CN103800915B - Associating carrier micelle of a kind of targeted integration element receptor and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of biomedicine, and relates to a combined administration micelle targeting integrin receptors and a preparation method thereof. The present invention synthesized the Pluronic-COOH modified by the terminal carboxyl group, and connected the RGD polypeptide with the carboxyl group through an amide bond to obtain the RGD peptide-modified Pluronic copolymer (RGD-Pluronic); the drug doxorubicin ( DOX) was copolymerized with Pluronic, and another drug paclitaxel (PTX) was entrapped by thin film hydration method to prepare mixed micelles (RGD-FP-DP). The invention can increase the drug loading capacity of the micelles, increase the drug concentration of the antineoplastic drug at the target site, reduce its accumulation at the non-target site, and effectively improve the therapeutic index of the drug.

Description

A combined drug-loaded micelle targeting integrin receptors and its preparation method

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a combined drug-loaded micelle targeting integrin receptors and a preparation method thereof.

Background technique

The prior art discloses that tumor tissue is different from normal tissue in that it has rich blood vessels, wide space between blood vessel walls, poor structural integrity, and lack of lymphatic drainage. Therefore, drug-loaded nanoparticles can be enriched in tumor tissue through the EPR effect. However, studies have found that after the nanoparticles enter the circulatory system, they are mainly taken up by the mononuclear phagocyte system (MPS), causing the drug to accumulate in the liver, spleen and lungs, which not only reduces the therapeutic index of the drug, but also causes damage to these organs. severe toxic side effects. Constructing a tumor-targeted drug delivery system, reducing the damage of anti-tumor drugs to normal organs, and realizing the targeted delivery of drugs have attracted more and more attention of researchers.

Integrin (Integrin) is one of the important members of the cell adhesion molecule family and the main receptor family of the extracellular matrix (ECM). Studies have shown that the expression of integrin αVβ3 is significantly up _- regulated in malignant tumors and endothelial cells stimulated and activated by pro-angiogenic growth factors secreted by tumor cells, while mature vascular endothelial cells and most normal tissues and organs in normal humans Integrin _αVβ3 was expressed at a low level. Studies have also shown that integrin _αVβ3 specifically recognizes and binds to the RGD (arginine-glycine-aspartic acid) sequence in the extracellular matrix ligand molecule and undergoes a conformational change to activate, mediate downstream signal transduction, and exert important regulatory role. Since all solid tumors or tumor cells are bound to be accompanied by tumor angiogenesis, integrin αVβ3, as a common substance in the process _of tumorigenesis and development, has certain specificity, so integrin _αVβ3 is a potential molecule for tumor therapy. target.

Polypeptides containing RGD sequences, such as linear RGD peptides, cyclic RGD peptides, and multimers of RGD peptides, have the advantages of small relative molecular weight, stability, easy preparation, and no immunogenicity. At present, some cyclic peptides containing RGD peptide sequences have entered clinical trials.

The practice of clinical tumor treatment shows that the combined administration of two or more drugs can reduce the total drug dosage, produce complementary effects and multiple effects, and is one of the effective strategies to improve drug efficacy and reduce drug toxicity. Drug systems with different properties can achieve ideal synergistic effects with the least amount of drugs in tumor treatment. The application of polymer materials to prepare micellar carriers for the delivery of anti-tumor drugs has become a hot spot in the research of drug delivery systems in recent years, but there is still the defect of low drug loading in micellar systems, and combined drug loading can make up for the drug loading of micelles Low defects, while exerting their respective effects and synergistic effects. Therefore, active targeting combined drug-loaded micelles have potential advantages in the construction of tumor-targeted drug delivery systems.

Contents of the invention

The purpose of the present invention is to overcome the defects of the prior art, and provide a combined drug-loaded micelle targeting integrin receptors and a preparation method thereof.

The present invention synthesized the Pluronic-COOH modified by the terminal carboxyl group, and connected the RGD polypeptide with the carboxyl group through an amide bond to obtain the RGD peptide-modified Pluronic copolymer (RGD-Pluronic); the drug doxorubicin ( DOX) was copolymerized with Pluronic, and another drug paclitaxel (PTX) was entrapped by thin film hydration method to prepare mixed micelles (RGD-FP-DP).

more specific,

In the present invention, an amphiphilic copolymer with an active carboxyl group and a preparation method thereof are provided.

In the present invention, an amphiphilic copolymer modified with an integrin receptor-targeted RGD peptide and a preparation method thereof are provided.

In the present invention, an amphiphilic copolymer with drug copolymerization and a preparation method thereof are provided.

