Proteinized lipid nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to the technical field of preparation of lipid nanoparticles, and particularly discloses proteinized lipid nanoparticles as well as a preparation method and application thereof. Dipalmitoyl phosphatidylcholine, cholesterol, triton X-100, absolute ethyl alcohol, a sodium citrate buffer solution and cell membrane protein are recombined to form cell membrane protein lipidosome with negative charges on the surface; the preparation method comprises the following steps: recombining 4-(N, N-dimethylamino) butyric acid (dilinoleyl) methyl ester, absolute ethyl alcohol, plasmids, triton X-100 and a sodium citrate buffer solution to obtain lipid nanoparticles with positive charges; and wrapping cell membrane protein liposome on the surface of the lipid nanoparticle by utilizing mutual attraction of positive and negative charges to obtain the proteinized lipid nanoparticle. The proteinized lipid nanoparticles can realize gene targeted delivery of specific cells such as suspension cells and primary cells, reduce non-specific uptake, and can be used as a gene delivery carrier.

Description

Technical Field

[0001] The present invention relates to the technical field of lipid nanoparticle preparation, and in particular to proteinized lipid nanoparticles and a preparation method and application thereof. Background Art

[0002] Lipid nanoparticles (LNPs) are one of the important technologies in gene delivery systems. They can effectively deliver genes into cells, where the contents of the carrier particles are released and translated into therapeutic proteins. Therefore, they have transformation prospects in the fields of gene delivery, vaccine design, immune cells, and stem cell therapy. Especially in the field of vaccine design, with the clinical application of the new crown mRNA vaccine, lipid nanoparticles (LNPs) as its delivery carrier have once again demonstrated their important position in the field of vaccine design. However, existing lipid nanoparticles (LNPs) do not have cell selectivity and cannot deliver genes to suspended cells and primary cells.

[0003] Therefore, there is a need to provide a new lipid nanoparticle for targeted gene delivery to specific cells. Summary of the invention

[0004] In order to provide a new lipid nanoparticle for targeted gene delivery to specific cells, the present invention provides a preparation method and application of protein lipid nanoparticles. The proteinized lipid nanoparticles (Proteolipidnanoparticles, PLNPs) prepared by the present invention can achieve targeted gene delivery to specific cells (including suspended cells and primary cells) by loading related proteins, while reducing non-specific uptake, and are gene delivery vectors with great transformation potential.

[0005] The invention provides a proteinized lipid nanoparticle, which comprises 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, anhydrous ethanol, a plasmid, Triton X-100, a sodium citrate buffer solution and a cell membrane protein liposome in a dosage ratio of 2mg-2.5mg: 1mL-2mL: 0.4mg-0.5mg: 0.1mg-0.2mg: 3mL-4mL: 5.5mg-12.0mg; the pH of the sodium citrate buffer solution is 5-6;

[0006] The cell membrane protein liposome comprises dipalmitoylphosphatidylcholine, cholesterol, Triton X-100, anhydrous ethanol, sodium citrate buffer solution and cell membrane protein in a dosage ratio of 4.75 mg to 8.75 mg: 1.15 mg to 1.5 mg: 0.5 mg to 1.5 mg: 1 mL to 2 mL: 2 mL to 3 mL: 1 mg;

[0007] Among them, dipalmitoylphosphatidylcholine, cholesterol, Triton X-100, anhydrous ethanol, sodium citrate buffer solution and cell membrane proteins are recombined to form cell membrane protein liposomes with negatively charged surface; 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, anhydrous ethanol, plasmid, Triton X-100 and sodium citrate buffer solution are recombined to obtain positively charged lipid nanoparticles; using the mutual attraction between positive and negative charges, a layer of negatively charged cell membrane protein liposomes is wrapped on the surface of the positively charged lipid nanoparticles to obtain proteinized lipid nanoparticles.

[0008] The present invention recombines dipalmitoylphosphatidylcholine, cholesterol, Triton X-100, anhydrous ethanol, sodium citrate buffer solution and cell membrane proteins to form cell membrane protein liposomes with negative charges on the surface; 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, anhydrous ethanol, plasmid, Triton X-100 and sodium citrate buffer solution are recombined to obtain positively charged lipid nanoparticles; using the mutual attraction between positive and negative charges, a layer of negatively charged cell membrane protein liposomes is wrapped on the surface of the positively charged lipid nanoparticles to obtain proteinized lipid nanoparticles. The prepared proteinized lipid nanoparticles ensure the stable loading of proteins on the basis of ensuring the effective loading of genes, so that they have the characteristics of highly targeted and precise delivery, and significantly improve the gene encapsulation rate and protein loading rate. The efficiency of GFP delivery to primary mouse mononuclear cells is significantly higher than that of LNPs and conventional gene delivery reagent polyethyleneimine (PEI).

[0009] Furthermore, the cell membrane protein is extracted from tumor cells.

