A polypeptide nanocarrier for targeted drug delivery and its preparation method and application

Abstract

The present invention relates to the field of preparation of targeted drug carriers, and provides a method for preparing a polypeptide nanocarrier for targeted drug delivery. DAG is used as a targeting modification molecule to transform PEG-PLGA nanoparticles to synthesize a new polypeptide carrier. In the preparation process, a solid-state synthesis method is selected to introduce carboxyl PEG-PLGA in a directional manner during the self-synthesis of DAG polypeptides. Amino acids are coupled to a resin containing activated amino groups, and then the Fomc protecting group of the main chain amino group is removed. The process is repeated for different amino acids to synthesize a linear polypeptide with protected side chain amino groups, so that the prepared PEG-PLGA-modified DAG cyclic peptide has high purity. The material has the advantages of good targeting, high drug transport efficiency and short drug release cycle, and is easy to promote and apply.

Description

Technical Field

[0001] The present invention relates to the field of preparation of targeted drug carriers, and in particular to a polypeptide nanocarrier for targeted drug delivery and a preparation method and application thereof. Background Art

[0002] In pharmaceutical engineering, improving drug targeting and extending its circulation time in the body are the keys to achieving efficient drug delivery and exerting drug efficacy. However, effective drug delivery across the blood-brain barrier (BBB) ​​remains a huge challenge. The lack of specific targeting modification molecules for the nervous system further complicates this problem.

[0003] Compared with traditional small molecule drugs, peptide drugs have many advantages, including high specificity and high affinity, low off-target effects, and the ability to effectively regulate complex biological pathways. Chinese patent CN202410384792.0 discloses a tri-zan gum carrier that can stably load fat-soluble active ingredients. The carrier uses an efficient emulsion delivery system to achieve slow release of active ingredients in the intestinal microenvironment, thereby improving bioavailability; by dissolving fat-soluble active substances in lipid materials as the oil phase, its lipid-carrying activity is increased. However, the carrier is not targeted and cannot achieve higher dosing concentrations in the central nervous system; at the same time, due to the inclusion of fat-soluble active substances, the molecular weight of the carrier will be larger and the spatial structure will also change, causing local mucosal barrier absorption disorders. In addition, due to the longer drug release cycle, the metabolic burden on the liver may increase and the drug utilization may be reduced. Chinese patent CN201310586482.9 discloses a Pep-1 peptide-modified glioma-targeted nano drug delivery system, which is composed of polymer nanoparticles prepared with amphiphilic polyethylene glycol-poly(lactic acid-glycolic acid) block copolymer as carrier material, paclitaxel encapsulated in the polymer nanoparticles, and Pep-1 polypeptide, a ligand modified on the surface of the polymer nanoparticles, which can improve the uptake and accumulation of drugs in the glioma site. However, Pep-1 is a straight-chain peptide, and as a transmembrane peptide, it has poor targeting selectivity and low drug transport efficiency.

[0004] Therefore, it is of great significance to develop a polypeptide nanocarrier for targeted drug delivery with good targeting, high drug transport efficiency and short drug release cycle. Summary of the invention

[0005] The purpose of the present invention is to solve the problems of the prior art and provide a polypeptide nanocarrier for targeted drug delivery and a preparation method and application thereof.

[0006] DAG (cDAGRKQKC) is a newly discovered cyclic peptide with the potential for targeted drug delivery in the neurovascular unit. β-sitosterol (BSIT) has been shown to have anti-inflammatory and anti-free radical effects and is a potential drug with brain protective effects.

[0007] The present invention uses DAG as a targeting modification molecule to transform PEG-PLGA nanoparticles and synthesize a new type of polypeptide carrier; targeted delivery to brain tissue is achieved by loading BSIT. In order to avoid the restriction of BBB on drug transport, the nose-to-brain administration method is preferably adopted. The results show that not only convenient and fast non-invasive drug administration is achieved, but also the brain targeting and delivery efficiency of the drug are significantly improved. At the same time, it is found that the use of BSIT-loaded DAG-PEG-PLGA nanoparticles (BSIT-DAG-PEG-PLGA NPs) for nasal drops for one week can significantly improve the cognitive impairment of carbon monoxide poisoning (COP) rats, improve motor and memory abilities, reduce neuronal apoptosis, and protect the structure and function of the nervous system.

[0008] At the same time, due to the small molecular weight and spatial conformation of DAG, it is more conducive to nasal-brain administration, and it can act as an interfering peptide to directly target and inhibit the active transport mechanism mediated by CaMKⅡ in brain tissue. Therefore, DAG-modified PEG-PLGA NPs can more efficiently pass through the nasal mucosa-brain barrier and achieve targeted drug delivery to the nervous system.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] A method for preparing a polypeptide nanocarrier for targeted drug delivery comprises the following steps:

[0011] Step 1, placing the amino resin in a reaction column, and sequentially performing an expansion treatment and a deprotection treatment;

[0012] Step 2, adding multiple activated amino acids to the amino acid values ​​in step 1 in sequence for coupling reaction, and removing the Fomc protecting groups respectively; wherein the coupling reaction sequence of the multiple activated amino acids is aspartic acid-alanine-glycine-arginine-lysine-glutamine-lysine-cysteine;

[0013] Step 3, mix carboxyl PEG-PLGA, EDC and NHS and dissolve them in DMF;

[0014] Step 4, adding the mixed solution obtained in step 3 to the resin-connected polypeptide obtained in step 2 for coupling treatment;

[0015] Step 5, adding the lysate to the solid phase resin containing the polypeptide to react, dissociating the polypeptide from the resin to obtain a linear DAG polypeptide containing a thiol group;

[0016] Step 6: Add hydrogen peroxide to the DMF solution of the thiol-containing linear DAG polypeptide, perform a cyclization reaction under ice bath conditions, precipitate, and wash to obtain a PEG-PLGA-modified DAG cyclic peptide.

