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Are smaller nanoparticles always better? Understand the biological effects of size-dependent aggregation of silver nanoparticles under biologically relevant conditions
Authors: Bélteky P, Rónavári A, Zakupszky D, Boka E, Igaz N, Szerencsés B, Pfeiffer I, Vágvölgyi C, Kiricsi M, Kónya Z
Péter Bélteky,1,* Andrea Rónavári,1,* Dalma Zakupszky,1 Eszter Boka,1 Nóra Igaz,2 Bettina Szerencsés,3 Ilona Pfeiffer,3 Csaba Vágvölgyi,3 Mónika Kiricsi of Environmental Chemistry, Hungary, Hungary Faculty of Science and Informatics, University of Szeged; 2 Department of Biochemistry and Molecular Biology, Faculty of Science and Information, University of Szeged, Hungary; 3 Department of Microbiology, Faculty of Science and Information, University of Szeged, Hungary; 4MTA-SZTE Reaction Kinetics and Surface Chemistry Research Group, Szeged, Hungary* These authors contributed equally to this work. Communication: Zoltán Kónya Department of Applied and Environment Chemistry, Faculty of Science and Informatics, University of Szeged, Rerrich Square 1, Szeged, H-6720, Hungary Phone +36 62 544620 Email [Email protection] Purpose: Silver nanoparticles (AgNPs) are one of the most commonly studied nanomaterials, especially due to their biomedical applications. However, due to the aggregation of nanoparticles, their excellent cytotoxicity and antibacterial activity are often compromised in biological media. In this work, the aggregation behavior and related biological activities of three different citrate-terminated silver nanoparticle samples with an average diameter of 10, 20, and 50 nm were studied. Method: Use transmission electron microscope to synthesize and characterize nanoparticles, evaluate their aggregation behavior at various pH values, NaCl, glucose and glutamine concentrations by dynamic light scattering and ultraviolet-visible spectroscopy. In addition, in the cell culture medium components such as Dulbecco improves the aggregation behavior in Eagle Medium and Fetal Calf Serum. Results: The results show that acidic pH and physiological electrolyte content generally induce micron-scale aggregation, which can be mediated by the formation of biomolecular corona. It is worth noting that larger particles exhibit higher resistance to external influences than their smaller counterparts. In vitro cytotoxicity and antibacterial tests were performed by treating cells with nanoparticle aggregates at different aggregation stages. Conclusion: Our results reveal a profound correlation between colloidal stability and the toxicity of AgNPs, as extreme aggregation leads to complete loss of biological activity. The higher degree of anti-aggregation observed for larger particles has a significant impact on in vitro toxicity, because such samples retain more antimicrobial and mammalian cell activity. These findings lead to the conclusion that, despite the general opinion in the relevant literature, targeting the smallest possible nanoparticles may not be the best course of action. Keywords: seed-mediated growth, colloidal stability, size-dependent aggregation behavior, aggregation damage toxicity
As the demand and output of nanomaterials continue to increase, more and more attention is paid to their biosafety or biological activity. Silver nanoparticles (AgNPs) are one of the most commonly synthesized, researched and utilized representatives of this class of materials because of their excellent catalytic, optical and biological properties. 1 It is generally believed that the unique characteristics of nanomaterials (including AgNPs) are mainly attributed to their large specific surface area. Therefore, the inevitably problem is any process that affects this key feature, such as particle size, surface coating Or aggregation, whether it will severely damage the properties of nanoparticles that are critical to specific applications.
