Development of optical pH nanosensors for biological insights into the intracellular trafficking of nanomedicines

The field of nanomedicine has progressed to a stage where a diverse set of materials are available for controlling how a drug is delivered in the body. Although these materials can be engineered to overcome many of the obstacles associated with drug delivery, the complexity of cellular trafficking mechanisms means controlling intracellular delivery remains a major challenge. The primary portal for the cellular internalisation of nanomedicines is endocytosis, which involves transport through a network of highly complex intracellular compartments undergoing a dynamic process of acidification. As a result, nanoparticle-based pH sensors offer a new perspective from which to investigate this process. In this study, ratiometric polyacrylamide pH nanosensors were utilised to probe fundamental aspects of intracellular trafficking with the view of developing biological insights to aid the rational design of nanomedicines. Nanosensors were fabricated with a dynamic range covering the entire range of the endocytic pathway (4.0 – 7.5), with sizes between 50 and 100 nm. Endocytic uptake of nanosensors was induced in four different cell types (HeLa, 3T3, MRC-5 and JAWS II) by increasing the surface charge on the nanosensor. Dynamic pH measurements were found to be highly sensitive to experimental methodology for performing ratiometric measurements, particularly image analysis. Consequently an optimised procedure for performing ratiometric measurements was developed, and subsequently validated by correlating pH measurements with intracellular location using 3D structured illumination microscopy (3D-SIM). Application of pH nanosensors in studies investigating fundamental aspects of intracellular trafficking resulted in three key findings: 1) HeLa, 3T3 and JAWS II cells process material in different ways with respect to the extent and rate of acidification in endocytic organelles, 2) surface charge does not affect the final intracellular location of polyacrylamide nanoparticles internalised by endocytosis, and 3) lipid-mediated transfection of siRNA is associated with a greater degree of lysosomal disruption compared to cationic polymer-mediated transfection, with the former observed to show increased toxicity. These findings represent biological insights, which can be utilised to provide a rational basis for tailoring the response of pH-sensitive nanomedicines to a specific cell type, tuning the physicochemical properties of a material for more efficient intracellular trafficking and optimising siRNA formulations for endo-lysosomal release

Development of optical pH nanosensors for biological insights into the intracellular trafficking of nanomedicines

The field of nanomedicine has progressed to a stage where a diverse set of materials are available for controlling how a drug is delivered in the body. Although these materials can be engineered to overcome many of the obstacles associated with drug delivery, the complexity of cellular trafficking mechanisms means controlling intracellular delivery remains a major challenge. The primary portal for the cellular internalisation of nanomedicines is endocytosis, which involves transport through a network of highly complex intracellular compartments undergoing a dynamic process of acidification. As a result, nanoparticle-based pH sensors offer a new perspective from which to investigate this process. In this study, ratiometric polyacrylamide pH nanosensors were utilised to probe fundamental aspects of intracellular trafficking with the view of developing biological insights to aid the rational design of nanomedicines. Nanosensors were fabricated with a dynamic range covering the entire range of the endocytic pathway (4.0 – 7.5), with sizes between 50 and 100 nm. Endocytic uptake of nanosensors was induced in four different cell types (HeLa, 3T3, MRC-5 and JAWS II) by increasing the surface charge on the nanosensor. Dynamic pH measurements were found to be highly sensitive to experimental methodology for performing ratiometric measurements, particularly image analysis. Consequently an optimised procedure for performing ratiometric measurements was developed, and subsequently validated by correlating pH measurements with intracellular location using 3D structured illumination microscopy (3D-SIM). Application of pH nanosensors in studies investigating fundamental aspects of intracellular trafficking resulted in three key findings: 1) HeLa, 3T3 and JAWS II cells process material in different ways with respect to the extent and rate of acidification in endocytic organelles, 2) surface charge does not affect the final intracellular location of polyacrylamide nanoparticles internalised by endocytosis, and 3) lipid-mediated transfection of siRNA is associated with a greater degree of lysosomal disruption compared to cationic polymer-mediated transfection, with the former observed to show increased toxicity. These findings represent biological insights, which can be utilised to provide a rational basis for tailoring the response of pH-sensitive nanomedicines to a specific cell type, tuning the physicochemical properties of a material for more efficient intracellular trafficking and optimising siRNA formulations for endo-lysosomal release

