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Strategies for improving mucosal drug delivery Within this review we will provide a comprehensive understanding in order to improve existing strategies and to develop new systems to lower the barrier for improving mucosal drug delivery. Mucosal administration of drugs achieves a therapeutical effect as the permeation of significant amounts of a drug is permitted through the absorption membrane. The absorption membrane relies on the mucosal layer and the epithelial tissue. In order to overcome barriers, drug delivery systems have to exhibit various functions and features, such as mucoadhesive and protective activity, solubility improving, permeation and uptake enhancing, and drug release controlling properties. This review also aims to provide an insight of well-distinguished strategies to date, as well as provide a focus on the enhancement of membrane permeability. Furthermore, since the development and functions of drug delivery systems exert a high influence on the ability of drug permeation through membrane, these considerations will also be discussed in this review. KEYWORDS: mucoadhesion n mucosal membrane n mucus n mucus penetration n nanodelivery n self-emulsifying drug delivery system
Nanotechnology has become a ‘buzzword’ in pharmaceutical sciences, and investigations are currently being undertaken to extend its appli cations in various models of pharmaceutical research. Over the last two decades, nanotechno logy has tremendously gained an influence in drug delivery research and a great number of nanoscale technologies/carriers have been, and still are being, investigated to date in order to enhance therapeutic efficacy of drugs [1–4]. An improvement of nanoscale technologies in ther apeutic efficacy might be caused by a number of factors, such as the improved solubility of hydrophobic drugs, the enhanced permeabil ity or transport of poorly permeable drugs, the modulation of biodistribution and drug disposi tion of drugs, the prevention of drug degradation in physiological milieu, and the ability to target delivery of drugs to the site of action. Classifica tion of nanoscale technologies can generally be divided into lipid-based nanocarriers, polymeric nanocarriers, inorganic nanocarriers and drug nanoparticles or nanosuspensions [5,6]. Lipid nanocarriers contain lipid core micelles, lipo somes, microemulsions, nanoemulsions, solid lipid nanoparticles and nanostructured lipid carriers, whereas polymeric nanocarriers contain polymeric micelles, polymeric nanoparticles and nanocapsules, dendrimers and polymer–drug nanoconjugates [3,7]. Inorganic nanocarri ers comprise nanostructures covering various inorganic metals, for instance gold particles, iron oxide (magnetic) nanoparticles, calcium
phosphate nanoparticles and quantum dots, whereas drugs in nanoparticulate form might be used as nanosuspensions [8]. Furthermore, nano carriers are intensively investigated for strategies to deliver drugs across the epithelia. By dem onstrating great potential in improving drug delivery by various routes of administration to mucosal surfaces, nanoscale delivery systems seem to be promising tools for further inves tigations [9]. Mucosal delivery systems establish promising features as they are patient compliant, nonpainful and have an accepted administra tion route. This review will provide an overview of conventional and novel strategies to improve our understanding for designing mucosal drug delivery systems, as well as recent developments in drug delivery for mucosal surfaces.
10.2217/NNM.13.178 © 2013 Future Medicine Ltd
Nanomedicine (2013) 8(12), 2061–2075
Flavia Laffleur1 & Andreas Bernkop‑Schnürch*1 Department of Pharmaceutical Technology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria *Author for correspondence: Tel.: +43 512 507 58600 Fax: +43 512 507 58699 [email protected] 1
Impact of the various mucosal membranes Understanding the importance of differ ent mucosa is essential for designing mucosal delivery systems. Therefore, an overview of the important key points will be given according to intestinal, buccal and vaginal mucosa. The human intestinal mucosa is composed of a simple layer of columnar epithelial cells as well as the underlying lamina propria and muscu lar mucosa [10]. Goblet cells synthesizing and releasing mucin, as well as other differentiated epithelial cell types, are present. The unstirred layer is located immediately above the epithelial cells. The tight junction is the component of the
part of
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2061
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Laffleur & Bernkop-Schnürch
Nanomedicine © Future Medicine Ltd
Mucus layer
Intestinal epithelial cells
Tight junctions Lamina propia
Figure 1. Components of the intestinal mucosal mechanical barrier. The mechanical barrier is made up of the mucus layer, intestinal epithelial cells, tight junctions between cells and the mucosal lamina propria.
