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Targeted nanoparticles with novel non-peptidic ligands for oral delivery
Anne des Rieux, Vincent Pourcelle, Patrice D. Cani, Jacqueline Marchand-Brynaert, Veronique Preat
PII: S0169-409X(13)00029-XDOI: doi: 10.1016/j.addr.2013.01.002Reference: ADR 12435
To appear in: Advanced Drug Delivery Reviews
Accepted date: 30 January 2013
Please cite this article as: Anne des Rieux, Vincent Pourcelle, Patrice D. Cani,Jacqueline Marchand-Brynaert, Veronique Preat, Targeted nanoparticles with novelnon-peptidic ligands for oral delivery, Advanced Drug Delivery Reviews (2013), doi:10.1016/j.addr.2013.01.002
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Targeted nanoparticles with novel non-peptidic ligands for oral
delivery
Anne des Rieux1, Vincent Pourcelle
2, Patrice D. Cani
3 Jacqueline Marchand-
Brynaert2, Véronique Préat
1*
1. Université catholique de Louvain, Louvain Drug Research Institute, Pharmaceutics and Drug Delivery
Research Group, Avenue E. Mounier, 73 B1.73.12, 1200 Brussels, Belgium
2. Université catholique de Louvain, Institute of Condensed Matter and Nanosciences, Laboratory of Solids,
Molecules and Reactivity, UCL L4.01.02, Place Louis Pasteur 1, 1348 Louvain-la-Neuve, Belgium
3. Université catholique de Louvain, Louvain Drug Research Institute, Metabolism and Nutrition Research
Group, Avenue E. Mounier, 73 B1.73.11, 1200 Brussels, Belgium
* corresponding author
Université catholique de Louvain
Louvain Drug Research Institute
Pharmaceutics and Drug Delivery Research Group
Avenue E. Mounier, 73 B1.73.12
1200 Brussels
Belgium
Tel : +32 2 764 73 09
Fax : +32 2 764 73 98
Email : [email protected]
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Abstract
Orally administered targeted nanoparticles have a large number of potential
biomedical applications and display several putative advantages for oral drug
delivery, such as the protection of fragile drugs or modification of drug
pharmacokinetics. These advantages notwithstanding, oral drug delivery by
nanoparticles remains challenging. The optimization of particle size and surface
properties and targeting by ligand grafting have been shown to enhance nanoparticle
transport across the intestinal epithelium. Here, different grafting strategies for non-
peptidic ligands, e.g., peptidomimetics, lectin mimetics, sugars and vitamins, that are
stable in the gastrointestinal tract are discussed. We demonstrate that the grafting of
these non-peptidic ligands allows nanoparticles to be targeted to M cells, enterocytes,
immune cells or L cells. We show that these grafted nanoparticles could be promising
vehicles for oral vaccination by targeting M cells or for the delivery of therapeutic
proteins. We suggest that targeting L cells could be useful for the treatment of type 2
diabetes or obesity.
Key words: Nanoparticles, oral delivery, ligand, non peptidic ligand, targeting
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Table of Content
1. INTRODUCTION ------------------------------------------------------------------------------- 4
1.1 CARRIERS AND GENERAL PROPERTIES -------------------------------------------------------------------- 4
1.2 MECHANISMS OF ORAL UPTAKE OF NANOPARTICLES ---------------------------------------------------- 5
1.3 TARGETING CELL RECEPTORS BY GRAFTING SPECIFIC LIGANDS TO THE NANOPARTICLE SURFACE ----- 7 1.3.1 Importance of receptor-mediated targeting ---------------------------------------------------------- 7 Limitations of peptidic and proteinic ligands ---------------------------------------------------------------- 7 1.3.2 ------------------------------------------------------------------------------------------------------------------------- 7 1.3.3 Advantages of non-peptidic small molecule ligands ------------------------------------------------ 8
2. GRAFTING OF NON-PEPTIDIC LIGANDS TO POLYMERS COMPOSING NANOPARTICLES --- 9
2.1 STRATEGIES FOR INTRODUCING NON-PEPTIDIC LIGANDS ONTO NANOPARTICLES ---------------------- 9
2.2 GRAFTING OF NON-PEPTIDIC LIGANDS TO A POLYMER ------------------------------------------------ 10
2.3 ANALYTICAL METHODS TO EVALUATE NON-PEPTIDIC LIGAND GRAFTING ----------------------------- 12
3. TARGETING OF NANOPARTICLES TO DIFFERENT INTESTINAL CELL TYPES --------------- 13
3.1 M CELLS -------------------------------------------------------------------------------------------------- 13
3.2 ENTEROCYTES -------------------------------------------------------------------------------------------- 17
3.3 GOBLET CELLS -------------------------------------------------------------------------------------------- 18
3.4 DENDRITIC CELLS ---------------------------------------------------------------------------------------- 18
3.5 ENTEROENDOCRINE CELLS AND THE IDENTIFICATION OF NOVEL POTENTIAL LIGANDS FOR
NANOPARTICLE DELIVERY --------------------------------------------------------------------------------------- 20
4. CONCLUSIONS REGARDING THE POTENTIAL BIOMEDICAL APPLICATIONS OF THE ORAL
DELIVERY OF NANOPARTICLES TARGETED WITH NON-PEPTIDIC LIGANDS ------------- 23
5. REFERENCES -------------------------------------------------------------------------------- 24
6. FIGURE LEGENDS --------------------------------------------------------------------------- 32
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1. Introduction
1.1 Carriers and general properties
The intestinal epithelium allows the absorption of nutrients, electrolytes and fluids
while acting as a defense system and an efficient barrier to macromolecules, toxins
and microorganisms. When particulate drug delivery systems are administered orally,
they are exposed to the harsh environment of the gastrointestinal tract. They must
cross the mucus layer before they come into contact with intestinal cells. Moreover,
they do not permeate easily across the intestinal barrier. However, fine-tuning the
size, shape and surface properties of the delivery system allows the enhancement of
drug-loaded nanoparticle (NP) absorption. A specific targeting effect can be achieved
by conjugating the ligands of receptors that are expressed at the apical site of
intestinal epithelial cells to the surfaces of the particulate delivery system. In other
words, the targeted delivery of drugs through the oral administration of particles
requires “smart” vehicles that are able to tolerate different conditions and cross
various barriers to entry via specific interactions with the targeted cell surface.
