Arginine Terminated, Chemically Designed Nanoparticle for Direct

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Arginine Terminated, Chemically Designed Nanoparticle for Direct Cell Translocation SANTU GHOSH, Prasanta Panja, Chumki Dalal, and Nikhil R. Jana ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00617 • Publication Date (Web): 29 Nov 2018 Downloaded from http://pubs.acs.org on November 30, 2018

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ACS Applied Bio Materials

Arginine Terminated, Chemically Designed Nanoparticle for Direct Cell Translocation Santu Ghosh, Prasanta Panja, 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]

Abstract: Direct cell translocation of nanomaterials is preferred over the endocytotic uptake for various sub-cellular targeting applications that can bypass the lysosomal trafficking/degradation. Although arginine rich cell penetrating peptides are routinely used for cell transfection of wider range materials from molecule to nanoparticle, the direct cell translocation of nanoparticle is not a routine approach, particularly due to their predominate endocytotic uptake. Here we report arginine terminated, designed nanoparticle of 15-30 nm hydrodynamic size that enters into cell via direct translocation. We found that direct cell translocation of nanoparticle is very efficient without localization at any specific sub-cellular compartment for 12-24 h. This study shows that nanomaterial can be chemically designed for direct cell translocation and for cytosolic delivery without any biomembrane-coated endosome that can be employed for subcellular targeting applications.

Keywords: nanoparticle, endocytosis, direct translocation, arginine, guanidinium, multivalency

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Introduction Arginine-rich cell penetrating peptides can efficiently enter into cell and transport wide range of cargoes including small molecules,1-3 macromolecules4-6 and nanoparticle.7-10 Extensive research shows that guanidinium groups present on arginine is actually responsible in this transport function.2,3 In addition arginine-terminated nanoparticle is used for cell delivery of macromolecules.11 According to the current understanding, the arginine-rich peptides enter into cell via two different mechanisms. In one mechanism, the arginine-rich peptides directly translocate through cell membrane via energy independent and non-endocytotic approach.12-15 The anionic sulphate/phosphate/carboxylate groups at the cell surface are involved in the electrostatic interaction with guanidinium groups of arginine and such interaction along with proton gradient around cell membrane drives this membrane translocation.12,15 In other mechanism arginine-modified polymer and nanoparticle enter into cell via energy dependent endocytosis16-20 that include lipid raft–dependent macropinocytosis,16 clathrin-mediated endocytosis and lipid raft-mediated endocytosis.20 However, factors responsible for this division between direct translocation versus endocytosis and the origin of different types of endocytosis (e.g. macropinocytosis, clathrin mediated endocytosis, lipid raft) are largely unsolved and direct cell translocation is not used as a routine approach. It is now well established that cellular endocytosis of foreign materials need a critical size requirements, typically > 10 nm.21 It is shown that nanoscale organization is critical prior to endocytosis of protein,22 nanoparticle enters into cell via clathrin-/lipid raft-/caveolae mediated endocytosis23 and larger size particle enters via macropinocytosis.24 Similarly, endocytotic uptake of arginine-rich peptides is reported to depend on their self-assembly property.25,26 In addition multivalency of arginine/guanidinium-based peptide is reported to influence the endocytosis processes of cargo material. For example, uptake of nanoparticle is shown to depend on the number of RGD peptide per nanoparticle with the maximum for 20 RGD,7 the uptake of nanoparticle is maximum for 10-20 TAT peptide per nanoparticle27 and lower TAT multivalency in the range of 10-20 offers efficient perinuclear targeting of nanoparticle via lipid raft endocytosis.20 These results suggest that nanometer length scale of cargo material and arginine multivalency has significant role in the cell uptake mechanism of arginine-rich cell penetrating peptides. Moreover cell uptake of these nanoparticles occur via energy dependent endocytosis processes, except in certain selected cases.10,11 2 ACS Paragon Plus Environment

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Here we report a designed nanoparticle that can enter into cell via energy independent direct translocation processes. Nanoparticle is 15-30 nm hydrodynamic size and terminated with multiple numbers of arginine. Compared to energy dependent endocytotic uptake of nanoparticle, the direct cell translocation reported here has the advantages of rapid and efficient cell uptake, longer cytosolic distribution and insignificant lysozomal trafficking. This study shows that appropriate size and surface chemistry of nanomaterial are critical for energy independent direct cell translocation. There are three distinct advantages of presented approach as compared to earlier reported arginine-rich peptide-based cell delivery and direct cell translocation of nanoparticle. First, nanoparticle is terminated with multiple numbers of molecular arginine as compared to argininerich peptide in most reported cases. Second, nanoparticle enters into cell via energy independent direct cell translocation as compared to commonly reported energy dependent endocytotic uptake.16-20 This direct cell translocation has significant advantage that offers direct cytosolic transfer of nanoparticle with insignificant endosomal/lysozomal trafficking. In contrast endocytotic uptake occurs via biomembrane-coated endosome and limits sub-cellular targeting applications. Third, presented direct cell translocation is simple as compared to reported direct cell translocation of nanomaterials that uses arginine-rich peptide with poly(disulfide) linkage10 or direct translocation of protein via self-assembly with arginine-terminated Au nanoparticle.11 Thus presented direct cell translocation approach is simple and can be easily adapted to various nanomaterials for the development of sub-cellular targeting nanoprobe and drug delivery carrier with minimum lysosomal degradation.

