Soluble microneedle containing tumor-derived total RNA (Ribonucleic Acid) nano vaccine as well as preparation method and application of soluble microneedle

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a soluble microneedle containing a tumor-derived total RNA (Ribonucleic Acid) nano vaccine as well as a preparation method and application of the soluble microneedle. The protamine, the TdRNA and the CpG are compounded by utilizing the characteristic that the protamine can be combined with the RNA in positive and negative electricity to form the nano vaccine for treating or preventing tumors. The synergistic effect of the TdRNA and CpG rich in tumor antigen broad-spectrum genetic codes subsequently drives the maturation of dendritic cells and the activation of cytotoxic T lymphocytes, so that strong anti-tumor immunity is generated, tumors are effectively treated or prevented, and the TdRNA and CpG compound has no obvious physiological toxicity and is high in safety. In addition, the RNA can be effectively protected by the mode of loading the RNA vaccine through the microneedle, the in-vitro degradation of the RNA is prevented, and the storage and transportation difficulty is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a soluble microneedle containing tumor-derived total RNA nanovaccine, and a preparation method and application thereof. Background Art

[0002] Cancer is one of the most deadly diseases in human beings today, with high morbidity and mortality rates. The emergence and development of immunotherapy has profoundly changed the landscape of cancer treatment, making it a viable approach to combat malignant tumors. Tumor vaccines have attracted great interest in the field of cancer immunotherapy. These vaccines enhance host immunity by exploiting differences in protein expression between tumor and normal cells, effectively present tumor antigens, and selectively activate cytotoxic T lymphocytes (CTLs) with high specificity and minimal adverse reactions. However, traditional tumor vaccines face major obstacles. For example, peptide vaccines derived from short peptides representing tumor antigen fragments need to bind to the patient's MHC molecules to elicit T cell immune responses against specific tumor antigens. The variability of the patient's MHC type limits its efficacy.

[0003] RNA vaccines have several advantages, such as they can bypass MHC restriction, induce immunogenicity without adjuvants, and are not integrated into the genome, which is safer. However, RNA vaccines targeting tumor-associated or specific epitopes may induce significant tumor-specific and systemic immunosuppression and promote immune escape. Tumor-derived total RNA (TdRNA) vaccines activate the immune system by utilizing total mRNA extracted from tumor cells, which encodes various tumor-specific antigens. Once internalized by dendritic cells, this mRNA is used to synthesize tumor-specific antigens and then presented on the surface of dendritic cells, which can activate T cells and B cells and trigger immune responses against tumor antigens. In addition, exogenous RNA can be directly recognized by pattern recognition receptors on DCs (such as TLR3, TLR7, and TLR8), which initiate signaling pathways that promote DC maturation and activation. This type of vaccine can trigger a broad immune response against multiple tumor antigens and enhance the ability of the immune system to recognize and eliminate tumor cells, which has great application potential and has shown preventive and therapeutic effects in various cancers.

[0004] However, TdRNA tumor vaccines face significant challenges in practical applications. The inherent instability of RNA molecules leads to their degradation before reaching antigen presenting cells (APCs), complicating the stimulation of specific tumor immune responses. Currently, the use of nanoparticle delivery systems is a promising strategy to improve the stability of RNA. For example, protamine is a cationic polypeptide composed mainly of arginine, which can encapsulate RNA through electrostatic interactions to produce positively charged nanoparticles. These particles are easily internalized by DCs, promoting their maturation. On the other hand, the vaccine delivery method plays a crucial role in determining the efficiency of the anti-tumor immune response elicited. Compared with intramuscular or subcutaneous injections, intradermal vaccination can significantly improve vaccine efficacy, but the discomfort caused by intradermal vaccination may lead to poor patient compliance. Microneedles (MNs) administration provides a minimally invasive and painless alternative for delivering vaccines to the epidermis or dermis. Microneedle arrays can evenly distribute vaccines on the skin, enhance immune cell activation over a larger area, and thus enhance immune responses. In addition, the ability to keep vaccine components in a dry state allows storage at room temperature, reduces dependence on cold chain logistics, and may enable self-administration. Therefore, microneedles have the potential to become a suitable vaccine delivery platform, but further optimization is still needed to improve the delivery efficiency of microneedles.