[0017] The present invention provides a method for preparing a polypeptide nanocarrier for targeted drug delivery, using DAG as a targeting modification molecule to transform PEG-PLGA nanoparticles to synthesize a new polypeptide carrier, and in the preparation process, a solid-state synthesis method is selected to introduce carboxyl PEG-PLGA in a directional manner during the self-synthesis of DAG polypeptide. Amino acids are coupled to a resin containing activated amino groups, and then the Fomc protecting group of the main chain amino group is removed, and the process is repeated for different amino acids to synthesize a linear polypeptide with protected side chain amino groups, so that the prepared PEG-PLGA-modified DAG cyclic peptide has high purity. The material has the advantages of good targeting, high drug transport efficiency and short drug release cycle, and is easy to promote and apply.

[0018] Furthermore, in step 2, the molar ratio of each activated amino acid to the amino resin is 4-6:1.

[0019] Furthermore, in step 3, the molar ratio of carboxyl PEG-PLGA, EDC and NHS is 0.8-1.2:1.2-1.8:1.2-1.8.

[0020] Furthermore, in step 4, the coupling treatment is carried out at room temperature for 3-5 hours.

[0021] Furthermore, in step 5, the reaction is carried out at room temperature and the reaction time is 3-5 hours.

[0022] Furthermore, in step 6, the cyclization reaction is carried out at room temperature, and the cyclization reaction time is 1-2h.

[0023] Furthermore, the method also includes using an esterifying agent to esterify the PEG-PLGA modified DAG cyclic peptide.

[0024] Another object of the present invention is to protect the product prepared by the above method.

[0025] For example, a polypeptide nanocarrier prepared by the above-mentioned method for preparing a polypeptide nanocarrier for targeted drug delivery.

[0026] The use of the above-mentioned polypeptide nanocarriers in the preparation of targeted drugs.

[0027] A brain tissue targeted drug, comprising the above-mentioned polypeptide nanocarrier and β-sitosterol;

[0028] Prepared by the following method:

[0029] The polypeptide nanocarrier and β-sitosterol are mixed and dissolved in tetrahydrofuran to form an organic phase; deionized water is used as the aqueous phase;

[0030] Under stirring conditions, the organic phase is injected into the aqueous phase and stirred to obtain a suspension of DAG-PEG-PLGA nanoparticles loaded with BSIT, which is a brain tissue targeting drug.

[0031] Including PEG-PLGA modified DAG cyclic peptide and β-sitosterol;

[0032] Preferably, it is prepared by the following method:

[0033] The PEG-PLGA modified DAG cyclic peptide and β-sitosterol were mixed and dissolved in tetrahydrofuran to form an organic phase; deionized water was used as the aqueous phase;

[0034] Under stirring conditions, the organic phase is injected into the aqueous phase and stirred to obtain a suspension of DAG-PEG-PLGA nanoparticles loaded with BSIT, which is a brain tissue targeting drug.

[0035] Based on (1) the nasal mucosa is a lipid structure, the absorption of drugs in the nasal mucosa is mainly a passive diffusion process, so fat-soluble drugs are easily absorbed. (2) Nasal mucosal absorption is closely related to molecular weight. The larger the molecular weight, the worse the absorption. Circular proteins and peptides are easier to absorb than linear ones. (3) The nasal mucosa is negatively charged, so positively charged drugs are easy to penetrate. Therefore, the present invention uses BSIT-DAG-PEG-PLGA NPs because of its smaller molecular weight, spatial structure and charge that are more in line with the characteristics of transnasal mucosal administration, and it is easier to pass through the mucosal barrier and deliver the drug directly to the brain, which can not only overcome the first-pass effect and improve drug utilization, but also avoid liver and kidney damage that may be caused by drugs.

[0036] 1. The present invention provides a method for preparing a polypeptide nanocarrier for targeted drug delivery. DAG is used as a targeting modification molecule to modify PEG-PLGA nanoparticles to synthesize a new type of polypeptide carrier. In the preparation process, a solid-state synthesis method is selected to introduce carboxyl PEG-PLGA in a directional manner during the self-synthesis of DAG polypeptide. Amino acids are coupled to a resin containing activated amino groups, and then the Fomc protecting group of the main chain amino group is removed. The process is repeated for different amino acids to synthesize a linear polypeptide with protected side chain amino groups, so that the prepared PEG-PLGA-modified DAG cyclic peptide has high purity. The material has the advantages of good targeting, high drug transport efficiency and short drug release cycle, and is easy to promote and apply.

[0037] 2. The BSIT-DAG-PEG-PLGA NPs provided by the present invention have a smaller molecular weight, a spatial structure and a charge that are more in line with the characteristics of transnasal mucosal administration, and are easier to pass through the mucosal barrier and deliver the drug directly to the brain, which can not only overcome the first-pass effect and improve drug utilization, but also avoid liver and kidney damage that may be caused by drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The synthesis flow chart of DAG-PEG-PLGA nanoparticles.

[0039] Figure 2 This is a characterization diagram of drug-loaded nanoparticles.

[0040] Figure 3 This is a graph showing the pharmacokinetic test results of drug-loaded nanoparticles.