[0010] The present invention also provides a method for preparing proteinized lipid nanoparticles, comprising the following steps:

[0011] Cell membrane protein extraction: extract membrane proteins from tumor cells in the logarithmic growth phase to obtain cell membrane proteins;

[0012] Preparation of cell membrane protein liposomes: dissolving dipalmitoylphosphatidylcholine, cholesterol and Triton X-100 in anhydrous ethanol, adding sodium citrate buffer solution and cell membrane protein, incubating and then obtaining cell membrane protein liposomes by extrusion filtration; the dosage ratio of dipalmitoylphosphatidylcholine, cholesterol, Triton X-100, anhydrous ethanol, sodium citrate buffer solution and cell membrane protein is 4.75mg-8.75mg: 1.15mg-1.5mg: 0.5mg-1.5mg: 1mL-2mL: 2mL-3mL: 1mg;

[0013] Preparation of proteinized lipid nanoparticles: 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester is dissolved in anhydrous ethanol to obtain Dlin-MC3-DMA solution; plasmid and Triton X-100 are dissolved in sodium citrate buffer solution to obtain plasmid solution; then the plasmid solution is mixed with the Dlin-MC3-DMA solution to obtain a mixed solution; then cell membrane protein liposomes are added to the mixed solution for ultrasonic vibration, and after incubation, a molecular weight cutoff of 3KD to 14KD is selected for dialysis to obtain proteinized lipid nanoparticles;

[0014] The dosage ratio of the 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, anhydrous ethanol, plasmid, Triton X-100, sodium citrate buffer solution and cell membrane protein liposome is 2mg-2.5mg: 1mL-2mL: 0.4mg-0.5mg: 0.1mg-0.2mg: 3mL-4mL: 5.5mg-12.0mg.

[0015] Furthermore, the dosage ratio of dipalmitoylphosphatidylcholine, cholesterol, Triton X-100, anhydrous ethanol, sodium citrate buffer solution and cell membrane protein is 7.5 mg: 1.5 mg: 1.0 mg: 1 mL: 2 mL: 1 mg.

[0016] Furthermore, the cell membrane protein concentration is 0.5 mg / mL to 2.0 mg / mL.

[0017] Furthermore, the cell membrane proteoliposomes are added to the mixed solution and incubated at 36° C. to 37° C. for 15 min to 20 min after ultrasonic shaking.

[0018] The present invention also provides an application of the proteinized lipid nanoparticles in preparing products for treating and / or preventing breast cancer.

[0019] Furthermore, the product is a full-antigen tumor vaccine.

[0020] Furthermore, the whole antigen tumor vaccine is used to inhibit the growth of tumor volume.

[0021] The present invention also provides a full antigen tumor vaccine, which uses monophosphoryl lipid A as an adjuvant to synthesize the PLNPs full antigen tumor vaccine; the construction process is as follows:

[0022] Dipalmitoylphosphatidylcholine, cholesterol, Triton X-100 and monophosphoryl lipid A are dissolved in 1 mL to 2 mL of anhydrous ethanol in a mass ratio of 4.75 mg to 8.75 mg: 1.15 mg to 1.5 mg: 0.5 mg to 1.5 mg: 0.050 mg to 0.080 mg, a sodium citrate buffer solution with a pH of 5 to 6 and cell membrane proteins are added, and after incubation, adjuvanted cell membrane protein liposomes are obtained by extrusion filtration; then, according to the preparation steps of proteinized lipid nanoparticles, the cell membrane protein liposomes containing the adjuvant are replaced by the cell membrane protein liposomes containing the adjuvant to prepare the adjuvanted PLNPs full antigen tumor vaccine.

[0023] The present invention obtains a full-antigen tumor vaccine by synthesizing the adjuvant monophosphoryl lipid A into proteinized lipid nanoparticles, which has a significant inhibitory effect on mouse tumor growth, inhibits tumor growth, and reduces the volume and weight of the tumor.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention recombines dipalmitoylphosphatidylcholine, cholesterol, Triton X-100, anhydrous ethanol, sodium citrate buffer solution and cell membrane proteins to form cell membrane proteoliposomes with negative charges on the surface; 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, anhydrous ethanol, plasmid, Triton X-100 and sodium citrate buffer solution are recombined to obtain positively charged lipid nanoparticles; and a layer of negatively charged cell membrane proteoliposome coat is wrapped on the surface of the positively charged lipid nanoparticles by utilizing the mutual attraction between positive and negative charges to obtain proteinized lipid nanoparticles. The proteinized lipid nanoparticles (PLNPs) prepared by the present invention are relatively traditional lipid nanoparticles. On the basis of ensuring the effective gene load, the proteinized lipid nanoparticles ensure the stable load of proteins, making them highly targeted and accurately delivered, significantly improving the gene encapsulation rate and protein loading rate, and the gene encapsulation rate and protein loading rate of PLNPs are both stable, and the PLNPs have low cytotoxicity. PLNPs have strong gene transfection ability, and the GFP delivery efficiency to primary mononuclear cells of mice is high. By studying the immune status of PLNPs full antigen vaccine, it was found that PLNPs full antigen vaccine can effectively activate antigen presentation of dendritic cells, and has a significant inhibitory effect on tumor cell activity. PLNPs full antigen vaccine combined with doxorubicin has a significant inhibitory effect on mouse tumor growth, inhibits tumor growth, and reduces tumor volume and weight. It can be seen that the proteinized lipid nanoparticles (PLNPs) prepared by the present invention have a stable and efficient gene encapsulation rate (93.63%) and protein loading rate (62.03%), which can significantly inhibit the proliferation and development of tumor cells, reduce tumor volume and mass, and achieve the effect of mass tumor.

[0026] The present invention prepares proteinized lipid nanoparticles (PLNPs), which provide technical support for targeted lipid nanoparticles and full-antigen tumor vaccines. The protein components in PLNPs give them specific functions, such as cell targeting, organ targeting, etc., so as to efficiently transfect genes of experimental suspended cells and provide technical support for in situ CAR immune cells in vivo. When facing tumors with unknown antigens, PLNPs can still achieve the construction of full-antigen tumor vaccines through cell membrane proteins, and the vaccine also has the functions of adjuvant loading and cytokine plasmid loading. The construction of full-antigen tumor vaccines is achieved, and a delivery carrier containing full tumor antigens and natural adjuvants is provided for the construction of tumor vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 Characterization of the physical and chemical properties of the PLNPs prepared in Example 1 and the LNPs prepared in Comparative Example 1;

[0029] In the figure, A is the average particle size test result of PLNPs and LNPs;

[0030] B is the Zeta potential test results of PLNPs and LNPs;

[0031] C is the polydispersity index (PDI) test results of PLNPs and LNPs.