[0005] Therefore, developing new drug delivery systems to enhance the immune effects of RNA vaccines, improve the stability and safety of medication, and reduce medication dosage is an urgent topic that needs to be tackled in current RNA vaccine research. Summary of the invention

[0006] The technical problem to be solved by the present invention is that the existing RNA stabilization method is complicated and cumbersome, and the RNA vaccine delivery method needs to be further optimized to improve the efficiency and shortcomings, and provides a soluble microneedle containing tumor-derived total RNA nanovaccine.

[0007] The purpose of the present invention is to provide a method for preparing the soluble microneedle containing the tumor-derived total RNA nanovaccine.

[0008] Another object of the present invention is to provide the application of the soluble microneedle containing the tumor-derived total RNA nanovaccine.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention provides a soluble microneedle containing a tumor-derived total RNA nanovaccine, wherein the tumor-derived total RNA nanovaccine is loaded on the needle tip of the soluble microneedle;

[0011] The tumor-derived total RNA nano-vaccine is a nanoparticle composed of a complex of tumor-derived total RNA (TdRNA), CpG and protamine.

[0012] The present invention utilizes the characteristic that protamine can combine with RNA with positive and negative charges, and compounds protamine, tumor-derived total RNA and CpG to form a nano vaccine for treating or preventing tumors. The synergistic effect of TdRNA and CpG rich in tumor antigen broad-spectrum genetic encoding subsequently drives the maturation of dendritic cells and the activation of cytotoxic T lymphocytes, thereby generating powerful anti-tumor immunity, effectively treating or preventing tumors, and having no significant physiological toxicity and high safety. In addition, this microneedle loading RNA vaccine method can effectively protect RNA, prevent RNA from in vitro degradation, and reduce the difficulty of storage and transportation. At the same time, a multi-step centrifugation method is adopted to concentrate the RNA vaccine at the microneedle tip, thereby improving the delivery efficiency of the microneedle. If the TdRNA therein is replaced with another type of RNA antigen, or CpG is replaced with another immune adjuvant, the anti-tumor effect will be greatly reduced.

[0013] Furthermore, CpG (also known as CpG ODN) is a synthetic oligodeoxynucleotide, the full name of which is capable of significantly enhancing the body's immune response, including innate immunity and acquired immunity. However, the degree of immune activation is often difficult to accurately control. Excessive immune activation may lead to adverse reactions or autoimmune diseases. The tumor-derived total RNA and CpG complex of the present invention can synergistically enhance the anti-tumor immune effect without significant physiological toxicity.

[0014] Furthermore, the nucleotide sequence of the CpG is TCCATGACGTTCCTGACGTT.

[0015] Preferably, the mass ratio of protamine, CpG and TdRNA is (1-4):(0.2-1):1, more preferably (2-4):(0.3-0.8):1.

[0016] Furthermore, the loading amount of the nano vaccine is 10 to 50 μg per square centimeter.

[0017] Further, the source of the TdRNA includes one or more of breast cancer, skin cancer, and liver cancer. Specifically, the TdRNA is extracted from cells of the above-listed cancers.

[0018] Furthermore, the skin cancer includes melanoma.

[0019] Specifically, the TdRNA extraction method comprises the following steps: obtaining tumor tissue containing total RNA, homogenizing, and extracting using an RNA extraction kit to obtain TdRNA. The obtained TdRNA is generally stored at -80°C for future use.

[0020] Furthermore, the base material of the needle tip is selected from one or more of hyaluronic acid, trehalose, sodium alginate and cellulose. These base materials have similar properties and can be used to successfully prepare the needle tip.

