Surface functionalization dependent subcellular localization of Superparamagnetic nanoparticle in plasma membrane and endosome
In this article, we elaborate the application of thermal decomposition based synthesis of Fe3O4 superparamagnetic nanoparticle (SPMNP) in subcellular fractionation context. Here, we performed surface functionalization of SPMNP with phospholipids and dimercaptosuccinic acid. Surprisingly, we observed surface functionalization dependent SPMNP localization in subcellular compartments such as plasma membrane, endosomes and lysosomes. By using SPMNP based subcellular localization with pulse–chase methodology, we could use SPMNP for high pure-high yield organelle (plasma membrane, endosomes and lysosome) fractionation. Further, SPMNP that are distinctly localized in subcellular compartments can be used as technology for subcellular fractionation that can complement existing tools for cell biology research. As a future perspective, isolated magnetic organelles can be extended to protein/protein complex purification for biochemical and structural biology studies.
KeywordsSuperparamagnetic nanoparticle (SPMNP) Plasma membrane Early or late endosomes Lysosomes
Plasma membrane, endosomes (early or late), and lysosomes are dynamic organelles that are known for their key role in cellular function such as cell signaling [1, 2, 3], intracellular [4, 5] and extracellular interaction [6, 7]. Interestingly, most drugs and therapeutic small molecules require cellular interaction for their biological action, and it is well known that high affinity with the cell surface results in effective mechanisms . Several drug candidates for cancer, neurodegenerative and metabolic diseases predominately interact with the proteins at plasma membrane, endosomes and lysosomes . For example, gamma-secretase protein complexes that play a key role in neurodegenerative disease are predominately localized in plasma membrane and endo-lysosomal compartments . Gamma-secretase substrate cleavage activity occurs at the cell surface and endosomal compartments . Hence, it is essential to identify proteins that interact with gamma secretase complex in those subcellular compartments during drug treatment or other perturbations. In order to identify such subtle mechanism, it is essential to characterize the biomolecular composition of plasma membrane, endosomes and lysosomes [12, 13, 14]. This is due to the dynamic nature of protein and lipid composition in a cell. For instance, 90% of cholesterol is present at the cell surface in normal cells and it has been observed that majority of the cholesterol is internalized in Presenilin double knockout (PSENdKO) cell lines . Hence, it is essential to isolate organelles such as plasma membrane, endosomes and lysosomes in order to identify subtle changes in protein or lipid composition compared to whole cell . Such an approach will facilitate the analysis of the composition and dynamics of subcellular compartments upon drug exposure. Major limiting factor in determining the biomolecular composition of these dynamic organelles are the difficulties in isolating these organelles with high purity and high yield . There are several existing methodologies such as density gradient centrifugation , antibody based immuno-precipitation , cationic silica based fractionation [20, 21] and streptavidin based magnetic fractionation [22, 23] that are used to isolate organelles such as plasma membrane with varied purity and yield. Similarly, nanoparticle based fractionation ; density gradient centrifugation  and antibody based immunoprecipitation  are used to isolate dynamic organelles such as endosome and lysosomes with limited purity. However, biomolecular composition of plasma membrane, endosomes and lysosomes are still incomplete for several cell types . SPMNPs based subcellular fractionation is an effective and simple methodology for isolating plasma membrane, early endosomes, late endosomes and lysosomes in any given eukaryotic cell . SPMNP based subcellular fractionation is considered to be superior as organelles are isolated under native physiological conditions devoid of detergents or acidic condition . In addition, SPMNP based subcellular fractionation are generic and can be applied to any living eukaryotic cell . However, synthesis and surface functionalization of SPMNP plays key role in its application. In this article, we show that thermal decomposition synthesis based SPMNP can be localized in two different subcellular compartments using two types of surface functionalization. We particularly show that phospholipid functionalized SPMNPs and dimercaptosuccinic acid (DMSA) functionalized SPMNPs can be used for plasma membrane and endosomal enrichment respectively. In addition, we present methodologies for thermal decomposition based SPMNP synthesis, surface functionalization of SPMNPs, bio-conjugation of SPMNPs with fluorescent phospholipid or by fluorescein-5-maleimide, pulse–chase paradigm and magnetic fractionation for subcellular organelles (plasma membrane, endosomes and lysosomes). As a proof of concept, here we focus on use of SPMNPs and its pulse chase dependent subcellular localization for subcellular fractionation in HeLa cells.
