Polymer-enabled delivery of pharmaceutical agents

Abstract

Disclosed herein are bioactive polymers for use in forming solutions and / or hydrogels to stabilize one or more pharmaceutically active agents before, during, or after administration. The polymer comprises: a first monomer for binding water; the second monomer is used for endowing the stent with mechanical properties; optionally, a third monomer for binding to a natural or synthetic peptide or protein (NSPP); and a fourth monomer for imparting a phase change behavior. Preferably, the first monomer is OEGMA; the second monomer is PLA / HEMA; the third monomer is NAS; and the fourth monomer is NIPAAm, and the polymer comprises: OEGMA in an amount of from about 1 mol% to about 15 mol%; pLA / HEMA in an amount of from 5 mol% to about 50 mol%; nAS in an amount from 0 to about 15 mol%; and NIPAAm in an amount of up to about 85 mol%.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480044203.2 (22) Application Date 2024.06.28 (30) Priority Data 2023902095 2023.06.30 AU (85) PCT International Application Entering National Phase Date 2025.12.30 (86) PCT International Application Application Data PCT / AU2024 / 050690 2024.06.28 (87) PCT International Application Publication Data WO2025 / 000039 EN 2025.01.02 (71) Applicant: Trimf IP Pte Ltd. Address: New South Wales, Australia (72) Inventors: A. Fatti, T. Abrams, W. Knox, S. Malikinia, P. Yang, D. Trayni, H. X. Ong, J. Al-Mazi (74) Patent Agency: Beijing Shifeng Intellectual Property Agency Co., Ltd. 11713 Patent Attorneys: Wang Jianxiu, Liu Xiaoli (51) Int.Cl. C08F 220 / 54 (2006.01) A61K 9 / 00 (2006.01) A61K 9 / 51 (2006.01) A61K 39 / 00 (2006.01) A61K 39 / 215 (2006.01) A61K 47 / 32 (2006.01) A61K 47 / 58 (2006.01) A61P 31 / 14 (2006.01) (54) Invention Title: Polymer-Assisted Drug Delivery (57) Abstract: This document discloses a bioactive polymer for forming solutions and / or hydrogels to stabilize one or more pharmaceutically active agents before, during, or after administration. The polymer comprises: a first monomer for binding water; a second monomer for imparting mechanical properties to the scaffold; optionally, a third monomer for binding to a natural or synthetic peptide or protein (NSPP); and a fourth monomer for imparting phase transition behavior. Preferably, the first monomer is OEGMA; the second monomer is PLA / HEMA; the third monomer is NAS; and the fourth monomer is NIPAAm, and the polymer comprises: about 1 mol% to about 15 mol% of OEGMA; 5 mol% to about 50 mol% of PLA / HEMA; 0 to about 15 mol% of NAS; and up to about 85 mol% of NIPAAm.Claims (3 pages), Description (33 pages), Drawings (20 pages), CN 121752616 A, 2026.03.27, CN 1 21 75 26 16 A 1. A polymer for forming a solution and / or hydrogel to stabilize one or more pharmaceutically active agents before, during, or after application, said polymer comprising: a first monomer for binding water; a second monomer for imparting mechanical properties to a scaffold; optionally, a third monomer for binding to a natural or synthetic peptide or protein (NSPP); and a fourth monomer for imparting phase transition behavior. 2. The polymer of claim 1, wherein the application is by intravascular, intramuscular, subcutaneous, inhaled respiratory tract, oral inhalation, or intranasal administration. 3. The polymer of claim 2, wherein the application is intranasal administration. 4. The polymer according to any one of the preceding claims, wherein the first monomer is one or more polyethers selected from: polyethylene glycol (PEG), oligomeric (ethylene glycol) (OEG), polyethylene oxide (PEO), polyethylene oxide-propylene oxide copolymer (PPO), copolymerized ethylene oxide block copolymers, or random copolymers thereof. 5. The polymer according to claim 4, wherein the first monomer is oligomeric (ethylene glycol) monomethyl ether methacrylate (OEGMA). 6. The polymer according to any one of the preceding claims, wherein the second monomer is methacrylate, or a random copolymer containing methacrylate, selected from: hydroxyethyl methacrylate (HEMA), hydroxyethyl methacrylate poly(lactic acid) copolymer (PLA / HEMA), poly(lactic acid), poly(caprolactone), poly(glycolic acid), poly(glycolic acid-lactide), poly(glycolic acid-lactide) copolymer, or poly(glycolic acid-caprolactone) copolymer. 7. The polymer according to claim 6, wherein the second monomer is hydroxyethyl methacrylate poly(lactic acid) (PLA / HEMA). 8. The polymer according to any one of the preceding claims, wherein the third monomer has an electrophilic functional group for binding with NSPP. 9. The polymer according to claim 8, wherein the third monomer is selected from: N-hydroxysulfonylsuccinimide (SNHS), N-hydroxyethoxylated succinimide (ENHS), and N-acryloyloxysuccinimide (NAS). 10. The polymer according to claim 9, wherein the third monomer is N-acryloyloxysuccinimide (NAS). 11. The polymer according to any one of the preceding claims, wherein the lower critical solution temperature (LCST) of the fourth monomer is less than about 37°C. 12. The polymer according to claim 11, wherein the fourth monomer is selected from: poly(ethylene oxide) / poly(propylene oxide) and poly(N-isopropylacrylamide) (PNIPAAm) homopolymers and copolymers.13. The polymer of claim 12, wherein the fourth monomer is (N-isopropylacrylamide) (NIPAAm). 14. The polymer of any preceding claim, wherein the polymer comprises about 1 mol% to about 15 mol% of a first monomer; about 5 mol% to about 50 mol% of a second monomer; about 0 mol% to about 15 mol% of a third monomer; and a fourth monomer to make up the remainder to 100% of the polymer. 15. The polymer of any preceding claim, wherein: the first monomer is OEGMA; the second monomer is PLA / HEMA; the third monomer is NAS; and the fourth monomer is NIPAAm, wherein the polymer comprises: about 1 mol% to about 15 mol% of OEGMA; 5 mol% to about 50 mol% of PLA / HEMA; 0 mol% to about 15 mol% of NAS; and up to about 85 mol% of NIPAAm. Claims 1 / 3 Page 2 CN 121752616 A 16. The polymer according to any one of the preceding claims, wherein the one or more pharmaceutically active agents comprise a vaccine originally limited to intramuscular injection. 17. The polymer according to claim 16, wherein the vaccine originally limited to intramuscular injection is a COVID-19 (SARS-CoV-2) vaccine. 18. The polymer according to claim 17, wherein the COVID-19 vaccine is a Pfizer-BioNTech (Comirnay / Tozinameran) vaccine. 19. The polymer according to any one of the preceding claims, wherein the polymer is present at a concentration greater than or equal to about 15 mg / mL. 20. The polymer according to claim 19, wherein the polymer is present at a concentration of about 25 mg / mL to about 35 mg / mL. 21. The polymer according to claim 20, wherein the polymer is present at a concentration of about 35 mg / mL.22. The polymer according to any one of the preceding claims, wherein the first monomer is OEGMA; the second monomer is PLA / HEMA; the third monomer is NAS; and the fourth monomer is NIPAAm; wherein the polymer comprises: about 1 mol% to about 15 mol% of OEGMA; 5 mol% to about 50 mol% of PLA / HEMA; 0 mol% to about 15 mol% of NAS; and up to about 85 mol% of NIPAAm; wherein the vaccine is an mRNA vaccine, preferably a Pfizer-BioNTech (Comirnay / Tozinameran) COVID-19 vaccine; wherein the polymer is present at a concentration of about 35 mg / mL; and wherein the polymer maintains the integrity of the mRNA at 37°C for up to 4 days. 23. A method for delivering one or more pharmaceutically active agents, the method comprising administering to a subject in need an effective concentration of one or more pharmaceutically active agents dispersed in a polymer defined according to any one of claims 1 to 22. 24. The method of claim 23, wherein the application is performed at ambient temperature, and the polymer transforms into its hydrogel form at a higher temperature (e.g., body temperature). 25. The method of claim 23 or 24, wherein the application is performed intranasally via nasal spray or deposition. 26. The method of any one of claims 23 to 25, wherein one or more pharmaceutically active ingredients may be dispersed in situ in the polymer, or pre-formulated and stored under conditions supporting the activity of the active ingredient. 27. The method of claim 26, wherein the conditions supporting the activity of the active ingredient include freeze-drying the formulation and then reconstituted it. 28. Use of the polymer as defined in any one of claims 1 to 22 in the production of a medicament for delivering one or more pharmaceutically active agents. 29. The use of claim 28, wherein the application is performed at ambient temperature, and the polymer transforms into its hydrogel form at a higher temperature (e.g., body temperature). 30. The use of claim 28 or 29, wherein the application is performed via nasal spray or deposition. 31. The use according to any one of claims 28 to 30, wherein one or more pharmaceutical active ingredients may be dispersed in situ in the polymer, or pre-formulated and stored under conditions supporting the activity of the active ingredient. 32. The use according to claim 31, wherein the conditions supporting the activity of the active ingredient comprise freeze-drying the formulation and then reconstituteing it.33. A kit capable of delivering one or more pharmaceutically active agents, the kit comprising a polymer as defined in any one of claims 1 to 22; an effective concentration of one or more pharmaceutically active agents stored under suitable conditions; and optionally, a specification regarding the addition of an effective concentration of the polymer to the one or more pharmaceutically active agents. 34. The kit of claim 33, wherein the polymer and the one or more pharmaceutically active agents are in a premixed form. Claims 3 / 3 Page 4 CN 121752616 A Polymer-Assisted Pharmaceutical Delivery Related Applications

[0001] This application claims Conventional priority to Australian Patent Application 2023902095, filed on June 30, 2023. The contents of AU'095 are incorporated herein by reference. Technical Field

[0002] The present invention relates to biocompatible polymers and the inventors’ unexpected discovery that such polymers stabilize certain pharmaceutical agents sufficiently for a period of time to allow them to be administered to a subject via a variety of delivery methods, such as intravascular, intramuscular, subcutaneous, oral inhalation, or intranasal administration.

[0003] The present invention also relates to methods capable of delivering one or more pharmaceutically active agents, and the use of biocompatible polymers in the production of medicaments for delivering one or more pharmaceutically active agents. Preferred embodiments relate to intranasal delivery.

[0004] In another embodiment, the present invention relates to kits capable of delivering one or more pharmaceutically active agents intravascularly, intramuscularly, subcutaneously, orally, or intranasally.

[0005] The present invention is intended to provide alternative routes of administration for vaccines or medicaments that were previously limited to intramuscular injection. It has been unexpectedly discovered that the stabilizing effect of polymers enables the delivery of one or more pharmaceutically active agents to a subject, where intravascular, intramuscular, subcutaneous, orally, or intranasal administration may have previously been infeasible.

[0006] The present invention is intended to provide alternative methods for the formulation, stabilization, production, and storage of vaccines or medicaments that were previously limited to cold transport, refrigeration, and complex production steps that may involve microfluidic devices or other expensive steps.

[0007] While the invention will be described below with reference to preferred embodiments thereof, those skilled in the art will understand that the spirit and scope of the invention may be embodied in many other forms. Background Art

[0008] Any discussion of the prior art throughout this specification should not be construed as an admission that such prior art is well-known or constitutes part of common general knowledge in the art.

[0009] The ongoing development of RNA-based active substances, including but not limited to vaccines for infectious disease control and cancer treatment, and the ongoing COVID-19 (SARS-CoV-2) pandemic, has driven public attention and interest in such technologies.Several COVID-19 vaccines (such as those from Pfizer-BioNTech and Moderna) use RNA to stimulate an immune response. When introduced into human tissues, the vaccine contains self-replicating RNA or messenger RNA (mRNA), both of which cause cells to express the SARS-CoV-2 spike protein. This teaches the body how to recognize and destroy the corresponding pathogen. RNA vaccines typically use nucleoside-modified messenger RNA. mRNA delivery is achieved by co-formulating the molecule with lipid nanoparticles (LNPs), which protect the RNA strand and facilitate its uptake by cells.

[0010] The stability of the LNPs and the incorporated RNA components is crucial for achieving the intended biological activity during the intended shelf life of the vaccine. This typically limits the limited shelf life of such vaccines at specific temperatures, during cold transport, and during storage. Such stringent requirements pose a challenge to the wider deployment of such technologies.

[0011] As previously stated, a preferred embodiment of the invention relates to a proprietary polymer of the applicant that can stabilize the active material in an mRNA vaccine (preferably a COVID-19 vaccine) under a variety of conditions, thereby enabling and supporting intranasal administration. Although the following discussion focuses on such implementations, those skilled in the art should understand that these are merely exemplary in the overall context of the present invention, page 1 / 33 of this specification, 5 CN 121752616 A.

[0012] All currently approved COVID-19 vaccines are administered via intramuscular injection. However, since this is not preferred or tolerated in a significant portion of the population, various other types of vaccine delivery methods have been investigated for future coronavirus vaccines. One such mode is intranasal delivery.

[0013] Intranasal vaccines target mucosal immunity in the nasal mucosa, which is the gateway for the virus to enter the body. These vaccines are designed to stimulate nasal immune factors, such as IgA. In addition to suppressing the virus, nasal vaccines offer the advantage of ease of administration because they do not involve needles (e.g., needle phobia). Nasal vaccines have been approved for influenza but not for COVID-19.

[0014] Carvalho's recent publication (Nature Medicine, Vol. 28, December 2022, pp. 2439-2440) points to the reasons why mRNA vaccines may be incompatible with intranasal delivery. More information is needed regarding the relevance of mucosal immune protection to understand how (or even whether) this will affect infection. Reactivity was mild to moderate. Antigen-specific mucosal antibody responses to intranasal vaccination were detectable in a small number of participants, rarely exceeding levels seen after SARS-CoV-2 infection. Systemic responses to intranasal vaccination were generally weaker compared to intramuscular ChAdOx1 nCoV-19 vaccination.Antigen-specific mucosal antibodies were detected in participants who received an intranasal mRNA vaccine followed by intramuscular vaccination. Seven participants developed symptoms of SARS-CoV-2 infection. In summary, this indicates that while the intranasal ChAdOx1 nCoV-19 formulation exhibits acceptable tolerability, it neither induced a sustained mucosal antibody response nor a strong systemic response.

[0015] Therefore, efforts continue to intensify to find suitable COVID-19 vaccines for intranasal administration, or delivery carriers that stabilize the active components before administration of existing vaccines or future formulations, or delivery systems that retain the active components over a large area of ​​the nasal mucosa to initiate and achieve an antibody response.

[0016] In another branch of the biomedical field, the applicant (Trimph IP Pty Ltd, of Sydney, Australia) has been actively filing a series of patents for biocompatible polymers for medical applications over the past decade. All patents and patent publications cited herein are incorporated herein by reference in their entirety.

[0017] WO 2013 / 091001 (PCT / AU2012 / 001566) relates to polymers, particularly polymers that can be used as hydrogels, and to the use of hydrogels for tissue repair or restoration. Specifically, the polymer and hydrogel of WO'001 can be used to repair or restore cartilage, particularly articular cartilage. The polymer comprises at least a monomer for binding water, a monomer for imparting mechanical properties, and a monomer for binding to extracellular proteins. The hydrogel comprises a polymer that comprises at least a monomer for binding water and a monomer for binding to extracellular proteins. The hydrogel is formed by crosslinking the polymer with extracellular matrix proteins.

[0018] The preferred polymer disclosed in WO'001 is poly(NIPAAm-co-NAS-co-(PLA / HEMA)-co-OEGMA), i.e., "PNPHO". The polymer PNPHO preferably comprises about 1 mol% to about 15 mol% of OEGMA, about 5 mol% to about 50 mol% of PLA / HEMA, up to 15 mol% of NAS, and the remainder in an amount (e.g., about 50 mol% to about 85 mol%) of NIPAAm to bring the polymer composition to 100%. For clarity, these percentages relate to the composition of the final polymer, not to the amount of feed used to form the polymer.

