Assessment of Silver-Nanoparticles-Induced Erythrocyte Cytotoxicity through Ion Transport Studies
Norma C. Adragnaa Praveen K. Allaa Ioana E. Pavel-Sizmoreb
Arathi S.L. Palurib Jerome Yaklicc Peter K. Laufa,d
aDepartment of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, OH, USA, bDepartment of Chemistry, College of Science and Mathematics, Wright State University, Dayton, OH, USA, cDepartment of Obstetrics and Gynecology, Boonshoft School of Medicine, Wright State University, Dayton, OH, USA dDepartment of Pathology, Boonshoft School of Medicine, Wright State University, Dayton, OH, USA
Key Words
Silver nanoparticles • Human adult and cord red blood cells • K+ and Rb+ transport • Nanoparticles functionalization • Hyperspectral imaging
Abstract
Background/Aims: Silver nanoparticles (AgNPs) are the most frequently used nanomaterials in industrial and biomedical applications. Their functionalization significantly impacts their properties and potential applications. Despite the need to produce, investigate and apply them, not much is known about the toxicity of silver nanoparticles to and their interaction with blood components, such as erythrocytes. Here, we report on the effect of two negatively charged AgNPs (Creighton, and Lee-Meisel) on ion transport in human red blood cells (HRBCs). Methods: HRBCs were obtained from blood of adult donors, which was either expired, fresh or refrigerated for variable lengths of time, and from fresh or refrigerated cord blood. Rb+ and K+ ions were measured by atomic emission and absorption spectrophotometry, respectively. Erythrocyte hemoglobin optical density (Hbc OD), was determined at 527 nm to estimate RBC volume in the same tubes in which Rb+ and K+ were measured. Cellular Rb+ uptake and intracellular K+ concentrations, [K]i, were calculated in mmol/L of original cells (LOC) per time. Rubidium, a potassium ion (K+) congener used to measure K+ influx, [K]i, and Hbc ODs were determined in the presence and absence of several concentrations (0-150 µg mL-1) of spherical AgNPs of an average diameter of 10 nm, at different time points (0-60 min). Results: Creighton AgNPs inhibited Rb+ influx and depleted the cells of K+ independently of the source and in a time and dose-dependent manner. In contrast, Lee-Meisel AgNPs caused ~ 50 % Rb+ influx inhibition and ~ 15 % K+ loss with larger interindividual variability than Creighton AgNPs. The loss of K+ from HRBCs was entirely accounted for by an increase in extracellular K+ concentration, [K]o. Enhanced dark field optical microscopy in conjunction with CytoViva® hyperspectral imaging helped visualize AgNPs internalized by HRBCs, thus pointing to a potential cause for their cytotoxic effects. Conclusion: These findings indicate that HRBC K+ homeostasis is an early and sensitive biomarker for AgNPs toxicity and is a function of their surface functionalization.
Introduction
Numerous studies have concentrated on the groundbreaking discoveries and applications of Nanotechnology, summarized by Roco et al. in a recent report [1] and further demonstrated by more recent findings [2, 3]. Silver nanoparticles (AgNPs) are the most frequently used nanomaterials for applications in Nanotechnology and Nanomedicine. The large surface area and high reactivity of AgNPs, in addition to their shape, are responsible for their bactericidal properties [4-6], which became of great interest in the early 2000s when multidrug-resistant bacteria were causing significant number of casualties in the human population [7-9]. This trend has continued developing with more promising results [2, 10-14]. In addition to the therapeutic properties of AgNPs, it became apparent that they also possess toxic effects in a large variety of systems, such as in mammalian germline stem cells [15], mammalian cells [16], spermatogonial stem cells [17], burn wounds [18], alveolar macrophages [19], human hepatoma cells [20], human lung cancer cells [21, 22], cultured keratinocytes [23], fibroblasts [24], and, in general, at the organismal, cellular and sub-cellular levels [2, 21, 22, 25-31]. Small nano-size particles are more likely to be internalized into cell lines than their large counterparts [32, 33]. Different types of NPs are known to induce cytotoxicity in various cell lines and animal models. AgNPs are extremely toxic because of their metallic silver and its high biological reactivity [34, 35]. Chronic exposure of keratinocytes to low doses of AgNPs to mimic more realistic exposure showed striking differences to acute studies [36]. In humans, colloidal nano silver has been shown to cause Argyria (a grayish discoloration of the skin [37].
