Freeze-Thaw Microfluidic System Produces "Themis" Nanocomplex for Cleaning Persisters-Infected Macrophages and Enhancing Uninfected Macrophages.

Macrophages are the primary effectors against potential pathogen infections. They can be "parasitized" by intracellular bacteria, serving as "accomplices", protecting intracellular bacteria and even switching them to persisters. Here, using a freeze-thaw strategy-based microfluidic chip, a "Themis" nanocomplex (TNC) is created. The TNC consists of Lactobacillus reuteri-derived membrane vesicles, heme, and vancomycin, which cleaned infected macrophages and enhanced uninfected macrophages. In infected macrophages, TNC releases heme that led to the reconstruction of the respiratory chain complexes of intracellular persisters, forcing them to regrow. The revived bacteria produces virulence factors that destroyed host macrophages (accomplices), thereby being externalized and becoming vulnerable to immune responses. In uninfected macrophages, TNC upregulates the TCA cycle and oxidative phosphorylation (OXPHOS), contributing to immunoenhancement. The combined effect of TNC of cleaning the accomplice (infected macrophages) and reinforcing uninfected macrophages provides a promising strategy for intracellular bacterial therapy.

Glucose-driven transformable complex eliminates biofilm and alleviates inflamm-aging for diabetic periodontitis therapy.

Diabetic periodontitis is a major complication of diabetes, which has a deep involvement in teeth loss and more serious systematic diseases, including Alzheimer's disease, atherosclerosis and cancers. Diabetic periodontitis is difficult to treat because of recalcitrant infection and hyperglycemia-induced tissue dysfunction. Current treatments fail to completely eliminate infection due to the diffusion-reaction inhibition of biofilm, and ignore the tissue dysfunction. Here, we design a glucose-driven transformable complex, composed of calcium alginate (CaAlg) hydrogel shell and Zeolitic imidazolate framework-8 (ZIF-8) core encapsulating Glucose oxidase (GOx)/Catalase (CAT) and Minocycline (MINO), named as CaAlg@MINO/GOx/CAT/ZIF-8 (CMGCZ). The reaction product of glucose-scavenging, gluconic acid, could dissolve ZIF-8 core and transform CMGCZ from inflexible to flexible, facilitating the complex to overcome the diffusion-reaction inhibition of biofilm. Meanwhile, reduced glucose concentration could ameliorate the pyroptosis of macrophages to decrease the secretion of pro-inflammatory factors, thereby reducing inflamm-aging to alleviate periodontal dysfunction.

Therapeutic Potential of Liraglutide for Diabetes-Periodontitis Comorbidity: Killing Two Birds with One Stone.

Background: The relationship between diabetes and periodontitis is bidirectional, and there is now consensus that periodontitis and diabetes are comorbid. There is a quest for a drug that can be used to treat both conditions simultaneously. This study evaluated the anti-inflammatory and osteoprotective effects of liraglutide (LIRA) on periodontitis in diabetic rats. Methods: Male Wistar rats (n = 46) were randomly divided into four groups: control group (n = 8), LIRA group (n = 8), diabetes-associated periodontitis+0.9% saline group (diabetic periodontitis (DP)+NaCl group, n = 15), and diabetes-associated periodontitis+LIRA group (DP+LIRA group, n = 15). LIRA treatment lasted for 4 weeks (300 µg/kg/d) after establishment of a rat model of DP. The expression of IL-6, TNF-α, and IL-1ß was detected by enzyme-linked immunosorbent assay. The morphological changes of periodontal tissues were observed by hematoxylin-eosin staining. The absorption of alveolar bone and its ultrastructural changes were observed by histomorphometry and microcomputed tomography. The expression of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) in alveolar bone was detected by immunohistochemistry. The levels of Runx2 mRNA and ALP mRNA in the gingival epithelium were examined by quantitative real-time polymerase chain reaction. Results: LIRA decreased alveolar bone resorption, improved the microstructure of alveolar bone, and reduced periodontal inflammation and damage (P < 0.05). LIRA also reduced blood glucose level and inhibited the secretion of serum IL-6, TNF-α, and IL-1ß (P < 0.05). In addition, after treatment with LIRA, the ratio of RANKL/OPG was reduced, and the expression levels of ALP mRNA and Runx2 mRNA were upregulated (P < 0.05). Conclusions: LIRA not only controls blood glucose level but also reduces inflammation and bone loss and enhances osteogenic differentiation in diabetes-associated periodontitis. Those indicate that LIRA may be used as a potential medicine for the adjunctive therapy of diabetes-periodontitis comorbidity.

