Galactose Multivalency Effect on the Cell Uptake Mechanism of

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C: Physical Processes in Nanomaterials and Nanostructures

Galactose Multivalency Effect on Cell Uptake Mechanism of Bioconjugated Nanoparticle Chumki Dalal, and Nikhil R. Jana J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b08047 • Publication Date (Web): 16 Oct 2018 Downloaded from http://pubs.acs.org on October 22, 2018

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The Journal of Physical Chemistry

Galactose Multivalency Effect on Cell Uptake Mechanism of Bioconjugated Nanoparticle

Chumki Dalal and Nikhil R. Jana* Centre for Advanced Materials and School of Materials Science, Indian Association for the Cultivation of Science, Kolkata 700 032, India *Address for correspondence to [email protected]

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ABSTRACT Galactose functionalized nanomaterials are commonly used for specific targeting/delivery of cancer cell, as galactose receptors are over-expressed in certain types of cancer cells. However, the role of galactose multivalency on cellular interaction, cell uptake mechanism and subcellular targeting are largely unexplored. Here we show that the receptor mediated cellular internalization of galactose terminated nanoparticle depends on galactose multivalency. We have synthesized galactose functionalized multivalent quantum dot (QD) of 15-20 nm hydrodynamic size with the average numbers of galactose per QD of 25, 50 and 80 [designated as QD(gal)25, QD(gal)50, and QD(gal)80] and investigated their uptake mechanism in galactose receptor over-expressed HepG2 cells. We found three distinct effect of galactose multivalency on nanoparticle uptake mechanism. First, cellular interaction and uptake kinetics of nanoparticle increases with increasing galactose multivalency. Second, cell uptake mechanism shifts from predominate lipid raft/caveolae- to predominate clathrin-mediated endocytosis as the nanoparticle multivalency increases from 25 to 50. Third, lower multivalent nanoparticle reside in cytoplasm for longer time (more than 12 h) but their endosomal/lysozomal trapping and exocytosis increases as galactose multivalency increases from 25 to 50. This work demonstrates the functional role of galactose multivalency in cellular processes and may be exploited for subcellular targeting applications.

INTRODUCTION 2 ACS Paragon Plus Environment

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Nanoparticles are widely used in variety of biomedical applications such as bioimaging/detection probe, photodynamic therapy and drug delivery carrier.1-3 As synthesized nanoparticles are transformed to functional nanoparticle and nanobioconjugate for all these applications.2-6 In general these nanoprobes are composed of inorganic core and molecular/polymeric shell along with multiple numbers of covalently attached affinity biomolecules at their surface.7-9 The inorganic core component provides optical/magnetic property, molecular/polymeric shell provides water dispersibility and affinity biomolecule offers interaction with biological interface. As nanoprobes have multiple numbers of affinity biomolecules at their surface, interaction with biological interface is essentially multivalent in nature.5,10 This type multivalent interaction is very common for nanoprobes as compared to conventionally used molecular probes that usually have single interacting point with biological interface.5,10 Earlier works show that multivalency of nanoprobes plays significant role in controlling biological labeling applications. In particular the control of nanoparticle multivalency in the range between 10-100 has significant impact in their cell labeling performance.10 It is shown that cellular interaction increases with increased multivalency

of

TAT

peptides11-13/folate14,15/oligonucleotides16/galactose17-21,

optimum

multivalency 10-25 are reported for RGD peptides22 /HER2 antibody23 and lower multivalency of < 10 often leads to insignificant cellular interaction.10 It is also reported that monovalent nanoprobe can reduce the clustering property of receptors at cell surface.24 However, multivalency effect of nanoprobes in biolabeling application are largely unexplored particularly due to difficulty in their synthesis and most of the commonly designed nanoprobes have uncontrolled multivalency. We have recently reported that nanoparticle multivalency influences their cell uptake mechanism and subcellular trafficking.13,15 We have shown that folate multivalency of < 20 offers modular interaction with folate receptors at cell surface and promotes their perinuclear trafficking via 3 ACS Paragon Plus Environment


