How to design the surface of peptide-loaded nanoparticles for efficient oral bioavailability?

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How to design the surface of peptide-loaded nanoparticles for ef ﬁ cient oral bioavailability?☆

Hélène Malhaire

^a,b

, Jean-Christophe Gimel

^a,b

, Emilie Roger

^a,b

, Jean-Pierre Benoît

^a,b

, Frédéric Lagarce

^a,b,c,

⁎

aINSERM U1066 Micro et nanomédecines biomimétiques, Angers, France

bUniversity of Angers, France

cPharmacy Department, Angers University Hospital, Angers, France

a b s t r a c t a r t i c l e i n f o

Article history:

Received 20 December 2015

Received in revised form 17 March 2016 Accepted 28 March 2016

Available online 4 April 2016

The oral administration of proteins is a current challenge to be faced in thefield of therapeutics. There is currently much interest in nanocarriers since they can enhance oral bioavailability. For lack of a clear definition, the key characteristics of nanoparticles have been highlighted. Specific surface area is one of these characteristics and represents a huge source of energy that can be used to control the biological fate of the carrier. The review discusses nanocarrier stability, mucus interaction and absorption through the intestinal epithelium. The protein corona, which has raised interest over the last decade, is also discussed. The universal ideal surface is a myth and over-coated carriers are not a solution either. Besides, common excipients can be useful on several targets. The suitable design should rather take into account the composition, structure and behavior of unmodified nanomaterials.

Keywords:

Protein and peptide drugs Nanoparticle

Speciﬁc surface area Protein corona Mucus interaction

Contents

1. Introduction . . . 321

2. What are the pitfalls of oral delivery for protein/peptide drugs? . . . 321

3. Why nanocarriers might help in addressing the challenges of orally administered proteins? . . . 321

4. Size, bulk, shape andﬂexibility . . . 322

5. Stability in the lumen . . . 322

6. Protease inhibitors . . . 322

7. Interaction with mucus . . . 323

7.1. Physiology . . . 323

7.2. Mucoadhesion . . . 324

7.3. Passive mucodiffusion . . . 324

7.4. Active mucodiffusion . . . 325

7.5. Back diffusion . . . 326

8. Crossing the cell barrier . . . 326

8.1. Paracellular passage enhancement . . . 326

8.2. Active targeting of endocytosis pathways or cells . . . 327

8.3. Permeation enhancer of the transcellular pathway . . . 328

8.4. Endosomal escape . . . 328

8.5. Efﬂux pumps . . . 329

9. Protein interaction . . . 330

10. Discussion . . . 330

11. Conclusion . . . 332

Acknowledgments . . . 332

References . . . 332

☆ This review is part of theAdvanced Drug Delivery Reviewstheme issue on“SI: Oral delivery of peptides”.

⁎ Corresponding author at: Inserm U 1066 MINT, 4 rue LArrey, 49033, Angers cedex 9, France. Tel.: +33 244688545; fax: +33 244688546.

E-mail address:frederic.lagarce@univ-angers.fr(F. Lagarce).

Contents lists available atScienceDirect

Advanced Drug Delivery Reviews

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / a d d r

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1. Introduction

With high efﬁciency, low toxicity and good tolerance, peptides and proteins offer advantageous and biocompatible solutions to treat various diseases. However, their fragility in biological environments, their hydrophilicity, and their large molecular weight, leading to poor permeability, drastically limit their systemic use, and most of them are administered by injection. This route of administration causes pain and trauma resulting in poor patient compliance. Since the oral route is non-invasive, painless, and preferred by patients, making proteins available for this administration route could improve their use in the proposed treatments. Nanoparticles (NP) might help in addressing this challenge, mainly for two reasons: (i) as a shield against a harsh biological environment and (ii) as a carrier that can cross epithelia.

Insulin has often been used as an example in thisﬁeld of research [1–5]. The protein is usually administered subcutaneously in diabetes patients. It is expected that oral application could improve life quality, compliance, and treatment efﬁcacy and reduce costs. Besides, the oral route is more in accordance to the physiological path of insulin which, once released from theβ-cells of the pancreas, goes into the portal vein and then directly to the liver where its action takes place[6–8].

A successful oral protein-loaded formulation is expected to reach a bioavailability level of above 5–10% for insulin for instance–instead of 0.5% obtained with insulin in solution–so that the protein, still in association with the formulation or not, can be transported at the right time to the right place for efﬁcient systemic biological activity [9–11]. In addition, this administration would have to be safe and reproducible at the required dose in the same individual[12]. Protein or peptide oral drug delivery is considered to be a major challenge of drug delivery. Hundreds of drug delivery systems (DDS) have been proposed to protect these large molecules in the gastrointestinal tract and to improve their absorption. This review will critically examine the literature and emphasize the interaction between the surface of the DDS and their biological environment, since this parameter is considered of primary importance in the performance of the DDS.

Thus, after a brief introduction on the pitfalls of the oral route, this paper will focus on the different aspects to be considered to improve the bioavailability of proteins or peptides through this administration route. Size, strategies to stabilize the nanocarriers, formation of protein corona in biologicalﬂuids (if the vector has no stealth properties), interaction with mucus, as well as active targeting will be covered. Finally, as all these points interact, the optimization of the overall surface design and the emergence of multifunctional vectors will be discussed.

2. What are the pitfalls of oral delivery for protein/peptide drugs?

The gastrointestinal tract (GIT) is in charge of the digestion process of food and is also the main barrier between the organism and potential pathogens. The GIT is composed of several sections that differ in chemical and enzymatic compositions as well as in pH and functions in the digestion process. The oral cavity is followed by the pharynx and the esophagus, which is connected to the stomach, where pepsin and gastric lipase, active at the low pH induced by hydrochloric acid secretion, help in digestion[13,14]. The gastric emptying drives the food bolus into the small intestine, where the main absorption process takes place at more neutral or slightly basic pH values. This latter section is composed of the duodenum that collects the bile from the liver and the gallbladder as well as the trypsin contained in pancreatic juice, the jejunum and the ileum. Then, what remains in the GIT goes to the large intestine, where salts and water are absorbed. Finally, the rectum, connected to the large intestine, collects the feces prior to their elimination.

Even if the digestion of proteins and peptides might start with salivary enzymes and mastication and then continues further in the stomach, the major damages are caused in the acidic environment of the stomach and continue in the small intestine. If one focuses further on this section, the intestinal barrier must be considered under three

aspects: (i) a chemical (mainly enzymatic) barrier, (ii) a physical barrier and (iii) a biological barrier[15,16]. Thefirst barrier is composed of di- gestive secretions (involving biliary salts that form micelles and interact with lipids), immune molecules, cell products such as cytokines, inflam- matory mediators and antimicrobial peptides, mainly produced by Paneth cells[17]. There are also possibilities of thiol/disulfide exchange reactions of peptide drugs with thiol rich food. This barrier includes the three classes of enzymes: (i) free enzymes in the lumen that break proteins into small peptides (up to eight amino acids) and free amino acids, (ii) brush border membrane-bound enzymes, which break these small peptides into di- and tripeptides that can more easily cross the cell membrane than free amino acids and (iii) cytosolic enzymes in the enterocytes that end the digestion of these di- and tripeptides in amino acids[18,19]. The mucus layer and the viscosity of the chyme or the digestivefluids are considered as the physical barrier. Finally, the biological barrier involves complex andfinely regulated biological mechanisms to cross the intestinal epithelium and the vascular endothelium[17,20]. The cell barrier consists of enterocytes, mucus- secreting goblet cells, M cells and Paneth cells. It is therefore unlikely that a peptide can successfully cross these barriers while preserving its integrity. Despite these pitfalls, it is interesting to target the small intestine since this represents an absorptive section of 200 m²covered by a thin layer of loosely adherent mucus[21].

3. Why nanocarriers might help in addressing the challenges of orally administered proteins?

There are various advantages of using nanocarriers: protection against chemical and enzymatic degradation, controlled release and targeting, tolerability and improved uptake and translocation resulting in better bioavailability and better therapeutic efﬁcacy[9]. The main particularity of these nanoparticulate systems is their size that may be linked to improved bioavailability through biological barriers[22–24].

