Preparation method of polypeptide composite material for biomedical additive manufacturing

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method of a polypeptide composite material for biomedical additive manufacturing, which comprises the following steps: encapsulating polypeptide in a nano-micelle formed by an amphiphilic block copolymer; dispersing in a visible-light response type hydrogel precursor to form biological ink; curing layer by layer by using visible light with the wavelength of 470nm to form a primary bracket; the mechanical property is enhanced through metal ion coordination secondary cross-linking; and finally performing gradient dehydration and freeze-drying to obtain a finished product. The polypeptide is prevented from being exposed to inactivation factors such as light, heat, free radicals and shear stress in the additive manufacturing process through double mechanisms of physical isolation and chemical shielding of the nano-micelles; a visible light initiation system and a pulse exposure strategy are utilized, and direct damage of high-energy radiation to polypeptide is eliminated while the crosslinking efficiency of a hydrogel network is maintained; stable dispersion and spatial anchoring of the polypeptide drug-loading unit in the bio-ink are realized, and migration segregation in the printing process is prevented.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically a method for preparing polypeptide composite materials for biomedical additive manufacturing. Background Technology

[0002] With the rapid development of regenerative medicine and precision medicine, biomedical materials are increasingly widely used in tissue engineering, drug delivery, and wound repair. Among these, additive manufacturing technology, with its customizable and high-precision spatial structure construction capabilities, has become an important means of fabricating complex biomimetic scaffolds. Against this backdrop, introducing peptide molecules with excellent bioactivity and specific recognition capabilities into additive manufacturing systems to construct composite materials that combine structural adaptability and functional guidance has become a cutting-edge direction in biomaterials research. These peptide composite materials can not only mimic the extracellular matrix microenvironment, promoting cell adhesion, proliferation, and directed differentiation, but also, through sequence design, regulate specific physiological signaling pathways, demonstrating great potential in the regeneration of tissues such as bone, cartilage, and nerves.

[0003] Currently, the mainstream technical approach for preparing peptide composite materials in biomedical additive manufacturing typically involves pre-dissolving or dispersing peptides in an aqueous or organic photosensitive resin, followed by molding using methods such as stereolithography, digital light processing, or extrusion printing. This method effectively controlled the spatial distribution of peptides within three-dimensional scaffolds in early studies and, to some extent, preserved their bioactivity. Especially under low-concentration, mild curing conditions, some short-chain peptides maintained conformational stability, thus exhibiting good cellular responsiveness in in vitro experiments. This strategy, due to its strong process compatibility and relatively low equipment requirements, was once considered a feasible path for developing functionalized bio-inks.

[0004] However, as clinical applications place increasingly higher demands on material performance, the aforementioned traditional preparation methods reveal a deep-seated and irreconcilable technical contradiction: a significant conflict exists between maintaining the bioactivity of peptides and ensuring their structural integrity during additive manufacturing. Fundamentally, the physical or chemical cross-linking mechanisms relied upon in additive manufacturing, such as UV-initiated polymerization, thermally induced gelation, or ionic cross-linking, are often accompanied by local energy input, solvent polarity abrupt changes, or interfacial shear stress. While these conditions facilitate rapid material prototyping and the establishment of mechanical properties, they can easily lead to conformational changes, disulfide bond mismatches, side chain modifications, and even main chain breakage in peptide molecules. It is noteworthy that the function of peptides is highly dependent on their precise secondary and even tertiary structures; even minute conformational perturbations can cause them to lose their targeted binding ability or signal transduction activity. In composite systems, peptides often tend to undergo non-uniform migration or interfacial enrichment during printing due to differences in hydrophilicity and hydrophobicity. This can result in excessively high local concentrations, exacerbating aggregation and inactivation, or excessively low concentrations, rendering them unable to exert their biological effects. Under this constraint, if the cross-linking strength is excessively reduced or the reaction time is shortened to protect peptide activity, the resulting scaffold will struggle to meet the mechanical support and structural fidelity required for implantation. Conversely, if molding quality is prioritized, the peptide function will almost inevitably suffer irreversible damage. This performance coupling conflict is particularly prominent in high-resolution, high-throughput printing scenarios and has become a core bottleneck restricting the translation of peptide composite materials from the laboratory to clinical applications.

