Efficient Subcellular Targeting to the Cell Nucleus of Quantum Dots

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Efficient subcellular targeting to the cell nucleus of quantum dots densely decorated with nuclear localization sequence peptide Amit Ranjan Maity, and David Stepensky ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b10295 • Publication Date (Web): 05 Jan 2016 Downloaded from http://pubs.acs.org on January 12, 2016

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ACS Applied Materials & Interfaces

Efficient subcellular targeting to the cell nucleus of quantum dots densely decorated with nuclear localization sequence peptide Amit Ranjan Maity and David Stepensky*

Department of Clinical Biochemistry and Pharmacology, The Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O.Box 653, Beer-Sheva 84105, Israel.

-1ACS Paragon Plus Environment


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ABSTRACT: Organelle-targeted drug delivery can enhance the efficiency of the intracellularlyacting drugs and reduce their toxicity. We generated core-shell type CdSe-ZnS quantum dots (QDs) densely decorated with NLS peptidic targeting residues using a 3-stage decoration approach and investigated their endocytosis and nuclear targeting efficiencies. The diameter of the generated QDs increased following the individual decoration stages (16.3, 18.9, and 21.9 nm), the ζ-potential became less negative (33.2, -17.5, and -11.9 mV), and characteristic changes appeared in the FTIR spectra following decoration with the linker and NLS peptides. Quantitative analysis of the last decoration stage revealed that 37.9% and 33.2% of the alkyne-modified NLS groups that were added to the reaction mix became covalently attached or adsorbed to the QDs surface, respectively. These numbers correspond to 63.6 and 55.7 peptides conjugated or adsorbed to a single QD (the surface density of 42 and 37 conjugated and adsorbed peptides per 1000 nm2 of QDs surface), which is higher than in majority of previous studies that reported decoration efficiencies of formulations intended for nuclear-targeted drug delivery. QDs decorated with NLS peptides undergo more efficient endocytosis, as compared to other investigated QDs formulations, and accumulated to a higher extent in the cell nucleus or in close vicinity to it (11.9%, 14.6%, and 56.1% of the QDs endocytosed by an average cell for the QD-COOH, QD-azide, and QD-NLS formulations, respectively). We conclude that dense decoration of QDs with NLS residues increased their endocytosis and led to their nuclear targeting (preferential accumulation in the cells nuclei or in close vicinity to them). The experimental system and research tools that were used in this study allow quantitative investigation of the mechanisms that govern the QDs nuclear targeting and their dependence on the formulation properties. These findings will contribute to development of subcellularly-targeted DDSs that will deliver specific drugs to the nuclei of the target cells and will enhance efficacy and reduce toxicity of these drugs.

KEYWORDS: subcellular targeting, quantum dots, peptidic targeting residues, nuclear localization sequence, Click chemistry, quantitative analysis of conjugation efficiency.


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INTRODUCTION Nucleus is site of action of numerous drugs, including some anticancer and immunosuppressive agents, steroids, DNA drugs, etc.1,

2

Inefficient penetration of

these drugs to the site of their desired action in the nucleus of target cells can limit their efficiency and can enhance their toxicity due to pharmacological activities at other locations. Therefore, encapsulation of drugs into specialized drug delivery systems (DDSs) targeted to the nucleus has been suggested as an approach that can enhance the desired drug effects and limit its toxicity (reviewed in

1, 3-5

). For this

purpose, nanoparticles, liposomes, and other types of DDSs can be used, and in many cases these DDSs are decorated with specific residues, such as nuclear localization sequences (NLS) or cell penetration peptides

3, 6

, that are expected to enhance the

DDSs targeting to the nucleus (i.e., to lead to preferential accumulation of the DDSs in the nucleus, as compared to other organelles). Dozens of studies reported endocytosis and accumulation of DDSs in the nucleus in different experimental settings (usually, in in vitro experiments with specific cell lines), and efficient targeting of DDSs to the nucleus was claimed in some of these studies. Unfortunately, in many cases these claims were based on qualitative data only (e.g., detection of DDSs in the nucleus of the studied cells using fluorescence-based or electron microscopy techniques)

7

. However, efficient

subcellular targeting of specific DDS to the nucleus implies its preferential accumulation in the nucleus, as compared to other organelles, and requires quantitative assessment of the intracellular biofate of the studied formulation. Limited volume of quantitative data on efficiency of nuclear DDSs targeting hampers the investigation of the mechanisms that govern the DDSs intracellular biofate and the factors that limit their efficiency. For instance, without quantitative characterization of the DDSs formulation properties and their intracellular disposition (distribution and elimination), conclusions can’t be reached regarding the preferred size of the DDSs, type/number/density of targeting residues, and other formulation properties that are associated with preferential DDSs accumulation in the nucleus. Moreover, mechanisms and factors that limit nuclear DDSs targeting (i.e., endocytosis, endosomal escape, intracellular trafficking mechanisms, interaction with nuclear vs. other membranes, etc.) cannot be identified without quantitative data on DDSs intracellular disposition, and approaches to overcome these limiting factors cannot be efficiently devised. -3ACS Paragon Plus Environment


