CN107157928B - Matrix metalloproteinase responsive polymer drug carrier and preparation method and application thereof - Google Patents

Abstract

The present invention provides a polymer drug carrier having a structure represented by formula (I) and a preparation method and application thereof. The polymer drug carrier provided by the present invention is used to carry a drug, and the obtained polymer drug can be highly effective in tumor tissue. Under the action of the expressed matrix metalloproteinase (MMP), the endocytosis of the tumor drug for the drug is increased, the drug efficacy is improved, and the biological safety is high.

Description

A matrix metalloproteinase responsive polymer drug carrier and preparation method thereof application

technical field

The invention relates to the field of polymer medicines, in particular to a matrix metalloproteinase responsive polymer medicine carrier and a preparation method and application thereof.

Background technique

Amphiphilic block polymers can self-assemble into micelles in aqueous solutions and have been widely used to encapsulate hydrophobic anticancer drugs. Compared with small-molecule anticancer drugs, polymer drug-loaded micelles can improve the pharmacokinetics of drugs and enhance the enrichment of drugs in tumor sites, thereby significantly improving the bioavailability of drugs and reducing toxic and side effects. Biocompatible and degradable polymer micelles have achieved great success in drug delivery, and some have been approved for clinical applications or are undergoing clinical trials. For example, Genexol-PM (polyethylene glycol-b-poly-D, L-lactide) (PEG-PDLLA) polymer-loaded PTX formulations showed better efficacy than solvent-borne paclitaxel (PTX). and lower toxicity, it has been approved in Korea for the treatment of metastatic breast cancer and non-small cell lung cancer. In addition, there are other amphiphilic block polymer micelles in clinical trials, such as NK105, NK911, NK012, SP1049C, BIND014.

On the other hand, however, the long blood circulation and higher tumor enrichment of polymeric micelles (eg, PEG-coated surfaces) also hindered tumor cell endocytosis. This is a disadvantage for most anticancer drugs that need to be endocytosed by cells to work. To solve this problem, various responsive smart polymer drug delivery systems have been designed to further improve the efficacy, but so far the polymer drug delivery systems that have entered the clinical stage are still very rare. The complex structure and unknown systemic toxicity of the polymer itself limit its further clinical application. Therefore, providing a polymer drug with low toxicity, easy availability and good curative effect is a technical problem to be solved at present.

SUMMARY OF THE INVENTION

In view of this, the technical problem to be solved by the present invention is to provide a matrix metalloproteinase (MMP) responsive polymer drug carrier and its preparation method and application. The polymer drug carrier provided by the present invention is not only easy to obtain, but also supports The polymer drug obtained after the drug has high efficacy and low toxicity.

The present invention provides a matrix metalloproteinase responsive polymer drug carrier, which has the structure shown in formula (I),

Among them, m is 40～230, n is 30～120;

The amino acid sequence of the Pep is GPLGVRGDG or GPLGVRG.

Preferably, the m is 80-150.

Preferably, the n ranges from 50 to 100.

The present invention also provides a preparation method of a matrix metalloproteinase-responsive polymer drug carrier, comprising:

1) PEG-N ₃ is reacted with alkynyl-Pep to obtain the PEG-Pep that the end is amino,

The amino acid sequence of the Pep is GPLGVRGDG or GPLGVRG;

2) the PEG-Pep that the end is amino and D, L-lactide are reacted, obtain the macromolecular drug carrier with the structure shown in formula (I);

However, m is 40-230, and n is 30-120.

Preferably, the catalyst for the reaction in step 1) is cuprous bromide and N,N,N',N,'N"-pentamethyldiethylenetriamine (PMDETA).

Preferably, the catalyst for the reaction in step 2) is one or more of stannous octoate, stannous acetate, titanium dioxide and aluminum chloride.

The present invention also provides a polymer drug, including a polymer drug carrier and an antitumor drug;

Wherein, the polymer drug carrier is the polymer drug carrier of the present invention.