AgNPs cause red blood cells (RBCs) deformation, agglutination and membrane damage [38]. AgNPs elicit RBCs hemolysis due to their large surface area, which facilitates an increased release of silver ion (Ag+) by direct interaction with RBCs [32, 39]. In fact, released Ag+ appears to be responsible for the AgNPs toxicity [40-42]. In human RBCs, AgNPs change the conformation of oxyhemoglobin to its deoxy form, which may be the result of a decrease in intracellular pH induced by the NPs [43]. In fish RBCs, decreasing the size of AgNPs from 100 to 15 nm increases their toxicity [44], although some studies have shown that the toxicity of Ag+ vs. AgNPs is dependent on their ratio [21]. In contrast, biogenic AgNPs possess bactericidal effects without eliciting the commonly known toxic effects of the non-biogenic [45]. Positively charged NPs are more toxic than the negatively charged [46] because the surface charge on RBCs is negative and thus possess higher affinity for the positively charged NPs. In human RBCs, internalized quantum dots (QDs) by D-penicillamine (DPA-QDs), enter the cells by a passive transport mechanism, and their toxicity is due to the cationic QDs [32].
However, few studies providing a detailed mechanism of AgNPs toxicity are available despite their abundant applications in industry and in AgNPs-based medicine, where they have been introduced into human tissue mostly upon administration by either injection or through skin exposure. These penetration routes will likely lead to interaction of AgNPs with blood tissue and its components [47, 48] and can be either beneficial or detrimental. For instance, AgNPs synergistically activate immune responses by human blood monocytes [49]. In contrast, AgNPs cause structural damage of erythrocytes and change erythrocyte rheology [38]. AgNPs also cause platelet aggregation and hemolysis of RBCs [50, 51].
Assessment of AgNPs toxic effects thus becomes a key task science must address given the fact that the predicted environmental concentrations already exceed the predicted no-effect AgNPs concentrations [52]. Furthermore, and in contrast to the abundance of information on AgNPs beneficial and toxic effects, relatively few studies have been reported on their effect on cellular function [53], and particularly on ion transport mechanisms in human RBCs [54, 55]. The importance of using blood and its components to assess both beneficial and toxic effects of AgNPs, and of nanomaterials in general, stems from the fact that once the NPs reach the circulating blood, one of the major tissues of the body with a plasma/erythrocyte ratio close to unity, they are the most likely compartments to become in contact with the NPs. Although white cells and platelets represent a significantly smaller blood compartment, NPs effects on them are certainly not negligible [51]. Most importantly, alterations of membrane permeability and ion transport are one of the early events leading to cell death, thus becoming a strategic point for applying maneuvers to reverse the NPs toxic effects.
The present study reports on the effect of two widely-used, negatively charged AgNPs on K+ transport metabolism in human adult and cord RBCs.
Materials and Methods
Chemicals
Tris-hydroxyamino methane (Tris) free base, sodium chloride (NaCl), magnesium chloride (MgCl2), sodium phosphate (NaPO4), sodium hydroxide (NaOH), and D-glucose were acquired from Fisher Scientific (Fair Lawn, NJ). The compound 3-morpholinopropane-1-sulfonic acid (MOPS) was from Sigma-Aldrich (St. Louis, MO); rubidium chloride (RbCl), 99.8 % (metals basis), from Alfa Aesar (Ward Hill, MA); cesium chloride (CsCl), from Life Technologies (Carlsbad, CA) and Acationox detergent from Baxter (Oxford labware (Illinois).
Silver Nanoparticles (AgNPs)
AgNPs were synthesized by the Creighton (CR) method [53, 56-58] and the Lee- Meisel (LM) method [59]. CR AgNPs were synthesized in an ice bath by the dropwise titration reduction of silver nitrate (50 mL of 1 mM AgNO3) with sodium borohydride (300 mL of 2 mM NaBH4). LM AgNPs were obtained under heating at about 90°C by the reduction of silver nitrate (1.7 mL of 1% AgNO3) with trisodium citrate (2 mL of 1% Na3C6H5O7) in 100 mL of water. Both reaction mixtures were stirred (~320 rpm) for 1 hour to ensure completion. The original colloid of CR AgNPs was size-selected, purified and concentrated by tangential flow filtration (TFF) (KrosFlo Research IIi)) using 500 kD (790 cm2, ~50 nm pore size) and 10 kD (790 cm2, ~5 nm pore size) modified polyether sulfone (mPES) filters and the retentate was collected [53, 56, 60-62]. For LM AgNPs, the colloid was concentrated by centrifugation and then filtered through a 10 kD filter to remove any free Ag+ present in the colloid (< 10 %) [53, 62]. Thus, the Ag+ core of CR AgNPs was protected by borate and the LM AgNPs by citrate ions. Both AgNPs were suspended in deionized water. Except for the capping agent, the average size (10 nm), shape (spherical) and color (deep brown) of CR and LM AgNPs were the same [53, 62].
