Clusterin affinity peptide and preparation method and application of long-circulating nanoparticles modified by Clusterin affinity peptide

Abstract

The invention discloses a Clusterin affinity peptide and a preparation method and application of long-circulating nanoparticles modified by the Clusterin affinity peptide, and relates to the technical field of biological medicines. According to the invention, the polypeptide with high affinity and selectivity with the Human Clusterin recombinant protein is obtained through multiple rounds of library panning enrichment by a phage display technology. When the polypeptide is applied to preparation of curcumin long-circulating nanoparticles, the half-life period of the medicine can be prolonged, and the acute lung injury treatment effect can be enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a preparation method and application of Clusterin affinity peptide and long-circulation nanoparticles modified therefrom. Background Art

[0002] Acute lung injury (ALI) is a common clinical syndrome characterized by diffuse infiltration of a large number of inflammatory cells in the lungs, increased pulmonary microvascular permeability, severe edema in the alveoli and lung parenchyma, and hypoxemia. To date, there is no safe and effective early intervention drug or powerful treatment to curb the progression of ALI. In recent years, curcumin has become an effective drug for the treatment of ALI, which can act through multiple cellular pathways and multiple molecular targets. However, due to its poor water solubility, instability, and easy degradation, curcumin has a short half-life in the body and low oral bioavailability, which limits its clinical application.

[0003] At present, nano drug carriers are widely used in the field of medicine. After the nanosystem enters the blood circulation, a layer of protein corona formed on the surface can cause its targeting specificity to weaken or even disappear. The fate of nanoparticles in vivo is closely related to the composition of their protein corona. In drug delivery systems, in order to reduce nonspecific cellular uptake, it is believed that the covalent connection of nanoparticles with polyethylene glycol (PEG) can reduce protein adsorption, thereby giving them a stealth effect. However, literature research has found that uptake experiments have shown that PEG nanoparticles that have not been incubated with plasma can be taken up in large amounts nonspecifically, while PEG nanoparticles have lower cellular uptake after incubation with plasma, and the protein corona formed on the surface of the incubated nanoparticles contains a large amount of Clusterin protein. Nanoparticles modified with poly (phosphoethylacetic acid) and incubated in high Clusterin show lower nonspecific adsorption than unincubated nanoparticles. It can be seen that the stealth effect of Clusterin plays a key role in both PEGylated and non-PEGylated nanoparticles.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of Clusterin affinity peptide and long-circulation nanoparticles modified therewith. The Clusterin affinity peptide is modified on the surface of the nanoparticles to increase the circulation time of the drug in the body, thereby improving the therapeutic effect of the drug.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a Clusterin affinity peptide, the amino acid sequence of which is an amino acid sequence shown in one of SEQ ID NO.1-8.

[0008] In some embodiments, the method for screening Clusterin affinity peptides comprises:

[0009] (1) Protein preparation: Preparation of Human Clusterin recombinant protein by eukaryotic expression can achieve a protein purity of more than 90%, meeting the requirements for library peptide screening;

[0010] (2) Peptide library screening: Using phage display technology, through multiple rounds of panning, peptides with high affinity and selectivity for Human Clusterin recombinant protein were obtained;

[0011] (3) Affinity detection: The affinity of the screened peptides and Human Clusterin protein is detected using biomembrane interferometry technology to screen peptides with appropriate affinity.

[0012] In a second aspect, the present invention also provides the use of the above Clusterin affinity peptide in improving the half-life of a drug.

[0013] After the polyethylene glycol-modified nanoparticles were incubated with plasma, the interaction between the surface adsorbed proteins and macrophages decreased, and the macrophages' uptake of it decreased, while the uptake of unincubated nanoparticles did not decrease. Subsequent studies have found that there are more clusterins (Clusterin, CLU) in the protein corona formed on the surface of the nanoparticles after incubation. Nanoparticles incubated in a clusterin-rich solution have weaker adsorption to opsonin proteins than those not incubated, and the probability of being engulfed and captured by macrophages is reduced. The stealth effect of clusterin in the protein corona plays a key role in both PEGylated and non-PEGylated nanoparticles. Based on the characteristics of CLU, in the present invention, the inventors used it as a target, screened high-affinity and selective affinity peptides through HumanClusterin recombinant protein, and modified them to the surface of nanoparticles, so that the nanoparticles modified with Clusterin affinity peptides have long circulation characteristics, increasing the probability of nanoparticles entering the inflammatory leakage site, thereby enhancing the efficacy.

[0014] In a third aspect, the present invention also provides a long-circulation nanoparticle, which includes a drug-loaded protein nanoparticle and a modified substance coupled to its surface; the modified substance includes the above-mentioned Clusterin affinity peptide.

[0015] In some embodiments, the drug-loaded protein nanoparticles include casein-loaded curcumin nanoparticles and albumin-loaded curcumin nanoparticles. It should be noted that the carrier of the drug-loaded protein nanoparticles is not limited to the casein and albumin mentioned above, but can also be other macromolecular substances with carrier functions; the drug in the drug-loaded protein nanoparticles is not limited to curcumin, but can also be other drugs, such as paclitaxel, cisplatin, doxorubicin, tripterygium wilfordii and other drugs.

