Polymer assisted drug delivery

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

Related Applications

[0001] This application claims convention priority from Australian patent application 2023902095 filed on 30 June 2023. The contents of AU’095 are incorporated herein by reference. TECHNICAL FIELD

[0002] The present invention relates to biocompatible polymers and the inventor’s unexpected discovery that such polymers stabilise certain pharmaceutical agents for a period of time sufficient to enable their administration to a subject by a variety of delivery modalities (e.g. intravascular, intramuscular, subcutaneous, oral inhalation or intranasal).

[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 manufacture of a medicament for the delivery of one or more pharmaceutically active agents. Preferred embodiments relate to intranasal delivery.

[0004] In another embodiment, the present invention relates to a kit capable of delivering one or more pharmaceutically active agents intravascularly, intramuscularly, subcutaneously, by oral inhalation or intranasally.

[0005] The present invention is intended to provide an alternative mode of administration for vaccines or pharmaceuticals that would otherwise be limited to delivery by intramuscular injection only. It has been unexpectedly discovered that the stabilising effect of the polymers enables the delivery of one or more pharmaceutically active agents to a subject where intravascular, intramuscular, subcutaneous, by oral inhalation or intranasal administration would otherwise not have been possible.

[0006] The present invention is intended to provide an alternative method for the formulation, stabilisation, production and storage of vaccines or pharmaceuticals that would otherwise be limited to cold transport, refrigeration and complex production steps that can involve microfluidic devices or other expensive steps.

[0007] While the present invention will be described hereinafter with reference to the preferred embodiments thereof, it is to be understood that the spirit and scope of the present invention can be embodied in many other forms. BACKGROUND

[0008] Any discussion of the background of the application herein is intended only to aid in understanding the invention and should not be taken as an admission that the prior art is prior art to the present invention.

[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 fueled public attention and interest in this technology. Several COVID-19 vaccines (such as those from Pfizer-BioNTech and Moderna) use RNA to stimulate an immune response. When introduced into human tissues, vaccines contain 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 LNPs and incorporated RNA components is crucial for achieving the intended biological activity within the expected shelf life of a vaccine. This typically limits the shelf life of such vaccines under specific temperatures, cold transport, and storage conditions. Such stringent requirements pose challenges to the wider deployment of such technologies.

[0011] As previously stated, preferred embodiments of the present invention relate to a proprietary polymer of the applicant that can stabilize the active substance in an mRNA vaccine (preferably a COVID-19 vaccine) under various conditions, thereby enabling and supporting intranasal administration. While the following discussion focuses on such embodiments, those skilled in the art will understand that these are merely exemplary in the overall context of the invention.

[0012] All currently approved COVID-19 vaccines are administered via intramuscular injection. However, because they are not preferred or tolerated in a significant portion of the population, Future coronavirus vaccines Various other types of vaccine delivery methods were studied. One of these methods is intranasal delivery.

[0013] Intranasal vaccines target mucosal immunity in the nasal mucosa, the gateway for viruses 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 are approved for influenza but not for COVID-19.

[0014] Carvalho's recent publications ( Nature MedicineThe study (Vol. 28, December 2022, pp. 2439-2440) pointed out the reasons why mRNA vaccines may be incompatible with intranasal delivery. Further understanding of the relevance of mucosal immune protection is needed to understand how (or even whether) this will affect infection. Reactivity was mild to moderate. Antigen-specific mucosal antibody responses to intranasal vaccination were detected 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 intranasal vaccination followed by intramuscular mRNA 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 characteristics, it neither induces a sustained mucosal antibody response nor a strong systemic response.

[0015] Therefore, efforts continue to be intensified 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 by way of 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 cross-linking the polymer with extracellular matrix proteins.

[0018] The preferred polymer disclosed in WO'001 is poly(NIPAAm- Co -NAS- Co -(PLA / HEMA)- Co-OEGMA), or "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 to 100% of the polymer composition (e.g., about 50 mol% to about 85 mol%) of NIPAAm. 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 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.

[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, comprising administering a therapeutically effective amount of a hydrogel formed from a bioactive polymer to the 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 to 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 wherein 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 an N-acryloyloxysuccinimide (NAS) unit; and the thermosetting monomer unit is an 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 the NSPP, and a fourth monomer for imparting phase transition behavior. Specifically, the composition, upon application in vivo or on the body surface, forms a viscous and highly fluid hydrogel, thereby facilitating tissue repair and regeneration. Therefore, WO'727 discloses a method for tissue repair and / or regeneration comprising applying the composition by injection or by aerosol administration to form a hydrogel at mammalian body temperature.

[0023] Finally, WO 2023 / 201397 (PCT / AU2023 / 050329) discloses a new polymer "PPHO", namely poly(N-isopropylacrylamide- Co -(polylactic acid / 2-hydroxymethyl acrylate)- Co -(Oligomer(ethylene glycol) / poly(NIPAAm-) Co -(PLA / HEMA)- Co -OEGMA).

[0024] (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 in an amount equal 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% 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 purpose of this invention is to overcome or improve at least one of the disadvantages 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 sufficiently long time before administration, or that can retain the formulation throughout the upper respiratory tract area for intranasal administration.

[0027] It is against this backdrop that the present invention was developed. Various embodiments of the present invention may reveal practical applications related to one or more of the aforementioned general needs.

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

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

[0030] In a broader sense, this invention relates to polymers at 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 to encapsulate and retain, when needed, one or more active pharmaceutical agents in vaccines or pharmaceuticals originally intended primarily for intramuscular delivery. It has been unexpectedly found that this polymer can stabilize one or more active pharmaceutical agents to extend their shelf life and / or allow them to be stored under less restrictive conditions.

[0031] The applicant's polymer, existing in solution or hydrogel form prior to administration, can stabilize the active substances in the vaccine 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 biological activity.

[0032] According to a first aspect of the invention, a polymer is provided for forming solutions and / or hydrogels to stabilize one or more pharmaceutical active agents before, during, or after application, the 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.

[0033] In the implementation plan, stabilizing one or more pharmaceutical active agents prior to application is to prevent their degradation, denaturation, shearing, or any chemical or physical changes that may affect their biological activity.

[0034] In the implementation plan, administration can be carried out via intravascular, intramuscular, subcutaneous, inhalation into the lungs, oral inhalation, or intranasal administration.

[0035] In the implementation plan, the application is intranasal.

[0036] In the implementation scheme, the first monomer is selected from: polyether, polyvinyl alcohol (PVA); poly(vinylpyrrolidone) (PVP); poly(amino acid) and dextran.

