CN113274560A

CN113274560A - Preparation method of anti-tumor composite material bracket loaded with 5-FU

Info

Publication number: CN113274560A
Application number: CN202110590593.1A
Authority: CN
Inventors: 朱沛志; 张思宸
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2021-05-28
Filing date: 2021-05-28
Publication date: 2021-08-20

Abstract

The invention relates to a preparation method of a 5-FU-loaded anti-tumor composite material scaffold in the technical field of composite materials. The preparation method comprises the following steps of uniformly mixing 0.5-2% by mass of fluorouracil (5-FU) powder and 98-99.5% by mass of Polycaprolactone (PCL) granules, extruding the mixture into wires with uniform diameters by an extruder, cooling the wires, and preparing the wires into the required composite material bracket by using a 3D printer; the fluorouracil and polycaprolactone composite material stent prepared by the method can fill the vacant part after the bone tumor is cut off in vivo, and the sustained-release fluorouracil kills the residual tumor cells, thereby being beneficial to treatment.

Description

Preparation method of anti-tumor composite material bracket loaded with 5-FU

Technical Field

The invention relates to the technical field of composite materials, in particular to a preparation method of a 5-FU-loaded anti-tumor composite material stent.

Background

Pentafluorouracil (5-FU), which is a pyrimidine fluoride, is a white or off-white crystal or crystalline powder, and belongs to anti-metabolism and anti-tumor drugs. 5-FU inhibits DNA synthesis by inhibiting thymidylate synthase. The action of this enzyme may transfer one carbon unit of formyltetrahydrofolate to deoxyuridine-phosphate to synthesize thymidine monophosphate. 5-FU also has a certain inhibitory effect on RNA synthesis. 5-fluorouracil can be injected intravenously or intracavity. 5-fluorouracil can generate F-dUMP and FUMP through different routes. The former can be covalently bound to the active center of thymidylate synthase, inhibiting the activity of thymidylate synthase, causing deoxynucleotide deficiency and DNA synthesis disorder. In addition, metabolites of 5-FU may also invade RNA and DNA in the form of pseudo metabolites, affect cellular functions, and cause cytotoxicity. 5-FU is an atypical cell cycle-specific drug that acts primarily on cells in other phases in addition to the S phase.

Polycaprolactone (PCL) is an organic high molecular polymer, is a functional polyester, has good biocompatibility, good organic polymer compatibility and good biodegradability, and can be used as a support material for cell growth. It is well compatible with biological cells in vivo, and cells can grow normally on their scaffolds and can be degraded into CO2 and H2O. Meanwhile, the material has good shape memory and temperature control properties, is widely applied to the fields of drug carriers, biomedical materials and the like, and also plays a great role in the field of bone tissue engineering.

Osteosarcoma is the most common malignant tumor of bones, and is a disease with high malignant tumor death rate in children and adolescents. It develops from a mesenchymal cell line and the rapid growth of tumors is due to the direct or indirect formation of tumor bone-like and bone tissue by the tumor through the cartilage stage. Common osteosarcomas originate from intraosseous or connective tissue. The latter is less frequent and the prognosis is slightly better.

In the prior art, after pathological diagnosis of osteosarcoma, i.e. before the start of chemical or radioactive therapy, resection of tumor tissue is an important step in osteosarcoma therapy. After removal of the tumor tissue, post-operative chemical or radioactive treatment is also required to treat residual tumor cells at the tumor growth site, which not only increases the physical burden on the patient, but also makes the surgical prognosis difficult. The invention is indicated to be urgently needed to invent an implant which can fill the vacant part after the bone tumor is removed, can kill the residual bone tumor cells and has corresponding mechanical property.

Disclosure of Invention

The invention provides a preparation method of a 5-FU-loaded anti-tumor composite material bracket, aiming at the problems that the vacant part behind the bone tumor can not be filled, and the tumor cells are remained at the tumor growth part, which is not beneficial to treatment in the prior art.

The invention aims to realize the preparation method of the 5-FU-loaded anti-tumor composite material scaffold, which comprises the following specific steps: uniformly mixing 0.5-2% of fluorouracil (5-FU) powder and 98-99.5% of Polycaprolactone (PCL) granules, extruding the mixture into wires with uniform diameter by an extruder, cooling the wires, and preparing the wires into the required composite material bracket by using a 3D printer.

