Multivalency Effect of TAT-Peptide-Functionalized Nanoparticle in

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Multivalency Effect of TAT Peptide Functionalized Nanoparticle in Cellular Endocytosis and SubCellular Trafficking Nikhil R. Jana, and Chumki Dalal J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b12182 • Publication Date (Web): 23 Mar 2017 Downloaded from http://pubs.acs.org on March 26, 2017

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The Journal of Physical Chemistry

Multivalency Effect of TAT Peptide Functionalized Nanoparticle in Cellular Endocytosis and Subcellular Trafficking Chumki Dalal 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: +9133-24732805.

1 ACS Paragon Plus Environment


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ABSTRACT.

Although

trans-activating

transcription

(TAT)-peptide

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functionalized

nanoparticle/polymer/liposome is widely used for cellular transfection applications, the multivalency (number of attached peptide per particle) effect on cell uptake mechanism and subcellular targeting performance is largely unexplored. Here we show that multivalency of nanoparticle controls the cellular interaction, cellular entry/exit mechanism and subcellular targeting performance. We have synthesized TAT peptide functionalized quantum dot (QD) of 30-35 nm hydrodynamic diameter with varied multivalency from 10 to 75 (e.g. QD(TAT)10, QD(TAT)20, QD(TAT)40, QD(TAT)75) and studied the role of multivalency in endocytosis and subcellular trafficking. We found that both low and high multivalent nanoparticles enter into cell predominantly via lipid-raft mediated endocytosis but the higher multivalency of 40 and 75 induces vesicular trapping followed by exocytosis within 12 h. In contrast lower multivalency of 10 and 20 offers efficient trafficking towards perinuclear region and Golgi apparatus. This work shows the functional role of nanoparticle multivalency in cellular uptake mechanism and importance of lower multivalency for efficient subcellular targeting.


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INTRODUCTION Nanoparticle-based bioimaging probes emerge as powerful alternative of conventional molecular probes due to their high brightness and photostability1-5 and allow monitoring of biochemical activity down to single molecule.6,7 Various nanoprobes have been successfully designed for imaging of biomolecule inside cell and to monitor their activity at subcellular length scale.1-7 These nanoprobes are composed of nanoparticle core, polymeric/molecular shell and covalently conjugated affinity biomolecules.7-9 As nanoprobe interacts with live cell via the affinity biomolecule and each nanoparticle has multiple number of these affinity biomolecule on their surface, the cell-nanoprobe interaction is essentially multivalent in nature.2,10 This is a significantly different situation as compared to molecular probe that usually has monovalency with single interacting site.2,10 However, there are very few example where multivalent interaction of nanoprobe has been studied and optimized for best performance. Examples include, multivalency dependent cellular uptake of nanoprobe functionalized with TAT peptide11,12/oligonucleotide13 /folate,14 optimum multivalency of 20 for cell uptake of RGD peptide functionalized nanoprobe15 and optimum multivalency of 11-23 for cell uptake of HER2 antibody functionalized nanoprobe.16 It is also reported that monovalency of nanoprobe reduces the clustering property of receptor at cell surface.17 In contrast multivalent interaction becomes insignificant if nanoparticle have very low multivalency (typically < 10 per particle) due to inaccessibility of affinity molecule by receptor.10 Thus control of nanoprobe multivalency typically in the range of 10-100 is crucial to evaluate/optimize their biological performance.10 Most of the currently available nanoprobes enter into cell predominately via clathrin-mediated endocytosis and trafficks to endosome/lysosome that restricts their subcellular targeting performance.4,8 We have recently showed that high multivalency (typically > 40) of folate functionalized nanoprobe induces clathrin-mediated endocytosis via strong interaction with cell 3 ACS Paragon Plus Environment


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through multiple receptors.18 In contrast lowering of multivalency to 10 offers modular interaction with cell surface receptors which eliminate the clathrin-mediated endocytosis pathway and

promotes perinuclear trafficking of nanoprobe via caveolae-mediated

endocytosis.18 This study indicates that lower multivalency of nanoprobe may be an important criteria for their efficient subcellular targeting. Here we have studied the multivalency effect of TAT-peptide functionalized nanoprobe on cellular interaction/uptake and found that lower multivalency in the range of 10-20 is ideal for efficient subcellular targeting. TAT-peptide is widely used for cell transfection and nuclear targeting of macromolecule19-23/nanoparticle2428