[0037] In the implementation scheme, 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 copolymer.

[0038] In the implementation scheme, the first monomer is oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA).

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

[0040] In the implementation scheme, 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 the implementation scheme, the second monomer is hydroxyethyl methacrylate poly(lactic acid) (PLA / HEMA).

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

[0043] In the embodiments, the third monomer is selected from: N-hydroxysulfosuccinimide (SNHS), N-hydroxyethoxysuccinimide (ENHS) and N-acryloyloxysuccinimide (NAS).

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

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

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

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

[0048] In the implementation scheme, the polymer contains a first monomer in an amount of about 1 mol% to about 15 mol%.

[0049] In the implementation scheme, the polymer contains a second monomer in an amount of about 5 mol% to about 50 mol%.

[0050] In the implementation scheme, the polymer contains a third monomer in an amount of about 0 mol% to about 15 mol%.

[0051] In the implementation scheme, the polymer contains a fourth monomer in an amount of about 50 mol% to about 85 mol%.

[0052] In one 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 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.

[0053] In the implementation scheme, 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.

[0054] In the implementation scheme, one or more pharmaceutically active agents comprise therapeutically active substances and / or vaccines that are originally limited to administration via intramuscular injection. Non-limiting examples include mRNA and siRNA vaccines, etc.

[0055] In the implementation plan, the vaccine originally limited to intramuscular injection was the COVID-19 (SARS-CoV-2) vaccine.

[0056] In the implementation plan, the COVID-19 vaccine is the Pfizer-BioNTech (Comirnaty / Tozinameran) vaccine.

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

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

[0059] In the implementation scheme, the polymer is present at a concentration of approximately 35 mg / mL.

[0060] In the implementation scheme, 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 in question is an mRNA vaccine, preferably the Pfizer-BioNTech (Comirnaty / Tozinameran) COVID-19 vaccine; The polymer was present at a concentration of approximately 35 mg / mL; and The polymer can maintain the integrity of mRNA for up to 4 days at 37°C.

[0061] Preferably, the polymer comprises: The first monomer used for binding water; A second monomer used to impart mechanical properties to the polymer; Optionally, a third monomer for binding to natural or synthetic peptides or proteins (NSPPs); and The fourth monomer used to impart phase transition behavior.

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

[0063] In the implementation scheme, 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 copolymer.

[0064] In the implementation scheme, the first monomer is oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA).

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

[0066] In the implementation scheme, 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.

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

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

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

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

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

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

[0073] In the implementation scheme, the fourth monomer is (N-isopropylacrylamide) (NIPAAm).

[0074] In the implementation scheme, the polymer contains a first monomer in an amount of about 1 mol% to about 15 mol%.

[0075] In the implementation scheme, the polymer contains a second monomer in an amount of about 5 mol% to about 50 mol%.

[0076] In the implementation scheme, the polymer contains a third monomer in an amount of about 0 mol% to about 15 mol%.

[0077] In the implementation scheme, the polymer contains a fourth monomer in an amount of about 50 mol% to about 85 mol%.

[0078] In one 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 the 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 the implementation scheme, 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 one embodiment, the application is carried out at ambient temperature, and the polymer transforms into a hydrogel form at a higher temperature (e.g., body temperature).

[0083] In the implementation plan, application is carried out via intranasal spray or deposition.

[0084] In the embodiments, 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.

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

[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 delivering one or more pharmaceutically active agents.

[0087] In the implementation scheme, the application is carried out under ambient conditions or substantially under ambient conditions, and then, upon reaching body temperature, a transformation into a hydrogel occurs.

[0088] In the implementation plan, administration is performed via intravascular, intramuscular injection, intranasal delivery, or any other route of administration.

[0089] In the embodiments, 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.

[0090] In the implementation plan, conditions that support the activity of the active ingredient include freeze-drying the formulation and then reconstituteing it.

[0091] According to a fourth aspect of the invention, a kit is provided for delivering one or more pharmaceutically active agents, the kit comprising a polymer as defined in a first aspect of the invention; 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.

[0092] In the implementation scheme, the kit further includes means for intranasal delivery of one or more pharmaceutical active agents and effective concentrations of polymers.

[0093] In the implementation scheme, the kit comprises a polymer and one or more pharmaceutically active agents in a premixed form.

[0094] Definitions and Terms In describing and claiming protection for this 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 "stability" for one or more pharmaceutical active agents aims to encompass the concept of "stability over shelf life." Therefore, by providing stabilizing effects, storage conditions (e.g., temperature) for the active agent can be simplified, allowing for longer vaccine storage. This primarily relates to pre-administration of the vaccine. Post-administration, particularly from the perspective of stability and sustained / controlled release, involves the delivery of polymers / vaccines via intravascular, intramuscular, subcutaneous, oral inhalation, intranasal delivery, and other methods.

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

[0097] "Preferred" and "ideally" refer to embodiments of the invention that may offer certain benefits under specific circumstances. However, other embodiments may also be preferred under 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 limits, 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 the integers that define the upper and lower limits and any integers contained within that range.

[0099] Except as provided in the operational examples or otherwise, all figures used herein to indicate the amount of an ingredient 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 otherwise 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 approximations, the values ​​listed in the specific examples are as accurate as possible. However, any value inevitably contains some errors due to the standard deviation present in its respective test measurement.

[0101] The following abbreviations are used in this instruction manual: Attached Figure Description

[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 The following stability at 4°C was demonstrated: (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 Zetasizer analysis of particle size of the formulation stored at 4°C on day 1 is shown. mRNA formulations with or without PNPHO, at a concentration of 5 mg / mL, formed TP005 (…). a ), 10 mg / mL to form TP010 ( b ) and 15 mg / mL form TP015 ( c ).

[0104] Figure 3 The following items were shown to be stable at 37°C: a Formulations containing 0, 0.15, and 0.35 TP and mRNA were prepared, followed by hydrogel formation (37°C) and redissolution after incubation at 4°C. The mRNA integrity was assessed at 24 h and 4 days ± with or without aerosolization using a VP7 Aptar nasal pump. b The mRNA levels of PBS and TP50 formulations were measured at the initial (day 0), 24 h (day 1), and 96 h (day 4) intervals, and the supernatant of TP035 formulation was sampled separately.

[0105] Figure 4 It showed ( a Nanoparticle forming ability of dialyzed and undialyzed PNPHO polymers at concentrations of 25 mg / mL and 35 mg / mL; bAdding and not adding PNPHO (25 mg / mL and 35 mg / mL of undialyzed PNPHO) Figure 4 b- i ) and dialysis-treated PNPHO ( Figure 4 b-ii Functional assay of mRNA-Lipofectamine.

