peak (DMSO-d6 at δ 2.50 ppm).
pBAE/siRNA complexes were prepared by mixing siRNA solution with polymer solution at varying polymer:siRNA w/w ratios. The solutions were vortexed and incubated at room temperature for 30 min before use. 20 μL of complexes solution at varying w/w ratios were mixed with 2 μL of 10× BlueJuice loading buffer. Next, the sample was loaded on 1% agarose gel containing SYBR gold gel stain. The measurement was conducted in 1× Tris-acetate-EDTA (TAE) buffer for 20 min under 100 V in a Sub-Cell system. siRNA bands were visualized under UV exposure using a G-box imaging system.
A subset of monomers depicted in
Six linear pBAEs using the monomers listed above, namely A1B1, A1B2, A1B3, A2B1, A2B2 and A2B3, were synthesized. 1H NMR was used to confirm the successful copolymerization of the monomers and chemical structure of the polymers. The siRNA binding efficiency of these six linear pBAEs was characterized using agarose gel electrophoresis assay to compare and identify the optimal monomer composition for efficient siRNA complexation (
Two linear pBAEs using B1 as diacrylate monomer showed poor siRNA condensation ability. Polymers using A2 as the amine monomer and B2 or B3 as diacrylate monomer resulted in slightly better siRNA complexation, which could stop siRNA migration at the polymer:siRNA weight ratio of 40:1. Among all, A1B3 gave the lowest weight ratios to fully retard the siRNA migration at w/w 25. Based on these gel retardation results, A1B3, A2B2 and A2B3 were selected to synthesize their corresponding hyperbranched pBAEs due to their slightly better siRNA complexation ability.
The disulfide monomers (SS) were firstly prepared according to the literature reported previously. Briefly, bis(2-hydroxylethyl) disulfide (2 g, 12.9 mmol) and TEA (9 mL, 64.8 mmol) were dissolved in 26 mL of anhydrous THF. Next, acryloyl chloride (6.287 mL, 77.8 mmol) dissolved in 4 mL of anhydrous THF was added dropwise into the mixture of the flask in an ice bath. The resulting heterogeneous mixture was stirred at 25° C. for 24 h under argon environment. After reaction, triethylamine hydrochloride precipitate was removed by filtration, and the solvent was removed by rotary evaporation. The product was dissolved in dichloromethane (DCM), washed three times with an aqueous solution of 0.1 M Na2CO3, two times with distilled water and once with brine. Finally, the product was dried using anhydrous MgSO4 and obtained by removing the solvent under reduced pressure.
To synthesize bioreducible h(A2B3)-1 (Scheme 1), TMPTA, A2 (amine monomer), B3 (diacrylate monomer) and SS (disulfide monomers) were dissolved in DMSO (500 mg/ml) and reacted at 90° C. for 48 h. The [vinyl]: [NH] of these reactions were set at 1.2:1. After 48 h reaction, the mixture was cooled to RT and diluted to 100 mg/ml with DMSO. 1-(2-aminoethyl)piperazine was added to end-cap the base polymer and react at RT for 24 h. The final polymers were purified by precipitation in diethyl ether three times, vacuum dried overnight, and then stored at −20° C.
The bioreducible h(A2B3)-1 with different disulfide percentage were characterized by 1H NMR. Their siRNA complexation ability was evaluated by gel electrophoresis assay. In vitro cytotoxicity was analyzed using MTT assay and siRNA transfection efficiency was studied according to the protocol above.
A subset of chosen monomers depicted in
To study the influence of branching density on gene delivery efficiency and cytotoxicity, two different ratios of TMPTA to diacrylate monomer were used to synthesize two hyperbranched pBAEs with different branching densities, namely as h(AB)-1 and h(AB)-2. The ratio of TMPTA to diacrylate for h(AB)-1 was 0.25:1 whereas the ratio of TMPTA to diacrylate for h(AB)-2 was 0.5:1. Generally, h(AB)-2 is more branched than h(AB)-1 due to its higher TMPTA molar ratio. The [vinyl]: [NH] ratio of these reactions was set at 1.2:1.
