Journal Pre-proof

A comprehensive review of the strategies to improve oral drug absorption with special
emphasis on the cellular and molecular mechanisms

Tanmay Padhye, Kavya Sree Maravajjala, Karnam Laxmi Swetha, Swati Sharma,
Aniruddha Roy

PII: S1773-2247(20)31467-2
DOI: https://doi.org/10.1016/j.jddst.2020.102178
Reference: JDDST 102178

To appear in: Journal of Drug Delivery Science and Technology

Received Date: 2 September 2020

Revised Date: 8 October 2020
Accepted Date: 21 October 2020

Please cite this article as: T. Padhye, K.S. Maravajjala, K.L. Swetha, S. Sharma, A. Roy, A
comprehensive review of the strategies to improve oral drug absorption with special emphasis on
the cellular and molecular mechanisms, Journal of Drug Delivery Science and Technology, https://
doi.org/10.1016/j.jddst.2020.102178.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.

© 2020 Elsevier B.V. All rights reserved.

Drug absorption pathways Permeation enhancers

Mucus
layer

Villi Cytoskeletal Increasing the

contraction Reduction of membrane fluidity;
tight junction
proteins Aqueous pore
Opening of formation in the
tight junctions membrane

f
oo
pr
-
re
Paracellular pathway Transcellular pathway

lP
Passive diffusion Active transport
Nanomedicine for oral delivery

na
Receptor-mediated Clathrin/ M cell-mediated
Macropinocytosis Lipid raft

ur
endocytosis Caveolae transport
ATP

Jo
Enterocytes ADP

Absorption into blood circulation Absorption into systemic circulation

Title:

A comprehensive review of the strategies to improve oral drug absorption with special
emphasis on the cellular and molecular mechanisms

Tanmay Padhye, Kavya Sree Maravajjala, Karnam Laxmi Swetha, Swati Sharma, Aniruddha
Roy#

Department of Pharmacy, Birla Institute of Technology & Science, Pilani, Vidya Vihar,
Rajasthan, India, 333031

of
#Corresponding author:

ro
Aniruddha Roy,
-p
E-mail: aniruddha.roy@pilani.bits-pilani.ac.in;
re
Phone: +91-1596 255643
Fax: +91-1596 244183
lP
na
ur
Jo

1
Abstract:
Oral drug delivery is the most convenient and cost-effective route of administration, due to
which most of the drugs are administered through the oral route. Apparently, oral delivery is
the easiest approach for drug administration; however, in reality, it is one of the most
complicated routes. Although the gastrointestinal (GI) tract offers a large surface area for
absorption, the GI epithelium is designed for the permeation of molecules with a specific set
of physicochemical properties only. Due to this, different drugs exhibit considerable
variabilities in absorption; one can show more than 90% absorption, while others may show
less than 10%. To improve the oral absorption of low bioavailable drugs, multiple strategies
have been developed, with variable success. To design a successful strategy, a thorough

of
understanding of the GI physiology and the mechanism of drug absorption is pivotal.

ro
Different parts of the GI tract have significant variations, all of which have a profound impact
on drug absorption. In this comprehensive review, we have done a cross-disciplinary
-p
exploration of the cellular physiology of different barriers present in the GI tract, how they
re
impact drug absorption, and also discussed the diverse strategies developed to overcome
absorption barriers present in the GI tract.
lP
na

Keywords:
ur

Oral drug delivery; Permeation barriers; Permeation enhancers; Enterocyte uptake;

Jo

Nanoparticles.

2
Contents:

1. Introduction
2. Mechanism of gastrointestinal drug absorption
2.1. Paracellular absorption
2.2. Transcellular absorption
3. Drug absorption barriers present in the GI tract
3.1. Gastro-intestinal epithelium
3.2. Mucus
3.3. Gut microbiota
4. Drug physicochemical parameters that influence oral absorption

of
5. Approaches towards enhancing intestinal permeability
5.1. Prodrugs

ro
5.2. Permeation Enhancers
-p
5.2.1. Paracellular permeation enhancers
re
5.2.2. Transcellular permeation enhancers
6. Use of mucoadhesive polymers for enhancing intestinal absorption
lP

7. Absorption enhancing techniques based on nanomedicine

8. Ion pair complex approach
na

9. Conclusion and perspectives

ur

Tables
Jo

References

3
1. Introduction

Oral delivery is the most preferred route of administration for most of the drug molecules due
to the advantages associated with it, including ease of delivery, self-administration, non-
invasiveness, safety, economic viability etc. However, oral delivery comes with significant
challenges as well, among which reduced bioavailability is the most significant one. As there
are multiple absorption barriers present in the gastrointestinal (GI) tract to prevent the entry
of any foreign materials in our body, only some selected types of molecules can pass through
it. Low molecular weight, non-polar molecules can pass relatively easily, however, for high
molecular weight or polar molecules, it provides a significant obstruction. Multiple
physicochemical properties of the drug molecule, including lipophilicity, ionic strength

of
(pKa), presence of rotatable bonds, hydrogen bonding ability, and molecular size regulate

ro
their absorption through the epithelial barrier of the GI tract. A precise balance between these

-p
properties has to be maintained to improve oral absorption.

Based on the solubility and permeability of a drug, biopharmaceutical classification system

re
(BCS) have been established which categorizes the drugs into four groups: Class I: high
lP

permeability, high solubility; Class II: high permeability, low solubility; Class III: low
permeability, high solubility; and Class IV: low permeability, low solubility (Figure 1) [1].
na

As solubility and permeability are the two major parameters influencing oral bioavailability
of a drug, concerted efforts have been made to improve both of them. There are many
ur

clinically successful strategies to enhance the solubility of a drug molecule [2], and, as a
Jo

result, a majority (41%) of the marketed drug molecules fall under the BCS class II category
[3]. However, enhancing permeability is more complicated. There are varieties of
permeability barriers present in the GI tract. Diverse formulation strategies have been
evaluated for crossing those barriers, with variable success. Though there are several reviews
previously published focusing on the formulation strategies for improving oral
bioavailability, a cross-disciplinary exploration of the mechanism of GI absorption, the
permeation barriers present in the GI tract, and the strategies to overcome the same is lacking.
For designing an effective oral delivery system, the researchers need to have a comprehensive
understanding of the GI physiology and the corresponding permeation barriers present in the
GI tract, along with strategies to overcome the same. In the present review, we are going to
focus initially on the GI physiology to understand the absorption pathway and the absorption
barriers present; then we will discuss different factors controlling permeability. Finally,

4
different approaches to improve the permeability will be discussed, with an emphasis on the
molecular mechanism of that approach.

of
ro
-p
re
lP
na
ur

Figure 1: Relationship between BCS classification and physicochemical characteristics of different

Jo

drugs. ARCS: Absorption rate control step; PSA: Polar surface area; clogP: Partition coefficients
calculated by considering the contribution of functional groups; MW: Molecular weight

2. Mechanism of gastrointestinal drug absorption

For systemic absorption, the orally administered drug has to permeate through the intestinal
epithelium. The outermost cells present in the intestinal epithelium are called enterocytes,
which acts as the primary absorptive cells. The penetration of molecules through the
enterocytes occurs via transcellular or paracellular transport (Figure 2). Paracellular pathway
involves drug diffusion through the tight junctions between the intestinal enterocytes. On the
other hand, in transcellular passage, the molecules have to pass through the enterocytes.
Depending on the drug's physicochemical properties, two primary mechanisms are involved
in the transcellular passage of drugs: passive diffusion and carrier-mediated transport. Passive

5
diffusion is an energy independent, concentration dependent process. In contrast, carrier-
mediated transport is an energy-dependent, concentration independent process, which
operates through a highly specific (in terms of molecular structure and binding site)
mechanism.

of
ro
-p
re
lP
na
ur
Jo

Figure 2: Mechanism of gastrointestinal drug absorption. Any molecule can be absorbed either by the
paracellular pathway where they cross the epithelial lining through the space between the enterocytes,
or they can take the transcellular route, where they pass through the enterocytes by crossing the apical
membrane and the basolateral membrane, consecutively. Transcellular absorption can be of two
different types, passive diffusion and active transport. In passive diffusion, absorption happens due to
simple diffusion of the drug through the membrane, whereas in active transport, the drug is taken up
by some specific uptake mechanism.
2.1. Paracellular absorption

Generally, drugs that are absorbed through the paracellular pathway are low molecular
weight drugs (MW < 250 Da) and polar (c log P < 0) [4]. It involves the passage of drugs
between the adjacent epithelial cells at the tight junctions, which majorly occurs through drug

6
diffusion via leak pathway or pore pathway [5]. Linnankoski et al. reported that the pore size
of the paracellular route varies from 5-15 Å [6]. As a net negative charge is found to be
associated with the junctional complex, positively charged molecules can permeate more
efficiently [7]. However, the paracellular pathway presents a limited absorption window and
accounts for only <0.1% of the total surface area of the intestinal mucosa. Besides, as the
drug travels from the jejunum towards the colon, the junctions between cells become tighter
[8]. Permeation enhancers and chelation of calcium are some strategies that are employed in
loosening the tight junctions to improve drug delivery using this route [9].

of
2.2. Transcellular absorption

ro
Majority of the drug molecules get absorbed through the transcellular pathway. It involves
the passage of the molecules across the epithelia, through intracellular transfer, to blood
-p
circulation. The transfer follows either passive partitioning from the intestinal lumen to the
re
systemic circulation, across the enterocyte cell membrane, or any other specific uptake
lP

mechanism via active transport. The physicochemical properties of the drug control the rate
of passive drug absorption. Mainly low molecular weight, lipophilic molecules are absorbed
na

through this route. A small number of molecules take the path of active uptake, which is a
more specific route depending on the recognition by the transporter proteins [10].
ur
Jo

3. Drug absorption barriers present in the GI tract

Any orally administered drug should be able to bypass the barriers present in the GI tract for
its systemic absorption. A brief discussion of the major absorption barriers is included below.

3.1. Gastro-intestinal epithelium

Significant differences exist between different parts of the GI mucosa in terms of cellular
structure and function, leading to region-dependent characteristics affecting drug absorption.

7
 of
ro
-p
re
lP
na
ur
Jo

Figure 3: Structural variability in different parts of the GI tract. A. Stomach B. Intestine C. Peyer’s
patches.

The most abundant cell type present in the GI tract are the enterocytes, which is uniformly
present in all part of the GI tract. Nevertheless, different specialized cells are also present in
different parts of the GI tract for specific functions (Figure 3). The stomach has numerous
gastric glands, which form a pit-like structure and secrete different substances into the lumen.
The openings of these gastric pits are lined by foveolar cells, which produce mucus to prevent
damage from the concentrated HCl present in the stomach. In the basal part of the gastric
gland, a large number of HCl-secreting parietal cells, pepsinogen secreting chief cells, and

8
enteroendocrine cells are present (Figure 3A). The enteroendocrine cells assist in the
secretion of HCl by the release of histamine.

The intestinal epithelium has an extensive villi structure, resulting in an absorptive surface
area of about 32 m2 [11]. The villi are primarily made up of the enterocytes, the primary
absorptive cells present in the GI tract. Epithelial invaginations, named ‘crypts of
Lieberkühn,’ are present surrounding the villi. Intestinal stem cells are present here, which
help in the constant regeneration of the intestinal epithelium [12]. A variable mucus layer is
present in the intestine: thin and discontinuous layer is observed in the small intestine [13],
whereas thick two-layered mucus is present in the colon [14]. This variability comes from the
distribution of mucus-producing goblet cells in the intestine, ranging from 4% in the

of
duodenum to 16% in the distal colon [15]. The intestinal epithelium also has specialized cells,

ro
named Paneth cells, which help in the production and secretion of antimicrobial peptides,
-p
including cryptdins and α-defensins [16] (Figure 3B). These antimicrobial peptides help to
maintain a healthy microbiome. Enteroendocrine cells are also present in the intestine, where
re
they regulate enzyme secretion, motility, and metabolism.
lP

Intestinal mucosa also has specialized lymphoid follicles, named as Peyer’s patches, to tackle
pathogenic bacteria in the GI tract. The follicular part of the Peyer’s patch lies beneath the
na

epithelial layer. A specialized enterocyte named M (microfold) cell is seen in the follicle-
ur

associated epithelium (Figure 3C). The major function of the M cell is to uptake extracellular
materials like macromolecules, bacteria, and viruses and transfer it at the basolateral side
Jo

[17]. The basal membrane in the M-cells forms an extracellular pocket, which is populated by
monocytes and lymphocytes. The formation of this pocket makes the cytoplasm thinner in the
apical surface, leading to a short transcellular pathway. Particles uptaken by M-cells are
generally delivered to the follicular immune cells and carried over in the lymphatic system
[18]. The advantage of lymphatic absorption is the avoidance of the first-pass metabolism in
the liver [19]. A large number of nanoparticle delivery system targets the M cells for efficient
oral delivery [20].

9
 of
ro
-p
Figure 4: Structure of the intestinal epithelial junctional complex. The intestinal epithelium is
re
consists of a single layer of enterocytes. Adjacent cells are strongly bound with each other by different
junctional complexes. As the junctional proteins are connected to the cytoskeletal proteins,
contraction of the cytoskeletal proteins leads to the opening of the junctional complex, facilitating
lP

paracellular absorption.
Intestinal epithelial tissue is developed from the pluripotent stem cells located at the bottom
na

of the villi, which forms 5 types of epithelial cells: enterocytes, goblet cells, endocrine cells,
Paneth cells, and M cells [21]. Monolayers of these cells make the intestinal epithelial barrier.
ur

The cohesion and polarity of these epithelial cells apical are maintained by the junctional
Jo

complex, which is comprised of tight junctions, adherens junctions, and desmosomes (Figure
4). Both tight junctions and adherens junctions are formed by the different concentrations of
occludin, claudin, junctional adhesion molecules (JAM), and tricellulin [22]. All of these are
transmembrane proteins, with extracellular loop structure. These loops bind to the
corresponding loops of a neighboring cell, creating a tight junction between the two cells
[23]. The intracellular domains of occludin and claudin are directly bound to the zonula
occludens 1, a peripheral scaffolding protein, which in turn is connected to cytoskeletal
proteins such as actin, myosin, and microtubules [24]. The tightness of the junctional
complex can be modulated by phosphorylation of these scaffolding proteins [25]. Claudins
are responsible for the charge selectivity of the paracellular pathway [26, 27]. JAM is
responsible for barrier function, where increased permeability is observed with JAM
deficiency [28]. Tricellulin maintains the structure and function of cellular contacts between
neighboring cells [29]. The junctional stability is maintained by desmosomes, one of the

10
strongest protein structures for cell-to-cell adhesion [30]. The tight junctions are dynamic
structures, as they regulate intestinal penetration of drug molecules across the epithelium. An
alteration of the balance in these proteins increases the mucosal permeability by allowing the
passage of endoluminal molecules into the deeper layers [31]. Some penetration enhancers
function by relaxing the tight junctions and facilitating the paracellular absorption [32].

3.2. Mucus

Mucus mainly acts as a shield for epithelial tissue, protecting it from digestive enzymes,
acids, harmful microorganisms, and antigens and also acts as a lubricant for intestinal

of
motility. The mucosal layer can be divided into two parts: the inner and outer layers. Both the

ro
layers are organized around glycosylated protein mucin, that forms a polymer like coat
secreted by foveolar cells and goblet cells [33]. The inner mucosal layer is very dense,
-p
blocking penetration. The inner mucus layer gradually expands its volume due to the
re
proteolytic activities by the host enzymes like proteases and glycosides, which allows it to get
lP

converted into the outer layer, which is less dense and easier to penetrate [34].

Mucus is critically responsible for modulating GI permeability. Mucus secretion varies

na

significantly along with the GI tract and is predominantly composed of 95% water and
remaining as glycoprotein (mucin), electrolytes etc. In the stomach, the key mucin proteins
ur

are MUC1, MUC5AC, and MUC6, whereas, in the small intestine and the colon, MUC2 is
Jo

the most abundant mucin [35]. GI mucus barrier decreases the permeability and diffusion of
drugs and other macromolecules.

Figure 5: Impact of mucus on drug absorption. The mesh structure produced by the mucus can
prevent the diffusion of larger molecules. The viscoelastic nature of the mucus can also impact drug
diffusion. Depending on the pH of the medium, nature of the mucus changes, influencing drug
interaction as well as stiffness of the mucus. Ionic strength and charge also have similar effects.
Adapted from: Physicochemical properties of mucus and their impact on transmucosal drug delivery
[36].

11
Mucus has multiple-barrier properties which can be categorized into 3 types: dynamic barrier,
steric barrier and interactive barrier [37]. The dynamic barrier is composed of continuously
secreting mucus; therefore, drugs need to diffuse in the upstream in order to reach and cross
the epithelium. In the steric barrier, there is a network of mucin which forms a size-exclusion
filter for larger compounds, making it a size-dependent barrier [38]. The interactive barrier is
dependent on the ability of mucin to form multiple low-affinity bonds via hydrophobic or
ionic interactions or hydrogen bond formation with drugs [37].

