Nanometer microsphere targeting multiple myeloma and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to multiple myeloma targeting nano microspheres and application thereof. The invention provides a delivery system for targeting multiple myeloma, the delivery system has different targeting molecules for bone targeting and cancer cell targeting, and multiple myeloma cells in a bone marrow microenvironment can be targeted. Furthermore, the delivery system is carried on the Selinexor, so that the selective delivery of the Selinexor on myeloma cells and affected bone tissues can be enhanced. In-vivo studies show that the delivery system significantly improves the treatment effect of MM. Malignant proliferation of myeloma cells is effectively inhibited, the bone density is increased, the bone microstructure is improved, and bone deterioration in an MM mouse model is relieved. The method for delivering the anti-MM drug through the dual-targeting nanoparticles represents an innovative strategy, MM cancer cell targeting and cancer microenvironment bone targeting are combined, and the method is never applied to MM treatment before.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a nano-microsphere targeting multiple myeloma and an application thereof. Background Art

[0002] Multiple myeloma (MM) is a malignant blood disease characterized by abnormal proliferation of clonal plasma cells. It is the third most common blood disease after leukemia and non-Hodgkin's lymphoma. MM is characterized by the unregulated proliferation of monoclonal plasma cells in the bone marrow, resulting in the production of immunoglobulins or immunoglobulin chains without complete function. MM is more common in the elderly, with a median age of 71 for men and 74 for women. The cause of this disease is still unclear and is associated with age, gender, family history, and lifestyle.

[0003] Common symptoms of MM include manifestations of bone marrow and related organ damage, namely "CRAB" symptoms: increased blood calcium, renal impairment, anemia, bone disease, and secondary amyloidosis and other related manifestations. MM is still incurable, and the goal of treatment is to control and relieve "CRAB" symptoms and improve the remission rate and disease-free survival of multiple myeloma patients. In the classification of MM, the treatment of relapsed / refractory multiple myeloma is particularly difficult. With the progress of experts' research on the pathogenesis in recent years, new drugs targeting the pathogenesis have emerged, and combination therapy based on new drugs has achieved better therapeutic effects in the treatment of patients with refractory relapsed multiple myeloma.

[0004] Although multiple myeloma (MM) is a blood disease, many cancer cells are rooted in the bone marrow. It is difficult for drugs to reach the lesions in the bones and to remove such lesions, which directly causes the refractory and easy relapse of MM. The drugs only circulate in the blood environment and cannot be enriched in the bone marrow to form an effective concentration, which will limit the therapeutic effect. How to enrich the drugs in the bone marrow is a problem faced by this field.

[0005] At present, general research is on drug delivery systems with single targets targeting cell surface receptors. The difficulty in treating multiple myeloma lies in its refractory nature and high recurrence rate. The original single-target strategy, such as targeting cell surface receptors, can eliminate cancer cells in the peripheral circulation system, but it is difficult to eliminate refractory lesions in the bone marrow because it is difficult to enter the bones and reach high effective concentrations in the bones to kill lesions in the bone marrow; if a drug delivery strategy targeting bones is used, it will also cause the drug to be delivered to the bones, but only adhere to the bone surface, and has no selectivity for cells in the bone marrow. It has a poor tendency to target cancer cells and is prone to accidentally injuring cells in the bones, causing bone damage. In addition, this single bone-targeting strategy has poor killing power against peripheral cancer cells. Summary of the invention

[0006] In order to solve the problem that the existing single-target drug delivery strategy is difficult to solve the refractory and high recurrence of multiple myeloma, the present invention creatively proposes a dual-system targeted MM treatment strategy to construct a drug delivery system that can target both MM cancer cells and MM deep bone marrow lesions, as follows:

[0007] The purpose of the first aspect of the present invention is to provide a delivery system targeting multiple myeloma.

[0008] The purpose of the second aspect of the present invention is to provide a method for preparing the multiple myeloma targeting delivery system of the first aspect of the present invention.

[0009] The third aspect of the present invention is to provide a pharmaceutical composition.

[0010] The purpose of the fourth aspect of the present invention is to provide use of the delivery system of the first aspect of the present invention and the pharmaceutical composition of the third aspect of the present invention in the preparation of a product for treating multiple myeloma.

[0011] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0012] The first aspect of the present invention provides a delivery system targeting multiple myeloma, characterized in that:

[0013] The delivery system includes a nanoskeleton, a targeting molecule and a drug;

[0014] The targeting molecules include bone targeting molecules and tumor targeting factors;

[0015] The bone targeting molecule includes pyrophosphate;

[0016] The tumor targeting factor includes at least one of folic acid and CD38.

[0017] In some embodiments of the present invention, the nanoskeleton comprises glycol chitosan nanoparticles.

[0018] In some embodiments of the present invention, the metal salt comprises an alkali metal salt or an alkaline earth metal salt.

[0019] In some embodiments of the present invention, the alkali metal salt includes at least one of a sodium salt and a potassium salt.

[0020] In some embodiments of the present invention, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt and aluminum salt.

[0021] In some embodiments of the present invention, the salt formed with the organic base includes a salt formed with the following organic bases: at least one of trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0022] In some embodiments of the present invention, the salt formed with the inorganic base includes a salt formed with the following inorganic bases: at least one of potassium hydroxide, barium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, copper hydroxide, iron hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, and potassium bicarbonate.

[0023] In some embodiments of the present invention, the salt formed with a basic amino acid comprises a salt formed with the following basic amino acids: at least one of arginine, lysine, and ornithine.

[0024] In some embodiments of the present invention, the delivery system further comprises a drug.

[0025] Preferably, the drug includes at least one of Selinexor, belamaf, and Melflufen.

[0026] Preferably, the drug is Selinexor.

[0027] In some embodiments of the present invention, the delivery system is specifically: GCNP@FA / PPi.

[0028] In some embodiments of the present invention, the delivery system is specifically: GCNP@CD38 / PPi.

[0029] The purpose of the second aspect of the present invention is to provide a method for preparing the delivery system targeting multiple myeloma according to the first aspect of the present invention, comprising the following steps:

[0030] The ethylene glycol chitosan is reacted with 2-(diisopropylamino)ethyl methacrylate and ammonium persulfate to obtain ethylene glycol chitosan nanoparticles;

[0031] The chitosan nanoparticles are mixed with tumor targeting molecules, and a catalyst is added to react to obtain tumor targeting chitosan nanoparticles;

[0032] The bone targeting molecule is mixed with the tumor targeting ethylene glycol chitosan nanoparticles, the pH is adjusted, and the mixture is reacted to obtain the dual targeting ethylene glycol chitosan nanoparticles having both bone targeting and tumor targeting.

[0033] In some embodiments of the present invention, in the steps: the ratio of ethylene glycol chitosan to 2-(diisopropylamino)ethyl methacrylate is (5-20):1 g / L; and the reaction is carried out in the dark.

[0034] In some embodiments of the present invention, in the step: the mass ratio of the tumor targeting factor to the chitosan nanoparticles is 1:(1-3).

[0035] In some embodiments of the present invention, the catalyst comprises HATU, HOAT.

[0036] In some embodiments of the present invention, in the step: the bone targeting factor is DA-PEG-PPI.