2 Experimental section
2.1 Thermal decomposition based synthesis of SPMNP
Using Sun and Zeng’s protocol , Magnetite (Fe3O4) nanoparticles with size from 3 to 20 nm can be produced using simple organic-phase synthesis. We prepared 6 nm Fe3O4 SPMNP by mixing 2 mmol of iron (III) acetylacetonate with 10 mmol of 1,2-hexadecanediol (10 mmol), 6 mmol of oleic acid and 6 mmol of oleyl amine in 20 ml of benzyl ether. We then magnetically stirred and heated the mixture to 200 °C for 2 h under N2 flow for synthesizing SPMNP seed. Reflux at 300 °C for 1 h resulted in SPMNP seeds growing to 6 nM SPMNP. After cooling to room temperature, we added ethanol and separated dark brown precipitate using magnet. We then dissolved dark brown material in hexane and centrifuged at 1000 rpm for 5 min to remove aggregates. Using phospholipids or DMSA as ligand, water dispersible SPMNP are generated in controlled fashion by retaining monodispersity and superparamagnetic properties.
2.2 Phospholipid based surface functionalization of SPMNP
Based on Dubertret et al protocol , we performed phospholipid (Pl) functionalization on SPMNP by ligand addition to generate Pl-SPMNP. Briefly we mixed 1:2 ratio of SPMNP with PEG functionalized Phospholipids in chloroform and vortexed for 4 h. The above reaction mixture was gently mixed and incubated for 3 h with gentle and continuous vortexing. After evaporating the chloroform and drying the particles using nitrogen gas, we dissolved the pellet in water (1 ml) by gentle shaking of the sample. We then centrifuged the above reaction mixture for 10 min at 5000 rpm at 21 °C in order to remove the major aggregates. Finally, we extracted the supernatant in a new 1.5 ml centrifuge tube and centrifuged it for 1 h at 5000 rpm at 21 °C in order to get monodispersity.
2.3 DMSA based surface functionalization of SPMNP
Using Thimiri Govinda Raj et al protocol , we performed ligand exchange on oleic acid functionalized SPMNP using DMSA as ligand to generate DMSA-SPMNP. We mixed 1:200 ratio of SPMNP with DMSA in 1:1 toluene and dimethyl sulfoxide (DMSO) solution. Then reaction mixture was vortexed for 48 h at room temperature. After 48 h, the black precipitate was pelleted through centrifugation at 5000 rpm for 10 min. Using glass pipette, the toluene and DMSA solvent mixture was discarded. The pellet was resuspended in appropriate volume of water by vigorous sonication. Finally SPMNP in water was adjusted to pH 7.
2.4 TMAOH and COO-TMACl based surface functionalization of SPMNP
Using Thimiri Govinda Raj et al protocol , we performed ligand exchange on oleic acid functionalized SPMNP using TMAOH and COO-TMACl as ligand to generate TMAOH or and COO-TMACl-SPMNP. We mixed 1:200 ratio of SPMNP with TMAOH or COO-TMACl in 1:1 toluene and methanol solution. Then reaction mixture was vortexed for 48 h at room temperature. After 48 h, the black precipitate was pelleted through centrifugation at 5000 rpm for 10 min. Using glass pipette, the toluene solvent mixture was discarded. The pellet was resuspended in appropriate volume of water by vigorous sonication. Finally SPMNP in water was adjusted to pH 7.
2.5 Generation of fluorescent SPMNP
We used two strategies to generate fluorescent SPMNP depending on the ligand functionalization (a) fluorescent Pl-SPMNP: 1:1:4 ratio of oleic acid functionalized SPMNP with PEG functionalized Phospholipids and fluorescent end grouped PEG functionalized Phospholipids respectively in chloroform was mixed. (b) Maleimide linker based conjugation of SPMNP : 1:25 molar ratio of SPMNP with fluorescein-5-maleimide in water at pH 7.0 was mixed. The reaction mixture was incubated in mild shaking for overnight at 4 °C. Using magnetic separation, unconjugated fluorescein-5-maleimide was removed. The magnetically purified fluorescein conjugated SPMNPs was resuspended in water at pH 7.0.