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caveolae-mediated endocytosis. In contrast higher folate multivalency ( > 40) induces stronger interaction with folate receptors that induces clathrin mediated endocytosis and lysosomal trafficking.15 Similarly, lower multivalency (< 20) of TAT peptide promotes trafficking of nanoprobes to perinuclear region via lipid-raft endocytosis but higher TAT multivalency (> 20) of nanoprobe induces exocytosis via larger vesicular trapping.13 Here we have studied the galactose multivalency effect on cell-nanoparticle interaction, cellular uptake mechanism and subcellular trafficking. Galactose functionalized nanoparticles are widely used for targeting cancer cells,25 targeted drug delivery26-29/protein delivery30/gene delivery31/SiRNA delivery32 application and in specific tissue engineering scaffold.33,33 It is reported that cellular interaction of nanoparticle depends on the number and density of galactose and the interaction increases with increasing galactose multivalency.20,21 Moreover it is shown that glycan density plays an important role in protein-ligand interaction, cell uptake and

intracellular

localization.34-36 However, role of

galactose multivalency in cellular uptake mechanism of nanoprobe are unexplored. Here we show that galactose multivalency in the range of 25-100 can influence the cellular interaction and uptake mechanism of nanoprobe. We have observed three distinct effect as the galactose multivalency is increased from 25 to 100. First, cellular interaction and uptake kinetics increases with increasing multivalency. Second, cell uptake mechanism of nanoprobe shifts from predominate lipid raft/caveolae-mediated endocytosis to predominate clathrin-mediated endocytosis. Third, residence of nanoprobe inside cell decreases with increasing galactose multivalency as exocytosis opens up with higher multivalency. EXPERIMENTAL SECTION Materials. Cadmium oxide, tri-octyl phosphine, tri-octyl phosphine oxide, stearic acid, zinc stearate, sulfur powder, selenium powder, poly(ethylene glycol) methacrylate, 3-sulfopropyl 4 ACS Paragon Plus Environment

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methacrylate,

N,N-methylene-bis(acryl

amide),

D-lactose

monohydrate,

sodium

cyanoborohydride, anthrone, chlorpromazine hydrochloride, genistein, amiloride hydrochloride, methyl-β-cyclodextrin, sucrose, sodium azide, deoxyglucose, nocodazole and Dulbecco’s modified Eagle’s medium (DMEM) culture medium were purchased from Sigma-Aldrich and used as received. Hoechst, NBD C6 ceramide (Golgi tracker), mitotracker red and lysotracker red were purchased from Life Technology. Synthesis of polyacrylate coated QD(NH2)120. CdSe-ZnS-based hydrophobic QD was synthesized using earlier reported method.37 Then hydrophobic QD was converted into hydrophilic QD via polyacrylate coating using our reported method.9,10 Here, we have used N-(3aminopropyl)-methacrylamide

hydrochloride

that

provides

primary

amine

group,

3-

sulfopropropyl methacrylate that offers anionic charge, poly(ethylene glycol) methacrylate that provides 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 average of ~120 primary amines per QD. Next, aqueous solution of ammonium persulfate was added to initiate the polymerization reaction. Then the reaction was continued for 15-20 min under inert atmosphere and ethanol was added to stop the polymerization reaction by precipitating QD. Resultant QD(NH2)120 was repeatedly washed with chloroform, ethanol and dimethyl formamide (DMF) and finally dissolved in distilled water. In order to remove the free polymer, the chloroform was added to this aqueous solution followed by shaking for 2-3 minute. After 2-3 h of waiting, some white precipitate was observed at chloroform-water interface. Supernatant QD solution was collected from top of the solutions and finally dialyzed against fresh water by using cellulose membrane (MWCO

12000 Da) to remove unreacted reagents. No free polymer is

observed in this solution which is confirmed by GPC. 5 ACS Paragon Plus Environment


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Synthesis of QD(gal)25, QD(gal)50 and QD(gal)80. In order to synthesize QD(gal)80, one mL aqueous solution of QD(NH2)120 (15 mg/mL) was prepared with the QD absorbance value of 0.8 at 590 nm. Next, 10 mg lactose was mixed followed by addition of 20 mg solid NaBH3CN and the solution was stirred for 10 h. Finally, solution was dialyzed against fresh water by using cellulose membrane (MWCO

