Metabolism score and machine learning models for the prediction of esophageal squamous cell carcinoma progression.

The incomplete prediction of prognosis in esophageal squamous cell carcinoma (ESCC) patients is attributed to various therapeutic interventions and complex prognostic factors. Consequently, there is a pressing demand for enhanced predictive biomarkers that can facilitate clinical management and treatment decisions. This study recruited 491 ESCC patients who underwent surgical treatment at Huashan Hospital, Fudan University. We incorporated 14 blood metabolic indicators and identified independent prognostic indicators for overall survival through univariate and multivariate analyses. Subsequently, a metabolism score formula was established based on the biochemical markers. We constructed a nomogram and machine learning models utilizing the metabolism score and clinically significant prognostic features, followed by an evaluation of their predictive accuracy and performance. We identified alkaline phosphatase, free fatty acids, homocysteine, lactate dehydrogenase, and triglycerides as independent prognostic indicators for ESCC. Subsequently, based on these five indicators, we established a metabolism score that serves as an independent prognostic factor in ESCC patients. By utilizing this metabolism score in conjunction with clinical features, a nomogram can precisely predict the prognosis of ESCC patients, achieving an area under the curve (AUC) of 0.89. The random forest (RF) model showed superior predictive ability (AUC = 0.90, accuracy = 86%, Matthews correlation coefficient = 0.55). Finally, we used an RF model with optimal performance to establish an online predictive tool. The metabolism score developed in this study serves as an independent prognostic indicator for ESCC patients.

Design and evaluation of curcumin-loaded poloxamer hydrogels as injectable depot formulations.

Poloxamer hydrogels are of interest as injectable depot delivery systems. However, their use for delivering hydrophobic drugs, such as curcumin, is limited due to poor loading capacity. Here, we evaluated the influence of incorporating hydrophobic medium chain triglycerides (MCT) or amphiphilic polyethylene glycol 400 (PEG400) on the physicochemical properties, drug loading, and in vitro compatibility of a curcumin-loaded poloxamer hydrogel. Poloxamer 407 and 188 hydrogel formulations (16:6 w/w) were prepared and MCT and PEG400 (saturated with curcumin) were added to these systems, either alone or in combination, up to a 10 % w/w additive solvent load. Formulation viscoelasticity, gelation behaviour, injectability, morphology and release profiles were assessed. The cytocompatibility of the formulations was also assessed on dermal fibroblasts (HDFn). Both additives increased curcumin loading into the formulation. Addition of MCT to the hydrogel significantly increased its gelation speed, while PEG400 had a less profound impact. Both additive solvents increased the force required to inject the formulation. PEG400 containing systems were single phase, whereas MCT addition created emulsion systems. All formulations released âˆ¼20-30 % of their loaded curcumin in a sustained fashion over 24 h. The modified hydrogel systems showed good biocompatibility on cells when administering up to ∼100-150 µM curcumin into the culture. This study addresses a key limitation in loading hydrophobic drugs into hydrogels and provides a strategy to enhance drug loading and performance of hydrogels by integrating additives such as MCT and PEG400 into the systems.

Fine-tuning of liposome integrity for differentiated transcytosis and enhanced antitumor efficacy.

Transcytosis-inducing nanomedicines have been developed to improve tumor extravasation. However, the fate during transcytosis across multicell layers and the structural integrity of the nanomedicines before reaching tumor cells could impact antitumor therapy. Here, a BAY 87-2243 (a hypoxia-inducible factor-1 inhibitor)-loaded liposomal system (HA-P-LBAY) modified by low molecular weight protamine (LMWP) and crosslinked by hyaluronic acid (HA) was constructed. This system could accomplish differentiate cellular transport in endothelial and tumor cells by fine-tuning its structural integrity, i.e. transcytosis across the endothelial cells while preserving structural integrity, facilitating subsequent retention and drug release within tumor cells via degradation-induced aggregation. In vitro cellular uptake and transwell studies demonstrated that HA-P-LBAY were internalized by endothelial cells (bEnd.3) via an active, caveolin and heparin sulfate proteoglycan (HSPG)-mediated endocytosis, and subsequently achieved transcytosis mainly through the ER/Golgi pathway. Moreover, the fluorescence resonance energy transfer (FRET) study showed that HA-crosslinking maintained higher integrity of HA-P-LBAY after transcytosis, more efficiently than electrostatic coating of HA (HA/P-LBAY). In addition, more HA-P-LBAY was retained in tumor cells (4T1) compared to HA/P-LBAY corresponding to its enhanced in vitro cytotoxicity. This may be attributed to better integrity of HA-P-LBAY post endothelial transcytosis and more degradation of HA in tumor cells, leading to more liposome aggregation and inhibition of their transcytosis, which was inferred by both TEM images and the HAase responsiveness assay proved by FRET. In vivo, HA-P-LBAY exhibited more potency in tumor suppression than the other formulations in both low and high permeability tumor models. This highlighted that fine-tuning of structural integrity of nanocarriers played a key role no matter whether the transcytosis of nanocarriers contributed to cellular transport. Collectively, this study provides a promising strategy for antitumor therapies by fine-tuning liposome integrity to achieve active trans-endothelial transport with structural integrity and selective aggregation for prolonged tumor retention.