Drug permeation through mucus also depends on viscoelasticity, pH, and ionic strength of the
mucus (Figure 5). Viscoelastic property of mucus is dependent on the presence of mucins,
DNA, lipids, and other mucus proteins [36]. Mucus pH varies throughout the GI tract, in

of
stomach mucus pH varies from 1 to 2 [39], while the pH of duodenal mucus is 6.1 and

ro
increases to 7 - 8 on reaching colon and rectum [40]. At acidic pH, conformational changes,
-p
from random coil to elongated structure, have been observed in the mucus layer [41]. This
elongated conformation can facilitate cross-linking in the mucin macromolecules, forming a
re
gel structure and preventing drug permeation [41].
lP

Mucin also undergoes swelling and hydration, which is mediated by electrostatic forces,
mostly by exchanging H+ and divalent Ca2+ with monovalent cations (Na+ and K+). Due to
na

the presence of sialic acid and sulfate moieties, mucin glycoproteins have a net-negative
ur

surface charge which can interact with cations [42]. It was shown that mucus viscosity
decreases with an increase in ion concentration, improving permeability [43, 44].
Jo

A detailed review of the impact of mucus on intestinal drug absorption was reported by Leal
et al. [36].

3.3. Gut microbiota

Gut microbiota plays many physiological functions as immune stimulation, barrier synthesis,
metabolism, etc. Alteration in the intestinal microbiota affects the pharmacokinetics of many
drugs. For example, prodrug loperamide oxide in the presence of gut microbiota converts to
active loperamide, which reduces the growth of enterocytes, leading to an increase in
intestinal permeability [45]. Digoxin, used as a cardiac glycoside in the treatment of
congestive heart failure, becomes inactive when the lactone ring is reduced, producing
dihydrodigoxin. It was observed that co-administration of antibiotics prevented

12
dihydrodigoxin production, indicating that gut microbiota may have a role in these
transformations [46]. It was later identified that gut residing Eggerthella lenta is the microbe
responsible for this transformation [47].

Change in the gut microbiota composition impairs the barrier function of epithelial junctions
as well as the mucus layer. The effect of gut microbiota on drug metabolism and
pharmacokinetics has been elaborately reviewed by Haiser and Turnbaugh [48] and Zhang et
al. [49].

4. Drug physicochemical parameters that influence oral absorption

of
Apart from the gut physiology, the physicochemical properties of a drug molecule play an

ro
equally important role in its absorption. Assessment of the physicochemical properties of a
-p
drug is an essential step while designing a formulation for oral delivery. In this section, we
will individually examine some of the major physicochemical properties, known as Lipinski’s
re
rule of five, influencing the membrane diffusion of drugs (Figure 6).
lP
na
ur
Jo

Figure 6: Different physicochemical parameters of the drug influence passive diffusion. A.

Molecular weight: (a) very low molecular weight polar molecules can pass through the aqueous
pores present on the cell membrane. (b) Low to medium molecular weight non-polar molecules can
pass through the membrane by passive diffusion. (c) High molecular weight molecules, irrespective of
polarity, can only permeate the cell membrane by receptor-mediated uptake. B, C. Lipophilicity and
Ionization: Lipophilic and non-ionic molecules (L, N) can only traverse the cell membrane by
passive diffusion, which route is non-accessible for hydrophilic and ionic molecules (H, I). D.
Solubility: Solubilization is highly important. Only solubilized molecules can be absorbed; insoluble
molecules cannot undergo diffusion due to the formation of large aggregates.

Molecular weight: It is one of the most significant parameters affecting the rate of passive
absorption of drugs across intestinal epithelia. Camenisch et al. conducted a study to analyze
the absorption of drug molecules in a weight range of 150-670 Dalton [50]. It was observed

13
that molecules less than 200 Dalton molecular weight can pass through the intestinal mucosa
via both paracellular and transcellular transport, depending on their lipophilicity. Paracellular
transport is preferred for hydrophilic molecules and transcellular pathway for lipophilic
molecules. Molecules with weight more than 500 Dalton exhibited reduced permeation as
diffusion was constrained, though they can adopt different endocytosis mechanisms for their
absorption (Figure 6A).

Hydrophobic nature of the molecule: Hydrophobicity is an important property of a

molecule concerning its absorption and distribution. Generally, two descriptors, logP, and
logD are used in defining the hydrophobicity of a molecule. LogP is the partition coefficient
of the molecule which is defined as the ratio of concentrations of the drug molecule in a

of
mixture of two immiscible solvents, usually water (polar) and n-octanol (non-polar).

ro
However, the partition coefficient takes into account only the unionized form of the

-p
molecule, and many molecules with pKa values lower or higher than biological pH are
present in their ionized form. To resolve this issue, LogP is substituted with logD
re
(Distribution coefficient). logD takes into consideration the concentration of both the ionized
lP

and unionized parts of the molecule present in the aqueous phase [51]. As the majority of
drug absorption is carried out through passive transcellular pathway, lipophilic drugs show
na

better absorption (Figure 6B). However, very high lipophilicity should be avoided because of
a range of factors including increased protein binding (which will reduce permeability),
ur

reduced solubility, and inappropriate tissue distribution.

Jo

Ionization: Only non-ionic molecules can be absorbed by the passive diffusion process
(Figure 6C). When administered orally, the extent of ionization of a drug molecule depends
on the specific pKa value of the drug and pH value of the medium. As different regions of the
GI tract have varying pH values, significant variation in the ionization is observed. Weak
bases are absorbed at a faster rate from the intestine (pH 7.0 – 8), and weak acids are
absorbed at a faster rate from the stomach (pH 1.2 – 2). Apart from the permeability,
ionization capacity also affects the steady-state of distribution of a drug in different
compartments, if there is any pH difference. [52].

Hydrogen bonding affinity of the drug: Calculating polar surface area (PSA) is one of the
efficient methods used in determining the H-bonding capacity of a drug molecule. Polar
surface area could be linked with a molecule's ability to permeate through the intestinal
membrane. PSA can be explained as sum total of polar atom’s surfaces in a compound

14
obtained by different computation techniques (CONCORD, topology of molecule,
conformational sampling etc.) [53] Palm et al. have demonstrated an inverse correlation
between dynamic PSA and permeability coefficients [54]. In a similar investigation, Kelder et
al. found an inverse relationship between the polar surface area and brain penetration [55].
They demonstrated that the CNS drugs which were orally absorbed had a PSA value less than
(60-70Å), and the orally absorbed non-CNS drugs displayed a PSA value less than 120Å.

Solubility: Solubility measures the amount of drug present in a saturated solution at a

specific pH and temperature. Temperature, pH, crystal lattice are some of the factors which
impact the equilibrium solubility. Solubilization of the drug is a prerequisite for absorption
through passive diffusion (Figure 6D). Nanoparticles, which are not real solutions, generally

of
absorbed via different endocytic pathways.

ro
Recently, several orally bioavailable compounds are discovered which venture beyond the
-p
Lipinski’s rule of five, demanding us to revise the aforementioned guidelines [56]. Doak et
re
al. have found out a set of 182 approved drugs for oral delivery that has a MW > 500 Da [56].
Important examples include roxithromycin, which has a molecular weight of 837 and PSA of
lP

218, however, shows 78% oral bioavailability [57]; josamycin (MW: 828, PSA: 207) shows
high oral bioavailability [58]; rifampicin (MW: 823, PSA: 224) with more than 90% oral
na

bioavailability [59], etc. These examples indicate that drug absorption through the oral route
ur

is more convoluted then presumed before. A thorough understanding of the physicochemical

properties of drugs would help us to extend the horizon of orally administered drugs.
Jo

In contrast to the small molecules discussed above, macromolecules like peptides/proteins

(MW>5kDa) do not follow the passive pathways to reach the systemic compartment, and
unlike small molecules, no molecular descriptor has been reported to predict their oral
bioavailability. Researchers are extensively working on exploring various strategies for oral
delivery of these macromolecules [60]. Techniques like site-specific delivery, use of
permeation enhancers, particulate-carrier systems and bioadhesive systems are being
investigated for peptide delivery. Some of these strategies are discussed in section 7 of this
review. For a more detailed perspective on the oral delivery of macromolecules, refer to the
following reviews [61-63].

15
5. Approaches towards enhancing intestinal permeability

5.1. Prodrugs

The most clinically successful strategy to improve the oral bioavailability of a poorly
permeable drug has been the use of prodrugs. Prodrugs are useful in overcoming several drug
absorption barriers such as poor solubility, instability, insufficient absorption, rapid first-pass
metabolism etc [64]. Prodrugs are designed to improve either the passive diffusion or active
transport of the drug molecule. As we have discussed previously, permeability is highly
influenced by the partition coefficient (log P) of the drug. Based on that, many researchers
have modified the parent drug to increase its lipophilicity in order to increase permeability.

of
Different strategies have been employed to increase the logP of a drug, including
modification of functional groups such as hydroxyl, carboxylic, carbonyl, amine, and

ro
phosphate groups, by making esters, phosphate esters, amides, and oximes [65]. Esters are
-p
one of the most common prodrugs which can enhance membrane partitioning of polar drugs
re
by converting the polar functional groups into a non-polar one. After absorption, the ester
bond can be easily hydrolyzed by the esterase found in the liver, blood, tissues and other
lP

organs. Temozolomide is one of the successful examples in this category. Temozolomide is a

derivative of MTIC (3-methyl-(triazen-1-yl) imidazole-4-carboxamide) which is an anti-
na

cancer drug generally prescribed for glioblastoma and anaplastic astrocytoma, it can be
administered orally and parenterally. Intracellularly, it transforms into an alkylating agent
ur

MTIC; which results in cell apoptosis [66] (Figure 7A). Another drug, Famciclovir, which is
Jo

used in the treatment of Herpes zoster, is a deoxy-diacetate ester analog of penciclovir. The
ester formation increases the lipophilicity and improves diffusion of penciclovir [64] (Figure
7B). Similar examples include adefovir [67, 68], cytarabine [69], mesalamine [70], (Figure
7C) etc.

Apart from making lipophilic prodrugs, modifying the drugs with active transport ligands is
another strategy that has been exploited by many researchers. A wide range of transporters
has been evaluated for this purpose, including bile acid transporters, monocarboxylate
transporter 1 (MCT-1), cholesterol transporter (ABCG5/8), organic cation transporter (OCT),
organic anion transporter (OAT), etc. [10, 71, 72]. Among different types of transporter
present in the intestine, amino acid transporters are one of the most abundant types [73].
Many clinically successful prodrugs have been developed targeting these amino acid
transporters to improve the absorption of polar drugs, like valacyclovir (Figure 7D),

16
valganciclovir, etc. Valacyclovir is a valyl ester prodrug of acyclovir, use of which can
increase the oral absorption of acyclovir 3 to 10-fold [74]. Gabapentin enacarbil (Figure 7E),
a prodrug of gabapentin, is absorbed by the MCT-1 transporter, present throughout the
intestinal tract [75], which showed very good bioavailability [76].

of
ro
-p
re
lP
na
ur
Jo

Figure 7: Different prodrugs used for enhancing oral absorption.

5.2. Permeation enhancers

When changing the physicochemical nature of the drug by the prodrug approach is not
feasible, the use of permeation enhancers can enhance the transport of poorly absorbed drugs.
A highly diverse set of compounds have shown to modify the epithelial barrier to increase
absorption [77, 78]. A list of such compounds with their mechanism of action is included in
Table 1. Permeation enhancers can function via both paracellular or transcellular routes.
Paracellular permeation enhancers act through the opening of the tight junctions, either by

17
changing intracellular signaling mechanisms (via Ca2+ signaling) or by direct disruption of
the adhesion proteins [79]. Transcellular permeation enhancers can either produce reversible
fluidization of the epithelial plasma membrane [78], or they can increase the membrane
interaction of the drug (e.g., by micelle/reverse micelle formation) to improve passive
transcellular permeation [80]. Though there is a significantly higher number of molecules that
exhibited transcellular permeation enhancing capacity than that of paracellular permeation
enhancer, more numbers of transcellular permeation enhancers have been tested clinically
[78]. This may be due to the intrinsic adverse effects associated with the non-specific
perturbation of the cell membrane seen with the transcellular permeation enhancers.

5.2.1. Paracellular permeation enhancers

of
Paracellular permeation enhancers can be categorized into 1st generation and 2nd generation;

ro
while 1st generation molecules act via modulation of intracellular signaling, 2nd generation
-p
molecules can disrupt the cell adhesion protein interaction at cell adhesion recognition (CAR)
re
sequences. Though there are several 2nd generation permeation enhancers developed [81], till
now, 1st generation molecules have shown more clinical viability [78]. The most promising
lP

among them are fatty acids. Among different types of fatty acids, medium-chain fatty acids
are mostly used for this purpose [77]. Lindmark et al. found improved absorption of
na

polyethylene glycols and mannitol from Caco-2 cells when sodium caprate (sodium salt of
ur

capric acid, C10) was used [82]. They have demonstrated that this is mediated through the
activation of phospholipase C-dependent inositol triphosphate/diacylglycerol pathway to
Jo

increase intracellular calcium [83]. Tomita et al. have further exhibited that the function of
sodium caprate is dependent on the calmodulin-dependent contraction of actin/myosin
filament [84]. Lindmark et al. have demonstrated that sodium caprate can enhance the
paracellular transport of molecules with an MW of < 1kDa, whereas the permeation of
molecules larger than 4kDa was found to be insignificant [85]. It is now utilized in certain
countries as an excipient in rectal suppositories [86]. Sodium caprate based excipient GIPET®
(Merrion Pharma, Ireland) has been tested clinically for absorption enhancing effect using
alendronate, desmopressin and low-molecular-weight heparin as the model drugs, which have
shown improved bioavailability with no serious adverse effects [87]. A sodium caprylate (C8)
based excipient, TPE® (Chiasma, Israel) has also exhibited good permeation enhancing
efficacy with octreotide in the monkey model [88].

18
Another group of fatty acid derivatives, N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC)
and N-(8-2hydroxy-5-chloro-benzoyl)-amino-caprylate (5-CNAC) have shown high efficacy
as permeation enhancers (Eligen®, Emisphere, USA). There is some debate regarding the
mode of action of these molecules. It has been proposed that they may make lipophilic
complexes by physical interactions with polar molecules, thereby improving passive
diffusion [78]. SAR analysis has indicated that lipophilicity [89] and hydrogen bonding
ability [90] may modulate the intestinal permeation enhancement. On the other hand, it has
been demonstrated that SNAC can increase membrane fluidity [91]. SNAC has been
clinically approved for the delivery of an oral vitamin B12 supplement [92].

EDTA (ethylenediaminetetraacetic acid) has also been extensively used as a paracellular

of
permeation enhancer. It has been demonstrated that EDTA depletes extracellular calcium by

ro
chelation and activates protein kinase C [93], resulting in the expansion of the paracellular
-p
route by the loosening of apical junctions via ROCK-II dependent activation of actomyosin,
increasing its contractility [94]. It has also been reported that depletion of extracellular Ca2+
re
by EDTA can disrupt Ca2+ dependent E-cadherin, leading to loosening of the tight junctions
lP

[95]. EDTA based excipient POD® (Oramed, Israel) has been clinically tested for oral
delivery of insulin, which exhibited good efficacy [96].
na

Quaternary ammonium compound derivative acylcarnitine (Peptilligence®, Enteris

ur

Biopharma, USA) have also exhibited permeation enhancing activity via reducing the
concentration of certain claudin subtypes like claudin 1, 4 and 5 [97]. Peptilligence® has
Jo

successfully completed a phase III clinical trial for the delivery of oral calcitonin [98]. It is
being evaluated for the oral delivery of leuprolide (phase II) and tobramycin (phase I) as well.

19
Figure 8: Mode of action of different paracellular permeation enhancers. Most of the paracellular
permeation enhancers act by modulating contraction of the cytoskeletal proteins. They may act
through PLC-IP3/DAG pathway (fatty acids), leading to cytoskeletal contraction and increased tight
junction permeability, or can deplete extracellular Ca2+ (EDTA), induces PKC which leads to
actomyosin contraction and loosening of the apical junction. Some can directly reduce the amount of
junctional proteins (acylcarnitine).