[0016] In some embodiments, the above-mentioned modifier also includes mPEG-SH. In the present invention, the modifier of the long-circulation nanoparticle contains a highly reactive chemical group, which can be the above-mentioned Clusterin affinity peptide, or mPEG-SH, or a substance with a highly reactive chemical group such as a thiol group or an amino group, all of which can effectively increase the drug's in vivo circulation time.

[0017] In a fourth aspect, the present invention also provides a method for preparing the long-circulation nanoparticles, comprising:

[0018] The protein and the chemical drug are ultrasonically mixed according to the mass ratio, and then added to a saturated salt solution. After mixing, a cross-linking agent is added, and after mixing again, the precipitate is separated and collected to obtain drug-loaded protein nanoparticles; the drug-loaded protein nanoparticles are then dispersed in a buffer solution containing a modifier, and long-circulation nanoparticles are obtained after incubation and separation.

[0019] In some embodiments, the above-mentioned modifiers include mPEG-SH and Clusterin affinity peptide.

[0020] In the present invention, the protein and the drug are formed into drug-loaded protein microparticles by using a saturated salt solution, and the drug-loaded protein microparticles are solidified by a cross-linking agent to form drug-loaded protein nanoparticles.

[0021] In some embodiments, the mass ratio of the drug-loaded protein nanoparticles to the modified substance is 10:1-1:10.

[0022] In some embodiments, the carrier protein comprises albumin and casein.

[0023] In some embodiments, the chemical drug comprises curcumin.

[0024] In some embodiments, the mass ratio of the above protein to the chemical drug is 5-20:1.

[0025] In some embodiments, the saturation of the saturated salt solution is 90-100%.

[0026] In some embodiments, the cross-linking agent includes tannic acid and ferric chloride.

[0027] In the drug-loaded protein nanoparticles of the present invention, the interior is protein microparticles encapsulating drugs, and the exterior is coated with tannic acid iron ion polymers.

[0028] Specifically, the preparation method of the long-circulation nanoparticles is as follows:

[0029] (1) The protein and curcumin were ultrasonically mixed at a mass ratio of 5:1-20:1, added to a saturated salt solution with a saturation of 100%-90%, vortexed for 30 seconds, tannic acid was added to 0.05-1 mg / mL, vortexed for 30 seconds, and ferric chloride was continued to be added to 1-50 mg / mL, vortexed for 30 seconds, and centrifuged at 4°C and 12000 rpm for 10 minutes to collect the precipitate, namely CurNP nanoparticles.

[0030] (2) CurNP nanoparticles were uniformly dispersed in PBS buffer containing mPEG-SH or Clusterin affinity peptide, with the ratio of Curcumin to mPEG-SH and Clusterin affinity peptide in the nanoparticles being 1:1. The nanoparticles were incubated with slow shaking at room temperature for 30 min, and collected by centrifugation at 4°C and 12,000 rpm for 10 min to obtain long-circulation nanoparticles.

[0031] In a fifth aspect, the present invention also provides the use of the long-circulation nanoparticles described above in the preparation of drugs for prolonging half-life.

[0032] In some embodiments of the present invention, by preparing Clusterin affinity peptide-modified curcumin long-circulation nanoparticles or mPEG-SH-modified curcumin long-circulation nanoparticles, and applying them to the preparation of nanomedicines, the obtained drugs have long-circulation characteristics and can better treat acute lung injury. Specifically, the drug can improve lung function (including vital capacity, forced vital capacity, forced expiratory volume in 200 milliseconds, dynamic lung compliance, static lung compliance), increase lung wet-to-dry weight ratio, reduce lung inflammation, etc.

[0033] The present invention has the following beneficial effects:

[0034] The present invention utilizes phage display technology to obtain polypeptides with high affinity and selectivity to Human Clusterin recombinant protein through three rounds of panning, and determines the affinity of the polypeptides and Human Clusterin protein through biomembrane interferometry technology to screen polypeptides with suitable affinity, and modifies the obtained polypeptides onto the surface of casein-loaded curcumin nanoparticles or albumin-loaded curcumin nanoparticles, which can prolong the circulation time of the drug in vivo, thereby improving the therapeutic effect of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0036] Figure 1 For enzyme digestion identification of the recombinant plasmid pKMD-CMV-Clusterin;

[0037] Figure 2 SDS-PAGE / WB results (A) and purity identification (B) for protein expression and purification;

[0038] Figure 3 The results of phage display library amplification and elution;

[0039] Figure 4 It is the signal diagram of the interaction between peptide and Clusterin;

[0040] Figure 5 The stability of nanoparticles (A: 7-day relative particle size change; B, C: 24-hour relative particle size and relative turbidity changes in serum);

[0041] Figure 6 is the mean plasma drug concentration-time curve (n=6);

[0042] Figure 7 HE staining pathological photos of rat lungs (A: normal group; B: sham operation group; C: model group; D: Cur group; E: CurNP group; F: CurNP-PEG group; G: CurNP-P14 group);

