Composite nano-material for dual targeting of glioblastoma and construction method of composite nano-material

Abstract

The invention discloses a composite nano-material for dual targeting of glioblastoma and a construction method thereof. The composite nano-material sequentially comprises modified mesoporous silica, a drug loading layer, a hybrid membrane layer and a specific modification layer from inside to outside, the hybrid membrane layer is obtained by combining lipidosome and a glioblastoma specific dendritic cell membrane. According to the composite nano material for dual targeting of glioblastoma, due to the existence of the hybrid membrane layer and the specific modification layer, dual targeting of glioblastoma cells can be achieved, meanwhile, no obvious immunogenicity exists, preloaded molecules or drugs can be efficiently released in the cells, and the targeting effect is good. The nano-material is expected to be developed into a novel nano-material for effectively treating glioblastoma.

Description

Technical Field

[0001] The present invention relates to the technical field of targeted drugs, and in particular to a composite nano material for dual-targeting glioblastoma and a construction method thereof. Background Art

[0002] Glioblastoma (GBM) is the most aggressive and most common primary malignant brain tumor in adults. The current standard of treatment is multimodal treatment, such as maximal surgical resection followed by radiotherapy and adjuvant chemotherapy with temozolomide (TMZ). However, due to the poor therapeutic targeting of TMZ, strong toxicity, easy drug resistance, and strong immune response, the prognosis of patients is still poor.

[0003] With the continuous optimization of nanosynthesis technology, the application of nanobiotechnology has provided a feasible solution for anti-tumor treatment. Among them, mesoporous silica nanoparticles (MSN) have become a star carrier due to their large surface area, strong loading capacity and good biocompatibility. MSN can not only deliver macromolecular drugs and peptides, but also transport DNA or RNA, playing an important role in anti-tumor, treatment of inflammation, improvement of myocardial infarction and osteoporosis. The surface properties of nanomaterials play a decisive role in their biological applications. However, since the charge carried by MSN is close to neutral, negatively charged and small-sized DNA and RNA are difficult to be efficiently delivered through MSN, and mesopores are prone to leakage while carrying drugs. Therefore, effectively blocking the pores of MSN can significantly increase the drug loading and thus treat diseases.

[0004] At present, MSNs are often encapsulated in liposomes to block the pores and increase biosafety. However, this strategy cannot solve the problem of nanoparticles' specific recognition of tumor cells. Although studies have shown that an appropriate amount of MSNs has no biological toxicity, the effects of excessive nanoparticles accumulated in the liver and kidneys on the body are still unclear. Therefore, it is crucial to develop highly specific nanoparticles. Summary of the invention

[0005] The purpose of the present invention is to provide a composite nanomaterial that can dual-target glioblastoma cells, can efficiently release pre-loaded molecules or drugs in cells, and has low immunogenicity.

[0006] In order to achieve the above-mentioned object, the present invention provides a composite nanomaterial for dual targeting of glioblastoma, which comprises, from the inside to the outside, modified mesoporous silica, a drug-loaded layer, a hybrid membrane layer, and a specific modification layer; The hybrid membrane layer is obtained by combining liposomes and glioblastoma-specific dendritic cell membranes.

[0007] Furthermore, the modified mesoporous silica is obtained by modifying mesoporous silica with a modifier; The modifier includes at least one of polyethyleneimine, (3-aminopropyl)triethoxysilane, and (3-aminopropyl)trimethoxysilane; The average particle size of the mesoporous silica is 70-100 nm.

[0008] Furthermore, the aptamer used in the specific modification layer is an aptamer with targeting property for Tau protein in nerve cells.

[0009] Furthermore, it is characterized in that the average particle size is 100-130nm.

