Drug screening on digital microfluidics for cancer precision medicine.

Drug screening based on in-vitro primary tumor cell culture has demonstrated potential in personalized cancer diagnosis. However, the limited number of tumor cells, especially from patients with early stage cancer, has hindered the widespread application of this technique. Hence, we developed a digital microfluidic system for drug screening using primary tumor cells and established a working protocol for precision medicine. Smart control logic was developed to increase the throughput of the system and decrease its footprint to parallelly screen three drugs on a 4 × 4 cm2 chip in a device measuring 23 × 16 × 3.5 cm3. We validated this method in an MDA-MB-231 breast cancer xenograft mouse model and liver cancer specimens from patients, demonstrating tumor suppression in mice/patients treated with drugs that were screened to be effective on individual primary tumor cells. Mice treated with drugs screened on-chip as ineffective exhibited similar results to those in the control groups. The effective drug identified through on-chip screening demonstrated consistency with the absence of mutations in their related genes determined via exome sequencing of individual tumors, further validating this protocol. Therefore, this technique and system may promote advances in precision medicine for cancer treatment and, eventually, for any disease.

Effects of early enteral nutrition in patients with severe burns: A systematic review.

BACKGROUND: Nutritional problems in the early stages of severe burns are prominent and seriously affect the clinical outcomes of patients. Our aim is to analyze the effects of early enteral nutrition (EEN) in patients with severe burns. METHODS: In this study, relevant articles were searched in 8 English and Chinese data, with a time limit from the creation of the database to June 2023. Two researchers independently completed the search, screening and quality assessment of the articles. We conducted a systematic review and meta-analysis of randomized controlled trials that examined EEN therapy in people with severe burns. We compared the effects of EEN and non-EEN therapy in severely burned patients. The outcomes were mortality, gastrointestinal complications, nutritional indicators, gastrointestinal hormones, sepsis, length of hospital stay and wound healing time. Categorical variables were expressed as OR and 95% CI was calculated, and continuous variables were expressed as MD and 95% CI was calculated. The protocol for this systematic review was registered in PROSPERO on May 12, 2023 (identifier CRD42023422895). RESULTS: Nineteen studies with a total of 1066 participants met the inclusion criteria. When comparing EEN therapy with non-EEN therapy, the experiment group had significantly lower mortality [OR = 0.39, 95% CI (0.20, 0.74), P = .004], lower wound healing time [MD = -10.77, 95% CI (-13.66,-7.88), P < .00001], fewer gastrointestinal complications [OR = 0.18, 95% CI (0.09, 0.36), P < .00001], lower rates of gastrointestinal hemorrhage [OR = 0.12, 95% CI (0.04, 0.36), P = .0001], lower rates of sepsis [OR = 0.40, 95% CI (0.24, 0.66), P = .0005], shorter length of hospital stay [MD = -12.08, 95% CI (-13.61, 9.19-10.56), P < .00001], and higher prealbumin levels [MD = 29.04, 95% CI (21.98, 36.10), P < .00001], higher total albumin levels [MD = 6.74, 95% CI (4.29, 9.19), P < .00001], and gastrin levels [MD = 15.93, 95% CI (10.12, 21.73), P < .00001]. However, there was no significant difference in albumin between the 2 groups [MD = 2.62, 95% CI (-0.30, 5.55), P = .08] or motilin levels [MD = 12.48, 95% CI (-43.59, 68.56), P = .66]. CONCLUSIONS: EEN plays an important role in the rehabilitation of patients with severe burns. EEN is beneficial to reduce complications and the length of hospital stay, maintain organ function, optimize the nutritional status of patients, promote wound healing, and improve the survival rate of patients.

Magnetic Particle Imaging: From Tracer Design to Biomedical Applications in Vasculature Abnormality.