[0021] Furthermore, the substrates of the needle body and the base of the soluble microneedle are selected from one or more of hyaluronic acid, polyvinyl pyrrolidone (PVP K90), cellulose, dextran and gelatin.

[0022] The method for preparing the soluble microneedle containing the tumor-derived total RNA nanovaccine protected by the present invention specifically comprises the following steps:

[0023] S1. Preparation of nano-vaccine containing tumor-derived total RNA;

[0024] S2, mixing the tumor-derived total RNA nanovaccine obtained in step S1 with the needle tip substrate evenly, and preparing the needle tip portion of the soluble microneedle by using a microneedle mold;

[0025] S3. Prepare the needle body part and the base part in sequence on the needle tip part obtained in step S2.

[0026] Furthermore, the method for preparing the nano vaccine containing tumor-derived total RNA specifically comprises the following steps: thoroughly mixing the protamine solution and the mixed solution containing TdRNA and CpG, and letting them stand to obtain the nano vaccine containing tumor-derived total RNA.

[0027] Furthermore, the mass concentration of the protamine solution is 1-4 mg / mL, more preferably 2 mg / mL.

[0028] Furthermore, the mass concentration of TdRNA in the mixed solution containing TdRNA and CpG is 0.5-1.5 mg / mL, preferably 1 mg / mL.

[0029] Furthermore, the mass concentration of CpG in the mixed solution containing TdRNA and CpG is 0.1-1 mg / mL, preferably 0.5 mg / mL.

[0030] Preferably, the concentration of the needle tip substrate is 50-100 mg / mL.

[0031] Furthermore, in step S2, the microneedle mold is a PDMS microneedle mold, and the morphological parameters are needle height 400-1500 μm, needle body diameter 300-500 μm, base diameter 5-30 mm, needle tip diameter less than 10 μm, and array number greater than 8×8.

[0032] Furthermore, in step S3, the base material concentration of the needle body is 300-400 mg / mL, and the base material concentration of the base is 300-400 mg / mL. The preparation is specifically as follows: the needle body is prepared on the needle tip, and the base is prepared on the needle body.

[0033] Specifically, the needle tip, needle body and base are respectively placed in a microneedle mold with a substrate solution, centrifuged and solidified; wherein the centrifugation speed is 2500-9000 rpm, and the time is 5-30 minutes.

[0034] The present invention protects the use of the soluble microneedle containing the tumor-derived total RNA nanovaccine in the preparation of cancer treatment drugs or medical devices.

[0035] Furthermore, the cancer includes one or more of breast cancer, skin cancer, and liver cancer.

[0036] Furthermore, the skin cancer includes melanoma.

[0037] The present invention has the following beneficial effects:

[0038] The present invention provides a soluble microneedle containing a tumor-derived total RNA nanovaccine, specifically, by utilizing the characteristic that protamine can combine with RNA with positive and negative charges, protamine, TdRNA and CpG are compounded to form a nanovaccine for treating or preventing tumors. The synergistic effect of TdRNA and CpG rich in a broad spectrum of genetically encoded tumor antigens subsequently drives the maturation of dendritic cells and the activation of cytotoxic T lymphocytes, thereby generating powerful anti-tumor immunity, effectively treating or preventing tumors, and having no significant physiological toxicity and high safety. In addition, this method of loading RNA vaccines on microneedles can effectively protect RNA, prevent RNA degradation in vitro, and reduce the difficulty of storage and transportation. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a statistical chart of the particle size measurement results of PTC NVs in Example 1.

[0040] Figure 2 This is a statistical diagram of the potential measurement results of PTC NVs in Example 1.

[0041] Figure 3 This is a transmission electron microscope scanning image of PTC NVs in Example 1.

[0042] Figure 4 This is a flow cytometer graph showing the activation effect of nanoparticles on BMDCs cells in Example 2.

[0043] Figure 5 This is a scanning electron microscope image of the soluble microneedle in Example 3.