[0041] Figure 4 This is a characterization diagram of the neuroprotective effect of drug-loaded nanoparticles. DETAILED DESCRIPTION

[0042] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0043] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0044] The present invention provides a method for preparing a polypeptide nanocarrier for targeted drug delivery, comprising the following steps:

[0045] Step 1, placing the amino resin in a reaction column, and sequentially performing an expansion treatment and a deprotection treatment;

[0046] Step 2, adding multiple activated amino acids to the amino acid values ​​in step 1 in sequence for coupling reaction, and removing the Fomc protecting groups respectively; wherein the coupling reaction sequence of the multiple activated amino acids is aspartic acid-alanine-glycine-arginine-lysine-glutamine-lysine-cysteine;

[0047] Step 3, mix carboxyl PEG-PLGA, EDC and NHS and dissolve them in DMF;

[0048] Step 4, adding the mixed solution obtained in step 3 to the resin-connected polypeptide obtained in step 2 for coupling treatment;

[0049] Step 5, adding the lysate to the solid phase resin containing the polypeptide to react, dissociating the polypeptide from the resin to obtain a linear DAG polypeptide containing a thiol group;

[0050] Step 6: Add hydrogen peroxide to the DMF solution of the thiol-containing linear DAG polypeptide, perform a cyclization reaction under ice bath conditions, precipitate, and wash to obtain a PEG-PLGA-modified DAG cyclic peptide.

[0051] The present invention provides a method for preparing a polypeptide nanocarrier for targeted drug delivery, using DAG as a targeting modification molecule to transform PEG-PLGA nanoparticles to synthesize a new polypeptide carrier, and in the preparation process, a solid-state synthesis method is selected to introduce carboxyl PEG-PLGA in a directional manner during the self-synthesis of DAG polypeptide. Amino acids are coupled to a resin containing activated amino groups, and then the Fomc protecting group of the main chain amino group is removed, and the process is repeated for different amino acids to synthesize a linear polypeptide with protected side chain amino groups, so that the prepared PEG-PLGA-modified DAG cyclic peptide has high purity. The material has the advantages of good targeting, high drug transport efficiency and short drug release cycle, and is easy to promote and apply.

[0052] In some embodiments, in step 2, the molar ratio of each activated amino acid to the amino resin is 4-6:1.

[0053] In some embodiments, in step 3, the molar ratio of carboxyl PEG-PLGA, EDC and NHS is 0.8-1.2:1.2-1.8:1.2-1.8.

[0054] In some embodiments, in step 4, the coupling treatment is carried out at room temperature for 3-5 hours.

[0055] In some embodiments, in step 5, the reaction is carried out at room temperature for 3-5 hours.

[0056] In some embodiments, in step 6, the cyclization reaction is carried out at room temperature, and the cyclization reaction time is 1-2 hours.

[0057] In some embodiments, the method further comprises esterifying the PEG-PLGA modified DAG cyclic peptide with an esterifying agent.

[0058] The following provides specific embodiments: Example

[0059] Some sources of ingredients:

[0060] β-Sitosterol (purity >98%). Catalog number: HY-N0171A. MedChemExpress, Inc., New Jersey, USA.

[0061] Carboxylated poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG-COOH, MW: 10000, 5000). Catalog number: R-PL1063-15KD. Ruixi Biotechnology Co., Ltd., Shaanxi Province, China.

[0062] Amino PEGA resin. Catalog number: 372109-59-6. Sigma-Aldrich. Company, Massachusetts, USA.

[0063] N,N-Dimethylformamide (DMF, purity 99.8%). Catalog number: 227056. Sigma-Aldrich. Company, Massachusetts, USA.

[0064] Fluorenylmethoxycarbonyl protected amino acids (Fmoc-AA) were purchased from CEM, North Carolina, USA.

[0065] Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU). Catalog number: 94790-37-1. Merck KGaA, Darmstadt, Germany.

[0066] 1-Hydroxybenzotriazole hydrate (HOBt). Catalog number: 123333-53-9. Merck KGaA, Darmstadt, Germany.

[0067] N,N-Diisopropylethylamine (DIPEA). Catalog number: 7087-68-5. Merck KGaA, Darmstadt, Germany.

[0068] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Catalog number: 22980. ThermoFisher Scientific, Massachusetts, USA.

[0069] N-Hydroxysuccinimide (NHS). Catalog number: 24500. Thermo Fisher Scientific, Massachusetts, USA.

[0070] Trifluoroacetic acid (TFA). Catalog number: T6508. Merck KGaA, Darmstadt, Germany.

[0071] Triisopropylsilane (TIS). Catalog number: 233781. Sigma-Aldrich. Inc., Massachusetts, USA.

[0072] Synthesis of DAG-PEG-PLGA Nanoparticles Loaded with β-Sitosterol

[0073] Synthesis of DAG peptide and PEG-PLGA directed modification

[0074] PEG-PLGA modification can increase the water solubility and stability of nanoparticles, reduce the chance of being recognized and cleared by the immune system, thereby prolonging the circulation time of the drug in the blood and improving the bioavailability of the drug. Given that commercially available DAG cyclic peptides have many reaction sites, the selection of any modified PEG will lead to the generation of many by-products, which will make subsequent purification and identification difficult. Therefore, we chose to use solid-state synthesis to introduce carboxyl PEG-PLGA in the process of synthesizing DAG peptides ourselves.

[0075] Amino acids are coupled to a resin containing activated amino groups, and then the Fomc protecting group of the main chain amino group is removed. The process is repeated for different amino acids to synthesize a linear polypeptide with protected side chain amino groups.

