On the mechanism of tissue-specific mRNA delivery
by selective organ targeting nanoparticles
Sean A. Dilliarda , Qiang Chenga,1 , and Daniel J. Siegwarta,1
a
Department of Biochemistry, Simmons Comprehensive Cancer Center, The University of Texas Southwestern Medical Center, Dallas, TX 75390
Edited by Joseph DeSimone, Department of Chemical Engineering, Stanford University, Stanford, CA; received May 19, 2021; accepted November 8, 2021
Lipid nanoparticles (LNPs) are a clinically mature technology for
the delivery of genetic medicines but have limited therapeutic
applications due to liver accumulation. Recently, our laboratory
developed selective organ targeting (SORT) nanoparticles that
expand the therapeutic applications of genetic medicines by
enabling delivery of messenger RNA (mRNA) and gene editing sys-
tems to non-liver tissues. SORT nanoparticles include a supplemen-
tal SORT molecule whose chemical structure determines the LNP’s
tissue-specific activity. To understand how SORT nanoparticles sur-
pass the delivery barrier of liver hepatocyte accumulation, we
studied the mechanistic factors which define their organ-targeting
properties. We discovered that the chemical nature of the added
SORT molecule controlled biodistribution, global/apparent pKa,
and serum protein interactions of SORT nanoparticles. Addition-
ally, we provide evidence for an endogenous targeting mechanism
whereby organ targeting occurs via 1) desorption of poly(ethylene
glycol) lipids from the LNP surface, 2) binding of distinct proteins
to the nanoparticle surface because of recognition of exposed
SORT molecules, and 3) subsequent interactions between surface-
bound proteins and cognate receptors highly expressed in specific
tissues. These findings establish a crucial link between the molecu-
lar composition of SORT nanoparticles and their unique and pre-
Downloaded from https://www.pnas.org by 124.219.109.112 on May 3, 2023 from IP address 124.219.109.112.
cise organ-targeting properties and suggest that the recruitment
of specific proteins to a nanoparticle’s surface can enable drug
delivery beyond the liver.
lipid nanoparticles j mRNA delivery j gene editing j endogenous targeting
N ucleic acids that enable gene silencing (1, 2), expression
(3–5), and editing (6–8) possess great potential for use as
genetic medicines in multiple clinical settings including cancer (9,
10), inherited genetic disorders (11, 12), and infectious diseases
(13–15). Due to the unfavorable pharmacokinetic properties of
nucleic acids, viral and nonviral delivery approaches are used to
facilitate nucleic acid delivery to target cells (16). Lipid nanopar-
ticles (LNPs) represent the most clinically mature nonviral plat-
form for the safe and efficacious delivery of genetic medicines.
Indeed, LNPs were an enabling technology for the US Food and
Drug Administration approval of the first small interfering RNA
(siRNA) drug, Onpattro, in 2018 (17) and the messenger RNA
(mRNA) vaccines currently being distributed for immunization
against the SARS-CoV-2 virus, the causative agent of the
COVID-19 pandemic (18, 19). Despite this progress, intrave-
nously (IV) administered LNPs typically accumulate in the liver
and are internalized by liver hepatocytes, thereby greatly limiting
the scope of their therapeutic applications (20).
Recently, our laboratory overcame this challenge through
the discovery of selective organ targeting (SORT), a strategy to
rationally design nanoparticles for the extrahepatic delivery of
mRNA and gene editing systems following IV administration
(Fig. 1A and SI Appendix, Table S1) (21–24). Conventional
LNPs are composed of four components: ionizable cationic lip-
ids, amphipathic phospholipids, cholesterol, and poly(ethylene
glycol) (PEG) lipids (25, 26). We found that augmenting con-
ventional four-component LNPs for mRNA delivery to the liver
with a fifth component (termed a SORT molecule) can alter
the LNPs’ in vivo organ-targeting properties and lead to the
PNAS 2021 Vol. 118 No. 52 e2109256118
extrahepatic delivery of mRNA. The SORT strategy was gener-
alizable to multiple classes of LNPs and SORT molecules.
Lung-, liver-, and spleen-targeting SORT LNPs could deliver
diverse cargoes (nucleic acids and proteins) to achieve gene
expression and CRISPR/Cas-based gene editing in therapeuti-
cally relevant cell types, including epithelial cells, endothelial
cells, B cells, T cells, and hepatocytes (21–24). However, the
mechanism of action remains undefined. Understanding the
mechanism which enables SORT is important for optimizing
delivery to currently targetable organs as well as extending the
SORT concept to other tissue and cell types. Moreover, mecha-
nistic understanding would establish a biological rationale for
overcoming the delivery barrier of liver accumulation that
currently hampers IV administered nanoparticles (27).
Herein, we identify and study mechanistic factors which could
ENGINEERING
explain the organ-targeting properties of SORT LNPs based on
three established principles that define the efficacy of liver-
targeting LNPs: biodistribution to the liver, an acid dissociation
constant (pKa) near 6.4, and the adsorption of apolipoprotein E
(ApoE) to the LNP surface (12, 28). We discovered that each of
these three factors (organ level biodistribution, apparent pKa,
MEDICAL SCIENCES
and serum protein adsorption) are distinct for SORT LNPs and
correlate with their tissue-targeting properties. Furthermore, we
provide evidence of a three-step mechanism for the functional
role of serum protein adsorption on tissue targeting that extends
Significance
Liver accumulation represents a significant barrier in the
development of therapeutically efficacious nanoparticle drug
delivery systems. Using a series of lipid nanoparticles with
distinct organ-targeting properties, we provide evidence for
a plausible mechanism of action for nanoparticle delivery to
non-liver tissues. Following intravenous injection, specific
proteins in the blood are recruited to the nanoparticle’s sur-
face based on its molecular composition and they endow it
with a unique biological identity that governs its ultimate
fate in the body. An innovative paradigm emerges from this
mechanistic understanding of nanoparticle delivery—endog-
enous targeting—wherein the molecular composition of a
nanoparticle is rationally engineered to interact with specific
proteins in the blood to overcome liver accumulation and to
target specific organs.
Author contributions: S.A.D., Q.C., and D.J.S. designed research; S.A.D. and Q.C.
performed research; S.A.D., Q.C., and D.J.S. analyzed data; D.J.S. supervised research;
and S.A.D. and D.J.S. wrote the paper.
Competing interest statement: D.J.S., Q.C., and the Regents of the University of Texas
System have filed patent applications related to this technology. D.J.S. is a cofounder
of/consultant with ReCode Therapeutics, which has licensed intellectual property from
the University of Texas Southwestern.
This article is a PNAS Direct Submission.
Published under the PNAS license.
1
To whom correspondence may be addressed. Email: Qiang.Cheng@UTSouthwestern.
edu or Daniel.Siegwart@UTSouthwestern.edu.
This article contains supporting information online at http://www.pnas.org/lookup/
suppl/doi:10.1073/pnas.2109256118/-/DCSupplemental.
Published December 21, 2021.
https://doi.org/10.1073/pnas.2109256118 j 1 of 10
 LNP biodistribution readout:
A LNP functional readout:
Luc mRNA translation to protein B Tracking of Cy5-mRNA C

Relative Fluorescence (%) Relative Fluorescence (%) Relative Fluorescence (%)

PBS 10% DODAP 20% DODAP 50% DODAP
Addition of 5th SORT molecule 80
Liver
Heart 60 Lung
Lung
40 Spleen

Conventional 4-Component LNP Liver Liver 20

18:1 DAP (DODAP) Spleen
SORT 0
5A2-SC8/DOPE/Chol/C14PEG/DODAP = Kidney Kidney 0 10 20 50
19/19/38/4/20 (mol. %) PBS 10% 18PA 20% 18PA 30% 18PA DODAP (%)

80
5A2-SC8
Liver
60 Lung
Spleen
Spleen 40
DOPE
18:1 PA (18PA)
SORT 20
Cholesterol 5A2-SC8/DOPE/Chol/C14PEG/18PA =
16.7/16.7/33.3/3.3/30 (mol. %) PBS 0% DOTAP 15% DOTAP 50% DOTAP 0
0 10 20 30
C14-PEG2K 18PA (%)
80
5A2-SC8/DOPE/Chol/C14PEG = Liver
23.8/23.8/47.6/4.8 (mol. %) 60 Lung
Lung Spleen
18:1 TAP (DOTAP) 40
SORT
5A2-SC8/DOPE/Chol/C14PEG/DOTAP = 20
11.9/11.9/23.8/2.4/50 (mol. %) ˜107 p/sec/cm2/sr
0
4.0 5.0 6.0 7.0 8.0 µW/cm2 0 15 50
DOTAP (%)