[0044] Figure 6 This is a macroscopic image of the soluble microneedles in Example 3.

[0045] Figure 7 This is a statistical graph of the RNA degradation rate results in the soluble microneedle group and the PBS group in Example 4.

[0046] Figure 8 This is a flow cytometric graph of the activation effect of soluble microneedles at different storage times on BMDCs cells in Example 4.

[0047] Fig. 9 Figure 5 shows the change in tumor volume in Example 5

[0048] Fig.10 This is a statistical diagram of tumor volume change data in Example 5.

[0049] Fig.11 This is a statistical graph of the weight change data of mice in Example 5.

[0050] Fig.12 This is the flow cytometry graph of DCs cells in mouse tumor tissue in Example 5.

[0051] Fig.13 This is a flow cytometry graph of T cells in mouse tumor tissue in Example 5. DETAILED DESCRIPTION

[0052] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0053] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0054] CpG ODN was purchased from Bio-Technology, with the sequence number being TCCATGACGTTCCTGACGTT.

[0055] The RNA extraction kit was purchased from New Saimei Biotechnology.

[0056] Example 1 Synthesis of Nanovaccines Containing Tumor-derived Total RNA (PTC NVs and PT NVs)

[0057] The preparation method of the nano vaccine containing tumor-derived total RNA (PTC NVs) specifically comprises the following steps:

[0058] Extraction of TdRNA: Mouse 4T1 tumors (breast cancer cell line) were removed, mouse tumors were collected, homogenized, and total RNA was extracted using a mouse tissue RNA extraction kit to obtain TdRNA, which was stored at -80°C for future use.

[0059] 200 μg of protamine (PRT) was dissolved in 100 μl of deionized water (RNase-free), 100 μg of TdRNA and 50 μg of CpG ODN were dissolved in 100 μl of deionized water (RNase-free), the resulting solutions were mixed and stirred at high speed for 2 min, and allowed to stand for 4 h to obtain PTC NVs nanoparticles.

[0060] In addition, different PTC NVs were prepared at different mass ratios (PRT:CpG:TdRNA=1:0.5:1, 3:0.5:1, 4:0.5:1).

[0061] Preparation of PT NVs (nanoparticles without CpG): The difference from PTC NVs nanoparticles is that they do not contain CpG, the mass (PRT: TdRNA) = 2:1, and the other step parameters are the same as the preparation process of PTC NVs nanoparticles.

[0062] The potential, particle size, SEM morphology and other characteristics of the obtained PTC NVs nanoparticles were measured. Figures 1 to 3 As shown in the figure, the obtained PTC NVs nanoparticles all have a certain positive charge ( Figure 1 ), the particle size is nanometer, less than 200nm ( Figure 2 ). With the increase of CpG content, the zeta potential gradually increased and tended to be stable. At the same time, the particle size gradually decreased and tended to be more stable. Among them, the PTC NVs with a mass ratio of 2:0.5:1 (PRT:CpG:TdRNA) had a higher zeta potential (about 12.61mv) and a smaller particle size (about 149.06nm) on the basis of a lower PRT content. The electron microscope image also supported this particle size result ( Figure 3 ). The PTC NVs prepared at this ratio use less PRT content (which can save reagent costs) and have a relatively high potential and a smaller particle size, so PTC NVs with this ratio were selected for subsequent experimental exploration.

[0063] Example 2 Activation of DCs by PTC NVs

[0064] In order to study the effect of PTC NVs (PRT:TdRNA=2:1) ​​obtained in Example 1 on dendritic cells, bone marrow-derived dendritic cells (BMDCs) were cultured and PTC NVs, PT NVs, Free TdRNA and PBS were added for co-incubation.

[0065] Test results such as Figure 4As shown, BMDCs treated with PT NVs and PTC NVs exhibited CD80 and CD86 expression, indicating that PT NVs could promote dendritic cell maturation, and their expression was further enhanced when PT NVs were used in combination with CpG (the combination of the two is PTC NVs).