Five hyperbranched pBAEs using the monomers listed above and two branching densities, namely h(A2B2)-1, h(A2B2)-2, h(A2B3)-1, h(A2B3)-2, and h(A1B3)-1, were synthesized. The successful synthesis of hyperbranched pBAEs for the chosen monomer compositions were confirmed by measurement of the 1H NMR. Their siRNA complexation ability was also evaluated by gel electrophoresis assay (
The gel retardation results demonstrated that all hyperbranched pBAEs could achieve lower weight ratio to completely retard siRNA mobility, as compared to their corresponding linear pBAEs. Linear polymer A2B2 stopped siRNA migration at the polymer:siRNA weight ratio (w/w) of 40. The introduction of hyperbranching structure enhance the siRNA complexation ability, which fully retard the siRNA migration at w/w 20 and 25 for h(A2B2)-1 and h(A2B2)-2, respectively. At w/w 40, the linear polymer A2B3 inhibited siRNA migration. However, by introducing a hyperbranching structure, the siRNA complexation ability improved greatly, leading to complete retardation of siRNA migration at w/w 20 and 25 for h(A2B3)-1 and h(A2B3)-2, respectively. In A1B3 series, the linear polymer A1B3 stopped siRNA migration at w/w 25. Nonetheless, with the incorporation of a hyperbranching structure, the siRNA complexation ability slightly increased, resulting in complete retardation of siRNA migration at w/w 20 for h(A1B3)-1.
These results demonstrated that incorporation of highly branched structure into the polymer backbone greatly enhances their electrostatic interaction with negatively charged siRNA, resulting in better siRNA complexation ability. This may be due to the increased cationic charge density of hyperbranched polymer as compared to their corresponding linear structure. Hyperbranched polymers have higher cationic charge density, hence providing larger capacity of electrostatic interaction with negative charged nucleic acid and subsequently enhancing the gene complexation ability.
HeLa cells (ATCC) and 3T3 cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 1% antibiotic antimycotic solution (100×) at 37° C. in a humidified and 5% CO2 incubators.
HeLa cells and 3T3 cells were seeded in 96-well plates at a density of 1×104 cells per well in 100 μL of DMEM complete medium. After 24 h incubation, the medium was removed and replaced by 100 μL of culture medium containing serial dilutions of polymers, continuing incubating the cells for 24 h. Thereafter, 50 μL of MTT solution (1 mg/ml) was added to each well and incubated for 2 h. Next, MTT solution was removed and 100 μL of isopropanol was added into each well. The absorbance of each well was measured by Tecan Spark microplate reader at 570 nm. The relative cell viability was calculated with the following equation: cell viability (%)=OD570(sample)/OD570(untreated)×100%. Data is means of triplicate measurements.
HeLa cells were seeded in 24-well plates at a density of 5×104 cells per well and then incubated for 24 h before transfection. 50 μL of polymer/siRNA complexes containing 15 pmol of Alexa Fluor® Red siRNA (Invitrogen) was prepared at w/w ratios of 20 and 30. The culture medium was removed and 450 μL of serum-free medium (Welgene) was added into the wells. 50 μL of complexes solution were added to each well to obtain a final RNA concentration of 30 nM and incubated for 24 h. Control experiments were performed with Lipo2k, as instructed by the vendor's manual (Invitrogen). After 24 h of transfection, the medium was removed; the cells were harvested using trypsin solution, washed with PBS, and fixed in 4% parafomaldehyde. The samples were measured by BD LSRFortessa X-20 flow cytometry. Untreated cells were used as control for background calibration. Mean fluorescence intensity (MFI) of PE-positive cells was measured and used for transfection and uptake analysis.