D DODAP SORT LNPs 18PA SORT LNPs DOTAP SORT LNPs

E
0%(mDLNP) 0%(mDLNP)
0%(mDLNP)
1.0 Lungs only
TNS fluorescence (%)

5%

TNS fluorescence (%)

TNS Fluorescence (%)

1.0 10% 1.0 5% Lungs + spleen

10%
20% 10% 15%
50% 20% 20% Spleen + lungs
30% Spleen only
80% 30% 0.5
0.5 0.5 40%
40% 50% Spleen + liver
60%
70%
Liver + spleen
0.0 0.0
80% Liver only
0.0 90%
3 5 7 9 11 3 5 7 9 11 3 5 7 9 11
pH pH pH

Fig. 1. SORT nanoparticles for tissue-specific mRNA delivery have unique biodistribution and ionization behavior. (A) By adding a fifth, supplemental
SORT molecule to a conventional, four-component LNP (mDLNP: 23.8 mol % 5A2-SC8, 23.8 mol % DOPE, 47.6 mol % cholesterol, and 4.8 mol % C14-
PEG2K), the tissue-specific activity of delivered mRNA changes based on the chemical structure of the included SORT molecule. An ionizable cationic lipid
(DODAP) enhances liver-specific mRNA translation (liver SORT: 19 mol % 5A2-SC8, 19 mol % DOPE, 38 mol % cholesterol, 4 mol % C14-PEG2K, and 20
mol % DODAP), an anionic lipid (18PA) results in spleen-specific mRNA translation (spleen SORT: 16.7 mol % 5A2-SC8, 16.7 mol % DOPE, 33.3 mol % cho-
lesterol, 3.3 mol % C14-PEG2K, and 30 mol % 18PA), and a cationic quaternary ammonium lipid (DOTAP) results in lung-specific mRNA translation (lung
Downloaded from https://www.pnas.org by 124.219.109.112 on May 3, 2023 from IP address 124.219.109.112.

SORT: 11.9 mol % 5A2-SC8, 11.9 mol % DOPE, 23.8 mol % cholesterol, 2.4 mol % C14-PEG2K, and 50 mol % DOTAP). (B) Ex vivo fluorescence of Cy5-
labeled mRNA in major organs extracted from C57BL/6 mice IV injected with SORT LNPs that incorporate increasing percentages of different SORT mole-
cules (0.5 mg/kg mRNA/body weight, 6 h). (C) Relative average Cy5 fluorescence measured in the liver, lung, and spleen as a function of SORT molecule
percent inclusion (0.5 mg/kg mRNA/body weight, n = 2). SORT molecules promote mRNA biodistribution to target organs. Data are shown as mean ±
SEM. (D) Representative TNS assay curves for determining the apparent pKa of SORT LNPs incorporating increasing percentages of ionizable cationic,
anionic, or permanently cationic lipid SORT molecules. Apparent pKa was defined as the point at which 50% of TNS fluorescence was achieved. (E) LNPs
were assigned a tissue specificity index based on the tissues in which functional luciferase mRNA was detected. Intermediate tissue specificity indexes rep-
resent LNPs in which mRNA activity was detected in multiple organs; these LNPs are plotted in the region of the organ for which higher activity was mea-
sured. For the 67 LNPs tested with the TNS assay, LNP apparent pKa was correlated with the specificity of luciferase mRNA tissue delivery.

the scope and application of the endogenous targeting mecha- they do not readily diffuse through anionic cellular membranes
nism defined for four-component LNP delivery to liver hepato- and are susceptible to degradation by ribonucleases (RNases)
cytes (12, 28). First, desorption of PEG lipids on the LNP surface in the blood (5). Ideally, LNPs will encapsulate and protect
exposes underlying SORT molecules in the LNP. Next, distinct mRNA from enzymatic degradation, enable accumulation in
serum proteins recognize the exposed SORT molecules and the target organ, facilitate receptor-mediated endocytosis into
adsorb to the LNP surface. Finally, surface-adsorbed proteins cells, and release mRNA from endosomes into the cytosol to
interact with cognate receptors expressed by cells in the target undergo translation into a functional protein (12). Through the
organs to facilitate functional mRNA delivery to those tissues. development of SORT LNPs, we showed that the chemical
The results indicate that the choice of SORT molecule gov- identity and amount of SORT molecule added to a four-
erns which proteins most avidly adsorb to the LNP surface, component LNP systematically alters tissue-specific protein
impacting the ultimate biological fate of the LNP (29). We envi- expression following mRNA delivery (21). The inclusion of
sion that this mechanistic understanding of SORT LNPs will ionizable cationic lipids enhanced liver targeting, anionic lipids
enable their optimization for therapeutic applications in the resulted in retargeting of delivery to the spleen, and per-
lung, liver, and spleen and lay the foundation for extending the manently cationic lipids bearing a quaternary ammonium
SORT platform to additional nanoparticle types, physiological headgroup retargeted delivery to the lungs (Fig. 1A and SI
tissues, and cell types. Furthermore, our findings suggest that Appendix, Table S1) (21). These experiments revealed how the
endogenous targeting—that is, tuning a nanoparticle’s molecu- choice of SORT molecule served as a design parameter to pre-
lar composition such that it binds specific proteins in the serum dictably dictate where mRNA would be translated to functional
to enable delivery to the target site—could be a generally useful proteins in vivo (21) but did not elucidate the organs where
strategy for engineering a wide array of nanomaterials capable SORT LNPs accumulated and the mechanism of how SORT
of extrahepatic delivery. molecules function. Biodistribution to the target organ should,
in principle, be a requisite step for targeted delivery.
Results Therefore, we first investigated the biodistribution of mRNA
SORT Molecules Alter LNP Biodistribution. mRNA therapeutics encapsulated in SORT LNPs to determine if there was a corre-
must overcome multiple barriers for intracellular delivery, as lation between the organs in which SORT LNPs accumulated

2 of 10 j PNAS Dilliard et al.

