Highly sensitive eight-channel light sensing system for biomedical applications.

We demonstrate the potential of an eight-channel light sensing platform system, named Black Box I (BBI), for rapid and highly sensitive measurement of low-level light using a nonradioactive optical readout. We developed, normalized, and characterized the photon sensitivities of the eight channels of the BBI using placental alkaline phosphatase (PLAP) as a model imaging reporter. We found that the BBI system had a statistically strong linear correlation with the reference IVIS Lumina II system. When we applied normalization constants, we were able to optimize the photomultiplier tubes (PMT) of all eight channels of the BBI (up to r2 = 0.998). We investigated the biomedical utilities of BBI by: (i) determining alkaline phosphatase activities in mouse plasma samples as a diagnostic secretory biomarker of cancer, and (ii) diagnosing cancer metastases in the organs of mice bearing triple negative breast cancer. We provide an important new addition to low-cost biomedical instruments intended for pre-clinical diagnostic imaging with high sensitivity, high sample throughput, portability, and rapid on-site analysis of low-level light.

BRET Sensors for Imaging Membrane Integrity of Microfluidically Generated Extracellular Vesicles.

Extracellular vesicles (EVs) derived from various cell lines have been extensively used as natural nanodelivery vehicles for drug, protein, and nucleic acid deliveries in therapeutic applications for cancer. Recently, we developed a microfluidic-based reconstruction strategy as a novel method to generate microRNA-loaded membrane vesicles for cancer therapy in vivo. We used EVs and cell membranes isolated from different source of cells for this reconstruction process. The microfluidic system produced reconstructed vesicles of uniform sizes with high microRNA loading efficiency independent of input membrane sources (EVs or cell membranes). To address the functional integrity of the membrane structure and of proteins in the reconstructed EVs, we introduce a membrane-insertable bioluminescence resonance energy transfer (BRET) sensor system. This sensor, with its membrane-insertable palmitoylation signal peptide sequence derived from a growth-associated protein 43 (GAP43), helps in trafficking the fusion protein to the cell membrane upon its expression in cells and allows for imaging reconstructed membrane vesicles using optical imaging. In this chapter, we detail the stepwise methods used for the engineering of cells using this sensor, isolation of EVs from the engineered cells, preparation of reconstructed EVs by microfluidic processing, and BRET imaging of reconstructed EVs for membrane integrity evaluation.

Compact Eight-Channel Light-Sensing System for Bioassays.

The present protocol introduces a new instrumental setup as a luminometer to simultaneously measure eight light samples with high sensitivity. The system consists of 8-channel photomultiplier tubes (8-PMTs) with different sensitivities to light. Therefore, it is critical to normalize the sensitivities of PMTs to light samples and integrate them as a system. We first introduce how to normalize the diverse light sensitivity among the PMTs using placental alkaline phosphatase (PLAP) as a model chemiluminescence light source. The normalized BBI system shows a statistically strong linear correlation graph to photon counts. The biomedical utility of this system is exemplified by (i) determining the alkaline phosphatase (AP) activities in mouse plasma samples as a cancer biomarker and (ii) diagnosing metastatic tissues during cancer progression using bioluminescent reporter.

Ultrasound-Guided Microbubble-Mediated Locoregional Delivery of Multiple MicroRNAs Improves Chemotherapy in Hepatocellular Carcinoma.

