GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy.

BACKGROUND: Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary treatment that has become a mainstay of advanced cancer treatment. Conventional glypican-3 (GPC3)-CAR-T cells have not produced ideal clinical outcomes in advanced hepatocellular carcinoma (HCC), and the mechanism is unclear. This study aims to investigate the clinical utility of novel GPC3-7-19-CAR-T cells constructed by our team and to explore the mechanisms underlying their antitumor effects. METHODS: We engineered a novel GPC3-targeting CAR including an anti-GPC3 scFv, CD3ζ, CD28 and 4-1BB that induces co-expression of IL-7 at a moderate level (500 pg/mL) and CCL19 at a high level (15000 pg /mL) and transduced it into human T cells. In vitro, cell killing efficacy was validated by the xCELLigence RTCA system, LDH nonradioactive cytotoxicity assay and was confirmed in primary HCC organoid models employing a 3D microfluid chip. In vivo, the antitumor capacity was assessed in a humanized NSG mouse xenograft model. Finally, we initiated a phase I clinical trial to evaluate the safety and effect of GPC3-7-19-CAR-T cells in the clinic. RESULTS: GPC3-7-19-CAR-T cells had 1.5-2 times higher killing efficiency than GPC3-CAR-T cells. The tumor formation rates in GPC3-7-19-CAR-T cells treated model were reduced (3/5vs.5/5), and the average tumor volumes were 0.74 cm3 ± 1.17 vs. 0.34 cm3 ± 0.25. Of note, increased proportion of CD4+ TEM and CD8+ TCM cells was infiltrated in GPC3-7-19-CAR-T cells group. GPC3-7-19-CAR-T cells obviously reversed the immunosuppressive tumor microenvironment (TME) by reducing polymorphonuclear (PMN)-myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells infiltration and recruiting more dendritic cells (DCs) to HCC xenograft tumor tissues. In one patient with advanced HCC, GPC3-7-19-CAR-T-cell treatment resulted in tumor reduction 56 days after intravenous infusion. CONCLUSIONS: In conclusion, GPC3-7-19-CAR-T cells achieved antitumor effects superior to those of conventional GPC3-CAR-T cells by reconstructing the TME induced by the dominant CD4+ TEM and CD8+ TCM cell subsets. Most importantly, GPC3-7-19-CAR-T cells exhibited good safety and antitumor efficacy in HCC patients in the clinic. ► Novel GPC3-7-19-CAR-T cells designed with mediate level of IL-7 secretion and high level of CCL19 secretion, which could recruit more mature DCs to assist killing on GPC3+HCCs. ►DC cells recruited by CCL19 could interact with CD4+ T cells and promote the differentiation of CD4+TEFF cells into CD4+TEM and CD8+TCM subsets, leading a better anti-tumor effect on GPC3+HCCs. ►Compared with conventional GPC3-CAR-T, GPC3-7-CCL19-CAR-T cells could reverse tumor immunosuppressive microenvironment by reducing PMN-MDSC and Treg cell infiltration.

Performance evaluation of a chemiluminescence microparticle immunoassay for CK-MB.

BACKGROUND: To verify and evaluate the performance characteristics of a creatine kinase phosphokinase isoenzymes MB (CK-MB) assay kit, which produced by Xiamen Innodx Biotech Co. Ltd. METHODS: Evaluation was carried out according to "Guidelines for principle of analysis performance evaluation of in vitro diagnostic reagent." The performance parameters included detection limit, linearity range, reportable range, recovery test, precision verification, interference test, cross-reactivity, matrix effect, and method comparison. RESULTS: The detection limit was 0.1 ng/mL. The assay had clinical linearity over range of 0.1 ng/mL-500 ng/mL. Reportable range was from 0.1 ng/mL to 1000 ng/mL. The average percent of recovery was 99.66%. The coefficient of variation (CV) for within-run and between-run of low CK-MB sample was 5.55% and 6.16%, respectively. As for high-level sample, it was 7.88% and 7.80%. In medical decision level, the relative deviation (Bias) of all interference tests was lower than 15%. When the sample had mild-hemolysis; hemoglobin ≤15 g/L; triglyceride ≤17 mmol/L; bilirubin ≤427.5 µmol/L; rheumatoid factor ≤206U/mL, there was no significant interference to be found. Moreover, assay kit had no cross-reaction with CK-MM and CK-BB. At last, total diagnostic accuracy of kit was 93.24%, when compared with refer kit. CONCLUSION: Overall the results of the verification study indicated the performance of kit is met the requirements of the clinical test.