Experimental Section Chemicals. Cadmium oxide, trioctyl phosphine, trioctyl phosphine oxide, stearic acid, zinc stearate, sulfur powder, selenium powder, N-(3-aminopropyl) methacrylamide hydrochloride (amine-acrylaye), poly(ethylene glycol) methacrylate (PEG-acrylate), glycidyl methacrylate, N,N-methylene-bis-(acrylamide) (bis-acrylate), L-arginine, glyceraldehyde, ethylene glycolbis(succinic acid N-hydroxysuccinimide ester) {NHS-PEG-NHS}, Dulbecco’s modified Eagle’s culture medium (DMEM), 2-deoxy-D-glucose (deoxy-glucose) and sodium azide (NaN3) amiloride

hydrochloride,

genistein,

sucrose,

chlorpromazine

hydrochloride,

methyl-β-

cyclodextrin and methyl thiazolyl diphenyl-tetrazolium bromide (MTT) were purchased from 3 ACS Paragon Plus Environment


Sigma-Aldrich and used as received. Hoechst and lysotracker red were purchased from life technology. Synthesis of arginine terminated quantum dot. Hydrophobic CdSe/ZnS core-shell quantum dots (QD) with red emission is transformed into arginine terminated QD via polyacrylate coating.28 In particular arginine conjugated acrylate (arginine-acrylate) is used as monomer during polyacrylate coating. (Supporting Information, Figure S1) At first arginine-acrylate is synthesized by mixing 15 µL glycidyl methacrylate (dissolved in 0.2 mL DMSO) with 17.4 mg arginine (dissolved 0.2 mL borate buffer of pH 9.0) under stirring. After overnight reaction, arginine-acrylate was precipitated by adding acetone. Finally, the precipitate was dissolved in 0.2 mL water and used for polyacylate coating. Next, hydrophobic QD with red emission was prepared, purified from excess surfactant and a stock solution is prepared in 2 mL cyclohexane. To this vial 1.5 mL Igepal and 4.5 mL cyclohexane were added. The QD concentration is adjusted by maintaining the absorbance of 0.40 at 571 nm. Next, 0.2 mL aqueous solution of arginine-acrylate, 36 µL PEG-acrylate and 1.5 mg bis-acrylate were added. The mixed solution was sonicated for 5 min and then stirred under nitrogen atmosphere for 15 min. Next, polymerization was initiated by adding 0.1 mL tetramethyl ethylenediamine and 5 mg ammonium persulfate (dissolved in 0.1 mL water). Polymerization was continued for 40 min and reaction was quenched by adding minimum ethanol to precipitate the particles. QDs were repeatedly washed with chloroform and ethanol and then dispersed in 2 mL double distilled water. Finally, polyacrylate-coated QD solution was dialyzed overnight against fresh water by using a cellulose membrane (MWCO ∼12 000 Da) to remove unreacted reagents. In other approach hydrophobic QD is transformed into polyacrylate coated QD and then conjugated with arginine. Hydrophobic QD is dissolved in Igepal-cyclohexane reverse micelle along with acrylate monomers, as described above. In one case 30 mg amine-acrylate and 1.5 mg bis-acrylate were used. In other case 36 µL PEG-acrylate, 30 mg amine-acrylate and 1.5 mg bisacrylate were used. The mixed solution was then sonicated for 5 min and then stirred under nitrogen atmosphere for 15 min and polymerization was initiated by adding 0.1 mL tetramethyl ethylenediamine and 5 mg ammonium persulfate (dissolved in 0.1 mL water). Polymerization was continued for 40 min and reaction was quenched by adding minimum ethanol to precipitate 4 ACS Paragon Plus Environment

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the particles. QDs were washed with chloroform and ethanol and dispersed in 2.0 mL double distilled water. Finally, polyacrylate-coated QD solution was dialyzed overnight against fresh water by using a cellulose membrane (MWCO ∼12 000 Da) to remove unreacted reagents. The resultant stock solution was used for arginine conjugation. Arginine was covalently conjugated with the surface exposed primary amine groups of polyacrylate coated QD using NHS-PEGNHS as coupling reagent. In brief, 0.15 mL borate buffer (pH 9.0) solution of arginine (12 mg/mL) and 0.15 mL DMF solution of NHS-PEG-NHS (31 mg/mL) were mixed and stirred for 2 min. Next, this mixture was added drop wise to 0.5 mL polyacrylate coated QD solution. The reaction was continued for overnight and finally the solution was dialyzed against fresh water to remove unbound reagents. In one of the case (where no PEG-acrylate is used) glyceraldehyde conjugation is used before dialysis to consume all the primary amines. Typically, 13 mg glyceraldehyde and 10 mg NaCNBH3 were added and reaction was continued for overnight. Finally, the solution was dialyzed overnight. Measuring the number of arginine per QD by estimating guanidinium groups. 9, 10phenanthrenequinone based fluorimetric titration method was used for the determination of arginine concentration.29 Firstly, ten different known concentrations of arginine in the range of 20-350 µM were prepared. Next, 50 µL of each solution was mixed with 150 µL of an ethanolic solution of 9, 10-phenanthrenequinone (10-6 M) and 25 µL of NaOH solution (2.0 N). These mixtures were incubated at 60 °C for 3 h and then 100 µL of each sample was mixed with 100 µL (1.2 N) HCl and allowed to stand for one hour at room temperature under dark. After that fluorescence intensity of each sample was measured at 397 nm (by exiting at 312 nm) in a fluorescence spectrometer and a calibration curve (Y = 3.15× 10-6 X - 93.68× 10-6 with R2 = 0.996, where Y is the arginine concentration in µM and X is the fluorescence intensity) was prepared by plotting the fluorescence intensities against the arginine concentration. Similarly, 50 µL of arginine conjugated QD solution was used to measure the fluorescence and arginine was estimated using the calibration curve. The molar concentration of QD was calculated using the equations reported earlier.30 In brief the diameter of QD was calculated using the peak position of the first excitation absorption peak (571 nm) and then molar extinction coefficient of QD was calculated for the respective diameter. Next, Lambert-Beer’s Law was used to estimate the molar concentration of QD. After knowing both the arginine and QD concentration, the number of arginine per QD has been determined from the molar ratio of arginine and QD. 5 ACS Paragon Plus Environment