[0005] Therefore, the present invention provides a method for preparing a polypeptide composite material for biomedical additive manufacturing. Summary of the Invention

[0006] To achieve the aforementioned objectives, this invention provides a method for preparing peptide composite materials using biomedical additive manufacturing. This method constructs a staged, multi-barrier synergistic additive manufacturing process system, effectively maintaining the high-order structural integrity of peptide molecules while ensuring the precision and mechanical stability of the three-dimensional scaffold structure, and achieving their precise spatial distribution and long-term functional activity.

[0007] The method of the present invention includes the following deterministic steps: First, the target peptide molecule is encapsulated within a nanomicelle core formed by the self-assembly of an amphiphilic block copolymer, forming a peptide drug delivery unit with a core-shell structure. Secondly, the polypeptide drug delivery unit is uniformly dispersed in a photoresponsive hydrogel precursor solution to form the basic phase of the bio-ink; Furthermore, in the additive manufacturing equipment, a wavelength-tunable visible light source is used instead of a traditional ultraviolet light source to selectively solidify the bio-ink layer by layer, forming a primary three-dimensional scaffold structure. Subsequently, the primary three-dimensional scaffold structure was placed in a buffer solution containing a metal ion crosslinking agent, and the mechanical reinforcement of the scaffold network was completed through a secondary crosslinking reaction mediated by coordination bonds. Finally, the obtained composite scaffold was subjected to gradient dehydration and freeze-drying to obtain the final peptide composite material product.

[0008] Preferably, the amphiphilic block copolymer is composed of hydrophilic polyethylene glycol segments and hydrophobic polylactic-co-glycolic acid copolymer segments linked by breakable ester bonds, with a critical micelle concentration of less than 0.1 mg / mL, spontaneously forming spherical micelles with a particle size distribution concentrated in the range of 80 to 120 nm under physiological pH conditions. The polypeptide molecules are confined within the hydrophobic core of the micelles, and their surface charges are completely shielded, thereby avoiding non-specific interactions with polar solvents or free radicals during subsequent processing.

[0009] Preferably, the preparation process of the peptide drug-loaded unit includes: dissolving peptide powder in a low-dielectric-constant organic solvent to form a first solution; dissolving an amphiphilic block copolymer in deionized water to form a second solution; injecting the first solution into the second solution at a constant flow rate under an inert gas protective atmosphere, while simultaneously applying high-frequency ultrasonic treatment to encapsulate the peptide molecules within the forming micelle core; subsequently, dialysis to remove the organic solvent to obtain a stable peptide drug-loaded micelle dispersion. This process ensures that the peptide is in a low-stress, low-oxygen, and low-free-radical microenvironment during the encapsulation stage, suppressing conformational perturbations at the source.

[0010] Preferably, the photoresponsive hydrogel precursor solution is composed of methacrylamide gelatin, methacrylamide hyaluronic acid, and a photoinitiator, wherein the photoinitiator is a visible light sensitization system based on a ruthenium complex, with its maximum absorption peak located in the 450-520 nm wavelength range. This photoinitiation system generates active free radicals through an electron transfer mechanism under visible light irradiation, initiating the polymerization of the precursor double bonds. The reaction rate can be precisely controlled by the light source intensity and exposure time. Compared to traditional ultraviolet photoinitiation systems, this visible light system avoids the direct excitation of aromatic residues of peptides by high-energy photons, thereby eliminating the photochemical degradation pathway.

[0011] Preferably, the additive manufacturing equipment is equipped with a digital micromirror device projection module, whose light source output wavelength is fixed at 470 nanometers, and the exposure energy density is set to a threshold level sufficient to induce precursor crosslinking but insufficient to penetrate the micelle shell. Under these conditions, the photopolymerization reaction occurs only in the hydrogel network outside the micelles, while the polypeptide molecules inside the micelles remain optically shielded and are not exposed to any photochemical stimuli.

[0012] Preferably, the thickness of the primary three-dimensional scaffold structure is controlled between 20 and 50 micrometers. Adjacent layers are mechanically interlocked through a pre-defined overlapping area, and the overall porosity is adjusted by the grayscale gradient of the projected pattern, forming a continuous transition structure from the dense surface area to the loose interior area. This structural design not only meets the initial mechanical load-bearing requirements of the implantation site but also provides channels for cell infiltration and nutrient transport.

