Knockout cancer by nano-delivered immunotherapy using perfusion-aided scaffold-based tumor-on-a-chip.

Cancer is a multifactorial disease produced by mutations in the oncogenes and tumor suppressor genes, which result in uncontrolled cell proliferation and resistance to cell death. Cancer progresses due to the escape of altered cells from immune monitoring, which is facilitated by the tumor's mutual interaction with its microenvironment. Understanding the mechanisms involved in immune surveillance evasion and the significance of the tumor microenvironment might thus aid in developing improved therapies. Although in vivo models are commonly utilized, they could be better for time, cost, and ethical concerns. As a result, it is critical to replicate an in vivo model and recreate the cellular and tissue-level functionalities. A 3D cell culture, which gives a 3D architecture similar to that found in vivo, is an appropriate model. Furthermore, numerous cell types can be cocultured, establishing cellular interactions between TME and tumor cells. Moreover, microfluidics perfusion can provide precision flow rates, thus simulating tissue/organ function. Immunotherapy can be used with the perfused 3D cell culture technique to help develop successful therapeutics. Immunotherapy employing nano delivery can target the spot and silence the responsible genes, ensuring treatment effectiveness while minimizing adverse effects. This study focuses on the importance of 3D cell culture in understanding the pathophysiology of 3D tumors and TME, the function of TME in drug resistance, tumor progression, and the development of advanced anticancer therapies for high-throughput drug screening.

Fabricating a low-temperature synthesized graphene-cellulose acetate-sodium alginate scaffold for the generation of ovarian cancer spheriod and its drug assessment.

3D cell culture can mimic tumor pathophysiology, which reflects cellular morphology and heterogeneity, strongly influencing gene expression, cell behavior, and intracellular signaling. It supports cell-cell and cell-matrix interaction, cell attachment, and proliferation, resulting in rapid and reliable drug screening models. We have generated an ovarian cancer spheroid in interconnected porous scaffolds. The scaffold is fabricated using low-temperature synthesized graphene, cellulose acetate, and sodium alginate. Graphene nanosheets enhance cell proliferation and aggregation, which aids in the formation of cancer spheroids. The spheroids are assessed after day 7 and 14 for the generation of reactive oxygen species (ROS), expression of the hypoxia inducing factor (HIF-1⍺) and vascular endothelial growth factor (VEGF). Production of ROS was observed due to the aggregated tumor mass, and enhanced production of HIF-1⍺ and VEGF results from a lack of oxygen and nutrition. Furthermore, the efficacy of anticancer drug doxorubicin at varying concentrations is assessed on ovarian cancer spheroids by studying the expression of caspase-3/7 at day 7 and 14. The current findings imply that the graphene-cellulose-alginate (GCA) scaffold generates a reliable ovarian cancer spheroid model to test the efficacy of the anticancer drug.

Active microfluidic reactor-assisted controlled synthesis of nanoparticles and related potential biomedical applications.

Fabricating high-performance nanoparticles (NPs) is currently a focus of researchers due to their manipulative size-dependent unique properties required to develop next-generation advanced systems. To harness the unique properties of NPs, maintaining identical characteristics throughout the processing and application process system is crucial to producing uniform-sized, or monodisperse, NPs. In this direction, mono-dispersity can be achieved by exerting extreme control over the reaction conditions during the NP synthesis process. Microfluidic technology offers a unique approach to control fluid conditions at the microscale and is thus well-positioned as an alternative strategy to synthesize NPs in reactors demonstrating micrometric dimensions and advanced size-controlled nanomaterial production. These microfluidic reactors can be broadly classified as active or passive based on their dependence on external energy sources. Passive microfluidic reactors, despite their lack of reliance on external energy, are frequently constrained in terms of their mixing efficacy when compared to active systems. However, despite several fundamental and technological advantages, this area of research as well as its application to the biological sciences is not well-discussed. To fill this gap, this review for the first time discusses various strategies for synthesizing NPs using active microfluidic reactors including acoustic, pressure, temperature, and magnetic assisted microfluidic reactors. Various established ways for achieving size control on NP synthesis in microfluidic reactors representing the applicability of micro-reaction technology in developing novel nanomaterials suitable for potential biomedical applications are presented in this review along with a comprehensive discussion about the challenges and prospects.

Application of dendrimer-based nanosensors in immunodiagnosis.