They might therefore help in addressing the challenges posed by the oral route. The NP platform includes liposomes, polymeric nanospheres and nanocapsules, fullerenes, micelles, lipid solid NPs, lipid nanostructured carriers, nanotubes, dendrimers, nanocage technology such cyclodextrine-based NPs and viral NPs. Even though there is a wide range of size distribution, shape, crystal structure, chemical composition, surface chemistry, surface charge and porosity–which are the main parameters of nanocarriers, NPs are commonly characterized by the European agency by a specific surface area above 60 m²/cm³ [25–28]. This equals a diameter of 100 nm when materials exhibit a density of 1. Since the regulatory agencies in the world do not agree on the same definition, there is a tolerance in pharmaceutical literature for up to 500 nm in diameter. Indeed, the surface-to-volume ratio increase leads to an increase of surface energy and renders NPs more biologically active. Besides, this surface area is thefirst layer in contact with the biological environment. Thus, it is obvious that a proper design of the NP surface may help in controlling its fatein vivo[29]. Numerous scientific papers report that surface properties are more important than mass in terms of the response of biological systems to NPs[30–34].

Surface modification can be achieved either by coating a component after formulation (polymers, surfactants, stabilizing agents, hydrophilic elements, etc.) or by including the modification during its formulation [33]. In the latter case, it takes place on the surface according to its structure and its affinity. This will affect the surface properties–zeta potential, hydrophilicity, therefore stability–and the biological fate of the system: stability in biologicalfluids, sensitivity to digestion, interaction with mucus and bioavailability.

Until now, there have only been a few peptide candidates for oral delivery for systemic use. Desmopressin and cyclosporine A are the only two available on the market and have poor but sufﬁcient bioavailability (N1% for desmopressin) or signiﬁcant variability after oral administration (from 10% to 89% for cyclosporine A)[35–37]. It is important to note that these two formulations are not based on nanotechnologies:

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commercial-cyclosporine-oral formulations are microemulsions and commercial-oral-desmopressin formulations are tablets. Oral, insulin- loaded formulations have failed due to poor bioavailability or side effects [10,38]. Even if recently some results have aroused interest, a commercial formulation is not yet at the clinical stage[39–41]. Besides, Card and Magnuson in their review of NP-based oral insulin delivery systems underlined the lack of information on the surface area[38]. Therefore, it is of interest to focus on what could be done to the NP surface to improve bioavailability through the oral route.

4. Size, bulk, shape andﬂexibility

Studies have shown that particle sizes ranging from 50 to 500 nm, depending on their composition, lead to interaction with the epithelial cells[22,42]. Other works have revealed size dependency that determines the intestinal uptake pathway[29]. Indeed, transcytosis increases when size decreases, and internalization in enterocytes favors NP sizes of 50–100 nm, whereas larger particles are more likely to be internalized by M cells [26,43]. But recently, findings have revealed that polymer-based microspheres from 500 nm to 5μm are likely to cross the intestinal epithelium through both phagocytosis in non-absorptive tissues and endocytosis in absorptive epithelia[44]. Particles with a diameter above 1μm were found mainly in the liver, the kidneys and the lungs, but in smaller quantities compared to particles with a diameter under 1μm. However, a study performed with silver NPs of different sizes has highlighted a critical size threshold with respect to proinflam- matory effects after repeated oral administration[45]. NPs of 22 nm, 42 nm and 71 nm increased serum levels of TGF-β and changed lymphocyte phenotypes in favor of B cells and NK cells. These immune responses were proportional to NP penetration into the tissues and were not observed for 323 nm NPs. This is in accordance with the fact that the smaller the NPs, the higher the surface energy[25]. Indeed, a controversial debate is taking place among scientists on the particle size effect. More and more people are linkingfine and ultrafine particles from 0.1 to 1μm, in food and in the environment, of being linked to the increase of autoimmune diseases such as Crohn's disease[46–51].

For the time being, there are only a few toxicity studies, and most of these studies on size dependency have been done with polystyrene NP, since it is a low-toxicity material. However, thefindings of these works are not always in agreement with studies performed on nanomaterials made with other components, and the size threshold as well as toxicity and efficacy may vary. For instance, the surface chemistry, more than bulk, will significantly impact the fate of the carrier and the pathway used to cross the epithelium. This relationship between composition and size is not the only issue to deal with. It should also be noted that it is challenging to compare the results since the assays performed are not standardized. In each case, even if the nature of the NP has to be considered, since it can influence the crossing through the mucus and the intestinal epithelium, the same balance between efficient crossing and a deleterious response should be observed: Smallest is not always the best.

Shape andﬂexibility may also impact the biological fate of the carrier.

Shape does not only impact drug release, as was demonstrated for tablets [52]. Works on HeLa cells have shown an uptake increase of rod-like particles, compared to spheres, cylinders and cubes[53]. Regarding the deformability of the carrier, since the analytical tools to study these parameters at a nanoscale are still being developed, there are only few articles emphasizing this criterion but there is no doubt that NP deformability plays a role in terms of bioavailability and drug delivery [54–57].

5. Stability in the lumen

The huge interface between NPs and biological matters presents non-negligible free energy. The system might evolve to diminish this interfacial free energy by decreasing the surface area throughﬂocculation

and aggregation/coalescence. Colloidal stability is nicely exposed in a recent book[58]. NP surface features may be designed to avoid these instability phenomena by creating an energy barrier that provides repulsion between the particles. On one hand, a neutral charge seems to be more immunocompatible, but electric charges on the surface can be used to balance Van der Waals forces and stabilize the drug delivery system (DDS) by electrostatic stabilization[58,59]. However, the limited data on toxicity lead to a debate on the consequence of a positive or negative surface charge, but all agree that it significantly impacts the interaction between NPs and biological matters and will thus modify the fate of the carrier[33]. Besides, the surface charge might vary with the composition of the biological environment and change along the tract. For that reason, electrostatic stabilization is difficult to predict and might not be the best option. On the other hand, nonionic macromolecules immobilized on the surface can provide steric stabilization. This phenomenon has been described by Heller to stabilize gold particles in aqueous dispersion with polyethylene glycol (PEG) polymers[60]. The macromolecule is composed of an anchored part and a stabilizing fragment. On one hand, the anchored part is insoluble in the dispersing phase and strongly immobilized on the surface (by adsorption, post-grafting or copolymerization of specific monomers during NP preparation). On the other hand, the stabilizing fragment is freely soluble and sufficiently stretched in the dispersing phase. The density of coating must be above a certain threshold; below this critical concentration, uncovered surfaces might trigger instability. With a proper covering rate, the lateral movement of the stabilizing species is limited and colloidal stability is carried out by a combination of two entropic phenomena: (i) with a local excessive concentration in macromolecules at the shell, the osmotic pressure increases, the entropy of water decreases, and the solvent is drained between the surfaces, which leads to repulsion—this is the mixing effect; and (ii) the volume restriction effect derives from the decrease in the degree of freedom of the chains when two particles are too close together, and elastic forces repel these particles (Fig. 1).

This steric stabilization is a thermodynamic process and might vary with temperature, since it inﬂuences the quality of the solvent for the stabilizing moiety of the macromolecule. PEG coating has been widely used to make the surface hydrophilic, to confer stealthiness to the system with respect to the immune system and to limit the interaction with biologicalﬂuids that might destabilize the system. Indeed, the colloidal stability of lipid nanocapsules obtained by the phase-inversion temperature method was explained by the PEG moiety of the surfactant exposed on the surface[61–64]. It has been highlighted that the charge that the PEG moiety might exhibit on the surface is not due to the macromolecule but rather due to the compensation of both water dipoles to PEG dipoles[64]. PEG coating has also been linked to a reduced lipolysis of lipid core NPs in the small intestine[65–69]. Finally, the two strategies may also be combined to provide electro-steric stabilization.