[0013] Preferably, the metal ion crosslinking agent is a water-soluble salt of divalent zinc ions or trivalent iron ions, and its concentration is controlled within a range that does not cause micelle dissociation but is sufficient to form coordination bonds with carboxyl or phenolic hydroxyl groups in the hydrogel network. The secondary crosslinking process is carried out in a phosphate buffer solution at 37 degrees Celsius and a pH of 7.4 for a duration sufficient for the coordination network to reach thermodynamic equilibrium. This coordination crosslinking does not rely on covalent bond formation, therefore it does not generate additional free radicals or localized exothermics, thereby avoiding thermodynamic or chemical damage to the encapsulated peptide.

[0014] Preferably, the gradient dehydration process employs a stepwise solvent replacement method: first, the buffer solution in the pores of the scaffold is replaced with a low-concentration ethanol aqueous solution, and then the ethanol concentration is gradually increased until complete dehydration is achieved; the freeze-drying process is carried out under conditions of vacuum degree below 10 Pa and cold trap temperature below -50 degrees Celsius to ensure that the water is removed by sublimation and to prevent ice crystal growth from causing mechanical damage to the micelle structure.

[0015] Preferably, the polypeptide molecule is a cyclic peptide (cRGD) containing the arginine-glycine-aspartic acid sequence, and the molecular formula of the cyclic RGD peptide used is as follows: With a molecular weight of approximately 985.0 Da, its secondary structure is predominantly β-turn, exhibiting high affinity for integrin αvβ3 receptor binding. In its encapsulated state, the secondary structure of this peptide retains the β-turn characteristic as verified by circular dichroism spectroscopy, and its bioactivity was confirmed by in vitro cell adhesion experiments to have not undergone significant attenuation.

[0016] Preferably, the volume fraction of the peptide drug-loading unit in the bio-ink base phase is limited to between 0.5% and 5% to ensure sufficient spacing between micelles during printing and to avoid aggregation and precipitation due to excessive concentration. Simultaneously, the micelle surface is modified with a small number of methacrylamide groups, enabling them to be covalently anchored to the hydrogel network and preventing migration and loss during subsequent crosslinking or washing steps.

[0017] Preferably, the visible light source uses a pulsed rather than continuous illumination mode, with each exposure cycle consisting of multiple short pulses. The pulse intervals are sufficient to allow the local temperature to return to ambient levels, thereby eliminating the cumulative thermal effect. This timing control strategy is implemented through a programmable logic controller built into the device, whose trigger signal is strictly synchronized with the platform's lifting and lowering movements.

[0018] Preferably, the ester bond connections of the amphiphilic block copolymer are designed to be hydrolyzable in a weakly acidic environment. Since the pH of the inflammatory microenvironment after scaffold implantation is typically below 6.5, the peptide release behavior is naturally coupled with the tissue repair process. In in vitro degradation simulation experiments, the peptide release was less than 10% within 72 hours at pH 7.4, while it exceeded 60% within 24 hours at pH 6.0, indicating that the system possesses pathologically responsive release capabilities.

[0019] Preferably, the primary three-dimensional scaffold structure undergoes a brief nitrogen purging treatment before secondary crosslinking to remove residual oxygen. Oxygen, as a free radical quencher, interferes with visible light-induced polymerization, leading to uneven local crosslinking. This pretreatment ensures a highly consistent crosslinking density throughout the scaffold network, thereby improving structural fidelity.

[0020] Preferably, the freeze-dried composite material is encapsulated in an aluminum foil composite bag and filled with nitrogen to isolate it from oxygen and moisture before storage. This packaging method ensures that the peptide maintains structural stability during its shelf life and avoids oxidation or hydrolytic degradation.

[0021] Preferably, the working chamber of the additive manufacturing equipment has built-in humidity and temperature sensors that provide real-time feedback of environmental parameters to the central control system. When a deviation from the preset range is detected, printing is automatically paused and an adjustment program is initiated. This closed-loop control mechanism ensures that the entire manufacturing process takes place in a strictly controlled microenvironment, eliminating the influence of external variables on peptide stability.

[0022] Preferably, the dispersion stability of the polypeptide drug-loading unit in the bio-ink is monitored by dynamic light scattering, and its particle size change rate is less than 5% throughout the printing process, indicating that the micelle structure remains intact under shear force. The inner diameter of the extrusion printhead is designed to be more than ten times the micelle particle size to reduce the shear rate during flow and avoid micelle breakage.

[0023] Preferably, the hydrogel precursor solution is filtered through a 0.22-micron filter membrane before use to remove any micron-sized impurity particles that may be present. If these particles are present in the ink, they can clog the nozzles or cause localized stress concentrations during printing, thereby indirectly affecting the uniformity of peptide distribution.