Nur77-IRF1 axis inhibits esophageal squamous cell carcinoma growth and improves anti-PD-1 treatment efficacy.

The nuclear receptor Nur77 plays paradoxical roles in numerous cancers. However, whether Nur77 inhibits esophageal squamous cell carcinoma (ESCC) growth and affects immunological responses against ESCC has not been determined. The functional role of Nur77 in ESCC was investigated in this study using human ESCC cell lines, quantitative real-time polymerase chain reaction (PCR), cell proliferation and colony formation assays, flow cytometry analysis, western blotting and animal models. The target gene controlled by Nur77 was verified using dual-luciferase reporter assays, chromatin immunoprecipitation analysis and functional rescue experiments. To examine the clinical importance of Nur77, 72 human primary ESCC tissues were subjected to immunohistochemistry. Taken together, these findings showed that, both in vitro and in vivo, Nur77 dramatically reduced ESCC cell growth and triggered apoptosis. Nur77 directly interacts with the interferon regulatory factor 1 (IRF1) promoter to inhibit its activity in ESCC. Pharmacological induction of Nur77 using cytosporone B (CsnB) inhibited ESCC cell proliferation and promoted apoptosis both in vitro and in vivo. Furthermore, CsnB increased CD8+ T-cell infiltration and cytotoxicity to inhibit the formation of ESCC tumors in an immunocompetent mouse model. In ESCC tissues, Nur77 expression was downregulated, and IRF1 expression was increased; moreover, their expression levels were negatively related. IRF1 and Nur77 were strongly correlated with overall survival. These findings suggested that Nur77 targets and regulates the IRF1/PD-L1 axis to serve as a tumor suppressor in ESCC. Graphical abstract of the regulatory mechanism of Nur77 overexpression downregulates IRF1 in the inhibition of ESCC progression and enhance anti-PD-1 therapy efficacy.

Fat mass and obesity-associated protein regulates RNA methylation associated with spatial cognitive dysfunction after chronic cerebral hypoperfusion.

RNA methylation can epigenetically regulate learning and memory. However, it is unclear whether RNA methylation plays a critical role in the pathophysiology of Vascular dementia (VD). Here, we report that expression of the fat mass and obesity associated gene (FTO), an RNA demethylase, is downregulated in the hippocampus in models of VD. Through prediction and dual-luciferase reporters validation studies, we observed that miRNA-711 was upregulated after VD and could bind to the 3'-untranslated region of FTO mRNA and regulate its expression in vitro. Methylated RNA immunoprecipitation (MeRIP)-qPCR assay and functional study confirmed that Syn1 was an important target gene of FTO. This suggests that FTO is an important regulator of Syn1. FTO upregulation by inhibition of miR-711 in the hippocampus relieves synaptic association protein and synapse deterioration in vivo, whereas FTO downregulation by miR-711 agomir in the hippocampus leads to aggravate the synapse deterioration. FTO upregulation by inhibition of miR-711 relieves cognitive impairment of rats VD model, whereas FTO downregulation by miR-711 deteriorate cognitive impairment. Our findings suggest that FTO is a regulator of a mechanism underlying RNA methylation associated with spatial cognitive dysfunction after chronic cerebral hypoperfusion.

Inhibition of autophagy-related protein 7 enhances anti-tumor immune response and improves efficacy of immune checkpoint blockade in microsatellite instability colorectal cancer.

BACKGROUND: The efficacy of anti-PD-1 therapy is primarily hindered by the limited T-cell immune response rate and immune evasion capacity of tumor cells. Autophagy-related protein 7 (ATG7) plays an important role in autophagy and it has been linked to cancer. However, the role of ATG7 in the effect of immune checkpoint blockade (ICB) treatment on high microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) CRC is still poorly understood. METHODS: In this study, patients from the cancer genome altas (TCGA) COAD/READ cohorts were used to investigate the biological mechanism driving ATG7 development. Several assays were conducted including the colony formation, cell viability, qRT-PCR, western blot, immunofluorescence, flow cytometry, ELISA, immunohistochemistry staining and in vivo tumorigenicity tests. RESULTS: We found that ATG7 plays a crucial role in MSI-H CRC. Its knockdown decreased tumor growth and caused an infiltration of CD8+ T effector cells in vivo. ATG7 inhibition restored surface major histocompatibility complex I (MHC-I) levels, causing improved antigen presentation and anti-tumor T cell response by activating reactive oxygen species (ROS)/NF-κB pathway. Meanwhile, ATG7 inhibition also suppressed cholesterol accumulation and augmentation of anti-tumor immune responses. Combining ATG7 inhibition and statins improved the therapeutic benefit of anti-PD-1 in MSI-H CRC. Importantly, CRC patients with high expression of both ATG7 and recombinant 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) experienced worse prognosis compared to those with low ATG7 and HMGCR expression. CONCLUSIONS: Inhibition of ATG7 leads to upregulation of MHC-I expression, augments immune response and suppresses cholesterol accumulation. These findings demonstrate that ATG7 inhibition has therapeutic potential and application of statins can increase the sensitivity to immune checkpoint inhibitors.

