Photo-Controlled Calcium Overload from Endogenous Sources for Tumor Therapy.

Designing reactive calcium-based nanogenerators to produce excess calcium ions (Ca2+ ) in tumor cells is an attractive tumor treatment method. However, nanogenerators that introduce exogenous Ca2+ are either overactive incapable of on-demand release, or excessively inert incapable of an overload of calcium rapidly. Herein, inspired by inherently diverse Ca2+ -regulating channels, a photo-controlled Ca2+ nanomodulator that fully utilizes endogenous Ca2+ from dual sources was designed to achieve Ca2+ overload in tumor cells. Specifically, mesoporous silica nanoparticles were used to co-load bifunctional indocyanine green as a photodynamic/photothermal agent and a thermal-sensitive nitric oxide (NO) donor (BNN-6). Thereafter, they were coated with hyaluronic acid, which served as a tumor cell-targeting unit and a gatekeeper. Under near-infrared light irradiation, the Ca2+ nanomodulator can generate reactive oxygen species that stimulate the transient receptor potential ankyrin subtype 1 channel to realize Ca2+ influx from extracellular environments. Simultaneously, the converted heat can induce BNN-6 decomposition to generate NO, which would open the ryanodine receptor channel in the endoplasmic reticulum and allow stored Ca2+ to leak. Both in vitro and in vivo experiments demonstrated that the combination of photo-controlled Ca2+ influx and release could enable Ca2+ overload in the cytoplasm and efficiently inhibit tumor growth.

A Peptide-Conjugated Probe with Cleavage-Induced Morphological Change for Treatment on Tumor Cell Membrane.

Despite the promising advancements of in situ forming nanoassembly for the inhibition of tumor growth and metastasis, the lack of sufficient triggering sites and hardly controlling the forming position restrict their further developments. Herein, a smart transformable peptide-conjugated probe (DMFA) with enzyme cleavage-induced morphological change is designed for treatment on the tumor cell membrane. Specifically, after self-assembling into nanoparticles and anchoring on the cell membrane with sufficient interaction sites rapidly and stably, DMFA will be efficiently cleaved into α-helix forming part (DP) and ß-sheet forming part (LFA) by overexpressed matrix metalloproteinase-2. Thus, the promoted Ca2+ influx by DP-induced cell membrane breakage and decreased Na+ /K+ -ATPase activity by LFA-assembled nanofibers wrapping the cells can inhibit PI3K-Akt signaling pathway, leading to the inhibition of tumor cell growth and metastasis. This peptide-conjugated probe undergoes in situ morphological transformation on the cell membrane, exhibiting great potential in tumor therapy.

Regulating the Membrane Affinity of Multi-module Probes to Address the Trade-off between Anchoring and Internalization.

Cell membrane transport is the first and crucial step for bioprobes to realize the diagnosis, imaging, and therapy in cells. However, during this transport, there is a trade-off between anchoring and internalization steps, which will seriously affect the membrane transport efficiency. In the past, because the interaction between probes and cell membrane is constant, this challenge is hard to solve. Here, we proposed a strategy to regulate the membrane affinity of multi-module probes that enabled probe to have strong affinity during cell membrane anchoring and weak affinity during internalization. Specifically, a multi-module probe defined as LK-M-NA was constructed, which consisted of three main parts, membrane-anchoring α-helix peptide (LK), anchoring regulator (M), and therapeutic module (NA). With the α-helix module, LK-M-NA was able to rapidly anchor on the cell membrane and the binding energy was -1450.90 kcal/mol. However, after pericellular cleavage by the highly active matrix metalloproteinase-2 , LK could be removed due to the breakage of M and the binding energy reduced to -869.95 kcal/mol. Thus, the internalization restriction caused by high affinity was relieved. Owing to the alterable affinity, the membrane transport efficiency of LK-M-NA increased to 14.58%, well addressing the trade-off problem.

Modular Peptide Probe for Protein Analysis.

The analysis and regulation of proteins are of great significance for the development of disease diagnosis and treatment. However, complicated analytical environment and complex protein structure severely limit the accuracy of their analysis results. Nowadays, ascribing to the editability and bioactivity of peptides, peptide-based probes could meet the requirements of good selectivity and high affinity to overcome the challenges. In this review, we summarize the advances in the use of modular peptide probes for proteins analysis. It focuses on how to design and optimize the structure of probes, as well as their performance. Then, the strategies and application to improve the analysis result of modular peptide probes are introduced. Finally, we also discuss current challenge and provide some ideas for the future direction for modular peptide probes, hoping to accelerate their clinical transformation.

