Preparation method and application of Newcastle disease virus microneedle delivery system

Abstract

The invention discloses a preparation method and application of a Newcastle disease virus microneedle delivery system. The delivery system entrapped with the Newcastle disease virus microneedle is easily absorbed by cells and has a good slow release effect, and experimental data also shows that the delivery system has a remarkable anti-tumor effect.

Description

TECHNICAL FIELD

[0001] The present application relates to a biological delivery system, in particular to a preparation method and application of a Newcastle disease virus microneedle delivery system. BACKGROUND

[0002] As one of the major diseases threatening global human health, liver cancer, the existing therapy such as chemotherapy, radiotherapy and the like produces many toxic side effects, which threatens the major organs, therefore, there is an urgent need for a new treatment regimen to change the status quo. As a drug delivery system, the microneedle avoids the gastrointestinal tract, thereby avoiding the transformation of the liver, so that the liver dysfunction and gastrointestinal irritation as side effects are very low. There are also other advantages, such as maintaining an effective drug delivery rate over time, a stable circulation rate, and the benefits of passive delivery systems and diffusion. Encapsulating anticancer drugs in liposomes can target drug delivery to tumor tissue and prevent damage to normal surrounding tissues, especially the availability of simultaneously targeting and killing cancer cells and tumor vasculature. Daniela Di Paolo made a related liposome anticancer research as early as 2008, Application of liposome antitumor drugs in neuroectodermal cancer therapy. A number of studies have shown that Newcastle disease virus (NDV) can be used as a safe oncolytic active substance for the treatment of various cancers. SUMMARY

[0003] Therefore, the first object of the present application is to provide a preparation method of a Newcastle disease virus microneedle delivery system, which is based on 2-hydroxy-2-methyl-1-phenyl-1-propanone to construct a porous microneedle, and then the Newcastle disease virus is combined with gelatin to encapsulate in the porous microneedle; the second object of the present application is to provide a microneedle delivery system of Newcastle disease virus prepared by the preparation method of the present application; the third object of the present application is to provide the application of the microneedle delivery system of Newcastle disease virus in the preparation of antitumor drugs.

[0004] The specific technical solutions adopted by the present application are as follows: The first aspect of the present application provides a preparation method of a Newcastle disease virus microneedle delivery system, comprising the following steps: (1) Preparation of GelMA porous microneedle: (I) Preparation of positive mold: Polyethylene glycol diacrylate and 2-hydroxy-2-methyl-1-phenyl-1-propanone are mixed and added to the microneedle mold, vacuumized to remove excess bubbles, and the cover glass is placed under ultraviolet light for molding. After washing and air drying, surface treatment is performed to obtain a mold with a hydrophobic surface; the mold with a hydrophobic surface is sealed with a sealing bag, dried, and ventilated, The dichloromethane and silane are mixed and loaded into a container, the container loaded with dichloromethane and silane and the mold with a hydrophobic surface are sealed and dried, the mold is taken out, the mold is placed flat in the center of a new container, the dichloromethane and silane mixture is poured into the new container, vacuum is drawn to remove excess bubbles, the container is cut open, the mold is removed, and a positive mold is obtained; (II) Preparation of GelMA: Take gelatin and anhydrous sodium carbonate, dissolve the anhydrous sodium carbonate in ultrapure water, then add the gelatin, fully dissolve and transfer to a conical flask, water bath stirring solution, fully dissolved to produce no precipitate, then slowly add methyl acrylate, continue water bath stirring reaction, after the reaction is completed, the mixed initial product solution is obtained. The mixed initial product solution is loaded into a dialysis bag, the dialysis bag is loaded into a container and dialyzed in a water bath, and the dialyzed reaction product, GelMA, is stored frozen. (III) Preparation of porous microneedles Take GelMA, add PBS buffer, heat in a water bath, and add 2-hydroxy-2-methyl-1-phenyl-1-propanone as a photoinitiator after complete dissolution to prepare a GelMA working solution, which is stored in the dark. Wash the positive mold and dry it, pour the GelMA working solution into the positive mold, vacuum and remove excess bubbles, place it under ultraviolet light to form microneedles, and store it in the freezer overnight. Take out the microneedles that have been frozen overnight and vacuum freeze-dry them to obtain GelMA porous microneedles. (2) Porous microneedle encapsulation of Newcastle disease virus: Take gelatin, add PBS buffer, heat and dissolve in a water bath to prepare a gelatin solution. When the gelatin solution reaches room temperature, add Newcastle disease virus and mix well to obtain a mixture. Immerse the GelMA porous microneedles in the mixture overnight. After light protection, freeze-dry for 4-6 hours to encapsulate the Newcastle disease virus.

[0005] Weigh 0.2 g of gelatin powder and add 10 mL of PBS buffer. Heat in a 65°C water bath and mix well during the process until the particles are dissolved. Prepare a 20% gelatin solution. When the 20% gelatin solution reaches room temperature, add 1 MOI of NDV and mix gently. Immerse the freeze-dried GelMA porous microneedles in the mixture overnight. Then wrap them with aluminum foil and place them in a freeze-dryer for 4-6 hours to encapsulate the Newcastle disease virus (NDV).

[0006] Further, mix polyethylene glycol diacrylate and 2-hydroxy-2-methyl-1-phenyl-1-propanone in a ratio of 100:1.

[0007] Further, perform surface treatment in a DT-01 type low-temperature plasma treatment instrument for 20 minutes to obtain a mold with a hydrophobic surface.

[0008] Further, the dichloromethane and silane are mixed in a ratio of 100:1 and loaded into a container.

[0009] Further, the drying is performed by placing in a constant temperature oven at 60-65°C overnight; the static ventilation is performed by placing in a fume hood for 30-60 min.

