Black phosphorus-based non-viral gene carrier, construction method and application thereof

Abstract

The application provides a construction method and application of a black phosphorus-based non-viral gene carrier which can cross the blood-brain barrier and has good stability, wherein the black phosphorus-based non-viral gene carrier is coated by a black phosphorus quantum dot core and a mannose-modified polydopamine. The black phosphorus-based non-viral gene carrier constructed by the application has good biocompatibility and biological metabolism, is beneficial to application in the biomedical field, and can realize gene therapy for Parkinson's disease. The disclosed black phosphorus-based non-viral gene carrier constructed by the application is mainly synthesized through condensation reaction, ultrasonic treatment and ice bath stirring. The preparation method of the application is simple, has good repeatability, and has good prospects for industrial production.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to a black phosphorus-based non-viral gene vector, its construction method, and its application. Background Technology

[0002] Gene therapy, a cutting-edge technology that treats major diseases such as hereditary diseases and tumors by introducing functional genes into target cells to correct or compensate for abnormal gene expression, has become an important development direction in the biomedical field. In gene therapy systems, gene vectors are the key core of the treatment. Naked therapeutic genes are difficult to enter cells autonomously, are easily degraded, and cannot achieve effective delivery and expression. Gene vectors, as delivery tools for therapeutic genes, can protect nucleic acids from damage, efficiently cross cell barriers, and target and mediate the stable expression of target genes within host cells. Their safety, delivery efficiency, and targeting directly affect the efficacy and clinical application of gene therapy. Therefore, developing safe, efficient, and highly targeted novel gene vectors is a key requirement for promoting breakthroughs in gene therapy technology and its clinical translation.

[0003] Black phosphorus (BP) has attracted widespread attention in the biomedical field due to its excellent biocompatibility and unique structure. However, its application in biomedicine still faces the following challenges: firstly, it is easily recognized and cleared by the immune system, and due to its insufficient targeting, it is difficult to reach the treatment site; secondly, it is easily oxidized and decomposed in vivo, making it difficult to exert its delivery function. Therefore, modifying black phosphorus nitride to construct nanocarriers with good stability, the ability to cross the blood-brain barrier, and nucleic acid drug delivery capabilities has significant research value. Summary of the Invention

[0004] The purpose of this application is to address the technical problems that existing technologies generally lack drugs targeting nucleic acids, which have difficulty crossing the blood-brain barrier, and that black phosphorus is easily oxidized and decomposed, thus limiting its application in the biomedical field.

[0005] To address the aforementioned technical problems, this application provides the following technical solution:

[0006] A method for preparing a black phosphorus-based nonviral gene vector includes the following steps:

[0007] S1: Dissolve block black phosphorus in NMP, sonicate under ice-water bath conditions, and centrifuge to obtain black phosphorus quantum dots (BPQDs);

[0008] S2: Disperse dopamine hydrochloride powder in water to obtain dopamine hydrochloride solution, add ammonia water, anhydrous ethanol and ultrapure water and stir at room temperature, and react in an open container to obtain polydopamine particle dispersion.

[0009] S3: Add acetone to the polydopamine sample dispersion obtained in S2 under rapid stirring, let stand, and then evaporate to dryness to obtain purified polydopamine particles (PDA).

[0010] S4: Disperse the PDA obtained in S3 in ethanol, add mannose, add acetic acid dropwise and heat to reflux, and obtain mannose-modified PDA through aldehyde-amine condensation reaction;

[0011] S5: Self-assemble the BPQDs obtained in S1 with the Man-PDA obtained in S4, stir in an ice-water bath for 2 hours to obtain MAN-PDA-BPQDs, which are mannose-modified polydopamine-encapsulated black phosphorus quantum dots.

[0012] Preferably, the ratio of the blocky black phosphorus to NMP in S1 is 1 mg: 1 mL.