The gene delivery capability of cationic polymers is dependent on molecular weight and branching of the polymer which directly affect their amino charge density (Fischer et al., Bioconjugate Chem. 2002, 13, 1124-1133). High molecular weight and branching of the polymer can enhance their gene transfection efficiency; however, they also increase the cytotoxicity due to the high density of amino groups and high molecular weight. The major concern which limits the application of cationic polymer in gene delivery is their toxicity both in vitro and in vivo (Pun & Davis, Bioconjugate Chem. 2002, 13, 630-9; Yhee et al., Bioconjugate Chem. 2013, 24, 1850-1860). Hence, polymer cytotoxicity has become an important consideration on selection of top-performing cationic polymers. To identify the top-performing hyperbranched pBAE polymer, the in vitro cytotoxicity of synthesized linear pBAEs and hyperbranched pBAEs was first evaluated in 3T3 fibroblasts and HeLa cells using MTT assay (
In A2B2 series, the incorporation of branching unit into the polymer backbone slightly increased the polymer toxicity of h(A2B2)-1. Although h(A2B2)-2 is more branched than h(A2B2)-1, the cytotoxicity is lower. In A2B3 series, the introduction of branching unit did not significantly affect the cytotoxicity. In 3T3 fibroblast cells, linear A2B3, hyperbranched h(A2B3)-1 and h(A2B3)-2 showed no cytotoxicity even at highest concentration. In HeLa cells, h(A2B3)-1 showed better biocompatibility with an IC50 value of 180 μg/mL as compared to A2B3 and h(A2B3)-1 polymer. Meanwhile, in A1B3 series, slightly increased polymer toxicity with reduced IC50 was observed in the hyperbranched h(A1B3)-1 as compared to the linear A1B3. Among all hyperbranched pBAEs, h(A2B3)-1 exhibited best biocompatibility with lowest cytotoxicity in both 3T3 cells (IC50>320 μg/mL) and HeLa cells (IC50=180 μg/mL).
Cellular uptake of the gene carrier is a key factor to achieve high gene transfection efficiency. Therefore, besides cytotoxicity, the in vitro siRNA uptake efficiency of the synthesized hyperbranched pBAEs was evaluated in HeLa cells (
h(A2B2) series showed the lowest uptake efficiency. On the other hand, the h(A1B3) and h(A2B3)-2 exhibited similar uptake efficiencies, which were slightly better than the h(A2B2) series. Meanwhile, h(A2B3)-1 showed the highest cellular uptake efficiency amongst the hyperbranched polymers, indicating that optimal branching degree in h(A2B3)-1 will promote better transfection capability than higher branching as in h(A2B3)-2. In particular, h(A2B3)-1 at a weight ratio of 30 showed the strongest mean fluorescence intensity, which was almost comparable with Lipo2k.
Based on the results above, h(A2B3)-1 with optimal monomer composition and branching degree was chosen to be the top-performing gene vector for siRNA delivery owing to its good biocompatibility, low cytotoxicity (with IC50 value up to 180 μg/mL), and good cellular uptake efficiency which are comparable with Lipo2k.
The particle size and zeta potential of the complexes were determined via DLS using a Malvern Zetasizer NanoZS. The complexes solution at varying w/w ratio was first prepared and diluted to 1 mL with sodium acetate buffer. The samples were measured with DLS immediately after preparation to obtain the particle size, polydispersity index (PDI) and zeta potential. All measurements were done in triplicates.
The chemical structure of the optimal hyperbranched pBAEs, h(A2B3)-1, is shown in
Comparison of In Vitro Cytotoxicity Between h(A2B3)-1 and Lipo2k
Lipo2k and h(A2B3)-1 with increasing concentration were added into HeLa cells and 3T3 cells and then incubated for 24 h. h(A2B3)-1 complexes containing 3 pmol of siRNA at polymer:siRNA w/w ratio of 30 were prepared with increasing concentration. Lipo2k/siRNA complexes at w/w ratio of 5 with increasing concentration was used as the standard for toxicity comparison. The complexes solution was added into the cells and incubated for 24 h. The cell viability (%) was then determined by the MTT assay after 24 h incubation.
