Abstract
Erythrocytes have been extensively investigated as drug delivery vehicles. However, many existing methods for loading therapeutic biomolecules into erythrocytes perturb the cell membrane, resulting in a loss of erythrocyte structural integrity and recognition by the host immune system. To overcome this challenge, we developed a novel nanoparticle carrier for transporting proteins into intact erythrocytes. We designed and characterized a nanoparticle where a poly-L-lysine-grafted-poly(ethylene) glycol copolymer was used to encapsulate a model protein, bovine serum albumin. The nanoparticle was functionalized with either an erythrocyte targeting peptide ERY1, the cell-penetrating peptide low molecular weight protamine, or a combination of the two peptides. The three resulting constructs, ENP, LNP, and ELNP, had a core/shell structure and, depending on functionalization, were approximately 30–90 nm in diameter and had negative surface charge. Esterase-like activity of the encapsulated protein was not affected by encapsulation. Under physiological conditions, nanoparticles caused no significant hemolysis and were resistant to dissociation by surfactants. The three nanoparticle constructs were evaluated using confocal microscopy and flow cytometry for their ability to bind and internalize within intact erythrocytes. LNPs showed significant binding and internalization into intact erythrocytes, with ~90% of erythrocytes containing particles after 24 h of co-incubation. Internalization occurred without adversely affecting erythrocyte membrane structure. Together, these results demonstrate that low molecular weight protamine–functionalized nanoparticles may serve as a universal platform to deliver protein therapeutics into erythrocytes.
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Abbreviations
- BNP:
-
bare/untargeted nanoparticles
- BSA:
-
bovine serum albumin
- CPP:
-
cell-penetrating peptides
- DLS:
-
dynamic light scattering
- DMSO:
-
dimethyl sulfoxide
- ELNP:
-
erythrocyte targeting peptide and low molecular weight protamine coated nanoparticles
- ENP:
-
erythrocyte targeting peptide coated nanoparticles
- ERY1:
-
erythrocyte targeting peptide
- ESEM:
-
environmental scanning electron microscope
- EtBr:
-
ethidium bromide
- FACS:
-
fluorescence-activated cell sorting
- FBS:
-
fetal bovine serum
- FITC-BSA:
-
fluorescein isothiocyanate–labeled bovine serum albumin
- FRET:
-
fluorescence resonance energy transfer
- GFP:
-
green fluorescent protein
- GPA:
-
glycophorin A
- LMWP:
-
low molecular weight protamine
- LNP:
-
low molecular weight protamine coated nanoparticles
- MAL:
-
maleimide
- MAL-PEG-NHS:
-
maleimide polyethylene glycol succinimidyl carboxymethyl ester
- mPEG-NHS:
-
N-hydroxylsuccinimide functionalized polyethylene glycol
- NP:
-
nanoparticle
- PALS:
-
phase analysis light scattering
- PBS:
-
phosphate-buffered saline
- PEG:
-
polyethylene glycol
- PLL:
-
poly-L-lysine
- PLL-g-PEG:
-
poly-L-lysine-grafted-poly(ethylene) glycol
- SDS PAGE:
-
sodium dodecyl sulfate polyacrylamide gel electrophoresis
- SEM:
-
scanning electron microscope
- TEM:
-
transmission electron microscope
- TRITC:
-
tetramethylrhodamine
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Acknowledgements
We thank Marie Montelongo and the Oklahoma Center for Respiratory and Infectious Diseases Center (GM103648) for providing core-facility support and technical expertise.
Funding
This work was funded by contract HDTRA1-13-1-0021 from the Defense Threat Reduction Agency (DTRA) and the Oklahoma State University Interdisciplinary Toxicology Program.
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Hikkaduwa Koralege, R.S., Sahoo, K., Flynn, N. et al. Erythrocytes internalize nanoparticles functionalized with low molecular weight protamine. J Nanopart Res 23, 96 (2021). https://doi.org/10.1007/s11051-021-05202-8
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DOI: https://doi.org/10.1007/s11051-021-05202-8