Up-conversion nanoparticle compound, preparation method thereof and cell regulation substrate

Abstract

The invention relates to an up-conversion nanoparticle compound, a preparation method thereof and a cell regulation substrate, the up-conversion nanoparticle compound comprises up-conversion nanoparticles, connecting molecules connected to the surfaces of the up-conversion nanoparticles, and light-operated isomeric molecules reversibly connected with the connecting molecules, rGD polypeptide is connected to the light-controlled isomeric molecule; wherein the upconversion nano-particles can convert near-infrared light irradiated on the upconversion nano-particles into ultraviolet light, and after the light-operated isomeric molecules connected with the RGD polypeptide are radiated by the ultraviolet light, the light-operated isomeric molecules and the RGD polypeptide fall off from the connecting molecules together. The up-conversion nanoparticle compound can be applied to a carrier for cell growth, and is irradiated by near-red light in the cell growth process to dynamically regulate and control cell adhesion, extension and differentiation.

Description

Technical Field

[0001] The present invention relates to the technical field of inorganic nanomaterial biological applications, and in particular to an upconversion nanoparticle complex and a preparation method thereof, as well as a cell regulation substrate having the upconversion nanoparticle complex. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cells with multidirectional differentiation potential. They can differentiate into various types of cells such as osteoblasts, chondrocytes, and adipocytes. Therefore, they have important application value in tissue engineering and regenerative medicine.

[0003] Dynamic regulation of cell-molecular interactions is fundamental to the diverse applications of mesenchymal stem cells. Among the many existing regulatory approaches, optical regulation has become a research hotspot in recent years due to its high spatiotemporal resolution, non-invasiveness, and reproducibility. Optical regulation involves triggering the release of bioactive molecules from materials through light, or influencing cell behavior through photodynamic effects.

[0004] Rare-Earth upconversion nanoparticles (UCNPs) are a class of nanomaterials that can upconvert near-infrared (NIR) light into high-energy visible light. They possess the advantages of high photostability, narrow-band emission, and low autofluorescence background. Currently, UCNPs are often used as light sources to convert NIR light into visible light of specific wavelengths, thereby activating functional groups bound to photoresponsive materials and enabling precise regulation of cellular behavior.

[0005] Chinese patent CN116716099A discloses a near-infrared light-responsive upconversion nanosubstrate, which includes a substrate and surface-modified upconversion nanoparticles connected to the substrate. The surface-modified upconversion nanoparticles include an upconversion nanoparticle as a core, silica coated on the outside of the upconversion nanoparticle, an RGD (arginine-glycine-aspartic acid) polypeptide connected to the silica, and a light-controlled bond-breaking small molecule coating the RGD polypeptide. The RGD polypeptide is a cell adhesion peptide that interacts with receptors on the cell membrane surface to mediate the adhesion of cells to the extracellular matrix. The received near-infrared light is converted into ultraviolet light by the modified upconversion nanoparticles. Under the irradiation of ultraviolet light, the light-controlled small molecule falls off the RGD polypeptide, exposing and activating the RGD polypeptide, thereby regulating the adhesion, extension and final differentiation behavior of cells in contact with the upconversion nanosubstrate. However, the regulation method disclosed in this patent involves irradiating the substrate with near-infrared light to adjust the exposure of the RGD polypeptide on the substrate before adding mesenchymal stem cells to the substrate. This is to regulate the degree of cell adhesion to the substrate after the addition of the mesenchymal stem cells, thereby achieving regulation of cell adhesion, extension, and ultimately cell differentiation. However, after the mesenchymal stem cells are added to the substrate, regulation is no longer possible, and therefore dynamic regulation of cells in the process of growth cannot be achieved. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to overcome the defects or shortcomings of the existing technology. On the one hand, it provides an upconversion nanoparticle complex, which can be used as a carrier for cell growth and dynamically regulate the adhesion, extension and differentiation behavior of cells during cell growth by being excited by near-infrared light.

[0007] An upconversion nanoparticle complex comprises an upconversion nanoparticle, wherein the surface of the upconversion nanoparticle is modified with a linker molecule, the linker molecule is connected to a photo-controlled isomer molecule, and the photo-controlled isomer molecule is connected to an RGD polypeptide; wherein the upconversion nanoparticle can convert near-infrared light irradiated thereon into ultraviolet light, and the photo-controlled isomer molecule connected to the RGD polypeptide falls off from the linker molecule together with the RGD polypeptide after being irradiated by the ultraviolet light.

