Polymers and micelles and compositions containing the same

Abstract

Complexes that include one or more diblock polymers and an oligonucleotide. The diblock polymers can be a cationic polymer, an anionic polymer, or a neutral polymer. The diblock polymer can include a hydrophobic block. Compositions that include a complex. Methods of making the complexes. Methods of using the complexes for transfecting a cell.

Description

POLYMERS AND MICELLES AND COMPOSITIONS CONTAINING THE SAMECROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 657,237, filed June 7, 2024, which is incorporated herein by reference in its entirety.SUMMARY

[0002] In one aspect, the present disclosure describes a complex that includes a cationic diblock polymer and an oligonucleotide cargo. The cationic diblock polymer includes an acrylate block and an acrylamide block. The acrylamide block includes a repeating group that includes a cationic containing group of the formula (i), (ii), (iii), or (iv):(') (»i) (Hi) (iv) . b is an integer from 0 to 5. z and z’ are independently an integer from 0 to 5. Q1and Q2are each independently a heteroatom or a group that includes a heteroatom.

[0003] In one or more embodiments, the complex further includes a neutral diblock polymer. The neutral diblock polymer includes an acrylate or methacrylate bock and a second block. The second block includes a -(O-CH2-CH2)- repeat unit.

[0004] In one or more embodiments, the complex further includes an anionic diblock polymer. The anionic diblock polymer includes an acrylate or methacrylate block and an acrylamide block. The acrylamide block includes repeating groups that include an anionic group.

[0005] In one or more embodiments, the anionic group includesor an ionized version thereof.

[0006] In one or more embodiments, the complex includes a cationic diblock polymer of the present disclosure and a neutral diblock polymer of the present disclosure.

[0007] In one or more embodiments, the complex includes a cationic diblock polymer of the present disclosure and an anionic diblock polymer of the present disclosure.

[0008] In one or more embodiments, the complex includes a cationic diblock polymer of the present disclosure, an anionic diblock polymer of the present disclosure, and a neutral polymer of the present disclosure.

[0009] In another aspect, the present disclosure describes compositions that include one or more complexes of the present disclosure. In one or more embodiments, a composition is a transfection composition.

[0010] In yet another aspect, the present disclosure describes a method of using a composition of the present disclosure. In one or more embodiments, the method is a method of transfecting a cell using a transfection composition of the present disclosure.BRIEF DESCRIPTION OF DRAWINGS

[0011] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0012] FIG. 1. Generation of diblock cationic amphiphiles from diblock modular scaffolds via post polymerization modification. The subsequently formulated micelles were complexedwith mRNA, biophysically characterized, and screened in vitro and in vivo to determine their delivery performance. The biological performance was evaluated via Shapley Additive exPlanations (SHAP) analysis and allied statistical tools to elucidate micelle-mRNA structure activity relationships.

[0013] FIG. 2. Scheme for synthesis of diblock scaffolds via reversible additionfragmentation chain-transfer (RAFT) polymerization and their subsequent modification with a library of ten different cationic modifiers (Al to A10).

[0014] FIG. 3. Diblock scaffold (polymers) characterization viaJH NMR (CDCI3 solvent) and size exclusion chromatography with multi-angle light scattering (SEC-MALS; THF Solvent).

[0015] FIG. 4. Micelle formulation in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer and micelle size (Dh) characterization via dynamic light scattering (DLS).

[0016] FIG. 5. Scheme representation of micelleplexation and a dye exclusion assay used to analyze mRNA binding efficiency of the micelles.

[0017] FIG. 6. Micelleplex size (Dh ,nm ) characterization via DLS measurements.

[0018] FIG. 7. Micelle - mRNA binding efficiency in PBS (micelleplex formulation media), OptiMEM (transfection media), and heparin (to demonstrate mRNA release efficiency from the micelleplexes).

[0019] FIG. 8. Heatmap showing the transfection efficiency (% of cells GFP positive) of the full micelle library upon transfection in HEK293-T cells.

[0020] FIG. 9. SHAP values of polymer features on transfection efficiency of the full micelle library upon transfection in HEK293-T cells. Higher SHAP value of a feature correlates to higher impact on the biological output. Similarly, the ordering of the features also displays their relative impact on the output. Each dot represents a polymer formulation, and the color corresponds to the normalized value of the respective feature (blue = low, purple = medium, red = high).

[0021] FIG. 10. Linear regression analysis to visualize the relative impact of individual amine type on transfection efficiency of the full micelle library upon transfection of HEK293-T cells. A positive impact factor correlates to a positive influence on biological output and vice versa.

[0022] FIG. 1 1 . Heatmap showing the cellular viability (% of cells GFP positive) of the full micelle library upon transfection of HEK293-T cells.

[0023] FIG. 12. SHAP values of polymer features on cellular viability of the full micelle library upon transfection of HEK293-T cells. A higher SHAP value of feature correlates to a higher impact on the biological output. Similarly, the ordering of the features also displays their relative impact on the output. Each dot represents a polymer formulation, and the color corresponds to the normalized value of the respective feature (blue = low, purple = medium, red=high).

[0024] FIG. 13. Linear regression analysis to visualize the relative impact of individual amine type on cellular viability of the full micelle library upon transfection of HEK293-T cells. A positive impact factor correlates to a positive influence on biological output and vice versa.

[0025] FIG. 14. Heatmap showing the mean fluorescence intensity of GFP expression (GFP MFI) of the full micelle library upon transfection of HEK293-T cells.

[0026] FIG. 15. SHAP values of polymer features on mean fluorescence intensity of GFP expression (GFP MFI) of the full micelle library upon transfection of HEK293-T cells. A higher SHAP value of feature correlates to a higher impact on the biological output. Similarly, the ordering of the features also displays their relative impact on the output. Each dot represents a polymer formulation, and the color corresponds to the normalized value of the respective feature (blue = low, purple = medium, red = high).

[0027] FIG. 16. Linear regression analysis to visualize the relative impact of individual amine type on mean fluorescence intensity of GFP expression (GFP MFI) of the full micelle library upon transfection in HEK293-T cells. A positive impact factor correlates to a positive influence on biological output and vice versa.

[0028] FIG. 17. Cy5 tagged mRNA was used to prepare micelleplexes that were subsequently transfected in HEK293-T cells, followed by quantification of Cy5 positive cells and Cy5 MFI at different time points via flow cytometry. Graphical representation of percentage of cells positive for Cy5 fluorescence indication successful internalization of mRNA loaded micelleplex and respective Cy5 MFI to quantify relative amount of Cy5 labelled mRNA internalized (t = 24h). U=Untreated, N= Naked mRNA, and J = JetPEI.

[0029] FIG. 18. HEK293-T cells were preincubated with the proton pump inhibitor Bafilomycin-Al (BAF). Reduction in GFP positive cells upon BAF treatment indicates a reliance of micelleplexes on endosomal acidification to escape the endosome.

[0030] FIG. 19. Percent protonation of Md library of amine as a gauge for buffering capacity of the respective amines.

[0031] FIG. 20. Percentage of cells positive for propidium iodide representative of membrane permeabilization ability of the micelles. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0032] FIG. 21. Percentage of cells positive for necrosis and / or apoptosis upon treatment with the respective micelleplexes. J = JetPEI.

[0033] FIG. 22. Correlation between membrane poration (PI positive), necrotic cell death and viability.

[0034] FIG. 23. Radar plot showing the relative impact of amine features on the biological interaction observed.

[0035] FIG. 24. In vivo luminescence images obtained via an IVIS imager. Single images shown for representation. Experiments performed in triplicates (n=3).

[0036] FIG. 25. Average luciferase radiance for the whole animal (n=3) from FIG. 24. Statistical significance by two-way ANOVA. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0037] FIG. 26. Average luciferase radiance for organs of the animals of FIG. 24. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0038] FIG. 27. Percentage of GFP positive cells upon GFP expressing mRNA transfection in fully serum supplemented cell culture media in HEK293-T cells. U=Untreated, N= Naked mRNA, J = JetPEI.

[0039] FIG. 28. Amphiphile physical and chemical properties of various diblock scaffolds.

[0040] FIG. 29.spectra of Poly(nBA)ii8 macro-CTA.

[0041] FIG. 30.spectra of Poly(nBA)iis-b-poly(PFPA)ioo / i5o / 2oo modular scaffolds.

[0042] FIG. 31. Pearson correlation coefficients between polymer features and micelleplex physical properties.

[0043] FIG. 32. Transfection efficiency and viability data for the Sh library at a 0.25 pg mRNA dose. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0044] FIG. 33. Transfection efficiency and viability data for the Sh library at a 0.5 pg mRNA dose. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0045] FIG. 34. Transfection efficiency and viability data for the Sh library at different a 0.75 gg mRNA dose. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0046] FIG. 35. Transfection efficiency and viability data for the Md library at different a 0.25 gg mRNA dose. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0047] FIG. 36. Transfection efficiency and viability data for the Md library at different a 0.5 gg mRNA dose. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0048] FIG. 37. Transfection efficiency and viability data for the Md library at different a 0.75 gg mRNA dose. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0049] FIG. 38. Transfection efficiency and viability data for the Lg library at different a 0.25 gg mRNA dose. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0050] FIG. 39. Transfection efficiency and viability data for the Lg library at different a 0.5 gg mRNA dose. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0051] FIG. 40. Transfection efficiency and viability data for the Lg library at different a 0.75 gg mRNA dose. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0052] FIG. 41. Correlation between transfection efficiency / GFP MFI and binding strength.

[0053] FIG. 42. SHAP correlation plots for micelleplex size -TE vs cLogP vs Binding efficiency.

[0054] FIG. 43. SHAP correlation plots for micelleplex size -V vs cLogP vs Binding efficiency.

[0055] FIG. 44. SHAP correlation plots for micelleplex size -MFI vs cLogP vs Binding efficiency.

[0056] FIG. 45. GFP expression and viability trends in A549 cells. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0057] FIG. 46. GFP expression and viability trends in HDFn cells. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0058] FIG. 47. GFP expression and viability trends in HuH7 cells. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0059] FIG. 48. Percent Cy5 tagged mRNA internalization in HEK-293T cells at different time points. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0060] FIG. 49. Mean fluorescence intensity (MFI) of Cy5 tagged mRNA internalization in HEK-293T cells at different time points. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0061] FIG. 50. Cy5-tagged mRNA uptake upon incubation with clathrin as an endocytosis inhibitor. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0062] FIG. 51. Cy5-tagged mRNA uptake upon incubation with caveolae as an endocytosis inhibitor. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0063] FIG. 52. Cy5-tagged mRNA uptake upon incubation with micropinocytosis as an endocytosis inhibitor. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0064] FIG. 53. Correlation between GFP expression decreases upon BAF incubation and amine pKa.

[0065] FIG. 54. Percent Cy5 tagged mRNA internalization upon transfection in presence of serum supplemented media. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0066] FIG. 55. Mean fluorescence intensity (MFI) of Cy5 tagged mRNA internalization upon transfection in presence of serum supplemented media. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0067] FIG. 56. Heatmap showing the cumulative transfection efficiency (% of cells GFP positive), data of the full micelle library upon transfection in HEK293-T cells.

[0068] FIG. 57. Heatmap showing the cumulative viability (% of cells GFP positive), data of the full micelle library upon transfection in HEK293-T cells.

[0069] FIG. 58. Heatmap showing the cumulative effective efficiency (% of cells GFP positive), data of the full micelle library upon transfection in HEK293-T cells. Effective efficiency is the transfection efficiency multiplied by the viability.

[0070] FIG. 59. Heatmap showing the GFP mean fluorescence intensity data of the full micelle library upon transfection in HEK293-T cells.

[0071] FIG. 60. The percent decrease in Cy5 mean fluorescence intensity (MFI) for various micelleplexes when incubated with cells in the presence of specific endocytosis pathway inhibitors. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0072] FIG. 61. Buffering capacity of polymers having the A7 or A8 cationic groups.Percentage of cells having destabilized membranes after treatment with a micelleplex having a polymer with the A7 cationic group or a polymer having the A8 cationic group.

[0073] FIG. 62. mRNA release efficiency and Cy5 mean fluorescence intensity for various micelleplexes having a polymer with the A7 cationic group or a polymer having the A8 cationic group.

[0074] FIG. 63. Images of cells treated with a micelleplexes formed from a polymer having A7 cationic groups and mRNA, a polymer having A7 cationic groups and pDNA, a polymer having A8 cationic groups and mRNA, or a polymer having A8 cationic groups and pDNA.

[0075] FIG. 64. Percentage of GFP positive cells after cells were treated with various micelleplexes in the presence of serum during transfection (serum) or after transfection (OptiMEM). U=Untreated, N= Naked mRNA, and J = JetPEI.

[0076] FIG. 65. Viability after treatment with various micelleplexes in the presence of serum during transfection (serum) or after transfection (OptiMEM). U=Untreated, N= Naked mRNA, and J = JetPEI.

[0077] FIG. 66. Effective efficiency after cells were treated with various micelleplexes in the presence of serum during transfection (serum) or after transfection (OptiMEM). U=Untreated, N= Naked mRNA, and J = JetPEI.

[0078] FIG. 67. Transfection efficiency of micelleplex in Hep2g cells. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0079] FIG. 68. Viability of Hep2g cells after treatment with various micelleplexes. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0080] FIG. 69. Effective efficiency of micelleplex in Hep2g cells. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0081] FIG. 70. Transfection efficiency of micelleplex in RAW 264.7 cells. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0082] FIG. 71. Viability of RAW 264.7 cells after treatment with various micelleplexes. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0083] FIG. 72. Effective efficiency of micelleplex in RAW 264.7 cells. U=Untreated, N= Naked mRNA, and J = JetPEI.

[0084] FIG. 73. Chemical structure of the three differently charged amphiphiles used for mixed micelle formulations.

[0085] FIG. 74. Mixed micelle formulation strategy. The amphiphiles are dissolved in a good solvent at the required ratios, dried via N2 stream to form the polymer film, and subsequently rehydrated with DI water to form micelles.

[0086] FIG. 75. Amphiphile ratio and naming for the different ternary mixed micelles. (D) Naming convention for ternary mixed

[0087] FIG. 76. DLS Hydrodynamic radii of micelles in water, micelleplexes in water, and micelleplexes in OptiMEM.

[0088] FIG. 77. TEM images showing morphology of APC 0 / 0 homomicelle and APC 10 / 0 binary PEG containing micelle (spherical micelles).

[0089] FIG. 78. TEM images showing morphology of and APC 10 / 10 and APC 10 / 20 ternary mixed micelles (spherical micelles).

[0090] FIG. 79. FIG. 77. TEM images showing morphology of APC 10 / 30 micelles (wormlike micelles).

[0091] FIG. 80. RiboGreen dye exclusion studies heatmap representing micelle-mRNA relative binding efficiency in water, and micelle-mRNA relative binding efficiency in OptiMEM, relative micelle mRNA binding strength (obtained through heparin incubation).

[0092] FIG. 81. Percentage of live cells positive for GFP expression upon full library transfection in HEK293T cells. U=Untreated, N = Naked mRNA, J = JetPEI (positive control).

[0093] FIG. 82. Mean fluorescence intensity (MFI) of GFP positive cells for full library transfection in HEK 293T cells. U=Untreated, N = Naked mRNA, J = JetPEI (positive control).

[0094] FIG. 83. Live cell mages representing the change in levels of GFP expression upon incremental anionic amphiphile incorporation. U=Untreated, N = Naked mRNA, J = JetPEI (positive control).

[0095] FIG. 84. Internalization studies with MNPs containing Cy5 tagged mRNA shows that high performing MNPs display higher mRNA consumption over time.

[0096] FIG. 85. Relative Cy5 MFI (MFI normalized to APC 0 / 0 values) as measured at 4h and 24h post transfection in HEK293T cells.

[0097] FIG. 86. Chemical structure so the four different anions used to study the impact of anion pKa on delivery performance.

[0098] FIG. 87. GFP MFI of the ternary mixed micelles containing different anions upon transfection in HEK293T. U=Untreated, N = Naked mRNA, J = JetPEI (positive control)

[0099] FIG. 88. Scheme for inhibition of proton-sponge mechanism endosomal escape via incubation in Bafilomycin-Al.

[0100] FIG. 89. Plot showing drastic decrease in GFP expression upon BAF-A1 incubation demonstrating the reliance on high endosomal escape efficiency for high performing MNPs. U=Untreated, N = Naked mRNA, J = JetPEI (positive control)

[0101] FIG. 90. Relative viabilities of HEK293T cells upon transfection with the full ternary micelle library as determined via CCK-8 assay. U=Untreated, N = Naked mRNA, J = JetPEI (positive control).

[0102] FIG. 91. Effective efficiencies (transfection efficiency*viability) of the different ternary mixed MNPs. U=Untreated, N = Naked mRNA, J = JetPEI (positive control)

[0103] FIG. 92. Scheme for MNPs transfection under (i) normal serum conditions, and (ii) serum conditions but with OptiMEM maturation.

[0104] FIG. 93. Percentage of HEK293T cells positive for GFP expression under two different serum-based transfection conditions. U=Untreated, N = Naked mRNA, J = JetPEI (positive control).

[0105] FIG. 94. GFP MFI of GFP positive cells under two different serum-based transfection conditions. U=Untreated, N = Naked mRNA, J = JetPEI (positive control).

[0106] FIG. 95. In vivo average luciferase radiance obtained via an IVIS imager for Flue mRNA delivery with ternary mixed MNPs via IV tail vein injection (n=3).

[0107] FIG. 96. Thoracic vs splenic Flue radiance distribution obtained from whole animal imaging. Data does not representative of actual organ level biodistribution.

[0108] FIG. 97. In vivo organ level average luciferase radiance obtained via an IVIS imager for Flue mRNA delivery with ternary mixed MNPs via IV tail vein injection (n=3).

[0109] FIG. 98. In vivo average luciferase radiance obtained via an IVIS imager for Flue mRNA delivery with ternary mixed MNPs via IV tail vein injection (n=3).

[0110] FIG. 99. In vivo total luciferase radiance obtained via an IVIS imager for Flue mRNA delivery with ternary mixed MNPs via IV tail vein injection (n=3).

[0111] FIG. 100. Average thoracic Flue radiance distribution obtained from whole animal imaging. Data not representative of actual organ level biodistribution.

[0112] FIG. 101. Average splenic Flue radiance distribution obtained from whole animal imaging. Data not representative of actual organ level biodistribution.

[0113] FIG. 102. Micelleplex size (Dh ,nm ) characterization via DLS measurements.

[0114] FIG. 103. Scheme for general in vitro transfection. GFP expressing mRNA is used to prepare micelleplexes that are transfected in HEK293-T cells, followed by quantification of GFP positive cells via flow cytometry.

[0115] FIG. 104. In vitro transfection efficiency (serum free). Delivery efficiency SHAP analysis.

[0116] FIG. 105. In vitro GFP mean fluorescence intensity. GFP MFI SHAP analysis.

[0117] FIG. 106. Endosomal escape inhibition. HEK293-T cells are preincubated withBafilomycin-Al, which is a proton pump inhibitor. Reduction in GFP positive cells upon BAF treatment indicates a reliance of micelleplexes on endosomal acidification to escape the endosome. Poly-L-Lysine (P) was used as negative control and JetPEI as positive control.Student’ s unpaired t-test was used to determine statistical significance

[0118] FIG. 107. Luciferase luminescence and Cy5 tagged mRNA biodistribution are highly localized in the lungs for A7 MNPs.

[0119] FIG. 108. SHAP analysis representing polymer feature dependencies of in vivo luciferase activity.

[0120] FIG. 109. Radar plot representing the average SHAP values of input parameters corresponding to the biological output (in vivo luciferase activity).

[0121] FIG. 110. Plasma cytokine levels 6-hours post-injection of MCP-1 and IFNg. Data points represent average of 2 technical repli-cates for n= 3 animals per group. Horizontal dotted line represents lower limit of quantification. Any values plotted below this line are extrapolated from the standard curve. Two-way ANOVA and Sidak's multiple comparisons test with alpha = 0.05 were used to identify statistically significant differences between groups. U=Untreated, N= Naked mRNA, J = JetPEI.

[0122] FIG. 111. Plasma cytokine levels 6-hours post-injection of TNFa and IL-2. Data points represent average of 2 technical repli-cates for n= 3 animals per group. Horizontal dotted line represents lower limit of quantification. Any values plotted below this line are extrapolated from the standard curve. Two-way ANOVA and Sidak's multiple comparisons test with alpha = 0.05 were used to identify statistically significant differences between groups. U=Untreated, N= Naked mRNA, J = JetPEI.

[0123] FIG. 112. Plasma cytokine levels 6-hours post-injection of IL-4 and IL-6. Data points represent average of 2 technical repli-cates for n= 3 animals per group. Horizontal dotted line represents lower limit of quantification. Any values plotted below this line are extrapolated from the standard curve. Two-way ANOVA and Sidak's multiple comparisons test with alpha = 0.05 were used to identify statistically significant differences between groups. U=Untreated, N= Naked mRNA, J = JetPEI.

[0124] FIG. 113. Percentage of cells positive for Cy5 fluorescence and respective Cy5 MFI for Md series of micelles transfection in presence of serum (DMEM + 10% FBS) (t = 24h). U=Untreated, N= Naked mRNA, J = JetPEI.

[0125] FIG. 114. SHAP analysis representing polymer feature dependencies of in vitro transfection efficiency in presence of serum.

[0126] FIG. 115. Radar plot representing the average SHAP values of input parameters corresponding to the biological output (transfection efficiency- serum present).DETAILED DESCRIPTION

[0127] Nucleic acid-based medicines have many important applications ranging from disease treatment to vaccine development. Antisense oligonucleotides (ASOs) are one type of pharmaceutical paradigm emerging as an innovative medicine for many diseases including cancer and spinal muscular atrophy. Some ASOs function by binding to RNA, such as mRNA, via Watson-Crick base pairing to modulate gene expression. For example, binding of an ASO to RNA can induce one or more mechanisms to inhibit or prevent the translation of RNA into a protein, induce ribonuclease H mediated decay of RNA, and / or modulate the splicing of pre- mRNA. ASOs were one of the first genetic therapeutics on the market because new ASOs can be readily developed with knowledge of the targeted mRNA sequence. For example, MILASEN is an ASO therapy developed specifically for one patient suffering from Batten disease, a fatal genetic disease-causing loss of vision, seizures, and dementia. The patient’s genome was sequenced to identify the exact mutation thought to cause the disease, and a specific ASO was developed (MILASEN) that improved patient health.

[0128] Messenger RNA (mRNA) therapeutics is another nucleic acid-based therapeutic that is a versatile platform for the development of next-generation therapeutics. For example, mRNA- based vaccines were approved to treat COVID- 19. As a therapeutic, mRNA works independentlyof the native genome and does not need to be translocated into the nucleus to fulfdl its functions. This results in rapid expression of foreign mRNA in cells into functional protein of interest, with low risk of mutagenesis, making it a more desirable nucleic acid therapeutic.

[0129] Although nucleic acid-based therapies have the potential to change the way diseases are treated, the physiological environment makes nucleic acid-based therapy delivery challenging. A nucleic acid-based therapy has many cellular barriers to cross, such as the cell membrane and the endosome, which can be exceedingly difficult due to the relatively large size and anionic nature of nucleic acid-based therapies. Additionally, for in vivo applications, the nucleic acid-based therapies should avoid extracellular barriers such as nuclease degradation, renal clearance, protein sequestration, among others, to even arrive at the cellular barriers.

[0130] Delivery vehicles are commonly used to facilitate delivery of oligonucleotide payloads through the cell membrane. Delivery vehicles include viral vectors and lipid-based systems such as liposomal nanoparticles. Viral vectors are expensive and a potentially immunogenic gene delivery system. While more economically tractable than viral vector systems, lipid-base systems face stability issues.

[0131] Polymer-based delivery systems are being explored as oligonucleotide payload delivery systems. Polymer-based delivery systems may have improved stability over lipid-based delivery systems because polymers are large molecules that are less dynamic, whereas lipid- based systems are complex and involve dynamic assemblies of many lipids with semi-stable structures. Cationic polymers bind to anionic oligonucleotides to form complexes such as polyplexes, which can facilitate delivery of many nucleic acid types. Synthesis of cationic polymers is modular, easy to scale up, and inexpensive. Cationic polymers bind to oligonucleotides through interpolyelectrolyte complexation driven by entropy, forming complexes. Linear cationic polymers such as poly(ethyleneimine) (PEI), poly(dimethylaminoethyl methacrylate) (PDMAEMA), poly( / ?-amino ester)s (PBAEs), and poly(amine-co-ester)s (PACEs), are the simplest polycation motifs and are the most explored as polymeric delivery platforms for oligonucleotides. However, these systems are typically colloidally unstable and have inefficient oligonucleotide loading. While many cationic polymer structures and architectures have been studied, cationic polymers are challenging to translate for clinical use. For example, serum instability of formulations is a challenge for nonviral formulations. Serum, a component of blood that includes many types of proteins, can causeaggregation, can cause burst release of oligonucleotide payloads, and / or can bind nonspecifically to delivery systems, causing issues with both in vitro and in vivo delivery. Methods of improving serum stability include incorporating hydrophilic sheath layers such as polyethylene glycol, carbohydrates, or zwitterionic moieties. However, these methods of improving serum stability can decrease interactions of the cationic polymer with the oligonucleotide payload and / or the interactions between the complex and a cell, both of which can result in decreased efficacy and / or efficiency of such cationic polymer delivery systems.

