CN112999356B - Scavenger receptor-A targeted fatty acid modified albumin nanoparticle and preparation method and application thereof - Google Patents

Scavenger receptor-A targeted fatty acid modified albumin nanoparticle and preparation method and application thereof Download PDF

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CN112999356B
CN112999356B CN201911313767.9A CN201911313767A CN112999356B CN 112999356 B CN112999356 B CN 112999356B CN 201911313767 A CN201911313767 A CN 201911313767A CN 112999356 B CN112999356 B CN 112999356B
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fatty acid
albumin
acid
pab
drug
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CN112999356A (en
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龚涛
龚婷
张志荣
宋旭
谭田田
张培
陈丹
孙逊
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Sichuan University
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    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
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    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
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Abstract

The invention relates to a scavenger receptor-A targeted fatty acid modified albumin and a preparation method and application thereof. The invention uses the carboxyl of fatty acid and the amino of the lysine residue of albumin to carry out acylation reaction to obtain the albumin modified by the fatty acid; and the albumin nano-particles modified by the fatty acid are successfully prepared by a solvent volatilization method, and other nano-preparations which are modified by the albumin modified by the fatty acid and have the scavenger receptor-A targeting effect, such as liposome, polymer nano-particles and micelle, are further successfully prepared. The invention takes the albumin modified by fatty acid as a new drug carrier or target, and can specifically deliver the drug to activated macrophage through the targeting effect of scavenger receptor-A. The prepared nano preparation has the advantages of stable structure, uniform particle size, wide drug loading range, high drug loading amount, strong specificity, good safety and biocompatibility, and great application value in the field of biological medicine, and can obviously improve the bioavailability of the drug.

Description

Scavenger receptor-A targeted fatty acid modified albumin nanoparticle and preparation method and application thereof
Technical Field
The present invention belongs to the field of biomedical material and medicine technology. More particularly, relates to a fatty acid modified albumin drug carrier, and a preparation method and application thereof.
Background
Macrophages are the most plastic cells of the hematopoietic system, present in all tissues, and are functionally diverse. They play a role in development, homeostasis, tissue repair and immunity, and are critical for the development and treatment of various diseases such as inflammation or tumors. Taking inflammation as an example, a large number of activated macrophages infiltrate during the development of diseases, and the macrophages can not only promote the activation of autoimmunity and the further development of inflammation and prolong the inflammation period, but also secrete various inflammatory cytokines to promote the malignant transformation and transfer of other cells at the inflammation part and further aggravate the disease degree. In the case of tumors, tumor-associated Macrophages (TAMs), which are selectively activated Macrophages present in large numbers at the tumor site, promote tumor growth, invasion and metastasis. Therefore, macrophages activated at the disease site are ideal targets for effective treatment of inflammation or tumors.
At present, more researches aiming at macrophage targeting are carried out, for example, chinese patent with publication No. CN109735570A provides an X inactive specificity transcript modified adipose-derived mesenchymal stem cell exosome targeting liver macrophage, but the preparation process is complex, a large amount of mouse and adult adipose-derived mesenchymal stem cells are needed, and the X inactive specificity transcript modified adipose-derived mesenchymal stem cell exosome targeting liver macrophage is only suitable for targeting liver macrophages, and the application in clinic and treatment of other diseases is limited. Chinese patent publication No. CN108178783A provides a tumor blood vessel and M1 type macrophage targeting peptide TCP-2, but its preparation cost is high, and its ability to target M1 type macrophages may limit its application in targeting tumor therapy to some extent, because M1 type macrophages have a promoting effect on tumor therapy. Other researches have also adopted natural high molecular compounds such as hyaluronic acid, mannose, folic acid or chondroitin sulfate to realize macrophage targeting, for example, chinese patent published as CN107335064A provides a chondroitin sulfate-modified nanoparticle targeted macrophage for treating ulcerative colitis, but such natural high molecular compounds can target macrophages, are also highly distributed in other main organs, especially the liver, and are not beneficial to guiding target cells and have potential hepatotoxicity.
Scavenger receptor-a (SR-a) is a receptor that is overexpressed on activated macrophages, with a high degree of activated macrophage infiltration in diseases such as inflammation, fibrosis, and tumors. SR-A is not distributed obviously in other organs, and mainly expresses endothelial cells under oxidative stress except activated macrophages. In addition, the existing SR-A targeting vectors are polyanionic molecules with strong electronegativity, and relevant contents for targeting macrophages by using the SR-A targeting vectors and treating diseases are not queried. Therefore, the novel carrier for realizing macrophage activation targeting by targeting SR-A through different mechanisms can overcome the defects and shortcomings of the prior art, make up for the vacancy of related researches, and can be widely applied to treatment of diseases such as inflammation, fibrosis and tumor.
Disclosure of Invention
The inventor unexpectedly discovers in research that the albumin modified by the fatty acid has stronger scavenger receptor-A targeting property, and is different from the existing SR-A targeting mechanism of polyanion molecules with strong electronegativity. Based on the analysis and experiments, the invention provides a drug carrier material which can activate macrophage targeting through SR-A targeting effect by using fatty acid modified albumin as a carrier, thereby realizing targeted therapy on inflammation, fibrosis or tumor.
It is a first object of the present invention to provide a drug carrier material.
The second purpose of the invention is to provide a preparation method of the SR-A targeted fatty acid modified albumin carrier material.
The third purpose of the invention is to provide a preparation containing the drug carrier material, and the preparation can be a nanoparticle preparation prepared by coating a drug with the fatty acid modified albumin carrier material, and can also be a nanoparticle preparation with SR-A targeting effect obtained by modifying polymer nanoparticles, liposomes, micelles and other nanoparticle preparations by using the fatty acid modified albumin as a targeting head.
The fourth purpose of the invention is to provide the application of the fatty acid modified albumin targeting carrier material in the preparation of nano-drug carriers with macrophage activating targeting function.
The above purpose of the invention is realized by the following technical scheme:
the invention relates to a scavenger receptor-A targeted drug carrier material, which is characterized in that the carrier material is fatty acid modified albumin obtained by connecting the amino group of a lysine residue of the albumin with the carboxyl group of fatty acid through acylation reaction.
The albumin is selected from bovine serum albumin and/or human serum albumin;
the fatty acid is a saturated or unsaturated fatty acid with 12-20 carbon atoms; further preferably one or more of myristic acid, palmitic acid, stearic acid, arachidic acid, oleic acid, linolenic acid, linoleic acid, and arachidonic acid.
In the present invention, the molar ratio of the albumin to the fatty acid is 1.
In the invention, the preparation method of the drug carrier material comprises the following steps: (1) Dissolving fatty acid in an organic solvent, adding Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), reacting for 2-24 hours at 0-37 ℃, and separating and purifying to obtain fatty acid succinimide ester; (2) Dissolving albumin in a buffer solution with the pH value of 8.0-10, dissolving fatty acid succinimide ester in an organic solvent, reacting for 12-24 hours at the temperature of 20-37 ℃ according to the molar ratio of the fatty acid succinimide ester to the albumin being 10-100, dialyzing, centrifuging and freeze-drying to obtain the albumin-containing liquid.