Blood
Concentrated red blood cells (RBCs) were donated by the Dayton Community Blood Center (DCBC), and human cord and adult blood by the Miami Valley Hospital (MVH), Dayton, OH. Packed RBCs from the DCBC were collected with citrate, phosphate, and dextrose (CPD) as anticoagulant and suspended in a preservation solution, mostly Optisol (AS-5, see below), to prolong storage [63, 64]. An additive solution was used to store RBCs up to 42 days at 2-6 °C, which contained adenine besides mannitol and NaCl, each at different concentrations, (AS) AS-1, AS-3 and AS-5 (http://www.mahasbtc.com/preservation-and-storage-blood). Cord blood (CB) was collected after C-sections at MVH and was a mixture of arterial and venous blood. The blood was collected into K+ ethylene diamine tetra-acetic acid (K2EDTA) vacutainer tubes and stored at 4° C until further use.
Ion transport determination
Whole blood (WB) obtained from MVH was collected in Vacutainer tubes and was used to determine the hematocrit (Hct) and optical density (OD) of packed cells (ODpcs). The ODpcs was obtained by diluting an aliquot of WB in hemolyzing solution (HS) (201X). ODs were measured in a Gilford Spectrophotometer (Gilford Systems, Oberlin, OH) at 527 nm, i.e., the isosbestic point for oxy- and met-hemoglobin (Hb) spectra [65]. The ODpcs for concentrated RBCs were calculated as described for cord blood. WB or concentrated RBCs were centrifuged at 10,000 rpm for 5 min to separate the RBCs from plasma and buffy coat that were aspirated and discarded. The packed RBCs remaining were collected and used for further studies.
For ion transport determination, several experimental solutions were required. Initial washing solution, composed of NaCl, 300 mOsM at room temperature (RT). Preincubation solution made of K+-free phosphate-buffered saline (PBS) containing (mM): 5 NaPO4 buffer, 5 Glucose, and NaCl to adjust osmolality to 295 mOsM, pH 7.4 at RT. Flux solutions containing (mM): 5 NaPO4 buffer, 10 RbCl, 5 Glucose, NaCl, pH 7.4, to adjust osmolality to 295 mOsM. Final washing solution containing (mM): 10 MOPS/Tris-MgCl2, pH 7.4, 4 °C, 295 mOsM. Hemolyzing solution containing (mM): 4 CsCl, 29 % NH4OH, and Acationox detergent.
Experimental Procedure
The RBC pellet obtained after WB or concentrated RBCs centrifugation was washed 4X with the initial washing solution at 37 °C, and spun until the centrifuge reached 10,000 rpm, at which point, centrifugation was stopped. The initial wash with isosmotic NaCl solution maintained the original cellular volume and served to remove anticoagulants and other chemicals. Then, RBCs were pre-incubated in PBS for 15 min at 37 °C, centrifuged in a Sorval RC5 ultracentrifuge at 10,000 rpm up and down, and the supernatant discarded. This step was also used to equilibrate RBCs with different chemicals, as needed, prior to exposure to the flux solutions. The technique for ion fluxes determination was adapted from previously published work [66] with some modifications for the NPs study as indicated next. RBCs were incubated at 37 °C in flux media as described above, and in the absence or presence of AgNPs for 1 h, swirling every 15 min. After 1 h of incubation, triplicates were prepared and washed 3X in final washing solution and spun down using an Eppendorf centrifuge (Eppendorf North America, Hauppauge, NY) at 14,000 rpm (18,188 g). Rb+ and K+ ions were extracted from RBCs with hemolyzing solution and measured by atomic emission and absorption spectrophotometry, respectively, using a Perkin Elmer 5000 atomic absorption spectrophotometer (Perkin Elmer Corporation, Waltham, MA). Hbc OD, was determined at 527 nm to estimate RBC volume in the same tubes in which Rb+ and K+ were measured. Cellular Rb+ uptake and [K]i were calculated in mmol/L of original cells (LOC) per time. Fig. 1 summarizes the steps described in this section.
Statement of Ethics
Concentrated red blood cells (RBCs) were donated by the Dayton Community Blood Center (DCBC), and human cord blood by the Miami Valley Hospital (MVH), Dayton, OH. Subjects (or their parents or guardians) have given their written informed consent and the study protocol was approved by the institute’s committee on human research under IRB # 06216, SC # 6034. Part of this work has been previously presented at the Experimental Biology Annual Meetings and published as abstracts in FASEB J. 2016, and 2017, and as a master’s thesis by one of the co-authors (P.K.A.).
Results
Two types of spherical AgNPs, average diameter of 5-10 nm with a narrow size distribution (1-40 nm) after filtration, and negatively charged (AgNPs-) were successfully synthesized by the CR and LM methods and used in this study [53, 62], see Materials and Methods for further details).
