Targeted medicine for treating prostatic cancer and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a targeted medicine for treating prostatic cancer and application thereof. The drug is an Apt-C-C-S-PEG-CHO anchored 293T exosome loaded with siRNA-JAG1 and siRNA-NOTCH3, the drug with an optimized molar ratio can target prostate cancer cells through peripheral veins, and the malignant progression of the prostate cancer can be more effectively inhibited.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a targeted drug for treating prostate cancer and its application. Background Art

[0002] Prostate cancer is one of the common types of malignant tumors in men, especially the incidence rate in elderly men has been showing an increasing trend year by year. Early prostate cancer usually responds well to androgen deprivation therapy or chemotherapy. However, as the disease progresses, tumor cells often develop tolerance and resistance to existing treatments, and ultimately evolve into castration-resistant prostate cancer. The characteristics of the disease at this time include an accelerated proliferation rate, limited treatment options, and a high risk of metastasis, which can easily lead to poor prognosis of patients.

[0003] In recent years, studies on the resistance mechanisms of advanced prostate cancer have shown that the androgen receptor signaling pathway still plays an important role in the occurrence and development of CRPC. However, conventional hormone therapy strategies are no longer able to fully inhibit the proliferation of these drug-resistant tumor cells. At the same time, genetic and epigenetic changes in tumors enable prostate cancer cells to continuously activate signaling pathways and expand their survival advantages in the microenvironment. These characteristics have led to limited efficacy of conventional treatment methods (including chemotherapy, radiotherapy, and new-generation androgen receptor pathway inhibitors), and a significant increase in the drug resistance incidence rate and recurrence rate.

[0004] To further improve the prognosis of patients with advanced prostate cancer and enhance the overall survival rate, more and more studies have turned their attention to biotherapy centered on molecular targets. With the development of multidisciplinary technologies such as genomics, transcriptomics, and proteomics, the molecular pathological characteristics of prostate cancer have been gradually analyzed, and new oncogenes or signaling pathways have been continuously discovered. On this basis, by identifying tumor-specific antigens or key molecular pathways and designing molecular targeted drugs or immunotherapy methods with higher specificity and fewer side effects, it has become the most promising direction in the comprehensive treatment of advanced prostate cancer and is also expected to break through the current problems of drug resistance and rapid progression. Summary of the Invention

[0005] The present invention mainly aims at the above technical problems and provides a targeted composition for treating prostate cancer and its preparation method.

[0006] The present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a targeted composition for treating prostate cancer, characterized in that the composition comprises the following components:

[0008] Exosomes, siRNA, and Apt-C-C-S-PEG-CHO, wherein the siRNA is siRNA targeting the JAG1 and / or NOTCH3 genes, the Apt has the sequence shown in SEQ ID NO.5, the siRNA is loaded into the exosomes, and the Apt-C-C-S-PEG-CHO is anchored to the exosomes.

[0009] Preferably, the aforementioned siRNA is two kinds of siRNA targeting the JAG1 and NOTCH3 genes.

[0010] Preferably, the sequences of the aforementioned siRNA are as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0011] Optionally, the aforementioned composition further contains a pharmaceutically acceptable carrier or excipient.

[0012] On the other hand, another object of the present invention is to provide an application of a composition in the preparation of a targeted drug for treating prostate cancer, characterized in that the composition is composed of the following components:

[0013] Exosomes, siRNA, and Apt-C-C-S-PEG-CHO, wherein the siRNA is siRNA targeting the JAG1 and / or NOTCH3 genes, the Apt has the sequence shown in SEQ ID NO.5, the siRNA is loaded into the exosomes, and the Apt-C-C-S-PEG-CHO is anchored to the exosomes.

[0014] Preferably, the aforementioned siRNA is two kinds of siRNA targeting the JAG1 and NOTCH3 genes.

[0015] Preferably, the sequences of the aforementioned siRNA are as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0016] Optionally, the aforementioned drug further contains a pharmaceutically acceptable carrier or excipient.