[0024] Preferably, the metal ion crosslinking agent is added slowly dropwise with gentle stirring to prevent excessively high local concentrations from neutralizing the surface charge of the micelles and causing aggregation. After crosslinking is complete, the scaffold is washed multiple times with buffer solution to thoroughly remove unreacted ions and avoid their toxic effects in vivo.

[0025] Preferably, the final product of the polypeptide composite material is sterilized by gamma irradiation before use, with the dose controlled below a threshold that does not damage the micelle structure or affect the polypeptide conformation. This sterilization method is suitable for heat-sensitive materials and has strong penetrating power, ensuring sterility inside the three-dimensional scaffold.

[0026] The beneficial effects of this invention are as follows: This invention discloses a method for preparing peptide composite materials for biomedical additive manufacturing. Through a dual mechanism of physical isolation and chemical shielding using nanomicelles, it avoids the peptides from being exposed to inactivating factors such as light, heat, free radicals, and shear stress during additive manufacturing. It utilizes a visible light initiation system and a pulsed exposure strategy to maintain the crosslinking efficiency of the hydrogel network while eliminating direct damage to the peptides from high-energy radiation. It achieves stable dispersion and spatial anchoring of the peptide drug-carrying units in the bio-ink, preventing migration and segregation during the printing process. It ensures that the scaffold structure achieves geometric accuracy and mechanical strength in both the primary photocuring and secondary ionic crosslinking stages, without interference between the two. Furthermore, through environmentally responsive micelle design, it dynamically matches the peptide release behavior with the tissue repair microenvironment, maintaining long-term biological function. Attached Figure Description

[0027] The invention will now be further described with reference to the accompanying drawings.

[0028] Figure 1 This is a schematic diagram of the overall process flow of the polypeptide composite material preparation method of the present invention; Figure 2 This is a schematic diagram of the core-shell structure of the polypeptide drug delivery unit nanomicelles in this invention; Figure 3 This is a schematic diagram of the final polypeptide composite material structure in this invention. Detailed Implementation

[0029] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0030] like Figures 1-3 As shown in the embodiment of the present invention, a method for preparing a peptide composite material for biomedical additive manufacturing is described, which prepares a peptide drug-carrying unit. The peptide drug-carrying unit is composed of a target peptide molecule encapsulated within a nanomicelle core formed by the self-assembly of an amphiphilic block copolymer, forming a nanocarrier with a well-defined core-shell structure. The amphiphilic block copolymer is composed of hydrophilic polyethylene glycol (PEG) segments and hydrophobic polylactic-co-glycolic acid (PLGA) segments linked by cleavable ester bonds, with a number-average molecular weight of 12,000 g / mol. The PEG segments account for 30% of the total mass, and the molar ratio of lactic acid to glycolic acid in the PLGA segments is 75:25. The critical micelle concentration (CMC) of this block copolymer in deionized water is 0.08 mg / mL, and it spontaneously forms spherical micelles with a particle size concentrated in the range of 80 to 120 nanometers under physiological pH (7.4) conditions, with a polydispersity index (PDI) of less than 0.15.

[0031] In some embodiments, the polypeptide molecule is a cyclic peptide (cRGD) containing the arginine-glycine-aspartic acid sequence, and the molecular formula of the cyclic RGD peptide used is [insert molecular formula here]. With a molecular weight of approximately 985.0 Da, its secondary structure is mainly β-turn, and it has a high affinity for integrin αvβ3 receptor binding ability. As mentioned above, the preparation process of the peptide drug delivery unit is as follows: 10 mg of cRGD powder is dissolved in 1 mL of anhydrous acetonitrile to form a first solution; 100 mg of the above-mentioned amphiphilic block copolymer is dissolved in 10 mL of deionized water to form a second solution; Under a nitrogen atmosphere, the first solution was injected into the second solution at a constant flow rate of 0.5 mL / min using a syringe pump, while simultaneously applying high-frequency sonication at 20 kHz and 150 W for 10 minutes. During sonication, acetonitrile, as a low-dielectric-constant organic solvent, caused PLGA segments to aggregate to form a hydrophobic core, in which cRGD molecules were encapsulated, while PEG segments extended outward to form a hydrophilic shell. The resulting mixture was then placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed against deionized water at 4°C for 48 hours, with the dialysate replaced every 6 hours to completely remove residual acetonitrile. A peptide-loaded micelle dispersion with a final concentration of 1 mg / mL was obtained. Dynamic light scattering (DLS) analysis showed an average particle size of 98.3 ± 4.2 nm and a Zeta potential of -8.7 ± 1.2 mV, indicating that the surface charge was effectively shielded.

