Other commonly used fractionation is antibody based pulldown assay. This assay makes use of magnetic beads that are tagged with antibodies selectively targeting the subcellular compartments [ 29 ].
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An example is the use of TOM22 antibodies conjugated with magnetic beads for mitochondria isolation [ 30 , 31 ]. This principle is also used for post-nanoparticle labeling based fractionation. Here, organelle-specific antibody conjugated nanoparticles are used to target fractionated subcellular compartment.
This technique is largely used in isolating compartments that are larger in size and less dynamic or more static such as ER, Golgi, nucleus, mitochondria and lysosomes [ 32 ]. However, the applicability of this technique is limited as it cannot be used to isolate intact cell membrane. Many methods have used charge based affinity to isolate eukaryotic cell membrane [ 33 ]. The cell membrane is negatively charged due to presence of anionic components such as proteins, lipids and carbohydrates.
Hence, conventional method such as cationic latex or silica beads is used for isolating cell membrane.
By using poly-lysine for crosslinking silica beads, cell membranes are isolated as cross-linked membrane layers which are disadvantage for performing functional studies [ 34 ]. An alternative method that has been used is biotin-streptavidin affinity fractionation. The main principle behind biotin-streptavidin affinity assay is based on selective binding of available lysine residues on cell surface protein by biotin molecule which is further captured by streptavidin [ 35 ]. These streptavidin tagged micro beads or magnetic beads are used to pull down cell membrane protein or protein complexes from cell fractionation.
By using pulse-chase method elaborated in the later part of the paper , biotin-streptavidin affinity can be used to isolate early, intermediate and late endosomal compartments [ 36 ]. Recently, nanoparticle based fractionation has emerged as the strategy to isolate dynamic subcellular compartments like cell membrane, endosomes and lysosome by using endocytosis machinery. There are different categories of endocytosis Fig.
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One of the main subsets is phagocytosis that mainly involves cellular uptake of beads of size 1— microns using phagosome [ 37 , 38 ]. In macropinocytosis, there is non-specific cellular uptake, which is mainly utilized by several nanoparticles—cell interactions [ 43 ]. In clathrin mediated endocytosis, ligand coupled nanoparticle endocytosis through receptor mediated uptake mechanism [ 44 , 45 ]. However, when clathrin mediated endocytosis was blocked using sucrose, nanoparticle uptake was not limited, and thereby showing that there is size dependent cellular uptake of nanoparticle via caveolin mediated endocytosis [ 47 ].
Nevertheless it is clear that surface coating and size of the nanoparticle are governing factors for selective endocytosis and cellular uptake. The surface functionalization, size, physical properties and endocytosis machinery of nanoparticle are key factors for nanobiotechnology strategy. The physical properties are dependent on the type of core—shell material [ 48 , 49 ]. It is possible to govern the magnetic properties of the nanoparticle by using iron oxide or cobalt-iron oxide as a core. Shell material surface coating which acts as an interface between core and biological environment governs the use of nanoparticle for different biological applications.
Nanoparticles that are synthesized in organic phase tend to be water insoluble.
They require an additional step of exchange with water soluble ligands for biological applications. Commonly used methods for synthesizing nanoparticle are a chemical precipitation method and b thermal decomposition method [ 51 ]. Thermal decomposition method is preferred for its high monodispersity in terms of its size and high quality yield. However, it requires additional step for water-soluble ligand exchange or addition [ 52 , 53 ] Fig.
Surface functionalization of nanoparticle determines the kinetics behind nanoparticle-cellular uptake. Addition of PEG group in supports biocompatibility [ 54 ] and the nanoparticle can be further functionalized by coupling with the endgroup Fig. By tagging fluorescent ligand, it is possible to perform live cell imaging and nanoparticle tracking for studying the receptor-ligand and nanoparticle-cell interaction. Depending on the target, an appropriate ligand can be selected and conjugated to the nanoparticle [ 55 ]. Ligand size and shape determines the surface area to volume ratio which is the governing factor for nanoparticle functionalization [ 56 ].
Another key factor is ligand selection. Ligand selection depends— a receptor that can be well internalized for cellular trafficking ; b ratio of ligand: nanoparticle to avoid nanoparticle crosslinking. First approach is to use monovalent avidin and target biotinylated protein. However, major limitation for bioconjugation is nanoparticle aggregation due to coupling reagent like glutaraldehyde. Although glutaraldehyde works very well for protein conjugation and crosslinking, there are tendency for reagent to result in multiple layer crosslinking among nanoparticles due to non-specific interaction and competitive affinity.
Such multilayer crosslinking results in increase of size and change in physical properties, thereby affecting organelle isolation. Pulse-chase methodology is a commonly used approach to study the mechanism of endocytosis. Generally, pulse-chase strategy for omics analysis includes five stages or phases wherein Phase-I: includes generation of water-soluble nanoparticle by existing thermal decomposition or chemical precipitation synthesis and quality control using characterization; Phase II: includes selective bioconjugation of nanoparticle for a selective pathway-specific cellular uptake.
Phase III: Pulse-Chase methodology is used to optimize pulse and chase period to selectively localize nanoparticle in vesicle. Phase IV: Magnetic separation strategy is used for subcellular compartmental enrichment along with ultracentrifugation. Phase V: Endosomal proteome using Mass Spectrometry analysis.