5.2.2. Transcellular permeation enhancers

Surface active agents as permeation enhancers: Improvement in the drug absorption by the
use of surfactants can be linked to fluidization and disintegration of membranes caused by
them (Figure 9A). Ionic surfactants are not very useful for this purpose as they cause toxicity
to the intestinal mucosa, and mostly non-ionic surfactants are used for this purpose [99].

of
Since surfactants are amphiphilic molecules, it is reported that the size and structure of both
the hydrophilic as well as lipophilic part influence the absorption enhancing activity [77].

ro
Non-ionic surfactant poly-oxyethylene esters and ethers have been extensively used in ocular
-p
drug delivery to enhance permeation [100]. Intestinal absorptivity of PABA (para-
aminobenzoic acid) was found to be augmented when poly-oxyethylene esters were used as
re
permeation enhancers [101]. Perninelli et al. studied a new sugar-based surface-active agent
lP

lactose oleate, the possible pathway could be the opening of tight junctions facilitating the
paracellular route; the transcellular path can also be involved [102].
na

Labrasol® (Gattefossé, France) is one commercially available PEG-based excipient which is

found to improve the oral bioavailability of drugs [103]. It is composed of free PEG as well as
ur

PEG ester of caprylic and capric acid. In pre-clinical studies, it was found to improve the
Jo

permeation of heparin [104], erythropoietin [105], and dextran [106].

As surfactants have membrane fluidizing effect, they can also impact other membrane
transportation processes and the dynamic property of the cell membrane. Danielsen et al.
have reported that a group of surfactants (1-decanoyl-rac-glycerol, nonaethylene glycol
monododecyl ether, and lauroyl-L-carnitine) can fuse brush border microvilli and block all
types of endocytic pathways [107].

Bile Acids: Attributing to their biocompatible nature, bile salts are extensively studied as
absorption enhancers. Bile acids are biological surface-active agents synthesized by the liver
from cholesterol. Bile acids are stored in the gall bladder and released when ingested food
arrives in the duodenum. They assist in increasing the pH of the medium, improve the
intestinal uptake of dietary fats by emulsification, and maintain cholesterol homeostasis
[108]. Only a small quantity of bile acids is newly synthesized as an effective recirculation

20
mechanism for bile acids is present. They are reabsorbed from the terminal part of the
intestine to get conveyed back to hepatocytes from where they are rereleased again, each
molecule can repeat it 4-12 times per day.
Bile acids can improve permeation through both the transcellular and paracellular pathways.
It has been shown that bile acids can increase the absorption of both hydrophilic and
hydrophobic drugs, by enhancing the fluidity of biological membranes in the former, and
assisting in the solubilization of the latter (Figure 9B) [109, 110]. When used with polar
drugs, bile acids act by enhancing both the paracellular and transcellular transport. Aqueous
channels are formed when bile acids are incorporated inside the biological membrane to form
reverse micelles, which assists in the transcellular permeation of polar moieties [109].

of
Increment in the paracellular transport is attributed to the opening of tight junctions, which

ro
may be the consequence of its binding with the calcium ion [109]. Certain bile acids hydroxyl
group at position 12 (like tauro-chenodeoxycholate, glycochenodeoxycholate) are reported to
-p
possess the property of Pgp-efflux inhibition, possibly due to alterations made by them in the
re
lipidic environment of the transporter protein [111]. Modifications are made in the structure
of bile acids to reduce their membranolytic effect and enhance the membrane permeation.
lP

Song et al. examined the absorption of salmon calcitonin with different bile salts and found
that sodium taurodeoxycholate shows the highest increment in permeability among all bile
na

salts, which delivered by forming a complex with the peptide [112, 113].
ur
Jo

Figure 9: Mode of action of different transcellular permeation enhancers. Two important examples of
transcellular permeation enhancers are surface-active agents and bile acids. A. Surface active agents

21
mostly act as a membrane destabilizing agent and produce either fluidization or disintegration in the
cell membrane, improving the transport of molecules through the membrane. B. Bile acids enhance
transcellular permeation through a variety of pathways. Due to their amphiphilic nature, bile acids can
improve the solubility of non-polar drugs by micelle formation and enhance their interaction with the
membrane. They can entrap polar drugs by forming reverse-micelles. Reverse-micelles can get
integrated into the membrane and form aqueous pores. They can also get integrated into the
membrane, increasing the fluidity of the membrane. Bile acids can also inhibit the function of P-gp,
preventing drug efflux.

Due to the presence of acidic groups, bile acids have also been utilized to make an ionic
complex with polar drugs to improve their absorption. Gaowa et al. have demonstrated
improvement in the absorption of an EGFR (epidermal growth factor receptor) targeted
hybrid peptide by making an electrostatic complex with bile acid [114]. Significant

of
improvement in the oral bioavailability of a highly polar xenobiotic drug ceftriaxone was also

ro
observed when it was complexed with deoxycholyethylamine [115].

-p
One formulation containing sodium ursodeoxycholate complexed with insulin (Capsulin®,
Proxima, UK) has exhibited high efficacy in humans [116]. It is currently undergoing a phase
re
IIb trial. Another sodium cholate based formulation of insulin (Insulin Tregopil®, Biocon,
lP

India) exhibited good efficacy in a phase I trial [117, 118].

A detailed review of different permeation enhancers has been reported by Maher et al. [78].
na

6. Use of mucoadhesive polymers for enhancing intestinal absorption

ur

Sustained-release, enhanced retention time, and delivery at a specific target site are some of
Jo

the benefits of using mucoadhesive agents for drug delivery [119]. Chitosan, a polycationic
polysaccharide, is one of the most exploited polymers for making mucoadhesive formulations
[120]. Multiple pathways have been discovered through which chitosan can show absorption
enhancing property. Due to positive charge, chitosan can interact with the negatively charged
mucus, which helps in the mucoadhesion of the chitosan-coated particles [121]. After binding
with the mucus, chitosan mostly enhances paracellular transport via a complex mechanism.
Ranaldi et al. have observed reversible opening of the tight junction and an increase in
paracellular fluxes with chitosan [122]. Rosenthal et al. demonstrated that the increase in
reversible paracellular transport with chitosan was associated with the activation of chloride
bicarbonate exchanger [123]. Activation of the chloride bicarbonate exchanger causes an
alteration in intracellular pH which can modulate the tight junction barrier, probably by

22
affecting scaffold proteins (ZO-1). Chitosan-based nanoparticles can also be absorbed via the
M-cells by endocytosis [124].
For its activity, chitosan needs to bind with the epithelial surface which requires a specific pH
environment. At higher pH, the deprotonation of chitosan weakens its binding and
permeation effect. To overcome this issue, derivatives of chitosan were synthesized (i.e.
Trimethyl chitosan and carboxymethyl chitosan) which exhibited improved activity [125-
127]. Besides chitosan, some anionic mucoadhesive polymers such as carbomer and
polycarbophil have also shown to enhance intestinal permeation [128]. Borchard et al.
compared between chitosan-glutamate and carbomer and found out that carbomer was more
efficient in opening tight junctions and improving paracellular transport [129].

of
Neutral polymers like PEG has also been exploited for their mucoadhesive property. It was

ro
demonstrated that while PEG of molecular weight less than 5 kDa enhances muco-
penetration, PEG of molecular weight 10 kDa shows mucoadhesive property [130, 131].
-p
Mahlert et al. showed PEGylated PLGA nanoparticles have a higher cellular uptake in
re
mucus-producing HT29 MTX cells compared to the chitosan-coated particles [132]. When
compared with negatively charged polystyrene nanoparticles, PEG-coated lipid nanocapsules
lP

exhibited faster diffusion through gastric mucin barrier [133].

na

7. Absorption enhancing techniques based on nanomedicine

ur

Nanomedicine has recently attracted considerable attention for oral drug delivery [134].
Jo

Employing nanoparticles as drug carriers have several advantages such as selective targeting,
sustained and controllable release, improved intracellular uptake, etc. However, oral delivery
of nano-drug carriers has to overcome several hurdles even before encountering the epithelial
cells for absorption. They have to remain stable in the harsh environment of the GI tract as
well as have to cross the mucosal barrier, which acts as a mesh and can prevent nanoparticle
interaction with the enterocytes [135]. For entering into the systemic circulation,
nanoparticles have to permeate through the apical membrane of the enterocytes, escape from
the endolysosomal degradation, and then exocytose from the basolateral membrane. Due to
the presence of so many barriers, designing a clinically successful oral nanoformulation is
highly challenging [136]. Currently, there is no FDA approved nanoformulation available for
oral delivery. Nevertheless, multiple advanced formulations have been developed and tested
at various stages of preclinical evaluation.

23
GI absorption of nanoparticles can occur via three possible mechanisms, transport through
the transcellular route, transport through the paracellular route, and transport via the M-cells
of the Peyer's patch. Nanoparticle size, charge, and surface properties have a significant
impact on endocytosis mechanisms and efficiency. Bannunah et al. have exhibited that
nanoparticles with positive charge show better uptake by the intestinal epithelial cells
compared to nanoparticles with negative charge [137]. A similar observation was made by Du
et al. as well [138]. Mechanism wise, positively charged nanoparticles are endocytosed via a
mixture of the micropinocytosis, clathrin-mediated pathway, and cholesterol-dependent
pathway, whereas negatively charged nanoparticles are uptaken mainly via lipid raft pathway
[137]. Among different shapes, rod-shaped particles were found to have a longer residence

of
time in the GI tract compared with spherical particles [139]. It was shown that smaller

ro
particles (less than 100 nm in diameter) can enter into enterocytes, whereas larger ones (more
than 500 nm in diameter) remain adhered to villi surfaces [140]. Banerjee et al. have
-p
demonstrated that intestinal cell uptake of nanoparticles is inversely related to their size, and
re
rod-shaped particles can be uptaken and transported more efficiently compared to spherical
particles, which further increases with active targeting [141]. Receptor-mediated endocytosis
lP

is a widely evaluated mechanism for nanoparticle uptake. Roger et al. have demonstrated that
na

folic acid-functionalized nanoparticles can improve oral absorption by folate-receptor

mediated endocytosis [142]. In another similar study, Pridgen et al. formulated nanoparticles
ur

that were aimed at targeting the neonatal Fc receptor (FcRn), which exhibited a two-fold
increase in transcellular transport of insulin compared to the non-functionalized ones [143].
Jo

Surface modification of nanoparticles with wheat germ agglutinin (WGA), which displays a
selectivity in binding with N-acetyl D-glucosamine and sialic acid present on the intestinal
epithelium can also improve through clathrin-mediated endocytosis process [144]. A dual
approach of mucoadhesion and galactose targeting has been exploited by Han et al. for oral
delivery of anti-VEGF (vascular endothelial growth factor) siRNA and anti-Survivin shRNA
[145]. They have used chitosan and cysteine in forming the nanoparticles which enhanced the
mucoadhesion and intestinal permeation. The nanoparticles were decorated with galactose
which promoted clathrin- and caveolin-mediated endocytosis, mostly via the M-cells.
However, the effect of this delivery on the enterocyte cells is not clear from the study. He et
al. have also evaluated a mannose functionalized trimethyl chitosan nanoparticles for the oral
delivery of Tumor necrosis factor-alpha (TNFα) siRNA, which exhibited enhanced M-cell
uptake through caveolae-mediated pathway as well as and macropinocytosis [146]. Lipid

24
nanoparticles (SLN (solid-lipid nanoparticles), NLC (nanostructured lipid carriers) etc.) have
been shown to use both caveolin and clathrin-dependent pathway for their uptake [147, 148].
The enterocytes are also capable of phagocytosis via a TLR4 mediated pathway [149]. In
table 2, we have listed some of the representative nanoparticles which had been evaluated to
be uptaken through these pathways.

After the nanoparticles get endocytosed, there are several possibilities for their fate. It may
get degraded in the lysosomes, or it may get accumulated in a cell organelle, or it is
exocytosed (Figure 10) [136]. To reach the systemic circulation, the nanocarriers need to be
designed in such a way that it can escape or bypasses the endolysosomal compartment.
Utilizing the intestinal bile acid pathway can be one such approach that can be employed for

of
this purpose. Samstein et al. reported that deoxycholic acid can improve the stability of

ro
PLGA nanoparticles in the GI lumen and increased its bioavailability [150]. Deoxycholic acid
-p
exploits the intestinal bile acid pathway to facilitate the cellular entry and intracellular
transfer of nanoparticles. There are two primary components of this pathway, namely,
re
sodium-dependent bile acid transporter (ASBT) present in the apical surface and ileal bile
lP

acid binding protein (IBABP) present in the cytosol. The former is associated with the
cellular entry, and the latter mediates the intracellular transfer of bile-acid conjugates. Fan et
na

al. utilized this pathway for improving the oral bioavailability of insulin [151]. ASBT-
regulated internalization helped the Deoxycholic acid nanoparticles (DNPs) to traverse inside
ur

the intestinal epithelium. Inside the cell, DNPs escape from lysosomal degradation due to the
Jo

membrane disrupting property of deoxycholic acid [152]. Lastly, due to the specificity of
IBABP towards bile acids, the DNPs gets transferred out of the enterocyte. Kim et al. have
demonstrated that nanoparticles delivered through this pathway transported to the systemic
circulation via the lymphatic system [153].

25
 of
ro
-p
Figure 10: Different nanoparticle uptake pathways active in the enterocytes. Receptor-mediated
endocytosis, caveolae or clathrin-mediated pathway, micropinocytosis, and phagocytosis are the
re
major uptake processes observed. In the conventional pathway (blue arrow), cargo from all these
uptake pathways may end up in the lysosome, leading to the metabolism of the cargo. An alternate
lP

pathway exists (red arrow), where endosomes may transport the cargo to the endoplasmic reticulum
through Golgi complex, from where they may get exocytosed by the secretory pathway.
na

The retrograde pathway, which leads endosomes to the endoplasmic reticulum/ Golgi
ur

apparatus can also be employed in avoiding lysosomal degradation. Some studies have
Jo

exhibited that caveolae-dependent uptake can bypass lysosomes [154-157]. It has been
demonstrated that the content of caveosomes is generally transported to the endoplasmic
reticulum or the Golgi apparatus [158-161]. Nanoparticles that can bypass the lysosomal
degradation and can enter the ER (endoplasmic reticulum) or Golgi, can get exocytosed via
vesicles of the conventional secretory pathway. Malik et al. did an extensive study on
caveolae dependent transcytosis. Transcytosis of caveolar vesicles is said to be controlled by
SNARE (soluble NSF attachment protein receptor) proteins. Vesicles anchor on the
basolateral membrane activates the SNARE machinery which results in the expulsion of
vesicular contents outside the cell [162-164]. One of the critical observations was that the
size-dependent nature of caveolar transport; small-sized nanoparticles (20-40 nm) were more
efficiently transcytosed than larger ones (100 nm) [162, 163]. Transcytosis can also be
dependent on the cytoskeleton and motor protein regulation [165, 166]. Another scheme to
evade the lysosomal degradation can be incorporating nanocarriers with endo-lysosome-

26
disrupting agents [167]. Fujiwara et al. demonstrated that a cell-penetrating peptide decorated
liposomes could accumulate in the Golgi apparatus, which implies that it had escaped the
lysosomal degradation [168].

M-cell mediated transport is another significant pathway through which nanoparticles can be
directly absorbed into the systemic circulation from the GI tract. M-cells are modified
enterocytes which have very high uptake capacity and are situated at the Peyer’s patches
present in the intestinal epithelium [18]. The most efficient way of targeting M-cells is
through active targeting. However, only a few of these receptors are currently known, and
many of them are co-expressed by neighboring enterocytes, jeopardizing the targeting
strategy [169]. The identified receptors include platelet-activating factor receptor (PAF), toll-

of
like receptors (TLR), apical glycoprotein (GP2) and α5β1 integrins [169, 170]. The ligands

ro
used for targeting these receptors include mannose and derivatives, lectin-binding ligands,
-p
wheat germ agglutinin (WGA), RGD (arginylglycylaspartic acid) peptide, LDV (Leu-Asp-
Val) derivative etc [18].
re
Various researchers have evaluated M-cell targeting to enhance the oral bioavailability of
lP

drugs. For example, Lubben et al. have shown enhanced uptake of chitosan microparticles in
the Peyer’s patches [171]. Liu et al. have developed a poly(N-(2-hydroxypropyl)
na

methacrylamide) with vitamin B12-modified chitosan in lipid polymeric nanoparticles which

ur

have exhibited absorption through Peyer's patch [172]. Yoo et al. have used a phage display
technique to select an M cell-targeting peptide and used that for making an M cell-targeted
Jo

chitosan nanoparticle [173].

