A quantum dot resonant biomaterial and its preparation method

Abstract

This invention discloses a quantum dot resonant biomaterial and its preparation method, belonging to the interdisciplinary field of biomaterials and nanophotonics. The material uses low-toxicity quantum dots of the same material and structure as the core component, forming dual quantum dot resonant units. Each quantum dot has a size of 2-10 nm, and the resonant units are fixed to a biodegradable carrier matrix at a spacing of 5-20 nm, covering a resonant wavelength range of 600-1100 nm. The quantum dot surface is coated with a three-layer composite structure consisting of a passivation layer, a biocompatible layer, and a functional layer, and the material contains various compliant biocompatible trace elements. It is prepared using microfluidic synthesis, multilayer surface modification, and self-assembled array molding. The resonant signal is 100-200 times higher than that of a single quantum dot, while also exhibiting high tissue penetration, biosafety, and stability. It can achieve biophotonic coherence modulation of diseased tissues and is suitable for medical and healthcare applications such as early tumor diagnosis, arthritis physiotherapy, and postoperative wound and bone defect repair.

Description

Technical Field

[0001] This invention relates to the field of interdisciplinary technology of biomaterials and nanophotonics, specifically to a biomaterial and its preparation method based on the theory of biophotonic coherence and utilizing the quantum dot resonance effect, which is applicable to medical and healthcare scenarios such as biomedical diagnosis, targeted therapy, and tissue repair. Background Technology

[0002] The coherence of biophoton radiation is closely related to the physiological state of living tissues, providing an important theoretical basis for the functional design of biomaterials. Quantum dots, as nanoscale luminescent materials, exhibit significant application potential in the field of biomaterials due to their high luminescence intensity, narrow emission linewidth, and tunable spectral characteristics. Among them, graphene quantum dots (GQDs), low-toxicity perovskite quantum dots, and group III-V InP quantum dots have become research hotspots in the field of medical biomaterials due to their biocompatibility advantages.

[0003] Existing quantum dot biomaterials primarily focus on single luminescent probe or drug carrier functions, relying mainly on photoluminescence properties for in vitro imaging or in vivo drug delivery. However, they face three major technological bottlenecks in practical medical applications: First, insufficient signal strength and limited penetration into deep tissues. The luminescent signal of conventional single quantum dots is easily attenuated by biological tissue scattering, making it difficult to accurately capture weak biophoton signals from diseased tissues, thus hindering deep tissue diagnosis and treatment. Second, they lack the ability to directionally regulate biophoton coherence, preventing the enhancement of therapeutic functions through quantum coherence superposition effects. They can only achieve single imaging or drug delivery functions, failing to achieve multi-functional synergy of "diagnosis-therapy-repair." Third, achieving both biostability and toxicity control is difficult. Some quantum dots, such as perovskite quantum dots, are prone to decomposition in physiological environments due to their ionic crystal structure, leading to toxic ion leakage. Conventional single passivation modifications cannot simultaneously meet the requirements of stability and biocompatibility, significantly limiting their widespread application in in vivo medicine.

[0004] Based on the above problems, combining the theory of biophotonic coherence with the quantum dot resonance effect, constructing novel biomaterials that combine high stability, biocompatibility, and functional synergy has become the key to breaking through the limitations of existing technologies. Summary of the Invention

[0005] This invention aims to address the technical problems of insufficient signal intensity, limited penetration into deep tissues, lack of directional modulation capability of biophotonic coherence, and difficulty in simultaneously achieving biostability and toxicity control in existing quantum dot biomaterials. It provides a biomaterial based on the quantum dot resonance effect and its preparation method. This material can precisely match the spontaneous emission frequency of biological tissues, achieving directional enhancement of resonance signal intensity. It can modulate the biophotonic coherence of diseased tissues through energy superposition effects, while also possessing excellent biosafety and environmental stability, making it suitable for customized needs in various healthcare scenarios.

[0006] II. Technical Solution

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A quantum dot resonant biomaterial comprises a core functional component, a surface modification layer, an auxiliary carrier matrix, and biocompatible trace elements. The core functional component is a dual-quantum dot resonant unit, which consists of two low-toxicity quantum dots of the same material and structure. The size of a single quantum dot is controlled within the range of 2-10 nm, with a size deviation of ≤0.5 nm for quantum dots from the same batch. The dual-quantum dot resonant units are fixed in the auxiliary carrier matrix at a spacing of 5-20 nm, and their resonant emission band covers 600-1100 nm, with the resonant frequency matching the biophoton radiation frequency of the target biological tissue.

[0008] The surface modification layer is a three-layer composite structure consisting of a passivation layer, a biocompatibility layer, and a functional layer that are sequentially coated on the outer surface of a single quantum dot from the inside out; the quantum dot resonant biomaterial also contains a variety of biocompatible trace elements, and the total content and in vitro release of each trace element meet the safety limits of the ICH Q3D guidelines.

[0009] Furthermore, the low-toxicity quantum dots are selected from graphene quantum dots, bilayer silica-coated perovskite quantum dots, or InP quantum dots, and their photoluminescence quantum yield is ≥85%.

[0010] Furthermore, the various biocompatible trace elements include, but are not limited to, Zn, Se, Te, Cd, Ga, In, Fe, Cu, Co, Mn, Cr, Mo, F, Si, Ti, Zr, Sn, B, V, Ni, Al, I, As, Ca, P, Mg, Sr, and Ba; wherein the content of elements such as Cd, As, and Ni meets the allowable daily exposure requirements for elements in categories 2A and 2B of the ICH Q3D guidelines, and the daily in vitro release of element F is controlled within the range of 0.5-1 mg.

