Dual-responsiveness nano-drug delivery system and preparation method thereof

Abstract

The invention discloses a dual-responsiveness nano-drug delivery system and a preparation method thereof.The delivery system comprises an inner layer and an outer layer, the inner layer is a calcium carbonate inner layer, the outer layer is a lipid bilayer membrane, the calcium carbonate inner layer is coated with a chemotherapeutic drug BSO, programmed cell death ligand-1 siRNA is adsorbed on the lipid bilayer membrane, and the lipid bilayer membrane is coated with a chemotherapeutic drug BSO. The lipid bilayer membrane comprises a transition layer and a lipid layer, the transition layer coats the periphery of the calcium carbonate inner core, the lipid layer coats the periphery of the transition layer, and the lipid layer comprises (2, 4, 6-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5 The cleaning agent is prepared from 2, 3-dioleoxyl propyl) trimethyl ammonium chloride, dioleoyl phosphatidylethanolamine, cholesterol hemisuccinate, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 and GE11-distearoyl phosphatidylethanolamine-polyethylene glycol 2000. The invention is an ideal drug delivery system in anti-tumor treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of nano drug delivery systems, in particular to a dual-responsive nano drug delivery system and a preparation method thereof. Background Art

[0002] Cancer is one of the main factors that seriously affect human health and threaten human life. Cancer, along with cardiovascular and cerebrovascular diseases and accidents, are the three major causes of death in all countries in the world today. The treatment of cancer remains a major clinical problem that the global medical field strives to overcome. Currently, chemotherapy combined with surgery is usually the core solution for treating most cancers. However, chemical drugs have large adverse reactions to cancer treatment, and most patients still die from adverse reactions to chemotherapy drugs and tumor recurrence and metastasis. Due to unclear targets, poor selectivity, poor host tolerance, low pharmacokinetics and bioavailability of chemotherapy drugs at the tumor site, and a large number of patients will develop resistance to a variety of anti-tumor drugs with different structures and mechanisms of action during treatment, chemotherapy alone can easily lead to negative effects such as cancer recurrence.

[0003] Small interfering RNA (siRNA) therapy is a new immunotherapy solution. The selective gene silencing function of siRNA also provides the possibility of treating cancers caused by abnormal expression of one or several genes, and siRNA provides effective and selective targeted delivery only in target tissues. Chemical drugs combined with siRNA therapy can overcome the negative effects of tumor cell resistance and cancer recurrence, creating a new platform for new cancer treatment.

[0004] siRNA is unstable. It is not only easily degraded by RNA nucleases (RNAase) in the air or blood, but also easily cleared by mononuclear macrophages in tissues and organs such as the liver and spleen. Secondly, due to the negative charge of siRNA, it is difficult for it to pass through the cell membrane and enter the cell. Thirdly, it is difficult to escape from the endosome and enter the cytoplasm to exert its effect.

[0005] How to successfully enter cells and efficiently escape is a bottleneck problem that limits the application of nucleic acid delivery. The development of efficient targeted delivery systems for chemotherapeutic drugs and nucleic acid drugs is the key to improving tumor treatment effects and achieving clinical transformation.

[0006] Therefore, in order to solve the above problems, a dual-responsive nano drug delivery system and a preparation method thereof are proposed. Summary of the invention

[0007] The object of the present invention is to provide a dual-responsive nano drug delivery system, which comprises an inner core and an outer layer, wherein the inner core is a calcium carbonate inner core and the outer layer is a lipid bilayer membrane, wherein the calcium carbonate inner core is loaded with a chemotherapeutic drug BSO, wherein the lipid bilayer membrane is adsorbed with a programmed cell death ligand-1 siRNA, wherein the lipid bilayer membrane comprises a transition layer coated on the periphery of the calcium carbonate inner core and a lipid layer coated on the periphery of the transition layer, wherein the lipid layer comprises (2,3-dioleyloxypropyl) trimethylammonium chloride, dioleoylphosphatidyl alcoholamine, cholesterol hemisuccinate, distearoylphosphatidylethanolamine-polyethylene glycol 2000, and GE11-distearoylphosphatidylethanolamine-polyethylene glycol 2000.