Profiling Metal Oxides with Lipids: Magnetic Liposomal Nanoparticles Displaying DNA and Proteins.

Metal oxides include many important materials with various surface properties. For biomedical and analytical applications, it is desirable to engineer their biocompatible interfaces. Herein, a phosphocholine liposome (DOPC) and its headgroup dipole flipped counterpart (DOCP) were mixed with ten common oxides. Using the calcein leakage assay, cryo-TEM, and ζ-potential measurement, these oxides were grouped into three types. The type 1 oxides (Fe3 O4 , TiO2 , ZrO2 , Y2 O3 , ITO, In2 O3 , and Mn2 O3 ) form supported bilayers only with DOCP. Type 2 (SiO2 ) forms supported bilayers only with DOPC; type 3 (ZnO and NiO) are cationic and damage lipid membranes. Magnetic Fe3 O4 nanoparticles were further studied for conjugation of fluorophores, proteins, and DNA to the supported DOCP bilayers via lipid headgroup labeling, covalent linking, or lipid insertion. Delivery of the conjugates to cells and selective DNA hybridization were demonstrated. This work provides a general solution for coating the type 1 oxides with a simple mixing in water, facilitating applications in biosensing, separation, and nanomedicine.

Tumor-Triggered Controlled Drug Release from Electrospun Fibers Using Inorganic Caps for Inhibiting Cancer Relapse.

A smart, tumor-trigged, controlled drug release using inorganic "caps" with CO3 (2-) functional groups in electrospun fibers is presented for inhibiting cancer relapse. When the drug-loaded intelligent electrospun fibers encounter pathological acidic environments, the inorganic gates react with the acids and produce CO2 gas, which enables water penetration into the core of the fibers to induce rapid drug release.

Modeling multi-component separation in hydrophobic interaction chromatography with improved parameter-by-parameter estimation method.

Mechanistic models are powerful tools for chromatographic process development and optimization. However, hydrophobic interaction chromatography (HIC) mechanistic models lack an effective and logical parameter estimation method, especially for multi-component system. In this study, a parameter-by-parameter method for multi-component system (called as mPbP-HIC) was derived based on the retention mechanism to estimate the six parameters of the Mollerup isotherm for HIC. The linear parameters (ks,i and keq,i) and nonlinear parameters (ni and qmax,i) of the isotherm can be estimated by the linear regression (LR) and the linear approximation (LA) steps, respectively. The remaining two parameters (kp,i and kkin,i) are obtained by the inverse method (IM). The proposed method was verified with a two-component model system. The results showed that the model could accurately predict the protein elution at a loading of 10 g/L. However, the elution curve fitting was unsatisfactory for high loadings (12 g/L and 14 g/L), which is mainly attributed to the demanding experimental conditions of the LA step and the potential large estimation error of the parameter qmax. Therefore, the inverse method was introduced to further calibrate the parameter qmax, thereby reducing the estimation error and improving the curve fitting. Moreover, the simplified linear approximation (SLA) was proposed by reasonable assumption, which provides the initial guess of qmax without solving any complex matrix and avoids the problem of matrix unsolvable. In the improved mPbP-HIC method, qmax would be initialized by the SLA and finally determined by the inverse method, and this strategy was named as SLA+IM. The experimental validation showed that the improved mPbP-HIC method has a better curve fitting, and the use of SLA+IM reduces the error accumulation effect. In process optimization, the parameters estimated by the improved mPbP-HIC method provided the model with excellent predictive ability and reasonable extrapolation. In conclusion, the SLA+IM strategy makes the improved mPbP-HIC method more rational and can be easily applied to the practical separation of protein mixture, which would accelerate the process development for HIC in downstream of biopharmaceuticals.

Comprehensive comparison of aroma profiles and chiral free and glycosidically bound volatiles in Fujian and Yunnan white teas.

