Accelerated Development of a COVID-19 Lateral Flow Test in an Academic Setting: Lessons Learned.

The COVID-19 pandemic further demonstrated the need for usable, reliable, and cost-effective point-of-care diagnostics that can be broadly deployed, ideally for self-testing at home. Antigen tests using more-detectable reporter labels (usually at the cost of reader complexity) achieve better diagnostic sensitivity, supporting the value of higher-analytical-sensitivity reporter technologies in lateral flow.We developed a new approach to simple, inexpensive lateral flow assays (LFAs) of great sensitivity, based on the glow stick peroxyoxalate chemistry widely used in emergency settings and in children's toys. At the peak of the COVID-19 pandemic, we had the opportunity to participate in the pandemic-driven NIH Rapid Acceleration of Diagnostics (RADx) initiative aiming to develop a deployable lateral flow diagnostic for SARS-CoV-2 nucleoprotein based on our novel glow stick-inspired light-emitting reporter technology. During this project, we screened more than 250 antibody pairs for analytical sensitivity and specificity directly in LFA format, using recombinant nucleoprotein and then gamma-irradiated virions spiked into negative nasal swab extracts. Membranes and other LFA materials and swabs and extraction reagent components also were screened and selected. Optimization of conjugate preparation and spraying as well as pretreatment/conditioning of the sample pad led to the final optimized LFA strip. Technology development also included optimization of excitation liquid enclosed in disposable droppers, design of a custom cartridge and smartphone-based reader, and app development, even a prototype reader usable with any mobile phone. Excellent preclinical performance was first demonstrated with contrived samples and then with leftover clinical samples. Moving beyond traditional academic focus areas, we were able to establish a quality management system (QMS), produce large numbers of customized LFA cassettes by contract injection molding, build in-house facilities to assemble and store thousands of complete tests for verification and validation and usability studies, and source kitting/packaging services and quality standard reagents and build partnerships for clinical translation, regulatory guidance, scale up, and market deployment. We were not able to bring this early stage technology to the point of commercialization within the limited time and resources available, but we did achieve strong proof-of-concept and advance translational aspects of the platform including initial high-performance LFAs, reading by the iPhone app using only a $2 plastic dark box with no lens, and convenient, usable excitation liquid packaging in droppers manufacturable in very large numbers.In this Account, we aim to provide a concise overview of our 18-month sprint toward the practical development of a deployable antigen lateral flow assay under pandemic conditions and the challenges and successes experienced by our team. We highlight what it takes to coach a technically savvy but commercially inexperienced academic team through the accelerated translation of an early stage technology into a useful product. Finally, we provide a guided tutorial and workflow to empower others interested in the rapid development of translatable LFAs.

Correction: Smartphone-read phage lateral flow assay for point-of-care detection of infection.

Correction for 'Smartphone-read phage lateral flow assay for point-of-care detection of infection' by Maede Chabi, et al., Analyst, 2023, 148, 839-848, https://doi.org/10.1039/D2AN01499H.

Protein A-Nanoluciferase fusion protein for generalized, sensitive detection of immunoglobulin G.

Detection and quantification of antibodies, especially immunoglobulin G (IgG), is a cornerstone of ELISAs, many diagnostics, and the development of antibody-based drugs. Current state-of-the-art immunoassay techniques for antibody detection require species-specific secondary antibodies and carefully-controlled bioconjugations. Poor conjugation efficiency degrades assay performance and increases the risk of clinical false positives due to non-specific binding. We developed a generic, highly-sensitive platform for IgG quantification by fusing the IgG-Fc binding Z domain of Staphylococcal Protein A with the ultrabright bioluminescence reporter Nanoluc-luciferase (Nluc). We demonstrated the application of this fusion protein in a sandwich IgG detection immunoassay using surface-bound antigens to capture target IgG and protein A-Nanoluc fusion as the detector. We optimized the platform's sensitivity by incorporating multiple repeats of the Z domain into the fusion protein constructs. Using rabbit and mouse anti-SARS-CoV-2 Nucleoprotein IgGs as model analytes, we performed ELISAs in two different formats, either with SARS-CoV-2 Nucleoprotein as the capture antigen or with polyclonal chicken IgY as the capture antibody. Using standard laboratory equipment, the platform enabled the quantitation of antibody analytes at concentrations as low as 10 pg/mL (67 fM).

Continuous monitoring of IgG using immobilized fluorescent reporters.