Further, in the above method for preparing a pharmaceutical carrier, the organic solvent in step (1) is selected from N, N-Dimethylformamide (DMF) or dimethyl sulfoxide (DMSO);
further, the reaction temperature in the step (1) is preferably 25-30 ℃; the reaction time in the step (1) is preferably 8 to 12 hours;
further, the buffer solution in the step (2) is selected from carbonate buffer solution, phosphate buffer solution, tris-hydrochloric acid buffer solution, glycine-sodium hydroxide buffer solution and boric acid-borax buffer solution, and is preferably carbonate buffer solution with pH of 8.3-9.2;
further, the organic solvent in step (2) is selected from DMF and/or DMSO, preferably DMF;
further, the molar ratio of the fatty acid succinimidyl ester to the albumin in the step (2) is preferably 10 to 20.
The invention also relates to a scavenger receptor-A targeted fatty acid modified albumin nanoparticle, which contains the scavenger receptor-A targeted drug carrier material and the drug loaded by the nanoparticle.
Preferably, the nanoparticle-loaded drug includes, but is not limited to, one or more of tripterine, curcumin, silibinin, glycyrrhetinic acid, paclitaxel, entecavir, dexamethasone, doxorubicin, pirarubicin, cabazitaxel, oxaliplatin, teniposide, tanshinone IIA, hydroxycamptothecin, mitoxantrone, gemcitabine, and trans-retinoic acid; further, the mass of the drug loaded on the nanoparticles is 0.1-20% of the mass of the drug carrier material.
Preferably, the nanoparticles may further comprise an oil for injection, preferably selected from soybean oil or medium chain triglycerides; further, the injectable oil is preferably used in an amount of 0 to 40% (wt%) of the amount of the fatty acid-modified albumin;
preferably, the nanoparticle may further comprise other pharmaceutically acceptable adjuvants, including but not limited to one or more of polyethylene glycol and polyethylene glycol derivatives, polyoxyethylene polyoxypropylene block copolymer, phospholipids for injection such as soybean phospholipid and egg yolk lecithin, and common additives for injection such as mannitol, lactose, glucose, maltose, trehalose, sucrose, sodium chloride, disodium hydrogen phosphate, and sodium dihydrogen phosphate
Preferably, the preparation method of the nanoparticle is characterized in that the drug carrier is dissolved in deionized water or deionized water in which the auxiliary materials except phospholipid for injection are dissolved, an organic phase is added, the organic phase is selected from an organic solvent, an organic solvent in which oil for injection is dissolved, an organic solvent in which oil for injection and drugs are dissolved, an organic solvent in which phospholipid compounds of drugs are dissolved or an organic solvent in which phospholipid compounds of oil for injection and drugs are dissolved, and the probe performs ultrasonic treatment to remove the organic solvent through rotary evaporation to obtain the nanoparticle.
The organic solvent is selected from one or more of dichloromethane, ethyl acetate and chloroform, and dichloromethane or ethyl acetate is preferred.
The particle size of the nanoparticle is preferably 50-200 nm.
Preferably, the invention provides a fatty acid modified albumin nanoparticle loaded with tripterine and having scavenger receptor-A targeting, which contains a carrier material of the drug targeted by scavenger receptor-A and tripterine with the dosage of 0-15% (wt%) of the carrier material.
The drug-loaded nanoparticles also contain injectable oil with the dosage of 0-40% (wt%) of the dosage of the fatty acid modified albumin; the injectable oil is preferably selected from soybean oil or medium chain triglycerides.
The drug-loaded nanoparticles can also contain pharmaceutical auxiliary materials with the dosage of 0-25% (wt%) of the dosage of the carrier material; the auxiliary material is selected from polyethylene glycol derivatives and polyoxyethylene polyoxypropylene block copolymers; preferably selected from the group consisting of DSPE-PEG2000,
Figure BDA0002325255530000041
HS 15 or polysorbate 80.
The preparation method of the nanoparticle comprises the steps of dissolving the carrier material in deionized water or deionized water containing the auxiliary materials, adding an organic solvent in which injectable oil and tripterine are dissolved, homogenizing under high pressure or ultrasonically emulsifying by a probe, and removing the organic solvent by rotary evaporation to obtain the nanoparticle.
The organic solvent is selected from dichloromethane, ethyl acetate and chloroform, and dichloromethane or ethyl acetate is preferred.
The invention also relates to a nanometer preparation with scavenger receptor-A targeting, which is characterized in that the nanometer preparation takes the carrier material as a target head and is used for modifying preparations such as polymer nanoparticles, liposomes, micelles and the like so as to prepare the preparations such as the polymer nanoparticles, the liposomes, the micelles and the like with scavenger receptor-A targeting. The nano preparation with the scavenger receptor-A targeting function comprises the fatty acid modified albumin, a liposome used for loading a medicament, a high-molecular nanoparticle or micelle and the like.
The liposome, the polymer nanoparticles and the micelles comprise one or more of liposome, glycolide-lactide copolymer nanoparticles, chitosan nanoparticles, zein (zein) nanoparticles, polydopamine nanoparticles, polylysine nanoparticles, dextran nanoparticles, diethylaminoethyl dextran nanoparticles, phospholipid cholate mixed micelles, polyethylene glycol-polyethylene imine micelles, polycaprolactone-polyethylene imine micelles and polycaprolactone-polyethylene glycol micelles.
Further, the mass ratio of the fatty acid modified albumin to the liposome or the polymer nanoparticle or the micelle is 4;
further, the preparation method of the nano preparation is characterized in that the carrier material is dissolved in deionized water, the liposome/polymer nano particle/micelle which does not carry or carries the medicine is added, and the mixture is stirred to obtain the nano preparation; further, the stirring temperature is 20-37 ℃;
further, the stirring time is 15min to 2 hours, preferably 30min to 1 hour.
The particle size of the nano preparation is preferably 90-200nm.
Advantageous effects
(1) The albumin modified by the fatty acid synthesized by the invention has strong specificity of scavenger receptor-A and good effect of targeting activated macrophage.
(2) Compared with the common albumin nanoparticles, the fatty acid modified albumin nanoparticles can effectively prolong the in vivo circulation time of the medicament and improve the bioavailability of the medicament.
(3) When the albumin nanoparticles modified by the fatty acid reach a target part by utilizing SR-A targeting effect and prolonged circulation time of the albumin nanoparticles, the albumin nanoparticles can be further absorbed by macrophages and other disease-related cells of the target part in a large amount through the SR-A targeting effect and increased lipophilicity, and the albumin nanoparticles are favorable for comprehensively improving the treatment effect of the loaded drugs.
(4) The fatty acid modified albumin nanoparticle can target activated macrophages accumulated in inflammation, fibrosis or tumor parts in large quantity through SR-A targeting action, can realize targeted treatment on the diseases, and has a wide application range.
(5) The albumin modified by the fatty acid can be prepared into nanoparticles with remarkable scavenger receptor-A targeting effect, can be physically modified on the surfaces of nano preparations such as liposome, polymer nanoparticles and micelle, plays the targeting effect of activated macrophages, and has a very wide application range.
(6) The preparation method of the albumin modified by the fatty acid has the advantages of simple reaction process, few reaction steps and good repeatability, and the preparation methods of the albumin nano drug-loaded nanoparticles modified by the fatty acid and the albumin nano preparation modified by the fatty acid are simple and good in repeatability, and the nanoparticles and the nano preparation have good safety and stability and have good application prospects and wide development space in the field of medicines.
Drawings
FIG. 1 is a graph of the fluorescence spectrum and circular dichroism spectrum of PAB and BSA.
