Fluorinated zwitterionic polyurethane gene vector compound and preparation method thereof
By designing a fluorinated zwitterionic polyurethane gene carrier complex, the problems of polymer carrier penetration and cell internalization in a mucous environment were solved, enabling efficient gene delivery and disease treatment.
Patent Information
- Application Number
- CN202511829975.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-05
- Publication Date
- 2026-03-03
AI Technical Summary
Existing polymer vectors have poor mucus penetration properties during gene delivery, and carrying positive charges is detrimental to cell internalization, thus limiting effective gene transfection.
A fluorinated zwitterionic polyurethane gene carrier complex is used to load genes and achieve efficient delivery in a mucus environment through intramolecular hydrogen bonding. The particle size is 100~200nm, and it has good mucus penetration performance and stability.
It improved gene transfection efficiency, overcame the obstacles of the mucus environment, reduced cytotoxicity, and enabled gene-level therapy for fibrosis and inflammation-related diseases.
Smart Images
Figure CN121588239A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a fluorinated zwitterionic polyurethane gene carrier complex and its preparation method, belonging to the field of biomedical polymer materials technology. Background Technology
[0002] Generally speaking, atomized nanosystems are considered an effective treatment for lung diseases due to their advantages such as high lung deposition rate, low therapeutic dose, safety, and good therapeutic effect. With in-depth research into atomized drug carriers, in addition to small molecule drugs, inhalable nanoparticles have also successfully loaded large molecules such as proteins, siRNAs, and miRNAs. Among these, miRNAs are 19-22 nucleotide sequences, a class of evolutionarily conserved non-coding small RNAs that can bind to the 3′ untranslated region (UTR) of target genes and inhibit the translation of target genes or the degradation of mRNA. Increasing evidence suggests that the occurrence of pulmonary fibrosis is closely related to some miRNAs; for example, miR-21 and miR-29 were upregulated and downregulated, respectively, in the lungs of mice with bleomycin (BLM)-induced pulmonary fibrosis. Therefore, the prospect of treating diseases at the gene level is very promising.
[0003] Currently, the development of various gene vectors is very rapid. Non-viral vectors, such as cationic liposomes, polymer vectors, dendritic macromolecules, and inorganic nanoparticles, can all achieve efficient gene delivery. Among these, polymer vectors have attracted much attention from researchers due to their ease of functionalization, low toxicity, and high stability. Polyurethane, short for polyurethane, is a condensation polymer synthesized from macromolecular polyols, diisocyanates, and chain extenders. Its chemical structure can be designed by controlling the types of monomers, giving it good mechanical properties and biocompatibility, making it one of the most widely used synthetic polymer biomaterials.
[0004] Therefore, combining polyurethane materials with nanotechnology to achieve gene loading and delivery shows broader promise in the treatment of various diseases. Summary of the Invention
[0005] One objective of this invention is to address the shortcomings of existing polymer carriers, such as poor mucus penetration performance and the fact that most carry a positive charge, which hinders cell internalization and limits effective gene transfection. This invention provides a fluorinated zwitterionic polyurethane gene carrier complex and its preparation method. The resulting gene carrier complex has a particle size of 100-200 nm, good stability, and excellent mucus penetration performance. This nanosystem enables efficient gene delivery.
[0006] Another objective of this invention is to provide applications of the aforementioned fluorinated zwitterionic polyurethane gene carrier complex. Many fibrosis-related diseases are accompanied by changes in gene expression at the gene level. This means that we can achieve effective treatment of diseases at the gene level by up- and down-regulating disease-related pathways. In the molecular structure of fluorinated zwitterionic polyurethane, the fluorinated side chains can achieve effective gene loading through intramolecular hydrogen bonds. In addition, the zwitterionic structure can help nanosystems overcome many mucus environments in the body, improve the effective delivery of genes to lesion sites, and ultimately achieve therapeutic effects on fibrosis or inflammation-related diseases.
[0007] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing a fluorinated zwitterionic polyurethane gene carrier complex includes the following steps: adding 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecano-1-octylthiol PFO and zwitterionic polyurethane PUSB4 to a solvent to carry out a fluorination grafting reaction to prepare fluorinated zwitterionic polyurethane PFSBU; mixing the fluorinated zwitterionic polyurethane PFSBU and a gene together in a solution environment, shaking and incubating, and after multiple washing and redispersing of the obtained particles, obtaining the fluorinated zwitterionic polyurethane gene carrier complex.
