Croda Pharma

Innovative lipidsfor nucleic aciddeliveryLipid-based therapeutic delivery systems have beenevolving for many years. From delivering naked mRNA, tousing liposomes, and now lipid nanoparticles, Croda
Pharma and Avanti Polar Lipids are here to provide you with themost innovative lipids for your nucleic acid delivery research. In
this article discover considerations for each component in anLNP, as well as some new alternatives to commonly used LNP
components such as PEG-lipids and cholesterol.

The history and evolution of nucleic
acid delivery systemsmRNA and liposomes were both first
discovered in the 1960s, and liposomes
were used to deliver mRNA into
eukaryotic cells in 1978.1,2 Even with theability to inject mRNA into cells, there
were several technical challenges toovercome before mRNA could be used
as a therapeutic, primarily the rapid
degradation of mRNA once injected into
the body. In 1990, naked mRNA wasinjected into the muscles of rats toprove that in vivo direct gene transfer
was possible.3 As early as 1995 the firstmRNA vaccine carrying cancer antigens
was reported.4 And more recently, lipid
nanoparticles (LNPs) have been used
to deliver mRNA capable of fighting
SARS-CoV-2, as well as CRISPR/Cas9 gene editing technologies.
Our understanding of mRNA and its
delivery has evolved substantially in thepast few decades - from naked mRNAdelivery to liposomal delivery and thenLNPs. We are far from where we
started and probably equally as far
away, if not farther, from where we areheaded.
Not only do delivery systems change,

Smart science to improve lives™

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but their individual components areconstantly evolving to meet currentchallenges and demands. To date,
LNPs have primarily been formulatedusing the following components:
Cationic Lipids (DOTAP, ALC-0315,DLin-MC3-DMA, and SM-102)
PEG-lipid Conjugates (DSPEPEG2000, DMG-PEG2000, DSGPEG2000, and ALC-0159)
Phospholipids (DSPC and DOPE)
Cholesterol
Recent research has been directed at
finding new, innovative ways to improve
encapsulation and transfection
efficiency, and stability, as well as
reduce systemic toxicity of LNP
therapeutics. Keep reading to see how
LNP components are evolving andwhere LNP technology is headed!
Considerations for future cationiclipids
In LNP formulations, cationic ionizable
lipids are used to complex negatively
charged mRNA. Aside from playing amajor role in the LNP formulation itself,
cationic lipids also serve a major role inthe biological administration of mRNA totarget cells. The RNA-loaded LNP fuseswith the cell membrane and is thendelivered into the cytosol. To be able toplay these roles efficiently, a cationicionizable lipid must be engineered witha suitable apparent acid dissociation
constant (pKa). The apparent pKa of aSmart science to improve lives™

cationic ionizable lipid is the likely pKa
at the LNP surface. Currently, thecationic ionizable lipids in FDAapproved therapeutics all have anapparent pKa between 6-7. This is
crucial for the cationic ionizable lipid tomaintain a neutral charge while insystemic circulation (pH above the pKaof the lipid, pH ~7.5), as well as its
ability to become positively charged in
the endosome (pH ~6.5) and facilitate
membrane fusion and subsequentcytosolic release.5
As crucial components in LNPs, newcationic ionizable lipids for nucleic aciddelivery are being investigated to
optimize mRNA complexation,
endosomal membrane fusion and cargo
release into the cytosol.

PEGylated lipids and the future ofstealth agents in LNPs
Historically, polyethylene glycol (PEG)lipids have been the polymer-lipid
conjugate of choice for LNP
formulations. PEG-lipids are used toprevent proteins from binding to LNPs
and increase systemic circulation times,
mediate indirect targeting capabilities ofLNPs, promote LNP self-assembly, andcontrol LNP size and stability. Theability of LNPs formulated with PEGlipids to bypass the reticulo-endothelial
clearance system and stay in systemic
circulation gives them their nickname 5
stealth agents”.

As crucial components
in LNPs, new cationicionizable lipids fornucleic acid delivery
are being investigatedto optimize mRNA
complexation,
endosomal membranefusion and cargo
release into the cytosol.

As great as PEG-lipids are, they alsopresent a few challenges. These
challenges are commonly referred to as
the “PEG dilemma”: 6
Steric hindrance of long PEG chainshinders the ability of a cell to uptake
the therapeutic
PEGylation also hinders endosomal
escape of nanoparticles, leading todecreased activity of the delivery
systemRepeated administration of
PEGylated systems can result in aphenomenon called accelerated
blood clearance (ABC)
Other alternatives to PEG-lipids in LNPs
are being explored, and recent work
has identified polysarcosine (pSar)lipids as a promising alternative. pSarlipids, a polymer-lipid conjugate basedon the amino acid sarcosine, havedemonstrated similar stealth properties
to PEG-lipids. Previous studies havealso shown polysarcosine lipids toinduce a lower immunogenic responsethan PEG-lipids when administered torabbits, zebrafish embryos, and mice.
And most recently, pSar-lipids were
evaluated in mRNA-LNPs anddisplayed high RNA transfection ability
and an improved safety profile. Avanti is7
now offering pSar-lipids with varying
polymeric chain lengths and lipid chain
lengths for your PEG-free mRNAdelivery research!