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In our previous studies, we developed experimental techniques for dense decoration of DDSs with targeting residues 8, tools for quantification of the decoration efficiency 9, and imaging-based techniques for quantification of DDSs intracellular disposition and targeting efficiency 10. The objective of this study was to apply these tools for quantitative analysis of nuclear targeting of a model DDS and of the limiting factors for nuclear targeting. To this end, we used quantum dots (QDs)-based formulation that possesses the following desired properties: a) easy preparation process that generates homogeneous nano-formulation with small particle size, b) surface properties (presence of surface carboxylic acid groups) that are compatible with the DDSs surface decoration approach that we applied in our previous studies 8, 9, c) unique optical properties that allow their sensitive detection and quantification using imaging-based tools that we developed in our previous studies

10

, and d) low

photo-bleaching and high chemical stability suitable for long-term experiments.


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MATERIALS AND METHODS Materials. Cadmium oxide, stearic acid, octadecyl amine, octadecene, zinc stearate, selenium powder, trioctylphosphine, trioctylphosphine oxide, sulphur powder, Igepal CO-520, 2-carboxyethyl acrylate, 3-sulfopropyl methacrylate potassium

salt,

N,N,N,N-tetramethylethylenediamine,

ammonium

persulphate,

methylenebisacrylamide, 11-azido-3,6,9-trioxaundecan-1-amine, penicillamine, N-(3(dimethylamino)propyl)-N-ethylcarbodiimide

hydrochloride

(EDC),

N-

hydroxysuccinimide (NHS), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Aldrich Israel Ltd. (Rehovot, Israel) and used as they received. All organic solvents were of analytical grade. Nuclear localization sequence peptide (PKKKRKVKAA) modified with alkyne group at its Cterminus was purchased from GL Biochem Ltd. (Shanghai, China).

Synthesis and surface decoration of quantum dots. Core-shell type CdSeZnS quantum dots were prepared using high temperature organometallic approach 11. Briefly, CdSe nanocrystals of desired size and fluorescence emission properties were prepared at 280oC under nitrogen atmosphere using octadecene as solvent, octadecyl amine and trioctyl phosphine oxide as capping agent. The obtained CdSe nanocrystals were purified by precipitation-redispersion method using acetone, ethanol and chloroform. Then, purified CdSe nanocrystals undergo ZnS shelling at 200oC under nitrogen atmosphere by alternative injection of zinc stearate and elemental sulphur. Then, CdSe-ZnS QDs were purified again using the same precipitation-redispersion method, and were resuspended in cyclohexane. Hydrophobic CdSe-ZnS QDs (QD-octadecyl) were transformed into waterdispersed hydrophilic QDs (QD-COOH) using reverse micelle-based polyacrylate coating chemistry (see Figure 1)

12

. In brief, aqueous solution of 2-carboxyethyl

acrylate, 3-sulfopropyl methacrylate potassium salt, methylenebisacrylamide was combined with hydrophobic QDs and Igepal CO-520-cyclohexane reverse micelles. Then polymerization was initiated under nitrogen atmosphere by adding ammonium persulphate. Polymerization was stopped after 20 minutes and particles were precipitated by addition of ethanol. The obtained polymer-coated QDs with surface carboxylic groups (QD-COOH) were purified using chloroform and ethanol and were dispersed in double distilled water.



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Linker (11-azido-3,6,9-trioxaundecan-1-amine) was conjugated to the carboxylic acid groups on the QDs surface using carbodiimide reaction (see Figure 1). EDC and NHS were added to dispersed QDs and the suspension was stirred for 30 minutes. Then the linker was added and stirring continued for 6 hours at room temperature. Unreacted reagents were removed from the QDs conjugated to the linker (QD-azide) by dialysis using 12 kD cut-off dialysis membrane. Nuclear localization signal (NLS) peptide was conjugated to the QDs (QDazide) using Click chemistry (see Figure 1) in presence of copper sulphate and sodium ascorbate catalysts. The reaction was performed for 2 hours at room temperature under constant stirring. Finally, NLS-conjugated QDs (QD-NLS) were dialyzed to remove any excess reagents and were used for further studies.