[0066] Example 3 Preparation of soluble microneedles containing PTC NVs or PT NVs

[0067] The preparation method of the soluble microneedle containing PTC NVs specifically comprises the following steps:

[0068] S1. Take 100 μg of the PTC NVs (PRT: TdRNA = 2:1) nanoparticles obtained in Example 1 and mix them evenly with 300 μL of a 70 mg / mL hyaluronic acid (HA) aqueous solution to obtain a HA solution containing PTC NVs;

[0069] S2, pouring the HA solution containing PTC NVs obtained in step S1 into the microneedle mold, centrifuging at 4000 rpm for 5 min, rotating 180° and centrifuging again for 5 min, scraping off excess liquid, centrifuging for 30 min, and drying until the solution solidifies to serve as the needle tip;

[0070] S3, prepare 300 mg / mL HA solution, and pour the prepared HA solution into the microneedle mold of step S2, centrifuge at 4000 rpm for 5 min, rotate 180° and centrifuge again for 5 min, scrape off excess liquid, centrifuge for 30 min, and dry until the solution solidifies to serve as the needle body;

[0071] S4. Prepare a 300 mg / mL PVP K90 aqueous solution, and pour the prepared PVP K90 aqueous solution into the microneedle mold of step S3, maintain the vacuum negative pressure for 15 minutes, extract the bubbles, scrape off the bubble-containing solution, add new PVP K90 aqueous solution, fill the entire mold, and dry until the solution solidifies as the base part to obtain soluble microneedles containing PTC NVs, referred to as PTC NVs@MNs.

[0072] According to the preparation process and parameters of PTC NVs@MNs, the PT NVs obtained in Example 1 were used to prepare soluble microneedles containing PT NVs (PT NVs@MNs).

[0073] The microneedle mold used is a PDMS microneedle mold, and its morphological parameters are: needle height 1200 μm, base diameter 10 mm, needle tip diameter less than 10 μm, and array number greater than 12×12.

[0074] The morphology of the obtained PTC NVs@MNs was characterized. Figures 5-6 shown.

[0075] Example 4 Testing of the RNA Protective Effect of Soluble Microtargets Containing PTC NVs

[0076] The RNA particles were dissociated using Trizol solution, and the concentration of RNA was determined using a micro-UV spectrophotometer. The concentration of RNA at different times was measured to evaluate the RNA degradation efficiency, and the protective effect of PTC NVs@MNs on the stability of TdRNA obtained in Example 1 was further evaluated.

[0077] The results are as follows Figure 7 As shown, TdRNA stored in PBS solution showed 93% degradation after 20 days, while PTC NVs encapsulated in MNs (microneedles) kept in a dry state at room temperature for several days showed only 1% degradation. Figure 8 As shown, the efficacy of the nanovaccine in promoting BMDC maturation remained essentially unchanged after 15 and 30 days of storage under dry conditions at room temperature, indicating that PTC NVs@MNs effectively protected the nanovaccine and preserved TdRNA activity for up to 30 days. These results confirm that the advanced multi-step centrifugation technology used to prepare the PTC NVs@MNs vaccine is effective in preventing RNA degradation and does not require strict storage conditions.

[0078] Example 5 The therapeutic effect of soluble microneedles containing PTC NVs on tumors

[0079] 1. Experimental materials:

[0080] Mouse breast cancer 4T1 cell line; 4-6 week-old Balb / c mice were purchased from Guangdong Provincial Animal Center. All animal operation procedures were carried out in accordance with the "Guidelines for the Care and Use of Laboratory Animals" of Sun Yat-sen University. All experiments involving animals strictly abide by the "Regulations on Animal Management in China" (1988, revised in 2017) and the "Guidelines for the Humane Treatment of Laboratory Animals in China" (MOST 2006).

