Biopharmaceutical Parameters to Consider in Order to Alter the Fate of Nanocarriers after Oral Delivery

Emilie Roger; Frederic Lagarce; Emmanuel Garcion; Jean-Pierre Benoit


Nanomedicine. 2012;5(2):287-306. 

In This Article

GI Tract & Barriers

The GI tract is considered to be the tube starting from the mouth and the esophagus, where it has only a transport role. Digestion then begins in the stomach and continues in the small intestine to the colon. Absorption essentially takes place in the small intestine.

Stability in the GI Medium

After oral administration, the first barrier met by nanocarriers is the GI lumen. The interaction between nanocarriers and the contents of the stomach or the intestine can lead to the degradation of particles. Consequently, nanocarriers need to be resistant and stable in the GI tract.[23] The stomach is characterized by an acidic pH (ranging between 1.5 and 5 for the fasted and fed state, respectively) and the presence of pepsin. In the small intestine, the pH is in the range of between 5.5 to 7 and several digestive enzymes such as trypsin, chymotrypsin, carboxypeptidase, amylase, lactase and lipase, as well as bile salts, are present.[24] Furthermore, the stability of nanocarriers can be affected by the residence time, the volume of fluid available in the GI tract and its motility. The in vitro models used in order to study the stability of nanocarriers were initially designed to evaluate the solubility of drugs in the GI tract.[25–28] They involved the use of biomimetic media that recapitulate the status of intestinal or gastric fluids in feeding or feeding-free conditions (Table 2).[29–40] Thus, these models are not totally suitable to study the stability of nanocarriers and the interpretation of the results has to be cautious.

The stability of different nanocarriers has been studied and in some cases modifications of the formulations were made in order to improve stability. First, the integrity of liposomes is controversial due to the possible degradation of these nanocarriers by the acidity of the stomach, bile salt and lipase.[41] Liposomes are structured in concentric bilayers of phospholipids. In the gastric medium, H+ cations can diffuse in the inner aqueous phase of liposomes and destabilize them. Moreover, the bile salt monomers are able to penetrate into liposomal lipid bilayers, and disrupt their vesicular structure.[42,43] Nevertheless, the increase of cholesterol quantities or the addition of saturated phospholipids improves the rigidity of liposomes and as a consequence decreases the possibility of enzymatic degradation.[44–47] Rowland et al. showed that liposomes composed of distearoylphosphatidylcholine/cholesterol were stable in presence of bile salts, pancreatic lipase and at low pH, on the contrary liposomes composed of dipalmitoylphosphatidylethanolamine/cholesterol/dicetylphosphate were unstable at low pH.[48] It was found that coating the surface of liposomes with a polymer, such as polyethyleneglycol (PEG), or with the sugar portion of mucin, protected them against bile salt destabilization and increased the stability of these colloidal carriers in the GI tract.[13,49–51] For example, Iwanaga et al. observed that uncoated liposomes released 20–50% of insulin in the presence of bile salts, whereas distearylphophoethanolamine-PEG-coated liposomes and mucin-coated liposomes released approximately 2 and 10% of insulin, respectively, within 6 h in the same medium.[51] It is interesting that liposomes coated with chitosan are more unstable than uncoated liposomes (retention of fluoresceine iso thiocyanate was approximately 20 vs 36% after 2 h exposure in simulated gastric fluid).[52] All the same, chitosan nanoparticles, poly(alkylcyanoacrylate) and calcium phosphate nanoparticles were rapidly degraded.[53–56] In the case of polylactide (PLA), poly(lactic-co-glycolic acid) (PLGA) and palmitic acid (PA) nanoparticles[57,58] or solid lipid nanoparticles (SLNs),[39,40] the presence of poloxamer or PEG at the surface improved stability of these nanoparticles. In fact, PLA, PLGA and PA polymers contain ester bonds that can be cleaved in an acidic environment or by enzymes.[59–61] Similarly, SLNs are particles made from lipids that are solid at room temperature and are degraded by the pancreatic lipase found in the intestinal fluid.[39,40] PEG and poloxamer form a sterically stabilizing crown on the surface of nanoparticles and, consequently, neither the acidic environment nor enzymes can affect their integrity. Likewise, the GI stability of polymer micelles[62] and lipid nanocapsules (LNCs)[16] were demonstrated in vitro in simulated fluid. LNCs were covered with PEG660 hydroxystearate (Solutol®),[63,64] which stabilized the particle structure. Many solutions have been found on a case-by-case basis to improve the stability of nanocarriers in the GI tract. No universal coating polymer was found to protect nanocarriers from degradation. Moreover, the in vivo realistic models remain to be validated. The fluorescence resonance energy transfer, already used for other stability studies,[65] is a very promising imaging technology in this field.