[0032] Figure 2 Transmission electron microscopy images of proteinized lipid nanoparticles (PLNPs) and lipid nanoparticles (LNPs);

[0033] In the figure, A is a transmission electron micrograph of proteinized lipid nanoparticles (PLNPs), which represent 200 nm and 50 nm from left to right;

[0034] B is a transmission electron microscopy image of lipid nanoparticles (LNPs), representing 200 nm and 50 nm from left to right.

[0035] Figure 3 Atomic force microscopy images of proteinized lipid nanoparticles (PLNPs) and lipid nanoparticles (LNPs);

[0036] In the figure, A is the atomic force microscopy detection image of proteinized lipid nanoparticles (PLNPs), from left to right, AFM-height (characterizing the surface roughness) and AFM-adhesion force (characterizing viscosity);

[0037] B is an atomic force microscopy image of lipid nanoparticles (LNPs), which shows AFM-height (characterizing surface roughness) and AFM-adhesion force (characterizing viscosity) from left to right.

[0038] Figure 4 UV-visible absorption spectra of proteinized lipid nanoparticles (PLNPs) and lipid nanoparticles (LNPs).

[0039] Figure 5 It is a statistical diagram of gene encapsulation efficiency and protein loading rate of proteinized lipid nanoparticles (PLNPs) and lipid nanoparticles (LNPs);

[0040] In the figure, A is the gene encapsulation efficiency of PLNPs and LNPs;

[0041] B is the protein loading rate of PLNPs and LNPs.

[0042] Figure 6 To test the gene encapsulation efficiency and protein loading stability of proteinized lipid nanoparticles (PLNPs) and lipid nanoparticles (LNPs);

[0043] In the figure, A is the gene encapsulation efficiency stability results of PLNPs and LNPs;

[0044] B is the protein loading stability results of PLNPs and LNPs.

[0045] Figure 7 To test the hydrated particle size and potential stability of proteinized lipid nanoparticles (PLNPs) and lipid nanoparticles (LNPs);

[0046] In the figure, A is the particle size stability test results of PLNPs and LNPs;

[0047] B is the PDI stability test results of PLNPs and LNPs;

[0048] C is the zeta potential stability test results of PLNPs and LNPs.

[0049] Figure 8 The cytotoxicity results of proteinized lipid nanoparticles (PLNPs) and lipid nanoparticles (LNPs).

[0050] Fig. 9 To detect the gene transfection ability of proteinized lipid nanoparticles (PLNPs);

[0051] In the figure, A, B, and C represent confocal images of cells after polyethyleneimine (PEI), LNPs, and PLNPs were delivered to mouse primary mononuclear cells, respectively.

[0052] Fig.10 The dendritic cell activation effect of PLNPs full antigen vaccine;

[0053] In the figure, A, B, C and D represent the dendritic cell activation effects of the control group, ovalbumin vaccine, 4T1 frozen-thawed cell vaccine and PLNPs full antigen vaccine, respectively.

[0054] Fig.11 The in vitro tumor cell killing effect of PLNPs full antigen vaccine.

[0055] Fig.12 This is the tumor growth curve of each group of mice in the in vivo tumor treatment experiment of PLNPs full antigen vaccine.

[0056] Fig.13 The tumor size and weight of each group of mice in the in vivo tumor treatment experiment of PLNPs full antigen vaccine;

[0057] A is a photo of the tumors in vitro of mice in each group who were killed on day 26;

[0058] B shows the comparison of tumor weights of mice in each group who were killed on day 26.

[0059] Fig.14 This is the tumor growth curve of each group of mice in the in vivo tumor immunoprevention experiment of PLNPs full antigen vaccine.

[0060] Fig.15 The tumor size and weight of each group of mice in the in vivo tumor immunoprevention experiment of PLNPs full antigen vaccine;

[0061] A is the in vitro photos of the tumors of mice in each group who were killed at 3 weeks;

[0062] B shows the comparison of tumor weights of mice in each group who were killed on day 26. DETAILED DESCRIPTION

[0063] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0064] Example 1: Preparation methods of proteinized lipid nanoparticles, PLNPs full antigen vaccine and 4T1 freeze-thawed cell vaccine.

[0065] 1. Preparation method of proteinized lipid nanoparticles

[0066] 1. Cell membrane protein extraction:

[0067] Mouse breast cancer 4T1 cells in the logarithmic growth phase were taken, and membrane proteins were extracted using a cell membrane protein extraction kit (Biyuntian P0033) to obtain cell membrane proteins, which were stored at 4 degrees for later use.

[0068] 2. Preparation of cell membrane protein liposomes (CMPliposomes):

[0069] Dissolve dipalmitoylphosphatidylcholine, cholesterol, and Triton X-100 in anhydrous ethanol, add sodium citrate buffer solution and cell membrane protein, and incubate and then obtain cell membrane protein liposomes by extrusion filtration. The specific method is as follows:

[0070] Dissolve dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), and Triton-X100 (Triton-X100) in 1 mL of anhydrous ethanol at a mass ratio of 7.5 mg: 1.5 mg: 1.0 mg, add 2 mL of sodium citrate buffer solution (pH = 5.5; 25 mM) and 1 mg (1.0 mg / mL) of prepared cell membrane protein at once, and incubate at 37 degrees for 30 minutes. First pass through a 400 nm filter squeezer 20 times, then pass through a 200 nm filter squeezer 20 times to obtain cell membrane protein liposomes (CMPliposomes), which are set aside. The obtained cell membrane protein liposomes contain cell membrane proteins and lipid molecules, especially cell membrane proteins with strong negative charges, which make them present negative charges in aqueous solution.