Among different types of nanoformulations developed, liposomes have the highest number of
clinical success [174]. However, oral delivery of liposomes is highly challenging due to its
instability in the GI tract. The presence of bile salts and lipolytic enzymes in the gastric juice
has damaging effects on the structural integrity of the liposomes, resulting in disruption of
membranes and leakage of the drug [175]. If they remain stable, intact liposomes can be
uptaken by the enterocytes via clathrin or caveolae-dependent endocytosis, macrocytosis or
membrane fusion [176]. Designing a liposome that survives in the GI tract is a highly
challenging task. Selecting appropriate lipid type, optimizing the composition and modulating
surface structure are some techniques investigated to improve the stability of liposomes [176].
Increased stability and reduced disruption of the liposomal membrane was observed when
bile salts were inserted inside lipid bilayers [177, 178]. Incorporating bile-salts in the bilayer

27
of liposome leads to the formation of bilosomes that can be utilized as an orally-stable drug
carrier. Niu et al. developed such a delivery system for oral administration of insulin, where
they incorporated bile salts in liposomes which have enclosed insulin in the aqueous core
[177]. Golocorbin-Kon et al. prepared bilosomes for oral delivery of cefotaxime and observed
a 9-fold increase in oral bioavailability than conventional liposomes [179]. For protecting the
liposomes from gastric acid, an enteric coating can be done using polymers like Eudragit
L100, Eudragit S100 [180].

8. Ion pair complex approach

of
The ionic nature of a drug molecule is one of the most significant absorption barriers. To

ro
improve the permeation of an ionic drug through the GI epithelium, one interesting approach
is to make a neutral complex of that molecule with a suitable counter-ion. This interaction
-p
depends on the ionic potential (positive or negative) of the two moieties involved, and in turn,
re
on the nature and pKa of the ionizable groups and on the pH of the medium and composition
lP

[181]. Ion pair complexation can effectively form non-ionic complexes, reducing the polarity
and positively influencing drug absorption through passive diffusion. Construction of an ion-
na

pair complex depends on the intermolecular hydrogen bonds, Van der Waals forces and
electrostatic interactions. The interplay between these interactive forces results in the creation
ur

of a temporary network between the polymers. The mechanism of drug uptake depends upon
the type of polymer chosen for complex formation. Selecting suitable polymers for complex
Jo

formation can increase the retention time at the absorption site, shield the drug from gastric
degradation, increase solubility, enhance paracellular transport and provide site-specific
delivery for the drug [182, 183].

Polyelectrolytes or ion-pairing agents are classified based on their ionic nature as anionic and
cationic polymers. Alginic acid, hyaluronic acid, carboxymethyl cellulose are some of the
examples of anionic ion-pairing agents. Chitosan and Eudragit are examples of cationic ion-
pairing agents [184].

Various scientists have evaluated different counter-ions to make ion-pair complexes with
different drugs. Negatively charged polymer λ-carrageenan was complexed with cationic
tropsium chloride (used as a model compound representing BCS class III drug), improvement
in bioavailability was observed which was attributed to the interaction between mucus and

28
the ion-pair complex [182]. Bigucchi et al. tried to achieve targeted delivery and improved
absorption for vancomycin by incorporating it in chitosan/pectin polyelectrolyte complexes
[185]. Complexes were prepared with varying proportions of chitosan and pectin to attain a
ratio suitable for colonic delivery. Increasing the ratio of pectin in complex formation
resulted in improved mucoadhesion and a better swelling property appropriate for colonic
delivery. In another study, Miller et al. increased log P values of two highly hydrophilic
antiviral agent zanamivir and oseltamivir by forming a polyelectrolyte complex with 1-
hydroxy-2-naphthoic acid (HNAP) [186]. Zanamivir-HNAP complex exhibited improved
permeability compared to the free drug in a rat jejunum assay [186]. Samiei et al. used the
ion-pair complex approach to improve penetration of alendronate using arginine and

of
phenazopyridine as the counter ion [187].

ro
Ionic complexation has also been exploited for the modulation of drug release behavior.
-p
Sumaila et al. developed a sustained release formulation of rifampicin in which rifampicin
was incorporated in a nanostructured ion-pair complex of low-molecular weight chitosan and
re
soy-lecithin which improved the permeability [188, 189]. Srinivas et al. developed a sustained
lP

release formulation of diclofenac sodium by forming a complex between carbopol and

polyvinyl pyrrolidone (PVP) [190]. The optimized formulation demonstrated comparable
na

dissolution profile with commercially available prolonged release product. Liu et al.
investigated an innovative approach for enhancing oral bioavailability of insulin by forming
ur

ion-pair complexes which imitate a virus envelope. In vivo studies showed the hypoglycemic
Jo

effect in rats and this response was amplified when SDS was used as a coating material for
polyelectrolyte complexes which protected it from enzymatic degradation and increased
permeation through mucus layer [191].

9. Conclusion and perspectives

As oral administration is the most preferable route for long term treatment, any drug
development approach is generally focused on molecules which can be delivered through this
route. As a result, experimental molecules with a particular set of physicochemical
parameters gets significant priority, which may eliminate a group of highly potential lead
molecules at the very early stage of drug discovery. Instead of the current approach of drugs
with inherent absorption property, if we can focus on the bioactivity only at the early design
stage, that will increase the spectrum of molecules to choose from. If the formulation

29
scientists have enough logistics in their arsenal for improving oral bioavailability, we can
have a multitude of drug candidates for different disorders.

Drug formulation as a scientific stratagem has come a long way since the time of tinctures
and lotions. In the case of oral delivery, we can now protect drug degradation at the extreme
gastric pH, we can precisely control drug release at a specific site in the GI tract, we can
make fast-dissolving or controlled release formulations, we can also make the formulation to
stay at a particular location in the GI tract for a prolonged period, slowly releasing the drug,
etc. These novel advancements have significantly impacted the clinical outcome of various
drugs delivered through the oral route [192, 193]. However, till now, we have achieved only
limited success in delivering drugs which have low permeability. Though some of the

of
permeation enhancers showed clinical success, more focused research is needed for

ro
overcoming the absorption barriers present in the intestinal epithelium. GI uptake is found out

-p
to be highly selective; some molecules can be absorbed very easily, whereas for others, it
presents a complex, multifaceted barrier. The complexities associated with these barriers
re
stem from their diversity. GI tract is composed of highly heterogeneous cells and tissues,
lP

which creates an extremely diversified barrier, changing the barrier property from one region
to another. Due to this reason, one uniform approach is not very effective, as that may be
na

applicable to a small fraction of the total GI surface only. A combination of multiple

approaches would be a potential strategy to target the majority of the GI surface to effectively
ur

enhance absorption. For example, Shan et al. have developed a self-assembled nanoparticle
Jo

system for oral delivery of insulin with a cell-penetrating peptide for efficient epithelial cell
penetration and a dissociable hydrophilic coating for mucus permeation [194]. The
nanoparticles exhibited a 20-fold increase in the absorption of insulin.

In terms of targeting multiple absorption barriers, one particular excipient, bile acids, stands
out among all. One significant advantage of bile acid is, as it is naturally present in the GI
tract, adverse effects would be minimal. As discussed previously, bile acids can improve the
oral absorption of a wide variety of drugs by diverse mechanisms. It not only can improve the
solubility of lipophilic drugs, it can also increase membrane interactions of hydrophilic
molecules. Additionally, it can alter the barrier property of the intestinal enterocytes by an
assorted mechanism, involving multiple absorption pathways. Furthermore, as discussed
previously, enterocyte uptake of bile acid formulations can bypass lysosomal degradation and
can be absorbed in the lymphatic system. This is highly beneficial to prevent drug

30
degradation in the absorption process as well as evading first-pass metabolism, predominant
for any orally administered drug.

Large varieties of absorption enhancing excipients have been developed. One major
challenge associated with the use of them is the reversibility of the membrane permeation
effect. As GI tract is thronged with different digestive enzymes, as well as myriad varieties of
bacteria and toxins etc., exposure to them for even a short period of time may turn out to be
damaging for the GI epithelium, which can even be fatal [195]. Thorough, long term testing
of such excipients has to be done for evaluating its effect on the integrity of the GI epithelium
for clinical use.

of
As different drugs exhibit low epithelial permeation for diverse reasons, a detailed analysis
needs to be done to understand the exact molecular reason and a customized formulation

ro
design needs to carried out. Without a comprehensive understanding of the precise pathway,
a cursory approach may be proved to be futile. -p
re
One important observation is, though some clinical success has been achieved using
permeation enhancers, none of the nanoparticle-mediated delivery was able to progress to
lP

clinical-stage yet. The primary reason might be associated with the pathway of absorption. As
na

discussed previously, nanoparticles are primarily up-taken by various endocytic mechanism

in the enterocytes, where they need to survive the lysosomal degradation and need to be
ur

exocytosed efficiently for their absorption. This is a substantially complicated process

compared to the action of permeation enhancers. Ultra-small nanoformulations with
Jo

permeation enhancers may be more successful in improving bioavailability. Nanoparticles

have the potential to penetrate through the mucus and co-release the encapsulated permeation
enhancer as well as the active drug in a localized fashion, which may effectively increase the
absorption.

Many of the current preclinical formulations aimed at enhancing oral bioavailability are
highly complex in nature. These complexities can introduce intricate variabilities which are
difficult to control, minimizing translational potential. A thorough understanding of the
physiology and cell biology of the GI epithelium would help us in understanding the natural
absorption process which we can modulate for favourable outcomes. There are tremendous
potentials for a well designed oral formulation and through a concerted conception of
physiology, cell biology, chemistry, and formulation design, we can achieve this "holy grail"
of the formulation scientists.

31
Type of the Mechanism of action Examples References
permeation
enhancer
Fatty acids • Modulating tight junction opening • Sodium caprate (C10) [82], [83],
by activation of phospholipase C- [GIPET®], Sodium [84], [85],
dependent inositol triphosphate/ caprylate (C8) [TPE®] [86], [87],
diacylglycerol pathway to increase [88], [89],
intracellular calcium [90], [91],
• Lipophilic complex formation, [92]
increasing membrane fluidity. • SNAC, 5-CNAC
[Eligen®] etc.
EDTA • Depletion of extracellular calcium, POD® [93], [94],

of
activation of protein kinase C [96]
Quaternary • Reducing the concentration of Acyl carnitine [97], [98]

ro
ammonium intracellular claudin, thereby [Peptilligence®]
compound opening of the tight junction.
Surfactants
membrane
-p
• Disintegration and fluidization of Polyoxyethylene esters and
ethers, Lactose oleate,
[100],
[101],
re
• Moderating tight junction opening Labrasol® etc. [102],
and facilitating paracellular route [103],
lP

[104],
[105],
[106]
na

Bile Acids • Creation of aqueous channels in the Sodium deoxycholate, [109],

membrane. taurocholate, tauro- [111], [112,
ur

lithocholate, sodium
• Reducing the resilience and 113], [114],
ursodeoxycholate [115],
viscosity of mucus layer.
[Capsulin®], Sodium
Jo

[116], [117,
• Promoting the paracellular transport cholate [Tregopil®] etc. 118]
by loosening up the tight junctions.
• Moderates expression of transporter
proteins
• Distortion of membrane integrity
• Improving solubility and membrane
interaction of both hydrophobic and
hydrophilic drugs through micelle
and reverse micelle formation.
Table 1: Examples of different types of permeation enhancers and their mode of action.

32
Uptake pathway Ligands used Cargo Comments References
Utilizing the Deoxycholic acid Insulin Escorts the nanoparticle [151]
Intestinal bile-acid modified outside the basolateral
pathway (ASBT- nanoparticles membrane and helps in
mediated escaping endolysosomal
endocytosis) degradation.

Transport through Galactose modified shRNA Use of chitosan and [145]

both caveolin and trimethyl chitosan- (Survivin) + cysteine enhanced the
clathrin dependent cysteine siRNA mucoadhesion and
pathway nanoparticles (VEGF) intestinal permeation.
Galactose as a ligand
promoted the clathrin- and
caveolin- mediated

of
endocytosis

ro
Saqiunavir Saqiunavir Caveolin and clathrin [148]
embedded in dependent pathway were
nanostructures lipid -p involved in both
carrier endocytosis and
transcytosis of
re
nanoparticles.
Peptide Ligand CSK Salmon Separate SLNs were [147]
lP

and IRQ calcitonin prepared with CSK and

IRQ, CSK possess goblet
cell targeting property
na

while IRQ is a cell

penetrating peptide.
Increased absorption of
ur

salmon calcitonin was

observed with peptide-
Jo

modified SLNs.
Folate- receptor Folic-acid Paclitaxel Folate receptors are [142, 196]
targeted expressed on the cell
transcytosis surface which uptake the
nanoparticles by caveolin-
mediated trancytotic
pathway.
Lectin-mediated Wheat germ Calcitonin Adhesive interaction occurs [144, 197]
uptake through agglutinin (WGA- with the biological surface
glycoprotein receptor targeted and the glycoprotein
receptor transport of receptors present on the
liposomes) intestinal epithelium and
the uptake of liposomes
occurs mostly by the
clathrin mediated
pathway.

33
 WGA, Ulex Insulin WGA remains stable in the [198]
eurapeous agglutinin GI tract and its receptors
1 and tomato lectin are present in diiferent
types of cells constituting
the intestinal membrane.

Biotin-receptor Liposome decorated Insulin Clathrin-mediated [199]

targeted with biotin- endocytosis is the
endocytosis conjugated DSPE (1, proposed pathway for
2-distearoyl-sn- uptake of biotin
glycero-3- functionalized liposomes.
phosphatidyl
ethanolamine)
Neonatal-Fc IgG Fc fragments Insulin Neonatal Fc receptor [143]
receptor targeted facilitates the transcytosis
transcytosis and of IgG across the cell.

of
fluid-phase
Funtionalized NPs crossed
pinocytosis

ro
the intestinal epithelium
more significantly than the
-p non-specific NPs (apprx.
11.2 folds more).
re
Uptake through Arginie-glycine- Ovalbumin RGD ligand gets attached [200]
M-cells aspartic acid (RGD to the β-1 integrin protein
peptide) on M-cells.
lP

CKS9 (M-cell Oral vaccine M-cell selectivity of the [173]

homing peptide) delivery, chitosan nanoparticles was
na

Bmpb enhanced by conjugating it

vaccine with CKS9 peptide. Intake
of peptide-conjugated NPs
ur

was observed to be 1.5

times more than the
Jo

conventional chitosan
nanoparticles.
Recombinant protein Oral CPE-30 is a peptide ligand [201]
(HA-HT) with CPE- vaccination binding to claudin-4
30 (Clostridium receptor. It was
perfringens incorporated in PLGA
enterotoxin) at C- nanoparticle which
terminus as claudin- exhibited an increases M-
4 targeting ligand. cell uptake.
Utilizing the Insulin Insulin was enclosed in [202]
vitamin B-12 dextran nanoparticles that
transport system are coated with Vitamin B-
(Intrinsic - 12 derivative, these NPs
receptor targeted take advantage of vit. B-12
endocytosis) uptake pathway and get
endocytosed via intrinsic
factor receptor to enter the
systemic circulation .
Table 2: Examples of selective nanoparticles used for oral delivery and their uptake pathway.

34
 References

[1] G.L. Amidon, H. Lennernas, V.P. Shah, J.R. Crison, A theoretical basis for a
biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in
vivo bioavailability, Pharm Res 12(3) (1995) 413-20 DOI: 10.1023/a:1016212804288.

[2] A. Singh, Z.A. Worku, G. Van den Mooter, Oral formulation strategies to improve
solubility of poorly water-soluble drugs, Expert opinion on drug delivery 8(10) (2011) 1361-
78 DOI: 10.1517/17425247.2011.606808.

[3] O. Wolk, R. Agbaria, A. Dahan, Provisional in-silico biopharmaceutics classification

(BCS) to guide oral drug product development, Drug design, development and therapy 8
(2014) 1563-75 DOI: 10.2147/DDDT.S68909.

[4] M. Laksitorini, V.D. Prasasty, P.K. Kiptoo, T.J. Siahaan, Pathways and progress in

of
improving drug delivery through the intestinal mucosa and blood-brain barriers, Therapeutic
delivery 5(10) (2014) 1143-63 DOI: 10.4155/tde.14.67.

ro
[5] L. Shen, C.R. Weber, D.R. Raleigh, D. Yu, J.R. Turner, Tight junction pore and leak
-p
pathways: a dynamic duo, Annual review of physiology 73 (2011) 283-309 DOI:
10.1146/annurev-physiol-012110-142150.
re
[6] J. Linnankoski, J. Makela, J. Palmgren, T. Mauriala, C. Vedin, A.L. Ungell, L. Lazorova,
P. Artursson, A. Urtti, M. Yliperttula, Paracellular porosity and pore size of the human
lP

intestinal epithelium in tissue and cell culture models, J Pharm Sci 99(4) (2010) 2166-75
DOI: 10.1002/jps.21961.
na

[7] J. Karlsson, A. Ungell, J. Grasjo, P. Artursson, Paracellular drug transport across

intestinal epithelia: influence of charge and induced water flux, Eur J Pharm Sci 9(1) (1999)
ur

47-56 DOI: 10.1016/s0928-0987(99)00041-x.