Spatial Order of Functional Modules Enabling Diverse Intracellular Performance of Fluorescent Probes.

To overcome a series of challenges in tumor therapy, modular-agent probes (MAPs) comprised of various functional modules have been proposed. Researchers have tried to optimize the MAPs by exploiting the new modules or increasing the numbers of module, while neglecting the configuration of various modules. Here, we focus on the different spatial arrangements of existing modules. By utilizing a tetraphenylethylene (TPE) derivative with stereochemical structure and dual modifiable end-group sites as small molecule scaffold, two MAPs with same modular agents (module T for enhancing the internalization of MAPs by tumor cells and module M for causing mitochondrial dysfunction) but different spatial arrangements (on the one side, TM-AIE, and two sides, T-AIE-M, of the molecule scaffold) are designed. T-AIE-M with larger RGD binding angle performed higher specificity, while TM-AIE characterizing longer α-helix structure displayed superior toxicity.

Sphingosine kinase 1 improves cutaneous wound healing in diabetic rats.

BACKGROUND: Diabetes is one of the most prevalent human metabolic diseases. Wound healing in diabetes is frequently impaired and treatment remains challenging. Sphingolipid metabolites play important roles in the regulation of glucose metabolism. SPK1 is the key enzyme in the sphingolipid metabolic pathway. S1P/SPK plays a pivotal role in the signalling pathways of diverse cellular processes including proliferation, differentiation, migration, apoptosis in diverse cell types. METHODS: To investigate the role of sphingosine kinase 1 (SPK1) in skin injury, plasmids containing the SPK1 gene (pcDNA3-FLAG-SPK1) were applied to cutaneous wounds on a streptozotocin-induced diabetic rat model over a 21-day period. The wound area and rate of wound healing were determined. The histopathological features of the healed wounds were also observed, and SPK1 expression in the skin was detected by immunohistochemistry. RESULTS: There was a significant decrease in wound area in diabetic rats treated with 125 and 60µg/wound pcDNA3-FLAG-SPK1 (P<0.001-0.01). The mean sizes of the wounds were 0.67±0.15cm(2), 0.83±0.18cm(2), and 1.09±0.23cm(2) in both treated and diabetic control group at the 7th day post-treatment respectively. In addition, wound healing in diabetic rats of test group was accelerated. At the 7th day, the mean rates of healing were 73.2±5.7% and 66±7.3% in test group of 125 and 60µg/wound respectively, and 55.4±9.9% in diabetic control group (P<0.001-0.01). Histology revealed that tissue sections from the treated diabetic rats contained more granulation tissue and capillaries than that of the control rats. There was high SPK1 expression in the skin of the treated diabetic rats. CONCLUSIONS: SPK1 gene therapy may represent a novel approach to cutaneous wound healing.

Treatment of chronical myocardial ischemia by adenovirus-mediated hepatocyte growth factor gene transfer in minipigs.

Growth factor gene transfer-induced therapeutic angiogenesis has become a novel approach for the treatment of myocardial ischemia. In order to provide a basis for the clinical application of an adenovirus with hepatocyte growth factor gene (Ad-HGF) in the treatment of myocardial ischemia, we established a minipig model of chronically ischemic myocardium in which an Ameroid constrictor was placed around the left circumflex branch of the coronary artery (LCX). A total of 18 minipigs were randomly divided into 3 groups: a surgery control group, a model group and an Ad-HGF treatment group implanted with Ameroid constrictor. Ad-HGF or the control agent was injected directly into the ischemic myocardium, and an improvement in heart function and blood supply were evaluated. The results showed that myocardial perfusion remarkably improved in the Ad-HGF group compared with that in both the control and model groups. Four weeks after the treatment, the density of newly formed blood vessels was higher and the number of collateral blood vessels was greater in the Ad-HGF group than in the model group. The area of myocardial ischemia reduced evidently and the left ventricular ejection fraction improved significantly in the Ad-HGF group. These results suggest that HGF gene therapy may become a novel approach in the treatment of chronically ischemic myocardium.

Cloning and Expression of the Gene Coding for the Fusion Protein rhGM-CSF/LIF.

A fusion gene was constructed in a plasmid in which the coding regions of human GM_CSF and LIF cDNAs were connected by a synthetic linker sequence encoding a short peptide G-S-G-G-S through DNA recombinant techniques. It was then subcloned into the pBV220 expression vector, and expressed in E. coli after transformation and temperature induction. The expressed protein named as rhGM-LIF was confirmed by Western blot. After purification, the determination of activities showed that rhGM-LIF exhibited both GM-CSF and LIF activities.