apical functional complex that exhibits the para cellular space in between epithelial cells. The lamina propria, located beneath the basement membrane, contains immune cells. A simple columnar epithelium covers the inner surface of the small intestine exhibiting villi that provide the majority of differentiated absorptive cells. The epithelial surface is expanded by villous thickness and crypts present between villi. The intestinal mucosal barrier consists mainly of a mechanical barrier, chemical barrier, microbial barrier and an immune barrier. The mechanical barrier (composed of the mucus layer, intestinal epithelial cells, tight junctions between cells and mucosal lamina propria), depicted in Figure 1, can effectively prevent xenobiotics from penetrating the intestinal mucosa and invading into deep tis sues. Buccal mucosa is composed of several layers of different cells. Numerous racemose, mucous, or serous glands are present in the submucous tissue of the cheeks. The maxillary artery sup plies blood to buccal mucosa. The blood flow is faster and richer (2.4 ml/min/cm 2) compared with the sublingual, gingival and palatal regions. Therefore, passive diffusion of drug molecules across the mucosa is facilitated. The thickness of the buccal mucosa is 500–800 µm and is rough textured and, thus, suitable for retentive delivery systems. The epithelium is approxi mately 40–50 cell layers thick. Buccal mucosa epithelium is nonkeratinized stratified squa mous epithelium. Its thickness is approximately 500–600 µm and its surface area is 50.2 cm2. The epithelial layer covers the lamina propia, which is located on the submucosa. Blood vessels and capillaries are present in the lamina propria opening to the internal jugular vein. The protec tion of the underlying tissue is the primary func tion of buccal epithelium. In nonkeratinized regions, lipid-based permeability barriers in the outer epithelial layers give protection to the underlying tissues against fluid loss and entry of 2062
Nanomedicine (2013) 8(12)
potentially harmful environmental agents. The rate and extent of drug absorption through the buccal mucosa is retarded by barriers, such as saliva, mucus, membrane-coating granules and the basement membrane. The main penetration barrier exists in the outermost quarter to third of the epithelium [11]. The vagina and ectocervix are covered by a nonkeratinized, stratified squa mous epithelium. The composition of the vagi nal mucosal layer exhibits multiple rugal folds, increasing the surface area. The squamous epi thelium is constantly renewed and desquamated. The squamous cells provide gap junction nex uses revealing an open channel system between adjacent cells. The endocervix is lined by a single layer of columnar epithelium. The major contri butions to cervical mucus consist of water and a matrix of mucins, which are high-molecularweight glycoproteins. Goblet cells within the columnar epithelium of the endocervix and the Bartholin’s glands provide the sources of mucus secretions. The menstrual cycle has a significant impact on the physical characteristics, compo sition and volume of mucus secretions of the endocervical epithelium. Cervical mucus shows a complex net-like structure resembling interlac ing microfibers. Orientation and pore size are influenced by circulating hormones. Mucuspenetrating nanoparticles mimicking the pore size of human cervicovaginal mucus show the average pore size to be 340 ± 70 nm. Mucus enables the maintainance of an unstirred layer adjacent to epithelial surfaces despite the shear ing action that occurs during vaginal inter course. The balance between the rate of secretion and the rate of shedding/degradation determines the depth of the unstirred layer. Drugs being delivered via vaginal administration that have targets within the mucosa, must move through and penetrate the unstirred layer before it is shed or degraded [12].
Barriers to overcome Mucus layer barrier The anatomy and physiology of the mucosa have been exhaustively described and reviewed in the literature [1,13,14]. The barrier based on the mucus layer is often undervalued in drug administra tion to mucosal surfaces. This barrier relies on the gel layer of mucus lining the mucosal epithe lia [15] and since it is approximately 83% water, it has been determined to be a main component of the mucus layer [16]. Mucus is bound to the apical site of the cell surface and its role is as a protective layer to the cells below. Mucus is a complex hydrogel composed of carbohydrates, future science group
Strategies for improving mucosal drug delivery
lipids, salts, proteins, antibodies, cellular debris and bacteria. The main component is mucin [17]. The mucus layer basically consists of glyco proteins of a relative molecular mass range of 1–40 × 106 kDa as depicted in Figure 2 . Mucin can be classified into either secreted or cellbound mucin [18]. The mucin consists of a lin ear protein core comprising high amounts of threonine and serine with glycosylation by side chains of oligosaccharides. Cysteine-rich sub domains are displayed on the protein core. The connection is provided by intra- and/or intermolecular disulfide bonds of theses cysteine-rich subdomains. Classification of mucins revealed them as membrane-bound and secretory classes. The membrane-bound mucins are attached to the epithelial cell layer and have a characteristic hydrophobic membrane-spanning domain. The secreted mucins are continuously released from cells and glands, and undergo a polymerization process promptly. This process principally relies on the formation of oxidative intermolecular disulfide bonds. Owing to this formation, the characteristic 3D network of the mucus layer is built up, exhibiting high viscosity and stability. Notwithstanding, a continuous erosion of the mucus layer, due to enzymes and mechanical surface challenges, must be taken in consider ation. Various factors, such as mucus secretagogs, stress or mechanical stimuli, impinge mucus secretion and mucus erosion. These provide a large turnover of mucus. The medium thickness of the mucus gel layer related to eyes, oral (buc cal) cavity and intestine is 40, 70 and between 80–200 µm, respectively, as shown in Table 1. The mucus membrane is the moist tissue lining organs and body cavities, such as the mouth, gut, rectum, genital area, nose and eye lids [13]. In Table 2 , mucus clearance time and rates are described from different mucosal origins. Tissue barrier Several pathways exist for molecules to reach the epithelium via overwhelming biological tissue barriers. Hydrophilic compounds, such as peptide drugs, prefer the paracellular route as the key route of absorbtion. Physical and chemical features of the drug determine if the compounds being administered obey transport via a transcellular or paracellular route. Pas sive diffusion through barriers via transcellular pathways is pursued by highly lipophilic com pounds, whereas hydrophilic and membraneimpermeable protein and peptide drugs ensue, to a greater degree, diffusion through the para cellular route, which is controlled by the tight future science group
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Loosely adherent layer rapid clearance
Firmly adherent layer slow clearance
Epithelium
Figure 2. Mucosal layers.
junctions. Only passive diffusion is involved in paracellular transport, whereas transcellular transport can take place by passive, facilitated or active processes. In general, passively trans ported hydrophilic compounds diffuse through the paracellular route while the more lipophilic compounds use the transcellular pathway. The flux of compounds via paracellular flux happens, in principle, by passive diffusion. Only a small number of drugs can be transported via active transport systems. For instance, the intestinal absorption of di- and tri-peptides is by active transport by the carrier-mediated oligopeptide transporter. For uptake through the mucus pathways, both paracellular and transcellular are favored; nevertheless, in many cases both pathways are involved in the absorption pro cess. Molecules exhibiting a radius greater than 15 Å show limited transport through the tight junctions, representing the restricted area for the paracellular route. For a better understanding of the tight junctions, a summary of the func tion of proteins that regulate and/or influence the gate fence area for the paracellular route is given in the ‘Enhanced permeation’ section. Claudins are another family of proteins bearing two extracellular loops. The originally identi fied proteins are claudin-1 and claudin-2, which exhibit a molecular mass of 22–24 kDa. In fact, claudin-1 and -2 provide no evidence for direct interaction with each other. Some claudins show the ability to mediate cell adhesion in a calciumindependent manner. Selection of ions to pass through the paracellular barrier seems to be the function of claudins. Enzymatic barrier The structures of the peptide or protein drugs that are administered make up the enzymatic barrier. Consequently, the proteases’ specifi cations are of importance when selecting the enzyme inhibitor(s) to ensure stability of the www.futuremedicine.com
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Table 1. Overview of thickness of various kinds of mucus according to Lai et al. Type of mucus
Average thickness Cited thickness (µm) (µm)
Ref.