[8] C. Chelakkot, J. Ghim, S.H. Ryu, Mechanisms regulating intestinal barrier integrity and
Jo

its pathological implications, Experimental & molecular medicine 50(8) (2018) 103 DOI:
10.1038/s12276-018-0126-x.

[9] T. Suzuki, Regulation of intestinal epithelial permeability by tight junctions, Cellular and
molecular life sciences 70(4) (2013) 631-659.

[10] J.H. Hamman, P.H. Demana, E.I. Olivier, Targeting receptors, transporters and site of
absorption to improve oral drug delivery, Drug target insights 2 (2007) 71-81.

[11] H.F. Helander, L. Fandriks, Surface area of the digestive tract - revisited, Scandinavian
journal of gastroenterology 49(6) (2014) 681-9 DOI: 10.3109/00365521.2014.898326.

[12] H. Clevers, The intestinal crypt, a prototype stem cell compartment, Cell 154(2) (2013)
274-84 DOI: 10.1016/j.cell.2013.07.004.

[13] B.H. Bajka, N.M. Rigby, K.L. Cross, A. Macierzanka, A.R. Mackie, The influence of
small intestinal mucus structure on particle transport ex vivo, Colloids and surfaces. B,
Biointerfaces 135 (2015) 73-80 DOI: 10.1016/j.colsurfb.2015.07.038.

35
[14] V. Strugala, P.W. Dettmar, J.P. Pearson, Thickness and continuity of the adherent
colonic mucus barrier in active and quiescent ulcerative colitis and Crohn's disease,
International journal of clinical practice 62(5) (2008) 762-9 DOI: 10.1111/j.1742-
1241.2007.01665.x.

[15] Y.S. Kim, S.B. Ho, Intestinal goblet cells and mucins in health and disease: recent
insights and progress, Current gastroenterology reports 12(5) (2010) 319-30 DOI:
10.1007/s11894-010-0131-2.

[16] N. Gassler, Paneth cells in intestinal physiology and pathophysiology, World journal of
gastrointestinal pathophysiology 8(4) (2017) 150-160 DOI: 10.4291/wjgp.v8.i4.150.

[17] Y. Yun, Y.W. Cho, K. Park, Nanoparticles for oral delivery: targeted nanoparticles with
peptidic ligands for oral protein delivery, Advanced drug delivery reviews 65(6) (2013) 822-
32 DOI: 10.1016/j.addr.2012.10.007.

of
[18] M. Shakweh, G. Ponchel, E. Fattal, Particle uptake by Peyer's patches: a pathway for

ro
drug and vaccine delivery, Expert opinion on drug delivery 1(1) (2004) 141-63 DOI:
10.1517/17425247.1.1.141.
-p
[19] J.A. Yanez, S.W. Wang, I.W. Knemeyer, M.A. Wirth, K.B. Alton, Intestinal lymphatic
transport for drug delivery, Advanced drug delivery reviews 63(10-11) (2011) 923-42 DOI:
re
10.1016/j.addr.2011.05.019.
lP

[20] R.S. Managuli, S.Y. Raut, M.S. Reddy, S. Mutalik, Targeting the intestinal lymphatic
system: a versatile path for enhanced oral bioavailability of drugs, Expert opinion on drug
delivery 15(8) (2018) 787-804 DOI: 10.1080/17425247.2018.1503249.
na

[21] N. Barker, J.H. van Es, J. Kuipers, P. Kujala, M. van den Born, M. Cozijnsen, A.
Haegebarth, J. Korving, H. Begthel, P.J. Peters, H. Clevers, Identification of stem cells in
ur

small intestine and colon by marker gene Lgr5, Nature 449(7165) (2007) 1003-7 DOI:
10.1038/nature06196.
Jo

[22] B. Lee, K.M. Moon, C.Y. Kim, Tight Junction in the Intestinal Epithelium: Its
Association with Diseases and Regulation by Phytochemicals, Journal of immunology
research 2018 (2018) 2645465 DOI: 10.1155/2018/2645465.

[23] H. Suzuki, T. Nishizawa, K. Tani, Y. Yamazaki, A. Tamura, R. Ishitani, N. Dohmae, S.

Tsukita, O. Nureki, Y. Fujiyoshi, Crystal structure of a claudin provides insight into the
architecture of tight junctions, Science 344(6181) (2014) 304-7 DOI:
10.1126/science.1248571.

[24] A.S. Fanning, C.M. Van Itallie, J.M. Anderson, Zonula occludens-1 and -2 regulate
apical cell structure and the zonula adherens cytoskeleton in polarized epithelia, Molecular
biology of the cell 23(4) (2012) 577-90 DOI: 10.1091/mbc.E11-09-0791.

[25] A. Taverner, R. Dondi, K. Almansour, F. Laurent, S.E. Owens, I.M. Eggleston, N.

Fotaki, R.J. Mrsny, Enhanced paracellular transport of insulin can be achieved via transient
induction of myosin light chain phosphorylation, Journal of controlled release : official
journal of the Controlled Release Society 210 (2015) 189-97 DOI:
10.1016/j.jconrel.2015.05.270.

36
[26] S. Tsukita, M. Furuse, M. Itoh, Multifunctional strands in tight junctions, Nat Rev Mol
Cell Biol 2(4) (2001) 285-93 DOI: 10.1038/35067088.

[27] O.R. Colegio, C.M. Van Itallie, H.J. McCrea, C. Rahner, J.M. Anderson, Claudins create
charge-selective channels in the paracellular pathway between epithelial cells, American
journal of physiology. Cell physiology 283(1) (2002) C142-7 DOI:
10.1152/ajpcell.00038.2002.

[28] K.J. Mandell, I.C. McCall, C.A. Parkos, Involvement of the junctional adhesion
molecule-1 (JAM1) homodimer interface in regulation of epithelial barrier function, J Biol
Chem 279(16) (2004) 16254-62 DOI: 10.1074/jbc.M309483200.

[29] J. Ikenouchi, M. Furuse, K. Furuse, H. Sasaki, S. Tsukita, S. Tsukita, Tricellulin

constitutes a novel barrier at tricellular contacts of epithelial cells, J Cell Biol 171(6) (2005)
939-45 DOI: 10.1083/jcb.200510043.

of
[30] A.P. Kowalczyk, K.J. Green, Structure, function, and regulation of desmosomes,

ro
Progress in molecular biology and translational science 116 (2013) 95-118 DOI:
10.1016/B978-0-12-394311-8.00005-4.
-p
[31] L. Shao, D. Serrano, L. Mayer, The role of epithelial cells in immune regulation in the
gut, Seminars in immunology 13(3) (2001) 163-76 DOI: 10.1006/smim.2000.0311.
re
[32] H.J. Lemmer, J.H. Hamman, Paracellular drug absorption enhancement through tight
lP

junction modulation, Expert Opin Drug Deliv 10(1) (2013) 103-14 DOI:
10.1517/17425247.2013.745509.
na

[33] M.E. Johansson, J.M. Larsson, G.C. Hansson, The two mucus layers of colon are
organized by the MUC2 mucin, whereas the outer layer is a legislator of host-microbial
interactions, Proc Natl Acad Sci U S A 108 Suppl 1 (2011) 4659-65 DOI:
ur

10.1073/pnas.1006451107.
Jo

[34] D.B. Subramani, M.E. Johansson, G. Dahlen, G.C. Hansson, Lactobacillus and
Bifidobacterium species do not secrete protease that cleaves the MUC2 mucin which
organises the colon mucus, Beneficial microbes 1(4) (2010) 343-50 DOI:
10.3920/BM2010.0039.

[35] J.F. Sicard, G. Le Bihan, P. Vogeleer, M. Jacques, J. Harel, Interactions of Intestinal

Bacteria with Components of the Intestinal Mucus, Frontiers in cellular and infection
microbiology 7 (2017) 387 DOI: 10.3389/fcimb.2017.00387.

[36] J. Leal, H.D.C. Smyth, D. Ghosh, Physicochemical properties of mucus and their impact
on transmucosal drug delivery, International journal of pharmaceutics 532(1) (2017) 555-572
DOI: 10.1016/j.ijpharm.2017.09.018.

[37] M. Boegh, H.M. Nielsen, Mucus as a barrier to drug delivery - understanding and
mimicking the barrier properties, Basic Clin Pharmacol Toxicol 116(3) (2015) 179-86 DOI:
10.1111/bcpt.12342.

[38] X. Murgia, P. Pawelzyk, U.F. Schaefer, C. Wagner, N. Willenbacher, C.M. Lehr, Size-
Limited Penetration of Nanoparticles into Porcine Respiratory Mucus after Aerosol
Deposition, Biomacromolecules 17(4) (2016) 1536-42 DOI: 10.1021/acs.biomac.6b00164.

37
[39] S. Schreiber, P. Scheid, Gastric mucus of the guinea pig: proton carrier and diffusion
barrier, Am J Physiol 272(1 Pt 1) (1997) G63-70 DOI: 10.1152/ajpgi.1997.272.1.G63.

[40] P. Langguth, V. Bohner, J. Heizmann, H.P. Merkle, S. Wolffram, G.L. Amidon, S.

Yamashita, The challenge of proteolytic enzymes in intestinal peptide delivery, Journal of
Controlled Release 46(1) (1997) 39-57 DOI: https://doi.org/10.1016/S0168-3659(96)01586-
6.

[41] X. Cao, R. Bansil, K.R. Bhaskar, B.S. Turner, J.T. LaMont, N. Niu, N.H. Afdhal, pH-
dependent conformational change of gastric mucin leads to sol-gel transition, Biophysical
journal 76(3) (1999) 1250-8 DOI: 10.1016/S0006-3495(99)77288-7.

[42] R. Shogren, T.A. Gerken, N. Jentoft, Role of glycosylation on the conformation and
chain dimensions of O-linked glycoproteins: light-scattering studies of ovine submaxillary
mucin, Biochemistry 28(13) (1989) 5525-36 DOI: 10.1021/bi00439a029.

of
[43] S.K. Lai, Y.Y. Wang, D. Wirtz, J. Hanes, Micro- and macrorheology of mucus, Adv

ro
Drug Deliv Rev 61(2) (2009) 86-100 DOI: 10.1016/j.addr.2008.09.012.

[44] R.S. Crowther, C. Marriott, S.L. James, Cation induced changes in the rheological
-p
properties of purified mucus glycoprotein gels, Biorheology 21(1-2) (1984) 253-63 DOI:
10.3233/bir-1984-211-227.
re
[45] K. Lavrijsen, D. van Dyck, J. van Houdt, J. Hendrickx, J. Monbaliu, R. Woestenborghs,
lP

W. Meuldermans, J. Heykants, Reduction of the prodrug loperamide oxide to its active drug
loperamide in the gut of rats, dogs, and humans, Drug Metab Dispos 23(3) (1995) 354-62.
na

[46] J. Lindenbaum, D.G. Rund, V.P. Butler, Jr., D. Tse-Eng, J.R. Saha, Inactivation of
digoxin by the gut flora: reversal by antibiotic therapy, N Engl J Med 305(14) (1981) 789-94
DOI: 10.1056/NEJM198110013051403.
ur

[47] J.R. Saha, V.P. Butler, Jr., H.C. Neu, J. Lindenbaum, Digoxin-inactivating bacteria:
Jo

identification in human gut flora, Science 220(4594) (1983) 325-7 DOI:

10.1126/science.6836275.

[48] H.J. Haiser, P.J. Turnbaugh, Developing a metagenomic view of xenobiotic metabolism,
Pharmacol Res 69(1) (2013) 21-31 DOI: 10.1016/j.phrs.2012.07.009.

[49] J. Zhang, J. Zhang, R. Wang, Gut microbiota modulates drug pharmacokinetics, Drug
Metab Rev 50(3) (2018) 357-368 DOI: 10.1080/03602532.2018.1497647.

[50] G. Camenisch, J. Alsenz, H. van de Waterbeemd, G. Folkers, Estimation of permeability

by passive diffusion through Caco-2 cell monolayers using the drugs' lipophilicity and
molecular weight, Eur J Pharm Sci 6(4) (1998) 317-24.

[51] H. Pham-The, I. Gonzalez-Alvarez, M. Bermejo, T. Garrigues, H. Le-Thi-Thu, M.A.

Cabrera-Perez, The Use of Rule-Based and QSPR Approaches in ADME Profiling: A Case
Study on Caco-2 Permeability, Molecular informatics 32(5-6) (2013) 459-79 DOI:
10.1002/minf.201200166.

[52] A.F. El-Kattan, Oral Bioavailability Assessment: Basics and Strategies for Drug
Discovery and Development, John Wiley & Sons2017.

38
[53] P. Ertl, B. Rohde, P. Selzer, Fast calculation of molecular polar surface area as a sum of
fragment-based contributions and its application to the prediction of drug transport properties,
J Med Chem 43(20) (2000) 3714-7 DOI: 10.1021/jm000942e.

[54] K. Palm, K. Luthman, A.L. Ungell, G. Strandlund, P. Artursson, Correlation of drug

absorption with molecular surface properties, J Pharm Sci 85(1) (1996) 32-9 DOI:
10.1021/js950285r.

[55] J. Kelder, P.D. Grootenhuis, D.M. Bayada, L.P. Delbressine, J.-P. Ploemen, Polar
molecular surface as a dominating determinant for oral absorption and brain penetration of
drugs, Pharmaceut Res 16(10) (1999) 1514-1519.

[56] B.C. Doak, B. Over, F. Giordanetto, J. Kihlberg, Oral druggable space beyond the rule
of 5: insights from drugs and clinical candidates, Chemistry & biology 21(9) (2014) 1115-42
DOI: 10.1016/j.chembiol.2014.08.013.

of
[57] M.J. Dolton, D.Z. D'Argenio, Population-based meta-analysis of roxithromycin

ro
pharmacokinetics: dosing implications of saturable absorption and protein binding, The
Journal of antimicrobial chemotherapy 72(4) (2017) 1129-1136 DOI: 10.1093/jac/dkw553.
-p
[58] M. Skinner, I. Kanfer, Comparative bioavailability of josamycin, a macrolide antibiotic,
from a tablet and solution and the influence of dissolution on in vivo release,
re
Biopharmaceutics & drug disposition 19(1) (1998) 21-9 DOI: 10.1002/(sici)1099-
081x(199801)19:1<21::aid-bdd69>3.0.co;2-g.
lP

[59] U. Loos, E. Musch, J.C. Jensen, G. Mikus, H.K. Schwabe, M. Eichelbaum,

Pharmacokinetics of oral and intravenous rifampicin during chronic administration, Klinische
na

Wochenschrift 63(23) (1985) 1205-11 DOI: 10.1007/BF01733779.

[60] V.K. Pawar, J.G. Meher, Y. Singh, M. Chaurasia, B. Surendar Reddy, M.K. Chourasia,
ur

Targeting of gastrointestinal tract for amended delivery of protein/peptide therapeutics:

strategies and industrial perspectives, J Control Release 196 (2014) 168-83 DOI:
Jo

10.1016/j.jconrel.2014.09.031.

[61] T.D. Brown, K.A. Whitehead, S. Mitragotri, Materials for oral delivery of proteins and
peptides, Nature Reviews Materials 5(2) (2020) 127-148 DOI: 10.1038/s41578-019-0156-6.

[62] M. Duran-Lobato, Z. Niu, M.J. Alonso, Oral Delivery of Biologics for Precision
Medicine, Adv Mater 32(13) (2020) e1901935 DOI: 10.1002/adma.201901935.

[63] E. Moroz, S. Matoori, J.C. Leroux, Oral delivery of macromolecular drugs: Where we
are after almost 100years of attempts, Adv Drug Deliv Rev 101 (2016) 108-121 DOI:
10.1016/j.addr.2016.01.010.

[64] V.J. Stella, Prodrug Approaches to Enhancing the Oral Delivery of Poorly Permeable
Drugs, in: V.J. Stella, R.T. Borchardt, M.J. Hageman, R. Oliyai, H. Maag, J.W. Tilley (Eds.),
Prodrugs: Challenges and Rewards Part 1, Springer New York, New York, NY, 2007, pp. 37-
82.

[65] K. Beaumont, R. Webster, I. Gardner, K. Dack, Design of ester prodrugs to enhance oral
absorption of poorly permeable compounds: challenges to the discovery scientist, Current
drug metabolism 4(6) (2003) 461-85 DOI: 10.2174/1389200033489253.

39
[66] M.F.G. Stevens, Chapter 5 - Temozolomide: From Cytotoxic to Molecularly Targeted
Agent, in: S. Neidle (Ed.), Cancer Drug Design and Discovery (Second Edition), Academic
Press, San Diego, 2014, pp. 145-164.

[67] K.C. Cundy, J.A. Fishback, J.P. Shaw, M.L. Lee, K.F. Soike, G.C. Visor, W.A. Lee,
Oral bioavailability of the antiretroviral agent 9-(2-phosphonylmethoxyethyl)adenine
(PMEA) from three formulations of the prodrug bis(pivaloyloxymethyl)-PMEA in fasted
male cynomolgus monkeys, Pharm Res 11(6) (1994) 839-43 DOI:
10.1023/a:1018925723889.

