Biomimetic photosensitizer nanocrystals trigger enhanced ferroptosis for improving cancer treatment.

As a novel non-apoptotic cell death pathway, ferroptosis can effectively enhance the antitumor effects of photodynamic therapy (PDT) by disrupting intracellular redox homeostasis. However, the reported nanocomposites that combined the PDT and ferroptosis are cumbersome to prepare, and the unfavorable tumor microenvironment also severely interferes with their tumor suppressive effects. To address this inherent barrier, this study attempted to explore photosensitizers that could activate ferroptosis pathway and found that the photosensitizer aloe-emodin (AE) could induce cellular ferroptosis based on its specific inhibiting activity to Glutathione S-transferase P1(GSTP1), a key protein for ferroptosis. Herein, we prepared AE@RBC/Fe nanocrystals (NCs) with synergistic PDT and ferroptosis therapeutic effects by one-step emulsification to obtain AE NCs cores and further modification of red blood cells (RBC) membranes and ferritin. Benefiting from the involvement of ferritin, the prepared AE@RBC/Fe NCs provide not only sufficient oxygen for oxygen-dependent PDT, but also Fe3+ for iron-dependent ferroptosis in tumor cells. Furthermore, the biomimetic surface functionalization facilitated the prolonged circulation and cancer targeting of AE@RBC/Fe NCs in vivo. The in vitro and in vivo results demonstrate that AE@RBC/Fe NCs exhibit significantly enhanced therapeutic effects for the combined two antitumor mechanisms and provide a promising prospect for achieving PDT/ferroptosis synergistic therapy.

Engineering HepG2 spheroids with injectable fiber fragments as predictable models for drug metabolism and tumor infiltration.

In vitro cell and tissue models are playing essential roles in the identification of active pharmaceutical ingredients. Though HepG2 cells have attractive profiles over primary hepatocytes in the availability and viability retention, the expression of metabolizing enzymes is quite low. In the current study, three-dimensional (3D) HepG2 spheroids with smaller sizes of 150 µm (3Ds) and bigger sizes of 300 µm (3Db) are engineered using injectable fiber fragments as the substrate. In contrast to two-dimensional (2D) culture, the enzyme activities for drug metabolisms are restored in 3Ds and the pathophysiological profiles of tumor tissues are rebuilt in 3Db spheroids. Compared with spheroid culture without fiber fragments, 3Ds spheroids show higher activities of metabolizing enzymes (CYP3A4, CYP2A9, and phase II) and higher sensitivities to enzyme inducers (rifampicin and glutathione) and inhibitors (ketoconazole and probenecid). The drug clearance and toxicity to 3Ds spheroids predict better the clinical observations and drug-drug interactions. In addition, compared to scaffold-free spheroid culture, stronger expressions of E-cadherin and hypoxia-inducible factor-1α (HIF-1α) and higher fibronectin secretions are determined in 3Db spheroids, displaying apparent hypoxic and apoptotic regions similar to those found in solid tumors. In contrast to the overestimated drug toxicity in other systems, the infiltrations of free drug and drug-loaded micelles are apparently restricted in 3Db spheroids, exhibiting drug resistance just like in tumor tissues. Thus, this study demonstrates HepG2 spheroids with different sizes as predictable and physiologically relevant models for high-throughput screening of drug metabolism and tumor infiltration.

Glucose and lipid metabolism screening models of hepatocyte spheroids after culture with injectable fiber fragments.