[0011] Furthermore, the passivation layer is a silicon dioxide layer or a mercaptopropionic acid layer with a thickness of 2-5 nm; the biocompatibility layer is a polyethylene glycol layer or a phospholipid micelle layer with a cytotoxic IC50 ≥ 50 μM; and the functional layer can be grafted with corresponding targeted functional molecules according to the application scenario.

[0012] Furthermore, the auxiliary carrier matrix is ​​selected from biodegradable nanofibers, water-soluble hydrogels, or hyaluronic acid, and its in vitro degradation rate can be controlled within the range of 1-30 days; the dual quantum dot resonant units are uniformly fixed in the auxiliary carrier matrix in a regular hexagonal array.

[0013] The above-mentioned method for preparing quantum dot resonant biomaterials includes the following steps: (1) Precise synthesis of quantum dots and trace element doping: core quantum dots are prepared by microfluidic synthesis technology, and core doping elements are introduced simultaneously by precursor doping method. The reaction temperature is controlled within the range of 80-180℃, and the element precursor concentration is within the range of 0.01-0.1mM, so that trace elements are uniformly dispersed inside the quantum dots. The size deviation of the obtained quantum dots is controlled to be ≤0.3nm, and the photoluminescence quantum yield is ≥85%; (2) Multilayer surface modification and trace element loading: a dense passivation layer is first coated on the surface of the quantum dots by sol-gel method, and the corresponding element precursor is added during the coating process; then a biocompatible layer is formed outside the passivation layer by covalent bonding, and the corresponding element precursor is mixed in during the bonding process. Organic complexes of the elements; finally, functional molecules are grafted onto the outer surface of the biocompatible layer through amide reaction, while loading targeted synergistic elements. The modified material is purified by centrifugation; (3) Assembly of dual quantum dot resonance units, carrier composite and material molding: The modified quantum dots are dispersed in the precursor solution of the auxiliary carrier matrix, and a salt solution of the matrix composite elements is added. After stirring evenly, the spacing between the two quantum dots is controlled to be 5-20 nm by photolithography micro-nano processing or self-assembly technology to form dual quantum dot resonance units and fix them in the carrier matrix. They are then cross-linked and cured or freeze-dried to form the material; (4) Performance calibration and screening: The resonance frequency, biocompatibility and stability of the material are detected, and the content, in vitro release rate and migration amount of each trace element are detected. The finished products that meet the requirements are screened.

[0014] Furthermore, when the core quantum dot is a graphene quantum dot, it is prepared by electrochemical exfoliation combined with hydrothermal modification; when the core quantum dot is an InP quantum dot, it is prepared by thermal injection.

[0015] Furthermore, in step (2), the centrifugal purification speed is 8000-12000 r / min; in step (3), the cross-linking curing temperature is 37-60℃, and when the auxiliary carrier matrix is ​​polylactic acid-hydroxyacetic acid copolymer, electrospinning technology is used for molding.

[0016] The aforementioned quantum dot resonant biomaterials can be applied to the preparation of healthcare products, including nanodiagnostic probes, therapeutic gels, wound repair dressings, and tissue engineering scaffold coatings. These products can be used for early tumor diagnosis, targeted therapy for osteoarthritis, postoperative wound repair, bone defect repair, and chronic diabetic wound repair. Various biocompatible trace elements can exert their bioactivity differently depending on the application scenario. For example, in bone repair scenarios, the ratio of Ca, P, and Sr can be enhanced, while in anti-inflammatory scenarios, the ratio of Zn, Se, and Mn can be enhanced.

[0017] Compared with existing technologies, this invention has the following significant advantages: First, significantly improved signal strength and deep penetration. Through the resonance effect of dual quantum dot resonant units, this invention achieves a 100-200 times increase in resonance signal strength compared to single quantum dots, effectively solving the problems of insufficient signal strength and limited deep tissue penetration in existing technologies. It can accurately capture weak biophoton signals from diseased tissues, with a maximum deep tissue penetration depth of up to 4.5 cm. Second, it possesses the ability to directionally regulate biophoton coherence. Based on the theory of biophoton coherence and cooperative radiation effect, this invention, through the energy superposition effect of dual quantum dot resonant units, can directionally regulate the biophoton coherence of diseased tissues, promoting the recovery of the physiological state of diseased tissues. This represents a technological breakthrough from a single imaging / drug delivery function to a multi-functional synergistic approach of "diagnosis-physiotherapy-repair." Third, excellent biosafety and environmental stability. This invention employs a three-layer composite structure design—a passivation layer, a biocompatibility layer, and a functional layer—to effectively prevent direct contact between quantum dots and biological media, inhibiting the leakage of toxic ions. The content and release of various trace elements strictly comply with the safety limits of the ICH Q3D guidelines. After degradation of the auxiliary carrier matrix, there are no toxic residues, and metabolites and trace elements can be safely excreted through the kidneys and feces, posing no risk of accumulation in the body. Fourth, its functions are customizable to adapt to various application scenarios. This invention allows for flexible adjustment of the type, size, resonant frequency, and trace element composition of quantum dots according to different application scenarios, enabling precise diagnosis and targeted treatment of different tissues and diseases. It has broad application prospects in multiple fields such as orthopedics, dermatology, tumor diagnosis, and rehabilitation therapy. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0019] The raw materials used in the following embodiments of the present invention are all commercially available analytical grade, and the detection equipment and methods used are uniformly standardized as follows: the size and morphology of quantum dots are characterized by a JEM-2100F transmission electron microscope (TEM) from Japan Electronics Corporation. Fluorescence properties and quantum yield were measured using a Hitachi F-7000 fluorescence spectrophotometer and an absolute quantum yield analyzer; resonance frequency and signal enhancement factor were measured using a self-made biophotonic coherence detection system with a photomultiplier tube (PMT). Biocompatibility was assessed using the CCK-8 assay to determine cell viability, and cytotoxicity was evaluated according to ISO 10993-5:2009. The content and release of trace elements were detected using a PerkinElmer ElanDRC-e inductively coupled plasma mass spectrometer (ICP-MS); the degradation performance was tested using an in vitro simulated physiological environment degradation test, in accordance with the standard GB / T16886.13-2017. The coherence order of biophotons is characterized by the second-order coherence detection method (g²(τ)). The closer g²(τ) is to 1, the higher the coherence order.

