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. 2020 Apr 21;5(17):9864–9869. doi: 10.1021/acsomega.0c00079

Size-Dependent Transmembrane Transport of Gold Nanocages

Yu Yang †, Qingrong Zhang †, Mingjun Cai ‡, Haijiao Xu ‡, Denghua Lu †, Yulin Liu †, Yanfeng Fu †, Guocheng Yang †, Yuping Shan †,*
PMCID: PMC7203911  PMID: 32391473

Abstract

graphic file with name ao0c00079_0005.jpg

Gold nanocages (Au NCs), as drug carriers, have been widely applied for cancer diagnosis and photothermal therapy (PTT). Transmembrane transporting efficacy of Au NCs is the fundamental and important issue for their use in PTT. Herein, we used a force tracing technique based on atomic force microscopy to track the dynamic transmembrane process of Au NCs at the single-particle level in real time. Meanwhile, we measured and compared the dynamic parameters of Au NCs with sizes of 50 and 100 nm usually used as nanodrug carriers of PTT. It is concluded that the 50 nm Au NC transmembrane transporting needs smaller force and shorter duration with a much faster speed. However, both the 50 and 100 nm Au NC transmembrane transporting depends on the caveolin-mediated endocytosis, clathrin-mediated endocytosis, and macropinocytosis, which was also confirmed by confocal fluorescence imaging. This report will provide a potential technique for screening nanodrug carriers from the perspective of transmembrane transporting efficacy.

Introduction

Nanostructure-mediated photothermal therapy (PTT) materials have been intensively investigated due to their advantages on cancer diagnosis and therapy.1 By utilizing nanocarriers, the efficacy of PTT was enhanced, which has been verified through imaging, therapeutic functions, and accumulation in tumor.2 Dai et al. prepared homogeneous polypyrrole (PPy) nanoparticles (NPs) with excellent PTT capacity in vitro, and subsequently, the photothermal effects of PPy were further studied in a mouse tumor.3 Zhu et al. also offered a new design method to coat red blood cell membranes on gold nanocages (Au NCs), which can potentially be used for targeting PTT and chemotherapy of cancer.4 The PTT efficacy of nanostructures is influenced by the size and shape that will affect the distribution of heat in the entire tumor.5 Yang et al. found that the PTT efficacy is size-dependent, and with the increase in carbon NP size, the absorption in near-infrared increases and the efficiency of the photothermal conversion increases.6 However, among the process of PTT, transmembrane transport of nanostructures is the first and critical step, which will tremendously affect the PTT efficacy.

There are currently two major subgroups of photothermal agents: carbon nanotubes and gold-based nanostructures including nanocages,7 nanorods,8 and nanostars.9 Au NCs were applied at the forefront of the nanostructure-mediated PTT material due to the easily modified surface,10 storing drug inside the nanocage for controlled release and accumulating within the tumor through enhanced permeability and retention effects.11 Unfortunately, how the size of Au NCs affects the PTT efficacy is still not clear, especially from the perspective of the transmembrane transporting process. The transmembrane transport of Au NCs is a dynamic process, and one technique with higher temporal–spatial resolution is necessary to track the dynamic process. Taking the advantages of high temporal–spatial resolution and single-particle tracking under physiological conditions, the force tracing technique on the basis of atomic force microscopy (AFM) has proven to be an excellent method for studying the transmembrane dynamic mechanism.12 Force tracing could detect force as low as 10 pN and track fast processes down to 10 μs, which is much faster than other force testing techniques (for example, magnetic forceps,13 optical tweezers,14 and glass microneedles15). The force tracing technique has been used to study the differences in transmembrane transporting dynamic parameters of gold nanoparticles (Au NPs, 5 nm, 10 nm, and 20 nm).16

Herein, the forcing tracing was used to study the transporting effect of larger Au NCs with different sizes by evaluating their transmembrane transporting dynamic parameters at the single-particle level under physiological conditions. The transmembrane force, duration, displacement, and average speed for different sizes of Au NCs were measured and compared. This report will provide a helpful technique to screen nanostructures with appropriate size as nanodrug carriers.

