CN107157928B

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

Info

Publication number: CN107157928B
Application number: CN201710355187.0A
Authority: CN
Inventors: 葛治伸; 柯文冬
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2017-05-18
Filing date: 2017-05-18
Publication date: 2020-02-07
Anticipated expiration: 2037-05-18
Also published as: CN107157928A

Abstract

The invention provides a polymer drug carrier with a structure shown in formula (I) and a preparation method and application thereof.

Description

Matrix metalloproteinase responsive polymer drug carrier and preparation method and application thereof

Technical Field

The invention relates to the field of high molecular drugs, in particular to a matrix metalloproteinase responsive polymer drug carrier, and a preparation method and application thereof.

Background

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, the polymer drug-loaded micelle can improve the pharmacokinetics of the drugs and improve the enrichment of the drugs in tumor parts, thereby obviously improving the bioavailability of the drugs and reducing toxic and side effects. Biodegradable polymeric micelles with biocompatibility have had great success in drug delivery, and some have been approved for clinical use or are undergoing clinical trials. For example, the Genexol-PM (polyethylene glycol-b-poly D, L-lactide) (PEG-PDLLA) polymer-loaded PTX formulation showed better therapeutic efficacy and lower toxicity compared to solvent-based Paclitaxel (PTX), which has been approved for the treatment of metastatic breast cancer and non-small cell lung cancer in korea. In addition, there are other amphiphilic block polymer micelles in clinical trials, such as NK105, NK911, NK012, SP1049C, BIND 014.

On the other hand, however, the long circulation of blood and the high tumor enrichment (e.g., PEG-coated surfaces) of polymeric micelles also hinder the endocytosis of tumor cells. This is a disadvantage for most anticancer drugs that require endocytosis to be effective. To solve this problem, various responsive smart polymer drug delivery systems have been designed to further improve the therapeutic effect, but polymer drug delivery systems that have entered the clinical stage so far have been scarce. The complex structure of the polymer itself and unknown systemic toxicity limit its further clinical application. Therefore, it is a technical problem to be solved at present to provide a polymer drug with low toxicity, easy availability and good therapeutic effect.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a Matrix Metalloproteinase (MMP) responsive polymer drug carrier, and a preparation method and an application thereof.

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

wherein m is 40-230, n is 30-120;

the amino acid sequence of Pep is GPLGVRGDG or GPLGVRG.

Preferably, m is 80-150.

Preferably, n is 50-100.

The invention also provides a preparation method of the matrix metalloproteinase responsive polymer drug carrier, which comprises the following steps:

1) mixing PEG-N₃Reacting with alkyl-Pep to obtain PEG-Pep with the end as amino,

the amino acid sequence of Pep is GPLGVRGDG or GPLGVRG;

2) reacting PEG-Pep with amino at the end with D, L-lactide to obtain a high molecular drug carrier with a structure shown in formula (I);

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

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

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

The invention also provides a high molecular drug, which comprises a high molecular drug carrier and an anti-tumor drug;

wherein, the macromolecular drug carrier is the macromolecular drug carrier provided by the invention.

Preferably, the anti-tumor drug is one or more of paclitaxel, camptothecin, cisplatin, nitrogen mustard and adriamycin.

The invention also provides a preparation method of the high molecular drug, which comprises the following steps:

the macromolecular drug carrier, the antitumor drug, the solvent and the buffer solution are reacted to obtain the macromolecular drug.

Preferably, the buffer solution is Phosphate Buffered Saline (PBS) at pH 7.4.

Compared with the prior art, the invention provides the polymer drug carrier with the structure shown in the formula (I), the polymer drug carrier is used for carrying drugs, the obtained polymer drug can increase the endocytosis of the tumor drug to the drugs, the drug effect is improved, and the biological safety is higher; the experimental result shows that compared with the existing molecular drug, the high molecular drug provided by the invention has the advantages that the curative effect is improved by 16 times; and the medicine is safe.