[0017] On the other hand, the present invention provides a method for preparing a targeted drug for treating prostate cancer, characterized in that the method includes preparing a composition mix-EXO-apt-siJAG1&NTOCH3, and the composition mix-EXO-apt-siJAG1&NTOCH3 includes: exosomes, siRNA, and Apt-C-C-S-PEG-CHO, wherein the siRNA is siRNA targeting the JAG1 and / or NOTCH3 genes, the Apt has the sequence shown in SEQ ID NO.5, the siRNA is loaded into the exosomes, and the Apt-C-C-S-PEG-CHO is anchored to the exosomes.

[0018] The specific method for preparing mix-EXO-apt-siJAG1&NTOCH3 is as follows:

[0019] 1. Design the E3 aptamer sequence (Apt): GGC UUU CGG GCU UUC GGC AAC AUC AGC CCC UCAGCC;

[0020] 2. The modified Apt sequence (Apt-C-C-S-S-C-C-OH), that is, attach Figure 2 the synthetic product in step a: synthesized by Genewiz;

[0021] 3. When using TCEP to decompose the modified Apt sequence to obtain Apt-SH, only need to mix well and let it stand for 1 hour (step b);

[0022] 4. When purifying Apt-SH, the purification reagents required are absolute ethanol and 3M sodium acetate, and the addition ratio of absolute ethanol to 3M sodium acetate is 3:1 (step c);

[0023] 5. After the purification of Apt-SH is completed, resuspend Apt-SH in a mixed buffer (that is, a mixed buffer of 2.5 mM MgCl2 and 1X PBSPH 7.4), heat at 70 °C for 10 min, and then quickly cool on ice for 10 min;

[0024] 6. Couple the obtained deprotected Apt-SH with Maleimide-PEG-Cholesterol at a molar ratio of 1:2, mix well and let it stand at 4 °C overnight (step d);

[0025] 5. Further dialysis can purify Apt-C-C-S-PEG-CHO. Place the material in a regenerated cellulose dialysis bag (2000), place it in distilled water and let it stand, change the water every 2 - 4 hours, and dialyze and purify for 24 hours (step d);

[0026] 6. Load siRNA into 293T exosomes: Just follow the steps of the exosome loading kit (step e);

[0027] 7. Prepare a mixed suspension of 293T exosomes and Apt-C-C-S-PEG-CHO, and let it stand overnight at 4 °C until Apt-C-C-S-PEG-CHO automatically anchors to the surface of the 293T exosome membrane (step f).

[0028] Preferably, mix EXO-apt-siJAG1 and EXO-apt-siNTOCH3 at a molar ratio of 1:0.85 to prepare mix-EXO-apt-siJAG1&NTOCH3.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. Targeted interference with the activity of the JAG1-NOTCH3 signaling pathway can more effectively inhibit the malignant progression of prostate cancer than inhibiting the expression levels of JAG1 or NOTCH3 genes alone.

[0031] 2. Design E3-siNOTCH3 and E3-siJAG1, with a preferred molar ratio of 1:0.85 as a mixed reagent, which enters the body through a peripheral vein, targets prostate cancer cells, and specifically interferes with JAG1 and NOTCH3 mRNA levels, showing a more effective anti-prostate cancer proliferation and metastasis effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The product, method and beneficial effects of the present invention are described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0033] Figure 1 siRNA-JAG1 and siRNA-NOTCH3 inhibited the mRNA expression of JAG1 and NOTCH3;

[0034] Figure 2 Schematic diagram of EXOApt-siRNA synthesis, detailing the synthesis steps and required materials.

[0035] Figure 3 Validation of EXOApt-siRNA synthesis results, including Figure 3 A: Western Blotting experiments proved that the extracted exosomes were from 293T cells, not 293T cells. 3B: RNA electrophoresis confirmed that compared with Line 4, Line 5 finally synthesized empty Apt-CCS-PEG-CHO. 3C: Immunofluorescence verified that EXOs (red) had been successfully loaded with fluorescent siRNA (green). 3D: Flow cytometry confirmed that EXOApt-si RNA can be specifically taken up by prostate cancer cells (PC3 and DU145) and has no specific contact with BPH cells.

[0036] Figure 4 Inhibitory effect of siRNA-loaded exosomes on prostate cancer cell growth.

[0037] Figure 5 Immunofluorescence staining of JAG1-CY5 and NOTCH3-CY3 in prostate cancer tissues.