The Dual Angiogenesis Effects via Nrf2/HO-1 Signaling Pathway of Melatonin Nanocomposite Scaffold on Promoting Diabetic Bone Defect Repair.

Purpose: Given the escalating prevalence of diabetes, the demand for specific bone graft materials is increasing, owing to the greater tendency towards bone defects and more difficult defect repair resulting from diabetic bone disease (DBD). Melatonin (MT), which is known for its potent antioxidant properties, has been shown to stimulate both osteogenesis and angiogenesis. Methods: MT was formulated into MT@PLGA nanoparticles (NPs), mixed with sodium alginate (SA) hydrogel, and contained within a 3D printing polycaprolactone/ß-Tricalcium phosphate (PCL/ß-TCP) scaffold. The osteogenic capacity of the MT nanocomposite scaffold under diabetic conditions was demonstrated via in vitro and in vivo studies and the underlying mechanisms were investigated. Results: Physicochemical characterization experiments confirmed the successful fabrication of the MT nanocomposite scaffold, which can achieve long-lasting sustained release of MT. The in vitro and in vivo studies demonstrated that the MT nanocomposite scaffold exhibited enhanced osteogenic capacity, which was elucidated by the dual angiogenesis effects activated through the NF-E2-related factor 2/Heme oxygenase 1 (Nrf2/HO-1) signaling pathway, including the enhancement of antioxidant enzyme activity to reduce the oxidative stress damage of vascular endothelial cells (VECs) and directly stimulating vascular endothelial growth factor (VEGF) production, which reversed the angiogenesis-osteogenesis uncoupling and promoted osteogenesis under diabetic conditions. Conclusion: This study demonstrated the research prospective and clinical implications of the MT nanocomposite scaffold as a novel bone graft for treating bone defect and enhancing bone fusion in diabetic individuals.

CDK12 inhibition upregulates ATG7 triggering autophagy via AKT/FOXO3 pathway and enhances anti-PD-1 efficacy in colorectal cancer.

As the world's fourth most deadly cancer, colorectal cancer (CRC) still needed the novel therapeutic drugs and target urgently. Although cyclin-dependent kinase 12 (CDK12) has been shown to be implicated in the malignancy of several types of cancer, its functional role and mechanism in CRC remain largely unknown. Here, we found that suppression of CDK12 inhibited tumor growth in CRC by inducing apoptosis. And CDK12 inhibition triggered autophagy by upregulating autophagy related gene 7 (ATG7) expression. Inhibition of autophagy by ATG7 knockdown and chloroquine (CQ) further decreased cell viability induced by CDK12 inhibition. Further mechanism exploration showed that CDK12 interacted with protein kinase B (AKT) regulated autophagy via AKT/forkhead box O3 (AKT/FOXO3) pathway. FOXO3 transcriptionally upregulated ATG7 expression and autophagy when CDK12 inhibition in CRC. Level of CDK12 and p-FOXO3/FOXO3 ratio were correlated with survival in CRC patients. Moreover, CDK12 inhibition improved the efficacy of anti-programmed cell death 1(PD-1) therapy in CRC murine models by enhancing CD8 + T cells infiltration. Thus, our study founded that CDK12 inhibition upregulates ATG7 triggering autophagy via AKT/FOXO3 pathway and enhances anti-PD-1 efficacy in CRC. We revealed the roles of CDK12/FOXO3/ATG7 in regulating CRC progression, suggesting potential biomarkers and therapeutic target for CRC.

Liposome-enabled bufalin and doxorubicin combination therapy for trastuzumab-resistant breast cancer with a focus on cancer stem cells.