[0010] The present invention also provides a method for preparing a composite nanomaterial for dual targeting of glioblastoma, comprising: The modifier solution and the mesoporous silica dispersion are mixed and reacted to obtain modified mesoporous silica; Adding drugs into the dispersion of modified mesoporous silica to form a drug-loaded layer on the surface of the modified mesoporous silica to obtain drug-loaded modified mesoporous silica; The liposomes and the glioblastoma-specific dendritic cell membrane are combined to obtain a hybrid membrane; the hybrid membrane is added to a dispersion of modified mesoporous silica loaded with drugs to react, a hybrid membrane layer is formed on the surface of the drug-loaded layer, and a composite dispersion is obtained after the reaction is completed; The aptamer solution is added into the composite dispersion for combination, a specific modification layer is formed on the surface of the hybrid membrane layer, and the insoluble matter is collected to obtain a composite nanomaterial with dual targeting of glioblastoma.

[0011] Further, the concentrations of the modifier solution and the mesoporous silica dispersion are 0.5-4 mg / mL and 1-3 mg / mL, respectively; The volume ratio of the modifier solution to the mesoporous silica dispersion is 1:0.8-1.5; The modifier includes at least one of polyethyleneimines.

[0012] Furthermore, the ratio of the modified mesoporous silica to the dispersed liquid is 200-300 µg:1 mL; and the mass of the modified mesoporous silica is 10-20 times the mass of the drug.

[0013] Furthermore, the method for preparing the glioblastoma-specific dendritic cell membrane is: The glioblastoma cells are lysed and then incubated to obtain lysate products; The lysate was incubated to obtain 10 6 -10 8dendritic cells, and isolated glioblastoma-specific dendritic cell membranes; The mass ratio of the liposome to the glioblastoma-specific dendritic cells is 0.5-1:1-2; The concentration of the drug-loaded modified mesoporous silica dispersion is 1-5 mg / mL; The volume ratio of the hybrid membrane to the dispersion of the modified mesoporous silica loaded with drugs is 0.1-0.3:1.

[0014] Further, the volume ratio of the aptamer solution to the composite dispersion is 1:30-100; The concentration of the aptamer solution is 50-200 µM; The aptamer is an aptamer with neuronal cell Tau protein targeting property.

[0015] It should be noted that the source of mesoporous silica in the present invention does not need to be strictly limited, and it can be prepared by itself or purchased, but its particle size must be 70-100 nm. For example, the present invention uses a modified Stöber method to synthesize mesoporous silica: Dissolve 0.5-2g of hexadecyltrimethylammonium bromide (CTAB) in 60mL of deionized water and stir for 30 minutes, add NaOH to adjust the pH to 10, add 10-30μL of triethanolamine (TEA) and stir the mixture at 95°C for 1 hour; add 1.5mL of tetraethyl silicate (TEOS) dropwise and continue stirring for 1 hour; centrifuge at 15000rpm for 45 minutes; reflux in a mixture of concentrated hydrochloric acid and methanol for 24 hours after centrifugation to remove the template agent, wash with deionized water 3 times, collect the precipitate by centrifugation and dry it in a vacuum drying oven at 60°C for 12 hours to obtain white powdery mesoporous silica.

[0016] In the present invention, liposomes are obtained according to conventional preparation methods. For example, 5-10 mg / mL of lecithin in chloroform solution and 0.5-1 mg / mL of cholesterol in chloroform solution are mixed and reacted to obtain liposomes.

[0017] In the present invention, the liquid used to disperse mesoporous silica, modified mesoporous silica or modified mesoporous silica loaded with drugs does not need to be strictly limited, and illustratively may be water, ethanol, phosphate buffer solution, etc.; the liquid used to dissolve the modifier and dilute the aptamer does not need to be strictly limited, and illustratively may be water, ethanol, acetone, etc.

[0018] In the present invention, the type of loaded drug does not need to be strictly limited, and can be siRNA or other drug molecules.

[0019] The present invention also provides the use of the above-mentioned dual-targeting glioblastoma composite nanomaterial in the preparation of glioblastoma drugs.