Insulin-loaded liposomes have also been stabilized by silica NPs thanks to the phenomenon called the Pickering effect[70]. The solid and inert NPs act as surfactants that stabilize the O/W interface by steric repulsion (Fig. 2). The study reports slowed-down enzymatic degradation and a sustained release of insulin but the release never exceeds 25% (even with uncoated liposomes).

Nevertheless, Andreani et al.[71,72]combined this Pickering effect with PEG coating, withoutﬁnding any improved release inin vitroand ex vivostudies.

6. Protease inhibitors

More than 25 years ago, research was already focusing on enzyme inhibitors to protect orally administered insulin[73]. It is still the case nowadays[74].

Bernkop-Schnürch provided an interesting review on this topic to overcome the low bioavailability of peptides and proteins by the oral route[75]. In this paper, three kinds of protease inhibitors were listed:

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(i) competitive inhibitors such as the Bowman-Birk Inhibitor, which takes the place of the substrate on the active site of the enzyme; (ii) a non-competitive inhibitor, which acts on another active site on the enzyme and reduces the enzyme reaction velocity; (iii) a compound that deprives the enzyme structure of essential metal ions, such as the complexing agent EDTA. It should be noted that in most cases, the three aforementioned mechanisms are involved. These compounds will be addressed in more detail within this ADDR Issue.

Some excipients can be versatile, and Lueßen et al.[76]confirmed what wasfirst described by Hutton et al.[77]in 1990: the capability of certain mucoadhesive polymers (i.e. poly(acrylate): polycarbophil and carbomer) to reduce the activity of endopeptidases (trypsin and α-chymotrypsin) and exopeptidases (carboxypeptidase A and cytosolic leucinaminopeptidase). In the experimental conditions, a deprivation of Ca²⁺ions for the trypsin structure was found responsible for its inacti- vation by these kinds of polymer[78]. The same observations were made withα-chymotrypsin, except that the required polymer concentrations had to be higher. Regarding zinc metalloprotease, such as carboxypeptidase A, the same inhibition was demonstrated, but a lower polymer concentration was required. The zinc depletion from the secondary structure of the enzyme explained the time-dependent in- activation. A higher dissociation constant of the cation out of the enzyme or a higher binding affinity of the poly(acrylates) for zinc compared to calcium was raised to explain the differences in concentration.

Bernkop-Schnürch reported that the dissociation constant of many chelators was much higher towards zinc than towards calcium[75].

Besides, as calcium ions play an important role in cell signaling, these chelation properties therefore display additional permeation- enhancing properties[79]. Bernkop-Schnürch's research group also demonstrated that mucoadhesive polymers exhibiting strong complexing

properties are also capable of a “far-distance inhibitory effect” on membrane-bound enzymes across the mucus layer[80]. This means that toxicity can be reduced by keeping this protease inhibitor in reduced amounts in the vectors to have local activity with a limited dilution for better efficiency on API protection. In addition, this could also prevent the disturbance of nutrient digestion. It has also been reported that local pH modification can impede enzymatic activity[81]. One component may act on several targets, and this is especially interesting since interfer- ence may lead to toxicity, undesirable effects, or loss of efficacy when too many components are in the formulation.

To conclude, protease inhibitors are controversial since their effects may induce toxicity. According to Bernkop-Schnürch, even if systemic toxic effects or intestinal mucosal damage can be excluded, the dis- turbed digestion of nutritive proteins and inhibitor-induced stimulation of protease secretion caused by a feed-back regulation, may induce toxicity[82]. To take advantage of these protease inhibitors and to limit their side effects on food digestion: (i) they can be associated with the protein in particles to permit their use in reasonable but efﬁcient concentrations and (ii) the proper protease inhibitor should be selected according to protein sensitivity to each enzyme, and the enzymes can be distributedin vivo. It is also important to note that the potential toxicity of these protease inhibitors has mainly to be considered for chronic treatments.

7. Interaction with mucus 7.1. Physiology

Mucus covers the gastrointestinal epithelium. To better apprehend the interaction with mucus and take advantage of this critical barrier,

Polymer entropy = elastic repulsion Water entropy =

(i) Mixing effect

(ii) Volume restriction effect

H20

H20 H20 H20

H20

Elastic forces osmotic pressurett

Fig. 1.Schematic representation of the mechanisms allowing the steric stabilization of coated nanoparticles.

Polymeric particle at the interface O/W Young’s equation: =

Fig. 2.Pickering emulsion.

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the structure, the composition, the functions and the behavior of mucus have to be taken into account[83]. This viscoelastic layer is usually composed of two underlayers: (i) a luminal mucus layer, which is on the luminal side and is rapidly cleared, and (ii) an adherent mucus layer in contact with the epithelium and that is cleared slowly[84].

This protective and lubricant assembly varies in thickness along the GIT in order to balance between protection capacities (i.e. against pathogens, potential damage caused by undigested food, or a harsh biological environment) and nutrient absorption. Thus, the small intestine can be considered as being covered by a loosely adherent mucus layer to allow better nutrient absorption[85], which lays on a moreﬁrmly adherent layer in contact with enterocytes apical side[86]. Water, proteins (mostly mucins), carbohydrates, lipids, electrolytes, immunoglobulins and DNA compose the mucus[21,84,86]. The main component of mucus is mucin. This glycoprotein exhibits a large molecular weight ranging from 0.5 to 20 MDa[87]. Several genes are responsible of many mucin types and the small intestine is mainly covered by MUC2 [21]. Both its composition and proportion evolve with diet and the physiopathological state of the individual[21,87].

Interaction with mucus includes: (i) mucoadhesion, which is due to a high afﬁnity between mucus and NPs; and (ii) mucodiffusion, which conversely lets the carrier diffuse through the mucus layer from the luminal compartment to the apical side of the epithelial cells. A lot of 2D- and 3D-models have been developed to characterize NP/mucus interactions and predictin vivobehavior of NPs with respect to mucus [88]. However, many parameters are involved and real physiological conditions are difﬁcult to mimic. Therefore, mucus models have to be standardized and carefully selected prior to studying NP performance.

7.2. Mucoadhesion

Mucoadhesive properties have been thoroughly studied and docu- mented since it has been assumed that they can improve the retention time of DDS, thus increasing local drug concentration, in an intensiﬁed contact with mucus, near the epithelial barrier and consequently improve drug bioavailability[72,89–96]. Different processes may be involved in mucoadhesion such as hydrogen bonding, macromolecule entanglement, electrostatic, hydrophobic or Van der Waals interactions[83, 84,86,92,97]. Any coating with components having good wettability and the capacity to diffuse and interpenetrate the mucus glycoproteins, will promote hydrogen bonding and macromolecule entanglement and will allow narrower contact between mucus and NPs[96]. Thus, ability to diffuse in mucus is, to a certain extent, related to mucoadhesive properties.

Electrostatic interactions might also take place between negatively charged sialic acid and sulfate residues of mucins, and positively charged nanocarriers[98,99]. Chitosan is the most described cationic mucoadhesive polymer. Bernkop-Schnürch summarized the different opportunities offered by these chitin derivatives[96]. Positive charges have thus shown good mucoadhesive properties, and it is argued that they show good afﬁnities for cell membranes too[100,101]. But it is also assumed that this particular interaction with phospholipid bilayers of cells is associated with cytotoxicity[100]. Toxicity has also been highlighted on Caco-2 cells with these positively charged particles, whereas modiﬁed PAMAM dendrimers with a negative charge were much less toxic[102]. Even if there is electrostatic repulsion between negatively charged mucins and negatively charged NPs, mucoadhesive properties might still be possible due to hydrogen bonding[71].

The capabilities of chitosan to enhance oral absorption related to its mucoadhesion properties, its ability to open tight junctions and to improve the paracellular passage, as well as its enzyme inhibitory effect have been well established. Many chemical modiﬁcations have been made on this versatile compound to improve complexing properties such as nitrilotriacetic acid-, EDTA- or diethylenetriaminepentaacetic acid-conjugation[95,103,104]. The Austrian team has also associated a thiol function on chitosan to generate thiomers. Indeed, thiomers have been described as a powerful mucoadhesive polymer but they also

display a good potential to inhibit some enzymes from the GIT, as mentioned previously[95,105,106].

