Protamine molecule and its use

Abstract

This invention relates to protamine molecules. More specifically, this invention relates to protamine molecules and their use in the delivery of payload molecules.

Description

[Technical Field]

[0001] This invention relates to protamine molecules. More specifically, this invention relates to protamine molecules and their use in the delivery of other molecules. [Background technology]

[0002] Protein drugs have gained a strong foothold in the global pharmaceutical market, holding a 20% share, due to their increased selectivity and extended half-lives compared to small molecule drugs.[1] While oral administration is sometimes preferred, the harsh gastrointestinal environment presents a particular biological barrier, which includes extreme acidity (pH 1.0–2.0) and active digestive enzymes, minimizing the oral absorption of protein drugs.[2] Limited by their inherently poor membrane permeability and stability in the gastrointestinal tract, protein and peptide drugs are often delivered via parenteral routes, e.g., intravenous (iv), subcutaneous (sc), and intramuscular (im) injections, which are associated with discomfort and injection site reactions.[3] This presents challenges with patient adherence, particularly in patients with chronic conditions requiring long-term drug therapy.[4]

[0003] Compared to oral delivery, the nasal environment is less harsh and allows for bypassing first-pass metabolism [5]. However, several barriers have been reported for nasal delivery of proteins and peptides, and these barriers include the mucosal layer, physical barriers that limit the diffusion of macromolecules, and epithelial barriers containing cell junctions that prevent paracellular penetration [2]. Disruption of adhesion junctions has been shown to result in increased penetration of pathogens, which can lead to rhinitis [7].

[0004] Sublingual administration has emerged as a promising option, characterized by increased patient preference [8], as well as reduced formulation requirements, such as pH and osmotic pressure [9]. Sublingual delivery also boasts convenience and rapid absorption, which is particularly beneficial in emergency situations [9]. However, the thick mucinous fluid secreted by the sublingual glands, and the tight epithelial layer beneath the mucus, minimize drug absorption, especially for macromolecules

[10] .

[0005] Various techniques have been developed to enhance the transmucosal and transcytotic delivery of biomolecules

[11] . Absorption enhancers, such as membrane lipids and proteins or surfactants that reconstitute the conformation of cell binding, have been shown to facilitate the transmucosal delivery of macromolecules

[12] and particles [13, 14]. However, absorption enhancers can risk irreversible binding damage in the epithelial layer

[15] .

[0006] Mucoadhesive materials, such as chitosan (CS), zein, and CS-N-arginine / alginate, can be incorporated into formulations to improve drug retention in the mucosa for enhanced absorption [16 - 18]. Due to the rapid mucociliary clearance in the nasal cavity (7 - 15 minutes), mucoadhesive materials and penetration enhancers are often used in drug formulations to enhance nasal absorption

[19] . However, these materials exhibit limited activity in penetrating the epithelium [20, 21].

[0007] Cell-penetrating peptides (CPPs) have been studied to improve the transmembrane delivery of macromolecules

[22] , which includes those via the nasal route

[78] . CPPs rich in arginine are used to facilitate the transmucosal delivery of macromolecules through the formation of transient pores in the cell membrane and the promotion of intracellular translocation

[23] .

[0008] CPPs contain up to 30 amino acids, the majority of which are positively charged, such as arginine and lysine. Commonly used CPPs include poly(arginine)8(R8), TAT, and iRGD. Cationic CPPs are involved in interactions with anionic cell membranes, which facilitates several mechanisms for intracellular delivery of macromolecules, including direct translocation across the membrane, transient pore formation in the membrane, actin rearrangement to reduce membrane mechanical tension and increase membrane permeability

[24] , and promotion of cytotubule formation and endocytosis [25, 26]. At low concentrations (≤5 μM), CPPs were translocated internally by cells, mainly via endocytosis, and accumulated in lysosomes, but rapid cytoplasmic release occurred at higher concentrations (≥10 μM)

[27] . Arginine-rich CPPs induced membrane multilayering and subsequently entered the cell via the formation of fusion pores

[28] . When CPP chemically attaches to proteins, conjugates at high concentrations (50–150 μM) have been shown to interact with anionic cytoskeletal components, resulting in irreversible actin and tubulin aggregation

[29] , followed by irreversible changes in the cytoskeleton and cellular ruffling [26, 30]. The intact actin cytoskeleton is required for intracellular translocation, and CPP can modify the actin cytoskeleton to influence cellular processes, including CPP-mediated intracellular delivery

[31] . In addition, actin rearrangement is a common mechanism of action for absorption enhancers for oral delivery

[13] . Recent studies have shown that cell membrane tension is regulated and maintained by the actin network. Decreased F-actin alignment induces membrane invagination and reduces membrane tension, which facilitates cellular endocytosis and nanoparticle penetration

[24] . Furthermore, reduced cortical tension is favorable for tubular formation, which enables the entry of macromolecules

[31] . F-actin aggregation has been observed in cells treated with arginine-rich CPP

[29] and is associated with increased transport of endosomes and lysosomes

[32] . Microtubule formation in cells is associated with CPP treatment and CPP-mediated endocytosis and intracellular drug delivery [31, 33].

[0009] However, CPP-mediated protein delivery can be limited by payload size and may be susceptible to endosomal entrapment [34, 35]. Fusing the protein payload to CPP increases mucosal adhesion and enhances endosomal escape [36, 37], but such methods require delicate chemistry or protein engineering to ensure consistent production of the conjugate and release of the active drug [38, 39, 77].

[0010] Protamine is an FDA-approved injectable pharmaceutical ingredient and an antidote for heparin overdose. Protamine is isolated from fish, usually salmon sperm, and has an average molecular weight of 4,000 - 5,000 Da. Protamine and low molecular weight protamine (LMWP) prepared by enzymatic digestion have been used to facilitate intracellular delivery of biomolecules including nucleic acids and proteins

[40] , but the strategy has focused on forming complexes with nucleic acids via charge-charge interactions or chemical conjugation with proteins to facilitate their delivery [41 - 44]. Protamine is arginine-rich and contains a nuclear translocation sequence [as45].

[0011] Chronic sinusitis with nasal polyps (CRSwNP) is classified as a type 2 inflammatory disease, affecting up to 4% of the population in the United States [46, 47]. If left untreated, the disease can lead to significant inflammation of the lower respiratory tract. Current standard treatments include sinus surgery, antibiotics, and oral corticosteroids

[48] , but recurrence rates are high and patients experience numerous side effects

[49] . IL-4 and IL-13 are major cytokines that mediate the progression of nasal polyposis, and therefore monoclonal antibodies (mAbs) that block the IL-4, IL-13, or both pathways have become emerging therapeutics for CRSwNP, with treatments including omalizumab, reslizumab, mepolizumab, and dupilumab

[50] . Dupilumab, administered via weekly subcutaneous injections for 16 weeks

[50] or twice-weekly subcutaneous injections for 52 weeks

[51] , blocks the IL-4 receptor α subunit (IL-4Rα). This results in inhibition of both the IL-4 and IL-13 inflammatory pathways

[52] , but these frequent, long-term injections cause injection site reactions in 40% of patients

[53] . In addition, high doses of the mAb are required to achieve effective local concentrations in nasal tissue. This not only increases the risk of systemic side effects but also increases the cost of the therapy

[54] . [Overview of the project]

[0012] This invention relates to protamine peptides and molecules, as well as their uses.

[0013] In one embodiment, a protamine molecule is provided comprising a first protamine peptide, wherein the first protamine peptide is conjugated to one or more of the following: a lipid, a hydrophobic moiety, a polymer containing an amino group, or an additional protamine peptide.

[0014] In some embodiments, the lipid may be a fatty acid, a lipid amine, or a lipid carboxyl, and may further comprise a linker, such as polyethylene glycol (PEG) or N-hydroxysuccinimide (NHS).

[0015] In some embodiments, the lipid may be palmitic acid, stearic acid, 1,2-phosphatidylethanolamine (PE), dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), DPPE-NHS, DSPE-NHS, or DOPE-NHS.

[0016] In some embodiments, the hydrophobic portion may be allyl glycidyl ether (AGE).

[0017] In some embodiments, the polymer containing the amino group may be poly(amidoamine) (PAMAM), polyethyleneimine (PEI), or polylysine (PLL).

[0018] In some embodiments, the polymer containing amino groups may include crosslinking agents, such as N-succinimidyl S-acetylthioacetate (SATA), or N-γ-maleimidobutyryloxysuccinimidone (GMBS), or succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).

[0019] In some embodiments, the protamine molecule may be protamine-stearic acid, protamine-PAMAM, protamine-DMPE, protamine-GMBS-PAMAM, protamine-DMPE, protamine-AGE, protamine-SATA-PAMAM, protamine-C18, protamine-C16, protamine-PEI, or protamine-PLL.

[0020] In some embodiments, the first protamine peptide may be conjugated to one or two additional protamine peptides, resulting in a protamine dimer or protamine trimer.

[0021] In some embodiments, the first protamine peptide and the additional protamine peptide may be the same or different.

[0022] In some embodiments, the first protamine peptide and / or additional protamine peptide may be a protamine salt, for example, a protamine sulfate from salmon.

[0023] In some embodiments, the first protamine peptide or additional protamine peptide may comprise any one of the amino acid sequences or conserved substitutions of SEQ ID NOs: 1-4.

[0024] In some embodiments, the protamine molecule may include an alpha-helix and / or a beta-sheet.

[0025] In some embodiments, protamine molecules can self-assemble into nanostructures.

[0026] In some embodiments, protamine molecules can increase density in the cell membrane, increase the concentration of protamine in the cell membrane, and / or increase the interaction between protamine and the cell membrane.

[0027] The protamine molecules described herein may be provided in a composition, for example, a pharmaceutical composition.

[0028] In an alternative embodiment, a method for delivering a payload molecule to a cell is provided, comprising contacting the cell with the payload molecule in combination with a protamine molecule described herein. In some embodiments, delivery may be non-parenteral delivery.

[0029] In an alternative embodiment, a method is provided for non-injection delivery of a large payload molecule to a cell, comprising contacting the cell with the payload molecule in combination with a protamine peptide or a protamine molecule.

[0030] In some embodiments, non-injectable delivery may be intranasal, sublingual, oral, buccal, rectal, vaginal, intravitreous, local, or into the skin, eye, brain, or lung. In some embodiments, non-injectable delivery may be transmucosal, for example, transepithelial or lamina propria, or transcellular. In some embodiments, transmucosal delivery is not by needle-based injection.

[0031] In some embodiments, the payload molecule may be delivered to the nucleus of a cell and / or substantially bypass the cell's lysosomes.

[0032] In some embodiments, the payload molecule may be a protein, peptide, peptide analog, or small molecule. In some embodiments, the payload molecule may be provided in a physical mixture with a protamine molecule. In some embodiments, the payload molecule may not form a complex with the protamine molecule. In some embodiments, the payload molecule may be an antibody, growth hormone, insulin, or semaglutide.

[0033] In an alternative embodiment, a method is provided for making a cell membrane permeable by contacting the cell membrane with a protamine peptide and / or protamine molecule as described herein. In some embodiments, the permeability may be transient and / or reversible.

[0034] In an alternative embodiment, a method for transfecting cells is provided, comprising contacting the cell membrane with a protamine peptide and / or protamine molecule as described herein.

[0035] In an alternative embodiment, a method is provided for treating or preventing a condition that would benefit from non-injectable delivery of a payload molecule, the method comprising administering the payload molecule together with the protamine peptide and / or protamine described herein to a subject in need thereof.

[0036] In some embodiments, the payload molecule may be administered simultaneously with or at different times to the protamine peptide and / or protamine molecule. In some embodiments, the protamine peptide and / or protamine molecule may be administered before the administration of the payload molecule.

[0037] In an alternative embodiment, the use of the protamine peptides and / or protamine molecules described herein is provided for treating or preventing conditions that would benefit from non-injectable delivery of the payload molecule.

[0038] In some embodiments, the condition may be diabetes, sinusitis, an eye condition, or a skin condition.

[0039] This summary does not necessarily describe all the features of the present invention.

[0040] These and other features of the present invention will become more apparent from the following description, in which the accompanying drawings will be referenced. [Brief explanation of the drawing]

[0041] [Figure 1] A and B are graphs showing the cytotoxicity of protamine in Caco2 (A) and RPMI2650 (B) cells. [Figure 2]This graph shows FITC-BSA uptake by RMPI2650 cells in the presence or absence of protamine from different sources. FITC-positive cells were quantified by FACS. (Protamine 1: Protamine sulfate from salmon, Protamine 2: Protamine sulfate from herring, Protamine 3: Protamine from salmon, Protamine 4: Protamine chloride from salmon). Data = mean ± SD. (n≧3). ****p<0.0001. [Figure 3] A–D are graphs showing intracellular delivery of FITC-BSA in RPMI2650 (A, C) and Caco2 (B, D) cells. Quantitative analysis by ImageJ of intracellular FITC-BSA (A, B) and FITC-BSA at different sites in the cytosol (C, D). Data = mean ± SD. (n≧5). Kruskal-Wallis test, followed by Dunnett's post-hoc test, was used to determine statistical significance (*p<0.05, **p<0.01). [Figure 4] A and B are graphs showing the delivery of Alexa488-anti-SIRT1 monoclonal antibody (mAb) to RPMI2650 cells in the presence or absence of protamine or R8. (A) Quantitative analysis of intracellular fluorescence of mAb (n≧4). Kruskal-Wallis test, followed by Dunnett's post-hoc test, was used to determine statistical significance (*p<0.05, **p<0.01). (B) Percentage of mAb fluorescence in lysosomes, nuclei, and cytosol, analyzed by ImageJ (n≧5). Data = mean ± SD. [Figure 5] A-C are graphs showing the actin cytoskeleton in RPMI2650 cells after different treatments. (A) Overall F-actin expression (n≧9). (B) Quantification of cell spread area by ImageJ (n≧6). (C) Quantification of % of cells with F-actin aggregates (n≧8). Significance determined by the Kruskal-Wallis test, followed by Dunnett's post-hoc test. Data = mean ± SD. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and ns, no significant difference. [Figure 6]Graphs A and B illustrate the characterization of microtubule formation in RPMI2650 after R8 and protamine treatment. (A) Number of microtubules per cell (n≧5), (B) Quantitative analysis of total microtubule length (n≧5). One-way ANOVA, followed by Tukey post-hoc test for significance. Data = mean ± SD. Statistical significance: *p<0.01, **p<0.01, ***p<0.001, and ns, no significant difference. [Figure 7] A and B are graphs showing transcellular delivery of FITC-BSA in vitro and in vivo. (A) Cells were dissociated from spheroids and analyzed by FACS (n≧4). (B) Plasma concentrations of Cy7-BSA after intranasal administration of Cy7-BSA in the presence of protamine or R8. Only saline and Cy7-BSA were included as controls (n≧6). Significance was determined by one-way ANOVA, followed by Tukey's post-hoc test. Data = mean ± SD. Statistical significance: **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 8] Graphs A-E show blood glucose (BG) levels in STZ-induced diabetic mice after intranasal (in) treatment with different protamine / insulin preparations. (A) Insulin was dissolved in water at pH 3.2, mixed with protamine in different ratios, and then administered intranasally (1 mg / kg insulin dose). (B) Insulin was dissolved in water at various pH levels, mixed with protamine in a 1:3 weight ratio (insulin:protamine), and then administered intranasally (1 mg / kg insulin dose). (C) The optimal protamine / insulin preparation was prepared as described above and delivered to mice in 0.5, 1, and 5 mg / kg. (D) Comparison of the BG-reducing effects of various preparations (1 mg / kg insulin dose). (E) Serum insulin levels (1 mg / kg insulin dose) at 0, 0.5, 1, 2, and 4 hours in STZ-induced diabetic mice after in treatment with various preparations. Data = mean ± SD (n≧4). Arrows indicate drug administration. [Figure 9] This graph shows the sublingual delivery of semaglutide. [Figure 10] This graph shows the sublingual delivery of semaglutide. [Figure 11] Graphs A-C show that sublingual delivery of GLP-1 / protamine provided better BG control compared to GLP-1 delivered IV or sublingually alone. [Figure 12] This graph shows the concentration of Cy7-BSA in plasma at different time points. [Figure 13] This graph shows the plasma concentrations of 647-dupilumab at different time points. [Figure 14] This graph shows the symptoms in a mouse model of chronic sinusitis. [Figure 15] This graph shows polypoid lesions and epithelial destruction in a mouse model of chronic sinusitis. [Figure 16] Graphs A-D show that protamine / CD124-treated mice exhibited reduced levels of INF-gamma, IL-1β, IL-17α, and TNF-α in the nose compared to PBS controls. [Figure 17] This graph shows the critical micelle concentration (CMC) of DMPE-protamine. [Figure 18] This graph shows the cellular uptake of CRISPR / Cas9-gRNA. [Figure 19] This is a schematic diagram showing a two-step reaction scheme for the preparation of protamine dimers. [Figure 20] This is a schematic diagram showing a two-step reaction scheme for the preparation of protamine-PAMAM. [Figure 21] This is a schematic diagram showing the reaction scheme for the preparation of protamine trimers. [Figure 22] This graph shows that protamine-PAMAM and (protamine)2 increased intracellular FITC-BSA delivery compared to protamine alone. [Figure 23] This graph shows that protamine-PAMAM and protamine-AGE increased the efficiency of GFP pDNA transfection in HEK 293T cells compared to protamine alone. [Figure 24] Graphs A-D show that DMPE-protamine, (protamine)2, and (protamine)3 preparations for insulin can reduce blood glucose levels more effectively than protamine / insulin preparations. [Figure 25] Graphs A-F show that PAMAM-protamine-SATA and protamine-AGE preparations for intranasal insulin delivery exhibited more consistent performance compared to protamine preparations in reducing BG in STZ diabetic mice. [Figure 26] This graph shows that protamine-AGE and PAMAM-AGE 0.35-protamine enhance the systemic absorption of Alexa647-dupilumab via intranasal delivery. [Figure 27] A and B are graphs showing the intracellular delivery of protamine sulfate by sequence as identified. [Figure 28] This graph shows sequences identified from protamine sulfate in lysosomes. [Figure 29] A and B are graphs showing Cas9 RNP / sgRNA delivery to HEK 293 GFP cells in the presence or absence of protamine or protamine-AGE. Quantitative analysis of intracellular fluorescence of EGFP and Cas9 RNP, respectively, was analyzed by Image J. Data = mean ± SD. (n≧3). *p<0.05, **p<0.01, ****p<0.0001. [Figure 30] This graph shows that protamine-based formulations enhanced nasal retention of mAbs. [Figure 31] A-G show the characterization of protamine and its derivatives. Synthetic schemes of C18-P(A) and P2(B). Mass spectra of protamine(C), C18-P(D), and P2(E). Insulin (5.7 kDa) was used as an internal standard. FTIR(F) and CD(G) spectra of protamine, C18-P, and P2. [Figure 32]This graph shows the cytotoxicity of protamine, C18-P, and P2 in human primary gingival keratinocytes. [Figure 33] Graphs A and B show the effects of protamine, C18-P, and P2 on IgG delivery to the cell membrane and intracellularly. Quantitative analysis by ImageJ of (A) intracellular AF647-IgG and (B) AF647-IgG co-localized with lysosomes. Data = mean ± SD. (n≧5). (**p<0.01, ****p<0.0001, no significant difference in ns). [Figure 34] This graph shows the uptake of AF647-IgG by primary human gingival keratinocytes in the presence or absence of protamine, C18-P, and P2. AF647-positive cells were quantified by FACS. Data = mean ± SD. (n≧3). ****p<0.0001. [Figure 35] A and B are graphs showing intracellular delivery of FITC-BSA in human primary gingival keratinocytes. (A) Quantitative analysis of intracellular FITC-BSA by Image J, and (B) FITC-BSA-positive cells quantified by FCM. Data = mean ± SD. (n≧5). (*p<0.05, **p<0.01, ****p<0.0001). [Figure 36] A and B are graphs showing spheroid penetration of AF647-IgG delivered by protamine, C18-P, and P2. (A) Heatmap of fluorescence intensity of AF647-IgG distributed around the spheroid. (B) Cells dissociated from the spheroid and analyzed for AF647-IgG+ cells by FCM. Data = mean ± SD. (n≧5). (**p<0.01, ****p<0.0001). [Figure 37] A and B are graphs showing spheroid penetration of FITC-BSA delivered by protamine, C18-P, and P2. (A) Heatmap of fluorescence intensity of FITC-BSA distributed around spheroids. (B) Cells dissociated from spheroids and analyzed for FITC-BSA+ cells by FACS. Data = mean ± SD. (n≧5). (**p<0.01, ****p<0.0001). [Figure 38] Graphs A and B show the gene editing efficiency mediated by Cas9-RNP delivered by protamine, C18-P, and P2 in eGFP HEK 293 cells. Cells were dissociated from spheroids, and Cas9-RNP-positive cells (A), as well as RNP+ and eGFP- cells (B), were quantified by FCM. Data = mean ± SD. (n≧5). (***p<0.001, ****p<0.0001). [Figure 39] A and B are graphs showing the gene editing efficiencies mediated by protamine C18-P and P2 in the eGFP HEK 293 cell line. (A) eGFP intensity from Figure 4A was quantified by Image J. (B) eGFP knockdown efficiency was analyzed by FCM. [Figure 40] Graphs A and B show the penetration of AF647-IgG delivered by protamine, C18-P, or P2 in human sublingual tissue replacements. Confocal images were analyzed in ImageJ for the overall fluorescence intensity of AF647-IgG (A) and the fluorescence intensity of AF647-IgG relative to the distance from the apical membrane (B). Data = mean ± SD. (n≧5). (**p<0.01, ***p<0.001). [Figure 41] A–C are graphs showing sublingual penetration of AF647-IgG delivered by protamine, C18-P, and P2 in mice. Fluorescence intensity of AF647-IgG collected at 0.5 hours (A), 2 hours (B), and 4 hours (C) in relation to the distance from the tissue surface in the sublingual tissue. [Figure 42]Graphs A and B show the effects of blood glucose (BG) control in mice. (A) Comparison of blood glucose levels in STZ-induced diabetic mice after sublingual (sub.) or subcutaneous (sc) treatment with various insulin preparations. Insulin was dissolved in water at pH 3.2, mixed with protamine, C18-P, or P2, and then administered sublingually (insulin dose = 5 mg / kg for sub. and 1 mg / kg for sc). Black arrows indicate preparation administration. (B) Comparison of BG control effects of various semaglutide preparations (drug dose = 60 μg / kg for sc and 500 μg / kg for sub.). Black arrows indicate preparation administration, and green arrows indicate glucose injection at 1.5 g / kg. Data = mean ± SD (n ≥ 4). Statistical analysis: protamine vs. C18-P (blue asterisk), protamine vs. P2 (green asterisk). *p<0.05, **p<0.01. [Figure 43] Graphs A-C show the pharmacokinetics of various sublingual (sub.) delivered protein formulations compared to subcutaneous (sc) protein administration. Plasma was collected at 0, 0.5, 2, and 4 hours. Proteins were lysed in PBS, mixed with protamine, C18-P, or P2, and then administered sublingually (doses of protamine, C18-P, and P2 were 6 mg / kg). (A) Recombinant human growth hormone (rhGH). (rhGH dose = 500 μg / kg). (B) Cy7-BSA. (Cy7-BSA dose = 20 mg / kg). (C) AF647-IgG. (AF647-IgG dose = 1.3 mg / kg). Data are presented as mean ± SD (n≧3). Statistical analysis of area under the curve (AUC): payload only vs. sc (black asterisk), payload only vs. C18-P (blue asterisk), payload only vs. P2 (green asterisk). *p<0.05, **p<0.01, and ****p<0.0001. No significant differences were observed between the CPP formulation and the sc control. [Figure 44]This graph shows the in vivo distribution of 111In-IgG delivered intranasally by different formulations. Quantitative analysis of the central nasal cavity as volume of interest (VOI) for both experimental groups. Results are provided as standardized uptake values ​​(SUV) + / - their standard deviations. [Figure 45] Graphs showing the in vivo efficacy of different αCD124 formulations in mice with moderate CRSwNP (A-F). (A) Number of times the nose was touched in 10 minutes for each mouse (N≧5). (B) Surface area ratio (airway / bone) obtained from micro-CT images. (C) Number of nasal polypoid lesions per mouse, counted from H&E images (N≧5). (D) Number of epithelial destructions per field of view (40x magnification), counted (N≧10). IgE concentrations in plasma (E) and nasal tissue homogenate (F) from moderate CRSwNP mice after different treatments (N≧3~7). [Figure 46] A-K are graphs showing the levels of type 2 inflammatory biomarkers in moderate CRSwNP mice treated with various formulations. [Figure 47] This is a schematic diagram showing the reaction scheme for the preparation of Dendri-P. [Figure 48] This graph shows the FTIR spectra of protamine, Nano-P, and Dendri-P. [Figure 49] A and B are graphs showing the penetration of AF-αCD124 delivered by protamine, Nano-P, and Dendri-P into nasal tissue in mice. Confocal images were analyzed by ImageJ for the overall fluorescence intensity of AF-αCD124 in nasal tissue (A) and the fluorescence intensity of AF-αCD124 relative to the distance from the apical membrane (B). Data = mean ± SD. (n≧5). (**p<0.01, ****p<0.0001). [Figure 50] This graph shows the quantification of epithelial thickness per field of view from different treatment groups. Each group consists of 4-7 mice with N≧15 fields of view. [Figure 51]Graphs A and B show the in vivo efficacy of different αCD124 formulations in severe CRSwNP mice. The number of nasal polyp-like lesions per field of view (A) (N≧4) and epithelial destruction per field of view (40x magnification) (b) (N≧10) were counted. [Figure 52] This graph shows eosinophil infiltration of paranasal sinus tissue in severe CRSwNP mice after treatment with various formulations. The graph shows the count of eosinophils per field of view in tissue sections collected from CRSwNP mice after various treatments. Fields of view N≧15 from 4-7 mice per group. [Figure 53] A and B are graphs showing Masson's trichrome staining and goblet cells in mouse nasal epithelium in nasal tissue of CRSwNP mice after various treatments. (A) Quantification of collagen area per field of view from different treatment groups. N≧15 fields of view from 4-7 mice per group. (B) Quantification of goblet cells (blue) per image field of view from different treatment groups. N≧15 fields of view from 4-7 mice per group. [Figure 54] A-N is a graph showing the levels of type 2 inflammatory biomarkers in CRSwNP mice treated with various formulations. [Figure 55] A-U are graphs showing the long-term in vivo safety evaluation of protamine, Nano-P, and Dendri-P in mice. (A) Organ / body weight ratios of different groups of experimental animals. (B-U) Effects of protamine, Nano-P, and Dendri-P on hematological and blood biochemical parameters. [Modes for carrying out the invention]

[0042] In general, this disclosure provides, in part, a protamine molecule and its uses.

