Evaluation of PAMAM dendrimers-stabilized gold nanoparticles: two-stage procedure synthesis and toxicity assessment in MCF-7 breast cancer cells

doi:10.21203/rs.3.rs-5280946/v1

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Evaluation of PAMAM dendrimers-stabilized gold nanoparticles: two-stage procedure synthesis and toxicity assessment in MCF-7 breast cancer cells

https://doi.org/10.21203/rs.3.rs-5280946/v1

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Gold nanoparticles stabilized with polyamidoamine dendrimers are one of the potential candidates for use as a contrast agent in computed tomography. This work demonstrates a rapid, two-step synthesis of such complexes, which are size-stable for up to 18 months. The first step of the synthesis involves a short 3-min sonication of gold (III) chloride hydrate with polyamidoamine dendrimers of 4th generation, while the second step uses microwaves to reduce gold (III) chloride hydrate with sodium citrate. Physicochemical characterization of the gold nanoparticle-polyamidoamine dendrimers complexes was performed using ultraviolet-visible spectroscopy, dynamic light scattering technique, infrared spectroscopy, atomic force microscopy and transmission electron microscopy. The toxicity of synthesized gold nanoparticles stabilized with polyamidoamine dendrimers on MCF-7 cell line has been studied using tetrazolium salt reduction test. The produced gold nanoparticles were administered to the MCF-7 cell culture line in two configurations: immediately after synthesis and after 18 months from synthesis. The cytotoxicity results were supplemented with studies of the influence of commercially available gold nanoparticles stabilized only with sodium citrate.

Gold nanoparticles synthesis

AuNPs

PAMAM G4 dendrimers

Atomic force microscopy

XTT tetrazolium salt reduction tests

cellular viability

• The developed synthesis method, based on sonication and microwave, enables rapid production of spherical and monodisperse gold nanoparticles stabilized with PAMAM G4 dendrimers.

• The gold nanoparticles stabilized with PAMAM G4 exhibit high size-stability up to 18 months.

• Synthesized gold nanoparticles stabilized with dendrimers and commercially available gold nanoparticles stabilized with sodium citrate show similar toxicity levels on breast cancer cells.

The field of cancer theranostics has seen the advent of nanoparticles, particularly gold nanoparticles (AuNPs), as a promising avenue for targeted and controlled drug delivery. The effective application of these nanostructures, as well as other nanostructures such as carbon nanotubes, dendrimers, liposomes, and micelles, hinges on their physicochemical properties such as size, shape, and surface characteristics [1, 2]. Among the different types of nanoparticles, gold nanoparticles have been demonstrated to inhibit proliferation and induce cell death on different types of cancer cell lines [3–8]. The versatility of these nanomaterials in terms of surface chemistry allows them to be coupled with molecular targeting agents like antibodies, thereby enabling them to specifically target tumor tissues and induce cell death. This targeted approach involves the use of cell surface receptors that can identify carcinoma cells, leading to the release of drugs that control cell cycle progression and trigger cell death.

In particular, AuNPs offer several advantages such as extended circulation time, easy modification with ligands that can be recognized by cancer cell surface receptors [9], and enhanced uptake through receptor-mediated endocytosis. This is in stark contrast to traditional cancer therapies like surgery and chemotherapy, which often fall short of expectations. AuNPs have found applications in targeted therapy for drug delivery [10], and their use in a controlled drug delivery system can significantly enhance the therapeutic effects while minimizing side effects. They have also been used as delivery vehicles in combined photothermal therapy [11] and for efficient drug delivery to cancer cells [12].

A significant amount of research has been conducted on the synthesis of AuNPs using polyamidoamine (PAMAM) dendrimers as stabilizers [13–15]. These dendrimers are excellent candidates for preparing metal nanoparticles due to their well-defined structure and robust stabilization properties. AuNPs/PAMAM complexes have shown potential for future applications in medicine, particularly in cancer cell imaging [16] and theranostics [17]. They have been used in computed tomography imaging as a contrast agent, demonstrating higher radiation absorption compared to clinically used iodine contrast agents [18]. Furthermore, these complexes can serve as carriers for drugs or genes. The attachment of various polymers/specific antibodies to gold nanoparticles stabilized with PAMAM dendrimers allows for high affinity for receptors on the surface of tumor cells, leading to their endocytosis at the tumor tissue. In conclusion, the use of AuNPs, particularly in conjunction with PAMAM dendrimers, opens up new possibilities in the field of cancer theranostics, offering a promising approach for targeted drug delivery and imaging.

