Tumor-Specific Catalysis-Mediated Enhanced Chemodynamic Therapy in Synergy with Mitophagy Inhibition Improves Therapeutic Efficacy in Endometrial Cancer

BackgroundChemodynamic therapy (CDT) relies on tumor microenvironment (e.g. high H2O2 level) responsive Fenton-like reactions to produce hydroxyl radicals (·OH) against tumors. However, endogenous H2O2 is insufficient for effective chemodynamic reactions.ResultsAn NAD(P)H: quinone oxidoreductase 1 (NQO1)highCatalase (CAT)low therapeutic window for the use of NQO1 bioactive drug β-lapachone (β-Lap) was firstly identified in endometrial cancer (EC). Accompanied by NADH depletion, β-Lap was catalyzed by NQO1 to produce excess H2O2 initiating oxidative stress, which selectively suppressed NQO1high EC cell proliferation, induced DNA double-strand breaks and promoted apoptosis. SiRNA-mediated NQO1 knockdown or dicoumarol rescued NQO1high EC cells from β-Lap-induced cytotoxicity. Arginine-glycine-aspartic acid (RGD)-functionalized iron-based metal organic frameworks-MOF(Fe) further promoted the conversion of accumulated H2O2 into highly oxidative ·OH, and in turn exacerbated the oxidative damage to RGD-positive target cells. Mitophagy inhibition by Mdivi-1 blocked a powerful antioxidant defense approach, ultimately ensuring the antitumor efficacy of stepwise amplified ROS signals. The tumor growth inhibition rate was about 85.92%.ConclusionsTumor specific chemotherapy in combination with CDT-triggered therapeutic modality presented unprecedented therapeutic advantages for the treatment of NQO1+ advanced type I or type II EC.

The Feasibility of Targeted Magnetic Iron Oxide Nanoagent for Noninvasive IgA Nephropathy Diagnosis

IgA nephropathy is the most common glomerular disease in the world and has become a serious threat to human health. Accurate and non-invasive molecular imaging to detect and recognize the IgA nephropathy is critical for the subsequent timely treatment; otherwise, it may progress to end-stage renal disease and lead to glomerular dysfunction. In this study, we have developed a sensitive, specific, and biocompatible integrin αvβ3-targeted superparamagnetic Fe3O4 nanoparticles (NPs) for the noninvasive magnetic resonance imaging (MRI) of integrin αvβ3, which is overexpressed in glomerular mesangial region of IgA nephropathy. The rat model of IgA nephropathy was successfully established and verified by biochemical tests and histological staining. Meanwhile, the clinical 18F-AlF-NOTA-PRGD2 probe molecule was utilized to visualize and further confirmed the IgA nephropathy in vivo via positron emission computed tomography. Subsequently, the Fe3O4 NPs were conjugated with arginine–glycine–aspartic acid (RGD) molecules (Fe3O4-RGD), and their integrin αvβ3-targeted T2-weighted imaging (T2WI) potential has been carefully evaluated. The Fe3O4-RGD demonstrated great relaxation in vivo. The T2WI signal of renal layers in the targeted group at 3 h after intravenous injection of Fe3O4-RGD was distinctly lower than baseline, indicating MRI signal decreased in the established IgA nephropathy rat model. Moreover, the TEM characterization and Prussian blue staining confirmed that the Fe3O4-RGD was located at the region of glomerulus and tubular interstitium. Moreover, no obvious signal decreased was detected in the untargeted Fe3O4 treated and normal groups. Collectively, our results establish the possibility of Fe3O4-RGD serving as a feasible MRI agent for the noninvasive diagnosis of IgA nephropathy.