[0020] Example 1: Bilayer silica-coated perovskite quantum dot resonant hydrogel material for arthritis physiotherapy

[0021] The hydrogel material of this embodiment, by mass percentage, comprises the following core components: 0.5 parts of formamidinium lead bromide (CsFAPbBr3) perovskite quantum dot pairs; a surface modification system including 2 parts of dense silica passivation layer, 5 parts of PEG2000 biocompatibility layer, and 0.2 parts of anti-inflammatory targeted peptide (TAIP) functional layer; 20 parts of medical-grade hyaluronic acid (molecular weight 1.2 million Da); and a trace element system containing various biocompatibility elements, including core doping elements Zn, Mn, and Co; passivation layer loading elements Ti, Zr, F, and Si; biocompatibility layer loading elements Ni, V, B, and Al; and matrix composite elements Mg, Li, Ba, Ca, P, and Sr. The content and release of each element comply with the safety limits of the ICH Q3D guidelines.

[0022] The preparation process in this embodiment is as follows: First, a cross-shaped channel microfluidic chip is used to accurately synthesize perovskite quantum dots and perform trace element doping. The chip has a channel width of 100 μm and a depth of 50 μm. An oil-phase precursor solution and an aqueous-phase doping precursor solution are prepared. The oil-phase precursor solution is an octadecene (ODE) solution containing 0.1 mmol / mL LPbBr2, 0.1 mmol / mL LCsBr, and 0.1 mmol / mL FABr, with oleic acid (OA) and oleylamine (OAm) added in a 1:1 volume ratio as ligands. The aqueous-phase doping precursor solution contains ZnCl2, MnCl2, and Co. Ultrapure aqueous solutions of Cl2 were prepared at concentrations of 0.05 mM, 0.03 mM, and 0.04 mM. The oil phase flow rate was controlled at 20 μL / min and the aqueous phase flow rate at 5 μL / min using a dual-channel syringe pump. After mixing, the mixture was introduced into a heated reaction zone at a controlled temperature of 120 °C and a reaction residence time of 15 s. The effluent was quenched in an ice bath, and then anhydrous ethanol was added. The mixture was centrifuged at 10000 r / min for 5 min. This purification process was repeated three times to obtain the target quantum dots. TEM characterization showed that the quantum dots had an average size of 8 nm, a batch-to-batch size deviation of ≤0.3 nm, and an absolute photoluminescence quantum yield of 88%.

[0023] After quantum dot synthesis, multilayer surface modification and trace element loading were performed. The purified quantum dots were first dispersed in a 4:1 mixture of cyclohexane and n-hexanol, emulsified with Triton X-100, and the pH was adjusted to 9.0 with ammonia. Tetrabutyl orthosilicate (TEOS) was added dropwise, along with tetrabutyl titanate, tetrabutyl zirconate, and ammonium fluoride precursors. The final concentrations of Ti, Zr, and F were controlled to be 0.02 mM, 0.02 mM, and 0.03 mM, respectively. The reaction was carried out at 25°C with stirring for 12 h, forming a dense passivation layer 2 nm thick on the quantum dot surface. After the reaction, the surface was demulsified with acetone, centrifuged at 10000 r / min for 5 min, and washed three times alternately with cyclohexane and anhydrous ethanol. The passivated quantum dots were then dispersed in PBS at pH 7.4. The reaction mixture was prepared by adding amino-modified PEG2000 and reacting it with silanol-amino covalent bonding at 25°C for 6 hours. During the reaction, boric acid, ammonium metavanadate, and nickel chloride organic complex were added to control the final concentrations of B, V, and Ni elements to be 0.02 mM, 0.015 mM, and 0.01 mM, respectively. After the reaction was completed, the mixture was purified by ultrafiltration three times using a 100 kDa ultrafiltration tube. Finally, the PEG-modified quantum dots were dispersed in MES buffer at pH 6.0, and EDC and NHS were added to activate the carboxyl groups at the PEG ends for 30 minutes. Then, the anti-inflammatory targeting peptide TAIP and sodium selenite were added to control the final concentration of Se element to be 0.02 mM. The mixture was stirred at 25°C in the dark for 4 hours and purified by centrifugation at 10000 r / min three times to obtain multilayer modified perovskite quantum dots.

[0024] Subsequent quantum dot pair assembly and hydrogel molding were carried out. First, medical hyaluronic acid powder was dissolved in sterile PBS buffer to prepare a 2% (w / w) hydrogel precursor solution. Then, the modified quantum dots were added to the precursor solution, and the final concentration of quantum dots was controlled at 0.5 mg / mL. After stirring evenly, sterile aqueous solutions of magnesium chloride, lithium chloride, barium chloride, calcium chloride, sodium dihydrogen phosphate, and strontium chloride were added to control the final concentrations of Mg, Li, Ba, Ca, P, and Sr elements to be 0.1 mM, 0.05 mM, 0.03 mM, 0.08 mM, 0.06 mM, and 0.02 mM, respectively. The remaining trace elements were added to make multiple elements. The mixture was stirred at low speed at 4℃ for 12 h to regulate the self-assembly of quantum dots into quantum dot pairs. TEM characterization showed that the average spacing of the quantum dot pairs was 8 nm, and they were uniformly dispersed in a regular hexagonal array. Then, 0.1% crosslinking agent BDDE was added, and after stirring and degassing, the mixture was injected into a mold and crosslinked and cured in a sterile environment at 37℃ for 2 h to obtain a flexible adhesive hydrogel with a thickness of 0.5 mm.