12000 Da) to remove unreacted reagents. In order to prepare QD(gal)25,

QD(NH2)120 was dispersed in DMF with the concentration of same as earlier (15 mg/mL). Next, 2 mg lactose was added followed by addition of 20 mg solid Na(CN)BH3 and solution was stirred for 1 h. Resultant QD(gal)25 was precipitated by centrifuge (14000 rpm) and dissolved in fresh water. Finally, QD(gal)25 solution was dialyzed (MWCO 12000 Da) against fresh water to remove excess reagents. QD(gal)50 was prepared using same condition except that the reaction time is increased to 4 h. Next, solution was dialyzed against fresh water by using cellulose membrane (MWCO 12000 Da) to remove unreacted reagents. Quantum yield (QY) measurement. The quantum yield of QD samples were measured using quinine sulfate (QY = 58 % at 354 nm excitation) as reference. The formula used for QY measurements is as follows(QY)sam= (QY)ref × [ (PL area / A)sam/ (PL area / A)ref] × η2sam / η2ref Where ‘sam’ indicates the sample, ‘ref’ indicates the standard, η is the refractive index of the solvent, PL area represents fluorescence area and A represents absorbance value. Estimation of the number of galactose per QD. Detection and quantification of galactose was performed using anthrone test.38 In brief, a stock solution of anthrone was prepared by dissolving 2 mg anthrone in 1 mL of 80 % sulphuric acid. Next, 100 µL of galactose solution was added to it, heated in boiling water bath for 10-15 min and cooled in ice bath. Then, absorbance at 622 nm was measured. A linear calibration curve was obtained by plotting the absorbance against 6 ACS Paragon Plus Environment

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concentration of galactose. The linear equation obtained was Y = 4528×X with R2 value 0.99. (X = concentration of galactose and Y = absorbance at 622 nm). Next, anthrone test was performed using galactose functionalized QD and the absorbance at 622 nm was measured and the concentration of galactose was determined using the above mentioned calibration curve. The concentration of QD in those solutions were separately determined using the molar extinction coefficient value of QD reported earlier.39 Finally, the number of galactose per QD was calculated from the ratio of molar concentration of galactose and QD. In second approach the number of galactose per QD was estimated by calculating the number of primary amines per QD before/after galactose functionalization, using the fluorescamine test. At first, fluorescamine based titration method has been used to calculate the number of primary amines per QD.10 The obtained calibration curve was as follows: Y= 0.65× 10-6 X – 12.81× 10-6 where X is the fluorescence intensity and Y is the primary amine concentration with R2 value of 0.99. Next, functional QD was treated with concentrated HCl followed by neutralizing with NaOH and treated with fluorescamine. Number of primary amine per QD was estimated before and after functionalization of galactose. Number of galactose per QD was calculated from the difference of number of primary amine before and after galactose functionalization. In third approach the number of galactose molecule per nanoparticle was calculated using gel permeation chromatography (GPC). Molecular weights of QD(NH2)120, QD(gal)25 and QD(gal)80 were determined via GPC and then increased molecular weight due to galactose conjugation was used to calculate the number of galactose per QD. Molecular weights of (QD(NH2)120, QD(gal)25 and QD(gal)80 were determined as 150kDa, 160 kDa and 180 kDa, respectively. Assuming that one galactose conjugation can increase ~ 326 Da molecular weight, the increase of 10 kDa accounts for ~30 galactose and increase of 30 kDa accounts for ~90 galactose. 7 ACS Paragon Plus Environment


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Cell labeling and imaging study. HepG2 cells (liver cancer cell) were used as galactose receptor over-expressed cells and HeLa cells (cervix cancer cell) were used as negative control. 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 cell imaging study cells were cultured in 24 well plates for overnight with 500 µL of serum free cell culture media and then 50-100 µL QD nanoprobe was added followed by 30 min to 3 h incubation. Next, cells were washed with PBS buffer to remove unbound particle and washed cells were used for imaging study. For localization study washed cells were further incubated with fresh media for next 6-24 h and used for imaging study. For colocalization study cells were incubated with nuclear probe/lysotracker red/Golgi tracker/mitotracker red for 10-30 min and washed cells were used for imaging. QD labeled cells were imaged in fluorescence microscope under different excitation and emission windows. UV excitation uses light of 330-385 nm band and emission band filter ranges from 400-800 nm, blue excitation uses light of 420-480 nm band and emission band filter ranges from 500-800 nm and green excitation uses light of 480-550 nm band and emission band filter ranges from 590-800 nm. For the colocalization study with lysotracker red/mitotraker red, we have used green emissive QD where blue excitation is used for collecting green fluorescence of QD and green excitation is used for collecting red fluorescence from lysotracker red/mitotracker red. For exocytosis study, cells were cultured in serum free cell culture media. Cells were incubated with QD nanoprobe for 3 h. Next, cells were washed with PBS buffer to remove unbound particles and kept for next 6-18 h with fresh media. At the intervals of 3 h, 6 h, 12 h and 18 h cells were incubated with trypsin-EDTA for 2-3 min and cells are isolated by centrifuge. Finally cells were dissolved in PBS buffer for flow cytometry study and at 10 % suprapure HNO3 for inductively coupled plasma atomic emission spectroscopy (ICP-AES) based study. 8 ACS Paragon Plus Environment

