Carrier-free self-assembled nanoparticles based on boron neutron capture therapy and their preparation method and application

Abstract

The present invention discloses a carrier-free self-assembled nanoparticle based on boron neutron capture therapy and its preparation method and application, comprising 10 B drug, polyphenol drug and metal ions self-assembled; 10 Drug B is a 10 B boron delivery agent; the polyphenol-containing drug is a compound containing phenolic hydroxyl groups; and the metal ion is a metal ion capable of coordinating with the phenolic hydroxyl groups. By utilizing the interaction between drug molecules, carrier-free self-assembled nanoparticles containing multiple drugs can be formed without the need for a carrier.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to carrier-free self-assembled nanoparticles based on boron neutron capture therapy, and a preparation method and application thereof. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Boron neutron capture therapy (BNCT) is a binary radiotherapy modality based on nuclear capture and fission reactions. 10 B) After being hit by thermal neutrons, nuclear capture and nuclear fission reactions occur, producing high-energy particles (α particles and 7 LI particles) are used to kill tumor cells by "point-targeted blasting." Compared with traditional radiotherapy, BNCT has great advantages such as precise tumor targeting, fewer side effects, and a shorter treatment cycle. The existing boron delivery agent, borophenylalanine (BPA), is a phenylalanine analog that can target tumors that highly express L-type amino acid transporter 1 (LAT1), thereby accumulating in tumor cells. However, BPA has poor water solubility and requires the use of large amounts of solubilizers in clinical practice. At the same time, clinical practice faces the challenge of real-time (non-invasive) detection of boron distribution and content in patients.

[0004] Nano drug delivery systems encapsulate drugs through carriers to improve problems such as poor water solubility of drugs. However, the drug loading capacity of existing carriers is low, which makes it difficult to meet the requirements of high-dose delivery by boron neutron capture. At the same time, there are problems such as complex preparation process and large toxic side effects of the carriers. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a carrier-free self-assembled nanoparticle based on boron neutron capture therapy and its preparation method and application. 10 The intermolecular interaction between drug B and polyphenol-containing drugs was used to construct boron drug-polyphenol self-assembled nanoparticles, and Gd 3+ Metal ions such as ions stabilize nanoparticles and also impart imaging capabilities. By utilizing the interaction between drug molecules, carrier-free self-assembled nanoparticles containing multiple drugs can be formed without the need for a carrier.

[0006] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0007] In the first aspect, the present invention provides a carrier-free self-assembled nanoparticle based on boron neutron capture therapy, comprising 10 Drug B, containing polyphenol drugs and metal ions, self-assembled;

[0008] The said 10Drug B is a 10 B Boron delivery agent;

[0009] The polyphenol-containing drug is a compound containing phenolic hydroxyl groups;

[0010] The metal ion is a metal ion that can coordinate with the phenolic hydroxyl group.

[0011] In some embodiments, the 10 The molar ratio of drug B to the polyphenol-containing drug is 0.5-6:1.

[0012] In some embodiments, the 10 Drug B is selected from one, two or more of BPA, 1-aminocyclobutane-carboxylic acid (ABCHC) or 1-amino-3-boronocyclopentacarboxylic acid (ABCPC).

[0013] In some embodiments, the polyphenol-containing drug is selected from one, two or more of epigallocatechin gallate (EGCG), chlorogenic acid or tannic acid.

[0014] In some embodiments, the metal ion is selected from Gd 3+ 、Fe 3+ or Sm 3+ One, two or more of.

[0015] Preferably, the 10 Drug B is BPA, the polyphenol-containing drug is epigallocatechin gallate, and the metal ion is selected from Gd 3+ In the present invention, it is named BPA-EGCG@Gd.

[0016] In a second aspect, the present invention provides a method for preparing carrier-free self-assembled nanoparticles based on boron neutron capture therapy, comprising the following steps:

[0017] will contain 10 Drug B and the polyphenol-containing drug are dissolved in an alkaline aqueous solution, to which the soluble salt of the metal ion is added, mixed evenly, and dialyzed to obtain the carrier-free self-assembled nanoparticles based on boron neutron capture therapy.

[0018] In some embodiments, the pH value of the alkaline aqueous solution is 7-14.

