A siRNA-delivering perifosine prodrug liposome

Abstract

The invention discloses a perifosine prodrug liposome for delivering siRNA. CLTX-PEG2000-DSPE, perifosine, DSPE-PEG2000 and cholesterol are respectively dissolved, added into anhydrous ethanol, and magnetically stirred in the dark to obtain a liposome mixed solution; DOTAP is dissolved into anhydrous ethanol to obtain a DOTAP solution, and siRNA is dissolved into nuclease-free water to obtain a siRNA solution, the DOTAP solution and the siRNA solution are vortex-mixed, and nuclease-free water is added to obtain a siRNA aqueous solution; and the liposome mixed solution is slowly dripped into the siRNA aqueous solution, and magnetically stirred in the dark to obtain C / P / D / siSTAT3 liposomes after preparation. The invention uses the drug perifosine as a raw material to synthesize a liposome shell, thereby reducing the amount of auxiliary materials used in the synthesis of the liposome.

Description

Technical Field

[0001] The invention relates to a perifosine prodrug liposome for delivering siRNA, and belongs to the field of drugs for treating glioblastoma. Background Art

[0002] Glioblastoma (GBM) is the most common primary malignant tumor of the central nervous system, with an incidence rate of up to 48%, which seriously threatens human life and health. The invasive growth characteristics of GBM in the brain make its treatment extremely challenging. Although modern medical technologies such as intraoperative navigation, intraoperative awakening and other microsurgery, as well as comprehensive treatment methods such as radiotherapy and chemotherapy under the guidance of molecular pathology have been widely used in the treatment of GBM, the median survival time of patients is still less than 14.6 months, and the 5-year survival rate is less than 5%. This severe situation makes the treatment of GBM a difficult problem that the medical community urgently needs to overcome.

[0003] Studies have confirmed that GBM is heterogeneous, and only a small portion of glioma stem cells (GSCs) are the root cause of GBM resistance to conventional treatment and recurrence, and they control the fate of glioma patients. These stem cells have strong self-renewal and tumorigenic abilities, and are the main cause of GBM resistance to radiotherapy and chemotherapy, immune escape, and recurrence. Existing treatment options, such as surgery and postoperative TMZ synchronous radiotherapy and chemotherapy, can kill a large number of differentiated non-stem glioblastoma cells (NSGCs) in a short period of time, but they are helpless against residual GSCs in the brain. Therefore, only by targeting both GSCs and NSGCs in GBM at the same time can we hope to achieve a radical cure for GBM.

[0004] Construct nanoliposome prodrugs of perifosine and siSTAT3, modify the surface with chlorotoxin, target blockade of Akt and STAT3, and deplete glioma stem cells. Problems to be solved: Perifosine has large oral side effects and low drug concentration in the brain. After perifosine is synthesized into liposomes, it can be used as a delivery carrier for nucleic acid drugs, such as small interfering RNA (siRNA), to achieve targeted inhibition of multiple pathways. Deficiencies of existing technologies: siRNA transfection reagents are only used as delivery carriers alone. Summary of the invention

[0005] In order to overcome the defects of the prior art, the present invention utilizes perifosine as a single drug to synthesize into liposomes, which can kill glioma cells while serving as a delivery carrier; specifically, the present invention is a perifosine prodrug liposome for delivering siRNA.

[0006] The present invention is achieved by the following technical scheme: a perifosine prodrug liposome for delivering siRNA, 5 mg, 10 mg of CLTX-PEG2000-DSPE, 10 mg of perifosine, 10 mg of DSPE-PEG2000 and 10 mg of cholesterol are taken and dissolved in 1 mL of anhydrous ethanol respectively to obtain four corresponding substance solutions, and 2 ul, 5 ul, 2 ul and 5 ul of the above four substance solutions are taken in turn, added to 300 μL of anhydrous ethanol, and magnetically stirred for 30 min in a dark place to obtain a liposome mixture;

[0007] Dissolve 100 mg of DOTAP in 2 mL of anhydrous ethanol to obtain a DOTAP solution, then dissolve 10 μmol of siRNA in 1 L of nuclease-free water to obtain a siRNA solution, take 0.6 μL of the DOTAP solution and 10 μL of the siRNA solution, vortex and mix, then add 1 mL of nuclease-free water to obtain a siRNA aqueous solution;

[0008] Then the liposome mixture was slowly dripped into the siRNA aqueous solution and magnetically stirred for 30 min in the dark, and C / P / D / siSTAT3 liposomes (chlorotoxin-modified perifosine / siRNA liposomes) were prepared;

[0009] The CLTX-PEG2000-DSPE is chlorotoxin-polyethylene glycol-1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

[0010] As a preferred embodiment of the perifosine prodrug liposome for delivering siRNA described in the present invention: DOTAP and siRNA in the siRNA aqueous solution are mixed at a nitrogen-to-phosphorus ratio of 10:1.

