A dual-targeting glioblastoma composite nanomaterial and a construction method thereof

Abstract

The application discloses a double-targeted glioma composite nanomaterial and a construction method thereof, wherein the composite nanomaterial comprises, from inside to outside, modified mesoporous silica, a drug loading layer, a hybrid membrane layer and a specific modification layer; and the hybrid membrane layer is obtained by combining liposomes and glioma-specific dendritic cell membranes. The double-targeted glioma composite nanomaterial can realize double targeting of glioma cells due to the existence of the hybrid membrane layer and the specific modification layer, has no obvious immunogenicity, can efficiently release preloaded molecules or drugs in cells, and is expected to develop into a new nanomaterial for more effective treatment of glioma.

Description

Technical Field

[0001] The present invention relates to the technical field of targeted drugs, and in particular to a composite nanomaterial with dual targeting of 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 care is multimodal therapy, including maximal surgical resection followed by radiotherapy and adjuvant chemotherapy with temozolomide (TMZ). However, due to TMZ's poor therapeutic targeting, potent toxicity, and potential for drug resistance, as well as its tendency to induce a strong immune response, the prognosis for patients remains poor.

[0003] With the continuous optimization of nanosynthesis processes, the application of nanobiotechnology has provided viable solutions for anti-tumor therapy. Mesoporous silica nanoparticles (MSNs) have become a prominent carrier due to their large surface area, strong loading capacity, and excellent biocompatibility. MSNs can not only deliver large-molecule drugs and peptides, but also transport DNA and RNA, playing a significant role in anti-tumor, inflammatory treatment, and improvement of myocardial infarction and osteoporosis. The surface properties of nanomaterials play a decisive role in their biological applications. However, due to the near-neutral charge of MSNs, negatively charged and small DNA and RNA are difficult to deliver efficiently through MSNs. While the mesopores carry drugs, they are also prone to leakage. Therefore, effectively blocking the pores of MSNs can significantly increase drug loading and thus treat diseases.

[0004] Currently, MSNs are often encapsulated in liposomes to block their pores and increase biosafety. However, this strategy cannot address the specific recognition of tumor cells by nanoparticles. Although studies have shown that moderate amounts of MSNs are biotoxic, the effects of excessive nanoparticle accumulation in the liver and kidneys on the body remain unclear. Therefore, the development of highly specific nanoparticles is crucial. Summary of the Invention

[0005] The purpose of the present invention is to provide a composite nanomaterial that can dual-target glioblastoma cells, 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 dual-targeting composite nanomaterial for 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;

[0007] The hybrid membrane layer is obtained by combining liposomes and glioblastoma-specific dendritic cell membranes.

[0008] Furthermore, the modified mesoporous silica is obtained by modifying mesoporous silica with a modifier;

[0009] The modifier includes at least one of polyethyleneimine, (3-aminopropyl)triethoxysilane, and (3-aminopropyl)trimethoxysilane;

[0010] The average particle size of the mesoporous silica is 70-100 nm.

[0011] Furthermore, the aptamer used in the specific modification layer is an aptamer that targets the Tau protein in nerve cells.

[0012] Furthermore, it is characterized in that the average particle size is 100-130nm.

[0013] The present invention also provides a method for preparing a dual-targeting glioblastoma composite nanomaterial, comprising:

[0014] The modifier solution and the mesoporous silica dispersion are mixed and reacted to obtain modified mesoporous silica;

[0015] Adding drugs to 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;

[0016] Combining liposomes with glioblastoma-specific dendritic cell membranes to obtain a hybrid membrane; adding the hybrid membrane to a dispersion of modified mesoporous silica loaded with drugs to react, forming a hybrid membrane layer on the surface of the drug-loaded layer; and completing the reaction to obtain a composite dispersion;

[0017] The aptamer solution is added to the composite dispersion for combination to form a specific modification layer on the surface of the hybrid membrane layer, and the insoluble matter is collected to obtain a composite nanomaterial with dual targeting of glioblastoma.

[0018] Furthermore, the concentrations of the modifier solution and the mesoporous silica dispersion are 0.5-4 mg / mL and 1-3 mg / mL, respectively;

[0019] The volume ratio of the modifier solution to the mesoporous silica dispersion is 1:0.8-1.5;

[0020] The modifier includes at least one of polyethyleneimine.