https://doi.org/10.1073/pnas.2109256118 On the mechanism of tissue-specific mRNA delivery by
selective organ targeting nanoparticles
 and their tissue-specific mRNA activity. We formulated liver,
lung, and spleen SORT LNPs as well as a representative four-
component dendrimer LNP for mRNA delivery to the liver
(mDLNP) (SI Appendix, Tables S1 and S7) to encapsulate Cy5-
labeled mRNA following the identical protocols and formula-
tion parameters used in our previous publications (11, 21). We
administered each LNP IV to C57BL/6 mice and tracked the
in vivo biodistribution of the Cy5 mRNA using fluorescence
imaging. The organs were excised and imaged ex vivo 6 h post-
injection (Fig. 1B). The average fluorescence produced by the
liver, spleen, and lungs was quantified (Fig. 1C). The incorpora-
tion of the ionizable cationic lipid 1,2-dioleoyl-3-dimethylam-
monium-propane (DODAP) to mDLNP resulted in an increase
in average Cy5 mRNA signal in the liver and a decrease of
average Cy5 mRNA signal in the spleen, with optimal liver bio-
distribution at 20% inclusion of DODAP (Fig. 1 B and C).
Meanwhile, increasing the percentage of anionic SORT mole-
cule 18PA added to the LNP increased average Cy5 mRNA sig-
nal in the spleen (Fig. 1 B and C). Similarly, as the permanently
cationic SORT molecule 1,2-dioleoyl-3-trimethylammonium-
propane (DOTAP) was included in the LNP in increasing pro-
portions, there was a progressive increase in Cy5 mRNA signal
in the lungs (Fig. 1 B and C). Importantly, these tracking stud-
ies quantify where the SORT LNPs biodistribute but not where
mRNA is translated to protein (functional delivery). These
studies demonstrate that inclusion of a SORT molecule in
mDLNP facilitates mRNA biodistribution to the target organ
but is not sufficient to explain tissue-specific mRNA activity.
SORT Molecules Alter Apparent LNP pKa. It is established that
global/apparent pKa plays an important role in determining the
potency of RNA delivery by LNPs. Indeed, siRNA delivery to
liver hepatocytes strongly depends on LNP pKa, with a pKa of
Downloaded from https://www.pnas.org by 124.219.109.112 on May 3, 2023 from IP address 124.219.109.112.
6.2 to 6.4 being optimal for gene silencing; LNPs with a pKa
outside of that narrow range were unable to functionally deliver
siRNA to the liver (30). Based on the link between pKa and cel-
lular delivery, we analyzed the apparent pKa of 67 different
SORT LNPs (SI Appendix, Table S2), all of which were effica-
cious for tissue-specific mRNA delivery in vivo (21), using the
6-(p-toluidino)-2-naphthalenesulfonic acid (TNS) assay (Fig. 1 D
and E and SI Appendix, Figs. S2 and S3). Because SORT involves
the inclusion of additional charged lipids, the resulting TNS titra-
tion curves capture the ionization behavior of more complex
mixed species LNPs. As the lower limit TNS fluorescence of 0%
was not measured for some SORT LNPs over the pH range of
buffers used in the TNS assay, we defined the apparent pKa as
the pH at which 50% normalized TNS fluorescence signal is mea-
sured (Fig. 1D).
When plotting relative pKa with respect to tissue-specific
activity, SORT LNPs grouped into defined ranges based on
their organ-targeting properties (Fig. 1E). Confirming the liter-
ature precedent, all liver-targeting SORT LNPs had an appar-
ent pKa within the well-established 6 to 7 range (30) (Fig. 1E).
Surprisingly, all lung-targeting SORT LNPs had a higher appar-
ent pKa (greater than 9) (Fig. 1E), while spleen-targeting LNPs
had a lower pKa between 2 and 6 (Fig. 1E). This result con-
trasts with the dogma for conventional LNP delivery to the liver
and contributes to the understanding of why SORT LNPs are
unconventional and can enable extrahepatic delivery. It is
important to note that the SORT LNPs tested here possess a
similar, near neutral zeta potential surface charge (SI Appendix,
Table S1). Thus, the addition of a SORT molecule to a multi-
component LNP alters its overall pKa that directly relates to
the organ-targeting properties of the LNP.
PEG Lipid Desorption Contributes to Effective mRNA Delivery
In Vivo. Conventional four-component LNPs are known to
enable functional RNA delivery into liver hepatocytes via an
Dilliard et al.
On the mechanism of tissue-specific mRNA delivery by
selective organ targeting nanoparticles
endogenous mechanism in which the desorption of PEG lipids
from the LNP surface enables ApoE to bind the LNP; this sub-
sequently enables receptor-mediated binding and uptake of the
LNPs by the low-density lipoprotein receptor (LDL-R) (12).
We hypothesized that SORT LNPs might operate by a similar
mechanism in which 1) PEG lipid desorbs from the LNP to
expose underlying SORT molecules, 2) distinct serum proteins
recognize the exposed SORT molecules and adsorb to the LNP
surface, and 3) these surface-adsorbed proteins interact with
cognate receptors which mediate uptake of the LNPs by cells in
target tissues (Fig. 2A). First, we established the role of the
PEG–lipid component on mRNA delivery efficacy in vivo.
LNPs incorporate a PEG lipid on the surface to promote colloi-
dal stability (26). Because the PEG–lipid molecules are nonco-
valently incorporated into the LNP, they spontaneously desorb
at a rate inversely proportional to the length of the PEG lipid’s
hydrophobic anchor (31, 32). PEG lipids on the LNP surface
can impair serum protein adsorption, resulting in reduced cel-
lular targeting and delivery efficacy (31). The shedding of PEG
lipid would be expected to expose the underlying SORT mole-
cules for recognition by serum proteins, promoting their bind-
ing of the SORT LNP in the blood.
To functionally characterize the consequences of PEG lipid
desorption on mRNA delivery, we measured the effects of
substituting DMG-PEG2000 (C14-PEG2K), a methoxy PEG
ENGINEERING
(mPEG) glyceride with 14-carbon-long alkyl tails, with DSG-
PEG2000 (C18-PEG2K), an mPEG glyceride with 18-carbon-
long alkyl tails, on the in vivo potency of SORT LNPs.
C18-PEG2K is expected to be less sheddable from the LNP
compared to C14-PEG2K due to its longer hydrophobic anchor
(31, 32). First, we measured the effects of increasing the PEG
MEDICAL SCIENCES
lipid anchor length on the delivery of luciferase mRNA by
injecting C57BL/6 mice IV at a dosage of 0.1 mg/kg mRNA and
imaging organ luminescence ex vivo at a timepoint of 6 h postin-
jection (Fig. 2B). Switching to C18-PEG2K significantly
reduced the total luminescence produced by luciferase mRNA
translated into a functional protein in target organs when com-
pared to C14-PEG2K for all SORT LNPs (Fig. 2C). To confirm
these findings, we delivered human erythropoietin (hEPO)
mRNA to target tissues in vivo using liver, spleen, or lung
SORT LNPs incorporating either C14-PEG2K or C18-PEG2K.
Because hEPO is a secreted protein, delivery efficacy can be
readily quantified by measuring hEPO levels in the serum (21).
We verified that serum hEPO concentration was lower in mice
treated with C18-PEG2K SORT LNPs compared to those
treated with C14-PEG2K SORT LNPs (Fig. 2D). Thus, we
quantitatively confirmed that SORT LNPs which incorporate a
PEG lipid with a longer hydrophobic anchor are less effective
for mRNA delivery to target organs. These studies suggest that
PEG lipid desorption is a necessary process for efficacious
mRNA delivery to target tissues by SORT LNPs.
SORT Molecule Choice Influences LNP–Protein Interactions in the
Serum. The second step of the hypothesized endogenous target-
ing mechanism for SORT LNPs involves the adsorption of
distinct proteins to the LNP surface to form unique protein
coronas (Fig. 2A). Following PEG lipid desorption, serum pro-
teins readily adsorb to the surface of IV administered LNPs,
forming an interfacial layer known as the “protein corona” that
defines their biological identity (29). Adsorption of ApoE has
been shown to drive liver targeting of conventional four-
component LNPs through binding of the LDL-R highly
expressed on hepatocytes (28). Given the strikingly different
organ-targeting capabilities of lung and spleen SORT LNPs, we
hypothesized that the set of serum proteins that bind the LNPs
would be distinct. We isolated the plasma proteins which bind
mDLNP, liver SORT, spleen SORT, and lung SORT using dif-
ferential centrifugation following ex vivo incubation with mouse
PNAS j 3 of 10
https://doi.org/10.1073/pnas.2109256118
 A E F Functional Classes
of Adsorbed Proteins G Isoelectric Point
H Top 5 Serum
Proteins Adsorbed
mDLNP Liver Spleen Lung
Mw SORT SORT SORT
250 15
150
2

% Abundance
1 2 3 100 ApoE
19 21
75 10 C1qA
16
50 IgκC
42 5
37
mDLNP IgHγ2B

25 C1qB
20 0
(kDa) 0 2 4 6 8 10 12 14
0 5 10 15

B C14-PEG2K C18-PEG2K
3.0
C Luciferase mRNA
✱
D hEpo mRNA
✱
15
ApoE

% Abundance
1.5×10 9 40000
Liver 6 10 C1qA
19

Serum hEPO
Luminescence
SORT 21

(mIU/mL)
30000
1×10 9 IgHµ
x107 20
20000 5
35 C1qB
5×10 8 Liver
10000 Alb
SORT 0
0 0 0 2 4 6 8 10 12 14
0.5 0 5 10 15

1.0 ✱ ✱ 25
1.5×10 8 5000
Spleen 3 β2-GPI

% Abundance
12 17 20
Luminescence

4000
SORT Serum hEPO
IgκC
1×10 8 (mIU/mL) 3000 15
x107 38 30 C1qA
2000 10
5×10 7
Spleen ApoE
1000 5
SORT IgHγ2B
0 0 0
0.05
0 2 4 6 8 10 12 14 0 10 20 30

Lung ✱
1.0 2×10 7 1500
15
SORT ✱
Vtn
Serum hEPO

% Abundance
Luminescence

1.5×10 7 14 18
(mIU/mL)

1000
10 Pon1
1×10 7 9
x106 ApoE
500 46 13
5×10 6 Lung 5 Alb
SORT
0 0 Clu
0.1 C14- C18- C14- C18- 0
PEG2K PEG2K PEG2K PEG2K 0 2 4 6 8 10 12 14 0 5 10 15
pI % Abundance