[0132] In one aspect, the present disclosure describes polymers. In one or more embodiments, a polymer includes cationic groups. A cationic group can be a part of a repeating unit. A polymer that includes cationic groups can be described as a cationic polymer, such as, for example, a cationic diblock polymer. In one or more embodiments, a polymer includes anionic groups. An anionic group can be a part of a repeating unit. A polymer that includes anionic groups can be described as an anionic polymer, such as, for example, an anionic diblock polymer. In one or more embodiments, a polymer does not include cationic or anionic groups in a repeating unit. A polymer that does not include cationic or anionic groups in a repeating unit can be described as a neutral polymer, such as, for example a neutral diblock polymer.

[0133] In another aspect, the present disclosure describes complexes that include one or more polymers of the present disclosure and a cargo. In one or more embodiments, a complex includes a polymer that has cationic groups, a polymer that has anionic groups, a neutral polymer, or any combination thereof. For example, in one or more embodiments, a complex includes a polymer having cationic groups. In one or more embodiments, a complex includes a polymer having anionic groups. In one or more embodiments, a complex includes a polymer having cationic groups and a neutral polymer. In one or more embodiments, a complex includes a polymer having cationic groups, a polymer having anionic groups, and a neutral polymer.

[0134] In another aspect, the present disclosure describes a composition that includes one or more of the complexes of the present disclosure.

[0135] In yet another aspect, the present disclosure describes methods of using a complex and / or a composition of the present disclosure. The complex and / or composition may be used as or in a delivery system for an oligonucleotide cargo. The delivery system may be used for in vitro and / or in vivo delivery of an oligonucleotide cargo. The complex may be a transfectioncomplex. A transfection complex is a complex used as or in a delivery system to deliver an oligonucleotide cargo to at least the cytosol of a cell.

[0136] A complex of the present disclosure includes an oligonucleotide cargo. As used herein, the terms “oligonucleotide” and “nucleic acid” are used interchangeably and refer to a polymer of two or more nucleotides. As use herein, the term “oligonucleotide cargo” refers to one or more oligonucleotides that are a part of a complex of the present disclosure. An oligonucleotide cargo may include a plurality of oligonucleotides. A plurality of oligonucleotides may include the same sequence or two or more different sequences.

[0137] An oligonucleotide of an oligonucleotide cargo may be single stranded, double stranded, or include one or more portions that are single stranded and one or more portions that are double stranded. Oligonucleotides include nucleosides linked through internucleoside linkages, also called backbone linkages. Nucleosides include a pentose sugar (e g., ribose or deoxyribose) and a nitrogenous base (nucleobase or base) covalently attached to the sugar. The canonical nucleobases found in DNA and / or RNA are adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). The canonical sugars found in DNA and / or RNA are deoxyribose (DNA) and ribose (RNA). A conical nucleoside linkage (also termed backbone linkage) is a phosphodiester bond. Oligonucleotide cargos may include canonical sugars, canonical bases, canonical internucleoside linkages, non-canonical sugars, non-canonical bases, non-canonical internucleoside linkages, or any combination thereof. Examples of non-canonical sugars include 2' modifications on a pentose or ribose such as 2'-O-methoxyethyl (2'0-M0E) and 2' methoxy (2’0-Me), locked sugars (also known as locked nucleic acids), and sugar alternatives such as methylenemorpholine rings. Examples of noncanonical backbone linkages include phosphorothioate linkages and phosphorodiamidate linkages. In some cases, a non-canonical sugar can be combined with a non-canonical backbone linkage, such as, for example in a phosphorodiamidate morpholino oligomer.

[0138] Examples of oligonucleotide cargos include, but are not limited to, plasmid DNA (pDNA), messenger RNA (mRNA), antisense oligonucleotides (ASOs), small interfering RNA (siRNA), micro-RNA (miRNA), guide RNA (gRNA), Cas9-gRNa complexes, aptamers, derivatives thereof, and combinations thereof.

[0139] In one or more embodiments, an oligonucleotide cargo includes an antisense oligonucleotide (ASO). An ASO is a single stranded oligonucleotide that is at least partiallycomplementary to a target nucleic acid. ASOs are generally single stranded oligonucleotides; however, in the complexes of the present disclosure, an ASO may exists as single-stranded oligonucleotide, a double stranded oligonucleotide, or have one or more single stranded regions and one or more double stranded regions. ASOs can be linear. ASOs can lack secondary structure. ASOs may include one or more non-canonical bases, one or more non-canonical sugars, one or more non-canonical intemucleoside linkages, or any combination thereof.

[0140] In one or more embodiments, the oligonucleotide cargo includes mRNA. mRNA is a single-stranded RNA molecule encoding at least a portion of a polypeptide. Once delivered to a cell, mRNA can be translated into at least a portion of a polypeptide. In one or more embodiments, an mRNA cargo can encode a gene editing protein of a gene editing system, such as a Cas9 system. In one or more embodiments, an mRNA cargo can encode a portion of protein configured to elicit an immune response in the host. In one or more embodiments, an mRNA cargo can be a part of a vaccine.

[0141] In one or more embodiments, an oligonucleotide cargo is not self-replicating. In one or more embodiments, an oligonucleotide cargo is not methylated; that is, any cytosine present in the oligonucleotide sequence is not a methylated cytosine. In one or more embodiments, an oligonucleotide cargo does not encode a gene product.

[0142] In one or more embodiments, an oligonucleotide cargo is self-replicating. In one or more embodiments, an oligonucleotide cargo is methylated; that is, one or more of the cytosines present in the oligonucleotide sequence may be a methylated cytosine. In one or more embodiments, an oligonucleotide cargo encodes a gene product.

[0143] An oligonucleotide cargo may have a variety of lengths. In one or more embodiments, the number of bases in an oligonucleotide cargo is 2 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 50 or more, 75 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 2000 or more, 3000 or more, 4000 or more, 5000 or more, 6000 or more, 7000 or more, 8000 or more, 9000 or more, 10000 or more, 20000 or more, 30000 or more, or 40000 or more.. In one or more embodiments, the number of bases in an oligonucleotide cargo is 50000 or less, 40000 or less, 30000 or less, 40000 or less, 30000 or less, 20000 or less, 10000 or less, 9000 or less, 8000 or less, 7000 or less, 6000 or less, 5000 or less, 4000 or less, 3000 or less, 2000 or less, 1000 or less, 900 or less, 800 or less, 700 or less, 600 orless, 500 or less, 400 or less, 300 or less, 200 or less, 100 or less, 75 or less, 50 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 15 or less, 10 or less, or 5 or less. The number of bases in an oligonucleotide cargo also can be characterized by a range having endpoints defined by any minimum the number of bases in an oligonucleotide cargo and any maximum the number of bases in an oligonucleotide cargo that is greater than the selected minimum number of bases in an oligonucleotide cargo.

[0144] In one or more embodiments, the number of bases in an oligonucleotide cargo is 5 to 1000. In one or more embodiments, the oligonucleotide cargo is an ASO and the number of bases in the ASO is 5 to 1000, 5 to 75, 5 to 50, 5 to 30, 10 to 50, 10 to 40, 10 to 13, 15 to 50, 15 to 40, 15 to 30, 15 to 25, 20 to 50, 20 to 40, or 20 to 30.

[0145] In one or more embodiments, the number of bases in an oligonucleotide cargo is 25 to 10000. In one or more embodiments, an oligonucleotide cargo is an mRNA and the number of bases in the mRNA is 25 to 10000, 50 to 5000, or 100 to 4000.

[0146] The sequence of an oligonucleotide cargo depends at least in part on the type of therapy. For example, in one or more embodiments, the sequence of an mRNA cargo can be designed to encode a polypeptide that can have a therapeutic effect. Designing an mRNA cargo can be a multistep process. The process may include identifying a polypeptide of interest, analyzing the mRNA sequence encoding the polypeptide of interest, and designing an mRNA cargo sequence encoding the polypeptide of interest.

[0147] In one or more embodiments where an oligonucleotide cargo is designed to hybridize or target a nucleic acid, designing an oligonucleotide cargo may be a multistep process. The process may include identifying a nucleic acid of interest, analyzing a transcript of the nucleic acid of interest, and identifying a particular region of the nucleic acid of interest to be targeted by the oligonucleotide. A nucleic acid of interest may be a pre-mRNA transcript, mRNA transcript, or a gene associated with a disease or disorder. Methods for designing, synthesizing, and screening oligonucleotide cargos are known.

[0148] The present disclosure describes polymers. One or more polymers of the present disclosure can be included in a complex of the present disclosure. Polymers are polymerized from one or more monomers. A polymer, or a portion of a polymer such as a block of a polymer, polymerized from a particular monomer may be described as “monomer name” polymer or poly(“monomer name”). For example, a polymer polymerized from n-butyl acrylate monomers,may be described as an n-butyl acrylate polymer or poly(n-butyl acrylate). Polymers functionalized post polymerization can be described from the identity of the repeating unit in the final polymer. Alternatively, polymers functionalized post-polymerization can be described from the perspective of the monomer that would have been used to form the post-functionalized repeating unit if the monomer was directly polymerized.

[0149] In one or more embodiments, a polymer of the present disclosure is a homopolymer or includes a homopolymer block. A homopolymer or a homopolymer block is a polymer polymerized from a single monomer. A homopolymer or homopolymer block formed from a single monomer can be functionalized post-polymerization to change the molecular composition of the repeating groups.

[0150] In one or more embodiments, a polymer of the present disclosure is a block polymer. A block polymer is a polymer that includes two or more polymer segments (blocks) joined by a covalent linkage. A block polymer may include two or more homopolymer blocks; two or more copolymer blocks; or one or more homopolymer blocks and one or more copolymer blocks that are joined by a covalent linkage. Block polymers can be synthesized by polymerizing the first block and then polymerizing the second block directly from one of the terminal groups of the first block.

[0151] In one or more embodiments, a polymer of the present disclosure is a diblock polymer. A diblock polymer is a polymer that includes two homopolymer blocks joined by a covalent linkage.Cationic Polymers

[0152] In one or more embodiments, polymers of the present disclosure include cationic groups. Such polymers can be described, for example, as cationic polymers. A cationic group can be a part of a repeat unit. The terms cationic group, cation group, and cation can refer to a chemical functionality that is a cation or is ionizable to a cation when exposed to various pHs. For example, when exposed to conditions having a pH lower than the pKa of the cationic group, the cationic group can be protonated resulting in a cation. For example, in one or more embodiments, a cationic group can be positively charged at conditions having a pH of 7.5 or lower. For example, a cationic group may be protonated at physiological pH (e.g., 7.0 -7.4) and / or at an acidic pH, such as the pH observed in an endosome (e g., pH 3 to 6.5). Cationic groups may be a part of a monomer used to form a polymer. For example, a monomer mayinclude a pendant cationic group that is not chemically modified during polymerization. Cationic groups may be appended to a polymer post polymerization. A cationic group can be a part of a repeat unit and such repeating units can be described as cationic repeating units.

[0153] In one or more embodiments, a cationic group includes an amine. As used herein, the term “amine” includes primary amines, secondary amines, tertiary amines, and quaternary ammonium salts. In one or more embodiments, a cationic group includes an amine that is a part of a ring.

[0154] In one or more embodiments, a cationic group includes a guanidine or a guanidinium group. Guanidine and guanidine group refer to the group -N=C(NR1R2)(NR3NR4) where each R (e.g., R1, R2, R3, and R4) are independently H or alkyl. A guanidinium group is a charged form of a guanidine group.

[0155] In one or more embodiments, a cationic polymer includes an acrylamide polymer block, the acrylamide polymer block including cationic groups. An acrylamide polymer (or block) includes the repeating group -CH2=CH-(C(=O)NHR) where R can include a cationic group. R can include a linker that separates an cationic group (when the acrylamide polymer of block includes cationic groups) from the amide of the acrylamide functionality. The linker can be a hydrocarbon linker. The hydrocarbon linker can include -(CH2)n- where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of acrylamide polymers or blocks of polymers that include cationic groups include, but are not limited to, amino ethyl acrylamide (AEA, AEAm), dimethyl amine ethyl acrylamide (DMA, DMAm), diethyl amine ethyl acrylamide (DEA, DEAm), trimethyl amine ethyl acrylamide (TMA, TMAm), morpholino ethyl acrylamide (MEA, MEAm), tri methyl ammonium ethylamine acrylamide (TMAEA, TMAEAm), and (1 -imidazolyl )propyl acrylamide (ImPAm).

[0156] In one or more embodiments, a cationic polymer of the present disclosure is an acrylamide polymer or includes an acrylamide block having the repeating group of Formula I:where RAincludes a cationic group, j’ is an integer from 0 to 5, and n is the number of repeating groups.

[0157] In Formula I, j’ is an integer from 0 to 5. For example, ]’ can be 0, 1, 2, 3, 4, or 5. In one or more embodiments, j ’ is 1. In one or more embodiments, j ’ is 2. In one or more embodiments, ]’ is 3. In one or more embodiments, ]’ is 4. In one or more embodiments, ]’ is 5.

[0158] RAincludes a cationic group. RAcan include at least two heteroatoms. One or more of the heteroatoms may be a part of a cationic group. For example, in one or more embodiments, RAincludes two cationic groups. In one or more embodiments, two or more heteroatoms make up the same cationic group. For example, a guanidinium group includes three nitrogen atoms. In one or more embodiments, RAincludes one cationic group and one or more additional heteroatoms that are not a part of a cationic group. In one or more embodiments, RAincludes at least one primary amine or at least one secondary amine.

[0159] In one or more embodiments where a polymer or block includes the repeating group of Formula I, RAcan be of Formula (i), (ii), (iii), or (iv):where b is an integer from 0 to 5, z is an integer from 0 to 5, z’ is an integer from 0 to 5, Q1is a heteroatom or a group that includes a heteroatom, Q2is a heteroatom or a group that includes a heteroatom.

[0160] In each of Formulas (i) to (iv), b can be 1, 2, 3, 4, or 5. In one or more embodiments, b is 1. In one or more embodiments b is 2. In one or more embodiments b is 3. In one or more embodiments b is 4. In one or more embodiments, b is 5. In one or more embodiments, RAis of Formula (i) and b is 2. In one or more embodiments, RAis of Formula (ii), and b is 2. In one or more embodiments, R is of Formula (ii), and b is 2.

[0161] Q1and Q2are each independently a heteroatom or a group that includes a heteroatom In one or more embodiments Q1and Q2are each independently N, NH, N b, NH3, O, or OH. In one or more embodiments Q1and Q2are the same or include the same heteroatom. For example,in one or more embodiments, RAis of Formula (i) and Q1is N and Q2is NH. In one or more embodiments, RAis of Formula (ii) and Q1is NH and Q2is NH3. In one or more embodiments, RAis of Formula (iv) and Q1is NH3 and Q2is NH3. In one or more embodiments, Q1and Q2are different. For example, in one or more embodiments RAis of Formula (i) and Q1is N and Q2is O. In one or more embodiments RAis of Formula (ii) and Q1is NH and Q2is OH. In one or more embodiments RAis of Formula (ii) and Q1is O and Q2is NH3. In one or more embodiments RAis of Formula (iv) and Q1is NH3 and Q2is OH.

[0162] In Formula (iv), z and z’ are each independently 1, 2, 3, 4, or 5. In one or more embodiments, z and z’ are the same. For example, in one or more embodiments, both z and z’ are 1. In one or more embodiments, both z and z’ are 2. In one or more embodiments, both z and z’ are 3. In one or more embodiments, both z and z’ are 4. In one or more embodiments, or both z and z’ are 5. In other embodiments, z and z’ are not the same. For example, in one or more embodiments, z is 1 and z’ is 2. In one or more embodiments, z is 1 and z’ is 3. In one or more embodiments, z is 1 and z’ 4. In one or more embodiments, z is 1 and z’ is 4. In one or more embodiments, z is 2 and z’ is 3. In one or more embodiments, z is 2 and z’ is 4. In one or more embodiments, z is 2 and z’ is 5. In one or more embodiments, z is 3 and z’ is 4. In one or more embodiments, z is 3 and z’ is 5. In one or more embodiments, or z is 4 and z’ is 5.

[0163] In some embodiments where a polymer or polymer block includes the repeating group of Formula I, RAcan be of Formula (A6), (A7), (A8), (A9), or (A10):

[0164] In one or more embodiments where a cationic polymer or a block of a cationic polymer includes the repeating group of Formula I, RAis of Formula (A6). In one or more embodiments where a cationic polymer or a block of a cationic polymer includes the repeatinggroup of Formula I, RAis of Formula (A7). In one or more embodiments where a cationic polymer or a block of a cationic polymer includes the repeating group of Formula I, RAis of Formula (A8). In one or more embodiments where a cationic polymer or a block of a cationic polymer includes the repeating group of Formula I, RAis of Formula (A9). In one or more embodiments where a cationic polymer or a block of a cationic polymer includes the repeating group of Formula I, RAis of Formula (A 10).

[0165] An acrylamide polymer or block can be formed from acrylamide monomers. An acrylamide polymer or block can be formed by reacting a polymer having a carboxylic acid or an ester with a compound that includes an amine capable of forming an amide bond. For example, an acrylamide polymer or block can be formed by modifying an acrylate polymer. Acrylate monomers may be polymerized to form an acrylate polymer having the acrylate functionality repeating groupthe ester is a labile ester. A compound having an amine capable of reacting with the ester of the acrylate functionality can react with the ester to displace Rxand form an amide bond thereby forming an acrylamide functionality. Rxcan be a group that allows for the ester to more easily react with an amine to form an amide. An example Rxgroup is pentafluorophenyl. An acrylamide polymer or segment formed this way may include residual acrylate functionalities that were not converted to acrylamide functionalities; that is, the polymer may include residualgroups. An acrylamide polymer, acrylamide or acrylamide block may refer to a polymer or polymer block that includes 80 mole percent (mol-%) or greater, 85 mol-% or greater, 90 mol-% or greater, 95 mol-% or greater, or 99 mol-% or greater acrylamide functionalities.

[0166] In one or embodiments, a polymer of the present disclosure is a diblock polymer where a block includes cationic groups. Such polymers can be described, for example, as cationic diblock polymers. A cationic diblock polymer may be amphiphilic; that is, the diblockpolymer can include a portion that is hydrophobic and a portion that is hydrophilic. For example, a cationic diblock polymer may include a homopolymer (block or segment) of hydrophobic repeating units and another homopolymer (block or segment) of hydrophilic repeating units. Hydrophilic repeating units can include cationic containing groups. A hydrophobic portion (block or segment) of a polymer may allow for micelle formation. Micelles may have a hydrophobic core and a hydrophilic surface. An oligonucleotide cargo can complex with a hydrophilic surface of the micelle.

[0167] In one or more embodiments, a polymer of the present disclosure is a cationic diblock polymer .A diblock polymer includes a first block and a second block. A first block of the diblock polymer can includes a first homopolymer block and a second block of the diblock polymer can include a second homopolymer block. The first homopolymer block of a cationic diblock polymer may include cationic groups. In one or more embodiments, a first homopolymer block of a cationic diblock polymer includes an acrylamide polymer block where the acrylamide polymer block includes cationic groups (e.g., a acrylamide block of the formula I).

[0168] A second homopolymer block of a cationic diblock polymer may be a polymer block that allows for the formation of micelles that include a diblock polymer (described herein). A second homopolymer block of a cationic diblock polymer may include a hydrophobic polymer block. In one or more embodiments, a second homopolymer block of a cationic diblock polymer includes a hydrophobic group. A hydrophobic group may be a part of the repeating unit. A hydrophobic group may be alkyl. An alkyl may be linear, branched, or cyclic. An alkyl may include 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Examples of hydrophobic alkyl groups include isopropyl, n-propyl, iso-butyl, n-butyl, and sec-butyl.

[0169] A second homopolymer block of a cationic diblock polymer may be an acrylate polymer block or an acrylamide polymer block. As such, a second homopolymer block may include a repeating acrylamide functionalityrepeating acrylatefunctionalityhydrophobic group described herein. Examples of acrylate polymers and polymer blocks include isopropyl acrylate; n-butyl acrylate; and sec-butyl acrylate. Examples of acrylamide polymers and polymer blocks include isopropyl acrylamide, n- butyl acrylamide, and sec-butyl acrylamide.

[0170] In one or more embodiments, a cationic diblock polymer is a is of Formula II:where j is an integer from 0 to 10; m is the number of acrylate repeating groups; n is the number of acrylamide repeating groups, the acrylamide repeating group being the cationic repeating group; and RAandj’ are as described herein.

[0171] j is an integer from 0 to 10. For example, j can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one or more embodiments] is 3, 4, or 5. In one or more embodiments] is 3. In one or more embodiments, j is 4. In one or more embodiments] is 5.

[0172] In one or more embodiments where a polymer is a diblock polymer having cationic groups, the diblock polymer may be of the formula [hydrophobic repeating uni t]m- [cationic repeating unit]nwere m and n describe the number or repeating groups in each block, m and n are each independently an integer from 1 to 1000. In one or more embodiments, m and z are each independently 1 or greater, 10 or greater, 25 or greater, 50 or greater, 75 or greater, 90 or greater, 100 or greater, 110 or greater, 120 or greater, 130 or greater, 140 or greater, 150 or greater, 160 or greater, 170 or greater, 180 or greater, 190 or greater, 200 or greater, 210 or greater, 220 or greater, 230 or greater, 340 or greater, 250 or greater, 260 or greater, 270 or greater, 280 or greater, 290 or greater, 300 or greater, 325 or greater, 350 or greater, 375 or greater, 400 orgreater, 425 or greater, 450 or greater, 475 or greater, 500 or greater, 600 or greater, 700 or greater, 800 or greater, or 900 or greater. In one or more embodiments, m and n are each independently 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 475 or less, 450 or less, 425 or less, 400 or less, 375 or less, 350 or less, 325 or less, 300 or less, 290 or less, 280 or less, 270 or less, 260 or less, 250 or less, 240 or less, 230 or less, 220 or less, 210 or less, 200 or less, 190 or less, 180 or less, 170 or less, 160 or less, 150 or less, 140 or less, 130 or less, 120 or less, 110 or less, 100 or less, 90 or less, 75 or less, 50 or less, 25 or less, or 10 or less.In one or more embodiments, m is 50 to 150 and n is 50 to 200. In one or more embodiments, m is 75 to 125 and n is 125 to 175. In one or more embodiments m is 90 to 120 and z is 90 to 120. In one or more embodiments, m is 90 to 120 and z is 140 to 160. In one or more embodiments, m is 90 to 120 and n is 190 to 210.

[0173] The ratio of hydrophobic repeating units to cationic repeating units may vary. For example, for cationic diblock polymer represented by the formula [hydrophobic repeating unit]m- [cationic repeating unit]n, the polymer may include 0.1 to 2 hydrophobic repeating units for every 1 cationic repeating unit.

[0174] In one or more embodiments, a polymer (e.g., a diblock polymer) can include more cationic repeating units than hydrophobic repeating units. For example, in one or more embodiments, a polymer includes 0.1 to 0.99 hydrophobic repeating units for every 1 cationic repeating unit. In one or more embodiments, a polymer includes 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, or 0.9 or more hydrophobic repeating units for every 1 cationic repeating unit. In one or more embodiments, a polymer includes 0.99 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, or 0.2 or less, hydrophobic repeating units for every 1 cationic repeating unit.

[0175] In one or more embodiments, a cationic diblock polymer includes fewer cationic repeating units than hydrophobic repeating units. In one or more embodiments, a cationic diblock polymer includes 1 or more, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, or 1.9 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or more hydrophobic repeating units for every 1 cationic repeating unit. In one or more embodiments, a cationic diblock polymer includes 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1.9 or less, 1.8or less, 1 .7 or less, 1 .6 or less, 1 .5 or less, 1 .4 or less, 1 .3 or less, 1 .2 or less, or 1 .1 or less hydrophobic repeating units for every 1 cationic repeating unit.

