Tracking the Single-Carbon-Dot Transmembrane Transport by Force

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Tracking the single carbon dots trans-membrane transporting by force tracing based on atomic force microscopy Denghua Lu, Xudong Yang, Qingrong Zhang, Ruixia Wang, Siyuan Zhou, Guocheng Yang, and Yuping Shan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01363 • Publication Date (Web): 21 Dec 2018 Downloaded from http://pubs.acs.org on December 24, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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ACS Biomaterials Science & Engineering

Tracking the single carbon dots trans-membrane transporting by force tracing based on atomic force microscopy Denghua Lua, Xudong Yanga, Qingrong Zhanga, Ruixia Wanga, Siyuan Zhoua, Guocheng Yanga, Yuping Shana* aSchool

of Chemistry and Life Science, Advanced Institute of Materials Science, Changchun

University of Technology, Changchun 130012, China, *Email: [email protected] KEYWORDS: carbon dots (CDs); trans-membrane transporting; force tracing; atomic force microscopy (AFM) Abstract: Carbon dots (CDs) evoke a great deal of attention on biomedicine due to its unique properties. As an emerging theranostic agent, CDs trans-membrane transporting process is the first and the most important step. Herein, trans-membrane dynamic process of transporting single CDs with folic acid into normal cells (Vero) and cancer cells (HeLa) were tracked respectively by means of force tracing technique based on atomic force microscopy. Meanwhile, the kinetic parameters of the trans-membrane process were measured and calculated. Interestingly, comparison between

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cancer cells (HeLa) and normal cells (Vero), the transporting force of single CDs with folic acid was smaller, and the transporting average speed was slower. Introduction Drug delivery systems (DDSs) based on nanoparticle has been studied for decades, being one of the most active multidisciplinary fields of research to date1. Of the several drug delivery platforms available, liposomes2, polymers3, and quantum dots are the most widely studied. As a sort of quantum dots, carbon dots have emerged as a potential alternatives to classical metalbased semiconductor quantum dots due to the abundance of their precursors4, ease of synthesis, strong fluorescence5, 6, favorable chemical stability, biocompatibility7, low toxicity8, 9 and high loading capacity10, especially, act as a platform for carrying several drugs and gene11. Kong et al. investigated the therapeutic activity of CD-DOX complex, and the results indicated that the CDDOX showed high efficacy on MCF-7 cells12. In the process of drug and gene delivery, the transmembrane process is the first and the most important step13, 14. However, its dynamic mechanism is still unclear. Although, Shi et al. had studied the dynamic process of carbon nanotubes entering cells, all of their experiments were performed on pure lipid bilayer and surfactants systems15. Therefore, these results obtained from the artificial bio-membrane model failed to reflect the real transporting dynamic process on living cells. In order to enhance the extensive application of CDs on biomedicine, a technique with high temporal-spatial resolution to track the trans-membrane dynamic process of transporting CDs into living cells is highly desirable. With high spatial resolution, atomic force spectroscopy (AFM) could detect the force down to 10 pN owing to extreme sensitivity of the AFM tip cantilever. Single molecule force spectroscopy (SMFS) technique based on AFM has widely been applied to study the interaction events of single molecule under near physiological condition16-20. In order to capture the dynamic


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trans-membrane process and detect important kinetic parameters, force tracing technique developed from SMFS by Wang et al. has been used to investigate the trans-membrane process of single virus particle21. Here, the technique was utilized to study the dynamic process of CDs trans-membrane transporting. Results and discussion With well adhesiveness22, Vero cell line is chosen to perform the force tracing measurement experiments. The CDs with diameter about 10 nm were prepared by hydrothermal reaction, and CDs surface are covered by folic acid and -NH2, the synthesis details and the characterization (Diameter distribution and FTIR spectra) are shown in supporting information (Figure S1 and S2). The AFM image confirms that the CDs are well dispersed (Figure S2A), ensuring the single particle experiments. CDs were covalently conjugated onto the AFM tip through a heterobifunctional aldehyde-PEG (poly (ethylene glycol))-NHS (N-Hydroxysuccinimide ester) linker23. The PEG linker was attached onto the AFM tip through the NHS group reacting with the 3-aminopropyltriethoxysilane (APTES)-aminated tip, and the aldehyde group from the other side of the PEG linker reacted with the -NH2 on CDs, the details are shown in Figure 1A. The length of PEG linker (30 nm) is suitable for measuring the dynamic process of CDs transporting into the living cells via cell membranes (approximately 20 nm)24, 25. Thus-modified AFM tip with low density of PEG linker has been validated effectively for detecting the activity at the single molecule level25-27. The principle of force tracing is shown in Figure 1B, the laser beam is reflected by the cantilever of AFM tip, and the photodetector records the laser position change that reflects the AFM tip cantilever deflection. To perform force tracing test, the AFM tip tethered with CDs was located onto the monolayer cell surface with the help of CCD camera (Figure S3). Then the