[0008] Compared with the prior art, the upconversion nanoparticle complex described in the present invention is modified with a linker molecule on the surface of the upconversion nanoparticle, and a photo-controlled isomer molecule connected to the linker molecule by host-guest interaction, and an RGD polypeptide that can adhere to cells is connected to the photo-controlled isomer molecule. The upconversion nanoparticle converts the received near-infrared light into ultraviolet light. Under the irradiation of ultraviolet light, the photo-controlled isomer molecule undergoes cis-trans isomerization, causing the photo-controlled isomer molecule to fall off from the linker molecule together with the RGD polypeptide connected thereto, thereby regulating the retention amount of the RGD polypeptide of the upconversion nanoparticle complex, and further regulating the adhesion, extension and differentiation behavior of cells in contact with the upconversion nanoparticle complex.

[0009] In one embodiment, the linker molecule is β-cyclodextrin, and the photoisomerizable molecule is a substance that can undergo cis-trans isomerization under ultraviolet light irradiation and separate from the linker molecule.

[0010] In one embodiment, the photoisomerizable molecule is an azobenzene analog.

[0011] In one embodiment, the upconversion nanoparticles include a first layer structure and a second layer structure coated on the outside of the first layer structure, wherein the first layer structure is doped with Tm ions and the second layer structure is doped with Nd ions.

[0012] In one embodiment, the upconversion nanoparticle complex further comprises silica coating the upconversion nanoparticles, and PEG connected to the surface of the silica, and the linker molecule is connected to the silica via PEG.

[0013] On the other hand, the present invention also provides a method for preparing an upconversion nanoparticle complex, which is characterized by comprising the following steps:

[0014] preparing upconversion nanoparticles;

[0015] connecting a linker molecule to the surface of the upconversion nanoparticle;

[0016] The light-controlled isomer molecule connected with the RGD polypeptide is connected to the connecting molecule to obtain an upconversion nanoparticle complex.

[0017] In one embodiment, the step of preparing upconversion nanoparticles comprises the following steps:

[0018] The preparation of upconversion nanoparticles comprises the following steps:

[0019] forming a first layer structure of the upconversion nanoparticles;

[0020] A second layer structure is coated on the outer side of the first layer structure of the upconversion nanoparticle to obtain a double-layer upconversion nanoparticle; wherein the first layer structure is doped with Tm ions and the second layer structure is doped with Nd ions.

[0021] In one embodiment, the step of attaching a linker molecule to the surface of the upconversion nanoparticle comprises the following steps:

[0022] Coating a layer of silicon dioxide on the surface of the upconversion nanoparticles;

[0023] Linker molecules are attached to the surface of silica-coated upconversion nanoparticles.

[0024] In one embodiment, the linker molecule is β-cyclodextrin, and the photoisomerizable molecule is a substance that can undergo cis-trans isomerization under ultraviolet light irradiation and separate from the linker molecule.

[0025] In another aspect, the present invention provides a cell regulation substrate, comprising a substrate, and any of the above-mentioned upconversion nanoparticle complexes, wherein the upconversion nanoparticle complex is connected to the surface of the substrate.

[0026] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic structural diagram of an embodiment of the upconversion nanoparticle complex of the present invention;

[0028] Figure 2 This is a schematic structural diagram of an embodiment of the cell regulation substrate of the present invention;

[0029] Figure 3 This is a TEM image of upconversion nanoparticles coated with silicon dioxide, prepared by the method for preparing the upconversion nanoparticle composite of the present invention;

[0030] Figure 4 The fluorescence spectrum of the upconversion nanoparticles coated with silicon dioxide and the upconversion nanoparticle complex prepared by the method for preparing the upconversion nanoparticle complex of the present invention;

[0031] Figure 5 This is a SEM scanning electron microscope image of the cell regulation substrate prepared by the cell regulation substrate preparation method of the present invention;

[0032] Figure 6 The ultraviolet spectrum of AAP-RGD released by the upconversion nanoparticle complex prepared by the preparation method of the upconversion nanoparticle complex of the present invention;

[0033] Figure 7The cell regulation substrate prepared by the cell regulation substrate preparation method of the present invention is illuminated at different near-infrared light intensities (from left to right corresponding to 0, 0.5, 1 and 2 W / cm 2 ) Water contact angle measurement spectrum under irradiation;

[0034] Figure 8 The cell regulation substrate prepared by the cell regulation substrate preparation method of the present invention is illuminated at different near-infrared light intensities (from left to right corresponding to 0, 0.5, 1 and 2 W / cm 2 ) Adhesion and extension of stem cells under irradiation;

[0035] Figure 9 The cell regulation substrate prepared by the cell regulation substrate preparation method of the present invention is illuminated at different near-infrared light intensities (from left to right corresponding to 0, 0.5, 1 and 2 W / cm 2 ) Vinculin immunofluorescence images in stem cells under irradiation;

[0036] Figure 10 The cell regulation substrate prepared by the cell regulation substrate preparation method of the present invention is illuminated at different near-infrared light intensities (from left to right corresponding to 0, 0.5, 1 and 2 W / cm 2 ) PCR results of stem cell multidirectional differentiation gene expression under irradiation (osteoblastic differentiation: ALP, OPN, Runx2; adipogenic differentiation: FABP4, C / EBPα, Adipoq);

[0037] Figure 11 The cell regulation substrate prepared by the cell regulation substrate preparation method of the present invention is illuminated at different near-infrared light intensities (from left to right corresponding to 0, 0.5, 1 and 2 W / cm 2 ) The results of ALP (osteogenic differentiation) and Oil Red (adipogenic differentiation) staining of multidirectional differentiation of stem cells under irradiation.