[0020] The inventive concept of the present invention is: using mesoporous silica nanoparticles modified by a modifier to improve the surface charge of mesoporous silica to better load therapeutic drugs, and then using a hybrid membrane formed by a mixture of liposomes and dendritic cell membranes to further encapsulate the nanocarrier; modifying the outermost layer with a nucleic acid aptamer Apt-Tau that has neuronal cell targeting. The composite nanomaterial specifically targets neurogenic cells through Apt-Tau, and specifically binds to glioblastoma cells through antigen presenting molecules on the surface of dendritic cell membranes, thereby achieving dual targeting of tumor cells. Subsequently, the principle of similar dissolves is used to enter the cell and release the preloaded drug, thereby achieving an anti-tumor effect.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The dual-targeting glioblastoma composite nanomaterial of the present invention can achieve dual targeting of glioblastoma cells due to the presence of the hybrid membrane layer and the specific modification layer, and at the same time has no obvious immunogenicity and can efficiently release pre-loaded molecules or drugs in cells, and is expected to develop into a more effective new nanomaterial for treating glioblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The Fourier transform infrared spectra of the intermediate product and the product prepared in Example 1 are shown; Figure 2 The intermediate product prepared in Example 1 and the average particle size characterization results of the product are shown; Figure 3 The Zeta potential analysis results of the intermediate product and the product prepared in Example 1 are shown; Figure 4 The projection electron microscope image of the product prepared in Example 1 is shown; Figure 5 The targeting effect diagram of the product prepared in Example 1 is shown; Figure 6 The targeting effect diagram of the product prepared in Example 2 is shown; Figure 7 The targeting effect diagram of the product prepared in Example 3 is shown; Figure 8 The immunogenicity evaluation diagram of the product prepared in Comparative Example 1 is shown; Fig. 9 The immunogenicity evaluation diagram of the product prepared in Example 1 is shown; Fig.10 A graph showing the immunogenicity evaluation of the product prepared in Example 2 is shown. DETAILED DESCRIPTION

[0024] The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in the present invention.

[0025] It should be noted that the glioblastoma cell line U87MG used in the examples of the present invention was purchased from Wuhan University Collection Center (CCTCC) with the collection number GDC0628.

[0026] The following will be combined with the specific embodiments of the present invention and the drawings of the specification 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.

[0027] Example 1 A method for preparing a composite nanomaterial for dual targeting of glioblastoma, comprising the following steps: S1. Preparation of modified mesoporous silica 1 g of CTAB was dissolved in 60 mL of deionized water and stirred for 30 minutes. NaOH was added to adjust the pH to 10. After adding 25 μL of TEA, the mixture was stirred at 95 °C for 1 hour. 1.5 mL of TEOS was added dropwise and continued to be stirred for 1 hour. The mixture was centrifuged at 15000 rpm for 45 minutes. After centrifugation, the mixture was refluxed in a mixture of concentrated hydrochloric acid and methanol for 24 hours to remove the template agent. The mixture was washed with deionized water for 3 times. The precipitate was collected by centrifugation and dried in a vacuum oven at 60 °C for 12 hours to obtain white powdered mesoporous silica (MSN). 10 mg of MSN was dispersed in 5 mL of deionized water, and 2.5 mg of polyethyleneimine (PEI) was dispersed in 5 mL of anhydrous ethanol. The two were ultrasonicated for 10 minutes respectively. The two were mixed and magnetically stirred for 30 minutes. After washing, they were freeze-dried to obtain MSN grafted with PEI (MSN-PEI). S2. Preparation of drug-loaded layer MSN-PEI was prepared into a 250µg / mL solution with sterile phosphate buffer solution (PBS), and then 15μg of siRNA was added and shaken on a vortex mixer for 6 hours to form a drug-loaded layer on the surface of MSN-PEI. After stopping the shaking, the insoluble matter was collected by centrifugation and washed three times with deionized water to obtain the drug-loaded modified mesoporous silica (MSN-PEI-Drug); S3. Preparation of hybrid membrane layer The liposomes were prepared by a thin film dispersion method: 100 mg of lecithin and 8 mg of cholesterol were dissolved in 1 mL and 10 mL of chloroform, respectively. After ultrasonic treatment for 10 minutes, the two were mixed and placed in a rotary evaporator at 37°C and 150 rpm until a honeycomb film was formed at the bottom of the bottle. 10 mL of deionized water was added for hydration for 20 minutes. The liquid obtained after the hydration was the liposome stock solution. The liposome stock solution was passed back and forth through a polycarbonate membrane with a pore size of 100 nm 10-20 times using a liposome extruder to obtain liposomes.