The general physicochemical properties required for orally delivered particles to
reach the attended sites of entry (e.g., gut-associated lymphoid tissue (GALT) or
enterocytes) or the desired sites of action (e.g., cancer cells or a zone of inflammation)
are beyond the scope of this paper and have been reviewed elsewhere [1-6]. This
review will focus on cell-NP-specific interactions that could be induced by specific
ligands that are present on the surfaces of the carriers. Nevertheless, we provide a
short overview of the influence of NP surface properties on their uptake by intestinal
cells because adequate surface chemistry promotes NP stability in gastrointestinal
(GI) tract fluids, increases their transit time in the gut and finally enhances their
ability to cross the mucosal barrier, including the mucus. This review will help to
identify the potential benefits of receptor targeting.
The nanometric particle size is of prime importance to allow the particle to cross the
mucus, avoid rapid clearance and finally enhance transmucosal transport. The
external shell should have a good hydrophilic and hydrophobic balance. Hydrophilic
molecules (i.e., PEGs, carbohydrates) are useful for the stabilization and diffusion of
the particles in the fluids and mucus, whereas a hydrophobic coating enhances cellular
and lymphatic uptake. The chemical functions of the materials play an important role
in the surface charge, the overall stability of the system and the non-specific
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interactions with surrounding media. Neutral surfaces facilitate the crossing of the
mucus barrier, whereas positively charged surfaces enhance interactions with the
mucus and negatively charged cell surfaces. In contrast, thiols, ammoniums and
lipophilic carbon chains reinforce adhesiveness and thus residency time by forming
disulfide bonds, electrostatic interactions and hydrophobic interactions, respectively.
Moreover, the presence of acidic or basic functional groups leads to pH-dependent
behaviors that are useful for targeting different portions of the GI tract.
In that context, degradable polymeric particles are particularly interesting [7]. Indeed,
polymers have well-defined properties (i.e., chain length, chemical functions, shape
and self-assembly) and can be derivatized using well-described chemistry. Therefore,
they constitute an interesting tool to produce tailored delivery vehicles. The most
widely described polymers for use in oral delivery are poly(glycolic acid) (PGA),
poly(lactic acid) (PLA) and their copolymers (e.g., PLGA, PLGA-PEG, and PLA-
PEG) [3,8], polyanhydrides [9], chitosan and its derivatives [10-12] and other
polysaccharides. As illustrated in Fig. 1, the surfaces of polymeric NPs can display
ligands that will bind to cell-specific receptors. Consequently, this review focuses
mainly on polymeric NPs.
1.2 Mechanisms of oral uptake of nanoparticles
The study of the mechanisms by which orally delivered drug loaded-NPs, whether
targeted or untargeted, are absorbed have attracted less attention than their design.
The design of new NPs for oral administration usually focuses on overcoming the
different barriers in the GI tract. The NPs and/or cargo must resist the harsh GI
environment, e.g., the low pH in the stomach and the degradative enzymes, but the
major barrier to their absorption remains the intestinal mucosa.
The first barrier that must be crossed is the mucus bilayer, which covers and protects
the epithelium. The NPs must adhere to and cross this highly viscoelastic layer, which
is continuously secreted and cleared. Mucoadhesive NPs, particularly positively
charged NPs, have been designed to promote strong interactions with the mucus and
prolong the retention time of the NPs at the mucosal surface. However, the
penetration of and diffusion through the mucus is also critical, as mucoadhesive NPs
can be trapped in the loosely adherent mucus and thus rendered vulnerable to rapid
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clearance [13]. NPs that penetrate the mucus and release drugs and/or drug-loaded
NPs closer to the epithelium have been engineered. Similar to viruses and bacteria,
NPs that are capable of penetrating the mucus should i) be small to allow their
diffusion in the mucin mesh; ii) have a non-mucoadhesive surface; and iii) be densely
coated, with a net neutrally charged hydrophilic surface [13]. The group of J. Hanes
has designed PEGylated muco-inert NPs that can penetrate the mucus. They found
that mucus-penetrating particles could be designed by carefully modifying their
surface properties, particularly by attaching a dense, low-molecular-weight PEG
[6,14]. In conclusion, a balance between minimal mucoadhesion or interactions with
the mucus and mucus penetration is required [6]. This also applies to targeted NPs,
which must be PEGylated to allow their penetration into the mucus and subsequent
access to the targeted receptor. Interestingly, the mucus layer is reduced at the surface
of M cells.
The second barrier to overcome for drug-loaded NPs is their generally limited cellular
uptake and translocation. In vitro models of the intestinal epithelium associated with
specific inhibitors or markers of endocytosis have been used to better understand the
fate of NPs in the intestines and optimize their design. It is generally accepted that
NPs do not diffuse through the paracellular route, although NP components, such as
chitosan, can affect tight junctions [1,6,12]. Rather, NPs can be taken up by
phagocytosis, which is restricted to M cells, or by pinocytosis. The uptake and
transport of particles by M cells is significantly higher than their transport by
enterocytes. Pinocytosis can occur by macropinocytosis, clathrin- or caveolae-
mediated endocytosis or clathrin- and caveolae-independent endocytosis [6,15,16].
The mechanism of uptake will affect subsequent intracellular trafficking and
transcytosis. The physicochemical properties of the particles influence their uptake,
e.g., the smaller the particle, the higher the uptake [1]. One strategy to enhance NP
uptake by intestinal cells is to conjugate a ligand to the NP surface that will favor cell-
NP interactions and enhance NP internalization, primarily through clathrin-mediated
endocytosis.
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1.3 Targeting cell receptors by grafting specific ligands to the nanoparticle
surface
1.3.1 Importance of receptor-mediated targeting
Despite the promising results obtained for NPs with appropriate size and surface
properties, the absorption and/or in vivo therapeutic efficiency of drugs following the
oral administration of drug-loaded NPs typically remains low [1-6,17]. The mucus
penetration, cellular uptake, NP trafficking inside the cells and biological fate of the
delivered drugs are still not optimal. Some studies have shown that conjugating the
NP surface with specific ligands for epithelial receptors or antibodies might enhance
the specific cellular uptake and transepithelial transport of the NP [1,6,18]. Moreover,
in the constantly moving environment of the gut, particles are rapidly cleared, and
thus, strong associations (e.g., receptor-ligand interactions) would favor the
accumulation of particles at their sites of action or absorption [2].
A large variety of epithelial cell receptors have been investigated as potential targets
of delivery systems [1,6,18]. Based on this knowledge, studies on ligand (or
antibody)-modified oral drug delivery systems have emerged in the last decade,
leading to promising in vitro results but unclear and often disappointing in vivo
conclusions.
