Pharmaceutical Research

, Volume 31, Issue 11, pp 3127–3135 | Cite as

Hemocompatibility Assessment of two siRNA Nanocarrier Formulations

  • Afrouz Yousefi
  • Marianne Lauwers
  • Reka Nemes
  • Thijs van Holten
  • Negar Babae
  • Mark Roest
  • Gert Storm
  • Raymond Schiffelers
  • Enrico Mastrobattista
Research Paper
First Online:
Received:
Accepted:

Abstract

Purpose

Since the discovery of RNAi and its therapeutic potential, carrier systems have been developed to deliver small RNAs (particularly siRNA) for modulation of gene expression at the post-transcriptional level. An important factor determining the fate and usability of these systems in vivo is interaction with blood components, blood cells, and the immune system. In this study, a lipid-based and a polymer-based carrier system containing siRNA have been investigated in vitro in terms of their hemocompatibility.

Methods

The nanocomplexes studied were Angiplex, a targeted lipid-based system, and pHPMA-MPPM polyplex, a formulation based on a cationic polymer. siVEGFR-2 was encapsulated in both carriers and activation of platelets, coagulation, and complement cascade as well as induction of platelet aggregation were evaluated in vitro.

Results

Both systems had been shown before to cause significant silencing in vitro. Our findings indicated that pHPMA-MPPM polyplex triggered high platelet activation and aggregation although it did not stimulate coagulation substantially. Angiplex, on the other hand, provoked insignificant activation and aggregation of platelets and activated coagulation minimally. Complement system activation by Angiplex was in general low but stronger than pHPMA-MPPM polyplex.

Conclusions

Taken together, these in vitro assays may help the selection of suitable carriers for systemic delivery of siRNA in early preclinical investigations and reduce the use of laboratory animals significantly.

Key words

Coagulation Complement activation Hemocompatibility Platelet activation siRNA delivery system 

Abbreviations

CHEMS

Cholesteryl hemisuccinate

C system

Complement system

DOPE

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

DSPE-PEG2000

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene-glycol)] with PEG molecular weight of 2,000 g.mol−1

DSPE-PEG2000-Mal

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000]

HBG

20 mM HEPES buffer containing 5% glucose at pH 7.4

HBS

20 mM HEPES and 150 mM NaCl

MPV

Mean platelet volume

PC

1,2-dioleyl-sn-glycero-3-phosphocholine

PE

1,2-dioleyl-sn-glycero-3 phosphoethanolamine

PGI2

Prostacyclin

pHPMA-MPPM

Poly((2-hydroxypropyl) methacrylamide 1-methyl-2-piperidine methanol

PS

1,2-dioleyl-sn-glycero-3-phospho-L-serine

HT

HEPES-Tyrode

SATA

N-succinimidyl S-acetylthioacetate

siVEGFR-2

Human siRNA against VEGFR2

TF

Tissue factor

TRAP-6

Thrombin receptor activator peptide 6

VEGFR2

Vascular endothelial growth factor receptor-2

vWF

Von Willebrand factor

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Notes

Acknowledgments and Disclosures

This research was financially supported by the Dutch Top Institute Pharma project T3-301.

Supplementary material

11095_2014_1405_Fig6_ESM.jpg (302 kb)
Supplementary Figure 1

Effect of concentration of lipoplexes on platelet activation after 1, 10 and 20 minutes of incubation with isolated platelets represented by mean fluorescence intensity (MFI) of (A) FITC-conjugated anti-fibrinogen and (B) PE-conjugated anti P-selectin (n = 3). siRNA concentrations were 0.5, 2, 5, 10 μg/ml. All values are subtracted from blank. (JPEG 301 kb)

11095_2014_1405_Fig7_ESM.jpg (290 kb)
Supplementary Figure 1

Effect of concentration of lipoplexes on platelet activation after 1, 10 and 20 minutes of incubation with isolated platelets represented by mean fluorescence intensity (MFI) of (A) FITC-conjugated anti-fibrinogen and (B) PE-conjugated anti P-selectin (n = 3). siRNA concentrations were 0.5, 2, 5, 10 μg/ml. All values are subtracted from blank. (JPEG 301 kb)

11095_2014_1405_Fig8_ESM.jpg (300 kb)
Supplementary Figure 2

Effect of concentration of lipoplexes on platelet activation after 1, 10, and 20 minutes of incubation with whole blood represented by mean fluorescence intensity (MFI) of (A) FITC-conjugated anti-fibrinogen and (B) PE-conjugated anti P-selectin (n = 3). siRNA concentrations were 0.5, 2, 5, 10 μg/ml. All values are subtracted from blank. (JPEG 299 kb)

11095_2014_1405_Fig9_ESM.jpg (351 kb)
Supplementary Figure 2

Effect of concentration of lipoplexes on platelet activation after 1, 10, and 20 minutes of incubation with whole blood represented by mean fluorescence intensity (MFI) of (A) FITC-conjugated anti-fibrinogen and (B) PE-conjugated anti P-selectin (n = 3). siRNA concentrations were 0.5, 2, 5, 10 μg/ml. All values are subtracted from blank. (JPEG 299 kb)

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Afrouz Yousefi
    • 1
  • Marianne Lauwers
    • 2
  • Reka Nemes
    • 1
  • Thijs van Holten
    • 3
  • Negar Babae
    • 1
  • Mark Roest
    • 3
  • Gert Storm
    • 1
  • Raymond Schiffelers
    • 3
  • Enrico Mastrobattista
    • 1
  1. 1.Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Faculty of ScienceUtrecht UniversityUtrechtThe Netherlands
  2. 2.Department of Pharmaceutics, Faculty of Pharmaceutical SciencesGhent UniversityGhentBelgium
  3. 3.Department of Clinical Chemistry and HematologyUniversity Medical Center UtrechtUtrechtThe Netherlands

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