A method for targeted accumulation of drugs in tumor cells

Abstract

The present application relates to the technical field of drug targeting accumulation, and particularly relates to a method for targeting accumulation of drugs in tumor cells. The method for targeting accumulation of drugs in tumor cells comprises: achieving the targeting accumulation of drugs in tumor cells through in-vitro shock wave treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug targeting accumulation, in particular to a method for targeting accumulation of drugs in tumor cells. BACKGROUND

[0002] In boron neutron capture therapy of tumor, boron neutron capture therapy (BNCT) utilizes 10 B atom captures thermal neutron to generate 10 B(n, α) 7 Li nuclear reaction to generate high linear energy transfer α particle ( 4 He) and recoil lithium nucleus 7 Li, which can achieve cell-level precise binary radiotherapy. In more than 2,000 clinical treatment cases of advanced tumors worldwide, BNCT shows superior treatment effect and future treatment advantage, and its treatment effect is gradually gaining wide recognition in the medical field.

[0003] BNCT relies on the nuclear reaction of thermal neutron and 10B atom to achieve precise killing of single tumor cells. To achieve effective BNCT treatment, it is necessary for 10 B compound to selectively target tumor tissue and reach a high enough accumulation concentration (>20ppm) and accumulate in tumor tissue for a sufficient time. For example, the boron targeting accumulation effect of boronophenylalanine used in clinical treatment is insufficient, and a large dose (500mg / kg) is required for administration, and a dose of 200mg / kg is usually required for intravenous infusion during neutron irradiation to maintain boron accumulation in tumor tissue, which brings great inconvenience to treatment. Therefore, the amount and time of boron accumulation in tumor tissue are crucial for the treatment plan and treatment effect of BNCT.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] Extracorporeal shock wave (hereinafter also referred to as "ESW") is a mechanical wave, which was first applied to ureteral lithotripsy in clinical practice. At present, it has become an effective clinical treatment method for bone and muscle diseases due to its characteristics of inducing new bone formation, improving bone tissue microstructure, reducing bone mass loss, and enhancing local bone quality, as well as good biocompatibility and good compatibility with blood vessels and nerves.

[0006] The present application finds that, compared with other mechanical waves (such as sound waves), ESW will cause a sudden change in the properties of energy-absorbing medium under the triple action of its unique radial compression, cavitation effect and layer splitting effect, which can promote the accumulation of drugs in tumor tissue.

[0007] Based on this, the present application has the following technical solutions:

[0008] In a first aspect, the present application provides a method for targeted accumulation of a drug in tumor cells, comprising: achieving targeted accumulation of a drug in tumor cells by in vitro shock wave treatment.

[0009] Preferably, the energy of the in vitro shock wave treatment is 1-4 Bar.

[0010] Preferably, the number of impacts of the in vitro shock wave treatment is 200-500.

[0011] In the present application, the in vitro shock wave treatment is effective when applied in vivo for more than 2000 times.

[0012] Preferably, the drug comprises one or more of a boron drug, doxorubicin, paclitaxel and cisplatin; the boron drug comprises BPA, BSH or modified BPA or BSH.

[0013] In the present application, BPA and BSH and modified BPA or BSH are commonly used boron drugs in BNCT treatment. Among them, BPA refers to (L)-4-dihydroxyboron phenylalanine, which is a boron-containing amino acid derivative; BSH refers to sulfydodecaborane dianion salt.

[0014] In the present application, the modified BPA or BSH can be PAMAM-BPA modified by polyethyleneimine (PAMAM) and the like.

[0015] In the present application, the above-mentioned drug can be made into a small molecule drug form, a high molecular drug form or a nano drug form according to actual needs, which is not limited here.

[0016] Further preferably, the drug comprises a boron drug.

[0017] In the specific implementation process, since the effective treatment of BNCT requires 10 The B compound selectively targets in tumor tissues and reaches a high enough accumulation concentration, i.e. the accumulation amount and time of the boron drug in the tumor tissue are crucial for the treatment of BNCT, therefore, the in vitro shock wave treatment is particularly suitable for the targeted accumulation of the boron drug.

