Green ionic self‐assembly and molecular recognition method for fabrication of polymeric chitosan nanomaterial and its application for active molecule delivery

Abstract

This invention presents an environmentally friendly method for synthesizing chitosan-based polymeric nanomaterials through a two-stage assembly: (i) ionic complexation between amino-protonated chitosan and nucleotide phosphates, followed by (ii) molecular-recognition-driven pairing of complementary nucleotide bases. The process forms nanostructures featuring nucleotide-paired hydrophobic micro-domains that enable high-efficiency encapsulation of hydrophobic active molecules (e.g., lutein, paracetamol, ibuprofen, sildenafil, melatonin, vitamins A, B, D, E, K) and hydrophobic-containing hydrophilic molecules (e.g., folic acid, B-group vitamins). These chitosan nanomaterials provide sustained release via muco-adhesive interactions, offering prolonged retention compared with conventional carriers. Sustainability advantages include aqueous, simple synthesis; absence of toxic solvents or chemicals; and scalable production using energy-efficient dialysis and lyophilization or low-temperature vacuum drying. The nanomaterials are applicable across diverse delivery platforms, including rapidly disintegrating tablets, topical creams, injectable CRISPR / Cas9 suspensions, and bio-adhesive sublingual sprays.

Description

Green Ionic Self‐Assembly and Molecular Recognition Method for Fabrication of Polymeric Chitosan Nanomaterial and its Application for Active Molecule Delivery

[0001] The present invention relates to a novel method for the preparation of chitosan nanomaterial through an ionic interaction process followed by self-assembly via a molecular recognition approach. This innovative method allows for the formation of nanomaterial with enhanced stability, nucleotide mimic-induced hydrophobic micro-domain and improved encapsulation efficiency of hydrophobic and hydrophobic-containing active molecules.

[0002] The invention further relates to the application of these chitosan nanomaterials as an advanced molecular recognition-based delivery system, owing to their nucleotide mimic-induced hydrophobic micro-domain. These nanomaterials have a high binding affinity for hydrophobic active molecules - including pharmaceuticals, bioactive compounds, and cosmetic actives.

[0003] A key advantage of this invention is its reliance on biocompatible and environmentally friendly preparation techniques, ensuring minimal toxicity and adherence to green chemistry principles. The resulting chitosan nanomaterial is highly versatile and can be formulated into various delivery systems, including solid, liquid, and cream-based formulations. This adaptability makes them suitable for a wide range of applications, such as drug delivery, cosmetic formulations, and gene therapy, providing a sustainable and efficient platform for active ingredient delivery.

[0004] In recent years, green chemistry and the use of mild, environmentally friendly preparation routes have gained popularity in the research and development of nanomaterials. Chitosan, a biodegradable and biocompatible polymer, has been widely used for various pharmaceutical applications, including drug and gene delivery systems.1-7

[0005] Among the various preparation techniques, spray drying is one of the commonly used methods. Chitosan is typically dissolved in aqueous acetic acid, and nanoparticles (NPs) are formed by atomizing the solution at air temperatures between 120°C and 150°C.8While effective, spray drying has its limitations when used to produce chitosan nanoparticles, including the use of acidic solvents and variability in particle size distribution.

[0006] Supercritical CO2-assisted solubilization and atomization (SCASA) is another pioneering green method, free of harmful acids and solvents, which employs water and CO2as the only components during preparation. However, SCASA requires extended processing time and often yields larger particles with a broad distribution, limiting its effectiveness for certain applications.9

[0007] My previous work, as described in patent WO08017839,10titled "Polymeric Micellar Clusters and Their Uses in Formulating Drugs," involves a method requiring multiple synthetic steps for the preparation of chitosan-based self-assembled polymeric micellar clusters via the hydrophobic interaction of long carbon chains conjugated to chitosan. While effective in certain applications, this approach is complex, relies heavily on synthetic chemistry, and does not fully align with environmentally friendly principles. Additionally, the multi-step process reduces the overall yield of the final product, limiting its scalability and broader application.

[0008] CN102908626B (Ma et al.) 11exploits G-C hydrogen bonding in chitosan-hyaluronic acid nanofiber films. However, dimethylformamide (DMF) solvent use and covalent attachment of cytosine’s amino group to HA induce steric hindrance, disrupting native G-C pairing and negating green credentials.