/drug29,30 and cellular internalization occurs predominantly via macropinocytosis25,28 or lipid-

raft endocytosis12,20,22 or clathrin-mediated endocytosis for some selected cases.21,23 Those study shows that nuclear targeting of nanoparticle is either partially successful19,26,27 or completely inefficient.20,21,23 Although there are some reports on multivalency effect on cellular uptake and uptake mechanism,11,12 there is no report of multivalency effect on subcellular targeting performance. Here we demonstrate three distinct effect of changing multivalency in the range of 10-75. First, cellular interaction and uptake kinetics increases with increased nanoprobe multivalency. Second, cellular entry and exit mechanism of nanoprobe is directed by their multivalency. Nanoprobes enter into cell predominantly via lipid-raft endocytosis which is not affected by changing multivalency. However, exocytosis of nanoprobe is initiated for the higher multivalent nanoprobe. Third, subcellular targeting and particularly asymmetric perinuclear trafficking (i.e. localization at one side of the perinuclear region) of nanoprobe becomes more efficient by lowering of their multivalency typically in the range of 10-20. EXPERIMENTAL SECTION


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Materials. Cadmium oxide, tri-octyl phosphine, tri-octyl phosphine oxide, stearic acid, zinc stearate, sulfur powder, selenium powder, poly(ethylene glycol) methacrylate, 3-sulfopropyl methacrylate, N,N-methylenebis(acryl amide), 4-malemido-butyric acid N-hydroxysuccinimide ester (MAL-but-NHS), chlorpromazine hydrochloride, genistein, amiloride hydrochloride, methyl-β-cyclodextrin, cytochalasin-D, nocodazole and Dulbecco’s modified Eagle’s medium (DMEM) culture medium were purchased from Sigma-Aldrich and used as received. TAT peptide (CGRKKRRQRRR), FITC-TAT peptide (CGRKKRRQRR (FITC) R), Hoechst, NBD C6 ceramide (Golgi tracker) and lysotracker red were purchased from Life Technology. Synthesis of polyacrylate coated QD with 125 primary amines per particle ([QD(NH2)125]. Hydrophobic CdSe-ZnS core-shell quantum dot (QD) was synthesized using the reported method.31 Then hydrophobic QD was converted into hydrophilic QD by using polyacrylate coating.10 In brief, hydrophobic QD and aqueous solution of acrylate monomers were dissolved in Igepal-cyclohexane reverse micelle. Here we used N-(3-aminopropyl)-methacrylamide hydrochloride that provides primary amine group, 3-sulfopropropyl methacrylate that offers anionic charge, poly(ethylene glycol) methacrylate that provide polyethylene glycol functionality and bis[2-(methacryloyloxy)ethyl]phosphate that functions as a cross-linker. Molar ratio of acrylate monomers was appropriately adjusted to produce 125 primary amines per QD. Next, aqueous solution of ammonium persulfate was added to initiate the polymerization reaction. Then, the reaction was continued for one hour under inert atmosphere and polymerization was stopped by precipitating QD via ethanol addition. Resultant QD(NH2)125 was repeatedly washed with chloroform and ethanol and finally dissolved in distilled water. Finally QD(NH2)125 solution was dialyzed against fresh distilled water by using cellulose membrane (MWCO ̴ 12000 Da) to remove unreacted reagents.



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Synthesis of TAT-peptide functionalized QD with different multivalency. [QD(TAT)10, QD(TAT)20, QD(TAT)40, QD(TAT)75]. TAT-peptide functionalized QD with varied multivalency has been synthesized. In order to prepare QD(TAT)40 and QD(TAT)75, one mL aqueous solution of QD(NH2)125 was prepared in borate buffer of pH 9. QD concentration was adjusted by keeping the absorbance value of 0.035 at 595 nm. Next, 35-70 µL of a dimethylformamide (DMF) solution of MAL-but-NHS (1 mg/mL) was mixed and after 10 min, 50-100 µL of TAT-peptide solution (4 mg/mL) was added and kept under stirring condition for next 1/12 h. Finally, solution was dialyzed (MWCO ̴ 12000 Da) against fresh water to remove excess reagents. Preparation of QD(TAT)10 involved similar reaction conditions except that reaction medium was changed from water to DMF. In brief, same concentration of QD(NH2)125 was dispersed in one mL DMF and mixed with 35 µL of DMF solution of MAL-but-NHS (1.0 mg/mL) followed by mixing of 50 µL of TAT-peptide solution (4 mg/mL) after 10 min. The reaction was continued for next 15 min and then QD(TAT)10 was precipitated by centrifuge and finally dissolved in fresh water. The QD(TAT)10 solution was then dialyzed (MWCO ̴12000Da) against fresh water to remove excess reagents. QD(TAT)20 was prepared using the same condition as of QD(TAT)10 except that reaction time was increased to 30 min. Estimation of number of TAT-peptide per QD. The number of TAT-peptide per QD was determined by using fluorescence property of FITC present in FITC-TAT peptide. At first solution of FITC-TAT of different concentrations were prepared in PBS buffer of pH ̴ 7.4. Then fluorescence intensity was measured at 520 nm by exciting the solutions at 460 nm. Next, a linear calibration curve was obtained by plotting the fluorescence intensity against TAT-FITC