[0010] Further, the mass-volume ratio of the gelatin, anhydrous sodium carbonate and ultrapure water is 20 g:10 g:200 mL.

[0011] Further, the water bath in step (I) and step (II) is a 50°C hot water bath.

[0012] Further, the conical flask is a conical flask with an A-type olive magnetic stirring rotor Further, the conical flask is wrapped with aluminum foil, 4 mL of methacrylic anhydride is slowly added into the conical flask, and the dropping is completed within 3 min.

[0013] Further, after adding the methacrylic anhydride, the stirring reaction in the water bath is continued for 1 hour, and during the reaction, the pH value is adjusted to 9 with 10% (w / v) sodium hydroxide solution every 10 min for 6 times.

[0014] Further, the water is changed 20 times during the dialysis, and the water is changed every 2 h on average.

[0015] Further, the water bath in step (III) is a 65°C hot water bath.

[0016] Further, the mass-volume ratio of the gelatin, PBS buffer and Newcastle disease virus is 0.2 g:10 mL:1 MOI.

[0017] Further, the water bath in step (2) is a 65°C hot water bath.

[0018] The second aspect of the present application provides a Newcastle disease virus microneedle delivery system prepared by the above preparation method.

[0019] The third aspect of the present application provides the use of the Newcastle disease virus microneedle delivery system, characterized by the use in preparing an anti-liver cancer drug.

[0020] The present application has the following beneficial effects: the Newcastle disease virus microneedle delivery system is easy to be absorbed by cells, has good sustained release effect, and experimental data also shows that it has significant anti-tumor effect. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The apparent image of the GelMA microneedle is shown; Figure 2 The killing effect of the Newcastle disease virus loaded by the porous microneedle of the biological delivery system on HepG2 was shown by the CCK8 method; Figure 3 The effect of the Newcastle disease virus loaded by the porous microneedle of the biological delivery system on L02 was shown (100x) ; Figure 4 The cytotoxic effect of the Newcastle disease virus loaded by the porous microneedle of the biological delivery system on HepG2 was shown (100x) ; Figure 5 The effect of the Newcastle disease virus loaded by the porous microneedle of the biological delivery system on the migration ability of HepG2 was shown (40x) ; Figure 6 The effect of the oncolytic virus loaded by the porous microneedle of the biological delivery system on the invasion ability of HepG2 was shown (200x) ; Figure 7 The apoptosis induction of the Newcastle disease virus loaded by the porous microneedle of the biological delivery system on HepG2 was shown by flow cytometry; Figure 8 The time change curve of the volume of the subcutaneous liver transplantation tumor and the body weight of the nude mice in the in vivo experiment of the naked mice was shown. DETAILED DESCRIPTION

[0022] In order to more clearly illustrate the present application, the present application is further described below in combination with preferred embodiments and drawings. Those skilled in the art should understand that the specific description below is illustrative rather than limiting, and should not limit the protection scope of the present application.

[0023] The materials in the following examples are all commonly used in the art, and can be obtained from commercial channels, unless otherwise specified.

[0024] Example 1 A preparation method of a GelMA porous microneedle, specifically comprising the following steps: (I) Preparation of a positive mold: Polyethylene glycol diacrylate and 2-hydroxy-2-methyl-1-phenyl-1-propanone were mixed in a ratio of 100:1.

[0025] The mixed solution of step (1) was added into a microneedle mold with a size of 15.8 mm x 15.8 mm by using a sample gun.

[0026] Put into a vacuum pump to extract vacuum, and remove the excess bubbles with tweezers.

[0027] Cover the cover glass, and form under ultraviolet light for a few seconds.

[0028] Wash with 75% alcohol, and naturally air dry, and put into a lunch box.

[0029] Put into the surface treatment in the DT-01 type low temperature plasma processing instrument for 20 min, obtain hydrophobic surface.

[0030] In the fume hood, dichloromethane and silane are mixed in a ratio of 100:1 and loaded into the container.

[0031] The container containing the mixture of dichloromethane and silane and the surface-treated mold are loaded into the lunch box, sealed with a sealing bag, and placed in a constant temperature oven at 60°C overnight.

[0032] The mold in the oven is taken out and placed in the fume hood for 30-60 min.

[0033] The mixed A and B silica gel solutions are mixed in a ratio of 1:1, the mold is taken out and placed flat in the center of the container, and the container is poured.

[0034] Vacuumize in the vacuum pump, and after no bubbles are generated, place flat in the 65°C oven overnight.

[0035] Cut open the container and demold.

[0036] (II) Preparation of GelMA Add 200 mL of distilled water to the beaker.

[0037] Weigh 20 g of gelatin (from pigskin) and 10 g of anhydrous sodium carbonate in a ratio of 2:1.

[0038] Dissolve the anhydrous sodium carbonate in the beaker with 200 mL of ultrapure water, add the gelatin, and dissolve thoroughly at 50°C until the solution becomes clear.

[0039] Transfer the above solution to a conical flask with an A-type olive magnetic stir rotor, stir the solution in a 50°C hot water bath, and dissolve thoroughly to the standard of no precipitation.

[0040] Wrap the bottle with aluminum foil, slowly add 4 mL of methacrylic anhydride to the conical flask, and control the time to be completed within 3 min.

[0041] Continue to stir the above solution in a 50°C hot water bath for 1 hour, and during the reaction, adjust the pH to 9 every 10 min with 10% (w / v) sodium hydroxide solution, use the pH standard colorimetric card as the pH reference, and add about 15-20 mL of sodium hydroxide solution in 6 times.

[0042] Under light-proof conditions, load the above reaction mixture solution into a dialysis bag, and clamp the dialysis bag with dialysis clamps on both sides.