HeLa cells were plated at a seeding density of 8×104 cells/well in 24-well plates and incubated for 18 h before pEGFP transfection. The cells were first transfected with Lipo2k/pEGFP complexes (1 μg of pEGFP plasmid DNA per well, using 1 μL Lipo2k) for 6 h in serum-free medium. Next, 50 μL of h(A2B3)-1/siRNA complexes containing 15 pmol of Silencer™ GFP siRNA (Invitrogen) or negative control siRNA (NC siRNA) was prepared at different weight ratios of 20, 30, 40 or 50. After transfection of pEGFP for 6 h, the medium was removed and replaced with 450 μL serum-free medium. 50 μL of complexes solution was added into the well to obtain a final RNA concentration of 30 nM and the cells were further incubated for 24 h. Control experiments were performed with Lipo2k, as instructed by the vendor's manual (Invitrogen). After 24 h of transfection, the medium was removed; the cells were harvested using trypsin solution, washed with PBS, and fixed in 4% parafomaldehyde. The samples were measured by BD LSRFortessa X-20 flow cytometry. Untreated cells were used as control for background calibration. Cell population percentage of FITC-positive cells was measured and used for transfection analysis.
The GFP silencing efficiency of h(A2B3)-1 was further investigated using confocal microscopy. The cells were transfected with complexes containing 15 pmol of Silencer™ GFP siRNA (Invitrogen) or NC siRNA for 24 h. After 24 h of transfection, the medium was removed and the cells were washed with PBS three times, fixed with 4% paraformaldehyde for 20 min and stained with DAPI solutions for 10 min. The cells were imaged under Zeiss LSM 800 confocal laser scanning microscope.
To demonstrate the good cytocompatibility and excellent siRNA transfection efficiency of h(A2B3)-1, in vitro cytotoxicity assay and Green Fluorescent Protein (GFP) silencing study were conducted with Lipo2k as positive control. As demonstrated in
Next, the siRNA transfection efficiency of h(A2B3)-1 was investigated using GFP siRNA as positive control and NC siRNA (scramble non-coding siRNA) as negative control. Lipo2k/siRNA at weight ratio of 5, 10 and 15 were used as the gold standard. As shown in
In comparison with commercial Lipo2k, h(A2B3)-1 has lower cytotoxicity and better siRNA knockdown efficiency, indicating a better safety profile and more potent siRNA transfection ability of h(A2B3)-1 polymer over commercial transfection reagent.
Animal procedures were approved by the Institutional Animal Care and Use Committee guidelines of Nanyang Technological University (IACUC, NTU, protocol number: A190004)
Cortices were isolated from newborn (within 24 h) Sprague-Dawley rats followed by removal of the cerebrum and hippocampus, as well as the meninges. The cortices were digested using a mixture of 1.2 U papain, 40 μg/mL of DNase, and 0.24 mg/mL of L-cysteine that were dissolved in DMEM with 1% Pen/Strep, at 37° C., 5% CO2 for 1 h.
The tissue digestion was stopped by adding DMEM Full medium (DMEM+10% FBS+1% Pen/Strep) digestion mix. All the remaining tissue pieces with medium were collected into a centrifuge tube. After centrifuging at 300 g for 5 min, the supernatant was discarded. Thereafter, the pellet was resuspended in DMEM Full medium and triturated through a 21 G and a 23 G needle for about 10 times sequentially. According to the established cell seeding density from our previous works (Zhang, Lin, & Chew, ACS Appl. Mater. Interfaces. 2021, 13, 55840-55850), cells from the cortices of 3 pups are seeded onto two PDL-(2.5 μg/mL) coated T75 flasks. After seeding, cells were cultured in DMEM Full medium at 37° C., 7.5% CO2. Medium was refreshed every 3 days until day 10 when a monolayer of astrocytes was formed.
