Design and Synthesis of Triphenylphosphonium Functionalized

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Design and Synthesis of Triphenylphosphonium Functionalized Nanoparticle Probe for Mitochondria Targeting and Imaging Atanu Chakraborty, and Nikhil R Jana J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/jp511870e • Publication Date (Web): 19 Jan 2015 Downloaded from http://pubs.acs.org on January 20, 2015

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The Journal of Physical Chemistry

Design and Synthesis of Triphenylphosphonium Functionalized Nanoparticle Probe for Mitochondria Targeting and Imaging

Atanu Chakraborty and Nikhil R. Jana* Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata700032, India *Corresponding author. E-mail: [email protected] Telephone: +91-33-24734971. Fax: +91-33-24732805

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ABSTRACT Although nanoparticle based various cellular imaging probes are reported as alternative of conventional molecular probes, the development of nanoparticle based sub-cellular imaging probes is challenging. Here we report inorganic nanoparticle based fluorescent probe of 3040 nm hydrodynamic diameter that can target and label mitochondia. Nanoprobe has pH responsive polyacrylate shell with both anionic and cationic surface charge and functionalized with triphenylphosphonium group. Optimized surface chemistry offers high cellular uptake via predominate caveolae mediated endocytosis and triphenylphosphonium group traffics them to mitochondria. This concept of surface chemistry can be extended to different nanoparticles and for development of other sub-cellular imaging nanoprobes.

Key words: nanoparticle, quantum dot, bioimaging, mitochondria imaging probe, endosomal escape


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INTRODUCTION Mitochondria are the power house of cell which is found in all kind of eukaryotic cells.1 It produces adenosine triphosphate (ATP) which is used as source of chemical energy for all type of biological activities.1 In addition it involves in various cell signalling processes, cell cycle, cell differentiation and cellular apoptosis.2 It has a typical size of 0.5-1.0 micrometer and every single cell may contain thousands of mitochondria. Mitochondria consists of outer and inner membrane with the highly negative potential (∆Ψ = -150 to -200 mV) of outer membrane and with the proton gradient across the inner membrane.3,4 Mitochondria dysfunction is associated with various human diseases such as cardiac problem,5 aging6 and neurodegenerative diseases.7,8 So labelling and imaging of mitochondria is an important aspect for understanding the cellular processes and in vitro diagnostic assay.9-29 Mitochondria targeting is also considered as therapeutic strategy for cancer14,16,18,21 that involves their destruction via delivering drugs/nanomaterials followed by therapy.17,20,22 Currently available mitochondria imaging probe include JC1 dye, MitoTracker, MitoFluor, Rhodamine and other dyes.27,28 Chemical structure of these probes is designed in such a way that they can bind with negatively charged mitochondrial membrane and provide fluorescence signal.27,28 However, these molecular probes have poor photostability with bleaching issue under continuous light exposure and thus development of photostable molecular probes are under development.29 In addition attempts have been made to develop nanoparticle based alternative probes having superior photostability that include quantum dot,19 carbon nanotube20 and gold nanoparticle.21 In these design nanoparticles are conjugated with small molecules14,15,24,26 or peptides21,23 for specific targeting of mitochondria. Among them triphenylphosphonium (TPP) is widely used for mitochondria targeting.9,10 TPP is a polar cationic molecule with high lipophilicity which make it suitable for penetrating the mitochondrial intermembrane potential barrier and have been used in different mitochondria