FIG. 2 shows SDS-PAGE of PAB and BSA with different degrees of substitution.
Figure 3 TEM characterization of nanoparticles.
Figure 4 in vitro stability of nanoparticles.
FIG. 5 results of experiments on the uptake and inhibition of uptake of DiD-BSA NPs and DiD-PAB NPs in macrophages and LPS-AR.
FIG. 6.DiD-BSA NPs and DiD-PAB NPs uptake in different cells except macrophages and LPS-AR.
FIG. 7 Co-localization of SR-A in PAB and LPS-AR.
FIG. 8 time-course of drug administration for CLT prodrugs, CLT-BSA NPs and CLT-PAB NPs.
FIG. 9 shows the distribution of nanoparticles in rats with rheumatoid arthritis model.
FIG. 10 shows co-localization of nanoparticles and SR-A at inflammatory joint site of rat model rheumatoid arthritis.
Figure 11. Variation of ankle thickness in rats in different treatment groups.
FIG. 12 serum TNF-. Alpha.and IL-1. Beta. Levels in rats of different groups after the treatment was completed.
Figure 13 distribution of nanoparticles in the pancreas of rats, a model of chronic pancreatitis and fibrosis.
FIG. 14 shows the distribution of nanoparticles at tumor sites in melanoma mice.
Detailed Description
The invention is further described with reference to the drawings and specific examples, which are not intended to limit the invention in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1
PAB, palmitic Acid (PA) -modified Bovine Serum Albumin (BSA), was synthesized. 0.63mM PA was weighed, and 1mL of N, N-Dimethylformamide (DMF) was added thereto, and after complete dissolution, 1.5eq of Dicyclohexylcarbodiimide (DCC) and 1.5eq of N-hydroxysuccinimide (NHS) were added thereto, and the mixture was stirred at room temperature for 12 hours to react, followed by recrystallization to obtain palmityl succinimide ester (PA-NHSE). 0.005mM BSA was weighed and added to 2mL sodium bicarbonate buffer (pH 8.47) to dissolve it completely. At the same time, 20eq of PA-NHSE was weighed and dissolved completely by adding 200uL of DMF. PA-NHSE in DMF was slowly added dropwise to BSA in sodium bicarbonate buffer solution under 37 ℃ water bath condition, and the reaction was stirred for 24 hours. And after the reaction is finished, filling the reaction solution into a dialysis bag, dialyzing the reaction solution for two days at room temperature by using deionized water, centrifuging the reaction solution for 10min at 10000rpm, collecting supernate, centrifuging the supernate for three times, and freeze-drying the supernate to obtain the final product PAB.
Example 2
And (4) synthesizing the PAB. Namely, palmitic Acid (PA) modified Bovine Serum Albumin (BSA). 0.63mM PA was weighed, added to 1mL of N, N-Dimethylformamide (DMF), and after complete dissolution, 1.5eq of Dicyclohexylcarbodiimide (DCC) and 1.5eq of N-hydroxysuccinimide (NHS) were added, and stirred in a water bath at 30 ℃ for reaction for 8 hours, followed by recrystallization to obtain palmityl succinimide ester (PA-NHSE). 0.005mM BSA was weighed and added to 2mL sodium bicarbonate buffer (pH 8.47) to dissolve it completely. At the same time, 10eq PA-NHSE was weighed and dissolved completely by adding 200uL DMF. PA-NHSE in DMF was slowly added dropwise to BSA in sodium bicarbonate buffer solution, and the reaction was stirred at room temperature for 24 hours. And after the reaction is finished, filling the reaction solution into a dialysis bag, dialyzing the reaction solution for two days at room temperature by using deionized water, centrifuging the reaction solution for 10min at 10000rpm, collecting supernate, centrifuging the supernate for three times, and freeze-drying the supernate to obtain a final product PAB.
Example 3
SAB, stearic Acid (SA) -modified Bovine Serum Albumin (BSA) was synthesized. 0.63mM SA is weighed, DMSO is added, 1.5eq Dicyclohexylcarbodiimide (DCC) and 2eq N-hydroxysuccinimide (NHS) are added after complete dissolution, the reaction is stirred for 10 hours under the condition of water bath at the temperature of 28 ℃, and the stearic acid succinimide ester (SA-NHSE) is obtained by recrystallization. 0.005mM BSA was weighed and added to 2mL sodium bicarbonate buffer (pH 8.47) to dissolve it completely. At the same time, 15eq SA-NHSE was weighed and dissolved completely by adding 200uL DMF. A DMF solution of SA-NHSE was slowly added dropwise to a sodium bicarbonate buffer solution of BSA under a water bath condition at 30 ℃ and the reaction was stirred for 16 hours. And after the reaction is finished, filling the reaction solution into a dialysis bag, dialyzing the reaction solution for two days at room temperature by using deionized water, centrifuging the reaction solution for 10min at 10000rpm, collecting supernate, centrifuging the supernate for three times, and freeze-drying the supernate to obtain the final product SAB.
Example 4
AAB, eicosanoid (AA) modified bovine serum albumin protein (BSA), was synthesized. Weighing 0.63mM AA, adding DMF, completely dissolving, adding 2eq Dicyclohexylcarbodiimide (DCC) and 2eq N-hydroxysuccinimide (NHS), stirring at room temperature for reaction for 12 hours, and recrystallizing to obtain the arachidic acid succinimide ester (AA-NHSE). 0.005mM BSA was weighed and added to 2mL sodium bicarbonate buffer (pH 8.47) to dissolve it completely. At the same time, 20eq of AA-NHSE was weighed and dissolved completely by adding 200uL of DMF. A DMF solution of AA-NHSE was slowly added dropwise to a buffered sodium bicarbonate solution of BSA under a water bath condition at 35 ℃ and the reaction was stirred for 20 hours. And after the reaction is finished, filling the reaction solution into a dialysis bag, dialyzing the reaction solution for two days at room temperature by using deionized water, centrifuging the reaction solution for 10min at 10000rpm, collecting supernate, centrifuging the supernate for three times, and freeze-drying the supernate to obtain a final product AAB.
Example 5
MAB, myristic Acid (MA) -modified Bovine Serum Albumin (BSA), was synthesized. Weighing 0.63mM MA, adding DMF, completely dissolving, adding 2eq Dicyclohexylcarbodiimide (DCC) and 2eq N-hydroxysuccinimide (NHS), stirring and reacting for 10 hours at room temperature under the condition of 28 ℃ water bath, and recrystallizing to obtain myristic acid succinimide ester (MA-NHSE). 0.005mM BSA was weighed and added to 2mL sodium bicarbonate buffer (pH 8.47) to dissolve it completely. At the same time, 20eq MA-NHSE was weighed and dissolved completely by adding 200uL DMF. The DMF solution of MA-NHSE was slowly added dropwise to the sodium bicarbonate buffer solution of BSA under 32 ℃ water bath condition, and the reaction was stirred for 24 hours. And after the reaction is finished, filling the reaction solution into a dialysis bag, dialyzing the reaction solution for two days at room temperature by using deionized water, centrifuging the reaction solution for 10min at 10000rpm, collecting supernate, centrifuging the supernate for three times, and freeze-drying the supernate to obtain the final product MAB.
Example 6
Preparation of palmitic acid modified albumin nanoparticles (PAB NPs). 80mg of PAB prepared in example 1 was dissolved in 4mL of deionized water to form an aqueous phase, 18mg of soybean oil was dissolved in 800uL of a mixed solvent of dichloromethane and ethyl acetate (volume ratio 1).