Breast cancer stem cells (BCSCs) play a key role in therapeutic resistance in breast cancer treatments and disease recurrence. This study aimed to develop a combination therapy loaded with pH-sensitive liposomes to kill both BCSCs and the okbulk cancer cells using trastuzumab-sensitive and resistant human epidermal growth factor receptor 2 positive (HER2+) breast cancer cell models. The anti-BCSCs effect and cytotoxicity of all-trans retinoic acid, salinomycin, and bufalin alone or in combination with doxorubicin were compared in HER2+ cell line BT-474 and a validated trastuzumab-resistant cell line, BT-474R. The most potent anti-BCSC agent was selected and loaded into a pH-sensitive liposome system. The effects of the liposomal combination on BCSCs and bulk cancer cells were assessed. Compared with BT-474, the aldehyde dehydrogenase positive BCSC population was elevated in BT-474R (3.9 vs. 23.1%). Bufalin was the most potent agent and suppressed tumorigenesis of BCSCs by ∼50%, and showed strong synergism with doxorubicin in both BT-474 and BT-474R cell lines. The liposomal combination of bufalin and doxorubicin significantly reduced the BCSC population size by 85%, and inhibited both tumorigenesis and self-renewal, although it had little effect on the migration and invasiveness. The cytotoxicity against the bulk cancer cells was also enhanced by the liposomal combination than either formulation alone in both cell lines (p < 0.001). The liposomal bufalin and doxorubicin combination therapy may effectively target both BCSCs and bulk cancer cells for a better outcome in trastuzumab-resistant HER2+ breast cancer.

Carfilzomib suppressed LDHA-mediated metabolic reprogramming by targeting ATF3 in esophageal squamous cell carcinoma.

Carfilzomib, a second-generation proteasome inhibitor, has been approved as a treatment for relapsed and/or refractory multiple myeloma. Nevertheless, the molecular mechanism by which Carfilzomib inhibits esophageal squamous cell carcinoma (ESCC) progression largely remains to be determined. In the present study, we found that Carfilzomib demonstrated potent anti-tumor activity against esophageal squamous cell carcinoma both in vitro and in vivo. Mechanistically, carfilzomib triggers mitochondrial apoptosis and reprograms cellular metabolism in ESCC cells. Moreover, it has been identified that activating transcription factor 3 (ATF3) plays a crucial cellular target role in ESCC cells treated with Carfilzomib. Overexpression of ATF3 effectively antagonized the effects of carfilzomib on ESCC cell proliferation, apoptosis, and metabolic reprogramming. Furthermore, the ATF3 protein is specifically bound to lactate dehydrogenase A (LDHA) to effectively suppress LDHA-mediated metabolic reprogramming in response to carfilzomib treatment. Research conducted in xenograft models demonstrates that ATF3 mediates the anti-tumor activity of Carfilzomib. The examination of human esophageal squamous cell carcinoma indicated that ATF3 and LDHA have the potential to function as innovative targets for therapeutic intervention in the treatment of ESCC. Our findings demonstrate the novel function of Carfilzomib in modulating ESCC metabolism and progression, highlighting the potential of Carfilzomib as a promising therapeutic agent for the treatment of ESCC.

Multidrug resistance protein 5 affects cell proliferation, migration and gemcitabine sensitivity in pancreatic cancer MIA Paca­2 and PANC­1 cells.

Gemcitabine­based chemotherapy has been widely adopted as the standard and preferred chemotherapy regimen for treating advanced pancreatic cancer. However, the contribution of multidrug resistance protein 5 (MRP5) to gemcitabine resistance and pancreatic cancer progression remains controversial. In the present study, the effect of silencing MRP5 on gemcitabine resistance and cell proliferation and migration of human pancreatic cancer MIA Paca­2 and PANC­1 cells was investigated by using short­hairpin RNA delivered by lentiviral vector transduction. The knockdown of MRP5 was confirmed on both mRNA and protein levels using qPCR and surface staining assays, respectively. MRP5­regulated gemcitabine sensitivity was assessed by MTT, PrestoBlue and apoptosis assays. The effect of MRP5 on pancreatic cancer cell proliferation and migration was determined using colony­formation, wound­healing and Transwell migration assays. The interaction of gemcitabine and cyclic guanosine monophosphate (cGMP) with MRP5 protein was explored using molecular docking. The results indicated that the MRP5 mRNA and protein levels were significantly reduced in all the MIA Paca­2 and PANC­1 clones. MRP5 affected gemcitabine cytotoxicity and the rate of gemcitabine­induced apoptosis. Silencing MRP5 decreased cell proliferation and migration in both MIA Paca­2 and PANC­1 cells. Docking studies showed high binding affinity of cGMP towards MRP5, indicating the potential of MRP5­mediated cGMP accumulation in the microenvironment. In conclusion, MRP5 has an important role in cancer proliferation and migration in addition to its drug efflux functions in two widely available pancreatic tumour cell lines (MIA Paca­2 and PANC­1).

Hydroxypropyl Methylcellulose Bioadhesive Hydrogels for Topical Application and Sustained Drug Release: The Effect of Polyvinylpyrrolidone on the Physicomechanical Properties of Hydrogel.