[0025] The hydrogel material prepared in this embodiment was systematically tested, and the comparison of its core performance indicators with those of Comparative Example 1 is shown in Table 1: Table 1 Comparison of Core Performance Testing between Example 1 and Comparative Example 1

[0026] To verify the practical application effect of the material in this embodiment, 12 volunteers with knee osteoarthritis were randomly divided into an experimental group and a control group, with 6 people in each group. The experimental group used the hydrogel patch of this embodiment applied to the affected knee joint twice a day for 30 minutes each time, while the control group used commercially available ordinary hyaluronic acid therapy patches. The usage methods were the same, and the intervention period was 2 weeks. The specific data of the experimental results are shown in Table 2: Table 2 Comparison of Clinical Application Effects of Example 1

[0027] Comparative Example 1 was set up as a control. The difference between this comparative example and the present embodiment is that a single perovskite quantum dot was used, no quantum dot pair structure was constructed, only a single layer of silica was used for modification, there was no PEG biocompatible layer and target functional layer, and no trace element system doping and loading were performed. The rest of the processes and parameters were completely the same.

[0028] Example 2: Graphene quantum dot nanofiber membrane for postoperative wound repair

[0029] Based on the aforementioned design concept of quantum dot resonant biomaterials, this embodiment provides a graphene quantum dot nanofiber membrane adapted for postoperative wound repair. By mass, the core formulation components are as follows: the core functional component is 0.3 parts of graphene quantum dots (GQDs); the surface modification system includes 1.5 parts of mercaptopropionic acid (MPA) passivation layer, 4 parts of phospholipid micelle biocompatibility layer, 0.15 parts of RGD healing-promoting peptide functional layer; the auxiliary carrier matrix is ​​18 parts of medical-grade PLGA (LA:GA=75:25, molecular weight 80,000 Da); and it is combined with a trace element system containing multiple biocompatible elements, wherein the core doping elements are Fe, Cu, and Mn; the passivation layer loading elements are F, Si, and Ti; the biocompatibility layer loading elements are B, Al, and V; and the matrix composite elements are Ca, P, Mg, Zn, Sr, etc. The content and release of each element comply with the safety limits of the ICH Q3D guidelines.

[0030] The preparation process of this embodiment is as follows: First, the precise synthesis and trace element doping of graphene quantum dots were completed by electrochemical exfoliation combined with hydrothermal modification. Highly oriented pyrolytic graphite (HOPG) was used as the working electrode, a platinum sheet as the counter electrode, an Ag / AgCl electrode as the reference electrode, and a 0.1M sodium sulfate aqueous solution as the electrolyte. A constant voltage of 10V was applied for electrochemical exfoliation for 2 hours to obtain a graphene oxide dispersion. Then, the dispersion was transferred to a hydrothermal reactor, and aqueous solutions of ferric chloride, copper chloride, and manganese chloride were added. The final concentrations of Fe, Cu, and Mn were controlled to be 0.03mM, 0.02mM, and 0.04mM, respectively. The hydrothermal reaction was carried out at 200℃ for 6 hours. After natural cooling, the mixture was filtered through a 0.22μm filter membrane and dialyzed through a 1000Da dialysis bag for 72 hours. After dialysis, the mixture was freeze-dried to obtain graphene quantum dot powder. TEM characterization showed that the average size of the quantum dots was 4nm, the batch size deviation was ≤0.2nm, and the absolute photoluminescence quantum yield was 91%.

[0031] After quantum dot synthesis, multilayer surface modification and trace element loading were performed. First, graphene quantum dots were dispersed in ultrapure water to prepare a 1 mg / mL dispersion. Mercaptopropionic acid was added to adjust the pH to 8.0, and the reaction was carried out with stirring in a 60℃ water bath for 4 hours. During the reaction, ammonium fluoride, TEOS, and tetrabutyl titanate were added, and the final concentrations of F, Si, and Ti were controlled to be 0.03 mM, 0.05 mM, and 0.02 mM, respectively. After the reaction was completed, the mixture was purified by dialysis for 48 hours. Subsequently, phospholipid micelles were prepared using a thin-film dispersion method, and soybean lecithin, cholesterol, and passivated quantum dots were mixed according to their mass... The mixture was dissolved in chloroform at a ratio of 10:2:1, and the lipid film was formed by rotary evaporation. After vacuum drying for 12 h, PBS buffer was added for hydration, and boric acid and aluminum chloride were added during the process. The final concentrations of B and Al were controlled to be 0.02 mM and 0.01 mM, respectively. The biocompatibility layer was constructed by sonication in a water bath at 37 °C for 30 min. Finally, maleimide-modified RGD peptide and potassium iodide were added to the micelle dispersion, and the final concentration of I was controlled to be 0.015 mM. The mixture was stirred at 25 °C in the dark for 3 h, and purified by ultrafiltration three times to obtain multilayer modified graphene quantum dots.