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concentration. The linear equation Y = 8×10-10 X with R2 value 0.98 was obtained.(Y = concentration of TAT-FITC and X= fluorescence intensity) Next, FITC-TAT conjugated QD solutions were treated with HCl to dissolve the QD, followed by neutralization with NaOH. Next, fluorescence intensity of FITC (present in the form of FITC-TAT) was measured and the concentration of FITC-TAT was determined using the above mentioned calibration curve. Separately, concentration of QD was determined by using the molar extinction coefficient value of QD reported earlier.32 Finally, the number of TAT-peptide per QD was calculated from the ratio of concentration of FITC-TAT and concentration of QD. In second approach, number of TAT-peptide per QD was calculated using gel permeation chromatography (GPC) based study. Molecular weight of QD(NH2)125 was determined before and after TAT conjugation via GPC. Next, increased molecular weight was accounted for the number of TAT-peptide or multivalency. For example, conjugation of each TAT accounts for increase of molecular weight of 1779 Da (molecular weight of FITC-TAT is 1499 Da + MALbut-NHS is 280 Da) and increase of molecular weight of 20 kDa accounts for 10 TAT-peptide and increase of molecular weight of 133 kDa accounts for 75 TAT-peptide. Cell labelling and imaging study. We have used four types of cells such as HeLa (cervix cancer cell), KB (mouth cancer cell), MCF-7 (breast cancer cell) and CHO (chinese hamster ovary cancer cell) in our study. Cells were cultured in DMEM media 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 in 24 well plates for overnight with 500 µL of serum free cell culture media and then 50-100 µL sample was added followed by 30 min to 3 h incubation. Next, cells were washed with PBS buffer to remove unbound particle. Then washed cells were used for imaging study. For localization study washed cells were further



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incubated with fresh media for next 24 h and used for imaging study. For colocalization study cells were incubated with nuclear probe, lysotracker red and Golgi tracker for 15-30 min and washed cells were used for imaging. Endocytosis inhibition study. In order to study the endocytosis uptake mechanism of QD samples, the cells were cultured in serum free DMEM media and pre-treated with different endocytosis inhibitors for one hour with their appropriate concentration. (Supporting Information, Table S1) We have used genistein (GEN) that blocks caveolae-mediated endocytosis, chlorpromazine (CHP) that is known to block clathrin-mediated endocytosis, methyl-β-cyclodextrin (MBCD) that blocks lipid-raft mediated endocytosis, amiloride (AMI) that blocks macropinocytosis, cytochalasin-D (Cyto-D) that inhibits the actin filament polymerization and nocodazole (NOC) that inhibit microtubule polymerization. Next, QD samples were added and incubated for another 2 h. Next, cells were washed with PBS buffer solution to remove the unbound QDs and treated with trypsin-EDTA for 2-3 min. Finally, detached cells were isolated by centrifuge and dispersed in PBS buffer and used for flow cytometry based study. Study of exocytosis. Cells were cultured in serum free DMEM media and incubated with nanoprobe QD(TAT)75 for 3 h. Next, cells were washed with PBS buffer to remove unbound particle and kept for next 12-24 h with fresh media. At 3 h and 24 h interval cells were incubated with trypsin-EDTA for 2-3 min and cells are isolated by centrifuge. Finally cells were dissolved in PBS buffer and 10% suprapure HNO3 for FACS (fluoresence-activated cell sorting) and inductively coupled plasma atomic emission spectroscopy (ICP-AES) based study, respectively. ICP-AES was also performed using supernatant of cell culture media using 10% suprapure HNO3.