Rationale: To assess treatment effects of 4 complementary miRNAs (miRNA-100/miRNA-122/antimiRNA-10b/antimiRNA-21) encapsulated in a biodegradable PLGA-PEG nanoparticle, administered by an ultrasound-guided microbubble-mediated targeted delivery (UGMMTD) approach in mouse models of hepatocellular carcinoma (HCC). Methods:In vitro apoptotic index was measured in HepG2 and Hepa1-6 HCC cells treated with various combinations of the 4 miRNAs with doxorubicin. Three promising combinations were further tested in vivo by using UGMMTD. 63 HepG2 xenografts in mice were randomized into: group 1, miRNA-122/antimiRNA-10b/antimiRNA-21/US/doxorubicin; group 2, miRNA-100/miRNA-122/antimiRNA-10b/antimiRNA-21/US/doxorubicin; group 3, miRNA-100/miRNA-122/antimiRNA-10b/US/doxorubicin; group 4, miRNA-122/anitmiRNA-10b/antimiRNA-21/doxorubicin; group 5, miRNA-100/miRNA-122/antimiRNA-10b/antimiRNA-21/doxorubicin; group 6, miRNA-100/miRNA-122/antimiRNA-10b/doxorubicin; group 7, doxorubicin only treatment; and group 8, without any treatment. Tumor volumes were measured through 18 days. H&E staining, TUNEL assay, and qRT-PCR quantification for delivered miRNAs were performed. Results:In vivo results showed that UGMMTD of miRNAs with doxorubicin in groups 1-3 significantly (P<0.05) delayed tumor growth compared to control without any treatment, and doxorubicin only from day 7 to 18. On qRT-PCR, levels of delivered miRNAs were significantly (P<0.05) higher in groups 1-3 upon UGMMTD treatment compared to controls. TUNEL assay showed that upon UGMMTD, significantly higher levels of apoptotic cell populations were observed in groups 1-3 compared to controls. Toxicity was not observed in various organs of different groups. Conclusions: UGMMTD of miRNA-100/miRNA-122/antimiRNA-10b/antimiRNA-21 combination improved therapeutic outcome of doxorubicin chemotherapy in mouse models of HCC by substantial inhibition of tumor growth and significant increase in apoptotic index.

Inhaled Gold Nano-star Carriers for Targeted Delivery of Triple Suicide Gene Therapy and Therapeutic MicroRNAs to Lung Metastases: Development and Validation in a Small Animal Model.

Pulmonary metastases pose significant treatment challenges for many cancers, including triple-negative breast cancer (TNBC). We developed and tested a novel suicide gene and therapeutic microRNAs (miRs) combination therapy against lung metastases in vivo in mouse models after intranasal delivery using nontoxic gold nanoparticles (AuNPs) formulated to carry these molecular therapeutics. We used AuNPs coated with chitosan-ß-cyclodextrin (CS-CD) and functionalized with a urokinase plasminogen activator (uPA) peptide to carry triple cancer suicide genes (thymidine kinase-p53-nitroreductase: TK-p53-NTR) plus therapeutic miRNAs (antimiR-21, antimiR-10b and miR-100). We synthesized three AuNPs: 20nm nanodots (AuND), and 20nm or 50nm nanostars (AuNS), then surface coated these with CS-CD using a microfluidic-optimized method. We sequentially coated the resulting positively charged AuNP-CS-CD core with synthetic miRNAs followed by TK-p53-NTR via electrostatic interactions, and added uPA peptide through CD-adamantane host-guest chemistry. A comparison of transfection efficiencies for different AuNPs showed that the 50nm AuNS allowed ∼4.16-fold higher gene transfection than other NPs. The intranasal delivery of uPA-AuNS-TK-p53-NTR-microRNAs NPs (pAuNS@TK-p53-NTR-miRs) in mice predominantly accumulated in lungs and facilitated ganciclovir and CB1954 prodrug-mediated gene therapy against TNBC lung metastases. This new nanosystem may serve as an adaptable-across-cancer-type, facile, and clinically scalable platform to allow future inhalational suicide gene-miR combination therapy for patients harboring pulmonary metastases.

High-Throughput Whole-Plate Imaging of Cells for Multiple Biological Applications.

Advanced multipurpose cell imaging systems along with integrated rapid quantitation software can enhance and expedite cancer cell culture studies in a variety of applications. Though accurate cell culture studies are an important and necessary component of nearly all cancer biomarker detection and therapy studies, the methods we currently use are of low-throughput, time consuming, and lack accuracy. Hence, it is important to improve several features of the assays to increase the accuracy of their quantitative outputs in most studies. In general, we perform cell culture analysis semimanually by counting a small aliquot of suspended cells using a hemocytometer or viewing a small area of cells on a plate using a bright-field microscope, and then extrapolate the counts or observations to estimate the values for the total numbers of cells. The fundamental problem with this process lies in using techniques, such as extrapolation, which inherently introduces intrasample variability while collecting the cells by enzymatic trypsinization for these assays that are affecting cell growth and other downstream assessments. Fluorescence (FL) microscopy-based assays are also used to image and count cells for various applications, including cell viability, proliferation, apoptosis, cell death, transfection efficiency, protein expression, stem cell properties, colony formation, cytotoxicity, drug dose-response, and treatment efficacy studies. These methods are not optimal for many researches, as they require real-time visualization under a microscope plus manual analysis to determine the final results. Owing to long exposure times for cells under fluorescent light of a microscope, the cells may be exposed to suboptimal conditions that affect cell growth, and with occasional photobleaching of the expressed FL probes. Alternatively, the use of cell imaging systems that integrate both advanced bright-field and FL imaging for cell counting and quantification can be useful. In this protocol, we discuss the advantages of a high-throughput cell imaging system using a whole-plate imaging format when used in various bioimaging studies by highlighting a few applications of the system. The system is designed to fundamentally improve the accuracy and time of cell culture analysis while also allowing us to perform the assay without trypsinization, thus avoiding the need to replicate multiple wells for monitoring cell growth over time.