[0081] 2. Experimental methods:

[0082] Tumor animal model establishment: The hair on the back of mice was removed and 50 μL (6×10 6) were injected into the back of mice, and the mice were divided into 4 groups, namely, PTC NVs (PRT: TdRNA = 2: 1) subcutaneous injection group (PTC NVsi.h.), PT NVs microneedle group (PT NVs@MNs prepared in Example 3), PTC NVs microneedle group (PTCNVs@MNs prepared in Example 3) and control group (Control, injected with the same amount of PBS). After 7 days of modeling, different groups were given different dosing treatments. Each microneedle was determined to contain 40μg of PTC NVs or 40μg of PT NVs. Each group was given drugs at this concentration, and the weight and tumor volume of the mice were recorded every two days.

[0083] 3. Experimental results

[0084] Results Figures 9-10 The therapeutic effect of the PTC NVs microneedle group was significantly better than that of the other injection groups, and the body weight of mice in each group did not decrease significantly ( Fig.11 ), proving that PTC NVs have no obvious physiological toxicity. Figures 12-13 It can be seen that flow cytometry analysis of the tumors of each group of mice showed that on day 14, the maturation rate of DCs in the tumors of the PTC NVs@MNs group increased by 23.50±3.90% compared with the Control group. + These results indicate that PTC NVs@MNs treatment effectively promotes DC maturation ( Fig.12 ) and T cells ( Fig.13 ) activation, significantly enhancing CD8 + T cells infiltrate the tumor and trigger a strong anti-tumor immune response.

[0085] Based on the above results, it can be proved that the soluble microneedles containing PTC NVs have no obvious physiological toxicity and have significant tumor inhibition effects.

[0086] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A soluble microneedle containing a tumor-derived total RNA nanovaccine, characterized in that: The tumor-derived total RNA nanovaccine is loaded on the needle tip of the soluble microneedle; The tumor-derived total RNA nano-vaccine is a nanoparticle composed of tumor-derived total RNA, CpG and protamine.

2. The soluble microneedle containing tumor-derived total RNA nanovaccine according to claim 1, characterized in that: The nucleotide sequence of the CpG is TCCATGACGTTCCTGACGTT.

3. The soluble microneedle containing tumor-derived total RNA nanovaccine according to claim 1, characterized in that: The mass ratio of protamine, CpG and tumor-derived total RNA is (1-4):(0.2-1):

1.

4. The soluble microneedle containing tumor-derived total RNA nanovaccine according to claim 1, characterized in that: The loading amount of the nano vaccine is 10 to 50 μg per square centimeter.

5. The soluble microneedle containing tumor-derived total RNA nanovaccine according to claim 1, characterized in that: The sources of the tumor-derived total RNA include one or more of breast cancer, skin tumor, and liver cancer.

6. The soluble microneedle containing tumor-derived total RNA nanovaccine according to claim 1, characterized in that: The base material of the needle tip part is selected from one or more of hyaluronic acid, trehalose, sodium alginate and cellulose.

7. The soluble microneedle containing tumor-derived total RNA nanovaccine according to claim 1, characterized in that: The substrates of the needle body and the base of the soluble microneedle are selected from one or more of hyaluronic acid, polyvinyl pyrrolidone, cellulose, dextran and gelatin.

8. The method for preparing the soluble microneedle containing tumor-derived total RNA nanovaccine according to any one of claims 1 to 7, characterized in that: The specific steps include: S1. Preparation of nano-vaccine containing tumor-derived total RNA; S2, mixing the tumor-derived total RNA nanovaccine obtained in step S1 with the needle tip substrate evenly, and preparing the needle tip portion of the soluble microneedle by using a microneedle mold; S3. Prepare the needle body part and the base part in sequence on the needle tip part obtained in step S2.

9. Use of the soluble microneedles containing tumor-derived total RNA nanovaccine according to any one of claims 1 to 7 in the preparation of cancer treatment drugs or medical devices.

10. The use according to claim 9, characterized in that: The cancer includes one or more of breast cancer, skin cancer, and liver cancer.

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