In the manufacture of therapeutic monoclonal antibodies, the clarified cell culture fluid (CCF) is typically loaded onto an initial protein A affinity capture column. Imperfect mass transfer and loading to maximum capacity can risk antibody breakthrough and loss of valuable product, but conservative underloading wastes expensive protein A resin. In addition, the effects of column fouling and ligand degradation require the frequent optimization of immunoglobulin G (IgG) loading to avoid wastage. Continuous real-time monitoring of IgG flowthrough is of great interest, therefore. We previously developed a fluorescence-based monitoring technology that allows batch mix-and-read mAb detection in the CCF. Here, we report the use of reporters immobilized on cyanogenbromide-activated Sepharose 4B resin for continuous detection of IgG in column breakthrough. The column effluent is continuously contacted with immobilized fluorescein-labeled Fc-binding ligands in a small monitoring column to produce an immediately-detectable change in fluorescence intensity. The technology allows rapid and reliable monitoring of IgG in a flowing stream of clarified CCF emerging from a protein A column, without prior sample preparation. We observed a significant change in fluorescence intensity at 0.5 g/L human IgG, sufficient to detect a 5% breakthrough of a 10 g/L load, within 18 s at a flow rate of 0.5 ml/min. The current small-scale technology is suitable for use in process development, but the chemistry should be readily adaptable to larger scale applications using fiber-optic sensors, and continuous IgG monitoring could be applicable in a variety of upstream and downstream process settings.

"Glow ELISA": sensitive immunoassay with minimal equipment and stable reagents.

Glow enzyme-linked immunosorbent assay (glow ELISA) uses inexpensive and shelf-stable glow stick reagents to chemically excite fluorescent reporters, obviating the need for excitation light sources, filters, and complex optics. It achieves excellent limits of detection while offering portability and equipment cost comparable to lateral flow immunoassays.

Smartphone-read phage lateral flow assay for point-of-care detection of infection.

The COVID-19 pandemic has highlighted the urgent need for sensitive, affordable, and widely accessible testing at the point of care. Here we demonstrate a new, universal LFA platform technology using M13 phage conjugated with antibodies and HRP enzymes that offers high analytical sensitivity and excellent performance in a complex clinical matrix. We also report its complete integration into a sensitive chemiluminescence-based smartphone-readable lateral flow assay for the detection of SARS-CoV-2 nucleoprotein. We screened 84 anti-nucleoprotein monoclonal antibody pairs in phage LFA and identified an antibody pair that gave an LoD of 25 pg mL-1 nucleoprotein in nasal swab extract using a FluorChem gel documentation system and 100 pg mL-1 when the test was imaged and analyzed by an in-house-developed smartphone reader. The smartphone-read LFA signals for positive clinical samples tested (N = 15, with known Ct) were statistically different (p < 0.001) from signals for negative clinical samples (N = 11). The phage LFA technology combined with smartphone chemiluminescence imaging can enable the timely development of ultrasensitive, affordable point-of-care testing platforms for SARS-CoV-2 and beyond.

Expression and Characterization of Intein-Cyclized Trimer of Staphylococcus aureus Protein A Domain Z.

Staphylococcus aureus protein A (SpA) is an IgG Fc-binding virulence factor that is widely used in antibody purification and as a scaffold to develop affinity molecules. A cyclized SpA Z domain could offer exopeptidase resistance, reduced chromatographic ligand leaching after single-site endopeptidase cleavage, and enhanced IgG binding properties by preorganization, potentially reducing conformational entropy loss upon binding. In this work, a Z domain trimer (Z3) was cyclized using protein intein splicing. Interactions of cyclic and linear Z3 with human IgG1 were characterized by differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). DSF showed a 5 ℃ increase in IgG1 melting temperature when bound by each Z3 variant. SPR showed the dissociation constants of linear and cyclized Z3 with IgG1 to be 2.9 nM and 3.3 nM, respectively. ITC gave association enthalpies for linear and cyclic Z3 with IgG1 of -33.0 kcal/mol and -32.7 kcal/mol, and -T∆S of association 21.2 kcal/mol and 21.6 kcal/mol, respectively. The compact cyclic Z3 protein contains 2 functional binding sites and exhibits carboxypeptidase Y-resistance. The results suggest cyclization as a potential approach toward more stable SpA-based affinity ligands, and this analysis may advance our understanding of protein engineering for ligand and drug development.

Hypoxia, Serum Starvation, and TNF-α Can Modify the Immunomodulation Potency of Human Adipose-Derived Stem Cells.