Other authors have reviewed more polymers that are interesting in terms of mucoadhesion[94,107]. Indeed, polymers represent the larg- est class of components suitable for this kind of interaction with mucus. Thus, any colloidal system coated with components exhibiting afﬁnities for mucus may be mucoadhesive. Although mucoadhesion is advantageous for local ocular delivery (to limit the fast clearance by tears and blinking) or for tablets or any other reservoir of NPs, it is not always sufﬁcient for oral NPs[108–110]. Indeed, if the formulation sticks to the luminal side of the mucus, it might be removed without crossing the epithelium.

7.3. Passive mucodiffusion

Maisel et al.[111]compared mucoadhesive particles (MAPs) and mucus-penetrating particles (MPPs) and found a better distribution of MPPs close to the epithelium. MAPS were composed of carboxylate- modiﬁed polystyrene, those particles were coated by 5 kDa PEG to ob- tain MPPs. MAPs were aggregated in the center of GI lumen, thus limiting contact with enterocytes. Conversely, MPPs were uniformly distributed in the lumen and some were even in close contact with the villi of the small intestine (Fig. 3,[111]).

The advantages of mucodiffusion have also been observed with chitosan nanolipoparticles. Li et al.[112]compared chitosan-based core shell corona (CSC) nanolipoparticles with chitosan nanolipoparticles for the oral administration of insulin. The CSC carrier consisted of chitosan nanoparticles covered with pluronic F127-lipid vesicles–the shell–and re- covered with polyethylene oxide to form thefinal corona that shielded the positive charges of chitosan. This complex carrier has shown improved penetration properties in mucus, followed by enhanced uptake in mucus-secreting HT29-MTX-E12 cells.In vivoevaluation in diabetic rats has revealed a 2.5-fold improved hypoglycemic effect with sustained release over the 12 h of the experiment as compared to chitosan nanolipoparticles. Besides, some researchers have investigated by multiple particle tracking, the influence of the surface charge and NP size on diffusion in mucus[113]. They observed a greater impact on the diffusion coefficient surface charge, compared to size variation, and the best results were obtained for neutral or near-neutral NP surfaces.

The same behavior has been observed with viruses and polioviruses.

The Norwalk virus, or human papillomavirus, overcomes the mucus layer thanks to its densely charged but globally neutral surface and their small size[114]. These features enable them to diffuse through the sieve of mucus and avoid being eliminated with the renewal of the loosely adherent layers[33,86,115,116]. For this reason, mucus- penetrating particles are arousing more and more interest.

Mucodiffusive coating might even counteract the size effect of nanocarriers. Indeed, PTX-loaded LNCs have shown mucodiffusion without size dependency[63]. Lai et al.[117]even highlighted that a PEG coating could decrease the interaction between NPs and mucus, and reverse the effect of polystyrene NP size in cervicovaginal mucus.

Even if the composition of cervicovaginal mucus differs from gastrointestinal mucus, it is interesting to observe that the mean Deffvalues increased for 200 and 500 nm particles as compared to 100 nm particles (Fig. 4,[117]).

These observations are even not in accordance with mucus mesh spacing, which was estimated to be less than 200 nm byﬂuorescence re- covery after photo-bleaching and electron microscopy[115,118,119].

However, in 2009, Cone reviewed the dynamic nature of mucus[120].

Indeed, it has been reported that mucins can aggregate together in larger cables, thus creating spacing between 100 and 1000 nm, according to the methods used[120–123]. This explains the heterogeneity of the mucus mesh porosity observed in native mucus[124].

Mucus issues are not limited to the oral route, and knowledge of its effects on nanocarrier diffusion can be gained from other routes. To avoid the entrapment of NPs in cervicovaginal mucus, thus limiting

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contact with the epithelium, Mert et al.[125]compared the behavior of PLGA NPs with different coatings. Uncoated material and coatings with PVA or with vitamin E coupled with 1 kDa PEG moiety were trapped in mucus, whereas a coating with vitamin E conjugated to 5 kDa poly(ethylene glycol) (VP5k) was able to help NPs penetrate human cervicovaginal mucus. It seems that these results were in reality due to a proper PEG coating, resulting in a slippery surface. Indeed, Wang et al.[126]highlighted a real PEG mucoadhesivity paradox:

PEG moieties that are too short (b2 kDa) do not allow efﬁcient mucodiffusion, whereas PEG moieties that are too long (N10 kDa) interpenetrate the mucin mesh network, resulting in entrapment of the NPs.

These experiments were carried out with cervicovaginal mucus, but similar results were observed with gastrointestinal mucus. Thus, 2 kDa PEG coating has provided nearly unimpeded diffusion through the mucus barrier[69]. No significant difference has been observed in in vitromucodiffusion of LNCs coated with PEG from 2 kDa to 5 kDa [127]. The apparent permeability coefficient has also highlighted the influence of coating density and 10% 2 kDa PEGylated SLN have shown better results than 20% or 5% 2 kDa PEGylated ones. Similarly, in spite of a difference in brush density, NP permeation in mucus did not significantly differ between 2 kDA PEG-PLGA NP and 5 kDA PEG-PLGA NP [128]. Self-nanoemulsifying drug delivery systems (SNEDDS) have also shown good mucodiffusion due to their hydrophobic surface, small size and shapeflexibility[119].

Advantages of both mucoadhesion and mucodiffusion can be combined in nanoparticles-in-microparticles (NiM)[110]. NPs with 700-nm diameter were prepared by nanoprecipitation and loaded into mucoadhesive, polyelectrolyte-complex alginate/chitosan microparticles. After oral administration in dogs, the oral bioavailability of the drug was found to be 47 times higher than normal, and the half-life was improved up to 95 times. There was no characterization of the NPs in terms of mucodiffusion, but this strategy seems promising.

7.4. Active mucodiffusion

If mucus is a critical barrier, why not disturb this layer to help NPs cross it? Takatsuka et al.[129]studied the co-administration of salmon

calcitonin (sCT) and Triton® X-100 as a permeation enhancer with a mucolytic agent, namely N-acetyl-L-cysteine (NAC), and evaluated the effect in Wistar rats after intra-intestinal administration. The results show that NAC was able to cleave the disulﬁde bonds in mucus and improve by a factor of 2.8 the drug–plasma concentrations as compared to an sCT solution, whereas the combination NAC with Triton X-100 had a 12.5-fold increased plasma concentration. These experiments also reveal damage of the intestinal epithelium, in correlation with enhanced bioavailability. Although the damage was reversible, the protection functions of the mucus were momentarily altered, leaving potential access to pathogens. Knowing this, are mucolytic agents relevant for oral route use, especially in chronic disease?

Recently, Müller et al.[130]investigated the proteolytic activity of another mucolytic, papain, when grafted on NPs on Sprague–Dawley rats. Papain-grafted poly(acrylic acid) (PAA) NPs exhibit a 3-fold higher diffusion level through the mucus layer, andin vivoevaluation revealed that more than 45% of these particles remained in the duodenum and jejunum. They attributed these results to the decreased mucus viscosity, resulting from mucoglycoprotein degradation by the grafted enzyme, and the ability of PAA, exhibiting negative charges, to diffuse through the mucus by repulsion with sialic acid residues. This degradation is ir- reversible, but the mucus layer is continuously renewed. The authors speciﬁed that damage was localized to a minor part of the mucus, since the enzyme is covalently bound to the polymer. Besides, it has been highlighted that papain had antioxidants, antibacterial, antifungal and anti-inﬂammatory activities to temporarily compensate for mucus loss[131]. Despite lower cytotoxicity on Caco-2 cells with papain- grafted PAA (up to 2 mg/ml), permeation studies, pulsed-gradient spin-echo NMR assessment of mucin mobility, and small-angle and spin-echo small-angle neutron scatterings, have revealed better mucodiffusion properties for a related molecule, bromelain, on bromelain-grafted NPs[132]. In their presence, overall gel strength of the mucus decreased as well as the maximum shear stress of the linear viscoelastic region[133]. However, there was no breakdown point change, compared to uncoated NPs dispersed in mucus. This observation assumes that NPs might more easily cross the mucus while the protection capabilities of mucus are preserved.

