ABT-263-loaded drug delivery system as well as preparation method and application thereof

Abstract

An ABT-263-loaded drug delivery system comprises a liposome and elemental gold, ABT-263 is loaded in the liposome, and the liposome is coated with the elemental gold to form a gold shell (Lipo-atABT263-atAu). Through verification, the senescent cell scavenger (ABT-263) and the golden shell liposome are integrated together to achieve the purposes of sensitizing radiation and continuously releasing drugs. In in-vitro experiments, the Lipo-ABT263-coated Au enhances cell apoptosis induced by radiation therapy, eliminates radiation-induced senescence tumor cells, and inhibits the tumor promoting effect of senescence-related secretion phenotypes. In an animal model, a drug delivery system remarkably inhibits tumor growth, skin fibrosis induced by radiotherapy is relieved through targeted aging fibroblasts, and systemic toxicity is not shown.

Description

Technical Field

[0001] The present invention relates to a composition, in particular to a nanotechnology-based composition for integrated targeted elimination of senescent cells, sensitization to radiotherapy, and inhibition of skin fibrosis. Background Art

[0002] Currently, radiotherapy is used to treat over 50% of malignant tumors, but it is plagued by issues such as radioresistance and off-target tissue damage. These issues are partially mediated by the generation of senescent cells. While some current anti-senescence drugs, such as ABT-263 (a Bcl-2 inhibitor), can eliminate senescent cells, they lack the ability to release them over a sustained period. Because the generation of senescent cells through radiotherapy is time-dependent, there is a particular need for a senescent remover that matches the rate of senescent cell generation after radiation. Summary of the Invention

[0003] One object of the present invention is to provide an ABT-263-loaded drug delivery system that can sustainably release ABT-263 and achieve synchronous targeted clearance of senescent cells.

[0004] Another object of the present invention is to provide an ABT-263-loaded drug delivery system that can sustainably release ABT-263 to enhance the sensitivity of tumor radiotherapy.

[0005] Another object of the present invention is to provide an ABT-263-loaded drug delivery system that can slowly release ABT-263, eliminate senescent fibroblasts, and inhibit skin fibrosis.

[0006] Another object of the present invention is to provide a method for preparing an ABT-263 drug delivery system to obtain a sustained-release ABT-263 drug delivery system.

[0007] A fifth object of the present invention is to provide an ABT-263 drug delivery system for use in preparing drugs for synchronously eliminating senescent cells, radiotherapy sensitizers, or inhibiting skin fibrosis.

[0008] After radiotherapy, the number of senescent cells increases with time, showing a time-dependent increase in senescent cells. Therefore, the amount of drug administered within 24 hours to eliminate senescent cells is crucial for the subsequent treatment effect. If the dosage is too small, senescent cells will accumulate significantly, while if the dosage is too large, the elimination of senescent cells will not be sustained.

[0009] Therefore, the present invention selects ABT-263 as the active ingredient and uses liposomes as the main carrier to achieve ABT-263 release behavior that meets the characteristic of time-dependent increase of senescent cells after radiotherapy. It not only maintains the amount of drug provided to eliminate senescent cells within 24 hours of administration, but also takes into account the continuity of administration (for example: 4 to 5 days), which is beneficial to protect normal tissues.

[0010] The invention discloses an ABT-263-loaded drug delivery system, comprising liposomes and elemental gold. ABT-263 is loaded in the liposomes, and the liposomes are covered with elemental gold to form a gold shell.

[0011] The encapsulation efficiency of the drug delivery system is greater than 90 wt% (for example, 93.11 wt%), and the loaded ABT-263 drug concentration is 3.662 μg / mL, which can achieve the effect of clearing senescent tumor cells and fibroblasts.

[0012] Liposomes have a nanometer particle size, for example, 180nm ± 10nm, and include phospholipids (such as lecithin, DSPC, and DOPC), cholesterol, and auxiliary additives (such as fatty acids and polyethylene glycol).

[0013] Another ABT-263-loaded drug delivery system also includes an antioxidant, such as vitamin E.

[0014] Another ABT-263-loaded drug delivery system further includes a lyoprotectant, such as sucrose and mannitol.

[0015] Another ABT-263-loaded drug delivery system further includes an osmotic pressure regulator, such as sodium chloride.

[0016] Another ABT-263-loaded drug delivery system includes lecithin, cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-thiol (DSPE-PEG-SH), with the amounts used being, for example, at a molar ratio of 10:4:1.

[0017] Another ABT-263-loaded drug delivery system further includes glutathione (GSH), which is attached to the surface of the liposome.