[0019] The preferred form of the polymer PNPHO is a polymer of formula (I), as shown below. Furthermore, x is in the range of 1-1000 and y is in the range of 1-1000, and m, n, p, and q are in the range of 1-20. Those skilled in the art will recognize that monomers A, B, C, and D can be present in the polymer in any order, provided that the desired water-binding, reinforcing, and / or crosslinking capabilities are achieved.Specification 2 / 33 pages 6 CN 121752616 A

[0020] (I) WO 2017 / 035587 (PCT / AU2016 / 050817) discloses biocompatible materials that can be used for tissue regeneration and repair, wherein the bioactive polymer can be in the form of a hydrogel, such as a thermoresponsive hydrogel. The bioactive polymer of WO'587 and the resulting hydrogel can be used for bone tissue regeneration. Therefore, this reference teaches a method for treating bone defects in mammals, the method comprising applying a therapeutically effective amount of a hydrogel formed from a bioactive polymer to a mammal to treat the bone defect.

[0021] WO 2017 / 015703 (PCT / AU2016 / 050653) discloses a polymer comprising at least one antibacterial / analgesic / anti-inflammatory monomer unit conjugated with at least three other monomer units that induce properties selected from the group consisting of: temperature activation, water solubility, mechanical strength, protein / polysaccharide binding capacity, and combinations thereof. Specifically, WO'703 discloses a polymer in which the water-soluble monomer unit is a hydrophilic ethylene glycol (OEGMA) unit; the monomer unit imparting mechanical strength is polylactic acid-co-2-hydroxy-ethyl methacrylate (PLA / HEMA); the protein-reactive monomer unit is N-acryloyloxysuccinimide (NAS) unit; and the thermosetting monomer unit is N-isopropylacrylamide (NIPAAm) unit. The antiseptic / analgesic / anti-inflammatory monomer unit comprises a methacrylate derivative of salicylic acid (5-HMA or 4-HMA, or a combination thereof).

[0022] WO 2021 / 119727 (PCT / AU2020 / 051332) teaches a composition comprising a polymer and a natural or synthetic peptide or protein (NSPP) (such as thymosin β-4). The polymer comprises a first monomer for binding water, a second monomer for imparting mechanical properties, a third monomer for binding NSPP, and a fourth monomer for imparting phase transition behavior. Specifically, the composition forms a viscous and highly fluid hydrogel upon application to the body or body surface, thereby aiding in tissue repair and regeneration. Therefore, WO'727 discloses a method for tissue repair and / or regeneration comprising applying the composition by injection or by application of an aerosol, thereby forming a hydrogel at mammalian body temperature.

[0023] Finally, WO 2023 / 201397 (PCT / AU2023 / 050329) discloses a novel polymer “PPHO”, namely poly(N-isopropylacrylamide-co-(polylactic acid / 2-hydroxymethyl acrylate)-co-(oligomeric (ethylene glycol) / poly(NIPAAm-co-(PLA / HEMA)-co-OEGMA).Specification 3 / 33 pages 7 CN 121752616 A

[0024] (II) The polymer PPHO preferably contains about 1 mol% to about 15 mol% of OEGMA, about 5 mol% to about 50 mol% of PLA / HEMA, and the remainder to 100% of the polymer composition (e.g., about 50 mol% to about 85 mol%) of NIPAAm. In a preferred embodiment, PPHO contains about 1 mol% to 15 mol% of OEGMA and / or about 15 mol% to 50 mol% of PLA / HEMA and / or about 50 mol% to 85 mol% of NIPAAM. As mentioned above, the percentages described herein relate to the composition of the final polymer, not to the amount of feed used to form the polymer. A representative polymer of PPHO (Formula (II)) is shown above.

[0025] The object of the present invention is to overcome or improve at least one disadvantage of the prior art, or to provide a useful alternative.

[0026] There is a general need in the art for an effective intranasal delivery solvent that can stabilize mRNA and / or LNP for a sufficient period of time before administration, or retain the formulation throughout the upper respiratory tract area for intranasal administration.

[0027] It is in this context that the present invention was developed. Various embodiments of the invention may reveal utility related to one or more of the above-mentioned general needs.

[0028] Specifically, the present invention can be used to provide physical and commercial alternatives for certain intramuscularly delivered pharmaceutical substances (e.g., COVID-19 vaccines).

[0029] While the invention will be described with reference to specific examples, those skilled in the art will understand that the invention can be embodied in many other forms. Summary of the Invention

[0030] In a broad sense, the present invention relates to polymers of effective concentrations for encapsulating and stabilizing one or more active pharmaceutical agents in vaccines or pharmaceuticals to improve the stability and shelf life of the active substance, and, when needed, encapsulating and retaining one or more active pharmaceutical agents in vaccines or pharmaceuticals that are originally limited to intramuscular delivery. Unexpectedly, the polymer was found to stabilize one or more active pharmaceutical agents, thereby extending their shelf life and / or allowing them to be stored under less restrictive conditions.

[0031] The applicant's polymer, present in solution or hydrogel form prior to administration, can stabilize the active substances in vaccines by preventing aggregation, thereby extending and / or improving the shelf life of such vaccines under relatively mild conditions (e.g., ambient temperature). If subsequently administered intranasally, it is able to adhere to / retain the active agent throughout the upper respiratory tract area (nasal cavity), resulting in better bioactivity.

[0032] According to a first aspect of the invention, a polymer is provided for forming solutions and / or hydrogels to stabilize one or more pharmaceutically active agents before, during, or after application, the polymer comprising: a first monomer for binding water; a second monomer for imparting mechanical properties to the scaffold; optionally, a third monomer for binding to a natural or synthetic peptide or protein (NSPP); and a fourth monomer for imparting phase transition behavior.

[0033] In embodiments, stabilizing one or more pharmaceutically active agents before application is to prevent their degradation, denaturation, shearing, or any chemical or physical changes that may affect their biological activity.

[0034] In embodiments, application can be performed intravascularly, intramuscularly, subcutaneously, by inhalation into the lungs, by oral inhalation, or by intranasal administration.

[0035] In embodiments, application is intranasal.

[0036] In embodiments, the first monomer is selected from: polyethers, polyvinyl alcohol (PVA); poly(vinylpyrrolidone) (PVP); poly(amino acids) and dextran.

[0037] In an embodiment, the polyether is selected from: polyethylene glycol (PEG), oligomeric (ethylene glycol) (OEG), polyethylene oxide (PEO), polyethylene oxide-propylene oxide copolymer (PPO), and copolymerized ethylene oxide block or random copolymers.

[0038] In an embodiment, the first monomer is oligomeric (ethylene glycol) monomethyl ether methacrylate (OEGMA).

[0039] In an embodiment, the second monomer is methacrylate, or a random copolymer containing methacrylate.

[0040] In an embodiment, the second monomer is selected from: hydroxyethyl methacrylate (HEMA), hydroxyethyl methacrylate poly(lactic acid) copolymer (PLA / HEMA), poly(lactic acid), poly(caprolactone), poly(glycolic acid), poly(glycolic acid-lactide), poly(glycolic acid-lactide) copolymer, or poly(glycolic acid-caprolactone) copolymer.

[0041] In an embodiment, the second monomer is hydroxyethyl methacrylate poly(lactic acid) (PLA / HEMA).

[0042] In an embodiment, the third monomer has an electrophilic functional group for binding NSPP.

[0043] In an embodiment, the third monomer is selected from: N-hydroxysulfonylsuccinimide (SNHS), N-hydroxyethoxylated succinimide (ENHS), and N-acryloyloxysuccinimide (NAS).

[0044] In an embodiment, the third monomer is N-acryloyloxysuccinimide (NAS).

[0045] In an embodiment, the lower critical solution temperature (LCST) of the fourth monomer is less than about 37°C.

[0046] In an embodiment, the fourth monomer is selected from: poly(ethylene oxide) / poly(propylene oxide) and poly(N-isopropylacrylamide) (PNIPAAm) homopolymers and copolymers.

[0047] In an embodiment, the fourth monomer is (N-isopropylacrylamide) (NIPAAm).

[0048] In an embodiment, the polymer contains about 1 mol% to about 15 mol% of the first monomer.

[0049] In an embodiment, the polymer contains about 5 mol% to about 50 mol% of the second monomer.

[0050] In an embodiment, the polymer contains about 0 mol% to about 15 mol% of the third monomer.

[0051] In an embodiment, the polymer contains about 50 mol% to about 85 mol% of the fourth monomer.

[0052] In an embodiment, the polymer contains: about 1 mol% to about 15 mol% of the first monomer; about 5 mol% to 50 mol% of the second monomer; 0 mol% to about 15 mol% of the third monomer; and the remainder to 100% of the fourth monomer.

[0053] In an embodiment, the first monomer is OEGMA; the second monomer is PLA / HEMA; the third monomer is NAS; and the fourth monomer is NIPAAm, wherein the polymer comprises: about 1 mol% to about 15 mol% of OEGMA; 5 mol% to about 50 mol% of PLA / HEMA; 0 mol% to about 15 mol% of NAS; and up to about 85 mol% of NIPAAm. Specification 5 / 33 page 9 CN 121752616 A

[0054] In an embodiment, one or more pharmaceutical active agents comprise therapeutically active substances and / or vaccines that are originally limited to administration by intramuscular injection. Non-limiting examples include mRNA and siRNA vaccines, etc.

[0055] In an embodiment, a vaccine that is originally limited to administration by intramuscular injection is a COVID-19 (SARS-CoV-2) vaccine.

[0056] In an embodiment, the COVID-19 vaccine is a Pfizer-BioNTech (Comirnaty / Tozinameran) vaccine.

[0057] In one embodiment, the polymer is present at a concentration greater than or equal to about 15 mg / mL.

[0058] In one embodiment, the polymer is present at a concentration of about 25 mg / mL to 35 mg / mL.

[0059] In one embodiment, the polymer is present at a concentration of about 35 mg / mL.

[0060] In an embodiment, the first monomer is OEGMA; the second monomer is PLA / HEMA; the third monomer is NAS; and the fourth monomer is NIPAAm; wherein the polymer comprises: about 1 mol% to about 15 mol% of OEGMA; 5 mol% to about 50 mol% of PLA / HEMA; 0 mol% to about 15 mol% of NAS; and up to about 85 mol% of NIPAAm; wherein the vaccine is an mRNA vaccine, preferably a Pfizer-BioNTech (Comirnay / Tozinameran) COVID-19 vaccine; wherein the polymer is present at a concentration of about 35 mg / mL; and wherein the polymer maintains the integrity of the mRNA at 37°C for up to 4 days.

[0061] Preferably, the polymer comprises: a first monomer for binding water; a second monomer for imparting mechanical properties to the polymer; optionally, a third monomer for binding to natural or synthetic peptides or proteins (NSPP); and a fourth monomer for imparting phase transition behavior.

[0062] In an embodiment, the first monomer is selected from: polyether, polyvinyl alcohol (PVA); poly(vinylpyrrolidone) (PVP); poly(amino acid) and dextran.

[0063] In an embodiment, the polyether is selected from: polyethylene glycol (PEG), oligomeric (ethylene glycol) (OEG), polyethylene oxide (PEO), polyethylene oxide-propylene oxide copolymer (PPO), copolymerized ethylene oxide block or random copolymer.

[0064] In an embodiment, the first monomer is oligomeric (ethylene glycol) monomethyl ether methacrylate (OEGMA).

[0065] In an embodiment, the second monomer is methacrylate, or a random copolymer containing methacrylate.

[0066] In an embodiment, the second monomer is selected from: hydroxyethyl methacrylate (HEMA), hydroxyethyl methacrylate poly(lactic acid) copolymer (PLA / HEMA), poly(lactic acid), poly(caprolactone), poly(glycolic acid), poly(glycolic acid-lactide), or poly(glycolic acid-lactide) copolymer.

[0067] In an embodiment, the second monomer is hydroxyethyl methacrylate poly(lactic acid) (PLA / HEMA).

[0068] In an embodiment, the third monomer has an electrophilic functional group for binding with NSPP.

[0069] In an embodiment, the third monomer is selected from: N-hydroxysulfosuccinimide (SNHS), N-hydroxyethoxysuccinimide (ENHS), and N-acryloyloxysuccinimide (NAS).

[0070] In an embodiment, the third monomer is N-acryloyloxysuccinimide (NAS).

[0071] In an embodiment, the lower critical solution temperature (LCST) of the fourth monomer is less than about 37°C.

[0072] In an embodiment, the fourth monomer is selected from: poly(ethylene oxide) / poly(propylene oxide) and poly(N-isopropylacrylamide) (PNIPAAm) homopolymers and copolymers.

[0073] In an embodiment, the fourth monomer is (N-isopropylacrylamide) (NIPAAm). Specification 6 / 33 pages 10 CN 121752616 A

[0074] In an embodiment, the polymer contains about 1 mol% to about 15 mol% of the first monomer.

[0075] In an embodiment, the polymer contains about 5 mol% to about 50 mol% of the second monomer.

[0076] In an embodiment, the polymer contains about 0 mol% to about 15 mol% of the third monomer.

[0077] In an embodiment, the polymer contains about 50 mol% to about 85 mol% of the fourth monomer.

[0078] In an embodiment, the polymer comprises: a first monomer in an amount of about 1 mol% to about 15 mol%; a second monomer in an amount of about 5 mol% to about 50 mol%; a third monomer in an amount of 0 mol% to about 15 mol%; and a fourth monomer to make up the remainder to 100% of the polymer.

[0079] In an embodiment, the first monomer is OEGMA; the second monomer is PLA / HEMA; the third monomer is NAS; and the fourth monomer is NIPAAm, wherein the polymer comprises: about 1 mol% to about 15 mol% of OEGMA; 5 mol% to about 50 mol% of PLA / HEMA; 0 mol% to about 15 mol% of NAS; and up to about 85 mol% of NIPAAm.

[0080] In an embodiment, the polymer comprises: about 5 mol% of OEGMA, about 7 mol% of HEMA-PLA, more than about 7 mol% of NAS, and about 81 mol% of NIPAAm.

[0081] According to a second aspect of the invention, a method for delivering one or more pharmaceutically active agents is provided, the method comprising administering to a subject in need an effective concentration of one or more pharmaceutically active agents dispersed in a polymer defined according to a first aspect of the invention.

[0082] In an embodiment, the administration is performed at ambient temperature, the polymer transforming into a hydrogel form at a higher temperature (e.g., body temperature).

[0083] In an embodiment, the administration is performed by intranasal spray or deposition.

[0084] In an embodiment, one or more pharmaceutically active ingredients may be dispersed in situ in the polymer, or pre-formulated and stored under conditions that support the activity of the active ingredient.

[0085] In an embodiment, conditions supporting the activity of the active ingredient include freeze-drying the formulation and then reconstituted.

[0086] According to a third aspect of the invention, the use of a polymer as defined in the first aspect of the invention is provided in the production of a medicament for the delivery of one or more pharmaceutically active agents.

[0087] In an embodiment, administration is performed under ambient conditions or substantially under ambient conditions, and then, upon reaching body temperature, a transformation into a hydrogel occurs.

[0088] In an embodiment, administration is performed via intravascular, intramuscular, or intranasal delivery or any other route of administration.

[0089] In an embodiment, one or more pharmaceutically active ingredients may be dispersed in situ in the polymer, or pre-formulated and stored under conditions supporting the activity of the active ingredient.

[0090] In an embodiment, conditions supporting the activity of the active ingredient include freeze-drying the formulation and then reconstituted it.

[0091] According to a fourth aspect of the invention, a kit for the delivery of one or more pharmaceutically active agents is provided, the kit comprising a polymer as defined in the first aspect of the invention; an effective concentration of one or more pharmaceutically active agents stored under appropriate conditions; and optionally, a description of adding an effective concentration of the polymer to one or more pharmaceutically active agents.