[0032] The subsequent assembly of quantum dot pairs and the formation of nanofiber membranes were carried out. First, PLGA powder was dissolved in hexafluoroisopropanol to prepare a spinning solution with a mass fraction of 10%. Then, the modified quantum dots were added to the spinning solution, and the final concentration of quantum dots was controlled at 0.3 mg / mL. After stirring evenly, aqueous solutions of calcium chloride, sodium dihydrogen phosphate, magnesium chloride, zinc chloride, and strontium chloride were added to control the final concentrations of Ca, P, Mg, Zn, and Sr elements to be 0.2 mM, 0.15 mM, 0.1 mM, 0.08 mM, and 0.03 mM, respectively. The remaining trace elements were added to make multiple elements. Subsequently, high-voltage electrospinning was used to form the membrane. The spinning voltage was set at 15 kV, the receiving distance was 15 cm, the injection speed was 0.8 mL / h, the ambient temperature was 25 ℃, and the relative humidity was 40%. During the spinning process, the spacing between quantum dot pairs was controlled by an electric field to be 12 nm, so that they were uniformly distributed in an array in the fiber. After spinning, the membrane was vacuum dried for 24 h, sterilized with ethylene oxide, and then sealed and stored to obtain a nanofiber membrane with a thickness of 0.2 mm.

[0033] The nanofiber membrane prepared in this embodiment was systematically tested, and the comparison of its core performance indicators with those of Comparative Example 2 is shown in Table 3. Table 3 Comparison of Core Performance Tests between Example 2 and Comparative Example 2

[0034] To verify the practical application effect of the material in this embodiment, a full-thickness skin defect model of SD rats was constructed, with a defect area of ​​2cm × 2cm. The rats were randomly divided into an experimental group and a control group, with 8 rats in each group. The experimental group was covered with the nanofiber membrane of this embodiment, while the control group was covered with commercially available medical PLGA dressing. The dressings were changed every 3 days. Specific experimental results are shown in Table 4. Table 4 Comparison of Repair Effects in Example 2 Rat Full-Thickness Skin Defect Model

[0035] Comparative Example 2 was set up as a control. The difference between this comparative example and the present embodiment is that a single graphene quantum dot was used, no quantum dot pair structure was constructed, only mercaptopropionic acid was used for passivation, there was no phospholipid micelle biocompatibility layer and RGD functional layer, and no trace element system doping and loading were performed. The other processes and parameters were completely the same.

[0036] Example 3: InP quantum dot porous titanium alloy scaffold coating for bone defect repair

[0037] Based on the core technical solution of this invention, this embodiment provides an InP quantum dot porous titanium alloy scaffold coating adapted for bone defect repair scenarios. By mass, the core formulation components are: 0.4 parts of indium phosphide (InP) quantum dot pair as the core functional component; 1.8 parts of silicon dioxide passivation layer, 4.5 parts of PEG4000 biocompatibility layer, 0.25 parts of BMP-2 osteogenic mimic peptide functional layer; 22 parts of medical-grade chitosan-hydroxyapatite composite matrix as the auxiliary carrier matrix; and a trace element system containing multiple biocompatibility elements, wherein the core doping elements are Zn, Se, and Mn; the passivation layer loading elements are Si, Ti, Zr, and F; the biocompatibility layer loading elements are Sr, Mg, and B; and the matrix composite elements are Ca, P, Fe, and Cu, etc. The content and release of each element comply with the safety limits of the ICH Q3D guidelines.

[0038] The preparation process of this embodiment is as follows: First, InP / ZnSe core-shell structured quantum dots were prepared by hot injection method and trace element doping was completed. 0.2 mmol of tris(dimethylamino)phosphine and 0.1 mmol of indium chloride were dissolved in 10 mL of octadecene, 3 mL of oleic acid was added, and the mixture was degassed under vacuum at 120 °C for 1 h to obtain the In precursor solution. At the same time, ZnCl2, SeO2, and MnCl2 were dissolved in octadecene to prepare the doping precursor solution. The final concentrations of Zn, Se, and Mn were controlled to be 0.06 mM, 0.04 mM, and 0.03 mM, respectively. Under nitrogen protection, the In precursor solution was heated to 180 °C and rapidly injected into the doping precursor solution. After holding the reaction at this temperature for 20 min, the reaction was quenched in an ice bath. The product was centrifuged at 12000 r / min for 8 min with anhydrous ethanol. The purification was repeated 4 times to obtain the target quantum dots. TEM characterization showed that the average size of the quantum dots was 6 nm, the batch size deviation was ≤0.3 nm, and the absolute photoluminescence quantum yield was 90%.

[0039] After the quantum dot synthesis was completed, multilayer surface modification and trace element loading were performed. First, an inverse microemulsion method was used to disperse InP quantum dots in cyclohexane. Triton X-100, n-hexanol, and ammonia were added to form a microemulsion. TEOS was then added dropwise, simultaneously with tetrabutyl titanate and ammonium fluoride. The final concentrations of Ti and F were controlled to be 0.03 mM and 0.04 mM, respectively. The reaction was stirred at 30°C for 10 h to form a 3 nm thick dense passivation layer. After demulsification, centrifugation, and washing, the passivated quantum dots were obtained. Subsequently, the amount of... The quantum dots were dispersed in PBS buffer, and silanized PEG4000 was added. The mixture was stirred at 25°C for 8 hours. During the reaction, strontium chloride, magnesium chloride, and boric acid were added to control the final concentrations of Sr, Mg, and B elements to be 0.05 mM, 0.08 mM, and 0.02 mM, respectively. The biocompatibility layer was constructed by ultrafiltration purification. Finally, the BMP-2 osteogenic mimic peptide was grafted onto the carboxyl group at the end of the PEG using the EDC / NHS activation method. The mixture was reacted at 25°C in the dark for 4 hours. After centrifugation purification, multilayer modified InP quantum dots were obtained.