[0038] Reference numerals:

[0039] 10. Substrate;

[0040] 20. Upconversion nanoparticle complex; 200. Upconversion nanoparticle; 202. Silica; 204. PEG; 206. Connector molecule; 208. Photoisomerizable molecule; 210. RGD polypeptide. DETAILED DESCRIPTION

[0041] Although there are currently UCNPs materials that can convert absorbed near-infrared light into ultraviolet light, and regulate the degree of adhesion between UCNP and the cells in contact with it through ultraviolet light radiation, thereby regulating the adhesion, extension and differentiation behavior of mesenchymal stem cells in contact with UCNP, this regulation is first exposed by light radiation to expose the RGD polypeptide on the UCNP, and then the exposure degree of the RGD polypeptide is adjusted to regulate the behavior of the cells in contact with the UCNP. In other words, after this UCNP comes into contact with the cells, it can no longer regulate the cells through light radiation.

[0042] Based on this, in order to achieve dynamic regulation, the present invention uses upconversion nanoparticles that convert absorbed near-infrared light into ultraviolet light as the core, and then modifies the surface of the upconversion nanoparticles with linker molecules and light-controlled isomer molecules connected to the linker molecules using host-guest interactions. The light-controlled isomer molecules are connected to RGD polypeptides that can adhere to cells. The upconversion nanoparticles convert the received near-infrared light into ultraviolet light. Under the irradiation of ultraviolet light, the light-controlled isomer molecules undergo cis-trans isomerization, causing them to fall off from the linker molecules, and the RGD polypeptide connected to the light-controlled isomer molecules also falls off. When the RGD polypeptide falls off with the light-controlled isomer molecules, the retention amount of the RGD polypeptide in the upconversion nanoparticle complex changes, causing the adhesion between the upconversion nanoparticle complex and the cells in contact with it to change, thereby affecting the morphology and differentiation direction of the cells, thereby achieving cell regulation.

[0043] In addition, since most existing UCNPs use near-infrared light with a wavelength of 980nm as an excitation light source, but near-infrared light with a wavelength of 980nm can cause a strong photothermal effect, its energy will be partially absorbed by biological tissues (such as hemoglobin) or substances (such as water) in the cells, resulting in local heating, which in turn has side effects on the cells. If the UCNPs material is regulated by near-infrared light during the culture process after the cells come into contact with the UCNPs material, it may damage the cells. Therefore, for this type of UCNPs material, the regulation of the UCNPs material can only be completed before the cells come into contact with the UCNPs material, and then the contact between the UCNPs material and the cells after regulation can be further regulated to regulate cell adhesion, extension, and differentiation.

[0044] To solve this problem, the present invention dopes the different layers of up-conversion nanoparticles with Nb ions that absorb 808nm near-infrared light and Tm ions that emit 365nm ultraviolet light, respectively, to achieve conversion of the absorbed near-infrared light with a wavelength of 808nm into ultraviolet light with a wavelength of 365nm. The reason why Nb ions and Tm ions are doped in different layers of up-conversion nanoparticles is that the inventors of the present invention found in their research that if Nb ions and Tm ions are doped in the same layer, the luminous intensity will be greatly reduced, resulting in the inability to excite 365nm ultraviolet light. Therefore, the present invention designs the up-conversion nanoparticles into a "core-shell" double-layer structure, which includes a first layer structure and a second layer structure coated on the outside of the first layer structure, and dopes the Nb ions and Tm ions in the different layer structures of the up-conversion nanoparticles to avoid the encounter of Nb ions and Tm ions causing a reduction in light intensity, so as to ultimately achieve conversion of the near-infrared light with a wavelength of 808nm into ultraviolet light with a wavelength of 365nm.

[0045] The solution of the present invention is described in detail below with reference to the accompanying drawings.

[0046] Figure 1 An exemplary structure of the upconversion nanoparticle complex of the present invention is shown. Figure 1 As shown, the upconversion nanoparticle complex 20 includes an upconversion nanoparticle 200 , the surface of the upconversion nanoparticle 200 is modified with a linker molecule 206 , a photoisomerization molecule 208 connected to the linker molecule 206 , and an RGD polypeptide 210 connected to the photoisomerization molecule 208 .

