Quantum Dot Based Designed Nanoprobe for Imaging Lipid Droplet

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Quantum Dot Based Designed Nanoprobe for Imaging Lipid Droplet Suman Mandal, and Nikhil R. Jana J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b07571 • Publication Date (Web): 03 Oct 2017 Downloaded from http://pubs.acs.org on October 4, 2017

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The Journal of Physical Chemistry

Quantum Dot Based Designed Nanoprobe for Imaging Lipid Droplet Suman Mandal and Nikhil R. Jana* Centre for Advanced Materials, Indian Association for the Cultivation of Science, 2A & 2B Raja S. C. Mullick Road, Kolkata-700032, India *Corresponding author. E-mail: [email protected].

1 ACS Paragon Plus Environment


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ABSTRACT. Lipid droplet is a dynamic sub-cellular organelle and its imaging is important for understanding their functional role toward the cellular scale origin of many diseases. However, currently available imaging probes are inefficient in monitoring their activity for longer time scale. Here we have designed a quantum dot based fluorescent nanoprobe that can be used to image lipid droplet for longer time. Nanoprobe has quantum dot core, polymer shell with zwitterionic surface charge and optimum lipophilic functionality. The nanoprobe has 30-40 nm hydrodynamic size with near zero surface charge at physiological pH, modular interaction with the cell membrane and enters into cell via predominate lipid-raft endocytosis and label lipid droplets. Presented nanoprobe can be used for understanding the functional role of lipid droplet towards various cellular functions.


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INTRODUCTION Lipid droplet is a dynamic sub-cellular organelle made of neutral lipid core and their surface is decorated with different proteins that are involved in lipid metabolism.1-3 The size of lipid droplet may vary from micrometer in adipocytes to nanometer in non-adipocyte cells. Lipid droplet is responsible for intracellular lipid storage, lipid metabolism and associated with various diseases or metabolic disorders such as obesity, cancer, diabetes, cardiovascular diseases, hypertension.1-3 With the increasing evidence of multifunctional role of lipid droplet, emphasis is given for monitoring of lipid droplet and its activity inside cell.4-7 Different approaches are developed for monitoring of lipid droplets that include immunohistochemistry of lipid droplet associated protein,6 Raman imaging8 and fluorescence imaging.9-17 Among them fluorescence imaging based monitoring of lipid droplet is most popular owing to the high sensitivity and simplicity.9-17 Several important fluorescent probes are reported for lipid droplets that include nile red,9 BODIPY dye,10 Seoul-Fluor11 and aggregation induced emission based molecular probes.12,15,16 Although these probes are routinely used and substantially enhance the understanding of the functional role of lipid droplets, they have limitations that need to be overcome. Limitations include photobleaching issue that restricts imaging of lipid droplet for more than a minute, small Stokes shift that causes self-absorption, broad emission, narrow emission window and low specificity.4-7 Aggregation induced emission based probes can solve the photobleaching issues but other issues remain mostly unresolved.12,15,16 In that respect quantum dot (QD) based fluorescent probe can be interesting as it has high photostability, large Stokes shift, narrow and tunable emission and wider excitation window (UV, blue and green excitation).18,19 However, designing QD based nanoprobe for lipid droplet is a challenging issue as it requires sub-cellular targeting property but the uptake of nanoprobe, that occurs via endocytosis, usually traffics them to endosome/lysosome.20



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We work on the development of nanoparticle-based imaging probe for cell and sub-cellular compartments.20-24 We have shown the importance of zwitterionic surface charge in modular cell-nanoparticle interaction and in controlling cellular endocytosis of nanoparticle.21 We have designed nanoprobe with appropriate surface chemistry in order to minimize clathrinmediated endocytosis and to minimize their endosomal/lysosomal trafficking.21-24 Using this principle we have designed nanoprobe that enters the cell via lipid-raft or caveolae-mediated endocytosis and label mitochondria,22 Golgi apparatus24 or traffics towards the perinuclear region.23 We envision that lipid droplet imaging probe may be designed using zwitterioniclipophilic nanoprobe. The zwitterionic-lipophilic nanoprobe should have modular interaction with cell membrane and specific interaction with the lipid-raft region of the membrane to induce