[0013] The ultrasonic treatment consisted of sonication for 6 hours in an ultrasonic cell disruption system under ice-water bath conditions, followed by centrifugation at 10,000 rpm for 1 hour.

[0014] Preferably, the concentration of the dopamine hydrochloride solution prepared in step S2 is 0.05 g / mL;

[0015] The ratio of dopamine hydrochloride powder, ammonia, anhydrous ethanol and ultrapure water was 50 mg: 0.5 mL: 10 mL: 23 mL. The stirring time at room temperature was 30 min, and the open reaction time was 24 h.

[0016] Preferably, the ratio of acetone to polydopamine particle dispersion in S3 is 1:(3-4), and the standing time is 12h.

[0017] Preferably, in step S4, the mass ratio of PDA to mannose is 1:(0.5-1), the amount of acetic acid added is 200 μL, the heating reflux temperature is 40-50℃, and the heating reflux time is 24-48 h.

[0018] Preferably, the mass ratio of BPQDs to Man-PDA in S5 is 1:(0.5-2).

[0019] This application also provides a black phosphorus-based non-viral gene vector, which is prepared using the preparation method described above.

[0020] Preferably, the black phosphorus-based non-viral gene vector MAN-PDA-BPQDs has a quantum dot structure, with black phosphorus quantum dots as the core and modified by mannose-modified polydopamine MAN-PDA.

[0021] Preferably, the planar size of the black phosphorus-based non-viral gene vector MAN-PDA-BPQDs is 2-10 nm.

[0022] This application also provides the application of a black phosphorus-based non-viral gene vector prepared by the above preparation method in the preparation of siRNA delivery vectors in BV2 cells.

[0023] Compared with the prior art, this application has at least the following beneficial effects:

[0024] This invention constructs a black phosphorus-based non-viral gene vector with good biocompatibility and biometabolism, which is beneficial for application in the biomedical field and can deliver siRNA.

[0025] Secondly, the black phosphorus-based non-viral gene vector MAN-PDA-BPQDs constructed in this application are mainly synthesized by condensation reaction, ultrasonic treatment, and ice bath stirring. The preparation method of this invention is simple, has good reproducibility, and has good prospects for industrial production.

[0026] Meanwhile, the black phosphorus-based nonviral gene vector provided in this application enhances its ability to cross the blood-brain barrier, making it a novel nonviral gene vector. Attached Figure Description

[0027] Figure 1 The image shows the X-ray photoelectron spectroscopy (XPS) spectrum of the black phosphorus quantum dots in Example 1 of this application.

[0028] Figure 2 This is the ultraviolet absorption spectrum of the black phosphorus-based non-viral gene vector MAN-PDA-BPQDs in one embodiment of the present invention.

[0029] Figure 3 This is a transmission electron microscopy image of the black phosphorus-based nonviral gene vector MAN-PDA-BPQDs in one embodiment of the present invention.

[0030] Figure 4 This is an example of the temperature variation of black phosphorus-based nonviral gene vector MAN-PDA-BPQDs at different concentrations in one embodiment of the present invention.

[0031] Figure 5 This is an agarose gel retardation diagram of siRNA by black phosphorus-based nonviral gene vectors MAN-PDA-BPQDs in one embodiment of the present invention.

[0032] Figure 6 This is a diagram of the response and release experiment of black phosphorus-based nonviral gene vector MAN-PDA-BPQDs in one embodiment of the present invention.

[0033] Figure 7 Fluorescence imaging of black phosphorus-based nonviral gene vector MAN-PDA-BPQDs negative siRNA-Cy5 delivered to BV2 cells at different time points in one embodiment of the present invention.

[0034] Figure 8 This is an in vivo imaging image of black phosphorus-based nonviral gene vector MAN-PDA-BPQDs negative siRNA-Cy5 injected into a PD model mouse via the tail vein, according to one embodiment of the invention. Detailed Implementation

[0035] This application provides a black phosphorus-based non-viral gene vector with good stability and the ability to cross the blood-brain barrier. The vector is prepared by coating mannose-modified polydopamine with black phosphorus quantum dots as the core.