In the present invention, micelles targeting integrin receptors and their preparation methods and applications are provided.

In the present invention, the above-mentioned micelle carrier targeting integrin receptors is applied to simultaneously load two or more hydrophilic and hydrophobic anti-tumor drugs, and a combined drug-loaded micelle with active targeting is obtained .

The object of the present invention is achieved through the following technical solutions:

An amphiphilic copolymer (Pluronic-COOH) with an active carboxyl group, an amphiphilic copolymer Pluronic-DOX of a polymer drug doxorubicin hydrochloride and a preparation method thereof, the synthesis method is as follows:

Among them, Pluronic commonly used models are PluronicL35, L43, L44, L61, L62, L64, F68, L81, P84, P85, F87, F88, L92, F98, L101, P103, P104, P105, F108, L121, P123 and F127; Drugs are usually water-soluble antineoplastic drugs.

In the present invention, the RGD peptide-modified amphiphilic copolymer with integrin receptor targeting has a general formula: RGD-X-Pluronic, wherein RGD-X is a linear RGD containing Arg-Gly-Asp sequence Peptides, cyclic RGD peptides, and multimers of RGD peptides, prepared by:

The above-mentioned amphiphilic integrin receptor-targeted RGD peptide-modified copolymer RGD-X-Pluronic, the common amphiphilic carrier Pluronic and Pluronic-DOX are mixed in a certain proportion, and co-dissolved in an organic solvent, Targeting micelles were prepared by thin film-hydration method. In the embodiments of the present invention, in the targeted micelles, the proportions of the preferred three components are as follows: RGD-X-Pluronic is 1%-20%, Pluronic is 89%-20%, Pluronic-DOX 10%-60%.

Hydrophobic antitumor drugs (such as paclitaxel (PTX)) and RGD-X-Pluronic, Pluronic, Pluronic-DOX are co-dissolved in organic solvents, prepared according to the film hydration method, and two antitumor drugs are loaded Drug-targeted micelles (such as PTX, DOX co-administered targeted micelles); the drug loading of selected hydrophobic anti-tumor drugs is 0.1%-20%.

In the embodiments of the present invention, the preparation method of micelles targeting integrin receptors loaded with drugs is preferred, which includes the steps of:

(1) Get each component by the following mass percentages: hydrophobic antitumor drug 0.1%-20%; RGD-X-Pluronic, Pluronic and Pluronic-DOX mixture 80-99.9%, RGD-X-Pluronic in this mixture is 1%-20%, Pluronic 89%-20%, Pluronic-DOX 10%-60%;

(2) Dissolve the above-mentioned components with an organic solvent, preferably dichloromethane or chloroform, ultrasonically dissolve the carrier material and the drug, remove the organic solvent by rotary evaporation, preferably at a temperature of 37 ° C, and vacuum dry overnight at room temperature , add deionized water, hydrate, preferably at a constant speed of 700rpm, 37°C water bath, stir for 30min, cool to room temperature, filter through a 0.22μm cellulose acetate membrane to obtain a drug-loaded targeted micelle solution, and lyophilize Save it later. The particle size measurement showed that the prepared micelles had a particle size of 10-400 nm, as shown in FIG. 3 .

In the present invention, the antitumor drug paclitaxel (PTX) has excellent antitumor efficacy, but its solubility is less than 1 μg·ml ^-1 , and its oral bioavailability is poor. Moreover, the existing paclitaxel injection taxol and PTX albumin conjugate All have different side effects; and for DOX is a cycle of non-specific anti-cancer chemotherapy drugs, clinically mainly used for the treatment of acute lymphoblastic leukemia, acute myelogenous leukemia, breast cancer, lung cancer, soft tissue sarcoma, liver cancer, etc., but in Serious adverse reactions often occur during chemotherapy; DOX has a low molecular weight and is easy to diffuse in the body, resulting in relatively even tissue distribution, causing toxic and side effects on normal tissues and affecting the anti-tumor effect; at the same time, according to clinical studies, DOX and PTX Combined administration has better anti-tumor effect than single administration, especially as a first-line treatment drug for breast cancer; RGD-X-Pluronic, a copolymer modified with amphiphilic RGD peptide, has a hydrophobic chain and can self-assemble to form a core - Shell-type nanocomposites, the hydrophobic core can be used as a miniature reservoir for fat-soluble drugs; the hydroxyl groups at both ends of Pluronic can be oxidized to carboxyl groups to modify hydrophilic drugs with amino groups, etc., the present invention modifies RGD peptides The block copolymer RGD-X-Pluronic was introduced into the combined drug delivery system containing PTX and DOX to prepare targeted micelles. It has been proved by experiments that the co-administered micellar system can effectively increase the concentration of the drug at the target site and reduce its toxic and side effects in non-target sites such as the liver and spleen, and can significantly improve the therapeutic index of the drug.