INTRODUCTION: Adipose-derived stem cells (ADSCs) are mesenchymal stem cells (MSCs) that are found in adipose tissues, which are easily obtained from liposuction procedures using an enzyme mixture. The adhering cells are then selectively cultivated. ADSCs have great potential in regenerative medicine because they are plentiful, easily accessible, and less invasive. They also have an impressive proliferation ability and can be differentiated into mesenchymal lineages and trans-differentiating into many other cell types. In particular, they have extraordinary abilities in immunomodulation. This study aimed to investigate the effects of culture conditions (hypoxia, starvation, and TNF-α treatment) on the immunomodulation of human ADSCs. METHODS: Human ADSCs were expanded in vitro in the standard condition before they were cultured in different stress conditions. ADSCs from passages fifth were confirmed as MSCs by some standard assays suggested by the International Society for Cell and Gene Therapy. These MSCs were used to culture in four different stress conditions: hypoxia, serum starvation, and TNF-α treatment in 48 h. After treatments, MSCs were used to evaluate their immunomodulation capacity using MSCs mixed lymphocyte reaction assay, and the concentrations of IDO, PGE2, IL-6, and IL-10 were secreted in the culture medium. RESULTS: In different stress conditions, ADSCs exhibited different responses related to their immunomodulation. In serum starvation, ADSCs exerted a strong secretion of IDO and PGE2, whereas they showed strong IL-6 secretion in the TNF-α-supplemented medium. When exposed to lymphocytes, ADSCs caused an increase in the ratio of regulatory T cells (Tregs), and co-culture lymphocytes with ADSCs induced in hypoxic malnutrition conditions increased the IL-10 level the most. In addition, when exposed to dendritic cells (DCs), ADSCs inhibited the mature marker expressions of the DCs. CONCLUSION: The current research showed that ADSCs change their immunomodulation properties to survive in in vitro culture environments. Treatment of ADSCs in the starvation medium for 48 h can increase the immunomodulation of ADSCs.

Neutral DNA-avidin nanoparticles as ultrasensitive reporters in immuno-PCR.

We have developed an immuno-PCR based diagnostic platform which couples detection antibodies to self-assembled, ultra-detectable DNA-avidin nanoparticles stabilized with poly(ethylene glycol) to link DNA amplification to target protein concentration. Electrostatic neutralization and cloaking of the PCR-amplifiable DNA labels by avidin and PEG coating reduces non-specific "stickiness" and enhances assay sensitivity. We further optimized the detectability of the nanoparticles by incorporating four repeats of a unique synthetic DNA PCR target into each nanoparticle. Using human chorionic gonadotropin hormone (hCG) as a model analyte, this platform was able to quantitate the target hCG protein in femtomolar concentrations using only standard laboratory equipment.

Synthesis and Bioactivity of Thiosemicarbazones Containing Adamantane Skeletons.

Reaction of 4-(1-adamantyl)-3-thiosemicarbazide (1) with numerous substituted acetophenones and benzaldehydes yielded the corresponding thiosemicarbazones containing adamantane skeletons. The synthesized compounds were evaluated for their in vitro activities against some Gram-positive and Gram-negative bacteria, and the fungus Candida albicans, and cytotoxicity against four cancer cell lines (Hep3B, HeLa, A549, and MCF-7). All of them showed good antifungal activity against Candida albicans. Compounds 2c, 2d, 2g, 2j and 3a, 3e, 3g displayed significant inhibitory activity against Enterococcus faecalis. Compounds 2a, 2e, 2h, 2k and 3j had moderate inhibitory potency against Staphylococcus aureus. Compounds 2a, 2e and 2g found so good inhibitory effect on Bacillus cereus. Compounds 2d and 2h, which contain (ortho) hydroxyl groups on the phenyl ring, were shown to be good candidates as potential agents for killing the tested cancer cell lines, i.e., Hep3B, A549, and MCF-7. Compounds 2a-c, 2f, 2g, 2j, 2k, 3g, and 3i were moderate inhibitors against MCF-7.

Sero-epidemiological status and risk factors of toxoplasmosis in pregnant women in Northern Vietnam.