Magnetic particle imaging (MPI) is an emerging non-invasive tomographic technique based on the response of magnetic nanoparticles (MNPs) to oscillating drive fields at the center of a static magnetic gradient. In contrast to magnetic resonance imaging (MRI), which is driven by uniform magnetic fields and projects the anatomic information of the subjects, MPI directly tracks and quantifies MNPs in vivo without background signals. Moreover, it does not require radioactive tracers and has no limitations on imaging depth. This article first introduces the basic principles of MPI and important features of MNPs for imaging sensitivity, spatial resolution, and targeted biodistribution. The latest research aiming to optimize the performance of MPI tracers is reviewed based on their material composition, physical properties, and surface modifications. While the unique advantages of MPI have led to a series of promising biomedical applications, recent development of MPI in investigating vascular abnormalities in cardiovascular and cerebrovascular systems, and cancer are also discussed. Finally, recent progress and challenges in the clinical translation of MPI are discussed to provide possible directions for future research and development.

High-level production of poly-γ-glutamic acid by a newly isolated Bacillus sp. YJY-8 and potential use in increasing the production of tomato.

Strain YJY-8, a new γ-polyglutamic acid producer, was separated from fermented soybean paste samples. The strain was identified as a genus of Bacillus by morphological and 16S rDNA sequence analysis and was named Bacillus sp. YJY-8. The optimal medium composition and cultural conditions were studied using a single-factor experiment and a response surface experiment. The optimized medium consisted of monosodium glutamate 70 g/L, glucose 54.3 g/L, glycerol 31.8 g/L, ammonium sulfate 11.1 g/L, yeast extract 3.2 g/L, tryptone 1.5 g/L, L-glutamic acid 6.8 g/L, MgSO4 7H2O 0.5 g/L, FeCl3 6H2O 0.02 g/L, KH2PO4 0.9 g/L, CaCl2 0.03 g/L, MnSO4 H2O 0.3 g/L, ammonium molybdate 0.02 g/L, pH 7.0. The optimal cultivation conditions were 35 °C and pH 7.0. Under the optimized conditions, after 48 hr of cultivation, the highest shaking flask fermentation level of γ-PGA reached 65.2 ± 0.36 g/L. In addition, through fed-batch fermentation in 30 L fermenters, the fermentation level of γ-PGA reached its highest level at 88.42 g/L and productivity was 1.23 g/(L hr) after 72 hr. Then, the effect of γ-PGA on tomato yield was investigated. At the seedling stage, the plant height and stem diameter of γ-PGA treated plants increased by 5.69 and 15.735% after spraying γ-PGA for 19 days. During the flowering and fruiting period, the stem diameter of the γ-PGA treatment group increased by 6.74%, with a maximum increase of 11.65%. The number of fruit branches increased by 0.56-16.29% and the number of fruit sets increased by 1.01-28.47%. At the fruit maturation stage, the yield of tomatoes increased by 10.51, 14.27, and 5.83%.

Digital Microfluidics with an On-Chip Drug Dispenser for Single or Combinational Drug Screening.

Rapid and accurate cancer drug screening is of great importance in precision medicine. However, the limited amount of tumor biopsy samples has hindered the application of traditional drug screening methods with microwell plates for individual patients. A microfluidic system provides an ideal platform for handling trace amounts of samples. This emerging platform has a good role in nucleic acid-related and cell related assays. Nevertheless, convenient drug dispensing remains a challenge for clinical on-chip cancer drug screening. Similar sized droplets are merged to add drugs for a desired screened concentration which significantly complicated the on-chip drug dispensing protocols. Here, we introduce a novel digital microfluidic system with a specially structured electrode (a drug dispenser) to dispense drugs by droplet electro-ejection under a high-voltage actuation signal, which can be conveniently adjusted by external electric controls. With this system, the screened drug concentrations span up to four orders of magnitude with small sample consumption. Various amounts of drugs can be delivered to the cell sample with desired amount in a flexible electric control. Moreover, single drug or combinatorial multidrug on-chip screening can be readily achieved. The drug response of normal MCF-10A breast cells and MDA-MB-231 breast tumor cells to two chemotherapeutic substances, cisplatin (Cis) and epirubicin (EP), was tested individually and in combination for proof-of-principle verification. The comparable on-chip and off-chip results confirmed the feasibility of our innovative DMF system for cancer drug screening.

Cancer drug screening with an on-chip multi-drug dispenser in digital microfluidics.

