1.

Introduction

Recently, nanodiamond (ND) has been proposed to be an effective nano-bio-probe utilizing its unique Raman^1,2 and fluorescence³ signals. Surface functionalized ND finds substantial progress for different applications such as conjugations of drugs for effective drug delivery, nanosurgery, and prosthetic devices for retinal implants.^4–6 The NDs have been proven to be an ideal biocompatible platform, while Raman and fluorescence signals serve as markers in various biomedical applications.^1–6

Raman spectroscopy is also suitable for investigating blood protein such as hemoglobin (Hb) and red blood cells (RBC).^7–9 Raman spectrum signal observed for Hb is strong but relatively complex; however, there are certain trivial footprints from Hb states, which can be used to draw important conclusions for studying the RBC.⁸ Particularly one can study the oxygenation and spin state dynamics of the heme (the active center of Hb) due to a selective enhancement of the Raman active vibrations in different oxygenation states. Owing to variations in the oxygenation states of Hb, the spectrum exhibits dramatic changes in the spin state regions (1500 to $1650{\mathrm{cm}}^{-1}$) and the methane C-H deformation region (1200 to $1250{\mathrm{cm}}^{-1}$) followed by $T\leftrightarrow R$ (tense-relaxed transition).⁷ These dramatic changes in the spin state regions and associated structural changes (at $T\leftrightarrow R$) of the heme and Hb globule can be studied in situ using Raman spectroscopy.⁸ Additionally, heme orientation is an important factor in RBCs’ functions. Raman spectroscopy has already been demonstrated as an imperative tool to study the heme ordering in functional RBC and the laser-induced effect^9,10 to monitor changes in the oxygenation state of human RBCs while they were placed under mechanical stress.¹¹

For the successful development of ND for bio-labeling and for drug delivery, the most critical feature for considering them for biomedical applications is their interaction mechanism with the RBC and ultimately with the heme. Even though previous studies have confirmed low in vitro cytotoxicity offered by the NDs, for the in vivo studies, studying ND interactions with living organisms as well as with organs and tissues, especially with blood, like integrative tissue, is necessary.^12,13

Among the constituents of blood, RBCs are major oxygen carriers for the circulating system, and Hb is the major protein component in RBC, which is responsible for the reversible oxygen carrying and storage. The RBCs do not reveal any phagocytosis and endocytosis, so other mechanisms including diffusion, transmembrane channels, adhesive interaction via electrostatic, Van der Waals and hydration forces, or steric interactions may be involved.¹⁴ Reports revealed that certain fine nanoparticles with size less than 200 nm can enter easily inside the RBC, and the larger ones are associated with the membrane.^14,15 Different effects of nanoparticles on blood/blood components, and biocompatibility have been discussed using mesoporous silica,¹⁶ gold nanoparticles,¹⁷ magnetite ${\mathrm{Fe}}_3{\mathrm{O}}_4$ nanoparticles,¹⁸ and ${\mathrm{TiO}}_2$.¹⁹ Mostly, hemolytic properties are used as one of the common tests for understanding nanoparticle interactions. Few studies¹² of ultrafine detonation nanodiamond with whole blood show some harmful effects on blood’s biochemical characteristics, but serious blood cell destruction was not observed. On the other hand, the attachment of polystyrene nanoparticles (100 to 1100 nm) with modified surfaces to RBC increases the circulation lifetime of these drug delivering particles.^20,21 However for the development of the in vivo bio applications of ND, serious study on ND interactions with blood (whole blood as well as blood components) is vital.

In the present work, we have studied the interaction of ND (5 and 100 nm) with human RBCs. Confocal microscopy has been used to observe ND attaching on the RBC membrane, penetration inside (only 5 nm ND) the RBC, and interaction of nanodiamond with the membrane. Raman spectroscopy and UV-visible absorption are used to characterize the effect of ND on the oxygenation and deoxygenation processes of RBC. Further, interaction of ND with RBC membrane is presented with the help of RBC deformability analysis. In addition, the ND hemolytic ability and cytotoxicity properties are also discussed.