In the present invention, using drug-resistant KBv cells as a model, the MTT method was used to investigate the growth inhibition of drug-resistant tumor cells by RGD-FP-DP, common micelles and free drugs. The inhibitory rate of tumor cells was significantly stronger than that of ordinary micelles and free drugs (as shown in Figure 4).

In the present invention, the red fluorescence of DOX is used as a probe to investigate the uptake of targeted micelles and ordinary micelles in drug-resistant KBv cells. The results show that the fluorescence intensity of drug-resistant cells in the targeted micelles group is significantly higher than that of Ordinary micelle group (as shown in Figure 5).

In the present invention, use the AnnexinV-APC/7-AAD apoptosis detection kit to detect the apoptosis caused by RGD-FP-DP, ordinary micelles and the mixed drug (Taxol+DOX) of Taxol and DOX, the results show that, The early apoptosis and late apoptosis of KBv cells induced by the RGD-FP-DP group were significantly stronger than those in the ordinary micelles and Taxol+DOX groups (as shown in Figure 6).

In the present invention, nude mice bearing KBv subcutaneous tumors were used as animal models, and fluorescent probe DiR-labeled targeting micelles and ordinary micelles were administered intravenously, and the distribution of nanocomposites in vivo was observed with a CRi live imager The results showed that the accumulation of targeted micelles in the tumor site was significantly higher than that of ordinary micelles, and the uptake of targeted micelles in the liver and spleen was significantly reduced (as shown in Figure 7).

In the present invention, nude mice bearing KBv subcutaneous tumors are used as animal models, and RGD-FP-DP, ordinary micelles and free drugs are administered intravenously, and pharmacodynamic evaluation in vivo is performed. The results showed that the drug effect of RGD-FP-DP on KBv-resistant tumors was significantly better than that of the other groups (as shown in Figure 8), and the dosage of the doxorubicin group was close to the maximum safe concentration of doxorubicin. Although the tumors were minimal, the mice lost a lot of body weight, indicating a high degree of toxicity.

The advantages of the combined drug-loaded micelles of the present invention are: it can increase the drug-loading capacity of the micelles, increase the drug concentration of anti-tumor drugs at target sites and reduce their accumulation at non-target sites, and can effectively improve the therapeutic index of drugs.

Description of drawings

Fig. 1 is the ¹ H-NMR spectrum of Pluronic, Pluronic-NHS and Pluronic-DOX.

Figure 2 is the full-wavelength UV scanning spectrum of DOX, PluronicP105-COOH and PluronicP105-DOX.

Figure 3 is the particle size distribution diagram (A) of RGD-FP-DP; transmission electron microscope (B) and atomic force microscope photos (C).

Figure 4 is the inhibition curve of RGD-FP-DP and ordinary micelles on the growth of KBv tumor cells.

Figure 5 is a comparison of the uptake of RGD-FP-DP and common micelles in KBv cells.

Figure 6 shows the apoptosis caused by the AnnexinV-APC/7-AAD cell apoptosis detection kit in the control group, the mixed drug of Taxol and DOX, ordinary micelles and RGD-FP-DP.

Figure 7 shows the distribution of fluorescent probe DiR-labeled targeting micelles and normal micelles in nude mice bearing KBv subcutaneous tumors.

Figure 8 is the photos of the tumors removed from the KBv tumor-bearing mice in each administration group after being sacrificed: normal saline (A), DOX (B), Taxol (C), Taxol+DOX (D), PF-DP (E) and RGD -FP-DP(F).

detailed description

The present invention will be described in detail below in conjunction with the examples. These examples are completely illustrative, and they are only used to describe the present invention in detail. The protection scope of the present invention is not limited to the following examples.

Example 1 Preparation of amphiphilic block copolymer Pluronic-COOH with active group carboxyl

Put 0.01MPluronic and 400ml acetone in a round-bottomed flask, slowly heat up until the solution is clear, let it cool to room temperature, add 17ml Jones reagent, magnetically stir overnight at room temperature, add 5mL isopropanol to quench the reaction. Add 12.6g of activated carbon and continue to stir for 2h, heat to 40°C, suction filter while hot to obtain a clear solution, evaporate under reduced pressure to obtain a white viscous substance, add precooled n-hexane to precipitate a solid, and dry in vacuo to obtain carboxylated Pluronic ( Pluronic-COOH). The purity of the product was analyzed by gel permeation chromatography (GPC), and the molecular weight of the product was calculated by the peak area ratio of the proton in the methylene group in the ¹ H-NMR spectrum.