[0018] The gold coating effectively increased the release of ABT-263 within 24 hours of administration while also significantly reducing the pH-dependent release of ABT-263. In a pH 7.4 buffer, Lipo@ABT263@Au released 36.08% of ABT-263 within 24 hours, and 48.68% at pH 5.5. This is comparable to the proportion of senescent tumor cells produced within 24 hours, demonstrating sufficient clearance for senescent cells.

[0019] The drug delivery system provided by the present invention integrates nanoliposomes, ABT-263, and a gold shell. The resulting Lipo@ABT263@Au is capable of slow and sustained release of ABT-263, with its release behavior synchronizing with the time-dependent elimination of senescent cells following radiotherapy. Clearing senescent tumor cells can overcome radioresistance, while clearing senescent fibroblasts can mitigate radiation-induced skin fibrosis. Furthermore, the outer gold shell further enhances radiotherapy sensitization. Therefore, the drug delivery system of the present invention can also be used as a sensitizer to enhance the efficacy of radiotherapy.

[0020] A method for preparing the drug delivery system of the present invention comprises:

[0021] A lipid solution is prepared by dissolving a lipid mixture consisting of phosphatidylcholine, cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-thiol (DSPE-PEG-SH) at a molar ratio of 10:4:1 in an organic solvent (e.g., chloroform);

[0022] Then, 100 μg of ABT-263 was added to the lipid solution. The organic solvent was removed from the lipid phase, and the residue was removed by vacuum freeze drying (e.g., 4 h). The formed lipid film was hydrated with deionized water and sonicated to obtain homogeneous ABT-263 nanoliposomes, denoted as Lipo@ABT263.

[0023] Then, Lipo@ABT263 was mixed with L-glutathione (mass ratio: 1:1) and mixed in the solution (e.g., 2 h). The L-glutathione-modified Lipo@ABT263 suspension was purified by affinity column (Sephadex G-15);

[0024] Subsequently, HAuCl4 solution (150 μL, 10 mM) was added thereto, and ascorbic acid (AA) was added at a [HAuCl4] / [ascorbic acid] molar ratio of 1:2 to reduce Au ions;

[0025] Finally, after overnight reaction at room temperature, the resulting solution was centrifuged (10,000 rpm, 15 minutes) and washed (eg, twice) to harvest the gold-shelled Lipo@ABT263, which was designated as Lipo@ABT263@Au. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1Figure 2 is the preparation and characterization results of Lipo@ABT263@Au; among them, a is the design schematic diagram of ABT-263 encapsulated and gold-shelled liposomes (Lipo@ABT263@Au), b is the transmission electron microscopy (TEM) field of view of liposomes, Lipo@ABT263, Lipo@Au and Lipo@ABT263@Au, c is the particle size distribution of liposomes, Lipo@ABT263, Lipo@Au and Lipo@ABT263@Au, d is the particle size distribution of liposomes, ABT-263, Lipo@Au and Li Ultraviolet-visible (UV-Vis) spectra of po@ABT263@Au, e is the Zeta potential diagram of liposomes, ABT-263, Lipo@ABT263, Lipo@Au and Lipo@ABT263@Au, f is the scanning transmission electron microscopy (STEM) field of view and elemental distribution (EDX) diagram of Lipo@ABT263 and Lipo@ABT263@Au (green: P, orange: O, yellow: S, blue: Au), g is the drug release curves of Lipo@ABT263 and Lipo@ABT263@Au in pH 7.4 buffer, h is the drug release curves of Lipo@ABT263@Au in pH 7.4 and pH 5.5 buffers, i is the drug release curves of Lipo@ABT263 in pH 7.4 and pH 5.5 buffers;

[0027] Figure 2 Figure 2 is a graph verifying the safety and biodistribution of Lipo@ABT263@Au in vivo; among them, a is a growth curve graph of mice receiving designated treatment, b is a HE staining graph of the heart, liver, lung, spleen and kidney of mice receiving designated treatment, c is a statistical graph of ALT, an indicator of liver and kidney function in each group of mice receiving designated treatment, d is a statistical graph of AST, an indicator of liver and kidney function in each group of mice receiving designated treatment, e is a statistical graph of UREA, an indicator of liver and kidney function in each group of mice receiving designated treatment, f is a statistical graph of liver and kidney function in each group of mice receiving designated treatment. UA statistical graph of the relevant indicator, g WBC statistical graph of the blood routine analysis indicators of each group of mice receiving designated treatment, h RBC statistical graph of the blood routine analysis indicators of each group of mice receiving designated treatment, i HB statistical graph of the blood routine analysis indicators of each group of mice receiving designated treatment, j PLT statistical graph of the liver blood routine analysis indicators of each group of mice receiving designated treatment, k is the biodistribution diagram of lipo@Au, Lipo@ABT263 or Lipo@ABT263@Au in the subcutaneous tumor model 5 minutes, 4 hours and 8 hours after intravenous injection of nanomedicine;