2.

Experimental Procedures

All experiments using human subjects were coherent to the national regulation of Human Subject Research (Taiwan) and the Declaration of Helsinki. To achieve the purpose, we had formally obtained informed written and signed consent for all volunteers; the PI, as well as the students who performed the experiments had taken lectures concerning ethical issues.

3.

Results and Discussion

Figure 1 shows the fluorescence and merging optical (bright field) images of RBC interactions with 5 and 100 nm sized cND. Nanodiamond’s intrinsic fluorescence has been used to image the locations of the cND. In this fluorescence imaging, the excitation wavelength used was 488 nm, and the fluorescence data were collected in the range of 491 to 539 nm. From Fig. 1(I) we observe that the 100 nm cND are localized on the RBC surface, whereas 5 nm cND penetrate inside the RBCs. Figure 1(II) depicts the $z$-scans of RBCs interacting with 100 nm cND of an individual RBC. It can be seen from the images of Fig. 1 (Ia to Ic), and particularly from the $z$-scans [Fig. 1(II)], that the cND with the particle size 100 nm interacts predominantly with the RBC membrane. As we already know from the earlier reports, a RBC does not reveal phagocytosis and endocytosis; however some nanoparticles with size less than 200 nm may penetrate the cell membrane,^14,15 so other mechanisms of nanoparticles-cell interaction can be involved. Contrary to this in our study, we do not observe 100 nm cND penetrations into a RBC, however penetration with smaller size (5 nm) were observed [Fig. 1(I)]. In addition, we observed that 5 nm cNDs induce the RBC aggregation even in PBS suspension [Fig. 1(II)]. Therefore, two important questions arise: first, how does the nanodiamond affect the RBC membrane; and second, can nanodiamond penetrate into the RBC and interact directly and affect the Hb state and functions? The present work is focused on the first question, and the second question will be considered in the course of future studies.

Fig. 1

Nanodiamond interaction with human red blood cells. (I) Fluorescence and bright field (BF) optical images of cND, RBCs and cND with sizes 100 and 5 nm at their interaction with RBCs. The cND fluorescence is excited with 488 nm and collected in the 491 to 539 nm range (shown in green). (II) A $z$-scan of 100 nm cND interacting with RBC. The images correspond to different positions along $z$-axis with 1 μm step. The cND fluorescence is excited with 543 nm and collected in range 562 to 659 nm (shown in green). The images indicate cNDs are attaching to the cell membrane of the RBC.

Results of cell viability, i.e., the WST-1 test on RBC interaction with 5 and 100 nm cND at concentrations of $5\mathrm{\mu g}/\mathrm{ml}$ and $50\mathrm{\mu g}/\mathrm{ml}$, are presented in Fig. 2. It is observed that RBC cells are viable for interaction with both the 5 nm and 100 nm cND. The nanodiamond hemolytic effect was estimated for 5 and 100 nm cND. Also, different concentrations, $5\mathrm{\mu g}/\mathrm{ml}$ and $50\mathrm{\mu g}/\mathrm{ml}$ of cND incubated with RBCs were used. It was further compared with the hemolysis effect for RBCs treated with 0.5% Triton $X$-100 and with PBS. The absorbance of the supernatant fluids of differently treated RBCs were measured and compared. Interestingly, it can be seen from Fig. 3 that the hemolytic effect of cND does not exceed 2% to 4% as compared with hemolysis created by Triton $X$-100, which is 100%. There were no differences within standard deviation between PBS- (control) and cND treated samples.

Fig. 2

RBC cell viability WST-1 test. Cell viability of RBC treated with normal saline (control) and cND’s (size 5 and 100 nm cND) with concentrations 5 and $50\mathrm{\mu g}/\mathrm{ml}$ in RBC suspension for 3 and 5 h. Data are reported as $\mathrm{mean}\pm \mathrm{standard}\mathrm{deviation}(\mathrm{SD})$. Three independent experiments with two replicates ($n=6$).