[0043] Accordingly, the present disclosure provides a protamine molecule comprising a polyvalent protamine peptide and a protamine peptide, wherein the polyvalent protamine peptide and the protamine peptide are conjugated to a polymer comprising a lipid, a hydrophobic moiety, and / or an amino group.

[0044] Protamine is a clinically used, arginine-rich peptide, an FDA-approved injectable pharmaceutical ingredient, and an antidote for heparin overdose.

[0045] As used herein, “protamine peptide” refers to protamine peptides isolated from natural sources or artificially synthesized protamine peptides, as well as fragments and / or mixtures thereof. In some embodiments, the protamine peptide may be in the form of a suitable salt, e.g., a sulfate, e.g., a sulfate from salmon. In some embodiments, the protamine peptide may be commercially available.

[0046] In some embodiments, protamine peptides isolated from natural sources ("natural protamine peptides") may be isolated from the sperm and / or fertilized eggs of fish, such as salmon (salmin), rainbow trout (iridine), herring (kurupein), sturgeon (sturin), or mackerel or tuna (chinnin).

[0047] Protamine peptides can generally be basic (pK a>10). In some embodiments, the protamine peptide may contain up to about 70% arginine or other basic amino acids, such as lysine. In some embodiments, the protamine peptide may have an amino acid length of about 30 to about 110, or any integer amino acid length between those two. In some embodiments, the protamine peptide may have an amino acid length of about 20 to about 150, or any integer amino acid length between those two. In some embodiments, the protamine peptide may consist of about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 135, 140, 145, 150 or more amino acids, and may contain at least 20%, 30%, 40%, 50%, 60%, 70% arginine and / or basic amino acids. In some embodiments, the protamine peptide may have 30 to 32 amino acids, of which 21 to 22 may be arginine. In some embodiments, the protamine peptide may have 32 amino acids, of which 21 may be arginine. In some embodiments, protamine peptides may have an average molecular weight of about 4,000 to about 5,000 Da. It is understood that the composition of natural protamine may vary depending on the source. Protamine nuclei and amino acid sequences have been identified from several species. In some embodiments, the protamine peptide may be mouse or human protamine.

[0048] In some embodiments, the protamine peptide is, without limitation, GenBank acceptance numbers AAG27965.1, AAG27962, AAG27961.1, AAG27960.1, AAG27964.1, AAG27963.1, AAG27959.1, AAG27952.1, AAG27958.1, AAG27956.1, AAG27954.1, AAG27953 It may have the amino acid sequence described in any one of the following: .1, AAG27957.1, AAG27951.1, AAG27955.1, AAG27950.1, AAC15630.1, AAC15629.1, AAC15628, AAC15627.1, AAB35760.1, AAB19741.1, AAB34978.1, AAB34977.1, NP_002752.1.

[0049] In some embodiments, the protamine peptide may be encoded, non-limitingly, by a nucleic acid sequence described in GenBank acceptance number X07511 or X01204.

[0050] In some embodiments, the protamine peptide may have the amino acid sequence or its conserved substitutions as described in any one of SEQ ID NOs: 1 to 4, as well as fragments and / or mixtures thereof.

[0051] As used herein, the terms “conservative amino acid substitution” or “conservative substitution” refer to a substitution of one amino acid at a given position in a peptide for another amino acid, wherein the substitution can be made without substantial loss of the relevant function.

[0052] Conservative substitutions may include the following: JPEG2026509413000001.jpg11897

[0053] When making such changes, substitutions of similar amino acid residues may be made based on the relative similarity of the side chain substituents, such as their size, charge, hydrophobicity, and hydrophilicity, and such substitutions may be assayed for their effects on the peptide function by conventional tests.

[0054] Peptides or peptide analogs can be synthesized by standard chemical techniques, for example, by automated synthesis using solution or solid-phase synthesis methodologies. Automated peptide synthesizers are commercially available and use techniques well known in the art. Peptides and peptide analogs can also be prepared using recombinant DNA techniques using standard methods.

[0055] As used herein, “protamine molecule” means a protamine peptide conjugated to another molecule, e.g., a lipid, a hydrophobic moiety, a polymer, or an additional protamine peptide. In some embodiments, the protamine molecules described herein may contain secondary structures, e.g., alpha-helices and / or beta-sheets. In some embodiments, the protamine molecules described herein may not be substantially in a random coil configuration, for example, less than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% may be in a random coil configuration. In this regard, and without being bound by any particular hypothesis, alpha-helices are associated with pore formation in cell membranes

[55] , beta-sheets with peptide self-assembly [56, 57], and random coils with disordered orientation

[58] .

[0056] The protamine molecules described herein may be capable of self-assembling into nanostructures, such as spheres or rods. Without being bound by any particular theory, such nanostructures may increase the local density and concentration of protamine molecules and / or increase the interaction between protamine molecules and cell membranes.

[0057] The protamine molecules described herein may exhibit increased interaction with cell membranes, and / or increased cell penetration, and / or increased cell permeability.

[0058] As used herein, “lipid” refers to a compound that is soluble in a nonpolar organic solvent. Those skilled in the art will understand that it is possible to readily determine suitable lipids or their conjugates for use in the protamine molecules described herein. In some embodiments, the lipids described herein contain an amine or carboxylate group. Suitable lipids include, but are not limited to, fatty acids, lipid-amines, lipid-carboxyl groups, etc. In some embodiments, the lipids described herein may be modified with a linker, e.g., polyethylene glycol (PEG)

[59] or N-hydroxysuccinimide (NHS), which results in lipid-PEG-amines, lipid-PEG-containing carboxyl groups, lipid-containing NHS, or lipid-PEG-containing NHS, etc. Lipid-conjugated protamine molecules may exhibit increased interaction with cell membranes, and / or increased cell penetration, and / or increased cell permeability. Exemplary lipids include, but are not limited to, palmitic acid, stearic acid, 1,2-phosphatidylethanolamine (PE), dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), DPPE-NHS, DSPE-NHS, and DOPE-NHS.

[0059] As used herein, “hydrophobic moiety” refers to a compound that is insoluble in water or other polar solvents. Those skilled in the art will understand that it is possible to readily determine suitable hydrophobic moieties for use in the protamine molecules described herein, for example, hydrophobic compounds having terminal double bonds. When conjugated to a substantially hydrophilic protamine peptide, hydrophobic moieties can create amphiphilic protamine molecules that can self-assemble into micelles. Without being bound by any particular hypothesis, such amphiphilic protamine molecules may exhibit increased interaction with cell membranes, and / or increased cell penetration, and / or increased cell permeability. Exemplary hydrophobic moieties include, non-limitingly, allyl glycidyl ethers (AGEs)

[60] .

[0060] Polyvalent protamine molecules may be prepared by conjugating protamine peptides. Without being constrained by any particular hypothesis, polyvalent protamine molecules can mimic protamine aggregation. In some embodiments, a first protamine peptide may be conjugated to a crosslinking agent and / or polymer, which may then be conjugated to additional protamine peptides. The protamine peptides may have the same amino acid sequence or different amino acid sequences. The protamine peptides may be from the same source and / or the same form (e.g., protamine sulfate from salmon) or from different sources.

[0061] As used herein, “crosslinking agent” refers to a compound capable of coupling proteins, such as protamine peptides, to prepare, for example, polyvalent protamine molecules. Those skilled in the art will understand that it is possible to readily determine suitable crosslinking agents for use in the protamine molecules described herein. Exemplary crosslinking agents include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA), N-γ-maleimidobutyryloxysuccinimid (GMBS), or succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).

[0062] As used herein, “polymer” refers to a compound composed of smaller repeating chemical units. As used herein, “dendrimer” refers to a structurally defined polybranched polymer that contains functional parts on its surface, enabling polyvalence. Those skilled in the art will understand that it is possible to readily determine suitable polymers or dendrimers for use in the protamine molecules described herein, for example, polymers or dendrimers containing amino groups. Thus, polyvalent protamine molecules may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or more protamine peptides, depending on the number of available amino groups in the polymer. Exemplary polymers include, but are not limited to, poly(amidoamine) (PAMAM) G1-G5, polyethyleneimine (PEI), or polylysine (PLL).

[0063] Without being constrained by any particular hypothesis, polyvalent protamine molecules may exhibit increased interaction with cell membranes, and / or increased cell penetration, and / or increased cell permeability, due to an increased number of charged amino acids (such as arginine and lysine).

[0064] Exemplary protamine molecules include, to a limited extent, protamine-GMBS-PAMAM, protamine-DMPE, protamine-AGE, protamine-SATA-PAMAM, protamine-C18, protamine-C16, protamine-PEI, protamine-PLL, and others.

[0065] As used herein, “conjugate” means to link a peptide to another molecule, such as a lipid, hydrophobic moiety, polymer, crosslinking agent, or peptide, by covalent chemical bonds to form a conjugated molecule. In some embodiments, as used herein, conjugation eliminates electrostatic interactions.

[0066] Protamine molecules may be prepared as described herein or as known in the art, and may be characterized by a variety of techniques, including, but not limited to, nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), transmission electron microscopy (TEM), atomic force microscopy (AFM), mass spectrometry (MS), and dynamic light scattering (DLS).

[0067] In some embodiments, the protamine molecule may be provided in a composition, for example, a composition suitable for cell transfection, cell penetration, cell permeation, or delivery to the cell nucleus. In some embodiments, the composition may be a pharmaceutical composition.

[0068] Therefore, the protamine molecules described herein may be provided alone or in combination with other compounds (e.g., “payload,” e.g., small molecules, proteins, peptides, or peptide analogs) in the presence of any suitable carrier, e.g., a carrier suitable for cell permeation, infiltration, or transfection, or delivery to the cell nucleus.

[0069] In alternative embodiments, the protamine molecules described herein may be provided alone or in combination with other compounds (e.g., “payload,” e.g., small molecules, proteins, peptides, or peptide analogs) in a form suitable for administration to subjects, e.g., mammals, e.g., humans, cattle, sheep, etc., in the presence of a pharmaceutically acceptable carrier. Conventional pharmaceutical practices may be used to provide formulations or compositions suitable for administration. In some embodiments, the pharmaceutical compositions described herein may explicitly exclude absorption enhancers.

[0070] In alternative embodiments, the protamine molecules described herein may be used without causing substantial toxicity. Toxicity may be determined using standard techniques, for example, by testing in cell cultures or experimental animals, and the therapeutic index, i.e., the ratio between LD50 (a dose lethal to 50% of the population) and LD100 (a dose lethal to 100% of the population), or ED 50 Alternatively, it may be determined by determining the semi-effective dose, i.e., the dose that produces the specified effect ("response") in 50% of the subjects under study.

[0071] In some embodiments, the protamine molecules described herein may be used in a method for delivering a payload molecule to a cell, which involves bringing the cell into contact with the payload molecule in combination with the protamine molecule.

[0072] In an alternative embodiment, the protamine peptide or protamine molecule described herein may be used in a method for non-injection delivery of a large payload molecule to a cell, wherein the cell is brought into contact with the payload molecule in combination with the protamine peptide or protamine molecule.

[0073] Any suitable route of delivery or administration, such as a non-injectable route, may be used. Non-injectable routes include intranasal, sublingual, oral, buccal, rectal, vaginal, intravitreous, or topical routes. Non-injectable delivery may target the skin, eyes, brain, lungs, etc. Non-injectable delivery may be transmucosal or transcellular (e.g., transepithelial or lamina propria). Transmucosal routes may include intranasal, sublingual, or intravaginal delivery. In some embodiments, non-injectable delivery routes include non-injectable (needle) transmucosal delivery of the payload molecule, such as via nasal, sublingual, and topical routes.

[0074] Parenteral routes include intravenous, subcutaneous, and intramuscular routes, such as by needle injection. In some embodiments, parenteral administration methods are explicitly excluded. In some embodiments, administration methods involving needle injection are explicitly excluded.

[0075] As used herein, “payload” molecule means any molecule that can be delivered to a cell in combination with a protamine peptide or protamine molecule as described herein. Therefore, payload molecules may, non-limitingly, include small molecules, proteins, peptides, or peptide analogs. In some embodiments, the payload molecule is not a nucleic acid molecule. In some embodiments, the payload molecule may have a size of approximately 0.8 to approximately 1000 KDa, or any range or value between them, for example, approximately 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 KDa, or approximately 50 to approximately 150 KDa, or approximately 150 to approximately 800 KDa, etc. In some embodiments, the payload molecule may be of a size of approximately 0.8 KDa to approximately 800 KDa, or any range or value between them, for example, approximately 0.8, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 KDa, or approximately 50 to approximately 150 KDa, or approximately 150 to approximately 800 KDa, etc. In some embodiments, the payload molecule may be about 20 kDa to about 150 kDa, or any range or value between them, such as about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 kDa, or about 20 to about 100 kDa, or about 50 to about 150 kDa, etc. In some embodiments, the “large” payload molecule may be at least about 3 kDa.In some embodiments, the “large” payload molecule may have a size of approximately 3 kDa to approximately 1000 kDa, or any range or value between them, for example, approximately 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 kDa, or approximately 50 to approximately 150 kDa, or approximately 150 to approximately 800 kDa, etc. In some embodiments, the “large” payload molecule may have a size of at least approximately 200 kDa. In some embodiments, the large payload molecule may be about 200 kDa to about 1000 kDa, or any range or value between them, for example, about 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 kDa, or about 250 to about 450 kDa, or about 550 to about 800 kDa, etc. In some embodiments, the payload molecule may be about 1000 kDa or larger. In some embodiments, the payload molecule may block the IL-4 pathway and / or the IL-13 pathway, for example, it may block a monoclonal antibody that blocks the IL-4 pathway and / or the IL-13 pathway. Exemplary payload molecules include, but are not limited to, growth hormone (e.g., human recombinant growth hormone (rhGH)), antibodies (e.g., monoclonal antibodies, e.g., omalizumab, reslizumab, mepolizumab, and dupilumab), immunoglobulin G (IgG)), insulin, or semaglutide.

[0076] In some embodiments, the large payload molecule may be delivered sublingually. In some embodiments, the payload molecule may be insulin, the protamine-to-insulin ratio may be at least 3:1, delivery may be intranasal, and / or the pH may be acidic. In some embodiments, the payload molecule may be insulin, the protamine-to-insulin ratio may be about 3:1 and about 10:1, delivery may be intranasal, and / or the pH may be acidic.

[0077] The payload molecule and the protamine peptide or protamine molecule may be combined by physical mixing. In some embodiments, the payload molecule may be released upon delivery, for example, after transcellular or transmucosal delivery, or after delivery to the cell nucleus. In some embodiments, the payload molecule and the protamine peptide or protamine molecule are not conjugated to each other. In some embodiments, the payload molecule and the protamine peptide or protamine molecule do not form a complex, for example, by covalent and / or electrostatic interactions. There may be some interactions between the payload molecule and the protamine peptide or protamine molecule, but it is understood that the interactions should not substantially hinder cell permeation, osmosis, or transfection, or delivery to the cell nucleus, or hinder the release of the payload. As used herein, “substantially hinder” means that the interaction between the payload molecule and the protamine peptide or protamine molecule is dynamic, i.e., not permanent.

[0078] The payload molecule and the protamine peptide or protamine molecule may be administered simultaneously (e.g., in the same composition or formulation) or separately (e.g., in different compositions or formulations). For example, in the case of topical administration, the protamine peptide or protamine molecule may be administered to the target skin, and the payload molecule may be administered thereafter. Similarly, cells may first come into contact with the protamine peptide or protamine molecule, and then come into contact with the payload molecule. When the protamine peptide or protamine molecule is provided separately from the payload molecule, it is understood that the timing of the provision of the payload molecule depends on the specific application and mode of delivery.

[0079] In some embodiments, the protamine peptides or protamine molecules described herein may be used in methods for making cell membranes permeable. Permeability may be transient and / or reversible.

[0080] In some embodiments, the protamine peptides or protamine molecules described herein may be used in methods for transfecting cells.

[0081] In some embodiments, the protamine peptide or protamine molecule described herein, optionally in combination with a payload molecule, may be delivered to the nucleus of a cell. In some embodiments, the protamine peptide or protamine molecule described herein in combination with a payload molecule may be delivered to the nucleus of a cell. In some embodiments, the protamine peptide or protamine molecule described herein in combination with a payload molecule may substantially bypass the lysosomes of a cell, for example, less than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the protamine peptide or protamine molecule in combination with the payload molecule may be found in the lysosomes of a cell. In some embodiments, the protamine peptides or protamine molecules described herein, optionally in combination with a payload molecule, may substantially bypass cellular lysosomes, for example, less than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the protamine peptides or protamine molecules, optionally in combination with a payload molecule, may be found in cellular lysosomes.

[0082] "Cells" can be any cells isolated from an animal, such as an invertebrate (e.g., an insect or bug) or a vertebrate, such as a mammal. For example, cells can non-limitingly include cells or tissues (e.g., from a biopsy or autopsy) from bone, brain, breast, colon, muscle, nerve, ovary, prostate, retina, skin, skeletal muscle, intestine, testes, heart, liver, lung, kidney, stomach, pancreas, uterus, adrenal gland, tonsil, spleen, soft tissue, blood, semen, etc. Cells can also include cultured cells, such as cells or cell lines prepared under experimental conditions.

[0083] In an alternative embodiment, the protamine peptide or protamine molecule described herein may be used in a method for treating a condition that would benefit from non-injectable delivery of a payload molecule, by administering the payload molecule together with the protamine peptide and / or protamine molecule to a subject in need thereof.

[0084] In some embodiments, the payload molecule may be administered simultaneously with or at different times to the protamine peptide and / or protamine molecule.

[0085] In some embodiments, the protamine peptide and / or protamine molecule may be administered before the administration of the payload molecule.

[0086] In some embodiments, the protamine peptide and / or protamine molecule and / or payload molecule may be administered in an effective dose. “Effective dose” includes a therapeutic effective dose or a prophylactic effective dose. “Therapeutic effective dose” means the amount that is effective in the dosage required to achieve the desired therapeutic outcome and is effective for the duration required therefor. The therapeutic effective dose may vary according to factors such as the disease state of the subject, age, sex, and weight, as well as the ability to induce the desired response in the individual. The dosage regimen may be adjusted to provide an optimal therapeutic response. The therapeutic effective dose is also the amount in which the therapeutically beneficial effect outweighs any toxic or adverse effects. “Prophylactic effective dose” means the amount that is effective in the dosage required to achieve the desired prophylactic outcome and is effective for the duration required therefor. Typically, prophylactic doses are used in subjects before or earlier in the disease, and thus the prophylactic effective dose may be less than the therapeutic effective dose. The therapeutic or prophylactic effective dose of the compound may be any value within the following ranges: 0.1 nM to 0.1 M, 0.1 nM to 0.05 M, 0.05 nM to 15 μM, or 0.01 nM to 10 μM.

[0087] It should be noted that dosage values ​​may vary depending on the severity of the condition being alleviated. For any particular subject, a specific drug regimen may be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition. The dosage ranges described herein are illustrative only and do not limit the dosage range that may be selected by the physician. The amount of active compound(s) in the composition may vary according to factors such as the individual's disease state, age, sex, and weight. For example, a single bolus may be administered, or divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. It may be advantageous to formulate the composition in unit dosage forms for ease of administration and uniformity of dosage.

[0088] It is understood that the condition may be any condition that benefits from non-injectable delivery of the payload molecule. In some embodiments, the condition that benefits from non-injectable delivery of the payload molecule may, non-limitingly, be a condition in which the standard mode of treatment or prophylaxis is by needle-based injection. In some embodiments, the condition that benefits from non-injectable delivery of the payload molecule may, non-limitingly, be a skin condition, e.g., psoriasis; an eye condition; an inflammatory condition, e.g., type 2 inflammatory disease, e.g., sinusitis, e.g., chronic sinusitis; diabetes mellitus, etc.

[0089] As used herein, subjects may include humans, non-human primates, rats, mice, cows, horses, pigs, sheep, goats, dogs, and cats. Subjects may include clinical patients, clinical trial volunteers, and laboratory animals. Subjects may be those suspected of having or at risk of having any of the conditions described herein, those diagnosed with any of the conditions described herein, or control subjects confirmed not to have any of the conditions described herein.

[0090] In some embodiments, protamine peptides and / or protamine molecules, or compositions thereof, may be provided in a first container of the kit, along with instructions for use. The first container may further contain the payload molecules described herein, along with instructions for use. Alternatively, the kit may contain a second container containing the payload molecules described herein, along with instructions for use.

[0091] This disclosure is further illustrated in the following embodiments. [Examples]

[0092] material and method Compounds and Antibodies: In research on protamine, Alexa Fluor 488 phalloidin, Alexa Fluor 647 protein labeling kit, LysoTracker Red DND-99, Tubulin Tracker, FITC conjugate of bovine serum albumin (FITC-BSA), and all ELISA kits were purchased from Thermo Fisher Scientific (Ottawa, ON, Canada). Rabbit polyclonal antibody (pAb) against E-cadherin was purchased from Abclonal. Goat anti-rabbit IgG (Alexa-Fluor-594 conjugate) was purchased from Elabscience. Human recombinant insulin, streptozotocin (STZ), Hoechst 33342, protamine, and FITC-insulin were purchased from Sigma-Aldrich. Dialysis membranes (molecular weight cutoff MWCO = 10 or 3.5 kDa) were purchased from Spectrum Laboratories (Waltham, MA). Hydrophobic fluorescent dye DiI (DiIC 18(3); 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine) and DiD (DiIC18(5); 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate) were purchased from Cedarlane Labs. Cyanine 7-NHS was purchased from Abcam. All other common laboratory chemicals were purchased from Fisher Scientific and VWR Scientific (Mississauga, ON, Canada).