BACKGROUND: In Vietnam, few studies have determined the epidemiological status of toxoplasmosis in pregnant women and no routine prenatal screening is in place. This study was conducted to evaluate the seroprevalence of this zoonotic parasitic infection in pregnant women in Northern Vietnam and to assess the association with awareness, risk factors and congenital toxoplasmosis. METHODS: Approximately 800 pregnant women were included in the study from two hospitals, one in Hanoi and one in Thai Binh province, which is known to have a dense cat population. Serological immunoglobulin G (IgG) and immunoglobulin M (IgM) detection was performed to estimate the seroprevalence of toxoplasmosis and sero-incidence of maternal and congenital toxoplasmosis. In addition, a survey was conducted about awareness, clinical history, presentation of signs and symptoms relating to toxoplasmosis and to detect biologically plausible and socio-demographic risk factors associated with toxoplasmosis. Associations with seroprevalence were assessed using univariable and multivariable analysis. RESULTS: The mean IgG seroprevalence after the full diagnostic process was 4.5% (95% confidence interval(CI): 2.7-7.0) and 5.8% (95% CI: 3.7-8.6) in Hanoi and Thai Binh hospital, respectively, and included one seroconversion diagnosed in Thai Binh hospital. Only 2.0% of the pregnant women in Hanoi hospital and 3.3% in Thai Binh hospital had heard about toxoplasmosis before this study. CONCLUSION: Since the percentage of seronegative, and thus susceptible, pregnant women was high and the awareness was low, we suggest to distribute information about toxoplasmosis and its prevention among women of child bearing age. Furthermore, future studies are recommended to investigate why such a low seroprevalence was seen in pregnant women in Northern Vietnam compared to other countries in South East Asia and globally.

PCB-Based Magnetometer as a Platform for Quantification of Lateral-Flow Assays.

This work presents a proof-of-concept demonstration of a novel inductive transducer, the femtoMag, that can be integrated with a lateral-flow assay (LFA) to provide detection and quantification of molecular biomarkers. The femtoMag transducer is manufactured using a low-cost printed circuit board (PCB) technology and can be controlled by relatively inexpensive electronics. It allows rapid high-precision quantification of the number (or amount) of superparamagnetic nanoparticle reporters along the length of an LFA test strip. It has a detection limit of 10-10 emu, which is equivalent to detecting 4 ng of superparamagnetic iron oxide (Fe3O4) nanoparticles. The femtoMag was used to quantify the hCG pregnancy hormone by quantifying the number of 200 nm magnetic reporters (superparamagnetic Fe3O4 nanoparticles embedded into a polymer matrix) immuno-captured within the test line of the LFA strip. A sensitivity of 100 pg/mL has been demonstrated. Upon further design and control electronics improvements, the sensitivity is projected to be better than 10 pg/mL. Analysis suggests that an average of 109 hCG molecules are needed to specifically bind 107 nanoparticles in the test line. The ratio of the number of hCG molecules in the sample to the number of reporters in the test line increases monotonically from 20 to 500 as the hCG concentration increases from 0.1 ng/mL to 10 ng/mL. The low-cost easy-to-use femtoMag platform offers high-sensitivity/high-precision target analyte quantification and promises to bring state-of-the-art medical diagnostic tests to the point of care.

Synthesis and Bioactivity of Hydrazide-Hydrazones with the 1-Adamantyl-Carbonyl Moiety.

Reaction of 1-adamantyl carbohydrazide (1) with various substituted benzaldehydes and acetophenones yielded the corresponding hydrazide-hydrazones with a 1-adamantane carbonyl moiety. The new synthesized compounds were tested for activities against some Gram-negative and Gram-positive bacteria, and the fungus Candida albicans. Compounds 4a, 4b, 5a, and 5c displayed potential antibacterial activity against tested Gram-positive bacteria and C. albicans, while compounds 4e and 5e possessed cytotoxicity against tested human cancer cell lines.

Ultrasensitive Magnetic Nanoparticle Detector for Biosensor Applications.

Ta/Ru/Co/Ru/Co/Cu/Co/Ni80Fe20/Ta spin-valve giant magnetoresistive (GMR) multilayers were deposited using UHV magnetron sputtering and optimized to achieve a 13% GMR ratio before patterning. The GMR multilayer was patterned into 12 sensor arrays using a combination of e-beam and optical lithographies. Arrays were constructed with 400 nm × 400 nm and 400 nm × 200 nm sensors for the detection of reporter nanoparticles. Nanoparticle detection was based on measuring the shift in high-to-low resistance switching field of the GMR sensors in the presence of magnetic particle(s). Due to shape anisotropy and the corresponding demag field, the resistance state switching fields were significantly larger and the switching field distribution significantly broader in the 400 nm × 200 nm sensors as compared to the 400 nm × 400 nm sensors. Thus, sensor arrays with 400 nm × 400 nm dimensions were used for the demonstration of particle detection. Detection of a single 225 nm Fe3O4 magnetic nanoparticle and a small number (~10) of 100 nm nanoparticles was demonstrated. With appropriate functionalization for biomolecular recognition, submicron GMR sensor arrays can serve as the basis of ultrasensitive chemical and biological sensors.

