3.1.1 Improving oral bioavailability with mucoadhesive particles
Small molecule drugs, proteins, or peptides can be encapsulated and protected from the harsh gastric environment by polymeric nanoparticles. Additionally, nanoparticle surface characteristics can be tailored to optimize mucoadhesion, cellular uptake, immune system interactions, and cell targeting. Particles synthesized from commonly used polymers, such as poly(lactic acid) (PLA), poly(sebacic acid) (PSA), poly(lactic-co-glycolic acid) (PLGA) and poly(acrylic acid) (PAA) may achieve mucoadhesion via hydrogen bonding, polymer entanglements with mucins, hydrophobic interactions, or a combination of these mechanisms [5
]. For example, 680–850 µm microspheres composed of mucoadhesive copolymers of fumaric acid and sebacic acid exhibited prolonged retention in the rat gut compared to a more weakly adhesive alginate particle based on fracture strength measurements and GI transit times; the copolymer formulation had ~50% increase in the area under the curve (AUC) for dicumarol as compared to the spray-dried drug and alginate formulation [43
]. Similarly, Mathiowitz et al demonstrated that encapsulating insulin in a similar mucoadhesive polymer blended ~200 nm nanoparticle led to a 7-fold decrease in deviation from fasting blood glucose concentration as compared to insulin solution in rats [44
]. The authors attributed this difference to reduced insulin degradation when it is encapsulated in the nanoparticles and to uptake of nanoparticles by Peyer’s patches. However, blood glucose levels were not statistically different for insulin solution as compared to saline only, which would indicate that the fragile protein was significantly degraded during GI transit. To determine whether mucoadhesion contributed to the improved blood glucose regulation, insulin encapsulated particles would have to be compared with ‘non-adhesive’ nanoparticles.
In addition to prolonging residence time, improved oral bioavailability can also be attributed to protection from proteolytic enzymes. Upon exposure to protease degradation, the stability of insulin and calcitonin was improved when encapsulated in polymeric nanoparticles [45
]. Alonso’s group has also observed that coating ~160 nm PLA nanoparticles with poly(ethylene glycol) (PEG) imparts additional protection against enzyme induced aggregation and degradation in simulated GI fluids in vitro
]. The zeta potential of uncoated PLA was −44 mV, which increased to −14 mV when the particles were coated with PEG (PLA-PEG), thus indicating a moderate surface coating of neutrally charged PEG; it is important to note that this partial coating would not likely be adequate to produce mucus penetrating particles, as was shown for cervicovaginal mucus [9
]. After oral gavage of 1 mL radio-labeled particles to rats, blood samples and lymph tissue were excised; 3–4 fold more radioactivity (a fraction of a percent of the original dose in total) was found in the blood at all time points up to 24 h in the case of PLA-PEG particles. The authors attributed this effect to increased uptake of PLA-PEG particles across the intestinal epithelium. However, it was shown that 5–15% of the radiolabel released from the particles after 4 h in intestinal and gastric fluid stimulants, indicating that a significant amount could release after 24 h. It is possible that the partial PEG coating on the PLA-PEG particles allowed a small fraction of nanoparticles to get in closer proximity of the epithelium before releasing the radiolabel, which could then be taken up into the bloodstream.
Another contributor to improved bioavailability is likely the direct uptake of particles by intestinal cells. Although the extent of absorption of particulate delivery systems by enterocytes versus M cells is controversial, it is clear that nanoparticle surface properties are very important for intestinal uptake. Evidence suggests that particle translocation can occur across enterocytes in the villus part of the intestine, but the overall endocytic activity is low [36
]. For instance, des Rieux et al. reported a thousand-fold increase in the transport of 200 and 500 nm polystyrene particles in vitro
when epithelial gut cells were co-cultured with cells that had been differentiated to achieve M cell-like features [48
]. Thus, extensive effort has been focused on elucidating optimal surface and size characteristics for uptake into the Peyer’s patches.