[0176] The amount of the cationic repeating unit in a cationic diblock polymer may vary. The mole percent (mol-%) of cationic repeating units in a polymer may be measured and calculated usingNMR via methods known in the art. In one or more embodiments, the amount of cationic repeating units in a polymer is 1 mol-% or greater, 5 mol-% or greater, 10 mol-% or greater, 15 mol-% or greater, 20 mol-% or greater, 25 mol-% or greater, 30 mol-% or greater, 60 mol-% or greater, 80 mol-% or greater. In one or more embodiments, the amount of cationic repeating units in a polymer is 100 mol-% or less, 80 mol-% or less, 60 mol-% or less, 30 mol-% or less, 25 mol-% or less, 20 mol-% or less, or 15 mol-% or less, 10 mol-% or less, or 5 mol-% or less. In one or more embodiments, the amount of cationic repeating units in a polymer is 1 mol-% to 100 mol-%, 1 mol-% to 80 mol-%, 1 mol-% to 60 mol-%, 1 mol-% to 30 mol-%, 1 mol-% to 25 mol-%, 1 mol-% to 20 mol-%, 1 mol-% to 15 mol-%, 1 mol-% to 10 mol- %, or 1 mol-% to 5 mol-%. In one or more embodiments, the amount of cationic repeating units in a polymer is 5 mol-% to 100 mol-%, 5 mol-% to 80 mol-%, 5 mol-% to 60 mol-%, 5 mol-% to 30 mol-%, 5 mol-% to 25 mol-%, 5 mol-% to 20 mol-%, 5 mol-% to 15 mol-%, or 5 mol-% to 10 mol-%. In one or more embodiments, the amount of cationic repeating units in a polymer is 10 mol-% to 100 mol-%, 10 mol-% to 80 mol-%, 10 mol-% to 60 mol-%, 10 mol-% to 30 mol-%, 10 mol-% to 25 mol-%, 10 mol-% to 20 mol-%, or 10 mol-% to 15 mol-%. In one or more embodiments, the amount of cationic repeating units in a polymer is 15 mol-% to 100 mol-%, 15 mol-% to 80 mol-%, 15 mol-% to 60 mol-%, 15 mol-% to 30 mol-%, 15 mol-% to 25 mol-%, or 15 mol-% to 20 mol-%. In one or more embodiments, the amount of cationic repeating units in a polymer is 20 mol-% to 100 mol-%, 20 mol-% to 80 mol-%, 20 mol-% to 60 mol-%, 20 mol-% to 30 mol-%, or 20 mol-% to 25 mol-%. In one or more embodiments, the amount of cationic repeating units in the polymer is 25 mol-% to 100 mol-%, 25 mol-% to 80 mol-%, 25 mol-% to 60 mol-%, or 25 mol-% to 30 mol-%. In one or more embodiments, the amount of cationic repeating units in a polymer is 30 mol-% to 100 mol-%, 30 mol-% to 80 mol-%, or 30 mol-% to 60 mol-%. In one or more embodiments, the amount of cationic repeating units in a polymer is 60 mol-% to 100 mol-% or 60 mol-% to 80 mol-%. In one or more embodiments, the amount of cationic repeating units in a polymer is 80 mol-% to 100 mol-%. In one or more embodiments,the amount of cationic repeating units in a polymer is 15 mol-% to 60 mol-%. In one or more embodiments, the amount of cationic repeating units in a polymer is 15 mol-% to 30 mol-%.

[0177] The acid dissociation constant (pKa) of a polymer that includes cationic groups may vary. The pKa of a polymer may affect the ability of the polymer to form complexes with an oligonucleotide cargo and / or the ability to release the oligonucleotide cargo from a complex. The pKa of a polymer may be determined using a potentiometric titration. In one or more embodiments, the pKa of a polymer is 5.0 or greater, 5.5 or greater, 6.0 or greater, 6.1 or greater, 6.2 or greater, 6.3 or greater, 6.4 or greater, 6.5 or greater, 6.6 or greater, 6.7 or greater, 6.8 or greater, 6.9 or greater, 7.0 or greater, 7.2 or greater, 7.5 or greater, 8.0 or greater, 8.5 or greater, 9.0 or greater, 10.0 or greater, 11.0 or greater, or 12.0 or greater. In one or more embodiments, the pKa of a polymer is 12.5 or less, 12.0 or less, 11.0 or less, 10.0 or less, 9.0 or less, 8.5 or less, 8.0 or less, 7.5 or less, 7.2 or less, 7.0 or less, 6.9 or less, 6.8 or less, 6.7 or less, 6.6 or less, 6.5 or less, 6.4 or less, 6.3 or less, 6.2 or less, 6.1 or less, 6.0 or less, or 5.5 or less. In one or more embodiments, a polymer has two pKa values. In some such embodiments, each of the two pKa values can be any pKa values described herein. For example, in one or more embodiment a polymer has two pKa values where the first pKa value is 5.5 to 6.0 and the second pKa value is 8.0 to 8.5.Anionic Polymers

[0178] In one or more embodiments, polymers of the present disclosure include anionic groups. A polymer that includes anionic groups can be described as an anionic polymer. The terms “anionic group,” “anion group,” and “anion” can refer to a chemical functionality is an anion or is ionizable to an anion when exposed to various pH values. For example, when exposed to conditions greater than the pKa of the anionic group, the anionic group can be deprotonated resulting in anion. In one or more embodiments, an anionic groups includes a carboxylic acid or a phosphoric acid ester. Anionic groups may be a part of a monomer used to form a polymer. For example, a monomer may include a pendant anionic group that is not chemically modified during polymerization. Anionic groups may be appended to a polymer post polymerization. An anionic group can be a part of a repeat unit and such repeat units can be described as anionic repeating units.

[0179] In one or more embodiments, an anionic polymer includes an acrylate polymer block, the acrylate polymer block including anionic groups. An acrylate polymer (or block) includes therepeating group -CH2=CH-(C(=O)O-RB) where RBcan include an anionic group or H. RBcan include a linker that separates an anionic group (when the acrylate polymer of block includes anionic groups) from the ester of the acrylate functionality. The linker can be a hydrocarbon linker. The hydrocarbon linker can include -(CH2)n- where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of acrylate polymers or blocks of polymers that include anionic groups include, but are not limited to, 2-carboxyethyl acrylate, succinic ethyl acrylate, benzyl acid ethyl acrylate, acrylic acid, and phosphate ethyl acrylate.

[0180] In one or more embodiments, an anionic polymer of the present disclosure is an acrylate polymer or includes an acrylate block having the repeating group of Formula I:where RBis H or includes an anionic group, j ” is an integer from 0 to 5, and q is the number of repeating groups having an anionic group.

[0181] In Formula V, j” is an integer from 0 to 5. For example, ]” can be 0, 1, 2, 3, 4, or 5. In one or more embodiments, j” is 1. In one or more embodiments, j ” is 2. In one or more embodiments, j ” is 3. In one or more embodiments, j” is 4. In one or more embodiments, ]’ is 5.

[0182] In one or more embodiments, RBincludes an anionic group. In one or more embodiments, RBincludes a carboxylic acid. In one or more embodiments RBincludes a benzyl acid. In one or more embodiments RBincludes a phosphoric acid ester. RBcan include atoms in addition to the anionic group.

[0183] In some embodiments where a polymer or polymer block includes the repeating group of Formula V, RBcan be of Formula (Bl), (B2), (B3), or (B4):

[0184] In one or more embodiments where an anionic polymer or block of an anionic polymer includes the repeating group of Formula V, RBis of Formula (Bl). In one or more embodiments where an anionic polymer or block of an anionic polymer includes the repeating group of Formula V, RBis of Formula (B2). In one or more embodiments where an anionic polymer or a block of an anionic polymer includes the repeating group of Formula V, RBis of Formula (B3). In one or more embodiments where an anionic polymer or block of an anionic polymer includes the repeating group of Formula V, RBis of Formula (B4).

[0185] Acrylate polymer, acrylate, or acrylate block may refer to a polymer or polymer block that includes 80 mole percent (mol-%) or greater, 85 mol-% or greater, 90 mol-% or greater, 95 mol-% or greater, or 99 mol-% or greater acrylate functionalities.

[0186] In one or embodiments, an anionic polymer of the present disclosure is a diblock polymer where a block includes anionic groups. Such polymers can be described as anionic diblock polymer. An anionic diblock polymer may be amphiphilic; that is, the diblock polymer can include a portion that is hydrophobic and a portion that is hydrophilic. In one or more embodiments, the hydrophilic portion includes anionic groups.

[0187] In one or more embodiments, an anionic polymer of the present disclosure is a diblock. In one or more embodiments, a first homopolymer block of an anionic diblock polymer includes anionic groups. In one or more embodiments, a first homopolymer block of an anionic diblock polymer includes an acrylate polymer block where the acrylate polymer block includes anionic groups (e.g., an acrylate block of the formula V).

[0188] A second homopolymer block of an anionic diblock polymer may be a polymer block that allows for the formation of micelles that include a diblock polymer (described herein). A second homopolymer block of an anionic diblock polymer may include a hydrophobic polymer block. In one or more embodiments, a second homopolymer block of an anionic diblock polymer includes a hydrophobic group. A hydrophobic group may be a part of the repeating unit. A hydrophobic group may be alkyl. An alkyl may be linear, branched, or cyclic. An alkyl may include 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Examples of hydrophobic alkyl groups include isopropyl, n-propyl, iso-butyl, n-butyl, and sec-butyl.

[0189] A second homopolymer block of an anionic diblock polymer may be an acrylate polymer block or an acrylamide polymer block. As such, a second homopolymer block may include a repeating acrylamide functionalityrepeating acrylate functionalitythe is a hydrophobic group described herein. Examples of acrylate polymers and polymer blocks include isopropyl acrylate; n-butyl acrylate; and secbutyl acrylate. Examples of acrylamide polymers and polymer blocks include isopropyl acrylamide; n-butyl acrylamide; and sec-butyl acrylamide.

[0190] In one or more embodiments, an anionic polymer is an anionic diblock polymer of Formula VI:or an ionized form thereo where j is an integer from 0 to 10; q is the number of acrylate repeating groups lacking the RBgroup; z is the number of anionic repeating groups, RBis any RDgroups disclosed herein, and j’ is disclosed herein.

[0191] j is an integer from 0 to 10. For example, j can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one or more embodiments] is 3, 4, or 5. In one or more embodiments] is 3. In one or more embodiments, j is 4. In one or more embodiments] is 5.

[0192] In one or more embodiments where a polymer is diblock polymer having anionic groups, the diblock polymer may be of the formula [hydrophobic repeating unit]q-[anionic repeating unit]z were q and z describe the number or repeating groups in each block, q and z are each independently an integer from 1 to 1000. In one or more embodiments, q and z are each independently 1 or greater, 10 or greater, 25 or greater, 50 or greater, 75 or greater, 90 or greater, 100 or greater, 110 or greater, 120 or greater, 130 or greater, 140 or greater, 150 or greater, 160 or greater, 170 or greater, 180 or greater, 190 or greater, 200 or greater, 210 or greater, 220 or greater, 230 or greater, 340 or greater, 250 or greater, 260 or greater, 270 or greater, 280 or greater, 290 or greater, 300 or greater, 325 or greater, 350 or greater, 375 or greater, 400 or greater, 425 or greater, 450 or greater, 475 or greater, 500 or greater, 600 or greater, 700 or greater, 800 or greater, or 900 or greater. In one or more embodiments, q and z are each independently 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 475 or less, 450 or less, 425 or less, 400 or less, 375 or less, 350 or less, 325 or less, 300 or less, 290 or less, 280 or less, 270 or less, 260 or less, 250 or less, 240 or less, 230 or less, 220 or less, 210 or less, 200 or less, 190 or less, 180 or less, 170 or less, 160 or less, 150 or less, 140 or less, 130 or less, 120 or less, 110 or less, 100 or less, 90 or less, 75 or less, 50 or less, 25 or less, or 10 or less.In one or more embodiments, q is 50 to 150 and z is 50 to 200. In one or more embodiments, q is 75 to 125 and z is 125 to 175. In one or more embodiments q is 90 to 120 and z is 90 to 120. Iz one or more embodiments, q is 90 to 120 and z is 140 to 160. In one or more embodiments, q is 90 to 120 and z is 190 to 210.

[0193] The ratio of hydrophobic repeating units to anionic repeating units may vary. For example, an anionic diblock polymer represented by the formula [hydrophobic repeating unit]q- [anionic repeating unit]z, may include 0.1 to 2 hydrophobic repeating units for every 1 anionic repeating unit.

[0194] In one or more embodiments, an anionic diblock polymer can include more anionic repeating units than hydrophobic repeating units. For example, in one or more embodiments, an anionic diblock polymer includes 0.1 to 0.99 hydrophobic repeating units for every 1 anionic repeating unit. In one or more embodiments, an anionic diblock polymer includes 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, or 0.9 or more hydrophobic repeating units for every 1 anionic repeating unit. In one or more embodiments, an anionic diblock polymer includes 0.99 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, or 0.2 or less, hydrophobic repeating units for every 1 anionic repeating unit.

[0195] In one or more embodiments, an anionic diblock polymer includes fewer anionic repeating units than hydrophobic repeating units. In one or more embodiments, an anionic diblock polymer includes 1 or more, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or more hydrophobic repeating units for every 1 anionic repeating unit. In one or more embodiments, an anionic diblock polymer includes 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 3 or less, 2 or less, 1.9 or less, 1.8 or less, 1.7 or less, 1.6 or less, 1.5 or less, 1.4 or less, 1.3 or less, 1.2 or less, or 1.1 or less hydrophobic repeating units for every 1 anionic repeating unit.

[0196] The amount of the anionic repeating unit in an anionic polymer (e.g., a diblock polymer) may vary. The mole percent (mol-%) of anionic repeating units in an anionic polymer may be measured and calculated usingJH NMR via methods known in the art. In one or more embodiments, the amount of anionic repeating units in an anionic polymer is 1 mol-% or greater, 5 mol-% or greater, 10 mol-% or greater, 15 mol-% or greater, 20 mol-% or greater, 25 mol-% orgreater, 30 mol-% or greater, 60 mol-% or greater, 80 mol-% or greater. In one or more embodiments, the amount of anionic repeating units in an anionic polymer is 100 mol-% or less, 80 mol-% or less, 60 mol-% or less, 30 mol-% or less, 25 mol-% or less, 20 mol-% or less, or 15 mol-% or less, 10 mol-% or less, or 5 mol-% or less. In one or more embodiments, the amount of anionic repeating units in an anionic polymer is 1 mol-% to 100 mol-%, 1 mol-% to 80 mol-%, 1 mol-% to 60 mol-%, 1 mol-% to 30 mol-%, lmol-% to 25 mol-%, 1 mol-% to 20 mol-%, 1 mol- % to 15 mol-%, 1 mol-% to 10 mol-%, or 1 mol-% to 5 mol-%. In one or more embodiments, the amount of anionic repeating units in an anionic polymer is 5 mol-% to 100 mol-%, 5 mol-% to 80 mol-%, 5 mol-% to 60 mol-%, 5 mol-% to 30 mol-%, 5 mol-% to 25 mol-%, 5 mol-% to 20 mol-%, 5 mol-% to 15 mol-%, or 5 mol-% to 10 mol-%. In one or more embodiments, the amount of anionic repeating units in an anionic polymer is 10 mol-% to 100 mol-%, 10 mol-% to 80 mol-%, 10 mol-% to 60 mol-%, 10 mol-% to 30 mol-%, 10 mol-% to 25 mol-%, 10 mol-% to 20 mol-%, or 10 mol-% to 15 mol-%. In one or more embodiments, the amount of anionic repeating units in an anionic polymer is 15 mol-% to 100 mol-%, 15 mol-% to 80 mol-%, 15 mol-% to 60 mol-%, 15 mol-% to 30 mol-%, 15 mol-% to 25 mol-%, or 15 mol-% to 20 mol-%. In one or more embodiments, the amount of anionic repeating units in an anionic polymer is 20 mol-% to 100 mol-%, 20 mol-% to 80 mol-%, 20 mol-% to 60 mol-%, 20 mol-% to 30 mol-%, or 20 mol-% to 25 mol-%. In one or more embodiments, the amount of anionic repeating units in an anionic polymer is 25 mol-% to 100 mol-%, 25 mol-% to 80 mol-%, 25 mol-% to 60 mol-%, or 25 mol-% to 30 mol-%. In one or more embodiments, the amount of anionic repeating units in an anionic polymer is 30 mol-% to 100 mol-%, 30 mol-% to 80 mol-%, or 30 mol-% to 60 mol-%. In one or more embodiments, the amount of anionic repeating units in an anionic polymer is 60 mol-% to 100 mol-% or 60 mol-% to 80 mol-%. In one or more embodiments, the amount of anionic repeating units in an anionic polymer is 80 mol-% to 100 mol-%. In one or more embodiments, the amount of anionic repeating units in an anionic polymer is 15 mol-% to 60 mol-%. In one or more embodiments, the amount of anionic repeating units in an anionic polymer is 15 mol-% to 30 mol-%.

[0197] In one or more embodiments, an anionic diblock polymer is of Formula VII, VIII, IX, or X:ionized for thereof where j, q, and z are described herein.

[0198] In one or more embodiments, an anionic diblock polymer is of Formula VII. In one or more embodiments, an anionic diblock polymer of Formula VIII. In one or more embodiments, an anionic diblock polymer is of Formula IX. In one or more embodiments, an anionic diblock polymer is of Formula X.

[0199] In one or more embodiments, an anionic polymer is an anionic diblock polymer of Formula VII where j is 3. In one or more embodiments, an anionic polymer is an anionic diblock polymer of Formula VIII where j is 3. In one or more embodiments, an anionic polymer is a diblock polymer of Formula IX where j is 3. In one or more embodiments, an anionic polymer is a diblock polymer of Formula X where j is 3.

[0200] In one or more embodiments, an anionic polymer is a diblock polymer having a poly(n-butyl acrylate) block and a poly(carboxyethyl acrylate) block. In one or more embodiments, an anionic polymer is a diblock polymer having a poly(n-butyl acrylate) block and a poly(succinic ethyl acrylate) block. In one or more embodiments, an anionic polymer is adiblock polymer having a poly(n-butyl acrylate) block and a poly(benzyl acid ethyl acrylate) block. In one or more embodiments, an anionic polymer is a diblock polymer having a poly(n- butyl acrylate) block and a poly(phosphate ethyl acrylate) block.

[0201] The acid dissociation constant (pKa) of a polymer that includes anionic groups may vary. The pKa of a polymer may affect the ability of the polymer to form complexes with an oligonucleotide cargo and / or the ability to release the oligonucleotide cargo from a complex. The pKa of a polymer may be determined using a potentiometric titration. In one or more embodiments, the pKa of a polymer is 2.0 or greater, 3.0 or greater, 4.0 or greater, 5.0 or greater, 6.0 or greater, or 7.0 or greater. In one or more embodiments, the pKa of a polymer is 8.0 or less, 7.0 or less, 6.0 or less, 5.0 or less, 4.0 or less, or 3.0 or less.Neutral Polymer

[0202] In one or more embodiments, polymers of the present disclosure are neutral. A neutral polymer does not include cationic groups or anionic groups in the repeating units of the polymer.

[0203] In one or more embodiments, a neutral polymer of the present disclosure includes poly(ethylene glycol) repeat units. In one or more embodiments, a neutral polymer of the present disclosure includes repeat units of the formula -(O-CH2-CH2)W)- where w is the number of repeating units, w can be, for example, 50 to 1000. w can be 50 or greater, 75 or greater, 100 or greater, 125 or greater, 150 or greater, 200 or greater, 225 or greater, 250 or greater, 275 or greater, 300 or greater, 325 or greater, 350 or greater, 375 or greater, 400 or greater, 425 or greater, 450 or greater, or 475 or greater, 500 or greater, 600 or greater, 700 or greater, 800 or greater, or 900 or greater, w can be 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 475 or less, 450 or less, 425 or less, 400 or less, 375 or less, 350 or less, 325 or less, 300 or less, 275 or less, 250 or less, 225 or less, 200 or less, 175 or less, 150 or less, 125 or less, 100 or less, or 75 or less. In one or more embodiments, w is 50 to 500, 50 to 400, 50 to 300, 100 to 500, 100 to 400, 100 to 300, 150 to 350, or 200 to 300.

[0204] In one or more embodiments, a neutral polymer of the present disclosure is a diblock polymer. Such polymers can be described as neutral diblock polymers. In one or more embodiments, a first homopolymer block of a neutral diblock polymer includes polyethylene glycol). A second homopolymer block of a neutral diblock polymer may be a polymer block that allows for the formation of micelles that include a diblock polymer (described herein). A secondhomopolymer block of a neutral diblock polymer may include a hydrophobic polymer block. In one or more embodiments, a second homopolymer block of a neutral diblock polymer includes a hydrophobic group. A hydrophobic group may be a part of the repeating unit. A hydrophobic group may be alkyl. An alkyl may be linear, branched, or cyclic. An alkyl may include 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Examples of hydrophobic alkyl groups include isopropyl, n-propyl, iso-butyl, n-butyl, and sec-butyl.

[0205] In one or more embodiments, a neutral diblock polymer is of the Formula (XX):where j is an integer from 0 to 10; and x and w are the number of repeat units, w can be, for example, any w disclosed herein.

[0206] j is an integer from 0 to 10. For example, j can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one or more embodiments] is 3, 4, or 5. In one or more embodiments] is 3. In one or more embodiments, j is 4. In one or more embodiments] is 5.

[0207] In one or more embodiments where a neutral polymer is diblock polymer having poly(ethylene glycol) (PEG) repeat units, the neutral diblock polymer may be of the formula [hydrophobic repeating unit]x-[PEG unit]w were x and w describe the number or repeating groups in each block, x and w are each independently an integer from 1 to 1000. In one or more embodiments, x and w are each independently 1 or greater, 10 or greater, 25 or greater, 50 or greater, 75 or greater, 90 or greater, 100 or greater, 110 or greater, 120 or greater, 130 or greater, 140 or greater, 150 or greater, 160 or greater, 170 or greater, 180 or greater, 190 or greater, 200 or greater, 210 or greater, 220 or greater, 230 or greater, 340 or greater, 250 or greater, 260 or greater, 270 or greater, 280 or greater, 290 or greater, 300 or greater, 325 or greater, 350 or greater, 375 or greater, 400 or greater, 425 or greater, 450 or greater, 475 or greater, 500 or greater, 600 or greater, 700 or greater, 800 or greater, or 900 or greater. In one or more embodiments, x and w are each independently 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 475 or less, 450 or less, 425 or less, 400 or less, 375 or less, 350 or less,325 or less, 300 or less, 290 or less, 280 or less, 270 or less, 260 or less, 250 or less, 240 or less,230 or less, 220 or less, 210 or less, 200 or less, 190 or less, 180 or less, 170 or less, 160 or less,150 or less, 140 or less, 130 or less, 120 or less, 110 or less, 100 or less, 90 or less, 75 or less, 50 or less, 25 or less, or 10 or less. In one or more embodiments, x is 50 to 150 and w is 50 to 200.In one or more embodiments, x is 75 to 125 and w is 125 to 175. In one or more embodiments x is 90 to 120 and w is 90 to 120. In one or more embodiments, q is 90 to 120 and z is 140 to 160. In one or more embodiments, x is 90 to 120 and w is 190 to 210.

[0208] The ratio of hydrophobic repeating units to PEG repeating units may vary. For example, for a neutral diblock polymer represented by the formula [hydrophobic repeating unit]x-[PEG repeating unit]w, the polymer may include 0.1 to 2 hydrophobic repeating units for every 1 PEG repeating unit.

[0209] In one or more embodiments, a neutral polymer (e.g., a neutral diblock polymer) can include more PEG repeating units than hydrophobic repeating units. For example, in one or more embodiments, a neutral polymer includes 0.1 to 0.99 hydrophobic repeating units for every 1 PEG repeating unit. In one or more embodiments, a neutral polymer includes 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, or 0.9 or more hydrophobic repeating units for every 1 PEG repeating unit. In one or more embodiments, a neutral polymer includes 0.99 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, or 0.2 or less, hydrophobic repeating units for every 1 PEG repeating unit.

[0210] In one or more embodiments, a neutral polymer (e.g., a neutral diblock polymer) includes fewer PEG repeating units than hydrophobic repeating units. In one or more embodiments, a neutral polymer includes 1 or more, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or more hydrophobic repeating units for every 1 PEG repeating unit. In one or more embodiments, a neutral polymer includes 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1.9 or less, 1.8 or less, 1.7 or less, 1.6 or less, 1.5 or less, 1.4 or less, 1.3 or less, 1.2 or less, or 1.1 or less hydrophobic repeating units for every 1 PEG repeating unit.