[0040] The subsequent assembly of quantum dot pairs and the coating of the scaffold were carried out. First, the 3D-printed porous titanium alloy scaffold was pretreated. This scaffold had a porosity of 65% and a pore size of 300-500 μm. It was ultrasonically cleaned sequentially with acetone, anhydrous ethanol, and ultrapure water for 15 min, followed by NaOH alkaline heat treatment for 24 h, and then rinsed and dried for later use. Next, chitosan was dissolved in a 2% acetic acid solution to prepare a 3% chitosan solution. Nano-hydroxyapatite powder was added, with a chitosan to hydroxyapatite mass ratio of 7:3. After stirring evenly, modified I was added. nP quantum dots were used, with a final concentration of 0.4 mg / mL. The quantum dots were arrayed with a spacing of 10 nm. Simultaneously, salt solutions of calcium, phosphorus, iron, and copper were added to supplement trace elements to obtain a coating precursor solution. Finally, the precursor solution was coated onto the scaffold surface using an dip-coating method at a lifting speed of 2 mm / s. The scaffold was dried at 40 °C and the coating was repeated 3 times to obtain a uniform coating with a thickness of 20 μm. The coating was then crosslinked and cured with glutaraldehyde vapor for 2 h. Residual crosslinking agent was rinsed off with sterile PBS, and the scaffold was sterilized with ethylene oxide and then sealed for storage.

[0041] The scaffold coating prepared in this embodiment was systematically tested, and the comparison of its core performance indicators with those of Comparative Example 3 is shown in Table 5. Table 5 Comparison of Core Performance Tests between Example 3 and Comparative Example 3

[0042] To verify the practical application effect of the material in this embodiment, a New Zealand rabbit radial bone critical bone defect model with a defect length of 15 mm was constructed. Rabbits were randomly divided into an experimental group and a control group, with 6 rabbits in each group. The experimental group received the coated scaffold of this embodiment, while the control group received an uncoated ordinary titanium alloy scaffold. Bone repair effects were evaluated by Micro-CT and histological examination at 4, 8, and 12 weeks post-operation. Specific experimental results are shown in Table 6. Table 6 Comparison of Repair Effects in Rabbit Radial Critical Bone Defect Model of Example 3

[0043] Comparative Example 3 was set up as a control. The difference between this comparative example and the present embodiment is that a single InP quantum dot was used, no quantum dot pair structure was constructed, only a single layer of silica was used for modification, there was no PEG biocompatibility layer and osteogenic targeting functional layer, and no trace element system doping and loading were performed. The rest of the processes and parameters were completely the same.

[0044] Example 4: Graphene quantum dot targeted nanoprobes for early tumor diagnosis

[0045] Based on the core principle of quantum dot resonance of the present invention, this embodiment provides a graphene quantum dot targeted nanoprobe adapted to the early diagnosis of tumors. By mass fraction, the core formulation components are: 0.2 parts of nitrogen-doped graphene quantum dots (N-GQDs) as the core functional component; 1.2 parts of a thioglycolic acid passivation layer; 3.5 parts of a DSPE-PEG2000 biocompatibility layer; and 0.3 parts of an RGD peptide + EpCAM antibody dual-targeting functional layer. It is also equipped with a trace element system containing a variety of biocompatibility elements, wherein the core doping elements are N, Fe, Cu, and Mn; the passivation layer loading elements are F and Si; and the biocompatibility layer loading elements are Se, I, and B. The content and release amount of each element meet the safety limits of the ICH Q3D guidelines.

[0046] The preparation process of this embodiment is as follows: First, nitrogen-doped graphene quantum dots were prepared by hydrothermal method and trace element doping was completed. Citric acid and urea were dissolved in ultrapure water at a mass ratio of 1:2 to prepare a 0.2 g / mL mixed solution. Aqueous solutions of ferric chloride, copper chloride, and manganese chloride were added, and the final concentrations of Fe, Cu, and Mn were controlled to be 0.02 mM, 0.01 mM, and 0.03 mM, respectively. After stirring evenly, the solution was transferred to a hydrothermal reactor and hydrothermally reacted at 180°C for 4 h. After natural cooling to room temperature, the product was filtered through a 0.22 μm filter membrane and dialyzed through a 500 Da dialysis bag for 48 h. After freeze-drying, nitrogen-doped graphene quantum dots were obtained. TEM characterization showed that the average size of the quantum dots was 3 nm, the batch size deviation was ≤0.2 nm, and the absolute photoluminescence quantum yield was 93%.

[0047] After quantum dot synthesis, multilayer surface modification and trace element loading were performed. First, N-GQDs were dispersed in ultrapure water, and mercaptoacetic acid was added to adjust the pH to 7.5. The reaction was carried out with stirring in a 50°C water bath for 3 hours. During the reaction, ammonium fluoride and TEOS were added, and the final concentrations of F and Si were controlled to be 0.02 mM and 0.04 mM, respectively. After the reaction was completed, the mixture was purified by dialysis for 24 hours. Subsequently, DSPE-PEG2000 and the passivated N-GQDs were dissolved in chloroform at a mass ratio of 5:1, and the mixture was rotary evaporated to form a film. After vacuum drying for 12 hours, the film was hydrated with PBS buffer. Sodium selenite, potassium iodide, and boric acid were added during the ultrasound process, and the final concentrations of Se, I, and B elements were controlled to be 0.03 mM, 0.02 mM, and 0.01 mM, respectively, to obtain PEGylated quantum dot micelles and complete the construction of the biocompatibility layer. Finally, RGD peptides and EpCAM monoclonal antibodies were grafted onto the PEG ends using the EDC / NHS activation method. The reaction was carried out at 25°C with light-protected stirring for 4 h. After ultrafiltration purification, dual-targeted modified graphene quantum dot pairs were obtained. The spacing between the quantum dot pairs was adjusted to 6 nm to precisely match the resonant frequency with the biophoton frequency of tumor tissue.

