TYPE Review
                                                                                                                   PUBLISHED 27 February 2023
                                                                                                                   DOI 10.3389/fimmu.2023.1129296




                                                The development of highly
OPEN ACCESS                                     dense highly protected
EDITED BY
Kirill Afonin,
University of North Carolina at Charlotte,
                                                surfactant ionizable lipid RNA
United States

REVIEWED BY
                                                loaded nanoparticles
Paolo Macor,
University of Trieste, Italy
Borislav Angelov,                               Ramon González-Rioja 1, Vivian A. Salazar 1, Neus G. Bastús 1,2
Institute of Physics (ASCR), Czechia
                                                and Victor Puntes 1,2,3,4*
*CORRESPONDENCE
Victor Puntes
                                                1
                                                  Institut Català de Nanociència i Nanotecnologia (ICN2), Consejo Superior de Investigaciones
   victor.puntes@icn2.cat                       Científicas (CSIC), The Barcelona Institute of Science and Technology (BIST), Universitat Autònoma de
                                                Barcelona (UAB), Barcelona, Spain, 2 Centro de Investigación Biomédica en Red (CIBER) en
SPECIALTY SECTION                               Bioingenierı´a, Biomateriales y Nanomedicina, Centro de Investigación Biomédica en Red en
This article was submitted to                   Bioingeniería Biomateriales y Nanomedicina (CIBER-BBN), Madrid, Spain, 3 Malalties Infeccioses,
Molecular Innate Immunity,                      Nanopartícules farmacocinétiques, Vall d’Hebron Institut de Recerca, Barcelona, Spain, 4 Institució
a section of the journal                        Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain
Frontiers in Immunology

RECEIVED 21 December 2022
ACCEPTED  14 February 2023
PUBLISHED 27 February 2023                      The long quest for efficient drug administration has been looking for a universal
CITATION                                        carrier that can precisely transport traditional drugs, new genomic and proteic
González-Rioja R, Salazar VA, Bastús NG       therapeutic agents. Today, researchers have found conditions to overcome the
and Puntes V (2023) The development of
highly dense highly protected surfactant        two main drug delivery dilemmas. On the one side, the versatility of the vehicle to
ionizable lipid RNA loaded nanoparticles.       efficiently load, protect and transport the drug and then release it at the target
Front. Immunol. 14:1129296.
                                                place. On the other hand, the questions related to the degree of PEGylation which
doi: 10.3389/fimmu.2023.1129296
                                                are needed to avoid nanoparticle (NP) aggregation and opsonization while
COPYRIGHT
© 2023 González-Rioja, Salazar, Bastús and    preventing cellular uptake. The development of different kinds of lipidic drug
Puntes. This is an open-access article          delivery vehicles and particles has resulted in the development of ionizable lipid
distributed under the terms of the Creative
                                                nanoparticles (iLNPs), which can overcome most of the typical drug delivery
Commons Attribution License (CC BY). The
use, distribution or reproduction in other      problems. Proof of their success is the late approval and massive administration
forums is permitted, provided the original      as the prophylactic vaccine for SARS-CoV-2. These ILNPs are built by electrostatic
author(s) and the copyright owner(s) are
credited and that the original publication in   aggregation of surfactants, the therapeutic agent, and lipids that self-segregate
this journal is cited, in accordance with       from an aqueous solution, forming nanoparticles stabilized with lipid polymers,
accepted academic practice. No use,
                                                such as PEG. These vehicles overcome previous limitations such as low loading
distribution or reproduction is permitted
which does not comply with these terms.         and high toxicity, likely thanks to low charge at the working pH and reduced size,
                                                and their entry into the cells via endocytosis rather than membrane perforation or
                                                fusion, always associated with higher toxicity. We herein revise their primary
                                                features, synthetic methods to prepare and characterize them, pharmacokinetic
                                                (administration, distribution, metabolization and excretion) aspects, and
                                                biodistribution and fate. Owing to their advantages, iLNPs are potential drug
                                                delivery systems to improve the management of various diseases and widely
                                                available for clinical use.

                                                KEYWORDS

                                                ionizable lipid nanoparticles, RNA-loading, drug delivery carriers, pharmacokinetics,
                                                biodistribution and clearance




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González-Rioja et al.                                                                                                   10.3389/fimmu.2023.1129296