Characterization of quantum dots. Samples of QDs suspensions were placed on copper grids and their morphology was studied using transmission electron microscopy (CM120 Super Twin TEM, Philips, operating at 120 kV). Size distribution of QDs and their zeta potential at pH 7.4 and 4.8 was measured using ZetaSizer Nano ZS, (Malvern Instruments, Malvern, UK). Fourier transform infrared spectra of solid and dry samples were measured using Nicolet FTIR spectrometer 6700 (Thermo Fisher Scientific, Inc.) using polarization modulation unit with sensitive liquid nitrogen cooled MCT-A detector. UV-visible absorption and photoluminescence of water-dispersed NLS-conjugated QDs were measured using Shimadzu

UV-3600

spectrophotometer

and

Shimadzu

RF-5301PC

spectrofluorophotometer, respectively (Shimadzu Scientific Instruments, Columbia, MD, USA).

Analysis of number of carboxylic groups on the QDs surface, and the buffering capacity of the QDs. Number of carboxylic acid functional groups on the QDs surface was estimated using reaction between these groups and methylene blue which leads to formation of colorless leucomethylene blue compound (the reduced form of methylene blue)

13

. Number of surface carboxylic acid groups on QDs was

determined from the change in absorbance of methylene blue (at 622 nm) following addition of QDs, based on the appropriate calibration curve 13. The buffering capacity of QDs (which is indicative of their endosomal escape behavior) was measured by analysis of pH changes during titration of QDs with 0.1N HCl 14. -6ACS Paragon Plus Environment

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Analysis of QDs decoration efficiency with NLS peptide residues. Conjugation reactions of QD-azide with NLS residues were set using all the required components, or part of them (see Table 1), using the previously-reported conditions 9. At the end of the reaction, penicillamine (chelator of Cu2+ ions) was added to reverse copper complexation with unreacted NLS peptides and to recover the free peptides 9. Concentration of unreacted NLS peptides in the supernatant was determined using high-performance liquid chromatography (HPLC) method utilizing a Waters liquid chromatography system (Waters Alliance 2695 system with 996 diode array detector) and Phenomenex Luna C18 column (5 µm, 150 x 4.6 mm). The mobile phase consisted of a gradient of 0.1% trifluoroacetic acid and acetonitrile with flow rate of 1 mL/min 9. The column and the samples were kept at 30oC and 15oC, respectively, the injected volume was 20 µl, and detection at 220 nm was applied. Concentration of NLS peptide in the analyzed supernatant was calculated from the obtained peak areas based on appropriate calibration curves. Number of NLS peptides per QD was calculated based on the results of HPLC-based analysis of reactions efficiency and molar extinction coefficient-based quantification of QDs concentrations. Surface occupied by a single NLS residue and number of these residues per 1000 nm2 surface area was calculated based on assumption that QDs have spherical shape (i.e., 21.91 nm diameter of QD-NLS corresponds to a surface area of 1508 nm2, according to the following formula: sphere surface area = 4πr2)

Analysis of QDs endocytosis and nuclear targeting efficiency. HeLa human cervical carcinoma cells were kept in DMEM supplemented with 5% fetal calf serum, 4.5 g/L D-glucose, 2 mM L-Glutamine, 100 IU/mL penicillin and 100 µg/mL streptomycin (all from Biological Industries Ltd., Beit Haemek, Israel). The cells were maintained in an incubator at 37oC in a humidified atmosphere with 5% CO2. The cells were plated on 24-well tissue culture plates (100,000 cells per well, with glass coverslips for the confocal imaging experiments). On the next day, approximately 1015 QDs

15

were added to each well (corresponding to 10 nM QDs

concentration) and the cells were returned to the incubator for 12 h. Then, the cells were extensively washed with PBS, harvested using trypsin solution (Biological



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Industries Ltd., Beit Haemek, Israel), and analyzed by FACS Canto II Flow Cytometer (Becton Dickinson Biosciences Ltd.). Alternatively, the cells grown on coverslips and incubated with QDs were extensively washed with PBS, stained with DiO membrane dye (Invitrogen Ltd.), fixed with 2.5% formaldehyde solution, washed with PBS, and mounted on glass slides using DAPI Fluoromount-G solution (SouthernBiotech, Birmingham, AL, USA). For analysis of QDs colocalization with the endo-lysosomal compartment, LysoTracker green DND-26 stain (Thermo Fisher Scientific Inc.) was added to HeLa cells during the last 4 h of their incubation with QDs (i.e., 8 h after QDs addition to the cells), and at the end of incubation the cells were washed with PBS (fixation not applied since it interferes with LysoTracker green staining) and mounted on glass slides using DAPI Fluoromount-G solution. Confocal images of the cells at the individual ﬂuorescence channels were sequentially collected using Olympus FV100-IX81 confocal microscope (Tokyo, Japan) equipped with 60x oil objective. The excitation and emission wavelengths for the individual fluorophores/markers were: DAPI (405 nm ex/425-475 nm em), DiO (488 nm ex/500-600 nm em), LysoTracker green (488 nm ex/500-600 nm em), and QDs (559 nm ex/575-675 nm em). All images were collected on the same day using a constant set of imaging parameters (that which were initially adjusted to keep fluorescence of all the samples within a linear range). The collected images were analyzed using a custom-written ‘IntraCell’ plugin for ImageJ software

8, 10

. The

borders of the individual cells and of their cell nuclei were identified based on DiO and DAPI staining, respectively, and the relative content of QDs inside the cells and their nuclei was determined (at least 35 cells for each type of QDs).