[0038] Figure 6 is the tumor growth, Figure 6 A: Gross picture of the mouse and tumor after the mouse was sacrificed.Figure 6 B: Growth curves of tumor volumes in each group. The final volumes of tumors in the EXO-apt-siJAG1 group and the EXO-apt-NOTCH3 group were significantly smaller than those in the EXO-apt-siRNA group, and the growth curves finally showed a trend of stable volume; the effect of tumor volume reduction in the mix-EXO-apt-siJAG1&NOTCH3 mixed reagent group was more obvious than that in the single-use groups, and a downward trend of tumor volume was presented. Figure 6 C: Gross pictures of ex vivo tumors. Figure 6 D: Comparison chart of tumor weights after stereotomy. The results were consistent with those of the tumor volume comparison. Detailed implementation manners

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0040] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0041] Example 1. Design of siRNA and its effect on inhibiting mRNA expression

[0042] To verify the inhibitory effect of siRNA-JAG1 and siRNA-NOTCH3 on mRNA expression, we added siRNA to prostate cancer cell lines PC3 and C4-2 for culture. After culture, PC3 and C4-2 RNA were extracted for PCR detection of the mRNA expression of JAG1 and NOTCH3. The results are as Figure 1 shown, and the inhibitory effect of si-RNA on JAG1 and NOTCH3 mRNA is significant.

[0043] Example 2. Construction of EXO-apt-siRNA

[0044] The main steps of the specific synthesis are as shown in the appendix Figure 2 shown. Among them, the most critical step in this synthesis is the synthesis of Apt-C-C-S-PEG-CHO and the loading of siRNA into EXOs.

[0045] Detailed description of the steps:

[0046] 1. Design the E3 aptamer sequence (Apt): GGC UUU CGG GCU UUC GGC AAC AUC AGC CCC UCAGCC;

[0047] 2. Modified Aptamer Sequence (Apt-C-C-S-S-C-C-OH), that is, attached Figure 2 The synthesis product in step a: Synthesized by GenScript;

[0048] 3. When using TCEP to decompose the modified Aptamer sequence to obtain Apt-SH, it only needs to be mixed well and then left standing for 1 hour (step b);

[0049] 4. When purifying Apt-SH, the purification reagents required are absolute ethanol and 3M sodium acetate, and the addition ratio of absolute ethanol to 3M sodium acetate is 3:1 (step c);

[0050] 5. After the purification of Apt-SH is completed, resuspend Apt-SH in a mixed buffer (that is, a mixed buffer of 2.5 mM MgCl2 and 1X PBSPH 7.4), heat at 70 °C for 10 min, and then quickly cool on ice for 10 min;

[0051] 6. Couple the obtained deprotected Apt-SH with Maleimide-PEG-Cholesterol at a molar ratio of 1:2, mix well and leave standing at 4 °C overnight (step d);

[0052] 5. Further dialysis can purify Apt-C-C-S-PEG-CHO. Place the material in a regenerated cellulose dialysis bag (2000), place it in distilled water and leave standing, change the water every 2 - 4 hours, and dialyze and purify for 24 hours (step d);

[0053] 6. Loading siRNA into 293T exosomes: Just follow the steps of the exosome loading kit (step e);

[0054] 7. Prepare a mixed suspension of 293T exosomes and Apt-C-C-S-PEG-CHO, leave at 4 °C overnight, and wait for Apt-C-C-S-PEG-CHO to automatically anchor to the surface of the 293T exosome membrane (step f).

[0055] The materials required for this synthetic compound are: E3 aptamer sequence, modified Aptamer sequence, TCEP, absolute ethanol, 3M sodium acetate, Maleimide-PEG-Cholesterol, MgCl2, 1XPBS, 293T exosomes, siRNA sequence of hnRNPA2B1 and exosome loading kit.

[0056] TCEP: Catalog number ED-8091, Yitao Biotech;

[0057] Absolute ethanol: Taken from the laboratory;

[0058] 3M sodium acetate: Catalog number BL541A, PH 5.2;

[0059] Maleimide-PEG-Cholesterol: Catalog number bqs129807-1g, Jinsong Biotech;

[0060] MgCl2: Catalog number M871940, Macklin Reagent;

[0061] 1X PBS: PH7.4, Catalog number KGL2206-500, KeyGen Biotech;

[0062] 293T exosomes: Extracted from the supernatant culture medium of 293T cells;

[0063] Exosome loading kit: Catalog number ELSR-06, from Enze Kangtai;

[0064] siRNA sequences of JAG1 and NOTCH3: The targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the JAG1 gene is shown in SEQ ID NO.3, and the targeting nucleotide sequence of the siRNA that specifically interferes with the NOTCH3 gene is shown in SEQ ID NO.4.