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To study the energy/cholesterol/microtubule dependency on exocytosis we have used mixture of sodium azide and deoxyglucose (that blocks energy dependent pathway) or methyl-β-cyclodextrin (inhibits cholesterol dependent pathway) or nocodazole (that inhibits microtubule polymerization). Typically, cells were incubated with QD nanoprobe for 3 h and then washed cells were further treated with above mentioned inhibitors and kept for next 18 h with fresh media. Finally, QD labeling was investigated by flow cytometry/ICP-AES. Endocytosis inhibition study. In order to investigate the endocytosis mechanism of nanoprobe, cells were cultured in serum free DMEM media and pre-treated with different endocytosis inhibitors for 1 h with their desired concentration. (Supporting Information, Table 1) We have used methyl-β-cyclodextrin (MBCD) that inhibits lipid-raft mediated endocytosis, genistein (GEN) that blocks caveolae-mediated endocytosis, chlorpromazine (CHP) and sucrose that blocks clathrinmediated endocytosis and amiloride (AMI) that blocks macropinocytosis. Next, nanoprobe solution was added into cell culture media and cells were incubated for next 1-2 h. Then, cells were washed with PBS buffer to remove unbound QD and treated with trypsin-EDTA for 2-3 min to detach cells from plate. Finally detached cells were isolated by centrifuge and dispersed in PBS buffer and used for flow cytometry based study. Instrumentation. UV-visible absorption spectra were measured using Shimadzu UV-2550 UVvisible spectrophotometer and fluorescence spectra were measured using BioTek SynergyMx microplate reader and Perkin Elmer LS 45. DLS and zeta potential were measured by Malvern Nano ZS instrument. Transmission electron microscope (TEM) study was carried out using FEI Tecnai G2 F20 microscope by putting a drop of nanoparticle solution on carbon coated copper grid. ICP-based quantification was performed using Optima 2100DV inductively coupled plasma atomic emission spectroscopy (ICP-AES, PerkinElmer). Fluorescence images were captured by 9 ACS Paragon Plus Environment


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Olympus IX 81 microscope and fluorescence based quantification study was done using BD Accuri C6 Flow Cytometer.

RESULTS Synthesis of nanoparticle with average galactose multivalency of 25, 50 and 80. Synthesis strategy of galactose functionalized QD nanoprobe with variable multivalency is shown in Scheme 1. Hydrophobic QD is transformed into hydrophilic primary amine terminated QD of 15-20 nm hydrodynamic size via polyacrylate coating. The average number of primary amine per QD is adjusted to ~120, using appropriate ratio of amine monomer and denoted as QD(NH2)120. (Supporting Information, Figure S1) The polyacrylate shell is also made of anionic sulfate and nonionic polyethylene glycol (PEG) group in addition to primary amine functional group so that non-specific interaction with cell surface is minimized. Next, primary amine groups present on polyacrylate shell surface have been used for galactose functionalization. Lactose has been used for galactose functionalization which is a disaccharide made of galactose and glucose. Reducing end of lactose is covalently attached with primary amines via reductive amination. Next, sodium cyanoborohydride is added to reduce the imine bond. We have synthesized galactose functionalized QD with average multivalency of 25, 50 and 80 and they are designated as QD(gal)25, QD(gal)50 and QD(gal)80.(Scheme 1) The number of galactose per QD has been controlled between 25 and 80 by changing reaction medium and reaction time. In one approach DMF is used as reaction medium where QD(NH2)120 insoluble and primary amine groups are partially accessible for the reaction.(Table 1) This approach produces QD(gal)25 and QD(gal)50 where the average multivalency is controlled by changing the reaction time. Higher multivalent