Example 7
Preparation of palmitic acid modified albumin nanoparticles (PAB NPs). 80mg of PAB prepared in example 1 is dissolved in 4mL of deionized water to form a water phase, 30mg of medium chain oil is dissolved in 800uL of a mixed solvent of dichloromethane and ethyl acetate (volume ratio is 1) to form an organic phase, the organic phase and the water phase are mixed, an ice-bath probe is used for ultrasonic treatment for 10min (power is 200W, work is 3s, and pause is 7 s), and the organic solvent is removed by rotary evaporation, so that PAB NPs are successfully prepared.
Example 8
Preparation of palmitic acid modified albumin nanoparticles (PAB NPs). 80mg of PAB prepared in example 2 was dissolved in 4mL of deionized water to form an aqueous phase, 800uL of an organic phase in a mixed solvent of dichloromethane and ethyl acetate (volume ratio 1) was added to the aqueous phase, and the PAB NPs were successfully prepared by removing the organic solvent by rotary evaporation with an ice-bath probe for 10min (power 200W, working time 3s, batch 7 s).
Example 9
Preparation of stearic acid modified albumin nanoparticles (SAB NPs). 80mg of SAB prepared in example 3 was dissolved in 4mL of deionized water to form an aqueous phase, 20mg of a mixture of soybean oil and medium-chain oil (mass ratio 1.
Example 10
And (3) preparing the tripterine (CLT) -loaded palmitic acid modified albumin nanoparticles (CLT-PAB NPs). 80mg of PAB prepared in example 1 is dissolved in 4mL of deionized water to form an aqueous phase, 2.8mg of CLT and 18mg of soybean oil are dissolved in 800uL of a mixed solvent of dichloromethane and ethyl acetate (volume ratio is 1) to form an organic phase, the organic phase and the aqueous phase are mixed, an ice-bath probe is used for ultrasonic treatment for 10min (power is 200W, work time is 3s, and pause time is 7 s), and the organic solvent is removed by rotary evaporation, so that CLT-PAB NPs are successfully prepared.
Example 11
Preparation of Curcumin (CUR) -loaded palmitic acid modified albumin nanoparticles (CUR-PAB NPs). 80mg of PAB prepared in example 1 is dissolved in 4mL of deionized water to form a water phase, 4mg of CUR and 28mg of soybean oil are dissolved in 400uL of ethyl acetate, 400uL of dichloromethane is added to form an organic phase, the organic phase and the water phase are mixed, an ice-bath probe is used for ultrasonic treatment for 10min (power 200W, working time 3s and intermittent time 7 s), and the organic solvent is removed by rotary evaporation to successfully prepare the CUR-PAB NPs.
Example 12
Preparation of Tripterine (CLT) -loaded long-circulating palmitic acid-modified albumin nanoparticles (CLT-PAB-PEG NPs). 80mg of PAB prepared in example 1 and 20mg of DSPE-PEG2000 were dissolved in 4mL of deionized water to form an aqueous phase, 2.8mg of CLT and 18mg of soybean oil were dissolved in 800uL of a mixed solvent of dichloromethane and ethyl acetate (volume ratio 1) to form an organic phase, the organic phase and the aqueous phase were mixed, an ice-bath probe was used for ultrasonic treatment for 10min (power 200W, working 3s, intermittent 7 s), and the organic solvent was removed by rotary evaporation, thereby successfully preparing CLT-PAB-PEG NPs.
Example 13
Preparation of Entecavir (ETV) -loaded palmitic acid modified albumin nanoparticles (ETV-PAB NPs). Dissolving 10mg of entecavir and 50mg of lecithin E80 in 5mL of a mixed solvent of isopropanol and ethanol with the volume ratio of 1, stirring for 1h under the condition of water bath at 55 ℃, and removing the organic solvent by rotary evaporation under reduced pressure to obtain the phospholipid complex of entecavir. Dissolving the obtained phospholipid complex and 30mg soybean oil in 1mL dichloromethane to obtain an organic phase, dissolving 200mg of PAB prepared in example 1 in 10mL deionized water to obtain an aqueous phase, mixing the organic phase and the aqueous phase, carrying out ultrasonic treatment on the organic phase and the aqueous phase for 10min by using an ice bath probe (power 200W, working time 3s and intermittent time 7 s), and carrying out rotary evaporation to remove the organic solvent to successfully prepare ETV-PAB NPs.
Example 14
Preparation of liposome (PAB-Lip) with scavenger receptor-A targeting effect. 26mg of soybean phospholipid (S100), 8mg of cholesterol (Chol) and 6mg of lysine-cholesterol ester (Chol-lys) are dissolved in a proper amount of mixed solvent of chloroform and methanol (volume ratio: 3). Adding 4mL of 250mol/L ammonium sulfate solution to hydrate the lipid membrane, performing ultrasonic treatment for 10min by using a probe (power of 200W, ultrasonic treatment for 5s and interval of 5 s), and passing through a Sephadex G-75 gel column to establish an ion gradient, wherein the mobile phase is 0.9 percent of NaCl solution, so as to obtain a blank liposome. 10mg of the PAB prepared in the example 1 is dissolved in 2mL of deionized water, the obtained PAB aqueous solution is added into the blank liposome, and the mixture is stirred at room temperature for 30 min-1 h at a constant speed, so that the PAB-Lip is successfully prepared.
Example 15
Preparation of Doxorubicin (DOX) carrying liposome (PAB-DOX-Lip) with targeting effect of scavenger receptor-A. The other conditions were the same as in example 14, with the only difference that the blank liposomes were replaced by DOX loaded liposomes. The DOX-loaded liposome is prepared by mixing blank liposome prepared in example 14 with DOX solution (drug-lipid ratio is 1: 10), and incubating for 1h in water bath at 37 ℃.
Example 16
Preparation of zein nanoparticles (PAB-zein NPs) with scavenger receptor-A targeting effect. 20mg of zein was dissolved in 2mL of 70% isopropyl alcohol. 4mg of the PAB prepared in example 1 was taken and dissolved in 10mL of deionized water. The zein solution in isopropanol was quickly poured into the PAB solution with stirring and stirring continued at room temperature for 30min. Then heated at 45 ℃ and purged with nitrogen to remove isopropanol, PAB-zein NPs were successfully prepared.
Example 17
Preparation of Curcumin (CUR) -loaded zein nanoparticles (PAB-CUR-zein NPs) with scavenger receptor-A targeting effect. The other conditions were the same as in example 16, except that the isopropyl alcohol solution of zein was replaced with a mixed isopropyl alcohol solution of zein and curcumin. The mixed isopropanol solution of zein and curcumin is prepared by completely dissolving 20mg of zein and 2.5mg of curcumin in 2mL of 70% isopropanol solution.
Example 18
Preparation of phospholipid bile salt mixed micelle (PAB micelles) with scavenger receptor-A targeting effect. 35mg of sodium deoxycholate and 25mg of phospholipid E80 were weighed and dissolved in 10mL of methanol. The methanol in the mixed solution was removed by rotary evaporation under a water bath condition of 30 ℃ to form a uniform film. And (3) taking 1.5mL of deionized water for hydrating the film formed by the phospholipid bile salt, and fully shaking to obtain a blank micelle. 10mg of the PAB prepared in the example 1 is dissolved in 1mL of deionized water, the obtained PAB aqueous solution is added into the blank micelle, and the mixture is stirred at a constant speed for 30min to 1h at room temperature, so that the PAB microorganisms are successfully prepared.