Results and Discussion

Human cervical cancer cells (HeLa cells) were used to track the dynamic transmembrane process of Au NCs in serum-free media. As shown in Figure S1, the Au NCs (red) could transport into HeLa cells through the cell membrane (green). To implement the transmembrane tracking, the Au NCs were covalently conjugated to the AFM tip via a heterobifunctional (SH-PEG-NHS, MW: 3400) linker, as illustrated in Figure 1A. The PEG linker was immobilized on the aminated AFM tip using the NHS terminus, and the −SH of the immobilized linker reacted with Au NCs through Au–S. The total length of the linker is approximately 30 nm, which is the adequate length to measure the internalization of Au NCs.17 As shown in Figure S2, it was demonstrated that Au NCs have successfully attached to the AFM tip. Wang et al. proposed a protein layer-lipid-protein island (PLLPI) model in which the thickness of the cell membrane is approximately 20 nm.18 According to the PLLPI model, the PEG linker is long enough to penetrate not only the lipid bilayer but also the layer of macromolecules connected to it on both the intra and extracellular sides. To perform the force tracing experiment, the Au NC-modified AFM tip was located on the cell surface with the help of CCD (Figure S3). After approaching to the cell surface, the force–distance curves were obtained to find out the contact point between the Au NC-modified AFM tip and the cell surface (Figure S4). Subsequently, the AFM tip was slowly moved to the contact point and slightly brought into contact with the cell surface via the AFM feedback system, and then the feedback system was turned off.12 The diagram of the force tracing setup is shown in Figure 1B, the photodetector will record the change of laser position caused by the cantilever deflection. Once the Au NCs on the AFM tip is internalized, the cantilever deflection caused by the internalization will be recorded by the PCI-DAQ card. The sampling rate of 20 kHz (the maximum sampling rate could be up to 1 MHz) was used for recording the dynamic transmembrane process.

Figure 1.

Figure 1

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Schematic diagram of the force tracing technique. (A) Representation of the AFM tip functionalization: the Au NCs were covalently conjugated to the AFM tip via a heterobifunctional PEG linker. (B) Diagram of the force tracing workflow.

The transmembrane dynamic process of 50 and 100 nm Au NCs was tracked. Figure 2A,B shows the typical transporting force tracing curves of 50 and 100 nm Au NCs, respectively, and the curves start from left to right. The transmembrane process on the curve can be described as: (i) The Au NCs attached on the AFM tip just located on the cell membrane in which the force tracing curve is flat. (ii) The AFM tip cantilever bended downward because of the Au NC internalization (red arrow),19 which could be converted into the transporting force of Au NCs. In addition, during the internalization process, a steeper increase (blue arrow in Figure 2A,B) was observed, which is not very clear due to the complex force applied on the AFM tip cantilever and the complicated cellular uptake process of Au NCs.20,21 Then, the AFM cantilever returned to its original flexion at which the force tracing curves returned to the level again.22 To confirm that the force signals resulted from the transmembrane transporting of Au NCs rather than from other factors, control experiments were performed on HeLa cells. As shown in Figure S5 no force signals were found in force tracing curves obtained by both the clean AFM tip and only the PEG linker-modified AFM tip.

Figure 2.

Figure 2

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Dynamic parameters of Au NCs transmembrane transporting on HeLa cells. (A, B) Typical force tracing curves for 50 and 100 nm Au NCs, respectively. (C, D) Force distribution for transporting 50 and 100 nm Au NCs, respectively. (E, F) Duration distribution for transporting 50 and 100 nm Au NCs, respectively. (n = 200).

Both the force and duration for transporting 50 and 100 nm Au NCs were directly measured from the force tracing curves. The force for 50 nm Au NCs ranged from 40 to 160 pN with a mean value of 77.2 ± 1.34 pN, and the force for 100 nm Au NCs was in the range of 40–180 pN with a mean value of 101.71 ± 2.01 pN, as shown in Figure 2C,D, respectively. The duration for 50 nm Au NCs was in the range of 70–550 ms with a mean value of 184.97 ± 5.62 ms, whereas the duration for 100 nm Au NCs varied from 80 to 1000 ms with a mean value of 426.63 ± 20.93 ms (Figure 2E,F). It is found that both the force and duration increased with the increase in Au NC size, and the size-dependent transmembrane dynamic parameters of Au NCs could be attributed to the interaction surface area. As the size of Au NCs increases, the interaction surface area between Au NCs and the cell membrane increases, and the local membrane curvature of the contacting area becomes smaller.21 In addition, the larger particles (100 nm Au NCs) may need larger force for rotation during the internalization.20,21

The displacement of the Au NCs during the transmembrane transporting was also calculated. As shown in Figure 3A, the modified Au NCs on the AFM tip located and brought into contact with the cell membrane before internalization. However, the internalization of Au NCs will cause the downward bending of the cantilever and the stretch of the PEG linker. Hence, the displacement D of Au NCs consists the bending distance of the AFM tip cantilever d and the extension length of the PEG linker Q.

graphic file with name ao0c00079_m001.jpg 1

Figure 3.