[0092] In an embodiment, the kit further comprises means for achieving intranasal delivery of one or more pharmaceutically active agents and an effective concentration of the polymer.

[0093] In an embodiment, the kit comprises a polymer and one or more pharmaceutical active agents in a premixed form.

[0094] Definitions and Terms In describing and claiming the invention, the following terms will be used according to the definitions listed below. It should also be understood that the terms used herein are for describing specific embodiments of the invention only and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0095] The term “stable” for one or more pharmaceutical active agents is intended to encompass the concept of “shelf life stability.” Thus, by providing stabilizing effects, storage conditions (e.g., temperature) for the active agent can be simplified, and the vaccine can be stored for a longer period. This is primarily related to pre-administration of the vaccine. After administration, particularly from the perspective of stability and sustained / controlled release, the application of the polymer / vaccine is via intravascular, intramuscular, subcutaneous, oral inhalation, intranasal delivery, etc.

[0096] Unless the context clearly specifies otherwise, throughout the specification and claims, the words “comprise”, “comprising”, etc., should be interpreted as inclusive rather than exclusive or exhaustive; that is, they should be interpreted as “including but not limited to”.

[0097] “Preferred” and “preferred” refer to embodiments of the invention that may provide certain benefits under specific circumstances.However, other embodiments may be preferred in the same or other circumstances. Furthermore, listing one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

[0098] As used herein, terms defining ranges or length limitations, such as “1 to 5,” mean any integer from 1 to 5, i.e., 1, 2, 3, 4, and 5. In other words, any range defined by two explicitly mentioned integers is intended to include and disclose integers defining upper and lower limits and any integers included within that range.

[0099] Except in operational embodiments or otherwise described, all figures used herein to indicate the amount of ingredients or reaction conditions should be understood to be modified in all cases by the term “about.” These examples are not intended to limit the scope of the invention. In the following, or where originally stated, “%” will mean “weight %,” “ratio” will mean “weight ratio,” and “parts” will mean “parts by weight.”

[0100] Although the numerical ranges and parameters illustrating the broad scope of the invention are approximate, the values ​​listed in the specific examples are as precise as possible. However, any numerical value inevitably contains some errors, which are caused by the standard deviation present in its respective test measurement.

[0101] The following abbreviations are used in this specification: Specification 8 / 33 page 12 CN 121752616 A Specification 9 / 33 page 13 CN 121752616 A Description of the Drawings

[0102] Preferred embodiments of the invention will now be described by way of example only, with reference to the accompanying drawings, in which: Figure 1 shows the stability of the following items at 4°C: (a) Formulations containing TP concentrations of 0.05, 0.10, and 0.15 ± mRNA; (b) Five-day stability study of mRNA integrity at TP concentrations of 0, 0.05, 0.10, and 0.15; and (c) Five-day stability study of mRNA content at TP concentrations of 0, 0.05, 0.10, and 0.15.

[0103] Figure 2 shows the Zetasizer analysis of particle size of the formulation stored at 4°C on day 1. mRNA formulations with or without added PNPHO formed TP005 (a) at a concentration of 5 mg / mL, TP010 (b) at a concentration of 10 mg / mL, and TP015 (c) at a concentration of 15 mg / mL.

[0104] Figure 3 shows the stability of the following at 37°C: (a) formulations containing 0, 0.15 and 0.35 TP and mRNA, with hydrogel formation (37°C) and redissolution after incubation at 4°C, and mRNA integrity was assessed at 24 h and 4 days ± with or without aerosolization using a VP7 Aptar nasal pump; (b) mRNA content was measured at the initial (day 0), 24 h (day 1) and 96 h (day 4) levels of PBS and TP50 formulations, with additional sampling of the supernatant of the TP035 formulation.

[0105] Figure 4 shows (a) the nanoparticle-forming ability of dialyzed and undialyzed PNPHO polymers at concentrations of 25 mg / mL and 35 mg / mL; (b) the functional assay of mRNA-Lipofectamine with and without PNPHO (25 mg / mL and 35 mg / mL undialyzed PNPHO (Figure 4b-i) and dialyzed PNPHO (Figure 4b-ii)).

[0106] Figure 5 shows micellar formation, in which the concentration-driven micellar formation potential of the PNPHO polymer was evaluated. To determine the critical micelle concentration (CMC) of PNPHO, the PNPHO polymer was serially diluted from 25 mg / mL.

[0107] Figure 6 shows the composite aggregation of nanoparticles. First, negatively charged bovine serum albumin (BSA, a model therapeutic agent) was added dropwise to the PNPHO solution (0.5 mg / mL) at a concentration of 1 mg / mL. Then, nanoparticles were created using chitosan by mixing magnetically mixed chitosan (1 mg / mL; pre-dissolved in 1% acetic acid) and PNPHO in ratios of 1:5, 1:1, and 5:1 to determine the optimal polymer:polymer ratio for forming nanocarriers.

[0108] Figure 7 shows the relationship between the hydrodynamic diameter and PDI of the PNPHO polymer at different concentrations. In this study, the CMC of the PNPHO polymer reached 0.5 mg / mL due to the initiation of self-aggregation of the monomer polymer. The low PDI (< 0.5) at PNPHO concentrations of 0.5 mg / mL or higher is sufficient to demonstrate this.

[0109] Figure 8 shows the hydrodynamic diameters of blank NP and BSA-encapsulated NP. Compared to blank NP, encapsulation of BSA as a model drug in the PNPHO:chitosan (1:1) formulation significantly reduced the particle size (p < 0.05) from 313.97 ± 43.71 nm to 112.41 ± 15.27 nm.

[0110] Figure 9 shows the drug deposition pattern in the silicon nasal cast. Within 20 min, the amount of drug deposited in the nasal cavity of the BSA-loaded PNPHO:chitosan 1:1 formulation gradually increased.In particular, the nasal turbinate region showed a relatively large amount of drug deposition, as indicated by the bright pink color from Sar-Gel.

[0111] Figure 10 shows the results of a qualitative study of nasal deposition at 0 min and 20 min time points using a commercially available nasal mold. The nasal mold deposition study using the BIVAX nasal spray device showed that the TP035 formulation can be deposited in a specific area (the olfactory region) and persist for up to 20 min after deposition.

[0112] Figure 11 shows the percentage size of particles containing and without mRNA in samples TP007, TP015, and TP035 (a), and a comparison of Z-ave (b) and Pdi (c).

[0113] Figure 12 shows the integrity of mRNA stored at 37°C for up to 4 days under static conditions (a) and after spraying (b).

[0114] Figure 13 shows the separation and semi-quantification of mRNA and LNP (lipofectamine) using the GPC method (a) and the preparation of different eGFP-mRNA / LNP formulations with / without TP or TL vectors. Control: a mixture of mRNA and LNP, a standard formulation without TP (b-i), a formulation A (b-ii) involving mRNA / LNP mixing followed by addition to TP solution, a formulation B (b-iii) involving the addition of lipofectamine to the TP vector followed by the addition of mRNA, and a formulation C involving the direct dissolution of mRNA in the TP vector followed by the addition of LNP.

[0115] Figure 14 shows the gel electrophoresis bands of the vector (without mRNA) (a) and containing mRNA (b).

[0116] Figure 15 shows the DLS results of TP000 (control; (a)), TP035 (b), TP050 (c), and TP100 (d)). Comparison of particle size measurement (e) and particle Pdi (f) after storage at 2–8 °C for 3 days.

[0117] Figure 16 shows mRNA encapsulation (a) and particle size measurement (b) of control (no vector; TP000) and TP035, TP050 and TP100 after storage at 2–8 °C for 14 days.

[0118] Figure 17 shows e-GFP expression in epithelial cells (a–i), epithelial cells with eGFP-mRNA (a–ii), epithelial cells with eGFP-mRNA + TP035 (a–iii), and epithelial cells with eGFP-mRNA + TP035 + lipfectomine (a–iv). In vivo imaging of cells was performed at the endpoint 48 hours (b–i), and fluorescence intensity at different time points (b–ii).

[0119] Figure 18 shows the TP000 nasal spray (control; carrier-free system) in an in vitro human model (a), significant nasal outflow (a-i and a-ii), and extremely low coverage of the upper region by the TP000 spray (a-iii). The TP035+ nasal spray (b) shows no nasal outflow (b-i) and extensive nasal surface coverage after TP035+ spraying (b-ii and b-iii).

[0120] Figure 19 shows the deposition patterns of TP000 (control) and TP035+ at 0, 5, 10, 15, and 20 minutes after application (a). Volume retention rates of TP000 and TP035+ at the application site (b). There was a statistically significant difference in volume retention between TP000 and TP035+ at all time points (p < 0.001). Nasal surface coverage after application (c). Regarding surface area coverage, there was a statistically significant difference between TP000 and TP035+ measured at 0 to 15 minutes (p < 0.001); there was no statistically significant difference at t=20.

[0121] Figure 20 shows the mRNA encapsulation efficiency of freshly prepared formulations without the carrier (TP000) and with the carrier (TP025+), as well as a formulation prepared with the carrier, freeze-dried, and reconstituted in water (TP025+FD). Detailed Description

[0122] The invention will now be described in more detail with reference to the accompanying examples and drawings. However, it should be understood that the following description is merely illustrative and should not be considered in any way as a general limitation of the invention described above.

[0123] Certain embodiments of the invention will now be referred to in detail. While the invention will be described in conjunction with embodiments, it should be understood that the purpose of the invention is not to limit the invention to these embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the invention as defined in the claims.

[0124] Those skilled in the art will recognize that many methods and materials similar to or equivalent to those described and materials herein can be used to practice the invention. The invention is by no means limited to the methods and materials described herein.

[0125] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more individual features mentioned in or apparent from the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0126] This document broadly discloses the use of biocompatible polymers for realizing intranasal administration of mRNA / LNP-based COVID-19 vaccines (particularly Pfizer-BioNTech (Comirnay) vaccines).

[0127] As used herein, the term "polymer" refers to a macromolecule (high molecular weight polymer) composed of repeating structural units (monomers). These subunits are typically linked by covalent chemical bonds. The polymer can be a linear polymer or a branched polymer. Preferably, the polymer of the present invention is a copolymer comprising three or more different monomers. For example, in one embodiment, the polymer of the present invention is illustrated by formulas (I) and (II) defined above.

[0128] As used herein, the term "monomer" refers to a structural unit that can be combined to form a polymer, but it can also be a polymer itself or a monomer or a derivative of a polymer. Such monomers are also referred to herein as "macromonomers". "Macromonomers" herein are polymers or oligomers that each have a terminal group as a monomer molecule, so that each polymer or oligomer molecule contributes only a single monomer unit to the chain of the product polymer.

[0129] The polymer of the present invention comprises: a first monomer for binding water; a second monomer for imparting mechanical properties to the polymer; optionally a third monomer for binding natural or synthetic peptides or proteins (NSPPs); and a fourth monomer for imparting phase transition behavior.

[0130] First Monomer: Water-Binding Monomer As discussed above, the advantages of the polymers of the present invention can be attributed at least in part to the specific components constituting the polymers of the present invention. A particularly advantageous property of the polymers of the present invention is their water-binding capacity. The presence of water in the polymers of the present invention provides an environment similar to the natural environment of damaged tissue (which facilitates tissue regeneration) and the required compressibility of the polymer.

[0131] Therefore, the preferred polymers used herein should contain monomers or units capable of binding water, which enables the formation of a plastic structure when the polymer is hydrated. Furthermore, the resulting structure should have the required compressibility and elasticity.

[0132] Those skilled in the art will understand that the polymers of the present invention require the presence of a certain proportion of water-binding monomers, sufficient to produce polymers that meet these requirements. Typically, the proportion of water-binding monomers in the polymer is about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50 (molar ratio of water-binding monomers to mechanical strength monomers). In fact, water-binding monomers need to make the polymer not only hydrophilic, but also to endow the polymer with a more significant water-binding capacity. Therefore, the polymers according to the present invention will have a water-binding capacity of about 70% to about 500%, about 80% to about 400%, about 90% to 300%, or about 100% to 200%.For example, the water-binding capacity of the polymer of the present invention is about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500%.

[0133] Examples of suitable water-binding monomers include those monomers that can be used to synthesize polymers, such as polyethers (e.g., alkylene oxides, such as polyethylene glycol (PEG), oligo(ethylene glycol) (OEG), polyethylene oxide (PEO), polyethylene oxide-polypropylene oxide copolymers (PPO), copolymers of ethylene oxide block or random copolymers, polyvinyl alcohol (PVA), poly(vinylpyrrolidone) (PVP), poly(amino acids), and dextrose. Polyethers, especially oligo(alkylene oxides) (e.g., OEG), are particularly preferred because they have the necessary water-binding capacity, are readily synthesized and / or available, and are inert because they elicit minimal or no immune response in the tissue to which they are placed.

[0134] Furthermore, various hydrophilic functional groups can be used to make the monomer (and the polymer formed therefrom) water-soluble. For example, water-soluble functional groups (such as phosphate, sulfate, quaternary ammonium, hydroxyl, amine, sulfonate and carboxylate) can be incorporated into the monomer to make it water-soluble. Specification 12 / 33 pages 16 CN 121752616 A

[0135] The monomer can also react with other compounds to form a “macromonomer”. Therefore, the first monomer can optionally be a macromonomer.

[0136] A preferred first monomer is oligomeric (ethylene glycol) monomethyl ether methacrylate (OEGMA), which is a hydrophilic monomer composed of two hydrophilic monomers: ethylene glycol and methacrylate.

[0137] Preferably, the polymer contains the first monomer in an amount of about 1 mol% to about 15 mol%. In various embodiments, the first monomer can be in the form of about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%. It exists in mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, or about 15 mol%.In various embodiments, the first monomer may be present in amounts of about 1 mol% to about 15 mol%, about 2 mol% to about 14 mol%, about 3 mol% to about 13 mol%, about 4 mol% to about 12 mol%, about 5 mol% to about 11 mol%, about 6 mol% to about 10 mol%, about 7 mol% to about 9 mol%, or about 8 mol%.

[0138] Second monomer: Monomer that imparts mechanical properties As discussed above, the advantageous properties of the polymers of the present invention can be attributed in part to the specific components constituting the polymers of the present invention. In embodiments, the polymers of the present invention are capable of providing additional mechanical properties and adhesiveness to the polymers of the present invention.

[0139] Those skilled in the art will understand that the polymers of the present invention require the presence of a certain proportion of monomers capable of imparting mechanical properties to the polymer, a proportion sufficient to produce a polymer having the desired mechanical properties. Typically, the ratio of "mechanical" monomers in the polymer is about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:20, about 1:30, about 1:40, or about 1:50, a molar ratio of water-bound monomers to mechanical strength monomers. Suitable examples of monomers that can impart mechanical properties (e.g., compressibility) to the polymer include acrylates (e.g., hydroxyethyl methacrylate (HEMA)), polyesters such as poly(lactic acid), poly(caprolactone), poly(glycolic acid), and their random copolymers such as poly(glycolic acid-co-lactic acid) and poly(glycolic acid-co-caprolactone).

[0140] The monomers can also react with other compounds to form "macromonomers". A preferred second monomer as a macromonomer is hydroxyethyl methacrylate poly(lactic acid) (PLA / HEMA).