[0076] Step 0. Pretreatment of amino resin support

[0077] First, the carrier amino resin is placed in the reaction column and soaked in dimethylformamide (DMF) for 30 minutes to fully swell the resin and prepare for the subsequent reaction. Then, a deprotection step is performed by adding a 20% solution of piperidine dissolved in DMF to the resin to remove the Fmoc protecting group on the resin. This process includes two deprotection steps, each lasting 10 minutes, followed by a thorough wash with a large amount of DMF to remove the remaining piperidine. After the deprotection step, the complete removal of the Fmoc group is confirmed by the Ninhydrin test.

[0078] Step 1. Solid phase-amino acid coupling

[0079] The Fmoc synthesis method was used for solid phase synthesis of peptides.

[0080] For amino groups on the amino acid side chain, tert-butyloxycarbonyl (BOC) protection is used. For thiol groups on the amino acid side chain, methylthioketone (Mtt) protection is used. Activate the amino acid before coupling. Select the required Fmoc-protected amino acid and dissolve it in dimethylformamide (DMF) to prepare a solution of approximately 0.1 M. Next, prepare HBTU and HOBt activation reagents at a concentration of 0.5 M, also using DMF as the solvent. Add DIPEA (diisopropylethylamine) at a concentration of 2.0 M as a basic catalyst to promote the coupling reaction.

[0081] The amino acid coupling stage is then carried out: the activated amino acid is added to the amino resin in a molar ratio of 5:1. Each coupling reaction is carried out at room temperature for 2 hours to ensure that the amino acid can fully react with the activated amino group on the resin.

[0082] Step 2. Fmoc deprotection

[0083] After the coupling is completed, the Fmoc protecting group is removed by treating with a DMF solution containing 20% ​​piperidine for 15 minutes. The resin is then washed three times with excess DMF, each time for 5 minutes, to ensure that all unreacted amino acids and byproducts are completely removed. After the washing step, the free amino group on the resin is tested by the Ninhydrin test to confirm whether the newly added amino acid is successfully coupled. The detection standard is that the condensation reaction is at least 99% for each amino acid coupling.

[0084] This process is repeated according to the peptide sequence until all amino acids are coupled in sequence. The order of synthesis is: D (aspartic acid) - A (alanine) - G (glycine) - R (arginine) - K (lysine) - Q (glutamine) - K (lysine) - C (cysteine).

[0085] In this experiment, a scrambled peptide was prepared as a control. Scrambled peptides are peptides whose amino acid composition is consistent with the target peptide, but the order of arrangement is random and disrupted. Such scrambled peptides are important tools in proteomics.

[0086] Step 3. Carboxyl PEG-PLGA modification

[0087] After the complete peptide chain is synthesized, only the terminal methionine contains a main chain amino group, so the modified commercially available carboxyl PEG-PLGA can be directional esterified with the main chain amino group to obtain a single PEG-PLGA modified DAG polypeptide. At this time, the polypeptide is still a straight chain and the side chain amino groups are protected by acid-sensitive groups.

[0088] Carboxyl-modified PEG-PLGA material was used, where the molecular weight of PEG was 10 kDa and the molecular weight of PLGA was 5 kDa. The carboxyl groups were fully activated before the reaction to improve the coupling efficiency. First, carboxyl PEG-PLGA was mixed with EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) at a molar ratio of 1:1.5:1.5 and dissolved in DMF. Then, PBS buffer containing the mixture was added to the resin-linked polypeptide. The mixture was mixed at room temperature and the reaction time was 4 hours to ensure sufficient coupling.

[0089] Step 4. Solid phase-peptide dissociation

[0090] After the synthesis is completed, the peptide can be dissociated from the solid phase support by treating the resin with a trifluoroacetic acid (TFA) mixture (containing 95% TFA, 2.5% TIS, and 2.5% water), and the acid-sensitive protecting groups on the side chain amino groups can be removed together without affecting the carboxyl PEG-PLGA condensed on the main chain amino groups. The cleavage mixture is directly added to the solid phase resin containing the peptide, and the amount of resin covered is sufficient to immerse the resin, and then the mixture is allowed to react at room temperature for 2 to 3 hours to ensure that the peptide is completely dissociated from the resin and all protecting groups are removed. Ideally, there is only one product, and the peptide material only needs to be precipitated and washed before the next ring closure treatment.

[0091] Step 5. Thiol reaction to form cyclic peptide

[0092] Hydrogen peroxide was selected as an oxidizing agent to allow the thiol group to react and form a disulfide bond. First, the linear DAG peptide containing thiol groups was dissolved in DMF. Next, 30% hydrogen peroxide was slowly added to the peptide solution, and the final concentration was adjusted to 5 mM to 10 mM. This operation was performed under an ice bath to control the reaction rate and prevent over-oxidation. The cyclization reaction was carried out at room temperature and lasted for 1 to 2 hours. After precipitation and washing, pure PEG-PLGA-modified DAG cyclic peptide was obtained.

[0093] Step 6. Esterification of carboxyl groups

[0094] To enhance the lipid solubility of DAG, the hydrophilic carboxyl groups in its external side chains were converted into lipophilic ester groups. The DAG cyclic peptide was dissolved in anhydrous methanol at a concentration of 10 mg / ml. EDC and NHS were added as esterification reagents. The molar ratio of carboxyl groups to EDC and NHS was 1:1.5:1.5. The reaction was carried out at room temperature with continuous stirring for 12 hours. The progress of the reaction could be monitored by periodic sampling and using high performance liquid chromatography (HPLC).

[0095] After the chemical modification is completed, an equal volume of anhydrous ether is added to the reaction solution for precipitation, and then centrifugation (5000 rpm, 10 minutes) is performed to collect the precipitate. The precipitate is washed several times with cold ether to remove unreacted substances and by-products. The washed precipitate is dried in a freeze vacuum dryer.