Diffusion Across Mucus

Prior to intestinal absorption, nanocarriers must diffuse across the unstirred water layer in spite of rapid transit. The unstirred water layer is a more or less stagnant layer of water, mucus and glycocalyx adjacent to the intestinal wall, and its thickness is approximately 30–100 µm.[66,67] Mucus is released by goblet cells and protects the epithelial surface by lubricant properties conferred by mucin, the main component of mucus. Mucin is a glycoprotein that significantly decreases the diffusion of small and large compounds and also creates a physical barrier to the absorption of particles. Owing to the hydrophobic domains of mucin, hydrophobic interaction represents an important mechanism by which mucus limits the transport of large entities.[68,69] For example, Behrens et al., demonstrated that hydrophobic polystyrene nanoparticles stayed anchored in mucus following their interaction with mucin and thus showed a lower association with epithelial cells.[70]

Nanocarriers with a positive surface charge can be transported more easily across mucus: the high number of sulfate sialic acid and sugar moieties in the carbohydrate part chains of the mucin molecule imparts a highly negative charge to mucin. Consequently, if the particles are positively charged, an electrostatic interaction with the negative charges of mucin favor the absorption of particles in the mucus layer and an increased internalization by epithelial cells, which are also negatively charged.[68] To illustrate this phenomenon, in situ studies of positively charged PLGA nanoparticles prepared with didodecyl dimethyl ammonium bromide have shown an increase of nanoparticle uptake in comparison with neutral or negatively charged PLGA nanoparticles prepared with polyvinyl alcohol.[71] Chitosan also conferred a positive charge to nanoparticles, which then adhered to the mucosal surface.[54,70,72] Bioadhesion in mucus leads to a higher residence time in the epithelial barrier area, thus providing a better probability for drug absorption. The relationship between surface properties and bioadhesion is described later in this review.

Transport Across the Intestinal Epithelium

Different mechanisms are used to transport nanocarriers across the intestinal barrier (Figure 2). Many transport studies across the intestinal barrier have been performed with different nanocarriers (Table 3).[73–99]

Figure 2.

Possible transcellular mechanism of nanocarrier uptake by the intestinal barrier.

Paracellular Transport Paracellular transport is the passive diffusion through intercellular spaces. Tight junctions are the closely associated areas of two cells that permit the formation of an almost impermeable barrier. Tight junctions are composed of a group of transmembrane and cytosolic proteins, principally including occludins, claudins, actin and zona occludens-1 proteins. Between these protein complexes, paracellular spaces are defined. Consequently, the paracellular transport between the epithelial cells is limited by the intercellular space whose pore diameter has been estimated to be between 3 and 10 Å.[100,101] In order to allow drug passage, tight junctions need to be opened.

Volkeimer introduced the concept of 'persorption' of particles, and suggested a passage of particles by the mechanical lesion of tight junctions.[102] Although the opening of tight junctions resulting in enhanced paracellular permeability is possible, intercellular spaces between epithelial cells remain limited in order to transport intact particles to the bloodstream.[103] Thus, paracellular transport of undamaged nanoparticles requires further elucidation and observed enhanced paracellular permeability must be more likely ascribed to the anticipated release of the drug from the nanocarrier. In some cases the nanocarriers itself or its components plays a role in tight junction disruption.