Drawings

FIG. 1 is a scheme showing the synthesis of a polymeric support according to the present invention;

FIG. 2 shows PEG-GPLGVRGDG-NH prepared in example₂And a GPC chart of PEG-GPLGVRGDG-PDLLA;

FIG. 3 shows PEG-GPLGVRGDG-NH₂And PEG-GPLGVRGDG-PDLLA¹H NMR chart;

FIG. 4 is a graph showing the ratio of the concentration of a high molecular weight drug to the intensity of the drug at 339nm and 332nm in the excitation spectrum using pyrene as a fluorescence probe(I₃₃₉/I₃₃₂) A correlation diagram of (1);

FIG. 5 shows the particle size characterization results of the polymer drug provided by the present invention;

FIG. 6 is a Gel Permeation Chromatography (GPC) result of co-culturing with MMP-2 at 1. mu.g/mL and P1 for various periods of time;

FIG. 7 shows the PEG release results of the co-culture of P1 nanoparticles, P2 nanoparticles, P3 nanoparticles and MMP-2;

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

FIG. 9 is a Transmission Electron Microscope (TEM) image of P1 nanoparticles before and after MMP-2 treatment;

FIG. 10 is a PTX release profile in the absence of MMP-2;

FIG. 11 is a PTX release profile in the presence of MMP-2;

FIG. 12 is the flow cytometry results for 4T1 cells;

FIG. 13 is a laser confocal microscope (CLSM) image of the polymeric drug of the present invention;

FIG. 14 shows the results of cytotoxicity evaluation of blank micelles;

FIG. 15 is an evaluation of cytotoxicity of different polymeric drugs at different PTX concentrations;

FIG. 16 is the change in blood at different times from the PTX content after intravenous injection of PTX loaded P1, P2 or P3;

FIG. 17 is the results of quantitative analysis of PTX in major organs and tumors after 12 and 24 hours of intravenous injection;

FIG. 18 is a biological profile of nanoparticles loaded with 1, 1 ' -diazadecyl-3, 3, 3 ', 3 ' -tetramethylmethylenecyanoside (DIR) fluorescent molecules in vivo after intravenous injection;

figure 19 is the results of the change in tumor size following intravenous injection of PTX-loaded nanoparticles and small molecule PTX;

FIG. 20 is a photograph of a typical tumor collected from different treatment groups on day 24;

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

FIG. 22 is the body weight change of mice;

fig. 23 is an i.v. staining of heart, liver, spleen, lung, kidney H & E, corresponding to PBS, PTX, P1, P2, and P3, respectively.

Detailed Description

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

wherein m is 40-230, n is 30-120;

the amino acid sequence of 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 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, most preferably 113-120; the n is preferably 50 to 110, and more preferably 60 to 100.

The invention also provides a preparation method of the polymer drug carrier, which comprises the following steps:

1) reacting the polyethylene glycol with the nitrified end group with the alkynyl functionalized polypeptide to obtain PEG-Pep with the amino at the tail end;

the amino acid sequence of Pep is GPLGVRGDG or GPLGVRG;

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

In accordance with the present invention, the terminal group of the present invention is also azidated polyethylene glycol (PEG-N)₃) With alkynyl functionReacting the esterified polypeptide (alkinyl-Pep, wherein alkynyl is connected to initial glycine) to obtain PEG-Pep with an amino terminal; the method has no special requirements on reaction conditions, and can be carried out by any method known in the field; wherein the catalyst for the reaction is preferably cuprous bromide and N, N', N "-Pentamethyldiethylenetriamine (PMDETA); the solvent of the reaction is preferably DMF; the reaction temperature is preferably 30-60 ℃; more preferably 40-50 ℃; in addition, in the present invention, Pep has an amino acid sequence of GPLGVRGDG or GPLGVRG.