the

lipid-raft

endocytosis

followed

by

sub-cellular

trafficking

without

endosomal/lysosomal entrapment. Additionally, lipophilic character of nanoprobe would offer strong interaction with the lipid droplet and labelling of such sub-cellular compartment. Here we have designed QD based nanoprobe for labeling of lipid droplet. The nanoprobe is zwitterionic in nature with lipophilic chemical functional groups. The zwitterionic functional groups offer near zero surface charge at physiological pH with modular interaction with the cell membrane and lipophilic functional groups offer selective interaction with the lipid-raft region of the cell membrane, inducing lipid-raft endocytosis. Nanoprobe selectively labels lipid droplet inside the cell without trafficking to endosome/lysosome. Compared to earlier reported lipid droplet imaging probe this probe is stable to continuous light exposure and can be used to monitor lipid droplet for longer time with the conventionally used fluorescence microscope via blue and green excitation. The efficiency of designed nanoprobe is determined by labeling/imaging lipid droplets in two different cell lines and using octyl or oleyl functionalized LQD and for each system labeling


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has been reproduced for > 10 times. The proposed nanoprobe design can be extended for specific delivery of drug to lipid droplet and regulating their biogenesis. EXPERIMENTAL SECTION Chemicals.

Poly(ethylene

glycol)

methacrylate,

3-sulfopropyl

methacrylate,

N,N-

methylenebis(acrylamide), octylamine, oleic acid, sodium borohydride, glutaraldehyde solution, cadmium oxide, trioctyl phosphine, trioctyl phosphine oxide, stearic acid, zinc stearate, sulfur powder, selenium powder, chlorpromazine hydrochloride, genistein, amiloride hydrochloride, methyl-β-cyclodextrin and nile red were purchased from Sigma-Aldrich and used as received. N-(3-aminopropyl) methacrylamide hydrochloride was purchased from Polyscience and used as received. Folate containing Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Sigma-Aldrich. Hoechst and Lysotracker red were purchased from Life Technology. Synthesis of QD nanoprobe. Hydrophobic red and green emissive QD (CdSe/ZnS) was synthesized by a previously reported method.22 Briefly, red emissive CdSe nanocrystal was synthesized at 280 °C in 1-octadecene solvent, and then ZnS shelling was performed at 220 °C. This hydrophobic QD was converted to hydrophilic QD (HQD) by polyacrylate coating following our previously reported method.21,22 Here three different acryl monomers were mainly used together to prepare hydrophilic QD such as poly(ethylene glycol) methacrylate, (N-(3-aminopropyl) methacrylamide and 3-sulfopropyl methacrylate. Lipophilic QD (LQD) was derived from HQD by covalent conjugation with octylamine via glutaraldehyde coupling. Typically, an equimolar mixture of glutaraldehyde and octylamine (or oleylamine) was added to the colloidal solution of HQD, and after one hour NaBH4 was added to reduce the imine bond and the reaction was continued for another 4 h. Next, the solution was dialyzed (with a molecular weight cut-off membrane of 12 000 Da) to remove the free