Conventional immunoassays such as ELISA and FLISA have been used for clinical diagnosis for a long time. These assays are complex, time-consuming, and uneconomical. They have been overwhelmed with newer and more efficient methods such as electrochemical and electrochemiluminescent immunosensors that are cost-effective and require less time. Immunosensor is a biosensor that consists of a signal transducer and a biologically interactive system such as antigen and antibody interaction. Recent advances in nanotechnology have seen numerous efforts towards the usage of nanoparticles such as dendrimers in immunoassays. Dendrimers are highly branched structures with a high density of active peripheral groups, expanding their wide range of applications in immunoassays. A vast number of peripheral groups enrich the sensitivity of the immunosensor by governing the orientation of the antibody on the sensor surface. The current review highlights recent progress and developments in applying dendrimers for different immunoassays and their applicability in analyzing various biomarkers in clinical disease diagnosis.

Development of nano-immunosensor with magnetic separation and electrical detection of Escherichia coli using antibody conjugated Fe3O4@Ppy.

Detection of bacterial pathogens is the need of the hour due to the increase in antibiotic resistance and the infusion of multi-drug-resistant parasites. The conventional strategies such as ELISA, PCR, and MNP based tests for the detection are efficient but they are cost, time, lab, and manpower intensive. Thus, warranting a simple and effective technique for rapid detection of bacterial pathogens. Magnetic nanoparticles (NPs) have proved to be better alternatives for separation of bacterial pathogens from a variety of sample sources. However, the use of magnetic NPs has not been successful in the detection of these parasites. The current work involves the coating of magnetic NPs (Fe3O4) with a conducting polymer (polypyrrole; Ppy) to facilitate simultaneous separation and detection. Electrical (conductivity) measurement was the mode of choice due to the sensitivity, accuracy, and ease it offers. To enhance the conductivity, carboxylic groups were expressed on the Fe3O4@Ppy complex and to ensure specificity, E. coli specific antibodies were conjugated. The resulting complex at various process parameters was characterized using FTIR, VSM, and SEM. SEM images were recorded to ensure bacterial separation at optimal process parameters. The impedance analysis and conductivity measurements were carried out for the sample volume of 15 µl. The bacterial suspension from 101-106 CFU ml-1 was successfully detected with a limit of detection of 10 CFU ml-1 within 10 min using a simplistic detection method.

High-quality quantum dots for multiplexed bioimaging: A critical review.

Bioimaging done using two or more fluorophores possessing different emission wavelengths can be termed as a multicolor/multiplexed bioimaging technique. Traditionally, images are captured sequentially using multiple fluorophores having specific excitation and emission. For this purpose, multifunctional nanoprobes, such as organic fluorophores, metallic nanoparticles, semiconductor quantum dots, and carbon dots (CDs) are used. Among these fluorophores, quantum dots (QDs) have emerged as an ideal probe for multiplexed bioimaging due to their unique property of size tunable emission. However, the usage of quantum dots in bioimaging is limited due to their toxicity. Furthermore, the reproducibility of optical properties is cynical. These desirable properties, along with enhancement in quantum efficiency, photostability, fluorescence lifetime, etc. can be achieved by stringent control over synthesis parameters. This review summarizes the desirable properties and synthesis methods of such superior QDs followed by their application in multiplexed imaging.

Applications of cobalt ferrite nanoparticles in biomedical nanotechnology.

Magnetic nanoparticles (MNPs) are very attractive especially for biomedical applications, among which, iron oxide nanoparticles have received substantial attention in the past decade due to the elemental composition that makes them biocompatible and degradable. However recently, other magnetic nanomaterials such as spinel ferrites that can provide improved magnetic properties such as coercivity and anisotropy without compromising on inherent advantages of iron oxide nanoparticles are being researched for better applicability of MNPs. Among various spinel ferrites, cobalt ferrite (CoFe2O4) nanoparticles (NPs) are one of the most explored MNPs. Therefore, the intention of this article is to provide a comprehensive review of CoFe2O4 NPs and their inherent properties that make them exceptional candidates, different synthesis methods that influence their properties, and applications of CoFe2O4 NPs and their relevant applications that have been considered in biotechnology and bioengineering.

Assessment of an Integrative Anticancer Treatment Using an in Vitro Perfusion-Enabled 3D Breast Tumor Model.