Analysis of QDs effect on the cell metabolic activity using MTT assay. Hela cells were plated on 96 well tissue culture plates (30,000 cells per well) and incubated overnight at 37oC in a humidified atmosphere with 5% CO2. Different concentrations of QDs or control solutions were added to the individual wells (in triplicates) and the plates were returned to the incubator for 12 hours. Then, the cells were washed carefully with PBS, 5 µL of 5mg/mL MTT solution were added to each well, and cells were returned to the incubator for 1 hour. The medium was carefully


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discarded, 200 µL DMSO were added to each well and UV absorbance at 570 nm of the individual wells was measured, and the relative metabolic activity of the samples (as compared to the negative control – tissue culture medium without QDs) was calculated.

Statistical analysis. The experiments were performed in triplicates, were repeated at least three times, and the outcomes of one representative data set are reported. The data are presented as mean ± standard deviation. Statistical differences in the studied parameters between the samples were analyzed using ANOVA with Tukey-Kramer or Dunnett’s post-test using Prism 5 software (GraphPad Software Inc.). P value of less than 0.05 was considered significant.



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RESULTS Characterization of QDs. We prepared CdSe-ZnS QDs (QD-octadecyl) and used a 3-stage approach to change their surface chemistry and to decorate them with linker and NLS peptide groups (see Figure 1). The QDs had spherical shape (see Figure 2A) and increased in size following each decoration stage. The initial diameter of the hydrophobic QDs (QD-octadecyl) was 5-6 nm, and following the coating with hydrophilic layer and decorations it increased to 16.3±6.2 nm, 18.9±6.6 nm and 21.9±6.6 nm (PDI values of 0.098, 0.07, and 0.07, respectively) for the QD-COOH, QD-azide and QD-NLS, respectively (see Figure 2B). This increase in QDs size was accompanied by change in the ζ−potential (-33.2 mV, -17.5 mV and -11.9 mV at pH 7.4 for the QD-COOH, QD-azide and QD-NLS, respectively). Coating and individual decoration steps induced characteristic changes in the FTIR spectra of QDs (see Figure 2C). Peaks at 2835-2930 cm-1 for QD-octadecyl are characteristic to C-H stretching of alkyl group and are consistent with presence of hydrophobic octadecyl groups on QDs surface. These peaks disappeared following polymeric shell coating, and peaks characteristic to C=O and –OH stretching (at 1720 cm-1 and 3250 cm-1, respectively) were found in the spectrum of QD-COOH. Decoration of these particles with azide-containing linker resulted in appearance of peaks characteristic to stretching of N3 and C=O of amide functional groups (at 2100 cm-1 and 1630 cm-1, respectively) in QD-azide formulation. After conjugation of the NLS peptide, peaks characteristic to N-H bending of –NH2 groups (1640 cm-1) appeared in the spectrum of QD-NLS. Spectra of QD-azide and QD-NLS retained the –COOH signature (C=O and –OH stretching at 1720 cm-1 and 3250 cm-1, respectively), consistent with presence of unreacted –COOH functional groups in QDazide and QD-NLS formulations. Analysis of the spectral properties of the fully decorated formulation (QDNLS) revealed that it has a broad absorption window with substantial absorbance in the 300-600 nm range, with a narrow emission spectrum centered at 620 nm (see Figure 2D).

Quantitative analysis of efficiency of QDs decoration with NLS residues. Analysis of the efficiency of the last decoration stage (interaction of QD-azide formulation with NLS-alkyne groups) revealed that small amounts of NLS-alkyne


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remained unreacted in the reaction mix or degraded during the reaction (see Table 1). Substantial amount of the NLS-alkyne groups (23.7% of the initial content) sedimented or formed complexes, but majority of these groups adsorbed or attached covalently to the QDs (33.2% and 37.9% of the initial content). Thus, on average, each QD-NLS particle contained 55.7 adsorbed and 63.7 conjugated NLS groups. Assuming that NLS groups are present at QDs surface only, these numbers correspond to the surface density of 37 adsorbed and 42 conjugated peptides per 1000 nm2.

Endocytosis and nuclear targeting of QDs. Surface decoration of QDs had profound influence of their interaction with HeLa cells. Functionalization with linker and NLS groups (QD-azide and QD-NLS, respectively) enhanced endocytosis efficiency of QDs (see Figure 3A and B). Moreover, decoration with NLS groups significantly affected the intracellular trafficking of QDs and increased their tendency to accumulate in the cell nucleus or in close vicinity to it (11.9%, 14.6%, and 56.1% of the QDs endocytosed by an average cell for the QD-COOH, QD-azide, and QDNLS formulations, respectively).