[0096] Through these steps, the lipid solubility of DAG cyclic peptide was effectively improved, the carboxyl group on the side chain was successfully converted into an ester group, and the primary amine was protonated, thereby enhancing its ability to penetrate biological membranes ( Figure 1 ).

[0097] DAG-PEG-PLGA nanoparticles encapsulating β-sitosterol

[0098] β-Sitosterol (BSIT) has significant antioxidant capacity and can reduce the damage of COP to nerve cells by inhibiting inflammation and scavenging free radicals. Studies have shown that BSIT can inhibit the apoptosis of nerve cells caused by brain damage and promote the regeneration and repair of nerve cells.

[0099] In this experiment, DAG-PEG-PLGA and BSIT were dissolved in tetrahydrofuran (THF) solution at a mass ratio of 4:1 to form an organic phase, and deionized water was used as the aqueous phase. The oil-water ratio was set to THF:H2O = 1:2, and the organic phase was slowly injected into the aqueous phase at a rotation speed of 400 rpm in a magnetic stirrer. Stirring was continued at room temperature for 4 hours until the organic solvent was completely evaporated to obtain a suspension of BSIT-loaded DAG-PEG-PLGA nanoparticles (BSIT-loaded DAG-PEG-PLGA NPs).

[0100] Nanoparticle characterization

[0101] Fourier transform infrared spectroscopy

[0102] Mix the newly synthesized nanoparticles with potassium bromide (KBr) powder in a ratio of 1:100 to ensure that the nanoparticles are evenly dispersed in the KBr. Place the mixed powder into a mold and press it using a hydraulic press. Use a pressure of about 10 tons and a pressing time of about 2 minutes to form a uniform and transparent KBr / nanoparticle flake. Check the flakes for cracks or uneven areas to ensure that the quality of the flakes is suitable for infrared spectroscopy analysis. Place the prepared sample flakes or films in the sample cell of the infrared spectrometer and use a Spectrum 100 FTIR spectrometer (PerkinElmer, Waltham, MA, USA) for background scanning to obtain the FTIR transmission spectrum of the nanoparticles. The scanning range is 400-4000 cm -1 , with a resolution of 2 cm -1 .

[0103] Transmission electron microscopy

[0104] Transmission electron microscopy (TEM) is an important tool for studying the morphology and size of drug-loaded nanoparticles. The nanoparticle solution was treated with ultrasound for 10 minutes to ensure that the nanoparticles were evenly dispersed in water. 2 μL of the nanoparticle solution was dropped onto a copper grid and allowed to dry naturally. Subsequently, 2% phosphotungstic acid stain was added for negative staining to increase the contrast. The sample was then loaded into a TEM sample holder to observe the size and morphology of the nanoparticles under TEM. The acquired images were subjected to size analysis to measure the diameter of the nanoparticles.

[0105] DLS particle size and zeta potential analysis

[0106] Dynamic light scattering (DLS) is based on Brownian motion. It detects the fluctuation of the intensity of scattered light from particles through laser detection, calculates the diffusion coefficient and derives the particle size and Zeta potential. The nanoparticles were diluted to a low concentration solution of 1 mg / ml and treated with ultrasound for 10 minutes to ensure that the nanoparticles were evenly dispersed in the solution. The particle size, polydispersity and potential of BSIT-loaded PEG-PLGA NPs and BSIT-loaded DAG-PEG-PLGA NPs in aqueous solution were detected using a nanoparticle size potential meter (Zetasizer Ultra, Malvern). The He-Ne laser source was selected for the experiment, the measurement wavelength was set to 633 nm, and the set temperature was controlled at 25°C. The measurement was repeated three times in the experiment to ensure the repeatability and accuracy of the data.

[0107] Cytotoxicity

[0108] The CCK-8 method was used to evaluate the cytotoxicity of nanoparticles to PC12 cells. Cells in logarithmic growth phase were plated at 5 × 10 3 The cells were seeded in a 96-well plate at a density of 100 cells / mL and cultured at 37°C and 5% CO2 for 24 hours. The next day, the culture medium was removed and culture medium solutions containing nanoparticles at different concentrations (1, 2, 4, 8, 16 mg / ml) were added to each well. The wells without PEG-PLGA nanoparticles served as the control group. Six replicate wells were set up for each concentration. After 24 hours of culture, the culture medium was removed and the cells were washed twice with PBS. CCK-8 reagent was added according to the experimental protocol provided by the reagent manufacturer, and the cells were incubated for 1 hour. The absorbance at a wavelength of 450 nm was read using an enzyme reader to calculate the cell viability. The formula for calculating cell viability is:

[0109] ;

[0110] Where A stands for absorbance.

[0111] Pharmacokinetics

[0112] In vitro cell delivery efficiency

[0113] First, the drug carrier was synthesized according to the above method. The experimental group used BSIT-loaded DAG-PEG-PLGANPs, and the negative control group used scrambled peptide (SP) to replace DAG peptide to construct the carrier. The rest of the synthesis method was exactly the same. Although the negative control SP has the same amino acid ratio as the DAG peptide, it does not have the sequence and ring structure of the DAG peptide, so it has no corresponding biological activity.

[0114] Subsequently, PC12 neurons in the logarithmic growth phase were cultured at 5 × 10 5 The cells were seeded in 6-well plates at a density of 100 cells / mL. After incubation at 37°C for 24 hours, the nanoparticles were labeled with FITC and added to each well. The concentration of FITC in each well was 5 μg. The cells were then incubated at 37°C for 4 hours. Afterwards, the cells were washed three times with PBS and treated with 2 μg / ml 4',6-diamidino-2-phenylindole (DAPI) solution at room temperature for 5 minutes. After washing again with PBS three times, the cells were fixed with 4% paraformaldehyde for 15 minutes. The paraformaldehyde solution was discarded and the cells were stored in 50% glycerol. The uptake of nanoparticles by cells was observed under a laser confocal microscope.