Introduction                                                                   nanoscale, allowing for the rapid and widespread development of
                                                                               new, more precise and more functional DDS. Interestingly, the
     The modern concept of drug delivery probably started with                 initial vehicles were called microspheres and then renamed
German Nobel laureate Paul Erlich’s “magic bullet” in 1907, a bullet           nanocarriers, even if some nanocarriers were bigger than
that cannot miss its target (1, 2) and became common with                      some microspheres.
penicillin in the early 20th century (3, 4). The initial idea was to               Among DDS, polymeric nanoparticles (NPs) were the first to be
kill prokaryotes leaving eukaryotes unharmed, thus reducing                    employed, showing significant therapeutic benefits but
damage to the body associated with uncontrolled biodistribution.               accompanied by polymeric toxicity, high cost, and lack of
Soon it was also developed to improve dosing and, with it,                     feasibility for scaling up. Besides, liposomes have traditionally
therapeutic effects. More recently, the concept was actualized for             been the more developed and implemented DDS. Lipid-based
chemotherapy due to its severe side effects, and thus, the firsts Drug          NPs made of lipids and surfactants (amphipathic molecules) are
Delivery Systems (DDS) were developed to improve the transport of              simple and safe, but there is typically a low degree of loading and
antitumoral drugs such as doxorubicin (5). Before, excipients                  structural fragility. They have become an up-and-coming delivery
allowed the drug to solubilize and properly reach their target, but            platform for hydrophobic and hydrophilic substances or a
their capacity was limited to solubility issues, with poor capabilities        combination of both (13, 14). These carriers can penetrate
in directing and protecting the drugs during the journey to the                abnormal tissue, remain for a long time and release their cargo
target. Similarly, if the development of DDS was initially intended            drugs, increasing drug efficacy. For example, in-vitro studies by
for solubilizing common drugs, it rapidly opened the possibility of            Wang et al. (15, 16) have shown the successful target delivery of
loading other substances, such as genetic material or proteins –               resveratrol, a poor aqueous solubility drug and curcumin
antibodies, enzymes, etc.-. These substances cannot be administered            hydrophobic polyphenol in breast cancer. Recently developed
in a free form since they are highly immunogenic and rapidly                   lipid-based formulations included micro and nanoemulsions, self-
biodegraded. Therefore, the full development of DDS will not only              emulsifying formulations, liposomes, lipid NPs and lipid-drug
optimize the pharmacology of current drugs but also dramatically               conjugates. Among the extensive range of lipid formulations, the
expand the pharmacopoeia we have available for the cure, which                 solid lipid nanoparticles (SLNs) (17–19), the nanostructured lipid
will have a clear impact on population health.                                 carried (NLCs) (14, 20), and the lipid-based nucleic acid
     It is important to note that therapeutic effectiveness strongly           therapeutics (21, 22) have probably centered the majority of the
depends on pharmacokinetic aspects, on how drugs travel and                    attention due to their successful activities toward multiples disease
interact through the body, reach their target, perform their intended          models. Other alternatives have been protein aggregates for
effect, are modified (metabolized), and excreted. Up to now,                    unsoluble chemotherapeutic agents such as paclitaxel in
pharmacokinetic principles were based on small drug properties,                Abraxane ® (23) or made of biological molecules such as
where balanced hydrophobicity and hydrophilicity allow it to reach             polylactic-co-glycolic acid (PGLA) NPs, which has been
all corners of the body (6) so that reaching the target was assured at         considered one of the first universal nanocarrier platforms (24, 25).
the expense of undesirable side effects. For example, after cisplatin              In this work, we refer to those NPs made of a mixture of lipids,
injection, a common chemotherapeutic agent, 60% goes to the                    ionizable surfactants, and nucleic acids, which spontaneously form
kidney causing nephrotoxicity, 36% irreversibly binds to albumin               NPs by the electrostatic and hydrophobic collapse in water. These
losing its activity, and only a small fraction of the remaining 4%             lead to highly dense and highly protective NPs, which can be
reaches the tumor cell’s DNA and performs its therapeutic action               singularized as ionizable lipid nanoparticles, iLNPs. These NPs
(7). In this scenario, protecting the drug and carrying it to the              can overcome the main biological barriers to cell transfection,
region of interest is a natural evolution of pharmacology.                     including protection from endonucleases, and RNases, and
     Together with the quest for efficient drug administration,                 selective targeting, when targeting moieties such as antibodies or
chemists and nanochemists have searched for a universal carrier                aptamers are included, to improve the contact with the targeted
that can accommodate many different substances. In addition, the               tissues or cells (26), cell internalization, and intracellular release.
carrier has to be safe, more than its loading. DDSs have to be so safe         Herein we focus on RNA-ionizable lipid NPs. RNA is not only a
that repeated administrations across one person’s life should not be           major player in genetic medicine that needs to be transported, but it
a problem. Also, for practical reasons, they have to be easy to                is also a model of macromolecule that has to be protected until
produce and easy to store.                                                     delivered inside the cells.
     As expected, DDS have been developed and exploited to                         From a historical perspective, ionizable lipids evolved from
enhance the delivery of drugs in treating several diseases showing             years of working with permanently charged cationic lipids for
potential benefits in terms of pharmaceutical flexibility, selectivity,          transfection. The mechanism by which these cationic lipids
dose reduction and minimization of adverse effects (8). In these               capture nucleic acids is through complexing them by ionic
platforms, different drugs, ligands and biomolecules can be                    interaction between the negatively charged phosphate groups on
combined by absorption (9), loading (10), coordination bonding                 the nucleic acid molecules and a positively charged group on the
(11), and entrapment (12) to perform different tasks. While DDS                lipid head, forming nucleotides-lipid complexes-, probably starting
popularization started in the 80s and developed as a full academic             in 1987 with DOTMA, the first bi-layer forming cationic lipid,
and technological discipline, nanotechnology irrupted in the 2000s             specifically designed and used for DNA transfection (27). However,
offering unprecedented control of mater structured at the                      due to its net positive charge, it presented unacceptable levels of


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González-Rioja et al.                                                                                                  10.3389/fimmu.2023.1129296