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reactants. In addition to that unreacted octylamine was removed via repeated extraction with chloroform. Estimation of the number of octyl groups per LQD. The number of octyl (or oleyl) groups per LQD was estimated by our reported method. In brief, the concentration of QD in LQD was measured by using the molar extinction coefficient of QD. Next, LQD was treated with HCl to dissolve the QD and neutralized by adding NaOH. Then fluorescamine based quantitative determination of free primary amine was done before and after conjugation with octyl group. The difference in primary amine concentration, before and after octylamine conjugation, was then correlated with the extent of octyl conjugation. Next, octyl groups per LQD were calculated using concentration ratio of QD and octyl group present in LQD. Imaging of lipid droplet using QD probe. HeLa and CHO cells were cultured in DMEM with 10 % heat inactivated fetal bovine serum (FBS) and 1% penicillin streptomycin at 37 °C and 5 % CO2 atmosphere. For the fluorescence microscopic study, cells were cultured overnight in a 4 well chamber slide with one mL medium. In order to induce lipid droplet, cells were incubated with the oleic acid solution for 6 h. Typically, 10 µL DMSO solution of oleic acid (4 mM) was added to one mL culture medium containing cells. After 6 h media was removed, washed with PBS buffer of pH 7.4 and incubated with fresh media. Next, cells with one mL culture media were incubated with 20-30 µL of QD nanoprobe solution for 1-2 h. In some control samples, this was followed by 2 µL DMSO solution of nile red (0.3 mM) for 5 min. After incubation, the medium was removed and washed with PBS buffer of pH 7.4 and incubated with fresh culture medium for next 1−24 h. The cells were imaged under the fluorescence microscope using appropriate excitation. Photobleaching and colocalization study. Lipid droplet induced cells were incubated with LQD for one hour. Next, cells were carefully washed with PBS buffer and fresh medium was added. Next, cells were incubated with nile red for 5 min and washed cells were again


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incubated with Hoechst dye for 20 min (for nucleus staining). Next, media was discarded, washed and 4 % paraformaldehyde fixative was added for 30 min. The fixed cells on chamber slides were imaged under green excitation (at 532 nm with 23 mw/cm2 power), blue excitation (at 495 nm with 26.8 mw/cm2 power) and UV excitation (at 352 nm) successively for nile red, LQD, Hoechst imaging. Photobleaching experiment was done by exciting LQD/nile red in their corresponding wavelength for 5 min and taking snapshots at 10-15 sec interval. Endocytosis mechanism study. Cells in DMEM medium were incubated with different endocytosis inhibitors for 45 min. Details of incubation time and dose were mentioned in Supporting Information, Table S1. Next, 30 µL LQD solution was added and the mixture was further incubated for 1 h. The cells were then washed with PBS buffer thrice to remove unbound LQD from the cell surface. Next, cells were treated with 100 µL trypsin-EDTA for 2 min and detached cells were isolated by centrifuge. Finally, cells were dispersed in PBS buffer and used for flow cytometric measurements. Typically, 200 µL of QD labeled dispersed cells of 0.1−0.5 million in number were used for each set. Cell viability study using MTT assay. HeLa cells were cultured in a 24 well plate in DMEM media. After that, cells were treated with different doses of samples for 24 h. After that cells were washed repeatedly with PBS buffer and fresh DMEM media was added. Next, each well with attached cells was treated with 50 µL of freshly prepared methyl thiazolyldiphenyl-tetrazolium bromide (MTT) solution (4-5 mg/mL) and incubated for 4 h. Then the supernatant was removed carefully leaving the formazan in the plate. This formazan was dissolved in SDS solution (8 g of SDS dissolved in 40 mL of DMF/H2O mixture), and absorbance was measured at 570 nm. Cell viability was measured assuming 100 % viability for control sample without any nanoparticle.



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Instrumentation. UV−visible absorption and fluorescence spectral studies were carried out with a Shimadzu UV-2550 UV-visible spectrophotometer and BioTek Synergy Mx microplate reader, respectively. Samples for the transmission electron microscopic (TEM) study were prepared by putting a drop of particle dispersion on carbon-coated copper grid, dried in air and observed with FEI Tecnai G2 F20 microscope using 200 kV electron source. The zeta potential and dynamic light scattering (DLS) were measured by Malvern Nano ZS instrument by taking dilute solution of nanoparticle in three different buffer solutions of pH 4.5, 7.4, and 9.0. Differential interference contrast and fluorescence images of live cells were performed using Olympus IX81 and Zeiss apotome microscope using image-pro plus version 7.0 software. Flow cytometry was studied using a BD Accuri C6 flow cytometer.