3.1.2 Optimizing mucoadhesive nanoparticle characteristics
Studying the effect of polymer hydrophobicity, Eldridge and coworkers determined in mice that 1–10 µm hydrophobic microspheres administered by oral gavage (polystyrene, polymethylmethacrylate, and polyhydroxybutrate) were taken up by Peyer’s patches more efficiently than less hydrophobic lactide and glycolide polymer particles. Particles composed of hydrophilic cellulose polymers were absorbed 100-fold less than hydrophobic particles, further supporting the interpretation that the major determinant for particle absorption by Peyer’s patches is particle hydrophobicity [49
]. However, it is also true that hydrophobic interactions play a major role in mucoadhesion [4
]. It is important to reemphasize that Peyer’s patches are relatively less protected by mucus, perhaps supporting their immune-sensory role for sampling lumenal contents [2
]. However, particles that adhere to GI mucus would not be able to efficiently reach the Peyer’s patches. It is possible that Peyer’s patch uptake could be optimized by a combination of mucus penetration and M-cell adhesion; unfortunately, the large hydrophilic particles used by Eldridge would likely be sterically trapped in GI mucus, thus eliminating the potential increase in hydrophilic particle uptake due to mucus penetration. Behrens and coworkers also determined that 200 nm hydrophobic polystyrene nanoparticles had enhanced uptake in Caco-2 cell culture, although the uptake was reduced 2-fold by the presence of mucus in MTX-E12 culture [50
]. Similar to Eldridge, Behrens suggests that although nanoparticle hydrophobicity enhances cell association and subsequent uptake, hydrophobicity could also be a major obstacle for mucus penetration. It is not clear whether cells in culture produce an intact mucus layer equivalent to the mucus barrier in vivo
, or whether culture buffers cause significant dilution of the mucus. Thus, it is possible that an intact mucus barrier would lead to a further decrease in hydrophobic particle uptake compared to a lack of a mucus barrier. However, it is clear that mucus affects nanoparticle uptake in cell culture models, so further testing of particle uptake in vivo
In addition to hydrophobicity/hydrophilicity, nanoparticle surface charge also affects oral drug delivery. The surface charge can be used to improve the proximity of nanoparticles to the epithelium to enhance drug absorption, as well as to increase particle uptake via Peyer’s patches. Many studies have indicated that mucoadhesive particles increase drug absorption compared to free drug; electrostatic interactions between a positively-charged particle surface and the extensive negatively charged sugar moieties on mucins are strongly mucoadhesive. For this reason, a considerable number of studies have been conducted using positively charged polymers such as chitosan for enhancing drug absorption [51
]. Coating PLGA nanoparticles with chitosan improved the absorption of tetanus toxoid [55
] and salmon calcitonin [56
]. It is of note that Schipper and coworkers observed that exposure of perfused rat ileal tissue to chitosan caused an increase in mucus secretion and only modest absorption-enhancing effects for atenolol. Upon further testing with mucus covered HT29-H goblet cells, the binding of chitosan to the epithelial surface and subsequent absorption-enhancing effects were significantly improved if the mucus layer was removed
prior to chitosan addition [57
]. It is likely that particle adhesion to the outer mucus surface limits drug absorption; the improvement when comparing delivery of encapsulated drug to free drug is likely due to sustained release from particles, decreased drug degradation during GI tract transit, etc. Utilizing a drug-loaded particle that can penetrate the mucus barrier and release drug closer to the epithelium might further improve drug absorption [see section 3.2]. A study by Jani and coworkers addressing particle uptake by Peyer’s patches found that neutral surface charge was ideal for uptake of 130 and 950 nm polystyrene particles [58
]. However, mucoadhesive particle uptake by Peyer’s patches is also limited by the presence of mucus [see section 3.2.1].
Aside from surface characteristics, size is an important characteristic for efficient uptake. Numerous studies investigating the effect of size have been conducted in various animal models and experimental systems, with the general consensus being that particles less than 1 µm in size can be transcytosed by M cells [48
]. However, the optimal size is also an area of contention and conflicting reports; care must be taken in interpreting uptake and translocation results due to the various limitations and the numerous experimental variables of in vitro
cell cultures and ex vivo
intestinal loop models [61
]. For example, Eyles and coworkers found that when 5 times the total volume of 870 nm polystyrene particles (0.5 mL compared to 0.1 mL) was administered by gavage to the rat stomach, almost 5-fold more polystyrene particles were found in the blood 15 minutes after dosing [62
]. This phenomenon is potentially important for explaining the large uptake seen in many experiments with mucoadhesive particles since dilution with a large fluid volume can degrade the mucus barrier in a way that may not be relevant for administering oral drugs in humans. Eyles and coworkers also observed in the same study that administering the particles in water (hypotonic) led to 5-fold greater uptake of particles into the blood 15 minutes after dosing as compared to particles administered in saline (isotonic) [62
]. In this case, significantly greater particle uptake by Peyer’s patches was probably caused by osmotically driven fluid absorption.