Microfluidics has been the most promising platform for drug screening with a limited number of cells. However, convenient on-chip preparation of a wide range of drug concentrations remains a large challenge and has restricted wide acceptance of microfluidics in precision medicine. In this paper, we report a digital microfluidic system with an innovative control structure and chip design for on-chip drug dispensing to generate concentrations that span three to four orders of magnitude, enabling single drug or combinatorial multi-drug screening with simple electronic control. Specifically, we utilize droplet ejection from a drug drop sitting on a special electrode, named a drug dispenser, under high-voltage pulse actuation to deliver the desired amount of drugs to be picked up by a cell suspension drop driven by low-voltage sine wave actuation. Our proof-of-principle validation for this technique as a convenient single and multi-drug screening involved testing of the drug toxicity of two chemotherapeutics, cisplatin (Cis) and epirubicin (EP), towards MDA-MB-231 breast cancer cells and MCF-10A normal breast cells. The results are consistent with those screened based on traditional 96-well plates. These findings demonstrate the reliability of the drug screening system with an on-chip drug dispenser. This system with fewer cancer cells, less drug consumption, a small footprint, and high scalability with regard to concentration could pave the way for drug screening on biopsied primary tumor cells for precision medicine or any concentration-related research.

Ferritin: A Multifunctional Nanoplatform for Biological Detection, Imaging Diagnosis, and Drug Delivery.

Ferritins are spherical iron storage proteins within cells that are composed of a combination of 24 subunits of two types, heavy-chain ferritin (HFn) and light-chain ferritin (LFn). They autoassemble naturally into a spherical hollow nanocage with an outer diameter of 12 nm and an interior cavity that is 8 nm in diameter. In recent years, with the constantly emerging safety issues and the concerns about unfavorable uniformity and indefinite in vivo behavior of traditional nanomedicines, the characteristics of native ferritin nanocages, such as the unique nanocage structure, excellent safety profile, and definite in vivo behavior, make ferritin-based formulations uniquely attractive for nanomedicine development. To date, a variety of cargo molecules, including therapeutic drugs (e.g., cisplatin, carboplatin, paclitaxel, curcumin, atropine, quercetin, gefitinib, daunomycin, epirubicin, doxorubicin, etc.), imaging agents (e.g., fluorescence dyes, radioisotopes, and MRI contrast agents), nucleic acids (e.g., siRNA and miRNA), and metal nanoparticles (e.g., Fe3O4, CeO2, AuPd, CuS, CoPt, FeCo, Ag, etc.) have been loaded into the interior cavity of ferritin nanocages for a broad range of biomedical applications from in vitro biosensing to targeted delivery of cargo molecules in living systems with the aid of modified targeting ligands either genetically or chemically. We reported that human HFn could selectively deliver a large amount of cargo into tumors in vivo via transferrin receptor 1 (TfR1)-mediated tumor-cell-specific targeting followed by rapid internalization. By the use of the intrinsic tumor-targeting property and unique nanocage structure of human HFn, a broad variety of cargo-loaded HFn formulations have been developed for biological analysis, imaging diagnosis, and medicine development. In view of the intrinsic tumor-targeting property, unique nanocage structure, lack of immunogenicity, and definite in vivo behavior, human HFn holds promise to promote therapeutic drugs, diagnostic imaging agents, and targeting moieties into multifunctional nanomedicines.Since the report of the intrinsic tumor-targeting property of human HFn, we have extensively explored human HFn as an ideal nanocarrier for tumor-targeted delivery of anticancer drugs, MRI contrast agents, inorganic nanoparticles, and radioisotopes. In particular, by the use of genetic tools, we also have genetically engineered human HFn nanocages to recognize a broader range of disease biomarkers. In this Account, we systematically review human ferritins from characterizing their tumor-binding property and understanding their mechanism and kinetics for cargo loading to exploring their biomedical applications. We finally discuss the prospect of ferritin-based formulations to become next-generation nanomedicines. We expect that ferritin formulations with unique physicochemical characteristics and intrinsic tumor-targeting property will attract broad interest in fundamental drug research and offer new opportunities for nanomedicine development.

A digital microfluidic system with 3D microstructures for single-cell culture.