Estimation of the number of primary amine per QD. Fluorescamine based titration method has been used to quantify the average number of primary amines per QD. First, fluorescamine test was performed using glycine as a standard in the range of 20-250 µM. Typically, 100 µL of glycine solution was added to 800 µL borate buffer solution of pH 9. Then 100 µL of acetone solution of fluorescamine (10-2 M) was mixed and the sample was transferred into a fluorimeter and fluorescence was measured at 493 nm (by exciting at 400 nm). A linear calibration curve was obtained by plotting the fluorescence intensity with respect to the concentration of glycine. The linear equation was obtained as follows: Y = 0.65× 10-6 X -12.81× 10-6 with R2 = 0.99. (Y is the glycine concentration in µM and X is the fluorescence intensity). Similarly, fluorescamine test was performed for polyacrylate coated QDs after dissolving it by adding few drops of concentrated HCl (1M), followed by neutralization with NaOH. The fluorescence intensity was then measured at 493 nm and primary amine concentration was calculated from the calibration curve. Separately, the concentration of QDs was measured by using the QD absorbance at 571 nm as described before.30 Next, the number of primary amines per QD has been determined from the molar ratio of primary amine and QDs. Cell labeling study. We have used three types of cells such as HeLa (human cervical cancer cell line), KB (mouth cancer cell line) and HT-22 (mouse hippocampal neuronal cell line) in our study. Cells were cultured in DMEM medium with 10 % heat activated fetal bovine serum (FBS) and 1 % penicillin streptomycin at 37 °C and 5 % CO2 atmosphere. Next, cells were cultured overnight in a 24-well plate with 500 L serum-free DMEM medium and then 10100 L QD sample was added followed by 1 h incubation. After incubation, cells were washed with PBS buffer solution and washed cells were used for imaging study or further incubated with fresh medium for 1-24 h for long term localization study. All the labeling/imaging experiments were repeated for more than 3 times using independently produced QD samples. For co-localization study, cells were cultured in 24 well plates for 24 h in serum free DMEM medium. Next, cells were incubated with QD samples for 1 h. Then, cells were washed and mixed with fresh media and kept for another 3-24 h for QD localization. Next, cells were incubated with lysotracker red for 30 min, and washed cells were imaged under microscope. For quantitative measurement, cells were treated with trypsin-EDTA and detached cells were 6 ACS Paragon Plus Environment

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isolated by centrifuge. Finally, cells were dispersed in PBS buffer and used for Flow Cytometry study. Energy dependent endocytosis study. Cells were seeded in a 24 well plate in DMEM medium with 10 % heat activated fetal bovine serum (FBS) and 1 % penicillin streptomycin at 37 °C and 5 % CO2 atmosphere. Next, cells were incubated with 500 μL of serum-free cell culture media at 37 °C in presence of NaN3 (10 mM final concentration) and deoxy-glucose (50 mM final concentration) for 1 h. Then cells were treated with sample and further incubated for 1 h. Next, the cells were washed three times with PBS buffer to remove unbound QD, imaged under microscope and followed the same procedure as described before for flow cytometry study. Temperature dependent endocytosis study. Prior to sample treatment, cells were incubated overnight with 500 μL of serum-free DMEM and then kept at this plate at 4 °C for 1 h in presence or absence of NaN3 (10 mM final concentration) and deoxy-glucose (50 mM final concentration). Next, cells were treated with 50 μL of sample and incubated for another 1 h at 4 °C. Then, cells were thoroughly washed with PBS buffer and follow the same procedure as described before for imaging and flow cytometry study. Endocytosis inhibition study. To study the endocytosis mechanism, cells were cultured overnight in a 24 well plate in serum free DMEM media. Next, cells were incubated for 1 h with different endocytosis inhibitors that are known to block different endocytosis mechanism. Typically, we have used chloropromazine (CHP, 50 μM) and sucrose (5 μM) that blocks clathrin-mediated endocytosis, genistein (GEN, 200 μM) that inhibits caveolae-based endocytosis, methyl-β-cyclodextrin (MBCD, 10 mM) that inhibits lipid raft endocytosis and amiloride (5 μM) that blocks macropinocytosis. Then, cells were treated with QD sample and incubated for another 1 h at 37 °C. Finally, cells were thoroughly washed with PBS buffer to remove the unbound QD from the cell surface 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. Cell viability assay. For cell viability study, HeLa cells were cultured in 24-well plates in cell culture media. After that, cells were treated with different doses the QD sample for 24 h and then washed through PBS buffer and fresh DMEM medium was added. Next, 50 μL of freshly prepared MTT solution (5 mg MTT in 1 mL deionized water) was added to each well and incubated for 4-5 h. Next, the supernatant was removed carefully leaving the formazon in the 7 ACS Paragon Plus Environment


plate and it was dissolved in sodium dodecyl sulfate (SDS) solution (8 g of SDS dissolved in 40 mL of DMF-H2O mixture). Finally, absorbance was measured at 570 nm. Cell viability was estimated assuming 100 % viability for the cells treated without any QD sample. Instrumentation. UV−visible absorption spectrums of all sample solutions were measured in Shimadzu UV-2550 UV−visible spectrophotometer using a quartz cell of 1 cm path length. Transmission electron microscopy (TEM) samples were prepared by putting a drop of a nanoparticle solution on a carbon-coated copper grid, dried in air and observed with a FEI Tecnai G2 F20 microscope. The hydrodynamic size and zeta potential of nanoparticles were measured by Malvern Nano ZS instrument. Fluorescence-based quantification studies were performed in Perkin Elmer fluorescence spectrometer model LS-45. Quantification of QD uptake in cell has been analyzed by BD Accuri C6 Flow Cytometer. Fluorescence images of cells were captured by Olympus IX 81 microscope using Image-Pro Plus v 7.0 software.