[0115] In vitro blood-brain barrier permeability

[0116] The in vitro BBB model was constructed by using Transwell plates. Briefly, mouse brain capillary endothelial cells (Shanghai Qincheng Biotechnology Co., Ltd., Cat no. 6380) were cultured at 2 × 10 5 Cells / well were seeded on the porous membrane of the Transwell top chamber and cultured in Dulbecco's modified Eagle's medium (DMEM) for 1 week. The endothelial cell resistance (TEER) of the endothelial cell porous membrane was measured by Millicell ERS (Millipore, USA). When the TEER value exceeded 200 Ωcm 2 The BBB model was considered successful when FITC-labeled nanoparticles (2 μg / ml) were dispersed in fresh culture medium and then added to the top chamber, and normal culture medium was added to the lower chamber of the Transwell. After incubation for 1, 2, 3, and 4 hours, the nanoparticle content in the culture medium in the basolateral chamber was measured by a fluorescence spectrophotometer to determine whether it had crossed the BBB.

[0117] Nanoparticles in vivo distribution and metabolism detection

[0118] Fluorescence tracing technology plays an important role in studying the distribution and metabolism of drugs in the body. This technology has high spatial resolution and is often used to study whether drugs can penetrate the BBB and enter the central nervous system. At the same time, fluorescence tracing technology helps evaluate the distribution of carriers and the drug release process in drug carrier research, providing support for the development of efficient targeted delivery systems.

[0119] In the study of drug distribution and metabolic distribution in vivo, we selected healthy, adult BALB / C mice (body weight 20-30 g). Two nanoparticles were labeled with Cy7 fluorescent dye (Sulfo-Cy7-E SE, Uelandy, Cat no. C5070), and the experimental method was referred to the supplier's protocol. When administering the drug by intranasal drops, the experimental mouse was first gently fixed with its head facing up, and the drug solution was dripped into the animal's nasal cavity through a pipette. Each animal was administered about 20 μl each time (adjusted according to the size of the animal, BSIT 2 mg / kg calculated according to the animal's weight), ensuring that the liquid was inhaled. After 30 minutes, isoflurane anesthesia was used, and fluorescent intravital imaging was performed using a small animal in vivo multi-mode imager (IVIS Spectrum) to track the range of drug diffusion.

[0120] In the metabolic distribution, two methods of administration were used: nasal drops and tail vein injection. The administration method and dosage of the nasal drops group were the same as before. Conventional injection techniques were used for the tail vein injection group. The amount of liquid injected into each animal was about 100 μl (BSIT 2 mg / kg calculated according to the animal's body weight), ensuring that the needle was inserted correctly and the liquid was pushed in smoothly during injection. After administration, ensure that the animal was resting quietly. Four hours after administration, the animals were anesthetized with isoflurane and quickly killed by dislocation to ensure that the animals were operated in a painless state. The animals were quickly dissected and the brain, heart, liver, spleen, kidney and lung were removed, and tissue damage was avoided as much as possible. The removed organs were quickly placed on ice to maintain tissue activity. The small animal in vivo multi-mode imager was used for imaging, and the fluorescence imaging system software was used for analysis to calculate the drug concentration in different organs and determine the distribution of the drug in each organ.

[0121] The excitation light for imaging is 746 nm and the emission light is 772 nm. The fluorescence efficiency (RE) target area is used to evaluate the drug concentration, and the calculation formula is:

[0122] ;

[0123] P / sec / cm² / sr represents the photon output intensity of a light source. It is measured in photons per square centimeter per solid angle per second.

[0124] μW / cm² stands for radiant power, which is the energy flux density per unit area of ​​a light source. It is measured in microwatts per square centimeter.

[0125] Evaluation of the therapeutic effect of BSIT-loaded nanoparticles in the treatment of COP brain injury

[0126] Carbon monoxide poisoning and nose-to-brain administration

[0127] COP is a common factor leading to brain damage. When CO enters the human body, it combines with hemoglobin to form carboxyhemoglobin (COHb), which causes hemoglobin to lose its oxygen-carrying capacity and triggers inflammation and oxidative stress, resulting in brain damage and cognitive impairment. This study used adult, healthy, male SD rats (body weight 200-220 g) and used BSIT-loaded DAG-PEG-PLGA NPS intervention therapy to treat neuronal damage by targeted delivery of the drug's active ingredient, β-sitosterol, to brain tissue.

[0128] SD rats were placed in a closed animal oxygen chamber and inhaled carbon monoxide (CO) gas at a concentration of 1000 ppm for 10 minutes, followed by CO gas at a concentration of 2000 ppm for 20 minutes. Rats with impaired consciousness and high HbCO concentrations (>40%) were considered to have successfully simulated acute severe CO poisoning.

[0129] The nasal mucosal targeted brain drug delivery method has the advantages of fast targeted transport speed, high targeting efficiency and avoidance of first-pass effect. In this animal experiment, the nasal instillation method was adopted. From 1 to 7 days after COP, the nasal instillation was performed once a day, and the dosage was calculated according to BSIT 2 mg / kg. The volume of each animal was about 30 μl per time.

