RNA - Image by Yikrazuul CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=10242870
During the COVID-19 pandemic,
the success of mRNA vaccines has
greatly propelled the development of mRNA therapeutics. mRNA is a negatively
charged nucleic acid that serves as a template for protein synthesis in
ribosomes. Despite its utility, the instability of mRNA necessitates
appropriate carriers for in vivo delivery. Currently, lipid
nanoparticles (LNPs) are the most mature approach for
protecting mRNA from degradation and enhancing its intracellular delivery. To
further optimize the therapeutic efficacy of mRNA, researchers have developed a
series of site-specific LNPs. Through local or systemic administration, these
site-specific LNPs can accumulate in specific organs, tissues, or cells,
allowing mRNA to be delivered to specific cells and enabling local or systemic
therapeutic effects. These methods not only improve the efficiency of mRNA
therapy but also reduce off-target adverse reactions.
Figure 1. Site-specific lipid nanoparticles for
mRNA delivery. (X, Xiao.; et al, 2023)
LNPs are composed of different lipids,
making quality control easier compared to other types of carriers such as
macromolecules or viruses. Additionally, it is easy to develop new LNPs by
altering lipid structures or compositions, increasing the versatility of LNPs.
Typically, LNPs consist of four types of lipids, including ionizable lipids,
helper lipids, cholesterol,
and PEGylated
lipids. Ionizable lipids are the most crucial component, responsible
for the encapsulation of nucleic acids during formulation and endosomal escape
after cellular uptake. The head groups of ionizable lipids carry a positive
charge under acidic conditions, allowing for electrostatic interactions with
negatively charged nucleic acids and enhancing encapsulation efficiency. The
hydrophobic alkyl chains of ionizable lipids are unsaturated, forming a
hexagonal lipid phase and enhancing the escape of mRNA from the LNP after cell
entry. Apart from ionizable lipids, the other three components are also
essential for LNPs. Helper lipids and cholesterol increase LNP stability and
enhance cellular internalization. PEGylated lipids are crucial for
improving LNP stability and prolonging circulation time, enhancing delivery
efficiency after intravenous injection.
LNP for
Localized Delivery of Specific Locations
Localized administration of LNP to specific
sites is crucial for the delivery of mRNA. Various administration routes have
been used to achieve site-specific delivery of LNP, including oral
administration, inhalation, and local injection (intramuscular, intratumoral,
and intracerebral injection).
1) Oral
administration is a widely used, convenient, and well-established route of
administration. However, it poses significant challenges for mRNA delivery due
to the susceptibility of mRNA molecules to nucleases and the harsh acidic
environment of the gastrointestinal tract. In this regard, researchers at the
Georgia State University Center for Diagnostics and Therapeutics prepared LNPs
loaded with IL-22 encoding mRNA for oral administration. The LNP consisted of
phosphatidic acid, monoolein, and dioleoyltrimethylammonium propane. In a mouse
model, oral administration of IL-22 mRNA-loaded LNP significantly increased
IL-22 expression in the colonic mucosa and accelerated the healing of colitis.
These results suggest that oral delivery of mRNA-loaded LNP is a viable
strategy for treating gastrointestinal diseases by reestablishing the
intestinal microenvironment.
2) Inhalation
is also a preferred route of administration. Due to its large absorptive
surface area and abundant pulmonary blood flow, inhaled drugs can rapidly
transfer to the bloodstream, thereby increasing their bioavailability. However,
achieving precise dosing through inhalation is challenging, and clearance in
the airways adds to the difficulty. Inhaled aerosols undergo two types of
clearance based on their size and deposition region. Aerosols larger than 5 μm
are cleared by mucociliary clearance, resulting in the clearance of over 80% of
the aerosols. In contrast, aerosols smaller than 5 μm are cleared by
macrophages. Nebulization is the most common method of inhalation. Although
advanced nebulization techniques can facilitate drug delivery to the lungs,
shear forces may disrupt the structure of nanoparticles, and physical barriers
in the airways can hinder their reach to the target. To address these issues,
researchers at the Georgia Institute of Technology’s Department of Biomedical
Engineering reported a screening approach to identify optimal LNP components
for mRNA delivery via nebulization. The results showed that a higher molar
ratio of PEG lipids in LNPs improves the performance of cationic assist lipids.
They prepared an mRNA-loaded LNP for pulmonary delivery, composed of a modified
PEI compound 7C1, cholesterol, DMG-PEG 2000,
and cationic lipid DOTAP.
A high percentage (55%) of DMG-PEG2000 enhanced pulmonary delivery of the LNP.
Subsequent studies found that this LNP, when loaded with mRNA encoding a
broadly neutralizing antibody against coagulase, could protect mice from H1N1
influenza infection.