3.1.3 Targeted mucoadhesive nanoparticle systems
In addition to “passive” targeting of lymphoid tissue, the use of targeting ligands to enhance particle uptake has been investigated using ligands that bind specific receptors expressed on enterocytes or on M cell surfaces. Coating nanoparticles with these ligands is intended to enhance the binding specificity and decrease the elimination rate due to mucus turnover [63
], although the extent to which these particles could penetrate the mucus barrier to adhere to enterocytes was unclear. Many different types of ligands have been described including lectins [65
], invasins [66
] and vitamin B12 derivatives [67
]. Lectins are naturally occurring proteins or glycoproteins that bind reversibly to specific sugars, and are involved in many cell recognition and adhesion processes. Several accounts have reported increases in lectin-conjugated particle uptake, thought to be caused by increasing interactions with mucus [68
] and epithelial cells [70
]. For example, Hussain et al. conjugated tomato lectin to 500 nm polystyrene particles administered by 0.1 mL oral gavage daily for 5 days [71
]. After washing the tissues with phosphate buffered saline, particles were extracted from intestinal tissue and Peyer’s patches; the authors determined that 12% of the dose was associated with the intestine (enterocytes) as compared to <1% associated with the Peyer’s patches. However, later experiments by Atuma et al. determined that not even suction could remove the adherent mucus layer in rat intestines [18
]. This would imply that the majority of particles were likely trapped in the adherent mucus layer, not absorbed by the enterocytes. Additionally, Hussain and coworkers determined that total systemic circulation of lectin-conjugated particles reached 23%, which was attributed to the fact that the majority of the intestinal surface is non-lymphoid tissue that was absorbing the particles [71
]. However, they did not take into consideration that the particles were administered in water, which has been shown to increase uptake by Peyer’s patches into systemic circulation [62
]. Uptake by M cells in the Peyer’s patches could be further increased by the presence of lectin, since three lectin types are known to bind to M cells in the rat [72
]. Indeed, when the binding was blocked by incubating the particles with a potent inhibitor of tomato lectin, the particle uptake was reduced to 0.5%, which is typical of polystyrene uptake by lymphoid tissue in rats [71
Wheat germ agglutinin (WGA) is another commonly used lectin, which targets N
-acetyl-D-glucosamine and sialic acid found ubiquitously throughout the intestinal tract [73
]. Yin and coworkers hypothesized that direct lectin interaction with the glycocalyx would make 200 nm PLGA nanoparticles less affected by the secreted mucus layer turnover, while also potentially triggering endocytosis by the intestinal epithelium [74
]. They believed that this effect led to increased bioavailability of encapsulated immunomodulating peptide (thymopentin) in immunosuppressed rats, as measured by a ~2-fold increase in the ratio of CD4+/CD8+ T cell populations as compared to uncoated PLGA nanoparticles, free thymopentin, and the control. Also, the WGA-coated PLGA particles administered orally had a similar effect as thymopentin solution administered by I.V. In another study further characterizing the effects of WGA-conjugated PLGA, Yin and coworkers, based on fluorescent photomicrographs, stated that these nanoparticles adhered to intestinal villous epithelium as well as Peyer’s patches after daily administration. This adhesion was found to increase with increasing surface density of WGA. However, fluorescent microscopy is insufficient for determining particle uptake by cells; confocal microscopy is more appropriate. As stated previously, washing the tissues prior to imaging does not remove the adherent mucus layer on the villous epithelium; it is more likely that the particles were trapped in this mucus layer on top of the epithelium rather than internalized. Systemic uptake was as high as 13% at 1 day and 15% after dosing for 7 days, which was 1.5–3 fold higher than unconjugated particles [75
]. The high overall systemic uptake of both coated and uncoated particles could have been due to large gavage volumes, but the specific value is not stated. It is possible that lectin interaction with Peyer’s patches also leads to enhanced uptake, because one potential drawback of lectin-based targeting for drug delivery applications is the potential for immunostimulatory effects [42
Immunostimulatory effects can be advantageous for vaccination, prompting the development of nanoparticles that induce M cell uptake. Taking inspiration from nature, it was noted that pathogenic bacteria such as Salmonella
species are able to invade the mucosal immune system via surface invasin proteins. These proteins both allow bacteria to adhere to the mucosa and be internalized by the epithelium [76
]. Salman and coworkers coated 280 nm poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA) nanoparticles with flagella-enriched Salmonella
extract, with the intention of increasing adhesion in the Peyer’s patches of rats [77
]. It was demonstrated that the coated particles exhibited competitive binding with Salmonella
dosed orally; the particles administered 30 mins prior decreased the epithelial binding of the bacterium. However, the gavage volume was not stated; a high volume could explain how these particles were able to pass through the mucus barrier and reach the epithelium. Hussain and coworkers coated 500 nm polystyrene nanoparticles with invasin-C192, which is found on the surface of Yersina
bacteria. After a single 0.1 mL gavage in rats, 13% of the invasin coated nanoparticles were found in the systemic circulation, compared to 2% of uncoated control particles. As a control, invasin coated nanoparticles were coated in porcine mucin, which would interfere with their cell adhesive capability, resulting in <2 % systemic uptake [76
]. The authors were unable to explain why coating the particles in porcine mucin would interfere with systemic uptake, but uptake was not decreased by interaction with mucin while the particles passed through the rat GI tract. It is possible that the relatively high density of M cells and relatively low mucin secretion in rats [see section 5] likely contributes to all of the studies described that result in such high particle uptake with mucoadhesive particles.