[0141] Preferably, the polymer contains about 1 mol% to about 50 mol% of the second monomer.In the implementation scheme, the second monomer can be approximately 1 mol%, approximately 2 mol%, approximately 3 mol%, approximately 4 mol%, approximately 5 mol%, approximately 6 mol%, approximately 7 mol%, approximately 8 mol%, approximately 9 mol%, approximately 10 mol%, approximately 11 mol%, approximately 12 mol%, approximately 13 mol%, approximately 14 mol%, approximately 15 mol%, approximately 16 mol%, approximately 17 mol%, approximately 18 mol%, approximately 19 mol%, approximately 20 mol%, approximately 21 mol%, approximately 22 mol%, approximately 23 mol%, approximately 24 mol%, approximately 25 mol%, approximately 26 mol%, approximately 27 mol%, approximately 28 mol%, approximately 29 mol%, approximately 30 mol%, approximately 31 mol%, approximately 32 mol%, approximately 33 mol%, approximately 34 mol%, approximately 35 mol%, approximately 36 mol%, approximately 37 mol%, approximately 38 mol%, approximately 39 mol%, approximately 40 mol%, approximately 41 mol%, approximately 42 It exists in mol%, approximately 43 mol%, approximately 44 mol%, approximately 45 mol%, approximately 46 mol%, approximately 47 mol%, approximately 48 mol%, approximately 49 mol%, or approximately 50 mol%. In various embodiments, the second monomer can be in the range of about 1 mol% to about 15 mol%, about 2 mol% to about 49 mol%, about 3 mol% to about 48 mol%, about 4 mol% to about 47 mol%, about 5 mol% to about 46 mol%, about 6 mol% to about 45 mol%, about 7 mol% to about 44 mol%, about 8 mol% to about 43 mol%, about 9 mol% to about 42 mol%, about 10 mol% to about 41 mol%, about 11 mol% to about 40 mol%, about 12 mol% to about 39 mol%, about 13 mol% to about 38 mol%, about 14 mol% to about 37 mol%, about 15 mol% to about 36 mol%, about 16 mol% to about 35 mol%, about 17 mol% to about 34 mol%, about 18 mol% to about 33 mol%, about 19 mol% to about 34 mol%. It exists in mol%, about 20 mol% to about 33 mol%, about 21 mol% to about 30 mol%, about 22 mol% to about 29 mol%, about 23 mol% to about 28 mol%, about 24 mol% to about 27 mol%, or about 25 mol% to about 26 mol%.

[0142] Those skilled in the art will understand that, since mechanical strength and adhesion are key factors in this invention, the range of amounts of the second monomer is wider than that of the other monomers.

[0143] Third monomer: NSPP-binding monomer As discussed above, the polymers used in this invention can optionally be formed by combining the polymer with NSPP. In order to effectively combine the polymer with NSPP, it is preferable to include monomers or units with crosslinking capabilities in the polymer.

[0144] Such crosslinking capability means that the polymer can bind with NSPP and thus crosslink NSPP to form a polymer containing NSPP. Alternatively, by a similar mechanism, NSPP acts as a crosslinking agent, thereby crosslinking the polymer to form a polymer.

[0145] In order to produce a polymer capable of binding with NSPP, those skilled in the art will understand that the polymers of this invention need to contain a certain proportion of monomers capable of binding with NSPP, which is sufficient to crosslink with NSPP to form a polymer in the presence of water. Typically, the ratio of "crosslinked" monomers in the polymer is about 15:1, about 10:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, or about 1:15 (crosslinked monomer: water-bound monomer).

[0146] Monomers capable of binding to NSPP typically have electrophilic or nucleophilic functional groups, such that, for example, nucleophilic functional groups on NSPP can react with electrophilic functional groups on monomers to form covalent bonds.

[0147] Thus, for example, if NSPP has nucleophilic functional groups (e.g., amines), the polymer can have electrophilic functional groups, such as N-hydroxysuccinimide (NHS). Other electrophilic functional groups suitable for the present invention are N-hydroxysulfosuccinimide (SNHS) and N-hydroxyethoxysuccinimide (ENHS). An example of such monomers is N-acryloyloxysuccinimide (NAS). On the other hand, if NSPP has electrophilic functional groups, the polymer may have nucleophilic functional groups, such as amines or thiols.

[0148] Preferably, the polymer contains up to 15 mol% of a third monomer. In various embodiments, the third monomer may be present in about 0 mol%, about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, or about 15 mol%.In various embodiments, the third monomer may be present in amounts of about 0 mol% to about 1 mol%, about 1 mol% to about 15 mol%, about 2 mol% to about 14 mol%, about 3 mol% to about 13 mol%, about 4 mol% to about 12 mol%, about 5 mol% to about 11 mol%, about 6 mol% to about 10 mol%, about 7 mol% to about 9 mol%, or about 8 mol%.

[0149] Those skilled in the art will understand that the polymer can be formed from a hydrophobic composition, and therefore the third monomer is optional in the polymer.

[0150] Fourth monomer: Phase change monomer In another embodiment of the invention, the polymer may further include a fourth monomer capable of imparting phase change properties to the polymer, thereby ensuring the stability of the polymer after application. Furthermore, these phase change properties enable the polymers of the present invention to form polymers whose various properties (e.g., viscosity) can be altered by changing factors (e.g., pH and temperature). These polymers are designed such that the low critical solution temperature (LCST) is below body temperature. Various thermoresponsive and injectable polymers, including poly(ethylene oxide) / poly(propylene oxide) and poly(N-isopropylacrylamide) (PNIPAAm) copolymers, are suitable for use in this invention.

[0151] Typically, the ratio of phase change monomers in the polymer is at least about 3:1 molar ratio of phase change monomer to water-bound monomer. This ratio can be increased to, for example, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 55:1, about 60:1, about 65:1, about 70:1 molar ratio, about 75:1, about 80:1, and about 85:1 (phase change monomer: water-bound monomer).

[0152] Preferably, the polymer contains a fourth monomer in an amount that makes up the remainder to 100% of the polymer composition.In the embodiment described on page 14 / 33 of CN 121752616 A, the mol% of the fourth monomer can be up to about 85%, preferably about 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, 60 mol%, 61 mol%, 62 mol%, 63 mol%, 64 mol%, 65 mol%, 66 mol%, 67 mol%, 68 mol%, 69 mol%, 70 mol%, 71 mol%, 72 mol%, 73 mol%, 74 mol%, 75 mol%, 76 mol%, 77 mol%, 78 mol%, 79 mol%, 80 mol%, 81 mol%, 82 mol%, 83 mol%, 84 mol%, or 85 mol%.

[0153] Other polymer properties Those skilled in the art will understand that polymers with a variety of different properties can be produced by combining different types of monomers. Furthermore, the properties of a polymer can be altered by incorporating specific monomers or functional groups into a pre-existing polymer. For example, copolymerization of HEMA monomer with other monomers (e.g., methyl methacrylate) can be used to modify properties such as swelling and mechanical properties. Monomers can also react with other compounds to form macromonomers (as defined above), which are then incorporated into the polymers of the present invention. For example, HEMA can react with lactide to form HEMA-polylactic acid polymer (PLA / HEMA), which itself can be used as a monomer in the polymers of the present invention. Furthermore, the monomer itself can be a combination of monomer units, which are then incorporated into the polymer. An example of such a monomer is oligomeric (ethylene glycol) monomethyl ether methacrylate (OEGMA), a hydrophilic monomer composed of two hydrophilic monomers: ethylene glycol and methacrylic acid.

[0154] Preferred polymers of the present invention can be further modified with one or more moieties and / or functional groups. Any moiety or functional group can be used according to the present invention. In some embodiments, the polymer can be modified with polyethylene glycol (PEG), carbohydrates, and / or acyclic polyacetals derived from polysaccharides. Furthermore, as discussed above, hydrophilic groups can be incorporated into the monomer (thereby incorporating it into the polymer) to improve the polymer's water-binding capacity.

[0155] In terms of sequence, the copolymer can be a block copolymer, graft copolymer, random copolymer, blend, mixture, and / or an adduct of any of the above polymers with other polymers. Generally, the polymers according to the invention are organic polymers. Preferably, the polymers of the invention are biocompatible. In embodiments, the polymer is biodegradable.In other embodiments, the polymer is biocompatible and biodegradable.

[0156] Preferred polymers of the present invention may also include other monomers in their structure. For example, the monomers may be polymers, such as poly(vinyl alcohol) (PVA), polyesters, acrylic polymers and ionic polymers, or monomers thereof.

[0157] If biodegradability or absorbability of the polymer is desired, one or more monomers having biodegradable linkages may be used. Optionally, or additionally, the monomers may be selected such that the reaction products between them form biodegradable linkages. For each method, the monomers and / or linkages may be selected such that the resulting biodegradable polymer will degrade or be absorbed within a desired time period, for example, from about 6 hours to about 6 months. Preferably, the monomers and / or linkages are selected such that when the polymer degrades under physiological conditions, the resulting products are non-toxic.

[0158] The biodegradable linkages may be chemically or enzymatically hydrolyzable or absorbable. Illustrative examples of chemically hydrolyzable biodegradable linkages include polymers, copolymers and oligomers of glycolide, lactide, caprolactone, dioxane, and trimethylene carbonate. Exemplary enzymatically hydrolyzable biodegradable linkages include peptide bonds that can be cleaved by metalloproteinases and collagenases. Other exemplary biodegradable linkages include polymers and copolymers of poly(hydroxy acids), poly(orthocarbonates), poly(anhydrides), poly(lactones), poly(amino acids), poly(carbonates), and poly(phosphonates).

[0159] In this invention, the chemical hydrolysis of lactide leads to an increase in the lower critical solution temperature (LCST) of the polymer (by reducing the overall hydrophobicity of the polymer), thereby improving its bioavailability.

[0160] Preferred Polymer The polymer preferably contains about 1 mol% to about 15 mol% of a first monomer. In various embodiments, the first monomer may be present in amounts of about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, or about 15 mol%. Preferably, the first monomer is OEGMA.

[0161] The polymer preferably contains a second monomer in amounts of about 5 mol% to about 50 mol%.In various implementation schemes, the second monomer can be in the form of about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 The polymer is present in amounts of about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, or about 50 mol%. Preferably, the second monomer is PLA / HEMA.

[0162] The polymer preferably contains up to 15 mol% of a third monomer. In various embodiments, the third monomer may be present in amounts of about 0 mol%, about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, or about 15 mol%. Preferably, the third monomer is NAS.

[0163] The polymer preferably contains a fourth monomer, for example, about 50 mol% to about 85 mol%, to make up the remainder to 100% of the polymer composition.In the implementation scheme, the mol% of the fourth monomer can be up to about 85%, preferably about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, about 60 mol%, about 61 mol%, about 62 mol%, about 63 mol%, about 64 mol%, about 65 mol%, about 66 mol%, about 67 mol%, about 68 mol%, about 69 mol%, about 70 mol%, about 71 mol%, about 72 mol%, about 73 mol%, about 74 mol%, about 75 mol%, about 76 mol%, about 77 mol%, about 78 mol%, about 79 mol%, about 80 mol%, about 81 mol%, about 82 mol%, about 83 mol%, about 84 mol%, or 85 mol%. Preferably, the fourth monomer is NIPAAm.

[0164] The percentages described herein relate to the composition of the final polymer, not to the amount of feed used to form the polymer.

[0165] In one embodiment, the polymer preferably comprises: about 1 mol% to about 15 mol% of a first monomer; about 5 mol% to about 50 mol% of a second monomer; up to 15 mol% of a third monomer; and up to about 85 mol% of a fourth monomer.

[0166] Preferably, the first monomer is OEGMA, the second monomer is PLA / HEMA, the third monomer is NAS, and the fourth monomer is NIPAAm.

[0167] In another embodiment, the polymer preferably comprises: about 7 mol% of a first monomer; about 30 mol% of a second monomer; about 7 mol% of a third monomer; and about 53 mol% of a fourth monomer.

[0168] Preferably, the first monomer is OEGMA, the second monomer is PLA / HEMA, the third monomer is NAS, and the fourth monomer is NIPAAm.

[0169] In one embodiment, the polymer of the present invention is a polymer of formula (I): (I) Wherein, on page 16 / 33 of the specification, 20 CN 121752616, AA is a first monomer (a water-bound monomer), for example, OEGMA; B is a second monomer (a monomer capable of imparting mechanical properties to the polymer), for example, PLA / HEMA; C is a third monomer (a monomer having a functional group that binds to NSPP), for example, NAS; and D is a fourth monomer (a monomer capable of imparting phase change properties to the polymer), for example, NIPAAm.

[0170] In various embodiments, m is an integer from 1 to 20; n is an integer from 1 to 20; p is an integer from 0 to 20; and q is an integer from 1 to 20.

[0171] An exemplary polymer of the present invention is shown in formula (I) as above, wherein A is the water-binding monomer OEGMA, B is the reinforcing monomer PLA / HEMA, C is the crosslinking agent NAS, D is the phase change monomer NIPAAm, and m, n, p, q, x, and y are as defined above.

[0172] Those skilled in the art will recognize that monomers A, B, C, and D can be present in the polymer in any order, provided that the desired water-binding, reinforcing, and / or crosslinking capabilities are achieved.

[0173] It has also been found that some monomers (e.g., PLA / HEMA), polyesters (e.g., poly(lactic acid), poly(caprolactone), poly(glycolic acid)) and their random copolymers (e.g., poly(glycolic acid-co-lactic acid) and poly(glycolic acid-co-caprolactone)), and other biodegradable and biocompatible polymers can improve the LCST of the preferred polymers used in this invention during in vivo degradation of the biodegradable segment (e.g., PLA), thereby leading to bioabsorption of the polymer. This invention also provides the additional advantage that the polymers used in this invention can be designed to be biodegradable in vivo.

[0174] The overall size of the preferred polymers used in this invention may vary depending on a variety of factors, such as the type of monomer incorporated into the polymer, the type of NSPP used to form the polymer, and the conditions under which the protein is coupled to the polymer. However, typically, the preferred polymers used in this invention can be molecules of about 1 kDa to about 100 kDa, about 5 kDa to about 60 kDa, or about 30 kDa.In various embodiments, the polymers of the invention can be about 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa, 27 kDa, 28 kDa, 29 kDa, 30 kDa, 31 kDa, 32 kDa, 33 kDa, 34 kDa, 35 kDa, 36 kDa, 37 kDa, 38 kDa, 39 kDa, 40 kDa, 41 kDa, 42 kDa, 43 kDa, 44 kDa, 45 kDa, 46 kDa, 47 kDa, 48 kDa, 49 kDa, 50 kDa, 51 kDa, 52 kDa, 53 kDa, 54 kDa, 55 kDa, 56 kDa, 57 kDa, 58 kDa, 59 kDa, 60 kDa, 61 kDa, 62 kDa, 63 kDa, 64 kDa, 65 kDa, 66 kDa, 67 kDa, 68 kDa, 69 kDa, 70 kDa, 71 kDa, 72 kDa, 73 kDa, 74 kDa, 75 kDa, 76 kDa, 77 kDa, 78 kDa, 79 kDa, 80 kDa, 81 kDa, 82 kDa, 83 kDa, 84 kDa, 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, 95 kDa, 96 kDa, 97 Molecules of kDa, 98 kDa, 99 kDa, or about 100 kDa.

[0175] PNPHO The preferred polymer of the present invention is poly(NIPAAm-co-NAS-co-(PLA / HEMA)-co-OEGMA), i.e., "PNPHO", for example, formula (I). The polymer PNPHO preferably contains about 1 mol% to about 15 mol% of OEGMA, about 5 mol% to about 50 mol% of PLA / HEMA, up to 15 mol% of NAS, and the remainder to 100% of the polymer composition (e.g., about 50 mol% to about 85 mol%) of NIPAAm.

[0176] The percentages described herein relate to the composition of the final polymer, not to the amount of feed used to form the polymer.

[0177] In one embodiment, preferably, the polymer comprises: about 3 mol% to about 8 mol% (e.g., about 4 mol% to about 6 mol%) of OEGMA; about 5 mol% to about 9 mol% (e.g., about 6 to about 8 mol%) of HEMA-PLA; at least about 7 mol% of NAS; and up to about 85 mol% (e.g., up to about 81 mol%) of NIPAAm.

[0178] In another embodiment, the polymer comprises: about 5 mol% of OEGMA; about 7 mol% of HEMA-PLA; about 7 mol% of NAS; and about 81 mol% of NIPAAm.

[0179] The preferred form of the polymer PNPHO used in this application is the polymer of formula (I), as shown above.