[0043] Figure 8 HE-stained pathological photos of the main organs of rats (A: normal group; B: sham operation group; C: model group; D: Cur group; E: CurNP group; F: CurNP-PEG group; G: CurNP-P14 group). DETAILED DESCRIPTION

[0044] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0045] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0046] Example 1

[0047] 1. Preparation of Clusterin

[0048] 1.1 Human Clusterin protein expression design

[0049] The gene sequence corresponding to the Human clusterin protein was amplified by PCR (the amino acid sequence is shown in SEQ ID NO.9), and the DNA fragment obtained by enzyme digestion and the pKMD-CMV expression vector were connected using ligase to construct the pKMD-CMV-Clusterin plasmid. Enzyme digestion was identified by 1% agarose gel electrophoresis.

[0050]

[0051] The bold part at the front is the signal peptide from human IL2, and the bold part at the back is 6xhis-flag, used to construct the pKMD-CMV expression vector.

[0052] The nucleotide sequence corresponding to the Human clusterin protein is shown in SEQ ID NO.10: the underlined portion is the target sequence, and the front and rear bold portions are the BamHI and XhoI restriction sites, respectively.

[0053]

[0054]

[0055] like Figure 1 As shown, after the recombinant plasmid pKMD-CMV-Clusterin was double-digested with restriction endonucleases BamHI and XhoI, a band of about 6000 bp and a band of about 1400 bp were obtained, which were consistent with the length of the target band (Clusterin: 1338 bp), indicating that Clusterin had been connected to the pKMD-CMV expression vector and the recombinant plasmid pKMD-CMV-Clusterin was successfully constructed.

[0056] 1.2 Transformation and extraction of recombinant expression plasmid

[0057] Thaw TOP10 E.coli chemical competent cells on ice, add 200ng of pKMD-CMV-Clusterin to the competent cells, mix gently, let stand on ice for 30min, then heat shock at 42℃ for 90s, and quickly let stand on ice for 5min. Add 900μL LB medium to the centrifuge tube, shake the bacteria at 37℃ and 225rpm for 4h. Take 100μL of bacterial solution and apply it to LB plate containing 30μg / mL ampicillin, and invert and culture at 37℃ for 12-16h. Pick white monoclonal colonies and inoculate them in 50mL LB medium containing 30μg / mL ampicillin, extract plasmids with a small extraction medium endotoxin-free kit, freeze-dry and concentrate, and re-dissolve the plasmids to a concentration of 1μg / μL.

[0058] 1.3 Cell transfection

[0059] One day before transfection, count the 293F cells in normal culture, take an appropriate amount of cell suspension and centrifuge, discard the supernatant and resuspend in fresh Expression Medium (containing 6mM L-glutamine) to 1×10 6 cells / mL (total volume 500mL×2), culture overnight at 37℃, 5% CO2, 125rpm. The next day, prepare the transfection complex. Add 40mL medium and 300μg plasmid DNA to a 50mL sterile centrifuge tube and vortex to mix. Add 600μL Lipofectin 2000 to the diluted DNA solution, vortex to mix, and incubate at room temperature for 10min. Add the transfection complex to 2×500mL of 293F culture, culture at 37℃, 5% CO2, 125rpm. After 4 days, centrifuge at 1000rpm for 5min to collect the expression supernatant, filter with 0.22μm filter membrane and use for protein purification.

[0060] 1.4 Nickel (Ni) column affinity chromatography purification

[0061] First, three buffer solutions were prepared: solution I (Binding Buffer, 20mM Na2HPO4, 0.15MNaCl, pH7.0), solution II (Washing Buffer, 0.02M Na2HPO4 (pH7.4), 0.5MNaCl, 20mM imidazole), and solution III (Elution Buffer, 0.02M Na2HPO4 (pH7.4), 0.5M NaCl, 300mM imidazole). A peristaltic pump and a UV absorption protein and nucleic acid detector were connected, and solution I was used to balance the Ni column at a flow rate of 2mL / min until it was ready for use; the cultured cell samples were centrifuged at 4000rpm and 4°C to collect the supernatant, and the supernatant was passed through the Ni column at a flow rate of 0.6mL / min. After loading, the Ni column was rinsed with solution II at 2mL / min until the UV returned to the baseline. The Ni column was rinsed with solution III until the sample was gradient eluted and the sample peak was collected. The collected proteins were identified by SDS-PAGE electrophoresis and Western Blot.

[0062] like Figure 2 As shown in (A), the collected cell supernatant in the SDS-PAGE electrophoresis contains all proteins, with many visible bands; the load flow-through is the liquid that flows out after the cell supernatant passes through the chromatography column, with more visible impurity protein bands; the washing liquid after washing the Ni column until the UV returns to the baseline does not contain protein or bands; the collected eluate contains the target protein after chromatography purification, namely the Human clusterin recombinant protein, with a single band and no obvious impurity bands. The WB results further show that the Human clusterin recombinant protein contained in the eluate specifically reacts with the Mouse anti-His tag antibody at a protein size of about 40kDa, with a single band and consistent with the expected size.