The 5-FU/PCL (methotrexate-polycaprolactone) composite material anti-tumor stent prepared by adopting a 3D printing technology and taking the pentafluorouracil powder and the medical-grade polycaprolactone as raw materials can slowly release a medicament in vivo to inhibit the growth of cancer cells, and the stent has mechanical properties matched with a loose bone. Due to good biodegradability, polycaprolactone has lower cytotoxicity when degraded in vivo.

Further, the method specifically comprises the following steps:

(1) grinding polycaprolactone: grinding polycaprolactone by a grinder, collecting the crushed polycaprolactone by a sieve for later use, and then collecting fluorouracil (5-FU) powder by a sieve for later use;

(2) mixing raw materials: uniformly mixing 0.5-2% of the polycaprolactone crushed in the step 1) with 98-99.5% of fluorouracil (5-FU) powder in percentage by mass, pouring the mixture into a feed inlet of an extruder, melting and extruding the material by the extruder to obtain a wire rod, and cooling the wire rod with water;

(3) 3D prints support: and (3) loading the wire rod prepared in the step 2) into a 3D printer, adjusting the printing parameters of the 3D printer, and starting printing after the wire rod is led into the three-dimensional model of the support to obtain the composite material support.

Further, the molecular weight of the polycaprolactone is 4-6 ten thousand, the intrinsic viscosity is 1-3 dL/g, the larger the molecular weight of the polycaprolactone is, the larger the crystallization rate is, the larger the melt viscosity of the system is, but the overlarge molecular weight can cause the difficulty in molecular diffusion, and the slow movement of a molecular chain from an overheated melt to a crystal growth surface can limit the crystallization rate.

Further, the sieving granularity is 100-200 meshes.

Further, the diameter range of the wire rod is 1.7-1.8 mm.

Further, in the step (2), the water cooling temperature is set to be 10-20 ℃.

Further, in the step (2), the extrusion temperature of the extruder is 70-75 ℃, and the rotation speed is 50 r/min.

Further, the printing parameters are that the temperature of the spray head is 70-75 ℃, the temperature of the printing bottom plate is 25-35 ℃, the printing speed is 15-25 mm/s, and the rotating speed of the cooling fan is 1500 rpm.

Drawings

FIG. 1 is a diagram of a sample of 5-FU/PCL composite antitumor scaffold prepared in example 1.

FIG. 2 is a graph showing the results of the CCK8 susceptibility test for the 5-FU/PCL composite prepared in example 1.

FIG. 3 is a graph showing the results of one day of growth of MG-63 (osteosarcoma) cells in three different environments.

FIG. 4 is a graph showing the results of experiments in which MG-63 (osteosarcoma) cells were grown in three different environments for three days.

FIG. 5 is a graph comparing the in vitro release results of 5-FU/PCL composites prepared in example 1, example 2 and example 3.

Detailed Description

Example 1 (1% by weight: 99% by weight of a 5-FU/PCL composite material holder)

Pentafluorouracil (5-FU), purchased from Meilunbio, used in the following examples; medical-grade PCL plastic is an imported medical-grade material, is purchased from Nature Works company, and is certified by FDA.

Grinding polycaprolactone by a grinder, sieving the crushed polycaprolactone by a 200-mesh screen, collecting the crushed polycaprolactone for later use, collecting powder by a 200-mesh screen with fluorouracil (5-FU) powder, respectively taking 1% of pentafluorouracil (5-FU) and 99% of PCL by mass percent, uniformly mixing to obtain a mixed material, then putting the mixed material into a feed inlet of an extruder, setting the melting temperature of the extruder to 73 ℃, the rotating speed to 50r/min, the extrusion diameter to 1.7mm, melting and extruding the material in the extruder to obtain a wire rod, cooling the wire rod by water at 20 ℃, drawing to obtain the wire rod, then loading the wire rod into a 3D printer, adjusting the printing parameters of the 3D printer, setting the printing parameters to be the nozzle temperature to 73 ℃, the printing bottom plate temperature to 30 ℃ and the printing speed to be 20 mm/s, and the rotating speed of the cooling fan is 1500 rpm, and printing is started after the three-dimensional model of the bracket is led in to obtain the composite bracket.