[68] T. Dando, G. Plosker, Adefovir dipivoxil: a review of its use in chronic hepatitis B,
Drugs 63(20) (2003) 2215-34 DOI: 10.2165/00003495-200363200-00007.

[69] J. Liu, J. Liu, D.J. Zhao, N.X. Ma, Y.X. Luan, Highly enhanced leukemia therapy and
oral bioavailability from a novel amphiphilic prodrug of cytarabine, Rsc Advances 6(42)

of
(2016) 35991-35999 DOI: 10.1039/c6ra02051h.

ro
[70] M. Kandula, K.B. Sunil Kumar, S. Palanichamy, A. Rampal, Discovery and preclinical
development of a novel prodrug conjugate of mesalamine with eicosapentaenoic acid and
caprylic acid for the treatment of inflammatory bowel diseases, International
-p
immunopharmacology 40 (2016) 443-451 DOI: 10.1016/j.intimp.2016.09.013.
re
[71] M.V. Varma, C.M. Ambler, M. Ullah, C.J. Rotter, H. Sun, J. Litchfield, K.S. Fenner,
A.F. El-Kattan, Targeting intestinal transporters for optimizing oral drug absorption, Current
lP

drug metabolism 11(9) (2010) 730-42 DOI: 10.2174/138920010794328850.

[72] B. Steffansen, C.U. Nielsen, B. Brodin, A.H. Eriksson, R. Andersen, S. Frokjaer,

na

Intestinal solute carriers: an overview of trends and strategies for improving oral drug
absorption, European journal of pharmaceutical sciences : official journal of the European
Federation for Pharmaceutical Sciences 21(1) (2004) 3-16 DOI: 10.1016/j.ejps.2003.10.010.
ur

[73] S. Broer, S.J. Fairweather, Amino Acid Transport Across the Mammalian Intestine,
Jo

Comprehensive Physiology 9(1) (2018) 343-373 DOI: 10.1002/cphy.c170041.

[74] L.M. Beauchamp, G.F. Orr, P. de Miranda, T. Bumette, T.A. Krenitsky, Amino Acid
Ester Prodrugs of Acyclovir, 3(3) (1992) 157-164 DOI: 10.1177/095632029200300305.

[75] R. Lal, J. Sukbuntherng, W. Luo, V. Vicente, R. Blumenthal, J. Ho, K.C. Cundy,

Clinical pharmacokinetic drug interaction studies of gabapentin enacarbil, a novel transported
prodrug of gabapentin, with naproxen and cimetidine, British journal of clinical
pharmacology 69(5) (2010) 498-507 DOI: 10.1111/j.1365-2125.2010.03616.x.

[76] D. Swearingen, G.M. Aronoff, S. Ciric, R. Lal, Pharmacokinetics of immediate release,

extended release, and gastric retentive gabapentin formulations in healthy adults,
International journal of clinical pharmacology and therapeutics 56(5) (2018) 231-238 DOI:
10.5414/CP203166.

[77] B.J. Aungst, Intestinal permeation enhancers, J Pharm Sci 89(4) (2000) 429-42 DOI:
10.1002/(SICI)1520-6017(200004)89:4<429::AID-JPS1>3.0.CO;2-J.

40
[78] S. Maher, R.J. Mrsny, D.J. Brayden, Intestinal permeation enhancers for oral peptide
delivery, Advanced drug delivery reviews 106(Pt B) (2016) 277-319 DOI:
10.1016/j.addr.2016.06.005.

[79] S. Maher, D.J. Brayden, L. Casettari, L. Illum, Application of Permeation Enhancers in

Oral Delivery of Macromolecules: An Update, Pharmaceutics 11(1) (2019) DOI:
10.3390/pharmaceutics11010041.

[80] O. Zupancic, A. Bernkop-Schnurch, Lipophilic peptide character - What oral barriers

fear the most, Journal of controlled release : official journal of the Controlled Release Society
255 (2017) 242-257 DOI: 10.1016/j.jconrel.2017.04.038.

[81] L. Gonzalez-Mariscal, S. Hernandez, J. Vega, Inventions designed to enhance drug

delivery across epithelial and endothelial cells through the paracellular pathway, Recent
patents on drug delivery & formulation 2(2) (2008) 145-76 DOI:

of
10.2174/187221108784534117.

ro
[82] T. Lindmark, N. Schipper, L. Lazorova, A.G. de Boer, P. Artursson, Absorption
enhancement in intestinal epithelial Caco-2 monolayers by sodium caprate: Assessment of
molecular weight dependence and demonstration of transport routes, J Drug Target 5(3)
-p
(1998) 215-223 DOI: Doi 10.3109/10611869808995876.
re
[83] T. Lindmark, Y. Kimura, P. Artursson, Absorption enhancement through intracellular
regulation of tight junction permeability by medium chain fatty acids in Caco-2 cells, The
lP

Journal of pharmacology and experimental therapeutics 284(1) (1998) 362-9.

[84] M. Tomita, M. Hayashi, S. Awazu, Absorption-enhancing mechanism of EDTA,

na

caprate, and decanoylcarnitine in Caco-2 cells, Journal of Pharmaceutical Sciences 85(6)

(1996) 608-611 DOI: Doi 10.1021/Js9504604.
ur

[85] T. Lindmark, N. Schipper, L. Lazorova, A.G. de Boer, P. Artursson, Absorption

enhancement in intestinal epithelial Caco-2 monolayers by sodium caprate: assessment of
Jo

molecular weight dependence and demonstration of transport routes, Journal of drug targeting
5(3) (1998) 215-23 DOI: 10.3109/10611869808995876.

[86] T. Lindmark, J.D. Soderholm, G. Olaison, G. Alvan, G. Ocklind, P. Artursson,

Mechanism of absorption enhancement in humans after rectal administration of ampicillin in
suppositories containing sodium caprate, Pharmaceut Res 14(7) (1997) 930-935 DOI: Doi
10.1023/A:1012112219578.

[87] T.W. Leonard, J. Lynch, M.J. McKenna, D.J. Brayden, Promoting absorption of drugs in
humans using medium-chain fatty acid-based solid dosage forms: GIPET, Expert opinion on
drug delivery 3(5) (2006) 685-92 DOI: 10.1517/17425247.3.5.685.

[88] S. Tuvia, D. Pelled, K. Marom, P. Salama, M. Levin-Arama, I. Karmeli, G.H. Idelson, I.

Landau, R. Mamluk, A novel suspension formulation enhances intestinal absorption of
macromolecules via transient and reversible transport mechanisms, Pharmaceutical research
31(8) (2014) 2010-21 DOI: 10.1007/s11095-014-1303-9.

[89] A. Leone-Bay, N. Santiago, D. Achan, K. Chaudhary, F. DeMorin, L. Falzarano, S.

Haas, S. Kalbag, D. Kaplan, H. Leipold, et al., N-acylated alpha-amino acids as novel oral

41
delivery agents for proteins, Journal of medicinal chemistry 38(21) (1995) 4263-9 DOI:
10.1021/jm00021a015.

[90] A. Leone-Bay, C. McInnes, N. Wang, F. DeMorin, D. Achan, C. Lercara, D. Sarubbi, S.

Haas, J. Press, E. Barantsevich, et al., Microsphere formation in a series of derivatized alpha-
amino acids: properties, molecular modeling, and oral delivery of salmon calcitonin, Journal
of medicinal chemistry 38(21) (1995) 4257-62 DOI: 10.1021/jm00021a014.

[91] A.W. Alani, J.R. Robinson, Mechanistic understanding of oral drug absorption
enhancement of cromolyn sodium by an amino acid derivative, Pharmaceutical research
25(1) (2008) 48-54 DOI: 10.1007/s11095-007-9438-6.

[92] M.C. Castelli, K. Friedman, J. Sherry, K. Brazzillo, L. Genoble, P. Bhargava, M.G.

Riley, Comparing the efficacy and tolerability of a new daily oral vitamin B12 formulation
and intermittent intramuscular vitamin B12 in normalizing low cobalamin levels: a

of
randomized, open-label, parallel-group study, Clinical therapeutics 33(3) (2011) 358-371 e2
DOI: 10.1016/j.clinthera.2011.03.003.

ro
[93] M. Tomita, M. Hayashi, S. Awazu, Absorption-enhancing mechanism of EDTA,
caprate, and decanoylcarnitine in Caco-2 cells, J Pharm Sci 85(6) (1996) 608-11 DOI:
-p
10.1021/js9504604.
re
[94] A.I. Ivanov, S.N. Samarin, M. Bachar, C.A. Parkos, A. Nusrat, Protein kinase C
activation disrupts epithelial apical junctions via ROCK-II dependent stimulation of
lP

actomyosin contractility, BMC cell biology 10 (2009) 36 DOI: 10.1186/1471-2121-10-36.

[95] C.B. Collares-Buzato, G.T. McEwan, M.A. Jepson, N.L. Simmons, B.H. Hirst,
na

Paracellular barrier and junctional protein distribution depend on basolateral extracellular

Ca2+ in cultured epithelia, Biochimica et biophysica acta 1222(2) (1994) 147-58 DOI:
10.1016/0167-4889(94)90163-5.
ur

[96] R. Eldor, E. Arbit, A. Corcos, M. Kidron, Glucose-reducing effect of the ORMD-0801

Jo

oral insulin preparation in patients with uncontrolled type 1 diabetes: a pilot study, PloS one
8(4) (2013) e59524 DOI: 10.1371/journal.pone.0059524.

[97] N. Doi, M. Tomita, M. Hayashi, Absorption Enhancement Effect of Acylcarnitines

through Changes in Tight Junction Protein in Caco-2 Cell Monolayers, Drug Metabolism and
Pharmacokinetics 26(2) (2011) 162-170 DOI: https://doi.org/10.2133/dmpk.DMPK-10-RG-
071.

[98] N. Binkley, M. Bolognese, A. Sidorowicz-Bialynicka, T. Vally, R. Trout, C. Miller, C.E.

Buben, J.P. Gilligan, D.S. Krause, I. Oral Calcitonin in Postmenopausal Osteoporosis, A
phase 3 trial of the efficacy and safety of oral recombinant calcitonin: the Oral Calcitonin in
Postmenopausal Osteoporosis (ORACAL) trial, Journal of bone and mineral research : the
official journal of the American Society for Bone and Mineral Research 27(8) (2012) 1821-9
DOI: 10.1002/jbmr.1602.

[99] E.S. Swenson, W.B. Milisen, W. Curatolo, Intestinal permeability enhancement:

efficacy, acute local toxicity, and reversibility, Pharmaceutical research 11(8) (1994) 1132-42
DOI: 10.1023/a:1018984731584.

42
[100] J. Jiao, Polyoxyethylated nonionic surfactants and their applications in topical ocular
drug delivery, Adv Drug Deliv Rev 60(15) (2008) 1663-73 DOI: 10.1016/j.addr.2008.09.002.

[101] K. Sakai, T.M. Kutsuna, T. Nishino, Y. Fujihara, N. Yata, Contribution of calcium ion
sequestration by polyoxyethylated nonionic surfactants to the enhanced colonic absorption of
p-aminobenzoic acid, J Pharm Sci 75(4) (1986) 387-90 DOI: 10.1002/jps.2600750414.

[102] D.R. Perinelli, S. Lucarini, L. Fagioli, R. Campana, D. Vllasaliu, A. Duranti, L.

Casettari, Lactose oleate as new biocompatible surfactant for pharmaceutical applications,
Eur J Pharm Biopharm 124 (2018) 55-62 DOI: 10.1016/j.ejpb.2017.12.008.

[103] F. McCartney, V. Jannin, S. Chevrier, H. Boulghobra, D.R. Hristov, N. Ritter, C.

Miolane, Y. Chavant, F. Demarne, D.J. Brayden, Labrasol(R) is an efficacious intestinal
permeation enhancer across rat intestine: Ex vivo and in vivo rat studies, Journal of
controlled release : official journal of the Controlled Release Society 310 (2019) 115-126

of
DOI: 10.1016/j.jconrel.2019.08.008.

ro
[104] S. Mori, A. Matsuura, Y.V. Rama Prasad, K. Takada, Studies on the intestinal
absorption of low molecular weight heparin using saturated fatty acids and their derivatives
as an absorption enhancer in rats, Biological & pharmaceutical bulletin 27(3) (2004) 418-21
-p
DOI: 10.1248/bpb.27.418.
re
[105] N. Venkatesan, K. Uchino, K. Amagase, Y. Ito, N. Shibata, K. Takada, Gastro-
intestinal patch system for the delivery of erythropoietin, Journal of controlled release :
lP

official journal of the Controlled Release Society 111(1-2) (2006) 19-26 DOI:
10.1016/j.jconrel.2005.11.009.
na

[106] K.A. Hamid, H. Katsumi, T. Sakane, A. Yamamoto, The effects of common

solubilizing agents on the intestinal membrane barrier functions and membrane toxicity in
rats, International journal of pharmaceutics 379(1) (2009) 100-8 DOI:
ur

10.1016/j.ijpharm.2009.06.018.
Jo

[107] E.M. Danielsen, G.H. Hansen, Intestinal surfactant permeation enhancers and their
interaction with enterocyte cell membranes in a mucosal explant system, Tissue barriers 5(3)
(2017) e1361900 DOI: 10.1080/21688370.2017.1361900.

[108] J.M. Ridlon, D.J. Kang, P.B. Hylemon, Bile salt biotransformations by human
intestinal bacteria, J Lipid Res 47(2) (2006) 241-259 DOI: 10.1194/jlr.R500013-JLR200.

[109] N. Pavlovic, S. Golocorbin-Kon, M. Ethanic, B. Stanimirov, H. Al-Salami, K. Stankov,

M. Mikov, Bile Acids and Their Derivatives as Potential Modifiers of Drug Release and
Pharmacokinetic Profiles, Frontiers in pharmacology 9 (2018) 1283 DOI:
10.3389/fphar.2018.01283.

[110] M. Stojančević, N. Pavlović, S. Goločorbin-Kon, M. Mikov, Application of bile acids

in drug formulation and delivery, Frontiers in Life Science 7(3-4) (2013) 112-122.

[111] R. Mazzanti, O. Fantappie, Y. Kamimoto, Z. Gatmaitan, P. Gentilini, I.M. Arias, Bile

acid inhibition of P-glycoprotein-mediated transport in multidrug-resistant cells and rat liver
canalicular membrane vesicles, Hepatology 20(1 Pt 1) (1994) 170-6 DOI: 10.1016/0270-
9139(94)90150-3.

43
[112] E. Moghimipour, A. Ameri, S. Handali, Absorption-Enhancing Effects of Bile Salts,
Molecules 20(8) (2015) 14451-14473 DOI: 10.3390/molecules200814451.

[113] K.H. Song, S.J. Chung, C.K. Shim, Enhanced intestinal absorption of salmon calcitonin
(sCT) from proliposomes containing bile salts, J Control Release 106(3) (2005) 298-308
DOI: 10.1016/j.jconrel.2005.05.016.

[114] A. Gaowa, T. Horibe, M. Kohno, K. Kawakami, Bile acid as an effective absorption

enhancer for oral delivery of epidermal growth factor receptor–targeted hybrid peptide,
Journal of pharmaceutical sciences 107(5) (2018) 1322-1329.

[115] S. Lee, S.K. Kim, D.Y. Lee, K. Park, T.S. Kumar, S.Y. Chae, Y. Byun, Cationic analog
of deoxycholate as an oral delivery carrier for ceftriaxone, Journal of Pharmaceutical
Sciences 94(11) (2005) 2541-2548 DOI: 10.1002/jps.20478.

of
[116] S.D. Luzio, G. Dunseath, A. Lockett, T.P. Broke-Smith, R.R. New, D.R. Owens, The
glucose lowering effect of an oral insulin (Capsulin) during an isoglycaemic clamp study in

ro
persons with type 2 diabetes, Diabetes, obesity & metabolism 12(1) (2010) 82-7 DOI:
10.1111/j.1463-1326.2009.01146.x.
-p
[117] A. Khedkar, H. Lebovitz, A. Fleming, A. Cherrington, V. Jose, S.N. Athalye, A.
Vishweswaramurthy, Pharmacokinetics and Pharmacodynamics of Insulin Tregopil in
re
Relation to Premeal Dosing Time, Between Meal Interval, and Meal Composition in Patients
With Type 2 Diabetes Mellitus, Clinical pharmacology in drug development 9(1) (2020) 74-
lP

86 DOI: 10.1002/cpdd.730.

[118] A. Khedkar, H. Iyer, A. Anand, M. Verma, S. Krishnamurthy, S. Savale, A. Atignal, A

na

dose range finding study of novel oral insulin (IN-105) under fed conditions in type 2
diabetes mellitus subjects, Diabetes, obesity & metabolism 12(8) (2010) 659-64 DOI:
10.1111/j.1463-1326.2010.01213.x.
ur

[119] T.M.M. Ways, W.M. Lau, V.V. Khutoryanskiy, Chitosan and Its Derivatives for
Jo

Application in Mucoadhesive Drug Delivery Systems, Polymers-Basel 10(3) (2018) DOI:

Artn 267
10.3390/Polym10030267.

