Novel chlorella-carrying 3D printing photo-thermal stent as well as preparation method and application thereof

Abstract

The invention discloses a novel chlorella-loaded 3D printing photo-thermal scaffold and a preparation method and application thereof.According to the novel chlorella-loaded 3D printing photo-thermal scaffold and the preparation method and application thereof, the characteristic that the tumor tissue local anoxic microenvironment of melanoma can be improved through the efficient photosynthetic oxygen release capacity of chlorella MA, so that recurrence and metastasis of the melanoma are more effectively inhibited is utilized, and through multiple tests, the chlorella-loaded 3D printing photo-thermal scaffold has a good application prospect. Chlorella MA and polydopamine (PDA) are reasonably integrated, related parameters are optimized, and the novel chlorella-loaded 3D printing photo-thermal stent prepared by using a microfluidic technology and a biological 3D printing technology has double effects of PTT and oxygen therapy. The chlorella-carrying 3D printing photo-thermal stent provided by the invention is complete in form, can stably release oxygen and has an excellent photo-thermal effect. In-vivo and in-vitro experiments prove that the chlorella-loaded 3D printing photo-thermal scaffold has a certain tumor killing effect and shows the potential of promoting normal cell proliferation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a novel Chlorella vulgaris-loaded 3D-printed photothermal scaffold and a preparation method and application thereof. BACKGROUND

[0002] Melanoma is a highly malignant tumor derived from melanocytes, with strong invasiveness and metastatic potential, induced by various factors such as environment (e.g., excessive sun exposure, ultraviolet radiation), genetics, aging, and decreased immune function, and is considered one of the most deadly types of skin cancer [1,2] . According to the American Journal of Dermatology, about 325,000 people worldwide were diagnosed with melanoma in 2020, and about 57,000 people died from the disease. It is estimated that by 2040, the number of newly diagnosed melanoma cases will increase by more than 50%, and the number of deaths will increase by about 68% [3] . Currently, surgical resection is a widely used and relatively effective treatment strategy for melanoma in clinical practice [4-6] . However, due to incomplete surgical resection, asymptomatic tumor tissue that has not been completely removed continues to remain in the surrounding tissue, gradually growing and spreading to distant sites, and about 80% of patients after resection have tumor recurrence or metastasis [7-9] . Clinically, this problem is mainly addressed through postoperative radiotherapy or chemotherapy. However, radiotherapy and chemotherapy, while killing tumor cells, also damage surrounding healthy tissues and organs, leading to a series of toxic side effects such as skin burns, nausea, vomiting, hair loss, anemia, or immune system damage. Some tumor cells may also develop resistance to chemotherapy drugs, reducing treatment effectiveness or even rendering it completely ineffective [10,11] . At the same time, melanoma resection often results in full-thickness skin defects and open cavities, leading to long recovery times and poor prognosis, making it a major challenge in postoperative management of melanoma. Therefore, there is an urgent need to develop new postoperative adjuvant therapy strategies for melanoma to improve treatment effectiveness, reduce recurrence rates, accelerate postoperative wound healing, and improve patient survival rates and quality of life.

[0003] Photothermal therapy (PTT) has been widely concerned by researchers due to its less invasiveness, high spatiotemporal selectivity and high repeatability. PTT is a kind of minimally invasive treatment method for treating diseases by using photothermal conversion effect. Long-wave radiation can be used to effectively penetrate the lesion to achieve the effect of auxiliary treatment of tumors. Photothermal agent is the core of PTT, which can convert light energy into heat energy under the irradiation of near-infrared (NIR) laser, and has a strong photothermal conversion effect. However, traditional photothermal agents have the disadvantages of phototoxicity, instability and low biocompatibility, which may cause damage to normal tissues after tumor resection, greatly limiting the development of PTT treatment. Therefore, researchers have developed a new type of photothermal agent, polydopamine (PDA). PDA particles, which have similar physicochemical properties to melanin, are self-polymerized from natural polyphenol substance dopamine in marine organisms under weak alkaline conditions. PDA has many unique properties, such as excellent biocompatibility, good biological adhesion, strong photothermal conversion and biological activity, and is considered to be a promising biophotothermal nanoparticle, which is a popular material in current PTT research. However, due to the small size of nanoparticle PDA, how to effectively construct a PDA photothermal conversion system, enhance the efficacy of PTT and repair the damaged wound after surgery is still a research difficulty in the auxiliary photothermal treatment of melanoma after surgery. SUMMARY

[0004] In view of the above, the present application aims to provide a novel chlorella-loaded 3D printed photothermal scaffold and a preparation method thereof. The present application prepares polydopamine (PDA)-chlorella (PDA-MA) through physical adsorption, and then uses microfluidic 3D printing technology to prepare a chlorella-loaded 3D printed photothermal scaffold using PDA-MA, sodium alginate and methacrylated gelatin as raw materials. The chlorella-loaded 3D printed photothermal scaffold is used for auxiliary photothermal treatment of melanoma after surgery, effectively preventing tumor recurrence after surgery.