[0009] CN105327356A (Tan et al.)12 fabricates magnetic nanogels by conjugating chitosan with adenine, complexing it with thymidine-heparin, and vacuum-drying. Organic solvents, chemical reactions, and slow processing render it non-green.

[0010] Sun et al.13 developed STAT3-targeted nanoparticles via: (i) chitosan protonation, (ii) dialysis / lyophilization, (iii) VCPH copolymer synthesis (vitamin E-chitosan-PEG-histidine), (iv) doxorubicin loading, and (v) pDNA complexation. Multi-step organic synthesis and purification impede scalability.

[0011] Pakornpadungsit et al.14form DNA-chitosan complexes using acetic acid, with freeze-dried outputs. Difficult-to-source DNA creates sterically crowded structures where chitosan chains block drug access to hydrophobic domains, while DNA purification hinders scale-up.

[0012] In contrast, the present invention streamlines the synthesis process by eliminating harsh conditions and enhancing production efficiency. The approach begins with the ionic binding of phosphate groups in single nucleotides to the amino groups of chitosan.15Subsequently, molecular recognition between complementary nucleotides—such as A-T, C-G, or A-U—drives the self-assembly process, resulting in a more practical and sustainable green method for polymeric chitosan nanomaterial formation.

[0013] The present invention provides a novel green technology for preparing chitosan nanomaterial using an ionic interaction process followed by self-assembly through a molecular recognition approach. This innovative technique enables the nucleotide-similarity hydrophobic micro-domain containing polymeric nanomaterial formation, enhancing its stability and encapsulation efficiency for hydrophobic and hydrophobic-containing active molecules.

[0014] This invention further introduces the application of these nucleotide-similarity hydrophobic micro-domain containing polymeric chitosan nanomaterial as an advanced molecular recognition-based delivery system. These nanomaterials potentially have high binding affinity for hydrophobic or hydrophobic containing active molecules, including pharmaceuticals, bioactive compounds, and cosmetic actives.

[0015] A distinguishing advantage of this method is its green technology and reliance on biocompatible and environmentally friendly preparation techniques, ensuring minimal toxicity and adherence to green chemistry principles. The resulting nucleotide-similarity hydrophobic micro-domain containing polymeric chitosan nanomaterial can be formulated into various delivery systems, including solid, liquid, and cream-based formulations, making them suitable for applications in drug delivery, cosmetics, and gene therapy.

[0016] This invention builds upon previous work described in patent WO08017839, which involved the preparation of chitosan-based self-assembled polymeric micellar clusters through a complex multi-step synthetic process. While effective, that approach relied heavily on synthetic chemistry, reducing overall yield and scalability while deviating from environmentally sustainable principles.The present invention applies water exclusive solvent system, no chemical synthesis steps, eliminates harsh conditions, and enhances production efficiency. The method involves an initial binding of single nucleotides via ionic interaction between the amino groups in chitosan and the phosphate groups in nucleotides,15followed by molecular recognition between complementary nucleotides to facilitate self-assembly. This approach offers a more practical, scalable, and sustainable alternative for nucleotide-similarity hydrophobic micro-domain containing polymeric nanomaterial formation, broadening its applicability in various industries.Detailed Description of the Invention:

[0017] The chitosan (CAS No.: 9012-76-4) employed in our formulation is characterized by:(a) A degree of deacetylation ranging from 70% to 95%; and(b) A molecular weight of 10–150 kDa.

[0018] The nucleotides comprise monophosphates, diphosphates, or triphosphates of adenine, thymine, cytosine, guanine, or uracil. To ensure the final nanomaterial retains a net positive charge, the molar ratio of amino groups in chitosan to the nucleotides in the final nanomaterial is maintained at >2:1 (monophosphates of adenine, thymine, cytosine, guanine, or uracil), >3:1 (diphosphates of adenine, thymine, cytosine, guanine, or uracil), or >4:1 (triphosphates of adenine, thymine, cytosine, guanine, or uracil).