1.3.2 Limitations of peptidic and proteinic ligands
In addition to the challenges presented by interactions with mucus and cells and the
poor uptake of NPs by cells, the chemical nature of ligands grafted onto the NP
surface can also play a role in the particle fate. In the GI tract, ligands present on the
NP surface are exposed to different harsh chemical conditions, such as the acidic pH
of the stomach and enzymatic digestion, among others. Moreover, most of the ligands
employed are biomacromolecules (e.g., antibodies and bacterial proteins) that can
diminish the progression of NP in the mucus by increasing their size and the
incidence of non-specific interactions, and they may also induce steric hindrance
when they approach the receptor surface [2]. Some of these biomolecules are also
immunogenic (Fc).
One critical consideration for ligands is the chemical method used for their
introduction in the drug delivery system. While it seems obvious that covalent
conjugation is mandatory, the chemistries that can be used and the chemical
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properties of both the carrier and the ligand are not necessarily easy to combine.
Biomacromolecules often have a large number of hydroxyl, amino or thiol functional
groups and are delicate compounds. In addition, the chemical characterization of the
obtained bioconjugates is a demanding task that requires an entire development
process in itself. It is thus difficult to determine whether a ligand is attached to the
particles by the desired chemistry, how much is bound and whether it is still bioactive
after conjugation [19,20].
Another limitation of targeted drug delivery systems is the accessibility and adequate
bioactive conformation of ligands in the biological media. These are indeed difficult
questions to address because during the formulation of the NP, it is challenging to
assess where the ligands will be located (i.e., outside or inside the particles).
Moreover, in the GI environment, the carrier can be reorganized, and the
conformation of the ligands can change. Several recent studies have attempted to
answer some of these questions. Most notably, the water solubility of the ligand
influences its localization in the NP and, consequently, its targeting efficiency. For
instance, in a self-assembling process, a hydrophilic ligand is more efficient than a
hydrophobic ligand because of its greater representation in the outer shell [21].
Furthermore, the nature of the ligands and their surface densities, might also play a
role in the level and nature of the immune response when antigen-loaded NPs are
targeted to dendritic cells [22]. These are controversial questions, and it is not yet
clear whether higher ligand density will lead to improved targeting because targeting
also depends strongly on the aforementioned surface characteristics of the NP [23],
the way ligands are tethered [24,25] and the affinity of the ligand for the targeted
receptor [22,25,26].
1.3.3 Advantages of non-peptidic small molecule ligands
Considering the above-mentioned limitations, the use of small molecule ligands (i.e.,
molecules of less than ~1500 Da), such as sugar derivatives, peptidomimetics or
metabolites, seems to have several advantages over peptidic conjugates: i) they are
chemically resistant to GI conditions (or could be modified for this purpose); ii) they
are usually conformationally stable; iii) they do not induce steric hindrance at the
receptor surface; iv) they can be conjugated to the NP through simple chemical
procedures, and the obtained conjugates can be more easily characterized; v) they are
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easier and less expensive to produce; and vi) they do not have immunogenic effects.
The main small molecule ligands that have been investigated in oral delivery are
mannose derivatives, RGD or LDV peptide sequence mimetics, lectin mimetics, fatty
acids and vitamins (see table 1).
2. Grafting of non-peptidic ligands to polymers composing nanoparticles
2.1 Strategies for introducing non-peptidic ligands onto nanoparticles
The conjugation of small ligands to a wide variety of biomaterials has been
extensively studied for applications ranging from tissue engineering to the conception
of targeted imaging agents. In targeted nanoparticulate delivery systems, particularly
those designed for oral administration, the conjugation methods are more limited.
Careful attention should be paid to the localization and density of the ligands.
Moreover, the biodegradable polymers employed can be degraded during the grafting
procedure. The chemical bonds should resist hydrolysis by GI pH and enzymes.
Consequently, the conjugation strategy should consider i) the chemical reaction used
for the anchorage (i.e., how to conjugate the ligands); ii) the localization of the ligand
along the polymer backbone (i.e., along the chain or at its end); and iii) the timing of
the conjugation step with respect to the overall NP synthesis process (i.e., before or
after NP formulation).
For instance, PLA, PLGA and their derivatives suffer from transamination and
hydrolysis reactions, even under very mild conditions [27]. Consequently, gentle,
selective and efficient chemistries must be used for their functionalization. Click
chemistry with copper-catalyzed azide alkyne cycloaddition (CuAAC) is the method
of choice for this type of polymer because it is a very tolerant reaction that occurs in
organic solvents without requiring amines.
Otherwise, modular approaches must be considered, such as our “clip and click”
strategy, in which PEG is first functionalized through “clip photochemistry” grafting
and then attached to a terpolymer of poly(lactide-co-glycolide-co--caprolactone) by
CuAAC [28,29]. A common strategy for attaching ligands to PLGA-based NPs is to
use poly(-caprolactone) (PCL) or PCL-PEG, which have the same properties as
PLGA but are more chemically resistant. Ligand-PCL conjugates can subsequently be
introduced to PLGA-based NPs. To ensure the protection of PLGA while using more
conventional chemistry (e.g., carbodiimide activation), some authors have worked
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directly with the particles after their formulation. This allows the PLGA core to be
protected against degradation and ensures that the ligands are present on the external
surface, but the risk of particle leakage and loss of payload cannot be avoided. Other
carriers used for conjugation with small ligands include reactive polymers (e.g.,
poly(anhydride)) and stable compounds that can tolerate typical chemical treatments
(i.e., PEGs, lipids, and polysaccharides).
The methods of conjugation will also influence the localization of the ligand along the
polymer backbone. However, it has not been clearly established that ligand
localization at the end of a polymer chain will generate better nanoparticle targeting
than a ligand grafted along the carrier backbone [24]. Moreover, the final availability
of the ligand may depend on the self-assembly process and the ligand characteristic
[21].
2.2 Grafting of non-peptidic ligands to a polymer
The strategies for ligand grafting are schematized in Fig. 2 and will mainly be
illustrated with PLGA and PCL, two biodegradable polymers that are commonly used
for nanoparticle formulation. Most authors have chosen chain-end approaches with
copolymers bearing reactive functions at the chain end of the hydrophilic block that
can react with the ligand via usual coupling methods (e.g., reductive amination,
ammonium formation, carbodiimide coupling, and polymerization initiated by
ligands). Recently however, several interesting methods have been developed to
obtain ligands that are grafted along the polymer chain (i.e., click chemistry, clip
chemistry and reactive polymers).
Reactive polymers can be grafted directly. Because poly(anhydride) (i.e., poly(methyl
vinyl ether-co-maleic anhydride)) is a reactive polymer, it forms naturally covalent
conjugates through the reaction of the amine functional group of modified VB12 with
the anhydride functional groups of the polymer (Fig. 2A) [30].