[120] M. Thanou, J.C. Verhoef, H.E. Junginger, Oral drug absorption enhancement by
chitosan and its derivatives, Advanced drug delivery reviews 52(2) (2001) 117-26 DOI:
10.1016/s0169-409x(01)00231-9.

[121] M.W. TM, W.M. Lau, V.V. Khutoryanskiy, Chitosan and Its Derivatives for
Application in Mucoadhesive Drug Delivery Systems, Polymers 10(3) (2018) DOI:
10.3390/polym10030267.

[122] G. Ranaldi, I. Marigliano, I. Vespignani, G. Perozzi, Y. Sambuy, The effect of chitosan

and other polycations on tight junction permeability in the human intestinal Caco-2 cell
line(1), The Journal of nutritional biochemistry 13(3) (2002) 157-167 DOI: 10.1016/s0955-
2863(01)00208-x.

[123] R. Rosenthal, D. Gunzel, C. Finger, S.M. Krug, J.F. Richter, J.D. Schulzke, M. Fromm,
S. Amasheh, The effect of chitosan on transcellular and paracellular mechanisms in the

44
intestinal epithelial barrier, Biomaterials 33(9) (2012) 2791-800 DOI:
10.1016/j.biomaterials.2011.12.034.

[124] I. Kadiyala, Y. Loo, K. Roy, J. Rice, K.W. Leong, Transport of chitosan-DNA

nanoparticles in human intestinal M-cell model versus normal intestinal enterocytes,
European journal of pharmaceutical sciences : official journal of the European Federation for
Pharmaceutical Sciences 39(1-3) (2010) 103-9 DOI: 10.1016/j.ejps.2009.11.002.

[125] B. Bellich, I. D'Agostino, S. Semeraro, A. Gamini, A. Cesaro, "The Good, the Bad and
the Ugly" of Chitosans, Mar Drugs 14(5) (2016) DOI: Artn 99
10.3390/Md14050099.

[126] M. Thanou, J.C. Verhoef, H.E. Junginger, Chitosan and its derivatives as intestinal
absorption enhancers, Adv Drug Deliv Rev 50 Suppl 1 (2001) S91-101 DOI: 10.1016/s0169-
409x(01)00180-6.

of
[127] A.F. Kotze, M.M. Thanou, H.L. Luessen, B.G. de Boer, J.C. Verhoef, H.E. Junginger,

ro
Effect of the degree of quaternization of N-trimethyl chitosan chloride on the permeability of
intestinal epithelial cells (Caco-2), European journal of pharmaceutics and biopharmaceutics :
-p
official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 47(3)
(1999) 269-74 DOI: 10.1016/s0939-6411(99)00006-5.
re
[128] H.L. Luessen, C.O. Rentel, A.F. Kotze, C.M. Lehr, A.G. deBoer, J.C. Verhoef, H.E.
lP

Junginger, Mucoadhesive polymers in peroral peptide drug delivery .4. Polycarbophil and
chitosan are potent enhancers of peptide transport across intestinal mucosae in vitro, J
Control Release 45(1) (1997) 15-23 DOI: Doi 10.1016/S0168-3659(96)01536-2.
na

[129] G. Borchard, H.L. Luessen, A.G. deBoer, J.C. Verhoef, C.M. Lehr, H.E. Junginger,
The potential of mucoadhesive polymers in enhancing intestinal peptide drug absorption .3.
ur

Effects of chitosan-glutamate and carbomer on epithelial tight junctions in vitro, Journal of

Controlled Release 39(2-3) (1996) 131-138 DOI: Doi 10.1016/0168-3659(95)00146-8.
Jo

[130] K. Maisel, M. Reddy, Q. Xu, S. Chattopadhyay, R. Cone, L.M. Ensign, J. Hanes,

Nanoparticles coated with high molecular weight PEG penetrate mucus and provide uniform
vaginal and colorectal distribution in vivo, Nanomedicine (Lond) 11(11) (2016) 1337-43
DOI: 10.2217/nnm-2016-0047.

[131] Y.Y. Wang, S.K. Lai, J.S. Suk, A. Pace, R. Cone, J. Hanes, Addressing the PEG
mucoadhesivity paradox to engineer nanoparticles that "slip" through the human mucus
barrier, Angewandte Chemie 47(50) (2008) 9726-9 DOI: 10.1002/anie.200803526.

[132] L. Mahlert, J. Anderski, D. Mulac, K. Langer, The impact of gastrointestinal mucus on

nanoparticle penetration - in vitro evaluation of mucus-penetrating nanoparticles for
photodynamic therapy, European journal of pharmaceutical sciences : official journal of the
European Federation for Pharmaceutical Sciences 133 (2019) 28-39 DOI:
10.1016/j.ejps.2019.03.010.

[133] H. Chen, E.D.H. Mansfield, A. Woods, V.V. Khutoryanskiy, B. Forbes, S.A. Jones,
Mucus penetrating properties of soft, distensible lipid nanocapsules, European journal of
pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur
Pharmazeutische Verfahrenstechnik e.V 139 (2019) 76-84 DOI: 10.1016/j.ejpb.2019.02.020.

45
[134] S. Vrignaud, J.P. Benoit, P. Saulnier, Strategies for the nanoencapsulation of
hydrophilic molecules in polymer-based nanoparticles, Biomaterials 32(33) (2011) 8593-604
DOI: 10.1016/j.biomaterials.2011.07.057.

[135] M. Boegh, H.M. Nielsen, Mucus as a Barrier to Drug Delivery – Understanding and
Mimicking the Barrier Properties, Basic & Clinical Pharmacology & Toxicology 116(3)
(2015) 179-186 DOI: 10.1111/bcpt.12342.

[136] J. Reinholz, K. Landfester, V. Mailander, The challenges of oral drug delivery via
nanocarriers, Drug delivery 25(1) (2018) 1694-1705 DOI: 10.1080/10717544.2018.1501119.

[137] A.M. Bannunah, D. Vllasaliu, J. Lord, S. Stolnik, Mechanisms of nanoparticle

internalization and transport across an intestinal epithelial cell model: effect of size and
surface charge, Molecular pharmaceutics 11(12) (2014) 4363-73 DOI: 10.1021/mp500439c.

of
[138] X.J. Du, J.L. Wang, S. Iqbal, H.J. Li, Z.T. Cao, Y.C. Wang, J.Z. Du, J. Wang, The
effect of surface charge on oral absorption of polymeric nanoparticles, Biomaterials science

ro
6(3) (2018) 642-650 DOI: 10.1039/c7bm01096f.

[139] Y. Zhao, Y. Wang, F. Ran, Y. Cui, C. Liu, Q. Zhao, Y. Gao, D. Wang, S. Wang, A
-p
comparison between sphere and rod nanoparticles regarding their in vivo biological behavior
and pharmacokinetics, Scientific reports 7(1) (2017) 4131 DOI: 10.1038/s41598-017-03834-
re
2.
lP

[140] F. Xia, W. Fan, S. Jiang, Y. Ma, Y. Lu, J. Qi, E. Ahmad, X. Dong, W. Zhao, W. Wu,
Size-Dependent Translocation of Nanoemulsions via Oral Delivery, ACS applied materials &
interfaces 9(26) (2017) 21660-21672 DOI: 10.1021/acsami.7b04916.
na

[141] A. Banerjee, J. Qi, R. Gogoi, J. Wong, S. Mitragotri, Role of nanoparticle size, shape
and surface chemistry in oral drug delivery, Journal of controlled release : official journal of
ur

the Controlled Release Society 238 (2016) 176-185 DOI: 10.1016/j.jconrel.2016.07.051.

Jo

[142] E. Roger, S. Kalscheuer, A. Kirtane, B.R. Guru, A.E. Grill, J. Whittum-Hudson, J.

Panyam, Folic acid functionalized nanoparticles for enhanced oral drug delivery, Molecular
pharmaceutics 9(7) (2012) 2103-10 DOI: 10.1021/mp2005388.

[143] E.M. Pridgen, F. Alexis, T.T. Kuo, E. Levy-Nissenbaum, R. Karnik, R.S. Blumberg, R.
Langer, O.C. Farokhzad, Transepithelial transport of Fc-targeted nanoparticles by the
neonatal fc receptor for oral delivery, Science translational medicine 5(213) (2013) 213ra167
DOI: 10.1126/scitranslmed.3007049.

[144] A. Makhlof, S. Fujimoto, Y. Tozuka, H. Takeuchi, In vitro and in vivo evaluation of

WGA-carbopol modified liposomes as carriers for oral peptide delivery, European journal of
pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur
Pharmazeutische Verfahrenstechnik e.V 77(2) (2011) 216-24 DOI:
10.1016/j.ejpb.2010.12.008.

[145] L. Han, C. Tang, C. Yin, Oral delivery of shRNA and siRNA via multifunctional
polymeric nanoparticles for synergistic cancer therapy, Biomaterials 35(15) (2014) 4589-600
DOI: 10.1016/j.biomaterials.2014.02.027.

46
[146] C. He, L. Yin, C. Tang, C. Yin, Multifunctional polymeric nanoparticles for oral
delivery of TNF-alpha siRNA to macrophages, Biomaterials 34(11) (2013) 2843-54 DOI:
10.1016/j.biomaterials.2013.01.033.

[147] T. Fan, C. Chen, H. Guo, J. Xu, J. Zhang, X. Zhu, Y. Yang, Z. Zhou, L. Li, Y. Huang,
Design and evaluation of solid lipid nanoparticles modified with peptide ligand for oral
delivery of protein drugs, European journal of pharmaceutics and biopharmaceutics : official
journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 88(2) (2014) 518-
28 DOI: 10.1016/j.ejpb.2014.06.011.

[148] A. Beloqui, M.A. Solinis, A.R. Gascon, A. del Pozo-Rodriguez, A. des Rieux, V. Preat,
Mechanism of transport of saquinavir-loaded nanostructured lipid carriers across the
intestinal barrier, Journal of controlled release : official journal of the Controlled Release
Society 166(2) (2013) 115-23 DOI: 10.1016/j.jconrel.2012.12.021.

of
[149] M.D. Neal, C. Leaphart, R. Levy, J. Prince, T.R. Billiar, S. Watkins, J. Li, S. Cetin, H.
Ford, A. Schreiber, D.J. Hackam, Enterocyte TLR4 mediates phagocytosis and translocation

ro
of bacteria across the intestinal barrier, Journal of immunology 176(5) (2006) 3070-9 DOI:
10.4049/jimmunol.176.5.3070.
-p
[150] R.M. Samstein, K. Perica, F. Balderrama, M. Look, T.M. Fahmy, The use of
deoxycholic acid to enhance the oral bioavailability of biodegradable nanoparticles,
re
Biomaterials 29(6) (2008) 703-708.
lP

[151] W. Fan, D. Xia, Q. Zhu, X. Li, S. He, C. Zhu, S. Guo, L. Hovgaard, M. Yang, Y. Gan,
Functional nanoparticles exploit the bile acid pathway to overcome multiple barriers of the
intestinal epithelium for oral insulin delivery, Biomaterials 151 (2018) 13-23 DOI:
na

10.1016/j.biomaterials.2017.10.022.

[152] T. Sousa, R.E. Castro, S.N. Pinto, A. Coutinho, S.D. Lucas, R. Moreira, C.M.
ur

Rodrigues, M. Prieto, F. Fernandes, Deoxycholic acid modulates cell death signaling through
changes in mitochondrial membrane properties, Journal of lipid research 56(11) (2015) 2158-
Jo

71 DOI: 10.1194/jlr.M062653.

[153] K.S. Kim, K. Suzuki, H. Cho, Y.S. Youn, Y.H. Bae, Oral Nanoparticles Exhibit
Specific High-Efficiency Intestinal Uptake and Lymphatic Transport, ACS nano 12(9) (2018)
8893-8900 DOI: 10.1021/acsnano.8b04315.

[154] T. Xia, M. Kovochich, M. Liong, J.I. Zink, A.E. Nel, Cationic polystyrene nanosphere
toxicity depends on cell-specific endocytic and mitochondrial injury pathways, ACS nano
2(1) (2008) 85-96 DOI: 10.1021/nn700256c.

[155] R.G. Parton, K. Simons, The multiple faces of caveolae, Nature reviews. Molecular cell
biology 8(3) (2007) 185-94 DOI: 10.1038/nrm2122.

[156] J. Sottile, J. Chandler, Fibronectin matrix turnover occurs through a caveolin-1-

dependent process, Molecular biology of the cell 16(2) (2005) 757-68 DOI:
10.1091/mbc.e04-08-0672.

[157] E.S. Seto, H.J. Bellen, T.E. Lloyd, When cell biology meets development: endocytic
regulation of signaling pathways, Genes & development 16(11) (2002) 1314-36 DOI:
10.1101/gad.989602.
47
[158] L.K. Medina-Kauwe, "Alternative" endocytic mechanisms exploited by pathogens: new
avenues for therapeutic delivery?, Advanced drug delivery reviews 59(8) (2007) 798-809
DOI: 10.1016/j.addr.2007.06.009.

[159] M.A. Yousuf, X. Zhou, S. Mukherjee, A.V. Chintakuntlawar, J.Y. Lee, M. Ramke, J.
Chodosh, J. Rajaiya, Caveolin-1 associated adenovirus entry into human corneal cells, PloS
one 8(10) (2013) e77462 DOI: 10.1371/journal.pone.0077462.

[160] W.I. Lencer, T.R. Hirst, R.K. Holmes, Membrane traffic and the cellular uptake of
cholera toxin, Biochimica et biophysica acta 1450(3) (1999) 177-90 DOI: 10.1016/s0167-
4889(99)00070-1.

[161] L. Pelkmans, J. Kartenbeck, A. Helenius, Caveolar endocytosis of simian virus 40

reveals a new two-step vesicular-transport pathway to the ER, Nature cell biology 3(5) (2001)
473-83 DOI: 10.1038/35074539.

of
[162] Z. Wang, C. Tiruppathi, J. Cho, R.D. Minshall, A.B. Malik, Delivery of nanoparticle:

ro
complexed drugs across the vascular endothelial barrier via caveolae, IUBMB life 63(8)
(2011) 659-67 DOI: 10.1002/iub.485.
-p
[163] Z. Wang, C. Tiruppathi, R.D. Minshall, A.B. Malik, Size and dynamics of caveolae
studied using nanoparticles in living endothelial cells, ACS nano 3(12) (2009) 4110-6 DOI:
re
10.1021/nn9012274.
lP

[164] S.A. Predescu, D.N. Predescu, K. Shimizu, I.K. Klein, A.B. Malik, Cholesterol-
dependent syntaxin-4 and SNAP-23 clustering regulates caveolar fusion with the endothelial
plasma membrane, The Journal of biological chemistry 280(44) (2005) 37130-8 DOI:
na

10.1074/jbc.M505659200.

[165] B. He, Z. Jia, W. Du, C. Yu, Y. Fan, W. Dai, L. Yuan, H. Zhang, X. Wang, J. Wang, X.
ur

Zhang, Q. Zhang, The transport pathways of polymer nanoparticles in MDCK epithelial cells,
Biomaterials 34(17) (2013) 4309-26 DOI: 10.1016/j.biomaterials.2013.01.100.
Jo

[166] J.A. Wang, T.F. Meyer, T. Rudel, Cytoskeleton and motor proteins are required for the
transcytosis of Neisseria gonorrhoeae through polarized epithelial cells, International journal
of medical microbiology : IJMM 298(3-4) (2008) 209-21 DOI: 10.1016/j.ijmm.2007.05.004.

[167] L. Rajendran, H.J. Knolker, K. Simons, Subcellular targeting strategies for drug design
and delivery, Nature reviews. Drug discovery 9(1) (2010) 29-42 DOI: 10.1038/nrd2897.

[168] T. Fujiwara, H. Akita, H. Harashima, Intracellular fate of octaarginine-modified

liposomes in polarized MDCK cells, Int J Pharm 386(1-2) (2010) 122-30 DOI:
10.1016/j.ijpharm.2009.11.005.

[169] R.P. Patel, P. Shah, K. Barve, N. Patel, J. Gandhi, Peyer’s Patch: Targeted Drug
Delivery for Therapeutics Benefits, in: A. Misra, A. Shahiwala (Eds.), Novel Drug Delivery
Technologies: Innovative Strategies for Drug Re-positioning, Springer Singapore, Singapore,
2019, pp. 121-149.