Example 19
Preparation of trans-retinoic acid-loaded phospholipid bile salt mixed micelle (RA-PAB micelles) with scavenger receptor-A targeting effect. The other conditions were the same as in example 18 except that 2mg of trans-Retinoic Acid (RA) was added to 10mL of methanol in addition to phospholipids and sodium deoxycholate to obtain RA-loaded micelles, and finally RA-PAB microorganisms were successfully prepared.
Comparative example 1
Preparation of bovine serum albumin nanoparticles (BSA NPs). The other conditions were the same as in example 6, with the only difference that: the PAB was replaced by BSA and BSA NPs were successfully prepared. The particle size was 113.3nm, which was uniform.
Comparative example 2
Preparation of Tripterine (CLT) -loaded bovine serum albumin nanoparticles (CLT-BSA NPs). Otherwise the conditions were the same as in example 10, with the only difference that the PAB prepared in example 1 was replaced by BSA, and CLT-BSA NPs were successfully prepared. The particle size is 116.5nm, and the drug loading is 2.4%.
Experimental example 1 fluorescence Spectroscopy and circular dichroism Spectroscopy of PAB
PAB and BSA prepared in example 1 were dissolved in PBS (pH 7.3) to give a final concentration of 8X 10 - 7 mol/L, analyzed with a fluorescence spectrophotometer while keeping the concentration at 2X 10 -6 The PAB PBS solution and the BSA PBS solution in mol/L were analyzed by a circular dichroism spectrometer, and the results are shown in FIG. 1.
FIG. 1a is a graph of the fluorescence spectra of PAB and BSA, where it can be seen that the maximum emission wavelength of PAB is significantly blue-shifted and the fluorescence intensity is reduced compared to BSA. FIG. 1b is a graph of the circular dichroism spectra of PAB and BSA, and it can be seen that both PAB and BSA have two distinct negative shoulders indicating alpha-helices, and that the peak intensity of PAB is greater than that of BSA, i.e., the number of alpha-helices of PAB is greater than that of BSA. This indicates that the PA successfully modifies BSA, and the microenvironment where the fluorophore (tryptophan) is located in the PAB structure is more lipophilic than BSA, so that the maximum emission wavelength of the fluorescence spectrum is blue-shifted and tends to fold, and the number of alpha-helices increases.
Experimental example 2 molecular weight and degree of substitution of PAB
The degree of substitution and molecular weight of PAB prepared in example 1 and example 2 were determined by the Indantrione method and SDS-PAGE, respectively, see FIG. 2 (Lane 1 for PAB in example 1, lane 2 for PAB in example 2, lane 3 for BSA, and Lane 4 for marker. The Indantrione method was carried out by 1) preparing a series of PAB/BSA solutions (0-10 mg/mL) with appropriate concentration gradients with deionized water 2) mixing 1mL of PAB/BSA solution with 1mL of acetic acid buffer (pH 5.4) and 1mL of ninhydrin developing solution 3) heating the three mixed solution in a water bath at 100 ℃ for 15min, followed by cooling to room temperature and standing for 5min 4) diluting with 3mL of 60% ethanol, and measuring the UV absorption of each sample at a wavelength of 570nm to calculate the degree of substitution. SDS-PAGE bands were prepared by conventional methods, and the samples were finally stained with Coomassie Brilliant blue, and the approximate molecular weight of the samples was determined by comparing the distance between the sample and the marker. The obtained substitution degrees and molecular weights were mutually confirmed, and finally it was found that the PABs prepared in examples 1 and 2 had substitution degrees of 17.53% and 9.81%, respectively, and molecular weights of 69.7KDa and 68.5KDa, respectively. The results indicate that PAB can be successfully obtained by modification of BSA with PA.
Experimental example 3 physicochemical Properties of the Nanodiulation
The PAB NPs of examples 6, 7 and 8, the SAB NPs of example 9, the PAB-Lip of example 14, the PAB-zein NPs of example 16 and the PAB microorganisms of example 18 were diluted and then the particle size, PDI and Zeta potential were measured by a laser particle sizer. As shown in table 1. The results show that PAB NPs, SAB NPs, PAB-Lip, PAB-zein NPs and PAB microorganisms with proper particle sizes and uniform distribution can be successfully prepared by the methods of examples 6 to 9 and 14, and examples 16 and 18.
TABLE 1 physicochemical characterization of the Nanodiulation
Figure BDA0002325255530000111
Experimental example 4 physicochemical Properties of drug-loaded NanoPrepration
After diluting the CLT-PAB NPs of example 10, the CUR-PAB NPs of example 11, the CLT-PAB-PEG NPs of example 12, the EVT-PAB NPs of example 13, the PAB-DOX-Lip of example 15, the PAB-CUR-zein NPs of example 17 and the RA-PAB microorganisms of example 19, the particle size, PDI and Zeta potentials were determined using a laser particle sizer, and the CLT or CUR content in the nanoparticles was determined using gel exclusion chromatography and HPLC. As shown in table 2. The results show that CLT-PAB NPs, CUR-PAB NPs, CLT-PAB-PEG NPs, EVT-PAB NPs, PAB-DOX-Lip, PAB-CUR-zein NPs and RA-PAB microorganisms with proper particle size, uniform distribution and drug loading capacity meeting the actual administration requirement can be successfully prepared by adopting the methods in examples 10-13, 15, 17 and 19.
TABLE 2 physicochemical characterization of drug-loaded nano-formulations
Figure BDA0002325255530000121
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Experimental example 5 TEM characterization of nanoparticles
The CLT-PAB NPs prepared in example 10 were negatively stained with 1% phosphotungstic acid, dropped onto a special copper mesh, naturally evaporated, and then the nanoparticle morphology was observed with a Transmission Electron Microscope (TEM) and photographs were taken.
FIG. 3 shows TEM characterization results, which shows that the nanoparticles are spherical, have a particle size of about 100nm, are uniformly distributed, and are consistent with the results measured by a laser particle sizer.
Experimental example 6 in vitro stability of nanoparticles
The change in particle size and PDI of the CLT-PAB NPs prepared in example 10 during storage at 4 ℃ and 37 ℃ was observed using a laser particle sizer to characterize the storage stability of the nanoparticles in vitro. As shown in fig. 4, the nanoparticles showed no significant change in particle size and PDI during storage for 2 weeks at either 4 ℃ or 37 ℃, indicating their good stability.
Experimental example 7 uptake and inhibition of uptake of nanoparticles in macrophages and lipopolysaccharide-activated macrophages
The inventors preliminarily determined the SR-a targeting effect of PAB by measuring uptake and inhibition of uptake of nanoparticles in macrophages (raw264.7) and Lipopolysaccharide (LPS) activated macrophages (LPS-AR).
The preparation method is similar to the embodiment 10 of the invention, 2.8mg of CLT in the prescription is replaced by 50 mu g of DiD, and the DiD-PAB NPs are prepared. DiD-labeled BSA NPs (DiD-BSA NPs) were prepared by simultaneously using the same method as in comparative example 2, except that 2.8mg of CLT in the formulation thereof was replaced with 50. Mu.g of DiD.
