A 3D bioprinted engineered green algal living material and method of making the same

Abstract

The application discloses a kind of 3D biological printing engineering green algae active material and preparation method thereof, active material includes the composite layer of a plurality of laminated settings, the composite layer includes: carrier layer, and its above biological ink layer;The mass ratio of carrier layer and biological ink layer is 1:1-1:2;The biological ink layer includes: hydrogel, culture medium and upconversion nanomaterial modified green algae cell;The emission wavelength of upconversion nanomaterial is in the excitation wavelength range of green algae chloroplast.This 3D biological printing engineering green algae active material of the application contains upconversion nanomaterial modified green algae cell, under near infrared light condition, upconversion nanomaterial can be excited to visible light by near infrared light, for green algae photosynthesis to produce oxygen;Chlorophyll a (photosensitizer) in green algae can be excited to excited state by the above visible light, and react with the above oxygen, produce reactive oxygen species (such as singlet oxygen 1 O2), induce cancer cell death.

Description

Technical Field

[0001] The present invention relates to the field of anti-tumor technology, and in particular to a 3D bio-printed engineered green algae active material and a preparation method thereof. Background Art

[0002] Photodynamic therapy (PDT) is a treatment technology based on photochemical reaction. Its basic elements are oxygen, photosensitizer and light: under the action of excitation light of a specific wavelength, the photosensitizer changes from the ground state to the excited state, and then reacts with oxygen molecules to produce reactive oxygen species (such as singlet oxygen 1 O2) interacts with nearby biomolecules (such as proteins, lipids, or DNA) to produce toxic photochemical products, which in turn induce cell apoptosis and local microvascular damage. As a photosensitive therapeutic technology different from traditional cancer treatments such as surgery, radiotherapy, and chemotherapy, PDT has the advantages of excellent physical targeting, no drug resistance, minimal trauma, and a short duration of action. It has been widely used in the clinical treatment of diseases such as superficial bladder cancer, lung cancer, esophageal cancer, and skin cancer.

[0003] However, most of the currently clinically approved photosensitizers use visible light as the excitation light source, and their tissue penetration ability is limited, which restricts the application of PDT in the treatment of deep tumors. In addition, malignant tumors are very likely to develop a hypoxic state due to abnormal vascular function and insufficient blood supply during their uncontrolled growth. Hypoxia is an important feature of most solid tumors, especially tumor areas that are far away from blood vessels. As a photosensitivity reaction involving oxygen, the efficacy of PDT is strongly dependent on the oxygen content in the tissue, resulting in very limited efficacy of PDT on hypoxic tumors. In addition, the large amount of oxygen consumption during the PDT process and the blockage of oxygen supply to the tumor site due to tumor vascular damage will further reduce the efficacy of PDT. Therefore, it is urgent to provide a material suitable for PDT treatment of hypoxic tumors to improve the clinical efficacy of PDT on deep, hypoxic tumors. Summary of the Invention

[0004] The purpose of the present invention is to provide a 3D bioprinted engineered green algae active material to solve the technical problem that existing PDT technology is difficult to treat hypoxic tumor areas.

[0005] To achieve the above objectives, the present invention provides a 3D bio-printed engineered green algae active material, which includes several stacked composite layers, wherein the composite layers include: a carrier layer and a bio-ink layer thereon; the mass ratio of the carrier layer to the bio-ink layer is 1:1-1:2; the bio-ink layer includes: a hydrogel, a culture medium, and green algae cells modified with an upconversion nanomaterial; wherein the emission wavelength of the upconversion nanomaterial is within the excitation wavelength range of the green algae cell chloroplasts.

[0006] Preferably, the upconversion nanomaterial is amination-modified NaYF4:Yb / Er.

[0007] Preferably, the green algae cells include at least one of Chlorella cells, Spirulina cells, and Microcystis aeruginosa cells.

[0008] Preferably, the mass ratio of the upconversion nanomaterial to the green algae cells is 1:25-1:10.

[0009] Preferably, the hydrogel is made of at least one of methacrylated gelatin, sodium alginate, and hyaluronic acid.

