A method for constructing a gene editing platform based on Prussian blue nanozyme for treating osteoporosis

Abstract

The present invention discloses a construction method HPB@RC‑ALN of a gene editing platform based on Prussian blue nanozyme for treating osteoporosis, the method includes synthesizing HMSNs, growing PB on the surface, carrying CRISPR / Cas9 plasmid, and combining ALN; HPB@RC‑ALN limits ROS generation, alleviates the aging of osteoblasts, and thus promotes bone formation. At the same time, HPB@RC‑ALN osteoblasts are equipped with CRISPER / Cas9 plasmids to achieve efficient transfection, accurately achieve the RANKL gene editing target, and knock out the RANKL gene in osteoblasts, so that the formation of osteoclasts is significantly inhibited. The gene editing platform based on nanozymes of the present invention realizes bidirectional regulation of osteoblasts and osteoclasts by saving cell aging and blocking the production of RANKL.

Description

Technical Field

[0001] The present invention relates to a method for constructing a gene editing platform, and in particular to a method for constructing a gene editing platform based on Prussian blue nanozyme for treating osteoporosis. Background Art

[0002] The osteoporotic microenvironment is characterized by the infiltration of inflammatory factors and the abnormal accumulation of reactive oxygen species (ROS). Recent studies have shown that the increase in mitochondrial-derived ROS is one of the key factors that induce osteoblast senescence, leading to significant redundancy in the osteogenesis process. At the same time, the secretion of receptor activator of nuclear factor kappa-B ligand (RANKL) secreted by osteoblasts in the osteoporotic microenvironment increases, stimulating the formation of mature osteoclasts from monocytic precursors, thereby significantly enhancing bone resorption activity. The secreted RANKL leads to increased bone resorption, resulting in a vicious osteoblast-osteoclast coupling mechanism in the osteoporotic process. Therefore, targeting ROS-induced osteoblast senescence and RANKL production should be a potential method to rescue osteoporosis.

[0003] Nanozymes are a class of nanomaterials with catalytic activities similar to those of natural enzymes, with advantages such as low cost, easy large-scale preparation, high stability and long-term storage. Among various nanozymes, Prussian blue nanozymes (PBNs) have been found to be ideal ROS scavengers because they have peroxidase (POD)-, superoxide dismutase (SOD)- and catalase (CAT)-like activities. The scavenging ability gives PBNs the potential to rescue osteoblast aging and restore osteogenesis. However, the single application of PBNs has problems such as short cycle time, poor selectivity and poor biocompatibility. In addition, the application of PBNs alone may not be able to completely reverse osteoporosis because the production of RANKL still activates osteoclasts. Therefore, the reduction of RANKL expression that interrupts the osteoblast-osteoclast malignant coupling mechanism is another aspect of alleviating osteoporosis. It has been reported that CRISPR / Cas9 plasmids enable targeted gene editing, which can effectively manipulate the RANKL gene in vivo to reduce its expression. Traditional gene delivery vectors rely on liposomes or viruses, but they are unlikely to carry PBNs at the same time to exert anti-ROS effects. Therefore, an efficient nanozyme / plasmid delivery platform is needed. Hollow mesoporous silica nanoparticles (HMSNs) are known to carry a large amount of reagents due to their high specific surface area, large pore volume, and easy modification. Compared with traditional viral gene vectors, HMSNs have the advantages of strong gene carrying capacity, good biocompatibility, low cost, and reduced cancer risk. Therefore, HMSNs are ideal gene vectors to improve the efficiency of RANKL knockout. At the same time, the simple modification of HMSNs makes it possible for PB to exert nanozyme activity after growing on their surface. Finally, it has been well demonstrated that alendronate (ALN)-modified nanomaterials have an affinity for bone tissue. Therefore, the nanozyme-based gene delivery system guided by ALN in vivo should exert its function more precisely. Summary of the invention

[0004] Purpose of the invention: The present invention provides a gene editing platform based on Prussian blue nanozyme for the treatment of osteoporosis.