With the rise of obesity, diabetes, and other metabolic diseases, in vitro hepatic cell and tissue models play an essential role in the identification of active pharmaceutical ingredients. Up to now, three-dimensional (3D) culture models have rarely focused on hepatic glucose and lipid metabolism. In addition, primary human liver cells suffer from limited availability and interdonor difference for establishing reproducible models. Thus, in the current study, the most available human liver cancer cell line (HepG2) and primary hepatocytes from rats (rPH) were proposed to construct 3D spheroids using injectable fiber fragments with galactose grafts (gSF) as the substrate. rPH and HepG2 spheroids show strong cell-cell and cell-fiber fragment interactions to promote the cell viability, albumin, and urea syntheses. Compared with HepG2 spheroids, rPH spheroids indicate stronger glucose metabolism abilities in terms of glucose consumption, intracellular glycogen content, gluconeogenesis rate, and sensitivity to glucose modulator hormones like insulin and glucagon. On the other hand, HepG2 spheroids display strong lipid metabolism abilities in producing significantly higher levels of total cholesterol and triglyceride. Compared with those without fiber fragments, the gSF-supported 3D culture establishes effective models for in vitro glucose (rPH spheroids) and lipid metabolisms (HepG2 spheroids). The screening models are confirmed from the respective enzyme activities and gene expressions and show significantly higher sensitivity and clinically related responses to hypoglycemic and lipid-lowering drugs. Thus, the culture configuration demonstrates a predictable in vitro platform for defining glucose and lipid metabolism profiles and screening therapeutic agents for metabolism disorders like diabetes and obesity.

Fibrous testing papers for fluorescence trace sensing and photodynamic destruction of antibiotic-resistant bacteria.

The increasing prevalence of antibiotic-resistant bacteria needs rapid identification and efficient destruction routes. This study proposes testing paper derived from electrospun fibrous mats and aggregation-induced emission (AIE) probes for trace sensing and simultaneous destruction of antibiotic-resistant E. coli. Aptamers are conjugated on fibers for selective capture of E. coli, and the capture capability can be regenerated via rinsing with salt solution. Hydroxyl tetraphenylethene (TPE) is linked with two cephalosporin molecules to construct TPE-Cep probes, and the fluorescence emission is turned on specifically in the presence of ß-lactamase, which is a critical marker for screening resistant bacteria. Fibrous mats are lit up only in the presence of antibiotic-resistant bacteria, and the fluorescence intensity changes could be statistically fitted into an equation for quantitative analysis. Fibrous strips display apparent color changes from blue to green for a visual readout of bacterial levels, and the limit of detection (LOD) is much lower than those of previous paper substrates. In addition, the TPE-Cep probes could produce reactive oxygen species (ROS) under room light illumination to kill the captured bacteria. Thus, the integration of aptamer-grafted electrospun fibers and functional AIE probes provides potential for selective capture, trace imaging and photodynamic destruction of antibiotic-resistant bacteria.

Macrophage Spheroids with Chronological Phenotype Shifting To Promote Therapeutic Angiogenesis in Critical Limb Ischemia.

Therapeutic angiogenesis becomes an essential approach to rescue ischemia and the cell-based therapy facilitates ischemia recovery via stimulation of paracrine signals or replacement of damaged cells. Macrophages participate in multiple processes of tissue repair, and the M1 and M2 phenotypes play distinct roles in the sequential stages of healing initiation, angiogenesis stabilization, and tissue maturation. In this study, macrophage spheroids (MøSs) are proposed to promote therapeutic angiogenesis in critical limb ischemia (CLI) via chronological shifting from M1 to M2 phenotypes. Uniform-sized MøSs are prepared by using electrosprayed microcapsules as the confined growth template, and fiber fragments are inoculated to achieve a local release of chrysin for macrophage phenotype manipulation. The macrophage polarization shifting is confirmed from the decreased CD86 expressions (M1 marker) from 64.7 to 31.7% and the concurrent increase in CD206 expressions (M2 marker) from 39.3 to 72.4%. Meanwhile, the tumor necrosis factor-α secretion from M1 is downregulated by around threefold, whereas the levels of interleukin-10 and transforming growth factor-ß1 from M2 increase by around threefold. The incubation with conditioned media from the MøS culture could promote the proliferation and migration of endothelial cells (ECs), showing a high healing rate of EC layers. The local treatment of hind-limb ischemia leads to a full recovery from ischemic to normal limbs with a high blood perfusion rate. Histological analysis shows few muscle degeneration and fibrosis areas and high capillary densities. As far as we know, this is the first attempt to develop uniform-sized MøSs and to undergo dynamic phenotypic shifting from early M1 to later M2 for angiogenesis promotion in the CLI treatment.