The study presents observations on anticancer therapeutic efficacy of magnetic fluid hyperthermia and a combination of hyperthermia and chemotherapy (i.e., integrative treatment) using an in vitro perfused and non-perfused 3D breast tumor model. The 3D in vitro breast tumor models were simulated using Comsol multiphysics, fabricated using specially designed chips, and treated with doxorubicin-loaded chitosan-coated La0.7Sr0.3MnO3 (DC-LSMO) nanoparticles for hyperthermia and combination therapy in both perfused and non-perfused conditions. Computation confirmed uniform heat distribution throughout the scaffold for both the models. The findings indicate that both hyperthermia and combination treatment could trigger apoptotic cell death in the perfused and non-perfused models in varying degrees. Specifically, the perfused tumors were more resistant to therapy than the non-perfused ones. The efficacy of anticancer treatment decreased with increasing physiological complexity of the tumor model. The combination (hyperthermia and chemotherapy) treatment showed enhanced efficacy over hyperthermia alone. This is a pilot study to investigate the effects of magnetic fluid hyperthermia-chemotherapy treatment using perfused and non-perfused 3D in vitro models of tumor. The feasibility of using 3D cell culture models for contributing to our understanding of cancer and its treatment was also determined as a part of this work.

A facile one-step method for cell lysis and DNA extraction of waterborne pathogens using a microchip.

Globally, waterborne organisms are the primary causative agents for the transmission of various forms of diarrheal diseases. For accurate diagnosis, molecular tools have gained considerable attention in the recent past. Molecular tools require DNA as the starting material for diagnosis, and hence, a prerequisite is the quality and integrity of DNA. To obtain high quality DNA rapidly, we have fabricated a microchip in poly(dimethyl siloxane) (PDMS) by soft lithography process. The microchip facilitated in-flow coating of chitosan on the magnetic nanoparticles, which under external mechanical vibration caused cell lysis and released DNA in the supernatant. The released DNA was captured by the nanoparticles owing to its positive charge (chitosan coating). The magnetic nanoparticle-DNA complex was then isolated from the in-flow matrix using permanent magnet, Further, removal of the cell debris, proteins, and carbohydrates was done using wash buffer. DNA extracted using the microchip was pure with absorbance (260/280) ratio of 1.77±0.04, as compared to 1.79±0.03 obtained by TRIzol method. The complete isolation of the DNA using the microchip took ~ 15min as against>2h with a TRIzol method. Six gram-negative waterborne pathogens were used to demonstrate the efficacy of the microchip based DNA extraction process. The integrity of the isolated DNA was assessed by amplifying the 16S rRNA gene using Com1 and Com2 universal primers. The presence of a band at 407bp on gel electrophoresis confirmed the amplified product. Further, the gel image was used for quantification of the amplified product using ImageJ software. Higher regression values obtained using microchip confirmed better quality and integrity of the extracted DNA as opposed to the conventional method. The lower (<2%) relative standard deviation values obtained from the data suggested that the microchip process was reproducible. The quality and integrity of the obtained DNA proved the simplicity, rapidity, and sensitivity of the microchip-assisted DNA extraction process.

In vitro and in vivo studies of a novel bacterial cellulose-based acellular bilayer nanocomposite scaffold for the repair of osteochondral defects.

Bacterial cellulose (BC) is a naturally occurring nanofibrous biomaterial which exhibits unique physical properties and is amenable to chemical modifications. To explore whether this versatile material can be used in the treatment of osteochondral defects (OCD), we developed and characterized novel BC-based nanocomposite scaffolds, for example, BC-hydroxyapatite (BC-HA) and BC-glycosaminoglycans (BC-GAG) that mimic bone and cartilage, respectively. In vitro biocompatibility of BC-HA and BC-GAG scaffolds was established using osteosarcoma cells, human articular chondrocytes, and human adipose-derived mesenchymal stem cells. On subcutaneous implantation, the scaffolds allowed tissue ingrowth and induced no adverse immunological reactions suggesting excellent in vivo biocompatibility. Implantation of acellular bilayered scaffolds in OCD created in rat knees induced progressive regeneration of cartilage tissue, deposition of extracellular matrix, and regeneration of subchondral bone by the host cells. The results of micro-CT revealed that bone mineral density and ratio of bone volume to tissue volume were significantly higher in animals receiving bilayered scaffold as compared to the control animals. To the best of our knowledge, this study proves for the first time, the functional performance of acellular BC-based bilayered scaffolds. Thus, this strategy has great potential for clinical translation and can be used in repair of OCD.

Nanoscale silver depositions inhibit microbial colonization and improve biocompatibility of titanium abutments.