[0180] Based on the previously defined formula I: A is oligo(ethylene glycol) monomethyl ether methacrylate OEGMA; B is hydroxyethyl methacrylate poly(lactic acid) (HEMA-PLA); C is N-acryloyloxysuccinimide (NAS); and D is N-isopropylacrylamide (NIPAAm).

[0181] The preferred form of the polymer PNPHO used in this application is a polymer of formula (I) as shown above. Furthermore, x is in the range of 1-1000 and y is in the range of 1-1000, and m, n, p and q are in the range of 1-20.

[0182] Those skilled in the art will recognize that monomers A, B, C and D can be present in the polymer in any order, provided that the desired water binding, reinforcement and / or crosslinking ability is achieved.

[0183] PPHO Another preferred polymer of the present invention is poly(NIPAAm-co-(PLA / HEMA)-co-OEGMA), i.e., "PPHO", for example, formula (II). The polymer PPHO preferably comprises about 1 mol% to about 15 mol% of OEGMA, about 5 mol% to about 50 mol% of PLA / HEMA, and the remainder is made up to 100% of the polymer composition (e.g., about 50 mol% to about 85 mol%) of NIPAAm.In a preferred embodiment, PPHO comprises about 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or about 15 mol% of OEGMA and / or about 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%. mol%, 47 mol%, 48 mol%, 49 mol%, or about 50 mol% PLA / HEMA and / or about 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, 60 mol%, 61 mol%, 62 mol%, 63 mol%, 64 mol%, 65 mol%, 66 mol%, 67 mol%, 68 mol%, 69 mol%, 70 mol%, 71 mol%, 72 mol%, 73 mol%, 74 mol%, 75 mol%, 76 mol%, 77 mol%, 78 mol%, 79 mol%, 80 mol%, 81 mol%, 82 mol%, 83 mol%, 84 mol%, or about 85 mol% of NIPAAM.

[0184] The percentages mentioned herein relate to the composition of the final polymer, not to the amount of feed used in forming the polymer.

[0185] The preferred form of the polymer PPHO used in this application is a polymer of formula (II), as shown below. Furthermore, x is in the range of 1-1000 and y is in the range of 1-1000, and m, n, and q are in the range of 1-20.

[0186] Those skilled in the art will recognize that monomers A, B, C, and D can be present in the polymer in any order, provided that the desired water-binding, reinforcing, and / or crosslinking capabilities are achieved.

[0187] Polymer Synthesis Those skilled in the art will know suitable methods for synthesizing the preferred polymers used in this invention.These include, for example, ring-opening polymerization, addition polymerization (including free radical polymerization), and condensation polymerization methods, as described on pages 18 / 33 of the specification, CN 121752616 A.

[0188] The following examples describe the formation of preferred polymers PNPHO and PPHO.

[0189] Excipients and Bioactive Agents The compositions and / or polymers of the present invention may include pharmaceutically acceptable excipients, and include any and all solvents, dispersion media, inert diluents or other liquid solvents, dispersing or suspending agents, granulators, surfactants, disintegrants, isotonic agents, thickeners or emulsifiers, preservatives, binders, lubricants, buffers, oils, etc., to suit a desired specific dosage form. Remington (Gennaro, A.R., Remington: The Science and Practice of Pharmacy, 21st Ed (2006) Lippincott Williams & Wilkins) discloses various excipients for formulating pharmaceutical compositions and known techniques for their preparation. Unless any conventional excipient is incompatible with a substance or its derivatives, for example by producing any undesirable biological effects or otherwise interacting harmfully with any other component of the pharmaceutical composition, its use is considered within the scope of this invention.

[0190] Excipients such as colorants, coating agents, sweeteners, flavorings, and fragrances may be present in the composition, as determined by the formulation person.

[0191] Bioactive agents or pharmaceutical compounds that may be added to the compositions and / or polymers of this invention include proteins, glycosaminoglycans, carbohydrates, nucleic acids, and inorganic and organic bioactive compounds such as enzymes, antibiotics, antitumor agents, local anesthetics, hormones, angiogenic agents, antiangiogenic agents, growth factors (e.g., insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), and transforming growth factor-b (TGFb)), antibodies, neurotransmitters, psychoactive drugs, anticancer drugs, chemotherapeutic drugs, drugs affecting reproductive organs, genes, and oligonucleotides.

[0192] Compositions containing multiple components, such as excipients and / or bioactive agents, can be prepared by combining the polymer of the present invention with NSPP, then combining the polymer with one or more other components, and then freeze-drying the resulting composition. This produces a ready-to-use polymer.

[0193] The amounts of polymer, NSPP, and bioactive agents present in the composition will necessarily depend on the specific drug and the condition to be treated. Those skilled in the art will recognize suitable reagents and amounts for treating the condition.

[0194] Compositions for forming hydrogels The present invention also relates to preferred compositions for forming the hydrogels used in the present invention.

[0195] The compositions of the present invention comprise a polymer and NSPP, the polymer comprising: a first water-binding monomer; and a second monomer imparting mechanical properties; optionally, a third monomer, the third monomer being an NSPP-binding monomer comprising functional groups capable of binding to NSPP; a fourth monomer capable of imparting hydrogel phase transition properties; wherein the natural or synthetic peptide or protein (NSPP) is thymosin β-4 or a functional homolog thereof; and wherein the binding of NSPP to the second monomer crosslinks the polymer, thereby enabling the composition to form a hydrogel upon contact with water.

[0196] As used herein, the term “composition” refers to a solid or liquid composition containing the above-described components. In some embodiments, the preferred compositions used in the present invention may also include other components, such as pharmaceutically acceptable excipients and bioactive agents (e.g., drugs, vitamins, and minerals), to aid in the repair and / or regeneration of target bone tissue, and / or to provide a method for targeted delivery of bioactive compounds.

[0197] Generally, the amount of polymer in the compositions used in the present invention is the amount required to achieve hydrogel formation. Specification 19 / 33 pages 23 CN 121752616 A

[0198] In some embodiments, the amount of polymer in the composition ranges from about 1% w / w to about 90% w / w, about 2% w / w to about 80% w / w, about 4% w / w to about 70% w / w, about 5% w / w to about 60% w / w, about 5% w / w to about 50% w / w, about 6% w / w to about 40% w / w, about 7% w / w to about 30% w / w, or about 8% w / w to about 20% w / w.

[0199] In some embodiments, the amount of polymer is: about 1% w / w, about 2% w / w, about 3% w / w, about 4% w / w, about 5% w / w, about 6% w / w, about 7% w / w, about 8% w / w, about 9% w / w, about 10% w / w, about 15% w / w, about 20% w / w, about 25% w / w, about 30% w / w, about 35% w / w, about 40% w / w, about 45% w / w, about 50% w / w, about 55% w / w, about 60% w / w, about 65% w / w, about 70% w / w, about 75% w / w, about 80% w / w, or higher. In some embodiments, the amount of polymer is about 85% w / w.

[0200] Generally, the solidity of the hydrogel increases with increasing polymer concentration in the composition.

[0201] Typically, the amount of NSPP in the composition of the present invention is the amount that achieves hydrogel formation.

[0202] In some embodiments, the amount of NSPP in the composition ranges from about 0.01% w / w to about 60% w / w, about 1% w / w to about 50% w / w, about 1% w / w to about 40% w / w, about 5% w / w to about 30% w / w, about 5% w / w to about 20% w / w, or about 5% w / w to about 10% w / w.

[0203] In some embodiments, the percentage of NSPP is about 1% w / w, about 2% w / w, about 3% w / w, about 4% w / w, about 5% w / w, about 6% w / w, about 7% w / w, about 8% w / w, about 9% w / w, about 10% w / w, about 20% w / w, about 30% w / w, about 40% w / w, about 50% w / w, or higher.

[0204] % w / w is based on the total weight of the composition before contact with water.

[0205] The polymer system serves as a carrier capable of loading mRNA and lipid nanoparticles (LNPs) and enhancing their structural and functional stability.

[0206] Standard laboratory equipment was used throughout the study. Nasal epithelial cells RPMI 2650 were purchased from ATCC (human squamous cell carcinoma; CCL-30). 1×MEM (minimum essential medium) was from Gibco (Ref. 11095-080, lot number 2444918). 1×Opti-MEM™ I serum-depleted medium (phenol red-free) was from Gibco (Ref. 11058021). Lipofectamine™ MessengerMAX™ transfection reagent was from ThermoFisher Scientific (Ref. LMRNA008). Clear, flat-bottomed, non-sterile 98-well immunochromatographic plates were from ThermoFisher Scientific. 6.5 mm Transwell® plates with 5.0 µm pores of sterile polycarbonate membrane inserts were from Corning (Ref. 3421). Spectramax iD3 multimode microplate reader was from Molecular Devices (USA). Gamma-ray sterilized PNPHO powder containing 81 mol% NIPAAm and 7 mol% PLA / HEMA was used. n=5, 7 mol% NAS and 5 mol% OEGMA.Gamma-ray sterilized PNPHO polymer (dialyzed to PBS via a 5 kDa cutoff membrane), TriLink Biotechnologies CleanCap EGFP mRNA (catalog number: L-7601), 1× PBS (phosphate-buffered saline) from Sigma-Aldrich (Ref. 806552, lot number RNBL1507), NanoDrop® 2000 spectrophotometer, Thermo Scientific™ 96-well black / transparent substrate, TC surface (catalog number: 165305).

[0207] The analysis was performed using a range of devices and apparatus, including a Molecular Devices Spectramax iD3 multimode microplate reader, Bio-Rad Gel DºC™ EZ Gel DºCumentation System, nvitrogen™ SYBR™ Green II RNA gel staining agent 10,000X concentrate (in DSMO*) (catalog number: S7568), Bioline 5× DNA loading buffer blue (catalog number: BIO-37045), Bioline agarose, general 100 g, powder, pure agarose, gel electrophoresis (catalog number: BIO-41025), 10× TAE buffer (0.4 M Tris, 0.01 M EDTA and 0.2 M acetic acid).

[0208] Preparation and Coding of TP and TL: PNPHO, dialyzed PNPHO polymer and / or PPHO, dialyzed PPHO polymer are dissolved in PBS and / or other buffer solutions (including Hartsman's DMEM, DPBS or other buffer solutions suitable for mRNA formulations) at 2 to 10 °C to obtain a clear single-phase solution. The resulting solution is coded as "TPXXX" (PNPHO) or "TLXXX" (PPHO), where the number "XXX" indicates the mass concentration of the polymer in the buffer solution. For example, TP050 indicates a concentration of 50 mg / mL of PNPHO polymer in PBS.

[0209] TP100 and TL100 are prepared using 1 × PBS or Opti-MEM medium and gently mixed overnight at 4 °C. The TP100 and TL100 solutions are stored at 4 °C until use. On the day of the experiment, the mRNA is mixed with an appropriate volume of TP100 and / or TL100 solution and PBS or Opti-MEM to create the desired TP or TL solution concentration.

[0210] For mRNA analysis, 10 μL of mRNA standards (1.95–4000 ng (final amount)) were loaded into a 2% (w / v) agarose gel incorporating SYBR™ Green II RNA gel dye at a ratio of 1:10000. The gel was run at 90 V for 40 min and imaged using the Gel DºC™ EZ Gel DºCumentation System in strong and weak band modes. The gel bands were quantified using ImageJ software. Quant-iT™ Ribogreen RNA assays were performed according to the manufacturer’s protocol (https: / / www.thermofisher.com / document-connect / document-connect.html?url=https: / / assets.thermofisher.com / TFS-Assets%2FLSG%2Fmanuals%2Fmp11490.pdf).

[0211] mRNA stability study 4°C – Integrity and content and nanoparticle potential: mRNA stability studies of RNA integrity and content were conducted using an Aptar VP7 5 mL nasal spray bottle, in which PBS-mRNA or TP-mRNA formulations were stored at 4°C and samples were taken daily (sprayed twice in a falcon tube and centrifuged at 100 × g for 5 minutes) for 5 days. mRNA content was analyzed by agarose gel electrophoresis. Nanoparticle potential was studied using a Zetasizer to measure whether any nanoparticles were detected in the formulation.

[0212] mRNA stability study 37°C – Integrity and content: mRNA stability studies of RNA content were conducted using 10 mL glass vials, in which PBS-mRNA or TP-mRNA formulations were stored at 37°C and samples (100 µL) were taken immediately (day 0), after 24 h (day 1), and after 96 h (day 4). After incubation at 37 °C, samples of the liquid supernatant formed on top of the hydrogel in the TP50 formulation were taken on days 1 and 4 to determine whether the mRNA was completely captured in the hydrogel layer or dispersed throughout the gel and liquid components. Samples were collected at designated times, and the gel was reconstituted into solution by incubation in a refrigerator for 1 h. Aerosol collection was then performed as described herein by direct pipetting and transfer to a VP7 Aptar pump. After sampling, the formulation was returned to the 37 °C glass vial for continued incubation. Samples were analyzed using agarose gel electrophoresis.

[0213] mRNA release studies of TP or TL formulations were conducted by placing Transwell inserts (5 µm, polycarbonate membrane) containing PBS-mRNA, TP-mRNA, or TL-mRNA formulations (100 µL) into 24-well plates and adding 0.5 mL of physiological saline to the basal chamber to measure sustained mRNA release. The plates were then incubated at 37°C. 100 µL samples were collected from the basal chamber at different time points, and an equal volume of fresh, preheated PBS was added after each sample collection. The assay was performed for up to 180 h. The collected samples were analyzed by the Quant-iT™ Ribogreen RNA assay to quantify the mRNA released by the formulation. These experiments were performed using a liquid dialysis polymer provided by the applicant.

[0214] mRNA-TP formulation transport studies via nasal epithelium were conducted by establishing nasal epithelium by growing RPMI2650 cells in an air-liquid interface culture using Transwell 24-well plates (5 µm, polycarbonate membrane). Using a micropipette, PBS-mRNA, TP-mRNA, or TL-mRNA formulation (10 μg mRNA in 100 µL) was deposited onto the nasal epithelium, with 500 µL of PBS in the basal chamber. 100 µL samples were collected from the basal chamber at different time points, and an equal volume of fresh, preheated PBS was added after each sample collection. After 4 h, the tip surface was washed with 400 µL of PBS to collect the formulation remaining on the nasal epithelium (“ON”). Quantification of “IN (intraepithelial)” samples was not attempted because cellular mRNA would interfere with EGFP mRNA quantification. mRNA quantification was performed using the Quant-iT™ Ribogreen RNA assay, page 21 / 33, CN 121752616 A, using the liquid dialysis polymer provided by the applicant.

[0215] mRNA function studies were performed using time-lapse microscopy to assess the function of EGFP mRNA by the production of green fluorescent protein (GFP) by cells. Using the RPMI 2650 basic cell model, cells were cultured in 24-well plates for 24 h. The cell culture medium was then replaced with Opti-MEM medium and incubated at 37°C for 2 h. The culture medium was then replaced again with the following: i. Control – 500 µL of fresh Opti-MEM medium; ii. mRNA alone – 500 ng of mRNA diluted to 500 µL with fresh Opti-MEM medium; iii. mRNA containing lipofectamine – 500 ng of mRNA mixed with 0.75 µL of lipofectamine and diluted to 500 µL with fresh Opti-MEM medium.

[0216] iv. mRNA containing TP025 / TL025—500 ng of mRNA was diluted to 500 µL with Opti-MEM medium containing 25 mg / mL PNPHO / PPHO.

[0217] v. mRNA containing Lipofectamine and TP025—500 ng of mRNA was mixed with 0.75 µL of lipofectamine and diluted to 500 µL with fresh Opti-MEM medium containing 25 mg / mL PNPHO. The corresponding mixtures for the PPHO polymer were obtained.