[0010] Preferably, the carrier layer is made of polycaprolactone and / or polylactic acid.

[0011] The present invention also provides a method for preparing the above-mentioned 3D bioprinted engineered green algae active material, which comprises the following steps:

[0012] Step 1, preparing a bio-ink precursor: mixing the hydrogel and the culture medium uniformly to obtain a hydrogel-culture medium mixture; filtering the hydrogel-culture medium mixture through a 0.22 μm microporous filter membrane to sterilize it, and adding green algae cells to obtain a bio-ink precursor; wherein the mass ratio of the hydrogel to the culture medium is 1:5-1:50; the final concentration of the green algae cells is 0.1-10×10 7 pcs / ml;

[0013] Step 2, 3D printing of the bio-ink layer and the carrier layer: adjusting the viscosity of the bio-ink precursor; loading the carrier material and the bio-ink precursor into two barrels of the 3D printer respectively, and printing the bio-ink layer and the carrier layer alternately layer by layer;

[0014] Step 3: Irradiating the bio-ink layer and the carrier layer to cross-link, thereby obtaining a 3D bio-printed engineered green algae active material.

[0015] Preferably, in step 1, the sterilization method is: filtering and sterilizing the hydrogel-culture medium mixture through a 0.22 μm microporous filter membrane.

[0016] Preferably, in step 2, the viscosity of the bio-ink precursor is 1-20 Pas.

[0017] Preferably, in step 3, the cross-linking method of the biological ink layer and the carrier layer is: immersing the carrier layer and the biological ink layer in a calcium chloride solution, adding a photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, and irradiating with blue light for 10-60s.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The present invention provides a 3D bioprinted engineered green algae active material, which comprises green algae cells modified with upconversion nanomaterials. Under near-infrared light conditions, the engineered green algae active material of the present invention produces the following reaction:

[0020] ① The upconversion nanomaterial is excited by near-infrared light (980nm) to produce visible light (660nm), which is used by green algae to photosynthesize and produce oxygen;

[0021] ② Chlorophyll a (photosensitizer) in green algae is excited by the above visible light and reacts with the above oxygen to produce reactive oxygen species (such as singlet oxygen 1 O2), the active oxygen species then reacts with cancer cells to produce toxic photochemical products, inducing cancer cell apoptosis, thereby solving the technical problem that existing PDT technology is difficult to treat in hypoxic tumor areas.

[0022] (2) The upconversion nanomaterial of the present invention has a synergistic effect with green algae cells. The upconversion nanomaterial of the present invention is amino-modified NaYF4:Yb / Er. The visible light band (660nm) generated by the upconversion nanomaterial after near-infrared light excitation is just suitable for the excitation of chlorophyll a.

[0023] (3) The present invention uses materials such as methacrylated gelatin, sodium alginate, and hyaluronic acid as components of the biological ink layer. These materials have good biocompatibility, and the engineered green algae active material produced will not produce a rejection reaction after being implanted into the body.

[0024] (4) The present invention uses polycaprolactone (PCL) and polylactic acid (PLA) as the carrier layer. These two materials are biodegradable and can be degraded and excreted after implantation into the body without any toxic residue. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Figure A is a field emission transmission electron microscope (TEM) photo of amination NaYF4:Yb / Er at high magnification. Figure 1 Figure B is a field emission transmission electron microscope (TEM) photo of amination NaYF4:Yb / Er at low magnification. Figure 1 Figure C shows the near-infrared absorption spectrum of amination NaYF4:Yb / Er. Figure 1 Figure D is the fluorescence emission (PL) spectrum (λ ex :980nm).

[0026] Figure 2 The near infrared light (980nm) and visible light laser (1.5W cm -2) under the action of engineered Spirulina (1×10 7 cells / ml) oxygen release curve.

[0027] Figure 3 This is a comparison of tumors in C57BL / 6 tumor-bearing mice on the 14th day after different treatments of the present invention. Figure 3 A is a photo of the tumor in C57BL / 6 tumor-bearing mice on the 14th day after different treatments. Figure 3 B is a box plot of tumor weight in C57BL / 6 tumor-bearing mice on the 14th day after different treatments. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below with reference to the embodiments.

