| Hydrogels are important biomaterials that have been widely used for cell cultrue, tissue engineering and regenerative medicine due to their high water content, tunable viscoelasticity and biocompatibility. Peptidic supramolecular hydrogelators can be prepared easily using solid-phase synthesis and display a high degree of chemical functionalities. Supramolecular hydrogels formed by peptides have several advantages over traditional polymer hydrogels, including dense signaling capacity, the absence of toxic cross-linking agents, inherent biocompatibility and biodegradation, sheer-thinning behavior or in vivo gelation, and facile chemical modification. It is also possible to use physiologically functional short peptides which can interact with biological systems to construct peptideic supramolecular hydrogels. Thus, these hydrogels are ideal biomaterials for extracellular matrix (ECM) in two-dimensional (2D) or three dimensional (3D) cell culture.Photoactive molecules have been widely used as photo switches in "smart" materials because their molecular structure and properties can be temporally and spatially regulated by light irradiation. Diaryl tetrazoles were reported as photoclick reaction substrates. Upon mild UV irradiation, the tetrazole moiety rapidly releases N2 and to form nitrilimine dipole intermediate, which then undergoes 1,3-dipolar cycloaddition reactions with olefins. The photoclick reaction has become an attractive bioorthogonal reaction in chemical biological study. In this dissertation, attracted great attention of researchers in chemical biology due to its numerous advantages such as good yield, high reaction rate, few byproducts and compatible with biological systems. In this dissertation, we constructed a variety of biocompatible photoresponsive supramolecular hydrogel systems byy linking different kinds of diaryl tetrazoles to the N terminal of short peptides. These biocompatible photoresponsive supramolecular hydrogels showed the potential to modulate microenvironment of the encapsulated cells by photo-irradiation.The first part of this dissertation focused on the construction of the photoresponsive hydrogel based on the intramolecular photoclick reaction. We prepared diaryl tetrazoles tet(Ⅰ) and tet(Ⅱ) that bearing an allyl ether substitution group on the ortho position of the pheny ring linked with the tetrazole N atom, which allows rapid intramolecular photo-click reaction upon mild UV irradiation. We then found hydrogelators from short peptides linked with Tet(Ⅰ) or Tet(Ⅱ). The photo-triggered tetrazole to pyrazoline transformation was found to induce not only a fluorescence turn-on response, but also can the disassembling of the hydrogel matrix by disturbing the balance between hydrophilic interaction and π-π stacking of the self-assembled system. Among those hydrogels, Tet(Ⅰ)-GFF and Tet(Ⅱ)-GFRGD gels show good mechanical strength and biocompatibility for 3D encapsulation and prolonged culture of live cells. Photo-modulation of cellular microenvironment was demonstrated not only for the cells grew on top of the gel, but also for stem cells encapsulated inside the hydrogels. Wherein, Tet (Ⅰ)-GFF hydrogel was successfully applied to 2D culture of C2C12 cells and the differentiation of C2C12 cells was controlled through photo induced release of horse serum encapsulated in the hydrogel. Tet (Ⅱ)-GFRGD hydrogel was used for the 3D culture of human mesenchymal stem cells (hMSCs). The spreading behavior of hMSCs was tuned by adjusting mechanical properties of the hydrogel with light irradiation. These work demonstrated that the photodegradable supramolecular hydrogels based on short peptides modified with diaryl tetrazoles which can undergo intramolecualr photoclick reactions have great potential in regulation of cellular microenvironment and behaviors.The second part of this dissertation constructed another type of photoresponsive hydrogel based on diaryl tetrazoles that were able to undergo intermolecular photoclick reactions with alkenes. To obtain the desirable photosensitivity of the supramolecular hydrogels, we firstly replaced the N terminal benzene ring of the diaryl tetrazoles with p-methoxyphenol and naphthalene to increase the HOMO orbital energy of the nitrilimine intermediates which can accelerate the dipolar cycloaddition reaction rate between nitrilimines and alkenes. Most of the diaryl tetrazole linked short peptides can form stable supramolecular hydrogels under phisiological conditions. Strongly hydrophilic p-styrene sulfonate was used as the other photoclick substrate to react with the tetrazole moiety on the hydrogelators. The hydrophilic pyrazoline moiety bearing the sulfonate group formed after the intermolecular photoclick reaction disturbed the balance between the hydrophobic and hydrophilic interactions of the supramolecular hydrogels, which led to rapid photodegradation of the supramolcular hydrogels. Among those hydrogels, MeO-Tet-GA hydrogel showed good biocompatibility and good mechanical strength for 3D culutre of mouse breast cancer cells (4Tls).The third part of this dissertation descibed a novel kind of two-photon patternable supramolecular hydrogels based on the intermolecular photoclick reactions between the diaryl tetrazoles linked short peptides and monomethyl fumurated modified biological functional molecules. Compared with single-photon excitation, two-photon exciation offers a superior spatiotemporal control of the photoclick reaction due to the reduced scattering of near infrared light in the hydrogels and significantly improves three dimensional localization of excitation. The N terminal benzene ring of the diaryl tetrazole was replaced with methoxy-naphthalene or acetyl-naphthalene and the C terminal benzene ring was replaced with phenoxy acetic acid respectively. These modifications can increase the electron density of the aromatic group which leads to the red shift of the excitation wavelength and the increasement of the two-photon absorption cross section. Two kinds of two-photon responsive diaryl tetrazoles, ie. TPI-COOH and TPII-COOH, were obtained and a variety of hydrogelators were get by modification of the N terminal of the short peptides with these two diaryl tetrazoles. Among them, TPI-GGF hydrogel was confirmed two-photon patterenable by using the femtosecond laser from the two-photon confocal laser scanning microscope. It is also suitable for the long term 3D culture of hMSCs and mouse neural stem cells (mNSCs). In order to study the effects of the chemical properties of the extracellular microenvironment on the differentiation behaviors of the stem cells, the monomethyl fumarate modified epitopes such as (RGDfK)-MMF and MMF-GIKVAV-OH were patterned into the TPI-GGF hydrogel encapsulated with stem cells through the biocompatible intermolecular photoclick reaction. The differentiation of hMSCs encapsulated in TPI-GGF hydrogel was promoted after photopatterning with cyclic RGD and the neurongenesis differentiation of mNSCs was accelerated by IKVAV modification. This part of the work showed that application of this kind of two-photon patternable supramolecular hydrogel will gain much insight into the simulation of complex cell microenvironment and the cell-matrix interactions. |
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