The invention also reacts PEG-Pep with amino at the end with D, L-lactide to obtain a high molecular drug carrier with the structure shown in formula (I); wherein, the catalyst of the reaction is preferably stannous octoate; the solvent for the reaction is preferably tetrahydrofuran; the reaction temperature is preferably 60-100 ℃, and more preferably 80-90 ℃.

wherein, the macromolecular drug carrier is the macromolecular drug carrier of the invention; the anti-tumor drug is preferably one or more of paclitaxel, camptothecin, hydrophobic platinum drugs, nitrogen mustard and adriamycin; more preferably paclitaxel.

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

The polymer drug carrier provided by the invention is used for carrying drugs, and the obtained polymer drug can increase the endocytosis of the tumor drug to the drug in tumor tissues with high MMP expression, so that the drug effect is improved, the biological safety is higher, and the polymer drug carrier has a good application prospect.

The following will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The compounds of formula (I) are indicated in the examples by the abbreviation PEG-Pep-PDLLA.

Examples

Preparation and characterization of macromolecular drugs

1) Process for the preparation of a polymeric support

PEG-GPLGVRGDG-NH₂Preparation of

Mixing PEG-N₃(0.3g, 0.06mmol), alkyl-Pep (0.062g, 0.072mmol), DMF (3mL) and PMDETA (31mg, 0.18mmol) were placed together in a 5mL sealed tube, evacuated in a freeze-cycle three times and cuprous bromide (26mg, 0.18mmol) was added under nitrogen, evacuated in a freeze-cycle two times, sealed under vacuum and left to react at 40 ℃ for 24 hours. After the reaction is finished, the reactant is added into 50mL of diethyl ether for precipitation, the obtained product is dried and dissolved in deionized water, and the solution is added into a dialysis membrane with the molecular weight of 3500 and dialyzed in flowing deionized water for 24 hours. Freeze-drying to obtain white powder product PEG-GPLGVRGDG-NH₂(ii) a The yield was 0.22g, 62.7%.

PEG-GPLGVRGDG-NH₂Initiator was dissolved in about 0.5mL of CH₂Cl₂Then, an excess of benzene is added. After lyophilization, an anhydrous white powder was obtained. Then, lyophilized PEG-GPLGVRGDG-NH₂(0.117g, 0.02mmol), D, L-lactide (0.288g, 2mmol), stannous octoate (30. mu.L, 10mg/mL benzene solution) were dissolved in 1mL of anhydrous tetrahydrofuran and 4mL of anhydrous benzene, added to a 10mL lock tube, lyophilized, added to 4mL of anhydrous tetrahydrofuran, locked, and reacted at 80 ℃ for 18 hours. The resulting polymer was then precipitated into 50mL of cold ether. The product was collected by centrifugation. The above dissolution-precipitation cycle was repeated twice. Drying the final product to obtain white pigmentThe yield was 0.26g of a colored solid powder, i.e., PEG-GPLGVRGDG-PDLLA, which is a polymer carrier, and 64% yield.

Similarly, PEG-GPLGVRG-PDLLA and PEG-PDLLA polymer carriers were synthesized using a similar synthesis method.

The specific preparation method of the polymer carrier is shown in figure 1, and figure 1 is a synthesis scheme of the polymer carrier.

The obtained polymer carrier was examined, and the results are shown in FIGS. 2 to 3, and FIG. 2 shows PEG-GPLGVRGDG-NH prepared in example₂And a GPC chart of PEG-GPLGVRGDG-PDLLA; FIG. 3 shows PEG-GPLGVRGDG-NH₂And PEG-GPLGVRGDG-PDLLA¹H NMR chart.

2) Preparation of high molecular medicine (medicine carrying micelle)

Dissolving PEG-GPLGVRGDG-PDLLA (10mg) and paclitaxel (1mg) in ethyl acetate (0.2ml), adding phosphate buffer solution (pH7.4)1ml, ultrasonic emulsifying at 90W power for 60 s, removing organic solvent under reduced pressure, centrifuging to remove un-embedded paclitaxel to obtain drug-loaded nanoparticles, i.e. high molecular drug (also called P1 nanoparticles or P1).