[0071] The present invention utilizes the lipid solubility of cell membrane proteins and dipalmitoylphosphatidylcholine, cholesterol, anhydrous ethanol, sodium citrate buffer solution and Triton X-100 to reorganize and form cell membrane protein liposomes, thereby effectively improving the membrane protein loading rate. Triton X-100 is added to the synthesis formula to regulate the flexibility of the cell membrane protein liposomes while stabilizing the membrane protein structure. It avoids the use of a large number of mechanical extrusion processes during the synthesis process to cause a large amount of membrane protein loss, thereby greatly improving the membrane protein loading rate of the cell membrane protein liposomes.

[0072] The cell membrane protein liposomes with a particle size of 400nm are removed, and when the particle size exceeds 200nm, the extruder can adjust the protein lipid nanoparticles to obtain cell membrane protein liposome protein lipid nanoparticles with uniform particle size.

[0073] 3. Preparation of proteinized lipid nanoparticles (PLNPs):

[0074] Proteinized lipid nanoparticles (PLNPs) were synthesized by ethanol injection method combined with ultrasonic oscillation method. The specific method is as follows:

[0075] Weigh 2.5 mg of 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (Dlin-MC3-DMA) and dissolve it in 1 mL of anhydrous ethanol to obtain Dlin-MC3-DMA solution. Dissolve 0.5 mg of green fluorescent protein (GFP) plasmid and 0.1 mg of Triton-X100 in 3 mL of sodium citrate buffer solution (pH = 5.5; 25 mM) to obtain a plasmid solution. Add the prepared plasmid solution to the Dlin-MC3-DMA solution and mix ultrasonically for 30 minutes to obtain a mixed solution. Subsequently, add 9.5 mg of cell membrane protein liposomes (CMPliposomes) prepared in the previous step to the mixed solution, oscillate ultrasonically at 100 W for 15 minutes, and incubate at 37 degrees for 15 minutes. Finally, place it in phosphate buffer (PBS) for 24 hours, with a molecular weight cutoff of 12KD, to obtain proteinized lipid nanoparticles (PLNPs), and store them at 4°C. Then, the physical and chemical properties and biological properties were characterized.

[0076] 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester was dissolved in anhydrous ethanol to obtain Dlin-MC3-DMA solution; plasmid and Triton X-100 were dissolved in sodium citrate buffer solution to obtain plasmid solution; and then the plasmid solution was mixed with the Dlin-MC3-DMA solution to obtain a mixed solution.

[0077] Dlin-MC3-DMA is protonated in a sodium citrate buffer solution at pH = 5.5 to obtain a positive charge; then the negatively charged plasmid solution is mixed with it to obtain lipid nanoparticles through the attraction of positive and negative charges. Triton X-100 in the plasmid solution can adjust the flexibility of lipid nanoparticles and prevent lipid nanoparticles from agglomerating to form overly large particles.

[0078] Then the cell membrane protein liposomes are added into the mixed solution for ultrasonic oscillation. The negatively charged cell membrane protein liposomes can wrap a layer of cell membrane protein liposome outer coat around the positively charged lipid nanoparticles through ultrasonic oscillation at the mixed interface of the aqueous phase and the organic phase.

[0079] Incubation at 37 degrees for 15 minutes helps the proteinized lipid nanoparticles to form stably without destroying the biological activity of cell membrane proteins.

[0080] Dialysis with a molecular weight cutoff of 3KD to 14KD can remove unreacted small molecules and obtain stable proteinized lipid nanoparticles.

[0081] It should be noted that the green fluorescent protein (GFP) plasmid can also be replaced by any other plasmid, such as interleukin 12b plasmid, interleukin 21 plasmid and chemokine CC-motif receptor 7 plasmid.

[0082] 2. Construction of PLNPs full antigen vaccine

[0083] According to the preparation method of proteinized lipid nanoparticles, the adjuvant monophosphoryl lipid A (MPLA) is synthesized into proteinized lipid nanoparticles to obtain PLNPs full antigen vaccine. Specifically, the steps include:

[0084] 1. Take mouse breast cancer 4T1 cells in the logarithmic growth phase, extract membrane protein using a cell membrane protein extraction kit (Biyuntian P0033), obtain cell membrane protein, and store it at 4 degrees for later use.

[0085] 2. Preparation of cell membrane protein liposomes (CMPliposomes) containing adjuvant:

[0086] Dissolve dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), Triton-X100 (Triton-X100), and monophosphoryl lipid A (MPLA) in 1 mL of anhydrous ethanol at a mass ratio of 7.5 mg: 1.5 mg: 1 mg: 0.075 mg, add 2 mL of sodium citrate buffer solution (pH = 5.5; 25 mM) and 1 mL (1.0 mg / mL) of prepared 4T1 cell membrane protein at once, and incubate at 37 degrees for 30 minutes. First pass through a 400 nm filter squeezer 20 times, then pass through a 200 nm filter squeezer 20 times to obtain cell membrane protein liposomes (CMPliposomes) containing adjuvants for later use.