[0005] The specific technical solutions adopted by the present application are as follows: The first aspect of the present application is to provide a preparation method of a novel chlorella-loaded 3D printed photothermal scaffold, comprising the following steps: (1) Preparation of polydopamine-chlorella (PDA-MA): mix chlorella (MA) solution and Tris-HCl buffer solution, add dopamine hydrochloride powder, shake thoroughly, and take out when the chlorella (MA) solution turns to gray black. Centrifuge and discard the supernatant to obtain engineered chlorella (MA) coated with polydopamine, i.e. polydopamine-chlorella; (2) A mixed solution of polyvinyl alcohol and calcium chloride is prepared as an inner phase, and a mixed dispersion solution of polydopamine-microalgae (PDA-MA), sodium alginate and methacrylated gelatin is prepared as an outer phase, the inner phase and the outer phase are injected into the inner phase inlet and the outer phase inlet of the microfluidic device through a syringe, 3D printing is carried out, and the new 3D-printed photothermal scaffold carrying microalgae is obtained after irradiation under an ultraviolet probe.

[0006] Further, the microfluidic device is a capillary microfluidic device, two cylindrical capillaries with the same specifications are prepared, one is used as an outer phase, and the other is used as an inner phase, the one end of the circular glass capillary is processed into a sharp cone using a tensile instrument, and the inner diameter is polished using sandpaper; the other one is pulled steadily and quickly under the heating of an airbrush, so that the middle part of the capillary becomes a filament-like pipeline; the inner phase capillary is inserted into the outer phase capillary, so that the inner and outer phase capillaries are horizontally and vertically centered, and are placed on a glass slide, the bottom end of the needle is covered to cover the capillary circular port, and the two liquid mixed hardening glue is packaged, and the capillary body is adhered to the glass slide by the two liquid mixed hardening glue.

[0007] Further, the microfluidic device is used to replace the original printing nozzle in the programmable 3D printer to perform 3D printing.

[0008] Further, the concentration of the Tris-HCl buffer solution is 0.15 g / mL.

[0009] Further, the pH value of the Tris-HCl buffer solution is 7.

[0010] Further, the MA solution and the Tris-HCl solution are mixed in a volume ratio of 1:1.

[0011] Further, the addition amount of dopamine hydrochloride is 0.5 mg / mL, and the concentration of the microalgae (MA) solution is 2 mg / mL.

[0012] Further, the concentrations of polyvinyl alcohol and calcium chloride in the polyvinyl alcohol and calcium chloride mixed solution dissolved in ultrapure water are 2% w / v and 0.8% w / v, respectively.

[0013] Further, the concentrations of sodium alginate and methacrylated gelatin dissolved in ultrapure water are 2% w / v and 10% w / v, respectively; and the volume concentration of PDA-MA is 2 × 10 8 million / mL.

[0014] The second aspect of the present application provides a new 3D-printed photothermal scaffold carrying microalgae, which is prepared by the preparation method described above.

[0015] A third aspect of the present invention provides the application of the above-mentioned novel Chlorella-carrying 3D-printed photothermal scaffold for use as an adjuvant therapy for postoperative melanoma treatment.

[0016] The beneficial effects of this invention are as follows: This invention utilizes the highly efficient photosynthetic oxygen release capacity of Chlorella macrophylla (MA) to improve the local hypoxic microenvironment of melanoma tumor tissue, thereby more effectively inhibiting melanoma recurrence and metastasis. Through multiple experiments, Chlorella macrophylla MA and polydopamine (PDA) are rationally integrated, and relevant parameters are optimized. A novel Chlorella macrophylla-loaded 3D-printed photothermal scaffold is prepared using microfluidic technology and bio-3D printing technology, exhibiting both PTT (phototherapy-to-tumor response) and oxygen therapy effects. The Chlorella macrophylla-loaded 3D-printed photothermal scaffold of this invention has a complete morphology, can stably release oxygen, and possesses excellent photothermal effects. In vitro and in vivo experiments have demonstrated that the Chlorella macrophylla-loaded 3D-printed photothermal scaffold has a certain tumor-killing effect, can effectively prevent postoperative tumor recurrence, and shows the potential to promote the proliferation of normal cells. Attached Figure Description