[0019] Preferably, the pH value of the alkaline aqueous solution is 8-11, more preferably 10. When the pH value is 10, the solubility of BPA is better and the self-assembly effect is better.

[0020] In some embodiments, the concentration of the metal ion is 0.1-0.5 mM, preferably 0.1-0.2 mM.

[0021] In some embodiments, 10 The concentration of drug B is 1-40 mg / mL, preferably 5-10 mg / mL.

[0022] In some embodiments, the concentration of the polyphenol-containing drug is 4.4-8.8 mg / mL.

[0023] In some embodiments, the method for preparing carrier-free self-assembled nanoparticles based on boron neutron capture therapy comprises the following steps: dissolving BPA and EGCG in an alkaline aqueous solution, respectively, adding the alkaline aqueous solution of BPA dropwise to the aqueous solution of EGCG under stirring, dialyzing the obtained aqueous solution with distilled water to remove free drugs and alkaline solution to obtain a brown-red nanoparticle solution, and refrigerating the nanoparticle aqueous solution at a low temperature (4° C.) away from light; or storing the freeze-dried powder at -20° C. after freeze-drying.

[0024] Preferably, the volume ratio of the BPA alkaline aqueous solution to the EGCG alkaline aqueous solution is 1:1-2, preferably 1:1.

[0025] In a third aspect, the present invention provides a pharmaceutical composition comprising the carrier-free self-assembled nanoparticles based on boron neutron capture therapy.

[0026] In some embodiments, the pharmaceutical composition further comprises at least one other pharmaceutically inactive ingredient or pharmaceutically inactive ingredient.

[0027] The inactive pharmaceutical ingredients may be carriers, excipients, and diluents commonly used in pharmacy. The inactive pharmaceutical ingredients such as carriers, excipients, and diluents that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards.

[0028] The fourth aspect of the present invention provides a drug carrier or a drug delivery system, wherein the drug carrier or the drug delivery system comprises at least the carrier-free self-assembled nanoparticles of the first aspect or the pharmaceutical composition of the third aspect.

[0029] The fifth aspect of the present invention provides the use of the carrier-free self-assembled nanoparticles of the first aspect, the pharmaceutical composition of the third aspect, the drug carrier or the drug delivery system of the fourth aspect in BNCT drug delivery.

[0030] The beneficial effects achieved by one or more embodiments of the present invention are as follows:

[0031] 1) The present invention prepares a carrier-free self-assembled nanoparticle for BNCT. The nanoparticle is carrier-free, has a high drug content, is simple to prepare, and has good biocompatibility, addressing the shortcomings of the existing drug BPA, which has poor water solubility and low drug loading;

[0032] 2) The nanoparticles prepared by the present invention have minimal toxicity and side effects, can effectively accumulate tumors at the cellular level, and have a certain magnetic resonance imaging effect. Therefore, they have good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0034] Figure 1 This is the particle size distribution diagram of BPA-EGCG@Gd nanoparticles in Example 1 of the present invention;

[0035] Figure 2 This is a picture of the appearance of the BPA-EGCG@Gd nanoparticle solution of Example 1 of the present invention;

[0036] Figure 3 This is a stability picture of the BPA-EGCG@Gd nanoparticle solution of Example 3 of the present invention;

[0037] Figure 4 This is a picture of the biosafety of BPA-EGCG@Gd nanoparticles in Example 1 of the present invention;

[0038] Figure 5 This is a picture of the cellular uptake of BPA-EGCG@Gd nanoparticles in Example 1 of the present invention;

[0039] Figure 6 This is a picture of in vitro nuclear magnetic resonance imaging of BPA-EGCG@Gd nanoparticles in Example 1 of the present invention. DETAILED DESCRIPTION

[0040] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0041] The pharmaceutical composition can be formulated into a pharmaceutical preparation for oral administration or injection, preferably for injection. The nanoparticles of the present invention have a particle size of 150-200 nm, are stable, and are suitable for intravenous injection. They can also be freeze-dried for easy storage and use. In some embodiments, the concentration of BPA in the intravenous injection of the present invention can reach 20 mg / ml or higher.

[0042] The drug can be used to prevent, treat and alleviate benign or malignant tumors or other indications for BNCT treatment; the cancers or tumors include but are not limited to melanoma, head and neck cancer, glioma, breast cancer, lung cancer, colon cancer, etc., including solid tumors and hematological tumors, preferably solid tumors, and BNCT indications are mostly solid tumors.