[0218] The 24-well plates were kept in a humidified chamber at 37°C, 5% CO2 atmosphere and 95% humidity and observed using a Nikon Eclipse Ti microscope (Nikon, Tokyo, Japan) with a Coolsnap ES2 camera. Images were taken every 2 h for 48 h using NIS-Elements (version 3.22.01) set to capture images in “phase” and “fluorescence” modes. Images were analyzed using Fiji ImageJ. A normal TP batch (STR03-075-2022-02-14-01) was used in this study.

[0219] Direct Exposure – mRNA Function Assay Using a Microplate Reader A microplate reader assay has been developed to measure the potency of EGFP mRNA. EGFP mRNA in RPMI cells was assessed by the green fluorescent protein (GFP) produced by the cells. Cells were cultured in 24-well plates for 24 h using an RPMI2650 basal cell model. The cell culture medium was replaced with Opti-MEM medium and incubated at 37°C for 2 h. Then, the medium was replaced with the following: i. Negative control – 0.75 µL of Lipofectamine diluted to 500 µL in fresh Opti-MEM medium ii. Positive control – 500 ng of mRNA was mixed with 0.75 µL of lipofectamine and diluted to 500 µL in fresh Opti-MEM medium.

[0220] iii. TP015 formulation—500 ng of mRNA was mixed with 0.75 µL of lipofectamine and diluted to 500 µL with fresh Opti-MEM medium containing 25 mg / mL polymer. The corresponding TL / PPHO formulation was prepared.

[0221] iv. TP035 formulation—500 ng of mRNA was mixed with 0.75 µL of lipofectamine and diluted to 500 µL with fresh Opti-MEM medium containing 35 mg / mL polymer. The corresponding TL / PPHO formulation was prepared.

[0222] After incubation at 37°C for 24 h in a 5% CO2 atmosphere, the microplates were read using a Spectramax iD3 microplate reader. The formulation was removed from the wells and washed once with 500 µL of cold PBS, and then read again (to eliminate any interference from TP / TL on the fluorescence signal).

[0223] Parallel mRNA function assays were performed using nanoparticle measurements. This study aimed to investigate whether TP / TL formulations containing mRNA and lipofectamine could form nanoparticles, and whether these nanoparticles could maintain the efficacy of the mRNA. Cells were cultured in 24-well plates for 24 h using an RPMI 2650 basal cell model. The cell culture medium was then replaced with Opti-MEM medium and incubated at 37°C for 2 h. Then, the culture medium was replaced with the following from page 22 / 33 of the instruction manual, CN 121752616 A: i. Negative control—Lipofectamine 0.75 µL diluted to 500 µL with fresh Opti-MEM medium ii. Positive control—500 ng mRNA was mixed with 0.75 µL lipofectamine and diluted to 500 µL with fresh Opti-MEM medium.

[0224] To indirectly expose the cells to the formulation, the culture medium on the cells was replaced with fresh Opti-MEM medium, and a Transwell insert (5 µm, polycarbonate membrane) was placed on top of the well carrying the following formulations: (i) TPO formulation—500 ng mRNA was mixed with 0.75 µL lipofectamine and diluted to 100 µL with fresh Opti-MEM. The corresponding TL / PPHO formulations were prepared.

[0225] (ii) TP015 formulation—500 ng of mRNA was mixed with 0.75 µL of lipofectamine and diluted to 100 µL with fresh Opti-MEM containing 15 mg / mL polymer. The corresponding TL / PPHO formulation was prepared.

[0226] (iii) TP030 formulation—500 ng of mRNA was mixed with 0.75 µL of lipofectamine and diluted to 100 µL with fresh Opti-MEM containing 30 mg / mL polymer.

[0227] Parallel 24-well plates were also set up for this cell experiment under identical conditions but without cells. After incubation at 37°C for 24 h in a 5% CO2 atmosphere, the Transwell insert was removed from all wells and the cell plates were measured using a fluorescent plate reader. The culture medium in the non-cell plates was analyzed using a Zetasizer.

[0228] Data are presented as mean ± standard deviation (STDev) of three independent experiments.Statistical analysis was performed using Prism software version 9.4.0 (GraphPad, San Diego, USA).

[0229] Results: The stability of 5 mRNA stored at 4°C and atomized using the VP7 Aptar nasal pump was analyzed by agarose gel electrophoresis to investigate the effect of adding PNPHO or PPHO on mRNA stability during storage at 2 to 10°C. For this analysis, TP005, TP010, and TP015 were prepared using 5 mg / mL, 10 mg / mL, and 15 mg / mL of PNPHO, respectively. The corresponding TL / PPHO formulations were prepared. The results showed that a concentration-dependent signal of PNPHO was observed in the gel near the wells, but it did not interfere with the mRNA signal, and aerosolization with the VP7 Aptar nasal spray did not cleave the mRNA.

[0230] The addition of low concentrations of PNPHO or PPHO, 5 mg / mL and 10 mg / mL, resulted in mRNA cleavage and instability, as shown by the diffuse degradation signal in Figure 2b. However, further addition of PNPHO polymers enhanced the stability of mRNA, as very clear and stable bands were observed in the formulation containing 15 mg / mL PNPHO (TP015). The amount of mRNA also remained stable close to 100% using TP015 (Figure 2c). These results confirm that at higher concentrations, the PNPHO polymer chains and their hydrophilic / hydrophobic interactions in the formulation provide protective properties.

[0231] The results in the figure show that the addition of PNPHO or PPHO to mRNA prevents the spontaneous aggregation of particles, leading to the formation of nanoparticles. This conclusion is drawn because, in the absence of PNPHO, the particle size of mRNA in PBS solution is greater than 100 d·nm, while with the addition of PNPHO, even at low concentrations, mRNA containing TP005, TP010, and TP015 can form nanoparticles (≤ 100 d·nm).

[0232] The results show that low concentrations of PNPHO or PPHO may have an adverse effect on the stability of mRNA, possibly due to the presence of small molecular weight PNPHO / PPHO segments in the formulation. However, at concentrations of 15 mg / mL or higher, the protective properties of PNPHO / PPHO and its role in the formation of mRNA nanoparticles lead to the formation of stable formulations. Therefore, for further research, polymer concentrations of 15 mg / mL or higher of PNPHO / PPHO were used.

[0233] The stability of mRNA at 37°C was further challenged at elevated temperatures based on the results of studies on the stability of mRNA with different concentrations of PNPHO / PPHO polymers.Therefore, mRNA formulations containing 15 mg / mL and 35 mg / mL PNPNO and PPHO were tested, according to the product manual, pages 23 / 33, CN 121752616 A, and mRNA stability was assessed using agarose gel electrophoresis. In the formulation containing 15 mg / mL PNPHO or PPHO, an opaque solution was formed when the temperature was raised to 37°C, while in the formulation containing 35 mg / mL PNPHO, a hydrogel layer was formed with the supernatant on top (Fig. a).

[0234] After incubation at 4°C for 1 h, the formulation was reconstituted, and the sample became a homogeneous solution as expected due to the reversibility of the polymer. No difference in mRNA integrity was observed between direct pipetting and nebulization of the formulation (Fig. a). On day 4, a significant decrease in mRNA content was observed in the original formulation (without polymer). However, the addition of PNPHO / PPHO polymer in the TP015 / TL015 and TP035 / TL015 formulations maintained mRNA integrity. Therefore, it was concluded that there was no significant difference in total mRNA content between day 0 and day 4 (p > 0.05) (Figure b). Furthermore, analysis of the TP035 / TL035 sample (supernatant above the hydrogel) showed the presence of mRNA in solution, indicating that the hydrogel did not selectively capture mRNA, which was dispersed in both the supernatant and the hydrogel.

[0235] Parallel mRNA function assays and nanoparticle analyses were performed using the TP / TL formulation to test lipofectamine-containing mRNA (mRNA + LNP) formulations, incorporating 25 mg / mL and 35 mg / mL of dialyzed or undialyzed PNPHO polymers (or their PPHO equivalents). The formulations containing and without PNPHO were challenged and stored at 37°C to assess the effect of PNPHO addition on the function of the incorporated mRNA. The results showed that dialysis of the PNPHO polymer resulted in the separation of polymer fragment groups, thus leading to different volume percentage groups (as shown in Figure a).

[0236] The effects of adding TP025 and TP035 (formed from dialyzed or undialyzed PNPHO) on eGFP-mRNA function after 24 h of storage were compared with the +control (fresh eGFP+LNP) formulation. The results in Figure b show that the addition of PNPHO polymers maintained mRNA function, as significantly higher (p < 0.001) GFP expression was recorded in both the TP025 and TP035 formulations formed from dialyzed and undialyzed PNPHO compared to the formulations without PNPHO incorporation.

[0237] In particular, formulations containing dialysis-contained PNPHO / PPHO polymers exhibited higher expression; after 24 h of storage, there was no significant difference in GFP expression between TP025 and TP035 and the + control (fresh eGFP-mRNA+LNP) (which is the maximally effective mRNA) (p > 0.05). Therefore, the results of these studies confirm that the mRNA-Lipofectamine-TP (or -TL) formulation can release functional nanoparticles into the culture medium, and the nanoparticles are able to induce the maximally effective mRNA in the basal RPMI2650 cell model and maintain functionality under elevated temperatures and harsher conditions.

[0238] mRNA and LNP delivery challenged the TP (prepared with PNPHO) and TL (PPHO) platform carrier systems as universal therapeutic delivery platforms by using different mRNA-based formulations. In the initial phase, GFP-mRNA was used and mixed with TP035, TP015, and TP007 (and their respective TL equivalents) to systematically investigate the effects and potential of the polymer platform carriers of the present invention. Subsequently, different mixing methods were investigated by changing the order of mRNA, LNP, and TP / TL incorporation, and the effect of the TP / TL vector on LNP encapsulation of mRNA was evaluated. TP050 and TP100 containing eGF-mRNA / LNP (and TL equivalents) were then formulated to ensure compatibility of the mRNA / LNP with higher polymer concentrations, which were expected to be more suitable for long-acting injectable formulations. Then, configurations more useful for nasal delivery were investigated, and the compatibility of the vectors with commercially available COVID-19 vaccines was evaluated. The effect of the vector layer on the bioactivity of the LNP and mRNA formulations was then investigated to ensure that the vectors did not impede their intended function.

[0239] Preservation of mRNA within the TP platform was achieved using eGFP-mRNA mixed with PNPHO solutions at 7 mg / mL, 15 mg / mL, and 35 mg / mL in PBS to obtain TP007+, TP015+, and TP035+, respectively. Equivalent “TLXXX+” formulations containing PPHO were obtained. Figure 11a shows the volume percentage of GFP-mRNA with and without different vector configurations. The results in Figure 11b show that the addition of the vector significantly reduced the Z-Ave (d / nm) value (p < 0.001), regardless of its solids content, i.e., for all three test configurations (p < 0.001). Similarly, the results in Figure 11c show that the polydispersity (Pdi) of the solution was significantly reduced after adding the vector platform (p < 0.01). These results indicate that the TP / TL platform carrier system can uniformly wrap mRNA and exhibits protective performance against mRNA sample aggregation.

[0240] To test the effect of adding a carrier system to mRNA-based therapies, different carrier configurations TP007+, TP015+, and TP035+ (and their TL equivalents) were collected and tested by agarose gel electrophoresis to assess the integrity of the mRNA components (Figure 14). All test samples were stored at 4°C for 5 days prior to analysis. During testing, the pH of all solutions was maintained at approximately 7.3. After spraying, TP007+ exhibited some degree of bubbling. Gel electrophoresis of the solutions using Sybr Green II staining showed good mRNA detection range; however, the carrier components were also stained on the larger molecular weight bands (Figure 14a). Nevertheless, the separation of the mRNA bands was effective and could be used to establish a standard curve for further quantification. The results in Figure 14b show significant mRNA cleavage in the TP007+ sample, while the degree of cleavage was not significant in the TP015+ and TP035+ samples. This result indicates that the protective properties of the polymer carrier are concentration-dependent. Therefore, the protective properties of the TP platform for mRNA are attributed to the charge of the polymer and its self-assembly ability at concentrations above its CMC.

[0241] To further evaluate the protective properties of the TP platform, TP015 and TP035 configuration + mRNA (and its TL equivalent) were challenged by raising the storage temperature to 37°C for 4 days. The integrity of the mRNA components was quantified by gel electrophoresis and compared with the original mRNA content. Samples were collected from the stored solution (before spraying) and after spray / hydrogel formation. The results in Figure 12 show that no shearing was observed in the integrity of mRNA in the control (without carrier), TP015, and TP035 after 0 hours and 24 hours of storage at 37°C. However, after 4 days of storage at 37°C, the integrity of mRNA samples containing carriers TP015 and TP035 was significantly higher (p < 0.001) than that of the control. For example, after 4 days of challenge, about 60% of the mRNA was present in the control group, while almost 100% of the mRNA was detected in the TP015 and TP035 groups. These results confirm that the TP / TL platform has protective properties against temperature fluctuations, thus reducing the stringency of storage conditions required for mRNA-based vaccines.

[0242] Incorporation of the TP / TL platform into mRNA / LNP formulations To evaluate the effect of incorporating the TP / TL platform into the mRNA / LNP production process, e-GPF mRNA containing lipofectamine was used as a model system. Quantification of mRNA was achieved by developing a GPC-MS (gel permeation chromatography) method and establishing a standard curve with R² = 0.99 (Figure 13a). This method was used to assess the amount of mRNA encapsulated within the LNP relative to the amount of leakage / leakage at different time points.Subsequently, the TP / TL platform was incorporated into the mRNA / LNP production process using three methods, and the results were compared with formulations without the polymer platform (TP000 / TL000; control). To incorporate the TP platform, a stock solution of TP100 was used, and after mixing it with eGFP-mRNA / LNP, the polymer platform concentration was reduced to 35 mg / mL, thus the formulation was labeled TP035+. These formulations included: Standard (TP000; control): Lipofectamine solution was mixed with mRNA to form eGFP-mRNA / LNP (Fig. 13b(i)).

[0243] Formulation (A): eGFP-mRNA / LNP was mixed for 5 minutes, followed by the addition of TP100 to form the final mixture (Fig. 13b(ii)).

[0244] Formulation (B): Lipofectamine was mixed with TP100, followed by the addition of eGFP-mRNA to the mixture (Fig. 13b(iii)).

[0245] Formulation (C): mRNA was added directly to the TP100 solution, followed by the addition of lipofectamine to form the final mixture (Fig. 13b(iv)).

[0246] Within 2 hours of formulation, the amount of mRNA leaking / leaking from the LNP was quantified and stored at 2–8°C to measure the encapsulation efficiency of the final formulation. The results showed that the control formulation had a relatively high encapsulation efficiency, with 81.2 ± 1.2% of the incorporated mRNA still encapsulated in the LNP (Fig. 13b(i)). However, by incorporating the TP platform into formulation A by mixing the formed mRNA / LNP with the TP platform, the encapsulation efficiency was significantly (p < 0.05) increased to 86.4 ± 2.1% (Fig. 13b(ii)). Subsequently, lipofectamine was mixed with the TP platform before inclusion in the mRNA specification (pages 25 / 33, CN 121752616 A) to further improve the encapsulation efficiency (Formulation B) to 92.4 ± 1.9% (Figure 13b (iii)). Finally, the mRNA was added directly to TP100 and then mixed with LNP, slightly improving the encapsulation efficiency to 95.7 ± 0.9%, with a p-value of 0.0491 (Figure 13b (iv)).

[0247] These results further confirm the compatibility of the PNPHO / PPHO polymer platform with mRNA and LNP formulations and can improve the stability and encapsulation efficiency of the final formulation. Next, the method of Formulation A was used because it directly incorporates the TP carrier system into the pre-prepared mRNA / LNP formulation (whether commercially available or under development). Therefore, although the results showed that Formulations B and C could achieve higher mRNA encapsulation efficiencies, the former was preferred due to its ease of production.

