Facile Deposition of Manganese Dioxide to Albumin-Bound Paclitaxel Nanoparticles for Modulation of Hypoxic Tumor Microenvironment To Improve Chemoradiation Therapy.

Tumor microenvironment with hypoxia and excess hydrogen peroxide (H2O2) tremendously limits the effect of chemoradiation therapy of colorectal cancer. For the first time, we developed a facile method to deposit manganese dioxide (MnO2) on the surface of albumin bound paclitaxel nanoparticles (ANPs-PTX) to obtain MnO2-functioned ANPs-PTX (MANPs-PTX). In the tumor microenvironment, MANPs-PTX could consume excess hydrogen peroxide (H2O2) to produce abundant oxygen for tumor oxygenation and improve chemoradiation therapy. Meanwhile, the released Mn2+ from MANPs-PTX had excellent T1 magnetic resonance imaging (MRI) performances for tumor detection. Notably, the obtained MANPs-PTX would be a promising theranostic agent and have potential clinical application prospects.

Enhancing osteosarcoma therapy through aluminium hydroxide nanosheets-enabled macrophage modulation.

Chemotherapy in osteosarcoma treatment has long been stagnating, leaving challenges in the treatment of patients with metastatic and recurrent osteosarcoma. Modulation of macrophages in the tumour microenvironment offers great opportunities to elicit a durable antitumour effect. Here, we employed aluminium hydroxide nanosheets (nAl) to co-deliver the chemotherapy drug doxorubicin (DOX) and immune modulator zoledronic acid (ZA). The hexagon nAl was obtained by a facile approach, with a high positive surface charge for the loading of ZA. With 37% and 8.5% payloads to ZA and DOX, the formed nAl/ZD showed efficient cell growth inhibition to LM8 osteosarcoma cells, and preferential M1 polarization induction to RAW 264.7 macrophage cells. Furthermore, enhanced antitumour effect was observed with nAl/ZD-enabled macrophage activation in the LM8/RAW 264.7 co-culture model. Our results may inspire new treatment strategies for osteosarcoma.

Enhanced Cartilage and Subchondral Bone Repair Using Carbon Nanotube-Doped Peptide Hydrogel-Polycaprolactone Composite Scaffolds.

A carbon nanotube-doped octapeptide self-assembled hydrogel (FEK/C) and a hydrogel-based polycaprolactone PCL composite scaffold (FEK/C3-S) were developed for cartilage and subchondral bone repair. The composite scaffold demonstrated modulated microstructure, mechanical properties, and conductivity by adjusting CNT concentration. In vitro evaluations showed enhanced cell proliferation, adhesion, and migration of articular cartilage cells, osteoblasts, and bone marrow mesenchymal stem cells. The composite scaffold exhibited good biocompatibility, low haemolysis rate, and high protein absorption capacity. It also promoted osteogenesis and chondrogenesis, with increased mineralization, alkaline phosphatase (ALP) activity, and glycosaminoglycan (GAG) secretion. The composite scaffold facilitated accelerated cartilage and subchondral bone regeneration in a rabbit knee joint defect model. Histological analysis revealed improved cartilage tissue formation and increased subchondral bone density. Notably, the FEK/C3-S composite scaffold exhibited the most significant cartilage and subchondral bone formation. The FEK/C3-S composite scaffold holds great promise for cartilage and subchondral bone repair. It offers enhanced mechanical support, conductivity, and bioactivity, leading to improved tissue regeneration. These findings contribute to the advancement of regenerative strategies for challenging musculoskeletal tissue defects.

Cell-free and cytokine-free self-assembling peptide hydrogel-polycaprolactone composite scaffolds for segmental bone defects.

Segmental bone defects over the self-healing threshold are a major challenge for orthopedics. Despite the advancements in clinical practice, traditional tissue engineering methods are limited by the addition of heterogeneous cells and cytokines, leading to carcinoma or other adverse effects. Here, we present a cell-free and cytokine-free strategy using an ECM-mimetic self-assembling peptide hydrogel (SAPH)- polycaprolactone (PCL) composite scaffold. The hydrophilic SAPH endows the rigid PCL scaffold with excellent biocompatibility and preference for osteogenesis induction. The autologous cells around the bone defect site immediately grew, proliferated, and secreted ECM and cytokines after contacting the implanted SAPH-PCL composite scaffold, and the bone repair of rabbit ulnar segmental bone defect was achieved in just six months. Quantitative proteomic analysis reveals that the SAPH-PCL composite scaffold accelerates osteoblastogenesis, osteoclastogenesis, and angiogenesis with moderate immune responses and negligible effects on pathological fibrosis. These findings have important implications for the potential clinical applications of the SAPH-PCL composite scaffold in patients with segmental bone defects and identify the mechanisms of action for accelerated segmental bone defect repair.