Preferably, the antitumor drug is one or more of paclitaxel, camptothecin, cisplatin, nitrogen mustard and doxorubicin.

The present invention also provides a preparation method of the polymer medicine of the present invention, comprising:

The polymer drug carrier, the antitumor drug, the solvent and the buffer solution according to the present invention are reacted to obtain the polymer drug.

Preferably, the buffer solution is phosphate buffered solution (PBS) with a pH of 7.4.

Compared with the prior art, the present invention provides a polymer drug carrier with a structure represented by formula (I). The polymer drug carrier provided by the present invention is used for carrying drugs, and the obtained polymer drug can increase tumor drugs. For the endocytosis of the drug, the drug efficacy is improved, and the biological safety is high; the experimental results show that the polymer drug provided by the present invention has a 16-fold improvement in curative effect compared with the existing molecular drug; and the drug is safe.

Description of drawings

Fig. 1 is the synthetic route diagram of the polymer carrier of the present invention;

Fig. ₂ is the GPC figure of PEG-GPLGVRGDG-NH and PEG-GPLGVRGDG-PDLLA prepared by the embodiment;

Figure 3 is the ¹ H NMR chart of PEG-GPLGVRGDG-NH ₂ and PEG-GPLGVRGDG-PDLLA;

Fig. 4 is a correlation diagram of the ratio (I ₃₃₉ /I ₃₃₂ ) of the concentration of the polymer drug to the intensity of the wavelength at 339 nm and 332 nm in the excitation light spectrum with pyrene as the fluorescent probe;

Fig. 5 is the particle size characterization result of the polymer drug provided by the present invention;

Figure 6 shows the results of gel permeation chromatography (GPC) co-cultured with 1 μg/mL of MMP-2 and P1 for different times;

Fig. 7 is the PEG release result after the co-culture of P1 nanoparticle, P2 nanoparticle, P3 nanoparticle and MMP-2 respectively;

Fig. 8 is a graph of changes in the size of P1 nanoparticles, P2 nanoparticles, and P3 nanoparticles in the presence of MMP-2;

Figure 9 is a transmission electron microscope (TEM) image of P1 nanoparticles before and after MMP-2 treatment;

Figure 10 is the PTX release curve in the absence of MMP-2;

Figure 11 is the PTX release curve in the presence of MMP-2;

Figure 12 is the flow cytometry results of 4T1 cells;

Figure 13 is a confocal laser microscope (CLSM) image of the polymer drug according to the present invention;

Figure 14 shows the cytotoxicity evaluation results of blank micelles;

Figure 15 is an evaluation of the cytotoxicity of different polymer drugs at different PTX concentrations;

Figure 16 shows the changes in blood and PTX content at different times after intravenous injection of PTX-loaded P1, P2 or P3;

Figure 17 shows the results of quantitative analysis of PTX in major organs and tumors after 12 and 24 hours of intravenous injection;

Figure 18 is a biodistribution diagram of the nanoparticles loaded with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanineiodide (DIR) fluorescent molecule after intravenous injection;

Figure 19 shows the results of changes in tumor size after intravenous injection of PTX-loaded nanoparticles and small molecule PTX;

Figure 20 is a photograph of typical tumors collected from different treatment groups on day 24;

Figure 21 shows the results of hematoxylin-eosin (H&E) staining and terminal deoxynucleotidyl transferase (TUNEL) fluorescence images of tumor sections;

Figure 22 is the change in body weight of mice;

Figure 23 shows H&E staining of heart, liver, spleen, lung, kidney corresponding to intravenous injection of PBS, PTX, P1, P2 and P3, respectively.

Detailed ways

The present invention provides a polymer drug carrier, which has the structure shown in formula (I),

Among them, m is 40～230, n is 30～120;

The amino acid sequence of the Pep is GPLGVRGDG (glycine-proline-leucine-glycine-valine-arginine-glycine-aspartic acid-glycine) or GPLGVRG (glycine-proline-leucine) -Glycine-Valine-Arginine-Glycine).