[0008] Preferably, the weight distribution ratio of (2,3-dioleyloxypropyl)trimethylammonium chloride, dioleoylphosphatidyl alcoholamine, cholesterol hemisuccinate, distearoylphosphatidylethanolamine-polyethylene glycol 2000, and GE11-distearoylphosphatidylethanolamine-polyethylene glycol 2000 is 40:20:35:4:1.

[0009] Preferably, the content of the chemotherapy drug BSO is 280-400 ug.

[0010] Preferably, the content of the programmed cell death ligand-1 siRNA is 10-30 ug.

[0011] Preferably, the calcium carbonate kernel has a calcium ion content of 50-200 ug, CO3 2- The content is 40-180mg.

[0012] Preferably, the steps of the preparation method include:

[0013] Step 1: prepare 5-10 ml of a solution of the above-mentioned commercially available (2,3-dioleyloxypropyl)trimethylammonium chloride, dioleoylphosphatidyl alcoholamine, cholesterol hemisuccinate, distearoylphosphatidylethanolamine-polyethylene glycol 2000, and GE11-distearoylphosphatidylethanolamine-polyethylene glycol 2000 in a ratio of 40:20:35:4:1;

[0014] Step 2: Select commercially available CaCl2 or other Ca-containing reagents other than CaCO3 and prepare 2.5M Ca 2+ Solution, choose commercially available K2CO3 or other solutions containing CO3 2- Reagent configuration CO3 2- Solution; select commercially available buthionine sulfoximine to prepare a 4 mg / ml solution;

[0015] Step 3: Place a small vial on a magnetic stirrer, add a magnet, add 0.8-1.4 ml of pure water under magnetic stirring at 400-1000 rpm, and add 1-5 ul of Ca while stirring under the action of the magnetic stirrer. 2+ The solution was then slowly injected with chemotherapy BSO 280-400ug under the action of a magnetic stirrer, and stirred at 20-30℃ for 3-10min;

[0016] Step 4: Then use the microfluidic method to inject the sample, using two 5ml syringes to add 0.8-1.2ml CO3 from the microfluidic injection pump at both ends. 2- Solution and 0.3-0.8 ml of lipid solution prepared in the proportion of step 1, turn on the microfluidic injection pump, set the syringe specification to 2 ml / 8.98 mm, and inject CO3 at a flow rate of 0.8-1.2 ml and an injection flow rate of 260-400 μl / min. 2- Phase; inject 0.3-0.8 ml lipid at a flow rate of 0.3-0.6 ml and an injection flow rate of 100-200 μl / min;

[0017] Step 5: Adjust the left and right limits of the injection pump to absorb CO3 2- The phase and lipid syringes are fixed to two syringe pumps respectively. The rubber tubes connected to the syringe pumps are inserted into the liquid surface being stirred in the vial on the magnetic stirrer. The two syringe pumps are started at the same time to start the injection. After the injection is completed, the preparation of the system for loading the evolutionary drug is completed.

[0018] Step 6: Finally, add 10-30ug programmed cell death ligand-1 siRNA at 20-30℃ and stir for 20-60min. The drug delivery system is ready. Use a dialysis bag with a molecular weight of 100000-20000Da to shake at 20-80rpm at 2-8℃ for 4-24h and collect the dialyzed drug delivery system.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The drug-loaded nano-delivery system provided by the present invention has a calcium carbonate core-lipid shell structure, and the chemical drug BSO and the nucleic acid drug are encapsulated in the lipid nanoparticles based on the calcium carbonate core, so that they have pH and hypoxia responsiveness, and the surface of the lipid nanoparticles based on the calcium carbonate core is covered with lipids modified with GE11 targeting peptides, so that the delivery system has high stability and cell entry efficiency during the drug delivery process, and then the nucleic acid drug can escape efficiently, so as to selectively target and transfect into cancer cells and induce efficient gene silencing effect, and it is an ideal drug delivery system in anti-tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1is a schematic diagram of the particle structure of the drug-loaded nano-delivery system prepared in Example 1;

[0022] Figure 2 is a morphology picture of the drug-loaded nano-delivery system particles prepared in Example 1 observed by TEM;

[0023] Figure 3 This is a particle size evaluation diagram of the drug-loaded nano-delivery system particles prepared in Example 1 using a laser particle size analyzer;