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targeting materials.9,10 TPP has been conjugated with nanoparticle,24,26 dendrimer,15 liposome14 and reported for targeted drug delivery and imaging of mitochondria. Nanoparticle based probes have high cellular uptake which can be tuned by surface chemistry.30 In addition multiple number of targeting molecules can be attached and cellular interaction can be tuned by controlling their number per particle.31 However, sub-cellular targeting of nanoprobes is more challenging as cellular entry of nanoprobe occurs via endocytosis that often traffic them to endozome/lysozome.30 This factor leads to inefficient mitochondria targeting for most of the designed nanoprobes.19,20,21 We are working on design and synthesis of new generation nanoparticle based cell imaging probes.30-32 These nanoparticle include fluorescent semiconductor nanoparticle,31 fluorescent gold and silver clusters,33 doped semiconductors nanoparticle,34 fluorescent carbon nanoparticle35 and fluorescent silicon nanoparticle.36 We transform as synthesized nanoparticle into water soluble and functional nanoparticle via coating and conjugation chemistry.32 We found that hydrodynamic size, surface charge and lipophilicity have significant impact to cell uptake, sub-cellular localization and cytotoxicity of nanoprobe.32 Here we have tuned this surface chemistry to develop mitochondria targeting nanoprobe. We have optimized the surface chemistry of nanoprobe with the aim of high cell uptake, low cytotoxicity and with the targeting property to mitochondria. In this design nanoparticle is coated with polyacrylate and covalently linked with TPP. The polyacrylate shell is decorated with polyethylene glycol and optimum ratio of SO3- and amine (both primary and secondary) functional groups that offers low non-specific binding with bio-environment, high cell uptake and induce for endosomal escape. Using this approach we have synthesized CdSe/ZnS-based quantum dot (QD) and iron oxide based nanoprobes and tested their mitochondria targeting property to different cell lines. We have found that optimized surface functional groups offer


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enhanced mitochondria targeting property of nanoprobes and lowers their lysozomal trafficking.

EXPERIMENTAL SECTION Chemicals. Poly(ethylene glycol) methacrylate, 3-sulfopropyl methacrylate, N-(3aminopropyl)

methacrylamide

formylmethyl-triphenylphosphonium

hydrochloride chloride

and

(TPP),

N,N-methylenebisacrylamide,

sodium

cyanoborohydride

and

fluorescein O-methacrylate were purchased from Sigma-Aldrich and used as received. N-(3aminopropyl) methacrylamide hydrochloride was purchased from Polyscience and used as received. Folate containing DMEM culture medium was purchased from Sigma-Aldrich. Chlorpromazine

hydrochloride,

genistein,

methyl-β-cyclodextrine

and

amiloride

hydrochloride were purchased from Sigma-Aldrich. Mitotracker orange, hoechst and lysotracker red were purchased from life technology. Synthesis of TPP Functionalized QD and γ-Fe2O3. High quality hydrophobic CdSe/ZnS based QD and hydrophobic γ-Fe2O3 were synthesized and transformed into polyacrylate coated nanoparticle following the previously reported methods.37-39 In brief green emissive CdSe nanocrystal was synthesized at 280 °C in 1-octadecene solvent and then ZnS shelling was performed at 220 °C.37 Similarly, highly monodispersed γ-Fe2O3 was synthesized by high temperature colloid-chemical technique using our previously reported method.38 These as synthesized hydrophobic QD and γ-Fe2O3 were transformed into hydrophilic nanoparticles by polyacrylate coating approach as reported previously.39 Briefly, hydrophobic nanoparticle and the hydrophilic acrylate monomers were dissolved in reverse micelle and then the polymerization was initiated in nitrogen atmosphere by adding persulfate. We have mainly used mixture of three acrylate monomers: poly-ethylene glycolmethacrylate, 3-sulfopropyl methacrylate and N-(3-aminopropyl) methacrylamide in their