[0021] 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.

[0022] Furthermore, the method for preparing the glioblastoma-specific dendritic cell membrane is:

[0023] The glioblastoma cells are lysed and then incubated to obtain lysate;

[0024] The lysate was incubated to obtain 10 6 -10 8 dendritic cells, and glioblastoma-specific dendritic cell membranes were isolated;

[0025] The mass ratio of the liposome to the glioblastoma-specific dendritic cells is 0.5-1:1-2;

[0026] The concentration of the drug-loaded modified mesoporous silica dispersion is 1-5 mg / mL;

[0027] The volume ratio of the hybrid membrane to the dispersion of the modified mesoporous silica loaded with drugs is 0.1-0.3:1.

[0028] Furthermore, the volume ratio of the aptamer solution to the composite dispersion is 1:30-100;

[0029] The concentration of the aptamer solution is 50-200 μM;

[0030] The aptamer is an aptamer with neuronal Tau protein targeting capability.

[0031] It should be noted that the source of mesoporous silica is not strictly limited in the present invention and can be prepared by oneself or purchased, but the particle size must be 70-100 nm. For example, the present invention uses a modified Stöber method to synthesize mesoporous silica:

[0032] 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 15,000rpm for 45 minutes. After centrifugation, reflux in a mixture of concentrated hydrochloric acid and methanol for 24 hours to remove the template agent. Wash with deionized water three 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.

[0033] In the present invention, liposomes are obtained according to conventional preparation methods. For example, 5-10 mg / mL chloroform solution of lecithin and 0.5-1 mg / mL chloroform solution of cholesterol are mixed to obtain liposomes.

[0034] 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 can 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 can be water, ethanol, acetone, etc.

[0035] In the present invention, the type of loaded drug does not need to be strictly limited, and can be siRNA or other drug molecules for example.

[0036] The present invention also provides the use of the dual-targeting glioblastoma composite nanomaterial in the preparation of glioblastoma drugs.

[0037] The inventive concept of this invention is to utilize modifiers to improve the surface charge of mesoporous silica nanoparticles for better drug loading. The nanocarriers are then encapsulated in a hybrid membrane composed of liposomes and dendritic cell membranes. The outermost layer is modified with a neuron-targeting nucleic acid aptamer, Apt-Tau. The composite nanomaterial specifically targets neurogenic cells through Apt-Tau and specifically binds to glioblastoma cells through antigen-presenting molecules on the dendritic cell membrane, achieving dual tumor cell targeting. Subsequently, the composite nanomaterial utilizes the principle of like dissolves like to enter the cell and release the preloaded drug, thereby achieving an anti-tumor effect.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 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. At the same time, it has no obvious immunogenicity and can efficiently release pre-loaded molecules or drugs in cells. It is expected to develop into a more effective new nanomaterial for treating glioblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any creative work.

[0041] Figure 1 The Fourier transform infrared spectra of the intermediate product and the product prepared in Example 1 are shown;

[0042] Figure 2 The intermediate product prepared in Example 1 and the average particle size characterization results of the product are shown;

[0043] Figure 3 The Zeta potential analysis results of the intermediate product and the product prepared in Example 1 are shown;

[0044] Figure 4 Shown is a transmission electron micrograph of the product prepared in Example 1;

[0045] Figure 5 The targeting effect diagram of the product prepared in Example 1 is shown;

[0046] Figure 6 The targeting effect diagram of the product prepared in Example 2 is shown;

[0047] Figure 7 The targeting effect diagram of the product prepared in Example 3 is shown;

[0048] Figure 8 Shown is a graph of the immunogenicity assessment of the product prepared in Comparative Example 1;

[0049] Figure 9 Shown is a graph of the immunogenicity assessment of the product prepared in Example 1;

[0050] Figure 10 A graph showing the immunogenicity evaluation of the product prepared in Example 3 is shown. DETAILED DESCRIPTION

[0051] The endpoints of the ranges and any values ​​disclosed herein 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 endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to form one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed in the present invention.

[0052] It should be noted that the glioblastoma cell line U87MG used in the examples of the present invention was purchased from the Wuhan University Collection Center (CCTCC) with the collection number GDC0628.

[0053] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the specific embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described 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 making creative efforts are within the scope of protection of the present invention.