Fig. 3.Particle trajectories in mucus and distribution in the small intestine or in colon of mucoadhesive particles versus mucus-penetrating particles.[111].

Fig. 4.Transport mode distributions of COOH- and PEG-modiﬁed nanoparticles in CV mucus: immobile particles (A), immobile and hindered particles (B), and diffusive particles (C).[117].

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Since DNA content in mucus may vary and result in a viscosity increase in diseases such as cysticﬁbrosis, DNase has been used as a mucolytic agent with more efﬁciency than NAC[134]. So far, it is used as a therapeutic agent but, just like NAC, it could be interesting to look at its absorption-enhancer capacities in nanoscale DDS.

Overall, mucolytic agents might be useful in diseases that express abnormal viscous mucus or to improve the efﬁcacy of cytoadhesion, but it should be kept in mind that they can also be deleterious[86,135].

7.5. Back diffusion

Crossing the mucus barrier might not be enough for efﬁcient oral bioavailability. Indeed, back diffusion may occur, thus decreasing the portion of NP in contact with the epithelium[119]. So far, three approaches are known to limit this phenomenon: thiomer formation, ligands targeting a glycosylated moiety, and particulate zeta potential variation in mucus.

Theﬁrst one involves pH-sensitive thiol functions on polymer, making them more reactive in disulﬁde-bond formation and mucoadhesion capacities when the pH increases near the cell membrane. This strategy has even been combined with a proteolytic enzyme such as papain [136]. This kind of system must be considered as a drug reservoir that increases drug concentration close to absorptive tissue, rather than as a carrier intended for transcytosis[119].

More specifically, ligand–receptor recognition with sugar residues from mucus and cells may also be used to improve mucoadhesion followed by cytoadhesion, which hinders back diffusion and enhances NP residence time[137,138]. Additionally, this interaction is less influ- enced by pH variability in GIT, but might be hindered by the sugar residues of food[137]. A reversible binding of glycosylated targets may occur (i) with mucin for mucoadhesion, leading to local and tem- porary retardation in transit; (ii) with glycocalyx coating the epithelium, for cytoadhesion, thus bringing the carrier closer to the absorptive site and (iii) with cell membrane receptors, mediating endocytosis, for cytoinvasion[137]. This latter point will be further addressed in the ap- propriate section. Two lectins are particularly interesting for the oral route, namely wheat germ agglutinin (WGA) and tomato lectin (TL) [139]. Thefirst one, a plant lectin, has shown binding capacities on Caco-2 cells as well as with HT-29 cells[140]. In addition, this low- toxic protein is insensitive to proteolytic degradation and is not neutral- ized by food[137]. For these reasons, it has been widely used to coat NPs for oral route use. WGA has been grafted onto lipid NPs[141].Ex vivoassays have confirmed the bioadhesion properties, andin vivopharmaco- dynamic studies after oral administration in rats have revealed a 2.7-fold increase in oral bioavailability of the drug. The ligand has been covalently linked to a mucoadhesive polymer and coated onto liposomes[142]. A hemagglutination test confirmed the preserved binding activity of WGA bound to carbopol, whilein vivoassays have shown bioadhesion in the duodenum and a significant decrease of calcium blood levels after the oral administration of calcitonin-loaded, WGA-modified liposomes. WGA has also been conjugated onto PLGA prior to preparing polymeric NPs by the double emulsion-solvent evap- oration technique[143]. Studies have confirmed bioadhesion and endocytosis in the small intestine as well as higher levels of uptake by Peyer's patches, resulting in a 1.4 to 3.1-fold increased systemic uptake of thymopentin as compared to unconjugated PLGA NPs. The same results were observed with tomato lectins (TL)[144]. Studies have shown ad- sorptive endocytosis and accumulation in enterocytes[145]. Unfortu- nately, since this glycoprotein resists enzymatic degradation, immune responses were associated to oral administration in mice with a high presence of specific serum IgG and a non-negligible level of IgA in the intestine[146].

The third approach to limit back diffusion in mucus consists of a zeta potential change of the surface while the carrier is migrating in mucus.

This strategy has been developed following the observation of sufﬁciently higher cell uptake by endocytosis of positively charged particles

compared to negatively charged ones, in spite of their mucoadhesive properties[119,147,148]. Indeed, positively charged NPs are more likely to interact with negatively charged cell membranes and initiate endocytosis [149]. A zeta potential change, from a negative value for mucodiffusion to a positive value in cell contact, might combine advantages of both these particle surfaces and improve oral bioavailability.

The selective activity of membrane-bound enzymes might be used to cleave a mucodiffusive moiety on the NP surface to reveal a cationic surface. This has been assessed by Bonengel et al.[150]. However, transport studies have to be performed to conﬁrm the expectations raised by this novel carrier.

8. Crossing the cell barrier

Further absorption enhancement through the intestinal cell barrier can be achieved by improving transport through different pathways, or by avoiding degradation in cells as well as by any leak due to efﬂux pumps in the apical side of the intestinal barrier[151]. To cross the intestinal cell barrier, protein/peptide drugs associated or not with NPs, or subfractions of NPs, can enter cells through simple diffusion, transporter or receptor-mediated transport. They can also diffuse through the paracellular pathway.

8.1. Paracellular passage enhancement

The paracellular route is restricted in surface (only 1% of the mucosal surface area) and by the tightness of the junctions between epithelial cells (only macromolecules or aggregates below 1 nm are small enough to cross the epithelium using the paracellular pathway)[33,152]. Per- meation enhancers can act on tight junctions (TJ) to open them, but it has been shown that opened TJs were less than 20 nm wide[153].

This limits the potential of this route for drug-loaded NPs but the transport of free proteins released from NPs or in crumbled NPs into systemic circulation, can be considered[154,155]. Zonula occludens toxins (ZOT), chitosan and thiolated polymers are also known to open TJs. ZOT act on speciﬁc intestinal receptors but not in the large intestine to reversibly increase epithelial permeability[156,157]. The mechanism under the TJ opening by chitosan has been further studied[158,159]. It was highlighted that the expression of claudin-4 at both protein and gene level was involved in TJ opening[159]. The opening was induced by a movement of claudin-4 from the membrane to the cytosolic compartment[158]. A C-terminal fragment ofClostridium perfringensenterotox- in (C-CPE) has been used as a modulator of claudin-4[160]. The inhibition of the barrier function of claudin-4 allowed the paracellular passage of dextran from 4 to 20 kDa with a theoretical Stokes radius of between 1.4 and 3.3 nm. Conversely, 40-kDa dextran with a theoretical Stokes radius of 4.5 nm was not absorbed. In addition, this transient- ly and reversible phenomenon implied c-Jun NH2-terminal kinase, a signaling protein belonging to the mitogen-activated protein kinase family[159]. Surfactants such as sodium lauryl sulfate or saponin, may also disrupt the intestinal barrier, but generally in a much more damaging manner than previous components[161]. Clausen and Bernkop-Schnürch have also demonstrated that the administration of carboxymethylcellulose-sodium (Na-CMC), a mucoadhesive polymer, combined with cysteine, a thiolated amino acid, can induce a reduction of transepithelial electrical resistance in an Ussing chamber, suggesting the opening of TJ to enhance the permeability of the drug through a paracellular pathway [162]. Other cationic compounds such as polyethyleneimine, anionic materials such as polyacrylic acid, or calcium chelators like diethylene triamine penta acetic and ethylene glycol tetra acetic acid (EGTA), have been used to improve the paracellular passage[7].

TJ opening has also been observed with PAMAM dendrimers [163,164]. Studies have revealed that at least a part of the NP had to be internalized within Caco-2 cells prior to opening TJ, suggesting cell trafﬁck- ing behind the mechanism of paracellular pathway enhancement[163].

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The improvement of peptide absorption is however limited. It is important to keep in mind that they also have some drawbacks such as potential toxicity. Similar to protease inhibitors, this class of compounds must rather be associated to the drug substance itself in order to circumscribe their action of protein passage across the intestinal barrier and should be limited to acute treatment.