Other groups have used materials that target particular areas of the GI tract based on their degradation kinetics by preserving the encapsulated material until reaching the target site. NiMOS (nanoparticles in microspheres oral system) contain pDNA inside type B gelatin nanoparticles, which are then encapsulated within poly(episilon-caprolactone) (PCL) microparticles [78
]. The outer PCL coat (2–5 µm in diameter) is degraded by intestinal lipases, which releases the encapsulated 200 nm nanoparticles. It is hypothesized that the nanoparticles are then taken up by the cells of the small and large intestine. In , green fluorescent protein- (GFP) expressing plasmid DNA was administered orally as either free plasmid, encapsulated in gelatin nanoparticles alone, or in the NiMOS system. It is evident that GFP expression occurred only in the small intestine of rats when the plasmid DNA was encapsulated in NiMOS [79
]. In a mouse model for TNBS-induced ulcerative colitis, NiMOS containing murine IL-10-expressing plasmid DNA were given in one oral dose. The expression of IL-10 acts to maintain immunological balance by inhibiting production of proinflammatory cytokines. The successful transfection, determined by significantly increased IL-10 mRNA transcript levels in the intestines, prevented progression of colitis as measured by colon length and weight, body weight, and myeloperoxidase (MPO) activity [80
]. These results are encouraging, but mucus in the human GI tract will likely be a more significant barrier than in the mouse [see section 5].
Figure 5 Qualitative enhanced green fluorescent protein (GFP) expression in the small and large intestinal tract of male Wistar rats cryosections after oral administrate of saline (A), naked GFP plasmid (B), gelatin nanoparticles encapsulating GFP plasmid (C) (more ...)
Another novel degradation-specific formulation being tested is siRNA encapsulated in a novel thioketal polymer. This polymer degrades in the presence of reactive oxygen species (ROS), which are present at relatively high concentrations in inflamed tissues. Specifically in the GI tract, biopsies taken from patients suffering from ulcerative colitis [81
], colon cancer [82
], and helicobacter pylori [83
] infections have 10–100 fold increased mucosal ROS concentrations. The particles are also coated with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a positively charged surfactant, increasing mucoadhesion in the intestines. Wilson and coworkers loaded the 600 nm particles with TNF-α-siRNA to treat mice with dextran sulfate sodium (DSS) induced ulcerative colitis [84
]; TNF-α is integral in the onset and persistence of intestinal inflammation. After receiving DSS or normal water for seven days, the mice were given daily 0.2 mL gavages for six days. On the seventh day, mice were sacrificed and assessed by histology, MPO activity, and weight loss. Indeed, the thioketal particles were associated with 2-fold lower MPO activity, improved histology, and reduced weight loss; however, it was clear that after day 3 of daily gavaging, the initial weight gain was beginning to reverse. At the time of sacrifice, the weight of the mice was almost back to the starting weight, and likely would have kept declining, whereas the weight of the control mice was increasing. It is likely that the effectiveness of this treatment was limited by the mucus barrier, considering the increased mucus secretion associated with inflammation. As inflammation progressed, the mucus barrier thickness would increase, sequestering the particles further from the epithelium.
Lamprecht et al. determined that polystyrene particles (0.1–10 µm) dosed by 0.5 mL gavage on two consecutive days selectively adhered to inflamed tissue in a rat model of TNBS-induced colitis [85
]. The authors determined seventy-two hours after the second gavage 0.1 µm particles had the highest deposition in inflamed tissue at 15% of the original dose (6.5 fold higher than deposition in controls) and that 1 and 10 µm particles had 3–4 fold increase in deposition over control tissues. Lamprecht went on to quantify the amount of particles remaining after mucus removal by ‘extensive’ washing of the tissues, which removed 62% of 0.1 µm, 69% of 1 µm, and 85% of 10 µm particles. Thus, the authors concluded that the nonspecific targeting to the inflamed epithelium was largely due to increased mucus secretion by the inflamed tissues.