Grafting of the RGD sequence to NPs has been investigated as a strategy to target
drug-loaded NP to integrins that are overexpressed by specific cells, particularly v3
in the angiogenic tumor endothelium [31] and 1 integrin in intestinal M cells of the
follicle-associated epithelium [32]. However, because RGD might be degraded in the
GI tract, non-peptidic analogues have also been investigated. RGD peptidomimetic
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(RGDp) sequences have been developed for potential applications in antiangiogenesis
treatment [33] or tissue engineering, but few have been used in targeted drug delivery
systems [34-38]. The works conducted by our group [32] [39] provide clear evidence
that RGDp-targeted PLGA NPs are superior to peptidic RGD-targeted NPs in
inducing an immune response against a model antigen (ovalbumin) in mice. These
RGDps are built on a tyrosine template [40] and mimic the bioactive conformation of
cRGDf(NMe)V inside the binding pocket of v3 integrin. These ligands were aimed
at biomaterial applications [41] and have a short oligo ethylene glycol (OEG) spacer
arm. They were conjugated via a “clip photochemistry” process to the PEG parts of a
PCL-PEG polymer before their incorporation with PLGA during the formulation of
the NPs [32,39,42]. The presence of the ligands in the shell of the NPs was confirmed
by surface chemistry analysis using X-ray photoelectron spectroscopy (XPS) [43].
Other targeting ligands, such as a Leu-Asp-Val (LDV) modified sequence and a LDV
peptidomimetic (LDVp) with an OEG spacer arm, initially designed to bind 41
integrins, have also been grafted onto PCL-PEG and incorporated into PLGA-based
NPs [39,44].
C-type lectins and mannose receptors are the most explored molecules for the
targeting of antigen-presenting cell (APC) receptors. Consequently, mannose-grafted
particles, intended to for dendritic cell or macrophage targeting, have been
investigated [45], including their activity when administered via the oral route. Recent
reviews [46-48] have more precisely detailed such chemistry. Thus, they will not be
described here, but some examples of the most relevant strategies should be
mentioned. For example, Rieger et al. produced PCL-PEG with a mannose residue on
the chain end of the PEG block [49,50]. They synthesized PCL-PEG terminating with
a secondary amine function that reacted with a mannose derivative equipped with a
small ethyl spacer ended by bromine to form an ammonium link. The obtained
copolymer easily formed positively charged micelles bearing mannose residues on
their surfaces (Fig. 2B). Similarly, Freichels et al. produced aldehyde-terminated
PCL-PEG that, through a reductive amination protocol with 2-aminoethyl-α -D-
mannopyroside, allowed for the production of end-capped mannosylated PCL-PEG
[51]. PCL-PEG micelles prepared with these modified polymers displayed a neutral
surface charge. Click chemistry can also be used to produce mannosylated PCL
bearing sugar residues along the entire polymer chain. For that purpose, Xu et al.
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produced PCL diblock polymers in which one block had azide functional groups that
could be engaged in a CuAAC reaction with propargylic sugar derivatives [52] (Fig.
2C). The large number of carbohydrates fixed on one block of the polymer confers
amphiphilic properties to this PCL-based polymer. We also successfully applied our
“clip photochemistry” process to the preparation of PCL-PEG grafted with mannose
(Fig. 2C) [42]. This mannosylated PCL-PEG was subsequently introduced into the
formulation of PLGA-based NPs [39].
Careful and demanding strategies are required to functionalize PLGA or PLA
polymers with mannose derivatives. For instance, Nagasaki used protected sugars to
induce the sequential ring-opening polymerization of ethylene oxide and DL-lactide
[53]. Deprotection yielded PLA-PEG chains that were end capped with carbohydrates
and demonstrated interesting micellization properties. Hamdy et al. synthesized
mannan-conjugated PLGA NPs by direct functionalization of the carboxylate
functions on the surface of preformed OVA-loaded NPs via a classical carbodiimide
protocol [54] (Fig. 2B). Similarly, Brandhonneur et al. produced PLGA particles
bearing different ligands (including lectin, RGD and mannose) and demonstrated their
enhanced uptake by macrophages in vitro [26]. The aforementioned “clip and click
strategy” allows the production of different functionalized PLGA-PEG with targeting
moieties randomly dispersed along the PEG block to produce mannosylated PLGA-
PEG copolymer with self-assembling properties [29].
2.3 Analytical methods to evaluate non-peptidic ligand grafting
Due to the uncertainties inherent in self-assembling processes, it is usually difficult to
predict where ligands will be localized. Consequently, new characterization methods
must be investigated in parallel with conjugation strategies.
The most common test to detect sugar moieties at the particle surface is to provoke
the aggregation of sugar-conjugated NPs in the presence of free soluble lectins and
further analyze them using isothermal titration calorimetry, enzyme-linked lectin
assays (ELLAs) [50], turbidimetry, surface plasmon resonance [55] or affinity column
retention assays [53]. NP aggregation upon binding to lectin is clear proof of the
presence of bioavailable sugar ligands. Quartz crystal microbalance with dissipation
(QCM-D) affinity binding assays have also been conducted, with mannosylated NPs
interacting with lectins immobilized on a QCM-D surface [29]. This test provides not
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only information about the availability of mannose for lectins on a surface but also an
opportunity to assess the relevance of the NP-receptor interaction under shear stress,
representing a “near to reality” evaluation of targeted NP behavior.
The chemical characterization of the NP surface can be performed by surface
chemistry techniques, such as X-ray photoelectron spectroscopy (XPS), but only
solids can be analyzed using this technique. NMR could be a good alternative for
studying particles in aqueous solution [20,56,57]. Indeed, in magnetic resonance, only
soluble components are detected. This provides valuable information regarding the
bioaccessibility of the constituents of the external shell of NPs, and the micellar
stability of the NP. Under appropriate conditions, NMR can also be used for
quantitative calculations of ligand accessibility and the lengths of the PEG chains
[58].
3. Targeting of nanoparticles to different intestinal cell types
Polymeric carriers protect antigens against degradation and inactivation in the harsh
gastro-intestinal environment and have the ability to enhance their transmucosal
transport [1,6,17]. Despite these advantages, the oral delivery of drug- or antigen-
loaded NPs remains challenging. The poor efficacy of these particulate systems has
been reported, possibly as a consequence of poor particle uptake. Thus, none of these
systems has reached the market. Methods for increasing the uptake and transcytosis of
orally delivered particles by specific cells represent a promising opportunity for
enhancing their efficacy. Due to their physiological functions, M cells are a
particularly attractive target for oral drug delivery [1,6,18,59,60]. Enterocytes, which
cover most of the gut surface and are responsible for absorption, could also be a good
target for NP uptake. Epithelial immune cells that sample particulate antigens and
microorganisms can also be exploited for NP uptake. Finally, endocrine cells, which
are rarely studied for NP targeting, could be potential new targets, mainly for local
drug delivery.