Results Arginine terminated nanoparticle with modular surface charge and guanidinium multivalency. We have used CdSe/ZnS-based core-shell QD which is well known for size dependent tunable emission colors from visible to near-infrared range, bright and stable emission and with insignificant photobleaching issue.9,10,20 Although the QD is selected as a model nanoparticle to study the cell uptake processes, the approach can be adapted to other nanoparticle. Synthesis approach of arginine conjugated QD is shown in Scheme 1. First hydrophobic QD of 4-5 nm size is prepared that is composed of red emitting CdSe core and ZnS shell. Next, hydrophobic QD is transformed into water soluble and polyacrylate coated QD with arginine termination. Polyacrylate coating is performed in reverse micelle where hydrophobic QD and acryl monomers are dissolved. Different acryl monomers are used such as amineacrylate that provides primary amines, arginine-acrylate that provides arginine termination, PEGacrylate that provides PEGylated surface and small amount (5 mole %) of bis-acrylate that cross link the polymer shell. In one approach, arginine-acrylate is used as the acryl monomer during polyacrylate coating along with PEG-acrylate. This approach produces QD-1 with PEGylated shell and surface terminated with arginine (average of 20 per QD). In other approach, hydrophobic QD is converted to polyacrylate coated QD with primary amine termination using the mixture of PEG-acrylate and amine-acrylate. Next, arginine is covalently conjugated using 8 ACS Paragon Plus Environment

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the primary amines at the polyacrylate shell. This approach produces QD-2 that has PEGylated shell terminated with arginine (average of 150 per QD) and primary amines (average of 750 per QD). In another approach, hydrophobic QD is converted to polyacrylate coated QD using the amine-acrylate. Next, arginine is covalently conjugated using the primary amines at the polyacrylate shell. Finally, remaining primary amines are reacted with glyceraldehydes. This approach produces QD-3 that is terminated with arginine (average of 100 per QD) and the polyacrylate shell contains secondary amine (but no primary amine) and glycerol (not PEG). Characteristic property of arginine terminated QDs are summarized in Table 1, Figure 1 and Supporting Information, Figure S2. All the particles have 15-30 nm hydrodynamic size, as observed from dynamic light scattering study. However, their surface chemistry is different. The QD-1 has PEGylated shell and average of 20 guanidinium (and 20 carboxylic acid groups, coming from arginine) per QD. The surface charge of QD-1 varies from positive to negative as the solution pH changes from acidic to basic (+12 mV at pH 4.5, +5 mV at pH 7.4 and -10 mV at pH 10.0) The QD-2 has PEGylated shell, average of 750 primary amines per QD and average of 150 guanidinium (and 150 carboxylic acid groups, coming from arginine) per QD. The surface charge of QD-2 varies from positive to negative as the solution pH changes from acidic to basic (+10 mV at pH 4.5, +4 mV at pH 7.4 and -5 mV at pH 10.0) The QD-3 has average of 700 secondary amines per QD and average of 100 guanidinium (and 100 carboxylic acid groups, coming from arginine) per QD. The surface charge of QD-3 vary from positive to negative as the solution pH varied from acidic to basic (+20 mV at pH 4.5, +6 mV at pH 7.4 and -11 mV at pH 10.0) These variations of surface charge of QD-1/QD-2/QD-3 is due to varied multivalency of cationic guanidinium, pH dependent protonation of primary/secondary amine and pH dependent formation of carboxylate anions. In particular at physiological pH of 7.4, the QD-1, QD-2 and QD-3 have low cationic charge. This low surface charge is particularly due to counter balance of cationic guanidinium by carboxylate anions. We have investigated the interaction of QD-1/QD-2/QD-3 with phosphate groups using the sodium hexametaphosphate based titration. (Supporting Information, Figure S3) Sodium hexametaphosphate is selected as it has multiple phosphate groups that can interact with QD and induces their aggregation. Typically, same amount of QD-1/QD-2/QD-3 solution (with micromolar

QD

concentration)

is

mixed

with

different

concentrations

of

sodium

hexametaphosphate in the range of 1-1000 µM. Next, zeta potential and colloidal stability of 9 ACS Paragon Plus Environment


QD-1/QD-2/QD-3 is investigated. Results show that zeta potential of all the QD decreases rapidly from positive to negative as the concentration of hexametaphosphate is increased from 1 µM to 100 µM. In addition QD-1 precipitates from colloidal solution at the intermediate hexametaphosphate where zeta potential value is close to zero. However, no such precipitation/aggregation is observed for QD-2/QD-3. These results suggest that all the QDs have strong interaction with phosphate groups and similar type interaction is expected with cell surface phosphate groups coming from phospholipids.12,15 MTT-based cytotoxicity assay of QDs are performed in wider dose showing good cell viability even at the highest tested dose (0.5 µM) as compared to the dose used (0.1 µM) in most of our experiments (0.1 µM). (Supporting Information, Figure S4) We have investigated the fluorescence stability and surface charge of designed QD in serum containing cell culture medium and co-existing substances in blood (e.g. Na+, K+, Fe3+, Ca2+, Zn2+, glutathione, L-cysteine, vitamin C). Results show that fluorescence of designed QD is reasonably stable with insignificant change is surface property. (Supporting Information, Figure S5) Direct cell translocation of arginine terminated nanoparticle. Cell labeling property of nanoparticles has been investigated by incubating with cells for different time interval, followed by washing to remove unbound particles and then imaging under fluorescence microscope. Results are summarized in Figure 2-6 and (Supporting Information, Figure S6-S12). Results show that all the QD samples can label cells within one hour but the distribution inside cytosol depends on the nature of QD surface chemistry. For example, QD-1 labels cells within one hour and in next 24 h do not have significant localization inside cell or no specific sub-cellular localization. (Figure 2, 3 and Supporting Information, Figure S6-S10) In contrast QD-2 and QD3 label cells within one hour and homogeneously distribute inside cytosol for 5 h (for QD-2) or 2 h (for QD-3) before localization near perinuclear region within 3 h (for QD-2) or 8 h (for QD-3). (Figure 4, 5) Entry of QD into cytosol is investigated by labeling of cells by QD and nuclear probe followed by fluorescence imaging at different Z-planes. Results show that emission of QD and emission of nuclear probe comes from same plane. This result suggests that QDs enters into cell cytoplasm and reside in the nuclear plane but do not colocalize with nucleus. (Supporting Information, Figure S11) Colocalization study shows that QD-2 and QD-3, that appear localize at perinuclear region after 3-8 h, do not localize with lysozome. (Supporting Information, Figure