[0008] In the above technical solution, the temperature of the fluorination grafting reaction is 25~50℃, and the total reaction time is 6~48h.
[0009] Furthermore, to construct a carrier with enhanced mucus penetration ability, an amphoteric polyurethane PUSB4 containing a higher content of SB-diol repeating units was used. It was prepared via a two-step synthesis procedure, the preparation method of which is as follows: First, hexamethylene diisocyanate (HDI), polypropylene fumarate (PPF), and 3-(methylbis(2-hydroxyethyl)amino)propane-1-sulfonate (SB-diol) are subjected to solution polymerization in a solvent, followed by chain extension reaction with ketethiodiamine (TK-NH2) to obtain zwitterionic polyurethane PUSB4, wherein the molar ratio of PPF to SB-diol is 1:2 to 1:4, and the solvent is dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF).
[0010] Furthermore, the mass ratio of PFO to PUSB4 is 5:1 to 2:1.
[0011] Furthermore, the solvent is one or a mixture of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), and dimethylacetamide (DMAC).
[0012] Furthermore, the solution environment is at least one of PVA, sodium cholate, PBS, and deionized water.
[0013] Furthermore, the gene is at least one of DNA, siRNA, and miRNA.
[0014] Furthermore, the mass concentration ratio of PFSBU to the gene is 30:1 to 2.5:1.
[0015] Furthermore, the oscillation incubation time is 5–30 min.
[0016] A fluorinated zwitterionic polyurethane gene carrier complex, prepared by any of the methods described above, wherein the gene is miRNA, the complex has a particle size of 100-200 nm, the particle size is stable after storage in water or atomization, and it can penetrate mucus.
[0017] The fluorinated zwitterionic polyurethane has a number-average molecular weight of 40–90 kDa. The corresponding grafting rate is 26%–42%, which can be used to screen gene transfection vectors with higher transfection efficiency.
[0018] By utilizing the above technical solution, compared with existing nanogene carriers, the fluorinated zwitterionic polyurethane gene carrier composite prepared by this invention has the following advantages: (1) The fluorinated zwitterionic polyurethane gene carrier is bound by intramolecular hydrogen bonds, rather than by traditional electrostatic adsorption. At the same time, the prepared fluorinated zwitterionic polyurethane gene carrier complex carries a negative charge, which is more conducive to cell endocytosis and greatly helps to improve transfection efficiency.
[0019] (2) The fluorinated zwitterionic polyurethane gene carrier has a small particle size and excellent stability, and the entire nanocarrier has excellent mucus penetration performance. The mucus environment in vivo usually hinders the effective delivery of genes by various gene carriers, but the fluorinated zwitterionic polyurethane gene carrier can overcome this problem well.
[0020] (3) In addition, the fluorinated zwitterionic polyurethane gene carrier itself has the effect of eliminating reactive oxygen species, which can reduce the concentration of various reactive oxygen species in the tissue microenvironment and alleviate oxidative stress damage. Therefore, it can achieve synergistic treatment of diseases with the delivered gene.
[0021] (4) Compared with currently commercialized vectors, the fluorinated zwitterionic polyurethane gene vector described above not only improves transfection efficiency but also possesses excellent biocompatibility. Compared with the commercial vector PEI, the fluorinated zwitterionic polyurethane gene vector can achieve effective gene transfection at a lower mass ratio. Moreover, this nanosystem does not exhibit significant cytotoxicity even at high concentrations. Attached Figure Description
[0022] Figure 1 The above are the 1H NMR spectra of the zwitterionic polyurethane PUSB4 prepared in Example 1 before and after fluorination. Figure 2 The NMR fluorine spectra of the zwitterionic polyurethane PUSB4 prepared in Example 1 before and after fluorination are shown. Figure 3 The fluorination grafting reaction route obtained in Example 1; Figure 4 The infrared spectra of the fluorinated graft obtained in Example 1 before and after grafting are shown. Figure 5 The XPS elemental analysis spectrum of the fluorinated zwitterionic polyurethane prepared in Example 1; Figure 6 Transmission electron microscopy image of the prepared fluorinated zwitterionic polyurethane gene carrier complex. Figure 7 The particle size stability of the fluorinated zwitterionic polyurethane gene carrier complex prepared in Example 1; Figure 8 The atomization stability of the fluorinated zwitterionic polyurethane gene carrier composite prepared in Example 1; Figure 9 The mucus penetration performance of the fluorinated zwitterionic polyurethane gene carrier composite prepared in Example 1. Detailed Implementation
[0023] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific examples.