toxicity at the doses necessary to produce therapeutically relevant          Ionizable lipid nanoparticles:
levels of transgene expression, especially in vivo animal models,
making its transition into clinical praxis impossible (28–30). In
                                                                             Structure, composition
addition, the positive net charge induces plasma protein adsorption          and characterization
and rapid clearance by the immune system, negatively affecting
transfection efficiency (31). They commonly present hemodynamic                   Ionizable Lipid NPs consist of nanometrically sized particles (39)
toxicities, such as the activation of the complement system and an           could be understood as a dense condensation of surfactants, lipids and
increase in blood coagulation time (32–34). Ionizable lipids entered         genetic material. These NPs can be synthesized quickly and efficiently
the field as an answer to this problem. These lipids are a pivotal            to encapsulate genetic material with high efficiency (high density) and
element of the iLNP systems (35) and are characterized by having             have sufficient stability and robustness to travel through the body to
an ionizable functional group in their polar head with an acid-              their destination without being degraded or opsonized, protecting their
dissociation constant (pKa) below 7.0 (36). Their pH-tunable                 cargo, and able to carry out an efficient cytoplasmic delivery of the
charge allows them to be neutral at physiological pH, minimizing             genetic material. Their standard composition consists of 5 different
their cationic burden and toxicity but be protonated at a lower pH           compounds: i) the genetic material which has to be delivered, ii) an
at the maturing, acidified endosome. Their cationic nature inside             ionizable cationic lipid to interact with the genetic material and render
the endosome will help its break and escape by enabling the                  it hydrophobic, iii) a helper amphipathic molecule, usually a
interaction with the anionic membrane lipids (and subsequent                 phospholipid such as DSPC, iv) a helper sterol lipid, in the majority
formation of non-by-layer phases), allowing cytosolic delivery.              of cases Cholesterol, to making the structure more robust, and v) a
Indeed, the ability to activate and deactivate the ionizable lipid           PEG-lipid at the particle surface for surface stabilization and to avoid
cationicity, when necessary, enables it to adapt to the needs of the         NP aggregation and opsonization. The ratio between these components
synthesis (charge on), distribution within the body (charge off) and         varies among different formulations, but typically it can be around:
escape from the endosome (charge on). These positively charged               ionizable lipid 50%mol (60%mass), phospholipid 10%mol (15%mass),
vesicles present the advantages of a liposome-mediated transfection          Cholesterol 38%mol (15%mass), PEG-lipid 1,5%mol (8%mass).
(e.g., fusion with the cell membrane, protection from degradation,               These components interact and spontaneously structure
digestion, opsonization, etc.) and of a cationic-mediated                    themselves through a self-assembly process based on the ethanol
transfection (e.g., complex formation with nucleic acids,                    injection method, which consists of rapid mixing of an ethanol
association with the negatively charged cell surface).                       phase, where the lipids are dissolved, into an aqueous phase, where
     Today, iLNPs have proven to be an efficient vehicle for                  the nucleic acids are dissolved in an acidic buffer. This rapid mixing
effectively delivering RNA inside the cells, opening the doors to            induces the sudden supersaturation of the lipidic molecules, which
gene therapy. Their rapid implantation in several medicines already          leads to burst nucleation and their assembly into NPs, trapping the
approved for human use is an unprecedented success within the                surfactants and genetic material (36). The process by which the
community of nanomedicine, drug delivery and gene therapy.                   genetic material is encapsulated in the lipid structure takes place in
Though, to date, only three different systems of RNA delivery are            the first steps of the synthesis process when the ethanolic phase is
approved by the FDA: antitumoral Patisiran and two RNA COVID-                mixed with the aqueous phase and self-assembly of lipids occurs.
19 vaccines (Pfizer/BionTech and Moderna) (37), and many others               The first force that drives this self-assembly is an electrostatic
are under clinical trial. Encouraged by the successful application of        interaction between the polar head of the positively-charged
the SarCov2 mRNA-lipid vaccines produced by Moderna and Pfizer                ionizable lipids at acidic pH and the negative charges of the
companies, the high biocompatibility of the lipid nanocarriers is            nucleic acid chains at the working pH (typically around 4), and
being explored to treat many other diseases. Thus, in a continuous           the second is the increase in polarity of the lipidic solvent by the
effort to cure other viral diseases, other mRNA vaccines are                 addition of water, expulsing lipidic material from the liquid phase
developing to fight against etiological agents such as                        into the NPs. Thus, as the polarity of the solvent progressively
Cytomegalovirus, Syncytial respiratory, or influenza viruses (Trial           increases, inverted micelle-like structures coalesce, interacting with
number: NCT05085366, NCT05127434, NCT04956575). In the                       the rest of the lipids and surfactants, which at the NP surface closes
field of cancer disease, the pharmaceutical companies Moderna                 the particle in a spherical form making the NPs soluble in water.
and BioNTech are advancing in the commercialization of a potent                  The PEG-lipid anchors to the NP surface’s lipidic domains
therapy based on mRNA vaccines for melanoma; their clinical trials           while extending its water-soluble part away from the NP, forming a
are in phases 1 and 2, respectively (Trial number: NCT0389788,               hydrophilic steric barrier that provides colloidal stability and
NCT04526899). The multifunctional characteristics of the iLNPs               prevents NP aggregation and opsonization (40). The whole phase
also have been advancing in treating solid tumors. In a lack of              mixing and complexation of the nucleic acid is done at pH<<pKa of
successful results compared to the CART cell therapy in liquid               the ionizable lipid so that it is cationic-charged nature, and the
tumors, the BioNTech company has developed an RNA-based                      entrapment efficiency of the nucleic acid is maximized. Afterwards,
CAR-T cell therapy to counter the accelerated growth in the                  once the synthesis is complete, the pH can be adjusted to
Gastric, Pancreatic, Ovarian, and Biliary Tract Tumors (Trial                physiological value since the nucleic acid is already complexed
number: NCT04503278) (38).                                                   and integrated inside the NP. In this way, a neutral charge of the




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vehicle is achieved and significantly minimizes the cationic burden                transfection efficiencies compared to higher substituted moieties,
and its related toxicity that the particles would experiment with                 which increase their steric hindrance as well as affecting the pKa
once they are administered (Figure 1).                                            (49–51). So it is the case of DLin-MC3-DMA, the ionizable lipid part of
     An essential part of this process is how efficient and fast the               the patisiran (Onpattro®) formulation. It is important in the history of
mixing between these two phases is, since it determines size, and                 the development of ionizable lipids and corresponding NPs since, when
size plays a decisive role in NP biodistribution, delivery efficiency,             first synthesized, it exhibited an improvement in the potency of more
and transfection potency (41). As shown by Belliveau et al.in 2012                than two orders of magnitude compared to the previous benchmark
(42) when investigating the influence of the flow rate on the iLNP                  formulation (DLinDMA), which allowed the TTR02 (later known as
particle size, the NP size decreased as the flow rate of the injection of          patisiran) formulation to transition into clinical development (45).
the ethanolic phase into the aqueous phase increased. Indeed, there               Notably, the structure and formulation of DLin-MC3-DMA laid the
is an universal tendency for NPs to decrease surface energy                       groundwork for further iLNPs development. Currently, the search for
(surface-to-volume ratio) by growth. Because of that, the PEG-                    new biodegradable, ionizable lipids with more potency or different
lipid is employed to reduce the surface energy and stabilize the NPs.             properties is at the center of research to advance iLNPs, and this effort
Multiple studies have shown that increasing the molar ratio of the                is yielding a large array of diverse and exciting types of iLNPs to adapt
PEG-lipid, by stabilizing NPs against aggregation yields                          them to organs and diseases (52). The structures and pKas of the
significantly smaller iLNPs, independent of other lipid                            ionizable lipids present in the approved iLNP formulations appear
components (41–43). This indicates that without PEG, or other                     in Figure 2.
similar biocompatible polymers, the NPs would continuously                             They share branched (bulky) lipophilic moieties and
aggregate and grow until complete phase separation.                               hydrophilic amino terminations. This geometry allows for a cone-
     Besides size, as discussed in the previous section, a determining            like conformation of the amphipathic molecules and of the cationic-
functional parameter is the charge, a fundamental aspect of iLNPs,                anionic lipid pair that occurs once the ionizable lipid interacts with
which should be positively charged during iLNPs formation to                      the lipids of the endosomal membrane. This non-by-layer, cone-like
allow nucleic acid complexation, neutral at physiological pH for its              conformation of the amphipathic molecules favors high surface
administration, and positively charged at the acidified, maturing                  curvature and is responsible for iLNP disruption. Once the
endosome for membrane disruption. The pKa value of the ionizable                  endosome and the iLNP have been disrupted, the pH goes back
lipid will be the factor that determines the charge on the iLNP under             to 7, and the iLNP release its cargo so that free mRNA can enter
the different pH conditions, which has to find a balance between                   ribosomes for expression. This disintegration is probably
(44, 45) i) being acidic during RNA trapping, ii) being neutral at                simultaneous with the endosomal disruption and mediated by the
physiological pH, to minimize toxicity and avoid rapid immune-                    many (negatively) charged and detergent-like (amphipathic)
clearance, iii) being as positively charged as possible at late                   compounds inside the cell –indeed, all proteins have hydrophilic
endosome stage to maximize the interaction with the endosome’s                    and hydrophobic domains- interfering with the amphipathic
membrane and its disruption. Also important to consider the effect                molecules of the iLNPs.
produced by absorbing protons during endosome acidification,                            Due to their reduced size and complex and unstable (dynamic)
inducing proton sponge effects.                                                   nature, their characterization can be a serious challenge, especially
     The pKa value in which this balance is optimal is not a universal            in the biological matrix (Figure 3). The standard parameters to
value for all lipids and depends on the iLNPs formulation and the                 evaluate include particle size, surface charge (z-potential), drug
nucleic acid sequences they carry. However, several studies                       content and surface state (composition and conformation). Particle
demonstrated that a pKas between 5,5 and 6,5 tend to show                         size, polydispersity index and charge analysis can be measured by
maximal potency in vivo (21, 46–48). Regarding their chemical                     dynamic light scattering (DLS) and associated z-potential with the
structure, as a rule of thumb, one can say that small head groups of              main advantage of not being time-consuming. Besides, electron
the ionizable lipid, such as dimethylamino-based, show higher                     microscopy allows for high-resolution observation of these NPs.




