Lipid nanoparticles are a promising delivery system for mRNA vaccines. They consist of four main components - ionizable lipid, helper lipid, cholesterol, and PEG lipid. Three common types are described: lipoplexes which have low stability and efficiency; lipid nanoparticles which have a core-shell structure and superior properties; and lipid-polymer hybrids which combine lipids and polymers. Key factors in lipid nanoparticle design include size, surface charge, encapsulation efficiency, and lipid ratios. Various preparation methods can be used but microfluidic mixing provides high reproducibility. Characterization of size, stability, and encapsulation efficiency is important for mRNA delivery optimization.

1. Lipid nanoparticles for mRNA vaccines
Presented by
Kaliannan Durairaj

2. Introduction
Colloidal particles ranging in size between 10 & 1000 nm are known as
nanoparticles
Types of nanoparticle as carrier for drug & diagnostic agents
Polymeric Nanoparticle
Nanocrystals
Polymeric micelles Ceramic NPS liposome
Fullerenes and dendrimers
Lipid nanoparticles
Magnetic nanoparticles
Gold Nanoshells
Carbon nanotubes
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3. Lipid-based Nanoparticles for mRNA Delivery
https://doi.org/10.3389/fchem.2020.589959
3

4.  It consists of cationic liposomes interacting with mRNA.
 This is the earliest lipid-based delivery system successfully employed to introduce
mRNA into target cells.
Major important concerns :
 Stability - Low
 Transfection efficiency - Low
 Poor customizable composition,
 Often formulated with an excess of cationic lipids not only to promote mRNA
binding but also to facilitate the interaction with the anionic phospholipids in the
plasma membrane and subsequently promote its uptake by endocytosis
Lipoplex
Lipid-based Nanoparticles For mRNA Delivery
https://doi.org/10.3389/fchem.2020.589959
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5. Lipid nanoparticle
 It consists of four ingredients such as Phospholipid/Helper lipid, Chelosterol,
Ionizable lipid, and PEG lipid.
 The morphology of lipid nanoparticles is not like a traditional liposome,
characterized by a lipid bilayer surrounding an aqueous core, indeed, they possess
an electron-dense core, where the ionizable lipids are organized into inverted
micelles around the encapsulated mRNA
Major important concerns :
 Superior stability
 Enhancing delivery efficiency
Lipid-based Nanoparticles For mRNA Delivery
https://doi.org/10.3389/fchem.2020.589959
5

6. Lipid-polymer Hybrid nanoparticle
Lipid bilayer Lipid monolayer
 It consists of a mRNA loaded with biodegradable polymeric nanoparticles and liposomes
 The lipids are organized into a lipid bilayer or lipid monolayer containing a mixture of ionizable lipids, helper lipids, and PEG-
lipid.
Major important concerns :
 Small size, high nucleic acid condensation efficiency
 large functionalizable surface that can be easily modified by the binding of different functional groups
 In addition, specific physicochemical properties of nanoparticle are potentially consequence in a different interaction of the
delivered mRNA with innate RNA sensors, consequently altering the immunogenicity and safety profile of lipopolyplex-based
immunotherapies
 Currently these types of nanomaterial under investigation in early-phase clinical trials.
https://doi.org/10.3389/fchem.2020.589959
Lipid-based Nanoparticles For mRNA Delivery
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7. History for the development of lipid nanoparticle
Development
of liposomes
– mRNA
formulations
1978
Development
of Cationic
LNP – mRNA
formulations
1989
Development
of liposomes –
mRNA
formulations as
AI vaccine
1993
Clinical trial
of LNP-mRNA
for cancer
immunothera
pies
2014
Clinical trial of
LNP-mRNA for
AI vaccine &
protein
replacement
therapies
2017
Onpattro the
first siRNA drug,
was approved by
the FDA & the
EMA
2018
COVID-19 mRNA
vaccine obtained
authorized by
multiple
countries
2020
Encapsulated
LNPs were
approved by
the FDA
1995 - 2012
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8. 3. Ionizable lipid
1. Phospholipid/Helper lipid
Head
Tail
2. Cholesterol
4. PEG-lipid
Lipid nanoparticle composition and their role
8

