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ORIGINAL ARTICLE
Year : 2021  |  Volume : 9  |  Issue : 2  |  Page : 117-121

Stability in erythrocyte fragility responses of hemoglobin genotypes exposed to nanosilver


Department of Physiology, School of Basic Medical Sciences, University of Benin, Benin City, Nigeria

Date of Submission27-Mar-2021
Date of Decision10-May-2021
Date of Acceptance13-May-2021
Date of Web Publication10-Aug-2021

Correspondence Address:
Dr. Ogechukwu Kalu Uche
Department of Physiology, School of Basic Medical Sciences, University of Benin, P.M.B. 1154, Benin City 300-001
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njecp.njecp_7_21

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  Abstract 


Background and Objective: Safety concerns have been expressed in the extensive applications of nanoparticles in nanomedicine and consumers' products. The aim of this study was to examine the impact of in vitro nanosilver (NS) exposure on erythrocytes membrane integrity during osmotic fragility (OF) reactivity in different hemoglobin genotypes (HbAA, HbAS, and HbSS). Materials and Methods: Blood sample was collected from 45 consenting male and female participants' age 20–30 years; comprising 15 (HbAA, HbAS, and HbSS). Red blood cells were separated, washed, and divided into three sets with each sample treated in triplicate with graded percentage concentrations of NaCl (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9). Two sets of the blood samples were preincubated with 1 ml and/or 2 ml of 10 ppm NS and 0.9 normal saline for 1 h, while the other set was exposed directly to access the capacity of erythrocyte hemoglobin genotypes to withstand osmotic stress. The absorbance from supernatants was recorded after 30 min incubation with standard spectrophotometer at 540 nm wavelength. The mean values of percentage hemolysis were plotted against the different NaCl concentrations. Results: The results showed that there was no significant difference (P < 0.05) in the OF response curves and mean OF (MOF) indices (concentration of the solution when 50% of the cells are hemolyzed) in the different genotypes. The MOF concentrations of the three genotypes were in the order: HbAA > HbAS > HbSS. The relative capacity of NS to stabilize erythrocyte membrane in the three genotypes was in the order HBSS > HBAS >HBAA. Conclusion: There was no undesirable NS effect on the erythrocyte OF responses in the different hemoglobin genotypes but a greater membrane stabilization effect in the HBSS.

Keywords: Erythrocyte osmotic fragility, hemoglobin genotypes, nanosilver


How to cite this article:
Uche OK, Oshomome AG. Stability in erythrocyte fragility responses of hemoglobin genotypes exposed to nanosilver. Niger J Exp Clin Biosci 2021;9:117-21

How to cite this URL:
Uche OK, Oshomome AG. Stability in erythrocyte fragility responses of hemoglobin genotypes exposed to nanosilver. Niger J Exp Clin Biosci [serial online] 2021 [cited 2021 Dec 4];9:117-21. Available from: https://www.njecbonline.org/text.asp?2021/9/2/117/323675




  Introduction Top


Nanoparticles (NPs) are heterogeneous substances with a size range of between 1 and 100 nm in at least one dimension and characterized by a high surface area-to-mass ratios, resulting in higher surface reactivity.[1],[2] Nanomaterials in the form of catheter, guidewires, dialyzers, oxygenators, cardiac pacemakers, vascular grafts, prosthetic valves, and industrial consumer products are widely used medical devices and biomaterials coming in direct contact with blood.[3] The applications of NPs have gained wide recognition and acceptance in current research areas of material science, consumer products as well as biomedical sciences due to their special properties of larger surface area-to-volume ratios and physiochemical properties, resulting in better reactivity and improved performance.[4]

Silver nanoparticles also known as nanosilver (NS) is the preferred nanomaterial of choice in nanotechnology owing to their antimicrobial potency and has been used clinically, well before the discovery of penicillin as well as its high propensity as antivirus, anti-inflammation, antibiofilm activities, and enhanced wound healing properties.[5],[6],[7] In addition, their unique small size provides for larger surface-to-volume ratio, resulting in increased membrane permeability across animal cell lines, increase bioavailability, biocompatibility, and longevity in circulation.[8]