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S12) These results clearly show that QD-1/QD-2/QD-3 enter into cell within one hour and then homogeneously distribute inside cytosol without localization into lysozome. In order to confirm the direct translocation of arginine terminated nanoparticle we have investigated the cell uptake efficiency of QD at low temperature (4 °C) and compared to that with 37 °C. Typically cells are kept at 4 °C for 1 h and then treated with sample for another 1 h. Next, washed cells are used to investigate cell uptake via fluorescence imaging and flow cytometry. (Figure 6, 7 and Supporting Information, Figure S13-S15) Flow cytometry-based quantitative cellular uptake shows that uptake of QD-1/QD-2/QD-3 is not significantly inhibited. (Figure 7) This result clearly suggests that endocytosis is not involved in cell uptake and it is an energy independent process. In order for direct proof of the energy independent uptake, cell uptake is studied in presence of sodium azide and deoxy-glucose that are known to inhibit adenosine triphosphate (ATP)-dependent processes. Fluorescence imaging and flow cytometrybased quantitative cellular uptake show that QD uptake is not significantly inhibited by NaN3 and deoxy-glucose. (Figure 6, 7) However, cell uptake is only partially inhibited for QD-1 sample under 4 °C and in presence of NaN3 and deoxy-glucose. (Figure 7) To investigate the origin of this difference of QD-1 with respect to QD-2 and QD-3, we have studied cell uptake mechanism of QD at 37 °C in presence of different endocytosis inhibitors that are known to block different endocytosis mechanism. Results show that uptake of QD-1/QD2/QD-3 is partially inhibited by MBCD and CHP/sucrose suggesting that nanoparticle can enter into cell via lipid raft and clathrin-mediated endocytosis pathway at 37 °C. (Supporting Information, Figure S16) Thus we may conclude that endocytosis is partially operative at 37 °C along with direct translocation but the endocytosis is completely blocked at 4 °C. So we may conclude that the difference of QD-1 with respect to QD-2 and QD-3 may be due to more involvement of lipid-raft endocytosis for QD-1. In another experiment, uptake mechanism is studied for control QD which is not functionalized with arginine. Results show that uptake of this nanoprobe is significantly inhibited by CHP/sucrose and MBCD, suggesting that they enter into cells via both lipid raft and clathrin-mediated endocytosis pathway. (Supporting Information, Figure S17) However, the cellular uptake of this control QD is significantly inhibited by treatment of NaN3, deoxy-glucose and at low temperature (4 °C) condition. Thus, we can conclude that direct translocation of nanoparticle is induced after arginine functionalization. All



these experimental data clearly suggest the direct translocation and only partial involvement of energy dependent endocytosis process in the cell uptake of QD-1/QD-2/QD-3. Control experiments show that nanoparticle size, surface chemistry and cell culture condition are very critical aspect for direct cell translocation of arginine terminated nanoparticle. We found that arginine terminated smaller nanoparticle of 15-20 nm size shows similar type direct translocation but arginine terminated 50-80 nm particle do not show this direct translocation (Figure 8 and Supporting Information, Figure 18) In addition nanoparticle surface should have significant number of arginine and without any phosphate/sulfate groups. If arginine is present along with phosphate/sulfate groups, colloidal stability of nanoparticle becomes poor, possibly due to interparticle interaction via guanidinium-phosphate/sulfate groups. (Supporting Information, Figure S19) Moreover, serum free cell culture media is required to avoid QD precipitation during cell labeling. (Supporting Information, Figure S20) If arginine is replaced with other cationic group such as ammonium, nanoparticle enters into cell via energy dependent endocytosis. (Supporting Information, Figure S21) These results reflect that presence of guanidinium groups on the nanoparticle surface is most critical and other cationic functional groups are unable to induce this direct cell translocation.

Discussion It is well known that nanoparticle enters into cell via endocytosis.20,21,23 In contrast here we show that arginine terminated nanoparticle enters into cell via direct translocation. In order to achieve this direct translocation two criteria are critical. First, nanoparticle should be small in size. Although small molecules are shown to enter by direct translocation via nanoscale assembly,12-15 direct cell translocation of nanoparticle is rarely reported.10,11 In one approach of direct cell translocation, arginine-based cell penetrating peptide with poly(disulfide) linkage is designed and cytosolic delivery of quantum dot is achieved via reductive depolymerization.10 In other approach arginine terminated 2 nm Au nanoparticle is used to make self-assembled submicron size particle with anionic protein and direct cell delivery of protein is achieved via lipid raftmediated processes.11 In the present work nanoparticle of 15-30 nm is used (that include 4-5 nm hard particle and 5-10 nm soft shell) and possibly this is the critical size below which direct translocation occurs over the endocytosis processes. The observed critical size range of 15-30 nm 12 ACS Paragon Plus Environment

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(with 4-5 nm hard particle and 5-10 nm soft shell) for direct cell translocation, that involves transfer of nanoparticle from bulk aqueous phase to organic-like cell membrane, is reasonable considering the fact that critical size of 10-20 nm is required for endocytosis of hard particle21 and size limit of 5-10 nm for solvent extraction of hard particles (extraction of hard particle from water to organic phase via surfactant capping).31 Second, nanoparticle surface chemistry and labeling conditions are critical to achieve the direct translocation. In particular nanoparticle surface should have significant number of arginine without any phosphate/sulfate groups and colloidal stability of guanidinium terminated nanoparticle need to be maintained in physiological condition in order to achieve this direct cell translocation. Based on our observation we propose the mechanism of cell translocation of nanoparticle. (Scheme 2) It involves interaction between nanoparticle and cell membrane via guanidinium group of nanoparticle and phosphate group of phospholipid coming from cell membrane. This interaction leads to the distortion of cell membrane followed by extraction of nanoparticle into cell membrane. Next, nanoparticles are transported to cytosol due to pH gradient present along the cell membrane. Once in cytosol, they are free to move and depending on their surface charge they may localize at certain compartments. The proposed mechanism is similar to earlier proposed mechanism for direct cell transport for small molecule.15 The direct cell translocation mechanism reported here has very important feature. The cell uptake of nanoparticle is extensive and without trafficking to endosome/lysosome. Thus the approach can be exploited for cell transfection of drugs without endosomal/lysosomal degradation. In addition approach can be used to design imaging nanoprobes for different sub-cellular compartments such as mitochondria, Golgi apparatus.