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Instrumentation. Fluorescence and UV-visible absorption spectral studies were done using BioTek SynergyMx microplate reader and Shimadzu UV-2550 UV-visible spectrophotometer, respectively. Transmission electron microscopic (TEM) study was carried out using FEI Tecnai G2 F20 microscope by putting a drop of nanoprobe solution on carbon coated copper grid. DLS and zeta potential were measured by Malvern Nano ZS instrument. GPC (Waters 515) equipped with Waters HSP gel column was used for determination of molecular weight. Fluorescence images of cells were captured by Olympus IX 81 microscope using Image-ProPlus v 7.0 software and Carl Zeiss Apotome Imager Z1 microscope. Fluorescence based quantification was studied using BD Accuri C6 Flow Cytometer and ICP-based quantification was performed using Optima 2100DV ICP-AES, Perkin Elmer. RESULTS AND DISCUSSION Synthesis of TAT-peptide functionalized QD with varied multivalency. Strategy for synthesis of TAT-peptide functionalized QD probes with low and high multivalency is shown in Scheme 1. Hydrophobic QD is transformed into primary amine terminated, hydrophilic QD of 30-35 nm hydrodynamic size via polyacrylate coating.10,31 The average number of primary amines per QD is adjusted to ̴ 125 using the appropriate ratio of acrylate monomers and designated as QD(NH2)125.10 In addition to primary amine, polyacrylate shell is also decorated with anionic sulfate and non-ionic polyethylene glycol (PEG) functional groups. Thus QD(NH2)125 has overall anionic surface charge that minimizes cellular interaction and cell uptake. The primary amines present on the surface of QD(NH2)125 have been used for covalent conjugation with TATpeptide. Thiol groups of cysteine terminated TAT-peptide is linked to primary amines of QD using MAL-but-NHS as conjugation reagent where MAL reacts with thiols and NHS reacts with amines. Typically, excess of MAL-but-NHS is added first to the solution of QD(NH2)125 for the



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reaction of the primary amines with NHS groups and excess MAL-but-NHS will be there in solution. Next, TAT-peptide solution (equivalent of MAL-but-NHS) is added to this mixture. At this stage thiols of TAT-peptide reacts with QD bound MAL groups or with free MAL-but-NHS. After this conjugation unreacted TAT-peptide, MAL-but-NHS and their coupled products are removed via dialysis. Characterization of this TAT-peptide functionalized nanoprobe is shown in Figure 1, Table 1 and Supporting Information, Figure S1, S2. Hydrodynamic size and zeta potential of nanoprobes are measured at different solution pH and in water. (Supporting Information, Figure S1, S2). Results show that low and high multivalent QD have almost similar hydrodynamic size at pH=7.4. However, hydrodynamic size of QD changes depending on solution pH or due to hydrophobic FITC. For example larger size associated with lowering of surface charge is observed for same QD sample when the pH becomes acidic. (Supporting Information, Figure S1) Similarly, QD probes becomes larger in size when functionalized with FITC-TAT as compared to TAT. (Supporting Information, Figure S2) Such larger size and high polydispersity of QD sample are linked to partial aggregation of particle, either due to lowering of surface charge or due to hydrophobic FITC. Another DLS-based study is done using serum free cell culture media. Results show that both low and high multivalent nanoprobes have similar hydrodynamic radii in cell culture media, meaning that they do not aggregate before interacting with cell.(Supporting Information, Figure S3) The number of TAT-peptide conjugated to each QD (multivalency) has been controlled by changing the reaction medium, reactant concentration and reaction time. Lower multivalent QDs [e.g. QD(TAT)10 and QD(TAT)20] are prepared in DMF where QD(NH2)125 is insoluble and primary amines are only partially accessible for the reaction with NHS groups. This approach produces QD with average multivalency of 10 or 20, depending on reaction time. Higher


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multivalent QD [e.g. QD(TAT)75 and QD(TAT)40] are prepared in water where QD(NH2)125 is completely soluble and primary amines are fully accessible for reaction with NHS groups. Under this condition TAT functionalized QD with average multivalency of 40 or 75 have been synthesized by varying the concentration of TAT-peptide and reaction time. The multivalency or the number of TAT-peptide per QD has been determined by two different approaches. (Figure 1) In first approach, FITC-TAT peptide is used for conjugation with QD(NH2)125 and then fluorescence property of FITC has been used for quantification of QD bound TAT. (Supporting Information, Figure S4) Separately, QD concentration is determined from the molar extinction coefficient of QD.32 Next, QD multivalency has been determined from the concentration ratio of TAT peptide and QD. In second approach, molecular weight of QD(NH2)125 has been determined before and after TAT conjugation via GPC. Next, increased molecular weight due to TAT conjugation has been accounted for the number of TAT bound to each QD. We have found that multivalency determined from these two approaches corroborates reasonably well and we are able to prepare multivalency of 10, 20, 40 and 75. Multivalency directed cellular interaction, endocytosis, exocytosis and subcellular trafficing. We have designed QD(NH2)125 with appropriate surface chemistry so that it has minimum non-specific interaction with cell.10 Control experiment shows that the uptake of QD(NH2)125 and QD(NH2)300 are insignificant as compared to TAT functionalized QDs. (Supporting Information, Figure S5, S6) This result suggests that TAT functionalization is responsible for cellular interaction of all the QDs. Results are summarized in Table 1. We have observed three distinct effect of QD multivalency. First, the cellular interaction and uptake kinetics of QD increases with increased multivalency. For example, when cells are incubated with same concentration of QD for 30 min, the cellular uptake increases with increased