Ocular [60]
Mucus layer
0.035
0.02–0.05
Tear film
5 40
3, 6–7 34–45
[60,61] [62]
Airway
15
7 30
[63,64] [65]
Bronchial
55
55 ± 5
[66]
Gastric
170
144 ± 52 192 ± 7
[67] [68]
Ileal
10
10
[69]
Respiratory
Gastrointestinal
Cecal
37
36.7 ± 7.2
[70]
AC
100
39.1 ± 9.9 (AC), 57.5 ± 14.5 (TC) and 69.6 ± 32.1 (DC) 79 ± 40 100–150 107 ± 48 (AC–TC) and 134 ± 68 (TC–DC) 110–160
[70] [69] [71] [72]
101.5 ± 80.3 155 ± 54
[70] [72]
Rectal
125
[73]
Gastrointestinal mucus generally exists as two layers, a basal ‘unstirred’ or ‘firmly adherent’ layer and a luminal ‘stirred’ or ‘sloppy’ layer [74–76]; however, most of the cited references report only one value for mucus layer thickness. Matsuo et al. report the thickness of both an ‘inner layer‘ and ‘outer layer’ of mucus; in the cecum, the inner layer was measured to be 5.6 ± 0.2 µm in the colon, 4.7 ± 1.4 µm for the AC, 7.0 ± 3.7 µm for the TC and 7.6 ± 3.4 µm for the DC; and 12.7 ± 6.0 µm in the rectum [70]. AC: Ascending colon; DC: Descending colon; TC: Transverse colon. Data taken from [22].
therapeutic agent in the intestine. However, it must not be forgotten that the quantity of coadministered inhibitors is also paramount to the intestinal stability of a peptide or protein drug. Comparisons between the enzymatic activity of luminally secreted and membrane-bound enzymes demonstrated that the total peptidase activity present in the lumen of the small intes tine of rats is 16-times greater than the total activity in the brush border membrane of the epithelial cells when using the B chain of insulin as a substrate [19]. This outcome might stem from the influence of different factors such as pancreatic stimula tion, the pH of the intestinal fluid, concentration of activating ions, as well as type and quantity of nutrients, which all lead to strongly differing enzymatic activity in the intestine. Developing drug delivery systems, where the main goal is to shield therapeutic peptides or proteins in a confined area of drug liberation and absorp tion, requires awareness of and experience with 2064
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the activity of brush border membrane-bound enzymes in this area, which are estimated to spread 0.5–2 cm². The data displaying the enzy matic activity of brush border membrane-bound proteases, however, are usually expressed in units per milligram or gram of protein isolated from the intestinal mucosa. Hence, gathering infor mation on the enzymatic activity in a given area to reveal the real adverse potential ‘evil’ (chal lenging) brush border membrane-bound pro teases is very difficult. Gaining data revealing the activity of brush border membrane-bound enzymes in centimeters squared would provide vital help for the development and evaluation of dosage forms that contain inhibitors. The enzymatic barrier not only contains luminally secreted and brush border membrane-bound enzymes, but also cytosolic enzymes as lyso zymes. Those enzymes play a more prominent role in transcellular peptide transport, which is a rather unlikely route for the passive diffusion of hydrophilic macromolecules [20]. future science group
Strategies for improving mucosal drug delivery
Strategies The mucosal route plays an important role in nanoscale delivery. The nanoscale systems have to penetrate or diffuse through the mucosal membrane to reach the epithelium as depicted in Figure 3A & B. The mucosal surfaces are lined by epithelial cells, establishing a barrier between the external environment and internal milieu. There are many strategies to follow in order to cross the mucus barrier. By learning lessons from studies of viruses, a strategy was achieved by Mrnsy [21]. Lai et al. revealed that viruses are able to penetrate the mucus [22]. First, viruses are small enough to avoid steric blockage due to the mucin mesh. Second, viruses possess muco adhesive less surfaces, and, furthermore, the lat ter are coated densely with negative and positive charges comprising a net-neutral, highly hydro philic surface. Moreover, Ensign and cowork ers developed mucus-penetrating particles composed of acyclovir monophosphate. When administered before a vaginal herpes simplex virus 2 challenge, acyclovir mucus penetrating particles protected 53% of mice compared with only 16% that were administered by soluble drugs [23]. Another strategy was described as the proteolytic enzyme strategy. By immobiliza tion of enzymes on the systems surface, these nanosystems show an improvement in paracel lular uptake [24]. As a consequence, the systems move across the epithelium entering the sys temic circulation. Furthermore, many of these enzymes, such as papain and trypsin, are known to improve the paracellular uptake of nanopar ticles in order to cleave the outer membrane pro tein substructures in the tight junctions. A large number of studies have determined the great efficacy of mucolytic agents, such as N-acetyl cysteine, to overcome the mucus barrier [25]. By breaking the disulfide bonds, N-acetylcysteine reduces the crosslinkage of mucin resulting in loss of viscosity. In the thiomeric strategy, the nanoscale sys tem, comprised of thiolated polymers, plays an important role by building up disulfide bonds via the thiol–disulfide exchange reaction with the cysteine-rich subdomains of the mucus. Consequently, thiomers remain concentrated on the absorption membrane without diffusing back [26]. Once the mucosal barrier is overcome, the epithelium exhibits two routes of transport, namely paracellular and transcellular transport. The paracellular route of transport is often limited by the presence of tight junctions and the transcellular route can be improved by the presence of various carrier mechanisms. Given future science group