[0093] Cell Culture: In the study on protamine, RPMI2650 (human nasal epithelium) and Caco2 (human intestinal epithelium) cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (100 units / mL penicillin, 100 μg / mL streptomycin, Gibco). All cells were cultured at 37°C under 5% CO2 and subcultured up to 15 times when confluence was reached.

[0094] XTT assay for cell viability: In a study on protamine, cells were placed in a 96-well plate in a 5 × 10⁶ configuration. 4 Cells were seeded at a density of 10 cells / well and incubated for 24 hours (37°C, 5% CO2, humidified). The medium was then replaced with fresh complete medium, and the cells were treated with protamine or R8 at different concentrations. After 24 hours of treatment, cell viability was analyzed by the XTT assay as previously described

[61] .

[0095] Cellular uptake and cellular changes of fluorescently tagged proteins in the presence of protamine or R8: In a study on protamine, cells were placed in a 12-well plate, 1 × 10⁶ per well. 5Cells were seeded in 1 mL of medium containing 10% FBS and incubated for 18–20 hours to achieve 70–80% confluence. For Alexa488-anti-SIRT1 monoclonal antibody (mAb) uptake studies, cell suspensions were first incubated with 1 μg / mL of membrane staining dye DiD at 37°C for 30 minutes, then centrifuged, washed three times with PBS, and subsequently seeded. The medium was replaced with serum-free medium containing 20 μg / mL of FITC-BSA or 1 μg / mL of Alexa488-anti-SIRT1 in the presence or absence of either 37.5 μg / mL of protamine or R8, at 12 or 37°C for 2–4 hours. Protamine from different sources was used, and their efficacy was compared. The different sources of protamine included protamine sulfate from salmon grade X, protamine sulfate from herring grade III, protamine sulfate from salmon grade IV, and protamine chloride from salmon. Cells were washed three times with PBS and then stained with Hoechst 33342 and Lysotracker, respectively, for the nucleus and lysosomes, according to the manufacturer's protocol. Stained (unfixed) cells were immediately imaged by confocal laser scanning microscopy (CLSM) and analyzed with ZEN software (Carl Zeiss, Oberkochen, Germany) and ImageJ.

[0096] For cellular transformation studies, cells were incubated with protamine or R8 at 37.5 μg / mL, washed three times with PBS, and then stained with Hoechst 33342 and Tubulin Tracker according to the manufacturer's protocol. Live cells were then imaged by CLSM. Alternatively, cells were fixed with 10% formalin at room temperature for 10 minutes, treated with 0.1% Triton X-100 in PBS at room temperature for 2 minutes, and washed with PBS. Cellular F-actin was stained with Alexa Fluor 488 phalloidin according to the manufacturer's protocol, then washed with PBS, placed on a glass slide with a drop of Fluoroshield (Sigma-Aldrich) containing DAPI, and imaged by CLSM. F-actin expression and cell spread area were quantified by ImageJ via cell fluorescence and morphological analysis, respectively. F-actin aggregation in dots was quantified, and cells containing 20 or more F-actin dots were counted as positive for actin aggregation. Microtubule quantification was performed using Fiji. Images were first processed using a Gaussian smoothing method with an intensity threshold of 140 (sigma = 0.100). Then, the "skeletonization" function was used to trace the microtubules and determine the amount and total length of microtubules per cell.

[0097] FITC-BSA uptake by 3D cell spheroids: In research on protamine, cell suspension (1 × 10⁻⁶ 5 First, the cells were incubated with 1 μg / mL of DiI at 37°C for 30 minutes and then stained with PBS. The cells were centrifuged at 400 g for 3 minutes and then washed three times with PBS. The cells were placed in a U-bottom 96-well plate (FaCellitate) at a rate of 1 × 10⁶. 4Seeds were seeded in one well. Two days later, spheroids were collected and treated with FITC-BSA at 40 μg / mL in the presence or absence of either protamine at 0.1–0.2 mg / mL or R8. Three hours later, spheroids were washed three times with PBS and then imaged with CLSM. Alternatively, spheroids were trypsinized for 5 minutes, resuspended in complete medium, centrifuged at 400 g for 3 minutes, washed three times with PBS, resuspended in PBS, and analyzed by fluorescence-activated cell sorting (FACS).

[0098] Cellular uptake of FITC-BSA in the presence of protamine from different sources: In a study on protamine, cells were taken in a 12-well plate, 1 × 10⁶ per well. 5 Cells were seeded in 1 mL of medium containing 10% FBS and incubated for 18–20 hours to achieve 70–80% confluence. The medium was replaced with serum-free medium containing 10 μg / mL FITC-BSA in the presence or absence of 37.5 μg / mL of protamine from different sources, including protamine sulfate from salmon grade X, herring grade III, salmon grade IV, and salmon chloride, at 37°C for 4 hours. Subsequently, the cells were trypsinized for 3 minutes, resuspended in complete medium, centrifuged at 400 g for 3 minutes, washed three times with PBS, resuspended in PBS, and analyzed by fluorescence-activated cell sorting (FACS).

[0099] Protamine uptake study. In the protamine study, protamine was first labeled with the Alexa Fluor 647 (AF647) protein labeling kit according to the manufacturer's protocol, and then added to culture medium at 37.5 μg / mL. Live cells were imaged under confocal laser scanning microscopy (CLSM) for 60 minutes.

[0100] Propidium iodide (PI) uptake study. In the study on protamine, cells were treated with 40 μM of PI, a membrane-impermeable dye, for 60 minutes, then protamine was added at a final concentration of 37.5 μg / mL, and the cells were imaged by CLSM in the live-cell imaging mode. Alternatively, cells were treated with AF647-protamine at 37.5 μg / mL for 10 minutes, washed three times with PBS, and incubated in complete medium for 2 minutes. Then, PI was added at a final concentration of 40 μM, and the cells were imaged by CLSM in the live-cell imaging mode.

[0101] Cell uptake of FITC-BSA at 12 or 37 °C: In the study on protamine, cells were seeded in a 12-well plate at 1 × 10 5 cells per well in 1 mL of medium containing 10% FBS and incubated for 18 - 20 hours to achieve 70 - 80% confluency. Cells were stained with Hoechst 33342, Lysotracker, and Tubulin Tracker for nuclei, lysosomes, and tubulin, respectively, according to the manufacturer's protocol. The medium was replaced with serum-free medium containing 20 μg / mL of FITC-BSA in the presence of 37.5 μg / mL of protamine at 12 or 37 °C for 4 hours. The stained (non-fixed) cells were immediately imaged by CLSM and analyzed with ZEN software (Carl Zeiss, Oberkochen, Germany) and Image J.

[0102] Animals: In the study on protamine, 4-week-old male CD-1 mice were obtained from Charles River Laboratories (Wilmington, MA). In vivo experiments were performed according to an established experimental protocol approved by the Animal Care Committee of the University of British Columbia (Vancouver, BC, Canada). Animal approval number: A22-0141.

[0103] Intranasal Delivery of Cy7-BSA: In a study on protamine, Cy7-BSA was synthesized by reacting cyanine 7-NHS and BSA in a molar ratio of 10:1. The reaction was carried out in Milli Q water at room temperature for 1 hour. The reaction solution was then dialyzed for 3 days and lyophilized. Cy7-BSA was mixed with protamine or R8 in water and delivered intranasally (in) to mice (dose = 20 mg / kg of Cy7-BSA; 3 mg / kg of protamine or R8). Conscious mice were restrained by the non-dominant hand, and a small droplet (approximately 5 μl) of the Cy7-BSA formulation was delivered near one nostril at a 45 dree angle via the tip of a 10 μl micropipette. After the droplet was inhaled, another droplet was administered to the other nostril. The procedure was repeated up to 5 times to deliver a total volume of formulation up to 24 μl. Only physiological saline and Cy7-BSA were included as controls. Blood was collected at 0.5 and 2 hours. Plasma was isolated by centrifugation (10,000 g, 5 min) and analyzed for Cy7 fluorescence using a plate reader.

[0104] Streptozotocin (STZ)-induced diabetic mouse model: In a study on protamine, mice were fasted for 6 hours, and then 200 mg / kg of STZ was administered via intravenous injection. Three days later, blood glucose (BG) was measured using a glucose meter (ONE Touch Ultra 2 blood monitoring system). Mice with BG levels of 300 mg / dL or higher were considered to have diabetes.

[0105] Preparation of insulin-protamine preparations: In studies on protamine, protamine was dissolved in water at a concentration of 10 mg / mL without adjusting the pH, and insulin was dissolved in water at a concentration of 5 mg / mL, with the pH adjusted to 3.2, 6.2, 7.4, 8.2, or 11.2 using 1N HCl or 1N NaOH. The protamine was then mixed with the insulin in weight ratios ranging from 0.2, 1.5, 2.5, 3.0, and 3.75 (w / w). The mixtures were used immediately.

[0106] Efficacy study of BG reduction in STZ-induced diabetic mice: In a study on protamine, BG in STZ diabetic mice was measured before treatment with one dose of either sc insulin or intranasal administration (in) of various protamine-insulin preparations at 0.5, 1, or 5 mg insulin / kg. The volume of protamine-insulin preparation for intranasal delivery was 10–20 μL per mouse. BG was determined at 0.5, 1, 2, 4, and 6 hours.

[0107] In a study on FITC-insulin penetration in the nostrils of diabetic mice: protamine, a protamine-FITC-insulin preparation was prepared as described above at a weight ratio of 3 and a pH of 3.2, and delivered to mice at a dose of 1 mg of FITC-insulin / kg. FITC-insulin dissolved in physiological saline was included as a control. After 2 hours, the mice were euthanized, their nasal turbinates were collected, and fixed in 10% formalin for 48 hours. The nasal turbinates were washed 10 times with PBS and decalcified in EDTA solution (143 g / L distilled water) for 1 week. The EDTA solution was replaced daily with fresh solution. The nasal turbinates were then incubated in PBS for 1 hour four times, followed by incubation in 30% sucrose for 24 hours, and then embedded in OCT. The samples were sectioned using Leica Cryostat. Sections were incubated in PBS for 10 minutes to remove OCT, followed by incubation in 10% formalin in PBS for 10 minutes. Next, sections were washed three times with PBS, permeabilized with 0.1% Triton X-100 for 5 minutes, and washed three times with PBS. Sections were blocked in 1% BSA in PBST (PBS + 0.1% Tween 20) for 45 minutes, and then washed five times with PBS. Phalloidin staining solution was applied to sections and incubated for 45 minutes, followed by five washes with PBS and incubation with E-cadherin rabbit pAb (abclonal, A3044) (1:200) at room temperature for 1 hour. Sections were washed five times with PBS (10 minutes each) and stained with goat anti-rabbit IgG (Alexa 594 conjugate) at 1 μg / mL at room temperature for 1 hour. The sections were washed five times with PBS (10 minutes each), stained with DAPI for 30 minutes, and then subjected to CLSM.

[0108] Pharmacokinetic studies: In studies on protamine, different insulin preparations were delivered to mice via either in or sc. Blood was collected at different time points in ethylenediaminetetraacetic acid (EDTA) coated tubes (Microvette, Sarstedt AG & Co., Numbrecht, Germany) via saphenous vein or cardiac puncture. Plasma was isolated by centrifugation (10,000 g, 5 min), and insulin was analyzed using a human insulin ELISA kit (Thermo) according to the manufacturer's protocol.

[0109] Histological analysis: In the protamine study, mice received a protamine-insulin mixture daily for 7 consecutive days, and were then euthanized one day after the final dose. The nasal turbinates, trachea, and lungs were collected, sectioned, stained with hematoxylin and eosin, imaged, and independently analyzed by Dr. Ian Walch, a committee-certified pathologist at UBC.

[0110] Statistical Analysis: In the study on protamine, the normality of the data distribution was first assessed using the Shapiro-Wilk test (p<0.05). For data that passed the test, a parametric one-way ANOVA with the Tukey post-hoc test (adjusted p-values) was used for multiple comparisons. For data that failed the Shapiro-Wilk test, group comparisons were performed using the non-parametric Kruskal-Wallis test, followed by the Dunnett multiple comparison post-hoc test. A p-value less than 0.05 was considered statistically significant. The data were plotted using GraphPad Prism version 8.0 (GraphPad Software). All data are presented as mean ± standard deviation (SD). All statistical analyses were performed using R software (version 4.0.2).

[0111] Compounds and Antibodies: In research on protamine derivatives, the Alexa Fluor 647 protein labeling kit, FITC conjugate of bovine serum albumin (FITC-BSA), EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), NHS (N-hydroxysuccinimide), 2-mercaptoethanol, Lysotracker® green DND-26, and all ELISA kits were purchased from Thermo Fisher Scientific (Ottawa, ON, Canada). Recombinant human growth hormone (rhGH), streptozotocin (STZ), Hoechst 33342, D-glucose, stearic acid, and protamine were purchased from Sigma-Aldrich. Ultra-LEAF® purified IgG was purchased from Biolegend. Dialysis membranes (molecular weight cutoff (MWCO) = 10 or 15 kDa) were purchased from Spectrum Laboratories (Waltham, MA). Hydrophobic fluorescent dye DiI(DiIC) 18 (3); 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine) was purchased from Cedarlane Labs. NHS-PEG-NHS was purchased from Biopharma PEG. Cyanine 7-NHS was purchased from Abcam. Pr OZEMPIC® (1.34 mg semaglutide / ml) was purchased from Novo Nordisk®. Type I bovine collagen was purchased from Advanced BioMatrix. 0.33 cm 2 Transwell inserts for 24-well plates with a growth area were purchased from Corning. Cas9-RFP-RNP and sgRNA were ordered from Integrated DNA Technologies. The sgRNA sequence was 5'-GUCGCCCUCGAACUUCACCU-3' (SEQ ID NO: 5). All other common laboratory chemicals were purchased from Fisher Scientific and VWR Scientific (Mississauga, ON, Canada).

[0112] Synthesis of protamine derivatives In a study on stearic acid conjugate protamine (C18-P): protamine derivatives, protamine (10 mg / ml) and EDC were dissolved in PBS in a 1:5 molar ratio and incubated for 15 minutes. Then, NHS was added to the protamine at a molar equivalent of 7.5, and the reaction was continued for 30 minutes. Subsequently, 2-mercaptoethanol was added to a final concentration of 20 mM to quench the EDC. Stearic acid was dissolved in chloroform (75 mg / ml) and added to the protamine at a molar equivalent of 10, and the reaction was allowed to proceed overnight. The product was dialyzed against Milli-Q water at room temperature, then filtered through a 0.22 μm filter and lyophilized.

[0113] Protamine dimer (P2): In studies on protamine derivatives, protamine (10 mg / ml) and NHS-PEG-NHS were dissolved in PBS (pH 7.2) in a molar ratio of 1:10. The solution was incubated at room temperature for 24 hours, followed by dialyzing in Milli-Q water for 48 hours, and then lyophilized.

[0114] Cell Culture: In the study of protamine derivatives, human primary gingival keratinocytes (PCS-200-014™, ATCC) were cultured in dermal cell basal medium (PCS-200-030™, ATCC) supplemented with a keratinocyte kit (PCS-200-040™, ATCC). Primary human gingival fibroblasts (PCS-201-018, ATCC) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The eGFP stable expression Flp-In™ T-REx™ 293 cell line (HEK 293 eGFP) was obtained from the laboratory of Dr. Colin Ross at UBC. The cells were cultured in DMEM supplemented with 10% FBS and 0.2 mg / mL hygromycin B. All cells were cultured at 37°C under 5% CO2 conditions, and subcultured up to 7 times when confluence was reached.

[0115] Cellular uptake of fluorescently tagged proteins in the presence of protamine, C18-P, or P2: In studies on protamine derivatives, cells were taken into a 12-well plate, 1 × 10⁶ per well. 5 Cells were seeded in 1 mL of culture medium for 18–20 hours to achieve 70–80% confluence. IgG was labeled with Alexa647 according to the manufacturer's protocol. The medium was replaced with serum-free medium containing 20 μg / mL FITC-BSA or 20 μg / mL AF647-IgG in the presence or absence of protamine (10 μg / mL), C18-P (10 μg / mL), or P2 (5 μg / mL), and subsequently incubated overnight at 37°C. Cells were washed three times with PBS and then stained with Hoechst 33342 and Lysotracker, respectively, for the nucleus and lysosomes, according to the manufacturer's protocol. Stained (unfixed) cells were immediately imaged by confocal laser scanning microscopy (CLSM) and analyzed with ZEN software (Carl Zeiss, Oberkochen, Germany) and ImageJ. Alternatively, cells were trypsinized for 5 minutes, resuspended in complete medium, centrifuged at 125 g for 10 minutes, washed three times with PBS, resuspended in PBS, and analyzed by flow cytometry (FCM). Alternatively, cells were trypsinized for 5 minutes, resuspended in complete medium, centrifuged at 125 g for 10 minutes, washed three times with PBS, resuspended in PBS, and analyzed by fluorescence-activated cell sorting (FACS).

[0116] XTT assay for cell viability: In a study on protamine derivatives, human primary gingival keratinocytes were placed in a 96-well plate in a 5 × 10⁶ field. 4 The cells were seeded at a density of 1 cell / well and incubated for 24 hours (37°C, 5% CO2, humidified). The medium was then replaced with fresh complete medium, and the cells were treated with protamine / C at different concentrations. 18 Cells were treated with either -P or P2. After 24 hours of treatment, cell viability was analyzed by the XTT assay as previously described.

[0117] Scanning electron microscopy (SEM): In research on protamine derivatives, primary human gingival keratinocytes (1 × 10 on a 20 mm coverslip) 5 Cells were incubated with protamine (10 μg / mL), C18-P (10 μg / mL), or P2 (5 μg / mL) at 37°C for 4 hours. The cells were washed three times with PBS, fixed with glutaraldehyde, dehydrated, and subsequently coated with iridium (10 nm thick) in a Leica EM high-vacuum sputter coater and dried in a Tousimis Samdri®-795 critical point dryer (30.98°C and 73.8 bar). Imaging was performed using a Helios NanoLab 650 focused ion bean SEM at 15 kV.

[0118] In a study on FITC-BSA and AF647-IgG penetration and cellular uptake in 3D cell spheroids, a primary human gingival keratinocyte cell suspension (1 × 10⁻¹⁶) was used. 5 First, the cells were incubated with 1 μg / mL of DiI at 37°C for 30 minutes and then stained with PBS. The cells were centrifuged at 125 g for 10 minutes and then washed three times with PBS. The cells were then placed in a U-bottom 96-well plate (FaCellitate), with 1 × 10⁶ cells per well. 4Cells were seeded individually. Two days later, spheroids were collected and treated with FITC-BSA or AF647-IgG at 20 g / mL in the presence or absence of either protamine, C18-P, or P2 at 0.1 mg / mL, and incubated overnight. The spheroids were then washed three times with PBS, and the distribution of FITC-BSA / AF647-IgG within the spheroids was analyzed using CLSM under Z-stack imaging with 20 μm spacing. The distance from the spheroid boundary to the center of the permeation fluorescence was analyzed using ImageJ. Alternatively, spheroids were trypsinized for 5 minutes, resuspended in complete medium, centrifuged at 125 g for 10 minutes, washed three times with PBS, resuspended in PBS, and analyzed by FCM. Alternatively, spheroids were trypsinized for 5 minutes, resuspended in complete medium, centrifuged at 125 g for 10 minutes, washed three times with PBS, resuspended in PBS, and analyzed by FACS.

[0119] Human sublingual tissue substitutes: In studies on protamine derivatives, human sublingual tissue substitutes were prepared according to a previously established procedure with minor modifications

[62] . In short, primary human gingival fibroblasts (2 × 10⁶) 4 cells / cm 2 ), FBS (9.5 μL / model), and type I bovine collagen were prepared at a neutral pH and placed in a 24-well plate (growth area = 0.33 cm²). 2 The mixture was poured into the transwell insert within the container. After incubation at room temperature for 1 hour, and then incubation at 37°C for another 1 hour, 200 μL of keratinocyte growth kit supplemented with skin cell basal medium was added, and the system was transferred to a 37°C incubator with 5% (v / v) CO2 and 95% (v / v) humidity for a further 2 hours. Subsequently, 2 × 10⁻⁶ 5 cells / cm 2 Primary human gingival keratinocytes were seeded onto a collagen matrix. After 24 hours, the model was elevated to the gas-liquid interface, and the culture medium was replaced with gingival keratinocyte differentiation medium. The tissue was cultured for 14 days (37°C, 5% CO2, and 95% humidity), with the medium changed every other day.

[0120] In a study on AF647-IgG infiltration into human sublingual tissue replacements using protamine derivatives, protamine, C18-P, or P2 were dissolved in 5% glucose at a concentration of 10 mg / mL. AF647-IgG was used at a concentration of 2 mg / mL in PBS. 8 μL of protamine or a derivative was mixed with 4 μL of AF647-IgG, and then the volume was increased to 20 μL in PBS. The preparation was applied to the apical side of the tissue. After 4 hours of treatment, the tissue was harvested and then embedded in OCT. After keeping the tissue frozen overnight in a -80°C freezer, the samples were sectioned to a thickness of 10 μm using Leica Cryostat. The sections were incubated in PBS for 10 minutes to remove the OCT, stained with DAPI for 30 minutes, and then subjected to CLSM.

[0121] Cas9-RNP / sgRNA delivery: In research on protamine derivatives, HEK 293 eGFP cells (2 × 10) 5 Cells were seeded in a confocal dish for 18–20 hours to achieve 70–80% confluence. The cells were then treated with either protamine (10 μg / mL), C18-P (10 μg / mL), or P2 (5 μg / mL) for 40 minutes. Next, 3.5 μL of Cas9-RNP-RFP (52 μmol / L) was diluted in 20 μL of nuclease-free water, then mixed with 2 μL of sgRNA (100 μmol / L), and incubated at room temperature for 5 minutes. The Cas9-RNP / sgRNA mixture was then added to the cells and incubated overnight. After 2 days, the cells were stained with Hoechst 33342 and imaged by CLSM. Alternatively, cells were trypsinized for 3 minutes, resuspended in complete medium, centrifuged at 1000g for 3 minutes, washed three times with PBS, resuspended in PBS, and analyzed by FCM. Alternatively, cells were trypsinized for 3 minutes, resuspended in complete medium, centrifuged at 1000g for 3 minutes, washed three times with PBS, resuspended in PBS, and analyzed by FACS.

[0122] The data were analyzed using FCS Express 7 (De Novo Software, Pasadena, CA, USA) and FlowJo (BD Ashland, OR, USA). Cells were first gated using a negative control (HEK 293 eGFP) to analyze only individual single cells. A second gate for cells with RFP violet (red channel) was then applied to identify Cas9-RFP-RNP positive cells. Finally, a gate for eGFP (green channel) was applied to calculate the percentage of green-negative cells.

[0123] Additionally, the cells were placed in a U-bottom 96-well plate (FaCellitate), with 1 × 10⁶ cells per well. 4 Cells were seeded individually. Two days later, spheroids were pretreated with 0.1 mg / mL protamine, C18-P, or P2 for 40 minutes, then a Cas9-RNP / sgRNA mixture was added and incubated for 48 hours. The spheroids were then washed three times with PBS, and the distribution of Cas9-RNP-RFP within the spheroids was analyzed using CLSM under Z-stack imaging with 10 μm spacing. Alternatively, spheroids were trypsinized for 5 minutes, resuspended in complete medium, centrifuged at 1000 g for 3 minutes, washed three times with PBS, resuspended in PBS, and analyzed by FCM. Alternatively, spheroids were trypsinized for 5 minutes, resuspended in complete medium, centrifuged at 1000 g for 3 minutes, washed three times with PBS, resuspended in PBS, and analyzed by FACS. The data were analyzed as described above.

[0124] Animals: For the study of protamine derivatives, 4-week-old male / female CD-1 mice were obtained from Charles River Laboratories (Wilmington, MA). In vivo experiments were conducted according to the protocol (A22-0141) approved by the Animal Care Committee of the University of British Columbia (Vancouver, BC, Canada).