[0211] The amount of PEG repeating unit in a neutral polymer (e.g., a diblock polymer) may vary. The mole percent (mol-%) of PEG repeating units in a neutral polymer may be measured and calculated usingNMR via methods known in the art. In one or more embodiments, theamount of PEG repeating units in a polymer is 1 mol-% or greater, 5 mol-% or greater, 10 mol-% or greater, 15 mol-% or greater, 20 mol-% or greater, 25 mol-% or greater, 30 mol-% or greater, 60 mol-% or greater, 80 mol-% or greater. In one or more embodiments, the amount of PEG repeating units in a polymer is 100 mol-% or less, 80 mol-% or less, 60 mol-% or less, 30 mol-% or less, 25 mol-% or less, 20 mol-% or less, or 15 mol-% or less, 10 mol-% or less, or 5 mol-% or less. In one or more embodiments, the amount of PEG repeating units in a polymer is 1 mol-% to 100 mol-%, 1 mol-% to 80 mol-%, 1 mol-% to 60 mol-%, 1 mol-% to 30 mol-%, 1 mol-% to 25 mol-%, 1 mol-% to 20 mol-%, 1 mol-% to 15 mol-%, 1 mol-% to 10 mol-%, or 1 mol-% to 5 mol-%. In one or more embodiments, the amount of PEG repeating units in a polymer is 5 mol- % to 100 mol-%, 5 mol-% to 80 mol-%, 5 mol-% to 60 mol-%, 5 mol-% to 30 mol-%, 5 mol-% to 25 mol-%, 5 mol-% to 20 mol-%, 5 mol-% to 15 mol-%, or 5 mol-% to 10 mol-%. In one or more embodiments, the amount of PEG repeating units in a polymer is 10 mol-% to 100 mol-%, 10 mol-% to 80 mol-%, 10 mol-% to 60 mol-%, 10 mol-% to 30 mol-%, 10 mol-% to 25 mol-%, 10 mol-% to 20 mol-%, or 10 mol-% to 15 mol-%. In one or more embodiments, the amount of PEG repeating units in a polymer is 15 mol-% to 100 mol-%, 15 mol-% to 80 mol-%, 15 mol-% to 60 mol-%, 15 mol-% to 30 mol-%, 15 mol-% to 25 mol-%, or 15 mol-% to 20 mol-%. In one or more embodiments, the amount of PEG repeating units in a polymer is 20 mol-% to 100 mol- %, 20 mol-% to 80 mol-%, 20 mol-% to 60 mol-%, 20 mol-% to 30 mol-%, or 20 mol-% to 25 mol-%. In one or more embodiments, the amount of PEG repeating units in the polymer is 25 mol-% to 100 mol-%, 25 mol-% to 80 mol-%, 25 mol-% to 60 mol-%, or 25 mol-% to 30 mol-%. In one or more embodiments, the amount of PEG repeating units in a polymer is 30 mol-% to 100 mol-%, 30 mol-% to 80 mol-%, or 30 mol-% to 60 mol-%. In one or more embodiments, the amount of PEG repeating units in a polymer is 60 mol-% to 100 mol-% or 60 mol-% to 80 mol- %. In one or more embodiments, the amount of PEG repeating units in a polymer is 80 mol-% to 100 mol-%. In one or more embodiments, the amount of PEG repeating units in a polymer is 15 mol-% to 60 mol-%. In one or more embodiments, the amount of PEG repeating units in a polymer is 15 mol-% to 30 mol-%.

[0212] In one or more embodiments, a neutral diblock polymer is of Formula XV:where x and w are defined herein.

[0213] In one or more embodiments, a neutral polymer is a diblock polymer having a poly(n- butyl methacrylate) block and a polyethylene glycol) block. In one or more embodiments, a neutral polymer is a diblock polymer having a poly(n-butyl acrylate) block and a poly(ethylene glycol) block.

[0214] The number-average molecular weight (Mn) of a polymer of the present disclosure may vary. Mn is calculated using the following equation:where Mi is the mean molecular size of range i and xiis the number fraction of the total number of polymer chains that are within Mi range. Mn may be determined using size exclusion chromatography with a multi-angle light scattering detector.

[0215] In one or more embodiments, the Mn of a polymer is 5 kilodalton (kDa) or greater, 10 kDa or greater, 20 kDa or greater, 25 kDa or greater, 30 kDa or greater, 40 kDa or greater, 50 kDa or greater, or 75 kDa or greater, 100 kDa or greater, 150 kDa or greater, 200 kDa or greater, or 250 kDa or greater. In one or more embodiments, the Mn of a polymer is 300 kDa or less, 250 kDa or less, 200 kDa or less, 150 kDa or less, 100 kDa or less, 75 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 25 kDa or less, 20 kDa or less, or 10 kDa or less. In one or more embodiments, the Mn of a polymer is 5 kDa to 100 kDa, 5 kDa to 75 kDa, 5 kDa to 50 kDa, 5 kDa to 40 kDa, 5 kDa to 30 kDa, 5 kDa to 25 kDa, 5 kDa to 20 kDa, or 5 kDa to 10 kDa. In one or more embodiments, the Mn of a polymer is 10 kDa to 100 kDa, 10 kDa to 75 kDa, 10 kDa to 50 kDa, 10 kDa to 40 kDa, 10 kDa to 30 kDa, 10 kDa to 25 kDa, or 10 kDa to 20kDa. In one or more embodiments, the Mn of a polymer is 20 kDa to 100 kDa, 20 kDa to 75 kDa, 20 kDa to 50 kDa, 20 kDa to 40 kDa, 20 kDa to 30 kDa, or 20 kDa to 25 kDa. In one or more embodiments, the Mn of a polymer is 25 kDa to 100 kDa, 25 kDa to 75 kDa, 25 kDa to 50 kDa, 25 kDa to 40 kDa, or 25 kDa to 30 kDa. In one or more embodiments, the Mn of a polymer is 30 kDa to 100 kDa, 30 kDa to 75 kDa, 30 kDa to 50 kDa, or 30 kDa to 40 kDa. In one or more embodiments, the Mn of a polymer is 40 kDa to 100 kDa, 40 kDa to 75 kDa, or 40 kDa to 50 kDa. In one or more embodiments, the Mn of a polymer is 50 kDa to 100 kDa or 50 kDa to 75 kDa. In one or more embodiments, the Mn of a polymer is 75 kDa to 100 kDa.

[0216] The weight-average molecular weight (Mw) of a polymer of the present disclosure may vary. Mw is calculated using the following equation:where Mi is the mean molecular size of range i and wiis the weight fraction of the total number of polymer chains that are within Mi range. Mw may be determined using size exclusion chromatography with a multi -angle light scattering detector.

[0217] In one or more embodiments, the Mw of ae polymer is 5 kilodalton (kDa) or greater, 10 kDa or greater, 20 kDa or greater, 25 kDa or greater, 30 kDa or greater, 40 kDa or greater, 50 kDa or greater, 75 kDa or greater, 100 kDa or greater, 150 kDa or greater, 200 kDa or greater, or 250 kDa or greater. In one or more embodiments, the Mw of a polymer is 300 kDa or less, 250 kDa or less, 200 kDa or less, 150 kDa or less, 100 kDa or less, 75 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 25 kDa or less, 20 kDa or less, or 10 kDa or less. In one or more embodiments, the Mw of a polymer is 5 kDa to 100 kDa, 5 kDa to 75 kDa, 5 kDa to 50 kDa, 5 kDa to 40 kDa, 5 kDa to 30 kDa, 5 kDa to 25 kDa, 5 kDa to 20 kDa, or 5 kDa to 10 kDa. In one or more embodiments, the Mw of the polymer is 10 kDa to 100 kDa, 10 kDa to 75 kDa, 10 kDa to 50 kDa, 10 kDa to 40 kDa, 10 kDa to 30 kDa, 10 kDa to 25 kDa, or 10 kDa to 20 kDa. In one or more embodiments, the Mw of a polymer is 20 kDa to 100 kDa, 20 kDa to 75 kDa, 20 kDa to 50 kDa, 20 kDa to 40 kDa, 20 kDa to 30 kDa, or 20 kDa to 25 kDa. In one or more embodiments, the Mw of a polymer is 25 kDa to 100 kDa, 25 kDa to 75 kDa, 25 kDa to 50 kDa, 25 kDa to 40 kDa, or 25 kDa to 30 kDa. In one or more embodiments, the Mw of a polymer is 30 kDa to 100 kDa, 30 kDa to 75 kDa, 30 kDa to 50 kDa, or 30 kDa to 40 kDa. In one or moreembodiments, the Mw of a polymer is 40 kDa to 100 kDa, 40 kDa to 75 kDa, or 40 kDa to 50 kDa. In one or more embodiments, the Mw of a polymer is 50 kDa to 100 kDa or 50 kDa to 75 kDa. In one or more embodiments, the Mw of a polymer is 75 kDa to 100 kDa.

[0218] The dispersity of the molecular weight of the polymer may affect the characteristics of the polymer. The molecular weight dispersity may be quantified as the dispersity DM). DM is the distribution of individual molecular masses of a polymer. D is calculated as the quotient of the mass average molecular weight (Mw) divided by the number-average molecular weight (Mn). The Mwand Mnmay be determined using various methods including viscometry, size exclusion chromatography, and mass spectrometry. Generally, a small DM is preferred. Although there is no desired lower limit, in practice the DM of a polymer may be 1.0 or greater, 1.1 or greater, 1.2 or greater, 1.3 or greater, 1.4 or greater, 1.5 or greater, 1.6 or greater, 1.7 or greater, 1.8 or greater, 1.9 or greater, 2.0 or greater, 2.1 or greater, 2.2 or greater, 2.3 or greater. 2.3 or greater, or 2.4 or greater. In one or more embodiments, the M of a polymer may be 2.5 or less, 2.2 or less, 2.0 or less, 1.8 or less, 1.7 or less, 1.6 or less, 1.5 or less, 1.4 or less, 1.3 or less, 1.2 or less, or 1.1 or less. In one or more embodiments, the DM for a polymer is 1.0 to 2.5, 1.0 to 2.2, 1.0 to 2.0, 1.0 to 1.8, 1.0 to 1.7, 1.0 to 1.6, 1.0 to 1.5, 1.0 to 1.4, 1.0 to 1.3, 1.0 to 1.2, or 1.0 to 1.1. In one or more embodiments, the DM for a polymer is 1.1 to 2.5, 1.1 to 2.2, 1.1 to 2.0, 1.1 to 1.8, 1.1 to 1.7, 1.1 to 1.6, 1.1 to 1.5, 1.1 to 1.4, 1.1 to 1.3, or 1.1 to 1.2. In one or more embodiments, the D for a polymer is 1.2 to 2.5, 1.2 to 2.2, 1.2 to 2.0, 1.2 to 1.8, 1.2 to 1.7, 1.2 to 1.6, 1.2 to 1.5, 1.2 to 1.4, or 1.2 to 1.3. In one or more embodiments, the M for a polymer is 1.3 to 2.5, 1.3 to 2.2, 1.3 to 2.0, 1.3 to 1.8, 1.3 to 1.7, 1.3 to 1.6, 1.3 to 1.5, or 1.3 to 1.4. In one or more embodiments, the M for a polymer is 1.4 to 2.5, 1.4 to 2.2, 1.4 to 2.0, 1.4 to 1.8, 1.4 to 1.7, 1.4 to 1.6, or 1.4 to 1.5. In one or more embodiments, the M for a polymer is 1.5 to 2.5, 1.5 to 2.2, 1.5 to 2.0, 1.5 to 1.8, 1.5 to 1.7, or 1.5 to 1.6. In one or more embodiments, the D for a polymer is 1.6 to 2.5, 1.6 to 2.2, 1.6 to 2.0, 1.6 to 1.8, or 1.6 to 1.7. In one or more embodiments, the M for a polymer is 1.7 to 2.5, 1.7 to 2.2, 1.7 to 2.0, or 1.7 to 1.8. In one or more embodiments, the for a polymer is 1.8 to 2.5, 1.8 to 2.2, or 1.8 to 2.0. In one or more embodiments, the M for a polymer is 2.0 to 2.5, 2.0 to 2.2, or 2.2 to 2.5.

[0219] Post-polymerization functionalization of reactive polymer scaffolds can provide a direct avenue for the combinatorial synthesis of large polymer libraries, facilitating comprehensive structure-activity relationship investigations. This methodology addresseschallenges associated with monomer incompatibility arising from differing reactivity ratios, a challenge often constraining accessible polymer sequences. The modification of poly (pentafluorophenol acrylate) polymers through primary amine-based nucleophilic substitution served as a strategy for generating parallel polymer libraries containing cationic blocks. Additionally, the modification results in generation of polyacrylamide blocks, which are generally considered to be hydrolytically stable and more hydrophilic than their acrylate counterparts and have been shown to have superior gene delivery performance.

[0220] A set of post-polymerization modifiable diblock copolymer scaffolds was synthesized with poly (n-butyl acrylate) (PnBA) serving as the hydrophobic block and poly (pentafluorophenol acrylate) as the modular block further functionalized with cationic containing groups. To introduce variability in the molecular weight of the hydrophilic block, pentafluorophenol acrylate was chain extended to different extents on a PnBA block, generating three distinct parent modular scaffolds. Varying the volume fraction of the hydrophilic block can alter the block copolymer assembly from micelle to vesicle or other shapes such as spherical or cylindrical morphologies. While the traditional characterization of amphiphile assembly geometry usually is described by the packing parameter (p), the synthetically accessible hydrophilic block fraction (fhydrophiiic) can serve as the primary predictor of assembly morphology. Generally, an fhydrophiiic value of approximately 0.5 is indicative of spherical micelle formation. Given the inevitable variation in molecular weights post-modification, fhydrophiiic based on repeat units was used to ensure consistency in analyses. In the three parent scaffolds generated, the PnBA block (hydrophobic) remained constant at 118 repeat units (RU), while the modular block varied with lengths of 100 RU (Sh Library, 0.46 fhydrophiiic), 150 RU (Md Library, 0.56 fhydrophiiic), and 200 RU (Lg Library, 0.63 fhydrophiiic) (FIGS. 2 and 3). Subsequently, these scaffolds were reacted with ten different primary amine-based modifiers to yield a series of thirty poly(nBA)-b-poly(cation) copolymers. The primary amine modifiers utilized featured nitrogenbased cationic functionalities, exhibiting a spectrum of properties that encompassed varied degrees of substitution (A1-A5), hydroxylation (A9-A10), and included specialty amines such as those with piperazine and guanido moieties (FIG. 2). Boc protection was applied to small molecules containing multiple reactive amine moieties to enhance selectivity reduce the likelihood and / or extend of crosslinking. Alongside amidating the reactive poly(pentafluorophenol acrylate) block, the primary amines can also cleave off the trithiocarb onateend-groups. To reduce the likelihood and / or extent of potential side reactions from resulting thiol end groups forming disulfide linkages, hydroxy ethyl acrylate was included as a thiol capping agent. The use of a poly (pentafluorophenol acrylate) as the modular unit allowed the ability to assess the extent of amidation via19F NMR spectroscopy. Complete conversion was confirmed by the disappearance of peaks corresponding to poly(pentafluorophenol acrylate) and the appearance of new signals corresponding to the pentafluorophenol product. After achieving complete conversion, the number-average molecular weight (Mn) and hydrophilic were calculated for the entire series, assuming a poly(n-butyl acrylate)ii8-b-poly(cationic)ioo / i5o / 2oo block composition (FIG. 29 and FIG. 30).

[0221] FIG. 2 shows the generic synthesis for synthesizing various poly(n-butyl acrylate)ns- b-poly(cationic)ioo 150 / 200 block composition. Briefly, n-butyl acrylate was first polymerized to form poly(nBa). Next, pentafluorophenol acrylate was polymerized off of the poly(nBa) block to form a diblock polymer of poly(nBa)-b-poly(PFPA). FIG. 3 shows the number of n-butyl acrylate repeat units, the number of pentafluoroacrylate repeating units, the average number molecular weight, the dispersity, and the fhydrophobicity for the poly(nBA)-ppoly(PFPA) of various sizes (Sh, Md, and Lg). The pentafluorophenol esters of the poly(PFPa) block were reacted with various amines having various R groups (A1-A10) to form the poly(hydrophobic block)-b- poly(cationic containing block) diblock polymer. FIG. 1 shows that different ratios of the n-butyl acrylate repeating unit and the cationic containing repeating unit were explored. A total of 30 polymers were synthesized. Each polymer in the series can be referred to as the cationic repeating unit name (e.g., Al to A10) and the size of the polymer (Sh, Md, Lg). For example, an Al Sh polymer is a polymer having 118 repeating units of n-butyl acrylate and 100 repeating units of Al. An Al Md polymer is a polymer having 118 repeating units of n-butyl acrylate and 150 repeating units of Al. An Al Lg polymer is a polymer having 118 repeating units of n-butyl acrylate and 200 repeating units of Al

[0222] The sidechain bulkiness (side chain is R (Al -A 10), A3) and hydrophilicity (cLogP) for each of the 30 polymers having cationic groups were predicted using an online platform, Molinspiration Property Calculation. Potentiometric titrations were conducted to determine the pKa values of the cationic groups in the polymers (FIG. 28). The observed pKa values ranged from 7.2 to 8.3, with the A7 polymers exhibiting two pKa values of 5.65 and 8.2. The pendant amine groups of A7 typically share a proton between them at higher pH levels, becomingindividually protonated as the pH decreases. Consequently, A7 also displayed a broad buffering region, indicating good buffering capacity. Conversely, the more hydrophobic amines showcased a narrower buffering range, which could be attributed to pKa suppression in polymers coupled with the presence of bulky substitutions. Furthermore, no correlation between corona length (number of cationic containing repeated groups) and pKa with amphiphiles (polymers) bearing similar pendant groups yielded similar pKas.

[0223] A set of cationic A7 amphiphiles, anionic carboxy ethyl acrylate (CEA)-derived amphiphiles, and neutral poly(ethylene glycol) (PEG)-based amphiphiles were synthesized (FIG. 73). Leveraging post-polymerization modification methodology, the cationic A7 amphiphiles were synthesized from the modular scaffolds poly(pentafluorophenyl acrylate)-block-poly(n- butyl acrylate) (PPFPAb-PnBA). These scaffolds were obtained through RAFT polymerization, and subsequently functionalized via amidation with an A7 amine derivative. Selectivity in modification was enhanced through Boc protection of amines in A7, and quantitative conversion was confirmed by the complete disappearance of poly(pentafluorophenyl acrylate) characteristic peaks, monitored via19F NMR spectroscopy. Concurrently, the anionic CEA amphiphiles were similarly synthesized through RAFT -mediated chain extension of carboxy ethyl acrylate monomer on a poly(n-butyl acrylate) backbone used for synthesis of A7 amphiphile, ensuring compositional consistency across amphiphiles. Finally, the neutral amphiphile was generated via the synthesis of a PEG-functionalized RAFT macro-chain transfer agent. Specifically, a PEG (10K) macro chain transfer agent was first synthesized via acid-alcohol coupling of polyethylene glycol with DDMAT. This PEG-functionalized macro-CTA facilitated subsequent RAFT polymerization of n-butyl acrylate, yielding the neutral PnBA-b-PEG amphiphile with matched hydrophobic chain length relative to the cationic and anionic amphiphiles. Each amphiphile possess hydrophilic blocks exhibiting consistent lengths of approximately 240-250 repeat units, ensuring uniformity in hydrophilic-to-hydrophobic ratios across the polymer library. Following synthesis, all amphiphiles underwent purification via dialysis, and purity was rigorously assessed using both nuclear magnetic resonance (NMR) spectroscopy and size-exclusion chromatography (SEC). Furthermore, potentiometric titration of these amphiphiles revealed distinct ionization behaviors; notably, the A7 amphiphile exhibited dual pKa values at 5.6 and 8.2, while the CEA amphiphile showed an acidic character with a single pKa around 5.5. The process yielded 3 amphiphiles: (1) the diblock poly(n-butyl acrylate)-poly(A7), abbreviated as A7 or A; (2) thediblock poly(n-butyl acrylate)-poly(carboxyethyl acrylate), abbreviated as CEA or C; and (3) the diblock poly(n-butyl acrylate)-poly(ethylene glycol), abbreviated as PEG, PEG- 1 OK or P.

[0224] In another aspect, the present disclosure describes complexes. A complex includes one or more polymers of the present disclosure and an oligonucleotide. A complex of the present disclosure includes a polymer of the present disclosure associated with an oligonucleotide cargo. The ratio of phosphate groups of the oligonucleotide cargo to the amine group (N / P ratio) of the polymer (e.g., amine cationic group) may vary. The N / P ratio of a polymer may affect the efficiency of the complex as a delivery system. As used herein, the N / P ratio refers to the N / P ratio of a single complex or the average N / P ratio of a plurality of complexes. It is understood that the N / P of a complex or a plurality of complexes is the predicted or calculated N / P of such complex or complexes. In one or more embodiments, the N / P ratio is 1 or greater, 3 or greater, 5 or greater, 6 or greater, 8 or greater, 10 or greater, 12 or greater, 14 or greater, 16 or greater, 18 or greater, 20 or greater, 25 or greater, or 30 or greater. In one or more embodiments, the N / P ratio is 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 18 or less, 16 or less, 14 or less, 12 or less, 10 or less, 8 or less, 6 or less, 5 or less, or 3 or less. In one or more embodiments, the N / P ratio is 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, or 1 to 3. In one or more embodiments, the N / P ratio is 3 to 40, 3 to 35, 3 to 30, 3 to 25, 3 to 20, 3 to 18, 3 to 16, 3 to 14, 3 to 12, 3 to 12, 3 to 10, 3 to 8, 3 to 6, or 3 to 5. In one or more embodiments, the N / P ratio is 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 18, 5 to 16, 5 to 14, 5 to 12, 5 to 12, 5 to 10, 5 to 8, or 5 to 6. In one or more embodiments, the N / P ratio is 6 to 40, 6 to 36, 6 to 30, 6 to 26, 6 to 20, 6 to 18, 6 to 16, 6 to 14, 6 to 12, 6 to 12, 6 to 10, or 6 to 8. In one or more embodiments, the N / P ratio is 8 to 40, 8 to 36, 8 to 30, 8 to 26, 8 to 20, 8 to 18, 8 to 16, 8 to 14, 8 to 12, 8 to 12, or 8 to 10. In one or more embodiments, the N / P ratio is 10 to 40, 10 to 36, 10 to 30, 10 to 26, 10 to 20, 10 to 18, 10 to 16, 10 to 14, or 10 to 12. In one or more embodiments, the N / P ratio is 12 to 40, 12 to 36, 12 to 30, 12 to 26, 12 to 20, 12 to 18, 12 to 16, or 12 to 14. In one or more embodiments, the N / P ratio is 14 to 40, 14 to 36, 14 to 30, 14 to 26, 14 to 20, 14 to 18, or 14 to 16. In one or more embodiments, the N / P ratio is 16 to 40, 16 to 36, 16 to 30, 16 to 26, 16 to 20, or 16 to 18. In one or more embodiments, the N / P ratio is 18 to 40, 18 to 36, 18 to 30, 18 to 26, or 18 to 20. In one or more embodiments, the N / P ratio is 20 to 40, 20 to 36, 20 to 30, or 20 to 26. In one or more embodiments, the N / P ratio is 25 to 40, 25 to 36, 25 to 30, 30 to 36, or 35 to 40.

[0225] A complex of the present disclosure generally includes a plurality of polymers associated with a cargo in a particular molecular configuration. The type of molecular configuration may be dependent at least in part on the type of polymers included in the complex. For example, a complex that includes homopolymers may be an unorganized complex of a plurality of homopolymer chains and a plurality of polynucleotide chains (oligonucleotide cargo), termed a polyplex. In a polyplex, the oligonucleotide can associate with (e.g., binds to) the polymer via electrostatic interactions between the negatively charged oligonucleotide backbone and the cationic groups.

[0226] In one or more embodiments, a complex that includes a diblock polymer may include or be a micelleplex. A micelleplex includes a micelle and an oligonucleotide cargo non- covalently bound to the exterior of the micelle. A micelle is supramolecular assembly of a plurality of amphipathic molecules organized such that the hydrophobic (or lipophilic) portion of the molecules are packed together forming a hydrophobic core and the hydrophilic portion of the molecules are displayed exterior to the hydrophobic core. For example, the hydrophobic block of a plurality of diblock polymers of the present disclosure may assembly together to form a hydrophobic core of a micelle while the block that includes the cationic groups, anionic groups, or PEG is displayed on the surface of the micelle. The cationic groups displayed on the micelle can associate with (e.g., bind to) an oligonucleotide cargo via electrostatic interactions between the negatively charged oligonucleotide backbone and the cationic and / or ionizable groups to form a micelleplex.

[0227] The size of a complex (e.g., a micelleplex or polyplex) may affect its efficacy and / or efficiency as an oligonucleotide delivery system. Complex size can be measured, for example, using dynamic light scattering. In one or more embodiments, a complex has a size of, or a plurality of complexes have an average size of 0.05 nm or greater, 0.5 nm or greater, 1 nm or greater, 10 nm or greater, 20 nm or greater, 30 nm or greater, 40 nm or greater, 50 nm or greater, 75 nm or greater, 100 nm or greater, 200 nm or greater, 300 nm or greater, 400 nm or greater, 500 nm or greater, 600 nm or greater, 700 nm or greater, 800 nm or greater, 900 nm or greater, 1000 nm or greater, or 1500 nm or greater. In one or more embodiments a complex has a size of, or a plurality of complexes have an average size of 2000 nm or less, 1500 nm or less, 1000 nm or less, 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, 500 nm or less, 200 nm orless, 100 nm or less, 75 nm or less, 50 nm or less, 40 nm or less, 30 nm or less, 20 nm or less, 10 nm or less, 1 nm or less, or 0.5 nm or less.