Despite the precise controllability of droplet samples in digital microfluidic (DMF) systems, their capability in isolating single cells for long-time culture is still limited: typically, only a few cells can be captured on an electrode. Although fabricating small-sized hydrophilic micropatches on an electrode aids single-cell capture, the actuation voltage for droplet transportation has to be significantly raised, resulting in a shorter lifetime for the DMF chip and a larger risk of damaging the cells. In this work, a DMF system with 3D microstructures engineered on-chip is proposed to form semi-closed micro-wells for efficient single-cell isolation and long-time culture. Our optimum results showed that approximately 20% of the micro-wells over a 30 × 30 array were occupied by isolated single cells. In addition, low-evaporation-temperature oil and surfactant aided the system in achieving a low droplet actuation voltage of 36V, which was 4 times lower than the typical 150 V, minimizing the potential damage to the cells in the droplets and to the DMF chip. To exemplify the technological advances, drug sensitivity tests were run in our DMF system to investigate the cell response of breast cancer cells (MDA-MB-231) and breast normal cells (MCF-10A) to a widely used chemotherapeutic drug, Cisplatin (Cis). The results on-chip were consistent with those screened in conventional 96-well plates. This novel, simple and robust single-cell trapping method has great potential in biological research at the single cell level.

A Novel and Robust Single-cell Trapping Method on Digital Microfluidics.

Due to cell heterogeneity, the differences among individual cells are averaged out in bulk analysis methods, especially in the analysis of primary tumor biopsy samples from patients. To deeply understand the cell-to-cell variation in a primary tumor, single-cell culture and analysis with limited amount of cells are in high demand. Microfluidics has been an optimum platform to address the issue given its small reaction volume requirements. Digital microfluidics, which utilizes an electric signal to manipulate individual droplets has shown promise in cell-culture with easy controls. In this work, we realize single cell trapping on digital microfluidic platform by fabricating 3D microstructures on-chip to form semi-closed micro-wells. With this design, 20% of 30 x 30 array can be occupied by isolated single cells. We also use a low evaporation silicon oil and a fluorinated surfactant to lower the droplet actuation voltage and prevent the drop from evaporation, while allowing cell respiration during the long term of culture (24 h). The main steps for single cell trapping on digital microfluidics, as illustrated in this protocol, include 3D microstructures design, 3D microstructures construction on chip and oil film with surfactant for single cell trapping on chip.

Turning On/Off the Anti-Tumor Effect of the Au Cluster via Atomically Controlling Its Molecular Size.

We reported two Au clusters with precisely controlled molecular size (Au5Peptide3 and Au22Peptide10) showing different antitumor effects. In vitro, both Au5Peptide3 and Au22Peptide10 were well taken up by human nasopharyngeal cancer cells (CNE1 cells). However, only Au5Peptide3 significantly induced CNE1 cell apoptosis. Further studies showed that CNE1 cells took up Au5Peptide3 (1.98 × 10-15 mol/cell), and 9% of them entered mitochondria (0.186 × 10-15 mol/cell). As a comparison, the uptake of Au22Peptide10 was only half the amount of Au5Peptide3 (1.11 × 10-15 mol/cell), and only 1% of them entered mitochondria (0.016 × 10-15 mol/cell). That gave 11.6-fold more Au5Peptide3 in mitochondria of CNE1 cells than Au22Peptide10. Further cell studies revealed that the antitumor effect may be due to the enrichment of Au5Peptide3 in mitochondria. Au5Peptide3 slightly decreased the Mcl-1 (antiapoptotic protein of mitochondria) and significantly increased the Puma (pro-apoptotic protein of mitochondria) expression level in CNE1 cells, which resulted in mitochondrial transmembrane potential change and triggered the caspase 9-caspase 3-PARP pathway to induce CNE1 cell apoptosis. In vivo, CNE1 tumor growth was significantly suppressed by Au5Peptide3 in the xenograft model after 3 weeks of intraperitoneal injection. The TUNEL and immuno-histochemical studies of tumor tissue verified that CNE1 cell apoptosis was mainly via the Puma and Mcl-1 apoptosis pathway in the xenograft model, which matched the aforementioned CNE1 cell studies in vitro. The discovery of Au5 but not Au22 suppressing tumor growth via the mitochondria target was a breakthrough in the nanomedical field, as this provided a robust approach to turn on/off the nanoparticles' medical properties via atomically controlling their sizes.

Targeted peptide-Au cluster binds to epidermal growth factor receptor (EGFR) in both active and inactive states: a clue for cancer inhibition through dual pathways.