According to the present invention, the amino acid sequence of Pep is GPLGVRG or GPLGVRGDG; the m is preferably 80-150, more preferably 100-140, most preferably 110-130, and most preferably 113-120; the n is preferably 50-110, more preferably 60-100.

The present invention also provides a preparation method of a polymer drug carrier, comprising:

1) react the polyethylene glycol of the azide end group with the alkynyl-functionalized polypeptide to obtain the PEG-Pep with the amino group at the end;

The amino acid sequence of the Pep is GPLGVRGDG or GPLGVRG;

2) the PEG-Pep that the end is amino and D, L-lactide are reacted, obtain the macromolecular drug carrier with the structure shown in formula (I);

However, m is 40-230, and n is 30-120.

According to the present invention, the present invention also reacts polyethylene glycol (PEG-N ₃ ) with an azide end group with an alkynyl functionalized polypeptide (alkynyl-Pep, wherein the alkynyl group is attached to the original glycine) to obtain PEG-Pep with an amino group at the end; the present invention has no special requirements for the conditions of the reaction, and any method known in the art can be used; wherein, the catalyst for the reaction is preferably cuprous bromide and N,N,N',N, 'N"-pentamethyldiethylenetriamine (PMDETA); the solvent of the reaction is preferably DMF; the temperature of the reaction is preferably 30-60°C; more preferably 40-50°C; , the amino acid sequence of Pep is GPLGVRGDG or GPLGVRG.

The present invention also reacts PEG-Pep whose terminal is amino group with D, L-lactide to obtain a polymer drug carrier with the structure shown in formula (I); wherein, the catalyst of the reaction is preferably stannous octoate; The solvent of the reaction is preferably tetrahydrofuran; the temperature of the reaction is preferably 60-100°C, more preferably 80-90°C.

The present invention also provides a polymer drug, including a polymer drug carrier and an antitumor drug;

Wherein, the polymer drug carrier is the polymer drug carrier of the present invention; the antitumor drug is preferably one or more of paclitaxel, camptothecin, hydrophobic platinum drugs, nitrogen mustard and doxorubicin species; more preferably paclitaxel.

The present invention also provides a preparation method of the polymer medicine of the present invention, comprising:

The polymer drug carrier, the antitumor drug, the solvent and the buffer solution according to the present invention are reacted to obtain the polymer drug. Wherein, the buffer solution is a phosphate buffer solution with a pH of 7.4; the solvent is preferably ethyl acetate; the reaction is preferably an ultrasonic reaction; the power of the ultrasonic is preferably 90-120W; the time of the ultrasonic is preferably 60 to 100 seconds.

The present invention provides a polymer drug carrier with a structure represented by formula (I). The polymer drug carrier provided by the present invention is used for carrying drugs, and the obtained polymer drug can increase tumor in tumor tissues with high MMP expression. The drug has a good application prospect for the endocytosis of the drug, improving the efficacy and high biological safety.

The following will clearly and completely describe the technical solutions of the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

In the examples, the compound of formula (I) is represented by the abbreviation PEG-Pep-PDLLA.

Example

1. Preparation and characterization of polymer drugs

1) Preparation method of polymer carrier

Preparation of PEG- _GPLGVRGDG -NH

PEG-N ₃ (0.3 g, 0.06 mmol), alkynyl-Pep (0.062 g, 0.072 mmol), DMF (3 mL) and PMDETA (31 mg, 0.18 mmol) were put together in a 5 mL sealed tube and refrigerated-circulated three times Then, cuprous bromide (26 mg, 0.18 mmol) was added under the protection of nitrogen, and the mixture was refrigerated and evacuated twice, sealed under vacuum, and placed at 40° C. to react for 24 hours. After the reaction was completed, the reactant was added to 50 mL of ether for precipitation, and the obtained product was dried and dissolved in deionized water, added to a dialysis membrane with a molecular weight of 3500, and dialyzed in flowing deionized water for 24 hours. After freeze-drying, a white powdery product was obtained, namely PEG-GPLGVRGDG-NH ₂ ; the yield was 0.22 g, and the yield was 62.7%.