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these circumstances, nanoscale drug delivery systems for various delivery methods might even pass across the epithelium by entering in the systemic circulation Figure 4 . Mucosal surfaces are the most common and convenient routes for delivering drugs into the body. However, macromolecular drugs, such as peptides and proteins, are unable to overcome the mucosal barriers and/or face degradation before reaching the systemic circulation. Muco sal delivery poses challenges for drug delivery. In Figur e 5 , strategies to overcome and solve these hindrances are provided. Drugs delivered through mucosal epithelia, such as the female reproductive, intraoral and GI tracts, encounter a mucosal membrane acting as a significant bar rier to drug penetration. Penetrating this layer would be beneficial to the delivery of drugs across mucosal tissues, such as gastrointestinal, vaginal or buccal routes. Solubility improvement Self-nanoemulsifying drug delivery systems (SNEDDS) or micro-emulsifying drug delivery systems (SMEDDS) spontaneously form emul sions upon contact with aqueous media, such as intestinal fluids, once they have permeated the mucus gel layer and reached the epithelium [27]. A simple strategy to generate a nanodelivery sys tem is nanoscale system based on the SNEDDS. Advantages and benefits of SNEDDS are eluci dated in Figure 6. The synthesis of this system bears one main advantage: it is a simple drug solution Table 2. General view of clearance time and clearance rate for various mucosae according to Lai et al. Type of mucus
Clearance time (min)
Clearance rate (mm/min)
Ref.
Ocular
5–7.7
ND
[77]
General
10–20
ND
[78]
Maxillary sinus
20–30
ND
[78]
Nasal
8.8 ND 20
ND 5–11 5
[79] [80] [81–83]
Tracheal
ND ND ND
4.1 ± 1.9 4.7 (range: 3.5–6) 15.5 (range: 4.5–30)†
[84] [84–90] [91–98]
Bronchial
ND
2.4 ± 0.5
[85]
1
[99]
Respiratory
Small airways
ND
Clearance rates were measured using a bronchoscopic or roentgenographic method, which generally results in higher measured rates of clearance, most likely due to the invasiveness of the procedures [97,98]. ND: Not determined. Data taken from [22]. †
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Mucus layer
Transcellular
Paracellular
Mucus layer
Transcellular
Paracellular Nanomedicine © Future Medicine Ltd
Figure 3. Two routes of drug transport in intestinal and buccal epithelium. (A & B) The paracellular route of transport is often limited by the presence of tight junctions and the transcellular route of transport can be improved by the presence of various carrier mechanisms. (B) Paracellular and transcellular routes of transport designated to the buccal mucosa.
process comprising the drug, organic solvents and coemulsifiers. Owing to their hydrophobic surfaces, the interactions of nanodroplets with the mucus layer are low [28]. Subsequently, such nanodelivery systems can rapidly pass across the mucus barrier. Additionally, the incorporation of macromolecular drugs in a lipophilic phase leads to better protection from intestinal fluid and enzymatic degradation [29]. SNEDDS might even improve the uptake of very-high-molecularweight DNA-based drugs by epithelial cells. The primary pitfall to this approach is delivery and attainment of therapeutically viable drug levels. Drugs solubilized in SNEDDS have a very high dissolution velocity in terms of the significantly induced spontaneous surface area:volume ratio afforded by the nanoemulsion [30]. This physical property leads to facilitated dissolution of lipo philic drugs. Furthermore, several components of SNEDDS can enhance membrane permeation of therapeutic agents, such as oily phases (oleic acid, monoglycerides of caprylic acid and propyl ene glycol esters of caprylic acid). Additionally, surfactants, such as polysorbate 80 and Labrasol® [31], as well as cosolvents comprising PEG400, Transcutol® [32] and alcohol, boost this effect of enhancement to permeate the membrane [33]. 2066
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Two main mechanisms render SNEDDS attrac tive for nanoscale delivery. On the one hand, SNEDDS overcome shortcomings of membranes and, on the other hand, they reduce first-pass metabolism due to the choice of components. This is due to the modulation and inhibition of the activity of CYP450, and metabolizing enzymes in the GI tract as shown with Witepsol® and coenzyme Q10 [34]. Additionally, the use of long-chain mono- and tri-glycerides promotes intestinal lymphatic transport. Furthermore, the use of nonionic surfactants such as Cremophor® EL [35] can simultaneously enhance perme ability and the uptake of drugs susceptible to P-glycoprotein-mediated efflux. Venkatesh and colleagues found that the optimum formulation of SMEDDS consisted of Capryol 90 (9.82%), Cremophor EL (70.72%), Labrasol (17.68%) and buparvaquone (1.78%). Emulsification time and the mean droplet size were found to be 1 min and 18.0 ± 0.25 nm, respectively. The cumula tive percentage of drug released in 90 min was 100% in both simulated gastric fluid and simu lated intestinal fluid. The calculated absolute oral bioavailability for buparvaquone was found to be 40.1% [36]. Chitneni et al. presented a study determin ing the intestinal absorption of sulpiride incor porated into SMEDDS by means of a singlepass intestinal perfusion method in rats, and to compare the effective permeability coefficient obtained with that of drug solution and micellar solution. The human intestinal permeability was predicted based on the rat effective permeability coefficient. The estimated human absorption of sulpiride for the SMEDDS dilutions was supe rior to that from sulpiride solution (p