[0043] The drugs of the present invention can be administered to the body in a known manner. For example, they can be delivered to the tissue of interest by intravenous systemic delivery or local injection (such as intratumoral or peritumoral injection). Such administration can be carried out via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be administered in the present invention can vary to a great extent depending on a variety of factors, such as the target cell, the type of organism or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0044] The subjects of drug administration can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc.

[0045] A method for treating tumors with BNCT, comprising administering a therapeutically effective dose of the aforementioned carrier-free self-assembling nanoparticles to a subject. The subject is an animal, preferably a mammal, and most preferably a human, that has been the subject of treatment, observation, or experiment. The term "therapeutically effective amount" refers to the amount of an active compound or agent, including a compound of the present invention, that elicits the biological or medical response sought by the researcher, veterinarian, physician, or other medical professional in a tissue system, animal, or human, including alleviation or partial alleviation of the symptoms of the disease, syndrome, condition, or disorder being treated. It should be understood that the optimal dosage and interval of administration of the active ingredient of the present invention are determined by its properties and external conditions such as the form, route, and site of administration, as well as the specific mammal being treated. This optimal dosage can be determined using conventional techniques. It should also be understood that the optimal course of treatment, i.e., the daily dosage of the compound over a specified period of time, can be determined using methods known in the art.

[0046] The present invention is further explained by the following examples, but is not intended to limit the present invention. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0047] Example 1 (Preparation of BPA-EGCG@Gd Nanoparticles)

[0048] Accurately weigh a certain amount of BPA and EGCG powder, dissolve them in water respectively, prepare 6 mg / ml and 4.4 mg / ml aqueous solutions, and adjust the pH to 10. Ultrasound is used to completely dissolve them. According to the volume ratio of 1:1, the BPA aqueous solution is added dropwise to the EGCG aqueous solution. After stirring for 2 hours, 0.19 mM GdCl3 aqueous solution is added dropwise and stirred for 1 hour to obtain a brown-red solution. Dialyze with distilled water for 4 hours to remove free drugs and alkaline substances to obtain BPA-EGCG@Gd nanoparticles. In addition, it can be freeze-dried for 2 days to obtain BPA-EGCG@Gd nanoparticle freeze-dried powder, which is dissolved with distilled water before use. Figure 1 As shown in Figure 2, the particle size of the BPA-EGCG@Gd nanoparticle solution is about 171 nm, with uniform size and good dispersion. Figure 2 shown.

[0049] Example 2 (BPA-EGCG@Gd nanoparticle stability verification)

[0050] Accurately weigh a certain amount of BPA and EGCG powder, dissolve them in water respectively, prepare 6mg / ml and 4.4mg / ml aqueous solutions, and adjust the pH to 10. Ultrasound to completely dissolve it. According to the volume ratio of 1:1, add the BPA aqueous solution dropwise to the EGCG aqueous solution. After stirring for 2 hours, add 0.19mM GdCl3 aqueous solution and stir for 1 hour to obtain a brown-red solution. Dialyze with distilled water for 4 hours to remove free drugs and alkaline substances to obtain BPA-EGCG@Gd nanoparticles. Take the above nanoparticle aqueous solution in a glass bottle and measure its nanoparticle size and potential within 0-7 days. Figure 3 As shown in the figure, there was no significant change in the particle size and potential of the nanoparticles from 0 to 7 days, indicating that the BPA-EGCG@Gd nanoparticles remained stable over a period of time.

[0051] Example 3 (BPA-EGCG@Gd Nanoparticle Biosafety Verification)

[0052] Cell Culture: Human umbilical vein endothelial cells (HUVECs) were selected for research. Frozen cells were cultured in DMEM supplemented with 1% penicillin-streptomycin solution and 10% fetal bovine serum at 37°C and 5% CO2. Cells were passaged when they reached the logarithmic growth phase and transferred to culture flasks at a ratio of 1 to 3 cells for further culture and cell counting.

[0053] Preparation of drug solution: Accurately weigh a certain amount of BPA-EGCG@Gd nanoparticles and use 1% penicillin-streptomycin solution and 10% fetal bovine serum in 1640 culture medium to prepare a 100-800 μg / ml nanoparticle culture medium solution for later use.