The PEG-GPLGVRGDG- _NH2 initiator was dissolved in about _0.5 mL of _CH2Cl2 , followed by the addition of excess benzene. An anhydrous white powder was obtained after lyophilization. Then, lyophilized PEG-GPLGVRGDG- _NH2 (0.117 g, 0.02 mmol), D,L-lactide (0.288 g, 2 mmol), stannous octoate (30 μL, 10 mg/mL solution in benzene) were dissolved in 1 ml of anhydrous To tetrahydrofuran and 4 ml of anhydrous benzene, add 10 ml of sealed tube, add 4 ml of anhydrous tetrahydrofuran after lyophilization, seal the sealed tube, and react at 80° C. for 18 hours. The resulting polymer was then precipitated into 50 mL of cold ether. The product is collected by centrifugation. The above dissolution-precipitation cycle was repeated twice. The final product was dried to obtain a white solid powder, namely the polymer carrier PEG-GPLGVRGDG-PDLLA, with a yield of 0.26 g and a yield of 64%.

Likewise, PEG-GPLGVRG-PDLLA and PEG-PDLLA polymeric carriers were synthesized using similar synthetic methods.

The specific preparation method of the polymer carrier is shown in Figure 1, which is a synthetic route diagram of the polymer carrier according to the present invention.

The obtained polymer carrier is detected, and the results are shown in Figures 2 to 3, Figure 2 is the GPC diagram of PEG-GPLGVRGDG-NH ₂ and PEG-GPLGVRGDG-PDLLA prepared in the example; Figure 3 is PEG-GPLGVRGDG-NH ₂ and ^1H NMR of PEG-GPLGVRGDG-PDLLA.

2) Preparation of polymer drugs (drug-loaded micelles)

PEG-GPLGVRGDG-PDLLA (10 mg) and paclitaxel (1 mg) were dissolved in ethyl acetate (0.2 ml), then 1 ml of phosphate buffer solution (pH 7.4) was added, and phacoemulsification was performed at a power of 90 W for 60 seconds, under reduced pressure The organic solvent is removed, and the unembedded paclitaxel is removed by centrifugation to obtain drug-loaded nanoparticles, that is, polymer drugs (also known as P1 nanoparticles or P1).

Similarly, paclitaxel was loaded on PEG-GPLGVRG-PDLLA and PEG-PDLLA by a similar synthetic method to obtain polymer drugs P2 and P3, respectively. In addition, the preparation of Nile Red and DIR-embedded nanoparticles (polymer drugs) was achieved by exchanging the above-mentioned PTX with the corresponding molecules.

By using pyrene as a fluorescent probe, the concentration of pyrene in the solution is 1×10 ^-7 M, the emission wavelength of the fluorescence test is 475nm, and the ratio of the excitation light intensity of the tested polymer drug at 339nm and 332nm is plotted against the polymer concentration, the results See Fig. 4, Fig. 4 is the correlation diagram of the ratio of the concentration of polymer drug and the intensity of the wavelength at 339nm and 332nm in the excitation light spectrum (I ₃₃₉ /I ₃₃₂ ) with pyrene as the fluorescent probe; it can be seen from the same species It can be concluded that the critical micelle concentrations of the polymer drugs in the present invention are 2.1×10 ^-3 mg/mL (P1), 2.5×10 ^-3 mg/mL (P2) and 3.3×10 ^-3 mg/mL (P2), respectively. P3).