[0048] The nanoprobes prepared in this embodiment were systematically tested, and the comparison of their core performance indicators with those of Comparative Example 4 is shown in Table 7. Table 7 Comparison of Core Performance Tests between Example 4 and Comparative Example 4

[0049] To verify the practical application effect of the probe in this embodiment, serum samples from 20 clinically diagnosed liver cancer patients and 20 healthy volunteers were selected. The probe in this embodiment was used to detect tumor exosomes, and a commercially available ELISA kit was used to detect AFP. The diagnostic efficacy was compared. Specific experimental results are shown in Table 8. Table 8. Comparison of diagnostic efficacy between Example 4 and conventional AFP testing.

[0050] Comparative Example 4 was set up as a control. The difference between this comparative example and the present embodiment is that it uses a single N-GQDs, does not construct a quantum dot pair structure, does not have targeted functional layer modification, and does not perform trace element system doping and loading. The other processes and parameters are completely the same.

[0051] Example 5: Perovskite quantum dot thermosensitive hydrogel for repairing chronic wounds in diabetic patients

[0052] Based on the technical solution of this invention, this embodiment provides a perovskite quantum dot thermosensitive hydrogel adapted to the repair of chronic diabetic wounds. By mass, the core formulation components are: 0.45 parts of cesium lead bromide (CsPbBr3) perovskite quantum dot pair as the core functional component; 2.2 parts of a bilayer silica passivation layer; 5.5 parts of a PEG-PLGA-PEG triblock copolymer biocompatibility layer; 0.22 parts of a bee venom peptide antimicrobial peptide functional layer; 25 parts of a poloxamer 407 thermosensitive hydrogel matrix as the auxiliary carrier matrix; and a trace element system containing multiple biocompatible elements, wherein the core doping elements are Zn, Mn, and Se; the passivation layer loading elements are Si, Ti, and F; the biocompatibility layer loading elements are Ag, Cu, and B; and the matrix composite elements are Ca, P, Mg, and Sr, etc. The content and release of each element comply with the safety limits of the ICH Q3D guidelines.

[0053] The preparation process in this embodiment is as follows: First, perovskite quantum dots are prepared using microfluidic synthesis technology and trace element doping is completed. An oil-phase precursor solution and an aqueous-phase doping precursor solution are prepared. The oil-phase precursor solution is an ODE solution containing 0.1 mmol / mL LpbBr2 and 0.1 mmol / mL LcsBr, with OA and OAm added as ligands in a 1:1 volume ratio. The aqueous-phase doping precursor solution is an aqueous solution containing ZnCl2, MnCl2, and Na2SeO3, with concentrations of 0.06%. mM, 0.04mM, and 0.03mM were used. A microfluidic chip with a channel width of 150μm was employed to control the oil phase flow rate at 25μL / min and the water phase flow rate at 6μL / min. The reaction temperature was 130℃, the reaction residence time was 12s, and the reaction was quenched in an ice bath. The product was centrifuged at 11000r / min for 6min and purified three times to obtain the target quantum dots. TEM characterization showed that the average size of the quantum dots was 7nm, the batch size deviation was ≤0.3nm, and the absolute photoluminescence quantum yield was 89%.

[0054] After quantum dot synthesis, multilayer surface modification and trace element loading were performed. First, a 2nm thick dense silica layer was coated using a reverse microemulsion method, during which TEOS, tetrabutyl titanate, and ammonium fluoride were added, and the final concentrations of Ti and F were controlled to be 0.025mM and 0.035mM, respectively. Then, a 3nm thick mesoporous silica layer was coated to obtain core-shell structured quantum dots, which effectively suppressed lead ion leakage. Subsequently, the quantum dots were dispersed in PBS buffer, and PEG-PLGA-PEG triblock copolymer was added. The reaction was stirred at 30℃ for 6h, during which silver nitrate, copper chloride, and boric acid were added, and the final concentrations of Ag, Cu, and B were controlled to be 0.01mM, 0.02mM, and 0.015mM, respectively. The biocompatibility layer was constructed by ultrafiltration purification. Finally, the bee venom antimicrobial peptide was grafted to the end of the copolymer using the EDC / NHS activation method, and the reaction was carried out at 25℃ in the dark for 3h. The multilayer modified perovskite quantum dots were obtained by centrifugation purification.

[0055] The subsequent assembly of quantum dot pairs and thermosensitive hydrogel formation were carried out. First, poloxamer 407 powder was dissolved in sterile PBS buffer at 4°C to prepare a 20% (w / w) solution, which was then refrigerated at 4°C. The modified quantum dots were then added to the precursor solution, and the final concentration of quantum dots was controlled at 0.45 mg / mL. The mixture was stirred at low speed at 4°C for 12 h to adjust the spacing between quantum dot pairs to 9 nm, dispersing them in a regular hexagonal array. Simultaneously, solutions of calcium, phosphorus, magnesium, strontium, and other salts were added to supplement trace elements to a variety of levels. The precursor solution was filtered through a 0.22 μm sterile filter membrane and then aseptically dispensed and refrigerated at 4°C. The solution was liquid at 4°C and rapidly transformed into a gel state above 32°C.

[0056] The thermosensitive hydrogel prepared in this embodiment was systematically tested, and the core performance indicators compared with those of Comparative Example 5 are shown in Table 9: Table 9 Comparison of Core Performance Tests between Example 5 and Comparative Example 5

[0057] To verify the practical application effect of the hydrogel in this embodiment, a diabetic full-thickness skin defect model was constructed in SD rats. The rats were randomly divided into an experimental group and a control group, with 8 rats in each group. The experimental group received the thermosensitive hydrogel applied to the wound, while the control group received commercially available diabetic wound dressings. Dressings were changed every two days. Specific experimental results are shown in Table 10. Table 10 Comparison of wound repair effects in diabetic rats in Example 5

[0058] Comparative Example 5 was set up as a control. The difference between this comparative example and the present embodiment is that a single perovskite quantum dot was used, no quantum dot pair structure was constructed, no antimicrobial peptide functional layer modification was performed, and no trace element system doping and loading were performed. The other processes and parameters were completely the same.