Fig. 3

Hemolytic effect of nanodiamond. After incubation with different concentration (5 and $50\mathrm{\mu g}/\mathrm{ml}$) of 5 and 100 nm cND for 3 h, the absorbance of the supernatant fluid of RBC was compared with 0.5% Triton $\times 100$ and normal saline treated RBC. The absorbance of Triton $\times 100$ groups was normalized to 100%. Data are reported as $\mathrm{mean}\pm \mathrm{standard}\mathrm{deviation}(\mathrm{SD})$. Three independent experiments with two replicates ($n=6$).

Previously, in-vitro studies on various nanoparticles demonstrated the nanoparticle-induced hemolysis,³¹ depending on the particles size, surface properties and experimental conditions.^19,32 It is known that nanoparticles can induce hemolysis directly affecting the membrane, or via oxidative stress (for example, ${\mathrm{TiO}}_2$).¹⁹ The adsorption of blood plasma proteins on NDs causes electrolyte and osmotic imbalance that results in cell destruction. Particularly, the RBC hemolysis during the interaction of whole blood with ultra-fine detonation nanodiamond was observed,¹² but was not estimated quantitatively. The present work shows quantitative information using 5 and 100 nm size cND. Figure 1 clearly demonstrates that cND is attached to RBC membrane, and a hemolysis test confirms the stability of the RBC membranes. Further, it is to be noted that in previous reports, the adsorption of blood plasma proteins on cND was analyzed, particularly, by varying pH, and it was shown that at pH near 7.4 the adsorbance of the main blood plasma proteins like albumin and immunoglobulin is low and varies for cND with various sizes and surface properties.^33,34

Figure 4 displays the Raman spectra of oxygenated and deoxygenated RBCs interacting with 100 nm sized cND. Similar spectra were observed for 5 nm cNDs as well (spectra not shown). The spectra have been compared with RBC’s spectra, which serve as reference. It is seen that the Raman spectrum does not reveal any structural transformations for RBCs (for Hb) due to the presence of cNDs. We have seen that cND can interact with RBC membrane and, correspondingly, interaction of cND with RBC membrane can change the membrane properties, and hence can change the conditions for gas transportation. This assumption is based on the fact that the penetration of oxygen (as well as ${\mathrm{CO}}_2$ and some other small molecules) through RBC membrane depends on the membrane structure and state.³⁵ Particularly, when the membrane fluidity is altered (for example chemically, with forming cholesterol-enriched membrane),³⁶ the membrane rearrangement changes the oxygen transport. To analyze the effect of cND on this RBC function, we study the process of oxygenation/deoxygenation.

Fig. 4

Nanodiamond effects on the Raman spectra of RBC. Comparison of Raman spectra of oxygenated and deoxygenated RBC at interaction with 100 nm cND. The numbers mark the positions of major Raman peaks of RBC (in ${\mathrm{cm}}^{-1}$).