Flow cytometry was used to quantify the cellular uptake of nanoparticles. And (4) taking Raw264.7 cells in the logarithmic growth phase, and digesting with 0.25% of pancreatin to obtain a cell suspension. Cells were seeded in 12-well cell culture plates at 1mL per well and incubated at 37 ℃ for 24h in the presence or absence of Lipopolysaccharide (LPS) (2. Mu.g/mL) for adherent growth. The medium was aspirated, washed 2 times with PBS, and DiD-PAB NPs and DiD-BSA NPs diluted with serum-free medium (DiD final concentration 0.4. Mu.g/mL) were added. Incubate at 37 ℃ for 2h. Removing the culture medium containing DiD by suction, washing with PBS for 3 times, digesting with 0.25% pancreatin, centrifuging at 2000rpm for 3min, removing the supernatant by suction, adding PBS solution to resuspend cells, repeating the centrifugal washing for 3 times, and finally placing the cells resuspended by PBS in a flow cytometer to measure the uptake of the preparation; simultaneously, flow cytometry is adopted to quantitatively analyze the uptake of the nanoparticles after being treated by different inhibitors in LPS-AR, and the operation process is as described before, and different inhibitors are firstly used for 1 hour before DiD-PAB NPs and DiD-BSA NPs which are diluted by serum-free culture medium are added. Inhibitors include 4 ℃ incubation conditions (energy suppression), amiloride (macropinocytosis), chlorpromazine (clathrin-mediated endocytosis), methyl- β -cyclodextrin (lipovalve/caveolin-mediated endocytosis), and dextran sulfate (DS, SR-a mediated pathway). The results are shown in figure 5 (figure 5a is the uptake of DiD-BSA NPs and DiD-PAB NPs in macrophages and LPS-AR, respectively, figure 5b and c are the uptake of DiD-PAB NPs and DiD-BSA NPs in LPS-AR after treatment with different inhibitors, respectively, all mean ± standard deviation, n =5,. P <0.05,. P < 0.001).
SR-A is expressed on the membrane of macrophage, and the expression level of SR-A is further increased remarkably in activated macrophage (LPS-AR). FIG. 5 results show that the uptake of DiD-PAB NPs is significantly higher than that of DiD-BSA NPs, especially in LPS-AR, both in Raw264.7 and LPS-AR; glucan sulfate (DS) is a specific inhibitor aiming at SR-A, and the uptake of DiD-PAB NPs in LPS-AR can be significantly inhibited by DS, which indicates that the uptake of DiD-PAB NPs by macrophages, particularly the uptake of activated macrophages, is mainly through an SR-A mediated pathway.
Experimental example 8 uptake of nanoparticles in macrophages, interleukin (IL) -4 activated macrophages, and other cells not expressing SR-A
The inventors further determined the SR-A targeting effect of PAB by measuring the uptake of nanoparticles in Raw264.7, IL-4 (80 ng/mL) activated macrophages (IL-4-AR), as well as human synovial fibroblasts (HFLS), human tubular epithelial cells (HK-2), and mouse melanoma cells (B16F 10) cells.
DiD-PAB NPs and DiD-BSA NPs were prepared as described in Experimental example 7. The uptake of DiD-PAB NPs and DiD-BSA NPs in several different cells was determined using the method described in Experimental example 7, with only two cells of Experimental example 7 being replaced with Raw264.7, IL-4 activated macrophages (IL-4-AR) and HFLS, HK-2 and B16F10 cells. The results are shown in figure 6 (figure 6a is the uptake in macrophages and IL-4-AR, figure 6B is the uptake in HFLS, HK-2 and B16F10, all expressed as mean ± standard deviation, n =5,. P < 0.001).
SR-A is expressed on Raw264.7 cell membrane, and the expression amount is further increased in IL-4-AR, while SR-A is hardly expressed in HFLS, HK-2 and B16F10 cells. FIG. 6 shows that the uptake of DiD-PAB NPs is significantly higher than that of DiD-BSA NPs in both Raw264.7 and IL-4-AR, and that the increased uptake of DiD-PAB NPs is more significant in IL-4-AR; whereas the uptake of DiD-PAB NPs in three other cells not expressing SR-A was very limited. The above experimental results further demonstrate the SR-A targeting effect of PAB.
Experimental example 9 Co-localization of PAB with SR-A in LPS-AR
The SR-A targeting effect of PAB was observed by examining the co-localization of PAB in example 1 with SR-A in LPS-AR.
PAB and BSA in example 1 were modified with FITC to give PAB-FITC and BSA-FITC. Inoculating Raw264.7 cells into the confocal dish, to 5% CO in the presence of LPS (2. Mu.g/mL) 2 And incubated at 37 ℃ for 24 hours, and then PAB-FITC solution was added to incubate the cells for 15min. Groups to which the BSA-FITC solution was added were set, and two groups were compared. The final concentration of BSA or PAB was 300. Mu.g/mL. The PAB-FITC or BSA-FITC containing media was removed and the cells were washed 3 times with PBS. 4% formalin fixed at room temperature for 20min, cells were washed with PBS, and SR-A on the cell membrane surface was stained with CD 204-antibody according to the instructions. After completion, incubation with anti-Alexa Fluor 594-labeled IgG continued. Cells were washed with PBS and stained for nuclei with DAPI at room temperature for 10 min. The cells were then washed 3 times with PBS and co-localized PAB and SR-A was observed using a confocal laser microscope. The results are shown in FIG. 7 (DAPI: nucleus, scale: 10 μm).
The result shows that PAB and SR-A have very obvious co-localization, while BSA-FITC on LPS-AR cell membrane has very weak fluorescence under the same concentration and culture condition, and has very obvious non-co-localization with SR-A, which indicates that PAB has SR-A targeting effect, and through the effect, PAB can realize the significantly higher uptake in LPS-AR than BSA.
Experimental example 10 in vivo pharmacokinetics study of nanoparticles
Healthy male SD rats were randomly divided into three groups, each group was n =5, free CLT was intravenously injected at a CLT administration dose of 2mg/kg, respectively, CLT-BSA NPs in comparative example 2, CLT-PAB NPs in example 10, and blood was taken through orbital venous plexus of each group of rats at 5,15,30,60,120,240,480,720,1440 and 2880min after administration, centrifuged at 5000rpm for 5min with heparin sodium as an anticoagulant, to obtain plasma samples, and finally CLT content in plasma was measured by LC-MS. The pharmacokinetic curves for each group are shown in figure 8 (all results are expressed as mean ± standard deviation, n = 5).
The result shows that compared with CLT-BSA NPs, the CLT-PAB NPs can obviously prolong the in vivo circulation time of the CLT, and the CLT concentration CLT-PAB NPs group at each time point is higher than that of the CLT-BSA NPs group, which indicates that compared with BSA, the PAB can effectively prolong the circulation time of the medicament and improve the bioavailability of the medicament.
Experimental example 11 distribution of nanoparticles in rheumatoid arthritis model rats
And (3) investigating the distribution condition of the nanoparticles in the rheumatoid arthritis rat body, and judging whether the PAB NPs can be obviously accumulated at the joint inflammation part.
DiD-PAB NPs and DiD-BSA NPs were prepared as described in Experimental example 7. An adjuvant-induced arthritis rat (AIA rat) model was established using healthy male SD rats, and rats were anesthetized at various time points after intravenous injection of free DiD, diD-PAB NPs and DiD-BSA NPs in AIA rats to examine the distribution of the three agents in the joints of AIA rats.