Heart Beat Monitoring And Wireless Data Logging Using Arm Cortex A8

Increasing importance in monitoring and logging the real time patient’s related data has led to the development of a new wireless data acquisition system. The basic concept presented in this paper is that we have made one type of wireless data logger[1] system using arm platform which is logging the real time heartbeat[2] data in one text file it is give the heartbeat rate with respect to time. Here we use UDOO[8] development board, UDOO is a single-board computer development platform that merges a dual or quad core ARM Free scale Cortex-A9 i.MX.6 CPU and an Arduino[4] compatible board embedded with a dedicated ARM Atmel SAM3X8E CPU. Physical data is converted in to the electrical form using Arduino UNO R3 and after processing these data it will be transmitted by the RFM. Same way at receiver side one RFM device is required to receive the data which was transmitted by the transmitter. Once the data is received, these data will be given to UDOO board. In UDOO board the data is processed and the data will be monitored on LCD or DVI Monitor

Wideband Flexible/Transparent Connected-Ground MIMO Antennas for Sub-6 GHz 5G and WLAN Applications

A flexible transparent wideband four-element MIMO antenna with a connected ground plane is proposed with numerical computation and experimental measurement studies. The optical transparency is obtained using flexible conductive oxide material AgHT-4 and Melinex substrate. The radiating elements are in the form of circular stub-loaded C-shaped resonators, which are positioned in a carefully structured flexible Melinex substrate with an interconnected partial ground plane structured in the form of an L-shaped resonator, attaining an overall antenna size of 0.33 lambda x 0.48 lambda at the lowest operating frequency. The proposed antenna spans over a -10 dB impedance bandwidth of 2.21-6 GHz (92.32%) with an isolation level greater than 15dB among all elements. The maximum gain is 0.53dBi with a minimum efficiency of 41%, respectively which is satisfactory considering flexible structure and sheet impedance of 4 Omega/sq. MIMO antenna parameters in terms of the envelope correlation coefficient (ECC) and diversity gain (DG) are also extracted where all the values are satisfactory for MIMO applications. The bending analysis of the proposed transparent MIMO antenna along the X and Y axis has revealed good performance in terms of scattering parameters and radiation pattern along with MIMO diversity performance. All of these technical points make the flexible MIMO antenna suitable for smart devices using sub-6 GHz 5G and WLAN band in IoT applications where visual clutter and co-site location issues need to be mitigated with the integration ease of conformal placement on the curved component/device surfaces

Lipid-peptide nanocomplexes for mRNA delivery in vitro and in vivo

Despite recent advances in the field of mRNA therapy, the lack of safe and efficacious delivery vehicles with pharmaceutically developable properties remains a major limitation. Here, we describe the systematic optimisation of lipid-peptide nanocomplexes for the delivery of mRNA in two murine cancer cell types, B16-F10 melanoma and CT26 colon carcinoma as well as NCI-H358 human lung bronchoalveolar cells. Different combinations of lipids and peptides were screened from an original lipid-peptide nanocomplex formulation for improved luciferase mRNA transfection in vitro by a multi-factorial screening approach. This led to the identification of key structural elements within the nanocomplex associated with substantial improvements in mRNA transfection efficiency included alkyl tail length of the cationic lipid, the fusogenic phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol. The peptide component (K16GACYGLPHKFCG) was further improved by the inclusion of a linker, RVRR, that is cleavable by the endosomal enzymes cathepsin B and furin, and a hydrophobic motif (X-S-X) between the mRNA packaging (K16) and receptor targeting domains (CYGLPHKFCG). Nanocomplex transfections of a murine B16-F10 melanoma tumour supported the inclusion of cholesterol for optimal transfection in vivo as well as in vitro. In vitro transfections were also performed with mRNA encoding interleukin-15 as a potential immunotherapy agent and again, the optimised formulation with the key structural elements demonstrated significantly higher expression than the original formulation. Physicochemical characterisation of the nanocomplexes over time indicated that the optimal formulation retained biophysical properties such as size, charge and mRNA complexation efficiency for 14 days upon storage at 4 °C without the need for additional stabilising agents. In summary, we have developed an efficacious lipid-peptide nanocomplex with promising pharmaceutical development properties for the delivery of therapeutic mRNA