Endosomal escape of QDs. Quantification of surface carboxylic acid groups using methylene blue revealed presence of 427, 414, and 522 –COOH groups on the surface of an average QD-azide, QD-NLS and QD-COOH particle, respectively (see Figure 4A). Furthermore, titration of QDs with 0.1N HCl revealed proton absorption behavior, in particular at the lysosomal pH range (pH 4-6), that was characteristic for all the studied QDs formulations (see Figure 4B). These data were consistent with reduced ζ−potentials of the studied QDs at lower pH (-25.7 mV, -12.9 mV and -8.4 mV at pH = 4.8 for the QD-COOH, QD-azide and QD-NLS, respectively; as compared to -33.2 mV, -17.5 mV and -11.9 mV at pH 7.4 for the same formulations; see above). Analysis of the confocal images of the cells incubated with QDs and with LysoTracker late endosome-lysosome stain (see Figure 4C) revealed low correlation between these fluorophores (Pearson’s correlation coefficient of 13.5%, 13.4% and 19.9% for the QD-COOH, QD-azide and QD-NLS, respectively), indicating that majority of the endocytosed QDs were located outside the endo-lysosomal system following 12 h incubation with HeLa cells.



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Effect of QDs on the cell metabolic activity. We determined the effects of prolonged cell incubation (for 12 h) with the investigated QDs on the cell metabolic activity using the MTT assay. The cells metabolic activity decreased significantly with increasing QDs concentrations (see Figure 5). At the QDs concentrations that were selected for analysis of their intracellular targeting (10 nM), the QD-azide and QD-NLS formulations exhibited limited effect on the cell metabolic activity (less than 15% reduction), while QD-COOH formulation did not affect the cell metabolic status.


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DISCUSSION Formulation properties of the generated QDs. We used a 3-stage approach for surface decoration of QDs. Polyacrylate coating (the 1st stage) is suitable for conversion of hydrophobic QDs to water-dispersible formulation with high colloidal stability. It allows control over the coating thickness and stability (by choosing the type

of

acrylate

monomers,

reaction

conditions,

and

content

of

the

methylenebisacrylamide monomer cross-linker) and over the type and number of surface groups (e.g., carboxylic acid groups) that can be used for subsequent conjugation stages. Conjugation reactions (the 2nd and 3rd stages) increased the size of QDs (see Figure 2B) and induced specific changes in their FTIR spectra (see Figure 2C), indicating successful attachment of the linker molecules and of the NLS peptides to the QDs surface. Both these reagents are positively charged at the physiological pH (amine and azide groups in the linker molecule, arginine and multiple lysine groups in the NLS peptide). Thus, substantial decline in the QDs ζ−potential following the conjugation reactions (see the Results) indicates successful attachment (covalent conjugation or adsorption) of multiple linker and NLS peptide groups to the QDs surface at each conjugation stage. Nevertheless, all the studied formulations (including the linker and NLS peptide-decorated QD-azide and QD-NLS formulations, respectively) contain substantial amounts of residual surface carboxylic acid groups. This conclusion is based on the characteristic FTIR signature of –COOH functional groups (see Figure 2C), less negative ζ−potentials of all the studied QDs formulations at lower pH values (see the Results), and the results of methylene blue titration (see Figure 4A). Presence of these surface carboxylic acid groups confers the investigated QDs formulations strong proton sponge behavior, as confirmed by the HCl titration data (see Figure 4B). Based on the size, charge, FTIR spectra, and other QDs’ characteristics, conclusions regarding the number of specific residues that become covalently attached or adsorbed to the surface QDs cannot be made. We applied an indirect HPLC-based analytical approach

9

to analyze the efficiency of QDs decoration with

NLS targeting residues, which is apparently one of the most important formulation parameters that can directly affect its intracellular targeting efficiency. This analysis revealed high efficiency of the Click reaction between the QD-azide and alkyne-