Fig. 2. Multiple steps are involved in the mechanism of SORT LNP tissue targeting, including formation of unique protein coronas. (A) A proposed three-
step endogenous targeting mechanism for tissue-specific mRNA delivery by SORT LNPs in which 1) PEG lipid desorption 2) enables distinct plasma proteins
to bind SORT LNPs, 3) resulting in cellular internalization in the target tissues by receptor-mediated uptake. (B) Ex vivo bioluminescence of major organs
Downloaded from https://www.pnas.org by 124.219.109.112 on May 3, 2023 from IP address 124.219.109.112.

excised from C57BL/6 mice IV injected with liver, spleen, and lung SORT LNPs incorporating either sheddable PEG lipids (C14-PEG2K) or less sheddable PEG
lipids (C18-PEG2K) (0.1 mg FLuc mRNA/kg body weight, 6 h). Total luminescence produced by each organ is reduced when less sheddable PEG lipid is
used, suggesting that PEG lipid desorption is a key process for efficacious mRNA delivery by SORT LNPs. (C) Quantification of total luminescence produced
by functional protein translated from FLuc mRNA in target organs of C57BL/6 mice IV injected with liver, spleen, and lung SORT LNPs incorporating either
C14- or C18-PEG2K (0.1 mg FLuc mRNA/kg body weight, 6 h). (D) ELISA quantification of serum hEPO in C57BL/6 mice treated with liver, spleen, or lung
SORT LNPs encapsulating hEPO mRNA (0.1 mg hEPO mRNA/kg body weight, 6 h). Using a less-sheddable PEG reduces SORT LNP potency. (E) SDS–PAGE of
the plasma proteins adsorbed to the surface of mDLNP, liver SORT, spleen SORT, and lung SORT LNPs. LNPs with different organ-targeting properties
bind distinct plasma proteins. (F) The average abundance of proteins with distinct biological functions in the protein coronas of mDLNP and liver, spleen,
and lung SORT LNPs. The choice of SORT molecule leads to large-scale differences in the functional ensemble of plasma proteins which bind the LNP. (G)
Isoelectric point distribution for the most enriched proteins which constitute 80% of the protein corona of the LNPs. A SORT molecule’s headgroup struc-
ture influences the pI distribution of the protein corona. (H) The top five most abundant plasma proteins that bind different SORT LNPs (n = 3). The
chemical structure of SORT molecule affects the number one plasma protein that is most highly enriched on the surface of SORT LNPs. Data are shown as
mean ± SEM. Statistical significance was determined using an unpaired two-tailed Student’s t test (*P < 0.05).

plasma (33). Treating four-component mDLNP as the base abundance) for each LNP (SI Appendix, Tables S3–S6). First, we
liver-targeting LNP composition, we used sodium dodecyl sul- assessed the biological function of these highly abundant pro-
fate (SDS)–polyacrylamide gel electrophoresis (PAGE) (SDS- teins. By clustering plasma proteins into the physiological classes
PAGE) to qualitatively study how the choice of SORT molecule of apolipoproteins, coagulation proteins, complement proteins,
impacted which proteins adsorbed to five-component SORT immune proteins, and other proteins (34), we discovered that the
LNPs (Fig. 2E). While the set of plasma proteins that adsorb to proportion of proteins in each class changed based on the choice
liver SORT looks qualitatively similar to that of mDLNP, the of SORT molecule, suggesting that the inclusion of a SORT mol-
plasma proteins that bind SORT LNPs targeting extrahepatic ecule results in large-scale differences in the ensemble of proteins
organs are markedly distinct (Fig. 2E). In particular, a key which bind an LNP (Fig. 2F). The distinct functions of these pro-
band is highly enriched for spleen SORT at an Mw of 54 kDa, teins may play a role in shaping an LNP’s endogenous identity
while a band near 65 kDa is highly enriched for lung SORT and subsequent fate in vivo.
(Fig. 2E). Thus, adding a SORT molecule to mDLNP alters the To better understand what drives these large-scale differ-
composition of the protein corona in accordance with the ences in the functional composition of the protein corona, we
SORT molecule’s chemical structure. studied how a SORT molecule’s chemical structure might affect
Unbiased mass spectrometry proteomics enabled identification which proteins bind to LNPs. While each SORT molecule
and quantification of which proteins bind SORT LNPs in the shares a common hydrophobic scaffold that enables the mole-
plasma. We found that over 900 different proteins adsorbed to cule to self-assemble into the LNP, they are distinguished by
SORT LNPs, but nearly 98% of these proteins are present at an the chemical structure and charge state of the headgroup.
abundance of less than 0.1%. We anticipate that the most abun- These molecular features may play a role in the differential
dant proteins are the ones most likely to be functionally impor- enrichment of proteins with distinct characteristics, possibly
tant; thus, we focused our subsequent analysis on the proteins through electrostatic forces which bring specific proteins into
that constitute the majority of the protein corona (80% proximity of the SORT LNP to facilitate further protein–LNP

4 of 10 j PNAS Dilliard et al.