[0024] Figure 4 This is a Zeta potential evaluation diagram of the drug-loaded nano-delivery system prepared in Example 1 using a laser particle size analyzer;

[0025] Figure 5 is the stability gel electrophoresis diagram of the drug-loaded nano-delivery system prepared in Example 1 in FBS;

[0026] Figure 6 This is a drug pH response release diagram of the drug-loaded nano-delivery system particles prepared in Example 1 measured by a laser particle size analyzer;

[0027] Figure 7 This is a diagram of the cell uptake of CT26 cells by the targeted drug-loaded nano-delivery system prepared in Example 1;

[0028] Figure 8 is a graph showing the cytotoxicity of the drug-loaded nano-delivery system prepared in Example 1 to CT26 cells;

[0029] Fig. 9 Graph showing the hypoxia response of the drug-loaded nano-delivery system to CT26 cells in Example 1;

[0030] Fig.10 It is a diagram of tumor tissue volume imaging and drug administration grouping after colorectal cancer-bearing mice were treated with each drug system in Example 1; DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In the following examples, the nucleic acid sequences of the sense strand and antisense strand of the mouse-derived PD-L1-siRNA are as follows:

[0033] siPDL1 positive strand (SEQ ID NO. 1): 5′-AGACGUAAGCAGUGUUGAATT-3′;

[0034] siPDL1 antisense strand (SEQ ID NO. 2): 5'-UUCAACACUG CUUACGUCUTT-3';

[0035] The nucleic acid sequences of the sense strand and antisense strand of the control siRNA are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively:

[0036] siNC sense strand (SEQ ID NO. 3): 5′-UUCUCCGAACGUGUCACGUTT-3′;

[0037] siNC antisense strand (SEQ ID NO. 4): 5'-ACGUGACACGUUCGGAGAATT-3'.

[0038] In the following examples, unless otherwise specified, the room temperature is 25±5°C.

[0039] Embodiment 1:

[0040] A dual-responsive nano drug delivery system comprises an inner core and an outer layer, wherein the inner core is a calcium carbonate inner core and the outer layer is a lipid bilayer membrane, wherein the calcium carbonate inner core is loaded with a chemotherapeutic drug BSO, wherein the lipid bilayer membrane is adsorbed with a programmed cell death ligand-1 siRNA, wherein the lipid bilayer membrane comprises a transition layer coated on the periphery of the calcium carbonate inner core and a lipid layer coated on the periphery of the transition layer, wherein the lipid layer comprises (2,3-dioleyloxypropyl)trimethylammonium chloride, dioleoylphosphatidyl alcoholamine, cholesterol hemisuccinate, distearoylphosphatidylethanolamine-polyethylene glycol 2000, and GE11-distearoylphosphatidylethanolamine-polyethylene glycol 2000.

[0041] Step 1: prepare 5-10 ml of a solution of the above-mentioned commercially available (2,3-dioleyloxypropyl)trimethylammonium chloride, dioleoylphosphatidyl alcoholamine, cholesterol hemisuccinate, distearoylphosphatidylethanolamine-polyethylene glycol 2000, and GE11-distearoylphosphatidylethanolamine-polyethylene glycol 2000 in a ratio of 40:20:35:4:1;

[0042] Step 2: Select commercially available CaCl2 or other reagents containing Ca other than CaCO3 to prepare a 2.5 M Ca2+ solution, select commercially available K2CO3 or other reagents containing CO32- to prepare a CO32- solution; select commercially available buthionine sulfoximine to prepare a 4 mg / ml solution;

[0043] Step 3: Place a small vial on a magnetic stirrer, add a magnet, add 1 ml of pure water under 600 rpm magnetic stirring, and add 2 ul of Ca 2+ The solution was then slowly injected with 360ug of the chemotherapy drug BSO under the action of a magnetic stirrer and stirred at room temperature for 5min;

[0044] Step 4: Then use the microfluidic method to inject the sample, use two 5ml syringes to add 1ml of CO3 from the microfluidic injection pump at both ends. 2- solution and 0.5 ml of lipid solution prepared in the above ratio; turn on the microfluidic injection pump, set the syringe specification to 2 ml / 8.98 mm, and inject CO3 at a flow rate of 1 ml and an injection flow rate of 300 μl / min. 2- Phase; inject 0.5 ml lipid at a flow rate of 0.5 ml and an injection flow rate of 150 μl / min;

[0045] Step 5: Adjust the left and right limits of the injection pump to absorb CO3 2- The phase and lipid syringes are fixed to two syringe pumps respectively. The rubber tubes connected to the syringe pumps are inserted into the liquid surface being stirred in the vial on the magnetic stirrer. The two syringe pumps are started at the same time to start the injection.