3) Local
injection refers to the administration of drugs to a small area of the body.
These drugs can affect not only the local site but also diffuse or transfer to
the bloodstream, exerting systemic therapeutic effects. The COVID-19 vaccine is
a typical example of a muscle injection that works systemically after
administration. Currently, more mRNA-based research is focusing on local
injections, which can provide targeted therapy at the injection site while
minimizing the potential for off-target effects. Researchers at Tel Aviv
University’s Laboratory of Precision NanoMedicine developed an LNP-based
CRISPR-Cas9 mRNA delivery system for the treatment of Duchenne Muscular
Dystrophy (DMD) caused by functional loss-of-function mutations in the
dystrophin gene. The CRISPR-Cas9 system can be used to restore dystrophin
protein expression and has a persistent effect, requiring an appropriate
delivery vehicle to deliver Cas9 mRNA and
sgRNA to target cells. The researchers synthesized ionizable lipids with a
triple hydrophobic alkyl tail and used them as components for formulating LNP
vesicles to deliver Cas9 mRNA and specific sgRNA. This LNP showed good local
therapeutic effects after intramuscular injection, while systemic therapeutic
effects were observed after limb perfusion in a DMD mouse model, representing a
promising carrier for delivering CRISPR-Cas9 gene editing tools.
Figure 2. Intratumoral injection of LNP loaded
with OX-40 mRNA to enhance cancer immunotherapy. (W, Q, Li.; et al. 2021)
Organ-Specific LNP for Administration
via Vein
Intravenous injection is another standard
route of administration, with a bioavailability of 100%. The bio-distribution
of LNPs after intravenous injection is crucial, as off-target delivery of mRNA
can lead to adverse reactions and greatly reduce therapeutic efficacy. In
recent years, a lot of research has been focused on organ-specific LNPs.
1) Liver
targeting: Commercially available LNPs carrying Onpattro siRNA primarily
accumulate in the liver after intravenous injection. The targeting mechanism is
achieved by using a 14-C lipid with DMG-PEG2000 in the LNP formulation. Due to
the short 14-C chain and weak binding to the LNP surface, this lipid quickly
dissociates from the LNP surface in circulation. Subsequently, apolipoprotein
E (ApoE) binds with the LNP to form a corona, which is recognized by
low-density lipoprotein receptors on liver cells and promotes LNP internalization.
Many studies have followed the prescription of Onpattro to prepare
liver-targeted LNPs. These mRNA-loaded LNPs deliver specific mRNA to the liver
for the treatment of various liver-related diseases, including infectious
diseases, liver fibrosis, cancer, and genetic disorders.
2) Spleen or
lung targeting LNP: Researchers at the University of Texas Southwestern Medical
Center reported that selective organ targeting capability can be achieved by
altering the composition and components of LNPs. They named this passive
targeting LNP as Selective Organ Targeting (SORT) nanoparticles. By adding a
fifth lipid to the existing prescription of traditional four-component LNPs and
adjusting the ratio of this lipid, selective targeting to different organs such
as the liver, lung, and spleen can be achieved. After in vivo screening, they
found that the traditional four-component LNPs mainly accumulate in the liver
and partially in the spleen, while the introduction of the fifth lipid changes
the distribution, which is dependent on the proportion of the fifth lipid. When
using a cationic
lipid as the fifth component, the distribution of LNPs in the
liver decreases as the proportion of the fifth lipid increases. Regarding the
distribution of LNPs in the lung and spleen, when the proportion of the
cationic lipid is higher than 50%, more than 90% of the LNPs accumulate in the
lung. If an anionic lipid is chosen as the fifth lipid, the amount of LNPs
distributed to the liver decreases with an increase in the proportion of the
anionic lipid, and there is almost no LNP distribution to the lung. For the
distribution of LNPs in the spleen, the maximum distribution in the spleen is
achieved when approximately 30% of anionic lipid is used. If the fifth lipid is
an ionizable cationic lipid, the distribution of LNPs to the liver increases
with an increase in the proportion of the fifth lipid and then decreases.
Addition of 20% ionizable cationic lipid achieves the maximum distribution to
the liver. In summary, the introduction of the fifth lipid greatly influences
the bio-distribution of LNPs in major organs, and 50% cationic SORT lipid, 30%
anionic SORT lipid, and 20% ionizable cationic SORT lipid promote the
distribution of LNPs in the lung, spleen, and liver, respectively.