RESULTS AND DISCUSSION Design and synthesis of lipophilic-zwitterionic quantum dot (LQD). Design and synthetic steps of lipophilic-zwitterionic quantum dot (LQD) are shown in Scheme 1. The LQD has red emitting QD core and polyacrylate shell with cationic, anionic and lipophilic functional groups. Synthesis of LQD involves preparation of hydrophobic QD nanoparticle, followed by their transformation into polyacrylate coated hydrophilic-zwitterionic QD (HQD) and finally functionalization with octylamine to get LQD. At first red emitting CdSe quantum dot is synthesized and capped with ZnS shell via high temperature organometallic approach.22 This ZnS capped CdSe is hydrophobic in nature due to hydrophobic surfactant capping. Next, they are transformed to polyacrylate coated hydrophilic quantum dot (HQD) using the reverse micelle-based approach.21-24 Different acrylate monomers are used to prepare HQD that offers good water solubility, zwitterionic surface charge and minimum non-specific interaction with the biological environment. We have used poly(ethylene glycol)


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methacrylate, 3-sulfopropyl methacrylate and N-(3-aminopropyl) methacrylamide that provide polyethylene glycol groups, SO3− groups,

primary amine groups, respectively.

Polyethylene glycol functional groups minimize the nonspecific binding with cellular environment, SO3− groups provide anionic surface charge, primary amines provide pH dependent cationic surface charge. The ratio of acrylate monomers is optimized (sulfopropyl methacrylate to aminopropyl methacrylamide as 1:2) in order to make the HQD with effective zwitterionic surface charge. Octylamine (or oleylamine) is then linked to HQD via glutaraldehyde coupling and subsequent reduction of imine bonds by sodium borohydride. Purification of the as-synthesized LQD is done by solvent extraction method using chloroform to remove hydrophobic ligands. Finally, extensive dialysis has been performed to remove free water soluble ligands. Presence of octyl (or oleyl) ligand on LQD surface has been characterized by FTIR and NMR spectroscopy. Additional vibrational band arises for LQD at 2822 cm-1 due to CH stretching frequency of octyl groups. (Figure 1d) Solution phase proton NMR spectroscopy also shows broad chemical shift around 2.2 ppm for long aliphatic chain protons. (Supporting Information, Figure S1) Presence of primary amines in LQD/HQD has been estimated via fluorescamine test and the number of primary amines per QD has been determined for both HQD and LQD. (see Experimental section and Supporting Information, Figure S2) The average number of primary amines per QD appears as ~550 for HQD and ~300 for LQD and the difference 250 is assumed due to consumption of primary amines for octyl conjugation. Considering the 1: 2 molar ratio of sulfopropyl methacrylate to aminopropyl methacrylamide used during polyacrylate coating and about 50 % of primary amines of coated nanoparticles are used for octyl (or oleyl) conjugation, about same numbers of sulfopropyl and primary amine groups exist in final nanoprobe.



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Physicochemical properties of LQD have been characterized by UV-visible, DLS, TEM, FTIR and NMR. (Figure 1, Supporting Information, Figure S1, S2) LQD shows typical absorption and emission spectra due to QD core. TEM image reveals the inorganic nanoparticle core of about 5-6 nm size. Hydrodynamic size of LQD is in the range of 30-40 nm which is slightly larger than HQD. The zwitterionic surface charge of HQD and LQD has been determined by measuring the Zeta potentials of their colloidal solutions at different solution pH. (Table 1) The Zeta potential values changes from positive to negative (typically from +15 mV to -24 mV) as the solution pH increased from 4.5 to 10.0 and the surface charge of LQD becomes near zero value at pH 7.4. The anionic surface charge can be explained due to sulfopropyl group (SO3-) that provide an anionic charge at all the tested pH. However, primary amines provide pH-dependent protonation (to produce ammonium cation) and the extent of protonation increases with decreasing pH. Thus Zeta potential becomes positive at pH 4.5 due to dominant cationic ammonium groups and at pH 7.4, the SO3- and ammonium cations counterbalance each other with resultant zero charge. Imaging lipid droplet by LQD. Cell labeling and lipid droplets labeling studies have been performed extensively using HeLa cell line. In addition, CHO cells have also been used in some selected cases. Cells are cultured overnight in oleic acid-supplemented medium for adequate lipid droplet induction inside cells. Next, cells are incubated with colloidal solution of LQD for adequate time (typically 6-12 h) to label cells followed by washing to remove unbound particles and then imaged under the fluorescence microscope. In control experiments, nile red is used to label lipid droplets, which is most commonly used fluorescent probe for lipid droplet. Results are summarized in Figure 2 and Figure 3. It shows that nile red labeled cells show the clear image of lipid droplets of 100-500 nm size, uniformly distributed in the cytoplasm and similar type labeling of lipid droplet is also observed by LQD labelled cells. In contrast, HQDs are concentrated in one side of the cell nucleus and