[0028] Preparation of glioblastoma-specific dendritic cell membranes (DCM): Collect 8 × 10 6 glioblastoma cells (U87MG) were diluted to 1.5 mL in a cryovial using PBS. The cryovial was placed in liquid nitrogen for 1 minute, and then heated in a 37°C water bath for 2 minutes. This process was repeated 3 times to obtain a U87MG lysate. The lysate was incubated for 10 7 Dendritic cells (DC2.4 cell line) were treated for 12 hours; the treated DC2.4 cell line was collected, washed three times with pre-cooled PBS, and centrifuged at 1000 rpm for 5 minutes. The D4 cell line was placed in an ice bath and ultrasonicated for 15 minutes, and centrifuged at 700g for 5 minutes at 4°C to obtain the cell membrane in the supernatant; ultracentrifuged at 50000 rpm for 30 minutes at 4°C, and hydrated with DNase / RNase-free water to obtain DCM.

[0029] The extracted dendritic cell membrane and liposome were mixed in a mass ratio of 1:1 to obtain a hybrid membrane; 1 mg of MSN-PEI-Drug was dispersed in 5 mL of deionized water, 1 mL of hybrid membrane was added, and incubated at room temperature for 1 hour to form a hybrid membrane layer on the surface of the drug-loaded layer to obtain a composite dispersion.

[0030] S4. Preparation of specific modification layer 100µM aptamer Apt-Tau was prepared using deionized water, and 20µL was added dropwise to 1ml of the composite dispersion and stirred at room temperature for 1 hour to form a specific modification layer on the surface of the hybrid membrane layer. After the reaction, the insoluble matter was collected and washed three times with deionized water to obtain a composite nanomaterial with dual targeting of glioblastoma.

[0031] Example 2 The method is basically the same as Example 1, except that in the step of preparing the hybrid membrane layer, the extracted dendritic cell membrane and liposome are mixed at a mass ratio of 0.5:1 to obtain the hybrid membrane.

[0032] Example 3 The method is basically the same as Example 1, except that in the step of preparing the hybrid membrane layer, the extracted dendritic cell membrane and liposome are mixed at a mass ratio of 2:1 to obtain the hybrid membrane.

[0033] Comparative Example 1 A method for preparing a composite nanomaterial for dual targeting of glioblastoma, comprising the following steps: S1. Preparation of modified mesoporous silica 1 g of CTAB was dissolved in 60 mL of deionized water and stirred for 30 minutes, NaOH was added to adjust the pH to 10, 25 μL of TEA was added and the mixture was stirred at 95°C for 1 hour; 1.5 mL of TEOS was added dropwise and the stirring was continued for 1 hour; the mixture was centrifuged at 15000 rpm for 45 minutes, and then refluxed in a mixture of concentrated hydrochloric acid and methanol for 24 hours to remove the template agent, and then washed with deionized water for 3 times. After centrifugation, the precipitate was collected and dried in a vacuum oven at 60°C for 12 hours to obtain MSN; 10 mg of MSN was dispersed in 5 mL of deionized water, and 2.5 mg of PEI was dispersed in 5 mL of anhydrous ethanol. The two were ultrasonicated for 10 minutes respectively. The two were mixed and magnetically stirred for 30 minutes. After washing, they were freeze-dried to obtain MSN-PEI. S2. Preparation of drug-loaded layer MSN-PEI was prepared into a 250 μg / mL solution with sterile PBS, and then 15 μg of siRNA was added and shaken on a vortex mixer for 6 hours to form a drug-loaded layer on the surface of MSN-PEI. After stopping the shaking, the insoluble matter was collected by centrifugation and washed three times with deionized water to obtain MSN-PEI-Drug; S3. Preparation of bonding film layer The liposomes were prepared by a thin film dispersion method: 100 mg of lecithin and 8 mg of cholesterol were dissolved in 1 mL and 10 mL of chloroform, respectively. After ultrasonic treatment for 10 minutes, the two were mixed and placed in a rotary evaporator at 37°C and 150 rpm until a honeycomb film was formed at the bottom of the bottle. 10 mL of deionized water was added for hydration for 20 minutes. The liquid obtained after the hydration was the liposome stock solution. The liposome stock solution was passed back and forth through a polycarbonate membrane with a pore size of 100 nm 10-20 times using a liposome extruder to obtain liposomes.