Hydrogels are homogeneous three-dimensional polymeric networks capable of holding large amounts of water and are widely used in topical formulations. Herein, the physicomechanical, rheological, bioadhesive, and drug-release properties of hydrogels containing hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone (PVP) were examined, and the intermolecular interactions between the polymers were explored. A three-level factorial design was used to form HPMC-PVP binary hydrogels. The physicomechanical properties of the binary hydrogels alongside the homopolymeric HPMC hydrogels were characterized using a texture analyzer. Rheological properties of the gels were studied using a cone and plate rheometer. The bioadhesiveness of selected binary hydrogels was tested on porcine skin. Hydrophilic benzophenone-4 was loaded into both homopolymeric and binary gels, and drug-release profiles were investigated over 24 h at 33 °C. Fourier transform infrared spectroscopy (FTIR) was used to understand the inter-molecular drug-gel interactions. Factorial design analysis supported the dominant role of the HPMC in determining the gel properties, rather than the PVP, with the effect of both polymer concentrations being non-linear. The addition of PVP to the HPMC gels improved adhesiveness without significantly affecting other properties such as hardness, shear-thinning feature, and viscosity, thereby improving bioadhesiveness for sustained skin retention without negatively impacting cosmetic acceptability or ease of use. The release of benzophenone-4 in the HPMC hydrogels followed zero-order kinetics, with benzophenone-4 release being significantly retarded by the presence of PVP, likely due to intermolecular interactions between the drug and the PVP polymer, as confirmed by the FTIR. The HPMC-PVP binary hydrogels demonstrate strong bioadhesiveness resulting from the addition of PVP with desirable shear-thinning properties that allow the formulation to have extended skin-retention times. The developed HPMC-PVP binary hydrogel is a promising sustained-release platform for topical drug delivery.

Rectal Bioavailability of Amoxicillin from Hollow-Type Suppositories: Effect of Chemical Form of Amoxicillin.

Rectal drug administration could offer advantages in the delivery of medicines for children by avoiding swallowability issues, improving stability and enabling administration by caregivers. This study aimed to evaluate the rectal bioavailability of hollow-type suppositories (HTS) and understand the effect of two chemical forms of amoxicillin: amoxicillin sodium (AS) or amoxicillin trihydrate (AMT). HTS were prepared by incorporating a lipophilic core containing the antibiotic with a polyethylene glycol (PEG) shell. Formulations were characterised in vitro, and the absolute bioavailability was determined in a rabbit model, while drug-base interactions were evaluated using X-ray diffraction crystallography (XRD), differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy. The in vitro amoxicillin release from AMT HTS was delayed, taking 27.3 ± 4.9 h to release 50% drug compared with 1.7 h for the AS HTS, likely due to solubility differences between AMT and AS. The presence of orthorhombic AMT and anhydrous AS crystals in respective HTS was confirmed via XRD and DSC. PEG shells were able to protect the drug chemical stability when stored at 25 °C/60% RH. Despite the difference in their in vitro release rates, a similar rectal bioavailability was found in both forms of amoxicillin (absolute bioavailability 68.2 ± 6.6% vs. 72.8 ± 32.2% for AMT HTS and AS HTS, respectively; p = 0.9682). Both HTS formulations showed little or no irritation to the rectal mucosa following a single dose.

Altered GC- and AT-biased genotypes of Ophiocordyceps sinensis in the stromal fertile portions and ascospores of natural Cordyceps sinensis.

OBJECTIVE: To examine multiple genotypes of Ophiocordyceps sinensis in a semi-quantitative manner in the stromal fertile portion (SFP) densely covered with numerous ascocarps and ascospores of natural Cordyceps sinensis and to outline the dynamic alterations of the coexisting O. sinensis genotypes in different developmental phases. METHODS: Mature Cordyceps sinensis specimens were harvested and continuously cultivated in our laboratory (altitude 2,254 m). The SFPs (with ascocarps) and fully and semi-ejected ascospores were collected for histological and molecular examinations. Biochip-based single nucleotide polymorphism (SNP) MALDI-TOF mass spectrometry (MS) was used to genotype multiple O. sinensis mutants in the SFPs and ascospores. RESULTS: Microscopic analysis revealed distinct morphologies of the SFPs (with ascocarps) before and after ascospore ejection and SFP of developmental failure, which, along with the fully and semi-ejected ascospores, were subjected to SNP MS genotyping analysis. Mass spectra showed the coexistence of GC- and AT-biased genotypes of O. sinensis that were genetically and phylogenetically distinct in the SFPs before and after ejection and of developmental failure and in fully and semi-ejected ascospores. The intensity ratios of MS peaks were dynamically altered in the SFPs and the fully and semi-ejected ascospores. Mass spectra also showed transversion mutation alleles of unknown upstream and downstream sequences with altered intensities in the SFPs and ascospores. Genotype #5 of AT-biased Cluster-A maintained a high intensity in all SFPs and ascospores. An MS peak with a high intensity containing AT-biased Genotypes #6 and #15 in pre-ejection SFPs was significantly attenuated after ascospore ejection. The abundance of Genotypes #5‒6 and #16 of AT-biased Cluster-A was differentially altered in the fully and semi-ejected ascospores that were collected from the same Cordyceps sinensis specimens. CONCLUSION: Multiple O. sinensis genotypes coexisted in different combinations with altered abundances in the SFPs prior to and after ejection, the SFP of developmental failure, and the two types of ascospores of Cordyceps sinensis, demonstrating their genomic independence. Metagenomic fungal members present in different combinations and with dynamic alterations play symbiotic roles in different compartments of natural Cordyceps sinensis.