[0228] The molecular configuration of a complex may affect its efficacy and / or efficiency as an oligonucleotide delivery system. Additionally, the configuration of the cells to which the complex is delivered may affect the complexes’ efficacy and / or efficiency as a delivery system. For example, in one or more embodiments, polyplexes may be able to more effectively delivery an oligonucleotide cargo to cells that are adhered to a surface as compared to micelleplexes. In one or more embodiments, micelleplexes may be able to more effectively delivery an oligonucleotide cargo to cells that free in solution (not adhered to a surface) as compared to polyplexes.

[0229] In embodiments where a complex is a micelleplex, the accessible hydrophilic block fraction (fhydrophiiic) of a polymer in the micelleplex may vary, fhydrophiiic is a measure of geometry. Generally, an fhydrophiiic value close to 0.5 indicates a spherical micelle. In one or more embodiment, fhydrophiiic of a polymer in a micelleplex may be 0.3 or greater, 0.35 or greater, 0.4 or greater, 0.45 or greater, 0.5 or greater, 0.55 or greater, 0.6 or greater, 0.65 or greater, 0.7 or greater, or 0.75 or greater. In one or more embodiment, fhydrophiiic of a polymer in a micelleplex may be 0.8 or less, 0.75 or less, 0.7 or less, 0.65 or less, 0.6 or less, 0.55 or less, 0.5 or less, 0.45 or less, 0.4 or less, or 0.35 or less.

[0230] A complex of the present disclosure may include more than one type of polymer. A type of polymer is the composition of the polymer, for example, including the cationic groups, anionic groups, or neutral groups. In one or more embodiments, a complex of the present disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different types of polymers. For example, a complex of the present disclosure may include a first polymer (e.g., homopolymer or diblock polymer) having a first cationic group and a second polymer (e.g., homopolymer or diblock polymer) having a second cationic group.

[0231] In one or more embodiments, a complex of the present disclosure includes a first polymer having a first cation group and a second polymer having a second cation group. In some such embodiments, the weight ratio or mole ratio of the first polymer to the second polymer can vary. In one or more embodiments, the weight ratio or mole ratio of the first polymer to the second polymer is 0.1 parts or greater of the first polymer for every 1 part of the second polymer; 0.2 parts or greater of the first polymer for every 1 part of the second polymer; 0.3 parts orgreater of the first polymer for every 1 part of the second polymer; 0.4 parts or greater of the first polymer for every 1 part of the second polymer; 0.5 parts or greater of the first polymer for every 1 part of the second polymer; 0.6 parts or greater of the first polymer for every 1 part of the second polymer; 0.7 parts or greater of the first polymer for every 1 part of the second polymer; 0.8 parts or greater of the first polymer for every 1 part of the second polymer; or 0.9 parts or greater of the first polymer for every 1 part of the second polymer. In one or more embodiments, the weight ratio or mole ratio of the first polymer to the second polymer is 1 part or less of the first polymer for every 1 part of the second polymer; 0.9 parts or less of the first polymer for every 1 part of the second polymer; 0.8 parts or less of the first polymer for every 1 part of the second polymer; 0.7 parts or less of the first polymer for every 1 part of the second polymer; 0.6 parts or less of the first polymer for every 1 part of the second polymer; 0.5 parts or less of the first polymer for every 1 part of the second polymer; 0.4 parts or less of the first polymer for every 1 part of the second polymer; 0.3 parts or less of the first polymer for every 1 part of the second polymer; or 0.2 parts or less of the first polymer for every 1 part of the second polymer.

[0232] In one or more embodiments, complex includes a first polymer that includes cationic groups (e.g., a cationic diblock polymer) and a second polymer that includes neutral groups (e.g., PEG repeating units; a neutral diblock polymer). In some such embodiments, the weight ratio or mole ratio of the first polymer (the polymer having cationic groups) to the second polymer (the polymer that includes PEG repeating units) can vary. In one or more embodiments, the weight ratio or mole ratio of the first polymer to the second polymer is 20 parts or less, 15 parts or less of the first polymer for every 1 part of the second polymer, 12.5 parts or less of the first polymer for every 1 part of the second polymer, 10 parts or less of the first polymer for every 1 part of the second polymer, 9 parts or less of the first polymer for every 1 part of the second polymer, 8 parts or less of the first polymer for every 1 part of the second polymer, 7 parts or less of the first polymer for every 1 part of the second polymer, 6 parts or less of the first polymer for every 1 part of the second polymer, 5 parts or less of the first polymer for every 1 part of the second polymer, 4 parts or less of the first polymer for every 1 part of the second polymer, 3 parts or less of the first polymer for every 1 part of the second polymer, or 2 parts or less of the first polymer for every 1 part of the second polymer. In one or more embodiments, the weight ratio or mole ratio of the first polymer to the second polymer is 2 parts or greater of the first polymer for every 1 part of the second polymer, 3 parts or greater of the first polymer for every 1 part of the secondpolymer, 4 parts or greater of the first polymer for every 1 part of the second polymer, 5 parts or greater of the first polymer for every 1 part of the second polymer, 6 parts or greater of the first polymer for every 1 part of the second polymer, 7 parts or greater of the first polymer for every 1 part of the second polymer, 8 parts or greater of the first polymer for every 1 part of the second polymer, 9 parts or greater of the first polymer for every 1 part of the second polymer, 10 parts or greater of the first polymer for every 1 part of the second polymer, 12.5 parts or greater of the first polymer for every 1 part of the second polymer, or 2 parts or greater of the first polymer for every 1 part of the second polymer.

[0233] In one or more embodiments, the weight or mole ratio of a first polymer having cationic groups (e.g., a cationic diblock polymer) to a second polymer having PEG groups (e.g., a neutral diblock polymer) is 5 parts to 20 parts of the first polymer for every 1 part of the second polymer. In one or more embodiments, the weight or mole ratio of a first polymer having cationic groups to a second polymer having PEG groups is 5 parts to 6 parts of the first polymer for every 1 part of the second polymer. In one or more embodiments, the weight or mole ratio of a first polymer having cationic groups to a second polymer having PEG groups is 9 parts to 12.5 parts of the first polymer for every 1 part of the second polymer. In one or more embodiments, the weight or mole ratio of a first polymer having cationic groups to a second polymer having PEG groups is 15 parts to 20 parts of the first polymer for every 1 part of the second polymer.

[0234] In one or more embodiments, complex includes a first polymer that includes cationic groups (e.g., a cationic diblock polymer) and a second polymer that includes anionic groups (e g., an anionic diblock polymer). In some such embodiments, the weight ratio or mole ratio of the first polymer (the polymer having cationic groups) to the second polymer (the polymer having anionic groups) can vary. In one or more embodiments, the weight or mole ratio of the first polymer (cationic group containing) to the second polymer (anionic group containing) can be 15 parts or less of the first polymer to every 1 part of the second polymer, 12.5 parts or less of the first polymer to every 1 part of the second polymer, 10 parts or less of the first polymer to every 1 part of the second polymer, 9 parts or less of the first polymer to every 1 part of the second polymer, 8 parts or less of the first polymer to every 1 part of the second polymer, 7 parts or less of the first polymer to every 1 part of the second polymer, 6 parts or less of the first polymer to every 1 part of the second polymer, 5 parts or less of the first polymer to every 1 part of the second polymer, 4 parts or less of the first polymer to every 1 part of the second polymer,3 parts or less of the first polymer to every 1 part of the second polymer, or 2 parts or less of the first polymer to every 1 part of the second polymer. In one or more embodiments, the weight or mole ratio of the first polymer (cationic group containing) to the second polymer (anionic group containing) can be 1 part or greater of the first polymer to every 1 part of the second polymer, 2 parts or greater of the first polymer to every 1 part of the second polymer, 3 parts or greater of the first polymer to every 1 part of the second polymer, 4 parts or greater of the first polymer to every 1 part of the second polymer, 5 parts or greater of the first polymer to every 1 part of the second polymer, 6 parts or greater of the first polymer to every 1 part of the second polymer, 7 parts or greater of the first polymer to every 1 part of the second polymer, 8 parts or greater of the first polymer to every 1 part of the second polymer, 9 parts or greater of the first polymer to every 1 part of the second polymer, 10 parts or greater of the first polymer to every 1 part of the second polymer, or 12.5 parts or greater of the first polymer to every 1 part of the second polymer.

[0235] In one or more embodiments, the weight or mole ratio of a first polymer (a polymer having cationic groups; e.g., a cationic diblock polymer) to a second polymer (a polymer that includes anionic groups; e.g., an anionic diblock polymer) can be 1 part to 10 parts of the first polymer to every 1 part of the second polymer. In one or more embodiments, one or more embodiments, the weight or mole ratio of a first polymer (a polymer having cationic groups) to a second polymer (a polymer that includes anionic groups) can be 1 part to 3 parts of the first polymer to every 1 part of the second polymer. In one or more embodiments, one or more embodiments, the weight or mole ratio of a first polymer (a polymer having cationic groups) to a second polymer (a polymer that includes anionic groups) can be 2 parts to 5 parts of the first polymer to every 1 part of the second polymer. In one or more embodiments, one or more embodiments, the weight or mole ratio of a first polymer (a polymer having cationic groups) to a second polymer (a polymer that includes anionic groups) can be 8 parts to 10 parts of the first polymer to every 1 part of the second polymer.

[0236] In one or more embodiments, a complex includes a first polymer that includes cationic groups (e.g., a cationic diblock polymer), a second polymer that includes neutral groups (e.g., PEG repeating units; e.g., a neutral diblock polymer), and a third polymer that includes anionic groups (e.g., an anionic diblock polymer). In some such embodiments, the weight ratio or mole ratio of the first polymer (the polymer having cationic groups) and the second polymer(the polymer that includes PEG repeating units) to the third polymer (the polymer that includes the anionic groups) can vary. Stated differently the weight or mole ratio of the first polymer plus the second polymer to the third polymer can vary. In one or more embodiments, the weight or mole ratio of the first polymer (cationic group containing) plus the second polymer (neutral) to the third polymer (anionic groups) can be 15 parts or less of the first polymer plus the second polymer to every 1 part of the third polymer, 12.5 parts or less of the first polymer plus the second polymer to every 1 part of the third polymer, 10 parts or less of the first polymer plus the second polymer to every 1 part of the third polymer, 9 parts or less of the first polymer plus the second polymer to every 1 part of the third polymer, 8 parts or less of the first polymer plus the second polymer to every 1 part of the third polymer, 7 parts or less of the first polymer plus the second polymer to every 1 part of the third polymer, 6 parts or less of the first polymer plus the second polymer to every 1 part of the third polymer, 5 parts or less of the first polymer plus the second polymer to every 1 part of the third polymer, 4 parts or less of the first polymer plus the second polymer to every 1 part of the third polymer, 3 parts or less of the first polymer plus the second polymer to every 1 part of the third polymer, or 2 parts or less of the first polymer plus the second polymer to every 1 part of the third polymer. In one or more embodiments, the weight or mole ratio of the first polymer (cationic group containing) plus the second polymer (neutral) to the third polymer can be 1 part or greater of the first polymer plus the second polymer to every 1 part of the third polymer, 2 parts or greater of the first polymer plus the second polymer to every 1 part of the third polymer, 3 parts or greater of the first polymer plus the second polymer to every 1 part of the third polymer, 4 parts or greater of the first polymer plus the second polymer to every 1 part of the third polymer, 5 parts or greater of the first polymer plus the second polymer to every 1 part of the third polymer, 6 parts or greater of the first polymer plus the second polymer to every 1 part of the third polymer, 7 parts or greater of the first polymer plus the second polymer to every 1 part of the third polymer, 8 parts or greater of the first polymer plus the second polymer to every 1 part of the third polymer, 9 parts or greater of the first polymer plus the second polymer to every 1 part of the third polymer, 10 parts or greater of the first polymer plus the second polymer to every 1 part of the third polymer, or 12.5 parts or greater of the first polymer plus the second polymer to every 1 part of the third polymer.

[0237] In one or more embodiments, the weight or mole ratio of a first polymer (a polymer having cationic groups; e.g., a cationic diblock polymer) and a second polymer (a polymer thatincludes PEG repeating units; e.g., a neutral diblock polymer) to a third polymer (a polymer that includes the anionic groups; e.g., an anionic diblock polymer) can be 1 part to 10 parts of the first polymer plus the second polymer to every 1 part of the third polymer. In one or more embodiments, the weight or mole ratio of a first polymer (a polymer having cationic groups) and a second polymer (a polymer that includes PEG repeating units) to a third polymer (a polymer that includes anionic groups) can be 1 part to 3 parts of the first polymer plus the second polymer to every 1 part of the third polymer. In one or more embodiments, the weight or mole ratio of a first polymer (a polymer having cationic groups) and a second polymer (a polymer that includes PEG repeating units) to a third polymer (a polymer that includes anionic groups) can be 2 parts to 5 parts of the first polymer plus the second polymer to every 1 part of the third polymer. In one or more embodiments, the weight or mole ratio of a first polymer (a polymer having cationic groups) and a second polymer (a polymer that includes PEG repeating units) to a third polymer (a polymer that includes anionic groups) can be 8 parts to 10 parts of the first polymer plus the second polymer to every 1 part of the third polymer.

[0238] The three amphiphiles: (1) the diblock poly(n-butyl acrylate)-poly(A7), abbreviated as A7 or A; (2) the diblock poly(n-butyl acrylate)-poly(carboxyethyl acrylate), abbreviated as CEA or C; and (3) the diblock poly(n-butyl acrylate)-poly(ethylene glycol), abbreviated as PEG, PEG- 1 OK or P were combined in various ratios to form mixed micelles. The self-assembly of the amphiphiles to generate mixed micelles was accomplished via an ultrasound-assisted thin-film hydration procedure, to optimize the uniform incorporation of the amphiphilic constituents (FIG. 74). Each amphiphile was individually dissolved in methanol, facilitating homogeneous mixtures upon aliquoting at specified molar ratios. Following mixing, the solvent was evaporated under a gentle nitrogen stream to yield a uniform polymer film. Subsequent hydration of the dried film in deionized water, coupled with ultrasound sonication, yielded micellar structures. To methodically explore ternary amphiphile compositions, a strategic approach was adopted by anchoring the internal molar ratio between the cationic A7 and neutral PEG amphiphiles at defined proportions: A7(100):PEG(0), A7(95):PEG(5), A7(90):PEG(10), and A7(85):PEG(15) (FIG. 75). Each of these binary mixtures was incrementally doped with 10%, 20%, and 30% molar ratios of the CEA amphiphile, yielding a comprehensive library of 16 distinct micellar formulations encompassing, 1 purely cationic A7 micelle (APC 0 / 0), 3 PEG containing binary mixtures (APC XX / 00), 3 CEA containing binary mixtures (APC 00 / YY) and 9 ternary mixtures(APC XX / YY) (FIG. 75). The nomenclature adopted for mixed micelle formulations is illustrated in FIG. 75. Briefly, micelle formulations are denoted as APC XX / YY, where 'A' corresponds to the A7 amphiphile, 'P' represents the PEG amphiphile, and 'C designates the CEA amphiphile, ‘XX’ corresponds to the molar percent of PEG in A7-PEG mixture, and ‘ YY’ corresponding to molar doping ratio of CEA. Importantly, practical limitations on amphiphile mixture solubility constrained maximum CEA incorporation to 30 mol-%, as higher doping led to precipitation upon hydration due to charge neutralization. Similarly, increased PEG incorporation beyond 15 mol-% was limited by aggregation-induced precipitation, thus establishing an upper bound for the A7:PEG ratio at 85: 15. Overall, these systematically designed micelles offer a versatile platform to precisely modulate micellar surface characteristics and systematically assess the biological impacts of amphiphilic composition.

[0239] Following the preparation of mixed micelles, dynamic light scattering (DLS) was employed to assess their hydrodynamic size distribution. As shown in FIG. 76, a clear trend emerged. Hydrodynamic radii increased progressively with the incremental incorporation of PEG and / or CEA amphiphiles. Specifically, binary formulations containing either PEG or CEA (APC XX / 00 and APC 00 / YY) exhibited hydrodynamic radii approximately twice as large as the APC 0 / 0 homomicelles, while ternary formulations showed up to a fourfold increase in size at their maximum measured values. The increase in size upon PEG incorporation aligns with literature precedence, as PEG’s high hydrophilicity enhances corona hydration leading to swelling, thereby expanding the hydrodynamic radius. In contrast, the size increase observed with CEA incorporation appears counterintuitive, as electrostatic complexation between anionic and cationic species should result in structural compaction. However, anionic amphiphiles such as CEA can facilitate partial charge neutralization through electrostatic interactions with the cationic A7 amphiphile. This interaction has been shown to produce a charge-neutral intermediate shell surrounding the hydrophobic core, while residual cationic A7 and neutral PEG moi eties contribute to a positively charged hydrophilic corona. Given that the molar ratio of anionic to cationic amphiphiles remained below unity, it is proposed that these ternary assemblies adopt a core-shell -corona micellar architecture. Furthermore, the charge neutral intermediate shell can undergo significant hydration-induced swelling, which likely accounts for the enlarged hydrodynamic radii observed in CEA-containing formulations.

[0240] To elucidate the underlying morphological transformations responsible for the observed size increase of the mixed micelles, transmission electron microscopy (TEM) was utilized to visualize the micellar structures directly. TEM imaging of the APC 10 / XX micelle series revealed a progressive morphological evolution with increasing CEA incorporation (FIGS. 77, 78, and 79). Initially, the native cationic A7 micelles (APC 0 / 0) and binary A7-PEG micelles (APC 10 / 0) predominantly exhibited spherical morphologies (FIG. 77). Furthermore, TEM imaging of APC 10 / 10 and APC 10 / 20 formulations provided direct evidence for the proposed structural rearrangement, revealing a well-defined core-shell-corona morphology (FIG. 78). Interestingly, while the APC 10 / 10 micelles retained a uniformly spherical shape, subtle micellemicelle associations became apparent in the APC 10 / 20 formulation. This behavior can be attributed to diminished corona charge-charge repulsion resulting from increased anionic incorporation and consequent reduced cationic density at the micelle surface. In the APC 10 / 30 formulation, micelle association effects became strikingly pronounced, driving a distinct morphological transition from discrete spherical structures to elongated wormlike assemblies (FIG. 78). This transformation may be driven by the elevated incorporation of anionic amphiphiles, which intensifies charge neutralization and significantly expands the volume of the neutral intermediate shell. As this shell swells, internal strain builds within the micellar assembly, destabilizing the spherical morphology. Simultaneously, the diminishing cationic surface charge density weakens electrostatic repulsions, further encouraging micelle-micelle fusion into extended wormlike structures. Overall, the transition from compact spherical micelles to elongated wormlike structures highlights the delicate balance of charge neutralization, hydration-driven swelling, and inter-micellar associations that dictate self-assembly behavior.

[0241] To validate the proposed core-shell-corona architecture, zeta potential measurements were conducted across all formulations of FIG. 75. Despite varying levels of anionic amphiphile incorporation, all micelles exhibited consistently high positive surface charges (~ +45-50 mV), suggesting that residual cationic A7 and PEG dominate the micelle surface, while CEA is primarily confined to the neutralized intermediate shell. Although this uniformity may appear counterintuitive, it still aligns with the proposed structure when considering that zeta potential is influenced not only by net surface charge, but also by factors such as double-layer thickness, ionic strength, and particle hydration. These physicochemical parameters can collectively modulate the electrokinetic behavior of micelles, allowing nanoparticles with differing chargedensities to display similar zeta potentials. Thus, the zeta potential data complements TEM findings and underscores the need to interpret surface charge measurements within the broader context of micelle architecture and physicochemical environment.

[0242] Subsequently, the biophysical characteristics governing micelle complexation with mRNA cargo were explored for the mixed micelles of FIG. 75. Complexes were formulated at a fixed nitrogen-to-phosphate (N / P) molar ratio of 10, representing the micelle nitrogen (N) content relative to mRNA phosphate groups (P). Upon complexation in aqueous conditions, dynamic light scattering (DLS) measurements revealed a subtle yet consistent decrease in the hydrodynamic diameter of the micelle-nucleic acid particles (MNPs) when compared to respective native micelles (Figure 2B). This reduction in size is attributed to efficient cargo compaction within the micellar corona, thereby resulting in more compact structures. However, dilution of the complexes into the transfection medium OptiMEM led to a pronounced increase in particle size, suggestive of MNP aggregation mediated by ionic interactions with salts present in the medium (FIG. 76). This aggregation phenomenon was particularly exacerbated in formulations containing higher levels of anionic amphiphile incorporation, as evidenced by the formation of aggregates extending into the micrometer scale. This behavior likely arises from increased charge neutralization, which reduces electrostatic repulsion and enhances susceptibility to salt-induced aggregation.

[0243] Effective mRNA delivery is can be influenced by micelle-mRNA binding affinity and the corresponding dynamics of cargo release. To investigate these parameters, two complementary dye exclusion assays employing RiboGreen dye were implemented. The initial RiboGreen assay conducted in pure water indicated consistently high mRNA complexation efficiencies (-98%) across all micelle formulations, independent of amphiphile composition (FIG. 80). Similarly, upon dilution into OptiMEM, an increase in fluorescence intensity — indicative of partial mRNA release — was observed; however, this release did not exhibit clear compositional dependence, maintaining an average binding efficiency of approximately 80-85% (FIG. 80). To further elucidate the relative strength of micelle-mRNA interactions under biologically relevant competitive conditions, heparan sulfate, an anionic glycosaminoglycan prevalent on cellular surfaces known to competitively displace nucleic acid cargo, was introduced. Employing a modified RiboGreen-based dye exclusion assay, the stability of micellepl exes in the presence of heparan sulfate was assayed. Despite the introduction of thiscompetitive agent, no discernible trends emerged regarding amphiphile composition and binding strength (FIG. 80). These results corroborate earlier zeta potential observations, which demonstrated uniformly positive surface charges (~ +45 mV) across all micellar formulations. Collectively, these data suggest that incorporation of PEG and anionic amphiphiles within the micelle architecture does not significantly compromise mRNA binding stability or complexation dynamics, highlighting the robust and consistent biophysical behavior of these mixed micelle formulations in mRNA delivery contexts.

[0244] In another experiment, homo micelles were formed using each of the 30 poly cationic amphiphiles described earlier herein. Each micelle was formed from a single polymer of the 30 cationic amphiphiles. The self-assembly of the 30 synthesized poly cationic amphiphiles (polymers containing a hydrophobic block and a cationic containing block) to form micelles was accomplished using an ultrasound-assisted direct dissolution technique. In summary, dry powders of the polymers were dissolved directly in MOPS (3-(N-morpholino)propanesulfonic acid) buffer at pH 7 and subjected to stirring at room temperature for 3 days, with 30-minute intervals of ultrasonication every 24 hours. The size distribution of the resultant micelles was assessed via dynamic light scattering, revealing intensity-weighted mean diameters (Z-Average value) ranging from 70 nm to 130 nm, displaying a narrow monomodal distribution across the entire library (FIG. 4). A noticeable inverse relationship was observed between the corona block length (length of the cationic containing block) and micelle size. For example, the Lg library consistently produced smaller micelles compared to counterparts with the same amine type. This trend may be attributed to an overall increase in hydrophilicity due to the increment in charge density associated with block length. Conversely, in comparing various amine modifications, an increase in amine hydrophobicity positively correlated with micelle size, which aligns with the assumption of hydrophilicity-associated size variation. Furthermore, the direct dissolution method typically results in the formation of nonergodic micelles, where size is significantly influenced by polymer characteristics and preparation techniques.

[0245] In another aspect, the present disclosure describes a composition that includes one or more complexes of the present disclosure. A composition may be a delivery system composition. In one or more embodiments, a delivery system is a transfection composition. A transfection composition is a composition that includes one or more components for transfection.Transfection is the in vivo or in vitro process of introducing an exogenous oligonucleotide (e.g., an oligonucleotide cargo) into a cell.

[0246] A transfection composition includes a plurality of complexes of the present disclosure. The plurality of complexes may include one complex species or two or more complex species. It is understood that a “complex species” refers to a complex that includes a specific oligonucleotide cargo and a specific polymer or polymers (e.g., a complex that includes a first polymer and a second polymer). A first complex species may differ from a second complex species by the oligonucleotide cargo, the polymer or polymers, or both.

[0247] In one or more embodiments, a transfection composition includes a first complex species and a second complex species. The amount of each complex in a transfection composition may vary. In one or more embodiments, the weight ratio or mole ratio of a first complex species to a second complex species is 0.1 parts or greater of the first complex species for every 1 part of the second complex species; 0.2 parts or greater of the first complex species for every 1 part of the second complex species; 0.3 parts or greater of the first complex species for every 1 part of the second complex species; 0.4 parts or greater of the first complex species for every 1 part of the second complex species; 0.5 parts or greater of the first complex species for every 1 part of the second complex species; 0.6 parts or greater of the first complex species for every 1 part of the second complex species; 0.7 parts or greater of the first complex species for every 1 part of the second complex species; 0.8 parts or greater of the first complex species for every 1 part of the second complex species; or 0.9 parts or greater of the first complex species for every 1 part of the second complex species. In one or more embodiments, the weight ratio or mole ratio of a first complex species to a second complex species is 1 part or less of the first complex species for every 1 part of the second complex species; 0.9 parts or less of the first complex species for every 1 part of the second complex species; 0.8 parts or less of the first complex species for every 1 part of the second complex species; 0.7 parts or less of the first complex species for every 1 part of the second complex species; 0.6 parts or less of the first complex species for every 1 part of the second complex species; 0.5 parts or less of the first complex species for every 1 part of the second complex species; 0.4 parts or less of the first complex species for every 1 part of the second complex species; 0.3 parts or less of the first complex species for every 1 part of the second complex species; or 0.2 parts or less of the first complex species for every 1 part of the second complex species.