Example 2 (0.5% by weight: 99.5% by weight of a 5-FU/PCL composite material holder)

Grinding polycaprolactone by a grinder, sieving the crushed polycaprolactone by a 100-mesh screen, collecting the crushed polycaprolactone for later use, then collecting powder by sieving fluorouracil (5-FU) powder by a 100-mesh screen, respectively taking 0.5% of pentafluorouracil (5-FU) and 99.5% of PCL by mass percent, uniformly mixing to obtain a mixed material, then pouring the mixed material into a feed inlet of an extruder, setting the melting temperature of the extruder to be 70 ℃, the rotating speed to be 50r/min, the extrusion diameter to be 1.75mm, melting and extruding the material in the extruder to obtain a wire rod, cooling the wire rod by water at 10 ℃, drawing to obtain the wire rod, then loading the wire rod into a 3D printer, adjusting the printing parameters of the 3D printer, setting the nozzle temperature to be 70 ℃, the printing bottom plate temperature to be 20 ℃, and the printing speed to be 15 mm/s, and the rotating speed of the cooling fan is 1500 rpm, and printing is started after the three-dimensional model of the bracket is led in to obtain the composite bracket.

Example 3 (2% by weight: 98% by weight of a 5-FU/PCL composite material holder)

Grinding polycaprolactone through a grinder, sieving the crushed polycaprolactone through a 200-mesh screen, collecting the crushed polycaprolactone for later use, collecting powder by passing fluorouracil (5-FU) powder through a 200-mesh screen, respectively taking 2% of pentafluorouracil (5-FU) and 98% of PCL by mass percent, uniformly mixing to obtain a mixed material, then pouring the mixed material into a feed inlet of an extruder, setting the melting temperature of the extruder to be 75 ℃, the rotating speed to be 50r/min, the extrusion diameter to be 1.8mm, melting and extruding the material in the extruder to obtain a wire rod, cooling the wire rod through 20 ℃ water, drawing the wire rod to obtain the wire rod, then loading the wire rod into a 3D printer, adjusting the printing parameters of the 3D printer, setting the printing parameters to be 75 ℃ of a spray head, 35 ℃ of a printing bottom plate and 25 mm/s of printing speed, and the rotating speed of the cooling fan is 1500 rpm, and printing is started after the three-dimensional model of the bracket is led in to obtain the composite bracket.

FIG. 1 is a sample diagram of the 5-FU/PCL composite material anti-tumor scaffold prepared in example 1, and it can be seen that the pores exist in the anti-tumor scaffold, the pore diameter is about 300 microns, and the existence of the porous pores accelerates the degradation of polycaprolactone in human body and also reduces the difficulty of drug release. And the pores provide convenience for the generation of new bone at the later stage. The support sample is not limited to be in a disc shape, and the specific shape can be automatically regulated according to CT data.

FIG. 2 is a graph showing the result of CCK8 (Cell Counting Kit-8 Cell Counting reagent) drug sensitivity test of 5-FU/PCL composite scaffold with 1% of pentafluorouracil prepared in example 1. MG-63 (osteosarcoma) cells were grown in three different environments for one, three and five days. As can be seen from the figure, MG-63 (osteosarcoma) cells could not grow normally in the 5-FU/PCL stent group, and the 5-FU/PCL anti-tumor stent slowly releases the drug to allow large area death of MG-63 cells.

FIGS. 3 and 4 are graphs showing the Live-Dead experimental results of the 5-FU/PCL composite scaffold with 1% of pentafluorouracil prepared in example 1. FIG. 3 is a graph showing the results of one day experiment of MG-63 (osteosarcoma) cells in three different environments, and FIGS. 3 (a), 3 (b) and 3 (c) show the growth of MG-63 (osteosarcoma) cells in pure nutrient solution, MG-63 (osteosarcoma) cells in PCL-containing nutrient solution and MG-63 (osteosarcoma) cells in the first day in 5-FU/PCL-containing nutrient solution, respectively, from which it can be seen that the growth density of MG-63 (osteosarcoma) cells in pure nutrient solution and MG-63 (osteosarcoma) cells in PCL-containing nutrient solution is higher in the same microscope, indicating that the cell growth is good, and FIG. 3 (c) shows that the growth density of MG-63 (osteosarcoma) cells in 5-FU/PCL-containing nutrient solution is low in the first day and the production is poor, FIG. 4 is a graph of the results of three days of experiments in which MG-63 (osteosarcoma) cells were grown in three different environments. FIG. 4 is a graph showing the results of one day experiment of MG-63 (osteosarcoma) cells grown in three different environments, and FIGS. 4 (a), 4 (b) and 4 (c) show the growth of MG-63 (osteosarcoma) cells in a pure nutrient solution, MG-63 (osteosarcoma) cells in a PCL-containing nutrient solution and MG-63 (osteosarcoma) cells in a 5-FU/PCL-containing nutrient solution on the third day, respectively, from which it can be seen that the growth density of MG-63 (osteosarcoma) cells in a pure nutrient solution and MG-63 (osteosarcoma) cells in a PCL-containing nutrient solution becomes larger than that of FIG. 3 under the same microscope, indicating benign growth of the cells, and FIG. 4 (c) shows that the growth density of MG-63 (osteosarcoma) cells in a 5-FU/PCL-containing nutrient solution on the third day becomes severely smaller, this is due to the slow release of pentafluorouracil, and MG-63 (osteosarcoma) cells were killed.