Data-independent acquisition-based proteomics analysis correlating type 2 diabetes mellitus with osteoarthritis in total knee arthroplasty patients.

BACKGROUND: To explore the effects of type 2 diabetes mellitus (T2DM) on osteoarthritis (OA), 12 bone tissue samples were obtained surgically from the human total knee arthroplasty patients and analyzed by quantitative proteomics. METHODS: Based on patient clinical histories, patient samples were assigned to diabetes mellitus osteoarthritis (DMOA) and OA groups. A data-independent acquisition method for data collection was used with proteomic data analysis to assess intergroup proteomic differences. Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genome pathway enrichment analysis were used to further find the correlation between T2DM and OA. RESULTS: GO functional analysis found 153 differentially expressed proteins between DMOA and OA groups, of which 92 differentially expressed proteins were significantly up-regulated and 61 were significantly down-regulated. Kyoto Encyclopedia of Genes and Genome pathway analysis found 180 pathways, including 9 pathways significantly enriched. Further data analysis revealed that 6 signaling pathways were closely associated with T2DM and OA. CONCLUSION: OA and DMOA onset and progression were closely related to synthesis and metabolism of extracellular matrix components (e.g., fibronectin, decorin, etc.). The effects of T2DM on OA occur though 2 major ways of oxidative stress and low-grade chronic inflammation, involving in 2 inhibited signaling pathways and 4 activated signaling pathways.

FEK self-assembled peptide hydrogels facilitate primary hepatocytes culture and pharmacokinetics screening.

A FEFEFKFK (FEK, F, phenylalaninyl; E, glutamyl; K, lysinyl)-based self-assembling peptide hydrogel (FEK-SAPH) was developed to replace sandwich culture (SC) for improved culture of primary hepatocytes in vitro. Under neutral conditions, FEK self-assembles to form ß-sheet nanofibers, which in turn form FEK-SAPH. For the culture of rat primary hepatocytes (RPH), the use of FEK-SAPH simplified operation steps and promoted excellent cell-cell interactions while maintaining the SC-related RPH polarity trend. Compared with SC, FEK-SAPH cultured RPH for 14 days, the bile duct network was formed, the secretion of albumin and urea was improved, and the metabolic clearance rate based on cytochrome P450 (CYPs) was comparable. In FEK-SAPH culture, the expression level of the biliary efflux transporter bile salt export pump increased by 230.7%, while the biliary excretion index value of deuterium-labeled sodium taurocholate (d8-TCA) differed slightly from the SC value (72% and 77%, respectively, p = .0195). The inhibitory effect of cholestasis drugs on FEK-SAPH was significantly higher than that of SC. In FEK-SAPH, hepatoprotective drugs were more effective in antagonizing hepatotoxicity induced by lithocholic acid (LCA). FEK-SAPH cultured RPH with hepatoprotective drugs can better recover from LCA-induced damage. In summary, FEK-SAPH can be used as a substitute for SC for pharmacokinetic screening to evaluate the drug absorption, disposition, metabolism, excretion, and toxicity (ADMET) in hepatocytes.

Peptide-drug conjugate-based novel molecular drug delivery system in cancer.

Drug delivery systems are generally believed to comprise drugs and excipients. A peptide-drug conjugate is a single molecule that can simultaneously play multiple roles in a drug delivery system, such as in vivo drug distribution, targeted release, and bioactivity functions. This molecule can be regarded as an integrated drug delivery system, so it is called a molecular drug delivery system. In the context of cancer therapy, a peptide-drug conjugate comprises a tumor-targeting peptide, a payload, and a linker. Tumor-targeting peptides specifically identify membrane receptors on tumor cells, improve drug-targeted therapeutic effects, and reduce toxic and side effects. Payloads with bioactive functions connect to tumor-targeting peptides through linkers. In this review, we explored ongoing clinical work on peptide-drug conjugates targeting various receptors. We discuss the binding mechanisms of tumor-targeting peptides and related receptors, as well as the limiting factors for peptide-drug conjugate-based molecular drug delivery systems.

Preparation and characterisation of bifunctional surface-modified silicone catheter in lumen.