After 10 days of culture, microglia, which attach most loosely, were removed by shaking on an orbital shaker at 37° C., 200 rpm for 1 h. Thereafter, the supernatant was discarded, and the flasks were refilled with fresh DMEM Full medium for another round of shaking at 37° C., 200 rpm for 16 h to detach all OPCs. The remaining microglial were then eliminated by differential adhesion, which was performed by incubating the supernatant in an uncoated Petri dish for 30 min. After that, the supernatant was collected for centrifugation at 300 g for 5 min to obtain purified OPCs. Serum-free OPC culture medium which consisted of DMEM:Neurobasal (50:50), 2% B27, 1% Pen/Strep, Glutamax, ITS, 10 ng/mL biotin, 5 μg/mL NAC, and SATO (100 μg/mL BSA, 60 ng/mL progesterone, 16 μg/mL putrescine, and 400 ng/mL T3 and T4).
For gene silencing studies, OPCs were seeded at a density of 1×105 cells in each well of 24-well plates. All the wells were coated with 2.5 μg/mL of PDL and washed with PBS in advance. OPCs were maintained at 37° C., 7.5% CO2 incubator for 24 h before transfection.
Transfection was conducted by adding 15 w/w of h(A2B3)-1/siRNA into each well to obtain a final concentration of 100 nM of RNA. As the positive control, commercial transfection reagent, TKO (Mirus Bio LLC, US), was used, which comprised of 1:1 volume ratio of 50 μM siRNA following manufacturer's protocol. To evaluate gene silencing outcomes, RNA was extracted after 16 h of transfection.
The digestion of cortices for neuron culture was stopped by adding NB medium with 10% FBS into the digestion mix. After centrifuging at 300 g for 5 min, the cell pellet was resuspended in Neuron seeding medium (NB medium, 10% FBS, 2% B27 supplement, 1% N2 supplement, 1% Pen/Strep, 1% Glutamax) and triturated with a 21 G needle for 10 times to obtain uniform cell suspension. Thereafter, the suspension was passed through a 70 μm cell strainer to remove unbroken tissue clumps. The filtered cell suspension was then used for cell culture. Cells were seeded at a density of 2×105/well on PDL-coated 24-well plates for gene silencing evaluation and 0.7×105/13 mm PDL-coated coverslip for cell uptake evaluation. After seeding overnight, Neurobasal Full medium (NB medium, 2% B27 supplement, 1% Pen/Strep, 1% Glutamax) was used to replace the Neuron seeding medium. Neurons were maintained at 37° C., 5% CO2 for 7 days before transfection, with medium change every 3 days. On Day 3, 10 μM of cytarabine was added to inhibit glial cells proliferation.
On Day 7, neurons were used for transfection. 10 w/w of h(A2B3)-1/siRNA or Cy5-labeled oligonucleotides (Cy5-ODN) was added into each well, to obtain a final concentration of 100 nM of siRNA. Commercial transfection reagent TKO (Mirus Bio LLC, US) was utilized as the positive control, which was used at 1:1 volume ratio to obtain a final concentration of 50 μM of siRNA. RNA was extracted after 16 h of transfection.
After 16 h of transfection, cells on coverslips were first washed with DPBS once and then fixed with 4% PFA for 15 min. Permeabilization and blocking were performed by incubating all samples in the same blocking buffer, which was composed of 1% BSA, 0.1% Triton in PBS for 1 h. All samples were incubated in the primary antibodies overnight at 4° C. After washing with PBS 3 times, all samples were incubated in secondary antibodies and DAPI at room temperature for 1 h before washing and mounting by covering the 13 mm round coverslip with stained cells on a 24 mm×50 mm coverslip. Details of the antibodies used are outlined in Table below. Thereafter, all samples were imaged under an inverted confocal microscope (Zeiss Airyscan Microscope-LSM800).