[0037] In some embodiments of the present invention, the mass ratio of the DA-PEG-PPI to the tumor-targeting chitosan nanoparticles is 1:(1-3).

[0038] In the second aspect of the present invention, GCNP@FA / PPi and GCNP@CD38 / PPi were prepared respectively.

[0039] For GCNP@FA / PPi, the preparation method is as follows:

[0040] Take 10 mg of DA-PEG-PPI, dissolve it in 1-3 mL of water, stir for 24-72 h, and self-oxidize the phenolic hydroxyl group in dopamine to carbonyl group (or add NaOH, let dopamine oxidize under alkaline conditions at pH = 8 for 1-3 h, and then add glacial acetic acid to neutralize it), then add 10-30 mg of GCNP and 10-30 mg of PDA-PEG-FA, and treat the mixture with 100 W ultrasound for 10 min. Stir evenly, add acetic acid dropwise to make the solution pH = 5, and allow the amino group in GCNP and the oxidized DA-PEG-PPI and the carbonyl / quinone group in PDA-PEG-FA to undergo Schiff base reaction. Stir at room temperature for 48 h, and then add NaOH dropwise to neutralize the pH to neutral, and then use a dialysis bag with a molecular weight MwCO of 14 kDa to dialyze with DD water for 2 days, freeze-dry, and obtain FA&PPi modified ethylene glycol chitosan GCNP@FA&PPI.

[0041] For GCNP@CD38 / PPi, the preparation method is as follows:

[0042] GCNP@CD38: Dissolve 20 mg GCNP in 1-3 mL PBS, add CD38 Pep (10-30) mg, catalyst HATU (5-10) mg, HOAT (1-5) mg, and treat the mixture with 100W ultrasound for 10 min and stir evenly. Stir at room temperature for (24-72) h to condense the amino group in ethylene glycol chitosan and the carboxyl group in the polypeptide into an amide bond. Then use a dialysis bag with a molecular weight MwCO of 14 kDa, dialyze with DD water for 2 days, and freeze-dry to obtain polypeptide-modified ethylene glycol chitosan GCNP@CD38.

[0043] GCNP@CD38&PPI: Take 10 mg DA-PEG-PPI, dissolve it in 2 mL DD water, stir for 48 h, so that the phenolic hydroxyl group in dopamine is self-oxidized to carbonyl group (or add NaOH, let dopamine oxidize for 2 h under alkaline conditions pH = 8, and then add glacial acetic acid to neutralize), then add GCNP@CD38 nanoparticles (10-30) mg, treat the mixture with 100 W ultrasound for 10 min, stir evenly, add acetic acid dropwise to make the solution pH = 5, and allow the amino group in GCNP@CD38 and the oxidized DA-PEG-PPI carbonyl group to undergo Schiff base reaction, stir at room temperature for (24-72) h, then use NaOH to add dropwise to neutralize the pH to neutral, and then use a dialysis bag with a molecular weight MwCO of 14 kDa to dialyze with DD water for 2 days, freeze-dry, and obtain CD38&PPi dual-modified ethylene glycol chitosan GCNP@CD38&PPI.

[0044] The third aspect of the present invention provides a pharmaceutical composition comprising GCNP@FA / PPi-Selinexor and GCNP@CD38 / PPi-Selinexor.

[0045] The fourth aspect of the present invention provides use of the multiple myeloma targeting delivery system of the first aspect of the present invention in the preparation of a product for treating multiple myeloma.

[0046] In some embodiments of the invention, the product comprises a pharmaceutical product.

[0047] Preferably, the drug comprises pharmaceutically acceptable excipients.

[0048] Preferably, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

[0049] Preferably, the drug is in the form of an oral preparation, an injection or a topical preparation. In particular, the oral preparation includes tablets, capsules, pills, powders, granules, syrups or solutions; the injection includes an injection liquid or a freeze-dried powder for injection; the topical preparation includes a cream, an ointment, a spray, an aerosol or a patch.

[0050] The pharmaceutical preparation is a delivery system targeting multiple myeloma of the first aspect of the present invention as an effective active ingredient, and includes other carrier components that are pharmaceutically acceptable. The carrier in the drug includes excipients, such as starch, water, etc.; lubricants, such as magnesium stearate, etc.; disintegrants, such as microcrystalline cellulose, etc.; fillers, such as lactose, etc.; binders, such as pregelatinized starch, dextrin, etc.; sweeteners; antioxidants; preservatives; flavoring agents; spices, etc.

[0051] The carrier selected when preparing oral preparations can be conventional pharmaceutical excipients such as starch, dextrin or cyclodextrin and various chemically modified cyclodextrins, sucrose, stearate, etc. When preparing freeze-dried powder injections, they can be prepared by aseptic spray drying, low-temperature vacuum drying, freeze drying, etc. The later preparation processes and equipment of each preparation belong to conventional techniques in the pharmaceutical field, and the present invention does not limit this.

[0052] The drugs described in the present invention are in the form of tablets, capsules, pills, powders, granules, syrups, solutions, injections, sprays, aerosols, patches, gels, and cataplasms, that is, pharmaceutical preparations include tablets, capsules, pills, powders, granules, syrups, solutions, injections, sprays, aerosols, patches, gels, cataplasms and the like, but are not limited to the above forms.

[0053] Furthermore, for the convenience of medication, the active ingredient targeted multiple myeloma delivery system can be processed into a specific dosage form with any one or more pharmaceutically acceptable excipients. These excipients can be diluents (such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol and microcrystalline cellulose, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, light magnesium oxide and calcium carbonate, etc.), wetting agents (such as water and ethanol, etc.), binders (such as hydroxypropyl methylcellulose, povidone, starch slurry and syrup, etc.), disintegrants (such as dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants and cross-linked polyvinylpyrrolidone, etc.), lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol and micropowder silica gel, etc.), colorants (such as titanium dioxide, sunset yellow, methylene blue and medicinal iron oxide, etc.), coating materials (such as acrylic resin, hydroxypropyl methylcellulose and povidone, etc.), solvents (such as water for injection, ethanol, propylene glycol and glycerol, etc.), acid-base regulators (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid and sodium tartrate, etc.), antioxidants (such as sodium sulfite, sodium pyrosulfite and sodium thiosulfate, etc.), antibacterial agents (such as phenol, benzyl alcohol and thimerosal, etc.), and isotonic regulators (such as sodium chloride and glucose, etc.).

[0054] The above-mentioned pharmaceutically acceptable excipients are generally recognized for this purpose and as inactive ingredients of medicaments. A compilation of pharmaceutically acceptable excipients can be found in reference books such as Handbook of Pharmaceutical Excipients (2nd edition, edited by A. Wade and PJ Weller; published by American Pharmaceutical Association, Washington and The Pharmaceutical 6Gess, London, 1994); Pharmacopoeia of the People's Republic of China - Catalogue of Pharmaceutical Excipients.