Similarly, paclitaxel was loaded on PEG-GPLGVRG-PDLLA and PEG-PDLLA by a similar synthesis method to obtain the high molecular drugs P2 and P3, respectively. Furthermore, the preparation of nile red and DIR embedded nanoparticles (polymeric drugs) was achieved by exchanging the above-mentioned PTX for the corresponding molecule.

By using pyrene as a fluorescent probe, the concentration of pyrene in the solution is 1X 10^-7M, the emission wavelength of the fluorescence test is 475nm, the ratio of the excitation light intensity of the tested high-molecular drug at 339nm and 332nm is plotted against the polymer concentration, the result is shown in FIG. 4, FIG. 4 is the ratio of the concentration of the high-molecular drug to the intensity thereof at 339nm and 332nm in the excitation light spectrum (I)₃₃₉/I₃₃₂) A correlation diagram of (1); from the same species, the critical micelle concentration of the polymer drug is 2.1 × 10^-3mg/mL(P1).2.5×10^-3mg/mL (P2) and 3.3X 10^-3mg/mL(P3)。

Characterizing the polymer drug by dynamic light scattering, and the results are shown in fig. 5, and fig. 5 is a particle size characterization result of the polymer drug provided by the present invention; as can be seen from the figure, the average particle size of the nanoparticles obtained by the invention is about 80nm, and the size is convenient for the nanoparticles to be enriched at tumor tissue parts. Furthermore, the drug loading rate and the encapsulation rate of the P1, P2 and P3 nanoparticles are measured to be almost the same. This indicates that the introduction of the polypeptide sequence in the middle of the block polymer has no great influence on the relevant properties of the polymer drug.

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

The polypeptide (GPLGVRGDG) used in the present invention has been shown to be cleaved by activated MMP-2 at a site between G and V. GPC characterization was performed after co-culturing P1 nanoparticles, P2 nanoparticles, and P3 nanoparticles with MMP-2 for various periods of time, respectively, and the results are shown in FIGS. 6 to 7, and FIG. 6 is Gel Permeation Chromatography (GPC) results of co-culturing MMP-2 and P1 at 1. mu.g/mL for various periods of time; FIG. 7 shows the PEG release results of the co-culture of P1 nanoparticles, P2 nanoparticles, P3 nanoparticles and MMP-2; as can be seen in FIG. 6, the GPC curve showed a new shoulder at 16.08 minutes after 1 hour of incubation, which corresponds to the PEG peak. This result demonstrates that MMP-2 results in the removal of PEG from the nanoparticles. As MMP-2 treatment time increased, the shoulder became stronger and it can be seen from fig. 7 that more than 70% of the PEG detached within 8 hours and finally, approximately 20% of the PEG remained on the nanoparticle surface.

While MMP-2 was cultured, we also used Dynamic Light Scattering (DLS) to study the size change of nanoparticles, and the results are shown in FIGS. 8 to 9, and FIG. 8 is a graph showing the change in size of P1 nanoparticles, P2 nanoparticles, and P3 nanoparticles in the presence of MMP-2; FIG. 9 is a Transmission Electron Microscope (TEM) image of P1 nanoparticles before and after MMP-2 treatment; as can be seen from fig. 8, the nanoparticle size did not change significantly within 24 hours; TEM measurements further confirmed that the depegylated nanoparticles remained relatively uniform in size, except for a few relatively large particles, as shown in figure 9. This phenomenon is probably due to the fact that 20% of PEG remains on the surface of the nanoparticles sufficient to stabilize them after 24 hours of MMP-2 culture. Control P2 also showed PEG detachment, whereas P3 was not responsive to MMP-2 (fig. 7). The above results indicate that MMP-2 is able to cleave peptide bonds efficiently, but without causing significant aggregation. It can be speculated that the residual peptide VRGDG is retained on the surface of the nanoparticle, which has great advantage for the endocytosis of tumor cells.

