microfluidic-buffer-exchange-for-interference-free-micronanoparticle

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Nanyang Technological University

Assessing biodistribution, fate, and function of implanted therapeutic cells in preclinical animal experiments is critical to realize safe, effective and efficient treatments for subsequent implementation within the clinic. Currently, tissue histology, the most prevalent analytical technique to meet this need, is limited by end-point analysis, high cost and long preparation time. Moreover, it is disadvantaged by an inability to monitor in real-time, qualitative interpretation and ethical issues arising from animal sacrifice. While genetic engineering techniques allow cells to express molecules with detectable signals (e.g., fluorescence, luminescence, T1 (spin-lattice)/T2 (spin-spin) contrast in magnetic resonance imaging, radionuclide), concerns arise regarding technical complexity, high-cost of genetic manipulation, as well as mutagenic cell dysfunction. Alternatively, cells can be labeled using nanoparticle-sensors-nanosensors that emit signals to identify cell location, status and function in a simple, cost-effective, and non-genetic manner. This review article provides the definition, classification, evolution, and applications of nanosensor technology and focuses on how they can be utilized in regenerative medicine. Several examples of direct applications include: (1) monitoring post-transplantation cell behavior, (2) revealing host response following foreign biomaterial implantation, and (3) optimization of cell bioprocess operating conditions. Incorporating nanosensors is expected to expedite the development of cell-based regenerative medicine therapeutics.

Nanosensors for regenerative medicine.

In fields such as cancer biology and regenerative medicine, obtaining information regarding cell bio-distribution, tropism, status, and other cellular functions are highly desired. Understanding cancer behaviors including metastasis is important for developing effective cancer treatments, while assessing the fate of therapeutic cells following implantation is critical to validate the efficacy and efficiency of the therapy. For visualization purposes with medical imaging modalities (e.g. magnetic resonance imaging), cells can be labeled with contrast agents (e.g. iron-oxide nanoparticles), which allows their identification from the surrounding environment. Despite the success of revealing cell biodistribution in vivo, most of the existing agents do not provide information about the status and functions of cells following transplantation. The emergence of aptamers, single-stranded RNA or DNA oligonucleotides of 15 to 60 bases in length, is a promising solution to address this need. When aptamers bind specifically to their cognate molecules, they undergo conformational changes which can be transduced into a change of imaging contrast (e.g. optical, magnetic resonance). Thus by monitoring this signal change, researchers can obtain information about the expression of the target molecules (e.g. mRNA, surface markers, cell metabolites), which offer clues regarding cell status/function in a non-invasive manner. In this review, we summarize recent efforts to utilize aptamers as biosensors for monitoring the status and function of transplanted cells. We focus on cancer cell tracking for cancer study, stem cell tracking for regenerative medicine, and immune cell (e.g. dendritic cells) tracking for immune therapy.

Aptamer technology for tracking cells' status & function.

Engineering cells with active-ingredient-loaded micro/nanoparticles is becoming increasingly popular for imaging and therapeutic applications. A critical yet inadequately addressed issue during its implementation concerns the significant number of particles that remain unbound following the engineering process, which inadvertently generate signals and impart transformative effects onto neighboring nontarget cells. Here we demonstrate that those unbound micro/nanoparticles remaining in solution can be efficiently separated from the particle-labeled cells by implementing a fast, continuous, and high-throughput Dean flow fractionation (DFF) microfluidic device. As proof-of-concept, we applied the DFF microfluidic device for buffer exchange to sort labeled suspension cells (THP-1) from unbound fluorescent dye and dye-loaded micro/nanoparticles. Compared to conventional centrifugation, the depletion efficiency of free dyes or particles was improved 20-fold and the mislabeling of nontarget bystander cells by free particles was minimized. The microfluidic device was adapted to further accommodate heterogeneous-sized mesenchymal stem cells (MSCs). Complete removal of unbound nanoparticles using DFF led to the usage of engineered MSCs without exerting off-target transformative effects on the functional properties of neighboring endothelial cells. Apart from its effectiveness in removing free particles, this strategy is also efficient and scalable. It could continuously process cell solutions with concentrations up to 10(7) cells·mL(-1) (cell densities commonly encountered during cell therapy) without observable loss of performance. Successful implementation of this technology is expected to pave the way for interference-free clinical application of micro/nanoparticle engineered cells.