[0018] Preferably, the tumor cells comprise K7M2 tumor cells, U2OS tumor cells, 4T1 tumor cells or HepG2 tumor cells.

[0019] Preferably, the method for targeted accumulation of a drug in tumor cells comprises:

[0020] S1: inoculating and culturing tumor cells in a culture medium;

[0021] S2: replace the culture medium in S1 with a culture medium containing a drug, and incubate;

[0022] S3: perform in vitro shock wave treatment on the tumor cells in S2.

[0023] In the present application, the culture medium in S1 is mainly composed of DMEM medium and fetal bovine serum (FBS); preferably, the specific formula is that DMEM medium accounts for 90-95%, and FBS accounts for 5-10%.

[0024] The culture medium containing a drug in S2 is the above-mentioned culture medium in S1 with the drug added; preferably, the concentration of the drug in the culture medium containing a drug is 0.001-100 mg / ml; preferably, the concentration of the drug is 0.1-10 mg / ml; for example, the concentration of the drug can be 0.1 mg / ml, 0.5 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml or 10 mg / ml, etc.

[0025] Preferably, the standard condition for culturing the tumor cells is about 37 degrees Celsius, and the gas phase composition is air and carbon dioxide; further preferably, the gas phase composition is air 95% and carbon dioxide 5%.

[0026] As a preference, the incubation time in S2 is within 48 h; preferably, the incubation time is within 24 h; for example, the incubation time can be 0.1 h, 0.2 h, 0.3 h, 0.4 h, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h or 24 h, etc.; it can also be a shorter time within 0.1 h, such as immediately performing in vitro shock wave treatment after replacing the culture medium.

[0027] As a preferred embodiment of the present application, the method for targeted accumulation of boron drugs in tumor cells comprises:

[0028] S1: inoculate and culture tumor cells in a culture medium;

[0029] S2: replace the culture medium in S1 with a culture medium containing a drug, and incubate for 24 h or less, and then perform in vitro shock wave treatment; the energy of the in vitro shock wave treatment is 1-4 Bar; the number of shocks is 200-500;

[0030] S3: wash the tumor cells with PBS.

[0031] In a second aspect, the present application provides use of extracorporeal shock wave in targeting accumulation of a drug in tumor cells.

[0032] In a third aspect, the present application provides use of the method for targeting accumulation of a drug in tumor cells in boron neutron capture therapy.

[0033] Based on the above technical solution, the present application has the following beneficial effects:

[0034] The present application can promote the targeting accumulation of a drug in tumor cells by extracorporeal shock wave treatment of the tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0036] Fig. 1 is a fluorescence photo of ESW promoting K7M2 tumor cells to uptake doxorubicin-loaded PLGA nanoparticles K7M2 in Example 1 and Comparative Example 1 of the present application, blue is the cell nucleus, and red is DOX fluorescence; the scale is 10 μm;

[0037] Fig. 2 is a flow cytometry analysis of ESW promoting K7M2 tumor cells to uptake doxorubicin-loaded PLGA nanoparticles K7M2 in Example 1 and Comparative Example 1 of the present application;

[0038] Fig. 3 is a statistical result of intracellular DOX fluorescence in Example 1 and Comparative Example 1 of the present application;

[0039] Fig. 4 is an effect of ESW enhancing K7M2 tumor cells to uptake PAMAM-BSH boron drugs in Example 2-7 of the present application;

[0040] Fig. 5 is an effect of ESW enhancing K7M2 tumor cells to uptake BPA boron drugs in Example 8-13 of the present application. DETAILED DESCRIPTION

[0041] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below in combination with the drawings in the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0042] Unless otherwise specified, the various raw materials used in the embodiments are all commercially available conventional raw materials, and the technical means used is the conventional means well known to those skilled in the art. In the present embodiment, the nano-drug is a PLGA nanoparticle, which is commercially available.