[0125] Streptozotocin (STZ)-induced diabetic mouse model: In a study on protamine derivatives, mice were fasted for 6 hours, and then injected intraperitoneally (ip) with 200 mg / kg of STZ. Three days later, blood glucose (BG) was measured using a glucose meter (ONE Touch Ultra 2 blood monitoring system). Mice with BG levels of 300 mg / dL or higher were considered to have diabetes.

[0126] Preparation of sublingual insulin preparations and animal studies: In studies on protamine derivatives, protamine, C18-P, or P2 were dissolved in water at 10 mg / mL, and insulin was dissolved in water at 20 mg / mL, with the pH adjusted to 3.2 with 1N HCl. The two solutions were mixed and used immediately for animal studies. The dose for protamine or derivative was 3 mg / kg, and the dose for insulin was 5 mg / kg. The insulin dose for subcutaneous (sc) controls was 1 mg / kg. Different insulin preparations were delivered to STZ mice via sc or sublingual (sub.). BG was measured before treatment and 0.5–6 hours after treatment.

[0127] Preparation of sublingual semaglutide preparations and animal studies: In studies on protamine derivatives, protamine, C18-P, or P2 was dissolved in water at 10 mg / mL and then mixed with Ozempic. The mixture was administered immediately. For sub-administration, the dose of protamine or derivative was 6 mg / kg, and the dose of semaglutide was 500 μg / kg. For sc controls, the dose of semaglutide was 60 μg / kg. After mice received different semaglutide preparations, a glucose solution was administered to the mice via ip injection at 1.5 mg / kg. Blood samples were collected at different time points and BG measurements were performed.

[0128] Pharmacokinetic Studies: In studies on protamine derivatives, C18-P or P2 was mixed with either rhGH, Cy7-BSA, or AF647-IgG to prepare formulations for sublingual delivery. The dose of the novel peptide was 6 mg / kg, while the doses of rhGH, Cy7-BSA, and AF647-IgG were 500 μg / kg, 20 mg / kg, and 1.3 mg / kg, respectively. Plasma was collected from mice and analyzed for payload concentration by ELISA (rhGH) or fluorescence plate reader (Cy7-BSA and AF647-IgG). Tissue penetration of AF647-IgG was analyzed by CLSM imaging of sublingual tissue sections collected from the base of the mouse tongue 4 hours after treatment.

[0129] Safety Analysis: In the study of protamine derivatives, mice received protamine or a derivative (6 mg / kg) via the sublingual route on days 0, 7, 14, and 21, and were euthanized on day 22. Whole blood was collected via cardiac puncture into ethylenediaminetetraacetic acid (EDTA) coated tubes (Microvette, Sarstedt AG & Co., Numbrecht, Germany). Plasma was isolated by blood centrifugation (1,000 g, 5 min). Whole blood was used for hematological analysis. Liver and kidney function were analyzed by performing blood chemistry analysis in UBC (IDEXX). Mouse body weight was monitored throughout the study. Major organs were collected, weighed, and stained with hematoxylin and eosin for imaging and toxicity analysis.

[0130] Statistical Analysis: In the study of protamine derivatives, two-group analysis was performed using a two-tailed independent Student's t-test. One-way ANOVA with Tukey's test was used for multiple comparisons. A p-value less than 0.05 was considered statistically significant. Data were plotted using GraphPad Prism version 9.0 (GraphPad Software). All data are presented as mean ± standard deviation (SD). All statistical analyses were performed using GraphPad Prism version 9.0.

[0131] Transmission electron microscopy (TEM): In studies on protamine derivatives, C18-P and P2 were prepared in deionized water at a concentration of 0.1 mg / mL. Before analysis, the samples were stained with 2% uranyl acetate (aqueous solution) and then deposited onto a TEM grid (Ted Pella) coated with 400-msh formvar. C18-P and P2 were then imaged using a 120 kV Tecnai Spirit electron microscope.

[0132] Atomic force microscopy (AFM): In studies on protamine derivatives, protamine, C18-P, and P2 were prepared in MilliQ at a concentration of 0.1 mg / mL. One drop of each sample solution was placed on a silicon wafer and dried overnight. Images were captured using a SUPERSHARPSILICON® silicon SPM sensor (SSS-NCL-10) under an Asylum Research intermolecular force probe 3D controller. The images were then analyzed using Gwyddion software.

[0133] Compounds and Antibodies: In research on protamine nanostructures, the Alexa Fluor 647 protein labeling kit, Trout's reagent (2-iminothiolane·HCl), Inject® Alum adjuvant, Pierce® Rapid Gold BCA Protein Assay Kit, ProcartaPlex® Mouse and Rat Mix & Match panel, and all ELISA kits were purchased from Thermo Fisher Scientific (Ottawa, ON, Canada). Streptozotocin (STZ), Hoechst 33342, allyl glycidyl ether (AGE), PAMAM G4, dimethyl sulfoxide (DMSO), albumin from chicken egg white (OVA), Staphylococcus aureus enterotoxin B (SEB), and protamine were purchased from Sigma-Aldrich. Ultra-LEAF® purified αCD124 was purchased from Biolegend (San Diego, USA). RIPA buffer (10×) was purchased from New England Biolabs (Ipswich, MA, USA). Dialysis membranes (fractionated molecular weight MWCO = 10 and 15 kDa) were purchased from Spectrum Laboratories (Waltham, MA). Hydrophobic fluorescent dye DiI (DiIC) 18 (3); 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine) was purchased from Cedarlane Labs (Burlington, ON, Canada). GMBS (N-γ-maleimidobutyryl-oxysuccinimide ester) was purchased from Biopharma PEG (Watertown, MA, USA). Indium-111 labeled IgG ( 111 In-IgG was provided by Dr. Urs Hafeli from UBC. All other common laboratory chemicals were purchased from Fisher Scientific and VWR Scientific (Mississauga, ON, Canada).

[0134] Synthesis of Protamine Nanostructures AGE-conjugated protamine (Nano-P): In a study on protamine nanostructures, allyl glycidyl ether (AGE, 1.5 g) was added dropwise to 20 mL of protamine solution (10 mg / ml in PBS) and stirred at 300 rpm for 3.5 hours at room temperature. The product was purified by dialysis (molecular weight cutoff, 10 kDa) in Milli-Q water for 48 hours. Nano-P was obtained after lyophilization.

[0135] PAMAM-Protamine (Dendri-P): In a study on the protamine nanostructure, 10 mL of protamine (10 mg / mL in PBS) and 1 mL of Trout's reagent (2-iminothiolane·HCl) (50 mg / mL in PBS) were mixed, followed by the addition of 1.2 mL of EDTA solution (50 mM). The mixture was incubated at room temperature for 1 hour, followed by dialysing against 5 mM EDTA for 1 hour to obtain solution A. 100 μL of GMBS (280 mg / mL in DMSO) and 3 mL of PAMAM G4 (4 mg / mL in PBS) were mixed. The mixture was incubated at room temperature for 1 hour, followed by dialysing against Milli Q water for 1 hour to obtain solution B. Solutions A and B were reacted at room temperature for 1 hour, followed by dialysing against Milli Q water for 3 days, and then lyophilized.

[0136] Transmission electron microscopy (TEM): In studies on protamine nanostructures, nano-P and Dendri-P were dissolved in deionized water at a concentration of 0.1 mg / mL. Before analysis, the samples were stained with 2% uranyl acetate (aqueous solution) and then deposited onto a TEM grid (Ted Pella) coated with 400-msh formvar. Nano-P and Dendri-P were then imaged using an 80 kV Tecnai Spirit electron microscope.

[0137] Animals: For the study of protamine nanostructures, 4-week-old female CD-1 and BALB / c mice were obtained from Charles River Laboratories (Wilmington, MA). In vivo experiments were performed according to established experimental protocols (A22-0141 and A20-0132) approved by the Animal Care Committee of the University of British Columbia (Vancouver, BC, Canada).

[0138] 111 In-IgG in vivo distribution: In research on protamine nanostructures, 111 In-IgG was mixed with protamine and then delivered intranasally to unsensitized 4-week-old CD-1 mice (dose = 111 In-IgG: 13 mg / kg; Protamine: 3 mg / kg). Conscious mice were restrained with their non-dominant hand and given small droplets (approximately 5 μL). 111 The in-IgG formulation was delivered via a 10 μL micropipette tip at a 45-degree angle near one nostril. After the droplet was inhaled, another droplet was administered into the other nostril. The procedure was repeated up to five times to deliver a total volume of formulation up to 24 μL. 12Mice were imaged by SPECT / CT at 0, 0.5, 2, 4, and 6 hours after intranasal administration under anesthesia. For SPECT / CT imaging sessions, mice were anesthetized using isoflurane (1–3% for maintenance, up to 5% for induction) delivered in oxygen from a high-precision vaporizer. The inventors used a VECTor / CT multimodal preclinical scanner (MILabs in the Netherlands) equipped with an ultra-high sensitivity (XUHS) pinhole collimator (2 mm) [63, 64]. Throughout the entire imaging process, the mice's respiratory rate and body temperature were continuously monitored. All animals recovered well after each imaging scan. After the final SPECT / CT scan at 24 hours post-administration, mice were kept under isoflurane and immediately euthanized by CO2 asphyxiation, followed by cardiac puncture. SPECT data were processed using the Pixel-based Ordered Subset Expectation Maximisation (POSEM) algorithm at 0.4 mm 3 The voxel size was used to reconstruct the dataset with 16 subsets and 6 iterations. The reconstruction was performed using the following method: 111 The procedure was performed using the 171 keV photoelectric peak of In with a 20% spectral width. Each SPECT scan was attenuated for injection time, and the recorded counts were adjusted using the attenuation rate determined from the CT scan at each time point. 111 Point source measurements of In samples were scanned to establish calibration coefficients, thereby relating counts / voxels to activity concentrations. In each SPECT image, the inventors manually outlined the volume of interest around the nasal cavity region to consider the activity concentration in this designated region. Subsequently, the inventors compared different groups by calculating mean standard uptake (SUV) using the formula SUV mean (g / mL) = [radioactivity concentration (MBq / mL)] / [injection dose (MBq) / animal body weight (g)]. The inventors used AMIDE software (version 1.0.6) to visualize and analyze all images

[65] .

[0139] In vivo distribution of AF-αCD124 in mice: In a study on protamine nanostructures, AF-αCD124 was mixed with protamine, Nano-P, or Dendri-P and then delivered intranasally to unsensitized 4-week-old CD-1 mice as described above (dose = 1.3 mg / kg for AF-αCD124; 3 mg / kg for protamine, Nano-P, or Dendri-P). After 2 hours, the mice were euthanized and their noses were collected. The noses were processed as previously reported. 13 In short, the nasal turbinates were washed with PBS and decalcified in EDTA solution (143 g / L in distilled water) for at least one week. The samples were transferred daily to fresh EDTA solution. The nasal turbinates were then incubated in PBS for 25 minutes four times, followed by incubation in 30% sucrose for 24 hours. These samples were then embedded in OCT and sectioned to a thickness of 10 μm using Leica Cryostat. The slides were then stained with DAPI for 30 minutes, followed by CLSM.

[0140] CRSwNP Model: In studies on protamine nanostructures, unsensitized 4-week-old BALB / c mice were systemically sensitized on days 0 and 5 by intraperitoneal injection of 25 μg of OVA mixed with 2 mg of aluminum hydroxide gel in 100 μl of saline. After general sensitization, 20 μl of 6% OVA in saline was administered intranasally to the mice daily from days 12 to 19. From days 19 to 47 or 107, 6% OVA was administered intranasally three times per week to establish moderate or severe CRSwNP models, respectively. After induction of allergic sinusitis, 6% OVA containing Staphylococcus aureus enterotoxin B (SEB, 20 ng) was administered intranasally three times per week for 8 weeks. Different formulations containing αCD124 were administered three times per week for 8 weeks, starting from the week of SEB treatment. For moderate CRSwNP, the dose of αCD124 was 1 mg / kg for both intranasal and subcutaneous routes. For severe CRSwNP, the dose was 2 mg / kg for the intranasal route and 25 mg / kg for the subcutaneous route. Protamine, Nano-P, and Dendri-P were administered at 3 mg / kg for both models. One day after the last treatment, mice were euthanized, whole blood was collected, and IgE analysis was performed. The noses were collected and imaged using a micro-CT specimen scanner (Scanco Medical μCT100) at 90 kVp, 0.2 mA, and then reconstructed at a voxel size of 17 μm. One-third of the nose was excised, homogenized in 500 μl of RIPA lysis buffer per 100 mg of tissue, and placed in homogenization tubes equipped with 0.5 mm zirconium oxide beads. The tube was placed in a Bullet Blender® (Next Advance) with the control set to speed 8 and time 5. The sample was then centrifuged at 16,000 rpm for 10 minutes at 4°C. The supernatant was collected, and the protein concentration was determined using the Pierce® Rapid Gold BCA Protein Assay Kit.Samples were assayed for mouse immunoglobulin E (IgE), eotaxin, thymic and activating regulatory chemokines (TARC), interleukin-13 (IL-13), tumor necrosis factor α (TNF-α), IL-2, IL-1β, thymic stromal lymphocyte necrosis factor (TSLP), IL-17A, IL-25, IL-33, interferon gamma (INF-γ), and IL-12p70 using ELISA or the ProcartaPlex® Mouse and Rat Mix & Match panel. Nasal remnants were decalcified as described above, sectioned to a thickness of 6 μm, and stained with hematoxylin and eosin (H&E), Giemsa, Masson's trichrome (MT), or Alcinablue-periodine Schiff (AB-PAS).

[0141] In vivo safety evaluation of protamine, Nano-P, and Dendri-P: In a study on protamine nanostructures, healthy mice received 3 mg / kg of protamine, Nano-P, or Dendri-P twice daily for one month, and were euthanized one day after the final dose. Whole blood was collected via cardiac puncture into ethylenediaminetetraacetic acid (EDTA) coated tubes (Microvette, Sarstedt AG & Co., Numbrecht, Germany). Plasma was isolated by centrifugation (10,000 × g, 5 min). Whole blood was used for hematological analysis. Liver and kidney function were analyzed via blood chemistry analysis in UBC (IDEXX). Body weight of each mouse was monitored throughout the study. Major organs, including the nose and trachea, were collected, weighed, and stained with hematoxylin and eosin for imaging and toxicity analysis. Animals were observed throughout the experiment, before, during, and immediately after each exposure, for nasal symptoms (nasal discharge)

[66] , tearing, eye irritation, excessive blinking, salivation, cyanosis, lethargy, piloerection, paralysis, skin irritation, erythema, nasal spasms, and directional and non-directional movement within the cage.

[66] A five-minute video of each mouse was recorded after the final dose of intranasal administration.

[0142] Statistical Analysis: In the study on protamine nanostructures, two-group analysis was performed using two-tailed independent Student's t-tests or Welch's t-tests. One-way ANOVA with Tukey's test was used for multiple comparisons. A p-value less than 0.05 was considered statistically significant. Data were plotted using GraphPad Prism version 9.0 (GraphPad Software). All data are presented as mean ± standard deviation (SD). All statistical analyses were performed using GraphPad Prism version 9.0.

[0143] result Characteristics of protamine-mediated cell penetration The inventors first demonstrated that protamine at concentrations below 37.5 μg / ml did not cause any toxicity to cells (Figures 1A-1B). Next, the inventors compared the cellular uptake of FITC-BSA in the presence of protamine from different sources at 37.5 μg / mL after 4 hours of incubation using FACS. As shown in Figure 2, FACS revealed that protamine 1 (protamine sulfate from salmon) showed superior activity in promoting FITC-BSA cell delivery compared to the other sources of protamine in this study, with approximately 85% of cells being FITC-positive with the protamine 1 formulation. Protamine 2 (protamine sulfate from herring) and 4 (protamine chloride from salmon) delivered FITC-BSA to 70-75% of cells, while protamine 3 (protamine from salmon) was ineffective. Based on the data above, protamine sulfate from salmon was used for the remainder of this study.

[0144] Next, the inventors utilized confocal live-cell imaging to examine cells during protamine treatment and characterize protamine-mediated cell delivery. Live-cell images of RPMI2650 and Caco2 cells were examined after incubation with AF647-protamine for up to 60 minutes under CLSM. Protamine was first labeled with the Alexa Flour 647 (AF647) protein labeling kit according to the manufacturer's protocol and then added to the culture medium at 37.5 μg / ml. The inventors found that AF647-labeled protamine adhered to the cell surface within 4–8 minutes after incubation, and adsorption to the cell surface increased over time. Protamine translocated to the nucleus at 20–30 minutes, and there was little accumulation of protamine in the cytosol at any given time. This suggests that protamine rapidly translocated from the cell membrane to the nucleus.

[0145] The uptake of the membrane-impermeable dye propidium iodide (PI) by RPMI2650 cells in the absence and presence of AF647-protamine under live CLSM conditions was investigated. PI uptake by RPMI2650 cells was not observed in the absence of protamine even after 60 minutes of incubation, while significant nuclear PI uptake was detected 2 minutes after protamine addition. The data further indicated that cell membrane permeability immediately increased upon protamine adsorption to the cell membrane, enabling PI to pass through the cell.

[0146] The cell membrane permeability of RPMI2650 to PI after protamine removal was investigated. Cells were treated with AF647-protamine for 10 minutes, followed by washing and fresh medium replacement. Cells were incubated with PI for up to 30 minutes and imaged under CLSM. The results showed that the effect of protamine on increased cell membrane permeability was reversible. When the protamine-containing medium was replaced with fresh medium, the cells became impermeable to PI again. Specifically, limited cell uptake of PI was detected after protamine removal, suggesting that the cell penetration effect of protamine was transient and reversible.

[0147] Protamine increased intracellular protein delivery compared to R8. Next, the inventors compared the delivery efficiency of FITC-BSA by protamine and R8 in cell cultures. Confocal microscopy images of cells treated with FITC-BSA in or without protamine or R8 were examined. Confocal images showed increased intracellular delivery of FITC-BSA when physically mixed with protamine, while the R8 effect was mild. Intracellular FITC-BSA fluorescence was quantified by ImageJ, demonstrating 20-fold (RPMI2650) and 5-fold (Caco2) increased intracellular delivery with protamine compared to R8 (Figures 3A-3B). Protamine facilitated nuclear delivery of FITC-BSA in both RPMI2650 and Caco2 cell lines, with approximately 60-75% of FITC-BSA delivered to the nucleus (Figures 3C-3D). Furthermore, in R8 / FITC-BSA treated cells, FITC-BSA was detected almost exclusively (approximately 95%) within lysosomes in both cell lines, while in protamine treated cells, lysosomes were hardly detected. The data suggest that protamine and R8 exhibit distinctive cell penetration mechanisms, and that protamine delivery was able to reduce lysosomal capture and biomolecule degradation.

[0148] In follow-up studies, confocal microscopy images of cells after treatment with mAbs in or without protamine or R8 were examined. An antibody targeting SIRT1 located in the nucleus (Alexa488-anti-SIRT1) showed that protamine improved intracellular delivery of this monoclonal antibody (mAb) in RPMI2650 cells 11-fold compared to R8 (Figure 4A), while free mAbs showed little to no cellular delivery. The nearly exclusive nuclear localization (approximately 80%) of this antibody delivered by protamine suggests that the antibody was delivered in an active form capable of targeting the antigen. In contrast, almost all Alexa488-anti-SIRT1 delivered by R8 accumulated in lysosomes. As shown in Figure 4B, over 95% of the mAbs were overlaid with lysosomes in R8-treated cells, over 80% of the mAbs in the cells accumulated in the nucleus in the protamine group, and less than 10% accumulated in lysosomes. Protamine delivery offered a significant advantage over R8 for this mAb, as it increased cellular uptake and improved delivery to target organelles.

[0149] Protamine showed increased activity in regulating cellular F-actin rearrangement and tubulation compared to R8. The inventors characterized and compared cellular actin and tubulation after R8 and protamine treatment. Confocal microscopy images of the cellular actin cytoskeleton after treatment with protamine or R8 were examined for F-actin elongation and increased cell elongation, or F-actin aggregation in dots. Cells were considered positive for actin aggregates when they expressed 20 or more F-actin dots. As shown in Figures 5A–5C, there were no significant differences in overall F-actin expression. However, cells treated with R8 and protamine showed increased elongation area. R8 and protamine increased cell elongation area by 1.5-fold and 2.3-fold, respectively, compared to the control. Protamine-treated cells showed the largest cytoplasm-to-nucleus ratio, and the cells were flattened. The data suggest that R8 and protamine-treated cells reorganized their intracellular actin to make the cells thinner, which could reduce membrane tension [13, 25] and increase membrane permeability. In the R8 treatment group, 40% of cells exhibited F-actin aggregates, whereas in the control and protamine treatment groups, only 10–15% of cells showed such aggregates. R8-treated cells showed increased actin aggregates and lysosomal accumulation of cargo. However, increased actin aggregation and lysosomal accumulation were not observed in protamine-treated cells, suggesting a different mechanism of action.

[0150] Confocal microscopy images of cellular microtubules after treatment with protamine or R8 were examined. TubulinTracker staining revealed characteristic morphologies, indicating that R8 and protamine appeared to affect tubulin polymerization into microtubules. To quantify tubule formation, the TubulinTracker-stained microtubule skeleton was quantitatively analyzed in Fiji to obtain the mean number of microtubules and total microtubule length per cell. Protamine increased the number of microtubules and total microtubule length per cell by approximately 2-fold and 3.5-fold, respectively, compared to the control, while R8 did not show such an effect (Figures 6A-6B). These data indicate that protamine demonstrated an enhanced effect in promoting cellular tubule formation, which is associated with mechanisms for transcellular delivery and CPP activity. The data are consistent with the results showing that protamine demonstrated increased intracellular delivery efficiency of BSA and anti-SIRT1.

[0151] Live-cell CLSM imaging of FITC-BSA uptake by RPMI2650 and Caco2 cells in the presence of protamine for 4 hours at 12 or 37°C was performed. The results showed that protamine-mediated cellular uptake of the protein was temperature-dependent (increased with higher temperatures), suggesting an energy-dependent mechanism. When tubular formation was reduced by decreasing the incubation temperature, the effect of protamine on intracellular delivery of FITC-BSA decreased, supporting the idea that tubular formation was one of the key mechanisms of protamine's CPP activity.

[0152] Protamine increased protein penetration in cell spheroids compared to R8. Next, the inventors investigated whether protamine can enhance FITC-BSA penetration into multiple cell layers compared to R8, using a cell spheroid model. RPMI2650 spheroids were incubated with FITC-BSA in or without protamine or R8 for 3 hours, washed, and imaged at different depths from the top by CLSM. Confocal microscopy analysis of FITC-BSA penetration in RPMI2650 spheroids in the presence of protamine or R8 was performed. Spheroids were imaged at different depths from the surface. Protamine-treated spheroids (DiI-labeled) showed significant FITC-BSA penetration, strongly indicating FITC signaling in all scanned layers, while FITC-BSA alone and R8 / FITC-BSA showed little spheroid penetration. Note that fluorescence in the spheroid core was undetectable due to limited photopenetration. R8 increased FITC-BSA delivery in 2D cell cultures compared to the protein-only group, but neither effect was observed in this 3D spheroid model. The results were further confirmed by FACS (Figure 7A), which showed that 94% of cells in the spheroids were FITC-positive in the protamine group, while less than 10% of cells in the FITC-BSA and R8 / FITC-BSA groups were associated with FITC fluorescence. Similar results were obtained in the Caco2 spheroid model, where confocal imaging of FITC-BSA penetration in Caco2 spheroids in the presence or absence of protamine or R8 was examined. Spheroids were imaged at different depths from the surface. The results showed that protamine increased FITC-BSA penetration into the spheroids compared to R8.

[0153] Protamine increased systemic absorption of proteins via the nasal pathway compared to R8. The inventors further investigated the effects of protamine in an in vivo model compared to R8. Cy7-BSA was mixed with protamine or R8 and delivered intranasally to mice. After 0.5–2 hours, the concentration of Cy7-BSA in plasma was measured by fluorescence. As shown in Figure 7B, at 2 hours, the protamine group showed a 3-fold increase in Cy7-BSA absorption compared to the R8 formulation and the protein-only groups. The results were consistent with those obtained in 2D culture and spheroid models.