Figure 3

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Displacement of the Au NCs during the transmembrane transporting process. (A) Diagram representation of the displacement caused by the extension of the PEG linker and the cantilever deflection. (B) 2D histogram of force–displacement of 50 nm Au NCs. (C) 2D histogram of force–displacement of 100 nm Au NCs; the scale on the right side represents the number of data corresponding to different colors.

The force-dependent extending behavior of the PEG linker can be most appropriately described by the extended worm-like chain (WLC) model following the equation23

graphic file with name ao0c00079_m002.jpg 2

where T represents the absolute temperature, kB represents the Boltzmann constant, Lp is the persistence length, k0 represents the enthalpic correction, Q represents the extension of the PEG linker, and L0 is the contour length. Based on previous report, the persistence length Lp is 3.8 ± 0.02 Å, and the enthalpic correction k0 is 1561 ± 33 pN. Given that the length of the PEG unit is 4.2 Å, the total estimated contour length L0 for PEG of 76-77 is nearly 321 Å. The bending distance d of the AFM tip cantilever can be calculated according to Hooke’s law:

graphic file with name ao0c00079_m003.jpg 3

where F is the force measured from the force tracing curves, and k is the spring constant of the AFM tip cantilever. The displacement D was obtained based on the above eqs 1, 2, and 3. The mean values of displacement were 29.76 and 31.09 nm for 50 and 100 nm Au NCs, respectively (Figure S6). The displacement of the 100 nm Au NCs is moderately greater than that of 50 nm Au NCs possibly due to the longer displacement required for the 100 nm Au NC rotation. Furthermore, the correlation between the displacement D and the transporting force was statistically compared. Figure 3B,C shows the force–displacement 2D histograms of 50 and 100 nm Au NCs, and the relationship between the transporting force and the displacement is plotted as exponential curves. The number on the right side of Figure 3B represents the number of data corresponding to different colors. The average speed of Au NC transmembrane transporting was also calculated by D/duration, and the average speed values for 50 and 100 nm Au NCs are 0.161 and 0.0728 μm/s, respectively. It is noted that the average speed of 50 nm Au NCs is faster than that of 100 nm Au NCs, which may be attributed to the larger size of the 100 nm Au NCs (which takes longer time) during internalization.24 Taken together, all the dynamic parameters including force, displacement, and speed are size-dependent during the transmembrane transporting process.

To further compare the transmembrane transporting of 50 and 100 nm Au NCs, a series of blocking experiments were performed. It is reported that HeLa cells uptake of Au NCs does not appear to follow any single specific pathway but use multiple pathways including caveole-dependent and clathrin-dependent endocytosis and macropinocytosis.25 Before performing the force tracing test, several drugs that block different pathways are used to incubate with cells.25 After blocking, most of the force signals disappeared, further confirming that the force signals in force tracing curves result from the transporting event (Figure S7). Methyl-β-cyclodextrin (M-β-CD) represents an inhibitor that depletes cholesterol by extracting cholesterol, which affects many pathways including macropinocytosis, clathrin-dependent or caveolin-dependent endocytosis, and lipid raft-dependent uptake.26 As shown in Figure 4A, the probability of force signals in force tracing curves for the control group (Au NCs, without the inhibit reagent incubation) was set to 100%. After incubating the cells with 10 mM M-β-CD at 37 °C for 30 min, the probability of force signals in force tracing curves decreased to 12.28 and 22.51% for 50 and 100 nm Au NCs, respectively. The Filipin inhibits the caveolin-dependent endocytosis via binding to the tiny components of glycolipids.27 After incubating the cells with Filipin (ultimate concentration of 5 μg/mL) for 30 min at 37 °C, the probability of force signals dramatically decreased to 11.98 and 16.80% for 50 and 100 nm Au NCs, respectively. Chlorpromazine (CPZ) is known to interfere with the clathrin-dependent pathway, which is ascribed to the blockage of the assembly of clathrin-coated pits.28 CPZ (ultimate concentration, 10 μg/mL) was coincubated with the cells for 30 min at 37 °C, and the probability of force signals reduced to 25.8 and 31.12% for 50 and 100 nm Au NCs, respectively. 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) specifically inhibiting the Na+/H+ pump located in the membrane led to inhibition of macropinocytosis.29 After treating the cells with 20 μg/mL (final concentration) EIPA for 1 h at 37 °C, most of the force signals disappeared, and the probability values of force signals were 20.40 and 29.63% for 50 and 100 nm Au NCs, respectively. We also measured the transporting force and duration of 100 nm Au NCs after inhibition, and comparing with those before inhibition, the values were not significantly different (Figures S8 and S9).