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multivalency. (Supporting Information, Figure S7) In addition lower multivalent nanoprobe QD(TAT)10 or QD(TAT)20 shows insignificant uptake in first 30 min and need at least one hour for significant labeling and uptake. In contrast QD(TAT)40 or QD(TAT)75 can label cells within 30 min. (Supporting Information, Figure S8) QD uptake kinetics has been further studied via Zstack fluorescence imageing. (Supporting Information, Figure S9, S10) Result shows that QD(TAT)75 reside at nuclear plane within 30 min but QD(TAT)10 cannot residing at nuclear plane within 30 min. (Supporting Information, Figure S9) Results also shows that both lower and higher multivalent nanoprobe reside at nuclear plane after 3 h. (Supporting Information, Figure S10) Flow cytometry study clearly shows that uptake kinetics of QD(TAT)10 is slower than QD(TAT)75. (Supporting Information, Figure S11) Second, subcellular trafficking and longer time cytosolic residence differs with the change of QD multivalency. (Figure 2 and Supporting Information, Figure S12) For example, QD(TAT)10 and QD(TAT)20 enter into cytosol and trafficks toward one side of perinuclear region within 12-24 h. This is clearly observed from fluorescence imaging of QD(TAT)10 labeled cells at different time interval. (Figure 2 and Supporting Information, Figure S12) In contrast QD(TAT)40 and QD(TAT)75 gradually disappear within 12-24 h, as observed from the lowering of QD fluorescence intensity in labeled cells. (Figure 2 and Supporting Information, Figure S12) This result indicates that lower multivalent QD remains inside cell for longer time but higher multivalent QD escapes from cell. Third, cellular entry and exit mechanism of QD depends on multivalency. It is observed that, irrespective of multivalency all the QDs enter into cell predominately via lipid-raft mediated endocytosis. (Figure 3 and Supporting Information, Figure S13) Typically, cells are pretreated with different endocytosis inhibitors followed by incubation with QD samples. Next, washed


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cells are used for quantification of QD labeling via flow cytometry. Results are shown for QD(TAT)10 and QD(TAT)75 as representative of lower and higher multivalency, respectively. It shows that uptake of both QD(TAT)10 and QD(TAT)75 is inhibited by MBCD and Cyto-D which suggests that their uptake occurs significantly via lipid-raft and actin filament dependent pathway. (Figure 3 and Supporting Information, Figure S13) We have observed that lower multivalent QD(TAT)10 remain inside cell for longer time (24 h or until cell division) but the higher multivalent QD(TAT)75 is exocytosized within 12 h. The exocytosis of QD(TAT)75 has been studied in detail using ICP based Cd estimation and flow cytometry based quantitative estimation of QD. (Figure 3 and Supporting Information, Figure S14) Flow cytometric study shows that emission from nanoprobe labeled cell is decreased after 24 h. Similarly, ICP study shows that concentration of Cd (present in QD) in labeled cells decreases after 24 h but increases in the culture medium. We have further investigated that exocytosis is microtubule dependent. (Supporting Information, Figure S15) Typically, QD(TAT)75 labeled cells are incubated with NOC that is known to inhibit the microtubule polymerization and we found that exocytosis process is completely inhibited. This result concludes that the exocytosis process is microtubule dependent. All these results indicate that both the lower and higher multivalent QD enter into cell via lipid-raft mediated endocytosis but in latter stage higher multivalent QD is readily exocytosized. Lower multivalency and lipid-raft endocytosis are essential for subcellular trafficking of nanoprobe. We have extensively studied the subcellular trafficking of QD(TAT)10 in different cell lines (e.g HeLa, KB, MCF-7 and CHO cell lines) and tried to correlate with the mechanism of uptake as we know that cellular uptake processes differs depending on cell type.33 We have identified two possible situations depending on cell type. In the first case (e.g HeLa, KB, MCF-