OBJECTIVES: The purpose of this study was to evaluate the coating of antimicrobial peptides (AMPs) and polyvinylpyrrolidone (PVP) to the surface of a silicone catheter to reduce bacterial growth and to increase hydrophilicity, respectively. METHODS: Surface characterisation was performed on bare silicone, AMP-coated, PVP-coated and AMP + PVP-coated silicone catheters using attenuated total reflectance-infrared (ATR-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS) and water contact angle. Antibacterial activity, antibacterial biofilm growth and XTT assay were performed on bare silicone, AMP-coated, PVP-coated and AMP + PVP-coated silicone catheters. Statistical analysis was performed by one-way ANOVA. RESULTS: The water contact angle of the AMP + PVP-coated silicone catheter was 21.37 ± 2.17° compared with 107.23 ± 0.96°, 74.40 ± 1.76° and 20.77 ± 0.32° for bare silicone, AMP-coated and PVP-coated silicone catheters. Based on in vitro antimicrobial tests, the AMP + PVP-coated silicone catheter had 6.2, 2.2 and 2.5 greater antibacterial activity than that of the bare silicone catheter against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, respectively. Moreover, bacterial biofilm growth on the surface of the AMP + PVP-coated silicone catheter was minimal as characterised by scanning electron microscopy. MTT assay showed that bare silicone, AMP-coated, AMP + PVP-coated and PVP-coated silicone catheters were non-cytotoxic to 3T3 and human colon cancer (Caco-2) cells. CONCLUSIONS: This work demonstrates that AMP + PVP-coated silicone catheters have potential clinical application prospects with improved hydrophilicity, excellent biocompatibility, antibacterial activity and a certain antibacterial biofilm effect.

Bifunctional liposomes reduce the chemotherapy resistance of doxorubicin induced by reactive oxygen species.

Doxorubicin (DOX) liposome is a widely used nano-medicine for colorectal cancer treatment. However, doxorubicin therapy increases the level of reactive oxygen species (ROS) in tumor cells, such as hydrogen peroxide (H2O2), which can stabilize hypoxia-inducible-factor-1α (HIF-1α). In a tumor hypoxic microenvironment, HIF-1 can up-regulate tumor-resistance related proteins, including P-glycoprotein (P-gp), glucose transporter 1 (GLUT-1), and matrix metalloproteinase 9 (MMP-9), leading to tumor tolerance to chemotherapy. The functional inhibition of HIF-1 can overcome this resistance and enhance the efficacy of tumor therapy. Here, we encapsulated one of the most effective HIF-1 inhibitors, acriflavine (ACF), and DOX in liposomes (DOX-ACF@Lipo) to construct bifunctional liposomes. ACF and DOX, released from DOX-ACF@Lipo, could effectively suppress the function of HIF-1 and the process of DNA replication, respectively. Consequently, the bifunctional liposome has great potential to be applied in clinics to overcome chemotherapy resistance induced by hypoxia.

Rheological and Mechanical Analyses of Felbinac Cataplasms by Using Box⁻Behnken Design.

Felbinac, an active pharmaceutical ingredient (API) used clinically for the treatment of osteoarthritis, has poor solubility. Felbinac cataplasm product design was investigated using rheological and mechanical analyses. Experiments using a response surface methodology based on Box⁻Behnken design (BBD) incorporated three independent variables: the proportions of partially neutralized polyacrylate (NP800), dihydroxyaluminum aminoacetate (DAAA), and felbinac. Statistically significant quadratic models obtained using BBD demonstrated optimal NP-800, DAAA, and felbinac cataplasm proportions of 4.78⁻5.75%, 0.30⁻0.59%, and 0.70⁻0.90%, respectively. Felbinac cataplasms exhibited "gel-like" mechanical property with predominantly elastic behavior. Rheological studies correlated increasing NP-800 and DAAA concentrations with increased complex modulus (G*) values that were inversely related to peeling strength. Frequency sweep and creep tests revealed decreasing tan θ values with increasing NP-800 and DAAA concentrations. G' and G" values were higher for higher NP-800 and DAAA levels, although G" values decreased with increasing DAAA concentration. Response surface methodology was applied to develop mathematical models. Variance analysis showed that the quadratic model effectively predicted felbinac and matrix material interactions, with two verification samples upholding model predictions. Relative errors between predicted and measured G* values were 3.28% and 1.10% and for peeling strength were 1.24% and 5.59%, respectively. In conclusion, rheological and mechanical analyses of felbinac cataplasms using BBD permits optimization of cataplasms as topical drug delivery vehicles.

Tumor Oxygenation and Hypoxia Inducible Factor-1 Functional Inhibition via a Reactive Oxygen Species Responsive Nanoplatform for Enhancing Radiation Therapy and Abscopal Effects.