The polymer drug is characterized by dynamic light scattering, and the results are shown in Figure 5, which is the particle size characterization result of the polymer drug provided by the present invention; it can be seen from the figure that the average particle size of the nanoparticles obtained by the present invention is 80 nm. This size facilitates the enrichment of nanoparticles at tumor tissue sites. Moreover, the drug loading and encapsulation efficiencies of P1, P2 and P3 nanoparticles were measured to be similar. This indicates that the introduction of the sequence of the polypeptide in the middle of the block polymer does not have a large impact on the relevant properties of the polymer drug.

3) Evaluation of the responsiveness of polymer drugs to MMP-2.

The polypeptide used in the present invention (GPLGVRGDG) has been shown to be cleaved at the site between G and V by activated MMP-2. After the P1 nanoparticles, P2 nanoparticles, and P3 nanoparticles were co-cultured with MMP-2 for different times, GPC characterization was carried out. Gel permeation chromatography (GPC) results of cultured at different times; Figure 7 shows the PEG release results after co-culture of P1 nanoparticles, P2 nanoparticles, P3 nanoparticles and MMP-2 respectively; as can be seen from Figure 6, cultured 1 The GPC curve after hours shows a new shoulder at 16.08 minutes, which corresponds to the PEG peak. This result demonstrates that MMP-2 leads to the removal of PEG from the nanoparticles. As the MMP-2 treatment time increased, the shoulder became stronger and more than 70% of the PEG was detached within 8 hours as can be seen from Figure 7, and finally, about 20% of the PEG was still on the nanoparticle surface.

While culturing MMP-2, we also used dynamic light scattering (DLS) to study the size change of nanoparticles. The results are shown in Figures 8 to 9. Figure 8 shows P1 nanoparticles and P2 nanoparticles in the presence of MMP-2. , P3 nanoparticles change in size; Figure 9 is the transmission electron microscope (TEM) images of P1 nanoparticles before and after MMP-2 treatment; as can be seen from Figure 8, the size of nanoparticles did not significantly change within 24 hours TEM measurements further confirmed that, as shown in Figure 9, except for a few relatively large particles, the dePEGylated nanoparticles still maintained a relatively uniform size. This phenomenon may be due to the fact that 20% PEG remained on the surface of the nanoparticles after 24 hours of MMP-2 incubation was sufficient to stabilize the nanoparticles. The control group P2 also showed PEG detachment, while P3 was not responsive to MMP-2 (Figure 7). The above results indicate that MMP-2 is able to efficiently cleave peptide bonds without causing significant aggregation. It can be speculated that the residual peptide VRGDG is retained on the surface of the nanoparticles, which has great advantages for tumor cell endocytosis.

Furthermore, to assess the effect of the presence of MMP-2 on drug release, we investigated the release behavior of PTX in the presence and absence of MMP-2. Figure 10 and Figure 11, Figure 10 is the PTX release curve in the absence of MMP-2; Figure 11 is the PTX release curve in the presence of MMP-2; as can be seen from the figure, P1, P2, and P3 loaded with PTX Similar release behavior was exhibited with or without MMP-2, with about 80% of PTX released within 72 hours.

4) Weak size change and PEG detachment by potent MMP-2 may promote cellular uptake. In addition, the residual peptide VRGDG on the nanoparticle surface could act as a targeting ligand to further promote the interaction between nanoparticles and tumor cells. To study the cellular uptake behavior of nanoparticles in the presence of MMP-2, equal amounts of Nile Red-loaded nanoparticles and 4T1 cells were co-cultured in the presence of MMP-2. Flow cytometry measurements were then performed to study nanoparticle endocytosis. The results are shown in Figures 12 to 13, Figure 12 is the flow cytometry results of 4T1 cells; Figure 13 is the confocal laser microscope (CLSM) image of the polymer drug according to the present invention; it can be seen from Figure 12 that P1 The nanoparticles exhibited almost three-fold higher cellular uptake compared to the P3 nanoparticles, while the P2 nanoparticles showed only a 1.2-fold increase in uptake, and under the action of MMP-2, the P2 nanoparticles although showed de-pegylation and The P1 nanoparticles not only exhibited de-pegylation, but also RGD residues were left on the surface of the nanoparticles. Therefore, P1 nanoparticles showed the highest cellular uptake. Furthermore, as can be seen from Figure 13, P1 nanoparticles exhibit significantly stronger Nile Red fluorescence intensity compared to P2 and P3 nanoparticles, exhibiting more efficient cellular internalization, thus, MMP-2-induced PEG removal and P1 Residual VRGDG peptides on the nanoparticle surface synergistically promote cellular uptake.

