PH-sensitive polyamino acid nano-micelle as well as preparation method and application thereof
By preparing pH-sensitive nanomicelles of polyethylene glycol-polylysine-polyphenylalanine triblock copolymer, the problems of slow drug release and toxic side effects of nanomicelles were solved, realizing the release and efficient delivery of tumor-targeted drugs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- FUZHOU UNIV
- Filing Date
- 2026-01-30
- Publication Date
- 2026-05-12
AI Technical Summary
Existing nanomicelles lack responsiveness to the tumor environment, resulting in slow drug release, failure to achieve targeted drug release, and toxic side effects on normal tissues.
pH-sensitive polyamino acid nanomicelles were prepared using a polyethylene glycol-polylysine-polyphenylalanine triblock copolymer. Targeted drug release was achieved by responding to changes in the tumor microenvironment via the polylysine moiety. A core-shell structure was formed using benzyloxycarbonyl group removal and self-assembly techniques.
This study achieved a significant increase in drug release rate under acidic conditions, reduced toxic side effects on normal tissues, and demonstrated that the nanomicelles can be effectively internalized by tumor cells, allowing the drug to be rapidly released into the cell nucleus, thus providing a new approach to drug sustained release.
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Figure CN122011370A_ABST
Abstract
Description
Technical Field
[0001] This application belongs to the fields of polymer chemistry and pharmaceutical technology, and in particular relates to a pH-sensitive polyamino acid nanomicelle, its preparation method and application. Background Technology
[0002] Chemotherapy is currently one of the main methods for treating malignant tumors in clinical practice. However, conventional chemotherapy drugs have poor selectivity; although they can inhibit tumor growth, they also damage normal tissues. The emergence of nanodelivery systems effectively avoids a series of problems associated with traditional drugs. Nanodelivery systems rely on the small size and surface effects of nanoparticles to improve drug absorption and achieve controlled drug release. Nanocarriers can also alter the distribution of drugs in the body, enhance drug concentration in the lesion area, and achieve targeted drug release through the enhanced permeability and retention effect (EPR effect) of tumor blood vessels, thereby reducing toxic side effects. The morphology of nanomicelles is usually spherical, cubic, or spindle-shaped, and by changing the specific surface area, they can be better captured by cell membranes. Nanomicelles have controllable size, good dispersibility, are conducive to circulation in the blood without precipitation, and can ensure the integrity of drugs when they reach specific tissues.
[0003] Polymer nanomicelles are functional systems with specific structures, formed by the self-assembly of amphoteric block polymers in aqueous or co-solution solutions. The main driving forces for self-assembly include hydrogen bonding, coordination bonds, van der Waals forces, electrochemical interactions, solvent effects, and hydrophilic and hydrophobic interactions. When the polymer concentration in aqueous solution is low, it is generally uniformly dispersed. When the polymer concentration exceeds its critical micelle concentration (CMC), hydrophobic segments bend to form a core to reduce the contact area with the aqueous phase, while hydrophilic segments cover the core to form a micelle shell. Typically, nanomicelles exhibit a spherical core-shell structure with a size ranging from 10 nm to 200 nm. The core can support hydrophobic chemotherapeutic drugs and promote their circulation in vivo through chemical bonding or electrostatic interactions. The hydrophilic shell prevents aggregation between different cores; some shells can be modified with active ligands to guide drug delivery to the lesion site, thereby reducing toxic side effects. Although traditional drug-loaded nanomicelles can achieve certain drug loading, they lack responsiveness to the tumor environment, leading to problems such as slow drug release. Summary of the Invention
[0004] This application is made in view of the above-mentioned issues, and its purpose is to provide a polyamino acid material with amphiphilic properties, wherein the polylysine moiety is a hydrophilic segment that responds to subtle changes in the tumor environment and is thus internalized by tumor cells to achieve targeted drug release in pH-sensitive polyamino acid nanomicelles.
[0005] The first aspect of this application provides a pH-sensitive polyamino acid nanomicelle, which is a triblock copolymer composed of polyethylene glycol (PEG), polylysine (PL-lysine), and polyphenylalanine (Phe). Specifically, the structure is as follows: the N-terminus is a PEG chain with 113 repeating units, connected to the next chain segment via amide bonds; the middle segment is a polylysine chain composed of 10 lysine residues, with amino groups (—NH2) on its side chains. The polylysine chain with lysine residues is formed by removing benzyloxycarbonyl groups from the polymer, exhibiting cationic properties; the C-terminus is a polyphenylalanine chain composed of n phenylalanine residues, where n = 10-30; the main chain of the entire molecule is a polyamino acid-polyethylene glycol block copolymer connected by peptide bonds (—CO—NH—), and this structure can be denoted as PEG. 113 -(Lys) 10 -(Phe) n It is a triblock copolymer of polyethylene glycol-polylysine-polyphenylalanine.