3.1 M cells
M cells are specialized epithelial cells that are located in the follicle-associated
epithelium (FAE) of Peyer’s patches or GALT [59-61] and are part of the mucosal
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immune system. They deliver samples of foreign material from the lumen to the
underlying organized mucosa lymphoid tissues to induce immune responses. They
have high transcytotic capabilities and are able to transport a broad range of materials,
such as bacteria, viruses, antigens and particles, from the intestinal lumen to the
underlying lymphoid tissues [1]. In addition, M cells have less glycocalyx and
reduced levels of membrane hydrolase activity, which can influence the fate of
protein-containing or protein-coated NPs, compared with normal epithelial cells.
Villous M cells located outside the FAE have also been observed [62], but the
transport of antigens and microorganisms across the intestinal mucosa is carried out
mainly by FAE-M cells [1]. Although they are less numerous than enterocytes, M
cells present enhanced transcytosis abilities, which makes them particularly
interesting for oral drug delivery applications. Therefore, M cells represent a potential
portal for the oral delivery of drug-loaded NPs, particularly peptides, proteins and for
the mucosal vaccination.
Although the role of M cells in particle uptake is well known, it is commonly believed
that their low proportions in the human GI tract (1% of the total intestinal surface) and
their variability among species (i.e., they represent 5% to 50% of the FAE surface and
express species-specific markers), individuals, physiological state and age decrease
the impact they could have on oral drug delivery [1]. However, in light of their
particle uptake capabilities, several groups have considered it worthwhile to work on
improving NP delivery through M cells, particularly with the aim of compensating for
the low number of M cells through more efficient targeting [1,18,32,39,59,60,63-69].
The main strategy has been to coat the NP surface with an M cell-targeting molecule.
This task is not trivial, especially considering the limited predictive value of the most
commonly employed mouse models and the difficulties in identifying markers that are
specific to human M cells.
The most investigated family of M cell-targeting molecules is the lectins. Lectins
constitute a structurally diverse group of proteins and glycoproteins that bind
reversibly and with relatively high affinity to specific carbohydrate residues present
on cell surface proteins or lipids [18]. In several studies, the Ulex europaeus
agglutinin-1 (UEA-1) lectin was grafted to the NP surface to target -L-fucose
residues expressed on the apical surface of M cells, yielding improvements in
nanoparticle transport across the intestinal barrier [66,67,70]. The conjugation of
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PLGA particles to Aleuria aurantia lectin (AAL) induced an increased expression of
IFN upon oral birch pollen immunotherapy [18] and effectively protected mice
against subcutaneous challenge with melanoma or prostate cancer cells [63].
However, the UEA-1 and AAL lectins, in addition to their potential immunogenicity,
are specifically expressed on mouse and not human M cells [60], limiting the
translation of these therapies to humans.
Many studies have been dedicated to the search for specific markers of human M
cells. Two lectins specific to the human FAE (galectin 9 and sialyl Lewis A antigen
(SLAA)) have been identified [65,71]. One anti-SLAA antibody out of the 41 tested
lectins and antibodies reacted strongly with human M cells and bound only weakly to
FAE enterocytes [59]. Galectin 9 has not been used as a targeting molecule for the
oral delivery of NP.
To target M cell receptors, it may be advantageous to replace lectins with small
molecule mimetics. Lambkin et al. identified UEA-1 lectin mimetics from a
combinatorial library [70,72]. These compounds are easy to synthesize and are based
on an oligo-lysine scaffold with 1 to 4 lysine units terminated by galloyl entities. The
lectin mimetics were coupled to a PEG construct through p-nitrophenyl-activated
esters to form a tetragalloyl-D-lysine dendrimer (TGDK) that was used to vectorize a
Rhesus CCR5-derived cyclopeptide antigen. Misumi et al. conducted in vivo studies
on macaques with this TGDK and found a significant stool IgA response and efficient
M cell transcytosis of the dendrimer, which induced neutralizing activity against SIV
infection [68]. TGDK was efficiently transported from the lumen of the intestinal tract
into Rhesus Peyer’s patches by M cells and then accumulated in germinal centers. In
addition, TGDK specifically bound to human M-like cells in vitro and was efficiently
transcytosed from the apical side to the basolateral side in the M-like cell model (Fig.
3).
M cells are considered the gateways for antigen entry to the underlying mucosal
tissues, and they are exploited by various enteric pathogens as a route of entry to the
underlying host tissue, predominantly through the hijacking of their endocytic
machinery [18]. The invasiveness of these viral and bacterial pathogens is mediated
by specific pathogen–host interactions, which could be adapted to deliver drug-loaded
NPs into the GALT [60,69]. Some important pathogen recognition receptors (PRRs),
such as Toll-like receptor-4 (TLR-4), platelet-activating factor receptor and 51
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integrin, are expressed on the surfaces of human and mouse M cells [32,39,73]. The
specific pathogen–host interactions are crucial for the translocation of bacteria across
the lumen. Consequently, targeting PRRs might be a suitable strategy for enhancing
the uptake of orally administered NPs by M cells [59].
For instance, M cells take up many enteropathogenic microorganisms, such as
Yersinia, via the high-affinity interaction between invasin and the 51 integrins that
are overexpressed at the apical pole of human M cells [32,74,75]. Interactions with
5 integrin occur mainly through RGD sequences. We have demonstrated that
grafting RGD to the PEG chains of PLGA-based NPs significantly increases the in
vitro transport of these NPs by human M-like cells [32] (Fig. 4A) and slightly
enhance the IgG immune response after oral immunization [32]. We hypothesized
that these phenomena were due to a partial degradation of the RGD peptide during its
trafficking through the GI tract. Therefore, an RGD peptidomimetic (RGDp) was
grafted onto PEGylated PLGA-based NPs. RGDp significantly increased the transport
of NPs across an in vitro model of human M cells (Fig. 4B), and intraduodenal
immunization with RGDp-labeled NPs elicited a higher production of IgG antibodies
than the intramuscular injection of free ovalbumin or the intraduodenal administration
of either non-targeted or RGD-NPs [39]. NPs conjugated to LDVp also exhibited
greater transport by M cells in vitro and showed promising immune responses
compared to untargeted NPs, suggesting that these LDV ligands might have bound to
1 integrins on the apical surface of M cells or other integrins homing lymphocytes in
the gut [76].
