Specific Targeting, Imaging, and Ablation of Tumor-Associated Macrophages by Theranostic Mannose-AIEgen Conjugates.

Tumor-associated macrophages (TAMs) that exist in tumor microenvironment promote tumor progression and have been suggested as a promising therapeutic target for cancer therapy in preclinical studies. Development of theranostic systems capable of specific targeting, imaging, and ablation of TAMs will offer clinical benefits. Here we constructed a theranostic probe, namely, TPE-Man, by attaching mannose moieties to a red-emissive and AIE (aggregation-induced emission)-active photosensitizer. TPE-Man can specifically recognize a mannose receptor that is overexpressed on TAMs by the sugar-receptor interaction and enables fluorescent visualization of the mannose-receptor-positive TAMs in high contrast. The histologic study of mouse tumor sections further verifies TPE-Man's excellent targeting specificity being comparable with the commercial mannose-receptor antibody. TAMs can be effectively eradicated upon exposure to white light irradiation via a photodynamic therapy effect. To our knowledge, this is the first small molecular theranostic probe for TAMs that revealed combined advantages of low cost, high targeting specificity, fluorescent light-up imaging, and efficient photodynamic ablation.

Spheroids of Endothelial Cells and Vascular Smooth Muscle Cells Promote Cell Migration in Hyaluronic Acid and Fibrinogen Composite Hydrogels.

Cell migration plays a pivotal role in many pathological and physiological processes. So far, most of the studies have been focused on 2-dimensional cell adhesion and migration. Herein, the migration behaviors of cell spheroids in 3D hydrogels obtained by polymerization of methacrylated hyaluronic acid (HA-MA) and fibrinogen (Fg) with different ratios were studied. The Fg could be released to the medium gradually along with time prolongation, achieving the dynamic change of hydrogel structures and properties. Three types of cell spheroids, i.e., endothelial cell (EC), smooth muscle cell (SMC), and EC-SMC spheroids, were prepared with 10,000 cells in each, whose diameters were about 343, 108, and 224 µm, respectively. The composite hydrogels with an intermediate ratio of Fg allowed the fastest 3D migration of cell spheroids. The ECs-SMCs migrated longest up to 3200 µm at day 14, whereas the SMC spheroids migrated slowest with a distance of only ~400 µm at the same period of time. The addition of free RGD or anti-CD44 could significantly reduce the migration distance, revealing that the cell-substrate interactions take the major roles and the migration is mesenchymal dependent. Moreover, addition of anti-N-cadherin and MMP inhibitors also slowed down the migration rate, demonstrating that the degradation of hydrogels and cell-cell interactions are also largely involved in the cell migration. RT-PCR measurement showed that expression of genes related to cell adhesion and antiapoptosis, and angiogenesis was all upregulated in the EC-SMC spheroids than single EC or SMC spheroids, suggesting that the use of composite cell spheroids is more promising to promote cell-substrate interactions and maintenance of cell functions.

Surface-Anchored Graphene Oxide Nanosheets on Cell-Scale Micropatterned Poly(d,l-lactide-co-caprolactone) Conduits Promote Peripheral Nerve Regeneration.

Regeneration and functional recovery of peripheral nerves remain formidable due to the inefficient physical and chemical cues in the available nerve guidance conduits (NGCs). Introducing micropatterns and bioactive substances into the inner wall of NGCs can effectively regulate the behavior of Schwann cells, the elongation of axons, and the phenotype of macrophages, thereby aiding the regeneration of injured nerve. In this study, linear micropatterns with ridges and grooves of 3/3, 5/5, 10/10, and 30/30 µm were created on poly(d,l-lactide-co-caprolactone) (PLCL) films following with surface aminolysis and electrostatic adsorption of graphene oxide (GO) nanosheets. The GO-modified micropatterns could significantly accelerate the collective migration of Schwann cells (SCs) and migration of SCs from their spheroids in vitro. Moreover, the SCs migrated directionally along the stripes with a fastest rate on the 3/3-GO film that had the largest cell adhesion force. The neurites of N2a cells were oriented along the micropatterns, and the macrophages tended to differentiate into the M2 type on the 3/3-GO film judged by the higher expression of Arg 1 and IL-10. The systematic histological and functional assessments of the regenerated nerves at 4 and 8 weeks post-surgery in vivo confirmed that the 3/3-GO NGCs had better performance to promote the nerve regeneration, and the CMAP, NCV, wet weight of gastrocnemius muscle, positive S100ß and NF200 area percentages, and average myelinated axon diameter were more close to those of the autograft group at 8 weeks. This type of NGCs thus has a great potential for nerve regeneration.

Unsaturated polyurethane films grafted with enantiomeric polylysine promotes macrophage polarization to a M2 phenotype through PI3K/Akt1/mTOR axis.