   FIGURE 1
   Main components of the iLNPs. The lipid components are dissolved in an ethanolic phase, and the nucleic acid is in an acidic buffer. When these two
   phases are efficiently mixed, the ionizable lipid gets protonated and electrostatically interacts with the anionic charges of the nucleic acids while the
   rest of the lipids self-assemble to form the iLNP structure. When the formation is complete, the pH of the batch can be brought to a value higher
   than the ionizable lipid pKa.




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   FIGURE 2
   Structure of the approved Ionizable lipids. From up to down DLin-MC3-DMA, Alc-0315 and SM-102, the ionizable lipids present in Onpattro,
   Comirnaty (BioNTech/Pifizer) and Spikevax (Moderna), respectively, with the pKas: 6.44, 6.09, 6.75, respectively. The number of hydrophobic tails has
   geometrical consequences in the structure of the iLNPs contributing to the determination of size and robustness.




However, they are frail in high vacuum and under the electron                       Information about the internal structure of the systems can be
beam, and therefore cryo-TEM is often employed, providing 2D                    obtained through X-ray (SAXS) and neutron (SANS) small angle
images of stable frozen-hydrated particles. Alternatively, low                  scattering (55). In the past decade, SAXS has been shown to be very
electron beam energy and staining also allow observation of the                 useful at providing information about the fine structure in self-
iLNPs morphology (53). These iLNPs can also be fluorescently-                    assembled soft matter materials, like iLNPs (56–59). Still, the full
labelled for their visualization and quantification in fluorescence               understanding of the nanoscale organization of the lipids and genetic
microscopy and spectroscopy techniques (54). Concentration and                  material in the interior of the particle remains yet to be achieved (60).
composition are also studied using thermogravimetry and                         It has been observed that depending on operational factors or
differential scanning calorimetric analysis (55).                               compositional changes, the synthesis yields different arrangements




   FIGURE 3
   Schematic representation of the main techniques for iLNPs characterization. Note that for the UV-vis absorption spectra, the signal corresponding to
   the nucleic acids present in the sample will account for the majority part of the signal.