Conclusion Here we report a designed nanoparticle that can enter into cell via energy independent direct translocation processes. Nanoparticle is terminated with multiple numbers of arginine with 1530 nm hydrodynamic size. The direct cell translocation demonstrated here is shown to have two distinct advantages over the energy dependent endocytotic uptake of nanoparticle. First, cell uptake of nanoparticle is very efficient. Second, nanoparticle neither localizes at any sub-cellular compartments for long time nor traffic to lysozome. This study shows that nanomaterial can be chemically designed with the capability of direct cell translocation and for different applications 13 ACS Paragon Plus Environment


such as drug delivery carrier or sub-cellular targeting with minimum lysosomal degradation/trafficking.

ASSOCIATED CONTENT Supporting Information Mass spectral characterization of arginine-acrylate, evidence of interaction of QD-1/QD-2/QD-3 with phosphate groups, additional cell imaging data on direct cell translocation of nanoparticle, colocalization with lysotraker and various control cell labeling experiments. This material is available free of charge via the Internet at http://pubs.acs.org. Notes The authors declare no competing financial interests. Acknowledgement. The authors acknowledge DST Nano Mission (Grant number SR/NM/NB/ 1009/2016) and CSIR (Grant number 02(0249)15/EMR-II) Government of India for financial assistance. SG, PP and CD acknowledge CSIR, India for providing research fellowship. References 1) Futaki, S.; Suzuki, T.; Ohashi, W.; Yagami, T.; Tanaka, S.; Ueda, K.; Sugiura, Y. Argininerich Peptides An Abundant Sourse of Membrane-Permeable Peptides Having Potential as Carriers for Intracellular Protein Delivery. J. Biol. Chem. 2001, 276, 5836−5840. 2) Futaki, S.; Nakase, I. Cell-Surface Interactions on Arginine-Rich Cell-Penetrating Peptides Allow for Multiplex Modes of Internalization. Acc. Chem. Res. 2017, 50, 2449−2456. 3) Walrant, A.; Cardon, S.; Burlina, F.; Sagan, S. Membrane Crossing and Membranotropic Activity of Cell-Penetrating Peptides: Dangerous Liaisons? Acc. Chem. Res. 2017, 50, 2968−2975.


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4) Takayama, K.; Tadokoro, A.; Pujals, S.; Nakase, I.; Giralt, E.; Futaki, S. Novel System to Achieve One-Pot Modification of Cargo Molecules with Oligoarginine Vectors for Intracellular Delivery. Bioconjugate Chem. 2009, 20, 249–257. 5) Liu, C.; Liu, X.; Rocchi, P.; Qu, F.; Iovanna, J. L.; Peng, L. Arginine-Terminated Generation 4 PAMAM Dendrimer as an Effective Nanovector for Functional siRNA Delivery in Vitro and in Vivo. Bioconjugate Chem. 2014, 25, 521−532. 6) Kojima, C.; Kameyama, R.; Yamada, M.; Ichikawa, M; Waku, T.; Handa, A.; Tanaka, N. Ovalbumin Delivery by Guanidine-Terminated Dendrimers Bearing an Amyloid-Promoting Peptide via Nanoparticle Formulation. Bioconjugate Chem. 2015, 26, 1804−1810. 7) Montet, X.; Funovics, M.; Montet-Abou, K.; Weissleder, R.; Josephson, L. Multivalent Effects of RGD Peptides Obtained by Nanoparticle Display. J. Med. Chem. 2006, 49, 6087−6093. 8) Delehanty, J. B.; Medintz, I. L.; Pons, T.; Brunel, F. M.; Dawson, P. E.; Mattoussi, H. SelfAssembled Quantum Dot-Peptide Bioconjugates for Selective Intracellular Delivery. Bioconjugate Chem. 2006, 17, 920−927. 9) Wei, Y.; Jana, N. R.; Tan, S. J.; Ying, J. Y. Surface Coating Directed Cellular Delivery of TatFunctionalized Quantum Dots. Bioconjugate Chem. 2009, 20, 1752–1758. 10) Derivery, E.; Bartolami, E.; Matile, S.; Gonzalez-Gaitan, M. Efficient Delivery of Quantum Dots into the Cytosol of Cells Using Cell-Penetrating Poly(disulfide)s. J. Am. Chem. Soc. 2017, 139, 10172−10175. 11) Mout, R.; Ray, M.; Tay, T.; Sasaki, K.; Tonga, G. Y.; Rotello, V. M. General Strategy for Direct Cytosolic Protein Delivery via Protein−Nanoparticle Co-engineering. ACS Nano 2017, 11, 6416−6421.



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12) Rothbard, J. B.; Jessop, T. C.; Lewis, R. S.; Murray, B. A.; Wender, P. A. Role of Membrane Potential and Hydrogen Bonding in the Mechanism of Translocation of Guanidinium-Rich Peptides into Cells. J. Am. Chem. Soc. 2004, 126, 9506−9507. 13) Takechi, Y.; Yoshii, H.; Tanaka, M; Kawakami, T.; Aimoto, S.; Saito, H. Physicochemical Mechanism for the Enhanced Ability of Lipid Membrane Penetration of Polyarginine. Langmuir 2011, 27, 7099–7107. 14)

Lin,

J.;

Alexander-Katz,

A.