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7) entry of QD(TAT)10 occurs via exclusive lipid-raft endocytosis and leads to asymmetric perinuclear localization. (Figure 2-4 and Supporting Information, Figure S13, S16) In the second case QD(TAT)10 enters into cell (CHO cell) via both lipid-raft and clathrin mediated endocytosis and such asymmetric perinuclear localization is restricted. (Figure 4 and Supporting Information, Figure S16) These results suggest that asymmetric perinuclear localization of nanoprobe requires exclusive lipid-raft mediated endocytosis. If nanoprobe enters via both clathrin and lipid-raft mediated endocytosis this asymmetric perinuclear trafficking is blocked, as clathrin-mediated endocytosis is faster and known to traffick them to endosome/lysozome. We have performed a control localization study in presence of endocytosis inhibitor that blocks the clathrin-mediated endocytosis. Results show that asymmetric perinuclear trafficking of QD(TAT)10 in CHO cell becomes successful after blocking the clathrin-mediated endocytosis by CHP. (Figure 5) Thus effective blocking of clathrin-mediated endocytosis can redirect nanoprobe toward the perinuclear region. Careful observation of QD(TAT)10 localization shows that they are concentrated at one side of the nucleus. So we have further investigated this subcellular compartment and the origin of such localization. At first we have labeled cells with nuclear probe and QD(TAT)10, performed higher magnification fluorescence imaging at different Z planes and confirmed that QD(TAT)10 localizes near perinuclear region and reside at nuclear plane. (Figure 6 and Supporting Information, Figure S17) Next, we have performed colocalization study of QD(TAT)10 and commercially available Golgi tracker. (Figure 7) We have used green emitting Golgi tracker and red emitting QD(TAT)10 and cells labeled with both probes and imaged under fluorescence microscope where Golgi tracker is excited with blue light and QD is excited with green light. Results show that QD(TAT)10 perfectly colocalize with Golgi tracker, suggesting


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that QD(TAT)10 is trafficked close to Golgi apparatus. We have also performed Z-stack fluorescence imageing showing that QD(TAT)10 and Golgi apparatus reside at same plane. (Supporting Information, Figure S18) Co-localization study is also performed usuing commercially available lysotracker red. (Supporting Information, Figure S19) Here we have used green emitting QD(TAT)10 and red emitting lysotracker red. Results show that nanoprobes are not localized with lysotracker, suggesting that nanoprobes are significantly localized at Golgi apparatus rather than lysosome. Presented result indicates that lipid-raft and actin filament dependent uptake of QD(TAT)10 possibly direct their anisotropic perinuclear trafficking and their localization near Golgi apparatus. This is further verified from microtubule dependent trafficking of QD(TAT)10. (Supporting Information, Figure S20) If cells are incubated with QD(TAT)10 followed by incubation with NOC (that inhibits the microtubule polymerization) such anisotropic perinuclear trafficking is completely disrupted. This result suggests that anisotropic perinuclear trafficking of QD(TAT)10 and localization near Golgi apparatus is a microtubule dependent process. It is well known that endocytosis mechanism dictates subcellular localization of external materials.33,34 In particular clathrin-mediated endocytosis trafficks the external material to acidic endosomal/lysosomal compartments but macropinocytosis/lipid-raft-mediated endocytosis trafficks them to the nucleus, endoplasmic reticulum, Golgi apparatus35,36 In addition microtubule

plays

an

macropinocytosis/lipid

important raft-mediated

role

for

nuclear/perinuclear

endocytosis

are preferred

trafficking.25,37

Thus

over clathrin-mediated

endocytosis for subcellular targeting application. In that respect TAT-peptide functionalized nanomaterials should have efficient subcellular targeting performance as they enter into cell predominantly via macropinocytosis25,28 or lipid-raft endocytosis.12,20-23 However, subcellular



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targeting of nanoparticle is either completely inefficient20,21 or only partially successful.19,26,27 and some studies show partial targeting of Golgi apparatus.38,39 Based on our results we propose cell uptake mechanism of TAT-peptide functionalized nanoparticle, role of nanoparticle multivalency in subcellular targeting performance and cause for inefficient subcellular targeting. (Scheme 2) Nanoprobes with low and high multivalency enter into cell via lipid-raft mediated endocytosis and actin filament dependent pathway. However, modular interaction of lower multivalent nanoprobe with cell membrane offers smaller vesicle formation followed by microtubule dependent trafficking toward Golgi apparatus. In contrast, higher multivalent nanoprobes strongly interact with cell membrane, produces heavily loaded vesicular trap and generate signals for microtubule dependent exocytosis. This mechanism shows that appropriate control of nanoprobe multivalency is essential for efficient subcellular targeting. As most of the earlier TAT peptide functionalized nanoparticls have uncontrolled or higher multivalency, they are inefficient for subcellular targeting. CONCLUSION We have shown that multivalency of TAT-peptide functionalized nanoparticle directs their interaction with cell as well as the subcellular targeting efficiency. In particular we have shown that there are two essential criteria for efficient subcellular targeting of TAT-peptide functionalized nanoparticle. First, multivalency of nanoparticle should be low, typically in the range of 10-20. Second, nanoparticle should enter into cell predominately via lipid-raft mediated endocytosis. Although higher multivalency increases the cellular interaction and uptake, it also induces exocytosis via larger vesicular trapping. These results show that fine tuning of nanoprobe multivalency is essential for the development of subcellular imaging nanoprobe. This