[0046] Step 6: After the injection is completed, the preparation of the drug-carrying system is completed. Finally, 13.3ug of programmed cell death ligand-1 siRNA is added at room temperature and stirred for 30 minutes. After the drug-carrying system is prepared, a 15000Da molecular weight dialysis bag is used to shake at 4°C at 30rpm for 12 hours, and the dialyzed drug-carrying system is collected.

[0047] As attached Figure 1 As shown, the drug components and lipid materials required for preparing the drug-loaded nano-delivery system, wherein

[0048] DOTAP—(2,3-dioleyloxypropyl)trimethylammonium chloride;

[0049] DOPE—dioleoylphosphatidylserine;

[0050] CHEMS—cholesterol hemisuccinate;

[0051] DSPE-PEG2000—distearoylphosphatidylethanolamine-polyethylene glycol 2000;

[0052] GE11-DSPE-PEG2000—GE11-distearoylphosphatidylethanolamine-polyethylene glycol 2000;

[0053] like Figure 2As shown, the co-delivery system prepared in Example 1 has a spherical structure with a smooth surface and uniform size. The black solid core body proves the successful preparation of the calcium carbonate core precipitation; the sample has good dispersion and no obvious aggregation phenomenon occurs. According to the ruler, the particle size is about 200nm;

[0054] like Figure 3 As shown, the optical density average particle size (%Intensity) of the co-delivery system prepared in Example 1 is 143.0±0.65 nm, and the average PDI is 0.17;

[0055] like Figure 4 As shown, the surface potential value of the co-delivery system prepared in Example 1 is -0.36±0.09mv, which is close to neutral;

[0056] like Figure 5 As shown, the nucleic acid encapsulation process was optimized by agarose gel electrophoresis. When the siPDL1 feed amount was 1 nmol, siPDL1 could be fully encapsulated in the nano drug delivery system particles provided by the present invention.

[0057] like Figure 6 As shown, the co-delivery system prepared in Example 1 rapidly dissolves and releases under pH 5.5 and has good pH responsiveness;

[0058] like Figure 7 As shown, the co-delivery system prepared in Example 1 has a significant increase in the cell uptake of CT26 cells and has good tumor targeting properties;

[0059] like Figure 8 As shown, the co-delivery system prepared in Example 1 has obvious cytotoxicity to the mouse colorectal cancer cell line CT26;

[0060] like Fig. 9 As shown, the co-delivery system prepared in Example 1 rapidly releases and relieves hypoxia under a hypoxic microenvironment and has good hypoxia responsiveness;

[0061] like Fig.10 As shown, the co-delivery system prepared in Example 1 was tested in mice. The mice were killed on DAY18 and the tumor tissues were taken to take pictures, showing that the system had a significant tumor growth inhibitory effect on mouse colorectal cancer.

[0062] Embodiment 2:

[0063] The co-delivery system was prepared according to the method of Example 1, except that BSO in step 3 was replaced by insulin drug.

[0064] Embodiment 3:

[0065] The co-delivery system was prepared according to the method of Example 1, except that GE11 in step 1 was replaced by RGD.

[0066] Embodiment 4:

[0067] The co-delivery system was prepared according to the method of Example 1, except that the programmed cell death ligand-1 siRNA, i.e., siPD-L1, in step six was replaced by sip53.

[0068] Embodiment 5:

[0069] The co-delivery system was prepared according to the method of Example 1, except that in step 1, the molar ratio of (2,3-dioleyloxypropyl)trimethylammonium chloride, dioleoylphosphatidyl alcoholamine, cholesterol hemisuccinate, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 was 5:2:4:1.

[0070] Utilizing the technical solution described in the present invention, or those skilled in the art designing a similar technical solution inspired by the technical solution of the present invention to achieve the above-mentioned technical effects, all fall within the protection scope of the present invention.