Indeed, different nanocarrier components have already demonstrated their capacity to open tight junctions.[104] Recently, a review[100,105] has described different polymers or surfactants such as anionic polyacrylate derivatives, poly(methacrylic) derivatives, cationic chitosan, chitosan derivatives (e.g., N-trimethyl chitosan), thiolated polymers, or thiomers, poly(amidoamine)[97] and their mechanisms to enhance the paracellular transport of drugs by the structural reorganization of tight junction-associated proteins or by reducing the extracellular Ca2+ concentration.[100]

In a recent publication, Sadeghi et al. compared the effect of free-soluble quaternized derivatives of chitosan and their incorporation in nanoparticles to open tight junctions.[106] They demonstrated that free polymer had much more effect on tight junction opening than chitosan entrapped in nanocarriers. Studies were performed on a Caco-2 cells model, a well established model to study the intestinal permeability of drugs.[107] This model express tight junctions of which quality can be controlled by measuring the transepithelial electrical resistance (TEER): a decrease of TEER reflects an increase of paracellular permeability.[108] In the presence of nanoparticles, the decrease of the transepithelial resistance was approximately 20% of the initial values, whereas in the presence of free chitosan derivative it went up to 55%. This result was correlated with the positive surface-charge density of polymers that was greater than that of nanoparticles, and consequently, free polymers could be fixed more abundantly on negative charges present in the interior of tight junction.

Sezgin et al. investigated the transport of PEG-phosphatidyl-ethanolamine (PE) micelles, and demonstrated the bioadhesion of micelles and their capacity to open tight junctions.[94] Nevertheless, the transport experiment showed a lack of drug passage through the intestinal barrier (~14 and 1% after 4 h for rhodamine-PE-labeled and meso-tetraphenyl-loaded micelles, respectively), which was due to the absence of the drug being released from the micellar core.

Consequently, two hypotheses could explain paracellular drug transport. Nanocarriers infiltrate the mucus layer, then break and release their components, which open up tight junctions, thus allowing the released drug to permeate through the paracellular route.[81] Alternatively, intact nanocarriers, such as micelles, adhere to tight junctions, open them up and then encapsulated drugs are released to be transported between paracellular cells. According to the second hypothesis, the drug remains encapsulated during the mucus layer transport to reach tight junctions, which is an advantage. However, in the first hypothesis, the drug is released in the mucus layer and needs to be stable and able to diffuse through the mucus. To date, no paracellular transport of intact nanocarriers has been demonstrated. It is worth noting that the opening of tight junctions is reversible,[78,81,90,96] but has consequences on their physiological functions when opened.[104] Indeed, when tight junctions are opened, toxins and biological pathogens can also be transported into the systemic circulation system.[109]

Transcellular transport After crossing the mucus, nanocarriers have to reach the surface of the enterocytes, where they could develop adhesive interactions and be 'translocated' by intestinal cells. For example, Yoncheva et al. demonstrated that pegylated nanoparticles interacted and bound directly with the cell surface rather than mucus components.[110] Moreover, the PEG-coating facilitated the transcellular transport of particles.

To cross the intestinal barrier, nanocarriers can be transported according to a number of different passive and active transcellular pathways. The latter divided in three main steps:

  • Uptake process at the apical side of cell

  • Transport through the cell

  • Release at the basolateral side of cell

These different transport stages across epithelial cells can be performed across enterocytes or M-cells. The intestinal epithelium is made up of different types of cells: absorptive cells or enterocytes, goblet cells (secreting mucus into the intestinal lumen), enteroendocrine cells (secreting hormones such as cholecystokinin and gastrin into the blood), paneth cells (secreting a number of antimicrobial molecules into the lumen) and M-cells.

Passive transcellular transport includes diffusion across cells. This transport by diffusion is only feasible for small molecules, due to the limited size of pores in the cell membrane that can be opened or closed by the conformational change of a membrane protein.[73,109] The rate of absorption is governed by Fick's Law, which depends, among other parameters, on the physicochemical properties of the molecule and on the concentration gradient across cells. Passive transport has been used to transport small, lipophilic molecules across the phospholipidic bilayer and the membrane-bound protein regions of the cell membrane.[23] Theoretically, supramolecular structures, such as nanocarriers, can not diffuse through cells by passive diffusion due to their size. In this instance, Mathot et al. demonstrated a decrease of Papp of polymer at 4°C and, consequently, suggested the passive diffusion of the polymer as well as the free drug fraction.[93,111] In this case, a release of encapsulated drug was suggested, which allowed its transport by passive diffusion.