Red blood cells (RBCs), also known as erythrocytes, are the most numerous type of blood cells essentially for carriage of respiratory gases for cellular metabolism and have been proposed as a prototypical cellular system regarding drug-mediated plasma membrane effects.[9] The mechanism by which NS interact with RBCs and other cellular elements has been reported to depend on their interactions with corona-like-capping agent by plasma protein cell adsorption and subsequent uptake by red cells and internalization into the cytoplasm with rapid biological activity.[10],[11] In hemoglobin genotypes, the adsorption process results in a remarkable retaining of the oxygenation properties of human adult hemoglobin and sickle cell hemoglobin, associated with an increase of the oxygen affinity.[12] Recently, NPs have attracted much attention as new scaffolds for hemoglobin-based oxygen carriers.[12] In view of the potential benefits of NPs to sickle cell anemia, nanomedical practice has proposed the use of nanorobots to repair damaged erythrocytes.[13]

Despite the wide acceptance and application of NPs, conflicting reports exist on the potential adverse effects and/or toxicity of NS particles on body organs, especially in relation to its high membrane permeate characteristics, hence the need to evaluate the hemocompatibility profile of NS. Hemocompatibility is a key criterion which limits the clinical applicability of blood-contacting biomaterials. Various human and animal experimental modules have been deployed in hemocompatibility evaluations. In this study, hemocompatibility following exposure to NS has been assessed by means of osmotic fragility (OF) reactivity responses in vitro. Resistance of RBC to hemolysis characterizes what is called the OF of the cell membrane. Functionally, OF is widely used to determine erythrocyte integrity and elucidate mechanisms of the influence of different factors on the osmotic properties of RBC membranes including shear stress and mechanical hemolysis, temperature, ultrasound effects, drugs, and irradiation.[14],[15] OF test is also useful in clinical diagnosis of certain hematological diseases such as hereditary spherocytosis, chronic liver disease, hematological anemia, glucose-6-phosphate dehydrogenase deficiency, and sickle cell anemia.[15] In vivo cell culture studies have shown conflicting data on toxic effects of NS in organ functions of animal models and a number of human cell cytotoxicity.[16],[17] The present study assessed and validated the hemocompatibility of NS impact on erythrocyte membrane integrity of hemoglobin genotypes (HbAA, HbAS, and HbSS) in vitro in young adults by characterization of OF responses.


  Materials and Methods Top


Subjects

Blood sample was collected from 45 consenting individuals, consisting of 15 samples from HbAA, 15 samples from HbAS, and 15 samples from HbSS. Both male and female apparently healthy asymptomatic participants within 18–25 years participated in the study after informed consent was obtained. The SCA participants were at stable states during the study.

Blood sampling

Five milliliters of blood was collected from each participant into ethylenediaminetetraacetic acid anticoagulant bottles and analyzed within 2 h of collection. Blood was centrifuged at 2500 rpm for 10 min in order to separate the erythrocytes from plasma. The erythrocytes were washed three times by methods as described by Tsakiris et al.,[18] with 5 ml of 0.9 phosphate buffer saline. Thereafter, erythrocytes were re-suspended in 3 ml of phosphate buffer saline and the test carried out with these washed and intact erythrocytes.

Fragility experimental protocols

The blood samples were divided into three sets for each genotype, with each sample set treated in triplicate with graded percentage concentrations of NaCl (0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, and 0.1) for control to assess their resistivity to osmotic stress and following preincubation with 1 ml and/or 2 ml 10 ppm NS at 37°C with 0.5 ml of blood in a water bath for 1 h, respectively, as described by Dacie and Lewis.[19] The optical absorbance was recorded with a standard spectrophotometer at 540 nm wavelength. Hemolysis in each tube was expressed as a percentage of the absorbance in distilled water. The average values recorded were plotted against the different NaCl concentrations.

Evaluation of fragility index and stabilization of erythrocytes

Mean OF (MOF) (concentration of the solution when 50% of the cells are hemolyzed) was graphically determined. While the relative capacity to stabilize or destabilize erythrocyte membrane was evaluated as a percentage of quotient of the difference between MOF values of the test and control samples to the control sample (Parpart et al. and Chikezie)[20],[21]

Thus,



Statistical analysis

Data analyses were done with GraphPad prism version 5.0 for Windows (GraphPad software, San Diego, CA, USA), followed by one-way analysis of variance. Values are presented as means ± standard error of the mean (SEM). P < 0.05 was considered statistically significant, while n-values denote number of animals in each experimental group.