Studies with Creighton (CR) AgNPs
Washed human cord red blood cells (RBCs) were incubated with CR AgNPs-, for 1 h at 37 °C (Fig. 2) under the following conditions: 1 = CONT, RBCs incubated in the absence of AgNPs- in a medium of the same osmolality as NPS; 2 = NPS, RBCs + medium + AgNPs-; 3 = NPSC, a AgNPs- control to take into account AgNPs potential interference in both cation and RBC volume determinations; 4 = Hydrogen peroxide (H2O2), a known oxidant and positive control in flux media + RBCs. The sample containing AgNPs showed a visible color change suggesting oxidation of RBC Hbc to MetHbc [67], which was spectrophotometrically confirmed by a decrease in absorbance of 10-15 % for peak A (Mean ± Range, 537.5 ± 1.5 nm, n = 2) and 10-30 % for peak B (Mean ± Range, 574.5 +/- 2.5 nm, n = 2) when compared to the CONT. The Hbc spectrum also showed an additional broad shoulder between 594.1 and 658.8 nm probably due to MetHb formation (Results not shown).
Effect of CR AgNPs on Rb+ influx, [K]i, and Hbc ODs in adult RBCs
Next, the effect of AgNPs on Rb+ influx and [K]i of human RBCs was studied in a pilot experiment (data not shown) on two day-expired RBCs from the DCBC. CR AgNPs completely inhibited Rb+ influx and depleted [K]i (p < 0.0001, n = 7, and p < 0.001, n = 7, respectively). This unexpected finding was thought to be due to the expired RBCs, which may have been leaky and prone to hemolysis. Alternatively, since AgNPs cause hemolysis of RBCs [51, 68], inhibition of Rb+ influx and the almost complete K+i loss was thought to be due to hemolysis. Contrary to this expectation, determination of Hbc ODs, a measure of the relative volume of RBCs for each condition, showed a significant difference in ODs between CONT and NPS (p < 0.0001, n = 12) indicating that the RBC volume in the latter was about 2-fold higher than that in CONT and almost at the level of the isotonic control (CONTC, see Fig. 3 below) dispelling doubts about RBC hemolysis as a cause of the results observed in expired RBCs and affirming that the observed [K]i depletion was indeed an effect caused by the AgNPs.
Exposure of fresh RBCs to AgNPs- at a concentration of 150 µg mL-1 inhibited ~ 100 % of Rb+ influx and caused a total [K]i loss (Fig. 3A and B, respectively) (p < 0.0001 vs. the control, n = 12). There was a statistically significant difference in Hbc ODs between CONT and NPS (p < 0.0001, n = 12) (Fig. 3C). The decrease in Hbc ODs in cells exposed to AgNPs appears to be in part due to cell swelling thus lowering the ODs with the hypoosmotic AgNPs suspension, and to some hemolysis. Furthermore, despite a lower Hbc ODs in AgNPs-exposed RBCs, the values are within those observed for expired adult RBCs, indicating presence of RBCs in all conditions tested. A statistically significant difference between CONTC and CONT (p < 0.001, n = 12) was also observed, which may be due to osmolality differences between these two media (Fig. 3C).
It was not clear from the transport data whether NPs that were adsorbed to the RBC surface through an electrostatic effect could attract the main intracellular cation, K+, and thus cause its depletion. In addition, in this case, the inhibition of Rb+ influx could have occurred by also binding of the extracellular cation to the adsorbed AgNPs. Another possibility is that AgNPs exerted their effect by penetrating the RBCs. This alternative is supported by studies on internalization of quantum dots (QD) as D-penicillamine (DPA)-QD in human RBCs [40].
The above question was answered by hyperspectral imaging with a CytoViva® instrument. Fig. 4A-D shows the CytoViva® optical image of a control RBC, an RBC exposed to CR AgNPs-, the hyperspectral image of the RBC exposed to the AgNPs- revealing the presence of the NPs taken up by the RBCs, and the hyperspectral signatures of AgNPs (green spectrum) and RBCs (red spectrum), respectively. For further details, see legend to Fig. 4. Thus, the unexpected effects observed in RBCs exposed to AgNPs- were indeed caused by their penetration into the cytosol instead of the sole adsorption onto the RBC surface. Therefore, the inhibition of Rb+ influx and complete loss of K+i may be due to other mechanisms caused by the AgNPs themselves.
Effect of CR AgNPs on Rb+ influx, [K]i, and Hbc ODs in cord RBCs
To rule out that results shown in the previous figures were a property of “adult” blood samples, i.e., due to “aging”, the effect of CR AgNPs- on Rb+ influx and [K]i was studied in human cord blood (Fig. 5). After incubation of cord RBCs with CR AgNPs for 60 min at 37 °C, Rb+ influx was completely inhibited (p < 0.0001, n = 6). In addition, Rb+ influx in CONT was higher than in CONTC (p < 0.005, n = 6) (Fig. 5A), probably due to a lower osmolality in the CONT, or to differences between cord and adult RBCs, or to interindividual variability within a given blood type, adult expired or fresh, and cord blood. Exposure of cord RBCs to CR AgNPs led to K+ depletion when compared to their control (CONT) (p < 0.00001, n = 6, Fig. 5B), and statistically significant difference was also found in [K]i between CONT and CONTC (p < 0.005, n = 6, Fig. 5B). Hbc ODs were about 2-fold higher in NPS with respect to its control (CONT, p < 0.00001, n = 6) (Fig. 5C), and closer to the level of CONTC. The borderline statistical difference in Hbc ODs between CONT and CONTC (p < 0.061, n = 6) could be due to some hemolysis induced by osmolality differences between these conditions. Inhibition of Rb+ influx and K+ depletion from RBCs (Fig. 5A & B) suggest a direct effect of the AgNPs on K+ metabolism in cord RBCs and independent of the donor’s age.