[0248] A transfection complex may further include a transfection medium. A transfection medium may be any suitable medium for contact with cells. In one or more embodiments, a transfection medium includes water. In one or more embodiments, a transfection medium includes salts and or agents or compounds that promote cell health and / or growth. In one or more embodiments, a transfection medium is a cell growth medium, such as, for example, Dulbecco’s Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), RPMI 1640 Medium, OPTIMEM I. In one or more embodiments, a transfection medium includes proteins. In other embodiments, a transfection medium does not include proteins. In one or more embodiments, a transfection medium is serum free. In other embodiments, a transfection medium includes serum.

[0249] The pH of a transfection composition may vary. In one or more embodiments, the pH of a transfection composition is 2 or greater, 3 or greater, 4 or greater, 5 or greater, 6 or greater, 7 or greater, or 8 or greater. In one or more embodiments, the pH of a transfection composition is 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, or 3 or less. In one or more embodiments, the pH of a transfection composition is 4 to 8, 5 to 8, 6 to 8, or 7 to 8.

[0250] The efficacy of mRNA delivery vectors balances their ability to efficiently complex with the cargo and facilitate its subsequent release. Early micelleplex-cargo dissociation leads to inefficient delivery while high compaction leads to poor mRNA release in the cytosol. Furthermore, transfection happens in complex media. The degree of mRNA compaction within the micelleplexes formed from the 30 synthesized polymers and mRNA, and its stability and lability were evaluated by using a dye exclusion assay and micelleplex size measurements.

[0251] The homo micelles formed using each of the 30 polycationic amphiphiles described earlier herein were investigated to analyze the impact of amphiphile identity (the 30 synthesized polymers) on micelle-mRNA binding and release dynamics. Two modified versions of the RiboGreen-based dye exclusion assay were used for this purpose. These assays mimicked alterations in micelleplex stability induced by transfection media and mRNA release mediated by cytosolic counterions. The assays were performed at N / P of 5 and 10. The micelle nitrogen (N) content to mRNA phosphate (P) content molar ratio (N / P) may influence micelleplex complexation dynamics and transfection efficiency. For the base dye exclusion assay, mRNA was first mixed with an intercalator dye RiboGreen, which fluoresces strongly upon binding with mRNA ( ex = 485 nm, and Xem = 528 nm). Subsequently, micelles were added at different N / Ps in PBS (pH 7.4) to induce micelleplex formation. Changes in mRNA compaction duringmicelleplex formation led to dye expulsion and a decrease in fluorescence signal, allowing for quantitative assessment of binding efficiency (following signal normalization with respect to naked mRNA fluorescence with RiboGreen) (FIG. 5). Dilution with OptiMEM post-base RiboGreen assay simulated transfection conditions, while incubation with heparan sulfate post- complexation elucidated mRNA release dynamics. Heparan is a naturally occurring anionic sulfonate-based polymer that has been shown to induce genetic material decomplexation in polyplexes. Decrease in micelleplex stability in transfection media and mRNA release due to heparan competitive binding are both indicated by an increase in RiboGreen fluorescence intensity due to exclusion of mRNA. Similarly, post complexation, DLS measurement were performed to obtain the micelleplex size distribution both in PBS and upon dilution with OptiMEM.

[0252] The base dye exclusion assay revealed a high degree of complexation (-98%) at both N / Ps for all micelles (FIG. 7). No clear correlation of micelle corona length and amine type with binding efficiency was observed. However, high degrees of binding are not always representative of the stability of the micelleplexes. Upon dilution with OptiMEM, a comparative increase in fluorescence was seen for N / P 5 formulations, indicative of more stable micelleplex formation at N / P 10 across the micelle library. Upon incubation with heparan, somewhat similar trends in release profile as the OptiMEM assay were observed but with higher mRNA release and some differences (FIG. 7).

[0253] Since complex crosstalk between polymer parameters may influence binding and release profiles, Pearson pairwise correlation values were computed to assess the relationships between the input features (FIG. 31). Greater amine substitution and hydrophobicity resulted in weaker mRNA binding indicated by high release efficiency in case of both OptiMEM and heparan incubation. As a notable exception, A7 micelleplexes showed strongest binding despite showing similar stability in OptiMEM as the bulkier A3-A5 micelleplexes. A9 and A10 showed comparable release efficiencies as their non-hydroxylated counterparts (Al and A2) despite having bulkier sidechains. This may be attributed to the emergence of H-bonding interactions with mRNA owing to the presence of hydroxyl groups, which negates the effect of bulky substitutions.

[0254] Micelleplex size is another parameter that may impact cellular internalization and functional delivery of mRNA. DLS measurements were performed on the micelleplexes at N / P 5and 10 both in the presence and absence of OptiMEM (FIG. 6 and FIG. 102). N / P 10 formulations afforded smaller micelleplexes that retained their size distribution over an hour. A correlation was seen with amine hydrophobicity. For example, more hydrophobic amines resulted in bigger micelleplex aggregates. Notably, the more hydrophilic A8 amphiphile typically resulted in large aggregates ranging into microns.

[0255] To study cargo delivery of micelleplexes formed from mRNA and the 30 polymers synthesized, a reporter assay, utilizing green fluorescent protein (GFP) encoding mRNA, to evaluate the transfection ability of our micelleplexes in vitro (FIG. 103). Given the transient nature of mRNA expression, relying solely on transfection efficiency may overlook nuances in delivery performance. Therefore, the geometric mean fluorescence intensity (MFI) of GFP expression within GFP positive population was assessed. GFP MFI serves as an indirect measure of delivery efficacy per cell, with the MFI directly correlating with the number of functional mRNA copies delivered and expressed. Therefore, while transfection efficiency offers a global perspective on delivery capability, GFP MFI complements this by offering a deeper understanding of the efficiency of mRNA release and expression at the cellular level. A flow cytometry-based protocol was employed to characterize the GFP positive cell population (transfection efficiency) and quantify geometric MFI as a metric for mRNA functionality. This analysis was complemented by a cytotoxicity assessment utilizing a CCK-8 viability assay, which relies on the metabolic activity of live cells to reduce a tetrazolium-based salt to formazan, resulting in a discernible color change that can be quantified through absorbance measurements at 450 nm. An initial screening was conducted in the HEK293T cell line, chosen for its high transfection efficiency, facilitating the explication of subtle variations in transfection capabilities among micelle formulations.

[0256] The GFP expression levels, viability profiles, transfection efficiency, and effective efficiency of individual micelleplexes were systematically evaluated at N / P ratios of 5 and 10 at three different mRNA dosages (0.25 pg, 0.5 pg, and 0.75 pg mRNA) FIGS. 8, 9, 10, 11, 12, 13, 14, 15, 16, 32, 33, 34, 35, 36, 37, 38, 39, 40, 56, 57, 58, and 59). A positive correlation between N / P ratio and mRNA dosage with GFP expression levels was observed. Viability exhibited a negative correlation between N / P ratio and mRNA dosage. This observed increase in payload expression upon increasing N / P can be ascribed to a delicate interplay between enhanced nucleic acid compaction and the presence of surplus free polymers capable of facilitating endosomalescape mechanisms. However, this effect is counterbalanced by a reduction in cell viability attributable to the necrotic effects of excessive cationic functionalities. Similarly, higher dosage of mRNA resulted in higher levels of expression and the associated higher dosage of micelles contributed to lower viability and vice versa. Hence, a balancing act was seen for a dosage with 0.5 pg of mRNA giving a good balance between viability and expression.

[0257] Beyond trends stemming from variations in N / P ratios and mRNA dosages, significant deviations in delivery performance were closely linked to the identity of the amine components within the micelleplexes (FIGS. 8, 9, 10, 11, 12, 13, 14, 15, and 16). Cursory analysis of the data indicated a consistent trend wherein an increase in the bulkiness of amine substitutions correlated with a decline in transfection efficiency and concurrent reduction in viability, particularly evident when comparing variants Al through A5. This observed phenomenon likely originates from the diminished binding efficiency of micelles bearing bulkier alkyl substitutions, coupled with the associated cytotoxicity attributed to the hydrophobic nature of these substitutions. Concurrently, a parallel trend was observed in GFP expression levels, as evidenced by a general decrease in GFP mean fluorescence intensity (MFI) with increasing bulkiness of amine side chains. Notably, although A3 micelles exhibited high transfection efficiencies, the corresponding GFP MFI was significantly lower compared to Al and A2 variants. Similarly, A6 micelles, characterized by a piperazine-based amine moiety with bulky substitutions, demonstrated a notably low GFP MFI. These outcomes can be ascribed to their suboptimal binding affinities, resulting in premature mRNA release and consequently fewer mRNA copies being effectively expressed, leading to lower MFIs. Interestingly, hydroxyl modification of primary and secondary amine-based pendant groups (A9 and A10) exerted a detrimental effect on viability without yielding a notable increase in GFP expression compared to their non-hydroxylated counterparts (Al and A2). However, the GFP MFI of A9 and A10 was comparatively higher than that of Al and A2, suggesting that hydroxyl modification may facilitate improved mRNA release in the cytosol, consequently leading to higher expression levels. Among the micelle variants, A7 micelles emerged as top performers in GFP expression, displaying robust transfection efficiency even at lower N / P ratios. The dual pKa exhibited by A7 can potentially increase its endosomal escape efficiency thus improving its functional mRNA delivery efficiency. A peculiar observation was the unexpectedly poor performance of guanido- based A8 micelles, failing to elicit GFP fluorescence despite their reported efficacy in pDNAdelivery systems. Despite the impact of amine identity on transgene expression levels, micelle corona length was found to be uncorrelated with performance. This observation differs from previous studies. For example, previous studies indicated a positive impact associated with an increase in polymer length and resulting high cation density. It is thought that the anticipated positive impact of cation density, often associated with longer polycations, is offset by the inherently dense corona architecture of the micelles. Consequently, this results in a charge density likely surpassing a threshold, beyond which the density-linked effect becomes negligible.

[0258] SHAP (SHapley Additive exPlanations) TreeExplainer analysis was used to explore the input design features and delivery outputs of the micelleplexes. SHAP is a game theoretic approach employed to extract the importance of a given model variable (polymer features) on a predicted output (delivery performance) based on initial learning data. The feature importance is indicated by the SHAP value magnitude, and the sign denotes the correlation with the model output. Polymer-dependent features considered as input variables include sidechain molecular volume (bulkiness), cLogP, binding efficiency, pKa, corona length, micelle size, and micelleplex size in OptiMEM (FIGS. 105 and 106) . These variables were modeled to assess their impact on dependent variables including transfection efficiency, viability, and GFP MFI (FIGS. 8, 9, 10, 11, 12, 13, 14, 15, and 16). The SHAP analysis revealed that micelleplex size exerts the greatest influence on determining transfection efficiency, viability, and GFP MFI. It is thought that the cumulative negative reciprocity of sidechain hydrophilicity and binding efficiency with micelleplex size inflates the impact of micelleplex size, as depicted in the SHAP dependency plot (FIGS. 42, 43, and 44). This heightened impact can be attributed to the hydrophilicity of pendant groups and correlated binding efficiency culminating in a tangible physical attribute - micelleplex size - experienced by the cells thus determining the transfection output. Furthermore, SHAP plots indicated a significant impact of binding efficiency on both transfection efficiency and GFP MFI. This correlation is represented in FIG. 41, wherein transfection efficiency demonstrates a linear dependence on binding efficiency, while GFP MFI exhibits a preference for intermediate values. This observation suggests that higher binding strength typically results in reduced decomplexation prior to cellular entry, thereby enhancing transfection efficiency. Conversely, an intermediate binding strength is optimal for ensuring cytosolic release of mRNA, leading to the observed higher GFP MFI. Finally, the SHAP analysis also demonstrated a poor dependence of corona length on affecting all biological outputs.

[0259] Transfection experiments using the Md library on various immortalized mammalian cells, including A549 (lung), HuH7 (liver), HDFn (fibroblasts), Hep2G2 (liver; FIGS. 67, 68 and 69), and RAW 264.7 (murine macrophage; FIGS. 70, 71, and 72) were done to explore the ability of different cationic groups to impact delivery. Given the variability in cell-dependent factors influencing uptake of foreign materials, differences in the total degree of uptake across the cell lines were observed. For example, A549 exhibited the highest susceptibility to transfection as compared to HEK293-T cells (FIG. 45, 46, and 47). In contrast, HuH7 and HDFn cells showed suppressed transfection levels compared to HEK-293T. Despite variations in expression efficiency among cell lines, trends in transfection efficiency and viability remained consistent to that in HEK293-T. Specifically, the Md A7 micelle formulation consistently demonstrated the highest transfection efficiency across all cell types. Furthermore, similar trends regarding the negative impact of amine bulkiness and hydrophobicity on cell viability across all cell lines were observed.

[0260] A series of cellular interaction studies including internalization pathway mechanism, endosomal escape efficiency, membrane destabilization, and mechanism of cell death induction were performed in an attempt to elucidate the reason behind various micelle performances. Since all three micelle libraries showed similar transfection performance which linearly correlated with N / P and dosage, the Md micelle library was chosen to perform the mechanistic assays at a median N / P of 7.5, and mRNA dosage of 0.5 pg per well in HEK293T cell line.

[0261] Cy5 labelled GFP expressing mRNA was used to probe the degree of internalization and quantified via flow cytometry (FIG. 17, 48, and 49). This experiment was done to understand if the differences in transfection performance across the amine moieties are attributed to differential mRNA internalization and their correlation to internalization pathways. Greater than 90% internalization across all ten different micelles at 4-hour mark post transfection induction was observed, which was sustained even at 24 and 48 hours. Since stochastic internalization of a single copy Cy5-mRNA can yield positive signal, the geometric mean fluorescence intensity of Cy5 as a gauge for relative amount of mRNA internalized was probed. The MFI change trend indicated peak internalization at 24 hours and greater than 50% decrease in Cy5 MFI at 48 hours across all ten micelles, likely due to mRNA metabolism over time. Comparing Cy5 MFI at 24 hours to amine identity, it was observed that micelles with poor binding affinities effectuate lower internalization of mRNA payloads. Even though multiplecomplex processes within the cytosolic environment ultimately determine mRNA expression levels, higher copy numbers of payloads may positively impact micelle performance and correlate to the transfection trends observed.

[0262] Soft nanoparticles such as polyplexes and micelleplexes are typically internalized by a combination of different endocytic and macropinocytotic routes. Charge induced membrane interaction and nanoparticle size typically dictate uptake pathway selectivity. To probe the route / s for uptake, cells were co-incubated with Cy5 labelled mRNA-micelle complexes and uptake pathway specific inhibitors. Clathrin and caveolae mediated endocytosis were probed by co-incubation with amantadine and filipin III respectively while dimethylamiloride was used for inhibiting micropinocytosis. The change in Cy5 MFI upon co-incubation with inhibitors revealed that all the micelleplexes, irrespective of amine type, primary undergo clathrin mediated endocytosis (CME) with some contribution form caveolar endocytosis and macropinocytosis pathways (FIGS. 50, 51, 52, 53, 60). While amine identity does not seem to impact the preferred uptake pathway, the dependence on CME pathway, which being an endolysosomal pathway indicates a downstream dependence of observed delivery performance on endosomal escape propensity.

[0263] Upon entry into the endolysosomal pathway, the micelleplexes encounter two potential outcomes: (i) successful translocation to the early endosome followed by subsequent release into the cytoplasm, or (ii) entrapment within the late endosome, leading to trafficking toward the lysosome. The latter scenario, characterized by acidification facilitated by vacuolar- type H+-adenosine triphosphate (V-ATPase) proton pumps, presents a significant risk of nucleic acid cargo degradation, and serves as a primary bottleneck in gene delivery processes. Aminebased polycations with pKa values closely aligned with the endosomal pH (< 6) exhibit buffering capacity, effectively functioning as "proton sponges" during ATPase driven endosomal acidification. This buffering phenomenon induces a rapid influx of chloride counter anions, resulting in swelling and eventual destabilization and rupture of the endosomal membrane. Additionally, because of micelleplex protonation during the buffering process, there is a potential for them to release micelles to maintain charge balance. Such free polycations have been observed to interact with and disrupt the endosomal lipid bilayer, leading to pore formation and subsequent release of the micelleplex. Hence, the interplay between excess free polycationic micelles and the buffering process can impact the efficiency of endosomal escape.

[0264] Various experiments were conducted to understand the dependency of micelles on these escape pathways and to analyze the impact of various amine properties on their endosomal escape efficiencies. First, a H+-ATPase inhibitor, Bafilomycin-Al (Baf-Al) was used to block the influx of protons and counterions into the endosome, thus negating the endosomal escape of micelleplexes by proton sponge mechanism. A significant decrease in GFP expression level upon Baf-Al treatment was observed across the different micelle types, indicating reliance on proton sponge mediated endosomal escape (FIG. 18). Since this pathway relies on the titratability of the amine, A7 micelle, exhibiting the broadest buffering region, had the highest drop in GFP expression (FIG. 19). A strong negative correlation was seen between endosomal escape efficiency (% change in GFP expression upon Baf-Al treatment) and pKa of the micelles indicating lower micelle pKas induced higher endosomal escape (FIG. 53). A significant decrease in GFP expression level upon Baf-Al treatment is found across micelle formulations, indicating a primary reliance on proton sponge-mediated endosomal escape (FIG. 106).

[0265] Next, a proxy assay was used to delve into the influence of amine on destabilizing the endosomal membrane (FIGS. 18, 19, 20, 21, and 22). Typically, cell membrane permeabilization is assessed by co-incubating cells with propidium iodide (PI), a dye selectively internalized into cells with compromised membranes. Consequently, the percentage of Pl-positive cells offers insight into the membrane permeabilization capacity of micelleplexes. Given that both the cell membrane and endosomal membrane share a similar composition, the extent of cell membrane permeabilization can provide valuable information regarding the capability of micelleplexes to disrupt endosomal membranes. Furthermore, the utilization of N / P ratio >1 ensures an ample presence of free micelles interacting with the cell membrane, thereby closely approximating the permeabilization occurring within endosomes. This strategic approach allows for a comprehensive assessment of the micelleplexes' ability to induce membrane disruption in both cellular compartments. Increasing the degree of substitution on the amines led to higher cell permeability to propidium iodide (PI) (FIG 18). Both excessive hydrophobicity and hydrophilicity contributed to heightened levels of membrane permeabilization. Hydroxylmodified amines such as A9 and A10 exhibited substantial PI internalization at par with the more hydrophobic counterparts like A4 and A5. Even though these two sets can potentially permeabilize the endosomal membrane to similar extents, differences in binding efficiencieslikely dictate the differences in expression observed. This suggests the presence of a balance between amine bulkiness and hydrophilicity for determining the degree of permeabilization.

[0266] A7 and A8 displayed comparatively lower PI internalization. Both A7 and A8 containing micelleplexes had comparatively low membrane poration ability (FIG. 60). As such, endosomal escape via endosomal membrane destabilization is comparatively low for A7 and A8 containing micelleplexes. It can be inferred that A7 primarily relies on the acidification route for escape from the endosome. Given the importance of titratability in facilitating cargo release and subsequent expression, this likely accounts for the observed high expression levels with A7. A7 has a broad buffering capacity range compared to A8 (FIG. 61). As such, A7 containing micelleplexes can escape the endosome by the endosomal buffering pathway. Conversely, A8's poor buffering capacity impedes efficient endosomal escape. Even if it escapes via membrane destabilization owing to free micelles, its high binding efficiency and limited titratability can hinder mRNA release, resulting in the observed poor expression levels.

[0267] Both A7 and A8 containing micelleplexes showed low mRNA release efficiency upon treatment with heparan (FIG. 62). In contrast to A8, the buffering capacity of A7 may facilitate counterion mediated mRNA release in the cytosol. High levels of Cy5 fluorescence in during both serum supplement transfection (DMEM + 10% FBS) and serum free transfection (OptiMEM) for A8 containing micelleplexes compared to A7 containing micelleplexes indicates that A8 binds to mRNA stronger than A7 in a serum environment thereby resulting in higher internalization. A8 has a low buffering capacity and hence endosomal escape primarily occurs via membrane destabilization (which is also comparatively low). Upon escape, mRNA release with aid from counterions and cytosolic proteins will be greatly reduced due to poor buffering and strong binding of mRNA to the polymer leading to poor transfection observed.

[0268] It is thought that A8 binds strongly to mRNA due to hydrogen bonding between the guanidine group and the bases of the mRNA. For example, the guanidine group on the polymer can mimic the hydrogen bonding of guanine. The hydrogen bonding between the polymer and the mRNA strengthens the association of the mRNA to the polymer. Although micelleplexes showed poor delivery of an mRNA cargo, they were able to transfect pDNA (FIG. 63; pDNA at an N / P of 5 with a 48 hour incubation). It is thought that the double-stranded pDNA mitigates hydrogen bonding between the polymer and the cargo thereby decreasing the affinity between the pDNA cargo and the polymer.

[0269] During the transfection process, a discernible amine-identity dependent cell viability trend was observed. Given the findings from PI internalization assays, indicative of pronounced membrane porosity, the intricate relationship between membrane destabilization and the consequential viability outcomes was further explored. To elucidate toxicity responses, an Annexin V assay was concomitantly conducted alongside PI internalization assay to delineate subpopulations undergoing apoptosis or necrosis. The Annexin protein exhibits a high affinity for phosphatidylserine, which is typically localized to the cytoplasmic leaflet of the phospholipid bilayer but gets exposed upon cell death. Consequently, necrotic cell death stemming from micelleplex-induced membrane destabilization would yield positive signals for both Annexin V (indicative of exposed phosphatidylserine) and PI (evidence of membrane poration). Conversely, cells undergoing apoptosis would exclusively exhibit positivity for Annexin V, delineating a distinct mechanistic pathway of programmed cell death induction. The correlation between necrosis levels and membrane destabilization as assessed by PI internalization was significant (FIGS. 21, 22, and 23), with apoptosis levels remaining consistent across the treatment library, suggesting that while some degree of membrane permeabilization is needed for effective transfection and endosomal escape, excessive destabilization can lead to heightened necrosis, compromising cell viability. Notably, micelles with more hydrophobic and bulky pendant amine groups showed increased necrosis in vitro, indicating their potential unsuitability for gene delivery applications. However, the Al, A2, and A7 micelles, which displayed intermediate reductions in cell viability, exhibited low necrosis levels, suggesting that their reduced viability may be due to cell growth slowdown rather than necrotic death.

[0270] The in vivo delivery performance of the library micelles through intravenous (IV) tail vein injections in BALB / c mice was explored. Given the poor correlation between micelle corona size and delivery performance observed in vitro, and further corroborated by SHAP analysis, the in vivo studies used the Md library to specifically probe the impact of amine substitution on delivery efficiency. To evaluate the delivery efficacy, luciferase-expressing mRNA as the reporter gene was employed, followed by screening for luciferase expression via imaging. Initial trials revealed that an N / P ratio of 10 and a dosage of 10 pg mRNA per mouse yielded good performance. Screening of the Md micelle library at these conditions demonstrated disparate in vivo performance to in vitro (in OptiMEM), with only Al, A7, and A9 micelles exhibiting significant luciferase signal, indicative of successful transfection (FIGS. 24 and 25).A7 micelles displayed the highest signal output, comparable to that of the commercial control JetPEI-in vivo at similar dosages. The signal for all three micelles was predominantly localized in the thoracic cavity, contrasting with the typical hepatic delivery and high abdominal localization observed with most liposomal and polymeric mRNA delivery vectors. Further investigation into organ biodistribution revealed lungs-specific luminescence for all three micelles, indicating organ-specific delivery (FIG. 26). The lungs specific luminescence recorded for A7 micelles was statistically highly significant compared to JetPEI-in vivo, thus indicating its high specificity. This was further validated by the delivery of Cy5-labeled mRNA and organ imaging, which confirmed that A7 micelles predominantly localized in the lungs (FIG. 107). The lungs are a prime target for gene delivery due to its importance in maintaining homeostasis as well as for treating chronic pathologies including cystic fibrosis and acute respiratory distress syndrome (ARDS).