[0011] As a preferred embodiment of the perifosine prodrug liposome for delivering siRNA according to the present invention: the preparation method of the CLTX-PEG2000-DSPE is as follows:

[0012] 5 mg of chlorotoxin (CLTX) and 9 mg of DSPE-PEG2000-PDP were added to 1 ml of DMSO solution, protected from light, and magnetically stirred for 96 h. The solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Kd, and then dialyzed in nuclease-free water for 48 h to obtain a dialysate, namely CLTX-PEG2000-DSPE. The liquid in the dialysis bag was lyophilized and stored at -20°C for later use;

[0013] The DSPE-PEG2000-PDP is 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000-thiopyridine.

[0014] As a preferred embodiment of the perifosine prodrug liposome for delivering siRNA according to the present invention, the reaction equation for the reaction of chlorotoxin (CLTX) with DSPE-PEG2000-PDP to generate CLTX-PEG2000-DSPE is as follows:

[0015] .

[0016] As a preferred embodiment of the siRNA delivery perifosine prodrug liposome described in the present invention: when the absorbance value of 2-thiopyridine in the dialysate at 343 nanometers is detected without significant change, the reaction is terminated. Among them, the absorbance value at 343 nanometers is 4.706 after 48 hours of reaction, 5.094 after 72 hours of reaction, 5.365 after 84 hours of reaction, and 5.37 after 96 hours of reaction. It is proved that chlorotoxin reacts with DSPE-PEG2000-PDP to generate CLTX-PEG2000-DSPE.

[0017] As a preferred embodiment of the perifosine prodrug liposome for delivering siRNA described in the present invention: the siRNA gene sequence is: Sense (5'-3') CAUCUGCCUAGAUCGGCUA, Antisense (5'-3'): UAGCCGAUCUAGGCAGAUG. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a schematic diagram of the synthesis of the targeted perifosine / siRNA nanoparticles in Example 1;

[0019] Figure 2 are transmission electron micrographs of perifosine / siRNA nanoparticles without targeting and perifosine / siRNA nanoparticles with targeting in Example 1 and Example 2;

[0020] Figure 3 The hydrodynamic diameter and surface potential of the non-targeted perifosine / siRNA nanoparticles and the targeted perifosine / siRNA nanoparticles in Example 1 and Example 2;

[0021] Figure 4 Agarose gel electrophoresis image showing the ability of the nanoparticles synthesized in Example 1 to load siRNA

[0022] Figure 5 The cytotoxicity of the nanoparticles synthesized in Example 1 to glioma stem cells was detected;

[0023] Figure 6 is a figure showing the luciferase detection of the inhibition of tumor growth by the perifosine / siRNA nanoparticles with targeting function prepared in Example 1 of the present invention;

[0024] Figure 7 It is the statistics of the fluorescence density value of the perifosine / siRNA nanoparticles with targeting function prepared in Example 1 to inhibit tumor growth.

[0025] The beneficial effects of the present invention are as follows: the present invention uses the drug perifosine as a raw material to synthesize the liposome shell, which reduces the amount of auxiliary materials used in the synthesis of liposomes. The perifosine liposome can be used as a delivery carrier for delivering other drugs, such as siRNA, other water-soluble drugs or fat-soluble drugs such as temozolomide. Specific implementation methods

[0026] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings.

[0027] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0028] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0029] Step 1: Preparation of targeted liposome shell: Dissolve 5 mg CLTX-PEG2000-DSPE (chlorotoxin-polyethylene glycol 2000-1,2-distearoyl-sn-glycero-3-phosphoethanolamine), 10 mg perifosine, 10 mg DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000), and 10 mg cholesterol in 1 mL of anhydrous ethanol for later use. Then take a predetermined volume (the volume of each substance is 2 ul, 5 ul, 2 ul, and 5 ul, respectively) and add it to 300 μL of anhydrous ethanol, and stir magnetically in the dark for 30 min.