Membranes Prepared from Recombinant RGD-Silk Fibroin as Substrates for Human Corneal Cells

A recombinant formulation of silk fibroin containing the arginine–glycine–aspartic acid (RGD) cell-binding motif (RGD-fibroin) offers potential advantages for the cultivation of corneal cells. Thus, we investigated the growth of corneal stromal cells and epithelial cells on surfaces created from RGD-fibroin, in comparison to the naturally occurring Bombyx mori silk fibroin. The attachment of cells was compared in the presence or absence of serum over a 90 min period and analyzed by quantification of dsDNA content. Stratification of epithelial cells on freestanding membranes was examined by confocal fluorescence microscopy and optimized through use of low molecular weight poly(ethylene glycol) (PEG; 300 Da) as a porogen, the enzyme horseradish peroxidase (HRP) as a crosslinking agent, and stromal cells grown on the opposing membrane surface. The RGD-fibroin reduced the tendency of stromal cell cultures to form clumps and encouraged the stratification of epithelial cells. PEG used in conjunction with HRP supported the fabrication of more permeable freestanding RGD-fibroin membranes, that provide an effective scaffold for stromal–epithelial co-cultures. Our studies encourage the use of RGD-fibroin for corneal cell culture. Further studies are required to confirm if the benefits of this formulation are due to changes in the expression of integrins, components of the extracellular matrix, or other events at the transcriptional level.

Engineering of Bio-Adhesive Ligand Containing Recombinant RGD and PHSRN Fibronectin Cell-Binding Domains in Fusion with a Colored Multi Affinity Tag: Simple Approach for Fragment Study from Expression to Adsorption

Engineering of biomimetic motives have emerged as promising approaches to improving cells’ binding properties of biomaterials for tissue engineering and regenerative medicine. In this study, a bio-adhesive ligand including cell-binding domains of human fibronectin (FN) was engineered using recombinant protein technology, a major extracellular matrix (ECM) protein that interacts with a variety of integrins cell-surface’s receptors and other ECM proteins through specific binding domains. 9th and 10th fibronectin type III repeat containing Arginine-Glycine-Aspartic acid (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) synergic site (FNIII9-10) were expressed in fusion with a Colored Multi Affinity Tag (CMAT) to develop a simplified production and characterization process. A recombinant fragment was produced in the bacterial system using E. coli with high yield purified protein by double affinity chromatography. Bio-adhesive surfaces were developed by passive coating of produced fragment onto non adhesive surfaces model. The recombinant fusion protein (CMAT-FNIII9/10) demonstrated an accurate monitoring capability during expression purification and adsorption assay. Finally, biological activity of recombinant FNIII9/10 was validated by cellular adhesion assay. Binding to α5β1 integrins were successfully validated using a produced fragment as a ligand. These results are robust supports to the rational development of bioactivation strategies for biomedical and biotechnological applications.

RGD-modified multifunctional nanoparticles encapsulating salvianolic acid A for targeted treatment of choroidal neovascularization

Background The development of alternative anti-angiogenesis therapy for choroidal neovascularization (CNV) remains a great challenge. Nanoparticle systems have emerged as a new form of drug delivery in ocular diseases. Here, we report the construction and characterization of arginine-glycine-aspartic acid (RGD)-conjugated polyethyleneimine (PEI) as a vehicle to load antioxidant salvianolic acid A (SAA) for targeted anti-angiogenesis therapy of CNV. In this study, PEI was consecutively modified with polyethylene glycol (PEG) conjugated RGD segments, 3-(4′-hydroxyphenyl) propionic acid-Osu (HPAO), and fluorescein isothiocyanate (FI), followed by acetylation of the remaining PEI surface amines to generate the multifunctional PEI vehicle PEI.NHAc-FI-HPAO-(PEG-RGD) (for short, RGD-PEI). The formed RGD-PEI was utilized as an effective vehicle platform to load SAA. Results We showed that RGD-PEI/SAA complexes displayed desirable water dispersibility, low cytotoxicity, and sustainable release of SAA under different pH conditions. It could be specifically taken up by retinal pigment epithelium (RPE) cells which highly expressed ɑvβ5 integrin receptors in vitro and selectively accumulated in CNV lesions in vivo. Moreover, the complexes displayed specific therapeutic efficacy in a mouse model of laser induced CNV, and the slow elimination of the complexes in the vitreous cavity was verified by SPECT imaging after 131I radiolabeling. The histological examinations further confirmed the biocompatibility of RGD-PEI/SAA. Conclusions The results suggest that the designed RGD-PEI/SAA complexes may be a potential alternative anti-angiogenesis therapy for posterior ocular neovascular diseases. Graphic abstract