[0130] Neural structure detection

[0131] Rats were anesthetized by intraperitoneal injection of 0.2 ml sodium pentobarbital, and the left ventricle was perfused with 150 ml saline and 150 ml paraformaldehyde in sequence. The heart was well fixed when it felt hard to the touch. The hair on the head was removed, the foramen magnum was cut along the midline of the parietal bone, the skull and olfactory bulb were removed, and the isolated brain tissue was obtained and immediately fixed in paraformaldehyde. After paraffin embedding, the brain was sliced ​​using a tissue slicer. For immunofluorescence staining, anti-NeuN antibody was used as the primary antibody (Cat no. ab177487, Abcam, 1:200 dilution). Alexa Fluor® 488-labeled goat anti-rabbit antibody was used as the secondary antibody (Cat no. ab150077, Abcam, 1:200 dilution).

[0132] Cognitive function testing

[0133] The Morris water maze test is a behavioral test widely used to assess the learning and memory abilities of animals. The water maze setup consists of a circular pool with a diameter of 1.6 m and a depth of 30 cm, divided into four quadrants. A transparent platform with a diameter of 10 cm was placed in one of the quadrants, with the surface of the platform 1 to 2 cm below the water surface. The water temperature was maintained in the range of 20 to 22 °C. In this experiment, the escape latency test was used to assess learning efficiency and memory formation, and the time spent in the quadrant where the platform was placed was used to assess the retention of spatial and long-term memory. The escape latency training lasted for 3 days, with multiple trials per day. In each trial, the rats were released from different quadrants, and the time required to find the platform was recorded. On the last day of training, the platform was removed for a probe test to observe the time the rats stayed in the quadrant where the platform was placed, with an observation period of 120 seconds. After each trial, the rats were quickly dried and placed in a warm environment to rest to mitigate the risk of hypothermia or infection.

[0134] result

[0135] Characterization of drug-loaded nanoparticles

[0136] Figure 2 The Fourier transform infrared spectra (FTIR) of BSIT loaded DAG-PEG-PLGA NPs and BSIT loaded PEG-PLGA NPs are shown in Figure 2. After DAG grafting, the presence of amino and amide bonds were observed. The infrared spectrum showed significant changes in the peak positions of the high wavenumber region (wavenumber 2940), amide I (wavenumber 1671), and amide II bands (wavenumber 1541), indicating that an amide formation reaction occurred between DAG and the COOH functional group, thus confirming the grafting of the peptide on the surface of PEG-PLGA NPs. This provides evidence for DAG peptide modification ( Figure 2 A in the figure).

[0137] TEM analysis confirmed that BSIT loaded DAG-PEG-PLGA NPs were spherical and uniform in morphology with a particle size of 112.5±7.5 nm ( Figure 2 B in the figure).

[0138] The particle size of BSIT loaded PEG-PLGA NPs measured by DLS was 97.2±3.0 nm, while the particle size of BSIT loaded DAG-PEG-PLGA NPs increased to 131.4±3.9 nm. The significant increase in particle size indicates the successful grafting of DAG to NPs. In addition, the particle size of the nanoparticles increased slightly after DAG modification, but was still below 200 nm. This small diameter is conducive to the penetration of drug-loaded nanoparticles through the BBB to achieve targeted drug delivery. The polydispersity index (PDI) of BSIT loaded DAG-PEG-PLGA NPs was measured to be 0.16, indicating that the particle size distribution was relatively uniform. In addition, the BSIT loaded DAG-PEG-PLGA NPs were positively charged, and the Zeta potential was measured to be 12.5±1.7mv ( Figure 2 C and D in Figure 4). The weak charge generated by chemical modification is theoretically more conducive to the carrier to cross the nasal mucosal barrier.

[0139] In vitro cytotoxicity experiments showed that BSIT loaded DAG-PEG-PLGA NPs had no significant toxic effects on PC12 cells in the concentration range of 0 ~ 16 mg / ml. The cell viability in the experiment was always above 80%, which met the non-cytotoxicity standard specified by the United States Pharmacopoeia. The viability of PC12 cells after 24 hours of incubation with BSIT loaded DAG-PEG-PLGA NPs is shown in the figure ( Figure 2 E in.

[0140] Pharmacokinetics

[0141] In order to detect the neuron targeting of NPs, we labeled NPs with FITC, used PC12 neurons as target cells, and detected them using a fluorescence microscope. The results showed that the proportion of PEG-PLGA NPs (SP-NPs) modified with scrambled peptides in the control group that could be phagocytosed by PC12 neurons was measured to be 33.9±11.1%. After modification with DAG peptide, the proportion of NPs phagocytosed was greatly increased to about 60.8±10.33%. This result shows that DAG peptide modification can enhance the neuron targeting ability of nanoparticles ( Figure 3 In the in vitro blood-brain barrier penetration experiment, we observed that compared with PEG-PLGANPs modified with scrambled peptides, PEG-PLGA NPs with DAG peptide as the target showed significantly higher cell layer penetration rate (P<0.05), indicating that DAG peptide plays a key role in the transport of BSIT across the BBB ( Figure 3 B in the figure).

[0142] In vivo drug distribution studies have found that nanoparticles labeled with Cy7 can be tracked through fluorescence effects. The fluorescence intensity is proportional to the concentration of the nanoparticles. Nanoparticles with DAG as the target head (DAG-NPs) can be more absorbed by animals after intranasal administration (IN) and diffuse to the tissues and organs of the animal's head within 30 minutes. Nanoparticles with scrambled peptides as the target head (SP-NPs) are difficult to be absorbed by animals and remain mainly in the nasal cavity after 30 minutes ( Figure 3 C in.