The Fujian and Yunnan provinces in China are the most representative origins of white tea. However, the key differences in the chemical constituents of the two white teas have rarely been revealed. In this study, a comprehensive comparison of the aroma profiles, chiral volatiles, and glycosidically bound volatiles (GBVs) in Fujian and Yunnan white teas was performed, and 174 volatiles and 28 enantiomers, including 22 volatiles and six GBVs, were identified. Linalool, linalyl-ß-primeveroside (LinPrim), and α-terpineol presented the opposite dominant configurations in Fujian and Yunnan white teas, and the chiral GBVs were firstly quantified with significant differences in the contents of R-LinPrim and ß-d-glucopyranosides of (2R, 5R)-linalool oxide A and (2R, 5S)-linalool oxide B. Moreover, discrimination functions for Fujian and Yunnan white teas were created using nine key variables with excellent reliability and efficiency. These results provide a new method for objectively distinguishing authentic white teas according to geographical origin.

Identification of autophagy-related genes as potential biomarkers for type 1 diabetes mellitus.

Background: Type 1 diabetes mellitus (T1DM) is a metabolic disease in which the autoimmune destruction of pancreatic islet ß-cells occurs. This study sought to investigate the role of autophagy-related genes and immune cells in the development of T1DM. Methods: We acquired the raw gene expression profiles of 302 T1DM and 422 normal control peripheral blood samples from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified using the Limma package, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. The Search Tool for the Retrieval of Interacting Genes/Proteins (https://string-db.org/) and Cytoscape autophagy genes were intersected with the DEGs for the immune cell analysis and the correlation analysis. Results: A total of 568 DEGs were identified in the T1DM and normal samples, of which 301 were upregulated and 267 were downregulated. The results of the functional and pathway enrichment analyses showed that the DEGs were closely associated with autophagy and immunity. Member RAS oncogene family (RAB11A), protein tyrosine phosphatase non-receptor type 11, lamin A/C, heat shock protein70, heat shock protein family A member 4, cluster of differentiation 8A, caspase 3 (CASP3), exportin 1, proto-oncogene, non-receptor tyrosine kinase, SMAD family member 4, and sirtuin 1 (SIRT1) were located at the center of the protein-protein interaction network as the core genes. The peripheral blood T cells were more elevated in the T1DM subjects than the normal subjects. RAB11A, CASP3, and SIRT1 are autophagy-associated genes. RAB11A and CASP3 were positively correlated with most immune cells, while SIRT1 was negatively correlated with most immune cells. Conclusions: Autophagy-related genes (i.e., RAB11A, CASP3, and SIRT1) and immune cells (i.e., T and B cells) may play important regulatory roles in the development of T1DM. Our findings provide novel insights into and potential targets for T1DM prediction and treatment.

Electrospun polymeric micro/nanofibrous scaffolds for long-term drug release and their biomedical applications.

Electrospun polymeric micro/nanofibrous scaffolds have been investigated extensively as drug delivery platforms capable of controlled and sustained release of therapeutic agents in situ. Such scaffolds exhibit excellent physicochemical and biological properties and can encapsulate and release various drugs in a controlled fashion. This article reviews recent advances in the design and manufacture of electrospun scaffolds for long-term drug release, placing particular emphasis on polymer selection, types of incorporated drugs and the latest drug-loading techniques. Finally, applications of such devices in traumatic or disease states requiring effective and sustained drug action are discussed and critically appraised in their biomedical context.

Cell infiltrative hydrogel fibrous scaffolds for accelerated wound healing.

Development of natural protein-based fibrous scaffolds with tunable physical properties and biocompatibility is highly desirable to construct three-dimensional (3D), fully cellularized scaffolds for wound healing. Herein, we demonstrated a simple and effective technique to construct electrospun 3D fibrous scaffolds for accelerated wound healing using a photocrosslinkable hydrogel based on gelatin methacryloyl (GelMA). We found that the physical properties of the photocrosslinkable hydrogel including water retention, stiffness, strength, elasticity and degradation can be tailored by changing the light exposure time. We further observed that the optimized hydrogel fibrous scaffolds which were soft and elastic could support cell adhesion, proliferation and migration into the whole scaffolds, facilitating regeneration and formation of cutaneous tissues within two weeks. Such tunable characteristics of the fibrous GelMA scaffolds distinguished them from other reported substrates developed for reconstruction of wound defects including glutaraldehyde-crosslinked gelatin or poly (lactic-co-glycolic acid) (PLGA), whose physical and chemical properties were difficult to modify to allow cell infiltration into the 3D scaffolds for tissue regeneration. We anticipate that the ability to become fully cellularized will make the engineered GelMA fibrous scaffolds suitable for widespread applications as skin substitutes or wound dressings. STATEMENT OF SIGNIFICANCE: In present study, we generate three-dimensional photocrosslinkable gelatin (GelMA)-based fibrous scaffolds with tunable physical and biological properties by using a combined photocrosslinking/electrospinning approach. The developed GelMA fibrous scaffolds can not only support cell viability and cell adhesion, but also facilitate cell migration and proliferation, accelerating regeneration and formation of cutaneous tissues. In addition, the physical properties of the engineered fibrous GelMA hydrogel including water retention capability, mechanical properties and biodegradability can be tuned to accommodate different patients' needs, making it a promising candidate for skin tissue engineering.