[0030] Step 2: DOTAP ((2,3-dioleoyl-propyl)-trimethylammonium chloride) was dissolved in anhydrous ethanol, and siRNA was dissolved in nuclease-free water. The ethanol solution of DOTAP and the solution of siRNA were mixed and magnetically stirred for 30 minutes. D / siRNA was obtained. The siRNA was siSTAT3 and NC siRNA (negative control group). The positively charged DOTAP and the negatively charged siRNA were adsorbed to each other.

[0031] Step 3: Preparation of targeted lipid nanoparticles encapsulating siRNA:

[0032] The mixture of step 1 was added dropwise to the mixture of step 2, and magnetic stirring was continued for 30 min. After preparation, chlorotoxin-modified perifosine / siRNA liposomes (C / P / D / siSTAT3 liposomes) were obtained. If the siRNA is NC siRNA (negative control group), C / P / D / NC siRNA was obtained. C refers to chlorotoxin, P refers to perifosine, and D refers to DOTAP. The synthesis diagram is shown in FIG. Figure 1 shown.

[0033] siRNA sequence Sense (5'-3') CAUCUGCCUAGAUCGGCUA,

[0034] Antisense (5'-3') UAGCCGAUCUAGGCAGAUG.

[0035] Step 1: Dissolve 10 mg perifosine, 10 mg DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000), and 10 mg cholesterol in 1 mL of anhydrous ethanol for later use. Then take a predetermined volume (5 ul, 2 ul, and 5 ul of each substance, respectively) and add it to 300 μL of anhydrous ethanol. Stir magnetically in the dark for 30 min.

[0036] Step 2: Add the mixture prepared in step 1 of Example 2 dropwise into another mixture prepared in step 2 of Example 1, and stir magnetically to obtain perifosine / siRNA liposomes (P / D / siSTAT3 liposomes). P refers to perifosine, and D refers to DOTAP.

[0037] Step 1: Dissolve 5 mg CLTX-PEG2000-DSPE (chlorotoxin-polyethylene glycol 2000-1,2-distearoyl-sn-glycero-3-phosphoethanolamine), 10 mg DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000), and 10 mg cholesterol in 1 mL of anhydrous ethanol for later use. Then take a predetermined volume (the volume of each substance is 2 ul, 5 ul, and 5 ul, respectively) and add it to 300 μL of anhydrous ethanol. Stir magnetically in the dark for 30 min.

[0038] Step 2: Add the mixture prepared in step 1 of Example 3 dropwise into the mixture prepared in step 2 of Example 1, and stir magnetically to obtain C / D / siRNA.

[0039] The morphology of the lipid nanoparticles in step 3 of Example 1 and step 2 of Example 1 was observed using a transmission electron microscope. The results showed uniformly dispersed spheres with a diameter of less than 200 nm. Figure 2 shown.

[0040] The hydrodynamic diameter and surface potential of the lipid nanoparticles in steps 3 and 5 of Example 1 were measured using a Malvern particle size analyzer. Figure 3 shown.

[0041] After the nanoliposomes were successfully synthesized, the ability of C / P / D-siSTAT3 to load siSTAT3 was tested by agarose gel electrophoresis. Figure 4 As shown in the figure, the band at N / P = 0 is the reference (Naked siSTAT3). As the N / P ratio increases, the color of the band gradually becomes lighter. When N / P = 10:1, the band disappears, indicating that the liposomes are fully loaded with siSTAT3. The nitrogen-phosphorus ratio (N / P) refers to the ratio of NH4+ in DOTAP to the phosphate group in siRNA. The results are shown in Figure 4 shown.

[0042] In this experiment, a brain glioma stem cell model was used to investigate the cytotoxicity of the targeted lipid nanomedicine in Example 1. Glioma stem cells were seeded at a density of 1×104 in a 96-well plate (the well plate was pre-coated with poly-lysine) and cultured for 24 h. Afterwards, the culture medium containing the targeted nanoliposome group, the non-targeted nanoliposome group, the PBS group, and the perifosine group was replaced with the culture medium and cultured for 24 h. 10 μL of CCK8 was added to the culture medium in each well and incubated at 37 °C for 4 h. The absorbance value was measured at a wavelength of 450 nm, and the cell survival rate was calculated with untreated cells as a reference; the targeted lipid nanomedicine group in Example 1 had the best therapeutic effect, and the results are shown as follows: Figure 5 shown.