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of iLNPs, such as multilamellar structures (61), Ia3d and Pm3n cubic           and tissues, provoking, in too many cases, the lack of -or
phases (62), and other types of non-lamellar structures where the              unexpected- biological results (65, 66). Note that the final size of
RNA molecules are inside aqueous cylinders (63).                               NP, which will reach the cells, is also determined by the interaction
                                                                               with the biological matrix, ultimately determining the distribution
                                                                               and kinetics of delivery.
Ionizable lipid nanoparticles:                                                     Several factors cause the aggregation of colloidal NPs; for
Pharmacokinetics                                                               instance, the initial concentration of NPs, their chemical nature
                                                                               and the ionic strength of the medium (67). The most widely
    It is well accepted that the potential use of iLNPs in medicine is         employed strategy to passivate the surface of nanosized objects, in
determined by the pharmacokinetic (administration, distribution,               general, has been modifying the NP surface with hydrophilic
metabolization and excretion) aspects that govern iLNP behavior,               polymers as polyethyleneglycol (PEG), which acts as a steric
which is different from previous drugs. Pharmacokinetics describes             barrier that minimizes interactions at the NP surface,
what the body does to the drug rather than what the drug does to               indispensable to stabilize the surface and allow NPs to exist
the body; the latter would be pharmacodynamics. The field of                    isolated in solution. This steric barrier “closes” the surface of the
pharmacokinetics has developed with the implantation of small-                 NP, provides colloidal stability and facilitates the small and narrow
molecule drugs as principal therapeutic agents. This is because                size distribution. However, the PEGylation of the NP surface
small molecule drugs can distribute across the body and enter inside           dramatically difficult the interaction with cellular membranes,
the cells. However, everything changes when we pretend to employ               reducing the necessary close contact required for endocytosis.
large and structured substances such as proteins, genetic material or          This double effect has been called the “PEG-dilemma” (68–71).
nanoparticles. Because of that, new pharmacokinetic models, which              Although alternative strategies are proposed, like cleavage of a PEG
describe the behavior of these materials once injected until they are          moiety (72), this problem is majorly addressed by a strategy based
excreted, have to be developed for the proper implementation of                on “reversible PEGylation”, where a lipid-containing PEG slowly
new medical substances and materials into the clinic practice, taking          detaches from the NP surface once administered. This allows to take
into account that the biochemical composition of NPs and its                   advantage of the disposing of the high PEG concentration needed
entrance route into the human body, determines the final activity               for small and monodisperse synthesis and distribution, and the
of these NPs (64). We focus on the existing clinical trials and in-vivo        lower PEG concentration needed to have a good cellular uptake at
experimental models using RNA-lipid carrier systems. Many DDS                  the moment when the NP reaches the target organ. Note that an
enter the body via inhalation, oral ingestion, topical (cutaneous and          increase in the concentration of PEG also influences its
ocular) application, and parenteral administration.                            conformation and protective effects, increasing the circulation
    Herein, we first analyze the different transformations iLNPs                time of the iLNPs (71). Indeed, a completely PEG-covered iLNP
may suffer, such as aggregation, interactions with proteins and                surface will dramatically inhibit the interaction with cells and serum
disintegration/dissolution, and then comment on their                          proteins, modifying circulation time and biodistribution (68, 71).
biodistribution and excretion since the latter strongly depends on             Such PEG-lipids remain integrated into the LNP structure during
the formers. These alterations significantly impact their behavior              formation and under storage conditions, but in the presence of a
and must be considered for their intended use in medicine.                     lipid sink like in plasma, these PEG-lipids are stripped off the
Therefore, research on iLNPs effects should strive to correlate                particle and into de medium, leaving the surface of the iLNP
with how they interact, evolve and are transformed during their                gradually unshielded (45). These PEG-lipids have short alkyl
exposure to the human body. That is, during their Administration,              chains -which act as “hydrophobic anchors”-allowing a
Distribution, Metabolization and Excretion (ADME) phases.                      reasonable desorption rate once they enter blood circulation (71).
                                                                               The proper balance between a fully protected surface for iLNP
                                                                               synthesis and distribution, and a relatively unshielded surface for
                                                                               interactions with cell membranes, is achieved by finding a
Transformations and metabolization                                             compromise between alkyl chain length and PEG-lipid
                                                                               surface concentration.
     Regarding nanosized objects in general, due to interactions                   Special mention deserves the interaction of NPs with proteins,
between NPs and components from the biological medium, NPs                     adjusted by the size and concentration of PEG at the NP surface.
are known to suffer different alterations when applied. Indeed, NPs            When NPs are administrated into the body, they first interact with
are intrinsically out of equilibrium, and transformations such as              biological fluids. Depending on the administration site, the
Ostwald ripening, NPs collapse, and over-grow always tend to                   biomolecules that will interact with the NPs can vary: from lung
occur. When administered, the NP’s environment radically                       surfactants when inhaled to the interstitial fluid when locally
changes from low electrolytic NP concentrated media to a media                 injected into blood plasma following intravenous administration
full of cells, proteins and electrolytes. The main alteration they may         (73). Proteins, which are the most important of these biomolecules,
suffer is the loose of colloidal stability and consequent aggregation.         will adsorb -to some extent- on the NP surface, especially as PEG is
This loss of colloidal stability and subsequent aggregation and                removed, coating the surface with a new layer that will define the
expulsion from the media has a dramatic effect on the abilities of             biological identity of the NP, the so-called “biomolecular corona”,
NPs to travel through the body and to be well dispersed in organs              or “protein corona” (74, 75) which can dramatically alter the surface


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properties of nanosized particles and determine their in vivo fate             characteristics of the surrounding media, including pH and ionic
(76). Already in 2004, it was reported that the presence of proteins           strength, as well as the presence of organic matter (85, 86). Thus,
in physiological media affects the entry and intracellular localization        while small NPs can be preserved in solution in the appropriate (as–
of NPs in cells, thus modulating their potential biological effects and        synthesized) conditions for a long time, they may also be prone to
toxicity. Later on, the formation of a protein corona on top of the            rapid degradation in physiological media. This is why iLNPs can be
NP surface was observed to control biodistribution, uptake and                 kept for a long time in storage conditions while in hours
biological response, transforming them from innocuous to toxic or              disintegrates once in the body. Indeed, it has been reported that it
vice versa. This surface coating can be formed by hundreds of                  is below c.a. 30 nm in diameter (87), where NPs cannot support the
biomolecules, including albumin, apolipoproteins,                              high surface energy anymore and tends to dissolve. The driving
immunoglobulins, coagulation factors, and many others (77).                    force behind dissolution strongly depends on the solubility of the
Some of these biomolecules might associate almost irreversibly                 constituent ions in a given environment and their concentration
with the iLNP surface, in either their native or denatured form,               gradients in the solution. This phenomenon, enhanced at the
affecting, de facto, all subsequent interactions. It has been proposed         nanoscale, is referred to as the Gibbs-Thomson effect, and in NPs
that the corona is comprised of both these tightly bound proteins              manifests as Ostwald ripening, where NPs in solution
(“hard” corona), which presumably bind directly to the iLNP                    spontaneously dissolve or grow due to concentration gradients,
surface with high affinity, and also a looser, more dynamic layer               becoming progressively larger and more polydisperse. Controlled
(“soft” corona) which constantly exchanges with proteins in the                release of matter from an NP is illustrated in the Noyes-Whitney
environment (78). The hard and soft corona are both considered                 equation, which relates the rate of dissolution to the properties of
relevant in determining iLNP interactions with cells (73).                     the components and the dissolution medium. If the released
    Traditionally, for biomedical applications, this corona has only           components are removed from the equilibrium because, for
been conceived as a disruptive effect that hinders the functionality           example, are used in competitive reactions or simple diluted in
of nanoparticles, provoking underestimated side effects like loss of           the body, the system is moved away from the saturation point,
colloidal stability, aggregation, sedimentation or rapid clearance by          reaches sink conditions, and the NP tends towards complete
the immune system. But the protein corona also plays an active role            dissolution. For a given mass, the kinetics of dissolution will be
in deciding the destination organ for delivery and accumulation of             proportional to the specific surface area and the coordination of the
the particles. For iLNPs, it has been shown that in Onpattro, there is         constituents at that surface (which decreases with size). NPs have to
a close relationship between the protein corona and the target organ           release their cargo at the appropriate rate and quantity: larger NPs
of delivery, mediated through the adsorption of Alipoprotein E onto            may release them too slowly and too much, while the smaller ones
the iLNP surface. Authors proposed that binding to ApoE will act as            may release them too fast and an insufficient amount of it. Thus, the
a highly effective targeting ligand by binding to lipoprotein                  reactivity of the NP has to be adjusted to persist more or less inside
receptors on the surface of hepatocytes, triggering the uptake by              the different parts of the body.
hepatocytes (45, 79). This relationship between the protein corona
and the biodistribution of NPs could allow the fate of the particles to
be actively altered. Indeed, the multifunctional physicochemical               Biodistribution and fate of iLNPs
properties of lipids can be designed to target different body tissues.
Min Qiu et al. (80) have achieved a lung-selective delivery in mice                The primary purpose of using these iLNPs is to cross natural
with the use of a series of ionizable lipids containing an amide bond          barriers, interact with the target cell, and deliver the treatment
in the tail which changes the interactions between plasma proteins             efficiently. The first natural barrier the iLNPs need to cross is the
in contrast with other types of lipids, like the ones with an ester            biological fluids, blood or lymph, sweat or tear, and the
bond in the PEG lipidic tail (as those present in the approved                 corresponding extracellular matrix, consisting of macromolecules
formulations), which easily accumulate in the liver (81). These are            and minerals that change in different tissues, compartments, and
exciting and promising results for improving the delivery of iLNP              health status (88). To overcome these barriers, some iLNPs can
beyond these organs, and other relationships between lipid                     interact with particular matrix components that facilitate the
composition and biodistribution should be carried out in the                   interaction with the target cell and the entrance by endocytosis
future. Active targeting by grafting a specific moiety, that is, the            (87). The second natural barrier is the mononuclear phagocyte
ligand of an over-expressed receptor onto the NP surface, is a very            system, capable of recognizing foreign substances and commensal
appealing strategy that, to this day, fails to impact the                      organisms when entering the body, labelling them with opsonins
biodistribution drastically. However, once the NPs have reached                and enhancing uptake by phagocytic cells, such as kupffer cells in
the organs, their uptake can be influenced by targeting moieties (75)           the liver. Basically, if NPs are recognized as a foreign substance, the
                                                                               innate immune response reduces their plasma half-life, decreasing
                                                                               the drug delivery efficiency to the target cell. However, the size range
Degradation and disintegration of iLNPs                                        of NPs is that of recognition of the immune system which is
                                                                               supramolecular structures and molecular patters of few tens of
    It is well-known that NPs can dissolve in certain dispersing               nm, mainly found is viruses and bacteria, in such a way that the
media (82–84). The extent of their dissolution depends not only on             immune response to NPs can be complex from tolerance, to pro-
their intrinsic properties, such as size and shape, but also on                inflammation to immunomodulation (89). As mentioned above, the