Embodiment 2 prepares Pluronic-DOX

Weigh 7.4 mM of Pluronic-COOH in the above Example 1, and dissolve it in 20 ml of N,N-dimethylformamide at room temperature. Add 1 g EDC and 2 g NHS to the solution of Pluronic-COOH. After reacting for 15 minutes, 2.8ml of 2-mercaptoethanol, 3.48g of DOX and 10mg of triethylamine were added, and stirring was continued at room temperature for 12 hours under nitrogen protection. After the reaction, dialyze with deionized water in the dark for 2 days (MWCO3500), and freeze-dry the concentrated solution to obtain Pluronic-DOX. The product was verified by ¹ H-NMR spectrum; the absorbance was measured by UV-Vis spectrophotometry, and the molar connection rate of DOX was calculated.

Embodiment 3 prepares the amphiphilic copolymer modified with RGD peptide

(1) Preparation of Pluronic-NHS by activating the carboxyl group with NHS:

Weigh 0.053mM of Pluronic-COOH, 22mg of DCC and 12.2mg of NHS in the above-mentioned Example 1 and dissolve them in 5ml of chloroform, and react at room temperature for 24h under the protection of nitrogen. After the reaction was completed, the solvent was removed under reduced pressure and precipitated with cold ether. The precipitate was vacuum-dried to constant weight to obtain Pluronic-NHS. The product is verified by 1H-NMR spectrum;

(2) Preparation of c(RGDyK)-Pluronic by cyclic RGD peptide (c(RGDyK)) modified amphiphilic copolymer;

Dissolve 0.0053mM of Pluronic-COOH in (1) above in 1ml of N,N-dimethylformamide to obtain solution A; dissolve 6.3mg (0.01mM) of c(RGDyK) peptide in 0.1M HEPES to obtain Solution B. Solution B was added dropwise to solution A, and the pH value was adjusted to 8.4 with N-methyl-morpholine. The reaction was carried out at room temperature for 24 hours. After the reaction was completed, the product was dialyzed in physiological saline (MWCO3500) for 48 hours to remove excess free c(RGDyK) peptide. After the dialysis was completed, the product c(RGDyK)-Pluronic was freeze-dried; the synthesized c(RGDyK) peptide-modified amphiphilic copolymer was verified by ¹ H-NMR spectrum.

Example 4

Put 3mgPTX, 100mgPluronic-DOX, 167mgPluronic and 30mgc(RGDyK)-Pluronic in a 50ml round bottom flask, add 5ml of dichloromethane to fully dissolve the carrier material and the drug, and evaporate the solvent to dryness by rotary evaporation at 37°C for 40min. Vacuum dry overnight at room temperature; add 5ml of deionized water, stir in a 37°C water bath at 700rpm for 30min at a constant speed, cool to room temperature, and filter with a 0.22μm cellulose acetate membrane to remove the unencapsulated drug to obtain RGD- FP-DP micelles, stored after lyophilization; the average particle size of RGD-FP-DP micelles is 28.33±3.61nm.

Example 5 Micellar Cell Uptake Experiment Targeting Integrin Receptors

The cultured KBv cells were digested with 0.25% trypsin, and 5×105 cells per well were seeded in a 24-well culture plate. Incubate for 24 hours at 37°C and 5% CO2; then discard the culture medium, RGD-FP-DP and ordinary micelles, and continue to incubate for 1 hour. After incubation, wash three times with pre-cooled PBS, and observe the micelles uptake by the cells under a fluorescence microscope; the results show that the fluorescence intensity of KBv cells that take up RGD-FP-DP is significantly higher than that of the ordinary micelles group.

Embodiment 6RGD-FP-DP and ordinary micelles are to KBv tumor cell growth inhibition experiment

Digest KBv cells with 0.25% trypsin, inoculate 5×10 ⁵ cells per well on a 96-well culture plate, and culture at 37°C and 5% CO ₂ for 24 hours; then discard the culture medium and add a certain concentration of paclitaxel solution (Taxol solution), DOX solution, Taxol+DOX, RGD-FP-DP and joint drug-loaded ordinary micelles (FP-DP), continue to incubate for 72h, after the end of the culture, add 5mg·ml ^-1 MTT to each well 20 μl solution, continue to culture at 37°C for 4 hours, stop the culture, discard the supernatant, add 200 μl DMSO to each well, shake for 10 minutes in the dark to fully dissolve the crystals, measure the OD value at 570nm with a microplate reader, and calculate the cell inhibition rate , the results showed that the inhibitory rate of RGD-FP-DP on KBv tumor cells was significantly stronger than that of ordinary micelles.