[0032] Furthermore, to enhance the anchoring ability of the peptide drug-carrying unit in the subsequent hydrogel network, a small amount of methacrylamide groups were modified on the micelle surface. Specifically, during the block copolymer synthesis stage, 5 mol% of methacrylic anhydride was introduced into the terminal hydroxyl groups of the PEG to carry polymerizable double bonds. ¹H NMR verification showed a methacrylamide degree of 4.8 mol%, sufficient for covalent cross-linking with the hydrogel precursor during photocuring, without significantly altering the micelle self-assembly behavior.

[0033] Next, the base phase of the bio-ink was prepared. The bio-ink consisted of polypeptide drug-carrying units uniformly dispersed in a photoresponsive hydrogel precursor solution. The precursor solution contained methacrylamide gelatin (GelMA), methacrylamide hyaluronic acid (HAMA), and a visible light-sensitized photoinitiating system. The GelMA had a substitution degree of 85%, the HAMA had a substitution degree of 60%, the mass ratio of the two was 3:1, and the total solid content was 8 wt%. The photoinitiation system was based on tris(2,2'-bipyridine)ruthenium(II) hexafluorophosphate ([Ru(bpy)3)). 2+The redox visible light initiator pair composed of N-vinylcarbazole (NVK) and N-vinylcarbazole has a maximum absorption peak at 490 nm. Under visible light irradiation, this system generates active free radicals through a single-electron transfer mechanism, initiating the free radical polymerization of methacryloyl groups.

[0034] The preparation steps for the bio-ink are as follows: Dissolve 80 mg GelMA and 26.7 mg HAMA together in 1 mL PBS buffer (pH 7.4), and stir in a 60°C water bath until completely dissolved; After cooling to room temperature, add 0.5 mg of [Ru(bpy)3]. 2+ Add 1.0 mg NVK and stir for 30 minutes in the dark; Subsequently, under light-protected conditions, the aforementioned peptide-loaded micelle dispersion was added at a volume fraction of 2%, and gently vortexed for 5 minutes to avoid generating bubbles. The resulting bio-ink had a viscosity of 28.5 ± 1.3 mPa·s (25℃, shear rate 100 s⁻¹). -1 The storage modulus G' is 12.3 Pa, which meets the rheological requirements for extrusion printing. After filtration through a 0.22 μm polyethersulfone filter membrane, it is immediately used for printing to remove micron-sized impurities that could clog the nozzle.

[0035] Next, additive manufacturing is performed. This invention employs digital light processing (DLP) technology. The equipment is equipped with a digital micromirror device (DMD) projection module, and the light source is an LED array with a wavelength of 470nm, with an adjustable output light intensity range of 1-50mW / cm². The printing platform is immersed in a bio-ink bath, and the primary three-dimensional scaffold structure is formed layer by layer through curing. The exposure energy density is set to 8mJ / cm², which has been experimentally determined to be a threshold level sufficient to induce sufficient cross-linking of the precursor (gel fraction > 90%) but insufficient to penetrate the micelle PEG shell (approximately 5nm thick). Under these conditions, the photopolymerization reaction occurs only in the aqueous phase region outside the micelles, while the peptide molecules inside the micelles remain optically shielded.

[0036] It should be noted that the light source adopts a pulsed irradiation mode. Each layer exposure cycle consists of 8 pulses, each lasting 0.5 seconds, with an interval of 0.8 seconds. This timing design ensures that the local temperature recovers to the ambient temperature (25±1℃) within the pulse interval, and the maximum temperature rise shown by infrared thermal imaging does not exceed 2.3℃, effectively eliminating the cumulative thermal effect. The device has a built-in programmable logic controller (PLC) to precisely synchronize the light source trigger signal with the platform lifting action. The layer thickness is controlled at 30μm, and a 10μm overlap area is set between adjacent layers to achieve mechanical interlocking. The projection pattern adopts a grayscale gradient design: the grayscale value of the surface area is 200 (corresponding to high cross-linking density), linearly decreasing to 50 towards the interior (corresponding to low cross-linking density), thus forming a gradient structure with a porosity that continuously transitions from 35% on the surface to 75% in the interior. This structure has been verified by micro-CT reconstruction, with a pore size distribution of 50-300μm and a connected pore ratio of >95%.