[0019] Preparation of Chitosan NanomaterialThe synthesis of nucleotide-similarity hydrophobic micro-domain containing polymeric chitosan nanomaterial can be prepared as the following multi-step protocol:Amino Protonation of ChitosanChitosan undergoes amino protonation via treatment with a diluted acid (e.g., glacial acetic acid, hydrochloric acid, or phosphoric acid).The protonated chitosan is dialyzed against water to eliminate excess acid, ensuring purity and optimal reactivity.The product is freeze-dried or vacuum-dried at temperatures ≤40°C to preserve structural integrity.Unlike conventional methyl quaternization methods (e.g., using methyl iodide10), this approach is simpler, more efficient, and eco-friendly.Self-Assembly of Base-Pair Hydrophobic Micro-Domain-Containing Chitosan Nanomaterial as per any of the following three assembly strategies:

[0020] (a) Sequential Assembly with a Single NucleotideAmino-protonated chitosan is dissolved in water, followed by the addition one kind or a few kinds of non-pair nucleotides. Ionic interactions between chitosan’s amino groups and the nucleotide’s phosphate groups drive spontaneous assembly.One kind or a few kinds of complementary nucleotides is introduced, inducing formation of hydrophobic micro-domain via molecular recognition of base pairing (e.g., A–T, C–G, or A–U).

[0021] (b) Preformed Base-Pair IntegrationTwo kinds of complementary nucleotides, or a few kinds of paired complementary nucleotides (e.g. A–T, C–G, or A–U at same molar ratio) are first dissolved in aqueous solution;Amino-protonated chitosan is subsequently introduced, facilitating ionic interactions between the phosphate groups of the nucleotide and the protonated amino groups of chitosan. Then, following the molecular recognition of complementary base pairs enables self-assembly into a polymeric nanomaterial containing hydrophobic micro-domains.

[0022] (c) Simultaneous Co-AssemblySeparately, the amino-protonated chitosan is ionically complexed with one or a few kinds of non-pair nucleotides of complementary nucleotides at same molar ratio (e.g., adenine - thymine, adenine - uracil, or cytosine -=+ guanine).The two complementary complexes interact via molecular recognition of base pairing after mixing, yielding hydrophobic base pair based micro-domains within the polymeric matrix.

[0023] The assembled nanomaterial is centrifuged to isolate the product, followed by freeze-drying or low-temperature vacuum drying (≤40°C) to ensure stability.Molecular Recognition and Encapsulation

[0024] Molecular recognition between dyes and DNA / RNA base-pair hydrophobic regions is well-documented.16-19The micro-hydrophobic areas within the polymeric chitosan nanomaterial mimic these base-pair regions found in DNA / RNA. This similarity enables the nanomaterial to exhibit molecular recognition properties like same area in the DNA / RNA, allowing them to encapsulate and deliver hydrophobic active molecules and hydrophobic-containing active compounds. These include, but are not limited to:Hydrophobic drugs (e.g., melatonin, sildenafil, ibuprofen, paracetamol)Amphiphilic bioactive moleculesVitamins (A, B, C, D, E, K)Cosmetic actives and fragrancesSunscreen agents (e.g., Octocrylene, Avobenzone, Homosalate, MBBT, Bemotrizinol, PBSA, DHHB)Lutein, folic acid, and other bioactive compounds

[0025] Additionally, these nucleotide-chitosan polymeric nanomaterials possess a positive charge, enabling them to function as antiseptic reagents. They can also bind to the membranes of mucosal cells, extending the retention time of active molecules and enhancing their delivery. Moreover, the positively charged nanomaterial can interact with negatively charged DNA and RNA, making them promising carriers for gene therapy.Applications of the Invention:

[0026] The nucleotide-similarity hydrophobic micro-domain containing polymeric chitosan nanomaterial can be applied using three primary methods:Pre-Loaded Formulation:Nanomaterial are first loaded with active pharmaceutical ingredients or active molecules and then incorporated into the final product.In-Situ Loading:Nanomaterial are applied without pre-loaded active pharmaceutical ingredients or active molecules, allowing the drug to be incorporated with chitosan-based nanomaterial directly during product formulation.Simultaneous Assembly and Loading:Amino-protonized chitosan, paired single nucleotides, and the active pharmaceutical ingredients or active molecules are formulated together into the final product, enabling concurrent nanomaterial formation, molecular recognition, and drug loading upon application to patients.

[0027] As mentioned previously, these nucleotide-chitosan polymeric nanomaterial can be utilized for the delivery of hydrophobic drugs, hydrophobic containing active molecules and fragrant compounds. The nanomaterial can encapsulate hydrophobic or hydrophobic containing amphiphilic active molecules within their hydrophobic micro-domain formed through molecularly recognized nucleotide pairs.