[0034] 1 mg of MSN-PEI-Drug was dispersed in 5 mL of deionized water, 1 mL of liposomes was added, and incubated at room temperature for 1 h to form a binding membrane layer (MSN-PEI-Drug@DCM-Lip) on the surface of the drug-loaded layer to obtain a composite dispersion.

[0035] S4. Preparation of specific modification layer Use deionized water to prepare 100µM aptamer Apt-Tau, take 20µL and add it to 1ml composite dispersion, stir at room temperature for 1 hour to form a specific modification layer on the surface of the hybrid membrane layer. After the reaction is completed, the insoluble matter is collected and washed 3 times with deionized water to obtain a composite nanomaterial (Nano-boosters) with dual targeting of glioblastoma.

[0036] Test Case The intermediates and products prepared in Example 1 were characterized by Fourier transform infrared spectroscopy. Figure 1 As shown, 1560 cm -1 The shift in the peak at indicates successful loading of the drug.

[0037] The particle size analysis results of the intermediate product and the product in the preparation of Example 1 are as follows: Figure 2 As shown, it can be seen that the average particle size of the prepared MSN is about 85nm, and there is no obvious change in the particle size after PEI modification and drug loading. The particle sizes of MSN-PEI-Drug@DCM-Lip introduced into the hybrid membrane layer and the final product Nano-boosters are similar and significantly larger, with an average particle size of about 125nm.

[0038] The Zata potential results of the intermediate product and the product in the preparation of Example 1 are as follows: Figure 3 As shown, it can be seen that the initial MSN is negatively charged, and the MSN-PEI obtained after PEI modification is positively charged. The other three groups are loaded with negatively charged drugs (siRNA) on the basis of MSN-PEI, so the overall potential changes from positive to negative again.

[0039] The transmission electron microscope image of the Nano-boosters prepared in Example 1 is as follows: Figure 4 As shown, it can be clearly seen that there is a film-like structure on the surface of MSN, which further indicates the successful preparation of the composite material.

[0040] Flow cytometry was used to show the targeting effect of Nano-boosters prepared in Examples 1 to 3 on glioblastoma cells. The results are as follows: Figure 5-Figure 7As shown, the data results are the proportion of fluorescently labeled Nano-boosters in glioma cells. It can be seen that compared with Example 2 (0.5:1) with a lower ratio of dendritic cell membrane to liposome, the higher ratios of Example 1 (1:1) and Example 3 (2:1) have better targeting effects. These results show that when the ratio of dendritic cell membrane is high, the content of immune co-stimulatory molecules and immune recognition molecules (such as MHC II, TLRs, etc.) on its surface is high, so the targeting recognition ability of target cells (glioblastoma cells) is stronger.