[0047] Specifically, the upconversion nanoparticle 200 has a double-layer structure, comprising a first layer and a second layer, with the second layer covering the first layer. The first layer is doped with Tm ions that emit ultraviolet light at a wavelength of 365 nm, while the second layer is doped with Nd ions that absorb near-infrared light at a wavelength of 808 nm. The double-layered upconversion nanoparticle 200 can use 808 nm near-infrared light as an excitation light source, converting the 808 nm near-infrared light into ultraviolet light at a wavelength of 365 nm for emission.

[0048] The connecting molecule 206 is specifically β-cyclodextrin, and the photoisomerization molecule 208 is an azobenzene analog. The azobenzene analog can be combined with β-cyclodextrin through host-guest interactions. Under the irradiation of 365nm ultraviolet light, the azobenzene analog undergoes cis-trans isomerization (converting from a trans structure to a cis structure). The new isomer formed has a reduced degree of binding to β-cyclodextrin, causing it to fall off from β-cyclodextrin, and the RGD polypeptide 210 connected to the azobenzene analog also falls off. In this embodiment, the photoisomerization molecule 208 is specifically a water-soluble arylazopyrazole (AAPs). Compared with azobenzene, water-soluble arylazopyrazoles have better water solubility and a more stable cis structure, which is beneficial for connection with the RGD polypeptide 210 and stability after connection. In subsequent cell regulation applications, it is also beneficial to reduce damage to cells.

[0049] When near-infrared light with a wavelength of 808 nm is used as the irradiation light source to illuminate the upconversion nanoparticle complex 20, the upconversion nanoparticle 200 converts the 808 nm near-infrared light into ultraviolet light with a wavelength of 365 nm. Under the irradiation of this ultraviolet light, the photo-controlled isomer molecule 208 undergoes cis-trans isomerization, and the connected RGD polypeptide 210 is detached from the connecting molecule 206. In this way, the retention amount of RGD polypeptide 210 on the upconversion nanoparticle complex 20 is regulated by near-infrared light. When the upconversion nanoparticle complex 20 is used to prepare a substrate for cell regulation, the adhesion, extension, and differentiation of cells growing on the substrate can be regulated by adjusting the retention amount of RGD polypeptide 210.

[0050] like Figure 1 As shown, the upconversion nanoparticle complex 20 of this embodiment further includes silica 202 coating the exterior of the upconversion nanoparticle 200, and PEG (Polyethylene Glycol) 204 attached to the surface of the silica 202, with a linker molecule 206 attached to the PEG. The coating of silica 202 and the attachment of PEG 204 to the surface of silica 202 enable the connection of the linker molecule 206 to the upconversion nanoparticle 200, thereby improving the biocompatibility of the upconversion nanoparticle complex 20 and significantly reducing its cytotoxicity.

[0051] Figure 2 An exemplary structure of the cell regulation substrate of the present invention is shown. Figure 2 As shown, the cell regulation substrate includes a substrate 10 and the above-mentioned upconversion nanoparticle complex 20, wherein the upconversion nanoparticle complex 20 is connected to the surface of the substrate 10. The material of the substrate 10 is not specifically limited, as long as it can provide physical support for the upconversion nanoparticle complex 20. Commonly used materials include glass, silicon, plastic, and other materials containing -OH groups on the surface.

[0052] The above-mentioned cell regulation substrate can be used to make a culture dish for cell culture. During use, mesenchymal stem cells are first added to the culture dish and cultured for a period of time. At this point, the upconversion nanoparticle complex 20 on the surface of the substrate 10 interacts with receptors on the cell membrane surface of the mesenchymal stem cells through the RGD polypeptide 210 on its surface, forming cell adhesion and allowing the mesenchymal stem cells to grow adherently. The mesenchymal stem cells in the culture dish are then irradiated with near-infrared light with a wavelength of 808 nm. The upconversion nanoparticle complex 20 on the substrate 10 converts the 808 nm near-infrared light into ultraviolet light with a wavelength of 365 nm. Under the irradiation of ultraviolet light, the photo-controlled isomer 208 undergoes cis-trans isomerization and detaches from the linker 206. The RGD polypeptide 210 attached to the photo-controlled isomer 208 also detaches, reducing the adhesion of the cell regulation substrate to the mesenchymal stem cells, thereby regulating the degree of cell adhesion. Without near-infrared light irradiation and UV light generation, the photoisomerizable molecules 208 do not shed due to cis-trans isomerization, and thus the RGD polypeptide 210 does not shed either. The amount of RGD polypeptide 210 retained on the surface of the upconversion nanoparticle 200 influences cell adhesion, extension, and ultimately differentiation. Therefore, by regulating the amount of RGD polypeptide 210 retained on the cell-regulating substrate, cell morphology and differentiation direction can be controlled.