Tumor-microenvironment-responsive poly-prodrug encapsulated semiconducting polymer nanosystem for phototherapy-boosted chemotherapy.

Phototherapy-induced hypoxia in the tumor microenvironment (TME) is responsible for diminished therapeutic efficacy. Designing an intelligent nanosystem capable of responding to hypoxia for TME-responsive drug delivery will, to some extent, improve the therapeutic efficacy and reduce side effects. Semiconducting polymers with high photothermal conversion efficiency and photostability have tremendous potential as phototheranostics. In this paper, hypoxia-activatable tirapazamine (TPZ) was conjugated onto poly(ethylene glycol) to form a pH-sensitive poly-prodrug, PEG-TPZ, that can be triggered by the low acidity of the TME to cleave the acylamide bond for controllable drug release. PEG-TPZ was then used to encapsulate a semiconducting polymer (TDPP) for NIR-II-fluorescence-imaging-guided synergistic therapy. The reactive oxygen species (ROS) generation and ultrahigh photothermal conversion efficiency (∼58.6%) of the TDPP@PEG-TPZ NPs leads to the destruction of the tumor blood vessels, thus further activating the hypoxia-induced chemotherapy of TPZ. As a result, effective tumor regression was achieved after laser irradiation.

Functionalisation of extracellular vesicles with cyclic-RGDyC potentially for glioblastoma targeted intracellular drug delivery.

With the intrinsic ability to cross the blood-brain barrier, small extracellular vesicles (sEVs) hold promise as endogenous brain-targeted drug delivery nano-platforms for glioblastoma (GBM) treatment. To increase GBM targetability, this study aimed to functionalise sEVs with cyclic arginine-glycine-aspartic acid-tyrosine-cysteine (cRGDyC), a ligand for integrin (αvß3) that is overexpressed in GBM cells. Firstly, the intrinsic cellular uptake of sEVs derived from GBM U87 and pancreatic cancer MIA PaCa-2 cells was investigated on the donor cells. To obtain functionalised sEVs (cRGDyC-sEVs), DSPE-mPEG2000-maleimide was incubated with the selected (U87) sEVs, and cRGDyC was subsequently conjugated to the maleimide groups via a thiol-maleimide coupling reaction. The GBM cell targetability and intracellular trafficking of cRGDyC-sEVs were evaluated on U87 cells by fluorescence and confocal microscopy, using unmodified sEVs as a reference. The cytotoxicity of doxorubicin-loaded vesicles (Dox@sEVs, Dox@cRGDyC-sEVs) was compared with a standard liposome formulation (Dox@Liposomes) and free Dox. Both U87 and MIA PaCa-2 cell-derived sEVs displayed tropism with the former being >4.9-fold more efficient to be internalised into U87. Therefore, the U87-derived sEVs were chosen for GBM-targeting. Approximately 4000 DSPE-mPEG2000-maleimide were inserted onto each sEV with cRGDyC conjugated to the maleimide group. The cell targetability of cRGDyC-sEVs to U87 cells improved 2.4-fold than natural sEVs. Despite their proneness to be colocalised with endosomes/lysosomes, both Dox@sEVs and Dox@cRGDyC-sEVs showed superior cytotoxicity to U87 GBM cells compared to Dox@Liposomes, particularly Dox@cRGDyC-sEVs. Overall, U87-derived sEVs were successufully conjugated with cRGDyC via a PEG linker, and cRGDyC-sEVs were demonstrated to be a potnetial integrin-targeting drug delivery vehicle for GBM treatment. Graphic abstract.

A simple and reliable isocratic high-performance chromatographic assay for the simultaneous determination of hydrophilic benzophenone-4 and lipophilic octocrylene in sunscreens.