[0043] Example 1 ESW enhances the uptake of K7M2 tumor cells to nano-drugs (loaded with doxorubicin)

[0044] The present embodiment provides a method for targeted accumulation of drugs in tumor cells, comprising:

[0045] K7M2 tumor cells were inoculated at 2*105 in a 35mm culture dish and placed in a 5% CO2, 37℃ incubator for 24 hours. The culture medium was aspirated and 1ml of 1mg / ml nano-drug containing doxorubicin was added. After 2h of incubation, ESW treatment (2Bar, 200 times) was performed, and the cells were washed with PBS for 3 times. The fluorescence intensity of doxorubicin in the cells was detected by fluorescence inverted microscope and flow cytometry, respectively.

[0046] The test results are shown in Table 1 Figs. 1-3 wherein, Figs. 1-3 The ESW+ nano-drug group in Table 1 is the experimental result of Example 1, the nano-drug group is the experimental result of Comparative Example 1, and the control group is the experimental result without ESW treatment and without adding drugs; nano-drug refers to nano-drug loaded with doxorubicin.

[0047] In the present application, ESW treatment also has a considerable targeted accumulation effect on other chemotherapeutic drugs other than doxorubicin, which will not be described here.

[0048] Example 2 ESW enhances the uptake of K7M2 tumor cells to PAMAM-BSH drugs

[0049] The present embodiment provides a method for targeted accumulation of drugs in tumor cells, comprising:

[0050] K7M2 tumor cells were seeded at 2*105 in 35mm dishes and incubated in a 5% CO2, 37°C incubator for 24 hours. The medium was removed and 1ml of 1mg / ml PAMAM-BSH containing medium was added. After 2 hours incubation, ESW treatment was performed (4 Bar, 500 times) and the cells were washed with PBS three times and collected. After cell counting, the boron content in the cells was detected by ICP-MS.

[0051] Example 3 ESW enhances the uptake of PAMAM-BSH drug by K7M2 tumor cells

[0052] The method for targeted accumulation of drugs in tumor cells provided in this example differs from that of Example 2 only in that the ESW treatment is performed under the following conditions: 4 Bar, 200 times.

[0053] Example 4 ESW enhances the uptake of PAMAM-BSH drug by K7M2 tumor cells

[0054] The method for targeted accumulation of drugs in tumor cells provided in this example differs from that of Example 2 only in that the ESW treatment is performed under the following conditions: 2 Bar, 500 times.

[0055] Example 5 ESW enhances the uptake of PAMAM-BSH drug by K7M2 tumor cells

[0056] The method for targeted accumulation of drugs in tumor cells provided in this example differs from that of Example 2 only in that the ESW treatment is performed under the following conditions: 2 Bar, 200 times.

[0057] Example 6 ESW enhances the uptake of PAMAM-BSH drug by K7M2 tumor cells

[0058] The method for targeted accumulation of drugs in tumor cells provided in this example differs from that of Example 2 only in that the ESW treatment is performed under the following conditions: 1 Bar, 500 times.

[0059] Example 7 ESW enhances the uptake of PAMAM-BSH drug by K7M2 tumor cells

[0060] The method for targeted accumulation of drugs in tumor cells provided in this example differs from that of Example 2 only in that the ESW treatment is performed under the following conditions: 1 Bar, 200 times.

[0061] The drug accumulation results of Examples 2-7 are shown in Table 1. Fig. 4 .

[0062] In the present application, ESW treatment also has a comparable targeted accumulation effect on other modified boron drugs other than PAMAM-BSH drug, which will not be described here.

[0063] Example 8 ESW enhances K7M2 tumor cell uptake of BPA drug

[0064] This example provides a method for targeted accumulation of a drug in tumor cells, comprising:

[0065] K7M2 tumor cells were seeded at 2*105 in 35mm dishes and incubated in a 5% CO2, 37°C incubator for 24 hours. 1ml of 1mg / ml fructose-BPA containing media was added and the cells were treated with ESW (4 Bar, 500 cycles). After 2H incubation, the cells were washed 3 times with PBS and harvested. After cell counting, the boron content in the cells was measured by ICP-MS.