Fibrous strips decorated with cleavable aggregation-induced emission probes for visual detection of Hg2.

The widespread contamination and high poisonousness have created significant concerns and thus demands for facile, rapid and selective monitoring of trace Hg2+. Inspired from the unique aggregation-induced emission (AIE) feature, in the current study, novel tetraphenylethylene (TPE) derivatives are prepared containing sulfonic groups for water solubility modulation and carboxyl dithioacetals for Hg2+ sensing. The TPE derivatives are grafted on electrospun fiber as test papers to initiate the AIE activities, while the Hg2+-specific cleavage of dithioacetal groups leads to the release of TPE derivatives and fluorescence turn-off. The decrease in the fluorescence intensities of fibrous mats could be fitted with Hg2+ levels for quantitative analysis, and the fibrous mats turn from green to bluish-green and then to blue in the presence of different Hg2+ levels. The limit of detection (LOD) reaches as low as 20 nM Hg2+, satisfying the threshold detection in drinking water, and the Hg2+ sensing indicates negligible interference from other metal ions and pH variations. The detected Hg2+ levels in lake water are consistent with the added amount with a recovery rate of over 98 %. It demonstrates a feasible strategy to integrate Hg2+-cleavable AIE probes on fibrous strips for real-time, highly specific and naked-eye detection of trace Hg2+.

3D spheroids generated on carbon nanotube-functionalized fibrous scaffolds for drug metabolism and toxicity screening.

The mechanical and electrical stimuli have a profound effect on the cellular behavior and function. In this study, a series of conductive nanofibrous scaffolds are developed by blend electrospinning of poly(styrene-co-maleic acid) (PSMA) and multiwalled-carbon nanotubes (CNTs), followed by grafting galactose as cell adhesion cues. When the mass ratios of CNTs to PSMA increase up to 5%, the alignment, Young's modulus and conductivity of fibrous scaffolds increase, whereas the average diameter, pore size and elongation at break decrease. Primary hepatocytes cultured on the scaffolds are self-assembled into 3D spheroids, which restores the hepatocyte polarity and sufficient expression of drug metabolism enzymes over an extended period of time. Among these conductive scaffolds, hepatocytes cultured on fibers containing 3% of CNTs (F3) show the highest clearance rates of model drugs, offering a better prediction of the in vivo data with a high correlation value. Moreover, the drug metabolism capability is maintained over 15 days and is more sensitive towards the inducers and inhibitors of metabolizing enzymes, demonstrating the applicability for drug-drug interaction studies. Thus, this culture system has been demonstrated as a reliable in vitro model for high-throughput screening of metabolism and toxicity in the early phases of drug development.

Identification of T-DNA Insertion Site and Flanking Sequence of a Genetically Modified Maize Event IE09S034 Using Next-Generation Sequencing Technology.

Molecular characteristics including information of insertion site, flanking sequence, and copy numbers are the base for the safety assessment and subsequent monitoring of genetically modified organisms (GMOs), which has to be revealed thoroughly in a case-by-case manner. Although both polymerase chain reaction (PCR)-based and next-generation sequencing (NGS)-based approaches are proven to be effective in the molecular characterization of most of GM events, they often fail to work with GM maize events, mainly due to the genome complexity. In this study, by using NGS, we successfully identified the 3' end T-DNA insertion site and flanking sequence of a GM maize event IE09S034, which were confirmed by PCR amplification and Sanger sequencing. Notably, insertions of unintended exogenous elements were revealed in this event although the single copy of target exogenous genes was also confirmed by digital PCR. The output of this study provides novel and important genetic evidence for the safety assessment and monitoring of GM maize event IE09S034.