Compact Wideband Four Element Optically Transparent MIMO Antenna for mm-Wave 5G Applications

A four-element compact wide-band optically transparent MIMO antenna with a full ground plane is proposed. The four elements transparent MIMO system has a compact size of 24x20 mm(2) with the undivided ground plane as most of the real-time systems demand a common reference. The complete antenna system achieves around 85% transparency due to a combination of AgHT-8 and Plexiglas which forms the transparent conductive patch/ground and substrate, respectively. The antenna geometry leads dual-band operation ranging from 24.10 - 27.18 GHz (Impedance bandwidth D 12%) and 33 - 44.13 GHz (Impedance bandwidth D 28.86%) targeting the mm-wave 5G applications. The 4-element antenna system achieves isolation between inter-elements > 16 dB and maximum gain value of greater than 3 dBi with more than 75% efficiency. The proposed transparent MIMO antenna is evaluated in terms of diversity gain (DG), envelope correlation coefficient (ECC), total active reflection coefficient (TARC), and mean effective gain (MEG) where decent MIMO performance with isolation more than >16 dB between the adjacent and other elements is achieved. Transparent MIMO antenna achieves directional patterns for the operating band with the value of DG > 9, ECC < 0.1, TARC value less than and the ratio of MEG within the agreed limit of +/- 3 dB conforming acceptable MIMO/diversity performance

Fluorescent nanosensors for intracellular measurements: synthesis, characterization, calibration, and measurement

Measurement of intracellular acidification is important for understanding fundamental biological pathways as well as developing effective therapeutic strategies. Fluorescent pH nanosensors are an enabling technology for real-time monitoring of intracellular acidification. The physicochemical characteristics of nanosensors can be engineered to target specific cellular compartments and respond to external stimuli. Therefore, nanosensors represent a versatile approach for probing biological pathways inside cells. The fundamental components of nanosensors comprise a pH-sensitive fluorophore (signal transducer) and a pH-insensitive reference fluorophore (internal standard) immobilized in an inert non-toxic matrix. The inert matrix prevents interference of cellular components with the sensing elements as well as minimizing potentially harmful effects of some fluorophores on cell function. Fluorescent nanosensors are synthesized using standard laboratory equipment and are detectable by non-invasive widely accessible imaging techniques. The outcomes of studies employing this technology are dependent on reliable methodology for performing measurements. In particular, special consideration must be given to conditions for sensor calibration, uptake conditions and parameters for image analysis. We describe procedures for: (1) synthesis and characterization of polyacrylamide and silica based nanosensors, (2) nanosensor calibration and (3) performing measurements using fluorescence microscopy