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Open

“Doors”

for

Cationic

Nanoparticles/Biomolecules: Insights into Uptake Kinetics. ACS Nano, 2013, 7, 10799–10808. 15) Herce, H. D.; Garcia, A. E.; Cardoso, M. C. Fundamental Molecular Mechanism for the Cellular Uptake of Guanidinium-Rich Molecules. J. Am. Chem. Soc. 2014, 136, 17459−17467. 16) Wadia, J. S.; Stan, R. V.; Dowdy, S. F. Transducible TAT-HA Fusogenic Peptide Enhances Escape of TAT-Fusion Proteins after Lipid Raft Macropinocytosis. Nat. Med. 2004, 10, 310−315. 17) Kawamura, K. S.; Sung, M; Bolewska-Pedyczak, E.; Garie´py, J. Probing the Impact of Valency on the Routing of Arginine-Rich Peptides into Eukaryotic Cells. Biochemistry 2006, 45, 1116−1127. 18) Ayman El-Sayed, A.; Harashima, H. Endocytosis of Gene Delivery Vectors: From Clathrindependent to Lipid Raft-mediated Endocytosis. Molecular Therapy, 2013, 21, 118–1130. 19) Lorents, A.; Säälik, P.; Langel, Ü.; Pooga, M. Arginine-Rich Cell-Penetrating Peptides Require Nucleolin and Cholesterol-Poor Subdomains for Translocation across Membranes. Bioconjugate Chem. 2018, 29, 1168−1177. 20) Dalal, C.; Jana, N. R. Multivalency Effect of TAT-Peptide-Functionalized Nanoparticle in Cellular Endocytosis and Subcellular Trafficking. J. Phys. Chem. B, 2017, 121, 2942–2951.


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21) Ding, H.-M.; Ma, Y.-Q. Theoretical and Computational Investigations of NanoparticleBiomembrane Interactions in Cellular Delivery. Small 2015, 11, 1055–1071. 22) Sharma, P.; Varma, R.; Sarasij, R. C.; Ira, G. K.; Krishnamoorthy, G.; Rao, M.; Mayor, S. Nanoscale Organization of Multiple GPI-Anchored Proteins in Living Cell Membranes. Cell 2004, 116, 577–589. 23) Conner, S.D.; Schmid, S. L.; Regulated Portals of Entry into the Cell. Nature 2003, 422, 37– 44. 24) Lim, J. P.; Gleeson, P. A. Macropinocytosis: An Endocytic Pathway for Internalising Large Gulps. Immunol. Cell Biol. 2011, 89, 836–843. 25) MacEwan, S. R.; Chilkoti, A. Digital Switching of Local Arginine Density in a Genetically Encoded Self-Assembled Polypeptide Nanoparticle Controls Cellular Uptake. Nano Lett. 2012, 12, 3322−3328. 26) Tesei, G.; Vazdar, M.; Jensen, M. R.; Cragnell, C.; Mason, P. E.; Heyda, J.; Skepo¨, M; Jungwirth, P.; Lund, M. Self-Association of a Highly Charged Arginine-Rich Cell-Penetrating Peptide. PNAS, 2017, 114, 11428–11433. 27) Zhao, M.; Kircher, M. F.; Josephson, L.; Weissleder, R. Differential Conjugation of Tat Peptide to Superparamagnetic Nanoparticles and Its Effect on Cellular Uptake. Bioconjugate Chem.2002, 13, 840–844. 28) Saha, A.; Basiruddin, SK.; Maity, A. R.; Jana, N. R. Synthesis of Nanobioconjugates with a Controlled Average Number of Biomolecules between 1 and 100 Per Nanoparticle and Observation of Multivalency Dependent Interaction with Proteins and Cells. Langmuir 2013, 29, 13917–13924.



29) Smith, R. E.; MacQuarrie, R. A. A Sensitive Fluorometric Method for the Determination of Arginine Using 9,10-Phenanthrenequinone. Anal. Biochem. 1978, 90, 246−255. 30) Yu, W. W.; Qu, L.; Guo, W.; Peng, X. Experimental Determination of the Extinction Coefficient of CdTe, CdSe, and CdS Nanocrystals. Chem. Mater. 2003, 15, 2854−2860. 31) Cheng, W; Wang, E. Size-Dependent Phase Transfer of Gold Nanoparticles from Water into Toluene by Tetraoctylammonium Cations: A Wholly Electrostatic Interaction . J. Phys. Chem. B 2004, 108, 24−26.


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Table 1: Property of arginine terminated nanoparticles.



Scheme 1. a) Synthetic approach in making arginine terminated nanoparticle. b) Schematic representation of chemical structure of arginine terminated QD-1, QD-2 and QD-3.

OH

NH

HN

OH

2

O

OH

OH OH

OH

OH

HN O OH

HN

NH

OH

O

OH

H2N NH2

OH OH

2N

NH2 H2N HN O

NH2 H2N HN O OH

QD-2/QD-3

NH

H

OH

OH

OH

H2N NH2 HN O

arginine conjugation

Polyacylate coated QD

OH

NH 2 H 2N HN O

b)

PEG-acrylate amine-acrylate

OH OH

NH 2 H 2N HN O

Hydrophobic QD

NH2

arginine-acrylate PEG-acrylate

QD-1

OH

OH

O

OH O

O

HN

H H2N N 2 HN

H2N NH2 HN

H2N NH2

OH

amine-acrylate

NH 2 H 2N HN O

OH

OH

OH

O H 2N NH HN 2

PEG -acrylate

HO O

NH2

OH OH

n

QD-3

O

N H

OH

O

OH

QD-2 O

OH

OH OH

OH

O

NH

NH 2 H 2N HN O

H O

H 2N NH H N 2

QD-1

HO

O

N H

arginine-acrylate


H N

HO NH2 NH2

OH

OH

a)

O H

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 20 of 30

O

H2N

arginine

H N

NH2 NH2

a) 1.5

b)

Emission Intensity (a.u.)

120

Absorbance

100 1.0

80 60

0.5

40 20

c)

240

450

500

550

600

160 120 80 40 500

20 10 0 0 20 40 60 80 100 Size (nm)

d) 200 0 µM 50 µM 70 µM 100 µM 150 µM 200 µM 250 µM 300 µM 400 µM 600 µM

400

30

0

Wavelength (nm)

200

0 300

650

Wavelength(nm)


0.0 400


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


Number (%)

Page 21 of 30

150 100 50 0 300

600

Control QD-1 QD-2 QD-3

400 500 Wavelength (nm)

600

Figure 1. a) UV-visible absorption and emission spectra of QD-1. b) TEM image of QD-1 along with hydrodynamic size (inset). c) Fluorescence spectral data of phenanthrenequinone based fluorimetric titration with varying arginine concentration. d) Fluorescence spectral data of QD-1, QD-2 and QD-3 after the phenanthrenequinone test, showing the presence of arginine. QD concentration is kept same for all samples and control represents QD without arginine. These fluorescence data are used to measure the number of arginine per QD.