[0087] 4. Construction of PLNPs full antigen vaccine: Weigh 2.5 mg of 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (Dlin-MC3-DMA) and dissolve it in 1 mL of anhydrous ethanol to obtain Dlin-MC3-DMA solution. Dissolve 0.5 mg of GFP plasmid (or IL12b plasmid) and 0.1 mg of Triton-X100 in 3 mL of sodium citrate buffer solution (pH = 5.5; 25 mM) to obtain a plasmid solution. Add the prepared plasmid solution to the Dlin-MC3-DMA solution and mix ultrasonically for 30 minutes to obtain a mixed solution. Subsequently, add the prepared adjuvant-containing cell membrane protein liposome (CMPliposome) to the mixed solution, ultrasonically vibrate for 15 minutes, and incubate at 37 degrees for 15 minutes. Finally, place it in phosphate buffer (PBS) for 24 hours, with a molecular weight cutoff of 12KD, to obtain a PLNPs full antigen tumor vaccine.

[0088] 3. Preparation method of 4T1 frozen-thawed cell vaccine

[0089] Preparation method of 4T1 frozen-thaw cell vaccine: Take 4T1 cells in the logarithmic growth phase, scrape them with a cell scraper, and resuspend them in 1 ml of phosphate buffered saline (PBS) to a density of 5*10 6 It was placed in liquid nitrogen for 5 minutes to freeze rapidly, then taken out and immediately placed in a 37-degree water bath for 5 minutes to thaw rapidly, and the above freezing and thawing process was repeated 15 times to finally obtain the 4T1 frozen-thawed cell vaccine.

[0090] Comparative Example 1: Lipid nanoparticles (LNPs) and preparation methods thereof.

[0091] Preparation steps of lipid nanoparticles (LNPs): 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (Dlin-MC3-DMA), dipalmitoylphosphatidylcholine (DPPC), and cholesterol (Chol) were dissolved in 1 mL of methanol at a mass ratio of 10:2:8 mg to obtain a lipid mixed solution, which was evaporated by a rotary evaporator with a circulating water bath at 45 degrees to obtain a lipid film. 0.5 mg of GFP (or IL12b plasmid) was dissolved in 6 mL of sodium citrate buffer solution (pH = 5.5; 25 mM) to obtain a plasmid solution. The prepared plasmid solution was added to the lipid film and ultrasonically mixed for 30 minutes to obtain the initial product of lipid nanoparticles (LNPs). Finally, it was dialyzed in phosphate buffer (PBS) for 24 hours with a molecular weight cutoff of 14 KD to obtain lipid nanoparticles (LNPs), which were stored at 4°C. The physical and chemical properties and biological properties were then characterized.

[0092] The products prepared in Example 1 and Comparative Example 1 were subjected to physical and chemical performance characterization, biosafety experimental research, cytotoxicity experiment, in vitro immune cell activation effect experiment, in vitro tumor cell killing effect experiment and in vivo tumor treatment effect comparative study. The specific methods and experimental results are as follows:

[0093] 1. Characterization of the physicochemical properties of proteinized lipid nanoparticles (PLNPs) and lipid nanoparticles (LNPs)

[0094] Hydration particle size and surface charge: tested by Malvern Zeta particle size detector, the results are as follows Figure 1 As shown in A, the average hydrated particle size of PLNPs is 199.2nm, which is larger than the average hydrated particle size of LNPs (150nm); the PDI results are as follows Figure 1 As shown in Figure C, the average PDI of PLNPs is 0.178, which is lower than the average PDI value of LNPs 0.245. PDI is a polydispersity index, which reflects the uniformity of particle size distribution. The lower the value, the more uniform the particle size distribution.

[0095] Zeta potential test results, such as Figure 1 As shown in Figure B, the average potential of LNPs is -1.92, while the average potential of PLNPs loaded with proteins decreases significantly to -11.93, which may be related to the presence of proteins in PLNPs.

[0096] Transmission electron microscopy detection of morphological characteristics of PLNPs and LNPs: The results are as follows Figure 2 As shown, the morphology of PLNPs and LNPs under electron microscopy is regular spheres; under high magnification, it can be observed that the outer layer of PLNPs is obviously covered with an outer membrane, which may be related to the protein lipid outer membrane carried by PLNPs.

[0097] Atomic force microscopy was used to detect the surface roughness and viscosity of PLNPs and LNPs. Figure 3 As shown in Figure 3, the surface roughness and viscosity of PLNPs were increased compared with those of LNPs, as detected by atomic force microscopy.

[0098] UV-visible absorption spectra: The UV-visible spectra of PLNPs and LNPs were detected by a micro-UV spectrophotometer (nanodrop). The results are as follows: Figure 4 LNPs have an absorption peak of plasmid at 260nm, while PLNPs have a weak absorption peak at 280nm in addition to the absorption peak of plasmid at 260nm. The absorption peak at 280nm is the absorption peak of cell membrane protein, which further confirms that PLNPs carry both plasmid and cell membrane protein.

[0099] 2. Gene encapsulation rate and protein loading rate detection

[0100] The plasmid encapsulation efficiency of PLNPs was detected by a micro-UV spectrophotometer (nanodrop), as Figure 5 As shown in A, the plasmid encapsulation efficiencies of PLNPs and LNPs were 93.63% and 83.98%, respectively. The protein loading rate of PLNPs was detected by BCA protein concentration assay kit (Biyuntian P0011). Figure 5 As shown in Figure B, the protein loading rate of PLNPs was 62.03%.