Controlled Fabrication of Bioactive Microtubes for Screening Anti-Tongue Squamous Cell Migration Drugs.

The treatment of tongue squamous cell carcinoma (TSCC) faces challenges because TSCC has an aggressive biological behavior and manifests usually as widespread metastatic disease. Therefore, it is particularly important to screen out and develop drugs that inhibit tumor invasion and metastasis. Two-dimensional (2D) cell culture has been used as in vitro models to study cellular biological behavior, but growing evidence now shows that the 2D systems can result in cell bioactivities that deviate appreciably the in vivo response. It is urgent to develop a novel 3D cell migration model in vitro to simulate the tumor microenvironment as much as possible and screen out effective anti-migration drugs. Sodium alginate, has a widely used cell encapsulation material, as significant advantages. We have designed a microfluidic device to fabricate a hollow alginate hydrogel microtube model. Based on the difference in liquid flow rate, TSCC cells (Cal27) were able to be evenly distributed in the hollow microtubes, which was confirmed though fluorescence microscope and laser scanning confocal microscope (LSCM). Our microfluidic device was cheap, and commercially available and could be assembled in a modular way, which are composed of a coaxial needle, silicone hose, and syringes. It was proved that the cells grow well in artificial microtubes with extracellular matrix (ECM) proteins by LSCM and flow cytometry. Periodic motility conferred a different motor state to the cells in the microtubes, more closely resembling the environment in vivo. The quantitative analysis of tumor cell migration could be achieved simply by determining the position of the cell in the microtube cross-section. We verified the anti-migration effects of three NSAIDs drugs (aspirin, indomethacin, and nimesulide) with artificial microtubes, obtaining the same results as conventional migration experiments. The results showed that among the three NSAIDs, nimesulide showed great anti-migration potential against TSCC cells. Our method holds great potential for application in the more efficient screening of anti-migration tumor drugs.

Bright Aggregation-Induced Emission Nanoparticles for Two-Photon Imaging and Localized Compound Therapy of Cancers.

Photodynamic therapy (PDT), a noninvasive therapeutic strategy for cancer treatment, which always suffers from the low reactive oxygen species (ROS) yield of traditional organic dyes. Herein, we present lipid-encapsulated aggregation-induced emission nanoparticles (AIE NPs) that have a high quantum yield (23%) and a maximum two-photon absorption (TPA) cross-section of 560 GM irradiated by near-infrared light (800 nm). The AIE NPs can serve as imaging agents for spatiotemporal imaging of tumor tissues with a penetration depth up to 505 µm on mice melanoma model. Importantly, the AIE NPs can simultaneously generate singlet oxygen (1O2) and highly toxic hydroxyl radicals (•OH) upon irradiation with 800 nm irradiation for photodynamic tumor ablation. In addition, the AIE NPs can be effectively cleared from the mouse body after the imaging and therapy. This study provides a strategy to develop theranostic agents for cancer image-guided PDT with high brightness, superior photostability, and high biosafety.

Nanocatalyst Complex Can Dephosphorylate Key Proteins in MAPK Pathway for Cancer Therapy.