[0053] In addition, the present invention provides a method for preparing the above-mentioned upconversion nanoparticle complex, comprising the following steps:

[0054] S1: Preparing upconversion nanoparticles 200, including the following steps:

[0055] S11: forming a first layer structure of upconversion nanoparticles 200;

[0056] Specifically, 1.59 mmol YCl3·6H2O, 0.4 mmol YbCl3·6H2O, and 0.01 mmol TmCl3·6H2O were respectively dissolved in 800 μl methanol, and added together with 15 mL oleic acid and 30 mL 1-18 ene into a conical flask. The mixture was stirred and slowly heated to promote the dissolution of the drugs. The solution was heated to 160°C and maintained for 1 hour until the drugs were completely dissolved to obtain a light yellow clear solution. The heating was stopped and the solution was stirred and cooled to room temperature. Then, 8 mmol NH4F and 5 mmol NaOH were weighed and dissolved in an appropriate amount of methanol, and the solution was added to the above yellow solution at room temperature and stirred for 1 hour to mix thoroughly. The mixture was then heated to 300°C and maintained for 1 hour to react to obtain a brown solution. After cooling, the product was washed with a mixture of cyclohexane and anhydrous ethanol to obtain the first layer structure of the upconversion nanoparticle 200, i.e., the "core" of the upconversion nanoparticle 200 was formed.

[0057] S12: coating a second layer structure on the outer side of the first layer structure of the upconversion nanoparticle 200;

[0058] Specifically, 1.59mmol YCl3·6H2O, 0.4mmol YbCl3·6H2O and 0.01mmol NdCl3·6H2O were dissolved in 800μl methanol, and added into a conical flask together with 15mL oleic acid and 30mL 1-18 ene. The mixture was stirred and heated slowly to promote dissolution until the temperature reached 160℃, and maintained for 1h until the above substances were completely dissolved to obtain a light yellow clear solution. The heating was stopped and the mixture was stirred and cooled to room temperature. Then 8mmol NH4F and 5mmol NaOH was dissolved in an appropriate amount of methanol, and 1 mmol of the first layer structure of the upconversion nanoparticle 200 obtained in step S12 was dissolved in cyclohexane and added to the above yellow solution at room temperature, stirred for 1 hour to fully mix; the mixed solution was then heated to 300°C and kept for 1 hour to react to obtain a brown solution. After cooling, the product was washed with a mixture of cyclohexane and anhydrous ethanol to form the "shell" of the upconversion nanoparticle 200, and finally a double-layer structure of the upconversion nanoparticle 200 consisting of the first layer structure and the second layer structure, namely UCNP, was obtained.

[0059] S2: Connecting the linker molecule 206 to the surface of the upconversion nanoparticle 200, including the following steps:

[0060] S21: coating a layer of silicon dioxide 202 on the surface of the upconversion nanoparticle 200;

[0061] Specifically, 0.25 mmol of the double-layer upconversion nanoparticles 200 obtained in step S1 was taken and dissolved in 20 mL of cyclohexane, and ultrasonicated for 1 hour to fully dissolve it; then 200 μL of ammonia water, 500 μL of deionized water, 500 μL of Triton, and 1 mL of n-hexane were added, and stirred thoroughly to make the solution present an alkaline environment brought by ammonia water and further promote the dispersion of the upconversion nanoparticles 200, and then 50 μL of tetraethyl silicate was added. After sufficient stirring, 10 μL of 3-aminopropyltriethyloxysilane was added, and stirred for another 6 hours. 5 mL of acetone was added and stirred for 10 minutes before stopping; then washed with anhydrous ethanol for three times to obtain silicon-coated upconversion nanoparticles 200 with silica 202 coated on the surface, namely UCNP@SiO2. Figure 3 Shows the TEM image of UCNP@SiO2, Figure 4 The fluorescence spectrum of UCNP@SiO2 is shown. Figure 4 It can be seen that UCNP@SiO2 can emit ultraviolet light with a wavelength of 365nm under the irradiation of near-infrared light with a wavelength of 808nm.

[0062] S22: connecting the linker molecule 206 to the surface of the silicon-coated upconversion nanoparticle 200;

[0063] Specifically, the connecting molecule 206 is β-cyclodextrin. 40 mg of the silicon-coated upconversion nanoparticles 200 prepared in step S2 is taken and dissolved in 10 mL of DMF. After sufficient dispersion, 10 mg of PEG-2000 is added and stirred at room temperature for 24 hours; then 20 mg of β-cyclodextrin (β-CD) is added and continued to stir. After sufficient reaction, the product is washed with anhydrous ethanol to obtain silicon-coated upconversion nanoparticles 200 connected with β-cyclodextrin, namely UCNP@SiO2-CD.