[0005] Technical solution: The method for constructing a gene editing platform based on Prussian blue nanozyme for treating osteoporosis comprises the following steps:

[0006] S1: Synthesis of hollow mesoporous silica nanoparticles HMSNs;

[0007] S2: Surface growth PB: HMSNs were dissolved in deionized water, PB was added, stirred, centrifuged, and dried to obtain HPB;

[0008] S3: HPB@RC loaded with CRISPR / Cas9 plasmid: HPB and CRISPR / Cas9 plasmid were mixed and stirred, and then centrifuged to obtain HPB@RC;

[0009] S4: Combining with PEG-ALN: Mix the HPB obtained in step S3 with PEG-ALN, stir in the dark, and finally form the HPB@RC-ALN gene editing platform.

[0010] Furthermore, the synthesis method of HMSNs in step S1 is as follows:

[0011] HMSNs were prepared by reverse microemulsion method, in which the aqueous phase was APS aqueous solution and the oil phase was a mixed solution of cetyltrimethylammonium bromide, cyclohexane, Triton X-100 and n-octanol. The aqueous phase was added to the oil phase to form an oil-in-water reverse microemulsion system. After stabilization, TEOS and NH 4 OH initiated the reaction, and acetone was added to terminate the reaction. A white precipitate was formed, which was washed three times to remove CTAB from the white precipitate and acetic acid from deionized water to obtain hollow mesoporous silicon nanoparticles HMSNs.

[0012] Further, the preparation method of PB in step S2 is as follows: FeCl 3 aqueous solution, and K 4 [Fe(CN) 6 ]3H2O solution was slowly mixed, stirred continuously, and centrifuged to dry to obtain PB.

[0013] Furthermore, the ratio of HPB to RC plasmid in step S3 is as follows: 0.5 mg RC plasmid is added to every 2 mg HPB.

[0014] Furthermore, in step S4, the gene editing is intracellular gene editing; the intracellular gene editing method is: resuspending the prepared gene nanoparticles with αMEM cell culture medium to obtain a nanoparticle resuspension, performing cell culture, and extracting RNA and protein from the cells to detect gene mutations.

[0015] The gene editing platform based on Prussian blue nanozyme is prepared by the preparation method.

[0016] The application of the gene editing platform based on Prussian blue nanozyme in the preparation of drugs for treating osteoporosis.

[0017] The present invention first constructs ALN-modified PB-HMSNs nanozyme and loads the RANKL-CRISPR / Cas9 (RC) gene editing system (HPB@RC-ALN). HPB-ALN nanozyme can remove ROS, reduce oxidative stress levels and reverse the abnormal metabolic state of the osteoporotic microenvironment. At the same time, the HPB-ALN nanozyme carrying the RC plasmid will knock out the RANKL gene and cut off the malignant coupling mechanism of osteoblasts and osteoclasts. The constructed complex can provide a novel and precise platform for osteoporosis management.

[0018] Beneficial effects: Compared with the prior art, the advantages of the present invention are: the present invention constructs ALN-modified PB-HMSNs nanozymes and loads the RANKL-CRISPR / Cas9 (RC) gene editing system (HPB@RC-ALN). HPB-ALN nanozymes remove ROS, reduce oxidative stress levels, and reverse the abnormal metabolic state of the osteoporotic microenvironment. At the same time, the HPB-ALN nanozyme carrying the RC plasmid knocks out the RANKL gene and cuts off the malignant coupling mechanism of osteoblasts and osteoclasts. The constructed complex may provide a new and precise platform for the treatment of osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A schematic diagram of the construction route of HPB@RC-ALN and its application mechanism in the treatment of osteoporosis according to an embodiment of the present invention;

[0020] Figure 2 This is a characterization result diagram of the gene nanoplatform of an embodiment of the present invention;