[0106] Figure 5 Micelle formation was demonstrated, in which the concentration-driven micelle 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 The composite aggregation of nanoparticles was demonstrated. First, negatively charged bovine serum albumin (BSA, a model therapeutic 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 nanocarriers.

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

[0109] Figure 8 The hydrodynamic diameters of blank NPs and BSA-encapsulated NPs are shown. Compared with blank NPs, 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 The drug deposition pattern in the silicon nasal cast is shown. Within 20 min, a gradual increase in the amount of drug deposited in the BSA-loaded PNPHO:chitosan 1:1 formulation within the nasal cavity is visible. In particular, the nasal turbinate region exhibits relatively abundant drug deposition, as indicated by the bright pink color from the Sar-Gel.

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

[0112] Figure 11 The percentage sizes of particles containing and without mRNA in samples TP007, TP015, and TP035 are shown (a), and comparisons of Z-ave (b) and Pdi (c).

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

[0114] Figure 13 The images show 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. Controls: a mixture of mRNA and LNP; a standard formulation without TP (bi); formulation A (b-ii) involving mRNA / LNP mixing followed by addition to a TP solution; formulation B (b-iii) involving the addition of lipofectamine to a TP vector followed by the addition of mRNA; and formulation C (c) involving the direct dissolution of mRNA in a TP vector followed by the addition of LNPs.

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

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

[0117] Figure 16 The mRNA encapsulation (a) and particle size measurements (b) of the control (no vector; TP000) and TP035, TP050 and TP100 after storage at 2-8°C for 14 days are shown.

[0118] Figure 17 The e-GFP expression of epithelial cells (a1), 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) are shown. Cells were in vivo imaged at the endpoint 48 hours (bi), and fluorescence intensity was observed at different time points (b-ii).

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

[0120] Figure 19 The deposition patterns of TP000 (control) and TP035+ formulations at 0, 5, 10, 15, and 20 minutes after application are shown (a). Volume retention rates of TP000 and TP035+ formulations at the application site are shown (b). Statistically significant differences in volume retention rates were observed between TP000 and TP035+ at all time points. p < 0.001). Nasal cavity surface area coverage after application (c). There was a statistically significant difference in surface area coverage 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 The mRNA encapsulation efficiency of freshly prepared formulations without the vector (TP000) and with the vector (TP025+) and a formulation prepared with the vector, freeze-dried and reconstituted in water (TP025+FD) is shown. Detailed Implementation

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

[0123] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with embodiments, it should be understood that the purpose of the invention is not to limit it 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 used in this invention 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 article extensively discloses the use of biocompatible polymers for enabling intranasal administration of mRNA / LNP-based COVID-19 vaccines, particularly Pfizer-BioNTech (Comirnaty) vaccines.

[0127] Polymer 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 combine to form a polymer, but it can also be a polymer itself, or a monomer, or a derivative of a polymer. This type of monomer is also referred to herein as a "macromonomer." In this context, a "macromonomer" is a polymer or oligomer that each has a terminal group as a monomer molecule, thus each polymer or oligomer molecule contributes only a single monomer unit to the chain of the resulting 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, an ability that allows the polymer to form a plastic structure when hydrated. Furthermore, the resulting structure should possess the required resistance to compression 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, or about 1:50 (molar ratio of water-binding monomers to mechanical strength monomers). In fact, the water-binding monomers are needed to not only make the polymer hydrophilic but also to impart 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 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 possess the necessary water-binding capacity, are readily synthesized and / or purchased, and are inert because they elicit minimal or no immune response in the tissues to which they are placed.

[0134] In addition, various hydrophilic functional groups can be used to make monomers (and polymers formed from these monomers) water-soluble. For example, water-soluble functional groups (such as phosphate, sulfate, quaternary ammonium, hydroxyl, amine, sulfonate, and carboxylate groups) can be incorporated into monomers to make them water-soluble.

[0135] Monomers can also react with other compounds to form "macromonomers". Therefore, the first monomer can optionally be a macromonomer.

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

[0137] Preferably, the polymer comprises a first monomer in an amount of about 1 mol% to about 15 mol%. In various embodiments, the first monomer may be present 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%, or about 15 mol%. In various embodiments, the first monomer may be present in the form 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: mechanical property conferring monomer 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.

[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 with the desired mechanical properties. Typically, the proportion of "mechanical" monomers in the polymer is approximately 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, or 1:50 (molar ratio of water-bound monomers to mechanical strength monomers). Suitable examples of monomers capable of imparting 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 monomer can also react with other compounds to form a "macromonomer". The preferred second monomer as a macromonomer is poly(lactic acid) hydroxyethyl methacrylate (PLA / HEMA).

[0141] Preferably, the polymer contains a second monomer in an amount of about 1 mol% to about 50 mol%. 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 the range of 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. For efficient combination of the polymer with NSPP, it is preferable to include monomers or units with crosslinking capabilities in the polymer.

[0144] This crosslinking ability means that the polymer can bind to NSPP and thus crosslink NSPP to form a polymer containing NSPP. Alternatively, through a similar mechanism, NSPP acts as a crosslinking agent, thereby causing the polymer to crosslink 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 polymer of the present invention needs to contain a certain proportion of monomers capable of binding with NSPP, such proportion being sufficient to crosslink with NSPP, thereby forming 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 that can bind to NSPP typically have electrophilic or nucleophilic functional groups, such that nucleophilic functional groups on NSPP can react with electrophilic functional groups on monomers to form covalent bonds.

[0147] Therefore, for example, if NSPP has a nucleophilic functional group (e.g., an amine), the polymer can have an electrophilic functional group, 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 an electrophilic functional group, the polymer can have a nucleophilic functional group, such as an amine or a thiol.

[0148] Preferably, the polymer 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%. 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 transition 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 the present invention.

[0151] Typically, the proportion 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 comprises a fourth monomer in an amount sufficient to make up the remainder to 100% of the polymer composition. In embodiments, 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 wide variety of 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 a HEMA-polylactic acid polymer (PLA / HEMA), which can itself be used as a monomer in the polymers of the present invention. Additionally, the monomer itself can be a combination of monomer units that 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] The preferred polymers of the present invention can be further modified with one or more moieties and / or functional groups. According to the present invention, any moieties or functional groups can be used. 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. Typically, the polymer according to the invention is an organic polymer. Preferably, the polymer of the invention is biocompatible. In embodiments, the polymer is biodegradable. In other embodiments, the polymer is both biocompatible and biodegradable.