Time course of Rb+ influx, [K]i, and Hbc ODs as a function of CR AgNPs-exposure in cord RBCs
Some reports on human RBCs indicate that AgNPs induce lysis and structural damage [38, 44, 50]. An indication that the effect of AgNPs on Rb+ influx and [K]i is due to the NPs and not to other factors was obtained from kinetic studies such as time-course and dose-dependence. Thus time-course studies were done under the conditions described for Fig. 3.
Rb+ uptake in CR AgNPs-exposed cord RBCs (NPS), increased by ~ 4-fold from 15 to 60 min incubation (Fig. 6A, p = 0.0000, n = 6 for NPS15 vs. NPS60 and p < 0.05 between NPS30 vs NPS60), whereas [K]i decreased by 2-fold in the same time period (Fig. 6B, p = 0.0000, n =6 for NPS15 or NPS30 vs NPS60). In addition, both Rb+ influx and [K]i were about 3-fold lower in NPS than in the CONT at 60 min (Fig. 6A and B, p < 0.0000, n = 6, respectively). In contrast, there was no statistically significant change in the Hbc ODs between 15, 30 and 60 min and the values were higher than the CONT at 60 min (Fig. 6C, p = 0.0001 for NPS60 vs CONT60) suggesting swelling in the absence but not in the presence of AgNPs. However, in contrast to former findings in expired or fresh adult or cord RBCs, where K+ loss was in the order of 100 % at 60 min exposure, in this patient’s RBCs, the [K]i decreased only by 60 % showing interindividual variability in the response of RBC K+ transport to AgNPs exposure.
Once the effect of CR AgNPs on Rb+ influx and [K]i was demonstrated to be time-dependent, it was important to determine whether their effect was also dose-dependent.
Dose-response of Rb+ influx, [K]i, and Hbc ODs as a function of CR AgNPs-exposure in cord RBCs
Fig. 7 shows a dose-response curve for Rb+ influx, [K]i and Hbc ODs as a function of CR AgNPs concentrations (30 – 150 µg mL-1) for CONT and NPS in cord RBCs. Rb+ influx was measured after incubation with CR AgNPs for 60 min at 37°C. RBCs exposure to the NPs induced more than 2-fold inhibition of Rb+ influx along the concentration range tested (p < 0.0001, n = 3, Fig. 7A). Moreover, the lowest concentration of 30 µg mL-1 already produced a full effect at which Rb+ influx was inhibited by more than 80 %. An additional 10 % inhibition in Rb+ influx was noticed at 150 µg mL-1, the highest concentration tested.
Like for Rb+ influx, cord RBC [K]i was measured in both CONT and NPS, the latter after incubation with increasing concentrations of CR AgNPs (30 – 150 µg mL-1) for 60 min at 37°C (Fig. 7B). Results show a concentration dependent effect of AgNPs on [K]i with about 30% inhibition at 30 µg mL-1 and depletion of RBC K+ at the highest concentration tested (150 µg mL-1), (p < 0.0001, n = 3, Fig. 7B). Thus, CR AgNPs induce a dose-dependent depletion of [K]i in cord RBCs, while [K]i decreased almost linearly by 70%. Furthermore, the kinetics of inhibition was different for Rb+ and K+, despite a dose response effect for the two cations.
The degree of hemolysis, i.e., Hbc ODs, as a function of AgNPs concentration, was determined in the samples already used for Fig. 7A and 7B (Fig. 7C). While the NPS ODs were relatively constant between 30–120 µg mL-1, the CONT ODs showed a decreasing linear relationship at concentrations of 60 µg mL-1 and higher, and at 120 µg mL-1 there was a statistically significant difference between CONT and NPS. Because the Hbc ODs of CONTC were the highest of the three conditions tested (Results not shown), the lower ODs in CONT and NSP are indicative of higher hemolysis or more swelling or both with the sequence CONT > NPS (Fig. 7C). At higher AgNPs concentration, both RBCs in the CONT and NPS conditions hemolyzed. Because the AgNPs were suspended in water, increasing the AgNPs concentrations would cause a decrease in the osmolality. To avoid a volume effect instead of an effect of the NPs, the osmolality was maintained constant along the concentration range tested.
In conclusion, CR AgNPs inhibited Rb+ influx and depleted RBCs of K+ in a time- and dose-dependent manner through a mechanism that needs further elucidation. Moreover, these results indicate that indeed CR-functionalized AgNPs are toxic to human RBCs.