Progress toward universal health coverage in Vietnam: Evidence on dispensing trends of diabetes medications from 2015 to 2021.

AIMS: This study aims to investigate the trends in treatment coverage through dispensing diabetes medications in Vietnam from 2015 to 2021. The findings will serve to inform health policies to mitigate the health burden of Type 2 diabetes mellitus (T2DM). METHODS: We collected information on major antidiabetic medicines from General Department of Vietnam Customs and payments for antidiabetics via the National Health Insurance Program. We applied ordinary least squares models, accounting for economic and health outcome characteristics, to estimate the association between the annual mass of medications and related factors. RESULTS: Nationally, the total mass/doses of all antidiabetic drugs increased rapidly from 2015 to 2021, based on both databases. Metformin was the most frequently prescribed medicine, with the total mass increasing nearly threefold over the study period. Gliclazide, a Sulfonylureas drug, ranked second. In the multivariate regression analysis, a one-unit increase in adults with diabetes (in 1,000 s) was associated with 0.11 % (95 %CI = 0.0005; 0.0076) and 0.13 % (95%CI = 0.0007; 0.0242) higher mass of Metformin and Glimepiride, respectively. CONCLUSION: Our data suggested that policies changes were related to significant increase in antidiabetic medication dispenses in Vietnam. The high treatment coverage indicates impressive progress in achieving universal health coverage in Vietnam, meeting the UN Sustainable Development Goal (SDG).

Glowstick-inspired smartphone-readable reporters for sensitive, multiplexed lateral flow immunoassays.

The COVID-19 pandemic has increased demand for point-of-care (POC) screening tests such as lateral flow assays (LFAs) and highlighted the need for sensitive and cost-effective POC diagnostic platforms. Here, we demonstrate an LFA platform using standard fluorescent nanoparticle reporters in which optical excitation is replaced by chemical excitation using the peroxyoxalate-based chemistry of inexpensive, shelf-stable glowsticks. The one-step chemi-excitation of fluorescent particles produces visible light readable by an unmodified smartphone, enhancing sensitivity while preserving simplicity and cost-effectiveness. Our Glow LFA detected the common model analyte human chorionic gonadotropin with a limit of detection (LoD) of 39 pg/mL-over ten times more sensitive than standard gold nanoparticles using the same antibodies. We also demonstrate its application to the detection of SARS-CoV-2 nucleoprotein at 100 pg/mL in nasal swab extract. Multiple fluorescent dyes can be chemi-excited by a single reagent, allowing for color multiplexing on a single LFA strip with a smartphone camera. The detection of three analytes on a single LFA test line was demonstrated using red, green, and blue fluorescent reporter particles, making glow LFA a promising platform for multiplexed detection.

Improving Visual Defect Detection and Localization in Industrial Thermal Images Using Autoencoders.

Reliable functionality in anomaly detection in thermal image datasets is crucial for defect detection of industrial products. Nevertheless, achieving reliable functionality is challenging, especially when datasets are image sequences captured during equipment runtime with a smooth transition from healthy to defective images. This causes contamination of healthy training data with defective samples. Anomaly detection methods based on autoencoders are susceptible to a slight violation of a clean training dataset and lead to challenging threshold determination for sample classification. This paper indicates that combining anomaly scores leads to better threshold determination that effectively separates healthy and defective data. Our research results show that our approach helps to overcome these challenges. The autoencoder models in our research are trained with healthy images optimizing two loss functions: mean squared error (MSE) and structural similarity index measure (SSIM). Anomaly score outputs are used for classification. Three anomaly scores are applied: MSE, SSIM, and kernel density estimation (KDE). The proposed method is trained and tested on the 32 × 32-sized thermal images, including one contaminated dataset. The model achieved the following average accuracies across the datasets: MSE, 95.33%; SSIM, 88.37%; and KDE, 92.81%. Using a combination of anomaly scores could assist in solving a low classification accuracy. The use of KDE improves performance when healthy training data are contaminated. The MSE+ and SSIM+ methods, as well as two parameters to control quantitative anomaly localization using SSIM, are introduced.

Development of a quantitative fluorescence lateral flow immunoassay (LFIA) prototype for point-of-need detection of anti-Müllerian hormone.