[0028] Figure 3Figure 1 is a graph verifying the radiosensitizing effect of Lipo@ABT263@Au in vitro and in vivo; wherein, a is a statistical analysis graph of the colony formation assay of OMM2.3, MUM2B and A375 cells treated with the specified treatment (n=3, data are expressed as mean ± standard deviation, two-tailed unpaired Student's t-test), b is a schematic diagram of the animal experiment process, c is a bioluminescence image of nude mice receiving the specified treatment, d is a statistical analysis graph of the bioluminescence signal (n=5. Data are expressed as mean ± standard deviation, two-tailed unpaired Student's t-test), e is a bright field photograph of the gross appearance of tumors isolated from mice in each group receiving the specified treatment, f is a statistical graph of the tumor weight of each group of test mice (n=5, data are expressed as mean ± standard deviation, two-tailed unpaired Student's t-test), g is a HE staining and Tunel staining image of the tumors in each group of mice receiving the specified treatment;

[0029] Figure 4 Figure 1 is a validation diagram of Lipo@ABT263@Au alleviating radiotherapy-induced skin fibrosis; wherein, a is the SA-β-gal staining diagram of each group of HDF cells treated with the specified treatment, b is the statistical diagram of the SA-β-gal staining experiment (n=3, data are expressed as mean ± standard deviation), c is the HE staining and Masson trichrome staining results of the skin around the tumor of each group of mice treated with the specified treatment, d is the γ-H2AX and p21 immunofluorescence staining diagram of the skin around the tumor of each group of mice receiving the specified treatment. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is described in detail below. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, and all of these should be included in the scope of the claims of the present invention.

[0031] The various test methods used in the following examples of the present invention are specifically described as follows:

[0032] 1) Characterization of ABT-263-loaded drug delivery systems

[0033] The morphology of the prepared nanomedicine was observed by TEM (JEM-2100, JEOL) and EDS.

[0034] Elemental mapping was performed in a JEOL JEMARM200F electron microscope at an accelerating voltage of 200 kV.

[0035] The size and zeta potential of the nanoparticles were measured using a Zetasizer (ZSU3200, Malvern).

[0036] The UV-visible absorption spectra were taken by a UV-visible spectrophotometer (UV-2450, Shimadzu).

[0037] The chemical composition of the nanomedicines was determined by XPS using a RBD-upgraded PHI-5000C ESCA system (Perkin-Elmer) at 250 W and 14.0 kV with Mg Ka radiation (hν = 1253.6 eV) at a detection angle of 54°.

[0038] 2) ABT-263 encapsulation efficiency and sustained release rate

[0039] 2mL of Lipo@ABT263@Au solution was placed in a dialysis bag (molecular weight of 350kD), and the dialysis bag was then immersed in a centrifuge tube containing 20mL of pH 5.5 and 7.4 PBS buffer respectively. At different time points (0, 1, 2, 4, 6, 9, 12, 24, 36, 48, 60, 72 and 96 hours), 1mL of PBS buffer was taken out and immediately refilled into the centrifuge tube with a new equal amount of PBS buffer. The sample was measured with a UV-visible spectrophotometer, and the drug release rate was quantified by the standard fitting curve of ABT-263 at an absorption wavelength of 330nm. According to the following equation, based on UV-visible absorbance, the drug loading and encapsulation efficiency were quantified as follows:

[0040] Drug loading (g / mg) = ABT-263 loading / nanodrug weight

[0041] Encapsulation efficiency (%) = (amount of ABT-263 loaded in the nanodrug / amount of ABT-263 drug injected) × 100

[0042] 3) In vivo experiments

[0043] Animal experiments were approved by the Animal Experimentation Ethics Committee of the Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. Four-week-old male BALB / c nude mice were used in a specific pathogen-free (SPF) animal room. A tumor transplant model was established by suspending 1 × 10^6 A375 cells in 100 μL of PBS and injecting them into the right axilla of nude mice. When the tumor volume reached approximately 100 mm, the tumor was resected and the tumor was resected. 3 At 10 mg / kg of Lipo@ABT263@Au was injected intravenously into nude mice, and 4 hours later, the tumor site was irradiated with 5 Gy of X-rays. The combined treatment was performed every three days for a total of four times. Changes in tumor volume were monitored over 21 days. At the end of the experiment, all nude mice were killed, and the tumors and surrounding skin tissues of each group were collected, weighed, fixed in formalin, embedded in paraffin, and sliced. Tumor sections were stained with HE and TUNEL according to standard procedures. At the end of the experiment, liver and kidney function were assessed by intraocular blood sampling of nude mice.