The epidermal growth factor receptor (EGFR) has become an important target protein in anticancer drug development. Meanwhile, peptide-Au cluster has been proposed as potential targeted nano-drug assembled by targeting peptide. Here, we designed and synthesized a novel peptide-Au cluster as Au10Peptide5 to target to EGFR. We found Au10Peptide5 could target to the natural binding sites of all EGFRs at membrane in both active and inactive states by molecular simulations. Its targeted ability was further verified by the co-localization and blocking experiments. We also study the configuration modifications of both active and inactive EGFRs after binding by Au10Peptide5. For active EGFR, the absorbed Au10Peptide5 might replace the natural ligand in EGFR endocytosis process. Then, the peptide-Au cluster in endochylema could inhibit the cancer relating enzyme activity including thioredoxin reductase1 (TrxR1) and induce the oxidative stress mediated apoptosis in tumor cells. For inactive EGFR, it was retained in inactive state by Au10Peptide5 binding to inhibit dimerization of EGFR for anticancer. Both pathways might be applied in anticancer drug development based on the theoretical and experimental study here.

Peptide-Au Cluster Probe: Precisely Detecting Epidermal Growth Factor Receptor of Three Tumor Cell Lines at a Single-Cell Level.

Alterations in protein (e.g., biomarkers) expression levels have a significant correlation with tumor development and prognosis; therefore, it is desired to develop precise methods to differentiate the expression level of proteins in tumor cell lines, especially at the single-cell level. Here, we report a precise and versatile approach of quantifying the protein expression levels of three tumor cell lines in situ using a peptide-Au cluster probe. The probe (Au5Peptide3) consists of a peptide with a specific cell membrane epidermal growth factor receptor (EGFR) targeting ability and an Au cluster for both cell membrane EGFR imaging using confocal microscopy and cell membrane EGFR counting by laser ablation inductively coupled plasma mass spectrometry. Utilizing the peptide-Au cluster probe, we successfully quantify the EGFR expression levels of SMMC-7721, KB, and HeLa cells at a single-cell level and differentiate the EGFR expression levels among these cell lines. The peptide-Au cluster probe, with the ability to differentiate the protein expression level of different cell lines, shows exceptional promise for providing reliable predictive and prognostic information of tumors at a single-cell level.

Atomic structure of a peptide coated gold nanocluster identified using theoretical and experimental studies.

Peptide coated gold nanoclusters (AuNCs) have a precise molecular formula and atomic structure, which are critical for their unique applications in targeting specific proteins either for protein analysis or drug design. To date, a study of the crystal structure of peptide coated AuNCs is absent primarily due to the difficulty of obtaining their crystalline phases in an experiment. Here we study a typical peptide coated AuNC (Au24Peptide8, Peptide = H2N-CCYKKKKQAGDV-COOH, Anal. Chem., 2015, 87, 2546) to figure out its atomic structure and electronic structure using a theoretical method for the first time. In this work, we identify the explicit configuration of the essential structure of Au24Peptide8, Au24(Cys-Cys)8, using density functional theory (DFT) computations and optical spectroscopic experiments, where Cys denotes cysteine without H bonded to S. As the first multidentate ligand binding AuNC, Au24(Cys-Cys)8 is characterized as a distorted Au13 core with Oh symmetry covered by two Au(Cys-Cys) and three Au3(Cys-Cys)2 staple motifs in its atomic structure. The most stable configuration of Au24(Cys-Cys)8 is confirmed by comparing its UV-vis absorption spectrum from time-dependent density-functional theory (TDDFT) calculations with optical absorption measurements, and these results are consistent with each other. Furthermore, we carry out frontier molecular orbital (FMO) calculations to elucidate that the electronic structure of Au24(Cys-Cys)8 is different from that of Au24(SR)20 as they have a different Au/S ratio, where SR represents alkylthiolate. Importantly, the different ligand coatings, Cys-Cys and SR, in Au24(Cys-Cys)8 and Au24(SR)20 cause the different Au/S ratios in the coated Au24. The reason is that the Au/S ratio is crucial in determining the size of the Au core of the ligand protected AuNC, and the size of the Au core corresponds to a specific electronic structure. By the adjustment of ligand coatings from alkylthiolate to peptide, the Au/S ratio could be controlled to generate different AuNCs with versatile electronic structures, optical properties and reaction stabilities. Therefore, we propose a universal approach to obtain a specific Au/S ratio of ligand coated AuNCs by adjusting the ligand composition, thus controlling the chemicophysical properties of AuNCs with ultimately the same number of Au atoms.