[0028] The resulting nucleotide-similarity hydrophobic micro-domain containing polymeric chitosan nanomaterial can be formulated into various delivery systems, including:Solid formulations:Tablets, hard capsules, and orally disintegrating tablets.Liquid, cream, or spray formulations:The chitosan nanoparticle-encapsulated functional actives can be re-dispersed into creams or liquid sprays for topical or oral administration.Examples:

[0029] Example 1: Sildenafil Oral Disintegrating Tablets (ODTs)

[0030] The nucleotide-similarity hydrophobic micro-domain containing polymeric chitosan nanomaterial is formulated with sildenafil into orally disintegrating tablets (ODTs). Each tablet contained 50 mg of encapsulated sildenafil, along with excipients including 50 mg sucrose, 50 mg mannitol, 80 mg microcrystalline cellulose, 20 mg crospovidone, 2.5 mg sucralose, 2 mg strawberry flavor, 3 mg magnesium stearate, and 2 mg colloidal silicon dioxide. The tablets were produced via direct compression, enabling rapid disintegration within 30 seconds, improving patient compliance and taste masking.

[0031] Example 2: Melatonin Oral Disintegrating Tablets

[0032] The nucleotide-similarity hydrophobic micro-domain containing polymeric chitosan nanomaterial is formulated with melatonin into orally disintegrating tablets (ODTs) for enhanced sleep support. Each tablet contained 5 mg encapsulated melatonin, combined with 50 mg sucrose, 50 mg mannitol, 80 mg microcrystalline cellulose, 20 mg crospovidone, 2.5 mg sucralose, 2 mg strawberry flavor, 3 mg magnesium stearate, and 2 mg colloidal silicon dioxide. The formulation facilitates rapid disintegration within 30 seconds, improving bioavailability and user experience.

[0033] Example 3: Ibuprofen Oral Disintegrating Tablets

[0034] The nucleotide-similarity hydrophobic micro-domain containing polymeric chitosan nanomaterial is formulated with ibuprofen into orally disintegrating tablets (ODTs) for pain relief and anti-inflammatory effects. Each tablet contained 200 mg encapsulated ibuprofen, 50 mg sucrose, 50 mg mannitol, 80 mg microcrystalline cellulose, 20 mg crospovidone, 2.5 mg sucralose, 2 mg strawberry flavor, 3 mg magnesium stearate, and 2 mg colloidal silicon dioxide. The formulation provided rapid disintegration within 30 seconds, improving patient compliance and onset of action.

[0035] Example 4: Paracetamol Oral Disintegrating Tablets

[0036] The nucleotide-similarity hydrophobic micro-domain containing polymeric chitosan nanomaterial is formulated with paracetamol into orally disintegrating tablets (ODTs) for fever and pain management. Each tablet contained 250 mg encapsulated paracetamol, along with 50 mg sucrose, 50 mg mannitol, 80 mg microcrystalline cellulose, 20 mg crospovidone, 2.5 mg sucralose, 2 mg strawberry flavor, 3 mg magnesium stearate, and 2 mg colloidal silicon dioxide. The formulation ensured rapid onset of action through fast disintegration within 30 seconds.

[0037] Example 5: Glutathione Oral Disintegrating Tablets

[0038] The nucleotide-similarity hydrophobic micro-domain containing polymeric chitosan nanomaterial is formulated with glutathione into orally disintegrating tablets (ODTs). Each tablet contained 50 mg glutathione, 60 mg mannitol, 40 mg sucrose, 80 mg microcrystalline cellulose, 20 mg crospovidone, 3 mg magnesium stearate, and 2 mg colloidal silicon dioxide. The tablets disintegrated within 20 seconds, ensuring rapid absorption and increased bioavailability.

[0039] Example 6: GLP-1 Oral Disintegrating Tablets

[0040] The nucleotide-similarity hydrophobic micro-domain containing polymeric chitosan nanomaterial is formulated with 2.4 mg GLP-1 (glucagon-like peptide-1) into orally disintegrating tablets (ODTs). Each tablet included 60 mg mannitol, 50 mg sucrose, 90 mg microcrystalline cellulose, 25 mg crospovidone, 3 mg magnesium stearate, and 2 mg colloidal silicon dioxide. The formulation provided rapid disintegration and effective GLP-1 delivery for diabetes management.

[0041] Example 7: Vitamin D Soft Gel

[0042] The nucleotide-similarity hydrophobic micro-domain containing polymeric chitosan nanomaterial is formulated with vitamin D into a soft gel formulation containing 10% olive oil, 2% lecithin, and 88% gelatin base. Each soft gel delivered a bioavailable dose of 25 mcg (1,000 IU) vitamin D3, improving solubility and absorption for bone health.