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unable to label lipid droplets. Control experiments show that if cells are not supplemented with oleic acid, such type of lipid droplet images is absent. In such case nile red shows homogeneous distribution all over the cells, LQDs shows perinuclear distribution and HQD shows insignificant labeling. (Supporting Information, Figure S3) A co-localization study has been performed using green fluorescent LQD (with blue excitation, 450-495 nm range) and red fluorescent nile red (with green excitation, 500-530 nm range) and merged images show that LQDs predominantly localize with nile red. (Figure 4) In order to investigate the general applicability of this approach, we have synthesized oleyl functionalized lipophilic QD for lipid droplet imaging. In addition, LQD based lipid droplet imaging has been extended to other cells. Figure 5 shows that oleyl functionalized LQD can label lipid droplet similar to octyl functionalized LQD. We have used oleic acid supplemented CHO cells for labeling of lipid droplet by LQD. Figure 6 clearly shows labeling of lipid droplet in CHO cells within 12 h of incubation. LQD enters the cell via lipid-raft endocytosis. Lipid droplet labeling mechanism by LQD has been investigated using time-dependent trafficking of LQD inside the cell, uptake study in presence of different endocytosis inhibitors and comparing with HQD uptake/localization. At first we show that cellular labelling is insignificant at 4 °C, suggesting the involvement of energy dependent processes. (Supporting Information, Figure S4) Next, time-dependent localization study of LQD/HQD has been performed using HeLa cells without oleic acid supplementation. Results show that HQDs enter into cell within 4 h, then localize inside cytoplasm but in next 15 h they exit from the cell possibly via exocytosis. (Supporting Information, Figure S5) In contrast LQD enter into cell within 2 h, localize inside cytoplasm within 4 h and stay longer inside the cell. (Supporting Information, Figure S6) Colocalization study of HQD/LQD with the nuclear probe and lysotracker red is performed at different time points using HeLa cells without oleic acid supplementation. (Supporting Information, Figure



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S7) These study show that at early stage they are possibly trafficked to lysosome and from there LQD localize near perinuclear region and HQD either exit from cell or localize near perinuclear region. These results indicate different types of mechanism for LQD and HQD and we have further investigated their uptake mechanism. Cellular uptake of LQD and HQD has been investigated in presence of different endocytosis inhibitors. (Supporting Information, Table S1) Typically, HeLa cells are incubated with endocytosis inhibitor followed by LQD/HQD and then washed cells are used for quantification of QD uptake via flow cytometry. HeLa cells with and without oleic acid supplement are used for this experiment. Results are summarized in Figure 7 and Supporting Information, Figure S8) Results show that uptake of HQD is significantly inhibited by chlorpromazine which is known to block clathrin-mediated endocytosis and this occurs for both oleic acid supplemented and non-supplemented cells. In contrast, cellular uptake of LQD is predominantly inhibited by methyl-ᵦ-cyclodextrin (that inhibits lipid-raft endocytosis) and also by genistein (that inhibits caveolae-mediated pathway) and this occurs for both oleic acid supplemented and non-supplemented cells. This results clearly suggest that HQDs enter into the cell via predominate clathrin-mediated endocytosis but LQDs enter into the cell via predominate lipid-raft-mediated endocytosis. Based on this observation we proposed labeling mechanism of lipid droplet by LQD. (Scheme 2) Zwitterionic surface property of LQD offers modular interaction with the cell membrane and lipophilic character of LQD offers preferential binding with cholesterol-rich lipid-raft region of membrane followed by lipid-raft endocytosis. This uptake mechanism minimizes their trafficking to endosome/lysosome. In contrast, HQD with cationic ammonium groups non-specifically interact with anionic plasma membrane and induces clathrin-mediated endocytosis. This uptake mechanism maximizes their trapping in endosome/lysosome and induces exocytosis.