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González-Rioja et al.                                                                                                  10.3389/fimmu.2023.1129296




lipid nanoparticle-surface functionalization with PEG minimizes               doxorubicin), where the inclusion of PEG-1,2-distearoyl-sn-
opsonization and therefore the immune response and increases                  glycero-3-phosphorylethanolamine (PEG-DSPE) extended
blood circulation time (90). Nonetheless, the activation of the               circulation time over 4- to a 16-fold enhancement of drug level
immune system has been found against some of the iLNPs                        (101). With this increased circulation time, the Doxil liposome
components as PEG. It has been reported that some patients can                formulation could accumulate more at the tumor site (101),
develop anti-PEG antibodies after a first dose of a PEGylated drug             achieving a more significant therapeutic effect (102, 103). Besides,
(anti-PEG immunoglobulin M (IgM)) (91), leading to rapid NPs                  blood and tissue porosity increases during inflammation, which
clearance in the liver and spleen, removing the drug from                     allows NPs to accumulate in those sites (104).
circulation (92). Fortunately, PEG immunogenicity is not so                        Once the NPs reach their target cells, they must enter and
prevalent and not so aggressive, while the rest of the approved               deliver the cargo. The cytoplasmatic membrane is very robust and
iLNPs components are safe.                                                    impermeable; things naturally enter either transported, typically
     The precise behavior of these materials during their full-life           ions and small molecules, or endocytosed, for proteins and larger
cycle inside the body is still relatively unknown, with controversy           objects. Endocytosis can be divided into pinocytosis (cell
about disparities between the in vitro and in vivo results.                   “drinking”) and phagocytosis (cell “eating”). Pinocytosis,
Additionally, subtle modifications of their nature -composition,               commonly termed endocytosis, is when a fraction of the
size, shape and surface state- may have or not have a strong                  membrane is invaginated, and whatever is on its surface or
influence on their behavior, affecting their interaction with                  around it is trapped. Endocytosis can be receptor-mediated or
proteins (93), aggregation state (94), chemical transformation and            receptor-independent when the cell membrane recycles (105).
degradation (95), and consequently biological responses (96). Once            Alternatively, substances like cationic detergents can permeate
they are stable and do not aggregate when administered, their                 through the membrane, and large liposomes can fuse with the
behavior is very different from small molecules, and they are                 membrane. Both pathways often show toxicity, especially
subordinated to the many-body barriers to protect integrity. In               membrane permeation, being endocytosis the most benign way to
addition to immunogenicity, which is somewhat tolerant to small               introduce substances inside the cell. For the case of iLNP the
molecules, the body is full of physical barriers. Considering                 successful endocytosis process is determined by their size, surface
intravenous administration, it is essential to note that the main             composition, and target cells (99). Once the iLNP reaches the target
blood vessels and capillaries in the body have a continuous lining of         cell and is up-taken by endocytosis (45), the cargo must be released
endothelial cells with pores of 6 nm. Besides, the fenestrated                and reach the cytoplasm. It is important to note that the
capillaries found in the intestine and some endocrine and                     relationship between cellular uptake of NPs and transfection
exocrine glands may have pores up to 50–60 nm, while                          efficiency is not trivial since this is determined by the ability to
discontinuous capillaries, as those found in the liver, spleen and            escape from the endosome. Once engulfed into the cell by the
the bone marrow, have pores between 100–1000 nm, which is where               endocytic process, the early endosomes mature into late endosomes
typically NPs are found (97). Special attention deserves the tight            and fuse with lysosomes decreasing pH and digesting its content for
junctions, including the blood-brain barrier, placenta and testis             recycling (106). When the pH becomes smaller than the ionizable
barrier, where pores smaller than 1 nm have been reported, where              lipid pKa, it becomes positively charged again, enabling an
the hydrophobic nature of LNPs seems to favor translocation (98).             electrostatic interaction between the iLNP and the negatively
In such conditions, small molecules can diffuse in-and-out from the           charged lipids of the cell membranes, as cationic lipids do.
blood vessels into the lymph, while the passive transport of large                 This interaction can have disruptive effects, first on the NP
objects, like proteins and NPs, through these porous is negligible,           structure and integrity, and second, on the endosome membrane,
and they tend to accumulate in organs of the mononuclear                      leading to its disruption (49, 107–109), promoting endosomal
phagocytic system, such as the liver and spleen, which are the two            escape and cytoplasmatic release of the nucleic acid cargo (107).
usual places of NPs fate and accumulation (97, 99).                           As the electrostatic interactions between ionizable lipid and RNA
     It is worth noting here that blood vessel and tissue permeability        increase as pH decreases, it is difficult to imagine the liberation of
is altered during the course of diseases, allowing for the passive            the cationic surfactant associated with the RNA from the NP,
accumulation of NPs in those areas. Indeed, this passive                      indicating the presence of an excess of ionizable lipids in the
accumulation can increase one order of magnitude the                          iLNP formulation. It has been proposed that endosome
concentration of the drug in tissue (11). For example, in solid               disruption is achieved by forming non-bi-layer phases due to the
tumors, their rapid growth results in leaky vessels with large pores          electrostatic interaction between the lipids. Of particular interest is
resulting from a defective angiogenic process, which facilitates NP           the HII inverted hexagonal structure since it’s been shown that this
accumulation in the absence of a functional lymphatic drain. This             phase is not compatible with bilayers, and, as lipids tend to adopt it
phenomenon, known as the Enhanced Permeability and Retention                  once they are mixed, membrane fusion and membrane disruption
effect (100), is widely reported in the literature and has been               events are more likely to occur (35, 49, 107, 109). Regarding
exploited to accumulate nanocarriers in tumors (101) passively.               endosomal disruption, another possible factor playing a
Note that protein aggregates and cell debris are naturally found in           significant role in endosomal disruption would be the proton
solid tumors due to the EPR effect. Thus, by increasing NP                    sponge effect. The proton sponge effect happens when during the
circulation times, this passive accumulation has been employed to             maturation of endosomes, HCl influxes to decrease pH for
deliver therapeutic doses of drugs. It is the case of Doxil (liposomal        digestion. The amine groups of the cationic lipid become