Example 7 Detection of apoptosis caused by RGD-FP-DP, FP-DP, Taxol, DOX and Taxol+DOX with AnnexinV-APC/7-AAD cell apoptosis detection kit

The KBv cells were digested with 0.25% trypsin, 5×10 ⁵ cells per well were inoculated on a 6-well culture plate, and cultured at 37°C and 5% CO ₂ for 24 hours; then the culture medium was discarded, and a certain concentration of each At the same time, the KBv cells added with blank 1640 culture medium were used as the negative control group, and incubated for 24 hours. After the incubation, the culture medium was collected, and the cells were washed by pre-cooled PBS centrifugation once, and an appropriate amount of 0.25% trypsin was added to digest the cells. Combine the culture medium collected above and the digested cell suspension, collect the cells by centrifugation, wash the cells 3 times with pre-cooled PBS and resuspend the cells in PBS for counting, refer to AnnexinV-APC/7-AAD Cell Apoptosis Detection Kit Method treatment, flow cytometry detection, the results showed that the early apoptosis and late apoptosis of KBv cells induced by the RGD-FP-DP group were significantly stronger than those of the ordinary micelles and the free drug groups.

Example 8

A suspension containing 5×10 ⁶ KBv cells was injected subcutaneously into the right hind limb of male balb/c nude mice. When the tumor grew to a diameter of about 7 mm, the fluorescent probe DiR-labeled targeting micelles and ordinary glue were administered through the tail vein. The distribution of nanocomplexes was observed on the CRI in vivo imager to investigate the active targeting of the targeted micelles at the tumor site. The results showed that the accumulation of the targeted micelles at the tumor site was significantly higher than that of the normal micelles. It shows that the uptake of targeted micelles in the liver and spleen is significantly reduced.

Example 9

Take a suspension containing 5×10 ⁶ KBv cells and inject it subcutaneously into the right hind limb of male balb/c nude mice. When the tumor grows to 50-100 mm ³ (denoted as day 0), the drug concentration is 10 mg·kg ^-1 , were given saline, DOX, Taxol, Taxol+DOX, FP-DP and RGD-FP-DP. On the 0th, 3rd, and 6th days, the mice were injected into the tail vein, and the mice were killed on the 10th day. The tumor was removed and weighed, and the tumor inhibition rate was calculated. The results showed that the KBv tumor inhibition rate of RGD-FP-DP was significantly higher than that of FP-DP and each free drug group produced good efficacy in treating drug-resistant tumors in vivo.

Claims (4)

1. the associating carrier micelle of a targeted integration element receptor, it is characterized in that, be made up of the component of following mass percent: hydrophobic antitumor drug 0.1%-20%, the RGD peptide with integrin receptor target modify amphipathic copolymer, be 80-99.9% with the block copolymer of medicine copolymerization and the mixture of common block copolymer, the amphipathic copolymer that in this mixture, RGD peptide is modified is 1%-20%, be 10%-60% with the block copolymer of medicine copolymerization, common block copolymer is 89%-20%; Be water solublity or hydrophobic drug with the medicine of copolymerization on the block copolymer of medicine copolymerization, be selected from amycin, doxorubicin hydrochloride, epirubicin, paclitaxel, Docetaxel, vincristine, vincristine sulfate, methotrexate or 9-nitrocamptothecin one or both or two or more; Associating medicine carrying mode is carried and copolymerization for wrapping.

2., by the associating carrier micelle of targeted integration element receptor according to claim 1, it is characterized in that, the described amphipathic nature block polymer with active group carboxyl, its molecular formula is Pluronic-COOH; Common block copolymer Pluronic, wherein, Pluronic represents various Pluronic model, comprises PluronicL35, L43, L44, L61, L62, L64, F68, L81, P84, P85, F87, F88, L92, F98, L101, P103, P104, P105, F108, L121, P123 and F127.

3. by the associating carrier micelle of targeted integration element receptor according to claim 1, it is characterized in that, its molecular formula of amphipathic copolymer that the described RGD peptide with integrin receptor target is modified is: RGD-X-PF-DP, wherein, RGD-X is the polymer of linear RGD peptide, ring-type RGD peptide and RGD peptide containing Arg-Gly-Asp sequence.

4. prepare a method for the associating carrier micelle of targeted integration according to claim 1 element receptor, it is characterized in that, it comprises: by described each component with organic solvent dissolution, adopts thin film/aquation legal system for targeting micelle.