The Clostridium perfringens enterotoxin (CPE) receptor (claudin 4) is a tight-junction
transmembrane protein that plays a role in establishing transepithelial electrical
resistance in the mucosal epithelium in addition to its function as a receptor for CPE
[77]. Claudin 4 is highly expressed in M cells and is conserved between mouse and
human Peyer’s patch. Targeting PLGA NPs to claudin 4 enhanced their in vivo uptake
and mucosal IgA responses [78].
Recently, Hase et al. [79,80] reported that glycoprotein 2 (GP2) is specifically
expressed at the apical surface of human and murine M cells and serves as a
transcytotic receptor for fimH+ bacteria (e.g., Escherichia coli, Salmonella enterica
and Yersinia). Thus, the GP2-dependent transcytosis pathway could provide a new
target for the development of M cell-targeted nanosystems.
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Reovirus in mice and poliovirus and HIV in humans use specific receptors to target
and cross the FAE [81-83]. Reovirus can invade intestinal M cells in rodents and
rabbits through interactions between its outer capsid protein σ1 with α(2,3)-linked
sialic acid containing glycoconjugates of the apical membrane [82]. The incorporation
of recombinant σ1 into liposomes or an OVA-σ1 fusion protein enhanced binding
to rat Peyer’s patch [84]. HIV-1 can adhere to M cells in mice and rabbits prior to its
endocytosis and transport across the epithelial barrier [85]. A lymphotropic (X4)
HIV-1 strain crosses M cell monolayers and infects the underlying CD4+ target cells.
This transport requires both the lactosyl cerebroside and CXCR4 receptors, which are
expressed on the apical surface of Caco-2 and M cells [81]. In contrast, a monotropic
(R5) HIV-1 strain is unable to cross M cell monolayers and infect underlying
monocytes. Caco-2 and M cells do not express CCR5, but the transfection of these
cells with CCR5 cDNA restores the transport of the R5 virus [81].
3.2 Enterocytes
The importance of enterocytes should not be overlooked; these cells vastly outnumber
M cells and can transcytose many macromolecules, such as cholera toxin (CT) and F4
fimbriae, as well as inert particles [86-89]. For instance, several Escherichia coli
strains express F4 fimbriae on their surface and bind to specific F4 receptors (F4Rs).
The expression of these receptors on the surface of porcine enterocytes is necessary to
induce a protective mucosal immunity following the oral administration of purified F4
fimbriae to piglets [88]. The conjugation of heterologous antigens to F4 fimbriae has
been shown to induce enhanced mucosal antigen-specific antibody responses upon
oral administration, but to date, no study evaluating the influence of F4 fimbriae
targeting on the oral transport of nanocarriers has been reported. The incorporation of
flagellin-rich Salmonella enteritidis extracts into Gantrez AN NPs induced
bioadhesion in the ileum during “Salmonella-like” gut colonization [90].
TLR-4, the receptor for LPS, mediates bacterial translocation through enterocytes
[91]. The rat enterocyte cell line IEC-6 internalized LPS-coated latex beads in a
TLR4-dependent manner, indicating that LPS-coated particles may provide yet
another alternative for the targeted delivery of NPs to the intestinal epithelium [18].
Some lectins (e.g., wheat germ agglutinin (WGA), concanavalin A (ConA) and
tomato-derived lectin) bind to enterocytes, with a relatively high affinity for the
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specific carbohydrate residues present on cell surface proteins or lipids [18,92-94].
They have been grafted onto the surfaces of nanoparticles for oral vaccines or drug
delivery.
Some authors have proposed using certain metabolic pathways as routes for NP
uptake. This strategy was attempted with vitamin B12 (VB12) or B1 (thiamine)-
coated NPs. VB12 forms a complex with the intrinsic factor (IF) in the stomach,
which is subsequently recognized by IF receptors on ileal epithelial cells, resulting in
the endocytosis of VB12. VB12-targeted micelles exhibited better in vitro uptake and
transport of a hydrophobic drug in a model intestinal cell monolayer in comparison to
untargeted micelles [30,95]. Similarly, Salman et al. also produced thiamine-
poly(anhydride) NPs, which could be advantageous because thiamine can be
administered at a higher daily dose than VB12. Encouraging immunization results
were also obtained with these thiamine-coated vehicles in mice, although no direct
comparison with VB12 NPs was made [96].
3.3 Goblet cells
Goblet cells represent the second largest population of intestinal epithelial cells, but
they are rarely chosen as a target for orally delivered nanocarriers. Recently, a CKS
peptide identified using a random phage display technique was found to have a
specific affinity for goblet cells [97]. Orally administered insulin-loaded trimethyl
chitosan chloride NPs that were modified with the CSK targeting peptide induced
enhanced transport via clathrin- and caveolae-dependent endocytosis and produced a
better hypoglycemic effect than non-targeted NPs [98].
Furthermore, Listeria monocytogenes is transcytosed across the intestinal barrier by
binding to E-cadherin, which is luminally accessible on goblet cells, suggesting that
targeting E-cadherin could be a promising strategy for delivering NP to goblet cells
[99].
3.4 Dendritic cells
Dendritic cells (DCs) represent the most potent APCs. They are found throughout the
intestine and can be divided into two major subsets, which can be distinguished based
on the expression of CD103 (the E chain of the E7 integrin), the receptor for the
epithelial cell adhesion molecule E-cadherin, and CX3CR1 [100]. Intestinal DCs have
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been proposed to be involved in the induction of protective immunity against
pathogens, tolerance to commensal bacteria and tolerance to food antigens and self-
antigens. Thus, they represent a potent target for oral vaccination strategies because
vaccine interactions with DCs can be enhanced by targeting DC surface molecules
[18,101]. There are very few, if any, DC-specific markers, but these cells possess a
broad spectrum of cell surface receptors that are involved in endocytosis and the
induction of immune responses, such as C-type lectins, scavenger receptors, TLRs
and Fc receptors (FcRs: FcgR, FcaR and FceR, which bind to IgG, IgA and IgE,
respectively) [102].
The outcome of the immune response can differ depending on the targeted receptor.
TLR ligands induce strong DC activation and thus have potent adjuvant properties.
The C-type lectin DEC-205 and mannose receptors are more involved in enhancing
endocytosis, although DC activation has been achieved with DEC-205 targeting
[101]. The FcRs are a family of membrane glycoproteins that bind the Fc fragment
of IgG and activate a signaling pathway that can regulate the adaptive immune
response when cross-linked with antigen-antibody immune complexes.
[26,54,100,103].
Mannan and other mannosylated structures enhance antigen endocytosis by DCs and
induce immune responses when grafted to a vaccine carrier surface [104,105].