[0143] In vivo drug metabolism studies have found that 4 hours after nasal instillation of DAG-NPs solution, a fluorescent signal was detected in the brain of the animal. The rest was mainly metabolized in the liver, and a small amount was metabolized in the kidneys. 4 hours after nasal instillation of SP-NPs, the fluorescence effect of brain tissue was significantly reduced compared with DAG-NPs. In animals administered intravenously (IV), we observed that the presence of drugs could be detected in all organs of the animals, and the fluorescence effects of the liver and kidneys were significantly higher than those of the nasal administration group. Statistical analysis showed that the brain drug level of mice in the DAG-NPs IN group was significantly higher than that in the SP-NPs IN group. Although the brain drug level of the DAG-NPs IV group was similar to that of the DAG-NPs IN group, more drugs accumulated in the liver and kidneys, and the liver-brain ratio of drug distribution was significantly lower than that of the latter ( Figure 3 D in.

[0144] This indicates that DAG can improve the brain targeting of NPs, and nanoparticles with DAG as a carrier (DAG-NPs) have a high blood-brain barrier penetration ability. Nasal administration of DAG-NPs and reducing the body's first-pass effect are the optimal drug delivery strategies.

[0145] Evaluation of the therapeutic efficacy of BSIT-loaded nanoparticles in the treatment of CO poisoning

[0146] Neurons are the main components of the nervous system, and hippocampal neurons are widely considered to be the structural basis for the transmission of neural electrical signals and the construction, storage and retrieval of memory information. The memory impairment and loss after CO poisoning are usually related to neuronal damage. BSIT has been shown to protect the nervous system through anti-inflammatory and anti-free radical methods, and is a potential drug for brain damage caused by CO poisoning.

[0147] In the immunofluorescence experiment, NeuN antibody was used to label neurons in the rat hippocampus. The results showed that DAG-targeted nanoparticles as carriers of BIST significantly increased the number of surviving neurons after CO poisoning, which may help repair cognitive function ( Figure 4 A in the figure).

[0148] Acute severe CO poisoning brain damage mainly affects the learning and memory ability of animals. The water maze test is often used to evaluate animal memory and motor function. The results showed that after treatment with the BIST vector with DAG as the target, the escape latency of CO poisoning rats in finding the platform was significantly shortened, and the time they stayed in the platform quadrant was significantly prolonged ( Figure 4 B in the figure).

[0149] These results indicate that DAG modification can increase the targeting ability of BSIT, thereby exerting a neuroprotective effect, not only reducing the toxic effects of CO on neurons in rats, but also helping to restore the learning and memory abilities of rats.

[0150] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a polypeptide nanocarrier for targeted drug delivery, characterized in that: The following steps are involved: Step 1, placing the amino resin in a reaction column, and sequentially performing an expansion treatment and a deprotection treatment; Step 2, adding multiple activated amino acids to the amino acid values ​​in step 1 in sequence for coupling reaction, and removing the Fomc protecting groups respectively; wherein the coupling reaction sequence of the multiple activated amino acids is aspartic acid-alanine-glycine-arginine-lysine-glutamine-lysine-cysteine; Step 3, mix carboxyl PEG-PLGA, EDC and NHS and dissolve them in DMF; Step 4, adding the mixed solution obtained in step 3 to the resin-connected polypeptide obtained in step 2 for coupling treatment; Step 5, adding the lysate to the solid phase resin containing the polypeptide to react, dissociating the polypeptide from the resin to obtain a linear DAG polypeptide containing a thiol group; Step 6: Add hydrogen peroxide to the DMF solution of the thiol-containing linear DAG polypeptide, perform a cyclization reaction under ice bath conditions, precipitate, and wash to obtain a PEG-PLGA-modified DAG cyclic peptide.

2. The method for preparing a polypeptide nanocarrier for targeted drug delivery according to claim 1, characterized in that: In the step 2, the molar ratio of each activated amino acid to the amino resin is 4-6:

1.

3. The method for preparing a polypeptide nanocarrier for targeted drug delivery according to claim 1, characterized in that: In the step 3, the molar ratio of carboxyl PEG-PLGA, EDC and NHS is 0.8-1.2:1.2-1.8:1.2-1.

8.

4. The method for preparing a polypeptide nanocarrier for targeted drug delivery according to claim 1, characterized in that: In the step 4, the coupling treatment is carried out at room temperature for 3-5 hours.

5. The method for preparing a polypeptide nanocarrier for targeted drug delivery according to claim 1, characterized in that: In the step 5, the reaction is carried out at room temperature and the reaction time is 3-5 hours.

6. The method for preparing a polypeptide nanocarrier for targeted drug delivery according to claim 1, characterized in that: In the step 6, the cyclization reaction is carried out at room temperature, and the cyclization reaction time is 1-2 hours.

7. The method for preparing a polypeptide nanocarrier for targeted drug delivery according to any one of claims 1 to 6, characterized in that: The method also includes esterifying the PEG-PLGA modified DAG cyclic peptide using an esterifying agent.

8. A polypeptide nanocarrier prepared by the method for preparing a polypeptide nanocarrier for targeted drug delivery according to any one of claims 1 to 7.

9. Use of the polypeptide nanocarrier as claimed in claim 8 in the preparation of targeted drugs.

10. A brain tissue targeting drug, characterized in that: comprising the polypeptide nanocarrier as claimed in claim 8 and β-sitosterol; Prepared by the following method: The polypeptide nanocarrier and β-sitosterol are mixed and dissolved in tetrahydrofuran to form an organic phase; deionized water is used as the aqueous phase; Under stirring conditions, the organic phase is injected into the aqueous phase and stirred to obtain a suspension of DAG-PEG-PLGA nanoparticles loaded with BSIT, which is a brain tissue targeting drug.

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