9. Molar ratio
0%
25%
50%
75%
100%
Molar
ratio
PEG-lipid
Inoizable lipid
Cholesterol
Structural lipid/Phospholipid
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10. Ionizable lipid
Five major structural classes of Ionizable lipids
Unsaturated
(containing unsaturated bond)
Multi-tail
(containing more than two tails)
Polymeric
(containing polymer or dendrimer)
Biodegradable
(containing biodegradable bond)
Branched-tail
(containing branched tail)
These unsaturated ionizable lipid
does not always correspond with
potent in vivo RNA delivery,
indicating that both rational
design and screening are
necessary
Multi-tail ionizable lipids often
have stable backbones and low
degradability, so their toxicity and
immunogenicity should always be
taken into consideration.
Polymeric lipids are increase their
complexity. Moreover, the toxic
polycation core and non
degradable backbone pose extra
hurdles for clinical translation
Despite the promise of disulfide
bond-containing ionizable lipids,
the difficulty of synthesis
and risk of premature release of
cargo. Even though, it is a key
future for the clinical translation
of ionizable lipid.
Increased tail branching is one of
the major features investigated
by LNP companies
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11. The highlighted Ionizable
lipids used in on-going
clinical trials have not
been publicly disclosed.
List of available Ionizable lipids
https://doi.org/10.1038/s41467-021-27493-0
Biodegradable, Multi-tail
& unsaturated structure
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12. Structural/Helper lipid Cholesterol PEG-lipid
These lipids can improve nanoparticle properties,
such as particle stability, delivery efficacy,
tolerability and Biodistribution.
1,2-distearoyl-snglycero-3-phosphocholine
(DSPC)
Phosphatidylcholine
Phosphatidylethanolamine
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
(DOPE)
PEG2000-DMG
Other available lipids
ALC-0159
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13. Ionizable lipids PEG lipids Helper lipids Cholesterol Molar ratio
Pfizer-BioNTech ALC-0315 ALC-0159 DSPC Cholesterol 46.3 : 9.4 : 42.7 : 1.6
Moderna SM-102 PEG2000-DMG DSPC Cholesterol 50: 10: 38.5: 1.5
CureVac ALC-0315 PEG2000-DMG DSPC Cholesterol 50: 10: 38.5: 1.5
Arcturus Lipid 2,2 (8,8) 4C CH3 Cholesterol 50: 10: 38.5: 1.5
Imperial College London A9 Cholesterol 50: 10: 38.5: 1.5
Chulalongkorn CL1 Cholesterol NA
List of Covid-19 mRNA vaccines and their LNP components with molar ratio
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14. Lipid nanoparticle essential characterization for mRNA vaccine
Several factors should be considered when selecting lipids and how they are formulated into LNP
0%
25%
50%
75%
100%
Molar
ratio
PEG-lipid
Inoizable lipid
Cholesterol
Structural lipid/Phospholipid
Lipid molar ratio
Size
(~60-80nm)
Polydispersity index
(~0.15-0.2)
Surface charge
(Positive) Encapsulation efficiency
(>90%)
pKa
(~6-7)
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15. Lipid nanoparticle preparation methods
Microfluidic mixing
devices
Herringbone
T-/Y- Junction mixers
Solvent injection Hand mixing
Cost
Scalability
EE (%)
Reproducibility
PDI Low Medium High High
High Low Low Low
High High Medium Low
High High Medium Low
High High Medium Low
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16. Schematic diagram LNP preparation instrumental setup
Herringbone
T-/Y- Junction mixers

17. Encapsulation Efficiency (%) =
𝐃𝟎−𝐃𝟏
𝐃𝟎
𝐗 100
Where,
D𝟎 = Lysed LNP concentration (encapsulated mRNA in
the solution)
D1 = Not lysed LNP concentration (unencapsulated
mRNA in the solution)
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Experiment methodology for RiboGreen™ RNA Assay

18. E-mail: kmdurairaj@gmail.com
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