BF

1h

F

BF

6h

F

BF

12 h

BF

24 h

1h

Page 22 of 30

M

1h

6h

M

6h

F

12 h

M

12 h

F

24 h

M

24 h

Figure 2. Fluorescence imaging of HeLa cells labeled with QD-1 showing that it labels cells within hour and in next 24 h homogeneously distribute inside cytosol without any significant localization inside cell. Typically colloidal sample is incubated with cells for one hour and washed cells are mixed with fresh culture medium for another 24 h before imaging under bright field (BF) or fluorescence (F) mode and then merged (M). Scale bar represents 25 μm.


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1h

F

6h

F

BF

12 h

BF

24 h

BF

BF

1h

M

1h

6h

M

6h

F

12 h

M

12 h

F

24 h

M

24 h

.

Figure 3. Fluorescence imaging of HT22 cells labeled with QD-1 showing that it labels cells within hour and in next 24 h mostly distribute inside cytosol without specific localization at any subcellular compartments. Typically, colloidal sample is incubated with cells for one hour and washed cells are mixed with fresh culture medium for another 24 h before imaging under bright field (BF) or fluorescence (F) mode and then merged (M). Scale bar represents 25 μm.



Page 24 of 30

BF

1h

F

1h

M

1h

BF

3h

F

3h

M

3h

BF

8h

F

8h

M

8h

Figure 4. Fluorescence imaging of HeLa cells labeled with QD-2 showing that it labels cells within hour and in next 5 h distribute inside cytoplasm, before localizing near perinuclear region within 8 h. Typically, colloidal sample is incubated with cells for one hour and washed cells are mixed with fresh culture medium for another 24 h before imaging under bright field (BF) or fluorescence (F) mode and then merged (M). Scale bar represents 25 μm.


Page 25 of 30 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


BF

15 min

F

15 min

M

15 min

BF

1h

F

1h

M

1h

BF

3h

F

3h

M

3h

Figure 5. Fluorescence imaging of HeLa cells labeled with QD-3 showing that it labels cells within 15 min and in next 2 h homogeneously distribute inside cytosol before localizing at perinuclear region in 3 h. Typically, colloidal sample is incubated with cells for 15 min and washed cells are mixed with fresh culture medium for another 24 h before imaging under bright field (BF) or fluorescence (F) mode and then merged. Scale bar represents 25 μm.



i)

a)

d)

c)

b) BF

BF

BF

BF

F

F

F

F

ii) a)

iii)

Page 26 of 30

c)

b) BF

F

BF

BF

F

F

F

b)

a)

d) BF

d)

c)

BF

BF

BF

BF

F

F

F

F

Figure 6. Bright field (BF) and fluorescence (F) image of HeLa cells labelled by i) QD-1, ii) QD-2 and iii) QD-3 at 37 ºC (a), at 4 ºC (b), at 37 ºC in presence of NaN3 and deoxy-glucose (c), and at 4 ºC in presence of NaN3 and deoxy-glucose (d). Results show that QD uptake is not significantly inhibited by NaN3, deoxy-glucose or at 4 ºC. Scale bar represents 50 µm.


Page 27 of 30

60 40 20 0

NaN3, 4 ºC 4 ºC + NaN3, Control deoxy-glucose deoxy-glucose

80 60 40 20 0

100

QD-2

Cellular uptake (%)

80

100

QD-1

Cellular uptake (%)

100

Cellular 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


Control NaN3, 4 ºC 4 ºC + NaN3, deoxy-glucose deoxy-glucose

QD-3

80 60 40 20 0

Control NaN3, 4 ºC 4 ºC + NaN3, deoxy-glucose deoxy-glucose

Figure 7. Flow cytometry-based quantitative cellular uptake of QD-1/QD-2/QD-3 in HeLa cells in presence of NaN3, deoxy-glucose and at 4 ºC with respect to control (37 ºC), showing that QD uptake is not significantly inhibited by NaN3, deoxy-glucose or at 4 ºC. The ± SD of three determinations (n = 3) are represented in error bars.



i) a)

b)

Page 28 of 30

d)

c)

BF

BF

BF

BF

F

F

F

F

b)

ii) a) BF

F

c)

d)

BF

BF

BF

F

F

F

Figure 8. Bright field (BF) and fluorescence (F) image of HeLa cells labelled by i) 15-20 nm arginine terminated QD and ii) 60-70 nm arginine terminated QD at 37 ºC (a), at 4 ºC (b), at 37 ºC in presence of NaN3 and deoxy-glucose (c), and at 4 ºC in presence of NaN3 and deoxyglucose (d). Results show that uptake of 60-70 nm QD is significantly inhibited by NaN3, deoxyglucose or at 4 ºC. However, no such inhibition is observed for 15-20 nm QD. Scale bar represents 50 µm.


Page 29 of 30

Scheme 2. Proposed mechanism of direct cell translocation of nanoparticle. In the first step nanoparticle from bulk solution interacts with phosphate groups of cell membrane phospholipid by using their guanidinium groups. As a result of this interaction, cell membrane is distorted and nanoparticle is extracted into cell membrane and the pH gradients present along the cell membrane transport them into cytosol.

R

R R R

− − − − − − −R − −

−−−

−−

−

R R R

−− −−−−−

− − − −−

−−

−−

− −− − − −

− − − −− − − − − − − direct penetration R R

R R R

− −−

− ≡ phosphate

R R R

R R

R ≡ arginine

R R R

R

−≡

+

−−

−− − − −− cytoplasm

−

R R R

− − − − − −−

−− − −−

−− − −−−

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 30 of 30

TOC Graphic

37 °C

4 °C media cell membrane direct penetration

50 μm

cytoplasm


50 μm

Arginine Terminated, Chemically Designed Nanoparticle for Direct

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