[0043] Example 8: Encapsulated Vitamin A for Skincare

[0044] The nucleotide-similarity hydrophobic micro-domain containing polymeric chitosan nanomaterial is formulated with vitamin A into a topical serum for enhanced stability and skin penetration. The formulation contained 1% encapsulated vitamin A, 5% hyaluronic acid, 3% glycerin, and 91% purified water. This encapsulation improved anti-aging efficacy, making it ideal for skincare applications.

[0045] Example 9: Vitamin E Topical Cream

[0046] The nucleotide-similarity hydrophobic micro-domain containing polymeric chitosan nanomaterial is formulated with vitamin E nanomaterial into a topical cream comprising 5% shea butter, 3% glycerin, 1% allantoin, and 91% purified water. The formulation provided antioxidant protection, improved skin hydration, and enhanced delivery efficiency.

[0047] Example 10: Eugenol Local Antiseptic Spray

[0048] A spray solution containing nucleotide-similarity hydrophobic micro-domain containing polymeric chitosan nanomaterial and eugenol was developed for antiseptic applications. The formulation included 1% eugenol, 0.5% glycerin, and 98.5% purified water. The spray has efficacy in dental antiseptics and men's health applications, including localized desensitization for premature ejaculation.

[0049] Example 11: CRISPR / Cas9 Gene Editing Delivery

[0050] The nucleotide-similarity hydrophobic micro-domain containing polymeric chitosan nanomaterial is loaded with Cas9 enzymes.

[0051] Example 12: mRNA-Based Vaccine Delivery

[0052] The nucleotide-similarity hydrophobic micro-domain containing polymeric chitosan nanomaterial is formulated with mRNA encoding a target antigen.

[0053] Example 13: Probiotic Encapsulation

[0054] The nucleotide-similarity hydrophobic micro-domain containing polymeric chitosan nanomaterial is formulated with Lactobacillus acidophilus for targeted gut delivery.Advantages of the Invention

[0055] My invention delivers a breakthrough green methodology:Solvent System: Exclusively aqueous (eliminating ALL organic solvents / acids)Process: Single-step self-assembly via preserved nucleotide base pairing (A-T / C-G)Functionality: Unobstructed hydrophobic microdomains for efficient drug loadingScalability: No purification, DNA, or covalent reactions, reduced steps

[0056] 1. Mansouri, S., Lavigne, P., Corsi, K., Benderdour, M., Beaumont, E., & Fernandes, J. C. (2004). Chitosan-DNA Nanomaterial as non-viral vectors in gene therapy: Strategies to improve transfection efficacy. European Journal of Pharmaceutics and Biopharmaceutics, 57(1), 1–8. https: / doi.org / 10.1016 / s0939-6411(03)00155-3

[0057] 2. Jayakumar, R., Chennazhi, K. P., Muzzarelli, R. A. A., Tamura, H., Nair, S. V., & Selvamurugan, N. (2010). Chitosan conjugated DNA Nanomaterial in gene therapy. Carbohydrate Polymers, 79(1), 1–8. https: / doi.org / 10.1016 / j.carbpol.2009.12.008

[0058] 3. Antoniou, V., Mourelatou, E. A., Galatou, E., Avgoustakis, K., & Hatziantoniou, S. (2024). Gene therapy with chitosan Nanomaterial: Modern formulation strategies for enhancing cancer cell transfection. Pharmaceutics, 16(7), 868. https: / doi.org / 10.3390 / pharmaceutics16070868

[0059] 4. Dong, L., Li, Y., Cong, H., Yu, B., & Shen, Y. (2024). A review of chitosan in gene therapy: Developments and challenges. Carbohydrate Polymers, 324, 121562. https: / doi.org / 10.1016 / j.carbpol.2023.121562

[0060] 5. Carrillo, C., Suñé, J. M., Pérez-Lozano, P., García-Montoya, E., Sarrate, R., Fàbregas, A., Miñarro, M., & Ticó, J. R. (2014). Chitosan Nanomaterial as non-viral gene delivery systems: Determination of loading efficiency. Biomedicine & Pharmacotherapy, 68(6), 775–783. https: / doi.org / 10.1016 / j.biopha.2014.07.009