[0101] Gene encapsulation rate and protein loading rate stability test of materials: Test the gene encapsulation rate and protein loading rate stability of PLNPs within a certain period of time. Figure 6 As shown in Figure A, the gene encapsulation efficiency of PLNPs was relatively stable within 28 days without significant changes; the gene encapsulation efficiency of LNPs fluctuated greatly within 28 days and was unstable. Figure 6 As shown in Figure B, the protein loading rate of PLNPs remained basically stable within 28 days, with a slight decrease.

[0102] Stability test of material hydration particle size and potential: Detect the changes in hydration particle size and potential stability of PLNPs over a certain period of time, such as Figure 7 As shown in Figures A and B, the particle size of PLNPs remained basically stable within 28 days, and the PDI increased slightly. Figure 7 As shown in C.

[0103] 3. Biosafety experiments of PLNPs

[0104] When mouse DC2.4 cells grow to 75%, they are passaged and inoculated into 96-well plates, 8,000 cells per well, 10% FBS DMEM, overnight. The next day, the solution was changed, and 0ug / mL, 1.375ug / mL, 3.75ug / mL, 7.5ug / mL, 10ug / mL (calibrated with the concentration of the plasmid) of LNPs and PLNPs were added, 3 replicates per group, and incubated for 24h. CCK8 detection reagent was used, 1uL of CCK8 reagent was added to each well, and after incubation for 2h, ABS was detected at 540nm on an ELISA instrument.

[0105] The results are as follows Figure 8 As shown, compared with the conventional gene delivery agent polyethyleneimine (PEI), LNPs and PLNPs have good biosafety and do not show cytotoxicity with increasing doses.

[0106] 4. Primary mononuclear cell transfection assay of PLNPs

[0107] Mouse mononuclear cells were extracted by lymph separation fluid and inoculated into confocal dish, 6000 cells per well, 10% FBS DMEM, 5ug (quantified by measured plasmid content) of LNPs and PLNPs were added, and incubated for 24h. Cell fluorescence was monitored by laser confocal microscope, parameters: excitation light 488nm, emission light 510nm.

[0108] The results are as follows Fig. 9 As shown, compared with conventional gene delivery agents polyethyleneimine (PEI) and LNPs, PLNPs have stronger gene transfection ability, and their GFP delivery efficiency to mouse primary mononuclear cells is higher, and more cells express green fluorescent protein.

[0109] 5. In vitro immune cell activation effect of PLNPs full antigen vaccine

[0110] Mouse dendritic cells DC2.4 were inoculated into 24-well plates, 10,000 cells per well, and cultured in 1640 with 10% FBS overnight. The next day, 5ug (quantified by the measured plasmid content) of LNP, PLNPs full antigen vaccine, 4T1 frozen-thaw cell vaccine, and 50ug of ovalbumin (OVA) were added and incubated for 24 hours. The expression changes of DC2.4 antigen presentation markers CD80 and CD86 were then detected by flow cytometry.

[0111] like Fig.10 As shown, CD80 in the control group + / CD86 + The percentage of double-positive dendritic cells was 6.56%, while the percentage of CD80 positive dendritic cells of PLNPs whole antigen vaccine, 4T1 frozen-thaw cell vaccine and ovalbumin vaccine was 2.34%. + / CD86 + The double positive dendritic cells were 39.84%, 34.90% and 32.13 respectively, which were significantly increased compared with the control. The above results show that PLNPs full antigen vaccine can effectively activate the antigen presentation of dendritic cells.

[0112] 6. In vitro tumor cell killing effect of PLNPs full antigen vaccine

[0113] Mouse dendritic cells DC2.4 were inoculated into 24-well plates, 10,000 cells per well, and cultured overnight in 10% FBS 1640. The next day, 5 ug (quantified by the measured plasmid content) of LNP and PLNPs full antigen vaccines were added and incubated for 48 hours.

[0114] T lymphocytes extracted from mouse spleen were added to each group, with the ratio of T lymphocytes to DC2.4 cells being 10: 1. After 48 hours of co-incubation, T lymphocytes were carefully aspirated, centrifuged, and resuspended in 10% FBS 1640 for later use.

[0115] Mouse breast cancer 4T1 cells in the logarithmic growth phase were seeded into 96-well plates, with 4000 cells per well in 3 replicates per group.

[0116] Each group of T lymphocytes obtained above was added to 4T1 cells and incubated for 48 hours, wherein the number of T lymphocytes: 4T1 cells was 10:1.

[0117] Using the CCK8 kit (Biyuntian-C0038), 1uL of CCK8 reagent was added to each well, and after incubation for 3h, ABS was detected at 540nm using an ELISA reader. The inhibition rate of tumor cells in each group of vaccines was detected.

[0118] The results are as follows Fig.11As shown, as the number of effector T cells in the PLNPs full antigen vaccine group increased, the activity of tumor cells decreased significantly, while this trend was observed in the control group and LNPs group.

[0119] 7. In vivo tumor treatment effect of PLNPs full antigen vaccine

[0120] BALB / c female mice were purchased from Jicui Yaokang Biotechnology Co., Ltd. and kept in an SPF environment. They were tumor-bearing at 6 weeks of age and the mouse breast cancer cell line 4T1 was used at 100,000 / cell / mouse to establish a mouse breast cancer model. Drug treatment was performed 1 week after tumor initiation and the mice were divided into a control group (Control), doxorubicin (DOX), 4T1 frozen-thawed cell vaccine combined with doxorubicin (4T1Frozen-thawed cells+DOX) group, and PLNPs full antigen vaccine combined with doxorubicin (PLNPs full antigen vaccine+DOX) group, with 5 mice in each group. The doxorubicin (DOX), 4T1 frozen-thawed cells vaccine combined with doxorubicin (4T1 Frozen-thawed cells+DOX) group, and PLNPs whole antigen vaccine combined with doxorubicin (PLNPs whole antigen vaccine+DOX) group were injected intratumorally with 0.1 mL, 0.5 mg / mL of doxorubicin (DOX) on days 1, 5, and 9, respectively; the 4T1 frozen-thawed cells vaccine combined with doxorubicin (4T1 Frozen-thawed cells+DOX) group, and PLNPs whole antigen vaccine combined with doxorubicin (PLNPs whole antigen vaccine+DOX) group were injected subcutaneously with 0.1 mL, 0.15 mg / mL (quantified by protein content) of 4T1 frozen-thawed cells vaccine and PLNPs whole antigen vaccine on days 3, 6, and 11, respectively. The size of the tumor in each group of mice was measured over time using a vernier caliper.