The effects of particle size and stabilizers are subjects that have been relatively well documented in the literature. For example, the generally accepted view is that smaller nanoparticles are more toxic than larger nanoparticles. 2 Consistent with general literature, our previous studies have demonstrated the size-dependent activity of nanosilver on mammalian cells and microorganisms. 3– 5 Surface coating is another attribute that has a broad influence on the properties of nanomaterials. Just by adding or modifying stabilizers on its surface, the same nanomaterial may have completely different physical, chemical, and biological properties. The application of capping agents is most often performed as part of nanoparticle synthesis. For example, citrate-terminated silver nanoparticles are one of the most relevant AgNPs in the research, which are synthesized by reducing silver salts in a selected stabilizer solution as the reaction medium. 6 Citrate can easily take advantage of its low cost, availability, biocompatibility, and strong affinity for silver, which can be reflected in various proposed interactions, from reversible surface adsorption to ionic interactions. Small molecules and polyatomic ions near 7,8, such as citrates, polymers, polyelectrolytes, and biological agents are also commonly used to stabilize nano-silver and perform unique functionalizations on it. 9-12
Although the possibility of altering the activity of nanoparticles by intentional surface capping is a very interesting area, the main role of this surface coating is negligible, providing colloidal stability for the nanoparticle system. The large specific surface area of ​​nanomaterials will produce large surface energy, which hinders the thermodynamic ability of the system to reach its minimum energy. 13 Without proper stabilization, this can lead to agglomeration of nanomaterials. Aggregation is the formation of aggregates of particles of various shapes and sizes that occurs when dispersed particles meet and current thermodynamic interactions allow the particles to adhere to each other. Therefore, stabilizers are used to prevent aggregation by introducing a sufficiently large repulsive force between the particles to counteract their thermodynamic attraction. 14
Although the subject of particle size and surface coverage has been thoroughly explored in the context of its regulation of biological activities triggered by nanoparticles, particle aggregation is a largely neglected area. There is almost no thorough study to solve the colloidal stability of nanoparticles under biologically relevant conditions. 10,15-17 In addition, this contribution is particularly rare, where the toxicity associated with aggregation has also been studied, even if it may cause adverse reactions, such as vascular thrombosis, or loss of desired characteristics, such as its toxicity, as shown in Figure 1.18, 19 shown. In fact, one of the few known mechanisms of silver nanoparticle resistance is related to aggregation, because certain E. coli and Pseudomonas aeruginosa strains are reported to reduce their nano-silver sensitivity by expressing the protein flagellin, flagellin. It has a high affinity for silver, thereby inducing aggregation. 20
There are several different mechanisms related to the toxicity of silver nanoparticles, and aggregation affects all of these mechanisms. The most discussed method of AgNP biological activity, sometimes referred to as the “Trojan Horse” mechanism, regards AgNPs as Ag+ carriers. 1,21 The Trojan horse mechanism can ensure a large increase in the local Ag+ concentration, which leads to the generation of ROS and membrane depolarization. 22-24 Aggregation may affect the release of Ag+, thereby affecting toxicity, because it reduces the effective active surface where silver ions may be oxidized and dissolved. However, AgNPs will not only exhibit toxicity through ion release. Many size and morphology-related interactions must be considered. Among them, the size and shape of the nanoparticle surface are the defining characteristics. 4,25 The collection of these mechanisms can be categorized as “induced toxicity mechanisms.” There are potentially many mitochondrial and surface membrane reactions that can damage organelles and cause cell death. 25-27 Since the formation of aggregates naturally affects the size and shape of silver-containing objects recognized by living systems, these interactions may also be affected.
In our previous paper on the aggregation of silver nanoparticles, we demonstrated an effective screening procedure consisting of chemical and in vitro biological experiments to study this problem. 19 Dynamic Light Scattering (DLS) is the preferred technique for these types of inspections because the material can scatter photons at a wavelength comparable to the size of its particles. Since the Brownian motion speed of particles in the liquid medium is related to the size, the change in the intensity of scattered light can be used to determine the average hydrodynamic diameter (Z-mean) of the liquid sample. 28 In addition, by applying a voltage to the sample, the zeta potential (ζ potential) of the nanoparticle can be measured similarly to the Z average value. 13,28 If the absolute value of the zeta potential is high enough (according to general guidelines> ±30 mV), it will generate strong electrostatic repulsion between the particles to counteract the aggregation. Characteristic surface plasmon resonance (SPR) is a unique optical phenomenon, mainly attributed to precious metal nanoparticles (mainly Au and Ag). 29​​ Based on the electronic oscillations (surface plasmons) of these materials on the nanoscale, it is known that spherical AgNPs have a characteristic UV-Vis absorption peak near 400 nm. 30 The intensity and wavelength shift of the particles are used to supplement the DLS results, as this method can be used to detect nanoparticle aggregation and surface adsorption of biomolecules.