Effect of cold storage on RBCs and interindividual variability
Fig. 2-8 indicate that CR AgNPs- cause significant changes in RBCs ion transport properties and in a time- and dose-dependent manner. These changes have different origins and demonstrated interindividual variability. A summary of these experiments is shown in Fig. 8A and B where the Rb+ influx and [K]i, respectively, were measured in fresh or stored adult and cord blood, under the different experimental conditions described above (Fig. 3, 5, and 7-8, i.e., CONTC, CONT and NPS). The most striking finding was that CR AgNPs- consistently inhibited Rb+ influx and depleted the cells of K+, independently of the blood source and time of storage in the cold. In some of the samples where RBCs were kept longer at 4 °C, the change in Rb+ influx and [K]i was variable and appeared to be more pronounced for Rb+ than for K+ (compare Fig. 8A and B). Hence CR AgNPs are highly toxic under the conditions tested in eight donors.
Functionalization has been shown to be an important feature of AgNPs that may significantly affect their toxicity and could render them amenable for diagnostic and therapeutic purposes [4-6, 49, 69-72]. Questions such as: Are all AgNPs as toxic as the CR NPs or do differentially functionalized AgNPs vary in their toxicity to ion transport in HRBCs? To this end, the effect of LM-functionalized AgNPs- was studied on RBC ion transport. These AgNPs, have citrate groups on the surface, in contrast to CR AgNPs functionalized with borate.
Effect of LM AgNPs on Rb+ influx, [K]i, and Hbc ODs in cord RBCs
In a parallel experiment with CR AgNPs- that inhibited ion movements by ~90% at 150 µg mL-1 (data not shown, see similar results in Fig. 3), Lee-Meisel AgNPs inhibited Rb+ influx by about 50 % (Fig. 9A) and K+ loss only by ~ 20 % (Fig. 9B), whereas there were no changes between the CONT and CONTC conditions (Fig. 9A & B). In addition, the Hbc ODs were practically unchanged in CONTC, CONT and NPS (Fig. 9C), indicating that no apparent hemolysis occurred by exposure of HRBCs to LM AgNPs. These results indicate that AgNPs functionalized with citrate are less toxic to RBCs K+ metabolism.
Time course of Rb+ uptake, [K]i, and Hbc ODs as a function of LM AgNPs-exposure in cord RBCs
Like the kinetic studies with CR AgNPs, the effect of LM AgNPs on Rb+ uptake, [K]i and Hbc ODs was studied in RBCs from cord blood at 15, 30, and 60 min in the CONT and NPS conditions (Fig. 10). Rb+ uptake in the control (CONT) was not a linear function of time, but rather increased by 2-fold between 15 and 60 min tending to saturate at the longer incubation times probably due to back-flux or to more than one mechanism of Rb+ uptake. Furthermore, LM AgNPs (NPS) inhibited Rb+ uptake from 30 % (p < 0.05) to 100 % (p < 0.0001) between 15 and 60 min vs. the control (Fig. 10A). In contrast, [K]i remained almost constant with time in both CONT and NPS, and the K+ loss, although statistically significant (p < 0.0005) was only 10 % of the [K]i in the control (Fig. 10B). Notably, LM functionalized AgNPs had no effect on Hbc ODs as a function of time between CONT and NPs (Fig. 10C). The decrease in Rb+ uptake caused by the LM AgNPs was comparable to that induced by CR AgNPs (Fig. 6), whereas the decrease in [K]i was about 3-fold less than with the latter (compare Fig. 6 and 10). These results clearly indicate that LM AgNPs are less toxic to cord RBCs than CR AgNPs under similar experimental conditions.
So far, the effect of two types of functionalized AgNPs, CR and LM, was studied under different conditions and in different types of human blood, i.e., adult, either expired or fresh, and cord blood. These studies indicate that, independently of the type of blood and storage conditions, CR but not LM AgNPs caused depletion of RBC [K]i. Further characterization of the mechanisms by which CR AgNPs alter ion transport involved studies on the effect of temperature (4 oC and 37 oC) on Rb+ influx and [K]i in AgNPs-treated RBCs from human cord blood (Results not shown). Data indicate that AgNPs alter Rb+ influx and [K]i through temperature-dependent pathways for both control (CONT) and experimental condition (NPS) and this effect was proportionally larger for Rb+ influx than for K+ loss. In addition, at 4 ºC, about 50 % of the Rb+ influx and 60 % of K+ loss was temperature-independent in NPS-treated RBCs when compared to their controls, suggesting ion movements through mechanisms that do not require metabolic energy.