https://doi.org/10.1073/pnas.2109256118 On the mechanism of tissue-specific mRNA delivery by
selective organ targeting nanoparticles
 interactions (34). A protein’s isoelectric point (pI) is defined as
the pH at which the protein molecule bears no net charge, pro-
viding an estimate of a protein’s charge state in the physiologi-
cal milieu. We determined the pI of the major proteins that
bound mDLNP and each SORT LNP using the bioinformatics
resource ExPASy (35). The inclusion of DODAP, an ionizable
cationic lipid, to mDLNP did not greatly alter the pI distribu-
tion of the major proteins which bound the LNP (Fig. 2G). In
contrast, the inclusion of 18PA, a lipid with an anionic head-
group, to mDLNP promoted the adsorption of plasma proteins
with a pI greater than physiological pH (Fig. 2G), while the
inclusion of DOTAP, a lipid with a cationic quaternary ammo-
nium headgroup, in mDLNP favored the enrichment of pro-
teins with a pI below physiological pH (Fig. 2G). Although all
SORT LNPs bound proteins across a broad range of pIs, the
nature of the headgroup of a chosen SORT molecule does
affect which proteins adsorb to the LNP.
We further analyzed the five most abundant proteins in the
protein corona of each LNP. It was discovered that the most
highly enriched protein in each protein corona was unique based
on the choice of SORT molecule (Fig. 2H). ApoE was the most
highly enriched plasma protein for mDLNP, a conventional four-
component LNP which targets the liver, on average composing
13.9% of mDLNP’s protein corona (Fig. 2H). This result agrees
with the established role of ApoE in RNA delivery to the liver
(28), supporting the validity of the experimental approach. Addi-
tionally, liver SORT most avidly bound ApoE at an average
abundance of 13.3%, representing a 55-fold enrichment com-
pared to native mouse plasma (Fig. 2H). In contrast, spleen
SORT was most highly enriched in β2-glycoprotein I (β2-GPI) at
an average abundance of 20.1% (125-fold higher than native
mouse plasma) (Fig. 2H), while lung SORT was most highly
enriched in vitronectin (Vtn) at an average abundance of 12.2%
Downloaded from https://www.pnas.org by 124.219.109.112 on May 3, 2023 from IP address 124.219.109.112.
(108-fold higher than native mouse plasma) (Fig. 2H). Thus,
SORT LNPs for extrahepatic mRNA most avidly bind proteins
distinct from ApoE compared to mDLNP. Furthermore, there
were additional distinctions in the top five plasma proteins bound
to SORT LNPs in terms of individual molecular species as well
as their physiological function (Fig. 2H). To further validate our
findings, we examined the protein coronas formed around SORT
LNPs in the plasma of an additional mouse strain and identified
the same key proteins highly enriched in the protein coronas of
SORT LNPs (SI Appendix, Tables S3–S6). Ultimately, SORT
LNPs bind low-abundance serum proteins to form unique protein
coronas with quantitatively distinct composition based on the
nature of the incorporated SORT molecule, which may impact
tissue-specific mRNA delivery.
Key Serum Proteins Define SORT LNP Cellular Targeting. One
mechanism by which surface-adsorbed proteins can endo-
genously target specific tissues is through interactions with
cognate cellular receptors, resulting in receptor-mediated endo-
cytosis of the LNP (36) (Fig. 2A). Having identified the serum
proteins which most avidly bind SORT LNPs, we functionally
characterized how these single proteins affect intracellular
delivery of mRNA by SORT LNPs. We incubated all SORT
LNPs with either ApoE, β2-GPI, or Vtn, the most highly
enriched serum proteins discovered to associate with SORT
LNPs in our proteomics studies and measured the effects of
these single proteins on LNP uptake and mRNA delivery effi-
cacy in vitro (Fig. 3A). First, we incubated SORT LNPs encap-
sulating Cy5 mRNA with ApoE and measured cellular uptake
in HuH-7 and Hep G2 cells, two cell lines which highly express
ApoE’s receptor, LDL-R. Intracellular accumulation of Cy5
mRNA was increased in HuH-7 and Hep G2 cells by a factor
of 2.4 (Fig. 3 B and C) and 6.5 (SI Appendix, Fig. S3), respec-
tively, when liver SORT LNPs were incubated with ApoE.
Next, we examined THP-1 macrophages, a cell line known to
Dilliard et al.
On the mechanism of tissue-specific mRNA delivery by
selective organ targeting nanoparticles
interact with β2-GPI–bound particles containing anionic phos-
pholipids (37), and found that incubation with β2-GPI
enhanced Cy5 mRNA uptake of spleen SORT LNPs by a factor
of 2.1 (Fig. 3 B and C). Finally, we examined A-498 and U-87
MG cells, which highly express Vtn’s receptor αvβ3 integrin.
Incubation with Vtn increased cellular uptake of lung SORT
LNPs at a factor of 23.2 (Fig. 3 B and C) and 4.2 (SI Appendix,
Fig. S4) times greater than the uncoated LNPs. Thus, the
choice of SORT molecule impacts which plasma proteins
adsorb to the LNP surface, and these proteins likely interact
with cognate receptors expressed by target cells to enhance cel-
lular uptake, promoting intracellular mRNA delivery.
Having determined that key proteins can enhance cellular
uptake by distinct receptors, we verified that these proteins
improve functional mRNA delivery by measuring the activity
of luciferase enzyme translated from mRNA delivered by
uncoated and coated SORT LNPs in vitro. Additionally, we
aimed to determine whether single protein coatings specifically
enhanced the delivery of the individual formulations which
most avidly bound those proteins. Cells were treated with liver,
spleen, and lung SORT LNPs, encapsulating luciferase mRNA,
that were incubated with increasing amounts of ApoE, β2-GPI,
or Vtn (Fig. 3D). Luciferase activity was enhanced in HuH-7
and Hep G2 cells by ApoE-coated liver SORT LNPs, whereas
luciferase activity in these cells was not improved by spleen or
ENGINEERING
lung SORT LNPs preincubated with ApoE (Fig. 3D and SI
Appendix, Fig. S3), suggesting that spleen and lung SORT
LNPs do not efficiently bind ApoE and enter LDL-R–
expressing cells. Thus, ApoE exclusively enhanced functional
mRNA delivery by liver SORT LNPs in LDL-R–expressing
cells. Similarly, incubating spleen SORT LNPs with β2-GPI
MEDICAL SCIENCES
exclusively enhanced functional mRNA delivery to THP-1 mac-
rophages (Fig. 3D), while incubating lung SORT LNPs with
Vtn exclusively enhanced functional mRNA delivery to A-498
and U-87 MG cells (Fig. 3D and SI Appendix, Fig. S4). Interest-
ingly, incubating lung SORT LNPs at a ratio of 1.0 g Vtn/g total
lipid resulted in diminished luciferase activity, suggesting that
excess (free in media) Vtn may be inhibiting αvβ3 integrin to
limit mRNA delivery. Collectively, these studies reveal that
unique interactions between specific SORT molecules and indi-
vidual serum proteins selectively promote SORT LNP targeting
to distinct cell types by enhancing cellular uptake.
Extrahepatic mRNA Delivery by SORT LNPs Occurs through an ApoE-
Independent Mechanism. The adsorption of ApoE to the surface
of conventional four-component LNPs is a crucial process neces-
sary for highly efficacious RNA delivery to liver hepatocytes (28).
mDLNP, which is a four-component LNP for mRNA delivery to
the liver, avidly binds to ApoE in the serum. However, the addi-
tion of a SORT molecule to mDLNP alters which serum proteins
adsorb to the LNP surface. The set of proteins which bind the
LNP might play a role in defining the organ-targeting properties
of SORT LNPs via an endogenous mechanism whereby surface-
adsorbed proteins interact with cognate receptors expressed by
cells in the target organ. Since lung and spleen SORT LNPs
include molecules (DOTAP and 18PA, respectively) that are not
common to conventional LNPs, we reasoned that differences in
organ targeting could be driven by differences in protein corona
composition. Genetically modified mice can be used to deplete
key proteins from the serum, preventing their adsorption to the
LNP surface and possibly impairing expected organ targeting. To
evaluate the plausibility of an endogenous targeting mechanism
in vivo, we investigated how the addition of a SORT molecule to
a conventional four-component LNP impacts the functional role
of ApoE on tissue-specific mRNA delivery using knockout mice.
We compared the delivery of luciferase mRNA by SORT
LNPs to target organs in B6.129P2-Apoetm1Unc/J (ApoE/)
mice, a genetic knockout model lacking ApoE expression, to
PNAS j 5 of 10
https://doi.org/10.1073/pnas.2109256118
 A
Cy5-mRNA tracking (cellular uptake)

Bioluminescence (mRNA translation)

SORT LNP Protein Cell Treatment

Coating

B C ✱✱✱✱
D 3×10 6
✱✱✱

Cy5 mRNA Fluorescence

1500000

Luminescence
Liver SORT
2×10 6

ApoE coating
HuH-7 Cells
uncoated

1000000

1×10 6
500000

0
0 g ApoE/g lipid

0. 0

0. 0
25
5
0

25

0. 0
5
0

25
5
0
0.
1.

0.
1.

0.
1.
uncoated ApoE

15000

Cy5 mRNA Fluorescence

100000 ✱

Spleen SORT

Luminescence
80000
β2-GPI coating

THP-1 Cells 10000

uncoated

60000 ✱

40000
5000
20000

0 0
uncoated β2-GPI g β2-GPI/g lipid

0. 0
25

0. 0
5
0

25

0. 0
5
0

25
5
0
0.
1.

0.
1.

0.
1.
400000 ✱✱✱✱ ✱✱✱✱
Cy5 mRNA Fluorescence

Lung SORT 1×10 7

A-498 Cells 300000 8×10 6

Luminescence
Vtn coating
uncoated

200000 6×10 6

4×10 6
100000
2×10 6
0
uncoated Vtn 0
g Vtn/g lipid

0. 0
25

0. 0
25

0. 0
5
0

5
0

25
5
0
0.
1.

0.
1.

0.
1.
Downloaded from https://www.pnas.org by 124.219.109.112 on May 3, 2023 from IP address 124.219.109.112.

Fig. 3. Distinct plasma proteins regulate SORT LNP uptake and efficacy in vitro. (A) SORT LNPs were preincubated with either ApoE, β2-GPI, or Vtn prior
to treating relevant cell lines to measure cellular uptake (Cy5 mRNA tracking) or functional mRNA delivery (bioluminescence). (B) Representative images
of cellular uptake of uncoated and coated SORT LNPs taken up by relevant cell types. Incubating a SORT LNP with the protein most avidly binds increases
mRNA uptake in cell lines expressing the cognate receptor (250 ng mRNA per well, 1.5 h). (Scale bar: 50 μm.) (C) Quantification of Cy5 mRNA fluorescence
in cells treated with uncoated or coated SORT LNPs (250 ng mRNA per well, 1.5 h, n = 10). Statistical significance was determined using an unpaired two-
tailed Student’s t test (****P < 0.0001, *P < 0.05). (D) Activity of functional luciferase protein translated from mRNA delivered by uncoated or protein-
coated SORT LNPs in relevant cell lines (25 ng mRNA, 24 h, n = 4). Statistical significance determined using one-way ANOVA with Brown–Forsythe test
(****P < 0.0001, ***P < 0.001, *P < 0.05). Individual proteins exclusively bind to specific SORT LNPs and enhance mRNA delivery only to cell lines express-
ing the cognate receptor. Data are shown as mean ± SEM.