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QD(gal)80 is prepared in water where QD(NH2)120 is soluble and primary amine groups are fully accessible for reaction. Property of galactose functionalized nanoparticles is summarized in Table 1, Figure 1a, Figure 1b and Supporting Information, Figure S2. TEM image of QD(NH2)120 shows the QD core of 3-5 nm size and dynamic light scattering (DLS) study shows 15-20 nm hydrodynamic size. Surface charge of these particles are negative (between -16 mV to -23 mV) mainly due to anionic sulfopropyl groups. Fluorescence quantum yield (QY) of QD is measured before and after galactose functionalization, using quinine sulfate as reference. In all cases QY is about 16 % and not affected by the galactose valency. This is particularly due to nature of QD having CdSe core with ZnS shell. The galactose bound to QD has been estimated using anthrone test.(Figure 1c and Supporting Information, Figure S3) Separately, QD concentration is determined from the molar extinction coefficient of QD. Next, the number of galactose per QD (i.e. galactose multivalency) has been determined from the concentration ratio of galactose and QD. In addition, the number of primary amine per QD has been estimated (by fluorescamine test) before and after the galactose functionalization. (Figure 1d) Next, the number of QD bound galactose is determined from the difference in the number of primary amines. Moreover, molecular weight of QD(NH2)120 has been determined via GPC, before and after the galactose functionalization. Next, the number of galactose per QD is determined from the difference of molecular weight. (Supporting Information, Figure S4) The biological activity of QD bound galactose has been tested using recinus communis agglutinin (RCA120) glycoprotein-based precipitation experiment. (Supporting Information, Figure S5) Galactose functionalized QD binds with RCA120 and leads to visible aggregation of QD from their solutions. Control experiment shows that QD without galactose functionalization, does not



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precipitate from solution. This result further concludes that galactose is successfully conjugated and their biological activity is retained. Increased galactose multivalency enhances receptor mediated cell uptake of nanoparticle. At first we have tested that galactose functionalized QD selectively label HepG2 cells which have over-expressed galactose receptors.

(Figure 2a) In one control experiment cell labeling is

performed using QD without any galactose functionalization and result show that QD cannot label HepG2 cell. In another control experiment galactose functionalized QD has been used to label HeLa cells which do not have over-expressed galactose receptors and result show that QD cannot label such cells. (Figure 2b) In another control experiment, labeling of galactose functionalized QD is performed in HepG2 cell in presence of free galactose molecule and result show that QD cannot label cells. (Figure 2c) All these control experiments conclude that the labeling of galactose functionalized QD to HepG2 cells is very selective and occurs via interaction with galactose receptors. Next, we have investigated the effect of galactose multivalency on cellular interaction and uptake kinetics. (Figure 3 and Supporting Information Figure S6, Figure S7) Typically, HepG2 cells are incubated with same concentration of QD(gal)25/QD(gal)50/QD(gal)80 for 30 min/1h/3h and washed cells are used for fluorescence imaging and flow cytometry study. Results show that QD(gal)25 need more than 1h to label cells but cell labeling by QD(gal)50/QD(gal)80 saturates within 30 min. Results clearly show that uptake of QD increases with increased galactose multivalency. Increased galactose multivalency shifts cell uptake mechanism of nanoparticle from predominate lipid raft/caveolae-mediated endocytosis to predominate clathrin-mediated endocytosis. We have studied the internalization of galactose functionalized QD in HepG2 cells


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at 4 ºC. (Supporting Information, Figure S8) Insignificant cellular uptake of nanoprobe confirms the energy dependent endocytosis mechanism. Next, we have investigated the galactose multivalency effect on cell uptake mechanism. (Figure 4a, 4b and Supporting Information, Figure S9) Typically, HepG2 cells are pretreated with endocytosis inhibitors of desired concentration followed by QD sample incubation. Next, washed cells are used for imaging and flow cytometry based study. It is observed that irrespective of nanoparticle multivalency, uptake of all the QDs is inhibited by MBCD. Additionally, uptake of QD(gal)25 is partially inhibited by GEN. In contrast, uptake of QD(gal)80 is significantly inhibited by CHP and sucrose. These results conclude that uptake of galactose functionalized nanoprobe occurs via cholesterol dependent lipid raft pathway. However, with increased galactose multivalency an additional clathrin-mediated endocytosis pathway opens up. In order to further investigate the endocytosis pathway of QD(gal)80, another control experiment has been performed using mixture of

inhibitors (CHP and MBCD).