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. FIG. 10 is a PTX release profile in the absence of MMP-2, as in FIGS. 10 and 11; FIG. 11 is a PTX release profile in the presence of MMP-2; as can be seen from the figure, PTX-loaded P1, P2, and P3 exhibited similar release behavior with or without MMP-2, with about 80% of the PTX released within 72 hours.

4) The weak size change and PEG detachment by potent MMP-2 are likely to promote cellular uptake. In addition, the residual peptide VRGDG remained on the surface of the nanoparticle can be used as a targeting ligand, and the interaction between the nanoparticle and the tumor cell is further promoted. To investigate 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 the endocytosis of the nanoparticles. The results are shown in FIGS. 12-13, and FIG. 12 is the flow cytometry results for 4T1 cells; FIG. 13 is a laser confocal microscope (CLSM) image of the polymeric drug of the present invention; as can be seen from fig. 12, the nanoparticles of P1 showed almost three times higher cellular uptake than the nanoparticles of P3, whereas the nanoparticles of P2 showed only a 1.2-fold increase in uptake, and under MMP-2, the nanoparticles of P2 showed not only depegylation but also the nanoparticles of P1 showed no pegylation, and RGD residues were left on the surface of the nanoparticles. Thus, the nanoparticles of P1 showed the highest cellular uptake. Furthermore, as can be seen from fig. 13, the P1 nanoparticles significantly stronger fluorescence intensity of nile red than the P2 and P3 nanoparticles showed more efficient cellular internalization, and thus, PEG removal by MMP-2 and residual VRGDG peptide on the surface of P1 nanoparticles synergistically promoted cellular uptake.

5) Effective cellular uptake has a key role in improving the curative effect of anticancer drugs. The cellular activity of 4T1 cells was determined by MTT method after different drug treatments. The specific method comprises the following steps:

4T1 cells were cultured at 1X 10⁴Density per well 100. mu.L DMEM medium containing FBS (10%) in 96-well plates at 37 ℃ in CO₂Incubate in atmosphere (5%) for 24 hours. Then, the old medium was replaced with 100 μ L of fresh medium, blank or PTX-loaded nanoparticles were added at different concentrations, and enzyme activator APMA activated MMP-2 was added, after 24 hours of culture the medium was completely aspirated, 100 μ L of fresh medium was added, and further culture was performed for another 48 hours. Cell activity was determined by 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2-H-tetrazolium bromide (MTT). mu.L of MTT solution (5mg/mL PBS solution) was added to each well and incubated at 37 ℃ for 4 hours in the dark. The medium is then aspirated again and DMSO (200 μ L) added and left for 30 minutes to dissolve the purple formazan crystals that form. Absorbance at 490nm was measured with a microplate reader. Half maximal Inhibitory Concentration (IC)₅₀) Values were calculated by GraphPad Prism software based on MTT results.

The results are shown in FIGS. 14 to 15, and FIG. 14 shows the results of cytotoxicity evaluation of blank micelles; figure 15 is an assessment of cytotoxicity of different polymeric drugs at different PTX concentrations, where p < 0.05. As can be seen from fig. 14, the blank nanoparticles showed little toxicity even at fairly high concentrations in the presence of MMP-2, indicating good biocompatibility of these block copolymers. As can be seen from fig. 15, PTX-loaded P1 nanoparticles exhibited significantly enhanced cytotoxicity compared to PTX-loaded P2 and P3 nanoparticles and small-molecule PTX. PTX-loaded P1 half Inhibitory Concentration (IC)₅₀) The value was 85ng/mL, the small molecule PTX was (147ng/mL), PTX-loaded P2 nanoparticles (560ng/mL), or P3 nanoparticles (1080 ng/mL).