Based on the information obtained, cell viability (MTT) and antibacterial assays are performed in a manner in which AgNP toxicity is described as a function of aggregation level, rather than (the most commonly used factor) nanoparticle concentration. This unique method allows us to demonstrate the profound importance of aggregation level in biological activity, because, for example, citrate-terminated AgNPs completely lose their biological activity within a few hours due to aggregation. 19
In the current work, we aim to greatly expand our previous contributions in the stability of bio-related colloids and their impact on biological activity by studying the effect of nanoparticle size on nanoparticle aggregation. This is undoubtedly one of the studies of nanoparticles. A higher-profile perspective and 31 To investigate this issue, a seed-mediated growth method was used to produce citrate-terminated AgNPs in three different size ranges (10, 20, and 50 nm). 6,32 as one of the most common methods. For nanomaterials that are widely and routinely used in medical applications, citrate-terminated AgNPs of different sizes are selected to study the possible size dependence of the aggregation-related biological properties of nanosilver. After synthesizing AgNPs of different sizes, we characterized the produced samples by transmission electron microscopy (TEM), and then examined the particles using the aforementioned screening procedure. In addition, in the presence of in vitro cell cultures Dulbecco’s Modified Eagle’s Medium (DMEM) and Fetal Bovine Serum (FBS), the size-dependent aggregation behavior and its behavior were evaluated at various pH values, NaCl, glucose, and glutamine concentrations. The characteristics of cytotoxicity are determined under comprehensive conditions. The scientific consensus indicates that in general, smaller particles are preferable; our investigation provides a chemical and biological platform to determine whether this is the case.
Three silver nanoparticles with different size ranges were prepared by the seed-mediated growth method proposed by Wan et al., with slight adjustments. 6 This method is based on chemical reduction, using silver nitrate (AgNO3) as the silver source, sodium borohydride (NaBH4) as the reducing agent, and sodium citrate as the stabilizer. First, prepare 75 mL of 9 mM citrate aqueous solution from sodium citrate dihydrate (Na3C6H5O7 x 2H2O) and heat to 70°C. Then, 2 mL of 1% w/v AgNO3 solution was added to the reaction medium, and then the freshly prepared sodium borohydride solution (2 mL 0.1% w/v) was poured into the mixture dropwise. The resulting yellow-brown suspension was kept at 70°C with vigorous stirring for 1 hour, and then cooled to room temperature. The resulting sample (referred to as AgNP-I from now on) is used as the basis for seed-mediated growth in the next synthesis step.
To synthesize a medium-sized particle suspension (denoted as AgNP-II), heat 90 mL 7.6 mM citrate solution to 80°C, mix it with 10 mL AgNP-I, and then mix 2 mL 1% w/v The AgNO3 solution was kept under vigorous mechanical stirring for 1 hour, and then the sample was cooled to room temperature.
For the largest particle (AgNP-III), repeat the same growth process, but in this case, use 10 mL of AgNP-II as the seed suspension. After the samples reach room temperature, they set their nominal Ag concentration based on the total AgNO3 content to 150 ppm by adding or evaporating additional solvent at 40°C, and finally store them at 4°C until further use.
Use FEI Tecnai G2 20 X-Twin Transmission Electron Microscope (TEM) (FEI Corporate Headquarters, Hillsboro, Oregon, USA) with 200 kV acceleration voltage to examine the morphological characteristics of nanoparticles and capture their electron diffraction (ED) pattern. At least 15 representative images (~750 particles) were evaluated using the ImageJ software package, and the resulting histograms (and all graphs in the entire study) were created in OriginPro 2018 (OriginLab, Northampton, MA, USA) 33, 34.
The average hydrodynamic diameter (Z-average), zeta potential (ζ-potential) and characteristic surface plasmon resonance (SPR) of the samples were measured to illustrate their initial colloidal properties. The average hydrodynamic diameter and zeta potential of the sample were measured by the Malvern Zetasizer Nano ZS instrument (Malvern Instruments, Malvern, UK) using disposable folded capillary cells at 37±0.1°C. Ocean Optics 355 DH-2000-BAL UV-Vis spectrophotometer (Halma PLC, Largo, FL, USA) was used to obtain characteristic SPR characteristics from the UV-Vis absorption spectra of samples in the range of 250-800 nm.