[0054] Example 1

[0055] A method for preparing a dual-targeting composite nanomaterial for glioblastoma, comprising the following steps:

[0056] S1. Preparation of modified mesoporous silica

[0057] 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 stirring continued for 1 hour. The mixture was centrifuged at 15,000 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 three 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).

[0058] 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, mixed, and magnetically stirred for 30 minutes. After washing, the mixture was freeze-dried to obtain MSN grafted with PEI (MSN-PEI).

[0059] S2. Preparation of drug-loaded layer

[0060] MSN-PEI was prepared into a 250 μg / mL solution using sterile phosphate buffered saline (PBS). 15 μg of siRNA was then added and the mixture was shaken on a vortex mixer for 6 hours to form a drug-loaded layer on the MSN-PEI surface. After stopping the shaking, the insoluble matter was collected by centrifugation and washed three times with deionized water to obtain drug-loaded modified mesoporous silica (MSN-PEI-Drug).

[0061] S3. Preparation of hybrid membrane layer

[0062] Liposomes were prepared using 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. The mixture was ultrasonicated for 10 minutes, and then 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 and hydrated for 20 minutes. The liquid obtained after 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 using a liposome extruder 10-20 times to obtain liposomes.

[0063] Preparation of glioblastoma-specific dendritic cell membranes (DCM): Collect 8 × 10 6 Glioblastoma cells (U87MG) were diluted to 1.5 mL in PBS in a cryovial. The cryovial was placed in liquid nitrogen for 1 minute, then removed and heated in a 37°C water bath for 2 minutes. This process was repeated 3 times to obtain U87MG lysate. The lysate was incubated for 107 Dendritic cells (DC2.4 cell line) were treated for 12 hours. The treated DC2.4 cells were collected, washed three times with pre-chilled PBS, and centrifuged at 1000 rpm for 5 minutes. D4 cells were sonicated on ice for 15 minutes and centrifuged at 700 g for 5 minutes at 4°C to obtain cell membranes from the supernatant. The supernatant was then ultracentrifuged at 50,000 rpm for 30 minutes at 4°C and hydrated with DNase- / RNase-free water to obtain DCM.

[0064] 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.

[0065] S4. Preparation of specific modification layer

[0066] 100µM aptamer Apt-Tau was prepared using deionized water, and 20µL was added dropwise to 1ml of the composite dispersion. The mixture was stirred at room temperature for 1 hour to form a specific modification layer on the surface of the hybrid membrane. After the reaction, the insoluble matter was collected and washed three times with deionized water to obtain a dual-targeting glioblastoma composite nanomaterial.

[0067] Example 2

[0068] 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.

[0069] Example 3

[0070] 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.

[0071] Comparative Example 1

[0072] A method for preparing a dual-targeting composite nanomaterial for glioblastoma, comprising the following steps:

[0073] S1. Preparation of modified mesoporous silica

[0074] 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 stirring continued for 1 hour. The mixture was centrifuged at 15,000 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 three times. The precipitate was collected by centrifugation and dried in a vacuum oven at 60°C for 12 hours to obtain MSN.

[0075] 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 mixture was ultrasonicated for 10 minutes, and then magnetically stirred for 30 minutes. After washing, the mixture was freeze-dried to obtain MSN-PEI.

[0076] S2. Preparation of drug-loaded layer

[0077] MSN-PEI was prepared into a 250µg / mL solution using sterile 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 MSN-PEI-Drug;

[0078] S3. Preparation of bonding film layer

[0079] Liposomes were prepared using 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. The mixture was ultrasonicated for 10 minutes, and then 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 and hydrated for 20 minutes. The liquid obtained after 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 using a liposome extruder 10-20 times to obtain liposomes.

[0080] 1 mg of MSN-PEI-Drug was dispersed in 5 mL of deionized water, 1 mL of liposomes was added, and the mixture was incubated at room temperature for 1 hour to form a binding membrane layer (MSN-PEI-Drug@DCM-Lip) on the surface of the drug-loaded layer to obtain a composite dispersion.