[0121] The results are as follows Fig.12 As shown, compared with the control group, doxorubicin (DOX), 4T1 frozen-thawed cell vaccine combined with doxorubicin (4T1Frozen-thawed cells+DOX) group, and PLNPs whole antigen vaccine combined with doxorubicin (PLNPs whole antigen vaccine+DOX) group had an inhibitory effect on mouse tumor growth; while the tumor growth of the PLNPs whole antigen vaccine combined with doxorubicin (PLNPs whole antigen vaccine+DOX) group was significantly inhibited, and the tumor volume was much smaller than that of the other three groups at 25 days. After 26 days, some mice (3 in each group) were killed, and the tumor size and weight of each group of mice were recorded by photographs and electronic balances.

[0122] like Fig.13As shown in A, compared with the control group, doxorubicin (DOX), 4T1 frozen-thawed cells vaccine combined with doxorubicin (4T1Frozen-thawed cells+DOX), and PLNPs whole antigen vaccine combined with doxorubicin (PLNPs whole antigen vaccine+DOX) groups all reduced the size of mouse tumors; and the tumor size of the PLNPs whole antigen vaccine combined with doxorubicin (PLNPs whole antigen vaccine+DOX) group was significantly reduced, much smaller than the other three groups. The tumor weighing results also confirmed the above results, as shown in Fig.13 As shown in B, the tumor weight of the PLNPs whole antigen vaccine combined with doxorubicin (PLNPs whole antigen vaccine+DOX) group was much smaller than that of the other three groups.

[0123] 8. In vivo tumor immune prevention effect of PLNPs full antigen vaccine

[0124] BALB / c female mice were purchased from Jicui Yaokang Biotechnology Co., Ltd. and raised in an SPF environment. Immunization was performed at 6 weeks of age and divided into a control group (Control), a 4T1 frozen-thawed cell vaccine (4T1Frozen-thawed cells), and a PLNPs whole antigen vaccine group, with 5 mice in each group. The control group (Control), a 4T1 frozen-thawed cell vaccine (4T1 Frozen-thawed cells), and a PLNPs whole antigen vaccine group were subcutaneously injected with 0.1 mL phosphate buffer (PBS), 0.1 mL, 0.15 mg / mL (concentration based on protein concentration) of 4T1 frozen-thawed cell vaccine (4T1 Frozen-thawed cells), and 0.1 mL, 0.15 mg / mL (concentration based on protein concentration) of PLNPs whole antigen vaccine on days 1, 5, and 10, respectively. Five days after each group of mice completed immune prevention, tumors were loaded and a mouse breast cancer cell line 4T1 was used at 50,000 / cell / mouse to prepare a mouse breast cancer model. The changes in the tumor size of mice in each group over time were measured using a vernier caliper.

[0125] The results are as follows Fig.14 As shown, compared with the control group, the 4T1 frozen-thaw cell vaccine group and the PLNPs whole antigen vaccine group both had an inhibitory effect on mouse tumor growth; and the tumor growth of the PLNPs whole antigen vaccine group was significantly inhibited, and the tumor volume at 20 days was much smaller than that of the other two groups.

[0126] After 3 weeks, some mice (3 mice in each group) were killed, and the tumor size and weight of mice in each group were recorded by photographs and electronic balance.

[0127] The results are as follows Fig.15As shown in A, compared with the control group, the 4T1 frozen-thaw cell vaccine group and the PLNPs full antigen vaccine group both reduced the size of mouse tumors; and the tumor size of the PLNPs full antigen vaccine group was significantly reduced, much smaller than the other two groups. The tumor weighing results also confirmed the above results, such as Fig.15 As shown in B, the tumor weight of the PLNPs full antigen vaccine group was much smaller than that of the other two groups.

[0128] In summary, the proteinized lipid nanoparticles (PLNPs) prepared by the present invention significantly improve the gene encapsulation rate and protein loading rate relative to traditional lipid nanoparticles, and the gene encapsulation rate and protein loading rate of PLNPs are both stable, and PLNPs have low cytotoxicity. PLNPs have strong gene transfection, and the efficiency of GFP delivery to primary mononuclear cells of mice is high. By studying the immune status of PLNPs full antigen vaccine, it was found that PLNPs full antigen vaccine can effectively activate the antigen presentation of dendritic cells, and has a significant inhibitory effect on the activity of tumor cells. PLNPs full antigen vaccine combined with doxorubicin has a significant inhibitory effect on mouse tumor growth, inhibits tumor growth, and reduces the volume and weight of tumors. It can be seen that the proteinized lipid nanoparticles (PLNPs) prepared by the present invention have a stable and efficient gene encapsulation rate (93.63%) and protein loading rate (62.03%), which can significantly inhibit the proliferation and development of tumor cells, reduce tumor volume and mass, and achieve the effect of mass tumor.