[0063] The purity of the collected Human clusterin recombinant protein was further identified. Figure 2 As shown in (B), the grayscale scan shows that the non-reduced purity of the protein is greater than 90%, indicating that the Human clusterin recombinant protein has been successfully expressed and prepared and can be used for the next step of library screening.

[0064] 2. Screening of Clusterin affinity peptide sequences

[0065] 2.1 Phage titer detection method

[0066] E. coli ER2738 strain was inoculated into LB medium (containing 30 μg / mL Tet antibiotic, the same below) for activation, and then inoculated into 5 mL fresh LB medium at a ratio of 1:100 and cultured until OD600nm Value 0.4-0.6. Weigh 1.25g IPTG and 1g X-Gal, dissolve in 25mL DMF and store at -20℃. Add 15g / L agar to 1L LB medium and add 1mL IPTG / X-Gal storage solution to prepare a plate and store at 4℃ in the dark. According to the 10-fold gradient dilution method, dilute the phage with LB culture. After the expanded host bacteria are evenly divided, 10μL of gradient phage diluted by LB is added to each tube, mixed and incubated at room temperature for 30min. Spread the infected host bacteria on the plate, culture overnight at 37℃, count the blue plaques, and the calculation formula is the number of plaques (pfu) = number of plaques × gradient dilution times × 10 2 .

[0067] 2.2 Phage expansion culture

[0068] E.coli ER2738 strain was inoculated into LB medium for activation and then inoculated into 5 mL of fresh LB medium at a ratio of 1:100 and cultured until OD 600nm The value is 0.4-0.6. Take 10 μL of phage and inoculate it into the above-mentioned expanded host strain, shake and culture at 200 rpm and 37°C for 3-6 hours. Centrifuge at 8000 rpm to collect the supernatant, which is the phage culture.

[0069] 2.3 Screening of 12-peptide phage display library

[0070] -96gIII sequencing primer, primer sequence (5'-3'): CCCTCATAGTTAGCGTAACG-SEQ ID NO.11.

[0071] 2.4 Selection of recombinant phage

[0072] Antigen coating: Dilute the purified Human clusterin recombinant protein to 4 μg / mL with PBS buffer and add to a 96-well ELISA plate, 100 μL per well (400 ng / well), three times in duplicate, and coat overnight at 4°C. PBS was used as a negative control.

[0073] Blocking: Discard the coating solution, add 150 μL 2% skimmed milk powder to each well, and block for 1 hour at room temperature.

[0074] Incubate phages: Wash 4 times with PBST, take the prepared phage solution and dilute it to 5 × 10 11 pfu / mL, add 100 μL to each well of the ELISA plate and incubate at room temperature for 2 h.

[0075] Elution: discard the phage sample, wash 10 times with PBST and then 5 times with PBS, add 100 μL of freshly prepared 0.1 M triethylamine to each well, let stand at room temperature for 10 min, aspirate the eluate and quickly neutralize with an equal volume of 1 M Tris-HCl (pH 7.4).

[0076] Determination of phage titer in the eluate: Determine the titer of the recombinant phage in the eluate according to the method in 2.1.

[0077] Concentration and purification of phage particles: Concentrate and purify phage particles according to method 2.2 for the next round of screening.

[0078] Repeat panning: Repeat the above steps twice to complete the second and third rounds of panning.

[0079] Human clusterin recombinant protein was used as the coating antigen to screen specific affinity peptides. During the screening process, the titer of recombinant phage in each round of elution was detected to evaluate the enrichment effect of specific peptide recombinant phage. Figure 3 As shown, after three rounds of library selection, the number of phages in each round increased significantly compared with the previous round, and the titer increased.

[0080] The screening results showed that with each round of affinity panning, phages with high affinity for Clusterin were continuously amplified, while phages with low affinity were continuously removed by panning, and ultimately phages with high affinity for Clusterin were significantly and efficiently enriched.

[0081] Twenty single clones were picked from the bacterial plate after the third round of screening, and the plasmids were extracted and sequenced after amplification and culture. As shown in Table 2, 8 new polypeptide sequences were obtained.

[0082] Table 1 Peptide library screening results

[0083]

[0084] 3. Affinity Testing

[0085] 3.1 Peptide dilution

[0086] After obtaining the correct polypeptide sequence, a biotinylated polypeptide was prepared by solid phase synthesis, and the prepared polypeptide was diluted to 1 μg / mL with PBS for solidification.

[0087] 3.2 Protein dilution

[0088] Human clusterin protein was diluted to 1 μM with 2×SD Buffer (PBS+0.02% BSA+0.04% Tween20) for subsequent detection.