Strategies for improving mucosal drug delivery Within this review we will provide a comprehensive understanding in order to improve existing strategies and to develop new systems to lower the barrier for improving mucosal drug delivery. Mucosal administration of drugs achieves a therapeutical effect as the permeation of significant amounts of a drug is permitted through the absorption membrane. The absorption membrane relies on the mucosal layer and the epithelial tissue. In order to overcome barriers, drug delivery systems have to exhibit various functions and features, such as mucoadhesive and protective activity, solubility improving, permeation and uptake enhancing, and drug release controlling properties. This review also aims to provide an insight of well-distinguished strategies to date, as well as provide a focus on the enhancement of membrane permeability. Furthermore, since the development and functions of drug delivery systems exert a high influence on the ability of drug permeation through membrane, these considerations will also be discussed in this review. KEYWORDS: mucoadhesion n mucosal membrane n mucus n mucus penetration n nanodelivery n self-emulsifying drug delivery system
Nanotechnology has become a ‘buzzword’ in pharmaceutical sciences, and investigations are currently being undertaken to extend its appli cations in various models of pharmaceutical research. Over the last two decades, nanotechno logy has tremendously gained an influence in drug delivery research and a great number of nanoscale technologies/carriers have been, and still are being, investigated to date in order to enhance therapeutic efficacy of drugs [1–4]. An improvement of nanoscale technologies in ther apeutic efficacy might be caused by a number of factors, such as the improved solubility of hydrophobic drugs, the enhanced permeabil ity or transport of poorly permeable drugs, the modulation of biodistribution and drug disposi tion of drugs, the prevention of drug degradation in physiological milieu, and the ability to target delivery of drugs to the site of action. Classifica tion of nanoscale technologies can generally be divided into lipid-based nanocarriers, polymeric nanocarriers, inorganic nanocarriers and drug nanoparticles or nanosuspensions [5,6]. Lipid nanocarriers contain lipid core micelles, lipo somes, microemulsions, nanoemulsions, solid lipid nanoparticles and nanostructured lipid carriers, whereas polymeric nanocarriers contain polymeric micelles, polymeric nanoparticles and nanocapsules, dendrimers and polymer–drug nanoconjugates [3,7]. Inorganic nanocarri ers comprise nanostructures covering various inorganic metals, for instance gold particles, iron oxide (magnetic) nanoparticles, calcium
phosphate nanoparticles and quantum dots, whereas drugs in nanoparticulate form might be used as nanosuspensions [8]. Furthermore, nano carriers are intensively investigated for strategies to deliver drugs across the epithelia. By dem onstrating great potential in improving drug delivery by various routes of administration to mucosal surfaces, nanoscale delivery systems seem to be promising tools for further inves tigations [9]. Mucosal delivery systems establish promising features as they are patient compliant, nonpainful and have an accepted administra tion route. This review will provide an overview of conventional and novel strategies to improve our understanding for designing mucosal drug delivery systems, as well as recent developments in drug delivery for mucosal surfaces.
10.2217/NNM.13.178 © 2013 Future Medicine Ltd
Nanomedicine (2013) 8(12), 2061–2075
Flavia Laffleur1 & Andreas Bernkop‑Schnürch*1 Department of Pharmaceutical Technology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria *Author for correspondence: Tel.: +43 512 507 58600 Fax: +43 512 507 58699 [email protected] 1
Impact of the various mucosal membranes Understanding the importance of differ ent mucosa is essential for designing mucosal delivery systems. Therefore, an overview of the important key points will be given according to intestinal, buccal and vaginal mucosa. The human intestinal mucosa is composed of a simple layer of columnar epithelial cells as well as the underlying lamina propria and muscu lar mucosa [10]. Goblet cells synthesizing and releasing mucin, as well as other differentiated epithelial cell types, are present. The unstirred layer is located immediately above the epithelial cells. The tight junction is the component of the
part of
ISSN 1743-5889
2061
Review
Laffleur & Bernkop-Schnürch
Nanomedicine © Future Medicine Ltd
Mucus layer
Intestinal epithelial cells
Tight junctions Lamina propia
Figure 1. Components of the intestinal mucosal mechanical barrier. The mechanical barrier is made up of the mucus layer, intestinal epithelial cells, tight junctions between cells and the mucosal lamina propria.