FIG. 5 is a graph comparing the results of in vitro release of 5-FU/PCL prepared in example 1, example 2 and example 3. And (3) respectively taking 1-2 printed sample supports with three concentrations into a centrifuge tube, then respectively adding 10ml of PBS buffer solution, fully oscillating, shaking up, marking, and then centrifuging. After standing for one day, ultraviolet tests are respectively carried out, whether the corresponding absorption peaks are normal or not is observed, the ultraviolet tests are carried out once every other day, the in vitro release result is shown in figure 4 after the 14 th day is detected. From the obtained sustained release curve, the 5-FU/PCL drug scaffolds with different content of pentafluorouracil prepared in the three examples have a certain burst release after several days, and then gradually release, and show a slow rising trend. The 5-FU/PCL drug stent with 0.5 percent of pentafluorouracil prepared in the example 2 has partial burst release on the third day, and then gradually becomes gentle, but still has a rising trend, and the whole release effect is general; the 5-FU/PCL drug stent with 1.0% of pentafluorouracil prepared in the example 1 can be released suddenly on the third day and then gradually, and starts to be released slowly after the fifth day, so that the stent has an obvious ascending trend and a better in-vitro release effect; the 5-FU/PCL drug scaffold with 2.0% of pentafluorouracil prepared in example 3 exhibited a large burst release beginning at the third day, gradually slowed down by the seventh day and then slowly released, and the overall release effect was improved over both samples. The in vitro release effect of the 5-FU/PCL medicament bracket is entirely satisfactory, when a tumor patient is clinically treated, the 5-FU/PCL medicament bracket firstly has certain burst release in the patient body, then slowly releases the anti-tumor medicament gradually, the concentration of the anti-tumor medicament can be gradually increased within a certain time, the proliferation of tumor cells is inhibited, and the good anti-tumor effect can be realized.

In conclusion, the 5-FU/PCL composite material has good anti-tumor property and mechanical property, can adapt to the complex body fluid environment in the human body to effectively control and kill tumor cells, and can resist strong external impact. The dosage of the pentafluorouracil can be regulated and controlled according to medical advice and individual conditions, so that the burden of postoperative drug chemotherapy of patients is relieved.

Claims

1. The preparation method of the 5-FU-loaded anti-tumor composite material stent is characterized by uniformly mixing 0.5-2 mass percent of fluorouracil powder and 98-99.5 mass percent of polycaprolactone granules, extruding the mixture into wires with uniform diameters by an extruder, cooling the wires, and preparing the required composite material stent by using a 3D printer.

2. The method for preparing the 5-FU-loaded anti-tumor composite material scaffold according to claim 1, comprising the following steps:

3. The preparation method of the 5-FU-loaded anti-tumor composite material scaffold according to claim 2, wherein the molecular weight of the polycaprolactone is 4-6 ten thousand, and the intrinsic viscosity is 1-3 dL/g.

4. The preparation method according to claim 2, wherein in the step (1), the sieving granularity is 100-200 meshes.

5. The method for preparing a 5-FU-loaded anti-tumor composite material scaffold according to claim 4, wherein in the step (2), the diameter of the wire is in the range of 1.7-1.8 mm.

6. The production method according to claim 2, wherein in the step (2), the water cooling temperature is set to 10 to 20 ℃.

7. The method for preparing the 5-FU-loaded anti-tumor composite material scaffold according to claim 6, wherein in the step (2), the extrusion temperature of the extruder is 70-75 ℃, and the rotation speed is 50 r/min.

8. The method for preparing the 5-FU-loaded anti-tumor composite material scaffold according to claim 7, wherein in the step (3), the printing parameters are that the temperature of the spray head is 70-75 ℃, the temperature of the printing bottom plate is 25-35 ℃, the printing speed is 15-25 mm/s, and the rotating speed of the cooling fan is 1500 rpm.