Transport by energy-dependent transcellular process should also be considered. Indeed, large objects such as macromolecules and particles are internalized by an active mechanism in which a portion of the membrane extends and envelops the object, drawing it into the cell to form a vacuole (cytosis). Active transport, which is illustrated by different energy-dependent mechanisms, is mostly used to transport drugs encapsulated in nanocarriers. This active transport has been described for M-cells and enterocytes. Thus, different studies performed at 4°C have demonstrated the transport of encapsulated drugs by an energy-dependent endocytic process. For example, El-Sayed et al. demonstrated a 38-fold decrease in the transport of fluoresceine iso thiocyanate-labeled dendrimers at 4°C after 150 min.[97] With nanoparticles, Win and Feng showed a reduction of uptake at 4°C down to 25–46% in comparison with 37°C.[112] These results suggest that the nanoparticles uptake could be due to energy-dependent processes, such as adsorptive endocytosis.


M-cells have been identified in the intestinal epithelium. They are specialized cells of the mucosa-associated lymphoid tissues (O-MALT) that consists of lymphoid follicles arranged to form distinct structures such as Peyer's patches. M-cells are characterized by their ability to transport antigens from the lumen of the small intestine to cells of the immune system. Indeed, the invagination of cells at the basolateral side contains lymphocytes and some macrophages,[113] which are then distributed throughout the whole body. M-cells can endocytose particles by different mechanisms (fluid phase endocytosis, adsorptive endocytosis and phagocytosis) and then exocytose particles across the basolateral membrane into the lymphoid tissue.[114] Due to the absorption property of M-cells, particle uptake by these cells has been described extensively.[103,115–128] Jung et al. suggested that nanoparticles with a negative charge combine with the hydrophobic surface and promote M-cell uptake.[103] Indeed, M-cells have sparse microvilli, glycocalyx and an absence of mucus, which relate to the hydrophilic property of the intestinal barrier.[121] In a recent review, des Rieux et al. summarize the properties of particle uptake by M-cells, specifically those exhibiting molecules on their surface to target M-cells.[116] Ulex europaeus agglutinin 1 ligand, Aleuria aurantica lectin, salmonella extract, invasin, RGD peptide, IgA, and ganglioside (GM1) are some specific ligands attached on particles, which could specifically target M-cells. Particles with sizes greater to 1 µm can also be taken up by M-cells. Box 1 lists nanocarriers that undergo M-cell uptake.[129–135]

M-cell transcytosis appears to be a target for the delivery of oral particulate-based vaccines, however it is limited since M-cells constitute less than 1% of the intestinal epithelial cell population.[119] The access of nanocarriers to systemic circulation is limited after M-cells transport as it is often trapped in the local lymph nodes.[136] Moreover, lymph flow is limited in comparison to blood flow (1:500), and is highly variable depending on the physiological conditions. Consequently, for drugs that need to reach the blood circulation, their transportation across absorptive epithelial cells (i.e., enterocytes) could be the most appropriate mechanism.


The transportation of nanocarriers by enterocytes has been extensively studied.[100] However, the underlying mechanisms responsible for transcellular transport are still unknown. As observed previously, the transcellular transport of particles is an energy-dependent process. Uptake by enterocytes is described by pinocytosis, which includes macropinocytosis, or endocytosis, which includes clathrine-mediated endocytosis, caveolae-mediated endocytosis and clathrine- and caveolae-independent endocytosis.[137,138] All of those vesicular transport mechanisms are tightly regulated and involve small GTPases regulated by accessory proteins for vesicular motility, docking, fusion or cytoskeleton reorganization.[139] Characterization of the intracellular transport profiles of nanocarriers is currently in research stages.