[0021] Figure 3 Zeta potential diagram of HPB@RC-ALN of an embodiment of the present invention;

[0022] Figure 4 This is an agarose gel electrophoresis diagram of HPB@RC-ALN of an embodiment of the present invention;

[0023] Figure 5 The bone targeting performance of the gene nanoplatform of the embodiment of the present invention;

[0024] Figure 6 This is a diagram of the gene nanoplatform of an embodiment of the present invention for removing reactive oxygen species and inhibiting aging (the scale bar is 200 μm);

[0025] Figure 7 This is a diagram of osteoinduction of the gene nanoplatform of an embodiment of the present invention;

[0026] Figure 8 This is a diagram showing the effects of the gene nanoplatform of an embodiment of the present invention on cell transfection and gene editing (scale is 100 μm);

[0027] Fig. 9 This is a graph showing the inhibition of osteoclast formation by the gene nanoplatform of an embodiment of the present invention;

[0028] Fig.10 This is a diagram showing the reversal effect of the gene nanoplatform of an embodiment of the present invention on osteoporosis in OVX mice. DETAILED DESCRIPTION

[0029] This embodiment provides a method for preparing a gene nanoplatform based on Prussian blue nanozyme. The structure of the gene editing system is as follows: Figure 1 As shown, the specific steps are as follows:

[0030] S1: Synthesis of HMSNs

[0031] HMSNs were prepared by reverse microemulsion method, in which the aqueous phase was 0.23% (v / v) APS aqueous solution, and the oil phase was a mixed solution of 15 mg hexadecyltrimethylammonium bromide, 72.6 mL cyclohexane, 17.88 g Triton X-100 and 17.6 mL n-octanol. The aqueous phase was slowly added to the oil phase under magnetic stirring to form an oil-in-water reverse microemulsion system, and 800 ul TEOS and 800 ul NH 4 OH initiated the reaction, and after stirring at room temperature for 24 h, 40 mL of acetone was added to terminate the reaction. The white precipitate was washed three times with ethanol and deionized water respectively. The white precipitate was stirred in 40 mL of acetic acid at room temperature for 4 h, CTAB was removed, and the acetic acid was removed by washing with deionized water three times to obtain hollow mesoporous silicon nanoparticles HMSNs.

[0032] S2: Surface growth PB

[0033] HMSNs (10.02 mg) were dissolved in 10.0 mL of deionized water by ultrasonication. 16.22 mL of FeCl 3 Aqueous solution (6.43 mg, 0.40 mmol) was stirred for 1 hour, and then K 4 [Fe(CN) 6 ].3H2O solution (8.45 mg, 0.20 mmol) was slowly dripped into the mixture, stirred continuously at 25°C for 0.5 h, and centrifuged to obtain HPB; transmission electron microscopy (TEM) images showed that HMSNs and HPB were uniformly dispersed spherical, with average sizes of 47 and 48 nm, respectively ( Figure 2 A, B). Energy dispersive X-ray spectroscopy (EDS) confirmed the presence of iron (Fe) in HPBs compared to HMSNs ( Figure 2 C, D). Elemental mapping further showed that PB was successfully combined with HMSN, and C, O, and Si elements were evenly distributed in the nanoframe ( Figure 2 E, F). Figure 2 As shown in GH, H and HMSNs were characterized by X-ray powder diffraction (XRD) and HPB, respectively, and it was found that both had good crystallinity and had a significant PB component in HPB (JCPDS Card No. 73-0687). In addition, X-ray photoelectron spectroscopy (XPS) also confirmed the distribution of Fe in HPB nanozymes ( Figure 2 I, J).