[0029] To address the technical challenge of existing PDT technology in treating hypoxic tumor areas, the present invention provides a 3D bioprinted engineered green algae active material. The material comprises a laminated composite layer comprising a carrier layer and a bio-ink layer thereon; the bio-ink layer comprises a hydrogel, a culture medium, and green algae cells modified with an upconversion nanomaterial; the mass ratio of the carrier layer to the bio-ink layer is 1:1-1:2. The emission wavelength of the upconversion nanomaterial is within the excitation wavelength range of the green algae cell chloroplasts.

[0030] Under near-infrared light conditions, the engineered green algae active material of the present invention produces the following reactions:

[0031] ① Upconversion nanomaterials are stimulated by near-infrared light to produce visible light, which is used by green algae to photosynthesize and produce oxygen;

[0032] ② Chlorophyll a (photosensitizer) in green algae is excited by the above visible light and reacts with the above oxygen to produce reactive oxygen species (such as singlet oxygen 1 O2), the reactive oxygen species then react with cancer cells to produce toxic photochemical products, inducing cancer cell apoptosis.

[0033] The green algae cells of the present invention can be Chlorella cells, Spirulina cells, Microcystis aeruginosa cells, etc. Experiments have shown that these cells can be modified with upconversion nanomaterials and produce reactive oxygen species under near-infrared light conditions.

[0034] In some embodiments, the green algae cells are Spirulina cells. To enable visible light excitation of chlorophyll a (a photosensitizer) within the green algae to an excited state, numerous upconversion nanomaterials were screened, ultimately identifying ammoniated NaYF4:Yb / Er, whose emission wavelength coincides with the excitation wavelength of chlorophyll a (a photosensitizer). Consequently, ammoniated NaYF4:Yb / Er was selected as the upconversion nanomaterial for modifying Spirulina.

[0035] like Figure 1 As shown in Figures A and B, field emission transmission electron microscope (TEM) photos of amination NaYF4: Yb / Er are shown. It can be seen from the figures that amination NaYF4: Yb / Er is a spherical structure with uniform particle size distribution and a size of about 25 nm. Figure 1 Figure C is the near-infrared absorption spectrum of amination NaYF4:Yb / Er. It can be seen from the figure that amination NaYF4:Yb / Er can strongly absorb near-infrared light around 980nm. Figure 1 Figure D shows the fluorescence emission (PL) spectrum of amination NaYF4:Yb / Er (λ ex :980nm), it can be seen from the figure that under 980nm light excitation, the amino NaYF4:Yb / Er produces strong emission spectra at 654nm and 660nm, and the emission spectrum is consistent with the visible light absorption spectrum of chlorophyll a.

[0036] Furthermore, the engineered green algae active material of the present invention can be made into a small-sized substance (about 1-2 mm in length, width and height) through 3D printing, and then implanted into the patient's tumor to induce tumor cell apoptosis.

[0037] Example

[0038] Preparation of Spirulina cells modified with upconversion nanomaterials

[0039] The spirulina used in this example was purchased from Shanghai Guangyu Biotechnology Co., Ltd., and the upconversion nanomaterial (aminated NaYF4: Yb / Er) was purchased from Hangzhou Fuwo Nanotechnology Co., Ltd.

[0040] Synthesis of Spirulina cells modified with upconversion nanomaterials: 10 mL of Spirulina (concentration of 1×10 7 / mL) was added with 600 μL of upconversion nanomaterial (concentration of 1 mg / mL), stirred in the dark at room temperature for 6 h, centrifuged (4000 rpm, 10 min), and then rinsed with PBS to obtain Spirulina cells modified with upconversion nanomaterials for later use.

[0041] The above experiment utilized the electrostatic interaction between the positive potential of amino-modified NaYF4:Yb / Er and the negative potential of the Spirulina cell surface to modify the upconversion nanomaterial on the Spirulina cell surface.

[0042] like Figure 2 As shown, under the excitation of visible light and near-infrared light, Spirulina cells modified with upconversion nanomaterials can carry out photosynthesis to produce oxygen.