[0271] SHAP analysis further revealed that the relative binding strength between micelles and mRNA is a factor influencing luciferase activity, followed by l<aand micelle size (FIGS. 108 and 109). This trend reinforces the critical role of binding strength on the stability of cargo against serum protein-induced decomplexation yet enabling effective cytoplasmic release. It also explains why A10 micelles, despite performing on par with A7 in vitro, failed in vivo. AlO’s weaker binding affinity relative to A7, which facilitated greater mRNA release and higher GFP MFI in vitro, likely caused premature decomplexation during circulation, ultimately compromising mRNA delivery. Nanoparticles with high / ?Ka(>8), particularly those containing nitrogenous moieties, are enriched with proteins having a pl (isoelectronic point) below physiological pH in their nanoparticle corona. These nitrogenous groups, such as quaternary ammonium species, facilitate the adsorption of vitronectin, which aids in binding with avfU integrin receptors on the endothelial cells within lung capillaries, ultimately directing tissuespecific delivery. Since Al, A7, and A9 micelles display a corona / ?Ka of around 8, a similar mechanism may account for the micelleplex targeting to the lungs to that previous study.

[0272] Injected mice displayed no discernable adverse events following micelle administration. In vivo tolerability assessments of Al, A7, and A9 micelles at N / P 10 with an mRNA dosage of 0.6 mg / kg revealed no significant alterations in ALT, AST, BUN, or creatinine serum levels compared to untreated controls 24 hours post-administration, indicating unaffected renal and hepatic functionality. To further probe the potential for micelle-induced tissue damageand inflammatory response, cytokine profiling was conducted. Plasma samples were collected 6 hours post-injection for analysis of pro-inflammatory cytokines including MCP-1, IFNy, TNFa, GM-CSF, IL-1 , IL-2, IL-4, IL-6, IL-10, and IL-12p70 (FIGS. 110, 111, and 112). Notably, JetPEI triggered marked elevations in MCP-1, IFNy, TNFa, and IL-4, relative to the PBS- injected controls, while the A7 MNP group exhibited a moderate MCP-1 increase, significantly lower than JetPEI but above control levels. No significant upregulation of IFNy, TNFa, or IL-4 was observed in the A7 cohort. Histopathological evaluation via hematoxylin and eosin staining revealed no differences in gross pathology across lung and liver tissues between the groups. Although modest inflammatory signatures and minor histological changes were detected, these are congruent with the delivery methodology employed. Elevated cytokines, particularly in the in 1 1’0- Jet PEI and A7 groups, are predominantly linked to monocyte and macrophage activation, which are abundant in pulmonary tissues. The heightened levels of IL-6 observed in the A7 group likely result from membrane permeabilization, triggering the release of damage-associated molecular patterns (DAMPs). These DAMPs, in turn, activate pattern recognition receptors (PRRs) on macrophages, initiating IL-6-mediated acute-phase inflammatory cascades. The preferential lung tropism exhibited by A7 MNPs likely produces the most membrane disruption. In contrast, the reduced delivery efficacy of Al and A9 in pulmonary tissues correlated with diminished IL-6 induction, indicative of less tissue perturbation. In vzvo-JetPEI, despite its lungspecificity, induced lower IL-6 levels than A7, likely due to its reduced membrane- permeabilizing capabilities. Collectively, these data highlight the favorable tolerability and robust lung-targeting capacity of micelle formulations for mRNA delivery.

[0273] Since proteins present in blood serum can affect micelleplex compaction, an in vitro transfection assays in HEK293-T cells in the presence of fully serum-supplemented transfection media was conducted to understand the observed disparity between in vitro and in vivo performance followed. FIGS. 64, 65, and 66 show a comparison of percent GPF positive cells, cell viability, and effective efficiency of the various Md library micelleplexes when transfection occurred in serum free media (OptiMEM) and serum supplemented media (serum). Flow analysis demonstrated high levels of GFP expression, at similar levels with serum-reduced conditions, particularly for A7, followed by diminished expression for Al and A9, and less than 5% expression for A2 and A10 (FIG. 27). This suggests a correlation between in vitro performance in serum and in vivo efficacy. The high binding efficiencies displayed by Al, A7,and A9, attributed to the presence of primary amines, likely contribute to lower micelleplex decomplexation in the presence of serum proteins, thereby enabling better transfection performance. This was further corroborated by Cy5 labelled mRNA uptake in presence of serum, wherein the high binding capable micelles effectuated higher mRNA internalization overall (FIGS. 54, 55, and 56).

[0274] SHAP analysis revealed that pl<a and micelle size influence the delivery efficiency under serum conditions, with binding efficiency ranking third (FIG. 114). The high binding efficiencies of Al, A7, and A9, attributed to their primary amine content, likely contribute to stable micelleplexes (lower mRNA decomplexation) in the presence of serum proteins, thereby enhancing delivery performance. This is further corroborated by Cy5-labeled mRNA uptake assays, which demonstrate that micelles with strong binding capabilities promote greater overall mRNA internalization in serum (FIG. 113).

[0275] The performance of A7 in both in vivo and in vitro (under serum) conditions, alongside the considerable activity of Al and A9, suggests a correlation between in vitro (under serum) and in vivo delivery. To explore this, multitask Gaussian Process (GP) models were applied, using BOTORCH to evaluate the predictive value of in vitro data for in vivo activity, incorporating data from both serum-supplemented and OptiMEM conditions. Input data were scaled using min-max normalization, while outputs were standardized with z-score normalization to prepare for multitask GP training. The multitask GP model, trained on both in vitro and in vivo datasets, enabled us to examine correlations across tasks. To assess the robustness of our predictive framework, the model was further trained on five different subsets of training data. Results showed that incorporating in vitro (serum present) data improved predictions for in vivo activity more effectively than incorporating in vitro (serum reduced) data. This enhancement was quantitatively supported by the task covariance matrix from the GP model, with a covariance of 0.935 ± 0.05 between in vitro (serum present) and in vivo activity, significantly higher than the covariance between in vitro (serum reduced) and in vivo activity (0.342 ± 0.09). Additionally, true vs. predicted plots validated that multitask models, especially with se-rum-based data, substantially improved predictions of amine-mediated trends in vivo.

[0276] In other experiments, the impact of the composition of the mixed micelleplexes formed from the micelle compositions of FIG. 75 on transfection was explored. To systematically elucidate the impact of mixed micelle composition on in vitro mRNA deliveryefficiency, a reporter gene assay was used. Specifically, green fluorescent protein (GFP)- encoding mRNA served as the cargo to evaluate the transfection capabilities of distinct micelleplex formulations. Transfection efficiency, defined by the percentage of GFP-positive cells, and geometric mean fluorescence intensity (MFI), indicative of functional mRNA expression per transfected cell, were quantified via flow cytometry. While transfection efficiency provides a global perspective on the overall ability of micelles to mediate cell entry and transfection, GFP MFI offers deeper insights into intracellular mRNA release efficiency and subsequent translation efficacy, directly correlating to the functional payload delivered per cell. Experiments were conducted in HEK293T cells at a fixed N / P ratio of 10. Recognizing that serum proteins commonly present in complete growth media can significantly influence MNP stability through aggregation or premature decomplexation, transfection was initially performed in serum-free OptiMEM for 4 hours. Post-incubation, cells were supplemented with complete growth medium to maintain cellular viability. This strategic experimental design facilitated precise delineation of micelle-specific delivery performance, independent of confounding effects introduced by serum proteins.

[0277] Analysis of transfection outcomes revealed clear compositional influences on micelle performance. Notably, incremental incorporation of the anionic CEA amphiphile displayed a robust positive correlation with GFP expression levels, whereas the inclusion of increasing proportions of PEG-based amphiphile exhibited a consistently negative correlation (FIGS. 81 ad 83). Formulations containing 30 mol-% CEA demonstrated significantly elevated transfection efficiency, achieving GFP-positive populations exceeding 95%, representing a substantial enhancement (-45%) compared to the native A7 homo-amphiphile micelle. This positive trend remained consistent across various ternary compositions, where higher anionic amphiphile doping uniformly correlated with increased GFP expression, typically resulting in improvements ranging from 30-40%. Conversely, increased PEG content within binary mixtures (APC XX / 00) systematically reduced GFP expression by approximately 10% relative to the A7 homopolymer micelles. Interestingly, deeper analysis via GFP MFI revealed even more pronounced effects attributable to micelle composition (FIG. 82). Incremental anionic amphiphile incorporation produced a significant and consistent enhancement in GFP MFI across all anion containing binary and ternary subsets, with optimal performance observed in formulations such as APC 0 / 30 and APC 5 / 30. These particular compositions displayed approximately eightfold increases inGFP MFI relative to the native A7 control. Conversely, increasing PEG amphiphile content negatively impacted GFP MFI, reinforcing the observed inverse relationship between PEG incorporation and mRNA delivery performance at the cellular level. Consequently, ternary compositions mirrored these trends, demonstrating reduced GFP MFI values with elevated PEG amphiphile content, yet maintaining a positive correlation with CEA incorporation. Importantly, formulations APC 0 / 30 and APC 5 / 30 not only exhibited superior performance relative to other micellar formulations but also markedly outperformed the commercial standard, JetPEI, in both transfection efficiency and GFP MFI. These findings were further validated in two additional cell lines, A549 and HuH7, where similar enhancements in GFP expression were observed, underscoring the broad applicability and robustness of these ternary mixed MNPs for efficient mRNA delivery across diverse cellular contexts. Together, these results highlight the role of micelle composition in modulating transfection outcomes, with anionic amphiphile incorporation encouraging gene expression.

[0278] To further probe the underlying mechanistic drivers governing these observed performance trends, cellular internalization assays were conducted. Uptake studies using Cy5- labeled GFP mRNA at 4 hours post-incubation demonstrated consistently high uptake efficiencies (>90%) across all micelleplex formulations, sustained even at 24 hours (FIGS. 84 and 85). Detailed Cy5 MFI analysis at the 4-hour timepoint revealed enhancements in mRNA internalization with increasing anionic amphiphile content. Specifically, formulation APC 0 / 30 exhibited approximately threefold greater Cy5 internalization compared to native APC 0 / 0 micelles, while formulations APC 5 / 30, APC 10 / 30, and APC 15 / 30 showed approximately twofold increases. Conversely, PEG incorporation within binary mixtures did not enhance internalization, correlating directly with their reduced transfection performance previously observed. For ternary mixed micelles, upon dilution with OptiMEM, enhanced aggregation was observed in formulations with higher anionic incorporation, which likely promoted accelerated MNP sedimentation onto cellular surfaces and facilitating enhanced uptake and subsequent high mRNA expression. Notably, relative Cy5 MFI levels measured at 24h post-transfection exhibited a marked reduction (>50%) for ternary formulations compared to Cy5 MFI levels at 4h timepoint. The relative levels of CY5 MFI were even lower by about 15-20% than native A7 homomicelle. This substantial decline in Cy5 fluorescence at the later timepoint strongly suggests more efficient intracellular release and subsequent translation-dependent catabolism of the delivered mRNA payload.

[0279] To investigate the impact of anionic amphiphile identity on delivery performance, the carboxyethyl acrylate (CEA) was replaces with other anionic amphiphiles, each featuring distinct chemical functionalities (FIG. 86). Three new amphiphiles were synthesized from the following monomers: succinic ethyl acrylate (SEA), benzyl acid ethyl acrylate (BEA), and phosphate ethyl acrylate (PEA). Given the delivery performance previously demonstrated by the APC 5 / XX micelle formulation incorporating CEA, analogous ternary micelle formulations were prepared with SEA, BEA, and PEA at an identical A7:PEG molar ratio of 95:5 with different amounts of anion doping (10-30%). Transfection assays in HEK293T cells revealed marked differences linked to anionic amphiphile identity (FIG. 87). SEA-containing ternary formulations exhibited transfection efficiency and GFP MFI trends closely resembling those observed with CEA, while formulations incorporating BEA or PEA demonstrated no significant performance enhancement over the native A7 homomicelle control. Potentiometric titration assays revealed that the distinct ionization properties of the anionic amphiphiles seemed to impact performance. SEA (pKa -5.9) exhibited a similar acidic profile to CEA (pKa -5.2), whereas BEA (pKa -7.2) and PEA (pKa -7.7) possessed substantially higher, near neutral pKa values. These findings strongly suggest that amphiphiles with acidic pKa values may play a key role in modulating pKa dependent cellular mechanism, which helps in improving delivery performance.

[0280] Given that cationic nanoparticles predominantly utilize endosome-mediated pathways for cellular internalization, successful nucleic acid delivery includes overcoming endosomal entrapment. A strategy to facilitate efficient endosomal escape involves enhancing the buffering capacity of delivery vehicles, thereby triggering the proton sponge effect. This mechanism describes how protonatable amine groups within nanoparticles become progressively protonated upon endosomal acidification, resulting in counterion influx (primarily Cl") and osmotic swelling, ultimately compromising endosomal integrity and enabling cargo release into the cytosol. For the mixed micelles, the combination of acidic CEA amphiphiles with A7 amphiphiles (which already display broad buffering capacity) may enhance endosomal escape via the proton sponge effect. To experimentally validate this hypothesis, bafilomycin-Al (Baf- Al), a potent inhibitor of V-ATPase proton pumps, was employed to block endosomal acidification (FIG. 88). Treatment with Baf-Al substantially reduced GFP MFI across all tested formulations, confirming the role of endosomal acidification — and consequently, the proton sponge mechanism — in mediating efficient cytosolic delivery (FIG. 89). Notably, micellescontaining anionic amphiphiles exhibited dramatic reductions (>90%) in GFP MFI following Baf-Al treatment, closely mirroring the behavior of the A7 homopolymer control. Detailed GFP MFI analysis further underscored this effect, revealing that the top-performing ternary micelle formulations (e.g., APC 0 / 30 and APC 5 / 30) experienced approximately seven-fold greater reductions in GFP MFI relative to A7 homomicelle control (APC 0 / 0). On the other hand, binary formulations containing only PEG as the neutral amphiphile demonstrated considerably lower sensitivity to Baf-Al treatment, experiencing only about a 50% reduction in GFP MFI. These results suggest that incorporating anionic amphiphiles significantly enhances the buffering capacity of mixed micelles, synergizing with the intrinsic buffering capabilities of polyamine- rich A7 amphiphile to amplify proton sponge-mediated endosomal escape. This enhanced buffering directly correlates with the substantial increase in cytosolic mRNA release, as evidenced by the pronounced elevation in GFP expression (MFI) observed for anionic- containing formulations.

[0281] Overall, the performance of ani on-doped mixed micelles can be attributed to a synergistic mechanism involving enhanced cellular uptake and endosomal escape. The anionic amphiphile drives micelle aggregation and sedimentation in culture media, promoting rapid and efficient cellular uptake. Once internalized, it’s low pKa values complements the broad buffering capacity of A7, synergistically enhancing the proton sponge effect to facilitate endosomal escape. This is evidenced by the sharp drop in Cy5 signal at later timepoints and the pronounced Baf-Al sensitivity, indicating efficient cytosolic release and rapid mRNA translation. Altogether, these findings establish that anion-driven modulation of micelle assembly, uptake, and endosomal trafficking can be used to achieve potent and broadly effective mRNA delivery with ternary mixed micelle systems.

[0282] To comprehensively evaluate how amphiphile composition in mixed micellepl exes influences cellular viability and overall delivery performance, CCK-8 viability assays were performed concurrently with the transfection experiments (FIGS. 90 and 91). The CCK-8 assay, based on the enzymatic reduction of a tetrazolium-based salt by NADH-dependent oxidoreductases present in viable cells, yields a quantifiable formazan product. The formation of formazan, detectable by its strong absorbance at 450 nm, allows the determination of relative viability by comparing MNP treated cell populations against untreated controls. Analysis of the CCK-8 results (FIG. 90) indicated that incorporation of anionic amphiphiles into binary micellesnegatively impacted cellular viability relative to native A7 homomicelles, reflecting potential cytotoxic effects associated with increased anionic incorporation. Conversely, binary micelles containing PEG amphiphiles demonstrate enhanced viability compared to both native A7 micelles and anionic binary mixtures. This observation suggests that PEG incorporation mitigates cytotoxicity through its ability to effectively shield excessive positive surface charges, thereby reducing detrimental cellular interactions. Finally, ternary micelles containing both PEG and anionic amphiphiles exhibited viability trends aligning closely with PEG-containing binary micelles, suggesting that PEG's protective effects persist within these more complex formulations.

[0283] Serum proteins are known to adversely impact the stability and performance of cationic polymer-based nucleic acid delivery systems by promoting polyplex destabilization, premature payload unpackaging, and reduced cellular internalization efficiency. To systematically investigate how amphiphile composition of mixed micelles influences serum resistance and overall transfection performance under physiological conditions, a series of in vitro transfection assays in H EK293T cells using OptiMEM supplemented with 10% fetal bovine serum (FBS) were conducted (FIG. 92, part (i)). Flow cytometry analysis revealed notable differences in performance under serum conditions compared to serum-free conditions (FIGS. 93 and 94). Incorporation of PEG amphiphiles into binary micelles (APC XX / 00) consistently enhanced transfection efficiency by approximately 20% relative to A7 homomicelles, although further incremental increases in PEG content did not yield additional improvements. Likewise, binary and ternary micelles containing anionic amphiphiles (APC 00 / XX and APC XX / XX) also exhibited improved transfection efficiency, achieving approximately a 20% increase over A7 micelles. Transfection efficiency for these anionic- containing formulations peaked at intermediate doping levels (APC XX / 10) and subsequently declined at higher anionic content (APC XX / 30). Nonetheless, all mixed micelle formulations, regardless of composition, outperformed the A7 homomicelle (APC 0 / 0) control under serum- supplemented conditions. However, when examining GFP MFI, a significant reduction (-80%) was observed across all formulations compared to serum-free conditions. Specifically, GFP MFI values, which ranged between approximately 500-4000 across the different mixed MNPs in the absence of serum, decreased dramatically to about 100-350 when serum was included. This reduction alludes to a substantial serum-mediated destabilization, resulting in diminishedintracellular payload delivery and expression. Binary PEG-containing micelles (APC XX / 00) showed negligible improvements in GFP MFI relative to the native A7 micelle control, whereas ternary micelles incorporating anionic amphiphiles demonstrated modest improvements, again peaking at intermediate anionic incorporation (APC XX / 10) before declining at higher doping levels. These results strongly suggest that excessive incorporation of PEG and anionic amphiphiles may overly neutralize the micelle surface charge, thereby compromising cargo compaction and promoting premature unpackaging in the presence of serum proteins.

[0284] To address serum -induced destabilization, an innovative strategy leveraging controlled micelleplex aggregation was used. Previously, rapid aggregation of mixed micelles was observed upon dilution into serum-free OptiMEM. Leveraging this behavior, a strategy where micelles were first allowed to aggregate in serum-free OptiMEM, after which serum- supplemented medium (OptiMEM with 20% FBS to achieve a final concentration of 10% total FBS) was introduced (FIG. 92, part (ii)) was used. By initially inducing micelle aggregation in serum-free conditions, followed by subsequent serum introduction, serum proteins may preferentially opsonize micellar aggregates rather than individual micelles, thereby preserving micelleplex stability. Implementing this strategy, transfection outcomes demonstrated performance trends closely resembling those of serum-free experiments, with significant improvements in transfection efficiency and GFP MFI observed for formulations incorporating higher levels of anionic amphiphiles (FIGS. 93 and 94). Particularly, the APC 10 / 30 formulation exhibited superior performance, surpassing both other tested formulations and the commercial JetPEI control. Viability remained high (>70%) across all formulations, though slight reductions were noted for formulations with higher anionic incorporation due to enhanced aggregate sedimentation. Attempts to experimentally verify serum-induced aggregate destabilization via dynamic light scattering (DLS) proved challenging, as interference from serum protein size distributions produced unreliable autocorrelation measurements. Nevertheless, robust transfection outcomes strongly support the effectiveness of our aggregation-based strategy (referred to as OptiMEM maturation), underscoring its potential to mitigate serum-induced performance reductions. Collectively, these findings emphasize the critical interplay between micelle composition, serum interactions, and controlled aggregation. Strategic aggregation of micelles prior to serum exposure presents a compelling approach for optimizing polymeric micelle-based nucleic acid delivery in physiologically relevant environments.

[0285] To systematically evaluate the in vivo performance and biodistribution of mixed micelleplex formulations, a comprehensive study was performed using luciferase-encoding mRNA. Mixed micelleplex were formulated at an N / P ratio of 10, and luciferase mRNA was administered intravenously at a dosage of 0 .5 m g / kg via tail-vein injection to immune- competent BALB / c mice. Luciferase expression, a direct marker of successful delivery and translation, was subsequently quantified by bioluminescence imaging to assess overall transfection efficiency and anatomical biodistribution. Quantitative analysis of whole-body bioluminescence revealed a clear dose-dependent relationship between micelle composition and luciferase expression (FIG. 95). Specifically, reducing the cationic amphiphile content via incremental PEG or anionic amphiphile incorporation led to a progressive decline in the average whole-body luminescence signal. Notably, however, despite this overall decrease, significant shifts in organ-specific biodistribution patterns emerged, highlighting distinct effects of amphiphile composition on nanoparticle organ tropism (FIG. 86). The native A7 homomicelles predominantly localized in the thoracic region, indicative of strong pulmonary tropism. Binary A7-PEG formulations (APC XX / 00) similarly displayed lung-specific luminescence, although with incrementally reduced intensity corresponding to increased PEG content. Conversely, micelles incorporating anionic amphiphiles exhibited a marked shift in biodistribution patterns. Intermediate anionic doping (APC XX / 10 and APC XX / 20 formulations) resulted in dual localization, with signals concurrently observed in both thoracic and splenic regions. Remarkably, formulations containing the highest anionic amphiphile content (APC XX / 30) exhibited pronounced splenic tropism, demonstrating significantly elevated splenic luminescence relative to lung signals (FIGS. 97, 98, 99, 100, and 101).

[0286] Similar shifts in organ tropism upon inclusion of anionic components have been previously reported in lipid nanoparticle (LNP) systems, suggesting that alterations in surface charge might influence nanoparticle biodistribution. However, zeta potential measurements in the present study consistently indicated a stable and uniformly positive surface charge (~+45 mV) across all tested micelle formulations, suggesting that surface charge alone is unlikely to drive the observed shift in organ tropism. Instead, it is proposed that micelleplex morphology plays a role. Previous TEM and DLS analyses revealed morphological transitions from smaller spherical micelles to larger elongated, wormlike structures with increasing anionic amphiphile incorporation. Consequently, the observed biodistribution shift may arise from morphologicalheterogeneity within micelle populations: smaller spherical micelles likely preferentially localize in pulmonary tissues, whereas larger wormlike aggregates exhibit enhanced sequestration in the spleen. This morphological heterogeneity effectively rationalizes the dual-site localization observed for intermediate anionic doping levels (APC XX / 10 and XX / 20), where a mixture of spherical and elongated structures coexist. It further explains the exclusive and pronounced splenic tropism of APC XX / 30 formulations, predominantly composed of elongated micelles. Collectively, these results underline the complex interplay between amphiphile composition, nanoparticle morphology, and resultant biodistribution patterns. They further emphasize the importance of morphological control as a potential factor for targeted organ-specific delivery.

[0287] Exploration of mixed micelleplexes revealed various insights. For example, in vitro assays revealed that incorporation of acidic anionic amphiphiles substantially enhanced transfection efficiency and GFP expression (MFI), attributed primarily to improved micelle aggregation-induced cellular uptake, efficient intracellular release, and enhanced endosomal escape via proton-sponge-mediated mechanisms. Incorporation of acidic amphiphiles with lower pKa values (e.g., CEA, SEA) outperformed amphiphiles with near-neutral pKa values (BEA, PEA). PEG incorporation positively impacted cell viability by mitigating cytotoxicity through charge shielding thereby. Ternary formulations combining PEG and acidic anionic amphiphiles (APC 10 / 30 and APC 15 / 30) demonstrated the highest effective efficiencies, reflecting a balance between viability and transfection performance. Introducing controlled pre-aggregation significantly improved serum resistance, preserving cargo stability, and ultimately restoring superior transfection efficiency and GFP expression levels comparable to serum-free conditions. Formulation APC 10 / 30 emerged as the top-performing candidate, surpassing both other mixed micelles and commercial standards such as JetPEI. In vivo biodistribution studies revealed a distinct shift in organ tropism with increasing anionic amphiphile incorporation. While cationic and PEG-containing micelles predominantly localized in the lung, anionic amphiphile incorporation markedly shifted organ tropism toward the spleen.

[0288] In another aspect, the present disclosure describes a method. The method includes forming a complex of the present disclosure. The method used to form the complex may depend at least in part on the polymer or polymers used to form the complex.

[0289] In one or more embodiments, the method includes mixing a polymer or polymers (e g., one or more different types of polymers) with an oligonucleotide cargo to create a firstmixture. The method may further include incubating the first mixture for a first period of time to form a second mixture comprising the complex. In some embodiments, the second mixture is a transfection composition that can be used to treat cells. For example, this method may be used to form a complex that includes a homopolymer. This method may be used to form a polyplex.