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molar ratio of 2:1:1. In the case of iron oxide nanoparticles we have used fluorescein Omethacrylate as additional monomer. In all cases a small amount (5 mol % of all monomers) of ethylene bisacrylamide was used as cross-linker. The reaction was continued for one hour and then particles were precipitated by adding ethanol. Particles were then solubilized in water and used as stock solution. Polymer coated nanoparticle solution was purified by dialysing the solution using molecular weight (MW=12000) cut-off filter. The primary amine groups of polymer coated nanoparticle were then used for linking with aldehyde group of TPP. Typically, 500 µL of polymer coated nanoparticle solution is taken in a 2 mL vial and mixed with 200 µL of formylmethyl-triphenylphosphonium chloride solution (10 mM) and stirred for one hour. Next, 2 mg of sodium cyanoborohydride was added and stirring was continued for another 6 hrs. Next, the solution was dialysed overnight by using molecular weight cut-off membrane filter (MW 12000 D) to remove unreacted reagents. The amines present in polymer coated nanoparticle was determined by fluorescamine based titration method reported earlier.31 Estimation of the Average Number of TPP Molecule per QD. The number of TPP molecules per QD had been estimated using following method: In a typical solution of QDTPP, QD concentration was estimated using molar extinction coefficient of QD.40 Next, concentration of TPP molecule was estimated using the absorbance of TPP molecule at 267 nm. At first a calibration curve is prepared using the absorbance of TPP at 267 nm against the known concentration of TPP molecule. Next, QD-TPP was dissolved by adding HCl followed by neutralization using NaOH and the absorbance of TPP was measured at 267 nm. Finally, concentration of TPP in this solution was determined using the fitting curve. The average number of TPP molecule per QD was calculated from the ratio of the concentration of TPP and that of QD and the value comes ~ 230.


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In Vitro Cell Labelling and Imaging: Cells were cultured in DMEM medium with 10% heat inactivated fetal bovine serum (FBS) and 1% penicillin streptomycin at 37 °C and 5% CO2 atmosphere. For fluorescence microscopic study cells were cultured overnight in 24 well plate with 500 µL medium. Next, about 10-100 µL sample was added to the cell culture medium and incubated for 1-8 hrs. After incubation for one hour, cells were washed twice with PBS buffer to remove the free unbound particles and used for imaging. Alternatively, cell were mixed with fresh medium for another 1-8 hrs and then used for imaging. Co-localization Study.

Cells were cultured in 24 well plates for 24 hrs in DMEM

medium. Next, cells were incubated with samples for one hour. Within this time the particles were attached with the cell membrane but did not internalize. Next, cells were washed and mixed with fresh media and kept for another 8 hrs for particle localization. Next, cells were incubated with mitotracker orange or lysotracker red for 10 minutes and washed cells were imaged under microscope. Cellular Uptake Mechanism Study. In order to understand the endocytosis mechanism of QD-TPP we have used different inhibitors that are known to block different pathways. (Table 1) First, cells were cultured in a 24 well plate for 24 hrs and then inhibitors of mentioned concentration were added and incubated for one hour. Next, QD-TPP was added and kept for another one hour. After that the cells were thoroughly washed with PBS buffer and removed from the wells by using trypsin and used for flow cytometry study. Table 1. The Inhibitors Used for the Study of Endocytosis Mechanism Inhibitors Concentration used Endocytosis pathway inhibition chloropromazine 50 µM clathrin based endocytosis amiloride 50 µM macropinocytosis genistein 200 µM caveolae based endocytosis methyl-β-cyclodextrin

10 mM


lipid raft/caveolae mediated endocytosis

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MTT Assay. For cell viability study cells were cultured in a 24 well plate in DMEM media. After that cells were treated with different doses of samples for 24 hrs and then washed thoroughly with PBS buffer and fresh DMEM media was added. Next, each well plate with attached cells were treated with 50 µL of freshly prepared methylthiazolyldiphenyltetrazolium bromide (MTT) solution (5 mg/mL) and incubated for 4 hrs. Then the supernatant was removed carefully leaving the formazon in the plate. These formazon was dissolved in SDS solution (8 gm SDS dissolved in 40 mL DMF-H2O mixture) and absorbance was measured at 570 nm. Instrumentation. All UV-visible spectra were measured in a Shimadzu UV-2550 spectrophotometer using a quartz cell of one cm path length and fluorescence spectra were measured with Fluoromax-4 spectrofluorometer (Horiba JobinYvon) using quartz cell of one cm path length. Fourier transform infrared spectroscopy on KBr pellets was performed using Shimadzu FT-IR 8400S instrument. Zeta potential and hydrodynamic size of nanoparticles were measured using Malvern (Nano ZS) instrument. TEM study was performed using FEI Tecnai G2 F20 microscope with a field-emission gun operating at 200 kV. Fluorescence image of cells were measured using Olympus IX 81 microscope attached with digital camera. Quantification of cell uptake has been performed by BD Accuri C6 Flow Cytometer.