To investigate the gene expression level change after transfection, qPCR was conducted to compare the GAPDH expression level in different group settings. The groups were named as following: h(A2B3)-siGAPDH: h(A2B3)-1 polymers with siGAPDH. h(A2B3)-siNeg: h(A2B3)-1 with non-functional scrambled siRNA. TKO-siGAPDH and TKO-siNeg: commercial transfection reagent TKO with siGAPDH and siNeg respectively. RNA was extracted using RNeasy Mini Kit (Qiagen). Thereafter, 100 ng of RNA was reverse-transcribed into cDNA using LunaScript RT SuperMix Kit. A total of 10 μL of reaction mix for qPCR, which was composed of 0.25 μL of 10 μM primer pair, 1 μL cDNA, 4.75 μL deionized water and 5 μL Luna Universal qPCR Master Mix, was used for qPCR, using the QuantStudio 6 Flex QRT PCR Machine (Applied Biosystems). The following thermal cycle condition was used: 95° C. for 60 s for initial denaturation; 95° C. for 15 s followed by 60° C. for 30 s for 40 cycles) and followed by melt curve analyses. Three repeats for each sample and each gene were analyzed to obviate technical variation. All the primers sequences are listed in the table below. Since the primers have similar amplification efficiency, the comparative delta Ct method was used with β-actin as the housekeeping gene.
To explore the potential application of h(A2B3)-1 in neural system gene silencing, transfection study was conducted on primary neural cells, specifically cortical neurons, and oligodendrocyte progenitor cells (OPCs). These cells play key roles in nerve regeneration and remyelination after traumatic injuries and degeneration.
Cortical neurons were stained with TUJ1 and DAPI after transfection with h(A2B3)-Cy5-labeled oligonucleotide (Cy5-ODN) and the cellular uptake of the h(A2B3)-1 complexes were examined under confocal microscope. As shown in
The gene silencing ability of h(A2B3)-1 complexes on primary cortical neurons and OPCs was evaluated by conducting cell transfection with commercial transfection reagent, TKO, as the positive control. We have previously shown that TKO effectively delivers small non-coding RNAs to primary neural cells (Diao, Low, Lu, & Chew, Biomaterials 2015, 70, 105-114; Milbreta et al., Mol. Ther. 2019, 27, 411-423; Zhang et al., Adv. Sci. 2019, 6, 1800808). As shown in
The results above showed that h(A2B3)-1 supported cell uptake of nucleic acids as well as downstream gene silencing. The extent of gene knockdown, however, was cell type dependent with OPCs showing better gene silencing outcomes regardless of transfection reagents used. One possibility for this difference may be the intrinsic difficulty to transfect neurons, due to the absence of cell cycle-dependent breakdown of the nuclear envelope in postmitotic cells (Huang & Chau, Mol. Pharmaceutics. 2021, 18, 377-385).
Interestingly, a trend of higher knockdown efficiency as observed in primary rat cortical neurons that were treated with h(A2B3)-1 vs. TKO. There are also many other gene carriers that have been reported in neural cells. For example, a type of amphiphilic 3-cyclodextrins, oligosaccharide-based molecules could achieve ˜40% knockdown effect in a rat striatal cell line at 100 nM final concentration of siRNA, also at a mass ratio of 10 (carrier: siRNA). But noteworthy, postmitotic cells are always much more difficult to be transfected than cell lines (Godinho, Ogier, Darcy, O'Driscoll, & Cryan, Mol. Pharmaceutics. 2013, 10, 640-649). In very recent research, which used three charged lipid nanoparticles (LNP) with different zeta potential to transfect retinal ganglia neurons. Results came out with 40, 35, and 32% knockdown efficiency with increasing surface charge at a concentration of 45 pmol/5×104 cells (Huang & Chau, Mol. Pharmaceutics. 2021, 18, 377-385). In comparison with our study, which used 12.5 pmol/5×104 cell, the knockdown efficiency is quite similar (˜34.8% in primary rat cortical neurons) even though three times lower amount of siRNA was delivered. As for OPCs, transfection with three particles made of polyornithine-based complexes, with different size and zeta potential at 100 nM siRNA, resulting in a range of decrease between 35% to 39% (Conejos-Sanchez et al., Nanoscale 2020, 12, 6285-6299). In comparison, the 53.4% knockdown efficiency of this h(A2B3)-1 polymer in our study reached an exciting degree which allows further in vivo applications.
h(A2B3)-1 polymers were dissolved and incubated in sodium acetate buffer (pH 5.0) and PBS buffer (pH 7.4) at 37° C. to induce the polymer degradation. At incubation time points of 2, 4, 8, 20 and 24 h, degraded polymers were used to test the siRNA complexation ability using gel electrophoresis assay and evaluate their post-degradation cytotoxicity using MTT assay. Polymer at 0 h of incubation time was used as the control and treated as undegraded polymers.