[0154] Protamine enabled systemic absorption of insulin via intranasal delivery, normalizing blood glucose levels in diabetic mice. Next, the inventors explored the medical utility of this non-invasive protein delivery technology and selected insulin for further study. The inventors initially focused on optimizing the protamine-insulin preparation by varying the ratio between protamine and insulin. Insulin was first dissolved in water at a pH of 3.2, mixed with different amounts of protamine, and then delivered to STZ diabetic mice. As shown in Figure 8A, no BG-reducing effect was detected when the protamine-to-insulin ratio was 2.5 or less. When the ratio increased to 3 or more, the preparation significantly reduced BG from 400 mg / dL to 200 mg / dL at 1 hour after delivery. BG further decreased to 100-150 mg / dL at 2-4 hours after delivery, but returned to 300 mg / dL at 6 hours. Therefore, the inventors used a protamine-to-insulin weight ratio of 3 for further study.

[0155] Next, the inventors focused on optimizing the pH of the formulation (Figure 8B). Although no significant difference was observed among the different pH levels due to large fluctuations in the data, the pH 3.2 formulation appeared to be the most effective, and the pH 3.2 formulation was selected to prepare the optimal formulation. Normal human nasal mucosa is in an acidic environment (approximately pH 5)

[67] , and nasal formulations with an acidic pH (2.5–3.5) have been demonstrated to be safe in clinical trials, including Taffix [68, 69] and preservative-free acidic saline nasal spray

[70] .

[0156] As shown in Figure 8C, the BG-reducing efficacy of the optimal protamine / insulin formulation was dose-dependent, with efficacy increasing with dose. Figure 8D shows that the BG-reducing effect of the optimal protamine / insulin formulation was comparable to that of sc-insulin delivery, although the effect of sc-insulin appeared statistically stronger and longer-lasting, maintaining BG at approximately 50 mg / dL for 2–6 hours after injection. It should be noted that the efficacy comparison in this study, based on the same low dose, showed that the effects of our nasal formulation and standard sc-insulin were statistically equivalent. In previous studies, much higher doses were required for non-injectable formulations for insulin to achieve significant activity in animals [13, 71]. This supports the high efficiency of protamine for intranasal delivery of insulin. Pharmacokinetic (PK) results are consistent with the efficacy data (Figure 8E). Sc-insulin was rapidly absorbed into plasma, reaching a peak concentration of 300 μIU / mL at 0.5 hours, and then rapidly decreasing to undetectable levels over 4 hours. The in protamine / insulin formulation produced a peak concentration of approximately 50 μIU / mL at 0.5–2 hours, and the concentration was below the detection limit at 4 hours. The area under the curve in the sc insulin group was 3.5 times higher than that in mice treated with in protamine / insulin. The relative nasal bioavailability of protamine-insulin was calculated by comparing the area under the curve (AUC) in Figure 8E, and the relative nasal bioavailability was 28.6%.

[0157] The optimal protamine formulation for insulin reported in this study differs significantly from clinically used intermediate-acting insulin (Neutral Protamine Hagedorn (NPH) insulin). Firstly, NPH insulin still requires sc-delivery, and the protamine in the NPH formulation is intended to control insulin release and produce an extended BG-reducing effect. Secondly, the protamine-to-insulin ratio in NPH is 0.2, while our formulation has a ratio of 3, achieving systemic absorption via nasal delivery. Thirdly, NPH has a neutral pH and its formulation is milky white in appearance. In contrast, our formulation has a pH of 3.2 and appears clear. Fourthly, in addition to insulin and protamine, the NPH formulation contains phenol for pH adjustment and zinc ions for controlled insulin release.

[0158] Protamine overcomes mucosal and epithelial barriers for intranasal delivery of insulin and remodels adhesive junctions in the nasal epithelium. FITC-insulin penetration in the nose was investigated in the presence and absence of protamine, at earlier and later stages of FITC-insulin distribution. The results indicate that protamine induced E-cadherin rearrangement in the epithelium, leading to loss of E-cadherin expression in some cell bindings. BG and PK data suggest that protamine facilitated systemic absorption of insulin, transporting it from the nasal cavity through the mucosa and epithelial barrier to the microvessels in the lamina propia layer. To provide visual evidence, nasal turbinates from mice were collected 2 hours after in-delivery of protamine / FITC-insulin. FITC-insulin alone was included as a control in this study. Different insulin absorption phases were observed within the same nasal turbinate samples. Early and later images are reported separately. In the absence of protamine, no FITC-insulin penetration into the mucosa (the layer above the epithelium) or epithelial (labeled with E-cadherin, a marker for adherent junctions) layer was detected. However, when mixed with protamine, FITC-insulin penetrated the mucosa and epithelial layers, reaching the lamina propria (the layer beneath the epithelium). Furthermore, some FITC-insulin was detected in the mucosa / epithelial layers in the protamine group during this early stage. It should also be noted that protamine inhibited E-cadherin expression in the epithelial layer, as E-cadherin was undetectable among some epithelial cells during this early stage. To quantify this phenomenon, the inventors counted the number of nuclei and E-cadherin loops in both groups. Nasal epithelium exposed to protamine had a significantly higher nucleus-to-E-cadherin inclusion ratio (1.4±0.28) than the saline-treated group (0.95±0.11). The data suggest that protamine was able to reduce epithelial adhesion and facilitate paraepithelial delivery of insulin. However, such effects were transient and reversible, and in the protamine group, E-cadherin expression in the epithelium returned to normal later in insulin absorption, when all FITC-insulin had left the epithelial layer and reached the lamina propria.However, in this later stage in the saline / FITC-insulin group, only low levels of FITC-insulin were detected in the mucosal layer, and there was no further penetration into the epithelium and lamina propria.

[0159] Short-term and long-term safety of protamine preparations Pathological analysis of the main airway tissue was performed on mice that received a single dose of protamine, mice that received protamine once daily for seven consecutive days, or mice that received PBS. The inventors also investigated the short-term (single dose) and long-term (once daily for seven consecutive days) safety of insulin / protamine preparations. One day after the final dose, the nasal cavity, trachea, and lungs were collected and sectioned. No abnormalities were found in the trachea, lungs, or nose in any of the groups by a committee-certified pathologist using H&E staining. The inventors also stained goblet cells in tissue, which overgrow and produce mucin in inflammatory conditions, using the periodate Schiff (PAS) method. Goblet cells were stained purple with PAS to distinguish them from other cell types with a columnar morphology. The images show that there was no increased proliferation of goblet cells and increased mucin expression in nasal tissue after protamine treatment compared to controls. PAS-positive goblet cells were not detected in the trachea and lungs. Toluidine blue was used to stain acidic mast cells, and the dye emitting a dark purple color in the granules within the acidic mast cells when it reacted with heparin and histamine, the main mediators in tissue inflammation. Significant mast cell infiltration in the nose, trachea, and lungs was not observed after protamine treatment compared to PBS controls. Finally, TNF-α, the main inflammatory cytokine, was stained in the tissue by immunohistochemistry, and no significant upregulation of TNF-α after protamine treatment was detected compared to PBS controls. All tissues showed background staining of TNF-α when compared to stained control samples without the primary antibody. Overall, the histological results support the absence of inflammatory toxicity induced by protamine treatment.

[0160] Protamine preparations were shown to offer several advantages over previously reported techniques for non-injectable transcellular and transmucosal delivery of proteins and peptides. First, the method was simple and required only physical mixing of protamine and protein cargo. Second, protamine is approved for human use by the FDA and has a proven safety record. Third, protamine facilitated transcellular and transmucosal penetration of proteins and peptides up to 110 kDa and had reduced lysosomal accumulation. Fourth, the protamine-mediated changes in cells were reversible and included transient increased membrane permeability and disruption of adhesion junctions in the nasal epithelium. Therefore, repeated treatment with protamine preparations was shown to be safe. Fifth, protamine-mediated intranasal delivery of insulin was effective, and its effect in diabetic animals was comparable to that of sc-insulin at the same dose.

[0161] The inventors demonstrated that protamine, through simple physical mixing, significantly increased intracellular protein delivery into cells in both 2D and 3D models compared to R8. Intracellularly, protamine-mediated delivery showed reduced accumulation in lysosomes and increased nuclear targeting compared to R8. Mechanistic studies revealed that protamine exerted a stronger effect than R8 on cellular actin rearrangement and tubulation associated with increased cell permeability and uptake. The protamine formulation was optimized for intranasal delivery of insulin, promoting systemic absorption. The optimal protamine / insulin formulation showed comparable BG-reducing activity compared to sc-insulin. Histological and confocal microscopy results indicated that protamine mediated rearrangement of adhesion junctions in the nasal epithelium, thereby facilitating insulin penetration into the lamina propia layer for systemic absorption. Repeated treatment with this formulation was safe in mouse models.

[0162] Protamine-enhancing FITC-BSA penetration in human skin Protamine sulfate from DAPI and salmon grade X was purchased from Sigma-Aldrich. FITC conjugate of poly(arginine) 8 (R8) and bovine serum albumin (FITC-BSA) was purchased from Thermo Fisher Scientific. Protamine solution (10 mg / ml) was mixed with FITC-BSA (2 mg / ml) in a weight ratio of 1.5:1 to a final volume of 100 μl, and the mixture was dropped onto the surface of human skin samples and incubated for 24 hours. The bottom of the human skin was moistened with liquid PBS. After 24 hours, the human skin was collected and frozen-sectioned. The sections were then stained with DAPI and imaged by confocal laser scanning microscopy (CLSM). The results showed that protamine enhanced FITC-BSA penetration in human skin.

[0163] Pig skin penetration FITC-CM-dextran (average molecular weight 150,000) and Sigma were dissolved in 5% glucose at a concentration of 2 mg / ml. Protamine was dissolved in 5% glucose at a concentration of 10 mg / ml. Hyaluronic acid was dissolved in water at a concentration of 0.1 mg / ml. FITC-BSA was dissolved in water at a concentration of 5 mg / ml.

[0164] Pig skin was inserted into the Franz cell device. The bottom of the Franz cell was filled with saline solution and maintained at 37°C. Solution A, 50 μL of FITC-CM-dextran + 45 μL of protamine + 25 μL of 5% glucose as a control; B, 50 μL of FITC-CM-dextran + 70 μL of 5% glucose as a control; C, 25 μL of FITC-CM-dextran + 75 μL of protamine + 25 μL of 0.1 mg / ml of hyaluronic acid as a control; D, 25 μL of FITC-CM-dextran + 75 μL of 5% glucose + 25 μL of 0.1 mg / ml of hyaluronic acid as a control; E, 20 μL of FITC-BSA + 90 μL of protamine; F, 20 μL of FITC-BSA + 90 μL of 5% glucose water or PBS as a control were applied to the top of pig skin and incubated for 24 hours.

[0165] Pig skin incubated with PBS for 24 hours was used as a control.

[0166] Pig skin incubated with A or B for 24 hours showed that FITC-CD-dextran was delivered to the epidermal layer by protamine, while FITC-CD-dextran alone did not show penetration.

[0167] Pig skin incubated with C or D for 24 hours showed that FITC-CD-dextran / HA was delivered to the epidermal layer by protamine, while FITC-CD-dextran / HA alone did not penetrate.

[0168] Pig skin incubated with E or F for 24 hours showed that FITC-BSA was delivered to the epidermal and dermal layers by protamine, while FITC-BSA alone did not penetrate.

[0169] Protamine enhanced sublingual delivery of semaglutide (a GLP-1 receptor agonist). Mice received glucose at a dose of 1.5 g / kg via i.p injection immediately after semaglutide delivery (glucose tolerance test, GTT). Blood glucose (BG) was monitored at 0, 5, 10, 20, 30, 50, 80, and 120 minutes. Another GTT was performed 1–3 days after semaglutide delivery.

[0170] Semaglutide delivered by sc, or sublingually delivered semaglutide mixed with protamine, effectively controlled BG for 2 days, while sublingually delivered semaglutide alone was ineffective (Figure 9).

[0171] The area under the BG curve was significantly lower for the semaglutide (sc) and semaglutide + protamine (sublingual) groups compared to the control group (Figure 10 and Table 1).

[0172] [Table 1]

[0173] Protamine-enhanced GLP-1 sublingual delivery Mice received 1.5 g / kg of glucose intravenously immediately after GLP-1 delivery (glucose tolerance test, GTT). Blood glucose (BG) was monitored at 0, 5, 10, 20, 30, 50, 80, and 120 minutes.

[0174] Sublingual delivery of GLP-1 / protamine provided better backgrowth control compared to GLP-1 delivered IV or sublingually (Figures 11A-11C).

[0175] Sublingual Cy7-BSA penetration Cy7-BSA was dissolved in water at a concentration of 60 mg / ml. Protamine was dissolved in water at a concentration of 10 mg / ml.

[0176] Cy7-BSA was mixed with protamine solution or water. The mixture was administered sublingually to mice. The mice were anesthetized during administration of the formulation and kept under anesthesia for an additional 5 minutes to allow for absorption.

[0177] The doses for protamine and Cy7-BSA were 6 mg / kg and 20 mg / kg, respectively. Mice were euthanized at 0, 0.5 hours, 2 hours, and 4 hours, respectively. Blood and tongues were collected. Plasma was isolated by centrifugation (10,000 g, 5 min) and analyzed for Cy7 fluorescence using a plate reader. Tongues were sectioned and analyzed by confocal imaging.

[0178] Protamine demonstrated enhanced sublingual delivery of Cy7-BSA, comparable to sc injection (Figure 12).

[0179] Protamine also enhanced Cy7-BSA penetration in the tongue at 0.5 hours.

[0180] Protamine demonstrated enhanced sublingual delivery of 647-dupilumab (Figure 13 and Table 2).

[0181] [Table 2]

[0182] Protamine enhanced the penetration and retention of 647-dupilumab in the tongue at 0.5, 2, 4, and 24 hours.

[0183] Lymph nodes were collected at 0.5, 2, 4, and 24 hours. 647-dupilumab delivered by sc or sublingual protamine showed accumulation in the lymph nodes.

[0184] A mouse model of chronic sinusitis with nasal polyps (CRSwNP). Unsensitized 4-week-old BALB / c mice were systemically sensitized on days 0 and 5 by intraperitoneal injection of 25 μg of OVA (grade V, Sigma, St. Louis, MO) mixed with 2 mg of aluminum hydroxide gel. After general sensitization, 3% OVA in sterile saline was administered intranasally to conscious mice daily from days 12 to 19. After induction of allergic sinusitis, 3% OVA in 40 μL of PBS containing Staphylococcus aureus enterotoxin B (SEB; 20 ng per mouse) was administered intranasally to conscious mice three times a week for 8 weeks. Different drug formulations were administered three times a week for 8 weeks, starting from the week of SEB treatment. The mice were then euthanized, and their noses were collected for analysis.

[0185] The treatment group included the following: Group A: Intranasal drip infusion of sterile PBS Group B: Intranasal drip infusion of CD124 (1.5 mg / kg) Group C: Intranasal drip infusion of protamine (3 mg / kg) + CD124 (1.5 mg / kg) Group D: Healthy mice

[0186] Symptoms were assessed by counting the number of rubbings that occurred within a 10-minute time frame 24 hours after the last dose. Protamine / CD124-treated mice showed a significant reduction in nasal rubbing compared to PBS controls, and CD124 in and CD124 sc showed no activity (Figure 14).

[0187] Polypoid lesions and epithelial destruction were counted from H&E-stained slides. Protamine / CD124-treated mice showed a significant reduction in the number of polypoid lesions and epithelial destruction compared to the CD124-only group (Figure 15).

[0188] The nasal cavities of mice were imaged using micro-CT. Protamine / CD124-treated mice showed the least degree of mucosal thickening and adhesion in the nasal tuberosities compared to treatment with PBS or CD124 alone.

[0189] ELISA results were obtained from nasal homogenization. Protamine / CD124-treated mice showed reduced levels of INF-gamma, IL-1β, IL-17α, and TNF-α in the nose compared to PBS controls (Figures 16A–16D).

[0190] Protamine lipid conjugate: DMPE-protamine material DMPE-PEG2k-NHS was purchased from Biochempeg Scientific Inc. Protamine sulfates from Hoechst 33342 and Salmon Grade X were purchased from Sigma-Aldrich. Dialysis membranes (molecular weight cutoff MWCO = 10 or 3.5 kDa) were purchased from Spectrum Laboratories (Waltham, MA). The hydrophobic fluorescent dye DiI (DiIC18(3); 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine) was purchased from Cedarlane Labs. All other common laboratory chemicals were purchased from Fisher Scientific and VWR Scientific (Mississauga, ON, Canada).

[0191] DMPE-Protamine DMPE-protamine was synthesized by reacting DMPE-PEG-NHS with protamine in a molar ratio of 10:1. The reaction was carried out in Milli Q water at room temperature for 1 hour. The reaction solution was then dialyzed for 3 days and freeze-dried. DMPE-protamine was characterized by NMR.

[0192] The critical micelle concentration (CMC) of DMPE-protamine was found to be 0.376 mg / ml, with a size of 107.4 ± 3.8 nm and a polydispersity index of 0.188 ± 0.02. The DMPE-protamine molecule self-assembled into nanoparticles in the aqueous phase (Figure 17).

[0193] Cellular uptake For the GFP-CRISPR / Cas9-gRNA uptake study, the cell suspension was first incubated with 1 μg / mL of the membrane stain DiI at 37°C for 30 minutes, then centrifuged, washed three times with PBS, and subsequently seeded. The cells (RPMI2650) were placed in a 12-well plate, with 1 × 10⁶ cells per well. 5 Cells were seeded in 1 mL of medium containing 10% FBS and incubated for 18–20 hours to achieve 70–80% confluence. The medium was replaced with serum-free medium containing 0.1 μM oligo(sgRNA) and 0.1 μM Cas9 protein in the presence or absence of either 37.5 μg / mL protamine or DMPE-protamine, at 37°C for 4 hours. Cells were washed three times with PBS and then stained with Hoechst 33342 and Lysotracker, respectively, for the nucleus and lysosomes, according to the manufacturer's protocol. Stained (unfixed) cells were immediately imaged by confocal laser scanning microscopy (CLSM) and analyzed with ZEN software (Carl Zeiss, Oberkochen, Germany) and Image J.

[0194] The results showed that DMPE-protamine demonstrated better nuclear delivery of CRISPR / Cas9-gRNA compared to protamine (Figure 18).

[0195] Polyvalent (or cross-linked) protamines: (protamine)2, (protamine)3, and PAMAM dendrimer-protamine conjugates material SULFO-SMCC and succinimidyl 4-maleimide butyrate (98%) (GMBS) were purchased from Biochempeg Scientific Inc. PAMAM G4 was purchased from Sigma. Trout's reagent (2-iminothiolane·HCl), EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodimide hydrochloride), NHS (N-hydroxysuccinimide), and 2-mercaptoethanol were purchased from Thermo. LysoTracker Red DND-99 and FITC conjugate of bovine serum albumin (FITC-BSA) were purchased from Thermo Fisher Scientific (Ottawa, ON, Canada). Hoechst 33342, anhydrous citric acid, and protamine sulfate from salmon grade X were purchased from Sigma-Aldrich. Dialysis membranes (molecular weight cutoff MWCO = 10 or 3.5 kDa) were purchased from Spectrum Laboratories (Waltham, MA). All other common laboratory chemicals were purchased from Fisher Scientific and VWR Scientific (Mississauga, ON, Canada).

[0196] (Protamine) 2 Protamine was crosslinked via a two-step reaction using Trout's reagent and SULFO-SMCC (Figure 19).

[0197] Solution A: Protamine and Trout's reagent (2-iminothiolane·HCl) were dissolved in 5 mM EDTA in a molar ratio of 1:20. The solution was incubated at room temperature for 1 hour, followed by dialyzing in 5 mM EDTA for 1 hour.

[0198] [ka]

[0199] Solution B: Protamine and SULFO-SMCC were dissolved in Milli Q water in a molar ratio of 1:10. The solution was incubated at room temperature for 1 hour, followed by dialyzing against Milli Q water for 1 hour.

[0200] Solution A was added to solution B and reacted at room temperature for 1 hour. The reaction solution was then dialyzed against Milli Q water for 3 days and freeze-dried.

[0201] (Protamine) 2 Protamine was crosslinked using the following NHS-PEG-NHS after a one-step reaction.

[0202] [ka]

[0203] Protamine and NHS-PEG-NHS were dissolved in PBS in a molar ratio of 1:10 (10 mg / ml protamine). The solution was incubated at room temperature for 24 hours, followed by dialyzing in water for 48 hours and lyophilization.

[0204] PAMAM Dendrimer-Protamine Protamine was linked to a PAMAM dendrimer (Figure 20).

[0205] Solution A: Protamine and Trout's reagent (2-iminothiolane·HCl) were dissolved in 5 mM EDTA in a molar ratio of 1:20. The solution was incubated at room temperature for 1 hour, followed by dialyzing in 5 mM EDTA for 1 hour.

[0206] [ka]

[0207] Solution B: GMBS was dissolved in dimethylacetamide (DMAC) and added to PAMAM G4 (10% in methanol) in a molar ratio of 640:1. The solution was incubated at room temperature for 1 hour, followed by dialyzing against Milli Q water for 1 hour.

[0208] Solution A was added to solution B and reacted at room temperature for 1 hour. The reaction solution was then dialyzed against Milli Q water for 3 days and freeze-dried.

[0209] (Protamine) 3 Protamine was ligated to citrate via an EDC / NHS reaction (Figure 21).

[0210] Solution A: 10 μmol of citric acid, 300 μmol of EDC, and 750 μmol of NHS were dissolved in 2 ml of water, and the pH was adjusted to 4.5-6 using 1 N NaOH. The mixture was allowed to react for 15 minutes. Next, 2-mercaptoethanol was added to a final concentration of 20 mM, the EDC was quenched, and then the pH was adjusted to 7.

[0211] Solution B: 200 mg of protamine was dissolved in PBS at a concentration of 10 mg / ml, and the pH was adjusted to 7-7.5.

[0212] Solution A was added to solution B and reacted at room temperature for 2 hours. Then, the reaction solution was dialyzed against Milli Q water for 2 days.

[0213] Cellular uptake Place the cells (RPMI2650) in a 12-well plate, 1 × 10⁶ per well. 5Cells were seeded in 1 mL of medium containing 10% FBS and incubated for 18–20 hours to achieve 70–80% confluence. The medium was replaced with fresh 10% FBS-containing medium containing 20 μg / mL FITC-BSA in the presence or absence of either 37.5 μg / mL protamine, (protamine)2, or PAMAM dendrimer-protamine, at 37°C for 2.5 hours. Cells were washed three times with PBS, then trypsinized for 3 minutes, resuspended in complete medium, centrifuged at 400 g for 3 minutes, washed three times with PBS, resuspended in PBS, and analyzed by fluorescence-activated cell sorting (FACS).

[0214] Second-generation protamines (protamine-PAMAM and (protamine)2) increased intracellular FITC-BSA delivery compared to protamine. (Figure 22)

[0215] Cellular uptake Place the cells (HEK 293T) in a 24-well plate, 5 × 10⁶ cells per well. 4 Cells were seeded in 0.5 mL of medium containing 10% FBS and incubated for 18–20 hours to achieve 50–60% confluence. The medium was replaced with fresh FBS-free medium containing 2 μg / mL of GFP pDNA in the presence or absence of 60 or 120 μg / mL of protamine, protamine-AGE, PAMAM, or PAMAM dendrimer-protamine, at 37°C for 48 hours. Cells were washed three times with PBS, then trypsinized for 3 minutes, resuspended in complete medium, centrifuged at 400 g for 3 minutes, washed three times with PBS, resuspended in PBS, and analyzed by fluorescence-activated cell sorting (FACS). (Slide 39)

[0216] Second-generation protamines (protamine-PAMAM and protamine-AGE) increased the efficiency of GFP pDNA transfection in HEK 293T cells compared to protamine alone (Figure 23).

[0217] Streptozotocin (STZ)-induced diabetic mouse model Mice were fasted for 6 hours and then injected i.p. with 200 mg / kg of STZ. Three days later, blood glucose (BG) was measured using a glucose meter (ONE Touch Ultra 2 Blood Monitoring System). Mice with BG of 300 mg / dL or higher were considered diabetic.