Although titanium dental implants are biocompatible, exhibit excellent corrosion resistance and high mechanical resistance, the material fails in providing resistance to infection because it exhibits poor antimicrobial activity. To address these issues, we deposited silver onto titanium abutments (Grade 5 titanium discs) using direct current (DC) sputtering and assessed the antimicrobial activity and biocompatibility of the modified implant material. Atomic absorption spectrometry and X-ray photoelectron spectroscopy were employed to investigate the concentration and elemental composition of the deposited silver. As expected, silver deposited using DC plasma was uniform and good control over the deposition could be achieved by varying the sputtering time. Moderate biocompatible responses (up to 69% viability) were observed in primary human gingival fibroblast cells incubated in the presence of Ti sputtered with Ag for 5min. Silver deposited titanium (Ti-Ag) showed excellent antibacterial effects on Pseudomonas aeruginosa and Streptococcus mutans at a very low concentration (Ag content 1.2 and 2.1µg/mm2). However, higher concentration of silver (6µg/mm2) was required to achieve a reduction in cell viability of Staphylococcus aureus and Candida albicans. The silver sputtered Ti abutments could maintain a long-term antibacterial activity as evidenced by the release of silver up to 22days in simulated body fluid. Our study illustrates that silver deposited titanium is indeed a promising candidate for soft tissue integration on dental abutments and prevents initial microbial adhesion.

Radio-frequency triggered heating and drug release using doxorubicin-loaded LSMO nanoparticles for bimodal treatment of breast cancer.

Radio-frequency responsive nanomaterials combined with drugs for simultaneous hyperthermia and drug delivery are potential anti-cancer agents. In this study, chitosan coated La0.7Sr0.3MnO3 nanoparticles (C-LSMO NPs) were synthesized and characterized by X-ray diffraction, dynamic light scattering, Fourier transform infra red spectroscopy, vibrating sample magnetometer, scanning electron and atomic force microscopy. Under low radio-frequency (365kHz, RF), C-LSMO NPs (90nm) showed good colloidal stability (+22mV), superparamagnetic nature (15.4 emu/g) and heating capacity (57.4W/g SAR value). Chitosan facilitated doxorubicin entrapment (76%) resulted in DC-LSMO NPs that showed drug release upon a 5min RF exposure. MCF-7 and MDA-MB-231 cancer cells responded to a 5min RF exposure in the presence of bimodal DC-LSMO NPs with a significant decrease in viability to 73% and 88% (Pearson correlation, r=1, P<0.01) respectively, as compared to hyperthermia alone. Internalization of DC-LSMO NPs via the endosomal pathway led to an efficient localization of doxorubicin within the cell nucleus. The ensuing DNA damage, heat shock protein induction, and caspase production triggered apoptotic cell death. Moreover, DC-LSMO NPs successfully restricted the migration of metastatic MDA-MB-231 cancer cells. These data suggest that DC-LSMO NPs are potential bimodal therapeutic agents for cancer treatment and hold promise against disease recurrence and drug resistance.

A high affinity phage-displayed peptide as a recognition probe for the detection of Salmonella Typhimurium.

Salmonellosis is one of the most common and widely distributed foodborne diseases. A sensitive and robust detection method of Salmonella Typhimurium (S. Typhimurium) in food can critically prevent a disease outbreak. In this work, the use of phage displayed peptides was explored for the detection of S. Typhimurium. A phage-displayed random dodecapeptide library was subjected to biopanning against lipopolysaccharide (LPS) of S. Typhimurium. The peptide NFMESLPRLGMH (pep49) derived from biopanning displayed a high affinity (25.8nM) for the LPS of S. Typhimurium and low cross-reactivity with other strains of Salmonella and related Gram-negative bacteria. Molecular insights into the interaction of pep49 with the LPS of S. Typhimurium was gleaned using atomistic molecular dynamics simulations and docking. It was deduced that the specificity of pep49 with S. Typhimurium LPS originated from the interactions of pep49 with abequose that is found only in the O-antigen of S. Typhimurium. Further, pep49 was able to detect S. Typhimurium at a LOD of 10(3) CFU/mL using ELISA, and may be a potential cost efficient alternative to antibodies.

Chitosan nanoparticles synthesis caught in action using microdroplet reactions.

The ionic gelation process for the synthesis of chitosan nanoparticles was carried out in microdroplet reactions. The synthesis could be stopped instantaneously at different time points by fast dilution of the reaction mixture with DI water. Using this simple technique, the effect of temperature and reactant concentrations on the size and distribution of the nanoparticles formed, as a function of time, could be investigated by DLS and SEM. Results obtained indicated very early (1-5 s) nucleation of the particles followed by growth. The concentration of reactants, reaction temperature as well as time, were found to (severally and collectively) determine the size of nanoparticles and their distribution. Nanoparticles obtained at 4 °C were smaller (60-80 nm) with narrower size distribution. Simulation experiments using Comsol software showed that at 4 °C 'droplet synthesis' of nanoparticles gets miniaturised to 'droplet-core synthesis', which is being reported for the first time.