[0290] In one or more embodiments, the method includes mixing a polymer or polymers in a pre-first mixture. The method may further include incubating the pre-first mixture for period of time to form a first mixture that includes micelles. Incubating the pre-first mixture allows at least a portion of the polymers to organize into one or more micelles. In one or more embodiments, the method includes incubating the first mixture with an oligonucleotide to form a second mixture, the second mixture including a plurality of complexes. Incubating the first mixture with the oligonucleotide allows at least a portion of the oligonucleotide to associate with the micelles to form micelleplexes. In some embodiments, the second mixture is a transfection composition that can be used to treat cells. For example, this method may be used to form a complex that includes a diblock polymer or two or more diblock polymers.

[0291] A pre-first mixture includes a carrier. A carrier may include water. A carrier may further include one or more additional components. For example, ae carrier may include a buffering agent and / or a salt. Examples of buffering agents include 3- (N- morpholino)propanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid, and (4-(2- hydroxy ethyl)- 1 -piperazineethanesulfonic acid).

[0292] A pre-first mixture may have one or more properties that may facilitate micelle formation. For example, a pre-first mixture may have a pH of 4 to 9, such as 6.5 to 7.5 or 7. A pre-first mixture may have an ionic strength of 10 mM to 1000 mM, such as 50 mM to 200 mM or 50 mM tolOO mM.

[0293] In one or more embodiments, the method may further include agitating a pre-mixture after incubation. For example, ultrasonication may be used to agitate a pre-mixture. In one or more embodiments, the method may further include incubating the agitated pre-mixture at an elevated temperature.

[0294] The absolute concentration of the one or more polymers and the absolute concentration of the oligonucleotide in the first mixtures of the methods presented herein may vary depending at least in part on the desired N / P ratio, the identity and properties of the polymeror polymers, the identity and properties of the oli onucleotide, the number of cells to be treated, other factors, or any combination thereof.

[0295] In one or more embodiments, a first mixture includes water. In one or more embodiments, a first mixture includes an aqueous solution of water and an acid. The acid is used to adjust the pH to a value of 1 to 5. A low pH may be beneficial to complex as a low pH may increase the probability that the amines of the polymer are protonated. Protonation of the amines gives the amines a positive charge which may increase electrostatic interactions between the polymer and the negatively charged backbone of the oligonucleotide. Any suitable acid or buffer may be included. Examples of acids include, but are not limited to, citric acid, acetic acid, hydrochloric acid, formic acid, lactic acid, uric acid, malic acid, tartaric acid, sulfuric acid, and the like. Examples of buffer include sodium acetate buffer, chloroacetate buffer, and the like.

[0296] The length of the first incubation period may affect the transfection efficiency of a complex. In one or more embodiments, the first period is 1 minute or greater, 15 minutes or greater, 20 minutes or greater, 25 minutes or greater, 30 minutes or greater, 40 min or greater, 1 hour or greater, or 5 hours or greater. In one or more embodiments, the first period is 5 hours or less, 1 hour or less, 40 minutes or less, 35 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, or 15 minutes or less. In one or more embodiments, the first period is 15 minutes to 40 minutes, 15 minutes to 35 minutes, 15 minutes to 30 minutes, 15 minutes to 25 minutes, or 15 minutes to 20 minutes. In one or more embodiments, the first period is 20 minutes to 40 minutes, 20 minutes to 35 minutes, 20 minutes to 30 minutes, or 20 minutes to 25 minutes. In one or more embodiments, the first period is 25 minutes to 40 minutes, 25 minutes to 35 minutes, or 25 minutes to 30 minutes. In one or more embodiments, the first period is 30 minutes to 40 minutes or 30 minutes to 35 minutes. In one or more embodiments, the first period is 25 minutes to 35 minutes.

[0297] In one or more embodiments, the method further includes diluting a second mixture with a dilution medium to create a third mixture. In some embodiments, a third mixture is a transfection composition that can be used to treat cells. In such embodiments, a dilution medium is a transfection medium.

[0298] A second mixture may be diluted with an amount of a medium such that the concentration of the complex in a third mixture is half is 99% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% orless, 1% or less 0.1% or less, 0.01% or less, 0.001% or less, 0.0001% or less of the concentration of the complex in the second mixture. In one or more embodiments, a second mixture may be diluted with an amount of a medium such that the concentration of the complex in a third mixture is 0.0001% or greater, 0.001% or greater, 0.01% or greater, 0.1% or greater, 1% or greater, 5% or greater, 10% or greater, 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, or 90% or greater of the concentration of the complex in the second mixture. In one or more embodiments, a second mixture may be diluted with an amount of a medium such that the concentration of the complex is 10% to 90%, 20% to 80%, or 40% to 60% of the concentration of the complex in the second mixture.

[0299] The length of the second period may affect the efficiency of transfection. In one or more embodiments, the second period is 1 min or greater, 15 minutes or greater, 20 minutes or greater, 25 minutes or greater, or 30 minutes or greater, 40 minutes or greater, 1 hour or greater, or 5 hours or greater. In one or more embodiments, the second period of time is 5 hours or less, 1 hour or less, 40 minutes or less, 35 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, or 15 minutes or less. In one or more embodiments, the second period is 15 minutes to 40 minutes, 15 minutes to 35 minutes, 15 minutes to 30 minutes, 15 minutes to 25 minutes, or 15 minutes to 20 minutes. In one or more embodiments, the second period is 20 minutes to 40 minutes, 20 minutes to 35 minutes, 20 minutes to 30 minutes, or 20 minutes to 25 minutes. In one or more embodiments, the second period is 25 minutes to 40 minutes, 25 minutes to 35 minutes, or 25 minutes to 30 minutes. In one or more embodiments, the second period is 30 minutes to 40 minutes or 30 minutes to 35 minutes. In one or more embodiments, the second period is 25 minutes to 35 minutes.

[0300] In another aspect, the present disclosure describes a method of transfecting a cell with a complex of the present disclosure. In some such embodiments, a complex is in a transfection composition of the present disclosure. A complex and / or transfection composition may include any components as disclosed herein and may be formed according to any method disclosed herein. The method includes contacting a complex and / or a transfection composition with a cell to form a transfection mixture. In one or more embodiments, a transfection composition is a third mixture following the second incubation time from the method of forming the complex described herein. The cell may be in vitro or in a subject, such as a human or a non-human animal.

[0301] The technique of contacting a complex and / or transfection composition with a cell may depend at least in part on if the cell is a part of an in vitro or in vivo system. In one or more embodiments where the cell is a part of an in vitro system, contacting the cell with the complex of a transfection composition may include placing the transfection composition on top of an adhered cell or in a mixture that includes non-adhered cells. In one or more embodiments where the cell is a part of an in vivo system, contacting a complex of a transfection composition with a cell may include administering the transfection composition to a subject. The subject may be a human or a non-human animal. Administration may be, for example, topical, enteral, or parenteral.

[0302] The pH of a complex or transfection composition used to treat the cells may affect the efficiency of transfection. For example, a pH that is too high or too low may kill the cells. In one or more embodiments, the pH of a complex or transfection composition is between 5 and 8. In one or more embodiments, the pH of a complex or transfection composition is between 7 and 8.

[0303] In one or more embodiments, the method further incudes incubating the transfection mixture for a transfection time. For example, in embodiments where the cell is a part of an in vitro system, a transfection composition may be in contact with the cell for a transfection time. In one or more embodiments, the transfection time is 1 minute or greater, 15 minutes or greater, 20 minutes of greater, 25 minutes or greater, 30 minutes or greater, 40 minutes or greater, 1 hour or greater, 5 hours or greater, 12 hours or greater, 24 hours or greater, 48 hours or greater, 64 hours or greater, or 96 hours or greater. In one or more embodiments, the transfection time of time is 96 hours or less, 64 hours or less, 48 hours or less, 24 hours or less, 12 hours or less, 5 hours or less, 1 hour or less, 40 minutes of less, 35 minutes or less, 30 minutes or less, 25 minutes or less, or 20 minutes or less. In one or more embodiments, the transfection time of time is 15 minutes to 40 minutes, 15 minutes to 35 minutes, 15 minutes to 30 minutes, 15 minutes to 25 minutes, or 15 minutes to 20 minutes. In one or more embodiments, transfection time is 20 minutes to 40 minutes, 20 minutes to 35 minutes, 20 minutes to 30 minutes, or 20 minutes to 25 minutes. In one or more embodiments, the transfection time is 25 minutes to 40 minutes, 25 minutes to 35 minutes, or 25 minutes to 30 minutes. In one or more embodiments, the transfection time is 30 minutes to 40 minutes or 30 minutes to 35 minutes. In one or more embodiments, the transfection time is 25 minutes to 35 minutes.

[0304] In one or more embodiments, the method further includes quenching the transfection mixture. For example, in one or more embodiments where the cell is a part of an in vitro system, the method further includes quenching the transfection mixture. Quenching the transfection mixture decreases the likelihood of transfection. Quenching the transfection mixture may increase cell survival during and / or after the transfection process. In one or more embodiments, quenching the transfection mixture includes adding serum-containing media to the transfection mixture to create a quenched mixture. The serum in the serum-containing media disrupts the ability of the transfection complex to be transfected into a cell.EXEMPLARY EMBODIMENTS

[0305] The following is a non-limiting list of exemplary embodiments.

[0306] Embodiment l is a complex that includes a cationic diblock polymer. The cationic diblock polymer includes an acrylate block and an acrylamide block. The arylamide block includes repeating groups that each include a cationic group (RA) of formula (i), (ii), (iii), or (iv):or an ionized form thereof, b is an integer from 0 to 5. z and z’ are each independently an integer from 0 to 5. Qi and Q2 are each independently a heteroatom or a group that includes a heteroatom. The complex also includes a oligonucleotide cargo.

[0307] Embodiment 2 is the complex of embodiment 1, where the acrylamide block includes the repeating group of Formula (I)ionized form, n is the number of acrylamide or cationic repeating units, j’ is an integer from 0 to 5. RAis the cationic containing group.

[0308] Embodiment 3 is the complex embodiment 2, where j’ is 2.

[0309] Embodiment 4 is the complex of any of embodiments 1 to 3, where the complex is or includes a micelle.

[0310] Embodiment 5 is the complex of any of embodiments 1 to 4, where Qi and Q2 are independently N, NH, NH2, NH3, O, or OH.

[0311] Embodiment 6 is the complex of any of embodiments 1 to 5, where the cationic group is of Formula (A6), (A7), (A8), (A9), or (A10):or an ionized form thereof.

[0312] Embodiment 7 is the complex of any of embodiments 1 to 6, where diblock polymeris of Formula (II) V’ / . j is an integer from 0 to 10. m is the number of acrylate repeating groups that do not include a cationic group, n is the number of repeating groups that include a cationic group. RAis the cationic containing group, j’ is an integer from 0 to 5

[0313] Embodiment 8 is the complex of any of embodiments 1 to 7, where the cationic diblock polymer includes fewer acrylamide repeating units (repeating units that include a cationic group) than acrylate repeating units (repeating units that do not include a cationic group).

[0314] Embodiment 9 is the complex of any of embodiments 1 to 7, where the cationic diblock polymer includes 1.1 to 2.0 acrylate repeating units (1.1 to 2.0 repeating units that do not include a cationic group) for every 1 acrylamide repeating group (for every 1 repeating unit that includes a cationic group).

[0315] Embodiment 10 is the complex of any of embodiments 1 to 7, where the cationic diblock polymer includes fewer acrylamide repeating units (fewer repeating units that have a cationic group) than acrylate repeating units (repeating units that do not include a cationic group).

[0316] Embodiment 11 is the complex of any of embodiments 1 to 7, where the diblock polymer includes 0.1 to 0.99 acrylate repeating units (0.1 to 0.99 repeating units not having a cationic group) for every 1 acrylamide repeating unit (every 1 repeating unit that does have a cationic group).

[0317] Embodiment 12 is the complex of any of embodiments 1 to 11, where the cationic diblock polymer has a pKa of 7.0 to 8.5.

[0318] Embodiment 13 is the complex of any of embodiments 1 to 11, where the cationic diblock polymer has two pKas.

[0319] Embodiment 14 is the complex of embodiments 13, where the first pKa is 5.5 to 6.0 and the second pKa is 8.0 to 8.5.

[0320] Embodiment 15 is the complex of any of embodiments 1 to 14, where the complex further includes a neutral diblock polymer. The neutral diblock polymer includes an acrylate or methacrylate block. The neutral diblock polymer further includes a second block that has -(O- CH2-CH2)- repeat units.

[0321] Embodiment 16 is the complex of embodiment 15, where the second block has is 50 to 500, 50 to 400, 50 to 300, 100 to 500, 100 to 400, 100 to 300, 150 to 350, or 200 to 300 -(O- CH2-CH2)- repeat units

[0322] Embodiment 17 is the complex of embodiment 15 or 16, where the neutral diblock polymer is of the Formula (XX):. j is an integer from 0 to 10. x is the number of acrylate repeating units, w is the number of-(O-CH2-CH2)w)- repeating units.

[0323] Embodiment 18 is the complex of any of embodiments 15 to 17, where the weight or mole ratio of the cationic diblock polymer to the neutral polymer in the complex is 5 parts to 6 parts of the cationic diblock polymer for every 1 part of the neutral diblock polymer, 9 parts to 12.5 parts of the cationic diblock polymer for every 1 part of the natural polymer, or 15 parts to 20 parts of the cationic diblock polymer for every 1 part of the neutral diblock polymer.

[0324] Embodiment 19 is the complex of any of claims 1 to 18, wherein the complex further includes an anionic diblock polymer. The anionic diblock polymer includes an acrylate or methacrylate block. The anionic diblock polymer further includes an acrylamide block having a repeating group that includes an anionic group.

[0325] Embodiment 20 is the complex of embodiment 19, wherein the anionic group is ofthe Formula (Bl), (B2), (B3), or (B4):, or an nionized version thereof.

[0326] Embodiment 21 is the complex of embodiment 19 or 20, where the anionic diblockpolymer is of Formula (VI): (VI) . j is an integer from 0 to 10. q is the number of acrylate repeating groups lacking an anionic containing group, z is the number of repeating groups includes the anionic containing group; and RBis the anionic group.

[0327] Embodiment 22 is the complex of embodiment 21, wherein RBis of Formula Bl, B2, B3, B4, or an ionized version thereof.

[0328] Embodiment 23 is the complex of embodiment 21 or 22, where j is 4.

[0329] Embodiment 24 is the complex of embodiment 19, where the anionic diblock polymer is of formula (VII), (VIII), (IX), or (X):or an ionized version thereof, j is an integer from 0 to 5. q is the number of acrylate repeating groups lacking an anionic group, z is the number of repeating groups containing an anionic group.

[0330] Embodiment 25 is the complex of any of embodiments 1 to 14, where the complex further includes the anionic diblock polymer of any of embodiment 19 to 24. In one or more embodiments, the complex does not include the neutral diblock polymer of any of embodiments 15 to 18.

[0331] Embodiment 26 is the complex of embodiment 25, where the weight or mole ratio of the cationic diblock polymer to the anionic polymer is 1 part to 10 parts of the cationic diblock polymer for every 1 part of the anionic diblock polymer; 1 part to 3 parts of the cationic diblock polymer for every 1 part of the anionic diblock polymer; 2 part to 5 parts of the cationic diblock polymer for every 1 part of the anionic diblock polymer; or 8 part to 10 parts of the cationic diblock polymer for every 1 part of the anionic diblock polymer.

[0332] Embodiment 27 is the complex of any of embodiments 1 to 14, where the complex further includes the neutral diblock polymer of any of embodiments 15 to 18 and the anionic diblock polymer of any of embodiment 19 to 24.

[0333] Embodiment 28 is the complex of embodiment 27, where the weight or mole ratio of the cationic diblock polymer and the neutral diblock polymer to the anionic polymer is 1 part to 10 parts of the cationic diblock polymer and the neutral diblock polymer for every 1 part of the anionic diblock polymer; 1 part to 3 parts of the cationic diblock polymer and the neutral diblock polymer for every 1 part of the anionic diblock polymer; 2 part to 5 parts of the cationic diblock polymer and the neutral diblock polymer for every 1 part of the anionic diblock polymer; or 8 part to 10 parts of the cationic diblock polymer and the neutral diblock polymer for every 1 part of the anionic diblock polymer.

[0334] Embodiment 29 is the complex of any of embodiments 1 to 28, where the oligonucleotide includes mRNA.

[0335] Embodiment 30 is the complex of any of embodiments 1 to 29, where the oligonucleotide includes DNA.

[0336] Embodiment 31 is the complex of embodiment 30, where the DNA is single stranded.

[0337] Embodiment 32 is the complex of embodiment 31, where the DNA is double stranded.

[0338] Embodiment 33 is the complex of any of embodiments 1 to 32, where the oligonucleotide includes a plurality of phosphate groups, the polymer includes a plurality of amines, and the ratio of moles of the amine to moles of the plurality of phosphate groups is 2 to 20.

[0339] Embodiment 34 is the complex of embodiment 33, where the ratio of moles of the amine to moles of the plurality of phosphate groups is 5 to 10.

[0340] Embodiment 35 is a method of forming the complex of any one of embodiments 1 to 34. The method includes mixing a micelle that includes the diblock polymer with the oligonucleotide and a carrier to create a first mixture. The method may further include incubating the first mixture for a period of time to form a second mixture that includes the micelleplex.

[0341] Embodiment 36 is the method of embodiment 35, where the method further includes mixing the diblock polymer and a second carrier in a pre-first mixture to form the micelle.

[0342] Embodiment 37 is a transfection composition that include the complex of any of embodiments 1 to 34.

[0343] Embodiment 38 is the transfection composition of embodiment 37, where the composition further includes a transfection medium.

[0344] Embodiment 39 is the transfection composition of embodiment 38, where the transfection medium includes water.

[0345] Embodiment 40 is the transfection composition of embodiment 37 or 38, where the transfection medium is serum free.

[0346] Embodiment 41 is a method of transfecting a cell. The method includes contacting the transfection composition of any of embodiments 37 to 40 with a cell.

[0347] Embodiment 42 is the method of embodiment 41, where contacting the transfection composition with a cell includes administering the transfection composition to a subject.

[0348] In the description and the claims, the term “and / or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended — i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by 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.).

[0349] In the preceding description, particular embodiments may be described in isolation for clarity. Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” “one or more embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment 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 embodiments. Thus, features described in the context of one embodiment may be combined with features described in the context of a different embodiment except where the features are necessarily mutually exclusive.

[0350] In several places throughout the above description, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. As used herein, “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” or the like are used in their open-ended inclusive sense, and generally mean “include, but not limited to,” “includes, but not limited to,” or “including, but not limited to.” Further, wherever embodiments are described herein with the language “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” and the like, otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. The term “consisting of’ means including, and limited to, that which follows the phrase “consisting of.” That is, “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. The term “consisting essentially of’ indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

[0351] For any method disclosed herein that includes discrete steps, the steps may be performed in any feasible order. And, as appropriate, any combination of two or more steps may be performed simultaneously.

[0352] As used herein, the word “exemplary” means to serve as an illustrative example and should not be construed as preferred or advantageous over other embodiments.

[0353] As used herein, the terms “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.

[0354] As used herein, the terms “formed from” and “polymerized from” are open ended and may include other components that may not be expressly described relative to the subject that is formed from or polymerized from the stated components. For example, a polymer formed from or polymerized from a particular monomer or monomers may include capping groups or other groups not expressly mentioned.

[0355] As used herein, “alkyl” or “alkyl group” refers to a fully saturated, straight, or branched hydrocarbon chain having from one to twelve carbon atoms, and which is attached to the rest of the molecule by a single bond.

[0356] When a group is present more than once in a formula described herein, each group is independently selected, whether specifically stated or not. For example, when more than one Y group is present in a formula, each Y group is independently selected. Furthermore, subgroups contained within these groups are also independently selected. For example, when each Y group contains an Rl, each R1 is also independently selected.

[0357] As used herein, the symbols “(hereinafter can be referred to as “a point of attachment bond or point of attachment”) when used in the context of a compound, denote a bond that is a point of attachment between two chemical entities, one of which is depicted as being attached to the point of attachment bond and the other of which is not depicted as being attached to the point of attachment bond. For example, “” and-XY” indicate that the chemical entity “XY” is bonded to another chemical entity via the point of attachment bond. The point of attachment of the chemical group to the compound may be described in several ways. For example, in some embodiments, the organic group may be described as the monovalent or radical of the respective functional group (e.g., alkyl for alkane, alkenyl for an alkene containing group). In some embodiments, where a general formula is shown with a covalent bond connecting the chemical group to a compound, the chemical group may be described as the common functional group that is or is contained within the chemical group. For example, if the chemical group R is described relative to the formula CH3CH2CH2-R, the chemical group may be described, for example, as an alkene.

Claims

CLAIMSWhat is claimed is:

1. A compl ex compri sing : a cationic diblock polymer comprising: an acrylate block; and an acrylamide block, the acrylamide block comprising a repeating unit comprising a cationic containing groups of the formula (i), (ii), (iii), or (iv)wherein: b is an integer from 0 to 5; z and z’ are independently an integer from 0 to 5; andQ1and Q2are each independently a heteroatom or a group that includes a heteroatom; and an oligonucleotide cargo.

2. The complex of claim 1, wherein the acrylamide block comprises the repeating unit of Formula (I)wherein n is the number of acrylamide repeating units, j ’ is an integer from 0 to 5, and R is the cationic containing group.

3. The complex of claim 1 or 2 wherein Q1and Q2are independently N, NH, NH2, NH3, O, or OH.

4. The complex of any one of claims 1 to 3, wherein the cationic containing group is of Formula (A6), (A7), (A8), (A9), or (A10):or an ionized version thereof.

5. The complex of any one of claims 1 to 4, wherein diblock polymer is of Formula (II)wherein: j is an integer from 0 to 10; m is the number of acrylate repeating units; n is the number of acrylamide repeating units;RAis the cationic group; and j ’ is an integer from 0 to 5.

6. The complex of any of claims 1 to 5, wherein complex further comprises a neutral diblock polymer comprising: an acrylate or methacrylate block; and a second block comprising -(O-CH2-CH2)- repeating unit.

7. The complex of claim 6, wherein the neutral diblock polymer is of the formulawherein j is an integer from 0 to 10, x is the number of acrylate repeating units and w is the number of-(O-CH2-CH2)w)- repeating units.

8. The complex of any of claims 1 to 7, wherein the complex further comprises an anionic diblock polymer comprising: an acrylate or methacrylate block; and an acrylamide polymer, the acrylamide polymer comprising a repeating unit comprising an anionic group.

9. The complex of claim 8, wherein the anionic group is of Formula:, or an nionized version thereof.

10. The complex of claims 8 or 9, wherein the anionic diblock polymer is of Formula:wherein: j is an integer from 0 to 10; q is the number of acrylate repeating units lacking an anionic group: z is the number of repeating units comprising an anionic group; and RDis the anionic containing group.11 . The complex of claim 10, wherein the anionic diblock polymer is of formulaor an ionized version thereof, wherein j is an integer from 0 to 5, q is the number of acrylate repeating units lacking an anionic group, and z is the number of repeating units containing an anionic group.

12. The complex of any of claims 1 to 5, wherein the complex further comprises the anionic diblock polymer of any of claims 8 to 11.

13. The complex of claim 12, wherein the weight or mole ratio of the cationic diblock polymer to the anionic polymer is 1 part to 10 parts of the cationic diblock polymer for every 1 part of the anionic diblock polymer; 1 part to 3 parts of the cationic diblock polymer for every 1 part of the anionic diblock polymer; 2 part to 5 parts of the cationic diblock polymer for every 1 part of the anionic diblock polymer; or 8 part to 10 parts of the cationic diblock polymer for every 1 part of the anionic diblock polymer.

14. The complex of any of claims 1 to 5, wherein the complex further comprises the neutral diblock polymer of any of claims 6 to 7 and the anionic diblock polymer of any of claims 8 to 11.

15. The complex of claim 14, wherein the weight or mole ratio of the cationic diblock polymer and the neutral diblock polymer to the anionic polymer is 1 part to 10 parts of the cationic diblock polymer and the neutral diblock polymer for every 1 part of the anionic diblock polymer; 1 part to 3 parts of the cationic diblock polymer and the neutral diblock polymer for every 1 part of the anionic diblock polymer; 2 part to 5 parts of the cationic diblock polymer and the neutral diblock polymer for every 1 part of the anionic diblock polymer; or 8 part to 10 parts of the cationic diblock polymer and the neutral diblock polymer for every 1 part of the anionic diblock polymer.

16. The complex of any one of claims 1 to 15, wherein the oligonucleotide comprises mRNA or DNA.

17. The complex of any one of claims 1 to 16, wherein the oligonucleotide comprises a plurality of phosphate groups, the polymer comprises a plurality of amines; and a ratio of moles of the amine to moles of the plurality of phosphate groups is 2 to 20 or 5 to 10.

18. A transfection composition comprising the complex of any one of claims 1 to 17.

19. The transfection composition of claim 18, wherein the composition further comprises a transfection medium.

20. A method of transfecting a cell, the method comprising contacting the transfection composition of any one of claims 18 to 19 with a cell.

Images