[0156] The 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 ionomers, or monomers thereof.

[0157] If biodegradability or absorbability of the polymer is desired, one or more monomers having biodegradable linkages can be used. Optionally, or additionally, the monomers can be selected such that the reaction products between them form biodegradable linkages. For each method, the monomers and / or linkages can 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 the resulting product is non-toxic when the polymer degrades under physiological conditions.

[0158] Biodegradable linkages can be chemically or enzymatically hydrolyzable or absorbable. Illustrative 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. Further 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 polymer's low critical solution temperature (LCST) (by reducing the overall hydrophobicity of the polymer), thereby enhancing its bioavailability.

[0160] Preferred polymers The polymer preferably contains a first monomer in an amount of about 1 mol% to about 15 mol%. 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 an amount of about 5 mol% to about 50 mol%. In various embodiments, 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 It is present in mol%, 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 in an amount that makes up the remainder to 100% of the polymer composition, for example, about 50 mol% to about 85 mol%. 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 mentioned in this article 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: 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 up to 15 mol%; and a fourth monomer in an amount of up to about 85 mol%.

[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) in A is the first monomer (water-bound monomer), for example, OEGMA; B is the second monomer (a monomer that can impart mechanical properties to the polymer), for example, PLA / HEMA; C is a third monomer (a monomer with a functional group that binds to NSPP), for example, NAS; and D is the fourth monomer (a monomer that can impart phase change properties to the polymer), for example, NIPAAm.

[0170] In various implementations, 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-bound 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] Also found were 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 the in vivo degradation of the biodegradable segment (e.g., PLA), thereby leading to the 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 kDa, 98 kDa, 99 A molecule of kDa or about 100 kDa.

[0175] PNPHO The preferred polymer of this invention is polyNIPAAm- 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 NIPAAm to make up the remainder to 100% of the polymer composition (e.g., about 50 mol% to about 85 mol%).

[0176] The percentages mentioned in this article 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: OEGMA in amounts of approximately 3 mol% to approximately 8 mol% (e.g., approximately 4 mol% to approximately 6 mol%); Amounts of HEMA-PLA ranging from approximately 5 mol% to approximately 9 mol% (e.g., approximately 6 to approximately 8 mol%); At least approximately 7 mol% of NAS; and A maximum of about 85 mol% (e.g., about 81 mol%) of NIPAAm.

[0178] In another embodiment, the polymer comprises: Approximately 5 mol% of OEGMA; Approximately 7 mol% of HEMA-PLA; Approximately 7 mol% of NAS; and Approximately 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 equation I: A is oligomeric (ethylene glycol) monomethyl ether methacrylate (OEGMA); B stands for hydroxyethyl methacrylate poly(lactic acid) (HEMA-PLA). C is N-acryloyloxysuccinimide (NAS); and D stands for N-isopropylacrylamide (NIPAAm).

[0181] The preferred form of the polymer PNPHO used in this application is the polymer of formula (I) 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, reinforcing, and / or crosslinking capabilities are 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 contains about 1 mol% to about 15 mol% of OEGMA, about 5 mol% to about 50 mol% of PLA / HEMA and NIPAAm to make up the remainder to 100% of the polymer composition (e.g., about 50 mol% to about 85 mol%). 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%, 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 in this article relate to the composition of the final polymer, not to the amount of feed used to form the polymer.

[0185] The preferred form of the polymer PPHO used in this application is the 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] Synthesis of polymers 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 polycondensation.

[0188] The following examples illustrate 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, dispersants 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, AR, 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. The use of any conventional excipient is considered within the scope of this invention unless it 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.

[0190] According to the formulation personnel's judgment, the composition may contain excipients such as colorants, coating agents, sweeteners, flavorings and fragrances.

[0191] Bioactive agents or pharmaceutical compounds that can be added to the compositions and / or polymers of the present invention include proteins, glycosaminoglycans, carbohydrates, nucleic acids, and inorganic and organic bioactive compounds, such as enzymes, antibiotics, antitumor agents, local anesthetics, hormones, angiogenic agents, anti-angiogenic 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 finally freeze-drying the resulting composition. This produces a ready-to-use polymer.

[0193] The amounts of polymers, 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 the appropriate agents and amounts for treating the condition.

[0194] Composition for forming hydrogel 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: First water-bound monomer; and The second monomer that imparts mechanical properties; Optionally, a third monomer is an NSPP-binding monomer containing functional groups capable of binding to NSPP. A fourth monomer capable of imparting phase transition properties to hydrogels; Among them, natural or synthetic peptides or proteins (NSPP) are thymosin β-4 or its functional homologs; Furthermore, the combination of NSPP with the second monomer crosslinks the polymer, 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, preferred compositions used in this invention may further 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] Typically, the amount of polymer in the composition used in this invention is the amount required to achieve hydrogel formation.

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

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

[0201] Typically, the amount of NSPP in the compositions of this invention is the amount required to achieve 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 implementations, the percentage of NSPP is approximately 1% w / w, approximately 2% w / w, approximately 3% w / w, approximately 4% w / w, approximately 5% w / w, approximately 6% w / w, approximately 7% w / w, approximately 8% w / w, approximately 9% w / w, approximately 10% w / w, approximately 20% w / w, approximately 30% w / w, approximately 40% w / w, approximately 50% w / w, or higher.

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

[0205] Polymer system as a carrier capable of loading mRNA and lipid nanoparticles (LNP) and enhancing their structural and functional stability. Preparation and coding of TP and TL

[0206] Standard laboratory equipment was used throughout the research process. 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, 7 mol% PLA / HEMAn = 5, and 7 mol% NIPAAm were used. 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] Analysis was performed using a range of devices and apparatus, including the Molecular Devices Spectramax iD3 multimode microplate reader, the Bio-Rad Gel DºC™ EZ Gel DºCumentation System, and nvitrogen. TM SYBR TM Green II RNA Gel Staining Reagent 10,000X Concentrate (in DSMO*) (Catalog No.: S7568), Bioline 5× DNA Loading Buffer Blue (Catalog No.: BIO-37045), Bioline Agarose, Universal 100 g, Powder, Pure Agarose, Gel Electrophoresis (Catalog No.: BIO-41025), 10× TAE Buffer (0.4 M Tris, 0.01 M EDTA and 0.2 M Acetic Acid).