apical functional complex that exhibits the para cellular space in between epithelial cells. The lamina propria, located beneath the basement membrane, contains immune cells. A simple columnar epithelium covers the inner surface of the small intestine exhibiting villi that provide the majority of differentiated absorptive cells. The epithelial surface is expanded by villous thickness and crypts present between villi. The intestinal mucosal barrier consists mainly of a mechanical barrier, chemical barrier, microbial barrier and an immune barrier. The mechanical barrier (composed of the mucus layer, intestinal epithelial cells, tight junctions between cells and mucosal lamina propria), depicted in Figure 1, can effectively prevent xenobiotics from penetrating the intestinal mucosa and invading into deep tis sues. Buccal mucosa is composed of several layers of different cells. Numerous racemose, mucous, or serous glands are present in the submucous tissue of the cheeks. The maxillary artery sup plies blood to buccal mucosa. The blood flow is faster and richer (2.4 ml/min/cm 2) compared with the sublingual, gingival and palatal regions. Therefore, passive diffusion of drug molecules across the mucosa is facilitated. The thickness of the buccal mucosa is 500–800 µm and is rough textured and, thus, suitable for retentive delivery systems. The epithelium is approxi mately 40–50 cell layers thick. Buccal mucosa epithelium is nonkeratinized stratified squa mous epithelium. Its thickness is approximately 500–600 µm and its surface area is 50.2 cm2. The epithelial layer covers the lamina propia, which is located on the submucosa. Blood vessels and capillaries are present in the lamina propria opening to the internal jugular vein. The protec tion of the underlying tissue is the primary func tion of buccal epithelium. In nonkeratinized regions, lipid-based permeability barriers in the outer epithelial layers give protection to the underlying tissues against fluid loss and entry of 2062
Nanomedicine (2013) 8(12)
potentially harmful environmental agents. The rate and extent of drug absorption through the buccal mucosa is retarded by barriers, such as saliva, mucus, membrane-coating granules and the basement membrane. The main penetration barrier exists in the outermost quarter to third of the epithelium [11]. The vagina and ectocervix are covered by a nonkeratinized, stratified squa mous epithelium. The composition of the vagi nal mucosal layer exhibits multiple rugal folds, increasing the surface area. The squamous epi thelium is constantly renewed and desquamated. The squamous cells provide gap junction nex uses revealing an open channel system between adjacent cells. The endocervix is lined by a single layer of columnar epithelium. The major contri butions to cervical mucus consist of water and a matrix of mucins, which are high-molecularweight glycoproteins. Goblet cells within the columnar epithelium of the endocervix and the Bartholin’s glands provide the sources of mucus secretions. The menstrual cycle has a significant impact on the physical characteristics, compo sition and volume of mucus secretions of the endocervical epithelium. Cervical mucus shows a complex net-like structure resembling interlac ing microfibers. Orientation and pore size are influenced by circulating hormones. Mucuspenetrating nanoparticles mimicking the pore size of human cervicovaginal mucus show the average pore size to be 340 ± 70 nm. Mucus enables the maintainance of an unstirred layer adjacent to epithelial surfaces despite the shear ing action that occurs during vaginal inter course. The balance between the rate of secretion and the rate of shedding/degradation determines the depth of the unstirred layer. Drugs being delivered via vaginal administration that have targets within the mucosa, must move through and penetrate the unstirred layer before it is shed or degraded [12].
Barriers to overcome Mucus layer barrier The anatomy and physiology of the mucosa have been exhaustively described and reviewed in the literature [1,13,14]. The barrier based on the mucus layer is often undervalued in drug administra tion to mucosal surfaces. This barrier relies on the gel layer of mucus lining the mucosal epithe lia [15] and since it is approximately 83% water, it has been determined to be a main component of the mucus layer [16]. Mucus is bound to the apical site of the cell surface and its role is as a protective layer to the cells below. Mucus is a complex hydrogel composed of carbohydrates, future science group
Strategies for improving mucosal drug delivery
lipids, salts, proteins, antibodies, cellular debris and bacteria. The main component is mucin [17]. The mucus layer basically consists of glyco proteins of a relative molecular mass range of 1–40 × 106 kDa as depicted in Figure 2 . Mucin can be classified into either secreted or cellbound mucin [18]. The mucin consists of a lin ear protein core comprising high amounts of threonine and serine with glycosylation by side chains of oligosaccharides. Cysteine-rich sub domains are displayed on the protein core. The connection is provided by intra- and/or intermolecular disulfide bonds of theses cysteine-rich subdomains. Classification of mucins revealed them as membrane-bound and secretory classes. The membrane-bound mucins are attached to the epithelial cell layer and have a characteristic hydrophobic membrane-spanning domain. The secreted mucins are continuously released from cells and glands, and undergo a polymerization process promptly. This process principally relies on the formation of oxidative intermolecular disulfide bonds. Owing to this formation, the characteristic 3D network of the mucus layer is built up, exhibiting high viscosity and stability. Notwithstanding, a continuous erosion of the mucus layer, due to enzymes and mechanical surface challenges, must be taken in consider ation. Various factors, such as mucus secretagogs, stress or mechanical stimuli, impinge mucus secretion and mucus erosion. These provide a large turnover of mucus. The medium thickness of the mucus gel layer related to eyes, oral (buc cal) cavity and intestine is 40, 70 and between 80–200 µm, respectively, as shown in Table 1. The mucus membrane is the moist tissue lining organs and body cavities, such as the mouth, gut, rectum, genital area, nose and eye lids [13]. In Table 2 , mucus clearance time and rates are described from different mucosal origins. Tissue barrier Several pathways exist for molecules to reach the epithelium via overwhelming biological tissue barriers. Hydrophilic compounds, such as peptide drugs, prefer the paracellular route as the key route of absorbtion. Physical and chemical features of the drug determine if the compounds being administered obey transport via a transcellular or paracellular route. Pas sive diffusion through barriers via transcellular pathways is pursued by highly lipophilic com pounds, whereas hydrophilic and membraneimpermeable protein and peptide drugs ensue, to a greater degree, diffusion through the para cellular route, which is controlled by the tight future science group
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Loosely adherent layer rapid clearance
Firmly adherent layer slow clearance
Epithelium
Figure 2. Mucosal layers.
junctions. Only passive diffusion is involved in paracellular transport, whereas transcellular transport can take place by passive, facilitated or active processes. In general, passively trans ported hydrophilic compounds diffuse through the paracellular route while the more lipophilic compounds use the transcellular pathway. The flux of compounds via paracellular flux happens, in principle, by passive diffusion. Only a small number of drugs can be transported via active transport systems. For instance, the intestinal absorption of di- and tri-peptides is by active transport by the carrier-mediated oligopeptide transporter. For uptake through the mucus pathways, both paracellular and transcellular are favored; nevertheless, in many cases both pathways are involved in the absorption pro cess. Molecules exhibiting a radius greater than 15 Å show limited transport through the tight junctions, representing the restricted area for the paracellular route. For a better understanding of the tight junctions, a summary of the func tion of proteins that regulate and/or influence the gate fence area for the paracellular route is given in the ‘Enhanced permeation’ section. Claudins are another family of proteins bearing two extracellular loops. The originally identi fied proteins are claudin-1 and claudin-2, which exhibit a molecular mass of 22–24 kDa. In fact, claudin-1 and -2 provide no evidence for direct interaction with each other. Some claudins show the ability to mediate cell adhesion in a calciumindependent manner. Selection of ions to pass through the paracellular barrier seems to be the function of claudins. Enzymatic barrier The structures of the peptide or protein drugs that are administered make up the enzymatic barrier. Consequently, the proteases’ specifi cations are of importance when selecting the enzyme inhibitor(s) to ensure stability of the www.futuremedicine.com
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Table 1. Overview of thickness of various kinds of mucus according to Lai et al. Type of mucus
Average thickness Cited thickness (µm) (µm)
Ref.