[0081] S4. Preparation of specific modification layer

[0082] 100µM aptamer Apt-Tau was prepared using deionized water, and 20µL was added dropwise to 1ml of the composite dispersion. The mixture was stirred at room temperature for 1 hour to form a specific modification layer on the surface of the hybrid membrane. After the reaction, the insoluble matter was collected and washed three times with deionized water to obtain dual-targeting glioblastoma composite nanomaterials (Nano-boosters).

[0083] Test Case

[0084] 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 drug loading.

[0085] 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 85 nm. There is no obvious change in the particle size after PEI modification and drug loading. The particle size of MSN-PEI-Drug@DCM-Lip introduced into the hybrid membrane layer is similar to that of the final product Nano-boosters and is significantly larger, with an average particle size of about 125 nm.

[0086] 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 after PEI modification, the MSN-PEI 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.

[0087] 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.

[0088] Flow cytometry was used to show the targeting effect of the Nano-boosters prepared in Examples 1 to 3 on glioblastoma cells. Figure 5-Figure 7 The data shown in Figure 2 shows the ratio of fluorescently labeled Nano-boosters in glioma cells. It can be seen that compared to Example 2 (0.5:1), which has a lower dendritic cell membrane to liposome ratio, the higher dendritic cell membrane to liposome ratios in Example 1 (1:1) and Example 3 (2:1) have better targeting effects. These results indicate that a higher dendritic cell membrane ratio results in higher levels of immune co-stimulatory and immune recognition molecules (such as MHC II and TLRs) on the dendritic cell surface, resulting in stronger targeting and recognition of target cells (glioblastoma cells).

[0089] The immunogenicity of the prepared Nano-boosters in vivo was also evaluated, and the results were as follows: Figures 8-10As shown, the data results are the proportion of inflammatory (M1) macrophages. It can be seen that Example 1, in which the ratio of dendritic cell membrane to liposomes is 1:1, has a lower proportion of inflammatory (M1) macrophages, close to that of Comparative Example 1, which does not include dendritic cell membranes. These results demonstrate that the present invention has successfully constructed a composite nanomaterial with strong targeting effects on glioblastoma cells and no obvious immunogenicity. Based on its ability to utilize the principle of like dissolves like to enter cells and release preloaded drugs, thereby achieving an anti-tumor effect, it is expected to be developed into a more effective new nanomaterial for the treatment of glioblastoma.

[0090] 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, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or 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 scope of protection of the present invention.

Claims

1. A dual-targeting composite nanomaterial for glioblastoma, characterized in that: From the inside to the outside, it includes modified mesoporous silica, drug-loaded layer, hybrid membrane layer, and specific modification layer; The preparation method of the dual-targeting glioblastoma composite nanomaterial comprises: The modifier solution and the mesoporous silica dispersion are mixed and reacted to obtain modified mesoporous silica; Adding drugs to a 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; Combining liposomes with glioblastoma-specific dendritic cell membranes to obtain a hybrid membrane; adding the hybrid membrane to a dispersion of modified mesoporous silica loaded with drugs to react, forming a hybrid membrane layer on the surface of the drug-loaded layer; and completing the reaction to obtain a composite dispersion; The aptamer solution is added to the composite dispersion for combination to form a specific modification layer on the surface of the hybrid membrane layer, and the insoluble matter is collected to obtain a composite nanomaterial with dual targeting of glioblastoma; The mass ratio of the liposome to the glioblastoma-specific dendritic cell membrane is 1:1; The aptamer used in the specific modification layer is an aptamer with targeting property for the Tau protein in nerve cells; 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.

2. The composite nanomaterial according to claim 1, characterized in that The average particle size of the mesoporous silica is 70-100 nm.

3. The composite nanomaterial according to claim 1, characterized in that Its average particle size is 100-130nm.

4. The composite nanomaterial according to claim 1, 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.

5. The composite nanomaterial according to claim 1, 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.

6. The composite nanomaterial according to claim 1, characterized in that The method for preparing the glioblastoma-specific dendritic cell membrane is as follows: The glioblastoma cells are lysed and then incubated to obtain lysate; The lysate was incubated to obtain 10 6 -10 8 dendritic cells, and glioblastoma-specific dendritic cell membranes were isolated; 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.

7. The composite nanomaterial according to claim 1, 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 Tau protein targeting capability.

8. Use of the dual-targeting glioblastoma composite nanomaterial according to any one of claims 1 to 7 in the preparation of a drug for treating glioblastoma.

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