Design And Synthesis Of Functional Dyes In Biomanufactur Photopolymerization Technology And Its Apllication In 3D Printing

The rapid development of 3D printing technology has significantly advanced biomanufacturing,particularly in the realm of photo-polymerization-based 3D printing.Notable progress has been achieved in scaffold fabrication,tissue repair,and organ printing.Functional dyes have been integral to this advancement.As crucial components of photo-polymerizable bio-inks,these dyes exhibit diverse functionalities.They act as visible light sensitizers,enhancing the biocompatibility of long-wavelength photo-polymerization;as light absorbers,they facilitate precise photo-polymerization through their specific absorption properties and photochemical reactions,thereby improving the technology’s adaptability and compatibility in biomanufacturing;and as fluorescent probes,they enable cellular-level imaging and tracking of the metabolism of transplanted active substances.However,the application of functional dyes faces several challenges,including the molecular toxicity and wavelength limitations of small molecule sensitizers,the need to balance enhanced photo-polymerization accuracy with the inhibition of polymerization activity when using traditional light absorbers,and issues with conventional fluorescent probes such as low signal-to-noise ratios,poor photostability,and aggregation-caused quenching（ACQ）effects.This paper focuses on the design,synthesis,and application of functional dyes in photo-polymerization technology for biomanufacturing and 3D printing.Our objective is to overcome the limitations of traditional dyes through the rational design of functional dye molecules and to leverage photo-polymerizable 3D printing technology to achieve high-precision printing of biomaterials,fluorescence imaging for tracking metabolically active biomaterials,and the development of long-wavelength photo-polymerizable cell encapsulation and 3D printing.The specific contributions of this paper include:（1）Addressing the aggregation-induced quenching（ACQ）effect commonly observed in traditional fluorophore dyes,we have developed a collagen-based macromolecular fluorescent probe dye,Col-SH-Cy,capable of participating in photo-polymerization.The chemical structure,molecular weight,and functionality of Col-SH-Cy were characterized using proton nuclear magnetic resonance（¹H NMR）,gel permeation chromatography（GPC）,and various UV-visible spectroscopic techniques.By introducing collagen macromolecules and thiol（SH）groups into Col-SH-Cy,we achieved an alteration in the aggregation state of the fluorescent molecules post-polymerization,exhibiting an Anti-ACQ effect.Investigation into the photopolymerization kinetics of the Col-SH-Cy and thiol-modified collagen（Col-SH）systems revealed that the introduction of SH groups eliminated oxygen inhibition during photopolymerization,thereby enhancing polymerization activity.Using Digital Light Processing（DLP）3D printing technology,we fabricated metabolizable collagen-based scaffolds containing Col-SH-Cy probe dyes and tracked the metabolism of the scaffolds in vivo in mice using infrared fluorescence imaging techniques.The results indicated no pathological lesions in mouse organ histopathological sections,demonstrating the biocompatibility of the macromolecular dye and the printed scaffolds.（2）In response to the lack of efficient photosensitizers for long-wavelength biomanufacturing using photopolymerization technology,this study designed and synthesized collagen-based macromolecular photosensitive dyes,Col-SH-CZ and Col-SH-TD,which contain carbazole（CZ）and thiadiazole（TDPA）moieties,respectively.Long-wavelength sensitization in biomanufacturing was achieved through photoinduced electron transfer（PET）interactions with iodonium salts（ONI）.The chemical structure,molecular weight,and functionality of Col-SH-CZ and Col-SH-TD were characterized using proton nuclear magnetic resonance（1H NMR）,gel permeation chromatography（GPC）,and various UV-visible spectroscopic techniques.Photopolymerization tests and dye system characterizations validated the photosensitivity of Col-SH-CZ and Col-SH-TD under green-to-red light.The photopolymerization mechanism,involving the sensitization of ONI by Col-SH-CZ and Col-SH-TD,was elucidated through steady-state photolysis,fluorescence quenching,and electrochemical tests.Cell toxicity and cell encapsulation experiments under different light initiation systems confirmed the superiority of the collagen-based photosensitizers/ONI systems for cell 3D encapsulation and bioprinting under green-to-red light.（3）In response to the issue of adding absorber dyes in bioprinting to enhance photopolymerization accuracy while suppressing polymerization activity,we designed and synthesized hemicyanine photosensitive dyes with D-π-A（donor-π-acceptor）structures using carbazole,triphenylamine,benzothiophene,and anthracene as electron-donating moieties:CZIN,TPAIN,BDTIN,and ANIN.The structures were characterized using techniques such as proton nuclear magnetic resonance（¹H NMR）,carbon-13 nuclear magnetic resonance(¹³C NMR),and high-resolution mass spectrometry（HRMS）.UV-visible absorption spectra and fluorescence emission spectra indicated that the absorption wavelengths of the four dyes covered the range of 400-600 nm.Photopolymerization experiments of the semi-phthalocyanine dye/iodonium salt（ONI）in a hydrogel system revealed that CZIN and TPAIN exhibited high photosensitization activity under green-to-red light.The sensitization mechanism between hemicyanine dyes and ONI was analyzed using cyclic voltammetry（CV）,fluorescence quenching experiments,electron paramagnetic resonance（EPR）experiments,and steady-state photolysis experiments,demonstrating the efficient electron transfer ability of CZIN and TPAIN with ONI.The influence of hemicyanine dyes on photopolymerization accuracy in the hydrogel system was experimentally tested,and the structure-activity relationship of the dyes in enhancing photopolymerization accuracy was analyzed.Additionally,it was found that the four hemicyanine dyes also exhibited recognition of alkalinity in aqueous solutions.（4）Addressing the challenges of high refractive index and severe light scattering in hydroxyapatite（HAP）photosensitive resin systems,hindering high-precision photopolymerization,we optimized the existing photo-initiation systems.By introducing the functional dye Sudan III,we effectively improved printing accuracy.We found that the Sudan dye not only underwent photo-induced cis-trans isomerization reactions to absorb light energy and enhance printing accuracy but also promoted initiator cleavage,thereby enhancing photopolymerization efficiency.Through printing of accuracy models and optimization of printing parameters,we successfully achieved high-precision printing of the HAP slurry system.By designing model unit structures,we obtained HAP ceramic scaffolds with complex structures.Through rat osteogenesis experiments,we analyzed the relationship between the structure of HAP ceramic scaffolds and their osteogenic effects.The results showed that cubic cross-linked ceramic scaffolds with greater internal connectivity and complex pore structures exhibited faster new bone formation rates and higher bone formation quality,further demonstrating the importance of high-precision 3D printing in the preparation of bone repair scaffolds.