Hypoxia, and hypoxia inducible factor-1 (HIF-1), can induce tumor resistance to radiation therapy. To overcome hypoxia-induced radiation resistance, recent studies have described nanosystems to improve tumor oxygenation for immobilizing DNA damage and simultaneously initiate oxygen-dependent HIF-1α degradation. However, HIF-1α degradation is incomplete during tumor oxygenation treatment alone. Therefore, tumor oxygenation combined with residual HIF-1 functional inhibition is crucial to optimizing therapeutic outcomes of radiotherapy. Here, a reactive oxygen species (ROS) responsive nanoplatform is reported to successfully add up tumor oxygenation and HIF-1 functional inhibition. This ROS responsive nanoplatform, based on manganese dioxide (MnO2) nanoparticles, delivers the HIF-1 inhibitor acriflavine and other hydrophilic cationic drugs to tumor tissues. After reacting with overexpressed hydrogen peroxide (H2O2) within tumor tissues, Mn2+ and oxygen molecules are released for magnetic resonance imaging and tumor oxygenation, respectively. Cooperating with the HIF-1 functional inhibition, the expression of tumor invasion-related signaling molecules (VEGF, MMP-9) is obviously decreased to reduce the risk of metastasis. Furthermore, the nanoplatform could relieve T-cell exhaustion via downregulation of PD-L1, whose effects are similar to the checkpoint inhibitor PD-L1 antibody, and subsequently activates tumor-specific immune responses against abscopal tumors. These therapeutic benefits including increased X-ray-induced damage, downregulated resistance, and T-cell exhaustion related proteins expression achieved synergistically the optimal inhibition of tumor growth. Overall, this designed ROS responsive nanoplatform is of great potential in the sensitization of radiation for combating primary and metastatic tumors.

Relative quantitation analysis of the substrate specificity of glutamyl endopeptidase with bovine α-caseins.

A bovine α-caseins' preparation digested with glutamyl endopeptidase (GE) at 37 and 50°C was quantitatively analysed with the isobaric tag for relative and absolute quantification (iTRAQ) technique using nano-LC-ESI-QTOF-MS/MS. Incubation temperature was shown to affect protein digestion. MS analysis of the digestion products indicated that phosphorylated peptides were less sensitive than non-phosphorylated peptides according to the MS intensities. GE hydrolysed Glu(51)-Tyr(52) and Glu(50)-Glu(51) in Glu(49)-Glu(50)-Glu(51)-Tyr(52) of bovine αs1-casein. The results herein helped to confirm the precise process of α-caseins' hydrolysis with GE, which is significant for quantifying the release of bio- and techno-functional peptides.

Investigation of the substrate specificity of glutamyl endopeptidase using purified bovine ß-casein and synthetic peptides.

Purified bovine ß-casein was digested with glutamyl endopeptidase (GE) at 37 and 50 °C for 4 h. The peptides generated were determined using nano-LC-ESI-qTOF-MS/MS. GE was highly specific and hydrolyzed peptide bonds in ß-casein predominantly on the carboxy terminal of Glu and Asp. Pro residues were not preferred, while Met was poorly preferred at the P1' position. Glu-Met hydrolysis was less preferred in comparison to Asp-Met hydrolysis. Five synthetic peptides corresponding to specific sequences in ß-casein were incubated with GE at 37 °C to further characterize the substrate specificity. MS analysis of the digestion products indicated that GE hydrolyzed Glu-Ser in Glu-Glu-Ser. Furthermore, hydrolysis of Glu-Met and Glu-Pro was observed. The presence of multiple-phosphorylated Ser residues upstream from the scissile bond did not appear to affect hydrolysis of Glu-Ser. The results herein are relevant to our understanding of the substrate specificity of GE and the peptides that may be expected during the hydrolysis of ß-casein.

Substrate specificity of glutamyl endopeptidase (GE): hydrolysis studies with a bovine α-casein preparation.

Glutamyl endopeptidase (GE) from Alcalase™ 2.4 L was purified using hydrophobic interaction (HIC) and ion-exchange (IEX) chromatography. The yield of GE obtained was approximately 42%. Bovine α-casein (containing α(s1)- and α(s2)-casein) was digested with GE at 37 and 50°C for 4h. Samples were withdrawn at various time intervals and the peptides generated were analysed using mass spectrometry. GE activity was highly specific and hydrolysed the peptide bond predominantly on the carboxy side of Glu residues while hydrolysis on the carboxyl side of Asp residues was also observed. Hydrolysis did not occur when Pro was at the P(1)' position. In Glu-Glu-X (X=Arg, Asn, Ile and Ser) and Glu-Glu-Glu-Lys sequences, hydrolysis of Glu-X and Glu-Lys was preferred. The results are relevant to our understanding of the hydrolytic specificity of Alcalase, a food-grade proteolytic preparation containing GE activity which is used in the generation of casein hydrolysates.