3) Bone
targeting LNP: In recent years, there has been an increasing incidence of
skeletal diseases and abnormalities, and the medical demand for novel
biomaterials that can target the bone microenvironment remains unmet. It has
been reported that LNPs loaded with siRNA can be systematically delivered to
the bone marrow, but passive diffusion still presents a challenge for
bone-targeted drug delivery. Inspired by the fact that ligand substitution can
achieve targeted LNP delivery, researchers at the University of Pennsylvania’s
Department of Bioengineering synthesized a series of lipids based on
bisphosphonates (BPs). These lipids can strongly bind to calcium ions on
hydroxyapatite on the bone surface through chelation and achieve long-term
retention in the bone microenvironment. They prepared LNPs loaded with luciferase mRNA
using different types of BP lipids and screened them through cell experiments.
The results showed that 490BPC14-LNP had the best transfection efficiency.
Additionally, bone morphogenetic protein-2 (BMP-2) mRNA was loaded into the
LNP, which exhibited excellent bone microenvironment targeting capability and
high expression of BMP-2 in bone tissue after intravenous injection.
Cell-Specific LNP for Systemic Delivery
To achieve more specific mRNA delivery
systems, cell-targeted LNPs have been developed that are designed to be
taken up by specific cells and induce protein expression in these cells.
Typically, this cell-specific uptake is mediated by ligand-receptor
interactions, but recent research has found that this interaction can also be
achieved by altering the lipid structure within the LNP.
1) White blood cell-targeted LNP
In order to achieve cell-specific mRNA
therapy for inflammatory bowel disease (IBD), researchers prepared
antibody-modified LNP to specifically deliver IL-10 mRNA to Ly6c+ inflammatory
white blood cells. Ly6c+ cells serve as targets for treating IBD, and the
immune-suppressive cytokine IL-10 has been reported to inhibit IBD. To induce
long-term IL-10 production, IL-10 mRNA-loaded LNP was first prepared, followed
by incubation with anchored secondary scFv enabling targeting (ASSET) micelles
at 4°C for 48 hours and subsequent incubation with anti-Ly6c monoclonal
antibody for 30 minutes. ASSET allows LNP to be bridged with targeting
antibodies under mild conditions. Upon intravenous injection in mice with
dextran sulfate sodium-induced colitis, this surface-modified LNP actively
targeted Ly6c+ white blood cells and induced IL-10 production, significantly
suppressing inflammation in the colon.
2) T cell-targeted LNP
Cardiac fibrosis is caused by excessive
production of extracellular matrix by cardiac fibroblasts. Limiting fibrosis
progression through anti-fibrotic therapy has been unsatisfactory for treating
cardiac fibrosis. Recently, researchers at the University of Pennsylvania,
developed a CAR-T therapy to remodel fibrosis by specifically delivering mRNA
to T cells. Fibroblast activation protein cardiac fibroblast activation protein
(FAP)-CAR, which recognizes FAP-positive cells and induces cell death, was
encoded into mRNA and then loaded into LNP modified with anti-CD5 antibodies to
achieve specific targeting of CD5+ T cells. FAP-CAR is expressed on the surface
of T cells and specifically recognizes FAP-positive cells, reducing cardiac
fibrosis. They also studied the therapeutic efficacy in a mouse model of
angiotensin II/phenylephrine-induced cardiac injury. Systemic administration of
CD5-targeted LNP loaded with FAP-CAR mRNA significantly reduced fibrosis and
improved heart function. This represents a great success of mRNA-loaded LNP in
treating heart disease.
Figure 3. Anti-CD5 antibody-modified LNP was
used for in vivo construction of CAR T cells specific for fibroblast activation
protein. (J, G, Rurik.; et al, 2022)
3) Kupffer cell and liver sinusoidal
endothelial cell (LSEC)-targeted LNP
Kupffer cells play an important role in
liver inflammation and immune tolerance, primarily by engulfing and clearing
particles. Increasing the size of LNP and modifying the surface with
hydrophobic molecules enhances the cellular uptake by Kupffer cells and
promotes immune regulation. LSECs are located in liver sinusoids and are
responsible for blood filtration, metabolic regulation, antigen presentation,
and lipid metabolism. To achieve cell-specific mRNA delivery to Kupffer cells
or LSECs, researchers at the Georgia Institute of Technology applied various
types of cholesterol in the formulation of LNP and found that the structure of
cholesterol greatly affected the targeting ability of LNP.
References
1. X, Xiao.; et al. Recent Advances in
Site-Specific Lipid Nanoparticles for mRNA Delivery. ACS Nanosci.
Au. 2023, 3(3): 192-203.
2. W, Q, Li.; et al. Biomimetic
Nanoparticles Deliver mRNAs Encoding Costimulatory Receptors And Enhance T Cell
Mediated Cancer Immunotherapy. Nature Communications. 2021,
12: 7264.
3. J, G, Rurik.; et al. CAR
T Cells Produced in vivo to Treat Cardiac
Injury. Science. 2022, 375(6576): 91-96
Author: By Carrier Tayloy, R&D director
of BOCSCI
Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)