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Advantages of designed nanoprobe. Besides common advantages of QD based nanoprobes, there are three other advantages of our designed nanoprobe. First, nanoprobe has high photostability compared to commonly used nile red. (Figure 8) Under continuous excitation, the fluorescence image snaps of nile red labeled lipid droplets becomes invisible within 30 sec due to photobleaching of nile red. In contrast, fluorescence image snaps of LQD labeled lipid droplets are visible for minutes and LQD fluorescence does not bleach. Second, nanoprobe has low toxicity, (Figure 9) good water solubility and labeling is highly reproducible via simple incubation with cell culture media. In contrast, lipid droplet imaging probes are water insoluble due to high hydrophobicity, their DMSO solution is added into cell culture media for imaging study and it is not clear if aggregated particle or molecular form is responsible for cell uptake/labeling. Third, designed nanoprobe labels lipid droplet via specific endocytosis mechanism and similar approach can be extended for the development of other sub-cellular nanoprobe. In contrast, molecular probe based lipid droplet imaging mechanism is larger unexplored. However, designed nanoprobe has limitation for in vivo imaging of lipid droplets as they can accumulate in organs due to larger hydrodynamic size compared to the threshold size (5-6 nm) requirement for renal clearance.25

CONCLUSION We have designed a quantum dot based fluorescent nanoprobe for imaging of lipid droplet inside the cell. Nanoprobe enters the cell via predominate lipid-raft endocytosis and labels lipid droplet without trafficking to endosome/lysosome. This work shows the importance of designed surface chemistry to control cellular endocytosis and sub-cellular trafficking of nanoprobe. In particular zwitterionic surface charge and optimum lipophilic functionality is critical for nanoprobe’s modular interaction with the cell membrane and to induce lipid-raft endocytosis. Developed nanoprobe has low cytotoxicity, can be used to image lipid droplet



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for longer time and can be used to understand the functional role of lipid droplet in the cellular scale origin of various diseases.

ASSOCIATED CONTENT Supporting Information Detailed characterization of LQD, experimental conditions of endocytosis inhibition study, control cell labeling data and colocalization study and flow cytometry data. This material is available free of charge via the Internet at http://pubs.acs.org. Notes The authors declare no competing financial interests. Acknowledgement. The authors acknowledge Department of Science and Technology (DST) (Grant number SR/NM/NB-1009/2016) and Council of Scientific and Industrial Research (CSIR) (Grant number 02(0249)/15/EMR-II), government of India for financial assistance. S.M. acknowledges CSIR, India for providing research fellowship.