[0059] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any technical solutions that adjust the type, size, spacing, surface modification system, carrier matrix, and trace element ratio of quantum dots to adapt to different healthcare scenarios, based on the core technical concept of the present invention, should be included within the scope of protection of the present invention.

Claims

1. A quantum dot resonant biomaterial, comprising a core functional component, a surface modification layer, and an auxiliary carrier matrix, characterized in that: The core functional component is a dual quantum dot resonance unit, which consists of two low-toxicity quantum dots of the same material and structure. The size of a single quantum dot is 2-10 nm, and the size deviation of quantum dots in the same batch is ≤0.5 nm. The dual quantum dot resonance unit is fixed in the auxiliary carrier matrix at a spacing of 5-20 nm, and its resonant emission band covers 600-1100 nm, and the resonant frequency is adapted to the biophoton radiation frequency of the target biological tissue. The surface modification layer is a three-layer composite structure consisting of a passivation layer, a biocompatibility layer, and a functional layer that are sequentially coated on the outer surface of a single quantum dot from the inside out. The quantum dot resonant biomaterial also contains a variety of biocompatible trace elements.

2. The quantum dot resonant biomaterial according to claim 1, characterized in that, The low-toxicity quantum dots are selected from graphene quantum dots, bilayer silica-coated perovskite quantum dots, or InP quantum dots, and the photoluminescence quantum yield of the low-toxicity quantum dots is ≥85%.

3. The quantum dot resonant biomaterial according to claim 1, characterized in that, The various biocompatible trace elements include, but are not limited to, Zn, Se, Te, Cd, Ga, In, Fe, Cu, Co, Mn, Cr, Mo, F, Si, Ti, Zr, Sn, B, V, Ni, Al, I, As, Ca, P, Mg, Sr, and Ba.

4. The quantum dot resonant biomaterial according to claim 3, characterized in that, The daily in vitro release of Cd, As, and Ni elements does not exceed the permissible daily exposure limit for the corresponding elements, and the daily in vitro release of F element is 0.5-1 mg.

5. The quantum dot resonant biomaterial according to claim 1, characterized in that, The passivation layer is a silicon dioxide layer or a mercaptopropionic acid layer with a thickness of 2-5 nm; the biocompatibility layer is a polyethylene glycol layer or a phospholipid micelle layer with a cytotoxic IC50 ≥ 50 μM; the functional layer is grafted with targeted functional molecules that match the application scenario.

6. The quantum dot resonant biomaterial according to claim 1, characterized in that, The auxiliary carrier matrix is ​​selected from biodegradable nanofibers, water-soluble hydrogels, or hyaluronic acid, and its in vitro degradation rate can be controlled within the range of 1-30 days; the dual quantum dot resonance units are uniformly fixed in the auxiliary carrier matrix in a regular hexagonal array.

7. A method for preparing a quantum dot resonant biomaterial according to any one of claims 1 to 6, characterized in that, Includes the following steps: (1) Precise synthesis of quantum dots and trace element doping: Core quantum dots were prepared using microfluidic synthesis technology, and core doping elements were introduced simultaneously through precursor doping. The reaction temperature was controlled at 80-180℃, and the element precursor concentration was 0.01-0.1mM, so that trace elements were uniformly dispersed inside the quantum dots. The size deviation of the obtained quantum dots was controlled to be ≤0.3nm, and the photoluminescence quantum yield was ≥85%. (2) Multilayer surface modification and trace element loading: First, a dense passivation layer is coated on the surface of quantum dots by sol-gel method, and corresponding element precursors are added during the coating process; then, a biocompatible layer is formed outside the passivation layer by covalent bonding, and corresponding organic complexes are mixed in during the bonding process; finally, functional molecules are grafted onto the outer surface of the biocompatible layer by amide reaction, and targeted synergistic elements are loaded at the same time. The modified material is purified by centrifugation. (3) Assembly of dual quantum dot resonant units, carrier composite and material forming: The modified quantum dots are dispersed in the precursor solution of the auxiliary carrier matrix, and a salt solution of the matrix composite element is added. After stirring evenly, the spacing between the two quantum dots is controlled to be 5-20nm by photolithography micro-nano processing or self-assembly technology to form dual quantum dot resonant units and fix them in the carrier matrix. They are then formed by cross-linking curing or freeze-drying. (4) Performance calibration and screening: The resonant frequency, biocompatibility and stability of the material are tested, and the content, in vitro release rate and migration of each trace element are tested to screen the finished products that meet the requirements.

8. The preparation method according to claim 7, characterized in that, When the core quantum dot is a graphene quantum dot, it is prepared by electrochemical exfoliation combined with hydrothermal modification; when the core quantum dot is an InP quantum dot, it is prepared by thermal injection.

9. The preparation method according to claim 7, characterized in that, In step (2), the centrifugation speed is 8000-12000 r / min; in step (3), the cross-linking curing temperature is 37-60℃, and when the auxiliary carrier matrix is ​​polylactic acid-hydroxyacetic acid copolymer, electrospinning technology is used for molding.

10. The application of quantum dot resonant biomaterials according to any one of claims 1 to 6 in the preparation of medical and health care products, wherein the medical and health care products include nanodiagnostic probes, physiotherapy gels, wound repair dressings, and tissue engineering scaffold coatings, and wherein the medical and health care products are used for early tumor diagnosis, targeted physiotherapy for osteoarthritis, postoperative wound repair, bone defect repair, and chronic diabetic wound repair.

Images