[0036] In one embodiment, the carrier is prepared as follows:

[0037] S1: Weigh out block black phosphorus, dissolve it in NMP, sonicate under ice-water bath conditions, and centrifuge to obtain black phosphorus quantum dots (BPQDs);

[0038] The ratio of the blocky black phosphorus to NMP is 1 mg: 1 mL.

[0039] The ultrasonic treatment was performed by sonicating the cells in an ice-water bath for 6 hours, followed by centrifugation at 10,000 rpm for 1 hour.

[0040] S2: Disperse dopamine hydrochloride powder in water to obtain dopamine hydrochloride solution, add ammonia water, anhydrous ethanol and ultrapure water and stir at room temperature, and react in an open container to obtain polydopamine particle dispersion.

[0041] The concentration of the prepared dopamine hydrochloride solution was 0.05 g / mL;

[0042] The ratio of dopamine hydrochloride powder, ammonia, anhydrous ethanol and ultrapure water was 50 mg: 0.5 mL: 10 mL: 23 mL. The stirring time at room temperature was 30 min, and the open reaction time was 24 h.

[0043] S3: Acetone is added to the polydopamine particle dispersion obtained in S2 under rapid stirring. The ratio of acetone to polydopamine particle dispersion is 1:(3-4). After standing, the dispersion is evaporated to obtain purified polydopamine particles PDA. In one embodiment, the standing time is 12h.

[0044] S4: Disperse the PDA obtained in S3 in ethanol, add mannose, the mass ratio of PDA to mannose is 1:(0.5-1), add 3-5 drops of acetic acid and heat to reflux. Preferably, the amount of acetic acid added is 200 μL, the reflux temperature is 40-50℃, and the reflux time is 24-48 h. Mannose-modified PDA (MAN-PDA) is obtained through aldehyde-amine condensation reaction.

[0045] S5: Self-assemble the BPQDs obtained in S1 with the Man-PDA obtained in S4, with a mass ratio of BPQDs to Man-PDA of 1:(0.5-2). Stir in an ice-water bath for 2 hours to obtain MAN-PDA-BPQDs, which are black phosphorus quantum dots coated with mannose-modified polydopamine.

[0046] Based on the above preparation method, this application provides black phosphorus-based non-viral gene vectors MAN-PDA-BPQDs, which are quantum dot structures with black phosphorus quantum dots as the core and modified with mannose-modified polydopamine MAN-PDA.

[0047] In one embodiment, the planar dimensions of the black phosphorus-based nonviral gene vector MAN-PDA-BPQDs are 2-10 nm.

[0048] Based on the above, this application also provides the application of black phosphorus-based nonviral gene vectors MAN-PDA-BPQDs in the preparation of siRNA delivery vectors in BV2 cells.

[0049] The above content will be explained in conjunction with specific verification experiments:

[0050] I. Experimental Materials and Sources

[0051]

[0052] II. Verification Experiment

[0053] Example 1

[0054] S1. Dissolve 30 mg of blocky black phosphorus in 30 mL of NMP, sonicate under ice-water bath conditions, and centrifuge to obtain black phosphorus quantum dots (BPQDs);

[0055] S2. Disperse 50 mg of dopamine hydrochloride powder in 1 mL of water, add 0.5 mL of ammonia, 10 mL of anhydrous ethanol and 23 mL of ultrapure water, stir at room temperature, and react in an open container to obtain a polydopamine particle dispersion.

[0056] S3. Add acetone to the polydopamine particle dispersion under rapid stirring. The ratio of acetone to polydopamine particle dispersion is 1:4. After standing, evaporate to dryness to obtain purified polydopamine particle PDA.