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modified NLS peptides, with 37.9% of the peptides that were added to the reaction tube forming covalent bond with the QDs, and additional 33.2% being adsorbed to the QDs (see Table 1). These reaction efficiencies correspond to 63.6 and 55.7 NLS peptides, conjugated and adsorbed, respectively, per single QD that was present in the reaction mix. Assuming globular shape of the QDs, these efficiencies indicate surface densities of 37 and 42 NLS peptides (conjugated and adsorbed, respectively) per 103 nm2 of QDs surface. Despite the availability of the research tools for quantitative analysis of DDSs and of their decoration with specific targeting residues, these tools are applied only by few researchers and are reported in some publications 7. Table 2 summarizes data from the studies that reported decoration efficiency with targeting residues of formulations intended for nuclear targeting. Based on these data, and the results obtained in our study, we can conclude that 3-stage decoration approach resulted in QDs densely decorated with NLS residues, and that the decoration efficiency is higher than in majority of previous studies that reported decoration efficiencies of formulations intended for nuclear-targeted drug delivery (see Table 2). The QDs that undergo all the decoration stages (QD-NLS) were characterized by a wide absorption spectrum that allowed efficient excitation by the laser wavelengths that are commonly used in FACS instruments and confocal microscopes (see Figure 2D). On the other hand, the fluorescence emission peak was sharp, and did not interfere with emission of other fluorophores that we used in our study (DAPI, LysoTracker green and DiO).

Endocytosis, endosomal escape and subcellular targeting of the generated QDs. Following incubation with HeLa cells in vitro, part of QDs undergo endocytosis, at the extent that differed between the formulations in the following order: QD-COOH < QD-azide < QD-NLS (see Figure 3A and B). This order is consistent with the differences in ζ−potential between the formulations and apparently reflects less efficient interaction with negatively charged cell membranes and lower endocytosis of particles with higher negative surface charge. It is also possible that differences in the QDs size (QD-COOH < QD-azide < QD-NLS, see Figure 2B and the Results) also affected to a certain extent efficiency of their endocytosis.


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All the three types of the investigated QDs formulations were able to escape efficiently from the endo-lysosome compartment (see Figure 4C and the Results). Apparently, this behavior was mediated by the large number of carboxylic acid groups (see Figure 4A and B) offering proton 'sponge' activity to the QDs surface 14. It is also possible that QDs-NLS endocytosis proceeded in part via lipid rafts directly into the cytosol (via NLS-cell membrane interactions), thus bypassing the endolysosomal compartment 16. In addition to more efficient endocytosis by HeLa cells, QD-NLS were characterized by enhanced accumulation in the cells’ nuclei or in close vicinity to them, as compared to other studied QDs formulations (see Figure 3C). The QDCOOH and QD-azide particles that were able to penetrate the cells, apparently escaped from late endosomes/lysosomes, but yet accumulated predominantly outside the nucleus (in the perinuclear region of the cytosol, at some distance from the nucleus), while less than 15% (on average) of the intracellular content these QDs were found in the nucleus or in close vicinity to it. On the other hand, more than 50% (on average) of QDs decorated with the NLS targeting residues (QD-NLS) were find in the cell nucleus or in close vicinity to it. This preferential accumulation of QD-NLS in the nuclei or in close vicinity to them indicates that the NLS peptides indeed acted as targeting residues, and that their amount on the QDs surface was sufficient for efficient subcellular targeting of QDs to the nucleus. The minimal amount (surface density) of NLS peptides that is required for efficient nuclear targeting of QDs and of other nano-formulations cannot be revealed from the results of our study and requires further more detailed investigation. The ‘IntraCell’ plugin for ImageJ software was not able to discriminate between the QDs inside the nuclei and the QDs that were located in close vicinity to the nuclei, apparently due to the limited resolution of confocal imaging technique (512 x 512 pixels images presented in Figures 3A and 4C; that correspond to ~82 µm voxel dimensions along the X and Y axes, and 2-3 times higher dimensions along the Z axis 17). These limitations of the ‘IntraCell’ plugin-based analysis can be overcome by changing its algorithm (e.g., by excluding the voxels that are identified as the nucleus border from the analysis), by collection and analysis of 3-dimensional confocal images (the Z-stacks, see the supporting information), or by electron microscopy analysis of experimental samples. In our future studies, we plan to



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explore these approaches for investigation of subcellular targeting efficiency of QDs decorated with targeting residues, and of other types of nano-formulations. QDs-NLS targeting to the nucleus in our study was mediated by the NLS of the SV40 large T antigen, which possesses strong nuclear targeting activity in different experimental systems

18, 19

. This NLS binds the nuclear pore complexes

(NPCs) via specific nuclear import proteins 20. NLS binding to NPCs may result to adsorption of QD-NLS particles to the nuclear membrane, and in some cases apparently leads to passage of some of these particles into the nucleus. It has been reported that NPCs form complex structures (a central ring sandwiched by two outer rings) with a ~20-50 nm diameter tube-like structure that allows passage of small molecular weight compounds (by simple diffusion) and of macromolecules or particles (via energy dependent mechanism mediated by import peptides like NLS) 21. Indeed, NLS-decorated particles with diameter of up to 90 nm are able to pass the NPCs in NLS-dependent fashion and accumulate in the nucleus 22-24}, and some of the QDs-NLS that were applied in our study (~25 nm average diameter in serum-free buffer, up to 70 nm diameter in serum-containing buffer, see Figure 2B and supporting information, respectively) are expected to follow the same trafficking pathway. Unfortunately, extent of nuclear targeted efficiency was not quantified in majority of studies that investigated (and in some cases also claimed) nuclear-targeted DDSs 7. Comparison of our findings to the data from two individual studies that reported DDSs nuclear targeting efficiency (see Table 2) indicated that our experimental approach was effective, and potentially can be superior to other approaches. The mechanisms of the QDs endocytosis and intracellular trafficking in our study require detailed investigation in future studies. For instance, enhanced endocytosis of QD-NLS, as compared to the QD-COOH and QD-azide formulations (see Figure 3B), can be attributed to ζ-potential-dependency of the cell uptake pathways (e.g., of clathrin-mediated endocytosis) interaction with lipid rafts