The immune system responds immediately to tissue trauma and to biomaterial implants under the participation of M1/M2 macrophages polarization. The surface properties of biomaterials can significantly influence the tissue repair progress through modulating the macrophage functions. In this study, the surface of poly(propylene fumarate) polyurethane films (PPFU) is grafted with a same density of enantiomeric poly-l-lysine (PPFU-g-PLL) and poly-d-lysine (PPFU-g-PDL), leading to a similar level of enhanced surface wettability for the PPFU-g-PLL and PPFU-g-PDL. The polylysine-grafted PPFU can restrict the M1 polarization, whereas promote M2 polarization of macrophages in vitro, judging from the secretion of cytokines and expression of key M1 and M2 related genes. Comparatively, the PPFU-g-PDL has a stronger effect in inducing M2 polarization in vivo, resulting in a thinner fibrous capsule surrounding the implant biomaterials. The CD44 and integrins of macrophages participate in the polarization process probably by activating focal adhesion kinase (FAK) and Rho-associated protein kinase (ROCK), and downstream PI3K/Akt1/mTOR signal axis to up regulate M2 related gene expression. This study confirms for the first time that polylysine coating is an effective method to regulate the immune response of biomaterials, and the polylysine-modified thermoplastic PPFU with the advantage to promote M2 polarization may be applied widely in regenerative medicine.

UV-Responsive Multilayers with Multiple Functions for Biofilm Destruction and Tissue Regeneration.

The increasing demands of surgical implantation highlight the significance of anti-infection of medical devices, especially antibiofilm contamination on the surface of implants. The biofilms developed by colonized microbes will largely hinder the adhesion of host cells, leading to failure in long-term applications. In this work, UV-responsive multilayers were fabricated by stepwise assembly of poly(pyrenemethyl acrylate- co-acrylic acid) (P(PA- co-AA)) micelles and chitosan on different types of substrates. Under UV irradiation, the cleavage of pyrene ester bonds in the P(PA- co-AA) molecules resulted in the increase of roughness and hydrophilicity of the multilayers. During this process, reactive oxygen species were generated in situ within 10 s, which destroyed the biofilms of Staphylococcus aureus, leading to the degradation of the bacterial matrix. The antibacterial rate was above 99.999%. The UV-irradiated multilayers allowed the attachment and proliferation of fibroblasts, endothelial cells, and smooth muscle cells, benefiting tissue integration of the implants. When poly(dimethylsiloxane) slices with the multilayers were implanted in vivo and irradiated by UV, the density of bacteria and the inflammatory level (judging from the number of neutrophils) decreased significantly. Moreover, formation of neo blood vessels surrounding the implants was observed after implantation for 7 days. These results reveal that the photoresponsive multilayers endow the implants with multifunctions of simultaneous antibiofilm and tissue integration, shedding light for applications in surface modification of implants in particular for long-term use.

Migration of endothelial cells into photo-responsive hydrogels with tunable modulus under the presence of pro-inflammatory macrophages.

Cell migration in three-dimensional environment is extremely important for tissue regeneration and other biological processes. In this work, a model system was developed to study how endothelial cells (ECs) migrate into photo-responsive hydrogels under the presence of pro-inflammatory macrophages. The hydrogel was synthesized from hyaluronic acid grafted with coumarin and methacrylate moieties by both carbon-carbon covalent linking and coumarin dimerization under UV irradiation at 365 nm. The structure of the hydrogel was conveniently modulated by UV irradiation at 254 nm to decompose the coumarin dimers, leading to the significant decrease of modulus and increase of swelling ratio and mesh size. Under the presence of M1 macrophages, ECs were induced to migrate into the hydrogels with a different degree. A significant larger net displacement of ECs was found in the softer hydrogel obtained by irradiation with UV at 254 nm than in the stiffer original one at day 7.

Migration of endothelial cells and mesenchymal stem cells into hyaluronic acid hydrogels with different moduli under induction of pro-inflammatory macrophages.

The design of hyaluronic acid (HA)-based and stimuli-responsive hydrogels to elicit highly controlled and tunable cell response and behaviors is a major field of interest in tissue engineering and regenerative medicine. The pH-responsive hydrogel can respond to pH variation during wound healing, which may in turn regulate the tissue regeneration process. In this study, a double-network hydrogel cross-linked with vinyl double bonds and Schiff base was prepared, whose properties were further adjusted by incubation in pH 7.4 and pH 5 buffers. The endothelial cells (ECs) migrated much deeper into the softer HA hydrogel pre-treated with pH 5 buffer than the stiffer hydrogel. By contrast, the mesenchymal stem cells (MSCs) migrated easily into the stiffer hydrogel. The ECs highly expressed RhoA and non-muscle myosin (NM) II genes in the softer hydrogel, which may facilitate amoeboid migration. Meanwhile, the MSCs were stiffer than the ECs, and highly expressed Rac1, RhoA, vinculin, NM II, hyaluronidase (HYAL) 2 and CD44 genes in the stiffer hydrogel, which facilitate mesenchymal migration. These results provide important clues for revealing the different migration strategies of the ECs and MSCs in HA hydrogels with different stiffness, and suggest that the mechanical properties and the network structure of hydrogels play an important role in regulating the three-dimensional migration process of these cells.