[0006] In any embodiment, the n of the triblock copolymer is 10, 20, or 30, corresponding to the structural formula PEG. 113 -(Lys) 10 -(Phe) 10 PEG 113 -(Lys) 10 -(Phe) 20 PEG 113 -(Lys) 10 -(Phe) 30 .
[0007] In any embodiment, the polylysine is L-Lys(Cbz)-NCA; the polyethylene glycol is mPEG. 113 -NH2; the polyphenylalanine is L-Phe-NCA.
[0008] A second aspect of this application provides a method for preparing pH-sensitive polyamino acid nanomicelles, comprising the following steps: 1) Synthesis process of copolymers L-Lys(Cbz)-NCA was dissolved in DMF; then, the system was evacuated and replaced three times with argon gas to maintain the system in an inert gas atmosphere; mPEG 113-NH2 was dissolved in anhydrous DMF and added to a flask; after reacting continuously at 35°C for 60 h, L-Phe-NCA was dissolved in DMF and slowly added to the system for the second stage of polymerization; the reaction was continued for another 60 h. After the entire process was completed, the mixture was concentrated by vacuum distillation, and a white powder solid was precipitated using diethyl ether as a poor solvent, named P-Cbz; the L-Lys(Cbz)-NCA:mPEG 113 The molar ratio of -NH2:L-Phe -NCA is 10:1:10-30; 2) Removal of benzyloxycarbonyl group Dissolve P-Cbz from step 1) in TFA; after stirring for 30 min, inject HBr / ACOH into the flask under ice bath conditions, and continue stirring for 4 h in a sealed state; then precipitate the target product P using diethyl ether as a poor solvent. The product is a pale yellow powder solid.
[0009] In any implementation, the L-Lys(Cbz)-NCA:mPEG 113 The molar ratio of -NH2:L-Phe-NCA was 10:1:10-10, yielding a white powder solid, named P1-Cbz, with P1 as the target product; the L-Lys(Cbz)-NCA:mPEG was obtained. 113 The molar ratio of -NH2:L-Phe-NCA was 10:1:10-20, yielding a white powder solid, named P12Cbz, with P2 as the target product; the L-Lys(Cbz)-NCA:mPEG... 113 The molar ratio of -NH2:L-Phe -NCA was 10:1:10-30, resulting in a white powder solid, named P3-Cbz, with P3 as the target product.
[0010] In any embodiment, the chemical synthesis process of the nanomicelles is as follows:
[0011] mPEG 113 -NH2 acts as an initiator for ring-opening polymerization, which is essential for mPEG. 113 The product is amination-terminated with -OH groups. A triblock polymer is obtained through stepwise polymerization, followed by hydrolysis to remove the benzyloxycarbonyl group, yielding the target polymer. The amount of reactants required for each polymerization reaction should not be excessive to avoid uneven molecular weight distribution.
[0012] Unreacted monomers or low-molecular-weight byproducts are dissolved in the ether, and the polymerization product precipitates out of the DMF.
[0013] A third aspect of this application provides an application of pH-sensitive polyamino acid nanomicelles for drug loading, wherein the nanomicelles or nanomicelles obtained by the above-described preparation method are loaded with doxorubicin (DOX) model drug.
[0014] In any embodiment, the nanomicelles are loaded with doxorubicin (DOX) model drug. DOX·HCl (8 mg) is dissolved in a certain amount of DMSO in a conical flask, and 5 μL of triethylamine is added and stirred for 5 h to remove hydrochloric acid. 30 mg of polyamino acid nanomicelles P is dissolved in a certain amount of DMF, transferred to a reaction flask, and stirred in the dark for 24 h. 5 mL of PBS solution (pH=7.4) is slowly added to the flask to complete the polymer self-assembly process. The mixed solution is then added to a dialysis bag for dialysis for 2 days. Finally, the solution is filtered using a 0.45 μm aqueous filter membrane to remove precipitates and impurities, and then lyophilized to obtain drug-loaded nanoparticles PM.
[0015] In any embodiment, the nanomicelles loaded with doxorubicin (DOX) model drug are chemically synthesized as follows:
[0016] 2.4 Drug loading process The drug loading process of the polymer was achieved through a specific method, and excess DOX could be removed by dialysis. DMSO, as an organic solvent, could effectively dissolve the synthesized polymer and DOX. Utilizing the water solubility of DMSO, the self-assembly effect of the drug-loaded polyamino acid was realized, thereby preparing nanomicelles. The other two (P2-M and P3-M) were obtained using the same method. By measuring the absorbance of the drug-loaded nanoparticle solution, the drug loading amount (LC) and drug loading efficiency (LE) could be calculated.