16, 26

25

, to NLS-mediated specific

}, or to combination of these factors. It is hard to

differentiate between these mechanisms (e.g., using the scrambled peptide approach, as we used in our previous study 8) since both of them are mediated, in part, by the composition of the NLS peptide (6 positive charges and +24 Kcal/mol hydrophobicity


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for the SV40 large T antigen NLS modulators

of

the

individual

27

). Nevertheless, use of specific inhibitors and

endocytosis

and

trafficking

pathways

(e.g.

chlorpromazine, methyl beta cyclodextrin, genistein and amiloride to block clathrinmediated, lipid raft-mediated, caveolae-mediated and macropinocytosis types of endocytosis, respectively) can be instrumental for revealing these mechanisms 28, 29}. An additional issue that requires clarification is the relative contribution of the conjugated vs. adsorbed NLS peptides to the endocytosis and nuclear targeting of QDNLS formulation. NLS peptides conjugation to the QD surface via the 3-stage decoration approach that was applied in this study leads to their ‘anchoring’ to the QDs surface via the C-terminal end which is compatible with NLS interaction with nuclear import proteins. On the other hand, NLS peptides that are adsorbed to the QDs surface (e.g., via electrostatic interactions of the NLS peptide positive charges with the carboxylic acid groups on the QDs) are expected to interact less efficiently with the nuclear import proteins (due to the improper conformation), and to be gradually eluted from the QDs surface. Thus, the adsorbed NLS peptides are expected to have a limited contribution to the endocytosis and nuclear targeting of QD-NLS formulation.

Future plans. The experimental system that is reported in this study (QDs decorated with NLS targeting residues using a 3-stage approach) and the available quantitative research tools make possible further detailed investigation of DDSs endocytosis and intracellular disposition and its dependence on the formulation properties. In our future studies, we plan to investigate the mechanisms that govern the QDs nuclear targeting and their limiting factors (mechanisms and efficiencies of endocytosis, endosomal escape, and intracellular trafficking). Based on the same experimental system that was used in this study, we will be able to: a) generate QDs decorated with different nuclear targeting residues, and analyze their relative efficiency and mechanisms of their effects on the QDs endocytosis and intracellular trafficking, b) determine the density of targeting residues on QDs surface that are required for efficient nuclear targeting, c) determine the relationship between the QDs size and surface charge and the nuclear targeting efficiency, d) investigate the differences in nuclear QDs targeting in cells of different origin, etc. These findings will contribute to development of subcellularly-targeted DDSs that will deliver specific drugs (such as anti-cancer agents and DNA drugs) to the nuclei of the target -17ACS Paragon Plus Environment


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cells and will enhance efficacy and reduce toxicity of these drugs. Similar experimental approach with appropriate analytical tools (fluorescence-based analysis of drugs or DDSs in non-continuous organelles 30, electron microscopy analysis, and so on) can be applied to target drugs to other intracellular locations, such as mitochondria (some anti-cancer agents, drugs for neurodegenerative diseases), endoplasmic reticulum (peptide and protein vaccines, drugs for protein folding diseases), cytosol (siRNA drugs, some antibiotics), etc.

CONCLUSIONS We generated QDs densely decorated with NLS peptidic targeting residues using a 3-stage decoration approach. Surface decoration of QDs significantly affected their endocytosis and intracellular trafficking. Specifically, dense decoration with NLS residues increased the endocytosis efficiency and enhanced accumulation of QDs in the cell nuclei or in close vicinity to them. The experimental system that is reported in this study is suitable for quantitative analysis of the mechanisms that govern the QDs nuclear targeting and their limiting factors (mechanisms and efficiencies of endocytosis, endosomal escape, and intracellular trafficking), and their dependence on the formulation properties (QDs size and charge, type and density of NLS residues, etc.). These findings will contribute to development of subcellularlytargeted DDSs that will deliver specific drugs (such as anti-cancer agents and DNA drugs) to the nuclei of the target cells and will enhance efficacy and reduce toxicity of these drugs.