Controlling phosphorylation processes of proteins is a facile way for manipulating cell fates. Herein, a synergistic therapeutic strategy utilizing a near-infrared (NIR)-responsive nanocatalyst (NC) complex is presented, which is comprised of photoactive NC and protein phosphatase 2A (PP2A), to synergistically inhibit hyperphosphorylation of mitogen-activated protein kinase (MAPK) pathway for cancer therapy, as an example of many biological processes this approach can apply to. NIR-triggered release of PP2A specially dephosphorylates and inactivates mitogen-activated protein kinase kinase (MAP2K, also known as MEK) and extracellular regulated protein kinases (ERK) in the MAPK pathway, meanwhile, the NIR-triggered activation of NC decreases the level of intracellular adenosine triphosphate to attenuate protein phosphorylation of MEK and ERK. The synergistic therapeutics effectively suppress melanoma progression by inhibiting hyperphosphorylation of the MAPK pathway. In addition, the nanocatalyst complex reduces the risk of drug-resistance through inhibiting a rebound of RAS-GTP. The NIR-responsive nanocatalyst complex paves a novel way for cancer therapeutics.

Thermo-triggered Release of CRISPR-Cas9 System by Lipid-Encapsulated Gold Nanoparticles for Tumor Therapy.

CRISPR/Cas9 system is a powerful toolbox for gene editing. However, the low delivery efficiency is still a big hurdle impeding its applications. Herein, we report a strategy to deliver Cas9-sgPlk-1 plasmids (CP) by a multifunctional vehicle for tumor therapy. We condensed CPs on TAT peptide-modified Au nanoparticles (AuNPs/CP, ACP) via electrostatic interactions, and coated lipids (DOTAP, DOPE, cholesterol, PEG2000-DSPE) on the ACP to form lipid-encapsulated, AuNPs-condensed CP (LACP). LACP can enter tumor cells and release CP into the cytosol by laser-triggered thermo-effects of the AuNPs; the CP can enter nuclei by TAT guidance, enabling effective knock-outs of target gene (Plk-1) of tumor (melanoma) and inhibition of the tumor both in vitro and in vivo. This AuNPs-condensed, lipid-encapsulated, and laser-controlled delivery system provides a versatile method for high efficiency CRISPR/Cas9 delivery and targeted gene editing for treatment of a wide spectrum of diseases.

Genome Editing for Cancer Therapy: Delivery of Cas9 Protein/sgRNA Plasmid via a Gold Nanocluster/Lipid Core-Shell Nanocarrier.

The type II bacterial clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 (CRISPR-associated protein) system (CRISPR-Cas9) is a powerful toolbox for gene-editing, however, the nonviral delivery of CRISPR-Cas9 to cells or tissues remains a key challenge. This paper reports a strategy to deliver Cas9 protein and single guide RNA (sgRNA) plasmid by a nanocarrier with a core of gold nanoclusters (GNs) and a shell of lipids. By modifying the GNs with HIV-1-transactivator of transcription peptide, the cargo (Cas9/sgRNA) can be delivered into cell nuclei. This strategy is utilized to treat melanoma by designing sgRNA targeting Polo-like kinase-1 (Plk1) of the tumor. The nanoparticle (polyethylene glycol-lipid/GNs/Cas9 protein/sgPlk1 plasmid, LGCP) leads to >70% down-regulation of Plk1 protein expression of A375 cells in vitro. Moreover, the LGCP suppresses melanoma progress by 75% on mice. Thus, this strategy can deliver protein-nucleic acid hybrid agents for gene therapy.

Gene regulation with carbon-based siRNA conjugates for cancer therapy.

We report fluorescent carbon nanoparticle (FCN)-based small interfering RNA (siRNA) conjugates (C-siRNA) for gene regulation and cancer therapy. The C-siRNA has a core of chitosan-derived FCN and a shell of siRNA, and can down-regulate the expression of polo-like kinase-1 (Plk1), a master regulator of mitosis, via siRNA targeting Plk1 (siPlk1), for cancer therapy. The required amount of the FCNs is only ∼1/30 of that of the gold nanoparticles in delivering equal amount of siRNA. The C-siPlk1 led to ∼80% knockdown of cellular Plk1 mRNA in A375 cells, and induced apoptosis of the A375 cells (31.9%) and MCF-7 cells (20.33%), much higher than those by commercial nonviral gene delivery vectors, such as Lipofectamine 2000 in both cell lines (apoptosis rate < 10%). After the C-siPlk1 was administrated to A375 tumor-bearing mice intravenously, the tumor volume was less than 1/11 of the control groups. The C-siRNA can thus be powerful tools for gene delivery and gene therapy.