Bacterial biofilm destruction by size/surface charge-adaptive micelles.

Biofilms formed by pathogenic bacteria are one of the most important reasons for multidrug resistance. One of the major limitations in the biofilm treatment is the existence of intensive matrices, which greatly block the diffusion of antimicrobial agents. In the current study, we designed poly(aspartamide)-derived micelles self-assembled from cationic copolymers with azithromycin-conjugated and pH-sensitive copolymers, followed by loading cis-aconityl-d-tyrosine (CA-Tyr) via electrostatic interactions. In response to the acidic microenvironment of the biofilm matrix, the hydrophilic transition of the pH-sensitive copolymers and the removal of CA-Tyr led to a sharp decrease in micelle size from 107 nm to 54 nm and a rapid shift in their zeta potential from -11.7 mV to +26.4 mV, which facilitated the penetration of the micelles into biofilms. The acid-labile release of d-tyrosine disintegrated the biofilm matrix, and the lipase-triggered release of azithromycin eradicated the bacteria in the biofilms. An in vitro test was performed on pre-established P. aeruginosa biofilms in microwells, while biofilms grown on catheters were surgically implanted in rats for in vivo evaluation. The results demonstrated the capabilities of the size/surface charge-adaptive micelles in the intensive infiltration in the biofilm matrix and spatiotemporal release of biofilm dispersion and antibacterial agents for the comprehensive treatment of biofilm-relevant infections.

Antibacterial Micelles with Vancomycin-Mediated Targeting and pH/Lipase-Triggered Release of Antibiotics.

Antibiotic delivery systems play an important role in increasing the efficacy while reducing the off-target toxicity and antibiotic resistance. Though bacterial infections share pathophysiological pathways similar to tumor tissues, few delivery systems have achieved bacterial targeting and on-demand release of antibiotics. In the current study, amphiphilic poly(ethylene glycol)-poly(ε-caprolactone) (PECL) copolymers are conjugated with vancomycin (VAN) as targeting ligands via pH-cleavable hydrazone bonds to obtain micelle carriers (Van-hyd-PECL). Subsequently, ciprofloxacin (CIP) is encapsulated to obtain Van-hyd-PECL/Cip micelles with an average size of 77 nm and a CIP loading amount of 4.5%. The poly(ethylene glycol) shells and the extension of VAN moieties on the micelle surface enhance the blood circulation and selective recognition of bacteria. The deshielding of VAN shells under acidic conditions disrupts the hydrophobic/hydrophilic balance leading to an increase in micelle sizes, which facilitates the degradation of poly(ε-caprolactone) by lipase overexpressed in the infection site and the release of encapsulated CIP for bacterial destruction. The micelle treatment has improved the survival of Pseudomonas aeruginosa-infected mice and reduced the bacterial burdens and alveolar injuries in lungs, compared with free drugs and micelles without inoculation of VAN moieties. Three doses of Van-hyd-PECL/Cip micelles further extend the animal survival, decrease the bacterial colonization in lungs, and almost restore the normal alveolar microstructure. In this regard, this study has demonstrated a strategy to enhance the bacterial targeting of micelles via an antibiotic (VAN) and to sequentially trigger the release of antibiotics (VAN and CIP) at the infection site.

Cardiomyocyte coculture on layered fibrous scaffolds assembled from micropatterned electrospun mats.