Remote Control of Time-Regulated Stretching of Ligand-Presenting Nanocoils In Situ Regulates the Cyclic Adhesion and Differentiation of Stem Cells.

Native extracellular matrix (ECM) can exhibit cyclic nanoscale stretching and shrinking of ligands to regulate complex cell-material interactions. Designing materials that allow cyclic control of changes in intrinsic ligand-presenting nanostructures in situ can emulate ECM dynamicity to regulate cellular adhesion. Unprecedented remote control of rapid, cyclic, and mechanical stretching ("ON") and shrinking ("OFF") of cell-adhesive RGD ligand-presenting magnetic nanocoils on a material surface in five repeated cycles are reported, thereby independently increasing and decreasing ligand pitch in nanocoils, respectively, without modulating ligand-presenting surface area per nanocoil. It is demonstrated that cyclic switching "ON" (ligand nanostretching) facilitates time-regulated integrin ligation, focal adhesion, spreading, YAP/TAZ mechanosensing, and differentiation of viable stem cells, both in vitro and in vivo. Fluorescence resonance energy transfer (FRET) imaging reveals magnetic switching "ON" (stretching) and "OFF" (shrinking) of the nanocoils inside animals. Versatile tuning of physical dimensions and elements of nanocoils by regulating electrodeposition conditions is also demonstrated. The study sheds novel insight into designing materials with connected ligand nanostructures that exhibit nanocoil-specific nano-spaced declustering, which is ineffective in nanowires, to facilitate cell adhesion. This unprecedented, independent, remote, and cytocompatible control of ligand nanopitch is promising for regulating the mechanosensing-mediated differentiation of stem cells in vivo.

Ultrasound-mediated delivery of miRNA-122 and anti-miRNA-21 therapeutically immunomodulates murine hepatocellular carcinoma in vivo.

Hepatocellular carcinoma (HCC) is the most common cause of cancer-related mortality, and patients with HCC show poor response to currently available treatments, which demands new therapies. We recently developed a synthetic microRNA-based molecularly targeted therapy for improving HCC response to chemotherapy by eliminating drug resistance. We used ultrasound-targeted microbubble destruction (UTMD) to locally deliver microRNA-loaded nanoparticles to HCC. Since the immune microenvironment plays a crucial role in HCC disease development and response to treatment, and UTMD and microRNAs have the potential to interfere with the immune system, in this study we analyzed the immunomodulatory effects of UTMD and miRNAs in HCC. We used an immunocompetent syngeneic HCC mouse model for the study. We conducted cytokine profiling in tumor, lymph nodes, and serum of animals within the first 24 h of treatment to analyze changes in the level of pro- and antitumoral cytokines. The results showed: (1) Hepa1-6 syngeneic tumors expressed HCC-related cytokines, (2) UTMD-microRNA combination therapy triggered transient cytokine storms, and (3) delivery of microRNA-122 and anti-microRNA-21 affected the immune microenvironment by decreasing the level of GM-CSF in tumors while modulating protumoral IL-1α, IL-1ß, IL-5, IL-6 and IL-17 and antitumoral IL-2 and IL-12 in tumor-proximal lymph nodes, and increasing IL-2 in the serum of tumor-bearing mice. Local delivery of targeted therapy by UTMD significantly reduced the concentration of IL-12 and IL-17 in lymph nodes of treated and contralateral tumors suggesting a systemic response. CONCLUSION: UTMD-mediated delivery of microRNA-122 and anti-microRNA-21 modulated the immune microenvironment of Hepa1-6 tumors at the level of cytokine expressions. Exploiting antitumoral immune effects could enhance the therapeutic efficacy of the proposed combination therapy for HCC.

Reconstructed Apoptotic Bodies as Targeted "Nano Decoys" to Treat Intracellular Bacterial Infections within Macrophages and Cancer Cells.