[0055] The beneficial effects of the present invention are:

[0056] The present invention provides a delivery system targeting multiple myeloma (MM), which is modified with two different targeting molecules for bone targeting and cancer cell targeting at the same time. Based on the dual targeting of cancer cells and cancer microenvironment, multiple myeloma cells in the bone marrow microenvironment can be targeted. Further, the delivery system is equipped with Selinexor, which can enhance the selective delivery of Selinexor to myeloma cells and affected bone tissue. In vivo studies have shown that the delivery system significantly improves the therapeutic effect of MM. The GCNP@CD38&PPI-selinexor and GCNP@FA&PPI-selinexor prepared by the present invention have excellent targeting to myeloma cells, can inhibit the reduction of spleen white pulp area caused by MM progression, inhibit the expression of spleen CD38 and CD138, and effectively inhibit the malignant proliferation of myeloma cells. Targeting the bone environment, GCNP@CD38&PPI-selinexor and GCNP@FA&PPI-selinexor significantly increase the bone mass of trabeculae, improve bone loss and bone microstructure deterioration in the late stage of MM. Compared with the Selinexor alone group, the dual-targeted delivery system significantly improved the therapeutic effect of MM. This dual-targeted nanoparticle delivery method of anti-MM drugs represents an innovative strategy that combines MM cancer cell targeting and cancer microenvironment bone targeting, which has never been applied to MM treatment before. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0058] Figure 1 This is a real picture of the preparation process of GCNP.

[0059] Figure 2 This is a diagram of the quality inspection results of the synthesis of CD38 polypeptide.

[0060] Figure 3 The peak diagram of Selinexor (left) and the standard curve diagram of Selinexor (right).

[0061] Figure 4 Particle size and potential results of GCNP, GCNP@FA&PPI-Selinexor and GCNP@CD38&PPI-Selinexor.

[0062] Figure 5 Transmission electron microscopy images of GCNP, GCNP@FA&PPI-Selinexor and GCNP@CD38&PPI-Selinexor.

[0063] Figure 6In the figure, A is the drug release result of Selinexor by GCNP; B is the proliferation inhibition result of Selinexor at different concentration gradients on multiple myeloma; C is the proliferation inhibition result of Selinexor, GCNP@FA&PPI-Selinexor, and GCNP@CD38&PPI-Selinexor on multiple myeloma; D is the fluorescence semi-quantitative analysis of targeted enrichment of U266 multiple myeloma by chitosan targeted nanoparticles labeled with Cy5.5; E Figure 1 is a fluorescence semi-quantitative analysis of the targeted enrichment of LC363 multiple myeloma cells by chitosan targeted nanoparticles labeled with Cy5.5; F is a fluorescence image of the targeted enrichment of multiple myeloma cells by U266 after chitosan targeted nanoparticles labeled with Cy5.5; G is a fluorescence image of the targeted enrichment of LC363 multiple myeloma cells by chitosan targeted nanoparticles labeled with Cy5.5; H is a fluorescence image of the endocytic uptake of LC363 multiple myeloma cells after chitosan targeted nanoparticles labeled with Cy5.5.

[0064] Figure 7 Fluorescence images of in vivo targeting verification of chitosan targeting nanoparticles labeled with Cy5.5.

[0065] Figure 8 A is the weight change results of animals in each group in Animal Experiment I; B&C are the weight ratios of spleen and liver of animals in each group in Animal Experiment I; D is the H&E staining image of tibia bone marrow cavity in Animal Experiment I, the H&E staining image of spleen in Animal Experiment I, the H&E staining image of liver in Animal Experiment I, the H&E staining image of stomach in Animal Experiment I; E is the immunofluorescence staining image of CD38 and CD138 of spleen in Animal Experiment I.

[0066] Fig. 9 A is the experimental plan in animal experiment I; B is the cross-sectional and longitudinal cross-sectional images of the tibial metaphysis after Micro CT 3D reconstruction in animal experiment I; CF are the changes in Micro-CT quantitative parameters of the tibia of each group of nude mice in animal experiment I.

[0067] Fig.10 A is the immunofluorescence staining of CD38 and CD138 of tibial bone marrow cavity cells in animal experiment I; B is the semi-quantitative analysis result of immunofluorescence of CD38 and CD138 of tibial bone marrow cavity cells in animal experiment I; C is the Elisa analysis of IL-6 concentration in animal serum in animal experiment I; D is the Elisa analysis of Igκ concentration in animal serum in animal experiment I.

[0068] Fig.11A is the experimental plan in animal experiment II; B is the Elisa analysis of Igλ concentration in animal serum in animal experiment II; C is the X-ray image of the right tibia of each group of animals in animal experiment II; D is the Micro CT 3D reconstructed image of the right tibia of each group of animals in animal experiment II; E is the change results of bone density of each group of animals in animal experiment II; FI is the change results of Micro-CT quantitative parameters of the tibia of each group of animals in animal experiment II. DETAILED DESCRIPTION

[0069] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0070] Example 1 Preparation of ethylene glycol chitosan nanoparticles and targeted preparations thereof

[0071] 1. Experimental Materials and Methods

[0072] 1). Preparation of dual-targeted GCNPs:

[0073] Polydopamine-polyethylene glycol-folic acid (PDA-PEG-FA) and dopamine-polyethylene glycol-pyrophosphate (DA-PEG-PPI) were synthesized by Xi'an Ruixi Biotechnology Co., Ltd. (China), and CD38 targeting peptide was synthesized by GenScript Biotech Co., Ltd. Chitosan nanoparticles (GCNPs) were prepared by copolymerization and grafting method according to the literature (Estrone-modified pH-sensitive Glycol Chitosan Nanoparticles for Drug Delivery in Breast Cancer, Acta Biomaterialia, Volume 73, June 2018, Pages 400-411. DOI: https: / / doi.org / 10.1016 / j.actbio.2018.04.020). The specific scheme is as follows: 2 g of glycol chitosan (GC; Shanghai McLean Biochemical Technology Co., Ltd.) was dissolved in 20 mL of deionized water, and then 200 μL of 2-(diisopropylamino)ethyl methacrylate (DPA; Sigma-Aldrich) and 3% w / v freshly prepared ammonium persulfate solution (200 μL) were added, and the reaction was stirred at 75 ° C for 3 h under light-proof conditions to finally obtain GCNP solution. Subsequently, the GCNP solution was loaded into a washed 14 kD specific molecular weight cutoff (MWCO) dialysis bag and dialyzed with double distilled water for 3 days under light-proof conditions. The dialysate was completely replaced at least 3 times during the dialysis process. Finally, the dialyzed solution was freeze-dried and the freeze-dried GCNP product was collected.

[0074] ① Preparation of CD38 targeting peptide modified polyethylene glycol-chitosan nanoparticles (GCNP@CD38)

[0075] Take 20mg GCNP and dissolve it with 2mL PBS, add 10mg CD38 Pep, add 6.9mg catalyst HATU, 3.6mg HOAT, and treat the mixture with 100W ultrasound for 10min and stir evenly. Stir at room temperature for 48h to condense the amino group in ethylene glycol chitosan and the carboxyl group in the polypeptide into an amide bond. Then use a dialysis bag with a molecular weight MwCO of 14kDa, dialyze with double distilled water (DD) for 2 days, freeze-dry, and obtain polypeptide-modified ethylene glycol chitosan GCNP@CD38.