Challenges remain in engineering cardiac tissues with functional and morphological properties similar to those of native myocardium. In the current study, micropatterned fibrous mats are obtained by deposition of electrospun fibers on lithographic collectors to reproduce the anisotropic structure of myocardium, and carbon nanotubes are included in fibers to provide conductivities at the same level of cardiac muscles. The patterned mats are assembled layer-by-layer into patterned scaffolds for coculture of primary cardiomyocytes (CMs) with cardiac fibroblasts (CFs) and endothelial cells (ECs). CMs are organized along the fibers with clear cardiac strips of sarcomeric α-actinin and belt-like connexin-43, showing strong cellular extraction forces and intercellular communications. Compared with square and rectangle patterns, honeycomb (Hc)-patterned scaffolds shows higher ultimate tensile strength and strain to failure. The finite element analysis indicates no apparent stress concentration under stress application in the two orthogonal directions. The Hc-patterned coculture demonstrates significantly higher CM viabilities, deeper penetrations of cells into scaffolds, stronger expression of troponin I, connexin-43 and sarcomeric α-actinin by CMs and more abundant formations of capillary-like networks by ECs than other scaffolds. CMs on Hc-patterned scaffolds display spontaneous beating rates at 101±12times/min after coculture for 5days and remain synchronously beating at 94±8times/min after 15days, which is close to those of adult and neonatal rats. The layered and patterned coculture strategy achieves the spatial arrangement of multiple types of cells and vascularization potential, providing a biomimetic strategy for engineering functional cardiac patches in vitro.

Micropatterned co-culture of cardiac myocytes on fibrous scaffolds for predictive screening of drug cardiotoxicities.

The spatial arrangement of cardiac myocytes (CMs) and other non-myocytes scaffolds, closely resembling native tissue, is essential to control the CM morphology and function for cardiac tissue regeneration. In the current study, micropatterned fibrous scaffolds were developed to establish a CM co-culture system with cardiac fibroblasts (CFs) and endothelial cells (ECs) as a potential in vitro drug screening model. To pursue a biomimetic approach to influence CM behaviors, strip, oval and wave-patterned mats were constructed by deposition of electrospun fibers on lithographic collectors, followed by precise stacking for cell co-cultures. CMs, CFs, and ECs were located on the patterned scaffolds with controlled cellular distribution in the respective regions and no across condition was found. Compared with those after strip and oval-patterned co-culture, CMs co-cultured on wave-patterned scaffolds displayed significantly greater cell viabilities, larger cell elongation ratios, stronger expressions of cardiac-specific Troponin I, connexin 43 and sarcomeric α-actinin and higher beating rates during 15 days of incubation. The responses of co-cultured CMs to quinidine, erythromycin and sotalol show good correlations with clinical observations in the beating rate and the prolongation of the contraction and relaxation time. The in vivo safety data reflected well with the concentrations for 50% of maximal effect (EC50) after drug treatment on co-cultured CMs, which was determined from the changes in the corrected field potential duration (FPDc) against the drug concentrations. During 15 days of patterned co-culture, the interbeat intervals and fluctuations of the CMs indicated quick changes in response to haloperidol treatment and sufficient restoration of the original beating profiles after drug removal. This study demonstrates the capabilities of micropatterned co-culture of CMs to establish the cardiac function as a reproducible and reliable platform for screening cardiac side effects of drugs.

Ratiometric fluorescent response of electrospun fibrous strips for real-time sensing of alkaline phosphatase in serum.

The development of rapid, convenient and reliable assays for monitoring alkaline phosphatase (ALP) levels is valuable for clinical diagnoses and biomedical research. In the current study, a ratiometric assay of ALP activity has been realized by covalent immobilization of fluorescein onto polyethylene terephthalate (PET) fibers, followed by electrostatic adsorption of bisquaternary ammonium salt of tetraphenylethene (TPE-2N+). In the absence of ALP, the complex formation between phosphorylated fluorescein and TPE-2N+ results in the aggregation-induced emission (AIE) of TPE at 471nm. While in the presence of ALP, the hydrolysis of phosphoesters leads to a gradual removal of TPE-2N+ and the restoration of fluorescein emission at 514nm. Fibers with surface amine densities of 30 nmol/mg show the most significant and almost linear increases in I514/I471 ratios from 0.73 to 3.05 with increasing ALP concentrations from 0 to 100 mU/mL. The ratiometric fluorescence responses result in color changes of fibrous strips from blue (TPE-2N+) to green (fluorescein) under an ultraviolet lamp in a matter of minutes. The color changes are more suitable for an eyeball detection of ALP levels ranging from 0 to 80 mU/mL, which is included in the concentration range of ALP in human serum considering the dilution factor if necessary. The ALP detection indicates no apparent interference by serum components and good agreement with enzyme-linked immunosorbent assay (ELISA). Thus, this is the first study on ratiometric fluorescent assay of serum ALP levels by fibrous strips, which offers a capacity to exploit electrospun fibrous mats and ratiometric responses for real-time assays of bioactive substances as self-test devices.