The oxygenation/deoxygenation analysis via Raman spectra (as seen in Fig. 4) can be explained as follows. The RBC Raman spectrum exhibits a number of peaks to characterize Hb at different states. The major differences in the spectra are attributed to changes in the spin state between the deoxygenated heme with Fe in high-spin ($S=2$) state and the oxygenated heme with low-spin ($S=1/2$) state Fe. Correspondingly, the spectrum is sensitive to the conformational changes in Hb molecules, i.e., Hb transition from relax ($R$) state (oxygenated Hb) to tense ($T$) state (deoxygenated Hb). The porphyrin perturbations in oxygenation-deoxygenation cycle are associated with the tense-relaxed ($T\leftrightarrow R$) state transition of hemoglobin (Hb) within a single red blood cell.⁷ Deoxy-Hb is in the $T$-state with high-spin iron ($S=2$; ${\mathrm{Fe}}^{2+}$); the main bands for this state are observed at 1606 to 1610 (${\nu }_{10}$),1580 to 1582 (${\nu }_{37}$), 1544 to 1547 (${\nu }_{11}$), and 1210 to $1215{\mathrm{cm}}^{-1}$ (${\nu }_5+{\nu }_8$). Oxy-Hb is in $R$-state with low-spin ${\mathrm{Fe}}^{3+}(S=1/2)$; some intense bands are observed at 1636 to 1640 (${\nu }_{10}$), 1562 to 1565 (${\nu }_2$), 1245 to 1259 (${\nu }_{13}$), and 1224 to $1226{\mathrm{cm}}^{-1}$ (${\nu }_5+{\nu }_8$) and a few other bands for carboxylated Hb (${\mathrm{Fe}}^{2+}$) are exhibited.⁸ The oxygenation degree of the samples can be monitored using the oxidation state marker bands (${\nu }_4$) in the ranges 1355 to $1380{\mathrm{cm}}^{-1}$ and 1500 to $1650{\mathrm{cm}}^{-1}$, which are sensitive to the spin and coordination state of the heme. To estimate the Hb oxygenated state we chose the intense and accentuated bands, which serve as reference for characterizing the low spin state of heme. In the deoxygenated RBCs the principal band is used at 1604 to $1608{\mathrm{cm}}^{-1}$, while for the oxygenated cells the bands used appear at 1638 to $1640{\mathrm{cm}}^{-1}$ and 1586 to $1588{\mathrm{cm}}^{-1}$. The intensity of the band at $755{\mathrm{cm}}^{-1}$ (${\nu }_{15}$, a pyrrole breathing mode) does not depend on Hb oxygenation state and serves as a reference point for the estimation of the degree of oxygenation.^37,38 Figure 5 shows the Raman spectra of a single RBC with 100 nm ND at various oxygenation states during the process of deoxygenation (with $N_2$ purging), which is further followed by oxygenation (in air).

Fig. 5

Raman spectra of RBC at various oxygenation states. Raman spectra are measured in-situ in the oxygenation/deoxygenation process of a single RBC cell. RBC interacting with 100 nm cND are gradually deoxygenated and then re-oxygenated by purging $N_2$ through medium (PBS, $\mathrm{pH}=7.4$) of RBC suspension. The Raman spectra were obtained with 532 nm wavelength laser excitation, laser power $\sim 1\mathrm{mW}$, and a $60\times$ water immersion objective. The nanodiamond concentration used was $20\mathrm{\mu g}/\mathrm{ml}$. The red boxes are indicating some affecting Raman peaks at various oxygenation degrees.

The cycle for deoxygenation followed by oxygenation is shown in Fig. 6. The oxygenation-deoxygenation cycle is characterized using the intensity ratio of peaks from $1640{\mathrm{cm}}^{-1}$ to $755{\mathrm{cm}}^{-1}$. In the measurements shown in Fig. 6(a) and 6(b), $20\mathrm{\mu g}/\mathrm{ml}$ of cND were used, and the curve for RBC sample is marked as solid squares, while the curve for RBC interacting with 100 nm [Fig. 6(a)] and 5 nm [Fig. 6(b)] cND is shown as red, solid circles. The intensity ratios are plotted as a function of oxygenation/deoxygenation time. It can be seen that the 100 nm cNDs does not greatly affect the deoxygenation dynamics of the RBC. However, some delay of the oxygenation is observed for the deoxygenated RBC with 100 nm cND, and the effect is more significant for 5 nm cND. Figure 6(c) shows that this effect of 100 nm cND on the RBC oxygenation/deoxygenation cycle is concentration dependent.

Fig. 6

Oxygenation dynamics of the RBC interacting with ND (A). The time dependence of the deoxygenation followed by oxygenation of (a) RBC (black squares) and RBC interacting with 100 nm cND (red dots); (b) RBC (black squares) and RBC interacting with 5 nm cNd (red dots). The concentrations of cND used was $20\mathrm{\mu g}/\mathrm{ml}$. (c) The concentration dependence of the deoxygenation and following oxygenation of (a) RBC (black squares) and RBC interacting with 100 nm cND in different concentrations (○): $20\mathrm{\mu g}/\mathrm{ml}$; (□): $100\mathrm{\mu g}/\mathrm{ml}$; (◊): $1000\mathrm{\mu g}/\mathrm{ml}$.