The specific method for establishing the AIA rat model comprises the following steps: rats were injected subcutaneously with complete Freund's adjuvant (100. Mu.l) containing 10mg/mL of heat-inactivated mycobacteria to the bottom of the tail. The progress of arthritis is monitored every day, obvious red swelling of soles and thick ankle joints can be observed on 14 days after molding, and the disability of the molding hind paws of part of rats can be observed when the ankle joints are thick, so that the success of molding is shown. AIA rats successfully modeled were randomly divided into 4 groups of N.S., free DiD, diD-BSA NPs, diD-PAB NPs, 5 animals each, and the DiD was administered to each group at an intravenous dose of 55 μ g/mL, and the rats were anesthetized at 2,4,8,12,24 and 48h after administration, and the distribution of the formulations in each group at the site of inflammation was observed using a biopsy apparatus. The experimental results are shown in FIG. 9.
The results show that both groups of nanoparticles accumulate at the joint area, but the accumulation of DiD-BSA NPs at the joint area can only last for 12h after administration, and almost no fluorescence is observed at the joint area by 24 h. The accumulation of DiD-PAB NPs at the joint site was higher at each time point than in the DiD-BSA NPs group and continued until 48h post-dose. Suggesting that PAB NPs may achieve higher accumulation at the joint site due to their SR-a targeting.
Experimental example 12 SR-A targeting effect of nanoparticles in rheumatoid arthritis model rats
The SR-A targeting effect of the PAB in the model rat body is further confirmed by investigating the co-localization of the SR-A at the inflammatory joint part of the nanoparticle and the rheumatoid arthritis model rat.
The preparation method of DiD-PAB NPs and DiD-BSA NPs, the model establishment method of AIA rats, and the grouping condition and administration dose of AIA rats were the same as those in Experimental example 11, but the rats were sacrificed one hour after administration, the inflamed ankle joint of each rat was fixed, decalcified, paraffin-embedded, sectioned, SR-A in the section was stained, and co-localization of DiD and SR-A in each group was observed with a laser confocal microscope. The results of the experiment are shown in FIG. 10 (DAPI: nucleus, scale: 50 μm).
The results show that 1h after administration, diD-PAB NPs can achieve significantly higher accumulation at the site of inflamed joints than DiD or DiD-BSA NPs, and that there is a very significant co-localization of DiD-PAB NPs and SR-A compared to the latter two. The above results further confirm the in vivo SR-a targeting effect of PAB.
Experimental example 13 preliminary pharmacodynamic evaluation of CLT-PAB NPs in AIA rat model
The treatment of AIA rats with CLT technical drug, CLT-BSA NPs and CLT-PAB NPs was examined in comparison. The preliminary findings were the thickness change at the inflamed joint site of each rat during the treatment and the levels of Tumor Necrosis Factor (TNF) -alpha and Interleukin (IL) -1 beta in the serum of each rat after the end of the treatment.
CLT-PAB NPs and CLT-BSA NPs were prepared according to example 10 and comparative example 2, respectively, and AIA rat models were constructed according to Experimental examples 11 or 12. AIA rats were randomly divided into 4 groups of 7 rats, and administration was started on day 14 after molding, wherein the dose of the CLT prodrug and CLT-BSA NPs was 1mg/mL, the dose of the CLT-PAB NPs was 0.6mg/mL, and the other group was intravenously injected with the same volume of physiological saline. The administration is once every other day for 4 times. The ankle thickness of the rats was recorded every other day from day 14 after molding to day 24 after molding. By day 24 after molding, blood samples were taken from each group of rats, serum was collected by centrifugation, and TNF-. Alpha.and IL-1. Beta.levels in serum were measured by ELISA kit according to the instructions. The results of the experiment are shown in figure 11 (all results are shown as mean ± sd, n =7, × p < 0.001) and figure 12 (all results are shown as mean ± sd, n =7, × p <0.05, × p < 0.001). The result shows that after the initial administration, the CLT-PAB NPs and the CLT-BSA NPs show obvious treatment effect, and after the treatment course is finished, the rat joint condition of the CLT-PAB NPs group is closer to that of a normal rat from the aspect of ankle joint thickness, the serum inflammatory factor level of the rats of the CLT-BSA NPs and the CLT-PAB NPs is equivalent to that of the normal rat, so that the CLT-BSA NPs and the CLT-PAB NPs have obvious treatment effect on the rheumatoid arthritis, the administration dosage of the CLT-PAB NPs is far lower than that of the CLT-BSA NPs, and compared with the CLT-BSA NPs, the CLT-PAB NPs have wider potential huge application prospect in the treatment of the rheumatoid arthritis, and the PAB also provides a brand-new carrier and thinking direction for the inflammation treatment.
Experimental example 14 distribution of nanoparticles in rats with chronic pancreatitis and fibrosis model
DiD-PAB NPs and DiD-BSA NPs were prepared as described in Experimental example 7. Establishing a rat model of chronic pancreatitis and fibrosis by using healthy male SD rats, killing the rats and dissecting out pancreas at different time points after DiD-PAB NPs and DiD-BSA NPs are injected into the bodies of the model rats intravenously, and investigating the distribution condition of two nanoparticles in the pancreas of the model rats.
The specific method for establishing the rat model of chronic pancreatitis and fibrosis comprises the following steps: a dibutyltin Dichloride (DBTC) solution with a concentration of 40mg/mL was prepared using a mixed solvent of absolute ethanol, glycerol and DMSO (volume ratio 1. The DBTC solution was injected from the tail vein of rats at 8 mg/kg. Pancreatitis symptoms occurred 4 days after tail vein injection, and fibrosis symptoms had occurred in rat pancreas by 7 days. The body weight of the rats gradually decreased after molding. Rats successfully molded are randomly divided into DiD-BSA NPs and DiD-PAB NPs 2 groups, each group comprises 5 mice, N.S. groups injected with physiological saline intravenously are used as blank controls, and the rats are injected with two kinds of nanoparticles intravenously with the administration dose of DiD of 45 ug/kg. The experiment was divided into three time points, 2,6 and 12 hours after dosing. Rats were sacrificed at preset time points after dosing, the pancreas was dissected out, and the fluorescence intensity of DiD in the pancreas was recorded with a biopsy machine. The experimental results are shown in FIG. 13.
The results show that both groups of nanoparticles accumulate in the diseased pancreas, but the accumulation of DiD-PAB NPs in the pancreas at each time point is significantly higher than that of the DiD-BSA NPs group. Also at 12 hours post-dose, the fluorescence of DiD-BSA NPs group pancreatic DiD was already weak, but that of DiD-PAB NPs group pancreatic DiD was still strong. It is suggested that PAB NPs may achieve higher accumulation at inflamed and fibrotic pancreatic sites due to their SR-a targeting.
Experimental example 15 distribution of nanoparticles in tumor sites of melanoma mice
DiD-PAB NPs and DiD-BSA NPs were prepared as described in Experimental example 7. Establishing a melanoma model by using male C57BL/6 mice, killing the mice 2 hours after injecting DiD-PAB NPs and DiD-BSA NPs from the tail vein of the tumor-bearing mice, separating tumor tissues, and investigating the distribution condition of two nanoparticles at the tumor part.