[0076] ② Preparation of dual-targeted GCNP nanoparticles

[0077] Type 1: PPI and FA dual-modified polyethylene glycol-chitosan nanoparticles (GCNP@FA&PPI) 10 mg DA-PEG-PPI was dissolved in 2 mL DD water and stirred for 48 h to allow the phenolic hydroxyl group in dopamine to self-oxidize to carbonyl (or add NaOH to allow dopamine to oxidize under alkaline conditions at pH = 8 for 2 h, and then add glacial acetic acid to neutralize), and then add GCNP 20 mg, PDA-PEG-FA 10 mg, the mixture was ultrasonically treated at 100 W for 10 min, stirred evenly, acetic acid was added dropwise to make the solution pH = 5, and the amino group in GCNP and oxidized DA-PEG-PPI and carbonyl / quinone group in PDA-PEG-FA underwent Schiff base reaction, stirred at room temperature for 48 h, and then NaOH was added dropwise to neutralize the pH to neutral, and then dialyzed with DD water for 2 days using a dialysis bag with a molecular weight MwCO of 14 kDa, and freeze-dried to obtain FA&PPi modified ethylene glycol chitosan GCNP@FA&PPI.

[0078] ③ Preparation of dual-targeted GCNP nanoparticles

[0079] Type 2: Polyethylene glycol-chitosan nanoparticles dually modified with PPI and CD38 peptide (GCNP@CD38&PPI) 10 mg DA-PEG-PPI was dissolved in 2 mL DD water and stirred for 48 h to allow the phenolic hydroxyl group in dopamine to self-oxidize to carbonyl group (or add NaOH to allow dopamine to oxidize for 2 h under alkaline conditions pH = 8, and then add glacial acetic acid to neutralize), then add 20 mg GCNP@CD38 nanoparticles, and the mixture was ultrasonically treated at 100 W for 10 min. After stirring evenly, acetic acid was added dropwise to make the solution pH = 5, so that the amino group in GCNP@CD38 and the oxidized DA-PEG-PPI carbonyl group underwent Schiff base reaction, stirred at room temperature for 48 h, and then NaOH was added dropwise to neutralize the pH to neutral, and then dialyzed with DD water for 2 days using a dialysis bag with a molecular weight MwCO of 14 kDa, and freeze-dried to obtain CD38&PPi dually modified ethylene glycol chitosan GCNP@CD38&PPI.

[0080] 2. Experimental results

[0081] The GCNPs successfully prepared by copolymerization and grafting of GC with PDA and AP are generally observed as follows: Figure 1 As shown, it appears as a light yellow clear liquid to the naked eye, and after freeze-drying it appears as a light yellow porous and loose floc, which is easily soluble in water.

[0082] Example 2 Preparation of Selinexor-loaded ethylene glycol chitosan drug delivery system targeting multiple myeloma

[0083] 1. Preparation process

[0084] Preparation of drug-loaded nanoparticles 2 mg of Selinexor was added to 1 mL of the targeted nanoformulation (prepared in Example 1), and after 100 W ultrasonic treatment for 20 minutes, 0.01 mL of DMSO was added to increase the solubility, and stirred at room temperature for 48 hours under light-proof conditions to complete the drug loading process. The mass ratios of the dual-targeted drug-loaded nanoparticles were GCNP@CD38:PPI:Selinexor=10:5:2 and GCNP:FA:PPI:Selinexor=10:5:5:2. The preparation of CD38 polypeptide was completed by Jiangsu GenScript Biotechnology Co., Ltd., and the test results are shown in Figure 2 shown.

[0085] 2. Characterization and evaluation

[0086] Encapsulation rate and release determination: The encapsulation rate and release of Selinexor-GCNP were determined by high performance liquid chromatography (HPLC, Agilent 1260, Agilent Technologies, USA). The morphology of GCNP nanoparticles and dual-targeted GCNPs was observed by transmission electron microscopy (TEM, HT7800, Hitachi, Japan). The particle size and zeta potential of the nanoparticles were analyzed by dynamic light scattering (DLS, Nicomp 380 analyzer, Particle Sizing Systems, USA). 3) Particle size and potential measurement experiments

[0087] 3. Experimental results

[0088] In order to more accurately calculate the encapsulation efficiency of GCNP for Selinexor, a standard curve of Selinexor was established using high performance liquid chromatography. Figure 3 As shown, the retention time of Selinexor is 3.506, and the correlation coefficient R2 value in the standard curve is 0.9995, with good correlation.

[0089] The particle size and potential measurement results are shown in Figure 4 , as shown in Table 1. The average particle size of GCNP is 157.70nm, and the Zeta potential is +2.760mV. After adding other targeting substances and loading Selinexor, the particle size of the entire targeted drug delivery system becomes larger, and the positive charge of the potential of GCNP becomes the negative charge of GCNP@FA&PPI-Selinexor and GCNP@CD38&PPI-Selinexor. The chitosan targeted drug delivery system carries a negative charge, which is conducive to cell adsorption. In order to better understand the entire targeted drug delivery system, the prepared GCNP, GCNP@FA&PPI-Selinexor and GCNP@CD38&PPI-Selinexor were negatively stained and photographed under a transmission electron microscope. The results are shown in Figure 5 shown.

[0090] Results of loading and release experiments. After selinexor was encapsulated by GCNPs, a light yellow transparent precipitate appeared after centrifugation at 10,000 rpm for 1 hour. The volume of the precipitate was 100 μL. The encapsulation efficiency of GCNPs loaded with selinexor was evaluated by high performance liquid chromatography (HPLC). According to the formula encapsulation rate (%) = (loaded drug amount / input drug amount) × 100%, the encapsulation rate ηER was calculated to be 62.68% by 2000-390.87 × 1.9) / 2000 × 100%; according to the formula release rate (%) = (released drug amount / loaded drug amount) × 100%, the drug release of selinexor from ethylene glycol chitosan drug delivery nanoparticles for 7 days was monitored by high performance liquid chromatography. The calculation results showed that selinexor had a stable and consistent release behavior from the drug-loaded GCNPs, with an average 7-day release rate of 36.61%. Figure 6 As shown in A.

[0091] Table 1 Particle size and potential of glycol chitosan and its targeted nanoformulations

[0092]

[0093]

[0094] Example 3 In vitro biological effects and in vitro and in vivo targeting evaluation of the glycol chitosan drug delivery system targeting multiple myeloma

[0095] 1. Experimental methods

[0096] 1) Inhibitory effect of Selinexor-loaded ethylene glycol chitosan targeted drug delivery system on U266 cells

[0097] ① Screening of effective concentration of Selinexor: U266 cells were cultured in T25 flasks and counted using a cell counting plate. 5 5mL of cells were cultured at a cell concentration of 100 / mL. After 48h, 5mL of new culture medium was slowly added to the bottle to continue culturing. After the cells were fully grown, the cells were moved to a 15mL centrifuge tube and centrifuged at 800rpm for 3 minutes. After centrifugation, the supernatant was removed and 1mL of culture medium was added for cell counting. 5000 cells were seeded in a 96-well plate per well, and 100μL of Selinexor at concentrations of 100ng / mL, 200ng / mL, 300ng / mL, and 500ng / mL was added, with 4 replicates per group. After 24 hours, 10μL CCK8 detection solution was added to each well and placed in a constant temperature cell culture incubator for 2 hours. The 96-well plate was placed in an ELISA reader, and data was detected using a wavelength of 450nm to obtain the effect of Selinexor on the cell viability of multiple myeloma cells.