(Supporting Information, Figure S10) Results shows that uptake of QD(gal)80 is completely inhibited in presence of both CHP and MBCD. Results clearly indicate that both lipid-raft and clathrin-mediated endocytosis is operative for QD(gal)80. Increased galactose multivalency stimulates exocytosis that decreases cytosolic residence of nanoparticle. We have observed that longer cytosolic residence of nanoprobe depends on galactose multivalency. (Figure 5 and Supporting Information, Figure S11 and Figure S12) Fluorescence imaging of labeled cells at different time interval shows that QD(gal)25 enters into cells and reside inside the cell for 18 h until cells are divided. In contrast emission of QD(gal)80 labeled cells decreases by 50 % after 6 h. We have ensured that decrease of this fluorescence is not due to fluorescence quenching effect of QD by lysosomal pH. The fluorescence stability of the galactose functionalized QDs has been studied in two different buffer solution of pH 7.4 and 4.5.



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(Supporting Information, Figure S13) No significant change of fluorescence intensity is observed, suggesting that fluorescence of galactose functionalized QD is stable in both cytosolic and lysosomal pH. Next, we have investigated the origin of such decrease in QD fluorescence via flow cytometry of labeled cells and ICP-AES-based Cd estimation inside/outside of the cells. (Figure 4c and Figure 4d) Results clearly show that emission of QD(gal)80 labeled cells decreases significantly (by 80 %) within 12 h but fluorescence of QD(gal)25 labeled cells decreases slightly (25 %) upto 18 h. Results also conclude that concentration of Cd of QD(gal)80 inside cell decreases gradually by 80 % and concentration of Cd in cell culture media increases by 50 %. In contrast, concentration of Cd of QD(gal)25 labeled cells slightly decreases (20 %) with time and no significant change of Cd is observed in culture media. These results indicate that QD(gal)80 enter into cells but then exocytosized within 12 h. In contrast QD(gal)25 resides inside cell for longer time. In order to investigate the role of clathrin-mediated endocytosis in the exocytosis processes, we have blocked the clathrin-mediated endocytosis of QD(gal)80 and observed their presence inside cell for longer time. Results show that fluorescence of QD(gal)80 labeled cells remain intact even after 18 h, suggesting the blocking of their exocytosis. (Supporting Information, Figure S14) In addition we have tested that exocytosis of QD(gal)80 occurs via energy dependent pathway. (Supporting Information, Figure S15) Typically, QD(gal)80 labeled cells are incubated with combination of sodium azide and deoxyglucose and found that exocytosis of QD(gal)80 is inhibited. Moreover, we have further investigated that exocytosis occurs via cholesterol and microtubule independent pathway. (Supporting Information, Figure S16 and Figure S17) In another control experiment we have investigated the pathway of exocytosis via time dependent colocalization study of QD(gal)80 with lysotracker red.(Supporting Information, Figure S18)


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Typically, cells are incubated with QD sample for 3 h and washed cells are incubated in fresh culture media for next 18 h and fluorescence imaging is performed at different time interval by treating with lysotracker red. Results show that QD(gal)80 is trapped at lysosome after 6 h, suggesting that excessive lysosomal trafficking of QD(gal)80 may drive their exocytosis. Lower galactose multivalency offers longer cytosolic residence of nanoparticle. We have extensively studied subcellular localization of QD(gal)25. (Figure 6) It is observed that QD(gal)25 is not localized at any subcellular compartment. First, cells are labeled with both QD(gal)25 and nuclear probe and fluorescence imaging is performed at different z-planes. Results show that QD(gal)25 resides in cytoplasm and does not localize at nucleus or at perinuclear region. (Supporting Information, Figure S19). Next, colocalization study has been performed using Golgi tracker, mitotracker red and lysotracker red. (Figure 6) Results show that QD(gal)25 does not colocalize with any of these tracker, suggesting that QD(gal)25 resides at cytoplasm without trafficking to any subcellular compartment.(Figure 6 and Supporting Information, Figure S20) Time dependent colocalization study of QD(gal)25 with lysotracker red is performed.(Supporting Information, Figure S21) Typically, cells are incubated with QD(gal)25 for 3 h and washed cells are further incubated with fresh media for next 18 h. Next, fluorescence imaging is performed to different time interval by treating with lysotracker red. Results show that QD(gal)25 does not colocalize with lysotracker red at any time point, suggesting that QD(gal)25 never trafficked at lysosome. This result concludes that lower galactose multivalency offers longer residence of nanoparticle inside cytosol, without trafficking to any subcellular compartments.