[0061] 6. Yanat, M., & Schroën, K. (2021). Preparation methods and applications of chitosan Nanomaterial: With an outlook toward reinforcement of biodegradable packaging. Reactive and Functional Polymers, 161, 104849. https: / doi.org / 10.1016 / j.reactfunctpolym.2021.104849

[0062] 7. Akdaşçi, E., Duman, H., Eker, F., Bechelany, M., & Karav, S. (2025). Chitosan and its Nanomaterial: A multifaceted approach to antibacterial applications. Nanomaterials, 15(2), 126. https: / doi.org / 10.3390 / nano15020126

[0063] 8. Demirbolat, G. M., & Degim, İ. T. (2013). Preparation of chitosan Nanomaterial by nano spray drying technology. FABAD Journal of Pharmaceutical Sciences, 38(3), 127–133.

[0064] 9. Misra, S. K., & Pathak, K. (2020). Supercritical fluid technology for solubilization of poorly water-soluble drugs via micro- and nanosized particle generation. ADMET & DMPK, 8(4), 355–374. https: / doi.org / 10.5599 / admet.811

[0065] 10. Ijeoma, F. U., Schatzlein, A. G., & Hou, X. (2008). Polymeric micellar clusters and their uses in formulating drugs (Patent No. WO2008 / 017839). World Intellectual Property Organization.

[0066] 11. Ma, G.-P., Wang, Z.-L., Nie, J., & Fang, D.-W. (2012). Method for preparing chitosan / transparent acid derivative nanofiber composite film through freezing drying technology (Patent No. CN102908626B). China National Intellectual Property Administration.

[0067] 12. Tan, H.-P., Fan, M., Yan, J.-X., Ma, Y., Qin, J.-C., & Li, X.-M. (2014). Magnetic polysaccharide nanometer gel material preparation method (Patent No. CN105327356A). China National Intellectual Property Administration.

[0068] 13. Sun, L., Sun, J., Li, C., Wu, K., Gu, Z., Guo, L., Zhou, Y., Han, B., & Chang, J. (2021). STAT3-specific nanocarrier for shRNA / drug dual delivery and tumor synergistic therapy.Bioactive Materials, 41, 137–157.

[0069] 14. Pakornpadungsit, P., Prasopdee, T., Mongkoldhumrongkul Swainson, N., Chworos, A., & Smitthipong, W. (2020). DNA:chitosan complex, known as a drug delivery system, can create a porous scaffold.Polymer Testing, 83, 106333. https: / doi.org / 10.1016 / j.polymertesting.2020.106333

[0070] 15. Amri, Y., Fajri, R., Fitriani, Novitasari, P., & Zulfajri, M. (2020). The effect of tripolyphosphate (TPP) volumes on the synthesis of chitosan Nanomaterial using ionic gelation. Proceedings of the 2nd International Conference on Science, Technology, and Modern Society (ICSTMS 2020) Advances in Social Science, Education and Humanities Research, volume 576, pp. 213–216. Atlantis Press.

[0071] 16. Dervan, P. B. (2001). Molecular recognition of DNA by small molecules. Bioorganic & Medicinal Chemistry, 9(8), 2215–2235. https: / doi.org / 10.1016 / S0968-0896(01)00066-5

[0072] 17. Lau, P. S., & Li, Y. (2011). Functional nucleic acids as molecular recognition elements for small organic and biological molecules. Current Organic Chemistry, 15(4), 557–575. https: / doi.org / 10.2174 / 138527211794474537

[0073] 18. Ihmels, H., & Otto, D. (2005). Intercalation of organic dye molecules into double-stranded DNA — General principles and recent developments. In F. Würthner (Ed.), Supramolecular Dye Chemistry (pp. 161–204). Springer.

[0074] 19. Agafontsev, A. M., Ravi, A., Shumilova, T. A., Oshchepkov, A. S., & Kataev, E. A. (2018). Molecular receptors for recognition and sensing of nucleotides. Chemistry – A European Journal, 25(11), 2684–2694. https: / doi.org / 10.1002 / chem.201802978