Second, evaluation of pharmacokinetics

The pharmacokinetics of polymeric drugs were evaluated in female 6-week-old CD-1(ICR) mice. Specifically, the method comprises the following steps:

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 10mg/kg PTX. At various time intervals, 100 μ L of blood samples were collected from the mouse orbit and loaded into heparinized tubes and 10mL of ethyl acetate was added. Followed by centrifugation at 10000 Xg for 5 minutes, separation of the organic solvent and addition of N₂And (5) drying. Determining paclitaxel content by reverse phase high performance liquid chromatography (RP-HPLC) (mobile phase: acetonitrile/H)₂O (3: 7V/V), flow rate of 1mL/min, UV-VIS detection wavelength fixed at 217 nm).

Results as shown in fig. 16, fig. 16 is the change in blood at different times from PTX content after intravenous injection of PTX loaded P1, P2 or P3; as can be seen from fig. 16, PTX-loaded P1, P2, and P3 nanoparticles showed similar relatively long blood circulation times after intravenous injection. Half-lives t of P1, P2 and P3_1/23.36h, 4.82h and 3.92h respectively. Long blood circulation can improve the enrichment of anticancer drugs at tumor sites.

Construction of H22 mouse tumor model

Given the importance of MMP enzymes in the development of various tumors, we selected the H22 tumor model of female CD-1(ICR) mice for the study of anti-tumor effects. To quantify the distribution of PTX amounts at the tumor site and major organs, PTX was extracted from tumor tissues and major organs after intravenous injection of various pharmaceutical formulations and analyzed by HPLC systems. Specifically, H22 cells (3X 10) suspended in 200. mu.L of PBS⁶) The right lower axilla of female 6-week-old CD-1(ICR) mice were injected subcutaneously. Tumor size was monitored using a digital vernier caliper and tumor volume (V) was determined from the equation V ═ a × b²The/2 calculation, where a and b represent the longest and shortest diameters of the tumor, respectively.

In addition, H22 tumor-bearing mice were used for in vivo distribution of the nanoparticles studied. When the tumor volume reaches 100mm³DIR-loaded P1, P2 or P3 nanoparticles were injected intravenously at a dose of 1 mg/kg. At different time intervals (1, 12, 24, 48 and 72 hours), mice were anesthetized and images were acquired with the IVIS small animal imaging system.

The results are shown in fig. 17 and fig. 18, fig. 17 is a quantitative analysis of PTX in major organs and tumors after 12 and 24 hours of intravenous injection; figure 18 is the in vivo biodistribution profile of DIR fluorescent molecule loaded nanoparticles after intravenous injection; as can be seen from fig. 17, drug enrichment of P1 nanoparticles was significantly enhanced at the tumor site at 24 hours, 1.47-fold increased over P2 nanoparticles, 2.01-fold (P < 0.05) P3 nanoparticles, while the distribution of the drug in the major organs showed no significant difference. In order to be able to see the condition of the nanoparticles in vivo, a hydrophobic near infrared fluorescence emitting Dye (DIR) was embedded in the nanoparticles for real-time monitoring. As shown in fig. 18, it can be seen from fig. 18 that the tumor site showed significant DIR fluorescence 1 hour after iv injection of three DIR-loaded nanoparticles, reaching a maximum at 24 hours. Also, P1 nanoparticles for DIR loading showed slightly stronger fluorescence at 48 hours.

Distribution in vivo

The efficacy of the PTX loaded nanoparticles was also evaluated using H22 tumor-bearing mice. 25 mice were divided into 5 groups, including PBS, small molecule PTX, PTX-loaded P1, P2, and P3 nanoparticles. When the tumor grows to about 100mm³In time, different pharmaceutical formulations were injected intravenously every two days at a dose of 10mg/kg for a total of three injections.

To quantify the accumulation of PTX in major organs and tumors, the corresponding organ and tumor tissues were collected after intravenous injection of a 10mg/kg dose of PTX. The samples were then washed with PBS, dried and weighed, and 0.5ml CH was added₃CN, and centrifuged at 10000 Xg for 10 minutes, and then the supernatant was further treated with 1mL of CHCl₃And (4) extracting. The organic phase was dried under a stream of nitrogen and the residue was dissolved in 100. mu.L of methanol and the amount of PTX was determined by RP-HPLC.