One of the aspects of the potential use of AuNPs/PAMAM complexes is the development of their repeatable synthesis, along with achieving long-term stability of the produced colloid. The aim of our work is development a rapid, two-step synthesis of gold nanoparticles stabilized with PAMAM dendrimers of 4th generation (G4), which are stable for a long period after synthesis, up to 18 months. The synthesis includes two steps - short sonication for up to 3 minutes, and then exposing the solution with a reducer to microwave waves for 10 second intervals, which results in the formation of stable nanoparticles colloids. PAMAM-stabilized gold nanoparticles were fully characterized just after synthesis, as well as after 18 months. The methods used to characterize nanoparticles include ultraviolet-visible spectroscopy (UV-Vis), Fourier Transform Infrared Spectroscopy (FTIR), dynamic light scattering (DLS), atomic force microscopy (AFM) and transmission electron microscopy (TEM). Additionally, the cytotoxicity of the produced colloids was assessed immediately after synthesis and after 18 months in comparison with commercially available sodium citrate-stabilized gold nanoparticles, which were tested on the MCF-7 breast cancer cell line using the tetrazolium salt reduction test (XTT).

Dendrimers used in this study were purchased from Sigma Aldrich (United States) as a methanol suspension. Polyamidoamine (PAMAM) dendrimers of 4th generation (cat. no. 412449) consist of an ethylenediamine core (2-carbon core) with 64 functional primary amino groups on the surface, respectively. These data refer to the specification sheets provided by the manufacturer. According to the manufacturer certificates the high purity of PAMAM solutions was confirmed by infrared spectrum and 13C NMR identity.

Au NPs stabilized with sodium citrate (cat. no. J67188 and J67284), defined as reference, were provided from Sigma Aldrich (United States). According to the manufacturer protocol, these gold nanoparticles are non-functionalized, stable in high salt (30mM NaCl) for at least 30 minutes.

2.1. Two - stage synthesis of AuNPs/PAMAM

Gold (III) chloride hydrate (HAuCl₄·3H₂O) (Sigma Aldrich, United States) was used as synthesis precursor to obtain gold nanoparticles. As a stabilizer, ethylenediamine core polyamidoamine dendrimers of 4th generation with 64 functional primary amino groups on the surface in 10% methanol solution (Sigma Aldrich, United States) were utilized. Sodium citrate (Chempur, Poland) was used as the reduction agent.

The method for preparing gold nanoparticles was based on a previous study by Avila-Salas et al. [19] with some modifications described below. The PAMAM G4 solution was dried at 60°C for 30 min to remove the methanol. Volumes of 0.25 mL of 1.5 mM, 1.8 mM and 2.0 mM HAuCl₄ solutions were mixed with 0.25 mL of an aqueous 0.1 mM PAMAM G4 solution and sonicated for 3 minutes at room temperature. In the second step, 0.25 mL sodium citrate was added to one third of gold molar concertation mixture of chloro-gold acid for reduction of Au complex. Then, the samples were placed in the microwave for three 10-second cycles. The resulting colloid turned to red, confirming the formation of gold nanoparticles. This two-step synthesis method, involving sonication and microwave irradiation, significantly reduces synthesis time to produces monodisperse gold nanoparticles that remain stable for up to 18 months compared to our previous work [20].

2.2. Ultraviolet-Visible Spectroscopy

Optical properties of AuNPs/PAMAM were revealed through UV-Vis spectra collected by Ultrospec 2100 Pro spectrophotometer (GE, United Kingdom). The measurements were performed at room temperature at various time intervals from the synthesis procedure (right after and 18 months after synthesis).