wild-type (WT) C57BL/6 mice containing normal levels of
ApoE in the serum. Luminescence was quantified to measure
functional luciferase mRNA delivery to the target organs of
each SORT LNP (Fig. 4). By eliminating ApoE from the
serum, mDLNP exhibited significantly reduced mRNA delivery
to the liver, with an average reduction of luciferase activity by
78% compared to WT C57BL/6 mice (Fig. 4). Liver SORT
LNPs, which also bind ApoE selectively from the serum, main-
tain this dependence on ApoE for mRNA delivery to the liver:
the knockout of ApoE significantly impaired liver targeting,
with an average reduction in luciferase activity by 87% com-
pared to WT C57BL/6 mice (Fig. 4). Thus, ApoE is indispens-
able for efficacious targeting of the liver by both mDLNP and
liver SORT LNPs. In stark contrast, mRNA delivery by spleen
SORT to the spleen was enhanced by a factor of 2.2 in ApoE/
mice, suggesting that ApoE plays an antagonistic role in effica-
cious mRNA delivery to the spleen (Fig. 4). Tissue-specific
mRNA delivery by lung SORT LNPs was not significantly differ-
ent in ApoE/ mice compared to WT mice, indicating that this
serum protein is not functionally important for modulating lung
targeting (Fig. 4). Together, these results indicate that mRNA
delivery is no longer an ApoE-dependent process upon inclusion
of either an anionic lipid or cationic lipid to mDLNP. Rather,
spleen SORT and lung SORT LNPs can enable extrahepatic
mRNA delivery via an ApoE-independent mechanism. It is
plausible that other serum proteins, such as those identified in
the proteomics study, may be responsible for endogenous target-
ing to the spleen and lungs in vivo.
Discussion
Clinical applications of genetic medicines are limited by the
availability of efficacious carriers for the intracellular delivery
of nucleic acid biomolecules to target tissues. Since IV adminis-
tered LNPs have been limited to targeting liver hepatocytes to
date, there is great need to develop drug delivery systems capa-
ble of extrahepatic targeting. Here, we identified mechanistic
factors which underly the tissue-targeting properties of SORT
LNPs for extrahepatic mRNA delivery. We found that a SORT
molecule’s chemical structure uniquely impacts an LNP’s bio-
distribution, apparent pKa, and serum protein interactions.
Furthermore, we provide evidence for a plausible endogenous
targeting mechanism involving PEG lipid desorption, serum
protein adsorption, and receptor binding followed by cellular
uptake to possibly explain the organ-targeting profiles of SORT
LNPs (Fig. 2A). Preferential uptake and activity of SORT LNPs
likely occurs in the organs which highly express the relevant cel-
lular receptors that favorably interact with serum proteins
enriched at the surface of SORT LNPs. Although we cannot
rule out additional mechanisms, we have identified multiple

6 of 10 j PNAS Dilliard et al.

https://doi.org/10.1073/pnas.2109256118 On the mechanism of tissue-specific mRNA delivery by
selective organ targeting nanoparticles
 A B C Target Organ
Wild-type (WT) ApoE-/-
Luminescence
5.0 ✱✱
mDLNP 2×10 8

Luminescence
1.5×10 8
x106
1×10 8

5×10 7

0.3
0
✱
Liver SORT 2.0 1.5×10 9

Luminescence
1×10 9
x107
5×10 8

0.2 0

✱
Spleen SORT 3.0 2.5×10 7

2×10 7

Luminescence
1.5×10 7
x106
1×10 7

5×10 6

0.3 0

1.0 2.5×10 7 ns

ENGINEERING
Lung SORT

Luminescence
2×10 7

1.5×10 7
x106
1×10 7

5×10 6

0.1 0
WT ApoE-/-

MEDICAL SCIENCES
Fig. 4. Extrahepatic mRNA delivery occurs via an ApoE-independent mechanism. (A) Ex vivo bioluminescence produced by functional protein translated
Downloaded from https://www.pnas.org by 124.219.109.112 on May 3, 2023 from IP address 124.219.109.112.

from FLuc mRNA in major organs excised from WT C57BL/6 mice IV injected with mDLNP or liver, spleen, or lung SORT LNPs (0.1 mg/kg FLuc mRNA, 6 h).
The role of ApoE on SORT LNP efficacy varies based on the chemical structure of the included SORT molecule. (B) Ex vivo bioluminescence produced by
functional protein translated from FLuc mRNA in major organs excised from ApoE/ mice IV injected with mDLNP or liver, spleen, or lung SORT LNPs (0.1
mg/kg FLuc mRNA, 6 h). (C) Quantification of total bioluminescence produced by target organs excised from WT and ApoE/ mice treated with mDLNP
or liver, spleen, or lung SORT LNPs (0.1 mg/kg FLuc mRNA, 6 h, n = 3). Data are shown as mean ± SEM. Statistical significance was determined using an
unpaired two-tailed Student’s t test (**P < 0.01, *P < 0.05, ns, P > 0.05). Elimination of ApoE from the serum using genetic knockout results in a marked
reduction of hepatic mRNA delivery by mDLNP and liver SORT LNPs. In contrast, spleen SORT LNPs have enhanced spleen targeting when ApoE is depleted
from the serum, while the efficacy of lung SORT LNPs is unaffected by ApoE elimination.

key factors which define the organ-targeting properties of
SORT LNPs.
The proposed mechanism may have similarity with that of
lipoproteins, which can be considered as a natural class of
nanoparticles for physiological cholesterol transport through
the blood. Acquisition of ApoE in the serum results in uptake
of lipoproteins by liver hepatocytes through receptor-mediated
endocytosis by LDL-R (38). This physiological function of
ApoE is leveraged by DLin-MC3-DMA LNPs for siRNA deliv-
ery to the liver; by binding ApoE in the serum, these nanopar-
ticles can endogenously target LDL-R highly expressed by liver
hepatocytes (12, 28). The hypothesized mechanism we provide
evidence for herein builds upon and extends the scope of this
endogenous targeting concept. Importantly, SORT LNPs
include classes of “out-of-the-box” supplemental molecules that
can tune an LNP’s molecular composition to promote the bind-
ing of distinct protein species to the LNP, which are not
typically observed in the protein corona, and enable mRNA
delivery to cells and organs beyond liver hepatocytes. Because
SORT LNPs include additional classes of molecules that have
not been included in LNPs before, the SORT platform expands
the toolbox of molecules available to control the protein corona
without loss of efficacy. Although a static ex vivo incubation
does not fully recapitulate the dynamic flow environment in
which the protein corona is formed in vivo, studies have not
identified significant differences in the relative abundance of
the key proteins we discovered to associate with SORT LNPs
during a static incubation versus a dynamic incubation (39–41).
It is known that the composition of the protein corona is
influenced by a nanoparticle’s surface chemistry (29, 42, 43).
Since SORT LNPs incorporate SORT molecules with distinct
chemical structures, SORT LNPs likely have different surface
chemistries underneath the PEG layer. The desorption of PEG
lipid from the LNP unveils this surface, enabling specific
plasma protein adsorption. It was recently shown that binding
of ApoE induces rearrangement of lipids in DLin-MC3-DMA
LNPs, promoting the migration of certain lipids from the core
to the shell (44). These observations suggest that protein
adsorption can alter the surface composition of SORT LNPs,
possibly generating unique nanodomains which further pro-
mote the adsorption of distinct proteins to the LNP surface.
The distinct surface chemistry of SORT LNPs can explain
which specific proteins bind to the LNP. β2-GPI is known to
interact with anionic phospholipids, including 18PA (45), and
Vtn has been associated with DOTAP (46). Furthermore,
ApoE, which facilitates liver targeting of LNPs, is highly
enriched in liver SORT LNPs (28). These results further sup-
port the proteomics findings.
Understanding the molecular interactions of these individual
proteins can illuminate why their enrichment might result in
the observed organ-targeting properties of SORT LNPs. Inter-
actions between ApoE and LDL-R drive hepatic accumulation