Macropinocytosis is an actin-dependent process that is not receptor mediated, and hence is not a specific transport system. It corresponds to the formation of large and heterogeneous dynamic vesicular structures at the cell surface, called macropinosomes, with sizes ranging from 0.5 to 2 µm.[140] Consequently, a suspension of particles can be internalized this way if they are smaller than 2 µm. In vitro experiments by Ragnarsson et al. have demonstrated the macropinocytosis transport of fluorescent carboxylated polystyrene nanoparticles.[141]

Clathrin-coated pit (CCP) is the best-studied receptor-mediated endocytosis mechanism. The assembly of clathrin molecules on the coated pit induces the invagination of the membrane and forms a clathrin-coated vesicle (120 nm) that requires the GTPase activity of dynamin and actin polymerization to form. This endocytosis pathway is initiated when the endocyted material interacts with the assembly polypeptide (AP)-2 and phosphoinositol biphosphate.[142] It was demonstrated that nanocarriers could stabilize the CCP.[143] The molecular basis of cargo captured by CCP was established with the characterization of endocytotic signaling within the cytoplasmic domain of the internalized cargo, as for example, a low-density lipoprotein receptor or a transferrin receptor that interacts directly with AP-2. Moreover, internalization of clathrin-coated vesicles was induced by triggering a receptor such as the EGF receptor.[142] Kitchens and colleagues demonstrated that poly(amidoamine) (PAMAM) dendrimers are internalized by a clathrin-dependent receptor mediated endocytosis process.[98,99,144] These macromolecular structures maintained a high degree of colocalization between clathrin and transferrin and induced rapid internalization by a clathrin-dependent endocytosis mechanism. Uptake experiments were also performed with chitosan nanoparticles, in the presence of chlorpromazine in Caco-2 cells. Chlorpromazine at a concentration of 6–10 µg/ml can induce the redistribution of clathrin and AP-2 from the whole surface and can reduce the number of CCPs at the cell surface. The resulting inhibition of clathrin-mediated endocytosis reduced the internalization of nanoparticles by up to 67.9%.[77]

Another transcellular pathway, caveolae-mediated endocytosis, was first observed 50 years ago, and was associated with cholesterol and sphingolipid-rich microdomains of the cell membrane.[137] The term 'lipid raft' refers to these putative membrane microdomains, which are defined as 'small heterogeneous membrane domains' enriched in cholesterol, glycosphingolipids, sphingomyelin, phospholipids, glycophosphatidylinositol-linked proteins and some membraner–spanning proteins.[145,146] The shape and structural organisation of caveolae-mediated endocytosis vesicles are characterized by the presence of a family of proteins called caveolins, that bind proteins present in intestinal cells.[147] Caveolins coat the cytoplasmic surface to form buds on the membrane, contrary to clathrin, which coats membrane invaginations of CCPs.[148] Moreover, caveolae appear to be remarkably uniform invaginations with dynamin localized at the neck and with a pit diameter estimated between 50 and 80 nm.[145,146,149] This diameter is smaller than clathrin-coated vesicles; consequently, the size of nanocarriers could be a parameter determining the endocytosis pathway.

Uptake by caveolae-mediated endocytosis can lead to caveolin-1-containing endosomes, called caveosomes.[150] Interestingly, simian virus (SV)40 trafficking via caveolae has been demonstrated. By binding to two lipid raft receptors, a virus can induce receptor clustering and a cascade of signaling events, including tyrosine kinase phosphorylation and protein kinase C activation, which contribute to caveolae formation and endocytosis.[151] In that sense, nanocarriers can be compared with viruses in terms of size and structure and this caveolae pathway could provide a possibility for their transport. Filipin is known to disrupt the caveolae structure by binding to sterols such as cholesterol and by disorganizing caveolin.[152] By using filipin and chlorpromazine, our group demonstrated on an in vitro Caco-2 cell model that LNCs were transported via both clathrin- and caveolae-dependent endocytosis.[91] Indeed, a decrease of approximately 20 and 65% of paclitaxel-encapsulated transport was measured in the presence of chlorpromazine and filipin, respectively. Similarly, Gao et al. demonstrated that wheat germ agglutinin (WGA)-conjugated nanoparticles were also absorbed via both of these mechanisms, which can be explained by the binding of WGA to N-acetyl-glucosamine or sialic acid on the cell surface.[86]

Finally, other endocytosis pathways, different from the three previously described and found to be associated with lipid rafts, were identified as clathrin- and caveolae-independent endocytosis. These poorly described pathways cannot be excluded for the transport of nanocarriers.