[0034] S3: Plasmid carrying CRISPR / Cas9

[0035] 2mgHPB and CRISPR / Cas9 plasmid (1000ng / ul, 500ul) were gently stirred overnight at 4°C and centrifuged to obtain HPB@RC. The average zeta potentials of HPB, HPB-ALN, and HPB@RC-ALN were 17.6, 18.2, and 1.94mV, respectively. The zeta potential in HPB@RC-aln was significantly reduced, indicating that the RC plasmid had been successfully loaded onto the HPB nanozyme ( Figure 3 HPB of different masses was mixed with plasmids, and the optimal mass ratio of the material to CRISPR / Cas9 plasmids was obtained by agarose gel electrophoresis. When the mass ratio of nanozyme to plasmid was 20:1, no obvious plasmid leakage was observed ( Figure 4 ).

[0036] S4: Binding of PEG-ALN

[0037] Mix 2 mg of HPB obtained in step S3 with 0.1 mg of PEG-ALN, stir slowly for 6 h in the dark, and finally form the HPB@RC-ALN gene editing platform, which can be stored at 4°C. Hydroxyapatite (HAP) was used as a material to simulate bone tissue in vitro to test the targeting efficiency of HBP-ALN. After HPB or HPB-aln were incubated on HAP for different times, the binding efficiency was evaluated by scanning electron microscopy. The data showed that both HPB and HPB-aln gradually bound to HAP in a time-dependent manner. However, compared with the HPB group, the number of HPB-aln nanoparticles observed on HAP increased significantly after 24, 48, and 72 h of co-culture ( Figure 5 AB). The results showed that the modification of HPB by ALN was successful and confirmed the skeleton targeting ability of ALN-modified HPB nanoparticles.

[0038] This example verifies the anti-ROS and anti-aging effects of the HPB@RC-ALN gene nanoplatform prepared above in vitro. 2 O 2 The solution (0-400 μM) was co-cultured with MC-3T3E1 cells for 24 hours, and it was found that the ROS accumulation and senescence of the cells increased in a concentration-dependent manner ( Figure 6 AC). When HPB, HPB-ALN or HPN@RC-ALN is added and mixed with H 2 O 2 When incubated with (400 μM) solution, the intracellular ROS and senescence levels decreased significantly ( Figure 6 DF). It is worth noting that the presence of the RC plasmid did not affect the anti-ROS and anti-aging abilities of the nanozyme ( Figure 6DF). These data indicate that HPB has good anti-ROS and anti-cell aging functions.

[0039] This example evaluates the effect of HPB@RC-ALN gene nanoplatform on osteogenic induction. 2 O 2 , H 2 O 2 +H、H 2 O 2 +HPB,H 2 O 2 +HPB-ALN or H 2 O 2 +HPB@RC-ALN were co-cultured in osteogenic induction medium. Figure 7 As shown in A, hydrogen peroxide solution significantly reduced ALP activity and calcium mineral deposition (ARS staining) in MC-3T3E1 cells, which was largely rescued in the presence of nanozymes. Quantitative polymerase chain reaction (qPCR) evaluation results showed that HPB, HPB-ALN, and HPB@RC-ALN constitutive bone marker genes (OSX, Runx2, cyanate, and ALP) were upregulated, reversing the pro-aging effects of hydrogen peroxide ( Figure 7 BE). The above data indicate that nanozymes can effectively rescue the inhibited osteogenesis by inhibiting the generation of ROS in the osteoporotic microenvironment.