Polymer-based nanoparticles for protein delivery: design, strategies and applications.

Therapeutic proteins have attracted significant attention as they perform vital roles in various biological processes. The delivery of therapeutic proteins to target sites is, however, challenging due to their intrinsic sensitivity to different environmental conditions. Polymeric nanoparticles (NPs) can offer not only physical protection from environmental stimuli but also targeted delivery of such proteins to specific sites. In particular, NPs containing charged polymers are preferred for many applications as they provide gentle protection through electrostatic interactions. Moreover, most organs exhibit a specific pH, and by tuning the extent of the electrostatic interactions and contact duration between the target organ and polymeric NPs, the intracellular uptake of the latter and thus long-term therapeutic efficacy can be optimized. In this article, we will critically discuss the design considerations of charged polymeric NPs, strategies for and routes of protein delivery and how these are influenced depending on the choice of delivery route.

Reinforcement of transvaginal repair using polypropylene mesh functionalized with basic fibroblast growth factor.

Numerous modifications have been developed over the past two decades seeking to improve the transvaginal repair in the pelvic organ prolapse (POP) by using polypropylene (PP) mesh implants. The hydrophobicity of PP, however, presents a great hindrance for translating potential technologies into viable clinical applications. In this study, by manipulating self-polymerization and strong adhesive characteristics of dopamine, we developed a facile method to enhance the transvaginal repair by modifying PP meshes with polydopamine (PDA), which allowed easy grafting of basic fibroblast growth factor (bFGF) onto the surface of PP. Such surface modification of PP meshes with bFGF was found to efficiently promote bioactivity without changing the morphology or mechanical properties of the PP meshes. Additionally, bFGF-modified PP meshes significantly promoted cell viability and adhesion compared to the unmodified PP. Ultimately, after three months of implantation, the bFGF-modified PP meshes exhibited improved tissue repair with greater degree of organization of deposited collagen, increased tensile strength and reduced inflammatory response. Overall, the surface-modified PP meshes will be highly practical as templates for transvaginal repair in the POP treatment.

Photocrosslinkable Gelatin Hydrogel for Epidermal Tissue Engineering.

Natural hydrogels are promising scaffolds to engineer epidermis. Currently, natural hydrogels used to support epidermal regeneration are mainly collagen- or gelatin-based, which mimic the natural dermal extracellular matrix but often suffer from insufficient and uncontrollable mechanical and degradation properties. In this study, a photocrosslinkable gelatin (i.e., gelatin methacrylamide (GelMA)) with tunable mechanical, degradation, and biological properties is used to engineer the epidermis for skin tissue engineering applications. The results reveal that the mechanical and degradation properties of the developed hydrogels can be readily modified by varying the hydrogel concentration, with elastic and compressive moduli tuned from a few kPa to a few hundred kPa, and the degradation times varied from a few days to several months. Additionally, hydrogels of all concentrations displayed excellent cell viability (>90%) with increasing cell adhesion and proliferation corresponding to increases in hydrogel concentrations. Furthermore, the hydrogels are found to support keratinocyte growth, differentiation, and stratification into a reconstructed multilayered epidermis with adequate barrier functions. The robust and tunable properties of GelMA hydrogels suggest that the keratinocyte laden hydrogels can be used as epidermal substitutes, wound dressings, or substrates to construct various in vitro skin models.

Self-coated interfacial layer at organic/inorganic phase for temporally controlling dual-drug delivery from electrospun fibers.