[0208] mRNA stability study 4°C - integrity and content and nanoparticle potential At 2 to 10 °C, 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) to obtain a clear, single-phase solution. The resulting solution is coded as "TPXXX" (PNPHO) or "TLXXX" (PPHO), where the three digits "XXX" represent 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] Prepare TP100 and TL100 using 1× PBS or Opti-MEM medium and gently mix overnight at 4°C. Store TP100 and TL100 solutions at 4°C until use. On the day of the experiment, mix mRNA with an appropriate volume of TP100 and / or TL100 solution along with 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 volume)) were loaded into a 2% (w / v) agarose gel, which was incorporated with SYBR at a ratio of 1:10000. TM Green II RNA gel dye was used. The gel was run at 90 volts for 40 minutes and then dried using Gel DºC. TM EZ Gel DºCumentation The system employs strong and weak band imaging modes. ImageJ software is used for quantification of the gel bands. Quant-iT TM 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 37°C - integrity and content mRNA stability studies for RNA integrity and content were performed using an Aptar VP7 5 mL nasal spray bottle. The PBS-mRNA or TP-mRNA formulation was stored in the spray bottle at 4°C, and samples were taken daily (sprayed twice into a Falcon tube and centrifuged at 100 × g for 5 minutes) for 5 days. mRNA content was analyzed by agarose gel electrophoresis. A Zetasizer was used to study nanoparticle potential, measuring the detection of any nanoparticles in the formulation.

[0212] mRNA release study of TP or TL formulations mRNA stability studies were performed using 10 mL glass vials. 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). Following incubation at 37°C, the liquid supernatant formed on top of the hydrogel in the TP50 formulation was sampled 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, as described herein, was then performed by direct pipetting and transfer to a VP7 Aptar pump. After sampling, the formulation was returned to the 37°C vial for further incubation. Samples were analyzed using agarose gel electrophoresis.

[0213] Transport study of mRNA-TP formulations through the nasal epithelium Sustained mRNA release was measured by placing a 5 µm polycarbonate membrane (5 µL) of Transwell insert containing 100 µL of PBS-mRNA, TP-mRNA, or TL-mRNA formulation into a 24-well plate and adding 0.5 mL of physiological saline to the basal chamber. The plate was then incubated at 37 °C. 100 µL samples were collected from the basal chamber at different time points, and an equal volume of freshly preheated PBS was added after each sample collection. The assay was performed for up to 180 h. Results were obtained via Quant-iT. TM Ribogreen RNA assays were performed on collected samples to quantify the mRNA released from the formulation. These experiments were conducted using a liquid dialysis polymer provided by the applicant.

[0214] mRNA function study using time-lapse microscopy Nasal epithelium was established by growing RPMI2650 cells in an air-liquid interface culture using Transwell 24-well plates (5 µm, polycarbonate membrane). PBS-mRNA, TP-mRNA, or TL-mRNA formulations (10 µg mRNA in 100 µL) were deposited onto the nasal epithelium using a micropipette, 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 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. Quant-iT was used. TM The Ribogreen RNA assay quantifies mRNA using a liquid dialysis polymer provided by the applicant.

[0215] Direct exposure - mRNA function assay using microplate plate reader The function of EGFP mRNA was assessed by cellular production of green fluorescent protein (GFP). Cells were cultured for 24 h in 24-well plates 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. The medium was then replaced again with the following: i. Control – 500 µL of fresh Opti-MEM medium ii. mRNA alone – Dilute 500 ng of mRNA to 500 µL using fresh Opti-MEM medium. iii. Lipofectamine-containing mRNA – Mix 500 ng of mRNA with 0.75 µL of lipofectamine and dilute to 500 µL with fresh Opti-MEM medium.

[0216] iv. mRNA containing TP025 / TL025 – Dilute 500 ng of mRNA to 500 µL in Opti-MEM medium containing 25 mg / mL PNPHO / PPHO.

[0217] v. mRNA containing Lipofectamine and TP025—Mix 500 ng of mRNA with 0.75 µL of Lipofectamine and dilute to 500 µL with fresh Opti-MEM medium containing 25 mg / mL PNPHO. Obtain the appropriate mixture targeting the PPHO polymer.

[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 captured every 2 hours for 48 hours using NIS-Elements (version 3.22.01) set to capture images in both "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] Parallel mRNA function assay using nanoparticle measurements A microplate reader assay has been developed to measure the potency of EGFP mRNA. EGFP mRNA in RPMI cells was assessed using cell-produced green fluorescent protein (GFP). 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. The medium was then replaced with the following: i. Negative control – Dilute 0.75 µL of Lipofectamine to 500 µL using fresh Opti-MEM medium. ii. Positive control – Mix 500 ng of mRNA with 0.75 µL of lipofectamine and dilute to 500 µL with fresh Opti-MEM medium.

[0220] iii. TP015 formulation – Mix 500 ng of mRNA with 0.75 µL of lipofectamine and dilute to 500 µL with fresh Opti-MEM medium containing 25 mg / mL polymer. Prepare the corresponding TL / PPHO formulation.

[0221] iv. TP035 formulation—Mix 500 ng of mRNA with 0.75 µL of lipofectamine and dilute to 500 µL with fresh Opti-MEM medium containing 35 mg / mL polymer. Prepare the corresponding TL / PPHO formulation.

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

[0223] Results: stability of 5 mRNA at 4°C storage and using VP7 Aptar nasal pump nebulization 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. 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 with the following: i. Negative control – Dilute 0.75 µL of Lipofectamine to 500 µL using fresh Opti-MEM medium. ii. Positive control – Mix 500 ng of mRNA with 0.75 µL of lipofectamine and dilute 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 wells carrying the following formulation: (i) TPO formulation – Mix 500 ng of mRNA with 0.75 µL of lipofectamine and dilute to 100 µL with fresh Opti-MEM. Prepare the corresponding TL / PPHO formulation.

[0225] (ii) TP015 formulation – Mix 500 ng of mRNA with 0.75 µL of lipofectamine and dilute to 100 µL with fresh Opti-MEM containing 15 mg / mL polymer. Prepare the corresponding TL / PPHO formulation.

[0226] (iii) TP030 formulation: Mix 500 ng of mRNA with 0.75 µL of lipofectamine and dilute 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 inserts were 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) from three independent experiments. Statistical analysis was performed using Prism software version 9.4.0 (GraphPad, San Diego, USA).