[0043] Load deionized water into a 2 L large beaker and dialyze in a 50°C hot water bath, change water 20 times during the process, change water every 2 h on average, and avoid light.

[0044] Pour the dialyzed reaction product into a dark disposable lunch box, not more than half the height. Put into the-20℃ refrigerator for short-term storage or freeze for 2 h and then vacuum freeze for 5 days.

[0045] Store the synthesis product into a 50 mL centrifuge tube, indicate the date, and store in the dark.

[0046] (III) Preparation of GelMA porous microneedle Weigh the GelMA sample, add PBS buffer, heat in a water bath at 65℃, and after complete dissolution, add 2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP) (as a photoinitiator) at a ratio of 100:1, prepare the GelMA working solvent, and store it in the dark with aluminum foil.

[0047] Add pure water to the ultrasonic cleaner and clean the male mold.

[0048] Dry the mold and wipe off the moisture.

[0049] Add GelMA working solvent to the mold, turn on the constant temperature vacuum pump at 50±5℃ to extract vacuum, and after extraction, use tweezers to remove excess bubbles.

[0050] Under the UV LED lamp light source, irradiate for a few seconds, the hydrogel is formed, and store it in the-20℃ refrigerator overnight.

[0051] Take out the hydrogel microneedle frozen overnight, freeze-dry in a vacuum freeze dryer, and obtain the GelMA porous microneedle.

[0052] As shown in Figure 1 The GelMA porous microneedle prepared in this embodiment has a needle length of 800 m, a needle diameter of 225 m, a needle tip distance of 650 m, a patch size of 15.8 mm x 15.8 mm, and a groove depth of 1.50 mm.

[0053] Example 2 A preparation method of a Newcastle disease virus microneedle delivery system, comprising the following steps: (1) Preparation of GelMA porous microneedle: prepared according to the method described in Example 1; (2) Newcastle disease virus (NDV) loaded in porous microneedle: weigh 0.2 g of gelatin powder, add 10 mL of PBS buffer, heat in a water bath at 65℃, and mix well during the process until the particles are dissolved, prepare a 20% gelatin solution, add 1 MOI of NDV after the 20% gelatin solution is cooled to room temperature, gently blow evenly, immerse the GelMA porous microneedle in the mixed solution overnight, then wrap it with aluminum foil and freeze-dry in a freeze dryer for 4-6 h to encapsulate the Newcastle disease virus (NDV).

[0054] Example 3 PCR verification of the long-acting performance of the NDV-releasing porous microneedle (Newcastle Disease Virus microneedle delivery system) to release NDV, the specific method is as follows: 1. Collect the release solution The NDV-loaded microneedle prepared in Example 2 was soaked in 2% PBS buffer, and the drug release solution was collected at 6 h, 12 h, 24 h and 48 h.

[0055] 2. Extract the RNA of the release solution 1. The entire process was operated on ice, the release solution was pipetted into an EP tube, 1 mL of trizol was added per tube, 0.2 mL of chloroform was added, and the solution was mixed by inverting and allowed to stand on ice for a few minutes. After a few minutes, the solution was layered.

[0056] Balance in a small desktop centrifuge at 4°C, then centrifuge at 12000 g for 15 min.

[0057] Pipette the supernatant into an EP tube to which 0.5 mL of isopropanol has been added, continue to mix well, and place the metal tube rack on ice for 10 min. Then centrifuge at 4°C, 12000 g for 10 min.

[0058] 75% ethanol was prepared with anhydrous ethanol as stock solution and DEPC water as diluent.

[0059] After centrifugation, a precipitate was observed, the supernatant was discarded, 1 mL of the prepared ethanol solution was added per tube, and the mixture was mixed by blowing with a 1 mL micropipette. Then transfer to a 4°C centrifuge and centrifuge at 10000 g for 5 min.

[0060] Discard the supernatant and air-dry by inverting on sterilized high-pressure handkerchief. Let stand at room temperature for a few minutes.

[0061] Add 40 μL of DEPC water per tube for dilution, and store at -80°C or on ice until use.

[0062] Adjust the zero with a small amount of DEPC water on the NanoDrop 2000 detection instrument, and then detect the RNA concentration.

[0063] 3. Reverse transcription 1. Label a set of PCR tubes, and the following steps are operated on ice.

[0064] Add 4 L of 4x gDNA wiper Mix to each PCR tube.

[0065] According to the RNA concentration detected by the NanoDrop 2000 detector, calculate the volume of the sample solution required to quantitate the RNA to 1000 ng, and supplement the solution volume to 16 L with DEPC water, and mix well.

[0066] PCR instrument setting program: 42℃, 2 min; 4℃, Hold. Mix the PCR tube and place it in the PCR instrument. Run the program.

[0067] Remove the PCR tube and place it in the 4℃ refrigerator for 3 min.

[0068] The PCR instrument is set to the program: 37℃, 15 min; 85℃, 5 s; 4℃, Hold. Add 5×HiScript III RT SuperMix 4 L / tube to the PCR tube, mix with a micropipette, tap the tube wall, and place it in the PCR instrument to start the program.

[0069] Dilute with 80 L DEPC water, mix well, and store in the -20℃ refrigerator.

[0070] 4. cDNA amplification of sample viruses 1. Label a set of PCR tubes for use. The following steps are performed on ice.

[0071] (2) Add the solutions in Table 1 below to the PCR tube step by step to prepare the PCR reaction system.

[0072] System components Amount (unit: L) Upstream primer 1 Downstream primer 1 ddH2O 9.5 2 x Tap Plus Master Mix II 12.5 cDNA 1 (3) Set the PCR reaction program: 59℃, 15 s, cycle 1 time; 72℃, 1 min, cycle 1 time; 98℃, 30 s; 59℃, 30 s, cycle 35 times; 72℃, 30 s; 98℃, 30 s, cycle 1 time. Tap the tube wall and then remove it, ready for machine.