5) Efficient cellular uptake plays a key role in improving the efficacy of anticancer drugs. The cell viability of 4T1 cells after different drug treatment was measured by MTT method. The specific method is as follows:

4T1 cells were seeded at a density of 1×10 ⁴ /well in a 96-well plate in 100 μL of DMEM medium containing FBS (10%) and cultured at 37° C. in a CO ₂ atmosphere (5%) for 24 hours. Then, the old medium was replaced with 100 μL of fresh medium, blank or PTX-loaded nanoparticles were added at different concentrations, and the enzyme activator APMA-activated MMP-2 was added. After culturing for 24 hours, the medium was completely aspirated and added. 100 μL of fresh medium was passed through for another 48 hours of incubation. Cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT). 20 μL of MTT solution (5 mg/mL in PBS) was added to each well and incubated at 37°C for 4 hours in the dark. Then the medium was aspirated and DMSO (200 μL) was added for 30 minutes to dissolve the formed purple formazan crystals. Measure the absorbance at 490 nm with a microplate reader. Half maximal inhibitory concentration ( _IC50 ) values were calculated by GraphPad Prism software from MTT results.

The results are shown in Figures 14 to 15, Figure 14 is the cytotoxicity evaluation results of blank micelles; Figure 15 is the cytotoxicity evaluation of different polymer drugs at different PTX concentrations, where *p<0.05. As can be seen from Figure 14, in the presence of MMP-2, the blank nanoparticles showed little toxicity even at fairly high concentrations, indicating the good biocompatibility of these block copolymers. As can be seen from Figure 15, the PTX-loaded P1 nanoparticles exhibited significantly enhanced cytotoxicity compared to the PTX-loaded P2 and P3 nanoparticles and the small molecule PTX. The half inhibitory concentration ( _IC50 ) value for P1 loaded with PTX was 85 ng/mL, for small molecule PTX (147 ng/mL), for PTX loaded P2 nanoparticles (560 ng/mL), or for P3 nanoparticles (1080 ng/mL).

The evaluation of pharmacokinetics

The pharmacokinetics of polymeric drugs were evaluated in female 6-week-old CD-1 (ICR) mice. specific:

Female 6-week-old CD-1 (ICR) mice were randomized into three groups (n=3). PTX-loaded P1, P2, or P3 nanoparticles were injected via tail vein at a dose of 10 mg/kg PTX. At various time intervals, 100 [mu]L blood samples were collected from mouse orbits into heparinized tubes and 10 mL of ethyl acetate was added. Following centrifugation at 10000 × g for 5 min, the organic solvent was separated and dried under _N2 . The content of paclitaxel was determined by reversed-phase high performance liquid chromatography (RP-HPLC) (mobile phase: acetonitrile/H ₂ O (3:7 V/V), flow rate 1 mL/min, UV-VIS detection wavelength fixed at 217 nm).

The results are shown in Figure 16. Figure 16 shows the changes in blood and PTX content at different times after intravenous injection of PTX-loaded P1, P2 or P3; it can be seen from Figure 16 that after intravenous injection, PTX-loaded P1 , P2 and P3 nanoparticles showed similar relatively long blood circulation times. The half-lives t _1/2 of P1, P2 and P3 were 3.36h, 4.82h and 3.92h, respectively. Longer blood circulation can enhance the enrichment of anticancer drugs at tumor sites.