[0024] Example 1 First, PPF diol and SB-diol diol were added to a three-necked flask at a molar ratio of 1:4 for dehydration, maintaining a dry nitrogen atmosphere inside the flask. Then, the temperature was lowered to 70 °C, and ultra-dry DMSO solvent was added. After complete dissolution, HDI was added in a molar ratio equal to 1.8 moles of the total active hydrogens. The reaction was continued for 3 h, followed by the addition of TK-NH2 in a molar ratio equal to 0.8 moles of the remaining active hydrogens, and a chain extension reaction was carried out for 4 h to obtain a polyurethane solution. Precipitation was performed in deionized water to remove unreacted small molecules, repeated three times. Finally, the solution was freeze-dried for 48 h to obtain dry zwitterionic polyurethane PUSB4. PFO and PUSB4 were then weighed at a mass concentration ratio of 2:1 and added to a single-necked flask. The mixture was heated to 40 °C and reacted for more than 12 h to obtain PFSBU. Figures 1-4 The images show the hydrogen NMR spectrum and fluorine NMR spectrum of the prepared zwitterionic polyurethane PUSB4 before and after fluorination, the grafting reaction route, the grafting infrared spectrum, and the XPS elemental analysis spectrum.
[0025] PFSBU (10 mg / mL) was dissolved in DMSO to obtain a stable solution. Then, PFSBU@miRNA NPs were prepared via nanoprecipitation through the self-assembly of the PFSBU carrier and miRNA. In short, DMSO solutions of PFSBU with different grafting degrees were added to the miRNA aqueous solution at a weight ratio of 10:1 (PFSBU: miRNA), and mixed and stirred at room temperature for 20 min. The mixture was then washed three times with ultrapure water, centrifuged to an appropriate volume using an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and collected. The particle size of the PFSBU@miRNA NPs was measured by dynamic light scattering, and the particle size of the prepared nanocarrier was found to be 150.7 nm. The morphology of the gene carrier complex was characterized using transmission electron microscopy by varying the PFSBU:miRNA weight ratio, and the results are shown below. Figure 6 As shown. Meanwhile, to verify the particle size stability of the nanocomposite, its stability in water and the particle size changes before and after atomization were statistically analyzed, and no significant differences were found, as shown. Figure 7 As shown in Figure 8. Furthermore, nanoparticles possess strong mucus-penetrating properties, such as... Figure 9 As shown.
[0026] Example 2 First, PPF diol and SB-diol diol were added to a three-necked flask at a molar ratio of 1:3 for dehydration, maintaining a dry nitrogen atmosphere inside the flask. Then, the temperature was lowered to 70 °C, and ultra-dry DMSO solvent was added. After complete dissolution, HDI was added in amounts twice the total number of active hydrogen atoms. The reaction was continued for 3 h, followed by the addition of TK-NH2 in amounts equal to the remaining total number of active hydrogen atoms, and the chain extension reaction was carried out for 6 h to obtain a polyurethane solution. Precipitation was performed in deionized water to remove unreacted small molecules, repeated three times. Finally, the solution was freeze-dried for 48 h to obtain dry zwitterionic polyurethane PUSB4. PFO and PUSB4 were then weighed at a mass concentration ratio of 2:1 and added to a single-necked flask, heated to 40 °C, and reacted for more than 24 h to obtain PFSBU.
[0027] PFSBU (10 mg / mL) was dissolved in DMSO to obtain a stable solution. Then, PFSBU@miRNA NPs were prepared via nanoprecipitation through the self-assembly of the PFSBU carrier and miRNA. In short, DMSO solutions of PFSBU with different grafting degrees were added to the miRNA aqueous solution at a weight ratio of 20:1 (PFSBU: miRNA), and mixed and stirred at room temperature for 20 min. The mixture was then washed three times with ultrapure water, centrifuged to an appropriate volume using an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and collected. The particle size of the PFSBU@miRNA NPs was measured by dynamic light scattering, and the particle size of the prepared nanocarrier was found to be 180.9 nm. Transmission electron microscopy characterized the morphology of the gene carrier complex as spherical, and it remained stable in aqueous solution. The particle size did not change significantly before and after atomization.