2.3. Dynamic Light Scattering Technique and Zeta Potential Measurements

Particle size distribution was measured by dynamic light scattering technique using Dyna Pro Nano Star (Wyatt, United States) device. Samples immediately after synthesis were placed in disposable cuvettes and measurements were made in five replications with 10 acquisitions of each. During the DLS measurements the following study conditions including temperature of 25°C, laser wavelength at 663 nm, laser power of 100 mW and scattering angle at 90° were fulfilled. Based on the obtained histograms and regularization analysis [21] the apparent hydrodynamic radius was designed for all populations.

Zeta potential measurements were performed by the Zetasizer Nano device (Malvern, United Kingdom). Measurements were performed in triplicate with 20 acquisitions each. AuNPs/PAMAM colloids were placed in disposable folded capillary zeta potential cells (Cat number DTS1070, Malvern, United Kingdom) and measured immediately and 18 months after synthesis. The measurement parameters were as follows: scattering angle 173°, wavelength 663 nm, laser power 4 mW and temperature 25°C. The study was conducted with automatic attenuation and automatic voltage selection. The calculations of zeta potential values were performed for the Au material for refractive index 0.2 and absorption 3.32 according to the Smoluchowski model.

2.4. Infra-Red Spectroscopy

Fourier Transform Infrared Spectroscopy measurements were performed using Nicolet iS50 FT-IR Thermo Scientific equipped with attenuated total reflectance (ATR) device, operated at 1 cm^− 1 resolution. The colloids were deposited on cleaved Si(001) wafers and left for drying at room temperature.

2.5. Atomic Force Microscopy

Visualization of AuNPs/PAMAM was performed using BioScope Resolve (Bruker, United States) with Peak Force Tapping mode in air using Scan Asyst Probe (Bruker, United States). AuNPs/PAMAM deposited on mica was prepared according to the procedure described in the paper [20]. The parameters were as follow: scan size of 3 × 3 µm and 1 × 1 µm, scan rates: 0.5 and 1 Hz, respectively.

2.6. Transmission Electron Microscopy

Transmission Electron Microscopy analysis was performed using Talos F200X (FEI, Netherlands) at the acceleration voltage of 200 kV. Selected colloid (sample defined as 1:18) was dropped onto carbon coated copper grids (300 mesh) and allowed to dry.

2.7. Cell culture and cytotoxicity measurements (XTT assay)

Human breast adenocarcinoma MCF-7 cell line (HTB-22™), Dulbecco’s Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), Penicillin/Streptomycin cocktail and 0.25% (w/v) Trypsin − 0.53 mM EDTA solution were obtained from ATCC. Phosphate buffered saline (PBS) was purchased from Gibco. The tetrazolium salt XTT, 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide, was purchased from Biological Industries. Breast cancer cells (MCF-7 line) were cultivated routinely in complete growth DMEM medium supplemented with 10% FBS. All cell culture reagents were used under sterile conditions. Cells were maintained in 75 cm² flasks in a humidified incubator set at 37°C with a 5% CO2 atmosphere. Subculturing occurred every 3–4 days when the first floating cells appeared in the medium and confluence was achieved at about 80–90%.

Cytotoxicity of the AuNPs/PAMAM G4 was performed using tetrazolium salt reduction tests as described by the supplier (Biological Industries, United States). Briefly, the MCF-7 cells were seeded on 96–well plates at a density of 10 000 cells/well. After 24 h of cell culture, the cell culture supernatants were replaced with suspensions of gold nanoparticles, at a final concentrations of 1, 2.5, 5, 10, 20, 25, 30, 40 and 50 µg/mL, suspended in the serum free cell culture medium. Additionally, analogous studies were performed for cells exposed to commercially available reference AuNPs with diameters of 10 and 30 nm. In parallel, control cells for all experiments were prepared, where the cell culture supernatant was replaced with the serum-free medium. After next 24 h of incubation, cell viability was assessed with XTT test. The light absorbance at 450 nm was measured for each well using a Victor X4 microplate reader (Perkin Elmer, United States). Cell viability data are expressed as percentages as compared to control cells. To illustrate the toxicity of selected nanoparticles, the concentrations that reduced cell viability to 90, 75 and 50 percent of control cells (untreated) were calculated and designated as EC₁₀, EC₂₅, EC₅₀, respectively.