RESULTS Design and Synthesis of Mitochondria Imaging Nanoprobe. Scheme 1 shows the design and steps for synthesis of TPP functionalized QD/γ-Fe2O3 (QD-TPP or γ-Fe2O3-TPP). Nanoprobe has QD or γ-Fe2O3 nanoparticle core with polyacrylate shell. The polyacrylate shell is decorated with different functional groups for different function. Polyethylene glycol functional groups lower the non-specific binding with cellular environment, sulfopropyl groups provide anionic surface charge, primary/secondary amines provide pH dependent


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cationic surface charge and induce proton sponge effect and TPP groups offer mitochondria targeting option. In case of γ-Fe2O3 fluorescein group is present in addition to other functional groups that would offer green emission to the nanoprobe. Synthesis

of

nanoprobe

involves preparation

of

hydrophobic

QD/γ-Fe2O3

nanoparticle37,38 followed by their transformation into polyacrylate coated nanoparticle39 and finally functionalization with TPP. Polyacrylate coating has been performed using the mixture of three acrylate monomers, namely, polyethyleneglycol-methacrylate, 3-sulfopropyl methacrylate and N-(3-aminopropyl) methacrylamide that provide polyethylene glycol, SO3and primary/secondary amine groups, respectively. We have adjusted the optimum ratio of functional groups using monomer molar ratio of 2:1:1. TPP functionalization of nanoprobe is then performed by reacting some of the primary amine groups with formylmethyltriphenylphosphonium chloride whose aldehyde group forms imine bond with primary amine groups. The unstable imine bond is then reduced by cyanoborohydride to make it stable. TPP functionalization of nanoprobe has been confirmed by UV-visible, NMR and FTIR spectroscopy. (Figure 1) TPP functionalized QD/γ-Fe2O3 shows characteristic absorption band of TPP at 267 nm (Figure 1a) and characteristic broad proton NMR signal at 7.7 ppm for phenyl hydrogen (Figure 1c). FTIR spectroscopy of QD before and after functionalization also provides the evidence for the TPP functionalization. After conjugation with TPP, the symmetry deformed vibration peak of NH3+ at 1532 cm-1 disappears and a new peak at 1439 cm-1 for phenyl-phosphorus linkage appear (Figure 1b) .The average number of TPP molecules attached per QD has been estimated by measuring the concentration of QD and TPP and then determining their ratio. The concentration of QD has been estimated using absorbance of QD and their known molar extinction coefficient.40 Absorbance of TPP at 267 nm has been used to measure the concentration of TPP. The average number of TPP molecule per QD has been estimated as ~ 230.