[0098] ②Inhibitory effect of Selinexor-loaded ethylene glycol chitosan targeted drug delivery system on U266 cells

[0099] U266 cells were cultured in T25 flasks and counted using a cell counting plate at 8 × 10 5 5 mL of cells were cultured at a cell concentration of 100 / mL. After 48 hours, 5 mL of new culture medium was slowly added to the bottle to continue culturing. After the cells were fully grown, the cells were transferred to a 15 mL centrifuge tube and centrifuged at 800 rpm for 3 minutes. After centrifugation, the supernatant was removed and 1 mL of culture medium was added for cell counting. 5,000 cells were seeded in a 96-well plate per well, and 100 μL of Selinexor, GCNP-Selinexor, GCNP@FA&PPI-Selinexor, and GCNP@CD38&PPI-Selinexor were added. There were 4 replicates in each group. After 48 hours, 10 μL of CCK8 detection solution was added to each well and placed in a constant temperature cell culture incubator for 2 hours. The 96-well plate was placed in an ELISA reader and data was detected using a wavelength of 450 nm.

[0100] 2) Cell targeted enrichment experiment

[0101] Cell culture: Human multiple myeloma cell lines (L363 and U266) purchased from ATCC were cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin. The cells were set at 37°C, CO 2 The concentration was 5% in an incubator.

[0102] 3) In vitro dual-targeted GCNPs targeted enrichment experiment:

[0103] L363 cells and U266 cells were cultured at 5 × 10 4Cells were seeded in 12-well plates for subsequent experiments. In order to fluorescently label the targeted nanoparticles, the fluorescent molecule Cy5.5 was added to GCNP and GCNP@FA&PPI at a concentration of 10 μg / mL to prepare 5 mL of Cy5.5 (10 μg / mL) labeled GCNP (0.5 mg / mL) and 3 mL of Cy5.5 (10 μg / mL) labeled GCNP@FA&PPI (0.5 mg / mL), respectively. The prepared fluorescently labeled nanoparticles were used to evaluate the targeted cumulative effect of targeted nanoparticles in multiple myeloma cells. The targeted nanoparticles and multiple myeloma cells were incubated overnight at a constant temperature of 37°C in the dark and cultured in 12-well plates for 24 hours. Finally, the cells were collected in a centrifuge tube, centrifuged at 800 rpm for 3 minutes, resuspended in 1 mL of PBS, and gently washed twice. After that, the cells were fixed with 500 μL of paraformaldehyde for 30 minutes, centrifuged and washed twice with PBS, and then resuspended with 500 μL of 0.1% Triton X-100 (5 μL + 5 mL PBS) for 5 minutes, and 500 μL of 1% bovine serum albumin (0.05 g), 5 μL of cytoskeleton staining solution and 5 mL of PBS were added after centrifugation, and each tube was incubated for 1 hour. After washing once with PBS, the cells were gently resuspended with DAPI staining solution (the volume of the staining solution was 3 times the volume of the sample), and after staining for 5 minutes, the extent of enrichment of targeted chitosan nanoparticles in L363 and U266 cells was observed using a confocal microscope (LSM900, Carl Zeiss Microscope Co., Ltd., Germany).

[0104] 4) In vivo targeting verification of ethylene glycol chitosan targeted nanoparticles

[0105] ① Fluorescence labeling of glycol chitosan targeted nanoparticles:

[0106] Preparation of fluorescently labeled glycol chitosan targeting nanoparticles GCNP-Cy5.5: Add 2.5 μL of cy5.5 (20 mg / mL) to 1 mL of GCNP (10 mg / mL), stir at room temperature and protect from light overnight to form GCNP loaded with Cy5.5 fluorescent molecules, and store at 4°C.

[0107] Preparation of fluorescently labeled dual-targeted glycol chitosan targeted nanoparticles GCNP@FA&PPI-Cy5.5: Add 2.5 μL of cy5.5 (20 mg / mL) to 1 mL of GCNP@FA&PPI (10 mg / mL), stir at room temperature and protect from light overnight to form GCNP@FA&PPI loaded with Cy5.5 fluorescent molecules, and store at 4°C.

[0108] Preparation of fluorescently labeled dual-targeted glycol chitosan targeting nanoparticles GCNP@CD38&PPI-Cy5.5: Add 2.5 μL of cy5.5 (20 mg / mL) to 1 mL of GCNP@CD38&PPI (10 mg / mL), stir at room temperature and protect from light overnight to form GCNP@CD38&PPI loaded with Cy5.5 fluorescent molecules, and store at 4°C.

[0109] Cy5.5 solution control: Add 2.5 μL of cy5.5 (20 mg / mL) to 1 mL of normal saline and store at 4°C away from light.

[0110] ② Evaluation of MM targeting and nanoparticle targeted distribution in vivo:

[0111] Fifteen 5-week-old male BALB / c nude mice were purchased, divided into 5 groups of 3 mice each. Three mice were selected as the blank control group, and 15 μL of normal saline was injected into the right tibial bone marrow cavity as the normal control group. The remaining mice were transplanted with 15 μL of U266 cells through the right tibial bone marrow cavity. ╳ 10 6 Cancer cells were detected in the right tibia of the blank group and the MM model group. After 2 weeks, the bone dissolution of the right tibia was observed by X-ray to establish the multiple myeloma model. The 12 mice were randomly divided into 4 groups, 3 mice in each group, and 50 μL of Cy5.5, fluorescently labeled GCNPI-Cy5.5, GCNP@FA&PPI-Cy5.5, and GCNP@CD38&PPI-Cy5.5 nanoparticles were injected into the tail vein. The nude mice were killed 7 days later, and the femur, tibia and major organs (femur, tibia, heart, liver, spleen, lung, and kidney) were taken for fluorescence imaging using a small animal fluorescence imaging system (BLT AniView 100; Guangzhou Bolu Teng Biotechnology Co., Ltd., China).

[0112] 2. Experimental results

[0113] 1) Inhibitory effect of chitosan targeted drug delivery system loaded with Selinexor on U266 cells

[0114] The results of CCK-8 cell viability analysis of U266 MM cells using different doses of selinexor showed that 100-500 ng / mL selinexor had a significant inhibitory effect on MM cells. Figure 6 As shown in Figure B, multiple concentrations of Selinexor have a good inhibitory effect on multiple myeloma cells. Compared with other drug concentrations, 200ng / mL of selinexor produced the most significant inhibitory effect (P<0.01). Subsequently, this example loaded this dose of selinexor (200ng) into dual-targeted GCNPs and evaluated its inhibitory effect on U266 cells. Figure 6 As shown in Figure C, Selinexor and chitosan targeted drug-loaded nanoparticles loaded with Selinexor can effectively inhibit the activity of U266 cells (P<0.05), confirming that the targeted drug delivery system will not affect the therapeutic effect of selinexor, among which the GCNP@CD38&PPI-selinexor group caused the most obvious decrease in U266 cell activity.