After being endocytosed, nanocarriers have to be transcytosed, that is, transported across cells to become available for blood transport to their therapeutic targets. In the case of transport across the enterocytes, nanocarriers can potentially enter by lymphatic or blood capillaries.

The main intracellular trafficking of macropinosome, caveosome or clathrin-coated vesicles is interesting. Clathrin-coated vesicles or macropinosomes are transported and fused with cytoplasmic or early endosomes (membrane-enclosed vesicles). From there, endosomal vesicles are acidified via a proton pump to become late endosomes, then lysosomes. Two mechanisms are possible to explain lysosome formation; either a part of the late endosomes compartment is transported to the Golgi area and matured to become lysosomes (membrane-enclosed vesicles containing acid hydrolases), or late endosomes fuse with already existing lysosomes. The contents of lysosomes could then be exocytosed into the intestinal lymphatic system.[153] Currently, the evolution of nanocarriers in lysosomal vesicles is unknown. In the lysosomal vesicles, the enzymatic destruction of molecules, including lipids, carbohydrates and proteins, can occur. Moreover, exocytosis by the apical side of late endosomes is also possible, and could limit the transport of carriers into the bloodstream. In the literature, the description of the intracellular trafficking of nanocarriers in intestinal cells has not yet been described. However, in other cell lines, it has been demonstrated that following their uptake into primary endosomes, a fraction of nanoparticles is released by exocytosis, while the other fraction of nanoparticles remains in primary endosomes and is then transported to secondary endosomes, which fuse with lysosomes.[154] Some nanocarriers described in the literature showed their ability to escape endolysosomes. For example, Panyam et al.[155] have demonstrated the rapid capacity of PLGA-nanoparticles to escape endolysosomal vesicles due to the reversal of nanoparticle's surface charge in the acidic medium of the endolysosomes.[156,157] The same authors suggested, in the case of nanoparticles localized in the cytoplasmic compartment, a possible exocytosis across the cell membrane.

Moreover, cationic liposomes can destabilize the endosomal membrane by electrostatic interactions, which induces the displacement of anionic lipids.[158] Consequently, there are three possibilities after the uptake of nanocarriers by clathrin-coated vesicles or macropinosomes. First, nanocarriers can be degraded and release their drug content in the endolysosomal vesicles. Second, nanocarriers can escape from the endolysosomal vesicles and undergo exocytosis to blood capillaries. In this case, key questions are: do the nanocarriers remain intact in the cytosol and how are they exocytosed? Finally, nanocarriers can remain intact in lysosomes and can be transported under the form of chylomicrons to the lymphatic system.

In the case of caveolae-coated vesicles, many studies have described a different transcytosis pathway through the cytoplasm that is distinct from endolysosomes.[145,159,160] Caveolae could deliver their content either to endosomes or to caveosomes and this could lead to direct exocytosis on the basolateral side of the enterocytes by an independent pathway.[152] This mechanism represents a refined permutation describing transcytosis and provides direct, specific transport across the cell. Consequently, caveolae-dependent endocytosis could be another hypothesis to explain the release of intact nanocarriers outside enterocytes in blood capillaries via the basolateral cell side. Sang Yoo et al. have demonstrated that salmon calcitonin-loaded PLGA nanoparticles could be transported across the intestinal barrier by a transcytosis mechanism based on caveolae-coated vesicles.[82]

However, it is important to notice that in the case of direct transport in the systemic circulation via the portal vein, traveling through the liver will take place and accordingly, a first-pass metabolism is highly probable. This is not the case if nanocarriers are transported via the lymphatic system.


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