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QD-TPP and γ-Fe2O3-TPP have high colloidal stability in water and in different buffer solutions. The size of the core QD ranges between 2-5 nm and size of core γ-Fe2O3 ranges between 3-6 nm as observed from TEM image (Figure 1d and 1e). The hydrodynamic diameter, that includes core inorganic nanoparticle and polyacrylate shell with functional groups, increases from 20-40 nm to 30-45 nm after TPP functionalization. (Figure 1f and Supporting Information, Figure S1) Role of SO3- and primary/secondary amines can be visualized from pH dependent change of zeta potential of nanoprobe. (Figure 2) The zeta potential changes from low positive value to moderate negative value as the solution pH changes from 4.5 to 9.0. The surface charge of nanoparticle with and without TPP functionalization is low positive/negative (- 2 mV to + 2 mV) at pH 4.5 but becomes high negative (- 15 mV to - 5 mV) at pH 7.4 and 9.0. This can be explained by the presence of sulfopropyl and primary-secondary amine groups on polyacrylate backbone. The sulfopropyl group provide anionic charge (SO3-) after deprotonation and primary-secondary amine provide cationic surface charge after protonation and extent of protonation depends on solution pH. The zeta potential of nanoprobe is negative at pH 7.4 and 9.0 due to dominant anionic SO3- groups over cationic ammonium groups. However, at pH 4.5 protonated ammonium groups dominate over anionic SO3- groups and the combined charge becomes positive. Effect of TPP functionalization is also observed from comparatively lower zeta potential value (particularly at pH 7.4 and 9.0) due to contribution from cationic phosphonium groups. Cellular Uptake and Mitochondria Targeting of Nanoprobe. Cell labelling property of nanoprobes has been investigated by incubating with cells for different time interval, followed by washing to remove unbound particles and then imaging under fluorescence microscope. It is generally observed that nanoparticles with or without TPP functionalization have strong interaction and high cell uptake. However, effect of TPP functionalization is


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observed in subcellular localization. Typical results are shown in Figure 3 and Supporting Information Figure S2-S4 for HeLa cells using QD and QD-TPP. It shows that within one hour both particles mainly bound with the cell membrane and then enter into cells in next 8 hrs. However, in longer incubation time QDs are observed to distribute all over the cells but QD-TPPs are seen to localize inside cells. This result indicates that designed coating offers high cellular uptake of QD but they do not localized inside cell. In contrast TPP functionalization with this QD induces sub-cellular localization of nanoparticle. Sub-cellular localization of QD-TPP and γ-Fe2O3-TPP has been tested in different cell lines and co-localization study has been performed with mitotracker and lysotracker probes. (Figure 4-6 and Supporting Information, Figure S5, S6) For example we have investigated the co-localization of nanoprobe in HeLa, CHO, HT29 and N2A cell lines and co-localization studies have been performed for both nanoprobes. We found that sub-cellular localization of nanoprobes is analogous to all the tested cell lines. (Figure 4) Co-localization study has been performed using mitotracker and lysotracker probes that are known to label mitochondria and lysozome, respectively. Results clearly show that nanoprobes significantly co-localize with mitochondria but do not co-localize with lysozome. (Figure 5, 6 and Supporting Information, S5, S6) This result indicates that designed nanoparticle offers enhanced mitochondria targeting of QD and iron oxide nanoparticle to various cell lines. In order to confirm that these nanoprobes are less toxic, cytotoxicity study has been performed using the conventional MTT assay (Supporting Information, S7) Results show that both probes are reasonably less toxic in the concentration range used for labelling experiments. However, in higher concentration toxicity is observed for QD-TPP and γ-Fe2O3-TPP (as compared to QD and γFe2O3) which is possibly due to their excessive binding with mitochondria and thus disturbing their function.