Analogous to Fig. 6, the oxygenation-deoxygenation cycle of RBC and RBC interacting with cND is characterized using the intensity ratio of peaks for oxygenated state at $1588{\mathrm{cm}}^{-1}$, and for deoxygenated state at $1555{\mathrm{cm}}^{-1}$ (attributed as ${\nu }_{19}$ or ${\nu }_{37}$).³⁷ The dependencies of the peak ratio $[\mathrm{PR}=I_{1588}/(I_{1555}+I_{1588})]$ on time, during deoxygenation followed by re-oxygenation, are presented in Fig. 7. Fittings were done using standard second order polynomial fit. From the detailed analysis, the result of this fit was found to be similar for the relations between the intensities of peaks at $1606{\mathrm{cm}}^{-1}$ to $1640{\mathrm{cm}}^{-1}$ (${\nu }_{10}$), 1212 to $1224{\mathrm{cm}}^{-1}$, (${\nu }_{13}$ or ${\nu }_{42}$), and 1358 to $1372{\mathrm{cm}}^{-1}$ (${\nu }_4$). It is to be mentioned that the degree of oxygenation (${\mathrm{SO}}_2$) can be expressed as ${\mathrm{SO}}_2=A\times \mathrm{PR}+B$, where $A$ and $B$ are the constants characterizing the conditions of the process; the details are mentioned elsewhere.³⁷ From Fig. 7, it also can be seen that some delay of the oxygenation of deoxygenated RBC is observed at the presence of cND, this delay being more significant for 5 nm cND and deeper deoxygenation.

Fig. 7

Oxygenation dynamics of the RBC interacting with ND (B). The peak intensities of the Raman bands for oxygenated ($I_{\mathrm{oxy}}$ at $1588{\mathrm{cm}}^{-1}$) and deoxygenated ($I_{\mathrm{deoxy}}$ at $1555{\mathrm{cm}}^{-1}$) are compared. The peak ratio (PR) was calculated using $[PR=I_{1588}/(I_{1555}+I_{1588})]$, and the dependencies of PR on time during deoxygenation and following re-oxygenation were fitted using standard second order polynomial fit. The results of fitting are shown: (a) $I-\text{RBC}$; $\mathrm{II}-\text{RBC}+100\mathrm{nm}$ ND; (b) $I-\text{RBC}$; $\mathrm{II}-\text{RBC}+5\mathrm{nm}$ cND; (c) $I-\text{RBC}$; $\mathrm{II}-\text{RBC}+5\mathrm{nm}$ ND; (d) $I-\text{RBC}$; $\mathrm{II}-\text{RBC}+5\mathrm{nm}$ cND.

We have repeated the same experiments several times ($N>10$) and found the same results. Data presented in Figs. 6 and 7 are from one of the experiments without averaging, since the experiments depend on the flow rate of $N_2$ gas, for example. Details that caused the delay deserve further investigation.

To confirm the oxygenation and deoxygenation of RBC, UV-visible absorption spectra of RBC suspensions, shown in Fig. 8, were measured. The figure depicts the comparison of spectra of the oxygenated and deoxygenated RBCs at the interaction with 100 nm cNDs. In this experiment, RBCs are suspended in PBS solution, the concentration of RBC was $1\mathrm{\mu l}/\mathrm{ml}$ and concentration of 100 cND suspended in $\text{RBC}+\text{PBS}$ suspension was $100\mathrm{\mu l}/\mathrm{ml}$. Spectra from RBC and RBC with cND revealed similar characteristic peaks. When RBCs are oxygenated, corresponding absorbance peaks appear at 542 and 576 nm; and for deoxygenated RBC, the characteristic absorption is observed at 556 nm. It is to be noted that the absorption of cND is negligible in the visible range. The figure shows that the interaction of cND with RBC does not affect RBC (more precisely, Hb inside the studied RBC) spectra, as well as the Raman spectra shown in Fig. 4.