During the whole experiment, three different measurement types related to colloidal stability were carried out at the same time. Use DLS to measure the average hydrodynamic diameter (Z average) and zeta potential (ζ potential) of the particles, because the Z average is related to the average size of the nanoparticle aggregates, and the zeta potential indicates whether the electrostatic repulsion in the system is strong enough to offset Van der Waals attraction between nanoparticles. Measurements are made in triplicate, and the standard deviation of Z mean and zeta potential is calculated by Zetasizer software. The characteristic SPR spectra of the particles are evaluated by UV-Vis spectroscopy, because changes in peak intensity and wavelength can indicate aggregation and surface interactions. 29,35 In fact, surface plasmon resonance in precious metals is so influential that it has led to new methods of analysis of biomolecules. 29,36,37 The concentration of AgNPs in the experimental mixture is about 10 ppm, and the purpose is to set the intensity of the maximum initial SPR absorption to 1. The experiment was carried out in a time-dependent manner at 0; 1.5; 3; 6; 12 and 24 hours under various biologically relevant conditions. More details describing the experiment can be seen in our previous work. 19 In short, various pH values ​​(3; 5; 7.2 and 9), different sodium chloride (10 mM; 50 mM; 150 mM), glucose (3.9 mM; 6.7 mM) and glutamine (4 mM) concentration, and also prepared Dulbecco’s Modified Eagle Medium (DMEM) and Fetal Bovine Serum (FBS) (in water and DMEM) as model systems, and studied their effects on the aggregation behavior of the synthesized silver nanoparticles. pH The values ​​of, NaCl, glucose, and glutamine are evaluated based on physiological concentrations, while the amounts of DMEM and FBS are the same as the levels used in the entire in vitro experiment. 38-42 All measurements were performed at pH 7.2 and 37°C with a constant background salt concentration of 10 mM NaCl to eliminate any long-distance particle interactions (except for certain pH and NaCl-related experiments, where these attributes are the variables under study). 28 The list of various conditions is summarized in Table 1. The experiment marked with † is used as a reference and corresponds to a sample containing 10 mM NaCl and pH 7.2.
Human prostate cancer cell line (DU145) and immortalized human keratinocytes (HaCaT) were obtained from ATCC (Manassas, VA, USA). Cells are routinely cultured in Dulbecco’s minimum essential medium Eagle (DMEM) containing 4.5 g/L glucose (Sigma-Aldrich, Saint Louis, MO, USA), supplemented with 10% FBS, 2 mM L-glutamine, 0.01 % Streptomycin and 0.005% Penicillin (Sigma-Aldrich, St. Louis, Missouri, USA). The cells are cultured in a 37°C incubator under 5% CO2 and 95% humidity.
In order to explore the changes in AgNP cytotoxicity caused by particle aggregation in a time-dependent manner, a two-step MTT assay was performed. First, the viability of the two cell types was measured after treatment with AgNP-I, AgNP-II and AgNP-III. To this end, the two types of cells were seeded into 96-well plates at a density of 10,000 cells/well and treated with three different sizes of silver nanoparticles in increasing concentrations on the second day. After 24 hours of treatment, the cells were washed with PBS and incubated with 0.5 mg/mL MTT reagent (SERVA, Heidelberg, Germany) diluted in culture medium for 1 hour at 37°C. Formazan crystals were dissolved in DMSO (Sigma-Aldrich, Saint Louis, MO, USA), and the absorption was measured at 570 nm using a Synergy HTX plate reader (BioTek-Hungary, Budapest, Hungary). The absorption value of the untreated control sample is considered to be 100% survival rate. Perform at least 3 experiments using four independent biological replicates. IC50 is calculated from a dose response curve based on vitality results.
Thereafter, in the second step, by incubating the particles with 150 mM NaCl for different periods of time (0, 1.5, 3, 6, 12, and 24 hours) before cell treatment, different aggregation states of silver nanoparticles were produced. Subsequently, the same MTT assay was performed as previously described to evaluate changes in cell viability affected by particle aggregation. Use GraphPad Prism 7 to evaluate the final result, calculate the statistical significance of the experiment by unpaired t-test, and mark its level as * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001) And **** (p ≤ 0.0001).
Three different sizes of silver nanoparticles (AgNP-I, AgNP-II and AgNP-III) were used for antibacterial susceptibility to Cryptococcus neoformans IFM 5844 (IFM; Research Center for Pathogenic Fungi and Microbial Toxicology, Chiba University) and Bacillus Test megaterium SZMC 6031 (SZMC: Szeged Microbiology Collection) and E. coli SZMC 0582 in RPMI 1640 medium (Sigma-Aldrich Co.). In order to evaluate the changes in antibacterial activity caused by the aggregation of particles, first, their minimum inhibitory concentration (MIC) was determined by microdilution in a 96-well microtiter plate. To 50 μL of standardized cell suspension (5 × 104 cells/mL in RPMI 1640 medium), add 50 μL of silver nanoparticle suspension and serially dilute twice the concentration (in the aforementioned medium, the range is 0 and 75 ppm, That is, the control sample contains 50 μL of cell suspension and 50 μL of medium without nanoparticles). Thereafter, the plate was incubated at 30°C for 48 hours, and the optical density of the culture was measured at 620 nm using a SPECTROstar Nano plate reader (BMG LabTech, Offenburg, Germany). The experiment was performed three times in triplicate.