[0017] Figure 1 SEM images of MA (a) and PDA-MA (b) are shown, with a scale bar of 1µm. Figure 2 The growth curve of PDA-MA over 14 days is shown. Figure 3 Representative optical micrographs of an empty 3D-printed scaffold (a) and a 3D-printed photothermal scaffold loaded with Chlorella (b) are shown, with a scale bar of 100 μm. Figure 4 The images shown are representative optical micrographs of an empty 3D-printed scaffold (a) and a 3D-printed photothermal scaffold loaded with Chlorella (b). The images within the dashed boxes are magnified images. The scale bar is 100 μm. Figure 5 The images show digital photographs (top) and optical micrographs (bottom) of Chlorella cultured on 3D-printed photothermal scaffolds for 5 days, with scale bars of 1 mm (top) and 300 μm (bottom). Figure 6 The MA concentration is shown as 1 × 10⁻⁶. 8 - 3 × 10 8 Quantitative analysis of oxygen changes in 3D-printed photothermal scaffolds of Chlorella under different loading conditions (a) and quantitative analysis of oxygen release from 3D-printed photothermal scaffolds of Chlorella under light (ON, yellow) and dark (OFF, gray) conditions (b). Figure 7 The images show thermal imaging of 3D-printed photothermal scaffolds loaded with Chlorella vulgaris under different power and different PDA-MA concentrations. Figure 8808 nm laser power is 0.50, 0.75, 1.00, 1.25, 1.50 w / cm 2 Quantitative analysis of temperature change of Chlorella-loaded 3D-printed photothermal scaffold (a) when 808 nm laser power is 0.50, 0.75, 1.00, 1.25, 1.50 w / cm 8 Quantitative analysis of temperature change of Chlorella-loaded 3D-printed photothermal scaffold (b) when PDA-MA concentration is 4 × 10 8 Quantitative analysis of temperature change of Chlorella-loaded 3D-printed photothermal scaffold (b) when PDA-MA concentration is 4 × 10

[0018] Figure 9 Fluorescence images of HSFs co-incubated with each group of materials under normoxic and hypoxic conditions (1% O2) and double-stained with hypoxia probe [Ru(dpp)3]Cl2 (red) and DAPI (blue), scale bar, 200 μm; Figure 10 Representative fluorescence staining images (a) and CCK-8 quantitative analysis (b) of B16F10 co-incubated with each group of materials under hypoxic conditions (1% O2), scale bar, 200 μm; Figure 11 Infrared images of B16F10 tumor-bearing mice after implantation of PBS, empty 3D-printed scaffold or Chlorella-loaded 3D-printed active photothermal scaffold into tumors and irradiation with 808 nm (1.00 W / cm 2 ) laser for 5 min; Figure 12 Growth of melanoma on the back of B16F10 tumor-bearing mice after different treatments for 10 days. DETAILED DESCRIPTION

[0019] In order to more clearly illustrate the present application, the present application will be further described below in conjunction 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.

[0020] In the following examples, the materials, unless otherwise specified, are commonly used in the art, and can be obtained from commercial channels.

[0021] Example 1 Preparation of polydopamine-Chlorella (PDA-MA) Take 15 g of Tris powder into a 100 mL beaker, then add 100 mL of ultrapure water, and stir with a glass rod until it is completely dissolved. Measure the pH of the above solution with a pH meter, and add dilute HC1 dropwise to the solution to adjust the pH of the solution to 7. The prepared Tris-HCl buffer solution is divided into glass bottles, and the key information such as concentration, pH value, preparation date, etc. is labeled clearly, and stored in a cool and dry place, avoiding direct sunlight and contamination.

[0022] The 10 mL of 1 mg / mL MA solution and the 10 mL of 2 mg / mL MA solution are mixed with 10 mL of Tris-HCl solution in a 1:1 volume ratio, then 10 mg of dopamine hydrochloride powder is added, and the solution is shaken at 50 rpm in a 23°C shaking table for 30 min. After the solution turns gray-black, it is taken out and centrifuged at 5000 rpm for 10 min at room temperature. The supernatant is discarded, and the engineered MA wrapped with polydopamine (PDA-MA) is obtained.

[0023] The engineered MA (PDA-MA) prepared from the 1 mg / mL MA solution, with PDA 1:1 wrapped on the surface of MA; The engineered MA (PDA-MA) prepared from the 2 mg / mL MA solution, with PDA 1:2 wrapped on the surface of MA; Morphology and performance characterization of PDA-MA: After the MA and the prepared PDA-MA are fixed in 4% paraformaldehyde, they are dehydrated with ethanol gradient of concentrations of 30%, 50%, 70%, 90%, and 100%, respectively, for 8 min each time. The surface morphology is characterized by scanning electron microscopy (SEM), as shown in Figure 1 The MA appears spherical under SEM, with no granular attachments on the surface. Due to the freeze-drying treatment, the skin is slightly shriveled. The PDA-MA appears spherical under SEM, with uneven granular protrusions on the surface, indicating that polydopamine is loaded on the surface of MA.

[0024] As shown in Figure 2 , the growth of MA wrapped with different concentrations of PDA is observed within 14 days, and the number of MA is calculated under high-power optical microscope to further explore the activity of PDA-MA. When PDA 1:1 is wrapped on the surface of MA, the growth of MA is severely limited. When PDA 1:2 is wrapped on the surface of MA, the growth of MA is slightly limited, but the overall growth trend and number are similar to those of pure MA.

[0025] Example 2 A new type of Chlorella-loaded 3D printing photothermal scaffold includes the following steps: (1) Preparation of polydopamine-Chlorella (PDA-MA) Take 15 g of Tris powder into a 100 mL beaker, then add 100 mL of ultrapure water, and stir with a glass rod until it is completely dissolved. Measure the pH of the above solution using a pH meter, and adjust the pH of the solution to 7 by adding dilute HC1 dropwise. The prepared Tris-HCl buffer solution is divided into glass bottles, and the key information such as concentration, pH value, preparation date, etc. is labeled clearly, and stored in a cool and dry place, avoiding direct sunlight and contamination.