[0040] This example also verifies the transfection and gene editing effects of the HPB@RC-ALN gene nanoplatform on cells. MC3T3-E1 cells were incubated with HPB@RC-ALN for 24, 48, and 72 hours. The transfection efficiency was evaluated by the green fluorescent protein (GFP) expressed and translated from the CRISPR / Cas9 plasmid as designed. The results showed that the transfection rate was time-dependent, with green fluorescence appearing after 24 hours of co-incubation and significantly increasing after 72 hours ( Figure 8 A). Flow cytometry analysis ( Figure 8 B, C) The transfection rate was further confirmed and quantified, showing that about 80% of the cells successfully carried the RC plasmid for 72 hours. The data showed that the synthesized HPB@RC-ALN had excellent cell transfection ability. Then, Western blot showed that when combined with H 2 O 2 When co-cultured with HPB@RC-ALN, the RANKL protein of MC3T3E1 cells decreased in a time-dependent manner ( Figure 8D, E), indicating that HPB@RC-ALN effectively inhibits RANKL secretion in the osteoporotic microenvironment. The gene editing ability of RC was further confirmed by using T7 endonuclease I (T7EI) assay. Notably, compared with the other groups, only HPB@RC-ALN was observed to disrupt the RANKL gene after 72 h of co-incubation with MC3T3-E1, and three bands appeared after T7E1 cleavage ( Figure 8 F). The expression of RANKL mRNA and protein levels in different groups were further analyzed by quantitative polymerase chain reaction (qPCR) and western blotting. RANKL expression increased after H2O2 stimulation of MC3T3-E1 cells, while RANKL expression decreased after the addition of HPB or HPB-ALN, which may be related to the excellent antioxidant effect of nanozymes. Notably, RANKL expression in the HPB@RC-ALN group was further reduced, indicating the effective gene editing function of RANKL by the RC plasmid carried in HPB@RC-ALN ( Figure 8 G and I).

[0041] This example also verifies that the HPB@RC-ALN gene nanoplatform inhibits osteoclast formation by blocking RANKL secretion in senescent osteoblasts. 2 O 2 , H 2 O 2 The supernatant was collected from the culture medium of MC3T3-E1 treated with HPB. 2 O 2 +HPB-ALN or H 2 O 2 +HPB@RC-ALN. Then, the supernatants from different groups were added to osteoclast induction medium containing reduced levels of RANKL (25 ng / mL). Fig. 9 As shown in AB, the control group failed to produce osteoclasts, while H 2 O 2 The supernatant of group H 2 O 2 Stimulating MC3T3-E1 cells to secrete more RANKL significantly induced osteoclast formation. However, after adding the supernatant of the HPB or HPB-ALN treatment group, the osteoclastogenesis process was partially alleviated. In addition, no osteoclast formation was observed after adding the supernatant of the HPB@RC-ALN treatment group, indicating that the RC released from the vector has an effective RANKL gene editing function. The expression of osteoclastogenesis-related markers (TRAP, CTSK, and OSCAR) was significantly reduced at the mRNA level in the HPB and HPB-ALN supernatant groups, and in the HPB@RC-ALN supernatant groups, the expression of osteoclastogenesis-related markers (TRAP, CTSK, and OSCAR) was significantly reduced at the mRNA level in the HPB and HPB-ALN supernatant groups. 2 O 2 +HPB@RC-ALN supernatant group was even lower ( Fig. 9 CE), which was attributed to the anti-ROS effect of nanoparticles and the RANKL gene editing effect of RC plasmid.

[0042] This example also verifies the reversal effect of HPB@RC-ALN gene nanoplatform on osteoporosis in OVX mice. In this example, the osteoporosis model was established by removing the bilateral ovaries of mice, and then the complex was locally injected into the femoral medullary cavity. The mice were killed 6 weeks after the end of the treatment, and the bone quality of the femurs of the mice was finally observed to determine the anti-osteoporosis effect of the nanozyme gene platform. The results are shown in the attached Fig.10 The integrity of the femurs in each group was observed by micro-computed tomography (micro-CT) and 3D reconstruction. As expected, OVX mice underwent severe bone loss ( Fig.10 A). In contrast, HPB- and HPB-ALN-treated mice exhibited elevated bone volume fraction (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and reduced trabecular separation (Tb.Sp). Due to the bone tissue-targeting properties of ALN, mice in the HPB-ALN group showed more bone mass than mice in the HPB group. Notably, mice treated with the HPB@RC-ALN group exhibited the greatest BV / TV, Tb.N, and Tb.Th among all groups, demonstrating the best osteoporosis rescue ability of HPB@RC-ALN ( Fig.10 BE). The colocalization of GFP and OCN observed in the mouse femur indicated that the RC plasmid successfully entered the osteoblasts of the mouse femur (Figure S3). DHE, SA-β-gal, OCN, and TRAP staining showed a consistent trend with that observed in micro-CT, with OCN expression significantly reduced in OVX mice, while HPB@RC-ALN showed the opposite trend ( Fig.10 F). SA-β-Gal staining showed that osteoblast senescence in OVX mice treated with nanozymes (HPB, HPB-ALN, and HPB@RC-ALN) was significantly reduced compared with the control group. In addition, TRAP staining showed that the number of osteoclasts in OVX mice treated with HPB@RC-ALN was almost restored to the same physical level as that of control mice ( Fig.10 F). These results suggest that the nanozyme gene platform may become a precise targeting strategy to rescue osteoporosis.