Mannosylated liposomes bind to DC-SIGN and the mannose receptor CD206,
resulting in enhanced antigen-specific cell proliferation relative to antigen alone or
non-targeted liposomes, and they protect mice from lethal challenge when delivered
intraperitoneally. Mannosylated, PEGylated PLGA NPs elicited higher antigen-
specific IgG serum responses in mice upon intraduodenal administration than non-
grafted NPs [39]. They also enhanced NP uptake in a mouse model of colitis [106].
Targeting intestinal DCs enhances the endocytosis of antigen-loaded carriers and can
thus improve immune responses, but most of the studies investigating this
phenomenon have been performed in vitro or in murine models. Therefore, further
studies are required to i) evaluate vaccine carriers targeting intestinal DCs of higher
species (pigs and primates), ii) analyze the behavior of antigen-loaded carriers in the
GI tract and iii) deepen our understanding of the interactions between vaccine carriers
and intestinal DCs.
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3.5 Enteroendocrine cells and the identification of novel potential ligands for
nanoparticle delivery
The enteroendocrine system constitutes the largest endocrine organ. Enteroendocrine
cells are scattered throughout the GI tract in the epithelium among enterocytes. These
cells are typically conically shaped, with a large base from which gut hormones are
released into the blood from secretory granules. These cells are distributed along the
GI tract, and the apical pole facing the gut lumen possesses microvilli. Strikingly,
these cells are among the least understood cells in the body. However, new molecular
genetic techniques have led to important advances, thereby highlighting novel aspects
of enteroendocrine biology [107]. Enteroendocrine cells represent approximately 1%
of all epithelial cells in the intestine and are subdivided into more than 10 different
cell types based on their main secretory products and localization along the GI tract
(e.g., ileal/colonic L cells). Multiple biological functions are physiologically regulated
by gut hormones (e.g., food intake, gastric emptying, gut motility, gut barrier function
and glucose metabolism). Among the enteroendocrine cells, L cells have attracted
particular interest because of the pleiotropic effects of their secreted peptides.
In the gut, the posttranslational processing of proglucagon in endocrine L cells gives
rise to the major proglucagon-derived peptides (GLP-1, GLP-2, oxyntomodulin and
glicentin) [108]. These peptides are rapidly secreted in response to food intake, and
their production is modulated according to the nutrient (i.e., lipid, carbohydrate, and
protein) [109]. L cells secrete another anorexigenic peptide, PYY. Similar to GLP-1
administration, PYY injection delays gastric emptying and pancreatic and gastric
secretions. GLP-2 is co-secreted by L cells with GLP-1. GLP-2 assists in the
maintenance of the physiological gut barrier function (i.e., protects against gut
microbiota) and facilitates the digestion and absorption of ingested nutrients. In
addition, GLP-2 regulates the stimulation of intestinal epithelial cell proliferation
[110] and constitutes a key target in the maintenance of gut barrier function.
Given the location of these cells (the ileum and colon), targeting L cells and thereby
the endogenous production of these hormones remains challenging. Therefore,
strategies devoted to targeting specific receptors involved in L cell physiology, thus
leading to the production of hormones, such as GLP-1, GLP-2 and PYY, could be a
promising potential method to locally influence the mechanisms involved in high-
impact diseases, such as diabetes, obesity and inflammatory diseases.
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Recent studies have shown that L cells express various G-protein-coupled receptors
(GPCRs) that are activated by a wide variety of endogenous ligands found in the gut
lumen. GPCRs are involved in a large number of physiological processes and could
serve as targets for coated NPs. Potential ligands for these receptors include specific
lipids from short-chain fatty acids (i.e., GPR43, GPR41 and GPR109a) [111,112] and
long-chain fatty acids (GPR40 and GPR120) [113]. Interestingly, GPR43 and GPR41
expression is well conserved across species (human, pigs, and rodents) [114]; both
receptors are expressed in L cells and have been shown to directly control GLP-1 and
PYY secretion (Fig. 5) [115]. Although a direct link between GPR43 or GPR41 and
GLP-2 secretion has not been established, we have demonstrated that changing the
composition of the gut microbiota using specific nutrients that increase Short-chain
fatty acids (SCFA) (fermentable carbohydrate) stimulate endogenous GLP-2
production (Fig. 5) and protect against gut permeability and associated inflammation
[116]. Moreover, a similar treatment increases PYY and GLP-1 secretion.
Bioactive lipids belonging to the N-acylethanolamine family that are part of the
endocannabinoid system [117,118] could also activate the L cell-specific receptor
GPR119, thereby increasing GLP-1, GLP-2 and PYY secretion. Finally, TGR5 (also
known as M-BAR, GPBAR-1 or GPR131) can be activated by bile acids [119].
SCFAs are present in the gut lumen (ileum and colon) at a high concentration
(approximately 100 mM) [120]. These fatty acids are mainly produced through the
metabolic activity of the gut microbiota (undigested carbohydrate fermentation). Over
the last 30 years, numerous roles have been attributed to SCFAs, including the
harvesting of energy from undigested food, the regulation of epithelial cell
proliferation, electrolyte uptake and smooth muscle contraction [112], and SCFAs
could thus be used as NP ligands to act on L cell metabolism.
Another potential target is the GPR109A receptor, which binds to the ketone body β-
hydroxybutyrate and butyrate [114,115]. The GPR109A receptor is highly expressed
in the gut lumen, in which the concentration of butyrate reaches approximately 20
mM, thereby activating this receptor. The intestinal GPR109A receptor could
therefore be targeted by butyrate-labeled NPs.
The postprandial satiety effect of dietary lipids and free fatty acids stimulates several
gut peptides that control food intake. Growing evidence suggests that these effects are
mediated through two different receptors, GPR40 and GPR120. Both receptors are
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activated by medium- to long-chain free fatty acids that stimulate gut peptide
secretion [113]. It is worth noting that similar strategies and ligands might be used to
target L cells (i.e., GLP-2 production) and improve gut barrier function.