[0026] Mix 10 mL of 2 mg / mL MA solution and 10 mL of Tris-HCl solution in a 1:1 ratio, then add 10 mg of dopamine hydrochloride powder, and shake at 50 rpm in a 23°C shaking table for 30 min. After the solution turns gray-black, remove it and centrifuge at 5000 rpm for 10 min at room temperature. Discard the supernatant to obtain polydopamine-coated engineered MA (PDA-MA).

[0027] (2) Preparation of microfluidic device Design and prepare a single-hole hollow microfluidic chip, the specific process is as follows: Take two cylindrical capillaries with the same specifications, with an inner diameter of 0.8 mm and an outer diameter of 1 mm. Select one of the capillaries as the outer phase, and stretch its one end in the tube stretcher with the parameters set to form a single-head conical shape. Under a light microscope, use sandpaper to polish the inner diameter to 300-400 μm, and the other end is cut to the appropriate length and polished smooth. Then ultrasonic clean the capillary to remove the debris inside, and dry the capillary. Take another capillary as the inner phase, and pull it steadily and quickly under the heating of the spray gun, so that the middle part of the capillary becomes a filamentous tube. Under a light microscope, measure the inner diameter of the filamentous tube, and mark the position with an inner diameter of 100-150 μm, and cut it off with a capillary knife. Place the treated inner and outer phase capillaries in parallel in the center of the glass slide, with the conical end of the outer phase capillary extending out of the glass slide, and the inner phase capillary inserted into the outer phase capillary, so that it is horizontally and vertically centered in the inner and outer phase capillaries. Use the bottom of the needle and two liquid mixed hardening glue to package the capillary round end and capillary body, and after overnight air drying, ultrasonic clean the debris and store in a dry and dust-free environment for use.

[0028] (3) Preparation of Chlorella-loaded 3D printed photothermal scaffold Use microfluidic-assisted bio-3D printing technology to prepare Chlorella-loaded 3D printed photothermal scaffold, the specific process is as follows: The polyvinyl alcohol and calcium chloride mixed solution was prepared as the inner phase, and the concentration of polyvinyl alcohol and calcium chloride in the inner phase was 2% w / v and 0.8% w / v respectively in ultrapure water; the PDA-MA, sodium alginate and methacrylated gelatin mixed dispersion was prepared as the outer phase, and the concentration of sodium alginate and methacrylated gelatin in the outer phase was 2% w / v and 10% w / v respectively in ultrapure water, and the volume concentration of PDA-MA was 0-10000 million / mL.

[0029] Two 2.5 mL syringes were used to suck the above-mentioned inner and outer phase solutions respectively and were fixed on two infusion pumps, and the syringe needles were connected to one end of two Teflon PFA tubes respectively, and the other end of the Teflon PFA tubes was connected to the inner and outer phase needles of the device. The infusion pump parameters were set to make the solutions in the syringes flow into the microfluidic device at a constant speed, and after the air in the device was discharged, the stable two-phase liquid could be observed to flow out, and the flowing two-phase liquid could be irradiated by an ultraviolet probe to quickly form smooth, homogeneous and tough hollow fibers. In the above process, the hollow fibers underwent two crosslinking reactions, namely the first ion crosslinking between calcium ions and alginate biopolymers, and the second photopolymerization crosslinking of the methacrylated gelatin component under ultraviolet irradiation.

[0030] The microfluidic device was used to replace the original printing nozzle in the programmable 3D printer, and the required wound model after melanoma surgery was established in the 3D Max or C4D software, and was imported into the programmable 3D printer, and various printing data were set. The printer data was adjusted according to the infusion pump and the state of the two-phase liquid, including speed, width and quadrant conversion, etc. When the flow rate of the two-phase fluid completely matched the moving speed of the microfluidic device printing head, the ultrafine hollow fibers of the microfluidic spinning could be stacked layer by layer into a 3D scaffold on a dry culture dish. The in-situ printed hollow scaffold could be picked up with tweezers and maintained its shape in air and liquid. Since the diffusion speed of calcium ions in the biopolymer fluid was slower than the printing speed, when the fiber layers were stacked into a 3D structure, the coagulation of the ultrafine hollow fibers was not complete, which helped the good connection of the fiber struts and the straight channels. After the printing was completed, the scaffold was irradiated under the ultraviolet probe for 30s, and a novel Chlorella-loaded 3D printed photo-thermal scaffold was obtained.