Figure 4.

Figure 4

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Blocking experiments. (A) Probability of force signals on HeLa cells before and after blocking with CPZ, EIPA, Filipin, and M-β-CD. The results were calculated by randomly selecting approximately 1000 force tracing curves (the number of force tracing curves with force signals divided by 1000). Data are the average of three independent measurements, and each set analyzed 1000 force tracing curves. (B) Average fluorescence intensity of HeLa cells coincubated with Au NCs before and after blocking with CPZ, EIPA, Filipin, and M-β-CD. Value is expressed as mean ± standard deviation.

Confocal fluorescence imaging was also performed to confirm the results (Figure S10). The amount of Au NCs that enter the cells was quantified as the average fluorescence intensity, and before inhibition, the average fluorescence intensity was set to 100%. After blocking with M-β-CD, the intensity decreased to 19.87 and 36.04% for 50 and 100 nm Au NCs, respectively. After blocking with Filipin, the intensity decreased to 15.63 and 26.53% for 50 and 100 nm Au NCs, respectively. After blocking with CPZ, the intensity values were only 28.80 and 32.10% for 50 and 100 nm Au NCs, respectively. EIPA inhibition resulted in the intensity reduction to 30.42 and 58.15% for 50 and 100 nm Au NCs, respectively (Figure 4B). It is worth noting that the data collected from the two techniques show a similar trend. Therefore, we can conclude that the HeLa cell transmembrane transport of 50 and 100 nm Au NCs is dependent on caveolin-dependent endocytosis, clathrin-dependent endocytosis, and macropinocytosis pathways.

In summary, the transmembrane transporting dynamic process of 50 and 100 nm Au NCs was successfully tracked by the force tracing technique at the single-particle level. The key dynamic parameters including force, duration, and average speed were measured and compared. The dynamic parameters of transmembrane transporting are size-dependent. For 50 nm Au NCs, transmembrane transporting needs smaller force, and particularly, the transmembrane transporting of 50 nm Au NCs takes shorter time with a much higher average speed. Blocking experiments showed that both the 50 and 100 nm Au NC trans-membrane transporting relied on caveolin-mediated endocytosis, clathrin-mediated endocytosis, and macropinocytosis. Our results will provide insight into the Au NC transmembrane transporting dynamic mechanism and may pave the way for designing appropriate nanostructures in nanomedicine applications from the perspective of transmembrane dynamics.

Materials and Methods

Materials and Cells

The Au NCs with good size dispersion (UV–vis spectrum of Au NCs was provided by the company) were purchased from Nanjing JCNANO Technology Co., Ltd. The surface of the Au NCs is coated with citrate to avoid aggregation. The remaining chemical reagents are of analytical grade and from Sigma-Aldrich (St. Louis, Missouri, USA). HeLa cells were purchased from Shanghai Institute of Biological Sciences. Force tracing experiments and fluorescence experiments were performed in the serum-free medium.

AFM Tip Modification

The AFM tips (MSCT, D-tip, Santa Barbara, CA) were first functionalized with APTES. The desiccator was purged with argon for 2 min, and 30 μL of APTES (99%) and 10 μL of N,N-diisopropylethylamine (99%) were placed into a small container at the bottom of the desiccator, leaving the AFM tips exposed to APTES vapor for 2 h. Subsequently, the PEG cross-linker (SH-PEG-NHS, MW: 3400) was conjugated with APTES in triethylamine and trichloromethane, as previously described.30 The AFM tips were then immersed in 100 μL of solution containing Au NCs for 1 h, and after functionalization, the AFM tips were washed with PBS three times and stored at 4 °C.