[0073] 5. Agarose gel electrophoresis 1. Clean the mold tank and rack, and comb, and let them air dry. Assemble the mold tank and rack.

[0074] Mix 10 mL of 50×TAE electrophoresis solution and 490 mL of high-purity water to prepare 1×TAE electrophoresis solution. Add 0.8 g of agarose powder and 80 mL of 1×TAE electrophoresis solution to a wide-mouth conical flask, and shake well. Heat in a microwave oven for 10 s, repeat 2-3 times, until the powder is completely dissolved.

[0075] Rinse the outside wall of the conical flask with tap water to cool it to a temperature that is not hot to the touch.

[0076] In a fume hood, add 4 L of nucleic acid dye to the conical flask, and gently shake the bottle to avoid air bubbles.

[0077] To avoid the formation of bubbles, pour the liquid slowly along one corner of the mold channel, and insert the comb immediately. Let the agarose gel stand in the fume hood for 30 min.

[0078] Gently pull out the comb, and transfer the rack to the electrophoresis tank. Add appropriate amount of 1 × TAE electrophoresis solution to the electrophoresis tank, so that the liquid covers the agarose gel.

[0079] Add 10 L of DL2000 Plus Marker to the left loading well in the agarose gel with a micropipette, and add 10 L of PCR product of the release solution of different time periods to the remaining loading wells.

[0080] Set the electrophoresis parameters: 150 V, 150 mA, 30 min, and start the electrophoresis.

[0081] After the electrophoresis is completed, the Loading Buffer can be seen below the agarose gel, but does not exceed the bottom of the agarose gel. Turn off the electrophoresis instrument.

[0082] Take out the agarose gel and place it in the dark room of the gel imaging system. Adjust the position to the center, and record the development results.

[0083] After 6 h of release of the porous microneedle loaded with NDV, obvious bands can be detected, which are about 136 bp, the bands of NDV genes. The same gene bands can be detected at 24 h and 12 h, and the band at 12 h is stronger than that at 24 h. The results of this time show that the porous microneedle has a long-acting release performance within 24 h as an NDV carrier.

[0084] Example 4 Effect of NDV-loaded porous microneedle (NDV microneedle delivery system) on human hepatoma cell HepG2 and normal cell line human liver cell L02: The cell lines used in this example, such as human hepatoma cell HepG2 and normal cell line human liver cell L02, are from the -80℃ refrigerator in our laboratory, and are cultured in DMEM containing 10% fetal bovine serum as the basic medium in a constant temperature cell incubator at 37℃ and 5% CO2.

[0085] Divide into four groups, respectively: PBS group, Material group (i.e. empty microneedle), NDV group (i.e. NDV alone), and GM10-Gel-NDV group (i.e. NDV-loaded porous microneedle prepared in Example 3); PBS group: Add an equal amount of PBS buffer to 2% FBS DMEM as a blank control; Material group: The lyophilized empty microneedles prepared in Example 1 were soaked in DMEM medium containing 2% FBS (extraction solution 0.1 g / mL) and incubated in a 37°C incubator for 24 h. After 24 h, the precipitate was discarded by centrifugation, and the supernatant was filtered with a 0.45 m needle filter for standby; NDV group: 1 MOI of NDV was mixed with DMEM medium containing 2% FBS; GM10-Gel-NDV group: The NDV-loaded porous microneedles containing 1 MOI of NDV prepared in Example 2 were released in DMEM containing 2% FBS; 1. CK8 experiment to detect the effect of NDV-loaded biological delivery system on HepG2 proliferation: The human liver cancer cells HepG2 in the logarithmic growth phase with a fusion degree close to 80% were taken and placed in a 96-well cell plate. The used cell culture medium was discarded, and the cells were gently blown with PBS buffer three times. The washing liquid was discarded, and the cells were digested with trypsin containing EDTA for several minutes. After observing the rounding or shrinking of the cells under a microscope, complete medium was added to terminate the digestion. The cells were blown off with a Pasteur pipette and collected into a 50 mL centrifuge tube. The centrifuge was balanced at 1000 rpm for 5 min. After centrifugation, the liquid was discarded, and PBS buffer was added for resuspension. The PBS-resuspended cell liquid was balanced and centrifuged at 1000 rpm for 5 min. This step was repeated three times. 10 L of resuspension liquid and 10 L of 0.4% trypan blue solution were taken, mixed evenly with a micropipette gun, and added to the counting pool of a counting plate under a low-power microscope. According to the count, the complete medium was added to adjust the cell density to 4×10 4 cell / mL. 0.1 mL of cell suspension was added to each well, and the cells were incubated in a cell culture incubator for cell adhesion and growth. After overnight incubation, the cells were grown adherently, the supernatant was discarded, and PBS buffer was added to wash the residual old medium in the plate. According to the experimental grouping, 100 L of 3.2 prepared solution was added per well. The plate was transferred to a carbon dioxide cell incubator for continued incubation for 24 h, 48 h, and 72 h. 10% CCK8 stock solution and 90% DMEM medium were used to prepare CCK8 working solution. After taking out the ninety-six-well plate, 100 L of freshly prepared CCK8 working solution was added to each well, and the plate was placed in the cell culture incubator for 1-2 h. The OD value at 450 nm in the incubated plate was detected using a microplate reader.