[0044] 4) Clone formation assay

[0045] The designated cells (1-2 × 10^3 cells, adjusted based on cell growth rate) were seeded in 6-well plates, and 2 mL of culture medium containing the designated drug or nanoparticles was added to each well. The medium was changed every 3 to 4 days. After 10-14 days of culture, colonies were stained with 0.25% crystal violet. Finally, colonies were photographed and counted using ImageJ software.

[0046] 5) β-galactosidase staining analysis

[0047] The treated cells were fixed with 4% paraformaldehyde and stained using a senescent cell histochemical staining kit (Sigma-Aldrich). Positive senescent cells were observed under a light microscope and quantitatively analyzed using ImageJ software.

[0048] 6) Statistical analysis

[0049] Statistical analysis was performed using GraphPad Prism 8 software. Data are presented as mean ± standard deviation (mean ± SD), and statistical significance between groups was calculated using a two-tailed unpaired t-test. P < 0.05 was considered statistically significant and is indicated by an asterisk (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0050] Example 1 Preparation and characterization results of ABT-263-loaded drug delivery system

[0051] To achieve sustained drug delivery, we encapsulated ABT-263 in liposomes (Lipo@ABT263). To further enhance radiosensitivity, we encapsulated Lipo@ABT263 with a gold shell ( Figure 1 a). The specific method is as follows:

[0052] ABT-263-encapsulated liposomes were prepared using a thin film hydration method. A lipid mixture consisting of phosphatidylcholine, cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-thiol (DSPE-PEG-SH) at a molar ratio of 10:4:1 was dissolved in chloroform using a round-bottom flask. Then, 100 μg of ABT-263 in chloroform was added to the lipid solution. The organic solvent was evaporated from the lipid phase, and the residue was removed by freeze-drying under vacuum for 4 h. The formed lipid film was hydrated with deionized water, and the sample was then sonicated using a VCX130 (Sonics, USA) to obtain homogenous Lipo@ABT263, which was stored at 4°C for further synthesis.

[0053] 1 mL of the prepared Lipo@ABT263 solution (1 mg / mL) was transferred to a glass vial, mixed with 20 μL of 50 mg / mL L-glutathione (GSH), and gently stirred for 2 h. The GSH-modified Lipo@ABT263 suspension was passed through a Sephadex G-15 column to remove unbound GSH. Subsequently, an aqueous solution of HAuCl₄ (150 μL, 10 mM) was added to the GSH-modified Lipo@ABT263 suspension, followed by the addition of ascorbic acid (AA) at a [HAuCl₄] / [ascorbic acid] molar ratio of 1:2 to reduce the Au ions. Finally, after an overnight reaction at room temperature, the resulting solution was centrifuged (10,000 rpm for 15 minutes) and washed twice with deionized water to harvest the gold-shelled Lipo@ABT263, i.e., Lipo@ABT263@Au. Control liposomes without ABT-263 or a gold shell were simultaneously prepared using the same method.

[0054] Transmission electron microscopy and DLS confirmed the uniformity of the nanoparticles (~180 nm) ( Figure 1 b and Figure 1 c), UV-visible spectroscopy confirmed the presence of liposomes, ABT-263, and gold nanoparticles ( Figure 1 d). Zeta potential measurements indicate stable colloidal properties ( Figure 1 e), in addition, elemental mapping confirmed the presence of P, O, and Au ( Figure 1 f). Drug release studies showed that in pH 7.4 buffer, Lipo@ABT263@Au released 36.08% of ABT-263 within 24 h, compared to 24.62% for Lipo@ABT263 ( Figure 1 g), the gold shell effectively increased the release of ABT-263 within 24 hours of administration. Acidic environment (pH = 5.5) enhanced the release of both drugs (48.68% vs. 39.72%) ( Figure 1 h,i). The gold nanoshell reduces the pH-dependent release difference, indicating enhanced tumor targeting of the nanodrug.

[0055] The above verification shows that this example successfully constructed and characterized the nanodrug Lipo@ABT263@Au.

[0056] Example 2 Safety and tumor targeting verification

[0057] The drug delivery system Lipo@ABT263@Au was injected into the tail vein of mice to verify its safety.