[0031] Example 3 Morphology characterization of Chlorella-loaded 3D printed photo-thermal scaffold The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown in Figure 3 The Chlorella-loaded 3D printed photo-thermal scaffold was prepared by using Example 3 (the volume concentration of PDA-MA in the outer phase was 0) and the Chlorella-loaded 3D printed photo-thermal scaffold The comparison results are shown inFigure 4 The freeze-dried empty 3D-printed scaffold surface was smooth, while the surface of the 3D-printed photothermal scaffold loaded with Chlorella was covered with PDA-MA. It was shown that PDA-MA was successfully loaded on the inner and outer walls of the 3D-printed photothermal scaffold loaded with Chlorella, and the system had high feasibility.

[0032] Example 4 Photosynthetic oxygen release performance test of the 3D-printed photothermal scaffold loaded with Chlorella The 3D-printed photothermal scaffold loaded with Chlorella was prepared by the method of Example 2 The distance between the LED lamp and the Petri dish of the 3D-printed photothermal scaffold loaded with Chlorella was fixed at 10 cm. The light intensity was set to 6000 lux per square meter, and then observation was performed every two days, and the growth of MA in the 3D-printed photothermal scaffold loaded with Chlorella was recorded using an optical microscope. At the same time, the dissolved oxygen content in the culture solution of the 3D-printed photothermal scaffold loaded with Chlorella under different experimental conditions was monitored in real time using a dissolved oxygen detector. In order to evaluate its photosensitivity, the 3D-printed photothermal scaffold loaded with Chlorella was exposed to light under the LED bulb for 60 min, and then immediately placed in a completely dark environment for 60 min, and the whole conversion culture process was repeated for 5 times.

[0033] As shown in Figure 5 , under continuous light (6000 lux), the 3D-printed photothermal scaffold loaded with Chlorella was cultured, and under a microscope, it was observed that MA increased significantly, and on the 5th day, the growth of MA completely covered the inside and outside of the whole sphere, which showed that MA in the 3D-printed photothermal scaffold loaded with Chlorella did not inactivate, and had strong survival and proliferation ability.

[0034] Example 4 Near-infrared photothermal response performance test of the 3D-printed photothermal scaffold loaded with Chlorella The 3D-printed photothermal scaffolds with different PDA-MA concentrations (0 × 10 8 , 1 × 10 8 , 2 × 10 8 , 3 × 10 8 , 4 × 10 8 / mL) were prepared by the method described in Example 2.

[0035] As shown in Figure 6 a, the photosynthetic oxygen release ability of the 3D-printed photothermal scaffold loaded with Chlorella with different PDA-MA concentrations was quantitatively evaluated using a dissolved oxygen detector. When the PDA-MA concentration was 2 × 10 8 / mL, the dissolved oxygen content (DO) reached the highest, which indicated that too little MA in the microspheres might not be able to release enough oxygen, and too much MA might not be conducive to its own growth and metabolism. As shown inFigure 6 When the Chlorella-loaded 3D-printed photothermal scaffold was exposed to sunlight, the scaffold continuously released oxygen, and when the Chlorella-loaded 3D-printed photothermal scaffold was transferred to the dark, the oxygen release amount of the scaffold decreased significantly, indicating that the Chlorella-loaded 3D-printed photothermal scaffold was highly sensitive to light / dark changes and could release more oxygen under light.

[0036] Each group of scaffolds was placed on an insulating stone plate and exposed to 808 nm laser for 5 min under different conditions, and the temperature change and thermal imaging images were recorded in real time by a thermal infrared imager. The specific process was as follows: The 3D-printed photothermal scaffolds prepared in Example 2 were irradiated with 808 nm laser at different power densities (0.50, 0.75, 1.00, 1.25, 1.50 W / cm 2 The 3D-printed photothermal scaffolds prepared in Example 2 were irradiated with 808 nm laser at different power densities (0.50, 0.75, 1.00, 1.25, 1.50 W / cm 8 The 3D-printed photothermal scaffolds prepared in Example 2 were irradiated with 808 nm laser at different power densities (0.50, 0.75, 1.00, 1.25, 1.50 W / cm 8 The 3D-printed photothermal scaffolds prepared in Example 2 were irradiated with 808 nm laser at different power densities (0.50, 0.75, 1.00, 1.25, 1.50 W / cm 8 The 3D-printed photothermal scaffolds prepared in Example 2 were irradiated with 808 nm laser at different power densities (0.50, 0.75, 1.00, 1.25, 1.50 W / cm 8 The 3D-printed photothermal scaffolds prepared in Example 2 were irradiated with 808 nm laser at different power densities (0.50, 0.75, 1.00, 1.25, 1.50 W / cm 8 The 3D-printed photothermal scaffolds prepared in Example 2 were irradiated with 808 nm laser at different power densities (0.50, 0.75, 1.00, 1.25, 1.50 W / cm 2 After determining the optimal power and concentration, the 3D-printed photothermal scaffold was irradiated with 808 nm laser at the power and concentration for 3 min (laser on), and then naturally cooled for 2 min (laser off). The temperature change of the scaffold was recorded in real time by a thermal infrared imager, and the above on / off cycle step was repeated 5 times to detect the photothermal stability of the 3D-printed photothermal scaffold.