[0086] The data were exported and the cell proliferation rate was calculated as follows: Figure 2, three curves represent the experimental results, there is no significant difference between the three groups after 24 h, the tumor cells have little effect. With the passage of time, 48 h and 72 h, although the Material group has no effect on tumor cells, but the difference between the groups at these two time periods is obvious, the use of NDV alone group and GM10-Gel-NDV group has a certain killing effect, and the killing effect of GM10-Gel-NDV group is significantly higher than that of Material group and NDV alone group. When incubated for 72 h after adding drugs, the relative growth rate of HepG2 in the Material group reached 92.65%±3.44%, the relative growth rate of HepG2 in the NDV group reached 63.62±8.22%, and the relative growth rate of HepG2 in the GM10-Gel-NDV group reached 30.62%±1.642%. It can be seen that the GM10-Gel-NDV group can inhibit the proliferation of HepG2.

[0087] Effect of NDV-loaded biological delivery system on the proliferation of HepG2 and L02 cells: HepG2 and L02 cells were plated in 6-well plates: logarithmic growth phase of two cells were digested with trypsin containing EDTA for 2-3 min, and the complete culture medium was added to terminate, and the cells were blown off and collected with a centrifuge tube. Centrifuged at 1000 rpm for 5 min. Discard the liquid after centrifugation, add PBS buffer, mix evenly with a pipette, and centrifuge after adjusting the volume. Repeat this process to wash the cells three times. Take 10 L of cell suspension and an equal amount of 0.4% trypan blue solution, mix them together, and then add them to the counting pool of the counting plate. Adjust the cell density to 1.5×10 5 cell / mL after adding complete culture medium. Take a small amount of PBS buffer to soak the bottom of the well plate, then discard the liquid, and take 2 mL of adjusted cell suspension into a single well of the 6-well plate. After standing for five minutes, observe the cell density under a microscope to about 35%, and place it in a cell culture incubator for cell adhesion culture.

[0088] 6-well plate of HepG2 and L02 cells with drug treatment: after the cells adhere, discard the old culture medium on the plate, add appropriate PBS buffer solution to wash the well plate, and repeat the washing twice. Record the cell state before adding drugs under an optical microscope. Discard the PBS buffer and add the solution of the above components, and place it in the incubator for continuous culture for 1 day, 2 days, and 3 days. Record the cell state every 24 h with a microscope imaging system.

[0089] The results are as follows: Figure 3As shown, the normal morphology of normal liver cells L02 before adding each group of drugs was full, like cobblestone-like, and evenly distributed in small groups. When adding drugs for 24 h, the L02 cells of each group were normal, full and almost no scattered dead cells were seen. Therefore, each group had almost no effect on L02 cells after 24 h of drug addition. After 48 h and 72 h of drug addition, single dead cells appeared in each group, and no large pieces of cells appeared, but there was no difference from the control group. The morphology of L02 cells in each group was still normal and full, so the biological delivery NDV had no effect on normal liver cells, i.e. it did not affect the growth of L02.

[0090] Using the same method, the killing effect of the tumor cells Figure 4 As shown, the tumor cells in the PBS group were not affected, and as the incubation time increased, single dead cells gradually appeared, and the living cells gradually grew in groups and eventually covered the field of view. After 24 h of drug addition, the HepG2 cells in the Material group had almost no change in morphology, but there were a few single scattered dead cells. The HepG2 cells in the NDV alone group showed a relatively swollen morphology and a small amount of death. As the virus began to infect tumor cells, it gradually replicated in the cell body, but no oncolysis and large piece cell death was found, because the amount of virus was small and did not reach the effect of dissolving cancer cells. The HepG cells in the GM10-Gel-NDV group gradually began to have blurred boundaries, and the cells were swollen and a small amount of cell death occurred. After 48 h of drug addition, the Material group still had no obvious change. The tumor cell morphology in the NDV alone group changed significantly, with blurred boundaries, cell shrinkage or virus lysis, and increased cell death. The HepG2 cells in the GM10-Gel-NDV group had unclear boundaries, increased shrunk cells, and virus-infected cells were lysed and died in large pieces. The proliferation of liver cancer cells was affected, and the cell proliferation was significantly lower than that of the normal control group. After 72 h of drug addition, a small amount of dead cells appeared in the Material group, but the cell morphology was still normal. A large number of cells died in the NDV group, but some cells still survived. Compared with the NDV group and the GM10-Gel-NDV group, the HepG2 cells in the GM10-Gel-NDV group were basically dead, the killing effect was most obvious, and the tumor cell proliferation was the lowest.

[0091] Effect of NDV loaded by the biological delivery system on HepG2 cell migration: The logarithmic growth phase of HepG2 was digested with trypsin containing EDTA, and the cell suspension was mixed with an equal amount of trypan blue staining solution and filled into a counting chamber. The cell density was adjusted to 6x10 5Cells per well: Wet each well of a 6-well plate with PBS solution, discard the PBS buffer, and add 2 mL of cell suspension to each well. Shake the plate in a cross pattern, let it stand for five minutes, and then incubate overnight in a cell culture incubator for cell adhesion and growth. Under a microscope, observe that the cell confluence in each well reaches over 80%. Using a 100 L sterile pipette tip, streak along a ruler perpendicular to the plate direction, applying consistent pressure. Discard the original culture solution, add an appropriate amount of PBS buffer to wash away excess floating cells, and repeat the PBS washing three times. Remove the plate, discard the old culture medium, wash with PBS solution, and add the respective solutions. Record the cell migration status after streaking using a microscope imaging system at 0 h after drug addition. Continue incubation for 24 h, 48 h, and 72 h. Record cell migration at the corresponding time points (24 h, 48 h, and 72 h after streaking) using a microscope imaging system and perform statistical analysis.