Tuning the conductivity and inner structure of electrospun fibers to promote cardiomyocyte elongation and synchronous beating.

The key to addressing the challenges facing cardiac tissue engineering is the integration of physical, chemical, and electrical cues into scaffolds. Aligned and conductive scaffolds have been fabricated as synthetic microenvironments to improve the function of cardiomyocytes. However, up to now, the influence of conductive capability and inner structure of fibrous scaffolds have not been determined on the cardiomyocyte morphologies and beating patterns. In the current study, highly aligned fibers were fabricated with loaded up to 6% of carbon nanotubes (CNTs) to modulate the electrical conductivity, while blend and coaxial electrospinning were utilized to create a bulk distribution of CNTs in fiber matrices and a spatial embedment in fiber cores, respectively. Conductive networks were formed in the fibrous scaffolds after the inoculation of over 3% CNTs, and the increase in the conductivity could maintain the cell viabilities, induce the cell elongation, enhance the production of sarcomeric α-actinin and troponin I, and promote the synchronous beating of cardiomyocytes. Although the conductivity of blend fibers is slightly higher than that of coaxial fibers with the same CNT loadings, the lower exposures to CNTs resulted in higher cell viability, elongation, extracellular matrix secretion and beating rates for cardiomyocytes on coaxial fibers. Taken altogether, core-sheath fibers with loaded 5% of CNTs in the fiber cores facilitated the cardiomyocyte growth with a production of organized contractile proteins and a pulsation frequency close to that of the atrium. It is suggested that electrospun scaffolds that couple conductivity and fibrous structure considerations may provide optimal stimuli to foster cell morphology and functions for myocardial regeneration or establishment of in vitro cardiomyocyte culture platform for drug screening.

Micropatterned coculture of hepatocytes on electrospun fibers as a potential in vitro model for predictive drug metabolism.

The liver is the major organ of importance to determine drug dispositions in the body, thus the development of hepatocyte culture systems is of great scientific and practical interests to provide reliable and predictable models for in vitro drug screening. In the current study, to address the challenges of a rapid function loss of primary hepatocytes, the coculture of hepatocytes with fibroblasts and endothelial cells (Hep-Fib-EC) was established on micropatterned fibrous scaffolds. Liver-specific functions, such as the albumin secretion and urea synthesis, were well maintained in the coculture system, accompanied by a rapid formation of multicellular hepatocyte spheroids. The activities of phase I (CYP3A11 and CYP2C9) and phase II enzymes indicated a gradual increase for cocultured hepatocytes, and a maximum level was achieved after 5 days and maintained throughout 15 days of culture. The metabolism testing on model drugs indicated that the scaled clearance rates for hepatocytes in the Hep-Fib-EC coculture system were significantly higher than those of other culture methods, and a linear regression analysis indicated good correlations between the observed data of rats and in vitro predicted values during 15 days of culture. In addition, the enzyme activities and drug clearance rates of hepatocytes in the Hep-Fib-EC coculture model experienced sensitive responsiveness to the inducers and inhibitors of metabolizing enzymes. These results demonstrated the feasibility of micropatterned coculture of hepatocytes as a potential in vitro testing model for the prediction of in vivo drug metabolism.

Tunable release of chemotherapeutic and vascular disrupting agents from injectable fiber fragments potentiates combination chemotherapy.