Dilliard et al. PNAS j 7 of 10

On the mechanism of tissue-specific mRNA delivery by https://doi.org/10.1073/pnas.2109256118
selective organ targeting nanoparticles
 of endogenous lipoproteins during cholesterol metabolism (38),
possibly explaining why ApoE adsorption is a hallmark of many
liver-targeting LNPs, including liver SORT. In a similar manner,
Vtn could bind its cognate receptor, αvβ3 integrin, which is
highly expressed by the pulmonary endothelium but not by liver
cells or other vascular beds (47–49), providing a plausible
explanation for why Vtn promotes lung specificity. Finally, β2-
GPI can bind phosphatidyl serine, an anionic lipid exposed by
senescent red blood cells to promote their filtration from the
circulation in the spleen (50), suggesting how β2-GPI could be
implicated in spleen SORT LNP targeting. These findings sup-
port the functional role of individual proteins on the organ-
targeting properties of SORT LNPs. We acknowledge that the
role of other highly enriched proteins in enhancing organ tar-
geting, such as albumin for liver SORT (51), should not be
discounted. Clusterin, highly enriched in lung SORT, could
potentially play a role in evading the mononuclear phagocyte
system to promote lung targeting (52, 53). Additionally, based
on the key role of ApoE in driving liver targeting, the reduced
binding of ApoE to spleen and lung SORT LNPs could pro-
mote extrahepatic mRNA delivery (54, 55), possibly because of
the displacement of ApoE by apolipoprotein C (53), which is
present in the protein coronas of spleen and lung SORT LNPs
but not liver SORT (SI Appendix, Tables S4–S6).
Our proposed mechanism suggests that tuning the molecular
composition of an LNP serves as a simple and effective method
for manipulating the endogenous ligands which bind the nano-
particle and govern its subsequent biological fate. Our work
clarifies that endogenous targeting via serum proteins acts as a
targeting mechanism that can be generalized to other LNP sys-
tems besides those currently utilized for nucleic acid delivery to
liver hepatocytes. With the key parameters for organ targeting
Downloaded from https://www.pnas.org by 124.219.109.112 on May 3, 2023 from IP address 124.219.109.112.
identified, spleen and lung SORT LNPs may be further opti-
mized for more efficacious tissue-specific delivery. Moving for-
ward, the function of highly enriched serum proteins with
respect to extrahepatic targeting in vivo should be further eluci-
dated. Additionally, identifying SORT molecules that bind
serum proteins distinct from those detailed within this manu-
script may serve as a worthwhile strategy for the discovery of
new LNPs which target other organs beyond the liver, spleen,
and lungs. We view endogenous targeting, that is, engineering
nanoparticle composition to facilitate interactions with distinct
serum proteins and thereby tissue-specific delivery, as an effec-
tive and broad paradigm for designing nanoparticles of various
material compositions to overcome liver accumulation and tar-
get extrahepatic organs.
Materials and Methods
Materials. 5A2-SC8 was synthesized and purified by following published pro-
tocols (25). DOTAP, 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (18PA),
DODAP, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-Dis-
tearoyl-rac-glycerol-methoxy(PEG MW 2000) (DSG-PEG2000) were purchased
from Avanti Polar Lipids. Cholesterol, sucrose, SDS, and phorbol 12-myristate
13-acetate were purchased from Sigma-Aldrich. DMG-PEG2000 was purchased
from NOF America Corporation. The ReadyPrep 2-D Cleanup Kit, 12% Mini-
PROTEAN TGX Precast Protein Gels, 2× Laemmli Buffer, and 10× Tris/Glycine/
SDS were purchased from Bio-Rad. SimplyBlue Safe Stain, Pierce 660 nm Pro-
tein Assay Reagent, Ionic Detergent Compatibility Reagent for Pierce 660 nm
Protein Assay Reagent, Hoechst 33342, Gibco VTN Recombinant Human Pro-
tein Truncated, and dynamic light scattering (DLS) Ultramicro cuvettes were
purchased from Thermo Fisher. Innovative Research CD1 Mouse Plasma
K2EDTA, Innovative Research C57BL/6 Mouse Plasma K2EDTA, Novus Biologi-
cals Recombinant Human ApoE4 Protein, and R&D Systems Recombinant
Mouse ApoH (β2-GPI) protein were purchased from Fisher Scientific. Pur-A-
Lyzer Midi Dialysis Kits (MWCO, 3.5 kDa) were purchased from Sigma-Aldrich.
CleanCap FLuc mRNA (5 moU), CleanCap Cyanine 5 FLuc mRNA (5 moU), and
CleanCap EPO mRNA (5 moU) were purchased from TriLink BioTechnologies.
D-Luciferin (sodium salt) was purchased from Gold Biotechnology. The hEPO
enzyme-linked immunosorbent assay (ELISA) kit was purchased from Abcam.
8 of 10 j PNAS
https://doi.org/10.1073/pnas.2109256118
The ONE-Glo + Tox Luciferase Reporter assay kit was purchased from Promega
Corporation. B6.129P2-Apoetm1Unc/J mice were acquired from the Jack-
son Laboratory.
Nanoparticle Preparation. RNA-loaded LNP formulations were prepared using
the ethanol dilution method. The mDLNP and the SORT formulations were
developed and reported in our previous papers (11, 21, 22, 25). Unless other-
wise stated, all lipids with specified molar ratios were dissolved in ethanol,
and RNA was dissolved in 10 mM citrate buffer (pH 4.0). The two solutions
were rapidly mixed at a ratio of 3:1 (mRNA:lipids, vol:vol) and then incubated
for 10 min at room temperature. To prepare SORT LNP formulations containing
anionic lipids, the anionic lipids were first dissolved in tetrahydrofuran and then
mixed with other lipid components in ethanol. All formulations were named
based on the additional lipids. All LNPs were prepared to have a 40:1 weight
ratio of lipids to mRNA. To prepare mDLNP, a mixture of 23.8 mol % 5A2-SC8,
23.8 mol % DOPE, 47.6 mol % cholesterol, and 4.8 mol % DMG-PEG2K was dis-
solved in ethanol, then mixed with mRNA, and diluted in 10 mM, pH 4.0 citrate
buffer at a volume ratio of 3:1 (mRNA:lipids). SORT LNPs were prepared by add-
ing the SORT molecule to the lipid solution at a given mole %. To prepare liver
SORT, a mixture of 19 mol % 5A2-SC8, 19 mol % DOPE, 38 mol % cholesterol, 4
mol % DMG-PEG2K, and 20 mol % DODAP was dissolved in ethanol, then
mixed with mRNA, and diluted in 10 mM, pH 4.0 citrate buffer at a volume ratio
of 3:1 (mRNA:lipids). To prepare spleen SORT, a mixture of 16.7 mol % 5A2-SC8,
16.7 mol % DOPE, 33.3 mol % cholesterol, 3.3 mol % C14-PEG2K, and 30 mol %
18PA in ethanol was prepared, then mixed with mRNA, and diluted in 10 mM,
pH 3.0 citrate buffer at a volume ratio of 3:1 (mRNA:lipids). To prepare lung
SORT, a mixture of 11.9 mol % 5A2-SC8, 11.9 mol % DOPE, 23.8 mol % choles-
terol, 2.4 mol % C14-PEG2K, and 50 mol % DOTAP was prepared in ethanol,
then mixed with mRNA, and diluted in 10 mM, pH 4.0 citrate buffer at a volume
ratio of 3:1 (mRNA:lipids). After 10 min of mixing the mRNA and lipid solutions,
the LNP formulations were diluted with 1× phosphate buffered saline (PBS) to
0.5 ng/μL mRNA for in vitro assays and characterization of physicochemical
properties. For in vivo experiments, the formulations were dialyzed (Pur-A-Lyzer
Midi Dialysis Kits, MWCO 3.5 kDa, Sigma-Aldrich) against 1× PBS for 2 h. After-
ward, LNPs were diluted with PBS to a final volume of 250 μL/mouse for
IV injections.
Characterization of mRNA Formulations. Size distribution, polydispersity
index, and zeta-potential were measured using DLS (Malvern MicroV model;
He-Ne laser, λ = 632 nm). To measure the apparent pKa of mRNA formula-
tions, the TNS assay was employed. mRNA formulations (60 μM total lipids)
and the TNS probe (2 μM) were incubated for 5 min with a series of buffers
containing 10 mM Hepes, 10 mM MES (4-morpholineethanesulfonic acid),
10 mM ammonium acetate, and 130 mM NaCl (the pH ranged from 2.5 to 11).
The mean fluorescence intensity of each well (black-bottom 96-well plate)
was measured by a Tecan plate reader with λEx = 321 nm and λEm = 445 nm,
and data were normalized to the value of pH 2.5. Typically, the apparent pKa
is defined by the pH at half-maximum fluorescence. Although this method