3.1 Characterization of AuNPs/PAMAM

Figure 1 shows the UV-Vis spectra with the absorbance peak about 525 nm for each sample post-synthesis and after 18 months. The measurements after 18 months revealed an increase in absorbance intensity for 1:15 and 1:20 samples. The absorbance intensity did not change for the 1:18 sample.

The apparent hydrodynamic distribution of the synthesized nanoparticles in suspensions 1:15, 1:18, 1:20 is shown in Fig. 2 at three different time points: immediately after synthesis (Fig. 2A), one month (Fig. 2B) and 18 months (Fig. 2C) after synthesis. For samples 1:15 and 1:18, the main population of nanostructures with an apparent hydrodynamic radius of about 9 nm was determined. Sample 1:15 also showed a second peak with an apparent radius of 2.2 nm. Also for the 1:20 sample, two populations of hydrodynamic radius were measured: approximately 1 nm and 15 nm. For all samples, value of polydispersity was low, ranging from 5.2–16.9%. Additionally, zeta potential measurements showed the following values: 43.0 (± 2.9) mV, 35.9 (± 3.4) mV, 30.3 (± 0.9) mV for samples 1:15, 1:18 and 1:20, respectively. Interestingly, after 18 months we showed lower zeta potential values for all synthesized colloids (Table 1).

Table 1

Zeta potential values for synthesized colloids just after production and after 18 months.
Samples	Zeta potential after synthesis [mV]	Zeta potential after 18 months [mV]
1:15	43.0 (± 2.9)	13.7 (± 0.7)
1:18	35.9 (± 3.4)	15.7 (± 0.7)
1:20	30.3 (± 0.9)	18.6 (± 0.6)

For the chemical characterization of AuNPs/PAMAM, the infrared spectroscopy was performed (Fig. 3). The FTIR spectra demonstrated broads vibration peak at the maximum at 3250 cm^− 1, 3070 cm^− 1, 2940 cm^− 1, 2840 cm^− 1, 1635 cm^− 1 and 1545 cm^− 1.

Figure 4 shows the AFM images of AuNPs/PAMAM samples deposited on a mica substrate. The upper images (Fig. 4A-C) show the surface topography for scan size of 3 × 3 µm. For a better visualization of AuNPs/PAMAM, images with a scan size of 1 × 1 µm (bottom images) are also included. From AFM images numerous nanostructures with sizes below 100 nm can be seen. During the sample drying of AuNPs/PAMAM on mica, the complexes changed, probably flattened (increase the size in X and Y directions) or aggregated. From the AFM images it can be concluded that the most separated nanoparticles and the most homogeneous surface over the entire scanning area were obtained for the 1:18 sample.

For TEM investigation (Fig. 5), the 1:18 sample was selected. The images show spherical and well dispersive nanoparticles with about 10 nm diameter.

3.2. Investigation of AuNPs/PAMAM cytotoxicity on MCF-7 cell line

The viability of MCF-7 cells exposed to AuNPs/PAMAM G4 of 1:18 sample immediately after synthesis and 18 months after synthesis as well as commercially available gold nanoparticles of 10 nm and 30 nm size is presented in Fig. 6. Au/PAMAM complexes immediately after synthesis are more toxic (noticeable above the concentration of 10 µg/ml). From the obtained viability graphs, EC₁₀, EC₂₅ and EC₅₀ concentrations were calculated, which are gathered in the Table 2. EC₁₀, EC₂₅, EC₅₀ correspond to the 90, 75 and 50% of the control cellular viability.

Table 2

Comparison of Au NPs concentrations. EC₁₀, EC₂₅, EC₅₀ correspond to the 90, 75 and 50% of the control cellular viability.
	AuNPs/PAMAM after synthesis [µg/ml]	AuNPs/PAMAM 18 months after synthesis [µg/ml]	Au 10 nm [µg/ml]	Au 30 nm [µg/ml]
EC₁₀	10 ± 2.3	15 ± 1.5	10.5 ± 1.9	12 ± 2.8
EC₂₅	15 ± 2.3	22 ± 1.7	15.5 ± 2.2	19 ± 3.4
EC₅₀	24.5 ± 2.8	33 ± 2.0	24 ± 2.7	31.5 ± 4.4