3. Construction of H22 mouse tumor model

Considering the importance of MMP enzymes in the development of various tumors, we selected the H22 tumor model of female CD-1(ICR) mice to study the antitumor effect. In order to quantify the distribution of PTX in tumor sites and major organs, after intravenous injection of different drug preparations, PTX was extracted from tumor tissues and major organs and analyzed by HPLC system. Specifically, H22 cells (3×10 ⁶ ) suspended in 200 μL of PBS were injected subcutaneously into the axilla of the right lower limb of female 6-week-old CD-1 (ICR) mice. Tumor size was monitored using a digital caliper, and tumor volume (V) was calculated from the equation V = axb2/ ² , where a and b represent the longest and shortest diameters of the tumor, respectively.

In addition, H22 tumor-bearing mice were used to study the in vivo distribution of nanoparticles. When the tumor volume reached 100 ^mm3 , DIR-loaded P1, P2 or P3 nanoparticles were injected intravenously at a dose of 1 mg/kg. At various time intervals (1, 12, 24, 48 and 72 hours), mice were anesthetized and images were acquired using the IVIS Small Animal Imaging System.

The results are shown in Figure 17 and Figure 18, Figure 17 is the quantitative analysis results of PTX in major organs and tumors after 12 and 24 hours of intravenous injection; Figure 18 is the in vivo biodistribution of nanoparticles loaded with DIR fluorescent molecules after intravenous injection; As can be seen in Figure 17, the drug enrichment of P1 nanoparticles at the tumor site was significantly enhanced at 24 hours, which was 1.47 times higher than that of P2 nanoparticles, and 2.01 times higher than that of P3 nanoparticles (*P<0.05), while the drug in the major organs was increased by 1.47 times and P3 nanoparticles. The distribution showed no significant difference. In order to be able to see the nanoparticles in vivo, hydrophobic near-infrared fluorescent emitting dyes (DIR) were embedded in the nanoparticles for real-time monitoring. As shown in Fig. 18, it can be seen from Fig. 18 that after 1 hour of intravenous injection of the three DIR-loaded nanoparticles, the tumor site exhibited obvious fluorescence of DIR, reaching a maximum value at 24 hours. Also, the DIR-loaded P1 nanoparticles showed slightly stronger fluorescence at 48 h.

4. Distribution in the body

The efficacy of PTX-loaded nanoparticles was also evaluated using H22 tumor-bearing mice. Twenty-five mice were divided into 5 groups including PBS, small molecule PTX, PTX-loaded P1, P2, and P3 nanoparticles. When tumors grew to about 100 ^mm3 , the different drug formulations were injected intravenously every two days at a dose of 10 mg/kg for a total of three injections.

To quantify the accumulation of PTX in major organs and tumors, the corresponding organs and tumor tissues were collected after intravenous injection of PTX at a dose of 10 mg/kg. The samples were then washed with PBS, then dried and weighed, 0.5 mL of _CH3CN was added, and centrifuged at 10,000 xg for 10 minutes, after which the supernatant was further extracted with 1 mL of _CHC13 . The organic phase was dried under nitrogen flow and the residue was dissolved in 100 μL of methanol and the amount of PTX was determined by RP-HPLC.

The results are shown in Figures 19 to 20. Figure 19 shows the results of changes in tumor size after intravenous injection of PTX-loaded nanoparticles and small molecule PTX; data are expressed as mean±SD, N=5, *P<0.05. Figure 20 is a photograph of typical tumors collected at day 24 from different treatment groups. As can be seen in Figures 19 and 20, tumors treated with PBS after 24 days showed a nearly 40-fold increase in volume, 25-fold for small molecule PTX, 16-fold for P3 nanoparticles, and 10-fold for P2 nanoparticles. In stark contrast, PTX-loaded P1 nanoparticles effectively inhibited tumor growth, with only a 1.9-fold increase in tumor size.