[0037] Before printing begins, the built-in temperature and humidity sensor in the working chamber monitors environmental parameters in real time, setting the temperature to 25℃ and the relative humidity to 60%. If a deviation exceeding ±2℃ or ±5%RH is detected, the system automatically pauses printing and initiates an adjustment program. In addition, the primary scaffold needs to be purged with nitrogen for 30 seconds before secondary crosslinking to remove residual oxygen. Oxygen, as a free radical quencher, at a concentration below 0.1 ppm, can ensure the uniformity of the polymerization reaction, and the coefficient of variation of the overall crosslinking density of the scaffold is <5%.

[0038] Subsequently, a secondary crosslinking enhancement was performed. The primary three-dimensional scaffold structure was transferred to a buffer solution containing a metal ion crosslinking agent. The crosslinking agent was a 5 mM aqueous solution of ZnCl2, and the solvent was phosphate-buffered saline (PBS) at pH 7.4. The crosslinking process was carried out in a constant temperature water bath at 37°C for 2 hours, which was determined by kinetic experiments to be the time required for the coordination network to reach thermodynamic equilibrium. Zn²⁺ ions react with the carboxyl groups (-COO₂) in the GelMA / HAMA network. - The phenolic hydroxyl group (-OH) forms a tetrahedral coordination structure, with a cross-linking point density of approximately one Zn per 100 sugar units. 2+ This coordination crosslinking does not involve the breaking or formation of covalent bonds, the enthalpy change ΔH < 5 kJ / mol, there is no significant exothermic reaction, and the temperature fluctuation at the center of the scaffold is < 0.5℃.

[0039] To prevent micelle aggregation due to excessively high local concentrations, the ZnCl2 solution was slowly added dropwise to the scaffold soaking solution at a rate of 0.1 mL / min using a peristaltic pump, while simultaneously maintaining magnetic stirring at 50 rpm. After crosslinking, the scaffold was washed five times with PBS (30 minutes each time). Inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed residual Zn. 2+ The concentration was <0.1 ppm, which is far below the cytotoxic threshold (10 ppm).

[0040] Finally, post-processing is performed to obtain the finished product. The post-processing includes gradient dehydration and freeze-drying. Gradient dehydration uses a stepwise replacement system of ethanol-water: the product is successively soaked in 10%, 30%, 50%, 70%, 90%, and 100% (v / v) ethanol-water solutions for 30 minutes each, with gentle centrifugation (500g, 1 minute) before each solution change to remove excess liquid. This process avoids micelle structure collapse due to sudden changes in surface tension. Subsequently, the completely dehydrated support was placed in a freeze dryer and dried for 24 hours under a vacuum of 8 Pa and a cold trap temperature of -55°C. Moisture was removed by sublimation, and scanning electron microscopy (SEM) showed that the micelles remained intact and spherical, with no signs of ice crystal damage.

[0041] The resulting polypeptide composite material was packaged in an aluminum foil composite bag, filled with high-purity nitrogen (O2 < 10 ppm, H2O < 50 ppm), heat-sealed, and stored at 4°C in the dark. Gamma ray sterilization was performed in a dedicated facility at a dose rate of 1 kGy / h, with a total dose of 15 kGy. Combined analysis by circular dichroism (CD) and high-performance liquid chromatography (HPLC) showed that the retention rate of the β-turn characteristic peak (negative peak at 228 nm) of the sterilized cRGD was >95%, and the purity of the main peak was >98%, indicating that both conformational and chemical integrity were maintained.

[0042] To verify the technical effect of the present invention, the following embodiments were designed for comparison with comparative examples.

[0043] Example 1: cRGD composite scaffolds were prepared using the above complete process flow, with a peptide drug delivery unit volume fraction of 2%, visible light wavelength of 470 nm, pulsed exposure, and Zn. 2+ Secondary crosslinking, gradient dehydration, and freeze drying Example 2: Except that the metal ion crosslinking agent was replaced with FeCl3 (3mM), the other conditions were the same as in Example 1.

[0044] Comparative Example 1: Omitting the nanomicelle encapsulation step, directly applying cRGD ( (The molecular weight is approximately 985.0 Da) was dissolved in bio-ink for printing, with the remaining conditions the same as in Example 1.

[0045] Comparative Example 2: A conventional ultraviolet light initiation system (Irgacure 2959, 365nm, 10mW / cm²) was used instead of the visible light system, with the other conditions the same as in Example 1.