[0092] like Figure 5 As shown in Figure A, at 0 h, the scratch widths of all groups were almost similar. After 24 h, the scratch widths of each group began to change. The PBS group showed the fastest cell migration and the narrowest scratch width, followed closely by the Material group, whose scratch width was also decreasing. The remaining two groups showed almost no change, with the scratch width remaining almost the same as before drug administration. After 48 h of drug administration, the PBS group healed the fastest, followed by the Material group. The scratches in the single-drug groups continued to heal slowly, while the scratches in the bio-delivered oncolytic virus group were the widest and healed the slowest. After 72 h of drug administration, the scratches in the PBS group (as a control group) were close to healing and had the narrowest width. The scratches in the Material group also showed a clear healing trend, with the width second only to the PBS group. The scratches in the NDV-only group also showed a slow decrease in width, but there was still some distance to go, and the healing was not obvious compared to the 0 h drug administration. The bio-delivered oncolytic virus group, i.e., the GM10-Gel-NDV group, showed the least obvious healing and the weakest trend, hindering the migration of HepG2 cells, and had the widest scratch width, which was significantly different from the control group. Figure 5 B. Statistical analysis yielded the following final HepG2 cell migration percentages after scratching in each group: PBS group: 69.95% ± 0.21%; Material group: 51.55% ± 0.50%; NDV group: 35.20% ± 2.25%; and biologically delivered oncolytic virus group: 23.69% ± 0.79%. Significant differences were found between the drug delivery group and the PBS group, Material group, and the drug-only group. P A value <0.01 was considered statistically significant. The results showed that the tumor cells in the bio-delivered oncolytic virus group migrated the slowest. The bio-delivered oncolytic virus group could affect tumor cell migration, inhibit the migration ability of HepG2 cells, and exhibited good anti-tumor effects.

[0093] The effect of NDV-loaded by a biological delivery system on HepG2 cell invasion: Remove Matrigel from the -20°C freezer and thaw overnight at 4°C. Pre-chill and sterilize pipette tips and serum-free DMEM medium. Mix serum-free DMEM medium and Matrigel at an 8:1 ratio on ice, avoiding air bubbles during mixing. Immediately add 100 μL of Matrigel dilution to the transwell chambers of a 24-well plate. Incubate the 24-well plate overnight at 37°C to promote gel formation. Digest HepG2 cells in the logarithmic growth phase with trypsin, collect the cells in centrifuge tubes, balance the liquid, and centrifuge. Discard the old culture medium, resuspend the cells in an appropriate amount of PBS buffer, balance the liquid again, and centrifuge at 1000 rpm for 5 min. Repeat the washing process with PBS three times. Count the cells from the cell suspension, resuspend the cells in serum-free DMEM complete medium, and adjust the cell density to 2.5 × 10⁶ cells / mL. 5 cell / mL. Add 1.5 mL of the adjusted cell suspension to an EP tube, balance and centrifuge at 1000 rpm for 5 min in a small benchtop centrifuge.

[0094] Preparation of drug-loaded solutions: The control group solution was serum-free DMEM medium. For the Material group, an extract was prepared by immersing lyophilized empty microneedles (0.1 g / mL extract) in serum-free DMEM medium and incubating for 24 h at 37°C. After 24 h, centrifugation was performed, and the supernatant was filtered using a 0.45 μm syringe filter. The virus solution was transferred to a centrifuge and centrifuged at 4°C for 15 min at 4000 rpm. The precipitate was discarded, and the supernatant was retained as the virus component. This component was filtered using a 0.45 μm syringe filter, and 1 MOI was used to prepare the virus group solution with serum-free DMEM medium. Following the same procedure as step 3.1, porous microneedles loaded with NDV were prepared. The microneedles were immersed in serum-free DMEM for 24 h to release the virus. The release solution was filtered into EP tubes. All these processes were performed in a clean bench at room temperature. The centrifuged liquid in the EP tubes was discarded, and the cells were fully resuspended in the above-mentioned component solutions. Add 700 μL of DMEM medium containing 10% FBS to the wells of a 24-well plate. Use forceps to pick up a Transwell chamber containing overnight gel and place it into the well. Aspirate 0.2 mL of cell suspension into the upper chamber, avoiding air bubbles. Transfer the chamber to a cell culture incubator and continue culturing for 48 h. Prepare cell fixation medium containing 4% paraformaldehyde. Dilute 1% crystal violet solution with PBS buffer to prepare 0.1% crystal violet staining solution. After 48 h of culture, remove the plate, discard the solution in the upper chamber, and use forceps to pick up the upper chamber and wash it in a beaker containing PBS solution, avoiding contact with the bottom of the chamber. Repeat this washing process three times. Immerse the chamber in cell fixation medium at room temperature for 20 min. Remove the chamber and wash it with PBS buffer to remove any remaining fixation medium. Repeat this washing process three times. Add 0.1% crystal violet staining solution to the upper chamber and stain for 10-15 min. Use forceps to pick up the upper chamber and wash it several times in a beaker containing PBS buffer, changing the PBS buffer as needed. Gently wipe away any excess staining solution from the upper chamber with a cotton swab. After air drying, immediately place it on a glass slide, take pictures and record the results under an inverted microscope, and take 3-5 fields of view to count and take the average value for quantitative analysis.

[0095] The results are as follows Figure 6 As shown in Figure A, cells in the PBS group penetrated the matrix gel in the small chambers to the bottom of the chambers. After crystal violet staining, they formed large, dense cell clusters, and their invasive ability was not hindered. Compared to the control group, the Material group showed a decrease in cells, but still contained large cell clusters at a moderate density. The NDV and GM10-Gel-NDV groups showed a significant reduction in cells, meaning fewer cells penetrated the matrix gel, indicating weakened cell invasiveness, especially in the experimental groups where NDV was encapsulated in porous microneedles. Statistical analysis (…) Figure 6B), the relative number of cells with invasion ability through the Matrigel of each group was: PBS group 208 ± 15.04, Material group 113 ± 2.517, NDV group 85.67 ± 3.18, GM10-Gel-NDV group 43.67 ± 3.93. The experiment showed that the NDV group and the GM10-Gel-NDV group inhibited the invasion ability of HepG2 cells, and the GM10-Gel-NDV group was the most obvious and the strongest.