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work also highlights the functional role of multivalency of nanoscale materials in controlling their cellular uptake mechanism and subcellular targeting.

Table 1. Property of TAT-peptide functionalized QD with low and high multivalency. Number of TAT peptide per QD is represented as mean ± SD of three determinations (n=3).

Nanoprobe

Hydrodynamic size (nm) ,charge (mV)

Abbreviation

Cell uptake pathway

Sub-cellular localization

lower multivalent (10±3,20±2)

30-35,-22

QD(TAT)10 , QD(TAT)20

lipid-raft

Golgi apparatus

higher multivalent (40±6,75±8)

35-45,-12

QD(TAT)40 , QD(TAT)75

lipid-raft

exocytosis



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Scheme 1. Synthesis strategy for TAT-peptide functionalized QD with low and high multivalency. Polyacrylate coated QD with 125 primary amine per particle [QD(NH2)125] is synthesized first and then conjugated with cysteine terminated TAT using MAL-but-NHS. Lower valent QDs [QD(TAT)10 and QD(TAT)20] are prepared in DMF where QDs are insoluble and primary amines are partially accessible. Higher valent QDs [QD(TAT)40 and QD(TAT)75] are prepared in water where QDs are soluble and primary amines are fully accessible.


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Figure 1. a) UV-visible and fluorescence property of QD(TAT)10 and QD(TAT)75 b) TEM image of QD(TAT)10, showing that the inorganic QD core of 4-5 nm size. c) Typical photoluminescence (PL) spectra of FITC for QD(TAT)10 and QD(TAT)75 that are prepared using FITC-TAT and with same QD concentration. QDs are dissolved by adding conc. HCl and neutralized by adding NaOH, prior to measuring FITC fluorescence. d) Molecular weight of QD before and after functionalization of TAT peptide, as determined by GPC. Results suggest that the molecular weight increases after functionalization of TAT-peptide and with increasing multivalency.



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Figure 2. Time dependent localization of QD(TAT)10 and QD(TAT)75 in HeLa cells. Cells are incubated with QD sample for 3 h and washed cells are further incubated with fresh culture media followed by imaging at different time point. Results show anisotropic perinuclear localization of QD(TAT)10 within 12 h and such localization remains for 24 h. In contrast, QD(TAT)75 label cells within 3 h but exocytosized within 12 h. Scale bar represents 50 µm.


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b) 100

QD(TAT)10

80 60 40 20

100

Cellular uptake (%)

Cellular uptake (%)

a)

0

c)

QD(TAT)75

80 60 40 20 0

d)

0.008

0.007 0.007

Cd in media

Cd in cell

Cd (mg/L)

0.006 0.006

Cd (mg/L)

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0.005 0.004 0.003

0.005 0.004 0.003

0.002

0.002

0.001

0.001

0.000

3h

12 h

24 h

0.000

3h

12 h

24 h

Figure 3. a,b) Flow cytometry based quantitative estimation of QD uptake in HeLa cell in the presence of different endocytosis inhibitors. Typically, cells are incubated with endocytosis inhibitors and followed by QD sample. Result shows that uptake of QD(TAT)10 and QD(TAT)75 is inhibited by both MBCD and Cyto-D. This result concludes that uptake of both QD(TAT)10 and QD(TAT)75 occurs via lipid-raft and actin filament dependent pathway. c,d) ICP based evidence of exocytosis of QD(TAT)75 in HeLa cell. Typically cells are incubated with QD for 3 h and kept for next 12/24 h with fresh media. Next, Cd is estimated in cell (c) and culture media(d). Results show that concentration of Cd inside cell decreases with time while it increases in culture media. The mean ± SD of three determinations (n=3) are represented in bars.