DISCUSSION



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Galactose receptors are over-expressed in certain cancer cell surface. These receptors interact with galactose, enter into cells during endocytosis and deliver bound galactose in the area surrounding nucleus. The internalized receptor then returns to the cell surface after delivering the galactose.40 It is shown that galactose functionalized nanoparticle can enters into cells via receptor mediated endocytosis.25-33 It is also shown that the number and density of galactose is important for their recognition with receptor17 and depending the galactose density, the clustering of receptor occurs at cell surface.17 Here we have designed a nanoprobe with varied galactose multivalency, typically in the range of 25-80 and observed their effect on cellular uptake performance. On the basis of our results we propose galactose multivalency dependent cell uptake mechanism and subcellular trafficking of galactose functionalized nanoparticle. (Scheme 2) Lower multivalent QD(folate)25 enters into cells via lipid-raft/caveolae mediated endocytosis pathway. The lower multivalency offers modular interaction with galactose receptor and directs the nanoprobe towards caveolae where galactose receptors are concentrated. This event leads to the modular clustering of galactose receptor that induces caveolae-mediated endocytosis, bypassing the lysosomal trapping and delivering to cytoplasm. In contrast, higher multivalent QD(gal)80 enters into cells via lipidraft/caveolae as well as clathrin-mediated endocytosis. Interaction of QD(gal)80 involves more numbers of galactose receptor due to increased galactose density at the nanoparticle surface.20,21 This leads to stronger interaction that induces clathrin-mediated endocytosis pathway followed by trapping with lysosome and exocytosis. The proposed mechanism can explain the inconsistency of receptor mediated endocytosis mechanism and subcellular targeting of galactose functionalized nanoparticle. Earlier work shows that galactose functionalized nanoparticles enter into cells via caveolae/lipid-raft, clathrin- or both caveolae and clathrin mediated endocytosis pathway.19,26,29 It is shown that most of the galactose 16 ACS Paragon Plus Environment

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functionalized nanoprobe is trapped in endosome/lysosome,20,25 and in some cases galactose functionalized nanoprobe reside at cytoplasm21,25 or localized at perinuclear region.32 In those cases multivalency effect of galactose is neither determined nor considered and possibly this effect contributed in the results. Although bioconjugated nanoparticles are widely used for biolabeling application the role of multivalency in cell uptake mechanism is largely unexplored. Here we have shown the effect of galactose multivalency on cell uptake mechanism of nanoparticle. We found that galactose multivalency controls the cellular interaction, uptake kinetics, cellular entry/exit mechanism and subcellular trafficking. In particular lower multivalency offers the advantage of bypassing lysosomal trapping of nanoparticle with longer residence in cytoplasm. This observation is similar to our earlier result where lower TAT-peptide multivalency offers efficient perinuclear targeting of nanoparticle but higher TAT-peptide multivalency induces their exocytosis and lower folate multivalency offers efficient perinuclear targeting of nanoparticle but higher folate multivalency induces lysosomal trapping. Thus lower multivalency in the range of 10-25 is critical for subcellular targeting of nanoparticle. In particular longtime cytosolic residence of biomacromolecule without trafficking to lysosome is the requirement for them to perform their biological function.25 CONCLUSION We have shown that galactose multivalency of nanoparticle directs their interaction with cell, cellular entry/exit mechanism and cytosolic residency. We found that galactose receptor mediated cellular internalization mechanism of nanoparticle shifts from lipid raft/caveolae- to clathrinmediated endocytosis, as the nanoparticle multivalency increases from 25 to 50. While lipid



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raft/caveolae-mediated uptake delivers nanoparticle into cytosol without trapping with lysosome, clathrin-mediated endocytosis traffics them to the lysosome followed by exocytosis. This work shows the functional role of galactose multivalency and importance of lower galactose multivalency (typically

Galactose Multivalency Effect on the Cell Uptake Mechanism of

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