[0017] The beneficial effects of this application are as follows: This invention prepares pH-sensitive drug-loaded nanomicelles by encapsulating the model drug doxorubicin (DOX) within an amphiphilic poly(amino acid) core-shell structure with good self-assembly properties. The compound's structure conforms to molecular design, with a narrow molecular weight distribution. The drug loading capacity (LC) and drug loading efficiency (LE) gradually increase with the elongation of the molecular chain, and the diameter of the nanomicelles also increases accordingly. In vitro drug release results show that the release rate of the nanomicelles is significantly higher under acidic conditions than under neutral conditions. The polymer is non-toxic to tumor cells, while the drug-loaded nanomicelles can effectively inhibit tumor cell proliferation. Furthermore, confocal laser scanning microscopy (CLSM) shows that the nanomicelles can be effectively internalized by tumor cells, and the drug can be rapidly released from the nanomicelles and passively diffuse into the cell nucleus. The synthesis of this type of material provides a new approach for the field of drug sustained release. Attached Figure Description
[0018] Figure 1 The images show the FT-IR spectra of three polymers (a) and the product (b) after deprotection (c) in Examples 1-3 of this application, and the 1H NMR spectra of polymer (c) and the product (d) after deprotection (c). Figure 2 The images show the average sizes of nanomicelles in Examples 1-3 of this application, P1-M(a), P2-M(b), P3-M(c) and TEM images, P1-M(d), P2-M(e), P3-M(f). Figure 3 The diagrams show the drug release behavior of nanomicelles at different pH values in Examples 1-3 of this application, P1-M(a), P2-M(b), and P3-M(c). Figure 4 These are cell compatibility diagrams of the polymers in Examples 1-3 of this application with L929 cells; Figure 5 This is a graph showing the relative cell viability of HeLa cells after co-culturing with drug-loaded nanomicelles in Examples 1-3 of this application. Figure 6 Cellular uptake behavior of HeLa cells after co-culturing with drug-loaded nanomicelles. Detailed Implementation
[0019] The following detailed description, with appropriate reference to the accompanying drawings, discloses an embodiment of a pH-sensitive polyamino acid nanomicelle, its preparation method, and its application. However, unnecessary details may be omitted. For example, detailed descriptions of well-known matters and repetitive descriptions of essentially the same structures may be omitted. This is to avoid unnecessarily lengthy descriptions and to facilitate understanding by those skilled in the art. Furthermore, the accompanying drawings and the following description are provided for the purpose of providing a full understanding of this application by those skilled in the art and are not intended to limit the subject matter of the claims.
[0020] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is expected that ranges of 60-110 and 80-120 are also included. Furthermore, if minimum range values of 1 and 2 are listed, and if maximum range values of 3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.
[0021] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.
[0022] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions.
[0023] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.
[0024] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.
[0025] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).
[0026] Polyamino acids (POAs) are natural or synthetic materials obtained by polymerizing one or more small-molecule amino acids. The side chains of POAs contain functional groups such as amino, carboxyl, or hydroxyl groups, which provide chemical sites for drug binding and structural modification. The ordered secondary structures of POAs (e.g., α-helices, β-sheets) influence the flexibility of the main chain, leading to differences in polymer properties and functions. POAs can achieve independent regulation of mechanical strength or other properties without affecting other functional expressions. By introducing different types of monomers into the structure, the hydrophilicity of the polymer can be adjusted, and the self-assembly effect of macromolecules can be used to load model drugs, enabling the application of POAs materials in drug sustained release.
[0027] In this application, a series of amphoteric polyamino acid materials were synthesized and loaded with the model drug doxorubicin (DOX) through physical intercalation and chemical bonding. DOX is a broad-spectrum antitumor drug, and this application aims to find a drug carrier to reduce damage to normal tissues during chemotherapy. Polyethylene glycol segments were used as the hydrophilic portion of the macromolecule, and polyphenylalanine was used as the hydrophobic portion of the structure. The polylysine portion not only serves as the main drug binding site but also forms a pH-sensitive point in the tumor microenvironment. By optimizing the formation mechanism and morphology-particle size relationship of the nanomicelles through the self-assembly behavior of the polymer, sustained drug release was achieved. Cell experiments demonstrated that the polymer is non-toxic and that the nanomicelles exhibit targeted release activity, providing a new approach to the field of sustained drug release.