This pulse-chase strategy was commonly used in radioactive labeling in the cell and this technique has now been extended to nanoparticle based subcellular compartmental isolation Fig. Briefly, pulse-chase strategy is used to govern receptor-mediated endocytosis of nanoparticle-ligand complex and has recently been extended to other endocytosis mechanisms [ 58 ]. This time frame allows nanoparticle to interact with the cell surface and its protein. Depending on whether the cells are adherent or in suspension, or it is receptor mediated or charge mediated, there is variation in the pulse time period required for cellular uptake [ 59 ].
After pulse period is performed, the chase is incubated at appropriate time period depending on the compartmental isolation. Chase period represents the time where the nanoparticle containing medium is replaced with fresh medium without nanoparticle. This supports streamlining nanoparticle internalization in the cell and accumulation of nanoparticle into a certain compartment of interest depending on the timeframe. However since endocytosis is dynamic in mechanism, it is relatively difficult to isolate highly pure early and late endosomes Fig.
This is mainly because lysosome is the endpoint for most of the endocytosis [ 60 ]. An advantage of using chase period is that it provides useful information for nanoparticle tracking. For this reason, fluorescence tagged nanoparticle is used for pulse—chase methodology and live-cell imaging [ 61 ]. By incubating with endocytic inhibitors for endosomal or lysosomal fusion, it is possible to limit the nanoparticle-cellular internalization and subcellular trafficking. For example, by limiting the endosome-lysosomal fusion using Latrunculin-A, it is possible to concentrate the nanoparticle in early or late endosomes [ 62 ].
It is also reported that the nanoparticle coupled ligand does not mimic the ligand cellular uptake and subcellular compartment localization. Few examples in the later part of this article elaborate on the deviation in cellular uptake. These examples are confirmed by using pulse-chase methodology, magnetic organelle isolation and live cell imaging using fluorescence tag [ 63 ]. For optimal use of pulse-chase method, it is important to establish a methodology for specific subcellular compartments.
Here, we describe two interesting nanoparticle based methodologies to isolate plasma membrane and endosomal compartments using affinity purification. After incubation, the nanoparticle containing supernatant is removed and washed twice with fresh ice-cold PBS. Detachment of cells using trypsin is not recommended as it might affect cell surface proteins which this method aims to isolate. Cell suspension is further homogenized using a homogenizing apparatus and buffers that maintain physiological conditions. The post nuclear fraction is passed through the magnetic field.
An additional washing step with homogenizing buffer can be included to further clear the unbound material. Further, the magnetic field is removed and bound fraction is eluted from the column. The pellet is resuspended in an appropriate amount of PBS for further analysis like mass spectrometry. Cell suspension is further homogenized as explained in the previous section. The post nuclear fraction is passed on the column in presence of magnetic field.
Here, unbound fraction is eluted while the magnetic fraction is further washed with the homogenizing buffer to further clear unbound material.
This is followed by the removal of magnetic field and elution of the bound fraction from the column. The pellet is resuspended in PBS for further analysis. Although nanoparticle-protein complex can be used for endosomal trafficking and for proteomics, there are illustrations, which show that nanoparticle-protein complexes are trafficked differently compared to the target protein complex.
For example, trafficking of ricin conjugated nanoparticle is reported to be unlike the ricin ligand where trafficking occurs from early endosomes EE , trans-Golgi network TGN and finally to endoplasmic reticulum ER. It is well known that transferrin is recycled to the cell surface via recycling endosomal compartments such as recycling endosome via early endosomes and multi-vesicular bodies. However, transferrin conjugated nanoparticle and Shiga toxin ricin conjugated nanoparticle are shown to traffic from early endosomes to late endosomes and finally accumulated at lysosomes.
It is also reported the Shiga toxin conjugated nanoparticle tend to accumulate at early endosomes while Shiga toxin traffic like ricin from EE to TGN and finally to ER. Both tables show that nanoparticle based methods hold many advantages as compared to existing methods. Using the isolation technology, several omics datasets for subcellular compartments can be generated for any given cell. There is an interesting aspect in the use of nanoparticle based method that is generic in nature.
Designer nanoparticle: nanobiotechnology tool for cell biology
Hence, the method can be applied to wild-type and diseased cell-type for example cancer cell for plasma membrane and endosomal compartmental isolation. By using the nanoparticle based subcellular compartmental isolation; one could potentially generate a complete and comprehensive plasma membrane or endosome or lysosome proteomics, glycomics and lipidomics for any cell type.
By the generated subcellular omics, nanobiotechnology can serve as a useful tool to build omics datasets for cancer biology using the bottom-up pyramid approach. Using the bottom-up pyramid approach in omics analysis, subcellular omics datasets including genomics, proteomics, lipidomics and glycomics can be compiled together and compared with the whole cell omics analysis.
This approach can also be used to generate several omics datasets in cancer biology that can enable the researchers to revisit the subcellular omics in order to understand the biological significance and functional relevance. For all such omics analysis studies, it is necessary to have an efficient, robust and high precision technology for subcellular compartmental isolation. The technology also needs optimization and fine tuning depending on its applicability with host cell system.
Using different nanobiotechnology tools for subcellular compartmental isolation, several high-throughput functional omics dataset like fluxomics, metabolomics, interactomics and localizomics can be generated. Using all these datasets, comprehensive Phenome and subcellular omics are generated that can by analyzed using nanotechnology for data storage studies. Further dataset thus gets larger for different diseases such as cancer, diabetes, infectious diseases, ageing related diseases, and neurodegenerative diseases. These dataset and nanotechnology based analytics can be used in drug development, pre-clinical studies, patent analytics, and other applications.
As a future perspective, the use of nanoparticle as nanobiotechnology tool is all set to be a game changer in the generation of Datasets for systems biology.