[0218] Preparation of insulin / DMPE-protamine and insulin / (protamine)2 formulations DMPE-protamine, (protamine)2, and (protamine)3 were dissolved in water at 3 - 4 mg / mL without adjusting the pH, and insulin was dissolved in water at 5 mg / mL with the pH adjusted to 3.2 using 1 N HCl. Then, DMPE-protamine, (protamine)2, and (protamine)3 were mixed with insulin at a weight ratio of 1.5 (w / w). The mixture was used immediately.

[0219] The results showed that DMPE-protamine, (protamine)2, and (protamine)3 formulations for insulin could reduce blood glucose levels better compared to protamine / insulin formulations.

[0220] Protamine and protamine-PAMAM increased insulin delivery to the brain via nasal delivery An Alexa Fluor 647 (AF647) protein labeling kit was purchased from Thermo Fisher Scientific. Human recombinant insulin, protamine sulfate from salmon grade X, DAPI, and FITC-insulin were purchased from Sigma-Aldrich. Dialysis membranes (fractional molecular weight MWCO - 10 or 3.5 kDa) were purchased from Spectrum Laboratories. All other general laboratory chemicals were purchased from Fisher Scientific and VWR Scientific.

[0221] Four-week-old female CD-1 mice were obtained from Charles River Laboratories (Wilmington, MA). In vivo experiments were performed according to established experimental protocols approved by the Animal Care Committee of the University of British Columbia.

[0222] Insulin was labeled with an AF647 protein labeling kit according to the manufacturer's protocol to produce 647-insulin. FITC-insulin or 647-insulin was mixed with protamine or protamine-PAMAM in water and delivered intranasally (in) to mice (dose = 1 mg / kg FITC-insulin or 647-insulin; 3 mg / kg protamine). Saline, FITC-insulin, and 647-insulin were included as controls. Brains were collected at 2 hours, frozen overnight, and then frozen-sectioned. Frozen sections were placed on glass slides with a drop of Fluorshield (Sigma-Aldrich) containing DAPI and imaged by CLSM. The results showed that protamine-PAMAM demonstrated enhanced efficiency for insulin delivery to the brain.

[0223] Preparation of insulin / DMPE-protamine and insulin / (protamine)2 formulations DMPE-protamine, (protamine)2, and (protamine)3 were dissolved in water at a concentration of 3-4 mg / mL without adjusting the pH. Insulin was dissolved in water at a concentration of 5 mg / mL, with the pH adjusted to 3.2 using 1N HCl. Then, DMPE-protamine, (protamine)2, and (protamine)3 were mixed with insulin in a weight ratio of 1.5 (w / w). The mixture was immediately administered intranasally. (Slide 41)

[0224] The results showed that the intranasal DMPE-protamine, (protamine)2, and (protamine)3 formulations for insulin demonstrated more consistent performance in reducing BG in STZ mice compared to the protamine group (Figures 24A-24D).

[0225] PAMAM-Protamine-SATA Solution A: Dissolve 1.10-15 mg of SATA (N-succinimidyl S-acetylthioacetate) in 50 μl of DMSO. 2. Dissolve protamine in MilliQ at a concentration of 10 mg / ml. Combine 10 mL of protamine solution with 50 μL of SATA solution. Incubate at room temperature for 30 minutes. 3. Dialysis was performed on MilliQ for 30 minutes. Combine 4.1 mL of SATA-modified (acetylated) protamine with 100 μL of deacetylated solution. Deacetylation solution: 0.5 M hydroxylamine, 25 mM EDTA in PBS, pH 7.2-7.5. Dissolve 1.74 g of hydroxylamine·HCl and EDTA (0.475 g of tetrasodium salt or 0.365 g of disodium salt) in 40 mL of MilliQ. Add ultrapure water to the final volume of 50 mL and adjust the pH to 7.2-7.5 with NaOH. 5. Dialysis was performed on MilliQ for 30 minutes. Solution B: GMBS was dissolved in DMSO and added to PAMAM G4 (10% in methanol) in a molar ratio of 10:1. The solution was incubated at room temperature for 1 hour, followed by dialyzing against Milli Q water for 30 minutes. Solution A was added to solution B and reacted at room temperature for 1 hour. The reaction solution was then dialyzed against Milli Q water for 3 days and freeze-dried.

[0226] Protamine-AGE: Allyl glycidyl ether (AGE, 1.5 g) was added dropwise to 15 mL of aqueous protamine solution (10 mg / ml) and reacted at 28°C for 3.5 hours. The product, protamine-AGE, was purified by dialysis in water for 48 hours (molecular weight cutoff, 10 kDa). Finally, protamine-AGE was obtained by lyophilization under vacuum. Protamine-AGE was characterized by NMR.

[0227] Protamine-2AGE: Allyl glycidyl ether (AGE, 1.84 g) was added dropwise to 18 mL of aqueous protamine solution (10 mg / ml, pH 8.56) and reacted at 28°C for 2.5 hours. The product, protamine-2AGE, was purified by dialysis in water for 48 hours (molecular weight cutoff, 10 kDa). Finally, protamine-2AGE was obtained by lyophilization under vacuum.

[0228] Streptozotocin (STZ)-induced diabetic mouse model Mice were fasted for 6 hours, and then 200 mg / kg of STZ was administered via intravenous injection. Three days later, blood glucose (BG) was measured using a glucose meter (ONE Touch Ultra 2 blood monitoring system). Mice with a BG of 300 mg / dL or higher were considered to have diabetes.

[0229] Preparation of insulin / PAMAM-protamine-SATA and insulin / protamine-AGE preparations PAMAM-Protamine-SATA and Protamine-AGE were dissolved in water at 10 mg / mL without pH adjustment, and insulin was dissolved in water at 5 mg / mL or 20 mg / mL, with the pH adjusted to 3.2 using 1N HCl. For intranasal delivery, the dose of PAMAM-Protamine-SATA and Protamine-AGE was 1.5 mg / kg, and the dose of insulin was 1 mg / kg. For sublingual delivery, the dose of PAMAM-Protamine-SATA and Protamine-AGE was 3 mg / kg, and the dose of insulin was 5 mg / kg. (Slide 47)

[0230] PAMAM-protamine-SATA and protamine-AGE formulations for intranasal insulin delivery showed more consistent performance in reducing BG in STZ diabetic mice compared to protamine formulations (Figures 25A-25F).

[0231] PAMAM-AGE-Protamine Solution A: Protamine and Trout's reagent (2-iminothiolane·HCl) were dissolved in 5 mM EDTA in a molar ratio of 1:20. The solution was incubated at room temperature for 1 hour, followed by dialyzing in 5 mM EDTA for 1 hour.

[0232] [ka]

[0233] Solution B: Allyl glycidyl ether (AGE, 1.5 g or 0.35 g) was dissolved in 7.5 mL of methanol / Milli-Q water (3 / 4, v / v). Next, 10% PAMAM G4 (700 μL) from the method was added dropwise to the AGE solution and reacted at 25°C for 3 hours. The product, PAMAM-AGE, was purified by dialysis in water for 1 hour (molecular weight cutoff, 10 kDa).

[0234] Solution A was added to solution B in the presence of the catalyst azobisisobutyronitrile (AIBN, 18 mg, 0.1 mmol). After reacting at 65°C for 24 hours, the mixture was transferred to a dialysis tube (molecular weight cutoff, 10 kDa) and then dialyzed against MilliQ for 48 hours. Finally, PAMAM-AGE was performed. 1.5 - Protamine and PAMAM-AGE 0.35 - Protamine was obtained using freeze-drying under vacuum.

[0235] Alexa647-Dupilumab Intranasal Delivery (IN): Dupilumab was first labeled with the Alexa Fluor 647 (AF647) protein labeling kit according to the manufacturer's protocol. Alexa647-Dupilumab was mixed with different protamine (prot) analogs in water and delivered intranasally (i.n.) to mice (dose = 3 mg / kg of protamine, protamine-AGE, PAMAM-AGE-protamine unless otherwise specified; 20 μL of 3 mg / ml Alexa647-Dupilumab). Only saline and Alexa647-Dupilumab were included as controls. Plasma was isolated by centrifugation of blood (10,000 g, 5 min) and analyzed for Alexa647 fluorescence using a plate reader 4 hours after intranasal delivery.

[0236] Protamine-AGE and PAMAM-AGE 0.35 - Protamine enhanced the tissue absorption of Alexa647-Dupilumab via intranasal delivery. (Figure 26)

[0237] Human Nasal Epithelial Cell (HNEpC) Model The HNEpC model was established by using the method developed by PromoCell (https: / / promocell.com / wp-content / uploads / 2022 / 07 / AppNote-HNEpC-and-HTEpC-ALI-_web.pdf).

[0238] After 10 days of culture, 8 μL of protamine or protamine-AGE (dissolved at 10 mg / mL in 5% glucose) plus 2 μL of Alexa647-Dupilumab (2 mg / mL) and 10 μL of 5% glucose were added dropwise onto the HNEpC model. After 2 hours, the HNEpC model was frozen and then sectioned for confocal microscopy analysis.

[0239] Protamine enhanced 647-dupilumab penetration in the HNEpC model compared to the 647-dupilumab-only group. Protamine-AGE further enhanced 647-dupilumab penetration compared to protamine alone.

[0240] Protamine enhanced 647-dupilumab penetration in the HNEpC model compared to the 647-dupilumab-only group. Protamine-AGE and PAMAM-protamine further enhanced 647-dupilumab penetration compared to protamine alone.

[0241] Cell uptake: Cells (RPMI2650) were placed in a 12-well plate, 1 × 10⁶ per well. 5 Cells were seeded in 1 mL of medium containing 10% FBS and incubated for 18–20 hours to achieve 70–80% confluence. The medium was replaced with fresh 10% FBS medium containing 20 μg / mL FITC-BSA in the presence or absence of either 37.5 μg / mL protamine or different CPP sequences isolated from protamine, at 37°C for 4 hours. Cells were washed three times with PBS, then stained with lysotracker for 30 minutes, followed by three washes with PBS. Subsequently, 2 mL of FBS-free medium was added, and the cells were observed under confocal observation. Alternatively, after 4 hours of incubation, cells were washed three times with PBS, then trypsinized for 3 minutes, resuspended in complete medium, centrifuged at 400 g for 3 minutes, washed three times with PBS, resuspended in PBS, and analyzed by fluorescence-activated cell sorting (FACS).

[0242] We examined different CPP sequences identified from protamine sulfate. S1:PRRRRRSSSRPIRRRRRPRASRRRRRGGRRRR(Sequence ID 1) S2:PRRRRSSRRPVRRRRRPRVSRRRRRRGGRRRR (Sequence ID 2) S3:PRRRRSSSRPVRRRRRPRVSRRRRRRGGRRRR(Sequence ID 3) S4:PRRRRASRRIRRRRRPRVSRRRRRGGRRRR (Sequence code 4) S2 and S4 were superior to S1 and S3 in delivering FITC-BSA into the cell nucleus. S2 and S4 were superior to S1 and S3 in delivering FITC-BSA into cells. (Figures 27A-27B) S2, S3, and S4 therapeutic cells showed fewer lysosomes compared to S1 cells. (Figure 28) Protamine-AGEs were characterized by NMR.

[0243] Protamine-AGE size was detected under PBS at pH 7.4. Particle size and zeta potential were determined using a particle analyzer (Zetasizer Nano-ZS, Malvern Instruments Ltd., Malvern, UK). The size results are expressed as the Z-mean diameter.

[0244] [Table 3]

[0245] Cas9 RNP / sgRNA delivery to HEK 293T EGFP cells: Cells (HEK 293T EGFP) were delivered in a 3.5 mm confocal dish, 1 × 10⁶ per well. 5Cells were seeded in 2 mL of medium containing 10% FBS and 0.2 mg / mL hygromycin B, and incubated for 18–20 hours to achieve 70–80% confluence. The medium was replaced with fresh FBS-free medium in or without 37.5 μg / mL protamine or protamine-AGE at 37°C for 40 minutes. 3.5 μL of Cas9-RNP-RFP (52 μM) was added to 20 μL of nuclease-free water, followed by 2 μL of sgRNA (100 μM), and then incubated for 10 minutes. The cell culture medium was replaced with FBS-free medium containing a Cas9-RNP-RFP / sgRNA mixture with either 37.5 μg / mL of protamine or protamine-AGE. The cells were kept at 37°C for 48 hours. Subsequently, the cells were treated with 2 μg / mL tetracycline hydrochloride for 24 hours to activate EGFP expression. The cells were washed three times with PBS, then 2 mL of FBS-free medium was added, and the cells were observed under confocal light. (Slide 62)

[0246] Protamine-AGE increased Cas9-RNP / sgRNA delivery for EGFP knockdown. (Figures 29A-29B)

[0247] Infiltration of 647-CD124 monocolonial antibody (mAb) into mouse noses 647-CD124 mAb was dissolved in PBS at a concentration of 2 mg / ml, and 20 μL of the mAb solution was mixed with different protamine solutions (protamine, protamine-2AGE, protamine-AGE, and PAMAM-protamine) to achieve a final protamine concentration of 3.3 mg / ml. The mixture was administered intranasally at doses of 1.3 mg / kg for the mAb and 3 mg / kg for the protamine. The total delivery volume was a maximum of 32 μL. 20 μL of the 647-CD124 mAb solution was administered subcutaneously as a control (dose = 1.3 mg / kg).

[0248] Mice were observed under IVIS at different time points (0, 5 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours). Mice were euthanized at 4 hours, their noses were collected, and sectioned for confocal imaging.

[0249] 647-CD124 retention in mouse noses Protamine-based formulations enhanced nasal retention of mAbs. (Figure 30)

[0250] [Table 4]

[0251] 647-CD124 penetration in mouse noses Second-generation protamine enhanced 647-CD124 mAb penetration in the mouse nose compared to protamine. The results were consistent with IVIS.

[0252] eye drops FITC-BSA was dissolved in water at a concentration of 5 mg / ml. Protamine, protamine-AGE, or PAMAM-protamine was dissolved in 5% glucose at a concentration of 10 mg / ml.

[0253] 5 μL of FITC-BSA solution was mixed with 22.5 μL of protamine, protamine-AGE, or PAMAM-protamine solution (or 5% glucose as a control) and applied to the eye. 5 μL of the mixture was applied to the eye each time. After 5 minutes, another drop was applied. The entire procedure was completed in 30 minutes.

[0254] After 2–24 hours, the eyes were collected and fixed in 10% formalin for 24 hours, then replaced with 10% sucrose water for 24 hours. Finally, the eyes were kept in 30% sucrose for 24 hours, then sectioned and imaged under confocal light.

[0255] Eyes were collected 2 hours after treatment. Protamine enhanced FTIC-BSA retention in mouse eyes.

[0256] Eyes collected 4 and 24 hours after treatment. Protamine-based formulations enhanced FITC-BSA penetration in the lens.

[0257] Synthesis and characterization of novel peptides C18-P was synthesized by linking a stereoacid to the terminal amino group of protamine via the EDC / NHS method (Figure 31A).

[0258] C18-P, protamine, and stearic acid, 1 1H NMR and 13 Characterized by 13C NMR. 1 ¹H NMR analysis showed a characteristic peak at 2.5 ppm, which corresponds to C18 from stearic acid. 13 The 13C NMR spectrum showed the presence of all stearic acid peaks, along with those from protamine. The shift of the protamine peak from 4.1 kDa (Figure 31C) to 4.4 kDa (Figure 31D) in MS indicated conjugation of stearic acid to protamine.

[0259] P2 was prepared by crosslinking the two terminal amino groups of protamine using NHS-PEG-NHS (Figure 31B).

[0260] P2, protamine, and NHS-PEG-NHS, 1 Characterized by 1H NMR. 1 A prominent 3.5 ppm peak in the 1H NMR spectrum confirmed the linkage of PEG to protamine, and the mass result showed a peak at 8.2 kDa (Figure 31E). These aggregate data affirmed the successful synthesis of C18-P and P2. FTIR spectroscopy revealed the reaction at 1300 cm⁻¹, indicating the amide bond. -1 This was further confirmed after the appearance of a peak (Figure 31F).

[0261] CD analysis, used to study the secondary structure of the novel peptide, revealed significant insights. Protamine exhibited a pattern consistent with existing literature

[72] , indicated by a major negative signal peak at 210 nm (Figure 31G). This suggested the presence of a random coil structure, which could be attributed to a high arginine content promoting positive charge-driven electrostatic repulsion. Modification with stearic acid or a PEG linker resulted in characteristic changes in the CD spectrum, which were analyzed by BeStSel to quantify secondary structures including α-helix, β-sheet, β-(anti)parallel, turn, and random coil (Table 5). The data confirmed that protamine was primarily in the random coil conformation, with α-helix and β-sheet structures being dominant in C18-P and P2. Without being constrained by any particular hypothesis, these structural changes could be attributed to the hydrophobic effect of the stearic acid tail or the effect of the PEG linker.

[0262] [Table 5]

[0263] When dissolved in water, protamine did not form aggregate structures as shown by AFM and DLS, while C18-P and P2 self-assembled into spherical and rod-shaped (400 nm × 5 nm) nanostructures as shown by TEM and AFM, respectively. DLS also confirmed the presence of nanoaggregates in samples prepared with C18-P and P2 (Table 6).

[0264] [Table 6]

[0265] These data are consistent with the increased presence of β-sheets in C18-P and P2. C18-P is an amphiphilic compound containing hydrophobic acyl chains and hydrophilic peptides, which are expected to self-assemble into micelles. The β-sheet content in P2 was similar to that of C18-P, and P2 exhibited a characteristic nanorod structure. Without being bound by any particular hypothesis, this difference may be due to steric hindrance from the PEG linker, which increased the separation between the two protamine molecules and reduced the density of the β-sheet structure.

[0266] C18-P and P2 increased intracellular protein delivery through increased pore formation in the cell membrane. The inventors first showed that neither C18-P nor protamine caused toxicity to primary human gingival keratinocytes at concentrations below 10 μg / ml, and that P2 needed to be reduced to 5 μg / ml (Figure 32). To investigate how protamine and its derivatives interacted with cells, human primary gingival keratinocytes were imaged by SEM after treatment at non-toxic concentrations. (SEM images of human primary gingival keratinocytes after treatment with protamine, C18-P, or P2 were examined.) In the untreated group, the cell membrane was relatively smooth with no large aggregates on the surface of any of the three. In the protamine-treated group, several small aggregates were found on the surface with pores (black areas) underneath. Increased amounts and sizes of aggregates and membrane pores were found in cells treated with C18-P and P2. SEM images were analyzed using ImageJ to better outline the membrane pores. Using ImageJ, we performed pore analysis in the cell membrane and quantified the pore area relative to the total cell membrane area. The percentage of pore area relative to the total cell surface quantified in the protamine treatment group was only 0.1%, compared to 1.4% and 2.0% in the C18-P and P2 groups, respectively. It should be noted that these numbers may have been underestimated because some pores may have been covered by aggregates and therefore not shown. Nevertheless, the data support our hypothesis that these structural modifications of protamine increased its cell permeability activity. Interestingly, protamine and its derivatives caused defects in the cell membrane but did not induce cytotoxicity, which may be because the effect was transient.

[0267] SEM data suggested that the novel peptide increased intracellular drug delivery compared to protamine. Next, AF647-IgG (150 kDa) was used as a model protein payload and incubated with human primary gingival keratinocytes for 12 hours in the presence of protamine, C18-P, or P2 at 5–10 μg / ml, followed by washing and confocal imaging. Confocal microscopy images of cells after treatment with AF647-IgG in the presence of protamine, C18-P, or P2 were examined, and C18-P and P2-treated cells showed increased IgG cytosolic delivery compared to protamine. ImageJ analysis revealed 8- and 21-fold increased IgG cellular uptake in C18-P and P2-treated cells compared to protamine, respectively (Figure 33A). In particular, IgG and derivatives delivered by protamine showed minimal overlay with lysosomes (Figure 33B), supporting the idea that membrane pore formation was the primary delivery mechanism. FCM data were consistent with confocal results (Figure 34). Similar results were obtained with a different payload, FITC-BSA (66kDa). Confocal microscopy images of cells treated with FITC-BSA in the presence or absence of protamine, C18-P, or P2 were examined. Quantitative analysis was performed (Figure 35).

[0268] In summary, these data suggest that the novel peptide facilitated intracellular delivery of proteins up to 150 kDa in size, bypassing lysosomal degradation.

[0269] C18-P and P2 enhanced protein penetration into cell spheroids. Next, the inventors investigated whether protamine and its derivatives could enhance protein penetration into multiple cell layers. Spheroids prepared from DiI-labeled human primary gingival keratinocytes were incubated overnight with AF647-IgG mixed with protamine, C18-P, or P2, then washed and imaged at a depth of 40 μm from the top of the spheroids by CLSM. Confocal microscopy analysis of AF647-IgG penetration in human primary gingival keratinocyte spheroids in the presence of protamine, C18-P, or P2 was performed. When the spheroids were imaged at a depth of 40 μm from the surface, delivery of free AF647-IgG into the spheroids was limited, and protamine significantly increased penetration. Spheroids treated with C18-P and P2 showed further enhanced uptake and penetration of AF647-IgG compared to protamine. Images were analyzed by ImageJ to compare AF647-IgG penetration within spheroids delivered by different CPPs. The majority of IgG was restricted to a distance of 30 μm from the periphery in the protamine group, while in the C18-P and P2 treatment groups, IgG was detected at up to 68 μm from the periphery (Figure 36A). To further confirm IgG delivery in spheroids, single cells were collected from the spheroids and analyzed by FCM. As shown in Figure 36B, AF647-positive cells represented 10.6%, 46.3%, 52.1%, and 71.3% of total cells in the PBS, protamine, C18-P, and P2 treatment groups, respectively. C18-P and P2 showed enhanced activity in promoting IgG penetration in spheroids compared to protamine, with P2 being superior to C18-P. Similar results were obtained with another protein payload, FITC-BSA. Confocal microscopy analysis of FITC-BSA penetration in human primary gingival keratinocyte spheroids in the presence of protamine, C18-P, or P2 was performed. Spheroids were imaged at a depth of 60 μm from the surface (Figures 37A-37B).

[0270] C18-P and P2 increased Cas9-RNP delivery in 2D and 3D cell models compared to protamine. Next, the inventors evaluated whether the protein payloads delivered by protamine and its derivatives remained active and exerted their biological activity in cell models. HEK293-eGFP cells were incubated with protamine, C18-P, or P2 for 40 minutes, followed by the addition of Cas9-RNA / sgRNA. The cells were cultured for a further 48 hours, then washed and imaged by CLSM. Confocal microscopy images of cells treated with Cas9-RNP-RFP / sgRNA in the presence of protamine, C18-P, or P2 showed that the delivery of free Cas9-RFP-RNP was limited, and eGFP was still strongly expressed. Protamine significantly increased RNP delivery and reduced eGFP expression compared to cells treated with free RNP. RNP delivery was further increased by C18-P and P2 compared to the protamine group, accompanied by further reduced eGFP expression (Figures 38A-38B).

[0271] Images were analyzed using ImageJ (Figures 39A-39B), which showed that the eGFP fluorescence intensities were 3118, 2058.4, 1180, and 938 a.u for free RNP, protamine, C18-P, and P2, respectively. FCM analysis showed that cells that were RNP+eGFP- represented 10.1%, 12.8%, 15.3%, and 30.2% of cells treated with free RNP, protamine, C18-P, and P2, respectively. In the 2D cell model, RNP delivered by CPP was active and effectively quenched eGFP, with delivery efficiency in the order of P2 > C18-P > protamine. Next, we investigated and compared these delivery systems in a cell spheroid model. The penetration of Cas9-RFP-RNP / sgRNA in eGFP HEK 293 spheroids in the presence of protamine, C18-P, or P2 was investigated. Cells were dissociated from the spheroids, and Cas9-RFP-RNP-positive cells (C), as well as RNP+ and eGFP- cells (D), were quantified by FCM. Data = mean ± SD. (n≧5). (***p<0.001, ****p<0.0001). Most RNPs were detected around the spheroids when treated with free RNP or protamine / RNP. The protamine group showed moderately improved RNP penetration compared to the free RNP group. On the other hand, cells treated with C18-P and P2 showed significantly enhanced RNP penetration into the spheroid core compared to protamine. To further confirm the delivery efficiency of RNP and eGFP knockdown in spheroids, single cells were collected and analyzed by FCM. As shown in Figure 38, the protamine-treated group showed a 1.6-fold increase in RNP uptake compared to the free RNP group, while C18-P and P2 increased delivery by 4 and 3.5-fold, respectively. Cells that were RNP+ and eGFP- in spheroids treated with free RNP, protamine, C18-P, and P2 accounted for 6.9%, 13.44%, 20.14%, and 20.34% of the total cells analyzed (Figure 38), demonstrating the superiority of C18-P and P2 in RNP delivery. No difference was found between C18-P and P2 in the spheroid model.

