Neutral liposomes as long-term antigen-releasing
activating macrophages nanoparticle vaccine carriers
Author: Timo Diederik s2496372
Co-authors: Pim Bastiaansen, Manuel Goncalvez, Marit van Arkel, Imke Nelen, Fleur Koutstaal, Joëlle
Zonneveld
Abstract
The concept of vaccination has been used for over 200 years. However, the concept of vaccination
has changed over the years. From administering an inactive or weakened virus to a less risky
antigen vaccine, a so called subunit vaccine. However, these types of vaccines do not induce a
high immunogenic response due to the relative size compared to viruses. Often an adjuvant is used
to stimulate the immunogenic response. Nanoparticles due to their similar virus-size are adjuvants.
The aim of this study is to evaluate four different kind of nanoparticles on their efficiency as
vaccine carriers, namely cationic, neutral and anionic liposomes as well as polymeric-based PLGA
particles. In order to study this, cell viability, macrophage activation, a release study and DLS
measurements were performed. Results showed that neutral liposomes are the most efficient as
vaccine carriers.
glycolide) polymer particles (PLGA) and
Introduction cationic, neutral and anionic liposomes. PLGA
nanoparticles have been reported to cross the
The first ever use of vaccination was blood-brain barrier and peptides or other
introduced back in 1796 by Edward Jenner.1 linkers can be attached on the surface to
The first traditional vaccines contained provide targeted delivery.7 The hydrolysed
inactive or weakened viruses. However, some products of PLGA are easily metabolized by
vaccines contained active viruses leading to the body.8 Liposomes are composed of one or
huge amounts of infection rates.2 Luckily, the more phospholipid bilayers and can also be
knowledge in molecular biology increased and loaded with proteins or peptides. They can be
subunits vaccines were developed in 1986.3 modified in a high variety of different lipid
These vaccines used individual proteins that composition changing the size and charge of
were part of viruses, reducing the risk on the particles.9
unwanted side-effects. However, these proteins
do not always activate an immune response The aim of this paper is to determine the
due to their relative small size compared to efficiency of the four nanoparticles as a
viruses. One way to overcome this is to add an vaccine carrier. Nanoparticles were loaded
adjuvant or encapsulate antigens in a with ovalbumin to mimic the antigen protein of
nanoparticle to stimulate the immune a virus and a dynamic light scattering assay
response.4,5 The use of nanoparticles not only was performed to determine the size, poly
increases the efficiency in vaccines but it also dispersity index (PDI) and zeta-potential.
reduces the toxicity of certain drugs resulting Furthermore, a release study was performed
in higher use for multiple purposes such as the together with a BCA-assay in order to
treatment for cancer.6 However, the determine the amount of antigen released at
characteristics of nanoparticles is greatly different timestamps. In addition a SDS-gel
dependent on the formulation of the particles. electrophoresis was performed to assure the
purity of the model antigen ovalbumin inside
This paper focuses on four kinds of the loaded particles. Lastly, the cell viability
nanoparticles, namely poly(D,L-lactide-co- was evaluated using a MTT-assay to determine
, the toxicity of the nanoparticles and also the Control particles were made using the same
macrophage activation on all particles was procedure but instead of using 50 µL of an
tested using a nitrite-assay. ovalbumin-solution, 50 µL of MilliQ was used.
Previous research has shown that cationic Preparation of the liposomes
liposomes are more potent than anionic and
neutral liposomes.10 However, they also seem Three different lipid-compositions all with 250
to be more toxic by inducing reactive oxygen µg of cholesterol (Sigma Aldrich) were made
species.11 Anionic liposomes have been using the following lipids:
reported with more effective and efficient Dimethyldioctadecylammonium (DDA), 1,2-
delivery.12 Lastly, PLGA particles seem less dioleoyl-sn-glycero-3-phosphocholine
capable as a vaccine carrier due to their short (DOPC), 1,2-dioleoyl-sn-glycero-3-
circulation life and high elimination rate due to phosphoethanolamine (DOPE) and 1,2-
their hydrophobic surface.13 However, PLGA dioleoyl-sn-glycero-3-phospho-L-serine
particles do induce a long-time effect due to (DOPS). All lipids were obtained at Avanti
their slow-release of loaded material.14 Overall, Polar Lipids. The composition of the
anionic liposomes seem to be generally the liposomes with these lipids are shown in Table
best compared to the other particles. 1
Dependent of the purposes of the vaccine
Table 1. Composition of the lipids used
either PLGA or anionic particles are the most for the liposomes.
efficient for vaccine carrier. Since PLGA DDA DOPC DOPE DOPS
induces a long-term effect it may be more Cationic 250 1500 - -
suitable for the use of a vaccine compared to µg µg
the anionic liposomes. Neutral - 1150 600 -
µg µg
Materials and methods Anionic - 1500 - 250
µg µg
Preparation of the PLGA nanoparticles
The lipid-composition was evaporated in a
25 mg of PLGA polymer (Sigma Aldrich) was rotary film evaporator at 60 C, 160 mbar and
added to 1 mL dichloromethane (Sigma 80 rpm. After full evaporation of the
Aldrich). The resulting mixture was added to chloroform, glass pearls were added and it was
50 µL of ovalbumin-solution (40 mg/mL, rehydrated by adding 500 µL ovalbumin (0.25
Sigma Aldrich) and was sonicated at 20 W for mg/mL in 10 mM of HEPES buffer pH=7.4).
15 seconds. 2 mL of 2% (w/v) PVA-solution The solution was put back in the rotary film
(Sigma Aldrich) was added drop by drop and evaporator at 1000 mbar until a white
once again the solution was sonicated at 20 W substance was formed. The collected samples
for 15 seconds. The emulsion was added to 25 were freeze-dried overnight. 500 µL of MilliQ
mL of 0.3% (w/v) PVA at 40 C and the was added to the freeze-dried samples and
organic layer was evaporated. In order to were sonicated at 20 W for 10 seconds. The
purify the particles, the remaining layer was samples were filtered in a vivaspin colom to
centrifuged at 10.000 rpm for 10 minutes and remove excess ovalbumin.
washed with MilliQ before returning into the
Control liposomes were made using the same
centrifuge for 30 minutes at 13.200 rpm. The
procedure but instead of using 500 µL of
pellets were dissolved into 1 mL of MilliQ and
ovalbumin, 500 µL of MilliQ was used.
were freeze-dried overnight and the samples
Furthermore, the control liposomes have not
were finally dissolved in MilliQ to achieve a
been filtered in a vivaspin.
concentration of 50 mg/mL.