[0089] 3.3SA chip (Streptavidin sensor) immobilized peptide detection protein

[0090] Add diluted protein solution and 50% PBS + 50% 2×SD Buffer for control to the sample plate of the biomolecular interaction analyzer, 200μL per well. Through program setting, the SA chip is first balanced in the buffer (Baseline), then immersed in the polypeptide solution to solidify (Loading), and the polypeptide binds to the surface of the SA chip to increase the thickness of its surface film layer. Then immerse in the buffer to make a baseline (Baseline), and then immerse in the protein diluent (Association), and increase the thickness of the SA chip film layer through specific binding between antigen and antibody. Finally, the SA chip is immersed in the buffer to dissociate (Dissociation), and the protein falls off the surface of the SA chip to reduce the thickness of its film layer. The signal and data of the interaction between polypeptide and protein, polypeptide and buffer are obtained by real-time monitoring of the thickness of the SA chip biofilm layer during the experiment, and the double subtraction calculation method of Fortebio data analysis 10.0 software is used, that is, the signal of polypeptide and buffer is subtracted from the signal of polypeptide and protein interaction. The data were processed by Align, Align Y: Yes, Step to Align: Association (the starting point of the binding signal was reset to zero, and the complete experimental process of the interaction was Baseline 300s, Binding 90s, Dissociation 180s), and finally the fitting model 1:1 was used to process the data to obtain the biomolecular binding kinetic constants of the peptide, including the affinity constant (K D ), binding constant (K a ), dissociation constant (K d ).

[0091] 8 peptides and 1 PEG were measured by biomolecular interaction analyzer, and the results were as follows Figure 4 The signals and data shown, as well as the biomolecule binding kinetic constants, are shown in Table 2.

[0092] Table 2 Affinity test results of peptides and Clusterin

[0093]

[0094] K D The value represents the strength of affinity, K a Represents the rate of product generation per unit time, K d Represents the percentage of product degradation into reactants per unit time, K D =K d / K a , K DThe value represents the strength of affinity, K D The smaller the value, the higher the affinity. In order to verify the long-circulation effect of the polypeptide, the present invention selects K D The relatively small peptide P14 was used to construct long-circulating nanoparticles and conduct subsequent experiments.

[0095] Example 2

[0096] 1. Preparation of curcumin-loaded casein nanoparticles

[0097] Curcumin (Cur) powder was dissolved in DMSO to prepare a 50 mg / mL Cur solution, and then 25 mg / mL sodium caseinate solution was added to prepare a 2 mg / mL Cur casein solution. Ice bath probe ultrasound (150w, ultrasound 3s, stop 1s, 10min) was used to prepare a casein-loaded Cur solution, which was dispersed 1:1 into a mixed solution of pure water and saturated ammonium sulfate solution in a ratio of 1:9, vortexed for 30s, tannic acid was added to 0.2 mg / mL, vortexed for 30s, and ferric chloride was continued to be added to 25 mg / mL, vortexed for 30s, and the precipitate, i.e., CurNP nanoparticles, was collected by centrifugation at 4°C and 12000rpm for 10min, and the free tannic acid and ferric chloride were washed with PBS buffer to remove.

[0098] 2. Preparation of long-circulating curcumin-loaded casein nanoparticles

[0099] CurNP nanoparticles were uniformly dispersed in PBS buffer containing mPEG-SH and polypeptide P14, with the ratio of Curcumin to mPEG-SH and polypeptide P14 in the nanoparticles being 1:1. The nanoparticles were incubated with slow shaking at room temperature for 30 min, and the nanoparticles were collected by centrifugation at 4°C and 12000 rpm for 10 min and washed with PBS buffer to remove free PEG / polypeptide.

[0100] 3. Characterization of long-circulating curcumin-loaded casein nanoparticles

[0101] The particle size, zeta potential and polydispersity index (PDI) of the prepared nanoparticles were analyzed using a particle size and surface potential analyzer.

[0102] The particle sizes of CurNP, CurNP-PEG, and CurNP-P14 were 146.3nm, 170.5nm, and 165.8nm, respectively, all of which were negatively charged and had similar Zeta potentials, indicating that the modification of mPEG-SH and polypeptide P14 had no significant effect on the particle size and Zeta potential of the nanoparticles. The PDI values ​​of CurNP, CurNP-PEG, and CurNP-P14 were all less than 0.3, indicating that the particle size distribution of the nanoparticles was relatively uniform, meeting the requirements of nanoparticle preparations, and could be used for subsequent experiments.

[0103] The validated liquid phase method for determining the content of Curcumin was used, and the chromatographic conditions were as follows:

[0104] Chromatographic column: Agela Ttechnologies, Innoval ODS-2, 150×4.6mm, 5μm

[0105] Mobile phase: 2% glacial acetic acid water: acetonitrile = 40:60

[0106] Flow rate: 1.0mL / min

[0107] Column temperature: 30°C

[0108] Detection wavelength: 426nm

[0109] Injection volume: 10 μL

[0110] Run time: 8 minutes

[0111] The peak area of ​​the standard was used as the calibration curve, and the encapsulation efficiencies of CurNP, CurNP-PEG, and CurNP-P14 were calculated to be 76.7%, 72.7%, and 76.3%, respectively.

[0112] The nanoparticle solutions (CurNP, CurNP-PEG, CurNP-P14) were stored in a 4°C refrigerator, and the particle sizes were measured at preset time points (0, 1, 2, 3, 4, 5, 6, and 7d). The particle size on the 0th day was used as the blank control, and the storage stability of the nanoparticles was investigated by observing the changes in particle size.