[0037] The results are shown in Figure 7 As shown in the results, the near-infrared photothermal responsiveness of the scaffold increased with the increase of the amount of PDA-MA, and the higher the power, the stronger the near-infrared photothermal responsiveness of the scaffold.

[0038] The near-infrared photothermal response performance of the 3D-printed photothermal scaffold was quantitatively analyzed, as shown in Figure 8 After the 3D-printed photothermal scaffold was irradiated with 808 nm laser, the scaffold rapidly increased in temperature and stabilized after 1 min. The 3D-printed photothermal scaffold containing the same concentration of PDA-MA was irradiated for 5 min at different powers: when the power was 0.50 w / cm 2 and 0.75 w / cm 2 , the temperature of the scaffold was lower than 40℃; when the power was 1.00 w / cm 2temperature of the scaffold was between 40-45 °C; when the power was 1.25 w / cm 2 and 1.50 w / cm 2 , the temperature of the scaffold exceeded 45 °C. At the same power, the 3D printed photothermal scaffolds containing different concentrations of PDA-MA were irradiated for 5 min: when the concentration of PDA-MA was 0 × 10 8 , 1 × 10 8 , 2 × 10 8 , 3 × 10 8 , and 4 × 10 8 , the temperature of the scaffold was below 40 °C; when the concentration of PDA-MA was 2 × 10 8 , the temperature of the scaffold was between 40-45 °C; when the concentration of PDA-MA was 3 × 10 8 , 4 × 10 5 , the temperature of the scaffold exceeded 45 °C. The optimal temperature for PTT to kill tumors was between 40-45 °C.

[0039] Example 5 In vitro cell hypoxia relief experiment of 3D printed photothermal scaffolds loaded with Chlorella Human skin fibroblasts (HSF, American Type Culture Collection) were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin double antibiotic. Cells in all experimental groups were incubated in a hypoxic cell incubator (1% O2) as needed to achieve a cell hypoxic state.

[0040] Different groups of 3D printed photothermal scaffolds were prepared, namely Ctrl group: control group, HF group: empty 3D printed scaffold, PDA-MA (dark) group: Chlorella-loaded 3D printed photothermal scaffold (2 × 10 5 , PDA-MA (light) group: Chlorella-loaded 3D printed photothermal scaffold (2 × 10 8 , cultured in light conditions.

[0041] The intracellular hypoxia relief effect of Chlorella-loaded 3D printed photothermal scaffolds was evaluated using a red fluorescent hypoxia indicator {[Ru(dpp)3]Cl2} that can be quenched by oxygen. Normoxic group (cells cultured under normal oxygen content) HSF were placed in 24-well plates (1 × 10 5 cells / well) and continuously incubated in a normoxic incubator for 12 h. Hypoxic group HSF were placed in 24-well plates (1 × 10 5 cells / well) and continuously incubated in a hypoxic environment for 12 h. Subsequently, empty 3D printed scaffolds and Chlorella-loaded 3D printed photothermal scaffolds (2 × 10 8Transwell chambers (cells / mL) were slowly placed into 24-well plates of HSF. Normo- and hypoxic groups were incubated for 12 h under normoxic and hypoxic conditions, respectively. The PDA-MA (light) group was continuously irradiated under a 6000 lux LED lamp for 12 h. After 24 h of incubation under these conditions, the Transwell chambers containing the scaffolds were removed from the 24-well plates, and the cell culture medium was replaced with fresh DMEM containing 8 μg / mL of hypoxic probe. Cells were incubated for another 4 h under normoxic and hypoxic conditions, respectively, and then the DMEM medium containing the hypoxic probe was discarded. Cells were then washed with PBS and fixed in 4% paraformaldehyde. Excess liquid was aspirated from the 24-well plates, and 4',6-diamidinyl-2-phenylindole (DAPI, blue) staining agent was added. Cell staining was observed under an inverted microscope.

[0042] The results are as follows Figure 9 As shown, the hypoxic fluorescence (red) in HSF co-cultured with the PDA-MA (light) group was significantly reduced. This is very close to the results observed under normoxic conditions. The hypoxic fluorescence in HSF co-cultured with the PDA-MA (dark) group was also slightly reduced, while the hypoxic fluorescence in HSF co-cultured with the Ctrl and HF groups remained almost unchanged. These results demonstrate that MA has the ability to release oxygen to alleviate cellular hypoxia. Under light conditions, the oxygen release capacity of MA is significantly enhanced, and it can even reverse the cellular hypoxic state.

[0043] Example 6 In vitro cell killing experiment using a 3D-printed photothermal scaffold loaded with Chlorella Mouse skin melanoma cells (B16F10, ATCC) were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin dual antibiotics. All experimental groups of cells were incubated in a hypoxic cell culture incubator (1% O2) as needed to achieve a hypoxic state.