Fig. 8

Absorption Spectra of RBC interacting with cND. UV-visible absorption of $\mathrm{RBC}+\mathrm{PBS}$ suspension at their oxygenation and deoxygenation in-situ, with $N_2$ purging; comparison with RBC suspension with 100 nm cND. The concentration of RBC is $1\mathrm{\mu l}/\mathrm{ml}$ and concentration of 100 cND is $100\mathrm{\mu l}/\mathrm{ml}$.

The RBC membrane structure and state determine the membrane permeability for gases like ${\mathrm{O}}_2$ and ${\mathrm{CO}}_2$, and, correspondingly, the ability of RBC for gas transportation. On the other hand, the membrane properties determine the blood rheology. The membrane state and rheological blood properties can be modified by cND interaction with blood. In the microscopic study, the aggregation of the RBC in suspension with $\text{cND}+\text{RBC}$ has been detected. Note however, that these RBC aggregates have irregular character in contrast to rouleaux aggregation that appears for the RBC in the whole blood. The RBC aggregation is an intrinsic process and characterizes the blood properties as whole, first of all, the blood microcirculation. Usually the aggregation is observed in whole blood, with 2D- or 3D-rouleaux formation. In the aggregation of RBC where the blood plasma constituents are involved, first of all fibrinogen and other large plasma proteins,³⁹ low density lipoproteins, as well as large polymers like dextran in blood plasma play the major roles.^40,41 According to the bridging model of the aggregation, the macromolecules participate in the bridges formation; and according to the depletion model, RBC aggregation occurs as a result of a lower localized macromolecules concentration near the cell surface. It leads to osmotic gradient and thus depletion interaction. Electrical and mechanical properties of the membrane also are considered as factors of the aggregation.⁴² Thus, changes of the aggregation conditions may be connected with formation of intercellular contacts facilitated by surface aggregated cND, as well as with changes in the membrane properties.^43,44

The influence of cND on the RBC’s mechanical properties is observed via variations of the cell deformability related to its membrane rigidity, which depends on the membrane structure and connects with the membrane functionality.^35,36,45,46 The dependencies of the cell deformability on the shear stress for normal RBC and RBC incubated with 100 nm cND in different concentrations are shown in Fig. 9. It can be seen from the figure that the deformability is decreased when RBCs interact with cND. The decrease in cell deformability (i.e., increasing stiffness or decrease in membrane fluidity) is seen to be more significant for the higher cND concentration. It can be seen that the membrane deformability changes due to the presence of cNDs; thereby the structure of the membrane may change. In the following paragraphs we discuss two issues; first, which structural changes of the membrane can be the reason for the variations in the oxygenation-deoxygenation dynamics, and second, why the effect of cND on oxygenation-deoxygenation cycle of RBCs looks more significant for deoxygenated RBC on the stage of beginning of oxygenation.

Fig. 9

Nanodiamond effect on RBC deformability. Dependences of deformability index of RBC on shear stress: (black square) control RBC suspension; (red dot) RBC with 100 nm cND, concentration $33\mathrm{\mu g}/\mathrm{ml}$; (blue triangle) RBC with 100 nm cND, concentration $100\mathrm{\mu g}/\mathrm{ml}$. The dotted data are mean values with root mean square deviations after averaging over $n=8$ blood samples from different individuals. The continuous curves are splines. Measurements were performed within two hours after blood drawing and after 1 h-long incubation of whole blood with cND.

The plasma membrane of erythrocytes plays a fundamental role in oxygen transport and in maintaining RBC metabolism.³⁵ The membrane structural changes created by cND interaction affect the RBC function, and thus can be reasons of the variations in the oxygenation-deoxygenation dynamics. Generally, it has been accepted that the oxygen can penetrate the RBC membrane via direct diffusion. In this case, the membrane permeability for oxygen is limited by the membrane molecules mobility. The mobility was determined by lipids constituting the membrane lipid bilayer with liquid-crystal-like structure, in which the membrane proteins are incorporated. The cND aggregation on the membrane can decrease the mobility of lipid molecules in the area of contact with cND surface as well as in surrounding areas due to some ordering of liquid-crystal structure of the membrane. One could assume that it decreases the oxygen transmembrane transport and correspondingly the rate of oxygenation and deoxygenation in RBC, although some doubts exist concerning the role of direct diffusion permeability of the RBC membrane for oxygen.^36,46 The role of channels and pores in the oxygen permeability is discussed, e.g., it was suggested that RBC membrane can modulate the function of hemoglobin, but the details are still not clear and incomplete.⁴⁷