Ocular [60]
Mucus layer
0.035
0.02–0.05
Tear film
5 40
3, 6–7 34–45
[60,61] [62]
Airway
15
7 30
[63,64] [65]
Bronchial
55
55 ± 5
[66]
Gastric
170
144 ± 52 192 ± 7
[67] [68]
Ileal
10
10
[69]
Respiratory
Gastrointestinal
Cecal
37
36.7 ± 7.2
[70]
AC
100
39.1 ± 9.9 (AC), 57.5 ± 14.5 (TC) and 69.6 ± 32.1 (DC) 79 ± 40 100–150 107 ± 48 (AC–TC) and 134 ± 68 (TC–DC) 110–160
[70] [69] [71] [72]
101.5 ± 80.3 155 ± 54
[70] [72]
Rectal
125
[73]
Gastrointestinal mucus generally exists as two layers, a basal ‘unstirred’ or ‘firmly adherent’ layer and a luminal ‘stirred’ or ‘sloppy’ layer [74–76]; however, most of the cited references report only one value for mucus layer thickness. Matsuo et al. report the thickness of both an ‘inner layer‘ and ‘outer layer’ of mucus; in the cecum, the inner layer was measured to be 5.6 ± 0.2 µm in the colon, 4.7 ± 1.4 µm for the AC, 7.0 ± 3.7 µm for the TC and 7.6 ± 3.4 µm for the DC; and 12.7 ± 6.0 µm in the rectum [70]. AC: Ascending colon; DC: Descending colon; TC: Transverse colon. Data taken from [22].
therapeutic agent in the intestine. However, it must not be forgotten that the quantity of coadministered inhibitors is also paramount to the intestinal stability of a peptide or protein drug. Comparisons between the enzymatic activity of luminally secreted and membrane-bound enzymes demonstrated that the total peptidase activity present in the lumen of the small intes tine of rats is 16-times greater than the total activity in the brush border membrane of the epithelial cells when using the B chain of insulin as a substrate [19]. This outcome might stem from the influence of different factors such as pancreatic stimula tion, the pH of the intestinal fluid, concentration of activating ions, as well as type and quantity of nutrients, which all lead to strongly differing enzymatic activity in the intestine. Developing drug delivery systems, where the main goal is to shield therapeutic peptides or proteins in a confined area of drug liberation and absorp tion, requires awareness of and experience with 2064
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the activity of brush border membrane-bound enzymes in this area, which are estimated to spread 0.5–2 cm². The data displaying the enzy matic activity of brush border membrane-bound proteases, however, are usually expressed in units per milligram or gram of protein isolated from the intestinal mucosa. Hence, gathering infor mation on the enzymatic activity in a given area to reveal the real adverse potential ‘evil’ (chal lenging) brush border membrane-bound pro teases is very difficult. Gaining data revealing the activity of brush border membrane-bound enzymes in centimeters squared would provide vital help for the development and evaluation of dosage forms that contain inhibitors. The enzymatic barrier not only contains luminally secreted and brush border membrane-bound enzymes, but also cytosolic enzymes as lyso zymes. Those enzymes play a more prominent role in transcellular peptide transport, which is a rather unlikely route for the passive diffusion of hydrophilic macromolecules [20]. future science group
Strategies for improving mucosal drug delivery
Strategies The mucosal route plays an important role in nanoscale delivery. The nanoscale systems have to penetrate or diffuse through the mucosal membrane to reach the epithelium as depicted in Figure 3A & B. The mucosal surfaces are lined by epithelial cells, establishing a barrier between the external environment and internal milieu. There are many strategies to follow in order to cross the mucus barrier. By learning lessons from studies of viruses, a strategy was achieved by Mrnsy [21]. Lai et al. revealed that viruses are able to penetrate the mucus [22]. First, viruses are small enough to avoid steric blockage due to the mucin mesh. Second, viruses possess muco adhesive less surfaces, and, furthermore, the lat ter are coated densely with negative and positive charges comprising a net-neutral, highly hydro philic surface. Moreover, Ensign and cowork ers developed mucus-penetrating particles composed of acyclovir monophosphate. When administered before a vaginal herpes simplex virus 2 challenge, acyclovir mucus penetrating particles protected 53% of mice compared with only 16% that were administered by soluble drugs [23]. Another strategy was described as the proteolytic enzyme strategy. By immobiliza tion of enzymes on the systems surface, these nanosystems show an improvement in paracel lular uptake [24]. As a consequence, the systems move across the epithelium entering the sys temic circulation. Furthermore, many of these enzymes, such as papain and trypsin, are known to improve the paracellular uptake of nanopar ticles in order to cleave the outer membrane pro tein substructures in the tight junctions. A large number of studies have determined the great efficacy of mucolytic agents, such as N-acetyl cysteine, to overcome the mucus barrier [25]. By breaking the disulfide bonds, N-acetylcysteine reduces the crosslinkage of mucin resulting in loss of viscosity. In the thiomeric strategy, the nanoscale sys tem, comprised of thiolated polymers, plays an important role by building up disulfide bonds via the thiol–disulfide exchange reaction with the cysteine-rich subdomains of the mucus. Consequently, thiomers remain concentrated on the absorption membrane without diffusing back [26]. Once the mucosal barrier is overcome, the epithelium exhibits two routes of transport, namely paracellular and transcellular transport. The paracellular route of transport is often limited by the presence of tight junctions and the transcellular route can be improved by the presence of various carrier mechanisms. Given future science group