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González-Rioja et al.                                                                                                    10.3389/fimmu.2023.1129296




protonated, capturing protons from the media that resist                       hydrolysis, allowing their degradation into different metabolites that
acidification. As a result, more protons are pumped into the                    are more easily excreted or harmless. The introduction of ester
endosomes, followed by passive entry of more chloride ions by                  bonds, stable at physiological pH, which are hydrolyzed by enzymes
osmosis, the consequent increase in ionic concentration, leading to            once inside the cell or tissues, is a widely used strategy to increase
osmotic water influx and swelling, and up to rupture of the                     the biodegradability of these lipids, reducing their accumulation
endosomes -and endolysosomes-, releasing their contents into the               and possible consequent side effects (52). DLin-MC3-DMA is
cytosol (110). Likely, membrane disruption and proton sponge                   primarily metabolized by hydrolysis to 4-dimethylaminobutyric
effects happen in parallel: while the excess of ionizable lipids               acid (DMBA) and excreted from the body through the urine
perturbs the membrane, RNA-bonded ionizable lipids scavenge                    (ONPATRO Assessment report EMA/554262/2018). On the other
H+ intended for acidification. Once the cargo is delivered, pH                  hand, PEG2000-C-DMG seems not to be extensively metabolized
returns to 7, quenching the cationic charge and leaving the                    and is suggested to be eliminated unchanged through the
genetic material ready for action. Finally, it is also important to            hepatobiliary tract to the feces (ONPATRO Assessment report
note the clear difference that exists typically in cellular uptake             EMA/554262/2018). In the case of ALC-0159, the PEG-lipid
between an in vitro system and an in vivo system, since in vitro               present in Comirnaty, the BioNtech Pfizer vaccine, the primary
conditions, the limitation of a low charge at physiological pH is              route of metabolism appears to be related to amide bond hydrolysis,
much more relaxed, allowing the use of ionizable lipids with higher            yielding N,Nditetradecylamine (COMIRNATY Assessment report
pKa, which can give a higher efficiency than in vivo (3). The                   EMA/707383/2020).
endosomal escape mechanism mediated by the iLNPs, is
illustrated in Figure 4.

                                                                               Current challenges for ionizable lipid
Excretion of iLNPs                                                             nanoparticles to be used in medicine
     Once the NP disintegrates, its components have to be processed                Regarding DDS, a common limitation is dosing. In systemic
and excreted from the body. DSPC and Cholesterol are both part of              delivery, taking into account that there is a limitation of volume that
cell membranes; therefore, degradation and metabolism of these                 can be injected in a single shot into the body (e.g. 10 mL/Kg for
products will occur integrated within the natural processes of the             intravenous injection in mammals), the therapeutic dose might not
cell. Nucleases metabolize the delivered genetic material to                   be reached unless iLNPs have been previously concentrated
nucleotides of various lengths. In the case of the ionizable lipids,           because, during synthesis and storage, high concentrations lead to
the three (DLin-MC3-DMA, SM-102 and ALC-0315) present in the                   larger and polydisperse particles. Besides, in vitro simple tests, as
approved formulations have ester bonds, which are degraded by                  those assessed in monolayer cell cultures, do not consider important




   FIGURE 4
   Schematic representation of endosomal scape mediated by iLNPs. 1) The first interaction between iLNPs and the target cell activates the endocytosis
   process, and 2) subsequently, the nanocarrier is encapsulated by the endosome. 3) As proton pumps reduce the pH of the endosome, the ionizable
   cationic lipid becomes progressively protonated (positively charged). As a result, it can interact with the endosome’s membrane anionic lipids to
   produce non-bilayer structures, disrupting the endosomal membrane and the iLNP structure and 4) releasing the mRNA into the cell cytoplasm.