[0064] S3: Connecting the photoisomerizable molecule 208 connected with the RGD polypeptide 210 to the connecting molecule 206;

[0065] Specifically, the photo-controlled isomer molecule 208 is an azobenzene analogue that can be reversibly linked to β-cyclodextrin through host-guest interaction. The azobenzene analogue used in this embodiment is a water-soluble arylazoazole (AAPs);

[0066] The UCNP@SiO2-CD prepared in step S2 was dissolved in 10 mL of DMSO. After sufficient dissolution, 1 mg of AAP connected to the RGD peptide 210 was added. After slow stirring for 12 h, the mixture was washed three times with anhydrous ethanol to obtain the upconversion nanoparticle complex 20, namely UCNP@SiO2-CD / AAP-RGD. Figure 4 The fluorescence spectrum of the upconversion nanoparticle complex 20UCNP@SiO2-CD / AAP-RGD is also shown. Figure 4 It can be seen that the photoisomerization molecule 208 connected with the RGD polypeptide 210 is successfully connected to the surface of the silicon-coated upconversion nanoparticle 200 .

[0067] Since the method of linking the RGD polypeptide 210 to the AAP belongs to the prior art and is not the technical focus of the present invention, the specific process will not be described in detail here.

[0068] Furthermore, the present invention also provides a method for preparing the above-mentioned cell regulation substrate, comprising the following steps:

[0069] Steps S1 to S3 are the steps for preparing the upconversion nanoparticle complex 20 , which will not be described in detail here.

[0070] Based on steps S1-S3, the following steps are performed:

[0071] S4: connecting the upconversion nanoparticle complex 20 to the surface of the substrate 10;

[0072] Specifically, a 10×10 mm glass plate was prepared as a substrate 10 to serve as a physical support for the upconversion nanoparticle complex 20. After cleaning, the substrate 10 was immersed in a piranha etchant (98% H2SO4 / 30% H2O2, 3 / 1) and horizontally shaken overnight, followed by rinsing with deionized water. 0.01% by volume of (3-mercaptopropyl)trimethoxysilane (MPTMS) was added to the substrate 10 and horizontally shaken for 8 hours. The substrate was then rinsed three times with anhydrous ethanol and then 0.5% by volume of diisopropylethylamine (DIPEA) was added.

[0073] Then, 5 g of the upconversion nanoparticle complex 20 prepared in step S3 was taken and dissolved in 5 mL of DMF, which was then added to the liquid-immersed substrate 10. After oscillation for 8 hours, the substrate 10 was washed with anhydrous ethanol and then deionized water. Finally, the substrate 10 with the upconversion nanoparticle complex 20 connected to the surface was obtained, i.e., the cell regulation substrate. Figure 5 The structure of the prepared cell regulation substrate under a scanning electron microscope is shown.

[0074] In order to verify the regulatory effects of the upconversion nanoparticle complex 20 and the cell regulatory substrate prepared by the above preparation method, the following verification experiments were performed:

[0075] (1) Light-controlled release experiment

[0076] The upconversion nanoparticle complex 20 prepared in step S3 of the above embodiment is dispersed in a PBS solution. The upconversion nanoparticles 200 in the upconversion nanoparticle complex 20 convert near-infrared light into ultraviolet light. The light-controlled isomer molecules 208 modified on the surface of the upconversion nanoparticles 200 undergo cis-trans isomerization under the action of ultraviolet light, fall off from the β-cyclodextrin cavity, and are thus separated from the upconversion nanoparticle complex 20. The ultraviolet absorption spectrum of AAP is detected by an ultraviolet spectrophotometer to quantify the amount of AAP released.

[0077] The results are as follows Figure 6 As shown, Figure 6 The four curves reflect 0, 0.5, 1 and 2 W / cm 2 The UV absorption spectrum of AAP retained on the upconversion nanoparticle complex 20 under near-infrared light (808 nm) illumination. Figure 6 It can be seen that with the increase of light intensity, the AAP retained on the upconversion nanoparticle complex decreased significantly, that is, the shedding of AAP intensified with the increase of light intensity.

[0078] Take the cell regulation substrate prepared in step S4 of the above embodiment and use 0, 0.5, 1 and 2 W / cm 2 The materials prepared by near-infrared light illumination were used to determine whether RGD could be regulated and shed under near-infrared light.

[0079] The results are as follows Figure 7 As shown, after the cell regulation substrate is irradiated by near-infrared light of different intensities, water droplets form different contact angles on the substrate 10 of the cell regulation substrate; the greater the light intensity, the greater the contact angle of the water droplet, indicating that the amount of RGD polypeptide 210 retained on the surface of the substrate 10 is less and the surface tension is smaller.