[0272] C18-P and P2 enhanced protein penetration in human sublingual tissue replacements. Keratinocytes in cell spheroids do not differentiate sufficiently to develop the structural barrier present in sublingual tissue that hinders drug penetration. Therefore, we then compared protein payload penetration mediated by protamine and derivatives in human sublingual tissue replacements. Sections of human sublingual tissue replacements stained with H&E were examined to confirm the successful establishment of human sublingual tissue replacements with characteristic anatomical layers, which included a basal layer of collagen-based stroma embedded with fibroblasts and an apical layer composed of stratified keratinocytes. Protamine, C18-P, or P2 mixed with AF-647-IgG was applied to the apical region. Tissue was then collected after 4 hours and imaged by CLSM. Confocal microscopy analysis of AF647-IgG tissue penetration in the presence of protamine, C18-P, or P2 revealed that delivery of free AF647-IgG into sublingual replacement material was limited, while protamine increased IgG adhesion to the tissue surface. C18-P and P2 facilitated IgG penetration into tissue compared to protamine. Images were analyzed using ImageJ (Figure 40A), showing that C18-P and P2 increased AF647-IgG delivery into tissue by 3.7-fold and 4.1-fold, respectively, compared to protamine. Furthermore, AF647-IgG delivered by protamine showed the furthest migration distance from the tissue surface at 60 μm, while migration distances in the C18-P and P2 treatment groups reached a maximum of 80 μm and 190 μm, respectively, and exhibited significantly increased fluorescence intensity within the tissue (Figure 40B). The data confirmed that both novel peptides were superior to protamine in enhancing IgG penetration in human sublingual tissue replacements, and the difference between C18-P and P2 was not significant.

[0273] C18-P and P2 enhanced sublingual penetration of AF647-IgG in mice compared to protamine. Next, the inventors compared sublingual penetration of AF647-IgG mediated by protamine, C18-P, and P2 in mice. AF647-IgG was mixed with protamine, C18-P, or P2 and delivered sublingually to mice under isoflurane anesthesia, and the mice were returned to their cages immediately after the procedure for recovery. The mice were then euthanized, and the base of the tongue containing sublingual tissue was collected, sectioned, and imaged by CLSM. Sublingual AF647-IgG alone was included as a control. Confocal microscopy analysis of AF647-IgG penetration in the presence of protamine, C18-P, or P2 was performed at 0.5, 2, and 4 hours after administration. In the absence of any delivery system, AF647-IgG penetration was not detected in the sublingual tissue. However, in the presence of protamine, C18-P, or P2, mucosal adhesion of AF647-IgG was significantly enhanced for at least 2 hours. In particular, increased sublingual penetration of IgG into the epithelium (C18-P and P2) and lamina propria (P2) was detected in the groups treated with C18-P and P2 compared to protamine. This was further demonstrated by the quantitative data in Figures 41A-41C, which showed that IgG delivered by protamine was found exclusively within 10 μm from the surface (i.e., in the mucus), while C18-P and P2 significantly enhanced tissue penetration, reaching up to 40 μm and 190 μm from the surface, respectively. Tissue penetration of IgG mediated by C18-P and P2 occurred as early as 0.5 hours after administration, lasted for at least 2 hours, and largely disappeared at 4 hours, which could be attributed to swallowing or sublingual absorption and degradation. In particular, P2 showed increased IgG penetration compared to C18-P, which could be attributed to increased mucosal adhesion mediated by hydrogen bonding between the PEG linker and the mucin carbohydrate.

[0274] C18-P and P2 enabled efficient sublingual delivery of insulin and semaglutide for enhanced blood glucose control in mice. To demonstrate the potential medical utility of the delivery technology, the inventors mixed protamine, C18-P, or P2 with either insulin or semaglutide and administered the formulations sublingually to mice, comparing their efficacy in controlling blood glucose. The sublingual dose for protamine and its derivatives was fixed at 3 mg / kg, while the doses for insulin and semaglutide were 5 mg / kg and 0.5 mg / kg, respectively. As shown in Figure 42A, sc-insulin at 1 mg / kg was effective in reducing BG in STZ-induced diabetic mice, with the effect occurring quickly (0.5–1 hour) and lasting for at least 6 hours, maintaining BG at approximately 100 mg / dL. Sublingual insulin at 5 mg / kg showed little efficacy, and the inclusion of protamine in the formulation did not significantly improve the effect. On the other hand, both the C18-P and P2 sublingual formulations effectively suppressed BG and had comparable onset, potency, and duration to sc-insulin. In the semaglutide study, 1.5 mg / kg of glucose was intraperitoneally injected into normal mice immediately after administration of semaglutide at 60 μg / kg (sc) or 500 μg / kg (sublingual). As shown in Figure 42B, blood glucose (BG) rapidly increased from approximately 150 mg / dL to 250 mg / dL in 0.25 hours after glucose injection, and then gradually decreased to baseline levels. When treated with sc semaglutide, BG increased to only 200 mg / dL and rapidly decreased to baseline at 50 minutes, and the BG control effect was maintained on day 2. The effect disappeared on day 3. Sublingual semaglutide showed efficacy in suppressing the rapid rise in BG during the first 50 minutes after glucose intake, but the effect was not significant after 1 hour. In contrast, sublingual protamine formulations showed no effect on controlling BG in the initial phase, although the effect began within 1 hour, and BG returned to baseline at 1.25 hours. However, the effect of sublingual protamine formulations on day 2 was only moderate. These results suggest that free semaglutide can be rapidly absorbed sublingually, but the absorption was too low to sustain efficacy beyond 1 hour.On the other hand, protamine could slow semaglutide absorption via drug binding, resulting in a delayed onset, and the degree of absorption was not optimal for maintaining the same level of efficacy on day 2. The superior C18-P and P2 formulations showed comparable onset, potency, and duration to sc-semaglutide, indicating that C18-P and P2 increased sublingual delivery of semaglutide compared to protamine. Additionally, no BG fluctuations were observed on day 2 in the groups treated with C18-P and P2 formulations. It should be noted that to achieve the same level of efficacy, the doses for the C18-P and P2 sublingual formulations were 5–8 times higher than the doses for the sc formulation. Without being bound by any particular hypothesis, this could be attributed to the rapid removal of the sublingual formulation by salivary dilution and swallowing. However, compared to Rybelsus®, an oral formulation of semaglutide, which requires up to a 100-fold increase in overall dose due to its low oral bioavailability (0.8%) and a 4-week daily dose to initiate function, the C18-P and P2 sublingual formulations offer significant advantages. Nausea as a side effect from semaglutide also presents a challenge for patients taking oral medication consistently.

[74]

[0275] C18-P and P2 enabled efficient sublingual absorption of proteins up to 150 kDa. After demonstrating that C18-P and P2 are superior to protamine in peptide delivery in mice, the inventors further investigated whether C18-P and P2 function for sublingual delivery of larger proteins in mice. Protein payloads containing recombinant human growth hormone (rhGH, 22 kDa), Cy7-BSA (66 kDa), and AF647-IgG (150 kDa) were mixed with either C18-P or P2 and delivered sublingually to mice. Plasma was then collected, and payload concentrations were measured using ELISA or fluorescence spectroscopy. As can be seen in Figures 43A–43C, sublingual absorption of all three protein payloads was limited in the absence of the delivery system, although Cy7-BSA showed a minimal but significant plasma concentration at 4 hours. Albumin has been shown to cross the epithelial barrier via gp60-mediated transcytosis

[75] . C18-P and P2 showed comparable activity in enhancing sublingual absorption of rhGH, Cy7-BSA, and AF647-IgG and had similar PK profiles. Both CPP sublingual formulations resulted in similar systemic absorption for both rhGH and Cy7-BSA as sc formulations, indicating efficient delivery. However, sublingual delivery of 150kDa IgG by these CPP formulations was inferior to that of sc formulations. Despite the fact that C18-P and P2 increased IgG penetration in mouse sublingual tissue (Figures 41A-41C), it is possible that a significant portion of the IgG penetrating the tissue was not absorbed systemically. While C18-P and P2 were effective in overcoming in vivo delivery barriers for smaller compounds, e.g., insulin, semagtide, rhGH, and BSA, further optimization was needed for larger molecules, e.g., IgG. Nevertheless, both CPPs enabled efficient sublingual absorption of macromolecules that had previously only been delivered by needle-based injection. In particular, the relative bioavailability (BA) of IgG for the C18-P and P2 groups was 51.2% and 63.2%, respectively, which were significantly higher than the 5% reported for other transmucosal delivery systems

[76] .

[0276] Safety studies of protamine, C18-P, and P2 Mice received sublingual CPP at a dose of 6 mg / kg once weekly for one month, and were then euthanized one day after the last dose. Blood and organs (including the base of the tongue (sublingual), heart, liver, spleen, lungs, kidneys, and trachea) were collected and analyzed. No significant differences were observed in the organ / body weight ratio of the treatment group compared to the saline-treated control group. RBC, hematocrit, hemoglobin, MCV, MCH, RDW, WBC, and lymphocyte values ​​were within the normal range in the treated animals, and no significant changes were observed compared to the saline-treated group. Liver and kidney function, as indicated by serum levels of urea (BUN), ALT, AST, and creatine kinase, was within the normal range. H&E staining of major tissues of interest was performed from mice that received protamine, C18-P, and P2. Histology of sublingual tissue and major organs showed no abnormalities compared to those collected from saline-treated mice. In summary, these data indicate that the three sublingually administered CPP formulations were safe in mice.

[0277] In summary, two novel peptides were synthesized. C18-P was a conjugate of protamine and stereoic acid, and P2 was a protamine dimer with a PEG linker. The content of α-helix and β-sheet conformations was 1.5–2 times higher in C18-P and P2 compared to protamine, resulting in increased peptide aggregation on the cell surface for cell membrane permeability and up to 21-fold increased cytosolic delivery compared to protamine. RNP delivery in 2D cell culture and spheroid models confirmed that protamine, C18-P, and P2 effectively overcame multiple membrane barrier layers to deliver payloads for gene knockdown, confirming that C18-P and P2 were superior to protamine. C18-P and P2 were more efficient than protamine in promoting IgG penetration in human sublingual tissue replacements and mouse sublingual tissue. In mice, C18-P and P2 enabled efficient sublingual absorption of insulin and semaglutide, resulting in enhanced BG control compared to protamine. Pharmacokinetic results supported the finding that C18-P and P2 also enabled efficient sublingual absorption of larger proteins up to 150 kDa in mice with relative BA > 50%. Finally, repeated treatment with these CPPs was safe in mice.

[0278] Protamine enhanced mAb retention in the nasal cavity. To demonstrate the ability of protamine to promote greater protein retention and permeation, the inventors introduced a study on mice. 111 In-IgG alone or with protamine and 111 SPECT / CT scans were acquired at 0.5 hours, 2 hours, 4 hours, and 6 hours after intranasal administration of an In-IgG mixture. 111In-IgG was mixed with saline or protamine and delivered intranasally to mice. The heads of the mice were scanned by SPECT / CT at 0.5, 2, 4, and 6 hours after intranasal administration, and the whole body was scanned at 6 hours. N=3. As shown in Figure 44, radioactivity was detected in the nasal cavity at 0.5 and 2 hours for both treatment groups. However, starting from 4 hours thereafter, the IgG-only group showed less radioactivity in the central nasal cavity compared to the protamine group. Transverse scanning images showed that the signal detected in the IgG-only group decreased and shifted over time from the region near the hard palate to the skull. This observation suggests that IgG remained on the surface of the nasal epithelium and was swallowed by the mice from 2 hours onwards. This was confirmed by the presence of IgG in the gastrointestinal tract of IgG-only mice at 6 hours. In contrast, in the protamine-treated mouse group, most of the IgG radioactivity was concentrated in the nasal septum without significant disappearance throughout the entire experimental time. Radioactivity was detected exclusively in the nasal cavity over the entire 0.5–6 hour period. Quantification of radioactivity in the central nasal cavity was performed using SPECT / CT images, and SUV in the specified region was determined. 平均 The values ​​were calculated. The signal of IgG administered alone rapidly decreased within 2 hours (Figure 44), decreasing from a standard uptake value (SUV) of 200–100 g / mL until the signal was almost gone after 4 hours. The protamine-containing radiolabeled IgG formulation in mice maintained its radioactivity and had an SUV of approximately 400 g / mL throughout the entire 6 hours (Figure 44). These results confirm that protamine enhanced the retention of IgG in the nasal cavity. Inspired by this mAb retention capacity, our subsequent experiments aimed to investigate whether protamine could be used for intranasal delivery of αCD124 to enhance its efficacy in the CRSwNP mouse model.

[0279] In vivo efficacy of intranasal protamine / αCD124 in moderate CRSwNP mice Progression of CRSwNP led to nasal itching, which in turn resulted in frequent nose touching

[79] . As shown in Figure 45A, the mean number of nose touches per 10 minutes in CRSwNP mice after PBS treatment was approximately 30, compared to approximately 15 in healthy mice. In protamine / αCD124-treated CRSwNP mice, the number of nose touches was significantly reduced to 25 per 10 minutes compared to the PBS, αCD124 in (approximately 35), and αCD124 sc (approximately 40) groups

[79] . There was no significant difference among the PBS, αCD124 in, and αCD124 sc groups.

[0280] The inventors further investigated the smoothness of the mucosal surface and the thickness of the mucus by imaging the nasal cavity using micro-CT. Micro-CT scans of the nose (N≧5) were performed from CRSwNP mice one day after the final dose, and diffuse mucosal thickening of the nasal septum, cribriform labyrinth, and maxillary sinus was observed in the PBS, αCD124 in, and sc treatment groups, while the protamine / αCD124 group showed a smooth, thin mucosal lining similar to that of the healthy control group. In addition, the nasal cavity was reconstructed into a 3D model to examine the airway (A 気道 ) and bone (A 骨 The surface area of ​​) was measured using a 3D Slicer. Reduced A 気道 / A 骨 The ratio indicates increased nasal obstruction. As shown in Figure 45B, the ratio in the protamine / αCD124 in group (0.49) was significantly higher than that in the PBS group (0.41), while other αCD124-treated mice (ratio of approximately 0.45) showed no improvement.

[0281] Representative H&E stained images of the paranasal sinuses treated with various formulations were examined for polypoid lesions, epithelial destruction, bone erosion, inflammatory infiltration into the ethmoid turbinates, and increased mucosal thickness. Invasive inflammation was induced in the nasal cavity of mice with CRSwNP, as evidenced by bone erosion (indicated by red arrows), inflammatory infiltration into the ethmoid turbinates, and increased mucosal thickness. These inflammatory features were significantly reduced in all αCD124 treatment groups, particularly in the protamine / αCD124 group. The number of nasal polypoid lesions (indicated by *) and epithelial destruction lesions (indicated by #) in these images was counted and compared. As shown in Figures 45C and 45D, protamine / αCD124-treated mice achieved approximately 50% reduction in nasal polyps and 50% reduction in epithelial destruction compared to the PBS-treated group, demonstrating superior efficacy compared to other αCD124 treatments. In the absence of protamine, intranasal delivery of αCD124 was ineffective, while scαCD124 treatment achieved a 25% reduction in epithelial destruction at the same dose.

[0282] Increased IgE production is typically found in CRSwNP patients, both in systemic circulation and within nasal polyps, attributable to active IL-4 and IL-13 pathways

[80] . Increased IgE production leads to eosinophil migration to inflammatory sites, which exacerbates inflammation. Blocking IL-4Rα with αCD124 has been shown to reduce IgE production

[81] . As shown in Figure 45E, protamine / αCD124 treatment significantly reduced systemic IgE levels compared to the αCD124-only group (in and sc) and significantly reduced nasal IgE compared to PBS-treated mice (Figure 45F). A decrease in IFN-γ in nasal tissue was also measured in the protamine / αCD124 group (Figure 46A). However, no significant differences were found for other type 2 inflammatory biomarkers (Figures 46B–46K). This could be because the 8-week treatment period was not long enough to produce enhanced efficacy

[82] or because the delivery of αCD124 was not optimal.

[0283] Nano-P and Dendri-P enhanced protein penetration in the nasal turbinates in mice. We created an amphiphilic conjugate (Nano-P) that can self-assemble into micelles by conjugating the hydrophobic AGE moiety to hydrophilic protamine. To synthesize this structure, the AGE was linked to the N-terminal amino group of protamine via amine-epoxide coupling.

[0284] Secondly, multiple protamine molecules were linked to a PAMAM dendrimer (Dendri-P). To synthesize Dendri-P, the primary N-terminal amino group of protamine was converted to a thiol group using Trout's reagent, and the surface amino group in PAMAM was reacted with GMBS to present a maleimide group. Subsequently, the thiol-protamine was conjugated to the maleimide-PAMAM via a Michael addition reaction (reaction scheme shown in Figure 47).

[0285] We confirmed the successful synthesis of these structures using several techniques. 1 1H NMR analysis revealed characteristic peaks at 2.85, 3.90, 4.1, 5.2, and 5.8 ppm, which corresponded to AGEs. For Dendri-P, a prominent peak at 3.4 ppm indicated the presence of 2-iminothiolane·HCl bound to protamine. Peaks b, c, and d indicated terminal sulfhydryl groups, and the presence of the GMBS linker was supported by peaks e, f, and g. Additionally, peaks observed at 3.2 ppm (methylene group in the branched chain of PAMAM) and 2.51 ppm (methylene group in the core and terminal chains of PAMAM) confirmed the linkage of PAMAM to protamine.

[0286] FTIR spectroscopy (Figure 48) determined Nano-P identity at 1050 cm⁻¹. -1 (CO extension), 1510cm -1 (C=C), and 3200cm -1 Further confirmation was made after the appearance of a peak at (-OH). 1300cm -1The peak indicated amide bonding in Dendri-P. Finally, TEM showed that Nano-P self-assembled into a spherical shape with an average diameter of approximately 60 nm, while Dendri-P exhibited a leaf-like structure with an average size of approximately 500 nm. (Regarding the morphology of Nano-P and Dendri-P under TEM, and the penetration of AF-αCD124 into nasal tissue in mice delivered by protamine, Nano-P, and Dendri-P). The results were consistent with DLS (Table 7).

[0287] [Table 7]

[0288] Two hours after administration of various AF-αCD124 formulations, nasal turbinates were collected from mice, then sectioned and imaged by CLSM. Confocal microscopy analysis of AF-αCD124 tissue penetration in the presence of protamine, Nano-P, or Dendri-P was performed. Confocal images were analyzed by ImageJ for the overall fluorescence intensity of AF-αCD124 in nasal tissue. In the sc injection group, AF-αCD124 was barely detectable in nasal tissue, indicating a limited distribution of AF-αCD124 from systemic circulation to the nasal turbinates. AF-αCD124 was barely detectable in tissue after intranasal delivery in groups without delivery systems, possibly due to efficient ciliary clearance. However, significant retention of AF-αCD124 in the mucus layer and penetration into the epithelial layer were observed in the protamine treatment group. In the Nano-P treatment group, AF-αCD124 was found in the epithelium (trace amounts) and lamina propria (large amounts) and was not significantly retained in the mucus, suggesting that Nano-P effectively penetrated the nasal delivery barrier, including mucus and epithelium. On the other hand, in Dendri-P treated mice, AF-αCD124 was found in the epithelium (large amounts) and lamina propria (trace amounts). Images were analyzed by ImageJ. The results shown in Figure 49A demonstrate that Nano-P and Dendri-P increased AF-αCD124 delivery into the nasal turbinates by 3.5 and 7.8 times, respectively, compared to protamine. In the Nano-P group, both longer distances up to 90 μm and higher concentrations of AF-αCD124 were measured compared to the protamine treatment group (Figure 49B). In contrast, no difference was observed between the protamine and Dendri-P groups in the furthest migration distance (60 μm) of AF-αCD124 from the tissue surface. However, Dendri-P showed significantly stronger AF-αCD124 intensity in the epithelial layer (2–35 μm) compared to protamine. Overall, these data suggest that protamine, Nano-P, and Dendri-P effectively overcame the mucous and epithelial delivery barriers for AF-αCD124.Nano-P demonstrated superior delivery of AF-αCD124 to the lamina propria (stronger AF-αCD124 intensity was observed at 25–90 μm), while Dendri-P showed enhanced epithelial retention. This may be due to differences in shape and size between Nano-P and Dendri-P. Nano-P is spherical with a small diameter of approximately 60 nm, which may have allowed it to penetrate deeper. Dendri-P, with an 8-fold increase in size (approximately 500 nm), may have limited its tissue penetration, resulting in AF-αCD124 accumulation in the epithelium. Since nasal polyps typically occur in the epithelium, Dendri-P may have offered an advantage over Nano-P for improving nasal histology.

[0289] In vivo efficacy of αCD124 formulations in severe CRSwNP mice Human patients are typically diagnosed in the advanced stage, which leads most patients to mAb therapy due to uncontrolled disease progression; therefore, we focused the remainder of our efficacy study on a severe CRSwNP model. In this severe model, increased amounts of mucus are produced in the nasal cavity, which can further hinder the penetration of the therapeutic agent, providing an opportunity to better distinguish between different delivery systems. To establish a severe CRSwNP model, mice were sensitized with OVA for 137 days. On day 137 after the first drop-infusion of OVA and SEB, αCD124 formulations were prepared by mixing αCD124 with protamine, Nano-P, or Dendri-P and then administered intranasally three times a week until the study endpoint (day 197). Subcutaneous administration of αCD124 three times per week was included as a control. Much higher mAb doses were used for sc delivery (25 mg / kg) compared to the intranasal formulation (2 mg / kg). 8 One day after the final dose, mice receiving different treatments were compared in terms of their disease burden.

[0290] Formulations containing Nano-P and Dendri-P alleviated symptoms in severe CRSwNP mice and reduced sinus turbidity compared to protamine formulations.

[0291] As shown in Table 8, the average number of nose touches in PBS-treated mice reached 111 in 10 minutes, compared to only 10 in healthy mice in the same period. The frequency of nose touches was significantly reduced to 50 in 10 minutes in the high-dose sc αCD124 group compared to PBS-treated mice. Intranasal αCD124 without any delivery system showed no significant activity, while intranasal αCD124 mixed with protamine, Nano-P, and Dendri-P reduced nose touches per 10 minutes to 37, 32, and 26, respectively. In this study, Dendri-P was superior to protamine in suppressing nose touches, while Nano-P and protamine showed no significant difference.

[0292] [Table 8]

[0293] Representative micro-CT scans of the paranasal sinuses, treated with various formulations and exhibiting uneven, diffusely swollen mucosa, were examined. Coronal CT scans revealed diffuse mucosal thickening in various parts of the nasal cavity in CRSwNP mice, with a large portion of the left nasal cavity obstructed due to extensive mucosal swelling and mucus filling. In the sc (high-dose) and inαCD124 treatment groups, diffuse mucosal thickening was found to vary in the nasal septum, cribriform labyrinth, and true maxillary sinus. These mice also showed uneven thickness across the entire mucosal surface, whereas healthy mice showed a smooth, thin mucosal lining. Reduced mucosal thickening was found in protamine / αCD124-treated mice compared to sc and inαCD124-treated mice. Further reductions in mucosal thickening were observed in Nano-P and Dendri-P-treated mice compared to the protamine-treated group.

[0294] Furthermore, CT images of the nasal cavity cross-section were sequentially collected and reconstructed into a three-dimensional (3D) model. 3D reconstructions of the paranasal sinuses were examined from mice treated with various formulations that showed obstruction. In healthy mice, the airways were well-defined and connected, while in PBS-treated CRSwNP mice, a significant portion of the nasal airway was obstructed. Moderate improvement in paranasal sinus airway morphology was observed with sc(high-dose)αCD124 and protamine / αCD124 treatment, while inαCD124 showed little effect. Nano-P and Dendri-P treatment preserved most of the nasal airway, which appeared to be superior to other treatments.