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these circumstances, nanoscale drug delivery systems for various delivery methods might even pass across the epithelium by entering in the systemic circulation Figure 4 . Mucosal surfaces are the most common and convenient routes for delivering drugs into the body. However, macromolecular drugs, such as peptides and proteins, are unable to overcome the mucosal barriers and/or face degradation before reaching the systemic circulation. Muco sal delivery poses challenges for drug delivery. In Figur e 5 , strategies to overcome and solve these hindrances are provided. Drugs delivered through mucosal epithelia, such as the female reproductive, intraoral and GI tracts, encounter a mucosal membrane acting as a significant bar rier to drug penetration. Penetrating this layer would be beneficial to the delivery of drugs across mucosal tissues, such as gastrointestinal, vaginal or buccal routes. Solubility improvement Self-nanoemulsifying drug delivery systems (SNEDDS) or micro-emulsifying drug delivery systems (SMEDDS) spontaneously form emul sions upon contact with aqueous media, such as intestinal fluids, once they have permeated the mucus gel layer and reached the epithelium [27]. A simple strategy to generate a nanodelivery sys tem is nanoscale system based on the SNEDDS. Advantages and benefits of SNEDDS are eluci dated in Figure 6. The synthesis of this system bears one main advantage: it is a simple drug solution Table 2. General view of clearance time and clearance rate for various mucosae according to Lai et al. Type of mucus
Clearance time (min)
Clearance rate (mm/min)
Ref.
Ocular
5–7.7
ND
[77]
General
10–20
ND
[78]
Maxillary sinus
20–30
ND
[78]
Nasal
8.8 ND 20
ND 5–11 5
[79] [80] [81–83]
Tracheal
ND ND ND
4.1 ± 1.9 4.7 (range: 3.5–6) 15.5 (range: 4.5–30)†
[84] [84–90] [91–98]
Bronchial
ND
2.4 ± 0.5
[85]
1
[99]
Respiratory
Small airways
ND
Clearance rates were measured using a bronchoscopic or roentgenographic method, which generally results in higher measured rates of clearance, most likely due to the invasiveness of the procedures [97,98]. ND: Not determined. Data taken from [22]. †
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Mucus layer
Transcellular
Paracellular
Mucus layer
Transcellular
Paracellular Nanomedicine © Future Medicine Ltd
Figure 3. Two routes of drug transport in intestinal and buccal epithelium. (A & B) The paracellular route of transport is often limited by the presence of tight junctions and the transcellular route of transport can be improved by the presence of various carrier mechanisms. (B) Paracellular and transcellular routes of transport designated to the buccal mucosa.
process comprising the drug, organic solvents and coemulsifiers. Owing to their hydrophobic surfaces, the interactions of nanodroplets with the mucus layer are low [28]. Subsequently, such nanodelivery systems can rapidly pass across the mucus barrier. Additionally, the incorporation of macromolecular drugs in a lipophilic phase leads to better protection from intestinal fluid and enzymatic degradation [29]. SNEDDS might even improve the uptake of very-high-molecularweight DNA-based drugs by epithelial cells. The primary pitfall to this approach is delivery and attainment of therapeutically viable drug levels. Drugs solubilized in SNEDDS have a very high dissolution velocity in terms of the significantly induced spontaneous surface area:volume ratio afforded by the nanoemulsion [30]. This physical property leads to facilitated dissolution of lipo philic drugs. Furthermore, several components of SNEDDS can enhance membrane permeation of therapeutic agents, such as oily phases (oleic acid, monoglycerides of caprylic acid and propyl ene glycol esters of caprylic acid). Additionally, surfactants, such as polysorbate 80 and Labrasol® [31], as well as cosolvents comprising PEG400, Transcutol® [32] and alcohol, boost this effect of enhancement to permeate the membrane [33]. 2066
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Two main mechanisms render SNEDDS attrac tive for nanoscale delivery. On the one hand, SNEDDS overcome shortcomings of membranes and, on the other hand, they reduce first-pass metabolism due to the choice of components. This is due to the modulation and inhibition of the activity of CYP450, and metabolizing enzymes in the GI tract as shown with Witepsol® and coenzyme Q10 [34]. Additionally, the use of long-chain mono- and tri-glycerides promotes intestinal lymphatic transport. Furthermore, the use of nonionic surfactants such as Cremophor® EL [35] can simultaneously enhance perme ability and the uptake of drugs susceptible to P-glycoprotein-mediated efflux. Venkatesh and colleagues found that the optimum formulation of SMEDDS consisted of Capryol 90 (9.82%), Cremophor EL (70.72%), Labrasol (17.68%) and buparvaquone (1.78%). Emulsification time and the mean droplet size were found to be 1 min and 18.0 ± 0.25 nm, respectively. The cumula tive percentage of drug released in 90 min was 100% in both simulated gastric fluid and simu lated intestinal fluid. The calculated absolute oral bioavailability for buparvaquone was found to be 40.1% [36]. Chitneni et al. presented a study determin ing the intestinal absorption of sulpiride incor porated into SMEDDS by means of a singlepass intestinal perfusion method in rats, and to compare the effective permeability coefficient obtained with that of drug solution and micellar solution. The human intestinal permeability was predicted based on the rat effective permeability coefficient. The estimated human absorption of sulpiride for the SMEDDS dilutions was supe rior to that from sulpiride solution (p