Implantable tissue engineering scaffolds with temporally programmable multi-drug release are recognized as promising tools to improve therapeutic effects. A good example would be one that exhibits initial anti-inflammatory and long-term anti-tumor activities after tumor resection. In this study, a new strategy for self-coated interfacial layer on drug-loaded mesoporous silica nanoparticles (MSNs) based on mussel-mimetic catecholamine polymer (polydopamine, PDA) layer was developed between inorganic and organic matrix for controlling drug release. When the interface PDA coated MSNs were encapsulated in electrospun poly(L-lactide) (PLLA) fibers, the release rates of drugs located inside/outside the interfacial layer could be finely controlled, with short-term release of anti-inflammation ibuprofen (IBU) for 30 days in absence of interfacial interactions and sustained long-term release of doxorubicin (DOX) for 90 days in presence of interfacial interactions to inhibit potential tumor recurrence. The DOX@MSN-PDA/IBU/PLLA hybrid fibrous scaffolds were further found to inhibit proliferation of inflammatory macrophages and cancerous HeLa cells, while supporting the normal stromal fibroblast adhesion and proliferation at different release stages. These results have suggested that the interfacial obstruction layer at the organic/inorganic phase was able to control the release of drugs inside (slow)/outside (rapid) the interfacial layer in a programmable manner. We believe such interface polymer strategy will find applications in where temporally controlled multi-drug delivery is needed.

Optimization of intrinsic and extrinsic tendon healing through controllable water-soluble mitomycin-C release from electrospun fibers by mediating adhesion-related gene expression.

To balance intrinsic and extrinsic healing during tendon repair is challenging in tendon surgery. We hypothesized that by mediating apoptotic gene and collagen synthesis of exogenous fibroblasts, the adhesion formation induced by extrinsic healing could be inhibited. With the maintenance of intrinsic healing, the tendon could be healed with proper function with no adhesion. In this study, we loaded hydrophilic mitomycin-C (MMC) into hyaluronan (HA) hydrosols, which were then encapsulated in poly(L-lactic acid) (PLLA) fibers by micro-sol electrospinning. This strategy successfully provided a controlled release of MMC to inhibit adhesion formations with no detrimental effect on intrinsic healing. We found that micro-sol electrospinning was an effective and facile approach to incorporate and control hydrophilic drug release from hydrophobic polyester fibers. MMC exhibited an initially rapid, and gradually steadier release during 40 days, and the release rates could be tuned by its concentration. In vitro studies revealed that low concentrations of MMC could inhibit fibroblast adhesion and proliferation. When lacerate tendons were healed using the MMC-HA loaded PLLA fibers in vivo, they exhibited comparable mechanical strength to the naturally healed tendons but with no significant presence of adhesion formation. We further identified the up-regulation of apoptotic protein Bax expression and down-regulation of proteins Bcl2, collage I, collagen III and α-SMA during the healing process associated with minimum adhesion formations. This approach presented here leverages new advances in drug delivery and nanotechnology and offers a promising strategy to balance intrinsic and extrinsic tendon healing through modulating genes associated with fibroblast apoptosis and collagen synthesis.

Levels of bisphenol-A in different paper products in Guangzhou, China, and assessment of human exposure via dermal contact.

Bisphenol A (BPA) is a chemical widely used both in plastics production as a food and beverage container and in thermal papers as a color developer. Dietary consumption is the main route of human exposure to BPA, but dermal absorption through handling different papers might be underestimated or ignored. In this study, BPA in different paper products, including different types of papers, receipts and Chinese currencies, were investigated. BPA was detected in receipts (n = 87) and Chinese currencies (n = 46) with concentrations of 0.17-2.675 × 10(4) µg per g paper and 0.09-288.55 µg per g paper, respectively. Except for parchment papers (n = 3), copy papers (n = 3) and food contact papers (n = 3), BPA was measured in all of the other paper products, with levels of 0.01-6.67 µg per g paper. BPA transferred from thermal papers to common papers increased with the increasing contact pressure. Compared with that in water, the migration speed of BPA was doubled in the synthetic sweat. Washing hands could reduce BPA dermal exposure, and washing hands with lotion was the most efficient way. However, about 19-47% of BPA was still found on hands after different washing methods. Dermal absorption via handling of receipts and papers was estimated to be 36.45 ng per day for the general population and 1.54 × 10(-3) to 248.73 µg per day for a cashier. These values are below the maximum doses recommended by the U.S. Environmental Protection Agency and the European Food Safety Authority. However, due to its uncertain adverse effects on human beings, long-term BPA exposure through dermal absorption should be paid more attention, particularly for some occupational populations.