Claims

A green method for preparing nucleotide-chitosan polymeric nanomaterial, consisting of:(a) amino-protonated chitosanTreating chitosan with a diluted acid selected from glacial acetic acid, hydrochloric acid, or phosphoric acid; dialyzing the protonated chitosan against water to remove residual acid; drying the dialyzed, protonated chitosan by freeze-drying or vacuum-drying at a temperature below 40°C;(b) Complexing of the amino-protonated chitosan with one kind nucleotide or mixture of a few kinds of non-paired nucleotides, followed by the addition of the same ratio of one kind of or mixture of a few kinds of complementary nucleotides to form base-pair hydrophobic micro-domains containing polymeric nanomaterial.The method of claim 1, wherein step (b) comprises one of the following assembly approaches:(i) dissolving the protonated chitosan in water, adding one kind of or mixture of a few kinds of non-pair nucleotides, and then introducing another protonated chitosan ionic interacted with its or the mixture of a few kinds of relative complementary nucleotides to form hydrophobic micro-domains via A-T, C-G, or A-U base pairing;(ii) pre-dissolving same molar ratio of two paired complementary nucleotides or mixture of a few kinds of paired complementary nucleotides (eg. A-T, C-G, or A-U base pairing) in aqueous solution before adding the protonated chitosan; or(iii) separately complexing the protonated chitosan with same molar ratio of any two complementary nucleotides (eg. A-T, C-G, or A-U base pairing) and mixing these two kinds of complexes to self-assemble into base-pair hydrophobic micro-domains containing chitosan-nucleotides polymeric nanomaterial.The method of claim 1, wherein the chitosan has:(a) a degree of deacetylation between 70% and 95%; and(b) a molecular weight of 10–150 kDa.The method of claim 1, wherein the nucleotides are selected from monophosphates, diphosphates, or triphosphates of adenine, thymine, cytosine, guanine, or uracil. To ensure the final nanomaterial retains a net positive charge, the molar ratio of amino groups in chitosan to the nucleotides is maintained at >2:1 (monophosphates of adenine, thymine, cytosine, guanine, or uracil), >3:1 (diphosphates of adenine, thymine, cytosine, guanine, or uracil), or >4:1 (triphosphates of adenine, thymine, cytosine, guanine, or uracil).The method of claim 1, wherein the assembled nanomaterial is centrifuged to isolate the product, followed by freeze-drying or low-temperature vacuum drying (≤40°C) to ensure stability.The method of claim 1, further comprising encapsulating a hydrophobic active molecule or hydrophobic-containing active molecule within the base-pair hydrophobic micro-domains of the nanomaterial via molecular recognition.A nucleotide-chitosan polymeric nanomaterial produced by the method of claim 1, consisting:(a) protonated chitosan ionically bound to nucleotides;(b) complementary nucleotide pairs forming hydrophobic micro-domains via A-T, C-G, or A-U base pairing; and(c) a positive surface charge which enables binding to mucosal membranes or negatively charged genetic material.The nanomaterial of claim 7, wherein the hydrophobic micro-domains encapsulate an active molecule selected from:(a) hydrophobic drugs, including melatonin, sildenafil, ibuprofen, paracetamol etc.;(b) cosmetic actives, including vitamins A, D, E, sunscreen agents, or fragrances; or(c) probiotics, including Lactobacillus acidophilus.A delivery system comprising the nanomaterial of claim 7, formulated as:(a) a solid dosage form selected from tablets, orally disintegrating tablets, hard capsules;(b) a topical formulation selected from creams, serums, or sprays; or(c) a liquid suspension for oral or injectable administration.The delivery system of claim 8, wherein the oral distinguish tablet form comprises:(a) of encapsulated active molecule;(b) excipients including sucrose, mannitol, microcrystalline cellulose, and crospovidone; and(c) a disintegration time of less than 30 seconds.A method for targeted delivery of an active molecule, comprising:(a) preparing the nanomaterial of claim 7;(b) loading the active molecule into the hydrophobic micro-domains via molecular recognition; and(c) administering the loaded nanomaterial to a subject,The method of claim 11, wherein the active molecule is delivered for:(a) therapeutic use, including pain relief, diabetes management, or sleep supporting;(b) cosmetic use, including anti-aging, UV protection, or skin hydration; or(c) antiseptic use, including dental or topical applications.Use of the nanomaterial of claim 7 as an antiseptic reagent wherein it binds to microbial membranes.The nanomaterial of claim 7 has a positively charged surface which can interact with mucosal surfaces, prolonging retention time and enhancing the therapeutic efficacy of encapsulated active molecules.Use of the nanomaterial of claim 7 in gene therapy, wherein the positive surface charge facilitates complexation with negatively charged DNA, RNA, or CRISPR / Cas9 components.