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In order to understand the origin of sub-cellular targeting property of nanoprobes, cellular uptake mechanism of these nanoprobes has been investigated. Typically, uptake of nanoprobes is studied in presence of chemicals that are known to block different endocytotic pathways. Typical results are shown in Figure 7 for HeLa and CHO cell lines using QD-TPP. It shows that uptake of QD-TPP is significantly decreased in both cells in presence of genistein which is known to block caveolae-mediated endocytosis. In addition the uptake of QD-TPP is decreased in HeLa cell but unaffected in CHO cell by methyl-β-cyclodextrin which is known to block the lipid raft-based endocytosis. In contrast, cell uptake of QD-TPP is insignificantly affected in both cells by chloropromazine and amiloride which are known to block the clathrin and macropinocytosis-based endocytosis, respectively. This result indicates that our nanoprobes enter into cell predominantly via caveolae or lipid raft-mediated pathways than through clathrin and macropinocytosis-based endocytosis. Thus predominant caveolae or lipid raft-mediated entry of nanoprobes is linked with their mitochondria targeting property.41,42 The observed differences in the mechanism of endocytosis in two different cells are linked with cell type dependence on endocytosis mechanism. Mitochondria synthesize ATP in association with generation of reactive oxygen species (ROS). In healthy cells this ROS concentration remains very low. When this ROS level is increased it leads to the decrease of mitochondria membrane potential. Typical mitochondrial membrane potential across the lipid bilayer is ~ -180 mV which can be as low as - 40 mV during excess ROS generation. Thus mitochondrial membrane potential is a measure of ROS level and health of mitochondria. We have measured the ROS level using mitotracker orange and QD-TPP and compared their performance. (Supporting Information, Figure S8) We have used H2O2 to generate high ROS level and then labelled mitochondria using mitotracker orange or QD-TPP. It is observed that the mitotracker orange lost its specificity to label the mitochondria and spread all over the cytoplasm of the cells. In contrast


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QD-TPP still label mitochondria without losing its specificity. This feature of QD-TPP is linked with the property of TPP. It has been previously shown that TPP functionalized molecular probe retain into the mitochondrial compartment even after destruction of the membrane potential.29

DISCUSSION There are several unique aspects of nanoprobe design that offer the successful mitochondria targeting. First, designed nanoprobes have low surface charge and high colloidal stability at physiological condition. Colloidal stability of nanoparticles is commonly associated with high surface charge (typically in the range of +/-20 to +/-50 mV) that inhibits particle-particle interaction.32 In contrast presented nanoparobes have low surface charge at physiological pH, typically in the range of -10 to +5 mV and colloidal stability is ensured by polyethylene glycol functional groups and presence of both cationic (ammonium) and anionic (SO3-) functional groups. Attempts have been made to synthesize zwitterionic nanoprobe43-49 and nanoprobe coated with polyethylene glycol functional groups50 in order to minimize nonspecific interaction with bio-environment but their transformation into sub-cellular nanoprobe is not yet demonstrated. Second, clathrin mediated endocytosis is commonly observed for cellular uptake of nanoprobe.30 This type of endocytosis leads to the trafficking of nanoparticle to endosome/lysozome and thus sub-cellular targeting is limited unless there is some kind of endosomal escape possibility.51,52 In contrast cell uptake of designed nanoprobe occurs mainly via caveolae-mediated endocytosis that can bypass the endosomal trafficking of material and thus have better option for sub-cellular targeting.41 As our designed nanoprobes enter into cell mainly via caveolae-mediated endocytosis they can successfully bypass


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endosome/lysozome and thus have better sub-cellular targeting option.41,42 Thus caveolaemediated endocytosis and TPP functionalization both are responsible for mitochondria targeting of nanoprobe. Longer time requirement of mitochondria targeting is possibly linked with caveolae mediated endocytosis pathways. It has been reported earlier that kinetics of caveolae mediated endocytosis is slow as compared to the clathrin mediated endocytosis41 and in our case it takes ~ 8 hrs. Third, nanoprobe surface functional groups are optimized for caveolae-mediated endocytosis. Nanoprobe surface is decorated with primary and secondary amine functional groups and SO3- functional groups. The primary and secondary amines on nanoparticle surface induce clathrin mediated endocytosis.30 However, pH-buffering function of amines groups can offer proton sponge effect and endosomal escape of nanoprobe.51 Anionic (SO3-) functional groups on the nanoparticle surface can partially block these properties of amine groups. This effect can be observed in the endocytosis mechanism of control QD (without TPP functionalization) which occurs via both clathrin and lipid raft mediated endocytosis. (Supporting Information, Figure S9) When QD is functionalized with TPP, the endocytosis mechanism is then shifted to caveolae-mediated endocytosis.