was useful for estimating LNP global/apparent pKa for most all LNPs, it could
not be used for SORT LNPs containing >40% permanently cationic lipid
because these LNPs are always charged. Therefore, we instead estimated the
relative pKa compared to base LNP formulation (no added SORT lipid) when
50% fluorescence normalized to the lowest fluorescence measurement was
produced. This alternative calculation did not change the pKa for most LNPs
but did allow the estimation of the pKa of SORT LNPs with >40% cationic lipid
that agreed with experimental results for tissue-selective RNA delivery. Thus,
we suggest that the standard TNS assay be used when LNPs contain a single
ionizable cationic lipid and the alternative 50% normalized signal method be
used for systems such as SORT that contain complex mixtures of multiple lipids
harboring a variety of charge states.
In Vivo Luc mRNA Biodistribution. All animal experiments were approved by
the Institution Animal Care and Use Committees of The University of Texas
Southwestern Medical Center and were consistent with local, state, and federal
regulations as applicable. C57BL/6 mice weighing 18 to 20 g were IV injected
with various SORT LNPs containing Cy5 FLuc mRNA at a dose of 0.5 mg/kg
mRNA. Ex vivo imaging (Cy5 channel) was performed 6 h postinjection. Average
fluorescence of the liver, lung, and spleen were measured using Living Image
Software (PerkinElmer) by drawing regions of interest around each organ. The
relative fluorescence for each organ was calculated as the following:
Faverage, organ  Fbackground
Frelative, organ ¼ × 100:
∑ðFaverage, organ  Fbackground Þ
PEG Lipid mRNA Delivery Efficacy. C57BL/6 mice weighing 18 to 20 g were IV
injected with liver, lung, and spleen SORT LNPs using either DMG-PEG2K or
Dilliard et al.
On the mechanism of tissue-specific mRNA delivery by
selective organ targeting nanoparticles
 DSG-PEG2K as the PEG lipid component at a dose of 0.1 mg/kg (n = 3). LNPs
encapsulated either FLuc mRNA or hEPO mRNA. For mice treated with
FLuc mRNA, after 6 h, mice were injected with D-Luciferin (150 mg/kg, intra-
peritoneal [IP]) and imaged by an IVIS Lumina system (PerkinElmer). Total
luminescence of target organs was quantified using Living Image Software
(PerkinElmer). For mice treated with hEPO mRNA, blood was withdrawn
retro-orbitally 6 h after, and serum was recovered. The serum concentration
of hEPO was quantified using an ELISA kit (Abcam) to quantify mRNA deliv-
ery efficacy.
Isolation of Plasma Proteins Adsorbed to SORT LNPs. SORT LNPs were pre-
pared according to the previously described method and were diluted to a
final lipid concentration of 1 g/L with 1× PBS. Mouse plasma was added to
each LNP solution at a 1:1 volume ratio and incubated for 15 min at 37 °C. A
0.7-M sucrose solution was prepared by dissolving solid sucrose in MilliQ
water. The LNP/plasma mixture was loaded onto a 0.7-M sucrose cushion of
equal volume to the mixture and centrifuged at 15,300 g and 4 °C for 1 h. The
supernatant was removed, and the pellet was washed with 1× PBS. Next, the
pellet was centrifuged at 15,300 g and 4 °C for 5 min, and the supernatant
was removed. Washing was performed twice more for a total of three washes.
Following the final wash, the pellet was resuspended in 2 weight % SDS.
Excess lipids were removed from each sample by following the protocol pro-
vided with the ReadyPrep 2-D Cleanup (Bio-Rad). The resulting pellet from the
cleanup step was resuspended in 2× Laemmli buffer. The concentration of
protein in each sample was quantified by Bradford Assay using the Pierce 660
nm Protein Assay Reagent mixed with Ionic Detergent Compatibility Reagent.
SDS–PAGE Characterization of Plasma Proteins. Plasma proteins isolated from
the surface of SORT LNPs were loaded onto a 10% Mini-PROTEAN TGX Precast
Protein Gel at a volume of 10 μL and separated at 200 V. Proteins were visual-
ized by staining the gel for 1 h with SimplyBlue Safe Stain. The gel was
destained using deionized (DI) water overnight and imaged with a Licor Scan-
ner the following day.
Preparation of Plasma Protein Samples for Mass Spectrometry. Plasma pro-
teins isolated from the surface of SORT LNPs were loaded onto a 12%
Mini-PROTEAN TGX Precast Protein Gel at a volume of 10 μL and run into
Downloaded from https://www.pnas.org by 124.219.109.112 on May 3, 2023 from IP address 124.219.109.112.
the gel 1 cm at 90 V. The gel was stained with SimplyBlue Safe Stain for 1
h to fix and visualize the proteins. After destaining for 1 h, the protein
bands were excised using a sterile razor blade and sliced into 1-mm3
cubes. The cubes were added to a 1.5-mL tube that had been rinsed with
1:1 MilliQ water:Ethanol and stored at 4 °C until being submitted to the
University of Texas Southwestern Proteomics Core for mass spectrometry
analysis. For the CD-1 plasma samples, a Thermo Fusion Lumos mass spec-
trometer was used to identify the constituents of the protein corona. For
the C57BL/6 samples, a Thermo QExactive HF mass spectrometer was used
to identify the constituents of the protein corona. The identified proteins
were ordered based on their abundance/Mw. For the proteins constitut-
ing 80% abundance of each protein corona, the proteins were grouped
into functional classes based on information provided by the UniProt
database. The pI of these proteins was computed using the bioinformatic
resource ExPASy (35).
In Vitro Luciferase Delivery. A-498, HuH-7, U-87 MG, or Hep G2 cells were
seeded into white-bottom 96-well plates at a density of 1 × 104 cells per well
and incubated at 37 °C overnight. Undifferentiated THP-1 cells were seeded
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On the mechanism of tissue-specific mRNA delivery by
selective organ targeting nanoparticles
into white-bottomed 96-well plates at a density of 2 × 104 cells per well and
treated with 100 nM phorbol 12-myristate 13-acetate overnight (to enable dif-
ferentiation into macrophages). All cells were cultured in complete medium
as recommended by the suppliers. Then, the old media was replaced with
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ENGINEERING
ImageJ software version 1.53c (NIH). Each treatment group was performed in
duplicates, and five images were taken for each well.
ApoE Knockout Mouse Experiments. SORT LNPs were prepared according to
the previously described method. B6.129P2-Apoetm1Unc/J mice weighing 18 to
20 g were IV injected with mDLNP and liver, lung, and spleen SORT LNPs at a
dosage of 0.1 mg/kg FLuc mRNA (n = 3). As a comparison, C57BL/6 mice weigh-
MEDICAL SCIENCES
ing 18 to 20 g were IV injected with mDLNP and liver, lung, and spleen SORT
LNPs at a dosage of 0.1 mg/kg FLuc mRNA (n = 3). After 6 h, mice were
injected with D-Luciferin (150 mg/kg, IP) and imaged by an IVIS Lumina system
(PerkinElmer). Total luminescence of target organs was quantified using Liv-
ing Image Software (PerkinElmer).
Data Availability. All study data are included in the article and/or SI Appendix.
ACKNOWLEDGMENTS. D.J.S. acknowledges grant support from the Cystic
Fibrosis Foundation (SIEGWA18XX0), the NIH National Institute of Biomedical
Imaging and Bioengineering (R01 EB025192-01A1), the Cancer Prevention
and Research Institute of Texas (RP190251), and the American Cancer Society
(RSG-17-012-01). We acknowledge the Southwestern Small Animal Imaging
Shared Resource, which is supported in part by the Harold C. Simmons Cancer
Center through a National Cancer Institute Cancer Center Support Grant (P30
CA142543). We acknowledge the University of Texas Southwestern Proteo-
mics Core for its assistance with the mass spectrometry proteomics experi-
ments. We especially thank Prof. Andrew Lemoff for insightful advice
regarding mass spectrometry experiments. S.A.D. acknowledges financial sup-
port from the NIH Pharmacological Sciences Training Grant (GM007062) and
the NIH Molecular Medicine Training Grant (GM109776). Cartoons for the fig-
ure were created using Biorender.com.
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