[0295] Epithelial thickening was further visualized using H&E. Figure 50 shows representative H&E images of paranasal sinus tissue after treatment with various formulations, illustrating increased epithelial thickening. Nasal mucosal thickness in healthy mice was approximately 20 μm, while that of CRSwNP mice increased at least fourfold. High-dose scαCD124 significantly reduced nasal epithelial thickness compared to mice receiving PBS or inαCD124 (without delivery system). The effect of protamine / αCD124 administered at 2 mg / kg was comparable to that of high-dose scαCD124 (25 mg / kg). Nano-P and Dendri-P formulations further reduced nasal epithelial thickening compared to protamine formulations. These results support the superior nasal penetration efficiency of the three protamine formulations compared to sc delivery, indicating that Nano-P and Dendri-P are more effective than protamine.

[0296] Nano-P and Dendri-P enhanced the anti-CRSwNP efficacy of αCD124 compared to protamine. Polypoid lesions and epithelial destruction were examined in H&E images of nasal tissue collected from severe CRSwNP mice after different treatments. (Representative images of H&E-stained sections of paranasal sinus tissue after various treatments showing classic polypoid lesions and epithelial destruction were examined.) As shown in Figure 51A, the number of polypoid lesions in PBS-treated CRSwNP mice was 1.3 times higher than in αCD124 sc-treated mice (30% reduction in polypoid lesions) and protamine-treated mice (32% reduction in polypoid lesions). αCD124 in, without any delivery system, did not exhibit any anti-polyposis effect. The data again confirmed that protamine-mediated intranasal delivery of αCD124 achieved similar effects to the sc-formulation, but at a dose 12.5 times lower. By using the same low dose of αCD124, the Nano-P and Dendri-P formulations achieved 47% and 58% reductions in polypoid lesions, respectively, compared to PBS-treated mice, which were superior to the protamine formulation. Similar results were obtained in epithelial disruption in nasal tissue. As shown in Figure 5C, scαCD124-treated mice had an average of 21 epithelial disruptions per field of view, which was a significant decrease compared to the average for PBS-treated mice (38 per field of view) and comparable to the average for protamine-treated mice (24 per field of view). The Nano-P and Dendri-P formulations further reduced the disruptions to 20 and 12 per field of view, respectively, and their efficacy was better than the protamine formulation. In particular, Dendri-P achieved a significantly lower number of epithelial disruptions compared to Nano-P. This may be due to increased delivery to the epithelial layer, as shown in Figure 51B.

[0297] CRSwNP is diagnosed by eosinophil enrichment in mucosal tissue. These cells release type 2 inflammatory cytokines, including IL-4 and IL-13, in the nasal epithelium, activating and transforming surrounding cells, which leads to increased inflammation and polyposis. Eosinophils in nasal tissue were stained using Giemsa staining. Representative Giemsa stained images of sinus tissue sections from CRSwNP mice after treatment with various formulations showing eosinophils were examined, and the number of eosinophils per field of view was identified and quantified using Trainable Weka Segmentation in ImageJ

[83] . Increased eosinophilic tissue infiltration was detected in PBS (37 per field of view) and αCD124 in (34 per field of view) compared to others. The high-dose scαCD124 treatment group showed reduced eosinophilic infiltration (24 per field of view), which was similar to the protamine group (27 per field of view). Nano-P and Dendri-P formulations were more effective than protamine, further reducing eosinophil counts to 17 and 16 per field of view, respectively (Figure 52).

[0298] Persistent tissue inflammation often results in increased extracellular matrix (ECM) deposition, which leads to increased tissue rigidity, impaired ciliary movement, and reduced mucus clearance. These pathological changes exacerbate inflammation and increase the chances of infection. We examined representative Masson trichrome stained images of sinus tissue sections from CRSwNP mice after various treatments, showing cartilage and collagen-based ECM. Masson trichrome (MT) was used to stain collagen deposition, which was isolated in trainable Weka segmentation in ImageJ

[83] (Figure 53A). In healthy mice, collagen deposition was barely detectable in nasal tissue, while in CRSwNP tissue, strong ECM staining was observed. High-dose scαCD124-treated mice showed a significant reduction in nasal tissue ECM compared to PBS-treated CRSwNP mice. Nasal ECM in the protamine group was 23% of that in PBS-treated mice and 43% of that in the αCD124 in group, suggesting that in protamine / αCD124 suppressed fibrosis of nasal tissue in CRSwNP mice. High-dose scαCD124 showed anti-fibrotic efficacy comparable to that of protamine preparations. In the Nano-P and Dendri-P groups, ECM area was significantly reduced to 11.6% and 14.5% of that in the PBS group, respectively, demonstrating superior efficacy compared to protamine preparations.

[0299] Goblet cell hyperplasia and excessive mucus secretion induced by type 2 eosinophilic inflammation are often characterized in CRSwNPs. Next, goblet cells were stained purple with arcinablue-periodine-Schiff (AB-PAS) staining. Representative PAS-stained images of paranasal sinus tissue from mice treated with various formulations were examined, and the number per field of view was quantified by ImageJ. Increased goblet cell counts were detected in the PBS (32 cells per field of view) and αCD124 in (30 cells per field of view) groups compared to the others. The high-dose scαCD124 treatment group showed reduced goblet cell staining (24 cells per field of view), similar to the protamine group (20 cells per field of view). Nano-P and Dendri-P formulations were more effective than high-dose scαCD124, further reducing goblet cell counts to 15 and 12 per field of view, respectively (Figure 53B). Dendri-P formulations were superior to protamine, which may be due to enhanced delivery and retention of αCD124 in the nasal epithelium.

[0300] Intranasal delivery of αCD124 via protamine, Nano-P, and Dendri-P suppressed type 2 inflammatory biomarkers in CRSwNP mice. Human patients with CRSwNP who received dupilumab showed reduced levels of IgE, eotaxin, IL-13

[84] , and thymic and activating regulatory chemokines (TARCs). Of these, IgE is a key mediator for eosinophil migration to polyps, which facilitates disease progression. As shown in Figures 54A–54B, CRSwNP mice had high IgE levels in plasma (approximately 100 ng / mL) and nasal tissue (approximately 400 ng / mL), while IgE levels in healthy mice were undetectable. Protamine, Nano-P, and Dendri-P formulations significantly reduced IgE levels in plasma and nasal tissue by 50–75%. In particular, in nasal tissue rather than plasma, Nano-P and Dendri-P formulations showed enhanced activity in reducing IgE release compared to protamine. This may be due to increased local delivery of αCD124 by Nano-P and Dendri-P. In the absence of a delivery system, αCD124 conferred by in or sc had no significant effect on IgE production in plasma or nasal tissue.

[0301] These three CPP formulations also demonstrated significant efficacy in suppressing type II inflammatory cytokines in nasal tissue. For example, treatment with protamine, Nano-P, and Dendri-P formulations significantly reduced eotaxin, IL-13, and TARC in nasal tissue by 25–100% (Figures 54C–54E). TARC and eotaxin are major chemokines that attract inflammatory cells, and the reduction in eotaxin and TARC in nasal tissue was consistent with reduced eosinophil infiltration. IL-13 is an inflammatory cytokine that promotes tissue remodeling, leading to excessive mucus secretion by goblet cells, subepithelial fibrosis, and increased airway hyperresponsiveness. IL-13 also promotes eosinophil migration by inducing the production of eosinophil-promoting factors, such as eotaxin, from type II helper T (Th2) cells and epithelial cells. Reduced eosinophil count, suppressed collagen deposition, and decreased goblet cell count in nasal tissue are consistent with reduced levels of IL-13.

[0302] TNF-α causes ciliated cell loss, epithelial metaplasia, and further accumulation of inflammatory cells in the subepithelial layer

[85] . IL-2 is associated with reduced olfactory function in CRS patients and is significantly elevated in CRSwNP patients [86-88]. As shown in Figures 54F-54G, levels of TNF-α and IL-2 in nasal tissue decreased by 50-75% after treatment with protamine, Nano-P, and Dendri-P preparations.

[0303] IL-25 and IL-33 are type 2 innate lymphoid cell (ILC2) activating cytokines that contribute to the production of IL-4 and IL-13, and INF-γ is involved in the persistence and exacerbation of inflammation

[89] . These cytokines and IL-12p70 are elevated in human CRSwNP patients. As shown in Figures 54H–54K, levels of IL-25, IL-33, INF-γ, and IL-12p70 in nasal tissue were reduced in the Nano-P and Dendri-P groups, but not in the other groups.

[0304] Elevated levels of IL-1β and IL-17A correlate with increased infiltration of inflammatory cells, e.g., eosinophils, in human CRSwNP patients [90, 91]. Additionally, IL-1β increases the secretion of chemokine (CC motif) ligand 5 (CCL5) from nasal polyp fibroblasts, contributing to glucocorticoid resistance, and thymic interstitial lymphocyte neoplastic factor (TSLP) promotes the production of IL-4 and IL-13, exacerbating type 2 inflammation

[92] . IL-1β, IL-17A, and TSLP were significantly reduced in the protamine, Nano-P, and Dendri-P groups compared to the PBS control (Figures 54L–54N)

[91] .

[0305] In the absence of a delivery system, αCD124 in did not suppress any of the cytokines compared to the PBS control. High-dose scαCD124 and in protamine / αCD124 reduced the tested cytokines by 5 / 12 and 8 / 12, respectively, while Nano-P and Dendri-P suppressed all 12 cytokines in nasal tissue. IL4Rα is a common receptor for both IL-4 and IL-13. Blocking IL4Rα in eosinophils, mast cells, basophils, goblet cells, lymphocytes, and ILC2s inactivates these inflammatory immune cells, resulting in reduced type II cytokine production and eosinophil recruitment. Both Nano-P and Dendri-P facilitate the penetration of αCD124 into the nasal mucosa, which blocks IL4Rα in inflammatory cells migrating to nasal tissue. Therefore, Nano-P and Dendri-P were more consistent than protamine in suppressing Th2 cytokines, and there was no difference between Nano-P and Dendri-P.

[0306] Nasal goblet cells are located in the epithelium, and goblet cell hyperplasia is characterized by loss of mucociliary clearance, accumulation of mucin gel, and ultimately, polyp formation in the nasal cavity

[93] . Dendri-P induces increased amounts of αCD124 accumulation in the epithelium compared to other protamine-based delivery systems, which may result in enhanced blockade of IL4Rα in goblet cells, which resulted in enhanced stabilization of goblet cells and improved nasal histology among different treatment groups.

[0307] The results indicate that the antipolyposis effect was highly dependent on the delivery of αCD124 to the nasal epithelium and lamina propria, blocking both the IL-4 and IL-13 pathways as well as type 2 inflammation, which resulted in reduced IgE production, eosinophil migration, goblet cell hyperplasia, and mucus secretion.

[0308] Safety of protamine, Nano-P, and Dendri-P after long-term use Mice received intranasal protamine, Nano-P, and Dendri-P at a dose of 3 mg / kg twice daily for one month, and no nasal symptoms were observed in any of the treated mice. Mice were euthanized one day after the final dose. Blood, as well as tissues including the nose, heart, liver, spleen, lungs, kidneys, and trachea, were collected and analyzed. There were no significant differences in organ weight between the treatment group and the control group (Figure 55A). Histology of nasal tissue and major organs showed no abnormalities. Representative images of H&E staining of main airway tissue sections from mice treated with protamine, Nano-P, or Dendri-P were examined. RBC, hematocrit, hemoglobin, MCV, MCH, RDW, WBC, and lymphocyte values ​​were within the normal range (Figures 55B-55U). Liver and kidney function, as indicated by serum levels of urea (BUN), ALT, AST, and creatine kinase, was also within the normal range (Figures 55B–55U). Overall, these data indicate that the three intranasal-conjugated formulations were safe in mice.

[0309] The results demonstrate a successful nasal delivery system for αCD124 to treat CRSwNP. The formulations were prepared by physically mixing αCD124 with a protamine-based delivery system, which provided convenience and flexibility for optimizing the drug-to-carrier ratio. αCD124 delivered by these protamine-based systems was administered at doses lower than 12.5 times, yet superior to the high-dose scαCD124, which is the standard therapy for CRSwNP. Furthermore, the inventors achieved antipolyposis within 8 weeks of treatment. Of these protamine-based formulations, the nanostructured Nano-P and Dendri-P enhanced the penetration of αCD124 into the nasal mucosa and thus improved the antipolyposis and type 2 inflammation effects. In particular, Nano-P facilitated increased penetration of αCD124 into the nasal lamina propria and appeared to be the most consistent in suppressing type 2 inflammation. Dendri-P promoted the accumulation of αCD124 in the nasal epithelium, the site of histological lesions, and therefore showed enhanced inhibition of epithelial destruction compared to other treatments.

[0310] In summary, high-dose scαCD124, the standard therapy for advanced CRSwNP, yielded significant but moderate efficacy in symptom relief, anti-polyposis, and anti-Th2 inflammation in severe CRSwNP mice. Nano-P and Dendri-P are nano-derivatives of protamine, synthesized to enhance nasal penetration of αCD124 for improved therapy of CRSwNP. Protamine increased αCD124 penetration into the nasal epithelium, while Nano-P and Dendri-P further enhanced penetration into the epithelium and lamina propria, respectively, compared to protamine. Three protamine-based delivery systems were physically mixed with αCD124 and intranasally administered at doses 12.5 times lower than high-dose scαCD124. The protamine formulations were generally comparable to high-dose scαCD124, but slightly more consistent in suppressing type 2 inflammation. Both nano-formulations performed better than protamine and high-dose scαCD124 in suppressing CRSwNP symptoms, polyposis, and type 2 inflammation. Nano-P showed increased penetration into the nasal lamina propria and demonstrated the most consistent efficacy in reducing type 2 inflammation, while Dendri-P tended to accumulate in the nasal epithelium and exhibited superior efficacy in improving nasal histology.

[0311] CPP intravitreal data + MM Protamine was dissolved in sterile water at a concentration of 10 mg / ml.

[0312] AF-647 IgG was dissolved in PBS at a concentration of 2 mg / ml.

[0313] Mice were anesthetized with isoflurane. A Neuros syringe (50 μL, Neuros syringe, Model 1705 RN, 33 gauge, needle tip shape 4 (65460-16|Laboratory|Hamilton Company)) was used.

[0314] A 33-gauge needle was positioned at a 90-degree angle to the sclera and injected to a depth of approximately 1 mm.

[0315] For IgG delivery, (10 μl of IgG + 5 μl of water or 5 μl of protamine) was slowly added over 30 seconds to allow for liquid diffusion.

[0316] Two hours later, the mice were euthanized and their eyeballs were collected. The eyes were then fixed overnight in 10% formalin.

[0317] The fixed eyes are cryopreserved in sucrose, followed by OCT embedding.

[0318] Place the eyes in PBS with 10% sucrose, usually overnight, until they are submerged.

[0319] Next, the eyes are placed in PBS with 30% sucrose until they are submerged, usually overnight.

[0320] The eye is embedded within the OCT. The sample was sectioned into 10 μm thick sections using a Leica Cryostat.

[0321] Protamine increases IgG delivery to the ganglion cell layer (GCL), inner granular layer (INL), retinal pigment epithelium (RPE), and choroid after intravitreous injection in mice.

[0322] 4-hour penetration of human skin replacements Protamine was dissolved in water at a concentration of 10 mg / ml.

[0323] Alexa647-IgG (AF-IgG) was dissolved in PBS at a concentration of 2 mg / ml.

[0324] In the protamine / AF-IgG treatment group, 40 µl of AF-IgG was mixed with 80 µl of protamine.

[0325] For the AF-IgG monotherapy group, 40 µl of AF-IgG was mixed with 80 µl of water.

[0326] The formulation was applied topically and incubated at 37°C for 4 hours.

[0327] Four hours later, the human skin replacement was mounted by OCT, sectioned at 10 μm, and subsequently stained with DAPI.

[0328] After 4 hours of IgG infiltration into healthy human skin replacement material, protamine enhanced AF-IgG retention and infiltration.

[0329] 4-hour penetration of psoriasis human skin replacement Protamine was dissolved in water at a concentration of 10 mg / ml.

[0330] Alexa647-IgG (AF-IgG) was dissolved in PBS at a concentration of 2 mg / ml.

[0331] In the protamine / AF-IgG treatment group, 60 µl of AF-IgG was mixed with 120 µl of protamine.

[0332] For the AF-IgG monotherapy group, 60 µl of AF-IgG was mixed with 120 µl of water.

[0333] The formulation was applied topically and incubated at 37°C for 4 hours.

[0334] Four hours later, the psoriasis-related human skin replacement tissue was mounted by OCT, sectioned at 10 μm, and subsequently stained with DAPI.

[0335] After 4 hours of IgG penetration of psoriasis-related human skin replacements, protamine enhanced AF-IgG retention and penetration.

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[0337] All references are incorporated herein by reference.

[0338] The present invention is described in relation to one or more embodiments. However, it will be obvious to those skilled in the art that several variations and modifications can be made without departing from the scope of the invention as defined in the claims. Thus, although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common technical knowledge of those skilled in the art. Such modifications include the substitution of known equivalents to any aspect of the invention in substantially the same manner to achieve the same results. Numerical ranges include the digits defining the range. It is understood that the elements enumerated in specific embodiments can be combined in any manner and in any number to produce additional embodiments. The various examples and preferred embodiments described herein should not be construed as limiting the invention to only the embodiments explicitly described. It should be understood that this description supports and encompasses embodiments that combine the explicitly described embodiments with any number of disclosed elements and / or preferred elements. Furthermore, any permutations and combinations of all elements described herein should be considered disclosed by the description herein unless the context indicates otherwise.

[0339] In this specification, the term “comprising” is used as an open-ended term and is substantially equivalent to the phrase “comprising but not limited to,” and the term “comprises” has the corresponding meaning. However, wherever the terms “comprising” or “comprises,” or variations of the same etymology, are used herein, it is understood that variations or modifications to “consisting” or “consists,” which exclude any unexpressed elements, steps, or components, or variations or modifications to “consisting essentially of” or “consists essentially of,” which limit the claimed invention to expressed materials or enumerated steps together with those that do not substantially affect the fundamental and novel features of the claimed invention, are also intended. "Approximately" means a variation (plus or minus) of 5% or less from a value or range, for example, 0.5%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%. The references made herein are not construed as an admission that such references constitute prior art of the present invention. All publications are incorporated herein by reference as specifically and individually indicated, and as if each individual publication were fully described herein. The present invention includes all embodiments and variations substantially described earlier with reference to examples and drawings.

Claims

1. A protamine molecule comprising a first protamine peptide, wherein the first protamine peptide is conjugated with one or more of the following: a lipid, a hydrophobic moiety, a polymer containing an amino group, or an additional protamine peptide.

2. The protamine molecule according to claim 1, wherein the lipid is a fatty acid, a lipid amine, or a lipid carboxyl.

3. The protamine molecule according to claim 2, wherein the lipid further comprises a linker.

4. The protamine molecule according to claim 3, wherein the linker is polyethylene glycol (PEG) or N-hydroxysuccinimide (NHS).

5. The protamine molecule according to claim 1, wherein the lipid is palmitic acid, stearic acid, 1,2-phosphatidylethanolamine (PE), dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), DPPE-NHS, DSPE-NHS, or DOPE-NHS.

6. The protamine molecule according to claim 1, wherein the hydrophobic portion is allyl glycidyl ether (AGE).

7. The protamine molecule according to claim 1, wherein the polymer containing an amino group is poly(amidoamine) (PAMAM), polyethyleneimine (PEI), or polylysine (PLL).

8. The protamine molecule according to claim 4, further comprising a crosslinking agent.

9. The protamine molecule according to claim 4, wherein the crosslinking agent is N-succinimidyl S-acetylthioacetate (SATA), or N-γ-maleimidobutyryloxysuccinimid (GMBS), or succinimimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).

10. The protamine molecule according to claim 1, wherein the protamine molecule is protamine-stearic acid, protamine-PAMAM, protamine-DMPE, protamine-GMBS-PAMAM, protamine-DMPE, protamine-AGE, protamine-SATA-PAMAM, protamine-C18, protamine-C16, protamine-PEI, or protamine-PLL.

11. The protamine molecule according to claim 1, wherein the first protamine peptide is conjugated to one or two additional protamine peptides, resulting in a protamine dimer or a protamine trimer.

12. The protamine molecule according to claim 1, wherein the first protamine peptide and the additional protamine peptide are the same or different.

13. The protamine molecule according to any one of claims 1 to 12, wherein the first protamine peptide and / or the additional protamine peptide is a protamine salt.

14. The protamine molecule according to claim 13, wherein the protamine salt is protamine sulfate derived from salmon.

15. The protamine molecule according to any one of claims 1 to 14, wherein the first protamine peptide or the additional protamine peptide comprises any one of the amino acid sequences or conserved substitutions of SEQ ID NOs: 1 to 4.

16. The protamine molecule according to any one of claims 1 to 15, wherein the protamine molecule comprises an alpha helix and / or a beta sheet.

17. The protamine molecule according to any one of claims 1 to 16, wherein the protamine molecule self-assembles into a nanostructure.

18. The protamine molecule according to any one of claims 1 to 17, wherein the protamine molecule increases density in the cell membrane, increases the concentration of protamine in the cell membrane, and / or increases the interaction between protamine and the cell membrane.

19. A composition comprising the protamine molecule described in any one of claims 1 to 18.

20. A pharmaceutical composition comprising the protamine molecule described in any one of claims 1 to 18.

21. A method for delivering a payload molecule to a cell, comprising contacting the cell with the payload molecule in combination with a protamine molecule according to any one of claims 1 to 18.

22. The method according to claim 21, wherein the delivery is a non-injection delivery.

23. A method for non-injection delivery of a large payload molecule to a cell, comprising contacting the cell with the payload molecule in combination with a protamine peptide or a protamine molecule.

24. The method according to claim 22 or 23, wherein the non-injectable delivery is intranasal, sublingual, oral, buccal, rectal, vaginal, intravitreal, local, or to the skin, eye, brain, or lung.

25. The method according to any one of claims 22 to 24, wherein the non-injectable delivery is transmucosal or transcellular.

26. The method according to claim 25, wherein the transmucosal delivery does not involve injection.

27. The method according to claim 25, wherein the transcellular delivery is transepithelial or to the lamina propria.

28. The method according to any one of claims 21 to 27, wherein the payload molecule is delivered to the nucleus of the cell and / or substantially bypasses the lysosome of the cell.

29. The method according to any one of claims 21 to 28, wherein the payload molecule is a protein, a peptide, a peptide analog, a nucleic acid molecule, or a small molecule.

30. The method according to any one of claims 21 to 29, wherein the payload molecule is provided in a physical mixture with the protamine molecule.

31. The method according to any one of claims 21 to 30, wherein the payload molecule does not form a complex with the protamine molecule.

32. The method according to any one of claims 21 to 31, wherein the payload molecule is an antibody, growth hormone, insulin, or semaglutide.

33. A method for making a cell membrane permeable, comprising contacting the cell membrane with a protamine peptide and / or protamine molecule according to any one of claims 1 to 18.

34. The method according to claim 33, wherein the permeability is transient and / or reversible.

35. A method for transfecting cells, comprising contacting the cell membrane with a protamine peptide and / or protamine molecule according to any one of claims 1 to 18.

36. A method for treating or preventing a condition that would benefit from non-injectable delivery of a payload molecule, comprising administering the payload molecule together with a protamine peptide and / or protamine molecule according to any one of claims 1 to 18 to a subject in need thereof.

37. The method according to claim 36, wherein the payload molecule is administered simultaneously with or at different times from the protamine peptide and / or the protamine molecule.

38. The method according to claim 36, wherein the protamine peptide and / or the protamine molecule is administered before the administration of the payload molecule.

39. The method according to any one of claims 36 to 38, wherein the condition is diabetes, sinusitis, an eye condition, or a skin condition.

40. Use of a protamine peptide and / or protamine molecule according to any one of claims 1 to 18 for treating or preventing a condition that would benefit from non-injectable delivery of a payload molecule.

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