CONCLUSIONS We have developed surface chemistry approach for transforming inorganic nanoparticles into mitochondria imaging nanoprobe of 30-40 nm hydrodynamic diameter. Nanoprobe is composed of pH responsive polyacrylate shell with both anionic and cationic surface charge and functionalized with triphenylphosphonium group. Balanced cationic-anionic surface charge offers high cellular uptake but with negligible cytotoxicity, pH responsive shell provides endosomal escape property and triphenylphosphonium group traffics them to


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mitochondria. This concept of surface chemistry can be extended for the development of other sub-cellular imaging nanoprobes.

Supporting Information Available: Nanoparticle characterization, time dependent localization study of QD and QD-TPP, Z-stacking image of labelled cells, cell viability data, endosomal escape study of probe and effect of H2O2 on mitochondria labelling. This material is available free of charge via the Internet at http://pubs.acs.org.

ASSOCIATED CONTENT Corresponding Author *E-mail: [email protected]. Notes. Authors declare no competing financial interest.

ACKNOWLEDGEMENTS NRJ would like to thank DST, DBT and CSIR, government of India for financial assistance. AC acknowledges CSIR, India for providing research fellowship.

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Page 22 of 30

Scheme 1. Synthetic strategy for polyacrylate coated nanoparticle (QD, γ-Fe2O3) and triphenylphosophonium (TPP) functionalized nanoparticle (QD-TPP, γ-Fe2O3-TPP). (For details of monomers used for polyacrylate coating please see experimental section.)

(hydrophobic γ-Fe2O3)

(hydrophobic QD)

polyacrylate coating

polyacrylate coating NH2

NH2 NH

NH NH

NH

NH

NH

H2N

H2N

NH2

NH2

(QD)

(γ-Fe2O3) O

NH

O

+

i)

+

i)

ii) NaBH3CN

ii) NaBH3CN

H N

H N

NH

NH

NH

NH

NH

NH

(QD-TPP)

(γ-Fe2O3-TPP) polyacrylate shell,

-

PEG,

SO3 ,

+ ,


22

2.0

Absorbance

a)

QD-TPP QD

1.5

TPP-CHO QD-TPP+HCl

1.0 0.5 0.0 300

400

500

600

700

800 500

QD-TPP

b)

c) TPP-CHO

QD

1439 cm-1

QD-TPP

QD

1532 cm-1 1000

1500

2000

2500

8

3000

6

Wavelnumber (cm-1)

Wavelength (nm)

ppm

4

2

0

f) d)

e) QD

Number (%)

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


% Transmitance (a.u.)

Page 23 of 30

QD-TPP

20

25

30

35

40

45

50

55

Size (nm)

Figure 1. a) UV-visible absorption spectra of TPP-CHO, polyacrylate coated QD, QD-TPP

and QD-TPP after dissolving QD with HCl. Presence of absorption band of TPP at 267 nm for QD-TPP suggests that TPP has been successfully conjugated with QD. b) FTIR spectra of QD and QD-TPP. The N-H stretching vibration of NH2 groups at 1532 cm-1 for QD disappears in QD-TPP and a new Ph-P band at 1439 cm-1 arises. c) Proton NMR spectra of QD, QD-TPP and TPP-CHO, showing the presence of phenylic hydrogen signal at 7.8 ppm for QD-TPP and the peak at 3.6 ppm is coming due to PEG molecules used for the polyacrylate coating. d) TEM images of QD-TPP. (scale bar represents 20 nm) e) TEM image of γ-Fe2O3-TPP. (scale bar represents 50 nm) f) Typical hydrodynamic size of QD and QDTPP as observed via DLS.