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AFM tip was engaged utilizing the PicoView software, and the force-distance curves were performed to determine the contact point (Figure S4) between cell membrane and the CDs attached on AFM tip. Then, the AFM tip was slowly moved to the contact point by virtue of the feedback system, and the feedback system was switched off. Once the CDs was internalized by cells, the AFM tip cantilever would bend downwards, and the vertical changes of AFM tip cantilever could be collected by a data acquisition card (PCI card). The (2 MS/s, 2 million data points per second) sampling rate of the PCI card is capable of monitoring the super-fast process down to 20 μs. In this report, the 20 kS/s sampling rate of data acquisition was applied, and the high frequency electronic noise was filtered by a 100 low pass filter. The typical force tracing curve is shown in Figure 2A, the curve starts from left to right. Initially, the CD tethered on AFM tip just locating on the cell membranes, the AFM tip cantilever kept a force balance position, and the left side of curve was flat. When the CD was internalized by the living cells, the AFM tip cantilever bended downwards and a sudden step in the curve (indicated by the red arrow) appeared. On the basis of the constant position mode of force tracing technique23, piezoelectric ceramics would drive the AFM tip cantilever to keep a force balance position after the internalization of CDs, and the force curve reached flat again (as indicated by the right parallel portion of the force tracing curve). Because of the early stage features of cellular uptake activity28, the transporting event does not happen occasionally, therefore positioning on different cells, thousands of the force tracing curves were collected. The duration and force for CDs transporting could be measured straightforwardly from the force tracing curves. The duration (the time of the intrenalization event take) distribution varies from 5 to 65 ms with the average value of 13±8.3 ms (Figure 2B).


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And the corresponding force distributes from 33 to 351 pN with the average value of 79±50 pN, as shown in Figure 2C. To understand the dynamic transporting process in-depth, another key parameter, the average speed during the process of CDs trans-membrane transporting was calculated (displacement divided by duration). Figure 3 displays that the displacement (D) of CD transporting includes the bending distance of the AFM tip cantilever (d) and the stretching length of the PEG linker (Q), as expressed by the following equation. D  d Q

(1)

According to the relationship between PEG stretching behavior and the force, the value of Q could be obtained by the extended worm-like chain (WLC) model29: 2

FLP 1  Q F  1 Q F  1       k BT 4  L0 k0  4 L0 k0

(2)

Where Lp is the persistence length, kB stands for the Boltzmann constant30, T refers to the absolute temperature, the extension of the PEG linker is Q, L0 is the contour length31, and k0 presents the enthalpic correction. Referring to the literature29, 32, the persistence Lp is 3.8±0.02 Å, and k0 is 1561±33 pN. Contour length L0 of the PEG linker we used is calculated as 324.45 Å = (PEG unit (4.2 Å) x 76 unit) + 5.25 Å (the length of terminus group). Then, the Q is calculated as 24.1 nm. Based on Hooke’s law, the bending distance d of AFM tip cantilever could be obtained from the following equation:

F  k d

(3)

Where F is the force of single CDs transporting into cell measured from the force tracing curves, and k represents the spring constant of the AFM tip cantilever. Thus, d is 2.26 nm calculated by