2.6 Subcellular barcoding—choice of SPMNP for pulse–chase paradigm
In order to isolate subcellular organelles, it is critical to select appropriate SPMNP that selectively localize at subcellular compartments. To select SPMNP that are cell surface bound or internalized in cell, magnetic cell isolation protocol with different pulse and chase conditions are to be performed. Adherent mammalian cells (ex: HeLa, MEFs) were incubated with different concentration of SPMNPs in medium at 4 or 37 °C for 15 min as pulse period. The cells were trypsinized and harvested at 1000 rpm for 10 min. Magnetic cell isolation was performed using the protocol for magnetic separation from Miltenyi Biotec.
2.7 Prussian blue staining
Adherent mammalian cells to 75% confluence were cultured in 6 well plates with coverslips. Further cells were incubated at 37 °C with SPMNPs for pulse period. After washing with PBS, cells were incubated at 37 °C in fresh medium devoid of SPMNP for chase period (0 and 240 min). Then cells were washed with ice-cold PBS three times and fixed with paraformaldehyde. Cells were incubated with fresh reagent- potassium ferrocyanide (2%): hydrochloric acid (2%) = 1:1 for 10 min and repeated with the fresh reagent. The cells were then rinsed with distilled water, counter stained with eosin for 30 min and washed again with tap water. The SPMNP (blue staining) localization was captured using light microscope.
2.8 Confocal analysis
Adherent mammalian cells to 75% confluence were cultured in 6 well plates with coverslips. Further cells were incubated at 37 °C with SPMNPs for pulse period. After washing with PBS and cell were incubated cells at 37 °C in fresh medium with lysotracker devoid of SPMNP for chase period (0 and 240 min). Then cells were washed with ice-cold PBS three times and fixed with paraformaldehyde. Cells were rinsed with PBS and SPMNP localization was captured using confocal microscope.
2.9 Western blot analysis on SPMNP based subcellular fractionation
Adherent mammalian cells to 75% confluence were cultured in 75 cm2 flask or in 8 × 10 cm culture-dishes at 37 °C and washed three times with warm PBS. The cells were then incubated with SPMNP in medium (0.2 mg/ml) for pulse time of 15 min at 37 °C in a mild shaking platform. To capture plasma membrane, cells with NH2-lipid-SPMNP in PBS (2 mg/ml) were incubated for pulse time of 15 min at 4 °C. Excess SPMNPs were removed by washing the cells with PBS, and a chase period of 0–15 h with fresh medium at 37 °C was performed (chase period—0 min to capture plasma membrane, chase period—15 min to 2 h to isolate endosomes and chase period 2–15 h to isolate lysosomes). The cells were harvested in PBS by centrifuging at 1000 rpm for 10 min. The cell pellet was resuspended in homogenizing buffer and homogenized with ball-bearing cell cracker (20 passages, clearance 10 or 15 µm). The cell debris was pelleted by centrifuging at 200g for 10 min and the supernatant as post nuclear supernatant fraction (PNS) was retained. The PNS was loaded in a PBS equilibrated LS column in the presence of magnetic field. The non-magnetic unbound fraction was aliquoted for quality control and western blot analysis. The column was washed in the presence of magnetic field with homogenizing buffer for three times. The bound fraction was removed by the magnetic field and using the plunger. The bound fraction was pelleted using ultracentrifugation in 55,000 rpm for 1 h at 4 °C. Western blot analysis was performed to study the magnetic fraction.
3 Result and discussion
3.1 Superparamagnetic nanoparticle (SPMNP) synthesis and surface functionalization
3.2 SPMNP-cell interaction
3.3 Subcellular fractionation
DBTGR performed all the major experiments with technical support from Anna University Trichy. DBTGR wrote the manuscript. Both authors read and approved final manuscript.
Figures are made through Servier Medical Art. The author thanks for funding from Not for Profit firms Envirotransgene® Biosolutions Global and Deepak Thimiri Consulting Group (DTCG®) Grenoble, France/Chennai, India. Authors thank Anna University Trichy, Chennai for technical help and instrumentation usage.
The authors declare that they have no competing interests. Deepak B. Thimiri Govinda Raj is an inventor of Plasma membrane isolation and Biarsenical nanoparticles for in vivo labeling in patent. Deepak B. Thimiri Govinda Raj is a consultant for Envirotransgene® Biosolutions Global and Deepak Thimiri Consulting Group (DTCG®).
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