[0028] Materials and Chemicals L-Phe-NCA and L-Lys(Cbz)-NCA were purchased from Sichuan Jiayinglai Technology Co., Ltd. (Sichuan, China); doxorubicin hydrochloride (DOX·HCl) and methoxy polyethylene glycol (mPEG113-OH) were purchased from Lanospharma Laboratories Co., Ltd. (Hong Kong, China); trifluoroacetic acid (TFA) and hydrobromic acid / acetic acid (HBr / HAC) were purchased from Shandong Jiwo Chemical Co., Ltd.; 4,6-diamino-2-phenylindole (DAPI), fetal bovine serum (FBS), and Cell Count Kit-8 (CCK-8) were purchased from Sigma-Aldrich Ltd. (Shanghai, China); diethyl ether, triethylamine (TEA), N,N-dimethylformamide (DMF), dichloromethane (DCM), and dimethyl sulfoxide (DMSO) were purchased from Adamas Ltd. (Hebei, China). All materials and solvents were purified using standard methods before use.
[0029] A method for preparing pH-sensitive polyamino acid nanomicelles as drug delivery systems, comprising the following steps: Synthesis of polyamino acids mPEG 113 -NH2 acts as an initiator for ring-opening polymerization, which is essential for mPEG. 113 The product is amination-terminated with -OH groups. A triblock polymer is obtained through stepwise polymerization, followed by hydrolysis to remove the benzyloxycarbonyl group, yielding the target polymer. The amount of reactants required for each polymerization reaction should not be excessive to avoid uneven molecular weight distribution.
[0030] 1) Synthesis process of copolymers L-Lys(Cbz)-NCA (0.524 g, 0.002 mol) was dissolved in DMF (6 mL). The system was then evacuated and replaced three times with argon gas to maintain an inert gas atmosphere. mPEG 113 -NH2 (1 g, 0.0002 mol) was dissolved in anhydrous DMF (6 mL) and added to a flask. After reacting continuously at 35 °C for 60 h, L-Phe-NCA (0.294 g, 0.002 mol) was dissolved in 6 mL DMF and slowly added to the system for the second stage of polymerization. The reaction was repeated for another 60 h. After the entire process was completed, the mixture was concentrated by vacuum distillation, and a white powder solid was precipitated using diethyl ether as a poor solvent, named P1-Cbz. Unreacted monomers or low-molecular-weight byproducts were dissolved in ether, and the polymerized product precipitated from the DMF. The preparation methods for the other two (P2-Cbz, P3-Cbz) were the same, except that the amount of L-Phe-NCA was adjusted according to the molecular design ratio. The yields of the three products were 81.4%, 78.3%, and 76.6%, respectively.
[0031] 2) Removal of benzyloxycarbonyl group The triblock copolymer was obtained by removing the benzyloxycarbonyl group from the polymer through acidic hydrolysis. P1-Cbz (0.5 g) was dissolved in 8 mL of TFA. After stirring for 30 min, HBr / ACOH was injected into the flask under ice bath conditions to avoid solution splashing or HBr evaporation. The mixed solution was kept sealed and stirred for another 4 h. The target product P1 was precipitated using diethyl ether as a poor solvent; the product was a pale yellow powder. The other two polyamino acids (P2 and P3) were obtained using the same method. The yields of the three products were 65.5%, 68.3%, and 70.2%, respectively. All products required storage in a low-temperature, dry environment.
[0032] Example The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.
[0033] Example 1 A method for preparing pH-sensitive polyamino acid nanomicelles includes the following steps: 1) Synthesis process of copolymers L-Lys(Cbz)-NCA (0.524 g, 0.002 mol) was dissolved in DMF (6 mL). The system was then evacuated and replaced three times with argon gas to maintain an inert gas atmosphere. mPEG 113 -NH2 (1 g, 0.0002 mol) was dissolved in anhydrous DMF (6 mL) and added to a flask. After reacting continuously at 35 °C for 60 h, L-Phe-NCA (0.294 g, 0.002 mol) was dissolved in 6 mL DMF and slowly added to the system for the second stage of polymerization. The reaction was continued for another 60 h. After the entire process was completed, the mixture was concentrated by vacuum distillation, and a white powder solid was precipitated using diethyl ether as a poor solvent, named P1-Cbz. The yield was 81.4%.
[0034] 2) Removal of benzyloxycarbonyl group P1-Cbz (0.5 g) was dissolved in 8 mL of TFA. After stirring for 30 min, HBr / ACOH was injected into the flask under ice bath conditions. The mixture was kept sealed and stirred for another 4 h. The target product P1 was precipitated using diethyl ether as a poor solvent; the product was a pale yellow powder. The yield was 65.5%. All products were stored in a cool, dry environment.