OBJECTIVE: This work aimed to develop a simple HPLC method for the simultaneous quantitative determination of the ultraviolet (UV) filters, hydrophilic benzophenone-4 and lipophilic octocrylene, in the presence of three other commonly used UV filters, avobenzone, octisalate and homosalate. METHODS: Reverse-phased HPLC was performed on a C18 column. A scouting gradient was initially used to determine the approximate mobile phase composition required for efficient analyte elution and separation before further optimization. The assay was validated with regard to specificity, linearity, intra- and inter-day accuracy and precision, limits of detection and limits of quantification. An ultrasound dispersion extraction method for the UV filters from a commercial sunscreen was developed, and the extraction efficiencies from spiked samples were calculated. RESULTS: An acetonitrile-methanol-water mixture (20:67:13, v/v/v), where the water component contained 0.2% trifluoroacetic acid (v/v), was found to be the optimal mobile phase at a flow rate of 1.0 mL/min. The assay was linear between 1.0-100 µg/mL for both benzophenone-4 and octocrylene (both correlation coefficients were above 0.999). There was no interference from the excipients of the sunscreen nor from the three other UV filters. The intra- and inter-day accuracy was between 90.0-104.6% for both analytes. Extraction recoveries from a spiked commercial sunscreen were between 95.4 ± 2.1% to 98.5 ± 2.1% for benzophenone-4, and between 87.3 ± 2.3% and 98.9 ± 3.1% for octocrylene. All validation parameters were within the acceptance criteria set out in the International Council for Harmonization (ICH) guidelines. The HPLC assay showed the extracted quantities of benzophenone-4 and octocrylene from the commercial sunscreen closely matched claimed quantities. CONCLUSION: The developed isocratic HPLC method was suitable for simultaneously determining the hydrophilic benzophenone-4 and lipophilic octocrylene in the presence of other commonly used UV filters. Additionally, the extraction method was simple and effective for accurately quantifying the UV filters in a commercial sunscreen.

OBJECTIF: Ces travaux visaient à développer une méthode de chromatographie en phase liquide à haute performance simple pour la détermination quantitative simultanée de la benzophénone-4 hydrophile et de l'octocrylène lipophile, des filtres ultraviolets (UV), en présence de trois autres filtres UV couramment utilisés, l'avobenzone, l'octisalate et l'homosalate. MÉTHODES: Une chromatographie en phase liquide à haute performance en phase inverse a été réalisée sur une colonne C18. Un gradient de référence a été initialement utilisé pour déterminer la composition approximative de la phase mobile requise pour une élution et une séparation efficace de l'analyte avant une optimisation plus poussée. Le dosage a été validé en termes de spécificité, de linéarité, d'exactitude et de précision intra- et inter-journalières, de limites de détection et de limites de quantification. Une méthode d'extraction par dispersion ultrasonique des filtres UV d'une crème solaire commerciale a été mise au point, et les efficacités d'extraction des échantillons artificiellement traités ont été calculées. RÉSULTATS: Un mélange acétonitrile-méthanol-eau (20:67:13, v/v/v), où la composante eau contenait 0,2 % d'acide trifluoroacétique (v/v), s'est avéré être la phase mobile optimale à un débit de 1,0 ml/min. Le dosage était linéaire entre 1,0 et 100 µg/ml pour la benzophénone-4 et l'octocrylène (les deux coefficients de corrélation étaient supérieurs à 0,999). Aucune interférence n'a été observée entre les excipients de l'écran solaire et les trois autres filtres UV. La précision intra et inter-journalière était comprise entre 90,0 et 104,6 % pour les deux analytes. Les récupérations par extraction à partir d'une crème solaire commerciale artificiellement traitée étaient comprises entre 95,4 % ± 2,1 % et 98,5 % ± 2,1 % pour la benzophénone-4, et entre 87,3 % ± 2,3 % et 98,9 % ± 3,1 % pour l'octocrylène. Tous les paramètres de validation étaient conformes aux critères d'acceptation définis dans les lignes directrices du Conseil international d'harmonisation (ICH). Le dosage par chromatographie en phase liquide à haute performance a montré que les quantités extraites de benzophénone-4 et d'octocrylène de la crème solaire commerciale correspondaient étroitement aux quantités revendiquées. CONCLUSION: La méthode de chromatographie en phase liquide à haute performance isocratique mise au point a permis de déterminer simultanément la benzophénone-4 hydrophile et l'octocrylène lipophile en présence d'autres filtres UV couramment utilisés. En outre, la méthode d'extraction était simple et efficace pour quantifier avec précision les filtres UV dans une crème solaire commerciale.

Differential coexistence of multiple genotypes of Ophiocordyceps sinensis in the stromata, ascocarps and ascospores of natural Cordyceps sinensis.