In order to verify the presence of AuNPs/PAMAM and their stability, UV-Vis and DLS techniques were used. UV-Vis spectra were collected right after synthesis and after 18 months storage in room temperature (Fig. 1). The specific band of UV-Vis spectra for the surface plasmon resonance (SPR) of gold nanoparticles occurred in broad range 500–550 nm [13]. The narrow absorbance peak at 525 nm suggested presence of spherical NPs of 4–50 nm diameter [22]. The absorbance intensity of the 1:18 sample remained unchanged, indicating high stability over 18 months of storage. In contrast, at ratios of 1:15 and 1:20, the intensity of the SPR peak increased after 18 months, suggesting an increase in the concentration of nanoparticles in the solutions over time [23].

The synthesized nanostructures should be considered as Au/PAMAM complexes, where the size and shape of nanostructures will be influenced by the size of both core of gold nanoparticles as well as shell of PAMAM dendrimers and sodium citrate as a stabilizer agents. The zeta potential values for colloids right after synthesis are very high, from 30.3 to 43.0 mV (Table 1). These results are consistent with the literature reports [24], where for AuNPs stabilized with PAMAM G4 dendrimers a value of about 41 (± 3) mV was obtained. Surprisingly, after 18 months the zeta potential values for the same colloids are 13.7, 15.7 and 18.6 mV, respectively, which indicates electrochemical changes for the produced nanoparticles and, consequently, may also affect their biological activity. From the DLS analysis (Fig. 2), it was determined that the apparent hydrodynamic radius of the gold nanoparticles of 1:18 sample, also after 18 months, was approximately 9 nm. For the 1:15 sample, we noticed main peak for radius of AuNPs of 9.7 nm and a second peak with an apparent radius of 2.2 nm, suggesting the presence of additional products such as stabilizers and synthesis by-products. Also for 1:20 sample, two populations of hydrodynamic radius were measured. The first peak had a size of about 1 nm, likely representing remaining products from the synthesis process, while the second peak likely corresponds to nanoparticles with a radius of 15 nm. In the case of all synthesis, stable colloids were obtained for up to 18 months, which is confirmed by DLS measurements. AFM imaging was performed for all samples. Spherical nanoparticles or their agglomerates below 100 nm were observed on the AFM images. However, the AFM technique has some disadvantages. AFM imaging is performed after drying the sample, firstly Au/PAMAM conjugates agglomerate with each other and also flatten (they increase their size in X and Y axes and decrease in Z axis) [25].

Based on the DLS results that the 1:18 sample is stable for up to 18 months, TEM imaging was performed for this case. Nanoparticles of about 10 nm (diameter) were observed on the TEM images. The size of the gold nanoparticles as measured from TEM images relates to the metallic core as low generation dendrimers are not visible in TEM. In literature we could found the TEM images showing that for the higher generation PAMAM dendrimers, like G8-G10, the metal nanoparticles are present inside the PAMAM structures [26]. The differences of nanostructures diameter values obtained by TEM and DLS techniques are due to possibility of observation of only gold core part of particles (TEM) and the whole complex of AuNPs/PAMAM (DLS)[20].

The FTIR spectra (Fig. 3) of AuNPs/PAMAM showed the presence of the dendrimers in all synthesised nanoparticles. The band at 3250 cm^− 1 correspond to the N-H stretching vibration arising from the PAMAM stabilizing layer. The next peak at 3070 cm^− 1 assign to the N–H bending vibration. The band at 2940 cm^− 1 and 2840 cm^− 1 indicated unsymmetrical and symmetrical stretching vibration of methylene [27]. The amide peaks at 1635 cm^− 1 and 1545 cm^− 1 (amide I and amide II, respectively) are characteristic of the dendrimer branches [28]. It confirm that PAMAM G4 is indeed formed on AuNPs.

Another aspect of our work was to investigate the toxicity of AuNPs/PAMAM on human breast adenocarcinoma cell line. The cytotoxicity of Au/PAMAM G4 on human breast cells was dose dependent [Figure 6]. Application of Au/PAMAM just after synthesis in a concentration of 10 µg/mL reduce cell viability to 90% in comparison to control (untreated) cells. After 18 months the AuNPs/PAMAM are less toxic, 90% of cell viability is achieved for 15 µg/mL.