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

[0230] Adding low concentrations of PNPHO or PPHO (5 mg / mL and 10 mg / mL) leads to mRNA cleavage and instability, such as... Figure 2 b The degradation signal was diffused in the middle. However, further addition of PNPHO polymer enhanced the stability of mRNA, as a very clear and stable band was observed in the formulation containing 15 mg / mL PNPHO (TP015). Using TP015, the amount of mRNA also remained stable close to 100%. Figure c These results confirm that, at higher concentrations, the PNPHO polymer chains and their hydrophilic / hydrophobic interactions in the formulation provide protective properties.

[0231] Stability of mRNA at 37°C storage The results The addition of PNPHO or PPHO to mRNA prevents the natural aggregation of particles, leading to the formation of nanoparticles. This conclusion is based on the fact that 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 showed that low concentrations of PNPHO or PPHO may adversely affect the stability of mRNA, possibly due to the presence of low molecular weight PNPHO / PPHO segments in the formulation. However, at concentrations increased to 15 mg / mL or higher, the protective properties of PNPHO / PPHO and its role in the formation of mRNA nanoparticles led to the formation of stable formulations. Therefore, for further research, polymer concentrations of 15 mg / mL or higher of PNPHO / PPHO are recommended.

[0233] Figure a Based on the results of studies on the mRNA stability of different concentrations of PNPHO / PPHO polymers, these formulations were further challenged at elevated temperatures. Therefore, mRNA formulations containing 15 mg / mL and 35 mg / mL of PNPNO and PPHO were tested, and mRNA stability was assessed using agarose gel electrophoresis. In the formulation containing 15 mg / mL of PNPHO or PPHO, an opaque solution formed at 37°C, while in the formulation containing 35 mg / mL of PNPHO, a hydrogel thin layer formed, with the supernatant phase as the upper layer. Figure a ).

[0234] After incubation at 4°C for 1 h, the formulation was reconstituted, and due to the reversibility of the polymer, the sample became a homogeneous solution as expected. No difference in mRNA integrity was observed between direct pipetting and nebulization of the formulation. Figure b On day 4, a significant decrease in mRNA levels was observed in the original formulation (without polymer). However, the addition of the 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 levels between day 0 and day 4. p > 0.05)( Parallel mRNA function assay and nanoparticle analysis using TP / TL formulations Furthermore, analysis of the TP035 / TL035 samples (the supernatant above the hydrogel) revealed the presence of mRNA in the solution, indicating that the hydrogel did not selectively capture the mRNA, which was dispersed in both the supernatant and the hydrogel.

[0235] Figure a A lipofectamine-containing mRNA (mRNA + LNP) formulation was tested, incorporating 25 mg / mL and 35 mg / mL of dialyzed or undialyzed PNPHO polymer (or its PPHO equivalent). 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. Results showed that dialysis of the PNPHO polymer led to the separation of polymer fragment groups, resulting in different volume percentage groups (e.g., ...). Figure b (As shown).

[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. mRNA and LNP deliveryThe results showed that the addition of PNPHO polymers preserved mRNA function, as significantly higher levels of TP025 and TP035 were recorded in both dialysis- and non-dialysis-incorporated PNPHO configurations compared to those without PNPHO incorporation. p GFP expression < 0.001).

[0237] In particular, formulations containing dialysis-contained PNPHO / PPHO polymers showed 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 most potent 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 maximum mRNA efficacy in the basal RPMI2650 cell model and maintain functionality under elevated temperatures and harsher conditions.

[0238] Preservation of mRNA within the TP platform The TP (formulated with PNPHO) and TL (PPHO) platform carrier systems were challenged as universal therapeutic delivery platforms 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 efficacy and potential of the polymer platform carriers of the present invention. Subsequently, different mixing methods were investigated by varying the order of mRNA, LNP, and TP / TL incorporation, and the effect of the TP / TL carrier on LNP encapsulation of mRNA was evaluated. Subsequently, TP050 and TP100 containing eGF-mRNA / LNP (and TL equivalents) were formulated to ensure compatibility of mRNA / LNP with higher polymer concentrations, anticipated to be more suitable for long-acting injectable formulations. Then, configurations more useful for nasal delivery were investigated, and the compatibility of the carriers with commercially available COVID-19 vaccines was evaluated. Finally, the effect of the carrier layers on the bioactivity of LNP and mRNA formulations was investigated to ensure that the carriers do not impede their intended function.

[0239] Figure 11 eGFP-mRNA was used and mixed with PNPHO solutions at concentrations of 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 11 a The graph shows the volume percentage of GFP-mRNA with and without different vector configurations. Figure 11 bThe results showed that adding the carrier system 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, Figure 14 c The results showed that the polydispersity (Pdi) of the solution was significantly reduced after the addition of the carrier platform. p <0.01). These results indicate that the TP / TL platform loading system can uniformly wrap mRNA and exhibits protective performance against mRNA sample aggregation.

[0240] To test the effects of adding a vector system to mRNA-based therapies, different vector configurations of 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. The pH of all solutions was maintained at approximately 7.3 during the testing period. Some bubbling occurred in TP007+ after spraying. Gel electrophoresis of the solutions using Sybr Green II staining reagent showed good mRNA detection range; however, the carrier component was also stained on the larger molecular weight bands. Figure 14 a In any case, the separation of mRNA bands is effective and can therefore be used to establish a standard curve for further quantification. Figure 12 b The results showed 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 against mRNA are attributed to the polymer's charge 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. Incorporation of TP / TL platform into mRNA / LNP formulations The results showed that after 0 and 24 hours of storage at 37°C, no splicing was observed in the mRNA integrity of the control (without vector), TP015, and TP035. However, after 4 days of storage at 37°C, the integrity of the mRNA samples containing vectors TP015 and TP035 was significantly improved. p< 0.001) higher than the control. For example, after 4 days of challenge, approximately 60% of mRNA was present in the control group, while almost 100% of 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] Figure 13 To evaluate the effectiveness of incorporating the TP / TL platform into the mRNA / LNP production process, e-GPF mRNA containing lipofectamine was used as a model system. A GPC-MS (gel permeation chromatography) method was developed and established. R 2 = 0.99 standard curve ( Figure 13 a This method was used to quantify mRNA. It was used to assess the amount of mRNA encapsulated within LNPs 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 ( Figure 13 b(i) ).

[0243] Formulation (A): Mix eGFP-mRNA / LNP for 5 minutes, then add TP100 to form the final mixture. (ii) b Figure 13 ).