Dynamic Titania Nanotube Surface Achieves UV-Triggered Charge Reversal and Enhances Cell Differentiation.

Stimuli-responsive biomaterials supply a promising solution to adapt to the complex physiological environment for different biomedical applications. In this study, a dynamic UV-triggered pH-responsive biosurface was constructed on titania nanotubes (TNTs) by loading photoacid generators, diphenyliodonium chloride, into the nanotubes, and grafting 2,3-dimethyl maleic anhydride (DMMA)-modified hyperbranched poly(l-lysine) (HBPLL) onto the surface. The local acidity was dramatically enhanced by UV irradiation for only 30 s, leading to the dissociation of DMMA and thereby the transformation of surface chemistry from negatively charged caboxyl groups to positively charged amino groups. The TNTs-HBPLL-DMMA substrate could better promote proliferation and spreading of rat bone mesenchymal stem cells (rBMSCs) after UV irradiation. The osteogenic differentiation of rBMSCs was enhanced because of the charge reversal in combination with the titania-based substrates.

A density gradient of VAPG peptides on a cell-resisting surface achieves selective adhesion and directional migration of smooth muscle cells over fibroblasts.

Selective adhesion and migration of smooth muscle cells (SMCs) over fibroblasts (FIBs) is required to prevent adventitia fibrosis in vascular regeneration. In this study, a uniform cell-resisting layer of poly(ethylene glycol) (PEG) with a density gradient of azide groups was generated on a substrate by immobilizing two kinds of PEG molecules in a gradient manner. A density gradient of alkynyl-functionalized Val-Ala-Pro-Gly (VAPG) peptides was then prepared on the PEG layer via click chemistry. The VAPG density gradient was characterized by fluorescence imaging, revealing the gradual enhancement of the fluorescent intensity along the substrate direction. The adhesion and mobility of SMCs were selectively enhanced on the VAPG density gradient, leading to directional migration toward the higher peptide density (up to 84%). In contrast, the adhesion and mobility of FIBs were significantly weakened. The net displacement of SMCs also significantly increased compared with that on tissue culture polystyrene (TCPS) and that of FIBs on the gradient. The mitogen-activated protein kinase (MAPK) signaling pathways related to cell migration were studied, showing higher expressions of functional proteins from SMCs on the VAPG-modified surface in a density-dependent manner. For the first time the selective adhesion and directional migration of SMCs over FIBs was achieved by an elaborative design of a gradient surface, leading to a new insight in design of novel vascular regenerative materials. STATEMENT OF SIGNIFICANCE: Selective cell adhesion and migration guided by regenerative biomaterials are extremely important for the regeneration of targeted tissues, which can avoid the drawbacks of incorrect and uncontrolled responses of tissue cells to implants. For example, selectivity of smooth muscle cells (SMCs) over fibroblasts (FIBs) is required to prevent adventitia fibrosis in vascular regeneration. Herein we prepare a uniform cell-repelling layer, on which SMCs-selective Val-Ala-Pro-Gly (VAPG) peptides are immobilized in a continuous manner. Selective adhesion and enhanced and directional migration of SMCs over FIBs are achieved by the interplay of cell-repelling layer and gradient SMCs-selective VAPG peptides, paving a new way for the design of novel vascular grafts with enhanced biological performance.

Mediating the invasion of smooth muscle cells into a cell-responsive hydrogel under the existence of immune cells.

Cell migration plays an important role in many physiological and biological processes, which is influenced by both physicochemical properties of surrounding matrix and signal gradient generated by neighboring/remote cells. Here we aim to develop a co-culture system of immune cells and smooth muscle cells (SMCs) based on the combination of Transwell and cell-responsive hydrogels. This model can be used to study the cell invasion into hydrogels in dynamic physiological conditions, with better mimicking of the in vivo microenvironment. Methacrylic anhydride-modified hyaluronic acid (MA-HA) macromolecules were crosslinked by matrix metalloproteinases (MMPs) sensitive peptides (MMP SP) to fabricate a cell-degradable hydrogel mimicking dynamic extracellular matrix (ECM). The migration of SMCs into the MMP-sensitive hydrogel was investigated under the existence of U937 cells, a type of macrophage-like cells. The invasion distance of SMCs in the MMP-sensitive hydrogels was much longer than that in the MMP-insensitive ones both in vitro and in vivo. The impact of hydrogel degradability and inductive signal gradient generated by U937 cells on cell invasion was compared, revealing that the degradability plays a major role in regulating cell invasion into the 3D hydrogels. Further mechanism investigation revealed that the expressions of cell migration-related genes and proteins were significantly up-regulated in the MMP-sensitive hydrogels compared to those in the MMP-insensitive hydrogels.