Claims

1. A dual-responsive nano drug delivery system, comprising an inner core and an outer layer, wherein the inner core is a calcium carbonate inner core and the outer layer is a lipid bilayer membrane, wherein the calcium carbonate inner core is loaded with a chemotherapeutic drug BSO, and the lipid bilayer membrane is adsorbed with programmed cell death ligand-1 siRNA, characterized in that: The lipid bilayer membrane comprises a transition layer coated on the periphery of the calcium carbonate inner core and a lipid layer coated on the periphery of the transition layer, wherein the lipid layer comprises (2,3-dioleyloxypropyl)trimethylammonium chloride, dioleoylphosphatidyl alcoholamine, cholesterol hemisuccinate, distearoylphosphatidylethanolamine-polyethylene glycol 2000, and GE11-distearoylphosphatidylethanolamine-polyethylene glycol 2000.

2. A dual-responsive nano drug delivery system according to claim 1, characterized in that: The weight distribution ratio of the (2,3-dioleyloxypropyl)trimethylammonium chloride, dioleoylphosphatidyl alcoholamine, cholesterol hemisuccinate, distearoylphosphatidylethanolamine-polyethylene glycol 2000, and GE11-distearoylphosphatidylethanolamine-polyethylene glycol 2000 is 40:20:35:4:

1.

3. A dual-responsive nano drug delivery system according to claim 1, characterized in that: The content of the chemotherapy drug BSO is 280-400ug.

4. A dual-responsive nano drug delivery system according to claim 1, characterized in that: The content of programmed cell death ligand-1 siRNA is 10-30ug.

5. A dual-responsive nano drug delivery system according to claim 1, characterized in that: The calcium carbonate kernel has a calcium ion content of 50-200 ug, CO3 2- The content is 40-180mg.

6. The method for preparing a dual-responsive nano drug delivery system according to claim 1, characterized in that: The steps of the preparation method include: Step 1: prepare 5-10 ml of a solution of the above-mentioned commercially available (2,3-dioleyloxypropyl)trimethylammonium chloride, dioleoylphosphatidyl alcoholamine, cholesterol hemisuccinate, distearoylphosphatidylethanolamine-polyethylene glycol 2000, and GE11-distearoylphosphatidylethanolamine-polyethylene glycol 2000 in a ratio of 40:20:35:4:1; Step 2: Select commercially available CaCl2 or other Ca-containing reagents other than CaCO3 and prepare 2.5M Ca 2+ Solution, choose commercially available K2CO3 or other solutions containing CO3 2- Reagent configuration CO3 2- Solution; select commercially available buthionine sulfoximine to prepare a 4 mg / ml solution; Step 3: Place a small vial on a magnetic stirrer, add a magnet, add 0.8-1.4 ml of pure water under magnetic stirring at 400-1000 rpm, and add 1-5 ul of Ca while stirring under the action of the magnetic stirrer. 2+ The solution was then slowly injected with chemotherapy BSO 280-400ug under the action of a magnetic stirrer, and stirred at 20-30℃ for 3-10min; Step 4: Then use the microfluidic method to inject the sample, using two 5ml syringes to add 0.8-1.2ml CO3 from the microfluidic injection pump at both ends. 2- Solution and 0.3-0.8 ml of lipid solution prepared in the proportion of step 1, turn on the microfluidic injection pump, set the syringe specification to 2 ml / 8.98 mm, and inject CO3 at a flow rate of 0.8-1.2 ml and an injection flow rate of 260-400 μl / min. 2- Phase; inject 0.3-0.8 ml lipid at a flow rate of 0.3-0.6 ml and an injection flow rate of 100-200 μl / min; Step 5: Adjust the left and right limits of the injection pump to absorb CO3 2- The phase and lipid syringes are fixed to two syringe pumps respectively. The rubber tubes connected to the syringe pumps are inserted into the liquid surface being stirred in the vial on the magnetic stirrer. The two syringe pumps are started at the same time to start the injection. After the injection is completed, the preparation of the system for loading the evolutionary drug is completed. Step 6: Finally, add 10-30ug programmed cell death ligand-1 siRNA at 20-30℃ and stir for 20-60min. The drug delivery system is ready. Use a dialysis bag with a molecular weight of 100000-20000Da to shake at 20-80rpm at 2-8℃ for 4-24h and collect the dialyzed drug delivery system.

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