Bioinstructive Layer-by-Layer-Coated Customizable 3D Printed Perfusable Microchannels Embedded in Photocrosslinkable Hydrogels for Vascular Tissue Engineering

The development of complex and large 3D vascularized tissue constructs remains the major goal of tissue engineering and regenerative medicine (TERM). To date, several strategies have been proposed to build functional and perfusable vascular networks in 3D tissue-engineered constructs to ensure the long-term cell survival and the functionality of the assembled tissues after implantation. However, none of them have been entirely successful in attaining a fully functional vascular network. Herein, we report an alternative approach to bioengineer 3D vascularized constructs by embedding bioinstructive 3D multilayered microchannels, developed by combining 3D printing with the layer-by-layer (LbL) assembly technology, in photopolymerizable hydrogels. Alginate (ALG) was chosen as the ink to produce customizable 3D sacrificial microstructures owing to its biocompatibility and structural similarity to the extracellular matrices of native tissues. ALG structures were further LbL coated with bioinstructive chitosan and arginine–glycine–aspartic acid-coupled ALG multilayers, embedded in shear-thinning photocrosslinkable xanthan gum hydrogels and exposed to a calcium-chelating solution to form perfusable multilayered microchannels, mimicking the biological barriers, such as the basement membrane, in which the endothelial cells were seeded, denoting an enhanced cell adhesion. The 3D constructs hold great promise for engineering a wide array of large-scale 3D vascularized tissue constructs for modular TERM strategies.

RGD-Modified Multifunctional Nanoparticles Encapsulating Salvianolic Acid A for Targeted Treatment of Choroidal Neovascularization

Background: The development of alternative anti-angiogenesis therapy for choroidal neovascularization (CNV) remains a great challenge. Nanoparticle systems have emerged as a new form of drug delivery in ocular diseases. Here, we report the construction and characterization of arginine-glycine-aspartic acid (RGD)-conjugated polyethyleneimine (PEI) as a vehicle to load antioxidant salvianolic acid A (SAA) for targeted anti-angiogenesis therapy of CNV. In this study, PEI was consecutively modified with polyethylene glycol (PEG) conjugated RGD segments, 3-(4’-hydroxyphenyl) propionic acid-Osu (HPAO), and fluorescein isothiocyanate (FI), followed by acetylation of the remaining PEI surface amines to generate the multifunctional PEI vehicle PEI.NHAc-FI-HPAO-(PEG-RGD) (for short, RGD-PEI). The formed RGD-PEI was utilized as an effective vehicle platform to load SAA. Results: We showed that RGD-PEI/SAA complexes displayed desirable water dispersibility, low cytotoxicity, and sustainable release of SAA under different pH conditions. It could be specifically taken up by ɑvβ5 integrin receptors expressing retinal pigment epithelium (RPE) cells in vitro and selectively accumulated in CNV lesions in vivo. Moreover, the complexes displayed specific therapeutic efficacy in a mouse model of laser induced CNV, and the slow elimination of the complexes in the vitreous cavity was verified by SPECT imaging after 131I radiolabeling. The histological examinations further confimed the biocompatibility of RGD-PEI/SAA.Conclusions: The results suggest that the designed RGD-PEI/SAA complexes may be a potential alternative anti-angiogenesis therapy for posterior ocular neovascular diseases.

Substrate ligand density modulates gap junction intercellular communication during mesenchymal cell condensation

Gap junction intercellular communication (GJIC) provides a continuous and efficient flow of biological information during tissue formation and is essential to sustain homeostasis and function in living organisms. Cell-matrix interactions have been widely addressed and their influence on tissue organization is recognized. However, how extracellular matrix (ECM) adhesion affects intercellular communication during tissue formation remains elusive. Here we use substrates with uneven nanopatterns of adhesive ligand arginine-glycine-aspartic acid (RGD) to control cell adhesion during mesenchymal condensation, a prevalent morphogenetic transition. We show that the establishment of GJIC is an adhesion-gated mechanism, which is dynamically regulated. Substrate effects continuously propagate into the forming tissue through actomyosin contraction, affecting both the 3D architecture and functionality of the GJIC network developing in prechondrogenic condensates.