Except that 50 μL of single aggregated nanoparticle samples were used at this time, the same procedure as previously described was used to examine the effect of aggregation on antibacterial activity on the aforementioned strains. Different aggregation states of silver nanoparticles are produced by incubating the particles with 150 mM NaCl for different periods of time (0, 1.5, 3, 6, 12, and 24 hours) before cell processing. A suspension supplemented with 50 μL of RPMI 1640 medium was used as a growth control, while in order to control toxicity, a suspension with non-aggregated nanoparticles was used. The experiment was performed three times in triplicate. Use GraphPad Prism 7 to evaluate the final result again, using the same statistical analysis as the MTT analysis.
The aggregation level of the smallest particles (AgNP-I) has been characterized, and the results were partially published in our previous work, but for a better comparison, all particles were thoroughly screened. The experimental data are collected and discussed in the following sections. Three sizes of AgNP. 19
Measurements performed by TEM, UV-Vis and DLS verified the successful synthesis of all AgNP samples (Figure 2A-D). According to the first row of Figure 2, the smallest particle (AgNP-I) shows a uniform spherical morphology with an average diameter of about 10 nm. The seed-mediated growth method also provides AgNP-II and AgNP-III with different size ranges with average particle diameters of approximately 20 nm and 50 nm, respectively. According to the standard deviation of the particle distribution, the sizes of the three samples do not overlap, which is important for their comparative analysis. By comparing the average aspect ratio and thinness ratio of TEM-based particle 2D projections, it is assumed that the sphericity of the particles is evaluated by ImageJ’s shape filter plug-in (Figure 2E). 43 According to the analysis of the shape of particles, their aspect ratio (large side/short side of the smallest bounding rectangle) is not affected by particle growth, and their thinness ratio (measured area of ​​the corresponding perfect circle/theoretical area) gradually decreases. This results in more and more polyhedral particles, which are perfectly round in theory, corresponding to a thinness ratio of 1.
Figure 2 Transmission electron microscope (TEM) image (A), electron diffraction (ED) pattern (B), size distribution histogram (C), characteristic ultraviolet-visible (UV-Vis) light absorption spectrum (D), and average fluid Citrate-terminated silver nanoparticles with mechanical diameter (Z-average), zeta potential, aspect ratio and thickness ratio (E) have three different size ranges: AgNP-I is 10 nm (top row), AgNP -II is 20 nm (middle row), AgNP-III (bottom row) is 50 nm.
Although the cyclic nature of the growth method affected the particle shape to some extent, resulting in the smaller sphericity of larger AgNPs, all three samples remained quasi-spherical. In addition, as shown in the electron diffraction pattern in Figure 2B, nano The crystallinity of the particles is not affected. The prominent diffraction ring-which can be correlated with the (111), (220), (200), and (311) Miller indices of silver-is very consistent with the scientific literature and our previous contributions. 9, 19,44 The fragmentation of the Debye-Scherrer ring of AgNP-II and AgNP-III is due to the fact that the ED image is captured at the same magnification, so as the particle size increases, the number of diffracted particles per unit area increases and decreases .
The size and shape of nanoparticles are known to affect biological activity. 3,45 Shape-dependent catalytic and biological activity can be explained by the fact that different shapes tend to proliferate certain crystal faces (having different Miller indices), and these crystal faces have different activities. 45,46 Since the prepared particles provide similar ED results corresponding to very similar crystal characteristics, it can be assumed that in our subsequent colloidal stability and biological activity experiments, any observed differences should be attributed to Nanoparticle size, not shape-related properties.
The UV-Vis results summarized in Figure 2D further emphasize the overwhelming spherical nature of the synthesized AgNP, because the SPR peaks of all three samples are around 400 nm, which is a characteristic value of spherical silver nanoparticles. 29,30 The captured spectra also confirmed the successful seed-mediated growth of nanosilver. As the particle size increases, the wavelength corresponding to the maximum light absorption of AgNP-II-more prominently-According to the literature, AgNP-III Experienced a redshift. 6,29
Regarding the initial colloidal stability of the AgNP system, DLS was used to measure the average hydrodynamic diameter and zeta potential of the particles at pH 7.2. The results depicted in Figure 2E show that AgNP-III has higher colloidal stability than AgNP-I or AgNP-II, because common guidelines indicate that a zeta potential of 30 mV absolute is necessary for long-term colloidal stability This finding is further supported when the Z average value (obtained as the average hydrodynamic diameter of free and aggregated particles) is compared with the primary particle size obtained by TEM, because the closer the two values ​​are, the milder the degree Gather in the sample. In fact, the Z average of AgNP-I and AgNP-II is reasonably higher than their main TEM-evaluated particle size, so compared with AgNP-III, these samples are predicted to be more likely to aggregate, where the highly negative zeta potential is accompanied by a close size The Z average value.