[0041] The immunogenicity of the prepared Nano-boosters in vivo was also evaluated. Figure 8-Figure 10 As shown, the data results are the proportion of inflammatory (M1) macrophages. It can be seen that Example 2, in which the ratio of dendritic cell membrane to liposome is 1:1, has a lower proportion of inflammatory (M1) macrophages, which is close to that of Comparative Example 1 without adding dendritic cell membrane. These results show that the present invention has successfully constructed a composite nanomaterial with strong targeting effect on glioblastoma cells and no obvious immunogenicity. Based on the ability to use the principle of similar dissolution to enter cells and release preloaded drugs, thereby achieving an anti-tumor effect, it is expected to develop into a more effective new nanomaterial for treating glioblastoma.

[0042] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A composite nanomaterial for dual targeting of glioblastoma, characterized in that: From inside to outside, it includes modified mesoporous silica, drug-loaded layer, hybrid membrane layer, and specific modification layer; The hybrid membrane layer is obtained by combining liposomes and glioblastoma-specific dendritic cell membranes.

2. The composite nanomaterial according to claim 1, characterized in that: The modified mesoporous silica is obtained by modifying mesoporous silica with a modifier; The modifier includes at least one of polyethyleneimine, (3-aminopropyl)triethoxysilane, and (3-aminopropyl)trimethoxysilane; The average particle size of the mesoporous silica is 70-100 nm.

3. The composite nanomaterial according to claim 1, characterized in that: The aptamer used in the specific modification layer is an aptamer with targeting property for the Tau protein of nerve cells.

4. The composite nanomaterial according to any one of claims 1 to 3, characterized in that: Its average particle size is 100-130nm.

5. A method for preparing a composite nanomaterial for dual targeting of glioblastoma, characterized in that: include, The modifier solution and the mesoporous silica dispersion are mixed and reacted to obtain modified mesoporous silica; Adding drugs into the dispersion of modified mesoporous silica to form a drug-loaded layer on the surface of the modified mesoporous silica to obtain drug-loaded modified mesoporous silica; The liposomes and the glioblastoma-specific dendritic cell membrane are combined to obtain a hybrid membrane; the hybrid membrane is added to a dispersion of modified mesoporous silica loaded with drugs to react, a hybrid membrane layer is formed on the surface of the drug-loaded layer, and a composite dispersion is obtained after the reaction is completed; The aptamer solution is added into the composite dispersion for combination, a specific modification layer is formed on the surface of the hybrid membrane layer, and the insoluble matter is collected to obtain a composite nanomaterial with dual targeting of glioblastoma.

6. The preparation method according to claim 5, characterized in that: The concentrations of the modifier solution and the mesoporous silica dispersion are 0.5-4 mg / mL and 1-3 mg / mL, respectively; The volume ratio of the modifier solution to the mesoporous silica dispersion is 1:0.8-1.5; The modifier includes at least one of polyethyleneimines.

7. The preparation method according to claim 5, characterized in that: The ratio of modified mesoporous silica to dispersed liquid is 200-300 µg:1 mL; the mass of the modified mesoporous silica is 10-20 times the mass of the drug.

8. The preparation method according to claim 5, characterized in that: The preparation method of the liposome is: The method for preparing the glioblastoma-specific dendritic cell membrane is as follows: The glioblastoma cells are lysed and then incubated to obtain lysate products; The lysate was incubated to obtain 10 6 -10 8 dendritic cells, and isolated glioblastoma-specific dendritic cell membranes; The mass ratio of the liposome to the glioblastoma-specific dendritic cells is 0.5-1:1-2; The concentration of the drug-loaded modified mesoporous silica dispersion is 1-5 mg / mL; The volume ratio of the hybrid membrane to the dispersion of the modified mesoporous silica loaded with drugs is 0.1-0.3:

1.

9. The preparation method according to claim 5, characterized in that: The volume ratio of the aptamer solution to the composite dispersion is 1:30-100; The concentration of the aptamer solution is 50-200 µM; The aptamer is an aptamer with neuronal cell Tau protein targeting property.

10. Use of the dual-targeting glioblastoma composite nanomaterial according to any one of claims 1 to 4 in the preparation of glioblastoma drugs.

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