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Polysarcosine-lipids
have demonstrated
similar stealthproperties to PEGylated
lipids and have been
evaluated in mRNALNPs, displaying highRNA transfection ability
and an improved safety
profile.7Cholesterol and its structural
analogs
Another major component of LNPs,
making up roughly 35-45% of the final
formulation, is cholesterol. Cholesterol
works as a stability enhancer by filling
gaps in the lipid layer and assists in thetransfection of RNA. Cholesterol must
be present to have an effective lipidbased delivery system but using too
much cholesterol can prevent particle
formation.
Since cholesterol makes up such alarge portion of an LNP formulation, it
makes sense that current research
efforts would attempt to find more
effective alternatives. With so manynaturally occurring analogs ofcholesterol available, evaluating thesubstitution of cholesterol with its
structural analogs was a great starting
point for a collaboration between
Moderna and Oregon State University.
They evaluated how three groups ofsteroids, 9,10-secosteroids, C-24 alkyl,
and pentacyclic steroids, affected theparticle size, mRNA encapsulation
efficiency, and transfection efficiency of
the resultant LNP formulations.7
Of the evaluated cholesterol analogs, βsitosterol proved to be head andshoulders above the rest. β-sitosterol
LNPs had a comparable particle size(diameter ~100 nm) and encapsulation
efficiency (~95% using 200 ng mRNA)as the cholesterol control. Where the βsitosterol-substituted LNPs stood out
was their transfection efficiency which
was several-fold higher than theSmart science to improve lives™

cholesterol control. So, comparable sizeand encapsulation efficiency, coupledwith markedly improved transfection
efficiency makes β-sitosterol an
intriguing alternative to cholesterol in
future RNA-LNPs. 8 Avanti now has βsitosterol (synthetic from plant sterol)available in the research catalog.

A long-term lipid innovations partner
Croda Pharma is a leading partner forthe development of excipients and thesupply of high-purity materials forpharmaceutical formulations. Thecompany is focused on empowering
biologics delivery through its Adjuvant
Systems and Small Molecule, Protein,
and Nucleic Acid Delivery platforms.
With a wide range of solutions for boththe human and animal health markets,
the pharmaceutical portfolio isunsurpassed in its excellence. Croda
Pharma's products, along with its inhouse formulation and regulatory
expertise, allow the company to meet its
customers' most demanding formulationneeds. The company is committed toenabling the next generation of drugdelivery systems.
Avanti Polar Lipids, now part of Croda
Pharma, has been supplying lipids fortherapeutic applications, including lipids
used for gene therapies, for more than
30 years. Researchers investigating
both viral vector and nonviral delivery
systems have been reliably provided
with high-quality, well-characterizedmaterials. Croda has added the ability
to supply lipids at commercial scale to
Avanti's development arm supporting
clinical programs.
In addition to supplying materials
currently in use for the delivery ofCRISPR/Cas9 gene-editing
components, Croda is pursuing - incooperation with its customers - efforts
to develop innovative lipid products that
can address some of the issues thathave arisen with LNPs for mRNA
vaccines around PEG lipids and othercomponents. Working collaboratively
with clients on lipids for these and nextgeneration gene-editing therapeutics is
how Avanti and Croda apply Smartscience to improve livesTM.

REFERENCES
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lymphocytes." Nature.
1978;274:923–924.
2. Ostro M.J., Giacomoni D., LavelleD., Paxton W., Dray S. Evidence fortranslation of rabbit globin mRNAafter liposome-mediated insertion
into a human cell line. Nature.
1978;274:921–923.
3. Wolff JA, Malone RW, Williams P,
Chong W, Acsadi G, Jani A, Felgner
PL. Direct gene transfer into mousemuscle in vivo. Science. 1990 Mar
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4. Conry RM, LoBuglio AF, Wright M,
Sumerel L, Pike MJ, Johanning F,Benjamin R, Lu D, Curiel DT.
Characterization of a messenger
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Cancer Res. 1995 Apr
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5. Hald Albertsen C, Kulkarni JA,
Witzigmann D, Lind M, Petersson K,Simonsen JB. The role of lipid
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Drug Deliv Rev. 2022
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6. Fang Y, Xue J, Gao S, Lu A, Yang
D, Jiang H, He Y, Shi K. Cleavable
PEGylation: a strategy forovercoming the "PEG dilemma" inefficient drug delivery. Drug Deliv.
2017 Dec;24(sup1):22-32.
7. Sara S. Nogueira, Anne Schlegel,Konrad Maxeiner, Benjamin Weber,Matthias Barz, Martin A. Schroer,Clement E. Blanchet, Dmitri I.
Svergun, Srinivas Ramishetti, DanPeer, Peter Langguth, Ugur Sahin,and Heinrich Haas. ACS Applied
Nano Materials 2020 3 (11), 1063410645.
8. Patel S, Ashwanikumar N,Robinson E, Xia Y, Mihai C, Griffith
JP 3rd, Hou S, Esposito AA, Ketova
T, Welsher K, Joyal JL, Almarsson
Ö, Sahay G. Naturally-occurring
cholesterol analogues in lipid
nanoparticles induce polymorphic
shape and enhance intracellulardelivery of mRNA. Nat Commun.
2020 Feb 20;11(1):983. Erratum in:
Nat Commun. 2020 Jul
6;11(1):3435.

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