[0065] Furthermore, Western Blotting experiments were used to prove that the extracted exosomes were from 293T cells rather than 293T cells ( Figure 3 A). RNA electrophoresis confirmed that compared with Line4, the final synthesized product of Line5 was the empty Apt-C-C-S-PEG-CHO ( Figure 3 B). Immunofluorescence verified that EXOs (red) had successfully loaded fluorescent siRNA (green) ( Figure 3 C). Flow cytometry confirmed that EXO Apt-siRNA could be specifically taken up by prostate cancer cells (PC3 and DU145) and had no specific contact with BPH cells ( Figure 3 D).

[0066] Example 3. Cell experiments to verify the inhibitory effect of mix-EXO-apt-siJAG1&NTOCH3 on tumor cells

[0067] Compare the effects of two loading methods on cell growth. The first is separate and independent loading, and after loading, they are mixed in proportion (EXO-apt-si-JAG1 AND EXO-apt-si-NTOCH3 groups); the second method is to mix the two siRNAs first and then load them into exosomes (mix-EXO-apt-siJAG1&NOTCH3 group. This loading mode can result in different siRNAs being loaded into the same exosome). Prostate cancer cells PC3 and macrophages THP-1 were co-cultured. The above siRNA mixture was added, and after co-culturing for 48 hours, CCK8 was used to evaluate cell growth, and a blank control was used as a reference (si-JAG1 and si-NTOCH3 in a molar ratio of 1:1). Results( Figure 4 A) Unexpectedly showed that under the same proportion, the mix-EXO-apt-siJAG1&NOTCH3 assembly loading method had a more obvious inhibitory effect on tumor cells than the EXO-apt-si-JAG1 AND EXO-apt-si-NTOCH3 groups, and the inhibitory effect was more than doubled. It can be seen that the loading and combination methods have an important impact on the inhibitory effect of tumor cells.

[0068] Using the method of mixing siRNAs and then loading them into exosomes (Mix-apt-siJAG1&NOTCH3), to further clarify the more efficient ratio of si-JAG1 and si-NOTCH3, we set seven groups according to the ratio of si-JAG1 to si-NOTCH3 (siJAG1:siNOTCH3 = blank control; 1:0.5; 1:0.7; 1:0.85; 1:1; 0.85:1; 0.7:1; 0.5:1). Prostate cancer cells PC3 and macrophages THP-1 were co-cultured. The above siRNA mixture was added, and after co-culturing for 48 hours, CCK8 was used to evaluate cell growth, and a blank control was used as a reference. Results( Figure 4 B) showed that the mixture obtained by exosome loading when siJAG1:siNOTCH3 = 1:0.85 had the best tumor inhibitory effect, and the inhibitory efficiency was more than doubled compared to the significant difference group. It can be seen that the ratio of different siRNAs also has an important impact on the tumor inhibitory effect.

[0069] Example 4 In vivo experiment of mix-EXO-apt-s iJAG1&NTOCH3 injected via peripheral blood

[0070] Detection of the inhibitory effect on gene expression after EXO-apt-siRNA enters the in-vivo prostate tissue through peripheral blood. TRAMP mice with prostate tumors were used. A mixed reagent (mix-EXO-apt-siJAG1&NTOCH3: EXO-apt-siJAG1 = 1 mmol / kg, EXO-apt-NOTCH3 = 0.85 mmol / kg) was injected into the peripheral blood once every 24 hours for 7 consecutive times. Then the prostate was dissected, frozen sections were made, and immunofluorescence staining was performed. The antibody combination was: JAG1-CY5, NOTCH3-CY3. The results are as Figure 5 shown: The expressions of JAG1 and NOTCH3 in the prostate cancer tissues of the treatment group were significantly lower than those in the control group. It can be seen that after mix-EXO-apt-siJAG1&NTOCH3 is injected into live animals through peripheral blood, it can stably exert an inhibitory effect in the prostate tissue.