Interference-free Micro/nanoparticle Cell Engineering by Use of High-Throughput Microfluidic Separation.

Nanoparticles are increasingly popular choices for labeling and tracking cells in biomedical applications such as cell therapy. However, all current types of nanoparticles fail to provide real-time, noninvasive monitoring of cell status and functions while often generating false positive signals. Herein, a nanosensor platform to track the real-time expression of specific biomarkers that correlate with cell status and functions is reported. Nanosensors are synthesized by encapsulating various sensor molecules within biodegradable polymeric nanoparticles. Upon intracellular entry, nanosensors reside within the cell cytoplasm, serving as a depot to continuously release sensor molecules for up to 30 days. In the absence of the target biomarkers, the released sensor molecules remain 'Off'. When the biomarker(s) is expressed, a detectable signal is generated (On). As a proof-of-concept, three nanosensor formulations were synthesized to monitor cell viability, secretion of nitric oxide, and β-actin mRNA expression.

A Nanoparticle-based Sensor Platform for Cell Tracking and Status/Function Assessment.

Graphene oxide (GO) is promising in the fight against pathogenic bacteria. However, the antibacterial activity of pristine GO is relatively low and concern over human cytotoxicity further limits its potential. This study demonstrates a general approach to address both issues. The developed approach synergistically combines the water shock treatment (i.e., a sudden decrease in environmental salinity) and the use of a biocompatible block copolymer (Pluronic F-127) as a synergist co-agent. Hypoosmotic stress induced by water shock makes gram-negative pathogens more susceptible to GO. Pluronic forms highly stable nanoassemblies with GO (Pluronic-GO) that can populate around bacterial envelopes favoring the interactions between GO and bacteria. The antibacterial activity of GO at a low concentration (50 μg mL(-1) ) increases from <30% to virtually complete killing (>99%) when complemented with water shock and Pluronic (5 mg mL(-1) ) at ≈2-2.5 h of exposure. Results suggest that the enhanced dispersion of GO and the osmotic pressure generated on bacterial envelopes by polymers together potentiate GO. Pluronic also significantly suppresses the toxicity of GO toward human fibroblast cells. Fundamentally, the results highlight the crucial role of physicochemical milieu in the antibacterial activity of GO. The demonstrated strategy has potentials for daily-life bacterial disinfection applications, as hypotonic Pluronic-GO mixture is both safe and effective.

Synergism of Water Shock and a Biocompatible Block Copolymer Potentiates the Antibacterial Activity of Graphene Oxide.

Assessing mesenchymal stem cell (MSC) differentiation status is crucial to verify therapeutic efficacy and optimize treatment procedures. Currently, this involves destructive methods including antibody-based protein detection and polymerase chain reaction gene analysis, or laborious and technically challenging genetic reporters. Development of noninvasive methods for real-time differentiation status assessment can greatly benefit MSC-based therapies. This report introduces a nanoparticle-based sensing platform that encapsulates two molecular beacon (MB) probes within the same biodegradable polymeric nanoparticles. One MB targets housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference, while another detects alkaline phosphatase (ALP), a functional biomarker. Following internalization, MBs are gradually released as the nanoparticle degrades. GAPDH MBs provide a stable reference signal throughout the monitoring period (18 days) regardless of differentiation induction. Meanwhile, ALP mRNA undergoes well-defined dynamics with peak expression observed during early stages of osteogenic differentiation. By normalizing ALP-MB signal with GAPDH-MB, changes in ALP expression can be monitored, to noninvasively validate osteogenic differentiation. As proof-of-concept, a dual-colored nanosensor is applied to validate MSC osteogenesis on 2D culture and polycaprolactone films containing osteo-inductive tricalcium phospate.

Nanosensors for Continuous and Noninvasive Monitoring of Mesenchymal Stem Cell Osteogenic Differentiation.