8.2. Active targeting of endocytosis pathways or cells

Enterocytes can internalize NPs by various forms of pinocytosis, namely clathrin-mediated endocytosis, the lipid raft/caveolae pathway, micropinocytosis, sometimes involving actins, protein tyrosine kinase or cyclooxygenase. As shown by He et al.[165], uncoated PLGA NPs might even use several paths at the same time (Fig. 5,[165]). Regulated by multiple mechanisms, the complexity of nano-bio interactions is beyond doubt, and size is not the only parameter that determines the path through the cell barrier.

Since some elements are capable of interacting with membrane- bound receptors prior to being internalized, one can promote a particular way of internalization. This strategy is triggered by the well-known ligand–receptor recognition and interaction and might limit DDS loss in dead ends like clathrin-mediated endocytosis which is destined for lysosomal degradation[166]. By decorating the NP surface with these compounds, it is assumed that the receptor-mediated pathway is improved and hence the bioavailability of the drug. This targeted receptor may be ubiquitous, i.e. widely distributed in the body, or be speciﬁc to some restricted cell lines.

Zhang and Wu have listed saccharides, vitamins, fatty acids and peptides used as ligands for oral, active targeting through vitamin, transferrin and hormone receptors on enterocytes[167]. A British group has highlighted that the gut-associated lymphoid tissue (GALT) was predominantly responsible for the uptake of unmodiﬁed polystyrene NPs and that the improved uptake following the grafting of tomato lectin onto the surface was the consequence of a shift from lymphoid tissue to absorptive intestinal tissues (14-fold more)[168,169]. As mentioned previously, lectins are glycoproteins resistant to enzymatic degradation and capable of speciﬁc recognition and binding to carbohydrate moieties of complex glycoconjugates such as intestinal glycoprotein and glycolipids of the apical membrane of epithelial cells and glycocalyx[140,170]. They have been shown to enhance the passage through the intestinal barrier by acting at several levels. Firstly, as

previously observed, lectins can specifically interact with glycocalyx that covers the absorptive tissue. Whereas lectin–carbohydrate interactions are specific, cytoinvasion is not specific[137]. Studies have shown WGA uptake through active transport using EGF-receptor recognition [171,172]. Whereas 3% of labeled WGA deposited on the apical side of confluent Caco-2 monolayers was found in the basolateral compartment of a Transwell® system after 3 days, the transcytosis of WGA- coated latex particles of 50 nm was improved by 20% as compared to uncoated NPs[137]. Moreover, transcytosis transport increased with increasing lectin density on the NP surface. Nevertheless, within 1 h, at least 50% of internalized free WGA was found in lysosomes[137].

Would the same apply to WGA-coated nanocarriers?

A transferrin receptor (TfR) speciﬁc 7peptide (7pep) has been conjugated to a PEG-b-PCL copolymer that was then used to prepare coumarin-loaded nanocarriers[173]. Live cell study and competition experiments have demonstrated clathrin-mediated internalization, related to the expression of TfR on Caco-2 cells. Whereas a part of the 7pep-M-C6 was found in late endosomes and lysosomes, another part was able to cross the cell monolayer through transcellular transcytosis.

A few years before, a study highlighted the role of GTPase in TfR- mediated internalization[174]. The presence of tyrphostin 8 (T-8), a GTPase inhibitor, with an insulin-transferrin (Tf) conjugate, was able to improve about 20-fold the TfR-mediated transcytosis of insulin on a Caco-2 cell monolayer. Although these results were obtained with a prodrug, it can help in optimizing the TfR-mediated transcytosis of NPs.

PAMAM dendrimers have also been used to improve oral bioavailability through transcytosis. Even if no speciﬁc ligand was grafted onto these G3.5 dendrimers, transcytosis has been reported through speciﬁcally clathrin-mediated endocytosis using dynamin-dependent mechanisms[163]. Dendrimers, internalized using caveolin-mediated endocytosis, were later found in lysosomes. NP surface characterization is lacking in order to better understand the biological interaction, but the anionic charge of the NPs might be responsible for the cell membrane disruption.

Finally, the transport capacity of enterocytes after receptor- mediated endocytosis is limited by cells[137]. Although enterocytes are widely distributed in the small intestine, this is the preferred pathway for high-potency drugs.

Since the intestinal epithelium is not only composed of enterocytes, M cells might be targeted as well through the phagocytic process or pinocytosis. Galectin 9 ligand, UEA-1 ligand, a lectin, integrin ligand

Fig. 5.Schematic diagram of the non-speciﬁc and partially energy-dependent transport of polymer nanoparticles in Caco-2 epithelial cells. Black solid arrows represent the pathways demonstrated in the study, and black dotted arrows indicate the proven pathways in previous reports. Red and green arrows in the diagram represent the proven regulation of actins and COX respectively[165].

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(RGD, LDV) and immunoglobulins have been used for this purpose. This targeting approach was inspired by the properties of immunoglobulins to easily cross the epithelial barrier[175]. Indeed, they managed to covalently conjugate polyclonal IgG Fc fragments to PEG moieties on nanoprecipitates using maleimidethiol chemistry, to speciﬁcally target the NPs to a transcytosis pathway. They thus obtained an absorption efﬁciency of 13.7% h with targeted NPs compared to 1.2% h with non- targeted NPs. In performing the same experiments with FcRn knockout mice, they demonstrated that this 11.5-fold improved bioavailability was directly related to the interaction between these Fc fragments grafted onto the NP and FcRn receptors. However, the authors moderat- ed their enthusiasm by keeping in mind that these receptors are widely distributed in many tissues, not limiting the effect to the intestine, and that the recognition could also result in a faster clearance from blood.

If NPs cross the intestinal barrier through M cells (although they only comprise 5% of the follice-associated epithelium in the Peyer's patch, they will be collected in mesenteric lymph nodes and will reach the blood poolviathe thoracic duct[4]. In the case of insulin oral delivery, this pathway might not be the most relevant one, since the advantage of oral administration is to target the liver in order to mimic the physiological secretion of insulin. However, the rationale behind the se- lection of this pathway is high transport activity despite a less-broad distribution in comparison to enterocytes[4,176].

Another cell type is more widely distributed than M cells. Goblet cells, responsible for mucus secretion, represent 10–24% of intestinal cells[159]. More recently, NP surfaces have been coated to target these cells[176]. Insulin has been loaded in trimethyl chitosan (TMC)- based NPs coated with a CSKSSDYQC (CSK) targeting peptide[177].

Although TMC is known to open TJ and to promote the paracellular pathway, uptake studies on the goblet-like HT29-MTX cell have revealed high internalization via clathrin- and caveolae-mediated endocytosis[177,178]. Transport studies on Caco-2/HT29-MTX co-cultured cell monolayers, andin vivoexperiments have confirmed these results, and orally administered, CSK-modified NPs were able to produce a 1.5-fold improvement in relative bioavailability compared with unmodified ones.“Pulse-chase”analysis seemed to reveal the implication of clathrin-mediated endocytosis in CSK-NP internalization [159].

The same kind of improved oral bioavailability was observed with CSK-coated SLNs[179].

To beneﬁt in the most advantageous way from this ligand–receptor interaction, initially the way the unmodiﬁed NPs cross the epithelium should be established in order to enhance the other pathways.

8.3. Permeation enhancer of the transcellular pathway

Carrier-mediated peptide transport systems might be involved in di- or tripeptide transport[180]. Unfortunately, it might not be sufficient to reach an efficient drug–plasma concentration. If so, in addition to the aforementioned strategies to increase oral drug bioavailability, other absorption enhancers might help in increasing cell membranefluidity and improve the transcellular pathway[181]. These compounds are either medium-chain fatty acids or contain medium-chain alkyl functional groups in their structure[181]. Their cytotoxicity is limited as long as the transmembrane ion gradient involved in cell function is preserved in the cell; their use might be considered if the effect is transient and if the tissue can renew itself. Does an NP surface made of these medium-size carbon chains or any other membrane disruptor act on membranefluidity?