[0044] Different groups of 3D-printed photothermal scaffolds were prepared: Ctrl group (control group), HF group (empty 3D-printed scaffold), and PDA-MA (dark) group (3D-printed photothermal scaffold loaded with Chlorella vulgaris, 2×10⁻⁶). 8 (cells / mL) were cultured in the dark. PDA-MA (light) group: Chlorella-loaded 3D-printed photothermal scaffold (2×10⁻⁶ cells / mL). 8 Chlorella cells / mL were cultured under light conditions. The PDA-MA (dark) + NIR group carried 2 × 10⁻⁶ Chlorella 3D-printed photothermal scaffolds. 8PDA-MA (light) + NIR group: Chlorella-loaded 3D-printed photothermal scaffold (2 x 10 8 PDA-MA (light) + NIR group: Chlorella-loaded 3D-printed photothermal scaffold (2 x 10

[0045] Mouse skin melanoma cells B16F10 were inoculated in 24-well culture plates (5.0 x 10 4 / well, 500 μL of medium) and incubated overnight in a hypoxic environment. After stable growth of the cells was observed under a microscope, Transwell chambers containing empty 3D-printed scaffolds and chlorella-loaded 3D-printed photothermal scaffolds (2 x 10 8 / well, 500 μL of medium) were slowly placed in the 24-well plates of mouse skin melanoma cells B16F10, and incubation was continued under hypoxic conditions. The PDA-MA (light) group was placed under a 6000 lux LED lamp for continuous irradiation for 12 h, the PDA-MA (dark) + NIR group was irradiated under 808 nm laser for 15 min, and the PDA-MA (light) + NIR group was placed under a 6000 lux LED lamp for continuous irradiation for 12 h and irradiated under 808 nm laser for 15 min. After 24 h, CCK-8 was used to detect tumor cell viability, i.e., an equal amount of CCK-8 reagent was added to each well of the 24-well plates of the cells in each group, the cells were allowed to contact the reagent fully, and the plates were then placed in a hypoxic incubator for continuous incubation for 30 min. Subsequently, an equal amount of CCK-8 reagent was aspirated into a 96-well plate, and the absorbance at 450 nm was measured using a microplate reader to observe the effect of different conditions on the viability of mouse skin melanoma cells B16F10.

[0046] The results are shown in FIG. 6. Figure 10As shown, the mouse skin melanoma cells B16F10 co-cultured with the Ctrl group and the HF group had no obvious changes and grew well. The mouse skin melanoma cells B16F10 co-cultured with the PDA-MA (dark) group and the PDA-MA (light) group showed a slight cell death phenomenon. The melanoma cells co-cultured with the PDA-MA (dark) + NIR group (the Chlorella-loaded 3D-printed photothermal scaffold was downloaded in the dark and irradiated under the 808 nm laser for 15 min) showed a more obvious death phenomenon. The melanoma cells co-cultured with the PDA-MA (light) + NIR group (the Chlorella-loaded 3D-printed photothermal scaffold was downloaded in the light and irradiated under the 808 nm laser for 15 min) showed a large area of death phenomenon, and the dead cells accounted for nearly 80% of the fluorescence field. The above results prove that the Chlorella-loaded 3D-printed photothermal scaffold can be warmed under the action of laser and kill tumor cells by PTT effect. At the same time, the oxygen released by MA can alleviate the hypoxic microenvironment of the tumor and cause further damage to the tumor cells, greatly enhancing the PTT effect.

[0047] Example 7 In vivo anti-tumor test of Chlorella-loaded 3D-printed photothermal scaffold The in situ postoperative mouse melanoma resection model was established by the method of subcutaneously injecting mouse skin melanoma cells B16F10 cells (2 × 10 6 cells in 150 mL PBS) into the right back of 5-7-week-old male C57BL / 6 mice. After 9 days of inoculation (tumor volume ≈ 300 mm 3 ), the mice were randomly divided into 6 groups (5 mice in each group), namely the Ctrl group: the control group, using PBS buffer to treat the melanoma resection site; the HF group: the melanoma resection site was placed with an empty 3D-printed scaffold; the PDA-MA (dark) group: the melanoma resection site was placed with a Chlorella-loaded 3D-printed photothermal scaffold (2 × 10 8 cells / mL) and raised in the dark; the PDA-MA (light) group: the melanoma resection site was placed with a Chlorella-loaded 3D-printed photothermal scaffold (2 × 10 8 cells / mL) and raised in the light; the PDA-MA (dark) + NIR group: the melanoma resection site was placed with a Chlorella-loaded 3D-printed photothermal scaffold (2 × 10 8 cells / mL) and the scaffold was irradiated with an 808 nm laser for 15 min, and then raised in the dark; the PDA-MA (light) + NIR group: the melanoma resection site was placed with a Chlorella-loaded 3D-printed photothermal scaffold (2 × 10 8The mice were treated with an 808 nm laser (number of tumors per mL) and irradiated for 15 min with a single laser, followed by feeding under light conditions. 90% of the mouse tumors were removed, leaving approximately 30 mm² of tumor volume. 3 Mice with residual tumors underwent different treatments as shown above, with empty 3D-printed scaffolds and Chlorella-loaded 3D-printed photothermal scaffolds implanted into the melanoma resection defects. Specifically, mice in the PDA-MA (light) group were continuously irradiated under a 6000 lux LED lamp for 12 hours daily; mice in the PDA-MA (dark) + NIR group were irradiated under an 808 nm laser for 15 minutes daily; and mice in the PDA-MA (light) + NIR group were irradiated under a 6000 lux LED lamp for 12 hours daily, followed by irradiation under an 808 nm laser for 15 minutes. During the experiment, thermal imaging was used to record temperature changes at the implanted scaffold sites in the tumor defect areas of the Ctrl, HF, PDA-MA (dark) + NIR, and PDA-MA (light) + NIR groups. After the experiment, tumor volume and body weight were measured every other day. Tumor volume was recorded using the following formula: 0.5 × major axis × minor axis. 2 .