pBAE is a class of cationic polymer which is biodegradable because of the hydrolysable ester bond along the polymer backbone. To study the polymer degradation of h(A2B3)-1, the polymers were incubated in PBS buffer (pH 7.4) and sodium acetate buffer (pH 5.0) for 2, 4, 8, 20, 24 h. After the designated time points of incubation, polymers were complexed with siRNA and tested for their siRNA condensation ability using gel electrophoresis assay. It is hypothesized that degraded polymers will lose their siRNA condensation ability because they are degraded into small molecules and cannot completely complex the nucleic acid.
Furthermore, the post-degradation cytotoxicity of h(A2B3)-1 was studied in HeLa cells using MTT assay. As shown in
The h(A2B3)-1 polymer showed good siRNA uptake and excellent siRNA transfection efficiency in the results above. However, the toxicity in the form of nanoparticles particularly at polymer concentrations above 40 μg/mL might become an issue to limit their application in the therapeutic field. Therefore, bioreducible disulfide bond was introduced into the polymer backbone to further improve the biocompatibility of h(A2B3)-1. It is well-known that the intracellular glutathione (GSH) concentration is about 2-10 mM whereas extracellular GSH level is only 1-10 μM. The disulfide bond can cleave spontaneously under the reductive environment with high GSH concentration while remain stable in extracellular environment with low GSH concentration.33, 47-49 Hence, introduction of disulfide bonds into the backbone of h(A2B3)-1 could make it become responsive to the intracellular reductive environment, induce spontaneous polymer degradation, decrease the cytotoxicity, and improve its biocompatibility.
The bioreducible h(A2B3)-1 was synthesized by the copolymerization of A2, B3, SS, and TMPTA via Michael addition approach (Scheme 1). Five disulfide-containing h(A2B3)-1, namely as S1, S2, S3, S4, and S5, with increasing mole percentage of SS monomer were prepared and characterized by 1H NMR (Figure S6). The measured mole percentage of disulfide content in S1 to S5 increased from 2.9% (S1), 6.8% (S2), 9.7% (S3), 19.8% (S4) to 26.6% (S5) respectively, which was calculated based on the integral ratios of the NMR spectra (Table S1). Next, their siRNA complexation ability was evaluated by gel electrophoresis assay. As shown in Figure S7, increasing SS % along the backbone of h(A2B3)-1 slightly affected the siRNA complexation. S1 with lowest disulfide percentage of 2.9% could retard the siRNA migration at weight ratio of 20 while S5 with highest disulfide percentage of 26.6% stopped the siRNA migration at weight ratio of 35.
To investigate the effect of disulfide bond on biocompatibility, in vitro cytotoxicity of all five bioreducible h(A2B3)-1 was studied in HeLa cells, with Lipo2k and non-reducible h(A2B3)-1 as control. As shown in
The siRNA transfection efficiency of bioreducible h(A2B3)-1 was also investigated using GFP siRNA. Lipo2k and non-reducible h(A2B3)-1 were used as controls. As shown in
Together with the cytotoxicity results, we concluded that the integration of disulfide features into the h(A2B3)-1 polymers can improve the biocompatibility but slightly reduce the siRNA transfection ability. With the aim to develop a siRNA delivery system which possesses good biocompatibility and efficient siRNA transfection efficiency simultaneously, S4 with disulfide percentage of 19.8% was chosen to be the optimal formulation among all bioreducible h(A2B3)-1 owing to its well balance performance between polymer cytotoxicity and siRNA silencing efficiency.
The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compositions and method steps disclosed herein are specifically described, other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments of the invention and are also disclosed. Other than in the examples, or where otherwise noted, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches
This application claims the benefit of U.S. Provisional Application 63/578,567, filed Aug. 24, 2023, the contents of which are hereby incorporated in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63578567 | Aug 2023 | US |