[0043] In summary, this example successfully constructed ALN-modified PB-HMSNs nanozyme and loaded the RANKL-CRISPR / Cas9 (RC) gene editing system (HPB@RC-ALN). HPB-ALN nanozyme can remove ROS, reduce oxidative stress levels, and reverse the abnormal metabolic state of the osteoporotic microenvironment. At the same time, the HPB-ALN nanozyme carrying the RC plasmid will knock out the RANKL gene and cut off the malignant coupling mechanism of osteoblasts and osteoclasts. The constructed complex may provide a new and precise platform for the treatment of osteoporosis.

Claims

1. A method for preparing a gene editing platform based on Prussian blue nanozyme for treating osteoporosis, It is characterized in that The steps include: S1: Synthesis of hollow mesoporous silica nanoparticles HMSNs; S2: Surface growth PB: HMSNs were dissolved in deionized water, PB was added, stirred, centrifuged, and dried to obtain HPB; S3: HPB@RC loaded with CRISPR / Cas9 plasmid: HPB and CRISPR / Cas9 plasmid were mixed at a mass ratio of 20:1 and centrifuged; S4: Combining with PEG-ALN: Mix the HPB obtained in step S3 with PEG-ALN, stir in the dark, and finally form the HPB@RC-ALN gene editing platform.

2. The method for preparing the gene editing platform based on Prussian blue nanozyme for treating osteoporosis according to claim 1, It is characterized in that The synthesis method of HMSNs in step S1 is as follows: HMSNs were prepared by reverse microemulsion method, in which the aqueous phase was APS aqueous solution and the oil phase was a mixed solution of cetyltrimethylammonium bromide, cyclohexane, Triton X-100 and n-octanol. The aqueous phase was added to the oil phase to form an oil-in-water reverse microemulsion system. After stabilization, TEOS and NH 4 OH initiated the reaction, and acetone was added to terminate the reaction. A white precipitate was formed, which was washed three times to remove CTAB from the white precipitate and acetic acid from deionized water to obtain hollow mesoporous silicon nanoparticles HMSNs.

3. The method for preparing the gene editing platform based on Prussian blue nanozyme for treating osteoporosis according to claim 1, It is characterized in that The preparation method of PB in step S2 is as follows: FeCl 3 aqueous solution, and K 4 [Fe(CN) 6 ]3H2O solution was slowly mixed, stirred continuously, and centrifuged to dry to obtain PB.

4. The method for preparing a gene editing platform based on Prussian blue nanozyme for treating osteoporosis according to claim 1, It is characterized in that In step S4, the gene editing is intracellular gene editing; the intracellular gene editing method is: resuspending the prepared gene nanoparticles with αMEM cell culture medium to obtain a nanoparticle resuspension, performing cell culture, and extracting RNA and protein from the cells to detect gene mutations.

5. A gene editing platform based on Prussian blue nanozyme prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the gene editing platform based on Prussian blue nanozyme as described in claim 5 in the preparation of drugs for treating osteoporosis.

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