Computational design of peptide-Au cluster probe for sensitive detection of α(IIb)ß3 integrin.

We have designed a novel peptide-Au cluster probe to specifically bind to αIIbß3 integrin. As indicated by molecular dynamics (MD) simulations, the binding mode of the native ligand of αIIbß3 integrin, γC peptide, can be realized by the designed probe. More importantly, the peptide-Au probe can provide multiple coating peptides to form additional salt bridges with protein, and the binding stability of the probe is comparable to the native ligand. The designed probe was then successfully synthesized. The specific binding in a cellular environment was validated by colocalization analysis of confocal microscopy. In addition, the binding affinity was confirmed by atomic force microscopy (AFM) based single molecule force spectroscopy. Our results suggest the combination of computational design and experimental verification can be a useful strategy for the development of nanoprobes.

Peptide-Conjugated Gold Nanoprobe: Intrinsic Nanozyme-Linked Immunsorbant Assay of Integrin Expression Level on Cell Membrane.

Precisely quantifying the membrane protein expression level on cell surfaces is of vital importance for early cancer diagnosis and efficient treatment. We demonstrate that gold nanoparticle bioconjugated by a rationally designed peptide as nanoprobe possesses selective labeling and accurate quantification capacity of integrin GPIIb/IIIa on the human erythroleukemia cell line. Through selective recognition and marking of integrin, two-photon photoluminescence of the nanoprobe is exploited for direct observation of protein spatial distribution on cell membrane. More importantly, utilizing intrinsic enzyme-like catalysis property of the nanoprobe, the expression level of integrin on human erythroleukemia cells can be quantitatively counted in an amplified and reliable colorimetric assay without cell lysis and protein extraction process. In addition, the analysis of the correlation between the gold nanoparticle and the membrane protein via relevant inductively coupled plasma mass spectrometry measurement verifies the reliability of the new analytical method. It is anticipated that this facile and efficient strategy holds a great promise for a rapid, precise, and reliable quantification of interested functional membrane proteins on the cell surface.

Facile approach to observe and quantify the α(IIb)ß3 integrin on a single-cell.

We report for the first time seeing and counting integrin α(IIb)ß3 on a single-cell level. The proposed method is based on the using of the Au cluster probe. With the fluorescent property of Au24 cluster and the specific targeting ability of peptide, our probe can directly visualize integrin α(IIb)ß3 on the membrane of human erythroleukemia cells (HEL) via confocal microscopy. On the basis of the accurate formula of our probe (Au24Peptide8), the number of integrin α(IIb)ß3 can be precisely counted by quantifying the gold content on a single HEL cell via laser ablation inductively coupled plasma mass spectrometry. Our results reveal that the number of integrin α(IIb)ß3 on a single cell varies from 5.75 × 10(-17) to 9.11 × 10(-17) mol, because of the heteroexpression levels of α(IIb)ß3 on individual cells.

Label-free Au cluster used for in vivo 2D and 3D computed tomography of murine kidneys.

Kidney disease is a worldwide health hazard. Noninvasive imaging modalities such as computed tomography are often used for diagnosis, to guide treatment, and to assess a disease state over the long-term. The physiology of the kidneys can be assessed with contrast agents. We present an albumin-stabilized Au cluster with red fluorescence and robust X-ray attenuation. In vivo studies revealed distribution of the Au clusters in the liver, spleen, and kidneys, with excretion mostly via the kidneys. Under optimal conditions, this agent can outline the anatomy of mouse kidneys on 2D and 3D computed tomography imaging, with clear visualization of the renal collecting system and ureters. This is a promising agent for kidney visualization and disease diagnosis.

Spatially marking and quantitatively counting membrane immunoglobulin M in live cells via Ag cluster-aptamer probes.

A probe composed of an aptamer and a silver cluster, where the aptamer targets mIgM of live cells and the silver cluster provides fluorescent imaging and mass quantification of mIgM of live cells, is presented. This new probe simultaneously provides accurate spatial and mass information of mIgM in live cells.