[0046] Comparative Example 3: The secondary cross-linking step was omitted, and the scaffold was formed solely by photocuring. The remaining conditions were the same as in Example 1.

[0047] The peptide structure integrity, bioactivity, and scaffold performance of the above samples were tested, and the results are shown in Table 1.

[0048] Table 1: Performance Comparison of Examples and Comparative Examples sample Polypeptide secondary structure retention rate (%) In vitro cell adhesion rate (%) Stent compression modulus (kPa) Release rate (%) at pH 7.4 over 72 hours Print fidelity (%) Example 1 96.2±1.8 92.5±3.1 185±12 8.7±1.2 98.3 Example 2 95.5±1.9 91.2±2.7 210±15 7.9±1.1 98.0 Comparative Example 1 62.3±4.5 58.4±5.2 180±11 42.6±3.8 97.8 Comparative Example 2 54.7±5.1 51.2±4.8 182±13 38.9±4.1 96.5 Comparative Example 3 95.8±1.7 92.0±2.8 85±8 8.5±1.3 92.1 Among them, the retention rate of peptide secondary structure was calculated by ellipticity at 228 nm using circular dichroism spectroscopy; the in vitro cell adhesion rate was determined by DAPI staining and counting of human umbilical vein endothelial cells (HUVECs) after 4 hours of culture on a scaffold; the compressive modulus was tested according to ISO 13314 standard; the release rate was determined in PBS at 37℃; and the printing fidelity was calculated by the volume overlap between the micro-CT reconstruction model and the CAD design model.

[0049] Data show that Examples 1-2 are significantly superior to the Comparative Examples in terms of peptide structure preservation, bioactivity, and mechanical properties. In Comparative Example 1, due to the lack of micellar protection, the peptide was exposed to an aqueous environment during printing, resulting in conformational development and non-specific adsorption. In Comparative Example 2, ultraviolet light directly excited aromatic residues of the peptide (such as tyrosine and tryptophan), initiating photo-oxidative degradation. Although Comparative Example 3 maintained peptide activity, the insufficient mechanical strength of the scaffold led to a decrease in printing fidelity.

[0050] Furthermore, the peptide release behavior in this invention exhibits pH responsiveness. In in vitro simulated degradation experiments, the scaffold from Example 1 was placed in PBS at pH 7.4 and pH 6.0, respectively, and cultured with shaking at 37°C. The cumulative peptide release was determined by HPLC, and the results conformed to the following piecewise function model: ; in, This represents the cumulative mass of the polypeptide released at time t (unit: μg). Total peptide loading in the scaffold (unit: μg). Release rate constant at pH 7.4 (unit: h) -1 ), The release rate constant at pH 6.0 (unit: h). -1 After nonlinear fitting, , This indicates that the release rate increases by approximately 11.4 times under acidic conditions. This behavior stems from the accelerated hydrolysis of the PLGA-PEG linking ester bonds in the micelles under weakly acidic conditions. ; in, Indicates ester bond concentration (unit: mol / L). The rate constant for acid-catalyzed hydrolysis is given by L / (mol·h). Hydrogen ion concentration (unit: mol / L); At pH 6.0, Compared to pH 7.4 ( The concentration of the peptides is about 25 times higher, which leads to accelerated micelle disintegration and premature release of peptides.

[0051] In summary, this invention achieves structural protection and functional maintenance of peptides throughout the entire additive manufacturing process through a multi-stage synergistic design involving nanomicelle encapsulation, visible light curing, secondary cross-linking with metal ions, and gradient dehydration. The various technical features work together to solve key technical challenges in 3D printing, such as the easy inactivation, uneven distribution, and uncontrollable release of bioactive molecules, providing a reliable technical path for the development of high-performance biomedical scaffolds.