The results are shown in fig. 19-20, fig. 19 is the results of tumor size changes after intravenous injection of PTX-loaded nanoparticles and small molecule PTX; data are presented as mean ± SD, N ═ 5, P < 0.05. Figure 20 is a photograph of typical tumors collected from different treatment groups on day 24. As can be seen from fig. 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, and 10-fold for P2. In contrast, PTX-loaded P1 nanoparticles effectively inhibited tumor growth, with only a 1.9-fold increase in tumor size.

Fifth, evaluation of antitumor Effect in animals

H22 tumor-bearing mice were randomly divided into 5 groups (n ═ 5). When the tumor volume reaches 100mm³Small molecule PTX, PTX-loaded P1, P2 and P3 nanoparticles were injected via tail vein at 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 a digital vernier caliper. Meanwhile, the body weight of the mice was recorded every two days. After the end of treatment (24 days), all mice were sacrificed and major organs (heart, liver, spleen, lung, kidney) and tumors were collected. The weight of the tumor was recorded, then the tumor and the organ were embedded in paraffin, and 5 μm paraffin sections were prepared. Slicing with hematoxylin and eosin (H)&E) And (6) dyeing. In addition, tumor sections were further stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit to identify apoptotic cells.

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

The results are shown in FIGS. 21 to 23, and FIG. 21 is a hematoxylin-eosin (H & E) staining result and a terminal deoxynucleotidyl transferase staining (TUNEL) fluorescence image of the tumor section; FIG. 22 is a graph of the body weight change of mice; FIG. 23 is an intravenous H & E staining of heart, liver, spleen, lung, kidney corresponding to PBS, PTX, P1, P2 and P3, respectively; wherein white arrows indicate liver injury. As can be seen from the figure, tumors treated with PTX-loaded P1 nanoparticles exhibited the strongest green fluorescence, indicating the most apoptosis. H & E staining also confirmed that the most significant efficacy was achieved by injection of PTX-loaded P1 nanoparticles. Furthermore, the body weight of five groups of mice did not change significantly during the treatment period; furthermore, as can be seen from fig. 13, the results of H & E staining of organs showed mainly liver damage caused by small molecule PTX, whereas the PTX loaded nanoparticle treated group had no apparent organ damage; these results show that the biosafety of P1, P2 and P3 nanoparticles is high.

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims

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

wherein m is 40-230, n is 50-100;

the amino acid sequence of Pep is GPLGVRGDG or GPLGVRG.

2. The polymeric drug carrier according to claim 1, wherein m is 80 to 150.

3. A preparation method of a matrix metalloproteinase responsive polymer drug carrier comprises the following steps:

1) reacting the end group azide polyethylene glycol with alkynyl functional polypeptide to obtain PEG-Pep with the end being amino,

the amino acid sequence of Pep is GPLGVRGDG or GPLGVRG;

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

4. The method according to claim 3, wherein the catalyst for the reaction in step 1) is cuprous bromide and N, N, N', N ", N" -pentamethyldiethylenetriamine.

5. The preparation method according to claim 3, wherein the catalyst for the reaction in step 2) is one or more of stannous octoate, stannous acetate, titanium dioxide and aluminum chloride.

6. A high molecular drug comprises a high molecular drug carrier and an antitumor drug;

wherein the polymeric drug carrier is the polymeric drug carrier according to any one of claims 1 to 3.

7. The polymeric drug of claim 6, wherein the anti-tumor drug is one or more of paclitaxel, camptothecin, hydrophobic platinum drugs, nitrogen mustard and doxorubicin.

8. A method for producing a polymer drug according to claim 6, comprising:

reacting the polymeric drug carrier according to any one of claims 1 to 2, an antitumor drug, a solvent and a buffer solution to obtain the polymeric drug.

9. The method according to claim 8, wherein the buffer solution is a phosphate buffer solution having a pH of 7.4.