OBJECTIVE: To examine the differential occurrence of Ophiocordyceps sinensis genotypes in the stroma, stromal fertile portion (SFP) densely covered with numerous ascocarps, and ascospores of natural Cordyceps sinensis. METHODS: Immature and mature C. sinensis specimens were harvested. Mature C. sinensis specimens were continuously cultivated in our laboratory (altitude 2,200 m). The SFPs (with ascocarps) and ascospores of C. sinensis were collected for microscopic and molecular analyses using species-/genotype-specific primers. Sequences of mutant genotypes of O. sinensis were aligned with that of Genotype #1 Hirsutella sinensis and compared phylogenetically using a Bayesian majority-rule method. RESULTS: Fully and semiejected ascospores were collected from the same specimens. The semiejected ascospores tightly adhered to the surface of the asci as observed by the naked eye and under optical and confocal microscopies. The multicellular heterokaryotic ascospores showed uneven staining of nuclei. The immature and mature stromata, SFPs (with ascocarps) and ascospores were found to differentially contain several GC- and AT-biased genotypes of O. sinensis, Samsoniella hepiali, and an AB067719-type fungus. The genotypes within AT-biased Cluster-A in the Bayesian tree occurred in all compartments of C. sinensis, but those within AT-biased Cluster-B were present in immature and mature stromata and SPFs but absent in the ascospores. Genotype #13 of O. sinensis was present in semi-ejected ascospores and Genotype #14 in fully ejected ascospores. GC-biased Genotypes #13-14 featured large DNA segment substitutions and genetic material recombination between the genomes of the parental fungi (H. sinensis and the AB067719-type fungus). These ascosporic offspring genotypes combined with varying abundances of S. hepiali in the 2 types of ascospores participated in the control of the development, maturation and ejection of the ascospores. CONCLUSION: Multiple genotypes of O. sinensis coexist differentially in the stromata, SFPs and 2 types of C. sinensis ascospores, along with S. hepiali and the AB067719-type fungus. The fungal components in different combinations and their dynamic alterations in the compartments of C. sinensis during maturation play symbiotic roles in the lifecycle of natural C. sinensis.

Stability of trastuzumab during nanomedicine formulation using SEC-HPLC coupled with polyacrylamide gel electrophoresis.

The anti-HER2 antibody trastuzumab has been proven to be an effective targeting ligand for drug delivery. This study investigates the structural integrity of trastuzumab under different stress factors in formulation development and its long-term stability. A validated size exclusion high performance liquid chromatographic (SEC-HPLC) method was first developed. The stability of trastuzumab (0.21-21 mg/ml) under stress conditions (mechanical, freeze-and-thaw, pH and temperature) and long-term storage in the presence of formulation excipients were monitored for up to 12 months, using both the SEC-HPLC method and sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The anti-proliferation activity of the reconstituted antibody stored at 4 °C against HER2+ BT-474 breast cells was also monitored over 12 months. The developed SEC-HPLC method was sensitive and accurate. Solutions of trastuzumab were resistant to mechanical stress and repeated freeze-and-thaw; but were unstable under acidic (pH 2.0 and 4.0) and alkaline (pH 10.0 and 12.0) environments. The samples degraded over 5 days at 60 °C, and within 24 h at 75 °C. Low temperature (-80 °C or 4 °C) and low concentration (0.21 mg/ml) favoured the long-term stability. The anti-proliferation activity was conserved at 4 °C for at least 12 months. This study provided valuable stability information in developing trastuzumab involved nano-formulation as well as in clinical settings.

Electrically responsive release of proteins from conducting polymer hydrogels.

Electrically modulated delivery of proteins provides an avenue to target local tissues specifically and tune the dose to the application. This approach prolongs and enhances activity at the target site whilst reducing off-target effects associated with systemic drug delivery. The work presented here explores an electrically active composite material comprising of a biocompatible hydrogel, gelatin methacryloyl (GelMA) and a conducting polymer, poly(3,4-ethylenedioxythiophene), generating a conducting polymer hydrogel. In this paper, the key characteristics of electroactivity, mechanical properties, and morphology are characterized using electrochemistry techniques, atomic force, and scanning electron microscopy. Cytocompatibility is established through exposure of human cells to the materials. By applying different electrical-stimuli, the short-term release profiles of a model protein can be controlled over 4 h, demonstrating tunable delivery patterns. This is followed by extended-release studies over 21 days which reveal a bimodal delivery mechanism influenced by both GelMA degradation and electrical stimulation events. This data demonstrates an electroactive and cytocompatible material suitable for the delivery of protein payloads over 3 weeks. This material is well suited for use as a treatment delivery platform in tissue engineering applications where targeted and spatio-temporal controlled delivery of therapeutic proteins is required. STATEMENT OF SIGNIFICANCE: Growth factor use in tissue engineering typically requires sustained and tunable delivery to generate optimal outcomes. While conducting polymer hydrogels (CPH) have been explored for the electrically responsive release of small bioactives, we report on a CPH capable of releasing a protein payload in response to electrical stimulus. The composite material combines the benefits of soft hydrogels acting as a drug reservoir and redox-active properties from the conducting polymer enabling electrical responsiveness. The CPH is able to sustain protein delivery over 3 weeks, with electrical stimulus used to modulate release. The described material is well suited as a treatment delivery platform to deliver large quantities of proteins in applications where spatio-temporal delivery patterns are paramount.