5. Evaluation of anti-tumor effect in animals

H22 tumor-bearing mice were randomly divided into 5 groups (n=5). When the tumor volume reached 100 ^mm3 , small molecule PTX, PTX-loaded P1, P2 and P3 nanoparticles were injected through the tail vein with a PTX dose of 10 mg/kg. Injections were given every two days for a total of three injections. Tumor size was monitored by measurement using digital vernier calipers. At the same time, the body weight of the mice was recorded every two days. After treatment (24 days), all mice were sacrificed and major organs (heart, liver, spleen, lung, kidney) and tumors were collected. Tumor weights were recorded, then tumors and organs were embedded in paraffin, and 5 μm paraffin sections were prepared. Sections were stained with hematoxylin and eosin (H&E). In addition, tumor sections were further stained with a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit to identify apoptotic cells.

Tumor sections were stained with H&E and TUNEL to evaluate apoptotic cells.

The results are shown in Figures 21 to 23. Figure 21 shows the results of hematoxylin-eosin (H&E) staining and terminal deoxynucleotidyl transferase (TUNEL) fluorescence images of tumor sections; Figure 22 shows the weight changes of mice; Figure 23 shows H&E staining of heart, liver, spleen, lung, and kidney, corresponding to intravenous injection of PBS, PTX, P1, P2 and P3, respectively; wherein, white arrows indicate liver damage. As can be seen from the figure, tumors treated with PTX-loaded P1 nanoparticles exhibited the strongest green fluorescence, which indicated the most apoptosis. H&E staining also confirmed that the most significant efficacy was achieved by injection of PTX-loaded P1 nanoparticles. In addition, the body weight of the five groups of mice did not change significantly during the treatment period; moreover, as can be seen from Figure 13, the results of H&E staining of the organs mainly showed liver damage caused by small molecule PTX, while the PTX-loaded nanoparticles treatment group did not Significant organ damage; these results show a high biosafety of P1, P2 and P3 nanoparticles.

The descriptions of the above embodiments are only used to help understand the method and the core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (9)

1. A matrix metalloproteinase responsive polymer drug carrier, having the structure shown in formula (I),

Among them, m is 40～230, n is 50～100; The amino acid sequence of the Pep is GPLGVRGDG or GPLGVRG. 2 . The polymer drug carrier according to claim 1 , wherein the m is 80-150. 3 . 3. A preparation method of a matrix metalloproteinase-responsive polymer drug carrier, comprising: 1) react the polyethylene glycol of the end group azide with the alkynyl-functionalized polypeptide to obtain the PEG-Pep with the amino group at the end, The amino acid sequence of the Pep is GPLGVRGDG or GPLGVRG; 2) the PEG-Pep that the end is amino and D, L-lactide are reacted, obtain the macromolecular drug carrier with the structure shown in formula (I); However, m is 40-230, and n is 30-120. 4. preparation method according to claim 3 is characterized in that, the catalyst of described step 1) reaction is cuprous bromide and N,N,N',N",N"-pentamethyldiethylene Triamine. 5. preparation method according to claim 3 is characterized in that, the catalyst of described step 2) reaction is one or more in stannous octoate, stannous acetate, titanium dioxide and aluminum chloride. 6. A polymer drug, comprising a polymer drug carrier and an antitumor drug; Wherein, the polymer drug carrier is the polymer drug carrier according to any one of claims 1 to 3. 7 . The polymer drug according to claim 6 , wherein the antitumor drug is one or more of paclitaxel, camptothecin, hydrophobic platinum drugs, nitrogen mustard and doxorubicin. 8 . 8. a preparation method of the described polymer medicine of claim 6, comprising: The polymer drug carrier, the antitumor drug, the solvent and the buffer solution according to any one of claims 1 to 2 are reacted to obtain the polymer drug. 9 . The preparation method according to claim 8 , wherein the buffer solution is a phosphate buffer solution with a pH of 7.4. 10 .

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