23


2

a)

pH

0

Zeta Potential (mV)

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

Page 24 of 30

4

5

6

7

8

9

10

-2 -4 -6 QD

-8

QD-TPP -10

γ-Fe2O3 γ-Fe2O3-TPP

-12 -14

pH

b) 6

4

+

+

NH2

+

10 H N

+

H3N

8

+

NH

+

Figure 2. a) pH dependent change of surface charge of QD, QD-TPP, γ-Fe2O3 and γ-Fe2O3-

TPP as measured by Zeta potential. b) Schematics showing the origin of pH dependent change of surface charge around nanoprobe due to the shifting of protonation-deprotonation equilibria of -NH2, -NH- and -SO3- groups.


24

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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


1h

QD

8 hrs

QD

1h

QD-TPP

8 hrs

QD-TPP

Figure 3. Fluorescence image of HeLa cells labelled with QD and QD-TPP for two different

incubation time (1 h and 8 hrs). Results show that QDs do not localize even for longer incubation times and spread all over the cytoplasm. In contrast QD-TPPs localize at longer incubation time. Scale bar represents 20 µm.


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Page 26 of 30

CHO F

F

F

Merged HeLa

HeLa

CHO CHO M

CHO

HT29 M

M

HT29

Figure 4. Fluorescence image of QD-TPP labelled different cells after longer incubation

time, showing the sub-cellular localization in all cells. Cells are incubated with QD-TPP for one hour and washed cells are further incubated with fresh media for another 8 hrs before imaging. Top row shows fluorescence image (F) and bottom row shows corresponding merged image with bright field (M). Scale bar represents 20 µm.


26

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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


QD-TPP

HeLa

mitotracker

HeLa

mitotracker

HT29

CHO merged

merged

mitotracker

N2A merged

HT29

CHO

QD-TPP

N2A

HT29

CHO 20µm mitotracker

HeLa

QD-TPP

QD-TPP

merged

N2A

20µm

Figure 5. Fluorescence imaging-based co-localization study of QD-TPP and mitotracker

orange in HeLa cells. Cells are labeled with QD-TPP and mitotracker orange and then QD is imaged under blue excitation (top panels) and mitotracker is imaged under green excitation (middle panels). Merged image (bottom panels) with yellow color indicates co-localization of two probes. Scale bar represents 20 µm.


27


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Page 28 of 30

QD-TPP

mitotracker

merged

γ-Fe2O3-TPP

mitotracker

merged

Figure 6. Higher magnification co-localized image of QD-TPP and γ-Fe2O3-TPP with

mitotracker orange in HeLa cells. Cells are labeled with QD-TPP or γ-Fe2O3-TPP along with mitotracker orange and then nanoprobe is imaged under blue excitation (left panel) and mitotracker is imaged under green excitation (middle panel). Merged image (right panel) with yellow color indicates co-localization of two probes. Scale bar represents 20 µm.


28

Page 29 of 30

b)

a)

400

300 200

Methyl β cyclodextrin Genistein Amiloride Chloropromazine QD-TPP

Relative cell count

Relative cell count

400

100 0 101

102

103

104

Methyl β cyclodextrin Genistein Amiloride Chloropromazine QD-TPP

300 200 100 0 101

105.2

Log fluorescence intensity

102

103

104

105.2

Log fluorescence intensity d)

100 75 50 25 0

100

% of Uptake

c)

% of 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


75 50 25 0

Figure 7. Flow cytometry analysis of uptake of QD-TPP in CHO (a, c) and HeLa (b, d) cells

in presence of different endocytosis inhibitors such as chloropromazine (CHP), genistein, methyl-β-cyclodextrin (MBCD) and amiloride. Log fluorescence intensity represents mean area of fluorescence signal collected by standard optical filters (585/40 nm).


29


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Page 30 of 30

Table of Content (TOC)

nucleus

caveosome

mitochondria


30

Design and Synthesis of Triphenylphosphonium Functionalized

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