[0115] 2) Evaluation of the targeting enrichment ability of ethylene glycol chitosan targeted drug delivery system on MM cells

[0116] In order to determine whether dual-targeted GCNPs tend to target MM cells, this example evaluates the targeting ability of GCNP@FA&PPi and GCNP@CD38&PPi nanoparticles. The dual-targeted glycol chitosan drug delivery system GCNPs were labeled with fluorescent dye Cy5.5 to evaluate its targeting on two MM cell lines (L363 and U266).

[0117] The results of targeted aggregation of ethylene glycol-targeted chitosan nanoparticles in U266 cells are as follows Figure 6 As shown, MM cells were pretreated with 1 μg / mL LPS for 48 hours to simulate inflammatory stimulation in the tumor microenvironment. The red fluorescence is the fluorescence of Cy5.5 or the fluorescence of targeted chitosan nanoparticles labeled with it. Compared with the Cy5.5 group, the amount of red fluorescence in the GCNP-Cy5.5, GCNP@FA&PPI-Cy5.5, and GCNP@CD38&PPI-Cy5.5 groups was greater, that is, the amount of nanoparticles targeted and accumulated in U266 cells was significantly greater. Compared with GCNP-Cy5.5, the amount of nanoparticles targeted and accumulated in U266 cells in the GCNP@FA&PPI-Cy5.5 and GCNP@CD38&PPI-Cy5.5 groups was significantly greater than that in the non-targeted GCNP-Cy5.5, and the targeting effect of nanoparticles in the GCNP@CD38&PPI-Cy5.5 group was more obvious ( Figure 6 D&F), which indicates that targeted nanoparticles are more likely to be enriched in targeted MM cancer cells. Similar results were also observed in another multiple myeloma cell line LC363 ( Figure 6 E&G), confirming that FA&PPi and CD38&PPi targeted modification increased MM cell uptake. High-resolution confocal images showed that the amount of nanoparticles in the GCNP@FA&PPI-Cy5.5 and GCNP@CD38&PPI-Cy5.5 groups that were endocytosed by LC363 cells was significantly greater than that of the non-targeted GCNP-Cy5.5 ( Figure 6 Middle H).

[0118] The targeting results of targeted chitosan nanoparticles in MM nude mice are as follows Figure 7As shown in the figure, compared with the Cy5.5 group, the cumulative fluorescence intensity of the tibial bone marrow cavity lesions in the GCNP-Cy5.5, GCNP@FA&PPI-Cy5.5 and GCNP@CD38&PPI-Cy5.5 groups was enhanced, indicating that the nanoparticles can be enriched in the lesions. Compared with the GCNP-Cy5.5 group, the fluorescence intensity of the bone marrow cavity lesions in the GCNP@FA&PPI-Cy5.5 and GCNP@CD38&PPI-Cy5.5 groups was stronger, indicating that the targeted modified nanoparticles can more effectively target the bone marrow lesions and increase the concentration of the drug in the lesions. Compared with the GCNP@FA&PPI-Cy5.5 group, the fluorescence intensity of the bone marrow cavity lesions in the GCNP@CD38&PPI-Cy5.5 group was not much different, but the cumulative fluorescence intensity in the spleen of the GCNP@CD38&PPI-Cy5.5 group was significantly enhanced.

[0119] Example 4 Evaluation of the therapeutic effect of the targeted glycol chitosan drug loaded with Selinexor

[0120] 1. Cell experiment

[0121] 1) Drug concentration screening

[0122] U266 cells were seeded in 96-well plates at 5,000 cells per well, and Selinexor was added at concentrations of 100 ng / mL, 200 ng / mL, 300 ng / mL, and 500 ng / mL (4 replicate wells per group). After 24 hours, 10 μL of CCK8 detection reagent was added to each well, and the absorbance of each well was measured at a wavelength of 450 nm after 2 hours.

[0123] 2) Inhibitory effect on U266 cells

[0124] U266 cells were seeded in 96-well plates at 5000 cells per well, and 100 μL of Selinexor, GCNP-Selinexor, GCNP@FA&PPI-Selinexor, and GCNP@CD38&PPI-Selinexor (Selinexor was 200 ng / mL) were added, with 4 replicate wells in each group. After 24 hours, 10 μL of CCK8 cell viability detection reagent (Biyuntian, China) was added to each well, and the absorbance of each well was measured at 450 nm using a microplate reader (BioTek, USA) 2 hours later.

[0125] 2. Animal experiments

[0126] Seventy-two 5-week-old male BALB / c nude mice (18-20 g) were purchased from Spencer (Beijing) Biotechnology Co., Ltd. (China). Two independent animal experiments were conducted, and each experiment was divided into 6 groups (n = 6 in each group), including control group (sham operation and tail vein injection of 0.9% saline), multiple myeloma (MM) model group (tail vein injection of 0.9% saline), MM model group gavage alone Selinexor, MM model group tail vein injection of GCNP-Selinexor group, MM model group tail vein injection of GCNP@FA&PPI-Selinexor group, MM model group tail vein injection of GCNP@CD38&PPI-Selinexor group.

[0127] Construction of MM model in animal experiment I: 15 μL of U266 cells were injected into the bone marrow cavity of the right tibia of mice. The control group underwent sham surgery and injected with 15 μL of normal saline.

[0128] Construction of MM model in animal experiment II: 7.5 μL of U266 cells were injected into the bone marrow cavity and the extraosseous cavity of the right tibia of mice (cells were suspended in 1×10 ^6 All animals were fed a standard diet and had free access to water, with a 12-h light-dark cycle and a temperature of 25°C ± 1°C.

[0129] Mice in animal experiment I were treated with dual-targeted GCNPs starting 1 week after MM cell implantation and were given once a week for 8 weeks. Mice in animal experiment II were treated starting 2 weeks after MM cell implantation and were given once a week for 3 weeks.

[0130] 1) In vivo cytotoxicity studies:

[0131] Animal Experiments The body weight of mice was measured weekly after the start of drug treatment. After the mice were killed at the end of the experiment, the liver and spleen were weighed, and serum was collected to analyze the levels of IL-6, Igκ, and Igλ in the serum using Elisa kits.

[0132] 2) X-ray imaging, bone density analysis and Micro-CT analysis:

[0133] Before the model was established and before the entire animal experiment, each group of mice was scanned by X-ray imaging. Nude mice were anesthetized with isoflurane, and after the mice entered the anesthetized state, they were placed in an X-ray irradiator (Faxitron, USA), and the X-rays were turned on and the mice were scanned. After the scan, the bone density data of the mice obtained by the X-ray imaging system were collected. After sacrifice, the femur and tibia of the mice were collected, the hind limb muscles were removed, and the knee joints were retained. The distal tibia specimens were fixed vertically along the long axis, keeping the epiphyseal direction of the specimens consistent, and scanned using the Skyscan micro-CT system (Skyscan1172, Bruker, Belgium). The selected scanning parameters were as follows: high resolution was selected, the image matrix was 2048×2048, the integration time was 200ms, the resolution was 10.4μm, the energy / intensity was 55kVp, 145μA, 8W, and the CT value was corrected to 1200mg HA / cm and 0° rotation. After scanning, the region of interest (ROI) of the tibia was analyzed, which was located 2 mm below the tibial growth plate, and the image information was segmented with a threshold of 100 to 255. The three-dimensional image reconstruction and quantitative analysis of bone tissue were performed using Skycan, the software provided by Micro-CT.