Cell Culture

The cells were cultured on cover glass slides in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum, 100 μg/mL penicillin, and 100 μg/mL streptomycin at 37 °C under an atmosphere of 5% CO2. Typically, cells need to be cultured for 1 to 2 days to achieve 75% coverage of cover glass slides for the force tracing experiments. Before performing the force tracing experiments, the adherent HeLa cells were sterilized with PBS (phosphate buffer, 1000 mL of PBS: 8.0 g of NaCl, 3.4785 g of Na2HPO4·12H2O, 0.2 g of KCl, 0.2 g of KH2PO4) three times and then washed with the serum-free DMEM medium once to remove cell metabolites and unattached cells.

Force Tracing Measurements

AFM 5500 (Agilent Technologies, Chandler, AZ) was used to acquire the force tracing curves. Before performing the force tracing test, the Au NCs attached to the AFM tip were slowly moved to the contact point using a proportional-integral (PI) control system with P = 0.001 and I = 0.001 (the error signal between the set point and the deviation of the cantilever is 2.0 V). The feedback system was then turned off, and the movement of the AFM tip was stopped. A small offset of the cantilever deflection signal was collected by a 16-bit DA/AD card (PCI-6361e, National Instruments). In this report, a 20 kHz data acquisition sampling rate was applied and the high-frequency electronic noise was filtered through a 100 low-pass filter. The sensitivity and the spring constant of the AFM tip are determined based on previous report.31 The force tracing tests were carried out in 2 mL of serum-free DMEM at 37 °C.

Blocking Experiments

In the blocking experiment, HeLa cells were incubated with EIPA (20 μg/mL) for 1 h, M-β-CD (10 μg/mL) for 30 min, CPZ (10 μg/mL) for 30 min, and Filipin for 30 min at 37 °C. In control experiments, the force tracing experiments were performed using clean AFM tips (unmodified tip) and AFM tips modified with only the PEG-linker.

Staining of Au NCs

For staining Au NCs, 0.4 mL of a 0.25 mM HS-PEG-NH2 (MW: 5 K) aqueous solution was added to 1 mL of Au NC aqueous suspension, 0.6 mL of H2O was added, and then the reaction mixture was immediately vortexed and kept at 4 °C overnight. After the reaction, the mixture was centrifuged at 14,000 rpm for 5 min and washed three times to obtain Au NCs@PEG particles. The Au NCs@PEG particles were reacted with excess Cy5 (2 μL, 10 mg/mL) for 3 h, and the resulting mixture was purified using a G-25 gel column to gain Au NCs@PEG@Cy5 particles.

Fluorescence Imaging

HeLa cells were cultured on glass dishes for at least 24 h. The Au NCs@PEG@Cy5 particles were coincubated with cells in the serum-free medium for 1 h at 37 °C, washed with PBS, and incubated with 5 mM DiO for 20 min at 25 °C. Excess DiO was washed with PBS three times. Fluorescence imaging was performed on a Leica TCS SP2 confocal microscope. Au NCs@PEG@Cy5 was excited with a 647 nm He-Ne laser, and DiO was excited with a 488 nm Ar-Kr laser. The fluorescence images were collected with a 100× 1.49 NA oil-immersion objective.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (nos. 21773017 and 21673023).

Glossary

Abbreviations

PTT

photothermal therapy

Ppy

polypyrrole

NPs

nanoparticles

Au NCs

gold nanocages

AFM

atomic force microscopy

SMFS

single-molecule force spectroscopy

PEG

poly(ethylene glycol)

APTES

3-aminopropyltriethoxysilane

WLC

worm-like chain

CPZ

chlorpromazine

M-β-CD

methyl-β-cyclodextrin

EIPA

5-(N-ethyl-N-isopropyl)amiloride

HeLa cells

cervical cancer cells

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c00079.

  • Fluorescence image of HeLa cells after incubation with Au NCs; finding out the contact point; optical image of the AFM tip cantilever located on the living cell surface; and blocking experiments (PDF)

Author Contributions

All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

Supplementary Material

ao0c00079_si_001.pdf (1.3MB, pdf)

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