[0035] Drug loading process of polyamino acid nanomicelles: DOX·HCl (8 mg) was dissolved in a certain amount of DMSO in an Erlenmeyer flask, and 5 μL of triethylamine was added and stirred for 5 h to achieve hydrochloric acid removal. Polyamino acid (P1) 30 mg was dissolved in a certain amount of DMF, transferred to a reaction flask, and stirred in the dark for 24 h. 5 mL of pH 7.4 PBS solution was slowly added to the flask to complete the polymer self-assembly process. The mixture was then dialyzed for 2 days. Finally, it was filtered through a 0.45 μm aqueous filter membrane to remove precipitates and impurities, and then lyophilized to obtain P1-M.
[0036] Example 2 A method for preparing pH-sensitive polyamino acid nanomicelles includes the following steps: 1) Synthesis process of copolymers L-Lys(Cbz)-NCA (0.524 g, 0.002 mol) was dissolved in DMF (6 mL). The system was then evacuated and replaced three times with argon gas to maintain an inert gas atmosphere. mPEG 113 -NH2 (1 g, 0.0002 mol) was dissolved in anhydrous DMF (6 mL) and added to a flask. After reacting continuously at 35 °C for 60 h, L-Phe-NCA (0.773 g, 0.004 mol) was dissolved in 12 mL of DMF and slowly added to the system for the second stage of polymerization. The reaction was continued for another 60 h. After the entire process was completed, the mixture was concentrated by vacuum distillation, and a white powder solid was precipitated using diethyl ether as a poor solvent, named P2-Cbz. The yield was 78.3%.
[0037] 2) Removal of benzyloxycarbonyl group P2-Cbz (0.5 g) was dissolved in 8 mL of TFA. After stirring for 30 min, HBr / ACOH was injected into the flask under ice bath conditions. The mixture was kept sealed and stirred for another 4 h. The target product P2 was precipitated using diethyl ether as a poor solvent; the product was a pale yellow powder. The yield was 68.3%. All products were stored in a low-temperature, dry environment.
[0038] Drug loading process of polyamino acid nanomicelles: DOX·HCl (8 mg) was dissolved in a certain amount of DMSO in an Erlenmeyer flask, and 5 μL of triethylamine was added and stirred for 5 h to achieve hydrochloric acid removal. Polyamino acid (P2) 30 mg was dissolved in a certain amount of DMF, transferred to a reaction flask, and stirred in the dark for 24 h. 5 mL of PBS solution (pH=7.4) was slowly added to the flask to complete the polymer self-assembly process. The mixed solution was then dialyzed in a dialysis bag for 2 days. Finally, the solution was filtered through a 0.45 μm aqueous filter membrane to remove precipitates and impurities, and then lyophilized to obtain P2-M.
[0039] Example 3 A method for preparing pH-sensitive polyamino acid nanomicelles includes the following steps: 1) Synthesis process of copolymers L-Lys(Cbz)-NCA (0.524 g, 0.002 mol) was dissolved in DMF (6 mL). The system was then evacuated and replaced three times with argon gas to maintain an inert gas atmosphere. mPEG 113 -NH2 (1 g, 0.0002 mol) was dissolved in anhydrous DMF (6 mL) and added to a flask. After reacting continuously at 35 °C for 60 h, L-Phe-NCA (1.159 g, 0.006 mol) was dissolved in 18 mL of DMF and slowly added to the system for the second stage of polymerization. The reaction was continued for another 60 h. After the entire process was completed, the mixture was concentrated by vacuum distillation, and a white powder solid was precipitated using diethyl ether as a poor solvent, named P3-Cbz. The yield was 76.6%.
[0040] 2) Removal of benzyloxycarbonyl group P3-Cbz (0.5 g) was dissolved in 8 mL of TFA. After stirring for 30 min, HBr / ACOH was injected into the flask under ice bath conditions. The mixture was kept sealed and stirred for another 4 h. The target product P3 was precipitated using diethyl ether as a poor solvent; the product was a pale yellow powder. The yield was 70.2%. All products were stored in a cool, dry environment.
[0041] Drug loading process of polyamino acid nanomicelles: DOX·HCl (8 mg) was dissolved in a certain amount of DMSO in an Erlenmeyer flask, and 5 μL of triethylamine was added and stirred for 5 h to achieve hydrochloric acid removal. Polyamino acid (P3) 30 mg was dissolved in a certain amount of DMF, transferred to a reaction flask, and stirred in the dark for 24 h. 5 mL of pH 7.4 PBS solution was slowly added to the flask to complete the polymer self-assembly process. The mixed solution was then added to a dialysis bag for dialyzing for 2 days. Finally, the solution was filtered through a 0.45 μm aqueous filter membrane to remove precipitates and impurities, and then lyophilized to obtain P3-M.