Bile salts (i.e. sodium glycocholate (SGC), sodium taurocholate (STC) and sodium deoxycholate (SDC)) have been incorporated in different liposomes for the oral administration of insulin[182,183]. The studies revealed stability enhancement and a bioavailability increase for insulin- loaded SGC-liposomes (up to 9% in diabetic rats, compared to the subcutaneous injection of free insulin). Indeed, SGC has been reported as both an enzyme inhibition and permeation enhancer[183–185]. Similar results were not observed when the permeation enhancer was co-

administered with insulin in a physical mixture. It might be worth eval- uating the pharmacokinetic proﬁle after the co-administration of free insulin and blank SGC-liposomes to better understand the mechanisms involved and to highlight the critical parameter of this strategy. Other similar excipients will be further discussed in another review in this special issue.

Cell-penetrating peptides might also help in addressing the challenge of poor oral bioavailability. Usually used for intracellular delivery, they might also be useful to cross the GIT barrier. A secretion peptide (Sec) has therefore been combined with penetratin (Pen) in PLAGA nanoparticles for the oral administration of insulin[186]. Whereas Pen facilitates the internalization step, Sec should contribute to the secretion step. Combining both peptide fragments should therefore facilitate transcellular transport. Indeed, insulin-loaded Sec-Pen-NP achieved a 2.63-fold increase of the pharmacological bioavailability of insulin after ileal segment administration in male Sprague–Dawley rats, in comparison with unmodiﬁed nanoparticles, when insulin-loaded Pen-NP sled to a 1.64-fold pharmacological bioavailability increase.

This latter strategy that confers a positive charge to the NPs provided bioavailability of 19.1%, compared to a subcutaneous injection. Other CPPs have been investigated. Polyarginine R8 has been used to prepare CPP-functionalized PEG-PLGA NP[187]. Bioavailability reached 3.1%, 10.2% and 13.9% for INS-NP, L-R8-NP and D-R8-NP, respectively, compared to the subcutaneous injection of free insulin. According to the authors, D-R8 promotes more efficient cell uptake than the L-form of the CPP and this enantiomer is also more resistant to enzymatic degradation. The ZP change from negative INS-NP to positive INS-R8-NP might also have played a role. In another study, two peptide ligands were used to modify salmon calcitonin-loaded solid lipid NPs (sCT SLN): CSKSSDYQC (CSK) and IRQRRRR (IRQ)[179]. The affinity for goblet cells has been reported for thefirst case, whereas the second case is known as being a CPP. Intraduodenal administration in rats revealed sCT bioavailability of 5.1%, 12.4% and 10.0% for sCT SLN, sCT CSK-SLN and sCT IRQ-SLN, respectively, compared to the intravenous injection of sCT.

Surprisingly, IRQ was able to promote the transport of sCT despite the size increase of sCT IRQ-SLN from 240 nm (sCT SLN) to 410 nm. It would be interesting to investigate the transport mechanisms of these CPP-modiﬁed NPs and highlight whether or not it helps free peptide transport or NP transport. Besides, this suggestion has been made in the literature. Since data are as yet insufﬁcient to clearly determine if this strategy promotes NP transport itself, this aspect is still controversial. An interesting review in this Issue will further discuss the rationale of using CPP.

8.4. Endosomal escape

If the NP enters epithelial cells by endocytosis, they might undergo the endosomal process. As mentioned previously, this may follow clathrin-mediated endocytosis[166,173]. If this pathway cannot be avoided, it may result in degradation and the loss of delivery efﬁciency [188]. This endosomal degradation might also affect protein integrity when it is released from the carrier.

Wagner and co-workers have developed a pH-responsive complex with an endosomolytic peptide, called melittin, and PEGylated polyethylenimine[189]. This conjugated compound has enhanced gene transfer by increasing the lytic activity at acidic pH, which triggered the destabilization of endosomal membranes such as bacterial endotoxins[188–190]. Other strategies involve a pH-buffering effect: an extensive inﬂow of ions and water into the endosome may lead to the rupture of the endosomal membrane and to the release of the drug into the cytosol. This proton-sponge effect can be achieved, for example, by histidine-rich molecules[188]. Cationic lipids and fusogenic peptides may also be used for endosomal escape. Indeed, the cationic lipids of liposomes may destabilize the endosomal membrane[191,192]. This strategy has also been used by Culli's group to provide electrical interaction with anionic endosomal membranes[193]. However, Wang et al.

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[194]described the“polycation dilemma”that consists of the interaction of these positively charged macromolecules with membrane phospholipids, leading to membrane disruption or with anionic serum protein, leading to their adsorption and/or aggregation [194,195].

However, if the aim of the DDS is to bring the API into systemic circulation, limiting endosome evolution in lysosomes might be more relevant than endosomal escape.

On the one hand, it can be interesting to avoid clathrin-dependent endocytosis and phagocytosis since they are intended for lysosomal degradation[166]. On the other hand, since homotypic fusion and the vacuole protein sorting (HOPS) complex are mandatory for endosome–lysosome tethering, their inhibition, for instance, could be considered to avoid this degradation process and hence increase the transcytosis of API[196,197].

8.5. Efﬂux pumps

Another phenomenon that might limit oral bioavailability through the transcellular route is the presence of efflux transporters. ATP- binding cassette (ABC) membrane proteins act against a concentration gradient with an energy-dependent process to transport various molecules out of the cells[198]. In intestinal epithelia, ABCB1 (Multi Drug Resistance/P-glycoprotein (P-gp) of subfamily ABCB), Multidrug Resistance Proteins (MRP) 2 of subfamily ABCC, and ABCG2 or Breast Cancer Resistance Protein (BCRP) of subfamily ABCG act as multi- specific drug efflux transporters[180,199–201]. Lipids, bile salts, xenobi- otics and peptides for antigen presentation may thus be cleared faster from cells[199]. Hydrophobic peptide drugs might therefore encounter this bioavailability limitation[202].

P-gp generally acts on hydrophobic, cationic molecules[180]. Some proteins have been found to be substrates of these efﬂux pumps[203].

Quantitative structure–activity relationship (QSAR) analysis methods suggest that BCRPs were likely to interact with structures having one amine bonded to one carbon of a heterocyclic ring and MRP2, like MRP1, is an organic anion transporter[180,204,205]. Research for oral administration has mainly focused on P-gp efﬂux pumps, which are the most likely to impede protein/peptide drug transcytosis.

Three strategies may help in addressing this challenge: (i) excipients may preferentially interact with this membrane protein and disrupt its activity, (ii) the use of a particular transcellular pathway may deprive

cell membranes of P-gp and limit API backﬂow or (iii) nanocarriers can mask the P-gp substrate to efﬂux pumps. These strategies might be combined.

P-gp inhibitors such as verapamil, promethazine and cyclosporine A have initially been proposed to reverse drug efﬂux but results for these quite small molecules have been disappointing. It has also been found that excipients currently used in pharmaceutical formulations may exhibit these P-gp inhibition properties without adding further toxicity.

Bansal et al.[198,206]have listed some of them: (i) surfactants like PEG-20 sorbitan monolaurate and PEG-80 sorbitan monolaurate, polyoxyl 35 castor oil, PEG-40 hydrogenated castor oil, polyoxyl 15 hydroxystearate, PEG-8 caprylic/capric glycerides, poloxamer or PEG monostearate; (ii) natural polymers such as anionic gum, xanthan gum or sodium alginate; (iii) synthetic polymers like PEG, third gener- ation polyamidoamine or thiomers; and (iv) lipids such as triglycerides of fractionated C8-C10 coconut oil fatty acids or neutral oil. Indeed, these excipients are capable of inhibition in various ways depending upon their nature. This was summarized inFig. 6. Therefore, NP degradation nearby the cells could release these intrinsic P-gp inhibitors, thus being advantageous for enhanced oral bioavailability of the free drug substance.