[0080] (2) Cell stretching experiment

[0081] Take the cell regulation substrate prepared in step S4 of the above embodiment and use 0, 0.5, 1 and 2 W / cm 2 The cell-regulating substrate prepared by irradiating it with near-infrared light was tested to determine whether it could regulate cell extension. Specifically, mesenchymal stem cells were first added to a culture dish containing the cell-regulating substrate and cultured for 12 hours to allow the cells to adhere and grow. The cell interface material was then irradiated with near-infrared light of varying intensities and cultured for another 24 hours. The cell membranes were then stained with a fluorescent dye and the staining results were observed using a laser confocal microscope.

[0082] The results are as follows Figure 8 As shown by Figure 8 It can be seen that with the increase of light intensity, the morphology of cells adhered to the cell regulatory substrate gradually changes from polygonal under low-power irradiation to nearly circular under high-power irradiation. This proves that with the increase of light intensity, RGD falls off together with AAP, and the traction force on the cells gradually weakens, indicating that the cell regulatory substrate can effectively regulate the cell extension morphology through light control.

[0083] (3) Cell pathway detection experiments

[0084] Take the cell regulation substrate prepared in step S4 of the above embodiment and use 0, 0.5, 1 and 2 W / cm 2 The cell regulation substrate prepared by irradiation with near-infrared light was used to determine whether it could affect the intracellular signaling pathway. Specifically, mesenchymal stem cells were first added to a culture dish with the cell regulation substrate and cultured for 12 hours to allow the cells to grow attached to the wall. The culture dish with the cell regulation substrate was then irradiated with near-infrared light of different intensities. After a further 48 hours of culture, the cells were fixed and immunofluorescence staining was performed on the samples using Vinculin primary antibody and phalloidin. Vinculin participates in the cell's force-chemical signal transduction by interacting with cytoskeletal proteins and cell adhesion molecules.

[0085] The results are as follows Figure 9 As shown by Figure 9 It can be seen that mesenchymal stem cells can be irradiated with low power (0W / cm 2) under the condition of 5% paraformaldehyde and 1% paraformaldehyde, the fluorescence intensity of Vinculin was significantly stronger, which further verified the regulation of cell extension by cell interface materials.

[0086] (4) Cell differentiation experiment

[0087] Take the cell regulation substrate prepared in step S4 of the above embodiment and use 0, 0.5, 1 and 2 W / cm 2 The cell regulatory substrate prepared by irradiation with near-infrared light was used to determine whether it could regulate cell differentiation. Specifically, mesenchymal stem cells were first added to a culture dish containing the cell interface material and cultured for 12 hours to allow the cells to adhere to the wall. The cell interface material was then irradiated with near-infrared light of varying intensities. The osteogenic / adipogenic induction medium was replaced. After 7 days of culture, the cells were digested, total RNA was extracted, and RT-PCR was performed to detect the expression of the stem cell osteogenic differentiation marker genes ALP, OPN, and Runx2, and the adipogenic differentiation marker genes FABP4, C / EBPα, and Adipoq.

[0088] The results are as follows Figure 10 As shown by Figure 10 It can be seen that when the light intensity is weak (0W / cm 2 ), the expression of osteogenic differentiation marker genes ALP, OPN and Runx2 was strong, indicating that mesenchymal stem cells tended to differentiate into osteoblasts; when the light intensity was strong (2W / cm 2 ), the adipogenic differentiation marker genes FABP4, C / EBPα and Adipoq were expressed strongly, indicating that stem cells tend to differentiate into adipocytes.

[0089] In order to further verify the effect of the above-mentioned cell regulation substrate on near-infrared light regulation of the multidirectional differentiation of mesenchymal stem cells, mesenchymal stem cells were first added to a culture dish with the cell regulation substrate and cultured for 12 hours to allow the cells to grow attached to the wall; then, the culture dish with the cell regulation substrate was irradiated with near-infrared light of different intensities, and the osteogenic / adipogenic induction culture medium was replaced. After continuing to culture for 10 days, alkaline phosphatase staining experiments (ALP) and oil red staining experiments (OR) were performed to verify the differentiation of the cells.

[0090] The results are as follows Figure 11 As shown by Figure 11 It can be seen that with the increase of light intensity, the content of red fat droplets gradually increased in the OR staining experiment, indicating that stem cells gradually differentiated towards adipocytes; when the light intensity was weak, the blue-purple color in the ALP staining experiment was deeper, indicating that the cells differentiated towards osteoblasts.