[0042] 1. Drug release process Five mL of drug-loaded concentrated nanobundles solution (P1-M) was placed in a dialysis bag containing 30 mL of PBS buffer at different pH values (pH=5.4, pH=6.4, and pH=7.4). The system temperature was set at 37 °C, and the solution was shaken at a constant speed of 100 rpm for 90 h in the dark. At the specified time, 4 mL of the release solution was taken to measure the absorbance, and an equal volume of fresh buffer was added. The absorbance intensity of the solution at 480–490 nm was measured, and the DOX concentration was calculated. All data were tested in triplicate and the average value was taken. The in vitro release behavior of P2-M and P3-M was studied using the same method.
[0043] 2. Cell compatibility evaluation 2.1 Cytotoxicity of Polyamino Acids The polymer was dissolved in a certain amount of DMSO and diluted to a specific medium concentration. Log-phase L929 cells were seeded in 96-well plates and cultured at a density of 5 × 10⁶ cells / well. 3 Cells / well. After 12 h of culture, the culture medium was aspirated, and polymer solutions of different concentrations were added for co-culture. After 24 h of continuous culture, the culture medium was aspirated, and 100 μL of culture medium and 10 μL of CCK-8 solution were added to the culture dish. After another 2 h of culture, the absorbance at 450 nm was measured using a microplate reader, and the relative cell viability was calculated.
[0044] 2.2 Cellular compatibility of nanomicelles The relevant procedures are similar to those in 2.6.1, except that HeLa cells are used instead of the co-cultured cells and the relative concentration is adjusted.
[0045] 2.3 In vitro cellular uptake HeLa cells were loaded at a rate of 2 × 10 5Initial cell / well density was seeded in 96-well plates and cultured for 12 h, followed by co-culture with medium containing drug-loaded nanomicelles at a DOX concentration of 10 µg / mL. After 3 h, the medium was removed, washed three times with fresh PBS, fixed with 4% paraformaldehyde for 15 min, stained with 1 μg / mL DAPI for 15 min, and the mixture was removed and washed with fresh PBS. Finally, the cell uptake behavior of the drug-loaded nanomicelles was observed using laser confocal microscopy (CLSM).
[0046] 3. Test Results 3.1 Structural Characterization of Polyamino Acids 3.1.1 FT-IR analysis The FT-IR spectra of the polymers are as follows: Figure 1 As shown in (a, b), the stretching vibration absorption peak of the NH group in the amide bond is at 3300 cm⁻¹. -1 Approximately 3050cm -1 This indicates the presence of a benzene ring. The stretching vibration absorption peaks of the methyl and methylene groups are at 2890 cm⁻¹. -1 and 2960cm -1 The bending vibration is around 1380cm. -1 and 1460cm -1 Around 1720 cm⁻¹. In the side-chain protecting group, the characteristic absorption peak of the ester carbonyl group appears at 1720 cm⁻¹. -1 The characteristic absorption peak of the amide carbonyl group appears at 1650 cm⁻¹. -1 After the deprotection reaction, the characteristic peak of the amide carbonyl group disappeared significantly, and the intensity of the benzyl ring peak decreased, indicating that the benzyloxycarbonyl group of the product had been successfully removed.
[0047] 3.1.2 1 HNMR analysis Figure 1 (c) is a preliminary synthesis of polyamino acids. 1 ¹H NMR spectra. The chemical shift of NH on the amide bond is 8.32 ppm, the chemical shift of CH on the benzene ring backbone is 7.33 ~ 7.11 ppm, and the chemical shift of CH on the methylene group attached to the benzene ring is 5.21 ppm. Other characteristic peaks, such as 3.55 ppm, 3.21 ppm, 2.89 ppm, and 1.54 ppm, all belong to the alkyl chain of the polyamino acid side chain. After the deprotection reaction, it can be seen that the integrated area of CH on the benzene ring is weakened, and the characteristic absorption peak of the group on the benzyloxycarbonyl group is also significantly weakened, proving that the product conforms to the molecular design.
[0048] 3.1.3 GPC Analysis GPC was used to describe the mass of the material and the distribution of the synthesized polymer. As shown in Table 1, the number-average molar mass of the three polymers increased with increasing phenylalanine content, and the PDI (partial dispersion index) was below 1.3 for all three, indicating a narrow molecular weight distribution. Based on... 1 The structure can be deduced from the integral area of HNMR.