[0048] Test results: like Figure 11 As shown, after 5 minutes of near-infrared radiation, the temperature of melanoma tissue treated with PBS and HF in the Ctrl and HF groups remained almost unchanged, while the temperature in the PDA-MA group reached approximately 44°C. This demonstrates that the 3D-printed active photothermal scaffold loaded with Chlorella exhibits a photothermal effect.

[0049] like Figure 12 As shown, the residual tumors on the backs of mice in the Ctrl and HF groups exhibited significant proliferation. The residual tumors in the PDA-MA (dark) and PDA-MA (light) groups also showed proliferation, but to a lesser extent than in the first two groups. The residual tumors on the backs of mice in the PDA-MA (dark) + NIR group did not show significant growth. However, the residual tumors on the backs of mice in the PDA-MA (light) + NIR group detached, exposing healthy muscle tissue. These results indicate that the 3D-printed active photothermal scaffold loaded with Chlorella exhibits a significant in vivo PTT effect.

[0050] 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 novel 3D-printed photothermal scaffold carrying Chlorella vulgaris, characterized in that, Includes the following steps: (1) Preparation of polydopamine-chlorella (PDA-MA): Chlorella (MA) solution and Tris-HCl buffer were mixed, dopamine hydrochloride powder was added, and the mixture was shaken thoroughly. After the chlorella (MA) solution turned gray-black, it was taken out, centrifuged, and the supernatant was discarded to obtain engineered chlorella (MA) encapsulated with polydopamine, namely polydopamine-chlorella; (2) Prepare a mixed solution of polyvinyl alcohol and calcium chloride as the inner phase, and prepare a mixed dispersion of polydopamine-chlorella (PDA-MA), sodium alginate and methacrylic anhydride gelatin as the outer phase. The inner and outer phases are injected into the inner phase inlet and outer phase inlet of the microfluidic device respectively through a syringe for 3D printing. After printing, the novel chlorella-carrying 3D printed photothermal scaffold is obtained by irradiation under an ultraviolet probe.

2. The preparation method according to claim 1, characterized in that, The microfluidic device is a capillary microfluidic device. Two cylindrical capillaries of the same specifications are prepared. One is used as the outer phase. One end of the circular glass capillary is processed into a pointed cone shape using a stretching instrument, and the inner diameter is polished with sandpaper. The other is used as the inner phase. It is stretched steadily and quickly under the heating of a spray gun to make the middle of the capillary into a filamentous tube. The inner phase capillary is inserted into the outer phase capillary, making it horizontally and vertically centered between the inner and outer phase capillaries. It is placed flat on a glass slide, and the bottom of the capillary is covered with the bottom of a needle and sealed with a two-liquid hardening adhesive. The capillary body is then glued to the glass slide with the two-liquid hardening adhesive.

3. The preparation method according to claim 1, characterized in that, Step (2) Replace the original printhead in the programmable 3D printer with a microfluidic device for 3D printing.

4. The preparation method according to claim 1, characterized in that, The Tris-HCl buffer solution has a concentration of 0.15 g / mL and a pH of 7.

5. The preparation method according to claim 1, characterized in that, The MA solution and Tris-HCl solution were mixed in equal volumes at a 1:1 ratio.

6. The preparation method according to claim 1, characterized in that, The amount of dopamine hydrochloride added is 0.5 mg / mL, and the concentration of the Chlorella (MA) solution is 2 mg / mL.

7. The preparation method according to claim 1, characterized in that, The concentrations of polyvinyl alcohol and calcium chloride dissolved in ultrapure water in the mixed solution of polyvinyl alcohol and calcium chloride are 2% w / v and 0.8% w / v, respectively.

8. The preparation method according to claim 1, characterized in that, The sodium alginate and methacrylic anhydride gelatin were dissolved in ultrapure water at concentrations of 2% w / v and 10% w / v, respectively; the PDA-MA volume concentration was 2 × 10⁻⁶. 8 10,000 / mL.

9. A novel 3D-printed photothermal scaffold carrying Chlorella vulgaris, characterized in that... It is prepared by the preparation method according to any one of claims 1-8.

10. The application of the novel Chlorella-carrying 3D-printed photothermal scaffold as described in claim 9, characterized in that, The novel Chlorella-loaded 3D-printed photothermal scaffold is used as an adjuvant therapy for postoperative melanoma treatment.

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