The interaction of proteins with ND may alter their structure and activity.^33,48,49 Presumably, if ND interacts extensively with the membrane proteins, it can modify the structure and functionality, including transport proteins involved in oxygen transport. As a result, interaction of ND with the membrane alters oxygen and other important substances concentrations in the RBC cytoplasm. Additionally, it has been already shown that the organic phosphate, i.e., 2,3-diphosphoglycerate (2, 3 DPG) in the RBC, binds with Hb (with $\beta$-sheets) and affects the Hb affinity to oxygen (oxygen binding capacity), facilitating the detaching ${\mathrm{O}}_2$ from Hb molecule.³⁵ It is important that 2, 3 DPG is involved in the interaction of Hb with membrane, particularly with the band three protein. Thus, varying membrane state can vary conditions of 2, 3 DPG interaction with the membrane; and correspondingly, the interaction between Hb and 2, 3 DPG changes. So, the predominant affinity of 2, 3 DPG to deoxy-Hb relatively oxy-Hb may be one of the reasons why the observed effect of cND on the oxygenation-deoxygenation cycle differs for different oxygenation states.

Connection between the oxygenation-deoxygenation of RBC and the RBC mechanical properties has been discussed⁵⁰ with consideration of RBC cell membrane fluctuations (CMF). They were observed involving local cell membrane displacements and bending deformability of the cell membrane. RBC deoxygenation results in a decrease of the CMF; when deoxygenated cells (revealing a small amplitude of displacement) are re-oxygenated, their amplitude of fluctuation is restored. The CMF are shown strongly metabolically dependent, and they are correlated with oxygenation-deoxygenation cyclic changes in metabolite concentrations. It is shown that hemoporphirine conformation (strongly connected with Hb’s ability to bind and release the ligands) is affected by Hb binding to the inner surface of erythrocyte membrane and membrane fluidity.³⁶ One can suppose that when the nanoparticle adsorption on the membrane increases the membrane rigidity, probably, the membrane transformation into a state with higher CMF can be obstructed. In turn, it may delay the beginning of re-oxygenation. Thus, probable mechanisms, involved in the interaction of ND with RBC and determining the ND influence on RBC are proposed. However, complete understanding needs further investigations in the same direction.

4.

Conclusion

In conclusion, interaction of 5 and 100 nm cNDs with RBCs has been studied. We show that the 100 nm cNDs are localized around RBCs, whereas 5 nm cND penetrate inside the RBCs. The 5 and 100 nm sized cND with 5 and $50\mathrm{\mu g}/\mathrm{ml}$ concentrations are nontoxic for RBCs. The hemolytic effect of cND does not exceed 2 to 4% with respect to 0.5% TritonX 100, which for Triton was normalized to 100%. Raman spectra for both sized cND do not show any transformations for the RBCs, thereby signifying no structural transformations in Hb. It is observed that the 5 and 100 nm cND does not affect significantly the deoxygenation dynamics. However some delay of the oxygenation is observed for the RBC deoxygenated in the presence of 100 nm cND and is concentration dependent. On the other hand, RBC with 5 nm sized cND show deeper deoxygenation followed by observably more delay of the oxygenation for deoxygenated RBC. Similar to Raman results, UV-visible absorption spectra confirmed that the interaction of cND with RBC does not affect Hb oxygenation state inside the studied RBC. Deformability analysis showed that deformability of RBCs decreases significantly for higher concentration of 100 nm cND. Finally, it is shown that 100 nm cND interact with the RBCs and can be used to study and manipulate the RBC’s membrane properties.