[0049] Table 1. GPC distribution of polymers
[0050] 3.2 Characterization of Dox-loaded nanomicelles 3.2.1 DLS Distribution The average particle sizes of the three dox-loaded nanomicelles were 19.49 nm, 32.83 nm, and 86.12 nm, respectively, showing a relatively narrow distribution. For example... Figure 2 As shown in (ac), the average size of the nanomicelles gradually increases with the elongation of the molecular chain. This is because hydrophobic polyphenylalanine can effectively promote the self-assembly behavior of macromolecules, thereby achieving greater drug loading and encapsulation efficiency. In addition, the size distribution of the three types of micelles is relatively narrow.
[0051] 3.2.2 TEM Images Figure 2 (df) shows TEM images of the three types of nanomicelles. As can be seen from the images, the nanomicelles are all spherically distributed. Due to concentration issues, a small amount of particle aggregation may occur, but overall the particle size distribution is relatively uniform. The experimental results are consistent with DLS, demonstrating that the size of DOX-loaded micelles increases slowly with increasing drug loading.
[0052] 3.3 Drug release from nanomicelles The LC values of the three types of nanomicelles were 12%, 18%, and 22%, respectively. The LE values were 45%, 52%, and 55%, respectively. The polymer side chains contained a large number of amino groups that could bind to DOX, and physical encapsulation also increased the drug loading of the model. The unique ratio of hydrophilic to hydrophobic segments could alter the molecular arrangement in response to changes in the external environment. This paper uses dialysis to study the release behavior of drug-loaded nanomicelles under different conditions. Figure 3 (ac) As can be seen, the nanomicelles are pH-sensitive, avoiding burst release under neutral conditions and achieving drug release under acidic conditions, exhibiting strong targeting. On one hand, under acidic conditions, the Schiff base structure in the nanomicelles is disrupted, and DOX dissociates through chemical bonds. On the other hand, the collapse of the core-shell structure leads to the release of more physically embedded drugs. Simultaneously, the "unwinding" effect of the macromolecular chains also disrupts intramolecular and intermolecular hydrogen bonds, resulting in an increased drug release rate.
[0053] The drug release model for spherical nanomicelles was established by Peppas et al., and the relevant formulas are as follows: M t / M ∞ = kt n
[0054] Table 2. Drug release kinetic parameters of nanomicelles under different conditions
[0055] k: rate constant; n: release index; R²: correlation coefficient As can be seen from the data in Table 2, drug release under acidic conditions not only increases in rate but also exhibits a dilatational effect. This is because acidic conditions lead to protonation of nanomicelles, and the structural changes cause drug release to exhibit multiple manifestations.
[0056] 3.4 Evaluation of cell compatibility 3.4.1 Cellular compatibility of polymers L929 cells are mouse epithelial fibroblasts, often used to investigate the cell compatibility of polymers, and their cell proliferation rate can be used to evaluate the cytotoxicity of the prepared materials. Figure 4 (ac) shows the compatibility effects of P1, P2, and P3. The results indicate that even at polymer concentrations as high as 500 μg / mL, the cell viability of the three materials remains above 82%, demonstrating that the synthesized materials have good cell affinity.
[0057] 3.4.2. Cell compatibility of DOX-loaded nanomicelles from Figure 5 As can be seen, the three drug-loaded nanomicelles exhibited significant cytotoxicity, with cell viability decreasing rapidly with increasing DOX concentration. DOX's mechanism of action involves inhibiting nucleic acid synthesis and mitosis by binding to cellular DNA. Previous studies have shown that the synthesized polyamino acids are non-toxic to normal cells. The results of this study indicate that nanomicelles can inhibit the proliferation of HeLa cells, mainly due to the effect of DOX coating, and the effect is more pronounced with increasing drug LC. Furthermore, the toxicity of nanomicelles is slightly lower than that of free DOX because nanomicelles only release the drug upon stimulation, while DOX can diffuse directly into the cell nucleus via passive diffusion. It can also be observed that the cellular inhibitory potential of the nanomicelles gradually increases with increasing drug loading.
[0058] 3.4.3. Cellular uptake behavior of nanomicelles DOX is a broad-spectrum anthracycline antibiotic used to treat tumors. Figure 6This shows the distribution of DOX in cells as observed by the CLSM method. The red area represents the distribution of DOX, the blue area represents the state after nuclear staining, and the overlapping area indicates that DOX is distributed in both the cytoplasm and the nucleus. The purple area becomes more prominent under magnification. These results demonstrate that drug release by nanomicelles is not a simple passive diffusion process. The results show that nanomicelles can efficiently enter cells through endocytosis, and fluorescent signals were found in both the nucleus and cytoplasm. The changes in drug concentration became more pronounced with prolonged co-culture time, indicating that the drug is released gradually in an α-type manner.
[0059] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.