[0043] Preparation of bio-ink precursors and support materials

[0044] Carrier material synthesis: polycaprolactone and polylactic acid particles are mixed evenly to obtain the carrier material.

[0045] Bio-ink precursor synthesis: At 50°C, the hydrogel was dissolved in Zarrouk medium (Shanghai Guangyu Biotechnology Co., Ltd.) to obtain a hydrogel-medium mixture. The mass ratio between the hydrogel and the medium was 1:5-1:50. After cooling to 37°C, the mixture was sterilized by filtration using a 0.22 μm microporous filter membrane. Then, 5×10 7 Spirulina modified with upconversion nanomaterials was mixed to a final concentration of 0.1-10×10 7 / mL to obtain the bio-ink precursor.

[0046] The hydrogel of this embodiment is prepared using methacrylated gelatin (GelMa, commercially available), sodium alginate (SA, commercially available), and hyaluronic acid (HA, commercially available). The mass ratio of hyaluronic acid, sodium alginate, and methacrylated gelatin is 1:1:18-1:9:10. The use of these three materials has the following two advantages:

[0047] (1) Methacrylated gelatin, sodium alginate, and hyaluronic acid have good biocompatibility. After being mixed with green algae cells, they can be used as 3D printed bio-inks and implanted into the body without causing rejection reactions.

[0048] (2) Due to the poor formability of hydrogels, they need to be cross-linked with a carrier to support the formation. Methacrylated gelatin, sodium alginate, and hyaluronic acid are photocurable materials. By adding a photoinitiator (blue light initiator: phenyl (2,4,6-trimethylbenzoyl) lithium phosphate), they can be cross-linked with the carrier after irradiation with blue light.

[0049] Preparation of 3D bioprinted engineered green algae active materials

[0050] 3D model design: Design a 3D printing 3D model using AutoCAD software, import the 3D model into the CAM system, and set the printing path so that the bio-ink layer and the carrier layer are printed alternately.

[0051] Carrier layer printing: The carrier material is loaded into a 150μm metal barrel of a 3D printer, melted at 85°C, and then 3D printed and extruded at 80°C and 600kPa.

[0052] Bio-ink layer printing: The bio-ink precursor was loaded into a 150 μm polytetrafluoroethylene cartridge of a 3D printer and the bio-ink layer was printed at room temperature and 50 kPa.

[0053] Among them, the bio-ink precursor should be adjusted to an appropriate viscosity (1-20 Pas) before 3D printing. If the viscosity of the bio-ink precursor is too high, Zarrouk's medium or hyaluronic acid can be added in an appropriate amount. If the viscosity is too low, methacrylated gelatin can be added in an appropriate amount.

[0054] Crosslinking of the carrier layer and the bio-ink layer: The alternately printed carrier layer and the bio-ink layer were immersed in a calcium chloride solution (5%, w / v), and the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate was added. The samples were crosslinked under blue light at room temperature for 10-60 seconds, washed three times with PBS solution, and placed in Zarrouk's medium for later use.

[0055] The following experimental examples illustrate the tumor-inhibiting effect of the 3D bioprinted engineered green algae active material of the present invention.

[0056] Experimental example

[0057] Eighteen C57BL / 6 tumor-bearing mice (MC38 transplanted tumors) were randomly divided into three groups, with six mice in each group.

[0058] Group 1 (3D bioprinted scaffold group): 3D printed scaffolds containing only carrier layer materials were punctured and implanted into the mouse tumor.

[0059] The second group (3D bioprinted green algae active scaffold group): 3D bioprinted scaffolds containing a carrier layer and a bio-ink layer were punctured and implanted into the mouse tumor, where the green algae cells in the bio-ink layer were not modified with upconversion nanomaterials.

[0060] The third group (3D bioprinted engineered green algae active scaffold group): The scaffold made of the 3D bioprinted engineered green algae active material of the present invention was punctured and implanted into the mouse tumor.

[0061] On the 1st, 2nd and 3rd day of treatment, the power density was 1.5W / cm 2 The three groups of mice were irradiated with 980nm near-infrared light for 20 minutes. The body weight of the mice was recorded every other day, the tumor volume was measured with a vernier caliper, and gross photos of the mice and the tumor area were taken.