[0248] TP / TL formulations with different administration routes were prepared using the mixing method of formulation A (described above), with PNPHO polymers at concentrations of 35 mg / mL, 50 mg / mL, and 100 mg / mL to prepare TP035+, TP050+, and TP100+ eGFP mRNA / LNP, respectively. The effect of the TP carrier on the particle size and stability of the formulations after storage at 2–8°C for 3, 7, and 14 days was investigated using DLS and GPC analysis techniques.

[0249] The results in Figure 15 show that at t=0, the particle size of the control mRNA / LNP formulation (TP000) was 88.7 ± 8.2 nm. The incorporation of the TP carrier significantly increased (p < 0.05) the particle sizes of TP035+, TP050+, and TP100+ to 123.9 ± 8.0 nm, 143.1 ± 4.0 nm, and 180.8 ± 9.7 nm, respectively. The results also showed that mixtures of TP and TL carrier systems could be formulated. Therefore, the TP035+ / TL050+ system exhibited a similar particle size to TP035+ (no statistical difference, p > 0.05).

[0250] This result further confirms that the TP and / or TL platforms form a protective layer around the compound due to their negative charge and hydrophilic / hydrophobic interactions. Regarding particle Pdi measurements, all configurations showed relatively stable Pdi, within a general range of 0.2–0.4. There was no statistically significant difference in Pdi measurements between the presence and absence of the TP / TL carrier; a slight increase in Pdi was observed in TP100 compared to TP000, attributed to the increased temperature during DLS measurements and the temperature-responsive nature of the carrier system.

[0251] In all formulations, the protective effect of the TP / TL carrier was evident after storage at 2–8°C for 3 days. DLS and GPC results showed that mRNA / LNP formulations ruptured in the absence of the vector, as evidenced by the <10 nm particle size and ~0% encapsulated mRNA content of TP000. After 3 days of storage, the encapsulated mRNA content in all formulations containing TP, TP / TL, and TL vectors was significantly higher (p < 0.001) than that in TP000. The encapsulated mRNA content in TP035+, TP050+, TL050+, TP035 / TL050+, and TP100 was ~70% to 80% (p > 0.05). Particle size measurements showed that after 3 days of storage, the increase in PNPHO / PPHO polymer content led to an increase in particle size from 71.41 ± 8.0 nm in TP035 to 117.5 ± 4.3 nm in TP050 and 167.5 ± 4.6 nm in TP100.

[0252] Similar results were obtained after incubation at 2-8°C for 7 days.In summary, the GPC results confirmed that dissociation of LNP / mRNA particles progressed in the control group, and no LNP / mRNA particle peaks appeared in the GPC results after 7 days of storage. However, in all three test formulations, mRNA / LNP containing TP, TL, or TP / TL vectors remained stable.

[0253] Long-term stability of formulations: The evaluation of the encapsulated mRNA content and particle size measurements of different formulations stored at 2-8°C for up to 14 days in Figure 16 confirmed that the presence of the vector system improved the stability of the formulations. TP035 was the most stable formulation, with more than 70% of the mRNA content still encapsulated after 14 days of formulation preparation and storage at 2-8°C. After 14 days, more than 50% of the mRNA content was still encapsulated; however, the particle size measurements at this time showed some degree of degradation.

[0254] This part of the results further confirms the findings in the preceding section and shows that the presence of the TP vector improves the stability of the mRNA / LNP formulations. Furthermore, PNPHO was successfully increased to 100 mg / mL, and even at such a high concentration, the mRNA / LNP ratio was more stable than that of formulations without a carrier system. The more stable properties of formulations with higher PNPHO content (including TP050 and TP100) are beneficial for the formulation of long-acting injectables in future studies.

[0255] Effect of Polymer Platform on mRNA and LNP Function To investigate the effect of TP035 on mRNA and LNP function, eGFP-mRNA was used, and GFP expression on epithelial cells was studied at different time points by live-cell imaging for up to 48 hours. This study was used to assess whether the addition of TP035 and its hydrophilic / hydrophobic interactions with LNP had any adverse effects on mRNA and LNP function. The results in Figures 17a(i) and 17a(ii) show that eGFP without LNP did not exhibit GFP expression characteristics on epithelial cells. As expected, the addition of TP035 did not affect eGFP expression (Fig. 17a(iii)). However, the addition of lipofectamine to eGFP-mRNA + TP035 resulted in expression in cells, as shown in Fig. 17a(iv) and Fig. 17b. The expression intensity observed in Fig. 17b(ii) was comparable to that achieved without the TP035 vector. The results confirm that the addition of TP035 did not adversely affect the effectiveness of eGFP-mRNA and the incorporated lipids.

[0256] In vitro human model nasal delivery of commercial formulations Fig. 18 plots the deposition patterns of a) a vector-free system and b) a TP035+-containing commercial vaccine formulations in a simulated human nasal model. In this in vitro evaluation, the test formulation was sprayed into a nasal model incubated at 37°C using a standard nozzle.In the control group (TP000), significant nasal outflow was observed immediately after application; this equates to ~60% of the spray volume or 150 µL of a 250 µL spray volume (Fig. 18a(i) and Fig. 18a(ii)). This significant volume loss and minimal reach of the formulation to the upper nasal cavity (Fig. 18a(iii)) are known drawbacks of nasal delivery, for example, of standard formulations without a carrier system. On the other hand, no nasal outflow was observed with the application of the TP035+ formulation (Fig. 18b(i)). Despite the presence of a carrier system in the TP035+ formulation and its relatively higher viscosity compared to the control group, aerosolization was successful, allowing the formulation to deposit on a larger surface area (Fig. 18b(ii) and Fig. 18b(iii)). The white arrows show the extensive coverage and rapid adhesion of the TP035+-containing formulation to the upper nasal cavity. The adhesiveness and immediate gelation of the carrier system prevented immediate nasopharyngeal outflow following application of the TP035+ formulation.

[0257] Figure 19a shows the deposition patterns of TP000 (control) and TP035+ formulations within 20 minutes after application. The results show that, due to the adhesiveness of the carrier system, the volume retained in the nasal cavity of the TP035+ group was significantly higher than that of the control group (TP000) at all test time points (i.e., 0, 5, 10, 15, and 20 minutes after application) (Figure 19b). In the TP035+ group, very little (< 10 µL) pharyngeal outflow was observed within 10 minutes after application. More importantly, the aqueous phase discharged during and after gelation of the TP035+ formulation after application caused the area to be gradually covered. Therefore, the nasal surface area of ​​the TP035+ formulation was significantly higher (p < 0.001) at all time points compared to the control.

[0258] These results confirm that the carrier system has a high potential for aerosolization with standard actuators, thereby enabling the formulation to pass through the nasal cavity. The resulting aerosol forms a viscous hydrogel layer, thereby preventing nasopharyngeal outflow. These findings further demonstrate the practicality of this innovative carrier system for nasal delivery applications.

[0259] Polymer nanocarriers pose a significant challenge to the treatment of neurological disorders by targeting the brain due to the presence of the blood-brain barrier (BBB). This can be explained by the tight endothelial cell junctions within the BBB, which prevent most molecules from freely transporting from the bloodstream to the brain. (6) To overcome this physiological barrier and enable the effective delivery of therapeutic molecules to the brain, intranasal administration of drugs is a suitable solution. This mechanism manipulates the olfactory and trigeminal nerves to transport drugs to the brain by bypassing the BBB. (7) PNPHO and PPHO are advanced biomaterials that can self-assemble and form nanocomposites to achieve more efficient and targeted naso-brain drug delivery. In this study, nanocarriers for drug loading were developed to enhance the penetration and delivery of therapeutic agents to the brain.These NPs, comprising two biocompatible polymers, were formulated in nasal formulations containing BSA (as a model drug) to bypass the BBB.

[0260] The determination of the critical micelle concentration (CMC) was used to assess the concentration-driven micelle formation potential of the PNPHO polymer (Figure). To determine the CMC of PNPHO, the PNPHO polymer was serially diluted from 25 mg / mL. A PNPHO stock solution was prepared by dissolving the polymer in phosphate-buffered saline (PBS) at pH 7.4. Subsequently, the stock solution and all diluted polymer samples were analyzed using dynamic light scattering (DLS) technology (n=3), a method established by Malvern Instrument Limited (UK) that is well-suited for determining the CMC.

[0261] Nanoparticles (NPs) made from anionic PNPHO and the cationic polymer chitosan were produced using composite aggregation. This manufacturing technique was employed to improve the stability of the NPs and to encapsulate the charged drug within the nanocomposite. First, negatively charged bovine serum albumin (BSA, a model treatment agent) was added dropwise to a PNPHO solution (0.5 mg / mL) at a concentration of 1 mg / mL. Then, nanoparticles were created using chitosan by magnetically mixing chitosan (1 mg / mL; pre-dissolved in 1% acetic acid) and PNPHO at ratios of 1:5, 1:1, and 5:1 to determine the optimal polymer:polymer ratio for forming the nanocarrier. A control formulation was also prepared in the absence of BSA loading.

[0262] The physicochemical properties of BSA-loaded NPs and control NPs were investigated using a Malvern Zetasizer (Worcestershire, UK) in terms of particle size, polydispersity index (PDI), and surface charge.

[0263] To quantify the encapsulation efficiency of BSA-loaded NPs, freshly prepared PNPHO:chitosan 1:1 samples were used to encapsulate BSA at three different concentrations (including 10, 500, and 1000 µg / mL). These formulations were loaded into an Amicon® filter (MWCO 30 kDa) and centrifuged at 13.3 k rpm for 15 minutes at 4 °C. The filtrate was analyzed by high performance liquid chromatography (HPLC) (Shimadzu, Japan) using a Symmetry™ C18 column (4.6 mm × 5 µm × 250 mm) from Phenomenex (California, USA).

[0264] The droplet size of the NPs sprayed from the Aptar VP7 nasal spray device was measured using laser diffraction.Prior to measurement, Malvern Panalytical Spraytec (Worcestershire, UK) was equilibrated at room temperature. 3 mL of the optimized pharmaceutical formulation (PNPHO:chitosan 1:1, containing 1000 µg / mL BSA) was loaded into the nasal pump for analysis. Upon startup, the device was initiated at a 45-degree angle (n=3) along the laser beam direction. A blank PNPHO:chitosan 1:1 formulation was also measured as a control.

[0265] To observe the spray patterns of the two BSA-loaded PNPHO:chitosan 1:1 NPs, a silicone nasal mold (Koken Co. Ltd., Bunkyo-ku, Tokyo, Japan) was used. An Aptar VP7 nasal spray pump was used to initiate a single spray to deposit the formulation into the nasal mold. Before the experiment, a thin layer of Sar-Gel water indicator paste was uniformly applied to the nasal mold using a clean brush. Images of the nasal mold were captured at 0, 5, 10, and 20 min.

[0266] All deposition studies were performed using an optimized BSA-loaded PNPHO:chitosan 1:1 NP nanoformulation pre-loaded into an Aptar VP7 nasal spray device with a minimum volume of 3 mL. To determine the drug deposition pattern, the Aptar VP7 device was initiated three times in in vitro nasal models, including a USP-approved nasal glass chamber and an Alberta Idealised Nasal Inlet (AINI). The flow rate was set to 15 L / min to represent nasal airflow. The amount of BSA deposited in each representative region was measured using the QuantiPro™ BCA assay kit (Sigma, Australia).

[0267] USP-approved nasal glass chamber. The percentage of drug deposition in the nanoformulation at initiation was evaluated using an FDA-approved glass chamber device (Copley, UK). The nasal glass chamber was attached to a next-generation impactor (NGI). Following initiation, the drug in the nasal spray was injected into the nasal model three times. At the end of the experiment, the drug deposited in the glass chamber was collected with 25 mL of Milli-Q, while 5 mL of Milli-Q water was added to the pharynx and NGI platform.

[0268] Alberta Idealised Nasal Inlet (AINI) was used for drug deposition. The deposition pattern of BSA model drugs was evaluated using an NGI equipped with AINI. The Aptar VP7 device was injected with AINI three times. To quantify the amount of BSA deposited in each nasal cavity component and NGI platform, 5 mL of Milli-Q water was used for rinsing, except for the nasopharynx where 10 mL of Milli-Q water was required for the AINI.

[0269] Reconstitution and Activity of Freeze-dried mRNA / LNP / TP The following embodiments tested the activity of the mRNA / LNP / polymer vector system of the present invention during freeze-drying, storage, and reconstitution.

[0270] A formulation containing eGFP mRNA / lipofectamine (labeled TP000) and an equivalent formulation containing 25 mg / mL of PNPHO polymer (labeled TP025+) were prepared. Five control samples (TP000) and ten TP025+ samples, each 200 µL in volume, were prepared. All samples were frozen at -20°C and then freeze-dried at -20°C for 36 hours. After the freeze-drying process, the resulting particles were collected back into their original containers (Eppendorf tubes). The samples without the vector (TP000) and those containing the vector (TP025+) were reconstituted with MilliQ water, and the mRNA content encapsulated after freeze-drying and reconstitution was compared with the original formulation (freshly prepared; without freeze-drying).

[0271] The mRNA encapsulation was quantified using a developed and previously outlined HPLC method, as shown in Figure 20. Results showed that, despite the presence of the carrier system, the freeze-dried particles could still be stored and reconstituted without affecting the overall encapsulation efficiency of the formulation; there was no statistically significant difference between the carrier-containing original formulation (TP025+) and the freeze-dried formulation (TP025+FD).

[0272] The study also confirmed that the freeze-drying process did not affect the particle size and distribution in the formulation; there was no statistically significant difference between the carrier-containing original formulation (TP025+) and the freeze-dried formulation (TP025+FD). The dried formulation could be stored at ambient temperature and reconstituted before application. This method, namely dry storage and in-situ reconstitution, may offer significant benefits for long-term storage and transportation of the product.

[0273] Results and Analysis: Determination of CMC: CMC is a key parameter for polymer self-assembly to form micelles for drug encapsulation. In this study, the CMC of the PNPHO polymer reached 0.5 mg / mL due to the initiation of monomer polymer self-aggregation. This is evidenced by low PDI (< 0.5) at PNPHO concentrations of 0.5 mg / mL or higher (Figure). A PDI < 0.5 also indicates the formation of micelles with a uniform particle size distribution. Furthermore, when the CMC of PNPHO was reached, particles with stable particle sizes and hydrodynamic diameters < 250 nm were observed. For other lower concentrations (below 0.5 mg / mL), the presence of unassociated monomers resulted in poor signal-to-noise ratios in DLS detection. As shown in the figure, samples with PNPHO concentrations < 0.5 mg / mL exhibited high PDI (> 0.5) or large particle sizes (> 1000 nm), indicating that the self-assembly process of NPs had not yet begun.

[0274] Based on initial CMC data, a PNPHO concentration of 0.5 mg / mL or higher is expected to be suitable for drug encapsulation resulting from the self-assembly process of NP formation. Surface charge measurements revealed that the PNPHO polymer was negatively charged at all tested concentrations. This surface property allows for subsequent chemical interactions with positively charged polymers, forming drug-encapsulated nanocomposites via electrostatic interactions. Furthermore, the inclusion of cationic polymers in nasal formulations further enhances the stability and integrity of the nanostructures, thereby effectively loading anionic drug molecules.

[0275] Dynamic light scattering analysis: Chitosan was selected as a model polymer with positive charge properties for formulation due to its good tolerability and permeability-enhancing effects. These properties are all important for the development of intranasal drug formulations. Compared to blank NPs, encapsulation with BSA as a model drug in a PNPHO:chitosan (1:1) formulation significantly reduced the particle size (p<0.05) from 313.97 ± 43.71 nm to 112.41 ± 15.27 nm (Figure). This indicates that BSA is a compatible model drug with a stabilizing effect on the nanocomposite.

[0276] For other NP formulations made from PNPHO:chitosan in ratios of 5:1 and 1:5, there was no significant effect on particle size (p > 0.05). Therefore, only PNPHO:chitosan in a 1:1 ratio was selected for further physicochemical and aerodynamic testing.