[0054] Cytotoxicity assay: HUVECs cells in the logarithmic growth phase were collected and 5×10 3 Cells were seeded at a density of 100 μg / ml in a 96-well plate overnight. The old culture medium was discarded, and 0-800 μg / ml of nanoparticle culture medium solution was added to each well. A well without BPA-EGCG@Gd (0 μg / ml nanoparticle culture medium solution) served as a positive control, and a well without cells was added as a negative control (n=3). After incubation for 24 hours, the drug-containing culture medium was removed, and 100 μl of culture medium and 10 μl of CCK-8 reagent were added to each well. The cells were incubated for 0.5-4 hours. The absorbance at 450 nm was measured using a microplate reader, and the cell viability (%) was calculated according to the following formula.

[0055] Cell survival rate (%) = [(As-Ab) / (Ac-Ab)] × 100%;

[0056] Where: As = absorbance of experimental wells [absorbance of wells containing cells (after incubation with BPA-EGCG@Gd), culture medium, and CCK-8]; Ab = absorbance of blank wells (absorbance of wells containing culture medium and CCK-8); Ac = absorbance of control wells [absorbance of wells containing cells (unincubated), culture medium, and CCK-8].

[0057] like Figure 4 As shown in the results, BPA-EGCG@Gd nanoparticles had no significant cytotoxicity to HUVECs cells and had good biosafety.

[0058] Example 4 (Verification of BPA-EGCG@Gd Cellular Uptake)

[0059] Cell Culture: Murine melanoma cell line B16F10 was selected for research. Frozen cells were cultured in 1640 medium supplemented with 1% penicillin-streptomycin solution and 10% fetal bovine serum at 37°C and 5% CO₂. Cells were passaged when they reached the logarithmic growth phase and transferred to culture flasks at a ratio of 1 to 4 for continued culture and cell counts were performed.

[0060] Drug solution preparation: Accurately weigh a certain amount of BPA-EGCG@Gd nanoparticles and Cy5-NH2, dissolve them in an EDC / NHS aqueous solution, stir for 12 hours, dialyze for 24 hours, and then lyophilize. Accurately weigh a certain amount of Cy5-modified BPA-EGCG@Gd nanoparticles and prepare a nanoparticle culture solution containing 600 μg / ml in 1640 medium containing 1% penicillin-streptomycin solution and 10% fetal bovine serum.

[0061] Cell uptake experiment: B16F10 cells in the logarithmic growth phase were collected and 2×10 5Cells were seeded at a high density in six-well plates overnight. At time points between 1 and 24 hours, 2 ml of a 600 μg / ml nanoparticle culture medium solution was added to each well. After incubation for various times, the cells were washed twice with PBS and digested with trypsin. The culture medium was then digested, centrifuged, and resuspended in PBS. Cellular uptake was measured using flow cytometry.

[0062] like Figure 5 As shown, the uptake behavior of BPA-EGCG@Gd nanoparticles by B16F10 cells showed a time-dependent characteristic, with the uptake time being prolonged and the fluorescence signal of the drug in the cells being enhanced.

[0063] Example 5 (Verification of in vitro and in vivo imaging effects of BPA-EGCG@Gd nanoparticles)

[0064] A certain amount of BPA-EGCG@Gd nanoparticles was weighed and prepared into a series of Gd concentration solutions of 0-0.4 mM using distilled water. The Gd concentrations were measured by nuclear magnetic resonance imaging. Figure 6 As shown, the magnetic resonance imaging of BPA-EGCG@Gd nanoparticles with different concentrations shows that BPA-EGCG@Gd nanoparticles can provide higher imaging contrast for magnetic resonance imaging.

[0065] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A carrier-free self-assembled nanoparticle based on boron neutron capture therapy, characterized in that: By Han 10 Drug B, containing polyphenol drugs and metal ions, self-assembled; The said 10 Drug B is a 10 B boron delivery agent; 10 Drug B is selected from para-boronophenylalanine; The polyphenol-containing drug is a compound containing phenolic hydroxyl groups; the polyphenol-containing drug is selected from one or both of epigallocatechin gallate and tannic acid; The metal ion is a metal ion capable of coordinating with a phenolic hydroxyl group; the metal ion is selected from Gd 3＋ 、Fe 3＋ or Sm 3＋ One, two or more of.