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Figure 4. Subcellular localization of QD(TAT)10 and flow cytometry based quantitative estimation of their uptake in presence of different endocytosis inhibitors. a, b) In KB and MCF-7 cell, uptake of QD(TAT)10 in inhibited by MBCD and Cyto-D, suggesting that the uptake occurs predominantly via lipid-raft and actin filament dependent pathway and QD has anisotropic perinuclear localization. c) In case of CHO cell, uptake is inhibited by CHP, MBCD and cyto-D, suggesting that uptake occurs via both clathrin- and lipid raft mediated endocytosis and QD fails to localize near perinuclear region. Cells are labeled with both nuclear probe and QD(TAT)10 and washed cell are imaged under differential interference contrast (DIC) mode or fluorescence (F) mode. Scale bar represents 20 µm. The mean ± SD of three determinations (n=3) are represented in bars.


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Figure 5. Anisotropic perinuclear localization of QD(TAT)10 in CHO cell in presence of CHP. At first cells are incubated with CHP inhibitor for 1 h followed by incubation with QD(TAT)10 for 3 h. Next, washed cells are further treated with fresh media and kept for further 24 h..Imaging results show that blocking of clathrin-mediated pathway offers QD(TAT)10 for anisotropic perinuclear localization. Red colour corresponds to QD and blue colour corresponds to nuclear probe. Scale bar represents 20 µm.



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Figure 6. Low and high magnification fluorescence images of QD(TAT)10 labelled HeLa cells. Typically, cells are labeled with QD and washed cells are further incubated with fresh media and kept for 24 h. Next, cells are incubated with nuclear probe for 10 min and washed cells are used for imaging. Merged images clearly show localization of QD(TAT)10 at asymmetric perinuclear region. Blue color corresponds to nuclear probe and red color corresponds to QD. Scale bar represents 20 µm.


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Figure 7. Colocalization study of QD(TAT)10 in HeLa and KB cell, showing that they localize predominantly at Golgi apparatus. Typically, cells are incubated with nanoprobe for 3h and then washed cells are further incubated with fresh media for 24 h. Next, cells are incubated with Golgi tracker for 30 min and washed cells are used for imaging. Blue excitation is used for imaging of Golgi tracker (green color) and green excitation is used for imaging of QD (red color).Yellow colour indicates colocalization of Golgi tracker and QD nanoprobe. Scale bar represents 20 µm.



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Scheme 2. Proposed endocytosis mechanism of low and high multivalent nanoprobe. Both nanoprobes enter into cell via lipid-raft mediated endocytosis and actin filament dependent pathway. However, modular interaction of lower multivalent nanoprobe with cell membrane offers smaller vesicle formation followed by microtubule dependent trafficking toward Golgi apparatus. In contrast, higher multivalent nanoprobes strongly interact with cell membrane, produces heavily loaded, larger vesicular trap and generate signals for microtubule dependent exocytosis.


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ACKNOWLEDGEMENT The authors acknowledge CSIR, government of India for financial assistance. (No. 02(0249)/15/EMR-II) C.D. acknowledge CSIR, India for providing research fellowship.

ASSOCIATED CONTENT Supporting Information. Details of endocytosis inhibitor concentration, property of functional nanoparticle, control cell labeling experiments and additional experimental results of multivalency effect. This material is available free of charge via the Internet at http://pubs.acs.org. Notes The authors declare no competing financial interests. References (1) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Quantum Dot Bioconjugates for Imaging, Labelling and Sensing. Nat. Mater. 2005, 4, 435-446. (2) Resch-Genger, U.; Grabolle, M.; Cavaliere-Jaricot, S.; Nitschke, R.; Nann, T. Quantum Dots Versus Organic Dyes as Fluorescent Labels. Nat. Methods 2008, 5, 763-775. (3) Murphy, C. J.; Gole, A. M.; Stone, J. W.; Sisco, P. N.; Alkilany, A. M.; Goldsmith, E. C.; Baxter, S. C. Gold Nanoparticles in Biology: Beyond Toxicity to Cellular Imaging. Acc. Chem. Res. 2008, 41, 1721-1730. (4) Jana, N. R. Design and Development of Quantum Dots and Other Nanoparticles Based Cellular Imaging Probe. Phys. Chem. Chem. Phys. 2011, 13, 385-396. (5) Ling, D.; Lee, N.; Hyeon, T. Chemical Synthesis and Assembly of Uniformly Sized Iron Oxide Nanoparticles for Medical Applications. Acc. Chem. Res. 2015, 48, 1276-1285.



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TOC Graphic


Multivalency Effect of TAT-Peptide-Functionalized Nanoparticle in

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