[0028] Example 3 First, PPF diol and SB-diol diol were added to a three-necked flask at a molar ratio of 1:3 for dehydration, maintaining a dry nitrogen atmosphere inside the flask. Then, the temperature was lowered to 70 °C, and ultra-dry DMSO solvent was added. After complete dissolution, HDI was added in a molar ratio equal to 1.8 times the total number of active hydrogens. The reaction was continued for 3 h, followed by the addition of TK-NH2 in a molar ratio equal to 0.8 times the remaining total number of active hydrogens, and a chain extension reaction was carried out for 6 h to obtain a polyurethane solution. Precipitation was performed in deionized water to remove unreacted small molecules, repeated three times. Finally, the solution was freeze-dried for 48 h to obtain dry zwitterionic polyurethane PUSB4. PFO and PUSB4 were then weighed at a mass concentration ratio of 2:1 and added to a single-necked flask, heated to 40 °C, and reacted for more than 24 h to obtain PFSBU.
[0029] PFSBU (10 mg / mL) was dissolved in DMSO to obtain a stable solution. Then, PFSBU@miRNA NPs were prepared via nanoprecipitation through the self-assembly of the PFSBU carrier and miRNA. In short, DMSO solutions of PFSBU with different grafting degrees were added to the miRNA aqueous solution at a weight ratio of 30:1 (PFSBU: miRNA), and mixed and stirred at room temperature for 20 min. The mixture was then washed three times with ultrapure water, centrifuged to an appropriate volume using an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and collected. The particle size of the PFSBU@miRNA NPs was measured by dynamic light scattering, and the particle size of the prepared nanocarrier was found to be 170.4 nm. Transmission electron microscopy characterized the morphology of the gene carrier complex as spherical, and it remained stable in aqueous solution. The particle size did not change significantly before and after atomization.
[0030] The embodiments described above are merely some preferred embodiments of the present invention, and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.
Claims
1. A method for preparing a fluorinated zwitterionic polyurethane gene carrier complex, characterized in that, The process includes the following: adding 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecylfluoro-1-octylthiol PFO and zwitterionic polyurethane PUSB4 to a solvent to carry out a fluorination grafting reaction to prepare fluorinated zwitterionic polyurethane PFSBU; mixing the fluorinated zwitterionic polyurethane PFSBU and the gene together in a solution environment, shaking and incubating, and after multiple washing and redispersing of the obtained particles, obtaining a fluorinated zwitterionic polyurethane gene carrier complex.
2. The method for preparing the fluorinated zwitterionic polyurethane gene carrier complex according to claim 1, characterized in that, The fluorination grafting reaction is carried out at a temperature of 25-50°C and the total reaction time is 6-48 hours.
3. The method for preparing the fluorinated zwitterionic polyurethane gene carrier complex according to claim 1, characterized in that, The zwitterionic polyurethane PUSB4 is prepared by the following method: First, hexamethylene diisocyanate (HDI), polypropylene fumarate (PPF), and 3-(methylbis(2-hydroxyethyl)amino)propane-1-sulfonate (SB-diol) are subjected to solution polymerization in a solvent. Then, chain extension is carried out using ketethiodiamine (TK-NH2) to obtain zwitterionic polyurethane (PUSB4), wherein the molar ratio of PPF to SB-diol is 1:2 to 1:
4. The solvent is dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF).
4. The method for preparing the fluorinated zwitterionic polyurethane gene carrier complex according to claim 1, characterized in that, The mass ratio of PFO to PUSB4 is 5:1 to 2:
1.
5. The method for preparing the fluorinated zwitterionic polyurethane gene carrier complex according to claim 1, characterized in that, The solvent is one or a mixture of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), and dimethylacetamide (DMAC).
6. The method for preparing the fluorinated zwitterionic polyurethane gene carrier complex according to claim 1, characterized in that, The solution environment is at least one of PVA, sodium cholate, PBS, and deionized water.
7. The method for preparing the fluorinated zwitterionic polyurethane gene carrier complex according to claim 1, characterized in that, The gene is at least one of DNA, siRNA, and miRNA.
8. The method for preparing the fluorinated zwitterionic polyurethane gene carrier complex according to claim 1, characterized in that, The mass concentration ratio of PFSBU to the gene is 30:1 to 2.5:
1.
9. The method for preparing the fluorinated zwitterionic polyurethane gene carrier complex according to claim 1, characterized in that, The oscillation incubation time is 5 to 30 minutes.
10. A fluorinated zwitterionic polyurethane gene carrier complex, characterized in that, Prepared by the method described in any one of claims 1-9, the gene is miRNA, the particle size of the complex is 100~200nm, the particle size is stable after storage in water or atomization, and it can penetrate mucus.