[0058] The body weight of mice remained stable throughout the experiment ( Figure 2 a), H&E staining showed no histopathological abnormalities in the main organs of mice ( Figure 2 b). The liver indexes (ALT, AST) and kidney indexes (creatinine, uric acid) in the blood of mice were comparable to those in the control group ( Figure 2 cf). Likewise, blood cell counts were not affected ( Figure 2 gj), the above experiments show that Lipo@ABT263@Au has good biocompatibility.

[0059] IVIS imaging showed that Cy5.5 labeling showed strong tumor accumulation at 5 minutes (5min), 4 hours (4h) and 8 hours (8h) after injection of nanomedicine. Figure 2 k), indicating that Lipo@ABT263@Au has good tumor targeting ability.

[0060] Example 3: Verification of radiotherapy sensitization effect

[0061] The cell colony formation assay showed that the Lipo@ABT263@Au combined with radiation treatment group had the strongest tumor inhibition effect ( Figure 3 a), thereby verifying the effectiveness of Lipo@ABT263@Au.

[0062] Similarly, in vivo experiments ( Figure 3 b) shows that mice receiving combined Lipo@ABT263@Au and irradiation showed the lowest tumor bioluminescence signal ( Figure 3 c, d), tumor volume ( Figure 3 e) and tumor weight ( Figure 3 f), a large number of TUNEL-positive cells indicated enhanced apoptosis ( Figure 3 g).

[0063] The above experiments show that Lipo@ABT263@Au has a good radiotherapy sensitization effect.

[0064] Example 4: Verification of Radiotherapy Sensitization Effect

[0065] First, SA-β-gal staining experiments showed that radiation significantly induced senescence in dermal fibroblasts, and ABT-263 effectively eliminated radiation-induced senescent cells ( Figure 4 a and Figure 4 b), thus it can be seen that Lipo@ABT263@Au has a protective effect on normal tissues.

[0066] In addition, H&E and Masson staining showed that skin fibrosis was reduced in mice treated with Lipo@ABT263@Au ( Figure 4 c), γ-H2AX and p21 levels were also low in the skin ( Figure 4d), These experiments demonstrate that Lipo@ABT263@Au can alleviate radiation-induced tissue damage.

Claims

1. A drug delivery system containing ABT-263, characterized in that: The method comprises liposomes and elemental gold, wherein ABT-263 is contained in the liposomes and the liposomes are covered with elemental gold to form a gold shell.

2. The ABT-263 drug delivery system according to claim 1, characterized in that: The liposome particle size is 180nm±10nm.

3. The ABT-263 drug delivery system according to claim 1, characterized in that The liposome comprises lecithin, cholesterol and 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-poly (ethylene glycol)-thiol.

4. The ABT-263 drug delivery system according to claim 3, characterized in that The liposomes were composed of phosphatidylcholine, cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-thiol in a molar ratio of 10:4:

1.

5. The ABT-263 drug delivery system according to claim 1, characterized in that The gold shell effectively reduced the pH-dependent release of ABT-263.

6. A method for preparing the ABT-263-loaded drug delivery system according to claim 1, comprising: The lipid mixture is dissolved in an organic solvent to prepare a lipid solution. Then, ABT-263 was added to the lipid solution, the organic solvent was removed from the lipid phase, and the residue was removed by vacuum freeze drying. The formed lipid film was hydrated with deionized water and sonicated to obtain homogeneous ABT-263 nanoliposomes, which were denoted as Lipo@ABT263. Then, Lipo@ABT263 and L-glutathione were mixed in a solution at a mass ratio of 1:1, and the L-glutathione-modified Lipo@ABT263 suspension was purified by affinity column; Subsequently, HAuCl4 solution was added, and ascorbic acid was added at a HAuCl4] / ascorbic acid molar ratio of 1:2 to reduce Au ions; Finally, after overnight reaction at room temperature, the resulting solution was centrifuged and washed to harvest the gold-shell-coated Lipo@ABT263, denoted as: Lipo@ABT263@Au.

7. Use of the ABT-263-loaded drug delivery system according to claim 1 in the preparation of a drug for eliminating senescent fibroblasts and inhibiting skin fibrosis, wherein after administration, ABT-263 is slowly released.

8. Use of the ABT-263-loaded drug delivery system according to claim 1 in the preparation of a drug for eliminating senescent tumor cells and overcoming radiotherapy resistance, wherein ABT-263 is sustained-released after administration.

9. Use of the ABT-263-loaded drug delivery system according to claim 1 in the preparation of a drug for reducing the time-dependent increase of senescent cells after radiotherapy, wherein ABT-263 is sustained-released after administration.

10. A sensitizer, characterized in that The invention comprises the ABT-263-loaded drug delivery system according to claim 1.

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