[0277] When NPs were prepared with different PNPHO:chitosan ratios, the polymer in a 1:1 ratio was found to have the most promising physicochemical properties. This was not observed in other formulations with polymer ratios of 5:1 and 1:5, and the high PDI indicated the formation of highly dispersed particles.

[0278] As expected, the 1:1 ratio of PNPHO:chitosan proved to be the optimal formulation because of the significant reduction in particle size. This result suggests that the addition of BSA produces NPs with a more compact nanostructure, which may be achieved through electrostatic interactions. (12) For NPs prepared at polymer ratios of 1:5 and 5:1 (PNPHO / chitosan), no significant changes in particle size were reported during the encapsulation of the complex with BSA, suggesting that the stabilizing effect of BSA exists only when the optimal ratio of polymer is present in the formulation.

[0279] BSA was chosen to encapsulate the drug into nanocomposites due to the promising performance observed in NPs with a PNPHO:chitosan ratio of 1:1. BSA is a common model protein due to its relatively low cost, easy availability, and charge compatibility. Its potential to form nanocomposites with polymers of opposite charge, such as chitosan, has been described in previous literature.(10), (11) After loading BSA into nanocomposites made of different PNPHO:chitosan ratios, the hydrodynamic diameter of the prepared particles was measured.

[0280] The results in Table 1 on the determination of encapsulation efficiency show that when PNPHO:chitosan was used in a 1:1 ratio for encapsulation of BSA (10, 500 or 1000 µg / mL), the encapsulation efficiency of all formulations was 98-100%. In addition, there was no significant difference in PDI of NP (p>0.05).

[0281] Table 1. Encapsulation efficiency and PDI of PNPHO:chitosan 1:1 NP loaded with different concentrations of BSA (as model protein)

[0282] The results in Table 2 on the laser diffraction measurement show the droplet size characteristics of the nasal formulations. According to the Dv10 of blank NP and BSA NP, no significant difference in droplet size was observed (p>0.05). In terms of Dv50 or median particle size, the droplet size of the BSA-loaded PNPHO:chitosan 1:1 formulation was 51.84 ± 1.40 µm. In contrast, for the control formulation, a significantly smaller median droplet size was recorded (36.76 ± 1.64 µm) (p<0.05). This droplet size pattern was also observed by Dv90 characterization, where the droplet size observed in the BSA-loaded formulation (140.84 ± 8.78 µm) was significantly larger than that in the blank nanoformulation (67.31 ± 3.09 µm) (p<0.05), indicating that the presence of protein drugs in the nanoformulation can affect the droplet properties of the nasal spray.

[0283] The encapsulation efficiency of the PNPHO:chitosan 1:1 formulation was consistently high, with at least 98% of the BSA encapsulated. The ability of the drug to be encapsulated in the nanocarrier was independent of the initial concentration of BSA added, ranging from 100 to 1000 µ g / mL. This can be explained by the saturation theory, which states that when proteins are encapsulated using electrostatic interactions, the encapsulation efficiency decreases only when the maximum protein concentration is loaded. (13) For successful intranasal drug delivery, nanocarriers with high encapsulation efficiency are important to deliver drugs efficiently to the desired brain regions with minimal waste. The increase in the encapsulation efficiency of NPs is proportional to the amount of drug reaching the CNS. (14) Since NPs loaded with BSA at all drug concentrations exhibited high encapsulation efficiency of 98–100%, droplet size characteristics and drug deposition patterns were further characterized using a 1:1 formulation of PNPHO:chitosan loaded with BSA (1000 µg / mL). The formulation with the highest initial drug concentration was more likely to achieve a therapeutic dose and produce an effective CNS effect in the brain.

[0284] Table 2. Droplet spray size data from Spraytec data, 30 / 33 pages, 34 CN 121752616 A

[0285] After 20 min of analysis, an increased amount of drug deposition was observed in the nasal cavity for the BSA-loaded PNPHO:chitosan 1:1 formulation. In particular, the nasal turbinate region showed a relatively large amount of drug deposition, as indicated by the bright pink of Sar-Gel (Figure).

[0286] In this study, the PNPHO polymer was characterized with respect to CMC to determine the minimum concentration required for NP formation. Subsequently, PNPHO with its CMC was used to form a nanocomposite by adding the cationic polymer chitosan via electrostatic interactions. This production method encapsulates a model therapeutic drug with a negatively charged surface. The effects of polymer ratio and initial drug concentration on the physicochemical properties of NP were determined. The data obtained indicate that the nanomedicine delivery platform is ready to load a variety of CNS drugs due to its promising physicochemical properties.

[0287] Deposition Studies of Pfizer Comirnaty Vaccine in Saline and TP035 Formulations The Pfizer-BioNTech Comirnaty vaccine is one of the most popular vaccines on the market for immunization against COVID-19 infection. While it has proven effective, there are still some limitations in terms of cold chain transport and storage requirements. This series of studies was conducted to determine whether the vaccine could be used in combination with PNPHO polymers (TP carrier system) or PPHO polymers (TL carrier system) for nasal delivery. Nasal mold deposition studies using the BIVAX nasal spray device (Figure) showed that the TP035 formulation could be specifically deposited in a specific area (the olfactory region), and the deposition lasted for up to 20 minutes after deposition. No significant dripping was observed during this period. The saline formulation deposited in a larger area of ​​the nasal cavity and further diffused within 20 minutes to cover most of the nasal cavity. Significant dripping was observed in the posterior throat. Similar results were observed with the TL035 formulation.

[0288] The strong retention / adhesion of the active substance throughout the upper respiratory tract indicates that the hydrogel properties of the polymer act as a solvent to "stick" the active substance to the site, preventing throat / nasal drip and thus maximizing the bioavailability of the active substance.

[0289] Industrial Applicability It should be understood that the present invention has readily applicable applications in the biomedical and vaccine fields. It can be seen that the biocompatible PNPHO or PPHO polymers provide an effective intranasal delivery solvent for existing mRNA / LNP COVID-19 vaccines, such as the popular Pfizer-BioNTech vaccine.

[0290] The main findings of this study are as follows: i. At any concentration above 0.5 mg / mL, PNPHO or PPHO polymers can form micelle-like structures (i.e., not in their hydrogel state under ambient conditions).

[0291] ii. Such micelles can be stabilized by ionic interactions. Those skilled in the art will note that examples related to BSA encapsulation efficiency are not critical; BSA examples are primarily intended to demonstrate the mechanism of action of the system in terms of its ionic interactions.

[0292] iii. The protective properties of PNPHO / PPHO (which may be reasonable based on its CMC) depend on concentration. Results showed that at low PNPHO concentrations (5 mg / mL and 10 mg / mL), the addition of the polymer led to mRNA cleavage, thus adversely affecting mRNA integrity. However, at concentrations of 15 mg / mL or higher, the addition of PNPHO / PPHO protected mRNA from cleavage.

[0293] iv. This protective property was further challenged at 37°C. The results showed that the addition of 35 mg / mL of PNPHO / PPHO polymer maintained the integrity of mRNA for at least 4 days under the more stringent conditions tested.

[0294] v. The function of mRNA+LNP with and without PNPHO was tested, showing that the addition of PNPHO at concentrations of 25 mg / mL and 35 mg / mL maintained the function of mRNA+LNP for at least 24 hours, as significantly higher eGFP expression was observed in the formulation with added PNPHO compared to the control (stored mRNA+LNP). A formulation was generated using dialyzed PNPHO (thus isolating fragmented PNPHO polymers) that could release functional nanoparticles into the culture medium to induce maximum efficacy.

[0295] vi. The results showed that the PNPHO / PPHO carrier system is compatible as a physical solvent system for intranasal delivery with certain commercial COVID-19 vaccines that may contain a mixture of mRNA and LNP.

[0296] References 32 / 33 pages 36 CN 121752616 A

[0297] 33 / 33 pages 37 CN 121752616 A Figure 1 Figure 1 / 20 pages 38 CN 121752616 A Figure 2 Figure 2 / 20 pages 39 CN 121752616 A Figure 2 (continued) Figure 3 / 20 pages 40 CN 121752616 A Figure 3(a) Figure 4 / 20 pages 41 CN 121752616 A Figure 3(b) Figure 5 / 20 pages 42 CN 121752616 A Figure 4 Figure 6 / 20 pages 43 CN 121752616 A Figure 5 Figure 6 Figure 7 / 20 pages 44 CN 121752616 A Figure 7 Figure 8 / 20 pages Page 45 CN 121752616 A Figure 8 Appendix 9 / 20 Page 46 CN 121752616 A Figure 9 Appendix 10 / 20 Page 47 CN 121752616 A Figure 10 Appendix 11 / 20 Page 48 CN 121752616 A Figure 11 Appendix 12 / 20 Page 49 CN 121752616 A Figure 12 Appendix 13 / 20 Page 50 CN 121752616 A Figure 13 Appendix 14 / 20 Page 51 CN 121752616 A Figure 14 Figure 15 Appendix 15 / 20 Page 52 CN 121752616 A Figure 16 Appendix 16 / 20 Page 53 CN 121752616 A Figure 17 Appendix 17 / 20 Page 54 CN 121752616 A Figure 18 Appendix to the Instruction Manual, Page 18 / 20, 55 CN 121752616 A Figure 19 Appendix to the Instruction Manual, Page 19 / 20, 56 CN 121752616 A Figure 20 Appendix to the Instruction Manual, Page 20 / 20, 57 CN 121752616 A.

Claims

1. A polymer for forming solutions and / or hydrogels to stabilize one or more pharmaceutically active agents before, during, or after application, said polymer comprising: The first monomer used for binding water; The second unit used to impart mechanical properties to the support; Optionally, a third monomer for binding to natural or synthetic peptides or proteins (NSPPs); and The fourth monomer used to impart phase transition behavior.

2. The polymer of claim 1, wherein the administration is performed via intravascular, intramuscular, subcutaneous, inhaled respiratory tract, oral inhalation, or intranasal administration.

3. The polymer according to claim 2, wherein the application is intranasal.

4. The polymer according to any one of the preceding claims, wherein the first monomer is one or more polyethers selected from: polyethylene glycol (PEG), oligomeric (ethylene glycol) (OEG), polyethylene oxide (PEO), polyethylene oxide-propylene oxide copolymer (PPO), copolymerized ethylene oxide block copolymers or random copolymers thereof.

5. The polymer according to claim 4, wherein the first monomer is oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA).

6. The polymer according to any one of the preceding claims, wherein the second monomer is a methacrylate or a random copolymer containing a methacrylate, selected from: hydroxyethyl methacrylate (HEMA), hydroxyethyl methacrylate poly(lactic acid) copolymer (PLA / HEMA), poly(lactic acid), poly(caprolactone), poly(glycolic acid), poly(glycolic acid-lactide), poly(glycolic acid-lactide) copolymer or poly(glycolic acid-caprolactone) copolymer.

7. The polymer according to claim 6, wherein the second monomer is hydroxyethyl methacrylate poly(lactic acid) (PLA / HEMA).

8. The polymer according to any one of the preceding claims, wherein the third monomer has an electrophilic functional group for binding with NSPP.

9. The polymer according to claim 8, wherein the third monomer is selected from: N-hydroxysulfosuccinimide (SNHS), N-hydroxyethoxysuccinimide (ENHS), and N-acryloyloxysuccinimide (NAS).

10. The polymer of claim 9, wherein the third monomer is N-acryloyloxysuccinimide (NAS).

11. The polymer according to any one of the preceding claims, wherein the lower critical solution temperature (LCST) of the fourth monomer is less than about 37°C.

12. The polymer of claim 11, wherein the fourth monomer is selected from: poly(ethylene oxide) / poly(propylene oxide) and poly(N-isopropylacrylamide) (PNIPAAm) homopolymers and copolymers.

13. The polymer according to claim 12, wherein the fourth monomer is (N-isopropylacrylamide) (NIPAAm).

14. The polymer according to any one of the preceding claims, wherein the polymer comprises a first monomer in an amount of about 1 mol% to about 15 mol%; a second monomer in an amount of about 5 mol% to about 50 mol%; a third monomer in an amount of about 0 mol% to about 15 mol%; and a fourth monomer to make up the remainder to 100% of the polymer.

15. The polymer according to any one of the preceding claims, wherein: The first monomer is OEGMA; the second monomer is PLA / HEMA; the third monomer is NAS; and the fourth monomer is NIPAAm. The polymer comprises: about 1 mol% to about 15 mol% of OEGMA; 5 mol% to about 50 mol% of PLA / HEMA; 0 mol% to about 15 mol% of NAS; and up to about 85 mol% of NIPAAm.

16. The polymer according to any one of the preceding claims, wherein the one or more pharmaceutically active agents comprise a vaccine that is originally limited to administration by intramuscular injection.

17. The polymer of claim 16, wherein the vaccine, which was originally limited to administration by intramuscular injection, is a COVID-19 (SARS-CoV-2) vaccine.

18. The polymer of claim 17, wherein the COVID-19 vaccine is a Pfizer-BioNTech (Comirnaty / Tozinameran) vaccine.

19. The polymer according to any one of the preceding claims, wherein the polymer is present at a concentration greater than or equal to about 15 mg / mL.

20. The polymer of claim 19, wherein the polymer is present at a concentration of about 25 mg / mL to about 35 mg / mL.

21. The polymer of claim 20, wherein the polymer is present at a concentration of about 35 mg / mL.

22. The polymer according to any one of the preceding claims, wherein The first monomer is OEGMA; the second monomer is PLA / HEMA; the third monomer is NAS; and the fourth monomer is NIPAAm; The polymer comprises: about 1 mol% to about 15 mol% of OEGMA; 5 mol% to about 50 mol% of PLA / HEMA; 0 mol% to about 15 mol% of NAS; and up to about 85 mol% of NIPAAm; The vaccine mentioned is an mRNA vaccine, preferably the Pfizer-BioNTech (Comirnay / Tozinameran) COVID-19 vaccine; The polymer is present at a concentration of approximately 35 mg / mL; and The polymer maintains the integrity of the mRNA for up to 4 days at 37°C.

23. A method for delivering one or more pharmaceutically active agents, the method comprising administering to a subject in need an effective concentration of one or more pharmaceutically active agents dispersed in a polymer as defined in any one of claims 1 to 22.

24. The method of claim 23, wherein the application is performed at ambient temperature, and the polymer transforms into its hydrogel form at a higher temperature (e.g., body temperature).

25. The method of claim 23 or 24, wherein the application is performed intranasally via intranasal spray or deposition.

26. The method according to any one of claims 23 to 25, wherein one or more pharmaceutical active ingredients may be dispersed in situ in the polymer, or pre-formulated and stored under conditions that support the activity of the active ingredient.

27. The method of claim 26, wherein the conditions supporting the activity of the active ingredient include freeze-drying the formulation and then reconstituteing it.

28. Use of the polymer as defined in any one of claims 1 to 22 in the production of a medicament for delivering one or more pharmaceutically active agents.

29. The use according to claim 28, wherein the application is performed at ambient temperature, and the polymer transforms into its hydrogel form at a higher temperature (e.g., body temperature).

30. The use according to claim 28 or 29, wherein the application is performed by intranasal spray or deposition.

31. The use according to any one of claims 28 to 30, wherein one or more pharmaceutical active ingredients may be dispersed in situ in the polymer, or pre-formulated and stored under conditions that support the activity of the active ingredients.

32. The use according to claim 31, wherein the conditions supporting the activity of the active ingredient include freeze-drying the formulation and then reconstituteing it.

33. A kit capable of delivering one or more pharmaceutically active agents, the kit comprising a polymer as defined in any one of claims 1 to 22; an effective concentration of one or more pharmaceutically active agents stored under suitable conditions; and optionally, instructions regarding the addition of an effective concentration of the polymer to the one or more pharmaceutically active agents.

34. The kit according to claim 33, wherein the polymer and the one or more pharmaceutical active agents are in a premixed form.

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