[0113] Fetal bovine serum was added to the nanoparticle solution (CurNP, CurNP-PEG, CurNP-P14) with a serum content of 10%. The particle size and turbidity (OD) of each group of nanoparticles were measured at the preset time points (0, 1, 2, 4, 8, 12 h and 24 h). 600 ), the particle size or turbidity at the 0th hour was used as the blank control, and the serum stability of the nanoparticles was investigated by observing the changes in particle size or turbidity.

[0114] like Figure 5 As shown in (A), overall, the particle size of each group of nanoparticles (CurNP, CurNP-PEG, CurNP-P14) changed slightly within 7 days, and the difference was not obvious, indicating that the nanoparticles had no obvious aggregation within 7 days and could remain relatively stable in vitro, making them easy to store for subsequent related experiments.

[0115] In serum stability tests, e.g. Figure 5As shown in (BC), overall, there was no significant change in the particle size and turbidity of each group of nanoparticles (CurNP, CurNP-PEG, CurNP-P14) in 10% fetal bovine serum within 24 hours, indicating that the nanoparticles can exist stably in serum, which is beneficial to their delivery in vivo.

[0116] Example 3

[0117] 1. Pharmacokinetic study of nanoparticles in rats

[0118] 24 rats were randomly divided into 4 groups according to body weight and gender, namely Cur, CurNP, CurNP-PEG, and CurNP-P14 groups, and the dosage was 3.0 mg / kg (calculated as Curcumin), and the drug was administered by single tail vein injection. Before and 2min, 5min, 10min, 20min, 30min, 45min, 1h, 1.5h, 2h, 4h, 8h, 12h, and 24h after administration, about 0.3mL of blood was collected from the jugular vein of the rats and put into a centrifuge tube anticoagulated with 0.5M EDTA-2K solution, and gently mixed. All blood samples were placed in an ice box and centrifuged at 3000rpm and 4℃ for 10min within half an hour, and the supernatant plasma samples were stored at -80℃.

[0119] 2. Processing methods of plasma samples

[0120] Take 50 μL of plasma sample (blank / drug-containing), add 10 μL of series concentration standard solution or 50% methanol water, add 10 μL of internal standard solution (250 ng / mL), add 600 μL of ethyl acetate, centrifuge at 12000 rpm and 4°C for 5 min, take 500 μL of supernatant and evaporate in a centrifugal concentrator under vacuum, add 100 μL of 50% acetonitrile water and vortex to mix, centrifuge at 12000 rpm and 4°C for 5 min, and take the supernatant for sampling and analysis.

[0121] 3. Chromatographic and mass spectrometry conditions

[0122] Chromatographic column: American Phenomenex, kinetex, C18, 50×2.1mm, 2.6μm

[0123] Mobile phase: water: acetonitrile = 40:60

[0124] Flow rate: 0.2mL / min

[0125] Injector temperature: 4°C

[0126] Column temperature: 40°C

[0127] Injection volume: 2 μL

[0128] Run time: 2 minutes

[0129] Electrospray ion source (ESI); negative ion mode (Negative ion); multi-stage reaction monitoring (MRM); spray gas temperature (TEM): 500℃; nebulizer gas (GS1): 50psi; heating auxiliary gas (GS2): 50psi; curtain gas (CUR): 35psi; collision gas (CAD): Medium; ion spray voltage (IS): -4500V; scan time (Scan time): 100ms.

[0130] like Figure 6 As shown in the figure, after rats were injected with Cur, CurNP, CurNP-PEG, and CurNP-P14 through the tail vein, the blood drug concentration reached a peak immediately after the administration, and the average peak concentrations were 1293.8 ng / mL, 2049.2 ng / mL, 3724.2 ng / mL, and 4494.2 ng / mL, respectively. Then, it dropped rapidly within 4 hours, and by 12 hours, it had dropped to close to or below the minimum limit of quantification. It can be seen from the average blood drug concentration-time curve of each group that the average blood drug concentration of Cur at each time point was significantly lower than that of the three nanoparticle groups, and CurNP-P14 had the highest blood drug concentration. Compared with the Cur group, the t 1 / 2 The Vd of CurNP-P14 group was significantly decreased compared with CurNP group. 1 / 2 The pharmacokinetic parameters of CurNP-PEG group and CurNP-P14 group were not significantly different. The experimental results show that CurNP-P14 has the best pharmacokinetic behavior. Compared with the free drug Curcumin, C max and AUC 0-t The in vivo exposure increased significantly, while Vd and CL decreased significantly. 1 / 2 The obvious extension indicates that the nanoparticles have certain long circulation characteristics. Although there is no significant difference in the pharmacokinetic parameters between CurNP-P14 and CurNP-PEG, CurNP-P14 is slightly better than CurNP-PEG overall.