Direct contact of AuNPs/PAMAM with cells in the medium as well as their penetration into the cells may influence the initiation of various processes leading to pathological changes as well as cell death. Metallic nanoparticles can accumulate on or just below the cell membrane [29–32]. The toxicity of the synthesized nanoparticles may be significantly influenced by PAMAM dendrimers. The cytotoxicity of dendrimers depends largely on the number and charge of surface functional groups. Cationic dendrimers are typically highly toxic, while anionic and uncharged dendrimers have little or no toxic effects. The cytotoxicity of cationic PAMAM dendrimers is thought to result from interactions between the positively charged dendrimers and the negatively charged cell surfaces [33, 34]. Based on the obtained results, after 18 months the AuNPs/PAMAM are less toxic (higher values of threshold concentrations EC₁₀, EC₂₅, EC₅₀), which may be related to the lower activity of polycationic groups of dendrimers. Also, the reduced zeta potential values after 18 months (Table 1) may suggest temporal changes related to the functional groups of PAMAM dendrimers. Moreover, the cytotoxicity of synthesized complexes were compere to toxicity of commercially available nanoparticles stabilized with sodium citrate with diameters of 10 and 30 nm. Surprisingly, AuNPs/PAMAM right after synthesis, are as toxic as commercially available nanoparticles stabilized with sodium citrate with a diameter of 10 nm (Table 2). It is worth noting that AuNPs/PAMAM conjugates, after a different synthesis model but of similar size, are much more toxic on human umbilical vein endothelial cells (HUVEC) [20] than the tested conjugates on breast adenocarcinoma MCF-7 cell line. This indicates both, that cancer lines are more resistant to the action of nanostructures, and may also be related to differences in the synthesis process of these conjugates and, consequently, their chemical activity and toxicity.

The presented method of synthesis allows to the preparation of spherical and well-separated, and long size-stable AuNPs/PAMAM in an aqueous solution. The synthesis of nanoparticles is based on two steps, the first is a short sonication and then the use of microwaves, which significantly shortens production time. The developed procedure is repeatable and enables the production of nanoparticles on a large scale. Additionally, XTT studies were performed to determine the effect of AuNPs/PAMAM on MCF-7 cells and, compared to commercially available nanoparticles stabilized only with sodium citrate, similar toxicity levels were observed.

In the future, the developed rapid synthesis may play a crucial role in medical and pharmaceutical sciences, for the use of AuNPs/PAMAM complexes in diagnostics/imaging as well as drug delivery systems.

Conflict of interests

The authors declare that no conflict of interests.

Author Contribution

Agnieszka M. Kołodziejczyk: Conceptualization, Investigation, Visualization, Writing – original draft, Writing–review & editing, Project administration, Funding acquisition.Magdalena M. Grala: Data curation, Formal analysis, Investigation, Writing – original draft. Piotr Komorowski: Funding acquisition.

Acknowledgements/Funding

This work is financed by The National Science Centre Poland, project title: “The influence of selected nanoparticles on the elastic properties of endothelial cells evaluated using atomic force microscopy” Agreement no. 2017/26/D/ST4/00918.

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Evaluation of PAMAM dendrimers-stabilized gold nanoparticles: two-stage procedure synthesis and toxicity assessment in MCF-7 breast cancer cells

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Abstract

Figures

Highlights

1. Introduction

2. Material and Methods

2.1. Two - stage synthesis of AuNPs/PAMAM

2.2. Ultraviolet-Visible Spectroscopy

2.3. Dynamic Light Scattering Technique and Zeta Potential Measurements

2.4. Infra-Red Spectroscopy

2.5. Atomic Force Microscopy

2.6. Transmission Electron Microscopy

2.7. Cell culture and cytotoxicity measurements (XTT assay)

3. Results

3.1 Characterization of AuNPs/PAMAM

3.2. Investigation of AuNPs/PAMAM cytotoxicity on MCF-7 cell line

4. Discussion

5. Summary

Declarations

Conflict of interests

Author Contribution

References

Additional Declarations

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