To develop standards of AgNPs toxicity, an important question is whether there is interindividual variability for the effect of the AgNPs- tested in this study, and of AgNPs in general. Indeed, as it is shown for human cord RBCs from a different patient, the effect of CR AgNPs- on K+ metabolism was less pronounced at 60 min in Fig. 6 than in Fig. 5 with only 60 % inhibition of Rb+ influx and [K]i vs. the control. Furthermore, Fig. 8 clearly shows interindividual variability for a sample of N = 8 patients, albeit CR AgNPs were toxic for RBCs from the patients studied. These are important findings indicating that some individuals may be more susceptible than others to the effect of AgNPs, thus making the task of establishing safe and acceptable limits for the use of AgNPs for either diagnostic or therapeutic purposes, more complex.
K+ concentration in supernatant, [K]o, after washing RBCs treated with CR or LM AgNPs
To assess whether K+ was lost from the cells into the media or remained adsorbed on the AgNPs surface after incubation in their presence, [K]o was determined in the supernatant fraction as a measure of the K+ loss from RBCs. The K+ loss from cells exposed to CR and LM AgNPs, decreased with increasing the number of washes, was higher in cells treated with CR than with LM AgNPs and followed the sequence NPSCR > CONTCR > NPSLM > CONTLM (Fig. 11). Furthermore, K+ loss induced by either CR or LM AgNPs could be accounted for by appearance of K+ in the extracellular space during the flux procedure as reflected by a rise in [K]o of the supernatants that were simultaneously collected after each wash. In other words, the difference in [K]i after 60 min exposure to NPs was equal to the total K+ loss determined in the supernatants (data not shown), indicating that K+ was not adsorbed on the NPs but it was released into the media. These results also support our findings (Fig. 3, 5, 7, 8, 9B, 10 and 11) of a higher toxicity of CR than LM NPs and demonstrate the important role of AgNPs functionalization for their biological effect.
Discussion
Nano-size particles cause more RBC hemolysis than larger particles due to their greater surface area which facilitates a larger release of Ag+ from AgNPs by their direct interaction with the RBCs. In fact, released Ag+ ions have been shown to be responsible for the AgNPs toxicity [40]. Small nano-size particles are more likely to be internalized into cell lines than the large ones [32, 33]. In various cell lines and animal models, different types of NPs are known to induce cytotoxicity. AgNPs are the most toxic NPs because of their metallic silver and high biological reactivity [34, 35]. Positively charged NPs are more toxic than the negatively charged [46], because the RBC surface charge is negative and thus possess higher affinity for the positively charged NPs. In human RBCs, QDs coated with D-penicillamine, a zwitterionic amino acid ligand (D-penicillamine DPA-QDs), enter the cells by a passive transport mechanism and their toxicity depends, in general, on their size (the smaller the more toxic) and charge (cationic QDs interacting stronger with cells than anionic counterparts) [32]. However, despite some cautious generalizations [32], numerous factors contribute to NPs toxicity, which depends on both the NPs physicochemical properties and the biological material with which they interact [46]. This is the case in the present study, where negatively charged AgNPs were expected to be less bioavailable and toxic than positive ones, as it was found in Vero 76 cells [53]. In contrast to expectations, AgNPs- induced complete [K]i depletion in HRBCs from all but one of the donors tested (Fig. 3B, 5B, 7B, and 8B), and this was independent of the type of cells, adult or cord, fresh or expired and of the time in cold storage (Fig. 3B, 5B, 7B, and 8B), but strongly dependent on the functionalization of the NPs. In fact, this study shows that CR AgNPs were more toxic than the LM (Fig. 3B, 5B, 7B-8B and 11 vs. Fig. 9-11), and this is supported by the lower Hbc ODs in CONT and NSP in Figure7C, which are indicative of higher hemolysis with the sequence CONT > NPS. Furthermore, LM AgNPs did not lyse RBCs (Fig. 10C) despite that the AgNPs-treated RBCs should have decreased their volume due to the inhibition of Rb+ uptake (Fig. 10A) and the increase in K+i loss (Fig. 10B), once more pointing to a less toxic effect of the latter when compared to CR AgNPs.
Furthermore, few studies providing a detailed mechanism of AgNPs toxicity are available, despite the abundant applications of AgNPs in industry and in AgNPs-based medicine where they have been introduced into human tissue upon administration by either injection or through skin exposure. These penetration routes will likely lead to interaction of AgNPs with blood tissue and its components [47, 48]. These interactions can be either beneficial or detrimental. For instance, AgNPs help to activate synergistically immune responses by human monocytes [49]. In contrast, AgNPs cause structural damage of erythrocytes and change erythrocyte rheology [38]. AgNPs also cause platelet aggregation and hemolysis of RBCs [50, 51].
Likewise, despite a large body of information described above, the mechanisms involving toxicity to RBCs remain to be elucidated [51]. To fill this gap, this study focused on the effect of AgNPs on RBC cellular ionic homeostasis and used differentially functionalized silver nanoparticles as experimental materials to treat RBCs. To achieve this goal, RBC Rb+ influx, [K]i, and Hbc ODs were determined in HRBCs under different experimental conditions, and as a function of NPs length of exposure, concentrations and functionalization.