[0131] Example 4

[0132] This example shows the therapeutic effect of nanoparticles on an animal model of acute lung injury

[0133] 1. Effects of Nanoparticles on Lung Function in Animal Models of Acute Lung Injury

[0134] Forty-two rats were randomly divided into 7 groups according to body weight and gender, namely normal group, sham operation group, model group, Cur group, CurNP group, CurNP-PEG group, and CurNP-P14 group, with 6 rats in each group, half of which were male and half were female.

[0135] Except for the normal group, animals in other groups underwent surgical modeling, that is, LPS solution was dripped into the animal's trachea using a syringe, and then the animal was stood up and shaken so that the injected liquid could be evenly distributed in the left and right lungs.

[0136] About 1 hour after modeling, the rats in the Cur group were injected with Curcumin solution through the tail vein, and the rats in the CurNP group, CurNP-PEG group, and CurNP-P14 group were injected with CurNP, CurNP-PEG, and CurNP-P14 solutions containing an equal amount of Curcumin (quantitated by liquid phase method) through the tail vein. The dosage was 3.0 mg / kg (calculated as Curcumin), and 0.9% sodium chloride injection was diluted to a volume of 1 mL per 100 g body weight (1 mg / mL). The normal group, sham operation group, and model group were injected with an equal volume of 0.9% sodium chloride injection. The drugs were administered continuously for 3 days, and the conditions of the rats were observed every day.

[0137] After continuous administration for 3 days, the rats were anesthetized by intraperitoneal injection of urethane solution on the 4th day. The anesthesia was successful when the anesthesia level reached no reaction when the animal's thigh was pinched. The anesthetized rats were tested using a small animal lung function test system. The test results of lung function-related indicators included vital capacity (VC), forced vital capacity (FVC), forced expiratory volume in 200 milliseconds (FEV200), dynamic lung compliance (Cdyn), and static lung compliance (Cchord).

[0138] The results showed that the Cchord and Cdyn of rats after modeling decreased, indicating that lung compliance was weakened, which may be caused by pulmonary edema. After treatment with CurNP-PEG and CurNP-P14, lung compliance was improved. VC, FVC and FEV200 of rats after modeling were significantly reduced, indicating that lung ventilation was reduced, which may be related to atelectasis caused by alveolar epithelial damage. After treatment with CurNP-PEG and CurNP-P14, VC, FVC and FEV200 were significantly increased.

[0139] 2. Effect of Nanoparticles on Lung Wet-to-Dry Weight Ratio in Animal Model of Acute Lung Injury

[0140] The left lower lobe was taken, dried with filter paper and weighed in wet weight, then placed in an oven at 60°C for 72 h, weighed in dry weight, and the lung wet-to-dry weight ratio was calculated using the formula: lung wet weight / lung dry weight × 100%.

[0141] The results showed that CurNP-PEG and CurNP-P14 significantly increased the lung wet-to-dry weight ratio of rats with acute lung injury compared with Cur and CurNP, and may also have a more significant effect on improving pulmonary edema.

[0142] Effects of nanoparticles on lung TNF-α and IL-6 levels in an animal model of acute lung injury

[0143] 100 mg of the right upper lung lobe of rats in each group was added to pre-cooled PBS, homogenized with an ice bath homogenizer, centrifuged at 12000 rpm and 4°C for 10 min, and the supernatant was collected. The supernatant was used to measure the levels of inflammatory factors according to the detection steps of TNF-α and IL-6 ELISA kits.

[0144] Compared with the normal group, there was no significant difference in TNF-α and IL-6 in the sham operation group, indicating that the surgery had no significant effect on the rat lungs; TNF-α and IL-6 in the model group increased significantly, indicating that the rat acute lung injury model was successfully established and inflammation occurred. Compared with the model group, there was no significant difference in TNF-α and IL-6 in the Cur group and CurNP group, and TNF-α and IL-6 in the CurNP-PEG group and CurNP-P14 group were significantly reduced, indicating that CurNP-PEG and CurNP-P14 can reduce lung inflammation in rats with acute lung injury.

[0145] Effects of Nanoparticles on Pulmonary Pathological Changes in Animal Models of Acute Lung Injury

[0146] The lung pathological sections of rats in each group were observed under a microscope. Figure 7 As shown in the figure, the alveolar structure of rats in the normal group and sham operation group was intact, the alveolar septum was thin, the alveolar cavity outline was clear, and there was no obvious inflammatory cell infiltration; the alveolar structure of rats in the model group was destroyed, partially collapsed or even disappeared, the alveolar septum was significantly widened, and a large number of inflammatory cells were observed in the alveolar septum. Compared with the model group, the degree of damage to the alveolar structure of rats in the Cur group and CurNP group was alleviated, the alveolar septum was reduced, a small amount of inflammatory cells were observed in the alveolar septum, and the pathological changes were improved. In contrast, the alveolar structure of rats in the CurNP-PEG group and CurNP-P14 group was relatively intact, the alveolar septum was not obviously thickened, a small amount of inflammatory cells were observed in the alveolar septum, and the pathological changes were significantly improved.

