Hemocompatibility of dextran-graft-polyacrylamide/zinc oxide nanosystems: hemolysis or eryptosis?

Abstract

Abstract Aim . In this study, blood compatibility of ZnO nanoparticles-polymer nanocomplex (D-PAA/ZnONPs( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" overflow="scroll"> <mml:msup> <mml:msub> <mml:mrow> <mml:mi mathvariant="normal">SO</mml:mi> </mml:mrow> <mml:mrow> <mml:mn>4</mml:mn> </mml:mrow> </mml:msub> <mml:mrow> <mml:mn>2</mml:mn> <mml:mo>−</mml:mo> </mml:mrow> </mml:msup> </mml:math> )) synthesized in situ into dextran-graft-polyacrylamide (D-PAA) using zinc sulphate as a precursor was tested using hemolysis, osmotic fragility and eryptosis assays. Materials and methods . Dose-dependent ability to induce eryptosis was assessed following 24 h incubation at concentrations of 0–800 mg l −1 analyzing hallmarks of eryptosis (cell shrinkage and phosphatidylserine externalization), as well as reactive oxygen species generation. Hemolysis was detected spectrophotometrically based on hemoglobin release following exposure to the D-PAA/ZnONPs( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" overflow="scroll"> <mml:msup> <mml:msub> <mml:mrow> <mml:mi mathvariant="normal">SO</mml:mi> </mml:mrow> <mml:mrow> <mml:mn>4</mml:mn> </mml:mrow> </mml:msub> <mml:mrow> <mml:mn>2</mml:mn> <mml:mo>−</mml:mo> </mml:mrow> </mml:msup> </mml:math> ) nanocomplex. Osmotic fragility test (OFT) involved detection of hemolysis of red blood cells exposed to 0.2% saline solution following incubation with the D-PAA/ZnONPs( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" overflow="scroll"> <mml:msup> <mml:msub> <mml:mrow> <mml:mi mathvariant="normal">SO</mml:mi> </mml:mrow> <mml:mrow> <mml:mn>4</mml:mn> </mml:mrow> </mml:msub> <mml:mrow> <mml:mn>2</mml:mn> <mml:mo>−</mml:mo> </mml:mrow> </mml:msup> </mml:math> ) nanocomplex. Additional incubation of the nanocomplex in the presence or absence of either ascorbic acid or EGTA was used to reveal the implication of oxidative stress- or Ca 2+ -mediated mechanisms in D-PAA/ZnONPs( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" overflow="scroll"> <mml:msup> <mml:msub> <mml:mrow> <mml:mi mathvariant="normal">SO</mml:mi> </mml:mrow> <mml:mrow> <mml:mn>4</mml:mn> </mml:mrow> </mml:msub> <mml:mrow> <mml:mn>2</mml:mn> <mml:mo>−</mml:mo> </mml:mrow> </mml:msup> </mml:math> ) nanocomplex-induced erythrotoxicity. Results . Hemocompatibility assessment of the D-PAA/ZnONPs( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" overflow="scroll"> <mml:msup> <mml:msub> <mml:mrow> <mml:mi mathvariant="normal">SO</mml:mi> </mml:mrow> <mml:mrow> <mml:mn>4</mml:mn> </mml:mrow> </mml:msub> <mml:mrow> <mml:mn>2</mml:mn> <mml:mo>−</mml:mo> </mml:mrow> </mml:msup> </mml:math> ) nanocomplex revealed that it induced hemolysis and reduced resistance of erythrocytes to osmotic stress at concentrations of above 400 and 200 mg l −1 , respectively. Oxidative stress- or Ca 2+ -mediated mechanisms were not involved in D-PAA/ZnONPs( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" overflow="scroll"> <mml:msup> <mml:msub> <mml:mrow> <mml:mi mathvariant="normal">SO</mml:mi> </mml:mrow> <mml:mrow> <mml:mn>4</mml:mn> </mml:mrow> </mml:msub> <mml:mrow> <mml:mn>2</mml:mn> <mml:mo>−</mml:mo> </mml:mrow> </mml:msup> </mml:math> ) nanocomplex-induced hemolysis. Strikingly, the D-PAA/ZnONPs( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" overflow="scroll"> <mml:msup> <mml:msub> <mml:mrow> <mml:mi mathvariant="normal">SO</mml:mi> </mml:mrow> <mml:mrow> <mml:mn>4</mml:mn> </mml:mrow> </mml:msub> <mml:mrow> <mml:mn>2</mml:mn> <mml:mo>−</mml:mo> </mml:mrow> </mml:msup> </mml:math> ) nanocomplex did not promote cell membrane scrambling, cell shrinkage and oxidative stress in red blood cells following the direct exposure for 24 h. Thus, the D-PAA/ZnONPs( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" overflow="scroll"> <mml:msup> <mml:msub> <mml:mrow> <mml:mi mathvariant="normal">SO</mml:mi> </mml:mrow> <mml:mrow> <mml:mn>4</mml:mn> </mml:mrow> </mml:msub> <mml:mrow> <mml:mn>2</mml:mn> <mml:mo>−</mml:mo> </mml:mrow> </mml:msup> </mml:math> ) nanocomplex did not induce eryptosis in vitro . Eryptosis is generally considered to occur earlier than hemolysis in response to stress in order to prevent hemolytic cell death. Counterintuitively, our data suggest that hemolysis can be triggered by nanomaterials prior to eryptosis indicating that eryptosis and hemolysis assays should be used in combination for testing blood compatibility of nanomaterials. Conclusions . The D-PAA/ZnONPs( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" overflow="scroll"> <mml:msup> <mml:msub> <mml:mrow> <mml:mi mathvariant="normal">SO</mml:mi> </mml:mrow> <mml:mrow> <mml:mn>4</mml:mn> </mml:mrow> </mml:

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