[0057] S4. Disperse the prepared PDA in 8 mL of ethanol, add 25 mg of mannose, the mass ratio of PDA to mannose is 1:1, and add 200 μL of acetic acid dropwise. Then heat and reflux to obtain mannose-modified PDA (MAN-PDA).

[0058] S5. Self-assemble the BPQDs obtained in S1 with the MAN-PDA obtained in S4, with the mass ratio of BPQDs to Man-PDA being 1:2, to obtain MAN-PDA-BPQDs, which are mannose-modified polydopamine-encapsulated black phosphorus quantum dots.

[0059] Please see Figures 1-3 The blocky black phosphorus described in this application contains phosphorus; and the MAN-PDA-BPQDs of this invention can be excited by near-infrared light; the above characterization shows that the black phosphorus-based non-viral gene vector MAN-PDA-BPQDs has been successfully prepared.

[0060] Example 2

[0061] Different concentrations of MAN-PDA-BPQDs materials were irradiated with an 808 nm power laser, and their temperature changes were recorded.

[0062] Please see Figure 4 , Figure 4 This indicates that the temperature of MAN-PDA-BPQDs increases with increasing concentration at different concentrations, but the temperature remains at a low level.

[0063] Example 3

[0064] MAN-PDA-BPQDs and siRNA of different mass ratios were incubated in a 37°C water bath for 0.5 h, and then the gel retardation results of MAN-PDA-BPQDs of different mass ratios were obtained by agarose gel retardation experiment.

[0065] Please see Figure 5 , Figure 5 The values ​​marked above represent the mass ratio of MAN-PDA-BPQDs to siRNA, derived from... Figure 5 As can be seen, the MAN-PDA-BPQDs prepared in Example 1 of this application can completely block the migration of siRNA in agarose at a mass ratio of 1:30. That is, the MAN-PDA-BPQDs prepared in this application can effectively condense siRNA for continued use as a non-viral gene vector.

[0066] Example 4

[0067] The MAN-PDA-BPQDs prepared in Example 1 were incubated with siRNA for 40 min to form a MAN-PDA-BPQDs / siRNA complex (MAN-PDA-BPQDs to siRNA mass ratio of 30:1). Then, the complex was irradiated with an 808 nm laser for different times, and the drug release results of MAN-PDA-BPQDs in response to siRNA were obtained by siRNA agarose gel retardation experiment.

[0068] See Figure 6 The black phosphorus-based non-viral gene vector MAN-PDA-BPQDs prepared in this invention exhibits partial siRNA release under 808 nm laser irradiation, with more siRNA released as the incubation time increases. Therefore, it can be concluded that the black phosphorus-based non-viral gene vector MAN-PDA-BPQDs prepared in this application possesses responsive drug release function for siRNA.

[0069] Example 4

[0070] The black phosphorus-based non-viral gene vector MAN-PDA-BPQDs prepared in Example 1 were incubated with rhodamine B at room temperature for 30 min, and then added to BV2 cells and cultured for 0 h, 2 h, 4 h, 6 h, and 8 h, respectively. The culture medium was then aspirated, washed 3-5 times with PBS, and photographed using a laser confocal microscope.

[0071] Please see Figure 7 ,Depend on Figure 7 It is evident that the black phosphorus-based nonviral gene vector MAN-PDA-BPQDs prepared in this invention can target BV2 cells.

[0072] Example 5

[0073] MAN-PDA-BPQDs prepared in Example 1 of this invention (3 mg / kg) were incubated with siRNA-Cy5 at room temperature for 30 min, and then injected intravenously into a PD mouse model. Analysis was performed using an in vivo imaging system 6 h after tail vein injection. Please refer to [link to relevant documentation]. Figure 8 ,Depend on Figure 8 It is evident that the MAN-PDA-BPQDs prepared in this invention have a PD-targeting effect, thereby achieving specific treatment for PD.