[0091] Compared to the prior art, the upconversion nanoparticle complex described in the present invention is based on an upconversion nanoparticle that converts absorbed near-infrared light into ultraviolet light. The surface of the upconversion nanoparticle is then modified with a linker molecule and a photoisomerization molecule connected to the linker molecule via host-guest interaction. The photoisomerization molecule is connected to an RGD polypeptide capable of cell adhesion. The upconversion nanoparticle converts the received near-infrared light into ultraviolet light. Under the irradiation of ultraviolet light, the photoisomerization molecule undergoes cis-trans isomerization, causing it to fall off from the linker molecule, and the RGD polypeptide attached to the photoisomerization molecule also falls off, thereby regulating the retention of the RGD polypeptide in the upconversion nanoparticle complex. When the upconversion nanoparticle complex is applied to cell regulation, by regulating the retention of the RGD polypeptide in the upconversion nanoparticle complex, the adhesion, extension, and differentiation behavior of cells in contact with the upconversion nanoparticle complex can be regulated. Furthermore, by setting up a double-layer structure of upconversion nanoparticles and placing Nb ions and Tm ions that can emit 365nm ultraviolet light in different layer structures of the upconversion nanoparticles, the upconversion nanoparticles can absorb near-infrared light with a wavelength of 808nm and convert it into ultraviolet light with a wavelength of 365nm, avoiding damage to the cells by irradiating the cells with a wavelength of 980nm near-infrared light during the dynamic regulation of cells.

[0092] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present application. The singular forms of "a", "said" and "the" used in the embodiments of the present application and the claims are also intended to include plural forms, unless the context clearly indicates other meanings. It should also be understood that, unless otherwise specified, "multiple" refers to two or more; the terms "first", "second", "third", etc. are only used to distinguish, and are not used to describe a specific order or sequence, nor can they be understood as indicating or implying relative importance. The term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items. When the above description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. In the description of the present application, for those of ordinary skill in the art, the specific meanings of the above terms in the present application can be understood according to the specific circumstances.

[0093] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. An upconversion nanoparticle complex, characterized in that: The invention comprises an upconversion nanoparticle, wherein the surface of the upconversion nanoparticle is modified with a connecting molecule, the connecting molecule is connected to a light-controlled isomer molecule, and the light-controlled isomer molecule is connected to an RGD polypeptide; The upconversion nanoparticles can convert near-infrared light irradiated thereon into ultraviolet light, and the light-controlled isomer molecules connected to the RGD polypeptide are detached from the connection molecules together with the RGD polypeptide after being irradiated by the ultraviolet light.

2. The upconversion nanoparticle complex according to claim 1, characterized in that: The connecting molecule is β-cyclodextrin, and the photoisomerization molecule is a substance that can undergo cis-trans isomerization under the irradiation of ultraviolet light and separate from the connecting molecule.

3. The upconversion nanoparticle complex according to claim 2, characterized in that: The photo-controlled isomerization molecule is an azobenzene analogue.

4. The upconversion nanoparticle complex according to claim 1, characterized in that: The upconversion nanoparticles include a first layer structure and a second layer structure coated on the outside of the first layer structure, wherein the first layer structure is doped with Tm ions and the second layer structure is doped with Nd ions.

5. The upconversion nanoparticle complex according to claim 1, characterized in that: The invention also includes silica coated on the upconversion nanoparticles and PEG connected to the surface of the silica, wherein the connecting molecule is connected to the silica through the PEG.

6. A method for preparing an upconversion nanoparticle complex, characterized in that: The following steps are involved: preparing upconversion nanoparticles; connecting a linker molecule to the surface of the upconversion nanoparticle; The light-controlled isomer molecule connected with the RGD polypeptide is connected to the connecting molecule to obtain an upconversion nanoparticle complex.

7. The method for preparing the conversion nanoparticle complex according to claim 6, characterized in that: The preparation of upconversion nanoparticles comprises the following steps: forming a first layer structure of the upconversion nanoparticles; Coating a second layer structure on the outer side of the first layer structure of the upconversion nanoparticle to obtain a double-layer structure upconversion nanoparticle; The first layer structure is doped with Tm ions, and the second layer structure is doped with Nd ions.

8. The method for preparing the conversion nanoparticle complex according to claim 6, characterized in that: The step of connecting the linker molecule to the surface of the upconversion nanoparticle comprises the following steps: Coating a layer of silicon dioxide on the surface of the upconversion nanoparticles; Linker molecules are attached to the surface of silica-coated upconversion nanoparticles.

9. The method for preparing the conversion nanoparticle complex according to claim 6, characterized in that: The connecting molecule is β-cyclodextrin, and the photoisomerization molecule is a substance that can undergo cis-trans isomerization under the irradiation of ultraviolet light and separate from the connecting molecule.

10. A cell regulation substrate, characterized in that: The invention comprises a substrate, and the upconversion nanoparticle complex according to any one of claims 1 to 5, wherein the upconversion nanoparticle complex is connected to the surface of the substrate.

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