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

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

[0246] Within 2 hours of preparation, 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. Results showed that the control formulation had a relatively high encapsulation efficiency, with 81.2 ± 1.2% of the incorporated mRNA still encapsulated within the LNP. Figure 13 b(i) However, incorporating the TP platform by mixing the formed mRNA / LNP with the TP platform in formulation A significantly ( p The encapsulation efficiency was increased to 86.4 ± 2.1% (< 0.05). Figure 13 b(ii) Subsequently, lipofectamine was mixed with the TP platform before mRNA incorporation to further improve the encapsulation efficiency (Formulation B) to 92.4 ± 1.9%. (iii) b Figure 13 Finally, the mRNA was added directly to TP100, followed by mixing with LNP, which slightly improved the encapsulation efficiency to 95.7 ± 0.9%. p The value is 0.0491 ( TP / TL configurations for different administration routes b(iv) ).

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

[0248] Figure 15 Using the mixing method of formulation A (described above), TP035+, TP050+, and TP100+ eGFP mRNA / LNP were formulated with PNPHO polymer at concentrations of 35 mg / mL, 50 mg / mL, and 100 mg / mL, respectively. The effects of the TP vector on the particle size and stability of the formulations after storage at 2–8°C for 3, 7, and 14 days were investigated using DLS and GPC analysis techniques.

[0249] Long-term stability of formulations The results showed 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 vector significantly increased ( pThe particle sizes of TP035+, TP050+, and TP100+ (< 0.05) reached 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 support systems could be formulated. Therefore, the TP035+ / TL050+ system exhibited similar particle sizes to TP035+ (no statistically significant 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 the Pdi measurements of the particles, all configurations showed relatively stable Pdi values, generally within the range of 0.2–0.4. There was no statistically significant difference in Pdi measurements between the presence and absence of the TP / TL support; a slight increase in Pdi was observed in TP100 compared to TP000, which is attributed to the increased temperature during DLS measurements and the temperature-responsive nature of the support system.

[0251] Among all formulations, the TP / TL vector exhibited significant protective effects 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 of TP000 and ~0% encapsulated mRNA content. After 3 days of storage, the encapsulated mRNA content was significantly higher in all formulations containing TP, TP / TL, and TL vectors. p < 0.001) is higher than TP000. The encapsulated mRNA content in TP035+, TP050+, TL050+, TP035 / TL050+ and TP100 is ~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 for TP035 to 117.5 ± 4.3 nm for TP050 and 167.5 ± 4.6 nm for 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 peak 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] Figure 16 Effect of polymer platform on mRNA and LNP function An evaluation of the encapsulated mRNA content and particle size measurements of different formulations stored at 2-8°C for up to 14 days confirmed that the presence of a carrier system improves the stability of the formulations. TP035 was the most stable formulation, with more than 70% of the mRNA still encapsulated after 14 days of formulation preparation and storage at 2-8°C. After 14 days, more than 50% of the mRNA was still encapsulated; however, particle size measurements at this point showed some degree of degradation.

[0254] This part of the study further confirms the findings in the preceding sections and demonstrates that the presence of the TP carrier improves the stability of the mRNA / LNP formulation. Furthermore, a PNPHO concentration of 100 mg / mL was successfully achieved, and even at this high concentration, the mRNA / LNP formulation was more stable than the formulation without a carrier. The increased stability of formulations with higher PNPHO content (including TP050 and TP100) is beneficial for the formulation of long-acting injectables in future studies.

[0255] Figure 17 To investigate the effects of TP035 on mRNA and LNP function, eGFP-mRNA was used, and GFP expression on epithelial cells was studied at different time points using live-cell imaging over a period of up to 48 hours. This study aimed to assess whether the addition of TP035 and its hydrophilic / hydrophobic interactions with LNPs had any adverse effects on mRNA and LNP function. Figure 17 a(i) and Figure 17 a(ii) The results showed that eGFP without LNP did not exhibit GFP expression characteristics on epithelial cells. As expected, the addition of TP035 did not affect eGFP expression. Figure 17 a(iii) However, adding lipofectamine to eGFP-mRNA + TP035 resulted in the expression of [a specific substance] in cells, such as [a specific substance]. Figure 17 a(iv) and Figure 17 b As shown. Nasal delivery of commercial formulations in an in vitro human model b(ii) The observed expression intensity was comparable to that achieved without the TP035 vector. These results confirm that the addition of TP035 did not adversely affect the effectiveness of eGFP-mRNA and the incorporated lipids.

[0256] Figure 18 Figure 18 Deposition patterns of a) carrier-free systems and b) commercial vaccine formulations containing TP035+ in simulated human nasal models were plotted. In this in vitro evaluation, the test formulation was sprayed into nasal models incubated at 37°C using a standard nozzle. In the control group (TP000), significant nasal outflow was observed immediately after application; this equated to ~60% of the spray volume or 150 µL of a 250 µL spray volume. Figure 18 a(i) and Figure 18 a(ii)This significant volume loss and the minimal extent to which the formulation reaches the upper nasal cavity ( Figure 18 a(iii) This is a known drawback of nasal delivery, for example, of standard formulations without a carrier system. On the other hand, no nasal discharge was observed with the application of the TP035+ formulation. Figure 18 b(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. Figure 18 b(ii) and Figure 19 b(iii) The white arrows indicate 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 prevent immediate nasopharyngeal drainage after application of the TP035+ formulation.

[0257] Figure 19 a The deposition patterns of TP000 (control) and TP035+ formulations within 20 minutes after application are shown. Results indicate that, due to the adhesiveness of the carrier system, the intranasal volume retained in the TP035+ group was significantly higher than that in the control group (TP000) at all test time points (i.e., 0, 5, 10, 15, and 20 minutes after application). Polymeric nanocarriers b In the TP035+ group, very little (< 10 µL) pharyngeal outflow was observed within 10 minutes of application. More importantly, the aqueous phase expelled during and after gelation of the TP035+ formulation caused gradual coverage of the nasal area. Therefore, compared with the control, the nasal surface area of ​​the TP035+ formulation was significantly larger at all time points. p < 0.001) higher.

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

[0259] Determination of critical micelle concentration (CMC) The presence of the blood-brain barrier (BBB) ​​makes targeted drug therapy for neurological disorders extremely challenging. This can be explained by the tight endothelial 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 is a suitable solution. This mechanism manipulates the olfactory and trigeminal nerves, transporting 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 into the brain. These NPs, comprising two biocompatible polymers, were formulated in a nasal formulation containing BSA (as a model drug) to bypass the BBB.

[0260] Figure Evaluation of the concentration-driven micelle formation potential of PNPHO polymers ( Reconstitution and viability of lyophilized mRNA / LNP / TP To determine the critical micelle concentration (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 CMC determination.