[0134] 3) Histology and immunofluorescence analysis:

[0135] Histological staining: The tibia tissue was decalcified with 12% EDTA, embedded in paraffin and sliced. 5 μm sections were dewaxed and hydrated with graded alcohol, stained with hematoxylin-eosin (Beijing Solebow Technology Co., Ltd., China), dehydrated with graded alcohol and sealed with neutral gum. The pathological changes of tibia bone tissue were observed and recorded by optical microscopy.

[0136] Immunofluorescence staining: After dewaxing, hydration, antigen retrieval and blocking, the tibia tissue sections were incubated with primary antibodies (anti-CD38 antibody (Abcam, ab235118, 1:100, USA), anti-CD138 antibody (Abcam, ab130405, 1:100, USA) and incubated overnight at 4°C. The next day, the sections were incubated with secondary antibodies (Alexa Fluor 488-conjugated antibody (Cell Signaling, 4412S, 1:200, USA) and Alexa Fluor 555-conjugated antibody (Cell Signaling, 4409S, 1:200, USA) for 1 hour, and then covered with anti-fluorescence quenching medium containing DAPI (Yeasen Biotech, 36308ES11, China). Immunofluorescence images were collected by confocal microscope system (LSM900, Carl Zeiss Mieroscopy Gmbh, Germany).

[0137] 3. Experimental results

[0138] 1) Effects of Selinexor-loaded targeted chitosan drug delivery system on spleen, liver and body weight in MM nude mice

[0139] Depend on Figure 8 As shown in Figure A, there was no significant change in body weight among the Control group, MM group and each treatment group.

[0140] Depend on Figure 8 As shown in Figures B&C, compared with the Control group, the liver and spleen weight ratios of the MM model group increased significantly. Compared with the MM group, the spleen weight ratios of the GCNP-Selinexor, GCNP@CD38&PPI-Selinexor, and GCNP@FA&PPI-Selinexor groups decreased significantly. Compared with the Selinexor group, there was no significant change in the spleen weight ratio and liver weight ratio of each nanoparticle group.

[0141] 2) Effects of Selinexor-loaded targeted chitosan drug delivery system on tibia bone marrow, liver, spleen, and stomach tissues in MM nude mice

[0142] like Figure 8 As shown in D, compared with the Control group, the number of bone marrow cavity cells in the MM group was significantly increased, and the cells grew in uneven aggregates, indicating that the model was successfully established. Compared with the MM group, there was no significant change in the bone marrow cavity cells in the Selinexor group and the GCNP-Selinexor group, and the cells grew in uneven aggregates; the number of bone marrow cavity cells in the GCNP@FA&PPI-Selinexor group and the GCNP@CD38&PPI-Selinexor group was significantly reduced, and the cells were evenly distributed.

[0143] Histological analysis of spleen histological images showed that compared with the healthy control, Selinexor and GCNP-Selinexor groups, MM progression caused a significant decrease in the MM spleen white pulp area, while GCNP@CD38&PPi and GCNP@FA&PPi treatment increased the spleen white pulp area compared with the MM model group. In the pathological analysis, the liver pathological sections of the MM group showed that the liver of the MM group had a certain enlargement and hepatocyte vacuolation, while none of the drug-treated groups showed obvious pathological changes in liver damage.

[0144] In the pathological section analysis, the gastric tissue in the Selinexor group became sparse and the surface mucosa was destroyed, while no obvious pathological changes were found in the gastric tissues of the other groups ( Figure 8(D). However, spleen immunofluorescence analysis showed that the expression of CD38 and CD138 in the spleen of MM mice was significantly increased. Selinexor and dual-targeted GCNPs treatment effectively inhibited the expression of CD38 and CD138, among which the inhibitory effect of the GCNP@CD38&PPI-Selinexor treatment group was the most obvious ( Figure 8 Middle E).

[0145] 3) Effects of Selinexor-loaded PEG-chitosan drug delivery system on Micro-CT quantitative parameters of tibia in MM nude mice

[0146] Fig. 9 Figure A shows the experimental plan of animal experiment I. Nine weeks after MM cell implantation, significant trabecular loss was observed in the bone marrow cavity of all MM mice. After drug treatment, quantitative μ-CT analysis showed that the trabecular number (Tb.N) and bone volume ratio (BV / TV) in the MM group were significantly reduced, and trabecular separation (Tb.Sp) was increased compared with the control group. These findings confirmed the successful establishment of the MM model and emphasized the severe deterioration of bone quality caused by MM cells ( Fig. 9 BF). It is worth noting that no significant improvement was observed in any CT parameters related to cancellous osteolysis in the selinexor-treated group compared with the MM group, indicating that oral selinexor is not effective in alleviating bone erosion in MM. Tb.Sp decreased significantly in the GCNP-Selinexor-treated group, but there were no significant changes in BV / TV, Tb.N and Tb.Th. This suggests that simply formulating selinexor into drug-loaded nanoparticles does not significantly alleviate osteolysis in the MM mouse model. In contrast, early intervention with GCNP@FA&PPI-Selinexor and GCNP@CD38&PPI-Selinexor significantly reduced MM cell aggregation and proliferation in the bone marrow, thereby significantly improving cancellous osteolysis in the tibia ( Fig. 9 BF). Compared with the MM group, the BV / TV of both the GCNP@FA&PPI-Selinexor and GCNP@CD38&PPI-Selinexor groups increased significantly. In addition, GCNP@CD38&PPI-Selinexor treatment resulted in an increase in Tb.Th compared with the MM group. Compared with the selinexor group, the BV / TV of both the GCNP@FA&PPI-Selinexor and GCNP@CD38&PPI-Selinexor groups increased significantly ( Fig. 9(Center CF). Micro-CT results showed that dual-targeted GCNPs treatment significantly alleviated bone degradation and increased trabecular bone mass. Specifically, GCNP@CD38&PPI-Selinexor treatment increased bone mass while further improving trabecular bone microarchitecture, providing a more comprehensive therapeutic benefit in combating MM-induced bone degradation.

[0147] 4) Effects of Selinexor-loaded PEG-chitosan drug delivery system on the expression of CD38 and CD138 in the tibia of MM nude mice and the concentrations of IL-6 and Igκ in serum

[0148] like Fig.10 The results showed that the expression levels of CD138 and CD38 in the bone marrow of MM mice were significantly higher than those in the normal group, indicating significant disease progression and cell aggregation. Treatment with GCNP-Selinexor, GCNP@FA&PPI-Selinexor, and GCNP@CD38&PPI-Selinexor significantly reduced the expression of CD138 and CD38 in the bone marrow of MM mice ( Fig.10 Serum analysis showed that the levels of interleukin 6 (IL-6) and immunoglobulin light chain (Igκ) in MM model mice were significantly increased ( Fig.10 C&D). Both GCNP@FA&PPI-Selinexor and GCNP@CD38&PPI-Selinexor treatments significantly reduced IL-6 and Igκ concentrations in the serum of MM mice compared with the MM group. In addition, IL-6 and Igκ levels were significantly lower in the GCNP@FA&PPI-Selinexor and GCNP@CD38&PPI-Selinexor groups compared with the Selinexor group. These findings demonstrate the high efficiency of these dual-targeted nanoparticles in inhibiting MM progression in mice. Based on these in vivo results, it was confirmed that dual-targeted GCNPs can effectively inhibit MM progression and alleviate trabecular bone deterioration through early drug intervention.