[0096] Effect of NDV loaded by biological delivery system on apoptosis of HepG2 cells: Take out the T75 culture bottle, and observe the cell fusion degree under a microscope. Discard the supernatant of HepG2 cells, add PBS buffer, and wash the cells three times. Add trypsin to digest the cells for 3-5 min, add complete culture medium to terminate digestion after the cells shrink, collect into a centrifuge tube, and centrifuge at 1000 rpm for 5 min. Discard the liquid after centrifugation, resuspend the cells with an appropriate amount of PBS buffer, and centrifuge at 1000 rpm for 5 min after adjustment. Repeat the PBS washing step three times. Take 10 L of cell suspension and mix with an equal amount of 0.4% trypan blue solution, and then add it to the counting pool of the counting plate. Count under a microscope. Add DMEM complete culture medium to adjust the cell density to 3x10 5 Take 6-well plates, soak the bottom of the plate with an appropriate amount of PBS buffer, discard the PBS solution, and add 2 mL of cell suspension to each well. Draw a cross on the plate, shake the cells, and transfer them to a carbon dioxide cell culture incubator for overnight adhesion growth. Discard the original culture medium in the well, add 2 mL of solution to each well according to the group, and transfer it to the cell culture incubator for 24 h, 48 h, and 72 h incubation.

[0097] Take out the plate at each time point: collect the solution in the well into a centrifuge tube, rinse with PBS buffer twice, add trypsin without EDTA to digest the cells in each well for 5-6 min, add complete culture medium to terminate digestion after the cells round, blow the cells gently, collect the corresponding 15 mL centrifuge tube, and centrifuge at 1500 rpm for 5 min. Discard the supernatant, add 5 mL of PBS buffer, and gently blow the cell suspension. Repeat this process three times to wash the cells. Discard the centrifuged liquid, add 1xBinding buffer to continue gently blowing the cells, adjust the cell density to 1x10 6cell / mL. The blank control group cells were divided into two parts, and one part was added with 500 L of the Yangshen reagent on ice for 30 min. After resuspension with PBS buffer, centrifugation was performed at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended with the same volume of 1x Binding buffer, and centrifugation was performed at 1500 rpm for 5 min. The positive double staining tube, PI single staining tube, FITC single staining tube, blank control untreated group, and four experimental groups were set, and 100 L of the adjusted cell suspension of each group was taken into the corresponding 5 mL flow tube. 5 L of Annexin V-FITC and 10 L of PI were added to the flow tube under light-proof conditions, and the flow tube was gently blown to be uniform, and incubated at room temperature for 15 min. 400 L of 1x Binding buffer solution was added to each tube, and gently mixed to avoid air bubbles, and flow cytometry detection was performed. The data was recorded, saved, and analyzed by flow cytometry.

[0098] The results are shown in Table 1. Figure 7 As shown in Table 1, the scatter plot statistical analysis of cell apoptosis showed that apoptosis occurred in each group to different degrees, and the PBS group and the Material group were the least, which were 2.5%±0.28% and 2.175%±0.1073% respectively, and the values of the other two groups were slightly higher, and the NDV group alone was 17.86%±0.50%, and the bio-delivery system loaded NDV group was 60.67%±0.26%. Moreover, comparison showed that the bio-delivery system loaded NDV in the experimental group induced apoptosis more strongly than the single drug group. Statistical analysis by software GraphPad Prism showed that the bio-delivery system loaded NDV group induced apoptosis significantly, and the anti-tumor effect was significant.

[0099] In summary, the NDV released by the Newcastle disease virus microneedle delivery system of the present application does not affect normal cells, but can replicate and express in tumor cells. Moreover, the NDV can obviously hinder the proliferation of HepG2 tumor cells, reduce the migration ability of HepG2 tumor cells, and inhibit the invasion ability of HepG2 tumor cells, and promote the apoptosis of HepG2 tumor cells, and the effect is more obvious than that of the single drug group (i.e. the NDV group).

[0100] Example 5 In vivo anti-cancer effect of the NDV porous microneedle (Newcastle disease virus microneedle delivery system): A Balb / c nude mouse human liver cancer subcutaneous metastasis tumor model was constructed, and the tumor diameter was about 5 mm. The tumor-bearing nude mice were randomly divided into four groups, and each group had 6 tumor-bearing nude mice. The treatment was performed once a day, and 200 L of PBS solution, Material group solution, NDV group solution, and GM10-Gel-NDV group solution were injected subcutaneously at the tumor site. Before each treatment, the size of the tumor and the body weight of the tumor-bearing nude mice were measured and recorded by using a vernier caliper. The long diameter (A, cm) and short diameter (B, cm) of the subcutaneous tumor were recorded, and the tumor size was calculated as A x B2 ×0.5, plotting a curve showing changes in tumor size and body weight. Results are as follows... Figure 8 As shown in Figure A, tumors in the PBS and Material groups grew rapidly, while the tumors treated with NDV alone and the GM10-Gel-NDV group showed changes over time. Tumor volume increased slowly in the first 14 days, and gradually decreased from days 14 to 22, showing better treatment efficacy than the control group. Furthermore, treatment with Newcastle disease virus via biological delivery to nude mice resulted in gradual tumor shrinkage, significantly more pronounced than in the NDV-only group. After 22 days of treatment, the average tumor volume for the four groups was: PBS group 1245 mm. 3 Material group 1185 mm 3 NDV group 529.3 mm 3 GM10-Gel-NDV group 284.7 mm 3 .like Figure 8 As shown in Figure B, the body weight of the nude mice in each group changed over time. The PBS group showed a gradual decrease in body weight, faster and at a lower rate than the other three groups. The Material group followed, showing a slight decrease in body weight during treatment, but still fluctuating within the normal range. The GM10-Gel-NDV and NDV groups showed no significant change in body weight during treatment, and their activity and physical condition were better than the PBS group.