[0052] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a polypeptide composite material for biomedical additive manufacturing, characterized in that, Includes the following steps: S1: Encapsulate the target polypeptide molecule within a nanomicelle core formed by the self-assembly of an amphiphilic block copolymer to form a polypeptide drug delivery unit with a core-shell structure; S2: The polypeptide drug delivery unit is uniformly dispersed in a photoresponsive hydrogel precursor solution to form the basic phase of the bio-ink; S3: In additive manufacturing equipment, a wavelength-tunable visible light source is used to selectively solidify the bio-ink layer by layer to form a primary three-dimensional scaffold structure. S4: The primary three-dimensional scaffold structure is placed in a buffer solution containing a metal ion crosslinking agent, and the mechanical reinforcement of the scaffold network is completed through a secondary crosslinking reaction mediated by coordination bonds. S5: The obtained composite scaffold is subjected to gradient dehydration and freeze-drying to obtain the final peptide composite material product; The amphiphilic block copolymer is composed of hydrophilic polyethylene glycol segments and hydrophobic polylactic acid-glycolic acid copolymer segments linked by cleavable ester bonds, spontaneously forming spherical micelles under physiological pH conditions. The polypeptide molecules are confined within the hydrophobic core of the micelles, and their surface charges are completely shielded. The photoresponsive hydrogel precursor solution contains methacrylamide gelatin, methacrylamide hyaluronic acid, and a visible light sensitized photoinitiation system based on ruthenium complexes. The wavelength of the visible light source is set to a threshold level sufficient to initiate precursor crosslinking but insufficient to penetrate the micelle shell, so that the photopolymerization reaction only occurs in the hydrogel network outside the micelles.

2. The preparation method according to claim 1, characterized in that, The preparation process of the peptide drug-loading unit includes: dissolving peptide powder in a low dielectric constant organic solvent to form a first solution, dissolving an amphiphilic block copolymer in deionized water to form a second solution, injecting the first solution into the second solution at a constant flow rate under inert gas protection, and simultaneously applying high-frequency ultrasonic treatment, followed by dialysis to remove the organic solvent, thereby obtaining a stable peptide drug-loading micelle dispersion; the surface of the micelles is modified with methacrylamide groups, enabling them to be covalently anchored to the hydrogel network.

3. The preparation method according to claim 1, characterized in that, The visible light source adopts a pulsed irradiation mode, and each exposure cycle consists of multiple short pulses. The pulse interval is sufficient to allow the local temperature to recover to the ambient level. The additive manufacturing equipment is equipped with a digital micromirror device projection module. There is an overlapping area between adjacent layers to achieve mechanical interlocking. The overall porosity is adjusted by the grayscale gradient of the projected pattern to form a continuous transition structure from the dense area on the surface to the loose area inside.

4. The preparation method according to claim 1, characterized in that, The metal ion crosslinking agent is a water-soluble salt of divalent zinc ions or trivalent iron ions, and its concentration is controlled within a range that does not cause micelle dissociation but is sufficient to form coordination bonds with carboxyl groups or phenolic hydroxyl groups in the hydrogel network; the secondary crosslinking is carried out in phosphate buffer for a duration required for the coordination network to reach thermodynamic equilibrium; the crosslinking agent is added slowly with gentle stirring, and after crosslinking is completed, it is washed with buffer replacement multiple times to remove unreacted ions.

5. The preparation method according to claim 1, characterized in that, The gradient dehydration process employs a stepwise solvent replacement method: the buffer solution in the pores of the scaffold is replaced with an aqueous ethanol solution.

6. The preparation method according to claim 1, characterized in that, The target polypeptide is a cyclic bioactive peptide containing an arginine-glycine-aspartic acid (RGD) sequence; in the encapsulated state, its secondary structure retains the β-turn characteristic, and its bioactivity does not significantly decrease; the volume fraction of the polypeptide drug-loading unit in the bio-ink base phase is 0.5%-5% to maintain micelle dispersion stability and prevent aggregation and precipitation.

7. The preparation method according to claim 1, characterized in that, Before the secondary crosslinking, the primary three-dimensional scaffold structure is purged with nitrogen to remove residual oxygen and ensure the uniformity of the photopolymerization reaction and the consistency of the crosslinking density.

8. The preparation method according to claim 1, characterized in that, The additive manufacturing equipment has a built-in temperature and humidity sensor in its working chamber, which feeds back environmental parameters to the central control system in real time. When the temperature or humidity deviates from the preset range, the printing is automatically paused and the adjustment program is started. The bio-ink is filtered through a filter membrane before use to remove micron-sized impurity particles.

9. The preparation method according to claim 1, characterized in that, The cleavable ester bonds in the amphiphilic block copolymer can be hydrolyzed in a weakly acidic environment, which enables the peptide release behavior to be dynamically coupled with the pH of the tissue repair microenvironment.

10. The preparation method according to claim 1, characterized in that, The finished polypeptide composite material is packaged in an aluminum foil composite bag and filled with nitrogen before storage. Before use, it is sterilized by gamma irradiation, with the irradiation dose controlled below the threshold that does not damage the micelle structure or affect the polypeptide conformation.

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