Staphylococcus aureus (S. aureus) is a highly pathogenic facultative anaerobe that in some instances resides as an intracellular bacterium within macrophages and cancer cells. This pathogen can establish secondary infection foci, resulting in recurrent systemic infections that are difficult to treat using systemic antibiotics. Here, we use reconstructed apoptotic bodies (ReApoBds) derived from cancer cells as "nano decoys" to deliver vancomycin intracellularly to kill S. aureus by targeting inherent "eat me" signaling of ApoBds. We prepared ReApoBds from different cancer cells (SKBR3, MDA-MB-231, HepG2, U87-MG, and LN229) and used them for vancomycin delivery. Physicochemical characterization showed ReApoBds size ranges from 80 to 150 nm and vancomycin encapsulation efficiency of 60 ± 2.56%. We demonstrate that the loaded vancomycin was able to kill intracellular S. aureus efficiently in an in vitro model of S. aureus infected RAW-264.7 macrophage cells, and U87-MG (p53-wt) and LN229 (p53-mt) cancer cells, compared to free-vancomycin treatment (P < 0.001). The vancomycin loaded ReApoBds treatment in S. aureus infected macrophages showed a two-log-order higher CFU reduction than the free-vancomycin treatment group. In vivo studies revealed that ReApoBds can specifically target macrophages and cancer cells. Vancomycin loaded ReApoBds have the potential to kill intracellular S. aureus infection in vivo in macrophages and cancer cells.

Fabrication of Nanofibrous Scaffold Grafted with Gelatin Functionalized Polystyrene Microspheres for Manifesting Nanomechanical Cues of Stretch Stimulated Fibroblast.

Cells sense mechanical cues in their microenvironments and effectively transduce them into intracellular signaling events, which subsequently orchestrate adaptive changes in cells cytoskeletal components and other adhesion proteins. Thus far, although diverse nanostructured scaffold systems have been utilized for evaluating such mechanical cues, most of them do not recapitulate the in vivo conditions (ECM matrix, 3D culture), which by themselves obscure natural cellular responses in the first place. To address this drawback, in the present work, a hybrid aligned nanofibrous scaffold grafted with gelatin functionalized polystyrene microspheres has been adapted for assessing fibroblast cells mechanobiology in response to mechanical stimulation by stretch cycles. In this work, with the help of a rationally designed hybrid scaffold, we present clear evidence of that fact that fibroblast cells when subjected to mechanical stress tend to manifest higher magnitude traction force on the underlying hybrid scaffold, which consequently introduces proportional distortion in orientation of aligned nanofibers scaffold as well as the polystyrene microspheres. A clear insight into sequestration of focal adhesion proteins and cellular cytoskeletal dynamics that actuate such changes was perceived and quantified by confocal microscopy, field emission-scanning electron microscopy, flow cytometer, and atomic force microscopy (peak force QNM). The mechanical stress induced alteration in regulation of genes associated with focal adhesion proteins and extracellular matrix proteins was also quantified by real-time polymerase chain reaction. In summary, the work presents a biologically relevant 4D hybrid scaffold and validates its implications for evaluation of cell mechanobiology.

Bioactive Core-Shell Nanofiber Hybrid Scaffold for Efficient Suicide Gene Transfection and Subsequent Time Resolved Delivery of Prodrug for Anticancer Therapy.

Nanofiber scaffold's ability to foster seemingly nonexistent interface with the cells enables them to effectively deliver various bioactive molecules to cells in the vicinity. Among such bioactive molecules, therapeutically active nucleic acid has been the most common candidate. In spite of such magnanimous efforts in this field, it remains a paradox that suicide gene delivery by nanofibers has never been sought for anticancer application. To investigate such a possibility, in the present work, a composite core-shell nanofiberous scaffold has been realized which could efficiently transfect suicide gene into cancer cells and simultaneously deliver prodrug, 5-Fluorocytosine (5-FC) in a controlled and sustained manner. The scaffold's ability to instigate apoptosis by suicide gene therapy in nonsmall lung cancer cells (A549) was ascertained at both phenotypic and genotypic levels. A cascade of events starting from suicide gene polyplex release from nanofibers, transfection, and expression of cytosine deaminase-uracil phosphoribosyltransferase (CD::UPRT) suicide gene by A549; subsequent prodrug release; and its metabolic conversion into toxic intermediates which finally culminates in host cells apoptosis has been monitored in a time-dependent manner. This work opens up new application avenues for nanofiber-based scaffolds which can effectively manage cancer prognosis.