[0071] Example 5. Animal experiment. mix-EXO-apt-siJAG1&NTOCH3 can effectively control and reduce the volume of prostate tumors

[0072] All animals involved in this experiment were approved by the Animal Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University. A subcutaneous xenograft model was established using male BALB / c nude mice. Five-week-old male nude mice were randomly divided into 4 groups: mix-EXO-apt-siJAG1&NOTCH3 group (n = 8), EXO-apt-siJAG1 group (n = 8), EXO-apt-NOTCH3 group (n = 8), and EXO-apt-siRNA group (n = 8). Approximately 2×10^6 PC3 cells were suspended in physiological saline and injected into the back of each mouse. The tumor growth was measured weekly, and the tumor volume was calculated according to the formula (length × width^2 × 0.6). All mice were euthanized after six weeks, the tumors were photographed and recorded, and the tumor weights were recorded. The results are as Figure 6 shown, Figure 6 A: Gross pictures of the mice and tumors after euthanasia. Figure 6 B: Tumor volume growth curves of each group. The final volumes of the tumors in the EXO-apt-siJAG1 group and the EXO-apt-NOTCH3 group alone were significantly smaller than those in the EXO-apt-siRNA group, and the growth curves finally showed a stable volume trend; the tumor volume reduction effect of mix-EXO-apt-siJAG1&NOTCH3 was more obvious than that of the single-use groups, and the tumor volume showed a downward trend. Figure 5 C: Gross pictures of the excised tumors. Figure 5 D: Comparative graph of the tumor weights after euthanasia. The results were consistent with the tumor volume comparison results.

[0073] In summary, the present invention first prepares 293T exosomes anchored with Apt-C-C-S-PEG-CHO and loaded with siRNA-JAG1 and siRNA-NOTCH3, and proves that the exosomes mixed with siRNA and having a specific molar ratio of siRNA can target prostate cancer cells through the peripheral vein, with better tumor inhibition effect.

[0074] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather should be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A targeted drug for treating prostate cancer, characterized in that: The composition comprises the following components: Exosomes, siRNA and Apt-CCS-PEG-CHO, wherein the siRNA is siRNA targeting JAG1 and / or NOTCH3 genes, the Apt is the sequence shown in SEQ ID NO.5, the siRNA is loaded in the exosomes, and the Apt-CCS-PEG-CHO is anchored on the exosomes.

2. The drug according to claim 1, characterized in that The siRNAs are two siRNAs targeting JAG1 and NOTCH3 genes.

3. The drug according to claim 1, characterized in that The sequences of the siRNA are shown in SEQ ID NO.3 and SEQ ID NO.

4.

4. The drug according to claim 1, characterized in that The medicine further contains a pharmaceutically acceptable carrier or excipient.

5. Use of a composition in the preparation of a targeted drug for treating prostate cancer, characterized in that: The composition comprises the following components: exosomes, siRNA and Apt-CCS-PEG-CHO, wherein the siRNA is siRNA targeting JAG1 and / or NOTCH3 genes, the Apt is a sequence as shown in SEQ ID NO.5, the siRNA is loaded in the exosomes, and the Apt-CCS-PEG-CHO is anchored on the exosomes.

6. The use according to claim 5, characterized in that The siRNAs are two siRNAs targeting JAG1 and NOTCH3 genes.

7. The use according to claim 5, characterized in that The sequences of the siRNA are shown in SEQ ID NO.3 and SEQ ID NO.

4.

8. The use according to claim 5, characterized in that The medicine further contains a pharmaceutically acceptable carrier or excipient.

9. A method for preparing a targeted drug for treating prostate cancer, characterized in that: The method includes preparing a composition mix-EXO-apt-siJAG1&NTOCH3, wherein the composition mix-EXO-apt-siJAG1&NTOCH3 includes: exosomes, siRNA and Apt-CCS-PEG-CHO, wherein the siRNA is an siRNA targeting JAG1 and / or NOTCH3 genes, the Apt is a sequence as shown in SEQ ID NO.5, the siRNA is loaded in the exosomes, and the Apt-CCS-PEG-CHO is anchored on the exosomes. .

10. The method according to any one of claims 7 to 9, characterized in that: The complement inhibitor can specifically block complement activation of the classical pathway.

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