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21) Chakraborty, A.; Jana, N. R. Clathrin to Lipid Raft-Endocytosis via Controlled Surface Chemistry and Efficient Perinuclear Targeting of Nanoparticle. J. Phys. Chem. Lett. 2015, 6, 3688–3697. 22) Chakraborty, A.; Jana, N. R. Design and Synthesis of Triphenylphosphonium Functionalized Nanoparticle Probe for Mitochondria Targeting and Iimaging. J. Phys. Chem. C 2015, 119, 2888–2895. 23) Dalal, C.; Saha, A.; Jana, N. R. Nanoparticle Multivalency Directed Shifting of Cellular Uptake Mechanism. J. Phys. Chem. C 2016, 120, 6778–6786. 24) Dalal, C.; Jana, N. R. Multivalency Effect of TAT-Peptide-Functionalized Nanoparticle in Cellular Endocytosis and Subcellular Trafficking. J. Phys. Chem. B 2017, 121, 2942–2951. 25) Choi, H. K.; Liu, W.; Misra, P.; Tanaka, E.; Zimmer, J. P.; Ipe, B. I.; Bawendi, M. G.; Frangioni, J. V. Renal Clearance of Nanoparticles. Nat. Biotechnol. 2007, 25, 1165–1170.

Table 1. Properties of HQD and LQDs used in this study. Nanoprobe

Hydrodynamic size 20-30 nm

Zeta potential (mV) at pH 4.5 7.4 10.0 +14 +08 -20

No. of lipophilic groups, primary amines# -----, 550±50

HQD

LQD (octyl)

30-40 nm

+12

LQD (oleyl)

30-40 nm

+15

250±50 (octyl), 300 ±50 250±50 (oleyl), 300±50

#

+03 +05

-14 -24

Endocytosis mechanism, specific labeling Clathrin mediated and exocytosis after 8h, don’t label lipid droplets Lipid-raft and no exocytosis, label lipid droplets Lipid-raft and no exocytosis, label lipid droplets

values are average of 3 experiments.



Scheme 1. Design and synthesis of lipophilic-zwitterionic-QD (LQD). Hydrophobic QD is converted to hydrophilic-zwitterionic QD (HQD) via polyacrylate coating with both SO3– and NH3+ groups in the polyacrylate shell. Next, a fraction of primary amines of HQD are covalently conjugated with octylamine to prepare LQD.

SO 3

polyacrylate coating

octylamine, glutaraldehyde

SO3 (HQD)

QD

SO 3

SO 3

SO 3

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SO3 (LQD)

polyacrylate cell


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Figure 1. Physicochemical properties of LQD. a) UV-visible absorption spectra and emission spectra of colloidal LQD, b) Hydrodynamic size distribution of colloidal LQD measured by DLS with 30-40 nm size, c) Transmission electron microscopic image of LQD showing the inorganic QDs core of 5-6 nm, d) FTIR spectra of LQD showing the additional vibrational stretching frequency at 2822 cm-1 after the octylamine functionalization.



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Figure 2. Fluorescence imaging of lipid droplet in HeLa cells by a) nile red (NR), b) LQD and c) HQD showing that LQD labels lipid droplets similar to NR, but HQD cannot label lipid droplets. First, cells are incubated with oleic acid for 6 h in order to induce lipid droplet. Next, washed cells are further incubated with fresh media along with HQD/LQD and then washed cells are incubated with fresh media for next 9 h prior to imaging. Cells are incubated with NR solution for 5 min for labeling with NR and incubated with Hoechst dye solution for 20 min for labeling cell nucleus. Red color corresponds to NR/HQD/LQD and blue color corresponds to nuclear probe. Scale bars represent 50 microns.


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Figure 3. High resolution image of cells labeled with nile red (NR) and LQD, showing clear view of lipid droplets and their cytosolic distributions. Labeling and imaging conditions are same as of Figure 2. Scale bars represent 10 microns.



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Figure 4. Co-localization of LQD and nile red (NR) in HeLa cells further confirming that LQDs are predominantly localized in lipid droplets. Typically, cells are incubated with LQD solution for 1 h, and washed cells are further incubated with fresh media for 8 h, Next, cells are incubated with NR solution for 5 min and washed cells are imaged under blue excitation for LQD and green excitation for NR. QD with green emission (under blue excitation) is used for this study so that LQD and NR have different excitation window. Merged image shows spotted yellow regions for lipid droplets. Scale bars represent 50 microns.