[170] E. Gullberg, A.V. Keita, S.Y. Salim, M. Andersson, K.D. Caldwell, J.D. Soderholm, P.
Artursson, Identification of cell adhesion molecules in the human follicle-associated
epithelium that improve nanoparticle uptake into the Peyer's patches, The Journal of

48
pharmacology and experimental therapeutics 319(2) (2006) 632-9 DOI:
10.1124/jpet.106.107847.

[171] I.M. van der Lubben, J.C. Verhoef, A.C. van Aelst, G. Borchard, H.E. Junginger,
Chitosan microparticles for oral vaccination: preparation, characterization and preliminary in
vivo uptake studies in murine Peyer's patches, Biomaterials 22(7) (2001) 687-94 DOI:
10.1016/s0142-9612(00)00231-3.

[172] Y. Liu, Z. Jiang, X. Hou, X. Xie, J. Shi, J. Shen, Y. He, Z. Wang, N. Feng, Functional
lipid polymeric nanoparticles for oral drug delivery: Rapid mucus penetration and improved
cell entry and cellular transport, Nanomedicine : nanotechnology, biology, and medicine 21
(2019) 102075 DOI: 10.1016/j.nano.2019.102075.

[173] M.K. Yoo, S.K. Kang, J.H. Choi, I.K. Park, H.S. Na, H.C. Lee, E.B. Kim, N.K. Lee,
J.W. Nah, Y.J. Choi, C.S. Cho, Targeted delivery of chitosan nanoparticles to Peyer's patch

of
using M cell-homing peptide selected by phage display technique, Biomaterials 31(30)
(2010) 7738-47 DOI: 10.1016/j.biomaterials.2010.06.059.

ro
[174] U. Bulbake, S. Doppalapudi, N. Kommineni, W. Khan, Liposomal Formulations in
Clinical Use: An Updated Review, Pharmaceutics 9(2) (2017)
-p DOI:
10.3390/pharmaceutics9020012.
re
[175] S. Hu, M. Niu, F. Hu, Y. Lu, J. Qi, Z. Yin, W. Wu, Integrity and stability of oral
liposomes containing bile salts studied in simulated and ex vivo gastrointestinal media,
lP

International journal of pharmaceutics 441(1-2) (2013) 693-700 DOI:

10.1016/j.ijpharm.2012.10.025.
na

[176] H. He, Y. Lu, J. Qi, Q. Zhu, Z. Chen, W. Wu, Adapting liposomes for oral drug
delivery, Acta pharmaceutica Sinica. B 9(1) (2019) 36-48 DOI: 10.1016/j.apsb.2018.06.005.
ur

[177] M. Niu, Y. Lu, L. Hovgaard, P. Guan, Y. Tan, R. Lian, J. Qi, W. Wu, Hypoglycemic
activity and oral bioavailability of insulin-loaded liposomes containing bile salts in rats: the
Jo

effect of cholate type, particle size and administered dose, European journal of pharmaceutics
and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische
Verfahrenstechnik e.V 81(2) (2012) 265-72 DOI: 10.1016/j.ejpb.2012.02.009.

[178] K.B. Choudhari, V. Labhasetwar, A.K. Dorle, Liposomes as a carrier for oral
administration of insulin: effect of formulation factors, Journal of microencapsulation 11(3)
(1994) 319-25 DOI: 10.3109/02652049409040461.

[179] S. Golocorbin-Kon, M. Mikov, M. Arafat, Z. Lepojevic, I. Mikov, M. Sahman-

Zaimovic, Z. Tomic, Cefotaxime pharmacokinetics after oral application in the form of
3alpha,7alpha-dihydroxy-12-keto-5beta-cholanate microvesicles in rat, European journal of
drug metabolism and pharmacokinetics 34(1) (2009) 31-6 DOI: 10.1007/BF03191381.

[180] K.M. Hosny, O.A. Ahmed, R.T. Al-Abdali, Enteric-coated alendronate sodium
nanoliposomes: a novel formula to overcome barriers for the treatment of osteoporosis,
Expert opinion on drug delivery 10(6) (2013) 741-6 DOI: 10.1517/17425247.2013.799136.

[181] S. De Robertis, M.C. Bonferoni, L. Elviri, G. Sandri, C. Caramella, R. Bettini,

Advances in oral controlled drug delivery: the role of drug-polymer and interpolymer non-

49
covalent interactions, Expert opinion on drug delivery 12(3) (2015) 441-53 DOI:
10.1517/17425247.2015.966685.

[182] C. Heinen, S. Reuss, S. Saaler-Reinhardt, P. Langguth, Mechanistic basis for

unexpected bioavailability enhancement of polyelectrolyte complexes incorporating BCS
class III drugs and carrageenans, European journal of pharmaceutics and biopharmaceutics :
official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 85(1)
(2013) 26-33 DOI: 10.1016/j.ejpb.2013.03.010.

[183] M. Thanou, S. Henderson, A. Kydonieus, C. Elson, N-sulfonato-N,O-

carboxymethylchitosan: a novel polymeric absorption enhancer for the oral delivery of
macromolecules, Journal of controlled release : official journal of the Controlled Release
Society 117(2) (2007) 171-8 DOI: 10.1016/j.jconrel.2006.11.002.

[184] M.E. Olivera, R.H. Manzo, F. Alovero, A.F. Jimenez-Kairuz, M.V. Ramírez-Rigo,

of
Chapter 13 - Polyelectrolyte-drug ionic complexes as nanostructured drug carriers to design
solid and liquid oral delivery systems, in: E. Andronescu, A.M. Grumezescu (Eds.),

ro
Nanostructures for Oral Medicine, Elsevier2017, pp. 365-408.

[185] F. Bigucci, B. Luppi, T. Cerchiara, M. Sorrenti, G. Bettinetti, L. Rodriguez, V. Zecchi,

-p
Chitosan/pectin polyelectrolyte complexes: selection of suitable preparative conditions for
colon-specific delivery of vancomycin, European journal of pharmaceutical sciences : official
re
journal of the European Federation for Pharmaceutical Sciences 35(5) (2008) 435-41 DOI:
10.1016/j.ejps.2008.09.004.
lP

[186] J.M. Miller, A. Dahan, D. Gupta, S. Varghese, G.L. Amidon, Enabling the intestinal
absorption of highly polar antiviral agents: ion-pair facilitated membrane permeation of
na

zanamivir heptyl ester and guanidino oseltamivir, Mol Pharm 7(4) (2010) 1223-34 DOI:
10.1021/mp100050d.
ur

[187] N. Samiei, S.M. Foroutan, F. Razipour, A. Zarghi, A. Shafaati, An investigation into

the ability of alendronate ion pairs to increase oral absorption, Int J Pharm 527(1-2) (2017)
Jo

184-190 DOI: 10.1016/j.ijpharm.2017.05.042.

[188] A. Sosnik, A.M. Carcaboso, R.J. Glisoni, M.A. Moretton, D.A. Chiappetta, New old
challenges in tuberculosis: potentially effective nanotechnologies in drug delivery, Adv Drug
Deliv Rev 62(4-5) (2010) 547-59 DOI: 10.1016/j.addr.2009.11.023.

[189] M. Sumaila, P. Ramburrun, P. Kumar, Y.E. Choonara, V. Pillay, Lipopolysaccharide

Polyelectrolyte Complex for Oral Delivery of an Anti-tubercular Drug, AAPS PharmSciTech
20(3) (2019) 107 DOI: 10.1208/s12249-019-1310-6.

[190] M. Srivastava, K. Kohli, M. Ali, Formulation development of novel in situ

nanoemulgel (NEG) of ketoprofen for the treatment of periodontitis, Drug delivery 23(1)
(2016) 154-66 DOI: 10.3109/10717544.2014.907842.

[191] C. Liu, H. Xu, Y. Sun, X. Zhang, H. Cheng, S. Mao, Design of Virus-Mimicking

Polyelectrolyte Complexes for Enhanced Oral Insulin Delivery, J Pharm Sci 108(10) (2019)
3408-3415 DOI: 10.1016/j.xphs.2019.05.034.

[192] B.F. Choonara, Y.E. Choonara, P. Kumar, D. Bijukumar, L.C. du Toit, V. Pillay, A
review of advanced oral drug delivery technologies facilitating the protection and absorption
50
of protein and peptide molecules, Biotechnology advances 32(7) (2014) 1269-1282 DOI:
10.1016/j.biotechadv.2014.07.006.

[193] A.C. Anselmo, S. Mitragotri, An overview of clinical and commercial impact of drug
delivery systems, Journal of controlled release : official journal of the Controlled Release
Society 190 (2014) 15-28 DOI: 10.1016/j.jconrel.2014.03.053.

[194] W. Shan, X. Zhu, M. Liu, L. Li, J. Zhong, W. Sun, Z. Zhang, Y. Huang, Overcoming
the diffusion barrier of mucus and absorption barrier of epithelium by self-assembled
nanoparticles for oral delivery of insulin, ACS nano 9(3) (2015) 2345-56 DOI:
10.1021/acsnano.5b00028.

[195] F. McCartney, J.P. Gleeson, D.J. Brayden, Safety concerns over the use of intestinal
permeation enhancers: A mini-review, Tissue barriers 4(2) (2016) e1176822 DOI:
10.1080/21688370.2016.1176822.

of
[196] L. Wan, X. Wang, W. Zhu, C. Zhang, A. Song, C. Sun, T. Jiang, S. Wang, Folate-

ro
polyethyleneimine functionalized mesoporous carbon nanoparticles for enhancing oral
bioavailability of paclitaxel, International journal of pharmaceutics 484(1-2) (2015) 207-17
DOI: 10.1016/j.ijpharm.2015.02.054. -p
[197] S.J. Cao, S. Xu, H.M. Wang, Y. Ling, J. Dong, R.D. Xia, X.H. Sun, Nanoparticles:
re
Oral Delivery for Protein and Peptide Drugs, AAPS PharmSciTech 20(5) (2019) 190 DOI:
10.1208/s12249-019-1325-z.
lP

[198] N. Zhang, Q.N. Ping, G.H. Huang, W.F. Xu, Investigation of lectin-modified insulin
liposomes as carriers for oral administration, International journal of pharmaceutics 294(1-2)
na

(2005) 247-59 DOI: 10.1016/j.ijpharm.2005.01.018.

[199] X. Zhang, J. Qi, Y. Lu, W. He, X. Li, W. Wu, Biotinylated liposomes as potential
ur

carriers for the oral delivery of insulin, Nanomedicine : nanotechnology, biology, and
medicine 10(1) (2014) 167-76 DOI: 10.1016/j.nano.2013.07.011.
Jo

[200] M. Garinot, V. Fievez, V. Pourcelle, F. Stoffelbach, A. des Rieux, L. Plapied, I. Theate,

H. Freichels, C. Jerome, J. Marchand-Brynaert, Y.J. Schneider, V. Preat, PEGylated PLGA-
based nanoparticles targeting M cells for oral vaccination, Journal of controlled release :
official journal of the Controlled Release Society 120(3) (2007) 195-204 DOI:
10.1016/j.jconrel.2007.04.021.

[201] T.E. Rajapaksa, M. Stover-Hamer, X. Fernandez, H.A. Eckelhoefer, D.D. Lo, Claudin
4-targeted protein incorporated into PLGA nanoparticles can mediate M cell targeted
delivery, Journal of controlled release : official journal of the Controlled Release Society
142(2) (2010) 196-205 DOI: 10.1016/j.jconrel.2009.10.033.

[202] K.B. Chalasani, G.J. Russell-Jones, A.K. Jain, P.V. Diwan, S.K. Jain, Effective oral
delivery of insulin in animal models using vitamin B12-coated dextran nanoparticles, Journal
of controlled release : official journal of the Controlled Release Society 122(2) (2007) 141-50
DOI: 10.1016/j.jconrel.2007.05.019.

51
Figure legends:
Figure 1: Relationship between BCS classification and physicochemical characteristics of
different drugs. ARCS: Absorption rate control step; PSA: Polar surface area; clogP: Partition
coefficients calculated by considering the contribution of functional groups; MW: Molecular
weight

Figure 2: Mechanism of gastrointestinal drug absorption. Any molecule can be absorbed

either by the paracellular pathway where they cross the epithelial lining through the space
between the enterocytes, or they can take the transcellular route, where they pass through the
enterocytes by crossing the apical membrane and the basolateral membrane, consecutively.
Transcellular absorption can be of two different types, passive diffusion and active transport.
In passive diffusion, absorption happens due to simple diffusion of the drug through the

of
membrane, whereas in active transport, the drug is taken up by some specific uptake
mechanism.

ro
Figure 3: Structural variability in different parts of the GI tract. A. Stomach B. Intestine C.
Peyer’s patches. -p
re
Figure 4: Structure of the intestinal epithelial junctional complex. The intestinal epithelium
is consists of a single layer of enterocytes. Adjacent cells are strongly bound with each other
lP

by different junctional complexes. As the junctional proteins are connected to the cytoskeletal
proteins, contraction of the cytoskeletal proteins leads to the opening of the junctional
complex, facilitating paracellular absorption.
na

Figure 5: Impact of mucus on drug absorption. The mesh structure produced by the mucus
ur

can prevent the diffusion of larger molecules. The viscoelastic nature of the mucus can also
impact drug diffusion. Depending on the pH of the medium, nature of the mucus changes,
Jo

influencing drug interaction as well as stiffness of the mucus. Ionic strength and charge also
have similar effects. Adapted from: Physicochemical properties of mucus and their impact on
transmucosal drug delivery [36].

Figure 6: Different physicochemical parameters of the drug influence passive diffusion. A.

Molecular weight: (a) very low molecular weight polar molecules can pass through the
aqueous pores present on the cell membrane. (b) Low to medium molecular weight non-polar
molecules can pass through the membrane by passive diffusion. (c) High molecular weight
molecules, irrespective of polarity, can only permeate the cell membrane by receptor-
mediated uptake. B, C. Lipophilicity and Ionization: Lipophilic and non-ionic molecules
(L, N) can only traverse the cell membrane by passive diffusion, which route is non-
accessible for hydrophilic and ionic molecules (H, I). D. Solubility: Solubilization is highly
important. Only solubilized molecules can be absorbed; insoluble molecules cannot undergo
diffusion due to the formation of large aggregates.
Figure 7: Different prodrugs used for enhancing oral absorption.

52
Figure 8: Mode of action of different paracellular permeation enhancers. Most of the
paracellular permeation enhancers act by modulating contraction of the cytoskeletal proteins.
They may act through PLC-IP3/DAG pathway (fatty acids), leading to cytoskeletal
contraction and increased tight junction permeability, or can deplete extracellular Ca2+
(EDTA), induces PKC which leads to actomyosin contraction and loosening of the apical
junction. Some can directly reduce the amount of junctional proteins (acylcarnitine).

Figure 9: Mode of action of different transcellular permeation enhancers. Two important

examples of transcellular permeation enhancers are surface-active agents and bile acids. A.
Surface active agents mostly act as a membrane destabilizing agent and produce either
fluidization or disintegration in the cell membrane, improving the transport of molecules
through the membrane. B. Bile acids enhance transcellular permeation through a variety of
pathways. Due to their amphiphilic nature, bile acids can improve the solubility of non-polar
drugs by micelle formation and enhance their interaction with the membrane. They can entrap

of
polar drugs by forming reverse-micelles. Reverse-micelles can get integrated into the

ro
membrane and form aqueous pores. They can also get integrated into the membrane,
increasing the fluidity of the membrane. Bile acids can also inhibit the function of P-gp,
preventing drug efflux. -p
re
Figure 10: Different nanoparticle uptake pathways active in the enterocytes. Receptor-
mediated endocytosis, caveolae or clathrin-mediated pathway, micropinocytosis, and
lP

phagocytosis are the major uptake processes observed. In the conventional pathway (blue
arrow), cargo from all these uptake pathways may end up in the lysosome, leading to the
metabolism of the cargo. An alternate pathway exists (red arrow), where endosomes may
na

transport the cargo to the endoplasmic reticulum through Golgi complex, from where they
may get exocytosed by the secretory pathway.
ur
Jo

53
Jo
ur
na
lP
re
-p
ro
of
Jo
ur
na
lP
re
-p
ro
of
Jo
ur
na
lP
re
-p
ro
of
Jo
ur
na
lP
re
-p
ro
of
Jo
ur
na
lP
re
-p
ro
of
Jo
ur
na
lP
re
-p
ro
of
Jo
ur
na
lP
re
-p
ro
of
Jo
ur
na
lP
re
-p
ro
of
Jo
ur
na
lP
re
-p
ro
of
Jo
ur
na
lP
re
-p
ro
of
Declaration of interests

The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

of
ro
-p
re
lP
na
ur
Jo