  Results Top


Results in [Figure 1] showed no significant difference in the fragility curves from cells incubated in NS compared with the control in the HBAA genotype. MOF (concentration of the solution when 50% of the cells are hemolysed) was in the order: 0.48 < 0.49 < 0.50 and showed no significant difference in OF indices.
Figure 1: Functional erythrocyte HBAA haemolysis curves in NS exposures

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N = 15; ± SEM P > 0.05

In [Figure 2], result of the hemolysate curves showed no significant difference in the osmotic resistance from cells incubated in NS compared with the control.
Figure 2: HBAS hemolysis curves in cells exposed to NS

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MOF was in the order: 0.49 < 0.51 and 0.51. N = 15; ± SEM P > 0.05

In [Figure 3], there was a leftward shift of the fragility curve from 1 ml NS-treated cells in 1 h compared with the control, indicating increase osmotic resistance. The tenet was the same in 2 ml NS-treated cells. MOF was in the order: 0.42 < 0.47 < 0.49; suggesting greater membrane stabilization effect in the HBSS following exposure to NS. N = 15; ± SEM P < 0.05.
Figure 3: HBSS hemolysis curves following exposure to NS

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  Discussion Top


In this study, we have employed two) protocols: red cells incubation with 1 ml and/or 2 ml 10 ppm NS at 37°C in a water bath for 1 h, respectively, to examine the impact of acute NS exposure on erythrocyte membrane integrity in fragility test reactivity on three different hemoglobin genotypes – HBAA, HBAS, and HBSS, respectively. There was no significant difference (P < 0.05) in the OF response curves and MOF indices (concentration of the solution when 50% of the cells are hemolyzed) in cells from the tests compared with cells of the control in the HBAA and HBAS genotypes [Figure 1] and [Figure 2]. However, there was a leftward shift of the fragility curve in 2 ml NS-treated cells of the HBSS genotype in 1 h compared with the control, indicating increase osmotic burst resistance. MOF in the three different genotypes studied was in the order: 0.42 < 0.47 < 0.49, suggesting greater membrane stabilization effect in the HBSS following exposure to NS. The data obtained from this study in the MOF of the three different genotypes [Figure 1],[Figure 2],[Figure 3] also indicated that the relative capacity of NS to stabilize erythrocyte membrane in the three genotypes was in the order HBSS > HBAS > HBAA. This observation corroborates with the reported potential benefits of NPs in sickle cell anemia, and the proposal for the use of nanorobots to repair damaged erythrocytes in nanomedical practice.[13]

The outcome of the present study does not support the notion of increased NS erythrocyte hemolytic effect as there were no observable differential changes in the hemolysate fragility response curves and mean osmotic concentration in NS-treated cells in HBAA and HBAS genotypes. On the contrary, the observation in increased resistance to lysis in cells from the HBSS genotype incubated with just 2 ml of 10 ppm NS for an hour is reasonable to think of greater NS particles protective potential effect in higher dosage and timeline exposure in sickle cell anemic patients. OF is defined by shifts in the hemolysis curves, which relates absorbance versus NaCl concentration, and is often established by determination of 50% of the hemolysis points.[15] In clinical practice, OF test is often performed to aid in the diagnosis of diseases associated with RBC membrane abnormalities.[22],[23] Some disease conditions linked to increased OF include hereditary spherocytosis and hypernatremia, while some linked to decreased fragility include chronic liver disease, iron deficiency anemia, thalassemia, polycythemia vera, and sickle cell anemia after splenectomy.[13] The increase in osmotic burst resistance following exposure to NS observed in the HBSS could be attributed to the postulation that the unique small size and physiochemical properties of NS particles enhanced rapid direct membrane permeability across cells for even distribution in blood.[24] From another perspective, comparative osmotic stability in the hemoglobin genotypes in NS medium may have sustained the relative tendency of the cells to retain more sodium ion (Na+) intracellularly with a concomitant loss of potassium ion (K+).[25]

RBCs are by far the most numerous type of blood cells in mammals. To date, erythrocytes have been studied as carriers of a wide range of drugs such as enzymes, antibiotics, anti-inflammatory, and antiviral drugs and for diagnostic purposes (e.g., magnetic resonance imaging).[26] Nevertheless, evidence that drugs and natural products can interfere with osmotic resilience of erythrocytes and hence their capacity and efficiency in drug delivery have been demonstrated.[26],[27],[28] Numerous membrane stabilizing/destabilizing agents may act by direct interaction with architectural membrane proteins and enzymes, thereby modifying their structure/function relationship that is necessary and required for membrane integrity.[29] Data from the present study, however, demonstrated somewhat similarity in the pattern of OF curves of the different hemoglobin genotypes exposed to NS which suggests a common mode of action and indication of a positive tendency in membrane stabilization of erythrocytes.


  Conclusion Top


In conclusion, there was no undesirable NS effect on erythrocytes OF responses in the different hemoglobin genotypes but a greater membrane stabilization effect in the HBSS genotype.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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  [Figure 1], [Figure 2], [Figure 3]



 

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