Bile acids are not only byproducts of cholesterol metabolism but also key metabolic
regulators that act through TGR5 [121]. For instance, Katsuma et al. discovered that
bile acids promote GLP-1 secretion through a TGR5-dependent mechanism [122],
thereby suggesting a novel role of bile acids in energy metabolism and glucose
homeostasis. Interestingly, a recent study has demonstrated the relevance of targeting
TGR5 in experimental colitis [123]. TGR5 activation has been shown to exert a
peripheral immune-modulatory effect in macrophages, and this study also showed that
TGR5 activation through a targeting strategy restores tight-junction protein
distribution, leading to reduced gut permeability [123]. For decades, ciprofloxacin has
been used for the treatment of Gram-negative bacterial infections occurring in the
context of Crohn’s disease. Cipriani et al have demonstrated that ciprofloxacin also
acts as a TGR5 agonist, thereby contributing to improvement of the inflammatory
status [123]. This last study supports the concept that the TGR5 receptor might be
targeted by not only specific bile acids but also synthetic molecules, such as
ciprofloxacin. While they are effective for treating infections in Crohn’s disease
patients, the wide use of antibiotics plays a minor role in the maintenance therapy for
these patients. Therefore, we propose that targeting TGR5 using NPs carrying a lower
dose of ciprofloxacin might be useful to target colon cells or macrophages to treat
inflammatory bowel disorder, preventing the major adverse effects linked to the
antibiotic activities of this compound.
In conclusion, although endogenous non-peptidic ligands have helped to identify the
different GPCRs described here, numerous synthetic agonists are also currently being
studied and may constitute a source of therapeutic agents that merit future
consideration [124]. Moreover, it is worth noting that in addition to their roles in the
control of gut peptide secretion, most of these receptors are also expressed in immune
cells, such as macrophages. Therefore, we propose that the oral delivery of NPs with
non-peptidic ligands targeting GPCRs of L cells could lead to new treatments, acting
locally on obesity, type 2 diabetes and intestinal inflammation. Thus, by exploring the
wide variety of L cell stimulation-dependent effects (e.g., GLP-1, GLP-2 and PYY
secretion) on GI tract physiology and peripheral metabolism (energy and glucose
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homeostasis), we propose to provide valuable insight into some novel beneficial
therapeutic targets.
4. Conclusions regarding the potential biomedical applications of the oral
delivery of nanoparticles targeted with non-peptidic ligands
Orally administered targeted NPs have a large number of potential biomedical
applications and exhibit several putative advantages for oral drug delivery, such as the
protection of fragile drugs or the potential for the modification of drug
pharmacokinetics. Despite these advantages, the oral delivery of drugs by NPs
remains challenging. To achieve efficient drug delivery, NPs must i) avoid rapid
mucus clearance; ii) penetrate the mucus layer; and iii) be extensively taken up by the
intestinal epithelium. The optimization of particle size and surface properties and the
targeting of specific cells by ligand grafting have been shown to enhance NP transport
across the intestinal epithelium.
In particular, targeted NPs with novel non-peptidic ligands for oral delivery have been
investigated. The main advantage of these particles is that their targeting properties
are improved by the use of ligands that are not degraded in the GI tract, unlike
peptidic/proteinic ligands, and are not limited to receptors, which interact only with
proteins. The use of targeted NPs will also depend on the type of cells/receptors
targeted by cell-specific targeting.
To the best of our knowledge, no clinical studies on the oral delivery of antigens or
drugs with targeted NPs are ongoing because until recently, the potential benefits of
these particles have been outweighed by the associated pitfalls. In particular, the
sophisticated design and associated high cost of the synthesis of the grafted polymer
and the high cost of NP production might be major limitations for their development
as pharmaceuticals. Due to their high cost of manufacture, targeted NPs must provide
significant added value for unmet pharmaceutical and medical needs. For example,
they will not be used for the solubilization of BCS class II or IV drugs; rather, their
use will likely be limited to punctual applications (e.g., vaccines) or the treatment of
diseases that are currently lacking satisfactory therapies. To a lesser extent, the lack of
control/robustness of NP absorption might also hinder their use in several biomedical
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applications. Thus, they should be used for applications that do not require a fine-
tuning of the delivered dose (e.g., vaccines and type 2 diabetes), and they would
likely not be suitable for insulin delivery for type 1 diabetes treatment.
Based on the literature review, we propose (as illustrated in Fig. 1) that the oral
delivery of NPs that are targeted using non-peptidic ligands would be useful to further
investigate i) oral vaccination targeting M cells; ii) the oral delivery of therapeutic
proteins and peptides by targeting enterocytes; and iii) the specific targeting of L cells
for the treatment of type 2 diabetes, obesity and inflammatory diseases.
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6. Figure legends
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Figure 2. Commonly used chemical principles for the conjugation of non-
peptidic ligands to (A) reactive polymers or (B) amphiphilic block copolymers. (C)
Chemical strategies for grafting ligands (or polymeric block-bearing ligands) along a
main polymer chain.
Figure 3. Tetragalloyl-D-lysine dendrimer (TGDK) targeting to M cells. (A) The
association of TGDK (red) with Rhesus Peyer’s patch follicle-associated epithelium.
(B) The penetration of TGDK (green) into Rhesus Peyer’s patch M cells (nuclear
staining by DAPI is shown in blue). (C) The transcytosis of TGDK through a Caco-
2/Raji B monolayer () and Caco-2 monolayer (o).
Figure 4. The influence of ligand grafting on nanoparticle transport across
monolayers of Caco-2 monoculture or Caco-2 and Raji B cell coculture,
mimicking the follicle-associated epithelium with M cells. Pegylated PLGA-based
nanoparticles of approximately 225 nm containing 15% PCL-PEG grafted to RGD, an
RGD peptidomimetic (RGDp), an LDV derivative (LDVd), an LDV peptidomimetic
(LDVp) or mannose (Man) were loaded with ovalbumin. Transport after (A) 90 min
of incubation at 37°C or (B) preincubation with (inhibitor +) or without (inhibitor -)
an anti-integrin 1 monoclonal antibody before adding nanoparticles for 90 min. The
number of particles transported is expressed as the apparent permeability coefficient
(Papp).
Figure 5. Schematic overview of the secretory function of L cells and the
receptors that can be targeted by nanoparticles with non-peptidic ligands.
Enteroendocrine L cells express a wide variety of GPCRs. The main endogenous and
physiological ligands of the different receptors are indicated at the top of the figure.
LCFA, long-chain fatty acid; SCFA: short-chain fatty acid; OEA:
oleoylethanolamide. Dietary lipids may be sensed by GPR40 and GRP120 expressed
on enteroendocrine cells. The gut microbiota contribute to the fermentation of various
non-digested compounds and produce specific metabolites, such as acetate,
propionate and butyrate, which have been described as specific ligands for GPR41, 43
and 109A receptors. Bioactive lipids, such as N-oleoylethanolamide, serve as ligands
of the GPR119 receptor. TGR5 (also known as M-BAR, GPBAR-1 or GPR131) is
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targeted by bile acids. Given the effects of gut peptides secreted by L cells (GLP-1,
GLP-2 and PYY), targeting L cells might help to treat obesity, type 2 diabetes and
inflammatory bowel diseases.
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Graphical Abstract