Synthesis of Monodisperse Chitosan Nanoparticles and in Situ Drug Loading Using Active Microreactor.

Chitosan nanoparticles are promising drug delivery vehicles. However, the conventional method of unregulated mixing during ionic gelation limits their application because of heterogeneity in size and physicochemical properties. Therefore, a detailed theoretical analysis of conventional and active microreactor models was simulated. This led to design and fabrication of a polydimethylsiloxane microreactor with magnetic micro needles for the synthesis of monodisperse chitosan nanoparticles. Chitosan nanoparticles synthesized conventionally, using 0.5 mg/mL chitosan, were 250 ± 27 nm with +29.8 ± 8 mV charge. Using similar parameters, the microreactor yielded small size particles (154 ± 20 nm) at optimized flow rate of 400 µL/min. Further optimization at 0.4 mg/mL chitosan concentration yielded particles (130 ± 9 nm) with higher charge (+39.8 ± 5 mV). The well-controlled microreactor-based mixing generated highly monodisperse particles with tunable properties including antifungal drug entrapment (80%), release rate, and effective activity (MIC, 1 µg/mL) against Candida.

Multiplexed detection of waterborne pathogens in circular microfluidics.

Microfluidic lab-on-a-chip presents an ideal solution for bacterial sensing and identification due to its advantages like large surface-to-volume ratio, requirement of low sample volume and multiplexing possibility. The present work deals with the development of an immunosensor chip using circular microchannels fabricated directly with microdimensional copper wire and permanent magnet for capture of Fe(3)O(4) magnetic nanoparticle (MNP) conjugate. The MNP facilitate capture of the antigen in a confined space and hence, enhanced fluorescence signal for detection. The multiplexed microfluidic chip permits visual detection and quantification of waterborne pathogens viz. Escherichia coli and Salmonella typhimurium simultaneously. CdTe quantum dots (QDs) with different emission wavelengths were conjugated with anti-E. coli and anti-S. typhimurium antibodies for concurrent fluorescence detection. The present technique provides an inexpensive yet powerful tool to image and quantify pathogens at low numbers with passage of large sample volumes.

Quantum dot based immunosensor using 3D circular microchannels fabricated in PDMS.

Microchannel is basic functional component of microfluidic chip and every step-forward of its construction technique has been receiving concern all over the world. The present work describes a novel, rapid and simple fabrication technique for building 3D microchannels in poly(dimethyl siloxane) (PDMS) elastomer. These microchannels were used for rapid detection of antigens (E. coli) by quantum dot (QD) based approach. Luminescent QD (CdTe) were synthesized by aqueous method and characterized using high resolution transmission electron microscopy (HRTEM), fluorescence spectroscopy and X-ray diffraction (XRD). The QDs were functionalized with anti-E. coli antibodies for immuno-detection. The reported process allowed easier and faster method of fabrication of circular 3D micochannels and demonstrated their potential use in an immuno-biosensor device.

Tailor-made functional surfaces: potential elastomeric biomaterials I.

In the present investigation, different functional monomers, like hydroxyethyl methacrylate, acrylic acid, N-vinyl pyrrolidone and glycidyl methacrylate, have been grafted onto the surface of EPDM film (approx. 200 microm) using simultaneous photo-grafting (lambda > or = 290 nm) and cold plasma-grafting techniques, to alter the surface properties, such as hydrophilicity and, therefore, biocompatibility. Here, we have carried out simultaneous plasma-grafting, unlike the conventional post plasma-grafting. The effect of different surface grafting techniques on the degree of surface modification and resultant biocompatibility has been investigated. The chemical changes on the polymer backbone are followed from the results of attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS), which shows the peaks corresponding to the functional groups of the monomers grafted onto the film surface. The morphology of the modified surfaces was investigated using scanning electron microscopy (SEM) technique. The induced hydrophilicity and resultant cell compatibility were followed from the water contact angle measurements and in vitro human carcinoma cell adhesion/proliferation tests, respectively. All the grafted samples exhibited variable cell compatibilities depending upon the type of monomer and their degree of grafting; however, always better than the neat samples. Hydroxyethyl methacrylate and acrylic acid showed exceptionally high cell compatibility in terms of cell adhesion and proliferation.