Föger et al.[207]have also reported P-gp inhibition with thiolated chitosan-based nanocarriers. They have shown properties of controlled release, better permeation through rat intestinal mucosa, and enhanced drug–plasma concentrations after oral administration, in comparison with drugs in solution, drugs in chitosan tablets, and in modified chitosan tablets. They have attributed these observations to improved mucoadhesion and to P-gp inhibition conferred by thiomers. However, the absolute bioavailability did not exceed 0.21%. They have claimed a special interest in using such polymers to overcome these efflux pumps, because of the greater difficulty for these macromolecules to cross the cell layer in comparison to smaller P-gp inhibitors, thus local- izing the action at the right place and limiting side effects[208,209]. But is the observed enhanced bioavailability due to real action on the P-gp or is it due to an improved mucoadhesion of the system in the early small intestine, where P-gp expression is reduced using another strategy? Beloqui et al.[210]developed a nanostructured lipid carrier with a P-gp substrate (i.e. saquinavir). They demonstrated that by improving clathrin-mediated transport, the P-gp could interact with caveolae which results in reduced efflux activity and better passage of the drug.

Fig. 6.Mechanism by which excipients tackle P-glycoprotein-mediated efﬂux[198].

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Indeed, P-gp is co-localized with Cav-1 in caveolae[211,212]. A study on P-gp in endothelial cells from the blood brain barrier has conﬁrmed the relationship between the phosphorylation state of caveolin-1 and the P-gp function [213]. However, as mentioned previously, clathrin- mediated endocytosis has been found to end in lysosomal degradation.

The same kind of relation between P-gp activity and the endocytic pathway was observed a few years ago in our laboratory[214,215]. Indeed, experiments have shown that lipid nanocapsule endocytosis could induce the formation of P-gp rich endocytic vesicles that deprive cell membranes of P-gp. In addition, the active endocytosis of the carrier may compete with P-gp for ATP and cholesterol bioavailability, two elements required for the efﬂux. P-gp efﬂuxes were thus limited by two different phenomena. As a result, colloidal particles were found in the basolateral compartment. Interestingly, Beloqui et al.[210]noticed that their results were dependent on NP size and on the method of preparation. This latter point might impact the NP surface design and change the internalization process.

Finally, it is also assumed that if the drug is a substrate of these efﬂux pumps, by increasing the amount of available substrate, saturation of the system may be achieved, resulting in an enhanced pathway for the drug. This way, any strategy that leads to an improved local concentration near the intestinal cell barrier, could overcome the P-gp efﬂux pumps.

9. Protein interaction

Once the intestinal barrier has been crossed, API-loaded NP or API-loaded crumbled NPs might face new pitfalls in the bloodstream. In- deed,in situbiotransformation occurs through the adsorption of various biomolecules by Van der Waals electrostatic interactions, hydrogen bonds, and hydrophobic and hydrophilic interactions[216]. For instance, it has been described that any hydrophobic NP surface in biologicalfluids is covered by surrounding proteins[70]. This can be an issue if the adsorbed proteins alter the functions of the NP surface and affect its biological fate. Plasmatic protein adsorption can provoke aggregation and destabilization; there can even be opsonin adsorption on the system with a faster clearance by the immune system[217–224]. This corona is not always a drawback since it can be correlated with low toxicity[223]. Besides, the adsorption of a dysopsonin such as albumin might even counteract opsonin adsorption[216]. A research group has focused on the influence of particle size and surface chemistry on in vivoprotein corona formation[32]. They distinguished two kinds of components: (i)“soft components”with a rapid, dynamic exchange between the NP surface and the biological medium and (ii)“hard coronas”, composed of biomolecules with high affinity for the NP surface. Size, shape, and surface chemistry have been reported to influence the amount, the composition and thein situevolution of the protein corona[216]. The physiopathological state of the patient also matters. Thefirst corona is difficult to characterize, whereas the second one can be considered as a long-life equilibrium protein signature which is more likely to impact cell transport and the other biological processes[216]. Besides, some consider that thesefingerprints can testify to the different biological processes undergone by the NP[225].

In a study, three different surfaces (carboxyl modified, unmodified and amine-modified surfaces) and two ranges of size (50 and 100 nm) with polystyrene NPs were compared, after incubation in human plasma for 1 h[32]. Important differences in protein corona composition were observed in terms of quantity and quality. Firstly, 100 nm NPs are more likely to be recognized by immunoglobulins and complement pathways, which are involved in immune responses, than 50 nm NPs, whatever the surface chemistry (except for an unmodified surface). Indeed, size variation impacts on the surface curvature and therefore on the adsorption phenomenon[216]. Secondly, an amine-modified surface is more likely to be recognized by apolipoproteins, which are involved, through high density, low density, or very low density lipoproteins, in the transportation of lipids and cholesterol in the blood pool. The authors

also highlighted that even minor changes might cause huge biological effects. It should be noticed that globular proteins may undergo denatur- ation when adsorbed in an oil–water interface, which can lead to covalent cross-linking through disulﬁde-bond formation[226,227]. This implies that biological proteins covalently bound to the NP might be non-functional. However, insulin has already been used to target insulin receptors of the blood brain barrier with injected NPs[228]. Since an improved antinociceptive effect was measured after the injection of lopinavir-loaded NPs in the tail vein, one can think that receptors of the BBB were still able to recognize grafted insulin. Unfortunately, as the hypoglycemic effect of insulin grafted onto the NPs has not been assessed, it is difﬁcult to determine if the integrity of the whole protein was preserved.

Troncoso et al.[229]usedβ-lactoglobulins to stabilize oil nanodroplets and highlighted that the lipid digestion rate decreases with particle radius (85–59 nm). They explained this phenomenon by differences in interfacial surface area, a thicker protein layer, and different structural organization ofβ-lactoglobulins at the interface of the smallest particles.

This results in more difﬁculties for bile salts and surfactants to displace the protein layer and let lipases penetrate into the NP. Is this effect due to the shield of the NP by a layer of biocompatible components, or does the protein play a full biological role?

As can be seen above, this protein corona can be anticipated as playing a role in the bioavailability of the DDS or it can be avoided with a hydrophilic and biocompatible shield that confers stealth properties. Indeed, protein adsorption, opsonization and removal from the circulation may occur when the surface is hydrophobic. A hydrophilic surface is therefore a good strategy to avoid this recognition, to limit this early clearance, and to improve circulation time. In addition to the numerous other functions, PEG is one of the most often used hydrophilic polymers to coat NPs and bring stealthiness to the carrier.

To conclude, even if a lot of pitfalls have been avoided so far to cross the epithelium and reach the bloodstream, as long as protein corona might be formed, thefinal goal has not yet been achieved and afinal trap must be overcome. Since the physiological parameters evolve with age, diet and the physiopathological state of the patient, knowing the composition of the biologicalfluids and the interaction that might be involved with the vector is essential to design the proper vector surface. In addition, intestinal barrier crossing might have altered the carrier. These changes have to be taken into account to successfully face this issue.

Even if most of the works on protein corona focus on the one formed in the bloodstream with serum proteins, it is assumed that the phenomenon may occur with proteins in the GIT. Recently, this interaction has been studied in the oral cavity and has shown a negative impact on therapeutic efﬁciency[224]. However, since this adsorption might be considered as a biocompatible shield, it is not excluded that NP uptake can beneﬁt from this corona if it is controlled.

10. Discussion

Considering the particular properties of NPs to cross epithelia and protect a therapeutic cargo, it is obvious that this technology might help overcome low oral bioavailability. Moreover, it has been highlighted that NPs exhibit a large specific surface area, corresponding to an equally significant energy that can be used to help in improving the biological fate of NPs[33]. This is even truer since it is thefirst layer in contact with biological material. Once a protein/peptide drug has been successfully encapsulated with a mild process that preserves its biological activity, it is of prime importance to properly design this NP surface.

A lot has been done to better understand the challenges we face, and we have now some ideas of the mechanisms resulting in low oral bioavailability[230]. The present review was aimed at highlighting recently developed strategies (seeTable 1) to overcome each step in the absorption of a peptide/protein from the intestinal environment to the bloodstream, including stability in lumen, enzymatic degradation,