[0043] Methods: Male BALB / C nude mice weighing 18 g were purchased from Beijing Weitong Lihua Company. The human glioma stem cell orthotopic transplantation model was used to evaluate the killing effect of targeted lipids / siRNA on orthotopic gliomas. Animals were allowed to adapt to the new environment for 1 week before transplantation of tumor cells. Ten days after tumor inoculation, the size of the tumor was determined by small animal in vivo imaging, and the nude mice were grouped (n=10). The PBS group, siRNA group, C / D / siRNA group, C / P / D / NC siRNA group, C / P / D / siRNA group, and perifosine group were injected through the tail vein. The drug was administered 12 days after tumor inoculation. The dosage of perifosine was 10 mg / Kg and that of siRNA was 1 mg / kg. The drug was administered every other day for 3 times. Luciferase in vivo imaging was performed on the 10th, 20th, and 27th days after tumor inoculation. The tumor volume was measured by luciferase imaging (see Figure 6 ) and fluorescence density values ​​(see Figure 7 ,* P <0.05,** P <0.01), and the results showed that the targeted perifosine / siRNA group had a significant tumor inhibitory effect compared with the perifosine group and other groups.

Claims

1. A perifosine prodrug liposome for delivering siRNA, characterized in that: Take 5 mg CLTX-PEG2000-DSPE, 10 mg perifosine, 10 mg DSPE-PEG2000 and 10 mg cholesterol, dissolve them in 1 mL of anhydrous ethanol, respectively, to obtain the corresponding four substance solutions, and take 2 ul, 5 ul, 2 ul and 5 ul of the above four substance solutions in turn, add them to 300 μL of anhydrous ethanol, and stir them magnetically in the dark for 30 min to obtain a liposome mixture; 100 mg of DOTAP was dissolved in 2 mL of anhydrous ethanol to obtain a DOTAP solution, and 10 μmol of siRNA was dissolved in 1 L of nuclease-free water to obtain a siRNA solution. 0.6 μL of the DOTAP solution and 10 μL of the siRNA solution were vortexed and mixed, and 1 mL of nuclease-free water was added to obtain a siRNA aqueous solution. Then the liposome mixture was slowly dripped into the siRNA aqueous solution and magnetically stirred for 30 min in the dark, and C / P / D / siSTAT3 liposomes (chlorotoxin-modified perifosine / siRNA liposomes) were prepared; The CLTX-PEG2000-DSPE is chlorotoxin-polyethylene glycol-1,2-distearoyl-sn-glycero-3-phosphoethanolamine; The reaction equation for the reaction of the chlorotoxin with DSPE-PEG2000-PDP to generate CLTX-PEG2000-DSPE is as follows: 。 2. The perifosine prodrug liposome for delivering siRNA according to claim 1, characterized in that: In the siRNA aqueous solution, DOTAP and siRNA are mixed at a nitrogen-to-phosphorus ratio of 10:

1.

3. The perifosine prodrug liposome for delivering siRNA according to claim 1, characterized in that: The preparation method of the CLTX-PEG2000-DSPE is as follows: 5 mg of chlorotoxin and 9 mg of DSPE-PEG2000-PDP were added to 1 ml of DMSO solution, protected from light, and magnetically stirred for 96 h. The solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Kd, and then dialyzed in nuclease-free water for 48 h to obtain a dialysate, namely CLTX-PEG2000-DSPE. The liquid in the dialysis bag was lyophilized and stored at -20°C for later use; The DSPE-PEG2000-PDP is 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000-thiopyridine.

4. The perifosine prodrug liposome for delivering siRNA according to claim 3, characterized in that: The absorbance of 2-mercaptopyridine in the dialysate at 343 nm was detected. When there was no significant change in the absorbance, the reaction was terminated, thereby proving that chlorotoxin reacted with DSPE-PEG2000-PDP to generate CLTX-PEG2000-DSPE.

5. The perifosine prodrug liposome for delivering siRNA according to claim 1, characterized in that: The siRNA gene sequence is: Sense (5'-3') CAUCUGCCUAGAUCGGCUA.

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