The explanation for this phenomenon can be twofold. On the one hand, the citrate concentration is maintained at a similar level in all synthesis steps, providing a relatively high amount of charged surface groups to prevent the specific surface area of ​​the growing particles from decreasing. However, according to Levak et al., small molecules like citrate can be easily exchanged by biomolecules on the surface of the nanoparticles. In this case, the colloidal stability will be determined by the corona of the biomolecules produced. 31 Because this behavior was also observed in our aggregation measurements (discussed in more detail later), citrate capping alone cannot explain this phenomenon.
On the other hand, the particle size is inversely proportional to the aggregation tendency at the nanometer level. This is mainly supported by the traditional Derjaguin-Landau-Verwey-Overbeek (DLVO) method, where particle attraction is described as the sum of attractive and repulsive forces between particles. According to He et al., the maximum value of the DLVO energy curve decreases with the size of the nanoparticles in the hematite nanoparticles, making it easier to reach the minimum primary energy, thereby promoting irreversible aggregation (condensation). 47 However, it is speculated that there are other aspects beyond the limitations of DLVO theory. Although van der Waals gravity and electrostatic double-layer repulsion are similar with increasing particle size, a review by Hotze et al. proposes that it has a stronger effect on aggregation than DLVO allows. 14 They believe that the surface curvature of nanoparticles can no longer be estimated as a flat surface, making mathematical estimation inapplicable. In addition, as the particle size decreases, the percentage of atoms present on the surface becomes higher, leading to electronic structure and surface charge behavior. And surface reactivity changes, which may lead to a decrease in the charge in the electric double layer and promote aggregation.
When comparing the DLS results of AgNP-I, AgNP-II, and AgNP-III in Figure 3, we observed that all three samples showed similar pH prompting aggregation. A heavily acidic environment (pH 3) shifts the zeta potential of the sample to 0 mV, causing particles to form micron-sized aggregates, while alkaline pH shifts its zeta potential to a larger negative value, where the particles form smaller aggregates (pH 5). And 7.2) ), or remain completely unaggregated (pH 9). Some important differences between the different samples were also observed. Throughout the experiment, AgNP-I proved to be the most sensitive to pH-induced zeta potential changes, because the zeta potential of these particles has been reduced at pH 7.2 compared to pH 9, while AgNP-II and AgNP-III only showed A considerable change in ζ is around pH 3. In addition, AgNP-II showed slower changes and moderate zeta potential, while AgNP-III showed the mildest behavior of the three, because the system showed the highest absolute zeta value and slow trend movement, indicating AgNP-III Most resistant to pH-induced aggregation. These results are consistent with the average hydrodynamic diameter measurement results. Considering the particle size of their primers, AgNP-I showed constant gradual aggregation at all pH values, most likely due to the 10 mM NaCl background, while AgNP-II and AgNP-III only showed significant at pH 3 Of gathering. The most interesting difference is that despite its large nanoparticle size, AgNP-III forms the smallest aggregates at pH 3 in 24 hours, highlighting its anti-aggregation properties. By dividing the average Z of AgNPs at pH 3 after 24 hours by the value of the prepared sample, it can be observed that the relative aggregate sizes of AgNP-I and AgNP-II have increased by 50 times, 42 times, and 22 times, respectively. III.
Figure 3 The dynamic light scattering results of the citrate-terminated silver nanoparticles sample with increasing size (10 nm: AgNP-I, 20 nm: AgNP-II and 50 nm: AgNP-III) are expressed as the average hydrodynamic diameter (Z average) (right) Under different pH conditions, the zeta potential (left) changes within 24 hours.