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Figure 5. Imaging of lipid droplet by oleyl functionalized LQD. HeLa cells are cultured overnight in oleic acid supplemented DMEM media in a 4 well chamber slide. Next, cells are washed and incubated with 30 µL oleyl functionalized LQD solution for 1 h. Washed cells are given fresh media and after 8 h cells are fixed and imaged under the fluorescence microscope. Red color corresponds to oleyl functionalized LQD and blue color is for nucleus



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stained with Hoechst dye. Results show that oleyl functionalized LQD shows similar performance as of octyl functionalized LQD. Scale bars represent 10 microns.

Figure 6. LQD based labeling of lipid droplet in CHO cells. Cells are cultured overnight in oleic acid supplemented DMEM media for lipid droplet induction. Next, cells are treated with 30 µL LQD for 1 h and washed cells are incubated with 50 µL Hoechst dye for 20 min. Finally, washed cells are incubated with fresh media for 2 h or 12 h and then imaged under the fluorescence microscope. Red color corresponds nanoprobe and blue color corresponds nuclear probe. Results show that LQDs label cells in 2 h and label lipid droplets within 8 h. Arrow indicates cytosolic lipid droplets. Scale bars represent 50 microns top images and 10 microns for bottom images.


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100

HQD, normal

100

% Uptake

% Uptake

60 40 20

LQD, normal

20

LQD, induced

80

% Uptake

40

40

100

80 60

60

0

0

100

HQD, induced

80

80

% Uptake

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60 40

20

20

0

0

Figure 7. Evidence of cellular entry of LQD via predominate lipid-raft endocytosis and entry of HQD via both clathrin and lipid-raft endocytosis. HeLa cells or oleic acid induced HeLa cells are labeled with LQD/HQD in presence of different endocytosis inhibitors and then nanoprobe uptake is quantified via flow cytometry using the fluorescence property of QD. About 20000 cells are used in each experiment and the area under the fluorescence intensity is used for uptake quantification. Uptake of LQDs is significantly blocked by MBCD/GEN but not by CHP, suggesting their uptake via lipid-raft endocytosis. In contrast, uptake of HQD is significantly blocked by CHP or both CHP and GEM, suggesting predominate clathrin meditate uptake along with partial lipid raft endocytosis. Results are mean ± SD of 3 experiments.



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Figure 8. Photostability of LQD probe over nile red under continuous irradiation. a) Fluorescence image snaps of nile red labeled lipid droplets under continuous green excitation, showing that fluorescence is lost within 30 sec and lipid droplets become invisible. b) Fluorescence image snaps of LQD labeled lipid droplets under continuous blue excitation, showing that LQD fluorescence does not bleach even up to 90 sec. HeLa cells are used for this experiment and scale bars represent 10 microns.


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LQD

100

HQD

80

% Cell viability

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60 40 20 0

0

20 40 60 80 -7 Concentration ( 10 M)

100

Figure 9. MTT based cell viability assay of HQD and LQD. HeLa cells are incubated with different concentration of HQD/LQD for 24 h and cell viability is measured assuming 100 % viability of control cells without any nanoparticle. Results show that cells viability is 70-80 % even in µM concentration range while 10-100 nanomolar concentrations are required for labeling experiments. Results are mean ± SD of 3 experiments.



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Scheme 2. Mechanism of lipid droplet labeling by LQD. Zwitterionic surface property and lipophilic character of LQD offers modular interaction with the cell membrane and preferential binding with cholesterol-rich lipid-raft region of membrane followed by lipid-raft endocytosis and labeling of lipid droplets. In contrast, HQD with cationic ammonium groups non-specifically interacts with the anionic plasma membrane, induces clathrin-mediated endocytosis and traffics toward endosome/lysosome.

Lipid-raft

Clathrin receptor

Cholesterol

Glycospingolipid

Transmembrane protein

(LQD) (HQD)

Clathrin pathway

Lipid-raft pathway

Endosome

Lysosome

Lipid droplet


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TOC Graphic


Quantum Dot Based Designed Nanoprobe for Imaging Lipid Droplet

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