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the equation (3). Consequently, the displacement D is 26.36 nm, and the average speed of single CDs transporting into cell is calculated as 2.03 μms-1 (26.36 nm/13 ms). In previous report, the trans-membrane transporting dynamic process of single nanoparticle/nanotube under 10 nm was simulated based on lipid bilayer, the duration is around 1 s and the speed is around 0.7 ms-1. Comparing with the report, the transporting speed of nanoparticle in this manuscript is much slower, since the living cell membrane is much complicated than the lipid bilayer model33, 34. The force tracing will provide a useful technique to track the dynamic process on living cell membranes in real time. The mechanism of CDs trans-membrane is another key point for the dynamic transporting process. Previous literatures reported that the primary pathway for cellular uptake of CDs is macropinocytosis, clathrin-dependent endocytosis and caveolae-dependent endocytosis8. Here blocking experiments were implemented by utilizing different specific biochemistry agents that block the different trans-membrane pathway (Figure 4). In blocking experiments, the cells were pre-treated with the blocking agents for some time before force tracing test. Cytoskeleton could induce deformation of plasma membrane35, thereby affecting the trans-membrane transporting process. Herein the cells were co-incubated with microfilament-depolymerizing drug CB (Cytochalasin B, final concentration of 2 μg mL-1) for 20 min at 37 C. After blocking, the probability of force tracing curves with force signal decreased from 8.7±3.3% (Control) to 3.0±1.7%. The results manifest that the actin contributes to the endocytosis process of CDs. Clathrin-mediated endocytosis (CME) is the main endocytosis pathway for nanoparticle and virus36. Chlorpromazine (CPZ), a biochemical inhibitor, could inhibit clathrin-mediated endocytosis by preventing the assembly of clathrin-coated pit (CCP) at plasma membrane37. In this study, after co-incubation of CPZ (ultimate concentration of 10 μg mL-1) with cells at 37 C


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for 30 min, the probability of force signal dramatically decreased to 1.6±0.3%. Cholesterol is very important to the caveolae/raft-dependent endocytosis38, 39. Thus, the chemical agents filipin, nystatin, and methyl-β-cyclodextrin (M-β-CD), sequestering or depleting cholesterol from plasma membrane, are usually used to inhibit caveolae/raft-dependent endocytosis38,

40.

We

pretreated the Vero cells with nystatin (final concentration of 0.03 mM, 37 C, 10 min), filipin (final concentration of 5 μg mL-1, 37 C, 10 min), and the M-β-CD (5 mM, 37 C, 10 min), respectively. As a result, the probability of force signal reduced to 1.5±0.2%, 0.5±0.2%, and 4.3±0.4%, respectively. Macropinocytosis is one of the major pathways for trans-membrane transporting41. We used EIPA (5-(N-Ethyl-N-isopropyl), Amiloride), interfering membrane Na+/H+ ATPase, to suppress the macropinocytosis. After treating the cells with 60 μM (ultimate concentration) of EIPA at 37 C for 1 h, most of the force signal vanished and the probability of remaining force signal was only 1.9±0.1%. These results manifested that CDs were transported into cells via macropinocytosis, caveolin-dependent endocytosis, and clathrin-dependent endocytosis. In addition, after blocking by one inhibitor (block one type of endocytosis pathway), the other endocytosis pathways may work and a few force signals could be detected, the force tracing curves before and after blocking are shown in supporting information Figure S5. In order to exclude the influence of the molecules on AFM tip, force tracing experiments were implemented by PEG linker modified AFM tip and clean AFM tip on normal Vero cell surface, and almost no force signal could be detected (Figure 4). Generally, the cancer cell lines express higher folic acid receptor (FA-receptor) on cell membrane, here the CDs with FA trans-membrane transporting process on cervical cancer cells (HeLa cell) with higher expression of FA-receptor was also tested. Figure 5A shows the typical force tracing curve of CDs trans-membrane transporting (more zoomed-in force tracing curves