[0129] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0130] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A proteinized lipid nanoparticle, characterized in that: The proteinized lipid nanoparticles include 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, anhydrous ethanol, plasmid, Triton X-100, sodium citrate buffer solution and cell membrane protein liposomes in a dosage ratio of 2mg-2.5mg: 1mL-2mL: 0.4mg-0.5mg: 0.1mg-0.2mg: 3mL-4mL: 5.5mg-12.0mg; the pH of the sodium citrate buffer solution is 5-6; The cell membrane protein liposome comprises dipalmitoylphosphatidylcholine, cholesterol, Triton X-100, anhydrous ethanol, sodium citrate buffer solution and cell membrane protein in a dosage ratio of 4.75 mg to 8.75 mg: 1.15 mg to 1.5 mg: 0.5 mg to 1.5 mg: 1 mL to 2 mL: 2 mL to 3 mL: 1 mg; Among them, dipalmitoylphosphatidylcholine, cholesterol, Triton X-100, anhydrous ethanol, sodium citrate buffer solution and cell membrane proteins are recombined to form cell membrane protein liposomes with negative surface charge; 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, anhydrous ethanol, plasmid, Triton X-100 and sodium citrate buffer solution are recombined to obtain positively charged lipid nanoparticles; utilizing the mutual attraction between positive and negative charges, a layer of cell membrane protein liposome coat is wrapped on the surface of lipid nanoparticles to obtain proteinized lipid nanoparticles.

2. The proteinized lipid nanoparticle according to claim 1, characterized in that The cell membrane protein is extracted from tumor cells.

3. A method for preparing proteinized lipid nanoparticles according to claim 1 or 2, characterized in that: The steps include: Cell membrane protein extraction: extract membrane proteins from tumor cells in the logarithmic growth phase to obtain cell membrane proteins; Preparation of cell membrane protein liposomes: dissolving dipalmitoylphosphatidylcholine, cholesterol and Triton X-100 in anhydrous ethanol, adding sodium citrate buffer solution and cell membrane protein, incubating and then filtering by extrusion to obtain cell membrane protein liposomes with negative surface charge; the dosage ratio of dipalmitoylphosphatidylcholine, cholesterol, Triton X-100, anhydrous ethanol, sodium citrate buffer solution and cell membrane protein is 4.75mg-8.75mg:1.15mg-1.5mg:0.5mg-1.5mg:1mL-2mL:2mL-3mL:1mg; Preparation of proteinized lipid nanoparticles: 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester is dissolved in anhydrous ethanol to obtain Dlin-MC3-DMA solution; plasmid and Triton X-100 are dissolved in sodium citrate buffer solution to obtain plasmid solution; then the plasmid solution is mixed with the Dlin-MC3-DMA solution to obtain a mixed solution; then cell membrane protein liposomes are added to the mixed solution for ultrasonic vibration, and after incubation, a molecular weight cutoff of 3KD to 14KD is selected for dialysis to obtain proteinized lipid nanoparticles; The dosage ratio of the 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, anhydrous ethanol, plasmid, Triton X-100, sodium citrate buffer solution and cell membrane protein liposome is 2mg-2.5mg: 1mL-2mL: 0.4mg-0.5mg: 0.1mg-0.2mg: 3mL-4mL: 5.5mg-12.0mg.

4. The method for preparing proteinized lipid nanoparticles according to claim 3, characterized in that: The dosage ratio of dipalmitoylphosphatidylcholine, cholesterol, Triton X-100, anhydrous ethanol, sodium citrate buffer solution and cell membrane protein is 7.5 mg: 1.5 mg: 1.0 mg: 1 mL: 2 mL: 1 mg.

5. The method for preparing proteinized lipid nanoparticles according to claim 3, characterized in that: The cell membrane protein concentration is 0.5 mg / mL to 2.0 mg / mL.

6. The method for preparing proteinized lipid nanoparticles according to claim 3, characterized in that: The cell membrane proteoliposomes are added to the mixed solution and incubated at 36° C. to 37° C. for 15 min to 20 min after ultrasonic shaking.

7. Use of the proteinized lipid nanoparticles according to claim 1 in preparing products for treating and / or preventing breast cancer.

8. Use of the proteinized lipid nanoparticles according to claim 7 in preparing a product for treating and / or preventing breast cancer, characterized in that: The product is a full-antigen tumor vaccine.

9. Use of the proteinized lipid nanoparticles according to claim 7 in preparing a product for treating and / or preventing breast cancer, characterized in that: The whole antigen tumor vaccine is used to inhibit the growth of tumor volume.

10. A full antigen tumor vaccine, characterized in that: The PLNPs full-antigen tumor vaccine was synthesized using monophosphoryl lipid A as an adjuvant; the construction process is as follows: Dipalmitoylphosphatidylcholine, cholesterol, Triton X-100 and monophosphoryl lipid A are dissolved in 1 mL to 2 mL of anhydrous ethanol according to a mass ratio of 4.75 mg to 8.75 mg: 1.15 mg to 1.5 mg: 0.5 mg to 1.5 mg: 0.050 mg to 0.080 mg, a sodium citrate buffer solution with a pH of 5 to 6 and the cell membrane protein of claim 2 are added, and after incubation, cell membrane protein liposomes containing an adjuvant are obtained by extrusion filtration; then, according to the preparation steps of proteinized lipid nanoparticles, the cell membrane protein liposomes containing an adjuvant are replaced by the cell membrane protein liposomes containing an adjuvant to prepare a PLNPs full-antigen tumor vaccine containing an adjuvant.

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