[0062] like Figure 3Figure 2 shows a comparison of tumor volumes in C57BL / 6 tumor-bearing mice on day 14 after different treatments. It can be seen that tumor growth in the third group of mice was significantly inhibited, demonstrating that the engineered green algae active material produced by 3D bioprinting has a significant tumor-suppressing effect.

[0063] In summary, the present invention provides a 3D bioprinted engineered green algae active material, which comprises green algae cells modified with upconversion nanomaterials. Under near-infrared light conditions, the upconversion nanomaterials will be excited to emit visible light for the green algae to produce oxygen through photosynthesis; the chlorophyll a (photosensitizer) in the green algae is excited to an excited state by visible light, and reacts with oxygen to produce reactive oxygen species (such as singlet oxygen). 1 O2), the active oxygen species then reacts with cancer cells to produce toxic photochemical products, inducing cancer cell apoptosis, thereby solving the technical problem that existing PDT technology is difficult to treat in hypoxic tumor areas.

[0064] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A 3D bioprinted engineered green algae active material, characterized in that: The method comprises a plurality of composite layers stacked together, wherein the composite layer comprises: a carrier layer and a bio-ink layer thereon; the mass ratio of the carrier layer to the bio-ink layer is 1:1-1:2; The bio-ink layer comprises: a hydrogel, a culture medium, and green algae cells modified with upconversion nanomaterials; wherein the hydrogel comprises: methacrylated gelatin, sodium alginate, hyaluronic acid, and a photoinitiator; the emission wavelength of the upconversion nanomaterial is within the excitation wavelength range of the chloroplasts of the green algae cells; the upconversion nanomaterial is amino-terminated NaYF4:Yb / Er; the green algae cells modified with the upconversion nanomaterials refer to electrostatic interactions between the positive potential of the amino-terminated NaYF4:Yb / Er and the negative potential of the green algae cell surface, thereby modifying the upconversion nanomaterial on the green algae cell surface; the green algae cells comprise at least one of Chlorella cells, Spirulina cells, and Microcystis aeruginosa cells; the carrier layer is made of polycaprolactone and / or polylactic acid, and the bio-ink layer and the carrier layer are cross-linked by blue light irradiation.

2. The 3D bioprinted engineered green algae active material according to claim 1, wherein: The mass ratio of the upconversion nanomaterial to the green algae cells is 1:25-1:

10.

3. The method for preparing the 3D bioprinted engineered green algae active material according to claim 1 or 2, characterized in that: The following steps are involved: Step 1, preparing a bio-ink precursor: mixing the hydrogel and the culture medium uniformly to obtain a hydrogel-culture medium mixture; sterilizing the hydrogel-culture medium mixture, and adding green algae cells modified with upconversion nanomaterials to obtain a bio-ink precursor; wherein the mass ratio of the hydrogel to the culture medium is 1:5-1:50; the final concentration of the green algae cells is 0.1-10×10 7 Pieces / ml; Step 2, 3D printing of the bio-ink layer and the carrier layer: adjusting the viscosity of the bio-ink precursor; loading the carrier material and the bio-ink precursor into two barrels of the 3D printer respectively, and printing the bio-ink layer and the carrier layer alternately layer by layer; Step 3: blue light irradiation is used to cross-link the bio-ink layer and the carrier layer to obtain a 3D bio-printed engineered green algae active material.

4. The method for preparing the 3D bioprinted engineered green algae active material according to claim 3, wherein: The mass ratio of hyaluronic acid, sodium alginate and methacrylated gelatin is 1:1:18-1:9:

10.

5. The method for preparing 3D bioprinted engineered green algae active materials according to claim 3, wherein: In step 2, the viscosity of the bio-ink precursor is 1-20 Pa·s.

6. The method for preparing 3D bioprinted engineered green algae active materials according to claim 3, wherein: In step 3, the cross-linking method of the bio-ink layer and the carrier layer is: immersing the carrier layer and the bio-ink layer in a calcium chloride solution, wherein the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, and irradiating with blue light for 10-60 s.

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