[0066] Example 9 ESW enhances K7M2 tumor cell uptake of BPA drug

[0067] This example provides a method for targeted accumulation of a drug in tumor cells, which differs from Example 8 only in that the ESW treatment is at 4 Bar, 200 cycles.

[0068] Example 10 ESW enhances K7M2 tumor cell uptake of BPA drug

[0069] This example provides a method for targeted accumulation of a drug in tumor cells, which differs from Example 8 only in that the ESW treatment is at 2 Bar, 500 cycles.

[0070] Example 11 ESW enhances K7M2 tumor cell uptake of BPA drug

[0071] This example provides a method for targeted accumulation of a drug in tumor cells, which differs from Example 8 only in that the ESW treatment is at 2 Bar, 200 cycles.

[0072] Example 12 ESW enhances K7M2 tumor cell uptake of BPA drug

[0073] This example provides a method for targeted accumulation of a drug in tumor cells, which differs from Example 8 only in that the ESW treatment is at 1 Bar, 500 cycles.

[0074] Example 13 ESW enhances K7M2 tumor cell uptake of BPA drug

[0075] This example provides a method for targeted accumulation of a drug in tumor cells, which differs from Example 8 only in that the ESW treatment is at 1 Bar, 200 cycles.

[0076] The drug accumulation results of Examples 8-13 are shown in Fig. 5 .

[0077] In this invention, ESW treatment also has a considerable targeted accumulation effect on boron drugs other than BPA drugs, which will not be elaborated here.

[0078] Comparative Example 1

[0079] This comparative example provides a method for targeted accumulation of drugs in tumor cells, which differs from Example 1 only in that: in vitro shock wave treatment is not performed.

[0080] The test results are shown in Figs. 1-3 .

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for targeted accumulation of drugs in in vitro tumor cells, characterized in that, include: Targeted accumulation of boron drugs in tumor cells is achieved through extracorporeal shock wave treatment with energy of 1-4 Bar. The tumor cells are: K7M2 tumor cells, U2OS tumor cells, 4T1 tumor cells, or HepG2 tumor cells. The boron drug is BPA, BSH, or modified BPA or BSH. The boron drug is a nano-drug.

2. The method for targeted accumulation of drugs in in vitro tumor cells according to claim 1, characterized in that, The number of shocks in the extracorporeal shock wave treatment is 200 to 500.

3. The method for targeted accumulation of drugs in in vitro tumor cells according to claim 1 or 2, characterized in that, include: S1: Inoculate and culture tumor cells in a culture medium; S2: Replace the culture medium in S1 with a culture medium containing boron and incubate it. S3: Treat the tumor cells in S2 with extracorporeal shock wave.

4. The method for targeted accumulation of drugs in in vitro tumor cells according to claim 3, characterized in that, The concentration of boron in the culture medium containing boron in S2 is 0.001~100 mg / mL.

5. The method for targeted accumulation of drugs in in vitro tumor cells according to claim 3, characterized in that, The concentration of boron in the culture medium containing boron in S2 is 0.1~10 mg / mL.

6. The method for targeted accumulation of drugs in in vitro tumor cells according to claim 4 or 5, characterized in that, The incubation time in S2 is within 48 hours.

7. The method for targeted accumulation of drugs in in vitro tumor cells according to claim 3, characterized in that, The incubation time in S2 is within 48 hours.

8. The method for targeted accumulation of drugs in in vitro tumor cells according to claim 4 or 5, characterized in that, The incubation time in S2 is within 24 hours.

9. The method for targeted accumulation of drugs in in vitro tumor cells according to claim 3, characterized in that, The incubation time in S2 is within 24 hours.

10. The method for targeted accumulation of drugs in in vitro tumor cells according to claim 1, characterized in that, include: S1: Inoculate and culture tumor cells in a culture medium; S2: Replace the culture medium in S1 with a culture medium containing boron, and incubate for no more than 24 hours, followed by in vitro shock wave treatment; the energy of the in vitro shock wave treatment is 1~4 Bar; the number of shocks is 200~500. S3: Wash tumor cells with PBS.

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