Lipid-peptide nanocomplexes for mRNA delivery in vitro and in vivo

Despite recent advances in the field of mRNA therapy, the lack of safe and efficacious delivery vehicles with pharmaceutically developable properties remains a major limitation. Here, we describe the systematic optimisation of lipid-peptide nanocomplexes for the delivery of mRNA in two murine cancer cell types, B16-F10 melanoma and CT26 colon carcinoma as well as NCI-H358 human lung bronchoalveolar cells. Different combinations of lipids and peptides were screened from an original lipid-peptide nanocomplex formulation for improved luciferase mRNA transfection in vitro by a multi-factorial screening approach. This led to the identification of key structural elements within the nanocomplex associated with substantial improvements in mRNA transfection efficiency included alkyl tail length of the cationic lipid, the fusogenic phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol. The peptide component (K16GACYGLPHKFCG) was further improved by the inclusion of a linker, RVRR, that is cleavable by the endosomal enzymes cathepsin B and furin, and a hydrophobic motif (X-S-X) between the mRNA packaging (K16) and receptor targeting domains (CYGLPHKFCG). Nanocomplex transfections of a murine B16-F10 melanoma tumour supported the inclusion of cholesterol for optimal transfection in vivo as well as in vitro. In vitro transfections were also performed with mRNA encoding interleukin-15 as a potential immunotherapy agent and again, the optimised formulation with the key structural elements demonstrated significantly higher expression than the original formulation. Physicochemical characterisation of the nanocomplexes over time indicated that the optimal formulation retained biophysical properties such as size, charge and mRNA complexation efficiency for 14 days upon storage at 4 °C without the need for additional stabilising agents. In summary, we have developed an efficacious lipid-peptide nanocomplex with promising pharmaceutical development properties for the delivery of therapeutic mRNA

Nanomedicines for the delivery of biologics

A special symposium of the Academy of Pharmaceutical Sciences Nanomedicines Focus Group reviewed the current status of the use of nanomedicines for the delivery of biologics drugs. This meeting was particularly timely with the recent approval of the first siRNA-containing product Onpattro™ (patisiran), which is formulated as a lipid nanoparticle for intravenous infusion, and the increasing interest in the use of nanomedicines for the oral delivery of biologics. The challenges in delivering such molecules were discussed with specific emphasis on the delivery both across and into cells. The latest developments in Molecular Envelope Technology® (Nanomerics Ltd, London, UK), liposomal drug delivery (both from an academic and industrial perspective), opportunities offered by the endocytic pathway, delivery using genetically engineered viral vectors (PsiOxus Technologies Ltd, Abingdon, UK), Transint™ technology (Applied Molecular Transport Inc., South San Francisco, CA, USA), which has the potential to deliver a wide range of macromolecules, and AstraZeneca’s initiatives in mRNA delivery were covered with a focus on their uses in difficult to treat diseases, including cancers. Preclinical data were presented for each of the technologies and where sufficiently advanced, plans for clinical studies as well as early clinical data. The meeting covered the work in progress in this exciting area and highlighted some key technologies to look out for in the future

Aperture-Fed Quad-Port Dual-Band Dielectric Resonator-MIMO Antenna for Sub-6 GHz 5G and WLAN Application

A four-port dielectric resonator-based connected ground multiple-input multiple-output (MIMO) antenna is designed. The presented antenna was excited through the aperture feeding technique. The dual bands are achieved by optimally feeding the rectangular dielectric resonator through engineered triangular slots. The antenna has operating modes of TE111X and TE111Y at 4.5 GHz and 5 GHz, respectively. It presents a 2 : 1 VSWR bandwidth of 2.64% (4.48 GHz-4.60 GHz) and 1.2% (4.96 GHz-5.04 GHz) in the lower and upper bands, respectively. The edge-to-edge distance between array elements is around 7.5 mm. The single antenna dimension is 30 mm x 30 mm, whereas the four-port antenna dimension is 60 mm x 60 mm. The optimum isolation was achieved by carefully placing the antenna elements on the substrate through multiple iterations. The antenna provides port isolation better than 20 dB at both resonances with full ground profile. The advantage of the antenna is that it provides fair antenna and MIMO parameters without additional isolation techniques. The antenna has efficiency in order of 88.02% and 86.31%. The peak gain is 7.67 dBi and 8.32 dBi at 4.5 GHz and 5 GHz, respectively. The optimum envelope correlation coefficient (ECC) is 0.037, channel capacity coss (CCL) is 0.2 bits/sec/Hz, diversity gain (DG) is 9.99 dB, and total active reflection coefficient (TARC) is -18.87. The antenna elements are orthogonally placed with adequate separation to achieve polarization diversity and spatial diversity. The antenna provides the utilization in Sub-6 GHz 5G and WLAN communication applications