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González-Rioja et al.                                                                                                    10.3389/fimmu.2023.1129296




factors such as organ vascularity, organ penetration and other                 diabetic retinopathy, occur at the retina level in the posterior part of
differential properties given by the organ microenvironment                    the eye. Here iLNPs provide sustained gene expression that could
(111), something that is not so critical for small molecule drugs              overcome these limitations (121). Recently, Wang and co-workers
but that is determinant for NP biodistribution and effects. Similarly,         employed an iLNP to generate a cell-specific gene delivery system
different in vivo models show variations between them, such as the             with sustained gene expression in the eye tissue (122).
different sizes of pores in vessels or different immunological
responses, which may result in different efficacy for the same NPs
depending on the model being used. Also, to be able to work with               Cutaneous application
animals with implanted tumors, SCID (Severe Compromised
Immunodeficiency) models are often used, which lack a fully                         As an external barrier, skin homeostasis is a complex process.
functional immune system, significantly when its size ranges                    Therefore, a broad spectrum of topical medication treats diverse
between virus and bacteria. In this context, 3D cell cultures have             cutaneous diseases. Herein, iLNPs are a promissory agent to
been proposed as suitable models to study the behavior of iLNPs in             improve drug penetration through the stratum corneum.
a particular environment due to the possibility of mimicking a                 Although to date, no iLNP has been reported for commercial
controlled extracellular matrix and different organ regions (112).             cutaneous applications, the efficient encapsulation of distinct
     Regarding the penetration and distribution inside organs, it is           cosmetic agents such as oils, vitamins, and antimycotic and anti-
known that macromolecular carriers fail to penetrate deep into                 age compounds are the most common pharmaceutical
organs and tumors and are generally accumulated just some                      approximations (123). As an approach to treating a chronic
micrometers away from the vessels that transported them (113–                  wound, Gainza and co-workers loaded the recombinant human
115). Furthermore, solid tumor penetration is also challenging for             epidermal growth factor (rhEGF) into an iLNP showing an essential
small molecules (111). Finally, as carriers, they could carry the              recovery in healing grade and wound maturity (124).
substance to the wrong place with high precision, producing
unexpected side effects. Also, regarding safety, as the vehicle aims
at universality, it will be applied repeatedly to the individual across
                                                                               Conclusions and future perspectives
their life, which may end up triggering pro-inflammatory immune
responses towards the iLNPs or some of their components, for
                                                                                   The iLNPs are a promissory concept to explore the efficient
example, PEG (116, 117). Beyond parenteral application, in the
                                                                               drug delivery of a broad range of substances, including genetic
following, we list some of the current iLNPs developments showing
                                                                               material, proteins and other NPs, decreasing collateral effects in
the universality of the vehicle’s ability to transport different
                                                                               healthy tissues and cells. However, despite the current knowledge
sequences through different organs and portals of entry:
                                                                               on the subject being scattered and too heterogeneous, many recent
                                                                               discoveries and advances preclude the inevitable success of iLNPs.
                                                                               Indeed it has been predicted that Nanoparticulate DDS will be the
Oral ingestion                                                                 most innovative and crucial cornerstones in pharmaceutical
                                                                               research, with a tremendous economic impact, where the
    The most preferred administration route for medical treatments
                                                                               possibility to adjust their physicochemical characteristics and
is oral delivery. Several studies suggest that the lipid nanoparticle
                                                                               increase the interaction with the target cell, and control the
composition enchases their biodisponibility in the gastrointestinal
                                                                               escape from the endosomal compartment allow for precision
mucosae following the natural entrance in the digestion process
                                                                               medicine. Thus, novel iLNPs will be developed with growing
(118). Additionally, studies on the siRNA- lipid nanoparticle
                                                                               interest and improved pharmacokinetic profiles compared to
stability showed that the LPNs remained potent and stable after
                                                                               standard drug delivery.
exposure to solutions with pH values as low as 1.2. However, future
                                                                                   They are simple to produce, can load hydrophobic and
research needs to increase cargo delivery and improve the
                                                                               hydrophilic substances, and are easily functionalizable with target
effectiveness once mucin is present in the intestines (119).
                                                                               moieties to ensure better precise delivery once an organ is reached.
Interestingly, new research conducted by Sung et al. (120),
                                                                               Indeed, encapsulating the genetic material into iLNPs is one of the
demonstrates the efficiency deliver of IL-22 mRNA -loaded iLPS
                                                                               fastest currently developing pharmaceutical technology. This
administrated by oral route in a mouse model of acute colitis. The
                                                                               success is the result of its clever design, which, in a biomimetic
results showed a high level of expression of interleukin 22, the
                                                                               way, adapts to the different barriers and biological difficulties
recovery of body weight, and an accelerated healing process in the
                                                                               encountered along the way. All in all, in the near future, iLNPs
colon tissues.
                                                                               may result in a quantum leap in medicine history.


Ocular applications
                                                                               Author contributions
    The main medicaments designed for ocular applications act in
the anterior part of the eye. However, some degenerative processes,               RG-R, VS, NB, and VP contributed to the design and
such as age-related macular degeneration, retinitis pigmentosa, and            implementation of the bibliographic research, the analysis and



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González-Rioja et al.                                                                                                                             10.3389/fimmu.2023.1129296




discussion of the literature and the writing of the manuscript. All                            Conflict of interest
authors contributed to the article and approved the submitted version.
                                                                                                   The authors declare that the research was conducted in the
                                                                                               absence of any commercial or financial relationships that could be
Acknowledgments                                                                                construed as a potential conflict of interest.

    We acknowledge financial support from the Spanish Ministerio                                Publisher’s note
de Ciencia, Innovació n y Universidades (MCIU) (RTI2018-099965-
B-I00, AEI/FEDER,UE) proyectos de I+D+i de programació n                                          All claims expressed in this article are solely those of the authors
conjunta internacional MCIN/AEI (CONCORD, PCI2019-                                             and do not necessarily represent those of their affiliated
103436) cofunded by the European Union and Generalitat de                                      organizations, or those of the publisher, the editors and the
Catalunya (2017-SGR-1431). ICN2 is supported by the Severo                                     reviewers. Any product that may be evaluated in this article, or
Ochoa program from Spanish MINECO (SEV-2017-0706) and is                                       claim that may be made by its manufacturer, is not guaranteed or
funded by the CERCA Programme/Generalitat de Catalunya.                                        endorsed by the publisher.




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