The specific method for establishing the melanoma mouse model comprises the following steps: depilating C57BL/6 mice of about 6 weeks old, and inoculating 5X 10 mice on the depilated flanks 5 The tumor volume was measured using an electronic vernier caliper for each B16F10 cell, and the formula was V = π/6 × a × B 2 Wherein a is the tumor major diameter and b is the tumor minor diameter. Tumor-bearing mice successfully modeled are randomly divided into DiD-BSA NPs and DiD-PAB NPs 2 groups, each group comprises 4 mice, N.S. groups injected with normal saline intravenously are used as blank controls, and two nanoparticles are injected into tail veins of the mice at the dosage of DiD of 45 ug/kg. Mice were sacrificed 2 hours after injection, tumor tissue was isolated and the fluorescence intensity of DiD in the tumor was recorded with a live imager. Experimental results fig. 14.
The results showed that the accumulation of DiD-PAB NPs at the tumor site 2 hours after administration was much higher than in the DiD-BSA NPs group. Suggesting that PAB NPs may achieve higher accumulation at the tumor site due to their SR-A targeting.

Claims (16)

1. The preparation method of the drug carrier is characterized by comprising the following steps:
(1) Dissolving fatty acid in an organic solvent, adding dicyclohexylcarbodiimide and N-hydroxysuccinimide, reacting for 2-24 hours at 0-37 ℃, and separating and purifying to obtain fatty acid succinimide ester; the organic solvent is selected from N, N-dimethylformamide and dimethyl sulfoxide;
(2) Dissolving albumin in a buffer solution with the pH value of 8.0-10, dissolving fatty acid succinimide ester in an organic solvent, reacting for 12-24 hours at the temperature of 20-37 ℃ according to the molar ratio of the fatty acid succinimide ester to the albumin of 10-100, dialyzing, centrifuging and freeze-drying to obtain the compound;
the drug carrier is fatty acid modified albumin obtained by connecting the amino group of the lysine residue of albumin with the carboxyl group of fatty acid through acylation reaction;
the albumin is selected from bovine serum albumin and/or human serum albumin;
the fatty acid is one or more of myristic acid, palmitic acid, stearic acid, arachidic acid, oleic acid, linolenic acid, linoleic acid and arachidonic acid;
the buffer solution is selected from carbonate buffer solution, phosphate buffer solution, tris-hydrochloric acid buffer solution, glycine-sodium hydroxide buffer solution and boric acid-borax buffer solution, and the organic solvent in the step (2) is selected from N, N-dimethylformamide and dimethyl sulfoxide.
2. The method for preparing a drug carrier according to claim 1, wherein the reaction temperature in step (1) is 20-37 ℃; the reaction time is 12 to 24 hours.
3. The method of claim 1, wherein the buffer is selected from the group consisting of carbonate buffers having a pH of 8.3 to 8.7.
4. The method for preparing a pharmaceutical carrier according to claim 1, wherein the molar ratio of fatty acid succinimidyl ester to albumin is 10-20.
5. A pharmaceutical carrier, characterized by being prepared by the preparation method of any one of claims 1 to 4.
6. A fatty acid modified albumin nanoparticle targeted by scavenger receptor-A comprises a drug carrier material targeted by scavenger receptor-A and a drug loaded on the nanoparticle; the mass of the drug loaded on the nanoparticles is 0.1-20% of the mass of the drug carrier material; the carrier material is fatty acid modified albumin obtained by connecting the amino group of lysine residue of albumin with the carboxyl group of fatty acid through acylation reaction; wherein the drug carrier material with scavenger receptor-A targeting is prepared by the preparation method of any one of claims 1 to 4;
the albumin is selected from bovine serum albumin and/or human serum albumin;
the fatty acid is one or more of myristic acid, palmitic acid, stearic acid, arachidic acid, oleic acid, linolenic acid, linoleic acid and arachidonic acid;
the particle size of the nanoparticles is 90-200nm.
7. A nanoparticle according to claim 6, wherein the nanoparticle-loaded drug is an anti-inflammatory or anti-cancer drug.
8. The nanoparticle according to claim 6, wherein the drug loaded on the nanoparticle is selected from one or more of celastrol, curcumin, silibinin, glycyrrhetinic acid, paclitaxel, entecavir, dexamethasone, doxorubicin, pirarubicin, cabazitaxel, oxaliplatin, teniposide, tanshinone IIA, hydroxycamptothecin, mitoxantrone, gemcitabine, and trans-retinoic acid.
9. A nanoparticle according to claim 6, further comprising an injection oil, a polyethylene glycol derivative, a polyoxyethylene polyoxypropylene block copolymer, an injection phospholipid, and an injection additive, wherein the injection additive is one or more selected from mannitol, lactose, glucose, maltose, trehalose, sucrose, sodium chloride, disodium hydrogen phosphate and sodium dihydrogen phosphate, and the injection oil is one or more selected from soybean oil, medium-chain fatty acid glyceride, tea oil and sesame oil; the phospholipid for injection is selected from one or more of soybean phospholipid and egg yolk lecithin; the dosage of the oil for injection is 0-40% (wt%) of the dosage of the albumin modified by the fatty acid.
10. A method for preparing nanoparticles according to any one of claims 6 to 9, characterized in that it is carried out by the following steps:
dissolving the drug carrier in deionized water or in deionized water in which other auxiliary materials except the phospholipid for injection are dissolved, adding an organic phase selected from an organic solvent, an organic solvent in which oil for injection is dissolved, an organic solvent in which oil for injection and the drug are dissolved, an organic solvent in which the drug is dissolved, an organic solvent in which a phospholipid compound of the drug is dissolved or an organic solvent in which a phospholipid compound of the oil for injection and the drug is dissolved, homogenizing under high pressure or ultrasonically emulsifying by a probe, and rotationally evaporating to remove the organic solvent to obtain the injection-resistant phospholipid-containing compound;
the organic solvent is selected from one or more of dichloromethane, ethyl acetate and chloroform.
11. The method according to claim 10, wherein the organic solvent is dichloromethane or ethyl acetate.
12. A nano preparation with a scavenger receptor-A targeting function is characterized in that the nano preparation takes the drug carrier of claim 5 as a target head to modify a high-molecular nanoparticle, a liposome or a micelle nano preparation, wherein the high-molecular nanoparticle is selected from one or more of glycolide-lactide copolymer nanoparticles, chitosan nanoparticles, zein nanoparticles, polydopamine nanoparticles, polylysine nanoparticles, dextran nanoparticles and diethylaminoethyl dextran nanoparticles, and the micelle is selected from one or more of phospholipid cholate mixed micelles, polyethylene glycol polyethyleneimine micelles, polycaprolactone polyethyleneimine micelles and polycaprolactone polyethylene glycol micelles.
13. A method for preparing the scavenger receptor-a targeted nano-formulation of claim 12, which is carried out by the following steps:
dissolving the drug carrier of claim 5 in deionized water, adding the liposome/polymer nanoparticle/micelle without or with drug, and stirring; the mass ratio of the drug carrier to the liposome or the polymer nanoparticle or the micelle is (4); the stirring temperature is 20-37 ℃; the stirring time is 15 min-2 h.
14. Use of a pharmaceutical carrier according to claim 5 for the preparation of a nano-formulation targeting scavenger receptor-a.
15. Use of a nanoparticle according to any one of claims 6 to 9 or a method of preparation according to any one of claims 10 to 11 for the preparation of a nanoparticle targeting scavenger receptor-a.
16. Use of a nano-formulation according to claim 12 or the method of preparation according to claim 13 for the preparation of a nano-formulation targeting scavenger receptor-a.
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