Objective: Anti-Müllerian Hormone (AMH) is a quantitative marker for ovarian reserve and is used to predict response during ovarian stimulation. Streamlining testing to the clinic or even to the physician's office would reduce inconvenience, turnaround time, patient stress and potentially also the total cost of testing, allowing for more frequent monitoring. In this paper, AMH is used as a model biomarker to describe the rational development and optimization of sensitive, quantitative, clinic-based rapid diagnostic tests. Design and Methods: We developed a one-step lateral-flow europium (III) chelate-based fluorescent immunoassay (LFIA) for the detection of AMH on a portable fluorescent reader, optimizing the capture/detection antibodies, running buffer, and reporter conjugates. Results: A panel of commercial calibrators was used to develop a standard curve to determine the analytical sensitivity (LOD = 0.41 ng/ml) and the analytical range (0.41-15.6 ng/ml) of the LFIA. Commercial controls were then tested to perform an initial evaluation of the prototype performance and showed a high degree of precision (Control I CV 2.18%; Control II CV 3.61%) and accuracy (Control I recovery 126%; Control II recovery 103%). Conclusions: This initial evaluation suggests that, in future clinical testing, the AMH LFIA will likely have the capability of distinguishing women with low ovarian reserve (<1 ng/ml AMH) from women with normal (1-4 ng/ml AMH) ovarian reserve. Furthermore, the LFIA demonstrated a wide linear range, indicating the assay's applicability to the detection of other health conditions such as PCOS, which requires AMH measurement at higher concentrations (>6 ng/ml).

Smartphone-readable RPA-LFA for the high-sensitivity detection of Leishmania kDNA using nanophosphor reporters.

Early diagnosis of infectious diseases improves outcomes by enabling earlier delivery of effective treatment, and helps prevent further transmission by undiagnosed persons. We demonstrated a proof-of-concept assay combining isothermal amplification and lateral flow assay (LFA) for early diagnosis of cutaneous leishmaniasis, a vector-borne infectious disease that affects ca. 700,000 to 1.2 million people annually. Conventional molecular diagnostic techniques based on polymerase chain reaction (PCR) require complex apparatus for temperature cycling. Recombinase polymerase amplification (RPA) is an isothermal DNA amplification method that has shown promise for use in low-resource settings. Combined with lateral flow assay as the readout, RPA-LFA can be used as a point-of-care diagnostic tool with high sensitivity and specificity, but reagent costs can be problematic. In this work, we developed a highly-sensitive smartphone-based RPA-LFA for the detection of Leishmania panamensis DNA using blue-emitting [(Sr0.625Ba0.375)1.96Eu0.01Dy0.03]MgSi2O7 (SBMSO) persistent luminescent nanophosphors as LFA reporters. The greater detectability of nanophosphors allows the use of a reduced volume of RPA reagents, potentially reducing the cost of RPA-LFA. The limit of detection (LOD) of RPA with gold nanoparticle-based LFA readout is estimated at 1 parasite per reaction, but LOD can be 100-fold better, 0.01 parasites per reaction, for LFA based on SBMSO. This approach may be useful for sensitive and cost-effective point-of-care diagnosis and contribute to improved clinical and economic outcomes, especially in resource-limited settings.

Oxidized Xanthan Gum Crosslinked NOCC: Hydrogel System and Their Biological Stability from Oxidation Levels of the Polymer.

Dynamic hydrogel systems from N,O-carboxymethyl chitosan (NOCC) are investigated in the past years, which has facilitated their widespread use in many biomedical engineering applications. However, the influence of the polymer's oxidation levels on the hydrogel biological properties is not fully investigated. In this study, chitosan is converted into NOCC and introduced to react spontaneously with oxidized xanthan gum (OXG) to form several injectable hydrogels with controlled degradability. Different oxidation levels of xanthan gum, as well as NOCC/OXG volume ratios, are trialed. The infrared spectroscopy spectra verify chemical modification on OXG and successful crosslinking. With increasing oxidation levels, more dialdehyde groups are introduced into the OXG, resulting in changes in physical properties including gelation, swelling, and self-healing efficiency. Under different volume ratios, the hydrogel shows a stable structure and rigidity with higher mechanical properties, and a slower degradation rate. The shear-thinning and self-healing properties of the hydrogels are confirmed. In vitro assays with L929 cells show the biocompatibility of all formulations although the use of a high amount of OXG15 and OXG25 limited the cell proliferation capacity. Findings in this study suggested a suitable amount of OXG at different oxidation levels in NOCC hydrogel systems for tissue engineering applications.