The observed pH-dependent aggregation also affected the characteristic surface plasmon resonance (SPR) of the AgNP samples, as evidenced by their UV-Vis spectra. According to Supplementary Figure S1, the aggregation of all three silver nanoparticle suspensions is followed by a reduction in the intensity of their SPR peaks and a moderate red shift. The extent of these changes as a function of pH is consistent with the degree of aggregation predicted by the DLS results, however, some interesting trends have been observed. Contrary to intuition, it turns out that the medium-sized AgNP-II is the most sensitive to SPR changes, while the other two samples are less sensitive. In SPR research, 50 nm is the theoretical particle size limit, which is used to distinguish particles based on their dielectric properties. Particles smaller than 50 nm (AgNP-I and AgNP-II) can be described as simple dielectric dipoles, while particles that reach or exceed this limit (AgNP-III) have more complex dielectric properties, and their resonance The band splits into multimodal changes. In the case of two smaller particle samples, AgNPs can be regarded as simple dipoles, and the plasma can easily overlap. As the particle size increases, this coupling essentially produces a larger plasma, which may explain the higher sensitivity observed. 29 However, for the largest particles, the simple dipole estimation is not valid when other coupling states may also occur, which can explain the decreased tendency of AgNP-III to indicate spectral changes. 29
Under our experimental conditions, it is proved that the pH value has a profound effect on the colloidal stability of citrate-coated silver nanoparticles of various sizes. In these systems, stability is provided by the negatively charged -COO- groups on the surface of AgNPs. The carboxylate functional group of the citrate ion is protonated in a large number of H+ ions, so the generated carboxyl group can no longer provide electrostatic repulsion between the particles, as shown in the top row of Figure 4. According to Le Chatelier’s principle, AgNP samples quickly aggregate at pH 3, but gradually become more and more stable as the pH increases.
Figure 4 Schematic mechanism of surface interaction defined by aggregation under different pH (top row), NaCl concentration (middle row), and biomolecules (bottom row).
According to Figure 5, the colloidal stability in AgNP suspensions of different sizes was also examined under increasing salt concentrations. Based on the zeta potential, the increased nanoparticle size in these citrate-terminated AgNP systems again provides enhanced resistance to external influences from NaCl. In AgNP-I, 10 mM NaCl is sufficient to induce mild aggregation, and a salt concentration of 50 mM provides very similar results. In AgNP-II and AgNP-III, 10 mM NaCl does not significantly affect the zeta potential because their values ​​remain at (AgNP-II) or below (AgNP-III) -30 mV. Increasing the NaCl concentration to 50 mM and finally to 150 mM NaCl is enough to significantly reduce the absolute value of the zeta potential in all samples, although larger particles retain more negative charge. These results are consistent with the expected average hydrodynamic diameter of AgNPs; the Z average trend lines measured on 10, 50, and 150 mM NaCl show different, gradually increasing values. Finally, micron-sized aggregates were detected in all three 150 mM experiments.
Figure 5 The dynamic light scattering results of the citrate-terminated silver nanoparticles sample with increasing size (10 nm: AgNP-I, 20 nm: AgNP-II and 50 nm: AgNP-III) are expressed as the average hydrodynamic diameter (Z average) (right) and zeta potential (left) change within 24 hours under different NaCl concentrations.
The UV-Vis results in Supplementary Figure S2 show that the SPR of 50 and 150 mM NaCl in all three samples has an instantaneous and significant decrease. This can be explained by DLS, because NaCl-based aggregation occurs faster than pH-dependent experiments, which is explained by the large difference between the early (0, 1.5, and 3 hours) measurements. In addition, increasing the salt concentration will also increase the relative permittivity of the experimental medium, which will have a profound effect on surface plasmon resonance. 29
The effect of NaCl is summarized in the middle row of Figure 4. In general, it can be concluded that increasing the concentration of sodium chloride has a similar effect as increasing the acidity, because Na+ ions have a tendency to coordinate around the carboxylate groups, suppressing negative zeta potential AgNPs. In addition, 150 mM NaCl produced micron-sized aggregates in all three samples, indicating that the physiological electrolyte concentration is detrimental to the colloidal stability of citrate-terminated AgNPs. By considering the critical condensing concentration (CCC) of NaCl on similar AgNP systems, these results can be cleverly placed in the relevant literature. Huynh et al. calculated that the CCC of NaCl for citrate-terminated silver nanoparticles with an average diameter of 71 nm was 47.6 mM, while El Badawy et al. observed that the CCC of 10 nm AgNPs with citrate coating was 70 mM. 10,16 In addition, the significantly high CCC of about 300 mM was measured by He et al., which caused their synthesis method to be different from the previously mentioned publication. 48 Although the current contribution is not aimed at a comprehensive analysis of these values, because our experimental conditions are increasing in the complexity of the entire study, the biologically relevant NaCl concentration of 50 mM, especially 150 mM NaCl, seems to be quite high. Induced coagulation, explaining the strong changes detected.