Claims
1. A pH-sensitive polyamino acid nanomicelle, characterized in that, The triblock copolymer, composed of polyethylene glycol, polylysine, and polyphenylalanine, has the following structure: The N-terminus is a polyethylene glycol chain with 113 repeating units, connected to the next segment via amide bonds; the middle segment is a polylysine chain consisting of 10 lysine residues, with amino groups in its side chains. The polylysine chain is formed by removing benzyloxycarbonyl groups from the polymer, exhibiting cationic properties; the C-terminus is a polyphenylalanine chain consisting of n phenylalanine residues, where n = 10-30; the entire molecule's backbone is a polyamino acid-polyethylene glycol block copolymer linked by peptide bonds. This structure can be denoted as PEG. 113 –(Lys) 10 –(Phe) n It is a triblock copolymer of polyethylene glycol-polylysine-polyphenylalanine.
2. The pH-sensitive polyamino acid nanomicelles according to claim 1, characterized in that, The triblock copolymer has n values of 10, 20, or 30, corresponding to the structural formula PEG. 113 –(Lys) 10 –(Phe) 10 PEG 113 –(Lys) 10 –(Phe) 20 PEG 113 –(Lys) 10 –(Phe) 30 .
3. The pH-sensitive polyamino acid nanomicelles according to claim 1, characterized in that, The polylysine is L-Lys(Cbz)-NCA; the polyethylene glycol is mPEG. 113 -NH2; the polyphenylalanine is L-Phe-NCA.
4. A method for preparing pH-sensitive polyamino acid nanomicelles, comprising the following steps: 1) Synthesis process of copolymers L-Lys(Cbz)-NCA was dissolved in DMF; then, the system was evacuated and replaced three times with argon gas to maintain the system in an inert gas atmosphere; mPEG 113 -NH2 was dissolved in anhydrous DMF and added to a flask; after reacting continuously at 35 °C for 60 h, L-Phe-NCA was dissolved in DMF and slowly added to the system for the second stage of polymerization; the reaction was continued for another 60 h. After the entire process was completed, the mixture was concentrated by vacuum distillation, and a white powder solid was precipitated using diethyl ether as a poor solvent, named P-Cbz; the L-Lys(Cbz)-NCA:mPEG 113 The molar ratio of -NH2:L-Phe -NCA is 10:1:10-30; 2) Removal of benzyloxycarbonyl group Dissolve P-Cbz from step 1) in TFA; after stirring for 30 min, inject HBr / ACOH into the flask under ice bath conditions, and continue stirring for 4 h in a sealed state; then precipitate the target product P using diethyl ether as a poor solvent. The product is a pale yellow powder solid.
5. A method for preparing pH-sensitive polyamino acid nanomicelles according to claim 4, characterized in that, The L-Lys(Cbz)-NCA:mPEG 113 The molar ratio of -NH2:L-Phe-NCA was 10:1:10-10, yielding a white powder solid, named P1-Cbz, with P1 as the target product; the L-Lys(Cbz)-NCA:mPEG was obtained. 113 The molar ratio of -NH2:L-Phe-NCA was 10:1:10-20, yielding a white powder solid, named P12Cbz, with P2 as the target product; the L-Lys(Cbz)-NCA:mPEG... 113 The molar ratio of -NH2:L-Phe -NCA was 10:1:10-30, resulting in a white powder solid, named P3-Cbz, with P3 as the target product.
6. The method for preparing pH-sensitive polyamino acid nanomicelles according to claim 4, characterized in that, The chemical synthesis process of the nanomicelles is as follows: 。 7. An application of a pH-sensitive polyamino acid nanomicelle drug loading, wherein the nanomicelles as described in any one of claims 1-3 or the nanomicelles prepared by any one of claims 4-6 are loaded with doxorubicin (DOX) model drug.
8. The application according to claim 7, characterized in that, The nanomicelles loaded with doxorubicin (DOX) model drug were prepared by dissolving 8 mg of DOX·HCl in a certain amount of DMSO in an Erlenmeyer flask, adding 5 μL of triethylamine and stirring for 5 h; dissolving 30 mg of polyamino acid nanomicelles P in a certain amount of DMF, transferring the solution to a reaction flask, and stirring in the dark for 24 h; and slowly adding 5 mL of PBS solution with pH=7.4 to the flask to complete the self-assembly process of the polymer. The mixed solution was then added to a dialysis bag for dialysis for two days. Finally, it was filtered using a 0.45 μm aqueous phase filter membrane to remove precipitates and impurities, and then freeze-dried to obtain drug-loaded nanoparticles (PM).
9. The application according to claim 8, characterized in that, The chemical synthesis process of the nanomicelles loaded with doxorubicin (DOX) model drug is as follows: 。