[0101] Although the above embodiments have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the above descriptions are merely embodiments of the present invention and do not limit the scope of patent protection of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A method for preparing a Newcastle disease virus microneedle delivery system, characterized in that, Includes the following steps: (1) Preparation of GelMA porous microneedles: (I) Preparation of positive mold:

1. Mix polyethylene glycol diacrylate and 2-hydroxy-2-methyl-1-phenyl-1-propanone, add to a microneedle mold, vacuum and remove excess air bubbles, place a coverslip under ultraviolet light to form, clean, air dry, and then perform surface treatment to obtain a mold with a hydrophobic surface; seal the hydrophobic mold with a sealing bag, dry, and allow to stand in a ventilated environment.

2. Mix dichloromethane and silane and put them into a container. Seal and dry the container containing dichloromethane and silane and the mold with a hydrophobic surface. Take out the mold and place it flat in the center of a new container. Pour the mixture of dichloromethane and silane into the new container, vacuum it, remove excess air bubbles, dry it, cut open the container, demold it, and obtain a positive mold. (II) Preparation of GelMA:

1. Take gelatin and anhydrous sodium carbonate. Dissolve the anhydrous sodium carbonate in ultrapure water, then add the gelatin. After it is fully dissolved, transfer it to an Erlenmeyer flask and stir the solution in a water bath. After it is fully dissolved and no precipitate is produced, slowly add methacrylic anhydride and continue to stir the reaction in a water bath. After the reaction is completed, a mixed initial solution is obtained.

2. Place the mixed initial solution into a dialysis bag, then place the dialysis bag into a container and perform dialysis in a water bath. Freeze the dialysis reaction product, GelMA. (III) Preparation of porous microneedles 1. Take GelMA, add PBS buffer, heat in a water bath until completely dissolved, then add 2-hydroxy-2-methyl-1-phenyl-1-propanone as a photoinitiator to prepare the working solvent for GelMA, and store it in the dark.

2. Clean and dry the positive mold, pour the GelMA working solvent into the positive mold, vacuum and remove excess air bubbles, place it under ultraviolet light to form microneedles, freeze overnight for storage, take out the frozen microneedles overnight, and vacuum freeze dry to obtain GelMA porous microneedles. (2) Newcastle disease virus encapsulated in porous microneedles: Take gelatin, add PBS buffer, heat in a water bath to dissolve, and prepare a gelatin solution. After the gelatin solution returns to room temperature, add Newcastle disease virus, blow and mix well to obtain a mixture. Immerse GelMA porous microneedles in the mixture overnight. After protection from light, freeze dry for 4-6 hours to encapsulate Newcastle disease virus. Weigh 0.2 g of gelatin powder, add 10 mL of PBS buffer, heat in a 65°C water bath, and shake and mix until the particles dissolve to obtain a 20% gelatin solution. After the 20% gelatin solution returns to room temperature, add 1 MOI of NDV, gently blow it to mix, immerse the microneedles prepared by lyophilized GelMA porous microneedles in the mixture overnight, then wrap them in aluminum foil and freeze-dry them in a freeze dryer for 4-6 hours to encapsulate Newcastle disease virus (NDV).

2. The method for preparing the Newcastle disease virus microneedle delivery system according to claim 1, characterized in that, Mix polyethylene glycol diacrylate and 2-hydroxy-2-methyl-1-phenyl-1-propanone in a ratio of 100:1; and / or, A mold with a hydrophobic surface is obtained by performing a 20-minute surface treatment in a DT-01 low-temperature plasma treatment system; and / or, The dichloromethane and silane are mixed in a 100:1 ratio and then placed in a container; and / or, The drying process involves placing the item in a constant temperature oven at 60-65℃ overnight; the static ventilation process involves placing the item in a fume hood for 30-60 minutes; and / or... The mass-to-volume ratio of the gelatin, anhydrous sodium carbonate, and ultrapure water is 20 g: 10 g: 200 mL; and / or, The water bath mentioned in steps (I) and (II) is a 50°C hot water bath; and / or, The conical flask is a conical flask equipped with a type A olive magnetic stirring rotor; and / or, Wrap the conical flask with aluminum foil, and slowly add 4 mL of methacrylic anhydride dropwise into the flask, completing the addition within 3 minutes; and / or, After adding methacrylic anhydride, the mixture was stirred continuously in a water bath for 1 hour. During the reaction, the pH was adjusted to 9 every 10 minutes using a 10% (w / v) sodium hydroxide solution, repeated 6 times; and / or, During dialysis, the water was changed 20 times, averaging once every 2 hours; and / or, The water bath mentioned in step (III) is a 65°C hot water bath.

3. The method for preparing the Newcastle disease virus microneedle delivery system according to claim 1, characterized in that, The mass-to-volume ratio of gelatin, PBS buffer, and Newcastle disease virus is 0.2 g: 10 mL: 1 MOI.

4. The method for preparing the Newcastle disease virus microneedle delivery system according to claim 1, characterized in that, The water bath mentioned in step (2) is a 65°C hot water bath.

5. A Newcastle disease virus microneedle delivery system, prepared by the preparation method according to any one of claims 1-4.

6. The application of the Newcastle disease virus microneedle delivery system according to claim 5, characterized in that, Application in the preparation of anti-liver cancer drugs.

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