Different types of human RBCs were used in the present study (adult or cord blood, expired or fresh, or stored refrigerated for different periods of time, from 0 to 14 days), and treated with two types of negatively charged functionalized AgNPs of an average diameter of 10 nm, Creighton and Lee Meisel. CR AgNPs inhibited Rb+ influx by 50% to 80 % (Fig. 3A, and 5A-7A) and depleted the cells of [K]i by 50 to 90 % (Fig. 3B and 5B-7B). LM AgNPs inhibited Rb influx by 50 % and depleted [K]i by an average of 15 % (Fig. 9 and 10), in contrast to CR AgNPs. Our results show inter-individual variability for [K]i in expired, fresh, or stored RBCs. However, it was not clear from the transport data whether adsorbed NPs on the RBC surface were responsible for both K+ loss and inhibition of Rb+ entry in the cells. In contrast to this argument, Fig. 4 shows that AgNPs exerted their effect by internalization of the NPs in the RBCs. This finding is further supported by studies on internalization of D-penicillamine (DPA)-QD in human RBCs [40]. The K+ loss observed upon cold storage (Fig. 8B) is further supported by findings of K+ loss when blood is maintained under refrigeration [73].
In summary, CR AgNPs inhibited Rb+ influx and K+i loss in a time- and dose-dependent manner (Fig. 6 and 7). LM AgNPs also altered RBC permeability to Rb+ and K+ in a time-dependent manner (Fig. 10). The K+ loss induced by either CR or LM AgNPs could be accounted for by appearance of K+ in the extracellular space during the flux procedure as reflected in [K]o collected after each wash (Fig. 11).
Based on the results described above, the present study shows that both CR and LM AgNPs altered K+ homeostasis in human RBCs. During these studies, however, it remained to be determined whether this effect was due to extracellular or internalized AgNPs in HRBCs. By using CytoViva® hyperspectral imaging, AgNPs were found to be internalized in human RBCs (Fig. 4 B-D), and thus their effect may have been due to their internalization (Fig. 4). Our findings in human RBCs agree with reports in human and nucleated fish RBCs where zwitterionic quantum dots and AgNPs toxicity is caused by internalization of the NPs [32, 33, 44].
Conclusion
CR and LM functionalized AgNPs were found to alter the K+ levels in RBCs in a time- and dose-dependent manner, and this effect appeared to be induced by AgNPs internalization, which led to a massive intracellular K+ loss that could be accounted for by its presence in the RBCs extracellular compartment. This is indeed an important finding indicative of potential and serious complications during AgNPs in vivo administration to patients receiving complex drug treatments that could enhance the NPs effect/s, and especially in patients suffering from cardiovascular diseases and kidney insufficiency for whom sudden changes in plasma K+ could lead to life threatening situations. Of equal importance, our findings show that alterations of membrane permeability and, consequently, ion transport are one of the early events leading to cell death (or hemolysis for RBCs), thus becoming a strategic point for applying maneuvers to reverse it.
Acknowledgements
Author Contributions: N.C.A. and P.K.L. conception and design of research; P.K.A. performed the experiments with initial help from N.C.A., P.K.A., N.C.A., and P.K.L., analyzed data; N.C.A., P.K.L. and P.K.A. interpreted results of experiments; P.K.A., and N.C.A. prepared figures; N.C.A. drafted manuscript; N.C.A., P.K.L. and P.K.A. edited and revised manuscript; N.C.A and P.K.L approved final version of manuscript. I.E.P-S and A.S.L.P. synthesized the AgNPs, provided important scientific and experimental input related to nanotechnology and the manuscript, and facilitated procurement of blood from the Dayton Community Blood Center. J.L.Y. procured cord blood from his patients at the Miami Valley Hospital, facilitated writing, submission, and approval of Informed Consent Forms, IRBs and IBCs, and participated in critical discussions about the clinical implications of the study.
Images and data illustrated in Fig. 4 were provided compliments of CytoViva®, Inc. www.cytoviva.com, info@cytoviva.com, 570 Devall Drive Suite 301, Auburn, AL 36832. Human adult blood was kindly provided by the Dayton Community Blood Center, and human cord blood by the Miami Valley Hospital, Dayton, OH. Dr. Marjorie Markopoulos participated in the initial stages of this study. Drs. Marjorie Markopoulos and Rose Maxwell facilitated the approval of IRBs, IBCs, amendments, and compliance with administrative regulations at Wright State University and MVH, respectively.
Funding Sources: This work was supported, in part, by the National Science Foundation (Awards # 1438340 and 1726095), Department of Pharmacology and Toxicology, Department of Chemistry, and Wright State Foundation.
Disclosure Statement
The authors report no conflict of interest.
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