[0149] 5) Selinexor-loaded dual-targeted GCNPs treatment reduces bone loss and bone microarchitecture deterioration in mice with advanced extramedullary MM

[0150] To evaluate the therapeutic effects of these dual-targeted GCNPs in more advanced MM models, animal experiment II was performed in this example, in which MM cells were injected into the tibial bone cavity and surrounding tissue. This approach enables the study of the effects of MM cells on bone marrow, cortical bone, and adjacent muscle tissue. U266 MM cells were introduced into the tibial cavity and surrounding skeletal muscle areas to create an advanced extramedullary MM mouse model. The experimental protocol is shown in Fig.11Middle A. Two weeks after MM cell implantation. The resulting extramedullary MM model showed a clear expansion of sarcomatoid tumors in the tibia, unlike earlier MM models. This suggests that a more complex MM model has been established in which cancer cell proliferation resembles solid tumors in the tibia. Dual-targeted GCNP drug treatment was initiated two weeks after MM cell injection and continued for three weeks ( Fig.11 Middle A). Serum ELISA data showed that the level of immunoglobulin light chain (Igλ) in MM model mice was significantly increased ( Fig.11 (B). Both GCNP@FA&PPI-Selinexor and GCNP@CD38&PPI-Selinexor treatments resulted in a significant decrease in Igλ concentrations compared with the MM group. In addition, IL-6 and Igλ levels were significantly reduced in the GCNP@FA&PPI-Selinexor and GCNP@CD38&PPI-Selinexor groups compared with the Selinexor group. These findings demonstrate the high efficiency of these dual-targeted nanoparticles in inhibiting MM progression in mice. X-ray and μ-CT imaging revealed severe bone erosion and damage not only in the metaphyseal cancellous bone but also in the cortical bone ( Fig.11 C&D). Micro-CT analysis showed a significant decrease in BV / TV and Th.N, while an increase in Th.Sp compared with healthy controls. In this late-stage model, MM mice exhibited significant bone erosion, with more severe erosion of cortical and trabecular bones than in early-stage MM models. Osteolytic lesions, a hallmark of MM, are common and are the main cause of bone pain and fractures in many patients. Animal Experiment II performed BMD analysis to monitor the progression of the model and was analyzed at the study endpoint after drug treatment. BMD was significantly decreased in mice with late-stage extramedullary MM ( Fig.11Middle E). Both GCNP@FA&PPI-Selinexor and GCNP@CD38&PPI-Selinexor treatment significantly improved BMD compared with the MM group. In addition, these dual-targeted GCNPs showed a significant increase in BMD compared with the Selinexor group. Although the trabecular number (Tb.N) increased in the Selinexor group, there was no significant change in trabecular separation (Tb.Sp), BV / TV, or trabecular thickness (Tb.Th) compared with the MM group. This suggests that standard oral Selinexor treatment alone is not sufficient to alleviate bone erosion and deterioration in the extramedullary MM model. Compared with the MM group, GCNP-Selinexor treatment significantly increased Tb.N and decreased Tb.Sp, but there was still no significant change in BV / TV or Tb.Th. Both Selinexor and GCNP-Selinexor treatment showed only slight improvements in resolving bone deterioration. In contrast, both GCNP@FA&PPI-Selinexor and GCNP@CD38&PPI-Selinexor treatments significantly increased Tb.N and BV / TV, while significantly decreasing Tb.Sp, compared with the MM group. In addition, BV / TV was significantly higher in the GCNP@FA&PPI-Selinexor and GCNP@CD38&PPI-Selinexor groups compared with the Selinexor group, indicating that dual-targeted GCNPs significantly enhanced the therapeutic efficacy of Selinexor and mitigated bone deterioration in an advanced extramedullary MM model ( Fig.11 (CI).

[0151] In summary, the present invention provides a delivery system targeting multiple myeloma, which has different targeting molecules for bone targeting and cancer cell targeting, and can target multiple myeloma cells in the bone marrow microenvironment based on the dual targeting of cells and microenvironment. Furthermore, the delivery system is equipped with Selinexor to enhance the selective delivery of Selinexor to myeloma cells and affected bone tissue. In vivo studies have shown that the delivery system significantly improves the therapeutic effect of MM. It effectively inhibits the malignant proliferation of myeloma cells, increases bone density, improves bone microstructure, and reduces bone deterioration in MM mouse models. This method of dual-targeted nanoparticle delivery of anti-MM drugs represents an innovative strategy that combines MM cancer cell targeting and bone targeting of the cancer microenvironment, which has never been used in MM treatment before.

Claims

1. A delivery system targeting multiple myeloma, characterized in that: The delivery system includes a nanoframe and a targeting molecule; The targeting molecules include bone targeting molecules and tumor targeting molecules; The bone targeting molecule includes pyrophosphate; The tumor targeting molecule includes at least one of folic acid and CD38.

2. The delivery system according to claim 1, characterized in that: The nano skeleton comprises ethylene glycol chitosan nanoparticles.

3. The delivery system according to claim 1, characterized in that: The pyrophosphate includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic base, a salt formed with an organic base, and a salt formed with a basic amino acid.

4. The delivery system according to claim 1, characterized in that: The delivery system also includes a drug; The drug includes at least one of Selinexor, belamaf, and Melflufen; Preferably, the drug is Selinexor.

5. The method for preparing the delivery system according to any one of claims 1 to 4, characterized in that: The following steps are involved: The ethylene glycol chitosan is reacted with 2-(diisopropylamino)ethyl methacrylate and ammonium persulfate to obtain ethylene glycol chitosan nanoparticles; The ethylene glycol chitosan nanoparticles are mixed with tumor targeting molecules, and a catalyst is added to react to obtain tumor targeting chitosan nanoparticles; The bone targeting molecule and the tumor targeting ethylene glycol chitosan nanoparticles are mixed, the pH is adjusted, and the mixture is reacted to obtain a dual-targeting ethylene glycol chitosan nanoparticle product.

6. The preparation method according to claim 5, characterized in that: In the steps: the ratio of ethylene glycol chitosan to 2-(diisopropylamino)ethyl methacrylate is (5-20): 1 g / L; The reaction was carried out in the dark.

7. The preparation method according to claim 5, characterized in that: In the steps: the mass ratio of the tumor targeting factor to the ethylene glycol chitosan nanoparticles is 1:(1-3); The catalysts include HATU and HOAT.

8. The preparation method according to claim 5, characterized in that: In the steps: the bone targeting factor is DA-PEG-PPI; The mass ratio of the DA-PEG-PPI to the tumor-targeting chitosan nanoparticles is 1:(1-3).

9. Use of the delivery system according to any one of claims 1 to 4 in the preparation of a product for treating multiple myeloma; the product comprises a medicine.

10. The use according to claim 9, characterized in that: The medicine includes pharmaceutically acceptable excipients.

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