Non-invasive and longitudinal monitoring of gene expression in living cells is essential for understanding and monitoring cellular activities. Herein, a smart magnetic nanosensor is constructed for the real-time, non-invasive and longitudinal monitoring of cellular mRNA expression through the layer-by-layer deposition of molecular beacons (MBs) and polyethylenimine on the iron oxide nanoparticles. The loading of MBs, responsible for the signal intensity and the tracking time, was easily tuned with the number of layers incorporated. The idea was firstly demonstrated with the magnetic nanosensors for Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) mRNA, which was efficiently internalized into the cells under the influence of magnetic field. This nanosensor allowed the continuous monitoring of the cellular GAPDH mRNA expression for one month. Then, this platform was further utilized to incorporate two kinds of MBs for Alkaline Phosphatase (ALP) and GAPDH mRNAs respectively. The multifunctional nanosensors permitted the simultaneous monitoring of the reference gene (GAPDH mRNA) and the early osteogenic differentiation marker (ALP mRNA) expression. By taking the fluorescence signal ratio between ALP mRNA MBs and GAPDH mRNA MBs, the dynamic osteogenic differentiation process of MSCs was accurately monitored.

Smart Magnetic Nanosensors Synthesized through Layer-by-Layer Deposition of Molecular Beacons for Non-Invasive and Longitudinal Monitoring of Cellular mRNA.

Biocompatible nanoparticles are good candidates to label bacteria for imaging and diagnosis purposes. A high labeling efficiency reduces the concentration of nanoparticles required for labeling and allows the labeled bacteria to be tracked for longer periods. This report explores the optimal labeling strategy for Pseudomonas aeruginosa, a common gram-negative opportunistic pathogen, with quantum dots. Three strategies including direct incubation, calcium chloride treatment, and heat shock are compared and the labeling efficiency is assessed through fluorescence microscopy and flow cytometry analysis. Of the three, heat shock is finally selected due to its comparable labeling efficiency and simplicity. Through the assay of the respiration rate of bacteria together with morphology analysis, the heat shock process does not show any negative effect over the cells activity even at sub-toxic concentrations.

Heat shock mediated labelling of Pseudomonas aeruginosa with quantum dots.

Nanoparticles are emerging transdermal delivery systems. Their size and surface properties determine their efficacy and efficiency to penetrate through the skin layers. This work utilizes three-dimensional (3D) bioprinting technology to generate a simplified artificial skin model to rapidly screen nanoparticles for their transdermal penetration ability. Specifically, this model is built through layer-by-layer alternate printing of blank collagen hydrogel and fibroblasts. Through controlling valve on-time, the spacing between printing lines could be accurately tuned, which could enable modulation of cell infiltration in the future. To confirm the effectiveness of this platform, a 3D construct with one layer of fibroblasts sandwiched between two layers of collagen hydrogel is used to screen silica nanoparticles with different surface charges for their penetration ability, with positively charged nanoparticles demonstrating deeper penetration, consistent with the observation from an existing study involving living skin tissue.

Article	Year
Nanosensors for regenerative medicine. Journal of biomedical nanotechnology\| PubMed ID: 25992416	2014
Aptamer technology for tracking cells' status & function. Molecular and cellular therapies\| PubMed ID: 26056599	2014
Interference-free Micro/nanoparticle Cell Engineering by Use of High-Throughput Microfluidic Separation. ACS applied materials & interfaces\| PubMed ID: 26355568	2015
A Nanoparticle-based Sensor Platform for Cell Tracking and Status/Function Assessment. Scientific reports\| PubMed ID: 26440504	2015
Synergism of Water Shock and a Biocompatible Block Copolymer Potentiates the Antibacterial Activity of Graphene Oxide. Small (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 26707949	2016
Nanosensors for Continuous and Noninvasive Monitoring of Mesenchymal Stem Cell Osteogenic Differentiation. Small (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 26756453	2016
Smart Magnetic Nanosensors Synthesized through Layer-by-Layer Deposition of Molecular Beacons for Non-Invasive and Longitudinal Monitoring of Cellular mRNA. ACS applied materials & interfaces\| PubMed ID: 26878880	2016
Heat shock mediated labelling of Pseudomonas aeruginosa with quantum dots. Colloids and surfaces. B, Biointerfaces\| PubMed ID: 26962762	2016
Layer-by-Layer 3D Constructs of Fibroblasts in Hydrogel for Examining Transdermal Penetration Capability of Nanoparticles. Journal of laboratory automation\| PubMed ID: 27325107	2016

Christian Wiraja

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