2. The carrier-free self-assembled nanoparticles based on boron neutron capture therapy according to claim 1, characterized in that: The said 10 The molar ratio of drug B to the polyphenol-containing drug is 0.5-6:

1.

3. The carrier-free self-assembled nanoparticles based on boron neutron capture therapy according to claim 1, characterized in that: The said 10 Drug B is para-boron phenylalanine, the polyphenol-containing drug is epigallocatechin gallate, and the metal ion is selected from Gd 3＋ .

4. The method for preparing carrier-free self-assembled nanoparticles based on boron neutron capture therapy according to any one of claims 1 to 3, characterized in that: The steps include: will contain 10 Drug B and the polyphenol-containing drug are dissolved in an alkaline aqueous solution, to which the soluble salt of the metal ion is added, mixed evenly, and dialyzed to obtain the carrier-free self-assembled nanoparticles based on boron neutron capture therapy.

5. The method for preparing carrier-free self-assembled nanoparticles based on boron neutron capture therapy according to claim 4, characterized in that: The pH value of the alkaline aqueous solution is 7-14.

6. The method for preparing carrier-free self-assembled nanoparticles based on boron neutron capture therapy according to claim 5, characterized in that: The pH value of the alkaline aqueous solution is 8-11.

7. The method for preparing carrier-free self-assembled nanoparticles based on boron neutron capture therapy according to claim 4, characterized in that: The concentration of metal ions is 0.1-0.5 mM.

8. The method for preparing carrier-free self-assembled nanoparticles based on boron neutron capture therapy according to claim 7, characterized in that: The concentration of metal ions is 0.1-0.2 mM.

9. The method for preparing carrier-free self-assembled nanoparticles based on boron neutron capture therapy according to claim 4, characterized in that: Contains 10 The concentration of drug B is 1-40 mg / mL.

10. The method for preparing carrier-free self-assembled nanoparticles based on boron neutron capture therapy according to claim 9, characterized in that: Contains 10 The concentration of drug B is 5-10 mg / mL.

11. The method for preparing carrier-free self-assembled nanoparticles based on boron neutron capture therapy according to claim 4, characterized in that: The concentration of polyphenol-containing drugs was 4.4-8.8 mg / mL.

12. The method for preparing carrier-free self-assembled nanoparticles based on boron neutron capture therapy according to claim 4, characterized in that: The method specifically comprises the following steps: dissolving p-boronophenylalanine and EGCG in an alkaline aqueous solution respectively, adding the alkaline aqueous solution of p-boronophenylalanine dropwise into the aqueous solution of EGCG under stirring conditions, dialyzing the obtained aqueous solution with distilled water to remove free drugs and the alkaline solution, and obtaining a brown-red nanoparticle solution.

13. The method for preparing carrier-free self-assembled nanoparticles based on boron neutron capture therapy according to claim 12, characterized in that: The volume ratio of the alkaline aqueous solution of boron phenylalanine to the alkaline aqueous solution of EGCG is 1:1-2.

14. The method for preparing carrier-free self-assembled nanoparticles based on boron neutron capture therapy according to claim 13, characterized in that: The volume ratio of the alkaline aqueous solution of borophenylalanine to the alkaline aqueous solution of EGCG is 1:

1.

15. A pharmaceutical composition, characterized in that: The invention comprises the carrier-free self-assembled nanoparticles based on boron neutron capture therapy according to any one of claims 1 to 3.

16. The pharmaceutical composition according to claim 15, characterized in that: The pharmaceutical composition further comprises at least one other pharmaceutically active ingredient or pharmaceutically inactive ingredient.

17. A drug carrier or drug delivery system, characterized in that: The drug carrier or drug delivery system comprises at least the carrier-free self-assembled nanoparticles based on boron neutron capture therapy according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 15 or 16.

18. Use of the carrier-free self-assembled nanoparticles based on boron neutron capture therapy according to any one of claims 1 to 3, or the pharmaceutical composition according to claim 15 or 16, or the drug carrier or drug delivery system according to claim 17 in the preparation of BNCT delivery drugs.

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