The next step in the polymerization experiment is to use simple but biologically relevant molecules to simulate nanoparticle-biomolecule interactions. Based on DLS (Figures 6 and 7) and UV-Vis results (Supplementary Figures S3 and S4), some general conclusions can be asserted. Under our experimental conditions, the studied molecules glucose and glutamine will not induce aggregation in any AgNP system, because the Z-mean trend is closely related to the corresponding reference measurement value. Although their presence does not affect aggregation, experimental results show that these molecules are partially adsorbed on the surface of AgNPs. The most prominent result supporting this view is the observed change in light absorption. Although AgNP-I does not exhibit meaningful wavelength or intensity changes, it can be observed more clearly by measuring larger particles, which is most likely due to the greater optical sensitivity mentioned earlier. Regardless of the concentration, glucose can cause a greater red shift after 1.5 hours compared with the control measurement, which is about 40 nm in AgNP-II and about 10 nm in AgNP-III, which proves the occurrence of surface interactions . Glutamine showed a similar trend, but the change was not so obvious. In addition, it is also worth mentioning that glutamine can reduce the absolute zeta potential of medium and large particles. However, since these zeta changes do not seem to affect the aggregation level, it can be speculated that even small biomolecules like glutamine can provide a certain degree of spatial repulsion between particles.
Figure 6 The dynamic light scattering results of citrate-terminated silver nanoparticle samples with increasing size (10 nm: AgNP-I, 20 nm: AgNP-II and 50 nm: AgNP-III) are expressed as the average hydrodynamic diameter (Z average) (right) Under external conditions of different glucose concentrations, the zeta potential (left) changes within 24 hours.
Figure 7 The dynamic light scattering results of the citrate-terminated silver nanoparticles sample with increasing size (10 nm: AgNP-I, 20 nm: AgNP-II and 50 nm: AgNP-III) are expressed as the average hydrodynamic diameter (Z average) (right) In the presence of glutamine, the zeta potential (left) changes within 24 hours.
In short, small biomolecules like glucose and glutamine do not affect colloidal stability at the measured concentration: although they affect the zeta potential and UV-Vis results to varying degrees, the Z average results are not consistent. This indicates that the surface adsorption of molecules inhibits electrostatic repulsion, but at the same time provides dimensional stability.
In order to link the previous results with the previous results and simulate biological conditions more skillfully, we selected some of the most commonly used cell culture components and used them as experimental conditions for studying the stability of AgNP colloids. In the entire in vitro experiment, one of the most important functions of DMEM as a medium is to establish the necessary osmotic conditions, but from a chemical point of view, it is a complex salt solution with a total ionic strength similar to 150 mM NaCl. 40 As for FBS, it is a complex mixture of biomolecules-mainly proteins-from the point of view of surface adsorption, it has some similarities with the experimental results of glucose and glutamine, despite the chemical composition and diversity Sex is much more complicated. 19 DLS and UV-The visible results shown in Figure 8 and Supplementary Figure S5, respectively, can be explained by examining the chemical composition of these materials and correlating them with the measurements in the previous section.
Figure 8 The dynamic light scattering results of the citrate-terminated silver nanoparticles sample with increasing size (10 nm: AgNP-I, 20 nm: AgNP-II and 50 nm: AgNP-III) are expressed as the average hydrodynamic diameter (Z average) (right) In the presence of cell culture components DMEM and FBS, zeta potential (left) changes within 24 hours.
The dilution of AgNPs of different sizes in DMEM has a similar effect on colloidal stability to that observed in the presence of high NaCl concentrations. The dispersion of AgNP in 50 v/v% DMEM showed that large-scale aggregation was detected with the increase of zeta potential and Z-average value and the sharp decrease of SPR intensity. It is worth noting that the maximum aggregate size induced by DMEM after 24 hours is inversely proportional to the size of primer nanoparticles.
The interaction between FBS and AgNP is similar to that observed in the presence of smaller molecules such as glucose and glutamine, but the effect is stronger. The Z average of the particles remains unaffected, while an increase in zeta potential is detected. The SPR peak showed a slight red shift, but perhaps more interestingly, the SPR intensity did not decrease as significantly as in the control measurement. These results can be explained by the innate adsorption of macromolecules on the surface of nanoparticles (bottom row in Figure 4), which is now understood as the formation of biomolecular corona in the body. 49