Cancer progression and metastasis relies much on vasculature networks in tumor microenvironment, and the combination treatment with chemotherapeutic drugs and vascular disrupting agents represents apparent clinical benefits. In the current study, fiber fragments with loadings of hydroxycamptothecin (HCPT) or combretastatin A-4 (CA4) were proposed for tumor inhibition and blood vessel disruption after local administration in tumors. To address challenges in balancing the disruption of tumor vessels and intratumoral uptake of chemotherapeutic agents, this study is focus on release tuning of HCPT and CA4 from the fiber fragment mixtures. Hydroxypropyl-ß-cyclodextrin (HPCD) was blended at ratios from 0 to 10% into CA4-loaded fiber fragments (Fc) to modulate CA4 release durations from 0.5 to 24days, and HCPT-loaded fiber fragments (Fh) indicated a sustained release for over 35days. In vitro cytotoxicity tests indicated a sequential inhibition on the endothelial and tumor cell growth, and the growth inhibition of tumor cells was more significant after treatment with mixtures of Fh and Fc containing 2% HPCD (Fc2) than that of other mixtures. In an orthotopic breast tumor model, compared with those of free CA4, or Fc with a fast or slow release of CA4, Fh/Fc mixtures with CA4 release durations from 2 to 12days indicated a lower tumor growth rate, a prolonged animal survival, a lower vessel density in tumors, and a less significant tumor metastasis. In addition, the tumor cell proliferation rate, hypoxia-inducible factor-1α expression within tumors, and the number of surface metastatic nodules in lungs were significantly lower after treatment with Fh/Fc2 mixtures with a CA4 release duration of 5days than those of other mixtures. It demonstrates the advantages of fiber fragment mixtures in independently modulating the release of multiple drugs and the essential role of release tuning of chemotherapeutic drugs and vascular disrupting agents in improving the therapeutic efficacy.

Collaborative trial for the validation of event-specific PCR detection methods of genetically modified papaya Huanong No.1.

For transferring the event-specific PCR methods of genetically modified papaya Huanong No.1 to other laboratories, we validated the previous developed PCR assays of Huanong No.1 according to the international standard organization (ISO) guidelines. A total of 11 laboratories participated and returned their test results in this trial. In qualitative PCR assay, the high specificity and limit of detection as low as 0.1% was confirmed. For the quantitative PCR assay, the limit of quantification was as low as 25 copies. The quantitative biases among ten blind samples were within the range between 0.21% and 10.04%. Furthermore, the measurement uncertainty of the quantitative PCR results was calculated within the range between 0.28% and 2.92% for these ten samples. All results demonstrated that the Huanong No.1 qualitative and quantitative PCR assays were creditable and applicable for identification and quantification of GM papaya Huanong No.1 in further routine lab analysis.

Spheroid culture of primary hepatocytes with short fibers as a predictable in vitro model for drug screening.

The establishment of a reliable in vitro liver model for drug screening remains challenging with respect to tethering the growth of hepatocyte spheroids and adapting to the current high-throughput system. In the current study, short fibers are utilized as scaffolds for the generation of size-controlled hepatocyte spheroids that recapitulate in vivo hepatic phenotypes and functions. The spheroid formation is modulated by the length and galactose/RGD grafts of short fibers, and short 50 µm long fibers motivate the spheroid formation with optimal hepatic function. Short fibers distribute throughout the entire spheroid for tethering hepatocyte growth to form compact spheroids. Compared with scaffold-free spheroid culture on agarose-coated plates, the spheroid culture with short fibers achieves higher clearance rates of model drugs and provides a better prediction of the in vivo drug clearance rate with a correlation value of 0.886. In addition, the drug metabolism capability is highly sensitive to the inducers and inhibitors of metabolizing enzymes, and the responsiveness is maintained during 20 days of culture, exhibiting an efficient in vitro model for determining drug-drug interactions. Therefore, the spheroid culture with short fibers provides an easily manipulated strategy to maintain hepatocyte functions for a prolonged period and enable ready deployment in conventional multiwell plates and diverse organ-on-a-chip devices for high-throughput drug screening.