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are shown in supporting information Figure S6), and the force curve is similar to that of on Vero cells. The duration of the transporting event varies from 8.6 to 20.8 ms with the average value of 12.3±2.8 ms (Figure 5B), and the corresponding force distributes from 20.2 to 72.1 pN with the mean value of 33±12 pN (Figure 5C). We also calculated the displacement and average speed of CDs during transporting on HeLa cell, the displacement D is 21.6 nm (Q + d = 20.6 nm + 1.0 nm), and the average speed is 1.76 μms-1 (21.6 nm/12.3 ms). It is found that the transporting duration for the both type cells is almost indentical. However, the transporting force for HeLa cell (33±12 pN) is smaller than that of Vero cells (79±50 pN). Meanwhile, it is noted that the corresponding average speed (1.76 μms-1) is slightly slower comparing with Vero cells (2.03 μms-1). Both the smaller transporting force and slower transporting speed may be attributed to the FA-receptor mediated endocytosis of CDs. HeLa cell is a cancer cell line with higher expression of FA-receptor, and the FA groups on the CDs will induce more FA-receptor mediated endocytosis to transport CDs42. The FA-receptor mediated endocytosis may need more time to perform the process of binding FA to specific FA-receptor, receptor clustering and internalization through coated vesicles into cell43. Furthermore, comparing with Vero cell, the shorter displacement D for transporting CDs on HeLa cell may be the characteristic of the FA-receptor mediated endocytosis. In summary, the trans-membrane dynamic process of transporting single CDs into HeLa cells and Vero cells were successfully tracked by force tracing technique, and the key kinetic parameters including force, duration and average speed were measured at single particle level. It is found that the trans-membrane transport event would be accomplished in around 13 ms with the average speed of ~2 μms-1. Interestingly, comparing HeLa cell to Vero cell, the transporting force is much smaller and the average speed is slightly slower. We deduced that the interaction


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between folic acid on CDs and folic acid receptor on HeLa cell membrane induced receptor mediated endocytosis, decreasing the transporting force. Meanwhile, it is because the interaction, the average speed is a little bit slower. Blocking experiment results indicated that CDs transmembrane transporting depended on caveolin-dependent endocytosis, clathrin-dependent endocytosis, and macropinocytosis. This report will prompt the further application of CDs on nanomedicine and provide a new way to screen nano-drug carriers. Methods Cell culture: African green monkey kidney cells (Vero cells) and Human cervical cancer cells (HeLa cells) were purchased from the Shanghai Institutes of Biological Sciences. Vero cells and HeLa cells were maintained in 5% CO2 and 95% air environment at 37 C, supplemented with Minimum Eagle Medium (MEM, BI) and Dulbecco's Modified Eagle Medium (DMEM, BI) respectively, with 10% Fetal Bovine

Serum

(FBS,

BI)

and

Penicillin

(10,000

units

mL-1),

Streptomycin (100 μg mL-1). The cells were sub-cultured for 2 or 3 days when 75% of the petri dish was covered by cells. The cells were washed with PBS (phosphate buffer solution) for three times and serum-free medium one time in sequence to remove cell debris and unattached cells before using in the force tracing experiments22. Modification

of AFM

tips

with

CDs: Firstly,

AFM tips (MSCT,

Bruker, USA) were soaking in freshly prepared piranha solution (H2SO4 : 30% H2O2, 3:1, v/v) for 1 h. Then, the AFM tips were successively cleaned with water and ethanol, and dried by argon,


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purged with O3 flow for 20 min to further remove the organic pollution

on

the

Subsequently,

tips

the

and

dried

tips

for

were

2

h

in

desiccator.

modified

by

3-

aminopropyltriethoxysilane (APTES) using vapor phase deposition method

for

1.5

h,

combined

(benzaldehyde-PEG76-NHS,

with

FW~3962,

the

heterobifunctional

SensoPath

PEG

Technologies,

Bozeman, 1mg mL-1) in methylbenzene with the presence of 0.5% triethylamine (v/v) for 2 h. Then, the tips were conjugated with CDs (6.6 mg mL-1) in PBS solution with the presence of 0.01 M NaCNBH3 for 1 h after drying by argon flow. To deactivate the aldehyde groups that didn’t react, 5 μL of 1 M ethanolamine was added

into

the

reaction

solution

and

reacted

for

10

min.

Finally, the AFM tips were washed by PBS solution for three times and stored at 4 C until use. Force tracing measurement: The force tracing measurement s were performed on AFM 5500 (Agilent Technologies, Chandler, AZ). The experiments were carried out at 37 C by temperature controller 325 (Agilent Technologies, Chandler, AZ). According to different cell lines (Vero cell or HeLa cell), 2 mL MEM or DMEM was added into the petri dish. To perform the force tracing experiments, AFM tip modified with CDs was right located onto the monolayer cells surface with the help of CCD camera, as illustrated in Figure

S3,

which

could

ensure

that

every

experiment

was

accomplished on the cell membrane surface.