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SUPPORTING INFORMATION QD formulations sizes and ζ–potentials in serum-containing medium; Z-stacks of HeLa cells incubated with QD-NLS formulation; colocalization of QDs with the late endosomes in the HeLa cells (PDF)

3D view of the QD-COOH particles in the HeLa cells (AVI)

3D view of the QD-azide particles in the HeLa cells (AVI)

3D view of the QD-NLS particles in the HeLa cells (AVI)

AUTHOR INFORMATION Corresponding Author *D.S. Tel: +972 8 6477381. E-mail: [email protected].

Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGEMENTS Dr. Amit Ranjan Maity receives post-doctoral scholarship for Outstanding Postdoctoral Fellows from China and India from the Planning and Budgeting Committee of the Israeli Council for Higher Education. We are grateful for Dr. Maya Bar Sadan and Dr. Pradipta Sankar Maiti (Dept. of Chemistry, Ben-Gurion University of the Negev) for providing high temperature synthesis equipment and for help with QDs synthesis.



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Table 1. The experimental setup and efficiency of Click chemistry-based decoration of QDs with the NLS peptide residues (the last stage of QDs decoration). Process

Reaction components added Process efficiency Estimated QDs to the tube decoration efficiency Peptide QD-azide Catalyst % of added No. of Surface area Number of peptides peptides per per peptide peptides per QD (nm2) 103 nm2

Unreacted (no incubation)

Yes

No

No

0.78

1.30

-

-

Degradation

Yes

No

No

4.4

7.3

-

-

Yes

No

Yes

23.7

39.8

-

-

Yes

Yes

No

33.2

55.7

27.06

37

Yes

Yes

Yes

37.9

63.6

23.17

42

Sedimentation/ Complexation Adsorption to QDs Covalent conjugation to QDs


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Table 2: Decoration with targeting residues and nuclear targeting efficiencies of formulations used for nuclear targeting in previous studies. Study

Formulation

Tkachenko Gold NP decorated et al., with BSA-NLS, ~21 2003 23 nm diameter QD-NLS, QDHoshino et mitochondria al., 2004 31 targeting peptide, 10100 nm PLGA NP-NLS Misra & loaded with Sahoo doxorubicin, 226 nm 2010 32 diameter

Pan et al., 2013 33

Doxorubicin-loaded mesoporous silica NP-TAT peptide, 57.49 nm diameter

Decoration with Number Estimated targeting residues of number of chemistry targeting targeting residues residues per DDS per 103 nm2 of DDS surface maleimide-thiol coupling

6±2

4.33

not reported

maleimide-thiol coupling

48

5.79

not reported

carbodiimide reaction

443

2.70

not reported

carbodiimide reaction

Kaplun & PLGA NP-NLS carbodiimide Stepensky targeting peptide, 350 reaction followed 2014 9 nm diameter by Click chemistry

Tang et QD-NLS, 3-11 nm al., 2014 34 diameter;

Nuclear targeting efficiency

zinc-amine interaction

~16-30% of the DDS localized in nucleus, as 106, 293 10.2, 28.2, measured by or 377 or 36.3 the Si content in the nuclei vs. the whole cells 28,80034,000

15-250


74.8-88.3

not reported

530-658

nuclear uptake of 30-60% of the DDS, in a NLS densitydependent fashion


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Figure 1. The consecutive stages of QDs surface decoration that were carried out in this study.


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Figure 2. QDs characterization at the different surface decoration stages: (A) morphology analysis by transmittance electron microscopy, (B) size distribution measurements using dynamic light scattering, (C) FTIR spectra of QDs formulations, and (D) absorption and emission spectra of QDs that undergo all the decoration stages (QD-NLS).



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Figure 3. Intracellular targeting efficiency of the investigated QDs formulations following their incubation in vitro with HeLa cells for 12 hours: (A) representative confocal images (red – QDs, blue – DAPI nuclear stain, green – DiO membrane stain), (B) QDs endocytosis analysis by flow cytometry (488/575 nm, ex/em), (C) relative amounts of individual formulations that accumulated in the nuclei of the individual cells or in vicinity to them. MFI – mean fluorescence intensity.


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Figure 4. Analysis of the number of surface carboxylic groups, proton ‘sponge’ activity and endosomal escape of the investigated QDs formulations. (A) Methylene blue-based estimation of the number of free carboxylic acid functional groups on QDs surface, calibration curve and analysis results, (B) Analysis of QDs proton absorption behavior by titration with 0.1N HCl, (C) Representative confocal images of HeLa cells incubated with the studied formulations and treated with LysoTracker green dye (red – QDs, blue – DAPI nuclear stain, green – LysoTracker green DND-26 late endosome/lysosome stain).



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Figure 5: Effect of 12 h incubation with QDs on the metabolic activity of HeLa cells measured using MTT assay. Tissue culture medium was used as negative control (100% viability). * - significantly different from the negative control, p

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