[0147] Example 5 In vivo safety study

[0148] 1. Hematology and blood biochemistry tests

[0149] Blood was collected from the abdominal aorta of each group of rats that completed the pulmonary function test in Example 4. About 1 mL of blood was collected using an EDTA-2K anticoagulant tube for routine blood tests, and about 1.8 mL of blood was collected using a sodium citrate anticoagulant tube for four coagulation tests. After blood collection, the tube was immediately inverted several times to fully mix the anticoagulant with the blood to prevent coagulation. About 2 mL of blood was collected using a vacuum blood collection tube (without anticoagulant) for blood biochemistry testing.

[0150] The blood routine test indicators include total red blood cell count (RBC), hemoglobin (HGB), red blood cell volume (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), total platelet count (PLT), reticulocyte count (Ret), total white blood cell count (WBC), and percentage of white blood cell classification (neutrophils (NEU), lymphocytes (LYM), monocytes (MON), eosinophils (EOS), and basophils (BOS)).

[0151] The four coagulation indicators include prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen content (Fbg).

[0152] Blood biochemical test indicators included alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatine kinase (CK), creatinine (CRE), total bilirubin (TBIL), glutamyl transferase (GGT), total protein (TP), albumin (ALB), blood urea nitrogen (Urea), total cholesterol (CHOL), blood glucose (GLU), triglycerides (TG), calcium (Ca) and phosphorus (P).

[0153] The results showed that the administration of nanoparticles in each group had no significant effect on the blood routine, coagulation, blood biochemistry and other indicators of rats.

[0154] 2. Organ Coefficients and Histopathological Examination

[0155] The brain, heart, liver, spleen and kidney of the rats obtained in Example 4 were weighed, and the organ coefficient was calculated using the formula: (organ weight / body weight)×100%. At the same time, the above organs were subjected to HE staining for histopathological examination.

[0156] The results showed that the administration of nanoparticles in each group had no significant effect on the organ coefficients of rats, and the HE staining pathological examination results of the brain, heart, liver, spleen and kidney of the rats in each group showed no obvious abnormalities ( Figure 8), indicating that the method of creating an ALI animal model by tracheal instillation of LPS did not cause pathological damage to the other major organs of the animals except the lungs, and also indicating that Curcumin, whether it is a free drug or prepared into nanoparticles and administered to animals by intravenous injection, would not cause organic damage to the major organs.

[0157] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Clusterin affinity peptide, characterized in that The amino acid sequence of the Clusterin affinity peptide is an amino acid sequence shown in one of SEQ ID NO.1-8.

2. The Clusterin affinity peptide according to claim 1, characterized in that The screening method of the Clusterin affinity peptide comprises: using phage display technology to perform multiple rounds of library panning and enrichment on the Human clusterin recombinant protein to obtain different polypeptide binding fragments, and then using biomembrane interferometry technology to determine the affinity of the polypeptide binding fragments with the Human Clusterin protein to screen out the Clusterin affinity peptide.

3. Use of the Clusterin affinity peptide as claimed in claim 1 or 2 in improving the half-life of a drug.

4. A long-circulation nanoparticle, characterized in that: Including drug-loaded protein nanoparticles and surface-coupled modifications thereof; The modified substance contains a chemical group with high reactivity, which includes the Clusterin affinity peptide according to claim 1 or 2.

5. The long-circulating nanoparticles according to claim 4, characterized in that: The drug-loaded protein nanoparticles include casein drug-loaded nanoparticles and albumin drug-loaded nanoparticles.

6. The long-circulating nanoparticles according to claim 5, characterized in that: The modification also includes mPEG-SH.

7. The method for preparing long-circulating nanoparticles according to any one of claims 4 to 6, characterized in that: include: The drug-loaded protein nanoparticles are dispersed in a buffer solution containing a modified substance, and the long-circulation nanoparticles are obtained after incubation and separation.

8. The preparation method according to claim 7, characterized in that: The mass ratio of the drug-loaded protein nanoparticles to the modified substance is 10:1-1:10; Preferably, the modification contains a highly reactive chemical group; Preferably, the modified substance comprises the Clusterin affinity peptide and mPEG-SH as described in claim 1 or 2.

9. The preparation method according to claim 8, characterized in that: The preparation method of the drug-loaded protein nanoparticles comprises: mixing a carrier protein with a chemical drug and adding the mixture into a saturated salt solution to form drug-loaded protein microparticles, and then adding a cross-linking agent to obtain the drug-loaded protein nanoparticles; Preferably, the carrier protein comprises albumin and casein; Preferably, the chemical drugs include curcumin, tripterine, paclitaxel, camptothecin and doxorubicin; Preferably, the mass ratio of the protein to the chemical drug is 5-20:1; Preferably, the saturation of the saturated salt solution is 90-100%; Preferably, the cross-linking agent comprises tannic acid and ferric chloride, the mass concentration of the tannic acid in the reaction system is 0.05-1 mg / mL, and the mass concentration of the ferric chloride in the reaction system is 1-50 mg / mL.

10. Use of the long-circulating nanoparticles according to claims 4-6 or the long-circulating nanoparticles obtained by the preparation method according to claims 7-9 in the preparation of drugs for prolonging half-life.

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