[0074] In summary, this application constructs a black phosphorus-based non-viral gene vector with good biocompatibility and biometabolability, which is beneficial for applications in the biomedical field. This black phosphorus-based non-viral gene vector can be used to deliver siRNA. Furthermore, the black phosphorus-based non-viral gene vector constructed in this application is mainly synthesized through condensation reaction, ultrasonic treatment, and ice bath stirring. The preparation method of this invention is simple, has good reproducibility, and shows promising prospects for industrial production. Simultaneously, the black phosphorus-based non-viral gene vector provided in this application enhances its ability to cross the blood-brain barrier, making it a novel non-viral gene vector.

Claims

1. A method for preparing a black phosphorus-based nonviral gene vector, characterized in that: Includes the following steps: S1: Dissolve blocky black phosphorus in NMP, sonicate under ice-water bath conditions, and then centrifuge to obtain black phosphorus quantum dots (BPQDs); S2: Disperse dopamine hydrochloride powder in water to obtain dopamine hydrochloride solution, add ammonia water, anhydrous ethanol and ultrapure water and stir at room temperature, and react in an open container to obtain polydopamine particle dispersion. S3: Add acetone to the polydopamine sample dispersion obtained in S2 under rapid stirring, let stand, and then evaporate to dryness to obtain purified polydopamine particles (PDA). S4: Disperse the PDA obtained in S3 in ethanol, add mannose, add acetic acid dropwise and heat to reflux, and obtain mannose-modified PDA through aldehyde-amine condensation reaction; S5: Self-assemble the BPQDs obtained in S1 with the Man-PDA obtained in S4, stir in an ice-water bath for 2 hours to obtain MAN-PDA-BPQDs, which are mannose-modified polydopamine-encapsulated black phosphorus quantum dots.

2. The method for preparing a black phosphorus-based non-viral gene vector according to claim 1, characterized in that: The concentration of black phosphorus in S1 is 1 mg / mL. The ultrasonic treatment involved sonication for 6 hours in an ice-water bath using an ultrasonic cell disruption system, followed by centrifugation at 10,000 rpm for 1 hour.

3. The method for preparing a black phosphorus-based non-viral gene vector according to claim 1, characterized in that: The concentration of the dopamine hydrochloride solution prepared in step S2 is 0.05 g / mL; The ratio of dopamine hydrochloride powder, ammonia, anhydrous ethanol and ultrapure water was 50 mg: 0.5 mL: 10 mL: 23 mL. The stirring time at room temperature was 30 min, and the open reaction time was 24 h.

4. The method for preparing a black phosphorus-based non-viral gene vector according to claim 1, characterized in that: The ratio of acetone to polydopamine particle dispersion in S3 is 1:(3-4), and the standing time is 12h.

5. The method for preparing a black phosphorus-based non-viral gene vector according to claim 1, characterized in that: In S4, the mass ratio of PDA to mannose is 1:(0.5-1), the amount of acetic acid added is 200μL, the heating reflux temperature is 40-50℃, and the heating reflux time is 24-48 h.

6. The method for preparing a black phosphorus-based non-viral gene vector according to claim 1, characterized in that: The mass ratio of BPQDs to Man-PDA in S5 is 1:(0.5-2).

7. A black phosphorus-based nonviral gene vector, characterized in that: It is prepared using the preparation method described in any one of claims 1-6.

8. The black phosphorus-based non-viral gene vector according to claim 7, characterized in that: The black phosphorus-based non-viral gene vector MAN-PDA-BPQDs has a quantum dot structure, with black phosphorus quantum dots as the core and modified by mannose-modified polydopamine MAN-PDA.

9. A black phosphorus-based non-viral gene vector according to claim 7, characterized in that: The planar dimensions of the black phosphorus-based non-viral gene vector MAN-PDA-BPQDs are 2-10 nm.

10. The application of a black phosphorus-based non-viral gene vector prepared by the methods described in claims 1-6 in the preparation of siRNA delivery vectors in BV2 cells.

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