[0261] Nanoparticles (NPs) made from anionic PNPHO and the cationic polymer chitosan were produced using a composite coagulation technique. This manufacturing technique was employed to improve the stability of the NPs and to encapsulate charged drugs within the nanocomposite. First, negatively charged bovine serum albumin (BSA, a model therapeutic 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. The optimal polymer:polymer ratio for forming the nanocarrier was determined 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. 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 (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 min 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 NPs expelled from the Aptar VP7 nasal spray device was measured using laser diffraction. Malvern Panalytical Spraytec (Worcestershire, UK) was equilibrated at room temperature prior to measurement. 3 mL of the optimized drug 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 two BSA-loaded PNPHO:chitosan 1:1 NP formulations, a silicone nasal mold (KokenCo. 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. Prior to the experiment, a thin layer of Sar-Gel water indicator paste was evenly applied to the nasal mold using a clean brush. Images of the nasal mold were taken 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 the Aptar VP7 nasal spray device with a minimum volume of 3 mL. To determine the drug deposition pattern, the Aptar VP7 device was activated three times in in vitro nasal models, including a USP-approved nasal glass chamber and the 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] A USP-approved nasal glass chamber was used. 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). Upon initiation, the drug in the nasal spray was injected three times into the nasal model. 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 throat 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 infused with AINI three times. To quantify the amount of BSA deposited in each nasal component and NGI platform, 5 mL of Milli-Q water was used for flushing, except for the nasopharynx where 10 mL of Milli-Q water was required for the AINI.

[0269] Figure 20 The following implementation schemes tested the activity of the mRNA / LNP / polymer carrier 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 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 that of the original formulation (freshly prepared; without freeze-drying).

[0271] The mRNA encapsulation was quantified and visualized using developed and previously outlined HPLC methods. Determination of CMC The 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 original formulation containing the carrier (TP025+) and the freeze-dried formulation (TP025+FD).

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

[0273] Results and Analysis Figure 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 further evidenced by the 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 size 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. Dynamic light scattering analysis As shown, samples with PNPHO concentrations <0.5 mg / mL exhibited high PDI (>0.5) or large particle size (>1000 nm), indicating that the self-assembly process of NPs had not yet begun.

[0274] Based on initial CMC data, PNPHO concentrations of 0.5 mg / mL or higher are expected to be suitable for drug encapsulation resulting from the self-assembly process of NP formation. Surface charge measurements revealed that the PNPHO polymer carried a negative charge 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 enabling the effective loading of anionic drug molecules.

[0275] Figure Chitosan was chosen as a model polymer with positive charge properties for formulation due to its good tolerability and permeability-enhancing effects. These properties are crucial for the development of intranasal drug formulations. Compared with blank NP, encapsulation with BSA as a model drug in 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. Determination of encapsulation efficiency This indicates that BSA is a compatible model drug and has a stabilizing effect on the nanocomposite.

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

[0277] When NPs were prepared with different PNPHO:chitosan ratios, the polymer at a ratio of 1:1 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, where high PDI indicated the formation of highly dispersed particles.

[0278] As expected, a 1:1 ratio of PNPHO:chitosan proved to be the optimal formulation due to a significant reduction in particle size. These results suggest that the addition of BSA produces NPs with a more compact nanostructure, likely 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, indicating 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 the nanocomposite due to the promising performance observed in NPs with a PNPHO:chitosan ratio of 1:1. BSA is a common model protein because of its relatively low cost, easy availability, and charge compatibility. Previous literature has described its potential to form nanocomposites with polymers of opposite charge, such as chitosan. (10),(11) After loading BSA into nanocomposites made of different PNPHO:chitosan ratios, the hydrodynamic diameter of the prepared particles was measured.

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

[0281] Encapsulation efficiency and PDI of PNPHO: chitosan 1:1 NPs loaded with different concentrations of BSA (as a model protein) Laser diffraction measurements

[0282] Table 2 Table 2.The results revealed the droplet size characteristics of the nasal formulation. No significant difference in droplet size was observed based on Dv10 for the blank NP and BSA NP (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. Conversely, 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 nanoformulations can influence the droplet properties of nasal sprays.

[0283] PNPHO: Chitosan 1:1 formulations consistently exhibit high encapsulation efficiency, with at least 98% of the BSA encapsulated. The ability of the drug to be encapsulated in the nanocarrier is independent of the initial concentration of BSA added, ranging from 100 to 1000 µg / mL. This can be explained by the saturation theory, which states that when protein encapsulation is performed using electrostatic interactions, the encapsulation efficiency decreases only after reaching the maximum protein concentration. (13) For successful intranasal drug delivery, nanocarriers with high encapsulation rates are crucial to efficiently deliver drugs to the desired brain regions with minimal waste. Increased encapsulation rate of nanocarriers is directly 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%, a 1:1 formulation of PNPHO:chitosan loaded with BSA (1000 µg / mL) was used to further characterize droplet size characteristics and drug deposition patterns. The formulation with the highest initial drug concentration was more likely to achieve a therapeutic dose and produce effective CNS effects in the brain.

[0284] Droplet spray size as shown by Spraytec data Siliceous nose mold

[0285] Figure After 20 minutes of analysis, increased drug deposition was observed in the nasal cavity for the BSA-loaded PNPHO:chitosan 1:1 formulation. Specifically, the nasal turbinate region showed relatively abundant drug deposition, as indicated by the bright pink color of Sar-Gel. Deposition study of Pfizer Comirnaty vaccine in physiological saline and TP035 formulation ).

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

[0287] Figure The Pfizer-BioNTech Comirnay vaccine is one of the most popular vaccines on the market for immunization against COVID-19 infection. While it has proven effective, some limitations remain regarding cold chain transportation 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 were conducted using the BIVAX nasal spray device (…). ​ The results showed that the TP035 formulation could specifically deposit in a particular area (the olfactory region), and the deposition lasted for up to 20 minutes after deposition. No significant leakage was observed during this period. The saline formulation, on the other hand, deposited in a larger area of ​​the nasal cavity and further diffused within 20 minutes to cover most of the nasal cavity. Significant leakage 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 fields of biomedicine and vaccines. It can be seen that 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 micellar-like structures (which are not in their hydrogel state under ambient conditions).

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

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

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

[0294] v. Testing the function of mRNA+LNP with and without PNPHO showed that adding 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 formulations 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 vector system, as a physical solvent system for intranasal delivery, is compatible with some commercial COVID-19 vaccines that may contain a mixture of mRNA and LNP.

[0296] References

[0297]

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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