The primary objective of this study is to examine whether the in vitro cellular transfection efficiency of PF14-mRNA nanoparticles is higher in medium supplemented with plasma or serum.
ID
Source
Brief title
Condition
- Other condition
Synonym
Health condition
Geen: het betreft fundamenteel onderzoek naar de werking van PepFect14-mRNA nanodeeltjes
Research involving
Sponsors and support
Intervention
Outcome measures
Primary outcome
The main parameters are the Cy5-mRNA uptake and EGFP expression after
transfection of cells with PF14-EGFP mRNA or PF14 with Cy5-labeled EGFP mRNA,
in the presence of plasma or serum. Cy5 and EGFP levels will be measured by
confocal laser scanning microscopy (CLSM).
Secondary outcome
N.a.
Background summary
Cell penetrating peptides (CPPs) are a potential novel platform for mRNA-based
therapies. An example of a specific cell penetrating peptide is PepFect14
(PF14), which is a polycationic oligopeptide that form nanoparticles with RNA
based on electrostatic interactions. After entering a cell, this RNA can be
released from the nanoparticles and subsequently translated by the cellular
protein synthesis machinery.
As of now, research into PF14-based mRNA delivery is still being conducted on
various delivery strategies, transfection efficiency and cytotoxicity, which is
takes place in a laboratory setting. In in vitro experiments in which cells are
transfected with PF14-mRNA, the cells are cultivated in medium supplemented
with 10% serum to provide necessary nutrients and growth hormones. However,
serum could contain endonucleases and serine proteases, which could be
detrimental for the PF14-mRNA nanoparticles since they consist largely of
oligopeptides and RNA. In contrast, plasma does not contain these activated
enzymes, and therefore we hypothesize that cellular transfection with PF14-mRNA
nanoparticles is more successful in medium supplemented with plasma than with
serum. In addition, studying the mRNA delivery in plasma instead of serum is
more physiologically relevant.
In order to qualitatively compare the transfection efficiency in the presence
of plasma or serum, we want to draw a small amount blood from a healthy
individual in order to collect fresh plasma. This way, we can avoid working
with plasma to which anticoagulants like EDTA, citrate or heparin have been
added, because of their cytotoxicity or detrimental effect on the PF14-mRNA
particles. Instead, we will use the anticoagulant aprotinin.
Study objective
The primary objective of this study is to examine whether the in vitro cellular
transfection efficiency of PF14-mRNA nanoparticles is higher in medium
supplemented with plasma or serum.
Study design
In vitro experimental study: collection of blood from a healthy donor for use
in in vitro research
Study burden and risks
The medical intervention, a venapuncture, is a standard procedure and causes
only minor inconvenience and negligible risks for the subject. In addition, the
procedure takes a very short period of time.
Geert Grooteplein Zuid 28
Nijmegen 6525GA
NL
Geert Grooteplein Zuid 28
Nijmegen 6525GA
NL
Listed location countries
Age
Inclusion criteria
Healthy indivuals, 18-60 years of age, bodyweight: above 50 kg. Being able and
willing to complete the informed consent process.
Exclusion criteria
Known to have a condition which results in more than minimal risk upon blood
drawing, e.g. hemophilia.
A medical condition which requires active medical intervention or monitoring to
avert serious danger to the individual's health or well-being.
Design
Recruitment
Followed up by the following (possibly more current) registration
No registrations found.
Other (possibly less up-to-date) registrations in this register
No registrations found.
In other registers
| Register | ID |
|---|---|
| CCMO | NL77254.091.21 |