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To reach the contact point, we moved the AFM tip down to the cell

membrane

slowly,

while

the

proportional-integral

(PI)

control system (P=0.001; I=0.001; the error signal between the set point and the deflection of the cantilever is 2.0 V) was open.

When

the

CDs

attached

AFM

tip

contacted

with

cell

membranes, the feedback system was turned off. The cantilever would bend downwards once the CDs was internalized by cell, and the deflection of the cantilever was recorded by a 16-bit DA/AD card (PCI-6361e, National Instruments, US), which was controlled by LabVIEW software. During the experiments, thousands of force tracing curves were collected at different cells and analyzed by LabVIEW (National Instruments Inc, Austin, Texas, USA).


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Figure 1. Diagram of the force tracing technique. (A) Schematic diagram of AFM tip functionalization. The CD was covalently conjugated to the AFM tip through a heterobifunctional PEG linker. (B) The workflow image of force tracing.

Figure 2. Force tracing measurement results on Vero cells. (A) The typical force tracing curve of transporting CD into the Vero cells. (B) The duration distribution of transporting CD through cell membranes. (C) The force distribution of transporting CD through cell membranes. (n=200)


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Figure 3. Schematic illustration of CD displacement during the transporting process. (A) The CDs attached AFM tip was placed on the cell surface and the AFM tip cantilever maintained balance. (B) The CD was internalized by the cell with the AFM tip cantilever bending (d) and the extension of PEG linker (Q).

Figure 4. Control and block experiments. (A) The force tracing curves carrying out on Vero cells by clean AFM tip (clean tip), PEG linker modified AFM tip (PEG), and CDs modified AFM tip on Vero cells treated by blocking agents (CB, EIPA, CPZ, Nystatin, Filipin and M-βCD). (B) The statistical counts of force tracing curves with force signal before (control) and after inhibitor treatment. The results are calculated by randomly choosing around one thousand force tracing curves (the number of force tracing curves with force signal divided by one thousand).


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The data are the average values of three sets of independent experimental measurements, and one thousand force curves were analysed for each single set. Values are represented by mean±standard deviation.

Figure 5. Kinetic parameters of transporting CDs into HeLa cells. (A) The red arrow points out the typical force signal. (B) The duration distribution histogram of transporting CDs into HeLa cells (C) The force distribution histogram of transporting CDs into HeLa cells. (n=100)

ASSOCIATED CONTENT Supporting Information Synthesis of FA-CDs; FTIR spectrum of chitosan, FA, and CDs with FA; AFM imaging of CDs; Diameter distribution histogram of CDs; Force tracing curves with force signal before and after blocking obtained on Vero cells. AUTHOR INFORMATION Corresponding Author *Email: [email protected]


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Author Contributions All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by the National Natural Science Foundation of China (no. 21773017, 31330082, and 21673023 to Y. S.), Jilin Provincial Science Research Foundation of China (no.20160520133JH to Y. S.). Notes The authors declare no competing financial interest. ABBREVIATIONS CDs: carbon dots; DDSs: drug delivery systems; AFM: atomic force spectroscopy; PEG: poly (ethylene glycol); APTES: 3-aminopropyltriethoxysilane; WLC: worm-like chain; CB: cytochalasin B; CME: clathrin-mediated endocytosis; CPZ: chlorpromazine; CCP: clathrincoated pit; M-β-CD: methyl-β-cyclodextrin; EIPA: (5-(N-Ethyl-N-isopropyl), Amiloride); HeLa cells: cervical cancer cells; FA-receptor: folic acid receptor. REFERENCES 1.

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Tracking the single carbon dots trans-membrane transporting by force tracing based on atomic force microscopy Denghua Lua, Xudong Yanga, Qingrong Zhanga, Ruixia Wanga, Siyuan Zhoua, Guocheng Yanga, Yuping Shana* aSchool

of Chemistry and Life Science, Advanced Institute of Materials Science, Changchun

University of Technology, Changchun 130012, China，*Email: [email protected]

Trans-membrane dynamic process of transporting single CDs into cells were tracked by means of force tracing technique based on atomic force microscopy.


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Tracking the Single-Carbon-Dot Transmembrane Transport by Force

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